Preface

Advances in medical oncology

Guest Editor

The specialty of medical oncology has traditionally been characterized by

steady, incremental gains in the eﬃcacy of therapy and only rarely by star-
tling breakthroughs. This is as true in veterinary medical oncology as it is in
the human specialty. The past decade in veterinary medical oncology has
produced modest advances in the treatment of several cancers. We have
added new compounds to our therapeutic arsenal and reﬁned the use of
standard anticancer chemotherapy drugs through improvements in dose and
scheduling of combination therapy. Early intervention has contributed to
this increased treatment success. Improved diagnostics, better understand-
ing of the biologic behavior of individual tumors provided by new prog-
nostic and predictive markers, and an attitudinal shift in the minds of
veterinary practitioners and the pet owning public have all contributed to
increased success in medical oncology. Patient outcomes have also been
improved through reﬁnements in multimodality therapy, combining the
expertise of specialists in medical, radiation, and surgical oncology. This
issue of Veterinary Clinics of North America: Small Animal Practice presents
updated information on the treatment of the diseases commonly encoun-
tered in veterinary oncology. I hope that the information provided will
prove useful to students, specialists in training, and practitioners.

Barbara E. Kitchell, DVM, PhD

Vet Clin Small Anim

33 (2003) xi–xiii

0195-5616/03/$ - see front matter

doi:10.1016/S0195-5616(03)00022-6

The commonly encountered tumors that impair the longevity and quality

of life of our patients are reviewed in the chapters by Drs. Fan, London,
Chun and de Lorimier, Rassnick, Smith, Hauck, Sorenmo, Henry, and
Klein. A great deal of the information in their reviews of the literature was
garnered from individual case reports, small case series, and retrospective
studies. I am sure these authors would agree that there is an urgent need for
prospective, appropriately blinded and randomized trials in veterinary med-
ical oncology. In the past few years, the ﬁrst large industry sponsored multi-
center trial with intent to achieve veterinary drug licensing was conducted,
and more such trials are ongoing or planned. The organization of multi-
center trial groups in the US, as well as the move into prospective work by
the venerable Veterinary Cooperative Oncology Group, promises to speed
progress in our specialty. The conduct of well-designed and ethically con-
ducted prospective trials is critical to our future.

Recent publications and abstracts describing the use of several agents

new to veterinary oncology are summarized here. The reader is advised to
consult veterinary medical oncology specialists for new information and
guidance regarding the use of these compounds. Clients should be informed
of the oﬀ-label status of all anticancer chemotherapy agents used in vet-
erinary medicine, and appropriate biosafety precautions should be
emphasized for all who come in contact with these drugs. Improved un-
derstanding of the mechanisms of drug resistance provided by Dr. Phil
Bergman’s chapter may prove helpful in explaining individual case out-
comes and in deﬁning future directions for therapy.

The past decade has seen molecularly targeted therapeutic approaches

advance as an intense focus in human medicine, with new compounds de-
veloped through research in traditional pharmacology, recombinant biolog-
ics, and immunotherapy. Some of these novel agents have changed the
standard of care for human patients with disseminated cancers, and more
hold promise in the near future. The exquisitely selective nature of molecular
targeting permits enhanced eﬃcacy while limiting adverse eﬀects. Unfortu-
nately, it is this precise selectivity in molecular targeting that makes the
translational use of these agents in clinical companion animal practice dif-
ﬁcult, if not impossible. The coming decade will be a challenging and
exciting one for veterinary medical oncology as we begin to move

en-

thusiastically into this new ﬁeld. Clearly, we have work to do.

I wish to thank all of the contributors for the their thorough and thought-

ful reviews. The residents in medical oncology at the University of Illinois,
especially Louis-Philippe de Lorimier and Amy Wiedemann, were in-
valuable for editorial input regarding both content and style. Finally, John
Vassallo at W.B. Saunders is a man of formidable patience, good humor, and

xii

B.E. Kitchell / Vet Clin Small Anim 33 (2003) xi–xiii

editorial skill. I owe him abundant thanks, a drink, and a case of Maalox for
putting up with me.

Barbara E. Kitchell, DVM, PhD

College of Veterinary Medicine

1008 West Hazelwood Drive

University of Illinois

Urbana, IL 61802, USA

E-mail address: kitchell@uiuc.edu

xiii

B.E. Kitchell / Vet Clin Small Anim 33 (2003) xi–xiii

Lymphoma updates

Timothy M. Fan, DVM

Department of Veterinary Clinical Medicine, Veterinary Teaching Hospital,

College of Veterinary Medicine, University of Illinois,

100 West Hazelwood Drive, Urbana, IL 61802, USA

Lymphoma arises from the malignant clonal expansion of lymphoretic-

ular cells. Primary lymphoid organs, such as the bone marrow and thymus,
as well as secondary lymphoid structures, including the lymph nodes, spleen,
and gut-associated lymphoid tissue (GALT), are potential sites of neoplastic
transformation. As a result of continuous lymphocyte traﬃcking, the origin
of lymphoma is not restricted solely to primary and secondary lymphoid
organs. Malignant transformation of lymphocytes can occur virtually
anywhere. Common extranodal sites of lymphoma include the skin, eye,
central nervous system, testis, and bone [1].

Lymphoma is reported to be the most common hematopoietic neoplasm

aﬀecting the dog and cat. In dogs, the incidence of lymphoma has been
reported to approach 0.1% in susceptible older individuals, with an annual
incidence rate of 84 per 100,000 dogs at risk [2]. The precise etiology of
lymphoma in the dog has not been identiﬁed. Several hypotheses have been
investigated but have not been deﬁnitely proven. Hypothesized etiologies for
canine lymphoma include retroviral infection, environmental contamination
with phenoxyacetic acid herbicides, magnetic ﬁeld exposure, chromosomal
abnormalities, and immune dysfunction [3–8].

In cats, feline leukemia virus (FeLV) has been identiﬁed as a biologic car-

cinogen resulting in malignant lymphocyte transformation [9,10]. Historical
epidemiologic investigations before the wide use of preventative FeLV vac-
cines estimated the annual incidence of feline lymphoma to be 200 per
100,000 cats at risk [11]. With the development of efﬁcacious FeLV vaccines
in conjunction with the practice of early detection and removal of viremic
cats from the general population, the incidence of FeLV-induced lymphoma
has been dramatically reduced [12].

Vet Clin Small Anim

33 (2003) 455–471

E-mail address:

t-fan@uiuc.edu

0195-5616

/03/$ - see front matter

doi:10.1016/S0195-5616(03)00005-6

Although lymphoma has been reported to occur in both dogs and cats,

response to therapy, disease-free intervals (DFIs), and survival times diﬀer
not only between species but among individuals of the same species. The
nonuniform response to treatment among dogs and cats with lymphoma is
caused, in part, by individual variances in host immunity and constitutional
status. A more signiﬁcant determinant of therapeutic response is dictated
by the variable biologic behavior of lymphoma. Some diﬀerences in bio-
logic behavior have been attributed to factors like anatomic location
and histologic grade [13]. An additional variable that seems to inﬂuence
the biologic behavior of lymphoma is immunophenotype [14,15]. Immuno-
phenotyping identiﬁes selected surface glycoproteins known as cluster of
differentiation (CD) antigens on lymphocytes and accessory immune cells.
Identifying CD antigens requires the application of antibodies speciﬁc for
differentiation antigens. Lymphocyte CD antigens serve multiple functions,
including intracellular signaling, cell-to-cell communication, and lympho-
cyte trafﬁcking [16].

Diagnostically important CD antigens for the characterization of

malignant lymphoma include CD3 and CD79. CD3 is a complex of ﬁve
polypeptides associated with the T-cell receptor (TCR). High-aﬃnity binding
of a TCR with its cognate major histocompatibility complex containing
processed peptide allows intracellular signaling to be mediated through the
CD3 complex [16]. Demonstration of the CD3 antigen on malignant
lymphocytes identiﬁes the lymphoma as being of T-lymphocyte origin.
Similarly, CD79 exists as a heterodimer associated with the B-cell receptor
(BCR) and is required for B-lymphocyte intracellular signal transduction
[16]. Demonstration of the CD79 antigen on malignant lymphocytes delin-
eates the malignant lymphoid population as being of B-lymphocyte origin.
T-cell lymphomas tend to be more biologically aggressive than B-cell
lymphomas, resulting in shorter remission and survival times [14,15]. Rarely,
malignant lymphocytes fail to demonstrate either CD3 or CD79 antigens. In
such instances, the cell of origin cannot be determined, and these lymphomas
are classiﬁed as being derived from null cells (nonreactive with any speciﬁc
lymphocyte antibody).

Treatment of lymphoma in the dog and cat is often rewarding. With con-

ventional chemotherapeutic protocols, most patients respond favorably.
Dogs treated with cyclophosphamide, doxorubicin, vincristine, and predni-
sone (CHOP) style protocols generally achieve a complete response rate of
80% to 90%, with expected median survival times of 1 year [17,18]. Although
chemotherapy is reasonably effective in the treatment of feline lymphoma,
response rates and survival times tend to be worse in cats than in dogs. Cats
treated with chemotherapy typically achieve response rates and survival
times of 50% to 70% and 4 to 6 months, respectively [19–21].

In conjunction with systemic chemotherapy, the implementation of

additional therapeutics, including surgery, radiation therapy, and biologic
response modiﬁers, may augment treatment success. Although a cure for

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/ Vet Clin Small Anim 33 (2003) 455–471

lymphoma remains elusive for most veterinary patients, the rapid accumu-
lation of knowledge elucidating the fundamentals of cancer biology inevi-
tably translates into the development of better diagnostic techniques, more
accurate prognostic indicators, and superior treatment options. The focus of
this article is to highlight some new developments in the diagnosis, prognosis,
and therapeutic management of lymphoma in the dog and cat.

New developments in canine lymphoma

New diagnostic tools

Immunoglobulin rearrangement

Nonpainful generalized peripheral lymphadenopathy is the most common

clinical manifestation of canine lymphoma [1]. Traditionally, the deﬁnitive
diagnosis of lymphoma has been made by either cytologic or histologic
assessment of affected sites. Fine-needle aspiration and cytologic evaluation
of enlarged peripheral lymph nodes usually provide a sufﬁcient quantity of
cells to identify a malignant population. Tissue biopsy of affected lymph
nodes by incisional or excisional techniques not only provides adequate
tissue sample size but preserves nodal architecture. Although cytologic and
histologic diagnosis is reliable and practical, in rare instances, these tech-
niques fail. Being able to identify a population of malignantly transformed
lymphocytes deﬁnitively would be valuable when cytologic or histologic
assessment is inconclusive.

Based on the monoclonality theory of cancer, all tumors originally arise

from a common progenitor transformed cell [22,23]. This theory also applies
to lymphoid malignancies; therefore, lymphoma should be considered a
clonal expansion of one malignant lymphocyte. Lymphocytes serve a critical
immune function by protecting the host from a diverse spectrum of
pathogens. For lymphocytes to execute their programmed effector functions,
they must recognize their cognate antigens through specialized antigen-
binding sites, which are unique to each individual lymphocyte [24]. In the
case of a T lymphocyte, the unique antigen-binding site is referred to as the
TCR. Likewise, in the case of a B lymphocyte, the antigen-binding site,
referred to as the BCR, is also distinctive. Because each TCR or BCR is
unique, isolating the DNA sequences coding these protein structures allows
for the differentiation of lymphocytes based on these DNA sequences [24].

The use of polymerase chain reaction (PCR) technology allows for the

ampliﬁcation of DNA sequences coding for either the TCR or BCR in
lymphocyte populations. In tissue containing lymphocytes derived from
multiple clones, such as is found in lymphoid hyperplasia, PCR ampliﬁcation
of the DNA encoding the antigen-binding region produces amplicons of
varying size. In lymphoid malignancies, however, PCR ampliﬁcation of the
DNA encoding the antigen-binding region produces a PCR product of
predominantly one size, indicating the presence of a clonally expanded

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/ Vet Clin Small Anim 33 (2003) 455–471

lymphocyte population [25]. Although this PCR technique is highly sensitive,
the methodology is relatively laborious and should only be used when the
clinician has a high index of suspicion for lymphoma and where conventional
cytologic and histologic diagnostic techniques have failed to be deﬁnitively
diagnostic.

Telomerase activity

Telomeres are long stretches of tandem hexanucleotide repeats

(TTAGGG) found at the 39 end of chromosomes. Functionally, telomeres
serve as protective ‘‘caps,’’ preventing deleterious chromosomal degradation
and recombination [26], Telomere length dictates the proliferative capacity of
normal cells. Subsequent to each replicative cycle, telomere length is reduced.
When terminal telomere length is reached, cellular replication is halted and
the cell enters into a state of cellular senescence [27]. One mechanism by
which malignantly transformed cells may circumvent reductions in telomere
length, thereby achieving cellular immortality, is by the activity of an enzyme
called telomerase. Telomerase is a ribonucleoprotein DNA polymerase, and
its RNA component provides the primer for telomere repeat synthesis. Most
normal somatic cells do not express telomerase, but this enzyme activity has
been detected in up to 90% of human cancers [28].

The utility of detecting telomerase activity as a diagnostic tool for canine

lymphoma has been investigated. Two studies in dogs measuring telomerase
activity by either enzyme-linked immunosorbent assay (ELISA) or a telomere
repeat ampliﬁcation protocol (TRAP) methodology failed to identify any
statistical diﬀerence in enzymatic activity between normal and malignantly
transformed lymph nodes [29,30]. Results from these two studies suggest that
telomerase activity may not be a valuable diagnostic marker for the discrimi-
nation of malignant lymphocytes from normal lymphoid cells. In a separate
investigation using a TRAP assay, however, strong telomerase activity was
identiﬁed in all malignant lymphoid tissues evaluated (n = 28), whereas only
weak telomerase activity could be demonstrated in few normal lymph nodes
(3 of 12 lymph nodes) [31]. Because of this divergence in results, additional
research is required to fully explore the potential for telomerase activity
as a diagnostic tool for detection of malignant transformation of canine
lymphocytes.

New prognostic markers

Because diﬀerent types of lymphocytes may undergo malignant trans-

formation, lymphomas manifest a wide spectrum of clinical and biologic
behaviors. Given the emotional, time, and ﬁnancial commitments required in
the course of lymphoma treatment, providing reliable information address-
ing response rates, DFIs, and survival times is important for owners in their
decision-making process. A client’s decision to treat a pet with traditional
chemotherapy may depend on the likelihood of achieving a favorable

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response. Advances in our understanding of cancer biology have led to the
discovery of new predictive markers that facilitate more accurate prognos-
tication of canine lymphoma.

Serum alpha 1-acid glycoprotein concentrations

The presence of infectious, neoplastic, or immune-mediated diseases

results in the liberation of inﬂammatory cytokines from immune cells.
Cytokines, such as interleukins, interferons, and tumor necrosis factor syn-
thesized and released by the host organism, can directly and indirectly
augment host immunity. Subsequent to cytokine release, hepatocytes
synthesize a variety of acute-phase proteins that enhance antigen-presenting
cell phagocytosis [32]. One acute-phase protein, serum alpha 1-acid
glycoprotein (AGP), has been demonstrated to have value in predicting
lymphoma relapse in the dog. In dogs with lymphoma, baseline serum AGP
levels are signiﬁcantly elevated when compared with those of healthy control
dogs. After complete remission induced by doxorubicin therapy, dogs with
lymphoma have serum AGP concentrations similar to those of healthy dogs.
Mean serum AGP concentrations 3 weeks before and at the time of relapse
are again signiﬁcantly elevated, achieving the concentration levels measured
before treatment [33]. Although the investigation of AGP has proven to be
a statistically signiﬁcant factor in predicting lymphoma relapse in dogs,
interpretative limitations exist. With any nonspeciﬁc inﬂammatory marker
like AGP, documented elevations warrant investigation for any systemic
inﬂammatory nidus, whether neoplastic or nonneoplastic.

Matrix metalloproteinase 2 and 9 expression

Multiple events are required for the successful distant spread of

a neoplasm. Being able to migrate through extracellular matrix (ECM) is
one prerequisite in the acquisition of a metastatic phenotype. Matrix
metalloproteinases (MMPs) are a family of zinc-dependent proteases capable
of degrading type IV collagen, resulting in ECM degradation [34]. Although
more than 20 distinct MMPs have been identiﬁed, MMP 2 and 9 have been
investigated most intensively for their potential roles in cancer metastasis. In
human cancer patients, MMP 2 and 9 expression is elevated in metastatic
neoplastic cells [34,35,36]. Similarly, alterations in MMP 2 and 9 levels have
been documented in canine neoplasms, including lymphoma, osteosarcoma,
and mast cell tumors [37–39]. In dogs with lymphoma, MMP 2 and 9 con-
centrations decreased signiﬁcantly after treatment with chemotherapy and
remained low until 6 to 12 weeks before recurrence of lymphoma. Higher
concentrations of pro-MMP 2 and pro-MMP 9 as well as active MMP 9 in
tumor tissue correlated with shorter remission times after doxorubicin
therapy [38]. Measurement of MMP activity by gelatin zymography or MMP
gene expression by microarray analysis is not commercially available,
thereby restricting MMP analysis as a predicative marker to research
settings.

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Monoclonal antibody C219 immunohistochemistry against P-glycoprotein

Although treatment with chemotherapy is intended to eradicate all cancer

cells, this is rarely the outcome in veterinary cancer patients. In part, failure to
cure dogs routinely with lymphoma stems from owner economic constraints
as well as from a lack of necessary support systems (ie, bone marrow
transplantation) required for managing the critically ill patient subsequent
to dose-intense chemotherapy. In addition to these exogenous limitations,
malignantly transformed lymphocytes may evade the cytotoxic eﬀects of
chemotherapy by the upregulation of resistance mechanisms.

Several resistance mechanisms have been documented in neoplastic cells,

including defective apoptotic pathways, enhanced free radical scavenging
capabilities, and upregulated DNA repair machinery [40]. Multidrug-
resistant (MDR) phenotype is used to describe neoplastic cells that are
resistant to the cytotoxic effects of certain antineoplastic agents. The
overexpression of a drug efﬂux pump called P-glycoprotein (P-gp) has been
examined in dogs with lymphoma [41,42]. Functionally, P-gp prevents the
retention of cytotoxic agents within neoplastic cells, allowing for escape
from lethal DNA damage.

The expression of P-gp on canine malignant lymphocytes has been dem-

onstrated by immunohistochemistry using the C219 monoclonal antibody.
The frequency of positively staining malignant lymphocytes at disease relapse
was signiﬁcantly higher than that found before the initiation of chemother-
apy. Furthermore, greater positive staining with C219 before initial
chemotherapy was inversely proportional to remission and survival times.
The frequency of positively staining malignant lymphocytes at relapse was
also inversely related to the time from relapse to death [43]. Several logical
deductions may be concluded from this investigation. First, P-gp expression
may be upregulated subsequent to chemotherapy exposure. Second, drug-
naive lymphomas with inherently greater expression of P-gp may be more
resistant to the cytotoxic effects of traditional chemotherapeutic agents,
especially doxorubicin and vincristine. Finally, overexpression of P-gp at
disease relapse may result in a poor response to rescue chemotherapeutic
agents and therefore shorter survival times. Although the expression of P-gp
maybe used as a surrogate marker for predicting response to therapy, DFIs,
and survival times, it should be emphasized that acquisition of an MDR
phenotype does not necessarily require overexpression of P-pg. Additional
mechanisms contributing to chemotherapeutic resistance, such as the
overexpression of glutathione-S-transferase, have also been demonstrated
in dogs with lymphoma. Functionally, glutathione-S-transferase catalyzes
the conjugation of glutathione with free radicals, thereby inactivating the
cytotoxic effects of several anticancer drugs, including doxorubicin,
cyclophosphamide, and platinum-based agents [40]. In dogs with lymphoma,
serum glutathione-S-transferase levels have been evaluated before disease
treatment, during treatment, and at disease relapse. Signiﬁcant elevations of
serum glutathione-S-transferase were present at the time of disease relapse,

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supporting the notion that additional mechanisms contribute to the
development of drug resistance in canine lymphoma [44].

Immunophenotyping: B5 antigen expression

Glycoproteins found on the surface of cells perform distinct functions,

including the mediation of cell-to-cell communication and the propagation of
intracellular signals. In lymphocytes, the identiﬁcation of speciﬁc surface
glycoproteins known as CD antigens allows cells to be classiﬁed into discrete
subsets. CD antigens can be identiﬁed by monoclonal antibodies; this process
is termed immunophenotyping. CD antigens identiﬁed on malignant lympho-
cytes that have prognostic importance are CD3 and CD79. Lymphomas
expressing CD3 but not CD79 are derived from T-cell lineage. Lymphomas
positive for CD79 but not CD3 are of B-cell origin. Multiple investigations
have consistently demonstrated dogs diagnosed with lymphoma of T-cell
lineage have shorter DFIs and survival times than dogs with B-cell
lymphoma [14,15,45]. In addition to CD3 and CD79, a nonimmunoglobulin
B-cell marker called B5 also seems to have prognostic value. The B5 antigen
is normally expressed on nonneoplastic B lymphocytes. Dogs with B-cell
lymphomas expressing lower than normal levels of the B5 antigen experience
shorter DFIs and survival times [15]. The exact function of the B5 antigen is
unknown. Investigating the functional properties of the B5 antigen in normal
B lymphocytes may provide an explanation for correlating reduced
expression with poorer clinical outcome in canine lymphoma.

New therapeutic modalities

In dogs, systemic chemotherapy for the management of multicentric

lymphoma is rewarding. With the use of multiagent doxorubicin-based
protocols, response rates are reported to exceed 90% [45,46]. Traditional
chemotherapeutic protocols are composed of two distinct treatment phases
referred to as ‘‘induction’’ and ‘‘maintenance.’’ During the induction phase,
aggressive weekly treatments are instituted with the intent of rapidly reducing
the burden of cancer cells. After induction therapy, maintenance chemo-
therapy may be used to thwart the rapid regrowth of residual cancer cells.
The necessity of maintenance chemotherapy in the successful management of
canine lymphoma has been challenged. Several investigations demonstrate
favorable DFIs and survival times in dogs treated for lymphoma by
aggressive induction protocols or radiation therapy. The use of either dose-
intense chemotherapy or adjunctive radiation therapy may offer a reasonable
alternative for the treatment of lymphoma without the use of chronic long-
term maintenance chemotherapy.

Chemotherapy treatment options that exclude maintenance therapy

In one study, dogs with multicentric lymphoma were treated with a dose-

intense modiﬁed version of the University of Wisconsin (UW)–Madison

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induction protocol without maintenance (Table 1). Compared with the
traditional UW-Madison regimen, the dose-intense protocol used a greater
dose and number of treatments of doxorubicin (37.5 mg

4 versus 30

3) and cyclophosphamide (250 mg/m

4 versus 200 mg/m

during a 25-week induction period. Interestingly, dogs treated with the dose-
intense induction protocol with no maintenance achieved statistically similar
DFIs and survival times as a historical control population of dogs treated
with the standard UW-Madison protocol (induction with maintenance).
Death because of treatment-related toxicity was signiﬁcantly higher in dogs
treated with the dose-intense version of the UW-Madison induction protocol
(odds ratio = 8.8) [45]. Results from this study support the notion that
aggressive induction therapy without maintenance can provide equivalent
DFIs and survival times when compared with more traditional chemother-
apeutic regimens that include maintenance therapy. Unfortunately, the high
incidence of treatment-induced morbidity and mortality makes this speciﬁc
dose-intense protocol unattractive to many veterinary oncologists.

In lieu of the unacceptable toxicity observed with this protocol, a second

investigation using a slightly diﬀerent dose-intense UW-Madison induction
protocol was evaluated. In this second study, the only diﬀerence between the
experimental dose-intense protocol and the traditional UW-Madison
regimen was the dose and number of treatments of cyclophosphamide
(250 mg

4 versus 200 mg/m

2) and the number of treatments of

doxorubicin (30 mg

4 versus 30 mg/m

3) during a 25-week induc-

tion period. Results demonstrated that dogs treated with the dose-intense
induction protocol without maintenance achieved statistically similar
DFIs and survival times as a historical control population of dogs treated
with the standard UW-Madison protocol (induction with maintenance).

Table 1
Comparison of University of Wisconsin–Madison canine maintenance-free lymphoma protocols

Week

1 2 3 4 5 6 7 8 9 11 13 15 17 19 21 23 25

Chun et al (2000)

-asparaginase (400 IU

/kg SC)

Vincristine (0.7 mg

IV)

Cyclophosphamide (250 mg

IV)

Doxorubicin (37.5 mg

IV)

Prednisone (2 mg

/kg PO q 24 h

7 d, then 1.5 mg/kg PO q 24 h 7 d, then 1.0 mg/kg PO q

24 h

7 d, then 0.5 mg/kg PO q 24 h 7 d, then stop)

Garrett et al (2002)

-asparaginase (400 IU

/kg SC)

Vincristine (0.7 mg

IV)

Cyclophosphamide (250 mg

IV)

Doxorubicin (30.0 mg

IV)

Prednisone (2 mg

/kg PO q 24 h

7 d, then 1.5 mg/kg PO q 24 h 7 d, then 1.0 mg/kg PO q

24 h

7 d then 0.5 mg/kg PO q 24 h 7 d, then stop)

Abbreviations:

IV, intravenous; PO, by month; q, every day; SC, subcutaneous.

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Furthermore, there was no statistical difference in treatment-associated
morbidity or mortality between the experimental and historical control
populations [46]. The results from this study suggest that moderately dose-
intense induction protocols without maintenance may be well tolerated and
can provide equivalent DFIs and survival times as traditional induction

maintenance protocols.

Half-body radiation therapy in lieu of maintenance chemotherapy

Malignant lymphocytes may undergo apoptosis after irreparable DNA

damage caused by ionizing radiation [47,48]. Despite the exquisite sensitivity
of lymphocytes to the cytotoxic effects of radiation, its use in veterinary
oncology has been traditionally reserved for ‘‘crisis’’ situations, where the
rapid reduction of localized tumor volume is necessary. In treating diffuse
lymphoid malignancies, such as canine multicentric lymphoma, delivering
radiation to the entire body would be required to maximize the therapeutic
response. Unfortunately, without autologous bone marrow transplanta-
tion (AuBMT) or other hematologic support, dogs treated with total body
irradiation (TBI) experience profound bone marrow suppression and high
mortality rates. Even with extremely low-dose TBI (total dose of 1 Gy at
10 cGy

/min), dogs with lymphoma experience grade III and IV thrombo-

cytopenia [49]. To derive the beneﬁts of TBI without the complications,
sequential half-body irradiation (HBI) of dogs with lymphoma in remission
has been investigated. In one study, 29 dogs were treated with HBI after
successful induction chemotherapy. HBI was delivered to the cranial half of
the body on week 8 and to the caudal half of the body on week 12. Side effects
of HBI included self-limiting alopecia, fever, and anorexia. Conclusions from
this study indicated that 800-cGy HBI is well tolerated and is as effective as
maintenance chemotherapy for dogs in remission with lymphoma [50]. In
a second study, 38 dogs with lymphoma were treated with HBI after
successful chemotherapy induction. After an 11-week induction protocol,
radiation was administered at a dose of 800 cGy per half body given in two
consecutive 400-cGy fractions. Cranial and caudal HBI was separated by a 3-
week interval and delivered on weeks 13 and 16, respectively. HBI resulted in
mild reductions in neutrophil and platelet numbers. Survival times of dogs
treated with HBI were comparable to those of dogs treated with traditional
maintenance chemotherapy [51].

New rescue therapies

Rescue chemotherapeutics

Although lymphoid neoplasms initially respond well to systemic chemo-

therapy, the development of resistant clones with eventual disease relapse is
common. Additional remissions after administration of rescue chemother-
apeutic agents are frequently achievable and may substantially prolong the
survival times of dogs and cats with lymphoma. The development of new

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rescue chemotherapeutics has been limited; however, the use of an oral and
inexpensive alkylating agent called lomustine has been demonstrated to be an
eﬀective rescue agent in dogs.

The safety and eﬃcacy of single-agent lomustine have been evaluated in

the treatment of canine lymphoma. Dogs with lymphoma relapse receiving
lomustine dosed at 90 mg

achieved a response rate of 28% and median

duration of remission of 86 days. The acute dose-limiting toxicity in this
study was neutropenia, and cumulative thrombocytopenia occurred in dogs
receiving continued long-term lomustine therapy [52]. Although lomustine
has not been formally reported to be an effective rescue agent for feline
lymphoma, anecdotal reports suggest lomustine’s efﬁcacy for the treatment
of various round cell neoplasms in the cat [53]. Cats may be safely dosed at
50 to 60 mg

every 6 weeks [54]. Alternatively, cats may be given a single

10-mg capsule every 21 days with a low incidence of hematologic side effects
[53].

Rescue radiation therapy

Malignant lymphocytes expressing MDR phenotypes are aﬀorded a

survival advantage when exposed to cytotoxic agents. Attempting to treat
resistant lymphocyte clones, even with novel chemotherapeutic agents, still
may result in disease progression. Because of the limitations of chemical
cytotoxic agents in conjunction with the inherent radiosensitivity of malig-
nant lymphocytes, the use of total lymphoid irradiation (TLI) for conﬁrmed
chemoresistant lymphoma has been investigated. Eleven dogs with conﬁrmed
MDR lymphoma were treated with total nodal irradiation. A total dose of
200 cGy given in six fractions over 2 weeks was administered to all aﬀected
peripheral lymph nodes. Complete remission was achieved in all treated
lymph nodes by the fourth radiation fraction. In the 9 dogs available for
follow up, the median survival time was 143 days [55].

Future therapies

Although aggressive chemotherapy and radiation therapy serve as excel-

lent treatment modalities for the management of lymphoma, newer treat-
ment options are continually being researched and developed. One type of
innovative therapy that demonstrates great promise is the selective targeting
of speciﬁc molecular proteins expressed on malignant lymphocytes. Trans-
lational use of these innovative therapies in dogs and cats may provide better
DFIs and survival times than what is currently achievable with existing
chemotherapy.

Recombinant immunotoxins

Lymphocytes express surface glycoproteins that serve as receptors for

soluble or membrane-bound cytokines. Binding of cytokines to their cognate
receptors elicits diverse cellular responses, including proliferation, diﬀeren-

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tiation, and apoptosis [56]. Interleukin-2 (IL-2) is a cytokine essential for the
initiation of a robust immune response. Both B and T lymphocytes express
the IL-2 receptor (IL-2R) on their plasma membrane surface. The receptor is
a heterotrimer consisting of three distinct subunits (a, b, and c). In the
inactive state, the IL-2R is composed of constitutively expressed b and c
subunits. After cellular activation, however, gene transcription with
subsequent protein translation of the a subunit is induced. Concurrent
expression of all three subunits confers high-afﬁnity IL-2 binding [57].

The expression of IL-2R has been identiﬁed on malignant T lymphocytes

and can be used as a molecular target for recombinant immunotoxins [58].
Denileukin diftitox (Ontak) is one such molecule, and consists of recom-
binant human IL-2 (rhIL-2) fused to a truncated diphtheria toxin [59]. This
provides a speciﬁc delivery system through which diphtheria toxin is delivered
to those malignant lymphocytes expressing the high-afﬁnity IL-2R. Both
intermediate and high-afﬁnity IL-2Rs have been demonstrated in canine T-
cell lymphomas and natural killer cell leukemias [60,61]. The translational
use of recombinant immunotoxins for the management of canine T-cell
malignancies remains to be investigated, and premature use of denileukin
diftitox should be discouraged. Without adequate pharmacokinetic studies
of denileukin diftitox in healthy and tumor-bearing dogs, the safe use of
denileukin diftitox cannot be supported.

Advances in immunotherapy

Monoclonal antibodies provide a method for the accurate delineation of

cell types. In addition to their diagnostic utility, monoclonal antibodies can
be used for the treatment of many cancers, including lymphoid malignancies
[62]. In human cancer patients, rituximab (Rituxan), a monoclonal antibody
recognizing the CD20 surface antigen found on B lymphocytes, is effective
for the treatment of B-lymphocyte malignancies [63]. Rituximab works
by means of several mechanisms, including enhancement of antibody-de-
pendent cellular cytotoxicity and intracellular calcium dysregulation [64,65].
Approximately 25% to 50% of human patients with B-cell malignancies
achieve a therapeutic response to rituximab [66]. Because rituximab’s
cytotoxic properties are partially dependent on host immunity, newer and
more potent monoclonal antibodies conjugated to radionuclides have been
developed. Currently, tositumomab (Bexxar) and ibritumomab tiuxetan
(Zevalin), two recombinant monoclonal antibodies against CD20 conjugated
to radioisotopes, are being evaluated for the treatment of B-lymphocytic
neoplasms in people [62].

Despite the eﬀectiveness of monoclonal-based therapies in people, several

pitfalls prevent their immediate translational use in dogs with B-lymphocyte
malignancies. One question that remains is the level of CD20 in the dog and
cat. Although some investigations support the identiﬁcation of CD20 in
canine B lymphocytes, the extracellular portion of this surface antigen that

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is responsible for monoclonal antibody binding has not been identiﬁed on
malignant canine B lymphocytes [67]. Even if CD20 were expressed on
canine B-cell lymphomas, subtle conformational differences between the
human and canine CD20 recognition epitope could have a negative impact
on monoclonal antibody binding.

In addition to questionable CD20 expression, several immunologic

limitations may stand in the way of using commercially available mono-
clonal antibodies. A portion of rituximab’s tumoricidal eﬀect is mediated
by antibody-dependent cellular cytotoxicity (ADCC). The favorable binding
kinetics of human phagocyte F

receptors and the F

fragment of rituximab

ensure optimal conditions for ADCC. Binding kinetics of canine phagocyte
F

receptors for rituximab may be of lower afﬁnity, resulting in an at-

tenuated ADCC response. Furthermore, because rituximab, tositumomab,
and ibritumomab tiuxetan are either humanized or chimeric proteins, their
prolonged use in the dog may result in the formation of neutralizing canine
antihuman antibodies. The presence of these neutralizing titers would limit
the use of humanized or chimeric monoclonal antibodies for the long-term
management of canine lymphoma.

New developments in feline lymphoma

Although signiﬁcant advances have been made in canine lymphoma, the

amount of information available for the diagnosis, prognosis, and treatment
of feline lymphoma remains disappointing. Even with aggressive chemo-
therapy, response rates and survival times in cats with lymphoma continue
to be inferior to results observed in the dog. In part, the lack of new
discoveries could be attributed to the decreased incidence of virally induced
lymphomas that resulted from the wide use of preventive FeLV vaccines.
Regardless, feline lymphoma remains a common and fatal hematopoietic
cancer. Continued research and investigation into the molecular under-
pinnings of feline lymphoma are required to achieve improved response
rates and survival times in aﬀected cats.

Newly identified risk factors

Chemical carcinogens present in cigarette smoke have been causatively

linked to the development of various cancers in people and domestic animals
[68–70]. In addition, an increased incidence of lung and nasal sinus cancers in
dogs has been weakly correlated with exposure to passive cigarette smoke
[71,72]. Recently, an epidemiologic study has demonstrated that cats exposed
to household environmental tobacco smoke have an increased relative risk of
developing malignant lymphoma. The relative risk of developing malignant
lymphoma increased with duration and quantity of environmental tobacco
smoke exposure [73].

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Inhibitor molecule p27Kip1 dysregulation

The cell cycle consists of four discrete phases G

, S, G

, and M. The

assembly of speciﬁc cyclins and their respective cyclin-dependent kinases
(Cdks) regulates orderly progression through each phase of the cell cycle [73].
Advancement of cells from G

to S requires G

cyclin

/Cdk complex for-

mation as well as passage through a restriction point called the G

check-

point. The G

checkpoint ensures that favorable conditions are available for

DNA replication. In normal cells, DNA damage, nutrient deﬁciency, and
other environmental stressors may prohibit cells from passing through the
G

checkpoint, resulting in cell cycle arrest [74]. The induction of cell cycle

arrest serves a protective function, allowing damaged DNA to be repaired
before DNA synthesis. The expression of an inhibitory protein class called
the Cip1

/Kip1 family promotes G

cell cycle arrest. Members of the

Cip1

/Kip1 family include p21, p27, and p57 [74]. Decreased expression of the

Cip1

/Kip1 inhibitory family may allow damaged cells to bypass the G

checkpoint, thus favoring the propagation of malignantly transformed cells.
In feline lymphoma, the expression of p27Kip1 has been investigated. The
expression of p27Kip1 was demonstrated by an immunohistochemical
method in normal lymphocytes of the follicular mantle zone as well as in
interfollicular small lymphocytes. Actively replicating lymphoblasts within
germinal centers did not stain for p27Kip1. Interestingly, feline T- and B-cell
lymphomas also failed to be immunolabeled. The results of this investigation
suggest that feline malignant lymphocytes may be able to bypass regulatory
checkpoints within the cell cycle [75].

Summary

Canine and feline lymphoma is a common hematopoietic malignancy that

generally responds well to systemic chemotherapy. In dogs, several recent
investigations have underscored the beneﬁcial eﬀects of adjunctive radiation
therapy for the treatment of multicentric lymphoma. With the emergence of
eﬀective immunotherapeutic agents against non-Hodgkin’s lymphoma in
people, some of these speciﬁc targeted immunotherapeutics may soon be
a viable option for treating lymphoid malignancies in dogs. Although the
eﬀective and durable treatment of feline lymphoma remains disappointing,
the identiﬁcation of environmental etiologic factors may help to shape future
recommendations for disease prevention. It is only reasonable to assume that
as our fundamental understanding of lymphoid malignancies grows, better
diagnostic tools, predictive markers, and therapeutic options will also emerge.

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Mast cell tumors in the dog

Cheryl A. London, DVM, PhD*,

Bernard Seguin, DVM, MS

Department of Surgical and Radiological Sciences, 2112 Tupper Hall, One Shields Avenue,

University of California at Davis, Davis, CA 95616, USA

In most species, neoplastic processes involving mast cells are relatively

uncommon. In contrast, mast cell tumors (MCTs) occur frequently in the pet
population, representing up to 20% of all cutaneous canine tumors [1–3]. As
such, a detailed comprehension of the diagnosis and treatment of mast cell
neoplasia in the dog is extremely important. Before discussing the speciﬁcs of
MCTs, however, it is ﬁrst necessary to understand normal mast cell biology.

Mast cell biology

Morphologic characteristics of mast cells

Mast cells are discrete round cells roughly one to three times the size of

a neutrophil. They possess a round to oval nucleus and distinct cytoplasmic
granules that stain with dyes, such as toluidine blue, Giemsa, and methylene
blue. Such staining is a result of the aﬃnity of these basic dyes for the acidic
proteoglycans contained in the mast cell granules [4–7]. Some of these dyes
assume a different color when bound by the granules than they do when
staining nuclear DNA, so that the granules are often called metachromatic.
Although mast cells may be visualized on hematoxylin and eosin–stained
sections, the dyes described previously are used to characterize mast cells
deﬁnitively. Moreover, mast cell granules may not be visualized with stains
like Diff-Quick (Dade Behring, Deerﬁeld, IL), precluding identiﬁcation on
some cytologic specimens [8].

Development of mast cells

Mast cells are derived from bone marrow precursor cells. They leave

the bone marrow in a presumed immature state and migrate to many

Vet Clin Small Anim

33 (2003) 473–489

* Correspondence.
E-mail address:

calondon@ucdavis.edu (C.A. London).

0195-5616

/03/$ - see front matter

doi:10.1016/S0195-5616(03)00003-2

tissues, particularly those having primary contact with foreign antigens
(eg, skin, respiratory and gastrointestinal tracts), where they then mature
into tissue mast cells [5]. Cytokines important in the development and
maturation of mast cells include interleukin (IL)-3, IL-6, IL-4, and stem
cell factor (SCF) among others [5]. The local tissue microenvironment
in which mast cells mature determines the subsequent functional ca-
pacities of these cells. For example, mast cells in the mucosa of the
gastrointestinal tract (labeled mucosal mast cells) have chondroitin sul-
fate as their major granule proteoglycan and contain little histamine. In
contrast, mast cells in the lung and serosa of body cavities (labeled tissue
mast cells) contain heparin as their major granule proteoglycan and pro-
duce large amounts of histamine [4]. Experiments in mice suggest that the
functional characteristics of mast cells are not ﬁxed, because granule
content can change if mast cells are moved from one environment to
another. In summary, the precise nature of the mast cell and the mediators
it can produce varies with its anatomic location and is probably regulated
by the local cytokine environment.

Function of mast cells

Mature mast cells bind IgE on their cell surface through expression of the

high-aﬃnity IgE receptor (FceRI). Mast cells also express receptors for
complement components (particularly C5a). The primary manner in which
mast cells are activated is by cross-linking of the FceRI-bound IgE on their
cell surface, leading to the release and production of various mediators
including the following [4,5]:

a. Contents of granules, such as histamine, heparin, chondroitin sulfate,

mast cell proteases, and others

b. Lipid mediators, such as prostaglandins, leukotrienes, and platelet-

activating factor

c. Cytokines, such as tumor necrosis factor-a, IL-3, IL-4, IL-5, and IL-6

The mediators described here lead to several reactions, including in-

creased vascular permeability, vasodilation, smooth muscle spasm, pruritus,
anticoagulation, and activation of eosinophils and neutrophils. Collectively,
these effects can lead to local hypersensitivity reactions, or more seriously,
to systemic hypersensitivity (anaphylactic shock) [4]. As such, mast cells
have primarily been associated with allergic reactions

/disorders. It is now

evident that mast cells seem to play an important role in the initiation of
innate immune responses. Experiments in mice demonstrated that mast cells
were important in initiating and sustaining neutrophil migration and ac-
tivation in response to bacteria [6]. Indeed, mast cells are often seen at sites
of inﬂammation as well as in reactive lymph nodes. Therefore, in addi-
tion to playing a role in the induction of pathologic allergic responses, mast
cells are critical players in protective immune responses.

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/ Vet Clin Small Anim 33 (2003) 473–489

Canine mast cell tumors

Incidence and signalment

As previously mentioned, the MCT is the most common skin tumor of the

dog and one of the most common malignant tumors noted in the canine
population. Whereas MCTs are usually found in older dogs (mean age of
approximately 8–9 years), they have also been reported in younger dogs [9–
12]. Several breeds seem to be at increased risk for the development of MCTs,
including dogs of bulldog descent (Boxer, Boston Terrier, and English
Bulldog), Labrador and Golden Retrievers, Cocker Spaniels, Schnauzers, and
Chinese Sharpeis [9,12–14]. No sex predilection has been reported.

Etiology

The etiopathogenesis of MCTs in the dog is unknown, as is the reason for

the extremely high incidence in this species. Although some studies have
suggested the possibility of a viral cause and MCTs have been transmitted
from dogs with solid tumors to susceptible laboratory dogs using tumor
tissue or extracts, there is no epidemiologic evidence to indicate horizontal
transmission of tumors [15]. Because most of the tumors arise in the skin, it
has been suggested that topical carcinogens may play a role in this disease.
No reports exist to imply such a cause, however, and there seems to be no
particular regional distribution of these tumors. The increased incidence of
MCTs in certain breeds suggests the possibility of an underlying genetic
cause [13]. Interestingly, although dogs of bulldog ancestry are at higher risk
for MCT development, it is generally accepted that MCTs in these dogs are
more likely to be benign [12].

As previously mentioned, SCF is an important growth factor for mast cells

[7]. The receptor for SCF is Kit, encoded by the proto-oncogene c-kit; SCF-Kit
interactions are required for the differentiation, survival, and function of mast
cells [7,16–21]. Mutations in c-kit leading to activation of Kit in the absence of
SCF binding have been demonstrated to occur in systemic mastocytosis in
people [22–28]. Additionally, Kit dysfunction has now been recognized in
a variety of other human cancers, including gastrointestinal stromal tumors,
small cell lung cancer, ovarian carcinoma, and gliomas [29–36]. Several
authors have recently identiﬁed the presence of activating mutations in the
proto-oncogene c-kit in canine MCTs. These mutations consist of internal
tandem duplications in the negative regulatory juxtamembrane domain of Kit
[37–40]. The high frequency of mutations in a gene known to play a role in
tumorigenesis suggests that aberrations in c-kit may be involved in the
development or progression of MCTs in dogs.

History and clinical signs

Although normal mast cells are found in abundance in the lungs and

gastrointestinal tract, the overwhelming majority of MCTs in the dog occur

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in the dermis and subcutaneous tissue [9,41]. Rarely, primary MCTs may
present in other sites, such as the oral cavity, nasopharynx, larynx, and
gastrointestinal tract [42,43]. Visceral MCT involving the spleen, liver, or
bone marrow (often referred to as disseminated mastocytosis) is usually
the result of systemic spread of an aggressive primary cutaneous MCT,
although it can occur as an independent syndrome [44,45]. Primary mast cell
leukemia in the dog is extremely rare.

Cutaneous MCTs usually occur as solitary nodules, although roughly

10% to 15% of dogs present with multiple tumors [46]. Approximately
50% of cutaneous MCTs occur on the trunk and perineal region, 40%
on the limbs, and 10% on the head and neck [2,46,47]. Perhaps most
importantly, the clinical appearance of MCTs can vary widely. Dermal
MCT may be well circumscribed, raised, and ﬁrm, or the surface maybe
erythematous and ulcerated; invasion into the subcutaneous tissue may be
present. MCTs arising in the subcutaneous tissue are often poorly cir-
cumscribed and may resemble lipomas. It is therefore not possible to
identify a cutaneous lesion as an MCT simply by its appearance.
Cutaneous MCTs may also be present for various lengths of time. In
general, MCTs that are slow growing and present for at least 6 months are
more likely to behave in a benign manner, whereas those that are rapidly
growing large tumors are more likely to behave in a malignant manner
[41]. The duration of the lesion does not always predict the subsequent
biologic behavior, however.

Clinical signs of MCTs are caused by the release of histamine, heparin,

and other vasoactive amines. Mechanical manipulation of the tumor during
physical examination can induce degranulation leading to erythema and
wheal formation (termed Darrier’s sign), and an owner occasionally reports
that the tumor seems to change in size over short periods [47]. Gastro-
intestinal ulceration is also a potential complication of MCTs; between 35%
and 83% of dogs with MCTs that underwent necropsy had evidence of
gastric ulcers, and plasma histamine concentrations were found to be ele-
vated in dogs with MCT [48,49]. Elevated histamine levels presumably lead
to stimulation of H2 receptors on parietal cells, excessive gastric acid pro-
duction, and the development of ulcers. Consequently, signs like vomiting,
anorexia, melena, and abdominal pain may be present. In one study, the
levels of histamine in dogs with MCTs were not related to clinical stage, his-
tologic grade, or tumor size [48].

Diagnosis

Cytologic evaluation of ﬁne-needle aspirates is probably the easiest

method to diagnose the presence of an MCT. The mast cells appear as
discrete round cells with a round to oval centrally placed nucleus that may
be diﬃcult to visualize because of the presence of granules. As mentioned
previously, mast cell granules may not stain with Diﬀ-Quik, leading to

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diﬃculty in making a deﬁnitive diagnosis by cytology. Additionally, poorly
diﬀerentiated malignant mast cells may contain few, if any, granules; in this
case, special stains (eg, toluidine blue, Giemsa) may be required. Other
round cell tumors that should be included in the diﬀerential diagnosis in-
clude lymphoma, plasmacytoma, histiocytoma, amelanotic melanoma, and
transmissible venereal tumor. Whereas the diagnosis of MCT can almost
always be made by ﬁne-needle aspiration cytology, excisional biopsy is
required for histologic grading of the tumor. It is important to note that
because wide surgical excision is the treatment most likely to cure most
MCTs, every eﬀort should be made to obtain a deﬁnitive diagnosis via
cytology before surgical intervention. Moreover, because any cutaneous
tumor may potentially be an MCT, ﬁne-needle aspiration should be
performed on all masses before removal. If cytologic diagnosis proves
diﬃcult, a needle or punch biopsy of the tumor can be obtained before
surgery. This may be preferable to a larger incisional biopsy, because local
release of mast cell mediators will signiﬁcantly inhibit healing and may result
in excessive bleeding.

Staging

Because any MCT is capable of metastasis, all dogs with MCTs should be

staged to determine the extent of their disease and overall health. This is
particularly important for dogs in which radiation therapy may be used in
the course of treatment. Some of or all the following procedures may be
indicated.

Complete blood cell count, biochemistry profile, urinalysis

These tests are part of a minimum database and should be included in the

workup of any animal suspected to have cancer. Dogs with MCTs may have
eosinophilia because of chemotactic factors and IL-5 produced by the mast
cells [4]. Anemia may be present secondary to hypersplenism or gastroin-
testinal bleeding. Occasionally, mast cells may be seen on a routine complete
blood cell count.

Buffy coat smear

A buﬀy coat smear is made by spinning peripheral blood in a micro-

hematocrit tube, breaking the tube at the buﬀy coat layer, and smearing the
concentrated leukocytes on a slide. The buﬀy coat is then carefully examined
for the presence of mast cells. For many years, examination of the buﬀy
coat for circulating mast cells has been used to screen dogs with MCTs for
the presence of systemic

/metastatic disease. It was originally believed that al-

though the buﬀy coat smear was not an extremely sensitive test, it was fairly
speciﬁc for mast cell neoplasia. It is now clear that this may not be the case,

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however, because several studies have demonstrated that dogs with many
diﬀerent kinds of disease, including pneumonia, parvovirus, pancreatitis,
skin disease, and gastrointestinal diseases, may have mast cells circulating
in the periphery [50–52]. In summary, the buffy coat smear is a relatively
easy test to perform; the ﬁnding of circulating mast cells in a patient
with cutaneous MCT warrants further investigation.

Bone marrow aspiration

In the normal bone marrow, mast cells are found infrequently. In one

study, of 51 bone marrow samples examined, only 2 contained a single mast
cell [52]. It should be pointed out that these marrow samples were obtained
from normal dogs, and it is possible that dogs with inﬂammatory disorders
may exhibit higher numbers of mast cells in the bone marrow [50,51]. In gen-
eral, bone marrow aspiration is considered to be a more sensitive indica-
tor of systemic involvement than the buffy coat smear [45].

Lymph node aspiration

All regional lymph nodes should be carefully examined for signs of en-

largement, and any suspicious nodes should be aspirated for cytologic exam-
ination. Additionally, because metastatic nodes may palpate within normal
size, it is recommended by some that all accessible regional lymph nodes be
examined by aspiration cytology. Mast cells may be present in normal
lymph nodes and are often found in reactive lymph nodes; thus, it may be
diﬃcult to determine if mast cells found on cytologic examination are neo-
plastic or part of the normal immunologic cellular repertoire. Indeed, 24% of
lymph node aspirates from normal Beagles contained mast cells [52]. There-
fore, lymph node biopsy with histopathology may be a better tech-
nique for diagnosis of lymph node metastasis.

Evaluation of the abdominal and thoracic cavities

Thoracic radiographs are always indicated as part of any staging pro-

cedure, although pulmonary involvement is uncommon in dogs with MCTs.
Abnormalities reported include lymphadenopathy (sternal and hilar), pleural
eﬀusion, and anterior mediastinal masses [45]. Evaluation of the abdomi-
nal cavity is important in dogs with MCTs, because spread to the liver and
spleen as well as other abdominal structures may be noted. Ultrasound is a
more sensitive diagnostic technique for evaluation of abdominal organs. Any
suspicious organ should be aspirated and examined by cytology. Some have
recommended that splenic and hepatic aspiration be performed as part of the
routine staging procedure, regardless of the presence of splenomegaly or
hepatomegaly. Because mast cells may be found normally in these organs,
however, the utility of this procedure is not clear. Evidence suggests that ﬁne-
needle aspiration of the liver and spleen should be performed if abnormalities
are detected in these organs during ultrasound examination or if the dog is
known to have a grade III MCT [53–55].

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Clinical staging system for canine mast cell tumors

The following is the current clinical staging system for canine mast cell

tumors:

0: single tumor, incompletely excised from dermis

I: single tumor conﬁned to dermis without regional lymph node

involvement

II: single tumor conﬁned to the dermis with regional lymph node

involvement

III: multiple dermal tumors or large inﬁltrating tumors, with or without

regional lymph node involvement

IV: any tumor with distant metastases or recurrence with metastases

(including blood or bone marrow involvement
substage a: no signs of systemic disease
substage b: signs of systemic disease

Prognostic factors

Canine MCTs possess a wide range of biologic behaviors ranging from

benign to extremely aggressive leading to metastasis and eventual death from
disease. Several prognostic factors have been identiﬁed that help to predict
the biologic behavior of an MCT as well as to direct the course of therapy.

Histologic grade

The histologic grade of an MCT is determined after incisional or

excisional biopsy of the tumor and cannot be assessed simply by cytologic
evaluation of ﬁne-needle aspirates. The grade of an MCT is determined by
the characteristics of the neoplastic cells (eg, degree of granulation, cytologic
and nuclear pleomorphism), number of mitotic ﬁgures, and extent of tumor
invasion into the underlying tissues (Table 1). Histologic grade is the most
consistent prognostic factor and correlates signiﬁcantly with survival, but it
does not predict the behavior of every tumor. The most commonly used
grading system is the one described by Patnaik et al [14]:

i. Well-differentiated tumors (grade I) are considered to behave in benign

manner, and complete surgical excision is usually curative. These
represent between 30% and 55% of all MCTs reported [14,41,46,56].
Several retrospective studies have demonstrated that 75% to 90% of
dogs do not die of their disease after deﬁnitive therapy [14,41,56,57].

ii. Intermediately differentiated tumors (grade II) represent between

25% and 45% of all MCTs reported, and their biologic behavior is
more difﬁcult to predict [14,41,46,56]. On histopathology, these
tumors exhibit invasion into the underlying subcutaneous tissue. As
a result, they may be more challenging to remove by surgical excision.
Historically, dogs with grade II MCT have a reported mean survival

time of 28 weeks after surgical removal, although the completeness of

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excision could not be assessed in this study [41]. More recently, it has
been shown that radiation therapy after incomplete excision of
solitary grade II MCTs can cure greater than 80% of affected
patients, indicating that adjuvant radiation therapy clearly improves
the survival times of dogs with these tumors [57,58]. It is important to
note that grade II MCTs have the ability to spread to local lymph
nodes as well as distant sites and that a proportion of dogs undergoing
deﬁnitive therapy (surgery and radiation) may go on to develop
metastatic disease. Furthermore, some dogs that present with grade II
MCTs already have evidence of metastatic disease, making appropri-
ate staging imperative for these dogs.

iii. Poorly differentiated tumors (grade III) represent between 20% and

40% of all MCTs reported [14,41,46,56]. They often behave in a
biologically aggressive manner, exhibiting metastasis early on in the
course of disease. The mean survival time of dogs with grade III MCT
has been reported as 18 weeks when treated with surgery alone [41]. In
one study, the percentage of dogs with grade III MCTs surviving at
1500 days was reported as 6%, and in another study, the percentage
of dogs surviving at 24 months was 7%, indicating that these tumors
are particularly malignant [14,59].

Table 1
Patnaik scheme for grading of canine mast cell tumors

Grade

Patnaik grade

Microscopic

Well differentiated

Well-diﬀerentiated mast cells with

clearly deﬁned cytoplasmic borders
with regular spherical, or ovoid
nuclei; mitotic ﬁgures are rare or
absent; granules are large, deep
staining, and plentiful; cells conﬁned
to the dermis and interfollicular
spaces

Intermediately differentiated

Cells closely packed with indistinct

cytoplasmic boundaries; nuclear

cytoplasmic ratio lower than
anaplastic; mitotic ﬁgures
infrequent; more granules than
anaplastic; neoplastic cells inﬁltrate
or replace the lower dermal and
subcutaneous tissue

Anaplastic undifferentiated

III

Highly cellular, undifferentiated

cytoplasmic boundaries, with
irregular size and shape of the
nuclei; frequent mitotic ﬁgures; low
number of cytoplasmic granules;
neoplastic tissue replaces the
subcutaneous and deep tissues

Data from

Patnaik AK, Ehler WJ, MacEwen EG. Canine cutaneous mast cell tumors:

morphologic grading and survival time in 83 dogs. Vet Pathol 1984;21:469–74.

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Clinical stage

Although the clinical staging scheme has been developed for prognostic

purposes, each increase in stage (0–IV) has not necessarily been proven to
worsen the prognosis. It has been demonstrated that when dogs are treated
with radiation therapy, those with stage 0 MCTs survived signiﬁcantly
longer than did dogs with stage I through III MCTs. In that study, no dog
had stage IV MCT; therefore, stage IV MCT could not be compared for
prognostic purposes [57]. In two other studies, the presence of mast cells in
the regional lymph node was a negative prognostic factor for survival and
disease-free interval on univariate analysis, suggesting that stage II has
a worse prognosis than stage I [60,61]. Dogs with stage III MCT with the
presence of multiple dermal masses may not necessarily have a worse prog-
nosis than dogs with stage I or II MCT. Indeed, in one study, dogs with mul-
tiple MCTs (stage III) did not have a worse prognosis than dogs with a
single MCT when treated with chemotherapy [60].

Anatomic location

MCTs that develop in the oral cavity, nail bed, or inguinal, preputial, and

perineal regions have been reported to behave in a more malignant fashion
regardless of histologic grade [57]. Deﬁnitive evidence for this in the liter-
ature is lacking, however. MCTs that originate in the viscera (eg, gastro-
intestinal tract, liver, spleen), bone marrow, or peripheral blood carry a grave
prognosis [45,62].

Growth rate

Tumors present for long periods (months to years) may be more likely to

behave in a benign manner. In one study, 83% of dogs with tumors present
for longer than 28 weeks before surgery survived for at least 30 weeks com-
pared with only 25% of dogs with tumors present for less than 28 weeks [41].
The same study demonstrated that most dogs surviving for longer than 30
weeks after surgery appeared to be cured.

Breed

Boxers have a high incidence of MCTs, but these tend to be more well

diﬀerentiated and carry a better prognosis [12,41]. Nevertheless, every MCT
should be treated as potentially malignant, regardless of breed.

Argyrophilic nucleolar straining organizing regions

Argyrophilic nucleolar staining organizing regions (AgNORs) are areas

present in the nucleus that take up silver stain and provide an indirect
measure of cell proliferation. This stain can be performed on formalin-ﬁxed
specimens or on cytologic samples. It has been demonstrated that the rel-
ative frequency of AgNORs correlates with histologic grade and is pre-
dictive of postsurgical outcome. The higher the AgNOR count, the poorer
is the prognosis [56,63,64].

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DNA ploidy

MCTs possessing an abnormal DNA content (aneuploid) exhibited

a trend toward shorter survival times; however, a signiﬁcant diﬀerence was
found between aneuploid and diploid tumors when comparing stage I and
non-stage I disease [65].

Proliferating cell nuclear antigen

Proliferating cell nuclear antigen (PCNA) is a protein required for DNA

synthesis; expression is associated with cell proliferation. In one study,
PCNA was signiﬁcantly higher in recurrent versus nonrecurrent tumors and
in metastatic versus nonmetastatic tumors, and it was a good predictor for
tumor recurrence at 6 months [56].

Ki-67

Ki-67 is composed of two protein subunits that are present in cells during

the active phases of the cell cycle but absent during rest. Levels of Ki-67 in
the nucleus seem to correlate with cell proliferation. In one study, the mean
number of Ki-67-positive nuclei per 1000 tumor nuclei was signiﬁcantly
higher for dogs that died of MCTs than for those that survived. For dogs
with grade II tumors, the number of Ki-67-positive nuclei per 1000 tumor
nuclei (<93 versus

93) was signiﬁcantly associated with outcome [59].

Other factors

Several other factors are currently under investigation to determine if

they may play a role in the aggressiveness of canine MCTs, including the
levels of active matrix metalloproteinase 2 and 9, tryptase, and chymase
[66,67]. Two recent studies evaluated the potential role of p53 in canine
MCTs; both found that although p53 overexpression was noted in some
MCTs, there was no obvious association with tumor behavior [68,69].

Treatment

The choice of treatment modalities used for a particular canine MCT

depends heavily on the prognostic indicators discussed previously, especially
the histologic grade and clinical stage.

Surgery

Wide surgical excision is indicated for all canine MCTs; although they

may feel like discrete masses, microscopically, most extend well beyond the
palpable borders. It is generally accepted that the lateral margins of excision
need to be at least 3 cm in each direction. Deep margins are as important as
the lateral margins and follow the concept of a quality margin as opposed to
a quantity margin. Collagen-dense and vascular-poor tissues tend to behave
as biologic barriers against cancer [70]. Therefore, the deep margin should
include a fascial plane that has not been invaded by the tumor and is
removed en bloc with the tumor. All the excised tissue should be submitted;

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the lateral and deep margins should be accurately labeled so that the
pathologist is able to identify speciﬁcally any areas of incomplete excision.
Careful examination of the histologic margins is imperative; however, even
histologically clean margins do not guarantee that a tumor will not recur.
This is particularly relevant for grade II and III tumors in which the
underlying tissues may be involved. Part of the difﬁculty in evaluating tumor
margins is that normal mast cells are present in all tissues, and it may be
difﬁcult for the pathologist to determine if a mast cell present at the tissue
margin represents a malignant cell or a normal cell. In one study, 83% of
dogs with grade I MCT, 44% of dogs with grade II MCT, and 6% of dogs
with grade III MCT were alive 1500 days after surgical excision [14]. In
another study, 100% of dogs with grade I MCT, 44% of dogs with grade II
MCT, and 7% of dogs with grade III MCT were alive 2 years after surgical
excision [59].

Complete surgical excision is likely to be curative for dogs with grade I

MCT. The need to perform adjuvant therapy for local control of the tumor
in dogs with completely excised grade II MCT seems to be unnecessary. Two
studies have reported a rate of local recurrence of 5% and 11% after
complete excision of grade II MCT [71,72]. Postsurgical treatment rec-
ommendations are as follows:

Grade I complete excision: no further therapy
Grade I incomplete excision: wider excision or radiation therapy if
surgery is not possible
Grade II complete excision: consider radiation therapy only if margins
are close
Grade II incomplete excision: wider excision or radiation therapy if
surgery is not possible
Grade III complete excision: chemotherapy
Grade III incomplete excision: chemotherapy with or without radiation
therapy

It has been advocated that the injection of deionized water in the surgical

site can decrease the likelihood of local recurrence after incomplete MCT
removal [73]. A more recent study found no beneﬁt in this procedure; as
such, the use of deionized water is currently not recommended [74].

Radiation therapy

Evidence suggests that radiation therapy is extremely eﬀective at elim-

inating remaining microscopic disease after incomplete excision of grade I
and II MCT ([90% 3-year control rate) [58,75]. Protocols that deliver the
total dose of radiation in a shorter period by administering more fractions
per week seem to provide better control of the MCTs [61]. Unfortu-
nately, dogs with grade III tumors do not fare as well; whereas the
radiation may be effective at preventing local recurrence of tumor, most dogs
eventually develop metastasis.

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Radiation therapy has been used to treat solid MCTs (macroscopic

disease) when surgery was not an option. Varying degrees of success have
been found; in one study, a 50% 1-year control rate was obtained using total
doses of 45 to 57 Gy [61]. Radiation therapy should not be used as the
primary therapeutic modality if surgical intervention is an option, however.
Palliative radiation has also been used to treat dogs with nonresectable high-
grade MCT. This may result in an improvement in quality of life but is
unlikely to signiﬁcantly increase survival time. Moreover, systemic effects of
mast cell degranulation after radiation may lead to vomiting, hypotension,
and gastrointestinal ulceration.

Chemotherapy

The use of adjuvant chemotherapy is indicated after excision of grade III

MCTs and metastatic MCTs as well as for nonresectable high-grade tumors.
It is important to note that radiation therapy (not chemotherapy) is the
treatment of choice for incompletely excised grade I and II MCTs. In
general, chemotherapy for macroscopic MCT has been unrewarding, and
long-term responses have not been demonstrated in well-controlled clinical
trials.

i. Corticosteroids: the exact mechanism of how corticosteroids kill

malignant mast cells is not known. The reported response rate of
canine MCT to prednisone is 20%, with reported remission times of
10 to 20 weeks [76]. Partial remissions are more common than
complete remissions, and at least some of the observed response may
be a result of a decrease in tumor-associated edema. Intralesional
corticosteroids have also been shown to be of some beneﬁt.

ii. CCNU (lomustine): a nitrosourea alkylating agent that has been used

to treat lymphoma and brain tumors in dogs, CCNU, was recently
found to have activity against canine MCT. A response rate of ap-
proximately 42% was noted when dogs with grade II and III MCTs
that had failed all other therapies were treated with CCNU [77].
As with prednisone, most of these were partial responses, and the
duration of response was only 79 days. Nevertheless, it does appear
that this drug has some efﬁcacy in treating MCT, and clinical trials
are currently underway to evaluate its efﬁcacy.

iii. Vinblastine: vinblastine has been reported to have efﬁcacy against

canine MCT in two separate studies. In the ﬁrst study, dogs with
grade II or III MCT with metastasis to the regional lymph node
underwent surgical removal of the primary tumor and involved lymph
node. They were then treated with a combination of vinblastine,
prednisone, and cyclophosphamide, resulting in a median survival
time of 18 months [78]. Interestingly, dogs with grade II or III MCT
and metastasis to the regional lymph node that did not undergo
surgery but were treated with the combination chemotherapy protocol

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lived for a median of 5 months. This would seem to indicate that
vinblastine is more useful in the face of microscopic rather than bulky
MCT. The second study was a retrospective analysis of dogs with
various forms of MCT treated with a combination of vinblastine and
prednisone [60]. This study revealed a 47% response rate, although
the retrospective nature of the analysis makes the data difﬁcult to
interpret. Interestingly, vincristine, a close relative of vinblastine,
appears to have little efﬁcacy in the treatment of canine MCT [79].
Studies are currently underway to further evaluate the utility of
vinblastine in the treatment of canine MCT.

iv. Miscellaneous drugs: in limited clinical reports, both

-asparaginase

and chlorambucil have been found to have activity against MCT.

Supportive care

Animals with large primary MCT, evidence of metastatic disease, or

systemic signs should be treated with medications to block some of or all the
eﬀects of histamine release.

a. H2 antagonists: because histamine stimulates gastric acid production

by parietal cells, MCT may cause gastrointestinal ulceration. To pre-
vent this, any of the standard H2 antagonists may be used, including
cimetidine, ranitidine, or famotidine. Alternatively, omeprazole may
be used; this is not a direct H2 antagonist but works by inhibiting
the proton pump on parietal cells necessary for the generation of gas-
tric acid.

b. H1 antagonists: massive mast cell degranulation can lead to hypo-

tensive shock and death. Therefore, patients with bulky mast cell dis-
ease should be placed on an H1 antagonist like diphenhydramine.

c. Miscellaneous: treatment with sucralfate is indicated for dogs with

evidence of gastrointestinal ulceration. Cyproheptadine and antihis-
taminic drugs with antiserotonin activity stabilize mast cells and may be
useful in the treatment of dogs with bulky mast cell disease.

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Update on the biology and management

of canine osteosarcoma

Ruthanne Chun, DVM

Louis-Philippe de Lorimier, DVM

Kansas State University College of Veterinary Medicine, Mosier Hall,

1800 Denison Avenue, Manhattan, KS 66506-5606, USA

Department of Veterinary Clinical Medicine, University of Illinois at Urbana-Champaign,

1008 W. Hazelwood Dr., Urbana, IL 61802, USA

Though the likelihood of developing osteosarcoma (OSA) remains

greatest in large to giant breed dogs, speciﬁc reasons for increased risk in
this population remain ill-deﬁned. This article will ﬁrst discuss the physical
and molecular changes that have been associated with the development of
osteosarcoma in dogs.

Risk factors and associations for the development of osteosarcoma

Ionizing radiation

Ionizing radiation, in both experimental and therapeutic settings, has

long been known to cause OSA in dogs [1–6]. One study described the
development of OSA in the radiation therapy (RT) ﬁeld 1.7 to 5 years after
completing therapy in 3

/87 dogs (3.4%) treated with megavoltage ir-

radiation for soft tissue sarcomas [2]. The fractionation scheme described in
this article was coarse, consisting of 10 fractions of 3.5 to 5 Gy over 3 weeks.
Late responding tissues, such as bone, are more sensitive to coarse fractions.
The mutagenic eﬀects of ionizing radiation are the likely cause of OSA as
a late complication. The same investigators evaluated an additional 67
beagle dogs randomized to receive either external beam RT (EBRT,
n

¼ 14), intraoperative RT (IORT, n ¼ 26) or EBRT þ IORT (n ¼ 27).

The fractionation schemes used in this study ranged widely, but all IORT
treatments were given as a single large dose. One dog in the IORT group

Vet Clin Small Anim

33 (2003) 491–516

* Corresponding author.
E-mail address:

chun@vet.k-state.edu (R. Chun).

0195-5616

/03/$ - see front matter

doi:10.1016

/S0195-5616(03)00021-4

and 7 dogs in the EBRT

þ IORT group developed OSA 4 or more years

after RT, supporting development of OSA as a late complication of coarse
fraction radiation therapy. Osteosarcoma in the RT ﬁeld has also been
described in two dogs treated for acanthomatous epulis [6,7]. These dogs
were treated with 250 kVp orthovoltage radiation; orthovoltage radiation
results in a bone-absorbed dose approximately 2 to 3 times the dose received
by adjacent soft tissues [8]. Because it delivers a more homogenous bone and
soft-tissue radiation dose, megavoltage irradiation used in a ﬁner fraction
scheme may minimize the development of secondary malignancies as a late
complication.

Bone infarcts and fatigue microdamage

Bone infarcts, whether spontaneous or associated with orthopedic

procedures such as arthroplasty, are rarely implicated in the development
of canine OSA [9–12]. The miniature Schnauzer breed is over-represented in
the literature for the development of OSA secondary to spontaneous bone
infarcts [10–13]. In humans, neoplasia associated with bone infarcts is
believed to be secondary to a defect in the chronic reparative process oc-
curring at the periphery of the infarct [9]. The pathogenesis of OSA in dogs
with bone infarcts remains unknown.

Fatigue microdamage of the metaphyses has been proposed as a potential

risk factor for OSA in large-breed dogs [14]. A study compared bone
microdamage of the distal radial metaphyseal plates of 10 small (<15 kg)
and 10 large (>25 kg) normal dogs, and it failed to detect a signiﬁcant
diﬀerence between the two groups. These authors concluded that such
microdamage might not be an important risk factor for OSA in large breeds
of dogs [14].

Gonadal hormone exposure

A recent report of 683 Rottweilers demonstrated that dogs subjected to

early gonadectomy (before 1 year of age) had a higher risk for bone sarcoma
[15]. This risk factor was found to be independent of adult height or body
weight. Early spay and neutering is still more beneﬁcial than detrimental
in terms of overall patient health, and the results of this study should
not unduly inﬂuence the decision to spay or neuter a canine patient of any
breed.

Genetic alterations

Alterations in the tumor-suppressor genes Rb and p53 are commonly

identiﬁed in human OSA patients, and they have been evaluated in dogs
with OSA [16–19]. The Rb gene family consists of Rb, p107, and p130. These
genes code for proteins with inhibitory functions in the regulation of
cell proliferation and diﬀerentiation. Human cancers frequently harbor

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mutations inactivating the Rb pathway. In human OSA, 60% or more have
Rb

-associated abnormalities [18]. By comparison, mutations in the related

genes p107 and p130 are rare. Normally, Rb protein regulates cell cycle
transit by controlling the availability and activity of speciﬁc members of the
E2F family of transcription factors. Inactivation of Rb protein through
phosphorylation results in release, and thereby activation, of E2F, which in
turn allows transcription of a variety of genes associated with cell repli-
cation. Normal Rb phosphorylation is controlled during mid-G1 by cyclins
and cyclin-dependent kinases (CDK). The CDK inhibitor p16

INK4A

re-

sponds to growth-inhibiting signals by blocking Rb phosphorylation, thus
preventing DNA synthesis [16,17]. Ultimately, aberrant Rb activity results in
unregulated activation of E2F-related gene transcription and, thus, un-
controlled cellular proliferation.

The p53 gene is a tumor suppressor that acts as a master regulator of cell

replication. It also plays a critical role in the cellular response to DNA
damage. Loss of functional p53 confers a selective growth advantage to
transformed cells. Under normal circumstances, damage to the DNA
template induces signaling through the interaction of p53 with the p21

WAF1

CIP1

pathway, which in turn causes cell cycle arrest at the G1 restriction

point by preventing hyperphosphorylation of Rb. This cell-cycle arrest
allows time for DNA repair, which is also partly mediated by the p53
protein. If DNA damage is too extensive for repairs to be completed within
a set time period, normal p53 triggers a cascade of events ultimately leading
to cellular apoptosis. Normal p53 protein is labile, and therefore rarely
detected in tissue sections by routine immunohistochemistry [17]. Accumu-
lation in malignant cells is generally caused by mutations in p53, which do
not allow triggering of downregulatory mechanisms such as those provided
by the mdm-2 gene. Increased expression of p53 may not be linked to
increased activity; in fact, excessive amounts of a nonfunctional protein are
usually produced. The p21

WAF1

/CIP1

gene codes for a CDK inhibitor that

blocks the G1-S transition phase of the cell cycle. This gene is one of a group
that depends on p53 for activation. When p53 activity is defective owing to
a mutation, aﬀected cells have unregulated replicative capability from lack
of activation of p21

WAF1

/CIP1

In a study investigating tumor suppressor gene expression in canine OSA,

5 cell lines were evaluated for abnormalities in Rb, p107, p130, mdm2,
p21

WAF1

/CIP1

, p53, and p16

INK4A

[17]. All 5 cell lines had defective p53

function, as evidenced by lack of induction of p21

WAF1

/CIP1

and mdm2. The

p53

mRNA and protein levels were elevated in 3 cell lines, and decreased in

the other 2. One cell line had increased p16 mRNA and protein levels, and it
signiﬁcantly reduced levels of Rb, p107, and p130 proteins. Another study
reported that 47% of 15 canine OSA samples analyzed had mutations
within the p53 gene at sites similar to those seen in human OSA [16]. In
a third study of 21 canine OSA samples, no gross gene alterations (ie,
translocations, ampliﬁcation, or deletions) in the structure of p53, Rb, or

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/ Vet Clin Small Anim 33 (2003) 491–516

mdm2

were identiﬁed by Southern blotting; however, p53 mutations were

detected by single-strand conformation polymorphism following ampliﬁca-
tion by polymerase chain reaction (PCR) in 8 of these samples [18]. Further
evaluation of Rb protein using Western blotting did not identify abnor-
malities in this protein. Thus, though both canine and human OSA patients
have p53 mutations at similar frequencies, the Rb gene does not appear to
play as important a role in canine OSA as it does in the human disease.

Another important tumor suppressor gene, PTEN, was evaluated in

a recent study [20]. Four of ﬁve canine OSA cell lines studied constitutively
expressed high levels of the phosphorylated form of Akt, an indirect
indicator of aberrant PTEN expression. Deletions, mutations, and down-
regulation of PTEN were observed in most of the cell lines, and in 10 of
15 tumors studied by immunohistochemistry. These ﬁndings suggest that
PTEN

mutations may play a role in the pathogenesis of canine OSA, as in

many human cancers.

A study evaluating the proto-oncogenes c-sis, c-myc, N-myc, and cH-ras

in 9 canine OSA and 17 normal canine tissue samples revealed signiﬁcant
ampliﬁcations of the c-sis and c-myc genes in tumor tissues [21]. This study
also showed similar levels of expression of the sis gene product, PDGF-b, in
human and canine OSA when analyzed by immunostaining. A more recent
study described overexpression of the sis oncogene in a canine OSA cell line,
and it found that all of the canine OSA cell lines tested in that study
contained PDGF receptors [22]. This suggests the possibility of an autocrine
growth factor loop participating in the pathogenesis of a subset of canine
OSAs.

The MET proto-oncogene was also investigated for mutation in canine

OSA by a group in Italy [23]. This oncogene is known to aﬀect metastatic
potential and invasiveness of certain cancers in humans, including OSA.
Five of seven samples of canine OSA examined by Northern blot expressed
high levels of the MET oncogene, and a lung metastasis from one of the
dogs expressed MET at higher levels than did the primary tumor.

A recent report described increased gene expression of growth hormone

(GH) in areas of active osteoid formation [24]. Expression of GH mRNA
was demonstrated in the metaphyseal area of the normal canine growth
plate, and in 25% of the canine OSA specimens studied. The authors of this
study concluded that locally produced GH is involved in osteoid formation
and could play a role in the growth of canine OSA.

Canine appendicular osteosarcoma

Appendicular OSA is the most common form of OSA seen in dogs. This

section will cover the typical incidence and signalment, history and physical
examination, prognostic factors, and treatment options for dogs with
appendicular OSA.

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/ Vet Clin Small Anim 33 (2003) 491–516

Incidence and signalment

Appendicular OSA is the most common of all primary bone tumors in

dogs, accounting for up to 75% to 85% of these lesions [25]. OSA tends to
occur in middle-aged to older dogs, with some reports showing another
incidence peak at 2 years of age (bimodal distribution) [25]. Though male
dogs are often reported to be over-represented, with a male to female ratio
of 1.5:1, this ﬁnding is not consistent among all publications. [26–31]. Large-
and giant-breed dogs including Irish setters, St. Bernards, Rottweilers, and
Doberman pinschers are at greater risk [15,25,26,28–30].

History and physical examination

Dogs with appendicular OSA most commonly are presented for acute to

chronic onset of lameness and limb swelling. Owners often report some
‘‘traumatic incident’’ associated with the onset of lameness. Dogs are usually
eating and drinking normally.

Weight-bearing or nonweight-bearing lameness may be observed, with

limb swelling typically localized away from the elbow or toward the knee.
Osteosarcoma occurs with equal frequency at the distal radius, proximal
humerus, and proximal tibia. The mass is usually ﬁrm and often painful on
palpation.

Prognostic factors

Though breed and sex have not been recognized to have prognostic

importance, young dogs with OSA appear to have shorter survival times
and biologically more aggressive disease [31]. The presence of detectable
metastatic lesions at the time of diagnosis is a recognized poor prognostic
factor, standard chemotherapy being ineﬀective at improving survival
in that setting [26,32,33]. A study found that, in addition to detectable
metastasis at presentation, a telangiectatic (vascular) subtype, rib or
scapular location, a higher body weight, and incompleteness of excision
were also negative prognostic factors for OSA of ﬂat or irregular bones
[32].

Elevated alkaline phosphatase (ALP) levels (total, bone or liver iso-

enzymes) are also negative prognostic factors [34,35]. Animals with elevated
levels have shorter survival times by approximately 50% (

170d versus

400d), even when treated aggressively with surgery and chemotherapy

[34,35]. One study reported that tumor located within the proximal humerus
and body weight of 40 or more kg were associated with a shorter disease free
interval (DFI) and survival [36]. Cited explanations for these factors were that
proximal humeral location potentially allows for more advanced local growth

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before detection, and larger dogs may receive a proportionately smaller
chemotherapy dose than small dogs, when treated on a meter-square body
surface area basis.

Percent tumor necrosis, induced by RT or chemotherapy before tumor

excision, is predictive of local tumor control when compared with dogs
treated with amputation alone [37]. In this study, 200 dogs were treated with
either nothing beyond surgery (n

¼ 100) or preoperative RT, IV cisplatin,

or intra-arterial (IA) cisplatin followed by a limb-sparing procedure in
a variety of combinations of these treatments. In spite of the varied treat-
ment protocols within each group, RT was concluded to be the best way to
induce tumor necrosis (82%); IA delivery (49%) reportedly increased tumor
necrosis over that seen with IV delivery (24%) of cisplatin. Overall, tumor
necrosis was strongly correlated with local tumor control, but there was no
correlation between tumor necrosis and time to metastasis. Another study
found a signiﬁcant direct correlation between survival time and percent
necrosis following 2 or 3 doses of doxorubicin [38]. In that study, percent
necrosis ranged from 0 to 87% (mean, 24.9%). Tumor necrosis is a well-
recognized prognostic factor in human OSA (cutoﬀ value for signiﬁcance is
90% necrosis post-therapy). Percent necrosis obtained after primary chemo-
therapy is used to modify postoperative chemotherapy drug protocols [38].

The use of scintigraphy to help predict time to metastasis in dogs with

OSA has been described [39]. Quantitative bone scintigraphy of the primary
tumor was performed before treatment in 25 dogs and following neo-
adjuvant therapy in 22 of these dogs. A signiﬁcant relationship was
identiﬁed between time to metastasis and (1) radiographic tumor area; (2)
pretreatment ratio of mean counts per pixel in tumor compared with the
counts in adjacent nontumor bone; and (3) the pre- versus post-treatment
ratio. A larger radiographic tumor area and high pretreatment tumor
activity were associated with earlier metastasis, as were tumors that had
a large decrease in uptake after treatment [39].

A recently described histologic grading system for OSA appears to

provide prognostic information [40,41]. A total of 166 dogs provided 166
primary and 34 metastatic OSA tumor samples. These were categorized into
one of three grades based on cellular pleomorphism, mitotic ﬁgures, tumor
matrix, tumor cellularity, and percent necrosis. The 25% of tumors graded
as 1 or 2 were associated with a signiﬁcantly longer disease-free and overall
survival than the 75% categorized as grade 3 tumors [40,41]. An earlier
study of 52 canine OSA samples showed that patients presenting with
detectable pulmonary metastases had increased microvascular density in the
primary tumor; it recognized this histopathologic ﬁnding to be a negative
prognostic indicator [42].

Finally, it has been reported that dogs with infections of their allografts

after limb-sparing surgery lived twice as long as dogs without infection
[43,44]. Nonspeciﬁc activation of the immune system has been advanced as
a potential explanation for the prolonged survival in infected patients.

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Diagnosis

A minimum database consisting of a complete blood count, serum

chemistry proﬁle, and urinalysis is recommended to screen for concurrent
disease, and to aid in establishing a prognosis by quantitating the serum
alkaline phosphatase concentration. Certain therapeutic decisions might
also be altered based on the results of such tests. For example, a patient with
marginal renal function might not be an ideal candidate for cisplatin
therapy, a known nephrotoxic agent, or could require a dose reduction of
a renally excreted drug such as carboplatin.

Diagnostic imaging plays an important role in staging dogs with OSA.

Radiographs of the primary lesion typically reveal bony proliferation and
lysis in the metaphyseal region of long bones. Thoracic radiographs reveal
gross metastatic disease in less than 10% of dogs at presentation. It is
well established, however, that more than 90% of these dogs will go on
to develop pulmonary metastatic disease if treated by surgery alone
[26,28,31,45]. Pulmonary metastatic lesions appear as discrete soft-tissue
nodules, and multiple lesions are common [46].

Nuclear scintigraphy is more sensitive than radiography for imaging

skeletal metastases because scintigraphy reveals changes in the rate of bone
remodeling that develop before structural changes can be detected ra-
diographically [47,48]. Thus, scintigraphy in OSA patients may identify
occult metastatic lesions in other bone sites or metachronous primary
lesions. Reports of using scintigraphy to stage OSA patients are contra-
dictory, however [47–50]. One study reported that 14 of 25 dogs with
appendicular or axial OSA showed suspicious lesions, of which 7 were
proven to be OSA by biopsy [49]. The remaining seven lesions were not
biopsied but were radiographically interpreted to be unrelated to the pri-
mary tumor. A larger study of 70 dogs with bone tumors (62 with ap-
pendicular OSA, 6 with ﬁbrosarcoma, and 2 with chondrosarcoma) found 1
soft tissue metastatic lesion (lymph node) in a dog with OSA, and 1 osseous
metastasis in a dog with an ulnar chondrosarcoma lesion [47]. The dis-
crepancy between these studies may be caused by the small numbers of
patients evaluated in the ﬁrst study. Also, animals in the ﬁrst study might
have been evaluated at later stages of disease. A third article commented on
the use of sequential scans to stage and monitor dogs with OSA [48].
Although no metastases were detected before treatment, 25

/26 dogs

eventually developed metastatic lesions. Of dogs with metastasis, 7

/12 with

bone lesions had radiographically occult metastases, supporting the use of
bone scans for monitoring OSA patients. Because bony radiographic
changes can lag behind scintigraphic changes, the use of scintigraphy to
screen OSA patients for occult disease is very sensitive [48]. The speciﬁcity of
this imaging modality is relatively low, however, and any scintigraphic
lesions beyond the primary tumor should ideally be veriﬁed by radiographs
or biopsy.

497

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Three recent studies evaluated diﬀerent imaging modalities for accuracy

regarding local extent of OSA, using histopathology as the gold standard for
comparison [51–53]. In a study of 20 dogs with distal radial OSA evaluated
before a limb-sparing procedure, nuclear scintigraphy was found to
overestimate the tumor length signiﬁcantly more than did radiography
[52]. Although this might provide a larger margin of safety when de-
termining the site of proximal osteotomy, it could also inappropriately
eliminate limb sparing as a treatment option for some patients. A second
study compared the use of MRI and CT to radiographs and histologic
evaluation in eight dogs with OSA [53]. In that study, MRI provided the
most accurate tumor estimate and was recognized as the best modality for
preoperative assessment of appendicular OSA, especially in the context of
a limb-sparing procedure. The last study compared MRI, CT, and radio-
graphs in 10 dogs with appendicular OSA, and failed to identify a superior
modality [51]. That study was performed on samples obtained post-
amputation, making it impossible to use injectable contrast agents for
enhancement of soft-tissue components.

Though biopsy remains the gold standard for diagnosis of OSA, ﬁne

needle aspirate via a 19-gauge needle may provide a diagnosis by less
invasive means [54]. Cells from lytic OSA lesions will readily exfoliate.
Aspirates often reveal large, immature mesenchymal cells that may have
intracytoplasmic or extracellular osteoid, which appears as an amorphous
pink matrix. In addition to malignant osteoblasts, benign osteoblasts and
osteoclasts may also be present. Fine needle aspiration of lytic bone lesions
may help to rule out fungal or bacterial osteomyelitis, where inﬂammatory
cells or organisms may be observed. A tissue biopsy can also be used to
provide preliminary cytologic information by touching or rolling the sample
on a slide. An ongoing study evaluates the usefulness of an ALP stain on
cytology samples to diﬀerentiate OSA from other sarcomas aﬀecting bones.
Preliminary results show that only OSA lesions, as veriﬁed by biopsy,
consistently stain positive for ALP, whereas other sarcomas do not retain
the stain (Dr. Anne M. Barger, personal communication).

Important risks associated with bone biopsy in OSA patients include

the potential for infection, nondiagnostic biopsy and uncommonly iatro-
genic fracture. At minimum, many patients will have increased swelling
and lameness following the biopsy. Biopsy is a painful procedure, and the
patients must be heavily sedated or anesthetized. Core biopsies may be
obtained using a Jamshidi bone core biopsy needle (Baxter Health Care
Corporation, Valencia, CA) or Michel’s trephine; larger samples can be
procured by open surgical biopsy. Several histologic subtypes (telangiectatic,
osteoblastic, chondroblastic, ﬁbroblastic) of osteosarcoma exist, but subtype
has yet to provide prognostic information. OSA arising from the periosteum
diﬀer in prognosis from those of medullary origin, however [43]. Periosteal
OSA is a high-grade, invasive, and metastatic disease that arises from the
periosteal surface of bone. Parosteal OSA also arises from the periosteal

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surface, but this lesion behaves in a manner more like that of a low-grade
soft-tissue sarcoma and is unlikely to invade bone locally or to metastasize
[43]. Histologically, paraosteal OSA lesions have a more benign appearance
than do those of intraosseous or periosteal OSA.

Biologic behavior

Appendicular OSA is a locally invasive, highly metastatic disease. The

pulmonary parenchyma is the most common metastatic site [29]. Lymph
nodes appear to be an uncommon site of metastases, with a reported rate
of 6.1% to 37% [26,29,31,55]. With the now common use of adjuvant
chemotherapy that provides excellent drug levels to pulmonary parenchyma,
bones and soft tissues are increasingly the site of OSA metastasis [55].

Treatment

Standard of care, deﬁned as the treatment option that results in the

longest median survival times, is surgical resection of the primary tumor
followed by 3 to 6 cycles of either a platinum- or doxorubicin-based
chemotherapy protocol [27,30,36,38,45,56,57]. Surgery alone, in dogs with
no evidence of metastatic disease, is associated with a median survival time
of 19 weeks [31].

Surgical options for appendicular OSA include amputation or a limb-

sparing procedure, in which the aﬀected bone is typically resected and
replaced by a normal bone allograft. The site that is most amenable to this
limb-sparing procedure is the distal radius [56,58,59]. Though it is tech-
nically possible to perform limb-sparing at other sites, patients typically
have poor limb function because the procedure requires arthrodesis of the
joint closest to the tumor [58,59]. Criteria for determining a patient’s
eligibility for limb-sparing surgery include tumor involving 50% or less of
the length of the bone, tumor not extending across the joint, and a patient
that is free from metastatic or concurrent disease [59]. Though the bone
allograft is the best described and most widely used technique, alternative
limb-sparing techniques have recently been reported and include ipsilateral
vascularized ulnar transposition autograft, pasteurized tumoral autograft,
bone transport osteogenesis, and intraoperative radiation techniques [43,
60–63].

There are also multiple reports of the use of combination chemother-

apy using doxorubicin and platinum-based chemotherapy drugs for the
treatment of canine OSA, and new protocols have been reported in abstract
form [28,44,64–67]. One report describes alternating doses of cisplatin and
doxorubicin as adjuvant to surgery in 19 dogs with OSA [28]. Survival times
in this study were similar to those reported for dogs treated with single agent
therapy (Table 1). A study evaluating 16 dogs treated with surgery and

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R. Chun, L.-P. de Lorimier

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ble

overvie

appe

ndicu

lar

osteosa

rcoma

therap

over

the

past

years

rgery

and

emothera

protoco

Autho

year

edian

survival

(1-

yr,

2-yr

survival)

alone

19)

Mauldin

1988

[28]

175d

(21%

0%)

(30

dl)

(60

d21

)

doses

19;

appe

ndicular,

axial)

300d

(37%

26%

)

cDDP

q28d

2–6

doses

19)

Shapiro

1988

[30]

301d

(unkn

own,

unkn

own)

cDDP

q28d

doses

until

tastasis

not

16)

Kraegel,

1991

[27]

413d

(62%

18%

)

alone

17)

Straw,

1991

[57]

119d

(11%

4%)

cDDP

q21d

doses

19)

262d

(38%

18%

)

cDDP

21d

preop

and

imm

ediately

postop

35)

282d

(43%

16%

)

alone

15)

Thomp

son,

1992

[45]

168d

(20%

unkno

wn)

cDDP

doses

and

stop

15)

290d

(33%

unkno

wn)

alone

162)

Spodn

ick,

1992

[31]

134d

(11.5%

/limb

spare

cDDP

q21d

1–6

doses

22;

amp,

limb

spa

re).

Surviv

data

not

divi

ded

for

type

Berg,

1992

[56]

325d

(45.5%

20.9%)

/limb

spare

wks

doses

35;

amp,

limb

spa

re).

Beca

use

2–3

doses

were

given

neoa

djuvant

surgery

only

patie

nts

with

minimal

clinical

sign

were

inc

luded;

surv

ival

date

not

divi

ded

for

types

Berg,

1995

[38]

366d

(50.5%

9.7%

)

carbo

300

q21d

48)

Bergman

1996

[36]

321d

(35.4%

unkno

wn)

(15–2

1–2

hour

before

cDDP)

cDDP

(60

)

doses

startin

53)

49)

days

postop;

dogs

died

after

ﬁrst

cycle.

Berg,

1997

[65]

postop

345

(48%,

28.3%,

3–yr

15.3%

)

10d

postop

330

6.2%,

27.5%

3–yr

18%

)

cDDP

dl)

(15

d2)

16).

Also

/empty

lipo

somes

Chun,

2000

[67]

540d

(68.7%

25%

)

bbrevia

tions:

amp

utation;

carbo

platin;

cDDP,

cisp

latin;

day

;

DOX,

doxo

rubicin;

postop,

stopera

tive;

preop,

preop

erative;

sx,

surgery

;

tx,

treated.

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R. Chun, L.-P. de Lorimier

/ Vet Clin Small Anim 33 (2003) 491–516

4 cycles of cisplatin

/doxorubicin, each given within a 24-hour period,

reported signiﬁcantly prolonged survival times (18.5-month median) [67]. A
report of 102 dogs treated with 3 cycles of the same protocol, however,
beginning either 2 or 10 days after surgery, documented median survivals of
11.5 months (beginning therapy 2 days postop) and 11 months (beginning
therapy 10 days postop) [65]. Potential reasons for the discrepancy in sur-
vival times between these studies include small numbers of dogs in the ﬁrst
study, and the fact that the dogs in the ﬁrst study also received empty
liposomes as part of the control arm of an immunotherapy study. Studies
have documented a greater combined immunomodulatory eﬀect of doxo-
rubicin and L-MTP-PE than that seen with either agent alone [68–71].
Though no studies have reported the immunostimulatory eﬀect of doxo-
rubicin with empty liposomes, it is possible that this combination could have
resulted in enhanced monocyte

/macrophage activity and, therefore, longer

survival time. In spite of some conﬂicting results, it remains clear that the
addition of chemotherapy increases survival times from a 4-month median
to an 18-month median at best (Table 1) [27,28,30,31,36,38,45,56,57,65,67].
Alternative routes for delivering chemotherapy and investigated innovative
therapies are brieﬂy described in Tables 2 and 3 [37,86–89,110–114,116].

The most recent reports discussing novel therapies for adjuvant medical

management of canine OSA failed to show improvement in survival times
[41,72,73]. A liposome-encapsulated form of cisplatin, while permitting a
higher dose of cisplatin to be safely administered to 20 dogs with OSA, did
not improve survival time when compared with the carboplatin-treated
control group [73]. A phase 2 clinical trial evaluation with the new third-
generation platinum compound lobaplatin in 28 dogs with OSA showed the
drug to be well tolerated, but the one year survival of 31.8% did not diﬀer
signiﬁcantly from the results obtained with accepted standard-of-care
therapy [41]. Finally, a randomized controlled trial combining a long-acting
insulin-like growth factor (IGF) suppressant (octreotide) with carboplatin in
23 dogs did not improve survival when compared with the carboplatin-
treated control group of 21 dogs, despite a signiﬁcant measurable decrease
in IGF levels [72].

It has been reported that interstitial ﬂuid pressure is greatly increased and

blood ﬂow decreased in OSA tissue, when compared with adjacent un-
aﬀected tissue [74]. This could partly explain poor drug delivery or response
to radiation in a subset of patients, and strategies to bypass this problem
should be evaluated.

Once metastatic disease is clinically detectable, chemotherapy is usually

ineﬀective [33]. Pulmonary metastatectomy is a well-described procedure
that can contribute to signiﬁcantly prolonged survival in dogs. Clinical
guidelines for determining patient eligibility for metastatectomy include:
primary tumor in complete and prolonged remission (ideally for >300 days);
1 to 2 radiographically detectable nodules only; no evidence of distant
metastatic disease at nonpulmonary sites; no evidence of life-limiting

501

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Table

Alternative

chem

othera

livery

rout

Treatm

ent

Autho

yea

Com

ments

Intra-a

rterial

cDDP

doses

limb

spare

16)

Powe

rs,

1991

[37]

49.1%

mean

tumor

necrosis

/14

80%)

Intra-a

rterial

cDDP

doses

/20–4

limb

spa

47)

83.7%

tum

necrosis

(31

/47

80%)

Goal

was

determ

ine

pre

dictive

valu

ercent

tumor

necrosis

DFI

and

ove

rall

survival

Intrame

dullary

cDDP

(via

Jam-Sh

idi

core

biops

instrum

ent)

1–6

doses

Hahn

1996

[111]

Case

7-mo

survival

Case

4.5-m

survival

(tum

present

6-mo

fore

tx)

Case

3-mo

survival

Case

1-mo

nth

surv

ival

Subcuta

neous

cDDP

50–7

saline

dose

Dernel

1997

[110]

Unac

ceptab

toxicity

Inhala

tion

emothera

using

paclitaxel

and

/or

DOX

q14

with

spe

cially

signed

aerosol

device

Hersh

ey,

1999

[112]

cases

metastati

OSA

respo

nses

PR,

Abbrevia

tions:

CR,

mplete

remissio

day;

xorubicin

;

IA,

intra

arter

ial;

mo,

month;

PR,

par

tial

remission;

rad

iation

therap

tx,

treatmen

502

R. Chun, L.-P. de Lorimier

/ Vet Clin Small Anim 33 (2003) 491–516

ble

Othe

repor

ted

eatment

Treat

ment

Auth

or,

year

Comments

Samar

ium

-153

ethylene-d

iam

ine-tetram

ethylen

phosph

onic

acid

(Sm

153

-EDTMP)

MBq

(1.0

mCi)

/kg

1–2

doses,

separa

ted

week

ttimer

1990

[87]

Axia

skeleto

8):

12-m

mean

survival.

Appe

ndicular

skel

eton

20):

surv

ival

site—

radi

/ulna

humeru

tibia

mo,

fem

Some

dogs

had

tastatic

lesions

that

respo

nded

therap

and

eac

eeks

apart

oe,

1996

[89]

Maxilla

OSA

with

and

maxillectomy,

surv

ival

mo.

MBq

/kg

mCi

/kg)

dose

ith

biopsy-

proven

appe

ndicular

OSA)

ilner,

1998

[88]

the

with

OSA

survival

and

response

were

noted.

36–5

/kg

1–4

doses

15,

appe

ndicular,

axial

)

Aas,

1999

[86]

also

with

surg

ery.

edian

survival

mo.

Lipo

some

encapsu

lated

muramyl-

tripeptid

ephosp

hatidyl-

ethanolam

ine

(L-M

TP-PE

)

116

dogs

mult

iple

stud

ies

ove

several

years

acEwe

1996

[114]

Longest

survivo

with

amp

cDDP

liposo

mes

(af

ter

cDDP):

14.4

media

Onl

dogs

NED

aft

received

L-MT

P-PE.

Nebu

lized

IL-2

liposo

inhalatio

therap

daily

15d

then

dail

15d

4),

daily

30d

anna,

1997

[113]

Study

dogs

metastati

primary

lung

cance

ith

OSA

had

and

mo.

cDDP

(60

1–6)

huma

cytot

oxic

T-cell

line

LL-104

-ir

radiated

T-cells

/kg

5d,

then

boosters

q30

mo)

23)

isonneau

1999

[116]

11.5

mo,

52.2%

year

survival,

8.7%

year

survival.

Only

dogs

NED

aft

received

T-c

ell

therap

(CD3

CD8

CD5

6þ

CD1

MHC

nrestricted

killer

activ

ity.

bbrevia

tions:

amp,

amp

utation;

cDDP,

cisp

latin;

CR,

comp

lete

rem

ission;

day;

Bq,

mega

cquerel;

mCi,

milliCurie;

month

;

NED

eviden

disease

;

OSA

osteosa

rcoma

;

tx,

eated.

503

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/ Vet Clin Small Anim 33 (2003) 491–516

intercurrent disease; and a long tumor doubling time (>30 days) with no
new lesions observed during this time period [46]. Though this last point
remains to be of proven importance for veterinary patients, it is a prudent
guideline to follow.

Palliative therapy

Reported survival times for canine patients treated with palliative intent

therapy range from 3 to 10 months [26,31,75–78]. Amputation alone or
administration of medications such as nonsteroidal anti-inﬂammatory drugs
or opioids may signiﬁcantly improve quality of life. Dogs treated with
amputation alone are most often euthanized because of pulmonary
metastatic disease, with a reported median survival time of 19 weeks [31].

The bisphosphonate drug alendronate has been anecdotally reported as

a palliative treatment in 2 dogs with OSA [78]. One dog had tibial disease
and survived 12 months after diagnosis; the other dog had maxillary disease
and survived 10 months after diagnosis. Both dogs were treated with 10 mg
alendronate orally, once daily. Bisphosphonates are antiresorptive agents
acting mainly by causing osteoclast apoptosis (cell death) and are used in
conditions such as osteoporosis and Paget’s disease in humans. The newer
and more potent aminobisphosphonates, such as pamidronate and zo-
ledronate, have been used to control hypercalcemia of malignancy and to
palliate for osteolytic bone pain in humans. Interestingly, recent in vitro
investigation of rat and human OSA cell lines, as well as of normal human
osteoblasts, show encouraging results for using aminobisphosphonates
as cytotoxic, cytostatic, or diﬀerentiating agents for OSA [79–83]. Ongoing
studies on canine OSA cell lines show similar preliminary ﬁndings, with
time- and dose-dependent cytotoxic and antiproliferative eﬀects (de
Lorimier, unpublished data). Canine in vivo studies will address safety
and potential eﬃcacy of such adjuvant therapy in the near future.

Palliative RT typically involves administering coarse fractions of 8 to 10

Gy of megavoltage irradiation, in 3 treatments at 0, 7, and 21 days [76,
77,84]. Palliative RT reportedly improves limb function and quality of life in
about 75% of patients for a median of 2 months duration [76,77,84]. A
recent report describing the use of 4 8-Gy fractions on days 0, 7, 14, and 21
showed response in 92% of sites treated, with a median duration of response
for appendicular sites of 94.5 days [75]. The median survival of these
patients was 313 days. Administration of radiation results in tumor cell
necrosis, replacement by ﬁbrous tissue, and formation and calciﬁcation of
woven bone, which is gradually replaced by lamellar bone. Immediate pain
relief has been attributed to a cytotoxic process aﬀecting normal bone cells
that release chemical mediators (prostaglandins, notably PGE

) in response

to the neoplastic process. These chemical mediators modulate pain per-
ception by stimulating nociceptors located in the periosteum. The later onset

504

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/ Vet Clin Small Anim 33 (2003) 491–516

of pain relief is felt to coincide with recalciﬁcation and repair of osteolytic
lesions [85].

The use of samarium has been described for both appendicular and axial

OSA in dogs (Table 3) [86–90]. Whether this treatment is deﬁnitive or
palliative remains to be fully determined. The radionuclide

153

Sm emits

both b particles, which provide the therapeutic eﬀect, as well as c particles,
which allow for visualization of lesions on bone scans. When bound to
ethylene-diamino-tetramethylene-phosphate (EDTMP) and administered
intravenously (IV), this agent is rapidly and preferentially taken up by
metabolically active bone. While the use of

153

Sm-EDTMP as an alternative

treatment for bone tumors has been well described [86–89], a standardized
protocol for veterinary use is not well established. In general, the agent is
given as an IV bolus at 37 MBq

/kg, and the dose may be repeated at 1- to

19-week intervals. The main toxicity is myelosuppression, which may
continue for up to 4 weeks post-therapy [87,88].

Canine axial osteosarcoma

Signalment

Medium- to large-breed dogs are most commonly aﬀected by axial OSA,

with a reported range in weights of less than 5 kg to 55 kg [25]. Middle-aged
dogs are most often reported to be aﬀected, with the main exception being
dogs with rib tumors. Dogs with rib OSA tend to be younger, although
reported mean and median ages range from 5 to 9 years [91–93]. Female
dogs are more commonly aﬀected than males (2.1:1) [91]. Golden and
Labrador retrievers, German Shepherds, Doberman Pinschers, and Irish
Setters are all over-represented, but the majority of reported dogs are mixed
breeds [91]. Up to 43% of all bone tumors in small (<15 kg) dogs are
osteosarcoma [94]. Unlike the disease in large-breed dogs, OSA in small
dogs tends to aﬀect the axial skeleton more often than the appendicular
skeleton, with 9

/16 (56%) cases being located on the axial skeleton in one

study [94].

History and physical examination

The history varies depending on aﬀected site. The tumor does not usually

grow rapidly, and clinical signs may be insidious. One article cited a range in
duration of signs before presentation extending from several days to 2 years
[91].

The most common sites of axial OSA are the mandible and maxilla; less

commonly aﬀected sites include spine, ribs, nasal cavity, and cranium
[84,91]. One study reported that 4

/37 primary spinal tumors were OSA,

whereas another described vertebral OSA in 14

/20 dogs with primary or

505

R. Chun, L.-P. de Lorimier

/ Vet Clin Small Anim 33 (2003) 491–516

metastatic tumors [55,95]. A total of 34 of 54 (63%) reported primary rib
tumors were OSA in one study, and 24 of 40 (60%) reported primary rib
tumors were OSA in another [92,93]. Thus, though primary bone tumors not
arising in the appendicular skeleton, mandible, or maxilla are rare, they are
likely to be diagnosed as OSA. Clinical signs will vary according to loca-
tion of the primary lesion. A visible or palpable mass, decreased appetite or
dysphagia, pain on opening the mouth, exophthalmia, ptyalism, epistaxis,
paraparesis, and dyspnea are among the observed ﬁndings.

Diagnosis

Diagnostic evaluation of dogs with axial skeletal OSA is similar to that

for dogs with appendicular OSA. A minimum database is advised to rule
out concurrent disease; the signiﬁcance of elevations of serum alkaline
phosphatase in dogs with axial disease is unknown. Diagnostic imaging of
the primary site and thorax is also recommended. Only 11% of axial skeletal
OSA patients have visible pulmonary metastatic disease at the time of
diagnosis (6

/54); however, this metastatic incidence may be site-dependent

[91]. One study of 116 dogs reported thoracic radiographs for 54 cases [91].
None of the dogs with maxillary (n

¼ 11), nasal cavity (n ¼ 4), or pelvic

¼ 2) OSA had lung metastases; however, lung metastases were observed

in dogs with mandibular (1

/19), spinal (1/6), rib (2/7), and cranium (2/5)

OSA. Myelography may be indicated to further delineate spinal tumors.
Vertebral OSA is typically an extradural lesion [95]. Advanced imaging
techniques, such as nuclear scintigraphy, CT, or MRI, can be extremely
valuable in the diagnostic evaluation of axial OSA and will help to plan
therapy. Abdominal radiographs or ultrasound to assess local extent of
disease or metastasis may be appropriate depending on the primary site.
Lymph node enlargement with histologic conﬁrmation of metastasis is
identiﬁed in 7% or fewer cases [91,96]. As with appendicular OSA, biopsy or
ﬁne needle aspirate is required for a diagnosis. Fluoroscopy or CT guidance
might prove valuable, especially for lesions of the spine.

Biologic behavior, prognosis, treatment

The biologic behavior of axial OSA has been postulated to be site-

dependent, with mandibular OSA being less likely to metastasize than OSA
originating in other sites [90]. Univariate analysis identiﬁed tumor loca-
tion on the mandible, complete surgical excision, and lower body weight
(<30 kg) as favorable prognostic factors in one study of 45 dogs [32]. In the
same article, surgical margins and body weight were the most signiﬁcant
prognostic factors based on multivariate analysis. A large retrospective
study described 1- and 2-year overall survival times of 26.3% and 18.4%,
respectively, for animals treated with surgery alone (38/116) [91]. Local

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recurrence was the cause of euthanasia in approximately 80% of cases,
whereas only 7% were euthanized because of metastatic disease. Because
local recurrence is the cause of euthanasia for the majority of cases,
metastases may not have time to manifest or to aﬀect quality of life in axial
OSA patients.

General treatment recommendations for axial OSA are surgical resection

followed by chemotherapy with a platinum- or doxorubicin-based protocol.
The exception is mandibular OSA, for which surgery alone may provide up
to 71% 1–year survivals, and for which chemotherapy has not yet
demonstrated any survival advantage [90].

An unfortunate tendency in the veterinary literature is to retrospectively

report large numbers of dogs treated by a variety of therapies. Because so
few dogs are treated with the same protocol, the reported disease-free and
overall survival times are diﬃcult to interpret. The following section at-
tempts to summarize usable data from reports studying bone tumors at
various anatomic sites.

Mandibular OSA has been described as having a lower metastatic rate

and better prognosis than appendicular OSA. One report of 12 dogs with
oral OSA (4 mandibular and 8 maxillary) treated with surgery alone
obtained a median 1–year survival of 42% [97]. Another article described 81
dogs with mandibular tumors, 9 of which were OSA [115]. In this study,
necropsies were performed in 5 dogs with OSA. Local recurrence was
documented in two of them, lymph node metastases were found in two, lung
metastases in four, and other sites of metastases in two. A third article
described 142 dogs treated surgically for mandibular tumors, 20 of which
were OSA [99]. These investigators reported that two dogs died because of
local recurrence, six dogs developed metastatic disease, and one dog died
from both local and distant disease. Straw and others documented 51 dogs
with mandibular OSA treated by surgery alone (n

¼ 32); surgery and

chemotherapy (n

¼ 10); surgery and radiation therapy (n ¼ 3); surgery,

radiation, and chemotherapy (n

¼ 4); or samarium (n ¼ 2) [90]. The dogs

treated with surgery alone had a 71% 1–year survival rate; there was no
apparent eﬀect on survival of various other treatment modalities. Histologic
grade was signiﬁcantly correlated with survival. Parameters measured for
grade included nuclear pleomorphism (0–3), mitotic rate (1–4), and percent
necrosis (0–3). Grade 1 tumors have a score of less than 6, grade 2 tumors
have a score of 6 to 8, and grade 3 tumors have a score more than 8.

Several studies have been published regarding dogs with maxillary OSA

[91,97,98,100]. A group reported 61 dogs with maxillary cancer. The 11 dogs
with OSA in this study were treated by surgery, with or without che-
motherapy, or radiation therapy [98]. Necropsy performed on 8 of 11 of the
dogs with OSA identiﬁed 3 with local recurrence and 4 with metastases (2 to
lung, and 2 elsewhere). All the dogs in the study, regardless of histologic
diagnosis, were divided into categories based on tumor location. Patients
with rostral lesions had better disease-free and overall survival times than

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those with caudal tumor locations. Respectively, 1- and 2-year survival rates
for lesions rostral to the canine tooth, from the canine to premolar 3, and
caudal to the third premolar were 81% and 68%, 35% and 24%, and 40%
and 20%, respectively. Two cited explanations for this site-associated re-
sponse include a diﬀerence in biologic behavior based on tumor location,
and earlier recognition of rostral tumors. Another explanation is improved
surgical access for complete excision in rostral tumors. In this same article,
there was no observed diﬀerence in survival times among patients with the
diﬀerent malignant tumors identiﬁed, and recurrence rates after ‘‘incom-
plete’’ and ‘‘complete’’ surgery were more than 50% and 40%, respectively.
Potential reasons cited for the high recurrence rate despite complete excision
included: (1) multifocal tumor; (2) development of new, unrelated tumor;
and (3) inadequate pathologic assessment. Overall median survival time for
dogs with maxillary OSA was 5 months, but these dogs received a variety of
treatment modalities [98]. Another study described 69 dogs treated with
hemimaxillectomy alone; 6 of these dogs had OSA and the median survival
time for them was 4.6 months [100].

The spine is an uncommonly reported site of primary OSA [55,91,95,101].

In 5 dogs treated by surgery alone, only one dog survived more than 3 days
[91]. This dog had a tail amputation and survived more than 2.5 years after
surgery. Another retrospective study of spinal tumors in dogs reported 2

dogs treated by surgery alone as having survival times of 21 and 300 days
[95]. A larger retrospective study identiﬁed 14 dogs with primary or met-
astatic vertebral OSA treated by surgery alone, radiation therapy and
chemotherapy, surgery and chemotherapy, or surgery, radiation, and che-
motherapy combined [55]. Although a number of factors varied greatly in
this study, the median survival time for dogs with OSA was 135 days.

Primary rib OSA accounted for 10% of axial OSA cases in one study [91].

In another study, 27% of dogs with rib OSA had metastases at diagnosis,
whereas 45% had metastases identiﬁed at necropsy [102]. Thus, rib OSA
may have a more aggressive behavior than those from other sites on the
axial skeleton. Reported sites of metastases included lungs (5), bone (3),
myocardium and lymph node (1), and lung and mediastinum (1) [96]. In
another study evaluating survival times in 34 dogs with primary rib tumors,
9 dogs with OSA received surgery and chemotherapy (either cisplatin alone
or alternating doses of cisplatin and doxorubicin), and 20 dogs received
surgery alone. Median DFI and survival were 225 days and 240 days for
surgery combined with chemotherapy, but only 60 and 90 days for surgery
alone [93]. A report of 40 dogs treated with en bloc rib resection alone for
primary rib tumors identiﬁed 24 dogs with OSA [92]. Twenty of the dogs
with OSA were followed up beyond 2 weeks postoperatively; 14 died or were
euthanized because of pulmonary metastases, and 4 were euthanized
because of both pulmonary metastases and local recurrence. The remaining
2 dogs were euthanized because of vertebral metastases. Overall median
survival time for the dogs with rib OSA in this study was 3.3 months.

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In a recent report on the use of 4 coarse fractions (8 Gy) of palliative

radiotherapy for OSA in 24 dogs, 9 tumors were of axial location [75]. All 9
dogs had a positive response to RT, with a median onset to diminution of
signs of 14 days, and median duration of 81.5 days. The survival times
ranged from 51 to 391 days, with a median of 162 days [75]. The ra-
dioisotope

153

Sm has been used for dogs with axial OSA, and, although

responses varied, good treatment response and pain control were obtained
in more than 50% of dogs treated [86,87,89]. A mean survival time of 12
months was reported for axial OSA in 8

153

Sm-treated dogs in one report

[87]. Because criteria establishing good candidates for

153

Sm therapy have

changed over time, future reports might provide improved response rates
and survival times.

Canine extraskeletal osteosarcoma and mammary gland osteosarcoma

Incidence and signalment

Canine extraskeletal osteosarcoma (ESOSA) is a rare tumor, accounting

for 0.13% of biopsy submissions (169

/130,754) and 12.6% of all OSA over

a 10-year period in 1 study [103]. In this retrospective study, mammary
gland OSA (MGOSA) was the most common site of ESOSA (108 of 169
cases) [103]. Of the remaining 61 cases, 10 were splenic, 13 originated within
the GI tract, 7 were in the urogenital tract, 6 were hepatic, 6 were cutaneous,
and 13 were in the subcutaneous tissues. In dogs, mixed malignant mam-
mary neoplasms (carcinosarcomas) that produce osteoid are fairly common,
but most arise in conjunction with adenocarcinomas [104]. Twenty of the
108 MGOSA patients had concurrent mammary gland tumors; 13 were
mixed adenomas, 6 were carcinomas, and 1 was a cystadenoma [103].
Although MGOSA comprised the majority of ESOSA in the aforemen-
tioned study, others have observed the spleen to be a more common site
[105–108]. Whereas 1 large retrospective study identiﬁed only 1 ESOSA in
1480 cases of splenic disease in dogs, 2 more recent studies of non-
angiomatous and nonlymphomatous neoplasms of the canine spleen
reported a higher incidence of this neoplasm [106–108]. Of these studies, 1
identiﬁed 8 cases of splenic ESOSA among 87 cases of primary splenic
mesenchymal tumors [106]. Another group reported 10 ESOSA out of 57
cases of splenic neoplasia [108]. In addition to the spleen and mammary
glands, two smaller retrospective studies of ESOSA in dogs described lung,
skin, axilla, mesenteric root, adrenal gland, eye, testicle, vagina, kidney,
intestine, gastric ligament, and liver as other primary sites [104,105]. Older
dogs are usually aﬀected, with mean and median ages of about 10 and 11
years, respectively [103,105]. Although smaller studies reported more
females than males, this has not been borne out in larger studies noting
that ESOSA has no sex predilection [103]. All reported cases of MGOSA
were in females (75% intact, 25% neutered) [103,105]. Though Beagles and

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Rottweilers were at higher risk for ESOSA, Miniature Poodles and German
Shepherds were at higher risk speciﬁcally for MGOSA [103].

History and physical examination

Reported clinical signs associated with ESOSA vary depending on the

location of the tumor and ranged from depression and lethargy to a distended
abdomen, mammary masses, diﬃculty breathing, weight loss, anorexia,
pyrexia, polyuria and polydipsia, vomiting, constipation, and adipsia [103,
105]. Duration of signs extended from 1 day to months before presentation.
Physical examination changes depend upon the site of the primary tumor.

Risk factors and associations

Beagles and Rottweilers have a 3.8 and 3.6 odds ratio, respectively, for

developing ESOSA [103]. Miniature Poodle and German Shepherd dogs
have a 2.7 and 2.2 odds ratio, respectively, for developing MGOSA [103].
Rare cases of esophageal OSA have been associated with Spirocerca lupi
infection [109]. Granulomas arise because of the migration and persistent
presence of larvae and adults within the esophagus; malignant trans-
formation of these lesions may then occur. Metastatic disease at the time
of initial presentation is a poor prognostic factor with ESOSA. The
signiﬁcance of elevated serum alkaline phosphatase levels in ESOSA is
unknown.

Diagnosis

Although no pathognomonic features will be revealed, a minimum

database is recommended to assess overall health status before recommend-
ing and initiating treatment. Radiographs of the thorax are indicated to
screen for metastatic disease. Thoracic radiographs made in 32

/61 cases of

ESOSA revealed lesions in 3

/32 at the time of diagnosis; 38/108 cases of

MGOSA had thoracic radiographs made, and 3

/38 had lesions at diagnosis

[103]. Abdominal radiographs and ultrasonography are indicated depending
on the site of the primary tumor. Many other neoplasms may be diﬀerentials
for MGOSA and ESOSA, making biopsy or ﬁne needle aspiration cytology
essential for diagnosis.

Biologic behavior

Similar to the more common forms of OSA, ESOSA, and MGOSA are

locally invasive and metastatic to lungs and other sites [103–106,108].
Lymph node involvement appears to be more common in ESOSA than in
primary bone OSA, being identiﬁed in 5

/11 dogs with ESOSA in a variety

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of locations and in 2

/7 dogs with MGOSA [103,104]. Liver metastases,

diagnosed in 7

/14 dogs with visceral or pulmonary primary sites of OSA, are

also more commonly reported for ESOSA than for disease of appendicular
or axial locations [105].

Treatment and prognosis

Only 83 of 169 cases had follow-up to death in 1 retrospective study [103].

A total of 72 out of 83 were euthanized or died because of the tumor. Dogs
with ESOSA had a median survival of 26 days, with death from suspected
local recurrence in 92%. Dogs with MGOSA had a median survival of 90
days with death from metastatic disease in 62.5% [103]. Because all of the
reports of ESOSA and MGOSA are retrospective, patients received a variety
of treatments. Based on the known biologic behavior of this tumor, we
recommend surgical resection, if possible, followed by a platinum- or
doxorubicin-based chemotherapy protocol, although improved survival
times with such adjuvant therapy remain to be reported.

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Medical management of soft tissue

sarcomas

Kenneth M. Rassnick, DVM

Comparative Cancer Program, Cornell University, College of Veterinary Medicine,

Box 31, Ithaca, NY 14853, USA

Soft tissue sarcomas (STS) represent a heterogeneous family of

malignancies that arise from mesenchymal tissues. The most common
histological types in veterinary patients include ﬁbrosarcoma, peripheral
nerve sheath tumor (schwannoma and neuroﬁbrosarcoma), malignant
ﬁbrous histiocytoma, and hemangiopericytoma.

Soft tissue sarcomas are locally invasive and inﬁltrative along fascial

planes by way of tendril-like microextensions. Because conservative surgical
resection rarely removes the entire tumor, local recurrence is common. Dogs
with STS having incomplete surgical margins are more than 10 times more
likely to have local recurrence than dogs with complete tumor-free surgical
margins [1].

Until recently, the metastatic potential of STS was diﬃcult to deﬁne. Many

older studies relied on conservative surgical excision alone to control these
tumors, and many patients died as a result of local recurrence soon after
surgery. Based on current information, the overall metastatic potential of
STS is fairly low. In dogs, reports of the metastatic rate of STS range from 8%
to 17% [1–3]. Table 1 summarizes the results of the metastatic rate of canine
STS in various studies. In one study of 75 dogs, 13 developed metastases to
lungs (8), lymph nodes (1), or lungs and lymph nodes (4). Median time to
detection of metastases was 365 days (range, 0 to 1444 days) [1].

Histopathologic grade is predictive of metastases and prognosis in dogs

with STS (Table 2). In the study by Kuntz et al [1], 4 of 31 (13%) dogs with
grade 1 tumors developed metastases, and only 2 of 27 (7%) with grade 2
tumors developed metastases, whereas 7 of 17 (41%) with grade 3 tumors
developed metastases. Overall median survival for dogs with STS treated by
surgery alone was 1416 days. Median survival for dogs with more than 19
mitotic ﬁgures per 10 high-power ﬁelds (HPF; n

¼ 11) was 236 days

Vet Clin Small Anim

33 (2003) 517–531

E-mail address:

kmr32@cornell.edu

0195-5616

/03/$ - see front matter

doi:10.1016

/S0195-5616(03)00019-6

compared with 1444 days (n

¼ 46) for dogs with less than 10 mitotic ﬁgures

per 10 HPF and 532 days for dogs with 10 to 19 mitotic ﬁgures per 10 HPF
(n

¼ 18). Percentage of histologic necrosis was also prognostic for survival

time. Dogs with greater than 10% necrosis were 2.78 times more likely to die
of tumor-related causes than dogs with less than 10% necrosis [1].

The true metastatic potential of STS in cats is controversial. Reported

metastatic rates have ranged from 0% up to 25% [4–7]. Table 1 summarizes
the results of the metastatic rate of feline STS in various studies. Recent
studies of cats with vaccine-associated sarcomas have included cats with
radical surgical procedures and often adjunctive treatment such as radiation
therapy. Local tumor control in these recent studies has improved, which
may in turn increase survival suﬃciently for occurrence and detection of

Table 1
Results of the metastatic rate of canine and feline soft tissue sarcomas in various studies

Investigator

Number evaluated

Patients
Metastases

Canine

Kuntz [1]

75 (all grades)

17%

31 (grade 1)

13%

27 (grade 2)

17 (grade 3)

41%

Forrest [2]

14%

McKnight [3]

Feline

Bregazzi [4]

Cohen [5]

12%

Hershey [7]

23%

Davidson [6]

26%

Cronin [8]

27%

Table 2
Variables used to determine histologic grade of canine soft tissue sarcomas

Score

Degree of
diﬀerentiation

Mitotic ﬁgures
per 10 HPF

Necrosis

Resemble normal adult

mesenchymal tissue

None

Speciﬁc histologic type

10–19

50%

Undiﬀerentiated

50%

Grade

Description

Cumulative score of <5

Cumulative score of 5 or 6

Cumulative score of >6

Abbreviation:

HPF, high-powered ﬁeld.

Data from

Kuntz CA, Dernell WS, Powers BE, et al. Prognostic factors for surgical

treatment of soft tissue sarcomas in dogs: 75 cases (1986–1996). J Am Vet Med Assoc 1997;211:
1147–51.

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metastases. Although metastases have been reported as early as 5 weeks
after surgery [8], feline STS metastases usually occur much later. Whether
histopathologic features predict prognosis for cats with STS is unclear.
Some investigators believe that the metastatic rate for vaccine-site sarcomas
may be higher than for sarcomas aﬀecting other sites. This has not been
conﬁrmed, however. When compared with nonvaccine sites, vaccine-
associated sarcomas typically have increased amounts of necrosis and
cavitations, peritumoral lymphoid aggregates, pleomorphism, multinucle-
ated giant cells, and increased mitotic activity [9,10]. Davidson et al
evaluated a histopathologic grading system based on mitotic activity and
diﬀerentiation [6]. When cats were treated by surgical excision alone, tumor
grade did not inﬂuence tumor-free interval or survival time signiﬁcantly.

Fortunately, most dogs and cats with STS will present without clinically

evident metastases. For this reason, adequate surgical resection with or
without adjuvant external beam irradiation remains the primary therapy for
STS. The greatest survival rates have been achieved in studies involving
radical resection [1,6,7]. If tumors are removed incompletely, however,
survival rates attained by use of radiation therapy adjunct to incomplete
surgical excision can equal or exceed those attained by wide surgical excision
[2–5].

Which patients are candidates for chemotherapy?

The overall metastatic rate of STS in dogs and cats is fairly low (Table 1).

Aggressive surgical resection or the combination of surgery and radiation
therapy represents the best chance for control. Nevertheless, many patients
with STS have a poorer prognosis and fail to be cured. For example, despite
adequate local control, the tumor may have metastasized early in the course
of disease. Also, several histologic types are included in the broad category
of STS but diﬀer from the most common types, either in location of
occurrence, or metastatic potential. Chemotherapy can be considered in the
following clinical situations:

1. Patients presenting with distant metastases.
2. Patients presenting with tumors that are inoperable because of tumor

size or location. In some patients, a measurable response to chemo-
therapy might allow for function-preserving surgical resection.

3. Patients with tumors that recur despite radical excision or radiation

therapy and are not amenable to retreatment.

4. Dogs presenting for treatment following surgical removal of a high-

grade (grade 3) STS.

5. Cats presenting for treatment following surgical removal or radiation

therapy for an injection-site sarcoma.

6. Dogs with hemangiosarcoma, synovial cell sarcoma, osteosarcoma, rhab-

domyosarcoma, lymphangiosarcoma, or liposarcoma. These histologic

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K.M. Rassnick

/ Vet Clin Small Anim 33 (2003) 517–531

types are included in the broad classiﬁcation of STS but diﬀer in their
metastatic potential. Readers should refer to appropriate literature to
determine speciﬁc features of histologic grade or clinical presentation that
may predict biological behavior.

7. Dogs and cats with STS of internal organs. Prognosis for patients with

STS of internal organs such as the spleen is often very poor. For
example, in one study of 65 dogs with splenic sarcomas, overall median
survival was 2 months [11]. Again, readers should refer to appropriate
literature to determine speciﬁc features of grade or location that may
correlate with prognosis.

8. Dogs with oral sarcomas. When treated with radiation therapy, median

survival for dogs with oral sarcomas is signiﬁcantly shorter than that of
dogs with sarcomas at other sites (1.5 years versus 6.2 years) [2]. Also,
histologically low-grade, yet biologically high-grade sarcomas occur in
the oral cavity of dogs. Often they are misdiagnosed as ﬁbromas or even
chronic inﬂammation. Despite deceptively benign histologic features,
these tumors tend to grow rapidly and invade deeper structures, includ-
ing bone. In one study of 25 dogs, 20% developed metastases to the
lungs or lymph nodes [12]. The metastatic rate in another report of dogs
with oral sarcomas was 54% (7 of 13) [13].

Single-agent chemotherapy

There are reports evaluating various chemotherapy agents to treat STS.

Most of these have been phase I and II studies or case reports and small case
series. There is a relative paucity of randomized trials, and many of these
have included only small numbers of patients. A further complicating factor
is the histologic heterogeneity of these tumors. With few exceptions, chemo-
therapy agents have not been evaluated against single histologic subtypes,
and studies enrolling all histologic types have not been large enough to allow
valid conclusions to be drawn from subgroup analysis. Although there are
relatively few agents with very impressive antitumor eﬃcacy against STS in
dogs and cats, it is important not to overlook the potential clinical utility of
certain commonly available drugs (Table 3).

Doxorubicin

Doxorubicin is considered to be the most active single agent to treat STS.

In people, doxorubicin has been reported to induce objective response rates
varying from 15% to 34% against STS, the majority of which are partial
responses [14]. In vitro sensitivity to doxorubicin has been examined for
various canine cell lines, including sarcomas [15]. In the study by Macy et al,
specimens from 34 canine tumors were grown in agar gel and exposed to
doxorubicin. Of the seven sarcoma cell lines examined, one was susceptible
to doxorubicin [15].

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The clinical antitumor eﬀect of two intravenous (IV) doses of doxorubicin

(30 mg

body surface area) 3 weeks apart has been documented in a multi-

institutional phase II study [16]. In that study, 157 dogs with nonlymphoid
malignant neoplasms were evaluated. Remissions were documented in 23%
(11 of 48) dogs with sarcomas. Complete responses (100% reduction in
tumor size) were observed in dogs with synovial cell sarcoma, undiﬀeren-
tiated sarcoma, liposarcoma, and neuroﬁbrosarcoma. Partial responses (at
least 50% reduction in tumor size) occurred in dogs with hemangiosarcoma,
osteosarcoma, synovial cell sarcoma, undiﬀerentiated sarcoma, ﬁbrosar-
coma, and one dog with an inﬁltrating lipoma [16].

Several investigators have evaluated the use of doxorubicin combined

with cyclophosphamide to treat malignancies [17–20]. Sorenmo et al treated
dogs with hemangiosarcoma every 3 weeks with doxorubicin (30 mg

IV,

day 0) and cyclophosphamide (50 to 75 mg

per os, days 2 to 5). Nine

dogs had measurable subcutaneous disease, and complete responses
occurred in two [18].

Further reports have documented the use of the doxorubicin

/cyclophos-

phamide combination. For example, the combination has been used to treat
dogs with synovial cell sarcoma. Tilmant treated a dog with synovial cell
sarcoma of the tarsus and noted a durable complete remission for greater than
3 years [19]. Vail treated two dogs with the combination, and neither tumor
responded [20].

The supplemental activity of cyclophosphamide in addition to doxorubi-

cin in the management of STS is controversial. For dogs with hemangio-
sarcoma, data suggest that single-agent doxorubicin may be equally eﬀective
as protocols containing doxorubicin and cyclophosphamide or doxorubicin
with vincristine and cyclophosphamide [21]. Most studies continue to be
conducted with the premise that doxorubicin represents the most active agent
and is the basis upon which combinations they should be built.

Doxorubicin also is considered to be the most eﬀective single antitumor

agent to treat STS in cats. Williams demonstrated signiﬁcant in vitro dose-
dependent eﬀects of doxorubicin on the viability of two vaccine-associated
feline sarcoma cell lines [22].

Table 3
Single-agent chemotherapy for canine and peline soft tissue sarcomas

Drug

Dosage

Route

Interval (days)

Canine

Doxorubicin

30 mg

14–21

Mitoxantrone

6 mg

Ifosfamide

375 mg

14–21

a,b

Feline

Doxorubicin

25 mg

Mitoxantrone

6.5 mg

Carboplatin

210 mg

21–28

Abbreviation:

IV, intravenous.

Dosing interval depends on adequate neutrophil recovery after treatment.

In conjunction with a speciﬁc protocol for diuresis and mesna administration.

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Phase II evaluations of single-agent doxorubicin in cats are lacking. The

combination of doxorubicin and cyclophosphamide has been examined,
however. Mauldin studied the use of doxorubicin (25 mg

IV day 0) and

cyclophosphamide (50 mg

per os, days 2 to 5) in 23 cats with malignant

nonhematopoietic tumors. Two of three cats with nonresectable oral ﬁbro-
sarcoma responded to treatment for 60 and 150 days, respectively [23].

Barber examined 12 cats, all with vaccine-associated sarcomas, treated

with the same chemotherapy combination. Overall response rate was 50% (6
of 12). Two of the 12 cats had major responses for 153 and 190 days,
respectively. Partial responses (at least 50%) occurred in four cats (33%).
Duration of partial response ranged from 41 to 144 days, with a median of
103 days. Of the seven cats in the study treated only with chemotherapy,
there was a signiﬁcant improvement in survival for those cats that responded.
Median survival of three cats responding to doxorubicin

/cyclophosphamide

combination therapy was 242 days, compared with only 83 days for the four
cats failing to respond [24].

Mitoxantrone

Mitoxantrone is an anthracenedione compound related to anthracyclines

such as doxorubicin. In people, mitoxantrone is minimally active against
STS. In a study of 115 patients with STS, response rate was only 1% [14]. A
dose escalation study of mitoxantrone in tumor-bearing dogs showed
activity against STS. Ogilvie et al used mitoxantrone to treat 126 dogs with
measurable tumors. Dosages ranged from 2.5 to 5 mg

IV. Further data

support that the maximally tolerated dosage of mitoxantrone in dogs is 6
mg

. Four of the 12 dogs with sarcomas responded in this study when

treated with mitoxantrone at 5 mg

. Dogs with ﬁbrosarcoma, chondro-

sarcoma, and hemangiopericytoma had measurable tumor reductions for 21
to 167 days [25].

Further evaluation of mitoxantrone for STS has been conducted by

Henry et al. In their study, dogs were treated with mitoxantrone (5 mg

IV, day 0) combined with cyclophosphamide (150 mg

IV, day 0) and

granulocyte colony stimulating factor (G-CSF). All six dogs with measur-
able sarcomas treated with the combination failed to respond. Tumor tissues
from dogs enrolled in this study were tested in vitro for sensitivity to the
mitoxantrone

/cyclophosphamide combination. Although the assay seemed

to predict response to the drugs, there was no correlation to clinical re-
sponse, since no dogs had measurable reduction of tumor [26].

There have been limited evaluations of mitoxantrone to treat STS in cats.

As with doxorubicin, mitoxantrone had signiﬁcant dose-dependent eﬀects
on the in vitro viability of two vaccine-associated feline sarcoma cell lines
[22]. In a study to evaluate in vivo toxicity and eﬃcacy of mitoxantrone, 7 of
87 cats had sarcomas [27]. Two of the seven cats had complete remissions
when treated with mitoxantrone. One cat with rhabdomyosarcoma had

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a durable complete remission for 120 days, and one cat with ﬁbrosarcoma
had a complete remission for 90 days [27].

Ifosfamide

Ifosfamide is an oxazophosphorine nitrogen mustard developed as an

isomer of the alkylating agent cyclophosphamide. As a result of minor
structural diﬀerences, ifosfamide has a diﬀerent spectrum of clinical anti-
tumor activity and toxicity than cyclophosphamide. Numerous clinical trials
in people have demonstrated that ifosfamide has major antitumor activity
against STS. Several phase II trials of ifosfamide in people with STS have
demonstrated response rates of 25% to 30% in untreated patients and 15% to
18% in patients previously treated with doxorubicin [28]. There has only been
one study to evaluate ifosfamide in tumor-bearing dogs. The eﬃcacy and
toxicity of ifosfamide were evaluated in 72 dogs with spontaneously occurring
tumors [29]. A dosage of 375 mg

was found to be safe in dogs. Treatments

can be given intravenously every 2 to 3 weeks, depending on hematopoietic
recovery. A speciﬁc protocol for saline ﬂuid diuresis to prevent renal toxicity
and mesna injections to prevent urothelial toxicity was used. In that study, 13
dogs had measurable sarcomas. Two of the 13 dogs had durable complete
remissions. One dog with metastatic cutaneous hemangiosarcoma responded
for more than 445 days after three consecutive treatments. The other dog had
metastatic leiomyosarcoma of the urinary bladder. Complete regression of
metastatic lymph nodes occurred after treatment with ifosfamide, and the dog
died of other causes more than 500 days later [29].

Phase I and II studies are underway to evaluate ifosfamide in cats

(Rassnick, unpublished data). Preliminary results indicate ifosfamide is safe
in cats, although the dosage and spectrum of toxicity are much diﬀerent
than that seen in dogs. As with dogs and people, ifosfamide appears active
against STS in cats. Although studies are still ongoing, complete and partial
responses have occurred in cats with injection-site sarcomas treated with
ifosfamide (Rassnick, unpublished data).

Other single-agent chemotherapy options

Other cytotoxic drugs may be useful to treat STS in dogs and cats.

Vincristine has been demonstrated to be eﬀective in 15% of people with STS
[14]. In vitro, vincristine has signiﬁcant dose-dependent eﬀects on the viability
of four vaccine-associated feline sarcoma cell lines [30]. There have been a few
reports of STS-bearing dogs and cats treated with vincristine. Hahn reported
a complete remission for 210 days in a cat with an oral ﬁbrosarcoma treated
solely with vincristine [31]. Vincristine was not successful in the treatment of
metastatic hemangiosarcoma in another cat, however [32].

Dacarbazine (DTIC) is an antitumor alkylating agent. In people, it often

is used with doxorubicin as ﬁrst-line therapy against sarcomas [14]. There

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have been anecdotal suggestions that dacarbazine may have eﬃcacy against
sarcomas in dogs.

Actinomycin-D is an important component of chemotherapy in the

treatment of pediatric rhabdomyosarcoma [14]. Information on actinomy-
cin-D as a single-agent in veterinary patients is limited, however. Hammer
evaluated actinomycin-D in 34 dogs. Two dogs in that study had measur-
able sarcomas, and neither dog had a positive response to treatment with
actinomycin-D [33].

Docetaxel is occasionally eﬀective against sarcomas in people. Response

rate is generally less than 15%, so use of the drug is limited [14]. Docetaxel
caused signiﬁcant inhibition of four vaccine-associated feline sarcoma cell
lines when tested in vitro [30]. There are no published reports of the use of
systemic docetaxel in dogs and cats. Polysorbate-80 is an inorganic solvent
necessary for IV docetaxel administration, and when given to dogs, severe
acute hypersensitivity reactions occur.

Finally, platinum-derived agents including cisplatin and carboplatin hold

some promise of clinical activity for the treatment of STS. In dogs, cisplatin
has signiﬁcant activity against bone sarcomas. The single-agent response
rate against other histologic subtypes has been poorly evaluated, however
[34]. Preliminary evidence to evaluate carboplatin in cats with STS suggests
activity; however, controlled studies are not available. Two cats that failed
treatment with doxorubicin subsequently were treated with carboplatin, and
no objective responses occurred [24].

New chemotherapy agents and approaches

Because of the relative lack of therapeutic eﬃcacy and signiﬁcant toxicity

of existing chemotherapy protocols, new agents, delivery methods, and
supportive measures to improve chemotherapy for STS are always areas
of research interest. For example, the most serious adverse eﬀects of
doxorubicin are myelosuppression and cardiotoxicity, and several strategies
have been used to limit these toxicities. Doxil, a pegylated liposomal form of
doxorubicin, is one such novel formulation. Liposomes containing small
fractions of polyethylene-glycol (PEG)-phospholipid (so-called ‘‘stealth’’
liposomes) avoid mononuclear phagocyte sequestration and result in
improved circulation time and a diﬀerent spectrum of toxicities. In a study
by Vail et al, 16 dogs with sarcomas were treated with Doxil. Responses were
observed in ﬁve dogs (31%), including complete remissions in dogs with
hemangiosarcoma and malignant ﬁbrous histiocytoma and partial remissions
in dogs with anaplastic sarcoma, ﬁbrosarcoma, and neuroﬁbrosarcoma [35].

FCE 23762-methoxymorpholino-doxorubicin is another new doxorubi-

cin analog. The drug was evaluated in 15 dogs with various STS including
osteosarcoma, undiﬀerentiated sarcoma, and lymphangiosarcoma. Three
dogs in this study achieved remission in response to treatment [36].

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Inhalational delivery of chemotherapy oﬀers several potential advantages

over oral and parenteral administration. These include locoregional delivery
to the lungs, avoidance of ﬁrst pass metabolic degradation by the liver, and
possibly fewer systemic adverse eﬀects. Hershey et al used an aerosol devise
designed to deliver paclitaxel and a new formation of doxorubicin to dogs
with primary and metastatic lung tumors. Two of three dogs with sarcomas
had measurable responses when treated with inhalational chemotherapy [37].

Another approach to limit systemic toxicities of drugs is through the use

of intratumoral or intracavitary implantation. OPLA (open cell polylactic
acid) is a porous biodegradable solid polymer that is impregnated with
cisplatin (OPLA-Pt). This local form of therapy can be placed into the wound
bed following resection of a tumor. Cisplatin concentrations within the
wound far exceed those attainable by IV administration, while obviating the
need for high systemic concentrations, thus avoiding systemic toxicities.
Dernell et al placed OPLA-Pt into the wound following marginal (histo-
logically incomplete) resection of STS in dogs. Local recurrence developed in
10 of 32 (31%) sites, possibly similar to the rate of recurrence after radiation
therapy. High tumor grade was associated with local tumor recurrence.
Unfortunately, OPLA-Pt had to be removed from 9 of 32 (28%) sites because
of local wound complications. This complication rate indicates the need for
further reﬁnement of the polymer

/cisplatin system before the full therapeutic

potential of this agent can be evaluated [38].

Combination and sequential chemotherapy

Several cytotoxic drugs have been identiﬁed to have single-agent clinical

activity against STS (Table 4). Clinical oncology has a long tradition of
mixing and matching individual chemotherapy drugs with documented
single-agent activities to construct chemotherapy protocols combining or
alternating drugs (sequential chemotherapy) on a biweekly or triweekly
schedule. Most studies have been undertaken with the premise that doxo-
rubicin represents the most active drug and is therefore the basis upon which
combination or sequential therapy should be built.

The combination of doxorubicin and cyclophosphamide for treatment of

STS has been reported frequently. The supplemental activity of cyclophos-
phamide in addition to doxorubicin in the management of STS remains
controversial. For example, in canine hemangiosarcoma, data suggest that
single-agent doxorubicin may be equally eﬀective as protocols containing
doxorubicin with cyclophosphamide. Deﬁnitive determination is not possible
in the absence of appropriately designed prospective randomized studies,
however [21].

Doxorubicin, cyclophosphamide, and vincristine (VAC protocol) have

been used extensively for adjunctive therapy of canine hemangiosarcoma
after surgery. Dogs are treated with doxorubicin (30 mg

IV, day 0),

cyclophosphamide (100 to 150 mg

per os, day 0), and with vincristine

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(0.75 mg

, days 7 and 14). The entire cycle is repeated every 21 days. In ﬁve

dogs with measurable hemangiosarcoma, no objective responses occurred;
however, the authors reported a median survival time of 172 days for the
15 dogs treated with the protocol [39]. Objective reductions in STS tumor
size when treated with VAC have been reported by other investigators.
For example, an atrial hemangiosarcoma responded for 16 weeks, and a
rhabdomyosarcoma of the bladder transiently responded for three weeks
[40,41]. Neutropenia, sepsis, and enterocolitis are signiﬁcant adverse eﬀects
of this protocol. Strict monitoring, appropriate supportive care, and overall
clinical caution are advised [17].

Because ifosfamide has been identiﬁed as an agent with antitumor

activity against STS, the author analyzed a biweekly, alternating protocol of
ifosfamide and doxorubicin in dogs. Dogs received ifosfamide (375 mg

IV) with saline diuresis and mesna on day 0 and doxorubicin (30 mg

IV)

on day 14. This cycle was repeated twice for a total of three doses of each
drug. The protocol was tolerated by dogs, but eﬃcacy against STS has not
been determined (Rassnick, unpublished data).

Ifosfamide has activity against feline STS (Rassnick, unpublished data),

but protocols to combine or alternate ifosfamide with other agents have not
been evaluated in cats.

A protocol alternating doxorubicin and carboplatin can be given to cats

safely. Cats can receive doxorubicin at 25 mg

IV on day 0 and carboplatin

at 190 mg

IV on day 21. The cycle can be repeated twice for a total of

three doses of each drug. Careful hematologic monitoring is warranted,
especially after treatment with carboplatin because of the potential for
prolonged neutropenia. Treatment may need to be delayed if neutrophil
recovery is not adequate before the next scheduled treatment. Investigation

Table 4
Combination and sequential chemotherapy options for canine and feline soft tissue sarcomas

Protocol

Drug

Dosage

Route Day

Cycle
interval

Number
of cycles

Canine AC

Doxorubicin

30 mg

21 days

Cyclophosphamide

75 mg

2–5

VAC

Doxorubicin

30 mg

21 days

Cyclophosphamide

100–150

Vincristine

0.75 mg

7, 14

Adria

/Ifos

Doxorubicin

30 mg

28 days

Ifosfamide

375 mg

Feline

Doxorubicin

20 mg

21 days

Cyclophosphamide

100 mg

Adria

/Carbo Doxorubicin

25 mg

42 days

Carboplatin

190 mg

Abbreviations:

IV, intravenous; PO, by mouth.

In conjunction with a speciﬁc protocol for diuresis and mesna administration.

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of the utility of this protocol to treat cats with STS is underway (Rassnick,
unpublished data).

Practical use of chemotherapy

Patients with measurable disease

Adequate surgical excision with or without radiation therapy remains the

principle treatment modality for dogs and cats with STS. Patients that may
qualify for chemotherapy, however, include those that present with dis-
tant metastatic disease, develop local recurrence despite aggressive local
therapies, or have nonresectable tumors.

Neoadjuvant or primary chemotherapy (chemotherapy before treatment

of the primary tumor with surgery or radiation therapy) increasingly has
been accepted as a modality that may assist in limiting the degree of radical
surgery. For patients that achieve a measurable response to preoperative
chemotherapy, a function-preserving surgical excision may be feasible.
Although neoadjuvant chemotherapy appears attractive in some situations,
caution remains appropriate, because one might do more harm to patients
by excessively decreasing local interventions aimed at achieving optimal
rates of local control. For example, if a tumor shrinks signiﬁcantly in
response to neoadjuvant chemotherapy, what should the surgical oncologist
plan as the volume of resection? Additionally, what volume of tissue should
be irradiated to obtain local control postoperatively? Additional experience
from clinical trials studying neoadjuvant chemotherapy and multimodality
approaches will be necessary before answers to these questions are clear.

In the measurable disease setting, generally two consecutive cycles of

single-agent chemotherapy are administered. If no response is observed, then
a diﬀerent chemotherapeutic should be considered. Response to therapy is
determined by measurable reduction (at least 50%) in size or extent of
disease. Tumors that do not show evidence of growth beyond 25% of the
original tumor volume, or volume reduction beyond 50%, are considered to
be in a state of ‘‘stable disease.’’ Because of the variable biologic behavior of
STS with regard to patterns of growth, it is diﬃcult to determine whether
disease stabilization in a given case is the result of treatment, or rather caused
by the natural course of tumor progression. Clinicians faced with stable
disease in the face of therapy must decide on a case-by-case basis whether the
chemotherapy should be continued. Randomized, prospective trials are
needed to establish the clinical signiﬁcance and patient beneﬁt achieved by
disease stabilization in light of toxicity risks and therapy costs.

Adjuvant chemotherapy

The principle objective of adjuvant chemotherapy following sur-

gical removal of the primary tumor or radiation therapy is to destroy

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micrometastatic disease and improve overall survival. The incorporation of
adjuvant chemotherapy in the management of canine appendicular osteo-
sarcoma and hemangiosarcoma is accepted. The clinically signiﬁcant beneﬁts
of adjuvant chemotherapy in the treatment of STS have not been clearly
established, however.

Although the response rates of most drugs in the advanced disease setting

is mild to moderate, adjuvant chemotherapy is based on the premise that
a reduced tumor burden might be eradicated more easily. In dogs, available
data suggest that low-grade STS is best managed with local therapies alone,
and that mortality caused by tumor in this population is low. In contrast, the
prognosis for patients with high-grade lesions is considerably worse, and
most of these patients ultimately die from disease. The biologic behavior
of oral sarcomas and of histologic STS subtypes such as osteosarcoma,
hemangiosarcoma, synovial cell sarcoma, and rhabdomyosarcoma may be
more aggressive. Early institution of chemotherapy may be indicated for
these patients, but continued investigation of systemic adjuvant therapies
is necessary. Commonly used adjuvant chemotherapy protocols for dogs
with STS include single-agent doxorubicin (ﬁve treatments), doxorubicin
combined with cyclophosphamide, VAC, or doxorubicin alternating with
ifosfamide (see Tables 1 and 2).

Options for adjuvant chemotherapy in cats with STS include single-agent

doxorubicin (ﬁve treatments), doxorubicin combined with cyclophospha-
mide, and doxorubicin alternating with carboplatin (see Tables 1 and 2). As
is the case with dogs, the use of adjuvant chemotherapy to treat cats with
STS remains controversial. In one study of 25 cats with vaccine-associated
sarcomas, 18 were treated with surgery and radiation therapy (58 Gy)
followed by doxorubicin (1 mg

/kg IV every 3 weeks for ﬁve treatments), and

seven cats were treated with surgery and radiation therapy only. The median
survival for the chemotherapy group was 674 days versus 842 days for the
group without chemotherapy. There was no signiﬁcant diﬀerence between the
groups. The power of the study (5%) was low, however, and more cats would
be needed in each treatment group to detect a diﬀerence, should one exist [4].

In another study of 76 cats with vaccine-associated sarcomas, 26 received

adjuvant chemotherapy of doxorubicin (20 mg

IV) and cyclophos-

phamide (100 mg

IV) every 3 weeks. As with the Bregazzi study [4], no

advantage for the chemotherapy group was detected. Recurrence rate
(41%), rate of metastasis (12%), and survival (730 days) for cats that
received chemotherapy were not signiﬁcantly diﬀerent from values of cats
that did not receive chemotherapy [5]. Also, as in the Bregazzi study,
analysis was limited. Although 26 cats received chemotherapy, many re-
ceived treatment after a recurrence or development of metastases. Power to
detect a diﬀerence and sample size was very small [5].

The role of adjuvant chemotherapy to treat STS in dogs and cats remains

undetermined. Further study is necessary to provide objective support for
the use of adjuvant chemotherapy in patients with resected sarcomas and to

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determine which regimens are most appropriate. The magnitude of any
expected beneﬁt and the costs, both ﬁnancial and in terms of toxicity, also
must be weighed before global recommendations can be made.

Summary

Local therapies for STS, including aggressive surgical resection and

radiation therapy, have improved dramatically over the last 10 years. This
has resulted in improvement of important clinical outcomes such as en-
hanced rates of local control and higher quality of life for patients. Systemic
therapy for STS, although eﬃcacious to a certain degree, remains
investigational. A major task for oncologists will be to measure accurately
the single-agent activity of drugs and to integrate promising agents into
multiagent regimens. This approach may allow signiﬁcant progress in the
management of STS in canine and feline patients.

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Hemangiosarcoma in dogs and cats

Annette N. Smith, DVM, MS

Department of Clinical Sciences, Veterinary Teaching Hospital,

Wire Road, Auburn University, AL 36849, USA

Incidence and patient factors

Hemangiosarcoma (HSA, including angiosarcoma and malignant he-

mangioendothelioma) is a highly malignant tumor derived from the
endothelial cell line and is characterized by early and aggressive metastasis
[1–15]. HSA is a common tumor type in dogs, comprising up to 5% to 7%
of noncutaneous primary malignant neoplasms [4,7–12,16–18]. In other
species, the incidence is much lower; in cats, it is reported as 0.5% to 2%
[19–22].

HSA is usually seen in older animals, although there are sporadic reports

of the tumor occurring in younger dogs and cats (C.M. Johannes et al,
unpublished data) [8,9,11,23–25]. The mean age of occurrence ranges from 8
to 13 years in the dog [1,8,9,11,26] and from 8 to 10.5 years in the cat (C.M.
Johannes et al, unpublished data) [19,21,25,27].

The breed that appears to be predisposed is the German Shepherd Dog,

also known as the Alsatian [9–12,26], although this breed was also the most
popular dog at the time of these studies. Other commonly reported breeds
include the Golden Retriever, Pointer, Boxer, Labrador Retriever, English
Setter, Great Dane, Poodle, and Siberian Husky [1,4,28]. Any large-breed
dog appears to be at increased risk [26,29,30]. Lightly pigmented, sparsely
haired dogs (Beagles, Bloodhounds, White Bulldogs, English Pointers,
Salukis, Dalmatians, and Whippets) have also been reported as predisposed
to the development of cutaneous and subcutaneous HSAs [24]. No speciﬁc
breeds have been identiﬁed in the cat as having a predisposition for HSA
development; most tumors are seen in the domestic shorthair cat
(C.M. Johannes et al, unpublished data) [19,21,25,27,31]. Cats with unpig-
mented skin may be predisposed to cutaneous tumor development in those
areas [31].

Vet Clin Small Anim

33 (2003) 533–552

E-mail address:

smith30@auburn.edu

0195-5616

/03/$ - see front matter

doi:10.1016/S0195-5616(03)00002-0

Sex predilection has been reported for male dogs [8,12], but some studies

have indicated no difference in occurrence between male and female dogs
[4,17], and in two studies, spayed female dogs were reported to be at
increased risk [11,32]. Because of the conﬂicting reports, some oncologists
have concluded that there is no true sex predilection [33,34]. One study
suggested a female predilection for splenic HSA in the cat [21], although the
male-to-female ratio was only 1:1.25 in 18 cats. Conversely, another study of
31 cats with HSA had more male cats than female cats affected, with a ratio
of 1.9:1 [25]. In a study of cutaneous vascular tumors, 12 of 15 cats were
male, and the authors suggested a male predilection [31].

Etiology

Cutaneous HSAs have been associated with ultraviolet light exposure in

dogs [24,35]. These tumors often arise on the ventral abdomen, where the
hair coat is sparse [24,36]. Breeds at risk are those with a light hair coat and
poor pigmentation, and the usual sites of occurrence are the prepuce and
ventral abdomen on glabrous skin [24]. Subcutaneous or hypodermal HSAs
do not seem to be associated with ultraviolet light exposure [24].

Other radiation sources can cause visceral HSA development. In dogs

exposed to intraoperative radiation therapy, dogs that received greater than
20 Gy seemed to be at more risk for sarcoma development [37]. One dog
developed an HSA of the bladder after 30 Gy had been administered to the
area 3.5 years previously [37]. Dogs that were exposed to aerosolized

144

SrC

developed primary pulmonary, bone, and liver HSAs at a high

rate and usually died of their tumor [38].

A breed predisposition suggests a genetic etiology. German Shepherd

Dogs are the most commonly reported breed with HSA [1,4,8,9,
12,15,18,26,39–42]. Other large-breed dogs are also overrepresented, espe-
cially Golden and Labrador Retrievers [5,18,41,42]. No speciﬁc genetic
mutations have been associated with HSA, although few genetic abnormal-
ities have been studied. In one study [43], the tumor suppressor protein p53
was investigated in feline tumors. Only one of seven HSAs demonstrated
p53 nuclear staining, with no evidence of cytoplasmic signals, suggesting
mutation of this gene is uncommon in feline HSA [43]. A progression of
cutaneous disease from hemangioma to HSA may occur, [24] reﬂecting
phenotypic development of more malignant tumor as a result of repeated
genetic damage. Subcutaneous or hypodermal HSAs do not seem to have
this progression [13,24].

The role of reproductive status and, therefore, hormonal status in dogs

and cats remains unclear at this time, although in one study of atrial HSA
[32], spayed female dogs had a greater than ﬁve times risk of developing this
tumor, and most cutaneous vascular tumors in cats have been reported in
male cats [31].

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Currently, few causes of feline cutaneous or visceral HSA have been

suggested. Sunlight exposure may be important in tumor development in
hypopigmented areas, especially of the head and pinnae [31]. Feline
leukemia virus infection has not been associated with this tumor’s devel-
opment [6].

Other carcinogens that have been linked to the development of HSAs in

people include thorium dioxide, arsenicals, and vinyl chloride [44,45], and
methylnitrosamine has been reported to cause HSA development in mink
[8]. These compounds could possibly be extrapolated to cause tumorigenesis
in companion animals.

Biologic behavior

HSA can arise in any tissue with blood vessels, but the most common

sites in dogs are the spleen (50%–65%), right atrium (3%–25%), sub-
cutaneous tissues (13%–17%), and liver (5%–6%) in the dog [1,4,9,16,46].
In the cat, the liver, spleen, mesentery, omentum, and subcutaneous tissues
are the most frequent primary sites (C.M. Johannes et al, unpublished data)
[19,21,25,47]. Other primary sites reported include the skin, lung, aorta,
kidney, oral cavity, muscle, bone, urinary bladder, intestine, tongue, pro-
state, vulva

/vagina, conjunctiva, and peritoneum in dogs [1,8,10,36,

48–52] and the nasal cavity, intestine, muscle, diaphragm, pancreas, lung,
kidney, bone, eye, and thoracic cavity in cats (C.M. Johannes et al,
unpublished data) [13,21,25,53–55]. HSA is the most common canine
primary cardiac tumor [4,16,32,56]. HSA is reported to comprise 2% to 3%
of all canine bone tumors [57] and 4% to 10.5% of primary bone tumors
in the vertebral column [58]. Some have suggested that HSA may be
overdiagnosed in bone tumors, however, because it can be difﬁcult to dif-
ferentiate a highly vascular area in another primary bone tumor from
HSA [48].

Overt metastasis is present in more than 80% of canine patients at clinical

presentation [5]. Splenic and atrial masses coexist in 25% of dogs with HSA
[15], although whether one site is primary or whether multicentric HSAs
have developed [3,8,9] is usually undetermined. Tumor spread to the liver,
omentum, mesentery, and lung [1,4,6,13,15,16,32] is most common in dogs,
although metastases have also been found in the kidney, muscle, peri-
toneum, lymph nodes, bone, adrenal glands, eye, prostate, and brain [1,5,6,
9,13,14,32,48,59]. HSA is the most common metastatic sarcoma in the
canine brain and is usually found in a cerebral location; 14% of patients
were reported to have brain metastasis in a study of 85 dogs with HSA [59].
Other metastatic sites were usually also present in these patients with brain
metastases [59]. In cats, metastatic disease can be found in the liver, intra-
abdominal lymph nodes, and lung (C.M. Johannes et al, unpublished data)
[25,27].

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A.N. Smith

/ Vet Clin Small Anim 33 (2003) 533–552

Death from treated visceral HSA is usually a result of metastatic disease

rather than local tumor recurrence [5]. Rupture of untreated atrial or
visceral organ tumors can also cause death as a result of cardiac tamponade
or intracavitary hemorrhage [4,5].

In cats, subcutaneous HSAs have a high recurrence rate after surgical

excision (C.M. Johannes et al, unpublished data) [19,25,31]. Cutaneous
HSAs (conﬁned to the dermis) may be easier to excise completely, and
recurrence is much less frequent (C.M. Johannes et al, unpublished data).
The incidence of metastasis is reported to be low in cutaneous HSA in dogs
and cats (C.M. Johannes et al, unpublished data) [24,25,31], but some
patients develop metastatic disease [24,27,60]. Conjunctival HSA also seems
to be more locally invasive than metastatic, because six dogs with follow-up
information ranging from 6 to 17 months had no evidence of recurrence or
metastasis after surgical intervention [61]. HSAs with hypodermal or
muscular involvement in dogs and cats seem to have a high recurrence rate
and incidence of metastasis (C.M. Johannes et al, unpublished data)
[25,31,60].

Clinical presentation

Clinical signs can be subtle in dogs with HSA, usually related to episodic

weakness or acute collapse associated with hemorrhage from a visceral
mass, followed by recovery when the blood is reabsorbed from the body
cavity [2,4,8,46]. Other signs associated with hypovolemia caused by
hemorrhage might include tachycardia, tachypnea, and mucous membrane
pallor [15,46]. Abdominal distention, anorexia, and weight loss may also
be noted [5]. If the bleeding tumor is associated with the heart, signs of
right heart failure caused by cardiac tamponade might include abdominal
distention, jugular pulses, mufﬂed heart sounds, and dyspnea [4,16,56].
Syncope, ataxia, and cyanosis can be seen with severe arrhythmias [4,62].
Subcutaneous masses can cause lameness or may be a primary complaint,
depending on tumor location and possibly the presence of active hemor-
rhage [3]. Primary osseous HSAs in the long bones can induce lameness
secondary to pathologic fractures or bone pain [29,48], and vertebral HSAs
can cause pain or paresis [58]. Active bleeding from tumor sites in locations
such as the urinary tract or nasal cavity may cause an owner to seek
veterinary attention [25,49,52]. Seizures can occur with HSA metastasis to
the brain [4,59]. Hemorrhage or petechiae formation on mucous membranes
in the presence of disseminated intravascular coagulation may also occur in
rare instances [7,63]. Finally, HSA that ruptures acutely and causes sudden
death may be an incidental ﬁnding at postmortem examination [5,46].

Cats with HSA also have a subtle clinical presentation, although the

onset of signs is generally acute (C.M. Johannes et al, unpublished data)
[21]. Subcutaneous or cutaneous masses with or without hemorrhage are

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a common complaint (C.M. Johannes et al, unpublished data) [25,27].
Visceral HSAs can cause lethargy, anorexia, vomiting, abdominal disten-
tion, weight loss, and possibly dyspnea [25,27,64]. A palpable abdominal
mass and pallor can be found on physical examination [25]. Mesenteric root
HSAs can cause chylous ascites as a result of obstruction

/disruption of the

lymphatic vessels [64].

Diagnosis and staging

A clinical staging system for canine HSA and tests for diagnosing

/staging

HSA are found in Boxes 1 and 2. Complete blood cell count (CBC)
abnormalities in dogs most commonly include evidence of a regenerative
anemia, including anisocytosis, polychromasia, increased red blood cell
(RBC) distribution width, and reticulocytosis [4,7]. Acanthocytosis has been
seen in up to 50% of dogs with HSA [40,65]. With microangiopathic
changes, schistocytes can also be seen. Nucleated red blood cells (nRBCs)
are often not removed from circulation by an abnormal spleen, elevating
their numbers in peripheral blood [4]. Other causes of increased circulating
nRBCs include hypoxemia, bone marrow inﬁltration by tumor, and extra-
medullary hematopoiesis [4]. A neutrophilic leukocytosis can also be found

Box 1. Clinical staging system for canine hemangiosarcoma

Primary tumor (T)
T0: no tumor evident
T1: tumor less than 5 cm in diameter, confined to organ, and

does not invade beyond dermis (cutaneous HSA)

T2: tumor greater than or equal to 5 cm in diameter or ruptured

or invasive into subcutaneous tissues (cutaneous HSA)

T3: tumor invades adjacent structures

Regional lymph nodes (N)
N0: no regional node involvement
N1: regional node involvement
N2: distant node involvement

Distant metastasis (M)
M0: no distant metastasis found
M1: distant metastasis confirmed

Stage I: T0 or T1, N0, M0

Stage II: T1 or T2, N0 or N1, M0

Stage III: T2 or T3, N0, N1, or N2, M1

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secondary to stress, tumor necrosis, or nonspeciﬁc bone marrow response
[1,4,7,14]. Thrombocytopenia is a common ﬁnding in dogs, occurring in
30% to 60% of dogs with HSA, secondary to immune-mediated processes,
sequestration, severe hemorrhage, or disseminated intravascular coagula-
tion (DIC) [66].

CBC abnormalities in cats usually include anemias and neutrophilic

leukocytosis (C.M. Johannes et al, unpublished data) [21,25,27]. Thrombo-
cytopenia can also occur (C.M. Johannes et al, unpublished data) [21,25,27].
The presence of nRBCs is rare [25], and fragmentation of RBCs has not
been reported in the cat [5].

Spontaneous hemorrhage may occur secondary to severe thrombocyto-

penias and DIC, which is often seen with canine HSA [63,66–68]. DIC is
associated with thrombocytopenia, elevations in prothrombin and partial
thromboplastin times, decreases in ﬁbrinogen and antithrombin III levels,
and elevations in ﬁbrin degradation products. DIC can be systemic or
localized to the tumor area [69]. DIC may resolve with therapy. The most
important treatment for DIC is tumor removal, but combinations of
heparin, plasma, or corticosteroids may prove beneﬁcial [63,68,69]. Cats
with visceral HSA have also been reported to be predisposed to DIC [27].

Serum chemistry abnormalities are usually not speciﬁc for HSA but can

reﬂect organ system involvement. For example, serum alkaline phosphatase
and alanine transferase elevations may reﬂect inﬁltration of the liver.
Hypoglycemia has been reported as a paraneoplastic syndrome [70].

Radiographs of the abdomen and thorax can demonstrate pleural or

peritoneal eﬀusion secondary to hemorrhage or right heart failure asso-
ciated with cardiac tamponade [4]. In 47% of dogs with cardiac HSA, an
abnormal cardiac silhouette can be seen [41]. Radiographic abnormalities
can include a globoid cardiac appearance secondary to pericardial effusion
or a soft tissue mass at the heart base [4,41].

Three-view thoracic radiographs (right and left lateral views plus

a ventrodorsal or dorsoventral view) are also necessary to evaluate the

Box 2. Tests for diagnosis/staging of hemangiosarcoma

Complete blood cell count
Serum chemistry panel
Urinalysis
Coagulation profile (prothrombin time, partial thromboplastin

time, fibrinogen, and fibrin degradation products (FDPs), with
or without antithrombin III)

Three-view thoracic radiographs
Abdominal ultrasound
Echocardiogram
Electrocardiography

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lung ﬁelds for evidence of metastatic disease for the highest detection
sensitivity [41]. Both a nodular pattern and a diffuse miliary pattern are
consistent with metastatic HSA, and the nodules can be ill deﬁned (most
frequently) or well circumscribed (less frequently) [71]. Occasionally, an
alveolar pattern consistent with intrapulmonary hemorrhage is seen [71].
Thoracic radiography has a sensitivity of 80% in detecting pulmonary
metastatic disease [41,71].

Abdominal radiographs may demonstrate splenomegaly, hepatomegaly,

or other intra-abdominal masses consistent with primary or metastatic HSA.
Generalized loss of detail often indicates hemoperitoneum [34]. Other
imaging should be performed as needed based on clinical signs, including
contrast studies or cystoscopy for the detection of renal or bladder masses
[49,52]. Osseous HSA usually has a lytic rather than proliferative radio-
graphic appearance, is often conﬁned to the medullary cavity, and may
involve the entire shaft of the bone [29]. MRI is preferred, and CT scans may
also be used to conﬁrm the presence of mass lesions within the cerebrum as
the cause of seizures in dogs with HSA. These advanced imaging modalities
are also useful for the diagnosis of subcutaneous masses and for planning
surgical and radiation therapy.

Pneumopericardiography and angiocardiography (selective and nonselec-

tive) have been used in the past to document space-occupying cardiac masses
[72], but these invasive procedures have largely been replaced by echocardi-
ography [73,74]. Echocardiography is considered more accurate than
radiography in cardiac HSA diagnosis and has been reported to have
a positive predictive value of 92% (11 of 12 cases) and a negative predictive
value of 64% (9 of 14 cases) in dogs with right atrial

/auricular cardiac masses

[73]. Some have suggested that nonselective angiography is a relatively simple,
safe, and quick procedure useful for imaging the right heart and delineating
masses, especially if echocardiography is equivocal or unavailable [75].
Endomyocardial biopsy has been used in a single case to diagnose a left
ventricular HSA [76], but this site is rare, and this invasive technique is not
used routinely to diagnose right auricular

/atrial masses.

Electrocardiographic abnormalities usually include nonlethal ventricular

arrhythmias associated with splenic disease rather than cardiac tumor
involvement [77,78]. Nevertheless, one report of right bundle branch block
caused by invasion of HSA locally into the right ventricle illustrates the
possibility of arrhythmias as a result of local tumor involvement of the
cardiac conduction system [62]. Pericardial effusion can result in decreased
complex amplitude or electric alternans [28]. Up to 40% of dogs with splenic
HSA have arrhythmias, including ventricular premature contractions
and ventricular tachycardia [78]. Arrhythmias may not be noted until
after splenectomy [78]. Pre-, peri-, and postsurgical arrhythmias have also
been reported in dogs with cardiac HSA, including atrial tachycardias [16].
Antiarrhythmic drug therapy may be necessary in some cases, and
anesthetic regimens should be chosen accordingly [16,78].

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Splenic HSA cannot be deﬁnitively diagnosed on the basis of clinical

signs. Splenic hematomas and hemangiomas as well as other benign diseases
have a similar clinical presentation as well as gross and ultrasonographic
appearance (Table 1) [11,18,42]. The presence of splenic rupture, nRBCs,
abnormal red blood cell morphology, or anemia is consistently found more
frequently in the presence of neoplasia [26]. One study suggested that the
presence of anemia and splenic rupture may be up to 69% accurate in
predicting the presence of splenic neoplasia [26]. Up to two thirds of dogs
with splenic masses have a malignant tumor, and of these dogs with
a malignancy, up to two thirds have HSA. The remaining patients have
benign disease that can be treated with a splenectomy and have an excellent
prognosis [11,18,26,39,42]. Ultrasonography may be helpful in detecting
metastatic disease in other organs [79].

Abdomino- or pericardiocentesis can be performed to obtain a ﬂuid

sample for analysis. Most eﬀusions are characteristic of intracavitary hemor-
rhage, with a high packed cell volume and a nonclotting sample [28,34]. On
cytologic examination, evidence of fresh hemorrhage (peripheral blood
components) or previous hemorrhage may be seen (hemosiderin-laden
macrophages). Idiopathic pericardial effusions can also be hemorrhagic, so
differentiation of neoplastic versus nonneoplastic processes is difﬁcult [80].
Protein concentrations and cell counts are also not different between ef-
fusions [80], although a pH greater than or equal to 7.0 has been reported to
be indicative of malignancy [81]. Sarcoma cells can also occasionally be

Table 1
Diﬀerential diagnoses for splenomegaly

/splenic nodules in the dog

Nonneoplastic disease

Neoplastic disease

Hyperplastic nodular enlargement

Benign

Hematoma

Fibroma

Vascular stasis

/congestion

Hemangioma

Thrombosis

/infarction

Lipoma

/myclolipoma

Torsion

Malignant

Traumatic rupture

Hemangiosarcoma

Abscessation

Fibrosarcoma

Extramedullary hematopoiesis

Leiomyosarcoma

Inﬂammation

/necrosis

Lymphosarcoma
Myxosarcoma
Osteosarcoma
Liposarcoma
Rhabdomyosarcoma
Chondrosarcoma
Mesenchymoma
Undifferentiated

/anaplastic sarcoma

Histiocytosis

/ﬁbrohistiocytic nodules

Mast cell tumor
Metastatic neoplasia

Data from

Refs. [18,39,82,108,109,111].

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apparent, indicating a presumptive diagnosis of HSA in about 25% of cases
[56], although false-positive results for malignancy have been reported in up
to 13% of cases [80]. False-negative results are quite common [16].

Aspiration cytology of masses is often obfuscated by hemorrhage, so this

technique is not considered to be sensitive in the diagnosis of HSA
[34,82,83]. Other causes of splenic masses or splenomegaly can be detected,
however, such as extramedullary hematopoiesis, primary hematopoietic
tumors (eg, lymphosarcoma, plasmacytoma), or metastatic disease (eg,
carcinoma, mast cell tumor) and may help to establish a diagnosis
[26,34,83,84]. The risk of splenic rupture or tumor seeding must be
considered when a decision is made to aspirate a splenic mass [83].

Histopathologic examination of tissues is required to diagnose HSA

deﬁnitively. An excisional biopsy is preferred, because it is both a diagnostic
and therapeutic procedure [5,33]. Multiple tissue samples or the entire mass
should be submitted, because most of a mass is usually composed of
hematomas, which can lead to an incorrect diagnosis in the absence of
representative tissues. Some authors advise slicing the spleen like a loaf
of bread to ﬁnd multiple samples of different textures and appearance,
although submission of the entire organ, if possible, is preferred [5]. At
exploratory surgery, not only the primary mass but any other suspicious
lesions within the spleen, liver, or omentum should be biopsied and
submitted for analysis. Pathologists can conﬁrm the origin of the tumor with
immunohistochemical staining for factor VIII-related antigen

/von Wille-

brand’s factor, which is highly speciﬁc for endothelial cells [85]. CD31 or
monoclonal antibody 3B5 may be even more useful in conﬁrming a vascular
endothelial cell origin, because up to 10% to 20% of HSAs are negative for
factor VIII [85–87].

Therapy

Published median survival times for dogs with HSA are listed in Table 2.

Surgery

Surgical removal of a bleeding mass provides relief of clinical signs for

a period and is the initial treatment of choice for HSA [5]. In some animals,
this cessation of clinical signs of hemorrhage lasts for several months, but,
ultimately, dogs die of metastatic disease [5]. Median survival for dogs with
splenic HSA treated with surgery alone is short, ranging from 19 to 86 days
[1,11,26,88].

Surgical resection of right auricular masses is possible in some cases

to palliate a bleeding tumor [28,89]. Pericardectomy has been suggested
to provide relief from signs of cardiac tamponade [56] but did not affect
recurrence of signs or survival in one study [90]. All these dogs had de-
tectable metastasis at the time of surgery, however. Thoracoscopic removal

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Table 2
Median survival times for dogs with hemangiosarcoma

Tumor site

Treatment

Number of
patients

Survival
(days)

Reference

Spleen

Surgery alone

Prymak et al [11]

Surgery alone

Brown et al [1]

Surgery alone

Johnson et al [26]

Stage I

Johnson et al [26]

Stage II

168

Johnson et al [26]

Stage III

Johnson et al [26]

Surgery alone

Wood et al [87]

Surgery + MBV

Brown et al [1]

Surgery + MBV

+ VMC

117

Brown et al [1]

Surgery + VAC

ChM

145

Hammer et al [95]

Surgery + VAC

140

Johnson et al [26]

Surgery + AC

180

Sorenmo et al [30]

Surgery + AC

141

Vail et al [101]

Stage I disease

166

Vail et al [101]

Stage II disease

Vail et al [101]

Surgery + AC

+ L-MTP-PE

273

Vail et al [101]

Right atrium

VAC alone

140

deMadron

et al [110]

Incisional biopsy

+ AC

113

Sorenmo et al [30]

Surgery

120

Aronsohn [16]

Surgery

Berg and

Wingﬁeld [56]

Cutaneous

/dermis

Surgery (stage I)

780

Ward et al [60]

Subcutaneous

VAC

ChM

425

Hammer et al [95]

Surgery (stage II)

172

Ward et al [60]

Surgery (stage III)

307

Ward et al [60]

Surgery + AC

211

Sorenmo et al [30]

Various sites

Surgery + AC

202

Sorenmo et al [30]

Stage I

250

Sorenmo et al [30]

Stage II

186

Sorenmo et al [30]

Stage III

Sorenmo et al [30]

Surgery + AC

+ minocycline

170

Sorenmo et al [102]

Various nonskin

sites

Surgery

(complete excision)
+ A

172

Ogilvie et al [94]

Surgery (incomplete

excision) + A

Ogilvie et al [94]

Abbreviations:

A, doxorubicin alone; AC, doxorubicin, cyclophosphamide; ChM, chloram-

bucil, methotrexate; L-MTP-PE, liposome-encapsulated muramyl tripeptide-phosphatidyletha-
nolamine; MBV, mixed bacterial vaccine; VAC, vincristine, doxorubicin, cyclophosphamide;
VMC, vincristine, methotrexate, cyclophosphamide.

Mean.

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of the pericardium has been described as an alternative to thoracotomy [91].
Patch grafts have been used to reconstruct the right atrium after aggressive
resection of large masses to obtain tumor-free margins [89]. Most dogs with
right atrial masses survive less than 4 months after surgery [23,89,90].

Cutaneous and subcutaneous HSAs can be removed surgically with wide

margins. Other primary or secondary HSA sites can also be surgically re-
moved depending on location (eg, bone tumors with amputation, renal tu-
mors with nephrectomy, bladder tumors with partial cystectomy). All
primary locations except superﬁcial cutaneous tumors carry a poor long-
term prognosis [60], but surgery can be useful for palliation.

Radiation

The use of radiation therapy has not been extensively studied in dogs and

cats with HSA, because the tumor is generally considered a systemic rather
than localized disease. Palliative radiation has been used at our institution
and others with good short-term results to control superﬁcial bleeding
masses not amenable to surgery and to relieve pain associated with tumors
within bone. Survival times of 12 to 42 weeks after 3 to 4 fractions up to
24 Gy total have been reported in four dogs with stage III disease, resulting
in stable disease (2 dogs), partial remission (1 dog), and complete (1 dog)
remission in one study [92]. Other investigators have reported poor
responses in two cats with large and unresectable cutaneous HSAs [93],
although the use of radiation therapy for control of cutaneous HSAs with
incomplete surgical resection, given their high recurrence rate, has been
suggested [5,33,93].

Chemotherapy

Chemotherapy for dogs with HSA is outlined in Table 3. Doxorubicin

has greatest chemotherapeutic efﬁcacy against canine HSA. Doxorubicin
has been used both as a single agent [94] and in combination with
cyclophosphamide [30], with or without the addition of vincristine [94].
Toxicities were frequent when the three-drug combination (vincristine,
doxorubicin, and cyclophosphamide [referred to as VAC]) using a high dose
of vincristine (0.75 mg

) was administered, necessitating hospitalization

for neutropenia or gastrointestinal toxicity in 7 of 15 patients [95]. Hos-
pitalization was only necessary in 3 of 16 dogs with the doxorubicin

cyclophosphamide protocol [30]. Survival times seem to be similar when
comparing doxorubicin alone versus combination chemotherapy [33,94],
although prospective trials to compare toxicity and efﬁcacy have not been
performed. As with all cancer treatment, balancing the risk of side effects
and the beneﬁts of therapy should be considered. In 1 dog, vincristine as
a single agent caused complete regression of presumed HSA metastatic lung
lesions after 12 weekly injections, which supports its use in combination

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protocols [96]. Ifosfamide as a single agent [97] and a combination of vin-
cristine, cyclophosphamide, and methotrexate (with a mixed bacterial
vaccine) [1] have also demonstrated some response (Table 4).

Chemotherapy delivery by novel routes has shown activity against HSA.

Inhalation chemotherapy with a special formulation of doxorubicin resulted
in partial remission of metastatic lung lesions with minimal side eﬀects in one
of three dogs with HSA [98]. The availability of the drug and specialized
administration equipment makes this therapy impractical currently but may
provide areas of future treatment of metastatic disease. Encapsulation of
doxorubicin in liposomes (Doxil; Sequus Pharmaceutical, Menlo Park, CA)
has been used to prevent clearance of the drug by the monophagocytic system,
thereby sustaining drug blood levels while decreasing systemic toxicity; this
therapy has been reported to have activity against canine HSA [99].

Table 3
Chemotherapy for dogs with hemangiosarcoma

Treatment day

Drug

Dose

Day 1

Doxorubicin (dilute to 0.5

/mL in saline)

30 mg

IV through

well-placed catheter slowly

Cyclophosphamide

100–200 mg

IV or

Days 8 and 15

50 mg

PO days 3–6

Vincristine

0.5–0.75 mg

Repeat cycle every 3 weeks for ﬁve to eight treatments.
Consider pretreatment with an antiemetic and antihistamine or corticosteroids to prevent

nausea and mast cell degranulation 20 minutes before doxorubicin administration.

Complete blood cell count with differential count before each chemotherapy injection.

Delay drug administration if neutrophil count is <2500–3000.

Can consider prophylactic antibiotic administration on days 1–8 after doxorubicin.
Dogs weighing <10 kg can be dosed with doxorubicin at 1 mg

/kg.

Consider echocardiogram periodically to monitor for possible myocardial necrosis as

evidenced by decreased fractional shortening or ejection fraction, especially at cumulative
doxorubicin doses >150–180 mg

Cats can be given 20–25 mg

or 1 mg

/kg of doxorubicin

cyclophosphamide at 100

IV every 3 weeks. Pretreatment, antibiotic, and blood monitoring as above. Monitor

renal function at cumulative doxorubicin doses of >80 mg

Abbreviations:

IV, intravenously; PO, per os.

Table 4
Vincristine, methotrexate, cyclophosphamide chemotherapy for dogs with hemangiosarcoma

Treatment day

Drug

Dose

Days 1, 8, 15, 22, 29, 36, 43,

50, 57, 64, 71, 78

Vincristine

0.0125 mg

/kg IV

Methotrexate

0.4–0.6 mg

/kg IV

Days 1–84

Cyclophosphamide

1 mg

/kg PO

Abbreviations:

IV, intravenously; PO, per os.

Data from

Brown NO, Patnaik AR, MacEwen EG. Canine hemangiosarcoma: retrospective

analysis of 104 cases. JAVMA 1985;186:56–8.

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Chemotherapy has not been extensively studied in cats with HSA, but

doxorubicin-based protocols have been recommended in this species,
especially for cases with visceral disease (C.M. Johannes et al, unpublished
data) [5,33]. Its use may also be necessary in cats with subcutaneous HSAs,
because recent studies have demonstrated a possibly higher metastatic
potential than previously believed (C.M. Johannes, unpublished data) [27].
No controlled studies have been conducted to evaluate therapy in cats with
HSA; however, several individual case reports are available in the literature.
In a single case report, a combination of doxorubicin, cyclophosphamide,
and vincristine resulted in survival of longer than 1 year, although the cat
eventually succumbed to metastatic disease [100]. Single-agent vincristine
therapy was ineffective in another cat that died within 2.5 months of diag-
nosis [27]. Other therapies used include VAC in one cat with an incompletely
resected cutaneous tumor; this cat survived for longer than 3.5 years. A cat
with an incompletely excised subcutaneous mass survived 440 days and was
euthanized for unrelated disease after being treated with mitoxantrone and
cyclophosphamide. Another cat with an incompletely resected subcutaneous
mass HSA received doxorubicin at a dose of 25 mg

intravenously every

3 weeks for four treatments and survived 290 days, ultimately dying of
probable metastatic thoracic disease. One cat with completely resected
colonic HSA received doxorubicin as described in the previous case and died
150 days later with suspected abdominal metastatic disease. Finally, one cat
died from progressive disease 60 days after incomplete surgical resection of
mesenteric HSA and carboplatin chemotherapy (C.M. Johannes et al,
unpublished data). Further controlled studies are necessary to determine the
utility of chemotherapy in cats with HSA.

Biologic therapy

Immunomodulators seem to be useful in prolonging survival times,

especially in combination with chemotherapy. A mixed bacterial vaccine [1]
and liposome-encapsulated muramyl tripeptide (L-MTP) [101] have both
been used to treat HSAs and have been associated with increased me-
dian survival times compared with those achieved by surgery alone. The
addition of chemotherapy (VMC) to the mixed bacterial vaccine increased
median survival further, to 117 days, although this increase was not statis-
tically different from splenectomy alone [1]. Splenectomy, L-MTP, and
doxorubicin

/cyclophosphamide chemotherapy resulted in 9.1-month median

survival times, which were signiﬁcantly prolonged (P

¼ 0.03) compared

with those achieved by splenectomy and chemotherapy alone (median

¼ 5.7

months) [101].

L-MTP is not currently used except on an experimental basis because of

lack of availability and high cost, but it or a similar formulation may be
commercially available in the future.

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Prognosis

Despite aggressive surgical, drug, or radiation therapy, median survival

times are short for almost all forms of primary HSA in cats and dogs. One-
year survival for dogs is less than 10% [2]. Stage of splenic disease at
diagnosis does not seem to affect survival according to several studies
[1,26,88], although in one study, dogs with hemoperitoneum associated with
splenic HSA (stage II–III) had a shorter median survival (17 days) after
splenectomy as opposed to dogs with stage I disease (median survival

121 days) [11]. Another study also suggested an improved length of survival
with lower disease stage (stage I disease

¼ 250 days, stage II disease ¼ 186

days, and stage III disease

¼ 87 days), although the differences were not

statistically signiﬁcant [30]. There seems to be no difference in survival
expectation between visceral and subcutaneous HSAs [30]. Only canine and
feline superﬁcial dermal HSAs (stage I) treated with surgery have a
consistent low metastatic potential and prolonged survival times of longer
than 1 year (median: 780 days in dogs,

36 months in cats). Occasionally,

metastasis is seen even with these more favorably located tumors (C.M.
Johannes et al, unpublished data) [19,24,21,25,27,60].

Future directions

Angiogenesis modiﬁers have garnered much media and scientiﬁc

attention in the last several years. Vascular development is essential for
primary and metastatic tumor growth beyond 1 mm

, so blockade of this

process would seem to be a viable antitumor therapy. Advantages of anti-
angiogenic agents include a purported lack of the development of drug
resistance and few systemic side effects. Most experts agree that angiogenic
inhibitors will not replace primary tumor therapy but will be useful in an
adjunctive setting. Antiangiogenic therapy will likely need to be maintained
throughout the life of the patient . As a tumor composed of blood vessels,
HSA would theoretically be the ideal candidate for this type of therapy. In
a single clinical trial, the antiangiogenic antibiotic minocycline did not show
improved patient survival when added to a standard surgery and che-
motherapy protocol [102]. The ideal antiangiogenic dose of this drug has not
been determined, however, and it is likely that multiple agents in a ‘‘cocktail’’
will be necessary to target multiple pathways for blood vessel development.
Other agents being investigated as anticancer therapy include the
antiangiogenic agents thalidomide, interferon, matrix metalloproteinases,
recombinant endogenous angiogenesis inhibitors, and antibodies directed
against receptors or factors involved in angiogenesis [103].

Diagnosis of HSA before surgery would be useful for veterinarians so as

to assist owners in making informed decisions before embarking on
extensive procedures in the face of a disease with a guarded to grave prog-
nosis. Vascular endothelial growth factor (VEGF), a proangiogenic agent

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related to endothelial cell proliferation, has been found to be increased in
tumor tissues in dogs [104]. In unpublished studies conducted by the author,
serum levels of VEGF seem to have some diagnostic utility for differentiating
HSA from hematoma or other benign disease processes. In another study,
plasma VEGF concentrations in dog with HSA were increased compared with
those of normal dogs, and in one dog, levels increased with disease progression
[105]. VEGF concentration was not useful as a predictor of tumor presence
when measured in body cavity effusions [106]. The role of platelet-derived
VEGF in this disease should be further investigated to determine the
usefulness of this factor in predicting tumor presence or as a monitoring device
for the presence of metastasis or recurrence.

Dietary therapy has been suggested to increase survival times in dogs

with lymphosarcoma, and patients with other tumor types may beneﬁt as
well [107]. Adequate caloric intake (1.1 [30 (patient weight in kilograms)+
70]

¼ kilocalories needed per day) with a highly bioavailable and palatable

food is likely most important, but suggested alterations in diet to combat
cancer cachexia might include low simple carbohydrate levels, moderate
good-quality protein levels, moderate fat levels, and adequate ﬁber levels
(both soluble and insoluble) [107]. N-3 fatty acids seem to be especially
important in managing the metabolic alterations seen in cancer patients
[107]. Vitamin E, arginine, cystine, and glutamine have also been suggested
as supplements to enhance immune and gastrointestinal function [107].
Further studies in this arena are necessary to elucidate the role of diets and
nutrition in the management of patients with HSA.

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Feline injection site sarcomas

Marlene Hauck, DVM, PhD

Department of Clinical Sciences, College of Veterinary Medicine,

North Carolina State University,

4700 Hillsborough Street, Raleigh, NC 27606, USA

Drs. Mattie Hendrick and Michael Goldschmidt and their colleagues at

the Laboratory of Pathology of the University of Pennsylvania School of
Veterinary Medicine deserve credit for initially recognizing the increasing
incidence of both reactions at the sites of rabies vaccinations and the for-
mation of sarcomas at sites commonly used for injection and vaccination
in cats [1,2]. Their letter to the editor in the Journal of the American
Veterinary Medical Association

in October 1991 alerted the profession to ‘‘the

possibility that ﬁbrosarcomas may arise at injection sites in cats.’’ They
suggested that this change was related to the enactment of a state law in
Pennsylvania requiring the rabies vaccination of cats, and they tracked an
increase in tumors arising at common vaccination sites (interscapular
region) versus tumors arising at nonvaccination sites (eg, head). Theirs was
the initial histopathologic description of the ﬁbrosarcomas as tumors with
a marked inﬂammatory component, including macrophages containing a
gray-brown foreign substance.

This letter and other commentary resulted in mixed responses among

veterinarians, with some contesting the proposed association of sarcoma
development with vaccinations [3]. A follow-up study of these sarcomas by
Hendrick and colleagues [4] demonstrated the foreign substance in the
macrophages to be aluminum by electron probe microanalysis. Because
aluminum is used as a component of vaccine adjuvants, the identiﬁcation
of aluminum within the macrophages was believed to be consistent with
residual adjuvant. Aluminum adjuvants have been reported to cause granu-
lomatous reactions in people [5].

Two changes occurred within the vaccine industry in the mid-1980s as well:

the ﬁrst killed rabies vaccine licensed for subcutaneous administration

Vet Clin Small Anim

33 (2003) 553–571

E-mail address:

marlene_hauck@ncsu.edu

0195-5616

/03/$ - see front matter

doi:10.1016/S0195-5616(03)00006-8

became available, as did a killed vaccine for feline leukemia virus (FeLV). To
evaluate further the suggested relation between sarcoma development and
vaccination, Dr. Philip Kass and others [6] undertook an epidemiologic study
using information about cats diagnosed with sarcomas at a large private
diagnostic laboratory. Sarcomas from sites typically used for vaccination
were compared with sarcomas developing at other sites (head, digit, and
forelimb). A total of 345 cats (185 sarcomas located at vaccination sites, 160
sarcomas located at nonvaccination sites) were included in an analysis of the
relation between vaccination status and tumor development. The ﬁndings
included an increased risk of sarcoma development, particularly in cats
vaccinated against FeLV but also with the administration of rabies vaccine.
The odds ratio for development of a sarcoma at a vaccination site after
vaccination with FeLV was 5.49 (95% conﬁdence interval [CI]: 1.98–15.24),
and the odds ratio after rabies vaccination was 1.99 (95% CI: 0.72–5.54). A
single vaccination given in the interscapular region resulted in a 50% higher
risk of developing a sarcoma at that site compared with cats that were not
vaccinated at that site, two vaccinations resulted in a 127% increased risk, and
three or four vaccinations given simultaneously at this site resulted in an
approximately 175% risk increase. The overall estimated risk for cats in this
population to develop ﬁbrosarcomas was relatively low, with roughly 20 cats
in 100,000 developing a sarcoma. Another interesting ﬁnding in this study was
a signiﬁcant increase in the percentage of cats diagnosed with ﬁbrosarcomas
at vaccination sites at the University of California–Davis Veterinary Medical
Teaching Hospital. The authors concluded that there was epidemiologic
support for a relation between vaccination and tumor development.

A similar retrospective study was undertaken at the University of

Pennsylvania and Tufts University under the direction of Dr. Mattie
Hendrick [7]. This group also compared cats with sarcomas arising at vac-
cination sites (n

¼ 181) with those arising at other sites (n ¼ 58). Cases were

drawn from the surgical pathology services at these two veterinary schools,
which provide services for approximately 2600 practices in the northeast. Of
the 181 tumors at sites used for vaccination, only 69 cats had a history of
receiving a vaccination at those sites. This study found that the cats with
sarcomas arising at sites of previous vaccination were signiﬁcantly younger
and their tumors were signiﬁcantly larger than those of cats with sarcomas
not associated with a previous vaccination. There were no differences
in the manufacturers of the vaccines used in the two groups. The authors
also noted that the tumors developing at the sites of vaccination recurred
more rapidly and more frequently than sarcomas at other sites and that the
interval from vaccination to sarcoma development ranged from 3 months to
3 years. Cats in the vaccine site sarcoma group were more likely to have
been vaccinated with FeLV than cats in the nonvaccine-associated sarcoma
group, although the reverse was true for rabies vaccination.

A commentary from a third large group of pathologists reported similar

ﬁndings [8]. The Animal Reference Pathology Division of the Associated

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/ Vet Clin Small Anim 33 (2003) 553–571

Regional and University Pathologists reported identifying 176 sarcomas
arising at common vaccination sites in cats with features similar to those
described in the initial reports: anaplastic sarcomas with bluish material in
the inﬂammatory macrophages associated with the tumors. In these cases,
the interval reported between vaccination and sarcoma development ranged
from several months to more than 3 years.

In an attempt to deﬁne better the actual prevalence of injection site

sarcomas, a study was undertaken with members of the American
Association of Feline Practitioners. Coyne and others [9] surveyed these
veterinarians concerning their feline patients in 1992. This year was chosen
because it was the ﬁrst year after the description of vaccine-associated
sarcomas and before the vaccine protocol recommendations designed to
decrease the incidence of injection site sarcomas. The overall incidence of
injection site sarcomas was estimated at 3.6 per 10,000 cats. If records only
from those practices using computerized medical records are considered
(19% of those responding), the prevalence of injection site sarcomas was
calculated as 2.7 per 10,000 cats. This is quite similar to the estimated
prevalence (based on polling 29 hospitals) of 1 to 2 per 10,000 cats [6]. One
of the criticisms of this study is that conﬁrmation of the diagnosis was
not required, so there may have been overreporting of this disease. In addi-
tion, only 21% of the practices contacted participated in the study, and if
veterinarians did not participate because they did not diagnosis injection site
sarcomas, the true prevalence may have been overestimated.

Although, initially, only FeLV and rabies vaccines were implicated in an

increased risk of sarcoma development at the site of vaccination, an
additional study from Canada suggested a role for killed vaccines including
panleukopenia and respiratory viruses [10]. The authors report 14 cases
from their practice with a documented history of receiving a killed
panleukopenia

/respiratory virus vaccination in the interscapular space.

These same cats had not been vaccinated for FeLV, and their rabies
vaccinations had been given in the caudal thigh. The incidence of injection
site sarcomas in this clinic was reported as 13 per 10,000 cats, which is
higher than those of the two studies discussed previously. In this practice,
changing the panleukopenia

/respiratory vaccine to a modiﬁed live product

decreased the number of interscapular sarcomas diagnosed. Although this
report is limited to a single practice, it does raise a question regarding the
ability of vaccines aside from FeLV and rabies to be tumorigenic. In closer
agreement with the higher incidence reported by Lester et al [10] is a project
using prospective monitoring of 2000 cats at a feline practice in
Philadelphia. Dr. Hendrick [11] has reported the diagnosis of ﬁve sarcomas
at the site of a previous rabies vaccination. The average interval between
vaccination and tumor development was 26 months. Dr. Macy at Colorado
State University’s Veterinary Teaching Hospital, in association with
Dr. Hendrick, reported that 2 cats developed injection site sarcomas from
a population of cats that received 548 doses of either FeLV or rabies vaccine

555

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/ Vet Clin Small Anim 33 (2003) 553–571

[12] One proposed reason for the wide variation in reported incidence is
possible regional differences, although there are currently no scientiﬁc data
to support this theory [13].

In an attempt to clarify this issue, a prospective study involving 40

veterinary practices in the Unites States and Canada was undertaken
[14]. These practices submitted monthly updates via the worldwide web on
the number cats vaccinated, the vaccination protocols followed, and the
development of reactions and sarcomas at the documented sites of vac-
cination. These 40 practices vaccinated more than 30,000 cats in the initial
2-year period of this study. Additional follow-up information was collected
for 1 year. In this group of cats, the incidence of injection site sarcomas was
0.63 per 10,000 cats vaccinated (95% CI: 0.081–2.3 per 10,000 cats).
Although additional sarcomas thought to be related to a previous
vaccination were diagnosed in these clinics over the 3-year study, they were
not included if they occurred as a result of a vaccine given before the
beginning of the study or if the information on the case was incomplete.

The incidence of vaccine site reactions was 11.8 per 10,000 vaccine doses

(95% CI: 9.3–14.9 per 10,000 cats). Of those cases with information on
the time to resolution of the reaction, most had resolved within 3 months
(>95%). Reactions were seen with all types of vaccinations administered
(with and without adjuvant). The frequency of vaccine site reactions was
higher in adjuvant vaccines compared with nonadjuvant vaccinations. Vac-
cines with multiple antigens, particularly FeLV, were also more likely to
cause a reaction at the site of injection.

It is worthwhile to note that none of these studies found any association

with gender or breed of cat and sarcoma development. There are several
potential explanations for the diﬀering estimates of prevalence of injection site
sarcomas. The highest estimates were derived from the results at one or two
practices and could reﬂect the incidence of sarcoma development with a single
vaccine manufacturer. No manufacturer-related eﬀect has been documented
in the larger epidemiologic studies; however, the low incidence of sarcoma
development may make diﬀerences between vaccine manufacturers diﬃcult to
detect. Alternatively, the higher incidence rates may be the result of statistical
chance, and these higher rates would be reduced if a larger number of cats were
followed. Other possible interactions that have not been addressed by the
studies discussed previously include the eﬀect of multiple vaccinations over the
lifetime of the cat, the potential for cats to develop sarcomas years after
a vaccination (which would be missed in studies of shorter duration, ie, 3
years), and the fact that some cats in these studies were followed for relatively
short periods [14]. These are not trivial issues to address, and they may require
extensive epidemiologic prospective studies to evaluate.

The United States Pharmacopeia (USP) is a private not-for-proﬁt organi-

zation that maintains a database of adverse reactions to veterinary products.
At the 1998 Veterinary Cancer Society meeting, Dr. Meyer presented a
description of the ﬁrst 169 cases of injection site sarcomas as collected by the

556

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USP (E. Kathryn Meyer, VMD, Rockville, MD, personal communication,
1998). Of the 243 vaccines potentially associated with these tumors, 41% (100)
were rabies; 33% (79) were feline rhinotracheitis virus, calicivirus, and
panleukopenia virus (FRVCP) plus or minus C 20% (49) were FeLV; and 6%
(15) were FRVCP plus or minus chlamydia (C)

/FeLV combinations. In 17

cats, the FRVCP vaccination was the last administered at the location of the
tumor, and in 3 cats, it was the only vaccination ever administered at that site.
Most cats (93%) developed their tumors within 4 years of vaccination. Of
these, 59% were diagnosed within 1 year of vaccination: 11 of these tumors
developed within 1 month of vaccination, 14 developed within 1 to 2 months,
and 11 cats developed tumors within 2 to 3 months.

It is of interest to note that this disease is not a problem exclusive to

North America. A recent survey in the United Kingdom documented 64
sarcomas with histologic characteristics of injection site sarcomas at the sites
of previous vaccinations [15]. These tumors were diagnosed over a 1-year
period through several commercial and university histopathology labora-
tories. The Australian Veterinary Journal has published a series of cases that
were consistent with injection site sarcomas as well [16].

An important part of the history of veterinarians’ response to this

problem is the formation of the Vaccine-Associated Feline Sarcoma Task
Force (VAFSTF) [17,18]. The American Association of Feline Practitioners,
American Animal Hospital Association, American Veterinary Medical
Association, and Veterinary Cancer Society joined resources to found the
VAFSTF. This task force was charged with the formation of a coordinated
mission of research and education with regard to this problem. In addition
to funding multiple research projects in the areas of epidemiology, etiology,
and treatment, the VAFSTF has issued vaccination protocol recommenda-
tions as well as recommendations on an appropriate response to a presumed
vaccine reaction granuloma. The VAFSTF has also played a large role in
the education of the public regarding injection site sarcomas.

Etiology

The ﬁrst step in understanding a new problem is to describe it in complete

detail. A thorough understanding of the histopathology of injection site
sarcomas also may suggest possible mechanisms of disease development.
The ﬁrst portion of this section discussing the pathogenesis of injection site
sarcomas is devoted to the histopathologic and cytologic descriptions of
these lesions and the questions that are raised on this basis. The second
portion discusses in greater depth the diﬀerent avenues of causation that
have been investigated.

Histopathology and cytology

Although not diagnostic of an injection site sarcoma, the characteristic

histopathologic appearance coupled with the appropriate history can suggest

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a high likelihood of this disease syndrome. Doddy and colleagues [19] re-
viewed cats diagnosed with sarcomas (including ﬁbrosarcoma, osteosarcoma,
malignant ﬁbrous histiocytoma, giant cell tumor of soft parts, myoﬁbro-
blastic sarcoma, rhabdomyosarcoma, leiomyosarcoma, chondrosarcoma,
sarcoma, and undifferentiated sarcoma) at the Indiana Animal Disease
Diagnostic Laboratory over a 6-year period. Of the 165 tumors analyzed, 104
arose at common vaccination sites, whereas 61 arose at other sites. The
histopathology of the sarcomas arising at vaccination sites differed from that
of sarcomas at other sites by their subcutaneous location, with necrosis being
more common, as well as an inﬂammatory inﬁltrate, increased mitotic index,
pleomorphism, and variable density of the extracellular matrix. The
inﬂammatory inﬁltrate was composed predominantly of lymphocytes, but
macrophages were also present. In a multivariate logistic regression analysis,
the vaccine site sarcomas were more likely to be subcutaneous and to
demonstrate an inﬂammatory component. Younger cats were found to be
more likely to develop a vaccine-associated sarcoma in this study as well.

These ﬁndings were consistent with the histopathologic appearance of

vaccine site sarcomas as reported by Hendrick et al [4] in 1992. In this report
and others, the sarcomas at vaccination sites are described as ‘‘surrounded
and partially inﬁltrated’’ by an inﬂammatory response composed of lym-
phocytes and macrophages [20]. The macrophages sometimes contained
a gray-brown material that was demonstrated to be aluminum. In addition,
this foreign material was described in postvaccinal granulomatous reactions
in cats and dogs [1]. Whereas inﬂammation was seen in 15% of sarcomas at
nonvaccination sites, 51% of the vaccine site sarcomas had an inﬂammatory
element.

On immunohistochemical marker staining, most tumors stained posi-

tively for vimentin, and reactivity to muscle markers was positive in most
cases as well [20]. A chondrosarcoma was positive for the S100 protein,
consistent with the diagnosis. Cytokeratin, CD34, factor VIII–related
antigen, epithelial membrane antigen, and factor 13a were negative. These
results are consistent with the surmised cell of origin for these tumors, the
ﬁbroblast

/myoﬁbroblast.

To characterize these tumors further, ultrastructural evaluation with

transmission electron microscopy was performed [21]. These studies
conﬁrmed the presence of myoﬁbroblasts, giant cells, ﬁbroblastoid cells,
and histiocyte-like cells within the tumors as well as intracellular particulate
material conﬁrmed by dispersive X-ray spectroscopy to be aluminum based.
Immunohistochemistry performed on a subset of these tumors was con-
sistent with the results discussed previously, with seven of seven staining posi-
tive for vimentin and one of seven staining positive for desmin.

Of particular interest are the similarities in the histopathologic ap-

pearance of injection site sarcomas and posttraumatic ocular sarcomas in
cats. Dubielzig and colleagues [22] described 13 cases of intraocular sar-
coma, and 11 of these cats had a history of ocular disease before tumor

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development. Diagnoses included ﬁbrosarcoma (8 cases), osteosarcoma (2
cases), and anaplastic sarcoma (3 cases). The breakdown of the lenticular
capsule was present in all 12 cats in which the entire globe was available.
The latent period to tumor diagnosis was often several years. These tumors
were anaplastic, with variable degrees of differentiation and regions of
granulation tissue and metaplasia [23]. A similar pathogenesis has been
suggested for these two phenomena.

The inﬂammatory component of injection site sarcomas led several

investigators to study the inﬂammation present at the site of vaccine reac-
tions in rats, cats, mink, and ferrets. It is interesting to recall that an
increased incidence of vaccine-site reactions noted by the pathologists at the
University of Pennsylvania was responsible, in part, for their recognition of
the association between vaccinations and sarcoma development [1,2]. The
histologic description of the injection site reactions was of a ‘‘focal
necrotizing granulomatous panniculitis’’ in the subcutaneous tissues.

Rats given one of ﬁve modiﬁed live vaccines with a variety of feline

pathogens failed to produce any injection site reactions [24]. Cats, however,
demonstrated a more vigorous inﬂammatory response to vaccination.
Schultze and colleagues [25] evaluated three standard feline vaccines in cats
for their ability to produce cytologic evidence of inﬂammation and to
characterize further that inﬂammation. Cats were vaccinated at four sites with
saline, FVRCP, FeLV (killed), and rabies virus (killed). Vaccination sites
were aspirated weekly, and the smears were evaluated cytologically. The site
of the saline injection yielded few cells or blood on aspirate. The smears from
the vaccination sites were more cellular, with the cells consisting of
lymphocytes, neutrophils, and macrophages, and they were similar at all
sites at week 1. By the second week, the cellularity at the sites of the rabies and
FeLV vaccinations was higher than at the site of the FVRCP vaccination,
with increased lymphocytes at the both the rabies and FeLV injection sites
compared with the FVRCP site and increased macrophages at the rabies site.
By the third week, the inﬂammation at the sites of the FeLV and FVRCP
vaccinations had resolved, although the numbers of white blood cells
continued to increase at the site of the rabies vaccination. A similar pattern
was seen at the fourth week. The most common cell type detected throughout
the evaluation period was lymphocytes. All cats developed palpable nodules
at the site of the rabies vaccination, whereas only one other vaccine site
reaction was noted, at the site of an FVRCP vaccination.

A comparison between early vaccine reactions of cats, mink, and ferrets

was performed, in part, because of a case report of an injection site sarcoma in
a ferret [26]. The investigators evaluated two killed rabies vaccines and one
modiﬁed live rabies vaccine (with or without the addition of neuraminidase
type V), two killed FeLV vaccines (one with aluminum in the adjuvant), 10%
aluminum ammonium sulfate (alum), and saline [27]. The injection sites were
examined histologically at times from 1 to 21 days. In this study, cats differed
from mink and ferrets in their response to the rabies vaccines, with increased

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collagen and lymphocytes in response to both and increased ﬁbroblasts as well
in response to one type of rabies vaccine. All three species responded similarly
to the FeLV vaccines. Granulomatous inﬂammation was seen in all species.
The authors conclude that the different response to vaccination may reﬂect
a subtle difference between these species. This difference may predispose cats’
inﬂammatory reactions to undergo neoplastic transformation.

A case report on a ﬁbrosarcoma arising at the site of a lufenuron injection

prompted investigation into injection reactions seen with this agent (Dennis
Macy, DVM, Fort Collins, CO, personal communication, 2000) [28]. Six
rats were injected with lufenuron subcutaneously, and the injection sites
were evaluated histologically 21 days later. All six rats developed injection
site reactions, but the nature of these reactions was different from those seen
in previous studies with vaccines. These reactive inﬁltrates were composed
predominantly of macrophages, without the increased numbers of neu-
trophils, lymphocytes, and plasma cells.

Current research on injection site sarcomas

The presence of the inﬂammatory capsule or inﬁltrate of injection site

sarcomas, which is seen much more rarely in noninjection site sarcomas,
suggested to some investigators that inﬂammation may play a role in on-
cogenesis. This suspicion also resulted in the studies of vaccine reactions
discussed previously. This hypothesis was ﬁrst proposed in 1992, with the
observation that some cases of injection site sarcoma were ‘‘transitional’’
foci of neoplastic cells within granulomatous inﬂammation [4]. This model
would be consistent with the development of ocular sarcomas at sites of
previous trauma or inﬂammation. There are several experimental models
demonstrating the role of inﬂammation in tumor promotion as well (for
a review, see the article by Macy [29]). It is hypothesized that the growth
factors elucidated at the site of inﬂammation interact with cells to result in
oncogenesis. For example, chickens infected with the Rous sarcoma virus
develop tumors at the site of injection. If a wound is made away from the
injection site, a tumor develops at the wounding site [30]. If, however,
inﬂammation at the wound is suppressed with methylprednisolone, no tumor
develops. The growth factors identiﬁed as sufﬁcient to cause this effect are
tumor growth factor-b (TGFb) and acidic and basic ﬁbroblastic growth
factors (aFGF and bFGF). This work is supportive of the concept that
inﬂammation is a necessary component of injection site tumor development.
It is also consistent with the observation that these tumors are seen more
commonly with killed vaccines (that contain more adjuvant) than with
modiﬁed live vaccines. To evaluate this hypothesis further, immunohisto-
chemistry was used to determine the expression of epidermal growth factor
(EGF) and its receptor, FGF and its receptor, platelet-derived growth factor
(PDGF) and its receptor, c-jun (AP-1), and TGFb in normal feline skin,
wounded skin, vaccine reactions, early malignant lesions, and injection site

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sarcomas as well as in sarcomas at other sites [31]. Sarcomas arising at
injection sites demonstrated consistent staining for EGF, PDGF, TGFb and
their receptors as well as for c-jun. Noninjection site sarcomas stained weakly
for these growth factors. The lymphocytes in the injection site tumors and
vaccine site reactions also stained positively for PDGF, although the lympho-
cytes of the noninjection site sarcomas and nonvaccine inﬂammation did
not stain for PDGF [32,33]. The strongest expression of the PDGF receptor
was in the adjacent sarcoma cells. Recently, the lymphoid follicles typically
seen along the periphery of the tumor have been shown on immunohisto-
chemistry to be composed of a high proportion of T cells (19%–87%) [34].

Additional studies of growth factor expression have been performed on

injection site sarcoma cell lines (J. Carew, Madison, WI, personal communi-
cation, 1999). The pattern of expression of PDGFa, PDGFb, PDGF
receptor, hepatocyte growth factor, c-met, EGF receptor, and insulin-like
growth factor-1 is being determined in 13 injection site sarcoma cell lines as
well as in 5 noninjection site sarcoma cell lines. In an extension of this de-
scriptive work, these investigators have also evaluated the ability of tyrosine
kinase inhibitor STI-571 to repress the proliferation of these cell lines in
vitro and to suppress receptor autophosphorylation (Ilene Kurzman, PhD,
Madison, WI, personal communication, 2001). STI-571 inhibited cell prolif-
eration and PDGF receptor autophosphorylation even in the presence of
PDGF.

In general, chronic inﬂammation is not thought to be capable of inducing

neoplastic transformation alone, and host factors are considered to be
essential in the transformation of cells. These factors could take the form of
infection with oncogenic viruses or genetic mutations present within host
cells that play a role in cancer initiation. This hypothesis is consistent with
the low incidence of tumor formation in the population of cats at risk.

The role of concurrent viral infection has been evaluated in cats with

injection site sarcomas. The transformative role of viruses in cats is well
known, particularly in the instance of FeLV. Another oncogenic virus, feline
sarcoma virus (FeSV), is a replication deﬁcient variant of FeLV that
requires coinfection with FeLV to become pathogenic. Previous retrospec-
tive studies failed to demonstrate an association between serologic evidence
of infection with FeLV or feline immunodeﬁciency virus (FIV) and injection
site sarcoma development [7]. Further elucidation of the potential role of
viral infection has been performed using direct analysis of tumor cells with
immunohistochemistry, polymerase chain reaction (PCR) and reverse
transcriptase PCR evaluation, and in situ hybridization of the tumor cell
DNA [35–40]. A number of potentially oncogenic viruses were investigated,
including FeLV, FIV, polyomavirus, papillomavirus, endogenous FeLV,
and feline foamy virus. Immunohistochemistry and PCR failed to detect the
presence of FeLV

/FeSV in 130 suspected injection site sarcomas [35]. When

testing for endogenous FeLV and FIV, 50 sarcomas from injection sites
were compared with 50 sarcomas from noninjection sites. The remaining

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three viruses were evaluated only on sarcomas of injection sites. When
comparing the presence of endogenous FeLV RNA, there was no difference
between injection site and noninjection site sarcomas [36]. No evidence of
infection with FIV, polyomavirus, papillomavirus, or feline foamy virus was
detected in any of the tumor samples [37–40]. Given these results, it is
unlikely that any of these viruses have a role in the development of injection
site sarcomas.

Other groups have investigated the role of mutations in tumor suppressor

genes, such as p53 (Elizabeth Hershey, DVM, Madison, WI, personal
communication, 1999; Sagarika Kanjilal, PhD, St. Paul, MN, personal
communication, 1999) [41,42]. In human tumors, p53 mutations have been
associated with initiation and progression of neoplasia. Mutations in p53
result in a longer half-life of p53 protein in the cell, which can be detected
using immunohistochemistry. In a series of 40 injection site sarcomas, 17
stained darkly for p53 expression in the nucleus, the nuclei of 8 tumors
stained palely, and 15 tumors did not stain [42]. Sequence analysis of the p53
gene in 8 tumors in which the nuclei stained darkly demonstrated single mis-
sense mutations in 5 tumors [41]. These mutations were not present in
the normal tissues, suggesting that although the abnormal p53 protein may
have contributed to cancer progression, it is unlikely that p53 mutations
were a factor in tumor development. In another study, 1 of 20 injection
site sarcomas had a point mutation in p53, and 7 of 18 had loss of
heterozygosity at this gene (Sagarika Kanjilal, PhD, St. Paul, MN, personal
communication, 1999).

The tools of genomics have only just been brought to the study of

injection site sarcomas. Identiﬁcation of chromosomal alterations in in-
jection site sarcomas may allow identiﬁcation of cats at risk for tumor
development and better understanding of the pathogenesis. The production
of feline chromosome-speciﬁc probes was the ﬁrst step for evaluating
the karyotype of injection site sarcomas [43]. The karyotypes of ﬁve
injection site sarcomas were evaluated using these probes, and all ﬁve were
‘‘highly aberrant’’ [44]. These abnormal chromosomal regions require
further evaluation before their signiﬁcance is known. Evaluation of early
evidence of DNA damage in vaccination reactions is also under current
investigation (J. Mac Law, DVM, PhD, Raleigh, NC, personal communi-
cation, 2002). The unique nature of this inducible tumor offers many
opportunities to study tumorigenesis that may be relevant to human cancer
as well [45].

The in vitro chemosensitivity of injection site sarcoma cell lines also has

been evaluated. Two cell lines were established from injection site sarcomas
and exposed to various levels of ﬁve chemotherapeutic agents [46]. The
drugs included carboplatin (Paraplatin), doxorubicin (Adriamycin), mitox-
antrone (Novantrone), methotrexate (Folex, Mexate), and vincristine (On-
covin). Of these agents, mitoxantrone and doxorubicin demonstrated the
highest degree of cytotoxicity. Recently, four injection site sarcoma cell lines

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were grown in the presence of vincristine and paclitaxel. Both drugs
showed signiﬁcant cytotoxicity at concentrations that may be achievable
in patients [47].

Clinical presentation and treatment

As mentioned previously, injection site sarcomas are typically diagnosed

in cats at a younger age than noninjection site sarcomas, and the tumors are
frequently larger at the time of diagnosis [7,19]. As veterinarians are alerted
to the possibility of sarcoma development at the site of a vaccination
reaction, the delay to diagnosis has hopefully decreased and the size of tu-
mors at initial presentation no longer may be as dramatic. It is important to
note that these tumors may develop after the injection of substances other
than vaccinations [28,48]. On presentation, these masses might have a cystic
cavity as a result of the large necrotic center that is a common histo-
pathologic aspect of injection site sarcomas. Often, strands of tumor tissue
extending to the underlying musculature or dorsal spinal processes can
be palpated.

A biopsy is necessary for deﬁnitive diagnosis. The degree of inﬂammation

associated with these tumors makes interpretation of a cytology sample
diﬃcult. Biopsies should be taken with the ﬁnal treatment protocol in mind,
with particular emphasis on placing the biopsy tract within tissue that is to
be removed at the time of surgical excision of the tumor. The current
recommendations of the VAFSTF for management of a mass that develops
at the site of a vaccine are to record the size and location and to perform
a biopsy if the mass (1) persists for more than 3 months, (2) is greater than
2 cm in diameter, or (3) is increasing in size 1 month after injection [17].

Currently, there are no known histopathologic prognostic indicators for

feline injection site sarcomas. Argyrophilic nucleolar organizer region
(AgNOR) staining has been shown to be a useful prognostic factor in several
canine tumors and was evaluated in 21 cats with injection site sarcomas
(Laura Garrett, DVM, Manhattan KS, personal communication, 2001). No
correlation was found between cats with fewer or greater numbers of
AgNORs than the median AgNOR count and the incidence of metastasis or
duration of survival. A grading scheme has been applied to injection site
sarcomas in cats, but no correlation with outcome has been documented [34].

The evaluation of a tumor for surgical excision or radiation therapy

requires more deﬁnitive evaluation than simple physical palpation of the
tumor. When contrast-enhanced CT images are used to determine the
tumor volume and are compared with tumor volumes based on physical
examination measurements, the inadequacy of the physical examination to
determine tumor extent becomes apparent. A study compared the two
methods of determining tumor volume [49]. Thirty-ﬁve cats with injection
site sarcomas had their tumor volumes measured by physical examination
and with CT (with and without contrast). The tumor volumes of 33 cats

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were larger when calculated using the postcontrast CT images. The median
tumor volume as measured on physical examination was 23.4 cm

, and

the median tumor volume as measured on postcontrast CT images was
57.2 cm

. The authors found that treatment recommendations were often

altered based on the CT images, further emphasizing the need for accurate
imaging before formulating a treatment plan.

In addition to careful evaluation of the extent of the primary tumor,

standard staging and health assessment are performed in feline patients with
injection site sarcomas. The most common sites of metastasis are the lungs,
followed by the lymph nodes and other internal organs [50]. The percentage
of cats with metastasis at the time of presentation is relatively low, with
studies reporting from 5% to 5.3% (Tetsuya Kobayashi, DVM, Raleigh,
NC, personal communication, 1999) [51]. The overall metastatic rate of cats
treated for injection site sarcomas has been reported to range from 0% to
28% [51,52]. In a retrospective study of 92 cats treated with multimodality
therapy at North Carolina State University, 21.7% (20 of 92 cats) developed
detectable metastasis after treatment. The median time to metastasis in these
20 cats was 155 days (range: 66–1022 days, unpublished data). The absolute
rate of metastasis may be higher, because not all cats were dead at the time
of analysis and not all cats were necropsied [50].

Surgical excision as the sole modality of treatment has been shown to be

inadequate in a number of studies, although the importance of surgery in
a multimodality approach is unquestionable. A retrospective analysis of 61
cats from two veterinary teaching hospitals treated with surgical excision
reported a median overall time to recurrence of 94 days, with only 11% of
cats remaining tumor-free for at least 1 year [51]. Surgery performed at the
referral institutions resulted in an increased median time to recurrence of
274 days. Cats with appendicular tumors (most commonly treated with
amputation) had a longer time to recurrence than cats with tumors at other
sites. Of the 5 cats that were long-term survivors (>1300 days), 4 were
treated with amputation. Interestingly, the median survival time for these 61
cats was 576 days, and many cats had multiple surgical procedures before
succumbing to their disease. Similar results with surgical excision were
reported from a third veterinary teaching hospital, with an overall median
disease-free interval (DFI) for cats treated with surgery alone of 124 days
(Carrie Wood. DVM, St. Paul, MN, personal communication, 1999). For
cats with complete excision, the median DFI was 221 days. A third study
had an overall median DFI of 10 months, and cats with complete tumor
excisions had a DFI of longer than 16 months [53].

There have been a few smaller studies evaluating more radical excision of

injection site sarcomas with promising results. Lidbetter et al [54] described
6 cats that underwent a lateral body wall resection for this tumor. Three cats
had preoperative radiation therapy. At a mean follow-up of 17.2 months, no
cats had evidence of recurrence. Kuntz [55] reported on a modiﬁed wide
local excision technique, in which 5-cm margins in all directions and two

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muscle planes deep were taken at surgery. Histopathologically, 23 of 24 cats
had complete excisions, and follow-up of these patients is continuing to
determine the effectiveness of this approach. At North Carolina State
University, surgeons currently excise 3- to 5-cm margins around injection
site sarcomas, removing as much of the surrounding tissue as possible while
still leaving the cats able to function relatively normally (Elizabeth Hardie,
DVM, PhD, Raleigh, NC, personal communication, 2002).

Limited evaluation has been done on the use of radiation therapy alone for

the treatment of injection site sarcomas, because radiation would not typically
be used as a single modality for palpable disease. A small study evaluating
hypofractionated radiation therapy (four weekly fractions of 8 Gray given
with palliative intent) with or without chemotherapy in cats with gross disease
demonstrated a median progression-free interval (PFI) of 210 days (Janean
Fidel, DVM, Zurich, Switzerland, personal communication, 2001).

The most promising outcomes achieved in treating cats with injection site

sarcomas have been the result of multimodality therapy, including radiation
therapy and surgery. Several types of radiation therapy have been evaluated in
combination with surgery, including orthovoltage (administered after
surgery, median DFI of 313 days) and brachytherapy (administered after
surgery, median DFI not yet achieved) as well as megavoltage (including
electron beam therapy) (Janet Burke, VMD, Philadelphia, PA, personal
communication, 1999) [56]. A recent retrospective study of 25 cats treated at
Colorado State University with surgery and megavoltage radiation, with or
without doxorubicin chemotherapy, reported an overall median survival of
701 days [52]. Seven cats received surgery, followed by 57-Gy radiation
therapy (19 daily fractions of 3 Gy). The remaining 18 cats were treated with
surgery, radiation therapy, and doxorubicin chemotherapy in the adjuvant
setting. Doxorubicin did not appear to improve median DFI, but the small size
of the groups and retrospective nature of this study make it difﬁcult to
determine the true impact of chemotherapy in this setting. These results
contrast with those presented by King and colleagues [57]. In this study, 61 cats
were treated with preoperative radiation therapy (60–62 Gy on a Monday

Wednesday

/Friday schedule over 7 weeks) and surgical excision, either with

or without ﬁve doxorubicin treatments. The DFI in the cats receiving
doxorubicin was prolonged as compared with the cats not receiving che-
motherapy (median of 360 days versus 162 days); however, there was no
difference in length of survival.

A retrospective study evaluating the eﬀect of the extent of surgery was

performed in 76 cats treated at Auburn University [58]. Nineteen of these
cats were treated with conservative resection of the palpable disease. All cats
received postoperative radiation therapy of 52 Gy (13 fractions of 4 Gy
given on a Monday

/Wednesday/Friday schedule) with 4- to 8-MeV elec-

trons. Median DFI for these cats was 405 days, with a median survival of
469 days from the start of radiation therapy. No difference in recurrence
rate was detected between cats treated with a conservative versus wide ﬁrst

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excision, although the statistical power of this comparison was low. Com-
pleteness of excision did not affect DFI in this group of cats. Cats that
had undergone multiple surgeries before radiation therapy had a decreased
DFI compared with cats with only a single sarcoma excision.

At the North Carolina Animal Cancer Program, we recently evaluated

outcome in 92 cats treated since 1985, with deﬁnitive preoperative radiation
therapy (16 daily fractions of 3 Gy) and wide surgical excision [50]. Thirty-
three of these cats were reported in a previous publication [59]. Of these 92
patients, 33 received chemotherapy: 19 received only carboplatin and 14
received other chemotherapy agents (doxorubicin, vincristine, cyclophos-
phamide [Cytoxan], with or without carboplatin). In this study, ﬁrst-event
analysis was used, which is a conservative statistical method where the
only animals censored are those that are alive and disease-free at the time
of analysis. The overall median time to ﬁrst event (death, metastasis, or
local recurrence) was 1.6 years. The completeness of surgical excision was
the only variable predictive of time to ﬁrst event. For cats with complete
surgical excision of their sarcoma, time to ﬁrst event was 2.7 years. Median
time to ﬁrst event was 0.8 years for cats with incomplete tumor resection.

The impact of chemotherapy on the treatment of injection site sarcomas

has yet to be clearly determined. Multiple chemotherapy agents have been
shown to have activity against injection site sarcomas, including carboplatin,
doxorubicin, and cyclophosphamide. Two phase I trials of carboplatin in cats
reported at least partial responses (>50% reduction in size) in cats with
injection site sarcomas, including 6 of 16 cats (37.5%) in one trial (William
Kisseberth, DVM, Madison, WI, personal communication, 1996; Carrie
Wood, DVM, North Grafton, MA, personal communication, 1996). An
additional 12 cats whose tumors were deemed unresectable were treated with
a combination of doxorubicin and cyclophosphamide in another study [60].
Six of the 12 cats had at least a partial response to this combination protocol,
although the responses were not durable (median of 125 days). The two
longest responses were in cats that achieved clinical complete remission on
this protocol. A case report from Europe also supports the assessment that
doxorubicin has activity against injection site sarcomas [61]. In this report,
a cat with an injection site sarcoma was presented to the Veterinary Teaching
Hospital in Vienna after the third excision of its tumor and was treated with
adjuvant doxorubicin. Recurrence of the tumor was delayed until more than
12 months after surgery and adjuvant doxorubicin, whereas the ﬁrst two
relapses had occurred by 2 and 3 weeks after surgery, respectively (Miriam
Kleiter, DVM, Raleigh, NC, personal communication, 2002).

Doxil is a liposome-encapsulated form of doxorubicin. The eﬃcacy of

Doxil in comparison to unencapsulated doxorubicin was evaluated in a
randomized trial in which these two formulations were given either to cats
with palpable disease or to cats that had incomplete surgical excisions of
their tumors [62]. The PFI in cats receiving chemotherapy was compared
with that in a historical control group of cats that were treated with surgery

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alone. The cats with microscopic disease that received either formulation of
doxorubicin had a signiﬁcant increase in their PFI (median of 388 days)
compared with the surgical control group’s PFI (median of 93 days).
No difference in PFI was seen between the two treatment groups. Some
cats receiving Doxil, however, developed a delayed nephrotoxicity, leading
the investigators to conclude that free doxorubicin was the preferred
formulation.

In the retrospective study at North Carolina State University, chemo-

therapy did not result in an increased time to ﬁrst event, although the 10 of
19 cats receiving carboplatin had not yet failed therapy, with a median
follow-up time in these 10 cats of 3.5 years [50]. The small sample size
necessitates the evaluation of this combination in a larger number of cats
before conclusions can be drawn; however, the initial results warrant further
investigation.

Additional prospective studies are required to address adequately the

role that chemotherapy, used in the adjuvant or neoadjuvant setting, should
have in the treatment of injection site sarcomas. The high rate of response,
even in phase I trials, suggests that chemotherapy may be a useful addition
to multimodality treatment.

The role of immunotherapy and novel approaches in the treatment of this

disease is unclear. Acemannan has been used to treat sarcomas in cats, but
no controlled studies have been performed to date [63]. The use of tyrosine
kinase inhibitors to block PDGF receptor signaling is an interesting ap-
proach to treatment and, possibly, prevention of injection site sarcomas,
but this modality of treatment is not yet being tested in the clinic. At North
Carolina State University, we have evaluated the use of local hyperthermia
to enhance liposome uptake in injection site sarcomas, and we were able to
demonstrate a signiﬁcant increase in liposome accumulation ranging from
2- to 13-fold in the heated tumors [64]. This approach may be used to im-
prove drug delivery to tumors. In conjunction with investigators at Duke
University, North Carolina State University and Colorado State University
are also evaluating interleukin-12 gene therapy in injection site sarcomas.
Much remains to be learned regarding the role of these novel approaches in
the treatment of this tumor.

Prevention

Injection site sarcomas are a challenge to the veterinary profession on

a number of levels: they are iatrogenic, diﬃcult to treat, and potentially
preventable. Thoughtful vaccination protocols to avoid the vaccination of
cats against diseases for which they are not at risk represent an often
recommended and relatively easy approach to integrate into routine well cat
care. The use of antibody titers to determine the requirement for booster
vaccinations is currently under evaluation, and initial results are promising
[65]. Although we can decrease the number of cats vaccinated against FeLV

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and use modiﬁed live FVRCP vaccines, vaccinating cats against rabies is
a matter of law in most states as well as being a signiﬁcant public health
concern. For the years 1998 through 2000, more than twice as many cats
as dogs tested positive for the rabies virus in the United States [66–68].
In a population survey from Pennsylvania during 1995, although 75% of
the bites were from dogs, people bitten by cats were six times more likely
to receive rabies postexposure prophylaxis (PEP) [69]. Dog bites accounted
for 30% of the PEP treatments, whereas cat bites accounted for 44% of
PEP—the largest group. When evaluating the percentage of owned animals
that were up to date on their rabies vaccination, 59% of dogs were vac-
cinated but only 41% of cats were current on their rabies vaccination.
Clearly, there is still room for improvement in the rabies vaccination of pet
cats in terms of the public health arena.

Following the recommendations of the VAFSTF on standardization of

vaccination protocols will help veterinarians to better correlate a given
vaccination and subsequent tumor development. These recommendations
include administering any vaccine containing rabies as distally as possible
in the right hind leg, administering FeLV vaccinations as distally as
possible in the left hind leg, and injecting FVRCP (with or without
chlamydia) vaccines in the right shoulder. Mixing of vaccinations is dis-
couraged. Documentation of the vaccination site as well as the manu-
facturer and lot or serial number is important if an adverse event develops.
All adverse reactions (including sarcoma development) would ideally be
reported to the USP (1-800-4-USP-PRN) for better population surveillance.
This problem is unlikely to go away on its own, and it is important
for veterinarians to play an active role in the prevention, monitoring,
and development of treatments for injection site sarcomas.

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Canine mammary gland tumors

Karin Sorenmo, DVM

University of Pennsylvania, School of Veterinary Medicine,

Department of Clinical Studies, 3900 Delancey Street,

Philadelphia, PA 19104, USA

Mammary tumors are the most common type of tumors in intact female

dogs. Several epidemiologic studies have been conducted over the years and
provide an estimate of the incidence of canine mammary gland tumors. A
survey from Alameda and Contra Costa counties in California from 1963
through 1966 found that mammary gland tumors comprised 13.4% of all
tumors in dogs and 41.7% of all tumors in intact female dogs [1]. The
incidence of canine mammary gland tumors in the United States, however,
has been reduced signiﬁcantly since that time because of the common
practice of performing ovariohysterectomy (OHE) at an early age.
Mammary gland tumors are much more common in many European
countries where ovariohysterectomy is not routinely performed. Recent data
from The Norwegian Canine Cancer Register reported a crude incidence of
malignant mammary gland tumors of 53.3% in female dogs of any breed [2].
This number appears high but may be inﬂuenced by the common use of
progestins to prevent estrus in this particular population. A previous study
from the same group found an increased risk, with an odds ratio of 2.32, of
mammary gland tumor development in dogs treated with progestins [3].
Estrogens and progesterones play a major role in normal mammary gland
development, but these hormones have also been implicated in tumor
development [4–7]. Estrogens are promoters of initiated cells in addition to
regulating the transcription of several nuclear proto-oncogenes [8–11].

Mammary gland tumors, both benign and malignant, express estrogen

receptors (ERs) [12,13]. In addition to being involved in the initial malignant
transformation, the ER may also represent a rationale therapeutic target in
canine mammary gland tumors, as in breast cancer in women. A woman’s
lifetime risk of developing breast cancer is inﬂuenced by the cumulative dose
of bioavailable estrogen exposure [100]. Endocrine therapy, speciﬁcally with

Vet Clin Small Anim 33

(2003) 573–596

E-mail address:

karins@vet.upenn.edu

0195-5616

/03/$ - see front matter

doi:10.1016

/S0195-5616(03)00020-2

anti-estrogens, is one of the most commonly prescribed treatments for
women with ER-positive breast cancer [9,12,14–17]. The situation is similar
in the dog. The duration of exposure to ovarian hormones early in life
determines the overall mammary cancer risk. The risk of developing
mammary gland tumors increases from 0.5% to 8%, and to 26%, depending
on whether the ovariohysterectomy is performed before the ﬁrst, second, or
any estrus thereafter, respectively [6]. Progestin treatment also increases the
risk of tumor development [3,5]. There is conﬂicting information in the
literature regarding whether the progesterone-induced tumors are benign,
malignant, or both. Earlier studies reported that dogs treated with
progestins developed mostly benign tumors or had a slightly higher risk
of all types of tumors [5,18–20]. A more recent study found, however, that
91% of dogs with mammary gland tumors that had been treated with
progestins had malignant tumors [3]. Mechanisms involved in pro-
gesterone-induced mammary gland tumors include an upregulation of
growth hormone (GH) production within the mammary gland [21,22]. GH
has direct growth stimulatory eﬀects on mammary tissue but also has
indirect eﬀects via insulin-like growth factor I (IGF-I) [7,21]. The insulin
growth factors play a crucial role both in normal cell proliferation and
malignant transformation; IGF-I has been shown to be involved in
tumorigenesis in many diﬀerent types of tumors, including mammary
gland tumors [23,24]. Women with low-serum IGF-I have a reduced risk
of developing breast cancer [25,26]. Transgenic mice, which overexpress
IGF-I, have abnormal mammary gland development and an increased
incidence of tumors; IGF-I interferes with apoptosis and is also a potent
mitogen for several breast cancer cell lines [27,28].

In addition to the hormonal inﬂuence on the risk for developing

mammary gland tumors, there are also other factors, such as genetic
predisposition and diet, which have been shown to contribute to tumor
development. Certain breeds are at increased risk of developing mammary
gland tumors. The breeds at increased mammary cancer risk vary somewhat
according to study and geographic location, but Toy and Miniature
Poodles, English Springer Spaniels, Brittany Spaniels, Cocker Spaniels,
English Setters, Pointers, German Shepherds, Maltese, Yorkshire Terriers,
and Dachshunds have been reported to have increased incidence of
mammary gland tumors in various studies [29–31]. The fact that certain
breeds have an increased risk of developing mammary gland tumors sug-
gests a genetic component. A common genetic mutation has not been
identiﬁed in dogs with mammary gland tumors, however. The tumor
suppression gene p53 is the most frequently mutated gene in human tumors,
and it has also been evaluated in canine mammary gland tumors. Several
studies have evaluated p53 in canine mammary gland tumors and have
reported variable results regarding overexpression and the frequency of
mutations. A recent study reported that 15

/20 mammary gland tumors

overexpressed mutant p53 protein [32]. Other studies have found a lower

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incidence of p53 mutations ranging from 15% to 63% of cases [33–35]. In
addition, the oncogene c-erbB2 has also been found to be overexpressed in
the majority of canine malignant mammary gland tumors evaluated, and
mutations in BCRA1 have also been documented in a few cases [36,37].
Germline mutations in BRCA1 and BRCA2 are thought to account for 5%
to 10% of all breast cancer cases in women, and they may also be involved
in the selected cases of mammary gland tumors in dogs [38]. Many diﬀerent
genetic alterations are probably involved in mammary gland tumorigenesis,
even though these various mutations may lead to convergent phenotypes
and result in clinically indistinguishable tumors. The importance of studying
these alterations lies in the hope that understanding and identifying the
mechanisms involved in malignant transformation may provide opportuni-
ties for prevention and treatment of cancer. An excellent example is the use
of trastuzumab (Herceptin

) in the treatment of breast cancer in women.

Trastuzumab is a humanized monoclonal antibody that binds to the
extracellular domain of the HER2

/neu (c-erbB2) receptor, which is a member

of the EGFr family of growth factor receptors. Trastuzumab has direct
antitumor eﬃcacy, improves survival in breast cancer patients with HER2

neu

overexpression, and is the ﬁrst successful example of molecularly

targeted therapy in the management of breast cancer in women [39,40].

Several studies have evaluated the eﬀect of various dietary factors and

obesity on the risk of developing mammary gland tumors in dogs. One study
reported that the risk of developing mammary gland tumors is signiﬁcantly
decreased in spayed dogs that were thin at 9 to 12 months of age, but it did
not ﬁnd an increased tumor risk in dogs that were obese 1 year before
mammary gland tumor development, or were fed a high-fat diet [41].
Another study by the same group evaluated the eﬀects of several dietary
factors on prognosis in dogs with mammary gland tumors and found that
dogs fed a low-fat, high-protein diet had a signiﬁcantly prolonged survival
compared with dogs fed a low-fat, low-protein diet. Survival was not
inﬂuenced by protein intake in dogs fed a high-fat diet [42]. A more recent
study conﬁrmed that obesity at 1 year of age and a high red-meat diet were
independent risk factors for developing mammary tumors and dysplasia
[43].

Obesity and a high-fat diet have also been linked to an increased breast

cancer risk in women. Obesity is associated with increased risk of breast
cancer in postmenopausal women. This may be caused by various factors
including hyperinsulinemia, increased levels of IGF-I, and decreased levels
of serum sex hormone–binding globulin (SHBG), which results in increased
free serum estrogen levels and, subsequently, an increased breast cancer risk
[44–46]. Immigrants from countries with low breast cancer rates, such as
Asian countries, often increase their breast cancer risk when adopting the
dietary habits of Western countries, which typically consists of high-fat diets
compared with traditional Eastern diets [5,47]. A woman’s risk of
developing breast cancer is inﬂuenced by the cumulative dose of estrogen

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exposure, and high-fat diets may contribute to an increased breast cancer
risk by increasing serum levels of bioavailable estrogens [9,10]. Women on
a low-fat diet have signiﬁcantly lower serum estrogen levels and decreased
breast cancer risk when compared with women on a high-fat diet. Several
independent retrospective studies as well as meta-analyses have conﬁrmed
that there is a direct association between dietary fat intake, serum estrogen
levels, and breast cancer risk [44,48,49].

Tumor types, histopathologic classiﬁcation, and biologic behavior

Though canine mammary gland tumors may be benign or malignant,

approximately 40% to 50% of these tumors are malignant [50,51]. Further
classiﬁcation may be performed according to tissue of origin (epithelial,
myoepithelial, or mesenchymal tissue), descriptive morphologic features,
and prognosis. The World Health Organization International Histological
Classiﬁcation of Mammary Tumors of the dog and the cat combines
histogenic and descriptive morphologic classiﬁcation, incorporating histo-
logic prognostic features that have been associated with increasing ma-
lignancy [52]. Most mammary gland tumors are of epithelial origin. Some,
however, can have mixed histology consisting of both epithelial and
myoepithelial tissue, with areas of cartilage and bone, and a few tumors are
of purely mesenchymal origin.

Epithelial tumors are often classiﬁed according to histopathologic

borders and diﬀerentiation. A carcinoma in situ is an epithelial tumor with
malignant features that has not invaded the basement membrane. These
lesions are often multicentric and can grow in pre-existing ducts or lobules.
Tubular carcinomas (adenocarcinoma) are the most common type of mam-
mary gland tumors in the dog; these tumors have retained some of the
original ductal or tubular morphology of the normal mammary gland. The
solid carcinomas are less diﬀerentiated; these tumors have lost the tubular

ductal structures and form solid sheets. Anaplastic mammary gland car-
cinomas are undiﬀerentiated, pleomorphic, and inﬁltrative epithelial tumors
that are not classiﬁable in any of the other categories of carcinomas [52].
Inﬂammatory mammary gland carcinomas are anaplastic carcinomas with
characteristic clinical and histopathologic features such as involvement of
the overlying skin with edema and pain, extensive inﬂammatory cell
inﬁltrate, malignant epithelial cells in the dermal lymphatics, and a rapid
clinical progression [53]. The histopathologic diﬀerentiation of epithelial
mammary gland tumors has an impact on prognosis, with a worsening of
prognosis associated with loss of diﬀerentiation. Carcinoma in situ and
adenocarcinomas have the best prognosis, and anaplastic and inﬂammatory
carcinomas have the worst prognosis [52].

Malignant myoeptheliomas, or spindle cell carcinomas, are malignant

tumors arising from the myoepithelial cells of mammary tissue, and they are

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quite rare in dogs. Diﬀerentiating between malignant myoepitheliomas and
ﬁbrosarcomas often requires immunohistochemical stains [29].

Primary mammary gland sarcomas are not common and are thought to

arise from pre-existing benign mixed tumors by malignant transformation, or
to arise from the interlobular stroma. The most common primary mammary
gland sarcomas include osteosarcomas and ﬁbrosarcomas. Other mammary
gland sarcomas occasionally encountered are chondrosarcomas and lipo-
sarcomas [29]. The mammary gland is the most common site of extraskeletal
soft tissue osteosarcoma according to a recent study [54]. The mammary gland
osteosarcomas behave as the appendicular osteosarcomas do, and they are
associated with early hematogenous metastasis and a short survival.

Mixed mammary gland tumors consist of both ductal and myoepithelial

cells with areas of cartilage and bone. The origin of the cartilage or bone in
these mixed tumors is controversial and may include metaplastic changes in
the epithelial cells, myoepithelial cells, or interstitial stromal cells. Malignant
mixed tumors, also called carcinosarcomas, are uncommon tumors in the
dog and are composed of both malignant epithelial cells and malignant
connective tissue elements. These tumors are most often a combination
of a carcinoma and an osteosarcoma [29]. The prognosis for dogs with
malignant mixed tumors is poor, and most dogs develop metastasis within
the ﬁrst year [55].

All malignant mammary gland tumors have the potential to metastasize.

The metastatic risk and pattern are inﬂuenced by the tumor type, histologic
diﬀerentiation, and several clinical prognostic factors. In general, malignant
epithelial tumors metastasize via the lymphatics to the regional lymph nodes
and the lungs, whereas the mesenchymal tumors typically metastasize by the
hematogenous route directly to the lungs [29]. Dogs with malignant mammary
gland tumors have a signiﬁcantly shorter survival time than dogs with benign
tumors. The overall 2-year survival has been reported to be between 25% and
40% with a mean survival time ranging from 4 to 17 months, but the survival is
inﬂuenced by multiple factors, and it can vary signiﬁcantly depending on
histologic type and diﬀerentiation, stage of disease, and type of treatment [52].
Dogs with small, well-diﬀerentiated malignant epithelial tumors may have an
excellent prognosis with surgical resection alone, and dogs with more un-
diﬀerentiated, advanced tumors have a guarded prognosis and may require
adjuvant therapy. There are currently no accepted guidelines or recommen-
dations for dogs in the latter group, however, and there are few reports
regarding the eﬀectiveness of adjuvant therapy in such dogs.

Clinical signs and physical exam

Dogs with mammary gland tumors are typically older, approximately 9

to 11 years old, sexually intact, or spayed later in life [5,29,30]. Most dogs
with mammary gland tumors are clinically healthy when they initially
present for evaluation of their tumors. The duration of the clinical signs also

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varies greatly from just a few days to many months. Several studies have
found that dogs with shorter duration of clinical signs have more aggressive
tumors and a worse prognosis than dogs with longer clinical histories
[19,53,56]. The tumor(s) may have been found by the owner or may be an
incidental ﬁnding during routine physical exam. Depending on the tumor
type and how soon it is detected, the tumors may be small, large, ulcerated,
ﬁxed, well circumscribed, or involving only one or multiple glands. The
caudal 4th and 5th mammary glands are more commonly involved than the
more cranial glands, but location does not appear to aﬀect prognosis [57,58].
It is not uncommon to ﬁnd more than one tumor in diﬀerent glands; more
than 60% of the cases have more than one tumor [55,56]. All of the
individual tumors should be biopsied because they may be of diﬀerent
histopathologic types [56]. Multiple tumors do not necessary imply a worse
prognosis. Rather, the prognosis is inﬂuenced by the size, type, and dif-
ferentiation of the individual tumors [52]. The regional lymph nodes may be
normal or palpably enlarged. Previous reports have found that 10% to
50% of dogs with mammary gland tumors have enlarged lymph nodes [31,52,
53]. Dogs with advanced metastatic disease or inﬂammatory mam-
mary carcinomas typically have systemic signs of illness when they are
diagnosed. Dogs with metastatic disease may present with nonspeciﬁc signs
such as fatigue, lethargy, and weight loss. The severity of these signs depends
on the extent and location of the metastases. Dogs with metastases may or
may not have obvious mammary gland tumors, depending on whether they
have had previous surgical resection of primary tumors. Most mammary
gland tumor metastases occur within 1 year of the initial surgery [6,59].
Dogs with inﬂammatory mammary gland carcinoma present with more
dramatic clinical signs. Typical clinical signs include extensive inﬂammation
of the involved mammary glands with edema and pain. These dogs therefore
may be misdiagnosed as having mastitis. They are often in poor clini-
cal condition and have generalized weakness, weight loss, polyuria and
polydipsia, and a high incidence of metastatic disease, both to the regional
lymph nodes and lungs. Prognosis is extremely poor with a mean survival of
25 days from diagnosis [53].

Diagnosis and staging

A surgical biopsy, typically an excisional biopsy, is recommended as the

initial diagnostic approach to dogs with mammary gland tumors. This
biopsy will provide tissue for histopathologic diagnosis and be therapeutic
for dogs with benign tumors. Dogs with small, well-diﬀerentiated malignant
tumors may be cured by excisional biopsy if the surgical borders are
complete. Fine needle aspirates may not always accurately diﬀerentiate
between malignant and benign epithelial tumors.

Complete staging requires blood work including complete blood count

(CBC), serum chemistry proﬁle, and urinalysis. Evaluation of the primary

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tumor, including size, type, and histologic diﬀerentiation; assessment of the
regional lymph nodes; and three-view thoracic radiographs, generally
completes the staging evaluation. The purpose of the staging is to evaluate
general health and to determine the extent of the tumor. Results from
staging assessment provide important prognostic information, which may
aﬀect the owner’s decision to treat. In addition, staging is also necessary for
treatment planning.

Blood work is normal in most dogs with mammary gland tumors, unless

they have other concurrent medical problems or nonspeciﬁc age-related
changes. A recent study evaluated hemostatic changes in dogs with ma-
lignant mammary gland tumors and found that two thirds of the dogs
had one or more hemostatic abnormalities, with an increased incidence in
dogs with stage III and IV disease. Of these, dogs with distant metastasis,
invasive or ﬁxed tumors, severe tumor necrosis, and inﬂammatory car-
cinomas were more likely to have coagulopathies. The clinical sig-
niﬁcance of these abnormalities is not clear, however [60]. Hypercalcemia
of malignancy is a not-infrequent complication of breast cancer in women.
This paraneoplastic manifestation may be caused by osteolytic bone
metastases or the production of PTHrP by the tumor cells. This ﬁnding is
rare in dogs with mammary carcinoma, however.

The status of the regional lymph node has a strong impact on survival in

dogs with mammary gland tumors [30,31,57]. Therefore, the regional lymph
nodes should be evaluated in all dogs with malignant tumors, so that systemic
treatment may be initiated in cases with regional lymph node metastasis. The
methods for assessing the regional lymph nodes include palpation, ﬁne needle
aspirates, Tru-cut biopsy

(Allegiance Healthcare Corporation, McGaw

Park, IL), or whole lymph node excision. Studies in human breast cancer
patients have found that physical exam alone is notoriously inaccurate in
determining axillary lymph node involvement; patients with palpably
enlarged lymph nodes may have reactive inﬂammatory changes, and
normal-appearing lymph nodes may harbor metastatic disease [61].

A study in veterinary medicine compared the sensitivity and speciﬁcity of

these four methods of evaluating lymph nodes in patients with various types
of solid tumors and found similar results. Palpation was inaccurate in
predicting lymph node metastasis, whereas cytology provided an accurate
method of assessing the draining lymph nodes, with a sensitivity of 100%
and a speciﬁcity of 96% [62]. This study included dogs with many diﬀerent
types of tumors, but it seems reasonable to assume that cytology would have
similar accuracy in dogs with mammary gland tumors. Fine needle aspirates
are usually easy to perform, are noninvasive, do not require sedation of the
patient, and provide quick and reliable results. Cytologic evaluation of the re-
gional lymph nodes should therefore be performed as the initial screening in
all dogs with malignant tumors. If the cytology is positive or questionable,
a complete excision of the involved lymph node may be considered. It is
controversial whether removing metastatic lymph nodes in cancer patients

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signiﬁcantly improves survival, but node resection may improve regional
tumor control and prevent signs associated with progressive lymph node
enlargement [63]. Eﬀective loco-regional control may also translate into
improvements in overall survival; results from human breast cancer studies
show signiﬁcant improvement in tumor-speciﬁc survival in women with
four or more positive axillary lymph nodes treated with both radiation
therapy and chemotherapy [64]. The 1st and 2nd cranial mammary glands
drain to the axillary lymph node on the ipsilateral side, and the 4th and
5th glands drain to their respective superﬁcial inguinal lymph nodes. The
lymphatic drainage of the 3rd mammary gland is most commonly to the
axillary lymph node, but this gland may also drain to the inguinal lymph
node. Both sites should be aspirated, therefore, in dogs with tumors in-
volving the 3rd gland [65].

All dogs with malignant mammary gland tumors should have three-view

thoracic radiographs taken. Radiography is still the standard diagnostic
method for evaluating veterinary patients for metastatic lung disease.
Conventional radiography may detect lung lesions ranging from 6 to 8 mm
in diameter. The ability to detect early metastasis can be improved by using
CT for metastasis as small as 4 mm in diameter. Early detection and
treatment of metastatic disease may have impact on response and outcome
in human patients, and CT has become the standard method of evaluat-
ing human cancer patients for lung metastasis [66]. The occurrence of
synchronous pulmonary metastatic disease at the time of initial diagnosis
was 35% according to an older study; however, a more recent study re-
ported a synchronous metastasis rate of only 6%. [31,59]. Changes in
veterinary medicine, as well as client commitment and awareness about
cancer in pets, may have led to earlier detection and, therefore, diagnosis of
dogs with early-stage disease. Dogs with clinically aggressive tumors should
have thoracic radiographs taken before surgical resection of the primary
tumors because the presence of pulmonary metastasis may inﬂuence the
decision to perform an extensive local resection. All dogs with malignant
mammary gland tumors should undergo thoracic imaging because distant
metastases are associated with a grave prognosis. The lungs are the most
common site for distant metastasis in dogs with malignant mammary gland
tumors, but additional diagnostic tests may be indicated, and other
anatomic sites may need to be assessed according to the speciﬁc clinical
signs of an individual patient. Metastasis to abdominal viscera or lymph
nodes may occasionally be observed. Abdominal ultrasonography or ra-
diography may be helpful to detect abdominal metastasis.

Mammary gland carcinomas are staged according to the World Health

Organization (WHO) TNM system or a modiﬁcation of this system [67,68].
The T designation describes the primary tumor, speciﬁcally the size of
the tumor, as follows: T0 (no evidence of tumor); T1 (less than 3 cm in
diameter); T2 (tumor between 3 and 5 cm); T3 (tumor larger than 5 cm);
and T4 (inﬂammatory carcinoma, any size). The N category describes

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involvement of the regional lymph nodes, where N0 is a histologic normal
lymph node, N1 is a positive ipsilateral lymph node, and N2 is bilateral
lymph node involvement. The M category represents distant metastasis; M0
indicates no metastasis, and M1 indicates the presence of distant metastasis.
The TNM system can be further organized as stage grouping (Table 1). Both
the TNM system by itself, or the modiﬁed stage grouping system, provide
valuable prognostic information that should be incorporated into the
treatment plan for dogs with mammary gland tumors.

Prognostic factors

Several prognostic factors have been identiﬁed in dogs with mammary

gland tumors including age, tumor size and stage, clinical behavior of the
tumor, tumor histopathologic type, tumor grade, estrogen receptor status,
microvessel density, and molecular genetic alterations.

Advanced age at diagnosis is reported to be a negative prognostic factor

in several veterinary studies [6,57]. Age has also been found to be a negative
prognostic factor in women with breast cancer, and older women are
reported to have shorter remissions and survival than younger women
[69,70]. However, age is usually not associated with a more malignant

Table 1
Canine mammary tumor staging modiﬁed (excluding inﬂammatory carcinoma)

T: Primary tumor

\3 cm maximum diameter

3–5 cm maximum diameter

[5 cm maximum diameter

N: Regional lymph node status

histologic or cytologic no metastasis

histologic or cytologic metastasis

M: Distant metastasis

No distant metastasis

Distant metastasis detected

Stage grouping

Stage
I

III

Any T

Any N

Modiﬁed from

The World Health Organization (WHO) Clinical Stage Classiﬁcation (Owen

LN. Classiﬁcation of tumors in domestic animals, 1st edition. Geneva: World Health
Organization; 1980); and Rutteman GR, Withrow SJ, MacEwen EG. Tumors of the mammary
gland. In SJ Withrow, EG MacEwen, editors. Small animal clinical oncology, 3rd edition.
Philadelphia: WB Saunders, 2001.

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phenotype and may therefore not be a negative prognostic factor in itself.
Rather, advanced age and concerns about toxicity may inﬂuence the type of
adjuvant therapy administered to older patients and therefore result in
suboptimal, less eﬀective treatments. This trend is not unique to breast
cancer patients and has been recognized in older cancer patients in general
[71]. Most of the veterinary studies showing that older dogs had a shorter
survival than younger dogs did not perform multi-variant analysis, did not
correlate outcome with tumor stage and treatment, or did not report on
cancer-speciﬁc deaths. Older dogs may be more likely to die from other
noncancer causes than younger dogs.

Tumor size has been found to be an independent prognostic factor in

many diﬀerent studies [30,31,58,72]. Tumors smaller than 3 cm in diameter
are associated with a signiﬁcantly better prognosis than tumors larger than
3 cm (Fig. 1). Information about tumor size should be readily available,
does not require additional diagnostic testing, and provides important
prognostic information that should be considered when deciding whether
additional therapy is needed.

Stage of disease using the WHO staging system, or a modiﬁed version of

this system, provides important prognostic information according to several
diﬀerent studies. As with many other solid tumors, dogs with mammary
gland tumors have a worsening prognosis with advancing stage [30,31].
Dogs with lymph node metastasis (stage IV disease) have a signiﬁcantly
shorter survival expectation than dogs with negative node staging, and dogs

Fig. 1. Size as a prognostic factor in canine mammary gland tumors. Kaplan-Meier disease-
free-interval curve according to tumor size in dogs with locally invasive malignant mammary
gland tumors. (From Kurzman ID, Gilbertson SR. Prognostic factors in canine mammary
tumors. Semin Vet Med Surg 1986;1:25–32; with permission.)

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with distant metastasis (stage V disease) have a worse prognosis than dogs
with negative staging or only regional lymph node metastasis (Figs. 2 and 3).

Tumor type, histologic diﬀerentiation, and grade have all been found to

have prognostic importance. In general, dogs with mammary gland sarcoma
have a worse prognosis than dogs with malignant epithelial tumors, except
for dogs with inﬂammatory carcionomas [54,57,58]. Primary mammary
gland sarcomas behave as high-grade sarcomas and are associated with
early hematogenous metastasis and high incidence of local recurrence.
According to the WHO Histological Classiﬁcation System, the biologic
behavior of the tumor corresponds with the histologic diﬀerentiation with
increasing malignancy from noninﬁltrating carcinoma (carcinoma in situ) to
complex carcinoma (two cell types), to simple carcinoma (one cell type),
tubulopapillary type, solid type, and to anaplastic carcinoma [52]. The
updated WHO staging system does not subclassify mammary gland car-
cinoma into ductal carcinomas, which arise from the small interlobular
or intralobular ductules, and alveolar carcinomas, which arise from the
alveolar epithelium. A study on lifetime morbidity and mortality of mam-
mary gland tumors in Beagles using a modiﬁed CRHL (Collaborative
Radiological Health Laboratory) classiﬁcation system found, however, that
the ductal carcinomas accounted for the majority of the fatalities in dogs
with malignant mammary gland carcinomas, even though they represented
only 19% of all carcinomas [55]. In addition to the histologic classiﬁcation
and diﬀerentiation of the tumors, there are other pathologic features

Fig. 2. Lymph node status as a prognostic factor in canine mammary gland tumors. Kaplan-
Meier disease-free-interval curve comparing disease-free- interval in dogs with lymph node
metastasis to disease-free interval in dogs without lymph metastasis. (From Kurzman ID,
Gilbertson SR. Prognostic factors in canine mammary tumors. Semin Vet Med Surg 1986;1:25–
32; with permission.)

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including vascular invasion, stromal invasion, absence of an inﬂammatory
response, the agyrophilic nucleolar organizer regions (AgNOR) index, and
a high histologic grade that may be associated with a guarded prognosis
[31,42,56–58,73–75].

The development of most mammary gland carcinomas is estrogen-

dependent, and the majority of canine mammary gland carcinomas express
ERs [6,8,9]. There is an inverse relationship between ER expression and
histologic diﬀerentiation. Benign tumors and well-diﬀerentiated tumors are
more likely to be ER-positive, whereas undiﬀerentiated, anaplastic tumors
are more likely to be ER-negative [8,9,76]. The association between
histologic grade and ER expression is further underscored by the fact that
there is an inverse correlation between ER expression and nuclear pro-
liferation indices as measured by proliferating cell nuclear antigen and
Ki-67 analysis. Tumors with high-proliferation indices had a more ag-
gressive clinical behavior with increased risk of metastasis [76] Estrogen
receptor expression has also been found to be associated with the hormonal
status of the dog. Younger, intact dogs were more likely to have ER-positive
tumors than older ovariohysterectomized dogs [8,76]. In addition to pro-
viding prognostic information, ER expression may also predict response
to hormonal therapy. Patients with ER-positive tumors are likely to respond
to estrogen ablation or ER blockage, and the decision whether to use
hormonal therapy in women with breast cancer is inﬂuenced by the tumor’s
receptor status. Estrogen receptor expression has traditionally been
evaluated using a standard dextran-coated charcoal biochemical assay but
can also be evaluated using immunohistochemistry on formalin-ﬁxed
tissues. There have been several recent studies successfully identifying ER
using immunohistochemistry for canine mammary gland tumors [77,78].

Fig. 3. WHO stage and prognosis in dogs with malignant mammary gland tumors. Survival
percentage according to TNM classiﬁcation and stage grouping. (From Yamagami T,
Kobayashi T, Takahashi K. Prognosis for canine malignant mammary tumors based on the
TNM and histologic classiﬁcation. J Vet Med Sci 1996;58(7):1079–83; with permission.)

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Angiogenesis is crucial for the growth and metastasis of most solid

tumors. Elevated serum levels of various tumor angiogenesis factors and
histopathologic evidence of increased neovascularization in the tumor,
measured as microvessel density, have been reported to have prognostic
importance in human cancer patients with various types of solid tumors [79–
81]. Microvessel density has been reported to be an important independent
prognostic factor in women with node-negative breast cancer [82,83].
Microvessel density has also been found to correlate with malignancy in
canine mammary gland tumors; malignant tumors have increased neo-
vascularization compared with benign tumors. Increased microvessel
density also correlates with risk of local recurrence, presence of lymph
node metastasis, and histologic diﬀerentiation in several studies [74,84,85].
In addition to the prognostic importance of the various angiogenesis assays,
results from these studies may also provide a rationale for the use of
antiangiogenic therapy in patients with increased microvessel density.

Molecular alterations, including p53 mutations and overexpression of

HER2

/neu (also called c-erbB2) have been associated with a more malignant

phenotype and a poor prognosis in women with breast cancer. Over-
expression and mutation of p53 have also been evaluated in many diﬀerent
veterinary studies. Results from these studies are discordant. In a few
studies, there was no signiﬁcant diﬀerence in outcome between dogs with
or without mutations, but other studies reported an increased risk of
recurrence and death in addition to a correlation between p53 mutations and
a high histologic grade of the tumors [34,35]. Overexpression of HER2

/neu

has been associated with resistance to endocrine therapy, shorter time
to relapse, and a low survival rate in women with breast cancer [35,39].
According to one study, this particular alteration is common in the dog, and
most malignant tumors overexpressed this oncogene, but there was no
correlation between expression and local invasion or metastasis [36].

Treatment of canine mammary gland tumors

Spontaneous tumors in dogs and cats are excellent models for human

tumors, and we often advocate using them to evaluate the eﬃcacy of new
and investigational therapies. The practice of clinical oncology in human
medicine is decades ahead of veterinary oncology, however, and we often
look to human oncology for guidance regarding therapy. The modalities
used in the treatment of women with breast cancer include surgery, ra-
diation therapy, hormonal therapy, or chemotherapy. The choice of
modality depends on results from many large prospective studies evaluating
the eﬃcacy of various treatments in patient groups stratiﬁed according to
stage and well-established prognostic factors. In general, the treatments
intensify with advancing clinical stage and increasing seriousness of the
prognostic factors. The Consensus Group on the treatment of women with

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breast cancer has provided speciﬁc recommendations regarding the use of
radiation therapy, chemotherapy, and hormonal therapy in women with
various stages of disease, which serve as guidelines to practicing oncologists
around the country [17].

This type of consensus recommendation does not exist in veterinary

medicine. Currently, surgery is the only accepted treatment for dogs with
mammary gland tumors, and there are no established guidelines for
treatment beyond surgery. Surgery is the mainstay of treatment for canine
mammary gland tumors and is the single most eﬀective modality for local
tumor control. The type of surgery does not appear to inﬂuence survival as
long as the entire tumor is removed with clean histologic margins [86,87].
The recommended surgical approach in dogs with mammary gland tumors
is to perform an excisional biopsy of the tumor; the size of the surgery,
ie, lumpectomy versus simple mastectomy, versus regional mastectomy,
depends on the size of the primary tumor [101]. An excisional biopsy will
provide tissues for histopathologic evaluation and may also provide local
control if the tumor margins are clean. Surgical excision can be curative
in dogs with stage I disease, and those with small, noninvasive, well-
diﬀerentiated carcinomas. Dogs with high-grade or larger tumors are likely
to develop metastatic disease, however, and may beneﬁt from additional
therapy [30,31].

Breast sparing surgery, followed by radiation therapy, is often used as an

alternative to radical mastectomy, or in addition to radical mastectomy in
women with large primary tumors or more than four positive axillary lymph
nodes. Studies have found signiﬁcant improvements in loco-regional control
and tumor-speciﬁc survival in women who received postmastectomy ra-
diation [17,64]. Radiation therapy has not been evaluated in treatment of
mammary gland tumors in dogs but may be a reasonable adjuvant to
surgery in dogs with primary mammary gland sarcomas because these
tumors often recur locally as well as at distant sites.

Both endocrine therapy and chemotherapy are used in women with high-

risk breast cancer. The use of endocrine therapy in the treatment of breast
cancer in women is empirical. Oophorectomy was ﬁrst used successfully to
induce regression of breast tumors 100 years ago [88]. Since that time,
endocrine therapy has evolved to include various types of medical and
surgical hormonal ablation. Today, medical hormonal ablation of various
types is commonly used to treat human breast cancer. The purpose of
hormonal therapy is to prevent further estrogen stimulation of breast cancer
cells. This can be achieved by several diﬀerent strategies, including blocking
receptors with speciﬁc estrogen receptor modulators (SERMS), which are
receptor antagonists such as tamoxifen. Suppression of estrogen synthesis
can be accomplished by administration of aromatase inhibitors, or by use of
luteinizing hormone-releasing hormone (LHRH) agonists. Tamoxifen is
currently the most commonly prescribed treatment for breast cancer in
postmenopausal women and is recommended as the ﬁrst antiestrogen

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approach by the National Consensus Group [17]. The newer agents, such as
the aromatase inhibitors and the LHRH agonists, are usually second-line
agents in women who have failed on tamoxifen. The decision whether to use
hormonal therapy in women with breast cancer depends on results from ER
analysis because only patients with ER-positive tumors are likely to beneﬁt
signiﬁcantly from such treatment. The development of mammary gland
tumors in the dog is also estrogen-dependent, and studies have found that
50% to 80% of malignant epithelial tumors express ER receptors [8,9].
These facts suggest that hormonal therapy may also be eﬀective in dogs with
mammary gland carcinomas, as it is in women. Most of the veterinary
literature evaluating the eﬀect of estrogen ablation in the form of OHE
has not found a survival beneﬁt in dogs with mammary gland tumors
[6,7,86,89]. A recent large retrospective study reported, however, that dogs
that underwent OHE concurrent with their tumor removal lived signiﬁ-
cantly longer than dogs that were treated with tumor removal alone. The
timing of the OHE in relation to the tumor surgery was important, and dogs
that were ovariohysterectomized concurrent with or less than 2 years before
their tumor surgery had the greatest beneﬁt from the OHE (Fig. 4) [90]. The
authors hypothesized that the sexual status of the dogs might aﬀect the
tumor receptor status, and dogs that were intact until their tumor surgery

Fig. 4. Survival in canine mammary gland tumors according to spay status and spay time.
Survival in intact dogs, dogs spayed more than 2 years prior to their tumor surgery, and dogs
spayed less than 2 years or concurrently with their tumor surgery. (From Sorenmo KU, Shofer
FS, Goldschmidt MH. Eﬀect of spaying and timing of spaying on survival of dogs with
mammary carcinoma. J Vet Intern Med 2000;14:266–70; with permission.)

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were more likely to have ER-positive tumors and, therefore, to beneﬁt the
most from hormonal modulation. Thus, more than 100 years after the initial
report in the Lancet, the beneﬁt of ovarian hormonal ablation may also have
been conﬁrmed in dogs with mammary gland tumors. The relationship
between the sexual status of the dog and the tumor ER status has been
conﬁrmed by other studies, and intact dogs have been demonstrated to be
more likely to have ER-positive tumors [8,76]. Information about ER status
is not routinely available in veterinary medicine and may therefore not be
available for inclusion in the treatment decisions. Ovariohysterectomy
should still be considered for intact dogs with malignant epithelial tumors.
Most of these tumors are ER-positive according to previous studies, and the
OHE may have other potential health beneﬁts in an older female dog. The
beneﬁt of OHE and tumor removal in dogs with mammary glad tumors
should ideally be evaluated by a prospective randomized clinical trial; The
Veterinary Hospital of the University of Pennsylvania is currently con-
ducting such a study in collaboration with The College of Veterinary
Medicine, University of Helsinki, Finland.

Chemotherapy is oﬀered to all women with breast cancer who are at risk

of failure from distant metastasis. Optimal selection of patients for adjuvant
chemotherapy requires accurate identiﬁcation of all patients at risk for
failing without including patients who do not need further treatment, thus
avoiding undertreatment as well as overtreatment. Recommendations re-
garding adjuvant treatment of early breast cancer have experienced major
changes in the past 25 years in human medicine. Since the mid-1970s, when
cyclophosphamide, methotrexate, and 5-ﬂuorouracil (CMF) resulted in
statistically signiﬁcant and clinically meaningful improvements in disease-
free and overall survival, the use of adjuvant chemotherapy has become
common practice in human breast cancer therapy [91,92]. Anthracyclines
such as doxorubicin and epirubicin have long been considered to be among
the most active available agents to treat breast cancer, and they have
become a core component of adjuvant regimens in the past decade.
Adjuvant chemotherapy regimens containing an anthracycline result in
statistically signiﬁcant improvements in survival compared with regimens
without an anthracycline [17,92,93].

Chemotherapy is also used in dogs with malignant mammary gland

tumors, both in the adjuvant setting as well as in dogs with gross metastatic
disease. There is, however, limited information regarding eﬃcacy of ad-
juvant chemotherapy in dogs with high-risk mammary gland tumors,
and only single case reports showing eﬃcacy of chemotherapy in dogs with
gross metastases [94]. It is encouraging to see objective regression of
pulmonary nodules in a dog with mammary gland tumor metastasis treated
with chemotherapy. Single case reports do not predict response rates in
a larger group of patients, however. Cases with positive responses to
chemotherapy, furthermore, may be more likely to be published than cases
with negative responses, and such reports may therefore lead to a general

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practice of treating patients with high-risk tumors with chemotherapy,
without further evaluating the beneﬁts and risks through randomized
prospective trials. There are no randomized studies on the eﬀect of adjuvant
chemotherapy in dogs with high-risk mammary gland tumors, but a recent
prospective study on 16 dogs with mammary gland carcinomas reported
signiﬁcant improvement in survival for dogs treated with a combination of
5-ﬂuorouracil and cyclophosphamide compared with dogs treated with
surgery alone (Fig. 5) [95]. Both groups of dogs underwent surgery for the
primary tumor and had stage III

/IV disease with similar tumor character-

istics. Eight of the sixteen dogs in this study were given chemotherapy based
on the owners’ decision, and the other eight dogs were treated with surgery
alone. All 16 of the dogs had high-risk tumors. The median survival was
only 6 months in the group treated with surgery alone compared with
24 months in the group treated with chemotherapy. This diﬀerence was
statistically signiﬁcant and provides encouraging data regarding the use of
adjuvant chemotherapy in dogs with mammary gland tumors. This is a small
study, the dogs were not randomized to treatment groups, and the results
may represent a type II error. Nevertheless, this study provides the basis for
additional larger prospective studies evaluating the beneﬁt of adjuvant
chemotherapy in dogs with negative prognostic factors. The drugs used in
this study were 5-ﬂuorouracil and cyclophosphamide, which were among
the ﬁrst chemotherapeutic drugs shown to have activity in breast cancer in

Fig. 5. Survival in dogs with high-risk mammary gland tumors treated with surgery alone
versus surgery and postoperative chemotherapy. (From Karayannopoulo M, Kaldrymidou E,
Constantinidis TC, et al. Adjuvant postoperative chemotherapy in bitches with mammary
cancer. J Vet Med Series A 2001;48(2):85–96; with permission.)

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women. Anthracycline-containing protocols are commonly used as ﬁrst-line
therapy in women with breast cancer today, and the use of anthracyclines,
speciﬁcally doxorubicin, may also be reasonable to evaluate further in the
adjuvant setting for canine mammary gland tumors. Doxorubicin was found
to be eﬀective in dogs with pulmonary metastasis, and a preliminary report
showed improved survival in dogs with high-risk mammary gland tumors
receiving adjuvant doxorubicin compared with dogs treated with surgery
alone [96,97]. Doxorubicin has also been found to have antitumor activity in
clonogenic assays, using established cell lines from canine mammary gland
tumors [98]. Primary mammary gland sarcomas are rare, and most studies
report that dogs with such tumors have a poor prognosis. A recent large
retrospective study reported a median survival of 90 days in dogs with
mammary gland osteosarcoma when treated with surgery alone [54].
Another study published around the same time evaluated the eﬀect of
chemotherapy in dogs with soft tissue osteosarcoma, including mammary
gland osteosarcomas, and found a signiﬁcant diﬀerence in survival between
dogs treated with chemotherapy versus the dogs treated with surgery alone.

Table 2
Proposed treatment guidelines for malignant canine mammary gland tumors based on tumor
size, stage, histopathological type, and diﬀerentiation

Tumor
size

Tumor
stage

Tumor
type

Histological
diﬀerentiation

Treatment
recommendations

\3 cm

Stage 1

Carcinoma

Well diﬀerentiated

Complete excision

Tubular

/papillary

OHE if intact

\3 cm

Stage 1

Carcinoma

Undiﬀerentiated

Complete excision
OHE if intact
Chemotherapy or clinical trial

>3 cm

Stages 2–3

Carcinoma

Any

Complete excision
OHE if intact
Chemotherapy or clinical trial

Any size

Stage 4

Carcinoma

Any

Complete excision

(including LN)

OHE if intact
Chemotherapy or clinical trial

Any size

Stage 5

Carcinoma

Any

ÿ surgery (if needed

for palliation)

Palliative chemotherapy

Any size

Any stage

Carcinoma

Inﬂammatory

ÿ surgery

Palliative treatments
Analgesics, anti-inﬂammatory
Chemotherapy or clinical trial

Any size

Stages 1–3

Sarcomas

Any

Complete (wide) excision

Carcinosarcomas

Radiation therapy if

incomplete excision

Chemotherapy or clinical trial

Abbreviation:

LN, lymph node; OHE, ovariohysterectomy.

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K. Sorenmo

/ Vet Clin Small Anim 33 (2003) 573–596

The drugs used in this study were those traditionally used to treat dogs with
appendicular osteosarcoma, such as cisplatin and doxorubicin [99].

Summary

The National Consensus Group recommends that all women with tumors

larger than 1 cm be oﬀered chemotherapy regardless of tumor histology of
lymph node status. This recommendation is to ensure that everyone at risk
for failing, even though the risk may be low in women with relatively small
tumors and favorable histology, has a choice and receives the beneﬁt of
adjuvant chemotherapy. This type of treatment recommendation may also
be made in dogs based on recognized, well-accepted prognostic factors such
as tumor size, stage, type, and histologic diﬀerentiation. Based on the
limited clinical information available in veterinary medicine, the drugs that
are eﬀective in human breast cancer, such as cyclophosphamide, 5-ﬂuo-
rouracil, and doxorubicin, may also have a role in the treatment of ma-
lignant mammary gland tumors in dogs. Randomized prospective studies
are needed, however, to evaluate the eﬃcacy of chemotherapy in dogs with
high-risk mammary gland tumors and to determine which drugs and
protocols are the most eﬃcacious. Until such studies are performed, the
treatment of canine mammary gland tumors will be based on the individual
oncologist’s understanding of tumor biology, experience, interpretation of
the available studies, and a little bit of gut-feeling. Table 2 is a proposal
for treatment guidelines for malignant canine mammary gland tumors
according to established prognostic factors, results from published vet-
erinary studies, and current recommendations for breast cancer treatment in
women.

Acknowledgments

The author would like to thank Katherine A. Kruger for her editorial

assistance.

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Management of transitional

cell carcinoma

Carolyn J. Henry, DVM, MS

a,b,

Department of Veterinary Clinical Sciences, College of Veterinary Medicine,

University of Missouri-Columbia, 379 East Campus Drive,

Columbia, MO 65211, USA

Division of Hematology and Oncology, Department of Medicine, School of Medicine,

University of Missouri-Columbia, Columbia, MO 65211, USA

Primary malignancy of the bladder is rare in dogs, comprising only 0.5%

to 1% of all canine cancers in published reports [1–3]. No large reviews of
canine bladder cancer incidence have been reported within the past decade.
Of the malignancies occurring in the canine bladder, transitional cell car-
cinoma (TCC) is the most common, accounting for approximately 50% to
75% of all reported cases [1–7].

Etiology and risk factors

The etiology of bladder cancer in dogs, as with other malignancies,

is likely multifactorial. Identiﬁed risk factors include obesity, exposure
to topical ﬂea and tick insecticides, exposure to marshes that have been
sprayed with mosquito control products, and possibly treatment with
cyclophosphamide [8–10]. Although early surveys indicated no gender pre-
disposition [2,11], subsequent reports document a female predisposition
[1,3,5,9,12,13]. One proposed explanation for the increased risk in females
is that male dogs urinate more frequently as a marking behavior, thus limit-
ing the contact time of bladder mucosa to carcinogens in the urine [5,14].
Further support for this theory, as opposed to a hormone-related cause of
carcinogenesis, came from laboratory studies indicating an ability to induce
bladder cancer in dogs, even when sex hormone levels were altered [15].
Certain breeds, including Shetland sheepdogs, beagles, collies, and various

Vet Clin Small Anim

33 (2003) 597–613

* Department of Veterinary Clinical Sciences, College of Veterinary Medicine, University

of Missouri-Columbia, 379 East Campus Drive, Columbia, MO 65211, USA.

E-mail address:

henryc@missouri.edu

0195-5616

/03/$ - see front matter

doi:10.1016

/S0195-5616(03)00032-9

terriers including Scottish, Airedale, and West Highland white and
Wirehaired fox terriers, appear to be predisposed to development of TCC
[1,5,9,13]. Of these, Scottish terriers and Shetland sheepdogs are the breeds
aﬀected most often [5,9,13].

Clinical presentation

Canine TCC occurs most commonly in older dogs, with a median age of

11 years [2,12,16,17]. The tumor may be seen in young dogs, however.
Therefore, TCC should not be excluded from a diﬀerential diagnosis simply
on the basis of patient age.

The bladder trigone is the most common site for TCC in dogs, often

leading to partial or complete urinary tract obstruction. The urethra and
prostate also are inﬁltrated with neoplastic cells often, either as advanced-
stage tumors inﬁltrating surrounding tissues or by way of metastatic foci. In
one report of 102 dogs with bladder TCC, 56% had concurrent urethral
involvement, and 29% of the male dogs had prostatic inﬁltration [9,13].
Some dogs diagnosed with primary prostatic carcinoma may, in fact,
represent misdiagnosed cases of TCC involving the prostatic urethra [18–20].

Dogs with TCC typically present with pollakiuria, stranguria, hematuria,

or tenesmus and may have a history of improvement with antibiotics. Severe
cases may present with bladder rupture, having associated signs including
acute abdominal pain and distension [4]. Occasionally, dogs are presented
for signs related to metastasis, rather than urinary signs. Hypertrophic
osteopathy (HPO) has been reported as a paraneoplastic syndrome in
association with TCC [21]. Although considered rare with TCC, the pre-
senting complaint of lameness and typical osteoproliferative lesions of HPO
on radiographs always warrants a search for underlying neoplastic or pri-
mary thoracic disease.

Physical examination should include abdominal palpation and rectal

examination. The bladder may be distended or have a palpable mass and wall
thickening. Urethral and trigonal thickening may be detectable upon pal-
pation in some cases. Physical exam may reveal no abnormalities, however, as
was noted for 30% of dogs with bladder and urethral tumors in one report [1].

Diagnosis and staging

Urinalysis

Urinalysis (UA) is often the ﬁrst test used to diagnose bladder TCC. One

must be cautious when obtaining urine for analysis, as TCC transplantation
has been reported in dogs following tumor manipulation with cystocentesis
or at the time of surgery [21–24]. Accordingly, the author cautions against
cystocentesis when TCC is suspected. Urinalysis results for dogs with TCC
may be indistinct from those noted with cystitis, including white blood cells,

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C.J. Henry

/ Vet Clin Small Anim 33 (2003) 597–613

red blood cells, and bacteria. Although urine sediment exam may reveal
tumor cells in at least 30% of cases [1,25], reactive, nonneoplastic tran-
sitional cells can look similar to TCC cells [26,27]. Cytocentrifugation of
freshly collected urine samples may be helpful in improving accuracy of
cytologic diagnosis. Cytologic evaluation should be supported by other
ﬁndings and conﬁrmed with histopathology when possible.

A veterinary bladder tumor antigen test (V-BTA Test; Alidex, Inc.,

Redmond, WA) is available and may serve as a quick screening test for
TCC. The V-BTA is a rapid latex agglutination dipstick test that uses anti-
bodies to a bladder tumor-associated glycoprotein complex detectable in
urine. The V-BTA test requires 0.5 mL of test urine, which should be spun
before testing. Additionally, the test should be run within 48 hours of
sample collection. The author and others evaluated the V-BTA in a pro-
spective, multisite study in which urine samples were collected from healthy
dogs and dogs with TCC, non-TCC urologic disease, and nonurologic
disease. A total of 229 specimens were analyzed, including 48 samples from
dogs with suspected (n

¼ 3) or conﬁrmed (n ¼ 45) TCC. Calculated

sensitivities were 88%, 87%, and 85% for all TCC cases, conﬁrmed TCC
cases, and conﬁrmed bladder TCC cases, respectively. Calculated speciﬁc-
ities were 84%, 41%, and 86% using samples from healthy dogs, dogs with
non-TCC urinary tract disease, and dogs with nonurinary disease,
respectively. Hematuria and proteinuria were associated with some false-
positive test results. These causes of false-positive test results have been
reported previously, as has glucosuria [28,29]. Two previous reports have
documented a 90% sensitivity for the bladder tumor antigen test [28,29].
The high sensitivity of the V-BTA test indicates that negative test results
correlate with absence of TCC. Thus, the clinical value of this test lies in its
ability to determine which dogs do not warrant further workup for TCC. As
with any screening test, positive results should prompt further evaluation to
conﬁrm a diagnosis.

Other urine screening tests that have been investigated for human bladder

cancer include the nuclear mitotic apparatus test (NMP22; Matritech,
Newton, MA), the Hemastix hematuria test (Bayer Corporation, Tarry-
town, NY), assays of telomerase, and assays (reverse transcriptase PCR or
immunoassay) of survivin, an inhibitor of apoptosis detected in 100% of
patients with new or recurrent bladder cancer. [30–32] Although these tests
(especially the latter) show great promise in human oncology, they remain
largely unexplored for the detection of canine bladder TCC.

Imaging and conﬁrmatory testing

Deﬁnitive diagnosis of TCC should be based on demonstration of

a bladder mass, along with cytological or, preferably, histopathological
demonstration of neoplastic cells from the mass. Demonstration of the mass
may be accomplished with imaging techniques such as radiography and

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ultrasonography or by direct tumor visualization by way of cystoscopy or
laparotomy. Double-contrast cystography is considered to be a reliable
method for detection of bladder masses in more than 95% of cases [1].
Ultrasonography of the bladder (cystosonography) is performed best when
the bladder is distended with urine or infused saline. Cystosonography oﬀers
the advantages of enhanced detection of intra-abdominal metastasis and the
ability to obtain biopsy samples by way of urinary catheterization under
ultrasound guidance. Visualization of the biopsy site by way of imaging may
improve yield, although one limitation of obtaining biopsy specimens by
catheterization techniques is the small sample size [33]. Alternatively, bi-
opsies may be obtained by cystoscopy or laparotomy.

In addition to verifying a diagnosis of TCC, tissue specimens may be

assigned a tumor grade using a modiﬁed World Health Organization (WHO)
system [34]. Tumor grade is based upon growth patterns, cell type, degree of
diﬀerentiation, and depth of invasion. In a published review applying this
grading system to tissues from bladder and urethral tumors of 110 dogs,
there were 58 transitional cell carcinomas and 42 tumors classiﬁed as TCC
with squamous or glandular metaplasia. Tumors were classiﬁed as grade 1, 2,
or 3, with grade 3 tumors being the most anaplastic. The authors concluded
that tumor grade and peritumoral desmoplasia were associated with survival.
A focal peritumoral lymphocytic inﬁltration was characteristic of transi-
tional cell carcinomas that metastasized. Considerable variability was noted
within single tissue samples, suggesting that obtaining biopsy specimens from
multiple sites is ideal for complete characterization of tumor tissue.

Tumor staging

Complete staging of bladder cancer in dogs requires evaluation of the

primary tumor and assessment for lymph node and distant metastasis. The
WHO clinical staging system (Table 1) uses degree of local tumor invasion
and metastasis to assign a clinical stage [35]. One recent abstract indicated
a correlation between bladder tumor stage and prognosis [36]. Median
survival times were 118 days for dogs with T3 tumors, compared with 218
days for dogs with T1 or T2 tumors. Those with N0 tumors had a median
survival time of 234 days, while those with N1 tumors had a median survival
time of 70 days. Distant metastasis was associated with a median survival
time of 105 days, compared with 203 days for dogs without distant
metastasis at the time of diagnosis. Complete tumor staging may provide
useful information for determining prognosis, developing a treatment plan,
and monitoring response to therapy.

Abdominal ultrasound is useful for staging, not only to assess tumor size

and degree of invasion within the bladder, but also for detecting intra-
abdominal metastasis. The metastatic rate for bladder TCC in dogs has been
reported to exceed 50% at the time of necropsy [1]. Reported metastatic sites
are listed in Table 2.

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Three-view thoracic radiographs are recommended to detect pulmonary

metastasis. Pulmonary metastatic disease from TCC may be misinterpreted,
unless one is familiar with the radiographic appearance of metastases with
this disease. Several distinct radiographic patterns have been described for
thoracic metastases of TCC and include nodular interstitial opacity, diﬀuse
unstructured interstitial opacity, cavitating pulmonary lesions, lobar in-
terstitial or alveolar inﬁltrates, multiple nodules, and normal pulmonary
opacity [37,38]. Hilar lymphadenopathy may be noted also. Dogs with
unstructured interstitial opacity have lesions described as a lacelike haze of
semidense diﬀuse opacity. This pattern can be easily misinterpreted as age-
related change. In one report, three of eight dogs with pulmonary metastasis
were incorrectly diagnosed as having normal age-related changes on
thoracic radiographs [38]. The cavitating pattern described with TCC is
likely caused by central necrosis of the metastatic lesions. This pattern also
has been noted uncommonly with metastatic transitional cell carcinoma in
people [39].

Treatment options for bladder transitional cell carcinoma

Surgery

The suitability of surgery for treatment of TCC depends upon the tumor

location and invasiveness and the client’s goals. In general, surgery is con-
sidered a palliative procedure, because of the high metastatic rate of canine
TCC, and because even grossly normal tissue may contain neoplastic or
preneoplastic tissue. Surgical options include partial cystectomy, permanent

Table 1
World Health Organization Clinical staging system (TNM) for canine bladder cancer

T: primary tumor

Carcinoma in situ

No evidence of primary tumor

Superﬁcial papillary tumor

Tumor invading bladder wall, with induration

Tumor invading neighboring organs (prostate, uterus, vagina, and

pelvic canal)

N: regional lymph nodes (internal and external iliac lymph nodes)

No regional lymph node involvement

Regional lymph node involvement

Regional lymph node and juxta-regional lymph node involvement

M: distant metastases

No evidence of distant metastasis

Distant metastasis present

Data from

Owen LN. TNM classiﬁcation of tumors in domestic animals. 1st edition.

Geneva: World Health Organization; 1980. p. 34.

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cystostomy catheter placement, or urinary diversion techniques such as total
cystectomy with ureterocolonic anastomosis [40,41]. Often, owners consid-
ering surgery choose the least invasive of these techniques, based on quality-
of-life and convenience issues.

Partial cystectomy

Partial cystectomy may be considered for dogs with localized bladder

tumors in areas amenable to resection with 1-cm to 2-cm margins of grossly
normal tissue. Potential postoperative complications include pollakiuria
caused by reduced bladder capacity and dehiscence of cystectomy suture
lines. Pollakiuria may resolve with time as the bladder size increases post-
surgery. Urinary catheters may be used in the acute postoperative period to
reduce tension on the cystectomy suture line and decrease the likelihood of
wound dehiscence. In one report of partial cystectomy for various urinary

Table 2
Metastatic sites reported for transitional cell carcinoma of the urinary bladder in dogs

Organ system

Site

References

Alimentary

Salivary glands

[2]

Esophagus

[2]

Liver

[2,19,71]

Intestines

[2]

Cardiovascular

Aorta

[2]

Heart

/pericardium

[2,19,82]

Central nervous system

Brain

[2,19]

Spinal cord

[19]

Eye

[82]

Endocrine

Thyroid

[82]

Adrenal

[2,21,82]

Pancreas

[82]

Genitourinary

Uterus

[2,3]

Prostate

[2,19,82]

Urethra

[2,3,19]

Ureter

[2,3,19]

Ovary

[2,19]

Vagina

[2]

Mammary gland

[19]

Musculoskeletal

Rib

[2,21]

Vertebrae

[2,19,71]

Humerus

[2]

Femur

[83]

Skull

[81]

Respiratory

Lungs

[2,3,19,21,71,80,82]

Reticuloendothelial

Sublumbar lymph nodes

[2,3,19,80]

Other lymph nodes

[2,19,71,82]

Spleen

[2,3,71,80]

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bladder tumors in 11 dogs, survival times ranged from 2 to 48 months and
the 1-year survival rate was 54.5% (6 of 11 dogs) [42]. The dog with the
longest survival had TCC treated with partial cystectomy on two diﬀerent
occasions 2 years apart and eventually was euthanized elsewhere for a pre-
sumed gastric torsion. One important ﬁnding in the study was that visual
assessment at the time of surgery was inaccurate for determining tumor-free
margins. Five dogs thought to have tumor-free margins based on gross
appearance of the tissue at the time of surgery subsequently were found to
have had incomplete excision when the tissue was examined by histopa-
thology. Therefore, if intraoperative evaluation of surgical margins by way
cytology or frozen section is not available, margins should be taken as
generously as possible.

Even with complete tumor excision, TCC recurrence is likely in dogs, as it

is in people following partial cystectomy. A review of the Purdue Compar-
ative Oncology Program Tumor Registry indicated a 106-day median
survival time for 42 dogs treated with surgery alone for localized TCC [43].
Recurrence rates of 30% to nearly 50% have been reported for people
undergoing partial or radical cystectomy, and the recurrence usually occurs
within 1 year [44,45]. This may be caused by microscopic tumors at the
surgical margins, or development of de novo tumors because of ﬁeld car-
cinogenesis. Because the etiology of bladder tumors is likely to involve
exposure of the bladder mucosa to carcinogenic substances, it has been
hypothesized that de novo tumors develop in the remaining nonexcised
bladder mucosa as a result of the same carcinogenic mechanisms that led to
initial tumor development, rather than a failure of surgical technique [42].

Permanent cystostomy catheter

Urinary outﬂow obstruction associated with bladder TCC may be

managed by placement of a permanent cystostomy catheter. This palliative
procedure is intended to relieve stranguria and to prevent secondary com-
plications associated with urinary outﬂow obstruction. Because ureteral
obstruction is not relieved by this procedure, excretory urography is recom-
mended before cystostomy catheter placement to ensure that palliation is
likely. In one report of cystostomy tube placement in seven dogs with known
or suspected TCC, six dogs had resolution of stranguria, and owners
reported satisfaction with the procedure [46]. Median survival time for these
dogs was 106 days. Cystostomy catheter placement predisposes dogs to
urinary tract infections, a complication noted in four of the seven dogs.
Therefore, periodic urinalysis and culture may be needed.

Ureterocolonic anastomosis

Although ureterocolonic anastomosis is dismissed by many clinicians as

being a labor-intensive procedure and impractical for most owners to
manage, it is possible to achieve excellent palliation of urinary symptoms
with this technique [47]. The procedure entails complete bladder resection,

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followed by end-to-side anastomosis of the ureters to the colonic mucosa.
Postoperative complications may include hyperchloremic acidosis and
related blood gas and electrolyte abnormalities, hydronephrosis, hydro-
ureter, obstruction at the anastomosis site, and pyelonephritis [40]. Owners
must be prepared to administer long-term antibiotics, adhere to recommen-
dations for dietary modiﬁcations for their pet including low-salt, low protein
diets with sodium bicarbonate supplementation, allow for frequent blood gas
and electrolyte monitoring, and be able to allow frequent access (every
4 hours) to the outdoors for urination. Despite these requirements, good
quality of life, urinary and fecal continence, and survival times up to 7 months
have been reported for dogs appropriately chosen for this surgery [40,47].

Bladder reconstruction

One relatively unexplored surgical option for TCC of the bladder in dogs

is radical cystectomy followed by bladder reconstruction. The author has
managed one case treated with surgical resection and use of a small in-
testinal submucosa (SIS) graft (Vet BioSISt; Cook Veterinary Products,
Bloomington, IN) to repair the bladder defect. Small intestinal submucosa
is an acellular biodegradable collagen-based material that is derived from
swine small intestines. The material has the ability to regenerate multi-
ple tissues, including ligaments, tendons, abdominal wall, skin, aorta, vena
cava, bladder transitional epithelium, smooth muscle, and peripheral nerves
[48–51]. The dog in the aforementioned case was treated in the adjuvant
setting with chemotherapy and a nonsteroidal anti-inﬂammatory agent and
had a survival time of 414 days. The ultrasonographic appearance of the
graft changed from hyperechoic to hypoechoic with time and eventually
resembled normal bladder wall thickness. The utility of SIS for regenerative
urinary bladder augmentation has been demonstrated in the preclinical
setting in normal dogs [48–50]. The ability of SIS to sustain or even en-
courage tumor growth at sites of incomplete excision, however, is a potential
risk that must be evaluated before the routine use of this product for
reconstruction of bladders with residual tumor cells after resection.

Medical therapy or chemotherapy for transitional cell carcinoma

Given its high rates of recurrence and metastasis, TCC of the canine

bladder is likely to require systemic chemotherapy from the outset or as
adjuvant therapy after surgery if one is to achieve long-term remissions or
cures. Several drugs and combination protocols have been evaluated for the
treatment of canine TCC.

Doxorubicin in combination therapy

Doxorubicin has shown signiﬁcant survival beneﬁt when used in

combination protocols to treat human bladder cancer in the neoadjuvant
setting [52]. It has not been widely used in veterinary medicine for this

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application, however. In one retrospective study of dogs with TCC,
doxorubicin and cyclophosphamide combination therapy was compared
with intravesicular thiotepa or surgery alone [53]. The doxorubicin and
cyclophosphamide combination provided a median survival time of 259
days, compared with 86 days with surgery alone and 57 days with intra-
vesicular thiotepa. Although no other reports have evaluated this protocol
critically for treatment of canine TCC, the overall survival times in the study
compare favorably to those obtained using other protocols. A retrospective
review of 25 dogs with unresectable urinary bladder carcinoma suggested
a survival advantage might exist when dogs receive an anthracycline drug
(doxorubicin or mitoxantrone) in addition to a platinum-based (cisplatin or
carboplatin) chemotherapy protocol, compared with those receiving only
a platinum compound [7]. Although this was not a randomized, prospective
trial, median survival for dogs in the platinum-only group was 132 days,
compared with 358 days for the anthracycline and platinum combination
group. In light of these preliminary results and the favorable responses in
human muscle-invasive bladder cancer to protocols containing methotrex-
ate, vinblastine, doxorubicin, and cisplatin (M-VAC), similar combination
protocols may warrant prospective investigation in dogs with TCC of the
bladder [52].

Piroxicam

The nonsteroidal anti-inﬂammatory (NSAID) piroxicam has shown

promise for the treatment of TCC, as monotherapy and in combination
protocols. Expression of cyclooxygenase-2 (COX-2) in canine bladder TCC
tissue, but not in normal bladder epithelium, suggests that COX-2 inhibition
by piroxicam may play a key role as a therapeutic adjuvant [54]. A recent
report, however, showed no association between tumor COX-2 expression
and response to piroxicam [55]. In vitro work has not shown evidence for
a direct cytotoxic eﬀect of piroxicam against canine TCC cell lines [56].
Despite the fact that its mechanism of action is not entirely clear, in a pro-
spective clinical trial, piroxicam provided responses in 6 of 34 (17%) dogs
with TCC for a median of 7 months [57]. Reports in the human oncology
literature have documented the chemopreventative and antitumor activity of
COX-2 inhibitors against bladder cancer, colorectal cancer, and other
carcinomas [58–67]. Piroxicam is administered at 0.3 mg

/kg daily by mouth.

As with other NSAIDs, adverse eﬀects may include gastrointestinal
irritation and nephrotoxicity. The prostaglandin analog misoprostol may
be used concurrently to protect against gastric ulceration.

Cisplatin

Despite its use to treat invasive bladder cancer in people and its apparent

cytotoxicity against canine transitional cell carcinoma in vitro [56], cisplatin
has been a disappointing treatment for canine TCC. Response rates have
been less than 25%, with median survival times of 6 months or less

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[16,17,43,68]. In a trial comparing single-agent cisplatin with the combina-
tion of cisplatin and piroxicam, none of the dogs that received cisplatin
alone experienced remission [43]. The combination resulted in a better
response rate than that seen with cisplatin monotherapy, but renal toxicity
was frequent (12 of 14 dogs, 87%) and dose limiting. Although the 71%
response rate attained with cisplatin and piroxicam therapy is promising,
unless dosage modiﬁcations permit less toxicity while maintaining eﬃcacy,
the author does not recommend use of these drugs together in clinical
practice.

Carboplatin

Despite its expense, carboplatin is considered an attractive alternative to

cisplatin for chemotherapy in dogs at risk of renal dysfunction and when the
6-hour diuresis protocol used with cisplatin is impractical. Two initial phase
II clinical trials of carboplatin in dogs with various tumor types reported
remissions in two of three dogs with TCC and 2 of 12 dogs with various
tumors types [69,70]. A prospective trial of 14 dogs with histologically-
proven bladder TCC treated with 300 mg

carboplatin every 3 weeks

resulted in no remissions [71]. A combination protocol using carboplatin
and piroxicam was evaluated subsequently and provided ﬁve partial re-
missions in 13 dogs [72]. Nephrotoxicity was not noted as a treatment
complication with this protocol. The 38% remission rate was an im-
provement over the lack of remissions reported with carboplatin single-
agent therapy [71]. Researchers noted that the median survival time of the
dogs treated with the combination protocol (93 days) was not better than
that achieved with either carboplatin (132 days) or piroxicam (180 days)
alone [72]. Most of the dogs (10 of 14) that failed carboplatin single-agent
therapy in the initial report went on to receive additional therapy
(piroxicam, mitoxantrone, or actinomycin D). For the dogs that received
additional therapy, median survival time was 204 days, compared with 25
days for those that did not. Accordingly, it is diﬃcult to compare results
beyond the initial response rates between the two studies.

Mitoxantrone

The anthracenedione mitoxantrone has known antitumor activity against

bladder cancer in people and dogs [7,73,74]. Because the toxicity proﬁle of
mitoxantrone does not include nephrotoxicity, one would anticipate that its
use in combination protocols with NSAIDs would be less likely to pre-
cipitate renal disease in dogs than would protocols featuring platinum
compounds. Results of a prospective study evaluating the tolerance and
eﬃcacy of mitoxantrone and piroxicam combination therapy for treatment
of canine bladder TCC have been determined subsequently [12]. Fifty-
ﬁve dogs were enrolled in the trial. The protocol included mitoxantrone
(5 mg

intravenously every 21 days) for four treatments and piroxicam

(0.3 mg

/kg per day) for the study duration. Tumor staging was performed

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at baseline, on day 42, and every 3 months after protocol completion.
Endpoints included time to treatment failure (TTF) and survival time (ST).
Data regarding response to therapy were available for 48 dogs and included
one complete response, 16 partial responses, 22 with disease stabilization
and nine with progressive disease, for an overall 35.4% measurable response
rate. Subjective clinical improvement as assessed by owners occurred in 75%
of treated dogs. Gastrointestinal toxicity and azotemia were the most com-
mon treatment complications, occurring in 18% and 10% of treated dogs,
respectively. The median TTF was 194 days, and the median ST was
350 days. Overall, the protocol was tolerated well and induced remission
more frequently than either drug as a single agent. This is the protocol used
by the author for initial therapy of canine TCC.

Radiation therapy

Radiation therapy, both intraoperative and postoperative, has been

described for the treatment of bladder TCC in dogs [1,75,76,84]. In one
report of 16 dogs treated with intraoperative radiation therapy, seven had
TCC of the bladder and were treated with total intraoperative electron doses
of 1000 cGy to 3160 cGy using a linear accelerator with 6 mV electrons [76].
Five of the seven dogs subsequently underwent postoperative radiation.
Survival times ranged from 1 month to 13 months. When ureters were included
in the intraoperative radiation ﬁelds, they tended to stenose and become
ﬁbrotic, leading to secondary hydroureter and hydronephrosis. Likewise,
ﬁelds encompassing most of the urinary bladder led to bladder ﬁbrosis, non-
distensible bladder tissue by 1 to 2 months, and urinary incontinence. These
results were inferior to an earlier report of intraoperative radiotherapy using
a

137

Cs teletherapy unit for 13 dogs with bladder neoplasia [75]. Although the

report included various bladder tumors, 11 of the 13 were TCC. The median
survival for dogs with TCC was 15 months and their 1-year, 18-month, and 2-
year survival rates were 54%, 27%, and 9%, respectively. A 46% recurrence
rate was noted, as were treatment complications including incontinence (46%
of cases), cystitis (38% of cases), and stranguria (15% of cases). None of the
dogs in the early report received external beam radiation after intraoperative
therapy, but the surgical excisions were more complete than those of the
subsequent report by Withrow et al. In a 1992 review, nine dogs with TCC
were treated with radiation and surgery [1]. Seven of the dogs had TCC of the
bladder; one had bladder and urethral TCC, and one had urethral
involvement only. Total radiation doses ranged from 0 cGy to 3000 cGy
intraoperatively and from 0 cGy to 4800 cGy as subsequent external beam
radiation. When all nine dogs with TCC were evaluated, the overall survival
times ranged from 30 days to 630 days, with a median survival of 105 days. All
dogs died of tumor-related causes. Treatment complications included in-
continence and bladder ﬁbrosis in six dogs. Based on these three reports,
a clear beneﬁt of external beam radiation over intraoperative radiation alone

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has not been demonstrated. Experimental bladder irradiation in normal dogs
and reported clinical experiences suggest that the total radiation dose
delivered to the canine ureters should be less than 3000 cGy to minimize the
likelihood of ureteral ﬁbrosis [75–77].

Combined radiation and cisplatin chemotherapy has been reported for

two dogs with bladder TCC. Both dogs were treated with Cobalt-60
teletherapy, which was fractionated using 400 cGy per fraction for total
doses of 4400 cGy and 4800 cGy. Intra-arterial cisplatin was administered at
a dose of 50 mg

, divided equally into three doses given 6 to 8 hours

before the ﬁrst three radiation fractions. Cisplatin was administered by the
same dose schedule again with the last three fractions, but was infused
intravenously, rather than by intra-arterial injection. Both dogs had
subjective clinical improvement and documented reduction in tumor size.
Survival times were 6 months and 7 months, respectively [68]. More re-
cently, the combination of mitoxantrone, piroxicam, and coarsely fraction-
ated radiation was evaluated in 10 dogs with TCC of the bladder [78]. The
protocol included six weekly 575 cGy fractions of radiation using a Cobalt-
60 unit, piroxicam (0.3 mg

/kg per day orally), and mitoxantrone (5 mg/m

intravenously every 21 days) until evidence of disease progression. Adverse
eﬀects were considered mild and consisted of dermatitis (n

¼ 1), hyper-

pigmentation (n

¼ 1), and mild urinary incontinence (n ¼ 4). No complete

or partial remissions were noted, but seven dogs had disease stabilization
and clinical improvement for 47 days to 320 days (median of 90 days). The
authors concluded that the protocol was tolerated well, and the severe
adverse eﬀects of bladder irradiation described in earlier reports were not
noted for these 10 dogs.

Photodynamic therapy

Photodynamic therapy (PDT) has been employed for treatment of bladder

cancer in people with variable results. Although an eﬀective protocol for PDT
of canine bladder cancer has not been established, investigations are ongoing.
A preclinical evaluation of bladder PDT using the prophotosensitizing agent
5-aminolevulinic acid, which is metabolized to protoporphyrin IX (PpIX)
after oral administration, demonstrated that PpIX ﬂuorescence was conﬁned
to the mucosa and spared the muscularis and serosa [79]. This is considered
a favorable ﬁnding, in that it will limit normal bladder tissue toxicity. Further
investigation of this treatment modality is warranted.

Summary

Canine TCC of the bladder is a disease for which early detection and

multimodality therapy are likely to produce the most favorable results.
Urine screening tests are being investigated as tools to permit earlier
detection. The possibility of tumor seeding must be considered when

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obtaining urine for analysis and when performing surgery. Because these
tumors tend to be very locally invasive at the time of diagnosis and are likely
to metastasize, cures are unlikely. Currently, combination protocols using
chemotherapy and the nonsteroidal anti-inﬂammatory agent piroxicam
show the most promise in producing tumor responses. Surgery and radi-
ation therapy are useful treatment modalities in select cases. Despite
advances in treatment of canine TCC, median survival times reported for
prospective clinical trials have never exceeded 1 year, regardless of the
treatment modality. Development of accurate tests for early tumor detection
could have a signiﬁcant impact on the success of treatment of this tumor in
canine patients.

Acknowledgments

The author would like to thank Ms. Allison Critchlow for her assistance

in preparing this manuscript.

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Multimodality therapy for head and neck

cancer

Mary K. Klein, DVM, MS

Southwest Veterinary Oncology, 141 East Fort Lowell Road, Tucson, AZ 85705, USA

For the purposes of this article, head and neck tumors include nasal

tumors, oral tumors, and tumors of the salivary glands, thyroid glands,
and ear canals. As a group, these tumors remain a treatment challenge in
both human and veterinary medicine. Generally, surgery is considered the
mainstay of treatment for head and neck tumors. Tumors that cannot be
completely resected and those associated with signiﬁcant metastatic poten-
tial are considered appropriate candidates for multimodality therapy.
Although there are now years of anecdotal experience in veterinary medicine
to indicate that multimodality approaches to these tumors increase control
rates, there are few studies to date that have accumulated enough cases to
make strong recommendations. The rate of development of distant metas-
tases can be reduced with systemic chemotherapy, but an overall eﬀect
on survival remains to be deﬁnitively shown. Those studies that are avail-
able are included in this review. In addition, several decades of informa-
tion accumulated through human clinical trials are summarized brieﬂy.

Nasal tumors

With the possible exception of extremely small well-deﬁned tumors,

surgery alone is not indicated in the treatment of nasal tumors. Most nasal
tumors are carcinomas or soft tissue sarcomas and are treated with radiation
therapy. Radiation therapy alone has increased reported median survival
times from the 3 to 7 months reported for surgery, chemotherapy, or im-
munotherapy [1–5] to 8 to 31 months [6–11]. Signiﬁcant variation exists
between protocols instituted and degree of staging employed. Elective
treatment of regional lymph nodes does not seem to be indicated, because

Vet Clin Small Anim

33 (2003) 615–628

E-mail address:

mkkl@mindspring.com

0195-5616

/03/$ - see front matter

doi:10.1016/S0195-5616(03)00018-4

regional spread of nasal tumors is unusual. All agree that there is un-
avoidable morbidity associated with the administration of conventional
external beam radiation therapy.

Side eﬀects of radiation therapy are usually separated into acute and late

eﬀects. Acute eﬀects are generally related to inﬂammatory reactions in the
tissues within the radiation ﬁeld. The temporary or permanent nature of
these eﬀects is usually related to dose and site. Late eﬀects develop over
several months to years but are irreversible and more likely to aﬀect the
patient’s quality of life. For example, ocular eﬀects that can be expected to
develop in the treatment of nasal tumors include cataracts and keratocon-
junctivitis sicca [12]. Although these side effects are quite acceptable for
most owners as the cost of controlling the tumor, they are discouraging
to deal with in the face of recurrent tumor within months of ﬁnishing
treatment. Improvements in survival times might make these side effects
more acceptable to owners, and minimizing side effects while increasing
expected survival duration would induce more owners to treat nasal tumors.

Modern imaging techniques, including CT and MRI, have dramatically

increased the accuracy of radiation therapy treatment planning for both
human and veterinary patients. Before the routine use of CT and MRI for
diagnosis and treatment planning, clinicians grossly underestimated the
extent of disease present in nasal tumor cases. It has been demonstrated that
using external landmarks without the beneﬁt of CT scans for radiation
planning would result in geographic misses in 90% of veterinary patients
[11,13]. Although the information gained from using these imaging
modalities usually increases the amount of tissue included in the treatment
ﬁeld, in general, the use of computerized treatment planning allows for
a decrease in the amount of normal tissue irradiated. Advanced imaging also
allows for more accurate evaluation of responses and their duration. The
greatest extent of regression seen on CT scans seems to occur 3 to 6 months
after the completion of treatment [14].

For external beam treatments, three-dimensional conformal radiation

and stereotactic radiotherapy are particularly exciting new areas. The
development of multileaf collimators and on-line portal imaging techniques
should make the delivery of three-dimensional radiation therapy more
eﬃcient. Intensity-modulated radiation is also being developed. All these
improvements decrease the amount of normal tissues irradiated and
improve the distribution of dose across tumor tissue. This should decrease
side eﬀects, but it remains to be seen whether these advances in radiation
planning and delivery translate into increased survival times with radiation
used as a single treatment modality. All these improvements have been
instituted in the treatment of most human patients. Unfortunately, only
small increases in tumor control have been noted, without a concomitant
positive impact on survival. Not until multimodality approaches were
instituted in human oncology were increases in survival documented [15].
It is important to note that only by improving the therapeutic ratio of

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radiation therapy with the technologic improvements listed previously were
radiation oncologists able to decrease morbidity sufﬁciently that the ad-
dition of other treatment modalities could be considered.

Most canine nasal tumor patients relapse in the mid- to caudal nasal

cavity. Boosting the dose there with conventional external beam radiother-
apy via shrinking ﬁelds added to morbidity but not to increased tumor
control in a previous study [16]. Although numbers were small in this study,
it is unlikely, given the human experience, that a signiﬁcant difference would
have been detected regardless of cohort size. Implementation of improve-
ments in treatment planning should allow for increased dose intensity to the
tumor and safe administration of multimodality approaches, including the
addition of surgery, radioprotectors, radiosensitizers, concurrent chemo-
therapy, immunotherapy, and targeted biologic therapies. The challenge
remains to ﬁnd a multimodality approach that successfully increases
survival times with acceptable morbidity. The radioprotector amifostine
has been shown to reduce the incidence of acute and chronic side effects in
human head and neck cancer patients treated with radiation and to allow
more patients to complete treatment without interruption [17]. The use of
radioprotectors, such as amifostine, has yet to be fully explored in veterinary
patients. Although surgery performed before megavoltage external beam
radiation therapy does not seem to inﬂuence outcome, the impact of surgery
after radiation therapy has not yet been assessed. Brachytherapy techniques
have also been successful in treating human head and neck tumors [15,84].
Because of the noncompliant nature of our patients, these techniques can be
difﬁcult to apply to veterinary head and neck cases. Several brachytherapy
techniques have been developed and successfully applied, however [18,19].

Rationale for chemoradiation therapy

The purpose of administering chemotherapy and radiotherapy together is

to take advantage of the radiosensitizing capability of many active
chemotherapeutic drugs for various tumor types and thereby increase
regional control rates as well as survival. Protracted radiation therapy as
a single modality treatment results in decreased local control rates, pre-
sumably because of accelerated repopulation of tumor cells surviving the
initial treatment [20]. The failure of induction chemotherapy to provide any
survival beneﬁt when compared with surgery or radiation alone in ran-
domized trials may have a similar cause. Signiﬁcant beneﬁts were not dem-
onstrated until chemotherapy was administered concurrently with the
radiation therapy. Administering cytotoxic drugs concurrent with radiation
has the potential to increase toxicity substantially, however, and necessitates
frequent interruptions in radiotherapy.

Mechanisms behind the synergy of chemoradiation therapy have been

postulated to include interference with sublethal damage repair, tumor cell
cycle synchronization, and prevention of the emergence of radioresistant or

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drug-resistant cells [21]. Cisplatin, carboplatin, paclitaxel, docetaxel, and
gemcitabine all have radiation-enhancing properties [15,22–24]. Cisplatin is
perhaps the most important chemotherapeutic agent for treating squamous
cell carcinoma of the head and neck in human patients [25] and is also the
only chemotherapeutic drug with signiﬁcant activity documented against
canine nasal carcinomas [26]. Carboplatin has signiﬁcantly less toxicity but
also lower response rates [27]. In human patients, ﬂuorouracil (5-FU) has
demonstrated synergy with cisplatin, leading to the establishment of the now
standard human treatment combination regimen of cisplatin plus 5-FU [28].
This combination has not been evaluated in canine patients. The optimal
schedule for radiosensitization has not been determined in human or
veterinary clinical trials. Nevertheless, the greatest survival beneﬁt observed
in most human studies is seen in the patient group receiving concurrent
cisplatin chemoradiation [29,30]. Because these trials have resulted in an
improvement in regional control that is profound enough to affect survival
by 20% to 30% on average, concurrent chemoradiation with cisplatin
is now considered the standard of care in human medicine [15]. Other
treatment modiﬁcations, such as altered fractionation with concomitant
boost or hyperfractionation with or without the addition of pre- or post-
radiation chemotherapy, only provided modest increases in regional control.
Interestingly, the longest survival times reported to date have resulted
from treating canine nasal tumors with external beam megavoltage radio-
therapy combined with the use of cisplatin as a slow-release formulation
likely to result in radiosensitizing doses [31]. This study yielded 1-year
survival rates of 81%, and the 2-year survival rate was 39%. These results
have not been duplicated using carboplatin as a radiosensitizer [32]. Con-
current chemotherapy with 5-FU

/cyclophosphamide or mitoxantrone or

preoperative surgery has not affected outcome in previous veterinary series,
but none of these compounds are documented to be good radiosensitizers
[6,7,9,33]. Phase I and II studies are in progress using radiation combined
with gemcitabine chemotherapy [35,36], but response rates are not yet
available.

It is important to note that increased toxicity, especially to mucous

membranes, was also documented in all these multimodality studies. Ag-
gressive support in the form of analgesics, oral care, and, on occasion,
gastrostomy tube placement is required, ideally at a treatment center
familiar with the expected severity of toxicity and potential complications.
Until we make improvements in limiting the morbidity associated with
chemoradiation, the biggest advantage is to patients with excellent per-
formance status and minimal tumor burden. Durable complete responses
and prolonged survival are probably possible in this small subset of patients,
based on the information gathered in human clinical trials [15]. In the
interim, new treatment modalities, such as immunotherapy, gene therapy
and biologically targeted compounds, should continue to be evaluated for
dogs with nasal sinus tumors.

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Oral tumors

Aggressive surgical techniques are well described in the literature and

allow for complete resection of a signiﬁcant percentage of oral tumors
[37,85,86]. The most common oral tumors in veterinary medicine include
dental tumors, ﬁbrosarcomas, melanomas, and squamous cell carcinomas.

Dental tumors

Common dental tumors in veterinary patients include epulides,

ameloblastoma, and other odontogenic tumors. Most dental tumors are
well controlled with surgery or radiation therapy even if they achieve fairly
large dimensions. Median survival times of 2 to 3 years are reported for both
surgery and radiation used as single modalities [38–43]. Except in the case of
extremely large tumors, single modality therapy is usually adequate.
Combination therapy should be reserved for large ameloblastomas that
are incompletely resected. These tumors should also be treated with radi-
ation therapy to minimize risk of recurrence.

Fibrosarcomas

Because of the low metastatic potential of oral ﬁbrosarcomas, most are

best treated by surgery wherever possible. Unresectable or incompletely
resected ﬁbrosarcomas require the application of multiple treatment mo-
dalities. Unfortunately, this situation occurs in a signiﬁcant percentage of
oral ﬁbrosarcoma cases.

In a summary of various papers on mandibulectomy or maxillectomy,

ﬁbrosarcomas were found to recur in greater than half of the cases, with
median survival times of approximately 11 months and 35% of the patients
alive at 1 year [37]. The administration of radiation therapy to those patients
with microscopic disease after resection seems to improve outcome, pro-
viding a median survival time of 540 days [44]. This is in contrast to
radiation alone, where control rates without surgical cytoreduction are
approximately 50% at 1 year [45]. Oral ﬁbrosarcomas had a statistically
signiﬁcant lower median survival time (540 days) when compared with
ﬁbrosarcomas located in other tumor sites (2270 days), indicating the
difﬁculty of effectively treating large portions of the oral cavity with high
doses of radiation therapy while avoiding unacceptable normal tissue
complications [44].

Radiation is usually delivered after surgery to dogs with oral ﬁbro-

sarcoma. There is evidence of a dose response. The human literature
indicates that patients who began radiation more than 6 weeks after surgery
and whose total therapy time extended beyond 12 to 13 weeks have worse
outcomes [15].

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Melanomas

Two important prognostic factors have been identiﬁed repeatedly in

studies of canine malignant melanoma: the size of the primary tumor
and the ability of the ﬁrst treatment intervention to control the disease
eﬀectively. Thus, any melanoma with a diameter larger than 2 cm is
considered to have signiﬁcant metastatic potential, and recurrence after
surgery is inevitably associated with aggressive behavior. These ﬁndings
should be interpreted as a caveat to treat aggressively the ﬁrst time. Patients
with small lesions (< 2 cm in diameter) that are completely resected have
a median survival time of 511 days in comparison to median survival times
of only 164 days for those patients with a tumor greater than 2 cm in diam-
eter or positive lymph node status [46,47].

Melanomas are also responsive to coarsely fractionated radiation therapy

[48–51]. Complete response rates ranging from 53% to 69% have been
reported, with overall median survival times ranging from 5 to 9.5 months.
Again, the size of the primary tumor is found to inﬂuence the survival time.
Dogs with less than stage II disease (primary tumor <2 cm in diameter) have
reported survival times ranging from 14.9 to 20 months in comparison to 5 to
6 months for cases with higher stage disease. Distant disease is the usual cause
of death in malignant melanoma, particularly when large tumors or early
metastasis is present. This highlights the need for multimodality treatment in
melanoma. Early studies with hyperthermia added to radiation therapy
demonstrated extremely high response rates; however, these response rates did
not translate into increased survival times, and hyperthermia is not routinely
available [52,53]. The addition of radiation therapy to surgery for malignant
melanomas is unlikely to increase overall survival times, because distant
disease is the most common cause of death in these cases [49,50].

Based on early studies indicating objective responses in measurable

melanomas, carboplatin has been added to the treatment regimen of ma-
lignant melanomas at many institutions across the country [54]. Results
from those studies are just beginning to be presented and must be cautiously
interpreted, because the data are not yet mature and there is substantial
variability in the protocols employed. One early study indicates that survival
time increased for all stages of malignant melanoma when chemotherapy
was added to the treatment regimen but only achieved statistical signiﬁcance
for dogs with stage III disease [50]. Large multi-institutional trials are re-
quired to elucidate fully any beneﬁt gained through the addition of chemo-
therapy to the treatment of canine oral melanoma.

Immunotherapy also shows promise in the treatment of oral melanomas. A

randomized study of 98 dogs treated surgically or by surgery in con-
junction with liposome muramyl tripeptide immunotherapy (L-MTP) showed
that those dogs with tumors less than 2 cm in diameter and lymph node
positivity had an 80% survival rate 2 years later in comparison to only
25% in the surgery alone arm. Unfortunately, L-MTP did not positively

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alter survival durations in those cases with larger tumors or metastatic
disease [55]. In vivo transfections of established tumors with immunostim-
ulatory genes can elicit antitumor activity and have been demonstrated
to induce complete and sustained local regression of large tumor burdens
in some canine melanoma patients [56]. Studies are currently underway to
combine immunotherapy with surgical resection. Further indication of the
role of immunotherapy in the manipulation of oral melanomas was pro-
vided by a phase I trial of human tyrosinase DNA vaccination [57]. As
these studies mature, adjuvant immunotherapy may replace the use of che-
motherapy in these tumors.

Osteosarcomas

Oral osteosarcoma is another tumor that challenges both local control

and the control of distant disease. With the possible exception of man-
dibular osteosarcoma, where surgery alone may prove curative [58], oral
osteosarcomas require both surgery to control the primary site and chemo-
therapy to address distant disease. In those animals in which complete
resection is not possible, it is logical to add deﬁnitive radiation therapy
to increase control rates. There are limited published data available to
provide information on the impact of adding adjunctive radiation on
local control rates or survival times other than evidence of activity in
vertebral tumors, however [59]. As for dogs with melanoma, the survival of
most of these animals is limited by the development of distant disease and
would not be expected to improve unless systemic therapy is combined with
effective local control.

Canine oral squamous cell carcinomas

The prognosis for these tumors seems to be quite site speciﬁc, with

tumors in the rostral oral cavity curable by surgery [37,60] or radiation
therapy alone [61]. Those of the caudal oral cavity, including the tonsil and
base of the tongue, are highly metastatic, and a multimodality approach is
indicated. A radiation dose response has been documented for these tumors,
with 1-year control rates of 46% in those cases receiving greater than 40 Gy
[61]. Median disease-free intervals of approximately 1 year are recorded
in response to radiation alone, with doses ranging from 38.5 to 57 Gy in
a variety of schema. Negative prognostic factors for survival include ad-
vanced age of the patient, caudal oral location, larger radiation portal size
requirement, and recurrent disease [62,63]. The addition of hyperthermia
increases control rates signiﬁcantly, but this modality is not routinely
available [53,64]. The addition of chemotherapy to treatment regimens for
canine oral squamous cell carcinoma of the caudal mandible or maxilla,
whether as an induction agent or concurrent radiosensitizer or in the

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adjuvant setting, awaits further study. Tonsillar squamous cell carcinomas
seem to be favorably affected by a multimodality approach. In a small
series of eight cases treated with radiation alone, median survival times of
110 days were reported [65]. Local recurrence was noted in only two of
the eight cases, but distant disease developed in ﬁve of the eight cases.
When surgery, radiation therapy, and chemotherapy were applied to a
similar patient population, the median survival time was increased to 270
days [66].

Feline oral squamous cell carcinomas have poor outcomes and remain

a therapeutic challenge. Patients left untreated or treated with any single
treatment modality have survival expectations of less than 3 months [67].
In those few cats with mandibular tumors that are amenable to surgical
resection followed by radiation, a median disease-free interval of 11 months
has been reported. Most of these cats suffered local recurrence [68]. The use
of etanidazole as a radiosensitizer resulted in a median survival time of 116
days [69]. Clearly, treatment of feline oral squamous cell carcinoma is an
area warranting further investigation.

Salivary gland tumors

Primary salivary gland neoplasias are rare in the cat and dog. Many

patients present with extracapsular extension of tumor, and the numerous
vital structures in close proximity to the salivary glands make aggressive
surgical removal diﬃcult. Incomplete removal invariably results in local
recurrence [70]. The addition of radiation therapy to surgical excision seems
to increase control rates and survival times [71,72]. Median survival times of
550 days for dogs and 516 days for cats have been reported with the addition
of radiation therapy. Radiation signiﬁcantly affected outcome in these cases,
but the role of chemotherapy remains to be deﬁned. Over half of the cats
presented with more advanced stages of primary tumor had metastatic
disease at the time of diagnosis [72].

Ear canal tumors

Many ceruminous gland adenocarcinomas and squamous cell carcinomas

of the ear are amenable to surgical resection via total ear canal ablation with
or without bulla osteotomy [73–75]. Most dogs live longer than 2 years when
treated with surgery alone, and most cats live longer than 1 year when
treated with surgery alone. In animals with incomplete tumor resection, the
addition of adjuvant radiation therapy seems to be of potential beneﬁt [76].
Because the metastatic potential of aural tumors is generally low, ranging
from 5% to 15%, chemotherapy is unlikely to have a major role in the
treatment of these cases [73].

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Thyroid tumors

Eighty to ninety percent of canine thyroid tumors can be expected to be

malignant. Although smaller and freely moveable tumors are amenable to
long-term control by surgery alone [77], larger nonresectable tumors have
been shown to be responsive to external beam radiation therapy [78].
Progression-free survival rates were 80% at 1 year and 72% at 3 years in one
radiation study. It often took many months for responses to be evident.
Twenty-eight percent of these dogs developed distant disease. Dogs with
bilateral disease were found to be at increased risk for metastatic disease
[78]. Responses to doxorubicin and cisplatin chemotherapy have been doc-
umented in canine thyroid carcinoma cases [79,80]. Thus, chemotherapy
may also contribute to the multimodality treatment of canine thyroid
carcinomas in combination with radiation therapy or surgery for those
animals at increased risk of developing metastatic disease [81]. Multi-
modality approaches have been shown to beneﬁt human anaplastic thyroid
carcinoma patients [34].

Biologically targeted therapies

The molecular and cellular pathways involved in the unregulated cell

growth that leads to head and neck tumors are complex. As clinical
researchers learn more, we can expect the development of biologically
targeted therapies. Three targeted therapies in human head and neck tumors
show early promising results. These include treatment with epidermal
growth factor receptor (EGFr) antagonists and cyclin-dependent kinase
(cdk) inhibitors and the administration of replication competent adeno-
viruses. The EGFr is a transmembrane glycoprotein that is a member of
the tyrosine kinase growth factor receptor family. Activation of this proto-
oncogene results in overexpression of the receptor and has been dem-
onstrated to occur in more than 90% of human squamous cell head
and neck tumors. Several monoclonal antibodies directed against epitopes
on the EGFr are in clinical development, including the chimeric antibody
C225 [82]. Enhanced toxicity is noted when this chimeric antibody is com-
bined with a number of chemotherapy agents, including cisplatin, as well
as when it is combined with radiotherapy. Phase I clinical trials are under-
way in human squamous cell carcinoma patients [83].

Summary

The reﬁnement of radiation therapy techniques should result in a decrease

in morbidity in canine and feline nasal carcinoma patients and should
further allow for the addition of adjuvant therapies. Patients with large oral
tumors that are incompletely excised should have radiation therapy added
to their treatment regimen. Tumors with signiﬁcant metastatic potential,

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such as melanoma, should be considered for addition of chemotherapy.
Carboplatin has activity in melanomas and is being added at several
institutions, but trial results are not yet available. Chemoradiation has
become the treatment of choice for human head and neck squamous cell car-
cinomas but remains largely unexplored in veterinary medicine. Hopefully,
development of chemoradiation will beneﬁt feline squamous cell car-
cinoma patients, because current treatment regimens are largely ineﬀective.
Immunotherapy agents and targeted biologic therapeutics seem to hold
promise for the future.

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New chemotherapy agents in

veterinary medicine

Antony S. Moore, BVSc, MVSc

Barbara E. Kitchell, DVM, PhD

Section of Oncology and Harrington Oncology Program, Tufts University School of

Veterinary Medicine, 200 Westboro Road, North Grafton, MA 01536, USA

Veterinary Cancer Care Clinic, University of Illinois College of Veterinary Medicine,

1008 West Hazelwood Drive, Urbana, IL 61802, USA

Several agents have been developed in recent years for anticancer therapy.

This article discusses experience with older agents lomustine and strepto-
zocin and newer agents ifosfamide and gemcitabine.

Lomustine

Mechanisms of action

Lomustine (cyclohexylchloroethylnitrosourea, CCNU, Ceenu) is a nitro-

sourea that is hydroxylated intracellularly to form an alkylating molecule
that creates DNA–DNA and DNA to protein cross-linkages. The key site
for DNA alkylation appears to be the O-6 methyl group of guanine. In
addition, an isocyanate compound is generated that can inhibit DNA
polymerase, DNA ligase, glutathione reductase, and enzymes involved in
RNA synthesis and processing. Hepatic microsomal metabolic transfor-
mation is responsible for deactivation of lomustine, and enhanced micro-
somal activity, such as would be seen with concurrent phenobarbital
administration, leads to a decrease in therapeutic eﬃcacy. Hepatic deg-
radation occurs rapidly and completely in most species, but this has not
been explored in cats. The plasma half life in dogs after intravenous (IV)
injection is approximately 15 minutes, and lomustine is detectable in the

Vet Clin Small Anim

33 (2003) 629–649

* Corresponding author.
E-mail address:

antony.moore@tufts.edu

0195-5616

/03/$ - see front matter

doi:10.1016

/S0195-5616(03)00033-0

cerebrospinal ﬂuid (CSF) within 10 minutes, reaching a plasma: CSF ratio
of 1:3 within 30 minutes [1].

Lomustine is highly lipophilic, and it is completely absorbed by the oral

route of administration. Lipid solubility is responsible for distribution
across biologic membranes including the blood-brain barrier. In contrast
to other alkylating agents, lomustine enters cells through passive diﬀusion.
Because cellular transport is passive, reduced drug entry into cells is not
a mechanism of resistance. Enhanced repair of alkylation at the O-6 methyl
group of guanine by the enzyme guanine-O-6-transferase is associated with
drug resistance to lomustine. Increased levels of intracellular thiol may
function to inactivate alkylating agents including lomustine. Glutathione
transferases are important in the denitrosation inactivation of CCNU [2].

Activity in people

The major indications for use of lomustine in people are treatment of

brain tumors, lymphoma (Hodgkin’s and non-Hodgkin’s), melanoma, and
renal and pulmonary carcinomas.

Preclinical studies

In preclinical studies using beagles, the most consistent toxicities involved

the bone marrow, liver, kidneys, and gastrointestinal (GI) tract. At high
doses, lomustine caused leukopenia and anemia during treatment. If the
animals did not die of sepsis or disseminated intravascular coagulation
(DIC), recovery of bone marrow was seen. Thrombocytopenia appeared to
be delayed, but reversible. Single doses of 200 mg

and above were

associated with 50% mortality, while doses of 60 mg

and below were

associated with only mild hematological toxicity when lomustine was given
by the oral or intravenous route [3,4]. Daily oral dosing of 25 to 200 mg

per day for 14 days was associated with almost 100% mortality, but
13 mg

per day for 14 days was associated with reversible leukopenia and

thrombocytopenia and reversible hepatic damage [3]. Studies of autologous
bone marrow support for high-dose chemotherapy allowed doses of 300 to
400 mg

to be given without lethal bone marrow toxicity. At doses of 600

, however, gastrointestinal toxicity was dose-limiting [5].

Delayed hepatotoxicity resulted in elevations of serum transaminase and

alkaline phosphatase activity and increased bromsulfophthalein (BSP) re-
tention. There was an inverse relationship between the size of the lomustine
dosage and the time to development of hepatotoxicity as signaled by eleva-
tions in serum alanine aminotransferase (ALT) activity. Hepatotoxicity was
seen as late as 1 month after dosing was discontinued but appeared to be
reversible at the lowest dosages (13 mg

per day for 14 days). Oral doses

of 6 mg

or less for 14 days caused only mild hepatic fatty changes [3].

Further investigation using serial liver biopsies following a single oral

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/ Vet Clin Small Anim 33 (2003) 629–649

lomustine dose of approximately 100 mg

showed glycogen depletion and

increased serum alkaline phosphatase (ALP) activity, noted 3 to 4 weeks
after dosing and continuing for more than 3 months. Histologic changes
were persistent, although serum alanine transferase (ALT) activity returned
to normal within 3 to 4 months [6].

Renal toxicity was uncommon in dogs but was seen at dosages of 150 to

200 mg

per day. In monkeys, renal toxicity is unpredictable and ir-

reversible at high doses [3]. Gastrointestinal [GI] toxicity was only seen at
the very highest dosages [3].

There are no preclinical studies of lomustine in cats.

Clinical applications in dogs

Dosage

Several doses of lomustine have been reported in diﬀerent settings. The

largest case series recommend clinical dosing of lomustine at 90 mg

orally every 4 weeks, with a dose reduction to 70 mg

every 4 weeks if

severe leukopenia (neutropenia; less than 500 cells per lL) occurs. The
neutrophil nadir is seen 6 to 7 days after dosing. It may be prudent to
prophylactically administer oral broad-spectrum antibiotics such as tri-
methoprim-sulfa combinations during the expected nadir from 5 until 10
days after dosing. The drug should be discontinued if thrombocytopenia or
increased serum creatinine or ALT activity is documented at the time
a dosage is due.

A lomustine dose of 60 to 80 mg

given every 6 to 8 weeks also has

been promoted for treatment of brain tumors in dogs. Lomustine treatment
at a dose of 50 mg

every 3 weeks has been reported in abstract form for

treatment of cutaneous lymphoma in dogs [10,11].

Toxicities

Myelosuppression is the major dose-limiting toxicity when lomustine is

administered to dogs in the clinical setting. When lomustine was given at
a dose of 100 mg

, acute neutropenia was dose-limiting, and this dosage

was abandoned as too toxic [7]. Although neutropenia following lower doses
can often be severe (less than 500 neutrophils per lL), it is rarely associated
with sepsis, possibly because of the lack of GI toxicity seen at these clinical
dosages. As discussed previously, oral broad-spectrum antibiotics such as
trimethoprim-sulfa combinations may be added prophylactically during the
expected neutrophil nadir from 5 to 10 days after dosing.

Thrombocytopenia may be documented at the nadir (7 to 14 days), but

platelet counts usually return to normal by the time the next dosage is
scheduled. Thrombocytopenia that persists at the time of next dose
necessitates dose delay. Persistent thrombocytopenia may be cause to dis-
continue the drug. Continued administration of lomustine to even mildly
thrombocytopenic dogs may result in dramatic worsening, with delayed and

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prolonged (months) thrombocytopenia that progresses even after the drug is
discontinued.

Idiopathic fevers that were not associated with neutropenia have been

observed in dogs receiving lomustine for relapsed lymphoma and mast cell
tumors, but this does not seem to be a common toxicity [7,8]. Hepatotoxicity
is uncommon at clinical doses, but this can be severe and progressive. In
a series of 180 dogs that were considered at true risk for developing
hepatotoxicity (more than one treatment, serum chemistry proﬁle monitor-
ing performed, and no evidence of pretreatment liver disease), 12 dogs
developed hepatotoxicity. Toxicity occurred after 2 to 10 doses of lomustine,
and was associated with clinical signs in 11 of the 12 dogs. Clinical signs seen
most commonly were loss of appetite and weight loss. The average time to
developing clinical signs of liver disease was 10 weeks after the last dose was
administered. Seven of the 12 dogs died from liver disease at a median of 7
weeks after diagnosis, while four dogs improved clinically [9]. Therefore, it is
recommended that a serum chemistry proﬁle be obtained before each
treatment with lomustine and that the drug be discontinued in the event of
elevated serum ALT activity. As in preclinical studies, it appears that the
liver changes seen in tumor-bearing dogs may be reversible if the drug is
discontinued at a low cumulative dosage.

Renal toxicity appears to be less common than hepatotoxicity at

recommended clinical doses in the dog (Kristal, personal communication,
2002). One dog developed acute fatal renal failure after receiving six doses of
CCNU. On necropsy, the renal lesion was membranoproliferative glomer-
ulonephritis [7]. Although nephrotoxicity is uncommon, dogs should be
monitored for evidence of renal damage during therapy with CCNU.

Eﬃcacy

A preliminary report of lomustine given at a dosage of 60 to 80 mg

every 6 to 8 weeks documented measurable brain tumor regressions in dogs,
and possibly prolonged remission in dogs that were treated adjunctively
following surgery [10]. Although this information has doubtless resulted in
treatment of other dogs, there have been no further reports on the eﬃcacy of
lomustine for brain tumors.

Forty-three dogs received lomustine at doses between 90 and 100mg

by mouth as treatment for relapsed and resistant lymphoma. These dogs
had received a median of ﬁve chemotherapy drugs for a median of 180 days
before rescue therapy with lomustine [7]. The overall response rate was 28%,
with a median remission duration of 86 days. The complete response rate
was low, however. Only 7% of dogs had a median complete remission of 110
days.

In a group of seven dogs with cutaneous lymphoma, the complete re-

sponse rate was 100% to lomustine treatment at a dose of 50 mg

every 3

weeks, with remission lasting 2 to 15 months. Those with epitheliotropic T

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cell lymphoma (mycosis fungoids) that had surgical excision followed by
chemotherapy with lomustine had the longest survival times [11].

Nineteen dogs with measurable mast cell tumors of various grades were

treated with lomustine at a dose of 90 mg

every 3 weeks [8]. One dog had

a long complete response (14 months), and the tumors in seven dogs reduced
by more than 50% in size for a median of nearly 3 months. The acute dose
limiting toxicity was neutropenia; 41% of these dogs had a neutrophil count
of less than 1000 neutrophils per lL during lomustine therapy. Following
these studies, clinical experience with longer-term therapy in the adjuvant
setting revealed an apparent cumulative myelosuppression in dogs, which
primarily aﬀected platelet production. It was reasoned that a 3-week inter-
treatment interval was too short to allow for full marrow recovery. As
a result, it is recommended that a dose of 90 mg

be administered no more

frequently than every 4 weeks, and that a complete blood count (CBC),
including a platelet count, be performed before each treatment. If the
platelet count is below normal at the time the next dose is due, then CCNU
should be discontinued. Continued therapy can lead to a marked and pro-
gressive drop in platelet numbers that continues for months after the drug is
discontinued.

Similarly, the occurrence of hepatotoxicity, while uncommon in the

clinical setting, appears to be cumulative, and often irreversible if not
recognized early. A serum chemistry proﬁle that includes serum alanine
transferase activity should be performed before each treatment, and CCNU
should be discontinued if the levels are elevated above normal.

Clinical applications in cats

Dosage

The recommended dose of lomustine for cats is 50 to 60 mg

orally

every 5 to 6 weeks [12]. The drug is reformulated as 2.5 mg capsules and
administered to the nearest 2.5 mg based on body size. This diﬀerence in
recommended dosage and administration interval between cats and dogs
reﬂects more the more signiﬁcant, prolonged, and severe neutropenia seen in
cats treated with lomustine. It is thought that cats are more similar to people
than to dogs with regard to lomustine toxicity. In people, hematologic
toxicities are delayed (neutrophil nadir at 3 to 4 weeks,) and complete
hematologic recovery is not seen until 6 to 7 weeks after therapy [13].

Toxicities

Lomustine at the recommended dosage reported previously resulted in

a median neutrophil count at the nadir of less than 1000 neutrophils per lL
[12]. In that study, more than half the cats experienced a neutrophil nadir
14 days after treatment, but nadirs also were seen from 7 to 28 days after
treatment. In addition, the neutrophil nadirs were often prolonged, with
a median of 7 days, but up to 14 days in individual cats. Information

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regarding platelet counts was less reliable, but the nadir appeared to occur
14 to 21 days after treatment [12].

Because of the formulation of lomustine capsules at a minimum size of 10

mg, another investigator administered 10 mg every 3 weeks to each cat
regardless of body size. This approach to dosing resulted in a range of doses
from 32 to 59 mg

in the series of cats studied [14]. In that study,

myelosuppression was rare, occurring in only 4% of patients, but there was
a trend to more severe neutropenia at higher cumulative doses. The low
incidence of toxicities in this study indicates that optimum therapeutic dose
intensity may not have been achieved by the 10 mg per cat approach. When
treating cats with lomustine, reformulation to achieve a uniform dose of 50
to 60 mg

is recommended.

There are insuﬃcient data to comment on the risk of hepatotoxicity,

renal damage, and thrombocytopenia in cats. It is recommended that pa-
tients be monitored as described for dogs, until further data become
available.

Eﬃcacy

There have been no studies of the eﬃcacy of CCNU in the treatment of

feline tumors. The toxicity studies previously documented responses in cats
with lymphoma [14,12], mast cell tumor [12], ﬁbrosarcoma [14], and multiple
myeloma [14]. The relatively modest response rates seen in these pilot studies
mean that it is unlikely that lomustine will have a similar therapeutic proﬁle
for feline malignancies as that documented in dogs.

Ifosfamide and mesna

Mechanisms of action

Ifosfamide (Ifex) is an oxazaphosphorine nitrogen mustard that, like its

isomeric parent compound cyclophosphamide, has no intrinsic alkylating
activity until it is metabolized hepatically to an active form.

The alkylating eﬀect of the active metabolites causes DNA interstrand

cross linking that correlates to toxicity; areas of active transcription appear
to be the most vulnerable to damage. Ifosfamide requires hepatic P450
mixed function oxidase for metabolism to acrolein and an active compound.
It is therefore at least theoretically possible that hepatic dysfunction may
lead to reduced activation and therefore to reduced eﬃcacy.

Without accompanying urothelial protection, the renally excreted

metabolites of ifosfamide cause urothelial toxicity (hemorrhagic cystitis) in
all patients. The metabolites of ifosfamide also can cause renal damage on
rare occasions. Identiﬁcation of the urothelial damaging metabolites leads to
the subsequent discovery of the protective drug mesna (sodium 2-mercapto-
ethane sulfonate). When administered intravenously, mesna rapidly forms

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a disulﬁde, which is eliminated rapidly and completely through glomerular
ﬁltration. During excretion, the disulﬁde is reduced to mesna. Mesna
(Mesnex) acts as an active thiol in the urinary tract to scavenge the urothelial
metabolites of the alkylating agent, thereby protecting the urinary system
epithelium.

Activity in people

Ifosfamide is one of the most active chemotherapeutics in the treatment

of soft tissue sarcomas and osteosarcomas in humans. Additionally,
ifosfamide has signiﬁcant activity in the treatment of urothelial transitional
cell carcinoma; germ cell tumors; pulmonary, ovarian, and breast car-
cinomas; and lymphoma. Ifosfamide has been demonstrated to have
superior antineoplastic activity when compared with cyclophosphamide in
many tumors in people.

Preclinical studies

Administration of ifosfamide at a dosage of 300 mg

daily for 4 days

resulted in the septic death of one dog at the neutrophil nadir on day 8;
however, three dogs survived [15]. Administration of a higher dosage (450
mg

daily for 4 days) to the remaining dogs resulted in sepsis in all

animals; however, all dogs recovered with supportive care. Continued
administration of the higher dosage at intervals of 3 weeks resulted in a 50%
mortality caused by sepsis. No urothelial toxicity was seen; all dogs received
mesna at a daily dose of 1.5 times the dose of ifosfamide and a further two
doses 8 and 16 hours after the last treatment. Ifosfamide was delivered in 1 L
of lactated Ringer’s solution over 90 minutes.

In separate dog studies, the highest nontoxic dose for ifosfamide given

daily for 5 days was 4.12 mg

/kg per day [16]. At a dose of 8.25 mg/kg per day

for 5 days, an intertreatment break of 9 days was insuﬃcient to allow full
hematological recovery, as evidenced by worsening leukopenia with
subsequent cycles of therapy [16]. Cystitis was common in dogs treated
every 3 weeks at a dosage of 10 to 20 mg

/kg for 6 months. These dogs did

not receive mesna. Leukopenia was characterized as moderate, although
some dogs developed pneumonia. Ifosfamide had suﬃcient activity when
given orally at a dose of 4.64 mg

/kg per day, 6 days a week for 26 weeks, to

result in marked neutropenia in most treated dogs [16].

Preclinical studies of mesna in the dog showed that single doses of 200

/kg and higher caused bradycardia and decreased blood pressure, and

death was seen at doses of 400 mg

/kg and above. When given IV daily for

6 weeks, intermittent vomiting and diarrhea was seen at doses between 100
and 300 mg

/kg. Oral dosing up to 2000 mg/kg had no eﬀect on dogs. These

studies emphasize the safety of mesna at the much lower, clinically relevant

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doses. The lack of interaction between mesna and ifosfamide activity (with
the exception of urothelial toxicity) has been conﬁrmed in dogs [17].

In the cat, ifosfamide administered at 100 mg

/kg IV did not result in

anaphylaxis [16], and mesna caused no adverse eﬀects up to the tested
dosage of 100 mg

/kg [17].

Clinical applications in dogs

Dosage

The recommended dosage of ifosfamide in dogs is 350 to 375 mg

IV every 2 to 3 weeks. The drug must be given with IV diuresis and mesna.
Mesna is administered at 20% of the ifosfamide dose, before treatment.
Diuresis is initiated at 18.3 mL

/kg per hour for 30 minutes, and then the

ifosfamide is given at the same ﬂuid rate over a further 30 minutes. Diuresis
is continued at the same rate for an additional 5 hours. Two additional
doses of mesna (20% of the ifosfamide dose each time) are given 2 and 5
hours after the ifosfamide infusion is completed during the post-treatment
diuresis phase.

Note: Mesna (Mesnex) is supplied with Ifex in the same package, and

does not have to be purchased as a separate drug.

Toxicities

A total of 134 doses of ifosfamide were given to 72 dogs in one study [18].

A dosage of 375 mg

was given to 37 evaluable dogs. Six dogs (16%)

developed neutropenia of less than 1000 cells

/lL, and three dogs developed

sepsis. Renal toxicity does not appear to be a substantial risk with this
protocol. Anecdotally, attempts to increase the dosage of ifosfamide to 400
mg

have failed because of severe neutropenia (Rassnick and Moore,

unpublished data).

When given according to the protocol described previously, no dog

reported in this study, nor any dogs treated subsequently by the author,
has developed hemorrhagic cystitis following ifosfamide administration.
A preclinical trial that did not use diuresis, but did administer mesna, also
did not result in hemorrhagic cystitis [15]. There have been no reported
GI toxicities reported in dogs treated with ifosfamide.

Eﬃcacy

In one study, complete responses were documented in 2 of 13 dogs with

measurable sarcomas. In this report, a dog with leiomyosarcoma had
a response of longer than 549 days, and a dog with hemangiosarcoma had
a response of longer than 445 days. Only 1 of 40 dogs treated for resistant
lymphoma was noted to have a partial response lasting 112 days. Seven dogs
with stage II splenic hemangiosarcoma lived a median of 147 days, which

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was not statistically diﬀerent from the survival of 86 days noted with surgery
alone.

The antitumor activity of ifosfamide in dogs does not appear to be as

high as it is in people with cancer. The reasons for this lack of eﬃcacy are
not clear.

Clinical applications in cats

Dosage

Clinical dosing of ifosfamide in cats is being investigated. From

preliminary work, it appears that cats tolerate higher ifosfamide dosages
than do dogs, possibly doses as high as 900 mg

when given with diuresis

as outlined previously (Rassnick, personal communication). The reader is
encouraged to conﬁrm this dosage before administering the drug to cats.

Toxicities

Myelosuppression appears to be the major dose-limiting toxicity in cats,

but this observation needs to be conﬁrmed through further studies. Other
toxicities seen include GI toxicity, probable idiosyncratic hypersensitivity
(swollen face and puﬀy eyes) in one cat, and renal failure in two cats, one of
which had pre-existing chronic renal failure. Ifosfamide is a nephrotoxic drug,
and careful case selection and monitoring of renal function are mandatory
before administration to cats (Rassnick, personal communication).

Eﬃcacy

Responses have been seen in cats with lymphoma and in cats treated for

soft tissue sarcomas. Therefore, ifosfamide and mesna may prove useful for
the treatment of sarcomas in cats (Rassnick, personal communication),
although further study is needed.

Streptozocin

Mechanisms of action

Streptozocin (streptozotocin, Zanosar) is a methylnitrosourea alkylating

agent that causes interstrand cross-linking in DNA. It is excreted primarily
in the urine. Streptozocin is directly cytotoxic to pancreatic beta cells. When
given by IV, streptozocin metabolism is rapid. Degradation occurs at an
estimated rate of 5 mg per minute in dogs [19], and the half-life in people is
15 minutes [20]. The drug is retained in the liver of dogs for many hours
after blood levels are undetectable [19].

Activity in people

Streptozocin is used for the treatment of unresectable or metastatic

insulinoma. Other diseases in people that have responded to streptozocin

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treatment include carcinoid, Hodgkin’s disease, lymphocytic lymphoma,
acute lymphoblastic leukemia, and synovial cell sarcoma [21–23].

Preclinical studies

In preclinical studies in dogs, the LD

of streptozocin was found to be

1500 mg

[24]. In acute toxicity studies in dogs, the major toxicoses were

diabetes mellitus, which developed after IV administration of a single dose
of 700 mg

, renal tubular damage, weight loss, and reversible hepatic

injury [25,26]. Streptozocin was reported to be a powerful emetogen in dogs,
with vomiting occurring 45 to 90 minutes after administration [27]. Toxic
eﬀects of streptozocin are dose-dependent, with vomiting and increases in
serum hepatic enzyme activities occurring at lower dosages (400 mg

and acute renal tubular damage occurring at higher dosages (greater than
700 mg

) [25,26].

There are no preclinical studies in cats.

Clinical applications in dogs

Dosage

Clinical use of streptozocin in two dogs was reported to result in acute

renal failure and death. These reports were ﬂawed, in that the drug was
administered without appropriate renal support [28,29]. Induction of
diuresis through administration of 0.9% NaCl has been reported to
ameliorate the renal toxic eﬀects of streptozocin in canine patients. This
eﬀect is attributed to protection of renal tubular macromolecules from the
alkylating eﬀects of streptozocin by reduced contact time between the drug
and the renal tubular epithelium [30].

Intravenous diuresis, using 0.9% NaCl at a rate of 18.3 mL

/kg per hour,

was administered for 3 hours before streptozocin administration. The dose
of streptozocin (500 mg

) was diluted to the appropriate volume and

administered over the subsequent 2 hours at the same calculated ﬂuid rate,
and 0.9% NaCl was administered for an additional 2 hours at the same rate
after streptozocin administration was completed. Butorphanol (0.4 mg

/kg)

was administered intramuscularly (IM) as an antiemetic immediately after
streptozocin administration was completed. Protection against vomiting
provided by butorphanol is less than complete. Treatments are repeated at
3-week intervals [30].

Toxicities

The major toxicity associated with streptozocin treatment in dogs is

proximal renal tubular necrosis, which is dose-related and cumulative.
Renal failure may develop. Less common toxicoses include nausea and
vomiting, which may be severe, and increases in serum hepatic enzyme
activities. Bone marrow toxicity is rare.

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In one study, 58 treatments were administered to 17 dogs with pancreatic

islet cell tumors [30]. Two dogs developed diabetes mellitus after receiving
ﬁve doses of streptozotocin. Only one dog developed azotemia during
treatment. This dog had pre-existing renal disease. Serum ALT activity
increased in some dogs, but decreased again when treatment was
discontinued. Hematologic toxicoses were rare and mild. Vomiting during
administration was seen following approximately 30% of streptozocin
treatments, and one dog was withdrawn from treatment by its owners
because of severe vomiting after each treatment. The high incidence of
vomiting after streptozocin treatment occurred despite pretreatment with
butorphanol. Butorphanol acts centrally to reduce nausea and has been
shown to reduce the risk of vomiting after cisplatin chemotherapy in dogs
[31] but was ineﬀective in preventing streptozocin-associated vomiting. It is
possible that other antiemetics such as dolasetron or ondansetron may be
more eﬀective in preventing vomiting in dogs receiving streptozocin. Acute
post-treatment hypoglycemia has been reported, presumably as a result of
streptozocin-induced beta cell degranulation. Clinicians should be aware
of this potential complication and be prepared to administer glucose if signs
of hypoglycemia occur.

Eﬃcacy

There was no measurable response in one dog with a nonfunctional islet

cell tumor. Despite renal toxicity seen in early case reports, evidence of
antineoplastic activity was seen in two dogs with insulinoma [28,29]. In
a more recent study, median duration of normoglycemia for 14 dogs with
stage II or III insulinoma treated with streptozocin was 163 days. This
patient cohort included dogs that had shown signs of tumor recurrence after
surgery and medical management. The normoglycemic period seen in this
study was not signiﬁcantly diﬀerent from that for dogs treated with surgery
and medical management alone (90 days). Two dogs had rapid resolution of
paraneoplastic peripheral neuropathy within 2 weeks of starting treatment,
however, and two others had measurable reductions in tumor size [30].

Based on the ﬁndings of these two reports, it would appear that

streptozocin has a role in the treatment of metastatic insulinoma in dogs.
Additional studies are required to better deﬁne the therapeutic potential of
this agent.

Clinical applications in cats

Streptozotocin administration has not been reported in cats.

Gemcitabine

Gemcitabine (2,2-diﬂuorodeoxycytidine, dFdC, Gemzar) is a diﬂuori-

nated pyrimidine analog of deoxycytidine, synthesized as an analog of

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cytarabine. This drug was the ﬁrst compound licensed based on antitumor
eﬃcacy and on improved quality-of-life scores in the pivotal study of people
with pancreatic carcinoma. The drug was licensed for human use in the 1996
[32,33].

Mechanism of action

The synthetic design used to create gemcitabine substitutes the hydrogens

of the 2-carbon position of the deoxyribose moiety with two ﬂuorine atoms.
This confers on the drug its DNA-directed biological activities [34].
Gemcitabine, like other nucleoside analogs, is hydrophilic and cannot tra-
verse cell membranes by passive diﬀusion. The prodrug gemcitabine enters
cells through the activity of specialized transporter systems (facilitated
nucleoside transport). Phosphorylation of gemcitabine is essential to its
biologic activity. The prodrug is metabolized intracellularly to the 59-
monophosphate form (dFdCMP) by deoxycytidine kinase. This intermediate
compound subsequently is phosphorylated by nucleoside monophosphate
and diphosphate kinases to the active diphosphate (dFdCDP) and tri-
phosphate (dFdCTP) nucleosides, respectively [34-37]. Gemcitabine also
increases its own intracellular concentration by a positive feedback loop of
activation. The drug requires intracellular phosphorylation by deoxycytidine
kinases to achieve the activated triphosphate form. This deoxycytidine kinase
then blocks ribonucleotide reductase function, which depletes the normal
cytidine base. Thus, greater incorporation of the gemcitabine in the DNA
occurs, as fewer molecules of the normal base are available to compete for
insertion (Fig. 1) [34–37].

The cytotoxic eﬀect of gemcitabine is attributed to a combination of two

actions of the diphosphate and triphosphate nucleosides, which results in
inhibition of DNA synthesis [34–37]. Following the incorporation of
dFdCTP into DNA, one additional nucleotide molecule is added to the
elongating DNA strands. After this normal base addition, DNA polymerase
e is unable to remove the dFdCTP and repair the growing DNA strands.
This ‘‘masked chain termination’’ results in inhibition of DNA synthesis and
induction of apoptosis. Gemcitabine exhibits cell phase speciﬁcity, primarily
killing cells undergoing DNA synthesis (S-phase) and also blocking the
progression of cells through the G1

/S phase boundary. Resistance appears

to be caused by reduced nucleoside transport into the cells and also to
decreased activity of the enzyme deoxycytidine kinase [38].

Activity in people

Gemcitabine is licensed in human medicine for treatment of pancreatic

carcinoma and nonsmall cell lung cancer [32,39], but its utility in com-
bination with other drugs has resulted in the expansion of the drug’s clinical
indication to include treatment of other GI [40], genitourinary [41,42], and

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respiratory carcinomas [39]. Preclinical studies and phase II trials document
the potential utility of gemcitabine in the combination treatment of various
lymphomas and soft tissue and bone sarcomas [43–45]. The drug appears to
be synergistic with platinum agents [41,46], anthracyclines, and alkylating
drugs [46,47] and is a very potent radiosensitizer [48–50]. Signiﬁcantly
enhanced radiation injury to normal tissues in the radiation ﬁeld have
limited its use as a radiosensitizer in people, and substantial dose reductions
are required over what are used in systemic chemotherapy [48,49]. Human
dose de-escalation studies were required for the use of gemcitabine as
a radiosensitizer in head and neck cancer, because of deep ulceration and
esophageal stricture. The estimated maximum tolerated dose in combination
with radiation therapy in people is between 10 to 50 mg

, which is less

than 5% of the human systemic cytotoxic dose [50].

Preclinical studies

A wide dose range of gemcitabine was administered to normal beagles in

published preclinical studies (3 to 10 mg

/kg, or 500 mg/m

once weekly for 3

weeks) [51,52]. Dogs metabolize gemcitabine by a two-compartment model.
The drug is deaminated to the uracil metabolite and subsequently cleared
through the kidneys. Plasma protein binding is negligible, and the plasma
half-life in the dogs is approximately 1.38 hours. Approximately 76% to
86% of the dose is detected as the uracil metabolite in the urine within the
ﬁrst 24 hours of administration [50,51]. Administration of gemcitabine at

Fig. 1. Molecular structure olf gemcitabine and mechanism of activation (Courtesy of Dr L-P
de Lorimier, modiﬁed from Abbruzzese JL. New applications of gemcitabine and future
directions in the management of pancreatic cancer. Cancer 2002;95:941–5; with permission.)

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a dosage of 350 mg

administered as a 1-hour IV infusion to normal

healthy beagles resulted in minimal toxicity [53,54]. Administration of
1200 mg

as an IV bolus was the estimated toxic dose (Horton, personal

communication).

There are no preclinical studies in cats.

General clinical applications

The ﬁrst veterinary administration of gemcitabine occurred at the

University of Illinois shortly after the drug became commercially available.
Results of the initial pilot study were reported by Boyce [55]. Doses used in
this study were derived from the ‘‘no toxic eﬀect’’ dose in preclinical studies
conducted at Lilly (Englehart, personal communication). Thus, the dose for
dogs was 60 mg

IV once weekly for a maximum of ﬁve weekly treat-

ments; 45 mg

was administered once weekly for ﬁve treatments to cats.

All doses were given as a 20-minute infusion. Most patients had previously
failed standard chemotherapy protocols and were assessed to have
progressive disease. If the drug was found to be eﬃcacious for a given
patient after the ﬁrst cycle, the cycle was repeated after a 2-week rest period.
Tumor types treated included hepatocellular carcinoma, biliary carcinoma,
lymphosarcoma, cholangiocarcinoma, pancreatic carcinoma, mammary
carcinoma, and bronchoalveolar carcinoma. Stable disease with prolonged
survival over that seen in historical controls was reported for most cases
treated. Partial responses and one complete response were seen in a dog with
multicentric hepatocellular carcinoma. In this initial study, toxicity was
minimal, and no animals died from treatment-related causes [55].

Walter et al reported on the use of gemcitabine as a rescue agent for

recurrent or refractory canine lymphoma. A dose of 275 mg

was ad-

ministered to ﬁve dogs as a 30-minute infusion every 2 weeks for up to ﬁve
cycles. No complete or partial responses were observed [56].

Kisseberth et al reported the results of a dose escalation study of 17 dogs

conducted at The Ohio State University. Escalation was performed in
cohorts of three, with 50% escalation in the succeeding cohort if no
signiﬁcant toxicities were observed. Doses administered ranged from 300 to
675 mg

(109 doses in total) administered as a 30-minute IV infusion of

drug in saline at a rate of 1 mL

/kg. Thirteen of these dogs received four or

more treatments. GI toxicity was evaluated for the ﬁrst four treatments,
with toxicity scores assigned as follows: 11 grade I; 5 grade II; 0 grade III;
and 1 grade IV (gastric carcinoma) in 61 evaluable treatments. Only one
grade I episode of neutropenia was reported. No complete or partial tumor
responses were observed during the ﬁrst 8 weeks in this group of dogs, with
the best response being stable disease in nine dogs. One dog with thyroid
carcinoma suﬀered bilateral retinal hemorrhages, which resolved without
treatment, and dosing was continued. These authors concluded that gem-
citabine could be safely administered at 675 mg

at 14-day intervals with

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minimal gastrointestinal and hematological toxicity. Further escalations
were not performed because of the single incident of unexplained retinal
hemorrhage [57].

Marconato et al reported on 33 dogs treated for conﬁrmed transitional

cell carcinoma of the urinary bladder. All dogs in this group were treated
concurrently with piroxicam. Dosing started at 800 mg

IV once weekly,

with planned treatment to continue for at least seven cycles. Infusion du-
ration was not reported. Dose escalation was attempted in three dogs. The
median number of doses administered was eight, with a range of 1 to 27
doses. Nausea or vomiting occurred in 65% of the patients treated at the 800
mg

dose, but GI toxicity was typically grade I to II. Neutropenia was the

most common hematological adverse eﬀect (nadir 3 to 7 days), and no
febrile episodes or sepsis were observed. Thrombocytopenia and anemia
were rare. Clinical improvement of stranguria, pollakiuria, and hematuria
was reported for all treated dogs [58].

Gemcitabine is being investigated as a radiosensitizer for dogs and cats.

An abstract presented by Ladue and Klein summarized the interim report of
the Veterinary Radiation Therapy Oncology Group trial of gemcitabine as
a radiosensitizer in canine sinonasal carcinoma and feline oral squamous cell
carcinoma. Fifteen dogs were entered from two radiation facilities. Radi-
ation therapy was administered to an average dose of 50 Gy in Monday
through Friday fractions of 3 to 3.2 Gy per fraction. The average number of
gemcitabine doses was ﬁve per patient, with a mean dose of 40 mg

Twelve of 15 dogs required dose delay or reduction because of myelo-
suppression or local tissue toxicity in the radiation ﬁeld. Results for these
dogs were not encouraging, as the median local disease control interval was
only 8.6 months. Nine dogs died of progressive disease. Three dogs were
alive with no evidence of disease at the time of the report (2, 8, and
29 months). Two dogs died of unrelated disease, while one was disease-free
but lost to follow-up at 18 months [59].

Clinical applications in dogs

Dosage

The optimal dose regimen for use of gemcitabine in tumor-bearing dogs

has yet to be established. Despite the common clinical use of this agent in
oncology practice for the past decade, controversy still exists as to the ap-
propriate dose and schedule for people with cancer [60]. One problem
associated with establishing eﬃcacy and toxicity of gemcitabine in tumor-
bearing dogs is that its eﬀects are dose- and schedule-dependent [61]. Using
an identical dose, but prolonging the infusion of drug from an IV bolus
injection to a 30-minute or longer duration, commonly results in increased
myelosuppression (Kitchell, unpublished data). One could theorize that this
also could provide increased anticancer eﬃcacy. There are several critical
limiting factors to be considered in gemcitabine administration, however. As

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a relatively cell cycle phase-speciﬁc drug, with maximum eﬀect seen during S
phase, it is likely that continuous IV infusion over a long period would result
in increased exposure of S phase cells to the toxic eﬀects of the drug. This
eﬃcacy is initially dependent upon the presence of appropriate nucleoside
transporters for intracellular uptake of the parent compound, however.
Subsequently, adequate activity of deoxycytidine kinase (rate-limiting step)
and other kinases in the tumor target cells is required to ensure appropriate
phosphorylation to the active forms. The steps of uptake and activation are
saturable; therefore, rapid infusion of high doses possibly could result in
rapid renal elimination, with no appreciable therapeutic impact [62,63]. The
intracellular accumulation of the triphosphate form of gemcitabine occurs
rapidly after infusion, which results in incorporation of the active agent into
the growing DNA chain during S phase and subsequent chain termination.
The added cytotoxic impact of accumulation of gemcitabine triphosphate
within the cell, with resultant inhibition of deoxycytidine kinase and
ribonucleotide reductase activity, occurs only with prolonged intracellular
concentration of the triphosphate form, which is achieved best by con-
tinuous long-term exposure [64]. The conceptual problem in designing dose-
ﬁnding studies for gemcitabine is that dose escalation and duration of
infusion (‘‘time-escalation’’) should be incorporated. Dose and duration of
infusion are under investigation in many clinical trials in people [40].

The main clinical utility of gemcitabine is likely not as a single agent, but

rather as a synergistic combination chemotherapy drug, by human medical
oncologists. There is no consensus for gemcitabine single-agent therapy or
as use to enhance the cytotoxic eﬀect of radiation therapy. This is clearly the
direction taken by dosing regimes in veterinary oncology. It is even more
daunting to contemplate combination therapy. It is clear that this agent is
synergistic or supra-additive with platinum agents, anthracyclines and
alkylating drugs, however. There is limited experience with administration
of gemcitabine and cisplatin or carboplatin in patients with treatment-
refractory carcinomas.

Toxicities

Myelosuppression is dose-limiting. Nausea and vomiting are rare and

generally mild. Although rare, additional toxicities observed in people
include mucositis and alopecia, along with transient rises in transaminases.
Eighteen percent of 979 people treated with single-agent gemcitabine ex-
perienced transient inﬂuenza-like symptoms, and mild fever was observed in
37% of patients. Renal insuﬃciency of undetermined etiology was rarely
reported, and 20% of patients had mild peripheral edema in the absence of
cardiac, hepatic, or renal failure [65]. Although few adverse eﬀects have been
reported in dogs, it is reasonable to assume that additional toxicities will be
added to the canine list as dose optimization is achieved.

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Eﬃcacy

Thus far, anecdotal responses of a partial nature have been the best

recorded for gemcitabine as a single agent. In most cases, disease stabi-
lization is achieved for variable periods of time. At the University of Illinois,
the authors have had brief complete responses in two dogs treated with
gemcitabine as third and fourth line rescue for high-grade refractory
lymphomas, which suggest the potential for addition of this agent into
combination protocols. Randomized control trials need to be designed to
evaluate this potential.

One case of biopsy conﬁrmed that multicentric hepatocellular carcinoma

treated with gemcitabine as a single agent at relatively low doses had
apparent complete remission based on follow-up ultrasonography. This
remission was not biopsy conﬁrmed and thus is suspect, however. Individual
cases have had prolonged survival over what might be predicted based on
historical reports of diseases such as pancreatic carcinoma and transitional
cell carcinoma.

Indications for use of gemcitabine in dogs would be expected to parallel

those in human oncology. Thus, randomized controlled trials of treatment
in combination protocols for advanced GI, genitourinary, and pulmonary
cancers seems the logical starting point for further clinical research.

Clinical applications in cats

Dosage

Most doses reported for gemcitabine use in cats are the radiosensitizing

doses.

Reported single agent doses range from 45 mg

given as a 30-minute

saline infusion [55], to the current single agent dose in use at the University of
Illinois of 250 to 275 mg

(Kitchell, unpublished data). The radiosensitizing

dose reported for cats ranges from 21 mg

in conjunction with full course

radiotherapy [59], to 25 mg

administered twice weekly in conjunction with

palliative radiation therapy of 6 Gy per fraction for six treatments [66].

Toxicities

Myelosuppression has been seen in cats treated with gemcitabine alone or

in combination with radiation therapy or carboplatinum.

Eﬃcacy

Of ﬁve cats treated with single-agent gemcitabine at the University of

Illinois between 1996 and 1999, two had stable disease, two had progressive
disease, and one was nonevaluable. The dose range was 60 to 125 mg

for

this group of cats, with one cat receiving a cumulative dose of 1907 mg
during the period of disease stabilization (Kitchell, unpublished data).

Ladue and Klein reported on a group of 10 cats with oral squamous cell

carcinoma treated with curative intent radiation therapy with gemcitabine

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radiosensitization [59]. In this trial, radiation was delivered in Monday
through Friday fractions of 3 to 3.1 Gy per fraction. Gemcitabine was
administered at an average dose of 21 mg

IV. Five cats required chemo-

therapy delay or dose reduction because of myelosuppression or unaccept-
able local tissue toxicity. Median local disease control was 3 months, with
a mean of 6 months. Six cats died of progressive local disease, while two died
of metastasis without local recurrence. Two cats were alive without evidence
of disease progression at the time of the report (1 and 32 months). Grades 2
to 3 local radiation toxicity was reported [59].

Eight cats with nonresectable squamous cell carcinoma of the oral cavity

were treated with palliative radiation therapy at 6 Gy fractions delivered
twice weekly for a total dose of 36 Gy. Low-dose gemcitabine (25 mg

)

was administered twice weekly in conjunction with this megavoltage ir-
radiation. The mean number of doses of radiation was 4.9, and a mean of
ﬁve doses of gemcitabine was given. In this population of cats, two had
complete responses, three had partial responses, and three were non-
responsive. Median duration of remission was 42.5 days for the cats that
responded, and median survival time was 111.5 days (range 11 to 234 days).
These data suggest that short-term therapeutic beneﬁt from radiation and
gemcitabine is possible in a palliative intent setting [66].

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Mechanisms of anticancer drug resistance

Philip J. Bergman, DVM, MS, PhD

Donaldson-Atwood Cancer Clinic and Flaherty Comparative Oncology Laboratory,

The Animal Medical Center, 510 East 62nd Street, New York, NY 10021, USA

For veterinarians involved in the treatment of pets with cancer via

chemotherapeutics, drug resistance is a common phenomenon and the most
common cause of treatment failure. For example, many chemotherapeutic
agents are capable of inducing rapid remissions in dogs with lymphoma;
however, the ability to treat these patients eﬀectively on relapse is routinely
signiﬁcantly impaired [1–6]. This inability to treat patients effectively after
relapse is likely the result of a multitude of resistance factors, which have
been elucidated over the last three decades. Multiple mechanisms of re-
sistance that represent a level of redundancy for protection of the cell and
the organism are likely present in most normal cells. Unfortunately, many
cancers have derived methods of employing these resistance mechanisms for
their own protection.

Drug resistance is generally categorized into intrinsic and acquired

forms, with probable overlap in mechanisms across these two categories.
For example, mechanisms of acquired drug resistance would include re-
duced drug accumulation, increased repair, increased detoxiﬁcation,
decreased apoptosis, and, lastly, alterations in drug targets, whereas cell
cycle–associated, cell adhesion–mediated, and other mechanisms would
represent intrinsic or de novo drug resistance mechanisms [7–15]. For
a complete list of mechanisms of classical acquired chemotherapy
resistance see Box 1.

In addition, resistance mechanisms can be categorized from organism

and cellular levels. Although there are many recognized processes re-
sponsible for overall resistance, there are many that we as clinicians can
easily circumvent, which include inappropriate underdosing of chemo-
therapeutics, improper scheduling of drugs, and inappropriate lengths of
time from diagnosis to the start of therapy. Others exist that we may not
presently have any control over, including poor absorption and erratic

Vet Clin Small Anim

33 (2003) 651–667

E-mail address:

philip.bergman@amcny.org

0195-5616

/03/$ - see front matter

doi:10.1016/S0195-5616(03)00004-4

bioavailability of oral drugs, decreased drug activation, increased repair of
drug damage, increased drug extrusion, and poor penetration of the drug
into the tumor as a result of carrier-mediated processes. Over the past two
decades, bench research has found mechanisms to circumvent some of these
previously untouchable resistance mechanisms; in fact, some have become
clinically relevant resistance reversal strategies. This overview focuses on the
major known cellular and molecular mechanisms of resistance. Recom-
mendations for minimizing the development of anticancer drug resistance
include the following:

Treat animals as soon as reasonably possible after diagnosis is conﬁrmed.
Use standardized and published chemotherapy protocols.
Adhere to the chemotherapy schedule whenever possible.

Box 1. Mechanisms of classical acquired chemotherapy
resistance

Reduced intracellular drug concentration
P-glycoprotein
Multidrug resistance–related protein
Lung resistance–related protein
Others

Drug target alterations
Topoisomerase II
Topoisomerase I

Drug detoxification
Glutathione
Glutathione-s-transferase
Dihydrofolate reductase
Thymidylate synthase
Aldehyde dehydrogenase
Others

Increased DNA damage repair
O6-alkylguanine-DNA alkyltransferase
Others (eg, mismatch repair, XPF and ERCC1)

Apoptosis/programmed cell death modulation
Bcl-2 family
p53, p21, p27, and others
Attenuated death receptor signaling (eg, tumor necrosis

factor-related apoptosis-inducing ligand [TRAIL])

Caspase based (eg, Caspase-8)
Others (eg, NF-jB, DNA repair)

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Use the recommended doses of chemotherapy in the protocol.
Use of proper mixing, handling, administration, and disposal techniques

is of paramount importance.

Whenever possible, do not use prednisone before a diagnosis is con-

ﬁrmed or before induction chemotherapy is instituted for dogs with
lymphoma.

Treat patients as soon as reasonably possible with the next appropriate

chemotherapy protocol once relapse has occurred.

P-glycoprotein

The most researched form of cellular drug resistance is the overexpression

of a plasma membrane protein given the name P-glycoprotein (Pgp
[P

¼ permeability]) or P-170 (protein molecular weight of 160–180 kd)

[16,17]. Resistance related to Pgp overexpression is one of the major factors
leading to the multidrug resistance (MDR) phenotype [9,18–20]. This is
the phenomenon whereby cancer cells become simultaneously resistant to
a variety of different drugs commonly used in veterinary medicine (eg,
doxorubicin, vincristine, actinomycin-

, mitoxantrone and others). Such

drugs are typically developed from natural sources and are hydrophobic
but otherwise have few similarities in their mechanisms of action or their
chemical structures [9]. Chemotherapeutics exhibiting cross-resistance in
MDR because of Pgp include the following:

Vincristine
Vinblastine
Doxorubicin
Daunorubicin
Mitoxantrone
Actinomycin-

Etoposide
Mitomycin-C
Taxol

/taxanes

Steroids (corticosteroids and sex steroids)
Others

Not only do cancer cells develop resistance to these chemotherapeutics,

but these same compounds induce the formation of the MDR phenotype.
Pgp genes have been found in bacteria, viruses, plants, insects, nematodes,
and mammals; such extreme evolutionary conservation signiﬁes the extreme
level of importance of this gene and its protein.

Pgp acts as a plasma membrane drug eﬄux pump that actively extrudes

drugs from cancer cells, thereby limiting the cytotoxicity of the drug at its
cellular site of action. Pgp is an adenosine triphosphate (ATP)–dependent
eﬄux pump that is a member of a family of ATP-binding cassette (ABC)
transporters [16]. Almost 50 ABC transporters have been discovered in

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humans to date. The normal function of Pgp is not completely known, but it
has been found to be expressed in normal human or canine adrenal gland,
kidney, liver, intestine, placenta, blood–brain barrier, lung, peripheral
blood

/bone marrow cells, and multiple fetal cell lines [9,16–18,20,21]. It is

therefore hypothesized that Pgp normally functions as a drug efﬂux pump.
For example, Pgp is located on the luminal surface of the endothelial cells in
the brain, thereby preventing cytotoxins from penetrating the brain across
the endothelium [22]. Mice that have been genetically modiﬁed to not
express Pgp (MDR ‘‘knockouts’’) are normally deﬁcient in Pgp or are given
Pgp inhibitors and are exquisitely sensitive to Pgp substrates, such as
ivermectin [23–28]. Interestingly, these mice and some Collies show similar
neurologic side effects on ivermectin administration [29–31]. Recent studies
have shown that a subpopulation of Collies sensitive to ivermectin ad-
ministration has a mutant Pgp and that 35% of Collies are homozygous for
the mutant allele of Pgp, which is consistent with previous reports sug-
gesting that 30% to 40% of Collies have sensitivity to ivermectin [30,32,33].
Studies are presently ongoing within this author’s laboratory to investigate
the immunohistochemical expression of Pgp in Collies compared with other
canine breeds.

In addition to numerous chemotherapy agents and ivermectin, there are

many other Pgp substrates that are commonly used therapeutic agents.
These include but are not limited to ondansetron, loperamide, itraconazole,
ketoconazole, cyclosporine, rifampin, phenobarbital, digoxin, doxycycline,
omeprazole, and many types of steroids, antibiotics, and antihistamines
[34–40]. Interestingly, Collies have been previously reported to be extremely
sensitive neurologically to loperamide, and this may be explained by loper-
amide being a known Pgp substrate [41,42]. Unfortunately, many of the
aforementioned agents are substrates as well as inducers of Pgp expression.
Therefore, the use of many of these agents may be inducing a drug-resistant
phenotype in our clinical patients, and additional research in this area is
strongly encouraged. Similarly, concomitant use of these agents at the time
of chemotherapy administration may change the pharmacokinetic and
toxicity proﬁle of chemotherapy agents or other Pgp substrates.

Many tumors derived from the normal anatomic locations that express

large amounts of Pgp are commonly intrinsically resistant to chemother-
apeutic agents because of increased Pgp expression [43–45]. The clinical
importance of MDR is demonstrated in human oncology by the fact that
increasing levels of Pgp expression positively correlate with a lack of re-
sponse or remission with appropriate forms of chemotherapy for a variety of
human malignancies [16,46].

A potential explanation for why dogs with lymphoma treated with

the chemotherapy protocol containing cyclophosphamide, vincristine, and
prednisone (COP) have a shorter remission than those treated with doxo-
rubicin alone may lie within the realm of MDR [3,47,48]. Two of the
drugs in the COP protocol are known to induce the MDR phenotype,

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whereas doxorubicin induces the MDR phenotype in single fashion.
Similarly, the use of corticosteroids before the initiation of chemotherapy
for canine lymphoma is thought to be a negative prognostic factor; however,
this is controversial [49]. This is likely related to the induction of Pgp
expression via prednisone, because steroids are well-known potent inducers
of Pgp expression and activity [50–53].

Three studies in veterinary medicine published in 1995 and 1996 dem-

onstrated the importance of Pgp in dogs with lymphoma. The ﬁrst study
used a Western blotting anti-Pgp methodology in membrane preparations
of lymphoma cells from 31 dogs with lymphoma [54]. These investigators
found positive expression of Pgp in 1 of 31 dogs and 3 of 8 dogs before the
initiation of chemotherapy and once resistant to chemotherapy, respectively.
Unfortunately, Western blotting methodologies are relatively insensitive
and laborious, and they generally require fresh cells. Similarly, Western
blotting probes for Pgp expression in the entire sample and some Pgp
antibodies may label Pgp in lymphoma cells as well as incorrectly label other
noncancerous tissues. For this reason and various others, a consensus
workshop on determination of Pgp expression in human cancers has
previously recommended the use of in situ technologies, such as im-
munohistochemistry, to allow for speciﬁc Pgp expression in the cancerous
tissue of choice [55].

In the second study, which was completed by this author and his

colleagues, the prevalence of positive staining of Pgp via immunohisto-
chemistry was ascertained in 58 dogs with lymphoma [56]. Dogs had
samples evaluated before the initiation of chemotherapy, at time of relapse,
at the time of necropsy, and at all three times in some dogs. Consistent with
previous ﬁndings in Pgp research in human oncology, Pgp expression levels
signiﬁcantly increased at relapse and necropsy when compared with levels at
the initiation of chemotherapy. This study also demonstrated that the level
of Pgp staining before initiation of chemotherapy was inversely correlated to
both remission and survival times, whereas the level of Pgp staining at
relapse was inversely correlated to the time from relapse to death. Therefore,
those patients with high pretreatment levels of Pgp had signiﬁcantly lower
remission times and survival times, and those patients with high Pgp levels
at relapse had signiﬁcantly decreased times from relapse to death. An ad-
ditional striking observation was that Pgp staining at the initiation of
chemotherapy was the most prognostic factor of any of those examined in
the study (including stage, substage, presence of hypercalcemia, age, weight,
and gender).

In the third study, expression of Pgp was again ascertained by im-

munohistochemistry, and pretreatment Pgp expression was found to be an
independent negative predictor of overall survival [57]. Additionally, Pgp
expression was greater after relapse when compared with pretreatment
samples. More recent studies in the in vitro setting show that canine cancer
cell lines can be induced to overexpress Pgp and be modulated by known

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Pgp inhibitors no differently than is seen in human in vitro systems [58,59].
Taken in concert, it seems that canine Pgp behaves similarly to human Pgp
and that Pgp expression determination in a prospective fashion for dogs
with lymphoma represents a viable pretreatment and intratreatment diag-
nostic tool. In addition, these works suggest that canine lymphoma
represents an excellent comparative model for human Pgp research. Future
studies in canine drug resistance incorporating determination of Pgp expres-
sion and the other aforementioned mechanisms in Box 1 performed in
a prospective fashion are hereby encouraged for lymphoma and other
neoplasms. Unfortunately, few data have been published concerning feline
Pgp; one study using a feline lymphoma cell line (FT-1) and its doxorubicin-
resistant subline (FT-1

/ADM) has shown upregulation of Pgp in FT-

/ADM with an MDR phenotype [60]. Interestingly, polymerase chain

reaction (PCR) cDNA ampliﬁcation of the feline Pgp showed 90% se-
quence identity to human Pgp, thereby continuing the theme of high
sequence identity across species for Pgp.

There are many methods for attempting reversal of Pgp-associated

MDR; however, to date, few have shown any signiﬁcant clinical beneﬁt
[16,61–63]. There are many compounds, such as verapamil, cyclosporine,
tamoxifen, and others, that competitively bind to Pgp to inhibit its efﬂux
actions [64]. Drugs known to modulate Pgp activity and

/or expression are

seen in Box 2.

Box 2. Drugs known to modulate P-glycoprotein
activity or expression

Verapamil
Quinine
Cyclosporine
Reserpine
Chloroquine
Trifluoperazine
Tamoxifen
Various surfactants (eg, Cremophor-EL)
Progesterone and other sex steroids
Corticosteroids
Loperamide
Ondansetron
Doxycycline
Itraconazole/ketoconazole
Digoxin
Omeprazole
Others (some antibiotics and antihistamines)

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A phase I trial of high-dose tamoxifen and chemotherapy in normal and

tumor-bearing dogs was published by the North Carolina State University
group and showed that plasma concentrations capable of reversing Pgp
could be safely achieved in dogs with mild to moderate toxicity [65].
Unfortunately, many of these compounds have either poor efﬁcacy (Pgp
substrates themselves) or severe unexpected toxicities at the levels necessary
for Pgp reversal; therefore, the search for less toxic resistance reversal
strategies continues [7]. Pgp inhibitors that are not Pgp substrates but retain
signiﬁcant sensitivity and speciﬁcity for Pgp, such as XR-9576, OC-1440935,
LY335979, and GF-120918, hold great promise and are presently being
evaluated in human clinical trials [16,66].

The dose-response curves of most cancer cells are extremely steep; thus,

one way to circumvent resistance is to use higher and higher doses of drugs.
Signiﬁcant toxicity, especially myelosuppression, is usually the result,
however. Similarly, although it is common for cancer cells to develop an
MDR phenotype, normal tissues continue their sensitivity to chemotherapy.
Because the bone marrow is invariably the dose-limiting toxicity for most
chemotherapeutics, investigators are transferring the genes that encode
Pgp (and other drug resistance genes) to bone marrow cells to confer
myeloprotection [67]. Pilot studies by this author and his colleagues at
Memorial Sloan-Kettering Cancer Center have shown that normal canine
hematopoietic stem cells (HSCs) can undergo drug resistance gene transfer
with concomitant in vitro myeloprotection to various chemotherapy agents
(unpublished observations). Strategies by other human and veterinary in-
vestigators to confer myeloprotection include the use of autologous HSC
infusion with high-dose chemotherapy [68,69].

Multidrug resistance–associated protein

Multiple studies have shown that when Pgp is not present, an MDR

phenotype is still possible; therefore, other resistance mechanisms are likely
at work in MDR. The second most frequently examined form of drug
resistance is that involving multidrug resistance–related protein (MRP)
[70–74]. MRP is a 190-kd protein that is similar in structure to Pgp.
Interestingly, MRP localizes to the cytoplasm in most normal tissues,
whereas plasma membrane localization is more common in neoplastic
tissues, suggesting an excretory function in the cancer cell. The normal
function of MRP has been identiﬁed as the carrier of glutathione S-
conjugates and bilirubin glucuronides in the cell and then excretion of these
conjugates to outside the cell [73]. Normal MRP seems to be most strongly
expressed in various epithelia, macrophages, the heart, and tissues with an
excretory function, such as the liver, kidney, and others, similar to the
normal expression of Pgp [72]. Therefore, MRP has been referred to by
many as the ‘‘toxic waste manager’’ of the cell for those toxins conjugated

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by the glutathione system, which, unfortunately, is a common detoxiﬁcation
system for many of the frequently used chemotherapy agents in veterinary
medicine (see section on glutathione). As a measure of MRP’s clinical
importance, rats and human beings with mutations in MRP have chronic
conjugated hyperbilirubinemia (Dubin-Johnson syndrome). Eight addi-
tional MRP-like proteins have been recently identiﬁed and are likely addi-
tional drug resistance mechanisms worthy of future investigation [74,75].

No studies have been published to date on MRP in veterinary oncology;

however, the prognostic signiﬁcance of MRP in human oncology is strong.
Many tumor cell lines coexpress Pgp and MRP, whereas others with an
MDR phenotype without Pgp expression can have strong MRP expression.
Acute leukemias, transitional cell carcinomas, and squamous cell carcino-
mas seem to be human neoplasms with the strongest MRP expression,
suggesting normal cells that undergo malignant transformation have
retained their MRP expression [76,77].

Lung resistance–related protein

Lung resistance–related protein (LRP) is a 110-kd protein originally

isolated from a human lung cancer cell line that is a distant relative of Pgp
and MRP [78]. The normal function of this protein is that of a major
‘‘vault’’ ribonucleoprotein, which regulates transport of substances between
the nucleus and cytoplasm [79]. This suggests LRP may be involved in the
transport of cytotoxic agents by redistributing drugs away from intracellular
targets. LRP has been detected in numerous cancer cell lines and clinical
specimens of many tumors, including acute myeloid leukemia, ovarian car-
cinoma, myeloma, and ﬁbrosarcoma [80]. No studies have been pub-
lished to date on the detection of LRP in veterinary patients, and no studies
have been published on reversal strategies to LRP-associated drug resistance.

Topoisomerases

DNA topoisomerases (Topo I and II) are essential enzymes that catalyze

various conformational changes in DNA necessary for normal steps in
DNA metabolism [81]. Topo II forms a transient DNA break followed by
DNA strand passage and then religation of the DNA. Inhibitors of Topo II
prevent the religation process by freezing the ‘‘cleavable complex’’ formed
between the enzyme and DNA, leading to cell death [82]. Drugs that tar-
get Topo II include actinomycin-

, epipodophyllotoxins (eg, etoposide),

anthracenediones (eg, mitoxantrone), anthracyclines (eg, doxorubicin,
daunorubicin), and other experimental agents. Chemotherapeutics involved
with Topo II–mediated MDR include all those in Pgp-associated MDR,
except the vinca alkaloids and taxanes. In contrast to Pgp-associated MDR,
chemoresistance associated with Topo II is the result of reduced expression
or activity of the enzyme [83,84].

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Glutathione system

The major role of glutathione S-transferase (GST) and glutathione

(GSH) is the detoxiﬁcation of cytotoxic drugs [85]. Many cancer cell lines
and clinical cancer specimens have increased production of GST and GSH
when compared with normal tissues [86,87]. It seems that doxorubicin,
chlorambucil, cyclophosphamide, melphalan, nitrosoureas, cisplatin, and
others are involved with GST

/GSH-mediated MDR [88,89]. Attempts at

reversal of GST

/GSH-mediated MDR with ethacrynic acid as a GST in-

hibitor or buthionine sulfoximine as a GSH synthesis inhibitor have been
met to date with poor efﬁcacy and signiﬁcant toxicity in human clinical trials
[87]. Two reports show the potential importance of the GSH system in
canine osteosarcoma and mammary tumor drug resistance [90,91].

Dihydrofolate reductase and thymidylate synthase

Ampliﬁcation of the dihydrofolate reductase (DHFR) gene leads to

subsequent overexpression of DHFR. DHFR is an enzyme responsible for
catalyzation of the reduction of dihydrofolate to tetrahydrofolate. DHFR
overexpression leads to a decreased concentration of the classic antifolate
chemotherapeutic methotrexate and represents one of the earliest forms of
drug resistance elucidated to date and the major cause of methotrexate
resistance [92]. Although other mechanisms of methotrexate resistance exist
(ie, antifolate transport modulation via reduced folate carriers, mutation of
target enzyme, polyglutamation modulation, thymidylate synthase), DHFR
overampliﬁcation remains the most important methodology of methotrexate
resistance investigated to date [93–96]. DHFR overampliﬁcation-based
methotrexate resistance may be superseded by massive doses of methotrex-
ate followed by leucovorin rescue. In addition, newer methotrexate
analogues, such as trimetrexate and edatrexate, may be useful, because
these compounds are believed to be more selectively inhibitory of DHFR
[97,98]. DHFR-based chemoresistance may have a limited role in veterinary
oncology, because methotrexate is not a commonly used veterinary che-
motherapy agent outside of some multiagent lymphoma protocols, and
there are no studies reporting the efﬁcacy of single-agent methotrexate
against lymphoma to date [99].

Others

Many antineoplastic drugs interact with DNA at the O

-position of

guanine to form extremely potent cytotoxic DNA adducts [100,101].
O

-alkylguanine-DNA alkyltransferase (O

-AT) is a DNA repair enzyme

encoded by the gene MGMT that has been recently implicated as
a mechanism of chemoresistance [102]. High levels of O

-AT have been

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associated with resistance to dacarbazine, streptozotocin, and nitrosoureas
(eg, CCNU, BCNU), presumably because this enzyme repairs the
chemotherapy-induced DNA damage [100,103]. A relatively new ‘‘methyl-
ating’’ chemotherapy agent, temozolomide, primarily induces O

-methyl-

guanine adducts, and high levels of O

-AT strongly predict temozolomide

resistance [104]. Phase II human clinical trials are ongoing to evaluate the
use of the O

-AT inhibitor O

-benzylguanine in concert with chemotherapy

agents that promote O

-guanine DNA adducts [100]. No veterinary studies

have been published to date on O

-AT–based chemoresistance.

The induction of DNA interstrand (between complementary strands of

DNA) and intrastrand crosslinks seems to be an important mechanism of
cytotoxicity for clinically relevant chemotherapy agents in the platinum class
and others [105]. Although reduced drug uptake and increased inactivation
by glutathione appear to be important mechanisms of platinum class
chemotherapy agents, it seems that DNA repair mechanisms, such as
overexpression of XPF and ERCC1 proteins, are of even greater importance
[106,107]. Interestingly, human cells derived from patients with xeroderma
pigmentosum are extremely sensitive to various chemotherapy agents,
radiation, and ultraviolet (UV) light, because these cells are nucleotide
excision repair deﬁcient [108]. The extreme sensitivity of these cells and
others like them is a result of myriad mutations in DNA repair genes,
including mismatch repair genes (eg, hMSH2, hMLH1); unfortunately,
cancer cells upregulate these same but nonmutated genes to confer che-
moresistance [106–110]. This has widespread implications for chemother-
apy agents that act on DNA and represents yet another mechanism for
cancer cells to escape the effects of various chemotherapy agents. No
veterinary studies have been published to date on DNA-repair–based
chemoresistance.

Aldehyde dehydrogenase (AD) is an additional mechanism of cellular

drug resistance to chemotherapy agents [111]. AD-based resistance seems
to be important for cyclophosphamide resistance in a variety of human
malignancies, including breast cancer, medulloblastomas, and leukemias
[112–114]. This method of chemoresistance is particularly intriguing,
because cyclophosphamide is a commonly used chemotherapy agent and
is not a Pgp substrate [16]. No veterinary studies have been published to
date on AD-based chemoresistance.

Apoptosis resistance–related multidrug resistance

Apoptosis, or programmed cell death, is an internally programmed

mechanism of cell death. Apoptosis is morphologically and biochemically
distinct from necrosis and permits noninﬂammatory single cell deletion
[115]. Most forms of chemotherapy and radiation kill cancer cells by in-
ducing them to undergo apoptosis [116,117]. Many normal endogenous

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activators and suppressors of apoptosis have recently been elucidated [13].
Unfortunately, cancer cells have developed mechanisms of upregulating or
activating these suppressors of apoptosis. Although much is left to be
learned about apoptosis and its relation to cancer biology, it seems that
apoptosis-resistant cancer cells are resistant to even the highest doses
possible of chemotherapeutics and radiation [8,118–121]. Therefore, based
on the fact that such apoptosis regulators lie profoundly downstream from
our therapeutic agents, a greater understanding of how apoptosis affects
resistance and cancer therapy in general is urgently needed. Such an
understanding should open new and likely extremely clinically relevant
therapeutic modalities for cancer as well as autoimmune, infectious, and
degenerative diseases.

Relatively little has been published to date on apoptosis and cancer in

veterinary medicine. The University of California–Davis group has found
that apoptotic and proliferation indexes as well as the proliferation

apoptotic ratio were predictive of relapse-free interval in dogs with lym-
phoma [122]. The Purdue University group has shown that urinary bladder
transitional cell carcinoma diagnosed in dogs that experience an antitumor
response to piroxicam was strongly associated with induction of apoptosis
and reduction in urine basic ﬁbroblastic growth factor concentration
[123,124]. Other studies published to date have involved in vitro apoptosis
investigations using cancer cell lines from veterinary patients, and we ea-
gerly await additional in vivo investigations [125–130].

Summary

Chemotherapy agents are extremely important in the treatment of liquid

malignancies, such as lymphoma, myeloma, and chronic lymphocytic
leukemia. In addition, chemotherapy agents have proven eﬀective in the
adjuvant treatment of solid tumors, such as osteosarcoma, hemangiosar-
coma, transitional cell carcinoma, and others. Unfortunately, chemotherapy
resistance in these situations is the most signiﬁcant cause of treatment
failure. Therefore, the ability to predict, treat, or circumvent resistance is
extremely likely to improve clinical outcomes. This article has reviewed the
most widely investigated forms of chemotherapy resistance, such as reduced
drug accumulation, increased DNA damage repair, decreased apoptosis,
and others; however, new mechanisms are being found at an alarming pace.
In addition, investigations to date have routinely centered on single-cell
mechanisms of drug resistance, and cancer is truly a three-dimensional
disease. The elucidation of mechanisms surrounding (1) how tumors interact
with their normal microenvironment, (2) how tumors interact in a three-
dimensional environment, and (3) a better understanding of basic tumor
physiology and biology may supersede in importance those previously elu-
cidated single-cell mechanisms of chemoresistance.

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Index

Note: Page numbers of article titles are in boldface type.

Abdominal cavity

evaluation of

in mast cell tumors in dogs

assessment, 478

Actinomycin-D

for soft tissue sarcomas, 524

Anastomosis(es)

uretercolonic

for bladder TCC, 603–604

Angiocardiography

for hemangiosarcoma in dogs

and cats assessment, 539

Anticancer drug resistance

dihydrofolate reductase in, 659
glutathione system in, 659
lung resistance–related, 659
mechanisms of, 651–667
multidrug apoptosis resistance,

660–661

multidrug resistance–associated

protein, 657–658

P-glycoprotein, 653–657
thymidylate synthase in, 659
topoisomerases in, 658

Apoptosis resistance–related

multidrug resistance, 660–661

Argyrophilic nucleolar straining

organizing regions

in mast cell tumors in dogs

assessment, 481

Aspiration

bone marrow

in mast cell tumors

in dogs assessment, 478

lymph node

in mast cell tumors in dogs

assessment, 478

Aspiration cytology

for hemangiosarcoma

in dogs and cats
assessment, 540

Axial canine osteosarcoma, 505–509. See

also Osteosarcoma, canine, axial.

B5 antigen expression

in canine lymphoma

prognosis, 461

Biochemistry proﬁle

in mast cell tumors in dogs

assessment, 477

Biologic therapy

for hemangiosarcoma in dogs and

cats, 545

Bladder

transitional cell carcinoma of,

597–613. See also Transitional
cell carcinoma, bladder.

Bladder reconstruction

for bladder TCC, 604

Bone infarcts

canine osteosarcoma

due to, 492

Bone marrow aspiration

in mast cell tumors in dogs

assessment, 478

Buﬀy coat smear

in mast cell tumors in dogs

assessment, 477–478

Cancer

head and neck. See Head and neck

cancer.

Canine appendicular osteosarcoma,

494–505. See also Osteosarcoma,
canine, appendicular.

Canine extraskeletal osteosarcoma,

509–511. See also Osteosarcoma,
canine, extraskeletal.

Vet Clin Small Anim

33 (2003) 669–675

0195-5616/03/$ - see front matter

doi:10.1016/S0195-5616(03)00051-2

Carboplatin

for bladder TCC, 606
for soft tissue sarcomas, 524

Cat(s)

hemangiosarcoma in, 533–552.

See also Hemangiosarcoma,
in dogs and cats.

injection site sarcomas in, 553–571.

See also Injection site sarcomas,
feline.

lymphoma in

inhibitor molecule p27Kip1

dysregulation in, 467

risk factors for, 466
updates related to, 466–467

Catheter(s)

permanent cystostomy

for bladder TCC, 603

Chemoradiation therapy

for head and neck cancer, 617–618

Chemotherapy

for canine lymphoma, 461–463
for hemangiosarcoma in dogs and

cats, 543–545

for mast cell tumors in dogs,

484–485

for soft tissue sarcomas

candidates for, 519–520
combination, 525–527
for patients with measurable

disease, 527

new agents, 524–525
practical uses, 527–529
sequential, 525–527
single-agent, 520–524

new agents, 629–649. See also

speciﬁc agents, e.g., Lomustine.

gemcitabine, 639–647
ifosfamide, 634–637
lomustine, 629–634
streptozocin, 637–639

rescue-related

for canine lymphoma,

463–464

Cisplatin

for bladder TCC, 605–606
for soft tissue sarcomas, 524

Complete blood count (CBC)

for hemangiosarcoma in dogs

and cats assessment, 538

in mast cell tumors in dogs

assessment, 477

Cystectomy

partial

for bladder TCC, 602–603

Cystostomy catheter

permanent

for bladder TCC, 603

Cytology

aspiration

for hemangiosarcoma in dogs

and cats assessment, 540

Dacarbazine

for soft tissue sarcomas, 523–524

Dental tumors

treatment of, 619

Dihydrofolate reductase

in anticancer drug resistance, 659

DNA ploidy

in mast cell tumors in dogs

assessment, 482

Docetaxel

for soft tissue sarcomas, 524

Dog(s)

hemangiosarcoma in, 533–552.

See also Hemangiosarcoma,
in dogs and cats.

lymphoma in

new developments in,

457–466. See also
Lymphoma(s), updates
related to, in dogs.

mammary gland tumors in,

573–596. See also Mammary
gland tumors, canine.

osteosarcoma in, 491–516.

Document Outline

00 Preface
01 Lymphoma updates
- Lymphoma updates
02 Mast cell tumors in the dog
- Mast cell tumors in the dog
03 Update on the biology and management of canine osteosarcoma
- Update on the biology and management of canine osteosarcoma
04 Medical management of soft tissue sarcomas
- Medical management of soft tissue sarcomas
05 Hemangiosarcoma in dogs and cats
- Hemangiosarcoma in dogs and cats
06 Feline injection site sarcomas
- Feline injection site sarcomas
07 Canine mammary gland tumors
- Canine mammary gland tumors
08 Management of transitional cell carcinoma
- Management of transitional cell carcinoma
09 Multimodality therapy for head and neck cancer
- Multimodality therapy for head and neck cancer
10 New chemotherapy agents in veterinary medicine
- New chemotherapy agents in veterinary medicine
11 Mechanisms of anticancer drug resistance
- Mechanisms of anticancer drug resistance
12 Index

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