ATM POLYMORPHISM IVS62

⫹60G⬎A IS NOT ASSOCIATED

WITH DISEASE AGGRESSIVENESS IN PROSTATE CANCER

ROBERT E. LEE BROWNING IV, HECHENG LI, ERIC T. SHINOHARA, QIUYIN CAI, HEIDI CHEN,

REGINA COURTNEY, CAROLYN CAO, WEI ZHENG,

AND

BO LU

ABSTRACT

Objectives. ATM is an important protein that protects the genome from double-stranded DNA breaks.
Mutations and polymorphisms in the ATM gene have been associated with an increased risk of certain types
of malignancies, especially breast cancer.
Methods. We analyzed tissue from 98 white patients with prostate cancer for the presence of
IVS62

⫹60G⬎A polymorphism in the ATM gene.

Results. The frequency of the homozygous IVS62

⫹60G/G, heterozygous IVS62⫹60G/A, and homozygous

IVS62

⫹60A/A was 18.37% (18 of 98), 30.61% (30 of 98), and 51.02% (50 of 98), respectively. Using

Fisher’s exact test, we found the polymorphism IVS62

⫹60G⬎A was not significantly related to age, tumor

grade, prostate-specific antigen level, or clinical stage (P

⬎0.05). No difference was found in relapse-free

survival between patients with IVS62

⫹60G/G and those with IVS62⫹60G/A or IVS62⫹60A/A (P ⫽ 0.4533).

Conclusions. The results of our study indicate that this ATM polymorphism is not associated with the
aggressiveness of prostate cancer in white men.

UROLOGY

67: 1320–1323, 2006. © 2006 Elsevier Inc.

P

rostate cancer has emerged as the third most
common cancer worldwide among men, ac-

counting for about 10% of all cancers in males.

1–3

One half million new cases of this slow-growing
cancer are reported each year.

4,5

To increase the

quality of treatment and to design better therapeu-
tic strategies, it would be beneficial to understand
and study markers that could predict the course
and outcome of the disease. Ataxia-telangiectasia
is a recessive disorder caused by a mutation in the
ATM gene and characterized by progressive neuro-
nal degeneration, immunologic deficiency, radio-
sensitivity, and an increased risk of cancer.

6

This

gene is located on chromosome 11Q22-2.

7

The

13-kb mRNA is assembled from 66 exons spread
across a 150-kb genomic region.

8,9

The protein

product of ATM is 350 kD, and it protects the ge-

nome from double-stranded breaks that arise from
endogenous and exogenous sources.

6

ATM acts at

different levels to maintain the integrity of the ge-
nome. It mediates arrest in the cell cycle at G

1

/S, S,

and G

2

/M to prevent the processing of damaged

DNA; it activates repair pathways; and, in cases of
extreme DNA damage, it activates apoptotic path-
ways. ATM mediates its biological effects by regu-
lating p53, BRCA1, and CHK2 through phosphor-
ylation of these downstream targets.

10

It has long

been speculated that changes in the ATM gene
such as single nucleotide polymorphisms, truncat-
ing, and missense mutations could lead to an in-
creased cancer risk. Borresen et al.

11

and Swift et

al.

12

have shown that persons heterozygous for

mutations in the ATM gene have an increased risk
of cancer, in particular breast cancer. The ATM
D1853N polymorphism has also been linked to an
increased risk of nonpolyposis colorectal cancer
when it occurs in conjunction with MLH1 and
MLH2 germline mutations.

13

In the present study,

we hypothesized that the ATM IVS62

⫹60G⬎A poly-

morphism would impair the function of the ATM
protein and, in turn, affect the aggressiveness of pros-
tate cancer. It should be noted that this single nucle-
otide polymorphism (SNP) has not been previously
studied in a white population, so the excepted fre-
quency of this polymorphism is unknown. To test

This work was supported by grants DOD PC031161 (B. Lu),
RO1CA90899 (W. Zheng), and UICC fellowship ICRETT1025
(H. Li).

From the Departments of Radiation Oncology and Medicine,

Vanderbilt University School of Medicine, Nashville, Tennessee

Reprint requests: Bo Lu, M.D., Ph.D., Department of Radiation

Oncology and Vanderbilt-Ingram Cancer Center, Vanderbilt Uni-
versity School of Medicine, Nashville, TN 37232. E-mail: bo.lu@
vanderbilt.edu

Submitted: August 13, 2005, accepted (with revisions): Decem-

ber 5, 2005

BASIC SCIENCE

© 2006 E

LSEVIER

I

NC

.

0090-4295/06/$32.00

1320

ALL RIGHTS RESERVED

doi:10.1016/j.urology.2005.12.012

our hypothesis, we assessed the ATM polymorphism
present in 98 white patients with prostate cancer to
determine whether IVS62

⫹60G⬎A correlated

with any of the well-known prognostic factors, as
well as relapse-free survival (RFS).

MATERIAL AND METHODS

P

ATIENT

P

OPULATION AND

C

LINICAL

D

ATA

The study population consisted of 98 white patients with

prostate cancer who underwent prostatectomy at Vanderbilt
Hospital in 1997. The institutional review board (No. 030986)
at Vanderbilt University School of Medicine approved the
study. All the patients had clinically localized prostate cancer
and were treated with radical prostatectomy as the primary
treatment. All patients had adenocarcinoma confirmed histo-
logically. The patients were followed up at Vanderbilt Hospital
or local hospitals, with a mean follow-up of 62 months. The
median patient age was 63 years (range 40 to 81). One pathol-
ogist reviewed all pathologic information, and the tumor dif-
ferentiation was evaluated using Gleason score criteria. The
clinical stage was classified according to the American Joint
Committee on Cancer TNM staging system.

14

T

ISSUE

P

REPARATION AND

DNA E

XTRACTION

Using a standard microtome with disposable blades, 5-

␮m-

thick sections of representative areas of the normal prostate
glands were cut from the paraffin-embedded blocks, stained
with hematoxylin-eosin, and examined under a microscope to
verify the absence of prostate cancer. A 5-

␮m-thick section

from each patient was used for DNA extraction. The section
was deparaffinized with xylene at room temperature for 30
minutes twice and then washed with 100% ethanol twice. Af-
ter the ethanol had completely evaporated, the tissue was com-
pletely lysed with proteinase K. Next, the QIAamp DNA Mini
Kit (QIAGEN, Valencia, Calif) was used to extract and purify
the DNA from the tissue according to the tissue protocol kit.

P

OLYMORPHISM

G

ENOTYPING

The allelic discrimination of the ATM gene IVS62

⫹60G/A

polymorphism was assessed with the ABI Prism 7900 HT Se-
quence Detection System. Polymerase chain reaction was per-
formed with a total volume of 5

␮L, which contained approx-

imately 2.5 ng DNA, 1

⫻ Taqman Universal PCR Master Mix,

each primer at a concentration of 900 nM, and each probe at a
concentration of 200 nM. The Taqman probes were as follows:
A allele specific, 5=-VIC-TCT TAC CAG GTA GAC TGT GTA
TCT CAT CAG GAA GTC ACT GAT GTG AAG AGC-NFQ-
3=; and G-allele specific, 5=-FAM- TCT TAC CAG GTA GAC
TGT GTA TCT CGT CAG GAA GTC ACT GAT GTG AAG
AGC-NFQ-3=. The thermal cycling conditions were as fol-
lows: 95°C for 10 minutes to activate the AmpliTaq Gold en-
zyme, followed by 40 cycles of 15 seconds at 95°C and 60
seconds at 60°C. The fluorescence levels were measured with
an ABI PRISM 7900 HT Sequence Detector and resulted in
clear identification of three genotypes of each polymorphism.

The laboratory staff was unaware of the identity of the men.

Quality controls were included in the genotyping assays. Each
384 well plate contained four water, eight CEPH 1347-02
DNA, and eight blinded quality control samples, and un-
blinded quality control samples. The blinded and unblinded
quality control samples were taken from the second tube of the
study samples included in this study.

S

TATISTICAL

A

NALYSIS

RFS was defined as the time between the date of the primary

surgery to the date of relapse or the date of late follow-up. The

Kaplan-Meier method was used to compute 5-year survival
rates, and the log-rank test was applied to test the difference in
survival across different genotypes. The association between
the ATM polymorphism and the other clinical and pathologic
characteristics was analyzed by Fisher’s exact test. P

⬍0.05

was considered statistically significant. All statistical analyses
were two-sided.

RESULTS

C

ORRELATION OF

IVS62

ⴙ60G/A P

OLYMORPHISM WITH

O

THER

C

LINICAL AND

P

ATHOLOGIC

P

ARAMETERS

With the Taqman SNP genotyping assay, the

concordance of the blinded samples was 100%.
Genotypes for IVS62

⫹60G/A were successfully de-

termined in 98 samples. The frequency of homozy-
gous IVS62

⫹60G, heterozygous IVS62⫹60G/A,

and homozygous IVS62

⫹60A/A was 18.37% (18 of

98), 30.61% (30 of 98), and 51.02% (50 of 98),
respectively. Using Fisher’s exact test, ATM poly-
morphism had no significant relationship with age
(P

⫽ 0.4972), prostate-specific antigen (PSA) level

(P

⫽ 0.5550), Gleason score (P ⫽ 0.6702), or clin-

ical stage (P

⫽ 0.2149;

Table I

). To increase the

power of the study, those who were homozygous
and heterozygous for the SNP were combined and
compared with the prognostic factors. We found
that the IVS62

⫹60G⬎A also had no significant re-

lationship with age (P

⫽ 0.7614), PSA level (P ⫽

0.2395), Gleason score (P

⫽ 0.7938), or clinical

stage (P

⫽ 0.9188) in this analysis (

Table II

).

F

IVE

-Y

EAR

RFS

The Kaplan-Meier method was used to compute

the survival rates, and the log-rank test was used to
test the difference in survival across different geno-
types. The 5-year RFS rate for this group was
87.71%. No significant difference was found be-
tween those with IVS62

⫹60G/G and those with

IVS62

⫹60G/A or IVS62⫹60A/A in RFS (log-rank

test, P

⫽ 0.4533;

Table III

). The combination of the

homozygous and heterozygous subjects for the
SNP had little effect on the correlation between
this ATM polymorphism and RFS (P

⫽ 0.4470;

Table IV

).

COMMENT

In the present study, we analyzed the IVS62

⫹

60G

⬎A polymorphism in 98 white patients with

prostate cancer and did not find any association be-
tween the polymorphism and the other clinical and
pathologic variables. We also did not find any sig-
nificant association between IVS62

⫹60G⬎A poly-

morphism and RFS. However, because of the low
numbers of subjects, the power of our study was
low. Also of note, in this group of patients, T stage,
patient age, and Gleason score were shown to be
true prognostics indicators. However, the PSA
level was not. With that in mind, the data trying to

UROLOGY 67 (6), 2006

1321

link PSA and the ATM polymorphism were not as
firm as one would like. Our group plans to expand
the number of subjects in the study to obtain a
more true measure.

ATM mutations and polymorphisms have been

shown to have a large impact on the cancer risk for
individuals that carry them. Breast cancer is by far
the most studied. Multiple studies have looked at
various changes among patients with breast cancer
to determine whether those changes affect the na-
ture of the cancer. This can be attributed to the
early observation by Swift

12

that family members of

patients with ataxia-telangiectasia have a greater
incidence of cancer, in particular, breast cancer.
This observation brought ATM into the spotlight
as a potential cancer gene. However, little research
to date has been conducted to investigate the rela-
tionship of changes in ATM and how they affect the
nature of prostate cancer. With the increased inci-
dence of prostate cancer worldwide, this field of
prostate cancer research is vastly understudied. To
our knowledge, only six polymorphisms have been
examined to identify whether any relationship ex-
ists between ATM polymorphisms and prostate
cancer. The first study to be conducted examined
five polymorphisms in ATM: 5557G

⬎A, 5558A⬎T,

IVS38-8T

⬎C, IVS38-15G⬎C, and 3161C⬎G. Only

TABLE I.

Associations of ATM polymorphism with other clinical and

pathologic parameters

Factor

IVS62

ⴙ60 Polymorphism (n)

IVS62

ⴙ60G/G

IVS62

ⴙ60A/A

IVS62

ⴙ60G/A

P Value

Age (yr)

ⱕ60

7 (20.59)

20 (58.82)

7 (20.59)

60–70

10 (19.23)

23 (44.23)

19 (36.54)

0.4972

⬎70

1 (8.33)

7 (58.33)

4 (33.33)

PSA (ng/mL)

ⱕ4

5 (29.41)

8 (47.6)

4 (23.53)

4–10

10 (19.23)

27 (51.92)

15 (28.85)

0.5550

⬎10

3 (10.34)

15 (51.72)

11 (37.93)

Gleason score

ⱕ6

12 (19.35)

33 (53.23)

17 (27.42)

⬎6

6 (16.67)

17 (47.22)

13 (36.11)

0.6702

Clinical stage

I

2 (22.22)

3 (33.33)

4 (44.44)

II

12 (17.91)

39 (58.21)

16 (23.88)

0.2149

III

4 (18.18)

8 (36.36)

10 (45.45)

Key: PSA

⫽ prostate-specific antigen.

Data in parentheses are percentages.

TABLE II.

Associations of ATM polymorphism

with other clinical and pathologic parameters

Factor

IVS62

ⴙ60 Polymorphism (n)

IVS62

ⴙ

60G/G

IVS62

ⴙ60A/Aⴙ

IVS62

ⴙ60G/A

P

Value

Age (y)

ⱕ60

7 (20.59)

27 (79.41)

60–70

10 (19.23)

42 (80.77)

0.7614

⬎70

1 (8.33)

11 (91.67)

PSA (ng/mL)

ⱕ4

5 (29.41)

12 (70.59)

4–10

10 (19.23)

42 (80.77)

0.2395

⬎10

3 (10.34)

26 (89.66)

Gleason score

ⱕ6

12 (19.35)

50 (80.65)

⬎6

6 (16.67)

30 (83.33)

0.7938

Clinical stage

I

2 (22.22)

7 (77.78)

II

12 (17.91)

55 (82.09)

0.9188

III

4 (18.18)

18 (81.82)

Key: PSA

⫽ prostate-specific antigen.

Data in parentheses are percentages.

TABLE III.

Association of ATM polymorphism

with recurrence

Genotype

Patients

(n)

Recurrence

(n)

P

Value

IVS62

⫹60G/G

18

2 (11.11)

IVS62

⫹60G/A

50

8 (44.44)

0.4533

IVS62

⫹60A/A

30

8 (44.44)

Data in parentheses are percentages.

TABLE IV.

Association of ATM polymorphism

with recurrence

Genotype

Patients

(n)

Recurrence

(n)

P

Value

IVS62

⫹60G/G

18

2 (11.11)

0.4470

IVS62

⫹60G/A⫹

IVS62

⫹60A/A

80

16 (88.88)

Data in parentheses are percentages.

1322

UROLOGY 67 (6), 2006

one of the five (3161C

⬎G) was associated with

prostate cancer.

15

At least 88 polymorphism sites

are in the ATM gene, with only a limited number
being examined to determine whether they affect
the risk or outcome of certain malignancies.

16

Our

group plans additional investigations into the rela-
tionship of ATM polymorphisms and prostate can-
cer. We are expanding this study to include more
subjects, as well as looking into investigating mul-
tiple ATM polymorphisms in the subjects we have
already included. An additional step that may be
considered in future studies is staining for ATM in
the prostatic tissue to evaluate whether protein ex-
pression is altered with certain SNPs. This may be
very helpful if an ATM polymorphism is identified
that affects the prognosis of prostate cancer. Be-
cause of its pivotal placement in the DNA surveil-
lance and repair pathway, any alteration of this
gene has the potential to increase the number of
cells with damaged genomes and thus increase the
cancer risk. As alterations in genes that function in
the same pathway as ATM become known it will be
worthwhile to determine whether ATM polymor-
phisms in conjunction with these alterations have
a significant effect on prostate cancer, similar to
their interactions in colorectal cancer. The large
size of the ATM gene and its multiple polymor-
phism sites introduces many constraints on its
study.

16

Therefore, careful study of each polymor-

phism and mutation of ATM should be considered
a valuable undertaking because any one of these
changes could be a possible indicator for the devel-
opment of malignancy.

CONCLUSIONS

In the present study, we examined one ATM

polymorphism IVS62

⫹60G⬎A. No association

was found in patients with prostate cancer who
possessed this polymorphism and Gleason score,
age at diagnosis, PSA level, clinical stage, or RFS.
This may have been because this polymorphism
is located on an intron of the ATM gene; thus,
one could hypothesize that this SNP may have no
effect on mRNA splicing or gene expression. Our
study was only designed to detect whether IVS
62

⫹60G⬎A is related to the prognosis of pa-

tients with prostate cancer. Further study of the
molecular consequences of this SNP would have

to be studied to determine its effect on carcino-
genesis. The results of our study have indicated
that the ATM polymorphism IVS62

⫹60G⬎A is

not associated with aggressiveness of disease in
white patients with prostate cancer.

REFERENCES

1. Parkin DM, Laara E, and Muir CS: Estimates of the

worldwide frequency of sixteen cancers in 1980. Int J Cancer
41: 184 –197, 1988.

2. Parkin DM, Pisani P, and Ferlay J: Estimates of the

world-wide incidence of 25 major cancers in 1990. Int J Can-
cer 80: 870 – 873, 1999.

3. Parkin DM, Bray FI, and Devesa SS: Cancer burden in

the year 2000: the global picture. Eur J Cancer 37(suppl):
S4 – 66, 2000.

4. Quinn M, and Babb P: Patterns and trends in prostate

cancer incidence, survival, prevalence and mortality. Part I:
international comparisons. BJU Int 90: 162–173, 2002.

5. Siegal JA, Yu E, and Brawer MK: Topography of neovas-

cularity in human prostate carcinoma. Cancer 75: 2545–2551,
1995.

6. Savitsky K, Bar-Shira A, Gilad S, et al: A single ataxia

telangiectasia gene with a product similar to PI-3 kinase. Sci-
ence 268: 1749 –1753, 1995.

7. Gatti RA, Berkel I, Boder E, et al: Localization of ataxia-

telangiectasia gene to chromosome 11q22-23. Nature 336:
577–580, 1988.

8. Uziel T, Savitsky K, Platzer M, et al: Genomic organiza-

tion of the ATM gene. Genomics 33: 317–320, 1996.

9. Platzer M, Rotman G, Bauer D, et al: Ataxia telangiec-

tasia locus: sequence analysis of 184 kb of human genomic
DNA containing the entire ATM gene. Genome Res 7: 592–
605, 1997.

10. Kastan MB, and Lim DS: The many substrates and func-

tions of ATM. Nat Rev Mol Cell Biol 1: 179 –186, 2000.

11. Borresen AL, Anderson TI, Treti S, et al: Breast cancer

and other cancers in Norwegian families with ataxia-telangi-
ectasia. Genes Chromosom Cancer 2: 339 –340, 1990.

12. Swift M, Morrell D, Massey RB, et al: Incidence of can-

cer in 161 families affected by ataxia-telangiectasia. N Engl
J Med 325: 1831–1836, 1991.

13. Maillet P, Chappuis PO, Vaudan G, et al: A polymor-

phism in the ATM gene modulates the penetrance of heredi-
tary non-polyposis colorectal cancer. Int J Cancer 88: 928 –
931, 2000.

14. American Joint Committee on Cancer: Prostate, in

AJCC Cancer Staging Manual, 6th ed. New York, Springer,
2002, pp 309 –316.

15. Angèle S, Falconer A, Edwards SM, et al: ATM polymor-

phisms as risk factors for prostate cancer development. Br J
Cancer 91: 783–787, 2004.

16. Thorstenson Y, Shen P, Tusher V, et al: Global analysis

of ATM polymorphism reveals significant functional con-
straint. Am J Hum Genet 69: 396 – 412, 2001.

UROLOGY 67 (6), 2006

1323