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PARK2<FRA6E and Afadin in breast cancer

A Letessief et al

A    6q26-<*27

B    ^a    No break    b    Monosomy

c PARK2 break with d PARK2 break with loss of 5’ region    loss of 3' region

Figurę I Break of tlić PA RK2 loeus in hreast eanccr. (Ai Map of the loeus. (a) Idcogram of chroinosotne 6 showing Ihc P. 1RK2 loeus al 6q26(clir6:16l 740081 163 119 211) and the AF-6 MLLT4 loeus al 6q27 (el»rf>:168046 404 168 183532). (b> The Iwo pools of BAC’ elones uscd as probes in I1SII cxpcrimcnts. To dctcci a break of PA RK2. tumors prcscnt on a IMA wcrc hybridi/ed using biotinylalcd and dig-labeled scqucnces in llie 5' (green) and 3' (redl regions of PARK2 rcvcaled aft er dctcction in green and red. respcctńcly. (e) l.nlarged PARK2 loeus sliowing exon intron organization and the iwo ovcrlapping BAC’ elones uscd as probes. (Bi L\amplcs of PARK2 status in breast eanccr detennined by I 1SII on IMA. (a) fu mor with intact PARK2 gene: two wihJ-typc eopiesof PARK2avc seen as clusters of green and red signals (arrows). (b) Tuinor with monosomy of PARK2 gene: only one wild-typc copy is scen and revealed by elustered green and red signals (arrow). (e) Tumor with break of PARK2 gene seen as a split of the elustered signals and disappcarancc of one green signal (arrow), suggesting the loss of the 5' region of the loeus. (d)Tuinor with break of PARK2 gene seen as a split of llie elustered signals and disappcarancc of one red signal (arrow ). suggesting the loss of the 3' region of the loeus.

canccrs. Moreover. AF-6 encoded product. Afadin. is expressed in norinal breast epithelium and is involvcd in epithclial celi architecture. We thus focused our atten-tion on Afadin. Wc lirst validatcd the two diflerent anti-Afadin antibodies (Tablc 2) on mammary celi lines by Western biot and IHC (data not shown).

We itwesiigalcd the expression of Afadin in breast tumors by IHC on TM A sections. Afadin was strongly exprcssed in the cytoplasm and at thecellular membranę of the epithelial cells of normal breast (Figurę 3Ba). As measured by the quick score (QS). two distinct levels of Afadin cxpression were obsersed in the 352 tumors with reliable results (Supplemenlary Table 1). In 301 tumors (85.5%). the level of Afadin was similar to that of normal breast tissue (Figurę 3Bb). In 51 tumors (14.5%). there was no Afadin ewpression (QS 0) (Figurę 3Bc).

Loss of Afadin ewpression correlates with PARK 2 break and prognosis

Loss of Afadin did not correlate with any of the studied histoclinical factors (Table 3). It tended to be associated with high pathological size (P 0.05389). Intercstingly, 36% of Afadin-negatise tumors (four cases) had break of PARK2. whercas only 5% of Afadin-positive tumors (seven cases) had break of PARK2 {P<0.001). Thus. loss of Afadin was associated with a PARK2 break. Other pathological mechanisms. such as mutations. intragenie delelions and epigenetic modilicalions. may explain loss of cxprcssion in the 64% of Afadin-ncgativc tumors without break of PARK2.

These results led us to examine the impact o!' Afadin expression on cli ni cal outeome. When we considcred the w hole populadon of patients analysed for Afadin ewpression (N 352). the 5-year MFS was 67.8% (55.3 82.9) for patients with an Afadin-negativc tumor. and 81.8% (77.4 86.5) for patients with an Afadin-positiee tumor (P 0.046) (Figurę 4a). When we considered the lymph node-negative populalion of patients (A'    181). the 5-year MFS was 72.8% (56.1

94.5) for patients with an Afadin-negative tumor. and 89.5% (84.5 94.7) for patients with an Afadin-positive tumor {P 0.0496) (Figurę 4b). Loss of Afadin was thus associated with poor outeome in tliis group of patients.

Oncogene