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CHEK2-Positive Breast in Young Polish Women Cezary Cybulski,1Bohdan Go¤ rski,1 Tomasz Huzarski,1 Tomasz Byrski,1Jacek Gronwald,1 Ta d e u s z D e˛bniak,1Dominika Wokołorczyk,1 Anna Jakubowska,1Elz˙bieta Kowalska,1 Oleg Oszurek,1Steven A. Narod,2 and Jan Lubin´ski1

Abstract Purpose: To investigate the contribution of CHEK2 to early-onset in Poland and to establish the characteristic features of these cancers. Experimental Design: We studied 3,228 women diagnosed with breast cancer under the age of 51 years and 5,496 population controls. CHEK2 mutations were detected by RFLP-PCR or allele-specific oligonucleotide-PCR assays. Clinical and pathologic features of CHEK2-positive cases and CHEK2-negative cases were compared. Results: AtruncatingCHEK2 (1100delC or IVS2+1G>A) was seen in 47 of 3,228 cases and in 34 of 5,496 controls (odds ratio, 2.4; P =0.0001).TheCHEK2 I157T missense mutation was presentin 207 of 3,228 cases, compared with264 of 5,496 controls (odds ratio, 1.4; P = 0.002). Breastcancers in women witha CHEK2 mutation were more commonly of lobular histology (21.5% versus 15.8%; P =0.05),ofsize>2cm(54.8%versus43.5%;P =0.01),orof multicentric origin (28.7% versus 19.5%; P = 0.01) than were cancers from women without a CHEK2 mutation. Bilateral cancers were equally common in both subgroups. Conclusion: Three founder alleles in CHEK2 contribute to early-onset breast cancer in Poland. Breasttumors which arise in carriers of CHEK2 mutations seem to be similar to those of breast cancers in the population at large.

CHEK2 encodes the human analogue of yeast checkpoint Materials and Methods kinases Cds1 and Rad53 (1, 2). Activation of these in response to DNA damage prevents cellular entry into . Patients. This study includes prospectively ascertained cases of A founder allele in CHEK2, 1100delC, has been reported to be invasive breast cancer diagnosed throughout Poland from 1996 to a low-penetrance breast cancer susceptibility allele in several 2003. The study was initiated in Szczecin in 1996 and was extended to studies, and in many ethnic groups (3–6). Other CHEK2 include Poznan˜ in 1997 and Olsztyn in 1998. Fifteen additional centers V variants (IVS2+1G>A, I157T, and S428F) have also been began recruiting subjects in 2001. All patients diagnosed at age 50 years before 2003 at the affiliated hospitals were eligible. Patients with suggested to confer increased breast cancer risks in different CHEK2 pure intraductal or intralobular cancer were excluded (ductal populations (7–9). Three founder mutations have in situ or lobular ) but patients with ductal carcinoma been described in Poland (1100delC, IVS2+1G>A, and I157T) in situ with microinvasion were included. Patients with or and one is present in the Ashkenazi Jewish population (S428F). lymphoma were excluded. Patients with a previous breast cancer were To investigate the contribution of CHEK2 mutations to early- included, provided the cancer was in the opposite breast. onset breast cancer and to establish the characteristic features A total of 4,316 incident cases of invasive breast cancer were of these cancers, we studied 3,228 early-onset breast cancer identified at the 18 different hospitals during the study period. Of these, cases and 5,496 population controls from Poland. Poland is 3,484 women accepted the invitation to participate (80.7%). Five well suited for this study because of the high frequency of patients withdrew their participation at a later time. A blood sample CHEK2 founder mutations and the relative genetic homogene- was obtained and DNA was successfully isolated from 3,474 cases. Among these, 198 women carried one of the three Polish founder ity of the Polish population. BRCA1 mutations (4153delA, 5328insC, and C61G) and were excluded from the present study. These cases are described elsewhere (10). The medical record and pathology report were reviewed locally by the physician associated with the study and relevant information was Authors’Affiliations: 1International Hereditary Cancer Center, Department of forwarded to the study center in Szczecin. Information was recorded on and Pathology, Pomeranian Medical University, Szczecin, Poland and age at diagnosis, stage, grade and lymph node status, estrogen- 2Centre for Research onWomen’s Health,Toronto, Ontario, Canada status, multicentricity, and bilaterality. This information was collected Received 1/23/06; revised 5/12/06; accepted 5/31/06. without knowledge of the mutation status of the individuals. Women The costs of publication of this article were defrayed in part by the payment of page with a previous contralateral breast cancer or with a current diagnosis of advertisement charges. This article must therefore be hereby marked in accordance bilateral cancer were considered to be bilateral. Patients with previous with 18 U.S.C. Section 1734 solely to indicate this fact. ipsilateral cancers were excluded. The study was approved by the Ethics Requests for reprints: Cezary Cybulski, International Hereditary Cancer Center, Department of Genetics and Pathology, Pomeranian Medical University, ul. Committee of the Pomeranian Medical University in Szczecin, Poland. Połabska 4, 70-115 Szczecin, Poland. Phone: 48-91-466-1532; Fax: 48-91-466- Pathology review. Tumor blocks and/or paraffin-embedded slides 1533; E-mail: [email protected]. were requested from the corresponding pathology centers. One or more F 2006 American Association for . specimens were obtained from 3,136 of the 3,474 patients. A central doi:10.1158/1078-0432.CCR-06-0158 pathology review was conducted in Szczecin by two pathologists

Clin Cancer Res 2006;12(16) August 15, 2006 4832 www.aacrjournals.org CHEK2-Positive Breast Cancers in Young Polish Women associated with the study. Pathologists were blinded to mutation status. with a truncating mutation was 1.8 years lower than of women Each case was reviewed with regard to histology (medullary, ductal, without a CHEK2 mutation (42.5 versus 44.3 years; P = 0.01). lobular, tubulo-lobular, other). The characteristics of the breast cancer cases in the 252 CHEK2 Mutation analysis. mutations were detected as previously women with a CHEK2 mutation are presented in Table 2 and described (11). compared with noncarriers. The distribution of histologic types Population controls. To estimate the frequency of the Polish founder CHEK2 mutations in the general population, three control groups were was similar in cases and controls. However, carriers of a combined. The first group consisted of 2,183 newborn children from mutation were slightly more likely to be diagnosed with lobular 10 hospitals throughout Poland (Szczecin, Białystok, Gorzo´w, cancer than were noncarriers (21.5% versus 15.8%; P = 0.05). Katowice, Wrocław, Poznan´ , Opole, Ło´dz´, and Rzeszo´w) between Intraductal cancers (ductal carcinoma in situ) with micro- 2003 and 2006. Samples of cord blood from unselected infants were were also more common in women with a CHEK2 forwarded to the study center in Szczecin. The second control group was mutation than in noncarriers (11.3% versus 7.2%; P = 0.06). taken from healthy adult patients (1,079 women and 817 men) of three However, several comparisons were made and these differences family doctors practicing in the Szczecin region. These individuals were were of marginal significance. selected randomly from the patient lists of family doctors. The Carriers and noncarriers were similar with respect to tumor third control group consisted of 1,417 young adults (705 women and size, but tumors of sizes >2 cm were slightly more common 712 men) from Szczecin who submitted blood for paternity testing. CHEK2 There was no association between the allele frequencies and either age among carriers of a mutation than in noncarriers P or sex in our control population. The frequency of a CHEK2 mutation (53.9% versus 43.5%; = 0.01) and tumors of sizes <1 cm were was 5.6% in 1,529 adult men and 5.4% in 1,784 adult women. less common in carriers compared with noncarriers (5.9% The frequency was 5.3% in 2,183 newborns, 5.5% in 2,056 controls versus 11.2%; P = 0.05). between the ages 15 and 50 years, and 5.4% in 1,257 controls of ages Carriers and noncarriers were similar with respect to estrogen >50 years. receptor status (65.1% versus 63.7%; P = 0.8) and lymph-node Cancer history was available for 1,074 women from family doctors status (45% positive versus 40.1%; P = 0.3). Bilateral tumors CHEK2 (age range, 15-91 years; mean 58.3 years). The frequency of a were equally common in both subgroups (2.3% versus 3.3%; mutation in the 1,074 cancer-free women was 5.5% (5.0% for the P = 0.6). In contrast, CHEK2-associated cancers were signifi- I157T and 0.5% for a truncating variant). CHEK2 cantly more likely to be multicentric (28.7%) than were tumors Statistical analysis. The prevalence of the three alleles in P cases and population controls was compared. Odds ratios (OR) were in noncarriers (19.5%; = 0.01). CHEK2 generated from two-by-two tables and statistical significance was The great majority of the women with a mutation assessed with the Fisher exact test. The ORs were used as estimates did not have a strong family of cancer—only 13.8% of the of relative risk. women with breast cancer with a CHEK2 mutation were from Because of the potential for to alter tumor character- a family with two or more first-degree relatives with breast istics, women who received neoadjuvant chemotherapy were excluded cancer. However, this was more frequent than reported by the from the analyses of histopathology type, size, nodal status, multi- noncarriers (8.9%) and the difference was statistically signifi- focality, and presence of estrogen receptors. However, they were cant (OR, 1.6; P = 0.02). retained for comparisons of bilaterality and family history.

Results Discussion

A CHEK2 mutation was identified in 252 of 3,228 women We identified a CHEK2 mutation in 7.8% of unselected early- with breast cancer (7.8%), including I157T (207 times), onset breast cancer patients in Poland and have confirmed the IVS2+1G>A (31 times), and 1100delC (16 times). Overall, contribution of three founder alleles to the burden of breast the OR for early-onset breast cancer in association with a cancer. Overall, the tumors that arise in carriers of CHEK2 CHEK2 mutation was 1.5 [95% confidence interval (95% CI), mutations seem to be similar to those of breast cancers in the 1.2-1.8; Table 1]. The OR was higher (OR, 2.4; 95% CI, 1.5-3.7) population at large. However, carriers of CHEK2 mutation were for women with a truncating mutation (1100delC or slightly more likely to develop lobular breast cancer than IVS2+1G>A) than for women with a missense mutation (OR, noncarriers (21.8% versus 15.8%; P = 0.05). Previous analyses 1.4; 95% CI, 1.1-1.6). The mean age of diagnosis in women show that neither BRCA1 nor BRCA2 is associated with an with a CHEK2 mutation was similar to that of the noncarrier increased frequency of lobular breast cancer (12, 13). However, cases (Table 2). However, the mean age of diagnosis of women increased frequency of lobular cancer was seen in non-BRCA1/2

Table 1. Prevalence of CHEK2 mutations in BRCA1-negative early-onset breast cancer cases and controls with corresponding ORs

Cases (N = 3,228) Controls (N = 5,496) OR (95% CI) P IVS2+1G>A 31 22 2.4 (1.4-4.2) 0.002 1100delC 16 12 2.3 (1.1-4.8) 0.04 Any truncating 47 34 2.4 (1.5-3.7) 0.0001 I157T 207 264 1.4 (1.1-1.6) 0.002 Total 252* 297* 1.5 (1.2-1.8) <0.0001

*Two cases and one control had both truncating and missense mutations.

www.aacrjournals.org4833 Clin Cancer Res 2006;12(16) August 15, 2006 Human Cancer Biology

Table 2. Comparison of CHEK2-positive and CHEK2-negative breast cancers cases

CHEK2 mutation negative CHEK2 mutation positive P Age (mean), y 44.3 44.2 0.7 Age group, y 20-30 1.7% (52 of 2,976) 3.6% (9 of 252) 0.07 31-40 17.2% (512 of 2,976) 15.9% (40 of 252) 0.7 41-50 81% (2,412 of 2,976) 80.5% (203 of 252) 0.9 Histology Ductal, low grade 26.9% (622 of 2,315) 29.3% (54 of 186) 0.6 Ductal, high grade 12.7% (294 of 2,315) 10.7% (20 of 186) 0.5 Medullary 4.9% (113 of 2,315) 2.1% (4 of 186) 0.1 Lobular 15.8% (366 of 2,315) 21.5% (40 of 186) 0.05 Tubulo-lobular 3.6% (83 of 2,315) 4.3% (8 of 186) 0.8 Ductal carcinoma in situ 7.2% (168 of 2,315) 11.3% (21 of 186) 0.06 Other 5.1% (118 of 2,315) 4.8% (9 of 186) 1.0 Missing or unknown 23.8% (551 of 2,315) 16.7% (31 of 186) 0.03 Preoperative chemotherapy 24.5% (661 of 2,693) 27.7% (66 of 238) 0.3 ER-positive 63.7% (1,048 of 1,646) 65.1% (97 of 149) 0.8 Size, cm <1 11.2% (193 of 1,728) 5.9% (9 of 152) 0.05 1-2 45.3% (783 of 1,728) 40.1% (61 of 152) 0.2 >2 43.5% (752 of 1,728) 53.9% (82 of 152) 0.01 Lymph nodes positive 40.1% (722 of 1,777) 45.0% (68 of 151) 0.3 Multicentric 19.5% (316 of 1,619) 28.7% (41 of 143) 0.01 Bilateral 3.3% (84 of 2,531) 2.3% (5 of 215) 0.6 Family history positive 8.9% (237 of 2,652) 13.8% (31 of 224) 0.02

NOTE: For all comparisons, except age, bilaterality, and family history, the cases with preoperative chemotherapy are excluded. Family history refers to a first-degree relative affected with breast cancer.

familial breast cancers, suggesting there may exist other breast unknown susceptibility in women with a family susceptibility genes that predispose specifically to bilateral breast cancer (17). Others have also suggested that lobular cancer (14). In addition, intraductal cancers (ductal CHEK2 might act in synergy with other cancer susceptibility carcinoma in situ) with microinvasion were more common in genes (3, 5, 18). In this scenario, predisposition to bilateral Polish women with a CHEK2 mutation than in noncarriers breast cancer might be a result of the combined effect of a (11.3% versus 7.2%; P = 0.06). In addition, CHEK2 mutation CHEK2 mutation and a mutation in a modifying . It is theo- carriers were more likely to present with tumors >2 cm or to retically possible that the prevalence of modifying alleles might have tumors of multicentric origin than were noncarriers (P = vary between the United Kingdom and Polish populations. 0.01). However, we have made numerous statistical compar- In previous studies, the CHEK2 1100delC mutation has been isons here and none of the associations was very strong. These found in excess in familial breast cancer patients (3–5, 15). We data need to be confirmed in other studies. found evidence for a higher prevalence of a CHEK2 mutation A recent report from the Netherlands described breast cancers among cases with a first-degree relative affected with breast in 34 CHEK2 1100delC carriers and in 102 noncarriers. They cancer than in cases with first-degree relative unaffected with found a significant difference between the two groups regarding breast cancer (OR, 1.6; 95% CI, 1.1-2.4; P = 0.02); however, the steroid receptor status—carriers more frequently had ER- effect was small and the great majority of patients with a positive tumors (91% versus 69%; P = 0.03; ref. 15). In our CHEK2 mutation in our series of unselected breast cancer larger group of CHEK2 mutation carriers, we saw a similar patients (86%) had no affected first-degree relative. prevalence of ER-positive tumors (65.1%) as in noncarriers Our results confirm that the I157T variant is pathogenic for (63.7%). breast cancer. It was originally reported that the I157T variant A recent follow-up study reported that patients with the does not seem to increase the risk of breast cancer (19, 20) and CHEK2 1100delC variant had a high risk of contralateral that CHEK2 carrying the I157T change has similar breast cancer (relative risk, 5.7; 95% CI, 1.7-19.7; ref. 15). This kinase activity, expression levels, and subcellular localization finding is supported by other studies that reported a six times as endogenous CHEK2 (21). The I157T missense variant is higher prevalence of CHEK2 1100delC carriers among patients localized in a functionally important domain of CHEK2 (the with bilateral breast cancer, as compared with patients with FHA domain) and protein with this mutation has been shown unilateral breast cancer (5, 16). Surprisingly, we saw a similar to be defective in its ability to bind and phosphorylate Cdc25A prevalence of bilateral cases among Polish women with a and to bind and BRCA1 (22–24). The I157T protein may CHEK2 mutation (2.3%), compared with those without a also have a dominant negative effect by forming heterodimers CHEK2 mutation (3.3%), but the numbers were small and we with wild-type CHEK2 (8). CHEK2 I157T variant has been restricted our analysis to early-onset cases. reported to be associated with increased breast cancer risk in A recent study from the United Kingdom suggested a Finland, Germany, and Belarus (8, 25). This variant has been multiplicative interaction between CHEK2 1100delC and other found to be associated with a susceptibility to other forms of

Clin Cancer Res 2006;12(16) August 15, 2006 4834 www.aacrjournals.org CHEK2-Positive Breast Cancers in Young Polish Women

cancer in Finland including prostate and colorectal cancers mutation carriers resemble the breast cancers that arise in the (26, 27). It has also been found to be associated with a population at large, but there is a suggested excess of lobular susceptibility to chronic lymphocytic in the United and multicentric tumors. Additional studies are required to Kingdom (28). In Poland, we saw a positive association confirm these initial findings. between the I157T variant and breast, prostate, colon, kidney, and thyroid cancers (7), and recently, bladder, endometrial, Acknowledgments and low-grade ovarian cancers.3 In conclusion, we estimate that f8% of early-onset breast We thank the following people who helped in this study: W. Domagala, M. cancers arise in Poland in women with a germ line CHEK2 Chosia,M.Ucin´ski, E. Grzybowska, D. Lange, B. Mika, A. Mackiewicz, A. Karczew- ska, J. Breborowicz, K. Lamperska, M. Stawicka, S. Gozdecka-Grodecka, mutation. Both missense and truncating mutations seem to M. Be˛benek, D. Sorokin, A.Wojnar, O. Haus, J. Sir,T.Mierzwa, S. Niepsuj, K. Gugała, be pathogenic. The minority of mutation-positive cases have S. Goz´dz´, J. Sygut, B. Kozak-Klonowska, B. Musiatowicz, M. Posmyk, R. Kordek, M. familial breast cancer. The tumors that arise in CHEK2 Morawiec, O. Zambrano, B.Was´ko, L. Fudali, D. Surdyka, K. Urban´ski, J. Mitus´,J. Rys´, M. Szwiec, A. Rozmiarek, I. Dziuba, P.Wandzel, R. Wis´niowski, C. Szczylik, A. Kozak, A. Kozłowski, R. Czajka, B. Czeszyn´ska, B. De˛bniak, G. Brembowicz, 3 Unpublished data. B. Kaluz˙ewski, A.Witek, A. Skret, A. Lenczewski, J.Wonton, and R. Gibaszek.

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