Human Cancer Biology Loss of Heterozygosity at the BRCA2 Locus Detected by Multiplex Ligation-Dependent Probe Amplification is Common in Prostate Cancers from Men with a Germline BRCA2 Mutation Amber J. Willems,1Sarah-Jane Dawson,2 Hema Samaratunga,4 Alessandro De Luca,5 Yoland C. Antill,2 John L. Hopper,3 Heather J. Thorne1 and kConFab Investigators Abstract Purpose: Prostate cancer risk is increased for men carrying a pathogenic germline mutation in BRCA2 , and perhaps BRCA1. Our primary aim was to test for loss of heterozygosity (LOH) at the locus of the mutation in prostate cancers from men who a carry pathogenic germline mutation in BRCA1 or BRCA2 , and to assess clinical and pathologic features of these tumors. Experimental Design: From 1,243 kConFab families: (a) 215 families carried a pathogenic BRCA1 mutation, whereas 188 families carried a pathogenic BRCA2 mutation; (b)ofthe158 men diagnosed with prostate cancer (from 137 families), 8 were confirmed to carry the family- specific BRCA1 mutation, whereas 20 were confirmed to carry the family-specific BRCA2 mutation; and (c) 10 cases were eliminated from analysis because no archival material was available. The final cohort comprised 4 and 14 men with a BRCA1 and BRCA2 mutation, respec- tively. We examined LOH at the BRCA1 and BRCA2 genes using multiplex ligation-dependent probe amplification of DNA from microdissected tumor. Results: LOH at BRCA2 was observed in 10 of 14 tumors from BRCA2 mutation carriers (71%), whereas no LOHat BRCA1 was observed in four tumors from BRCA1 mutation carriers (P =0.02). Under the assumption that LOH occurs only because the cancer was caused by the germline mutation, carriers of BRCA2 mutations are at 3.5-fold (95% confidence interval, 1.8-12) increased risk of prostate cancer. A high Gleason was the only distinct clinical feature. Conclusions: These observations are consistent with the idea that BRCA2 ,butnotBRCA1, is a tumor suppressor of prostate cancer. Prostate cancer, a common cancer diagnosed in Western men, high individual risk has been unsuccessful, perhaps due to is a major cause of morbidity and mortality. Risk factors genetic heterogeneity. Studies are under way to find common include family history, especially if relatives are diagnosed at genetic variants associated with a small increased risk (2). a young age (1). To date, the search for genes associated with a Rare germline mutations in the breast cancer predisposition genes, BRCA1 and, in particular, BRCA2, have been implicated in susceptibility to prostate cancer (3). The issue is complicated Authors’Affiliations: 1Kathleen Cuningham Consortium for Research into Familial by the fact that, except in populations with founder mutations, Breast Cancer (kConFab), Research Department, Peter MacCallum Cancer Centre; only indirect estimates of the prevalence of germline mutations 2Department of Haematology and Medical Oncology, Peter MacCallum Cancer in unaffected men are available. These estimates have wide 3 Centre; and Centre for Molecular, Environmental, Genetic and Analytic confidence intervals (that have not necessarily been correctly Epidemiology, School of Population Health,The University of Melbourne, Victoria, BRCA2 Australia; 4Department of Anatomical Pathology, Sullivan Nicolaides Pathology, reported). The prevalence of mutations in selected Taringa, Queensland, Australia; and 5Centre for Translational and Applied series of men with prostate cancer is low, around 1% to 2%, but Genomics, British Columbia CancerAgency,Vancouver, BC, Canada these estimates are imprecise (4, 5). In addition, estimates of Received 12/20/07; revised 2/17/08; accepted 2/18/08. penetrance from mutation testing case series have not been Grant support: Peter Mac Foundation; the National Breast Cancer Foundation; collected systematically and have used information from the National Health and Medical Research Council (grants 145684, 288704, and 454508); and by the Queensland Cancer Fund; the Cancer Councils of New relatives (6), leading to potential biases. The best data have South Wales,Victoria,Tasmania, and South Australia; and the Cancer Foundation of come from modified segregation analysis of mutation-carrying Western Australia. families identified from studying multiple-case breast and The costs of publication of this article were defrayed in part by the payment of page ovarian cancer families. These data suggest that the risk of charges. This article must therefore be hereby marked advertisement in accordance prostate cancer is 4.7-fold higher [95% confidence interval with 18 U.S.C. Section 1734 solely to indicate this fact. BRCA2 Note: Y.C. Antill is supported by a RACP Research Fellowship. J.L. Hopper is an (95% CI), 3.5-6.2] in men carrying germline mutations Australia Fellow of the National Health and Medical Research Council. than in the general population. This relative risk (RR) is esti- Requests for reprints: HeatherThorne, kConFab, Research Department, Peter mated to be higher in carriers when younger than 65 years MacCallum Cancer Centre, Locked Bag 1, A’Beckett Street, Victoria, Australia (RR, 7.3; 95% CI, 4.7-11.5; ref. 3). 3002. Phone: 61-3-9187-1501; Fax: 61-3-9656-1457; E-mail: Heather.Thorne@ BRCA1 petermac.org. In the context of breast and ovarian cancers, and F 2008 American Association for Cancer Research. BRCA2 are classic tumor suppressor genes as first described doi:10.1158/1078-0432.CCR-07-5237 by Knudson (7) for retinoblastoma. Breast tumors arising in www.aacrjournals.org 2953 Clin Cancer Res 2008;14(10) May 15, 2008 Downloaded from clincancerres.aacrjournals.org on October 1, 2021. © 2008 American Association for Cancer Research. Human Cancer Biology mutation carriers usually display inactivation of both copies of Eligibility for inclusion in this prostate cancer study required each the corresponding gene (8–13); one allele is inactivated as a participant to be a carrier of a germline pathogenic mutation in either result of the inherited germline mutation, whereas the second BRCA1 or BRCA2, have a confirmed diagnosis of prostate cancer, and allele is somatically inactivated, typically as a consequence for there to be access to archival tumor material. Mutation status was of deletion (8–11) but less frequently by epigenetic changes confirmed by either a predictive clinical mutation test or by extended genotyping within a family by the kConFab laboratory. (12–15). Whatever the mechanism, the absence of a functional All prostate cancers were verified through the State or Centralised wild-type allele from tumors fits the Knudson 2-hit model and Cancer Registries or by a diagnostic pathology report. All participants suggests a causal link between the presence of a germline gave informed consent and approval for the study was obtained from BRCA1 or BRCA2 mutation and the development of breast or the human research ethics committee at the Peter MacCallum Cancer ovarian malignancy. Centre. BRCA1 and BRCA2 mutation carriers are on average at 10 or more times population risk of breast cancer, and more so for Nucleic acid isolation ovarian cancer (16). Therefore, although the proportion of LOH analysis. Archival blocks for all tissues used were obtained breast and ovarian cancers in mutation carriers that arise from pathology laboratories in Australia and New Zealand. A 3- to A independent of them being a mutation carrier is <10%, it is still 5- m section stained with H&E was marked by a pathologist for tumor rich (>95%) regions. Tumor cells were microdissected from an overlaid >0%. That is, women who carry germline mutations may still unstained section. DNA was extracted from unstained sections of the develop breast or ovarian cancers via other pathways. Although formalin-fixed paraffin-embedded tissue using the DNeasy blood & rare, the proportion that does is a reflection of the extent of tissue kit (Qiagen) and a modification of the protocol by Wu et al. (23), an increase in cancer risk experienced by carriers. In other which involves a 3-d incubation at 56jC with the addition of 240 Ag words, if the increased risk of cancer in mutation carriers is proteinase K at 24-h intervals. RR-fold, then 100 Â RR / (RR + 1) % of carriers who develop Methylation analysis. The DNA from the paraffin-embedded tis- cancer will have done so through being a carrier. On average, sues was extracted using the Puregene DNA Purification kit (Gentra the tumors of 100 / (RR + 1) % of carriers would not necessarily Systems) according to the manufacturer’s instructions for paraffin- have the same biology or pathology as those that have been embedded tissue. caused by the germline mutation. We shall use the reverse of LOH testing this argument to estimate the increased risk of prostate cancers Analysis of LOH was done using the multiplex ligation-dependent for germline mutation carriers under some assumptions. probe amplification (MLPA) technique, which allows for relative To date, only two small studies have reported on loss of quantification of multiple DNA fragments in a single reaction (24). BRCA2 heterozygosity (LOH) in prostate cancers from muta- The BRCA1 and BRCA2 MLPA kits (MRC-Holland) were used as tion carriers (9, 17). In the first study, LOH analysis was done described by the manufacturer, using 55- to 100-ng tumor DNA. MLPA on prostate cancers from four brothers, at least three of whom reactions were analyzed on the ABI 3100 Prism Genetic Analyser carried a family-specific BRCA2 mutation. Two tumors showed (Applied Biosystems), and dosage profiles were assembled using the loss of the wild-type allele for at least one informative GeneMarker software (v.1.2; SoftGenetics) by comparison to a reference microsatellite marker, whereas heterozygosity was retained in sample without large genomic rearrangements in BRCA1 or BRCA2.As the remaining two tumors (17). The second study identified an additional quality assurance measure, samples known to carry large BRCA1 BRCA2 LOH in six of seven (86%) prostate cancers from men with genomic rearrangements ( del exon 5 and del exons 14_16) were also included in each set of MLPA reactions.