SHORT REPORT SOMATIC MUTATIONS IN THE BRCA2 GENE AND HIGH FREQUENCY OF ALLELIC LOSS OF BRCA2 IN SPORADIC MALE BREAST CANCER Eliza KWIATKOWSKA 1 , Marek TERESIAK 2 , Danuta BREBOROWICZ 3 and Andrzej MACKIEWICZ 1 * 1 Department of Cancer Immunology, University School of Medical Sciences and Great Poland Cancer Center, Poznan, Poland 2 Department of Surgery II, Great Poland Cancer Center, Poznan, Poland 3 Department of Pathomorphology, Great Poland Cancer Center, Poznan, Poland Breast cancer occurs rarely in men and risk factors for the disease include germline mutations of the BRCA2 gene. High frequency of allelic loss at the BRCA2 locus has been reported in sporadic breast tumors, but somatic mutations of BRCA2 are very rare. Here we report the first case of somatic BRCA2 mutation in male breast cancer with demonstrated loss of heterozygosity. We analyzed a series of 27 archival samples from male breast cancer patients for BRCA2 mutations and loss of heterozygosity at BRCA2 locus. The mutation analysis of BRCA2 gene was performed using SSCA-HA and sequenc- ing methods. PCR was used to detect LOH at 3 highly poly- morphic microsatellite markers spanning BRCA2 region on 13q by comparing the allelic pattern in matched tumor and blood DNA samples. In this study LOH at the BRCA2 locus was observed in 82.6% of informative cases, confirming pre- vious observations on high frequency of LOH affecting the BRCA2 region in male breast cancer. We identified 5 somatic BRCA2 mutations in a set of 23 sporadic male breast cancers (21%). Two silent and 1 missense alterations were novel BRCA2 variants. Here we also report first somatic frameshift BRCA2 mutation in male breast cancer 8138del5. In 3 tumors with somatic BRCA2 alterations, 1 missense, 1 silent and frameshift LOH at chromosome 13q12-13 were detected and losses involved a wild–type allele of BRCA2 gene. © 2002 Wiley-Liss, Inc. Key words: male breast cancer; BRCA2; germline mutations; so- matic mutations; loss of heterozygosity Carcinoma of the male breast is an uncommon disease, account- ing for less than 1% of all malignancies detected in men. The most important risk factor for the disease in both male and female breast cancer seems to be inherited predisposition. Two genes, BRCA1 and BRCA2 account for the disease in large majority of breast cancer families. 1 Unlike BRCA1 mutations, germline mutations of BRCA2 are involved in the development of a considerable number of male breast cancer. 2–5 LOH on chromosome 13q12-13 was reported in 20 – 60% of spo- radic breast tumors. 6–8 Such high frequency of allelic loss at BRCA2 locus points to an important role of this gene in the development and progression of sporadic breast cancer. However, in sporadic breast cancers somatic mutations of BRCA2, like BRCA1, are very rare. Here we report the first case of somatic BRCA2 mutation in male breast cancer with demonstrated loss of heterozygosity. We analyzed a series of 27 archival samples from male breast cancer patients for BRCA2 mutations and loss of heterozygosity at BRCA2 locus. Four patients were previously identified as BRCA2 mutation carriers (4T, 8T, 37T and 38T), whereas the other 23 tumors studied were considered sporadic since no germline BRCA2 mutations were found in these male breast cancer patients. 9 Family history data were obtained from each patient. Two mutation carriers (4T and 38T) and 2 patients with sporadic breast cancer (13T and 22T) had a family history of breast cancer in one first- (38T, 13T, 22T) or second-degree (4T) relative. There was no case with a family history of ovarian cancer. Beside breast cancer, relatives were also af- fected with cancer of the lung, prostate (1 case), stomach, liver, brain, skin, larynx and female genital tract. None of the 27 patients had evidence of familial breast cancer. DNA was isolated from paraffin-embedded tumor tissues and peripheral blood leukocytes of each patient using Wizard Genomic DNA Purification Kit (Promega, Madison, WI). The entire coding region of BRCA2 gene and exon/intron splice junctions were amplified from genomic DNA with 63 primer pairs, and the length of amplified fragments varied from 136 –300 base pairs. The mutation analysis of BRCA2 gene was performed using the SSCA-HA method. PCR products from variant conformers de- tected in SSCA-HA analyses were purified and subsequently sequenced in both directions using fmol DNA Sequencing Sys- tem (Promega). PCR was used to detect LOH at 3 highly polymorphic microsatellite markers spanning the BRCA2 region on the long arm of chromosome 13 by comparing the allelic pattern in matched tumor and blood DNA samples. D13S260 and D13S171 markers are located centromeric to the BRCA2 gene, whereas D13S267 is located telomeric. PCR was run in a total volume of 5 l1 PCR buffer (Promega), 200 nM of each deoxynucleotide triphosphate, 1.5 mM MgCl 2 ,1 M Cy5- labeled forward primer, 1 M reverse primer with 50 ng of DNA and 0.1 unit of Taq DNA polymerase. The annealing temperatures were adapted to each primer pair. Thirty-five cycles of amplification (10 sec at 94°C, 10 sec at 50 –55°C, 20 sec at 72°C) were followed by a final extension step for 7 min at 72°C. Two microliters of amplified products were separated in denaturing polyacrylamide gel ReproGel High Resolution (Pharmacia, Uppsala, Sweden) run on an AlfExpress II DNA Sequencer. AlleleLink version 1.0 software was used for quan- tification of PCR products. Blood lymphocytes DNA samples heterozygous at a given locus were considered to be “informa- tive,” whereas homozygotes were considered “uninformative.” LOH was evaluated only in informative cases. LOH was con- sidered to occur when the intensity of tumor tissue band was reduced by more than 50% when compared to normal tissue band. SSCP-HA analyses revealed 5 somatic BRCA2 alterations in breast tumor tissues but not in blood lymphocytes of patients 16T, 21T, 23T, 24T and 29T. Sequencing of variant alleles from these patients demonstrated 1 frameshift mutation and 4 point mutations. The frameshift mutation detected in patient 16T was a 5-bp dele- tion at nucleotide 8138, resulting in termination codon at position 2639. Among the point mutations, 2 were missense variants of the BRCA2 gene: Thr1915Met (5972C/T, patient 21T) and novel vari- ant Cys1580Tyr (4967G/A, patient 23T), whereas the remaining 2 were novel silent variants: 8727G/A (Lys2833Lys, patient 24T) and 8706C/T (Tyr2826Tyr, patient 29T). In cases were somatic *Correspondence to: Great Poland Cancer Center, Department of Cancer Immunology, Garbary 15, 61-866 Poznan, Poland. Fax: +48-61-8528502. E-mail: amac@amu.edu.pl Received 16 August 2001; Revised 25 October 2001; Accepted 7 De- cember 2001 Published online 20 February 2002 Int. J. Cancer: 98, 943–945 (2002) © 2002 Wiley-Liss, Inc. DOI 10.1002/ijc.10289 Publication of the International Union Against Cancer