Patricia Machado

Screening for a BRCA2 Rearrangement in High-Risk Breast/Ovarian Cancer Families: Evidence for a Founder Effect and Analysis of the Associated Phenotypes

PURPOSE BRCA2 rearrangements are rare genetic events. A large BRCA2 genomic insertion was recurrently observed in our participants, and we sought to characterize it at the molecular and phenotypic level. PATIENTS AND METHODS We studied 210 high-risk breast/ovarian cancer families. Fifty-three probands were fully screened for BRCA1/2 mutations, and three of 53 had a large insertion in exon 3 of BRCA2. This finding was analyzed by polymerase chain reaction (PCR), reverse transcriptase PCR (RT-PCR), and sequencing. An additional 157 consecutive families were screened for this mutation by a three-step PCR method. Phenotype and haplotype analysis was also performed. RESULTS Sixteen BRCA mutations were observed in 19 of 53 patients (36% detection rate). A recurrent Alu motif insertion in position c.156_157 was observed after sequencing of an abnormal fragment obtained after the amplification of BRCA2 exon 3. RT-PCR revealed exon 3 skipping. Screening of this rearrangement identified 14 additional families (out of 157). In total, 17 (8%) of 210 high-risk families ascertained in our clinic were positive for this mutation. Segregation of a common haplotype (from D13S260 to D13S1695) confirmed a common origin, estimated to have occurred 2,400 to 2,600 years ago. The following four cancer phenotypes were observed in the 17 positive families: female breast (n = 9), male breast (n = 4), breast/ovarian (n = 2), and heterogeneous (n = 2). Male breast cancer was more frequently observed in c.156_157insAlu-positive families compared with negative families (23% v 12%, respectively), and 33% of all male breast cancer families with an identified BRCA mutation were c.156_157insAlu positive. CONCLUSION c.156_157insAlu is a founder mutation of Portuguese origin and is the most frequent BRCA2 rearrangement described to date.

Familial breast/ovarian cancer and BRCA1/2 genetic screening : The role of immunohistochemistry as an additional method in the selection of patients

Only 20–25% of families screened for BRCA1/2 mutations are found positive. Because only a positive result is informative, we studied the role of BRCA1/2 immunohistochemistry as an additional method for patient selection. From 53 high-risk-affected probands, 18 (34%) had available paraffin blocks of their tumors and were selected for this study. Mutation screening was done by conformation-sensitive gel electrophoresis and multiplex ligation-dependent probe amplification. For immunohistochemistry, 21 neoplastic specimens (15 breast carcinomas, 5 ovary neoplasms, and 1 rectal adenocarcinoma) were analyzed with BRCA1 (monoclonal antibody, Ab-1, oncogene) and BRCA2 (polyclonal antibody, Ab-2, oncogene) antibodies. Absence of the BRCA1 protein was confirmed in negative tumors by Western blotting. Seven patients were positive for BRCA1/2 mutations: 5 for BRCA1 and 2 for BRCA2. Four out of five positive patients had tumors negative for BRCA1 immunostaining, and the remaining 13 BRCA1-negative patients had positive BRCA1 immunostaining in all tumor samples. Sensitivity to predict for BRCA1 mutation carriers was 80%, and specificity was 100%, with a positive predictive value of 100% and a negative predictive value of 93%. This correlation was statistically significant (p=0.001). No correlation was observed for BRCA2. If larger studies confirm these results, high-risk patients with BRCA1-negative tumors should be screened first for this gene.

Caution should be taken in the methodology used to confirm c.156_157insAlu BRCA2 mutation

To the Editor We read with great interest the article by Peixoto et al. [1] about the screening of the c.156_157insAlu BRCA2 mutation in Northern Portugal. As we previously reported, [2] this BRCA2 rearrangement first described by Teugels et al. [3] in a Portuguese family living in Belgium, will surely be the explanation for breast and ovarian cancer risk in several families of Portuguese ancestry. Most of these families have been, as reported by Peixoto et al. [1], considered negative by the usual screening methods. We are writing this letter to comment about the methodology that must be used to confirm the splicing effect of the c.156_157insAlu mutation. Peixoto et al. report RT–PCR data in their paper, that corroborates our own data concerning the segregation of c.156_157insAlu BRCA2 mutation and cancer in individuals from positive families, but this should not be taken as the most appropriate methodology for the confirmation of this rearrangement. The primers used for their RT–PCR reactions amplify not only the pathogenic Alu-mediated splicing product, lacking exon 3, but also a physiologic, ubiquitous splicing product, also exon 3-deficient, observed in individuals with sporadic cancers and healthy persons [4–8]. Although both splicing products lack exon 3, only the one associated with c.156_157insAlu mutation includes at least the first part of BRCA2 exon 10 and this distinguishes them [9]. Primers like the ones used by Peixoto et al., located in exons 1 and 6 of BRCA2 cDNA, amplify (as shown in Fig. 1a of their report) three fragments in all samples, both c.156_157insAlu carriers and wild type carriers. As a confirmation step, RT–PCR is expected to amplify only true positive samples, originating from really positive c.156_157insAlu carriers. Although the authors explain the three bands observed, as a rule, for diagnosis, we should avoid relying on subjective observations like band intensity. For reasons of length, we did not include the reference to the ubiquitous exon 3-defficient transcript in our original paper [2] but we did discuss it when, very pertinently, Diez et al. [4] wrote a letter raising doubts about our methodology to confirm c.156_157insAlu as a pathogenic BRCA2 mutation. They were very well aware of previous data showing a physiological BRCA2 transcript lacking exon 3 [5–8]. Their questions allowed us, in a reply [9], to explain that indeed, we had tried several primers for our RT–PCR confirmation step and eventually chose primers 1FcDNA/ 10RcDNA, first described by Nordling et al. [10], because these never amplify the ubiquitous splicing transcript. As in the case of our families, and in the case of the BRCA2 family described by Nordling et al., they only amplify a BRCA2-mutated associated transcript. Our three-step-PCR (patent pending) was optimized and every step decided after several studies. The first two steps described by Peixoto et al., are so similar to our own that we believe that no false positive was reported: from our experience, with a total of 29 c.156_156insAlu families already diagnosed, if the first two steps are positive the third always confirms it. But BRCA1 and BRCA2 molecular diagnosis has implications for individuals and their families at clinical, emotional, legal and ethical levels. No doubt must remain in the methodologies for screening, P. Machado F. Vaz (&) Molecular Biology Department, Instituto Portugues de Oncologia de Lisboa, Francisco Gentil, Lisbon, Portugal e-mail: fvaz@ipolisboa.min-saude.pt

Men seeking counselling in a Breast Cancer Risk Evaluation Clinic

Background Hereditary breast and ovary cancer syndrome affects both genders but little is known about the uptake of genetic services by men. The objective of this study is to characterise the male population counselled through a multidisciplinary breast/ovarian program. Methods Descriptive analysis of male patients counselled from January 2000 to December 2015. Data in this analysis include new cancer diagnoses during prospective follow up. Results From 4,320 families registered, 362 male patients were identified: 236 (65.2%) from hereditary cancer families (HCF) and 126 (34.8%) from non-HCF. In HCF, 121 patients (51.3%) were mutation carriers (MC): BRCA2 – 102 (84.3%), BRCA1 – 16 (13.2%), CHEK2 – 1 (0.8%) and TP53 – 2 (1.7%). Non-HCF included 126 patients: 85 (67.5%) belonged to families without pathogenic mutations or with variants of unknown clinical significance; 22 (17.5%) refused testing after counselling and 19 (15.0%) did not meet criteria for testing. Both HCF and non-HCF included patients with previous cancer diagnoses: HCF- Breast Cancer (BC) - 18; prostate cancer (PC) - 13; melanoma - 1; others - 7) and non-HCF (BC - 77; PC - 20; gastric cancer (GC) - 1; melanoma - 8; bladder cancer - 1; others - 22). From the 121 MC identified (including the TP53 and CHEK2 carriers), 97 patients (80.2%) adhered to prospective surveillance. With a median follow-up of 36.9 months, 17 cancers were diagnosed in 14 patients, PC being the most frequently diagnosed neoplasia (5 cases). Eleven patients (78.6%) are alive and three patients died of advanced cancer (2 with GC, 1 with disseminated adenocarcinoma). Conclusion We observed a high adherence to counselling, genetic testing and active surveillance by men belonging to hereditary BC families. Male carriers of pathogenic DNA variants are at risk for several cancers and should be included in prospective follow-up studies.