Fátima Vaz

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.

Meningeal carcinomatosis and uterine carcinoma: three different clinical settings and review of the literature.

Introduction: Leptomeningeal carcinomatosis is a rare metastatic event in gynecological neoplasias, and most cases occur in ovarian cancer. It is extremely infrequent in cervical cancer, and so far, there are not any reports of this complication in association with endometrial cancer. Patients and Methods: We report a case of leptomeningeal carcinomatosis secondary to endometrial carcinoma and 2 complex cervix cancer cases. A MEDLINE search was done to review all published cases of this complication in gynecological cancer to identify predictive factors for this diagnosis. Results and Discussion: Leptomeningeal carcinomatosis is usually diagnosed late in the course of the disease, and most reports concern ovarian cancer patients. The number of cases describing this neurologic complication in cervix cancer is increasing. Gadolinium-enhanced magnetic resonance imaging may be necessary for this diagnosis, because cerebrospinal fluid analysis results may be negative. Most cervix cases had squamous cell (8/14) or neuroendocrine histologic subtype (3/14), and when reported, differentiation was usually poor. The case we report of endometrial carcinoma, unique in the literature, is a serous adenocarcinoma. Conclusions: A high index of suspicion is necessary, and leptomeningeal carcinomatosis should be considered in patients with unexplained neurologic symptoms whose gynecologic tumors are poorly undifferentiated or have a serous component.

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 attending a breast cancer risk evaluation clinic.

CTRC-AACR San Antonio Breast Cancer Symposium: 2008 Abstracts Abstract #1103 Background - Counselling for hereditary breast cancer risk has been mostly directed to women and their female counterparts. However, BRCA1/2 male carriers are at risk for several cancers and their motivation for counselling and follow up is mostly unknown. Material and methods - Review of individual and family records from all male pts counseled in our clinic from Jan2001-Dec2007. Results: eighty one male pts were counseled, 32 previously affected with cancer (mean age 61yrs) and 49 healthy men (mean 41 yrs). Mostly, all affected pts were refered to or invited to as probands for BRCA2 screenning. All but 2 of these accepted screening after counseling, being the concern with their offspring the main motivation for testing. With 3 exceptions all unaffected men were unvited for counselling due to the identification of BRCA2 mutations in their families: 6/49 refused or postponed genetic testing after counselling (mean age 39 yrs). All male BRCA carriers (total of 32) are BRCA2 positive: 12 index probands affected with Breast (10 cases, 3 bilateral) or prostate cancer ( 2 cases) and 20 healthy carriers under increased surveillance (yearly observations at the Breast Clinic + Dermatology and Urology). Psychosocial support is also available but only one male BRCA2 carrier, a prostate cancer survivor with previous depressive symptons, has this support regularly. Male carriers did adhere to increased surveillance and 5 neoplasias were diagnosed in 4 BRCA2 male breast cancer survivors: 3 cases of asymptomatic prostate cancer (Gleasons 9, 7 and 6 respectivly) 1 case of contralateral breast cancer and 1 case of gastric cancer. All 4 pts are alive without evidence of disease, after appropriate treatment. Conclusions: men belonging to BRCA families are at risk for several cancers and seem to adhere to genetic testing and surveillance. Guidelines for hereditary breast cancer management should be comphreensive and include orientations to counsell male individuals at risk. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 1103.

The Use of Sentinel Lymph Node Biopsy in BRCA1/2 Mutation Carriers Undergoing Prophylactic Mastectomy: A Retrospective Consecutive Case-Series Study

Introduction Sentinel lymph node biopsy in prophylactic mastectomy is controversial. It avoids lymphadenectomy in occult carcinoma but is associated with increased morbidity. Women with BRCA mutations have a higher incidence of occult carcinoma and our objective was to assess the clinical utility of sentinel lymph node biopsy when these women undergo prophylactic mastectomy. Materials and Methods Seven-year retrospective consecutive case-series study of women, with a BRCA deleterious mutation, admitted to prophylactic mastectomy, at our center. Breast MRI < 6 months before surgery was routine, unless contraindicated. Results Fifty-seven patients (43% BRCA1; 57% BRCA2) underwent 80 prophylactic mastectomies. 72% of patients had had breast cancer treated before prophylactic mastectomy or synchronously to it. The occult carcinoma incidence was 5%, and half of the cases were invasive. SLNB was performed in 19% of the prophylactic mastectomies; none of these had tumor invasion. Women with invasive carcinoma who had not undergone sentinel lymph node biopsy were followed closely with axillary ultrasound. The median follow-up was 37 months, with no local recurrence; 1 patient died of primary tumor systemic relapse. Conclusions Our data do not support this procedure for routine (in agreement with previous literature), in this high risk for occult carcinoma population.

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.

Abstract P4-12-14: Distress as a measure of the psychological impact after disclosure of a BRCA1/2 positive test result

Introduction and objectives- A positive result after BRCA1/2 screening can represent a difficult psychological experience. Previous studies have shown that the psychological outcomes following BRCA1/2 testing vary according to the previous individual and family experiences of each person. Objectives of this study were to measure the distress caused by the disclosure of a positive BRCA1/2 test result and to analyse the degree of BRCA1/2 carriers retention of information, transmitted at the post-test counselling interview. Material and Methods-This is a prospective study. All consecutive individuals with a BRCA1/2 positive test were invited to participate, after the post-test counselling visit. Participation involved signing an informed consent form and agreeing to a structured post-test phone interview, one week and one month after disclosure of the test result. Phone interviews were done by nurses trained by the Psychological Unit of our centre. Measure instruments: 1) Emotional thermometer (ET) - analogical scale ranging from 0 (no distress) to 10 (maximum distress), measures distress during the previous week 2) Distress questionnaire (DQ)- 13 items to evaluate depression, anxiety and loss of emotional control, with a global score ranging from 3 (no distress) and 45 (maximum distress). 3) Knowledge of disease status and understanding of the individualized risk management plan- additional 4 items included at the end of DQ. Subgroup analysis was performed according to age, sex, previous cancer diagnosis and offspring existence using Wilcoxon rank sum test with continuity correction. Results-From 177 eligible carriers, 28 were not included (14 for logistical reasons; 2 deaths; 1 refused; 3 progressive symptomatic disease ). A total of 149 carriers were included: 120 women (81%) and 29 men (19%); median age 43 yrs (21-74); 67 (45%) with a previous cancer diagnosis and 82 (55%) healthy at risk; 42 (28%) had no offspring and 102 (68%) were professionally active. The mean distress scores were 3.07 (SD 2.72) and 20.13 (SD 7.88) for ET and DQ instruments, respectively. Using the NCCN (2013) guidelines for ET classification, we found that 95 (64%) of our carriers did not have clinically significant distress. For the DQ (using a cut-off Subgroup analysis: A statistically significant difference was found with both ET and DQ for higher distress levels in women than men (p=0.006 and p than 50yrs), previous cancer diagnosis or with vs no offspring. Levels of knowledge and understanding of individual risk management were high (average 18.7; maximum 20) and no correlation was found with distress levels. Twenty-eight (19%) carriers were found in need of specialized psychological/psychiatric support and were appropriately referred. Conclusions-In our BRCA1/2 carrier population clinically significant distress was not frequent and only 19% needed specialized psychological/psychiatric support. Distress was higher in women than in men. Retention of information given during counselling was high, and there was no correlation between information retention and distress levels. Citation Format: Joana Parreira, Susana Esteves, Fatima Vaz, Carla Simoes, Paula Rodrigues, Ana Luis, Ana Clara, Sandra Bento, Maria Jesus Moura. Distress as a measure of the psychological impact after disclosure of a BRCA1/2 positive test result [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P4-12-14.

Abstract P2-12-12: Breast cancer in young women: Fertility preservation as a component of treatment planning and discussion

Background: as part of education and informed consent before cancer therapy, health care providers should address the possibility of infertility with patients (pts) treated during their reproductive years, and be prepared to discuss fertility preservation options and/or to refer all potential patients to Reproductive Specialists. I here we describe the recently established referral protocol of our institution with a fertility preservation centre. Both institutions belong to the Portuguese National Health System. Methods: the referral protocol includes female pts, ≤ 39 years old, with a diagnosis of breast or gynecological cancer or sarcoma with indication to curative intention chemotherapy. The possibility of fertility preservation is primarily discussed at the cancer centre and only interested pts are referred to the reproductive clinic, where specialized counseling is provided. Fertility preservation procedures (FPPs) (oocyte, embryo and/or ovarian tissue cryopreservation) are conditional to a previous evaluation of the ovarian pool (OP). OP markers are: antral follicle count, follicle stimulating hormone (FSH) and anti-mullerian hormone (AMH). Only good prognosis cases are eligible for FPPs. Results: After primary discussion with eligible breast cancer (BC) pts, 17 were referred to the reproductive clinic: median age at BC diagnosis was 34 yrs (29-38), median previous pregnancies number of 0 (0-2) and median number of children of 0 (0-2). Marital status: most (64%) pts had a companion but 36% did not. All pts except 1 were candidates for immediate chemotherapy, either in the adjuvant or neoadjuvant setting. In the adjuvant setting (9 pts), median time from surgery until starting systemic treatment was 61.5 (43-146) days (aprox. 8,8 weeks); the case with a 146 day interval before chemotherapy was due to surgical complications and not to FPP. In the neoadjuvant setting (5 pts), median time between multidisciplinary decision and systemic treatment beginning was 17 (11-24) days (2,4 weeks). One patient was proposed to hormone therapy only after BC surgery and underwent FPP before systemic treatment. The timing for primary discussion of FPP was in multidisciplinary decision consultation. After counselling at the reproductive clinic, 2 pts declined FPP (%). For the other 13 pts oocyte cryopreservation was the FPP mostly used, although in one case embryo cryopreservation was performed. One case of ovarian hyperstimulation syndrome was observed but this FPP complication resolved without sequela and this event did not cause delay in chemotherapy treatment. Conclusions: our collaboration protocol allows for an efficacious referral of BC pts seeking fertility preservation counselling. Although BC patients may be focused initially on their cancer diagnosis, health care providers are encouraged to advise them regarding potential threats to fertility. Formal collaboration between cancer and reproductive centres, like the one described here, are crucial so as to allow for the widest array of options for fertility preservation and to prevent delay in cancer treatment. We intend to follow up these patients in order to realistically understand the impact of this practice in fertility and quality of life of cancer patients. Citation Format: Hugo Nunes, Fatima Vaz, Margarida Brito, Claudia Melo, Ana Teresa Almeida Santos, Antonio Moreira. Breast cancer in young women: Fertility preservation as a component of treatment planning and discussion [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P2-12-12.

898PPRIMARY CYTOREDUCTIVE SURGERY (PCS) VS NEOADJUVANT CHEMOTHERAPY (NACT) FOR ADVANCED OVARIAN CARCINOMA (AOC): DECISION CRITERIA AND EFFICACY OUTCOMES

ABSTRACT Aim: PCS is the standard of care for aOC but NACT and interval debulking surgery (IDS) are acceptable treatment options for patients (pts) with stages IIIC and IV OC. Our aims are to characterize OC pts treated with PCS or NACT in our centre and to analyse decision criteria and efficacy outcomes of both strategies. Methods: All pts with ovarian tumours registered at the Southern Portuguese Cancer Registry (ROR-Sul) between 2006-2011 were reviewed for age at diagnosis, histology, FIGO stage, treatment (none, surgery and/or CT), progression free survival (PFS), overall survival (OS), criteria for NACT, number of platinum based CT cycles and residual macroscopic disease. Results: From 345 pts registered with ovarian tumours, 258 were epithelial ovarian cancers (EOC) (75%). EOC pts had a median age of 62 yrs (10-90); 64 (25%) were treated with surgery alone, 53 (21%) with NACT (stages IIIB-3, IIIC-15, IV-29) and 94 (37%) with PCS (25% stages IA-IC; 23% IIA-IIIA; 51% IIIB-IV).Other pts (17%) were not treated at our centre (only pathology review, multidisciplinary decision or death before treatment). For similar stages (IIIB-IV), median age was higher for NACT pts (64 yrs, 36-90) vs 59 yrs (33-82); median number of CT cycles was 8 (NACT) and 6 (PCS). Nine pts in the NACT group progressed and died before IDS. Complete resection of macroscopic disease was achieved in 24 (55%) of NACT pts and in 17 (36%) of PCS pts. Median OS and PFS were 29,2 and 8,1 months (NACT) and 38,1 months and 10,6 months (PCS). Bad prognosis pathology (mucinous/clear cell) was observed in 10 pts (3 NACT, 7 PCS). NACT decision was based on radiological criteria (74% of cases) such as implants >2cm outside the pelvis, lymphadenopathies above renal hilum, pre-sacred retroperitoneal disease or liver metastasis. In 14 pts (26%), comorbidities contraindicated upfront surgery. Conclusions: NACT was mostly decided for older pts with contraindications for upfront surgery and/or with radiologically determined unresectable disease. Observed OS was similar to that observed in trials with more fit pts. OS for the PDS group is in line with previous reports of single centre studies with experience in OC treatment. Disclosure: All authors have declared no conflicts of interest.