Yannick Bidet

Molecular Analysis of the Breast Cancer Genes BRCA1 and BRCA2 Using Amplicon-Based Massive Parallel Pyrosequencing

The aim of this study was to implement the massively parallel sequencing technology for diagnostic applications. We evaluated an amplicon-based method for the analysis of the BRCA1 and BRCA2 genes on the Roche 454 GS-FLX sequencer, to identify disease-causing mutations in breast and/or ovarian cancer patients. A first evaluation relied on the analysis of DNA fragments containing known mutations. Secondly, the entire coding regions of the BRCA1 and BRCA2 genes were interrogated in more than 400 patient samples, using a multiplex PCR-based assay. Variants were filtered on the basis of their frequency (20%) and sequencing depth (>25×). Special attention was given to sequencing accuracy in homopolymers. In the initial evaluation, all known heterozygous mutations were detected. The percentage of mutant reads ranged from 22% to 62%. For the multiplex assay, 95% sensitivity and 91% specificity were obtained. In addition, we were able to reliably distinguish mutations from noise through the analysis of the raw signal intensities in homopolymers. This work presents an evaluation of the next-generation sequencing for use in diagnostics, based on a relatively high number of samples and experiments. We anticipate that the technique would further improve, and would allow reducing the costs per analysis and the turn-around time, to benefit patients who undergo BRCA molecular testing.

BRCA Mutations Increase Fertility in Families at Hereditary Breast/Ovarian Cancer Risk.

Background Deleterious mutations in the BRCA genes are responsible for a small, but significant, proportion of breast and ovarian cancers (5 - 10 %). Proof of de novo mutations in hereditary breast/ovarian cancer (HBOC) families is rare, in contrast to founder mutations, thousands of years old, that may be carried by as much as 1 % of a population. Thus, if mutations favoring cancer survive selection pressure through time, they must provide advantages that compensate for the loss of life expectancy. Method This hypothesis was tested within 2,150 HBOC families encompassing 96,325 individuals. Parameters included counts of breast/ovarian cancer, age at diagnosis, male breast cancer and other cancer locations. As expected, well-known clinical parameters discriminated between BRCA-mutated families and others: young age at breast cancer, ovarian cancer, pancreatic cancer and male breast cancer. The major fertility differences concerned men in BRCA-mutated families: they had lower first and mean age at paternity, and fewer remained childless. For women in BRCA families, the miscarriage rate was lower. In a logistic regression including clinical factors, the different miscarriage rate and mens mean age at paternity remained significant. Results Fertility advantages were confirmed in a subgroup of 746 BRCA mutation carriers and 483 non-carriers from BRCA mutated families. In particular, female carriers were less often nulliparous (9.1 % of carriers versus 16.0 %, p = 0.003) and had more children (1.8 ± 1.4 SD versus 1.5 ± 1.3, p = 0.002) as well as male carriers (1.7 ± 1.3 versus 1.4 ± 1.3, p = 0.024). Conclusion Although BRCA mutations shorten the reproductive period due to cancer mortality, they compensate by improving fertility both in male and female carriers.

BRCA1 and BRCA2 Mutations in Ethnic Lebanese Arab Women With High Hereditary Risk Breast Cancer

PURPOSE Breast cancer is the most common malignancy among women in Lebanon and in Arab countries, with 50% of cases presenting before the age of 50 years. METHODS Between 2009 and 2012, 250 Lebanese women with breast cancer who were considered to be at high risk of carrying BRCA1 or BRCA2 mutations because of presentation at young age and/or positive family history (FH) of breast or ovarian cancer were recruited. Clinical data were analyzed statistically. Coding exons and intron-exon boundaries of BRCA1 and BRCA2 were sequenced from peripheral blood DNA. All patients were tested for BRCA1 rearrangements using multiplex ligation-dependent probe amplification (MLPA). BRCA2 MLPA was done in selected cases. RESULTS Overall, 14 of 250 patients (5.6%) carried a deleterious BRCA mutation (7 BRCA1, 7 BRCA2) and 31 (12.4%) carried a variant of uncertain significance. Eight of 74 patients (10.8%) aged ≤40 years with positive FH and only 1 of 74 patients (1.4%) aged ≤40 years without FH had a mutated BRCA. Four of 75 patients (5.3%) aged 41-50 years with FH had a deleterious mutation. Only 1 of 27 patients aged >50 years at diagnosis had a BRCA mutation. All seven patients with BRCA1 mutations had grade 3 infiltrating ductal carcinoma and triple-negative breast cancer. Nine BRCA1 and 17 BRCA2 common haplotypes were observed. CONCLUSION Prevalence of deleterious BRCA mutations is lower than expected and does not support the hypothesis that BRCA mutations alone cause the observed high percentage of breast cancer in young women of Lebanese and Arab descent. Studies to search for other genetic mutations are recommended.

Improved Efficiency and Reliability of NGS Amplicon Sequencing Data Analysis for Genetic Diagnostic Procedures Using AGSA Software

Screening for BRCA mutations in women with familial risk of breast or ovarian cancer is an ideal situation for high-throughput sequencing, providing large amounts of low cost data. However, 454, Roche, and Ion Torrent, Thermo Fisher, technologies produce homopolymer-associated indel errors, complicating their use in routine diagnostics. We developed software, named AGSA, which helps to detect false positive mutations in homopolymeric sequences. Seventy-two familial breast cancer cases were analysed in parallel by amplicon 454 pyrosequencing and Sanger dideoxy sequencing for genetic variations of the BRCA genes. All 565 variants detected by dideoxy sequencing were also detected by pyrosequencing. Furthermore, pyrosequencing detected 42 variants that were missed with Sanger technique. Six amplicons contained homopolymer tracts in the coding sequence that were systematically misread by the software supplied by Roche. Read data plotted as histograms by AGSA software aided the analysis considerably and allowed validation of the majority of homopolymers. As an optimisation, additional 250 patients were analysed using microfluidic amplification of regions of interest (Access Array Fluidigm) of the BRCA genes, followed by 454 sequencing and AGSA analysis. AGSA complements a complete line of high-throughput diagnostic sequence analysis, reducing time and costs while increasing reliability, notably for homopolymer tracts.

Early Onset Multiple Primary Tumors in Atypical Presentation of Cowden Syndrome Identified by Whole-Exome-Sequencing

A family with an aggregation of rare early onset multiple primary tumors has been managed in our oncogenetics department: the proband developed four early onset carcinomas between ages 31 and 33 years, including acral melanoma, bilateral clear cell renal carcinoma (RC), and follicular variant of papillary thyroid carcinoma. The proband’s parent developed orbital lymphoma and small intestine mucosa-associated lymphoid tissue (MALT) lymphoma between 40 and 50 years old. Whole-exome-sequencing (WES) of the nuclear family (proband, parents, and sibling) identified in the proband a de novo deleterious heterozygous mutation c.1003C > T (p.Arg335∗) in the phosphatase and tensin homolog (PTEN) gene. Furthermore, WES allowed analysis of the nuclear family’s genetic background, and identified deleterious variants in two candidate modifier genes: CEACAM1 and MIB2. CEACAM1, a tumor suppressor gene, presents loss of expression in clear cell RC and is involved in proliferation of B cells. It could explain in part the phenotype of proband’s parent and the occurrence of clear cell RC in the proband. Deleterious mutations in the MIB2 gene are associated with melanoma invasion, and could explain the occurrence of melanoma in the proband. Cowden syndrome is a hereditary autosomal dominant disorder associated with increased risk of muco-cutaneous features, hamartomatous tumors, and cancer. This atypical presentation, including absence of muco-cutaneous lesions, four primary early onset tumors and bilateral clear cell RC, has not been described before. This encourages including the PTEN gene in panel testing in the context of early onset RC, whatever the histological subtype. Further studies are required to determine the implication of CEACAM1 and MIB2 in the severity of Cowden syndrome in our proband and occurrence of early onset MALT lymphoma in a parent.

Abstract 5224: Next-generation sequencing of a panel of genes contributing to breast cancer risk

BRCA mutations account for a minority of hereditary breast cancer risk families. Many other genes have been identified, with rare but highly penetrant mutations, or more frequent mutations giving modest breast cancer risk. Next generation sequencing has made it possible to explore a wide sample of genes for their contribution to breast cancer risk. We have sequenced gene panels in two populations of non-BRCA breast cancer families. First, 47 French families were selected for triple-negative breast cancer in the index case and >1 first degree relative with breast cancer. A panel of 33 genes involved in cancer risk, DNA repair, and/or partners of BRCA1 or BRCA2 was sequenced using DNA capture and GS-Flex sequencing. Second, a Lebanese cohort of 25 non-BRCA cases with Manchester scores >18 was sequenced on a MiSeq after capture of a similar panel of 37 genes. BRCA1 and BRCA2 were included in both panels to confirm previously obtained results. Large rearrangements were detected using MLPA. Sequence alignment and variant identification was performed using a combination of commercial software and an in-house GATK-based pipeline. Variants were described as neutral or likely neutral if the MAF in online databases exceeded 1%, if in silico analyses suggested neutrality, and if splicing prediction algorithms agreed on the absence of any significant effect. NGS analysis confirmed the absence of BRCA mutations. Deleterious mutations were observed in three Lebanese cases: two with a deletion in PALB2 and one stop mutation in ATM. Nine additional Lebanese cases and four from the French TNBC group were found to carry variants of unknown significance (VUS) in one or more genes. Most cases were found to have one VUS; two TNBC cases had more than one VUS and one Lebanese case had a VUS and a PALB2 mutation. The cases with deleterious mutations had Manchester scores of 56, 22 and 19. Scores for cases with VUSs ranged from 18 to 45 (average 23), and those with only neutral or likely neutral variants ranged from 18 to 27 (average 21.8). These differences are not significant, but additional cases are under study and will be presented. No difference was observed between French TNBC families with or without VUSs. DNA capture of gene panels is a quick and efficient way of analyzing multiple genes involved in hereditary cancer risk. Our panels focused on a limited number of genes involved in DNA repair and/or direct interactions with the BRCA genes: the small number of genes allowed us to analyze a maximum number of patients per experiment on two relatively low-throughput machines. Although the two cohorts are small, they show that the number of variants requiring follow-up in non-BRCA genes is low. The absence of deleterious mutations in genes involved in homologous recombination repair in the TNBC cohort showed that although genes of this pathway are candidates for tumor sensitivity to PARP inhibitors, the candidates we tested may not be frequently constitutionally mutated. Citation Format: Nancy A. Uhrhammer, Nagi El Saghir, Yannick Bidet, Manon Delahaye-Sourdeix, Flora Ponelle, Marie Ollier, Sandrine Viala, Firas Kreidieh, Nathalie Khoueiry-Zgheib, Yves-Jean Bignon. Next-generation sequencing of a panel of genes contributing to breast cancer risk. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 5224.

Abstract P1-03-06: BRCA1 and BRCA2 mutations in ethnic Lebanese Arab high risk women for hereditary breast cancer

Background: Breast cancer is the most common malignancy in women in Lebanon and Arab countries with 50% of cases below age 50. The incidence of hereditary breast cancer in Lebanese and Arab women is unknown. Methods: 250 Lebanese women with breast cancer, of young age with or without family history, were recruited at the American University of Beirut Medical Center (AUBMC) between 2009 and 2012. Study was approved by IRB. All signed an informed consent. Risk assessment questionnaire, medical chart review, and whole blood were collected. Coding exons and intron-exon boundaries of BRCA1 and BRCA2 were sequenced. Full BRCA gene sequencing was performed at Institut Jean Perrin, France. Study was funded in part by an Ethnic Research Initiative (ERI) grant awarded by GSK. Results: 14 out of the 250 patients (5.6%) had a deleterious BRCA mutation (7 BRCA1, 7 BRCA2) and 31 (12.4%) had a variant of uncertain significance (VUS). Table 1 shows deleterious BRCA mutations based on age group and FH. All 7 BRCA1 mutation carriers had a positive family history, were between 32 and 48 years of age, and had Grade 3 IDC with negative ER, PR, HER2 receptors (TNBC). Six BRCA2 mutation carriers had IDC with positive hormone receptors (HR) and 2 had HER2-positive disease. We found 31 VUS. One VUS (BRCA2) was seen in two sisters with breast cancer. One VUS (BRCA2) was seen in 4 patients and another in 2 patients, while 2 VUS (BRCA1) mutations were seen in 2 sets of 2 patients. The significance of these VUS cannot be ascertained at this time. Haplotype analysis is ongoing. Conclusions: This is the first large study of ethnic Lebanese Arab women with breast cancer. The prevalence of BRCA deleterious mutations in women with breast cancer who are considered high risk of carrying a BRCA mutation is 5.6% in our total cohort, while in patients ≤40 with positive FH it is 10.6%. Those numbers are lower than expected from US and European populations. Tumor grade and pathology characteristics in this patient population correlated with that previously documented for BRCA1 (TNBC) and BRCA2 (positive HR) associated breast cancers. Our data supports use of young age together with positive FH should be used to select patients for counseling and BRCA testing in Lebanon and Arab countries with resource-sensitive guidelines. Several VUS were found in patients and sisters with breast cancer. The finding that 94.4% of high risk patients had no deleterious BRCA mutations suggests the need to look for alternate gene mutations and other factors that may contribute to the development of breast cancer in these high risk patients. Conclusions regarding haplotypes and diversity will be reported at the meeting. Citation Format: Nagi El Saghir, Nancy Uhrhammer, Hussein Assi, Katia Khoury, Stephanie Decousous, Yannick Bidet, Sara Jaber, Raghid Charara, Rania Farhat, Ziad Salem, Ali Shamseddine, Arafat Tfayli, Jaber Abbas, Faek Jamali, Muhieddine Seoud, Deborah Armstrong, Yves-Jean Bignon, Nathalie Zgheib. BRCA1 and BRCA2 mutations in ethnic Lebanese Arab high risk women for hereditary breast cancer [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 P1-03-06.

Abstract 4669: Response to the anti-EGFR antibody panitumumab combined with standard neoadjuvant chemotherapy in triple negative breast cancer (TNBC): the immune and IGFR pathways.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Background: EGFR overexpression is one of the hallmarks of the “basal-like” TNBC definition by immunohistochemistry (IHC) (Nielsen, 2004). In a phase II neoadjuvant clinical trial targeting EGFR in TNBC, we investigated various biomarkers to better identify an EGFR-sensitive population for potential further regimen development. Methods: Sixty patients (pts) with stage II-IIIA TNBC were prospectively included. Systemic treatment (ST) consisted of the anti-EGFR antibody panitumumab combined with FEC 100, followed by 4 cycles of docetaxel. All pts underwent surgery after ST completion. Patient characteristics: median tumor size: 40 mm (20-120); invasive ductal carcinoma: 96.7%; SBR grade III: 71.7%; complete pathological response (pCR) rate: 46.8% (Chevalliers classification). Paraffin-embedded and frozen tumor samples were collected before and after ST for biologic studies. Germinal BRCA1 mutations, and EGFR, KRAS, BRAF and PI3KCA somatic mutations were analyzed by NGS. EGFR, IGF-1R, MET, cytokeratins 5/6 and 8/18, PTEN, P-cadherin, ALDH1, Ki-67, p53, tumoral FOXP3 expression and the number of FOXP3+ or CD8+ tumor-infiltrating lymphocytes (TILs) were evaluated by IHC. Biopsies and surgical samples were analysed using Affimetrix arrays. Results : By univariate analysis, high CD8+ TILs was response-predictive (pCR rates: CD8 TILs: 84% high vs 10% low; p=3.4.10−6). Tumor FOXP3 expression and high FOXP3 TILs tended to be predictive (pCR rates: tumor FOXP3: 77% positive vs 36% negative, p=0.07; FOXP3 TILs: 66% high vs 37% low, p= 0.08). High IGF-1R expressors responded better than low expressors (80% vs 33%, respectively, p=0.028). As could be expected, a positive correlation between tumor FOXP3 expression and FOXP3 TILs was found (p = 2.7.10−4, r = 0.56). Surprisingly, a positive correlation was found between FOXP3 TILs and CD8+ TILs (p = 2.10−5, r = 0.59) and between tumor FOXP3 and CD8+ TILs (p = 6.3.10−3, r = 0.43). Comparison of EGFR, IGF-1R and Her3 in biopsies versus surgical samples showed reduced EGFR levels in non-responders (p = 0.037), while Her3 (p = 0.049) and IGF-1R (p = 0.08) increased. Sequencing revealed BRCA1 mutations in 10% of pts. No difference of response (pCR) was observed between mutated patients and others (p=0.91). Somatic mutations of PI3K were observed in 6 pts. No mutations were observed in BRAF, KRAS, or EGFR. Analysis of Affymetrix arrays for gene expression is underway and will be presented. Conclusions : Interestingly, the CD8+ TIL count seems to predict the response to panitumumab. Tumor FOXP3 expression and high FOXP3 TILs also tended to be predictive. Tumor levels of IGF-1R seem to play a determinant role in TNBC response to anti-EGFR antibodies, in concordance with our observations in a head-and-neck cancer cohort (Clin Canc Res 2012). Confirmatory and mechanistic studies of those biomarkers are warranted. Citation Format: Frederique Penault-llorca, Catherine Abrial, Marie-Melanie Dauplat, Maud Privat, Nancy Uhrhammer, Alexis Desrichard, Yannick Bidet, Nina Radosevic-Robin, Anne Cayre, Cecile Aube, Fabrice Kwiatkowski, Nassera Chalabi, Yves-Jean Bignon, Philippe Chollet, Jean-Marc Nabholtz. Response to the anti-EGFR antibody panitumumab combined with standard neoadjuvant chemotherapy in triple negative breast cancer (TNBC): the immune and IGFR pathways. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4669. doi:10.1158/1538-7445.AM2013-4669

Abstract P1-08-34: Is it possible to predict the efficacy of a combination of cetuximab plus docetaxel in patients with operable, triple negative breast cancer (TNBC)? Final biomarker results from a phase II neoadjuvant trial

Background: TNBC is a heterogenous group of tumors for some of which the Epithelial Growth Factor Receptor pathway (EGFR) may play an important role. We evaluated the efficacy and toxicity of an anti-EGFR antibody (cetuximab) combined with docetaxel, which were given to the TNBC patients (pts) in the neoadjuvant setting. A biomarker analysis accompanied this trial, aiming to identify predictors of response. Methods: 35 patients with stage II-IIIA TN breast disease were prospectively included in this multicentre pilot study. Systemic therapy (ST) consisted of 6 cycles of docetaxel (100 mg/m 2 ) each 3 weeks, in combination with weekly cetuximab (first dose 400mg/m 2 then 250 mg/m 2 /week) for 6 cycles. All patients underwent surgery at completion of ST. Patient characteristics : mean age 48 [28-67]; TNM: T1: 3%, T2: 73%, T3: 24%; N0: 61%, N1-N2: 39%; mean tumor size 40 mm [15-100]; SBR: grade III: 73%, grade II: 27%. The median number of cycles was for docetaxel 6 [1-6] and for cetuximab 15 [1-18]. Pathological complete response (pCR) rate was 24% according to the Chevallier and Sataloff classifications; 28% if we consider breast pCR only. Overall clinical response rate was 57% (22% CR). Conservative surgery was performed in 75% of cases. The main side effect was skin toxicity: grade II: 39%, grade III: 36%, grade IV: 3%. Other side effects were: neutropenia grade IV: 12.7%, febrile neutropenia: 1.3%, hand-foot syndrome grade III: 3%, grade II: 3%, ungueal toxicity grade III: 3%, grade II: 33%. Paraffin-embedded and frozen samples were systematically collected before and after ST for biomarker studies. Germinal BRCA1 mutations and EGFR, KRAS, BRAF and PI3KCA somatic mutations were analyzed by NGS. EGFR, MET, cytokeratins 5/6 and 8/18, PTEN, P-cadherin, ALDH1, Ki67, p53 and the number of FOXP3+ or CD8+ tumor-infiltrating lymphocytes (TIL) were evaluated by immunohistochemistry. Results: The biomarker analysis was interpretable on the samples from 21 pts (3 pCR and 18 non-pCR). We applied the ROC curve to identify the best cut-off value for Ki67, EGFR, MET, cytokeratin 5/6 and 8/18, p53, ALDH1, PTEN, P-cadherin and the FOXP3+ or CD8+ TIL counts. None of these biomarkers was predictive of pCR except for the CD8+/FOXP3+ TIL count ratio. pCR rate was higher in the pts with the ratio equal or higher than 2.75 than in the others (43% versus 0%, p = 0.047). BRCA1 mutations were detected in 16% of pts. PI3K and EGFR somatic mutations were observed in 1 and 3 patients, respectively. The presence of the mutations was not predictive of pCR. Conclusion: Similarly to the previously reported trial by our group (SABCS 2012, abstract 1081), the immune component of the tumor microenvironment plays a very important role in the TNBC response to cytotoxic therapy. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P1-08-34.

Abstract 2602: CHEK2 contribution to hereditary breast cancer in non-BRCA famillies

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Mutations in the BRCA1 and BRCA2 genes are responsible for only a part of hereditary breast cancer (HBC). The origins of “non BRCA” HBC families may be attributed in part to mutations in genes giving moderate risk, such as CHEK2. We investigated the contribution of CHEK2mutations to non-BRCA HBC by direct sequencing of its entire coding sequence. Fifteen mutations were discovered among 507 non-BRCAHBC cases and four among 513 controls. The frequency of CHEK2variants was significantly higher among cases (p= 0.0076), and gave an OR for breast cancer of 4.72 for deleterious mutation carriers. We then used both in silico tools and in vitro kinase activity to evaluate recombinant mutant proteins. Tumor characteristics and tumor grade of paraffin-embedded tissue blocks from 8 CHEK2 mutated patients were evaluated by histology. To further characterize those tumors, breast cancer immunohistochemical markers such as hormone receptors, HER2 and P53 were assessed. Because the mechanisms of tumorigenesis in association with CHEK2 variants are still unclear, we performed genetic and epigenetic analysis of those tumors. Three relevant SNPs spanning the CHEK2 gene locus were used to determine loss of heterozygosity (LOH). Also, the proximal CpG islands of the CHEK2 gene were investigated for hypermethylation. Our results suggest a contribution of CHEK2 mutations to non-BRCA HBC, though the usefulness of moderate penetrance genes for genetic counseling remains controversial. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2602. doi:1538-7445.AM2012-2602