Uncovering the clinical relevance of unclassified variants in DNA repair genes: a focus on BRCA negative Tunisian cancer families

Introduction: Recent advances in sequencing technologies have significantly increased our capability to acquire large amounts of genetic data. However, the clinical relevance of the generated data continues to be challenging particularly with the identification of Variants of Uncertain Significance (VUSs) whose pathogenicity remains unclear. In the current report, we aim to evaluate the clinical relevance and the pathogenicity of VUSs in DNA repair genes among Tunisian breast cancer families.Methods: A total of 67 unsolved breast cancer cases have been investigated. The pathogenicity of VUSs identified within 26 DNA repair genes was assessed using different in silico prediction tools including SIFT, PolyPhen2, Align-GVGD and VarSEAK. Effects on the 3D structure were evaluated using the stability predictor DynaMut and molecular dynamics simulation with NAMD. Family segregation analysis was also performed.Results: Among a total of 37 VUSs identified, 11 variants are likely deleterious affecting ATM, BLM, CHEK2, ERCC3, FANCC, FANCG, MSH2, PMS2 and RAD50 genes. The BLM variant, c.3254dupT, is novel and seems to be associated with increased risk of breast, endometrial and colon cancer. Moreover, c.6115G>A in ATM and c.592+3A>T in CHEK2 were of keen interest identified in families with multiple breast cancer cases and their familial cosegregation with disease has been also confirmed. In addition, functional in silico analyses revealed that the ATM variant may lead to protein immobilization and rigidification thus decreasing its activity. We have also shown that FANCC and FANCG variants may lead to protein destabilization and alteration of the structure compactness which may affect FANCC and FANCG protein activity.Conclusion: Our findings revealed that VUSs in DNA repair genes might be associated with increased cancer risk and highlight the need for variant reclassification for better disease management. This will help to improve the genetic diagnosis and therapeutic strategies of cancer patients not only in Tunisia but also in neighboring countries

Relapse profile of early breast cancer according to immunohistochemical subtypes: guidance for patient’s follow up?

Purpose: To analyze the profile of annual recurrence rate (ARR) of patients with early breast cancer (BC) in Tunisia. Patients and methods: We classified 293 patients with histologically confirmed early BC relapsing after 1 year of follow up into three subgroups: hormone receptor (HR)+ ‘HR’ [estrogen receptor (ER)+ or progesterone receptor (PR)+ and human epidermal growth factor receptor 2 (HER2)–], triple negative ‘TN’ (ER–, PR– and HER2 score 0/1 or fluorescence in situ hybridization (FISH)/chromogenic in situ hybridization negative) and HER2 overexpression ‘HER2’ (HER2+). ARR was restricted to follow-up contribution of each specified time interval. The HR group was the reference group for comparison. Results: A higher proportion of patients who were up to 35 years old (18% versus 10%, p = 0.04), and patients with obesity (46% versus 26%, p = 0.045) was seen in the TN group. Median time to relapse (MTR) was shorter in TN and HER2 groups than in HR patients (20 and 29 months compared with 56 months, respectively, p < 0.001). In the HR group, the ARR was 22%, 16% and 10% at 3, 4 and 5 years respectively, becoming less than 3% at 7 years. In the TN group, 71% of patients relapsed during the first 2 years and the ARR declined rapidly to less than 1.5% after 4 years. In the HER2 group, the ARR peaked at 2 years (29%) and decreased significantly to 7% and 3% at 5 and 7 years respectively. Adjuvant trastuzumab delayed the MTR from 24 to 34 months ( p = 0.022). Conclusion: The relapse risk in Tunisian patients is higher in young women and patients with HER2+ and TN tumors. A long and close follow up is recommended for patients with HR and HER2. Conversely, we suggest that follow up in patients with TN could be spaced after 4 years (ARR being <1.5% after this period)

Inflammatory breast cancer in 210�patients: A retrospective study on epidemiological, anatomo‑clinical features and therapeutic results

International audienceTo report epidemiological and anatomo-clinical features within a retrospective series of inflammatory breast cancer and to evaluate prognostic factors. This retrospective study included 210 Tunisian patients presenting a clinically diagnosed IBC, treated at the Institute Salah Azaiez (ISA) of Tunis, Tunisia, from 2008 to 2013. We collected data on epidemiology, anatomo-clinical and biological features and histologic response to neoadjuvant therapy. Overall and disease-free survivals were calculated by Kaplan-Meier method and compared by log-rank tests and Cox's models were used to identify prognostic factors impacting survival. The 210 IBC patients had a median age of 42 years (24-62) and 15% of them were aged less than 35 years. Mean age at menarche was 13 years and 45% had their 1st childbirth before 23 years. On histology, grades III represented 42% of cases, hormone receptors were negative in 59%, HER2 over-expressed in 32, 25% of our IBC cases had a triple negative profile and Ki-67 was >20% in 53% of cases. High pathological grade III was significantly correlated to TN subtype (58%) (Fisher's exact test, P=7.5×10-3). Further, high Ki-67 expression (>20%) was evident in the TN subtype (84%) (Fisher's exact test, P=3.7×10-4). After neoadjuvant therapy (and trastuzumab in 88 and 69% of HER2+ patients, respectively), we observed 49% of objective clinical responses and 35% of pathological complete response (pCR) and >3 axillary lymph nodes were invaded on a resected tumor in 55% of cases. Overall survival (OS) was associated with age at menarche (Wald-test, P=2.2×10-2) and metastases at diagnosis (Wald-test, P=2.4×10-2). Reaching a pCR was correlated with a better metastasis-free survival (MFS), (Fisher's exact test, P=3.6×10-2)

Germline copy number variations in BRCA1/2 negative families: Role in the molecular etiology of hereditary breast cancer in Tunisia.

Hereditary breast cancer accounts for 5-10% of all breast cancer cases. So far, known genetic risk factors account for only 50% of the breast cancer genetic component and almost a quarter of hereditary cases are carriers of pathogenic mutations in BRCA1/2 genes. Hence, the genetic basis for a significant fraction of familial cases remains unsolved. This missing heritability may be explained in part by Copy Number Variations (CNVs). We herein aimed to evaluate the contribution of CNVs to hereditary breast cancer in Tunisia. Whole exome sequencing was performed for 9 BRCA negative cases with a strong family history of breast cancer and 10 matched controls. CNVs were called using the ExomeDepth R-package and investigated by pathway analysis and web-based bioinformatic tools. Overall, 483 CNVs have been identified in breast cancer patients. Rare CNVs affecting cancer genes were detected, of special interest were those disrupting APC2, POU5F1, DOCK8, KANSL1, TMTC3 and the mismatch repair gene PMS2. In addition, common CNVs known to be associated with breast cancer risk have also been identified including CNVs on APOBECA/B, UGT2B17 and GSTT1 genes. Whereas those disrupting SULT1A1 and UGT2B15 seem to correlate with good clinical response to tamoxifen. Our study revealed new insights regarding CNVs and breast cancer risk in the Tunisian population. These findings suggest that rare and common CNVs may contribute to disease susceptibility. Those affecting mismatch repair genes are of interest and require additional attention since it may help to select candidates for immunotherapy leading to better outcomes

Family specific genetic predisposition to breast cancer: results from Tunisian whole exome sequenced breast cancer cases.

International audienceBACKGROUND:A family history of breast cancer has long been thought to indicate the presence of inherited genetic events that predispose to this disease. In North Africa, many specific epidemio-genetic characteristics have been observed in breast cancer families when compared to Western populations. Despite these specificities, the majority of breast cancer genetics studies performed in North Africa remain restricted to the investigation of the BRCA1 and BRCA2 genes. Thus, comprehensive data at a whole exome or whole genome level from local patients are lacking.METHODS:A whole exome sequencing (WES) of seven breast cancer Tunisian families have been performed using a family-based approach. We focused our analysis on BC-TN-F001 family that included two affected members that have been sequenced using WES. Relevant variants identified in BC-TN-F001 have been confirmed using Sanger sequencing. Then, we conducted an integrative analysis by combining our results with those from other WES studies in order to figure out the genetic transmission model of the newly identified genes. Biological network construction and protein-protein interactions analyses have been performed to decipher the molecular mechanisms likely accounting for the role of these genes in breast cancer risk.RESULTS:Sequencing, filtering strategies, and validation analysis have been achieved. For BC-TN-F001, no deleterious mutations have been identified on known breast cancer genes. However, 373 heterozygous, exonic and rare variants have been identified on other candidate genes. After applying several filters, 12 relevant high-risk variants have been selected. Our results showed that these variants seem to be inherited in a family specific model. This hypothesis has been confirmed following a thorough analysis of the reported WES studies. Enriched biological process and protein-protein interaction networks resulted in the identification of four novel breast cancer candidate genes namely MMS19, DNAH3, POLK and KATB6.CONCLUSIONS:In this first WES application on Tunisian breast cancer patients, we highlighted the impact of next generation sequencing technologies in the identification of novel breast cancer candidate genes which may bring new insights into the biological mechanisms of breast carcinogenesis. Our findings showed that the breast cancer predisposition in non-BRCA families may be ethnic and/or family specific