BRCA1/2 variants and copy number alterations status in non familial triple negative breast cancer and high grade serous ovarian cancer.

BACKGROUND: While the role of BRCA1/2 genes in familial breast and ovarian cancer is well established, their implication in the sporadic form of both cancers is still controversial. With the development of poly (ADP-ribose) polymerase (PARP) inhibitors, the exact relationship between BRCA1/2 genes and sporadic triple negative breast cancer/high grade serous carcinoma (TNBC/HGSC) needs to be further investigated. Therefore, we conducted a study in which we analyze BRCA1/2 point mutations and copy number alterations in Moroccan patients suffering from TNBC/HGSC. METHODS: To achieve our goal, we analyzed BRCA1/2 genes in the FFPE tissue blocks and blood samples of 65 TNBC/HGSC selected patients, using next generation sequencing technology. RESULTS: From the 65 successfully sequenced patients in our cohort, we detected five-point variants in six different patients, four variants were classified as pathogenic and one of unknown significance. Regarding copy number alterations we detected one copy number loss in BRCA1 gene and one copy number gain in BRCA2 gene. The genetic screening of BRCA1/2 genes using these patients' genomic DNA indicated that five harbored a germline genetic alteration. While three harbored a somatic genetic alteration. To the best of our knowledge, three-point variants detected in our study have never been reported before. CONCLUSION: According to the results found in the present study, in a population without a family history of cancer, the possibility of a BRCA1/2 somatic pathogenic variant in high grade serous carcinoma is 7%. While for Triple negative breast cancer somatic point variants and copy number alterations seems to be a very rare genetic event.

Next-generation sequencing of BRCA1 and BRCA2 genes in Moroccan prostate cancer patients with positive family history.

Prostate cancer is the most common male cancer in Morocco. Although sporadic forms account for a large proportion of patients, familial forms of prostate cancer are observed in 20% of cases and about 5% are due to hereditary transmission. Indeed, germline mutations in BRCA1/2 genes have been associated with prostate cancer risk. However, the spectrum of these mutations was not investigated in Moroccan Prostate cancer patients. Thereby, the aim of this study was to characterize and to estimate the prevalence of germline BRCA1/2 mutations and large rearrangements in Moroccan patients with familial prostate cancer. The entire coding regions and intron/exon boundaries of BRCA1 and BRCA2 genes have been analyzed by next generation sequencing (NGS) in a total of 30 familial prostate cancer patients. Three pathogenic mutations were detected in four unrelated patients (13.3%). One BRCA1 mutation (c.1953_1956delGAAA) and two BRCA2 mutations (c.7234_7235insG and BRCA2ΔE12). In addition, sixty-three distinct polymorphisms and unclassified variants have been found. Early identification of germline BRCA1/2 mutations may be relevant for the management of Moroccan prostate cancer patients.

Large scale genomic analysis of 3067 SARS-CoV-2 genomes reveals a clonal geo-distribution and a rich genetic variations of hotspots mutations.

In late December 2019, an emerging viral infection COVID-19 was identified in Wuhan, China, and became a global pandemic. Characterization of the genetic variants of SARS-CoV-2 is crucial in following and evaluating it spread across countries. In this study, we collected and analyzed 3,067 SARS-CoV-2 genomes isolated from 55 countries during the first three months after the onset of this virus. Using comparative genomics analysis, we traced the profiles of the whole-genome mutations and compared the frequency of each mutation in the studied population. The accumulation of mutations during the epidemic period with their geographic locations was also monitored. The results showed 782 variants sites, of which 512 (65.47%) had a non-synonymous effect. Frequencies of mutated alleles revealed the presence of 68 recurrent mutations, including ten hotspot non-synonymous mutations with a prevalence higher than 0.10 in this population and distributed in six SARS-CoV-2 genes. The distribution of these recurrent mutations on the world map revealed that certain genotypes are specific to geographic locations. We also identified co-occurring mutations resulting in the presence of several haplotypes. Moreover, evolution over time has shown a mechanism of mutation co-accumulation which might affect the severity and spread of the SARS-CoV-2. The phylogentic analysis identified two major Clades C1 and C2 harboring mutations L3606F and G614D, respectively and both emerging for the first time in China. On the other hand, analysis of the selective pressure revealed the presence of negatively selected residues that could be taken into considerations as therapeutic targets. We have also created an inclusive unified database (http://covid-19.medbiotech.ma) that lists all of the genetic variants of the SARS-CoV-2 genomes found in this study with phylogeographic analysis around the world.

Screening of BRCA1/2 genes mutations and copy number variations in patients with high risk for hereditary breast and ovarian cancer syndrome (HBOC).

BACKGROUND: Hereditary breast and ovarian cancer (HBOC) is an autosomal dominant inherited cancer susceptibility disorder. Both BRCA1 and BRCA2 genes are considered as high penetrance genes of this syndrome. The identification of BRCA1/2 genetic alterations before cancer development, grant patients the chance to benefit from various medical cancer prevention approaches. Therefore, the appearance of recent advanced technologies in molecular analysis such as next generation sequencing has simplified full BRCA1/2 analysis. Many attempts took place in hope of understanding the molecular germline spectrum of these two genes in Moroccan HBOC patients. However, most of the past projects focused only on young breast cancer cases, lacked ovarian cancer cases in their cohort and only a limited number of these studies were able to analyze the entire exons or copy number variations for both genes. In attempt of gaining more information regarding the molecular profile of BRCA1/2 in HBOC, we conducted a study in which we analyze their molecular profile on selected Moroccan patients suspected of having HBOC syndrome. METHODS: In this study we obtained blood samples from 64 selected Moroccan patients, who suffered from Breast and/or ovarian cancer and had a strong family history for cancer. To analyze BRCA1/2 punctual variants and copy number variations, we used the Ion Personal Genome Machine (PGM) and Oncomine BRCA1/2 research assay panel. Afterward, we correlated the molecular results with the clinic-pathologic data using IBM SPSS Statistics ver 2. RESULTS: From the 64 selected cases, Forty-six had breast cancer, fifteen had ovarian cancer and three had both breast and ovarian cancer. The molecular analysis revealed that 18 patients from the 64 harbored a pathogenic variant (28%). Twelve had six different BRCA1 pathogenic variants and six had six different BRCA2 pathogenic variants. In this study, we report four pathogenic variants that to the best of our knowledge has never been reported in the Moroccan population before. Regarding copy number variation analysis, No CNV was detected in both genes for all the 64 successfully sequenced and analyzed patients in our cohort. CONCLUSION: Work like the present has an important implication on public health and science. It is critical that molecular profiling studies are performed on underserved and understudied population like Morocco.

EGFR, BRCA1, BRCA2 and TP53 genetic profile in Moroccan triple negative breast cancer cases.

Triple negative breast cancer account for 10% to 20% of all newly diagnosed breast cancer cases, this subtype is well known for its lack of estrogen, progesterone and HER2 expression unlike the other subtypes of breast cancer that usually express at least one of the three. The absence of a specific biomarker for TNBC has made his treatment very challenging and his death rates very high compared to the other subtypes. Therefore, in morocco, many studies have been conducted in the hope of finding a specific biomarker for TNBC, but none of these studies has analyzed the EGFR protein expression and its gene molecular profile and correlated the EGFR analyses results with the genetic profile of other genes. In this study, we analyzed EGFR protein expression and the molecular profile of EGFR, BRCA1, BRCA2 and TP53 genes in 47 TNBC patients. We conducted a retrospective study of 47 Moroccan patients diagnosed with triple negative breast cancer between early 2013 and 2016. In this study, we have analyzed the EGFR. Protein expression, for all the 47 TNBC patients using pharmDx Kit. Then we used the Ion Personal Genome Machine (PGM) and Ion Ampliseq BRCA1/2 panel and hotspot Cancer panel to analyze the molecular profile of BRCA1/2 genes and the hotspot regions of TP53 and EGFR genes. The statistical analysis was performed using IBM SPSS Statistics ver. From the 47 analyzed patients using EGFR pharmDx Kit only 16 (34%) had EGFR overexpression while 31 (66%), patients were normal, moreover, From the 47 TNBC patients, only 39 underwent Mutational analysis of EGFR, BRCA1/2, and TP53 genes. One patient harbored a BRCA1 mutation c.798_799delTT (p.Ser267Lys). While for TP53 gene, 16 patients out of 39 (41%) presented hotspot mutations, seven of them harbored c.743G>A (p.Arg248Gln) mutation, six patients harbored exon 6 mutations from which five harbored the mutation c.659A>G (p.Tyr220Cys) and one the mutation c.817C>T (p.Arg273Cys), and finally, three patients harbored the mutation c.524G>A (p.Arg175His). Regarding BRCA2 and EGFR sequencing results, no mutations or other genetic alterations were detected in 39 patients that were successfully sequenced. Statistical analysis revealed the absence of any correlations.

Detection of PIK3/AKT pathway in Moroccan population with triple negative breast cancer.

BACKGROUND: Triple Negative Breast Cancer (TNBC) is an aggressive form of breast cancer, that represents 10-20% of all breast carcinomas and characterized by the lack of a specific cell surface marker compared to other breast cancer subtypes. Due to the absence of molecular markers for TNBC his treatment options remains limited, without proven targeted therapies, which emphasize the need for discovering molecular markers that could be targeted for patient treatment, An important number of TNBC cases harbor aberrations in the phosphoinositide 3-kinase (PI3K) pathway, leading to constitutive activation of the downstream signaling pathway. Among mechanisms of PI3K enhancement, PIK3CA mutations are most frequently (~ 30%) observed, along with protein loss of PTEN and AKT activation by phosphorylation (pAkt). Therefore, we propose to analyze clinocopathologic and molecular characteristics of PI3K/AKT/PTEN pathway in Moroccan triple negative breast cancer patients. METHODS: We conducted a retrospective study of 39 patients diagnosed with triple negative breast cancer between early 2013 and 2016. In this study, we used the Ion Personal Genome Machine (PGM) and Ion Torrent Ampliseq Cancer panel to sequence hotspot regions from PIK3CA, AKT and PTEN genes to identify genetic mutations in 39 samples of TNBC subtype from Moroccan patients and to correlate the results with clinical-pathologic data. RESULTS: All patients were female with a median age of 46 years from (34-65). Most patients have had invasive ductal carcinoma (84.6%) and 69.2% of them were grade III SBR. Among the 39, 9 were right sided tumor patients and the remaining 30 were left-sided. Mutational analysis of PIK3CA gene was achieved in all TNBC patients. PIK3CA hotspot mutations were detected in 5/39 of TNBC (13%), in detail, among these 5 TNBC patients, one harbored mutation in exons 9 and four in exon 20. CONCLUSION: The PI3KCA gene is highly activated and plays a crucial role in the pathogenesis of TNBC more, therefore, may be a potential therapeutic target to improve outcomes in patients.

First application of next-generation sequencing in Moroccan breast/ovarian cancer families and report of a novel frameshift mutation of the BRCA1 gene.

At present, breast cancer is the most common type of cancer in females. The majority of cases are sporadic, but 5-10% are due to an inherited predisposition to develop breast and ovarian cancers, which are transmitted as an autosomal dominant form with incomplete penetrance. The beneficial effects of clinical genetic testing, including next generation sequencing (NGS) for BRCA1/2 mutations, is major; in particular, it benefits the care of patients and the counseling of relatives that are at risk of breast cancer, in order to reduce breast cancer mortality. BRCA genetic testing was performed in 15 patients with breast cancer and a family with positivity for the heterozygous c.6428C>A mutation of the BRCA2 gene. Informed consent was obtained from all the subjects. Genomic DNAs were extracted and the NGS for genes was performed using the Ion Torrent Personal Genome Machine (PGM) with a 316 chip. The reads were aligned with the human reference HG19 genome to elucidate variants in the BRCA1 and BRCA2 genes. Mutations detected by the PGM platform were confirmed by target direct Sanger sequencing on a second patient DNA sample. In total, 4 BRCA variants were identified in 6 families by NGS. Of these, 3 mutations had been previously reported: c.2126insA of BRCA1, and c.1310_1313delAAGA and c.7235insG of BRCA2. The fourth variant, c.3453delT in BRCA1, has, to the best of our knowledge, never been previously reported. The present study is the first to apply NGS of the BRCA1 and BRCA2 genes to a Moroccan population, prompting additional investigation into local founder mutations and variant characteristics in the region. The variants with no clear clinical significance may present a diagnostic challenge when performing targeted resequencing. These results confirm that an NGS approach based on Ampliseq libraries and PGM sequencing is a highly efficient, speedy and high-throughput mutation detection method, which may be preferable in lower income countries.