Abstract
Background:Pathogenic germline mutations in BRCA1/2 constitute the majority of hereditary breast and/or ovarian cancers worldwide. Incidence and mortality rate of breast and ovarian cancers in Pakistani women is high. Thus, to establish the diagnosis for targeted therapy in Pakistan, we conducted Next-generation sequencing-based germline testing for the detection of BRCA1/2 oncogenic variants associated with breast and ovarian cancer subtype. Methods:Peripheral blood of 24 women, diagnosed with breast and epithelial ovarian cancers, was taken from the recruited cases with the consent of performing germline genetic testing. DNA was isolated from the peripheral blood and subjected to indexed BRCA Panel libraries. Targeted NGS was performed for all coding regions and splicing sites of BRCA1 and BRCA2 genes using AmpliSeq for Illumina BRCA Panel and Illumina MiSeq sequencer (placed at AFIP). Analysis of the sequencing results has been done by using Illumina bioinformatics tools. Results:We detected 421 variants having a quality score of 100 in all cases under study. The list of identified variants in BRCA1 and BRCA2 genes was narrowed down after filtering out those which did not pass q30 and those with a minor allele frequency (MAF) > 0.05 based on gnomAD browser. To classify these variants, clinical significance was predicted using external curated databases. As a result, we interpreted (n = 4) 16.7% pathogenic variants in BRCA1 and (n = 6) 25% variants of uncertain significance (VUS) in both genes. Descriptive statistics depicted that the age and BMI of BRCA positive cases are less than BRCA negative cases. Conclusion:Our findings exhibit an initial report for the NGS based cancer genetic testing in Pakistan. This will enable us to pursue screening and diagnosis of hereditary BRCA mutation utilizing the latest state-of-the-art technique locally available in Pakistan ultimately resulting in targeted cancer therapy.
Highlights
In spite of rigorous research and considerable progress in precise diagnosis for the prediction of targeted therapy of breast and ovarian cancers worldwide, these malignancies remain a major health problem and are a predominant cause of death in women
To establish the diagnosis for targeted therapy in Pakistan, we conducted Next-generation sequencing-based germline testing for the detection of BRCA1/2 oncogenic variants associated with breast and ovarian cancer subtype
Cases diagnosed with Triple negative breast cancer, Early onset breast carcinoma, High grade serous carcinoma and Endometrioid carcinoma were included
Summary
In spite of rigorous research and considerable progress in precise diagnosis for the prediction of targeted therapy of breast and ovarian cancers worldwide, these malignancies remain a major health problem and are a predominant cause of death in women. Previous investigations have confirmed that the BRCA-mutated cancer patients are sensitive to platinum chemotherapy. These cases show a good prognosis after treatment with poly (ADPribose) polymerase inhibitors (Vidula and Bardia, 2017; Taylor and Eskander, 2018). According to National Comprehensive Cancer Network (NCCN) guidelines, breast carcinoma patients with triple negative immunohistochemical markers; Estrogen, Progesterone and HER2/ Neu, with age equal or less than 60 should be screened for BRCA mutations/variants. No NGS-based genetic testing of breast and ovarian cancer has been performed and reported indigenously rather the sample are outsourced to foreign laboratories. NGS system in a clinical laboratory i.e. Armed force institute of pathology (AFIP) Pakistan, for the genetic testing of breast and ovarian cancer subtypes. All exons and splice site regions of BRCA1 and BRCA2 genes were screened by targeted generation sequencing with the objective of detecting germline pathogenic variants cases under study
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