BackgroundNext generation sequencing has a potential to revolutionize the management of cancer patients within the framework of precision oncology. Nevertheless, lack of standardization decelerated entering of the technology into the clinical testing space. Here we dissected a number of common problems of NGS diagnostics in oncology and introduced ways they can be resolved.MethodsDNA was extracted from 26 formalin fixed paraffin embedded (FFPE) specimens and processed with the TrueSeq Amplicon Cancer Panel (Illumina Inc, San Diego, California) targeting 48 cancer-related genes and sequenced in single run. Sequencing data were comparatively analyzed by several bioinformatics pipelines.ResultsLibraries yielded sufficient coverage to detect even low prevalent mutations. We found that the number of FFPE sequence artifacts significantly correlates with pre-normalization concentration of libraries (rank correlation −0.81; p < 1e−10), thus, contributing to sample-specific variant detection cut-offs. Surprisingly, extensive validation of EGFR mutation calls by a combination of aligners and variant callers resulted in identification of two false negatives and one false positive that were due to complexity of underlying genomic change, confirmed by Sanger sequencing. Additionally, the study of the non-EGFR amplicons revealed 33 confirmed unique mutations in 17 genes, with TP53 being the most frequently mutated. Clinical relevance of these finding is discussed.ConclusionsReporting of entire mutational spectrum revealed by targeted sequencing is questionable, at least until the clinically-driven guidelines on reporting of somatic mutations are established. The standardization of sequencing protocols, especially their data analysis components, requires assay-, disease-, and, in many cases, even sample-specific customization that could be performed only in cooperation with clinicians.

Background: About 30%  of cases of hereditary breast cancer (BC) are associated with the BRCA1 and BRCA2 gene mutations. The absence of the programs of mandatory genetic screening for hereditary BRCA-associated BC in Russia, as well as of an algorithm for molecular genetic testing does not allow fully accomplishing the necessary preventive, diagnostic and medical measures.Aim: To elaborate an algorithm for molecular genetic testing of BC patients in order to improve the efficacy of identification of the hereditary nature of the disease.Materials and methods: The study is based on the analysis of the results of molecular genetic testing of 3826 BC patients aged from 22 to 90 years, who were examined and treated in the Russian Research Center of Roentgenoradiology (Moscow) from 2010 to 2016. At the first stage of the study, germinal mutation in the BRCA1 and BRCA2 genes prevalent in the Russian population were identified by the real-time polymerase chain reaction (PCR). At the second stage, we searched for rare genetic variants of these genes by the ‘next generation sequencing’ (NGS) method.Results: The real-time PCR (the first stage) showed that the prevalence of the most typical for the Russian population mutations in the BRCA1 gene, associated with BC risk, was 3.5% (132/3826 BC patients). No carriers of the BRCA2 mutations were identified. Based on the analysis of a  questionnaire survey and primary medical documentation, a group of 717 patients was selected from the total cohort, who had clinical features of the hereditary disease (CFHD). In this group, the BRCA1 and BRCA2 gene mutations were found in 126 patients (17.6%). At the second stage, a group of 193 patients with CFHD and no BRCA1 and BRCA2 mutations prevalent in the Russian population was investigated by NGS. Rare pathogenic mutations of these genes were found in 27  patients (14%). In total, it may be concluded that at least 30% of the BC patients with CFHD have germinal mutations in the BRCA1 and BRCA2 genes. Based on the data obtained, we have developed the algorithm of molecular genetic testing of BC patients aimed at identification of the hereditary nature of the disease.Conclusion: The high frequency of mutations in the BRCA1 and BRCA2 genes found in this study in BC patients with CFHD confirms the necessity of genetic testing for this hereditary disease. The information on its hereditary nature allows for the introduction of essential therapy modification with a personalized approach. Regular follow-up of patients with hereditary BC and prevention of new BC cases and other cancers (ovarian, gastric, pancreatic and prostate cancer, as well as melanoma) in their relatives with BRCA1 and BRCA2 mutations have to be implemented by a multidisciplinary team (specialists in mammology, gynecology, oncology, medical genetics, chemotherapy and psychotherapy).

"Standard" diagnostic panels allow identification of only a few of BRCA1 and BRCA2 gene mutations most common in a population. Therefore, tests relying on such panels may return false negative results, since the coding regions of these genes may have other defects. For breast cancer (BC) patients, false negative test results may translate into selection of inadequate therapy by their doctors. This study aimed to identify the features of BRCA-associated breast cancer in the population of the Russian Federation. The study included breast cancer patients (n = 4440). At the first stage, all patients were screened for the eight most common BRCA1 and BRCA2 genes mutations with the help of real-time PCR. Next, patients that exhibited clinical signs of a hereditary disease (CSHD) in the absence of common mutations (n = 290) had the entire coding regions of BRCA1 and BRCA2 genes studied with next generation sequencing (NGS). "Standard" mutations in the BRCA1 and BRCA2 genes were identified in 169 (3.8%) cases. In the CSHD group, such mutations were revealed in 15.4% of cases. NGS uncovered 33 rare pathogenic BRCA1 and BRCA2 gene mutations in 40 out of 290 breast cancer patients (13.8%). It was concluded that among the residents of the Russian Federation, the range of pathogenic variants of BRCA-associated breast cancer is wide, and it stretches beyond the mutations considered by the "standard" diagnostic panels. Analysis of the entire coding regions of BRCA1 and BRCA2 genes allows increasing efficiency of detection of germline mutations in breast cancer patients at least twofold.

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