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Activating mutations of EGFR are associated with sensitivity of non-small cell lung cancer (NSCLC ) to tyrosine kinase inhibitors (TKI). Liquid biopsy using circulating cell-free tumor DNA (cfDNA) is proposed in cases when formalin fixed paraffin embedded (FFPE) tumor tissue is not available and for monitoring of EGFR status. In the study we evaluated new qPC R assay for EGFR mutations in plasma cfDNA. Sensitivity of the assay was 1 % of the mutant allele for L858R, L861Q, S768I mutations and deletions in exon 19, and 5 % of the mutant allele for G719X or T790M mutations Before surgery, mutation was detected in plasma of 4 out of 7 patients (57 %) with mutant EGFR in FFPE tumor tissue. Mutations found in cfDNA completely matched those found in tumor tissue in 2 cases. In one case with G719X and S768I mutations in FFPE tissue, only S768I was found in cfDNA. In another case, T790M was detected in plasma in addition to L858R that was present in tumor tissue. No EGFR mutations were detected in plasma DNA from 12 healthy volunteers and 13 cases of NSCLC with wt EGFR suggesting 100 % specificity of the assay. Liquid biopsy detected EGFR mutations in cfDNA in 8 of 16 cases of NSCLC with mutant EGFR being under therapy with TKI. Among them, 7 cases had mutations in liquid biopsy that matched those in tumor tissue and another case had T790M in addition to L858R. In 3 cases increased mutant allele frequency was detected 212 months before clinical progression.

PCR clamping/wild-type blocking PCR with non-extendable locked nucleic acid (LNA) oligonucleotides is used for sensitive detection of somatic mutations in tumors. Various versions of the technique use different DNA polymerases and LNA oligonucleotides with and without additional phosphorothioate modifications. Here we studied requirements for successful PCR clamping with LNA oligonucleotides and Taq DNA polymerase for analysis of mutations in KRAS and BRAF genes by means of real-time PCR and Sanger sequencing. We found that addition of phosphorothioate linkages at the 5’-end of LNA oligonucleotide to protect from 5’- exonuclease activity of Taq DNA polymerase did not improve clamping. For most target sequences, efficient clamping was observed at melting temperature of LNA oligonucleotide 20‑25°C above annealing/extension temperature of the PCR with a 2-step protocol. Under such conditions, simple and sensitive detection of mutations in KRAS and BRAF genes was feasible using real-time PCR with TaqMan probes or Sanger sequencing.

Aim of the study. Aim of the study was to estimate the occurrence of pathogenic mutations in the BRCA1 gene in Russian breast cancer patients.Material and methods. Complete coding sequence of the BRCA1 gene of 445 early onset breast cancer patients (under 40 years) from Novosibirsk region (Russia) were analyzed by targeted Next Generation Sequencing (NGS) using Ion Torrent platform. Results. Forty (9%) carriers of various pathogenic mutations were revealed. Thirty five (7,9%) patients carried 5382insC mutation, described earlier as a founder mutation for Slavic population. Five (1.1%) patients carried various pathogenic mutations, namely C61G, 462delCC, E143X, 4153delA, and IVS18+1G>T. Besides, 29 genetic variants with no clinical significance or with unknown clinical significance were detected in BRCA1 gene among 445 early onset breast cancer patients. Conclusions. Data on the frequency of genetic variations in the BRCA1 gene among early onset breast cancer patients in the Novosibirsk Region (Russia) were obtained. Proportion of the 5382insC mutation is 87.5% of all pathogenic mutations in the BRCA1 gene found in patients.

The BRCA1 5382insC and CHEK2 1100 delC mutations were analyzed in 3850 of non-selected breast cancer patients residing in Novosibirsk region, Russia. One hundred seventy probands, BRCA1 5382insC or CHEK2 1100delC mutation carriers, were found. The study demonstrated that more than 80 % of probands informed relatives about their hereditary or familial cancer risk. Fifty-nine BRCA1 5382insC and CHEK2 1100 delC mutation carriers were found among 144 proband’s relatives. Mutation carriers or their relatives were interviewed two years after starting the project. At least 18 % of the mutation carriers reported primary tumor or recurrence of the tumor 2 years after starting the project.

Mutations in KRAS and BRAF genes in 80 colorectal cancer (CRC) samples from Russian patients were tested using two methods: 1) allele-specific real-time PCR (as-rt PCR) and 2) wild-type blocking PCR with Sanger sequencing (WTBS). Material and methods. Sections of fresh frozen or formalin-fixed paraffin embedded tumor tissue from 80 patients were used in the study. Tumor tissue content was determined on H&E stained sections. Samples were first tested by as-rtPCR for common mutations of the KRAS gene (G12C, G12S, G12R, G12V, G12D, G12A, G13D) and mutations BRAFV600E. After that samples were evaluated in PCR with oligonucleotide blocking amplification of wild-type DNA for enrichment with mutant allele followed by Sanger sequencing of the PCR DNA (WTBS method). Results. In 5 (6.3 %) cases samples had low tumor tissue content (