Candidate gene analysis of BRCA1/2 mutation-negative high-risk Russian breast cancer patients.

Twenty one DNA repair genes were analyzed in a group of 95 BC patients, who displayed clinical features of hereditary disease predisposition but turned out to be negative for mutations in BRCA1 and BRCA2 entire coding region as well as for founder disease-predisposing alleles in CHEK2, NBN/NBS1 and ATM genes. Full-length sequencing of CHEK2 and NBN/NBS1 failed to identify non-founder mutations. The analysis of TP53 revealed a woman carrying the R282W allele; further testing of additional 108 BC patients characterized by a very young age at onset (35 years or earlier) detected one more carrier of the TP53 germ-line defect. In addition, this study confirmed non-random occurrence of PALB2 truncating mutations in Russian hereditary BC patients. None of the studied cases carried germ-line defects in recently discovered hereditary BC genes, BRIP1, FANCC, MRE11A and RAD51C. The analysis of genes with yet unproven BC-predisposing significance (BARD1, BRD7, CHEK1, DDB2, ERCC1, EXO1, FANCG, PARP1, PARP2, RAD51, RNF8, WRN) identified single women carrying a protein-truncating allele, WRN R1406X. DNA sequencing of another set of 95 hereditary BC cases failed to reveal additional WRN heterozygous genotypes. Since WRN is functionally similar to the known BC-predisposing gene, BLM, it deserves to be analyzed in future hereditary BC studies. Furthermore, this investigation revealed a number of rare missense germ-line variants, which are classified as probably protein-damaging by online in silico tools and therefore may require further consideration.

Novel ALK fusion partners in lung cancer.

Detection of ALK rearrangements in patients with non-small cell lung cancer (NSCLC) presents a significant technical challenge due to the existence of multiple translocation partners and break-points. To improve the performance of PCR-based tests, we utilized the combination of 2 assays, i.e. the variant-specific PCR for the 5 most common ALK rearrangements and the test for unbalanced 5'/3'-end ALK expression. Overall, convincing evidence for the presence of ALK translocation was obtained for 34/400 (8.5%) cases, including 14 EML4ex13/ALKex20, 12 EML4ex6/ALKex20, 3 EML4ex18/ALKex20, 2 EML4ex20/ALKex20 variants and 3 tumors with novel translocation partners. 386 (96.5%) out of 400 EGFR mutation-negative NSCLCs were concordant for both tests, being either positive (n = 26) or negative (n = 360) for ALK translocation; 49 of these samples (6 ALK+, 43 ALK-) were further evaluated by FISH, and there were no instances of disagreement. Among the 14 (3.5%) "discordant" tumors, 5 demonstrated ALK translocation by the first but not by the second PCR assay, and 9 had unbalanced ALK expression in the absence of known ALK fusion variants. 5 samples from the latter group were subjected to FISH, and the presence of translocation was confirmed in 2 cases. Next generation sequencing analysis of these 2 samples identified novel translocation partners, DCTN1 and SQSTM1; furthermore, the DCTN1/ALK fusion was also found in another NSCLC sample with unbalanced 5'/3'-end ALK expression, indicating a recurrent nature of this translocation. We conclude that the combination of 2 different PCR tests is a viable approach for the diagnostics of ALK rearrangements. Systematic typing of ALK fusions is likely to reveal new NSCLC-specific ALK partners.

Pattern of clinically relevant mutations in consecutive series of Russian colorectal cancer patients.

One hundred and ninety-five consecutive surgically treated Russian colorectal cancer (CRC) patients were retrospectively analyzed for the presence of mutations in KRAS, NRAS, BRAF and PIK3CA genes as well as for the microsatellite instability status. Comparison between high-resolution melting analysis, co-amplification at lower denaturation temperature PCR, DNA sequencing and allele-specific PCR for the detection of KRAS codon 12/13 mutations revealed that none of these methods alone provided satisfactory results in 100 % of the analyzed cases; this experience supports the use of more than one mutation-detecting technique at least in some circumstances. KRAS codon 12/13 substitutions were detected in 70 (35.9 %) CRC cases. Other mutations in the RAS/RAF genes occurred in 22 (11.3 %) cases and included rare KRAS (n = 6), NRAS (n = 8) and BRAF (n = 8) alterations. 5 BRAF mutations affected codon 600, while the remaining 3 potentially functional substitutions were located in the position 594. Twenty-four (12.3 %) CRC cases carried mutations in the PIK3CA, and 18 of these tumors also contained activating alteration in the RAS/RAF genes (p = 0.007). Only 3 (1.5 %) CRC cases showed high-level microsatellite instability (MSI-H) as determined by a panel of mononucleotide markers. Overall, the distribution of potentially predictive mutations in Russian CRC cases is similar to the one observed in other patient series of European descent. Noticeable occurrence of D594G mutation in BRAF oncogene and low frequency of MSI-H may deserve specific attention.