Ethnicity-specific BRCA1, BRCA2, PALB2, and ATM pathogenic alleles in breast and ovarian cancer patients from the North Caucasus.

BACKGROUND: Mountain areas of the North Caucasus host several large ethnic communities that have preserved their national identity over the centuries. METHODS: This study involved high-grade serous ovarian cancer (HGSOC) and breast cancer (BC) patients from Dagestan (HGSOC: 37; BC: 198), Kabardino-Balkaria (HGSOC: 68; BC: 155), North Ossetia (HGSOC: 51; BC: 104), Chechnya (HGSOC: 68; BC: 79), Ingushetia (HGSOC: 19; BC: 103), Karachay-Cherkessia (HGSOC: 13; BC: 47), and several Armenian settlements (HGSOC: 16; BC: 101). The group of BC patients was enriched by young-onset and/or family history-positive and/or bilateral and/or receptor triple-negative cases. The entire coding region of BRCA1, BRCA2, PALB2, and ATM genes was analyzed by next-generation sequencing. RESULTS: A significant contribution of BRCA1/2 pathogenic variants (PVs) to HGSOC and BC development was observed across all North Caucasus regions (HGSOC: 19-39%; BC: 6-13%). Founder alleles were identified in all ethnic groups studied, e.g., BRCA1 c.3629_3630delAG in Chechens, BRCA2 c.6341delC in North Ossetians, BRCA2 c.5351dupA in Ingush, and BRCA1 c.2907_2910delTAAA in Karachays. Some BRCA1/2 alleles, particularly BRCA2 c.9895C > T, were shared by several nationalities. ATM PVs were detected in 14 patients, with c.1673delG and c.8876_8879delACTG alleles occurring twice each. PALB2 heterozygosity was observed in 5 subjects, with one variant seen in 2 unrelated women. CONCLUSION: This study adds to the evidence for the global-wide contribution of BRCA1/2 genes to HGSOC and BC morbidity, although the spectrum of their PVs is a subject of ethnicity-specific variations. The data on founder BRCA1/2 alleles may be considered when adjusting the BRCA1/2 testing procedure to the ethnic origin of patients.

KRAS, NRAS, BRAF, HER2 and MSI Status in a Large Consecutive Series of Colorectal Carcinomas.

This study aimed to analyze clinical and regional factors influencing the distribution of actionable genetic alterations in a large consecutive series of colorectal carcinomas (CRCs). KRAS, NRAS and BRAF mutations, HER2 amplification and overexpression, and microsatellite instability (MSI) were tested in 8355 CRC samples. KRAS mutations were detected in 4137/8355 (49.5%) CRCs, with 3913 belonging to 10 common substitutions affecting codons 12/13/61/146, 174 being represented by 21 rare hot-spot variants, and 35 located outside the "hot" codons. KRAS Q61K substitution, which leads to the aberrant splicing of the gene, was accompanied by the second function-rescuing mutation in all 19 tumors analyzed. NRAS mutations were detected in 389/8355 (4.7%) CRCs (379 hot-spot and 10 non-hot-spot substitutions). BRAF mutations were identified in 556/8355 (6.7%) CRCs (codon 600: 510; codons 594-596: 38; codons 597-602: 8). The frequency of HER2 activation and MSI was 99/8008 (1.2%) and 432/8355 (5.2%), respectively. Some of the above events demonstrated differences in distribution according to patients' age and gender. In contrast to other genetic alterations, BRAF mutation frequencies were subject to geographic variation, with a relatively low incidence in areas with an apparently warmer climate (83/1726 (4.8%) in Southern Russia and North Caucasus vs. 473/6629 (7.1%) in other regions of Russia, p = 0.0007). The simultaneous presence of two drug targets, BRAF mutation and MSI, was observed in 117/8355 cases (1.4%). Combined alterations of two driver genes were detected in 28/8355 (0.3%) tumors (KRAS/NRAS: 8; KRAS/BRAF: 4; KRAS/HER2: 12; NRAS/HER2: 4). This study demonstrates that a substantial portion of RAS alterations is represented by atypical mutations, KRAS Q61K substitution is always accompanied by the second gene-rescuing mutation, BRAF mutation frequency is a subject to geographical variations, and a small fraction of CRCs has simultaneous alterations in more than one driver gene.

Rapid and Cost-Efficient Detection of RET Rearrangements in a Large Consecutive Series of Lung Carcinomas.

RET-kinase-activating gene rearrangements occur in approximately 1-2% of non-small-cell lung carcinomas (NSCLCs). Their reliable detection requires next-generation sequencing (NGS), while conventional methods, such as immunohistochemistry (IHC), fluorescence in situ hybridization (FISH) or variant-specific PCR, have significant limitations. We developed an assay that compares the level of RNA transcripts corresponding to 5'- and 3'-end portions of the RET gene; this test relies on the fact that RET translocations result in the upregulation of the kinase domain of the gene and, therefore, the 5'/3'-end expression imbalance. The present study included 16,106 consecutive NSCLC patients, 14,449 (89.7%) of whom passed cDNA quality control. The 5'/3'-end unbalanced RET expression was observed in 184 (1.3%) tumors, 169 of which had a sufficient amount of material for the identification of translocation variants. Variant-specific PCR revealed RET rearrangements in 155/169 (91.7%) tumors. RNA quality was sufficient for RNA-based NGS in 10 cases, 8 of which carried exceptionally rare or novel (HOOK1::RET and ZC3H7A::RET) RET translocations. We also applied variant-specific PCR for eight common RET rearrangements in 4680 tumors, which emerged negative upon the 5'/3'-end unbalanced expression test; 33 (0.7%) of these NSCLCs showed RET fusion. While the combination of the analysis of 5'/3'-end RET expression imbalance and variant-specific PCR allowed identification of RET translocations in approximately 2% of consecutive NSCLCs, this estimate approached 120/2361 (5.1%) in EGFR/KRAS/ALK/ROS1/BRAF/MET-negative carcinomas. RET-rearranged tumors obtained from females, but not males, had a decreased level of expression of thymidylate synthase (p < 0.00001), which is a known predictive marker of the efficacy of pemetrexed. The results of our study provide a viable alternative for RET testing in facilities that do not have access to NGS due to cost or technical limitations.

Somatic loss of the remaining allele occurs approximately in half of CHEK2-driven breast cancers and is accompanied by a border-line increase of chromosomal instability.

PURPOSE: Germline mutations in CHEK2 gene represent the second most frequent cause of hereditary breast cancer (BC) after BRCA1/2 lesions. This study aimed to identify the molecular characteristics of CHEK2-driven BCs. METHODS: Loss of heterozygosity (LOH) for the remaining CHEK2 allele was examined in 50 CHEK2-driven BCs using allele-specific PCR assays for the germline mutations and analysis of surrounding single-nucleotide polymorphisms (SNPs). Paired tumor and normal DNA samples from 25 cases were subjected to next-generation sequencing analysis. RESULTS: CHEK2 LOH was detected in 28/50 (56%) BCs. LOH involved the wild-type allele in 24 BCs, mutant CHEK2 copy was deleted in 3 carcinomas, while in one case the origin of the deleted allele could not be identified. Somatic PIK3CA and TP53 mutations were present in 13/25 (52%) and 4/25 (16%) tumors, respectively. Genomic features of homologous recombination deficiency (HRD), including the HRD score ≥ 42, the predominance of BRCA-related mutational signature 3, and the high proportion of long (≥ 5 bp) indels, were observed only in 1/20 (5%) BC analyzed for chromosomal instability. Tumors with the deleted wild-type CHEK2 allele differed from LOH-negative cases by elevated HRD scores (median 23 vs. 7, p = 0.010) and higher numbers of chromosomal segments affected by copy number aberrations (p = 0.008). CONCLUSION: Somatic loss of the wild-type CHEK2 allele is observed in approximately half of CHEK2-driven BCs. Tumors without CHEK2 LOH are chromosomally stable. BCs with LOH demonstrate some signs of chromosomal instability; however, its degree is significantly lower as compared to BRCA1/2-associated cancers.

PCR-based analysis of PD-L1 RNA expression in lung cancer: comparison with commonly used immunohistochemical assays.

BACKGROUND: PD-L1 testing is currently performed by immunohistochemistry (IHC). We questioned whether the results of PCR-based measurement of PD-L1 RNA expression correlate with IHC scores obtained by different commercial assays. MATERIALS AND METHODS: 167 consecutive non-squamous non-small cell lung carcinomas (NSCLCs) were analyzed for PD-L1 RNA expression and 22C3, SP263, and SP142 IHC scoring using recommended cut-offs. RNA expression was divided into low, moderate, and high categories. RESULTS: RNA and protein expression demonstrated moderate correlation as continuous variables. Using prespecified RNA cut-offs, PCR testing showed a high negative predictive value towards the IHC analysis: the share of PD-L1 protein-negative tumors among cases classified as PD-L1-low by the PCR test reached 92-99% for all three antibodies. Meanwhile, about half of cases with moderate to high PD-L1 RNA expression had IHC staining in less than 1% tumor cells as determined by 22C3 or SP263 antibodies. Among the 51 discordant cases, which had <1% tumor staining by both 22C3 and SP263 clones but high RNA level, 29 (57%) showed ≥1% positive immune cells by SP263 and/or 22C3, 14 cases (27%) had detectable IHC expression in 0.1-0.9% tumor or immune cells by SP263 and/or 22C3, and 8 (16%) were entirely negative by IHC. CONCLUSION: Some NSCLCs demonstrate readily detectable PD-L1 expression on the level of RNA, but fall below commonly accepted cut-offs by IHC. It remains to be studied whether these discrepancies are attributed to technical or biological reasons. Clinical sensitivity of these tumors to immune therapy deserves additional investigations.

Set7/9 controls proliferation and genotoxic drug resistance of NSCLC cells.

The SET domain containing lysine-specific methyltransferase, Set7/9, covalently attaches methyl moieties to a variety of histone and non-histone substrates. Among the substrates of Set7/9 are: p53, NF-kB, PARP1, E2F1, and other transcription factors that regulate many vital processes in the cell. Through the post-translational regulation of these critical master-regulators Set7/9 is involved in regulation of cell proliferation, cancer progression, and DNA damage response. Noteworthy, the role of Set7/9 in tumorigenesis is contradictory and apparently depends on the cellular context. In this study, we investigated the effect of Set7/9 on tumorigenic characteristics of lung cancer cells. We showed that CRISPR/Cas9-mediated knock-out of Set7/9 in A549 and its shRNA-mediated knock-down in H1299 NSCLC cell lines both augment the proliferation rate of tumor cells compared to the matching wild-type cells. Mechanistically, ablation of Set7/9 increased the expression of cyclin A2 and D1 genes thereby promoting the accumulation of cells in S phase. Furthermore, knockout of Set7/9 decreased the expression of E-cadherin, whose product is critical for cell-cell interactions. Accordingly, this led to the increased migration of lung cancer cells. Finally, both ablation or pharmacological inhibition of Set7/9 enzymatic methyltransferase activity by the selective inhibitor (R)-PFI-2 sensitized NSCLC cells to genotoxic drug, doxorubicin. This effect was also recapitulated on patients-derived NSCLC cell lines. Taken together, our results suggest that Set7/9 plays anti-proliferative and DNA damage-protective roles in NSCLC cells and hence represents an attractive target for anti-cancer chemotherapy.

Exome sequencing study of Russian breast cancer patients suggests a predisposing role for USP39.

PURPOSE: Germline variants in known breast cancer (BC) predisposing genes explain less than half of hereditary BC cases. This study aimed to identify missing genetic determinants of BC. METHODS: Whole exome sequencing (WES) of lymphocyte DNA was performed for 49 Russian patients with clinical signs of genetic BC predisposition, who lacked Slavic founder mutations in BRCA1, BRCA2, CHEK2, and NBS1 genes. RESULTS: Bioinformatic analysis of WES data was allowed to compile a list of 229 candidate mutations. 79 of these mutations were subjected to a three-stage case-control analysis. The initial two stages, which involved up to 797 high-risk BC patients, 1504 consecutive BC cases, and 1081 healthy women, indicated a potentially BC-predisposing role for 6 candidates, i.e., USP39 c.*208G > C, PZP p.Arg680Ter, LEPREL1 p.Pro636Ser, SLIT3 p.Arg154Cys, CREB3 p.Lys157Glu, and ING1 p.Pro319Leu. USP39 c.*208G > C was strongly associated with triple-negative breast tumors (p = 0.0001). In the third replication stage, we genotyped the truncating variant of PZP (rs145240281) and the potential splice variant of USP39 (rs112653307) in three independent cohorts of Russian, Byelorussian, and German ancestry, comprising a total of 3216 cases and 2525 controls. The data obtained for USP39 rs112653307 supported the association identified in the initial stages (the combined OR 1.72, p = 0.035). CONCLUSIONS: This study suggests the role of a rare splicing variant in BC susceptibility. USP39 encodes an ubiquitin-specific peptidase that regulates cancer-relevant tumor suppressors including CHEK2. Further epidemiological and functional studies involving these gene variants are warranted.

Pathways and targeting avenues of BRAF in non-small cell lung cancer.

INTRODUCTION: BRAF is a serine-threonine kinase implicated in the regulation of MAPK signaling cascade. BRAF mutation-driven activation occurs in approximately 2-4% of treatment-naive non-small cell carcinomas (NSCLCs). BRAF upregulation is also often observed in tumors with acquired resistance to receptor tyrosine kinase inhibitors (TKIs). AREAS COVERED: This review describes the spectrum of BRAF mutations and their functional roles, discusses treatment options available for BRAF p.V600 and non-V600 mutated NSCLCs, and identifies some gaps in the current knowledge. EXPERT OPINION: Administration of combined BRAF/MEK inhibitors usually produces significant, although often a short-term, benefit to NSCLC patients with BRAF V600 (class 1) mutations. There are no established treatments for BRAF class 2 (L597, K601, G464, G469A/V/R/S, fusions, etc.) and class 3 (D594, G596, G466, etc.) mutants, which account for up to two-thirds of BRAF-driven NSCLCs. Many important issues related to the use of immune therapy for the management of BRAF-mutated NSCLC deserve further investigation. The rare occurrence of BRAF mutations in NSCLC is compensated by high overall incidence of lung cancer disease; therefore, clinical studies on BRAF-associated NSCLC are feasible.

Response to trametinib, hydroxychloroquine, and bevacizumab in a young woman with NRAS-mutated metastatic intrahepatic cholangiocarcinoma: a case report.

Systemic chemotherapy is the main treatment option for patients with advanced intrahepatic cholangiocarcinoma (iCCA), however, its efficacy is limited. Herein, we report a young patient with NRAS-mutated chemoresistant metastatic iCCA, who received second-line therapy with a combination of trametinib (MEK1/2 inhibitor), hydroxychloroquine (autophagy inhibitor), and bevacizumab (angiogenesis inhibitor). A significant response was achieved during therapy, resulting in a 25% decrease in the size of tumor lesions after 2 months of treatment and an improvement in the patient's condition. The duration of this response was 4 months, but the patient died 10 months after the initiation of this triple therapy. This case report and the analysis of other available studies warrant further investigations on combined MEK and autophagy inhibition in RAS-mutated tumors.

Strong founder effect for BRCA1 c.3629_3630delAG pathogenic variant in Chechen patients with breast or ovarian cancer.

Coding sequences of BRCA1, BRCA2, ATM, TP53, and PALB2 genes were analyzed in 68 consecutive Chechen patients with high-grade serous ovarian cancer (HGSOC). Pathogenic BRCA1/2 variants were identified in 15 (22%) out of 68 HGSOC cases. Nine out of ten patients with BRCA1 pathogenic alleles carried the same deletion (c.3629_3630delAG), and three out of five BRCA2 heterozygotes had Q3299X allele. The analysis of 49 consecutive patients with triple-negative breast cancer (TNBC) revealed 3 (6%) additional BRCA1 heterozygotes. All women with BRCA1 c.3629_3630delAG allele also carried linked c.1067G>A (Q356R) single nucleotide polymorphism, indicating that this is a genuine founder variant but not a mutational hotspot. An ATM truncating allele was detected in one HGSOC patient. There were no women with TP53 or PALB2 germline alterations. Genetic analysis of non-selected HGSOC patients is an efficient tool for the identification of ethnicity-specific BRCA1/2 pathogenic variants.

Lack of Response to Imatinib in Melanoma Carrying Rare KIT Mutation p.T632I.

Approximately 15% of acral and mucous melanomas carry activating mutations in KIT oncogene. There is a diversity of spectrum of KIT mutations, with some of them rendering tumors responsive to imatinib, while others being imatinib-resistant or not studied yet. Here we present an acral melanoma patient with KIT р.T632I mutation, who failed to respond to imatinib.

First-Line Cetuximab Monotherapy in KRAS/NRAS/BRAF Mutation-Negative Colorectal Cancer Patients.

BACKGROUND: Colorectal carcinomas (CRCs) are sensitive to treatment by anti-epidermal growth factor receptor (EGFR) antibodies only if they do not carry activating mutations in down-stream EGFR targets (KRAS/NRAS/BRAF). Most clinical trials for chemo-naive CRC patients involved combination of targeted agents and chemotherapy, while single-agent cetuximab or panitumumab studies included either heavily pretreated patients or subjects who were not selected on the basis of molecular tests. We hypothesized that anti-EGFR therapy would have significant efficacy in chemo-naive patients with KRAS/NRAS/BRAF mutation-negative CRC. METHODS: Nineteen patients were prospectively included in the study. RESULTS: Two (11%) patients experienced partial response (PR) and 11 (58%) subjects showed stable disease (SD). Median time to progression approached 6.1 months (range 1.6-15.0 months). Cetuximab efficacy did not correlate with RNA expression of EGFR and insulin-like growth factor 2 (IGF2). Only one tumor carried PIK3CA mutation, and this CRC responded to cetuximab. Exome analysis of patients with progressive disease (PD) revealed 1 CRC with high-level microsatellite instability and 1 instance of HER2 oncogene amplification; 3 of 4 remaining patients with PD had allergic reactions to cetuximab, while none of the subjects with PR or SD had this complication. Comparison with 19 retrospective KRAS/NRAS/BRAF mutation-negative patients receiving first-line fluoropyrimidines revealed no advantages or disadvantages of cetuximab therapy. CONCLUSIONS: Cetuximab demonstrates only modest efficacy when given as a first-line monotherapy to KRAS/NRAS/BRAF mutation-negative CRC patients. It is of question, why meticulous patient selection, which was undertaken in the current study, did not result in the improvement of outcomes of single-agent cetuximab treatment.

Survival Outcomes in EGFR Mutation-Positive Lung Cancer Patients Treated with Gefitinib until or beyond Progression.

BACKGROUND: Discontinuation of gefitinib treatment is often accompanied by a disease flare. Some studies have demonstrated a benefit of the use of epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKI) beyond progression; however, long-term results of these investigations remain limited. PATIENTS AND METHODS: We observed 70 patients with EGFR-mutated (EGFR-M+) non-small cell lung cancer (NSCLC) receiving single-agent gefitinib in a routine clinical setting; 56 patients were experiencing RECIST progression at the time of the analysis. RESULTS: There was a significant increase (p = 0.00001) in overall survival (OS) in patients continuing on gefitinib beyond progression (n = 21; median duration of continued gefitinib use: 4.2 months; median OS: not reached; expected OS: 29.7 months) as compared to those who stopped gefitinib treatment upon disease progression (n = 35; median OS: 14.0 months). The association between extended gefitinib use and improved OS remained true in multivariate Cox regression analysis (hazard ratio = 4.49, 95% confidence interval 1.25-16.09; p = 0.021). Patient selection bias constitutes an essential limitation of this clinical observational study, given that patients with a more favorable disease course and/or high initial tumor sensitivity to TKI treatment were more likely to be considered for prolonged gefitinib use. CONCLUSION: This study confirms that continued administration of gefitinib beyond progression is a viable treatment option for some patients with EGFR-M+ NSCLC, in particular those who cannot be rescued by novel EGFR mutation-specific inhibitors such as osimertinib.