Structural implications of amyloidogenic rare variants Ser282Leu and Gln356Arg identified in h-BRCA1.

Preliminary studies have shown BRCA1 (170-1600) residues to be intrinsically disordered with unknown structural details. However, thousands of clinically reported variants have been identified in this central region of BRCA1. Therefore, we aimed to characterize h-BRCA1(260-553) to assess the structural basis for pathogenicity of two rare missense variants Ser282Leu, Gln356Arg identified from the Indian and Russian populations respectively. Small-angle X-ray scattering analysis revealed WT scores Rg -32 Å, Dmax -93 Å, and Rflex-51% which are partially disordered, whereas Ser282Leu variant displayed a higher degree of disorderedness and Gln356Arg was observed to be aggregated. WT protein also possesses an inherent propensity to undergo a disorder-to-order transition in the presence of cruciform DNA and 2,2,2-Trifluoroethanol (TFE). An increased alpha-helical pattern was observed with increasing concentration of TFE for the Gln356Arg mutant whereas Ser282Leu mutant showed significant differences only at the highest TFE concentration. Furthermore, higher thermal shift was observed for WT-DNA complex compared to the Gln356Arg and Ser282Leu protein-DNA complex. Moreover, mature amyloid-like fibrils were observed with 30 µM thioflavin T (ThT) at 37°C for Ser282Leu and Gln356Arg proteins while the WT protein exists in a protofibril state as observed by TEM. Gln356Arg formed higher-order aggregates with amyloidogenesis over time as monitored by ThT fluorescence. In addition, computational analyses confirmed larger conformational fluctuations for Ser282Leu and Gln356Arg mutants than for the WT. The global structural alterations caused by these variants provide a mechanistic approach for further classification of the variants of uncertain clinical significance in BRCA1 into amyloidogenic variants which may have a significant role in disease pathogenesis.

Molecular Analysis of Biliary Tract Cancers with the Custom 3' RACE-Based NGS Panel.

The technique 3' rapid amplification of cDNA ends (3' RACE) allows for detection of translocations with unknown gene partners located at the 3' end of the chimeric transcript. We composed a 3' RACE-based RNA sequencing panel for the analysis of FGFR1-4 gene rearrangements, detection of activating mutations located within FGFR1-4, IDH1/2, ERBB2 (HER2), KRAS, NRAS, BRAF, and PIK3CA genes, and measurement of the expression of ERBB2, PD-L1, and FGFR1-4 transcripts. This NGS panel was utilized for the molecular profiling of 168 biliary tract carcinomas (BTCs), including 83 intrahepatic cholangiocarcinomas (iCCAs), 44 extrahepatic cholangiocarcinomas (eCCAs), and 41 gallbladder adenocarcinomas (GBAs). The NGS failure rate was 3/168 (1.8%). iCCAs, but not other categories of BTCs, were characterized by frequent FGFR2 alterations (17/82, 20.7%) and IDH1/2 mutations (23/82, 28%). Other potentially druggable events included ERBB2 amplifications or mutations (7/165, 4.2% of all successfully analyzed BTCs) and BRAF p.V600E mutations (3/165, 1.8%). In addition to NGS, we analyzed microsatellite instability (MSI) using the standard five markers and revealed this event in 3/158 (1.9%) BTCs. There were no instances of ALK, ROS1, RET, and NTRK1-3 gene rearrangements or MET exon 14 skipping mutations. Parallel analysis of 47 iCCA samples with the Illumina TruSight Tumor 170 kit confirmed good performance of our NGS panel. In conclusion, targeted RNA sequencing coupled with the 3' RACE technology is an efficient tool for the molecular diagnostics of BTCs.

Hereditary cancer syndromes.

Hereditary cancer syndromes (HCSs) are arguably the most frequent category of Mendelian genetic diseases, as at least 2% of presumably healthy subjects carry highly-penetrant tumor-predisposing pathogenic variants (PVs). Hereditary breast-ovarian cancer and Lynch syndrome make the highest contribution to cancer morbidity; in addition, there are several dozen less frequent types of familial tumors. The development of the majority albeit not all hereditary malignancies involves two-hit mechanism, i.e. the somatic inactivation of the remaining copy of the affected gene. Earlier studies on cancer families suggested nearly fatal penetrance for the majority of HCS genes; however, population-based investigations and especially large-scale next-generation sequencing data sets demonstrate that the presence of some highly-penetrant PVs is often compatible with healthy status. Hereditary cancer research initially focused mainly on cancer detection and prevention. Recent studies identified multiple HCS-specific drug vulnerabilities, which translated into the development of highly efficient therapeutic options.

Changes in the concentration of EGFR-mutated plasma DNA in the first hours of targeted therapy allow the prediction of tumor response in patients with EGFR-driven lung cancer.

PURPOSE: This study aimed to analyze changes in the plasma concentration of EGFR-mutated circulating tumor DNA (ctDNA) occurring immediately after the start of therapy with EGFR tyrosine kinase inhibitors (TKIs). METHODS: Serial plasma samples were collected from 30 patients with EGFR-driven non-small cell lung cancer before intake of the first tablet and at 0.5, 1, 2, 3, 6, 12, 24, 36 and 48 h after the start of the therapy. The content of EGFR alleles (exon 19 deletions or L858R) in ctDNA was measured by ddPCR. RESULTS: ctDNA was detected at base-line in 25/30 (83%) subjects. Twelve (50%) out of 24 informative patients showed > 25% reduction of the ctDNA content at 48 h time point; all these patients demonstrated disease control after 4 and 8-12 weeks of therapy. The remaining 12 individuals showed either stable content of EGFR-mutated ctDNA (n = 5) or the elevation of ctDNA concentration (n = 7). 10 of 12 patients with elevated or stable ctDNA level achieved an objective response at 4 weeks, but only 5 of 10 evaluable patients still demonstrated disease control at 8-12 weeks (p = 0.032, when compared to the group with ctDNA decrease). The decline of the amount of circulating EGFR mutant copies at 48 h also correlated with longer progression-free survival (14.7 months vs. 8.5 months, p = 0.013). CONCLUSION: Comparison of concentration of EGFR-mutated ctDNA at base-line and at 48 h after the start of therapy is predictive for the duration of TKI efficacy.

Content of circulating tumor DNA depends on the tumor type and the dynamics of tumor size, but is not influenced significantly by physical exercise, time of the day or recent meal.

PURPOSE: This study aimed to investigate factors, which influence the content of circulating tumor DNA (ctDNA). METHODS: 398 serial plasma samples were collected within 1-7 consecutive days from patients with EGFR-mutated lung cancer (n = 13), RAS/RAF-mutated colorectal cancer (n = 54) and BRAF-mutated melanoma (n = 17), who presented with measurable tumor disease. The amount of ctDNA was determined by ddPCR. RESULTS: Among 82 patients, who donated 2-6 serial plasma samples, 42 subjects were classified as ctDNA-positive; only 22% cases were mutation-positive across all consecutive tests, while 24/82 (29%) patients showed presence of mutated ctDNA in some but not all blood draws. Subjects with progressing tumors had higher probability of being detected ctDNA-positive as compared to patients, who responded to therapy or had stable disease (39/55 (71%) vs. 4/24 (17%); p = 0.0001). Our study failed to reveal the impact of the time of the day, recent meal or prior physical exercise on the results of ctDNA testing. CONCLUSIONS: Presence of ctDNA in plasma is particularly characteristic for patients, who experience clinical progression of tumor disease. Consecutive plasma tests may occasionally provide discordant data; thus, the repetition of analysis may be advised in certain cases in order to ensure the validity of negative ctDNA result.

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.

Biophysical evaluation to categorize pathogenicity of cancer-predisposing mutations identified in the BARD1 BRCT domain.

The BRCT domain of BARD1 (BARD1 BRCT) is involved in many cellular processes such as DNA damage repair (DDR) and cell-cycle checkpoint regulation. BARD1 BRCT performs tumor suppressor function by recruiting BRCA1 at DNA damage site via interactions with other DNA damage repair (DDR) proteins. Considering the importance of the BRCT domain in genomic integrity, we decided to evaluate reported mutations of BARD1 BRCT Cys645Arg, Val695Leu, and Ser761Asn for their pathogenicity. To explore the effect of the mutation on the structure and function, BARD1 BRCT wild-type proteins and the mutant proteins were studied using different biochemical, biophysical and in silico techniques. Comparative fluorescence, circular dichroism (CD) spectroscopy and limited proteolysis studies demonstrate the well-folded structural conformation of wild-type and mutant proteins. However, thermal and chemical denaturation studies revealed similarity in the folding pattern of BARD1 BRCT wild-type and Cys645Arg mutant proteins, whereas there was a significant loss in the thermodynamic stability of Val695Leu and Ser761Asn mutants. Molecular dynamics (MD) simulation studies on wild-type and mutant protein structures indicate the loss in structural integrity of mutants compared with the wild-type protein.