Eva Machackova

Polygenic risk scores (PRS) for epithelial ovarian cancer (EOC) have the potential to improve risk stratification. Joint estimation of Single Nucleotide Polymorphism (SNP) effects in models could improve predictive performance over standard approaches of PRS construction. Here, we implemented computationally efficient, penalized, logistic regression models (lasso, elastic net, stepwise) to individual level genotype data and a Bayesian framework with continuous shrinkage, “select and shrink for summary statistics” (S4), to summary level data for epithelial non-mucinous ovarian cancer risk prediction. We developed the models in a dataset consisting of 23,564 non-mucinous EOC cases and 40,138 controls participating in the Ovarian Cancer Association Consortium (OCAC) and validated the best models in three populations of different ancestries: prospective data from 198,101 women of European ancestries; 7,669 women of East Asian ancestries; 1,072 women of African ancestries, and in 18,915 BRCA1 and 12,337 BRCA2 pathogenic variant carriers of European ancestries. In the external validation data, the model with the strongest association for non-mucinous EOC risk derived from the OCAC model development data was the S4 model (27,240 SNPs) with odds ratios (OR) of 1.38 (95% CI: 1.28–1.48, AUC: 0.588) per unit standard deviation, in women of European ancestries; 1.14 (95% CI: 1.08–1.19, AUC: 0.538) in women of East Asian ancestries; 1.38 (95% CI: 1.21–1.58, AUC: 0.593) in women of African ancestries; hazard ratios of 1.36 (95% CI: 1.29–1.43, AUC: 0.592) in BRCA1 pathogenic variant carriers and 1.49 (95% CI: 1.35–1.64, AUC: 0.624) in BRCA2 pathogenic variant carriers. Incorporation of the S4 PRS in risk prediction models for ovarian cancer may have clinical utility in ovarian cancer prevention programs.

Cutaneous melanoma is the deadliest skin malignity with a rising prevalence worldwide. Patients carrying germline mutations in melanoma-susceptibility genes face an increased risk of melanoma and other cancers. To assess the spectrum of germline variants, we analyzed 264 Czech melanoma patients indicated for testing due to early melanoma (at POT1 and CHEK2 variants were characterized by functional assays. Mutations in clinically relevant genes were significantly more frequent in melanoma patients than in controls (31/264; 11.7% vs. 58/1479; 3.9%; p = 2.0 × 10⁻⁶). A total of 9 patients (3.4%) carried mutations in high-to-moderate melanoma risk genes (CDKN2A, POT1, ACD) and 22 (8.3%) patients in other cancer syndrome genes (NBN, BRCA1/2, CHEK2, ATM, WRN, RB1). Mutations in high-to-moderate melanoma risk genes (OR = 52.2; 95%CI 6.6–413.1; p = 3.2 × 10⁻⁷) and in other cancer syndrome genes (OR = 2.3; 95%CI 1.4–3.8; p = 0.003) were significantly associated with melanoma risk. We found an increased potential to carry these mutations (OR = 2.9; 95%CI 1.2–6.8) in patients with double primary melanoma, melanoma and other primary cancer, but not in patients with early age at onset. The analysis revealed affected genes in Czech melanoma patients and identified individuals who may benefit from genetic testing and future surveillance management of mutation carriers.

Ovarian cancer (OC) is the deadliest gynecologic malignancy with a substantial proportion of hereditary cases and a frequent association with breast cancer (BC). Genetic testing facilitates treatment and preventive strategies reducing OC mortality in mutation carriers. However, the prevalence of germline mutations varies among populations and many rarely mutated OC predisposition genes remain to be identified. We aimed to analyze 219 genes in 1333 Czech OC patients and 2278 population-matched controls using next-generation sequencing. We revealed germline mutations in 18 OC/BC predisposition genes in 32.0% of patients and in 2.5% of controls. Mutations in BRCA1/BRCA2, RAD51C/RAD51D, BARD1, and mismatch repair genes conferred high OC risk (OR > 5). Mutations in BRIP1 and NBN were associated with moderate risk (both OR = 3.5). BRCA1/2 mutations dominated in almost all clinicopathological subgroups including sporadic borderline tumors of ovary (BTO). Analysis of remaining 201 genes revealed somatic mosaics in PPM1D and germline mutations in SHPRH and NAT1 associating with a high/moderate OC risk significantly; however, further studies are warranted to delineate their contribution to OC development in other populations. Our findings demonstrate the high proportion of patients with hereditary OC in Slavic population justifying genetic testing in all patients with OC, including BTO.

Gastric adenocarcinoma and proximal polyposis of the stomach (GAPPS) is a rare variant of familial adenomatous polyposis. It is an autosomal-dominant cancer-predisposition syndrome with massive polyposis of the stomach and a significant risk of gastric adenocarcinoma. Li et al., 2016, described point mutations in the Ying Yang 1 binding site of the APC gene 1B promoter associated with...

Background: Deleterious mutations in the BRCA1 and BRCA2 genes account for a considerable proportion of dominantly inherited breast and ovarian cancer susceptibility. The laboratory interpretation has always been dependent on the information available at the time of the report conclusion. The aim of this study has been to review the results from the BRCA testing at Masaryk Memorial Cancer...

Germline mutations in checkpoint kinase 2 (CHEK2), a multiple cancer‐predisposing gene, increase breast cancer (BC) risk; however, risk estimates differ substantially in published studies. We analyzed germline CHEK2 variants in 1,928 high‐risk Czech breast/ovarian cancer (BC/OC) patients and 3,360 population‐matched controls (PMCs). For a functional classification of VUS, we developed a complementation assay in human nontransformed RPE1‐CHEK2‐knockout cells quantifying CHK2‐specific phosphorylation of endogenous protein KAP1. We identified 10 truncations in 46 (2.39%) patients and in 11 (0.33%) PMC (p = 1.1 × 10⁻¹⁴). Two types of large intragenic rearrangements (LGR) were found in 20/46 mutation carriers. Truncations significantly increased unilateral BC risk (OR = 7.94; 95%CI 3.90–17.47; p = 1.1 × 10⁻¹⁴) and were more frequent in patients with bilateral BC (4/149; 2.68%; p = 0.003), double primary BC/OC (3/79; 3.80%; p = 0.004), male BC (3/48; 6.25%; p = 8.6 × 10⁻⁴), but not with OC (3/354; 0.85%; p = 0.14). Additionally, we found 26 missense VUS in 88 (4.56%) patients and 131 (3.90%) PMC (p = 0.22). Using our functional assay, 11 variants identified in 15 (0.78%) patients and 6 (0.18%) PMC were scored deleterious (p = 0.002). Frequencies of functionally intermediate and neutral variants did not differ between patients and PMC. Functionally deleterious CHEK2 missense variants significantly increased BC risk (OR = 3.90; 95%CI 1.24–13.35; p = 0.009) and marginally OC risk (OR = 4.77; 95%CI 0.77–22.47; p = 0.047); however, carriers low frequency will require evaluation in larger studies. Our study highlights importance of LGR detection for CHEK2 analysis, careful consideration of ethnicity in both cases and controls for risk estimates, and demonstrates promising potential of newly developed human nontransformed cell line assay for functional CHEK2 VUS classification.

Heterozygous germline BRCA2 mutations predispose to breast, ovarian, pancreatic and other types of cancer. The presence of a pathogenic mutation in patients or their family members warrants close surveillance or prophylactic surgery. Besides clearly pathogenic mutations, variants leading only to a single amino acid substitution are often identified. The influence of such variants on cancer risk is often unknown, making their presence a major clinical problem. When genetic methods are insufficient to classify these variants, functional assays with various cellular models are performed. We developed and applied a new syngeneic model of human cancer cells to test all variants of unknown significance in exon 18 identified by genetic testing of high-risk cancer patients in the Czech Republic, via introduction of constructs containing each of these variants into the wild-type allele of BRCA2-heterozygous DLD1 cells (BRCA2^wt/Δex11). We found unaffected DNA repair function of BRCA2 in cell lines BRCA2^{7997G>C/Δex11}, BRCA2^{8111C>T/Δex11}, BRCA2^{8149G>T/Δex11}, BRCA2^{8182G>A/Δex11}, and BRCA2^{8182G>T/Δex11}, whereas the cell line BRCA2^{8168A>G/Δex11} and the nonsense mutation carrying line BRCA2^{8305G>T/Δex11} did affect protein function. Targeting the BRCA2 wild-type allele with a construct carrying the variant c.7988A> G resulted in incorporation exclusively into the already defective allele in all viable clones, strongly suggesting a detrimental phenotype. Our model thus offers a valuable tool for the functional evaluation of unclassified variants in the BRCA2 gene and provides a stable and distributable cellular resource for further research.

Carriers of mutations in hereditary cancer predisposition genes represent a small but clinically important subgroup of oncology patients. The identification of causal germline mutations determines follow-up management, treatment options and genetic counselling in patients’ families. Targeted next-generation sequencing-based analyses using cancer-specific panels in high-risk individuals have been rapidly adopted by diagnostic laboratories. While the use of diagnosis-specific panels is straightforward in typical cases, individuals with unusual phenotypes from families with overlapping criteria require multiple panel testing. Moreover, narrow gene panels are limited by our currently incomplete knowledge about possible genetic dispositions. We have designed a multi-gene panel called CZECANCA (CZEch CAncer paNel for Clinical Application) for a sequencing analysis of 219 cancer-susceptibility and candidate predisposition genes associated with frequent hereditary cancers. The bioanalytical and bioinformatics pipeline was validated on a set of internal and commercially available DNA controls showing high coverage uniformity, sensitivity, specificity and accuracy. The panel demonstrates a reliable detection of both single nucleotide and copy number variants. Inter-laboratory, intra- and inter-run replicates confirmed the robustness of our approach. The objective of CZECANCA is a nationwide consolidation of cancer-predisposition genetic testing across various clinical indications with savings in costs, human labor and turnaround time. Moreover, the unified diagnostics will enable the integration and analysis of genotypes with associated phenotypes in a national database improving the clinical interpretation of variants.

The prevalence and spectrum of germline mutations in BRCA1 and BRCA2 have been reported in single populations, with the majority of reports focused on White in Europe and North America. The Consortium of Investigators of Modifiers of BRCA1/2 (CIMBA) has assembled data on 18,435 families with BRCA1 mutations and 11,351 families with BRCA2 mutations ascertained from 69 centers in 49 countries on six continents. This study comprehensively describes the characteristics of the 1,650 unique BRCA1 and 1,731 unique BRCA2 deleterious (disease‐associated) mutations identified in the CIMBA database. We observed substantial variation in mutation type and frequency by geographical region and race/ethnicity. In addition to known founder mutations, mutations of relatively high frequency were identified in specific racial/ethnic or geographic groups that may reflect founder mutations and which could be used in targeted (panel) first pass genotyping for specific populations. Knowledge of the population‐specific mutational spectrum in BRCA1 and BRCA2 could inform efficient strategies for genetic testing and may justify a more broad‐based oncogenetic testing in some populations.