Population-specific validation and comparison of the performance of 77- and 313-variant polygenic risk scores for breast cancer risk prediction.

BACKGROUND: The polygenic risk score (PRS) allows the quantification of the polygenic effect of many low-penetrance alleles on the risk of breast cancer (BC). This study aimed to evaluate the performance of two sets comprising 77 or 313 low-penetrance loci (PRS77 and PRS313) in patients with BC in the Czech population. METHODS: In a retrospective case-control study, variants were genotyped from both the PRS77 and PRS313 sets in 1329 patients with BC and 1324 noncancer controls, all women without germline pathogenic variants in BC predisposition genes. Odds ratios (ORs) were calculated according to the categorical PRS in individual deciles. Weighted Cox regression analysis was used to estimate the hazard ratio (HR) per standard deviation (SD) increase in PRS. RESULTS: The distributions of standardized PRSs in patients and controls were significantly different (p < 2.2 × 10-16) with both sets. PRS313 outperformed PRS77 in categorical and continuous PRS analyses. For patients in the highest 2.5% of PRS313, the risk reached an OR of 3.05 (95% CI, 1.66-5.89; p = 1.76 × 10-4). The continuous risk was estimated as an HRper SD of 1.64 (95% CI, 1.49-1.81; p < 2.0 × 10-16), which resulted in an absolute risk of 21.03% at age 80 years for individuals in the 95th percentile of PRS313. Discordant categorization into PRS deciles was observed in 248 individuals (9.3%). CONCLUSIONS: Both PRS77 and PRS313 are able to stratify individuals according to their BC risk in the Czech population. PRS313 shows better discriminatory ability. The results support the potential clinical utility of using PRS313 in individualized BC risk prediction.

PRDM1 rs2185379, unlike BRCA1, is not a prognostic marker in patients with advanced ovarian cancer.

BACKGROUND: Ovarian cancer (OC) is mostly diagnosed in advanced stages with high incidence-to-mortality rate. Nevertheless, some patients achieve long-term disease-free survival. However, the prognostic markers have not been well established. OBJECTIVE: The primary objective of this study was to analyse the association of the suggested prognostic marker rs2185379 in PRDM1 with long-term survival in a large independent cohort of advanced OC patients. METHODS: We genotyped 545 well-characterized advanced OC patients. All patients were tested for OC predisposition. The effect of PRDM1 rs2185379 and other monitored clinicopathological and genetic variables on survival were analysed. RESULTS: The univariate analysis revealed no significant effect of PRDM1 rs2185379 on survival whereas significantly worse prognosis was observed in postmenopausal patients (HR = 2.49; 95%CI 1.90-3.26; p= 4.14 × 10 - 11) with mortality linearly increasing with age (HR = 1.05 per year; 95%CI 1.04-1.07; p= 2 × 10 - 6), in patients diagnosed with non-high-grade serous OC (HR = 0.44; 95%CI 0.32-0.60; p= 1.95 × 10 - 7) and in patients carrying a gBRCA1 pathogenic variant (HR = 0.65; 95%CI 0.48-0.87; p= 4.53 × 10 - 3). The multivariate analysis interrogating the effect of PRDM1 rs2185379 with other significant prognostic factors revealed marginal association of PRDM1 rs2185379 with worse survival in postmenopausal women (HR = 1.54; 95%CI 1.01-2.38; p= 0.046). CONCLUSIONS: Unlike age at diagnosis, OC histology or gBRCA1 status, rs2185379 in PRDM1 is unlikely a marker of long-term survival in patients with advance OC.

A deep intronic recurrent CHEK2 variant c.1009-118_1009-87delinsC affects pre-mRNA splicing and contributes to hereditary breast cancer predisposition.

Germline CHEK2 pathogenic variants confer an increased risk of female breast cancer (FBC). Here we describe a recurrent germline intronic variant c.1009-118_1009-87delinsC, which showed a splice acceptor shift in RNA analysis, introducing a premature stop codon (p.Tyr337PhefsTer37). The variant was found in 21/10,204 (0.21%) Czech FBC patients compared to 1/3250 (0.03%) controls (p = 0.04) and in 4/3639 (0.11%) FBC patients from an independent German dataset. In addition, we found this variant in 5/2966 (0.17%) Czech (but none of the 443 German) ovarian cancer patients, three of whom developed early-onset tumors. Based on these observations, we classified this variant as likely pathogenic.

ENIGMA CHEK2gether Project: A Comprehensive Study Identifies Functionally Impaired CHEK2 Germline Missense Variants Associated with Increased Breast Cancer Risk.

PURPOSE: Germline pathogenic variants in CHEK2 confer moderately elevated breast cancer risk (odds ratio, OR ∼ 2.5), qualifying carriers for enhanced breast cancer screening. Besides pathogenic variants, dozens of missense CHEK2 variants of uncertain significance (VUS) have been identified, hampering the clinical utility of germline genetic testing (GGT). EXPERIMENTAL DESIGN: We collected 460 CHEK2 missense VUS identified by the ENIGMA consortium in 15 countries. Their functional characterization was performed using CHEK2-complementation assays quantifying KAP1 phosphorylation and CHK2 autophosphorylation in human RPE1-CHEK2-knockout cells. Concordant results in both functional assays were used to categorize CHEK2 VUS from 12 ENIGMA case-control datasets, including 73,048 female patients with breast cancer and 88,658 ethnicity-matched controls. RESULTS: A total of 430/460 VUS were successfully analyzed, of which 340 (79.1%) were concordant in both functional assays and categorized as functionally impaired (N = 102), functionally intermediate (N = 12), or functionally wild-type (WT)-like (N = 226). We then examined their association with breast cancer risk in the case-control analysis. The OR and 95% CI (confidence intervals) for carriers of functionally impaired, intermediate, and WT-like variants were 2.83 (95% CI, 2.35-3.41), 1.57 (95% CI, 1.41-1.75), and 1.19 (95% CI, 1.08-1.31), respectively. The meta-analysis of population-specific datasets showed similar results. CONCLUSIONS: We determined the functional consequences for the majority of CHEK2 missense VUS found in patients with breast cancer (3,660/4,436; 82.5%). Carriers of functionally impaired missense variants accounted for 0.5% of patients with breast cancer and were associated with a moderate risk similar to that of truncating CHEK2 variants. In contrast, 2.2% of all patients with breast cancer carried functionally wild-type/intermediate missense variants with no clinically relevant breast cancer risk in heterozygous carriers.

Germline multigene panel testing of patients with endometrial cancer.

Endometrial cancer (EC) is the most common gynecological malignancy in developed countries. The present study aimed to determine the frequency of germline pathogenic variants (PV) in patients with EC. In this multicenter retrospective cohort study, germline genetic testing (GGT) was performed in 527 patients with EC using a next generation sequencing panel targeting 226 genes, including 5 Lynch syndrome (LS) and 14 hereditary breast and ovarian cancer (HBOC) predisposition genes, and 207 candidate predisposition genes. Gene-level risks were calculated using 1,662 population-matched controls (PMCs). Patients were sub-categorized to fulfill GGT criteria for LS, HBOC, both or none. A total of 60 patients (11.4%) carried PV in LS (5.1%) and HBOC (6.6%) predisposition genes, including two carriers of double PV. PV in LS genes conferred a significantly higher EC risk [odds ratio (OR), 22.4; 95% CI, 7.8-64.3; P=1.8×10-17] than the most frequently altered HBOC genes BRCA1 (OR, 3.9; 95% CI, 1.6-9.5; P=0.001), BRCA2 (OR, 7.4; 95% CI, 1.9-28.9; P=0.002) and CHEK2 (OR, 3.2; 95% CI, 1.0-9.9; P=0.04). Furthermore, >6% of patients with EC not fulfilling LS or HBOC GGT indication criteria carried a PV in a clinically relevant gene. Carriers of PV in LS genes had a significantly lower age of EC onset than non-carriers (P=0.01). Another 11.0% of patients carried PV in a candidate gene (the most frequent were FANCA and MUTYH); however, their individual frequencies did not differ from PMCs (except for aggregated frequency of loss-of-function variants in POLE/POLD1; OR, 10.44; 95% CI, 1.1-100.5; P=0.012). The present study demonstrated the importance of GGT in patients with EC. The increased risk of EC of PV carriers in HBOC genes suggests that the diagnosis of EC should be included in the HBOC GGT criteria.

Low Frequency of Cancer-Predisposition Gene Mutations in Liver Transplant Candidates with Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC) mainly stems from liver cirrhosis and its genetic predisposition is believed to be rare. However, two recent studies describe pathogenic/likely pathogenic germline variants (PV) in cancer-predisposition genes (CPG). As the risk of de novo tumors might be increased in PV carriers, especially in immunosuppressed patients after a liver transplantation, we analyzed the prevalence of germline CPG variants in HCC patients considered for liver transplantation. Using the panel NGS targeting 226 CPGs, we analyzed germline DNA from 334 Czech HCC patients and 1662 population-matched controls. We identified 48 PVs in 35 genes in 47/334 patients (14.1%). However, only 7/334 (2.1%) patients carried a PV in an established CPG (PMS2, 4×NBN, FH or RET). Only the PV carriers in two MRN complex genes (NBN and RAD50) were significantly more frequent among patients over controls. We found no differences in clinicopathological characteristics between carriers and non-carriers. Our study indicated that the genetic component of HCC is rare. The HCC diagnosis itself does not meet criteria for routine germline CPG genetic testing. However, a low proportion of PV carriers may benefit from a tailored follow-up or targeted therapy and germline testing could be considered in liver transplant recipients.

Prevalence of Germline Pathogenic Variants in Cancer Predisposing Genes in Czech and Belgian Pancreatic Cancer Patients.

(1) Background: The proportion and spectrum of germline pathogenic variants (PV) associated with an increased risk for pancreatic ductal adenocarcinoma (PDAC) varies among populations. (2) Methods: We analyzed 72 Belgian and 226 Czech PDAC patients by multigene panel testing. The prevalence of pathogenic variants (PV) in relation to personal/family cancer history were evaluated. PDAC risks were calculated using both gnomAD-NFE and population-matched controls. (3) Results: In 35/298 (11.7%) patients a PV in an established PDAC-predisposition gene was found. BRCA1/2 PV conferred a high risk in both populations, ATM and Lynch genes only in the Belgian subgroup. PV in other known PDAC-predisposition genes were rarer. Interestingly, a high frequency of CHEK2 PV was observed in both patient populations. PV in PDAC-predisposition genes were more frequent in patients with (i) multiple primary cancers (12/38; 32%), (ii) relatives with PDAC (15/56; 27%), (iii) relatives with breast/ovarian/colorectal cancer or melanoma (15/86; 17%) but more rare in sporadic PDAC (5/149; 3.4%). PV in homologous recombination genes were associated with improved overall survival (HR = 0.51; 95% CI 0.34-0.77). (4) Conclusions: Our analysis emphasizes the value of multigene panel testing in PDAC patients, especially in individuals with a positive family cancer history, and underlines the importance of population-matched controls for risk assessment.

Computed tomography angiographic parameters of pulmonary artery as prognostic factors of residual pulmonary hypertension after pulmonary endarterectomy.

OBJECTIVES: This study aimed to retrospectively assess using computed tomography pulmonary angiography (CTPA) for predicting residual pulmonary hypertension (RPH) in patients with chronic thromboembolic pulmonary hypertension (CTEPH) after pulmonary endarterectomy (PEA). METHODS: We retrospectively analyzed data of 131 patients with CTEPH who underwent PEA in our center (2008-2015). We measured several diameters of the pulmonary artery and thoracic aorta preoperatively. We evaluated the relationship between these measurements (and their indices) and signs of RPH represented by pulmonary artery systolic pressure (PASP) estimated by echocardiography. RESULTS: Significant correlations were observed between the aortopulmonary index and prediction of any residual hypertension and moderate/severe hypertension 1 year after PEA, and any residual hypertension and severe hypertension 2 years after PEA. The aortopulmonary index was significantly related to a reduction in PASP 1 year after the operation. A lower aortopulmonary index (≤0.88 for the ascending aorta and ≤0.64 for the descending aorta) predicted lower RPH. CONCLUSIONS: Preoperative CTPA parameters can be used to assess the risk of RPH after PEA. The aortopulmonary index has significant predictive value for RPH and a reduction in PASP after PEA. Lower values of the aortopulmonary index suggest a better outcome after PEA.

Processing of emotionally ambiguous stimuli in eating disorders: an fMRI pilot study.

PURPOSE: People with eating disorders (EDs) have difficulties understanding their own emotions and recognizing the emotions of others, especially in ambiguous settings. We examined the neuronal mechanisms underlying the emotion processing of ambiguous interpersonal stimuli in EDs and healthy controls (HCs). METHODS: The fMRI data were acquired by a blocked experimental design with 28 women (14 EDs) during the visual presentation of a modified Thematic Apperception Test. RESULTS: EDs showed very strong associations between experienced and inferred emotions evoked by the stimuli; no such relationship was found in HCs. HCs displayed elevated left anterior insula activity during the mentalizing condition; EDs showed increased activity in the right supramarginal gyrus and medial prefrontal cortex. CONCLUSION: The two groups seem to apply different strategies for judging emotionally ambiguous stimuli, albeit resulting in equivalent judgments. We assume that activity in the supramarginal gyrus and insula in EDs is linked with suppressing their own perspective while considering emotional states, probably due to alexithymia and the lack of awareness of their own mental states. We hypothesize that the strong correlation between experienced and inferred emotions in EDs could reflect their tendency to use others as a reference point for perceiving themselves and gaining information about their affective state. LEVEL OF EVIDENCE: No level of evidence, this is a basic science study.

CHEK2 Germline Variants in Cancer Predisposition: Stalemate Rather than Checkmate.

Germline alterations in many genes coding for proteins regulating DNA repair and DNA damage response (DDR) to DNA double-strand breaks (DDSB) have been recognized as pathogenic factors in hereditary cancer predisposition. The ATM-CHEK2-p53 axis has been documented as a backbone for DDR and hypothesized as a barrier against cancer initiation. However, although CHK2 kinase coded by the CHEK2 gene expedites the DDR signal, its function in activation of p53-dependent cell cycle arrest is dispensable. CHEK2 mutations rank among the most frequent germline alterations revealed by germline genetic testing for various hereditary cancer predispositions, but their interpretation is not trivial. From the perspective of interpretation of germline CHEK2 variants, we review the current knowledge related to the structure of the CHEK2 gene, the function of CHK2 kinase, and the clinical significance of CHEK2 germline mutations in patients with hereditary breast, prostate, kidney, thyroid, and colon cancers.

Identification of Germline Mutations in Melanoma Patients with Early Onset, Double Primary Tumors, or Family Cancer History by NGS Analysis of 217 Genes.

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 <25 years) or the presence of multiple primary melanoma/melanoma and other cancer in their personal and/or family history. All patients were analyzed by panel next-generation sequencing targeting 217 genes in four groups: high-to-moderate melanoma risk genes, low melanoma risk genes, cancer syndrome genes, and other genes with an uncertain melanoma risk. Population frequencies were assessed in 1479 population-matched controls. Selected 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.

Multigene Panel Germline Testing of 1333 Czech Patients with Ovarian Cancer.

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.

Comprehensive Assessment of BARD1 Messenger Ribonucleic Acid Splicing With Implications for Variant Classification.

Introduction: Case-control analyses have shown BARD1 variants to be associated with up to >2-fold increase in risk of breast cancer, and potentially greater risk of triple negative breast cancer. BARD1 is included in several gene sequencing panels currently marketed for the prediction of risk of cancer, however there are no gene-specific guidelines for the classification of BARD1 variants. We present the most comprehensive assessment of BARD1 messenger RNA splicing, and demonstrate the application of these data for the classification of truncating and splice site variants according to American College of Medical Genetics and Genomics and the Association for Molecular Pathology (ACMG/AMP) guidelines. Methods: Nanopore sequencing, short-read RNA-seq (whole transcriptome and targeted), and capillary electrophoresis analysis were performed by four laboratories to investigate alternative BARD1 splicing in blood, breast, and fimbriae/ovary related specimens from non-cancer affected tissues. Splicing data were also collated from published studies of nine different tissues. The impact of the findings for PVS1 annotation was assessed for truncating and splice site variants. Results: We identified 62 naturally occurring alternative spliced BARD1 splicing events, including 19 novel events found by next generation sequencing and/or reverse transcription PCR analysis performed for this study. Quantitative analysis showed that naturally occurring splicing events causing loss of clinically relevant domains or nonsense mediated decay can constitute up to 11.9% of overlapping natural junctions, suggesting that aberrant splicing can be tolerated up to this level. Nanopore sequencing of whole BARD1 transcripts characterized 16 alternative isoforms from healthy controls, revealing that the most complex transcripts combined only two alternative splicing events. Bioinformatic analysis of ClinVar submitted variants at or near BARD1 splice sites suggest that all consensus splice site variants in BARD1 should be considered likely pathogenic, with the possible exception of variants at the donor site of exon 5. Conclusions: No BARD1 candidate rescue transcripts were identified in this study, indicating that all premature translation-termination codons variants can be annotated as PVS1. Furthermore, our analysis suggests that all donor and acceptor (IVS+/-1,2) variants can be considered PVS1 or PVS1_strong, with the exception of variants targeting the exon 5 donor site, that we recommend considering as PVS1_moderate.

A comprehensive evaluation of pathogenic mutations in primary cutaneous melanomas, including the identification of novel loss-of-function variants.

The most common histological subtypes of cutaneous melanoma include superficial spreading and nodular melanoma. However, the spectrum of somatic mutations developed in those lesions and all potential druggable targets have not yet been fully elucidated. We present the results of a sequence capture NGS analysis of 114 primary nodular and superficial spreading melanomas identifying driver mutations using biostatistical, immunohistochemical and/or functional approach. The spectrum and frequency of pathogenic or likely pathogenic variants were identified across 54 evaluated genes, including 59 novel mutations, and the newly identified TP53 loss-of-function mutations p.(L194P) and p.(R280K). Frequently mutated genes most commonly affected the MAPK pathway, followed by chromatin remodeling, and cell cycle regulation. Frequent aberrations were also detected in the genes coding for proteins involved in DNA repair and the regulation and modification of cellular tight junctions. Furthermore, relatively frequent mutations were described in KDR and MET, which represent potential clinically important targets. Those results suggest that with the development of new therapeutic possibilities, not only BRAF testing, but complex molecular testing of cutaneous melanoma may become an integral part of the decision process concerning the treatment of patients with melanoma.

Truncated PPM1D impairs stem cell response to genotoxic stress and promotes growth of APC-deficient tumors in the mouse colon.

Protein phosphatase magnesium-dependent 1 delta (PPM1D) terminates cell response to genotoxic stress by negatively regulating the tumor suppressor p53 and other targets at chromatin. Mutations in the exon 6 of the PPM1D result in production of a highly stable, C-terminally truncated PPM1D. These gain-of-function PPM1D mutations are present in various human cancers but their role in tumorigenesis remains unresolved. Here we show that truncated PPM1D impairs activation of the cell cycle checkpoints in human non-transformed RPE cells and allows proliferation in the presence of DNA damage. Next, we developed a mouse model by introducing a truncating mutation in the PPM1D locus and tested contribution of the oncogenic PPM1DT allele to colon tumorigenesis. We found that p53 pathway was suppressed in colon stem cells harboring PPM1DT resulting in proliferation advantage under genotoxic stress condition. In addition, truncated PPM1D promoted tumor growth in the colon in Apcmin mice and diminished survival. Moreover, tumor organoids derived from colon of the ApcminPpm1dT/+ mice were less sensitive to 5-fluorouracil when compared to ApcminPpm1d+/+and the sensitivity to 5-fluorouracil was restored by inhibition of PPM1D. Finally, we screened colorectal cancer patients and identified recurrent somatic PPM1D mutations in a fraction of colon adenocarcinomas that are p53 proficient and show defects in mismatch DNA repair. In summary, we provide the first in vivo evidence that truncated PPM1D can promote tumor growth and modulate sensitivity to chemotherapy.

Germline CHEK2 Gene Mutations in Hereditary Breast Cancer Predisposition - Mutation Types and their Biological and Clinical Relevance.

BACKGROUND: Hereditary mutations in the CHEK2 gene (which encodes CHK2 kinase) contribute to a moderately increased risk of breast cancer (BC) and other cancers. Large variations in the frequency of CHEK2 mutations and the occurrence of variants of unknown clinical significance (VUS) complicate estimation of cancer risk in carriers of germline CHEK2 mutations. PATIENTS AND METHODS: We performed mutation analysis of 1,526 high-risk Czech BC patients and 3,360 Czech controls. Functional analysis was performed for identified VUS using a model system based on a human RPE1-CHEK2-KO cell line harboring biallelic inactivation of endogenous CHEK2. RESULTS: The frequency of ten truncating CHEK2 variants differed markedly between BC patients (2.26%) and controls (0.11%; p = 4.1 × 1012). We also found 23 different missense variants in 4.5% patients and in 4.0% of controls. The most common was p.I157T, which was found in patients and controls with the same frequency. Functional analysis identified nine functionally deleterious VUS, another nine functionally neutral VUS, and four intermediate VUS (including p.I157T). We found that carriers of truncating CHEK2 mutations had a high BC risk (OR 8.19; 95% CI 4.11-17.75), and that carriers of functionally deleterious missense variants had a moderate risk (OR 4.06; 95% CI, 1.37-13.39). Carriers of these mutations developed BC at 44.4 and 50.7 years, respectively. Functionally neutral and functionally intermediate missense variants did not increase the BC risk. BC in CHEK2 mutation carriers was frequently ER-positive and of higher grade. Notably, carriers of CHEK2 mutations developed second cancers more frequently than BRCA1/BRCA2/PALB2/p53 or mutation non-carriers. CONCLUSION: Hereditary CHEK2 mutations contribute to the development of hereditary BC. The associated cancer risk in mutation carriers increases with the number of affected individuals in a family. Annual follow-up with breast ultrasound, mammography, or magnetic resonance imaging is recommended for asymptomatic mutation carriers from the age of 40. Surgical prevention and specific follow-up of other tumors should be considered based on family cancer history. The work was supported by grants from the Czech Health Research Council of the Ministry of Health of the Czech Republic NR 15-28830A, 16-29959A, NV19-03-00279, projects of the PROGRES Q28/LF1, GAUK 762216, SVV2019 / 260367, PRIMUS/17/MED/9, UNCE/MED/016, Progress Q26, LQ1604 NPU II and project AVČR Qualitas. The analysis of a set of unselected controls was made possible by the existence and support of the scientific infrastructure of the National Center for Medical Genomics (LM2015091) and its project aimed at creating a reference database of genetic variants of the Czech Republic (CZ.02.1.01/0.0/0.0/16_013/0001634). The authors declare they have no potential conflicts of interest concerning drugs, products, or services used in the study. The Editorial Board declares that the manuscript met the ICMJE recommendation for biomedical papers. Submitted: 2. 4. 2019 Accepted: 14. 5. 2019.

Contribution of Massive Parallel Sequencing to Diagnosis of Hereditary Ovarian Cancer in the Czech Republic.

BACKGROUND: Ovarian cancer is a disease with high mortality. Approximately 1,000 women are diagnosed with ovarian cancer in the Czech Republic annually. Women harboring a mutation in cancer-predisposing genes face an increased risk of tumor development. Mutations in BRCA1, BRCA2, BRIP1, and Lynch syndrome genes (RAD51C, RAD51D, and STK11) are associated with a high risk of ovarian cancer, and mutations in ATM, CHEK2, NBN, PALB2, and BARD1 appear to increase the risk. Our aim was to examine the frequency of mutations in cancer-predisposing genes in the Czech Republic. MATERIALS AND METHODS: We analyzed 1,057 individuals including ovarian cancer patients and 617 non-cancer controls using CZECANCA panel next-generation sequencing on the Illumina platform. Pathogenic mutations in high-risk genes, including CNVs, were detected in 30.6% of patients. The mutation frequency reached 25.0% and 18.2% in subgroups of unselected ovarian cancer patients and patients with a negative family cancer history, respectively. The most frequently mutated genes were BRCA1 and BRCA2. The overall frequency of mutations in non-BRCA genes was comparable to that in BRCA2. The mutation frequency in ovarian cancer patients aged >70 years was three times higher than that in patients diagnosed before the age of 30. CONCLUSION: Ovarian cancer is a heterogeneous disease with a high proportion of hereditary cases. The lack of efficient screening for early diagnosis emphasizes the importance of identifying carriers of mutations in ovarian cancer-predisposing genes; this is because proper follow-up and prevention strategies can reduce overall ovarian cancer-related mortality. This work was supported by grants AZV 15-27695A, SVV2019/260367, PROGRES Q28/LF1. The authors declare they have no potential conflicts of interest concerning drugs, products, or services used in the study. The Editorial Board declares that the manuscript met the ICMJE recommendation for biomedical papers. Submitted: 7. 3. 2019 Accepted: 24. 4. 2019.

Estrogen Receptor Status Oppositely Modifies Breast Cancer Prognosis in BRCA1/BRCA2 Mutation Carriers Versus Non-Carriers.

Breast cancer (BC) prognosis in BRCA1 and BRCA2 mutation carriers has been reported contradictorily, and the significance of variables influencing prognosis in sporadic BC is not established in BC patients with hereditary BRCA1/BRCA2 mutations. In this retrospective cohort study, we analyzed the effect of clinicopathological characteristics on BC prognosis (disease-free survival [DFS] and disease-specific survival [DSS]) in hereditary BRCA1/BRCA2 mutation carriers. We enrolled 234 BRCA1/BRCA2 mutation carriers and 899 non-carriers, of whom 191 carriers and 680 non-carriers, with complete data, were available for survival analyses. We found that patients with ER-positive tumors developed disease recurrence 2.3-times more likely when they carried a BRCA1/BRCA2 mutation (23/60; 38.3% ER-positive carriers vs. 74/445; 16.6% ER-positive non-carriers; p < 0.001). ER-positive mutation carriers also had a 3.4-times higher risk of death due to BC compared with ER-positive non-carriers (13/60; 21.7% vs. 28/445; 6.3%; p < 0.001). Moreover, prognosis in ER-negative BRCA1/BRCA2 mutation carriers was comparable with that in ER-positive non-carriers. Our study demonstrates that ER-positivity worsens BC prognosis in BRCA1/BRCA2 mutation carriers, while prognosis for carriers with ER-negative tumors (including early-onset) is significantly better and comparable with that in ER-positive, older BC non-carriers. These observations indicate that BRCA1/BRCA2 mutation carriers with ER-positive BC represent high-risk patients.

Identification of deleterious germline CHEK2 mutations and their association with breast and ovarian 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.