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PDF file - 118K, Supplementary Table S1. PCR and sequencing primers. Supplementary Table S2. Identified missense variants in PALB2 gene not previously reported. Supplementary Table S3. Frequency of mutations in the BRCA1, BRCA2 and PALB2 genes identified in our high-risk patients.

Background: Several reports indicate that inherited mutations in the PALB2 gene predispose to breast cancer. However, there is little agreement about the clinical relevance and usefulness of mutation screening in this gene. We analyzed the prevalence and spectrum of germline mutations in PALB2 to estimate their contribution to hereditary breast and/or ovarian cancer in the Czech Republic.Methods: The entire PALB2 coding region was sequenced in 409 breast/ovarian cancer patients negative for BRCA1 and BRCA2 mutations. Testing for large genomic rearrangements (LGR) was performed by multiplex ligation-dependent probe amplification (MLPA) analysis.Results: We have identified 13 different pathogenic alterations including 10 truncating mutations and three LGRs in 16 of 409 patients (3.9%), whereas one truncating mutation was found in a group of 1,226 controls (0.08%; P = 2.6 × 10−9). Three novel LGRs included deletions involving exons 7–8 and 9–10, respectively, and a duplication spanning exons 9–11. Five frameshift and two nonsense mutations were novel, whereas three truncating mutations were described previously. The only recurrent mutation was the c.172_175delTTGT detected in four unrelated breast cancer individuals.Conclusions: Our analyses demonstrated the significant role of the PALB2 gene in breast cancer susceptibility. The highest frequency of PALB2 mutations (comparable with that previously reported for BRCA2) was found in a subgroup of patients with hereditary breast cancer (HBC) (13/235; 5.5%).Impact: Our results show that mutation analysis of the PALB2 gene, including the analysis of LGRs, is primarily indicated in patients with HBC in case of their BRCA1 and BRCA2 negativity. Cancer Epidemiol Biomarkers Prev; 22(12); 2323–32. ©2013 AACR.

PURPOSE To estimate age-specific relative and absolute cancer risks of breast cancer and to estimate risks of ovarian, pancreatic, male breast, prostate, and colorectal cancers associated with germline PALB2 pathogenic variants (PVs) because these risks have not been extensively characterized. METHODS We analyzed data from 524 families with PALB2 PVs from 21 countries. Complex segregation analysis was used to estimate relative risks (RRs; relative to country-specific population incidences) and absolute risks of cancers. The models allowed for residual familial aggregation of breast and ovarian cancer and were adjusted for the family-specific ascertainment schemes. RESULTS We found associations between PALB2 PVs and risk of female breast cancer (RR, 7.18; 95% CI, 5.82 to 8.85; P = 6.5 × 10−76), ovarian cancer (RR, 2.91; 95% CI, 1.40 to 6.04; P = 4.1 × 10−3), pancreatic cancer (RR, 2.37; 95% CI, 1.24 to 4.50; P = 8.7 × 10−3), and male breast cancer (RR, 7.34; 95% CI, 1.28 to 42.18; P = 2.6 × 10−2). There was no evidence for increased risks of prostate or colorectal cancer. The breast cancer RRs declined with age ( P for trend = 2.0 × 10−3). After adjusting for family ascertainment, breast cancer risk estimates on the basis of multiple case families were similar to the estimates from families ascertained through population-based studies ( P for difference = .41). On the basis of the combined data, the estimated risks to age 80 years were 53% (95% CI, 44% to 63%) for female breast cancer, 5% (95% CI, 2% to 10%) for ovarian cancer, 2%-3% (95% CI females, 1% to 4%; 95% CI males, 2% to 5%) for pancreatic cancer, and 1% (95% CI, 0.2% to 5%) for male breast cancer. CONCLUSION These results confirm PALB2 as a major breast cancer susceptibility gene and establish substantial associations between germline PALB2 PVs and ovarian, pancreatic, and male breast cancers. These findings will facilitate incorporation of PALB2 into risk prediction models and optimize the clinical cancer risk management of PALB2 PV carriers.

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.

(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.

Hepatocyte nuclear factor-1-beta (HNF1B) is a transcription factor and putative biomarker of solid tumours. Recently, we have revealed a variety of HNF1B mRNA alternative splicing variants (ASVs) with unknown, but potentially regulatory, functions. The aim of our work was to quantify the most common variants and compare their expression in tumour and non-tumour tissues of the large intestine, prostate, and kidney. The HNF1B mRNA variants 3p, Δ7, Δ7–8, and Δ8 were expressed across all the analysed tissues in 28.2–33.5%, 1.5–2%, 0.8–1.7%, and 2.3–6.9% of overall HNF1B mRNA expression, respectively, and occurred individually or in combination. The quantitative changes of ASVs between tumour and non-tumour tissue were observed for the large intestine (3p, Δ7–8), prostate (3p), and kidney samples (Δ7). Decreased expression of the overall HNF1B mRNA in the large intestine and prostate cancer samples compared with the corresponding non-tumour samples was observed (p = 0.019 and p = 0.047, respectively). The decreased mRNA expression correlated with decreased protein expression in large intestine carcinomas (p

Familial non-autoimmune hyperthyroidism is a rare disease caused by germline activating variants in the thyroid-stimulating hormone receptor (TSHR) gene. The c.1856A G (p.Asp619Gly) pathogenic variant has been described in cases of toxic adenoma but never before, to our knowledge, in a case of familial non-autoimmune hyperthyroidism.A 3-year-old boy was admitted for acute gastroenteritis presenting with goiter and tall stature. Laboratory findings revealed peripheral hyperthyroidism and negativity for thyroid autoantibodies. Antithyroid drug treatment was effective, but relapses occurred shortly after attempts to decrease the drug dose. As the boy's father and paternal grandmother also experienced relapsing hyperthyroidism manifesting in early childhood, genetic testing of TSHR was indicated. The c.1856A G (p.Asp619Gly) pathogenic variant was found in all three affected family members. Functional in vitro characterization of the variant verified that it enhances constitutional activation of the receptor, leading to increased production of cyclic adenosine monophosphate. Total thyroidectomy was indicated in the boy due to an unsatisfactory prognosis. Due to persistent positive thyroglobulin serum concentration, a diagnostic radioiodine scan was performed approximately 2 years later. Residual thyroid tissue was revealed; therefore, radioiodine ablative therapy was performed. Despite adequate thyroxine substitution over a long period of follow-up, TSH remained suppressed.Unlike Graves' disease, familial non-autoimmune hyperthyroidism cases present with antithyroid drug-dependence. Not ultrasound but positive thyroglobulin serum concentration indicated residual thyroid tissue. Early detection of residual thyroid tissue and radioiodine ablation prevented the subject from experiencing relapsing hyperthyroidism and undergoing unnecessary repeated surgery. Life-long hormone substitution should be adjusted to free thyroxine rather than TSH serum concentrations.

Ovarian cancer is one of the most common gynecologic cancers with the highest mortality rate over a long period. Genetic predisposition to ovarian cancer is unusually high. In the Czech Republic, causal mutation in any ovarian cancer predisposition gene is identified in approximately 30% of the ovarian cancer patients. Therefore, according to the current guidelines, all ovarian cancer patients should be provided with genetic testing. The BRCA1 and BRCA2 are the two major ovarian cancer predisposition genes. Nevertheless, mutations in other predisposition genes, including RAD51C and RAD51D, are associated with high ovarian cancer risk. Mutations in RAD51C and RAD51D are found in 1% of ovarian cancer patients in each respective gene. Currently, identification of germline mutation in RAD51C and RAD51D is primarily of preventive importance but it potentially could make a prognostic difference. The aim of this review is to summarize the recent RAD51C and RAD51D knowledge, including the biological function, cancer risks associated with germline mutations, and recommendations for mutation carriers.

We report a meta-analysis of breast, prostate, ovarian, and endometrial cancer genome-wide association data (effective sample size: 237,483 cases/317,006 controls). This identified 465 independent lead variants (P−8) across 192 genomic regions. Four lead variants were >1Mb from previously identified risk loci for the four cancers and an additional 23 lead variant-cancer associations were novel for one of the cancers. Bayesian models supported pleiotropic effects involving at least two cancers at 222/465 lead variants in 118/192 regions. Gene-level association analysis identified 13 shared susceptibility genes (P−6) in 13 regions not previously implicated in any of the four cancers and not uncovered by our variant-level meta-analysis. Several lead variants had opposite effects across cancers, including a cluster of such variants in the TP53 pathway. Fifty-four lead variants were associated with blood cell traits and suggested genetic overlaps with clonal hematopoiesis. Our study highlights the remarkable pervasiveness of pleiotropy across hormone-related cancers, further illuminating their shared genetic and mechanistic origins at variant- and gene-level resolution.

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 &gt, 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.

PURPOSE: To estimate age-specific relative and absolute cancer risks of breast cancer and to estimate risks of ovarian, pancreatic, male breast, prostate, and colorectal cancers associated with germline PALB2 pathogenic variants (PVs) because these risks have not been extensively characterized. METHODS: We analyzed data from 524 families with PALB2 PVs from 21 countries. Complex segregation analysis was used to estimate relative risks (RRs; relative to country-specific population incidences) and absolute risks of cancers. The models allowed for residual familial aggregation of breast and ovarian cancer and were adjusted for the family-specific ascertainment schemes. RESULTS: We found associations between PALB2 PVs and risk of female breast cancer (RR, 7.18; 95% CI, 5.82 to 8.85; P = 6.5 × 10(−76)), ovarian cancer (RR, 2.91; 95% CI, 1.40 to 6.04; P = 4.1 × 10(−3)), pancreatic cancer (RR, 2.37; 95% CI, 1.24 to 4.50; P = 8.7 × 10(−3)), and male breast cancer (RR, 7.34; 95% CI, 1.28 to 42.18; P = 2.6 × 10(−2)). There was no evidence for increased risks of prostate or colorectal cancer. The breast cancer RRs declined with age (P for trend = 2.0 × 10(−3)). After adjusting for family ascertainment, breast cancer risk estimates on the basis of multiple case families were similar to the estimates from families ascertained through population-based studies (P for difference = .41). On the basis of the combined data, the estimated risks to age 80 years were 53% (95% CI, 44% to 63%) for female breast cancer, 5% (95% CI, 2% to 10%) for ovarian cancer, 2%-3% (95% CI females, 1% to 4%; 95% CI males, 2% to 5%) for pancreatic cancer, and 1% (95% CI, 0.2% to 5%) for male breast cancer. CONCLUSION: These results confirm PALB2 as a major breast cancer susceptibility gene and establish substantial associations between germline PALB2 PVs and ovarian, pancreatic, and male breast cancers. These findings will facilitate incorporation of PALB2 into risk prediction models and optimize the clinical cancer risk management of PALB2 PV carriers.

Alternative pre-mRNA splicing increases transcriptome plasticity by forming naturally-occurring alternative splicing variants (ASVs). Alterations of splicing processes, caused by DNA mutations, result in aberrant splicing and the formation of aberrant mRNA isoforms. Analyses of hereditary cancer predisposition genes reveal many DNA variants with unknown clinical significance (VUS) that potentially affect pre-mRNA splicing. Therefore, a comprehensive description of ASVs is an essential prerequisite for the interpretation of germline VUS in high-risk individuals. To identify ASVs in a gene of interest, we have proposed an approach based on multiplex PCR (mPCR) amplification of all theoretically possible exon-exon junctions and subsequent characterization of size-selected and pooled mPCR products by next-generation sequencing (NGS). The efficiency of this method is illustrated by a comprehensive analysis of BRCA1 ASVs in human leukocytes, normal mammary, and adipose tissues and stable cell lines. We revealed 94 BRCA1 ASVs, including 29 variants present in all tested samples. While differences in the qualitative expression of BRCA1 ASVs among the analyzed human tissues were minor, larger differences were detected between tissue and cell line samples. Compared with other ASV analysis methods, this approach represents a highly sensitive and rapid alternative for the identification of ASVs in any gene of interest.

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 PPM1D(T) allele to colon tumorigenesis. We found that p53 pathway was suppressed in colon stem cells harboring PPM1D(T) resulting in proliferation advantage under genotoxic stress condition. In addition, truncated PPM1D promoted tumor growth in the colon in Apc(min) mice and diminished survival. Moreover, tumor organoids derived from colon of the Apc(min)Ppm1d(T/+) mice were less sensitive to 5-fluorouracil when compared to Apc(min)Ppm1d(+/+)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. Funding Agencies|Czech Science FoundationGrant Agency of the Czech Republic [16-19437S, 18-09709S]; Academy of Sciences of the Czech RepublicCzech Academy of Sciences [RVO 68378050]; EEA Czech-Norwegian Research Programme-Norwegian Financial Mechanism 2009-2014 (PHOSCAN) [7F14061]; Czech Centre for Phenogenomics [LM2015040]; project: Higher quality and capacity for transgenic models [OP RDI CZ.1.05/2.1.0019.0395]; Biotechnology and Biomedicine Centre of the Academy of Sciences [CZ.1.05/1.1.00/02.0109]; Charles University [CZ.1.05/1.1.00/02.0109]; Worldwide Cancer Research foundation [14-1176]; National Sustainability Program I (NPU I) [LO1503]; Swedish Cancer Foundation; Swedish Research CouncilSwedish Research Council; Health Research Council in South-East Sweden; Research Council of NorwayResearch Council of Norway [179571, 250993]; Norwegian Cancer SocietyNorwegian Cancer Society [182759-2016]; South-Eastern Regional Health Authorities

Purpose To describe a snapshot of international genetic testing practices, specifically regarding the use of multigene panels, for hereditary breast/ovarian cancers. We conducted a survey through the Evidence-Based Network for the Interpretation of Germline Mutant Alleles (ENIGMA) consortium, covering questions about 16 non- BRCA1/ 2 genes. Methods Data were collected via in-person and paper/electronic surveys. ENIGMA members from around the world were invited to participate. Additional information was collected via country networks in the United Kingdom and in Italy. Results Responses from 61 cancer genetics practices across 20 countries showed that 16 genes were tested by > 50% of the centers, but only six ( PALB2, TP53, PTEN, CHEK2, ATM, and BRIP1) were tested regularly. US centers tested the genes most often, whereas United Kingdom and Italian centers with no direct ENIGMA affiliation at the time of the survey were the least likely to regularly test them. Most centers tested the 16 genes through multigene panels; some centers tested TP53, PTEN, and other cancer syndrome–associated genes individually. Most centers reported (likely) pathogenic variants to patients and would test family members for such variants. Gene-specific guidelines for breast and ovarian cancer risk management were limited and differed among countries, especially with regard to starting age and type of imaging and risk-reducing surgery recommendations. Conclusion Currently, a small number of genes beyond BRCA1/ 2 are routinely analyzed worldwide, and management guidelines are limited and largely based on expert opinion. To attain clinical implementation of multigene panel testing through evidence-based management practices, it is paramount that clinicians (and patients) participate in international initiatives that share panel testing data, interpret sequence variants, and collect prospective data to underpin risk estimates and evaluate the outcome of risk intervention strategies.

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 AVCR 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.

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.

An inherited predisposition to breast cancer underlies 5-10% of breast tumors. High-risk BRCA1 and BRCA2 genes result in an 85% lifetime risk of breast cancer and a 20-60% lifetime risk of ovarian cancer. Next-generation sequencing or massive parallel sequencing are now established testing methods that enable screening for many genes that predispose to heterogeneous hereditary cancer syndromes (22 genes are required by the health insurance companies). In addition to BRCA1 and BRCA2, inherited mutations in other genes predispose to breast and/or ovarian cancer. High-risk breast cancer genes include TP53, STK11, CDH1, PTEN, PALB2, and NF1, while moderate-risk (2-4 times increased risk) breast cancer genes include ATM, CHEK2, and NBN. Moderate risk is also suggested for Lynch syndrome, MUTYH, BRIP1, RAD51C, RAD51D, BARD1, FANCA, FANCC, FANCM, BLM, WRN genes. In heterozygotes for other recessive syndromes the risk of developing breast cancer is subject to current research. Low-risk genes are (mostly) irrelevant from a clinical perspective. Other genes that increase the risk of ovarian cancer include the genes for Lynch syndrome, the BRIP1, RAD51C and RAD51D genes. Preventive care should be proposed based on assumed cumulative breast cancer risk (see http: //www.mamo.cz): a risk ofgt;20% for BRCA1/2, TP53, PTEN, STK11, CDH1, PALB2, CHEK2, ATM, and NF1; and a risk of 10-20% for BRIP1, RAD51C, RAD51B, BARD1, FANCA, FANCC, FANCM, NBN, BLM, and WRN. The genetic risk should be assessed by a geneticist and be based on inherited mutations and empirical risk according to family history. Prophylactic mastectomy is considered for high-risk gene carriers but not for moderate-risk gene carriers; however, it may be considered if there is an underlying family history, a risk of parenchyma of the mammary gland, or other risk factors. Ovarian cancer risk increases significantly in carriers of the BRIP1, RAD51C, and RAD51D genes. For prevention of ovarian cancer, prophylactic salpingo-oophorectomy is an important component of preventive care. In ovarian cancer families with no identified risk germline mutation, preventive salpingo-oophorectomy is not routinely recommended but may be considered as the only efficient method of prevention due to the increased empirical risk (4 times) of ovarian cancer in first-degree relatives. Supported by the grant project MH CZ - RVO (MMCI, 00209805), AZV 15-27695A and AZV 16-29959A. 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: 17. 5. 2019 Accepted: 31. 5. 2019.

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.

Pancreatic ductal adenocarcinoma (PDAC) has the worst prognosis among common solid cancer diagnoses. It has been shown that up to 10% of PDAC cases have a familial component. Characterization of PDAC-susceptibility genes could reveal high-risk individuals and patients that may benefit from tailored therapy. Hereditary mutations in PALB2 (Partner and Localizer of BRCA2) gene has been shown to predispose, namely to PDAC and breast cancers; however, frequencies of mutations vary among distinct geographical populations. Using the combination of sequencing, high-resolution melting and multiplex ligation-dependent probe amplification analyses, we screened the entire PALB2 gene in 152 unselected Czech PDAC patients. Truncating mutations were identified in three (2.0%) patients. Genotyping of found PALB2 variants in 1226 control samples revealed one carrier of PALB2 truncating variant (0.08%; P = 0.005). The mean age at PDAC diagnosis was significantly lower among PALB2 mutation carriers (51 years) than in non-carriers (63 years; P = 0.016). Only one patient carrying germline PALB2 mutation had a positive family breast cancer history. Our results indicate that hereditary PALB2 mutation represents clinically considerable genetic factor increasing PDAC susceptibility in our population and that analysis of PALB2 should be considered not only in PDAC patients with familial history of breast or pancreatic cancers but also in younger PDAC patients without family cancer history.

Hereditary breast cancer comprises a minor but clinically meaningful breast cancer (BC) subgroup. Mutations in the major BC-susceptibility genes are important prognostic and predictive markers; however, their carriers represent only 25% of high-risk BC patients. To further characterize variants influencing BC risk, we performed SOLiD sequencing of 581 genes in 325 BC patients (negatively tested in previous BRCA1/BRCA2/PALB2 analyses). In 105 (32%) patients, we identified and confirmed 127 truncating variants (89 unique; nonsense, frameshift indels, and splice site), 19 patients harbored more than one truncation. Forty-six (36 unique) truncating variants in 25 DNA repair genes were found in 41 (12%) patients, including 16 variants in the Fanconi anemia (FA) genes. The most frequent variant in FA genes was c.1096_1099dupATTA in FANCL that also show a borderline association with increased BC risk in subsequent analysis of enlarged groups of BC patients and controls. Another 81 (53 unique) truncating variants were identified in 48 non-DNA repair genes in 74 patients (23%) including 16 patients carrying variants in genes coding proteins of estrogen metabolism/signaling. Our results highlight the importance of mutations in the FA genes' family, and indicate that estrogen metabolism genes may reveal a novel candidate genetic component for BC susceptibility.

PP2C family serine/threonine phosphatase WIP1 acts as a negative regulator of the tumor suppressor p53 and is implicated in silencing of cellular responses to genotoxic stress. Chromosomal locus 17q23 carrying the PPM1D (coding for WIP1) is commonly amplified in breast carcinomas and WIP1 was proposed as potential pharmacological target. Here we employed a cellular model with knocked out PPM1D to validate the specificity and efficiency of GSK2830371, novel small molecule inhibitor of WIP1. We have found that GSK2830371 increased activation of the DNA damage response pathway to a comparable level as the loss of PPM1D. In addition, GSK2830371 did not affect proliferation of cells lacking PPM1D but significantly supressed proliferation of breast cancer cells with amplified PPM1D. Over time cells treated with GSK2830371 accumulated in G1 and G2 phases of the cell cycle in a p21-dependent manner and were prone to induction of senescence by a low dose of MDM2 antagonist nutlin-3. In addition, combined treatment with GSK2830371 and doxorubicin or nutlin-3 potentiated cell death through a strong induction of p53 pathway and activation of caspase 9. We conclude that efficient inhibition of WIP1 by GSK2830371 sensitizes breast cancer cells with amplified PPM1D and wild type p53 to chemotherapy.

BACKGROUND The PALB2 (FANCN) gene was identified as a component of endogenous BRCA2 complex that encodes a DNA repair protein participating along with BRCA1 and BRCA 2 proteins in DNA double-strand break repair. Hereditary PALB2 mutations are associated with an increased risk of breast and pancreatic cancers in heterozygotes. Breast cancer risk for PALB2 mutation carriers has recently been estimated at 33-58% depending on family history of breast cancer; pancreatic cancer risk in carriers of PALB2 mutations has not been precisely quantified, yet. MATERIALS AND RESULTS Results of a study identifying PALB2 mutations in high-risk, BRCA1/2-negative, breast and/or ovarian cancer patients in the Czech Republic indicate that the frequency of hereditary PALB2 mutations in our population is quite high. Interestingly, almost 20% of all recognized mutations comprised large genomic rearrangements. The highest proportion of PALB2 mutations (comparable with the number of mutations reported for BRCA2) was found in a subgroup of hereditary breast cancer patients (5.5%). Frequency of mutations in an independent group of Czech unselected pancreatic cancer patients was approximately 1.3%. CONCLUSION Considering the frequency of pathogenic, hereditary PALB2 mutations in our population, their phenotypic similarity to BRCA2, and expected risk of breast cancer associated with PALB2 mutations, its screen-ing (including large genomic rearrangements) should be encouraged in patients from hereditary breast cancer families. The follow-up of pathogenic PALB2 mutation carriers should be similar to that in BRCA2 mutation carriers, enabling early diagnosis, prevention, and possible targeted therapy. Preventive surgical interventions for the carriers could be considered in case of strong family cancer history and evident segregation of a pathogenic mutation with a tumor phenotype. Additional analysis of various cancer patient populations and further meta-analyses will be necessary for accurate assessment of PALB2 gene penetrance and its significance for the risk of pancreatic and other cancers.

Hepatocyte nuclear factor 1-beta (HNF-1-beta) is a transcription factor involved in cancerogenesis of various tumors, including endometrioid carcinoma. We performed comprehensive analysis of HNF-1-beta in lesions of the endometrium, including protein expression and genetic and epigenetic changes. Expression of HNF-1-beta was analyzed immunohistochemically in 320 cases including both tumor and non-tumor endometrial lesions. Promoter methylation and genetic variants were evaluated, using bisulphite and direct sequencing, in 30 (18 fresh frozen, 12 FFPE tumors) endometrioid carcinomas (ECs) and 15 ovarian clear cell carcinomas (OCCCs) as a control group. We detected expression of HNF-1-beta in 28 % of ECs (51/180 cases), 26 % of serous carcinoma (7/27 cases), 83 % of endometrial clear cell carcinoma (15/18 cases), 93 % of hyperplastic polyps with atypias (13/14 cases), 100 % of hyperplastic polyps without atypias (16/16 cases), 88 % of hyperplasias with atypias (14/16 cases), 91 % of hyperplasias without atypias (10/11 cases), and in ≥80 % of different normal endometrium samples. The control group of OCCCs showed HNF-1-beta expression in 95 % (18/19 cases). Methylation in promoter region was detected in 13.3 % (4/30) of ECs, but not in corresponding normal tissue where available, nor in OCCCs (0/15 cases). Mutation analysis revealed truncating variant c.454C > T (p.Gln152X) in one EC and missense variant c.848C > T (p.Ala283Val) was detected in one OCCC. In conclusion, expression of HNF-1-beta was detected in various extents in all types of lesions analyzed, nevertheless its strong expression was mostly limited to clear cell carcinomas. Biological significance of genetic and epigenetic changes needs further investigation.

Germline protein truncating variants (PTVs) in the FANCM gene have been associated with a 2&ndash, 4-fold increased breast cancer risk in case-control studies conducted in different European populations. However, the distribution and the frequency of FANCM PTVs in Europe have never been investigated. In the present study, we collected the data of 114 European female breast cancer cases with FANCM PTVs ascertained in 20 centers from 13 European countries. We identified 27 different FANCM PTVs. The p.Gln1701* PTV is the most common PTV in Northern Europe with a maximum frequency in Finland and a lower relative frequency in Southern Europe. On the contrary, p.Arg1931* seems to be the most common PTV in Southern Europe. We also showed that p.Arg658*, the third most common PTV, is more frequent in Central Europe, and p.Gln498Thrfs*7 is probably a founder variant from Lithuania. Of the 23 rare or unique FANCM PTVs, 15 have not been previously reported. We provide here the initial spectrum of FANCM PTVs in European breast cancer cases.

Background: Several reports indicate that inherited mutations in the PALB2 gene predispose to breast cancer. However, there is little agreement about the clinical relevance and usefulness of mutation screening in this gene. We analyzed the prevalence and spectrum of germline mutations in PALB2 to estimate their contribution to hereditary breast and/or ovarian cancer in the Czech Republic. Methods: The entire PALB2 coding region was sequenced in 409 breast/ovarian cancer patients negative for BRCA1 and BRCA2 mutations. Testing for large genomic rearrangements (LGR) was performed by multiplex ligation-dependent probe amplification (MLPA) analysis. Results: We have identified 13 different pathogenic alterations including 10 truncating mutations and three LGRs in 16 of 409 patients (3.9%), whereas one truncating mutation was found in a group of 1,226 controls (0.08%; P = 2.6 × 10−9). Three novel LGRs included deletions involving exons 7–8 and 9–10, respectively, and a duplication spanning exons 9–11. Five frameshift and two nonsense mutations were novel, whereas three truncating mutations were described previously. The only recurrent mutation was the c.172_175delTTGT detected in four unrelated breast cancer individuals. Conclusions: Our analyses demonstrated the significant role of the PALB2 gene in breast cancer susceptibility. The highest frequency of PALB2 mutations (comparable with that previously reported for BRCA2) was found in a subgroup of patients with hereditary breast cancer (HBC) (13/235; 5.5%). Impact: Our results show that mutation analysis of the PALB2 gene, including the analysis of LGRs, is primarily indicated in patients with HBC in case of their BRCA1 and BRCA2 negativity. Cancer Epidemiol Biomarkers Prev; 22(12); 2323–32. ©2013 AACR.

Mutations in PPM1D/Wip1 phosphatase impair the DNA damage-induced checkpoint and may predispose cells to tumorigenesis., The DNA damage response (DDR) pathway and its core component tumor suppressor p53 block cell cycle progression after genotoxic stress and represent an intrinsic barrier preventing cancer development. The serine/threonine phosphatase PPM1D/Wip1 inactivates p53 and promotes termination of the DDR pathway. Wip1 has been suggested to act as an oncogene in a subset of tumors that retain wild-type p53. In this paper, we have identified novel gain-of-function mutations in exon 6 of PPM1D that result in expression of C-terminally truncated Wip1. Remarkably, mutations in PPM1D are present not only in the tumors but also in other tissues of breast and colorectal cancer patients, indicating that they arise early in development or affect the germline. We show that mutations in PPM1D affect the DDR pathway and propose that they could predispose to cancer.

Alternative pre-mRNA splicing is a fundamental post-transcriptional regulatory mechanism. Cancer-specific misregulation of the splicing process may lead to formation of irregular alternative splicing variants (ASVs) with a potentially negative impact on cellular homeostasis. Alternative splicing of BRCA1 pre-mRNA can give rise to BRCA1 protein isoforms that possess dramatically altered biological activities compared with full-length wild-type BRCA1. During the screening of high-risk breast cancer (BC) families we ascertained numerous BRCA1 ASVs, however, their clinical significance for BC development is largely unknown. In this study, we examined the influence of the BRCA1Δ17–19 ASV, which lacks a portion of the BRCT domain, on DNA repair capacity using human MCF-7 BC cell clones with stably modified BRCA1 expression. Our results show that overexpression of BRCA1Δ17–19 impairs homologous recombination repair (sensitizes cells to mitomycin C), delays repair of ionizing radiation-induced DNA damage and dynamics of the ionizing radiation-induced foci (IRIF) formation, and undermines also the non-homologous end joining repair (NHEJ) activity. Mechanistically, BRCA1Δ17–19 cannot interact with the partner proteins Abraxas and CtIP, thus preventing interactions known to be critical for processing of DNA lesions. We propose that the observed inability of BRCA1Δ17–19 to functionally replace wtBRCA1 in repair of DNA double-strand breaks (DDSB) reflects impaired capacity to form the BRCA1-A and -C repair complexes. Our findings indicate that expression of BRCA1Δ17–19 may negatively influence genome stability by reducing the DDSB repair velocity, thereby contributing to enhanced probability of cancer development in the affected families.

Pancreatic ductal adenocarcinoma (PDAC) is the sixth most frequent cancer type in the Czech Republic with a poor prognosis that could be improved by an early detection and subsequent surgical treatment combined with chemotherapy. Genetic factors play an important role in PDAC risk. We previously identified one PDAC patient harboring the Slavic founder deleterious mutation c.657del5 in the NBN gene, using a panel next-generation sequencing (NGS). A subsequent analysis of 241 unselected PDAC patients revealed other mutation carriers. The overall frequency of c.657del5 in unselected PDAC patients (5/241; 2.07%) significantly differed from that in non-cancer controls (2/915; 0.2%; P = 0.006). The result indicates that the NBN c.657del5 variant represents a novel PDAC-susceptibility allele increasing PDAC risk (OR = 9.7; 95% CI: 1.9 to 50.2). The increased risk of PDAC in follow-up recommendations for NBN mutation carriers should be considered if other studies also confirm an increased frequency of c.657del5 carriers in PDAC patients from other populations.

Individuals with hereditary cancer syndromes form a minor but clinically important subgroup of oncology patients, comprising several thousand cases in the Czech Republic annually. In these patients, the identification of pathogenic mutations in cancer susceptibility genes has an important predictive and, in some cases, prognostic value. It also enables rational preventive strategies in asymptomatic carriers from affected families. More than 150 cancer susceptibility genes have been described so far; however, mutations in most of them are very rare, occurring with substantial population variability, and hence their clinical interpretation is very complicated. Diagnostics of mutations in cancer susceptibility genes have benefited from the broad availability of next-generation sequencing analyses using targeted gene panels. In order to rationalize the diagnostics of hereditary cancer syndromes in the Czech Republic, we have prepared the sequence capture panel "CZECANCA", targeting 219 cancer susceptibility genes. Besides more than 50 clinically important high- and moderate-penetrance susceptibility genes, the panel also targets less common candidate genes with uncertain clinical relevance. Alongside the panel design, we have optimized the analytical and bioinformatics pipeline, which will facilitate establishing a collective nationwide database of genotypes and clinical data from the analyzed individuals. The key objective of this project is to provide diagnostic laboratories in the Czech Republic with a reliable procedure and collective database improving the clinical utility of next-generation sequencing analyses in high-risk patients, which would help improve the interpretation of rare or population-specific variants in cancer susceptibility genes.

Alterations in dihydropyrimidine dehydrogenase gene (DPYD) coding for the key enzyme (DPD) of fluoropyrimidines (FPs) catabolism contribute to the development of serious FPs-related toxicity. We performed mutation analysis of DPYD based on cDNA sequencing in 76 predominantly colorectal cancer patients treated by FPs with early development of high (grade 3-4) hematological and/or gastrointestinal toxicity. Six previously described [85T>C (C29R), 496A>G (M166V), 775A>G (K259E), 1601G>A (S534N), 1627A>G (I543V), IVS14+1G>A, 2194G>A (V732I)] and two novel [187A>G (K63E) and 1050 G>A (R357H)] non-synonymous DPYD variants were found in 56/76 (73.7%) high-toxicity patients. Subsequently, these alterations were analyzed in 48 patients with excellent long-term tolerance of FPs and in 243 controls and were detected in 37/48 (77.1%) and 166/243 (68.3%) cases, respectively. Analysis of these alterations as risk factors for development of toxicity in pooled FPs-treated population demonstrated that C29R negatively correlated with overall gastrointestinal toxicity (OR = 0.48; 95%CI 0.23-1.0) and M166V in women protected against overall hematological toxicity and neutropenia (both OR = 0.26; 95%CI 0.07-0.89), whereas IVS14+1G>A (found in five high-toxicity patients only) increased risk of mucositis in overall population (OR = 7.0; 95%CI 1.1-44.53), and thrombocytopenia in women (OR = 10.8; 95%CI 1.24-93.98). Moreover, we identified a strong association of V732I with leucopenia (OR = 8.17; 95%CI 2.44 - 27.31) and neutropenia (OR=2.78; 95% CI 1.03-7.51). Our data enabled characterization of "high risk" haplotypes (carriers of IVS14+1G>A or V732 lacking M166V) representing small (22% female and 11% male patients), population in high risk of serious hematological toxicity development, and in patients with "lower risk" that unlikely develop serious hematological toxicity [carriers of M166V without IVS14+1G>A and V732I in females (32% women), and non-carriers of C29R, M166V, IVS14+1G>A, and V732I in males (46% men)]. Our results indicate that genotyping of several DPYD variants may lead to stratification of patients with respect to the risk of serious hematological toxicity development during FPs treatment.

Summary Objective To study the influence of chronic malnutrition in patients with anorexia nervosa on endocrine function of adipose tissue on both circulating and subcutaneous fat mRNA expression level. Patients and design A total of 12 patients with anorexia nervosa and 18 normal weight age-matched women underwent anthropometric examination, single blood drawing and subcutaneous adipose tissue biopsy. Measurements Serum concentrations of high-sensitive CRP (hsCRP), leptin, soluble leptin receptor, adiponectin, resistin, interleukin-6 and insulin were measured by Luminex, enzyme-linked immunosorbent assay (ELISA) and radioimmunoassay (RIA) kits. Subcutaneous adipose tissue mRNA expression of the same adipokines, adiponectin receptors 1 and 2 and immunocompetent cells marker CD68 was measured by real-time polymerase chain reaction (PCR). Results Decreased body fat content of patients with anorexia nervosa was accompanied by reduced hsCRP, leptin and increased adiponectin and soluble leptin receptor. Resistin, interleukin-6 and insulin levels did not differ from those of the control group. Fat mRNA adiponectin, leptin, interleukin-6 and CD68 expression was reduced, resistin mRNA expression was increased and adiponectin receptor 1 and 2 expression were unchanged as compared to the control group. Conclusions Local perturbations in resistin, adiponectin and interleukin-6 mRNA expression in subcutaneous adipose tissue are not reflected by its circulating levels. These changes could be involved in some local metabolic disturbances in subcutaneous adipose tissue of anorexia nervosa patients.

Atypical polypoid adenomyoma (APA) is an uncommon uterine lesion that commonly recurs after local excision and is occasionally associated with or precedes the development of atypical hyperplasia or endometrioid adenocarcinoma. Despite the fact that about 230 cases have been reported in the literature, only 2 studies of 6 and of 7 cases have investigated the molecular aspects; as such, molecular alterations that occur in APA remain largely unknown. We undertook a comprehensive immunohistochemical and molecular analysis of 21 cases of APA in 17 patients (including 4 recurrent/persistent lesions). The analyzed genes were PTEN and TP53 (by fluorescence in situ hybridization) and KRAS, BRAF, EGFR, and NRAS (all by polymerase chain reaction). Immunohistochemical staining was performed for PTEN, p53, mTOR, β-catenin, HNF-1β, and GLUT1 and for the mismatch-repair proteins MLH-1, MSH-2, MSH-6, and PMS-2. In most cases, there was nuclear expression of β-catenin in squamous morules and expression of HNF-1β, mTOR, and GLUT1 in the glandular component. All cases exhibited "wild-type" expression of p53. A common finding was loss of PTEN expression (6/19 cases). In 1 of these cases, loss of PTEN expression was accompanied by PTEN deletion. Mutation of the KRAS gene was found in 5/19 cases. Intact mismatch-repair protein expression was present in all cases, and TP53 abnormalities or mutations of EGFR, NRAS, or BRAF genes were not found. Given the association with atypical hyperplasia and endometrioid adenocarcinoma and the shared immunohistochemical and molecular features, we feel that, conceptually, APA is best regarded as analogous to a localized form of atypical hyperplasia.

Recent studies have conferred that the RAD51C and RAD51D genes, which code for the essential proteins involved in homologous recombination, are ovarian cancer (OC) susceptibility genes that may explain genetic risks in high-risk patients. We performed a mutation analysis in 171 high-risk BRCA1 and BRCA2 negative OC patients, to evaluate the frequency of hereditary RAD51C and RAD51D variants in Czech population. The analysis involved direct sequencing, high resolution melting and multiple ligation-dependent probe analysis. We identified two (1.2%) and three (1.8%) inactivating germline mutations in both respective genes, two of which (c.379_380insG, p.P127Rfs*28 in RAD51C and c.879delG, p.C294Vfs*16 in RAD51D) were novel. Interestingly, an indicative family cancer history was not present in four carriers. Moreover, the ages at the OC diagnoses in identified mutation carriers were substantially lower than those reported in previous studies (four carriers were younger than 45 years). Further, we also described rare missense variants, two in RAD51C and one in RAD51D whose clinical significance needs to be verified. Truncating mutations and rare missense variants ascertained in OC patients were not detected in 1226 control samples. Although the cumulative frequency of RAD51C and RAD51D truncating mutations in our patients was lower than that of the BRCA1 and BRCA2 genes, it may explain OC susceptibility in approximately 3% of high-risk OC patients. Therefore, an RAD51C and RAD51D analysis should be implemented into the comprehensive multi-gene testing for high-risk OC patients, including early-onset OC patients without a family cancer history.