Publications by authors named "Jan Hojny"

18 Publications

  • Page 1 of 1

Gynecological lesions in hereditary cancer predisposition syndromes.

Cesk Patol 2021 ;57(2):96-104

Hereditary tumor syndromes with a possible manifestation in the female internal genital tract represent a heterogeneous group of diseases. The two most common entities are the hereditary breast and ovarian cancer syndrome, and the Lynch syndrome. The less common syndromes include the rhabdoid tumor predisposition syndrome, Cowden syndrome, tuberous sclerosis complex, DICER1 syndrome, nevoid basal cell carcinoma syndrome, Peutz-Jeghers syndrome, von Hippel-Lindau disease, and hereditary leiomyomatosis and renal cell cancer syndrome. The goal of this manuscript is to provide a comprehensive overview of those hereditary tumor syndromes which can manifest in the area of the female genital system, with an emphasis on their summary, the characteristics of the tumors which can develop in association with these syndromes, and the approach to the processing of prophylactically removed tissues and organs. The issue of Lynch syndrome screening is also discussed.
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July 2021

A comprehensive analysis of the expression, epigenetic and genetic changes of HNF1B and ECI2 in 122 cases of high-grade serous ovarian carcinoma.

Oncol Lett 2021 Mar 6;21(3):185. Epub 2021 Jan 6.

Institute of Biology and Medical Genetics, First Faculty of Medicine, Charles University and General University Hospital in Prague, 12800 Prague, Czech Republic.

High-grade serous ovarian cancer (HGSC) is the most common subtype of ovarian cancer, with a poor prognosis; however, most studies concerning ovarian carcinoma have focused mainly on clear cell carcinoma. The involvement of hepatocyte nuclear factor 1β (HNF1B) in the carcinogenesis of HGSC has not yet been fully elucidated. To the best of our knowledge, the present study is the first to analyse the expression of the possible downstream target of HNF1B, enoyl-CoA (Δ) isomerase 2 (ECI2), in HGSC. The present study performed a comprehensive analysis of HNF1B mRNA and protein expression, and epigenetic and genetic changes, as well as an analysis of ECI2 mRNA and protein expression in 122 cases of HGSC. HNF1B protein expression was detected in 28/122 cases, and was positively associated with lymphovascular invasion (P=0.025). Protein expression of ECI2 was detected in 115/122 cases, but no associations with clinicopathological variables were revealed. Therefore, ECI2 does not seem to function as a suitable prognostic marker for HGSC. In the sample set, a positive correlation between HNF1B and ECI2 protein expression was detected (P=0.005). HNF1B mRNA was also positively correlated with HNF1B protein expression (P=0.001). promoter methylation was detected in 26/67 (38.8%) of cases. A novel pathogenic somatic mutation was detected in 1/61 (1.6%) of the analysed HGSC cases. No other correlations between the examined SNPs (rs4430796, rs757210 and rs7405776), HNF1B promoter methylation, HNF1B/ECI2 expression or clinicopathological characteristics were found.
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http://dx.doi.org/10.3892/ol.2021.12446DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7816296PMC
March 2021

Analysis of expression, epigenetic, and genetic changes of HNF1B in 130 kidney tumours.

Sci Rep 2020 10 13;10(1):17151. Epub 2020 Oct 13.

Institute of Pathology, First Faculty of Medicine, Charles University and General University Hospital in Prague, Studnickova 2, 12800, Prague 2, Czech Republic.

Hepatocyte nuclear factor 1 beta (HNF1B) is a transcription factor which plays a crucial role in nephronogenesis, and its germline mutations have been associated with kidney developmental disorders. However, the effects of HNF1B somatic exonic mutations and its role in the pathogenesis of kidney tumours has not yet been elucidated. Depending on the type of the tumour HNF1B may act as a tumour suppressor or oncogene, although the exact mechanism by which HNF1B participates in the process of cancerogenesis is unknown. Using an immunohistochemical approach, and methylation and mutation analysis, we have investigated the expression, epigenetic, and genetic changes of HNF1B in 130 cases of renal tumours (121 renal cell carcinomas, 9 oncocytomas). In the subset of clear cell renal cell carcinoma (ccRCC), decreased HNF1B expression was associated with a higher tumour grade and higher T stage. The mutation analysis revealed no mutations in the analysed samples. Promoter methylation was detected in two ccRCCs and one oncocytoma. The results of our work on a limited sample set suggest that while in papillary renal cell carcinoma HNF1B functions as an oncogene, in ccRCC and chRCC it may act in a tumour suppressive fashion.
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http://dx.doi.org/10.1038/s41598-020-74059-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7555858PMC
October 2020

HNF1B, EZH2 and ECI2 in prostate carcinoma. Molecular, immunohistochemical and clinico-pathological study.

Sci Rep 2020 09 1;10(1):14365. Epub 2020 Sep 1.

Department of Pediatrics and Adolescent Medicine, First Faculty of Medicine, Charles University and General University Hospital in Prague, Prague 2, Czech Republic.

Hepatocyte nuclear factor 1 beta (HNF1B) is a tissue specific transcription factor, which seems to play an important role in the carcinogenesis of several tumors. In our study we focused on analyzing HNF1B in prostate carcinoma (PC) and adenomyomatous hyperplasia (AH), as well as its possible relation to the upstream gene EZH2 and downstream gene ECI2. The results of our study showed that on an immunohistochemical level, the expression of HNF1B was low in PC, did not differ between PC and AH, and did not correlate with any clinical outcomes. In PC, mutations of HNF1B gene were rare, but the methylation of its promotor was a common finding and was positively correlated with Gleason score and stage. The relationship between HNF1B and EZH2/ECI2 was equivocal, but EZH2 and ECI2 were positively correlated on both mRNA and protein level. The expression of EZH2 was associated with poor prognosis. ECI2 did not correlate with any clinical outcomes. Our results support the oncosuppressive role of HNF1B in PC, which may be silenced by promotor methylation and other mechanisms, but not by gene mutation. The high expression of EZH2 (especially) and ECI2 in PC seems to be a potential therapeutic target.
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http://dx.doi.org/10.1038/s41598-020-71427-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7463257PMC
September 2020

Microscopic extraovarian sex cord proliferation: report of a case with bilateral Fallopian tube involvement and a comprehensive molecular analysis.

Pol J Pathol 2020 ;71(2):175-180

Institute of Pathology, First Faculty of Medicine, Charles University and General University Hospital in Prague, Czech Republic.

We report a case of a 58-year-old female with microscopic extraovarian sex cord proliferations affecting both Fallopian tubes. Molecular analysis showed likely pathogenic germline missense mutations of the KDM5A and KMT2D genes. However, mutations of other genes, including FOXL2 and STK11, were not detected. Our case represents the 12th case of extraovarian sex cord proliferation reported in the literature to date. This is the first time that a molecular genetic analysis of the lesion has been performed, and it showed a wild-type FOXL2 gene, which represents another argument supporting the estimated benign nature of these rare lesions.
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http://dx.doi.org/10.5114/pjp.2020.97023DOI Listing
October 2020

Ovarian mesonephric-like adenocarcinoma arising in serous borderline tumor: a case report with complex morphological and molecular analysis.

Diagn Pathol 2020 Jul 21;15(1):91. Epub 2020 Jul 21.

Gynecologic Oncology Center, Department of Obstetrics and Gynecology, First Faculty of Medicine, Charles University and General University Hospital in Prague, Apolinarska 18, 12808, Prague 2, Czech Republic.

Background: Mesonephric-like adenocarcinoma (M-LAC) is a rare, recently described tumor occurring in the uterine corpus and ovary, which shares the same morphological and immunohistochemical features with the more common mesonephric adenocarcinoma (MAC), which mostly arises the uterine cervix. Despite the similarities between these tumors, the histogenesis of M-LAC is still disputable.

Case Presentation: Sixty-one-year-old woman presented with an advanced tumor of the left ovary with intraabdominal spread and liver metastases. After receiving 5 cycles of neoadjuvant chemotherapy, she underwent a hysterectomy with bilateral salpingo-oophorectomy, and resection of the liver metastasis, omentum, and appendix. Histologically, the ovarian tumor consisted of two components, whose morphology and immunohistochemical results were typical of either a serous borderline tumor (immunohistochemical positivity for PAX8, WT1, ER and PR) or a mesonephric-like carcinoma (immunohistochemical positivity for PAX8, TTF1 and GATA3). Only the component of the mesonephric-like adenocarcinoma metastasized to the omentum and liver. A molecular analysis with a panel of 271 genes (size 1020 kbp) was performed separately on samples from the borderline tumor, primary ovarian mesonephric-like adenocarcinoma, and liver metastasis. The results showed the clonal origin of all samples, which shared the same KRAS (NM_004985.3:c.34G > T, p.(G12C)) and PIK3CA (NM_006218.2:c.1633G > A, p.(E545K)) somatic mutations. Moreover, in the sample from the primary mesonephric-like carcinoma and its liver metastasis a likely pathogenic somatic MYCN mutation (NM_005378.4:c.131C > T, p.(P44L) was found. In all samples, the deletion of exons 9-10 in the CHEK2 gene was present, which is in concordance with the previously performed genetic testing of the blood specimen which revealed the hereditary CHEK2 mutation in this patient.

Conclusions: Our result support the theory that at least some mesonephric-like ovarian adenocarcinomas are of Müllerian origin. The serous borderline tumor seems to be a precursor of mesonephric-like adenocarcinoma, which has been proven in our case by both tumors sharing the same mutations, and the presence of cumulative molecular aberrations in the mesonephric-like adenocarcinoma.
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http://dx.doi.org/10.1186/s13000-020-01012-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7372838PMC
July 2020

Results of targeted next-generation sequencing in children with cystic kidney diseases often change the clinical diagnosis.

PLoS One 2020 23;15(6):e0235071. Epub 2020 Jun 23.

Institute of Biology and Medical Genetics, First Faculty of Medicine, Charles University and General University Hospital in Prague, Prague, Czech Republic.

Cystic kidney diseases are a very heterogeneous group of chronic kidney diseases. The diagnosis is usually based on clinical and ultrasound characteristics and the final diagnosis is often difficult to be made. Next-generation sequencing (NGS) may help the clinicians to find the correct final diagnosis. The aim of our study was to test the diagnostic yield of NGS and its ability to improve the diagnosis precision in a heterogeneous group of children with cystic kidney diseases. Next-generation sequencing of genes responsible for the formation of cystic kidneys was performed in 31 unrelated patients with various clinically diagnosed cystic kidney diseases gathered at the Department of Pediatrics of Motol University Hospital in Prague between 2013 and 2018. The underlying pathogenic variants were detected in 71% of patients (n = 22), no or only one (in case of autosomal recessive inheritance) pathogenic variant was found in 29% of patients (n = 9). The result of NGS correlated with the clinical diagnosis made before the NGS in 55% of patients (n = 17), in the remaining 14 children (45%) the result of NGS revealed another type of cystic kidney disease that was suspected clinically before or did not find causal mutation in suspected genes. The most common unexpected findings were variants in nephronophthisis (NPHP) genes in children with clinically suspected autosomal recessive polycystic kidney disease (ARPKD, n = 4). Overall, 24 pathogenic or probably pathogenic variants were detected in the PKHD1 gene, 8 variants in the TMEM67 gene, 4 variants in the PKD1 gene, 2 variants in the HNF1B gene and 2 variants in BBS1 and NPHP1 genes, respectively. NGS is a valuable tool in the diagnostics of various forms of cystic kidney diseases. Its results changed the clinically based diagnoses in 16% (n = 5) of the children.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0235071PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7310724PMC
September 2020

Expression, Epigenetic, and Genetic Changes of HNF1B in Colorectal Lesions: an Analysis of 145 Cases.

Pathol Oncol Res 2020 Oct 1;26(4):2337-2350. Epub 2020 Jun 1.

Institute of Pathology, First Faculty of Medicine, Charles University and General University Hospital in Prague, Studnickova 2, 12800, Prague 2, Czech Republic.

Hepatocyte nuclear factor 1 beta (HNF1B) is transcription factor which plays a crucial role in the regulation of the development of several organs, but also seems to be implicated in the development of certain tumours, especially the subset of clear cell carcinomas of the ovary and kidney. Depending on the type of the tumour, HNF1B may act as either a tumour suppressor or an oncogene, although the exact mechanism by which HNF1B participates in the process of cancerogenesis is unknown. Using immunohistochemical approach and methylation and mutation analysis, we have investigated the expression, epigenetic, and genetic changes of HNF1B on 40 cases of colorectal adenomas and 105 cases of colorectal carcinomas. The expression of HNF1B was correlated with the benign or malignant behaviour of the lesion, given that carcinomas showed significantly lower levels of expression compared to adenomas. In carcinomas, lower levels of HNF1B expression were associated with recurrence and shortened disease-free survival. The mutation analysis revealed three somatic mutations (two frameshift and one nonsense) in the carcinoma sample set. Promoter methylation was detected in three carcinomas. These results suggest that in colorectal cancer, HNF1B may play a part in the pathogenesis and act in a tumour suppressive fashion.
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http://dx.doi.org/10.1007/s12253-020-00830-2DOI Listing
October 2020

Identification of novel HNF1B mRNA splicing variants and their qualitative and semi-quantitative profile in selected healthy and tumour tissues.

Sci Rep 2020 04 24;10(1):6958. Epub 2020 Apr 24.

Institute of Pathology, First Faculty of Medicine, Charles University and General University Hospital in Prague, Prague, 12808, Czech Republic.

Hepatocyte nuclear factor-1-beta (HNF1B) is a transcription factor crucial for the development of several tissues, and a promising biomarker of certain solid tumours. Thus far, two HNF1B alternative splicing variants (ASVs) have been described, however, the complete spectrum, prevalence and role of HNF1B ASVs in tumorigenesis are unclear. Considering the equivocal data about HNF1B ASVs and expression presented in literature, our aim was to characterize the spectrum of HNF1B mRNA splicing variants across different tissues. Here, we characterize HNF1B ASVs with high sensitivity in carcinomas of the uterine corpus, large intestine, kidney, pancreas, and prostate, with selected paired healthy tissues, using the previously described multiplex PCR and NGS approach. We identified 45 ASVs, of which 43 were novel. The spectrum and relative quantity of expressed ASVs mRNA differed among the analysed tissue types. Two known (3p, Δ7_8) and two novel (Δ7, Δ8) ASVs with unknown biological functions were detected in all the analysed tissues in a higher proportion. Our study reveals the wide spectrum of HNF1B ASVs in selected tissues. Characterization of the HNF1B ASVs is an important prerequisite for further expression studies to delineate the HNF1B splicing pattern, potential ASVs functional impact, and eventual refinement of HNF1B's biomarker role.
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http://dx.doi.org/10.1038/s41598-020-63733-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7181708PMC
April 2020

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

Sci Rep 2019 11 19;9(1):17050. Epub 2019 Nov 19.

Institute of Pathology, First Faculty of Medicine, Charles University and General University Hospital in Prague, Prague, Czech Republic.

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.
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http://dx.doi.org/10.1038/s41598-019-53636-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6863855PMC
November 2019

Leiomyoma with Bizarre Nuclei: a Study of 108 Cases Focusing on Clinicopathological Features, Morphology, and Fumarate Hydratase Alterations.

Pathol Oncol Res 2020 Jul 31;26(3):1527-1537. Epub 2019 Aug 31.

Institute of Pathology, First Faculty of Medicine, Charles University and General University Hospital in Prague, Studnickova 2, 12800, Prague 2, Czech Republic.

Leiomyoma with bizarre nuclei (LBN) is an uncommon variant of uterine smooth muscle neoplasm. Involvement of fumarate hydratase (FH) has been suggested in the pathogenesis of a subset of LBN. The goal of our study is to assess the clinicopathological, morphological, immunohistochemical and molecular findings focusing on FH in LBNs (n = 108) and compare it with the findings in usual leiomyomas (UL; n = 50) and leiomyosarcomas (LMS; n = 42). Immunohistochemically, loss of FH expression was found in 67/108 of LBN, 1/50 of UL and in no LMS. Class 4/5 FH mutations were detected in 15/53 LBN with sufficient DNA quality for molecular analysis. Pathogenic variants of the FH gene were detected in neither UL nor LMS. Local recurrence after surgery was present in 18/92 of LBN patients, 7 of which were histologically verified and 2 of which were found to be LBN. Our results confirmed that LBN behave in a benign fashion, although they may relapse. FH gene mutations were a common finding only in LBN, but not in UL and LMS. Immunohistochemistry with an antibody against FH seems to have a good sensitivity (87%) and moderate specificity (58%) with regard to predicting FH gene mutations and could be used as a screening method in tumors with features suggestive of FH alterations to identify patients who are at risk for the FH aberrations.
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http://dx.doi.org/10.1007/s12253-019-00739-5DOI Listing
July 2020

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

Klin Onkol 2019 ;32(Supplementum2):36-50

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.
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http://dx.doi.org/10.14735/amko2019S36DOI Listing
January 2020

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

Int J Cancer 2019 10 20;145(7):1782-1797. Epub 2019 May 20.

Institute of Biochemistry and Experimental Oncology, First Faculty of Medicine, Charles University, Prague, Czech Republic.

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 ). 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 ) 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 ), 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.
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http://dx.doi.org/10.1002/ijc.32385DOI Listing
October 2019

Synchronous endometrioid endometrial and ovarian carcinomas are biologically related: A clinico-pathological and molecular (next generation sequencing) study of 22 cases.

Oncol Lett 2019 Feb 20;17(2):2207-2214. Epub 2018 Dec 20.

Institute of Pathology, First Faculty of Medicine, Charles University and General University Hospital in Prague, 12800 Prague, Czech Republic.

The criteria for distinction between independent primary tumors and metastasis from one site to the other in synchronous endometrioid endometrial and ovarian carcinoma (SEO) has been a matter of dispute for a long time. In our study we performed a comprehensive clinico-pathological and molecular analysis of 22 cases of SEO. Based on conventional clinico-pathological criteria the cases were classified as independent primary tumors (10 cases) and metastasis from one location to the other (12 cases). All tumors were analyzed by NGS with a panel of 73 genes (219 kbp). Clonal origin was confirmed in all cases by at least one shared mutation in and . Two patients carried germline pathogenic mutation in cancer-predisposing genes or . Microsatellite instable phenotype was detected in 5/22 (22.7%) SEO, but in one case only in the endometrial tumor. In conclusion, our results showed that all 22 SEOs were clonally related, irrespectively of their clinico-pathological features. Even low grade and low stage tumors classified as independent primaries, according to the conventional morphological criteria, have a clonal origin. From the practical point of view, only the conventional morphological criteria should be used for the classification (staging) of these tumors. However, molecular profiling of these tumors may have prognostic and predictive meaning.
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http://dx.doi.org/10.3892/ol.2018.9855DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6341770PMC
February 2019

Germline mutation in the TP53 gene in uveal melanoma.

Sci Rep 2018 05 16;8(1):7618. Epub 2018 May 16.

Institute of Pathology, First Faculty of Medicine, Charles University and General University Hospital in Prague, Prague, Czech Republic.

We performed comprehensive molecular analysis of five cases of metastasizing uveal malignant melanoma (UM) (fresh-frozen samples) with an NGS panel of 73 genes. A likely pathogenic germline TP53 mutation c.760A > G (p.I254V) was found in two tumor samples and matched nontumor tissue. In three cases, pathogenic BAP1 mutation was detected together with germline missense variants of uncertain significance in ATM. All cases carried recurrent activating GNAQ or GNA11 mutation. Moreover, we analyzed samples from another 16 patients with primary UM by direct Sanger sequencing focusing only on TP53 coding region. No other germline TP53 mutation was detected in these samples. Germline TP53 mutation, usually associated with Li-Fraumeni syndrome, is a rare event in UM. To the best of our knowledge, only one family with germline TP53 mutation has previously been described. In our study, we detected TP53 mutation in two patients without known family relationship. The identification of germline aberrations in TP53 or BAP1 is important to identify patients with Li-Fraumeni syndrome or BAP1 cancer syndrome, which is also crucial for proper genetic counseling.
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http://dx.doi.org/10.1038/s41598-018-26040-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5955881PMC
May 2018

Multiplex PCR and NGS-based identification of mRNA splicing variants: Analysis of BRCA1 splicing pattern as a model.

Gene 2017 Dec 14;637:41-49. Epub 2017 Sep 14.

Institute of Biochemistry and Experimental Oncology, First Faculty of Medicine, Charles University, Prague 12853, Czech Republic; Institute of Biology and Medical Genetics, First Faculty of Medicine, Charles University and General University Hospital in Prague, Prague 120 00, Czech Republic. Electronic address:

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.
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http://dx.doi.org/10.1016/j.gene.2017.09.025DOI Listing
December 2017

Association of Germline CHEK2 Gene Variants with Risk and Prognosis of Non-Hodgkin Lymphoma.

PLoS One 2015 27;10(10):e0140819. Epub 2015 Oct 27.

Institute of Biochemistry and Experimental Oncology, First Faculty of Medicine, Charles University in Prague, Prague, Czech Republic.

The checkpoint kinase 2 gene (CHEK2) codes for the CHK2 protein, an important mediator of the DNA damage response pathway. The CHEK2 gene has been recognized as a multi-cancer susceptibility gene; however, its role in non-Hodgkin lymphoma (NHL) remains unclear. We performed mutation analysis of the entire CHEK2 coding sequence in 340 NHL patients using denaturing high-performance liquid chromatography (DHPLC) and multiplex ligation-dependent probe amplification (MLPA). Identified hereditary variants were genotyped in 445 non-cancer controls. The influence of CHEK2 variants on disease risk was statistically evaluated. Identified CHEK2 germline variants included four truncating mutations (found in five patients and no control; P = 0.02) and nine missense variants (found in 21 patients and 12 controls; P = 0.02). Carriers of non-synonymous variants had an increased risk of NHL development [odds ratio (OR) 2.86; 95% confidence interval (CI) 1.42-5.79] and an unfavorable prognosis [hazard ratio (HR) of progression-free survival (PFS) 2.1; 95% CI 1.12-4.05]. In contrast, the most frequent intronic variant c.319+43dupA (identified in 22% of patients and 31% of controls) was associated with a decreased NHL risk (OR = 0.62; 95% CI 0.45-0.86), but its positive prognostic effect was limited to NHL patients with diffuse large B-cell lymphoma (DLBCL) treated by conventional chemotherapy without rituximab (HR-PFS 0.4; 94% CI 0.17-0.74). Our results show that germ-line CHEK2 mutations affecting protein coding sequence confer a moderately-increased risk of NHL, they are associated with an unfavorable NHL prognosis, and they may represent a valuable predictive biomarker for patients with DLBCL.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0140819PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4624763PMC
June 2016

Expression of human BRCA1Δ17-19 alternative splicing variant with a truncated BRCT domain in MCF-7 cells results in impaired assembly of DNA repair complexes and aberrant DNA damage response.

Cell Signal 2013 May 14;25(5):1186-93. Epub 2013 Feb 14.

Institute of Biochemistry and Experimental Oncology, First Faculty of Medicine, Charles University in Prague, U Nemocnice 5, CZ-128 53 Prague 2, Czech Republic.

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.
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http://dx.doi.org/10.1016/j.cellsig.2013.02.008DOI Listing
May 2013
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