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Introduction: The Institute of Oncology Ljubljana (IOL) provides genetic assessment for cancer patients who require test results for treatment planning and whenever there is a risk of a hereditary cancer syndrome. In this study, we aimed to analyse the trend of referrals of cancer patients and to assess the pathogenic / likely pathogenic variant (PV/LPV) detection rate in tested individuals according to family history. Methods: In the Department of Clinical Cancer Genetics, IOL, we performed data analysis using descriptive statistical methods and the Microsoft Excel tool. It was based on the number of referrals, patients’ family history data and their genetic test results for germline PV/LPV, focusing on specific cancer site (breast, ovaries, pancreas, and prostate) between 01/01/2020 and 30/06/2024. Results: In a period of 4.5 years, 4,252 patients were referred for genetic counselling, in particular breast cancer patients (3,074). On average, it took 4.3 years from cancer diagnosis to germline genetic testing. 16.5% of all patients tested were diagnosed with germline PV/LPV. Germline PV/LPV were mostly found in ovarian cancer patients (25.2%), followed by pancreatic cancer patients (16.2%), breast cancer patients (16.2%), and prostate cancer patients (11.1%). PV/LPV were most frequent in the genes BRCA1/2. The percentage of patients with a positive family history who tested positive was 19.5%, whereas the percentage of patients with a negative family history was 12.4%. Conclusion: The survey proves the importance of germline genetic testing for cancer patients and their relatives. In fact, 16.5% of patients tested positive for germline PV/LPV and the results were used for treatment planning and prevention. Therefore, it would be reasonable to consider introducing universal genetic testing for certain cancer patients in a safe clinical setting and to be able to identify all those at higher cancer risk.

We assessed the prevalence, localization, type and outcome of occult cancer at risk-reducing salpingo-oophorectomy or salpingectomy (RRSO) in asymptomatic carriers of pathogenic or likely pathogenic BRCA1/2 variants and high-risk BRCA1/2 negative women.

In Slovenia like in other countries, till recently, personal history of epithelial ovarian cancer (EOC) has not been included among indications for genetic counselling. Recent studies reported up to 17% rate of germinal BRCA1/2 mutation (gBRCA1/2m) within the age group under 50 years at diagnosis. The original aim of this study was to invite to the genetic counselling still living patients with EOC under 45 years, to offer gBRCA1/2m testing and to perform analysis of gBRCA1/2m rate and of clinico-pathologic characteristics. Later, we added also the data of previously genetically tested patients with EOC aged 45 to 49 years.

Abstract Background A single nucleotide polymorphism located in the 3'-untranslated region of the KRAS oncogene (KRAS variant; rs61764370) disrupts a let-7 miRNA binding and was recently reported to act as a genetic marker for increased risk of developing human cancers. We aimed to investigate an association of the KRAS variant with sporadic and familial breast cancer and breast tumor characteristics. Methods Genotyping was accomplished in 530 sporadic postmenopausal breast cancer cases, 165 familial breast cancer cases (including N = 29, who test positive for BRCA1/2 mutations) and 270 postmenopausal control women using the flurogenic 5' nuclease assay. Information on hormone replacement therapy (HRT) use and tumor characteristics in sporadic breast cancer cases was ascertained from a postal questionnaire and pathology reports, respectively. Associations between the KRAS genotype and breast cancer or breast tumor characteristics were assessed using chi-square test and logistic regression models. Results No evidence of association was observed between the KRAS variant and risk of sporadic and familial breast cancer - either among BRCA carriers or non-BRCA carriers. The KRAS variant was statistically significantly more often associated with human epidermal growth factor receptor 2 (HER2) - positive tumors and tumors of higher histopathologic grade. However, both associations were detected only in HRT users. Conclusion Our data do not support the hypothesis that the KRAS variant rs61764370 is implicated in the aetiology of sporadic or of familial breast cancer. In postmenopausal women using HRT, the KRAS variant might lead to HER2 overexpressed and poorly-differentiated breast tumors, both indicators of a worse prognosis.

Abstract Background The BRCA1 and BRCA2 mutation spectrum and mutation detection rates according to different family histories were investigated in 521 subjects from 322 unrelated Slovenian cancer families with breast and/or ovarian cancer. Methods The BRCA1 and BRCA2 genes were screened using DGGE, PTT, HRM, MLPA and direct sequencing. Results Eighteen different mutations were found in BRCA1 and 13 in BRCA2 gene. Mutations in one or other gene were found in 96 unrelated families. The mutation detection rates were the highest in the families with at least one breast and at least one ovarian cancer - 42% for BRCA1 and 8% for BRCA2. The mutation detection rate observed in the families with at least two breast cancers with disease onset before the age of 50 years and no ovarian cancer was 23% for BRCA1 and 13% for BRCA2. The mutation detection rate in the families with at least two breast cancers and only one with the disease onset before the age of 50 years was 11% for BRCA1 and 8% for BRCA2. In the families with at least two breast cancers, all of them with disease onset over the age of 50 years, the detection rate was 5% for BRCA2 and 0% for BRCA1. Conclusion Among the mutations detected in Slovenian population, 5 mutations in BRCA1 and 4 mutations in BRCA2 have not been described in other populations until now. The most frequent mutations in our population were c.181T > G, c.1687C > T, c.5266dupC and c.844_850dupTCATTAC in BRCA1 gene and c.7806-2A > G, c.5291C > G and c.3978insTGCT in BRCA2 gene (detected in 69% of BRCA1 and BRCA2 positive families).

Abstract Background An ideal tumor vaccine should activate both effector and memory immune response against tumor-specific antigens. Beside the CD8+ T cells that play a central role in the generation of a protective immune response and of long-term memory, dendritic cells (DCs) are important for the induction, coordination and regulation of the adaptive immune response. The DCs can conduct all of the elements of the immune orchestra and are therefore a fundamental target and tool for vaccination. The present study was aimed at assessing the ability of tumor vaccine composed of C-class CpG ODNs and irradiated melanoma tumor cells B16F1 followed by two additional injections of CpG ODNs to induce the generation of a functional long-term memory response in experimental tumor model in mice (i.p. B16F1). Results It has been shown that the functional memory response in vaccinated mice persists for at least 60 days after the last vaccination. Repeated vaccination also improves the survival of experimental animals compared to single vaccination, whereas the proportion of animals totally protected from the development of aggressive i.p. B16F1 tumors after vaccination repeated three times varies between 88.9%-100.0%. Additionally, the long-term immune memory and tumor protection is maintained over a prolonged period of time of at least 8 months. Finally, it has been demonstrated that following the vaccination the tumor-specific memory cells predominantly reside in bone marrow and peritoneal tissue and are in a more active state than their splenic counterparts. Conclusions In this study we demonstrated that tumor vaccine composed of C-class CpG ODNs and irradiated tumor cells followed by two additional injections of CpG ODNs induces a long-term immunity against aggressive B16F1 tumors.

Competing Interests: Declarations. Ethics approval: All participants provided written informed consent. The present study was approved by the National Ethics Committee and the Institutional Ethics Committee of the Institute of Oncology Ljubljana (0120–591/2020/3 on the 20th of January 2021). Research was conducted according to the 1975 Helsinki Declaration as revised in 1983 and the procedures used met the ethical standards of these bodies. Competing interests: The authors declare no competing interests.

Objective: In patients with platinum-sensitive relapsed ovarian cancer (PSROC) harboring pathogenic/likely pathogenic variants (PV) in BRCA1 and BRCA2 genes, olaparib maintenance monotherapy (OMT) is a viable option. Our study aimed to evaluate the impact of different BRCA1/2 PV in survival outcomes and safety of OMT in BRCA1/2-mutated PSROC patients, focusing on the type and location of PV., Methods: We assessed the outcomes of 100 BRCA1/2-mutated PSROC patients treated at our institute, analyzing progression-free survival (PFS) and overall survival (OS). Germline and tumor BRCA1/2 genotyping was conducted using Illumina's next-generation sequencing (NGS)., Results: PFS and OS were significantly shorter in PSROC patients with PV in BRCA1 compared to those with PV in BRCA2 (PFS:14.0 vs. 38.8 months, p = 0.007, OS: 21.8 vs. 62.0 months, p = 0.011). Notably, there was a significant difference in PFS based on the intragenic location of BRCA1 PV, with shorter PFS in patients with 1st/2nd relapse, harboring PV in BRCA1 RING domain compared to those with PV in the DNA binding domain (DBD) and BRCT domains (12.4 vs. 23.0 months, p = 0.046). No differences in PFS and OS were observed between patients with germline versus somatic BRCA1/2 PV (PFS:14.9 vs.19.3, p = 0.316, OS: not reached vs. 25.8 months; p = 0.224). However, there were significant differences in the reasons for OMT discontinuation between patients with germline and somatic BRCA1/2 PV, primarily due to adverse side effects., Conclusions: In summary, the type and location of BRCA1 and BRCA2 PV provide additional insight into the expected survival outcomes of olaparib MT in PSROC patients., Trial Registration Number: ISRCTN42408038, Name of registry: ISRCTN registry, Date of registration: 24/11/2015., Competing Interests: Declaration of competing interest The authors declare that they have no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Context.—: Small cell carcinoma of the ovary, hypercalcemic type (SCCOHT) is a rare and lethal tumor, characterized by hypercalcemia and early onset and associated with germline and somatic SMARCA4 variants., Objective.—: To identify all known cases of SCCOHT in the Slovenian population from 1991 to 2021 and present genetic testing results, histopathologic findings, and clinical data for these patients. We also estimate the incidence of SCCOHT., Design.—: We conducted a retrospective analysis of hospital medical records and data from the Slovenian Cancer Registry in order to identify cases of SCCOHT and obtain relevant clinical data. Histopathologic review of tumor samples with assessment of immunohistochemical staining for SMARCA4/BRG1 was undertaken to confirm the diagnosis of SCCOHT. Germline and somatic genetic analyses were performed using targeted next-generation sequencing., Results.—: Between 1991 and 2021, we identified 7 cases of SCCOHT in a population of 2 million. Genetic causes were determined in all cases. Two novel germline loss-of-function variants in SMARCA4 LRG_878t1:c.1423_1429delTACCTCA p.(Tyr475Ilefs*24) and LRG_878t1:c.3216-1G>T were identified. At diagnosis, patients were ages 21 to 41 and had International Federation of Gynecology and Obstetrics, or FIGO, stage IA-III disease. Outcomes were poor, with 6 of 7 patients dying of disease-related complications within 27 months from diagnosis. One patient had stable disease for 12 months while receiving immunotherapy., Conclusions.—: We present genetic, histopathologic, and clinical characteristics for all cases of SCCOHT identified in the Slovenian population during a 30-year period. We report 2 novel germline SMARCA4 variants, possibly associated with high penetrance. We estimate the minimal incidence of SCCOHT to be 0.12 per 1 million per year., Competing Interests: The authors have no relevant financial interest in the products or companies described in this article., (© 2024 College of American Pathologists.)

Background: High-grade serous carcinoma (HGSC) is the most common and aggressive type of ovarian cancer, and it is often associated with ascites at presentation. The objective of this study was to evaluate the accuracy of cytopathology to identify immunophenotypic features of HGSC and BRCA1/2 mutations from ascites., Methods: The study included 45 patients with histologically confirmed primary HGSC and malignant ascites. Immunocytochemistry (ICC) staining for PAX8, WT1, P53, P16, and Ki-67 was performed on cytospins and cytoblocks prepared from ascites. Next-generation sequencing (NGS) was used to detect germline/somatic BRCA1/2 mutations in the ascites. Both ICC and NGS results were compared with immunohistochemistry (IHC) and NGS results from tissue blocks of primary tumor. Cronbach α and χ 2 statistics, respectively, were used., Results: ICC/IHC results for PAX8, WT1, P53, and P16 showed good reliability between cytospins, cytoblocks, and tissue blocks (α > 0.75), whereas poor reliability and significant differences were observed for Ki-67 between ascites and tissue blocks (α < 0.26; p < .001 [Kruskal-Wallis]). For germline BRCA1/2 mutations, 100% concordance was confirmed, but only 14% concordance was confirmed for somatic mutations., Conclusions: These results demonstrate that cytopathology is an accurate method for identifying immunophenotypic features of HGSC and detecting germline BRCA1/2 mutations from ascites. However, further investigation is required for assessing the proliferation activity of HGSC in ascites and for detecting somatic BRCA1/2 mutations., (© 2022 The Authors. Cancer Cytopathology published by Wiley Periodicals LLC on behalf of American Cancer Society.)

BAP1 cancer syndrome is a rare and highly penetrant hereditary cancer predisposition. Uveal melanoma, mesothelioma, renal cell carcinoma (RCC) and cutaneous melanoma are considered BAP1 cancer syndrome core cancers, whereas association with breast cancer has previously been suggested but not confirmed so far. In view of BAP1 immunomodulatory functions, BAP1 alterations could prove useful as possible biomarkers of response to immunotherapy in patients with BAP1-associated cancers. We present a case of a patient with BAP1 cancer syndrome who developed a metastatic breast cancer with loss of BAP1 demonstrated on immunohistochemistry. She carried a germline BAP1 likely pathogenic variant (c.898&#95;899delAG p.(Arg300Glyfs*6)). In addition, tumor tissue sequencing identified a concurrent somatic variant in BAP1 (partial deletion of exon 12) and a low tumor mutational burden. As her triple negative tumor was shown to be PD-L1 positive, the patient was treated with combination of atezolizumab and nab-paclitaxel. She had a complete and sustained response to immunotherapy even after discontinuation of nab-paclitaxel. This case strengthens the evidence for including breast cancer in the BAP1 cancer syndrome tumor spectrum with implications for future cancer prevention programs. It also indicates immune checkpoint inhibitors might prove to be an effective treatment for BAP1-deficient breast cancer., (© 2022. The Author(s).)

In patients with gastrointestinal stromal tumors (GIST), it has become mandatory to determine the driver mutation in order to predict the response to standard treatment with tyrosine kinase inhibitors (TKI). A total of 10‑15% of all GIST lack activating mutations in KIT proto‑oncogene, receptor tyrosine kinase ( KIT )/platelet‑derived growth factor receptor alpha ( PDGFRA ) and have been classified as KIT/PDGFRA wild‑type (WT) GIST. They are characterized by poor response to TKI. From a group of 119 metastatic GIST patients, 17 patients with KIT/PDGFRA/BRAF WT GIST as determined by reverse transcription‑quantitative (RT‑q) PCR and Sanger sequencing were profiled by a targeted next‑generation sequencing (NGS) approach and their treatment outcome was assessed. In the present study, 41.2% of patients as KIT/PDGFRA/BRAF WT GIST examined with RT‑qPCR and Sanger sequencing were confirmed to be carriers of pathogenic KIT/PDGFRA mutations by NGS and were responsive to TKI. The percentage of genuinely KIT/PDGFRA WT GIST in the present study thereby dropped from the initial 14.3% detected with the RT‑qPCR and Sanger sequencing to 7.6% after NGS. Their outcome was universally poor. The reliability of RT‑qPCR and direct Sanger sequencing results in this setting is therefore insufficient and it is recommended that NGS becomes a requirement for treatment decision at least in KIT/PDGFRA/BRAF WT GIST as determined by RT‑qPCR and Sanger sequencing.

Pathogenic/likely pathogenic variants in susceptibility genes that interrupt RNA splicing are a well-documented mechanism of hereditary cancer syndromes development. However, if RNA studies are not performed, most of the variants beyond the canonical GT-AG splice site are characterized as variants of uncertain significance (VUS). To decrease the VUS burden, we have bioinformatically evaluated all novel VUS detected in 732 consecutive patients tested in the routine genetic counseling process. Twelve VUS that were predicted to cause splicing defects were selected for mRNA analysis. Here, we report a functional characterization of 12 variants located beyond the first two intronic nucleotides using RNAseq in APC , ATM , FH , LZTR1 , MSH6 , PALB2 , RAD51C , and TP53 genes. Based on the analysis of mRNA, we have successfully reclassified 50% of investigated variants. 25% of variants were downgraded to likely benign, whereas 25% were upgraded to likely pathogenic leading to improved clinical management of the patient and the family members.

Detection of germline and somatic pathogenic/likely pathogenic variants (PV/LPV) in BRCA genes is at the moment a prerequisite for use of PARP inhibitors in different treatment settings of different tumors. The aim of our study was to determine the most appropriate testing workflow in epithelial ovarian cancer (EOC) patients using germline and tumor genotyping of BRCA and other hereditary breast and/or ovarian cancer (HBOC) susceptibility genes. Consecutive patients with advanced non-mucinous EOC, who responded to platinum-based chemotherapy, were included in the study. DNA extracted from blood and FFPE tumor tissue were genotyped using NGS panels TruSightCancer/Hereditary and TruSight Tumor 170. Among 170 EOC patients, 21.8% had BRCA germline or somatic PV/LPV, and additionally 6.4% had PV/LPV in other HBOC genes. Sensitivity of tumor genotyping for detection of germline PV/LPV was 96.2% for BRCA genes and 93.3% for HBOC genes. With germline genotyping-only strategy, 58.8% of HBOC PV/LPV and 68.4% of BRCA PV/LPV were detected. By tumor genotyping-only strategy, 96.1% of HBOC PV/LPV and 97.4% of BRCA PV/LPV were detected. Genotyping of tumor first, followed by germline genotyping seems to be a reasonable approach for detection of PV/LPV in breast and/or ovarian cancer susceptibility genes in non-mucinous EOC patients.

Purpose: To analyze the prevalence of pathogenic/likely pathogenic variants (P/LPVs) in BRCA1 and BRCA2 genes in the largest cohort of Slovenian male breast cancer (MBC) patients to date and to explore a possible correlation between the Slovenian founder variant BRCA2:c.7806-2A > G and predisposition to MBC., Methods: We performed a retrospective analysis of 81 MBC cases who underwent genetic counseling and/or testing between January 1999 and May 2020. To explore a possible genotype-phenotype correlation, we performed additional analyses of 203 unrelated families with P/LPVs in BRCA2 and 177 cases of female breast cancer (FBC) in carriers of P/LPVs in BRCA2., Results: Detection rate of P/LPVs in the BRCA1 and BRCA2 genes was 24.7% (20/81) with 95% of them in BRCA2 gene. The only two recurrent P/LPVs were BRCA2:c.7806-2A > G and BRCA2:c.3975&#95;3978dupTGCT (9 and 5 MBC cases, respectively). In families with BRCA2:c.7806-2A > G, the incidence of MBC cases was higher compared to families with other P/LPVs in BRCA2; however, the difference did not reach statistical significance (17.8% vs. 8.9%, p = 0.105). BRCA2:c.7806-2A > G was detected in both families with multiple cases of MBC. This splice-site variant represented a significantly higher proportion of all BRCA2 P/LPVs detected in MBC carriers compared to FBC carriers (47.4% vs. 26%, p = 0.049)., Conclusion: We observed a high mutation detection rate and conclude this may be due to the prevalent BRCA2:c.7806-2A > G variant in Slovenia. Our results indicate a possible association between this variant and higher risk of breast cancer in males compared to other identified P/LPVs in BRCA2., (© 2021. The Author(s).)

RNA sequencing is a promising technique for detecting normal and aberrant RNA isoforms. Here, we present a new single-gene, straightforward 1-day hands-on protocol for detection of splicing alterations with deep RNA sequencing from blood. We have validated our method's accuracy by detecting previously published normal splicing isoforms of STK11 gene. Additionally, the same technique was used to provide the first comprehensive catalogue of naturally occurring alternative splicing events of the NBN gene in blood. Furthermore, we demonstrate that our approach can be used for detection of splicing impairment caused by genetic variants. Therefore, we were able to reclassify three variants of uncertain significance: NBN :c.584G>A, STK11 :c.863-5&#95;863-3delCTC and STK11 :c.615G>A. Due to the simplicity of our approach, it can be incorporated into any molecular diagnostics laboratory for determination of variant's impact on splicing.

Background: Currently, data on pathogenic variants in the CHEK2 gene and their impact on cancer risk are lacking. This study aimed to explore the characteristics of breast cancer (BC) patients from families with CHEK2 pathogenic variants in Slovenia., Methods: In the years 2014 to 2019, CHEK2 pathogenic variants/likely pathogenic variants (PV/LPVs) were found in probands from 50 different families who underwent genetic counseling and testing using a multigene panel at the authors' institution. Altogether, the study enrolled 75 individuals from 50 CHEK2 families who were carriers of a CHEK2 PV/LPV. The clinical data on 41 BC patients with CHEK2 PV/LPV and other carriers of CHEK2 PV/LPV from Slovenia were collected and analyzed., Results: Breast cancer was diagnosed in 41 of 75 CHEK2 PV/LPV carriers (40 females, 1 male). The mean age at BC diagnosis was 42.8 years (range, 21-63 years), and 27 (65.8%) of the 41 of patients with BC had a positive family history for BC. Contralateral BC (CBC) was observed in 8 (19.5%) of the 41 patients (mean age, 55.6 years). Of 12 patients with human epidermal growth factor receptor 2 (HER2)-positive tumor type, a c.444+1G > A PV/LPV was detected in 4 patients, c.349A > G in 3 patients, deletion of exons 9-10 in 3 patients, deletion of exon 8 in 1 patient, and c.1427C > T PV/LPV in 1 patient., Conclusion: Bilateral BC was diagnosed in as many as 19.5% of the Slovenian BC patients with CHEK2 PV/LPVs. Breast cancer associated with a germline CHEK2 PV/LPV occurs in younger patients compared with sporadic BC.

Background: The incidence and risk factors for the development of high-grade dysplasia (HG-D) and laryngeal squamous cell carcinoma (LSCC) were assessed in patients with laryngeal squamous cell papillomas (LSP)., Methods: Clinical data, human papillomaviruses (HPV) typing, HPV E6/E7 mRNA in situ hybridization, and sequencing of host genes in LSP biopsies of 163 patients were analyzed., Results: Progression to HG-D and LSCC was identified in 21.5% and 4.3% of LSP patients, respectively. A more advanced age at LSP onset and lack of HPV infection were detected as risk factors for the development of HG-D and LSCC (P < .05). The identification of HG-D was associated with its progression to LSCC (P < .05). Host gene mutations were identified in 3 of 7 patients with LSCC., Conclusions: The histological monitoring of LSP and HPV typing are necessary for early detection of epithelial changes. Further research is needed to elucidate the role of host gene mutations in LSCC transformation., (© 2020 Wiley Periodicals LLC.)

In total, ~85% of malignant gastrointestinal stromal tumours (GISTs) harbour activating mutations in one of the genes KIT or PDGFRA, while 10‑15% of all GISTs have no detectable KIT or PDGFRA mutations, but could have alterations in genes of the succinate dehydrogenase complex or in BRAF, PIK3CA or rarely RAS family genes. The clinical benefit of tyrosine kinase inhibitors, such as imatinib, depends on the GIST genotype, therefore molecular characterization of GIST has a crucial role in overall management of GIST. The aim of the present study was to molecularly characterize a cohort of 70 patients with metastatic GISTs from the Slovenian Cancer Registry (National Cancer Registry) treated between January 2002 and December 2011. Exons 9, 11, 13 and 17 of the KIT gene and exons 12, 14 and 18 of the PDGFRA gene were analysed by direct Sanger sequencing. All KIT/PDGFRA wild‑type GISTs were tested for the presence of mutations in hot spot regions of KRAS, NRAS, BRAF, PIK3CA and AKT1 genes. Novel variants were characterized and classified using Cancer Genome Interpreter and according to The American College of Medical Genetics and Genomics/Association for Molecular Pathology guidelines. In total, 60 (85.7%) patients had mutations in KIT and 2 (2.9%) in PDGFRA. Whereas, 8 (11.4%) patients with GIST had no mutation in either of the analysed genes. The majority of GIST cases (n=52) had a mutation in KIT exon 11, where 40 different mutations were detected. Eight of the variants were novel: c.1652&#95;1672del, c.1653&#95;1660delinsAA, c.1665&#95;1672delinsCC, c.1668&#95;1686del, c.1676&#95;1720del, c.1715&#95;1756dup, c.1721&#95;1765dup, and c.1722&#95;1766dup. Mutation frequencies of KIT and PDGFRA genes observed in Slovenian patients are comparable with those in other European populations. In the present group of patients analysed, the most frequently mutated region was exon 11 in the KIT gene, responsible for coding juxtamembrane domain of KIT protein. In this region, eight novel mutations were identified and classified as likely pathogenic driver variants. In addition, the present study identified 6 patients with secondary KIT mutation and 1 patient with double mutant GIST, who had two different mutations in PDGFRA exon 14.

Background: In a patient with a germline BRCA2 pathogenic variant with breast cancer, an adnexal mass can represent either a metachronous primary tumor or a metastasis of the breast cancer. A clear distinction between those two possibilities is crucial since treatments differ substantially and so does survival of the patient., Case Presentation: We present a case of a 47-year-old patient with bilateral breast carcinoma with a germline BRCA2 pathogenic variant. The first manifestation of the disease was a lump in her left breast in 1998, histological report was invasive ductal carcinoma, triple-negative. She was treated with surgery, chemotherapy and radiotherapy. In 2011 a new occult carcinoma was found in her right axilla, however the specimen was estrogen receptor (ER) and progesterone receptor (PgR) positive. She was treated as a new primary occult carcinoma of the right breast with surgery, radiotherapy and adjuvant hormonal treatment. In 2016 a mass in the left adnexa was found with imaging techniques. She underwent surgery as if it was primary ovarian cancer, yet histology revealed it was a metastasis of a triple-negative breast carcinoma in the fimbrial part of the left Fallopian tube. She received adjuvant chemotherapy after surgery and is now in complete remission., Conclusion: We present an interesting and quite rare case of two primary breast carcinomas in a patient with a known BRCA2 pathogenic variant with metastasis in the fimbrial part of the left Fallopian tube. We conclude that there were two primary breast tumours and the one from 2011 spread into the fimbrial part of the left Fallopian tube in 2016. Despite the fact that molecular analyses could not confirm the joint tumour origin, we believe that there was a receptor status conversion over time explaining different receptor status. The possibility of a triple-negative metastasis from the tumour treated in 1998 is less probable. With both of aforementioned possibilities being prognostically unfavourable, the patients' outcome is so far excellent and she was in complete remission at the time of writing this article., Competing Interests: Competing interestsThe authors declare that they have no competing interests., (© The Author(s) 2020.)

The diagnostics of Lynch syndrome (LS) is focused on the detection of DNA mismatch repair (MMR) system deficiency. MMR deficiency can be detected on tumor tissue by microsatellite instability (MSI) using molecular genetic test or by loss of expression of one of the four proteins (MLH1, MSH2, MSH6, and PMS2) involved in the MMR system using immunohistochemistry (IHC) staining. According to the National Comprehensive Cancer Network (NCCN) guidelines, definitive diagnosis of LS requires the identification of the germline pathogenic variant in one of the MMR genes. In the report, we are presenting interesting novel MLH1 in-frame deletion LRG&#95;216t1:c.2236&#95;2247delCTGCCTGATCTA p.(Leu746&#95;Leu749del) associated with LS. The variant appears to be associated with uncommon isolated loss of PMS2 immunohistochemistry protein staining (expression) in tumor tissue instead of MLH1 and PMS2 protein loss, which is commonly seen with pathogenic variants in MLH1 . The variant was classified as likely pathogenic, based on segregation analysis and molecular characterization of blood and tumor samples. According to the American College of Medical Genetics (ACMG) guidelines, the following evidence categories of PM1, PM2, PM4, and PP1 moderate have been used for classification of the novel variant. By detecting and classifying the novel MLH1 variant as likely pathogenic, we confirmed the LS in this family., Competing Interests: The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

Neurofibromatosis type I (NF1) is one of the most common autosomal dominant disorders, since the estimated incidence is one in 3,500 births. In this study, we present bioinformatical and functional characterization of two novel splicing NF1 variants, detected in NF1 patients. Patient 1, carrying NF1 :c.122A>T, which introduces a new exonic 5' donor splice site, was diagnosed with hormone-positive, Her-2-negative breast cancer at the age of 47. She had an atypical presentation of NF1, with few café-au-lait spots and no Lisch nodules. Patient developed a hemothorax due to subclavian artery rupture, which has previously been described as an extremely rare complication of NF1. Patient 2, carrying NF1 :c.7395-17T>G that creates a new intronic 3' acceptor splice site, had quite a typical clinical presentation of NF1: formations on her tongue in the region of her left metacarpal bones and on her left foot, plexiform neurofibroma in her pelvis, several café-au-lait spots, and axillary freckling. She was also diagnosed with cognitive impairment. In the report, we are presenting two novel variants which were successfully classified based on NGS and mRNA analysis. Based on results of mRNA analysis, both variants were classified as likely pathogenic according to ACMG guidelines applying evidence categories PS3, PM2, PP3, and PP1 supporting. By characterizing those two novel NF1 splicing variants, we have confirmed the neurofibromatosis type I phenotype in the two probands.

Background: High-grade serous ovarian cancer is a detrimental disease. Treatment options in patients with a recurrent disease are dependent on BRCA1/2 mutation status since only patients with known BRCA mutation are eligible for treatment with poly(ADP-ribose) polymerase inhibitors (PARPi). The aim of this study was to compare concordance of BRCA mutation analyses from cytological samples (CS) with BRCA mutation analyses from histological formalin fixed paraffin embedded (FFPE) samples., Methods: Mutation analysis of BRCA1 and BRCA2 genes was performed in 44 women diagnosed with primary or recurrent high-grade ovarian cancer from three different samples: blood, cytological sample (ascites, pleural effusion and enlarged lymph nodes) and tumor tissue. Results from all three samples were compared., Results: Among 44 patients, there were 15 germline mutations and two somatic mutations. A 100% concordance was found between cytological and histologic samples., Conclusion: There is a 100% concordance in BRCA mutation testing between cytological and histologic samples. BRCA mutation testing from CS could replace testing from FFPE tissue in clinical decision making in ovarian cancer patients., Trial Registration: The study was retrospectively registered at ISRCTN registry on 24/11/2015 - ISRCTN42408038 .

Mutations in breast cancer susceptibility gene 1 (BRCA1) lead to defects in a number of cellular pathways including DNA damage repair and transcriptional regulation, resulting in the elevated genome instability and predisposing to breast and ovarian cancers. We report a novel mutation LRG&#95;292t1:c.4356delA,p.(Ala1453Glnfs*3) in the 12th exon of BRCA1, in the splice site region near the donor site of intron 12. It is a frameshift mutation with the termination codon generated on the third amino acid position from the site of deletion. Human Splice Finder 3.0 and MutationTaster have assessed this variation as disease causing, based on the alteration of splicing, creation of premature stop codon and other potential alterations initiated by nucleotide deletion. Among the most important alterations are frameshift and splice site changes (score of the newly created donor splice site: 0.82). c.4356delA was associated with two ovarian cancer cases in two families of Slavic origin. It was detected by next generation sequencing, and confirmed with Sanger sequencing in both cases. Because of the fact that it changes the reading frame of the protein, novel mutation c.4356delA p.(Ala1453Glnfs*3) in BRCA1 gene might be of clinical significance for hereditary ovarian cancer. Further functional as well as segregation analyses within the families are necessary for appropriate clinical classification of this variant. Since it has been detected in two ovarian cancer patients of Slavic origin, it is worth investigating founder effect of this mutation in Slavic populations.

In our previous studies, it has been demonstrated that in more than 80% of mice long-lasting antitumor immunity has been established following intraperitoneal (i.p.) vaccination with tumor vaccine composed of irradiated syngeneic tumor cells and CpG ODNs class C. The aim of this study was, therefore, to investigate molecular mechanisms through which this vaccine triggers the immunity and to define genes particularly involved in this process. Changes in gene expression were followed in mononuclear cells isolated from peritoneal lavages, spleens and bone marrow samples. The expression of 84 genes significant for T-cell and B-cell activation as well as genes engaged in activation of macrophages, NK cells and DCs was determined using the RT 2 - Profiler PCR array. It has been observed that this tumor vaccine induces the up-regulation of genes involved in activation, proliferation and survival of memory T-cells (Cd8a, Cd8b1, Prlr, Was, Cxcl12, Il12, Sftpd, Tnfrsf13c, Il15, Il18), and prevents the activation of genes involved in generation of Treg and induction of immune tolerance (Sit1, Sla2, Cd1d1, Pdcd1lg2, Pawr, Socs5, Il27, Il4). We may conclude based on results of gene expression analysis, that tumor vaccine fine-tunes the proportion of cytotoxic to regulatory lymphocytes having an important impact on the induction and maintenance of memory cells in bone marrow., (Copyright © 2016 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

In the present study, the detection of tumor-specific KRAS proto-oncogene, GTPase ( KRAS ) and B-Raf proto-oncogene, serine/threonine kinase ( BRAF ) mutations in the peripheral blood of colorectal cancer (CRC) patients at all stages and adenomas was used for the estimation of disease stage prior to surgery and for residual disease following surgery. A total of 65 CRC patients were enrolled. The primary tumor tested positive for the specific mutations ( KRAS mutations in codons 12, 13, 61, 117 or 146 and BRAF mutations in codon 600) in 35 patients. In all these patients, the specimen of normal bowel resected with the tumor was also tested for the presence of the same mutations in order to exclude the germ-line mutations. Only patients who tested positive for the specific mutation in the primary tumor were included in further analysis for the presence of tumor-specific mutation in the peripheral blood. No statistically significant differences were found between the detection rates of tumor mutations in the blood and different tumor stages (P=0.491). However, statistically significant differences in the proportions of patients with detected tumor-specific DNA mutations in the peripheral blood were found when comparing the groups of patients with R0 and R2 resections (P=0.038). Tumor-specific DNA mutations in the peripheral blood were more frequently detected in the patients with an incomplete surgical clearance of the tumor due to macroscopic residual disease (R2 resections). Therefore, the study concludes that the follow-up of somatic KRAS - and BRAF -mutated DNA in the peripheral blood of CRC patients may be useful in assessing the surgical clearance of the disease.

The estimated proportion of hereditary breast and ovarian cancers among all breast and ovarian cancer cases is 5-10%. According to the literature, inherited mutations in the BRCA1 and BRCA2 tumour-suppressor genes, account for the majority of hereditary breast and ovarian cancer cases. The aim of this report is to present novel mutations that have not yet been described in the literature and pathogenic BRCA1 and BRCA2 mutations which have been detected in HBOC families for the first time in the last three years. In the period between January 2009 and December 2011, 559 individuals from 379 families affected with breast and/or ovarian cancer were screened for mutations in the BRCA1 and BRCA2 genes. Three novel mutations were detected: one in BRCA1 - c.1193C>A (p.Ser398*) and two in BRCA2 - c.5101C>T (p.Gln1701*) and c.5433&#95;5436delGGAA (p.Glu1811Aspfs*3). These novel mutations are located in the exons 11 of BRCA1 or BRCA2 and encode truncated proteins. Two of them are nonsense while one is a frameshift mutation. Also, 11 previously known pathogenic mutations were detected for the first time in the HBOC families studied here (three in BRCA1 and eight in BRCA2). All, except one cause premature formation of stop codons leading to truncation of the respective BRCA1 or BRCA2 proteins.

Background: Some tumour suppressor genes (BRCA2) and mismatch repair genes (MSH2, MLH1) are correlated with an increased risk for male breast cancer., Case Report: Our patient developed secondary breast cancer after the treatment for Hodgkin's disease in childhood. DNA was isolated from the patients' blood and screened for mutations, polymorphisms and variants in BRCA1, BRCA2, p53, CDKN2A, MLH1 and MSH2 genes. We found no mutations but common polymorphisms, and three variants in mismatch repair genes., Conclusions: Nucleotide variants c.2006-6T>C and p.G322D in MSH2 might be correlated with male breast cancer.

Association between long-term hormone replacement therapy (HRT) use and increased risk of breast cancer is still under debate. Functionally relevant genetic variants within the estrogen metabolic pathway may alter exposure to exogenous sex hormones and affect the risk of postmenopausal breast cancer. We investigated the associations of common polymorphisms in 4 genes encoding key proteins of the estrogen metabolic pathway, duration of HRT use and their interactions with breast cancer risk. We studied 530 breast cancer cases and 270 controls of the same age and ethnicity participating in a case-control study of postmenopausal women. Duration of HRT use was ascertained through a postal questionnaire. Genotyping was conducted for CYP1B1 (rs1056836), COMT (rs4680), GSTP1 (rs1695) and MnSOD (rs4880) polymorphisms by PCR-based RFLP and TaqMan® allelic discrimination method. Adjusted odds ratios and 95% confidence intervals were calculated using logistic regression analysis. HRT use was significantly associated with decreased breast cancer risk (p<0.001). None of the polymorphisms studied was associated with breast cancer risk. A significant interaction was observed between MnSOD 47T>C and HRT use (pinteraction=0.036); the risk of breast cancer associated with long-term vs. short-term HRT use was decreased in women homozygous for the wild-type allele and increased in women with at least one variant allele of the MnSOD 47T>C polymorphism. Our results suggest that MnSOD 47T>C polymorphism in interaction with long-term HRT use may modify the risk of breast cancer.

Objective: Estrogen plays a key role in breast cancer development and functionally relevant genetic variants within the estrogen metabolic pathway are prime candidates for a possible association with breast cancer risk. We investigated the independent and the combined effects of commonly occurring polymorphisms in four genes encoding key proteins of estrogen metabolic pathway on their potential contribution to breast cancer risk., Methods: We studied 530 breast cancer cases and 270 controls of the same age and ethnicity participating in a case-control study of postmenopausal women. Genotyping was conducted for CYP1B1 (rs1056836), COMT (rs4680), GSTP1 (rs1695), and MnSOD (rs4880) polymorphisms by polymerase chain reaction based restriction fragment length polymorphism and TaqMan allelic discrimination method. Adjusted ORs and 95% CIs were calculated using logistic regression., Results: None of the 4 genetic variants examined contributed to breast cancer risk individually. When the combined effects of the risk genotypes were investigated, significant associations were observed among women with two high-risk genotypes in CYP1B1 and COMT (OR, 2.0; 95% CI, 1.1 to 3.5) and two high-risk genotypes in COMT and MnSOD (OR, 2.0; 95% CI, 1.0 to 3.8), compared to those with low-risk genotypes., Conclusion: Our results suggest that individual susceptibility to breast cancer incidence may be increased by combined effects of the high-risk genotypes in CYP1B1, COMT, and MnSOD estrogen metabolic genes.

Only properly mature dendritic cells (DCs) in the presence of tumor antigens accomplish to activate all of the elements of the immune network and have the potential to induce tumor-specific effectors and memory T cells. In the current study, we firstly aimed to investigate the in vivo maturation of antigen presenting cells (APCs) at the molecular level by following the expression of CD11c, CD86 and MyD88 genes in the mixture of mononuclear cells after treatment of mice with a tumor vaccine composed of C-class CpG oligodeoxynucletides (CpG ODN) and irradiated melanoma B16F1 tumor cells. The second objective was to define whether the tumor vaccine induces generation of memory T cells (CD44hiCD62Llo/hiCD27hi). Finally, based on gene expression pattern we aimed to determine the tissue distribution and homing of the (mature) APCs and memory cells after vaccination. We demonstrated that by tumor vaccine the APCs (including DCs) are manipulated in vivo. By this kind of vaccine, the differentiation and maturation of APCs is triggered primarily in the spleen and is subsequently followed by the migration of these APCs to the bone marrow. Once in the bone marrow, these APCs play a crucial role in the development and maintenance of long-lived memory T cells capable of preventing a relapse of malignant disease. In conclusion, our results provide insight into the nature and scope of the antitumor immune response elicited by this kind of tumor vaccine in vivo. We showed that the maturation of APCs is a prerequisite for the generation of effective long-term antitumor immunity.

Published data on the association between methylenetetrahydrofolate reductase (MTHFR) gene polymorphisms and breast cancer risk are inconclusive. We investigated the independent and the combined effects of two commonly occurring polymorphisms, MTHFR 677C>T (rs1801133) and MTHFR 1298A>C (rs1801131), as well as their interaction with the use of hormone replacement therapy (HRT), to determine their potential contribution to breast cancer risk. We studied 530 breast cancer cases and 270 controls of the same age and ethnicity participating in a case-control study of postmenopausal women. The duration of HRT use was ascertained through a postal questionnaire. Genotyping was conducted by TaqMan® allelic discrimination. Adjusted odds ratios and 95% confidence intervals were calculated using logistic regression. No significant association was observed between either the individual or the combined MTHFR genotypes and the risk of postmenopausal breast cancer. Additionally, no effects resulting from the interaction between MTHFR genotypes and HRT use were detected. Therefore, our data do not support the hypothesis that genetic variation in the MTHFR gene is implicated in the aetiology of postmenopausal breast cancer.

Until now, the anti-tumor efficacy of synthetic oligodeoxynucleotides containing CpG motifs (CpG ODNs) has been reported in a number of preventive and therapeutic tumor models. Predominately class B CpG ODNs were used, relatively little has been reported regarding the class C CpG ODNs. The present study was, therefore, aimed at assessing the ability of CpG ODNs class C applied as a single agent and in combination with radiotherapy to induce the anti-tumor immunity in an experimental tumor model in mice (subcutaneous [s.c.] B16F1). Class C CpG ODNs applied three times as a single agent efficiently delayed the growth of s.c. B16F1 tumors. The combined therapy (CpG ODNs and tumor irradiation) remarkably enhanced the anti-tumor effect. The peritumoral (p.t.) application of CpG ODNs in combination with irradiation increased the number of dendritic cells (DCs) at the tumor site and improved the antigen loading and maturation of DCs. In conclusion, the combined therapy with CpG ODNs and irradiation creates a unique in situ DCs vaccine that could be easily applicable without prior knowledge of tumor antigens.

The present study was aimed at assessing the ability of tumor vaccine - CpG ODN class C in combination with irradiated melanoma tumor cells B16F1 to trigger the antitumor immunity in experimental tumor model in mice (i.p. B16F1) as well as at evaluating some of its mechanisms of action. A significant preventive antitumor immunity was achieved with the vaccine and two additional injections of CpG ODN (the proportion of protected mice was between 75% and 100%). In more than 80% of survivors, a long-lasting immunity was triggered. The therapy with the vaccine and two additional injections of CpG ODN significantly prolonged survival of tumor bearing mice. The rates of cured mice were 21.1% and 11.1% in mice with smaller or larger tumor masses, respectively. The mechanism of stimulation of the immune system by this kind of vaccine is likely to be through the augmentation of APC maturation (a significantly increased proportion of CD86+ CD11c+ was determined in vaccinated mice), consequent activation of T lymphocytes (the proportions of CD25+ and CD69+ splenic lymphocytes increased after the exposure to activated DCs) and establishment of memory cells. In conclusion, vaccine composed of CpG ODN class C and irradiated tumor cells powerfully triggers the immune system bringing about both preventive and therapeutic effects.

Understanding of immunological processes at the molecular level in the cancer-bearing organism is the prerequisite for development of specific tumor vaccines. Most of these vaccines are aimed at enhancing the immunogenicity of the antigens presented by the tumor cells. Since dendritic cells (DC) are potent antigen presenting cells (APC) in the organism, they are considered as a powerful tool to deliver the signals essential for the activation of immune system. In an attempt to clarify the functional importance of DC in the process of anti-tumor vaccination, we characterized the effect of DC activated with a classical tumor vaccine (mixture of irradiated B16F1 tumor cells and MVE-2) on the activation of T lymphocytes. The T lymphocyte activation was assessed by determination of expression of CD25, CD69, and intracellular IFNgamma and IL4 production. Activated DC significantly increased the proportion of CD25+ and CD69+ cells as well as IFNgamma+ and IL4+ cells among CD3+ T lymphocytes. On the other hand, the direct effect of the tumor vaccine on T lymphocytes was just an increment in the proportion of IL4+ T lymphocytes. With the results of in vivo experiments, the phagocytic cells (including DC) were proved to be essential for establishing an active protection against tumor cells (tumor development), but more importantly, also for formation of the memory cell pool. These data indicate that DC loaded with tumor antigens are required for effective stimulation of T lymphocytes, and that the phagocytic cells (including DC) are essential for the anti-tumor immunity triggered by this kind of vaccine.

The crucial step in cellular immortalisation seems to be the activation of telomerase, the enzyme that synthesizes telomere repeat ending sequences. Since the telomerase activity has been detected in almost all types of cancer tissue it has been proposed as new reliable tumor marker. This study was conducted to evaluate the significance of appearance of circulating RNA for telomerase subunits hTR and hTERT in the plasma of cancer patients. Seven healthy volunteers, 25 primary breast cancer patients (stage I-III), 29 patients with advanced malignant melanoma (stage III-IV), and 4 patients with advanced thyroid cancer (stage III-IV) were included in the study. The total RNA was extracted from plasma samples, reverse transcribed to cDNAs, and specific cDNAs for hTR and hTERT were amplified by semi-nested PCR. In healthy volunteers, the control GAPDH was positive in all, hTR was positive in 3 cases, and hTERT was negative in all 7 tested cases. Among 25 breast cancer patients, hTR was positive in 23, and hTERT in 12 patients. Two cases, that were hTR and hTERT negative, were at the same time negative also for GAPDH. Of the 29 patients with malignant melanoma, 24 were positive for hTR, and 17 for hTERT. Again, the 5 patients that were negative for hTR and hTERT, were negative also for the control GAPDH. In all plasma samples from thyroid cancer patients, hTR and hTERT were positive. In conclusion, the RT-PCR followed by semi-nested PCR is a highly sensitive method for detection of circulating RNA in the plasma. Among the telomerase subunits, only hTERT could serve as an unspecific tumor marker in the plasma of cancer patients.