Abstract Introduction: Thyroid carcinoma is the most common endocrine cancer. Some somatic mutations in genes (BRAF, NRAS and TERT) involved in key signaling pathways and genome stability have been recently identified to play an important role in its development. Very little research has been done on their frequency and clinical relevance in Bulgarian patients with papillary thyroid cancer (PTC). This study is focused on investigating somatic mutation frequency in Bulgarian patients with PTC and their association with clinicopathologic features. Material and Methods: The study included 50 PTC from Bulgarian patients analyzed for mutations in BRAF (V600E), NRAS (Q61K), single nucleotide polymorphism (SNP) rs2853669 and TERT (C228T and C250T) genes by Sanger sequencing. The results were interpreted using Benchling and SeqScape software, and statistical analysis performed with SPSS. Results: In the studied PTC group BRAF(V600E) and TERT (C228T) mutations were found with frequency of 24% and 2%, respectively. Co-occurrence of both mutations was found in 1 patient (2%). The mutations Q61K (NRAS), and C250T (TERT) were not detected. The SNP rs2853669 was found in 18 patients (52.9%). Correlation analysis with the clinical characteristics of the patients revealed statistically significant association with larger size of the tumor for BRAF(V600E) and smaller tumor size for rs2853669. Conclusion: In the present pilot study, we found that BRAF(V600E) and rs2853669 in TERT are common among PCT patients. While the presence of BRAF V600E mutation was associated with large tumors, the presence of rs2853669 in TERT was found in the majority of PCT below 2 cm. More extensive molecular genetic analysis of TERT, BRAF or RAS mutations in larger sample is needed to further elucidate the clinically important diagnostic and prognostic biomarkers for thyroid cancer.

Globally, the diffuse goiter affects more than 10% of the population and in some regions is endemic. Thyroid nodules are found in approximately 5% of the population using the oldest method for thyroid examination – palpation. When performing ultrasound screening, this percentage increases significantly and reaches between 20 and 75% of the total population. Thyroid carcinoma is a rare malignancy and accounts for up to 1% of all malignant tumors. It is the most common endocrine cancer and is clinically manifested as a thyroid nodule. Somatic mutations play an important role in its development. Differentiation of benign and malignant thyroid nodules is of great importance due to the different therapeutic approach. Therefore, new diagnostic tools are sought to help distinguish the two. Despite the progress in our knowledge of carcinogenesis in recent years, a number of key issues still remain unanswered. The establishment of new rare somatic mutations can improve pre-surgical diagnosis and optimize post-operative strategies for the treatment of thyroid carcinoma. Next-generation sequencing (NGS) allows for extensive mutation and genome rearrangements tracking. The results obtained with NGS provide the basis for the development of new approach for systematic genetic screening, at prevention, early diagnosis, accurate prognosis, and targeted therapy of this disorder.

Background Over the past decade an increase in the incidence and severity of multiple primary neoplasias has been observed. The main causes of multiple primary tumors (MPT) are genetic factors, environmental factors, infections with oncogenic viruses, etc. The aim of the current study was to explore the role of genes associated with familial cancers in MPT development. Methods The study included 12 MPT patients, of which 6 women with metahronous/synchronous breast and ovarian tumors; and 6 men who developed primary tumors with different localisation: bladder/bile ducts; rectum/pancreas; prostate/colon; prostate/sigma; sigma/stomach; palate/larynx+hypopharynx/tongue, respectively. Seventy five (9/12) of the patients had family history of cancer and 50% (6/12) early onset ( Results A total of 82 variants were found of which 18.3% were evaluated as clinically significant. Among selected variants 33.3% (5/15) were pathogenic, 13.3% (2/15) likely pathogenic and 53.3% (8/15) variants of uncertain significance (VUSs). Pathogenic/likely pathogenic variants were detected in the genes BRCA1 (20%), MLH1 (13.3%), BRCA2 (6.7%) and CDH1 (6.7%) while VUSs in PMS1, GPC3, DIS3L2, PRF1, STK11, DICER1, RET, and MSH6, respectively. Conclusions Overall, the genetic cause of MPT was found in 58.3% (7/12) of the patients. Further research is needed to evaluate the functional effect of all VUSs. Legal entity responsible for the study Medical University of Sofia. Funding Medical University Sofia; National Science Fund, Ministry of Education and Science, Bulgaria. Grants D-71/03.05.2018/MU-Sofia; KP-06-OPR03/1719.12.2018/NSF; DUNK01-2/2009/NSF, MES Bulgaria. Disclosure All authors have declared no conflicts of interest.

Introduction According to the National Cancer Register in Bulgaria more than 800 women develop ovarian cancer annually. The most frequent histological type, representing about three quarters of these cases is high-grade serous ovarian carcinoma (HS-OC). About 25% of the HG-OC are supposed to be caused by mutations in the BRCA1 and BRCA2 genes. Material and methods We have screened 80 Bulgarian patients with high grade serous ovarian cancer (HS-OC), disease progression and platinum sensitivity for germline mutations in the BRCA1/2 genes. The mutation analysis was performed by Next Generation Sequencing (NGS) with Ion Torrent PGM System, Sanger Sequencing and MLPA (Multiplex Ligation Dependent Probe Amplification). All identified pathogenic variants with NGS were confirmed by direct sequencing. Results and discussions In total 26 (32.5%) pathogenic mutations were found of which 23 (28.75%) in BRCA1: three recurrent c.5263_5264insC (7/80), c.2019delA (3/80), c.5333–1G>A (2/80), the rest appearing just once c.139T>C, c.139T>G, c.181T>G, c.3496delG, c.4391delC, c.5212G>A, c.5497G>A, c.5533_5534insT, deletion of exons 3–7. In BRCA2 only 4 (5%) mutations were found: c.3545_3546delTT, c.8059_8063delGTTCT, c.8674A>T, c.9294C>G. The most prevalent mutation in the study group observed with frequency of 8.75% was c.5263_5264insC, followed by c.2019delA (3.75%) and c.5333–1G>T (2.50%) in BRCA1. The recurrent mutations account for 57.7% of all detected mutations. Conclusion Twenty six (32.5%) of the HS-OC patients in our study were carriers of germline pathogenic mutations. These results are relevant to the clinical practice and personalised treatment of patients with OC. The BRCA1 and BRCA2 mutations are related to survival and chemotherapy response. The mutation carriers, detected by NGS sequencing, could benefit from therapy with PARP inhibitor. Acknowledgements DUNK01-2/2009/NSF, Ministry of Education and Science, Bulgaria, AstraZeneca LTD, Bulgaria

Abstract Background. Breast cancer is the most commonly diagnosed malignancy and the most frequent cause of death in women due to cancer. About 5% to 10% of breast cancers are thought to be hereditary. Pathogenic mutations in BRCA1/2 genes across Hereditary Breast and Ovarian Cancer (HBOC) patients estimates are at around 15-20%. Other less common genes have also been associated with an increased risk of developing breast cancer, such as mutations in the TP53, PTEN, RAD51C, CDH1, ATM, CHEK2 or PALB2 tumor suppression genes. NGS based sequencing panels allow fast and simultaneous screening of large number of high- and low-penetrance susceptibility genes in these patients. Methods. In the current study we included a group of 31 Bulgarian female breast cancer patients, selected following the strict BCLC and NCCN criteria for hereditary cancer. All of them were prescreened by direct sequencing and MLPA analysis, and tested negative for pathogenic mutations in BRCA1 and BRCA2 genes. Next generation target resequencing using a panel of 94 cancer related genes (Illumina TruSight cancer panel) was performed to explore the hereditary component beyond BRCA1/2 genes in these patients. All detected mutations and variants of unknown clinical significance (VUSs) were confirmed by Sanger sequencing method. Results. Pathogenic and likely pathogenic mutations were found in 14 out of 31 BRCA1/2 negative patients: 1 new frameshift mutation in ATM gene; 6 new likely pathogenic missense mutations in PTCH1, RAD51C, MET, MUTYH, ATM and CHEK2; 7 previously reported pathogenic missense variants in WRN, ERCC4, PALB2, PRF1, RET, SDHB and AIP genes. In addition 27 VUSs (one new splice donor variant in ALK gene and 26 missense variants) were found. Conclusions. The use of next generation target resequencing with TruSight Cancer panel lead to identification of clinically relevant pathogenic variants in 45% of the investigated patients. This could be the preferred diagnostic method in HBOC patients, carefully selected according the strict BCLC and NCCN criteria. Citation Format: Dacheva D, Dodova R, Mitkova A, Kamenarova K, Tzveova R, Popov I, Vlahova A, Taushanova – Hadjieva M, Valev S, Dikov T, Timcheva K, Christova S, Mitev V, Kaneva R. Exploration of the diagnostic utility of next generation sequencing with TruSight cancer panel for BRCA negative hereditary breast and ovarian cancer patients. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P2-09-18.

Results: Aberrant methylation of the BRCA1 promoter was detected in 33 of 241 (13.7%) unselected ovarian carcinomas.Methylation was more frequent in the BRCA1 mutation-negative (15.8%, 32/203) than in mutation-positive tumors (2.6%, 1/38, P = 0.0365).Among the 38 mutation carriers examined, only one demonstrated aberrant BRCA1 promoter methylation.Methylation was more common in tumors with LOH at the BRCA1 locus (23.1%, 28/121) than in tumors with retention of heterozygosity (5%, 2/40, P = 0.0166).Aberrant methylation was observed in 3 of 13 (23.1%)undifferentiated, 3 of 17 (17.6%)other types, 24 of 169 (14.2%) serous, 2 of 18 (11.1%)endometrioid and one of 13 (7.7%)clear cell carcinomas.None of the 11 mucinous tumors had methylated BRCA1 promoter.There was no association between promoter methylation and serous histology.In addition, we found no differences in the frequency of BRCA1 methylation according to patient's age, disease stage or tumor grade.Conclusions: Our findings suggest that promoter methylation may be an alternate mechanism of gene inactivation to mutation; in association with LOH, it may lead to biallelic gene silencing in sporadic cancers.

The erythroleukemia developed by spi-1/PU.1 transgenic mice is a multistage process characterized by an early arrest of the proerythroblast differentiation followed later on by malignant transformation. Herein, we report the presence of acquired mutations in the SCF receptor gene (Kit) in 86% of tumors isolated during the late stage of the disease. Kit mutations affect codon 814 or 818. Ectopic expression of Kit mutants in nonmalignant proerythroblasts confers erythropoietin independence and tumorigenicity to cells. Using PP1, PP2, and imatinib mesylate, we show that Kit mutants are responsible for the autonomous expansion of malignant cells via Erk1/2 and PI3K/Akt activations. These findings represent a proof of principle for oncogenic cooperativity between one proliferative and one differentiation blocking event for the development of an overt leukemia.

WWOX is a tumour suppressor gene involved in various tumours including breast cancer. High chromosomal abnormalities in a genomic region spanned by WWOX are associated with the fact that this gene covers approximately 1 million base pairs of the second most affected among common chromosomal fragile sites FRA16D. We evaluated WWOX expression levels in breast cancer samples in association with diagnostics–prognostics markers.We performed quantitative real-time RT-PCR to analyse levels of expression of WWOX in 132 cases of breast cancer. We evaluated the relationship between WWOX mRNA levels, clinico-pathological factors, expression of aberrant WWOXΔ6-8 mRNA and other cancer related genes.Expression of WWOX was higher in patients younger than 50 years old, in ER and PR positive tumours vs negative for those receptors and tumours without lymph node metastasis vs LN+. WWOX mRNA levels were also higher in tumours with higher apoptotic index (Bcl2/Bax ratio). Negative associations were found between WWOX expression and cytokeratins 5/6 and 17 (P<0.05). High level expression of WWOX was also associated with better disease free survival. Presence of WWOXΔ6-8 transcripts were accompanied with lower WWOX wild type mRNA level.Reduced WWOX expression commonly observed in various neoplasias in cases of breast cancer is associated with markers of bad prognosis. Our findings reveal additional evidence that WWOX may be involved in steroid (estrogens) metabolism and signaling pathways. WWOX can be considered as a new target for gene therapy development due to the association of high WWOX expression with improved disease free survival.