OBJECTIVE: For safe implementation and broader application of fecal microbiota transplantation (FMT), quality controlled stool banking is a must. Establishing a stool bank is a complex, time-consuming. and expensive process, making it a real challenge in an Eastern European country. We aimed to establish the first stool bank in Eastern Europe - in Bulgaria. SUBJECTS AND METHODS: A multidisciplinary team of gastroenterologists, microbiologists, infectionists, and geneticists was set up. We used a questionnaire based on the First European FMT Consensus in order to recruit possible stool donors. Laboratory blood and stool tests were performed on all potential donors. RESULTS: Between October 2018 and April 2019, 112 donor volunteers completed a questionnaire; 70 (62.5%) were excluded, mainly because of age above 50, an unhealthy BMI, and risk behavior. Fourty-two (37.5%) donor candidates were invited for laboratory testing of blood and feces, of which 12 (28.6%) passed this screening. Of 12 donors, 4 (33%) failed at the following screening test, which is performed every 3-6 months. Finally, 8 (7.14%) active donors were enrolled. Ten successful FMTs were performed on patients with recurrent Clostridium difficile infection. CONCLUSIONS: Even though we found many healthy volunteers, only a low percentage (7.14%) of them were suitable to become feces donors. Establishing a stool bank in an Eastern European country is essential for making FMT safe and more popular as a treatment method, finding further implementation and regulation of FMT and supporting physicians offering this treatment to their patients.

The process of neoangiogenesis is one of the classic hallmarks of a cancer. Its intricate mechanisms have long been one of the major domains in cancer research and a hope for a therapeutic breakthrough. Last decade a new subgroup of non-coding RNA molecules was reported called microRNAs. Literally hundreds of new molecules in this class are being uncovered as pivotal regulators in virtually all intracellular processes. The aim of this study is to classify and review those microRNA molecules that have a role in the processes of tumor angiogenesis and map their places in the regulatory framework of the classical proangiogenic genes and their canonical cascades.

The aim of this study is to evaluate which dysregulated angiomiRs compose the specific proangiogenic microRNA signature of advanced laryngeal cancer and review the literature. Thirty-six samples from twelve patients with advanced laryngeal carcinoma were collected. Total RNA was extracted and microRNA global profiling was performed using Agilent Technologies Microarray Kit. Fifty-nine microRNAs were found to have significantly different expression levels. Eleven microRNAs from the whole group were sorted as regulators of tumor angiogenesis (angiomiRs): seven were up-regulated-miR-1246, miR-181b 5p, miR-18a 5p, miR-21 3p, miR-210 3p, miR-503 5p, miR-93 5p and four were down-regulated-miR148a 5p, miR-145 5p, miR-204 5p, miR-125b 5p. For none of those microRNAs we found heterogeneity in tumor tissue. We are the first to report the specific proangiogenic microRNA signature in advanced laryngeal carcinoma and we confirm and amplify findings from previous studies that expand our perception of a specific "molecular state" of angiogenesis that is distinctive only for laryngeal cancer.

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.

Laryngeal squamous cell carcinoma (LSCC) is one of the most common cancers of the head and neck. LSCC is characterized by a high frequency in Bulgaria and Europe, 8.9/100 000 3.2/100 000, respectively. No change was observed in the survival, despite the introduction of modern approaches in treatment. It is therefore necessary to establish biomarkers that can be successfully applied in clinical and medical-biological practice for the diagnosis and therapy of patients with LSCC. Micro RNAs are small non-coding sequences that are actively involved in the regulation of cellular processes. Altered expression levels are commonly seen in cancers of the head and neck, including the LSCC, in both tissue samples and in samples of plasma, serum and saliva. It was found that micro RNAs may have oncogenic or tumor suppressor functions in the carcinogenesis of LSCC, which makes them attractive as molecular biomarkers. In this review we summarize current knowledge of the altered expression levels of micro RNAs in patients with LSCC and in malignant cell lines, and their potential as noninvasive biomarkers in clinical practice and their role in the processes of resistance to treatment.

Introduction Non-coding RNAs are become one of the most investigated biomarkers in non-invasive diagnostics of cancer diseases. In this study we explore the potential of three miRNAs and one lncRNA as biomarkers in advanced laryngeal cancer (LSCC). Material and methods Plasma samples were obtained from 22 patients with advanced LSCC and 21 healthy controls. Relative expression of miR-145–5 p, miR-196a-5p, miR-222–3 p and lncRNA MALAT1 was performed with RT-qPCR. Data was analysed by SPSS 17.0. A p-value less than 0.05 was considered statistically significant. Results and discussions Our data showed significant ectopic expressions of miR-145–5 p (p=0.022), miR-222–3 p (p=0.013), miR-196a-5p (p=0.055), and lncRNA MALAT1 (p=0.004) in LSCC patients. Decreased expression levels of miR-145–5 p were obtained in 16 patients (mean RQ=0.828;SD=1.42,n=22), miR-222–3 p in 13 patients (mean RQ=0.59;SD=0.88;n=22) and lncRNA MALAT1 in 9 patients (mean RQ=0.67;SD=0.42;n=22). Upregulated levels of miR-196a-5p were reach in 17 patients (mean RQ=2.99;SD=1.43;n=22). Association analysis between markers and TNM stage, differentiation, tumour localization, tobacco and alcohol use was performed. Significant relation between lncRNA MALAT1 with nodal metastasis (p=0.049) and differentiation stage (p=0.020) were reached, as highest expression levels were obtained in those patients with nodal metastasis and poor differentiation tumour. None of the miRNAs showed significant association with any of clinicopathological characteristics. In order to evaluate the potential of included in the study markers as discriminative markers for LCSS patients, we used ROC analysis. The strongest significant potential was combination of miR-145–5 p, miR-196a-5p and lncRNa MALAT1 with AUC=0.870 (p=0.0001,95%CI:0.758–0.982; 86.4% sensitivity and 71.4% specificity). Separately, the ROC results showed for miR-222–3 p: AUC=0.703 (p=0.022,95%CI:0.547–0.860), miR-145–5 p: AUC=721(p=0.013 95% CI:0.570–0.872), miR-196a-5p: AUC=0.671 (p=0.055, 95% CI:0.4860.856), and lncRNA MALAT1: AUC=0.753 (p=0.004, 95% CI: 0.601–0.905). Conclusion Overexpression of included in the study miRNAs and lncRNA might take role in cancerogenesis of LSCC. In addition, lncRNA MALAT was associated with advanced tumorogenesis. The fusion of miR-145–5 p, miR-196a-5p and MALAT1 could be potential marker for non invasive diagnostic of LSCC. Grants:This work was supported by Project№ 8553/12.12.2016/Contract №D-137/MU-Sofia; DUNK01/2/2009/NSF.

Introduction MicroRNAs (MiRNAs) can help understanding the carcinogenesis of lung cancer and serve as potential diagnostic biomarkers for differentiating adenocarcinoma (ADC) and squamous cell lung carcinoma (SCC). The aim of the present study is to analyse and compare the expression patterns of miR-21, miR-197, miR-205, miR-375, miR-495 in ADC and SCC samples. Material and methods Fresh frozen tissue samples from 50 non-small cell lung cancer (NSCLC) patients (25ADC, 25SCC) and adjacent normal tissues were examined. The expression of miR-21, miR-197, miR-205, miR-375, miR-495 were evaluated by RT-qPCR. The statistical analysis was performed by SPSSv20. Results and discussions We found overexpression of: miR-21 in 60% (12ADC and 18SCC), miR-205 in 62% (13ADC and 18SCC), miR-197 in 14% (5ADC and 2SCC), miR-375 in 30% (11ADC and 4SCC) and miR-495 in 40% (13ADC and 7SCC) of the samples. The following miRNAs demonstrated decreased expression: miR-21 in 10% (5ADC and 5SCC), miR-205 in 10% (7ADC and 3SCC), miR-197 in 42% (11ADC and 10SCC), miR-375 in 42% (6ADC and 15SCC) and miR-495 in 30% (9ADC and 6SCC) of the tumours. Statistically significant (p ROC analysis was performed for distinguishing ADC from SCC. Only miR-205 (AUC=0,672, p=0,036) and miR-375 (AUC=0,692, p=0,019) showed statistically significant results independently. The combination of these two miRNAs (AUC=0,771; p=0,001) could distinguish between ADC and SCC with 80% sensitivity and 76% specificity. With the same sensitivity and specificity the combination of miR-205 and miR-375 and miR-495 (AUC=0,776; p=0,001) and the combination of miR-205 and miR-197 and miR-375 (AUC=0,774; p=0,001) could also discriminate between ADC and SCC. Conclusion It has been suggested that miR-205 appeared to be a promising diagnostic biomarker for SCC. Our results show that different panels of miRNAs including miR-205, have better potential to discriminate between ADC and SCC. However further analysis of enlarged sample is necessary to ascertain their diagnostic potential in NSCLC. Acknowledgements This work was supported by Grants №D-138/Contract №8554/12.12.2016/MU-Sofia;DUNK01/2/2009/NSF.

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

Адрес за кореспонденция: С. Гирагосян, sggiragosyan@abv.bg ***** Address for correspondence: S. Giragosyan, sggiragosyan@abv.bg