Protective effects of Sanicula europaea extracts on nuclear DNA damage

EACR24 Poster Sessions / European Journal of Cancer 61, Suppl. 1 (2016) S9–S218 206 Mutation screening of Bulgarian hereditary breast and ovarian cancer patients with multi-gene cancer panel A. Mitkova1 , R. Dodova1 , D. Pencheva1 , A. Vlahova2 , M. TaushanovaHadjieva3 , S. Valev3 , K. Timcheva3 , S. Christova2 , V. Mitev1 , R. Kaneva1 . 1 Medical University of Sofia, Molecular Medicine Center- Department of Medical Chemistry and Biochemistry- Medical Faculty, Sofia, Bulgaria, 2 Medical University of Sofia, General and Clinical Pathology Clinic- University Hospital “Alexandrovska”/Department of General and Clinical PathologyMedical Faculty, Sofia, Bulgaria, 3 Nadezhda Women’s Health Hospital, Clinic of Medical Oncology Chemotherapy, Sofia, Bulgaria Background: It has been estimated that about 5% to 10% of breast cancers and 15 to 20% of ovarian cancers are hereditary. Germline mutations in the highly penetrant cancer susceptible genes BRCA1/2 account for the majority of the cases with Hereditary Breast and Ovarian Cancer (HBOC). Other less common genes have been also associated with high (TP53, PALB2, PTEN), moderate (CHEK2, ATM, NF1, NBN), elevated, but imprecise (CDH1, STK11) breast and ovarian cancer risk (MMR genes, RAD51D, BRIP1). Advances in next generation sequencing technologies make massive parallel sequencing more feasible and afford an efficient hereditary gene panel testing of HBOC patients. Methods: In the present study we included a group of 75 Bulgarian high-risk HBOC patients, selected following the recognised BCLC and NCCN criteria for genetic testing. The mutation screening was performed by NGS in panel of 94 cancer related genes (TruSight Cancer Sequencing Panel, Illumina) on a MiSeq platform (Illumina). All detected mutations and variants of unknown clinical significance (VUSs) were confirmed by Sanger sequencing. Results: Twenty six pathogenic mutations (15 frameshift, 8 missense, one nonsense, one splice, one 5 UTR) and one likely pathogenic missense variant were found in 31 out of 75 HBOC patients. Damaging BRCA1/2 mutations (12 frameshift, two missense, one splice variant) were observed in 58% (18/31) of the mutation carriers, followed by two missense mutations in AIP (12.9%, 4/31) and RET (9.7%, 3/31), respectively. Pathogenic variants in other cancer related genes were detected in 29% (9/31) of the carriers: 3 frameshifts in ATM, BLM and WT, one nonsense mutation in MLH1, one 5 UTR in BULB1B and 5 missense mutations in SDHD, NBN, TSC1, CHEK2 and MUTYH. Six of the patients harboured two pathogenic variants in two different genes. In addition 215 VUSs were found. Conclusions: The mutation screening of Bulgarian HBOC patients with TruSight Cancer Sequencing Panel lead to identification of clinically relevant pathogenic variants in 41.33% of the cases. It is evident that hereditary cancer multi-gene panel testing offers new opportunities in the diagnosis and clinical management of cancer susceptibility, with improved power to identify more HBOC patients who are carrying undiagnosed pathogenic mutations. While panel testing is more likely to provide a more complete capture of an individual’s genetic landscape, there are several unanswered questions and areas of future research. As the number of tested genes increases, the likelihood of detecting VUSs increases as well. Another challenge for the genetic counselling is the interpretation of pathogenic variants in cancer related genes that have never been associated with HBOC syndrome. Acknowledgements: This work was supported by Grant No. 95, Contract No. 7-C/2015 Medical University of Sofia, Bulgaria. No conflict of interest. 207 Protective effects of Sanicula europaea extracts on nuclear DNA damage ˘ 1 , L. Dalyan1 , N. Arda1 . 1 Istanbul E. Onay U¸car1 , M. Pekmez1 , E. Mertoglu University, Molecular Biology and Genetics, Istanbul, Turkey Background: Sanicula europaea is widely used for the treatment of many diseases in traditional and complementary therapies. It is known that Sanicula europaea extracts have antiviral, antifungal and antimicrobial activities. The aim of the present study is to examine the comparative effects of different Sanicula europaea extracts on H2 O2 -induced DNA damage in HeLa cells. Material and Method: In this study, the effects of ethanolic and water extracts of Sanicula europaea are compared. Oxidative damage in two specific nuclear regions [transcriptionally active APEX (apurinic/aprimidinic endonucleases) gene and transcriptionally inactive insulin gene] are assessed by QPCR assay. The HeLa cells are pretreated with the extracts for 48 hours, followed by the treatment with 750 mM H2 O2 for 1 hour. Intracellular ROS level of the cells are evaluated by using a fluorescent probe, 2,7-dichlorofluorescein diacetate (DCFH-DA). Results: DNA damage was significantly induced by H2 O2 while it was inhibited by Sanicula europaea extracts. All extracts completely protected against nuclear DNA damage, especially the ethanolic extracts were significantly inhibited DNA damage. While the intracellular ROS level increased in HeLa cells treated with H2 O2 , on the other hand ROS level decreased in the cells pretreated with Sanicula europaea extracts. Our results suggest that the extracts of Sanicula europaea can prevent oxidative DNA damage. Conclusion: Taken together, our results suggest that Sanicula europaea extracts can protect against DNA damage via direct inhibition of ROS formation

S37

and/or indirectly by other mechanisms. Therefore, dietary intake of Sanicula europaea extract may lower the risk of oxidative stress-mediated diseases such as some cancers via reduction of oxidative DNA damage and intracellular levels of ROS. No conflict of interest. 208 Prognostic significance of TERT promoter mutations and association with other common genomic aberrations in Bulgarian glioblastoma patients G. Stancheva1 , T. Goranova1 , M. Laleva2 , A. Mitkova1 , G. Poptodorov2 , N. Velinov2 , M. Kamenova3 , V. Mitev1 , N. Gabrovsky2 , R. Kaneva1 . 1 Medical University of Sofia, Molecular Medicine Center- Department of Medical Chemistry and Biochemistry, Sofia, Bulgaria, 2 University Multiprofile Hospital for Active Treatment and Emergency Medicine “N.I. Pirogov”, Department of Neurosurgery, Sofia, Bulgaria, 3 University Multiprofile Hospital for Active Treatment and Emergency Medicine “N.I. Pirogov”, Department of Pathology, Sofia, Bulgaria Background: In the recent years many comprehensive studies were focused on genomic characteristics of gliomas. This led to the discovery of a variety of genes that were not associated with glial carcinogenesis before. Mutations in genes encoding isocitrate dehydrogenase isoforms 1 (IDH1) and 2 (IDH2) have been found in a large proportion of gliomas. IDH1/2 genetic alterations occur early in tumour progression of brain neoplasias, but rarely in other solid tumours and are associated with better outcome. Two mutations (c.1–124G>A and c.1–146G>A) affecting the promoter region of the telomerase reverse transcriptase (TERT) gene, have been recently associated with poor prognosis as well as with EGFR amplification, CDKN2A homozygous deletion and loss of 10q chromosome harbouring PTEN. Materials and Methods: To determine the prevalence of pTERT and IDH1/2 mutations in Bulgarian patients and their impact on prognosis, 30 primary glioblastomas were examined by sequencing for mutations in exon 4 of IDH1/2 as well as in the promoter region of TERT. Further alterations in EGFR, TP53, PTEN and CDKN2A genes were investigated by MLPA kit P105. Results: The two known hot spot mutations c.1–124G>A and c.1–146G>A in TERT promoter were found in 13 (43%) and 7 (23%) patients, respectively. Both TERT mutations have tendency of association with higher age of onset (median age 59.9 vs 44 in non-mutated cases; p = 0.142) while c.1– 146G>A was significantly associated with worse patient survival (median survival 4.27 months vs 16.43 in non-mutated cases; p = 0.05). IDH1 genetic alteration R132H was observed in 6 (20%) tumour samples. Mutations in IDH2 were not detected. The IDH1 mutation was found in younger patients (median age 43 vs 60.5 in non-mutated cases; p = 0.04) and was associated with an increased overall survival (median survival 28.4 months vs 8.07 in non-mutated cases; p = 0.002). In 56.6% (17) of the patients EGFR amplification was observed whereas PTEN, CDKN2A and TP53 deletions were identified in 63.3% (19), 43% (13) and 13% (4) of the patients, respectively. Patients with PTEN deletions revealed significant worse survival than patients without such aberrations (median survival 18.07 months vs 8.33 in non-mutated cases; p = 0.023). Multivariate regression analysis determined the IDH1 mutation as an independent prognostic factor of better survival (p = 0.023, HR = 0.104) whereas the mutation c.1–146G>A in the TERT promoter (p = 0.026, HR = 2.347) as such decisive for worse survival. Conclusions: The genetic alteration affecting IDH1 and the c.1–146G>A mutation in the promoter region of TERT could be used as an independent specific marker for prognosis of Bulgarian patients with primary glioblastoma. Acknowledgements: This work was supported by Grants: 66/2015, Medical University of Sofia and DUNK01/2/2009, National Science Fund, Ministry of Education and Science, Bulgaria. No conflict of interest. 209 Genome-wide sequencing identifies genetic relationship between first and late-onset second cancers in aristolochic acid nephropathy patients X. Castells1 , M. Ardin1 , S. Rorive2,3 , N. Broeders4 , A. Heguy5 , P.P. Bringuier6 , T. Quackels7 , T. Roumeguere7 , J. Nortier4 , J. Zavadil1 . 1 International Agency for Research on Cancer, Molecular Mechanisms and Biomarkers Group, Lyon, France, 2 Universite´ Libre de Bruxelles, Department of Pathology − ´ Erasme University Hospital, Brussels, Belgium, 3 Academie Universitaire Wallonie-Bruxelles, DIAPath − Center for Microscopy and Molecular Imaging 4 CMMI, Gosselies, Belgium, Universite´ Libre de Bruxelles, Department of Nephrology − Erasme University Hospital, Brussels, Belgium, 5 New York University Langone Medical Center, Genome Technology Center, New York, ˆ Edouard Herriot, Laboratoire de Biologie des Tumeurs- Service USA, 6 Hopital Central d’Anatomie et Cytologie Pathologiques, Lyon, France, 7 Universite´ Libre de Bruxelles, Department of Urology − Erasme University Hospital, Brussels, Belgium Introduction: Exposure to aristolochic acid (AA, IARC Group 1 carcinogen) present in some Chinese traditional medicines leads to aristolochic acid