C.S.P. Lima

PO-275 Effect of ionising radiation in FaDu cell line- preliminary results

Introduction Head and neck cancer includes malignancies usually originated in upper aerodigestive tract mucosa. Despite hypopharyngeal cancer has a low incidence, patients have poor prognosis, advanced stage and distant metastization. A low overall survival of these cancer might be explained by the presence of cancer stem cells (CSC). Identification of a CSCs markers panel could help to overcome this barrier allowing to develop target drugs. This study aims to evaluate and characterise the expression of proliferation, adhesion and CSC markers in a hypopharyngeal cell line (FaDu) before and after ionising radiation exposure. Material and methods Paraffin cell blocks were performed in order to evaluate by immunohistochemistry (IHC), epithelial characterisation (cytokeratin CAM 5.2, leukocyte common antigen (LCA), vimentin), tumoral aggressiveness (P16, P53, OCT4 and SALL4), proliferation (Ki-67) and CSC markers (EpCAM, CD10, CD44, CD117, CD133, beta-catenin). Also, 14 subtypes of high-risk HPV (31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, 68, 16, and 18), EBV, Akt and Wnt expressions were analysed. Radiotherapy was performed with 0.5 × 106 cells/mL, exposed to increasing doses of X-ray (0.5 to 10 Gy), except for control cells. Clonogenic assay and IHC were realised. Treated cells for IHC were fixed with alcohol 96%, 48 hour after treatment and posteriorly analysed the following antigens Ki-67, EpCAM, CD133, CD44, beta-catenin, CD117, CD10, Akt, Wnt, OCT4 and P53. Results and discussions FaDu cell line showed positive immunostaining for cytokeratin, vimentin and negative to LCA. Relatively to tumoral aggressiveness, cells only expressed positively P16, while P53, OCT4 and SALL4 were negative. About proliferation it was observed Ki-67+. Between analysed stemness markers just CD44, CD133 and beta-catenin were expressed. Among HPV subtypes analysed only HPV18 was stained positive and EBV-. Adding, control cells expressed Akt +and Wnt +in normal conditions. Preliminary results related to ionising radiation effects showed that cell survival is X-ray dose dependent. Moreover, it was observed Wnt expression alterations with X-ray exposure. Conclusion These results demonstrate that FaDu survival is affected by ionising radiation exposure. This may be associated with Wnt expression which is altered after irradiation, highlighting that this molecule is involved in cell fate determination, cell polarity, migration and cell proliferation.

Impact of drug formulation and free platinum/cisplatin ratio on hypersensitivity reactions to cisplatin: formulation matters.

Use of cisplatin can induce type I hypersensitivity reactions that may also be linked to the quality of the drug utilized. We observed cases of hypersensitivity that appeared to be associated with the brand of cisplatin used. The aim of this study was to compare two different brands of cisplatin in relation to type I hypersensitivity reactions.

278PASSOCIATION OF ERP29 GENETIC POLYMORPHISM WITH BREAST CANCER RISK AND PROGNOSIS

ABSTRACT Aim: ERP29, a tumor suppressor gene, was related with the onset of tumors. The influence of ERP29 c.*293A > G (rs7114) polymorphism in breast cancer (BC) risk and prognosis has never been performed before and was the aim of this study. Methods: We analyzed 737 BC patients and 742 healthy women. The BC patients were treated by conventional procedures. ERP29 c.*293A > G genotypes were obtained from genomic DNA by TaqMan® genotyping assays. The expression of ERP29 mRNA was determined by quantitative PCR using total RNA from blood leukocytes of healthy individuals with distinct genotypes (15 individuals with AA, 17 with AG, and six with GG). Overall survival (OS) was obtained from date of first diagnosis until the date of death or last follow-up. The differences in frequencies of distinct genotypes in patients and controls were analyzed by logistic regression model, serving to obtain age, ethnic origin and adjusted crude odds ratios (ORs), considering a confidence interval (CI) of 95%. OS time was calculated using the Kaplan-Meier estimate probabilities and differences between survival curves were analyzed by the log-rank test. Statistical significance was established at a P Results: The frequency of ERP29 AG + GG combined genotypes was higher in BC patients than in controls (36.5% vs. 30.7%, P= 0.03). Carriers of G allele were under a 1.32 (95% CI: 1.02-1.70)-fold increased risk for the tumor. The mRNA expression was lower in carriers of G allele than those with ERP29 AA genotype (0.75 arbitrary units (AUs) vs. 1.30 AUs; P= 0.008). The median of observation of BC patients enrolled in the study was 54 months (1-325). OS of BC patients with the ERP29 AG + GG combined genotypes was higher than that observed in those with the ERP29 AA wild genotype (67.5% vs. 59.0% at 120 months of follow up; P= 0.04). Conclusions: Our data suggest, for the first time, that ERP29 c.*293A > G polymorphism alters the risk and prognosis of BC possibly due to variation in the protein production. Disclosure: All authors have declared no conflicts of interest.

1119PPOLYMORPHISMS IN THE APOPTOSIS PATHWAY IN CUTANEOUS MELANOMA: RECURRENCE AND SURVIVAL

ABSTRACT Aim: Cutaneous melanoma (CM) is notorious for its poor prognosis and resistance to conventional chemotherapy. Genes involved in the apoptosis pathway, such as P53, (tumor suppressor gene), MDM2 (p53 inhibitor), BAX (proapoptotic) and BCL2 (anti-apoptotic) are important for CM growth and survival. The aim of this study was to evaluate whether P53 Arg72Pro, MDM2 T309G, BAX G(-248)A and BCL2 C(-938)A polymorphisms, involved with inherited variations in apoptosis, are associated with relapse free survival (RFS) and overall survival (OS) of CM patients. Methods: Our analysis included 234 consecutive CM patients at diagnosis seen at our University Hospital from December 1989 to November 2013. Genomic DNA from peripheral blood of patients was analyzed by polymerase chain reaction followed by enzymatic digestion for discrimination of pertinent genotypes. RFS and OS were calculated using the univariate Kaplan-Meier estimate probabilities, and differences between survival curves were analysed by the log-rank test. RFS was defined as the time to beginning of treatment until the date of the first relapse. OS was calculated from date of first diagnosis until the date of death or last follow-up. Results: The median period of observation of patients in study was 50 months (9-192) for RFS and 58 months (range: 9-285) for OS. We observed at 120 months of segment, that the RFS was higher in patients with BCL2 CC + CA plus MDM2 TT + TG than patients with BCL2 AA plus MDM2 GG genotypes (54% versus 37%, P = 0.01). The OS in CM patients with the BAX GA + AA was higher than in those with the GG genotypes (100% versus 80%, P = 0.01). Moreover, the OS was also higher in patients with BAX GA + AA plus BCL2 CC + CA combined genotype than in those with BAX GG plus BCL2 AA (100% versus 78%, P = 0.02) and BAX GA + AA plus MDM2 TT + TG than BAX GG plus MDM2 GG (100% versus 71%, P = 0.001). Conclusions: The data suggest, for the first time, that BAX G(-248)A polymorphism may independent or jointly with BCL2 C(-938)A and MDM2 T309G polymorphisms modulate RFS and OS in CM patients. Additional studies will provide some promising guidance for clinical management and tailored or personalized therapeutics in treating for CM. Disclosure: All authors have declared no conflicts of interest.

959PBAX AND BCL-2 POLYMORPHISMS MODULATING AGGRESSIVENESS AND PROGNOSIS IN DIFFUSE LARGE B-CELL LYMPHOMA

ABSTRACT Aim: Apoptosis, with participation of the pro-apoptotic BAX and the anti-apoptotic BCL-2 proteins, play a key role in outcome of patients with diffuse large B cell lymphoma (DLBCL). The ability to induce apoptosis is variable in humans, since several proteins enrolled in the process are encoded by polymorphic genes. The G wild allele of the BAX G(-248)A and the variant A allele of the BCL2 C(-717)A single nucleotide polymorphisms (SNPs) are related to lower transcriptional activity and higher BCL-2 protein expression, respectively, compared with the A variant and C wild alleles. Since the roles of these SNPs in clinical aspects and prognosis of DLBCL are still unknown, investigation of these were the aims of the present study. Methods: Our analysis included 154 consecutive DLBCL patients at diagnosis seen at the University Hospital from December 2007 to March 2014. Genomic DNA from peripheral blood was analyzed by polymerase chain reaction followed by enzymatic digestion for discrimination of distinct genotypes of each SNP. Multivariate analysis using the logistic regression model served to assess the associations between genotypes and clinical aspects. Overall survival (OS) was calculated using the Kaplan-Meier estimate probabilities, and differences between survival curves were analysed by the log-rank test. Results: The frequencies of BAX GG and BAX GG plus BCL-2 AA genotypes were higher in patients with stage IV tumors compared to those with tumors of I + II + III stages (95% vs. 77%, P = 0.01; 94% vs. 54%, P = 0.02), respectively. The median time of observation of patients was 22 months (range: 1-75). On univariate analysis, the presence of B symptoms (68% vs. 87%, P = 0.02), bone marrow involvement (53% vs. 79%, P = 0.009), high LDH levels (64% vs. 81%, P = 0.03), high risk disease (52% vs. 76%, P = 0.002), and stage IV (61% vs. 81% P = 0.01) were predictive of worse outcome at 24 months of follow up. Moreover, at the same time, patients with BCL2 CA + AA genotypes had worse outcome than others (68% vs. 88%, P = 0.04). Conclusions: Our data indicate, for the first time, that inherited abnormalities in intrinsic apoptosis pathway, related to the BAX G(-238)A and BCL2 C(-717)A SNPs, influence aggressiveness and outcome of DLBCL patients. Disclosure: All authors have declared no conflicts of interest.