J.E. Choi

Association of a common AGO1 variant with lung cancer risk: a two-stage case-control study.

Based on the important role of microRNA (miRNA) biosynthesis genes in carcinogenesis, we hypothesized that polymorphisms in the miRNA biosynthesis genes may modulate susceptibility to lung cancer. To test this hypothesis, we conducted a two‐stage study to evaluate the associations between single nucleotide polymorphisms (SNPs) in the miRNA biosynthesis genes and the risk of lung cancer. In stage 1 of the study, 24 SNPs in the 11 miRNA biosynthesis genes (DROSHA, DGCR8, RAN, XPO5, DICER, AGO1, AGO2, HIWI, GEMIN3, GEMIN4, and TRBP) were genotyped in 100 lung cancer patients and 100 healthy controls using a sequenome mass spectrometry‐based genotyping assay. One promising SNP (AGO1 rs636832A > G) was selected for stage 2 of the study, and genotyped by a melting‐curve analysis using fluorescence‐labeled hybridization probes in an independent set of 552 cases and 552 controls. The AGO1 rs636832A > G exhibited highly consistent results between the two stages of the study. In combined analysis, the 636832A > G was associated with a significantly decreased risk of lung cancer in a dose‐dependent manner (Ptrend = 6.0 × 10−4). Individuals with at least one rs636832G allele were at a significantly decreased risk of lung cancer compared with those with the AA genotype (adjusted odds ratio = 0.67, 95% confidence interval = 0.53–0.84, P = 4.0 × 10−4). This finding suggests that the AGO1 rs636832A > G might be a useful marker for determining the susceptibility to lung cancer and that the AGO1 gene might be involved in the development of lung cancer.

Polymorphisms in the FAS and FASL Genes and Survival of Early Stage Non–small Cell Lung Cancer

Purpose: This study was conducted to investigate the impact of functional polymorphisms in the FAS and FASL genes on the survival of early stage non–small cell lung cancer (NSCLC) patients. Experimental Design: Three hundred and thirty-eight consecutive patients with surgically resected NSCLC were enrolled. The FAS -1377G>A (rs2234767) and -670A>G (rs1800682) and FASL -844C>T (rs763110) polymorphisms were investigated. Immunohistochemistry was used to assess FAS protein expression in tumors. The genotype and haplotype associations with survival were analyzed using Cox proportional hazards model, Kaplan-Meier method, and the log-rank test. Results: Patients with the GG and combined AG + GG genotypes of the FAS -670A>G locus had a significantly decreased survival when compared with patients with the AA genotype [adjusted hazard ratio = 1.71, 95% confidence interval (95% CI) = 1.06-2.77, and P = 0.03; and adjusted hazard ratio = 1.48, 95% CI = 1.01-2.20, and P = 0.047, respectively]. In addition, the FAS -1377G/-670G and -1377A/-670G haplotypes exhibited a significantly lower survival compared with the -1377G/-670A haplotype (adjusted hazard ratio = 1.87, 95% CI = 1.20-2.91, and P = 0.006; and adjusted hazard ratio = 1.31, 95% CI = 1.05-1.65, P = 0.02, respectively). Strongly positive FAS immunostaining was significantly less frequent in patients with the FAS -670 AG + GG genotype than in patients with the -670 AA genotype (4.5% versus 10.8%; P = 0.04). Conclusion: The FAS -670A>G polymorphism may affect survival in early-stage NSCLC. The analysis of the FAS -670A>G polymorphism can help identify patients at high risk for a poor disease outcome.

Epigenetic inactivation of Homeobox A5 gene in nonsmall cell lung cancer and its relationship with clinicopathological features.

Promoter methylation is an important mechanism in gene silencing and is a key epigenetic event in cancer development. Homeobox A5 (HOXA5) is a master regulator of the morphogenesis and cell differentiation to be implicated as a tumor suppressor gene in breast cancer, but its role in lung cancer is still unknown. In this study, we have investigated the methylation status of the promoter region of the HOXA5 gene in nonsmall cell lung cancers (NSCLCs) using nested and standard methylation‐specific PCR (MSP) and correlated the methylation status with clinicopathological features. With standard MSP analysis, HOXA5 methylation were found in 113 (81.3%) of 139 NSCLCs and 72 (51.8%) in their corresponding nonmalignant lung tissues. RT‐PCR and immunohistochemical analysis showed that HOXA5 methylation correlates with gene expression. Moreover, in the patients with stage I disease, HOXA5 methylation was more frequent in smokers than in never‐smokes (P = 0.01). There was no influence of HOXA5 methylation on survival in all NSCLCs or at stages II–IV. However, in the patients with stage I disease, HOXA5 methylation was associated with a borderline significantly worse survival (P = 0.09). These findings suggest that downregulation of the HOXA5 gene by aberrant promoter methylation occurs in the vast majority of NSCLCs and that it may play a role in the pathogenesis of NSCLC. Additional studies with larger sample sizes are required to evaluate the prognostic value of HOXA5 methylation in patients with stage I NSCLC.

A genetic variation in microRNA target site of KRT81 gene is associated with survival in early stage non-small cell lung cancer

BACKGROUND MicroRNAs (miRNAs) have a key role in carcinogenesis through negative regulation of their target genes. Therefore, genetic variations in miRNAs or their target sites may affect miRNA-mRNA interactions, thereby result in altered expression of target genes. This study was conducted to investigate the associations between single-nucleotide polymorphisms (SNP) located in the miRNA target sites (poly-miRTSs) and survival of patients with early-stage non-small-cell lung cancer (NSCLC). METHODS Using public SNP database and miRNA target sites prediction program, 354 poly-miRTSs were selected for genotyping. Among these, 154 SNPs applicable to Sequenoms MassARRAY platform were investigated in 357 patients. A replication study was carried out on an independent patient population (n = 479). Renilla luciferase assay and reverse transcription-polymerase chain reaction were conducted to examine functional relevance of potentially functional poly-miRTSs. RESULTS Of the 154 SNPs analyzed in a discovery set, 14 SNPs were significantly associated with survival outcomes. Among these, KRT81 rs3660G>C was found to be associated with survival outcomes in the validation cohort. In the combined analysis, patients with the rs3660 GC + CC genotype had a significantly better overall survival compared with those with GG genotype [adjusted hazard ratio (aHR) for OS, 0.65; 95% confidence interval (CI) 0.50-0.85; P = 0.001]. An increased expression of the reporter gene for the C allele of rs3660 compared with the G allele was observed by luciferase assay. Consistently, the C allele was associated with higher relative expression level of KRT81 in tumor tissues. CONCLUSION The rs3660G>C affects KRT81 expression and thus influences survival in early-stage NSCLC. The analysis of the rs3660G>C polymorphism may be useful to identify patients at high risk of a poor disease outcome.BACKGROUND MicroRNAs (miRNAs) have a key role in carcinogenesis through negative regulation of their target genes. Therefore, genetic variations in miRNAs or their target sites may affect miRNA-mRNA interactions, thereby result in altered expression of target genes. This study was conducted to investigate the associations between single-nucleotide polymorphisms (SNP) located in the miRNA target sites (poly-miRTSs) and survival of patients with early-stage non-small-cell lung cancer (NSCLC). METHODS Using public SNP database and miRNA target sites prediction program, 354 poly-miRTSs were selected for genotyping. Among these, 154 SNPs applicable to Sequenoms MassARRAY platform were investigated in 357 patients. A replication study was carried out on an independent patient population (n = 479). Renilla luciferase assay and reverse transcription-polymerase chain reaction were conducted to examine functional relevance of potentially functional poly-miRTSs. RESULTS Of the 154 SNPs analyzed in a discovery set, 14 SNPs were significantly associated with survival outcomes. Among these, KRT81 rs3660G>C was found to be associated with survival outcomes in the validation cohort. In the combined analysis, patients with the rs3660 GC + CC genotype had a significantly better overall survival compared with those with GG genotype [adjusted hazard ratio (aHR) for OS, 0.65; 95% confidence interval (CI) 0.50-0.85; P = 0.001]. An increased expression of the reporter gene for the C allele of rs3660 compared with the G allele was observed by luciferase assay. Consistently, the C allele was associated with higher relative expression level of KRT81 in tumor tissues. CONCLUSION The rs3660G>C affects KRT81 expression and thus influences survival in early-stage NSCLC. The analysis of the rs3660G>C polymorphism may be useful to identify patients at high risk of a poor disease outcome.

155P: PD-L1 polymorphism can predict clinical outcomes of non-small cell lung cancer patients treated with first-line paclitaxel–cisplatin chemotherapy

QuantStudio® 3D Digital PCR System (ABI, USA). For realtime PCR was used a thermal cycler Rotor-Gene 6000 (Qiagen, Germany), PCR was performed in a final volume of 20ml, reaction mixture contained 70mM Tris-HCl (pH 8.8), 16.6mM ammonium sulphate, 0.01% Tween-20, 2mM magnesium chloride, 200 nM of each dNTP, 500 nM of primers, 250 nM of fluorescent probe, 1000 nM of blocking probe, 1.5 units Taq-DNA polymerase. Probes used in fluorescent dyes – FAM and VIC, quenchers – BHQ-1 and BHQ-2. Sets of oligonucleotides were designed and produced by “Testgen Ltd” (Russia). Results: By comparing of the EGFR mutations status in plasma and tumor samples concordance was 88.7% [95% CI 85%, 92%], sensitivity – 83.3% [95% CI 76%, 90%], specificity – 100%, PPV – 100%. The analytical sensitivity of dPCR was 0.1% Analytical specificity – 99.5%. Analytical sensitivity for real-time PCR was 0.2%, the analytical specificity – 99.7%. Conclusions: Digital PCR has demonstrated the best analytical sensitivity, wherein concordance with real-time PCR was 100%. Legal entity responsible for the study: Kazan Clinical Oncology Center, Kazan, RU Funding: Kazan Clinical Oncology Center, Kazan, RU Disclosure: All authors have declared no conflicts of interest.

107P: A functional polymorphism located in intron of EGFR affects survival outcome of early-stage non-small cell lung cancer

Background: Radiation pneumonitis (RP) is the most frequent acute pulmonary toxicity following stereotactic body radiation therapy (SBRT) for lung cancer. Here we investigate clinical and dosimetric factors associated with symptomatic RP in stage I non-small cell lung cancer (NSCLC) patients treated with SBRT. Methods: A total of 67 patients with stage I NSCLC who received SBRT at our institution were enrolled and their clinicopathological parameters and dosimetric parameters were recorded and analyzed. Results: The median follow-up period was 26.4 months (range, 7–48 months). In univariable analysis, tumor size (P = 0.041), mean lung dose (MLD; P = 0.028), V2.5 (P = 0.024), V5 (P = 0.014), V10 (P = 0.004), V20 (P = 0.024), V30 (P = 0.020), V40 (P = 0.040), V50 (P = 0.040) were associated with symptomatic RP. In multivariable logistic regression analysis, V10 (P = 0.049) was significantly associated with symptomatic RP. Conclusions: In conclusion, this study found that tumor size, MLD and V2.5–50 were risk factors markedly associated with symptomatic RP.V10 was the most significant factor and planning constraints should be optimized to minimize the lung dose V10.For both central and peripheral stage I lung cancer, rates of RP grade 2 was low after SBRT with appropriate fraction dose and it has no correlation with the location of radiation field. Clinical trial identification: ChiCTR-OPN-15006864 (Chinese Clinical Trial Registry) Legal entity responsible for the study: N/A Funding: N/A Disclosure: All authors have declared no conflicts of interest.

185PASSOCIATION OF POLYMORPHISMS IN THE MICRORNA TARGET SITES AND SURVIVAL OF PATIENTS IN EARLY-STAGE NON-SMALL-CELL LUNG CANCER

ABSTRACT Aim: MicroRNAs (miRNAs) have a key role in carcinogenesis through the negatve regulation of their target genes. Therefore, genetic variations in miRNAs or miRNA target sites may affect miRNA-mRNA interactions, thereby result in altered expression of target genes. This study was conducted to investigate the associations between single nucleotide polymorphisms (SNPs) located in the miRNA target sites (poly-miRTSs) and survival of patients with early-stage non-small cell lung cancer (NSCLC). Methods: Using public SNP database and miRNA target sites prediction program, 354 poly-miRTSs were selected for genotyping. Among these, only 154 SNPs could be applied to Sequenoms MassARRAY platform and investigated in 357 patients. A replication study was performed on an independent patient population (n = 479). Renilla luciferase assay and reverse transcription-PCR were conducted to examine functional relevance of potentially functional poly-miRTSs. Results: Of the 154 SNPs analyzed in the discovery set, 14 SNPs were significantly associated with survival outcomes. Among these, KRT81 rs3660C > G was found to be associated with survival outcomes in the validation cohort. In combined analysis, patients with the rs3660 GC + CC genotype had a significantly better overall survival (OS) compared with those with GG genotype (adjusted hazard ratio for OS, 0.65; 95% confidence interval 0.50-0.85; p = 0.001). An increased expression of the reporter gene for the C allele of rs3660 compared with the G allele was observed by luciferase assay. Relative expression level of KRT81 in tumor tissues was significantly higher in CC genotype compared with GG or GC genotypes. Conclusions: The rs3660G > C affects KRT81 expression and thus influences survival in early-stage NSCLC. The analysis of the rs3660G > C polymorphism may be useful to identify patients at high risk of a poor disease outcome. Disclosure: All authors have declared no conflicts of interest.