Multiple imputation and clinico-serological models to predict human papillomavirus status in oropharyngeal carcinoma: An alternative when tissue is unavailable.

In cancer epidemiological studies, determination of human papillomavirus (HPV) in oropharyngeal squamous cell carcinoma (OPSCC) typically depends on the availability of tumor tissue testing, and/or tumor tissue access. Identifying alternative methods for estimating HPV status can improve the quality of such studies when tissue is unavailable. We developed multiple predictive models for tumor HPV status and prognosis by combining both clinico-epidemiological variables and either serological multiplex assays of HPV or multiple imputation of HPV status (HPVmi ). Sensitivity, specificity and accuracy of these methods compared to either p16 immunostaining (p16 IHC) or survival were assessed. When compared to a reference of tumor tissue p16 IHC in 783 OPSCC patients, the clinic-HPVsero model incorporating a composite of 20 HPV serological antibodies (HPVsero ) and 4 clinical factors (c-index: 0.96) performed better than using HPVsero (c-index: 0.92) or HPVmi (c-index: 0.76) alone. However, the model that contained a single HPV16 E6 antibody combined with four clinical variables, performed extremely well (clinic-s1-16E6; c-index: 0.95). When defining HPV status by HPVsero , s1-16E6, HPVmi or through p16 IHC, each of these definitions demonstrated improved overall and disease-free survival in HPV-positive OPSCC patients, when compared to HPV-negative patients (adjusted hazard ratios between 0.25 and 0.63). Our study demonstrates that when blood samples are available, a model that utilizes a single s1-16E6 antibody combined with several clinical features has excellent test performance characteristics to estimate HPV status and prognosis. When neither blood nor tumor tissue is available, multiple imputation, calibrated on local population characteristics, remains a viable, but suboptimal option.

Association of two BRM promoter polymorphisms and smoking status with malignant pleural mesothelioma risk and prognosis.

Brahma (BRM), of the SWI/SNF complex, has two 6 to 7 bp insertion promoter polymorphisms (BRM-741/BRM-1321) that cause epigenetic BRM suppression, and are associated with risk of multiple cancers. BRM polymorphisms were genotyped in malignant pleural mesothelioma (MPM) cases and asbestos-exposed controls. Multivariable logistic regression (risk) and Cox regression (prognosis) were performed, including stratified analyses by smoking status to investigate the effect of polymorphisms on MPM risk and prognosis. Although there was no significant association overall between BRM-741/BRM-1321 and risk in patients with MPM, a differential effect by smoking status was observed (P-interaction < .001), where homozygous variants were protective (aOR of 0.18-0.28) in ever smokers, while never smokers had increased risk when carrying homozygous variants (aOR of 2.7-4.4). While there was no association between BRM polymorphisms and OS in ever-smokers, the aHR of carrying homozygous-variants of BRM-741, BRM-1321 or both were 4.0 to 8.6 in never-smokers when compared to wild-type carriers. Mechanistically, lower mRNA expression of BRM was associated with poorer general cancer prognosis. Electrophoretic mobility shift assays and chromatin immunoprecipitation experiments (ChIP) revealed high BRM insertion variant homology to MEF2 regulatory binding sites. ChIP experimentation confirmed MEF2 binding only occurs in the presence of insertion variants. DNA-affinity purification assays revealed YWHA scaffold proteins as vital to BRM mRNA expression. Never-smokers who carry BRM homozygous variants have an increased chance of developing MPM, which results in worse prognosis. In contrast, in ever-smokers, there may be a protective effect, with no difference in overall survival. Mechanisms for the interaction between BRM and smoking require further study.

Two BRM promoter polymorphisms predict poor survival in patients with hepatocellular carcinoma.

Polymorphisms in the promoter of the BRM gene, a critical subunit of the chromatin remodeling SWI/SNF complex, have previously been implicated in risk and prognosis in Caucasian-predominant lung, head and neck, esophageal, and pancreatic cancers, and in hepatocellular cancers in Asians. We investigated the role of these polymorphisms in hepatocellular carcinoma (HCC) risk and prognosis. HCC cases were recruited in a comprehensive cancer center while the matched controls were recruited from family practice units from the same catchment area. For risk analyses, unconditional logistic regression analyses were performed in HCC patients and matched healthy controls. Overall survival analyses were performed using Cox proportional hazard models, Kaplan-Meier curves, and log-rank tests. In 266 HCC cases and 536 controls, no association between either BRM promoter polymorphism (BRM-741 or BRM-1321) and risk of HCC was identified (P > 0.10 for all comparisons). There was significant worsening of overall survival as the number of variant alleles increased: BRM-741 per variant allele adjusted hazards ratio (aHR) 5.77, 95% confidence interval (CI) 2.89-11.54 and BRM-1321 per variant allele aHR 4.09, 95%CI 2.22-7.51. The effects of these two polymorphisms were at least additive, where individuals who were double homozygotes for the variant alleles had a 45-fold increase in risk of death when compared to those who were double wild-type for the two polymorphisms. Two BRM promoter polymorphisms were strongly associated with HCC prognosis but were not associated with increased HCC susceptibility. The association was strongest in double homozygotes for the allele variants.

BRM polymorphisms, pancreatic cancer risk and survival.

Variant alleles of two promoter polymorphisms in the BRM gene (BRM-741, BRM-1321), create MEF2D transcription binding sites that lead to epigenetic silencing of BRM, the key catalytic component of the SWI/SNF chromatin remodeling complex. BRM suppression can be reversed pharmacologically.(1) Our group and others have reported associations with lung, head and neck, hepatocellular cancer risk,(1-3) and with lung and esophageal cancer prognosis (ASCO 2013; abstract 11057 & 4077). Herein, we assessed risk and survival associations with pancreatic cancer. A provincial population-based case-control study was conducted with 623 histologically confirmed pancreatic adenocarcinoma cases and 1,192 age/gender distribution-matched controls.(4) Survival of cases was obtained through the Ontario Cancer Registry. Logistic and Cox proportional hazard regression models were fitted, adjusting for relevant covariates. Median age was 65 y; 52% were male; Stage I (8%), II (55%), III (14%), IV (23%); 53% after curative resection, 79% after chemotherapy; and 83% had died. In the risk analysis, adjusted odds ratios (aOR) were 1.01 (95% CI: 0.1-2.0) and 0.96 (95% CI: 0.7-1.3) for the homozygotes of BRM-741 and BRM-1321, respectively; aOR of double-homozygotes was 1.11 (95% CI: 0.80-1.53), compared to the double-wildtype. For the survival analysis, adjusted hazard ratios (aHR) were 2.19 (95% CI: 1.9-2.5) for BRM-741 and 1.94 (95% CI: 1.7-2.2) for BRM-1321, per unit increase in variant alleles. Compared with the double-wildtype, aHR for carrying no, one, and two double-homozygotes were 2.14 (95% CI: 1.6-2.8), 4.17 (95% CI: 3.0-5.7), 8.03 (95% CI: 5.7-11.4), respectively. In conclusion, two functional promoter BRM polymorphisms were not associated with pancreatic adenocarcinoma risk, but are strongly associated with survival.

Discovery and validation of vascular endothelial growth factor (VEGF) pathway polymorphisms in esophageal adenocarcinoma outcome.

Polymorphisms in the vascular endothelial growth factor (VEGF)/angiogenesis pathway have been implicated previously in cancer risk, prognosis and response to therapy including in esophageal adenocarcinoma. Prior esophageal adenocarcinoma studies focused on using candidate polymorphisms, limiting the discovery of novel polymorphisms. Here, we applied the tagSNP (single nucleotide polymorphism) approach to identify new VEGF pathway polymorphisms associated with esophageal adenocarcinoma prognosis and validated them in an independent cohort of esophageal adenocarcinoma patients. In 231 esophageal adenocarcinoma patients of all stages/treatment plans, 58 genetic polymorphisms (18 KDR, 7 VEGFA and 33 FLT1) selected through tagging and assessment of predicted function were genotyped. Cox-proportional hazard models adjusted for important socio-demographic and clinico-pathological factors were applied to assess the association of genetic polymorphisms with overall survival (OS) and progression-free survival (PFS). Significantly associated polymorphisms were then validated in an independent cohort of 137 esophageal adenocarcinoma patients. Among the 231 discovery cohort patients, 86% were male, median diagnosis age was 64 years, 34% were metastatic at diagnosis and median OS and PFS were 20 and 12 months, respectively. KDR rs17709898 was found significantly associated with PFS (adjusted hazard ratio, aHR = 0.69, 95% confidence interval (CI): 0.53-0.90; P = 5.9E-3). FLT1 rs3794405 and rs678714 were significantly associated with OS (aHR = 1.44, 95% CI: 1.04-1.99; P = 0.03 and aHR = 1.50, 95% CI: 1.01-2.24; P = 0.045, respectively). No VEGFA polymorphisms were found significantly associated with either outcome. Upon validation, FLT1 rs3794405 remained strongly associated with OS (aHR = 1.59, 95% CI: 1.04-2.44; P = 0.03). FLT1 rs3794405 is significantly associated with OS in esophageal adenocarcinoma, whereby each variant allele confers a 45-60% increased risk of mortality. Validation and evaluation of this association in other cancer sites are warranted.

Association of two BRM promoter polymorphisms with head and neck squamous cell carcinoma risk.

The SWI/SNF chromatin remodeling complex is an important regulator of gene expression that has been linked to cancer development. Expression of Brahma (BRM), a critical catalytic subunit of SWI/SNF, is lost in a variety of solid tumors. Two novel BRM promoter polymorphisms (BRM-741 and BRM-1321) have been correlated with BRM loss and elevated cancer risk. The aim(s) of this study were to examine BRM expression in head and neck squamous cell carcinoma (HNSCC) and to correlate BRM polymorphisms with HNSCC risk. BRM expression studies were performed on eight HNSCC cell lines and 76 surgically resected tumor samples. A case-control study was conducted on 668 HNSCC patients (oral cavity, oropharynx, larynx and hypopharynx) and 700 healthy matched controls. BRM expression was lost in 25% of cell lines and 16% of tumors. The homozygous genotype of each polymorphism was significantly associated with increased HNSCC risk [BRM-741: adjusted odds ratio (aOR) 1.75, 95% CI 1.2-2.3, P < 0.001; BRM-1321: aOR 1.65, 95% CI 1.2-2.2, P < 0.001]. Individuals that were homozygous for both BRM polymorphisms had a more than 2-fold increase in the risk of HNSCC (aOR 2.23, 95% CI 1.5-3.4, P < 0.001). A particularly elevated risk was seen within the oropharynx, human papillomavirus-positive subgroup for carriers of both homozygous variants (aOR 3.09, 95% CI 1.5-6.8, P = 0.004). BRM promoter polymorphisms appear to act as susceptibility markers of HNSCC with potential utility in screening, prevention and treatment.

Genetic sequence variants in vitamin D metabolism pathway genes, serum vitamin D level and outcome in head and neck cancer patients.

Although some studies have reported associations between serum vitamin D level and prognosis in several cancers, others have found associations between genetic sequence variants (GSVs) in the vitamin D metabolism pathway genes and outcomes in various cancers including head and neck cancer (HNC). We comprehensively evaluated the association and interaction of GSVs in vitamin D metabolism pathway genes and their regulatory effects on circulatory serum vitamin D level in HNC outcome. We systemically evaluated the association of 89 tagging and candidate-based GSVs in six major vitamin D metabolism pathway genes (VDR, GC, CYP24A1, CYP27A1, CYP27B1 and CYP2R1) and the circulating serum vitamin D level with overall survival (OS) and second primary cancer (SPC) in 522 Stages I-II radiation-treated patients with HNC. For OS: median follow-up time was 8 years; for SPC, 4.4 years. The most common subsite was the larynx (84%). Three hundred and twelve patients were alive at the end of follow-up for OS. SPCs were diagnosed in 108 patients and were primarily of lung (46%). Serum vitamin D levels were significantly lower in patients carrying the minor alleles of GC:rs4588 and CYP2R1:rs10500804. CYP24A1:rs2296241 was significantly associated with OS and CYP2R1:rs1993116 was with SPC. These two GSVs remained significantly associated after adjusting for serum vitamin D level and important clinical factors. GSVs in the vitamin D metabolism pathway genes were associated with disease outcomes in HNC patients; however, these GSVs are different from those affecting serum vitamin D levels.

Analysis of prognostic germline polymorphisms in patients with advanced hepatocellular carcinoma.

Background: The prognosis of hepatocellular carcinoma (HCC) is influenced by both tumor and patient specific factors. Current therapies of advanced HCC target angiogenesis and immune evasion, however there are no clinically useful biomarkers to guide clinicians. Methods: Our aim in this retrospective cohort study was to validate single nucleotide polymorphisms (SNPs) prognostic of outcome in advanced HCC from the literature, and to analyze exploratory SNPs chosen from evaluation of the HCC tumor immune microenvironment. Using a database of patients with HCC treated with sorafenib, blood samples were genotyped, clinical variables were retrospectively collected, and SNPs were analyzed for association with progression-free survival (PFS) and overall survival (OS). A subsequent analysis was conducted to determine if identified SNPs were prognostic in trans arterial chemoembolization (TACE) treated patients. Results: Literature review identified 7 SNPs in vascular endothelial growth factor (VEGF), eNOS, angiopoietin 2 (ANGPT2) and vascular endothelial growth factor receptor 2 (VEGFR2), however none were externally validated in our dataset. Of the 35 exploratory immunomodulatory SNPs, the following were associated with PFS or OS: CCL2 C-C motif ligand 2 (CCL2) (rs1024611), interleukin-10 (IL-10) (rs1800896), cytotoxic T-lymphocyte antigen-4 (CTLA-4) (rs231775) and NFKB1 (rs28362491). Conclusions: SNPs identified by literature review to be prognostic in sorafenib treated patients with advanced HCC were not validated in our dataset. Our findings suggest potentially important prognostic implications of SNPs in VEGFR2, CCL2, IL-10, CTLA-4 and NFKB1 that deserve further study.

Genetic sequence variants and the development of secondary primary cancers in patients with head and neck cancers.

BACKGROUND: Secondary primary cancers (SPCs), a major cause of morbidity and mortality in head and neck cancers (HNCs), are commonly associated with field cancerization. We comprehensively evaluated 23 germline sequence variants (from published literature) in 17 genes from 7 biological pathways associated with the HNC survival. Because cancer prognosis correlates with disease aggressiveness, the factors that determine aggressive disease may influence field cancerization process to favor SPC development. We thus hypothesized that the same sequence variants associated with HNC survival can also be associated with SPC. METHODS: Germline DNA from 531 stage I-II radiation-treated HNC patients (originally recruited for an alpha-tocopherol/beta-carotene placebo-controlled secondary prevention clinical trial) were genotyped, and analyzed using Cox proportional hazards models, stratified by treatment arm, adjusting for clinical prognostic factors. RESULTS: The majority of SPCs were of lung and HNCs. Median follow-up time was 5 years. SPCs were diagnosed in 21% of patients. The 5-year SPC-free survival was 79%. All but 1 evaluated sequence variant were not associated with SPC. There was a strong association of the DNA (cytosine-5-)-methyltransferase 3 beta (DNMT3B) sequence variant, DNMT3B:C149T (rs2424913) with SPC: the adjusted hazard ratio (aHR) for TT versus CC was 2.23 (1.32-3.78; P = .003), whereas each variant T allele was associated with an aHR of 1.49 (1.15-1.95; P = .003). CONCLUSIONS: A functional sequence variant in DNMT3B is associated with the development of SPCs in HNC early stage patients treated with radiation. Aberrant DNA methylation may be an important modulator of SPC development in at-risk individuals with HNCs.

Cell-free DNA methylation-defined prognostic subgroups in small-cell lung cancer identified by leukocyte methylation subtraction.

Small-cell lung cancer (SCLC) methylome is understudied. Here, we comprehensively profile SCLC using cell-free methylated DNA immunoprecipitation followed by sequencing (cfMeDIP-seq). Cell-free DNA (cfDNA) from plasma of 74 patients with SCLC pre-treatment and from 20 non-cancer participants, genomic DNA (gDNA) from peripheral blood leukocytes from the same 74 patients, and 7 accompanying circulating tumor cell-derived xenografts (CDXs) underwent cfMeDIP-seq. Peripheral blood leukocyte methylation (PRIME) subtraction to improve tumor specificity. SCLC cfDNA methylation is distinct from non-cancer but correlates with CDX tumor methylation. PRIME and k-means consensus identified two methylome clusters with prognostic associations that related to axon guidance, neuroactive ligand-receptor interaction, pluripotency of stem cells, and differentially methylated at long noncoding RNA and other repeats features. We comprehensively profiled the SCLC methylome in a large patient cohort and identified methylome clusters with prognostic associations. Our work demonstrates the potential of liquid biopsies in examining SCLC biology encoded in the methylome.

Association between Genetic Variants and Cisplatin-Induced Nephrotoxicity: A Genome-Wide Approach and Validation Study.

This study aims to evaluate genetic risk factors for cisplatin-induced nephrotoxicity by investigating not previously studied genetic risk variants and further examining previously reported genetic associations. A genome-wide study (GWAS) was conducted in genetically estimated Europeans in a discovery cohort of cisplatin-treated adults from Toronto, Canada, followed by a candidate gene approach in a validation cohort from the Netherlands. In addition, previously reported genetic associations were further examined in both the discovery and validation cohorts. The outcome, nephrotoxicity, was assessed in two ways: (i) decreased estimated glomerular filtration rate (eGFR), calculated using the Chronic Kidney Disease Epidemiology Collaboration formula (CKD-EPI) and (ii) increased serum creatinine according to the Common Terminology Criteria for Adverse Events v4.03 for acute kidney injury (AKI-CTCAE). Four different Illumina arrays were used for genotyping. Standard quality control was applied for pre- and post-genotype imputation data. In the discovery cohort (n = 608), five single-nucleotide polymorphisms (SNPs) reached genome-wide significance. The A allele in rs4388268 (minor allele frequency = 0.23), an intronic variant of the BACH2 gene, was consistently associated with increased risk of cisplatin-induced nephrotoxicity in both definitions, meeting genome-wide significance (ß = -8.4, 95% CI -11.4--5.4, p = 3.9 × 10-8) for decreased eGFR and reaching suggestive association (OR = 3.9, 95% CI 2.3-6.7, p = 7.4 × 10-7) by AKI-CTCAE. In the validation cohort of 149 patients, this variant was identified with the same direction of effect (eGFR: ß = -1.5, 95% CI -5.3-2.4, AKI-CTCAE: OR = 1.7, 95% CI 0.8-3.5). Findings of our previously published candidate gene study could not be confirmed after correction for multiple testing. Genetic predisposition of BACH2 (rs4388268) might be important in the development of cisplatin-induced nephrotoxicity, indicating opportunities for mechanistic understanding, tailored therapy and preventive strategies.

Circulating miR-16-5p, miR-92a-3p, and miR-451a in Plasma from Lung Cancer Patients: Potential Application in Early Detection and a Regulatory Role in Tumorigenesis Pathways.

BACKGROUND: Micro(mi)RNAs, potent gene expression regulators associated with tumorigenesis, are stable, abundant circulating molecules, and detectable in plasma. Thus, miRNAs could potentially be useful in early lung cancer detection. We aimed to identify circulating miRNA signatures in plasma from patients with lung adenocarcinoma (LUAD) and lung squamous cell carcinoma (LUSC), and to verify whether miRNAs regulate lung oncogenesis pathways. METHODS: RNA isolated from 139 plasma samples (40 LUAD, 38 LUSC; 61 healthy/non-diseased individuals) were divided into discovery (38 patients; 21 controls for expression quantification using an 800-miRNA panel; Nanostring nCounter®) and validation (40 patients; 40 controls; TaqMan® RT-qPCR) cohorts. Elastic net, Maximizing-R-Square Analysis (MARSA), and C-Statistics were applied for miRNA signature identification. RESULTS: When compared to healthy individuals, 580 of 606 deregulated miRNAs in LUAD and 221 of 226 deregulated miRNAs in LUSC had significantly increased levels. Among the 10 most significantly overexpressed miRNAs, 6 were common to patients with LUAD and LUSC. Further analysis identified three signatures composed of 12 miRNAs. Signatures included miRNAs commonly overexpressed in patient plasma. Enriched pathways included target genes modulated by three miRNAs in the C-Statistics signature: miR-16-5p, miR-92a-3p, and miR-451a. CONCLUSIONS: The 3-miRNA signature (miR-16-5p, miR-92a-3p, miR-451a) had high specificity (100%) and sensitivity (84%) to predict cancer (LUAD and LUSC). These miRNAs are predicted to modulate genes and pathways with known roles in lung tumorigenesis, including EGFR, K-RAS, and PI3K/AKT signaling, suggesting that the 3-miRNA signature is biologically relevant in adenocarcinoma and squamous cell carcinoma of the lung.

Correction: Hedgehog inhibition mediates radiation sensitivity in mouse xenograft models of human esophageal adenocarcinoma.

[This corrects the article DOI: 10.1371/journal.pone.0194809.].

Two functional indel polymorphisms in the promoter region of the Brahma gene (BRM) and disease risk and progression-free survival in colorectal cancer.

BACKGROUND AND OBJECTIVE: The Brahma gene (BRM) encodes a catalytic ATPase subunit of the Switch/Sucrose non-fermentable (SWI/SNF) complex, which modulates gene expression and many important cellular processes. Two indel polymorphisms in the promoter region of BRM (BRM-741 and BRM-1321) are associated with its reduced expression and the risk of susceptibility or survival outcomes in multiple solid cancers. In this study, we have examined these variants in relation to susceptibility and survival outcomes in colorectal cancer. METHODS: Genotypes were obtained using TaqMan assays in 427 cases and 408 controls. Multivariate logistic and Cox regression models were fitted to examine the associations of the BRM-741 and BRM-1321 genotypes adjusting for relevant covariates. Sub-group analyses based on tumor location and patient sex were also performed. In all analyses, indels were examined individually as well as in combination. RESULTS: Our results showed that there was no association between the BRM polymorphisms and the risk of colorectal cancer. However, genotype combinations of the BRM-741 and BRM-1321 variants were associated with the risk of colon cancer. Particularly, patients having at least one variant allele had increased risk of colon cancer when compared to patients with the double wild-type genotype. In the survival analyses, BRM-741 heterozygosity was associated with longer progression-free survival time in the colorectal cancer patients. A stronger association was detected in the male patients under the recessive genetic model where the homozygosity for the variant allele of BRM-741 was associated with shorter progression-free survival time. CONCLUSIONS: Our analyses suggest that BRM-741 and BRM-1321 indels are associated with the risk of developing colon cancer and the BRM-741 indel is associated with the disease progression in colorectal cancer patients, especially in the male patients. Although our results show a different relationship between these indels and colorectal cancer compared to other cancer sites, they also suggest that BRM and its promoter variants may have biological roles in susceptibility and survival outcomes in colorectal cancers. Performing further analyses in additional and larger cohorts are needed to confirm our conclusions.

Hedgehog inhibition mediates radiation sensitivity in mouse xenograft models of human esophageal adenocarcinoma.

BACKGROUND: The Hedgehog (Hh) signaling pathway is active in esophageal adenocarcinoma (EAC). We used a patient-derived murine xenograft (PDX) model of EAC to evaluate tumour response to conventional treatment with radiation/chemoradiation with or without Hh inhibition. Our goal was to determine the potential radioresistance effects of Hh signaling and radiosensitization by Hh inhibitors. METHODS: PDX models were treated with radiation, chemotherapy or combined chemoradiation. Tumour response was measured by growth delay. Hh transcript levels (qRT-PCR) were compared among frozen tumours from treated and control mice. 5E1, a monoclonal SHH antibody, or LDE225, a clinical SMO inhibitor (Novartis®) inhibited Hh signaling. RESULTS: Precision irradiation significantly delayed xenograft tumour growth in all 7 PDX models. Combined chemoradiation further delayed growth relative to either modality alone in three of six PDX models. Following irradiation, two of three PDX models demonstrated sustained up-regulation of Hh transcripts. Combined LDE225 and radiation, and 5E1 alone delayed growth relative to either treatment alone in a Hh-responsive PDX model, but not in a non-responsive model. CONCLUSION: Hh signaling mediates the radiation response in some EAC PDX models, and inhibition of this pathway may augment the efficacy of radiation in tumours that are Hh dependent.

Association of BRM promoter polymorphisms and esophageal adenocarcinoma outcome.

PURPOSE: Brahma (BRM) is a critical catalytic subunit of the SWI/SNF chromatin remodeling complex; expression of BRM is commonly lost in various cancer types. BRM promoter polymorphisms (BRM-741; BRM-1321) are associated with loss of BRM expression, and with cancer risk/survival. We evaluated these two polymorphisms in the overall survival (OS) of esophageal adenocarcinoma (EAC) patients. RESULTS: Of 270 patients, 37% were stage IV. Minor allele frequencies were 47-49%; 15% were double-homozygotes. When compared to the wild-type genotype, the homozygous variant of BRM-741 carried an adjusted OS hazard ratio (aHR) of 1.64 (95% CI:1.1-2.4); for BRM-1321, the aHR was 2.09 (95% CI:1.4-3.0). Compared to the double wild-type, carrying homozygous variants of both promoter polymorphisms (double-homozygote) yielded an aHR of 2.21 (95% CI:1.4-3.6). Directions/magnitudes of associations were similar in subsets by age, gender, smoking status, use of platinum agents, and disease stage, and for progression-free survival. MATERIALS AND METHODS: In a cohort of EAC patients of all stages (84% male; median age of 64 years), two BRM polymorphisms were genotyped. Cox proportional hazards models, adjusted for known prognostic variables, estimated the association of polymorphisms with OS. CONCLUSIONS: BRM polymorphisms were associated with OS in EAC in this study. Validation studies are warranted.

Validation of microRNA pathway polymorphisms in esophageal adenocarcinoma survival.

Polymorphisms in miRNA and miRNA pathway genes have been previously associated with cancer risk and outcome, but have not been studied in esophageal adenocarcinoma outcomes. Here, we evaluate candidate miRNA pathway polymorphisms in esophageal adenocarcinoma prognosis and attempt to validate them in an independent cohort of esophageal adenocarcinoma patients. Among 231 esophageal adenocarcinoma patients of all stages/treatment plans, 38 candidate genetic polymorphisms (17 biogenesis, 9 miRNA targets, 5 pri-miRNA, 7 pre-miRNA) were genotyped and analyzed. Cox proportional hazard models adjusted for sociodemographic and clinicopathological covariates helped assess the association of genetic polymorphisms with overall survival (OS) and progression-free survival (PFS). Significantly associated polymorphisms were then evaluated in an independent cohort of 137 esophageal adenocarcinoma patients. Among the 231 discovery cohort patients, 86% were male, median diagnosis age was 64 years, 34% were metastatic at diagnosis, and median OS and PFS were 20 and 12 months, respectively. GEMIN3 rs197412 (aHR = 1.37, 95%CI: [1.04-1.80]; P = 0.02), hsa-mir-124-1 rs531564 (aHR = 0.60, 95% CI: [0.53-0.90]; P = 0.05), and KIAA0423 rs1053667 (aHR = 0.51, 95% CI: [0.28-0.96]; P = 0.04) were found associated with OS. Furthermore, GEMIN3 rs197412 (aHR = 1.33, 95% CI: [1.03-1.74]; P = 0.03) and KRT81 rs3660 (aHR = 1.29, 95% CI: [1.01-1.64]; P = 0.04) were found associated with PFS. Although none of these polymorphisms were significant in the second cohort, hsa-mir-124-1 rs531564 and KIAA0423 rs1053667 had trends in the same direction; when both cohorts were combined together, GEMIN3 rs197412, hsa-mir-124-1 rs531564, and KIAA0423 rs1053667 remained significantly associated with OS. We demonstrate the association of multiple miRNA pathway polymorphisms with esophageal adenocarcinoma prognosis in a discovery cohort of patients, which did not validate in a separate cohort but had consistent associations in the pooled cohort. Larger studies are required to confirm/validate the prognostic value of these polymorphisms in esophageal adenocarcinoma.

BRM Promoter Polymorphisms and Survival of Advanced Non-Small Cell Lung Cancer Patients in the Princess Margaret Cohort and CCTG BR.24 Trial.

Introduction: BRM, a key catalytic subunit of the SWI/SNF chromatin remodeling complex, is a putative tumor susceptibility gene that is silenced in 15% of non-small cell lung cancer (NSCLC). Two novel BRM promoter polymorphisms (BRM-741 and BRM-1321) are associated with reversible epigenetic silencing of BRM protein expression.Experimental Design: Advanced NSCLC patients from the Princess Margaret (PM) cohort study and from the CCTG BR.24 clinical trial were genotyped for BRM promoter polymorphisms. Associations of BRM variants with survival were assessed using log-rank tests, the method of Kaplan and Meier, and Cox proportional hazards models. Promoter swap, luciferase assays, and chromatin immunoprecipitation (ChIP) experiments evaluated polymorphism function. In silico analysis of publicly available gene expression datasets with outcome were performed.Results: Carrying the homozygous variants of both polymorphisms ("double homozygotes", DH) when compared with those carrying the double wild-type was associated with worse overall survival, with an adjusted hazard ratios (aHR) of 2.74 (95% CI, 1.9-4.0). This was confirmed in the BR.24 trial (aHR, 8.97; 95% CI, 3.3-18.5). Lower BRM gene expression (by RNA-Seq or microarray) was associated with worse outcome (P < 0.04). ChIP and promoter swap experiments confirmed binding of MEF2D and HDAC9 only to homozygotes of each polymorphism, associated with reduced promoter activity in the DH.Conclusions: Epigenetic regulatory molecules bind to two BRM promoter sequence variants but not to their wild-type sequences. These variants are associated with adverse overall and progression-free survival. Decreased BRM gene expression, seen with these variants, is also associated with worse overall survival. Clin Cancer Res; 23(10); 2460-70. ©2016 AACR.

ABCC2 polymorphisms and survival in the Princess Margaret cohort study and the NCIC clinical trials group BR.24 trial of platinum-treated advanced stage non-small cell lung cancer patients.

BACKGROUND: The drug transporter ABCC2 is upregulated in non-small cell lung cancer (NSCLC) and implicated in platinum resistance. We evaluated the association between germline polymorphisms in the ABCC2 gene and survival outcomes of platinum-treated advanced NSCLC patients. MATERIAL AND METHODS: Ten candidate and tagging germline polymorphisms in the ABCC2 gene were genotyped in a discovery cohort of 170 platinum-treated stage IV NSCLC patients from the Princess Margaret Cancer Centre. Associations with overall survival were assessed using multivariate Cox proportional hazard models adjusted for prognostic variables. To validate our results, we analyzed the association of the two top polymorphisms in the ABCC2 gene on survival outcomes of 219 stage IIIB-IV NSCLC patients enrolled on the NCIC Clinical Trials Group BR.24 clinical trial. RESULTS: Only one polymorphism was validated across both cohorts for an association with overall survival: the A allele of the ABCC2 polymorphism, rs8187710 (4544G>A), was associated with adverse overall survival (adjusted hazard ratio [aHR] 2.22; 95% CI: 1.2-4.0; p=0.009) among our stage IV NSCLC patients. A significant association with overall survival (aHR 1.73; 95% CI: 1.0-2.9; p=0.036) was observed for the same ABCC2 polymorphism in the BR.24 validation cohort. No other ABCC2 polymorphisms were associated with outcome. CONCLUSION: The ABCC2 polymorphism, rs8187710 (4544G>A), is associated with overall survival in platinum-treated advanced NSCLC patients. Additional studies are needed to evaluate the predictive versus prognostic nature of this relationship, and to explore the functional effect of this polymorphism on the pharmacokinetics of platinum drugs.

A genome-wide association study of non-HPV-related head and neck squamous cell carcinoma identifies prognostic genetic sequence variants in the MAP-kinase and hormone pathways.

BACKGROUND: Carcinomas of the oral cavity, pharynx and larynx are referred to as head and neck cancers (HNC); together they account for 2-3% of all newly diagnosed cancers in North America. Between 40-50% of HNC are early diagnosed at stages I-II. The 5-year and 10-year relative survival rates are 61% and 50%, respectively. Germline genetic sequence variants (GSV) have become increasingly found to have prognostic implications in a variety of cancers. Identifying these variants may have important clinical and biological implications. METHODS: We conducted a genome-wide association study (GWAS) in 531 Stage I-II radiation-treated HNC patients (originally recruited for α-tocopherol/ß-carotene placebo-controlled secondary prevention study) and used a replication cohort of 566 HNC patients of all stages, of mostly non-HPV-related cancers. Survival rates were estimated by the Kaplan-Meier method. Cox proportional hazards models adjusted for potential clinical factors and principal components were used to test for associations between the GSV and overall survival (OS) in these tumors. RESULTS: The median follow-up time for OS was 9.21 years (GWAS cohort) and 2.37 years (replication cohort). In both cohorts, CACNA2D1:rs2299187, ESRRG:rs946465 and ESRRG:rs1416612 were each individually significantly associated with survival. In silico analysis of ESRRG:rs946465 identifies that it produces a splice variant in ESRRG. Variant alleles of CACNA2D1:rs2299187 and ESRRG:rs946465 were associated with higher expression of the corresponding protein. CONCLUSIONS: Putatively functional polymorphisms in the MAP-Kinase and estrogen pathways, identified through GWAS and replicated in an independent dataset were associated with the survival of HNC patients.