Abstract 2256: Novel prognostic targets identified using integrated analysis of copy number and expression data in esophageal adenocarcinoma

Abstract

Abstract Introduction: The incidence of esophageal adenocarcinoma (EAC) has increased 6-fold in the west over the last 30 years, with a dismal survival rate of less than 20%. Understanding the genomic aberrations and biology of this cancer may enhance disease interventions. Aims: i) To identify novel molecular targets with clinical relevance using an integrated approach in analyzing genome-wide copy number and expression data of EAC. ii) To correlate reduced expression of molecular targets with promoter hypermethylation in EAC. Methods: 30K oligonucleotide array comparative genomic hybridization (CGH) and gene expression microarray profiling were performed on 56 chemo-naive EAC resection samples with long-term clinical follow-up data (median follow up: 20 months, range 0.5-137) to identify regions of copy number changes leading to differential expression levels. Fluorescence in situ hybridization (FISH) assays were carried out on touch-imprint slides of frozen tumor samples to confirm amplification status. Immunohistochemistry (IHC) assays on tissuemicroarrays (TMAs) on the same (n=56) and an independent (n=371; median follow-up: 17 months, range 0.5-193) cohort of EAC samples were used for subsequent validation at the protein level. Kaplan-Meier survival analyses were performed based on IHC data to determine prognostic significance of novel molecular targets. An Illumina Infinium Array was used to investigate the methylation status of 24 independent EAC tumors, compared with 24 premalignant Barrett's Esophagus (BE) samples. Results: Integrated array CGH data with matched gene expression microarray data highlighted 4 genes with copy number gains (log2 ratio>0.32) and increased expression (fold-change>6.5): EGFR, WT1, NEIL2 and MTMR9. FISH assays confirmed amplifications of these genes (more than 6 copies/centromere) in 11% of EAC samples and IHC assays (n=371 independent cases) demonstrated protein over-expression (IHC scores above 2): EGFR (10%), WT1 (20%), NEIL2 (14%) and MTMR9 (25%), corroborating findings from the integrated analysis. Each of these targets individually had prognostic significance in our cohort (P<0.060). Interestingly, reduced expressions of WT1 (P=0.037) and NEIL2 (P=0.051) were associated with poorer prognosis in EAC. In order to investigate the mechanism of reduced expression of these targets which might have contributed to a worse prognosis, methylation array data was utilized from an independent dataset. This showed increased methylation of WT1 in EAC (63%) compared to BE (55%; t-test P=0.028) and normal controls (50%), but no difference in methylation status of NEIL2 between disease states. Conclusion: Integration of array CGH and gene expression data identified novel molecular targrets in EAC and reduced expression of WT1 may be secondary to methylation. These genes have prognostic potential and require further study. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2256. doi:10.1158/1538-7445.AM2011-2256