348. identifying novel molecular subtypes of esophageal adenocarcinoma using laser capture microdissected rna-seq samples

Abstract Background Treatment options for esophageal adenocarcinoma (EAC) are limited by a lack of disease stratification methods. In other cancers, gene expression profiling has been successfully used to identify prognostically-relevant and treatment-susceptible molecular subtypes. Previous efforts to subtype EAC have been hindered by low biopsy and resection tumour cellularity. We hypothesize that laser-capture microdissection (LCM) tumour cell enrichment will permit the classification EAC tumours into multiple molecular subtypes that will be important for future therapeutic strategies. Methods Treatment naïve patient samples (N = 52) were collected from primary biopsies (N = 37), resections (N = 10) and metastatic biopsies (N = 5). Samples were laser-capture microdissected to enrich tumour cells followed by total RNA-seq. Gene expression was quantified using salmon and non-negative matrix factorization with 10 components was used to identify gene expression programs. The components identified were validated on a publicly available normal tissue cohort (N = 13 esophagus, gastric, intestinal, Barrett’s samples) and the TCGA EAC cohort (N = 80). We applied NMF with 4 components to the normal tissue (K = cohort) to identify normal tissue gene signatures to validate our RNA-seq data. Results We verified that our RNA-seq samples were depleted for normal tissue using the four NMF signatures from normal tissue cohort and, as expected, our samples were depleted for gene expression from the gastric and esophagus tissues compared to TCGA. Next, we explored the gene expression programs from our tumour samples and identified seven tumour-intrinsic components and three tumour microenvironmental components. The top 50 genes from each tumour intrinsic components were used with consensus cluster plus to identify K = 6 subtypes (Figure 1). Interestingly, two of these subtypes would be difficult to separate from normal tissue contamination without the use of LCM. Conclusion We have successfully used LCM to deplete normal tissue gene expression from our esophageal adenocarcinoma cohort and identified six molecular subtypes. We are currently evaluating the treatment and clinical implications of these subtypes and aiming to accumulate an additional (N = 50) EAC RNA-seq samples.