Abstract 830: DNA methylation cytometry reveals cancer survival related to cell composition
Authors: Salas Lucas A. , Kelsey Karl T. , Koestler Devin C. , Wiencke John K. , Christensen Brock C. 
Published: August 2019

Abstract
Tumor microenvironments are heterogeneous and include epithelial cells, stroma, blood vessels and leukocytes. The interactions between normal and tumor epithelial cells result in responses including angiogenesis, immune regulation and tumor growth. Although flow cytometry and single cell sequencing have been used to investigate cellular composition in tumors, these approaches are dependent upon appropriate substrates that may not be routinely available. Recently, the use of molecular markers of cell type from mRNA (CIBERSORT) and DNA methylation (MethylCIBERSORT) have been used to infer specific cell proportions in tumors. Immune cell type DNA methylation signatures have been well-established for whole blood deconvolution but a challenge in tumors is the potential for carcinoma cells to have signal closely related to nontumor epithelial cells, and heterogeneity of potential tumor distinguishing deconvolution biomarkers. We aim to offer an alternative reference-based approach for tumor deconvolution with DNA methylation using ten normal cell components that we apply to TCGA tumor samples and test the relation of sample composition with patient survival. Ten magnetic sorted cell types with Illumina EPIC DNA methylation data were used as references (B cells, CD4T, CD8T and NK, monocytes, neutrophils, eosinophils, epithelial, endothelial and stromal cells). In total 1256 CpGs were selected for the reference library which was applied to deconvolute 17 TCGA tumor types (n=6417: BLCA, BRCA, COAD, CESC, ESCA, HNSC, KIRC, KIRP, LIHC, LUAD, LUSC, PAAD, PRAD, SKCM, STAD, THCA, UCEC). The most abundant cells were epithelial (median:36.6%), endothelial (median: 17.1%), CD8T (median: 12.1%) stromal cells (median:10.3%), and CD4T (median: 4.4%). We calculated three cell ratios: CD8T to epithelial (CD8T:epith), CD4T to epithelial (CD4T:epith) and endothelial to epithelial cells (vasc:epith) and fit Cox proportional hazards models stratified by cancer stage (I, II, III and IV), and adjusted for sex, age at diagnosis, and the three ratios. For all cancers, increased CD8T:epith was associated with worse survival HR: 1.39 (95%CI: 1.15, 1.68). For KIRC, increased CD8T:epith increased the risk of death HR: 1.54 (95%CI: 1.06, 2.22), whereas survival was improved for BRCA HR: 0.15 (95%CI: 0.03, 0.68), and CESC HR: 0.19 95%CI: 0.04, 0.90. A strong effect of increased vasc:epith ratio on risk of death was observed in BLCA HR: 8.03 (95%CI: 3.01, 21.4), and the CD4T:epith ratio reduced the risk of death HR: 0.11 95%CI: 0.02, 0.56. DNA methylation deconvolution is a promising tool for cancer prognosis and offers several technical and cost advantages. However, additional work to further stratify leukocyte signals by cell state such as CD8 T-cell exhaustion, and CD4T Treg infiltration is required.
Citation Format: Lucas A. Salas, Karl T. Kelsey, Devin C. Koestler, John K. Wiencke, Brock C. Christensen. DNA methylation cytometry reveals cancer survival related to cell composition [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 830.