Abstract 548: RNAseq analysis of infiltrating immune cells in liver cancer
Authors: Bhuvaneshwar Krithika , Smith Coleman I. , Kroemer Alexander H. , He Aiwu Ruth , Gusev Yuriy 
Published: July 2017

Abstract
Hepatocellular carcinoma (HCC) has emerged as second most common cause of cancer deaths worldwide. During the last 10 years, there has been a clear delineation of landscape of genetic alterations in HCC and deregulated pathways in HCC. However, the treatment for patients with advanced HCC is limited despite of great effort developing therapeutic targeting the deregulated pathways in HCC. Recent studies reveal a direct causal relationship between cancer & immune dysfunction, whereby tumor cells and their microenvironment are able to evade immune attack by exploiting various immunoregulatory mechanisms in a process termed cancer immune editing.
Methods In this poster, the objective is to perform exploratory analysis of TCGA liver cancer data to see if immune infiltrates matter, and if they offer anti-tumor immunity to a cell. For this purpose, we analyzed RNA-seq data for a cohort of 75 liver cancer samples from TCGA collection. We obtained the gene expression data from a pre-selected group of specific markers for infiltrating lymphocytes (several subtypes), and explored the association of expression of these markers with clinical outcome.We downloaded raw RNA-seq data from the TCGA Liver cancer collection from 75 patients. These included 25 patients who had Hepatitis B virus (HBV), 25 patients who had Hepatitis C virus (HCV) and 25 patients who had both viruses. After processing of raw data, we extracted isoform expression (TPM values) from specific markers for infiltrating lymphocytes. This data was stratified into ‘high’ and ‘low’ expression based on median cutoff.We compared the ‘high’ and ‘low’ expression groups of patients by performing differential expression & pathway analysis, to see if the differentially expressed results were linked to immune pathways. We then performed survival analysis tests (Log rank, Cox regression) and Kaplan Meier (KM) survival graphs to explore the association with overall survival outcome.
Results We found 14 of 75 HCC cases expressed CD8B isoform, while 61 of 75 HCC cases did not express CD8B isoform. The Log Rank survival test showed a significant association between the expression of CD8B isoform and overall survival (Chisq= 5.2 on 1 degrees of freedom, p= 0.0222). The survival test using a Cox model on the same CDBB isoform, showed that samples that expressed CD8B isoform were at higher risk of event compared to those that did not express the isoform. Other markers that showed good separation of survival curves included CD3 & CD8A.
Conclusion Additional immune cell subtype specific transcripts are being tested. Based on our preliminary analysis, we saw that most of the affected pathways were highly relevant to lymphocyte signaling & immune response and infiltration. Hence, exploring infiltrating lymphocytes can give evidence of immune surveillance against HCC. Testing immune cell specific transcript in tumor samples may service as predictor to treatment targeting immune evasion in cancer patients.
Citation Format: Krithika Bhuvaneshwar, Coleman I. Smith, Alexander H. Kroemer, Aiwu Ruth He, Yuriy Gusev. RNAseq analysis of infiltrating immune cells in liver cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 548. doi:10.1158/1538-7445.AM2017-548