Abstract 1625: NGS reveals specimen characteristics have minimal impact on immune gene expression signature
Authors: Conroy Jeffrey , Glenn Sean , Burgher Blake , Pabla Sarabjot , Qin Maochun , Andreas Jon , Giamo Vincent , Ernstoff Marc , Nesline Mary , He Ji , Gardner Mark , Morrison Carl 
Published: July 2017

Abstract
Background: Therapeutic antibodies targeting immune checkpoint molecules have been approved by the FDA for the treatment of several types of cancer. Currently, evaluation of the tumor checkpoint blockade is limited to FDA-approved IHC assays measuring PD-L1 ligand status which is subjective and not analytically robust. As the number of antibodies targeting immune checkpoints expands, assays that can evaluate additional biomarkers in tumor specimens are needed to accurately predict patient response to these drugs. To address these issues, a custom immune response NGS assay was developed to measure the transcript level of 54 genes involving T-cell receptor signaling (TCRS) and tumor infiltrating lymphocytes (TILs) in solid tumors of various characteristics including heterogeneity, disease, biopsy type and age. As part of the study, we evaluated the impact these variable have on the immune gene expression signature and their role as possible assay interferents.
Methods: Studies were designed to evaluate the analytical performance of a targeted RNA-seq assay for FFPE samples from NSCLC, melanoma, RCC, HNSCC, kidney and bladder cancer. To assess degree of assay tolerance to the wide range of specimen characteristics that are inherent in tumors, samples and mixed samples of various histopathologic characteristics were included. PCA and unsupervised clustering was performed on samples with checkpoint inhibition, TCRS and TILs genes to reveal sample groups with three distinct immune signatures (low, indeterminate and high). Further correlation and over-representation analysis was performed to determine impact of specimen characteristics on these three immune signatures.
Results: Immune signatures were maintained for the majority of characteristics studied within a specified range. As expected, only TIL status was significantly associated with the high expression group. Other factors including architecture, neoplastic content, percent necrosis, stroma quality/quantity, T-Path, PMR, specimen type, tissue amount and specimen age were not over-represented in any immune signature.
Conclusion: Tumor samples harbor a mixture of potential assay interferents including variable benign, neoplastic and immune cells populations with both naïve and reactive stroma contributing to a complex tumor microenvironment that is difficult to catalogue prior to testing. Our study demonstrates that the immune signature present in the tumor microenvironment is sufficiently strong to withstand a wide range of tumor heterogeneity, thereby reducing the need of extensive tissue macrodissection and the exclusion of samples previously thought to be non-evaluable.
Citation Format: Jeffrey Conroy, Sean Glenn, Blake Burgher, Sarabjot Pabla, Maochun Qin, Jon Andreas, Vincent Giamo, Marc Ernstoff, Mary Nesline, Ji He, Mark Gardner, Carl Morrison. NGS reveals specimen characteristics have minimal impact on immune gene expression signature [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 1625. doi:10.1158/1538-7445.AM2017-1625