Abstract 3994: Using NGS to characterize 40 NSCLC tumor with gene expression and tumor infiltrating T cell repertoire profiling from FFPE and matched Fresh Frozen samples
Authors: Mongan Ann , Lowman Geoffrey , Ku Yuan-Chieh , Looney Timothy , Lin Lifeng , Peng Xinzhan , Hyland Fiona 
Published: July 2017

Abstract
The tumor microenvironment (TME) is made up of stromal cells, immune cells, signaling molecules, and blood vessels surrounding the tumor cells. It has emerged as a key factor in multiple stages of cancer progression, immune-escaping, and disease progression. The composition and activity of TME co-evolve with tumor cells and may likely affect how the cancer responses to immunotherapy. A clear understanding of the functional effect and evolution of the TME necessitates a comprehensive approach to identify key immune cells as well as to characterize signaling and inflammatory (immune-editing) activities at the tumor site. Here we are describing the use of a targeted NGS panel to detect aggregated gene expression and a novel AmpliSeqTM approach to profile the relative abundance of different T cell clones at the TME. Specifically, we measured the expression of 395 relevant genes that capture interferon and chemokine signaling, T and B cell activation, checkpoint pathway, tumor proliferation, and antigen presentation. By looking at the expression of markers specific to different effector cell types, this gene panel offers a high-level view of the composition of different lymphocyte infiltrates. Complementarily, the TCR sequencing assay provides an estimate of T cell diversity and therefore offering a different dimension of the immune response. We studied 40 NSCLC tumor samples, of which we have matched FFPE and fresh frozen specimens. With a targeted panel, we could detect expression of transcripts present as few as 50 copies in 10 ng of total RNA as input. Across 40 NSCLC samples, we were able to measure expression of many low expressing cytokines such as IL2, IL21, IL23, IFNG and TNF. We observed strong co-expression pattern among genes involved in type II interferon signaling, indicating that they’re informative of T cell activation. More specifically, we found strong correlation between CD8 expression and other T cell co-stimulatory receptors (CD28, CD80, CD86, CD40), suggesting that expression of these genes can be reliable surrogates for the protein counterparts as markers for CD8+ T cells. TCRβ was sequenced for each of the matched fresh frozen sample in duplicates. Clonotype abundance of replicates was highly correlated with each other, indicating that the assay was reliable, and the samples were sequenced to appropriated depth. We identified 2000-10,000 unique clones in each tumor sample; with the diversity index moderately correlated with the percentage of tumor infiltrating lymphocytes provided by pathology review. Together, these two assays provide a convenient means to characterize T cells and their activities in combating tumor cells, thereby offering insights into how that tumor may respond to a particular immunotherapy.
Note: This abstract was not presented at the meeting.
Citation Format: Ann Mongan, Geoffrey Lowman, Yuan-Chieh Ku, Timothy Looney, Lifeng Lin, Xinzhan Peng, Fiona Hyland. Using NGS to characterize 40 NSCLC tumor with gene expression and tumor infiltrating T cell repertoire profiling from FFPE and matched Fresh Frozen samples [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 3994. doi:10.1158/1538-7445.AM2017-3994