Abstract B99: Immune profiling of NSCLC tumors and matching normal lung samples by multicolor flow cytometry
Authors: Ivanova Elena , Jones Robert E. , Awad Mark M. , Bittinger Mark A. , Kulkarni Meghana M. , Koyama Shohei , Almonte Christina G. , Santos Abigail A. , English Jessie M. , Barlow Julianne , Richards William G. , Hammerman Peter S. , Rodig Scott J. , Bueno Raphael , Wong Kwok-Kin 
Published: May 2016

Abstract
With the recent advancements in cancer immunotherapy, understanding of tumor microenvironment becomes crucial to progressing oncology research. Standard method for tumor immune characterization today is traditional IHC technique with 1-2 color staining which presents certain limitations. In order to simultaneously identify immune infiltrate cellular components and parallel it with the analysis of expression of activation/suppression markers in various subpopulations, we developed a multicolor flow cytometry pipeline for freshly resected tumors.
Thirty one individual biomarkers were used in several antibody staining panels with suffficientdesigned redundancy to allow crossover and address intra-experiment variability. Aliquots of the samples were frozen and stained on separate days to test for inter-assay variability.
We performed flow cytometry analysis of 23 NSCLC tumors (17 adenocarcinoma, 1 adenosquamous and 5 squamous carcinoma subtypes) and matcheding normal lung tissue. Unsupervised clustering ofby 70 parameters, including percentages of cellular subtypes as well as modulator marker expression, showed distinct segregation of normal and tumor tissue samples. The immune infiltrate in resected tumors exhibited significantly decreased CD66b+ granulocytes and CD56+ NK cells (especially cytotoxic CD56+CD16+ NK cells) and highly increased CD3+ T and CD19+ B cells when compared to flow analysis of normal lung tissue. Adeno and squamous carcinomas were not segregated into separate groups by clustering analysis but rather joined together to form 3 subgroups defined by exhaustion/activation marker expression.
Grouping the tumor samples by the expression of the clinically relevant PD-L1 marker in immune cells indicated that “high PD-L1” tumors tend to have highly activated T cells populations. This finding correlates with the latest views on high PD-1/PD-L1 expressing immune cells being “negatively regulated” rather than “exhausted” as the result of their extensive interaction with cancer cells.
As we continue to characterize the immune profile of NSCLC tumors and paired normal lung, in conjunction with genetic and clinical information, we aim to further understanding molecular and clinical correlates that influence the tumor microenvironment and to ultimately of the mechanisms of immune response to overcome both innate and acquired resistance to immune therapy agents.
Citation Format: Elena Ivanova, Robert E. Jones, Mark M. Awad, Mark A. Bittinger, Meghana M. Kulkarni, Shohei Koyama, Christina G. Almonte, Abigail A. Santos, Jessie M. English, Julianne Barlow, William G. Richards, Peter S. Hammerman, Scott J. Rodig, Raphael Bueno, Kwok-Kin Wong. Immune profiling of NSCLC tumors and matching normal lung samples by multicolor flow cytometry. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr B99.