Abstract 5123: Multiplex in situ immunoprofiling and survival outcomes in non-small cell lung cancer
Authors: Lin Steven H. , Mansfield James R. , Rao Xiayu , Jiang Wen , Fang Penny , Roman Kristin , Wang Chichung , Komaki Ritsuko , Hahn Stephen , Swisher Stephen G. , Fujimoto Junya , Wistuba Ignacio , Wang Jing , Hoyt Clifford C. 
Published: July 2016

Abstract
The clinical activity of immune checkpoint blockade (“immunotherapies”) in non-small cell lung cancer (NSCLC) has highlighted the importance of the immune system in controlling cancer growth. Immune cells play a central role in determining response to immunotherapies, yet obtaining phenotypic information about the various immune cells in intact tissue sections and measuring cell-to-cell interactions is challenging. For this study, we applied multiplex immunofluorescence imaging to simultaneously resolve both phenotypic and morphologic information in order to determine the immune subset in the tumor -microenvironment that dictated the prognosis of NSCLC. One-hundred-twenty nine non-metastatic NSCLC tumors were studied using Tissue Microarray (TMA), with each tumor represented in triplicate. Majority of the patients were females (56%), had stage 2 (11%) or stage 3 (84%) disease, and all patients had primary resection and postoperative radiotherapy. TMA sections were stained for CD4, CD8, CD68, FOXP3, CD274 (PD-L1) and cytokeratin, with a DAPI counterstain, using a sequential multiplex IHC approach involving antibody stripping and tyramide signal amplification. Slides were imaged using an automated multispectral microscope. We determined cellular phenotype counts and per-cell marker expression in tumor and stromal areas in each core of the TMA, using machine learning-based pattern recognition and cell classification approaches. On average, 4,770 cells were analyzed in each TMA core. The densities of phenotyped cells (counts/mm2) for tumor and stroma were calculated. We analyzed the tumor to stroma ratios in each core for each of the immune subsets (CD8, CD4, FoxP3+ Treg, and CD68+ macrophages). In multivariate analysis, after controlling for patient and tumor characteristics, high CD4 tumor:stroma ratio predicted significantly for poorer overall survival (OS) (5 year OS of CD4 high: 27% vs. CD4 medium/low: 50%, p = 0.001). High CD4 content also correlated significantly to high FoxP3+ Treg levels (p&lt;0.0001, Fisher's Exact test) and lower PD-L1 expression in the tumor (p = 0.038). This initial data suggests the immunosuppressive tumor microenvironment, as reflected by high tumor CD4 content, portends to poor prognosis in NSCLC patients. This is an example of assessments enabled by data sets such as this, that include per-cell expression of checkpoint proteins that retain the exact cellular locations of the various cell phenotypes within the microenvironment context. We will present analyses that explore more in depth on the immuno-biology cell-to-cell interactions, including associations between PD-L1 expression and outcomes. This study highlights the power of multiplex immunofluorescence characterization to unravel the complex immunologic network within the tumor microenvironment that may not only be prognostic but also predictive of immunotherapy responsiveness.
Citation Format: Steven H. Lin, James R. Mansfield, Xiayu Rao, Wen Jiang, Penny Fang, Kristin Roman, Chichung Wang, Ritsuko Komaki, Stephen Hahn, Stephen G. Swisher, Junya Fujimoto, Ignacio Wistuba, Jing Wang, Clifford C. Hoyt. Multiplex in situ immunoprofiling and survival outcomes in non-small cell lung cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 5123.