Abstract 1174: Characterization of the immunologic intra-tumor heterogeneity in early stages of non-small cell lung carcinoma using multiplex immunofluorescence and image analysis approaches
Authors: Cruz Alejandro Francisco , Parra Edwin R. , Jiang Mei , Fujimoto Junya , Chow Chi-Wan , Rodriguez-Canales Jaime , Behrens Carmen , Kalhor Neda , Weissferdt Annikka , Heymach John , Swisher Stephen , Sepesi Boris , Lee J. Jack , Moran Cesar , Futreal P. Andrew , Zhang Jianjun , Wistuba Ignacio I. 
Published: August 2018

Abstract
Introduction. Recurrence of non-small cell lung carcinoma (NSCLC) is associated with genetic and epigenetic intra-tumor heterogeneity (ITH). The interaction between malignant cells, stromal cells, and tumor-associated immune-cells (TAICs), such as T-cell lymphocytes (TCLs) and tumor-associated macrophages (TAMs), is important for progression of NSCLC and the characterization of the immunologic ITH might be relevant to predict recurrence in surgically treated patients at early stages of NSCLC. The aim of this study was to characterize the immunologic ITH of primary NSCLC tumors at early stages using image analysis and multiplex immunofluorescence (mIF) approaches.
Material and methods. Eight cases of stage IA and 8 cases of stage IB surgically resected NSCLC (11 adenocarcinomas, ADCs; and 5 squamous-cell carcinomas, SCCs) with a history of early recurrence were selected for this preliminary analysis. Formalin-fixed, paraffin-embedded (FFPE) blocks were obtained and consecutive sections were stained with two panels of mIF for immune profiling, panel 1: pan-cytokeratin (AE1/AE3), PD-L1, PD-1, CD3, CD8, and CD68; panel 2: AE1/AE3, CD3, CD8, granzyme-B (GB), CD45RO, and FOXP3. Three not adjacent, intra-tumor regions (3mm2 each) per case were randomly selected after gridding the whole tumor section. A total of 41 intra-tumor regions were scanned by Vectra multispectral-microscope (PerkinElmer) and analyzed using InForm-software (PerkinElmer). TAICs were quantified in the epithelial and stromal compartments from each intra-tumor region.
Results. The median density of TCLs and TAMs were 1527 cells/mm2 and 635 cells/mm2, respectively, without significant differences between histologic subtypes. TCLs were predominantly concentered in the stromal compartment (median, 2222 cells/mm2) when compared with epithelial compartment (median, 332 cells/mm2). The percentage and density of TCLs and TAMs varied 4 and 8 times, respectively, between cases and regions. Non-cytotoxic T-cells and inactive cytotoxic T-cells were the most prevalent phenotypes. Higher density of TAMs and antigen-experienced TCLs were observed in stage IB than stage IA in the primary tumor of patients with NSCLC.
Conclusion. The characterization of the immunologic ITH of NSCLC is able by mIF and image analysis with FFPE tumor tissue. There is a variability of TAICs densities between regions from the same tumor and different subpopulations were observed. TAMs and exhausted T-cells were more prominent in stage IB (tumor &gt;3cm) suggesting these cells may play an important role in recurrence. Ongoing studies with a larger cohort and comparison with non-recurrent surgically treated patients are warranted. Supported in part by CPRIT RP160668 grant
Citation Format: Alejandro Francisco Cruz, Edwin R. Parra, Mei Jiang, Junya Fujimoto, Chi-Wan Chow, Jaime Rodriguez-Canales, Carmen Behrens, Neda Kalhor, Annikka Weissferdt, John Heymach, Stephen Swisher, Boris Sepesi, J. Jack Lee, Cesar Moran, P. Andrew Futreal, Jianjun Zhang, Ignacio I. Wistuba. Characterization of the immunologic intra-tumor heterogeneity in early stages of non-small cell lung carcinoma using multiplex immunofluorescence and image analysis approaches [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1174.