Abstract 2758: Multiplex immunofluorescence (mIF) reveals differences in tumor immune microenvironment between molecularly-defined subsets of small cell lung cancer
Authors: Rocha Pedro , Stewart C. Allison , Parra Edwin , Solis Luisa M. , Gay Carl M. , Cardnell Robert J. , Uraoka Naohiro , Francisco-Cruz Alejandro , Dejima Hitoshi , Xi Yuanxin , Diao Lixia , Wang Jing , Negrao Marcelo V. , Zhang Jianjun , Wistuba Ignacio , Gibbons Don L. , Byers Lauren A. 
Published: July 2021

Abstract
Introduction: Despite the recent approval of immune checkpoints inhibitors (ICI) as a treatment option in the extensive-stage small cell lung cancer (SCLC) setting, survival has not significantly changed in the last decades. Recent scientific efforts have led to the identification of 4 major subtypes defined by expression of three transcription factors: ASCL1 (SCLC-A), NEUROD1 (SCLC-N), POU2F3 (SCLC-P) with the fourth subtype characterized by increased expression of immune genes (Inflamed subtype - SCLC-I). Transcriptomic results, along with recent immunotherapy trials, suggest that modulation of tumor immune microenvironment (TIME) could potential be critical to achieving clinical responses in a subset of patients, hence a comprehensive study of the TIME in SCLC is imperative. Here we report the feasibility of a multiplex immunofluorescence (mIF) methodology to analyze the TIME in SCLC.
Methods: FFPE sections from surgically resected SCLC (N=4, one representative case across all SCLC subtypes) were identified from the ICON Project at UT MD Anderson Cancer Center. We used mIF to identify and quantify immune markers grouped into two 6-antibody panels: Panel 1: cytokeratin (CK, AE1/AE3), CD3, CD8, PD-1, PD-L1 and CD68; Panel 2: CK, CD20, granzyme B, FOXP3, CD45RO, and CD57. Finally, genomic (WES), transcriptomic (RNA sequencing) and proteomic (RPPA) data from these cases were integrated with the mIF data.
Results: SCLC molecular subtypes (SCLC-A, N, P, I) were classified using transcriptomic and proteomic data. Analysis of TIME unveils a higher immune cell infiltration within SCLC-I subtype compared with the other cases representing, immune “cold” SCLC subtypes. SCLC-I subtype showed a 2-13 folder higher (range) immune cell density than SCLC-A, N and P subtypes (measured as a median of cell density). Specifically, T cells (CD3+) (695 and 242 cells/mm2, for SCLC-I and the median for the other subtypes respectively), T cytotoxic cells (CD3+ CD8+) (206 and 105), activated T cells (CD3+ CD8+ granzyme+) (20 and 2), antigen experienced ‘like' T cells (CD3+ PD-1+) (17 and 0), memory T cells (CD3+ CD45RO+) (328 and 91) and macrophages (CD68+) (773 and 57). PD-L1 expression in malignant cells did not show significant differences within the 4 SCLC subtypes. However, PD-L1 expression in macrophages was significantly higher in the SCLC-I subtype, suggesting an increase of IFN-gamma in the TIME.
Conclusions: TIME study show the use of mIF in SCLC tumors to be feasible, and could potentially provide key information towards the identification of SCLC patients that could benefit from ICI. For the first time we complemented transcriptomic data from SCLC tumors with mIF analysis unveiling the complex interplay of the host immune response and malignant cells. Our preliminary results warrant further studies to explore the role of TIME in immunotherapeutic response in SCLC.
Citation Format: Pedro Rocha, C. Allison Stewart, Edwin Parra, Luisa M. Solis, Carl M. Gay, Robert J. Cardnell, Naohiro Uraoka, Alejandro Francisco-Cruz, Hitoshi Dejima, Yuanxin Xi, Lixia Diao, Jing Wang, Marcelo V. Negrao, Jianjun Zhang, Ignacio Wistuba, Don L. Gibbons, Lauren A. Byers. Multiplex immunofluorescence (mIF) reveals differences in tumor immune microenvironment between molecularly-defined subsets of small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2758.