Abstract 4321: Integrating multiplex immunohistochemistry (IHC) and transcriptomics for characterization of association between spatial heterogeneity of lymphocytic infiltration and TGFβ pathway activity in solid tumors
Authors: Theilhaber Joachim , Pollard Jack , Pomponio Robert , Sanicola-Nadel Michele , Caron Anne , Naimi Souâd , Wiederschain Dmitri , Lin Tun Tun , Wang Rui 
Published: September 2020

Background: Increasing evidence suggests that the presence and spatial localization of tumor infiltrating lymphocytes (TILs) influence the disease course of solid malignancies and associate with response to immunotherapies. The tumor microenvironment (TME) can be classified into three phenotypes (‘topography'): immune inflamed, immune excluded and immune desert based on the localization and distribution of TILs. Yet, there is lack of comprehensive understanding of molecular mechanisms underlying these phenotypes. Due to the important prognostic role of TILs, development of an accurate and robust approach to quantify these immune topographies is of great need. Herein, we report an integrative approach to assess the distribution of TILs and characterize associated molecular features.

Method: A multiplex fluorescent IHC assay was developed for PanCK/CD3/CD8 and image analysis was optimized to detect localization of CD3+ and CD8+ lymphocytes on a collection of procured patient biopsies including Bladder Cancer (n=20), CRC (n=29), HNSCC (n=19), Gastric Cancer (n=18) and Ovarian Cancer (n=19). Pathologist annotations were used to determine tumor and stroma regions and the spatial localization of lymphocyte subsets, which led to the assignment of each case to one of the three infiltration phenotypes. RNAseq was performed on CRC samples (n=29) to further investigate the prominent biological features associated with infiltration phenotypes, as well as the correlation between these phenotypes and TGFβ activation signature derived from differential gene analysis of TGFβ-stimulated versus naïve cancer cell lines.

Result: The systematic profiling of spatial distribution of T lymphocytes in biopsies (N=105) across five solid tumors revealed distinct phenotypes of immune inflamed, excluded and desert. The image analysis further suggested how these topographies differ between cancer types, i.e., CRC and Bladder cancer tend to have more immune-excluded pattern. The TGFβ activation signature, when applied to CRC, indicated TGFβ pathway activation was negatively correlated with TILs infiltration and discriminated significantly between the three infiltration phenotypes (p=0.014).

Conclusion: We developed a multiplex fluorescent IHC method for assessing the spatial distribution of TILs. The integrative investigation with both IHC and RNA-seq results further suggested the significant association between TGFβ pathway activity and infiltration phenotypes. Importantly, the current study provided evidence for developing quantitative approach to measure immune topographies, which may serve as stratification biomarkers for patients with solid tumors undergoing TGF-β inhibitor therapy.

Citation Format: Joachim Theilhaber, Jack Pollard, Robert Pomponio, Michele Sanicola-Nadel, Anne Caron, Souâd Naimi, Dmitri Wiederschain, Tun Tun Lin, Rui Wang. Integrating multiplex immunohistochemistry (IHC) and transcriptomics for characterization of association between spatial heterogeneity of lymphocytic infiltration and TGFβ pathway activity in solid tumors [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4321.