Abstract 1571: Inter- and intra- tumorheterogeneity of the microenvironment in pulmonary adenocarcinoma
Authors: Kazdal Daniel , Budczies Jan , Kirchner Martina , Kluck Klaus , Allgäuer Michael , Neumann Olaf , Brandt Regine , Rempel Eugen , Ploeger Carolin , von Winterfeld Moritz , Schneider Marc , Dietz Steffen , Winter Hauke , Muley Thomas , Sülmann Holger , Meister Michael , Herth Felix , Heussel Claus , Penzel Roland , Endris Volker , Schirmacher Peter , Thomas Michael , Christopoulos Petros , Stenzinger Albrecht 
Published: September 2020

Abstract
BACKGROUND: Recent findings indicate that the currently approved predictive biomarker for immune checkpoint blockade (ICB), i.e. PD-L1 expression, is imperfect. Hence, additional biomarkers are warranted, and ICB-related gene expression profiling (GEP) was advocated as a promising approach enabling comprehensive interrogation of the ICB-effector compartment. Here, we assessed inter- and intra-tumor heterogeneity (ITH) of the immunological microenvironment using a comprehensive gene expression profiling approach (770 genes) to further elucidate the biological basis for new diagnostic applications.
METHODS: All formalin-fixed paraffin-embedded tissue specimens used for this study were derived from surgically resected lung adenocarinomas at the Thoraxklinik at Heidelberg University Hospital and diagnosed (according to 2015 WHO Classification of lung tumors) at the Institute of Pathology at Heidelberg University Hospital. To evaluate the significance of ITH on gene expression profiling, multi-regional gene expression analysis (2-4 samples per tumor) was performed for 24 ADC using the NanoString IO 360 Panel. ANOVA was used to compare inter-tumor variance to intra-tumor variance of mRNA expression. The F statistics was calculated as ratio of these quantities. A list of genes with significantly higher inter-tumor variance was isolated using the F-test. Multiple testing was addressed using the Benjamini-Hochberg method to control the false discovery rate (FDR).
RESULTS: In an unsupervised hierarchical clustering analysis of the entire set of 770 genes, the two to four segments of each of the tumors clustered together. For 752 of the 770 genes (97.7%) the inter-tumor variance was significantly higher than the intra-tumor variance (FDR&lt;5%). For 257 of the 770 genes (33.4%) the inter-tumor variance was more than ten-fold higher than the intra-tumor variance.
CONCLUSION: In our preliminary analysis of ICB-GEP, variance of expression levels between tumors was substantial and exceeded ITH for the majority of analyzed genes. In-depth analyses are ongoing to further delineate the influence on gene signatures and ICB related pathways.
Citation Format: Daniel Kazdal, Jan Budczies, Martina Kirchner, Klaus Kluck, Michael Allgäuer, Olaf Neumann, Regine Brandt, Eugen Rempel, Carolin Ploeger, Moritz von Winterfeld, Marc Schneider, Steffen Dietz, Hauke Winter, Thomas Muley, Holger Sülmann, Michael Meister, Felix Herth, Claus Heussel, Roland Penzel, Volker Endris, Peter Schirmacher, Michael Thomas, Petros Christopoulos, Albrecht Stenzinger. Inter- and intra- tumorheterogeneity of the microenvironment in pulmonary adenocarcinoma [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 1571.