Large-scale transcriptomic profiling of the tumor immune microenvironment in ALK+ lung cancer.
Authors: Lam Vincent K. , Balan Archana , Zhu Qingfeng , Murray Joseph Christopher , Marrone Kristen A. , Scott Susan Combs , Feliciano Josephine Louella , Hann Christine L. , Ettinger David S. , Smith Kellie Nicole , Forde Patrick M. , Brahmer Julie R. , Levy Benjamin Philip , Elliott Andrew , VanderWalde Ari , Oberley Matthew James , Liu Stephen V. , Ma Patrick C. , Anders Robert A. , Anagnostou Valsamo 
Published: June 2023

9020 Background: Patients (pts) with ALK+ Non-Small Cell Lung Cancer (NSCLC) do not derive significant clinical benefit from immune checkpoint inhibitors. To better understand this lack of immunotherapy sensitivity, we aimed to characterize major immune components of the tumor microenvironment (TME) by comprehensive transcriptomic and immunohistochemistry (IHC) analyses. Methods: We analyzed NGS data from 5490 NSCLC pts that underwent DNA (592 Gene Panel, NextSeq, or WES, NovaSeq) and RNA (NovaSeq, WTS) sequencing at Caris Life Sciences (Phoenix, AZ). 374 ALK-rearranged cases were evaluated, along with 3169 KRAS-mut ( STK11/ KEAP1-wt) and 1947 EGFR-mut cases serving as comparators with known heterogenous and inert immune TMEs, respectively. PD-L1 (22C3) was evaluated by IHC. Immune cell fractions were inferred using quanTIseq. Gene expression profiles were analyzed for a T cell-inflamed signature (TIS) predictive of response to immunotherapy and for other immune modulatory genes such as IFNG, GZMB, TGFB1, and those of the adenosine pathway ( CD73/NT5E, CD39/ENTPD1, ADORA1, ADORA2A/B). A significant difference between genomic subgroups was defined as fold-change > 1.2. In an independent cohort of 14 ALK+ NSCLC pts, density and spatial organization of CD4+ and CD8+ T cells, Tregs, major myeloid lineage cells, PDL1, and CD73 were assessed by quantitative IHC. Results: ALK+ tumors were associated with high PD-L1 (≥50%) expression (40% vs 47% for KRAS-mut vs 18% for EGFR-mut, p < 0.001) and low TMB (median 3 mut/MB vs 9 for KRAS-mut vs 4 for EGFR-mut, p < 0.001). The abundance of CD8+ T cells (fold-change -1.3, p < 0.001), Tregs (fold-change -1.2, p < 0.001), M2 macrophages (fold-change 1.2, p < 0.001), and CD4+ T cells (fold-change 1.9, p < 0.001) differed from KRAS-mut; notably, similar to EGFR-mut. In ALK+ tumors, IFNG (fold-change -1.5, p < 0.001), GZMB (fold-change -1.6, p < 0.001), TGFB (fold-change -1.3, p < 0.001), LAG-3 (fold-change -1.4 p < 0.001), CD73/NT53 (fold-change -1.7 p < 0.001) , and ADORA2A (fold-change -1.4, p < 0.001) were decreased while ADORA1 (fold-change 1.3, p < 0.001) was increased compared to KRAS-mut. EML4-ALK comprised 94.7% of the ALK+ tumors and distribution of EML4-ALK variants was consistent with prior reports (e.g. v1 35.6%, v3 35.1%). Immune cell fractions and immune-related gene expression did not vary significantly between major variant subgroups (v1 vs non-v1, and v3 vs non-v3, p > 0.05). Conclusions: To our knowledge this is the largest transcriptomic analysis of the ALK+ NSCLC TME. Despite high levels of PD-L1, ALK+ tumors exhibit multiple features of an inert immune TME, primarily characterized by low TMB and decreased CD8+ T cells and immune activation markers. Our findings suggest that, while immunosuppressive factors such as M2 macrophages and adenosine signaling may also be targeted, strategies to enhance immunogenicity are critical for an effective immune response in ALK+ NSCLC.