Comprehensive characterization of neurotransmitters and neuronal signaling (NT) pathway alterations in colorectal cancer (CRC).
Authors: Battaglin Francesca , Xiu Joanne , Zeng Jia , Baca Yasmine , Jayachandran Priya , Kawanishi Natsuko , Arai Hiroyuki , Deshpande Krutika , Goldberg Richard M. , Lockhart A. Craig , Hwang Jimmy J. , Seeber Andreas , Zhang Wu , Mumenthaler Shannon M. , Shields Anthony Frank , Marshall John , Korn Wolfgang Michael , Neman Josh , Lenz Heinz-Josef 
Published: June 2021

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Background: Aberrant NT signaling has been shown to activate uncontrolled proliferation and dissemination in several gastrointestinal cancer types. Neurotransmitters have been shown to affect endothelial cells and immune cells in the tumor microenvironment to promote tumor progression. We previously showed that single nucleotide polymorphisms in the dopamine and GABA pathways are associated with outcome in patients with metastatic CRC receiving first-line treatment. Here we further evaluated the distribution and molecular context of NT pathway alterations in CRC. Methods: A total of 7,595 CRC tumors tested at Caris Life Sciences (Phoenix, AZ) with NextGen Sequencing on DNA (Next Seq, 592 genes or NovaSeq, WES) and RNA (NovaSeq, WTS) were analyzed. ssGSEA (single-sample gene set enrichment analysis) was used to calculate pathway enrichment scores (ES) of 7 NT gene sets (GABA, nicotinic, muscarinic, dopamine (DA), reelin, glial cell line-derived neurotrophic factor and neurotrophins). X2/Fisher-Exact was used for comparison and significance was determined as p-value adjusted for multiple comparison of ( q) < 0.05. Results: ES based on sample sites showed a substantial heterogeneity in NT enrichment. Notably, when compared to primary tumors, all 7 gene sets were significantly enriched in brain metastases (mets; ES ratio 1.14-1.55), while abdomen, liver, and peritoneal mets displayed significant decreases in most NT gene sets. DA was enriched in ovarian and lung mets (ES ratio: 1.18 and 1.09, respectively), the latter also showing increased neurotrophins ES (1.06) (all q < 0.05). When investigating primary tumors grouped according to overall ES by unsupervised clustering, right-sided and CMS4 CRCs were more prevalent in the high ES cluster compared to the low ES cluster (32 vs 29%, P = 0.02 and 46 vs 30%, P < 0.001, respectively). In addition, tumors in the high ES cluster showed lower prevalence of TMB-H (≥ 10mt/MB) (7 vs 10%), MSI-H (6 vs 10%) and PD-L1 (2 vs 6%), while higher CNA rates were noted in 9 genes (all q < 0.05). High ES tumors showed significant positive associations with microenvironment infiltration of B cells, T cells (NK, CD4+ and CD8+ T cells, but not Treg), M2 Macrophages, Myeloid Dendritic Cell, Neutrophils, and an inverse association with M1 Macrophages, regardless of MSI status ( q < 0.05). Conclusions: This is the first and most extensive molecular profiling study to investigate NT signaling pathway alterations in CRC. Our data show a distinct distribution of pathway enrichment according to metastatic site, distinct molecular features in high vs low ES clusters in primary tumors (including CMS subtypes, TMB, MSI and PD-L1 rates), and differential immune cell infiltration. These findings support the role of NT signaling in the metastatic spread of CRC and modulation of tumor immune microenvironment.