Abstract 664: Integrated scRNAseq and spatial transcriptomic analyses identify immune correlates of response to combined BRAF/MEK/PD-1 inhibition in metastatic colorectal cancer

Abstract Background: Our clinical trial of combined BRAF, MEK, and PD-1 inhibition showed a 24% response rate in BRAF V600E mutant colorectal cancer (CRC) (PMID: 36702949). However, the mechanism underlying this response is unknown. Methods: We analyzed 327,000 immune and stromal cells from 23 paired scRNAseq profiles of pre- and day 15 on-treatment tumor biopsies. Progression-free survival of 6 months or greater was defined as a clinical response. We performed hierarchical clustering on the scRNAseq data and tracked the TCR repertoire in tumor and blood. Spatial transcriptomic mRNA microscopy (Xenium) was run on 16 biopsies and single cell cluster assignments were mapped onto the microscopy image. Results: Analysis of scRNAseq data revealed that responders had significant increases in CXCL10+ macrophages and MHCII+ and HOBIT+ CD8 T cells. Prior analysis of the tumor epithelial cells showed that responders had inhibition of the MAPK pathway (consistent with BRAF/MEK blockade) and increased interferon-stimulated gene (ISG) scores. In contrast, the combination therapy induced more modest shifts in MAPK gene expression scores in immune cells. ISG scores increased across immune cells. Integration of scRNAseq T cell clusters with TCR clones found a greater influx of new clones (as a fraction of all clones in the activated CD8 T cell clusters) on-treatment in mismatch repair proficient (MMRp) responders versus non-responders (p=0.02), with higher normalized counts per new clone in responders versus non-responders (p=0.03). Within the blood, TCRs from Tregs and activated CD8 T cells (identified based on scRNAseq signatures in tumor-infiltrating lymphocytes) displayed a burst at two weeks on-treatment but this clonotypic expansion diminished over the next couple of weeks. Using spatial transcriptomic to localize the populations identified by scRNAseq, we found that SPP1+ macrophages and Tregs were diffusely distributed in tumor areas whereas CXCL10+ macrophages and CD8 T cells formed clusters near the tumor-stromal interface, consistent with tumor immunity hubs. Conclusions: Altogether, our work suggests that MAPK inhibition in combination with PD1 blockade can induce anti-tumor immune T cell responses, even in MMRp CRC. However, our T cell clonal analysis and subsequent patient cancer progression indicate this response is not sustained. Future work will focus on developing ways to increase the durability of this response, possibly by therapeutically targeting immunity hubs. Ethics approval: This study was approved by the DF/HCC IRB as protocol 18-144 (see ClinicalTrials.gov NCT03668431). Citation Format: Hongcheng Yao, Alexander Tang, Ryan Corcoran, Nir Hacohen, Ilya Korsunsky, Jonathan H. Chen. Integrated scRNAseq and spatial transcriptomic analyses identify immune correlates of response to combined BRAF/MEK/PD-1 inhibition in metastatic colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 664.