Abstract 6555: scRNAseq and TCR repertoire analysis identifies immune correlates of response to combined BRAF/MEK/PD1 inhibition in a phase 2 trial
Authors: Yao Hongcheng , Jorgji Vjola , Tang Alexander L. , Lieb David , Chao Sherry X. , Stein Daniel J. , Spurrell Maxwell , Elmelech Liad , Tian Jun , Sade-Feldman Moshe , Pelka Karin , Corcoran Ryan B. , Hacohen Nir , Korsunsky Ilya , Chen Jonathan 
Published: March 2024

Abstract
Background: Our recent clinical trial of combined BRAF, MEK, and PD-1 inhibition showed a 24% response rate in BRAF V600E mutant colon cancer (PMID: 36702949). Analysis of epithelial single-cell RNA sequencing data showed an increase in interferon-stimulated genes (ISG) and inhibition of the MAPK pathway in responders. MAPK inhibition in organoids also induced ISGs. These data suggest a synergy between MAPK inhibition and induction of immune responses.
Methods: We analyzed over 100,000 immune and stromal cells from 23 paired scRNAseq profiles of pre- and day 15 on-treatment tumor biopsies. Progression free survival greater than 6 months was defined as a clinical response. We performed traditional differentially expressed gene and gene program analyses, and tracked TCR repertoire in tumor and blood. We developed a novel approach based on supervised clustering (PMID: 34675423) to identify fine-grained cell subtypes associated with response. We applied supervised clustering separately to T and myeloid cells, and tested the association of each cluster’s abundance with response.
Results: We applied supervised clustering to T cells and identified a CD74+IFNG+ CD8 T cell cluster that increased in only responders (p=0.0078). In the myeloid compartment, we identified two populations of CXCL10+ macrophages: one whose abundance increased in all patients and a CCL3+CCL4+ activation state expanded (p=0.01) only in responders. Pathway analysis of the expanded populations suggests an enrichment of ISGs in both CD74+IFNG+ CD8 T cells and CXCL10+CCL3+CCL4+ macrophages, with specific enrichment of interferon-gamma responsive genes GBP1 and GBP4 in the macrophages.
T cell analysis yielded two responders with marked clonal expansion within the CXCL13+ population, which likely represent tumor-specific T cells. Within the blood, these CXCL13+ clonotypes underwent a short-lived burst at day 15, yet represented only a minor population. We saw concomitant expansion of both CXCL13+ CD4 and CD8 populations within patients, suggesting CXCL13+ CD4s might provide help to the CXCL13+ CD8 T cells. The same two responders had increased stem-like TCF7+PD1+ CD8 T cells previously implicated in immunotherapy response and an increase in CCL19+ fibroblasts associated with stem-immunity hubs.
Conclusions: Our analysis of pre- and on-therapy biopsy scRNAseq data from our clinical trial of MAPK and PD-1 inhibition found expansion of ISG enriched CD74+IFNG+ CD8 T cells and CXCL10+CCL3+CCL4+ macrophages in responders. Both populations are enriched in ISGs: this coordinated expansion may result from a shared, interferon-rich microenvironment. Ongoing work will explore this hypothesis using spatial transcriptomics to identify these activated cells and their microenvironments.
Ethics approval
"This study was approved by the DF/HCC IRB as protocol 18-144 (see also ClinicalTrials.gov NCT03668431).
Citation Format: Hongcheng Yao, Vjola Jorgji, Alexander L. Tang, David Lieb, Sherry X. Chao, Daniel J. Stein, Maxwell Spurrell, Liad Elmelech, Jun Tian, Moshe Sade-Feldman, Karin Pelka, Ryan B. Corcoran, Nir Hacohen, Ilya Korsunsky, Jonathan Chen. scRNAseq and TCR repertoire analysis identifies immune correlates of response to combined BRAF/MEK/PD1 inhibition in a phase 2 trial [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6555.