Abstract 2260: Characterizing tumor stromal evolution after neoadjuvant anti-PD-1 therapy in Merkel cell carcinoma
Authors: Sunshine Joel C. , McMiller Tracee L. , Skaist Alyza , Zhang Yan , Berger Alan , Schuebel Kornel , Meyers Jennifer , Deutsch Julie S. , Ogurtsova Aleksandra , Engle Elizabeth L. , Cope Leslie , Taube Janis M. , Topalian Suzanne L. 
Published: April 2023

Abstract
Background: Neoadjuvant anti-PD-1-based therapies have shown promise in several cancers, including Merkel cell carcinoma (MCC), a rare and aggressive skin cancer. We have shown that on-treatment specimens from patients responding to anti-PD-1 are characterized histologically by exuberant proliferative fibrosis, neovascularization, and robust inflammation including diverse immune cell types. Understanding how the stroma participates in the process of tumor regression may open therapeutic opportunities by redirecting stromal remodeling in treatment-refractory tumors.
Methods: To evaluate stromal changes in neoadjuvant treated MCC, 13 on-treatment FFPE tumor specimens from patients with resectable MCC who received anti-PD-1 for ~4 weeks before surgery (NCT02488759) were subjected to laser capture microdissection to collect stromal tissue and isolate RNA. We conducted multiplex qRT-PCR targeting 125 unique mRNAs representing TME-relevant immune cell subsets, immune checkpoint pathways, and immune-modulating transcription factors and cytokines, followed by whole transcriptome RNA sequencing. Differential gene expression was considered significant if the magnitude of the signed fold-change was ≥2 and the p-value was ≤0.1 (Benjamini-Hochberg adjusted for RNAseq). Gene set enrichment analysis (GSEA) was performed with R Topper. Pathologic response was scored on H&E staining as pathologic complete response (pCR, n=6), major pathologic response (MPR, ≤10% residual viable tumor [RVT]; n=4), or non-MPR/pCR (&gt;10% RVT; n=3).
Results: Stratifying by response status (6 pCR vs 3 non-MPR/pCR) demonstrated that pCR stroma in MCC is distinguished by upregulation of molecules associated with neovascularization (VEGFA, EGR2) and M1 and M2 macrophages (IL1A, ARG1) via qRT-PCR, and mesenchymal cells (fibroblasts, myocytes, adipocytes) and extracellular matrix formation via RNAseq. GSEA demonstrated a strong association of pCR with both myocyte and fibroblast differentiation and functional pathways, suggesting a role for myofibroblasts in tissue remodeling during anti-PD-1-mediated tumor regression. In contrast, the stroma of non-MPR/pCR tumors showed evidence for ongoing IFNg-mediated inflammation, as evidenced by upregulation of IFNg, IL-27, STAT1, CXCL10 and LAG-3 via qRT-PCR; these findings were supported by RNAseq, which also showed upregulation of genes associated with inflammation (e.g., PLXNC1, TRPV2, LYZ, LAIR1, HLA-A, B2M, CD4). Similar results were obtained when MPRs were grouped with pCRs (n=10) vs non-MPR/pCR (n=3).
Conclusion: These findings demonstrate that improved response to anti-PD-1 therapy is associated with a shift in the tumor stroma from an inflammatory to a proliferative myofibroblastic phenotype, recapitulating the stages of normal wound healing. This suggests a potential functional role for myofibroblasts in that critical transition.
Citation Format: Joel C. Sunshine, Tracee L. McMiller, Alyza Skaist, Yan Zhang, Alan Berger, Kornel Schuebel, Jennifer Meyers, Julie S. Deutsch, Aleksandra Ogurtsova, Elizabeth L. Engle, Leslie Cope, Janis M. Taube, Suzanne L. Topalian. Characterizing tumor stromal evolution after neoadjuvant anti-PD-1 therapy in Merkel cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2260.