Sherman, Will A.

Abstract Purpose: Digital proteomic profiling was performed to identify spatial context in relationship to patient response and survival after anti–PD-1 therapy in metastatic colorectal cancer. Experimental Design: Primary colorectal cancers with deficient mismatch repair from patients treated with anti–PD-1 antibodies were analyzed (N = 30) using digital spatial profiling (GeoMx nCounter). At the invasive margin, 71 proteins were profiled in 10 regions of interest/slide that were segmented into 3 compartments labeled with pan-cytokeratin (epithelia), CD45 (stromal cells), and SYTO13 (nuclei). In an independent cohort (n = 13), digital spatial profiling data and single-cell transcriptomic data were analyzed. Differential protein abundance, after Benjamini–Hochberg correction, was examined by clinical response and progression-free survival (PFS) using multivariable Cox regression. Results: Protein abundance varied significantly between epithelial and stromal compartments. Nonresponders to anti–PD-1 showed higher fibronectin and smooth muscle actin abundance in the epithelial compartment that was associated with significantly shorter PFS (adjusted HR: 6.49 and 4.52, respectively; P < 0.05). In CD45+ stroma, increased expression of proteins related to T cells (CD3 and CD4), NK cells (CD56), antigen presentation (CD40), immune activation (CD27, ICOS), and apoptosis (GZMA) were found in responders (vs nonresponders) to anti–PD-1; each marker was significantly associated with longer patient PFS (0.02 < adjusted HR < 0.17; P < 0.05). In a separate cohort, consistent results by compartment were found for fibronectin and CD56. Gene expression data revealed that fibronectin and smooth muscle actin were primarily derived from cancer-associated fibroblasts. Conclusions: Spatially resolved protein profiles within microenvironments of deficient mismatch repair colorectal cancers can influence patient response and survival after anti–PD-1, highlighting their potential clinical significance.

3520 Background: In patients with metastatic colorectal cancer (CRC) and deficient DNA mismatch repair (dMMR), we previously found transcriptomic signatures reflecting stromal compartment composition and proliferative capacity that can predict the efficacy of immune checkpoint blockade (ICB). To identify proteomic markers of response and/or resistance to ICB, we performed spatially resolved proteomic analysis of the tumor microenvironment. Methods: Patients with surgically resected, primary dMMR metastatic CRCs received pembrolizumab (Mayo Clinic, N=30). In FFPE tissues, Digital Spatial Profiling of 71 proteins was performed using panels covering immune cells, immune activation, cell death, PI3/AKT signaling, MAPK signaling, and pan-tumor markers (GeoMx nCounter). In ten regions of interest/slide (~600 µm2) at the tumor invasive margin, protein expression was determined in four cellular compartments: epithelium (panCK+), immune cells (CD45+; leucocyte common antigen), stroma (panCK-), and nonimmune stroma (panCK-/CD45-). After normalization, protein abundance per compartment was analyzed by response (RECIST 1.1) [OmicVerse Python]. Differentially expressed proteins (binary) were examined by progression-free survival (PFS) using multivariable Cox regression with covariate adjustment (Lifelines Python). Results: Pre-treatment panels including 71 proteins were analyzed by treatment response and PFS after PD-1 inhibition. Proteins found to be significantly associated with response (p< 0.01) and PFS are described. Across all compartments, responders showed upregulation of NK cells and T cell subsets (CD3+, CD4+, CD8+), and ICOS; strongest association was found for CD4+: HRadj: 0.16; 95%CI: 0.08-0.31; p< 0.001. Nonresponders showed higher phospho-JNK (HRadj: 2.09; 95%CI: 1.28-3.42; p=0.003) and SMA. In the epithelial compartment, upregulation of the Treg marker CD25 and reduced caspase-9 were observed in responders. Among responders, the stromal compartment was highly enriched in HLA-DR, dendritic cells (CD11c), PD-L1, immune activation markers (CD40, CD44, CD80, CD27), CD95/Fas, and GZMB. In the stromal compartment, downregulation of BCL6 and phospho-GSK3B was observed in responders. The immune compartment of responders showed increased beta2-microglobulin and Ki-67, while the non-immune stromal compartment showed increase in the macrophage marker CD68. Importantly, proteins found to be upregulated in responders were significantly and independently associated with longer PFS whereas the opposite was true for nonresponders. Conclusions: In metastatic dMMR CRCs, spatially defined protein markers were significantly associated with anti-PD-1 response, and independently associated with patient PFS (strongest predictive utility for CD4 in immune stroma and phospho-JNK in epithelium).