Spatially resolved, multi-region proteomics for prediction of immunotherapy outcome in deficient mismatch repair metastatic colorectal cancer

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 (CRC). Experimental Design. Primary CRCs with deficient mismatch repair (d-MMR) 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 (PanCK; epithelia), CD45 (stromal cells) and SYTO13 (nuclei). In an independent cohort (n=13), DSP data and single cell transcriptomic data were analyzed. Differential protein abundance, after Benjamini-Hochberg correction, was examined by response and progression-free survival (PFS) using multivariable Cox regression. Results. Protein abundance varied significantly between epithelial and stromal compartments. Nonresponders (NR) to anti-PD-1 showed higher fibronectin and smooth muscle actin (SMA) abundance in the epithelial compartment that were associated with significantly shorter PFS (HRadj: 6.49 and 4.52, respectively; p < 0.05). In CD45+ stroma, increased expression of proteins related to T-cells (CD3, CD4), NK cells (CD56), antigen presentation (CD40), immune activation (CD27, ICOS), and apoptosis (GZMA) were found in responders (vs NR) to anti-PD-1; each marker was significantly associated with longer patient PFS (0.02 <HRadj < 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 SMA were primarily derived from cancer-associated fibroblasts. Conclusions. Spatially resolved protein profiles within microenvironments of d-MMR CRCs can influence patient response and survival after anti-PD-1 highlighting their potential clinical significance.