Spatially Resolved, Multi-region Proteomics for Prediction of Immunotherapy Outcome in Deficient Mismatch Repair Metastatic Colorectal Cancer

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.