Predictive biomarker discovery for immune checkpoint inhibitor in metastatic MMRd endometrial cancer through deep proteomic profiling of FFPE tissue samples

Mismtach repair deficiency (MMRd) status is a robust predictive biomarker for immune checkpoint inhibitor (ICI) in endometrial cancer (EC). Nevertheless half of the selected patients do not respond to the therapy. Most studies exploring biomarkers of response to ICI have focused on tumor or immune cell factors. In the present study, we aimed to identify novel response biomarker by exploring the proteomic profiles of baseline patient FFPE samples with known treatment outcome.

Clinical data and outcomes of metastatic MMRd EC patients, treated with ICI at Gustave Roussy Institute (2016-2021), were retrospectively collected. Patients were classified as responders/Rs (complete response/CR, partial response/PR, or stable disease/SD ≥12 months) or non-responders/NRs (progressive disease/PD or SD <12 months). 25 Pre-ICI treatment FFPE tumor samples (17 Rs and 8 NRs) were subjected to Biognosys UltraDeep TrueDiscovery™ data-independent acquisition (DIA)-Mass Spectrometry (MS) analysis and resulted data were analyzed using Spectronaut software.

In total, 11"187 proteins were quantified in all samples with an average of 10101 proteins quantified per sample. Further in-depth analysis of the data unveiled that extracellular matrix modeling is one of the major pathways driving the separation of Rs and NRs. For instance, multiple proteins from the collagen family are significantly upregulated in the NRs.

To further validate this finding, several orthogonal technologies were employed to investigate the relationship between collagen and response to ICI: 1) Collagen density was determined on whole FFPE slides using Masson"s trichrome and quantified by Qupath as % positive surface in the intratumoral area. 2) Second-harmonic generation (SHG) microscopy combined with polarimetry (p-SHG) was used to estimate collagen content and describe the spatial orientation of fibrillar collagen in the intratumoral area. 3) intraepithelial (ie) and stromal (s) CD3+ and CD8+ cells were quantified by Qupath.

The spatial imaging results indicate that collagen density in the intratumoral area was significantly higher in NRs compared to Rs. Intratumoral collagen density was positively correlated with collagen type V expression, and negatively correlated with ie and s CD3+ cell and ie and s CD8+ cell infiltration. SHG confirmed that fibrillar collagen content within the tumor area was significantly greater in NRs and p-SHG revealed that NR tumors tended to show a highly disorganized collagen matrix.

Taken together, MS-based discovery proteomics was instrumental in identifying ECM as one of the drivers of treatment response. Two different spatial approaches consistently demonstrated that increased collagen expression or density may serve as a potential predictive biomarker of resistance to ICI in MMRd metastatic EC, contributing to create an immunosuppressive tumor microenvironment (TME).