Letter to the Editor: Reovirus as an immunomodulatory adjuvant in KRAS-mutant colorectal cancer - translational insights and future directions

Table 1 Proposed testable hypotheses and designs to overcome peripheral blood limitations

Core hypothesis (key question)	Proposed design (main steps)	Expected outcomes
Does peripheral blood IFN-γ elevation correspond to enhanced infiltration of KRAS-mutant-specific CD8+ T cells in the TME, thereby reversing the immunologically cold state in MSS-type CRC?	Multicenter cohort (n > 50, KRAS-mutant mCRC); Matched peripheral blood + tumor biopsies (baseline, days 8/15); scRNA-seq + TCR-seq (IFN-γ gene signatures vs KRAS-specific clones); Flow cytometry to detect CD8+PD-1+ cells	Correlation coefficient > 0.7 (peripheral IFN-γ vs TME T-cell metrics), confirming immune hot conversion
Does reovirus (pelareorep) regulate the PI3K/AKT pathway (phosphatase and tensin homolog pseudogene 1 increase, rapamycin-insensitive companion of mammalian target of rapamycin decrease) to suppress VEGF expression in the TME, leading to vascular normalization and increased immune cell recruitment?	KRAS-mutant PDX models + clinical biopsies; multiplex immunofluorescence (co-localization of VEGF/PI3K with CD31+ vessels); scRNA-seq (endothelial subpopulation changes and immune scores)	> 30% reduction in VEGF expression post-treatment, accompanied by increased TME immune scores and vascular normalization
Biomarker-driven phase II trial to validate TME immune conversion and efficacy in KRAS-mutant MSS mCRC	Multicenter Simon two-stage phase II trial (n = 40-60); patient population: KRAS-mutant (prioritizing G12/G13 subtypes), MSS mCRC, after second-line failure, Eastern Cooperative Oncology Group 0-1; stratification: Baseline TME signature (high/Low VEGF; low/high IFN-γ); intervention: Pelareorep (3 × 1010 tissue culture infectious dose 50/day × 5 days, every 28 days) + folinic acid, fluorouracil, and irinotecan + bevacizumab ± PD-1 inhibitor; primary endpoint: TME immune conversion rate (> 50% shift to “hot” phenotype, assessed by scRNA-seq); biomarkers: Mandatory biopsies + spatial transcriptomics (e.g., GeoMx) to monitor PI3K/AKT heterogeneity and IFN-γ signatures; statistics: Kaplan-Meier survival analysis + Wilcoxon paired test (80% power, α = 0.05)	Confirmation of TME immune hot conversion, ORR ≥ 35% (vs historical approximately 15%), improved progression-free survival, supporting advancement to phase III

IFN: Interferon-gamma; TME: Tumor microenvironment; MSS: Microsatellite stable; CRC: Colorectal cancer; mCRC: Metastatic colorectal cancer; scRNA-seq: Single-cell RNA sequencing; TCR-seq: T-cell receptor sequencing; PD-1: Programmed cell death protein 1; PI3K/AKT: Phosphatidylinositol 3-kinase/protein kinase B; VEGF: Vascular endothelial growth factor; PDX: Patient-derived xenograft; ORR: Objective response rate.