Converting cold to hot: Strategies to sensitize microsatellite-stable colorectal cancer to immunotherapy

Table 3 Candidate biomarkers for patient selection and monitoring in microsatellite-stable/proficient mismatch repair colorectal cancer

Biomarker	Assay/readout	Rationale in MSS CRC	Clinical application and limitations	Ref.
MSIH/dMMR	IHC (MLH1/MSH2/MSH6/PMS2), PCR, NGS	High neoantigen load and inflamed TME	Established selection for PD-1 (± CTLA-4); rare in metastatic CRC	Gandini et al[130]; Ambrosini et al[131]
TMB-high; POLE/POLD1 ultra-mutation	NGS panel; TMB cutoffs	May confer immunogenicity despite MSS	Identifies small ICI-sensitive subset; platform/cutoff heterogeneity	Xue et al[132]; Domingo et al[135]
PD-L1 expression	IHC (TPS/CPS; immune cell staining)	Surrogate of immune activation, but weak predictor in CRC	Limited standalone utility; may contribute in combination contexts	Nakamura et al[137]
Immunoscore/TIL density (CD3/CD8)	Standardized IHC quantification (center + invasive margin)	Captures pre-existing antitumor immunity; enriches for ICI-responsive biology	Potential for patient selection (e.g., POCHI); requires harmonization and cutoffs	Yamaguchi et al[60]; Esmail et al[134]
Inflamed vs excluded gene signatures (IFN-γ; TGF-β/stromal)	RNA-seq/nano string signatures	Defines immune-hot vs immune-excluded states and matches mechanisms	Trial stratification; resource-intensive; signatures not yet standardized	Mortezaee and Majidpoor[27]; Li et al[28]; Esmail et al[134]
Liver metastasis status	Imaging; metastatic pattern	Liver promotes systemic immune tolerance and attenuates ICI benefit	Negative predictor in multiple ICI + TKI datasets; guides patient selection/Locoregional consolidation	Fakih et al[66]; Fakih et al[70]; Taïeb et al[138]
ctDNA kinetics	Plasma NGS (VAF dynamics; MRD)	Early molecular response/resistance signal	Monitoring and adaptive strategies; assay and threshold variability	Hou et al[140]
Host inflammatory/nutrition indices	NLR, albumin, ECOG	Correlates with myeloid skewing and immune fitness	Adjunct prognostic/predictive markers (not standalone)	Zhang et al[68]
Gut microbiome features	Metagenomics; targeted qPCR (e.g., Fusobacterium)	Microbiota shapes systemic immunity and therapy response/toxicity	Exploratory; actionable interventions (diet/FMT) under evaluation	Luo et al[33]; Luu et al[34]; Ramachandran et al[139]

MSI-H: Microsatellite instability-high; dMMR: Deficient mismatch repair; TMB: Tumor mutational burden; PD-L1: Programmed death-ligand 1; TIL: Tumor-infiltrating lymphocyte; CD: Cluster of differentiation; IFN: Interferon; TGF-β: Transforming growth factor-β; ctDNA: Circulating tumor DNA; IHC: Immunohistochemistry; NGS: Next-generation sequencing; PCR: Polymerase chain reaction; qPCR: Quantitative polymerase chain reaction; CPS: Combined positive score; TPS: Tumor proportion score; RNA-seq: RNA-sequencing; MRD: Minimal residual disease; VAF: Variant allele frequency; NLR: Neutrophil-to-lymphocyte ratio; ECOG: Eastern Cooperative Oncology Group; TME: Tumor microenvironment; MSS: Microsatellite-stable; CRC: Colorectal cancer; ICI: Immune checkpoint inhibitor; PD-1: Programmed cell death protein 1; CTLA-4: Cytotoxic T-lymphocyte-associated protein-4; TKI: Tyrosine kinase inhibitor; FMT: Fecal microbiota transplantation.