In the setting of localized colon cancer (CC), circulating tumor DNA (ctDNA) monitoring in plasma has shown potential for detecting minimal residual disease (MRD) and predicting a higher risk of recurrence. With the tumor-only sequencing approach, however, germline variants may be misidentified as somatic variations, precluding the possibility of tracking in up to 11% of patients due to a lack of known somatic mutations. In this study, we assess the potential value of adding white blood cells (WBCs) to tumor tissue sequencing to enhance the accuracy of sequencing results. A total of 148 patients diagnosed with localized CC were prospectively recruited at the Hospital Clínico Universitario in Valencia (Spain). Employing a custom 29-gene panel, sequencing was conducted on tumor tissue, plasma and corresponding WBCs. Droplet digital PCR and amplicon-based NGS were performed on plasma samples post-surgery to track MRD. Oncogenic somatic variants were identified by annotating with COSMIC, OncoKB and an internal repository of pathogenic mutations database. A variant prioritization analysis, mainly characterized by the match of oncogenic mutations with the evidence levels defined in OncoKB, was carried out to select specific targeted therapies. Utilizing paired tumor and WBCs sequencing, we identified somatic mutations in all patients (100%) within our cohort, compared to 89% using only tumor tissue. Consequently, the top 10 most frequently mutated genes for plasma monitoring were altered. The sequencing of WBCs identified 9% of patients with pathogenic mutations in the germline, with APC and TP53 being the most frequently mutated genes. Additionally, mutations in genes related to clonal hematopoiesis of indeterminate potential were detected in 27% of the cohort, with TP53, KRAS, and KMT2C being the most frequently altered genes. There were no observed differences in the sensitivity of monitoring MRD using ddPCR or amplicon-based NGS (p = 1). Ultimately, 41% of the patients harbored potentially targetable alterations at diagnosis. The germline testing method not only enhanced sequencing results and raised the proportion of patients eligible for plasma monitoring, but also uncovered the existence of pathogenic germline variations, thereby aiding in the identification of patients at a higher risk of hereditary cancer syndromes.

Background/Objectives: MSS-CRC comprises a heterogeneous group of tumors generally considered “immune cold” due to limited neoantigen generation and T-cell exclusion or inactivation. Current evidence indicates that the composition of T and B immune cells within the tumor microenvironment represents a prognostically relevant factor, significantly associated with both tumor expression profiles and molecular subtypes. Methods: We conducted an exploratory analysis to identify prognostically relevant immune cell components in this group of tumors and to investigate corresponding differences in RNA-based bulk expression and high-resolution spatial transcriptomic profiles. Results: A total of 254 cases of localized mismatch repair-proficient colorectal cancer cases were evaluated. Our findings revealed PD-L1 expression as a robust, independent prognostic biomarker associated with favorable outcomes in this specific population. Bulk RNA expression analysis showed that PD-L1–negative tumors exhibited an expression profile consistent with abundant cancer-associated fibroblast infiltration, increased matrix stiffness, and impaired immune activation—features aligned with tumor progression and poorer clinical outcomes. In contrast, PD-L1–positive tumors displayed stromal programs enriched in immune activation and controlled remodeling, consistent with an immunologically active microenvironment. Spatial transcriptomics added an additional layer of evidence, revealing that epithelial-to-mesenchymal transition–related programs can dominate stromal niches in PD-L1–negative tumors, particularly within macrophage-enriched stromal regions. Conclusions: Our observations suggest a crosstalk link between PD-L1 expression on immune cells and immune-activated vs mesenchymal-dominant states driven within tumor-associated macrophage-enriched stromal niches. These results provide insight into the biological mechanisms underlying disease progression and highlight tumor-associated macrophages as a potential therapeutic target to overcome immune resistance particularly in PD-L1–negative MSS-CRC tumors.

Background/Objectives: MSS-CRC comprises a heterogeneous group of tumors generally considered “immune cold” due to limited neoantigen generation and T-cell exclusion or inactivation. Current evidence indicates that the composition of T and B immune cells within the tumor microenvironment represents a prognostically relevant factor, significantly associated with both tumor expression profiles and molecular subtypes. Methods: We conducted an exploratory analysis to identify prognostically relevant immune cell components in this group of tumors and to investigate corresponding differences in RNA-based bulk expression and high-resolution spatial transcriptomic profiles. Results: A total of 254 localized mismatch repair-proficient colorectal cancer cases were evaluated. Our findings revealed PD-L1 expression as a robust independent prognostic biomarker associated with favorable outcomes in this specific population. Bulk RNA expression analysis showed that PD-L1-negative tumors exhibited an expression profile consistent with abundant cancer-associated fibroblast infiltration, increased matrix stiffness, and impaired immune activation—features consistent with tumor progression and poorer clinical outcomes. In contrast, PD-L1-positive tumors displayed stromal programs enriched in immune activation and controlled remodeling, consistent with an immunologically active microenvironment. Spatial transcriptomics added an additional layer of evidence, revealing that epithelial to mesenchymal transition-related programs can dominate stromal niches in PD-L1-negative tumors, particularly within macrophage-enriched stromal regions. Conclusions: Our observations suggest an association between PD-L1 expression on immune cells and immune-activated versus mesenchymal-dominant states, potentially occurring within macrophage-enriched stromal niches. These results provide insight into the biological mechanisms underlying disease progression and highlight tumor-associated macrophages as a potential therapeutic target to overcome immune resistance, particularly in PD-L1-negative MSS-CRC tumors.