The role of Comprehensive Genomic Profiling in the molecular characterization of colorectal carcinoma

In recent years, the development and clinical implementation of targeted therapies and immunotherapies have significantly changed the therapeutic approach to metastatic colorectal carcinoma (mCRC), leading to substantial improvements in patient outcomes. In this context, molecular characterization plays a central role in guiding treatment decisions and enabling a precision medicine approach. Currently, the main predictive biomarkers used in clinical practice include activating mutations in the RAS and BRAF genes, NTRK gene rearrangements, and HER2 amplification for targeted therapies, while mismatch repair (MMR) status and microsatellite instability (MSI) are essential for immunotherapy selection. However, current diagnostic algorithms, mainly based on small-to-medium NGS panels or single-gene assays, may not fully capture the molecular complexity of mCRC, particularly in advanced disease. In this setting, comprehensive genomic profiling (CGP), based on the simultaneous analysis of hundreds of genes using large NGS panels, represents a promising tool for achieving a broader and more in-depth molecular characterization, with potential diagnostic, prognostic, and therapeutic implications. Nevertheless, the actual clinical utility of CGP in routine mCRC management remains to be fully established. The aim of this study was to evaluate the role of CGP in the molecular characterization of mCRC compared with standard first-level testing, with a focus on identifying additional potentially targetable alterations in both RAS/BRAF wild-type and mutated tumors. Furthermore, the study aimed to identify genomic alterations associated with resistance to therapies, particularly anti-EGFR agents, and to assess their impact on clinical outcomes, including progression-free survival (PFS) and overall survival (OS). Finally, the utility of CGP in evaluating predictive biomarkers of response to immunotherapy was investigated, with particular attention to MSI/MMR status, tumor mutational burden (TMB), and POLE/POLD1 mutations. A retrospective, single-center cohort of 138 patients with mCRC was analyzed. Among these, 77 cases (55.8%) were RAS/BRAF wild-type, while 61 cases (44.2%) harbored mutations in these genes. In the RAS/BRAF wild-type group, CGP identified pathogenic variants in all cases and at least one potentially targetable alteration in 30% of patients, mainly involving the PIK3CA gene, as well as BRAF, MDM2, and PTEN. Similarly, in RAS/BRAF-mutated tumors, additional potentially targetable alterations were identified in 31% of cases, with a higher prevalence in PIK3CA and PTEN, highlighting the ability of CGP to expand therapeutic options even in this subgroup. Regarding immunotherapy, CGP showed high concordance with standard methods in the assessment of MSI/MMR status while providing additional clinically relevant information, such as TMB and mutations in MMR genes. This integrated approach allowed the identification of additional patients potentially eligible for immunotherapy and facilitated the interpretation of discordant cases across different methodologies. Survival analyses in RAS/BRAF wild-type patients treated with anti-EGFR antibodies demonstrated that the presence of resistance-associated genomic alterations was significantly associated with shorter progression-free survival (PFS), whereas no significant differences in overall survival (OS) were observed. This association was also confirmed in the subgroup of left-sided colon and rectal tumors. In RAS/BRAF-mutated patients treated with systemic chemotherapy combined with bevacizumab, the presence of resistance-associated alterations was associated with improved PFS in patients receiving triplet chemotherapy compared with doublet regimens, whereas in patients without such alterations, doublet chemotherapy showed comparable or more favorable outcomes. Overall, the results of this study indicate that a more comprehensive molecular characterization through CGP may improve the clinical management of patients with mCRC. In particular, CGP enables the identification of a substantial proportion of additional targetable alterations, refines patient selection for targeted therapies, and enhances the understanding of resistance mechanisms. Moreover, the ability of CGP to assess, in addition to MSI status, other predictive biomarkers of response to immunotherapy, such as TMB and POLE/POLD1 mutations, may contribute to increasing the identification of patients eligible for immune checkpoint inhibitor treatment. Although limited by the retrospective design and the relatively small sample size, these findings support the potential role of CGP in clinical practice and highlight the need for further studies to evaluate its impact on clinical outcomes and cost-effectiveness.