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Elucidating the Role of KRAS, NRAS, and BRAF Mutations and Microsatellite Instability in Colorectal Cancer via Next-Generation Sequencing
by
Marta Rada Rodríguez
1,
Bárbara Angulo Biedma
2,
Irene Rodríguez Pérez
3 and
Javier Azúa Romeo
4,* 
1
Department of Human Anatomy and Histology, Universidad de Zaragoza, Calle Pedro Cerbuna, 50009 Zaragoza, Spain
2
Area of Molecular Biology, Analiza, 28008 Madrid, Spain
3
Area of Pathology, Analiza, 28008 Madrid, Spain
4
Chief of Pathology, Analiza, 28008 Madrid, Spain
*
Author to whom correspondence should be addressed.
Cancers 2025, 17(13), 2071; https://doi.org/10.3390/cancers17132071
Submission received: 20 May 2025
/
Revised: 11 June 2025
/
Accepted: 16 June 2025
/
Published: 20 June 2025
(This article belongs to the Special Issue Significance of KRAS Gene Mutations in Colorectal Cancer (2nd Edition))
Simple Summary
Background: Next-generation sequencing (NGS) in sporadic colorectal adenocarcinoma (CRC) represents an ambitious diagnostic approach that may pose considerable challenges. There are limitations regarding the indication and availability of this technique, which may indirectly influence the outcome of the underlying disease. Objective: To identify and analyze the frequency of mutations in commonly altered genes in this pathology, assess their correlation, and highlight the need for a personalized therapeutic approach based on the molecular profile of the tumor. Additionally, we evaluated the importance of early genomic analysis using NGS in comparison to other techniques (real-time PCR and immunohistochemistry), aiming to optimize diagnosis in terms of turnaround time, tissue preservation, and cost-effectiveness. Our findings reinforce the role of KRAS as the most prevalent mutation, typically associated with microsatellite-stable tumors, whereas a mutated BRAF gene is more frequently associated with MSI.
Abstract
Methods: We retrospectively and cross-sectionally reviewed the cases of 648 patients with a histological diagnosis of colon adenocarcinoma. Of these, 166 had partial molecular studies, and 42 cases were selected based on the availability of the genetic markers targeted in this study. We analyzed the frequency of mutations in these genes, as well as their correlation with microsatellite instability (MSI). Results: A high mutation rate was found in the KRAS gene (52.4%). NRAS mutations were less frequent (8.9%), whereas BRAF mutations were observed in 20.8% of cases. This allowed us to identify a patient subgroup with MSI, representing 12.1% of cases. Among the 42 patients analyzed for KRAS, NRAS, BRAF, and MSI mutations, a significant association was observed between KRAS mutations and microsatellite stability, while no association was found between NRAS mutations and MSI. BRAF mutations showed a statistically significant association with MSI (p < 0.05), with the most common mutation being c.1799T > A, p.Val600Glu. The objective of this study is to demonstrate that the NGS-based method for evaluating MSI is rigorously valid compared to the results obtained using IHC and PCR. Conclusions: Comprehensive NGS profiling from the start improves diagnostic efficiency by saving time, tissue, and costs compared to gene-by-gene analysis. It also enables better molecular characterization and facilitates tailored therapeutic strategies, particularly in identifying candidates for targeted therapy and immunotherapy. This approach supports efficient tumor classification based on using KRAS, BRAF, NTRK, ERBB2, and PIK3CA as key markers, along with MSI status. We recommend that, if initial NGS is not feasible, start with KRAS analysis, then test BRAF and MSI if no mutation is found.
