Evaluating the Utility of Fresh Tissue in Molecular Diagnostics of Colorectal Cancer.

Background: Molecular diagnostics has become a critical component of precision oncology in solid tumors, including colorectal cancer, yet the use of formalin-fixed, paraffin-embedded (FFPE) tissue often suffers from DNA degradation that compromises sequencing quality. This study aimed to evaluate the feasibility and effectiveness of using fresh, intraoperatively collected tumor tissue for next-generation sequencing-based molecular diagnostics in colorectal cancer. Methods: Tissue samples from 24 patients undergoing colorectal tumor resection were obtained based on macroscopic evaluation and tested with a custom gene panel. Sequencing metrics, mutation profiles, and correlations with clinical and pathological features were analyzed. Results: All samples yielded high-quality sequencing data. Oncogenic or likely oncogenic variants were detected in 21 out of 24 samples (87.5%), predominantly affecting genes frequently involved in colorectal cancer carcinogenesis, including APC, TP53, and KRAS. In three cases, no typical mutations were found despite visual confirmation of tumor tissue during surgery, which may be attributed to insufficient tumor cellularity or molecular alterations beyond the panel's scope. Conclusions: The results support the use of fresh tissue as a high-quality source for molecular diagnostics, capable of reducing turnaround time and avoiding formalin-induced artifacts. However, the findings also highlight the diagnostic risk of relying solely on macroscopic tumor assessment without histological confirmation. Overall, fresh tissue-based testing represents a promising yet currently investigational approach that can enhance molecular diagnostics in colorectal cancer.