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

## TL;DR

This study shows that fresh tissue from colorectal cancer surgeries can provide high-quality DNA for molecular testing, potentially improving diagnostic accuracy and speed.

## Contribution

The study demonstrates the feasibility of using fresh tissue for molecular diagnostics in colorectal cancer, offering a potential alternative to FFPE tissue.

## Key findings

- All 24 fresh tissue samples provided high-quality DNA suitable for sequencing.
- Oncogenic mutations were identified in 87.5% of samples, primarily in APC, TP53, and KRAS genes.
- Three samples lacked typical mutations, possibly due to low tumor cellularity or limitations of the gene panel.

## Abstract

Colorectal cancer is a common oncological disease in which treatment decisions increasingly rely on detailed molecular testing of the tumor. In daily practice, such tests are usually performed on formalin-fixed, paraffin-embedded (FFPE) tissue, but fixation can damage DNA and reduce the quality of sequencing results. In this study, we explored whether small pieces of fresh tumor tissue collected directly during surgery could be used instead. We analyzed samples from 24 patients with colorectal cancer using a multigene next-generation sequencing panel. All fresh samples provided high-quality DNA and robust sequencing data, and cancer-driving mutations were identified in most tumors, mainly in APC, TP53 and KRAS genes. Our findings indicate that fresh tissue is a promising source of high-quality material for molecular diagnostics and may help shorten turnaround time, but careful control of tumor cell content and further methodological refinement are needed before this approach can be safely implemented in routine practice.

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.