Determining Minimal Residual Disease Using ctDNA Deep Sequencing

Neoadjuvant chemotherapy is the standard of care for locally advanced gastrointestinal tumors. However, not all patients respond to preoperative treatment. Early identification of disease progression during neoadjuvant chemotherapy or diagnosis of early disease relapse during adjuvant therapy is essential for modifying the treatment strategy. The aim of the project is to validate ctDNA as a molecular biomarker of disease relapse/progression.

The persistence of tumor cells after primary treatment of cancer is a key prognostic indicator of the disease's future course. In solid tumors, minimal residual disease (MRD) may involve tumor cells in the blood, small metastases in the body, or a small portion of the primary tumor that remains after treatment. Detection of MRD at a stage when the tumor cannot be detected by morphological imaging techniques is becoming an important diagnostic indicator that could better stratify patients after primary treatment in the future. MRD determination should thus serve in the future both to indicate further treatment and to monitor treatment response. For the successful clinical application of MRD determination, it is crucial to establish a sufficiently sensitive and specific method based on the detection of circulating tumor DNA (ctDNA). Tumor ctDNA is a component of circulating free DNA (cfDNA), which is released into the plasma from apoptotic or necrotic cells in the form of fragments with an average length of approximately 170 bp. The release of DNA from cells into the bloodstream can be influenced by a number of factors, which is reflected in the high variability of total cfDNA concentration, which can range from 1 to 100,000 fragments per milliliter of plasma. Therefore, to quantify MRD, it is crucial to determine both the total concentration of cfDNA and the proportion of tumor ctDNA. To reliably distinguish tumor ctDNA from non-tumor cfDNA, the detection of mutations identified in the primary tumor is currently used. For this purpose, PCR or digital PCR detecting specific mutations using fluorescent probes can be used. An alternative to PCR is mutation detection using next-generation sequencing (NGS), which allows for the detection of multiple different mutations in parallel. A disadvantage of MRD determination using commercial NGS kits designed for liquid biopsies is the high financial cost and the time-consuming preparation of sequencing libraries. We hypothesize that deep sequencing of cfDNA targeting mutations identified in the primary tumor can significantly increase both the specificity and sensitivity of MRD detection from liquid biopsies. Deep sequencing of 2-3 genomic regions with known mutations should enable the parallel detection of MRD in multiple patients while reducing the cost of this type of testing compared to commercially available options.

• Monitoring ctDNA levels [Time frame: Through study completion, an average of 24 months.]
• Variant allele frequency (VAF) of detected driver mutations in cfDNA [Time frame: Through study completion, an average of 24 months.]