Sequencing paired tumor DNA and white blood cells improves circulating tumor DNA tracking and detects pathogenic germline variants in localized colon cancer.

BACKGROUND: In the setting of localized colon cancer (CC), circulating tumor DNA (ctDNA) monitoring in plasma has shown potential for detecting minimal residual disease (MRD) and predicting a higher risk of recurrence. With the tumor-only sequencing approach, however, germline variants may be misidentified as somatic variations, precluding the possibility of tracking in up to 11% of patients due to a lack of known somatic mutations. In this study, we assess the potential value of adding white blood cells (WBCs) to tumor tissue sequencing to enhance the accuracy of sequencing results. PATIENTS AND METHODS: A total of 148 patients diagnosed with localized CC were prospectively recruited at the Hospital Clínico Universitario in Valencia (Spain). Employing a custom 29-gene panel, sequencing was conducted on tumor tissue, plasma and corresponding WBCs. Droplet digital PCR and amplicon-based NGS were performed on plasma samples post-surgery to track MRD. Oncogenic somatic variants were identified by annotating with COSMIC, OncoKB and an internal repository of pathogenic mutations database. A variant prioritization analysis, mainly characterized by the match of oncogenic mutations with the evidence levels defined in OncoKB, was carried out to select specific targeted therapies. RESULTS: Utilizing paired tumor and WBCs sequencing, we identified somatic mutations in all patients (100%) within our cohort, compared to 89% using only tumor tissue. Consequently, the top 10 most frequently mutated genes for plasma monitoring were altered. The sequencing of WBCs identified 9% of patients with pathogenic mutations in the germline, with APC and TP53 being the most frequently mutated genes. Additionally, mutations in genes related to clonal hematopoiesis of indeterminate potential were detected in 27% of the cohort, with TP53, KRAS, and KMT2C being the most frequently altered genes. There were no observed differences in the sensitivity of monitoring MRD using ddPCR or amplicon-based NGS (p = 1). Ultimately, 41% of the patients harbored potentially targetable alterations at diagnosis. CONCLUSION: The germline testing method not only enhanced sequencing results and raised the proportion of patients eligible for plasma monitoring, but also uncovered the existence of pathogenic germline variations, thereby aiding in the identification of patients at a higher risk of hereditary cancer syndromes.

Targeted next-generation sequencing of circulating-tumor DNA for tracking minimal residual disease in localized colon cancer.

BACKGROUND: A high percentage of patients diagnosed with localized colon cancer (CC) will relapse after curative treatment. Although pathological staging currently guides our treatment decisions, there are no biomarkers determining minimal residual disease (MRD) and patients are at risk of being undertreated or even overtreated with chemotherapy in this setting. Circulating-tumor DNA (ctDNA) can to be a useful tool to better detect risk of relapse. PATIENTS AND METHODS: One hundred and fifty patients diagnosed with localized CC were prospectively enrolled in our study. Tumor tissue from those patients was sequenced by a custom-targeted next-generation sequencing (NGS) panel to characterize somatic mutations. A minimum variant allele frequency (VAF) of 5% was applied for variant filtering. Orthogonal droplet digital PCR (ddPCR) validation was carried out. We selected known variants with higher VAF to track ctDNA in the plasma samples by ddPCR. RESULTS: NGS found known pathological mutations in 132 (88%) primary tumors. ddPCR showed high concordance with NGS (r = 0.77) for VAF in primary tumors. Detection of ctDNA after surgery and in serial plasma samples during follow-up were associated with poorer disease-free survival (DFS) [hazard ratio (HR), 17.56; log-rank P = 0.0014 and HR, 11.33; log-rank P = 0.0001, respectively]. Tracking at least two variants in plasma increased the ability to identify MRD to 87.5%. ctDNA was the only significantly independent predictor of DFS in multivariable analysis. In patients treated with adjuvant chemotherapy, presence of ctDNA after therapy was associated with early relapse (HR 10.02; log-rank P < 0.0001). Detection of ctDNA at follow-up preceded radiological recurrence with a median lead time of 11.5 months. CONCLUSIONS: Plasma postoperative ctDNA detected MRD and identified patients at high risk of relapse in localized CC. Mutation tracking with more than one variant in serial plasma samples improved our accuracy in predicting MRD.