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Introduction: Both quantitative and molecular changes in ctDNA can hold important information when treating metastatic colorectal cancer (mCRC), but its clinical utility is yet to be established. Before conducting a large-scale randomized trial, it is essential to test feasibility. This study investigates whether ctDNA is feasible for detecting patients who will benefit from treatment with epidermal growth factor receptor inhibitors and the prognostic value of circulating tumor DNA (ctDNA) response., Materials and Methods: Patients with mCRC, who were considered for systemic palliative treatment and were eligible for ctDNA analysis. Mutational testing on cell-free DNA (cfDNA) was done by ddPCR. ctDNA response from baseline to the third treatment cycle was evaluated in patients with detectable ctDNA at baseline. ctDNA maximum response was defined as undetectable ctDNA at the third treatment cycle, ctDNA partial response as any decrease in the ctDNA level, and ctDNA progression as any increase in the ctDNA level., Results: Forty-nine patients were included. The time to test results for mutational testing on cfDNA was significantly shorter than on tumor tissue (p < .001). Progression-free survival were 11.2 months (reference group), 7.5 months (HR = 10.7, p= .02), and 4.6 months (HR = 11.4, p= .02) in patients with ctDNA maximum response, partial response, and progression, respectively. Overall survival was 31.2 months (reference group), 15.2 months (HR = 4.1, p= .03), and 9.0 months (HR = 2.6, p= .03) in patients with ctDNA maximum response, partial response, and progression, respectively., Conclusion: Pretreatment mutational testing on cfDNA in daily clinic is feasible and can be applied in randomized clinical trials evaluating the clinical utility of ctDNA. Early dynamics in ctDNA during systemic treatment hold prognostic value., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: Cell free DNA (cfDNA) has shown promising utility as prognostic biomarker for patients with colorectal cancer (CRC), with an ongoing need to optimize and validate the laboratory methodology. Here, we report our optimization and validation of a direct fluorescent assay and display the potential utility in patients with colorectal cancer., Methods: Plasma cfDNA was analyzed by a direct fluorescent assay (DFA) and compared to quantification by droplet digital PCR (ddPCR). For clinical validation, baseline blood samples were available for a total of 273 patients from six different Nordic trials, covering patients with locally advanced rectal cancer (n = 176, cohorts A + B), liver limited metastatic CRC (n = 75C + D) and wide spread metastatic CRC (n = 22 E + F)., Results: Validating the DFA analysis with ddPCR revealed a strong correlation with an R2 of 0.81. For the clinical cohorts, the levels of cfDNA were: 0.8 ng/uL (95%CI 0.75-0.83) (A + B), 0.93 ng/uL (95%CI 0.86-1.02) (C + D) and 1.2 ng/uL (95%CI 0.85-1.47) (E + F), respectively (p < 0.01). All cohorts of colorectal cancer had higher levels of cell free DNA than healthy individuals (n = 94) (p < 0.01)., Conclusion: Analysis of cell free DNA by a direct fluorescent assay could be an attractive laboratory option for a rapid inexpensive quantification of cell free DNA., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Background: Treatment of patients with locally advanced rectal cancer (LARC) is based on a combination of chemo-radiotherapy (CRT) and surgery. The rate of distant recurrences remains over 25%. Circulating cell-free DNA (cfDNA) in plasma is a mixture of normal and cancer-specific DNA segments and is a promising biomarker in patients with colorectal cancer. The aim of our study was to investigate plasma cfDNA as a prognostic marker for outcome in patients with LARC treated with neoadjuvant CRT and surgery., Patients and Methods: In total, 123 patients with LARC were included in 2 biomarker studies. Patients were treated with neoadjuvant CRT before TME surgery. Fifty-two (42%) of the patients received induction chemotherapy with capecitabine + oxaliplatin. Total cfDNA was measured by direct fluorescent assay in EDTA plasma samples obtained at baseline, after induction chemotherapy, and after CRT. Serial samples 5 years after surgery were collected in 51 patients (41%)., Results: Median follow-up was 55 months. Distant or local recurrence was seen in 30.9% of the patients. Patients with baseline cfDNA levels above the 75th quartile had a higher risk of local or distant recurrence and shorter time to recurrence compared with patients with plasma cfDNA below the 75th percentile (HR = 2.48, 95% CI: 1.3-4.8, P = 0.007). The same applied to disease-free survival (DFS) (HR = 2.43, 95% CI: 1.27-4.7, P = 0.015). In multivariate analysis, a high cfDNA level was significantly associated with time to progression and DFS. During follow-up, the association remained significant regardless of time point for sample analysis., Conclusion: We have demonstrated an association between a high baseline plasma level of cfDNA and increased risk of recurrence, shorter time to recurrence, and shorter DFS in patients with LARC. Consequently, cfDNA could potentially improve pre- and post-treatment risk assessment and facilitate individualized therapy for patients with LARC.