Methodological development and biological observations of cell free DNA with a simple direct fluorescent assay in colorectal cancer.

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.

Circulating cell-free DNA as predictor of treatment failure after neoadjuvant chemo-radiotherapy before surgery in patients with locally advanced rectal cancer.

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.

Induction chemotherapy with capecitabine and oxaliplatin followed by chemoradiotherapy before total mesorectal excision in patients with locally advanced rectal cancer.

BACKGROUND: Preoperative chemoradiation in patients with locally advanced rectal cancer has no impact on overall survival (OS) and distant recurrences. The aim of the study was to evaluate local downstaging, toxicity and long-term outcome in patients with locally advanced rectal cancer after induction therapy with capecitabine and oxaliplatin (CAPEOX) followed by radiotherapy concomitant with capecitabine [chemoradiotherapy (CRT)] before total mesorectal excision (TME). PATIENTS AND METHODS: Patients with T4 tumors, all T3N+ tumors or T3 tumors involving or with a distance ≤1 mm to the mesorectal fascia were included. Patients were planned for two cycles of CAPEOX followed by radiotherapy concomitant with capecitabine. TME was carried out 6 weeks after the completion of CRT. RESULTS: Of 84 consecutively admitted patients starting induction CAPEOX, 77 patients underwent surgery. R0 resection was seen in 94% and T downstaging in 69%. In the intention-to-treat group, pathological complete response was seen in 23%. Five-year disease-free survival (DFS) and OS were 63% [95% confidence interval (CI), 52.2% to 73.7%] and 67% (95% CI, 56.1% to 77.3%), respectively. Grade 3/4 toxicity was seen in 18%, and four deaths occurred within 2 months of therapy. CONCLUSION: Induction chemotherapy before CRT and surgery showed a high local control rate and promising long-term outcome as OS and DFS.