Circulating free DNA as biomarker and source for mutation detection in metastatic colorectal cancer

Abstract

Background: Circulating cell-free DNA (cfDNA) in plasma has shown potential as biomarker in various cancers and could become an importance source for tumour mutation detection. The objectives of our study were to establish a normal range of cfDNA in a cohort of healthy individuals and to compare this with four cohorts of metastatic colorectal cancer (mCRC) patients. We also investigated the prognostic value of cfDNA and analysed the tumour-specific KRAS mutations in the plasma.

Methods: The study was a prospective biomarker evaluation in four consecutive Phase II trials, including 229 patients with chemotherapy refractory mCRC and 100 healthy individuals. Plasma was obtained from an EDTA blood-sample, and the total number of DNA alleles and KRAS mutated alleles were assessed using an in-house ARMS-qPCR as previously described.

Results: Median cfDNA levels were higher in mCRC compared to controls (p < 0.0001). ROC analysis revealed an AUC of 0.9486 (p<0.00001). Data showed impaired OS with increasing levels of baseline cfDNA both when categorising patients by quartiles of cfDNA and into low or high cfDNA groups based on the upper normal range of the control group (Median OS 10.2 (8.3-11.7) and 5.2 (4.6-5.9) months, respectively, HR 1.78, p = 0.0006). Multivariate analysis confirmed an independent prognostic value of cfDNA (HR 1.5 (95% CI 1.3-1.7) for each increase in the cfDNA quartile). The overall concordance of KRAS mutations in plasma and tissue was high (85%).

Conclusions: These data confirm the prognostic value of cfDNA measurement in plasma and utility for mutation detection with the method presented.

Fig 1. A and B. Cell free DNA concentrations in colorectal cancer cohorts and healthy controls.

A depicts Box and whisker plots with 25%, 50%, 75% percentiles, upper and lower adjacent values and outliers (dots). Horizontally the four clinical trial cohorts and control group, vertically the cfDNA concentration on a logarithmic scale. C cetuximab study, GemCap GemCap cohort, PG PG study cohort, T TIRASMUS study cohort, Controls healthy control group. B Shows a receiver operating curve (ROC) estimating the performance of cfDNA to discriminate between colorectal cancer patients and controls. The AUC was 0.9486, (95% CI 0.9182–0.9679, p<0.00001).

Fig 2. Kaplan Major Plots of OS probabilities.

A. Patients are grouped by quartiles of cfDNA (from the right) lowest, second lowest, second highest and highest quartile of cfDNA. The median OS according to cfDNA quartiles were; 10.2 months (95% CI 8.9–12.8), 7.8 months (5.7–9.3), 5.0 months (4.3–6.0) 3.5 months (3.0–3.9), respectively. B. Patients are grouped by the upper normal range as defined by mean cfDNA+2SD in the control group (7100 alleles per ml.) Low risk group (full line), Median OS, 10.2 months (8.3–11.7). High risk group (dotted line) Median OS, 5.2 months (4.6–5.9). HR 1.78, p = 0.0006.

Fig 3. Scatter plot of correlation between cfDNA and pKRAS concentrations.

This plot illustrates the strong correlation between the total number of DNA alleles and the number of mutated alleles in the patients with KRAS mutations detected. The spearman rank correlation was calculated to 0.86 and r² = 0.97 (p < 0.0000). The different symbols represent the individual cohorts of cancer patients. (Square PG correlation = 0.9, circle cetuximab correlation = 0.88, triangle GemCap correlation = 86, pentagon TIRASMUS correlation = 0.67).