Pre-treatment characteristics and cell-free DNA levels in all cohorts.

<p>Data have been rounded to nearest 100.</p><p># total number with available samples,</p><p>* only cancer patients</p><p>**One sample excluded because of visible hemolysis</p><p>*** All p-values for differences between each cancer cohort and the control group</p><p>Pre-treatment characteristics and cell-free DNA levels in all cohorts.</p

Multivariate analysis of overall survival.

<p>^† Reference group</p><p>* PS = Performance Status (ECOG)</p><p>** Entered as a continuos variable</p><p>The risk ratio refers to moving from the reference group to the other group or changing one step in parameters entered as continuous variables.</p><p>Multivariate analysis of overall survival.</p

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

<p><b>A</b> 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>B</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).</p

Kaplan Major Plots of OS probabilities.

<p>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.</p

Laser micro-dissection.

<p>Areas of tumour cells have been removed (arrows). Areas to be removed from the stromal tissue have been marked (red lines).</p

Selected reference gene candidates from the Expression Suite data-analysis.

<p>Selected reference gene candidates from the Expression Suite data-analysis.</p

Stability values for reference gene candidates according to Norm Finder.

<p>Stability values for reference gene candidates according to Norm Finder.</p

Overview of experiments.

<p>Experiments outlined in A) were used to compare dual and prolonged single centrifugation (experiment 1) and to compare qPCR and ddPCR with respect to precision and repeatability (part of experiment 4). Experiments outlined in B) were used to investigate the correlation between qPCR (single assays) and ddPCR (part of experiment 4), correlation between single TaqMan assays and TaqMan Low Density Array (experiment 3) and correlation between TaqMan assays performed with microRNA purified from standard plasma and PPP, respectively (experiment 2).</p

Comparison of two centrifugation protocols to produce platelet-poor plasma.

<p>Comparison of two centrifugation protocols to produce platelet-poor plasma.</p

Relative microRNA levels and order of blood draw.

<p>Plots showing the relative microRNA levels in 30 PPP samples from a healthy volunteer in order of blood draw. Samples with odd numbers were prepared by dual centrifugation (red triangles) and samples with even numbers by a prolonged single centrifugation (blue squares). MicroRNA-levels were normalized to cel-miR-39 (plots A, D and G), miR-16 (plots B and E) or the mean of cel-miR-39 and miR-16 (plots C and F). In all cases, we found no significant differences (t-test) in the mean microRNA-levels between the first 10 tubes and the last 10 tubes drawn (p>0.05).</p