Specificity of metabolic colorectal cancer biomarkers in serum through effect size

Introduction: Colorectal cancer is one of the most diagnosed cancers, leading to numerous deaths. In addition to existing screening methods, metabolic profiling could help both to diagnose and to understand the various states of the disease.

Objectives: Find specific candidate biomarkers (CB) in serum of patients with colorectal cancer (CRC), in comparison to the situation after remission (R-CRC), evaluated on distinct patients.

Methods: All serum samples were analyzed using comprehensive two-dimensional gas chromatography (GC × GC) coupled to high resolution time of flight mass spectrometry (TOF-MS) through an optimized and validated untargeted analytical method regulated by a quality control (QC) system. First, we used a specific multi-approaches data (pre)processing workflow to highlight, annotate and assess the performances of the most altered metabolites between CRC patients (n = 18) and healthy control samples (HC, n = 19) specifically matched for age and gender, two of the most influential confounding factors. On the contrary, due to the difficulty to control for all clinical and demographic traits when sampling small cohorts, the samples from patients in remission (n = 17) were not matched. Because of the consequent risk of bias, the usual null hypothesis significance tests (NHST) could not be applied reliably. Therefore, we compared the R-CRC samples to another specifically matched group of healthy controls (R-HC, n = 17), and used this comparison to indirectly address the difference between patients with colorectal cancer and patients in remission through a measure called effect size (ES) whose methodological aspects were investigated.

Results: 24 candidate biomarkers were found significantly altered and able to discriminate the CRC and HC samples efficiently (Receiver Operating Characteristic (ROC) area under the curve (AUC) of 0.86, sensitivity and specificity of 0.72 and 0.78). 10 of those were found to have signals close to healthy levels in the R-CRC samples and were therefore specific to colorectal cancer. In the point-biserial case studied here, r-like (strength of association) and d-like (standardized mean difference) ES were directly convertible and only linear and rank-based ES were different. We therefore used and recommend Hedges' g, Spearman's rho and Kendall's tau, along with an unstandardized ES. The confidence intervals, that quantify the uncertainty of the measure, were well represented through scatterplots and distribution curves.

Conclusion: The candidate biomarkers found, along with their specificity, could help for the detection of colorectal cancer, the diagnosis of remission, and for the understanding of its pathophysiology, after proper validation on independent cohorts. The effect size, here applied on a MS global profiling data set, is an ideal complement to NHST and a useful tool to compare and combine distinct cohorts, within a study as well as between studies (meta-analysis).