Analysis of organ-enriched microRNAs in plasma as an approach to development of Universal Screening Test: feasibility study

Abstract

Background: Early disease detection with a minimally invasive screening test will significantly increase effectiveness and decrease the cost of treatment. Here we propose a framework of a novel approach - Universal Screening Test (UST) for the detection of pathological processes in a particular organ system, organ, or tissue by RT-qPCR analysis of circulating cell-free miRNAs in plasma. As the first step towards assessing the feasibility of this concept, the present study was designed to analyze whether the same microRNAs (miRNAs) can detect various diseases of a particular organ system.

Methods: RNA was extracted from plasma using Trizol treatment and silica binding. Levels of miRNAs were measured by single target RT-qPCR. The following innovations have been tested and proven effective: (i) the use of organ system/organ/tissue-enriched miRNAs; (ii) the use of miRNAs associated with broad disease categories, such as cancer and inflammation, in combination with the organ-enriched miRNAs; and (iii) the use of "miRNA pairs" for selecting miRNA combinations with the highest sensitivity and specificity.

Results: Here we report biomarker miRNA pairs effectively differentiating (i) patients with pulmonary system diseases (asthma, pneumonia and non-small cell lung cancer) and gastrointestinal (GI) system diseases (Crohn's disease, stages I/II esophageal, gastric and colon cancers) from controls, each with 95% accuracy; (ii) patients with a pathology of the pulmonary system from patients with a pathology of the GI system with 94% accuracy; and (iii) cancer patients (stages I/II esophageal, gastric, colon cancers, or non-small cell lung cancer) from patients with inflammatory diseases (asthma, pneumonia, or Crohn's disease) with 93%-95% accuracy.

Conclusions: The results obtained in the present study, along with the data reported by us and others previously, are encouraging and lay the ground for further investigation of the described approach for UST development.

Figure 1

Differentiation of GI pathologies from controls by miRNA biomarker pairs. The concentrations of miRNAs in plasma samples of patients with four GI pathologies, and healthy donors were measured by RT-qPCR and the ratios of levels of various miRNAs were calculated as 2^-ΔCq x 100. A-E – box-plots. Here and in other figures with box and whisker plots the results are presented in the log₁₀ scale. The upper and lower limits of the boxes and the lines inside the boxes indicate the 75th and 25th percentiles and the median, respectively. The upper and lower horizontal bars denote the 90th and 10th percentiles, respectively. The points indicate assay values located outside of 80% data. A-D – individual pathologies (10 patients in each group) against controls (30 subjects); E – combined GI pathologies (40 patients total) against controls (30 subjects). F –ROC curves of differentiation between patients with four GI pathologies and controls obtained with different biomarker pairs. The areas under the ROC curves (AUC) are reported. Sensitivity, specificity and accuracy for each miRNA pair are calculated for the “cutoff” point (indicated as a dot on each plot) – the value of the ratio where the accuracy of predictions is the highest (see Methods, ref. 14).

Figure 2

Differentiation of Pulmonary system (PS) pathologies from controls by miRNA biomarker pairs. The concentrations of miRNAs in plasma samples from patients with three PS pathologies, and from healthy donors were measured by RT-qPCR and the ratios of various miRNAs were calculated as 2^-ΔCq x 100. A-E – box-plots; A-D – individual pathologies (10 patients in each group) against controls (20 subjects); E – combined PS pathologies (30 patients total) against controls (20 subjects). F –ROC curves of differentiation between patients with three pathologies and controls obtained with different biomarker pairs. The statistical analysis is performed as in Figure 1.

Figure 3

Differentiation of GI pathologies fromPS pathologies by miRNA biomarker pairs. The concentrations of miRNAs in plasma samples from patients with GI pathologies (40 patients total) and pulmonary system pathologies (30 patients total) were measured by RT-qPCR and the ratios of various miRNAs were calculated as 2^-ΔCq x100. A, B – box-plots. C, D –ROC curves for differentiation between patients with the four GI and the three pulmonary system (PS) pathologies obtained with different biomarker pairs. All statistical analyses are performed as in Figure 1. E, F – 2D-graphs comparing biomarker miRNA pairs from A, C and B, D, respectively.

Figure 4

Differentiation of all cancers from all inflammatory pathologies by miRNA biomarker pairs. The concentrations of miRNAs in plasma samples from patients with cancers of four organs (40 patients total) and three inflammatory diseases (30 patients total) were measured by RT-qPCR and the ratios of various miRNAs were calculated as 2^-ΔCq x 100. A, B – box-plots. C, D – Receiver-Operating Characteristic (ROC) curves of differentiation between patients with cancers of the four organs and patients with any of the three inflammatory diseases obtained with different biomarker pairs. All statistical analyses are performed as in Figure 1. E, F – 2D-graphs comparing biomarker miRNA pairs from A, C and B, D, respectively.

Figure 5

The UST workflow.