The goal of medical screening is the detection of a disease in subjects at asymptomatic stages, which leads to more effective treatment of the disease. Two major considerations limiting application of screening tests are economical and psychological in nature. From economical perspective, the factors limiting the utility of screening tests are: (i) cost, especially when large populations are screened for rare diseases; and (ii) practically unavoidable false positive results with additional expenses for subsequent diagnostic investigations. The latter issue also leads to negative psychological effects, such as unwarranted stress and anxiety.
To address these issues, we propose a novel approach to screening, namely Universal Screening Test (UST), to detect pathologic processes in organ systems, organs and tissues. Such a test will not diagnose specific diseases, but rather will detect the presence of a pathology in a particular organ or organ system. Additional diagnostic tests will then be applied to a limited number of subjects pre-selected by UST. The present paper describes an initial study demonstrating feasibility of detection of various pathologies of a particular organ system by analysis of levels of circulating cell-free miRNAs in plasma.
There have been numerous attempts to develop screening and diagnostic tests based on analysis of circulating miRNAs. In many cases samples from patients with a particular disease were successfully differentiated from controls [5, 19, 24, 30]. However, such findings by themselves do not necessarily mean that the tests specifically detect the disease. Many miRNAs are associated with a common pathology type, e.g. cancer, inflammation, or hypoxia and the same circulating miRNAs have been described as potential biomarkers of different diseases. For example, changes of miR-155 concentrations were found in the bloodstream of patients with breast, esophageal, lung, pancreatic cancers and lymphomas [25, 27]. Level of miR-21 increases in plasma/serum of patients with osteosarcoma, bladder, esophageal, gastric, lung, breast, colorectal cancers, neck squamous cell carcinoma and other tumors [25–27]. Some miRNAs are associated with both cancer and inflammation [29, 32–35]. A large number of similar examples can be found in literature. In summary, a mere differentiation of patients with a disease from control subjects is not sufficient for specific detection of a disease in a clinical setting. Additional information, such as localization of a disease in a particular organ, is required.
Here we proposed to analyze organ/organ system-enriched miRNAs to differentiate pathological processes of different organs, based on a hypothesis that changes in their concentrations in plasma will most likely be associated with a disease of a respective organ or organ system. We also included in the study a number of miRNAs involved in such broad classes of pathologies as carcinogenesis and inflammation, to find out whether analysis of the levels of these miRNAs along with the levels of organ-enriched miRNAs can provide more specific information about pathology.
In the current study analysis of miRNA ratios (miRNA biomarker pairs) was performed to account for numerous factors that affect detectable levels of various miRNAs: miRNA stability in plasma, effectiveness of its purification, potential RT-qPCR inhibition, etc. Further, calculation of ratios of two miRNAs could increase sensitivity and specificity of a biomarker panel, since expression and secretion of different miRNAs may be affected differently (e.g. changed in opposite directions) by pathology.
Two organ systems and seven pathologies were used to test the proposed approach. The results obtained can be summarized as follows: (1) analysis of organ system-enriched miRNA levels in plasma effectively detected patients with the pathologies studied: asthma, pneumonia and NSCLC in pulmonary system and Crohn’s disease, esophageal, gastric and colon cancers in GI system; it is important to note that all 30 cancers of the GI system and 4 out of 10 NSCLC were in stages I/II (see Additional file 1: Table S1), providing a dataset with relatively early stages of pathology; (2) miRNA pairs successfully differentiating patients with pulmonary system diseases from patients with the GI system diseases have been identified; and (3) cancer patients were effectively distinguished from patients with inflammatory diseases.
We have recently demonstrated that a similar approach can be used for early detection of MCI, a syndrome characteristic of early stages of various neurodegenerative diseases, including Alzheimer’s and Parkinson’s diseases . Two sets of biomarker miRNA pairs capable of differentiating MCI from age matched controls with 82%-92% accuracy were found. These biomarkers were then successfully validated in a larger study with independent cohorts of plasma samples, and accuracy obtained in this validation study was 87% - 96% (poster presented in 2013 Alzheimer's Association International Conference; Aging, manuscript in press). In addition, in a small retrospective longitudinal study we analyzed plasma samples collected from the same patients at different time points for up to 5 years and demonstrated that MCI can be detected 1–5 years prior to clinical diagnosis . The values of miRNA biomarker pairs in plasma samples collected from same subjects over the course of 1 to 5 years were very consistent.
The most common approach to search for circulating miRNA biomarkers of various diseases is miRNA array analysis of numerous miRNAs in serum or plasma. However, the concentrations of many organ-enriched miRNAs in bodily fluids are too low to be detected by miRNA arrays and, as a result, they can be missed in such studies. Nonetheless, some of these miRNAs are detectable and, in fact, these miRNAs are among the most promising biomarkers due to increase of their levels in plasma or serum. Circulating liver-enriched miR-122 and heart-enriched miR-1, miR-133, miR-208 and miR-499 represent good examples since their concentrations have been found to be significantly higher in the bloodstream of patients with various diseases of respective organs. Interestingly, in some instances concentrations of these miRNAs in organ tissues are decreased due to pathology but their levels in plasma or serum go up , which additionally supports the idea of using organ-enriched miRNAs for detecting pathology of respective organs.
Thus, the data obtained in the present study and the results of literature analysis indicate that the idea of Universal Screening Test presented herein is viable. Naturally, larger studies involving patients with diseases of GI and pulmonary systems, as well as accurately matched control subjects, including cohorts with different age, gender, and ethnicity, are necessary for further validation of the approach. Longitudinal studies and analysis of precancerous conditions, such as adenomatous polyps, will help clarify how early various organ pathologies can be detected. It also would be important to compare levels of these miRNA biomarkers in subjects before, during and after treatment in order to evaluate their applicability to disease and treatment monitoring. Further, studies with additional miRNAs and patients with pathologies of other organs should be performed.
Currently, data on miRNA expression in various organs, tissues and cell types are still limited. Some miRNAs can be enriched in several organs. Further, plasma levels of miRNAs not enriched in a certain organ may increase due to higher expression or secretion caused by pathology. The increase in concentration of circulating miR-192 in patients with pathologies of pulmonary system reported here (see Additional file 1: Figure S2) provides a good example of such an increase caused by pathology. We believe that a potential ambiguity with respect to organs or organ systems affected by pathology, as inferred from analysis of certain miRNAs, such as miR-192, can be overcome by measuring levels of several miRNAs enriched in each organ system, organ and tissue.
Figure 5 presents a diagram of the proposed UST workflow. Briefly, after measuring miRNA concentrations in a bodily fluid, pathologies in one or several organ systems are detected based on calculations of miRNA ratios. When possible, a pathology type (e.g. cancer, inflammation) is determined. If pathology is detected, specific diagnostic tests should be performed.