miRNAs in lung cancer
Studying complex fingerprints in patient’s blood cells by microarray experiments
The role of miRNAs in pathogenesis and the power to associate expression changes with disease states underscores their value as molecular biomarkers: The expression of miRNAs is highly specific for tissues and developmental stages and has also been proven to allow for molecular classification of tumors. miRNAs are not only found in tissues but also in human blood, both in mononuclear blood cells and as free circulating nucleic acids, highlighting their potential as stable blood-based markers for the detection of cancer or other human diseases. This study investigated whether there is a sufficient number of miRNAs deregulated in blood cells of lung cancer patients to be able to distinguish between cancer patients and controls. Using the fully automated Geniom Real Time Analyzer platform, the miRNA biomarker expression in 17 blood cell samples of patients with non-small cell lung carcinomas (NSCLC) and in 19 blood samples of healthy controls were analyzed. 27 miRNAs have been found to be significantly deregulated in blood cells of lung cancer patients as compared to the controls. Using a subset of 24 miRNA biomarkers allowed for discriminating between blood cell samples of tumor patients and controls with an accuracy of 95.4% [94.9%-95.9%], a specifity of 98.1% [97.3%-98.8%], and a sensitivity of 92.5% [91.8%-92.5%]. These findings support the idea that neoplasia leads to a deregulation of miRNA expression in blood cells of cancer patients compared to blood cells of healthy individuals, and that miRNA biomarker signatures can be used for blood-based diagnosis of human diseases such as cancers.
Experimental setup
We synthesized 866 human miRNAs and miRNA* sequences as annotated in the Sanger miRBase onto a microarray designed by febit biomed gmbh.
Using the fully automated Geniom RT Analyzer platform, we analyzed the miRNA expression in 17 blood cell samples of patients with non-small cell lung carcinomas (NSCLC) and in 19 blood samples of healthy controls.
miRNA Profiles
Figure 1: The bar-chart shows for 15 of the 27 deregulated miRNAs the median value of cancer samples and normal samples. Here, blue bars correspond to cancer samples while red bars to controls.
Figure 2: Blood-based biomarker test reveals deregulated expression of microRNAs in lung carcinoma patients vs. healthy controls. Back to back histograms of two example miRNAs. Blue bars correspond to lung cancer blood samples, red bars to controls blood samples.
Results
Using t-test, we detected 27 miRNAs significantly deregulated in blood cells of lung cancer patients as compared to the controls. Some of these miRNAs were validated using qRT-PCR. To estimate the value of each deregulated miRNA, we grouped all miRNAs according to their diagnostic information that was measured by Mutual Information. Using a subset of 24 miRNAs, a radial basis function Support Vector Machine allowed for discriminating between blood cell samples of tumor patients and controls with an accuracy of 95.4% [94.9%-95.9%], a specificity of 98.1% [97.3%-98.8%], and a sensitivity of 92.5% [91.8%-92.5%].
Figure 3: Validation of blood-based biomarker test. miRNA expression results were verified using quantitative RT-PCR. Scatterplot of fold quotients of qRT-PCR (x-axis) and Geniom RT Analyzer microarray experiments (y-axis).
Conclusion
This is the first evidence for the diagnostic potential of miRNA expression profiles in peripheral blood cells of cancer patients and healthy individuals. It remains to be seen whether other cancers and other non-cancer diseases also show a specific miRNA expression pattern that might be used to tell these diseases apart form controls and possibly apart from each other.
We are optimistic that the high specificity and sensitivity of the miRNAs deregulated in blood cells of lung carcinoma patients together with the high reproducibility of the applied technique will open avenues for applying this approach in prospective trials of a non-invasive diagnostic test.