Clinical significance of circulating miR-126 quantification in malignant mesothelioma patients
Graphical abstract
Highlights
► Relative qRT-PCR detects circulating miRNAs with the confidentiality interval < 5%. ► Endogenous and exogenous control normalization improve the precision of the assay. ► The sample dilution enhances miRNA extraction reducing the coefficient of variation. ► Circulating miRNA-126 represent a sensitive diagnosis and prognosis marker for MM.
Introduction
Cancers, such as malignant mesothelioma (MM), are often diagnosed at a late stage with concomitant poor prognosis [1], [2], [3]. MM is an extremely aggressive tumour, highly resistant to chemotherapy and to radiotherapy. Significant advances in MM treatment will imply an early diagnosis to select candidates for therapy with curative intent [4]. Therefore, the development of minimal invasive tests for the early detection and monitoring of malignancies is of paramount clinical importance. Focus has been on finding tumour markers in biological fluids like blood that can be used in association with therapy for non-invasive detection of MM.
Micro-RNAs (miRNAs) present a novel arena for molecular diagnosis of cancer [5], [6]. Accumulating reports suggest that circulating miRNAs are present in blood and have the potential as biomarkers for several malignancies [7], [8]. We and others have found that deregulated miRNAs can differentiate MM tissue from normal mesothelium (NM) tissue [9], [10], [11], [12], [13]. In our model, miR-126 was found to be significantly downregulated in the malignant tissue. Further, expression of miR-126 can be easily evaluated in serum, and its level in association with a specific marker of MM, soluble mesothelin-related peptides, can be used to identify the disease at relatively early stages [13].
A prerequisite for developing circulating miRNA-based diagnosis is the ability to evaluate the levels of molecular biomarkers in plasma and/or serum with sufficient sensitivity and precision to be clinically relevant. These challenges have been met by innovative solutions based on quantitative RT-PCR (qRT-PCR). The clinical effectiveness of circulating nucleic acid as biomarkers is affected by a range of variables including pre-analytical factors involved in specimen collection, processing factors influencing RNA extraction efficiency, as well as the technical issues involved in successful qRT-PCR and data analysis. Therefore, a standardized method is required. In this study, an optimised method for the detection of circulating miRNA in serum has been performed and clinically validated. Both the endogenous and exogenous controls have been used for normalization. Accuracy and precision of the method were evaluated, and the relative qRT-PCR analysis was compared with absolute qRT-PCR analysis. The method was validated by detecting serum miR-126 in MM and non-small-cell lung cancer (NSCLC) patients and its comparison with healthy controls. The clinical performance of miR-126 was tested for its correlation with the prognosis of individual MM patients.
Section snippets
Sample collection
Serum instead of plasma was used to avoid anti-coagulant interferences and the risk of cell contamination. The MM patients (n = 45, age 67.7 ± 8.9, 31 males and 14 females), including smokers 21/45 (47%), ex-smokers 9/45 (20%) and non-smokers 15/45 (33%) and NSCLC patients (n = 20, age 69.6 ± 8.1, 15 males and 5 females), including smokers 5/20 (25%), ex-smokers 6/20 (30%) and non-smokers 9/20 (45%) were recruited, from November 2007 to December 2011, at the Oncology Clinic of the University Hospital
Normalization of controls
There are several well established protocols for normalization of gene expression measurements in different samples. Most of them utilize a housekeeping gene. Similar approaches can be applied to normalization of cellular miRNA assessment. However, there are currently no known extra-cellular housekeeping genes for circulating miRNAs. To find an endogenous control that can be used to reliability quantify the expression of miRNAs of interest in the serum, we examined the levels of U6 in serum by
Discussion
Several reports have proposed the detection of circulating tumour-specific miRNAs as a potentially valuable tool for early cancer detection in order to predict the clinical cancer behaviour and/or its therapeutic response [13], [14], [15], [16], [17], [18], [19], [21]. However, the clinical significance in the use of miRNA as biomarkers has not been evaluated thus far. In our previous study, miR-126 was found expressed in the serum of MM patients in low levels with respect to the healthy
Conclusions
The intra- and inter-assay analysis revealed that the relative qRT-PCR is a more reliable method. The use of an endogenous invariant control and an exogenous synthetic control as well sample dilution markedly improved the accuracy and precision of the assay with a CV less than 5%. Although currently there are some difficulties with using miRNAs as cancer biomarkers such as unclearly cut reference range and the lack of large-sample data, our study extends the potential of miR-126 to serve as
Acknowledgments
Financial assistance from the National Institute against Occupational Injury Insurance (INAIL) is thankfully acknowledged. J.N. was supported in part by grants from the Australian Research Council and the Grant Agency of the Czech Republic (204/08/0811).
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