Differential expression of microRNA in serum fractions and association of Argonaute 1 microRNAs with heart failure.

The serum or plasma microRNA (miRNA) molecules have been suggested as diagnostic and prognostic biomarkers, in various pathological conditions. However, these molecules are also found in different serum fractions, such as exosomes and Argonaute (Ago) protein complexes. Ago1 is the predominant Ago protein expressed in heart tissue. The objective of the study was to examine the hypothesis that Ago1-associated miRNAs may be more relevant to cardiac disease and heart failure compared with the serum. In total, 84 miRNA molecules were screened for their expression in the whole serum, exosomes and Ago1, and Ago2 complexes. Ago1-bound miR-222-3p, miR-497-5p and miR-21-5p were significantly higher, and let-7a-5p was significantly lower in HF patients compared with healthy controls, whereas no such difference was observed for those markers in the serum samples among the groups. A combination of these 4 miRNAs into an Ago1-HF score provided a ROC curve with an AUC of 1, demonstrating clear discrimination between heart failure patients and healthy individuals. Ago1 fraction might be a better and more specific platform for identifying HF-related miRNAs compared with the whole serum.

Multicentre validation of a microRNA-based assay for diagnosing indeterminate thyroid nodules utilising fine needle aspirate smears.

AIMS: The distinction between benign and malignant thyroid nodules has important therapeutic implications. Our objective was to develop an assay that could classify indeterminate thyroid nodules as benign or suspicious, using routinely prepared fine needle aspirate (FNA) cytology smears. METHODS: A training set of 375 FNA smears was used to develop the microRNA-based assay, which was validated using a blinded, multicentre, retrospective cohort of 201 smears. Final diagnosis of the validation samples was determined based on corresponding surgical specimens, reviewed by the contributing institute pathologist and two independent pathologists. Validation samples were from adult patients (≥18 years) with nodule size >0.5 cm, and a final diagnosis confirmed by at least one of the two blinded, independent pathologists. The developed assay, RosettaGX Reveal, differentiates benign from malignant thyroid nodules, using quantitative RT-PCR. RESULTS: Test performance on the 189 samples that passed quality control: negative predictive value: 91% (95% CI 84% to 96%); sensitivity: 85% (CI 74% to 93%); specificity: 72% (CI 63% to 79%). Performance for cases in which all three reviewing pathologists were in agreement regarding the final diagnosis (n=150): negative predictive value: 99% (CI 94% to 100%); sensitivity: 98% (CI 87% to 100%); specificity: 78% (CI 69% to 85%). CONCLUSIONS: A novel assay utilising microRNA expression in cytology smears was developed. The assay distinguishes benign from malignant thyroid nodules using a single FNA stained smear, and does not require fresh tissue or special collection and shipment conditions. This assay offers a valuable tool for the preoperative classification of thyroid samples with indeterminate cytology.

Specific MicroRNAs Differentiate Adrenocortical Adenomas from Carcinomas and Correlate With Weiss Histopathologic System.

MicroRNAs (miRs) play a central role in regulating gene expression and are strongly associated with cancer development. This study sought to determine if adrenocortical carcinomas can be differentiated from adenomas by their miR profiles and to correlate the findings with the histologic Weiss system for identifying malignancy in adrenocortical tumors (ACTs). Forty-six primary and 2 recurrent ACTs retrieved from the files of the pathology department of a tertiary medical center were evaluated blindly for the Weiss criteria. High-quality RNA was extracted, and miR expression was evaluated with microarrays and quantitative reverse-transcriptase polymerase chain reaction. The Weiss system defined 17 tumors as carcinomas and 29 as adenomas. On microarray analysis, over a dozen miRs were upregulated or downregulated in carcinomas compared with adenomas. Upregulation of miR-503 was the best single discriminator of malignancy. The combination of miR-34a and miR-497 underexpression discriminated carcinomas from adenomas with 100% sensitivity and 96% specificity. Statistical analysis revealed a high level of correspondence between the Weiss system and miR expression. In conclusion, miR expression can accurately identify malignant ACTs with equal efficiency to the Weiss system. miR analysis may have added value in tumors with borderline features that are difficult to interpret histopathologically.

Relation of reduced expression of MiR-150 in platelets to atrial fibrillation in patients with chronic systolic heart failure.

Atrial fibrillation (AF) is associated with poor prognosis in patients with heart failure (HF). Although platelets play an important role in rendering a prothrombotic state in AF, the exact mechanism by which the effect is mediated is still debated. MicroRNAs (miRNAs), which have been shown to be involved in a variety of cardiovascular conditions, are abundant in platelets and in a cell-free form in the circulation. In the present study, we performed a genome-wide screen for miRNA expression in platelets of patients with systolic HF and in controls without cardiac disease, in pursuit of specific miRNAs that are associated with the presence of AF. MiRNA expression was measured in platelets from 50 patients with systolic HF and 50 controls, of which, samples from 41 patients with HF and 35 controls were used in the final analysis because of a quality control process. MiR-150 expression was 3.2-fold lower (p = 0.0003) in platelets of patients with HF with AF relative to those without AF. A similar effect was seen in serum samples from the same patients, in which miR-150 levels were 1.5-fold lower (p = 0.004) in patients with HF with AF. Furthermore, the serum levels of miR-150 were correlated to platelet levels in patients with AF (r = 0.65, p = 0.0087). In conclusion, miR-150 expression levels in platelets of patients with systolic HF with AF are significantly reduced and correlated to the cell-free circulating levels of this miRNA.

A diagnostic assay based on microRNA expression accurately identifies malignant pleural mesothelioma.

The definitive identification of malignant pleural mesothelioma (MPM) has significant clinical implications, yet other malignancies often involve the lung pleura, confounding the diagnosis of MPM. In the absence of accurate markers, MPM can be difficult to distinguish from peripheral lung adenocarcinoma and metastatic epithelial cancers. MicroRNA expression is tissue-specific and highly informative for identifying tumor origin. We identified microRNA biomarkers for the differential diagnosis of MPM and developed a standardized microRNA-based assay. Formalin-fixed, paraffin-embedded samples of 33 MPM and 210 carcinomas were used for assay development. Using microarrays, we identified microRNAs differentially expressed between MPM and various carcinomas. Hsa-miR-193-3p was overexpressed in MPM, while hsa-miR-200c and hsa-miR-192 were overexpressed in peripheral lung adenocarcinoma and carcinomas that frequently metastasize to lung pleura. We developed a standardized diagnostic assay based on the expression of these microRNAs. The assay reached a sensitivity of 100% and a specificity of 94% in a blinded validation set of 68 samples from the lung and pleura. This diagnostic assay can provide a useful tool in the differential diagnosis of MPM from other malignancies in the pleura.

Differential diagnosis of hepatocellular carcinoma from metastatic tumors in the liver using microRNA expression.

Distinguishing hepatocellular carcinoma from metastatic tumors in the liver is of great practical importance, with significant therapeutic and prognostic implications. This differential diagnosis can be difficult because metastatic cancers in the liver, especially adenocarcinomas, may mimic the morphology and immunoexpression of hepatocellular carcinoma. Biomarkers that are specifically expressed in either hepatocellular carcinoma or metastatic adenocarcinoma can therefore be useful diagnostic tools. To find such biomarkers, we studied microRNA expression in 144 tumor samples using custom microarrays. Hsa-miR-141 and hsa-miR-200c, microRNAs that promote epithelial phenotypes, had significantly higher levels in non-hepatic epithelial tumors. In contrast, endothelial-associated hsa-miR-126 showed higher expression levels in hepatocellular carcinomas. Combinations of these microRNAs accurately identified primary hepatocellular carcinoma from metastatic adenocarcinoma in the liver. These findings were validated using quantitative real-time PCR to measure microRNA expression in additional samples. Thus, the tissue-specific expression patterns of microRNAs make them useful biomarkers for the diagnosis of liver malignancies.

Validation of a microRNA-based qRT-PCR test for accurate identification of tumor tissue origin.

Identification of the tissue of origin of a tumor is vital to its management. Previous studies showed tissue-specific expression patterns of microRNA and suggested that microRNA profiling would be useful in addressing this diagnostic challenge. MicroRNAs are well preserved in formalin-fixed, paraffin-embedded (FFPE) samples, further supporting this approach. To develop a standardized assay for identification of the tissue origin of FFPE tumor samples, we used microarray data from 504 tumor samples to select a shortlist of 104 microRNA biomarker candidates. These 104 microRNAs were profiled by proprietary quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) on 356 FFPE tumor samples. A total of 48 microRNAs were chosen from this list of candidates and used to train a classifier. We developed a clinical test for the identification of the tumor tissue of origin based on a standardized protocol and defined the classification criteria. The test measures expression levels of 48 microRNAs by qRT-PCR, and predicts the tissue of origin among 25 possible classes, corresponding to 17 distinct tissues and organs. The biologically motivated classifier combines the predictions generated by a binary decision tree and K-nearest neighbors (KNN). The classifier was validated on an independent, blinded set of 204 FFPE tumor samples, including nearly 100 metastatic tumor samples. The test predictions correctly identified the reference diagnosis in 85% of the cases. In 66% of the cases the two algorithm predictions (tree and KNN) agreed on a single-tissue origin, which was identical to the reference diagnosis in 90% of cases. Thus, a qRT-PCR test based on the expression profile of 48 tissue-specific microRNAs allows accurate identification of the tumor tissue of origin.

Diagnostic assay based on hsa-miR-205 expression distinguishes squamous from nonsquamous non-small-cell lung carcinoma.

PURPOSE: Recent advances in treatment of lung cancer require greater accuracy in the subclassification of non-small-cell lung cancer (NSCLC). Targeted therapies which inhibit tumor angiogenesis pose higher risk for adverse response in cases of squamous cell carcinoma. Interobserver variability and the lack of specific, standardized assays limit the current abilities to adequately stratify patients for such treatments. In this study, we set out to identify specific microRNA biomarkers for the identification of squamous cell carcinoma, and to use such markers for the development of a standardized assay. PATIENTS AND METHODS: High-throughput microarray was used to measure microRNA expression levels in 122 adenocarcinoma and squamous NSCLC samples. A quantitative real-time polymerase chain reaction (qRT-PCR) platform was used to verify findings in an independent set of 20 NSCLC formalin-fixed, paraffin-embedded (FFPE) samples, and to develop a diagnostic assay using an additional set of 27 NSCLC FFPE samples. The assay was validated using an independent blinded cohort consisting of 79 NSCLC FFPE samples. RESULTS: We identified hsa-miR-205 as a highly specific marker for squamous cell lung carcinoma. A microRNA-based qRT-PCR assay that measures expression of hsa-miR-205 reached sensitivity of 96% and specificity of 90% in the identification of squamous cell lung carcinomas in an independent blinded validation set. CONCLUSION: Hsa-miR-205 is a highly accurate marker for lung cancer of squamous histology. The standardized diagnostic assay presented here can provide highly accurate subclassification of NSCLC patients.

Serum microRNAs are promising novel biomarkers.

BACKGROUND: Circulating nucleic acids (CNAs) offer unique opportunities for early diagnosis of clinical conditions. Here we show that microRNAs, a family of small non-coding regulatory RNAs involved in human development and pathology, are present in bodily fluids and represent new effective biomarkers. METHODS AND RESULTS: After developing protocols for extracting and quantifying microRNAs in serum and other body fluids, the serum microRNA profiles of several healthy individuals were determined and found to be similar, validating the robustness of our methods. To address the possibility that the abundance of specific microRNAs might change during physiological or pathological conditions, serum microRNA levels in pregnant and non pregnant women were compared. In sera from pregnant women, microRNAs associated with human placenta were significantly elevated and their levels correlated with pregnancy stage. CONCLUSIONS AND SIGNIFICANCE: Considering the central role of microRNAs in development and disease, our results highlight the medically relevant potential of determining microRNA levels in serum and other body fluids. Thus, microRNAs are a new class of CNAs that promise to serve as useful clinical biomarkers.

MicroRNAs accurately identify cancer tissue origin.

MicroRNAs (miRNAs) belong to a class of noncoding, regulatory RNAs that is involved in oncogenesis and shows remarkable tissue specificity. Their potential for tumor classification suggests they may be used in identifying the tissue in which cancers of unknown primary origin arose, a major clinical problem. We measured miRNA expression levels in 400 paraffin-embedded and fresh-frozen samples from 22 different tumor tissues and metastases. We used miRNA microarray data of 253 samples to construct a transparent classifier based on 48 miRNAs. Two-thirds of samples were classified with high confidence, with accuracy >90%. In an independent blinded test-set of 83 samples, overall high-confidence accuracy reached 89%. Classification accuracy reached 100% for most tissue classes, including 131 metastatic samples. We further validated the utility of the miRNA biomarkers by quantitative RT-PCR using 65 additional blinded test samples. Our findings demonstrate the effectiveness of miRNAs as biomarkers for tracing the tissue of origin of cancers of unknown primary origin.