MiR-Score: A novel 6-microRNA signature that predicts survival outcomes in patients with malignant pleural mesothelioma

Background Prognosis of malignant pleural mesothelioma (MPM) is poor, and predicting the outcomes of treatment is difficult. Here we investigate the potential of microRNA expression to estimate prognosis of MPM patients. Methods Candidate microRNAs from microarray profiling of tumor samples from 8 long (median: 53.7 months) and 8 short (median: 6.4 months) survivors following extrapleural pneumonectomy (EPP) were validated by RT-qPCR in 48 additional EPP samples. Kaplan–Meier log ranking was used to further explore the association between microRNA expression and overall survival (OS). Binary logistic regression was used to construct a microRNA signature (miR-Score) that was able to predict an OS of ≥20 months. Performance of the miR-Score was evaluated by receiver operating characteristic (ROC) curve analysis and validated in a series of 43 tumor samples from patients who underwent palliative surgery [pleurectomy/decortication (P/D)]. Results The miR-Score, using expression data of six microRNAs (miR-21-5p, -23a-3p, -30e-5p, -221-3p, -222-3p, and -31-5p), enabled prediction of long survival with an accuracy of 92.3% for EPP and 71.9% for palliative P/D. Hazard ratios for score-negative patients were 4.12 (95% CI: 2.03–8.37) for EPP and 1.93 (95% CI: 1.01–3.69) for P/D. Importantly, adding the miR-Score to a set of clinical selection criteria (histology, age, gender) increased predictive accuracy in the independent validation set from 76.3% for clinical factors only to 87.3%. Conclusions This study has identified a novel 6-microRNA signature (miR-Score) that can accurately predict prognosis of MPM patients

Validation of a minimal panel of antibodies for the diagnosis of malignant pleural mesothelioma

AIMS: We previously established the use of a minimal panel of antibodies as sufficient to diagnose most epithelial malignant mesothelioma (MPM). We aimed to validate this approach and investigate the utility of a D2-40 antibody. METHODS: A series of 80 MPM patients selected for surgery and 21 consecutive patients with pleural metastatic carcinoma were included. A minimal panel of antibodies, consisting of calretinin, BG8 and CD15, and D2-40 was investigated. RESULTS: There were 61 epithelial and 19 biphasic MPM as well as 12 metastatic lung, six breast (5 ductal adenocarcinomas, 1 mixed ductal/lobular adenocarcinoma), two serous papillary ovarian carcinomas and one moderately differentiated colorectal adenocarcinoma. The sensitivity of positive calretinin labelling to confirm the diagnosis of MPM was 97.5%, while the 'diagnostic sensitivities' of lack of labelling for BG8 and CD15 were 91.3% and 97.5%, respectively. The use of calretinin, BG8 and CD15 resulted in correct classification in 97.5% of all MPMs. All MPM cases investigated showed at least focal positive D2-40 labelling. CONCLUSIONS: We have validated the usefulness of a minimal panel of antibodies with calretinin, BG8 and CD15 as the initial step to the diagnosis of MPM. D2-40 emerged as a helpful diagnostic tool for cases where our initial approach failed to conclusively diagnose MPM

Validation of tissue microarray technology in malignant pleural mesothelioma

AIMS: Tissue microarray (TMA) technology has been utilised for assessment of cancers including malignant pleural mesothelioma (MPM). Given the intralesional heterogeneity of MPM, it is questionable if TMAs can adequately represent MPMs. We here investigate the validity of TMAs for MPM. METHODS: TMAs were constructed from at least five cores for each of 80 archival tumours processed by two centres between 1994 and 2009. The percentage of cases correctly subtyped on TMAs compared with whole sections, in relation to the number of cores analysed, was calculated. Immunohistochemical labelling for calretinin and D2-40 was performed on TMAs and whole sections. To evaluate the validity of quantitative immunohistochemistry, percentages of positive cells were recorded and two-way analysis of variance (ANOVA) performed. RESULTS: Five cores were assessable for 91% of patients. Four cores were sufficient to reach concordance with the whole-section result in 98% of cases for calretinin and 99% for D2-40. The correlation of the quantitative scores between the whole section and TMA cores was statistically significant (D2-40, rho = 0.84, p < 2.2e-16; calretinin, rho = 0.65, p = 7.9e-11). Neither the origin nor age of the blocks affected the results. CONCLUSION: If a minimum of four cores is used, TMA is an appropriate method for immunohistochemistry in MPM

Increased circulating miR-625-3p

INTRODUCTION: We investigated the ability of cell-free microRNAs (miRNAs) in plasma and serum to serve as a biomarker for malignant mesothelioma (MM). METHODS: Using miRNA microarrays, we profiled plasma samples from MM patients and healthy controls. miRNAs with significantly different abundance between cases and controls were validated in a larger series of MM patients and in an independent series of MM patients using quantitative real-time polymerase chain reaction. Levels of candidate miRNAs were also quantified in MM tumor samples. RESULTS: We compared cell-free miRNA profiles in plasma from MM patients with healthy controls. Reviewing 90 miRNAs previously reported to be associated with MM, we found that the levels of two miRNAs, miR-29c* and miR-92a, were elevated in plasma samples from MM patients. In addition, we identified 15 novel miRNAs present at significantly higher levels in the plasma of MM patients. Further analysis of candidate miRNAs by real time-quantitative polymerase chain reaction confirmed that one of them, miR-625-3p, was present in significantly higher concentration in plasma/serum from MM patients and was able to discriminate between cases and controls, in both the original and the independent series of patients. MiR-625-3p was also found to be up-regulated in tumor specimens from a group of 18 MM patients, who underwent extrapleural pneumonectomy. CONCLUSION: Our data confirm the potential of miR-29c* and miR-92a as candidate tumor markers and reveal that miR-625-3p is a promising novel diagnostic marker for MM