Are differentially expressed microRNAs useful in the diagnostics of malignant pleural mesothelioma?

Malignant pleural mesothelioma (MPM) is radiologically and histologically difficult to distinguish from reactive mesothelial proliferations (RMPs), partly because proposed MPMmarkers have not shown enough specificity and reproducibility or need further validation (1–3). MicroRNAs (miRs) are small non-coding RNA-strands (~22 nt) post-transcriptionally regulating gene-expression and playing increasingly evident roles in several diseases (4). MiRs might potentially be attractive biomarkers for MPM, as MiR-expression in other cancers has shown prognostic and diagnostic significance (5). Furthermore, miRs are more stable and much smaller than mRNAs, thus they can be reliably detected in formalin-fixed paraffin-embedded (FFPE) tissues (6). However, it is unclear whether currently published miR-data may provide biomarker candidates for differentiating MPM from RMP, as it has been implied (7–11). The only study published to date comparing miR-expression in patientmatched MPM and adjacent non-neoplastic pleura (NP) samples, was based on few analyzed miRs (7). Recently, more extensive in vitro analysis compared miR-expression in normal mesothelial cell-cultures and commercially available MPM-cell-lines (8). Other comprehensive studies in vivo instead focused on miRsignatures differentiating MPM histological subtypes (epithelioid, sarcomatoid, and biphasic) or discriminating MPM from lung adenocarcinoma (9–11). For instance, Busacca et al. reported that the expression of miR-17-5p and miR-30c, already known to be related to other malignancies (5), was associated with mesothelioma subtypes (11). Others recently confirmed miR-17-5p up-regulation in MPM-cell-lines, in association with reduced expression of one of its targets, the cyclin-dependent kinase-inhibitor (CDKI) p21 (8, 12). In contrast miR-221 and -222, known negative regulators of the CDKI p27 and the tumor suppressor PTEN, were reported as down-regulated in human MPM cell lines compared to non-tumorous immortalized mesothelial cells (11). This is somehow surprising given that loss of p27 and PTEN expression is frequent and associated with poor prognosis in MPM patients (4,12,13) To test whether miR-17-5p, -30c, -221, and 222 may be useful in differentiating MPM from RMP in vivo, we quantified and compared their expression in FFPE epithelioid MPM (stages I-IV) and NP tissue-samples collected from 13 patients (11 male patients, 2 female patients, ages 49–69) (Table 1) during extra-pleural pneumonectomy preceded by 1–3 cycles of chemotherapy (vinorelbine/cisplatin or gemcitabine/carboplatin). We quantified these miRs and reference RNU6B by realtime-PCR-based TaqMan MicroRNA Assays (Applied Biosystems, Foster City, CA, USA) according to manufacturer’s instructions using 7500 Real-Time PCR System (Applied Biosystems). Threshold-cycles (Ct) were determined with related system-software (SDS v1.2.2, Applied Biosystems) and PCR-data analyzed comparatively with RNU6B as normalizer. Statistically significant (p < 0.05) differences between independent or paired groups were detected by nonparametric Mann–Whitney and Wilcoxon matched pairs tests, respectively.