APMIS 120: 767–769
Ó 2012 The Authors APMIS Ó 2012 APMIS DOI 10.1111/j.1600-0463.2012.02891.x
Letter to the Editor 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.
Table 1. Clinical features of the patients included in the study and pathological characteristics of the analyzed MPM tissue specimens Patient Gender Stage1 Histotype Age (years) No. 1 Epithelioid 61 M T3N0M0 2 Epithelioid 51 M T3N0Mx 3 Epithelioid 63 M T4N1Mx 4 Epithelioid 64 F T3N0Mx 5 Epithelioid 62 M T4NxMx 6 Epithelioid 69 M T2N0Mx 7 Epithelioid 66 M T1N0M0 8 Epithelioid 46 M T4N1M0 9 Epithelioid 68 M T3N0M0 10 Epithelioid 62 M T2N0M0 11 Epithelioid 56 M T3N0M0 12 Epithelioid 49 F T3N0Mx 13 Epithelioid 69 M T3N2Mx 1 Stage according to the recommendations of the International Mesothelioma Interest Group. M, Metastases; N, Lymph nodes; T, Tumor.
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We found significant down-regulation of miR-17-5p and up-regulation of miR-221, but no differential expression of miR-30c and miR-222 in MPM- as compared to NP-specimens (Fig. 1 A–D). Moreover, miR-17-5p and miR-221 expression-pattern was opposite of that previously reported in MPM cell lines (8, 11), highlighting the difficulties in comparing in vitro results to in vivo tissue-samples. To rule out that the deregulation of miR-17-5p and miR-221 was caused by pre-surgical chemotherapy, we compared their expression in six surgical tissue-samples and in six patientmatched diagnostic biopsies collected prior to any treatment, and found no significant differential expression, We further validated that chemotherapy did not affect these miRs’ expression by observing comparable miR-
expression-levels in surgical samples and diagnostic biopsies from six independent MPM patients (Fig. 1 A–D). According to the International Mesothelioma Interest Group (IMIG)’s recommendations, an MPM-marker should have sensitivity or specificity greater than 80% (1). To test if miR-175p or miR-221 could fulfill these requirements we generated receiver-operating-characteristics (ROC)-curves using the RT-qPCR-data. As evidenced by area-under-the-curves (AUCs) 0.05, the IMIG-criteria for a suitable mesothelioma-marker were not met (Figure 1.E-F). Altogether our data indicate that although miR-17-5p, miR-30c, and miR-221/ 222 are reportedly specific for mesothelioma subtypes or differentially expressed in MPMcell-lines vs normal mesothelium-cell-lines, and
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Fig. 1. (A–D) Box plots of hsa-miR-17-5p, -30c, 221, and hsa-miR-222 expression in malignant pleural mesothelioma (MPM), adjacent non-neoplastic pleura (NP), and in a separate group of 12 diagnostic biopsies (DB). Total RNA fractions were purified from FFPE samples with RecoverAllTM Total Nucleic Acid Isolation Kit (Ambion). TaqMan® MicroRNA Assays (Part no. 4427975, Applied Biosystems) was used for the quantification of hsa-miR-17-5p (Assay ID: 000393), hsa-miR-30c (Assay ID: 000419), hsa-miR-221 (Assay ID: 000524) hsa-miR-222 (Assay ID: 002276) and reference RNU6B (Assay ID: 001093). RNU6B was used for normalization and 2 DCt values are shown. The boxes mark the interval between the 25th and 75th percentiles, the lines inside the boxes the median, and the whiskers the 10th and 90th percentiles. Filled circles outside the boxes are the minimum and maximum values. p-values are calculated with Mann–Whitney test or Wilcoxon matched pairs test. (E–F) ROC curves of miR-17-5p and miR-221 created using RT-qPCR data. Corresponding area-under-the-curve (AUC), 95% confidence interval (CI) and p-values are depicted. *p < 0.05 is considered statistically significant. Depiction of p < 0.05* in figure 1C represents the rounded number of an exact p-value of 0.0494.
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© 2012 The Authors APMIS © 2012 APMIS
LETTER TO THE EDITOR
able to play a pathogenic role in MPM (8–11), they may not be suitable diagnostic markers for differentiating MPM from RMP. This conclusion, however, requires corroboration in a larger set of samples. Although other miRs reported to be specifically dysregulated in MPM-cell-lines might eventually prove to be diagnostic biomarkers, we believe that for identification of miR-candidates potentially useful in differential diagnosis between MPM and RMP, a broad RT-qPCRscreen of comparative miR-expression in surgical samples from MPM and NP is needed. MiR-candidates can be subsequently validated directly on tissue sections by in situ-hybridization-techniques, to fully assess their histopathological diagnostic value and pathogenic role. This study was funded by donations from Dansk Kræftforsknings Fond, Jeppe Juhl & Hustrus Mindelegat and Kommunehospitalets 100 A˚rs Jubilæumsfond.
MORTEN ANDERSEN1 MORTEN GRAUSLUND,1 MARWA MUHAMMADALI,1 JESPER RAVN,2 JENS BENN SØRENSEN,3 CLAUS B. ANDERSEN1 and ERIC SANTONI-RUGIU1 Departments of 1Pathology, 2Thoracic Surgery, and 3Oncology, Rigshospitalet, Copenhagen University Hospital, Copenhagen, Denmark E-mail:
[email protected] REFERENCES 1. Husain AN, Colby TV, Ordo´n˜ez NG, Krausz T, Borczuk A, Cagle PT, et al. Guidelines for pathologic diagnosis of malignant mesothelioma: a consensus statement from the International Mesothelioma Interest Group. Arch Pathol Lab Med 2009;133:1317–31. 2. Shi M, Fraire AE, Chu P, Cornejo K, Woda BA, Dresser KA, et al. Oncofetal protein IMP3, a new diagnostic biomarker to distinguish malignant mesothelioma from reactive mesothelial proliferation. Am J Surg Pathol 2011;35: 878–82.
© 2012 The Authors APMIS © 2012 APMIS
3. Zimling ZG, Jørgensen A, Santoni-Rugiu E. The diagnostic value of immunohistochemically detected methylthioadenosine phosphorylase deficiency in malignant pleural mesotheliomas. Histopathology 2012;60:e96–105. 4. Sayed D, Abdellatif M. MicroRNAs in development and disease. Physiol Rev 2011;91:827–87. 5. Volinia S, Calin GA, Liu CG, Ambs S, Cimmino A, Petrocca F, et al. A microRNA expression signature of human solid tumors defines cancer gene targets. Proc Natl Acad Sci USA 2006;103:2257–61. 6. Siebolts U, Varnholt H, Drebber U, Dienes HP, Wickenhauser C, Odenthal M. Tissues from routine pathology archives are suitable for microRNA analyses by quantitative PCR. J Clin Pathol 2009;62:84–8. 7. Santarelli L, Strafella E, Staffolani S, Amati M, Emanuelli M, Sartini D, et al. Association of MiR-126 with soluble mesothelin-related peptides, a marker for malignant mesothelioma. PLoS One 2011;6:e18232. 8. Balatti V, Maniero S, Ferracin M, Veronese A, Negrini M, Ferrocci G, et al. MicroRNAs dysregulation in human malignant pleural mesothelioma. J Thorac Oncol 2011;6:844–51. 9. Benjamin H, Lebanony D, Rosenwald S, Cohen L, Gibori H, Barabash N, et al. A diagnostic assay based on microRNA expression accurately identifies malignant pleural mesothelioma. J Mol Diagn 2010;12:771–9. 10. Gee GV, Koestler DC, Christensen BC, Sugarbaker DJ, Ugolini D, Ivaldi GP, et al. Downregulated microRNAs in the differential diagnosis of malignant pleural mesothelioma. Int J Cancer 2010;127:2859–69. 11. Busacca S, Germano S, De Cecco L, Rinaldi M, Comoglio F, Favero F, et al. MicroRNA signature of malignant mesothelioma with potential diagnostic and prognostic implications. Am J Respir Cell Mol Biol 2010;42:312–9. 12. Mineo TC, Ambrogi V, Cufari ME, Pompeo E. May cyclooxygenase-2 (COX-2), p21 and p27 expression affect prognosis and therapeutic strategy of patients with malignant pleural mesothelioma? Eur J Cardiothorac Surg 2010;38: 245–52. 13. Opitz I, Soltermann A, Abaecherli M, Hinterberger M, Probst-Hensch N, Stahel R, et al. PTEN expression is a strong predictor of survival in mesothelioma patients. Eur J Cardiothorac Surg 2008;33:502–6.
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