Foss KM, Sima C, Ugolini D, Neri M, Allen KE, Weiss GJ.

*Translational Genomics Research Institute, Phoenix, Arizona; †Department of Oncology, Biology and Genetics, University of Genoa, Genoa; ‡Unit of Epidemiology, Biostatistics and Clinical Trials, National Cancer Research Institute, Genoa; §Rehabilitative Pneumology, IRCCS San Raffaele Pisana, Rome, Italy; and ∥Virginia G. Piper Cancer Center at Scottsdale Healthcare, Scottsdale, Arizona.

Abstract

Introduction: The ability to diagnose non-small cell lung cancer (NSCLC) at an early stage may lead to improved survival. The aim of this study was to identify differentially expressed serum-based microRNAs (miRNAs) between patients with early-stage NSCLC and controls. These miRNAs may serve as biomarkers for NSCLC early detection.

Methods: miRNA profiling was performed on total RNA extracted from serum obtained from 22 individuals (11 controls and 11 patients with early-stage NSCLC). Quantitative polymerase chain reaction (qPCR) was used to validate the profiling results in the discovery set and in a validation set of 31 controls and 22 patients with early-stage NSCLC. Additionally, six matched plasma samples (four NSCLC cases and two controls) and three serum mesothelioma samples were analyzed by qPCR. Receiver operating characteristic curves were generated for each possible combination of the miRNAs measured by qPCR.

Results: The expression of hsa-miR-1254 and hsa-miR-574-5p was significantly increased in the early-stage NSCLC samples with respect to the controls. Receiver operating characteristic curves plotting these two miRNAs were able to discriminate early-stage NSCLC samples from controls with 82% and 77% of sensitivity and specificity, respectively, in the discovery cohort and with 73% and 71% of sensitivity and specificity, respectively, in the validation cohort. The mesothelioma and plasma samples did not seem to classify into either NSCLC or control groups.

Conclusions: Serum miRNAs are differentially expressed between patients with early-stage NSCLC and controls. The utility of miR-1254 and miR-574-5p serum-based biomarkers as minimally invasive screening and triage tools for subsequent diagnostic evaluation warrants additional validation.

Marini V, Michelazzi L, Cioé A, Fucile C, Spigno F, Robbiano L.

Department of Internal Medicine, Division of Clinical Pharmacology and Toxicology, University of Genoa, Viale Benedetto XV, 2, I-16132 Genoa, Italy.

Abstract

Inhalation of asbestos, a mineral extensively used in a variety of applications, is strongly associated with malignant mesothelioma (MM), a fatal cancer of the pleura. Soluble mesothelin-related peptides (SMRP) are a promising biomarker suggested for the screening of MM in healthy asbestos-exposed subjects. In the present study a comparison of micronucleus (Mn) frequencies in peripheral blood lymphocytes (PBL) between 44 asbestos-exposed and 22 control individuals has been performed, and the correlation with serum SMRP has been examined. SMRP levels were found to be significantly higher in subjects exposed to asbestos and in their various subgroups than in controls. Concerning micro-nucleated lymphocytes, a statistically significant difference from controls was seen in the percentages of both micro-nucleated mononucleated lymphocytes (MnMNL) and micro-nucleated binucleated lymphocytes (MnBNL), but the difference was markedly higher for the percentage of micro-nucleated polynucleated lymphocytes (MnPNL). With respect to the correlation between the frequency of the three types of micronucleated lymphocytes and serum-SMRP values of asbestos-exposed subjects, it was statistically significant for MnMNL, but not for MnBNL and MnPNL.

Expression of GATA-6 transcription factor in pleural malignant mesothelioma and metastatic pulmonary adenocarcinoma.

Lindholm PM, Soini Y, Myllärniemi M, Knuutila S, Heikinheimo M, Kinnula VL, Salmenkivi K.

Department of Medicine, University of Helsinki and Helsinki University Central Hospital, Finland.

Abstract

Background: Malignant mesothelioma (MM) is a highly aggressive tumour with poor prognosis and limited response to therapy. New markers for the prediction of prognosis in MM and in pulmonary adenocarcinoma of the pleura are valuable. GATA-6 belongs to a six member zinc finger transcription factor family named after their recognition motif W-GATA-R. AIM: To clarify the distribution and possible function of GATA-6 transcription factor in MM and in pleural metastasis of lung adenocarcinomas.

Methods: 63 pleural MM and 36 pleural metastatic pulmonary adenocarcinomas were studied for GATA-6 expression by immunohistochemistry using tissue microarrays. Expression of GATA-6 was examined in relation to thyroid transcription factor-1 expression, survival, proliferation and apoptosis.

Results: Nuclear immunoreactivity for GATA-6 was stronger and more frequent in MM than in metastatic pleural adenocarcinoma. Prognosis was better in patients with GATA-6 expression when compared to those with no GATA-6 expression (p = 0.002); in the subgroup analysis the difference was significant in epithelial and sarcomatous mesothelioma. GATA-6 was not associated with spontaneous proliferation or apoptosis of the tumour cells in situ.

Conclusion: Results suggest that GATA-6 plays a role in pleural malignancies, predicting longer survival in subgroups of MM.

Greillier L, Baas P, Welch JJ, Hasan B, Passioukov A.

European Organisation for Research and Treatment of Cancer (EORTC), Headquarters, Brussels, Belgium. laurent.greillier@mail.ap-hm.fr

Abstract

Malignant pleural mesothelioma (MPM) is an aggressive tumor with poor prognosis, whose main etiology is exposure to asbestos fibers. The incidence of MPM is anticipated to increase worldwide during the first half of this century. For various reasons, MPM is difficult to diagnose and is notoriously refractory to most treatments. However, recently two active chemotherapy regimens have been demonstrated to significantly increase survival in patients with MPM, and several therapeutic agents and strategies are currently under evaluation.

Researchers have actively sought MPM biomarkers for more than 20 years. Biomarkers would be helpful in managing three clinical aspects of MPM: early diagnosis, prognosis, and treatment outcome prediction. The aims of the present review are to summarize the published and recently presented data on MPM biomarkers and to identify the prospects for future translational research projects.

Among the ‘classical’ diagnostic biomarkers measured in biological fluids, such as cytokeratins and cell surface antigens, none discriminate patients with MPM from those with other malignancies and nonmalignant diseases. Osteopontin, soluble mesothelin, and megakaryocyte potentiating factor (MPF) appear to be the most promising of the recent biomarkers, but are still subject to some limitations. Osteopontin lacks specificity for mesothelioma, while both soluble mesothelin and MPF lack sensitivity for detecting non-epithelial subtypes. Panels consisting of a small set of biomarkers do not improve the diagnostic yield, and results from molecular profiling are too preliminary to be brought into daily clinical practice. While a large number of biomarkers have been assessed in biological fluids and tumor tissue for their prognostic value, none have had a widespread impact on clinical practice. In contrast, data concerning predictive biomarkers are very limited, even though they are most interesting from the perspective of clinicians.

Additional prospective studies, in large and independent samples of patients, with rigorous statistical methodology and standardized laboratory techniques are now warranted to validate and define the precise value of diagnostic and prognostic MPM biomarkers. Future research efforts should focus on biomarkers predictive of the efficacy and toxicity of standard chemotherapy. Translational research should be systematically incorporated into the design of clinical trials assessing new targeted agents in MPM.

Scherpereel A, Grigoriu BD, Astoul P.

Service de Pneumologie et Oncologie Thoracique, Hôpital Calmette, CHRU de Lille, Lille, France. a-scherpereel@chru-lille.fr

Abstract

Malignant pleural mesothelioma (MPM) is a serious issue worldwide because of its increasing incidence and poor prognosis despite real recent improvements in the disease management. Most of the patients are diagnosed late in the course of the disease when radical treatment is no more an option. Therefore an earlier diagnosis of MPM is needed to significantly increase the survival of patients. Some soluble markers, including soluble mesothelin and osteopontin, have been previously proposed for MPM diagnosis but none has been validated yet. Soluble mesothelin, assessed in blood and in pleural effusion, seems to be the most promising candidate. However, even if it has a good diagnostic and prognostic value, it is quite specific for the epithelioid subtype, the most frequent one of mesothelioma, thus limiting its usefulness in practice. Despite sometimes a good sensitivity, other potential markers as osteopontin are of little interest for MPM diagnosis because of a low specificity. In conclusion, the present data do not justify the use of biology for MPM diagnosis in routine yet but rather suggest a need for a continuing evaluation of soluble mesothelin in clinical studies and the search for other potential tumor markers.

Keywords: Mesothelioma, Tumor marker, Biology, Thoracoscopy, Pleural cancer

Inami K, Kajino K, Abe M, Hagiwara Y, Maeda M, Suyama M, Watanabe S, Hino O.

Department of Pathology and Oncology, Juntendo University School of Medicine, Tokyo 113-8421, Japan.

Abstract

ERC/mesothelin gene (MSLN) encodes a precursor protein, which is cleaved by proteases to generate N-ERC/mesothelin and C-ERC/mesothelin. N-ERC/mesothelin is a soluble protein, also known as megakaryocyte-potentiating factor, which is released into extracellular space. N-ERC/mesothelin is known to be a serum marker of mesothelioma. We have previously developed an enzyme-linked immunosorbent assay system for N-ERC/mesothelin, which can detect mesothelioma. C-ERC/mesothelin is expressed in normal mesothelial cell, pancreatic cancers, ovarian cancers, mesotheliomas and some other cancers. Pancreatic ductal carcinoma remains a fatal disease because its diagnosis often occurs very late. In this study, we examined ERC/mesothelin expression in human pancreatic cancer cell lines (MIA-PaCa2, PK-1, KP-3, TCC-PAN2, PK-59 and PK-45H) by reverse transcription-polymerase chain reaction and immunoblotting and N-ERC/mesothelin concentration in the supernatant of cultured cancer cells by the ELISA system.
We also investigated C-ERC/mesothlein expression in human pancreatic ductal carcinoma tissues by immunostaining using 5B2 anti-mesothelin monoclonal antibody and N-ERC/mesothelin concentration in sera obtained from patients with pancreatic ductal carcinoma via ELISA. In vitro, N-ERC/mesothelin concentration in cell culture medium nearly correlated with the expression level of C-ERC/mesothelin. Although C-ERC/mesothelin was frequently expressed in human pancreatic ductal carcinoma, serum N-ERC/mesothelin concentration of cancer patients was equivalent to healthy controls. N-ERC/mesothelin was not useful as a serum marker of pancreatic ductal carcinoma, but because of frequent expression, C-ERC/mesothelin might be useful as a target of molecular imaging and immunotherapy.

Schneider J, Hoffmann H, Dienemann H, Herth FJ, Meister M, Muley T.

Institute and Policlinic for Occupational and Social Medicine, Justus-Liebig Universität, Giessen, Germany.

Abstract

Introduction and Methods: We investigated the diagnostic and prognostic value of soluble mesothelin-related proteins (SMRP) in sera from patients with newly diagnosed malignant pleural mesothelioma (MPM) (n = 100), MPM patients at tumor relapse (n = 29), primary lung cancer (n = 139), and benign asbestosis (n = 75) using Mesomark-enzyme-linked immunosorbent assay kit (Fujirebio Diagnostics, Malvern, PA).

Results: SMRP concentrations were significantly higher in MPM compared with benign asbestosis (p < 0.001) or lung cancer (p < 0.001). The median values were 1.4 nM, 0.9 nM, and 0.8 nM, respectively. The best statistical cutoff was found to be 1.35 nM resulting in a sensitivity of 53% and a specificity of 82.7%. Receiver operating characteristics curves gave an area under curve of 0.72 for the discrimination between MPM and non-MPM patients (p < 0.001). No significant differences in SMRP levels were found among histologies and stages of MPM. The highest median SMRP levels (4.2 nM) were measured in 29 MPM patients with relapse/progression (75.8% > 1.35 nM). Univariately, SMRP discriminated significantly (p < 0.003) between favorable (n = 71, median survival: 17.1 month; 1-year survival: 63.1%) and worse prognosis (n = 20, median survival: 8.4 months, 1-year survival: 32%) at 3.5 nM. In multivariate analysis, histology, therapy, and SMRP were shown to be independent prognostic
factors in all MPM patients (hazard ratio for SMRP: 1.96; p = 0.025). Nevertheless, subtype-driven reanalysis showed only a trend in epithelial MPM.

Conclusion: In conclusion, SMRP add limited information to the diagnosis of MPM. Nevertheless, SMRP might be a useful measure in treatment and monitoring of MPM. The prognostic impact of SMRP in MPM is not conclusive and needs further evaluation.

Hasegawa S, Koshikawa M, Takahashi I, Hachiya M, Furukawa T, Akashi M, Yoshida S, Saga T.

Diagnostic Imaging Group, Molecular Imaging Center, National Institute of Radiological Sciences, 4-9-1 Anagawa, Inage-ku, Chiba 263-8555, Japan. shase@nirs.go.jp

Abstract

Background and Aim: Molecular diagnostics and therapeutics of human mesothelioma using disease-related markers present major challenges in clinical practice. To identify biochemical alternations that would be markers of human mesothelioma, we measured the intracellular steady-state levels of biologically important trace metals such as manganese (Mn), copper (Cu), and zinc (Zn) in a human mesothelial cell line, MeT-5A, and in five human mesothelioma cell lines (MSTO-211H, NCI-H226, NCI-H2052, NCI-H2452, ACC-MESO-1) by inductively coupled plasma-mass spectrometry (ICP-MS). We also aimed to investigate whether the alterations were related to the intracellular status of metal-containing superoxide dismutase (SOD).

Results: There were no significant differences in the contents of the trace metals among MeT-5A, MSTO-211H, and ACC-MESO-1 cells. However, each of the other three mesothelioma cell lines had a unique characteristic in terms of the intracellular amounts of the metals; NCI-H226
contained an extremely high level of Mn, an amount 7.3-fold higher than that in MeT-5A. NCI-H2052 had significantly higher amounts of Cu (3.4-fold) and Zn (1.3-fold) compared with MeT-5A. NCI-H2452 contained about 5.8-fold the amount of Cu and 2.5-fold that of Mn compared with MeT-5A. As for the intracellular levels of copper/zinc-SOD (Cu/Zn-SOD) and manganese-SOD (Mn-SOD), those of Cu/Zn-SOD were relatively unchanged among the cells tested, and no notable correlation with Cu or Zn contents was observed. On the other hand, all mesothelioma cells highly expressed Mn-SOD compared with MeT-5A, and a very high expression of the enzyme with a robust activity was observed in the two mesothelioma cells (NCI-H226, NCI-H2452) containing a large amount of Mn.

Conclusions: In comparison with MeT-5A human mesothelial cells, some human mesothelioma cells had significantly higher amounts of Mn or Cu and one mesothelioma cell had a significantly higher amount of Zn. Interestingly, all mesothelioma cells overexpressed Mn-SOD compared with MeT-5A, and the cells whose Mn-SOD activity was increased contained higher amounts of Mn. It seemed that intracellular Mn content was positively correlated with Mn-SOD, suggesting that the intracellular Mn level is associated with Mn-SOD activity. These biochemical signatures could be potential disease-related markers of mesothelioma.

Keywords: Mesothelioma; Trace element; Superoxide dismutase

Creaney J, Yeoman D, Demelker Y, Segal A, Musk AW, Skates SJ, Robinson BW.

National Research Centre for Asbestos Related Diseases, Western Australian Institute of Medical Research, School of Medicine and Pharmacology, University of Western Australia, Sir Charles Gairdner Hospital, Nedlands, Australia. creaneyj@cyllene.uwa.edu.au

Abstract

Introduction: There is increasing interest in identifying a blood-based marker for the asbestos-related tumor, malignant mesothelioma. Three potential markers for mesothelioma are mesothelin, megakaryocyte potentiating factor (MPF) and osteopontin. The purpose of the current study was to directly compare these biomarkers in the same sample population, determining their sensitivity and specificity in establishing a diagnosis, and to determine if diagnostic accuracy for mesothelioma is improved by combining the data from all three markers.

Methods: Serum levels of mesothelin, MPF and osteopontin were determined by commercially available assays in 66 samples from patients with pleural malignant mesothelioma, 20 healthy individuals, 21 patients with asbestos-related lung or pleural disease, 30 patients presenting with benign pleural effusions and 30 patients with other malignancies.

Results: Serum levels of the three markers were elevated in mesothelioma patients. At a level of specificity of 95% relative to healthy controls and patients with benign asbestos related disease, the sensitivity for mesothelioma was 34% for MPF, 47% for osteopontin and 73% for mesothelin. Osteopontin and MPF were unable to differentiate patients with mesothelioma from patients with other malignancies or those presenting with transudative pleural effusions. Combining the data from the three biomarkers using a logistic regression model did not improve sensitivity for detecting mesothelioma above that of the mesothelin marker alone.

Conclusion: Serum mesothelin remains the most specific marker for the diagnosis of mesothelioma.

Amati M, Tomasetti M, Mariotti L, Tarquini LM, Valentino M, Santarelli L.

Department of Molecular Pathology and Innovative Therapies, Institute of Occupational Medicine, Polytechnic University of Marche, Ancona, Italy.

Abstract

Epidemiological studies have shown that mortality from malignant mesothelioma (MM) and lung cancer have increased with increasing cumulative exposure to asbestos. To investigate whether tumour-related biomarkers can contribute towards the evaluation of the carcinogenic risk in populations exposed to asbestos, the DNA adduct 8-hydroxy-2′-deoxyguanosine (8OHdG), interleukine-6 (IL-6), platelet-derived growth factor (PDGF-BB), hepatocyte growth factor (HGF), basic fibroblast growth factor (bFGF), vascular endothelial growth factor (VEGFbeta) and soluble mesothelin-related peptides (SMRPs) were analysed in a cohort of workers differently exposed to asbestos fibres at the workplace. To document biomarker levels in an unexposed population, 54 age-matched subjects were enrolled. A total of 119 subjects with a history of occupational exposure to asbestos underwent clinical examination and were interviewed by trained personnel, responding to a detailed questionnaire related to duration of asbestos exposure, smoking, and occupational task. According to the occupational tasks, asbestos-exposed subjects were analysed for their asbestos cumulative dose and the association with the biomarkers was evaluated. Among the occupational groups, maintenance workers, pipe fitters and electricians were exposed to a higher cumulative dose of asbestos fibres. Exposure to asbestos significantly increased the steady-state content of 8OHdG in DNA. Elevated levels of 8OHdG and IL-6 best reflected a high level of SMRPs, which is related to cell transformation. Subjects heavily exposed to asbestos [>60(ff/cm(3))xyears] showed also a higher level of angiogenic factors. A combination of angiogenic biomarkers with a specific mesothelioma-biomarker such as SMRPs could be used for close surveillance of workers with a history of asbestos exposure.