Malignant mesothelioma (MM) is strongly associated with a previous asbestos exposure. To improve timely detection of MM in asbestos workers, better screening tools – like minimally-invasive biomarkers – are desirable. Between 2008 and 2018 2,769 patients with benign asbestos-related diseases were recruited to participate in annual screens. Using a nested case-control design the protein markers calretinin and mesothelin were determined by enzyme-linked immunosorbent assays in prediagnostic plasma samples of 34 MM cases as well as 136 matched controls from the cohort. Conditional on a pre-defined specificity of 98% for calretinin and 99% for mesothelin the markers reached individual sensitivities of 31% and 23%, respectively, when including the incident cases with samples taken between one and 15 months before diagnosis. The combination of both markers increased the sensitivity to 46% at 98% specificity. Marker complementation increased with earlier sampling. The marker combination improves the sensitivity of the individual markers, indicating a useful complementation and suggesting that additional markers may further improve the performance. This is the first prospective cohort study to evaluate a detection of MM by calretinin and its combination with mesothelin up to about a year before clinical diagnosis. Whether an earlier diagnosis will result in reduced mortality has yet to be demonstrated.

BackgroundCalretinin is one of the well-established immunohistochemical markers in the diagnostics of malignant mesothelioma (MM). Its utility as a diagnostic tool in human blood, however, is scarcely investigated. The aim of this study was to develop an enzyme-linked immunosorbent assay (ELISA) for human calretinin in blood and to assess its usefulness as a potential minimally invasive diagnostic marker for MM.MethodsInitially, attempts were made to establish an assay using commercially available antibodies and to optimize it by including a biotin-streptavidin complex into the assay protocol. Subsequently, a novel ELISA based on polyclonal antibodies raised in rabbit immunized with human recombinant calretinin was developed. The assay performance in human serum and plasma (EDTA/heparin) and the influence of calcium concentrations on antibody recognition were studied. Stability of spiked-in calretinin in EDTA plasma under different storage conditions was also examined. In preliminary studies serum and plasma samples from 97 healthy volunteers, 35 asbestos-exposed workers, and 42 MM patients were analyzed.ResultsThe mean detection range of the new ELISA was 0.12 to 8.97 ng/ml calretinin. The assay demonstrated markedly lower background and significantly higher sensitivity compared to the initially contrived assay that used commercial antibodies. Recovery rate experiments confirmed dependence of calretinin antibody recognition on calcium concentration. Calcium adjustment is necessary for calretinin measurement in EDTA plasma. Spiked-in calretinin revealed high stability in EDTA plasma when stored at room temperature, 4°C, or after repeated freeze/thaw cycles. Median calretinin values in healthy volunteers, asbestos workers, and MM patients were 0.20, 0.33, and 0.84 ng/ml, respectively (p < 0.0001 for healthy vs. MM, p = 0.0036 for healthy vs. asbestos-exposed, p < 0.0001 for asbestos-exposed vs. MM). Median values in patients with epithelioid and biphasic MM were similar. No influence of age, gender, smoking status, or type of medium (plasma/serum) on calretinin values was found.ConclusionsThe novel assay is highly sensitive and applicable to human serum and plasma. Calretinin appears to be a promising marker for the blood-based detection of MM and might complement other markers. However, further studies are required to prove its usefulness in the diagnosis of MM patients.

BackgroundFor the detection of malignant mesothelioma no single biomarker with reasonable sensitivity and specificity has been described so far. Mesothelin, the most prominent blood-based biomarker, is characterized by high specificity but low sensitivity. It might be reasonable to combine biomarkers of different molecular classes in order to improve the overall performance. The aim of this study was to assess the performance of the combination of mesothelin and miR-103a-3p as blood-based biomarker for mesothelioma.Methods/Principal FindingsMesothelin concentration in plasma and miR-103a-3p levels in the cellular blood fraction were analyzed in 43 male mesothelioma patients and 52 male controls formerly exposed to asbestos. For the discrimination of epithelioid and biphasic mesothelioma from asbestos-exposed controls mesothelin and miR-103a-3p showed 74% and 89% sensitivity and 85% and 63% specificity, respectively. For the combination of mesothelin and miR-103a-3p a sensitivity of 95% and a specificity of 81% were calculated.Conclusions/SignificanceThe results of this study show that the combination of mesothelin and miR-103a-3p improves the diagnostic performance of individual blood-based biomarker to detect malignant mesothelioma. The obtained results indicate that the use of biomarkers of different molecular classes might be a reasonable approach to assemble a biomarker panel.

BackgroundMalignant mesothelioma (MM) is a deadly cancer mainly caused by previous exposure to asbestos. With a latency period up to 50 years the incidence of MM is still increasing, even in countries that banned asbestos. Secondary prevention has been established to provide persons at risk regular health examinations. An earlier detection with tumor markers might improve therapeutic options. Previously, we have developed a new blood-based assay for the protein marker calretinin. Aim of this study was the verification of the assay in an independent study population and comparison with the established marker mesothelin.MethodsFor a case-control study in men, a total of 163 cases of pleural MM and 163 controls were available from Australia, another 36 cases and 72 controls were recruited in Germany. All controls had asbestosis and/or plaques. Calretinin and mesothelin were determined by ELISA (enzyme-linked immunosorbent assay) in serum or plasma collected prior to therapy. We estimated the performance of both markers and tested factors potentially influencing marker concentrations like age, sample storage time, and MM subtype.ResultsCalretinin was able to detect all major subtypes except for sarcomatoid MM. Calretinin showed a similar performance in Australian and German men. At a pre-defined specificity of 95% the sensitivity of calretinin reached 71% and that of mesothelin 69%, when excluding sarcomatoid MM. At 97% specificity, the combination with calretinin increased the sensitivity of mesothelin from 66% to 75%. Sample storage time did not influence the results. In controls the concentrations of calretinin increased 1.87-fold (95% CI 1.10–3.20) per 10 years of age and slightly more for mesothelin (2.28, 95% CI 1.30–4.00).ConclusionsCalretinin could be verified as a blood-based marker for MM. The assay is robust and shows a performance that is comparable to that of mesothelin. Retrospective analyses would not be limited by storage time. The high specificity supports a combination of calretinin with other markers. Calretinin is specific for epithelioid and biphasic MM but not the rarer sarcomatoid form. Molecular markers like calretinin and mesothelin are promising tools to improve and supplement the diagnosis of MM and warrant further validation in a prospective study.Electronic supplementary materialThe online version of this article (doi:10.1186/s12885-017-3375-5) contains supplementary material, which is available to authorized users.

Urine-based biomarkers for non-invasive diagnosis of bladder cancer are urgently needed. No single marker with sufficient sensitivity and specificity has been described so far. Thus, a combination of markers appears to be a promising approach. The aim of this case-control study was to evaluate the performance of an in-house developed enzyme-linked immunosorbent assay (ELISA) for survivin, the UBC® Rapid test, and the combination of both assays. A total of 290 patients were recruited. Due to prior bladder cancer, 46 patients were excluded. Urine samples were available from 111 patients with bladder cancer and 133 clinical controls without urologic diseases. Antibodies generated from recombinant survivin were utilized to develop a sandwich ELISA. The ELISA and the UBC® Rapid test were applied to all urine samples. Receiver operating characteristic (ROC) analysis was used to evaluate marker performance. The survivin ELISA exhibited a sensitivity of 35% with a specificity of 98%. The UBC® Rapid test showed a sensitivity of 56% and a specificity of 96%. Combination of both assays increased the sensitivity to 66% with a specificity of 95%. For high-grade tumors, the combination showed a sensitivity of 82% and a specificity of 95%. The new survivin ELISA and the UBC® Rapid test are both able to detect bladder cancer, especially high-grade tumors. However, the performance of each individual marker is moderate and efforts to improve the survivin assay should be pursued. A combination of both assays confirmed the benefit of using marker panels. The results need further testing in a prospective study and with a high-risk population.