[Show abstract][Hide abstract] ABSTRACT: For 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.
Mesothelin 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.
The 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.
[Show abstract][Hide abstract] ABSTRACT: This article describes the principles of marker research with prospective studies along with examples for diagnostic tumor markers. A plethora of biomarkers have been claimed as useful for the early detection of cancer. However, disappointingly few biomarkers were approved for the detection of unrecognized disease, and even approved markers may lack a sound validation phase. Prospective studies aimed at the early detection of cancer are costly and long-lasting and therefore the bottleneck in marker research. They enroll a large number of clinically asymptomatic subjects and follow-up on incident cases. As invasive procedures cannot be applied to collect tissue samples from the target organ, biomarkers can only be determined in easily accessible body fluids. Marker levels increase during cancer development, with samples collected closer to the occurrence of symptoms or a clinical diagnosis being more informative than earlier samples. Only prospective designs allow the serial collection of pre-diagnostic samples. Their storage in a biobank Upgrades cohort studies to serve for both, marker discovery and validation. Population-based cohort studies, which may collect a wealth of data, are commonly conducted with just one baseline investigation lacking serial samples. However, they can provide valuable information about factors that influence the marker level. Screening programs can be employed to archive serial samples but require significant efforts to collect samples and auxiliary data for marker research. Randomized controlled trials have the highest level of evidence in assessing a biomarker's benefit against usual care and present the most stringent design for the validation of promising markers as well as for the discovery of new markers. In summary, all kinds of prospective studies can benefit from a biobank as they can serve as a platform for biomarker research. This article is part of a Special Issue entitled: Biomarkers: A Proteomic Challenge.
No preview · Article · Dec 2013 · Biochimica et Biophysica Acta
[Show abstract][Hide abstract] ABSTRACT: The long noncoding RNA MALAT1 (metastasis-associated lung adenocarcinoma transcript 1) is described as a potential biomarker for NSCLC (non-small cell lung cancer). Diagnostic biomarkers need to be detectable in easily accessible body fluids, should be characterized by high specificity, sufficient sensitivity, and robustness against influencing factors. The aim of this study was to evaluate the performance of MALAT1 as a blood based biomarker for NSCLC.
MALAT1 was shown to be detectable in the cellular fraction of peripheral human blood, showing different expression levels between cancer patients and cancer-free controls. For the discrimination of NSCLC patients from cancer-free controls a sensitivity of 56% was calculated conditional on a high specificity of 96%. No impact of tumor stage, age, gender, and smoking status on MALAT1 levels could be observed, but results based on small numbers.
The results of this study indicate that MALAT1 complies with key characteristics of diagnostic biomarkers, i.e., minimal invasiveness, high specificity, and robustness. Due to its relatively low sensitivity MALAT1 might not be feasible as a single biomarker for the diagnosis of NSCLC in the cellular fraction of blood. Alternatively, MALAT1 might be applicable as a complementary biomarker within a panel in order to improve the entire diagnostic performance.
Full-text · Article · Dec 2013 · BMC Research Notes
[Show abstract][Hide abstract] ABSTRACT: UROtsa is an authentic, immortalized human urothelial cell line that is used to study the effects of metals and other toxic substances, mostly in the context of bladder cancer carcinogenesis. Unusual properties on the molecular level of a provided UROtsa cell line stock prompted us to verify its identity.
UROtsa cell line stocks from different sources were tested on several molecular levels and compared with other cell lines. MicroRNA and mRNA expression was determined by Real-Time PCR. Chromosome numbers were checked and PCR of different regions of the large T-antigen was performed. DNA methylation of RARB, PGR, RASSF1, CDH1, FHIT, ESR1, C1QTNF6, PTGS2, SOCS3, MGMT, and LINE1 was analyzed by pyrosequencing and compared with results from the cell lines RT4, T24, HeLa, BEAS-2B, and HepG2. Finally, short tandem repeat (STR) profiling was applied.
All tested UROtsa cell line stocks lacked large T-antigen. STR analysis unequivocally identified our main UROtsa stock as the bladder cancer cell line T24, which was different from two authentic UROtsa stocks that served as controls. Analysis of DNA methylation patterns and RNA expression confirmed their differences. Methylation pattern and mRNA expression of the contaminating T24 cell line showed moderate changes even after long-term culture of up to 56 weeks, whereas miRNAs and chromosome numbers varied markedly.
It is important to check the identity of cell lines, especially those that are not distributed by major cell banks. However, for some cell lines STR profiles are not available. Therefore, new cell lines should either be submitted to cell banks or at least their STR profile determined and published as part of their initial characterization. Our results should help to improve the identification of UROtsa and other cells on different molecular levels and provide information on the use of urothelial cells for long-term experiments.
[Show abstract][Hide abstract] ABSTRACT: To date, no biomarkers with reasonable sensitivity and specificity for the early detection of malignant mesothelioma have been described. The use of microRNAs (miRNAs) as minimally-invasive biomarkers has opened new opportunities for the diagnosis of cancer, primarily because they exhibit tumor-specific expression profiles and have been commonly observed in blood of both cancer patients and healthy controls. The aim of this pilot study was to identify miRNAs in the cellular fraction of human peripheral blood as potential novel biomarkers for the detection of malignant mesothelioma.
Using oligonucleotide microarrays for biomarker identification the miRNA levels in the cellular fraction of human peripheral blood of mesothelioma patients and asbestos-exposed controls were analyzed. Using a threefold expression change in combination with a significance level of p<0.05, miR-103 was identified as a potential biomarker for malignant mesothelioma. Quantitative real-time PCR (qRT-PCR) was used for validation of miR-103 in 23 malignant mesothelioma patients, 17 asbestos-exposed controls, and 25 controls from the general population. For discrimination of mesothelioma patients from asbestos-exposed controls a sensitivity of 83% and a specificity of 71% were calculated, and for discrimination of mesothelioma patients from the general population a sensitivity of 78% and a specificity of 76%.
The results of this pilot study show that miR-103 is characterized by a promising sensitivity and specificity and might be a potential minimally-invasive biomarker for the diagnosis of mesothelioma. In addition, our results support the concept of using the cellular fraction of human blood for biomarker discovery. However, for early detection of malignant mesothelioma the feasibility of miR-103 alone or in combination with other biomarkers needs to be analyzed in a prospective study.
[Show abstract][Hide abstract] ABSTRACT: In contrast to the temporary administration of arsenic in cancer therapy (e.g. in the form of arsenic trioxide), chronic exposure to low doses can cause bladder cancer and other cancers. Especially the trivalent arsenic species MMA(III) (monomethylarsonous acid) and DMA(III) (dimethylarsinous acid) are known to be highly toxic. In the present study we analysed the soluble, intracellular biotransformation products of MMA(III) in methylating HepG2 (hepatocytes) and non-methylating UROtsa cells (urothelial cells) after various times of exposure. As most of the intracellulararsenic is bound to cellular structures and proteins the soluble arsenicmetabolites can hardly be speciated and even less quantified. Using an improved isolation procedure and HPLC-ICP/MS, we investigated the time-resolved biotransformation of MMA(III) and detected and quantified MMA(V) (monomethylarsonic acid) as an oxidation product of MMA(III) and, to a minor degree, DMA(V) (dimethylarsenic acid) as a methylation and oxidation product of MMA(III) in the lysates of HepG2 cells. In contrast, only MMA(V) but no DMA(V) was detected in the lysates of UROtsa cells. We conclude from our study that MMA(III) is taken up by HepG2 and UROtsa cells and immediately oxidized to MMA(V). Only in HepG2 cellsMMA(V) is finally methylated to DMA(V) over time. The new method might help to advance the analysis of metabolic pathways of arsenic in mammalian cells.
No preview · Article · Dec 2011 · Journal of Analytical Atomic Spectrometry
[Show abstract][Hide abstract] ABSTRACT: The aim of the study is to examine the cancer-predictive values of SMRP (soluble mesothelin-related peptides), CA125, and CYFRA21-1 as potential tumor markers for lung cancer and malignant mesothelioma in a cohort of workers formerly exposed to asbestos. A voluntary surveillance program has been established for German workers with former asbestos exposure. A subgroup of 626 subjects with a mean age of 63 years (range 53-70 years) at baseline was enrolled in an extended health examination program with high-resolution computer tomography (HRCT) of the chest and blood drawing between 1993 and 1997. Serum concentrations of SMRP, CA125, and CYFRA21-1 were measured in archived serum samples in 2005 and 2006. A mortality follow-up was conducted through 2007. So far, 12 cases with lung cancer and 20 cases with malignant mesothelioma have been observed in this cohort. The average time between sample collection and diagnosis was 4.7 years. Analyzed biomarkers showed low sensitivities (5-25%) and positive predictive values (4-30%) for both cancer sites. Marker combinations resulted in sensitivities between 5 and 50% and positive predictive values ranging from 3 to 14%. Even in those cases, where biomarker concentrations were available within 36 months before diagnosis, no trend for increasing biomarker levels was observed. The analyzed tumor markers were characterized by high specificities, but low sensitivities. SMRP, CA125, and CYFRA21-1 alone or in combination were less suitable to serve as predictors for the diagnosis of lung cancer or malignant mesothelioma. However, a prospective study with annual sampling might reveal a better predictive value of these markers.
No preview · Article · Mar 2011 · Archives of Toxicology
[Show abstract][Hide abstract] ABSTRACT: In this study we evaluate the suitability of two methods of RNA conservation in blood samples, PAXgene and RNAlater, in combination with variable shipping conditions for their application in multicenter studies and biobanking. RNA yield, integrity, and purity as well as levels of selected mRNA and microRNA species were analyzed in peripheral human blood samples stabilized by PAXgene or RNAlater and shipped on dry ice or at ambient temperatures from the study centers to the central analysis laboratory. Both examined systems were clearly appropriate for RNA stabilization in human blood independently of the shipping conditions. The isolated RNA is characterized by good quantity and quality and well suited for downstream applications like quantitative RT-PCR analysis of mRNA and microRNA. Superior yield and integrity values were received using RNAlater. It would be reasonable to consider the production and approval of blood collection tubes prefilled with RNAlater to facilitate the use of this excellent RNA stabilization system in large studies.
[Show abstract][Hide abstract] ABSTRACT: Calretinin 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.
Initially, 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.
The 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 degrees 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.
The 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.
[Show abstract][Hide abstract] ABSTRACT: The purpose of this analysis was to evaluate if serum levels of potential tumor markers for the diagnosis of malignant mesothelioma and lung cancer are affected by confounding factors in a surveillance cohort of workers formerly exposed to asbestos. SMRP, CA125, and CYFRA21-1 concentrations were determined in about 1,700 serum samples from 627 workers formerly exposed to asbestos. The impact of factors that could modify the concentrations of the tumor markers was examined with linear mixed models. SMRP values increased with age 1.02-fold (95% CI 1.01-1.03) and serum creatinine concentration 1.32-fold (95% CI 1.20-1.45). Levels differed by study centers and were higher after 40 years of asbestos exposure. CA125 levels increased with longer storage of the samples. CYFRA21-1 values correlated with age 1.02-fold (95% CI 1.01-1.02), serum creatinine 1.21-fold (95% CI 1.14-1.30) and varied by study centers due to differences in sample handling. Tumor marker concentrations are influenced by subject-related factors, sample handling, and storage. These factors need to be taken into account in screening routine.
Full-text · Article · Jan 2010 · Biomarker insights
[Show abstract][Hide abstract] ABSTRACT: The objective of this analysis was the estimation of the cancer risks of asbestos and asbestosis in a surveillance cohort of high-exposed German workers. A group of 576 asbestos workers was selected for high-resolution computer tomography of the chest in 1993-1997. A mortality follow-up was conducted through 2007. Standardised mortality ratios (SMRs) were calculated and Poisson regression was performed to assess mesothelioma risks. A high risk was observed for pleural mesothelioma (SMR 28.10, 95% CI 15.73-46.36) that decreased after cessation of exposure (RR 0.1; 95% CI 0.0–0.6 for ≥30 vs. <30 years after last exposure). Asbestosis was a significant risk factor for mesothelioma (RR 6.0, 95% CI 2.4-14.7). Mesothelioma mortality was still in excess in former asbestos workers although decreasing after cessation of exposure. Fibrosis was associated with subsequent malignancy.
No preview · Article · Jan 2010 · International Journal of Hygiene and Environmental Health
[Show abstract][Hide abstract] ABSTRACT: SMRP (soluble mesothelin-related peptides) is a promising marker for detection of malignant mesotheliomas (MM) in serum that has not yet been validated in appropriate epidemiological studies. Field studies might not always provide optimal conditions for storage and transport of samples, and follow-up studies have to rely on sample integrity. Proper validation of the marker would require sufficient stability of the antigen and robustness of the assay. SMRP concentrations were evaluated in serum samples of 98 healthy donors, using the MESOMARK ELISA kit. The SMRP distribution in the healthy study population was determined and biological and pre-analytical variations were examined regarding their influence on SMRP concentrations. For diagnostic decisions a best statistical and unbiased cut-off between 1.5 and 1.6 nmol/L was determined (95th percentile). No age- or gender-specific differences could be observed. SMRP exhibits excellent stability regarding short-term storage, long-term storage, and repeated freeze/thaw cycles. Scientific studies as well as real life applications that employ SMRP would not be limited by sample stability issues.
No preview · Article · Feb 2007 · Cancer biomarkers: section A of Disease markers