Profiling Tumor-Associated Markers for Early Detection of Malignant Mesothelioma: An Epidemiologic Study

163 Profiling Tumor-Associated Markers for Early Detection of Malignant Mesothelioma: An Epidemiologic Study Monica Amati, 1 Marco Tomasetti, 1 Mario Scartozzi, 2 Laura Mariotti, 1 Renata Alleva, 3 Elettra Pignotti, 3 Battista Borghi, 3 Matteo Valentino, 1 Mario Governa, 1 Jiri Neuzil, 4,5 and Lory Santarelli 1 1 Department of Molecular Pathology and Innovative Therapies, Clinic of Occupational Medicine, Polytechnic University of Marche, Ancona, Italy; 2 Department of Medical Oncology, Hospital University of Ancona, Ancona, Italy; 3 Department of Anaesthesiology, IRCCS Orthopaedic Institute Rizzoli, Bologna, Italy; 4 Apoptosis Research Group, School of Medical Science, Griffith University, Southport, Queensland, Australia; and 5 Molecular Therapy Group, Institute of Molecular Genetics, Academy of Sciences of the Czech Republic, Prague, Czech Republic Abstract Improved detection methods for diagnosis of asymptomatic malignant mesothelioma (MM) are essential for an early and reliable detection and treatment of this type of neoplastic disease. Thus, focus has been on finding tumor markers in the blood that can be used for noninvasive detection of MM. Ninety-four asbestosexposed subjects defined at high risk, 22 patients with MM, and 54 healthy subjects were recruited for evaluation of the clinical significance of 8-hydroxy-2 -deoxyguanosine (8OHdG) in WBCs and plasma concentrations of soluble mesothelin-related peptides (SMRPs), angiogenic factors [platelet-derived growth factor B, hepatocyte growth factor, basic fibroblast growth factor, and vascular endothelial growth factor B (VEGFB)], and matrix proteases [matrix metalloproteinase (MMP) 2, MMP9, tissue inhibitor of metalloproteinase (TIMP) 1, and TIMP2] for potential early detection of MM. The area under receiver operating characteristic (ROC) curves indicate that 8OHdG levels can discriminate asbestosexposed subjects from healthy controls but not from MM patients. Significant area under ROC curve values were found for SMRPs, discriminating asbestos-exposed subjects from MM patients but not from healthy controls. Except for platelet-derived growth factor B, the hepatocyte growth factor, basic fibroblast growth factor, and VEGFB can significantly differentiate high-risk individuals from healthy control and cancer groups. No diagnostic value was observed for MMP2, MMP9, TIMP1, and TIMP2. In addition to the diagnostic performance defined by the ROC analysis, the sensitivity and specificity results of markers with clinical significance were calculated at defined cutoffs. The combination of 8OHdG, VEGFB, and SMRPs best distinguished the individual groups, suggesting a potential indicator of early and advanced MM cancers. The combination of blood biomarkers and radiographic findings could be used to stratify the risk of mesothelioma in asbestos-exposed populations. (Cancer Epidemiol Biomarkers Prev 2008;17(1):163 70) Introduction Received 7/3/07; revised 9/3/07; accepted 11/6/07. Grant support: Regione Marche (Italy) and Polytechnic University of Marche (Ancona, Italy) research grant. J. Neuzil was supported by Dust Diseases Board of Australia and the Grant Agency of the Academy of Sciences of the Czech Republic. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Note: M. Amati and M. Tomasetti contributed equally to this work. Requests for reprints: Marco Tomasetti, Department of Molecular Pathology and Innovative Therapies, Polytechnic University of Marche, via Tronto 10/A, Torrette di Ancona, 60020 Ancona, Italy. Phone: 39-071-2206062; Fax: 39-071-2206182. E-mail: m.tomasetti@univpm.it Copyright D 2008 American Association for Cancer Research. doi:10.1158/1055-9965.epi-07-0607 Malignant mesothelioma (MM) is an aggressive tumor of serosal cavities, which is resistant to conventional therapy, surgery, or radiation. MM remains a universally fatal disease of increasing incidence worldwide (1). Patient survival from presentation is <12months (2). Occupational hazard, particularly asbestos exposure, is the main factor involved in MM pathogenesis (3, 4). Asbestos-induced cell damage is mediated to some extent by iron-catalyzed formation of toxic oxygen radicals (5, 6), which induce DNA strand breaks (7) and oxidant-induced base modifications. 8-Hydroxy-2 -deoxyguanosine (8OHdG), a major product of such oxidative damage (8), causes G!T and A!C transversions (9, 10). These substitutions have been reported as the sites of spontaneous oncogene expression and may be largely responsible for the onset of carcinogenesis and cell proliferation, ultimately leading to cancer manifestation (8-11). Most of the necessary mutations occur early during cancer development, also resulting in processes such as chronic inflammation, together providing the environment to expand and select malignant clones. Tumor growth and metastasis are angiogenesisdependent events. Like other tumors, MMs induce the vascular stroma to grow (12). Current results indicate that the switch to the angiogenic phenotype depends on a net balance between positive and negative angiogenic factors released by the tumor cells (13). To date, many angiogenic factors, such as the hepatocyte growth factor (HGF), the fibroblast growth factor family members (FGF), the vascular endothelial growth factor (VEGF), or the platelet-derived growth factor (PDGF), have been identified and shown to be produced by a variety of different tumor cells, including those of MM (14-17). Functionally active growth factors induce mesothelioma cell migration and matrix metalloproteinase (MMP) production (14, 18). MMPs belong to the group of extracellular

164 Biomarkers in Early Detection of Malignant Mesothelioma matrix degradation enzymes. The balance of secreted MMPs and their specific inhibitors (TIMPs) plays an important role in maintaining connective tissue homeostasis in normal tissue (19). Activated MMP2and MMP9 are described to have an effect in MM carcinogenesis (20). Thus, determination of these mediators in blood plasma could be used for noninvasive early diagnosis of MM. Recently, soluble mesothelin-related peptides (SMRPs) have been suggested as a promising biomarker for MM (21-23). Mesothelin is normally expressed at low levels in mesothelial cells. Its overexpression was observed in cancers including MM (21-24). SMRPs can be detected in serum and are highly increased in the blood of patients with MM (21-23) and ovarian tumors (23). In the present study, the levels of 8OHdG in circulating WBCs, plasma concentrations of SMRPs, a panel of angiogenic factors [PDGFh, HGF, basic FGF (bfgf), and VEGFh], as well as MMPs (MMP2and MMP9) and their inhibitors (TIMP1 and TIMP2) were evaluated in a cohort of asbestos-exposed, high-risk subjects, in patients with MM, and in healthy controls. The potential clinical relevance of these markers was evaluated for detection of the cancer risk and thus may help in the prevention and management of the disease. Materials and Methods Study Groups. Three groups of subjects were included in the study: 94 asbestos-exposed subjects (high risk), 54 aged-matched controls (no exposure to asbestos), and 22 patients with MM. All subjects filled in a questionnaire including their informed consent and provided a blood sample. The study was carried out according to the Helsinki Declaration and approved by the Ethical Committee of the Polytechnic University of Marche. The demographic characteristics are shown in Table 1. WBC 8OHdG as well as plasma angiogenic factors (VEGFh, HGF, bfgf, and PDGFh), MMPs (MMP2and MMP9) and their inhibitors (TIMP1 and TIMP2), and SMRP levels were determined in the patients samples. Subjects with Asbestos Exposure. To recruit asbestosexposed subjects, 500 invitation letters were sent to subjects who worked or were working at the shipbuilding industry. From November 2004 to September 2005, 94 male subjects (response rate, 19%; mean age, 61.3 F 7.4 years) with history of asbestos exposure were enrolled at the Institute of Occupational Medicine, Polytechnic University of Marche (Ancona, Italy). The subjects had been exposed to asbestos dust on average for more than 20 years. Smokers (12%), ex-smokers (52%), and nonsmokers (36%) were examined. Each subject underwent lung function analysis, chest radiography, and high-resolution computed tomography. Evidence of asbestos-related diseases (fibrosis and pleural plaques) was found in 28 (24%) subjects. Subjects with No Exposure to Asbestos. The control group consisted of 54 aged-matched subjects (mean age, 63.0 F 7.8 years; 33 males and 21 females) recruited from November 2004 to January 2007 (response rate, 70%). The subjects were undergoing screening radiography for chemoprevention at the Pneumology Clinic of the University Hospital of Ancona (Ancona, Italy). None of them had ever been exposed to asbestos as documented by their occupational histories. All subjects had normal chest radiographs. Subjects with MM. Blood samples were collected from 22 patients (mean age, 68.7 F 7.9; 18 males and 4 females) diagnosed for MM, who were recruited, from November 2004 to January 2007, at the Oncology Clinic of the University Hospital of Ancona, with a response rate of 90%. Exclusion criteria were the presence or suspicion of any infectious disease and previous radical surgery, radiotherapy, as well as chemotherapy for MM. Pathologic diagnosis was done on pleural biopsies obtained by thoracoscopy or thoracotomy. Tumors were classified as epithelial in 11, mixed in 5, and sarcomatoid in 6 patients. WBC 8OHdG Analysis. The levels of 8OHdG were determined in WBCs using the fluorometric OxyDNA assay kit (Calbiochem) according to the manufacturer s instructions. Whole blood (7 ml) collected into EDTA tubes was immediately centrifuged at 1,500 g (20jC, 15 min). The buffy coat was removed, placed in a 15 ml Falcon tube, and resuspended in 4 ml PBS. The suspension was then layered onto 4 ml Lympholyte-H (Cedarlane) and centrifuged at 1,000 g (20jC, 30 min). The resulting cloudy layer was collected and placed in a 15 ml Falcon tube filled with PBS and centrifuged at 230 g (20jC, 5 min). After removing the supernatant, the pellet was resuspended in 500 AL PBS/500 AL of2% paraformaldehyde and incubated on ice for 15 min. Two washes in PBS were then made, and after centrifugation, the cells were resuspended in 50 AL of the blocking solution for 1 h at 37jC. The blocking solution was removed and 100 AL of the fluorescent probe for 8OHdG were added and the samples were incubated for 1 h. After washing, the cells were resuspended in the fluorescence-activated cell sorting buffer and analyzed by flow cytometry (FACSCalibur, BD PharMingen Italy). The results are expressed as mean fluorescence intensity (arbitrary units). SMRP Assay. Plasma levels of SMRPs were determined using a sandwich-type ELISA assay (Mesomark, Schering) according to the manufacturer s instructions Table 1. Demographic characteristics of recruited subjects Characteristics Control group (n = 54) Asbestos-exposed group (n = 94) MM group (n = 22) Age (y) 63.0 F 7.8 61.3 F 7.4 68.7 F 7.9 Sex (M/F) 33/21 94/0 18/4 Smoking history Nonsmokers 41 (80%) 34 (36%) 6 (27%) Ex-smokers 5 (9%) 49 (52%) 11 (50%) Smokers 8 (15%) 11 (12%) 5 (23%)

Cancer Epidemiology, Biomarkers & Prevention 165 Table 2. Levels of plasma biomarkers Control group (n = 54) Asbestos-exposed group (n = 118) MM group (n =22) SMRPs (nmol/l) 1.1 F 1.5 1.7 F 6.0 10.9 F 16.2*, c Min-max 0.2-7.8 0.2-44.1 1.0-57.6 8-OHdG (AU) 5.1 F 2.1 8.4 F 3.8 b 9.2 F 4.7 c Min-max 2.1-10.0 2.7-26.4 3.3-17.8 MMP2(ng/mL) 601.5 F 31.8 475.7 F 24.5 487.7 F 26.4 Min-max 233-1,690 149-1,120 112-1,848 MMP9 (ng/ml) 244.0 F 243.5 163.2 F 132.0 220.6 F 250.7 Min-max 12-1,022 22-582 10-1,008 TIMP1 (ng/ml) 318.7 F 250.2 221.5 F 103.4 243.5 F 60.6 Min-max 91-1,004 53-655 161-300 TIMP2(ng/mL) 276.3 F 431.0 203.0 F 255.1 114.5 F 49.6 Min-max 68-1,765 38-1,479 39-200 PDGFh (ng/ml) 19.1 F 19.0 21.3 F 21.4 b 47.4 F 30.1*, c Min-max 0.5-520.3-81 3.0-106 HGF (ng/ml) 5.0 F 3.5 7.4 F 5.1 b 15.0 F 8.5*, c Min-max 1.4-13.7 1.4-21.3 2.2-33.0 bfgf (ng/ml) 0.6 F 0.4 0.8 F 0.4 b 1.4 F 0.8*, c Min-max 0.1-1.5 0.1-2.1 0.3-3.2 VEGFh (ng/ml) 0.5 F 0.20.7 F 0.3 b 1.7 F 1.7*, c Min-max 0.1-1.0 0.2-2.2 0.1-6.0 NOTE: Values are presented as mean F SD and within a minimum-maximum range. Statistical differences among the groups were calculated by the nonparametric Kruskal-Wallis test. *P = 0.00005, MM group versus asbestos-exposed group. cp = 0.00005, MM group versus control group. bp = 0.00005, asbestos-exposed group versus control group. and the results are expressed in nmol/l. Briefly, 100 AL of standard and plasma samples (1:100 dilution) were added to a 96-well microtiter plate coated with specific antibodies against SMRPs and then incubated at room temperature for 60 min. After washing, the plate was incubated with a secondary horseradish peroxidase conjugated antibody. The detection process involved addition of 100 AL of the substrate (3,3,5,5 -tetramethylbenzidine) to all wells and the absorbance was read at 405 nm using an ELISA plate reader (Sunrise, Tecan). Concentrations of SMRPs were extrapolated from the standard curve and expressed in nmol/l. Human Angiogenesis and MMP Arrays. Human angiogenesis (PDGFh, HGF, bfgf, and VEGFh) and human MMP (MMP2, MMP9, TIMP1, and TIMP2) arrays were analyzed by multiplex sandwich ELISA (Search- Light, Pierce Biotechnology) according to the manufacturer s instructions. Each well of the microplate was prespotted with target protein-specific antibodies. These antibodies capture the specific target protein in the standard and plasma samples added to the plate (50 AL of 1:5 diluted plasma). Unbound proteins were washed away and biotinylated detecting antibodies were added. After washing, antibody streptavidin-horseradish peroxidase was used for detection. Each sample was tested in duplicate and the results are expressed in ng/ml. Statistical Analysis. All data are presented as mean F SD and within a minimum-maximum range. Comparisons between groups were done using Mann-Whitney U test for unpaired samples and Kruskal-Wallis analysis for multiple comparisons, and the rank correlation coefficient according to Spearman was used. Multiple regression analysis was used to estimate the influence of independent variables such as age, smoking, fibrotic changes and pleural plaques, as well as duration of exposure to asbestos on the markers studied (dependent variable). Receiver operating characteristic (ROC) curves were plotted to quantify the marker performance. ROC curves correlate the sensitivity of a diagnostic test within the entire range of the possible false-positive rate. The area under the ROC curve (AUC) indicates the average sensitivity of a marker over the entire ROC curve. The best statistical cutoff was calculated by minimizing the distance between the point with specificity = 1 and sensitivity = 1 and the intercept on the ROC curve. AUC values are reported with their 95% confidence intervals. The usage of biomarker combinations in MM diagnosis was assayed with logistic regression. This method was applied to specify a probability, which depends on several factors. Statistical calculations were done using the Statistical Package for the Social Sciences statistical package version 12.0F (SPSS). Statistical differences of at least P < 0.05 were considered statistically significant. Results Concentrations of Biomarkers in the Groups. The plasma biomarker concentrations detected in the three groups are summarized in Table 2. High levels of 8OHdG were observed in WBCs of the asbestos-exposed group and MM group compared with subjects without exposure to asbestos (healthy controls). Plasma SMRP concentrations of asbestos-exposed subjects were not significantly different from the age-matched subjects. Conversely, patients with MM showed high levels of SMRPs when compared with both the asbestos-exposed group and the control group. As listed in Table 2, the mean plasma levels of PDGFh (P < 0.001), HGF (P < 0.0001), bfgf (P < 0.0001), and VEGFh (P < 0.0001) were significantly increased in the asbestos-exposed group and more in the MM group compared with the control group. The levels of MMP2, MMP9, TIMP1, and TIMP2 as well as the MMPs to TIMPs ratio were not different between groups (data not shown). None of the biomarkers was influenced

166 Biomarkers in Early Detection of Malignant Mesothelioma Table 3. Correlation coefficients according to Spearman between biomarkers SMRPs 8OHdG MMP2MMP9 TIMP1 TIMP2PDGFh HGF bfgf VEGFh SMRPs 1.00 0.06 0.20 0.020.25* 0.13 0.47 c 0.42 c 0.28* 0.23* 8OHdG 1.00 0.17 0.15 0.08 0.03 0.01 0.06 0.020.05 MMP21.00 0.44 c 0.51 c 0.76 c 0.39 c 0.45 c 0.44 c 0.16 MMP9 1.00 0.56 c 0.45 c 0.26* 0.12 0.20 0.04 TIMP1 1.00 0.62 c 0.01 0.00 0.08 0.09 TIMP2 1.00 0.23* 0.29* 0.27* 0.06 PDGFh 1.00 0.81 c 0.78 c 0.46 c HGF 1.00 0.90 c 0.65 c bfgf 1.00 0.76 c VEGFh 1.00 NOTE: Correlation coefficients were determined according to Spearman test. The levels of 8OHdG were expressed as arbitrary units, SMRPs as nmol/l, and MMP2, MMP9, TIMP1, TIMP2, PDGFh, HGF, bfgf, and VEGFh as ng/ml. Correlations with P < 0.05 were considered statistically significant. Abbreviation: AU, arbitrary units. *P < 0.05. cp < 0.01. by sex, age, smoking habits, and the presence or absence of pleural plaques and lung fibrosis (data not shown). Biomarker Correlations. Table 3 presents the Spearman correlation coefficients among the analyzed biomarkers. A positive correlation was observed between MMP2and MMP9 and between their inhibitors TIMP1 and TIMP2. The angiogenic factors (PDGFh, HGF, bfgf, and VEGFh) positively correlated with each other. MMP2 and the inhibitor TIMP2negatively correlated with the PDGFh, HGF, and bfgf levels. Only a negative correlation was found between MMP9 and PDGFh. Notably, no correlations were found between 8OHdG and any of the markers tested. The SMRPs positively correlated with TIMP1 and with all angiogenic factors evaluated. Diagnostic Validity of the Single and Combined Markers. The ROC curves were generated to analyze the diagnostic values of individual markers (Figs. 1 and 2). SMRPs represent a marker with the highest AUC, allowing to discriminate between patients with MM and both the control subjects (AUC = 0.920 F 0.030; P = 0.0001) and the asbestos-exposed subjects (AUC = 0.927 F 0.022; P = 0.0001). An AUC curve that did not reach statistical significance was observed by comparing the asbestos-exposed subjects with the control subjects (AUC = 0.459 F 0.042; P = 0.502). The WBC 8OHdG level was found to be appropriated to evaluate the asbestos exposure. ROC analyses comparing the subjects with those without asbestos exposure showed an AUC of 0.775 F 0.037 (P = 0.001). The AUC for discriminating between patients affected by MM and the control agematched subjects was 0.788 F 0.090 (P = 0.004). An AUC not statistically significant was found between asbestosexposed subjects and patients with MM (AUC = 0.556 F 0.110; P = 0.536; Fig. 1). No diagnostic value was observed for MMP2, MMP9, TIMP1, and TIMP2. The AUC values were not statistically significant to differentiate the three groups (data not shown). As observed for SMRPs, the PDGFh levels distinguished MM patients from both the control subjects (AUC = 0.783 F 0.065; P = 0.001) and the asbestos-exposed subjects (AUC = 0.765 F 0.061; P = 0.001) but not the asbestos-exposed subjects from the control group (AUC = 0.534 F 0.071; P = 0.632). Conversely, HGF, bfgf, and VEGFh significantly discriminated the asbestos-exposed subjects from the Figure 1. ROC curves for 8OHdG and SMRPs. The AUCs were determined for 8OHdG and SMRPs, discriminating agematched control subjects (Ctrl) from asbestos-exposed subjects (Exp), asbestos-exposed subjects from MM patients, and age-matched control subjects from MM patients. Differences with P < 0.05 were considered statistically significant.

Cancer Epidemiology, Biomarkers & Prevention 167 Figure 2. ROC curves for PDGFh, HGF, bfgf, and VEGFh. The AUCs were determined for PDGFh, HGF, bfgf, and VEGFh, discriminating age-matched control subjects (Ctrl) from asbestos-exposed subjects (Exp), asbestos-exposed subjects from MM patients, and age-matched control subjects from MM patients. Differences with P < 0.05 were considered statistically significant. controls and the MM patients and the latter from the controls (Fig. 2). In addition to the diagnostic performance defined by the AUCs of the ROC analyzed, the sensitivity and specificity results of selected markers were calculated at defined cutoffs. Table 4 presents the diagnostic sensitivity and specificity (90% limits) of the SMRPs, 8OHdG, PDGFh, HGF, bfgf, and VEGFh to distinguish between the healthy persons and the asbestos-exposed subjects, the asbestos-exposed subjects and the MM patients, and the healthy persons and the MM patients. To evaluate whether a marker combination may increase the predictive value for early detection of MM, the logistic regression analysis was done. No increase in the accuracy of determining the disease was observed by the Wald test. The SMRPs alone highly discriminate the MM patients from the healthy controls and the asbestos-exposed subjects. Additionally, the logistic regression analysis revealed that VEGFh can increase the discriminative power of 8OHdG in determining the subjects with the risk to develop the disease (control group versus asbestos-exposed group; P = 0.0001, Wald test). The probability of the risk to develop the disease can be calculated by the formula P = exp ( 6.6 + 1.698 X 1 + 0.001358 X 2 ) / 1 + exp ( 6.6 + 1.698 X 1 + 0.001358 X 2 ), with X 1 = 8OHdG WBC level and X 2 = VEGFh plasma level. Accordingly, the AUC increases from 0.775 F 0.037 for 8OHdG and 0.714 F 0.062for VEGFh to 0.925 F 0.035 for the 8OHdG/VEGFh combination. The increase in sensitivity and specificity is reflected by the ROC curves in Fig. 3A. For better visualization, dot plots are shown for the SMRPs and the 8OHdG/VEGFh combination (Fig. 3B). Discussion Biomarker or a combination of several biomarkers that could predict the development of MM or detect the disease in its early stages in the population with high risk would be of paramount importance, particularly given the fact that there is as yet no cure for MM. In the present study, levels of the DNA adduct 8OHdG and factors involved in tumor growth (PDGFh, HGF, bfgf, and VEGFh), progression (MMP2, MMP9, TIMP1, and TIMP2), and cell transformation (SMRPs) were measured

168 Biomarkers in Early Detection of Malignant Mesothelioma Table 4. Diagnostic sensitivities and specificities of MMPs, TIMPs, and PDGFB, HGF, bfgf, and VEGFB to distinguish between healthy persons and asbestos-exposed subjects, asbestos-exposed subjects and MM cancer patients, and healthy persons and MM cancer patients Marker Ctrl vs Exp Exp vs MM Ctrl vs MM ng/ml Sensitivity Specificity ng/ml Sensitivity Specificity ng/ml Sensitivity Specificity SMRPs (nmol/l) 0.290 0 1.0 90 79 1.0 90 78 1.9 9 90 1.7 7290 1.9 68 90 8OHdG (AU) 4.4 90 48 3.9 90 4 3.9 90 37 7.9 43 90 12.7 20 90 7.9 60 90 PDGFh (ng/ml) 0.8 90 18 4.3 90 38 3.3 90 45 45.1 14 90 50.245 90 45.1 45 90 HGF (ng/ml) 1.8 90 21 4.0 90 36 4.2 90 48 9.3 3290 14.7 35 90 9.3 75 90 bfgf (ng/ml) 0.3 90 31 0.5 90 34 0.5 90 52 1.1 30 90 1.4 45 90 1.1 50 90 VEGFh (ng/ml) 0.3 90 34 0.3 90 20.3 90 17 0.7 34 90 1.0 60 90 0.7 70 90 NOTE: Data result from ROC analysis done with 54 control subjects, 94 asbestos-exposed subjects, and 22 MM patients. The cutoffs correspond to the values at 90% sensitivity and specificity as indicated. Abbreviations: Ctrl, control subjects; Exp, asbestos-exposed subjects. in healthy subjects, in asbestos-exposed subjects defined as at high risk, and in patients with MM. 8OHdG is an indicator of oxidative DNA damage induced by reactive oxygen species (11). It has been widely used as a biomarker for detecting oxidative stress and oxidative DNA damage in both animal and human studies (25). We found that asbestos-exposed subjects and patients with MM showed comparably high WBC 8OHdG levels differing significantly from those in agematched controls. A multiple regression analysis revealed that the age, smoking status, and fibrotic changes and pleural plaques were not the most important factors influencing the 8OHdG levels. The increased generation of 8OHdG indicates that high levels of reactive oxygen species are produced in the WBCs of subjects exposed to asbestos. It has been proposed that reactive oxygen species are critical for the development of asbestosrelated diseases (7, 8, 10), and oxidative damage to the DNA of WBCs may be induced as a response to increased oxidative stress in the pleural surface of subjects chronically exposed to asbestos (26, 27). To evaluate whether the 8OHdG levels would be useful in predicting MM in asbestos workers, the ROC analysis was done. We found that the biomarker 8OHdG significantly discriminated the asbestos-exposed subjects from the age-matched controls but not from MM patients (cf Fig. 1). It is noteworthy that the 8OHdG levels were not evaluated in the target (mesothelial) cells but in the surrogate cells (WBCs). Thus, analysis of 8OHdG provides information only about the systemic status. The suitability of measuring 8OHdG as a biomarker of oxidative DNA damage depends on a variety of variables, which affect the interpretation of the data. These variables include the steady state between the mature and the newly differentiated or dying WBCs, DNA repair, and cell division and turnover (25). The levels of 8OHdG found in WBCs depend not only on the life span of the cells but also on the recovery of these adducts and individual blood count variability. The value of the 8OHdG levels for predicting cancer on an individual basis is therefore questionable. However, in agreement with other authors (26, 27), our results support the notion that the biomarker 8OHdG detects oxidative DNA damage in humans caused by exposure to asbestos fibers, which are involved in the etiology of MM, but they cannot be used to discriminate between asbestos-exposed individuals with and without MM. Recently, plasma SMRPs have been proposed as a suitable marker for MM diagnosis (21-23). The release of SMRPs into blood is linked to cell transformation; thus, it could be used as a diagnostic biomarker of MM. In our study, MM patients showed higher plasma SMRP levels relative to the asbestos-exposed and the control subjects. The ROC analysis revealed that the SMRP levels can discriminate MM patients from both the asbestos-exposed and the asbestos-unexposed subjects but do not discriminate the asbestos-exposed individuals from the agematched controls (cf Fig. 1). Thus, the level of SMRPs in plasma can be proposed as a biomarker suitable for diagnosis of existing MM but not to predict the disease. Differently as found for 8OHdG and SMRPs, the growth factors HGF, bfgf, and VEGFh can significantly differentiate the high-risk individuals from the healthy controls and the cancer group (cf Fig. 2). Such an observation may be important when determining the risk status of individuals with no physical manifestation of the disease. Using the statistical ROC program, we calculated the different sensitivities and specificities of markers with clinical significance (cf Figs. 1 and 2; Table 4). The best indicator of MM was SMRPs with high sensitivity and specificity. Although lacking such high sensitivity and specificity, the levels of 8OHdG, HGF, bfgf, and VEGFh alone can distinguish high-risk subjects from healthy persons and MM patients. We found that combination of the exposure marker 8OHdG with the growth factor VEGFh highly increased the sensibility and specificity to discriminate the high-risk populations from the healthy controls (cf Fig. 3A and B). We found a significantly increased discriminative capability by the use of a combination of 8OHdG and VEGFh compared with the use of single variables. Positive correlation between SMRPs and plasma growth factors suggests involvement of growth factors in the development, growth, and progression of

Cancer Epidemiology, Biomarkers & Prevention 169 MM (cf Table 3). VEGFh is of particular interest because reduction/blockage of members of the VEGF system has been suggested to be of therapeutic value in MM (28, 29). Dot plots for marker combinations were used to evaluate the best marker combinations to distinguish the high-risk population from the healthy persons and the MM patients. The combination of SMRPs with 8OHdG and VEGFh was found to be the best to distinguish the individual groups, suggesting a potential diagnostic indicator for patients in the early stages of MM. The importance of the changes in the levels of 8OHdG and SMRPs as well as growth factors in MM has been previously described in the literature, but here, we have for the first time evaluated their clinical relevance for their potential use for both diagnostic and screening purposes. However, the value of our biomarkers as indicators of both prediction and clinical presentation of MM needs to be validated in prospective studies in larger subject populations in which the exposed subjects Figure 3. ROC curves of 8OHdG and VEGFh and combination of 8OHdG and VEGFh as a marker to predict MM. A. The AUCs were determined for 8OHdG and VEGFh alone and in combination, discriminating age-matched control subjects (Ctrl) from asbestos-exposed subjects (Exp). B. Scatter plot of SMRPs and combination of 8OHdG and VEGFh, discriminating asbestos-exposed subjects (open circles) from agematched control subjects (closed circles). will be followed for an adequate period of time. Accordingly, a longitudinal study on our asbestos-exposed group is under way. 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