152 Pre-diagnostic soluble mesothelin related protein, CA125, CYFRA 21-1 and risk of mesothelioma: A case-control study

Saturday, October 21, 2006 / Poster discussion session: The high-risk individual III enzymes Glutathione S Transferase Theta 1 (GSTT1) and Mu 1 (GSTM1) were associated with MPM in a case-control study. Methods: We identified 106 incident MPM cases recruited through the International Mesothelioma Program based at Brigham and Women’s Hospital from 2004 to present. As controls, 113 individuals with occupational asbestos exposure were selected from an ongoing population-based study of head and neck cancer in the greater Boston area. Genotyping was performed using PCR and PCR-RFLP methods on DNA extracted from whole blood. Tests for association were performed with Chi square analysis and logistic regression models controlling for age and gender. Results: The GSTM1 null genotype was not associated with risk of MPM (OR, 0.7; 95% CI, 0.4-1.2). However, individuals with homozygous deletion for GSTT1 had a significantly reduced risk of disease (OR, 0.4; 95% CI, 0.2-0.9). In addition, individuals homozygous for the variant allele at XRCC1-399 also had a significantly decreased risk of MPM (OR, 0.4; 95% CI, 0.2-0.9). Conclusions: Our study suggests that XRCC1-399 homozygous variant carriers are at diminished risk for this asbestos-associated disease. In addition, while the GSTT1 null genotype was also associated with less risk, previous small studies have not reported similar significant associations. Consequently, further studies of larger sample size and of additional target genes are necessary to both confirm these results, and help identify other potential risk alleles for MPM.

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Effects of long-term supplementation with retinol on plasma, malignant mesothelioma, lung cancer and potential side-effects in the Wittenoom cohort

H. Alfonso 1 , N. de Klerk 1 , A. Reid 1 , G. Ambrosini 1 , N. Olsen 1 , J. Beilby 1 , B. Musk 2 . 1 University of Western Australia, Nedlands, Australia; 2 Sir Charles Gairdner Hospital, Perth, Australia Background: In 1989 workers from the Wittenoom crocidolite industry who had been followed up since 1975 were invited to join a cancer prevention program using supplemental vitamin A. Participants attend a clinic annually where blood is collected for retinol assay, liver function tests and creatinine, a questionnaire on potential side effects is completed and to receive a year’s supply of vitamin A. Aims: 1) To record trends in time in plasma retinol. 2) To examine the relationship between plasma retinol and the incidence of mesothelioma or lung cancer. 3) To establish the association between plasma retinol and liver function tests. 4) To determine the relationship between plasma retinol and potential side effects. Methods: Non-fasting blood was collected for biochemical assays. Plasma retinol was measured by HPLC. Incident cases of disease were obtained from the Cancer Registry of WA. Associations were examined using general linear mixed models and time-dependent repeated Cox Regression Results: 2106 people were selected for this study, providing 16213 measurements of plasma retinol. Average duration of follow-up was 7.6y (sd 5.5y). Plasma retinol increased by 0.059 (95% CI 0.052, 0.065) mmol/L/y of participation, after adjusting for potential confounders. There were 75 incident cases of mesothelioma and 47 cases of lung cancer. For each additional unit of increase in plasma retinol the risk of developing mesothelioma was reduced by 44% and for each additional unit of increase the risk of lung cancer was reduced by 60% (p < 0.001). Levels of plasma retinol were inversely associated with levels of creatinine and positively associated with levels of all liver function tests. The frequency of severe side effects did not differ substantially along quartiles of plasma retinol. Conclusions: This study shows that the risk of mesothelioma and lung cancer is inversely associated with levels of plasma retinol and any potential side effects are minimal.

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Predictive and/or diagnostic significance of serum soluble mesothelin related proteins (SMR) in asbestos-related pleural malignant mesothelioma: Follow-up of a large population of workers previously exposed to asbestos

A. Cristaudo 1 , R. Foddis 1 , A. Vivaldi 1 , G. Guglielmi 1 , V. Gattini 1 , R. Buselli 1 , N. Dipalma 1 , R. Filiberti 2 , L. Mutti 3 , R. Puntoni 2 . 1 Occupational Preventive Medicine, Pisa, Italy; 2 National Cancer Resarch Institute, Genova, Italy; 3 Local Health Unit 11, Vercelli, Italy Background: Due to the occupational exposure to asbestos fibres at least a quarter of a million of men are expected to die of pleural malignant mesothelioma (MM) in the next few decades in Western Europe (Peto J, et al., Br J Cancer, 1999). Despite this dramatic scenario, effective biological tests playing a predictive role on the risk to develop MM among exposed individuals are not available yet. Quite recently determination of serum SMR has been suggested to be a useful marker for diagnosis of MM and to monitor disease recurrence after therapy. The aim of this study was to verify if the significant association

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between SMR and MM found by other authors (Robinson BW et al., Lancet, 2003) could be confirmed with a larger scale survey, and finally to evaluate if serum SMR might also prove helpful for screening asbestos-exposed individuals for early evidence of MM. Therefore, we carried out a case-control study and, at the same time, we started a follow-up of hundreds of workers previously exposed to asbestos, using a protocol including both radiological tests (chest XR, low dose CT, and eventually PET) and biological markers such as serum SMR. Methods: Sera samples from 100 patients with MM, 300 workers previously exposed to asbestos, and 100 patients affected by chronic lung disease have been analyzed for SMR titration, using an ELISA test (provided by Shering). SMR measurements have been repeated periodically in followed-up asbestos exposed workers. A questionnaire aimed at assessing the level of asbestos exposure was administered directly to the patients or to their relatives. Results and Conclusions: Our data allow us to confirm the association between MM and high levels of SMR, and suggest that SMR could be usefully added to other exams in medical surveillance protocols of previously exposed workers.

151

Discovery and validation of novel serological biomarkers of mesothelioma

A. Vachani 1 , R. Carroll 1 , S. Hoffman 1 , G. Tan 2 , L. Echan 2 , H.Y. Tang 2 , S. Albelda 1 , D. Speicher 2 . 1 University of Pennsylvania, Philadelphia, USA; 2 Wistar Institute, Philadelphia, USA The overall goal of this project is to develop and validate new blood tests that could be used to make an early diagnosis of mesothelioma or to predict clinical outcome. Development of minimally invasive, economical serological assays have great potential to: 1) decrease morbidity, mortality, and health care costs due to mesothelioma by reliably detecting early stage disease in high risk populations, 2) provide a molecular basis for predicting clinical outcome, and 3) provide improved methods of monitoring relapse. In this project, we will take advantage of exciting new proteomics technologies to discover novel diagnostic biomarkers of mesothelioma. The underlying hypothesis of this study is that a SCID mouse/human tumor model can be used to detect human proteins released from the tumor and that a portion of these putative biomarkers will correlate with disease in human plasma. The plasma proteome is highly complex. Proteins are present over a large dynamic range of concentrations, and biomarkers of disease are often low abundance. We will utilize a xenograft mouse model containing human mesothelioma tumors coupled with a four-dimensional protein separation strategy to overcome sample complexity and improve detection of lower abundance proteins. This separation strategy includes top-3 major protein depletion, ZOOM-IEF pre-fractionation, 1D SDS PAGE/grid-like slicing of the entire gel, and trypsin digestion followed by nano-HPLC to an LTQ FT MS/MS. Proteins from the mouse plasma will identified by matching MS/MS spectra with theoretical spectra from protein sequence databases. Human proteins identified in mouse plasma will represent candidate protein biomarkers for mesothelioma. To date, two newly created human mesothelioma cell lines (M30, B-111) have been injected into the flanks of SCID mice, and plasma/serum and tumors have been harvested at various tumor volumes. Further studies are ongoing.

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Pre-diagnostic soluble mesothelin related protein, CA125, CYFRA 21-1 and risk of mesothelioma: A case-control study

O.D. Røe 1,2 , J. Creaney 3 , S. Lundgren 1,2 , E. Larsson 1 , H. Sandech 1 , P. Boffetta 4 , T.I. Nilsen 2 , B. Robinson 3 , K. Kjærheim 5 . 1 St. Olavs University Hospital,Trondheim, Norway; 2 Norwegian University of Science and Technology, Trondheim, Norway; 3 University of Western Australia, Perth, Australia; 4 International Agency for Research on Cancer, Lyon, France; 5 The Cancer Registry of Norway, Oslo, Norway Background: Malignant mesothelioma is usually diagnosed in a late stage, and there are no effective means to screen populations at risk. Recently SMRP (Soluble Mesothelin Related Protein) has been suggested as a possible marker of pre-clinical disease, even a few years before symptoms. Examination of preclinical sera in this case-control designed study may elucidate biomarker biology and possible clinical usefulness. Methods: We examined bio-bank sera on levels of SMRP, CA125 and CYFRA 21-1 from 49 subjects that developed mesothelioma 1-30 years later and 147 cancer-free, matched controls. Odds Ratio (OR) on mesothelioma risk related to biomarker levels, timelag and asbestos exposure were calculated. Results: Biomarker level at lagtime <10yrs, 10-20 yrs and >20 yrs was not significantly associated to increased OR for mesothelioma. Exploring the OR of mesothelioma associated with tertiles of tumor markers, only the second tertile of SMRP was significantly elevated with OR= 2.41 (95% CI 1.01-5.75), while CA125 had an OR= 1.37 (95% CI 0.59-3.21) and CYFRA 21-1 an inverse relation with OR=0.32 (95% CI 0.10-1.07). The regression analysis on the tumor

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Saturday, October 21, 2006 / Poster Session: The high-risk individual III

markers adjusted to asbestos exposure showed a trend of correlation between increasing asbestos exposure and SMRP but only CA125 reached statistical significance for those highest exposures (p=0.049, 95% CI1.00-9.45). CA125 and CYFRA 21-1 levels were significantly elevated in female versus male controls. Female cases were significantly younger than the male and none were occupationally asbestos-exposed. Conclusions: Low-grade elevation of SMRP and CA125 can preceed mesothelioma many years before clinical tumor, but do not significantly predict mesothelioma. These markers should therefore not be used as a screening test based on our results. There is an increased risk of mesothelioma in asbestos-exposed men with elevated CA125.

Results: One hundred subjects (99M, 1F) were scanned between March and September 2005, with a mean age of 60.6 years (50-81), and an average exposure to asbestos of 24.2 years. 65 subjects scored 0 (65%). 35 individuals (35%) demonstrated features of parenchymal lung disease with scores ranging from 1-25: 11/35 showed interlobular septal thickening, 6/35 ground glass opacities, 3/35 honeycombing, 4/35 architectural distortion, 6/35 subpleural lines, 18/35 parenchymal bands, 8/35 round atelectasis. There was lower lobe predominance with 50.4% in both lower lobes, 49.6% in the remaining lungs. 17subjects (17%) demonstrated centrilobular emphysema. Conclusions: LDCT can demonstrate parenchymal lung disease in this subgroup with a history of asbestos exposure. The presence of subpleural lines, septal thickening, honeycomb formation, architectural distortion and ground glass opacities, although non specific, may entertain asbestosis as an etiology and thus require further diagnostic work up.

Saturday, October 21, 2006 POSTER SESSION

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The high-risk individual III 153

Analysis of NAT2 genotypes as risk factors for asbestosrelated malignant mesothelioma (MM) in a general population study

M. Betti, A. Biava, M. Bertolotti, D. Ferrante, D. Mirabelli, C. Magnani, I. Dianzani. Università del Piemonte Orientale, Novara, Italy Background: We have recently observed an association between polymorphisms in DNA repair genes and asbestos-associated MM in a panel of 81 MM patients and 110 age and sex-matched controls, all residents at Casale Monferrato, an Italian town highly exposed to asbestos pollution (Dianzani et al. Mutation Res 599, 124, 2006). Our data indicated that genetic factors are involved in MM development. Two other studies observed an association between MM and metabolic polymorphisms. One of them showed an increased risk of MM in Italian subjects carrying the NAT2 fast acetylator genotypes (Neri et al. Mutation Res 14, 1741, 2005). Conversely, a Finnish study observed that the NAT2 slow acetylator phenotype was associated with an increased risk of MM in combination with a deletion of the GSTM1 gene (Hirvonen et al. Cancer Res 55, 2981, 1995). The opposite results obtained in Finland and Italy could be ascribed to random chance, but a role has been hypothesized for the fact that different types of asbestos have been used in the two countries (Neri et al. Int J Hyg Environ Health 209, 393, 2006). Methods: We have thus decided to analyse NAT2 genotypes on our panel of patients and controls by using the SNaPshot technique.Unconditional multivariable logistic regression was used to estimate odds ratios (ORs) and 95% confidence intervals (CIs). Results: NAT2 fast acetylator genotypes showed an increased OR, both in asbestos-exposed subjects (OR = 1.49; 95% CI = 0.78–2.88) and in the entire population (OR = 1.33; 95% CI = 0.71–2.52). Conclusions: Although our data are not statistically significant, they seem to confirm those observed in the other Italian study. We are presently extending our analyses on a larger panel of patients and controls (154 patients and 202 controls).

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Low-dose computed tomography in workers previously exposed to asbestos: Detection of parenchymal lung disease

D. Patsios 1 , H. Bayanati 2 , M. Johnston 3 , N. Paul 1 , H. Roberts 1 . 1 UHN and Mount Sinai, Toronto, Canada; 2 Princess Margaret Hospital, Toronto, Canada; 3 UHN, Toronto, Canada Background: We screen prior asbestos workers for the early detection of lung cancer and mesothelioma using low-dose computed tomography (LDCT). For this presentation we analyze the presence of diffuse parenchymal abnormalities. Methods: Subjects studied had a history of asbestos exposure at least 20 years prior to enrollment and/or demonstration of pleural plaques on chest radiographs. A baseline LDCT of the chest was performed using a multidetector CT scanner with collimation of 1.25mm, 60mAs and 120kV. Focal parenchymal and pleural masses or plaques are reported separatedly. For this analysis, we noted the following 8 interstitial patterns according to their presence, lobar location and extent: inter- and intralobular septal thickening, subpleural ground glass opacities, honeycombing, architectural distortion, subpleural curvilinear lines, parenchymal bands, round atelectasis. In each of the 5 lobes, each pattern was scored as 0 (absence), 1 (within 1 cm from the pleura), 2 (within 2 cm from the pleura) and 3 (more than 2 cm from the pleura), with a maximum score of 120. The presence of emphysema was noted separately.

Low-dose computed tomography in prior asbestos-exposed workers: Assessment of pleural plaques and screening for lung cancer and malignant mesothelioma

H. Bayanati, I. Sitartchouk, D. Patsios, A. Pereira, G. Dong, A. Kale, N. Paul, M. Johnston, K. Uy, M. de Perrot, H. Roberts. University Health Network, Toronto, Canada Background: Asbestos exposure is a known risk factor for lung cancer and malignant pleural mesothelioma (MPM). We use low-dose computed tomography (LDCT) for screening and report on the pleuropulmonary abnormalities. Methods: Since May 2005, we have enrolled 300 individuals with asbestos exposure at least 20 years ago and/or documented pleural plaques. A LDCT is performed (1.25 mm, 60 mA, 120 kV). We note the appearance of nodules as well as the characteristics of pleural plaques. Indeterminate nodules and suspicious pleural plaques are followed in 3–6 months. Results: To date, 297 men and 3 women are enrolled, averaging 62 years of age (range 35-83 years). 166 (55.3%) are former smoker, 72 (24.0%) are current smoker and 62 (20.7%) are life-time non-smoker. One or more pulmonary nodules were detected in 200 (66.7%) patients. 47 nodules were ≥ 5 mm. On follow-up, 2 nodules had grown and both proved to be malignant, one stage 1 adenocarcinoma and one stage 1 squamous cell carcinoma. In 3 cases an immediate biopsy revealed one benign pleural plaque, one mesothelioma and one extensive small cell carcinoma. One biopsy for a highly suspicious central 19 mm nodule is pending. All cases of malignancy were heavy smokers with an average of 36 pack-years. 232 individuals (77.3%) had pleural plaques. In 37 (15.9%) there were lobular and in 23 (9.9%) they had asymmetric distribution. Of those asymmetrical plaques, 20 (87%) were more prominent on the left side. 82% (102/125) of smokers with more than 20 pack/year of smoking had pleural plaques, compared to 72% (44/61) of non-smokers. This difference is statistically significant (p=0.01). Conclusions: We have detected four bronchogenic carcinomas and one advanced MPM, all were heavy smokers. In cases with asymmetric pleural plaques, they are more commonly seen on the left side. Pleural plaques following asbestos exposure are more common in heavy smokers.

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Soluble mesothelin-related protein (SMRP) is a useful biomarker for malignant mesothelioma in Japan

K. Fukuoka, A. Uesaka, K. Kuribayashi, M. Miyake, S. Miyata, T. Nakajima, S. Iida, S. Nobuyama, K. Tamura, S. Yamada, A. Murakami, T. Nakano. Hyogo College of Medicine, Japan Background: Malignant mesothelioma is a highly aggressive, incurable neoplasm associated with asbestos exposure. It arises primary from the surface mesothelial cells of the pleura, and less commonly, of the pericardium or peritoneum. Retrospective studies of small numbers of patients with pleural mesothelioma have attempted to identify biomarkers that predate symptoms in a high-risk population. Mesothelin is a 40-kDa cell surface glycoprotein that is highly expressed in mesotheliomas. In this study, we retrospectively investigate that a soluble variant form of mesothelin, termed as soluble mesothelin-related protein (SMRP), could be a useful biomarker for malignant mesothelioma. Methods: Serum samples were collected from patients with the following diseases: 97 patients with mesothelioma, including 93 pleural, and 4 peritoneal origin; control sera from 72 healthy adults with no apparent history of occupational asbestos exposure. Serum SMRP levels were determined in a double determinant ELISA using two antibodies (OV569 and 4H3). The cut-off value is set at 1.5 nM. In 19 patients, expression of mesothelin protein in neoplastic tissues was evaluated immunohistochemistry. Results: The serum levels of SMRP in the patients with mesothelioma had significantly elevated, as compared with those in non-asbestos-exposed controls (5.7 nM vs 0.6 nM). SMRP has a sensitivity of 56% for pleural mesothelioma (52/93), and a specificity of 96%. According to each histologic subtype, the positive rate of SMRP showed as follows; 63% in epithelioid, 17% in sarcomatoid,