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Discovery and Validation of Potential Genomic-Based Biomarkers for Asbestos Related Neoplasms

This study aims to discover and validate potential genomic-based biomarkers for asbestos-related neoplasms, specifically pleural mesothelioma. The American Australian Mesothelioma Consortium and NYU Mesothelioma Biomarker Discovery Laboratory are collaborating to identify novel markers for early detection and improved prognosis. The study includes the analysis of serum, plasma, urine, and tissue samples from mesothelioma patients, as well as control cohorts. This research holds promise for the development of effective diagnostic tools and targeted therapies.

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Discovery and Validation of Potential Genomic-Based Biomarkers for Asbestos Related Neoplasms

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  1. Discovery and Validation of Potential Genomic-Based Biomarkers for Asbestos Related Neoplasms American Australian Mesothelioma Consortium and NYU Mesothelioma Biomarker Discovery Laboratory

  2. Mesothelioma Biomarkers and Their Validation • Introduction • SMRP and Osteopontin • Biomarkers in Progress • US Validation Trial Update • The Cappadochian Studies

  3. American Australian Mesothelioma Consortium • NYU School of Medicine (NYU) • Harvey I. Pass MD, PI • University of Western Australia (UWA) • Bruce Robinson MD, PhD, Co-PI • Peter MacCallum Cancer Institute (PMCC) • David Bowtell PhD • Andrew Holloway, PhD • Fujirebio Diagnostics, Inc (FDI)

  4. Asbestos-Related Thoracic Cancers • Pleural Mesothelioma • 2500 in United States • 15-30 year latency period • Median Survival 6-13 months • Uniformly fatal when diagnosed after symptoms • $54 billion in asbestos-related claims and the estimated future liability ranges from $145 to $210 billion.

  5. Mesothelioma ArchivesNYU • 221MPM tumors, snap frozen • 63 corresponding normal peritoneum • 249 sera • 34 plasma • 120 pleural effusion • 136 urine • Complete clinical demographics • 85 Asbestos exposed • All with serum, plasma, and urine • Complete clinical demographics • Over 200 lung cancers, snap frozen • Corresponding normal lung • Corresponding serum (all); 60 with plasma • Complete clinical demographics • 62 high risk for lung cancer (chemoprevention trial) • All with serum and plasma • Complete clinical demographics

  6. Novel Markers for Mesothelioma • Ready for Validation • SMRP (MesoMark™) • Partnership with Fujirebio Diagnostics, Malvern Pennslvania • Osteopontin • Studies in Progress • MMP1 and MMP9 • HAPLN1 (CRTL-1)

  7. Mesothelin • MAb K1 demonstrated selective staining of MPM tissue and cell lines • Pastan et al: 1992 • Willingham et al: 1992 • The cloned cDNA from an ovarian cDNA library encoded an antigen recognized by K1: • a 40-kDa glycoprotein (mesothelin) present on the surface of mesothelial cells, MPMs, and ovarian cancers with a 69 kDa precursor

  8. 8 years later….

  9. Serum Mesothelin Related Peptide(SMRP, Mesothelin Variant 1)) • Same N-terminal amino acid sequence as mesothelin and megakaryocyte potentiating factor. • Most likely originates as a portion of the extracellular domain of membrane-bound mesothelin • Non-Quantitative “sandwich ELISA” developed with antibodies 569 and 4HR Scholler N: Proc.Natl.Acad.Sci.U.S.A, 96:11531-11536, 1999 Onda M: Clin. Canc. Res., 15: 4225-4231, 2006.

  10. SMRP (Mesothelin Variant I) Antibody 569 • The antibody 569 stained 42/62 (68%) MPMs and 7/74 (10%) adenocarcinomas. All MPMs stained in a membranous pattern, and positive staining was seen in mainly epithelial components. (x200) Mesothelioma (x200) Adenocarcinoma

  11. SMRP and Mesothelioma • 84% sensitivity • 100% specificity when compared with other pleural diseases • 95% specificity when compared with other lung tumors • 83% when compared with people with asbestos exposure Not perfect Robinson, B.: Lancet, 362: 1612-1616, 2003.

  12. Validation of SMRP in the American Cohort

  13. Methods • Patient Population • Serum • 90 MPM • 170 NSCLC • 66 Asbestos-exposed volunteers from the Center for Occupational and Environmental Medicine • 409 normal volunteers • Pleural Effusion • 45 MPM • 20 Other Cancers • 30 Benign • SMRP • MesoMark™ duplicate samples • Statistical Analysis • ROC curves • Kruskal-Wallis and ANOVA

  14. Serum Demographics

  15. P<0.001 P=0.173 P<0.001 P=0.044 P=0.210 P<0.003

  16. Serum SMRP:Age/Sex Matched Controls (n=50) P<0.01

  17. Serum SMRP Mesothelioma Histology

  18. Serum SMRP and MPM Stage P=0.02 P=0.0001 P<0.0001

  19. Serum SMRP PerformanceMPM vs Normal (n=409) AUC = 0.94 95% CI = 0.910 to 0.955

  20. AUC = 0.805 95% CI = 0.734 to 0.864 AUC = 0.741 95% CI = 0.630 to 0.834 C D

  21. FDA and SMRP • January 2007: limited indication reference laboratory for the “monitoring” of treatment of mesothelioma

  22. SMRP and Treatment Monitoring NO RECURRENCE INTRATHORACIC RECURRENCE INTRAABDOMINAL RECURRENCE

  23. SMRP Conclusions • SMRP is a reasonable single marker for mesothelioma • The exact ranges for asbestos exposed cohorts must be studied in greater numbers of patients and in different geographies • This should be done in the context of an EDRN validation trial as an initial step

  24. Genomic Discovery of Biomarkers • Hypothesis • Affymetrix and Ingenuity Pathway Analyses can predict extracellular/secreted proteins which differ between normal mesothelium and early stage mesothelioma • Specific Aims • Discover new markers in serum and plasma • Validate these markers using appropriate control cohorts

  25. Methods for DiscoveryDifferences between Normal and Mesothelioma: All Genes • Specimens • 8 normal peritoneum • 7 Stage 1 mesothelioma • Platform • Affymetrix U133Plus • Analysis • dCHIP crossed with SAM • 453 genes which were significantly different

  26. U133 Plus Unsupervised Clustering: Peritoneum vs Stage I MPM: All Genes Significantly Different

  27. Identification of secreted proteins • 8 NP and 7 Stage I MPM were then compared for differences in 2036 genes which code for extracellular or secreted proteins (NetAffx™) • 669 genes were different (p<0.01) • These 669 genes were then inputted into Ingenuity Pathway analyses which selected 330 genes for the analysis. • 35 focus geneswere chosen for the networks

  28. 16 fold elevation Osteopontin

  29. Actual Expression for OPN in MPM

  30. Osteopontin Levels and Environmental Cancers:Test Populations • 48 normal sera • 66 asbestos-exposed • 72 mesothelioma sera What happens to Osteopontin in Asbestos Exposed Individuals?

  31. Published Data: Serum OPN and MPM • Serum OPN rises with duration of exposure and severity of radiographic asbestos changes • Promising distinction between asbestos exposed individuals and mesotheliomas Pass H, Lott D, Lonardo F, et al: Asbestos Exposure, Pleural Mesothelioma, and Serum Osteopontin Levels. NEJM 2005:353;1564-1573

  32. Osteopontin New Initiatives • Is this reproducible in plasma? • Can you distinguish MPM from lung cancer?

  33. Why Plasma? • Serum worked but could be erroneous. • Follow-up series of investigations to • test plasma osteopontin as a biomarker (34) • Measure levels in asbestos exposed (45), lung cancer (60), and smokers with dysplasia (56)

  34. Plasma Osteopontin Levels: Thoracic Malignancies And Controls NS P<0.0001 P<0.0001 P<0.0001 Plasma osteopontin looks as promising as serum osteopontin for MPM

  35. ROC Curves: Tumors vs Controls: Tumors vs Tumors Area under ROC curve = 0.988 95% CI = 0.947 to 0.998 Area under ROC curve = 0.783 95% CI = 0.686 to 0.861 Area under ROC curve = 0.980 95% CI = 0.920 to 0.997

  36. Osteopontin Conclusions • Both serum and plasma osteopontin are elevated in MPM compared to high risk asbestos controls • Plasma Osteopontin levels are also elevated in Lung Cancer and could be confused with MPM • Need other markers to distinguish between the two • The exact ranges for asbestos exposed cohorts must be studied in greater numbers of patients and in different geographies • This should be done in the context of an EDRN validation trial as an initial step

  37. What about other markers?MMP1 and MMP9

  38. Plasma OPN and MMP9 in mesotheliomas and asbestos exposed cohorts 450+51 287+38 38+4 51+10

  39. MMP9 and OPN: MPM and Asbestos Exposed CohortsMatched Plasma Specimens

  40. Moreover, MMP9 is elevated in lung cancer • MMP9 is NOT elevated in MPM • MMP9 IS elevated in lung cancer • Possible better discrimination between the two by combining with osteopontin?

  41. What about other markers?

  42. 23 fold elevation

  43. Microarray CRTL1/HAPLN1 expression data in mesothelioma patients

  44. HAPLN1 differential expression in mesothelioma and normal pleura samples (RT-PCR) 143T 144T 219T 322T 342T 351T 367T 374T 143N 166T 172T 249T 291N 318T 336N Loading control, PPIA

  45. T N TN TN TN TN N N 978T 997 NYU11 821 930 978 155 128 HAPLN1 PPIA Expression of HAPLN in matched tissues (normal pleura/mesothelioma)

  46. Mesothelioma, HAPLN1 antibodies (Genosis)

  47. Preparations for validation of SMRP,osteopontin

  48. Plans for EDRN Validation • Every two week conference calls • Harvey Pass, BDL • Mark Thornquist, DMCC • Jackie Dahlgreen, DMCC • Karl Krueger, NCI • Protocol Formulation • Definition of Ranges for Controls • ROC vs MPM • Retrospective/prospective studies

  49. Validation Trial • Phase I • Identification and assemblage of representative cohorts of individuals • with MPM • no malignancies but increased risk for MM due to asbestos exposure • (optionally) lung malignancies other than MM.

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