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Trent Hummel, Yonatan Mahler, Shyra Miller, Timothy Cripe, Walter Jessen,

Microarray gene expression utilized to identify putative serum biomarkers of NF1 associated tumors Connective Tissue Oncology Society November 14, 2008. Trent Hummel, Yonatan Mahler, Shyra Miller, Timothy Cripe, Walter Jessen, Bruce Aronow, Nancy Ratner Cincinnati, OH, USA. Background.

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Trent Hummel, Yonatan Mahler, Shyra Miller, Timothy Cripe, Walter Jessen,

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  1. Microarray gene expression utilized to identify putative serum biomarkers of NF1associated tumorsConnective Tissue Oncology SocietyNovember 14, 2008 Trent Hummel, Yonatan Mahler, Shyra Miller, Timothy Cripe, Walter Jessen, Bruce Aronow, Nancy Ratner Cincinnati, OH, USA

  2. Background • 30% of NF1 patients develop plexiform neurofibromas (PNF) – these typically arise in childhood • Benign tumors involving larger nerves • Can invade critical structures causing death and significant morbidity • Irregular borders make growth calculations difficult with CT and MRI scans • Surgery is the only reliable treatment • Plexiform neurofibromas can undergo transformation to malignant peripheral nerve sheath tumors (MPNST) Huson SM, et al. Brain 1988 Rasmussen SA, et al Am J Epidemiol 2000

  3. Background • Malignant peripheral nerve sheath tumors (MPNST) • Highly aggressive sarcomas • 5 year survival approximately 30% • Lifetime risk in NF1 patients is 8-13% • No reliable method to detect transformation of plexiform neurofibromas to MPNSTs Huson SM, et al. Brain 1988 Rasmussen SA, et al Am J Epidemiol 2000

  4. Hypotheses • There are unique serum proteins that mark malignant peripheral nerve sheath tumors • There are unique proteins in the peripheral blood of NF1 patients with PNF that correlate with tumor burden/growth.

  5. NHSC 45 Cell culture samples dNFSC +/- 10 Normal human Schwann cell dNFSC -/- 2 Dermal neurofibroma NF1 +/- Schwann cell pNFSC 9 Dermal neurofibroma NF1 -/- Schwann cell 11 Plexiform neurofibroma Schwann cell MPNST cell 13 MPNST cell line 32 Tumor samples dNF 13 Dermal neurofibroma pNF 13 Plexiform neurofibroma MPNST 6 MPNST NF1 Microarray Consortium86 microarrays in 9 batches(77 samples plus 9 references) • Gene expression from 10 independent cultures of primary human Schwann cells were compared to 67 NF1 tumor samples • Included cultured neurofibroma Schwann cells and primary tumors on an Affymetrix platform NF1 Microarray Consortium - Nancy Ratner, Shyra Miller, Bruce Aronow, Walter Jessen, Marco Giovannini, Anat Stemmer-Rachamimov, Peggy Wallace, Conxi Lazaro, and Grier Page

  6. 6.0 0.0 Normal dNFSC +/- dNF dNFSC -/- pNF pNFSC MPNST MPNST cell • The degree of differential expression is expressed by color • Each row represents a single gene expression profile • Genes in blue exhibit down regulation • Genes in red signify upregulated expression as compared to normal human Schwann cells. • For this project, genes that demonstrated differential upregulation between NHSC and neurofibromas and/or MPNSTs were chosen for further analysis.

  7. Methods Filter these up-regulated genes through a database of secreted proteins Validate candidates with quantitative RT-PCR Confirm secretion of proteins in cell media Test candidates in human model

  8. Methods • Using microarray data from NF1 Microarray Consortium (Miller S, et al. submitted) • Compared differentially expressed probesets with the Secreted Protein Database • List of probesets that encode secreted proteins that may be released into the extracellular milieu Chen Y, et al. Nucleic Acids Res. 2005

  9. 9473 genes differentially expressedbetween cell culture classesNormal Human Schwann cell dermal NFSC, plexiform NFSC,MPNST cell line (FDR=0.05) 3721 genes from the Secreted Protein Database with a confidence rank of 0-3 1664 of 3721 genes upregulated from Normal Human Schwann cell to Neurofibromaor Normal Human Schwann cell to MPNST Methods • Analysis yielded 666 genes that encode putative secreted proteins • Differentially upregulated between NHSC and neurofibromas and/or MPNST • Subsequent cluster analysis across both tumors and cell lines reduced the number of candidates to 132 • Alternatively spliced forms of genes were removed • 92 genes that encode putative secreted proteins and that are upregulated in MPNST and/or neurofibromas as compared to normal human Schwann cells

  10. Top Candidates • Initial Candidates Selected base on: • Robust expression in microarray data in all cell lines when compared to NHSC • Commercially available reagents • Adrenomedullin (ADM)

  11. Adrenomedullin • Adrenomedullin (ADM) is a secreted peptide (52 AA) • Stimulates angiogenesis • ADM knockout mice die at E14 because of severe vascular defects • ADM expression correlates with vascularity in RCC and breast carcinomas • Hypoxia up-regulates ADM in human glioma cell lines • GBM Xenograft tumor model - anti-ADM antibody greatly reduced both tumor growth and vascularity Zhao et al 1998 Jougasaki and Burnett, 2000 Kitamuro et al, 2000 Garayoa et al, 2000 Shindo et al., 2001 Oehler et al., 2002 Ouafik et al., 2002

  12. Validation of Array Data with RT-PCR • Quantitative RT-PCR confirms microarray data in MPNST cell lines and NFSC (NF1+/- and -/-) cell lines as compared to Normal Human Schwann cells • Greater than 3 fold change considered significant

  13. ADM in Conditioned Media Results using a competitive ELISA adrenomedullin detection kit (Phoenix Pharmaceuticals) following the manufacturer’s protocol

  14. Methods Filter these up-regulated genes through a database of secreted proteins Validate candidates with quantitative RT-PCR Confirm secretion of proteins in cell media Test candidates in human model

  15. Characteristics *4/5 MPNST serum samples courtesy of Victor Mautner, 1/5 was a 16 y/o who had serum collected for presumed PNF and found to have MPNST.

  16. ADM elevated in patients with NF1 regardless of tumor status • No difference in ADM concentration when comparing plexiform neurofibroma tumor burden, tumor location, age, or gender • Adrenomedullin is a potential serum biomarker of NF1 • Increased serum levels of ADM may correlate with presence of MPNST, though small sample size limits the power of this analysis. 4/5 MPNST samples courtesy Victor Mautner

  17. Conclusions • Feasible to utilize microarray gene expression data to generate a list of putative secreted biomarkers • Confirming expression with secondary technique such as RT-PCR • Testing protein expression/secretion in cell lines and subsequently in human serum • Adrenomedullin • Potential biomarker for NF1 • Also potential serum biomarker for NF1 related MPNSTs

  18. Future Directions • Continue investigating list of 92 genes • Obtain more human MPNST serum samples • Further investigate the biological effects of adrenomedullin on MPNST cell lines • Envision a panel of proteins used to delineate between growth of plexiform neurofibromas and transformation to malignant peripheral nerve sheath tumors • Add to clinical decision making • Add to treatment decisions regarding therapy

  19. Thanks to… • Nancy Ratner • Shyra Miller • Members of NF1 Microarray consortium - Bruce Aronow, Walter Jessen, Marco Giovannini, Anat Stemmer-Rachamimov, Peggy Wallace, Conxi Lazaro, and Grier Page • Jon Williams • John Perentesis • Elizabeth Schorry • Tim Cripe • Yoni Mahler

  20. SPD • Entries ranked according to prediction confidence (rank 0 -3) Chen Y, et al. Nucleic Acids Res. 2005

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