1. CTOS 11.13.08 Growth Factor Pathway Activation in Chordoma Carolyn Hoban; Dafydd Thomas; David Lucas; Laurence Baker, University of Michigan, Departments of Internal Medicine, Division of Oncology/Hematology; And Pathology, Ann Arbor, Michigan

2. CTOS 11.13.08 Backgound:  Tumor microenvironment of bone and nervous system  Usually slow growing tumors believed to arise from remnants of notochordal tissues  Challenging to treat with surgery, radiation and/or chemotherapy regimens due to location of the tumor.  Frequently recur and may have distant metastasis in late stage of disease. Factors regulating recurrence have not been determined. Chordoma

3. CTOS 11.13.08 Translational focus  To determine expression levels and activation of key growth factor pathways immunohistochemically using quantitative methods.  Retrospective analysis of routinely processed paraffin sections of chordoma clinical specimens.

4. CTOS 11.13.08  34 cases of Chordoma obtained from UM pathology archives with IRB approval  3 tumor cores /pt /TMA  Diagnostic confirmation upon review by pathologists (Lucas)  M:F = 14:14; Age: avg. 52 (Range 11-81)  Anatomic location:  Clival (n=11); Sacral (n=9)  Primary and recurrent tumors Chordoma Clinical Specimens

5. CTOS 11.13.08  Low to intermediate-grade tumor, resembling notochord  Clusters of large polymorphous cells in myxoid matrix  Pleomorphic & hyperchromatic nuclei  Mucin filled vacuoles ‘physaliferous’  Positive IHC for Cytokeratin19, S100, EMA Chordoma Histology

6. CTOS 11.13.08 IHC: Identification of activated pathways & drug targets Brachyury* [Chordoma marker] RTK: HER family PDGFR  and , IGF1R, c-Ret, c-kit, c-src, mTOR, AKT, and MAPK 2008 Tirabosco: Am J Surg Pathol, 2005 Henderson Genome Biology 2006 Vujovic J. Paht.

7. CTOS 11.13.08 TMA 8387 TMA 6971 TMA 6983TMA 6974 Chordoma TMA: IHC scores Brachyury: high high frequency 100% high intensity 3+  c-kit: neg/ low  HER family 1.EGFR mod 2.EGFRVIIIneg 3.Her2 : neg/ low Fasig et al. ckit low (33%); EGFR (67%) Weinberger et al. Her2 focal staining 123

8. CTOS 11.13.08 IHC scores: GF receptor profile  PDGF_R: high  c-ret: high (GDNF pathway)  IGF1R  Phospho-tyr IGF1R TMA 6977 PDGF-R  PDGF-R  IGF1R pY 1147 -IGF1R TMA 6985

9. CTOS 11.13.08  Automated Quantitative Analysis (AQUA)  Advantages:  Automated, high-throughput,  Objective (r/o intra/inter-observer concordance; lesion heterogeneity)  Fluorescence-based Ab used in IHC  Multiplex:  Antigen of interest  Tumor- and stromal-specific marker  Subcellular location (nuclei, cytoplasm,PM)  Automated scoring and data analysis  Activation index (phospho:total GF-receptor) Quantitative IHC

10. CTOS 11.13.08 Stroma Tumor Necrosis Cytokeratin Cy3 Nuclear Component Tumor Mask AQUA analysis

11. CTOS 11.13.08 IGF1R, c-ret, PDGFR pathways are activated AQUA score

12. CTOS 11.13.08 Challenge: optimal pathway inhibition Ab to RTK: Small mol inh: PI3K/ AKT mTOR …….. Which drugs? Combinations? Ab to RTK: Small mol inh: PI3K/ AKT mTOR …….. Which drugs? Combinations?

13. CTOS 11.13.08 From Clinical evidence to Preclinical models

14. CTOS 11.13.08 In vitro models (chordoma cell lines)  U-CH1 (Scheil et al 2001) [courtesy of Chordoma Foundation, Josh Sommer; Mike Kelly, Duke Univ)  Morphological features: physaliferous, round nuclei, mucinous, (BrU,CK, S100 & EMA positive)  Other cell lines in development In vivo models (chordoma/ microenvironment)  UCH-1 cells with stable expression of luciferase  Developing BLI- Xenograft lines (passage in SCID mice)  Plan to evaluate drugs that inhibit GF pathways in vivo Preclinical models of chordoma

15. CTOS 11.13.08 Used data obtained from clinical specimens to build our experimental models:  Rank cell lines with similar features to clinical specimens (molecular markers, immunopositive GF pathway activation)  Test drug combinations to optimally inhibit key pathways, in vitro (cell kill) and in vivo (OS)  Support nomination of optimal combinations of drugs into clinical research  Translation Clinic-lab-Clinic Conclusion

16. CTOS 11.13.08  The team:  University of Michigan Multidisciplinary Sarcoma group  Collaborators  Patients & friends (the village) Financial support from  Stefan L. Harris Fund Contact: carhoban@umich.edu  The team:  University of Michigan Multidisciplinary Sarcoma group  Collaborators  Patients & friends (the village) Financial support from  Stefan L. Harris Fund Contact: carhoban@umich.edu Thank you

17. CTOS 11.13.08 A model for the mechanism by which PTHrP regulates growth plate chondrocytes through the Gli transcription factors. PTHrP acts to regulate the progression of growth plate chondrocytes from a proliferative to the hypertrophic zones by regulating the activity of the Gli transcription factors. It is able to do this by both regulating hedgehog ligand activity and by regulating the processing of Gli3 in a hedgehog ligand independent, PKA dependent, manner. Does this work in Neural crest lineage, would it control chordoma development Hedgehog pathway in neural crest

18. CTOS 11.13.08 p-MAPK mTOR p-AKT P-mTOR Quantitative analysis of signalling molecules