1. CTOS-VENICE NOVEMBER 2006 University of Washington Sarcoma Service MULTIPLE PET TRACERS IN STS TREATMENT RESPONSE UNIVERSITY OF WASHINGYON SEATTLE CANCER CARE ALLIANCE CHILDRENS HOSPITAL SEATTLE, WASHINGTON 1 University of Washington Medical Center and 2 Children’s Hospital and Regional Medical Center, Seattle, Washington, USA E.U. CONRAD MD, J.F. EARY MD, J.E. BUTRYNSKI MD, J.M. LINK PhD, A. M. CIZIK MPH, M MUZI MS, and K.A. KROHN PhD supported by NIH NCI CA 42045, S10 RR017229-01

2. CTOS-VENICE NOVEMBER 2006 University of Washington Sarcoma Service SARCOMA GRADING & SUBTYPES French Protocol 1997 MitosesDifferentiationNecrosis Grade Discrepancies = 34.6% “Intermediate” Malignancies Were Omitted! Guillou L, Coindre JM, Bonichon F, et al: Comparative Study of the National Cancer Institute and French Federation of Cancer Centers Sarcoma Group Grading Systems in a Population of 410 Adult Patients with Soft-Tissue Sarcoma, J Clin Oncol, 15(1): 350–62, 1997. Deyrup AT and Weiss SW. Grading of Soft Tissue Sarcomas: the challenge of providing precise information in an imprecise world. Histopathology 48: 43-50, 2006. NCI vs FRENCH SARCOMA PATH GRADE

3. CTOS-VENICE NOVEMBER 2006 University of Washington Sarcoma Service Sarcoma Patient Survival PET “GRADE” Eary JF, O’Sullivan, F, Powitan Y, Kingshuk RC, Vernon C, Bruckner JD, and Conrad III EU: Sarcoma tumor FDG uptake measured by PET and patient outcome: a retrospective analysis. European Journal of Nuclear Medicine, 29(9): 1149–1154, 2002. Overall Survival Disease-free Survival p<0.003p<0.001 SUV < 6.0 ?

4. CTOS-VENICE NOVEMBER 2006 University of Washington Sarcoma Service Biopsy - Sampling Error in Large Tumors High-grade Bx: Myxoid & round-cell liposarcoma; no necrosis. Interm’-grade Bx: Myxoid liposarcoma; no round-cell areas, no necrosis. Necrotic Bx: No viable tumor identified. GRADING “VARIANCE”?

5. CTOS-VENICE NOVEMBER 2006 University of Washington Sarcoma Service UW NEOADJUVANT CHEMOTHERAPY PET PRIOR TO BIOPSY & RESECTION FDG PETBiopsyChemo 1Chemo 2 FDG PETChemo 3Chemo 4 FDG PET Chemo 3Chemo 4 FDG PETResectionPostop FDG PET ResectionPostopChemo

6. CTOS-VENICE NOVEMBER 2006 University of Washington Sarcoma Service FDG PET SURVIVAL: SOFT TISSUE vs BONE vs CARTILAGE Eary JF, O’Sullivan, F, Powitan Y, Kingshuk RC, Vernon C, Bruckner JD, and Conrad III EU: Sarcoma tumor FDG uptake measured by PET and patient outcome: a retrospective analysis. European Journal of Nuclear Medicine, 29(9): 1149–1154, 2002. Tumor TypeMean+SDMean+95%CIMedianLog-rank (Months)(Months)SUV max (p value) Evaluating Overall Survival Cartilage23.9+16.323.9+7.413.90.008 Bone17.6+16.417.6+4.5610.00.505 Soft Tissue18.7+15.718.7+2.685.60.002 Overall18.9+16.018.9+2.186.00.003 Evaluating Disease-free Survival Cartilage17.7+15.817.7+7.003.90.103 Bone11.2+13.811.2+3.8510.00.483 Soft Tissue14.5+14.814.5+2.525.60.005 Overall14.0+14.714.0+2.006.00.001

7. CTOS-VENICE NOVEMBER 2006 University of Washington Sarcoma Service Prognostic Factors for DFS Variable Exp (Coef) 95% C.I. P-Value SUV max response (>40%) 0.2860.104–0.7900.009 Tumor size (<10 cm) 0.6030.250–1.4570.261 Tumor grade (Gr 3) 0.6580.253–1.7110.378 Residual viable tumor (<5%) 0.6280.184–2.1410.433 Location (extremity) 0.8860.366–2.1420.789 Schuetze SM, Rubin BP, Vernon C, Hawkins DS, Bruckner JD, Conrad EU, Eary JF. Use of PET in Localized Extremity Soft Tissue Sarcoma Treated with Neoadjuvant Chemotherapy. CANCER 103: 329-348. 2004. > 40% < 40%

8. CTOS-VENICE NOVEMBER 2006 University of Washington Sarcoma Service PET vs”RECIST” ASCO 2005 Schuetze Eary,Conrad et al ASCO 2005 Schuetze Eary,Conrad et al Prospective series ( n= 33) Prospective series ( n= 33) 2 cycles(poor resp) vs 4 cycles(good resp) 2 cycles(poor resp) vs 4 cycles(good resp) MRI vs PET(40%) vs Histologic Response MRI vs PET(40%) vs Histologic Response FDG PET vs Path p=0.092 FDG PET vs Path p=0.092 RECIST did not predict Path p=0.002 RECIST did not predict Path p=0.002

9. CTOS-VENICE NOVEMBER 2006 University of Washington Sarcoma Service Factors in Response and Resistance Proliferative Rate Thymidine & Analogs Glycolytic Rate FDG Hypoxia FMISO, EF1, ATSM Efflux Pumps MIBI, Verapamil, Colchicine

10. CTOS-VENICE NOVEMBER 2006 University of Washington Sarcoma Service MULTIPLE IMAGING FDG—Water—Thymidine FDG PERFUSION (H2O) THYMIDINE PRE CHEMO POST CHEMO PREOP Pre-Chemo PET Post-Chemo PET

11. CTOS-VENICE NOVEMBER 2006 University of Washington Sarcoma Service Verapamil Model P-glycoprotein Substrate (similar to Adriamycin) P-glycoprotein Substrate (similar to Adriamycin) Quantify Serial Scans vs. Patient Baseline Quantify Serial Scans vs. Patient Baseline P-glycoprotein Inactivated by Cyclosporin,etc. P-glycoprotein Inactivated by Cyclosporin,etc. Does Chemo Induce P-gp Activity ? Does Chemo Induce P-gp Activity ? Hendrikse NH, de Vries EGE, Franssen EJF, Vaalburg W, van der Graaf WTA. C-11 Verapamil kinetics in human tissue. EJCP 2001. Hendrikse NH, de Vries EGE, Franssen EJF, Vaalburg W, van der Graaf WTA. C-11 Verapamil kinetics in human tissue. EJCP 2001.

12. CTOS-VENICE NOVEMBER 2006 University of Washington Sarcoma Service UW Verapamil (Preliminary Studies ) PrimateHuman (Brain) Brain P-gp Blocked P-gP Active CYCLOSPORIN

13. CTOS-VENICE NOVEMBER 2006 University of Washington Sarcoma Service Specific Aims Specific Aim 1: Correlate [C-11]thymidine uptake in sarcoma patients with histologic, immunohistochemical, and gene expression profiles. Specific Aim 1: Correlate [C-11]thymidine uptake in sarcoma patients with histologic, immunohistochemical, and gene expression profiles. Specific Aim 2: Perform pre-and post-chemotherapy hypoxia imaging with [F-18] fluoromisonidazole (FMISO) and correlate imaging results with tissue hypoxia markers. Specific Aim 2: Perform pre-and post-chemotherapy hypoxia imaging with [F-18] fluoromisonidazole (FMISO) and correlate imaging results with tissue hypoxia markers. Specific Aim 3: Quantitate the efflux of [C-11] verapamil in sarcoma tumors in vivo and correlate with tissue molecular markers for p-glycoprotein pump and drug-resistance markers. Specific Aim 3: Quantitate the efflux of [C-11] verapamil in sarcoma tumors in vivo and correlate with tissue molecular markers for p-glycoprotein pump and drug-resistance markers.

14. CTOS-VENICE NOVEMBER 2006 University of Washington Sarcoma Service METHODOLOGY:METHODOLOGY: Chemo 1 A Biopsy 3X Chemo Thymidine2 Thymidine 3 FMISO 2 FMISO 3 Water 1Water 2 Resection Followed by XRT Then follow- up Imaging Study 1 Imaging Study 2 Imaging Study 3 FMISO 1 Chemo +/- Thymidine 1 Verapamil 1 Verapamil 2 METHODS: SYNTHESIS AGENTS& IMAGING DATA COLLECTION &ANALYSIS MODELING BLOOD FLOW

15. CTOS-VENICE NOVEMBER 2006 University of Washington Sarcoma Service RESULTS-MULTIPLE TRACERS Patient ID # GenderAgeDiagnosisGradeLocationSize Metastatic Disease at presentation 501F48 Pleomorphic liposarcoma 3 L proximal medial thigh 8 cm No 502F55 Myfibroblastic fibrosarcoma 2 R forearm 7 cm Indeterminate 503M54 Myofibroblastic sarcoma 2 L pelvis to proximal thigh 10 cm LN+ 504F18 Alveolar rhabdomyosarcom 3 R forearm 15 cm LN+, Lung, Spine

16. CTOS-VENICE NOVEMBER 2006 University of Washington Sarcoma Service Pre-Neoadjuvant Chemotherapy on MRI Status Post-2 Cycles Neoadjuvant Chemotherapy on MRI 501501

17. CTOS-VENICE NOVEMBER 2006 University of Washington Sarcoma Service Pre-Neoadjuvant Chemotherapy on MRI Status Post-2 Cycles Neoadjuvant Chemotherapy on MRI 502502

18. CTOS-VENICE NOVEMBER 2006 University of Washington Sarcoma Service Pre-Neoadjuvant Chemotherapy on MRI Status Post-2 Cycles Neoadjuvant Chemotherapy on MRI 503503

19. CTOS-VENICE NOVEMBER 2006 University of Washington Sarcoma Service Pre-Neoadjuvant Chemotherapy on MRI Status Post-2 Cycles Neoadjuvant Chemotherapy on MRI 504504

20. CTOS-VENICE NOVEMBER 2006 University of Washington Sarcoma Service Results: SUV max

21. CTOS-VENICE NOVEMBER 2006 University of Washington Sarcoma Service FDG -48% Blood Flow -7% Thymidine -32% Verapamil -27% FMISO -5% PRE THERAPYPOST THERAPY Liposarcoma

22. CTOS-VENICE NOVEMBER 2006 University of Washington Sarcoma Service Pre-Neoadjuvant Chemotherapy on MRI Status Post-2 Weeks Neoadjuvant Chemotherapy on MRI 503503 FDG +257% TdR -3% FMISO +164% Poor Responder - MFH

23. CTOS-VENICE NOVEMBER 2006 University of Washington Sarcoma Service Gene Array Data Results – Pending Nielsen TO, West RB…, and van de Rijn M, et al: Molecular characterisation of soft tissue tumors: a gene expression study. The Lancet, 359: 1301–1307, 2002. Gene Array Correlation with F-MISO, Verapmil and Thymidine Tumor Heterogenity Mapping

24. CTOS-VENICE NOVEMBER 2006 University of Washington Sarcoma Service Water (20-80s) Verapamil (5-25m) Pre-Therapy Post-Therapy T2 coronal MRI MULTIPLE PET TRACERS:

25. CTOS-VENICE NOVEMBER 2006 University of Washington Sarcoma Service SARCOMA CHALLENGES- ASSESSING GRADE : 1. CLINICAL “GRADE”: 1. CLINICAL “GRADE”: Soft Tissue Density, Depth, Size Soft Tissue Density, Depth, Size 2. MRI “GRADE”: 2. MRI “GRADE”: Size. T2(fluid), inflam zone, Size. T2(fluid), inflam zone, “Heterogeneity”(density/necrosis) “Heterogeneity”(density/necrosis) 3. PET “SUV” GRADE: 3. PET “SUV” GRADE: INITIAL/PRE-CHEMO vs POST- INITIAL/PRE-CHEMO vs POST- CHEMO CHEMO “FINAL GRADE” ? “FINAL GRADE” ?

26. CTOS-VENICE NOVEMBER 2006 University of Washington Sarcoma Service CTOS IN SEATTLE 2007 !