1. Gastrointestinal Stromal Tumors in Pediatric Patients MSKCC Grand Rounds May 1, 2008 Katherine Janeway, MD

2. Gastrointestinal stromal tumor (GIST) Most common mesenchymal neoplasm of the gastrointestinal tract  Still rare Annual incidence estimates 6.8 to 14.5/million  40% high risk or malignant features  1,500-4,000 cases per year  Osteosarcoma 660 new cases per year Extremely rare in children Resistant to standard chemotherapy and radiation  Response rate to doxorubicin <5%  Median survival in metastatic GIST 20 months

3. Activating mutations in KIT 80% of patients have GISTs with KIT mutations 5% of patients have GISTs with PDGFRA mutations 10-15% of patients have GISTs without PDGFRA or KIT mutations

4. Demetri, et al NEJM 2002 Dramatic responses to imatinib Gold et al, Ann Surg Oncol, 2006 Blanke, JCO, 2008

5. What about GIST in pediatric patients? Epidemiology and clinical features? Treatment?  Receptor tyrosine kinase inhibitors KIT and PDGFRA transforming roles?  Mutation analysis  Protein activation Are the transforming events different?  Genetic progression mechanisms  Succinate dehydrogenase  IGF1R

6. CharacteristicAdult GISTPediatric GIST AgePeak 60 Gender distributionEqual Morphology70% spindle cell KIT IHCPositive (except PDGFRA mutant) Natural historyMedian survival 20 mos ModelsCell lines, xenografts, transgenics Epidemiology and clinical features of GIST in pediatric patients Median 12 75% Female 70% epithelioid Positive None More indolent

7. Genetic Syndromes Carney Triad  GIST, paraganglioma, pulmonary chondroma Germline KIT or PDGFRA mutations NF-1

8. KIT and PDGFRA mutations in pediatric GIST Background  31 cases in the literature  Mutation rate in KIT and PDGFRA approximately 15% Objective  To determine whether the incidence of KIT / PDGFRA mutations in a large group of pediatric patients is similar to that reported previously in smaller patient groups

9. KIT and PDGFRA mutations in pediatric GIST Patients 27 patients age less than 25 with confirmed GIST KIT and PDGFRA mutation analysis KIT exons 9, 11, 13 and 17 and PDGFRA exons 12 and 18 were PCR amplified and screened for mutations by high performance liquid chromatography The entire KIT coding sequence was PCR amplified from cDNA and sequenced using the ABI 3730 xl sequencer

10. KIT and PDGFRA mutations in pediatric GIST Three of 27 patients (11%) with KIT or PDGFRA mutation  KIT exon 11 homozygous deletion VV559-560 (17 yo)  KIT exon 9 heterozygous AY502-503 tandem duplication (22 yo)  PDGFRA exon 18 D842V point mutation (14 yo) Four patients with GISTs lacking mutations on genomic DNA sequencing also lacked KIT mutations upon sequencing of the entire coding region from cDNA

11. KIT and PDGFRA mutations in pediatric GIST Most pediatric GISTs are KIT/PDGFRA - wildtype  Our 27 cases plus 31 previously published genotyped cases: 15% of pediatric GISTs harbor KIT or PDGFRA mutations PediatricAdult KIT exon 11 5% 66% KIT exon 9 9% 10% PDGFRA 3% 7%

12. KIT and downstream signaling in pediatric GISTs Background  KIT is expressed in pediatric GISTs KIT IHC positive Objective  To correlate KIT / PDGFRA genotype with the activation status of KIT, PDGFRA and downstream signaling intermediates Rationale  To inform approach to investigation and use of receptor tyrosine kinase inhibitors

13. Patients 27 patients age less than 25 with confirmed GIST  Subset of 15 patients with cryopreserved specimens Western blotting Whole cell lysates from cryopreserved tumors were separated by gel electrophoresis using 4 to 12% Bis- Tris gels and blotted to nitrocellulose membranes. Immunostains were detected by enhanced chemiluminesence. KIT and downstream signaling in pediatric GISTs

15. Pediatric KIT-wildtype GISTs display KIT expression and activation at levels comparable with KIT-mutant pediatric and adult GISTs  Mechanism is unclear Therapeutic inhibitors of KIT activation, particularly wildtype-KIT activation and inhibitors of signaling molecules downstream of KIT have the potential to be active in pediatric KIT-wildtype GIST KIT and downstream signaling in pediatric GISTs

16. Imatinib treatment of pediatric GIST AgeGenderDiseaseSurgeryIndicationResponse 15MHighCompleteAdjuvantNED 1.3 years 13MMetastaticIncompleteAdvanced dzSD 8 mos 9FMetastaticCompleteAdjuvantNo follow-up 11F2x3 cmNoneGastricSD (4 weeks) 13F6x3cmCompleteAdvanced dzPD (paraganglioma) 15FMetastatic / recurrent IncompleteAdvanced dzPD 10FMetastatic / recurrent IncompleteAdvanced dzSD 12 months Prakash S, et al J Pediatr Hematol Oncol, 2005 Durham MM, et al. J Pediatr Surg, 2004 Cypriano MS, et al. Cancer, 2004 Sauseng, et al. Pediatr Hematol Oncol, 2007 Hayashi Y, et al. Pediatr Surg Int, 2005

17. Imatinib response by genotype Heinrich, et al JCO, 2003 Exon 11 Exon 9 Wildtype

18. Sunitinib in advanced, IM resistant GIST Demetri, et al, Lancet Oncology, 2006 Sunitinib: Inhibitor of KIT, Flt-3, PDGFRβ, PDGFRα, Ret, CSF- 1R and VEFRs

19. Sunitinib response by genotype Heinrich MC, 2006 ASCO Annual Meeting

20. Sunitinib treatment of advanced pediatric GIST resistant to imatinib Objectives:  To determine the efficacy of sunitinib in pediatric patients with GIST resistant to imatinib  To characterize the side effect profile of sunitinib in pediatric patients Methods  Reviewed pediatric patients treated as part of the sunitinib compassionate use protocol Primary Objective: Availability Secondary objective: further characterize toxicity and outcomes

21. Compassionate use trial Eligibility Criteria  Confirmed diagnosis of GIST not amenable to cure with standard therapy  Not eligible for other ongoing trials of Sunitinib  Potential to derive benefit from Sunitinib therapy  Failed prior treatment with Imatinib disease progression or toxicity  1 week since last imatinib dose  Resolution of all toxicities of prior therapy  Adequate hepatic and bone marrow function Administration  6 week cycles, taken orally once daily for 4 weeks followed by 2 weeks off therapy  Dose levels: 25mg daily, 37.5 mg daily or 50 mg daily

22. Patient characteristics PathologyGenotype CaseAgeGenderSiteKIT IHC MorphologyPrior therapyKITPDGFRA 117FemaleMetastatic+EpithelioidIMWT 210FemaleGastric+EpithelioidIMWT 316FemaleMetastatic+EpithelioidIMWT 416MaleMetastatic+EpithelioidIMWT 516FemaleMetastatic+SpindleIM, investigational RTK inhibitor WT 616MaleMetastatic+MixedIMd/n/a 714Femaled/n/a IMd/n/a

23. Response to sunitinib Time to progression (months) CaseDaily DoseResponseImatinibSunitinibSunitinib vs. imatinib 150 mgSD<17+6 237.5 mg, reduced to 25 mgPR14>21+7 325 mgSD128-4 450 mgSD1618+2 550 mgSD<1>18+17 650 mgPD<1 0 750 mg, reduced to 37.5 mg then 25 mg SD1218+6

24. Response to imatinib therapy

25. Treatment related adverse events Adverse event Any grade N (%) Grade 3 or 4 N (%) Hematologic4 (57%)1 (14%) Gastrointestinal4 (57%)1 (14%) Fatigue4 (57%)1 (14%) Hepatic1 (14%)0 Musculoskeletal4 (57%)0 Hypothyroidism2 (29%)0 Hair hypopigmentation4 (57%)0 Headache2 (29%)0 Anorexia1 (14%)0

26. Sunitinib treatment of advanced pediatric GIST resistant to imatinib 1. Sunitinib treatment has antitumor activity and acceptable tolerability in this group of pediatric patients with GIST following failure of Imatinib. 2. Duration of response was greater with sunitinib that with prior imatinib in the majority of patients

27. Questions to ponder 1. Is there sufficient data to inform a recommendation about whether imatinib or sunitinib should be used as first-line therapy in pediatric patients with unresectable GIST? 2.Why is sunitinib effective in pediatric GIST? 3.What can be done when patients progress on sunitinib? Nilotinib: With Novartis will review compassionate access program

28. A word about surgery It is still a very important treatment modality in GIST  This is especially true in patients without KIT mutations

29. What about GIST in pediatric patients? Epidemiology and clinical features? Treatment?  Receptor tyrosine kinase inhibitors  Surgery KIT and PDGFRA transforming roles?  Mutation analysis  Protein activation Are the transforming events different?  Genetic progression mechanisms  Succinate dehydrogenase  IGF1R

30. Genetic progression mechanisms in pediatric GIST Background  In adult GISTs typical chromosomal aberrations follow KIT and PDGFRA mutation Changes are the same regardless of mutation type Loss of 14q, 22q, 1p  Prior karyotypes of pediatric GIST normal diploid Objective  To define the chromosomal aberrations in pediatric GIST in relation to those seen in adult GIST

31. Genetic progression mechanisms in pediatric GIST Patients 27 patients age less than 25 with confirmed GIST  Subset of 15 patients with cryopreserved specimens SNP assay DNAs were isolated from cryopreserved tumor and analyzed using an Affymetrix 10K SNP array at the DFCI Microarray Core Facility

33. Mechanisms of genetic progression are significantly different in pediatric KIT-wildtype GISTs versus pediatric and adult KIT-mutant GISTs  Biologically different tumors Another question to ponder  Are there in fact no genetic changes or is the mechanism not detectable by SNP arrays Epigenetic phenomena? Balanced translocations? Microsatellite instability? Genetic progression mechanisms in pediatric GIST

34. Pediatric GISTs are biologically distinct Prakash, et al J Pediatr Hematol Oncol, 2005

35. So what might be other oncogenic events be? Carney-Stratakis syndrome  Occurrence of GIST and paraganglioma Autosomal dominant Incomplete penetrance

36. Succinate Dehydrogenase aka CYBL Gottlieb and Tomlinson, Nature Reviews Cancer, 2005

37. SDH Mutations in Paraganglioma and Pheochromocytoma Familial paraganglioma  Primarily head and neck, can be intra-abdominal  10% of paragangliomas  Germline mutations in SDHB, SDHC, SDHD SDHB mutations can be associated with phechromocytoma and renal cell carcinoma Mutations missense in conserved AAs or nonsense All tumors show loss of the normal allele Sporadic paraganglioma  10% of patients with germline mutations in SDH Amar L JCO 23(34): 8812, 2005

38. More Succinate Dehydrogenase Mutations in Carney-Stratakis Syndrome 9 families, 11 patients  Age 9 to 37  In 7 families, germline mutations (or deletion) of SDHB, SDHC or SDHD  Of note, 2 patients had no significant family history McWhinney, et al NEJM, 2007

39. Metabolism Cancer Connection: HIF 1 Alpha Selak, et al, Cancer Cell, 2005

40. More questions to ponder What about SDH in sporadic GISTs in general and pediatric GISTs in particular?  Allelic imbalance in one of the SDH genes in 70% of sporadic GISTs Ferrando, et al, AACR 2007 abstract 3654

41. IGF1R KIT PKC theta Adult KIT mutant ped KIT mut GIST Ped KIT wildtype GIST Pediatric GIST and Insulin Like Growth Factor 1 Receptor (IGF1R) Could IGF1R antibody be effective in pediatric GIST?

42. Conclusions Despite lack of mutations, KIT is an important therapeutic target in pediatric GIST  Inhibitors of wildtype KIT likely to be more effective Pediatric GISTs are biologically distinct from adult GISTs  SDH and IGF1R are potentially involved in transformation It is feasible to study a rare pediatric tumor  Especially of there is a good comparison group  NIH GIST clinic

43. Thanks! Dana Farber Cancer Institute  George Demetri  Karen Albritton  Travis Quigley Brigham and Women’s Hospital  Jonathan Fletcher  Bernadette Liegl Oregon Health Sciences University  Mike Heinrich  Chris Corless Children’s Hospital, Boston  Antonio Perez-Atayde  Harry Kozakewich Patients, parents Gina Z. D’Amato, Chris R. Garrett, Paolo Pedrazzoli, Salvatore Siena, Joel Picus, Dana C. Matthews, James E. Butrynski, Marcus Schlemmer