From micro GIST to GIST: How can it happen? (Why doesn t it happen?)

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1 Brigham and Women s Hospital, Harvard Medical School From micro GIST to GIST: How can it happen? (Why doesn t it happen?) Jonathan A. Fletcher, M.D. Dept of Pathology Brigham and Women s Hospital Dana-Farber Cancer Institute Boston Dana-Farber/Harvard Cancer Center

2 microgist (<1cm) Found in 30% of the general population EG junction, spindle cell type Dr. Cher-Wei Liang

3 Why Study MicroGIST??? One of the most common benign tumors Only common benign tumor that results from TK genomic mutation Define events responsible for progression from TK-mutant ICC hyperplasia to microgist from microgist to clinical GIST Characterize barriers to malignant progression

4 P FAK PAK1 P Adhesion, motility GRB2 P SOS SHC Oncogenic KIT/PDGFRA DOK RAS-GAP P PI3K P JAK P RAS-GDP RAS-GTP AKT P STATs P P SAPK P MAPK P MEK1/2 BCLX L RAF BCLX L mtor BAD P P BAD ETV1 Mitochondria

5 Mutation Status in Small (<2cm) vs. Clinically-overt GISTs Genotype Small GISTs (N = 135) Overt GISTs (N = 101) P value Mutant 74% 84% KIT exon 11 46% 61% Drs. Sabrina Rossi, Roberta Maestro, Paolo dei Tos, AJSP, 2010

6 Relevant Risk Parameters for Primary GIST Including Molecular Data Parameters Lower risk Higher risk Surgery R0 R1, rupture Location Stomach Size < 5 cm > 5 cm Mitotic count < 5/50 HPFs > 5/50 HPFs Small bowel, rectal or other location Mutated gene KIT mutational type PDGFRA Duplication/ Insertion in exon 11 KIT Wildtype (non-pdgfr, non-kit) Deletion in exon 11 (esp. in codons W557 and K558); Duplication/insertion in exon 9 Wardelmann et al., Virchows Archiv 2007

7 Worrisome KIT mutations in benign microgists Exon 9 (4mm) Ex 11 del (3mm) Ex 11 homozygous (3mm) Dr. Cher-wei Liang

8 acgh Microdissect ion of tumor tissue WGA x 4 times Pool Cohybridization Result Microdissecti on of normal tissue WGA x 4 times Pool Cohybridization Cher-Wei Liang, MD

9 MicroGIST Genome Screen DELETIONS 1p 14q 22 GIST Genetic Progression KIT or PDGFRA mutation 14 22q 1p

10 FISH evaluation of chr 14 deletion in viable vs. hyalinized regions of microgists (N = 11) Cellular region Hyalinized region Chr 14 deletion evaluated in 200 cells from 11 microgists 5 of 11 microgists (45%) had chr 14 deletion present equally in cellular and hyalinized components

11 Pseudo-amplification of Biologically Crucial GIST Genes DOG1(ANO1, TMEM16A): RS 0.52 Cher-Wei Liang, MD

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13 DOG1 Pseudo-amplification: exonic and intronic Courtesy of Cher-Wei Liang, MD

14 MicroGISTs and clinical GISTs share gene expression biomarkers Top 30 active-transcription regions in microgists Top 30 Neighbors Top 30 Genes Clinical GIST Non-GIST Sarcomas IGF2 (0.78), DOG1 (0.52), CD34 (0.50), SPRY4 (0.45), KIT (0.32), PRKCQ (0.27), PROM1 (0.29), ETV1 (0.24)

15 Despite KIT & PDGFRA mutations, and GIST biology : MicroGISTs struggle to grow B Calcification Hyalinization Dr. Cher-wei Liang

16 KIT exon 11 deletions in clinical GISTs vs. microgists Distribution of KIT E11 Deletion, clinical GISTs J Lasota & M Miettinen, Histopathology 2008 Distribution of KIT exon 11 deletion in 110 microgists (30, reference) collected from 6 series. CL Corless et al., AJP 2002 K Kawanowa et al., Hum P 2006 A Agaimy et al., AJSP 2007 A Agaimy et al., AJSP 2008 S Rossi et al., AJSP 2010 Distribution of KIT Ex11 deletions, microgists (34 cases from present series, and 76 cases from previously published series)

17 MicroGIST Mutations Expressed in KIT-negative GIST48B SCF (KIT-LG) dose response Cher-wei Liang

18 MicroGIST Mutations Expressed in KIT-negative GIST48B SCF (KIT-LG) dose response Cher-wei Liang

19 MicroGIST Mutations Expressed in KIT-negative GIST48B SCF (KIT-LG) dose response Cher-wei Liang

20 MicroGIST Mutations Expressed in KIT-negative GIST48B SCF (KIT-LG) dose response Cher-wei Liang

21 MicroGIST Mutations Expressed in KIT-negative GIST48B SCF (KIT-LG) time response Cher-wei Liang

22 KIT-ligand (SCF): strong expression in smooth muscle; weakto-no expression in microgist Green: Desmin Red: SCF Blue: DAPI

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24 Dr. Jason Hornick MicroGISTs have infiltrative borders (90.7%) and smooth muscle markers (49%) RED = Desmin Brown = DOG1 GREEN = Desmin RED = DOG1 BLUE = DAPI

25 Dr. Cher-Wei Liang Some microgists are smooth muscledependent tumors

26 Cell cycle dysregulation in GIST Low-risk GIST (N = 16) TP53 MDM2 CDKN2A RB1 ATM None %mut % High-risk GIST (N = 24) TP53 MDM2 CDKN2A RB1 ATM None %mut %

27 Conclusions #1 MicroGISTs are on a biologic continuum with malignant GISTs But, they virtually never progress to malignancy And, they very often involute Certain KIT & PDGFRA mutations cannot sustain malignant progression

28 Conclusions #2 Progression from microgist to clinical problem Ligand-independence or autocrine/paracrine Further cell cycle dysregulation Other genetic hits (14q, 22q, 1p..)

29 Acknowledgements Brigham and Women s Hospital Department of Pathology Cher-wei Liang Yuexiang Wang Adrián Mariño Enríquez Stephen Swank Meijun Zhu Cheng-Han Lee Christopher D.M. Fletcher Jason Hornick Division of Surgical Oncology Chandrajit P. Raut Dana Farber Sarcoma Program George D. Demetri Andrew Wagner Memorial Sloan Kettering Cancer Center Cristina Antonescu Virginia and Daniel K.Ludwig Trust for Cancer Research, GIST Cancer Research Fund, The LifeRaft Group, National Institutes of Health (1P50CA127003). Treviso Angelo Paolo dei Tos Sabrina Rossi Aviano Roberta Maestro