Glivec en KIT: immuunhistochemie en mutatie analyse in gastro-intestinale stromacel tumoren. Judith V.M.G. Bovee Patholoog LUMC

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1 Glivec en KIT: immuunhistochemie en mutatie analyse in gastro-intestinale stromacel tumoren Judith V.M.G. Bovee Patholoog LUMC

2 Case 60 year old female Pain and discomfort upper abdomen MRI: tumor small bowel wall

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8 Differential diagnosis Gastro-intestinal stromal cell tumour Leiomyoma / leiomyosarcoma des-, CD117+ des+, CD117- Schwannoma MPNST Spindle cell carcinoma Desmoid type fibromatosis Melanoma S100++, CD117- CD117- keratin+, CD117-, CD34- CD117-, nuclear beta-catenin S100+, focal CD117+

9 ; definition Mesenchymal tumor Adjacent to / vicinity of the GI-tract Characteristic histomorphology 95% expression of the KIT receptor (CD- 117) Previously often diagnosed as a type of soft tissue sarcoma

10 GIST; origin Recapitulation of interstitial cells of Cajal Pacemaker cells of the gut wall Combines (incomplete) myoid and neural phenotype

11 Tumor location Stomach 40-50% Small bowel 30% Rectum/ Colon 10% Omentum/Mesentry 7% Esophagus 1-2% Peritoneum Rare Metastasis Liver, rarely to lymph nodes and almost never to the lungs

12 Symptoms Often asymptomatic Often non-specific symptoms At diagnosis > 30% were found evident malignant due to: Widespread intra-abdominal abdominal and liver metastasis Tumor infiltration in surrounding organs

13 Clinical features Most frequent mesenchymal tumor of the GI tract (esp. stomach and small bowel) Equal male : female ratio Highest incidence 5th 7th decade 5 yr survival 50-65% after surgery <35% at incomplete resection or metastases

14 Risk assessment Any GIST is potentially malignant Metastatic likelyhood given by size and mitotic activity Size (cm) Mitoses Very Low Risk < 2 < 5/50HPF Low Risk 2-5 5/50HPF Intermediate Risk High Risk > 5 > 10 Any size 6-10/50HPF 5/50HPF > 5/50HPF Any MR > 10/50HPF NIH Consensus Conference Hum Pathol 2002;33:459 Ann Oncol 2005;16:566

15 Macroscopy submucosal lesion normal overlying mucosa occasionally central ulcer with bleeding biopsies often not sufficient tissue

16 Histological spectrum Spindle Cell Type (70%) Epithelioid Cell Type (20%) Mixed Spindle and Epithelial Cell Type (10%)

17 Spindle Cell GIST Epithelioid GIST

18 Immunohistochemistry CD117 (kit) 95% CD % Sm-actin 30-40% S100 5% Keratin 1-2% Desmin 1-2%

19 Immunohistochemistry

20 CD117 in GIST

21 CD117 staining patterns Membranous, Cytoplasmic, or Dot-like

22 CD117 J Clin Pathol 2001:54;96-103

23 Antigen retrieval and CD117 Antigen retrieval: percentage positive cases in series presumed GIST raises Staining intensity raises variety of unrelated tumors are getting positive without a therapeutic meaning (material a.o. from Eur J Cancer 2003;39:2006) International consensus: no AR for CD117 in GIST KIT mutation analysis useful for morphologic GIST in which KIT is negative

24 EORTC Phase III trial (62005) False positive (GIST) diagnosis in +/- 6% GIST misdiagnosed as: melanoma, clear cell sarcoma, leiomyosarcoma, carcinoma, reticulum cell sarcoma, glomus tumor, desmoid fibromatosis, sarcoma nos Lancet 2004;364:1127 J Clin Oncol 2005;23:5795

25 Incidence Exact incidence unknown The Netherlands ~12/million Availability of immunohisto- chemistry Awareness of therapeutical options

26 Case CD34

27 Case Mutation analysis is indicated CD117

28 Mutations 85-90% KIT activating mutations 4q11-21 Type III tyrosine kinase growth factor receptor % 15% KIT mutation negative 35% mutation PDGFRA Receptor tyrosine kinase Kit (CD117) expression low / absent

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30 Imatinib (Glivec /Gleevec/STI571) Pre-glivec Glivec idem: PDGFR-A Bcr-abl Signal Transduction Pathways Activated c-kit Receptor ATP binds to kinase portion of receptor Nucleus Cell membrane Signal Transduction Pathways Inhibited Blocks ATP binding

31 Proof of concept study with Imatinib (april 2001)

32 Mutations in KIT

33 Mutations in the juxtamembrane domain (exon 11) 20-92% of mutations Function juxtamembrane domain: inhibit receptor dimerization in absence of SCF Mutations: disrupt this function, allowing ligand independent receptor dimerization Corless et al, JCO 2004 H Heterozygous deletion V559-E561

34 Mutations in the extracellular domain (exon 9) ~10% of mutations AY duplication / insertion 95% associated with small intestine More aggressive Mechanism of action not determined; disrupts antidimerization motif in extracellular domain? Corless et al, JCO 2004

35 Mutations in the kinase I domain (exon 13) % of mutations K642E Ligand independent activation V654A in secondary resistance to imatinib Corless et al, JCO 2004

36 Mutations in the activation loop (exon 17) 0.6% of mutations N822K and N822H Constitutive activation of the kinase domain D820Y in familial GIST Mutations codon 816 in Mast cell disease Seminoma/dysgerminoma AML Sinonasal NK/T cell lymphoma Corless et al, JCO 2004

37 Mutations in PDGFR % of mutations Juxtamembrane (exon 12) Activation loop (exon 18) Associated with epithelioid phenotype Substitution involving codon D842 (exon 18) (62%): resistant to Imatinib Corless et al, JCO 2005 Corless et al, JCO 2004 T R GAC GTC D842V

38 Molecular diagnostics LUMC mutation analysis: direct sequencing of KIT exon 9, 11, 13, 17 PDGFRA exon 12, 18 2 steps: GIST1: KIT exon 9, 11, PDGFRA exon 12, 18 GIST2: KIT exon 13, 17

39 Indication mutation analysis LUMC CD117 negative GIST some will respond to imatinib Primary and secondary resistant GIST Exon 9 mutations: mg Alternatives to imatinib are being developed

40 Response to Imatinib Clinical response to imatinib in ~80% of GIST patients Depends on exonic location of KIT mutation 10-20% of GIST patients are primary resistant Secondary resistance after 2 years (median) KIT exon 11: exon 9: exon 13: exon 17: PDGFRA : no mutations: 84% PR 48% PR 100%PR 50% PR 0-67% PR 0% PR, 35% SD Heinrich et al, JCO 2003

41 Response to Imatinib Event free survival Overall survival Heinrich et al, JCO 2003

42 Molecular classification of GIST Corless et al, JCO 2004

43 Secondary resistance to Imatinib; molecular mechanism Additional point mutations in KIT kinase domains (can vary between metastases in one patient) (48%) In PDGFRA D842V (4%) Amplification of KIT or PDGFRA (8%) Loss of KIT expression; KIT independent mechanism of resistance (8%) Debiec-Rychter et al, Gastroenterology 2005

44 H Exon 11 REV Aanvulling: MD nummer: Percentage tumor: 70 % DNA concentratie tumor: 98.8 ng/ul Er is een Kit mutatie gevonden in exon 11: heterozygote deletie van W557(TGG)- V559(GTT)-> C(TGT) m.b.v. sequentie analyse. Er is geen mutatie gevonden in exon 9 van Kit en ook niet in exon 12 en 18 van PDGFR-alpha. Gezien de secundaire Gleevec resistentie zal nog mutatie analyse worden verricht voor exon 13 en 17 van KIT om te zien of er een tweede mutatie is opgetreden. Hierover volgt nader bericht.

45 Exon 13 H e Aanvulling: MD nummer: , (DNA MD nummer: ). Percentage tumor: 70% DNA concentratie tumor: 98.8 ng/ul Er is een Kit mutatie gevonden in exon 13: V654A (GTG --> GCG), m.b.v. sequentie analyse. Er is geen mutatie gevonden in exon 17 van Kit. Er is derhalve een tweede mutatie gevonden in exon 13; in dit exon, dat codeert voor een van de twee tyrosine kinase domeinen, worden vaak secundaire mutaties beschreven welke kunnen leiden tot gleevec resistentie (Wardelmann et al, Clin Cancer Res :1743-9).

46 Secondary KIT mutations in imatinib resistance Heinrich et al, JCO 2006

47 Secondary resistance to Imatinib Sunitinib (Sutent) (multi-targeted KIT inhibitor) can be effective: Alternatives are being developed (pre-clinical studies): More potent kinase inhibitors (nilotinib) Flavopiridol: KIT transcriptional repression Demetri et al, Lancet 2006 Inhibition of the KIT chaperone HSP90 degradation KIT / PDGFRA Fletcher and Rubin, Curr Opin Gen Develop 2007