TERAPEUTIC CONSEQUENCES FROM MOLECOLAR BIOLOGY FOR GIST PATIENTS AFFECTED BY NEUROFIBROMATOSIS TYPE 1 Mussi C, Schildhaus HU, Gronchi A, Wardelmann E, Hohenberger P CTOS-Seattle, November 2007 supported by Conticanet Mannheim University Hospital, Germany Bonn University Hospital, Germany Istituto Nazionale Tumori, Italy

BACKGROUND Patients affected by Neurofibromatosis type 1 have an increased risk of GIST developing NF-1 associated GISTs seem to be wild type for KIT/PDGFRA mutations This subset of GIST has likely a different oncogenic molecular mechanism Lack of data on imatinib and other tyrosine kinases inhibitors activity in this different setting

QUESTIONS Should these patients enrolled in the ongoing trials of imatinib? Should this decision taken on the basis of the molecular analysis?

PATIENTS 28 PATIENTS OPERATED 13 MALES 15 FEMALES M:F=0,87:1 Median age 57 (range 28-72)

DIAGNOSIS NEUROFIBROMATOSIS TYPE 1 >2 following criteria: > 6 cafe-au-lait macules (>5mm before puberty, >15 mm after) skin-fold freckles (groin, axilla, neck base) > neurofibromas (1 plexiform) skeletal dysplasia (orbital or tibial) Lisch nodules (>2 iris amartomas) optic glioma family history

DIAGNOSIS GIST The diagnosis was confirmed histologically in terms of morphology and immunophenotyping Seven tumors were reclassified as GIST after pathologic review 2 MPNST 1 Schwannoma, 1 Neurofibroma 2 Leiomyosarcoma 1 Leiomyoma All tumors were sympthomatic except one

PRESENTATION PRESENT SERIES SPORADIC GIST Age 57 yrs (28-72) M:F=0,87:1 LOCALIZED 82% METASTATIC 18% RISK STRATIFICATION High 30,5% Intermediate 39% Low/very low 30,5% Age 60 yrs (20-80) M:F=1,3:1 LOCALIZED 50-85% METASTATIC 15-50% RISK STRATIFICATION High 23-35% Intermediate 20% Low/very low 45% 70%

SITE PRESENT SERIES SPORADIC GIST 25% 14% 68% Other: 11% 60% 30% Other: 10% 7%

NUMBERS OF TUMORS PRESENT SERIES SPORADIC GIST MULTIPLE TUMORS 43 %Occasional

IMMUNOCHEMISTRY PRESENT SERIES SPORADIC GIST CD 117 pos100% CD 34 pos 86% S-100 pos 19% Actine pos 24% PDGFRA pos37,5% BCL-2 pos 43% CD 117 pos 95% CD 34 pos 70% S-100 pos10% Actine pos25% PDGFRA pos 70% BCL-2 pos 20-93%

PATHOLOGY PRESENT SERIES SPORADIC GIST Spindle cell75% Epithelioid 15% Mixed 10% Skenoid fiber 33% Associated Cajal cell hyperplasia 21% Spindle cell38-77% Epithelioid 8-38% Mixed 14-23% Skenoid fiber 34%

MOLECULAR ANALYSIS c-KIT exons 9, 11,13,17 PDGFRA exon 12, 14, 18 DNA extracted from paraffin embedded microdissected sections was sequenced for: 25 PTS

MOLECULAR ANALYSIS exon 11 (V560del) exon 9 (A504_Y505) Exon 18 (D842V) PRIMARY MUTATIONS

MOLECULAR ANALYSIS Exon 17 D820Nmutation in metastatic lesions after gleevec therapy SECONDARY MUTATION

MOLECULAR ANALYSIS Series N° of Pts KIT mut PDGFRA mut NF1 mut Kinoshita, None None 2/3 pts Cheng, ex 11 None NS Anderson, None None NS Takazawa, ex 11, 1 ex 13 1 ex 12, 1 ex 18 NS Yantiss, ex 11 None NS Miettinen, None None NS Maertens, polimorphisms 5 silents 3/3 pts Nemoto, None None 1/1 pts Guillaud, None None NS Lee, None None NS Steward, None None 1/2 pts Kang, None None NS Present series 25 1 ex 11, 1 ex 9 1 ex 18 NS ________________________________________________________________ 10 KIT-PDGFRA Mutations / 90 patients analysed = 11, 1%

MOLECULAR ANALYSIS SPORADIC GISTNF-1 ASSOCIATED GIST KIT Mutations 7,8% Exon 11 5,6% Exon 9 1,1% Exon 13 1,1% Exon 17 0% PDGFRA Mutations 3,3% Exon 12 1,1% Exon 14 0% Exon 18 2,2% KIT Mutations 80% Exon 11 67,5% Exon 9 11% Exon 13 0,9% Exon 17 0,5% PDGFRA Mutations 7,5% Exon 12 0,9% Exon 14 0,3% Exon 18 6,3%

CLINICAL OUTCOME Prospective periodical assessment Five patients had other maligniancies (2 gastrointestinal carcinoid; 1 cutaneous basalioma; 1 brain meningioma; 2 uterus carcinoma; 1 adrenal pheocromocytoma; 1 breast cancer) 8 patients develloped local recurrence or metastasis Six patients died of disease

SURVIVAL 5-year EFS 46,9% (median 48 months) 5-year DSS 54,3% (median NR) Post-event median survival 34 months Multiple tumors had a better outcome

IMATINIB THERAPY: resectable GIST Pts Primary SettingTrial ImatinibEFS Status Localized postop.EORTC 400mg/d 11 NED Localized postop.SSGVIII/ 400mg/d 8 NED AIO 3 Syncronous postop. / 400mg/d 22 NED resectable metastasis 4 Multiplepostop. / 400mg/d 45 NED recurrent tumors 5Localized postop. EORTCControl14 NED Arm

IMATINIB THERAPY: advanced GIST Pts Site Risk Metastasis Molecular Resp. Post IM Status (prim.) analysisSurv (EORTC trial) ExGI H liver, WT PD 22 DOD peritoneal 2 Small H liver, WT PD 19 DOD Bowel peritoneal 3 Stomach I liver, EX 18 SD 22 DOD peritoneal 4 Small H peritoneal WT prim; PD 10 DOD Bowel Secondary ex Median survival after imatinib onset 21 months

IMATINIB THERAPY: ex vivo Kit phosporylation is stem cell factor dependent The MAPK pathway is more active then in sporadic GIST JAK-STAT 3 and P13K-AKT are less active then in sporadic GIST The MAPK phosporylation cannot be complete shut down by imatinb and the effect is not dose dependent

IMATINIB THERAPY: neurofibrin deficit is associated with high levels of Kit expression

IMATINIB THERAPY: in vivo

…IN BRIEF The incidence of GIST in NF-1 is unknown, but symptomatic tumors are often high or intermediate risk (70%) Most tumors are wild type for KIT/PDGFRA mutations (89%) The oncogenic mechanism causing the MAPK pathway activation and KIT overexpression is related to the neurofibrin deficit

CONCLUSIONS The molecular analysis is always raccomended to individuate sporadic mutation to clarify the prognostic meaning of mutations in this subset of GIST

CONCLUSIONS Further studies are necessary to clarify the effecacy of IM and other inhibitors of tyrosine kinases in this setting

CONCLUSIONS Localized wild type GIST should not be elegible for adjuvant trials The molecular analysis should be done before the enrollement

CONCLUSIONS Local advanced GIST enrolled in trials of preoperative imatinib should be carefully surveilled

CONCLUSIONS Metastatic GIST could benefit from imatinib treatment Sunitinib could be the first alternative in non responder tumors

CONCLUSIONS The future treatent of this subset of GIST is likely dependent from further investigations of the molecular pathways activated by neurofibrin as new molecular targets

THANKS!