Spindle Cell Tumours of the Gastro-Intestinal Tract Geraint T Williams Pathology Department Wales College of Medicine Cardiff University

GI Spindle Cell Tumours early 1990s Leiomyomas Epithelioid leiomyomas Leiomyosarcomas Neurofibromas Schwannomas Malignant Schwannomas

Immunostaining early 1990s Desmin Smooth muscle actin Smooth muscle myosin S-100 protein Neurofilament PGP9.5

GI Spindle Cell Tumours mid 1990s Many tumours with equivocal immunostaining: GISTs GANTs STUMPs SITSFs OSTs, GaSTs, DuSTs, JeSTs, ISTs, CoSTs, ReSTs, ASTs

Gastrointestinal Stromal Tumours 2000s Immunopositivity for: c-kit (KIT, CD117) CD34 bcl-2 nestin protein kinase C-theta

Leiomyoma Oesophagus, stomach, small bowel Usually <0.5 cm, polypoid and asymptomatic Most related to muscularis mucosae Oesophageal leiomyomas in MEN1 LOH at 11q13 Microleiomyomas at oesophago-gastric junction in 8% population Diffuse oesophageal leiomyomatosis associated with Alports-type nephropathy Peritoneal leiomyomatosis

Leiomyosarcoma Very rare Oesophagus, stomach, small bowel Expresses desmin and/or smooth muscle actin Usually high grade (>10 mitoses/10 HPFs)

Ganglioneuromatosis Diffuse submucosal and myenteric: May cause motility disorders/megacolon NF-1 Multiple endocrine neoplasia IIb Shekitka et al 1994 Am J Surg Pathol 18: 250-7 Smith et al 1999; Gut 45: 143-6

Ganglioneuromatosis Diffuse submucosal and myenteric: May cause motility disorders/megacolon NF-1 Multiple endocrine neoplasia IIb Polypoid mucosal: Juvenile polyposis Cowden’s syndrome

Gastrointestinal “Schwannoma” Benign Not associated with NF-1 No NF2 lesions Majority gastric, occasionally oesophageal or colonic, virtually never in small bowel Intramural or polypoid No necrosis, haemorrhage or cystic change Sarlomo-Rikala & Miettinen 1995 Histopathology 27: 355–60 Hou et al 2005 Histopathology in press

Gastrointestinal “Schwannoma” Typically brisk lymphoid reaction, usually as a peritumoural lymphoid cuff, often with germinal centres Mainly spindle, rarely epithelioid Verocay bodies unusual Significant nuclear atypia but mitoses very sparse

Gastrointestinal “Schwannoma” S-100 positive GFAP and nestin usually positive Occasional CD34 positive cell KIT, SMA, CK, NF, desmin negative

Mucosal epithelioid nerve sheath tumours Small polyps Epithelioid cells in nests and whorls Intranuclear pseudoinclusions S-100 positive KIT negative Lewin et al 2005 Am J Surg Pathol 29: 1310

Benign fibroblastic polyps of the colon Solitary Bland monotonous mucosal spindle cell proliferation Vimentin positive only Eslami-Varzanehet al 2004 Am J Surg Pathol 28: 374

Gastrointestinal Stromal Tumours Immunopositivity for: vimentin 95-100% nestin 90-100% CD34 70-85% (low in SI) smooth muscle actin 20-40% (high in SI) heavy caldesmon 60-80% connexin 43 most SI, rare in stomach desmin 5-20% S-100 0-15% (mainly SI) cytokeratin rare

KIT (CD117) Receptor for Stem cell factor Trans-membrane tyrosine kinase growth factor receptor Expressed on Haemopoietic stem cells Mast cells Melanocytes Breast epithelium Interstitial cells of Cajal (pacemaker cells)

Interstitial cells of Cajal Gut pacemaker cells Form intramural network Develop from intrinsic gut mesenchyme Common precursor with smooth muscle cells Express KIT and nestin

Interstitial cells of Cajal Similar cells now described in Pancreas Portal vein Fallopian tube Myometrium Breast

KIT Mutations in GISTs Activating mutations exon 11, occasionally in exon 9 ~ 85% of GISTs More frequent in malignant GISTs Different mutations (exon 17) in mast cell tumours Hirota et al 1998 Science 279:577

KIT mutations in GISTs “Early” event in tumorigenesis Transfection into cell lines leads to transformation by autophosphorylation of KIT ligand-independent tyrosine kinase activity cell proliferation Hirota et al 1998 Science 279:577 Rubin et al 2001 Cancer Res 61:8118

KIT Mutations in GISTs Present in 72% 80% exon 11 17% exon 9 Exon 9 more frequent in aggressive small bowel GISTs Exon 11 nearly all spindle cell Penzel et al 2005 J Clin Pathol 58:634

Familial GISTs Germline mutations of KIT Multiple tumours Cutaneous hyperpigmentation Hyperplasia of Cajal cells GI Motility disorders Some overlap with NF-1 Hirota et al 1998 Nat Genet 19: 323 Chompret et al 2004 Gastroenterology 126: 318

Chromosomal abnormalities in GISTs Most tumours: 14q, 22q deletion Malignant tumours: 1p, 9p deletion 8q and 17q amplification

Gastrointestinal Stromal Tumours (GISTs) Incidence 14.5/million/year (prevalence 129/million) 5th-7th decade Decreasing frequency down GI tract Pedunculated, dumb-bell or ulcerated May arise in mesentery, omentum, retroperitoneum Prediction of behaviour unreliable Predisposition: Familial Neurofibromatosis Carney’s triad Nilsson et al 2005 Cancer 103:821

GISTs in NF-1 Multiple tumours Mainly in small intestine Spindle cell, low grade Often skeinoid fibres Often S-100 positive KIT and PDGFRA mutations uncommon Background Cajal cell hyperplasia Andersson J et al 2005 Am J Surg Pathol 29: 1170-6 Takazawa et al 2005 Am J Surg Pathol 29: 755-63

Carney Syndromes Carney J A The triad of gastric epithelioid leiomyosarcoma, pulmonary chondroma and functioning extra-adrenal paraganglioma: a 5-year review. Medicine 1983; 62: 159–169 Carney J A, Stratakis C A Familial paraganglioma and gastric stromal sarcoma: a new syndrome distinct from the Carney triad. Am J Med Genet 2002; 108: 132-139

Gastrointestinal Stromal Tumours (GISTs) Small tumours found incidentally Symptomatic Obstruction Bleeding

Malignant GISTs Approximately 30-45% Notoriously difficult to predict Intra-abdominal spread, especially multinodular peritoneal seeding Distant metastases: Liver Lung Bone

GISTs - Outcome Resectable Unresectable, metastatic 10 year survival 30-50% Unresectable, metastatic median survival 12 months no response to conventional chemotherapy

Predictors of Malignant Behaviour in GISTs Uncertain malignant potential Low malignant potential High malignant potential

Major Predictors of Malignant Behaviour in GISTs Size (>5 cm) Mitotic counts (>5/50 HPF)

Risk of Aggressive Behaviour (NIH) Fletcher et al 2002 Hum Pathol 33: 459-465

Behaviour of GISTs Low malignant potential 56% 1% Frequency Tumour- Median related deaths survival (months) Low malignant potential 56% 1% High malignant potential 29% 63% 40 Overtly malignant 15% 83% 16 Nilsson et al 2005 Cancer 103:821

Other Predictors of Malignant Behaviour in GISTs Site (stomach vs intestine)

Gastric GISTs Miettinen et al 2005 Am J Surg Pathol 29: 52-68

Other Predictors of Malignant Behaviour in GISTs Site (stomach vs intestine) Histological type (epithelioid>spindle) Cellularity and pleomorphism Invasive pattern

Other Predictors of Malignant Behaviour in GISTs Chromosome 9q deletion 1530ins6 mutation of KIT (intestine) P16 loss P53 positivity (gastric GISTs) Lasota et al 2003 Hum Pathol 34: 1306 Gunawan et al 2004 J Pathol 202:421 Feakins 2005 Histopathology 46: 270 Schneider-Stock et al 2005 Clin Cancer Res 11: 638

Gastrointestinal Stromal Tumours (GISTs) Spindle cell Epithelioid Round cell Clear cell Plasmacytoid Pleomorphic

Differential Diagnosis Inflammatory fibroid polyp Inflammatory myofibroblastic tumour Solitary fibrous tumour Mesenteric fibromatosis Dedifferentiated liposarcoma

Inflammatory Fibroid Polyp Stomach, ileum, proximal colon Usually presents with intussusception Centred on submucosa Distinctive histology: Thin-walled blood vessels Onion-skin arrangement of palisaded spindle cells around larger blood vessels Oedema Eosinophils

Inflammatory Fibroid Polyp CD34 positive fascin positive bcl-2 negative KIT negative CD99 negative Pantanowitz et al 2004 Am J Surg Pathol 28: 107

Inflammatory Myofibroblastic Tumour Loose mixture of fibroblasts, myofibroblasts and inflammatory cells May be ganglion-like cells CD34 negative ALK-1 positive May be KIT positive ? tumour of fibroblastic reticulum cells Nonaka et al 2005 Histopathology 46: 604

Solitary Fibrous Tumour Wide spectrum histologically Fascicular or storiform pattern Ectatic vessels CD34 positive Bcl-2 positive KIT negative CD99 positive

Mesenteric Fibromatosis May be KIT positive (depending on antibody) beta-catenin positive (nuclear) CD34 negative Montgomery al 2002 Am J Surg Pathol 26: 1296

Dedifferentiated Liposarcoma mdm2 positive cdk4 positive S-100 positive may be KIT positive

Differential Diagnosis Kaposi’s sarcoma KIT negative Angiosarcoma CD31 positive Occasionally KIT positive Mesothelioma Rare weak positivity for KIT

Differential Diagnosis Sarcomatoid carcinoma / carcinosarcoma Rarely KIT positive (GISTs may show perinuclear dot staining for CAM5.2) Small cell carcinoma Metastases: melanoma seminoma myeloproliferative lesions, mast cell tumours breast, ovarian, nasopharyngeal, colorectal carcinoma

Problems with KIT immunostaining Different antibodies with different sensitivity and specificity (e.g. mesenteric fibromatosis) Pre-treatment affects staining Expensive Level of expression variable Granular cytoplasmic staining alone unreliable Membranous and/or paranuclear dot reliable Use normal stomach as control watch for aberrant expression in smooth muscle

KIT-negative GISTs Epithelioid morphology commoner Usually CD34 and Protein kinase C-theta positive Usually have 14q and 22q deletions (and 1p deletions in malignant tumours) Debiec-Rychter et al 2004 J Pathol 202:430

KIT-negative GISTs ~30% have activating mutations of Platelet derived growth factor receptor-alpha (PDGFRA, tyrosine kinase) Exon 18, occasionally in exon 12 KIT and PDGFRA mutations mutually exclusive Heinrich et al 2003 Science 299:708 Debiec-Rychter et al 2004 J Pathol 202:430 Medeiros et al 2004 Am J Surg Pathol 28: 889

PDGFRA mutation in GISTs Autophosphorylation and ligand-independent activation of tyrosine kinase receptor Heinrich et al 2003 Science 299:708

PDGFRA mutant, KIT-negative GISTs Mainly gastric Mainly epithelioid Tumour giant cells Usually low mitotic rate 83% benign behaving Lasota et al 2004 Lab Invest 84:874 Pauls et al 2005 Histopathology 46:166 Penzel et al 2005 J Clin Pathol 58:634

PDGFRA immunostaining 8/125 GISTs (all 8 KIT-negative) 4/15 intra-abdominal desmoids 0/12 leiomyomas 0/8 leiomyosarcomas 0/3 schwannomas 0/2 solitary fibrous tumours 0/1 inflammatory fibroid polyp 0/1 inflammatory myofibroblastic tumour Rossi et al 2005 Histopathology 46:522

DOG-1 “Discovered on GIST-1” Identified through gene expression profiling Expressed in 136/139 GISTs irrespective of KIT or PDGFRA mutation status 0/17 fibromatosis 0/3 schwannomas 4/438 non-GISTs synovial sarcoma, leiomyosaroma, fibrosarcoma, Ewings sarcoma/PNET West et al 2004 Am J Pathol 165:107

Imatinib (ST1571; Glivec) Tyrosine kinase inhibitor developed as inhibitor of PDGF receptor Powerful inhibitor of ABL tyrosine kinases Effective treatment for chronic myeloid leukaemia (ABL and BCR-ABL) Dramatic response of malignant GIST with relatively mild toxicity Joenssu et al 2001 NEJM 344:1052

Imatinib (ST1571; Glivec) EORTC Phase I study in advanced cases showed inhibition of tumour growth in 32/36 cases >50% volume reduction in 19 patients side effects (nausea, vomiting, oedema, rash) limited treatment in 5 patients response seen within 2 months Similar findings in US Study of 36 patients van Oosterom et al 2001 Lancet 358:1421

Imatinib (ST1571; Glivec) Phase III study: 946 patients 5% complete response 47% partial response 32% stable median time to best response 107 days 73% free from progression at 12 months serious side effects 37% Verweij J et al 2004 Lancet 364: 1127

Imatinib (ST1571; Glivec) Complete response very unusual Partial response rates 85 Exon 11 KIT mutations 83.5% 23 Exon 9 KIT mutations 47.8% “Escape” with time in some cases associated with novel KIT mutations (exon 17) Heinrich et al 2003 J Clin Oncol 21: 4342 Chen et al 2004 Cancer Res 64: 5913 Antonescu et al 2005 Clin Cancer Res 11: 4182

NICE proposals - Imatinib 400mg/day for KIT-positive unresectable or metastatic disease Continue only if response is achieved within 12 weeks

Response to Imatinib South West Oncology Group Assessed by CT, MRI or PET Complete response Partial response Stable disease Progressive disease Unknown

NICE proposals - Imatinib 400mg/day for KIT-positive unresectable of metastatic disease Continue only if response is achieved within 12 weeks For responders continue until development of progressive disease Increased dose not recommended in non-responders £19,000 per year

CSTI571-B2222 Phase II, 147 patients, 91% KIT +ve Unresectable or metastatic GIST 400mg or 600mg/day for median 21 months Survival 88% at 1 year 78% at 2 years No CR, 66% PR, 17% stable, 12% progressive Improved performance status 15% major adverse events, 10% withdrew Resistance in 16 pts, 3 primary, 13 secondary

(SU11248) Multi-targeted tyrosine kinase inhibitor Objective response or stable disease in 26/48 progressing tumours on imatinib Particular benefit in exon 9 mutants Demetri et al 2004 ASCO Abstract 3001

Imatinib-treated GISTs Myxoid, gelatinous consistency; cystic change Decreased cellularity Stromal hyalinisation More epithelioid Loss of KIT and/or CD34 immunoreactivity Acquired desmin immunoreactivity Novel KIT mutations especially exon 17 Antonescu et al 2005 Clin Cancer Res 11: 4182 Loughrey et al 2005 J Clin Pathol 58: 779 Pauwels et al 2005 Histopathology 47: 41

Conclusions Diagnosis of GI spindle cell tumours, and GIST in particular, is important Should be made by experienced pathologists with access to quality-assured KIT immunostaining Molecular diagnosis likely to become important Managed by MDT Surgery is first line therapy Imatinib should be considered for patients with advanced or unresectable tumours Participate in trials