Gastrointestinal Stromal Tumors. Review on Morphology, Molecular Pathology, Prognosis, and Differential Diagnosis

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1 Gastrointestinal Stromal Tumors Review on Morphology, Molecular Pathology, Prognosis, and Differential Diagnosis Markku Miettinen, MD; Jerzy Lasota, MD This review summarizes the definition, histogenesis, molecular pathogenesis, clinical background, histopathologic spectrum, assessment of biologic potential, and differential diagnosis of gastrointestinal stromal tumors (GISTs), mesenchymal tumors specific to the gastrointestinal (GI) tract. Proper identification of GIST has become very important since the availability of specific, pathogenesis-targeted treatment with the Kit/platelet-derived growth factor receptor alpha (PDGFRA) tyrosine kinase inhibitor imatinib mesylate (Gleevec, Novartis Pharma, New Hanover, NJ [in later text imatinib]) and newer agents as a targeted oncologic therapy now widely used for metastatic and unresectable GISTs. 1 Accepted for publication March 10, From the Department of Soft Tissue Pathology, Armed Forces Institute of Pathology, Washington, DC. The authors have no relevant financial interest in the products or companies described in this article. Reprints: Markku Miettinen, MD, Armed Forces Institute of Pathology, Department of Soft Tissue Pathology, th St, NW, Bldg 54, Room G090, Washington, DC ( miettinen@afip.osd.mil). Context. Gastrointestinal stromal tumors (GISTs) are specific, generally Kit (CD117)-positive, mesenchymal tumors of the gastrointestinal tract encompassing a majority of tumors previously considered gastrointestinal smooth muscle tumors. They are believed to originate from interstitial cells of Cajal or related stem cells. Objective. To review current clinicopathologically relevant information on GIST. Data Sources. Literature in Medline and authors own experience. Conclusions. GISTs usually occur in older adults (median age years) and rarely in children in the second decade ( 1%) throughout the gastrointestinal tract: 60% in stomach, 35% in small intestine, and less than 5% in rectum, esophagus, omentum, and mesentery; most GISTs in the latter 2 sites are metastatic. Five percent of GISTs occur in patients with neurofibromatosis type 1 syndrome (multiple small intestinal tumors) and in Carney triad (gastric epithelioid GISTs in young females). Familial GISTs occur in patients with inheritable germline Kit or plateletderived growth factor receptor alpha (PDGFRA) mutations. Histologically GISTs vary from spindle cell tumors to epithelioid and pleomorphic tumors. Most GISTs (95%) express Kit (CD117), CD34 (70%), and heavy caldesmon (80%), whereas 25% are positive for smooth muscle actin and less than 5% for desmin. Tumor size and mitotic activity are best predictive prognostic features; small intestinal tumors behave more aggressively than gastric tumors with similar parameters. Mutually exclusive gain-of-function Kit or PDGFRA mutations occur in a majority of GISTs representing in-frame deletions, point mutations, duplications and insertions. Mutations in Kit juxtamembrane domain (exon 11) are the most common in GISTs of all sites, whereas rare Kit extracellular domain (exon 9) Ala 502 -Tyr 503 duplication is specific for intestinal GISTs. Mutations in PDGFRA have been identified in juxtamembrane (exon 12) and tyrosine kinase domains (exons 14 and 18), nearly exclusively in gastric GISTs, mostly in epithelioid variants. Some Kit and PDGFRA mutations have a prognostic value. Kit/PDGFRA tyrosine kinase inhibitor imatinib has been successfully used in the treatment of metastatic GISTs for more than 5 years. However, primary and acquired secondary resistance linked to certain types of Kit and PDGFRA mutations is limiting long-term success necessitating the use of alternative treatments. (Arch Pathol Lab Med. 2006;130: ) DEFINITION AND TERMINOLOGY Gastrointestinal stromal tumors are here defined as specific, generally Kit (CD117)-positive and Kit or PDGFRA mutation-driven mesenchymal tumors of the GI tract with a set of characteristic histologic features including spindle cell, epithelioid, and rarely pleomorphic morphology. GISTs include tumors with a wide spectrum of biologic potential at all sites of their occurrence. 2 Gastrointestinal stromal tumors comprise a majority of tumors previously diagnosed as GI leiomyomas, leiomyoblastomas, and leiomyosarcomas and include many tumors previously considered neurofibromas or schwannomas. Esophageal intramural leiomyomas and colorectal muscularis mucosae leiomyomas are the 2 most important examples of true smooth muscle tumors. Intramural smooth muscle and nerve sheath tumors occur sporadically throughout the GI tract. GI autonomic nerve tumor is now understood as a variant of GIST, based on its similarity with GIST by histology, Kit positivity, and Kit mutations. 3 CLINICAL ASPECTS The most common presentation of GIST is GI bleeding. This may be acute (melena or hematemesis) or chronic 1466 Arch Pathol Lab Med Vol 130, October 2006 Gastrointestinal Stromal Tumors Miettinen & Lasota

2 insidious bleeding leading to anemia. In older times, the tumor diagnosis was often delayed for years after the first bleeding episode. Tumor rupture, GI obstruction, or appendicitis-like pain can cause an acute abdomen. Smaller GISTs are often incidental findings during surgery, radiologic studies, or endoscopy; the latter applies especially to environments where mass screening for gastric carcinoma is being practiced. Approximately 20% to 25% of gastric and 40% to 50% of small intestinal GISTs are clinically malignant. Metastases commonly develop in the abdominal cavity and liver; rarely in bones, soft tissues, and skin; and extremely rarely in lymph nodes and lungs. Metastases can develop 10 to 15 or more years after primary surgery necessitating long-term clinical follow-up. In general, complete excision is the main treatment. Patients whose tumors are unresectable or who have metastatic disease are treated with Kit/PDGFRA tyrosine kinase inhibitors, such as imatinib, also used in chronic myelogenous leukemia by its ability to inhibit Bcr-Abl tyrosine kinase. This oral treatment ( mg/day) is generally well-tolerated, and the majority of patients achieve complete or partial remission. Long-term success is limited by development of imatinib resistance via secondary mutations or clonal selection. In such cases, other inhibitors of Kit/PDGFRA receptors or downstream targets, such as protein kinase theta, have been applied. 1,4 HISTOGENESIS Gastrointestinal stromal tumors are believed to originate from interstitial cells of Cajal or their stem cell-like precursors. 5,6 Cajal cells are intermediates between the GI autonomic nervous system and smooth muscle cells regulating GI motility and autonomic nerve function. 7,8 They are Kit and Kit-ligand (stem cell factor) positive and dependent cells, located around the myenteric plexus and in the muscularis propria throughout the GI tract. Cajal cells are or include a subset of multipotential stem cell-like cells that can differentiate into smooth muscle cells if Kit signaling is disrupted. 9 Kit or stem cell factor deficient mice lack Cajal cells and have intestinal dysmotility. 10 Constitutional activation of KIT by mutations causes Cajal cell proliferation and ultimately GIST, as illustrated by transgenic mouse models with introduced constitutional KIT-activating mutations. 11,12 KIT mutations in GIST were originally reported by Kitamura and Hirota 13 in 1998 based on analogy with the mast cell system, in which KIT deficiency caused lack of mast cells and activating mutations caused mast cell neoplasia. 13 GIST PATHOGENESIS: KIT AND PDGFRA MUTATIONS Mutually exclusive mutations in Kit or PDGFRA receptor tyrosine kinase proteins observed in more than 80% of GISTs are central in sporadic GIST pathogenesis. These mutations are somatic and present only in tumor tissue, whereas similar, constitutional mutations in familial GISTs are present in all cells of the body and are inheritable. Mutations cause functional changes in Kit and PDGFRA proteins, usually leading to ligand-independent dimerization and constitutional activation. 13,14 KIT and PDGFRA genes encode for similarly named highly homologous receptor tyrosine kinase proteins. Both genes are located pericentromerically at 4q12 having probably evolved as a duplication of an ancestral gene. 15 The corresponding proteins have structural characteristics of type III receptor tyrosine kinase family (Figure 1). 16 Activating mutations of KIT and PDGFRA permit the phosphorylation of the receptor tyrosine kinases perpetuating the receptor-initiated signal and causing activation of the downstream effectors. The end result of such activation is increase in cellular proliferation and decrease in apoptosis, ultimately leading into neoplasia, probably following other, currently unknown genetic events. 5,13,14 In addition to receptor tyrosine kinase mutations, other genetic changes have been detected in GISTs. Among these, losses of potential tumor suppressor genes in chromosome arms 14q and 22q are often early, possibly pathogenetically important, changes. 17,18 However, no specific genes have been identified so far. Although 1 copy of the NF2 gene in 22q is often lost, the NF2 gene does not seem to be involved in a biallelic manner in GIST pathogenesis, 19, 20 as is observed in schwannian nerve sheath tumors. KIT Mutations Four different regions of KIT have been found to be mutated in sporadic GISTs. They are, in decreasing order of frequency, exon 11, exon 9, exon 13, and exon 17. Most Kit-mutant proteins are sensitive to imatinib. However, exon 17 Kit-mutants in GIST are primarily resistant, and exon 9 Kit-mutants are less sensitive than exon 11 mutants. 21 Schematic distribution and types of KIT mutations in GISTs are shown in Figure 1. Most KIT mutations in GIST involve exon 11, the juxtamembrane domain. This region, just inside the cell membrane, is a helical domain of KIT apparently representing an inhibitory region regulating the KIT activation. 22 Approximately 60% to 70% of KIT exon 11 mutations are in-frame deletions of 1 to several codons. A majority of these mutations involve the proximal part of KIT exon 11 and cluster between codons Gln 550 and Glu ,14,23,24 It has been suggested that loss of Trp 557 and Lys 558, the most common simple deletion in GISTs, might be associated with poor clinical outcome Missense point mutations in KIT exon 11 occur in 20% to 30% of GISTs. They involve almost exclusively 3 codons, Trp 557,Val 559,andVal 560 in the proximal part and codon Leu 576 in the distal part of exon 11. Gastric GISTs with exon 11 missense mutations seem to have a better prognosis than tumors with exon 11 deletions, but no similar difference has been found for small intestinal GISTs. 28,29 Internal tandem duplications of 1 up to more than 20 codons occur in the distal part of KIT exon 11. These relatively rare mutations often occur in gastric tumors and are associated with favorable outcome. 30,31 Exon 9 encoding the end of the extracellular domain is the second most often involved region of KIT. Nearly all mutations have been identical 6-nucleotide duplications encoding Ala 502 -Tyr 503 originally reported by Lux et al. 32 This mutation was reported with a frequency of 5% to 13%. However, its apparent frequency depends on the ratio of intestinal to gastric tumors studied, because this mutation is nearly specific to intestinal GISTs. 14,31,33,34 Although most small intestinal tumors with such mutation have been malignant, no significant difference was found between the behavior of GISTs with exon 9 and exon 11 mutations, indicating that the apparent association with malignant behavior is related to the worse prognosis of small intestinal GISTs in general. 29 Recently, isolated cases Arch Pathol Lab Med Vol 130, October 2006 Gastrointestinal Stromal Tumors Miettinen & Lasota 1467

3 Figure 1. Schematic representation of KIT and platelet-derived growth factor receptor alpha (PDGFRA) molecules and KIT and PDGFRA mutations in gastrointestinal stromal tumor (GIST). EC indicates extracellular domain; TM, transmembrane domain; JM, juxtamembrane domain; TK1 and TK2, tyrosine kinase domains; KI, kinase insert; pm, point mutation; ins, insertion; itd, internal tandem duplication; and del, deletion. with duplication of 9 nucleotides encoding Phe 506 -Ala Phe 508 have been reported. 21 Missense mutations resulting in substitution of Glu for Lys 642 in exon 13 encoding the tyrosine kinase 1 domain have been reported with a low frequency ( 1% 2%) based on 2 large series. 32,33 This mutation seems to be associated with malignant tumor behavior. It is sensitive to imatinib, which abolished the phosphorylated status of KIT in a cell line. 35 Only a few GISTs with mutations involving exon 17 encoding the catalytic tyrosine kinase 2 (phosphotransferase) domain of KIT have been reported. 36 Seminoma and mastocytoma more commonly have KIT exon 17 mutations. 14 PDGFRA Mutations Three different regions of PDGFRA have been found to be mutated in GISTs. They are, in decreasing order of frequency, exon 18, exon 12, and exon These regions correspond to KIT mutational hotspots exon 17, 11, and 13. Schematic distribution and types of PDGFRA mutations are shown in Figure 1. PDGFRA mutations show a strong predilection to gastric GISTs with epithelioid morphology. However, a few nongastric PDGFRA-mutant GISTs have also been reported, especially in the duodenum. 39,41 Initially, PDGFRA mutations were reported in 35% of KIT mutation-negative GISTs. 37 However, the apparent frequency of these mutations depends on the ratio of gastric to intestinal and spindle to epithelioid GISTs studied. A majority ( 80%) of PDGFRA mutations occur in exon 18 and are missense mutations leading to Val for Asp 842 substitution. This mutation is resistant to imatinib. 41 More recently, missense mutations affecting exon 14 have been reported. 42,43 These mutations lead to Lys or Tyr for Asn 659 substitution and seem to be associated with low Kit expression and unexpectedly favorable prognosis. 43 Mutations in PDGFRA exon 12 are rare. The majority of them represent missense mutations leading to Asp for Val 561 substitution, but inframe deletions and insertions have also been reported around codon Val 561. Secondary Mutations Acquired During Imatinib Treatment Many patients enjoying imatinib-induced remission ultimately develop metastases with acquired drug resistance, usually based on secondary, imatinib-resistant mutations in Kit or PDGFRA tyrosine kinase (TK1 and TK2) domains Several distinctive secondary point mutations have been recently identified and shown to affect the same gene allele as the primary mutations OCCURRENCE OF GIST The annual incidence of GIST has been estimated in population-based studies as 14.5 per million in Sweden 48 and as 11 per million in Iceland. 49 Assuming the same incidence, 3300 to 4350 new GISTs would be expected annually in the United States. The prevalence of GIST is higher because many patients live years with the disease. Gastrointestinal stromal tumor has a predilection for adults older than 50 years, with the median ages in the largest series varying slightly around 60 years. There is no clear sex predilection, but malignant GISTs may be slightly more common in men. The proportion of patients 1468 Arch Pathol Lab Med Vol 130, October 2006 Gastrointestinal Stromal Tumors Miettinen & Lasota

4 younger than 40 years varies between 5% and 20% in different sites. Gastrointestinal stromal tumors are very rare in children ( 1%). In this age group they usually occur in the second decade with a marked female predominance, are located in the stomach, and often have epithelioid morphology. These tumors may have a different pathogenesis than adult GISTs, because neither KIT nor PDGFRA mutations seem to be present. 50,51 Gastrointestinal stromal tumors occur throughout the GI tract from the lower esophagus to the anus. The most common sites are stomach (60%), jejunum and ileum (30%), duodenum (5%), and colorectum ( 5%). Only small numbers of cases ( 1%) have been reported in the esophagus and appendix. 2 Gastrointestinal stromal tumors outside the GI tract in the abdomen, especially in the omentum, mesenteries, and retroperitoneum, are usually metastatic or possibly detached from their GI tract origin. However, a small number of apparent primary tumors have been reported in these sites Some GISTs are diagnosed as disseminated abdominal tumors. GIST Syndromes Less than 5% of GISTs are associated with 1 of the 3 tumor syndromes: neurofibromatosis type 1 (NF1), Carney triad, and familial GIST syndrome, in order of decreasing frequency. GISTs occur in connection with NF1 syndrome by a nonrandom association In the Armed Forces Institute of Pathology series, 6% of duodenal GISTs occurred in NF1 patients. This means 180-fold enrichment of NF1 patients among duodenal GIST patients, compared with the occurrence in the general population. 55 In NF1 patients, GIST has a high predilection to small intestine, tumors are often multiple, and the majority are small, mitotically inactive, and clinically indolent. Diffuse Cajal cell hyperplasia is often present. Some NF1 patients, however, develop a malignant GIST; formerly these tumors were often believed to be malignant schwannomas. Different from other GISTs is the apparent lack of KIT and PDGFRA mutations in the largest studies, although some authors have found KIT mutations in occasional NF1-associated GISTs. 56,60 The Carney triad, described by Carney, includes gastric GIST together with paraganglioma, pulmonary chondroma, or both; it has been suggested that GIST with paraganglioma may constitute a separate tumor syndrome. 61,62 Gastrointestinal stromal tumors in the triad (and paraganglioma syndrome) seem to occur exclusively in the stomach. They usually have epithelioid morphology and often present in young age, including children, with a strong female predominance (85%). A majority of tumors have an indolent behavior, but even patients who develop liver metastases can survive for years with stable disease. In the largest published series, there was only 13% of tumor-related mortality. 61 These studies were conducted prior to the availability of imatinib. Twelve families with familial GIST have been reported worldwide Typical of all of them is autosomal dominant transmission of constitutional, heterozygous, activating KIT (11 families) or PDGFRA (1 family) mutation. Most common is missense KIT mutation substituting Val for Ala 559. These patients typically develop multiple, sometimes diffuse GISTs, usually not until middle age. Tumor behavior varies from indolent to eventually sarcomatous, and additional manifestations of KIT activation, such as cutaneous hyperpigmentation, mastocytosis, and dysphagia, may be present. The pathogenetic role of KIT mutation is supported by 2 transgenic animal models, in which introduction of KIT mutations ( knock in mutants ) similar to those in human GISTs into germline gives an inheritable GIST syndrome similar to human familial GIST. In 1 of these models, an exon 11 KIT mutation (KIT Val 558 ) was introduced, 11 and in another model, the syndrome resulted from introduction of a KIT exon 13 point mutation substituting Glu for Lys In familial GIST, polyclonal Cajal cell proliferations have been detected coexisting with monoclonal GISTs, 75 suggesting that additional genetic changes are necessary for clonal expansion and malignant transformation. PATHOLOGIC SPECTRUM OF GISTS Gastrointestinal stromal tumors have a wide clinicopathologic spectrum in most sites ranging from minute incidental nodules to large tumors. The gross appearances are quite variable. Microscopic features are site dependent, but most GISTs are spindle cell tumors, and a minority has epithelioid or mixed spindle, epithelioid, or rarely pleomorphic histology. Tumor size and mitotic activity are key parameters in assessing the biologic potential and are discussed in detail later. Gross Patterns of GIST Small GISTs often form solid subserosal, intramural, or less commonly polypoid intraluminal masses. A majority of larger GISTs form external, sometimes pedunculated masses attached to outer aspect of gut involving the muscular layers. Many larger tumors are centrally cystic, and some develop a diverticulum-like appearance with the external tumor communicating with the lumen by a fistula tract. Some GISTs have an asymmetric hourglasslike pattern with a smaller internal and a larger external component. Histologic Patterns of Gastric GISTs Up to 70% of gastric GISTs can be histologically subclassified into 8 subtypes, 4 of them describing spindle cell and 4 of them describing epithelioid tumors. The biologic potential of these subtypes form a continuum; their specific recognition helps to identify the morphologic spectrum GISTs. 28 Sclerosing spindle cell GISTs are usually small, mitotically inactive, relatively paucicellular tumors, in which the cells are dispersed in a prominently collagenous, sometimes calcified matrix (Figure 2, A). Palisading-vacuolated subtype shows nuclear palisading resembling that of peripheral schwannomas, and prominent perinuclear vacuolization is a typical feature. These tumors usually have low mitotic counts but can reach a size of more than 10 cm. This subtype is the most common among gastric GISTs (Figure 2, B). Hypercellular subtype contains densely packed, uniform spindle cells lacking significant atypia and mitotic activity (Figure 2, C). Sarcomatous spindle cell tumors have marked mitotic activity (generally 20 per highpower field [HPF]; 4 per 10 HPFs) and diffuse atypia manifested by nuclear enlargement and hyperchromasia without much pleomorphism (Figure 2, D). Sclerosing epithelioid variant contains polygonal tumors cells set in a syncytial pattern in a variably sclerosing Arch Pathol Lab Med Vol 130, October 2006 Gastrointestinal Stromal Tumors Miettinen & Lasota 1469

5 Figure 2. Histologic spectrum of gastric gastrointestinal stromal tumor (GIST). A, Sclerosing spindle cell GIST. B, Palisaded-vacuolated spindle cell GIST. C, Hypercellular spindle cell GIST. D, Sarcomatous spindle cell GIST. E, Sclerosing epithelioid GIST with a syncytial pattern. F, Epithelioid GIST with a dyscohesive pattern. G, Hypercellular epithelioid GIST. H, Sarcomatous epithelioid GIST (A through H, hematoxylin-eosin, original magnifications 120) Arch Pathol Lab Med Vol 130, October 2006 Gastrointestinal Stromal Tumors Miettinen & Lasota

6 Figure 3. Histologic spectrum of small intestinal gastrointestinal stromal tumor (GIST). A, Example rich in skeinoid fibers. B, Tumor with abundant pools of cell processes. C, Sarcomatous spindle cell GIST. D, An unusual pleomorphic sarcomatous GIST (A through D, hematoxylin-eosin, original magnifications 120). stroma without visible cell borders (Figure 2, E). These tumors have low mitotic rate, but focal atypia and multinucleation is common. Dyscohesive epithelioid subtype features epithelioid cells surrounded by a lacunar space and sharp cell borders (Figure 2, F). Focal nuclear pleomorphism is common in these tumors and is not worrisome unless accompanied by an elevated mitotic rate. Hypercellular (Figure 2, G) and sarcomatous (Figure 2, H) subtypes represent highly cellular tumors with closely apposed cells, typically with well-defined cell borders; the former is characterized by low mitotic activity, whereas the latter shows marked mitotic activity, often more than 20 per 50 HPFs. Histology of Small Intestinal GISTs These tumors do not form distinctive histologic subtypes as gastric GISTs do. Most small intestinal GISTs are composed of spindle cells, and nearly one half (40% 50%) of them contain microscopically distinctive, round, oval, or elongated eosinophilic and periodic acid-schiff positive aggregates of extracellular collagen fibers (Figure 3, A). These have been named as skeinoid fibers by their lamellar, concentric, ultrastructural appearance. 76 Such structures are especially seen in the nonmalignant examples and have been found as a statistically favorable prognostic feature. 29,77 Other patterns of small intestinal GIST include tumors with anuclear areas resembling neuropil material (Figure 3, B). These areas represent collections of entangled cell processes that are typically more prominent in small intestinal than in gastric GISTs, based on ultrastructural studies. 78 Despite the commonly malignant course, only a minority of small intestinal GISTs have histologically sarcomatous features with high mitotic activity (Figure 3, C) and pleomorphic forms are rare (Figure 3, D). The epithelioid pattern in small intestinal GISTs is significantly linked with malignant tumors. It differs both morphologically and clinically from the gastric epithelioid GISTs and probably represents a morphologic manifestation of tumor progression rather than a distinct histologic subtype. Histologic Features of GISTs of Other Sites Most GISTs of sites other than stomach and small intestine are spindle cell tumors. The rarely reported appendiceal GISTs resemble the small intestinal tumors by Arch Pathol Lab Med Vol 130, October 2006 Gastrointestinal Stromal Tumors Miettinen & Lasota 1471

7 Figure 4. Examples of Kit positivity in gastrointestinal stromal tumor (GIST). A, Relatively weak and patchy positivity in gastric epithelioid GIST. B, Tumor with Kit positive and negative areas. C, Strongly positive tumor with cytoplasmic and perinuclear staining. D, Spindle cell GIST with pronounced positivity in areas with cell processes (A through D, immunostains counterstained with hematoxylin-eosin, original magnifications 240). the common content of skeinoid fibers, which are also seen in some colonic but not in rectal GISTs. Rectal spindle cell GISTs can show hyalinized-calcified or palisading nuclear pattern somewhat similar to those seen in gastric tumors, and malignant examples can have a leiomyosarcoma-like fascicular pattern. Epithelioid mitotically inactive GISTs similar to those often seen in stomach are rarely observed in the rectum. In this location, they can be clinically indolent, similar to their gastric counterparts. 79 The GISTs in the omentum can have spindle cell and epithelioid features resembling those of gastric GISTs, whereas mesenteric GISTs often have features resembling the small intestinal tumors, including the presence of skeinoid fibers in some examples. 52 These findings raise the possibility that omental and mesenteric GISTs are derived from stomach and small intestine, respectively, representing tumors that for some reason have detached from their GI origin during their development. IMMUNOHISTOCHEMICAL FEATURES OF GIST The key feature of GIST is positivity for the Kit (CD117) receptor tyrosine kinase, observed in more than 95% of GISTs. Although Kit positivity is a major defining feature for GIST, it is no longer considered an absolute requirement. Kit expression in GIST is a constitutional feature and not a consequence of mutation. Other, commonly expressed but less GIST-specific antigens are CD34 and nestin. Gastrointestinal stromal tumors are variably positive for smooth muscle markers (smooth muscle actin [SMA], heavy caldesmon, calponin, and embryonic smooth muscle myosin) but are generally negative for desmin. Positivity for S100 protein is rare, and glial fibrillary acidic protein is absent. Keratin 18, and to lesser degree, keratin 8, are occasionally expressed. There is no rationale to separate GISTs into myoid or neural types based on expression of smooth muscle markers and S100 protein. However, GISTs positive for smooth muscle markers and S100 protein have to be separated from true smooth muscle and nerve sheath tumors. Kit and PDGFRA Representative examples of Kit immunostaining in GIST are shown in Figure 4. Kit positivity in GISTs is typically strong and global, often apparently pancytoplasmic. Membrane staining is best observed in epithelioid GISTs, 1472 Arch Pathol Lab Med Vol 130, October 2006 Gastrointestinal Stromal Tumors Miettinen & Lasota

8 especially in dyscohesive, hypercellular, and sarcomatous variants. Some epithelioid GISTs of the stomach are less uniformly and sometimes only weakly positive (Figure 4, A) or even negative for Kit. Many of these tumors have PDGFRA mutations. Some GISTs may have Kit positive and negative areas (Figure 4, B) or perinuclear Kit-positive dots ( Golgi-zone pattern ) (Figure 4, C). In some small intestinal GISTs, the areas rich in cell processes show greater Kit positivity than the cell bodies (Figure 4, D). According to our experience, the best KIT antibodies currently available for formalin-fixed and paraffin-embedded tissue are polyclonal ones, whereas most monoclonal Kit antibodies react inconsistently with GISTs. Specific Kit antibodies should react with normal Kit-positive components, such as mast cells and interstitial cells of Cajal, and not with normal smooth muscle cells or fibroblasts. Although a definitional feature, Kit positivity as such is not sufficient for the diagnosis. Other tumors that are consistently Kit-positive are mastocytoma, seminoma, pulmonary small cell carcinoma, and (blastic) extramedullary myeloid tumor (granulocytic sarcoma). However, by their overall clinicopathologic features, these tumors can hardly be confused with GISTs. Other abdominal or GI tumors that are variably KIT positive include metastatic melanoma and the related clear cell sarcoma (30% 50%), Ewing sarcoma family of tumors (50%), childhood neuroblastoma (30%), angiosarcoma (50%), and some carcinomas. 80 Data on PDGFRA expression are scant, and many available antibodies are not reliable on paraffin-embedded tissue. However, some studies suggest that PDGFRA can be a diagnostic immunohistochemical marker. 81 Other GIST Markers Protein kinase theta is a downstream effector in the KIT signaling pathway activated following KIT activation and suggested as a therapeutic kinase target. 82 It has been also suggested as an immunohistochemical marker for GIST, and in 1 study, all 26 GISTs examined were positive for this marker. 83 In our experience, positive staining is often weak and less distinctive than Kit positivity. DOG1, a new gene so named being discovered on GIST and encoding for a protein of unknown function, seems to be expressed in GIST independent of mutation type and is absent in non-gists. 84 Experience is limited, and antibodies are not yet generally available. Approximately 80% to 85% of gastric GISTs and 50% of small intestinal GISTs are positive for CD34, the hematopoietic progenitor cell antigen also expressed in endothelial cells, subsets of fibroblasts, and many neoplasms related to these cell types. Gastrointestinal stromal tumors of esophagus and rectum are nearly consistently CD34- positive (95% 100%). CD34 expression has not been a significant prognostic factor in gastric and small intestinal GISTs. 28,29 Muscle Cell Markers Approximately 30% of GISTs, more often small intestinal than gastric tumors, are positive for SMA, whose expression is sometimes reciprocal with that of CD34. The SMA positivity varies from focal to extensive and can be equally prominent as Kit positivity in these tumors. Infiltration of GISTs between normal smooth muscle fibers results in numerous entrapped SMA (and desmin) positive intratumoral spindle cells and should not be confused with muscle marker positivity. SMA positivity has been a statistically significant favorable prognostic factor in gastric and small intestinal GISTs. 28,29 Positivity for desmin, the muscle type intermediate filament protein, is rare in GISTs of all sites, and this occurs more commonly in esophageal and gastric GISTs and very rarely in intestinal GISTs. Desmin positivity is more common in gastric epithelioid GISTs and is usually focal. 28 Sequential pretreatment and posttreatment biopsies have suggested that imatinib treatment may induce desmin expression in some tumors. 85 Embryonic form of smooth muscle myosin 86 and heavy caldesmon, an actin-binding cytoskeletal protein, 87 are smooth muscle antigens commonly expressed in GISTs. The expression of myoid antigens in GIST may be a reflection of multipotentiality of the ancestral Cajal cells or their relatedness to smooth muscle precursor cells. Neural and Other Markers S100 protein expression is relatively rare in GISTs but seems to be more common in small intestinal than in gastric GISTs. 29 Based on a small number of cases, S100 protein positivity seemed to be an adverse prognostic factor in gastric 28 but not in small intestinal GISTs. 29 Gastrointestinal stromal tumors are consistently positive for nestin, a type VI intermediate filament protein typical of many stem cells, including those of nervous and muscular systems. 88 However, nestin is equally often present in GI schwannomas suggesting its incomplete specificity for GISTs. 89 Gastrointestinal stromal tumors are positive for vimentin. They are uniformly negative for glial fibrillary acidic protein, which helps to separate them from GI schwannomas, tumors that are typically glial fibrillary acidic protein positive. 89 Neurofilament 68 occurs in a subset of GISTs ( 10%). 55 The biologic significance of this observation is presently unclear. Keratin positivity can be seen in GISTs with antibodies reacting with keratin 18 and, to lesser degree, to keratin 8, but keratins 7, 13, 14, 17, 19, and 20 are not present. Therefore, antibody cocktails such as AE1/AE3 would usually give negative results (including possible rare positivity because of potential detection of keratin 8). Keratin 18 positivity may be more common in malignant GISTs. 89 EVALUATION OF BIOLOGIC POTENTIAL OF GISTS Gastrointestinal stromal tumors form a continuum in biologic potential at all sites of their occurrence. Older studies on GI smooth muscle tumors (largely representing GISTs in the present terminology) usually reported separately on benign and malignant tumors, creating an illusion of a clear separation between benign and malignant tumors. Because criteria of separation were not precisely stated, it is difficult to gain an understanding on predictive parameters based on older data. Cancer hospital series emphasize clinically malignant tumors because of referral bias, potentially leaving an impression of the generally malignant nature of GISTs. Recent large series ( 500 cases each) have evaluated the behavior of large numbers of GISTs from the most common sites, stomach and small intestine, in relation to prognostic parameters. 28,29 These data antedating application of imatinib give insight into the natural history of the disease and may help target adjuvant treatments to proper patient populations. The most powerful and most widely examined criteria Arch Pathol Lab Med Vol 130, October 2006 Gastrointestinal Stromal Tumors Miettinen & Lasota 1473

9 Table 1. Prognosis of Gastrointestinal Stromal Tumor (GIST) Based on Long-Term Follow-Up of Observation of 1684 Patients in Armed Forces Institute Studies Prior to Imatinib* Tumor Parameters Patients With Progressive Disease During Follow-Up and Characterization of Malignant Potential, % Group Size, cm Mitotic Rate per 50 HPFs Gastric GISTs Small Intestinal GISTs Very low if any 0 Very low if any Low 4.3 Low 3a Low 24 Intermediate 3b Intermediate 52 High Low 50 High Intermediate 73 High 6a High 85 High 6b High 90 High * Note significantly worse prognosis in small intestinal GISTs. Based on data from Miettinen et al. 28,29 HPFs indicates high-power fields. Denotes tumor categories with very small numbers of cases insufficient for prediction of malignant potential. Table 2. Tumors That Have Been Separated From Gastrointestinal Stromal Tumor* True smooth muscle tumors Leiomyoma Intramural Of muscularis mucosae origin Uterine-type leiomyoma in women Glomus tumor Leiomyosarcoma Nerve sheath and melanocytic tumor GI schwannoma Metastatic melanoma Primary GI clear cell sarcoma Fibroblastic tumor Desmoid Inflammatory myofibroblastic tumor Inflammatory fibroid polyp Undifferentiated sarcoma * GI indicates gastrointestinal. for evaluating biologic potential of GIST are tumor size and mitotic activity, the latter typically expressed per 50 HPFs ( 40) (totaling 5 mm 2 ). 28,29 If modern wide-field microscopes with wide-field eyepieces are used, the mitotic count should be adjusted to correspond to the same total area; this equals approximately 25 fields with a 40 magnification. Because size and mitotic rate parameters are universally applicable, they probably should be recorded for all GISTs and included in the pathology report. There is a well-established relationship between these parameters and tumor behavior based on several large clinicopathologic studies. The parameters should be applied differently in gastric and small intestinal GISTs, because of the higher malignant potential of small intestinal GISTs. The reasons for the greater biologic potential of small intestinal versus gastric GISTs are unknown. Small intestinal GISTs have a more aggressive behavior than gastric GISTs with similar size and mitosis parameters, especially including tumors more than 5 cm with low mitotic rates. 29 In addition, small intestinal GISTs tend to be larger and more advanced when diagnosed. Suggested guidelines for evaluating biologic potential of gastric and small intestinal GISTs are summarized in Table 1. These criteria are based on long-term follow-up of more than 1600 patients. 28,29 They differ from the proposed consensus criteria 90 in that separate criteria are given for gastric and small intestinal tumors. The consensus criteria are not based on specific data sets and they significantly overestimate the biologic potential of gastric GISTs, especially the potential of large tumors with low mitotic rate. There are less data on GISTs of rare sites, such as esophagus, colon, and rectum. However, in terms of prognosis, tumors at these sites should probably be evaluated similar to intestinal GISTs. There is no specific grading or staging system for GISTs. The French Federation of Cancer Centers grading system, 91 widely applied for soft tissue sarcomas, is not relevant for GISTs, because specific modes of targeted treatments are used. Also, the mitosis thresholds in that system are too high for GISTs. In this system, a majority of GISTs would be low or intermediate grade, despite high-grade tumor behavior. Other significant pathologic findings that can assist in evaluation of malignant potential of GIST are peritoneal nodules, invasion to peritoneal fat, attachment or involvement or surrounding organs, and mucosal invasion. Proliferation markers (Ki-67 analogs) might be useful in assessing the proliferative rate. However, they have not been proven superior to mitotic counting. 92,93 It is possible that such potentially more objective parameters, such as proliferation indices based on sophisticated morphometric evaluation, could be used in the future in the assessment of biologic potential, if sufficient data on their correlation with prognosis are obtained. Loss of p16 cell cycle regulator has been shown as an unfavorable parameter in GIST. 94 HISTOLOGIC DIFFERENTIAL DIAGNOSIS OF GIST Gastrointestinal (true) smooth muscle tumors, nerve sheath tumors, desmoids, inflammatory myofibroblastic tumors, inflammatory fibroid polyps, and undifferentiated sarcomas are tumors most commonly confused with GISTs (Table 2). These tumors have been almost uniformly reported as Kit negative, and many have other, distinctive markers that are generally not expressed in GISTs (Figure 5). Occasionally, poorly-differentiated carcinomas and his- Figure 5. Important differential diagnoses for gastrointestinal stromal tumor (GIST) with illustrative immunostainings. A, Uterine-type leiomyoma surrounding rectum (hematoxylin-eosin, original magnification 150). B, Estrogen receptor positivity in a uterine-type peri-intestinal leiomyoma 1474 Arch Pathol Lab Med Vol 130, October 2006 Gastrointestinal Stromal Tumors Miettinen & Lasota

10 (immunoperoxidase, original magnification 240). C, Gastric schwannoma with a microtrabecular pattern (hematoxylin-eosin, original magnification 150). D, Strong S100 protein positivity in gastric schwannoma (immunoperoxidase, original magnification 240). E, Mesenteric desmoid with myofibroblasts in collagenous stromal punctuated by prominent vessels (hematoxylin-eosin, original magnification 120). F, Nuclear betacatenin positivity in desmoid (immunoperoxidase, original magnification 240). G, Inflammatory myofibroblastic tumor is characterized by spindled plump myofibroblasts and diffusely sprinkled lymphocytes and plasma cells (hematoxylin-eosin, original magnification 120). H, Anaplastic lymphoma kinase positivity in inflammatory myofibroblastic tumor (immunoperoxidase, original magnification 240). Arch Pathol Lab Med Vol 130, October 2006 Gastrointestinal Stromal Tumors Miettinen & Lasota 1475

11 tiocytic sarcoma can also enter into differential diagnosis that is greatly aided by immunohistochemistry. Smooth Muscle Tumors In the GI tract, smooth muscle tumors generally segregate into benign (leiomyomas) and malignant (leiomyosarcomas), in contrast with the continuum in biologic potential observed in GISTs. Intramural leiomyomas most often occur in the esophagus in the GI tract, and in fact, in this location, leiomyomas are far more common than GISTs at least by a margin of 3:1. These tumors often occur in young patients. Intramural leiomyomas are rare in the stomach and small intestine but may be relatively more common in the colon. Muscularis mucosae leiomyomas form small, usually less than 1-cm intraluminal polyps that are most often diagnosed in the colon and rectum. 95 They are composed of mature, occasionally focally atypical, smooth muscle cells that are phenotypically similar to those in other leiomyomas. Uterine-type leiomyomas occur around intestines and in the retroperitoneum in women. These tumors are histologically similar to uterine leiomyomas (Figure 5, A). They are immunohistochemically positive for SMA, desmin, and estrogen (Figure 5, B) and progesterone receptors. 29 Recognition of these tumors separate from GISTs is significant for management, because a more conservative approach can be taken, especially for tumors located in the anorectal region. Glomus tumors are rare in the GI tract and occur almost exclusively in the stomach. They are usually benign and are histologically and immunohistochemically similar to peripheral glomus tumors being positive for SMA and negative for desmin, S100 protein, and Kit. 2 Primary leiomyosarcomas are rare in the GI tract. In our experience, they occur in the intestine with a frequency of 2% to 10% of that of GIST, whereas they are extremely rare in the stomach. These tumors often form polypoid intraluminal masses, and if completely excised, they can have a better prognosis than a GIST with similar tumor size and mitosis parameters. 79 The diagnosis of leiomyosarcoma is based on histologic similarity to peripheral leiomyosarcoma, immunohistochemical positivity for SMA and desmin, and negativity for Kit and lack of KIT and PDGFRA mutations. 96 Schwannoma, Melanoma, and Related Tumors Gastrointestinal schwannomas usually occur in the stomach (60% 70%) or colon (20% 30%) of older adults, and they are rare in small intestine and other GI locations. 97,98 Nearly all gastric and most intestinal schwannomas are relatively small ( 5 cm), intramural spindle cell tumors with low mitotic activity less than 5 per 50 HPFs, focally to extensively surrounded by a lymphoid cuff. These tumors are composed of bundles of spindle cells with focal atypia and often form a microtrabecular pattern intermingled with fibrovascular septa (Figure 5, C). Epithelioid variants occasionally present as colonic mucosal polyps. All these lesions are S100 (Figure 5, D) and glial fibrillary acidic protein positive. Metastatic amelanotic melanoma or the closely related primary clear cell sarcoma involving the GI tract can simulate a GIST when forming an intramural mass. Some GI clear cell sarcomas are distinctive in containing osteoclastic giant cells; their prominent S100 positivity and t(12;22) translocation with EWS-ATF1 gene fusions are diagnostic features, whereas these tumors seem to lack HMB45, in contrast with peripheral clear cell sarcomas. 99 Fibroblastic Tumors Intra-abdominal desmoid can involve the GI tract in GIST-like manner, and it occurs sporadically and in connection with Gardner syndrome. This tumor is distinctive as a whitish, hard mass and is histologically rich in collagen and mildly dilated, prominent vessels (Figure 5, E). Although (focal) cytoplasmic Kit positivity has been reported in desmoids, 100 studies on a purified polyclonal antibody have not shown similar reactivity. 101 Immunohistochemical demonstration of nuclear beta-catenin positivity (Figure 5, F) may be diagnostically helpful, although this is not seen in all cases. 102 Inflammatory myofibroblastic tumor (previously often designated as inflammatory pseudotumor or inflammatory fibrosarcoma) typically occurs in children and young adults. 103 The prototypic form occurs in abdomen and can clinically and grossly simulate a GIST, especially when forming an intramural GI tract mass. Histologically, these tumors are composed of large spindled cells with amphophilic cytoplasm intermingled with lymphoplasmacytic infiltration and streaks of fibrosis (Figure 5, G). The tumor cells are negative for Kit and CD34 but can be actin positive. These tumors are often positive for anaplastic lymphoma kinase (ALK) generally showing pancytoplasmic staining (Figure 5, H). A rearrangement of the ALK gene in chromosome 2p23 by a number of gene fusion-type translocations activating the ALK gene is believed to be a central pathogenetic event and diagnostic marker. 104 Inflammatory fibroid polyp is in our experience a heterogeneous group of lesions typically forming ulcerated intraluminal polyps. They most commonly occur in the small intestine, especially ileum, of adult patients and often cause an intussusception. Histologically the lesions are highly vascular with a loose somewhat granulation tissuelike texture and often contain eosinophilic granulocytes. Some, especially gastric tumors of this type, are CD34 positive. 105 Undifferentiated sarcomas form a heterogeneous group that can involve any segment of the GI tract, but they seem to be more common in the intestines than in the stomach. 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