Genetic Alterations in Gastrointestinal Stromal Tumors: Implications for Diagnosis and Management Christopher Corless, MD, PhD Department of Pathology and Knight Diagnostic Laboratories Oregon Health & Science University, Portland, Oregon Email: [email protected] www.knightdxlabs.com Introduction Based on a number of population studies the annual incidence of new GIST cases in the United States is estimated between 3,500 and 6,250. The tumors occur primarily in older patients of either sex, and more than 80% are localized at the time of diagnosis. Following surgical resection, however, up to 50% of patients will suffer a recurrence, which may be local or diffuse throughout the abdomen, and most of these patients will have progression involving the liver, lung and other distant sites. Prior to the advent of targeted therapies, the prognosis for advanced GISTs was poor due to their inherent resistance to both chemotherapy and radiation therapy. As most GISTs are dependent on mutations that activate a receptor tyrosine kinase, they have long served as a model for the impact of molecularly targeted therapies in solid tumors. Clinical trials conducted during the past 15 years have established roles for imatinib and other KIT kinase inhibitors in the treatment of GIST, both in the adjuvant and advanced disease settings. Clinical and pathologic features GISTs most commonly arise in the stomach (60%) and small intestine (25%), but they also occur in the colon, rectum, esophagus, mesentery, omentum and retroperitoneum (15% together). Fatigue, abdominal pain, dysphagia, satiety, and bowel obstruction are among the presenting clinical symptoms. Anemia related to mucosal bleeding hemorrhage is also common. The tumors are generally well circumscribed, have a fleshy pink or tan cut surface, and may show areas of hemorrhagic necrosis and cystic degeneration. They range from 1 cm to more than 40 cm, with an average of approximately 5 cm. GISTs show a wide spectrum of morphologies, from bland spindle cell proliferations to highly cellular epithelioid tumors with significant nuclear pleomorphism. Skeinoid fibers and foci of calcification are common in lower grade lesions. The two main immunohistochemical markers for GIST are CD117 (KIT) and DOG1 (ANO1), which are usually co-expressed. If a tumor is negative for both these markers, the diagnosis of GIST should be questioned. CD34 is expressed in about 70% of the tumors. Smooth muscle actin and muscle specific actin are variably expressed in GISTs, while desmin is usually absent. As discussed below, immunostaining for SDHB is important in identifying GISTs deficient in succinate dehydrogenase activity; such tumors retain CD117 positivity. Oncogenic mutations in GISTs As summarized in Table 1, GISTs can be divided into a number of molecular subtypes. In the broadest terms, the tumors fall into two main groups: those with a mutation in a gene acting primarily through the MAP kinase pathway (e.g. KIT or PDGFRA), and those that have loss of succinate dehydrogenase (SDH) activity. Table 2 provides details on the therapeutic implications of the various oncogenic mutations found in GISTs.

2 KIT Mutations Hirota and colleagues were the first to show KIT expression in GISTs, and to identify KIT gene mutations as the main oncogenic driver in the majority of tumors. Approximately 75% of GISTs harbor a KIT mutation that leads to constitutive activation of the kinase. The functional importance of KIT mutations in GIST development is supported by the following observations: 1) phosphorylated (activated) KIT is detectable in GIST tumor extracts; 2) mutant KIT is oncogenic, supporting the growth of stably transfected BA/F3 cells in nude mice; and 3) mutant forms of KIT expressed in transfected cell lines show constitutive kinase activity in the absence of SCF ligand. Tumor extracts from KIT-mutant GISTs demonstrate activation of downstream signaling pathways, including the MAP kinase pathway (RAF, MEK, ERK), the PI3 kinase/AKT pathway, and STAT3 (Figure 1A). ETV1 is an important regulator of GIST-specific gene expression during tumorigenesis, and KIT signaling serves to maintain ETV1 activity. Two-thirds of GISTs harbor mutations in KIT exon 11, which encodes the juxtamembrane domain. In-frame deletions, insertions, substitutions, or combinations thereof alter the ability of this domain to repress kinase activity. Deletions involving codons 557 and/or 558 are associated with more aggressive behavior than other exon 11 mutations, but mitotic index is prognostically more important than mutation status. Significantly, all KIT exon 11 mutations are sensitive to imatinib and other KIT kinase inhibitors (Table 2). Between 7% and 10% of GISTs have a mutation in an extracellular domain encoded by KIT exon 9. These mutations are thought to mimic the conformational change that the extracellular KIT receptor undergoes when ligand is bound. Importantly, the kinase domain in exon 9-mutant KIT is essentially the same as in wild-type KIT, and therefore requires a higher dose of imatinib than exon 11mutant KIT. Interestingly, these mutations occur in tumors arising in the small and large intestine, but are very rarely seen in gastric GISTs. Primary mutations in the kinase domain are uncommon. They include mutations of the activation loop (exon 17) and the ATP binding region (exon 13) that serve to stabilize the active conformation of the kinase. PDGFRA Mutations PDGFRA is structurally similar to KIT, and GIST-related mutations in the PDGFRA gene affect the same protein domains: juxtamembrane (exon 12), ATP binding (exon 14), and the activation loop (exon 18) (Table 1). Consistent with their extensive functional overlap, KIT and PDGFRA mutations are mutually exclusive in GISTs. Of note, the most common PDGFRA mutation (D842V in exon 18), is fully resistant to imatinib and all other FDA-approved KIT/PDGFRA kinase inhibitors (Table 2). As in the case of KIT, mutant PDGFRA expressed in transfected cell lines shows constitutive kinase activity in the absence of its ligand, PDGF-A. In addition, both KIT- and PDGFRA-mutant tumors are immunopositive for DOG1 and protein kinase C theta (PKCθ), and both genotypes are associated with other cytogenetic changes that are distinctive for GIST. Despite these similarities, PDGFRA-mutant GISTs show variable (sometimes negative) expression of CD117, as well as a striking predilection for the stomach and a generally lower potential for malignancy. PDGFRA-mutant GISTs are not reliably distinguishable from KIT-mutant GISTs based on morphology alone. Other MAPK pathway mutations GISTs that are negative for KIT and PDGFRA mutations, commonly referred to as ‘wild-type’ GISTs, are a heterogeneous group (Table 1). Most can be classified as SDH-deficient GISTs (discussed below), but a small subset harbors mutations in genes important in the MAP kinase

3 pathway, functioning downstream of KIT/PDGFRA (Figure 1b). Among these is the BRAF V600E mutation that is common in papillary thyroid carcinoma and melanoma. BRAF-mutant GIST can respond to BRAF and MEK inhibitors. Patients with type I neurofibromatosis are at increased risk for GISTs in the small bowel; correspondingly, some wild-type GISTs have loss-of-function mutations in NF1. Alterations of HRAS, NRAS and PIK3CA gene mutations are rare and their therapeutic management has yet to be established (Table 2). Gene fusions that activate NTRK1, NTRK3 or FGFR1 have recently been uncovered in wildtype GISTs. Interestingly, all cases identified to date have been non-gastric. A patient with an NTRK1 fusion-positive GIST responded very well to treatment in a TRK inhibitor trial, suggesting that all wild-type, non-gastric GISTs should be screened for gene fusions by FISH or next-generation sequencing. SDH-Deficient GISTs The majority of KIT/PDGFRA wild-type GISTs shows loss of succinate dehydrogenase (SDH), which is a heterotetrameric enzyme encoded by SDHA, SDHB, SDHC and SDHD. This complex serves to oxidize succinate to fumarate as part of the mitochondrial Krebs cycle. Loss of any of these subunits through gene mutation/deletion or promoter methylation destabilizes the complex and causes the accumulation of succinate, which in turn leads to upregulation of HIF1a transcriptional activity and a marked decrease in DNA demethylation by TET2 (Figure 1b). In most cases of SDH-deficient GIST there is a germline abnormality in one of the SDH genes, and a second hit leads to the DNA hypermethylation and tumor development. Such patients are at risk not only for GIST, but also for paragangliomas and other tumors (Carney-Stratakis syndrome). Regardless of which gene is affected, SDH-deficient GISTs are readily identified by their lack of immunostaining for SDHB, which is unstable when the SDH tetramer cannot assemble. SDHA staining may be absent specifically in tumors with SDHA mutations. SDH-deficient GISTs arising in the pediatric setting characteristically show hypermethylation of the SDHC gene promoter, the cause for which is unknown but is not heritable. These patients may manifest part or all of Carney’s Triad (GIST, pulmonary chondroma, paraganglioma). From a clinical perspective, important features of SDH-deficient GISTs include their restriction to the stomach, multinodular/infiltrative growth pattern, and relatively slow growth but high predilection for recurrence and metastasis over time. Importantly, these tumors do not respond well to imatinib (Table 2). Sunitinib and regorafenib appear to be more effective than imatinib, possibly due to cross-inhibition of VEGFR, but the optimal management of these tumors beyond repeated surgical resections has not been firmly established. The origin of GISTs Interstitial cells of Cajal The interstitial cells of Cajal (ICCs), which serve as pacemakers for peristaltic contractions, share many features with GISTs. Both express high levels of PKCθ, nestin, DOG1 and the ETS family transcription factor ETV1. Moreover, mice engineered to express mutant KIT develop diffuse hyperplasia of the interstitial cells of Cajal as well as GIST-like tumors. This histologic picture is similar to that seen in individuals with inherited KIT-activating mutations. Micro-GISTs Minute nodular growths (1 to 10 mm) of ICC cells are present in between 2.9% and 35% of stomachs thoroughly examined after surgical removal or at autopsy. These so-called micro-GISTs are mitotically inactive, suggesting tumorigenic arrest. However, the type and frequency of KIT mutations in micro-GISTs is essentially the same as in clinically significant tumors, and sub-centimeter GISTs

4 with PDGFRA mutations have also been reported. These observations suggest that kinase gene mutations occur very early in GIST tumorigenesis, but that the mutations are not sufficient for progression to an oncologically threatening lesion. The large pool of micro-GISTs in the general population likely explains the multiple reported cases in which two or more genotypically distinct GISTs are found in a patient during a single surgical procedure. Assessing GIST Prognosis Imatinib is approved by the FDA and EMEA for use in the adjuvant setting, and it is common for patients to take the drug for 3 years or even longer to prevent disease recurrence. Therefore, it is imperative that the prognosis of a newly resected GIST be predicted as accurately as possible. While a KIT or PDGFRA mutation may set the initial course of most GISTs, prognosis at the time of clinical presentation is clearly dependent on other genetic events, such as loss of CDKN2A (encoding p16) or TP53. Unfortunately, our knowledge of these additional mutations remains limited, and current recommendations for assessing prognosis are based on tumor size, tumor location, and mitotic index (mitoses per 5 mm2). The most widely used risk assessment scheme was developed by Miettinen, Lasota and colleagues, whose considerable efforts in studying the outcome of patients prior to the advent of imatinib have provided the most complete data available (Table 3). It should be noted that tumor rupture, either before or during surgery, is another important negative prognostic factor, as is incomplete resection (particularly in the setting of a rectal GIST). Responses to kinase inhibitor therapy Disease persistence In an attempt to determine the optimal duration of imatinib therapy for advanced, unresectable GIST, a trial conducted in France randomized patients who had good control of their disease during 3 years of imatinib treatment to either continue or to discontinue the drug. For those continuing treatment, progression-free survival over the next two years was 80%, but for those who stopped therapy it was only 16%. Thus, GIST cells persist even in the face of longstanding KIT suppression. This is supported by histological analyses of resected tumors after 1 to 31 months of imatinib treatment, where histologic responses range from 90%, but persistent tumor cells are always present. Primary resistance Primary resistance to kinase inhibitor therapy can be defined as progression within the first 6 months of treatment. Based on data from phase II and phase III trials of imatinib, tumor response correlates with the underlying kinase genotype (Table 2). Exon 11-mutant KIT is highly sensitive to imatinib, with an IC 50 of 10 cm

10%

52%

34%

57%

Mitotic

≤ 2 cm

(None)

(High)

Insuff. data

54%

Index

> 2 ≤ 5 cm

16%

73%

50%

52%

> 5 ≤ 10 cm

55%

85%

Insuff. data

Insuff. data

> 10 cm

86%

90%

86%

71%

≤5 per 5 mm2

>5 per 5 mm2

The risk of disease recurrence or metastasis can be estimated based on three parameters defined in this table: 1) the mitotic index (either ≤5 mitoses per 5 mm2 or > 5 mitoses per 5 mm2); 2) tumor size (largest diameter); and 3) tumor site of origin. The table is based on data published by Miettinen and Lasota (Arch Pathol Lab Med 2006;130:1466–1478).

9

KIT KIT / PDGFRA

P

P

P

SOS

P

SHC

GRB2

RAF

RAS

STAT

P

P

PDK

PI3K

MEK P

P

P

P

P

P

PTEN

P

AKT

S6K P mTOR P

ERK ↑ JUN

↑ ETV1, ↑ CDK4, ↑Cyclin D1, ↓p16

Figure 1A. KIT and PDGFRA cell signaling pathways. Dimerization of KIT or PDGFRA leads to signaling through the MAP kinase pathway (RAF, MEK, ERK), and the PI3 kinase pathway (AKT, mTOR, S6 kinase). In addition, STAT3 is activated. The collective impact favors an increase in cell metabolism, cell cycle progression and a decreased sensitivity to apoptosis.

10

KIT

NF1 RAS

BRAF

SDIA SDIB SDIC SDID

Prolyl hydroxylase

P

MEK HIF1a

P

ERK

Succinate

Proteosome Complex

↑ ETV1

HIF1a

VEGF, IGF1, IGF2

DNA demethylation

Figure 1B. Cell signaling in KIT/PDGFRA ‘wild-type’ GISTs. Mutations in NF1, RAS genes or BRAF lead to increased signaling through the MAP kinases MEK and ERK, promoting cell growth. Loss of the mitochondrial succinate dehydrogenase complex, through mutations in SDHA, SDHB, SDHC or SDHD, leads to an accumulation of succinate, which inhibits prolyl hydroxylase-mediated degradation of HIF1α through the proteosome complex. This results in upregulated transcription of a number of genes, including VEGF, IGF1 and IGF2. Succinate also inhibits demethylation of DNA by TET2 through increasing α-ketoglutarate levels.

11

Recommended Literature Note: Due to space considerations, it is not possible to provide a complete bibliography of GIST publications. Apologies are extended to any authors whose work is not included on the list below. 1.

Kindblom LG, Remotti HE, Aldenborg F, Meis-Kindblom JM. Gastrointestinal pacemaker cell tumor (GIPACT): Gastrointestinal stromal tumors show phenotypic characteristics of the interstitial cells of Cajal. Am.J.Pathol 152, 1259-1269. 1998.

2.

Hirota S, Isozaki K, Moriyama Y, Hashimoto K, Nishida T, et al. Gain-of-function mutations of c-kit in human gastrointestinal stromal tumors. Science 279, 577-580. 1998.

3.

Heinrich MC, Corless CL, Duensing A, McGreevey L, Chen CJ, Joseph N, Singer S, Griffith DJ, Haley A, Town A, Demetri GD, Fletcher CD, Fletcher JA. PDGFRA Activating Mutations in Gastrointestinal Stromal Tumors. Science 2003 Jan 31;299(5607):708-10.

4.

Hirota S, Ohashi A, Nishida T, Isozaki K, Kinoshita K, Shinomura Y, Kitamura Y. Gain-of-function mutations of platelet-derived growth factor receptor alpha gene in gastrointestinal stromal tumors. Gastroenterology 2003 Sep;125(3):660-7.

5.

Corless CL, Schroeder A, Griffith D, Town A, McGreevey L, Harrell P, Shiraga S, Bainbridge T, Morich J, Heinrich MC. PDGFRA Mutations In Gastrointestinal Stromal Tumors: Frequency, Spectrum and In Vitro Sensitivity To Imatinib. J Clin Oncol 2005 May 31;23:5357-64.

6.

Heinrich MC, Griffith DJ, Druker BJ, Wait CL, Ott KA, Zigler AJ. Inhibition of c-kit receptor tyrosine kinase activity by STI 571, a selective tyrosine kinase inhibitor. Blood 2000 Aug 1;96(3):925-32.

7.

West RB, Corless CL, Chen X, Rubin BP, Subramanian S, Montgomery K, Zhu S, Ball CA, Nielsen TO, Patel R, Goldblum JR, Brown PO, Heinrich MC, Van de RM. The novel marker, DOG1, is expressed ubiquitously in gastrointestinal stromal tumors irrespective of KIT or PDGFRA mutation status. Am J Pathol 2004 Jul;165(1):107-13.

8.

Sommer G, Agosti V, Ehlers I, Rossi F, Corbacioglu S, Farkas J, Moore M, Manova K, Antonescu CR, Besmer P. Gastrointestinal stromal tumors in a mouse model by targeted mutation of the Kit receptor tyrosine kinase. Proc Natl Acad Sci U S A 2003 May 27;100(11):6706-11.

9.

Rossi F, Ehlers I, Agosti V, Socci ND, Viale A, Sommer G, Yozgat Y, Manova K, Antonescu CR, Besmer P. Oncogenic Kit signaling and therapeutic intervention in a mouse model of gastrointestinal stromal tumor. Proc Natl Acad Sci U S A 2006 Aug 22;103(34):12843-8.

10.

Chi P, et al. ETV1 is a lineage survival factor that cooperates with KIT in gastrointestinal stromal tumours. Nature 2010;467:849-53.

11.

Heinrich MC, Corless CL, Demetri GD, Blanke CD, von Mehren M, Joensuu H, McGreevey LS, Chen CJ, Van den Abbeele AD, Druker BJ, Kiese B, Eisenberg B, Roberts PJ, Singer S, Fletcher CD, Silberman S, Dimitrijevic S, Fletcher JA. Kinase mutations and imatinib response in patients with metastatic gastrointestinal stromal tumor. J Clin Oncol 2003 Dec 1;21(23):4342-9.

12.

Antonescu CR, Besmer P, Guo T, Arkun K, Hom G, Koryotowski B, Leversha MA, Jeffrey PD, Desantis D, Singer S, Brennan MF, Maki RG, DeMatteo RP. Acquired resistance to imatinib in gastrointestinal stromal tumor occurs through secondary gene mutation. Clin Cancer Res 2005 Jun 1;11(11):4182-90.

13.

Andersson J, Sihto H, Meis-Kindblom JM, Joensuu H, Nupponen N, Kindblom LG. NF1-associated gastrointestinal stromal tumors have unique clinical, phenotypic, and genotypic characteristics. Am J Surg Pathol 2005 Sep;29(9):1170-6.

12 14.

Maertens O, Prenen H, Debiec-Rychter M, Wozniak A, Sciot R, Pauwels P, De W, I, Vermeesch JR, de RT, De PA, Speleman F, van OA, Messiaen L, Legius E. Molecular pathogenesis of multiple gastrointestinal stromal tumors in NF1 patients. Hum Mol Genet 2006 Mar 15;15(6):1015-23.

15.

Prakash S, Sarran L, Socci N, DeMatteo RP, Eisenstat J, Greco AM, Maki RG, Wexler LH, LaQuaglia MP, Besmer P, Antonescu CR. Gastrointestinal stromal tumors in children and young adults: a clinicopathologic, molecular, and genomic study of 15 cases and review of the literature. J Pediatr Hematol Oncol 2005 Apr;27(4):179-87.

16.

Killian JK, Miettinen M, Walker RL, Wang Y, Zhu YJ, Waterfall JJ, Noyes N, Retnakumar P, Yang Z, Smith WI Jr, Killian MS, Lau CC, Pineda M, Walling J, Stevenson H, Smith C, Wang Z, Lasota J, Kim SY, Boikos SA, Helman LJ, Meltzer PS. Recurrent epimutation of SDHC in gastrointestinal stromal tumors. Sci Transl Med. 2014;6(268):268ra177

17.

Pantaleo MA, et al. SDHA Loss-of-Function Mutations in KIT-PDGFRA Wild-Type Gastrointestinal Stromal Tumors Identified by Massively Parallel Sequencing. J Natl Cancer Inst 2011;103:983-7.

18.

Dwight T, Benn DE, Clarkson A, Vilain R, Lipton L, Robinson BG, Clifton-Bligh RJ, Gill AJ. Loss of SDHA Expression Identifies SDHA Mutations in Succinate Dehydrogenase-deficient Gastrointestinal Stromal Tumors. Am J Surg Pathol 2012 Oct 10.

19.

Janeway KA, et al. Defects in succinate dehydrogenase in gastrointestinal stromal tumors lacking KIT and PDGFRA mutations. Proc Natl Acad Sci U S A 2011;108:314-8.

20.

Italiano A, et al. SDHA loss of function mutations in a subset of young adult wild-type gastrointestinal stromal tumors. BMC Cancer 2012;14(12):408.

21.

Wagner AJ, Remillard SP, Zhang YX, Doyle LA, George S, Hornick JL. Loss of expression of SDHA predicts SDHA mutations in gastrointestinal stromal tumors. Mod Pathol 2012 Oct 7;153.

22.

Carney JA. Gastric stromal sarcoma, pulmonary chondroma, and extra-adrenal paraganglioma (Carney Triad): natural history, adrenocortical component, and possible familial occurrence. Mayo Clin Proc 1999 Jun;74(6):543-52.

23.

Isozaki K, Terris B, Belghiti J, Schiffman S, Hirota S, Vanderwinden J-M. Germline-activating mutation in the kinase domain of KIT gene in familial gastrointestinal stromal tumors. Am J Pathol 2000 Nov;157(5):1581-5.

24.

Maeyama H, Hidaka E, Ota H, Minami S, Kajiyama M, Kuraishi A, Mori H, Matsuda Y, Wada S, Sodeyama H, Nakata S, Kawamura N, Hata S, Watanabe M, Iijima Y, Katsuyama T. Familial Gastrointestinal Stromal Tumor With Hyperpigmentation: Association With a Germline Mutation of the c-kit Gene. Gastroenterology 2001;120(1):210-5.

25.

Nishida T, Hirota S, Taniguchi M, Hashimoto K, Isozaki K, Nakamura H, Kanakura Y, Tanaka T, Takabayashi A, Matsuda H, Kitamura Y. Familial gastrointestinal stromal tumours with germline mutation of the KIT gene. Nature Genetics 1998 Aug;19(4):323-4.

26.

Corless CL, McGreevey L, Haley A, Town A, Heinrich MC. KIT mutations are common in incidental gastrointestinal stromal tumors one centimeter or less in size. Am J Pathol 2002 May;160(5):1567-72.

27.

Yamamoto H, et al. Neurofibromatosis type 1-related gastrointestinal stromal tumors: a special reference to loss of heterozygosity at 14q and 22q. J Cancer Res Clin Oncol 2009;135:791-8.

28.

Ricci R, Arena V, Castri F, Martini M, Maggiano N, Murazio M, Pacelli F, Potenza AE, Vecchio FM, Larocca LM. Role of p16/INK4a in gastrointestinal stromal tumor progression. Am J Clin Pathol 2004 Jul;122(1):35-43.

29.

Joensuu H, Roberts PJ, Sarlomo-Rikala M, Andersson LC, Tervahartiala P, Tuveson D, Silberman S, Capdeville R, Dimitrijevic S, Druker B, Demetri GD. Effect of the tyrosine kinase inhibitor STI571 in a patient with a metastatic gastrointestinal stromal tumor. N Engl J Med 2001 Apr 5;344(14):1052-6.

30.

Demetri GD, von Mehren M, Blanke CD, Van den Abbeele AD, Eisenberg B, Roberts PJ, Heinrich MC, Tuveson DA, Singer S, Janicek M, Fletcher JA, Silverman SG, Silberman SL, Capdeville R, Kiese B, Peng B, Dimitrijevic S,

13 Druker BJ, Corless C, Fletcher CD, Joensuu H. Efficacy and safety of imatinib mesylate in advanced gastrointestinal stromal tumors. N Engl J Med 2002 Aug 15;347(7):472-80. 31.

Blanke CD, Rankin C, Demetri GD, Ryan CW, von MM, Benjamin RS, Raymond AK, Bramwell VH, Baker LH, Maki RG, Tanaka M, Hecht JR, Heinrich MC, Fletcher CD, Crowley JJ, Borden EC. Phase III randomized, intergroup trial assessing imatinib mesylate at two dose levels in patients with unresectable or metastatic gastrointestinal stromal tumors expressing the kit receptor tyrosine kinase: S0033. J Clin Oncol 2008 Feb 1;26(4):626-32.

32.

Gastrointestinal Stromal Tumor Meta-Analysis Group (MetaGIST). Comparison of two doses of imatinib for the treatment of unresectable or metastatic gastrointestinal stromal tumors: a meta-analysis of 1,640 patients. J Clin Oncol 2010;28:1247-53.

33.

DeMatteo RP, et al. Adjuvant imatinib mesylate after resection of localised, primary gastrointestinal stromal tumour: a randomised, double-blind, placebo-controlled trial. Lancet 2009;373:1097-104.

34.

Miettinen M, Sobin LH, Lasota J. Gastrointestinal Stromal Tumors of the Stomach: A Clinicopathologic, Immunohistochemical, and Molecular Genetic Study of 1765 Cases With Long-term Follow-up. Am J Surg Pathol 2005 Jan;29(1):52-68.

35.

Miettinen M, Makhlouf H, Sobin LH, Lasota J. Gastrointestinal Stromal Tumors of the Jejunum and Ileum: A Clinicopathologic, Immunohistochemical, and Molecular Genetic Study of 906 Cases Before Imatinib With Long-term Follow-up. Am J Surg Pathol 2006 Apr;30(4):477-89.

36.

Le CA, et al. Discontinuation of imatinib in patients with advanced gastrointestinal stromal tumours after 3 years of treatment: an open-label multicentre randomised phase 3 trial. Lancet Oncol 2010;11:942-9.

37.

Heinrich MC, Owzar K, Corless CL, Hollis D, Borden EC, Fletcher CD, Ryan CW, von MM, Blanke CD, Rankin C, Benjamin RS, Bramwell VH, Demetri GD, Bertagnolli MM, Fletcher JA. Correlation of kinase genotype and clinical outcome in the North American Intergroup Phase III Trial of imatinib mesylate for treatment of advanced gastrointestinal stromal tumor: CALGB 150105 Study by Cancer and Leukemia Group B and Southwest Oncology Group. J Clin Oncol 2008 Nov 20;26(33):5360-7.

38.

Heinrich MC, Maki RG, Corless CL, Antonescu CR, Harlow A, Griffith D, Town A, Mckinley A, Ou WB, Fletcher JA, Fletcher CD, Huang X, Cohen DP, Baum CM, Demetri GD. Primary and secondary kinase genotypes correlate with the biological and clinical activity of sunitinib in imatinib-resistant gastrointestinal stromal tumor. J Clin Oncol 2008 Nov 20;26(33):5352-9.

39.

Biron P, et al. Outcome of patients (pts) with PDGFRA D842V mutant gastrointestinal stromal tumor (GIST) treated with imatinib (IM) for advanced disease. J Clin Oncol 2010;28:15s.

40.

Chen LL, Trent JC, Wu EF, Fuller GN, Ramdas L, Zhang W, Raymond AK, Prieto VG, Oyedeji CO, Hunt KK, Pollock RE, Feig BW, Hayes KJ, Choi H, Macapinlac HA, Hittelman W, Velasco MA, Patel S, Burgess MA, Benjamin RS, Frazier ML. A missense mutation in KIT kinase domain 1 correlates with imatinib resistance in gastrointestinal stromal tumors. Cancer Res 2004 Sep 1;64(17):5913-9.

41.

Debiec-Rychter M, van OA, Marynen P. Mechanisms of resistance to imatinib mesylate in gastrointestinal stromal tumors and activity of the PKC412 inhibitor against imatinib-resistant mutants. Gastroenterology 2005 Feb;128(2):270-9.

42.

Heinrich MC, Corless CL, Blanke CD, Demetri GD, Joensuu H, Roberts PJ, Eisenberg BL, von Mehren M, Fletcher CD, Sandau K, McDougall K, Ou WB, Chen CJ, Fletcher JA. Molecular correlates of imatinib resistance in gastrointestinal stromal tumors. J Clin Oncol 2006 Oct 10;24(29):4764-74.

43.

Wakai T, Kanda T, Hirota S, Ohashi A, Shirai Y, Hatakeyama K. Late resistance to imatinib therapy in a metastatic gastrointestinal stromal tumour is associated with a second KIT mutation. Br J Cancer 2004 Jun 1;90(11):2059-61.

14 44.

Wardelmann E, Thomas N, Merkelbach-Bruse S, Pauls K, Speidel N, Buttner R, Bihl H, Leutner CC, Heinicke T, Hohenberger P. Acquired resistance to imatinib in gastrointestinal stromal tumours caused by multiple KIT mutations. Lancet Oncol 2005 Apr;6(4):249-51.

45.

Liegl B, Kepten I, Le C, Zhu M, Demetri G, Heinrich M, Fletcher C, Corless C, Fletcher J. Heterogeneity of kinase inhibitor resistance mechanisms in GIST. J Pathol 2008 Sep;216(1):64-74.

46.

Demetri GD, Van Oosterom AT, Garrett CR, Blackstein ME, Shah MH, Verweij J, McArthur G, Judson IR, Heinrich MC, Morgan JA, Desai J, Fletcher CD, George S, Bello CL, Huang X, Baum CM, Casali PG. Efficacy and safety of sunitinib in patients with advanced gastrointestinal stromal tumour after failure of imatinib: a randomised controlled trial. Lancet 2006 Oct 14;368(9544):1329-38.

47.

Demetri GD, Reichardt P, Kang Y, et al. Efficacy and safety of regorafenib for advanced gastrointestinal stromal tumours after failure of imatinib and sunitinib (GRID): an international, multicentre, randomised, placebo-controlled, phase 3 trial. Lancet 2013 Jan 26;381(9863):295-302.