GIST under imatinib therapy

Seminars in Diagnostic Pathology (2006) 23, 84-90

GIST under imatinib therapy Raf Sciot, MD, PhD,a Maria Debiec-Rychter, MDb From the Department of aPathology, University Hospital, Catholic University of Leuven, Leuven, Belgium; and the b Department of Human Genetics, University Hospital, Catholic University of Leuven, Leuven, Belgium. KEYWORDS GIST; Imatinib; Phenotype; Genotype; Treatment effect

The prognosis of patients with a GIST improved significantly since the introduction of imatinib mesylate treatment, leading to disease control in 70% to 85% of patients. The response depends on the presence/ absence and type of mutations in the KIT or Platelet derived growth factor receptor. Unfortunately, we are increasingly faced with the problem of resistance to imatinib treatment, mainly secondary resistance, which by definition occurs after at least 6 months of initial response to the drug. The effects of imatinib on a GIST are still in full exploration and this review focuses upon the available data on the phenotype and genotype of a GIST treated with imatinib. Two settings are elaborated separately, a responding/stable GIST, and a resistant GIST. In addition, the attention will be drawn to remarkable (immuno)phenotypic changes that can occur in a GIST under imatinib treatment. © 2006 Elsevier Inc. All rights reserved.

In March 2000, a patient with GIST metastatic to the liver was granted compassionate use of imatinib mesylate (Glivec, Gleevec, Novartis).1 The remarkable clinical response of this patient to imatinib prompted several multiinstitutional phase I/II/III studies to further study the activ2-7 ity of imatinib in unresectable or metastatic GIST. Based on these studies, it is clear that imatinib reliably achieves disease control in 70% to 85% of patients and the median progression-free survival is in the range of 20 to 24 months. Many trials are now in course, which are considering the possibility of using the drug in an adjuvant or neoadjuvant setting.8 Most GISTs carry activating mutations in KIT that are associated with constitutive activation and receptor phosphorylation. Many patients with a GIST without KIT mutations have mutations in the platelet-derived growth factor receptor-alpha (PDGFRA).9 The molecular base of the success story of imatinib relates to the fact that it targets KIT, but also PDGFRs, BCR-ABL, and ARG, by competitively blocking ATP binding to the ATP pocket of the kinase domains. This binding prevents phosphorylation and Address reprint requests and correspondence: Raf Sciot, MD, PhD, Department of Pathology, University Hospital St. Rafaël, Minderbroedersstraat 12, B-3000 Leuven, Belgium. E-mail address: [email protected].

0740-2570/$ -see front matter © 2006 Elsevier Inc. All rights reserved. doi:10.1053/j.semdp.2006.08.005

activation of downstream signaling pathways.10 The likelihood of an objective clinical response to imatinib correlates with the presence or absence and type of mutations in KIT or PDGFRA.7,9 A general drawback with the imatinib mesylate therapy is the relative ease by which tumors can develop resistance to this drug when applied as a single agent. Primary resistance, defined as complete absence of response from the beginning of the treatment, is seen in about 10% to 15% of patients.3,9 Secondary resistance, which by definition occurs after at least 6 months of initial response to the drug, is increasingly observed. In this review, we will focus on the effect of imatinib on the phenotype and the genotype of a GIST. Two settings will be elaborated separately: a responding/stable GIST and a nonresponding GIST. In addition, the attention will be drawn to (immuno)phenotypic changes of GIST under imatinib treatment.

Responding/stable GIST These two situations are not considered separately because it is often very difficult to objectively define response in a GIST patient treated with imatinib. GISTs treated with

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GIST Under Imatinib Therapy

Figure 1 A 57-year-old male patient treated with imatinib for 24 months. Surgery was performed because of a duodeno-colonic fistulization. Resection specimen shows residual tissue with a myxofibrous appearance, and, in this instance a prominent calcified aspect. Arrow indicates the lumen of the colon.

imatinib can stay the same size when a patient is receiving incredible benefit. In fact, in some instances, the tumor can actually increase in size, eg, due to intratumoral hemorrhage. In addition, patients with “stable disease,” and those that had minor shrinkage that didn’t qualify as a response, had survival rates that were very similar to patients achieving a “partial response.”11 The information on the histopathological changes in patients successfully treated with imatinib is quite limited, but the overall changes in these patients are very similar. A variable, but often prominent loss of tumor cells is seen and this loss is replaced by a myxohyaline to fibrinoid stroma (Figures 1 and 2). In addition, stromal hemorrhage and variable amounts of macrophages and myofibroblasts in the context of scarring are present. Proliferation markers such as mitotic count and Mib1 labeling are usually decreased.12 The majority of patients with a clinical response or stable disease also show an obvious histologic response, albeit minimal in few patients.12,13 The degree of response does not correlate with the duration of imatinib therapy, with possible variable grade and heterogeneity of response patterns within different tumor areas or tumoral lesions of individual patients. A pathologic complete response is exceedingly rare. Investigators from the M.D. Anderson Cancer Center reported 2 patients with a pathologic complete response in a series of 17 patients (12%) in lesions surgically resected after treatment with imatinib.14 In another analysis of patients who underwent surgery following response to imatinib therapy, Bauer and coworkers showed that 1 of 12 patients (8%) showed a complete response.12 Chacon and coworkers and Melichar and coworkers each reported complete response in 1 patient with metastatic disease.15,16 Finally, Högenauer and coworkers described a GIST with complete remission after 5 months of therapy, albeit based on negative true cut biopsies only.17 It is clear that the pathological assessment of imatinib pretreated GIST represents a challenge, especially in large tumors where numerous sections need to be screened to detect viable tumor cells. In addition, KIT

85 expression may be lost under treatment with imatinib, which obviously may lead to a false-negative pathologic complete response. In concordance with the histopathological analyses, data from most clinical studies show that many patients responding to imatinib eventually progress, indicating that the effect of the drug on residual GIST cells is cytostatic rather than cytotoxic.18 In our experience, GISTs under imatinib treatment rarely show frank tumor cell necrosis, but rather areas of myxohyaline stroma.19 This finding suggests that inhibition of KIT mainly induces apoptotic pathways. In this respect, Duensing and coworkers reported that KIT inhibition in vitro strongly affects the AKT/MTOR survival pathway, resulting in decreased proliferation and increased apoptosis.20 Imatinib-induced apoptosis of tumor cells also occurs in vivo, but, interestingly, seems to be accompanied by apoptosis of endothelial cells.21 The finding of a decreased blood flow and blood vessel density in GISTs of patients treated with imatinib further underscores the antivascular effect. The authors also describe a decreased phopsphorylation of KIT and thus inhibition of activation of KIT in the tumor-associated endothelium. It is of note that the antitumoral and anti-vascular effects occur within 1 week of treatment.21 Thus, imatinib probably not only targets tumor cells, but also tumor-related vessels. This effect is most likely mediated through the activation of PDGFRs in vascular pericytes of tumor vessels.22-24 This topic will undoubtedly be a major scope of research in the field of efficacy of various tyrosine kinase inhibitors. In this respect, a recent observation on sunitinib (Sutent, Pfizer), a novel FDA-approved drug for the treatment of imatinib-resistant GIST patients, is of particular note. Like imatinib mesylate, sunitinb inhibits the kinase activity of KIT and PDGFRs, exerting in addition activity against other targets, most notably the vascular endothelial growth factor receptor (VEGFR) and the FLT3 receptor tyrosine kinases. Seandel and coworkers describe a dramatic clinical response to sunitinb in an imatinib-resistant GIST patient, with elimination of the majority of the tumor cells; however, feeding blood vessels were still present after 12 months of therapy.25 Thus, the main target of sunitinib seems to be the tumor cells and not the vessels. Mounting evidence accumulates that different tumor genotypes correlate with a varying response rate to imatinib and that some GISTs are thereby inherently resistant to the treatment. Those GISTs with juxtamembrane region KIT mutations (exon 11) generally respond well to imatinib, whereas GISTs without demonstrable KIT mutations typically do not respond. The GISTs with KIT mutations in the extracellular region (exon 9) have an intermediate response rate to imatinib with approximately 25% to 50% of patients showing a major response.7,9 The varying responses of patients with different KIT mutations contrast with the uniform in vitro sensitivity of these mutant receptors to the drug. Therefore, the dependence of GISTs on KIT signaling

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Figure 2 A 73-year-old female patient was diagnosed with a gastric “leiomyosarcoma” in 1997. After surgery, she received chemotherapy and developed multiple abdominal and liver metastases in 2001. Revision of the primary tumor diagnosis was a CD117 positive spindle cell GIST (whole mount view in a, detail in c). She received 2 ⫻ 500 mg imatinib and improved spectacularly. After 6 weeks of treatment, she was operated on for partial necrosis of a bowel segment. The removed residual tumoral masses showed an impressive depletion of tumor cells, and prominent myxohyaline to fibrinoid changes (whole mount of a colon wall location in b, details and CD117 immunostaining in d and e).

for survival and proliferation is probably incomplete and complex. The still limited clinical data about the sufficiency of imatinib in patients with tumors expressing different PDGFRA isoforms indicate a heterogeneous response, with PDGFRA exon 12 and 14 mutants responding the best. Importantly, however, most isoforms with a substitution involving codon 842 in PDGFRA exon 18, including the

most frequent D842V, are primarily resistant to imatinib.7,9,19 Not much is known about secondary KIT or PDGFRA mutations in responding/stable lesions. The preliminary studies on a very limited number of tumors indicate lack of secondary KIT mutations in surgical biopsies of patients who underwent debulking surgery during the responding or stable phase of the disease.1,34 Moreover, no

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GIST Under Imatinib Therapy

copy number abnormalities of KIT were detected in responsive/stable tumors.1 In line with this report, we have found only base-line original KIT mutations and no evidence of KIT amplification in six tumors that were resected in the stable phase of therapy (unpublished observation).

Resistant GIST Patients with primary resistance usually lack any histological evidence of response to imatinib. This resistance is generally multifocal and is mainly seen in PDGFRA exon 18 mutants and in the majority of wild-type GISTs. Oncologists are increasingly faced with secondary resistance. Acquired, secondary resistance to imatinib mesylate develops particularly in more advanced tumors and in patients who are followed over longer time periods, especially since imatinib is unable to eradicate all KIT-positive cells in most patients. The clinical pattern of disease progression varies among patients, reflecting the clonal evolution of resistant GISTs. Some patients experience generalized disease progression, similar to that seen with primary drug resistance. The progression in others is more limited in nature, implying only one or more tumor nodule(s), although other tumoral lesions may still respond. The acquired resistance is commonly mirrored by the lack of histological response, but tumor cells may lose their KIT expression. Several mechanisms explain imatinib resistance in GISTs patients.26,28 The principle mechanism of resistance

Table 1

87 involves the selection of cancer cells with secondary mutations in the targeted kinase, KIT or PDGFRA. Less common is overexpression of KIT due to kinase amplification or switching of GIST dependence from KIT to a different, still unidentified alternative kinase. The imatinib-resistant, secondary mutations which are described until now are listed in Table 1. They always involve the kinase domains of the receptor coded by exons 13, 14, and 17 of KIT,13,27,29-34 or exon 18 of PDGFRA,19,26 directly preventing or weakening the interaction with the inhibitor. Importantly, multiple secondary mutations may be found in evolving tumor sub27,29 clones of different tumor residues in the same patient. The vast majority of secondary mutations are missense substitutions, while in-frame size-altering mutations are only sporadically detected (M.D-R., unpublished data).16 The most common recurrent mutations are V654A and T670I substitutions. The T670I mutation in KIT is homologous to the T790I mutation in EGFR and to the T315I mutation in BCR-ABL, and all three mutations confer resistance to clinical-stage ATP-competitive kinase inhibitors. As shown in Table 1, secondary kinase mutations are more frequently reported in GISTs with primary KIT exon 11 mutations (60-65%) than exon 9 mutations (16-35%).35 Since the former are on average longer on imatinib treatment before progression than the latter, the probability of a secondary mutation increases with the duration of imatinib treatment. In our experience, in a series of 94 imatinibresistant GIST specimens, 25% and 65% revealed a primary KIT mutation in exons 9 and 11, respectively. Only 11% of the former versus 66% of the latter displayed a secondary KIT mutation, with a majority (55%) located in exon 17 of

Type and frequency of the secondary KIT mutations in imatinib-resistant GISTs according to published data Secondary KIT mutation

References 30

26

Chen et al., 2004; Debiec-Rychter et al., 2005; Tamborini et al.,33 2006; Antonescu et al.,13 2005; Wardelmann et al.,27 2005 Tamborini et al.,32 2004; Debiec-Rychter et al.,26 2005; Wardelmann et al.,29 2006 Wardelmann et al.,29 2006 Wardelmann et al.,29 2006 Debiec-Rychter et al.,26 2005 Debiec-Rychter et al.,26 2005 Wardelmann et al.,29 2006 Grimpen et al.,31 2005 Grimpen et al.,31 2005 Wardelmann et al.,29 2006 Debiec-Rychter et al.,26 2005 Debiec-Rychter et al.,26 2005; Antonescu et al.,13 2005; Wardelmann et al.,29 2006 Debiec-Rychter et al.,26 2005; Antonescu et al.,13 2005; Wardelmannn et al.,29 2006 Wakai et al.,34 2004; Antonescu et al.,13 2005; Wardelmann et al.,29 2006

Primary KIT mutation In exon 9 (%)

In exon 11 (%)

Total (%)

V654A

2

16

18

T670I

1

8

9

1 1 1 1 1 1 1 2

1 1 1 1 1 1 1 2 1 3

3

3

T670E S709F D716N D816G D816E D816H Del815R_816D D820E D820G D820Y

1 1 1 1

N822K Y823D Not detected Total

14 (66.7) 21 (25.3)

4 22 (35.4) 62 (74.7)

4 36 (43.4) 83 (100)

88 the gene, including hitherto not reported KIT del820D and N822Y isoforms (unpublished observations). Two of the reported secondary mutations, D820Y and N822K, were described previously as the primary mutations in imatinibnaïve GISTs,5,9 being likely activating mutations in KIT that also directly confer resistance to imatinib. Remarkably, in two of the resistant samples, we have observed homozygosity of the primary KIT mutation associated with the heterozygosity of resistant V654A or D816G alleles, indicating the reduplication of the mutant KIT allele before progression. Interestingly, we were not able to detect any imatinib-resistant mutations in three pediatric GISTs that progressed during treatment after initial response to the drug, suggesting different mechanisms of resistance in this group of patients. This observation requires confirmation on a larger group of tumors. Notably, the resistance of the PDGFRA D842V isoform to imatinib (a relative frequent cause of primary resistance) may add also to the acquired resistance, being reported as a secondary mutation in tumors originally harboring KIT exon 11 and PDGFRA exon 12 isoforms.19,26

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(Immuno)phenotypic change under imatinib treatment In the majority of GISTs, no dramatic change in the cellular phenotype is seen under imatinib treatment. Nevertheless, we found striking histopathological changes in 4 of 51 patients of which tissue was available before and after prolonged (⬎1 year) treatment with imatinib. Three of these patients were reported before.36 Briefly, the first case showed a predominant spindle cell phenotype in the original tumor. However, all rapidly progressive lesions, which had developed under imatinib treatment, lost their spindle cell character, being replaced by an epithelioid type of proliferation that resembled more a carcinomatous, melanoma-like, or a histiocytic proliferation. In the second case, a resected metastatic liver lesion, which was clinically stable under imatinib treatment, showed a tubulopapillary growth pattern, quite different from the primary duodenal tumor that was a classical spindle cell GIST. From a histological point of view, a carcinoma or even mesothelioma could be considered. In case three, a progressive liver metastasis showed

Figure 3 Phenotypic change of GIST. Before treatment, the tumor corresponded to a spindle cell CD117 immunopositive GIST (a, b). Under imatinib treatment, the progressive tumor became epithelioid, pleomorphic, and mitotically active (c).

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GIST Under Imatinib Therapy

an epithelioid cell morphology, with a strong eosinophilic staining of the cytoplasm. Again, the morphologic picture of this lesion differed substantially from the primary spindle cell GIST. In all three patients, the tumor biopsies taken during sustained imatinib treatment revealed complete loss of KIT immunoreactivity, which in two cases was also accompanied by the loss of CD34 reactivity. Moreover, in the progressive lesion of patient 3, a remarkable desmin immunopositivity in nearly all tumor cells was encountered, although the original GIST was completely negative for desmin. Interestingly, KIT mutational analysis revealed the presence of distinct exon 11 mutant isoforms in all three cases, while the same genotype was sustained in the base line and on-therapy tumor specimens, proving the common origin of analyzed specimens. The fourth, yet unreported case concerned a 47-year-old male patient with a GIST of the colon and hepatic metastases. The primary tumor was a classical spindle cell GIST with strong KIT expression (Figure 3a and b). He received 800 mg imatinib a day with a partial response, but after 164 weeks of treatment, the metastatic lesions became progressive. Histology showed a mitotically very active epithelioid neoplasm with loss of KIT expression (Figure 3c). Genotyping of the resistant lesion indicated only the presence of the primary KIT p.del552M_554E mutation, which had also been originally detected in the pretreatment tumor biopsy of this patient. Thus, GISTs under imatinib treatment might undergo remarkable (immuno)phenotypic changes, which are not necessary corroborated by new genotypic changes. This situation may be observed in a subset of either responsive or progressive tumors that, due to therapy, lost their dependence from KIT, most likely throughout cessation of KIT signaling. Because of the mimic with other tumor types, this feature creates a differential diagnostic challenge, of which the pathologist should be aware.

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89 7. Debiec-Rychter M, Sciot R, Le Cesne A, et al: KIT mutations and dose selection for imatinib in patients with advanced gastrointestinal stromal tumours. Eur J Cancer 42:1093-1103, 2006 8. Blay JY, Bonvalot S, Casali P, et al: Consensus meeting for the managament of gastrointestinal stromal tumors. Report of the GIST consensus conference of 20-21 March 2004, under the auspices of ESMO. Ann Oncol 16:566-578, 2005 9. Heinrich MC, Corless CL, Demetri GD, et al: Kinase mutations and imatinib response in patients with metastatic gastrointestinal stromal tumor. J Clin Oncol 21:4342-4349, 2003 10. Tornillo L, Terracciano LM: An update on molecular genetics of gastrointestinal stromal tumours. J Clin Pathol 59:557-563, 2006 11. Blanke CD, Demitri GD, Von Mehren MC, et al: Long-term follow-up of a phase II randomized trial in advanced gastrointestinal stromal tumor (GIST) patients treated with imatinib mesylate. J Clin Oncol 24:9528, 2006 12. Bauer S, Hartmann JT, de Wit M, et al: Resection of residual disease in patients with metastatic gastrointestinal stromal tumors responding to treatment with imatinib. Int J Cancer 117:316-325, 2005 13. Antonescu CR, Besmer P, Guo T, et al: Acquired resistance to imatinib in gastrointestinal stromal tumor occurs through secondary gene mutation. Clin Cancer Res 11:4182-4190, 2005 14. Scaife CL, Hunt KK, Patel SR, et al: Is there a role for surgery in patients with “unresectable” cKit⫹ gastrointestinal stromal tumors treated with imatinib mesylate? Am J Surg 186:665-669, 2003 15. Chacon M, Roca E, Huertas E, et al: CASE 3. Pathologic complete remission of metastatic gastrointestinal stromal tumor after imatinib mesylate. J Clin Oncol 23:1580-1582, 2005 16. Melichar B, Voboril Z, Nozicka J, et al: Pathological complete response in advanced gastrointestinal stromal tumor after imatinib therapy. Intern Med 44:1163-1168, 2005 17. Hogenauer C, Langner C, Lipp RW, et al: Complete remission of a metastatic gastrointestinal stromal tumour with the tyrosine kinase inhibitor imatinib (STI571): effect of low dosage in an advanced tumour with exon 11 mutation. Eur J Gastroenterol Hepatol 15:323327, 2003 18. Bauer S, Lang H, Schutte J, et al: Complete remission with imatinib in metastatic gastrointestinal stromal tumors. J Clin Oncol 23:6800-6801, 2005 19. Corless CL, Schroeder A, Griffith D, et al: PDGFRA mutations in gastrointestinal stromal tumors: frequency, spectrum and in vitro sensitivity to imatinib. J Clin Oncol 23:1-8, 2005 20. Duensing A, Medeiros F, McConarty B, et al: Mechanisms of oncogenic KIT signal transduction in primary gastrointestinal stromal tumors (GISTs). Oncogene 23:3999-4006, 2004 21. Trent JC, Choi K, Hunt H, et al: Apoptotic and anti-vascular activity of imatinib in GIST patients. J Clin Oncol 23:9001, 2005 22. Ostman A: PDGF receptors-mediators of autocrine tumor growth and regulators of tumor vasculature and stroma. Cytokine Growth Factor Rev 15:275-286, 2004 23. Bagley RG, Weber W, Rouleau C, et al: Pericytes and endothelial precursor cells: cellular interactions and contributions to malignanacy. Cancer Res 65:9741-9750, 2005 24. Song S, Ewald AJ, Stallcup W, et al: PDGFR␤⫹ perivascular progenitor cells in tumours regulate pericyte differentiation and vascular survival. Nat Cell Biol 7:870-879, 2005 25. Seandal M, Shia J, Linkov I, et al: The activity of sunitinib (SU11248) against gastrointestinal stromal tumor (GIST) appears to be distinct from its anti-angiogenic effects. Clin Cancer Res 2006, in press 26. Debiec-Rychter M, Cools J, Dumez H, et al: Mechanisms of resistance to imatinib mesylate in gastrointestinal stromal tumors and activity of the PKC412 inhibitor against imatinib-resistant mutants. Gastroenterology 128:270-279, 2005 27. Wardelmann E, Thomas N, Merkelbach-Bruse S, et al: Acquired resistance to imatinib in gastrointestinal stromal tumours caused by multiple KIT mutations. Lancet Oncol 6:249-51, 2005

90 28. Fletcher JA, Corless CL, Dimitrijevic S, et al: Mechanisms of resistance to imatinib mesylate (IM) in advanced gastrointestinal stromal tumors (GIST). Proc Am Soc Clin Oncol 22:815, 2003 29. Wardelmann E, Merkelbach-Bruse S, Pauls K, et al: Polyclonal evolution of multiple secondary KIT mutations in gastrointestinal stromal tumors under treatment with imatinib mesylate. Clin Cancer Res 12:1743-1749, 2006 30. Chen LL, Trent JC, Wu EF, et al: A missense mutation in KIT kinase domain 1 correlates with imatinib resistance in gastrointestinal stromal tumors. Cancer Res 64:5913-5919, 2004 31. Grimpen F, Yip D, McArthur G, et al: Resistance to imatinib, lowgrade FDG-avidity on PET, and acquired KIT exon 17 mutation in gastrointestinal stromal tumour. Lancet Oncol 6:724-727, 2005 32. Tamborini E, Bonadiman L, Greco A, et al: A new mutation in the KIT ATP pocket causes acquiered resistance to imatinib in a

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