KIT-negative gastrointestinal stromal tumors: proo... - ResearchGate

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Medeiros et al Am J Surg Pathol • Volume 28, Number 7, July 2004 FIGURE 1. Epithelioid GIST (A) with negative KIT immunohistochemistry (B). Note the positive mast cells. myosarcoma, melanoma, schwannoma, and carcinoma. Notably, the cytogenetic profiles obtained in four tumors were classic for GIST. Each of the karyotypes was noncomplex and featured loss of material from the long arm of chromosome 14, which is the most frequent cytogenetic aberration in GIST. 10 TABLE 2. Cytogenetic Profiles of Four KIT-Negative GISTs Case No. Cytogenetic Profile 1 47,XY,add(1)(p11),add(1)(p13),+8,del(14)(q22),−19,r(19) (p13q13),add(21)(p11),+mar1 2 43–44,X,−X,i(8)(q10),add(11)(p15),−14,−15,−21,+1–2rings 3 45,XX,add(1)(p32),del(14)(q22p32),−22 5 46,XY,der(1)t(1;13)(p11;q11),der(3)t(1;3)(p32,q11), +i(8)(q10),−14 FIGURE 2. Expression of phosphorylated and total receptor tyrosine kinases in KIT-positive and KIT-negative GISTs. KITpositive GISTs, both with KIT exon 11 mutations, are in lanes 1 to 2. KIT-negative GISTs include two with PDGFRA mutations (lanes 3–4) and one with KIT exon 11 mutation (lane 5). Phosphorylated and total KIT are expressed strongly in the KITpositive/KIT-mutant control GISTs, whereas phosphorylated and total PDGFRA are expressed strongly in the KITnegative/PDGFRA-mutant GISTs (case nos. 1 and 23). KIT and PDGFRA are not demonstrably expressed in the KITnegative/KIT-mutant GIST (case no. 4). The PI3-K stain provides evidence for approximate equivalency in loading of intracellular proteins. By contrast, leiomyosarcomas, and particularly those of higher histologic grade, typically have extremely complex karyotypes. Most GISTs are characterized by oncogenic mutations of KIT and constitutive activation of the KIT receptor tyrosine kinase, which seems to drive tumor formation. However, a small subset of GISTs has been identified that lacks detectable KIT mutations. 17,20 Recently, activating mutations in the related receptor tyrosine kinase PDGFRA were reported in 35% to 67% of GISTs lacking KIT mutations. 9,11 We evaluated PDGFRA and KIT mutations in the 25 cases in our series and found that the majority exhibited mutations of the PDGFRA gene, most frequently involving exon 18. All pure epithelioid tumors in this series harbored PDGFRA mutations. However, we also identified four tumors with KIT mutations despite the 892 © 2004 Lippincott Williams & Wilkins
Am J Surg Pathol • Volume 28, Number 7, July 2004 KIT-Negative GISTs consistent negativity of these tumors for KIT by both immunohistochemistry and immunoblotting. No tumors had mutations involving KIT and PDGFRA concomitantly, corroborating previous evidence that these are mutually exclusive transforming equivalents in GIST oncogenesis. 9 Three tumors otherwise indistinguishable from the others in this study had no detectable KIT or PDGFRA mutations, suggesting that some KIT-negative GISTs arise through alternative oncogenic mechanisms. The biologic basis for the absence of KIT expression, in the subgroup of GISTs identified in our study, is unclear. Most of the tumors contained PDGFRA mutations, and as discussed above, such mutations appear to be alternate, or “either/or” transforming mechanisms, compared with the more common KIT oncogenic mutations in GISTs. In PDGFRA-mutant tumors, KIT expression may simply be superfluous and therefore down-regulated, as mutant PDGFRA appears to act as an oncogenic substitute for KIT in these cases. It would have been of interest, if feasible, to assess the PDGFRA-mutant cases immunohistochemically for evidence of PDGFRA protein expression. However, multiple trials using a variety of currently available antibodies (in both our Boston and Portland laboratories) have failed to identify any commercial antibody that yields meaningful and reproducible results with a clean background. Thus, we do not think that these presently available antibodies have clinical utility. More puzzling are the four tumors containing KIT gene mutations but in which KIT protein expression was not demonstrated. It is counterintuitive that the neoplastic GIST cells would select for a genomic KIT mutation in the absence of the functional protein product of that gene. One possibility is that these tumors originated through a KITactivated pathway and then later became independent as a result of additional secondary mutations. 6 Another possibility is that these discordant genomic and protein findings could be related to technical factors. For example, deletion of the KIT C-terminus, containing the epitope against which the DAKO KIT antibody was raised, could lead to false-negative immunostaining results. On the other hand, C-terminal truncation cannot account for the negative KIT immunohistochemistry in all KIT-negative GISTs, because we corroborated absence of KIT protein expression, using antibodies to a kinase domain epitope, by immunoblotting in three PDGFRA-mutant or KITmutant cases with available frozen tissue. Finally, it is conceivable that routine immunohistochemistry is insufficiently sensitive to detect the lowest biologically relevant levels of KIT expression in all tumors. We recently reported a case of a KITmutant GIST in which KIT expression was virtually absent, excluding the patient from a clinical trial. The tumor became PET negative and showed shrinkage on CT scan when the patient was treated with imatinib off protocol. 1 Based on this anecdotal example, it is possible that other KIT-mutant GISTs that lack KIT expression, such as the 4 cases in the present series, are imatinib sensitive. In summary, we report that a small subset of GISTs lack KIT expression but have otherwise typical clinical, histopathologic, and cytogenetic features. Most of these tumors have at least partly or totally epithelioid cytomorphology and harbor PDGFRA mutations, but some examples have KIT mutations despite the absence of demonstrable KIT protein expression. Given that most KIT-negative GISTs contain PDGFRA or KIT mutations, one might expect that patients with such tumors could potentially respond to therapeutic PDGFRA and KIT inhibition with imatinib. 8 Even acknowledging that the commonest PDGFRA D842V mutation is intrinsically imatinib resistant, 8,11 approximately 30% of the PDGFRA mutations identified are known to be potentially imatinib sensitive, 8 providing a greater hope of treatment response than with any other currently approved therapy. Therefore, it is important for pathologists and oncologists to be aware that, in the context of otherwise typical morphology, a GIST diagnosis should not be precluded on the basis of negative immunohistochemical staining for KIT and that such patients should not a priori be denied imatinib therapy. Where testing is available, the finding of KIT or PDGFRA mutations not only may help to confirm or enable the diagnosis of a KIT-negative GIST, but can also provide important prognostic information for patients in whom imatinib therapy is being considered. 8 ACKNOWLEDGMENTS The authors thank Troy Bainridge, Laura McGreevey, Andrea Haley, Ajia Town, Maureen Thyne, and Catherine Quigley for excellent technical assistance. REFERENCES 1. Bauer S, Corless CL, Heinrich MC, et al. Response to imatinib mesylate of a gastrointestinal stromal tumor with very low expression of KIT. Cancer Chemother Pharmacol. 2003;51:261–265. 2. Corless CL, McGreevey L, Haley A, et al. KIT mutations are common in incidental gastrointestinal stromal tumors one centimeter or less in size. Am J Pathol. 2002;160:1567–1572. 3. DeMatteo RP, Lewis JJ, Leung D, et al. Two hundred gastrointestinal stromal tumors: recurrence patterns and prognostic factors for survival. Ann Surg. 2000;231:51–58. 4. Demetri GD, von Mehren M, Blanke CD, et al. Efficacy and safety of imatinib mesylate in advanced gastrointestinal stromal tumors. N Engl J Med. 2002;347:472–480. 5. Fletcher CD, Berman JJ, Corless C, et al. Diagnosis of gastrointestinal stromal tumors: a consensus approach. Hum Pathol. 2002;33:459–465. 6. Fletcher JA, Corless C, Dimitrijevic S, et al. Mechanisms of resistance to imatinib mesylate (IM) in advanced gastrointestinal stromal tumor (GIST) [Abstract]. Proc Am Soc Clin Oncol. 2003;22:815. 7. Fletcher JA, Kozakewich HP, Hoffer FA, et al. Diagnostic relevance of clonal cytogenetic aberrations in malignant soft-tissue tumors. N Engl J Med. 1991;324:436–442. 8. Heinrich MC, Corless CL, Demetri GD, et al. Kinase mutations and imatinib response in patients with metastatic gastrointestinal stromal tumor. J Clin Oncol. 2003;21:4342–4349. 9. Heinrich MC, Corless CL, Duensing A, et al. PDGFRA activating mutations in gastrointestinal stromal tumors. Science. 2003;299:708–710. 10. Heinrich MC, Rubin BP, Longley BJ, et al. Biology and genetic aspects of gastrointestinal stromal tumors: KIT activation and cytogenetic alterations. Hum Pathol. 2002;33:484–495. 11. Hirota S, Ohashi A, Nishida T, et al. Gain-of-function mutations of plate- © 2004 Lippincott Williams & Wilkins 893
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