Molecular Analysis of c-Kit and PDGFRA in GISTs Diagnosed by EUS
Molecular Analysis of c-Kit and PDGFRA in GISTs Diagnosed by EUS
Gastrointestinal stromal tumors (GISTs) are characterized by overexpression and mutations of c-Kit. Approximately 80% of c-Kit mutations occur in exon 11, being a response factor to imatinib (Gleevec) therapy. Mutations of platelet-derived growth factor receptor-a (PDGFRA) are observed in a subset of GISTs lacking c-Kit mutations.
We aimed to assess whether c-Kit and PDGFRA mutation analysis of GISTs obtained by endoscopic ultrasound–guided fine-needle aspiration (EUS-FNA) could be routinely performed. Mutation analysis of c-Kit hotspot exons (9, 11, 13, and 17) and PDGFRA hotspot exons (12 and 18) was performed in aspirates of 33 GISTs and 18 non-GIST mesenchymal tumors.
Of the GIST cases, 19 (58%) of 33 contained a mutation in exon 11, 1 (3%) in exon 9, and none in exons 13 and 17. No activating c-Kit mutations were identified in non-GIST cases. No PDGFRA mutation was detected.
Mutation analysis is possible in these FNA cell blocks and can assist in the diagnosis and therapeutic decisions in GIST cases.
Gastrointestinal stromal tumors (GIST) are the most common mesenchymal tumors of the gastrointestinal tract, with an annual incidence of 10 to 20 cases per million. GISTs are rarely found outside the gastrointestinal tract, being most commonly found in the stomach (40%-70%), small intestine (20%-50%), colon and rectum (5%-15%), and esophagus (<2%). These tumors are thought to arise from interstitial cells of Cajal owing to their similar positive immunoreactivity for CD34 and CD117 (c-Kit), and to their lack of immunoreactivity for desmin and S-100 protein. Interstitial cells of Cajal are spindle-shaped cells located throughout the gastrointestinal tract. They are specialized cells of the enteric nervous system forming a network in the myenteric plexus layer, which primarily controls gastrointestinal tract motility. Before 1998, almost all GISTs were misdiagnosed as smooth muscle tumors such as leiomyomas, leiomyosarcomas, or leiomyoblastomas. In 1998, Hirota et al described the expression of c-Kit protein as an incontestable feature of GISTs, further separating them from other gastrointestinal mesenchymal tumors. At present, GIST is considered to be a specific category different from true smooth muscle tumors and neurogenic tumors.
c-Kit belongs to the class III receptor tyrosine kinases (RTKs), together with platelet-derived growth factor receptor-a (PDGFRA), colony-stimulating factor-1 receptor (CSF-1-R), vascular endothelial growth factor receptors 1 and 2 (Flt-1 and Flk-1, respectively), Flk-2, and Flt-4. The RTKs are characterized by the presence of an extracellular domain, a transmembrane domain, a juxtamembrane domain, and an intracellular domain where the 2 kinase domains are lodged. Activation of c-Kit occurs when by the binding of stem cell factor, the receptor homodimerizes and experiences conformational transformations that lead to the activation of the kinase domains. These, in turn, transphosphorylate the tyrosine residues of the opposing homodimerized receptor, allowing its association with substrates of various kinds. Like the majority of RTKs, c-Kit has been attributed many physiologic functions such as cell survival, proliferation, differentiation, adhesion, and apoptosis by signaling through the MAP kinase, PI3-kinase, and JAK/STAT pathways. c-Kit signaling is essential for normal erythropoiesis, lymphopoiesis, gametogenesis, and melanogenesis and for the correct development and function of mast cells. A dysfunctional activation of this RTK, therefore, has been involved in diverse neoplasias such as mastocytosis/mast cell leukemia, germ cell tumors, small cell lung carcinoma, acute myeloid leukemia, neuroblastoma, melanoma, ovarian carcinoma, and breast carcinoma, besides GISTs.
Mutations of the c-Kit oncogene are the major genetic alterations in GISTs. There is a broad spectrum of c-Kit mutations in GISTs, ranging from 30% to 90%, and c-Kit status can constitute a prognostic factor for survival. Most of the mutations are located in the juxtamembrane domain (exon 11), followed by the extracellular domain (exon 9), and seldom are in the kinase domains (exon 13 and 17). Recently, Heinrich et al showed that about 35% of GISTs lacking c-Kit mutations have intragenic activation mutations in PDGFRA, the most common, located in exons 12, 14, and 18. Mutations of c-Kit and PDGFRA seem to be mutually exclusive oncogenic events in GISTs.
Until recently, the prognosis of patients with GISTs was poor owing to its frequent recurrence and resistance to chemotherapy and radiotherapy regimens. The development and current treatment with specific RTK inhibitors is changing this scenario. Imatinib mesylate (Gleevec) is a selective inhibitor of RTKs; by competing with the adenosine triphosphate for its binding site, preventing further phosphorylation of signaling molecules downstream of the receptor, it is responsible for abnormal viability and proliferation signals in these cells. Several studies have linked different responses to the drug with c-Kit alterations. In particular, tumors harboring exon 11 c-Kit mutations are more likely to respond to imatinib therapy than those with exon 9 c-Kit mutations or no detectable mutation. On the other hand, some mutations, such as D816V, are linked to imatinib response. Currently, imatinib is used for the treatment of patients with c-Kit (CD117)-positive GIST with unresectable and/or metastatic malignant tumor. Besides imatinib, another RTK inhibitor, sunitinib (Sutent), has been recently approved for the treatment of patients with GIST whose disease has progressed or who are unable to tolerate treatment with imatinib.
It is a fact that obtaining an accurate diagnosis is of utmost importance for correct treatment; an earlier diagnosis is crucial for prompt therapy. Endoscopic ultrasound–guided fine-needle aspiration (EUS-FNA) biopsy has been increasingly used for the assessment of diverse intra-abdominal and intrathoracic tumors.
EUS-FNA biopsy not only allows for a meticulous representation of extramural and intramural structures of the gastrointestinal tract but also permits tissue sampling from masses in these locations. In the present study, we aimed to perform molecular analysis of c-Kit and PDGFRA genes in formalin-fixed, paraffin-embedded cell blocks obtained by EUS-FNA biopsy from GISTs and non-GISTs. The feasibility of such analysis would lead to a more precise diagnosis and therapeutic decision in routine management of patients with GISTs (particularly patients with recurrent or imatinib-resistant tumors).
Abstract and Introduction
Abstract
Gastrointestinal stromal tumors (GISTs) are characterized by overexpression and mutations of c-Kit. Approximately 80% of c-Kit mutations occur in exon 11, being a response factor to imatinib (Gleevec) therapy. Mutations of platelet-derived growth factor receptor-a (PDGFRA) are observed in a subset of GISTs lacking c-Kit mutations.
We aimed to assess whether c-Kit and PDGFRA mutation analysis of GISTs obtained by endoscopic ultrasound–guided fine-needle aspiration (EUS-FNA) could be routinely performed. Mutation analysis of c-Kit hotspot exons (9, 11, 13, and 17) and PDGFRA hotspot exons (12 and 18) was performed in aspirates of 33 GISTs and 18 non-GIST mesenchymal tumors.
Of the GIST cases, 19 (58%) of 33 contained a mutation in exon 11, 1 (3%) in exon 9, and none in exons 13 and 17. No activating c-Kit mutations were identified in non-GIST cases. No PDGFRA mutation was detected.
Mutation analysis is possible in these FNA cell blocks and can assist in the diagnosis and therapeutic decisions in GIST cases.
Introduction
Gastrointestinal stromal tumors (GIST) are the most common mesenchymal tumors of the gastrointestinal tract, with an annual incidence of 10 to 20 cases per million. GISTs are rarely found outside the gastrointestinal tract, being most commonly found in the stomach (40%-70%), small intestine (20%-50%), colon and rectum (5%-15%), and esophagus (<2%). These tumors are thought to arise from interstitial cells of Cajal owing to their similar positive immunoreactivity for CD34 and CD117 (c-Kit), and to their lack of immunoreactivity for desmin and S-100 protein. Interstitial cells of Cajal are spindle-shaped cells located throughout the gastrointestinal tract. They are specialized cells of the enteric nervous system forming a network in the myenteric plexus layer, which primarily controls gastrointestinal tract motility. Before 1998, almost all GISTs were misdiagnosed as smooth muscle tumors such as leiomyomas, leiomyosarcomas, or leiomyoblastomas. In 1998, Hirota et al described the expression of c-Kit protein as an incontestable feature of GISTs, further separating them from other gastrointestinal mesenchymal tumors. At present, GIST is considered to be a specific category different from true smooth muscle tumors and neurogenic tumors.
c-Kit belongs to the class III receptor tyrosine kinases (RTKs), together with platelet-derived growth factor receptor-a (PDGFRA), colony-stimulating factor-1 receptor (CSF-1-R), vascular endothelial growth factor receptors 1 and 2 (Flt-1 and Flk-1, respectively), Flk-2, and Flt-4. The RTKs are characterized by the presence of an extracellular domain, a transmembrane domain, a juxtamembrane domain, and an intracellular domain where the 2 kinase domains are lodged. Activation of c-Kit occurs when by the binding of stem cell factor, the receptor homodimerizes and experiences conformational transformations that lead to the activation of the kinase domains. These, in turn, transphosphorylate the tyrosine residues of the opposing homodimerized receptor, allowing its association with substrates of various kinds. Like the majority of RTKs, c-Kit has been attributed many physiologic functions such as cell survival, proliferation, differentiation, adhesion, and apoptosis by signaling through the MAP kinase, PI3-kinase, and JAK/STAT pathways. c-Kit signaling is essential for normal erythropoiesis, lymphopoiesis, gametogenesis, and melanogenesis and for the correct development and function of mast cells. A dysfunctional activation of this RTK, therefore, has been involved in diverse neoplasias such as mastocytosis/mast cell leukemia, germ cell tumors, small cell lung carcinoma, acute myeloid leukemia, neuroblastoma, melanoma, ovarian carcinoma, and breast carcinoma, besides GISTs.
Mutations of the c-Kit oncogene are the major genetic alterations in GISTs. There is a broad spectrum of c-Kit mutations in GISTs, ranging from 30% to 90%, and c-Kit status can constitute a prognostic factor for survival. Most of the mutations are located in the juxtamembrane domain (exon 11), followed by the extracellular domain (exon 9), and seldom are in the kinase domains (exon 13 and 17). Recently, Heinrich et al showed that about 35% of GISTs lacking c-Kit mutations have intragenic activation mutations in PDGFRA, the most common, located in exons 12, 14, and 18. Mutations of c-Kit and PDGFRA seem to be mutually exclusive oncogenic events in GISTs.
Until recently, the prognosis of patients with GISTs was poor owing to its frequent recurrence and resistance to chemotherapy and radiotherapy regimens. The development and current treatment with specific RTK inhibitors is changing this scenario. Imatinib mesylate (Gleevec) is a selective inhibitor of RTKs; by competing with the adenosine triphosphate for its binding site, preventing further phosphorylation of signaling molecules downstream of the receptor, it is responsible for abnormal viability and proliferation signals in these cells. Several studies have linked different responses to the drug with c-Kit alterations. In particular, tumors harboring exon 11 c-Kit mutations are more likely to respond to imatinib therapy than those with exon 9 c-Kit mutations or no detectable mutation. On the other hand, some mutations, such as D816V, are linked to imatinib response. Currently, imatinib is used for the treatment of patients with c-Kit (CD117)-positive GIST with unresectable and/or metastatic malignant tumor. Besides imatinib, another RTK inhibitor, sunitinib (Sutent), has been recently approved for the treatment of patients with GIST whose disease has progressed or who are unable to tolerate treatment with imatinib.
It is a fact that obtaining an accurate diagnosis is of utmost importance for correct treatment; an earlier diagnosis is crucial for prompt therapy. Endoscopic ultrasound–guided fine-needle aspiration (EUS-FNA) biopsy has been increasingly used for the assessment of diverse intra-abdominal and intrathoracic tumors.
EUS-FNA biopsy not only allows for a meticulous representation of extramural and intramural structures of the gastrointestinal tract but also permits tissue sampling from masses in these locations. In the present study, we aimed to perform molecular analysis of c-Kit and PDGFRA genes in formalin-fixed, paraffin-embedded cell blocks obtained by EUS-FNA biopsy from GISTs and non-GISTs. The feasibility of such analysis would lead to a more precise diagnosis and therapeutic decision in routine management of patients with GISTs (particularly patients with recurrent or imatinib-resistant tumors).