Detection of c-kit-activating mutations in gastrointestinal stromal tumors by high-resolution amplicon melting analysis

High-resolution amplicon melting analysis was used to scan for c-kit-activating mutations in exons 9, 11, 13, and 17 in 29 neoplasms diagnosed as gastrointestinal stromal tumors (GISTs). Immunohistochemically, 7 of 29 did not show strong CD 17 positivity and might represent true smooth muscle tumors or c-kit-negative GISTs. No c-kit-activating mutations were detected in the 7 CD117- cases by high-resolution amplicon melting analysis or direct DNA sequencing. Alterations in the remaining 22 CD117+ cases included 13 (59%) in exon 11, 2 (9%) in exon 9, 1 (5%) in exon 13, and none in exon 17. The genetic alterations consisted of point mutations and in-frame insertions, duplications, and deletions. In exon 11, 7 (54%) of 13 alterations have not been described previously. In 2 cases, the identical exon 11 mutation was observed in the primary tumor and a metastatic/recurrent lesion. In all cases, direct DNA sequencing confirmed that polymerase chain reaction products with an abnormal melting curve contained a mutation and products with a normal melting curve, a normal DNA sequence. High-resolution melting analysis can be used to scan DNA for potential c-kit-activating mutations and can aid in the diagnosis of GISTs.     

A c-kit-negative gastrointestinal stromal tumor with a platelet-derived growth factor receptor alpha mutation.

Gastrointestinal stromal tumors (GISTs) are well-recognized mesenchymal neoplasms of the intestinal tract. A diagnosis of GIST is not always possible using IHC techniques for detection of c-kit. The authors describe a 64-year-old man who presented with an upper abdominal quadrant mass. Histology showed a predominantly epithelioid neoplasm with focal "spindle cell" areas. IHC studies were positive for muscle markers and negative for c-kit. The morphologic and immunophenotypic appearance could be compatible with either a smooth muscle tumor or a GIST. Because of the differences in treatment protocols and prognosis between these two entities, molecular studies to detect c-kit or platelet-derived growth factor receptor (PDGFR) activating mutations were performed. No mutations were found in the c-kit gene, but a mutation was detected in the PDGFR gene. This additional molecular study allowed the authors to formulate the precise diagnosis of a c-kit-negative GIST with strong smooth muscle marker expression.     

Immunohistochemical profile and c-kit mutations in gastrointestinal stromal tumors

Gastrointestinal stromal tumors (GISTs) are low-grade sarcomas arising from the interstitial cells of Cajal, harboring mutation of c-kit. We investigated the morphological, immunohistochemical, and molecular profile of 55 GISTs to establish the prevalence of mutations, their clinical significance, and diagnostic utility. c-kit mutations were investigated by evaluating the entire coding sequence of the gene with non-radioisotopic PCR-SSCP, and characterized with fluorescent cycle sequencing. Mutations were detected in 39 tumors (71%), the majority (67%) involving exon 11. Two tumors showed exon 9 mutations (one tumor located in the small intestine and one in the stomach), whereas two cases showed a polymorphism at the splicing site of exon/intron 1 present in healthy blood donors with a 3% frequency. CD117 was expressed in 53 tumors (96%); CD34 was positive in 42 cases (76%); 42 cases (76%) expressed both CD117 and CD34. c-kit mutations were similarly distributed in stromal tumors at low risk of aggressive behavior (78%), intermediate risk (66%), and high risk (71%). Fifteen tumors expressing CD117 showed wild-type kit gene, and on histological grounds, they were equally distributed among epithelioid and spindle cell morphology. One case neither expressed CD117 nor did it show c-kit mutation. Data suggest that both immunohistochemical and molecular evaluation may be useful in tumors likely to be classified as GISTs; molecular analysis appears valuable to support the diagnosis and to identify cases that can benefit from recent novel therapeutic tools.     

Expression of platelet-derived growth factor receptor in low-grade endometrial stromal sarcomas in the absence of activating mutations

AIMS: To investigate platelet-derived growth factor receptor (PDGFR)alpha and PDGFRbeta expression and a mutational analysis of PDGFRalpha (exons 11, 12, 17 and 18) and PDGFRbeta (exon 12) genes in endometrial stromal sarcomas (ESS). Gastrointestinal stromal tumours (GISTs), which have somatic mutations of the transmembrane tyrosine kinase receptor, respond to tyrosine kinase inhibitors, which act through an inhibitory effect on class 3 receptor tyrosine kinase members such as PDGFRalpha, PDGFRbeta and c-kit. METHODS AND RESULTS: The immunohistochemical expression of PDGFRalpha and PDGFRbeta was investigated in 37 archival c-kit- ESS. Staining was scored as negative (0-10% positive tumour cells) and positive (weakly positive 11-50% positive cells; strongly positive > 50% positive cells). PDGFRalpha was expressed in 24/37 ESS [65%; strongly by 19/37 (51.5%) and weakly by 5/37 ESS (13.5%)]. ESS tumour cells were negative for PDGFRbeta, but endothelial cells stained positive. A mutational analysis of PDGFRalpha (exons 11, 12, 17 and 18) and PDGFRbeta (exon 12) genes on frozen metastatic ESS from three patients detected no mutations leading to amino acid changes in the mature protein. CONCLUSIONS: Patients with PDGFRalpha+ ESS may benefit from treatment with tyrosine kinase inhibitors by blocking autocrine and paracrine stimulation loops, blocking neovascularization and enhancing the effects of chemotherapy.     

CD117阴性的胃肠道间质瘤的超微结构特点及基因突变检测

目的探讨CD117阴性的胃肠道间质瘤（GIST）的超微结构及c-kit和血小板源性生长因子受体A（PDGFRA）基因的突变情况。方法用免疫组织化学方法（EnVision法和SP法）从101例GIST中筛选到6例CD117阴性的GIST,观察了6例CD117阴性的胃肠道间质瘤的电镜变化,用PCR直接测序的方法检测6例CD117阴性的胃肠道间质瘤的c-kit基因外显子9、11、13、17和PDGFRA基因外显子12和18突变。结果电镜下观察到6例GIST的超微结构特点与卡哈尔细胞相似,通过PCR直接测序检测揭示c-kit基因9、11、13和17外显子均无突变,而3例GIST的PDGFRA有突变,其中2例D842V突变,1例R841S突变。结论PDGFRA基因的突变可能是CD117阴性的胃肠道间质瘤发生的重要分子基础。     

A new case of Carney triad: gastrointestinal stromal tumours and leiomyoma of the oesophagus do not show activating mutations of KIT and platelet-derived growth factor receptor alpha

The Carney triad is a rare syndrome of unknown aetiology, with synchronous or metachronous appearance of rare neoplasms: gastrointestinal stromal tumours (GISTs), pulmonary chondromas and extra-adrenal paragangliomas. In most cases, the Carney triad is incomplete. The combination encountered typically, GISTs and pulmonary chondromas, was also seen in our patient, a 22-year-old woman. She was diagnosed with the triad after Billroth II gastrectomy for histologically proved gastric GISTs. The diagnosis of pulmonary chondromas was confirmed by transthoracic, computed tomography-guided needle biopsy. An oesophageal leiomyoma was resected 2 years after the initial diagnosis, on suspicion of paraganglioma. The clinical course of the patient has been uneventful since. The last follow-up was carried out 6 years after the initial diagnosis. On histological examination, the cells of gastric GIST were partly positive for CD34, whereas CD117 was expressed in all areas in variable intensity and S-100 protein was negative. The oesophageal tumour was classified as leiomyoma due to strong immunopositivity for smooth muscle actin and desmin, being negative for CD34 and CD117. Two different gastric GIST lesions as well as the oesophageal leiomyoma and normal tissue were analysed for activating mutations in common hot spots of KIT (exon 9 and 11) and platelet-derived growth factor receptor alpha (exon 18), but in all probes wild-type sequences were found. These results are in accordance with the first published analyses of GIST lesions from Carney patients.     

Myxoid epithelioid gastrointestinal stromal tumor (GIST) with mast cell infiltrations: a subtype of GIST with mutations of platelet-derived growth factor receptor alpha gene

We analyzed 30 gastrointestinal stromal tumors (GISTs) that were immunohistochemically weak or negative for KIT. Histologically, all 30 GISTs consisted of epithelioid tumor cells in at least a part of the tumor. The tumor cells showed different morphologies and arranged themselves in different histological patterns. In 20 of the 30 GISTs, round or oval epithelioid tumor cells often showed a less cohesive pattern of growth and showed eosinophilic cytoplasm and peripherally placed nuclei with myxoid stroma, whereas in the remaining 10 cases, tumor cells were arranged in a more cohesive pattern without myxoid stroma. The former type of tumors is called myxoid epithelioid GISTs in this study. Subsequent mutational analyses showed that the platelet-derived growth factor receptor alpha (PDGFRA) gene mutations in exon 12 or exon 18 were identified in 20 (66.7%) of the 30 GISTs, and especially in 18 (90%) of the 20 myxoid epithelioid GISTs. Moreover, 17 (85%) of the 20 myxoid epithelioid GISTs were accompanied by mast cell infiltrations within the tumor nodules. In the remaining cases, 2 (6.7%) of the 30 GISTs had c-kit gene mutations in exon 11, and no mutation was found in 8 (26.7%) of 30 GISTs. None of the patients with myxoid epithelioid GISTs died of disease. These results suggest that myxoid epithelioid GISTs are a distinct subtype of GISTs that are closely correlated with the PDGFRA gene mutation and that recognition of such histological characteristics should be helpful for molecular subclassification of GISTs that are important for molecular targeting therapy by imatinib mesylate (STI571).     

Detection of epidermal growth factor receptor and human epidermal growth factor receptor 2 activating mutations in lung adenocarcinoma by high-resolution melting amplicon analysis: correlation with gene copy number, protein expression, and hormone receptor expression

Activating mutations in the epidermal growth factor receptor (EGFR) (7p12.3-p12.1) and in the human epidermal growth factor receptor 2 (HER2) (17q21.1) characterize a subset of lung carcinomas. These mutations may relate to the response of the tumor to the tyrosine kinase inhibitors gefitinib and erlotinib. High-resolution melting amplicon analysis is a screening technique that has been shown to be able to detect missense mutations as well as deletions and insertions in tumor DNA isolated from paraffin-embedded tissue sections. In this study, we used high-resolution melting amplicon analysis to screen for EGFR and human HER2 activating mutations in 39 patients with primary lung adenocarcinoma. There were 20 cases that showed bronchioloalveolar histology and 19 cases that did not. The EGFR exons screened were exons 18, 19, 20, and 21, and the HER2 exons screened were exons 19 and 20. Six (15%) of the 39 patients had tumors that contained EGFR activating mutations. Four of the mutations were in adenocarcinomas, which had some bronchioloalveolar features, and 2 mutations were in tumors without bronchioloalveolar features. The EGFR mutations were in exon 19 (2 cases), exon 20 (2 cases), and exon 21 (1 case). One case contained mutations in both exons 18 and 20. One (2.6%) of the 39 patients had a tumor that contained an HER2 activating mutation, and the mutation was located in exon 20. Two of the 6 EGFR mutation-positive cases showed polysomy for chromosome 7, and each one showed overexpression of EGFR as determined by immunohistochemical staining. The other EGFR mutation-positive cases did not show EGFR overexpression and appeared disomic for chromosome 7. The HER2 mutation-positive case was in an adenocarcinoma with bronchioloalveolar features. This tumor did not show overexpression of HER2 and was disomic for chromosome 17. For the non-EGFR mutation-positive cases, 4 (13%) of 32 evaluated cases showed polysomy for chromosome 7 and EGFR. No case showed EGFR gene amplification. Polysomy for chromosome 7 was not related to EGFR overexpression as estimated by immunohistochemistry. Estrogen and progesterone receptor expression was not strong in any of the cases and did not correlate with the presence of EGFR or HER2 mutations.     

A gastrointestinal stromal tumor of the stomach morphologically resembling a neurofibroma: demonstration of a novel platelet-derived growth factor receptor alpha exon 18 mutation

Gastrointestinal stromal tumors are increasingly being recognized because of their characteristic expression of KIT (CD 117). Most KIT-positive gastrointestinal stromal tumors have activating mutations in the c-kit gene. A subgroup of gastrointestinal stromal tumors are negative for KIT expression, and in these tumors, activating mutations in platelet-derived growth factor receptor α are common. Most platelet-derived growth factor receptor α mutation–positive gastrointestinal stromal tumors show an epithelioid histology and are located in the stomach. Herein, we describe an unusual gastric stromal tumor. The tumor was negative for KIT expression and the morphology did not show an epithelioid pattern but rather was composed of bland spindle cells reminiscent of a neurofibroma. Molecular analysis revealed a somatic mutation in platelet-derived growth factor receptor α exon 18 (D842F). Aside from demonstrating a new platelet-derived growth factor receptor α mutation, this case illustrates the usefulness of molecular testing as a diagnostic tool and clearly indicates the wide range of morphology that can be observed in gastrointestinal stromal tumors.     

c-kit蛋白和血小板源生长因子受体α在卵巢上皮性肿瘤中的表达

目的探讨c-kit蛋白、血小板源生长因子受体α(PDGFRα)在卵巢上皮性肿瘤中的表达及其与卵巢癌临床病理的关系.方法采用免疫组织化学法(SP法)检测c-kit蛋白、PDGFRα,分析c-kit蛋白、PDGFRα在卵巢上皮性肿瘤中的表达与肿瘤分型、分化程度及国际妇产科联盟(FIGO)分期的关系.结果(1)c-kit蛋白在卵巢上皮性癌中表达的阳性率为50.70%,明显高于正常卵巢组织(10.00%)及良性卵巢肿瘤(20.00%)(P均<0.01).PDGFRα在卵巢上皮性癌中表达的阳性率为63.38%,明显高于正常卵巢组织(15.00%)及良性卵巢肿瘤(25.00%)(P均<0.01).(2)c-kit蛋白在浆液性卵巢癌中表达的阳性率高于黏液性卵巢癌(57.63%比16.67%,P<0.05),而PDGFRα在浆液性及黏液性卵巢癌中表达阳性率筹异无统计学意义(P>0.05).低分化卵巢癌组c-kit蛋白及PDGFRa表达的阳性率明显高于高中分化卵巢癌组(69.23%比40.00%,80.77%比53.33%,P<0.05).在Ⅲ～Ⅳ期与Ⅰ～Ⅱ期卵巢癌中c-kit蛋白及PDGFRα表达的阳性率差异无统计学意义(P>0.05).(3)卵巢上皮性癌的c-kit蛋白和PDGFRα表达无相关性(P>0.05).结论 c-kit蛋白与PDGFRα的表达与卵巢上皮性癌细胞的分化程度相关联.对c-kit蛋白与PDGFRα的检测可用于判断卵巢上皮性肿瘤的恶性程度及其预后.     

Inflammatory fibroid polyps harbour mutations in the platelet-derived growth factor receptor alpha (PDGFRA) gene

Inflammatory fibroid polyps (IFPs) are mesenchymal tumours which arise in the submucosa and mucosa of the gastrointestinal tract. To date, the pathogenesis is unknown and IFPs are considered reactive and non-neoplastic lesions. Investigating a series of 23 IFPs, we made the observation that the tumours consistently express PDGFRA. To further elucidate the pathogenetic role of PDGFRA, we performed mutational analyses of exons 10, 12, 14, and 18. As IFPs are characterized by an inflammatory infiltrate rich in eosinophils, we used fluorescence in situ hybridization in a subset of tumours to investigate a possible FIP1L1-PDGFRA translocation which is known as the cause of hypereosinophilic syndrome (HES). Sixteen IFPs (70%) harboured activating mutations in exons 12 and 18, respectively: V561D (n = 1), R560SDelta561-567 (n = 1), Delta559-561D591H (n = 1), S566RDelta567-571 (n = 3), D842V (n = 7), D842I (n = 1), Delta842-845 (n = 1), and Delta845-848 (n = 1). These mutations equal pathogenic mutations detected in gastrointestinal stromal tumours previously. Activating mutations in exons 10 and 14 were not noted. None of the cases revealed the FIP1L1-PDGFRA translocation. Considering the remarkable number of activating mutations detected in our series, we conclude that the vast majority of IFPs harbour gain-of-function mutations in the PDGFRA gene. The presence of PDGFRA mutations questions the reactive nature of IFPs and raises the possibility of a neoplastic process. Copyright (c) 2008 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Detection of c-kit exons 11- and 17-activating mutations in testicular seminomas by high-resolution melting amplicon analysis.

A subgroup of testicular seminomas has been reported to contain activating mutations in KIT, the transmembrane tyrosine kinase receptor encoded by the c-kit gene. Most mutations are in exon 17, although exon 11-activating mutations have recently been described. For patients refractory to standard therapeutic protocols for seminoma, the presence of c-kit-activating mutations in some of these neoplasms might suggest an alternative therapy with KIT targeting drugs. We used the novel mutation scanning technique, high-resolution melting amplicon analysis, to screen a series of 22 testicular seminomas for c-kit-activating mutations. Four cases (18%) had exon 17-activating mutations and these included D816Y, D816V, Y823N and one case that contained both D816E and D820H. A single case (5%) had an exon 11-activating mutation. Interestingly, the exon 11-activating mutation was L576P, the same mutation that characterizes the rare c-kit mutation-positive cases of malignant melanoma. Fluorescence in situ hybridization (FISH) for c-kit suggested that most seminomas are probably polysomic for c-kit and there was not a significant difference in c-kit FISH characteristics between the mutation-positive and mutation-negative cases. The use of high-resolution melting amplicon analysis as a screening technique will allow for the rapid identification of patients with testicular seminomas whose tumors contain c-kit-activating mutations. This could benefit patients whose tumors are refractory to standard therapeutic protocols.     

c-kit mutations in gastrointestinal stromal tumors occur preferentially in the spindle rather than in the epithelioid cell variant.

Gastrointestinal stromal tumors (GISTs) coexpress CD34 and the Kit tyrosine-kinase receptor (CD117). A subset of GISTs carry gain-of-function mutations of the c-kit proto-oncogene in its juxtamembrane domain. The relationship between the mutational status and histological as well as immunohistochemical features has not been assessed in detail. 36 GISTs and 14 other gastrointestinal mesenchymal tumors were investigated for their morphology and immunophenotype as well as for the presence of c-kit mutations. DNA was extracted from formalin-fixed, paraffin-embedded tissue. Exons 9, 11, 13, and 17 of c-kit were analyzed by SSCP. Bands with altered mobility were excised, reamplified, and sequenced. C-kit mutations in Exon 11 encoding the juxtamembrane domain were identified in 19 cases (52.8%), with deletions in 12 cases, insertions in 3 cases (2 of these as duplications), and point mutations in 4 cases. The mutations clustered between Codons 553 and 561, pinpointing the critical region for deregulated Kit receptor activation. In both Exons 9 and 13, single mutations could be identified, whereas no mutations were found in Exon 17. There were c-kit mutations in 66.6% of benign GISTs (14/21), 83.3% of the malignant (5/6), and 40% of the cases of intermediate malignancy (2/5). A low frequency of mutations in benign GISTs, as reported previously by other researchers, could not be observed in our panel. Interestingly, all GISTs with c-kit mutations displayed a spindle cell phenotype, whereas mutations were absent in all 7 tumors with an epithelioid component (P =.03). This finding suggests a relationship between c-kit mutation and histological subtype in GISTs.     

The value of c-kit mutational analysis in a cytokeratin positive gastrointestinal stromal tumour

The expression of cytokeratins in gastrointestinal stromal tumours (GISTs) is rare and may lead to diagnostic confusion when it occurs. This report describes a metastatic GIST that stained strongly for cytokeratins, CD117, and CD34 in a patient who was previously diagnosed with gastric epithelioid angiosarcoma. A review of both tumours showed the same histological and immunohistochemical profiles, and c-kit molecular analysis revealed an insertional mutation at codon 558 of exon 11 in both tumours. Thus, pathologists should be aware that GISTs can occasionally express cytokeratins, and that c-kit mutational investigations may have a key diagnostic role and may prevent diagnostic mistakes that could have important clinical implications.     

KIT and PDGFRA mutations in gastrointestinal stromal tumors (GISTs)

Mutually exclusive KIT and PDGFRA mutations are central events in GIST pathogenesis, and their understanding is becoming increasingly important, because specific treatment targeting oncogenic KIT and PDGFRA activation (especially imatinib mesylate) has become available. KIT mutations in GIST are clustered in four exons. Most common are exon 11 (juxtamembrane domain) mutations that include deletions, point mutations (affecting a few codons), and duplications (mostly in the 3' region). The latter mutations most often occur in gastric GISTs. Among gastric GISTs, tumors with deletions are more aggressive than those with point mutations; this does not seem to hold true in small intestinal GISTs. Exon 9 mutations (5-10%) usually are 2-codon 502-503 duplications, and these occur predominantly in intestinal versus gastric GISTs. Lesser imatinib sensitivity of these tumors has been noted. Kinase domain mutations are very rare; GISTs with such mutations are variably sensitive to imatinib. PDGFRA mutations usually occur in gastric GISTs, especially in the epithelioid variants; their overall frequency is approximately 30% to 40% of KIT mutation negative GISTs. Most common is exon 18 mutation leading Asp842Val at the protein level. This mutation causes imatinib resistance. Exon 12 and 14 mutations are rare. Most mutations are somatic (in tumor tissue only), but patients with familial GIST syndrome have consitutitonal KIT/PDGFRA mutations; >10 families have been reported worldwide with mutations generally similar to those in sporadic GISTs. GISTs in neurofibromatosis 1 patients, children, and Carney triad seem to lack GIST-specific KIT and PDGFRA mutations and may have a different disease mechanism. Secondary mutations usually occur in KIT kinase domains in patients after imatinib treatment resulting in resistance to this drug. Mutation genotyping is a tool in GIST diagnosis and in assessment of sensitivity to kinase inhibitors. This is a US government work. There are no restrictions on its use.     

Mutation in the platelet-derived growth factor receptor alpha inhibits adeno-associated virus type 5 transduction

Due to its non-pathogenic lifecycle, little is known about the cellular determinants of infection by adeno-associated virus (AAV). To identify these critical cellular factors, we took advantage of the gene transfer abilities of AAV in combination with a forward genetic selection to identify proteins critical for transduction by this virus. AAV serotype 5 (AAV5) vectors encoding the furin gene were used to transduce furin-deficient cells followed by selection with furin-dependent toxins. A population of cells specifically resistant to AAV5 transduction was identified and sequence analysis suggested all had a single amino acid mutation in the leader sequence of the platelet-derived growth factor receptor alpha (PDGFRα) gene. Characterization of this mutation suggested it inhibited PDGFRα trafficking resulting in limited expression on the plasma membrane. Mutagenesis and transfection experiments confirmed the effect of this mutation on PDGFRα trafficking, and the AAV5 resistant phenotype could be rescued by transfection with wild type PDGFRα.