胃肠道间质瘤中c-kit和PDGFRA基因的突变及其意义

背景与目的:胃肠道间质瘤(GIST)是最常见的胃肠道间叶源性肿瘤,GIST中c-kit和PDGFRA基因突变及突变位点与甲磺酸伊马替尼(格列卫)治疗疗效有关,而突变与生物学行为的关系一直存有争议.本研究探讨GIST中c-kit和PDGFRA基因的突变情况及意义.方法:用PCR扩增和直接测序方法检测50例GIST中c-kit外显子9、11、13、17及PDGFRA基因外显子12、18的突变.结果:50例GIST中仅检测出c-kit外显子11突变,突变率为54%(27/50),均为杂合性突变.其中缺失性突变16例(59%)、点突变10例(37%),伴有点突变的缺失性突变1例.突变位点几乎均集中在5'端"热点"区(96%).CD117阴性的2例GIST中1例检测到c-kit外显子11突变.外显子11突变在不同组织学类型之间比较,差异有显著性(P＜0.05),而在不同原发部位及不同侵袭危险性之间比较,差异均无显著性(P＞0.05).结论:c-kit突变是GIST的普遍现象,突变位点有集中趋势,以梭形细胞型突变最常见.突变检测不能作为生物学行为判断参考指标.     

A missense mutation in KIT kinase domain 1 correlates with imatinib resistance in gastrointestinal stromal tumors

KIT gain of function mutations play an important role in the pathogenesis of gastrointestinal stromal tumors (GISTs). Imatinib is a selective tyrosine kinase inhibitor of ABL, platelet-derived growth factor receptor (PDGFR), and KIT and represents a new paradigm of targeted therapy against GISTs. Here we report for the first time that, after imatinib treatment, an additional specific and novel KIT mutation occurs in GISTs as they develop resistance to the drug. We studied 12 GIST patients with initial near-complete response to imatinib. Seven harbored mutations in KIT exon 11, and 5 harbored mutations in exon 9. Within 31 months, six imatinib-resistant rapidly progressive peritoneal implants (metastatic foci) developed in five patients. Quiescent residual GISTs persisted in seven patients. All six rapidly progressive imatinib-resistant implants from five patients show an identical novel KIT missense mutation, 1982T-->C, that resulted in Val654Ala in KIT tyrosine kinase domain 1. This novel mutation has never been reported before, is not present in pre-imatinib or post-imatinib residual quiescent GISTs, and is strongly correlated with imatinib resistance. Allelic-specific sequencing data show that this new mutation occurs in the allele that harbors original activation mutation of KIT.     

Deletion of Trp-557 and Lys-558 in the juxtamembrane domain of the c-kit protooncogene is associated with metastatic behavior of gastrointestinal stromal tumors

Gastrointestinal stromal tumors (GISTs) typically express high levels of the Kit-receptor. The majority of GISTs carry mutations in the c-kit protooncogene clustering in exon 11. The significance of c-kit mutations for the biological behavior of GISTs is still under discussion. We evaluated 55 sporadic GISTs with available follow-up data for c-kit mutations in the juxtamembrane domain and detected mutations in 35 cases (63.6%). We found a mutational hotspot in codons 557 (tryptophan) and 558 (lysine) preferentially in histomorphologically malignant tumors. In the group of GISTs carrying c-kit mutations, 16 of 21 malignant, but only 3 of 8 benign GISTs and 3 of 6 lesions with uncertain malignant potential, carried mutations of Trp-557 and/or Lys-558. We investigated whether mutations in these 2 amino acids had an impact on biological behavior. Trp-557 and/or Lys-558 were mutated in all 15 metastatic GISTs carrying c-kit mutations but only in a minority of nonmetastatic tumors. A combined deletion of Trp-557 and Lys-558 occurred exclusively in 8 metastatic GISTs. We conclude that in addition to histomorphological evaluation determination of mutations in exon 11 may be an additional parameter for predicting the metastatic risk of GISTs and may be important for the decision that patients will need close clinical follow-up or further adjuvant treatment with kit antagonists.     

Rectal gastrointestinal stromal tumors associated with a novel germline KIT mutation

Somatic, activating mutations of KIT or PDGFRA are early oncogenic events in the majority of sporadic gastrointestinal stromal tumors (GISTs). Also a number of families with GISTs have been described in recent years. The familial GIST syndrome is a rare autosomal dominant disorder with high penetrance and diverse manifestations associated mostly with germline KIT mutations. In this report, we show a novel germline mutation in the juxtamembrane domain of KIT, identified in 2 brothers, both presenting with recurrent, high risk/malignant rectal GISTs. The KIT p.Q575_P577delinsH mutation was found in tumor samples as well as in peripheral blood leukocytes from both patients, proving that the mutation was indeed inherited. Besides rectal GISTs, no other features characteristic for the familial GIST syndrome was observed in either brother or any of their first-degree relatives. The patients were treated with imatinib, achieving either long-term partial response or stable disease. This observation confirms that imatinib can be successfully used in familial GISTs, as it is used in the sporadic advanced tumors, and that tumors bearing a KIT p.Q575_P577delinsH mutation are responsive to imatinib treatment.     

白血病Flt3基因近膜区突变的检测和分析

目的：检测白血病Flt3基因近膜区突变,探讨与白血病发生、发展的关系。方法：采用PCR、PCR-SSCP和DNA测序方法,对60例白血病患者骨髓标本Flt3基因外显子14、15进行检测,以20名健康志愿者骨髓标本作为对照。结果：在60例白血病患者中,发现8例Flt3-ITD突变,3例Flt3-L576P点突变,其中1例合并有Flt3基因内含子14缺失突变。结论：白血病Flt3-L567P点突变是近膜区新发现的点突变,可能与白血病的发生、发展相关。     

Imatinib inhibits various types of activating mutant kit found in gastrointestinal stromal tumors

Mutations of proto-oncogene c-KIT in gastrointestinal stromal tumors (GISTs) are considered to cause a constitutive activation of KIT responsible for their oncogenesis. Imatinib has therapeutic potential for GISTs because of its inhibitory effect on KIT kinase activity. To investigate the effect of Imatinib on various c-KIT mutations found in GISTs, we examined kinase activity of KIT, cell proliferation and tumorigenicity of transfectants with various c-KIT mutations. Murine lymphoid Ba/F3 cells transfected with one of the three types of mutants (KIT(del559-560), KIT(642Glu), and KIT(820Tyr)) or wild-type KIT were used for the experiments. Phosphorylation of KIT, mitogen-activated protein (MAP) and Akt was studied by immunoblotting with or without immunoprecipitation. In vitro studies on cell proliferation using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenylcetrazolium bromide colorimetric assay and in vivo tumorigenicity assay using nude mice were also carried out. Imatinib could inhibit the KIT, MAP and Akt phosphorylation of all the transfectants but had a weaker effect on KIT(820Tyr). Imatinib potently inhibited the proliferation of cells transfected with KIT(820Tyr) at the concentration of 10 microM whereas it inhibited the other 3 types at 1 microM. Moreover, Imatinib could inhibit the tumor formation in nude mice transplanted with transfectants. In various types of activating mutant KIT, Imatinib could inhibit the constitutive activation of KIT signal transduction and cell proliferation both in vitro and in vivo although the effect of Imatinib on KIT(820Tyr) was weaker than that on KIT(del559-560) or KIT(642Glu).     

胃肠道间质瘤中原癌基因c-kit突变及其蛋白表达

目的：探讨c-kit基因蛋白表达及基因突变在胃肠道间质瘤（GIST）发病中的作用及其与临床病理,预后的关系。方法：采用免疫组化和聚合酶链反应单链构象多态性技术（PCR－SSCP）方法,检测82例GIST中c-kit蛋白的表达及c-kit基因exon 11突变的情况。结果：全组c-kit蛋白的阳性率为97．6％（80／82）,c-kit基因突变率为41．5％（34／82）,良性GIST c-kit蛋白表达率为95．0％（19／20）,c-kit基因突变为阴性;恶性GIST c-kit蛋白表达率为98．4％（61／62）,c-kit基因突变率为54．8％（34．62）,与基因突变阴性病例相比,突变阳性组的GIST容易出现邻近组织的侵袭,转移或者复发,结论：c-kit蛋白是GIST的重要诊断指标;c-kit基因突变在GIST发生发展中可能发挥重要作用,可作为判断GIST患者预后的指标之一。     

Prognostic significance of c-kit mutation in localized gastrointestinal stromal tumors.

PURPOSE: Constitutive mutational activation of c-kit has been found to be associated with the pathogenesis of gastrointestinal stromal tumors (GISTs). The prognostic significance of c-kit mutations, however, is still controversial. EXPERIMENTAL DESIGN: We examined 86 patients curatively resected for localized GIST. Genomic DNA was extracted from paraffin-embedded tumor tissues. Exons 9, 11, 13, and 17 of the c-kit gene were amplified by PCR and sequenced. RESULTS: Mutations in exon 11 were detected in 61 tumors, and mutations in exon 9 were observed in three tumors, whereas no mutations were detected in exons 13 or 17. The overall c-kit mutation frequency was 74%. Amino acid alterations in the 61 tumors with exon 11 mutations were deletion in 33 tumors, substitution in 20, both deletion and substitution in 4, insertion in 1, and duplication in 3. Histologically, tumors with c-kit mutations showed higher mitotic counts and higher cellularity. The 5-year relapse-free survival (RFS) in patients having GISTs with c-kit mutations was 21%, compared with 60% in those without c-kit mutations. Significantly higher RFS rates were observed in patients with tumors having mitotic counts < 5 mitoses/50 high power field, spindle-cell histology, tumor size < 5 cm, or gastric GISTs. Multivariate analyses indicated association of poorer RFS with a higher mitotic count > or = 5 of 50 high power fields; odds ratio (OR) = 3.0], presence of c-kit mutations (OR = 5.6), and a larger tumor size (> or = 5 cm; OR = 4.2). CONCLUSIONS: The presence of c-kit mutation, along with high mitotic count and larger tumor size, was an independent factor for poor prognosis in patients with localized GISTs.     

胃肠道间质瘤c-kit基因突变的研究

目的　探讨c kit基因在胃肠道间质瘤 (GIST)中的突变状况。方法　用PCR扩增和基因测序的方法 ,检测 5 2例GIST及 2 8例对照肿瘤c kit基因第 11号外显子序列 ,其中 30例GIST另检测了c kit基因第 9和第 13号外显子序列。结果　 2 5例恶性GIST中 ,14例有c kit基因 11号外显子突变 (5 6 .0 % ) ;2 7例良性及交界性GIST中 ,仅 2例有突变 (7.4 % )。良恶性GIST中 ,c kit基因突变的差异有显著性 (χ2 =14 .39,P <0 .0 1)。 5 2例GIST中 ,14例为杂合性突变 ,2例为纯合性突变。突变方式有点突变和片段的缺失或重复等 ,缺失和重复的片段为 3～ 4 8bp不等 ,碱基数是 3的倍数。原发及复发组织突变方式相同 ,突变病例瘤旁正常组织及伴发的腺癌无突变。对照肿瘤无c kit基因突变。GIST中c kit基因 11号外显子的突变位点多不固定 ,但有集中趋势 ,点突变和片段的缺失集中在5 5 0～ 5 70密码子 ,片段的重复集中在 5 70～ 5 85密码子。结论　 11号外显子的突变是恶性GIST的分子生物学机制之一 ,可作为辅助判断GIST良恶性的参考指标。c kit基因突变提示GIST是不同于消化道平滑肌瘤及神经鞘瘤的独立疾病。     

胃肠道间质瘤c-kit基因突变及其临床意义

胃肠道间质瘤（GIST）是一组独立起源于胃肠道间质干细胞的肿瘤，其主要发病机制是c-kit基因突变导致c-kit蛋白（CD117）活化，进而引发细胞增殖失控及凋亡抑制。目前研究显示c-kit基因的突变状态和类型不仅与GIST的恶性程度及预后密切相关，而且还是GIST对甲磺酸伊马替尼（STI571）治疗反应的预测因子，在GIST的临床诊治中具有重要意义。     

Effect of c-kit mutation on prognosis of gastrointestinal stromal tumors.

Gastrointestinal stromal tumor (GIST) is the most common mesenchymal tumor of the gastrointestinal tract. Gain-of-function mutations in the juxtamembrane domain of the c-kit gene have been found in several GISTs. In this study, we examined the correlation between the presence of c-kit mutation and prognosis in 124 cases of GIST. DNA samples were extracted from paraffin sections. Exon 11 of the c-kit gene encoding the juxtamembrane domain and exon 17 encoding the kinase domain were amplified by PCR and sequenced. Most GISTs (89%) express the KIT protein, and missense mutations of exon 11 were found in 71 of 124 GISTs (57%). No mutations were detectable in exon 17. These 71 mutation-positive GISTs were larger in size and had more frequently invaded adjacent tissues than did the 53 mutation-negative GISTs. Histologically, the mutation-positive GISTs showed higher mitotic figures and more necrosis and hemorrhage. The patients with mutation-positive GISTs showed more frequent recurrences (P = 0.0005) and higher mortality (P = 0.0001) than did those with mutation-negative GISTs. The c-kit mutation was an independent prognostic factor for overall and cause-specific survival of the patients with GISTs. These results suggest that GISTs may be divided into mutation-positive and -negative subtypes. The prognosis was worse in patients with mutation-positive GISTs than in those with mutation-negative GISTs. Thus, mutation of the c-kit gene may be a good prognostic marker of GISTs.     

Mutations of c-kit JM domain are found in a minority of human gastrointestinal stromal tumors

The c-kit gene encodes a transmembrane receptor kinase (KIT) which is expressed in the majority of human gastrointestinal stromal tumors (GISTs), a subtype of gastrointestinal mesenchymal neoplasms. A previous study identified mutations in the juxtamembrane (JM) domain of c-kit in five of six GISTs (Science 279: 577, 1998). To better define the frequency and spectrum of c-kit gene mutations in mesenchymal neoplasms of the GI tract that had been characterized for KIT protein expression, we examined archived tissue samples for mutations in the JM domain by PCR amplification and DNA sequencing. c-kit JM domain mutations were found in nine of 56 mesenchymal tumors (46 GISTs, eight leiomyomas, two leiomyosarcomas) and occurred exclusively in GISTs (21%). Seven of the nine mutations consisted of intragenic deletions of one to 19 codons. There was one insertion mutation that added 12 codons and one missense mutation (Val560Asp). None of the mutations disrupted the downstream reading frame of the gene. The single missense mutation (Val560Asp) is very similar to the only other missense mutation reported in GISTs (Val599Asp). Of the 46 GISTs, 43 were strongly positive for KIT protein expression and negative for diffuse expression of desmin. Neither KIT expression nor gene mutations were found in gastrointestinal leiomyomas or leiomyosarcomas. We conclude that mutation of the c-kit JM domain does not occur in gastrointestinal mesenchymal neoplasms with well developed-smooth muscle differentiation, and is restricted to GISTs. However, since these mutations are only found in a minority of GISTs, further investigation into the mechanisms of c-kit gene activation in this group of neoplasms is warranted.     

Germline mutation of the PTEN gene in a Japanese patient with Cowden's disease

Cowden's disease (CD) is an autosomal dominant disorder which confers a high susceptibility to diverse benign and malignant tumors. The PTEN (phosphatase and tensin homologue deleted in chromosome ten) gene has been identified as a tumor suppressor gene responsible for cancers of the endometrium, ovary, prostate, and glioblastomas. Recently, germline mutations of this gene were also found in patients with CD, and it is now recognized as a gene responsible for this disease. We identified a germline nonsense mutation at codon 130 in exon 5 of PTEN in a 56-year-old Japanese woman with CD. The patient had adenoid facies and mucocutaneous lesions including multiple facial papules, acral keratoses on neck and shoulders, palmoplantar keratoses, multiple oral papillomas, scrotal tongue, mucosal and cutaneous hemangiomas, and a sclerotic fibroma on the arm. She also had benign and malignant polypoid neoplasms throughout the entire digestive tract, including adenocarcinoma of the colon and submucosal lipomas of the rectum, as well as bilateral breast carcinomas, multinodular goiters, an ovarian cyst with a fibroma-like nodule, hepatic hemangiomas, and abdominal hernia. We searched CD cases with the same genotypic PTEN mutation as the present case and compared their phenotypes. Further studies will disclose a better understanding of the role of mutation in the PTEN gene in the course of tumorigenesis of both benign and malignant tumors developed in patients with CD.     

白血病Flt3基因编码的近膜区点突变的检测和内含子14缺失的分析

 目的　对白血病Flt3基因编码的近膜区突变点进行检测,研究其与白血病发生、发展的关系。方法　应用聚合酶链反应（PCR）、聚合酶链反应单链构象多态性（PCR／SSCP）和DNA测序方法,对60例白血病患者外周血和部分骨髓标本Flt3基因外显子14、15进行检测。结果　在60例白血病患者中,3例白血病Flt3-L576P发生点突变,其中1例合并有Flt3基因内含子14缺失突变。结论　白血病Flt3-L567点突变是近膜区（JM）新发现的点突变,其发生与临床白血病的发生、发展具有相关性。     

Pleomorphic characteristics of a germ-line KIT mutation in a large kindred with gastrointestinal stromal tumors, hyperpigmentation, and dysphagia.

PURPOSE: Somatic mutations that result in the activation of the growth factor receptor KIT are commonly found in gastrointestinal stromal tumors (GISTs). Six families have been reported in which a germ-line mutation in KIT is associated with an autosomal dominant predisposition to the development of GISTs. Hyperpigmentation, urticaria pigmentosa, and dysphagia have been described in some, but not all, families. Preliminary correlations between the site of mutation and the clinical phenotype have been proposed, but the strength of these associations is not defined. DESIGN: A large kindred with multiple GISTs, hyperpigmentation, and dysphagia was identified after the index case presented with multiple GISTs. A germ-line mutation in KIT (W557R) was identified in an affected cousin, after which a large family meeting was held and testing offered. Clinical data were obtained by interview and, whenever possible, medical record documentation. RESULTS: To date, 19 individuals have been tested, and the mutation has been shown to cosegregate with the syndrome. The phenotypic expression, however, is variable. GISTs, often presenting as upper gastrointestinal bleeding, and hyperpigmentation are common, but not diagnosed in all documented or obligate carriers. Dysphagia is a less prevalent complaint. The diagnosis of GISTs appears to be made at a younger age in more recent generations. Metastatic disease is uncommon. CONCLUSIONS: A germ-line mutation in KIT resulting in an amino acid substitution in the juxtamembrane region is associated with a syndrome of GIST, hyperpigmentation, and dysphagia, although the prominence of each component varies.     

c-KIT mutation analysis for diagnosis of gastrointestinal stromal tumors in fine needle aspiration specimens

BACKGROUND: Gastrointestinal stromal tumors (GISTs) are a specific clinicopathologic entity characterized by activating mutations in the c-KIT gene. These mutations are associated with increased immunohistochemical staining for the c-KIT protein. Because many GISTs display a definitive response to the KIT inhibitor imatinib, accurate diagnosis of these neoplasms is of great clinical importance. GISTs are the most common mesenchymal neoplasms of the digestive tract and show lineage differentiation along the lines of the interstitial cells of Cajal. The cytomorphology of GISTs has been well described, but the cytologic features are not entirely specific and immunohistochemical staining is frequently used to confirm the diagnosis. However, KIT protein positivity established by immunohistochemistry (IHC) may be unreliable in some instances because of a small number of KIT-negative GISTs and immunohistochemical KIT positivity in some non-GIST spindle cell neoplasms of the gastrointestinal tract. Antigen retrieval may be the cause of this spurious positivity. Recently, the demonstration of KIT and PDGFRA mutations has been shown to be more reliable in establishing the diagnosis of GIST than IHC. METHODS: High resolution amplicon melting analysis was performed in a series of eight gastrointestinal stromal tumors sampled by fine needle aspiration (FNA). RESULTS: In five of eight cases, adequate material was obtained for mutational analysis. In four of these, mutations in the c-KIT gene were detected, and in the fifth case a mutation in the PDGFRA gene was detected. CONCLUSIONS: It appears that high resolution amplicon melting analysis can be successfully performed on material obtained by FNA and will show either KIT for PDGFR mutations in the majority of GIST FNA specimens.