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。     

CD117阴性胃肠道间质瘤的临床病理及免疫组织化学研究

目的 探讨免疫组织化学在形态学典型、免疫组织化学CD117阴性胃肠道间质瘤(GIST)诊断中的意义.方法 对10例CD117阴性、形态学典型的GIST进行c-kit基因第9、11、13、17号外显子及血小板源性生长因子受体α(PDGFRA)基因第12和18号外显子的基因检测,同时所有病例均进行CD117、CD34、平滑肌肌动蛋白(SMA)、结蛋白、S-100蛋白、WT-1、DOG-1 的免疫组织化学染色(EnVision法).结果 10例中8例完成c-kit及PDGFRA基因的检测,仅1例有c-kit基因第9号外显子突变,余未发现基因突变.10例CD117阴性的病例9例CD34阳性,2例SMA局灶阳性.结蛋白和S-100蛋白均阴性.DOG1弥漫阳性者5例,1例弥漫弱阳性,2例局灶阳性,2例阴性.4例WT-1弥漫阳性,2例局灶阳性,1例有散在肿瘤细胞阳性,3例阴性.结论 对胃肠道及胃肠道外形态学典型、但CD117阴性的GIST病例,联合应用多种免疫组织化学标记有助于诊断.DOG-1和WT-1可作为补充加入到CD117阴性GIST的诊断中.     

胃肠道间质瘤c-Kit及PDGFRA基因突变与临床病理特征、免疫表型及预后的关系

目的探讨胃肠道间质瘤(gastrointestinal stromal tumors,GIST)中c-Kit/PDGFRA基因突变与临床病理特征、免疫表型及预后的关系。方法采用直接测序法检测185例GIST标本中c-Kit和PDGFRA基因突变类型。结果 c-Kit基因突变中,外显子11突变最常见,以缺失突变、点突变及混合突变为主,并见3例双外显子突变。外显子9在小肠GIST中的突变率显著高于胃GIST,且均为A502-Y503串联重复突变,提示小肠GIST有独特的基因型。PDGFRA在上皮样细胞型和混合细胞型GIST中的突变率显著高于梭形细胞型GIST。c-Kit基因外显子11缺失突变的中高危GIST易出现术后复发转移,且复发转移患者中发生二次突变率较高,表现为c-Kit基因外显子13、14、17或18点突变,以13号外显子V654A最为常见。CD117和DOG1的表达与基因突变之间无相关性,不能作为预测疾病基因突变的指标。单因素分析结果显示,肿瘤直径、核分裂象、Ki-67增殖指数、危险程度分级、术后复发和转移是影响GIST预后的重要因素,术后靶向药物治疗可一定程度改善预后。c-Kit基因外显子11缺失突变和外显子9突变的GIST患者预后较差。结论 c-Kit基因9号外显子在小肠GIST中突变率较高,PDGFRA基因在上皮样细胞型和混合细胞型GIST中突变率较高。c-Kit基因外显子11缺失突变的中高危GIST易发生二次突变导致术后复发转移,外显子11缺失突变可作为GIST预后不良的独立影响因素之一。     

PDGFRA强阳性表达是诊断GIST较为特异性标志

胃肠道间质肿瘤(GIST)是腹腔最常见的间叶性肿瘤,来自于Cajal间质细胞或其前体细胞。大多数GISTs有c-kit癌基因的突变,c-kit(CD117)阳性被认为是诊断GIST的金标准,也是格列为治疗GIST的必要条件。近来研究发现PDGFRA基因突变作为另一个癌变机制可见于部分缺乏c-kit突变的GIST中,然而,PDGFRA免疫组化检测在GIST诊断中的价值还未见报道。作者应用免疫组化方法检测了39例GISTs和20例其它腹腔内间叶性肿瘤中PDGFRA的表达,同时进行了CD117免疫组化染色和PDGFRA和c-kit基因的突变分析。39例GISTs中,胃24例、小肠10例、大…     

结外鼻型NK/T细胞淋巴瘤c-kit基因突变分析

目的分析鼻型NK／T细胞淋巴瘤（N—NK／T—L）组织中c—kit基因突变和表达情况，探讨N．NK／T-L可能的特异分子标记物。方法研究组为36例N-NK／T-L，对照组包括11例同部位B细胞淋巴瘤（BCL）和5例颈部非特殊性外周T细胞淋巴瘤（PTCL）；采用免疫组化EnVision法对淋巴瘤进行c—kit产物CD117标记；采用巢式PCR法对31例N—NK／T—L、11例BCL和5例PTCL肿瘤组织c—kit基因11和17号外显子片段进行扩增，PCR纯化产物测序和序列分析。结果N—NK／T—L组织学表现为凝固性坏死，不同大小和异型的肿瘤性淋巴细胞呈破坏性生长浸润于炎性背景中。36例N．NK／T-L免疫组化表达阳性率为：CD45RO75．0％、CD3e72．2％、TIA83．3％、GranzymeB61．1％、CD5680．5％、CD3030．6％，而全部病例CD117阴性。对照组5例PTCL均为TIA1阳性，仅1例GranzymeB阳性，而11例BCL仅CD20和CD79et阳性，其余标记均阴性。GranzymeB表达与N-NK／T-L肿瘤细胞的异型性具有相关性（P〈0．05）。尽管所有病例CD117染色阴性，但c—kit基因11和17外显子扩增序列分析表明8例（8／31，25．8％）N-NK／T—L病例具有基因突变，其中分别有4例涉及11外显子和17外显子；对照组11例BCL和5例PTCL中分别有1例具有11外显子突变。所有突变均为碱基替代错义突变，热点涉及571、572和821位点。结论上述结果提示除组织学特征外，GranzymeB可能是更可靠的N-NK／T—L诊断和鉴别诊断指标。c-kit错义突变可发生于约1／4的N—NK／T—L病例中，该突变可能引起c-kit的功能失调。     

CD117阴性胃肠道间质瘤的临床病理及免疫组织化学研究

目的 探讨免疫组织化学在形态学典型、免疫组织化学CD117阴性胃肠道间质瘤(GIST)诊断中的意义.方法 对10例CD117阴性、形态学典型的GIST进行c-kit基因第9、11、13、17号外显子及血小板源性生长因子受体α(PDGFRA)基因第12和18号外显子的基因检测,同时所有病例均进行CD117、CD34、平滑肌肌动蛋白(SMA)、结蛋白、S-100蛋白、WT-1、DOG-1 的免疫组织化学染色(EnVision法).结果 10例中8例完成c-kit及PDGFRA基因的检测,仅1例有c-kit基因第9号外显子突变,余未发现基因突变.10例CD117阴性的病例9例CD34阳性,2例SMA局灶阳性.结蛋白和S-100蛋白均阴性.DOG1弥漫阳性者5例,1例弥漫弱阳性,2例局灶阳性,2例阴性.4例WT-1弥漫阳性,2例局灶阳性,1例有散在肿瘤细胞阳性,3例阴性.结论 对胃肠道及胃肠道外形态学典型、但CD117阴性的GIST病例,联合应用多种免疫组织化学标记有助于诊断.DOG-1和WT-1可作为补充加入到CD117阴性GIST的诊断中.

Abstract：

Objective To study the immunophenotype and c-kit or platelet derived growth factor receptor alpha(PDGFRA)gene mutations in CD117-negative gastrointestinal stromal tumors(GISTs).Methods Ten cases of GISTs with typical histologic features but no CD117 expression were retrieved from the archival of Department of Pathology,Peking Union Medical College Hospital,China.The Cages were further evaluated for the presence of c-kit exons 9.11, 13 and 17 mutations and PDGFRA exons 12 and 18mutations.DNA was extracted from the paraffin-embedded tuinor tissue.The PCR products were sequenced directly for the mutations.An immunohistochemical study for CD117,CD34,smooth muscle actin,desmin,S-100 protein.WT-1 and DOC-1 Was also performed.Results Eight of the 10 Cases had the mutation tests completed.C-kit mumfion in exon 9 Wag detected in only one case.Amongst the 10 cases studied, CD34Wag expressed in 9 cases. Smooth muscle actin was focally positive in 2 cases.None of them expressed desmin or S-100 protein.DOG-1 and WT-1 were diffusely positive in 5 and 4 Cages.respectively.In addition.DOG1 Was diffusely but weakly positive in 1 case and focally expressed in 2 cages.Three cases were focally positive for WT-1.Conclusion Pathologic diagnosis of CD117-negative GISTs can be facilitated with the application of a panel of immunohistochemical markers.including DOG-1 and WT-1.     

DOG1是一种敏感和特异性的胃肠道间质瘤的标记物

胃肠道间质瘤（GIST）是胃肠道最常见的间叶性肿瘤，大多数含有受体酪氨酸激酶基因c-KIT或PDGFRA的活化突变。GIST的诊断主要依赖于免疫组化KIT／CD117蛋白的表达，然而有4％～15％的GIST不表达CD117，导致诊断困难。DOG1是应用基因表达谱技术发现的一种高度表达于GIST的基因，作者采用2种新的鼠单克隆DOGI抗体（DOG1．1和DOG1．3），     

胃肠道间质瘤的分子分型与靶向治疗

胃肠道间质瘤(gastrointestinal stromal tumors, GIST)是胃肠道最常见的间叶源性肿瘤,通常c-kit(CD117)免疫表型阳性,对传统的化学治疗和放射治疗不敏感.该类肿瘤一般具有c-kit基因(75%～85%)或PDGFRA基因(5%～10%)的突变[1-2].这两个基因编码两个功能相似的酪氨酸激酶蛋白受体,其突变造成配体非依赖性的c-kit或PDGFRA受体二聚化,导致信号通路不需诱导持续激活,在GIST发病过程中发挥关键作用[3-4].c-kit或PDGFRA基因在GIST中的突变有一定的热点区域和规律,并且与GIST的临床病理学特征及预后存在非随机的联系.     

PDGFRA突变的胃肠道间质肿瘤的诊断形态学特征

胃肠道间质肿瘤(GIST)是胃肠道最常见的间叶性肿瘤,其中胃是最常发生的部位。尽管KIT蛋白的表达或c-kit基因突变被证明是诊断GIST的特征性标记,并在其病理发生中起着重要作用,但仍有一部分GIST不表达KIT或c-kit野生型。近来研究发现PDGFRA活化突变在大多数KIT阴性的GIST中被检测到,并发现PDGFRA突变的GIST形态学特征不同于c-kit突变的GIST。为了证实这种发现,作者检测了60例胃GIST中c-kit和PDGFRA的突变状态。60例胃GIST中,男27例,女33例,年龄12~92岁,平均63.8岁,其中仅有1例发生于21岁前。52例有肿瘤大小的记录,直…     

小肠的恶性胃肠道间质瘤伴淋巴结转移病理分析

目的 探讨伴淋巴结转移的小肠恶性胃肠道间质瘤的临床病理特征、c-kit基因突变情况,以及对甲磺酸伊马替尼(imatinib mesylate,Glivec)的治疗反应.方法 对2例发生在小肠伴发淋巴结转移的胃肠道间质瘤进行光镜观察、免疫组织化学标志及基因突变分析并随访甲磺酸伊马替尼的治疗效果.结果 2例均为小肠浆膜面肿块,镜下观察肿瘤均以梭形细胞为主,伴有少量上皮样细胞,呈多结节状,并出现大片凝固性坏死;免疫组织化学标志肿瘤细胞CD117阳性,基因突变检测发现均存在c-kit基因第11号外显子的突变.例1显示第11号外显子559～569位点杂合性缺失,伴570、571位点TACATA杂合性突变为GACAGA;例2显示第11号外显子559～565杂合性缺失.结论 小肠胃肠道间质瘤伴淋巴结转移是一种少见病变,需要同发生在此处的其他恶性软组织肿瘤鉴别;该肿瘤对甲磺酸伊马替尼的治疗效果取决于c-kit基因的具体突变类型.     

Mutations in Exon 11 of c-Kit Occur Preferentially in Malignant versus Benign Gastrointestinal Stromal Tumors and Do Not Occur in Leiomyomas or Leiomyosarcomas

Gastrointestinal stromal tumors (GISTs) comprise the largest subset of mesenchymal tumors of the gastrointestinal tract. These neoplasms differ histologically and immunohistochemically from typical leiomyomas and leiomyosarcomas. Most GISTs express CD34 and CD117 (c-kit protein) but not desmin. Recently, gain-of-function mutations of c-kit proto-oncogene have been shown in five solitary GISTs and in tumors and leukocytes from a family with multiple GISTs. An in-frame deletion or a point mutation in exon 11 of c-kit was detected in these cases. Stable transfection of the mutant c-kit complementary DNA was also shown to induce malignant transformation of murine lymphoid cells, suggesting that the c-kit mutations contribute to tumor development. In this study, we evaluated 43 GISTs and 14 smooth muscle tumors for mutations in the exon 11 of c-kit by a PCR-assay. Half of the malignant GISTs (12/24) and only one benign GIST (1/19) revealed mutant bands. No mutant bands were found in 3 leiomyomas and 11 leiomyosarcomas. Sequence analysis confirmed the presence of an in-frame deletion of 3–21 bp in all 13 GISTs with mutant bands. Wild-type bands from 8 malignant and 11 benign GISTs and 7 smooth muscle tumors without mutant bands were cloned and sequenced. Additional mutations were found in 3 malignant and 2 benign GISTs. There were no mutations in 3 leiomyomas and 4 leiomyosarcomas. The mutation status of exon 11 did not correlate with immunohistochemically detectable expression of the CD117, as virtually all GISTs with or without such mutations showed CD117 immunoreactivity. The c-kit mutations occur preferentially in malignant GISTs and might be a clinically useful adjunct marker in the evaluation of GISTs. The conservation of the c-kit mutation pattern, observed in consecutive lesions from the same patients, suggests that these mutations might be useful tumor markers in monitoring recurrence or minimal residual disease.     

Strong PDGFRA positivity is seen in GISTs but not in other intra-abdominal mesenchymal tumors: Immunohistochemical and mutational analyses.

Mutation of the platelet-derived growth factor receptor-alpha (PDGFRA) gene has been well documented as an alternative oncogenic mechanism in a subset of gastrointestinal stromal tumors (GISTs) lacking c-kit mutations. However, the role of PDGFRA immunohistochemistry in the diagnosis of GISTs has not been well studied. We investigated PDGFRA immunoreactivity in GISTs and in other intra-abdominal mesenchymal tumors, and correlated PDGFRA expression with CD117 positivity and with the mutational status of PDGFRA and c-kit genes. In addition, expression of phosphorylated AKT, an activated downstream molecule in the PDGFRA and c-kit signaling pathways, was correlated with PDGFRA and CD117 status. A total of 39 GISTs and 20 other mesenchymal tumors in the abdomen were included in this study. Thirty-five of 39 GIST cases (89.7%) were positive for PDGFRA and 19 of these 35 positive cases were strongly positive. Five of 20 non-GIST lesions (25%) were positive for PDGFRA, but none of these cases were strongly positive. With one exception, PDGFRA-positive cases were also positive for CD117. Phosphorylated AKT positivity was not associated with the immunoreactivity or mutation of PDGFRA and c-kit, suggesting that the activation of AKT is probably independent of the activation of PDGFRA and c-kit in GISTs. Of 14 GISTs assayed, 4 had mutations in c-kit at exons 11 or 17, and 4 had mutations in PDGFRA at exons 12 or 18. Three of 4 GIST cases with PDGFRA mutations show epithelioid morphology and strong PDGFRA immunoreactivity with prominent perinuclear dotlike accentuation (so-called Golgi pattern). In conclusion, strong PDGFRA positivity with Golgi pattern is a useful adjunct in the diagnosis of GISTs with PDGFRA mutation.     

High-resolution melting amplicon analysis as a method to detect c-kit and platelet-derived growth factor receptor alpha activating mutations in gastrointestinal stromal tumors

High-resolution melting amplicon analysis (HRMAA) was used to detect c-kit and platelet-derived growth factor receptor alpha (PDGFRA) activating mutations in 96 gastrointestinal stromal tumors (GISTs). HRMAA detected mutations in 87 GISTs (91%). Of the 87 cases, 69 (79%) contained c-kit mutations and 18 (21%), PDGFRA mutations. One c-kit mutation-positive case contained an exon 9 mutation, ins FY at codon 503, that has not been previously described. One PDGFRA mutation-positive case contained mutation D842V del 843, also not previously described. Of 18 PDGFRA mutation-positive cases, 3 (17%) were strongly positive for kit expression as measured by CD117 immunohistochemical analysis. Of 69 c-kit mutation-positive cases, 66 (96%) showed strong kit immunohistochemical expression, but 3 (4%) showed negative to weak CD117 expression. Of 96 cases, 9 (9%) were wild type for c-kit and PDGFRA. Of the wild-type cases, 8 still showed strong immunohistochemical kit expression, whereas 1 showed weak kit expression. GISTs with PDGFRA mutations were found in the stomach, omentum, and peritoneum but not the small intestine. GISTs with c-kit exon 9 mutations were found primarily in the small intestine. HRMAA is a sensitive technique that can be used to rapidly identify c-kit and PDGFRA activating mutations in GISTs.     

C-kit mutations in gastrointestinal stromal tumours

AIMS: Gastrointestinal stromal tumours (GISTs), once assumed to be of smooth muscle origin, generally express CD117 and CD34, similar to the interstitial cells of Cajal. Assessment of malignant potential in GISTs is problematic, especially on small biopsies. Some recent data indicate that mutations in the juxtamembrane domain (exon 11) of the c-kit (CD117) proto-oncogene may be associated with a worse prognosis. In this study, the frequency of c-kit exon 11 mutations has been determined in a series of 18 gut stromal tumours. METHODS: Immunophenotype was assessed by immunoperoxidase stains for smooth muscle actin, desmin, S100, CD34 and CD117, and each tumour classified as being of low, uncertain (intermediate) or high malignant potential based on standard histological criteria. DNA from each tumour was extracted from fresh (n = 5) or formalin-fixed, paraffin-embedded (n= 13) tissues using the direct lysis method. Exon 11 was amplified by PCR and sequencing of both sense and antisense strands was performed on two occasions using an ABI 377 sequencer. RESULTS: Mutations in exon 11 were detected in three of 14 confirmed GISTs, two being point mutations at codon 560 and one a 3-bp deletion resulting in the in-frame deletion of glutamine at codon 561. All three tumours were of high or intermediate malignant potential histologically. Three other 'high risk' primary GISTs and a metastatic GIST deposit were negative for exon 11 mutations. CONCLUSIONS: Data on this relatively small cohort of Australian patients indicate that c-kit exon 11 mutation analysis does not correlate well with histological assessment of malignant potential, and cannot be regarded as a reliable objective marker for poor prognosis in GISTs.     

Imatinib in the management of multiple gastrointestinal stromal tumors associated with a germline KIT K642E mutation

Gastrointestinal stromal tumors (GISTs) are the most common mesenchymal tumors of the gut and are distinguished by expression of CD117 (c-Kit). Oncogenic mutations in the KIT or PDGFRA gene are detected in approximately 85% of sporadic GISTs. In recent years, examples of familial GIST have been reported in which germline mutations of KIT or PDGFRA result in multiple GISTs, skin disorders, and other abnormalities. The most common germline mutations are in KIT exon 11, mutations in exons 8 and 17 have also been described, and there are 2 families with germline PDGFRA mutations. We present a case in which a germline KIT exon 13 mutation (K642E) was discovered in a patient with multiple GISTs of rectum, small intestine, and esophagus, as well as diffuse hyperplasia of the interstitial cells of Cajal. To our knowledge, this is only the second germline example of this particular mutation. The patient's esophageal tumors were stabilized with imatinib.     

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-alpha (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/     

Heterogeneity of kinase inhibitor resistance mechanisms in GIST

Most GIST patients develop clinical resistance to KIT/PDGFRA tyrosine kinase inhibitors (TKI). However, it is unclear whether clinical resistance results from single or multiple molecular mechanisms in each patient. KIT and PDGFRA mutations were evaluated in 53 GIST metastases obtained from 14 patients who underwent surgical debulking after progression on imatinib or sunitinib. To interrogate possible resistance mechanisms across a broad biological spectrum of GISTs, inter- and intra-lesional heterogeneity of molecular drug-resistance mechanisms were evaluated in the following: conventional KIT (CD117)-positive GISTs with KIT mutations in exon 9, 11 or 13; KIT-negative GISTs; GISTs with unusual morphology; and KIT/PDGFRA wild-type GISTs. Genomic KIT and PDGFRA mutations were characterized systematically, using complementary techniques including D-HPLC for KIT exons 9, 11-18 and PDGFRA exons 12, 14, 18, and mutation-specific PCR (V654A, D820G, N822K, Y823D). Primary KIT oncogenic mutations were found in 11/14 patients (79%). Of these, 9/11 (83%), had secondary drug-resistant KIT mutations, including six (67%) with two to five different secondary mutations in separate metastases, and three (34%) with two secondary KIT mutations in the same metastasis. The secondary mutations clustered in the KIT ATP binding pocket and kinase catalytic regions. FISH analyses revealed KIT amplicons in 2/10 metastases lacking secondary KIT mutations. This study demonstrates extensive intra- and inter-lesional heterogeneity of resistance mutations and gene amplification in patients with clinically progressing GIST. KIT kinase resistance mutations were not found in KIT/PDGFRA wild-type GISTs or in KIT-mutant GISTs showing unusual morphology and/or loss of KIT expression by IHC, indicating that resistance mechanisms are fundamentally different in these tumours. Our observations underscore the heterogeneity of clinical TKI resistance, and highlight the therapeutic challenges involved in salvaging patients after clinical progression on TKI monotherapies.     

A great majority of GISTs with PDGFRA mutations represent gastric tumors of low or no malignant potential

Gastrointestinal stromal tumors (GISTs) are KIT expressing spindle cell, epithelioid and rarely pleomorphic mesenchymal tumors. The majority of GISTs show gain-of-function KIT mutations. However, GISTs without KIT mutations and GISTs with weak or lack of immunohistochemical KIT expression have also been reported. Recently, gain-of-function mutations in exon 18 (activation loop) and exon 12 (juxtamembrane domain) of the PDGFRA were identified in such tumors. The purpose of this study was to test the hypothesis that PDGFRA mutation may define a specific clinicopathologic subgroup of GISTs. A total of 447 KIT exon 11 (juxtamembrane domain) mutation-negative GISTs were studied. DNA samples were obtained from formaldehyde-fixed paraffin-embedded tissues. Genomic sequences of PDGFRA exons 18 and 12 were evaluated for the mutations by PCR amplification and direct sequencing. PDGFRA exon 18 mutations were identified in 122 of 346 (35.3%) gastric GISTs and two of 75 (2.7%) intestinal GISTs. A great majority of these mutations represented simple T to A missense mutation at the codon 842 leading to substitution of the valine for aspartic acid (D842 V). However, in-frame deletions and deletions with point mutations clustering between codons 841-847 were found in approximately 23% of all exon 18 mutations. Mutations in PDGFRA exon 12 were found only in 10 of 170 (5.8%) gastric and one of 54 (1.9%) intestinal GISTs negative for KIT exon 11 and PDGFRA exon 18 mutations. There were seven substitutions of aspartic acid for valine at codon 561 (V561D) and four in-frame deletions with point mutations clustering between codons 566 and 571. The majority of GISTs with PDGFRA mutations had pure or predominant epithelioid morphology. Low mitotic activity, < or =5 mitoses/50HPF was detected in 81% of analyzed GISTs including larger, >5 cm tumors. Based on long-term follow-up (average 135 months), a majority (83.5%) of GISTs with PDGFRA mutations followed a benign course.     

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.