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.     

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。     

神经纤维瘤病Ⅰ型伴发胃肠道外间质瘤临床病理及分子遗传学分析

目的 探讨神经纤维瘤病1型(neurofibromatosis type 1,NF1)伴发的胃肠道外间质瘤(extragastrointestinal stromal tumor,EGIST)的临床及病理形态、免疫表型及基因表型的特征.方法 光镜观察1例NF1伴发的EGIST的形态特征,采用免疫组化EnVision两步法检测CD117、CD34、PDGFRA、SMA、S-100、DOG-1、desmin及Ki-67在肿瘤组织内的表达,DNA序列分析kit和PDGFRA基因表型.结果 镜下肿瘤细胞为梭形略呈编织状排列,细胞中～重度异型,可见多灶性、凝固性坏死.免疫组化标记显示肿瘤细胞CD117和PDGFRA阳性,而CD34、SMA、S-100及desmin均为阴性,Ki-67高表达,增殖指数约20%.DNA序列分析显示kit第9、11、13和17外显子和PDGFRA第12/18外显子均为野生型,未发生突变.患者术后未予以放、化疗,目前身体状况良好.结论 NF1伴发的胃肠道间质瘤(gastrointestinal stromal tumor,GIST)多为多发性,主要见于小肠,组织形态以梭形细胞为主,DNA序列分析大多显示kit和PDGFRA均为野生型,危险度偏低.本例肿块位于腹膜后,其细胞增殖活性高,属高度危险性肿瘤,此类病变可能存在其它发病机制.     

165例胃肠道间质瘤中c-kit和PDGFRA基因突变的检测和临床诊断意义

目的探讨在中国较大样本的胃肠道问质瘤（GIST）中c-kit基因和PDGFRA基因的突变状况,为进一步的生物靶向治疗提供依据。方法用免疫组织化学EnVision法、聚合酶链反应（PCR）扩增和直接测序的方法,检测165例GIST c-kit基因9、11、13和17号外显子突变以及PDGFRA基因12和18号外显子突变。结果病理组织学诊断的165例GIST病例中有155例（94％）免疫组织化学显示CD117阳性。在CD117阳性的GIST中,c-kit基因总突变率为76．1％（118／155）：分别为11号外显子67．1％（104／155）、9号外显子7．1％（11／155）、13号外显子1．3％（2／155）和17号外显子0．6％（1／155）。绝大多数为杂合性突变,少数为纯合性突变。11号外显子的突变位点多集中在5’端的经典热点,其次为3’端的框内串联重复。后者主要以核分裂象少的老年女性胃部病例多见。9号外显子突变代表一类发生在年轻男性体积较大的小肠病变。13号外显子发现一处新的突变点L641P。PDGFRA基因突变见于50％（5／10）CD117阴性病例,均为18号外显子突变,包括常见的D842V点突变和一个框内843-846处IMHD缺失伴有S847T的新突变。PDGFRA基因突变多见于发生在后腹膜／网膜的具有高度侵袭危险性的病例。结论中国GIST病例大多数存在c-kit基因和PDGFRA基因的突变,且在基因突变类型和肿瘤原发部位问有非随机的联系。除了发现几个新的突变形式外,国人的GIST似乎和西方国家有些不同的突变特点。靶向治疗需要基因突变分型的启示和指导。     

GIST外科治疗新探

GIST(胃肠道间质瘤)是一类特殊的,通常CD117免疫表型阳性的胃肠道最常见的间叶源性肿瘤.组织学上由梭形细胞、上皮样细胞、偶或多形性细胞排列成束状或弥漫状图像,免疫表型上表达c-kit基因蛋白产物KIT,由突变的c-kit或血小板源生长因子受体(PDGFRA)基因驱动.本文主要运用文献资料法、逻辑推理法以及笔者多年工作经验,针对GIST外科治疗进行论述,希望能进一步促进GIST治疗手段的进步.     

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…     

胃原发中度危险性胃肠道间质瘤36例临床病理观察

目的：探讨胃原发的中度危险性胃肠道间质瘤(gastrointestinal stromal tumors,GIST)临床病理特征,提高对改组患者的诊治水平。方法：回顾性分析36例胃原发中度危险性胃肠道间质瘤的临床病理资料。结果：36例患者年龄21~69岁,平均55岁,男女比例为0.64。临床主要表现为腹痛及黑便。发生在胃体部28例,胃底部5例,胃窦部3例,肿瘤大小平均7.5 cm (2.5~10 cm)。组织学表现为中等密集的肿瘤细胞流水样或弥漫分布,肿瘤细胞梭形或上皮样或二者混合,核分裂象平均4.8/50HPF(2~10/50HPF),免疫组织化学染色CD117、PDGFRA、DOG-1、CD34阳性表达率分别为91.7%、88.9%、80.6%和97.2%,Ki67指数平均为6.5%(2%~8%)。随访时间8~50个月,患者1、3、5年生存率分别为97.2%、88.9%、83.3%,3例死于肿瘤复发、2例死于转移、1例死于并发症。所有患者均接受手术治疗,其中15例c-kit基因11外显子检测阳性的患者中8例术后接受伊马替尼治疗,其预后好于未接受伊马替尼治疗的患者,差异有统计学意义(P<0.05)。结论：胃原发中度危险性GIST确诊需靠病理形态学及免疫组织化学,c-kit基因检测阳性的患者手术后辅助伊马替尼治疗可明显改善患者预后。     

胃肠道间质瘤病理及CD117和CD34表达与意义

目的：对本院病案资料中GIST重新分类，加 强对胃肠道间质瘤的认识。方法：用CD117、CD34、α-SMA、S-100等抗 体对56例GIST进行标记、分类，根据GIST的临床病理及形态学特点，正确诊断，并探讨 GIST的临床特点。结果:56例GIST CD117、CD34弥漫强表达，抗体 阳性率分别为CD117（50/56，89.3%）、CD34（37/56，66.1%），部分病例局灶表达α-SM A、S-100，阳性率分别为（17/56，30.4%）、（4/56，7.1%），结蛋白desmin均阴性。其 中良性及交界性29例，恶性27例。本组病例中胃及小肠GIST共达91.1％，其它部位少见。 结论：CD117、CD34在GIST表达显著，可作为GIST诊断的一个辅助指标。 胃和小肠GIST最常见，胃镜等影像学无特异性，诊断主要依靠病理诊断。组织病理不仅有助 于诊断，而且有助于预后的判断。     

29例上皮样胃肠间质瘤的临床病理特征

目的:探讨29例上皮样胃肠间质瘤(gastrointestinal stromal tumors,GIST)的组织学形态和免疫组织化学特点以及诊断和鉴别诊断,对临床正确诊断治疗和判断预后具有十分重要的临床意义.方法:回顾性分析安阳市肿瘤医院病理科2009年月3月至2016年8月321例完整切除GIST标本,经筛查并重新阅片诊断上皮样GIST 29例.结果:29例上皮样GIST,发生胃部15例,小肠2例,肠系膜3例,直肠4例,腹腔3例,腹膜后1例,盆腔1例.恶性GIST 25例,良性4例.瘤细胞丰富,胞质嗜酸或透明,部分瘤细胞核质比高,核大小不等具有多形性,核分裂较多,可伴有多灶凝固性坏死,间质多数伴有黏液样变性.组织结构形成器官样、片状、巢状及腺泡状等.免疫组织化学CD34,DOG-1在上皮样GIST均弥漫阳性(阳性率100%),CD117阳性率(86%).结论:上皮样GIST发生部位广泛,形态多变,易误诊其他上皮样分化的肿瘤;免疫组织化学CD34,DOG-1,CD117在上皮样GIST诊断及鉴别诊断中具有重要价值;如肿物较大、细胞丰富、核分裂多、间质黏液样变性;绝大多数要考虑恶性GIST.     

Gastrointestinal stromal tumors (GISTs): CD117, DOG-1 and PKCθ expression. Is there any advantage in using several markers?

Gastrointestinal stromal tumors (GISTs) are the most common mesenchymal tumors of the digestive tract. Expression of CD117, DOG1 and PKCθ was investigated immunohistochemically in a series of 99 paraffin-embedded GISTs in order to determine the sensitivity and diagnostic value of these markers. KIT exons 9, 11, 13 and 17 and PDGFRA exons 12 and 18 were amplified by PCR and sequenced. A total of 94/99 (94%) GISTs stained positive for CD117, 81/99 (82%) for PKCθ and 90/99 (91%) for DOG-1. A significant correlation was noted between CD117 and DOG-1 expression (p=0.0001). All three markers were expressed in 74% (73/99) of GISTs. Of the five CD117-negative cases, two were PKCθ-negative/DOG1-negative and had mutations in KIT exon 11. Two were PKCθ-positive/DOG1-positive and had mutations in PDGFRA (one each in exons 12 and 18), and one was DOG1-negative/PKCθ-positive, with a PDGFRA exon 18 mutation. The most sensitive marker was CD117, followed by DOG-1 and PKCθ. Although PKCθ was less sensitive, and its staining is more challenging and difficult to interpret, the use of this marker is highly recommended, particularly in CD117-negative/DOG-1-negative GISTs.     

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.     

Use of tissue microarrays and immunohistochemistry to standardize the diagnosis of gastrointestinal stromal tumors

We assessed the concordance among seven general pathologists with respect to histologic diagnosis and interpretation of c-kit proto-oncogene (KIT) and platelet-derived growth factor receptor alpha (PDGFRA) immunostaining of 36 cases of primary spindle-cell tumor, predominantly of the gastrointestinal tract, mesentery, and retroperitoneum, based on review of a tissue microarray (TMA) subjected to immunohistochemistry with antibodies to KIT/CD117, PDGFRA, vimentin, desmin, smooth muscle action, CD34, and S-100 protein. Tumors included 20 molecularly analyzed gastrointestinal stromal tumors (GISTs), 4 leiomyosarcomas, 4 schwannomas, 4 desmoid-type fibromatoses, and 4 solitary fibrous tumors. The mean overall concordance with original diagnosis for each histologic type was 91.1%, with a mean kappa value of 0.91. With respect to PDGFRA immunostaining, the four GISTs with PDGFRA mutation were interpreted as cytoplasm positive, but the 16 GISTs with c-kit mutation were interpreted as weak or positive. These results indicate that the overall concordance with original diagnosis in mesenchymal tumors with the use of immunohistochemical panels is high, despite the use of TMAs. To some extent, PDGFRA immunophenotyping may be useful in GISTs with PDGFRA mutation, but it was not highly reproducible or specific. Therefore, in KIT-negative or weakly positive GISTs, mutation analysis will be required.     

Extragastrointestinal stromal tumor of the urinary wall bladder: case report and review of the literature

Most mesenchymal tumours of the gastrointestinal tract are now referred to as gastrointestinal stromal tumours (GISTs). These tumours typically express c-kit (CD117) and CD34; 30-50% are (often focally) positive for alpha-smooth muscle actin, and all are negative for desmin and S100 protein. Recently, mutations in exon 11 of the c-kit gene have been identified as a molecular genetic marker for the subset of GISTs. In this report, we describe a mesenchymal tumour removed from the pelvic cavity of a 34-year-old man. The tumour was strongly attached to the external wall of the urinary bladder. The neoplasm grossly resembled a leiomyoma, and was histologically composed of sheets of spindle cells with a dense collagenous background. The mitotic activity was low (less then 1 per 50 high-power fields). Immunohistochemically, tumour cells were negative for alpha-smooth muscle actin and desmin and positive for CD117 and CD34. This case illustrates that tumours which are phenotypically and genotypically similar to GISTs may present in sites other than the tubular gastrointestinal tract.     

Loss of DOG-1 expression associated with shift from spindled to epithelioid morphology in gastric gastrointestinal stromal tumors with KIT and platelet-derived growth factor receptor α mutations

Most gastric gastrointestinal stromal tumors (GISTs) display spindle cell morphology and coexpress CD117 (KIT), DOG-1, and CD34. Secondary loss of DOG-1 has not been reported. We present two gastric GISTs which showed loss of DOG-1 in the epithelioid component but retained its expression in the minor spindle cell component. Patients were a 67-year-old man and an 80-year-old woman with 4.8-cm and 3.5-cm gastric GISTs harboring mutations in KIT exon 11 (c.1729_1758dup30; p.P577_R586dup) and platelet-derived growth factor receptor α (PDGFRA) exon 18 (c.2527_2538del12; p.I843_D846del), respectively. Both were predominantly epithelioid with a minor microscopic spindle cell component (3-12 mm). The spindle cell component was CD117⁺CD34⁺DOG-1⁺ in both cases. The epithelioid component in case 1 was CD117⁺CD34⁺DOG-1^?. In case 2, the epithelioid component strongly expressed PDGFRA (dot-like) but lost CD117, CD34, and DOG-1. These cases confirm the immunophenotypic heterogeneity as secondary events in GIST. Loss of DOG-1 in KIT-negative PDGFRA mutants should not preclude diagnosis.     

Spindle cell tumor of urinary bladder serosa with phenotypic and genotypic features of gastrointestinal stromal tumor

Most mesenchymal tumors of the gastrointestinal tract are now referred to as gastrointestinal stromal tumors (GISTs). The tumors differ from ordinary leiomyomas and schwannomas in several respects: the GISTs typically express c-kit protein (CD117) and CD34, 30% to 50% of them are (often focally) positive for alpha-smooth muscle actin, and all are negative for desmin and S100 protein. Recently, mutations in the exon 11 of the c-kit gene have been identified and confirmed as a molecular genetic marker for the subset of GISTs. In this report, we describe a mesenchymal tumor removed from the pelvic cavity of a 52-year-old woman, who is alive without disease 36 months after the surgery. The 5-cm tumor was densely attached to the external aspect of the urinary bladder but was attached to small intestine by only filmy adhesions. The tumor grossly resembled a leiomyoma and was histologically composed of sheets of spindle cells with a dense collagenous background. The mitotic activity was low, less then 1 per 50 high-power fields. Immunohistochemically, the tumor cells were negative for alpha-smooth muscle actin and desmin and positive for CD117 and CD34. Molecular genetic analysis of the exon 11 of the c-kit gene revealed a point mutation in the region commonly mutated in GISTs. This mutation substituted T for A in the codon 557, leading to the change of amino acid sequence (tryptophan for arginine) of the KIT protein. This case illustrates that tumors phenotypically and genotypically similar to GISTs may present in sites other than the tubular gastrointestinal tract.