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.     

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.     

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.     

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.     

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。     

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.     

Mutations in exons 9 and 13 of KIT gene are rare events in gastrointestinal stromal tumors. A study of 200 cases

Gastrointestinal stromal tumors (GISTs), the most common mesenchymal tumors of the gastrointestinal tract, typically express the KIT protein. Activating mutations in the juxtamembrane domain (exon 11) of the c-kit gene have been shown in a subset of GISTs. These mutations lead into ligand-independent activation of the tyrosine kinase of c-kit, and have a transforming effect in vitro. Several groups have studied the clinical implication of the c-kit mutation status of exon 11 in GISTs and a possible relationship between c-kit mutations and malignant behavior has been established. Recently, a 1530ins6 mutation in exon 9 and missense mutations, 1945A>G in exon 13 of the c-kit gene were reported. The frequency and clinical importance of these findings are unknown. In this study we evaluated 200 GISTs for the presence of mutations in exons 9 and 13 of c-kit. Six cases revealed 1530ins6 mutation in exon 9 and two cases 1945A>G mutation in exon 13. All tumors with mutations in exon 9 and 13 lacked mutations in exon 11 of c-kit. None of the analyzed tumors had more than one type of c-kit mutation. All but one of the eight tumors with mutations in exon 9 or 13 of the c-kit gene were histologically and clinically malignant. All four of six cases with exon 9 mutation of which location of primary tumor was known, were small intestinal, suggesting that this type of mutation could preferentially occur in small intestinal tumors. Exon 9 and 13 mutations seem to be rare, and they cover only a small portion (8%) of the balance of GISTs that do not have mutations in exon 11 of c-kit. This finding indicates that other genetic alterations may activate c-kit in GISTs, or that KIT is not activated by mutations in all cases.     

Nestin and caveolin-1 in the diagnosis of GISTs

Gastrointestinal stromal tumors (GISTs) are the most common primary mesenchymal neoplasias of the gastrointestinal tract, typically expressing c-kit (CD117) and CD34. Recently, it was reported that nestin and caveolin-1 are also expressed in some human sarcomas, GISTs included. We performed a retrospective study on formalin fixed, paraffin embedded samples from 81 cases of confirmed GISTs, aiming to characterize their immunohistochemical profile, including nestin and caveolin-1 expressions. Tissue samples were evaluated immunohistochemically for CD117, CD34, nestin and caveolin-1. The patients (M:F 36:45), aged 46 to 84 years, had spindle cell type GISTs in 56.7% of cases, epithelioid in 30.8% and mixed pattern in 12.3%. Immunohistochemically, CD117 was positive in 88.9% of GISTs, CD34 in 85.1%, nestin in 77.7% and caveolin-1 in 71.6% of the tumors. Of nine c-kit negative GISTs, 66.7% expressed nestin, the same as caveolin-1 and 44.5% expressed both nestin and caveolin-1. Statistical analysis using Kendall's and Spearman's tests revealed significant correlations between nestin and caveolin-1 expressions (p=0.024). Our results suggest that nestin and caveolin-1 could be considered sensitive markers in GISTs, together with CD117 and CD34, for diagnostic purposes. Their significant expression in CD117 negative GISTs could represent an immunohistochemical alternative in establishing the diagnosis of these tumors.     

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.     

PKCtheta expression in gastrointestinal stromal tumor.

Gastrointestinal stromal tumor is characterized by a gain of function mutation of KIT gene and the expression of c-kit protein, but in 5% of cases, c-kit expression is negative although histological findings of gastrointestinal stromal tumor are most suspicious. The existence of c-kit-negative gastrointestinal stromal tumors points to the need of additional markers for making the diagnosis. In this study, we studied the expression of PKCtheta and correlated their expression with other immunohistochemical profiles of gastrointestinal stromal tumors and evaluated their usability as a diagnostic marker. For this purpose, 220 gastrointestinal stromal tumors were immunohistochemically stained for PKCtheta, c-kit, CD34, alpha-smooth muscle actin and S-100 protein. Additionally, genetic studies of KIT and PDGFRA genes were performed using c-kit-negative or PKCtheta-negative cases. All the 220 masses were either PKCtheta-positive or c-kit-positive. PKCtheta was positive in 212 (96%) cases and c-kit was positive in 216 (98%) cases in the cytoplasm of tumor cells with a diffuse staining pattern. Out of 212 PKCtheta-positive GISTs, 208 (98%) cases were c-kit-positive, 174 (82%) cases were CD34-positive, 62 (29%) cases were SMA-positive and S-100 protein was positive in 54 cases (26%). Genetic analyses on eight PKCtheta-negative cases showed exon 11 mutations of KIT gene in four cases. Two PKCtheta-positive and c-kit-negative GISTs showed mutations of PDGFRA gene. Our study shows that PKCtheta is a useful marker and it may play a role in the development of gastrointestinal stromal tumors. Together with c-kit, PKCtheta immunostaining can be used as an important diagnostic tool in the pathologic diagnosis of gastrointestinal stromal tumors with its high specificity and sensitivity.     

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。     

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.     

食管间质瘤与平滑肌肿瘤对照性研究

目的 探讨食管间质瘤与平滑肌肿瘤临床病理、免疫组织化学及分子生物学特点。方法 24例食管间叶源性肿瘤用CD117、CD34等一组抗体重新进行分类,部分病例同时测定c-kit基因11外显子序列。结果 此组肿瘤分别为间质瘤3例（交界性1例、恶性2例）,年龄71、56、60岁,均为男性,瘤体直径4、8、14cm,源于固有肌层。瘤细胞梭形,上皮样多角形及印戒样,呈交叉束状、栅栏状及弥漫片状排列,免疫表型为CD117、CD34弥漫强阳性。平滑肌瘤20例,年龄30－60岁,平均41.6岁,男性12例,女性8例,15例源于固有肌层,直径0.8-10.5cm（平均4.5cm),5例源于黏膜肌层,直径为0.2-1.0cm(平均0.6cm)。平滑肌肉瘤1例,男性,61例,瘤体直径5cm,源于黏膜肌层。平滑肌（肉）瘤胞质丰富,嗜伊红,交叉束状排列,免疫表型为平滑肌肌动蛋白、肌特异性肌动蛋白、结蛋白弥漫强阳性。恶性间质瘤有c-kit基因11外显子的突变,平滑肌瘤无突变。结论 食管间叶源性肿瘤仍以平滑肌瘤多见,可发生与胃肠道间质瘤相同形态与免疫表型的间质瘤,典型平滑肌肉瘤极为罕见,食管间质瘤与平滑肌瘤具有不同的临床病理学及分子生物学特征。     

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.     

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.     

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.     

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.     

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.     

Gastrointestinal stromal tumor: consistent CD117 immunostaining for diagnosis,and prognostic classification based on tumor size and MIB-1 grade

Gain-of-function c-kit gene mutations and immunoreactivity of the c-kit protein CD117 in many gastrointestinal stromal tumors (GISTs) seem to support the idea that GISTs form a biologically distinct entity. In this study, the clinicopathologic features of 171 cases of GIST at a single institution were investigated for accurate diagnosis, and their relative risk for mortality was estimated by multivariate analysis. A GIST was defined diagnostically as a mesenchymal spindle or epithelioid cell lesion arising in the wall of the gastrointestinal tract with consistent immunoreactivity for CD117. The 171 patients with GISTs comprised 96 males (56.1%) and 75 females (43.9%), with a mean age of 59.4 years. One hundred and forty-five tumors (84.8%) occurred in the stomach, 18 (10.5%) in the small intestine, 6 (3.5%) in the rectum, and 2 (1.2%) in the esophagus. The median tumor size was 4.5 cm (range, 1.2 to 38 cm). Spindle-cell GISTs were present in 132 cases (77.2%); mixed GISTs, in 25 cases (14.6%); and epithelioid GISTs, in 14 cases (8.2%). Ten cases (55.6%) of spindled small intestine GIST contained eosinophilic skeinoid fibers. Immunoreactivity for CD34, h-caldesmon, alpha-smooth-muscle actin (SMA), desmin, and S-100 was observed in 156 (91.2%), 131 (76.6%), 46 (26.9%), 7 (4.1%), and 14 (8.2%) tumors, respectively. The percentage of CD34 positivity (38.8%) was low, in contrast with the high percentage of reactivity for SMA (77.8%) and S-100 (44.4%) in small intestine GISTs. By our histologic grading system using tumor differentiation, MIB-1 score, and necrosis, 129 tumors (75.4%) were classified as low grade and 42 tumors (24.6%) were classified as high grade. With a median follow-up period of 83.5 months for 122 living patients, the 5-year and 10-year survival rates were 81.7% and 67.4%, respectively. Multivariate analysis showed that both tumor size >10 cm and high grade were significantly associated with a poor outcome. As a result, GISTs >10 cm or high grade, 5 to 10 cm and low grade, and < or =5 cm and low grade were regarded as high risk, intermediate risk, and low risk for mortality, respectively. In conclusion, it is important to recognize GISTs that have a specific molecular pathogenesis and to separate them from other mesenchymal tumors with optimal immunostaining for CD117 when making a diagnosis and prognostic classification based on tumor size and MIB-1 grade.     

胃肠道间质瘤的光镜、免疫组织化学和超微结构的观察

目的 研究胃肠道间质瘤（GISTs）的光镜,电镜形态特点和免疫组织化学在诊断中的价值,探讨肿瘤的组织来源和分型。方法 对GISTs进行光镜和超微结构的观察,用EnVision二步法免疫组织化学方法检测波形蛋白,CD117（c-kit),CD34等8种抗原标记物在肿瘤中的表达情况。结果 65例GISTs占同期消化系统间叶性肿瘤的85．5％（65／76）;其中梭形细胞为主的有46例,伴有上皮样细胞的有13例,单纯由上皮样细胞组成的有6例,瘤细胞呈长,短梭形和圆形,胞质弱嗜酸,常见核端空泡,有时呈印戒样或透明细胞样;排列呈旋涡状,栅栏状或弥漫性巢状。超微结构表现出树枝样突起,神经内分泌颗粒,桥粒样连接等神经分化特点,或（和）胞质内出现密斑,密体等肌性分化。免疫组织化学显示肿瘤组织中抗原标记物表达阳性率波表蛋白为100％（65／65）,CD11793．8％（61／65）,CD3478．5％（51／65）。结论 GISTs是消化道最常见的间叶性肿瘤,光镜形态与真性肌源性和神经源性肿瘤极为相似,电镜和CD117,CD34等免疫标记物配合使用可对其做作出正确诊断,GISTs可能起源于多潜能的,卡哈尔间质细胞样的前体细胞。