Gastrointestinal stromal tumours (GISTs) negative for KIT (CD117 antigen) immunoreactivity

Gastrointestinal stromal tumours (GISTs) are currently defined as mesenchymal tumours of the gastrointestinal tract that express KIT receptor tyrosine kinase. However, a small subgroup of tumours that fulfil the clinical and morphological criteria for GISTs lack KIT expression. So far, the biological features of these tumours have rarely been addressed. The present study describes seven gastrointestinal stromal neoplasms that presented clinicopathological features typical of GISTs but showed absence of CD117 expression as detected by immunohistochemistry. The tumours originated from the stomach (n = 5), duodenum (n = 1), and colon (n = 1), showing histologically either predominantly epithelioid (n = 3), mixed spindled and epithelioid (n = 2), or anaplastic/spindle cell (n = 2) type features. CD34 and alpha-smooth muscle actin (alpha-SMA) positivity was present in four and three tumours, respectively. Chromosomal analysis was performed in two cases, both showing losses of chromosomes 14, 22, and 1p, which is the characteristic feature of GISTs. Dual-colour interphase fluorescence in situ hybridization (FISH) analysis, utilizing chromosome 1p-, 14-, and 22-specific probes, revealed a similar cytogenetic profile in the remaining five tumour specimens. Mutational analysis of exons 9, 11, 13, and 17 of KIT, and exons 12 and 18 of PDGFRA was performed in all cases by denaturing high-pressure liquid chromatography (DHPLC) pre-screening, followed by direct sequencing. None of the tumours showed KIT mutant isoforms. Three tumours harboured PDGFRA exon 18 activating mutations; two were Asp --> Val(842) missense substitutions and one was a DIM842-844 amino acid deletion. KIT and PKC theta (protein activated in interstitial cells of Cajal and GISTs) expression was determined by western immunoblotting of the total cell lysates from three tumour biopsies. None of these three tumours expressed KIT, while all specimens showed expression of PKC theta protein. These findings indicate that there is a subgroup of KIT-negative GISTs that exhibit the same morphological, cytogenetic, and molecular features as KIT-positive tumours. While intragenic PDGFRA activating mutations are present in some of these tumours, the oncogenic events underlying the pathogenesis of the others remain unknown.     

Targeted ultradeep next‐generation sequencing as a method for KIT D816V mutation analysis in mastocytosis

Next‐generation sequencing (NGS) is becoming increasingly used for diagnostic mutation analysis in myeloid neoplasms and may also represent a feasible technique in mastocytosis. However, detection of the KIT D816V mutation requires a highly sensitive method in most patients due to the typically low mutation levels. In this study, we established an NGS‐based KIT mutation analysis and analyzed the sensitivity of D816V detection using the Ion Torrent platform. Eighty‐two individual NGS analyses were included in the study. All samples were also analyzed using highly sensitive KIT D816V mutation‐specific qPCR. Measurements of the background level in D816V‐negative samples supported a cutoff for positivity of 0.2% in three different NGS panels. Clinical samples from patients with SM that tested positive using qPCR with a D816V allele burden >0.2% also tested positive using NGS. Samples that tested positive using qPCR with an allele burden <0.2% tested negative using NGS. We thereby demonstrate that caution should be taken when using the potentially very sensitive NGS technique for KIT D816V mutation analysis in mastocytosis, as many patients with SM have D816V mutation levels below the detection limit of NGS. A dedicated and highly sensitive KIT D816V mutation analysis therefore remains important in mastocytosis diagnostics.     

High expression of the RNA-binding protein RBPMS2 in gastrointestinal stromal tumors

Gastrointestinal stromal tumors (GISTs) are the most common mesenchymal neoplasms of the gastrointestinal tract and are often associated with KIT or PDGFRA gene mutations. GIST cells might arise from the interstitial cells of Cajal (ICCs) or from a mesenchymal precursor that is common to ICCs and smooth muscle cells (SMCs). Here, we analyzed the mRNA and protein expression of RNA-Binding Protein with Multiple Splicing-2 (RBPMS2), an early marker of gastrointestinal SMC precursors, in human GISTs (n = 23) by in situ hybridization, quantitative RT-PCR analysis and immunohistochemistry. The mean RBPMS2 mRNA level in GISTs was 42-fold higher than in control gastrointestinal samples (p < 0.001). RBPMS2 expression was not correlated with KIT and PDGFRA expression levels, but was higher in GISTs harboring KIT mutations than in tumors with wild type KIT and PDGFRA or in GISTs with PDGFRA mutations that were characterized by the lowest RBPMS2 levels. Moreover, RBPMS2 levels were 64-fold higher in GIST samples with high risk of aggressive behavior than in adult control gastrointestinal samples and 6.2-fold higher in high risk than in low risk GIST specimens. RBPMS2 protein level was high in 87% of the studied GISTs independently of their histological classification. Finally, by inhibiting the KIT signaling pathway in GIST882 cells, we show that RBPMS2 expression is independent of KIT activation. In conclusion, RBPMS2 is up-regulated in GISTs compared to normal adult gastrointestinal tissues, indicating that RBPMS2 might represent a new diagnostic marker for GISTs and a potential target for cancer therapy.     

酪氨酸激酶受体KIT在小鼠结肠黏膜上皮的表达及其功能

目的 探讨酪氨酸激酶受体KIT在小鼠结肠黏膜上皮的表达及作用。 方法 应用Western blotting和RT-PCR技术检测c-kit基因及KIT
蛋白在野生型C57BL/6小鼠结肠黏膜上皮的表达;用免疫荧光染色显示野生型C57BL/6小鼠以及乙基亚硝酸钠（ENU）诱变的c-kit基因点突变纯
合子小鼠（Wads ^m/m）结肠黏膜上皮KIT阳性细胞的部位;腹腔注射5-溴脱氧尿嘧啶核苷（BrdU）（30 mg/kg）观察对比野生型以及Wads m/m
小鼠结肠黏膜上皮BrdU掺入情况及动态变化,分析KIT在结肠黏膜上皮更新过程的作用。 结果 RT-PCR检测结果表明,在野生型小鼠结肠黏膜组
织表达 c-kit基因, Western blotting证明在肠黏膜组织存在KIT蛋白;免疫荧光染色显示,KIT阳性细胞位于结肠黏膜上皮,主要位于肠腺隐
窝基底部,但是Wads ^m/m小鼠KIT阳性细胞数以及KIT蛋白表达量较野生型小鼠明显减少;BrdU掺入实验发现,Wads ^m/m小鼠远端结肠黏膜上皮
BrdU摄入和更新速度较野生型小鼠明显减慢。结论 结肠黏膜上皮表达KIT蛋白与结肠黏膜上皮的更新密切相关。     

Primary small cell carcinoma of prostate without immunoreactive neuroendocrine proteins but with expressions of KIT and platelet‐derived growth factor‐α

Primary small cell carcinoma of the prostate is extremely rare. Herein reported is a case of primary small cell carcinoma of the prostate with immunohistochemical examination of KIT and platelet‐derived growth factor‐α. The present case is unique in that the small cell carcinoma did not express neuroendocrine antigens. A 68‐year‐old man was found to have high serum prostate‐specific antigen, and biopsy showed malignant small tumor cells fulfilling the small cell carcinoma criteria of the World Health Organization. Immunohistochemically, tumor cells were positive for pan‐cytokeratin, KIT, platelet‐derived growth factor‐α, p53, Ki‐67 labeling = 65%, prostate‐specific antigen and alpha‐methylacyl‐CoA racemase. Tumor cells were negative for vimentin, CD56, synaptophysin, chromogranin and neuron‐specific enolase. Imaging modalities showed multiple metastases, and the patient was treated by chemotherapy. The present report is the fifth with immunohistochemistry of prostatic small cell carcinoma.     

KIT exon 11 codon 557/558 deletion/insertion mutations define a subset of gastrointestinal stromal tumors with malignant potential

AIM： To study the association of the frequency and pattern of KIT and PDGFRA mutations and clinicopathological factors in a group of patients with gastrointestinal stromal tumors （GIST）. METHODS： Thirty patients with GIST were examined. Exons 9, 11, 13, and 17 of the KIT and exons 12 and 18 of the PDGFRA gene were analyzed for the presence of mutations by PCR amplification and direct sequencing. RESULTS： KIT or PDGFRA mutations were detected in 21 of the 30 patients （70%）. Sixteen patients had mutations within KIT exon 11, three within KIT exon 9, and two within PDGFRA exon 18. GISTs with KIT exon 9 mutations were predominantly located in the small intestine, showed a spindle cell phenotype, and were assessed as potentially malignant. GISTs with KIT exon 11 mutations were located in the stomach and intestine, showed mainly a spindle cell phenotype, and were scored as potentially malignant （P 〈 0.05）. Tumors with KIT exon 11 codon 557/558 deletion/insertion mutations were found to be associated with a potentially malignant clinical behaviour （P 〈 0.003）. GISTs with PDGFRA mutations located in stomach showed a mixed cell phenotype and were classified as of very low or low moderate malignant potential. CONCLUSION： Determination of KIT and PDGFRA mutations should be additional parameters for the better prediction of GISTs clinical behaviour. Tumors with deletion/insertion mutations affecting codons 557/558 of the KIT gene seem to represent a distinct subset of malignant GISTs.     

Risk assessment and pathological reporting of gastrointestinal stromal tumour

Gastrointestinal stromal tumour (GIST) is the most common mesenchymal neoplasm of the GI tract. GISTs form a biological continuum ranging from benign incidentally detected minute lesions (stromal tumourlets) of no clinical significance to large and highly malignant sarcomatous neoplasms. The disease is characterized by indistinct borders between biologically different subsets within its spectrum leading to unpredictable course of the disease in the majority of cases. Availability of effective tyrosine kinase inhibitors underlined the urgent need for a reliable risk assessment system to reliably identify those patients who are at a significant risk for disease relapse and would thus profit from currently available effective targeted molecular therapy. While several risk systems have been established for GIST over the last decade, evidence is growing that no single system is optimal for all patients. Accordingly, an individualized risk system integrating classical parameters (site, size, mitotic index), status of serosal mechanical barrier covering the tumour (presence of tumour rupture or serosal penetration) as well as several other still debatable histological (coagulative necrosis, venous invasion, mucosal infiltration), biomarkers (proliferative index, p16 expression, etc.), and molecular (specific mutation types, adverse chromosomal aberrations, etc.) prognosticators would be of superb value for selecting the most appropriate patient treatment.     

Primary cutaneous neuroendocrine tumor (atypical carcinoid) expressing KIT and PDGFRA with myoepithelial differentiation: a case report with immunohistochemical and molecular genetic studies

Primary cutaneous neuroendocrine tumors (NET) except for Merkel cell carcinoma have rarely been reported. Herein reported is a very unique case of primary cutaneous NET with immunohistochemical markers of myoepitheliomas. A 47-year-old woman presented a tumor measuring 0.8x0.9x0.6 cm of the face. The tumor was excised completely with wide margins. Morphologically, the tumor was located in the dermis, and the tumor was composed of epithelioid cells arranged in trabecular, sinusoidal, rosette, ribbon-like, and cord-like patterns. Focal areas show tubular formations. The tumor cells were homogenous, and their nuclei showed hyperchromasia but no apparent histological features of malignancy were seen. The stroma was very scant. No invasive features were seen. Immunohistochemically, the tumor cells were strongly positive for cytokeratin (CK) 34BE12, CD5/6, CK14, NCAM (CD56), p63, and KIT (CD117), and moderately positive for CK AE1/3, p53, chromogranin, synaptophysin, neuron-specific enolase (NSE), PDGFRA, CA19-9, and Ki-67 antigen (labeling index=23%). The tumor cells were negative for CK CAM5.2, CK7, CK8, CK18,CK19,CK20, EMA, vimentin, CEA, HMB45, S100 protein, α-smooth muscle antigen, desmin, CD34, GFAP, neurofilaments, CD99 (MIC2), CD45, CD57, ErbB2, TTF-1, MUC1, MUC2, MUC5AC, and MUC6. Mucins examined by d-PAS and Alcian blue techniques were negative. A genetic analysis using PCR-direct sequencing method in paraffin sections identified no mutations of KIT (exons 9, 11, 13 and 17) and PDGFRA (exons 12 and 18) genes. Imaging modalities including CT and MRI identified no tumor in the body. The clinicians thought that the tumor was cured. She was a sailor and immediately visited other countries; therefore the follow-up could not be done.     

Circulating KIT D816V mutation-positive non-mast cells in peripheral blood are characteristic of indolent systemic mastocytosis

It is presently accepted that the KIT D816V mutation is detectable in tissues with neoplastic mast cells in most patients with indolent systemic mastocytosis. In this study, neoplastic mast cells were detected in bone marrow, but not in peripheral blood, by flow cytometry in all 25 included cases of indolent systemic mastocytosis. However, the KIT D816V mutation was detected using mutation-specific qPCR in both bone marrow and peripheral blood in all 25 cases, demonstrating for the first time that the KIT D816V mutation is consistently present in non-mast cells in indolent systemic mastocytosis and that these cells are circulating in peripheral blood.