C-kit Gene Research Articles

Molecular mechanism of gastrointestinal stromal tumors and progress in drug research

The functional mutation of c-kit and platelet-derived growth factor receptor α (PDGFRA) which encode proto-oncogene receptor tyrosine kinase are the crucial pathogeneses of gastrointestinal stromal tumors(GISTs). 80%-85% c-kit gene mutation including exon 11,exon 9,exon 13,exon 17 and 5%-10% PDGFRA gene mutation such as exon 18, exon 12 are examined in GISTs. Neither of c-kit or PDGFRA gene mutation are called wide type GISTs. The pathogeneses of wild type GISTs are not clear. The deficiency of succinate dehydrogenase B(SDHB)-related insulin-like growth factor 1(IGF-1R) activation, BRAF gene mutation and neurofibromatosis type 1 may be related to progression of wild type GISTs. More than half of metastatic GISTs patients receiving imatinib treatment can develop to c-kit secondary mutations, which are responsible for secondary resistance. However, the reasons of imatinib resistance in GISTs without c-kit secondary mutation need to be explored. At present, many clinical trials are ongoing to evaluate new drugs in GISTs treatment, including nilotinib, masitinib, pazopanib, dovitinib, ponatinib, dasatinib, crenolanib, linsitinib and immunotherapy, which may bring resistance GISTs treatment to new hope. Next generation sequencing (NGS) and liquid biopsy will be very important in GISTs research and clinical practice.

Molecular Characterization of Pediatric Acute Myeloid Leukemia: Results of a Multicentric Study in Brazil

The biological characterization of childhood acute myeloid leukemia (c-AML) is an important outcome predictor. In Brazil, very little is known about the frequency of AML subgroups, although c-AML accounts for about 18% of leukemias. We carried out this study to investigate the contribution of type I and II gene mutations in the probability of overall survival (pOS) of c-AML in Brazil. Seven hundred and three de novo pediatric AML cases (2000-2015) were assessed throughout a multicentric network study. Mutations in hotspot regions of FLT3, NRAS, KRAS, PTPN11, and c-KIT genes were analyzed as well as fusion genes (RUNX1-RUNX1T1, MLL/KMT2A-r, CBFβ-MYH11, and PML-RARα) associated with AML. Patients were treated out of the clinical trial although following the BFM-AML2004 protocol. Acute promyelocytic leukemia (APL) was treated differently. AML with Down syndrome was excluded. There were significant differences in gene mutations among age ranges (≤2years-old; >2-10years old and ≥11years old) and the nonrandom association between type I/II mutations. Lower white blood cell count (≤50 × 109/L) was associated with RUNX1-RUNX1T1, whereas higher WBC with CBFβ-MYH11 (p <0.05). Cumulative pOS in 5years was 37.7±2.8% for total AMLs and 59.8±6.2% for APL (p=0.03). pOS differences were observed between Brazilian regions. The South-Southeast regions had a better 5-year pOS, whereas the Midwest region presented the poorest pOS (23.7 ±4.9%). PTPN11 mutations conferred an adverse prognosis as an independent prognostic factor. Identification of genetic subgroups contributes to the molecular epidemiology and biology of AML worldwide, reflecting the profile of pediatric AML cases in Brazil.

Canine lymphomas: DNA changes in tumour genes

Lymphoma is the most frequent hematopoietic neoplasm in dog, and it shares some clinical, cytological, histopathological and molecular similarities with humans. In our study, DNA changes in TP53 , c-KIT and N-RAS were detected in lymphoma tissues of cross breed bitch and Bernese Mountain Dog bitch. One transversion A/G at nucleotide position 138 in exon 5 in TP53 gene and one transversion A/G at nucleotide position 35 in exon 8 of c-KIT gene were observed in donor 2. It seems that these genes did not play a key role in canine lymphoma formation and progression.

Disruption of c-Kit Signaling in Kit(W-sh/W-sh) Growing Mice Increases Bone Turnover.

c-Kit tyrosine kinase receptor has been identified as a regulator of bone homeostasis. The c-Kit loss-of-function mutations in WBB6F1/J-KitW/W-v mice result in low bone mass. However, these mice are sterile and it is unclear whether the observed skeletal phenotype is secondary to a sex hormone deficiency. In contrast, C57BL/6J-KitW-sh/W-sh (Wsh/Wsh) mice, which carry an inversion mutation affecting the transcriptional regulatory elements of the c-Kit gene, are fertile. Here, we showed that Wsh/Wsh mice exhibited osteopenia with elevated bone resorption and bone formation at 6- and 9-week-old. The c-Kit Wsh mutation increased osteoclast differentiation, the number of committed osteoprogenitors, alkaline phosphatase activity and mineralization. c-Kit was expressed in both osteoclasts and osteoblasts, and c-Kit expression was decreased in Wsh/Wshosteoclasts, but not osteoblasts, suggesting an indirect effect of c-Kit on bone formation. Furthermore, the osteoclast-derived coupling factor Wnt10b mRNA was increased in Wsh/Wsh osteoclasts. Conditioned medium from Wsh/Wsh osteoclasts had elevated Wnt10b protein levels and induced increased alkaline phosphatase activity and mineralization in osteoblast cultures. Antagonizing Wnt10b signaling with DKK1 or Wnt10b antibody inhibited these effects. Our data suggest that c-Kit negatively regulates bone turnover, and disrupted c-Kit signaling couples increased bone resorption with bone formation through osteoclast-derived Wnt 10 b.

Frequency and clinical features of ASXL2 gene mutation in acute myeloid leukemia patients with AML1- ETO fusion gene positive

目的探讨伴AML1-ETO融合基因的急性髓系白血病（AML）患者ASXL2基因突变情况、突变阳性患者临床特征及ASXL2基因突变与c-kit基因突变的关系。方法采用PCR扩增产物片段直接测序分析法，检测59例伴AML1-ETO融合基因初发AML患者ASXL2基因第11、12外显子编码区突变情况，比较ASXL2基因突变阳性和阴性组患者的临床特征、生存及c-kit基因突变情况。结果59例患者中7例存在ASXL2突变，突变率为11.9%。ASXL2基因突变阳性组患者初诊时外周血红蛋白浓度中位数为56.2（38.0~72.0）g/L，显著低于ASXL2突变阴性组患者的69.0（37.2~154.0）g/L，差异有统计学意义（P=0.038）；外周血WBC、PLT、嗜酸粒细胞比例、骨髓原始细胞比例与ASXL2突变阴性组相比，差异均无统计学意义（P值均>0.05）。两组均未见肝、脾、中枢神经系统浸润；淋巴结不同程度肿大，但ASXL2基因突变阳性、阴性两组间差异无统计学意义（P=0.859）。免疫表型分析显示：ASXL2基因突变阳性组CD33表达显著低于阴性组（P=0.033）；两组患者均未表达cCD3，CD117、cMPO、HLA-DR、CD34、CD38、CD13、CD44、CD15、CD64、CD11b、CD56、CD19、cCD79a、CD7两组表达差异均无统计学意义（P值均>0.05）。ASXL2基因突变阳性与阴性组患者总缓解率、总生存时间差异均无统计学意义（P值分别为0.577、0.631）。两组c-kit基因突变检出率分别为14.3％和29.4%，差异无统计学意义（P=0.697）。结论该组伴AML1-ETO融合基因AML患者ASXL2基因突变率为11.9%。ASXL2突变阳性患者外周血红蛋白浓度、CD33表达方面呈现一定的临床特征。ASXL2基因突变与c-kit基因变突可能没有特定的关联性。

Receptor tyrosine kinase (c-Kit) inhibitors: a potential therapeutic target in cancer cells.

c-Kit, a receptor tyrosine kinase, is involved in intracellular signaling, and the mutated form of c-Kit plays a crucial role in occurrence of some cancers. The function of c-Kit has led to the concept that inhibiting c-Kit kinase activity can be a target for cancer therapy. The promising results of inhibition of c-Kit for treatment of cancers have been observed in some cancers such as gastrointestinal stromal tumor, acute myeloid leukemia, melanoma, and other tumors, and these results have encouraged attempts toward improvement of using c-Kit as a capable target for cancer therapy. This paper presents the findings of previous studies regarding c-Kit as a receptor tyrosine kinase and an oncogene, as well as its gene targets and signaling pathways in normal and cancer cells. The c-Kit gene location, protein structure, and the role of c-Kit in normal cell have been discussed. Comprehending the molecular mechanism underlying c-Kit-mediated tumorogenesis is consequently essential and may lead to the identification of future novel drug targets. The potential mechanisms by which c-Kit induces cellular transformation have been described. This study aims to elucidate the function of c-Kit for future cancer therapy. In addition, it has c-Kit inhibitor drug properties and their functions have been listed in tables and demonstrated in schematic pictures. This review also has collected previous studies that targeted c-Kit as a novel strategy for cancer therapy. This paper further emphasizes the advantages of this approach, as well as the limitations that must be addressed in the future. Finally, although c-Kit is an attractive target for cancer therapy, based on the outcomes of treatment of patients with c-Kit inhibitors, it is unlikely that Kit inhibitors alone can lead to cure. It seems that c-Kit mutations alone are not sufficient for tumorogenesis, but do play a crucial role in cancer occurrence.

Analysis of common gene mutations and cytogenetics in core binding factor related acute myeloid leukemia

Objective To assess the prevalence of c-Kit and FLT3 gene mutations in core binding factor related acute myeloid leukemia (CBF-AML) and analyze the karyotype characteristics of the CBF-AML patients. Methods Mutations of c-Kit, FLT3-ITD and FLT3-TKD were detected by genomic DNA PCR and sequencing, and the karyotype changes were analyzed in 48 newly diagnosed CBF-AML patients. Results c-Kit aberrations were detected in 13(27.1%) out of 48 patients, including 5 cases with exon 8 mutation and 8 cases with exon 17 mutation. c-Kit was more prominent in t(8; 21) AML patients than in inv(16) AML patients [(33.3% (9/27) vs 19.0% (4/21), P<0.05]. Only 1 case (2.1%) had FLT3-ITD mutation (FLT3-ITD+) and 3 cases(6.3%) had FLT3-TKD mutation (FLT3-TKD+). Prevalence of RUNX1-RUNX1T1 with additional chromosome abnormality was as high as 25.9%(7/27), in which sex chromosome elimination was the most common one, while prevalence of CBFβ-MYH11 with additional chromosome abnormality was low. Conclusion c-Kit gene mutations and RUNX1-RUNX1T1 additional chromosome abnormalities are common in patients with CBF-AML and would be helpful for individualized treatment studies. Key words: Leukemia, myeloid, acute; Core binding factor; c-Kit; FLT3; Cytogenetics

Abstract 3107: A single ddPCR assay to detect KIT exon 11 mutations in tumor and cell free plasma DNA of patients with gastrointestinal stromal tumors

Abstract . Introduction Gastrointestinal stromal tumors (GIST) are rare mesenchymal tumors of the gastrointestinal tract. These tumors are characterized by genomic mutations in the c-KIT gene that drive tumor growth and are target for specific inhibitors. Mutations in exon 11 (single nucleotide variations and deletions) are detected in approximately 70% of GIST. Although unique for each patient, most variations cluster in two different mutational hotspots each 25bp in size. The aim of this study was to develop and test a single quantitative digital droplet PCR (ddPCR) assay to detect exon 11 c-KIT mutations in both formalin fixed paraffin embedded (FFPE) tissue and serial collected cell-free plasma DNA (cfDNA) of GIST patients for diagnostic and treatment response monitoring. Materials and methods The drop-off ddPCR assay for detection of exon 11 mutations consists of two fluorescent (FAM and HEX) labeled probes that each cover one of the 2 mutation hotspots. Since double exon 11 mutations are very rare in GIST, one probe will anneal to the non-mutated sequence (reference probe), while the other probe will not anneal. A decrease of one of the 2 fluorescent signals is considered as the presence of a mutation (“drop-off”). For validation, ddPCR results from tumor biopsies were compared with data from next generation sequencing (NGS) using the IonTorrent platform. Pretreatment FFPE tumor material was obtained from 29 (18 exon 11 mutated, 11 non-exon 11 mutated) GIST patients treated in 2014 and 2015. Plasma samples were collected at baseline (before start of treatment with imatinib) and at consecutive time points during treatment. Results Using the drop-off ddPCR assay on the same DNA used for NGS, mutations in 17 of the 18 samples were detected. Comparison of the allelic fraction of the mutation shows that ddPCR analysis is very similar to NGS. In 11 control samples, who were wild-type (4 samples) or had mutations other than c-KIT exon 11 (7 samples) no loss of signal was detected. CfDNA was analyzed of a 72-year old representative patient with metastatic GIST carrying a c-KIT deletion in exon 11 with a fractional abundance (FA) in tissue of 91%. We detected the mutation using the drop-off ddPCR assay in the plasma sample collected before start of imatinib treatment (FA 16%). Two weeks after initiation of imatinib a rise in mutant allelic frequency was observed (FA 61%) and a decrease after 4 weeks (FA 3%) of therapy. Conclusion The detection of multiple exon 11 mutations/deletions using a single ddPCR assay could be used as a rapid prescreening method for the detection of c-KIT mutations in FFPE tissue in GIST patients. Because of the quantitative nature of this assay, ddPCR analysis might be used to monitor response to treatment. The relevance of this drop-off assay for treatment decision making seems promising and is currently validated on a large series of cfDNA samples and a prospective study in GIST patients (NCT02331914). Citation Format: Pieter A. Boonstra, Marco Tibbesma, Lisette J. Bosman, Ron H.J. Mathijssen, Frits van Coevorden, Neeltje Steeghs, Albert J.H. Suurmeijer, Arja ter Elst, Jourik A. Gietema, Anna K.L. Reyners, Ed M.D. Schuuring, Dutch GIST Consortium. A single ddPCR assay to detect KIT exon 11 mutations in tumor and cell free plasma DNA of patients with gastrointestinal stromal tumors. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3107.

Abstract 3341: Microarray analysis of gastrointestinal stromal tumor-originated spheroids

Abstract Background: Although the main cause of gastrointestinal stromal tumor (GIST) is gain-of-function mutations in the c-kit gene in the interstitial cells of Cajal, concomitant genetic or epigenetic changes other than c-kit are thought to occur during tumor progression. Although sequential therapy with tyrosine kinase inhibitors (TKIs), imatinib mesylate, sunitinib malate and regorafenib, is an effective and standard treatment strategy for recurrent or metastatic GISTs, it is difficult to cure recurrent GISTs with TKIs alone. It has been reported that viable cells are often seen in tumors resected in response to imatinib therapy and they may cause refractory tumors after interruption of imatinib in responding patients with advanced GISTs. Therefore targeting these persistent clones, which may have stem cell characteristics, can be a novel treatment strategy to cure recurrent or metastatic GISTs. In this study, we generated spheroids from resected GIST and performed microarray analysis to identify gene sets typically expressed in GIST-originated spheroid (GISTOS). Methods: According to the previous report of cancer tissue originated spheroid (CTOS) (Kondo J et al. PNAS 108:6235-40, 2011), GISTOS was prepared from fresh samples of 6 primary gastric GISTs. Total RNAs were extracted from 6 GISTOS and their original tumors and utilized for cDNA microarray analysis. None of the patients had received imatinib therapy before surgery. Gene expression levels were compared between GISTOSs and their original tumors. Results: Hierarchical clustering revealed two distinct clusters, and distinguished GISTOS from original tumors. Gene set enrichment analysis (GSEA) revealed that the gene sets CISPLATIN_RESISTSNCE, METASTASIS_STROMAs and TUMOR_INVASIVENESS_2D were upregulated and gene sets CELL_CYCLE_G1_S and MIR21_TARGETS were downregulated in GISTOSs compared with original tumors. These gene sets appear to reflect distinctive features of GISTOS that consists of enriched stem cell populations. Conclusions: We generated GIST-originated spheroids. Microarray analysis revealed gene sets up-regulated and down-regulated in GISTOS. These gene sets appear to reflect distinctive features of GISTOS and may serve as novel molecular markers of stem cells in GIST. Citation Format: Hirotoshi Kikuchi, Shinichiro Miyazaki, Tomohiro Murakami, Tomohiro Matsumoto, Yusuke Ozaki, Yoshihiro Hiramatsu, Kinji Kamiya, Manabu Ohta, Takanori Sakaguchi, Hiroyuki Konno. Microarray analysis of gastrointestinal stromal tumor-originated spheroids. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3341.

Internal associations and dynamic expression of c-kit and nanog genes in ventricular remodelling induced by adriamycin.

The present study aimed to investigate the dynamic expression of the c-kit and nanog genes in rats with left ventricular remodelling induced by adriamycin (ADR), and explore its internal association and mechanism of action. Sprague-Dawley male rats were randomly divided into a normal control group and a heart failure model group. Heart failure was induced by a single intraperitoneal injection of ADR (4 mg/kg) weekly for six weeks. The normal control group was given the same amount of saline. At the eighth week, rat cardiac function was examined to demonstrate the formation of heart failure. The rat hearts were harvested frozen and sectioned, and the expression levels of the nanog and c-kit genes in the myocardial tissue samples were detected using immunohistochemistry, immunofluorescence and reverse transcription-polymerase chain reaction (RT-PCR). Hematoxylin and eosin staining demonstrated various pathological changes in the myocardial cells in the heart failure model group, whereas myocardial infarction was not observed in the normal control group. Immunohistochemistry and immunofluorescence demonstrated that nanog-positive cells were predominantly expressed in the vascular endothelium, with a few myocardial cells and stem cells in normal myocardium. The expression levels of c-kit and nanog in the myocardium of the rats with heart failure decreased significantly. c-kit-positive cells clustered together in the epicardium and its vicinity, and c-kit expression significantly decreased in the myocardium of rats with heart failure, as compared with normal rats. In both groups, some cells co-expressed both the c-kit and nanog genes. The RT-PCR results demonstrated that the expression levels of the two genes in the heart failure model group were significantly lower compared with those in the normal control group (P<0.05). In conclusion, the c-kit- and nanog-positive stem cells decreased in the myocardium of the rats with left ventricular remodelling induced by ADR. Their abnormal expression was significantly correlated with left ventricular remodelling, thereby indicating an internal association (influences of two indexes in the experimental group and control group) between them.

Activated c-Kit receptor in the heart promotes cardiac repair and regeneration after injury.

The role of endogenous c-Kit receptor activation on cardiac cell homeostasis and repair remains largely unexplored. Transgenic mice carrying an activating point mutation (TgD814Y) in the kinase domain of the c-Kit gene were generated. c-KitTgD814Y receptor was expressed in the heart during embryonic development and postnatal life, in a similar timing and expression pattern to that of the endogenous gene, but not in the hematopoietic compartment allowing the study of a cardiac-specific phenotype. c-KitTgD814Y mutation produced a constitutive active c-Kit receptor in cardiac tissue and cells from transgenic mice as demonstrated by the increased phosphorylation of ERK1/2 and AKT, which are the main downstream molecular effectors of c-Kit receptor signaling. In adult transgenic hearts, cardiac morphology, size and total c-Kit+ cardiac cell number was not different compared with wt mice. However, when c-KitTgD814Y mice were subjected to transmural necrotic heart damage by cryoinjury (CI), all transgenic survived, compared with half of wt mice. In the sub-acute phase after CI, transgenic and wt mice showed similar heart damage. However, 9 days after CI, transgenic mice exhibited an increased number of c-Kit+CD31+ endothelial progenitor cells surrounding the necrotic area. At later follow-up, a consistent reduction of fibrotic area, increased capillary density and increased cardiomyocyte replenishment rate (as established by BrdU incorporation) were observed in transgenic compared with wt mice. Consistently, CD45−c-Kit+ cardiac stem cells isolated from transgenic c-KitTgD814Y mice showed an enhanced endothelial and cardiomyocyte differentiation potential compared with cells isolated from the wt. Constitutive activation of c-Kit receptor in mice is associated with an increased cardiac myogenic and vasculogenic reparative potential after injury, with a significant improvement of survival.

Frequency of c-kit mutation and prognosis in t(8;21) acute myeloid leukemia patients with trisomy 4

Objective To investigate the frequency of c-kit mutation and prognosis in t(8;21) acute myeloid leukemia (AML) patients with trisomy 4. Methods A total of 145 de novo t(8;21) AML patients from February 2005 to January 2013 were analyzed retrospectively. Detection of exons 8 and 17 mutation of c-kit by PCR and cytogenetic analysis by R-banding technologies were performed on bone marrow samples of all patients at diagnosis. Clinical data were collected and analyzed statistically. Results Among 145 t(8;21) AML patients, 12 cases (8.3 %) were trisomy 4, 91.7 % (11/12) of them were identified with c-kit mutation, which was significantly higher than that without trisomy 4 [26.3 % (35/133), P < 0.01]. The follow-up data showed that the patients with trisomy 4 were correlated with the lower overall survival (OS) rate (15 % vs 56 %, P< 0.01) and disease-free survival (DFS) rate (0 vs 51 % , P < 0.01) when compared with patients without trisomy 4. Furthermore, the subgroup of patients with both trisomy 4 and c-kit mutation had a worse OS and DFS (P < 0.05). Conclusions Trisomy 4 is associated with high frequency of c-kit mutation and demonstrates poor prognosis in t(8;21) AML patients. Trisomy 4 or it combined with c-kit gene mutation is the main influencing factor on the survival of the patients with t(8; 21) AML. Key words: Leukemia, myeloid, acute; Trisomy 4; c-kit; t(8;21); Prognosis

P-186 The molecular pathology characteristics of c-Kit and PDGFRA gene mutation in gastrointestinal stromal tumors in China

P-186 The molecular pathology characteristics of c-Kit and PDGFRA gene mutation in gastrointestinal stromal tumors in China

Tumor B7-H1 regulates cancer stem cell generation and virulence

Abstract Cancer stem cells (CSC) resist chemotherapy and radiation, leading to treatment failures and reduced patient survival. Thus, CSC are clinically relevant therapy targets. B7-H1 is an immune checkpoint molecule and a target for αB7-H1 immunotherapy. We generated control (native B7-H1+) and B7-H1lo B16 melanoma lines by shRNA. B16*ctrl cultures were 4.2% CSCs (CD44+CD133+CD24+) vs. 0.19% in B16*B7-H1lo. CSC from B16*B7-H1lo cultures formed fewer and smaller (&amp;lt;50 μm) microspheres vs. many, large (100–150 μm) microspheres from control CSC. B16*ctrl CSC were more tumorigenic in mice than B7-H1lo CSC. B7-H1 expression was 2-fold higher on CSC vs. non-CSC. αB7-H1 reduced CSC numbers in B16*B7-H1lo cultures (~4-fold) but not control cultures. PD-1 expression was 2-fold higher in B16*B7-H1lo non-CSC vs B16*ctrl non-CSC. PD-1 was 5-fold higher in B7-H1lo CSC vs. B7-H1lo non-CSC, but equal in B16*ctrl CSC vs. non-CSC, suggesting PD-1 regulation by B7-H1. αPD-1 reduced CSC only in B7-H1*ctrl cultures (18.9% to 2.4%). 0.1 ng/ml IFN-γ for 48 hours reduced total cell counts in control vs. B7-H1lo cultures (by 55% and 65%, respectively). Strikingly, IFN-γ increased B7-H1lo CSC numbers 9.5-fold vs. 5.6-fold for control CSC. In parallel work, we tested ID8agg ovarian cancer cells. CSC (CD24+CD44+) were 12.2% vs. 2.2% in ID8*ctrl vs. ID8agg*B7-H1lo culture. B7-H1 and PD-1 expression was similar to B16. RNA-seq of total ID8agg cells showed significantly lower expression of CSC associated genes c-Kit, Lin28A and CD24 in B7-H1lo cells. Classic CSC genes were too low to read. B7-H1 is a candidate CSC regulator. It affects CSC PD-1 expression and alters αB7-H1, αPD-1 and IFN-γ effects differentially in CSC vs non-CSC suggesting novel pathogenic mechanisms.

Detection and clinical research of FMS-like tyrosine kinase-3, NPM1 and c-kit genes in bone marrow slides of patients with acute myeloid leukemia

Objective To study the FMS-like tyrosine kinase-3 (FLT3) gene, NPM1 gene and c-kit gene mutations in acute myeloid leukemia (AML) by extracting DNA from the storage of bone marrow slides, and to investigate the relationship between the three gene mutations and clinical features in AML. Methods The bone marrow slides of 55 patients diagnosed with AML were enrolled in this study. The PCR, DNA sequencing and molecular cloning were used to detect and analyse the FLT3-ITD, NPM1 and c-kit gene mutations. Patients' remission, progression and survival time were also recorded. Results The DNA was successfully extracted from the bone marrow slides with-20 ℃ frozen storage without Wright stained, chemically fixed, and room temperature storage Wright stained discoloured by phenol∶chloroform∶isoamyl alcohol method, which can be used in PCR, direct sequencing and molecular cloning sequencing analysis. 10 of the 55 cases (18.2 %) were FLT3-ITD positive, including 9 cases with heterozygous mutations and 1 case with homozygous mutation. FLT3-ITD positive group had lower complete remission (CR) rate, shorter event-free survival (EFS) time and overall survival (OS) time than the negative group (P<0.05). 9 of the 55 cases (16.4 %) had NPM1 heterozygous gene mutations, all belonging to type A. The EFS rate of the patients with NPM1 mutation was higher in 10 months and the OS rate was higher in 19 months (P<0.05). 3 of 9 NPM1 mutations patients were FLT3-ITD positive. The CR rates of the four groups after initial remission induction therapy in order were NPM1+ FLT3-ITD-, NPM1- FLT3-ITD-, NPM1- FLT3-ITD+, NPM1+ FLT3-ITD+ (P<0.05). Besides, NPM1- FLT3-ITD+ was a risk factor affecting the OS (RR=1.250, P=0.005). 2 of the 55 cases (3.6 %) had c-kit gene mutations, namely mutant D816H and mutant D816V. The c-kit gene mutations were not found in patients with FLT3-ITD and NPM1 mutations. Conclusions The FLT3-ITD mutation is a poor prognosis molecular marker in AML, and NPM1 mutation is a good factor for the prognosis. NPM1- FLT3-ITD+ is a risk factor affecting OS. The rate of c-kit gene mutation is low in AML, without the overlap of FLT3 and NPM1 mutations. Key words: Leukemia, myeloid, acute; Bone marrow slides; Gene, FLT3-ITD; Gene, NPM1; Gene, c-kit; Prognosis

Expression and mutation of c-Kit in intracranial germ cell tumors: A single-centre retrospective study of 30 cases in China.

Although primary central nervous system (CNS) germ cell tumors (GCTs) are one of the most treatable types of malignant brain tumor, a subset of patients remain resistant to standard chemotherapy. Gain-of-function mutations of the c-Kit gene, and KIT protein expression, have been observed in a number of GCTs, including testicular seminoma, ovarian dysgerminoma and mediastinal seminoma in various ethnic groups. Although a small number of studies have reported the role of c-Kit in CNS GCTs, few have focused on Chinese patients exhibiting CNS GCTs. In the present study, the frequency and location of c-Kit mutations and KIT protein expression levels in CNS GCTs were investigated in 30 patients, between January 1994 and October 2014. Immunohistochemical assays suggested that KIT protein expression was present in 59.1% patients (66.7% in males and 42.9% in females); however, no statistically significant correlation was identified between KIT protein expression and patient clinicopathological features. By performing PCR amplification and direct sequencing, 4 mutational hot spots of the c-Kit gene (exons 9, 11, 13 and 17) were examined, and c-Kit gene mutation was identified in 1/17 (5.9%) CNS germinoma cases. This mutation was located in exon 11 at codon 557–558 WK (Tryptophan-Lysine). No c-Kit gene mutations were detected in non-germinomatous GCTs. Imatinib, a tyrosine kinase inhibitor, may be an effective treatment against standard chemotherapy-resistant CNS germinoma patients exhibiting c-Kit mutations.

Detection of novel polymorphisms in the ckit gene of canine patients with lymphoma, melanoma, haemangiosarcoma, and osteosarcoma.

Tyrosine kinase inhibitors (TKIs) that specifically target cKIT represent a therapeutic approach for non-resectable canine mast cell tumours (MCTs) grade II/III. The therapeutic benefit of TKIs has been investigated in other tumours based on clinical response rates and identification of gain-of-function mutations. In the present study, cKIT expression in 14 dogs with osteosarcoma, melanoma, haemangiosarcoma, lymphoma, and fibrosarcoma was analysed. Tissue samples were used for cKIT sequencing to (I) detect the cKIT transcript and to (II) identify gain-of-function mutations. The cKIT transcript was detected in ten patients. Four novel amino acid substitutions and five silent polymorphisms were identified. Furthermore, an insertion mutation (GNSK) was discovered in the tissue, but not in the blood sample of one dog. CKIT expression was identified in a variety of canine tumours and, therefore, TKIs might have a broader therapeutic indication apart from treatment of MCTs. Further investigations will be necessary to localize the cKIT protein in the respective tumours and to evaluate the functional consequence of the cKIT variants identified in the present study.

Clinicopathologic features of succinate dehydrogenase-deficient gastrointestinal stromal tumor

To investigate clinicopathologic features of succinate dehydrogenase-deficient gastrointestinal stromal tumors (SDH-deficient GIST). Immunohistochemical EnVision technique was used to assess the expression of succinate dehydrogenase subunit B (SDHB) in 192 cases of GIST. Cases of SDH-deficient GIST were further evaluated for the presence of CKIT exons 9, 11, 13 and 17 mutations and PDGFRA exons 12 and 18 mutations with clinical followed-up data. Seven of the 192 cases showed SDHB-deficiency (3.6%, 7/192). The patients ranged in age from 35 to 84 years (median=56 years; mean=60 years). Four were male and three were female. Six tumors involved stomach and one involved mesentery. Histopathologic features of SDHB-deficient GIST included four cases of mixed-cell type and three of epithelioid cell type. The tumors commonly involved muscularis propria of the stomach as multiple nodules, creating a plexiform pattern. The tumors had high cellularity with cytoplasmic vacuolization. Five cases developed lymph node metastases including one also metastasizing to liver and pancreas. Two cases showed no evidence of metastasis. None of the 7 cases of the SDHB-deficient GIST had CKIT exons 9, 11, 13 and 17 mutations and PDGFRA exons 12 and 18 mutations. Three of the seven SDHB-deficient GIST cases had followed-up data: two did not recur and one died after 24 months of surgery of unknown cause. SDHB-deficient GIST has characteristic clinicopathologic features with wide-type CKIT gene and a favorable prognosis.

犬の消化管間質腫瘍(GIST)の臨床的特徴とc-kit遺伝子変異

犬の消化管間質腫瘍(GIST)の臨床的特徴とc-kit遺伝子変異

Relationship of c-Kit Gene and Microsatellit Instability in the Pathogenesis of Wilms Tumors.

Relationship of c-Kit Gene and Microsatellit Instability in the Pathogenesis of Wilms Tumors.