C-kit Mutation Research Articles

C-Kit deficiency impairs nitric oxide signaling in smooth muscle cells

IntroductionReceptor tyrosine kinases have been implicated in various vascular remodeling processes and cardiovascular disease. However, their role in the regulation of vascular tone is poorly understood. Herein, we evaluate the contribution of c-Kit signaling to vasoactive responses. MethodsThe vascular reactivity of mesenteric arteries was assessed under isobaric conditions in c-Kit deficient (KitW/W−v) and littermate control mice (Kit+/+) using pressure myography. Protein levels of soluble guanylyl cyclase beta 1 (sGCβ1) were quantified by Western blot. Mean arterial pressure was measured after high salt (8% NaCl) diet treatment using the tail-cuff method. ResultsSmooth muscle cells (SMCs) from c-Kit deficient mice showed a 5-fold downregulation of sGCβ1 compared to controls. Endothelium-dependent relaxation of mesenteric arteries demonstrated a predominance of prostanoid vs. nitric oxide (NO) signaling in both animal groups. The dependence on prostanoid-induced dilation was higher in c-Kit mutant mice than in controls, as indicated by a significant impairment in vasorelaxation with indomethacin with respect to the latter. Endothelium-independent relaxation showed significant dysfunction of NO signaling in c-Kit deficient SMCs compared to controls. Mesenteric artery dilation was rescued by addition of a cGMP analog, but not with a NO donor, indicating a deficiency in cGMP production in c-Kit deficient SMCs. Finally, c-Kit deficient mice developed higher blood pressure on an 8% NaCl diet compared to their control littermates. Conclusionc-Kit deficiency inhibits NO signaling in SMCs. The existence of this c-Kit/sGC signaling axis may be relevant for vascular reactivity and remodeling.

Role of TGF-β1 and C-Kit Mutations in the Development of Hepatocellular Carcinoma in Hepatitis C Virus-Infected Patients: in vitro Study.

Transforming growth factor beta (TGF-β) acts as a tumor-suppressing cytokine in healthy tissues and non-malignant tumors. Yet, in malignancy, TGF-β can exert the opposite effects that can promote proliferation of cancer cells. C-Kit plays a prominent role in stem cell activation and liver regeneration after injury. However, little is known about the cross-talk between TGF-β and C-Kit and its role in the progression of hepatocellular carcinoma (HCC). Here, we studied the effect of increasing doses of TGF-β1 on CD44+CD90+ liver stem cells (LSCs) and C-Kit gene expression in malignant and adjacent non-malignant liver tissues excised from 32 HCC patients. The percentage of LSCs in malignant tumors was two times higher compared to their counterparts from the non-malignant tissues. When treated with increasing doses of TGF-β1, proliferation of both malignant and non-malignant LSCs was progressively suppressed, but low TGF-β1 dose failed to suppress the growth of malignant LSCs. Moreover, C-Kit exons 9 and 11 were expressed in malignant LSCs, but not in their non-malignant counterparts. Analysis of C-Kit detected mutations in exon 9 (but not in exon 11) in some malignant liver cells resulting in the changes in the amino acid sequence and dysregulation of protein structure and function. Interestingly, in malignant liver cells, mutations in exon 9 were associated with high-viremia hepatitis C virus (HCV), and expression of this exon was not suppressed by the TGF-β1 treatment at all doses. To our knowledge, this is the first report that mutations in the C-Kit gene in HCC patients are associated with high- viremia HCV. Our study emphasizes the need for investigation of the TGF-β1 level and C-Kit mutations in patients with chronic HCV for HCC prevention and better therapy management.

Characterization of mutational pattern of patients with core-binding factor acute myeloid leukemia

To characterize the mutational profile of patients with core-binding factor acute myeloid leukemia (CBF-AML). A total of 81 acute myeloid leukemia patients were recruited, which included 36 cases of CBF-AML and 45 cases of cytogenetically normal acute myeloid leukemia (CN-AML) . Mutations of FLT3-ITD, FLT3-TKD, NPM1, c-KIT, NRAS, KRAS, TET2, IDH1/2, RUNX1, DNMT3A, GATA2, ASjXL1, TP53, PTPN11, JAK2V617F, SETBP1 and CEBPA genes were simultaneously detected by DNA-based PCR and Sanger sequencing. Over all, mutations were detected in 68 patients (83.9%), with the most common ones including double CEBPA mutations (n=17), followed by NPM1 (n=15), c-KIT (n=11), NRAS (n=10), TET2 (n=9), FLT3-TKD (n=9), FLT3-ITD (n=8), IDH1 (n=7), RUNX1 (n=7), KRAS (n=7), DNMT3A (n=6), IDH2 (n=4), and GATA2 (n=4) mutations. AML1-ETO and CBFβ-MYH11 fusions were present in 21 and 15 patients, respectively. Coexistence of ≥2 mutations was more common in CN-AML comparing with CBF-AML. The mutation rate of NPM1, FLT3-ITD, DNMT3A, IDH1 and CEBPA double mutations were higher in patients with CN-AML. NRAS, c-KIT and KRAS mutations were identified more frequently in patients with CBF-AML (P<0.05). Based on the function, aberration of genes involved in DNA methylation, NPM1 proteins and transcription predominated in CN-AML, while tyrosine kinase receptor signaling and RAS pathways have predominated in CBF-AML. The genomic landscape of CBF-AML patients has differed from that of CN-AML patients. Synergy of fusion genes with particular mutations may impact the clinical phenotype and prognosis of patients.

Abstract 1777: Correlation between different C-KIT exon mutation and clinical outcome to Imatinib Mesylate treatment in patients with gastro-intestinal stromal tumors (GIST)

Abstract Aim: C-KIT exon mutations were assessed in patients with gastrointestinal stromal tumor (GIST) in relation to patients’ characteristics, response to treatment and survival rates. Methods: C-KIT mutations were assessed in the representative formalin fixed paraffinembedded tissues (FFPETs) of 89 patients with GIST compared to 35 normal control (NC) subjects using the Real Time PCR (Rt-PCR) and immunohistochemistry (IHC). Results: C-KIT mutations were detected in 61/89 (68.5%) patients, compared to none of the NC subjects (P=&amp;lt;0.001). Most mutations were detected in exon 11 (50.8%), followed by exon 9 (24.5%), exon 13 (19.6%), exon 7 (4.1%), and exon 12 (only 1%). C-KIT protein expression was detected in 69 (77.5%) patients compared to none of the NC subjects (p=&amp;lt;0.001). Significant measure of agreement was detected between C-KIT expression by IHC and and RT-PCR (K=0.774, p=0.0001). The presence of C-KIT mutations associated significantly with large tumor size (P=0.01), High mitotic rate, lymph nodes metastasis and high tumor risk (p=0.001, for all). Patients with exon 11 mutations 26/31 (83.9%) had a better response to treatment compared to those with exon nine 7 (46.7%) and exon 13 mutation 1/12 (8.3%; P=0.001). Exon 11 mutations associated significantly with better PFS and OS compared to those with exon 9 and 13 mutations (P= 0.001, P= 0.0001; respectively). On univariate analysis, Exon 13 mutations was the most significantly associated with reduced PFS (P=0.0001), whereas advanced disease stage, absence of adequate safety margins and the site of exon mutation associated significantly with reduced OS. (P=0.012, P=0.05 and P=0.005; respectively. Conclusion: GIST patients with C-KIT exon 11 mutations have a better response to imatinib treatment (predictive value), better OS and lower risk of disease progression than those with exon 9 and 13 mutations. Our specified mutations in exons 9&amp; 13 mutations could be used as an independent prognostic factor for GIST patients. Citation Format: Abeer A. Bahnnasy, Ola Khorshid, Mona S. Abdellateif, Ghada Y. Zakaria, Nasr M. Allahloubi, Auhood Nassar, Abdel-Rahman N. Zekri. Correlation between different C-KIT exon mutation and clinical outcome to Imatinib Mesylate treatment in patients with gastro-intestinal stromal tumors (GIST) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1777.

Abstract 2447: Accurate prediction of the signaling pathway activity elicited by variants of uncertain significance (VUS) and their drug sensitivity using deep convolutional neural networks on florescent microscopy images of cells

Abstract Sequencing based methods to tailor the treatment of cancer patients are at the cutting edge of personalized medicine and have remarkably advanced in the past few years. However, a large number of mutations that are found when sequencing tumors are poorly characterized in the literature and are thus labeled as VUS. Because of their uncertain significance, it is not possible to assign them a function and hence are overlooked when designing treatment regimens, and this can have deleterious effects. Here we report the development of a Deep Convolutional Neural Network (DCNN) platform trained using Deep Learning algorithms, that is able to accurately predict the activity elicited by specific mutations. The DCNN is based on a high-throughput platform to predict the activation of signaling pathways elicited by the specific mutations via changes to the intra-cellular localization of fluorescently labeled protein reporters. For our training set we used images of 6 billion cells expressing either WT or mutations with known activity of the analyzed genes. After training, the network was presented with images of cells transfected with VUS that the network has never seen before. We show that the network was able to correctly predict the level of activity of a large number of VUS in KRAS, BRAF and cKIT and on multiple signaling pathways, specifically the MAPK and JAK-STAT pathways. These findings were further confirmed by both biochemical assays as well as literature search. Furthermore, images of cells expressing known mutations of BRAF and cKIT and treated with increasing doses of the targeted treatments Vemurafenib and Imatinib, respectively, were analyzed by the network and we were able to generate high resolution dose response curves in specific signaling pathways. Lastly, we present the predicted dose response curves in specific signaling pathways of a variety of VUS which have never been characterized before and show that different VUS respond differently to the tested drugs. Overall, we provide evidence that this novel tool enables robust and highly accurate calculation of activity profiles of a wide range of genes and signaling pathways. Each mutation is highly specific in the magnitude of activity it elicits. The high degree of resolution allows the investigation of the efficacy of different targeted therapies on a wide range of mutations. Citation Format: Lior Zimmerman, Arie Aizenman, Yutti Daitsh, Zohar Barbash, Gabi Tarcic, Michael Vidne. Accurate prediction of the signaling pathway activity elicited by variants of uncertain significance (VUS) and their drug sensitivity using deep convolutional neural networks on florescent microscopy images of cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2447.

Abstract 693: Deep learning analysis of circulating tumor DNA identified a pan-tumor molecular subtype with enhanced response to durvalumab (anti-PDL1)

Abstract Tumor mutation burden (TMB) has emerged as a promising predictive biomarker for anti-PD1/L1; however, whether somatic mutations in specific genes can sensitize to immunotherapy is poorly understood. We used deep learning to identify molecular subtypes from mutations detectable in circulating tumor DNA (ctDNA) and associated these with outcomes with anti-PDL1 (durvalumab) and durvalumab + anti-CTLA4 (tremelimumab) therapy. Patient blood samples from 4 nonrandomized phase 2 trials evaluating durvalumab (NCT01693562, 1L+ solid tumors; NCT02087423, 3L+ NSCLC) or durvalumab+ tremelimumab (NCT02000947, 2L+ NSCLC; NCT02261220, 2L+ solid tumors) were sequenced with the Guardant 360 assay of 73 genes. CtDNA mutations were examined pre-treatment in a discovery cohort of 818 patients composed of &amp;gt;15 tumor types and validated in a cohort of 94 patients composed of &amp;gt;5 tumor types. Deep learning neuron network was computed using Tensorflow to identify 4 unique molecular subgroups. Kaplan-Meier analysis was calculated and objective response rates (ORR) were assessed with RECISIST v1.1. The subgroups consisted of: DS1 (N = 215/818, 26%) enriched in RB1 mutations (17%) and low TMB, DS2 (N = 237/818, 29%) enriched in ARID1A (33%), NFE2L2 (8%), and cKIT (13%) mutations, DS3 (N = 251/818, 31%) enriched in EGFR, ERBB2, BRAF and STK11 mutations, and DS4 (N = 115/818, 14%) enriched in TERT, FGFR3, and PIK3CA mutations. In the discovery cohort, the DS2 subgroup had most improved outcomes (mOS=24.6 months, mPFS=3.7 months, ORR=27%) compared to the other 3 subgroups. DS1 subgroup had the worst outcomes (mOS=6.3 months, mPFS=1.6 months, ORR=10%), while the biomarker evaluable population (BEP, n=818) had ORR=17%. In the validation cohort, DS2 also had the most improved outcomes (mOS=not reached, mPFS=13.2 months, and ORR=64%), with the BEP (n=94) having an ORR=37%. PD-L1 was not correlated with any specific molecular subgroup, while an IFNg mRNA signature trended with correlation with subgroup DS2. Deep learning analysis of ctDNA identified a molecular subgroup with enhanced response to anti-PDL1 treatment in multiple tumor types. Citation Format: Song Wu, Sriram Sridhar, Han Si, Michael Kuziora, Judson Englert, Shaad E. Abdullah, Philip Dennis, Feng Xiao, Guozhi Gao, Koustubh Ranade, Brandon W. Higgs. Deep learning analysis of circulating tumor DNA identified a pan-tumor molecular subtype with enhanced response to durvalumab (anti-PDL1) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 693.

Exclusive Bee Venom Allergy: Risk Factors and Outcome of Immunotherapy

Introduction: Venom immunotherapy (VIT) is considered to be the gold-standard treatment for patients with Hymenoptera venom allergy. Data regarding VIT in bee venom (BV) allergic patients are scarce. Aim: The aim of this study was to evaluate the outcome of VIT in patients with exclusive BV allergy and to try to define risk factors for VIT-induced systemic reactions (VIT-ISR) and VIT failure. Methods: This is a retrospective study including data from all BV allergic patients that were treated by VIT in the Allergy Unit at the Meir Medical Center in the years 1995–2018. Results: Two hundred and forty-seven patients with exclusive BV allergy were included; 206 (83.4%) preferred to undergo rush buildup. Sixty-nine patients (27.9%) had at least 1 reaction during buildup, with the c-kit mutation being the only significant risk factor (100 vs. 28.9%, p = 0.02). Female gender (25.4 vs. 13.3%, p = 0.04), conventional buildup schedule (26.8 vs. 14.1%, p = 0.04), and c-kit mutation (100 vs. 16.8%, p < 0.01) but not tryptase level were found to be significantly more frequent in recurrent reactors. Females (20.3 vs. 9%, p = 0.03), patients with severe systemic reaction to the index sting (24.3 vs. 9.5%, p = 0.004), and c-kit mutation (66 vs. 12%, p = 0.05) but not tryptase level were found to be risk factors for severe systemic reactions. Conclusion: Despite the considerably high rate of VIT-ISR in patients with exclusive BV allergy, VIT can be performed safely and efficiently. C-kit mutation, and not basal serum tryptase level, seems to be a preferable biomarker for VIT-ISR in these patients.

Discovery of 2-(4-Chloro-3-(trifluoromethyl)phenyl)-N-(4-((6,7-dimethoxyquinolin-4-yl)oxy)phenyl)acetamide (CHMFL-KIT-64) as a Novel Orally Available Potent Inhibitor against Broad-Spectrum Mutants of c-KIT Kinase for Gastrointestinal Stromal Tumors.

Starting from our previously developed c-KIT kinase inhibitor CHMFL-KIT-8140, through a type II kinase inhibitor binding element hybrid design approach, we discovered a novel c-KIT kinase inhibitor compound 18 (CHMFL-KIT-64), which is potent against c-KIT wt and a broad spectrum of drug-resistant mutants with improved bioavailability. 18 exhibits single-digit nM potency against c-KIT kinase and c-KIT T670I mutants in the biochemical assay and displays great potencies against most of the gain-of-function mutations in the juxtamembrane domain, drug-resistant mutations in the ATP binding pocket (except V654A), and activation loops (except D816V). In addition, 18 exhibits a good in vivo pharmacokinetic (PK) profile in different species including mice, rats, and dogs. It also displays good in vivo antitumor efficacy in the c-KIT T670I, D820G, and Y823D mutant-mediated mice models as well as in the c-KIT wt patient primary cells which are known to be imatinib-resistant. The potent activity against a broad spectrum of clinically important c-KIT mutants combining the good in vivo PK/pharmacodynamic properties of 18 indicates that it might be a new potential therapeutic candidate for gastrointestinal stromal tumors.

Homoharringtonine synergy with oridonin in treatment of t(8; 21) acute myeloid leukemia.

Collaboration of c-KIT mutations with AML1-ETO (AE) has been demonstrated to induce t(8; 21) acute myeloid leukemia (AML). Targeted therapies designed to eliminate AE and c-KIT oncoproteins may facilitate effective treatment of t(8; 21) AML. Homoharringtonine (HHT) features activity against tumor cells harboring c-KIT mutations, whereas oridonin can induce t(8; 21) AML cell apoptosis and AE cleavage. Therefore, studies should explore the efficacy of combination therapy with oridonin and HHT in t(8; 21) AML. In this study, we investigated the synergistic effects and mechanism of oridonin combined with HHT in t(8; 21) AML cell line and mouse model. The two drugs synergistically inhibited cell viability and induced significant mitochondrial membrane potential loss and apoptosis. Oridonin and HHT induced significant downregulation of c-KIT and its downstream signaling pathways and promoted AE cleavage. HHT increased intracellular oridonin concentration by modulating the expressions of MRP1 and MDR1, thus enhancing the effects of oridonin. The combination of oridonin and HHT prolonged t(8; 21) leukemia mouse survival. In conclusion, oridonin and HHTexert synergistic effects against t(8; 21) leukemia in vivo and in vitro, thereby indicating that their combination may be an effective therapy for t(8; 21) leukemia.

Gastrointestinal Stromal Tumors of the Stomach and Esophagus.

Gastrointestinal stromal tumors (GISTs) arise anywhere along the gastrointestinal tract, most commonly as a result of c-kit or PDGFRA proto-oncogene mutations. Surgical resection is an important component of treatment. However, molecular profiling of GISTs has provided many insights into adjuvant and neoadjuvant therapy options. Imatinib, the most frequently studied medical therapy, has been shown in numerous studies to provide benefit to patients in both the neoadjuvant and adjuvant setting. Interval imaging is an important component of the treatment of GISTs and national surveillance recommendations should be followed.

Mutational status and thromboembolic risk in lung, gastrointestinal and breast cancer patients.

e23147 Background: Cancer-Associated Thrombosis (CAT) is one of the most threatening complications of cancer. Recent evidences suggested a link between the molecular profile of solid tumors and the incidence of CAT. The aim of this study was to explore the relationship between the mutational status of breast, lung and gastrointestinal cancer patients and the risk of CAT. Methods: We retrospectively evaluated the molecular profile, analysed as per clinical practice, of all consecutive patients hospitalized at the National Cancer Institute’s Department in Milan from October 2017 to November 2018. Patients with previous thromboembolic events and patients under anticoagulant therapy at cancer diagnosis were excluded. Due to death as competing risk, the Fine and Gray proportional regression model was used to detect statistical association and estimate relative risk. Results: The resulting cohort consisted of 484 patients, of whom 47% had gastrointestinal cancers, 18% had lung cancer and 15% had breast cancer. Molecular investigations were available for 375 (77%) patients; in particular, a 50-gene Next Generation Sequencing (NGS) panel was performed on 148 (31%) patients. After a median follow up of 17 months, 118 patients (24%) exhibited clinical manifestations of thrombosis (i.e. deep vein thrombosis, pulmonary thromboembolism, splanchnic thrombosis, disseminated intravascular coagulation, arterial thrombosis) and 117 (24%) patients deceased without thrombotic events. A statistically significant association was observed between incidence of CAT and presence of TP53 (HR 0.50, p = 0.04), c-KIT (HR 4.30, p = 0.041), and SMAD4 (HR 3.19, p = 0.029) mutations. No significant association was detected for KRAS and MET gene mutations, even if HRs were &gt;2. Conclusions: In this study, the mutational status of TP53, SMAD4 and c-KIT genes was statistically associated to the risk of thrombosis. Due to methodological limits and low prevalence of mutations, large prospective studies are warranted, with the aim of better defining the role of oncogenes in CAT risk.

Outcomes of melanoma patients with brain metastases receiving immune checkpoint inhibitor (ICI) therapy.

e21028 Background: The effect of ICI therapy on brain metastasis (mets) in melanoma patients has been described, however the impact of genomic alterations and local treatment (trt) including radiation therapy (RT) on CNS outcomes and overall survival (OS) has not yet been explored. Methods: Under institutional IRB approval, we reviewed our electronic medical record to identify patients (pts) with metastatic melanoma and brain mets who received trt with ICI. Trt history, CNS responses as indicated by the treating physician and available genomic data were recorded. OS was estimated using a multivariable Cox regression model adjusting for genomic alteration, age, ICI regimen and brain mets. Results: A total of 49 pts were identified (65% male). Thirty seven pts had testing for tumor genomic alterations (alt, hotspot and comprehensive genomic testing); BRAFV600E( 30%), cKIT(5%), NRAS(16%), no alt (38%). Prior to initiation of ICI, 78% had known CNS mets, of these, 61% received RT. There was no difference in median OS for pts who started RT prior to ICI compared to those without RT (mOS = 75.71 vs 49.4 weeks, p = 0.75). Median OS was improved for those pts who received RT &gt; 90 days prior to initiation of ICI trt however this was not statistically significant (45.7 vs 136 weeks, p = 0.12) and HR = 0.45 (95% CI 0.08, 2.36). OS was not significantly different in pts with genomic alterations compared with those without alterations (p = 0.264). Of the pts with known brain mets at the time of ICI start, 34% had progressive disease at 3 months and 11% were felt to have pseudoprogression. 18% survived to undergo 6-month disease assessment, of these 12% had further progression. All pts with cKIT mutations survived to 6-month disease assessment. 75% of pts with known brain mets developed new lesions, 15% had increased mass size. Pts who developed new brain mets while on trt with ICI had worse survival than those with preexisting brain mets and developed new brain lesions (28 vs 58 weeks, p = 0.05). 6% of all pts developed CNS immune related adverse effects. Conclusions: Most patients with melanoma and known brain metastasis prior to ICI developed new brain lesions as a site of metastatic spread. The development of new brain mets was associated with worse outcomes. Neither genomic alterations nor RT prior to ICI start were associated with improved survival.

Clinical Diagnosis of Gastrointestinal Stromal Tumor (GIST): From the Molecular Genetic Point of View.

Gastrointestinal stromal tumors (GISTs) originating from the interstitial cells of Cajal are mesenchymal tumors of the gastrointestinal tract and have been found to harbor c-KIT mutations and KIT (CD117) expression since 1998. Later, PDGFRA mutations, SDH alterations, and other drive mutations were identified in GISTs. In addition, more and more protein markers such as DOG1, PKCθ were found to be expressed in GISTs which might help clinicians diagnose CD117-negative GISTs. Therefore, we plan to comprehensively review the molecular markers and genetics of GISTs and provide clinicians useful information in diagnostic and therapeutic strategies of GISTs. Twenty years after the discovery of KIT in GISTs, the diagnosis of GISTs became much more accurate by using immunohistochemical (IHC) panel (CD117/DOG1) and molecular analysis (KIT/PDGFRA), both of which constitute the gold standard of diagnosis in GISTs. The accurately molecular diagnosis of GISTs guides clinicians to precision medicine and provides optimal treatment for the patients with GISTs. Successful treatment in GISTs prolongs the survival of GIST patients and causes GISTs to become a chronic disease. In the future, the development of effective treatment for GISTs resistant to imatinib/sunitinib/regorafenib and KIT/PDGFRA-WT GISTs will be the challenge for GISTs.

Discovery of ( E)- N1-(3-Fluorophenyl)- N3-(3-(2-(pyridin-2-yl)vinyl)-1 H-indazol-6-yl)malonamide (CHMFL-KIT-033) as a Novel c-KIT T670I Mutant Selective Kinase Inhibitor for Gastrointestinal Stromal Tumors (GISTs).

Gain-of-function mutations of c-KIT kinase play crucial pathological roles for the gastrointestinal stromal tumors (GISTs). Despite the success of imatinib as the first-line treatment of GISTs, dozens of drug-acquired resistant mutations emerge, and c-KIT T670I is one of the most common mutants among them. Although several kinase inhibitors are capable of overcoming the T670I mutant, none of them can achieve the selectivity over the c-KIT wild-type (wt), which also plays important roles in a variety of physiological functions such as hematopoiesis. Starting from axitinib, through fragment hybrid type II kinase inhibitor design approach, we have discovered a novel inhibitor 24, which not only exhibits potent activity to c-KIT T670I mutant but also achieves 12-fold selectivity over c-KIT wt. Compound 24 displays good antiproliferative effects against c-KIT T670I mutant-driven GIST cell lines (GIST-T1/T670I and GIST-5R) and also exhibits suitable in vivo pharmacokinetic profiles as well as dose-dependent antitumor efficacy. This study provides a proof of concept for developing a c-KIT mutant selective inhibitor that theoretically can render a better therapeutic window.

Factors Affecting Disease-Free Survival in Operated Nonmetastatic Gastrointestinal Stromal Tumors

BackgroundPossibly originating from interstitial Cajal cells, gastrointestinal stromal tumors (GISTs) have variable biological behaviors. In this study, we aimed to examine the factors affecting the disease-free survival (DFS) in patients with GIST who underwent operation. Material and methodsThe study included the patients who were followed up and treated for GIST in our oncology clinic between 2002 and 2017. The Armed Forces Institute of Pathology criteria (Miettinen risk score) were used for risk stratification of patients. ResultsSeventy-four patients were included to the study, where female patients constituted 52.7%, and the median age was 56 (range: 24-83) y. Most common primary tumor location was the stomach (51.4%), followed by the small intestine (33.8%), colorectum (10.8%), and retroperitoneum (4.1%). Miettinen risk score showed 12 patients (16.7%) at very low risk, 15 patients (20.8%) at low risk, 18 patients (25%) at intermediate risk, and 27 patients (37.5%) at high risk. DFS was significantly lower in patients with small intestine involvement than in cases with stomach involvement (P = 0.004). DFS was significantly lower in patients at high risk than in patients with no high risk (P = 0.034). Small intestine localization (hazard ratio [HR], 8.98; 95% confidence interval [CI], 1.14-8.18), high-risk score (HR, 5.16; 95% CI, 1.42-12.75), c-kit positivity (HR, 0.24; 95% CI, 0.13-0.69), and adjuvant therapy (HR, 0.37; 95% CI, 0.20-0.92) were found to be the most significant factors affecting DFS. ConclusionsOur study showed negative effects of small intestine localization and high-risk category and positive effects of c-kit positivity and adjuvant therapy on DFS in patients with GIST who underwent operation. When a decision will be made in favor of adjuvant therapy, tumor localization and c-kit mutation should also be considered in addition to risk score.

Discovery of Conformational Control Inhibitors Switching off the Activated c-KIT and Targeting a Broad Range of Clinically Relevant c-KIT Mutants.

Drug resistance due to acquired mutations that constitutively activate c-KIT is a significant challenge in the treatment of patients with gastrointestinal stromal tumors (GISTs). Herein, we identified 1-(5-ethyl-isoxazol-3-yl)-3-(4-{2-[6-(4-ethylpiperazin-1-yl)pyrimidin-4-ylamino]-thiazol-5-yl}phenyl)urea (10a) as a potent inhibitor against unactivated and activated c-KIT. The binding of 10a induced rearrangements of the DFG motif, αC-helix, juxtamembrane domain, and the activation loop to switch the activated c-KIT back to its structurally inactive state. To the best of our knowledge, it is the first structural evidence demonstrating how a compound can inhibit the activated c-KIT by switching back to its inactive state through a sequence of conformational changes. Moreover, 10a can effectively inhibit various c-KIT mutants and the proliferation of several GIST cell lines. The distinct binding features and superior inhibitory potency of 10a, together with its excellent efficacy in the xenograft model, establish 10a as worthy of further clinical evaluation in the advanced GISTs.

Role for targeted resection in the multidisciplinary treatment of metastatic gastrointestinal stromal tumor.

The management of advanced gastrointestinal stromal tumors (GISTs) has evolved in the modern era due to the discovery of c-kit mutations and the development of tyrosine kinase inhibitors (TKIs). Until the advent of TKIs such as imatinib, the median survival reported for patients with advanced GIST was 19 months. Although surgery is the treatment of choice for resectable primary GIST, its role in cases of recurrence and metastasis remains to be unclear. This review outlines the potential beneficial role of repeat surgical resection in the multidisciplinary treatment of advanced GIST in the era of TKIs.

Efficacy Evaluation of Imatinib for the Treatment of Melanoma: Evidence From a Retrospective Study.

Melanoma is an aggressive malignancy with a poor prognosis. Current studies show that imatinib treatment is a promising approach in treating advanced melanoma patients harboring c-Kit mutations or amplifications. We retrospectively analyzed the clinical medical records of 78 patients with metastatic melanoma harboring c-Kit mutations or amplifications. These patients were treated with imatinib at a dose of 400 mg/day continuously unless intolerable toxicities or disease progression occurred. Endpoints for exploration included overall survival (OS), progression-free survival (PFS), objective response rate (ORR), and disease of control rate (DCR). The median OS and PFS of all patients were 13.1 and 4.2 months, respectively. ORR and DCR were 21.8% and 60.3%, respectively. The survival time of patients who achieved partial response or stable disease was significantly superior to those with disease progression. Cox regression analysis showed that patients with M1c stage, subtype of cutaneous melanoma, or elevated LDH level (>upper limit of normal) had higher hazard ratios for overall survival. Our study, combined with those studies targeting patients with a c-Kit alteration, validates the role of imatinib as an important and promising therapeutic agent in the treatment of patients with advanced melanoma.

Prognostic factors for survival in patients with mucosal and ocular melanoma treated with ipilimumab: Turkish Oncology Group study.

To evaluate prognostic factors associated with the use of ipilimumab in patients with mucosal and uveal melanoma. In this multicenter, retrospective study, 31 patients with uveal and mucosal melanoma diagnosed between 2010 and 2017 were enrolled. Patients' characteristics, metastatic disease sites, treatment before ipilimumab therapy, performance status, hemoglobin, lactate dehydrogenase levels, B-RAF and c-kit mutation status, toxicity, and survival data were assessed for patients with mucosal and uveal melanoma. SPSS version 17 was used for statistical analysis. Kaplan-Meier method was used for survival analysis. The log-rank test was used for univariate analyses. The Cox regression analysis was used to test the association between multivariate variables and survival. The p-value of less than 0.05 was considered statistically significant. Twenty patients had uveal and eleven patients had mucosal melanoma. The median overall survival was seven months (95% confidence interval: 1.1-12.7). In univariate analysis, while bone metastasis, anemia, high lactate dehydrogenase level, and more metastatic sites were associated with lower overall survival, better treatment response and administration of ipilimumab in first or second lines were associated with favorable overall survival. In multivariate analysis, only treatment response status and administration of ipilimumab in first or second lines were found to be significant as independent prognostic factors for survival. Ipilimumab therapy may be associated with increased survival, but this retrospective small N study makes that hard to definitely conclude.

Prevalence and prognostic value of c-kit and TP53 mutations in canine mast cell tumours

Cutaneous mast cell tumours (MCT) are among the most frequent malignancies in dogs. Their clinical behaviour is highly variable and, with the exception of mutations in the c-kit gene, little is known about their genetic aetiology. The mutational status of the c-kit exons 8, 9 and 11, and exons 5–8 of the TP53 gene was analysed to find markers for molecular stratification of MCTs and predictors of clinical outcome. Mutations in the c-kit gene were detected in 19.5% (n = 8/41) samples and their presence was significantly associated with the high histopathological grade (P = 0.038). Mutations in the DNA binding domain of the TP53 gene were found in 14.6% (n = 6/41) of the analysed MCTs, and their frequency was similar in low and high grade MCTs (P > 0.05). TP53 mutations were not useful prognostic factors in this sample of canine cutaneous MCTs.