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The maintenance of hematopoietic stem cells (HSCs) is a complex process involving numerous cell-extrinsic and -intrinsic regulators. The first member of the cyclin-dependent kinase family of inhibitors to be identified, p21, has been reported to perform a wide range of critical biological functions, including cell cycle regulation, transcription, differentiation, and so on. Given the previous inconsistent results regarding the functions of p21 in HSCs in a p21-knockout mouse model, we employed p21 -tdTomato (tdT) mice to further elucidate its role in HSCs during homeostasis. The results showed that p21-tdT+ HSCs exhibited increased self-renewal capacity compared to p21-tdT− HSCs. Zbtb18, a transcriptional repressor, was upregulated in p21-tdT+ HSCs, and its knockdown significantly impaired the reconstitution capability of HSCs. Furthermore, p21 interacted with ZBTB18 to co-repress the expression of cKit in HSCs and thus regulated the self-renewal of HSCs. Our data provide novel insights into the physiological role and mechanisms of p21 in HSCs during homeostasis independent of its conventional role as a cell cycle inhibitor. [ABSTRACT FROM AUTHOR]

Aims In hypoxia, endothelial cells (ECs) proliferate, migrate, and form new vasculature in a process called angiogenesis. Recent studies have suggested that ECs rely on glycolysis to meet metabolic needs for angiogenesis in ischaemic tissues, and several studies have investigated the molecular mechanisms integrating angiogenesis and endothelial metabolism. Here, we investigated the role of stem cell factor (SCF) and its receptor, cKIT, in regulating endothelial glycolysis during hypoxia-driven angiogenesis. Methods and results SCF and cKIT signalling increased the glucose uptake, lactate production, and glycolysis in human ECs under hypoxia. Mechanistically, SCF and cKIT signalling enhanced the expression of genes encoding glucose transporter 1 (GLUT1) and glycolytic enzymes via Akt- and ERK1/2-dependent increased translation of hypoxia inducible factor 1A (HIF1A). In hypoxic conditions, reduction of glycolysis and HIF-1α expression using chemical inhibitors significantly reduced the SCF-induced in vitro angiogenesis in human ECs. Compared with normal mice, mice with oxygen-induced retinopathy (OIR), characterized by ischaemia-driven pathological retinal neovascularization, displayed increased levels of SCF, cKIT, HIF-1α, GLUT1, and glycolytic enzymes in the retina. Moreover, cKIT-positive neovessels in the retina of mice with OIR showed elevated expression of GLUT1 and glycolytic enzymes. Further, blocking SCF and cKIT signalling using anti-SCF neutralizing IgG and cKIT mutant mice significantly reduced the expression of HIF-1α, GLUT1, and glycolytic enzymes and decreased the pathological neovascularization in the retina of mice with OIR. Conclusion We demonstrated that SCF and cKIT signalling regulate angiogenesis by controlling endothelial glycolysis in hypoxia and elucidated the SCF/cKIT/HIF-1α axis as a novel metabolic regulation pathway during hypoxia-driven pathological angiogenesis. [ABSTRACT FROM AUTHOR]

Background: Mutations in cKIT or PDGFRA are found in up to 90% of patients with gastrointestinal stromal tumors (GISTs). Previously, we described the design, validation, and clinical performance of a digital droplet (dd)PCR assay panel for the detection of imatinib-sensitive cKIT and PDFGRA mutations in circulating tumor (ct)DNA. In this study, we developed and validated a set of ddPCR assays for the detection of cKIT mutations mediating resistance to cKIT kinase inhibitors in ctDNA. In addition, we cross-validated these assays using next generation sequencing (NGS). Methods: We designed and validated five new ddPCR assays to cover the most frequent cKIT mutations mediating imatinib resistance in GISTs. For the most abundant imatinib-resistance-mediating mutations in exon 17, a drop-off, probe-based assay was designed. Dilution series (of decreasing mutant (MUT) allele frequency spiked into wildtype DNA) were conducted to determine the limit of detection (LoD). Empty controls, single wildtype controls, and samples from healthy individuals were tested to assess specificity and limit of blank (LoB). For clinical validation, we measured cKIT mutations in three patients and validated results using NGS. Results: Technical validation demonstrated good analytical sensitivity, with a LoD ranging between 0.006% and 0.16% and a LoB ranging from 2.5 to 6.7 MUT fragments/mL. When the ddPCR assays were applied to three patients, the abundance of ctDNA in serial plasma samples reflected the individual disease course, detected disease activity, and indicated resistance mutations before imaging indicated progression. Digital droplet PCR showed good correlation to NGS for individual mutations, with a higher sensitivity of detection. Conclusions: This set of ddPCR assays, together with our previous set of cKIT and PDGFRA mutations assays, allows for dynamic monitoring of cKIT and PDGFRA mutations during treatment. Together with NGS, the GIST ddPCR panel will complement imaging of GISTs for early response evaluation and early detection of relapse, and thus it might facilitate personalized decision-making. [ABSTRACT FROM AUTHOR]

The existence of rare cancer cells that sporadically acquire drug-tolerance through epigenetic mechanisms is proposed as one mechanism that drives cancer therapy failure. Here we provide evidence that specific microenvironments impose non-sporadic expression of proteins related to epithelial plasticity and drug resistance. Microarrays of robotically printed combinatorial microenvironments of known composition were used to make cell-based functional associations between microenvironments, which were design-inspired by normal and tumor-burdened breast tissues, and cell phenotypes. We hypothesized that specific combinations of microenvironment constituents non-sporadically impose the induction of the AXL and cKIT receptor tyrosine kinase proteins, which are known to be involved in epithelial plasticity and drug-tolerance, in an isogenic human mammary epithelial cell (HMEC) malignant progression series. Dimension reduction analysis reveals type I collagen as a dominant feature, inducing expression of both markers in pre-stasis finite lifespan HMECs, and transformed non-malignant and malignant immortal cell lines. Basement membrane-associated matrix proteins, laminin-111 and type IV collagen, suppress AXL and cKIT expression in pre-stasis and non-malignant cells. However, AXL and cKIT are not suppressed by laminin-111 in malignant cells. General linear models identified key factors, osteopontin, IL-8, and type VIα3 collagen, which significantly upregulated AXL and cKIT, as well as a plasticity-related gene expression program that is often observed in stem cells and in epithelial-to-mesenchymal-transition. These factors are co-located with AXL-expressing cells in situ in normal and breast cancer tissues, and associated with resistance to paclitaxel. A greater diversity of microenvironments induced AXL and cKIT expression consistent with plasticity and drug-tolerant phenotypes in tumorigenic cells compared to normal or immortal cells, suggesting a reduced perception of microenvironment specificity in malignant cells. Microenvironment-imposed reprogramming could explain why resistant cells are seemingly persistent and rapidly adaptable to multiple classes of drugs. These results support the notion that specific microenvironments drive drug-tolerant cellular phenotypes and suggest a novel interventional avenue for preventing acquired therapy resistance.

BackgroundAdenoid cystic carcinoma (ACC) is a subtype of malignant salivary gland tumors (MSGT), in which 90% of cases express cKIT. Dasatinib is a potent and selective inhibitor of five oncogenic protein tyrosine kinases (PTKs)/kinase families including cKIT. We conducted a phase II study to determine the antitumor activity of dasatinib in ACC and non-ACC MSGT.Patients and methodsIn a two-stage design, patients with progressive, recurrent/metastatic ACC (+cKIT) and non-ACC MSGT (separate cohort) were treated with dasatinib 70 mg p.o. b.i.d. Response was assessed every 8 weeks using RECIST.ResultsOf 54 patients: 40 ACC, 14 non-ACC (1, ineligible excluded); M:F = 28 : 26, median age 56 years (range 20-82 years), ECOG performance status 0 : 1 : 2 = 24 : 28 : 2, prior radiation: 44, prior chemotherapy: 21. The most frequent adverse events (AEs) (as % of patients, worst grade 2 or higher) were: fatigue (28%), nausea (19%), headache (15%), lymphopenia (7%), dyspnea (11%), alanine aminotransferase increased (7%), anorexia (7%), vomiting (7%), alkaline phosphatase increased (6%), diarrhea (6%), neutropenia (6%), and noncardiac chest pain (6%). No grade 4 AE occurred, 15 patients experienced a grade 3 AE, primarily dyspnea (5) and fatigue (4), and cardiac toxicity (1 prolonged QTc). Among ACC patients, best response to dasatinib: 1 patient (2.5%) had partial response, 20 patients (50%) had stable disease (SD) (3-14 months), 12 patients (30%) had PD, 2 withdrew, 3 discontinued therapy due to AE, and 2 died before cycle 2. Median progression-free survival was 4.8 months. Median overall survival was 14.5 months. For 14 assessable non-ACC patients, none had objective response, triggering early stopping rule. Seven had SD (range 1-7 months), 4 PD, 2 discontinued therapy due to AE, and 1 died before cycle 2.ConclusionAlthough there was only one objective response, dasatinib is well tolerated, with tumor stabilization achieved by 50% of ACC patients. Dasatinib demonstrated no activity in non-ACC MSGT.

While eosinophilic esophagitis (EOE) is defined by histologic presence of eosinophils, a few studies have established the presence of mast cells in EOE and even shown their correlation with symptom persistence despite resolution of eosinophils. Expression of aberrant mast cell markers CD25 and CD2 have not been studied in EOE. This study quantifies the number of hotspot cells per high power field expressing CKIT/CD117, tryptase, CD25, CD2 and CD3 by immunohistochemical stains in endoscopic esophageal biopsies of the following three cohorts: (1) established and histologically confirmed EOE, (2) suspected EOE with biopsies negative for eosinophils, and (3) no history of or suspicion for EOE with histologically unremarkable biopsies. In this study, mast cells were highlighted by CKIT and tryptase in EOE, and not seen in other clinically mimicking cases. There were also significantly higher densities of CD25 and pan-T-cell marker staining in EOE cases. These findings suggest an inflammatory cellular milieu in EOE, beyond just eosinophils, that can be demonstrated by immunohistochemistry, and that invite further study into the role that these cells may play in EOE. • Mast cells by CKIT and tryptase found in eosinophilic esophagitis • Significant CD25 expression found in eosinophilic esophagitis • Significant T-cells by CD2 and CD3 found in eosinophilic esophagitis [ABSTRACT FROM AUTHOR]

Amaç: Bu çalışmanın amacı pediatrik medullbolastom olgularında p53, ERBB2, c-Kit ve Bcl-2 ekspresyonunun klinik ve prognostik öneminin araştırılmasıdır.Gereç ve Yöntem: Toplam 29 medulloblastom olgusu demografik, klinik, histopatolojik özellikler ve immünhistokimyasal olarak p53, ERBB2, c-Kit ve Bcl-2 ekspresyonlarının prognostik anlamı açısından değerlendirmeye alındı. Bulgular: Ortanca tanı yaşı 69 aydı (21-122 ay). Ortalama takip süresi 54 ay (2-209 ay) olarak belirlendi. Ondört olgu klasik (%48), 13 olgu nodüler/dezmoplastik (%45) ve 2 olgu anaplastik (%7) olarak değerlendirildi. İmmünhistokimyasal olarak 28 olguda c-Kit, 10 olguda Bcl-2, 9 olguda p53 ve 2 olguda ERBB2 pozitifliği gösterildi. Genel sağkalım oranı %62 ve hastalıksız sağkalım oranı %52 olarak bulundu. Bcl-2 ekspresyonunun nodüler/dezmoplastik alt tipte arttığı gösterildi. Klinik, histopatolojik ve immünhistokimyasal özelliklerin herhangi birisinin prognoz üzerinde etkisi gösterilemedi.Sonuç: Bu çalışma medulloblastom tedavisinde kombine tedavi protokollerinin kullanılmaya başladığı ilk yılları yansıtmaktadır. Sağkalım oranları tanı yıllarına göre literatür ile benzer saptanmıştır. Değerlendirilen klinik, histopatoloijk ve immünhistokimyasal özelliklerin prognozla ilişkisi gösterilemese de, gelecekte moleküler ve immünhistokimyasal yöntemlerin birlikte kullanıldığı çalışmalarla medulloblastom olgularında prognostik faktörlerin saptanmasının mümkün olacağı düşüncesindeyiz.

Stem cell factor (SCF) and its receptor, cKIT, are novel regulators of pathological neovascularization in the eye, which suggests that inhibition of SCF/cKIT signaling may be a novel pharmacological strategy for treating neovascular age-related macular degeneration (AMD). This study evaluated the therapeutic potential of a newly developed fully human monoclonal antibody targeting cKIT, NN2101, in a murine model of neovascular AMD. In hypoxic human endothelial cells, NN2101 substantially inhibited the SCF-induced increase in angiogenesis and activation of the cKIT signaling pathway. In a murine model of neovascular AMD, intravitreal injection of NN2101 substantially inhibited the SCF/cKIT-mediated choroidal neovascularization (CNV), with efficacy comparable to aflibercept, a vascular endothelial growth factor inhibitor. A combined intravitreal injection of NN2101 and aflibercept resulted in an additive therapeutic effect on CNV. NN2101 neither caused ocular toxicity nor interfered with the early retinal vascular development in mice. Ocular pharmacokinetic analysis in rabbits indicated that NN2101 demonstrated a pharmacokinetic profile suitable for intravitreal injection. These findings provide the first evidence of the potential use of the anti-cKIT blocking antibody, NN2101, as an alternative or additive therapeutic for the treatment of neovascular AMD.

Although stem cell factor (SCF)/cKIT interaction plays key functions in erythropoiesis, cKIT signaling in human erythroid cells is still poorly defined. To provide new insights into cKIT-mediated erythroid expansion in development and disease, we performed phosphoproteomic profiling of primary erythroid progenitors from adult blood (AB), cord blood (CB), and Polycythemia Vera (PV) at steady-state and upon SCF stimulation. While AB and CB, respectively, activated transient or sustained canonical cKIT-signaling, PV showed a non-canonical signaling including increased mTOR and ERK1 and decreased DEPTOR. Accordingly, screening of FDA-approved compounds showed increased PV sensitivity to JAK, cKIT, and MEK inhibitors. Moreover, differently from AB and CB, in PV the mature 145kDa-cKIT constitutively associated with the tetraspanin CD63 and was not endocytosed upon SCF stimulation, contributing to unrestrained cKIT signaling. These results identify a clinically exploitable variegation of cKIT signaling/metabolism that may contribute to the great erythroid output occurring during development and in PV.

Although stem cell factor (SCF)/cKIT interaction plays key functions in erythropoiesis, cKIT signaling in human erythroid cells is still poorly defined. To provide new insights into cKIT-mediated erythroid expansion in development and disease, we performed phosphoproteomic profiling of primary erythroid progenitors from adult blood (AB), cord blood (CB), and Polycythemia Vera (PV) at steady-state and upon SCF stimulation. While AB and CB, respectively, activated transient or sustained canonical cKIT-signaling, PV showed a non-canonical signaling including increased mTOR and ERK1 and decreased DEPTOR. Accordingly, screening of FDA-approved compounds showed increased PV sensitivity to JAK, cKIT, and MEK inhibitors. Moreover, differently from AB and CB, in PV the mature 145kDa-cKIT constitutively associated with the tetraspanin CD63 and was not endocytosed upon SCF stimulation, contributing to unrestrained cKIT signaling. These results identify a clinically exploitable variegation of cKIT signaling/metabolism that may contribute to the great erythroid output occurring during development and in PV. [ABSTRACT FROM AUTHOR]

The majority of the tumors in the gastrointestinal (GI) tract of rats and mice, with spindle cell morphology, are diagnosed as smooth muscle tumors (SMTs). Similarly, several decades ago human GI tumors with spindle cell morphology were also diagnosed as SMTs. However, later investigations identified most of these tumors in humans as gastrointestinal stromal tumors (GISTs). The GISTs are considered to arise from the interstitial cells of Cajal located throughout the GI tract. Positive immunohistochemical staining with CKIT antibody is a well-accepted diagnostic marker for GISTs in humans. Since there is a considerable overlap between the histomorphology of SMTs and GISTs, it is not possible to distinguish them on hematoxylin and eosin stained sections. As a result, GISTs are not routinely diagnosed in toxicological studies. The current study was designed to evaluate the tumors diagnosed as leiomyoma or leiomyosarcoma in the National Toxicology Program's 2-year bioassays using CKIT, smooth muscle actin, and desmin immunohistochemistry. The results demonstrate that most of the mouse SMTs diagnosed as leiomyoma or leiomyosarcoma are likely GISTs, whereas in rats the tumors are likely SMTs and not GISTs. [ABSTRACT FROM AUTHOR]

Background: cKIT kinase overexpression and gain-of-function mutations are the critical pathogenesis of gastrointestinal stromal tumors (GISTs). Although the multiple kinase inhibitors such as imatinib, sunitinib, and regorafenib have been approved for GISTs, the acquisition of polyclonal secondary resistance mutations in KIT is still a limitation for GIST treatment. Here we explored the KIT inhibitory activity of axitinib in preclinical models and describe initial characterization of its activity in GIST patient-derived primary cells. Methods: The activities of axitinib against mutant KIT were evaluated using protein-based assay and a panel of engineered and GIST-derived cell lines. The binding modes of axitinib-KIT/KIT mutants were analyzed. Four primary cells derived from GIST patients were also used to assess the drug response of axitinib. Results: Axitinib exhibited potent activities against a variety of cKIT associated primary and secondary mutations. It displayed better activity against cKIT wild-type, cKIT V559D/A/G, and L576P primary gain-of-function mutations than imatinib, sunitinib, and regorafenib. In addition, it could inhibit imatinib resistant cKIT T670I and V654A mutants in vitro and in vivo GIST preclinical models. Conclusion: Our results provide the basis for extending the application of axitinib to GISTs patients who are unresponsive or intolerant to the current therapies. [ABSTRACT FROM AUTHOR]

The existence of rare cancer cells that sporadically acquire drug-tolerance through epigenetic mechanisms is proposed as one mechanism that drives cancer therapy failure. Here we provide evidence that specific microenvironments impose non-sporadic expression of proteins related to epithelial plasticity and drug resistance. Microarrays of robotically printed combinatorial microenvironments of known composition were used to make cell-based functional associations between microenvironments, which were design-inspired by normal and tumor-burdened breast tissues, and cell phenotypes. We hypothesized that specific combinations of microenvironment constituents non-sporadically impose the induction of the AXL and cKIT receptor tyrosine kinase proteins, which are known to be involved in epithelial plasticity and drug-tolerance, in an isogenic human mammary epithelial cell (HMEC) malignant progression series. Dimension reduction analysis reveals type I collagen as a dominant feature, inducing expression of both markers in pre-stasis finite lifespan HMECs, and transformed non-malignant and malignant immortal cell lines. Basement membrane-associated matrix proteins, laminin-111 and type IV collagen, suppress AXL and cKIT expression in pre-stasis and non-malignant cells. However, AXL and cKIT are not suppressed by laminin-111 in malignant cells. General linear models identified key factors, osteopontin, IL-8, and type VIα3 collagen, which significantly upregulated AXL and cKIT, as well as a plasticity-related gene expression program that is often observed in stem cells and in epithelial-to-mesenchymal-transition. These factors are co-located with AXL-expressing cells in situ in normal and breast cancer tissues, and associated with resistance to paclitaxel. A greater diversity of microenvironments induced AXL and cKIT expression consistent with plasticity and drug-tolerant phenotypes in tumorigenic cells compared to normal or immortal cells, suggesting a reduced perception of microenvironment specificity in malignant cells. Microenvironment-imposed reprogramming could explain why resistant cells are seemingly persistent and rapidly adaptable to multiple classes of drugs. These results support the notion that specific microenvironments drive drug-tolerant cellular phenotypes and suggest a novel interventional avenue for preventing acquired therapy resistance.

Clinical evidence in oncology argues for the advantages of performing molecular analysis of blood biomarkers to provide information about systemic changes and tumor heterogeneity. Whereas the diagnostic value of cell-free circulating DNA (fcDNA) has successfully been demonstrated in several studies, DNA enclosed in extracellular vesicles (EV) has only recently been described, and its potential diagnostic value is unclear. We established a protocol for separation of EV and fc fractions and tested for presence of mutant BRAFV600E mediating resistance to Vemurafenib and cKITD816V mediating resistance to Imatinib in blood of patients with melanoma and mastocytosis. Our results show that EV contain significantly higher amounts of total DNA as compared to the fc fraction. However, about ten-fold higher copy numbers of the wild type and mutant BRAF and cKIT were detected in the fcDNA fraction supporting its diagnostic value and pointing to differences in fc and EV DNA content. [ABSTRACT FROM AUTHOR]

The major goal in regenerative medicine is to repair and restore injured, diseased or aged tissue function, thereby promoting general health. As such, the field of regenerative medicine has great translational potential in undertaking many of the health concerns and needs that we currently face. In particular, hematopoietic and vascular systems supply oxygen and nutrients and thus play critical roles in tissue development and tissue regeneration. Additionally, tissue vasculature serves as a tissue stem cell niche and thus contributes to tissue homeostasis. Notably, hematopoietic and vascular systems are sensitive to injury and subject to regeneration. As such, successful hematopoietic and vascular regeneration is prerequisite for efficient tissue repair and organismal survival and health. Recent studies have established that the interplay among the ETS transcription factor ETV2, vascular endothelial growth factor, and its receptor VEGFR2/FLK1 is essential for hematopoietic and vascular development. Emerging studies also support the role of these three factors and possible interplay in hematopoietic and vascular regeneration. Comprehensive understanding of the molecular mechanisms involved in the regulation and function of these three factors may lead to more effective approaches in promoting tissue repair and regeneration. Developmental Dynamics 246:318-327, 2017. © 2016 Wiley Periodicals, Inc. [ABSTRACT FROM AUTHOR]

Gastrointestinal stromal tumors are the most frequent mesenchymal tumors of the gastrointestinal tract. The discovery that these tumors, formerly thought of smooth muscle origin, are indeed better characterized by specific activating mutation in genes coding for the receptor tyrosine kinases CKIT and PDGFRA and that these mutations are strongly predictive for the response to targeted therapy with receptor tyrosine kinase inhibitors has made GISTs the typical example of the integration of basic molecular knowledge in the daily clinical activity. The information on the mutational status of these tumors is essential to predict (and subsequently to plan) the therapy. As resistant cases are frequently wild-type, other possible oncogenic events, defining other entities, have been discovered (e.g. succinil dehydrogenase mutation/dysregulation, insuline growth factor expression, mutations in the RAS-RAF-MAPK pathway). The classification of disease must nowadays rely on the integration of the clinico-morphological characteristics with the molecular data.

Patients with metastatic melanoma have historically had dismal outcomes. The last several years has seen the emergence of effective immune and targeted therapies for metastatic melanoma. Targeted therapies have primarily impacted the 40–50 % of patients with BRAFV600 mutated melanoma. The remainder of patients with advanced melanoma harbor a wide spectrum of mutations other than BRAFV600 that are associated with unique pathophysiological, prognostic, and therapeutic implications. The treatment of this subset of patients is a challenging problem. In recent years, preclinical and early clinical studies have suggested that inhibitors of mitogen activated protein kinase (MAPK) pathway and parallel signaling networks may have activity in treatment of BRAFV600 wild-type (WT) melanoma. In this review, we will discuss available and developing therapies for BRAF WT patients with metastatic melanoma, particularly focusing on molecular targeted options for various genetically defined melanoma subsets. [ABSTRACT FROM AUTHOR]

Background. Ipilimumab improves overall survival (OS) in advanced melanoma. Acral melanoma is an uncommon clinical subtype of this disease associated with poor prognosis. The clinical activity of ipilimumab has not been well-defined in advanced acral melanoma. Methods. We retrospectively reviewed the demographics, treatment history, and clinical outcomes for all patients with acral melanoma treated with ipilimumab from two academic centers between February 2006 and June 2013. Using Cox proportional hazards models, we assessed for factors that correlated with OS. Results. A total of 35 patients with acral melanoma received ipilimumab. Melanomas arose on volar surfaces (n = 28) and subungual sites (n = 7); stage M1c disease was present in 54%, and 45% had elevated serum lactate dehydrogenase (LDH). Best response by RECIST 1.1 criteria was complete response in 1 patient, partial response in 3, and stable disease (SD) in 4 for an objective response rate (ORR) of 11.4% and a clinical benefit rate (ORR + SD) at 24 weeks of 22.9%. Median progression-free survival was 2.5 months (95% confidence interval [Cl]: 2.3-2.7 months); median OS was 16.7 months (95% Cl: 10.9-22.5 months). Normal LDH and absolute lymphocyte count ≥1,000 at 7 weeks predicted longer OS. Immune-related adverse events (irAEs) were noted in 16 patients including 7 with grade 3/4 irAEs (20%). Conclusion. Ipilimumab is clinically active in acral melanoma with similar ORR and OS compared with unselected melanoma populations. Ipilimumab remains a viable therapeutic option for patients with advanced acral melanoma. [ABSTRACT FROM AUTHOR]

Gastrointestinal stromal tumors (GIST) are the most common sarcomas of the gastrointestinal tract, with transformation typically driven by activating mutations of cKIT and less commonly platelet-derived growth factor receptor alpha (PDGFRA). Successful targeting of tyrosine-protein kinase Kit with imatinib, a tyrosine kinase inhibitor, has had a major impact in the survival of patients with GIST in both the adjuvant and metastatic setting. A recent modification of treatment guidelines for patients with localized, high-risk GIST extended the adjuvant treatment duration from 1 year to 3 years. In this paper, we review the clinical data of patients with GIST treated in the Oncology Outpatient Unit of "Attikon" University Hospital and aim to assess which patients are eligible for prolongation of adjuvant imatinib therapy as currently suggested by treatment recommendations. [ABSTRACT FROM AUTHOR]

Background 5-Hydroxytryptamine (5-HT, serotonin) is an important regulator of colonic motility and secretion; yet the role of serotonergic neurons in the colon is controversial. Methods We used immunohistochemical techniques to examine their projections throughout the enteric nervous system and interstitial cells of Cajal ( ICC) networks in the murine proximal to mid colon. Key Results Serotonergic neurons, which were mainly calbindin positive, occurred only in myenteric ganglia (1 per 3 ganglia). They were larger than n NOS neurons but similar in size to Dogiel Type II ( AH) neurons. 5- HT neurons, appeared to make numerous varicose contacts with each other, most n NOS neurons, Dogiel Type II/ AH neurons and glial cells. 5- HT, calbindin and n NOS nerve fibers also formed a thin perimuscular nerve plexus that was associated with ganglia, which contained both n NOS positive and negative neurons, which lay directly upon the submucosal pacemaker ICC network. Neurons in perimuscular ganglia were surrounded by 5- HT varicosities. Submucous ganglia contained n NOS positive and negative neurons, and calbindin positive neurons, which also appeared richly supplied by serotonergic nerve varicosities. Serotonergic nerve fibers ran along submucosal arterioles, but not veins. Varicosities of serotonergic nerve fibers were closely associated with pacemaker ICC networks and with intramuscular ICC ( ICC- IM). 5- HT2B receptors were found on a subpopulation of non-5- HT containing myenteric neurons and their varicosities, pacemaker ICC- MY and ICC- IM. Conclusions & Inferences Myenteric serotonergic neurons, whose axons exhibit considerable divergence, regulate the entire enteric nervous system and are important in coordinating motility with secretion. They are not just interneurons, as regularly assumed, but possibly also motor neurons to ICC and blood vessels, and some may even be sensory neurons. [ABSTRACT FROM AUTHOR]

Mast cells, sentinel immune cells, are most abundantly expressed in vascularized tissues that interface the external environment, such as the skin and ocular surface. Our previous reports have shown mast cells reside closely with vascular endothelial cells and mediate the pathogenic angiogenic response. However, the contribution of mast cells and their underlying mechanisms on lymphangiogenesis have not been fully deciphered. Using a murine model of inflammatory corneal angiogenesis, we observed adjacent migration of activated mast cells with new lymph vessel growth. Our in vitro co-culture assays demonstrate that mast cells express high levels of of VEGF-D and directly promote lymphatic endothelial cell tube formation and proliferation. Moreover, our loss-of-function approaches, using mast cell knockout mice and cromolyn-mediated mast cell inhibition, showed mast cell deficiency suppresses the induction of inflammatory lymphangiogenesis and VEGF-D expression at the ocular surface following corneal tissue insult. Our findings suggest blockade of mast cells as a potential therapeutic strategy to inhibit pathological lymphangiogenesis. • Activation and infiltration of mast cells into the cornea during inflammatory lymphangiogenesis • Mast cells directly promote lymphatic endothelial cell proliferation and tube formation. • Genetic deletion of mast cells abrogates pathological corneal lymphangiogenesis. • Pharmacological blockade of mast cell activation suppresses inflammatory lymphangiogenesis. [ABSTRACT FROM AUTHOR]

Background: Racial and geographic factors seem to affect the incidence of cutaneous and mucosal melanoma. Objective: To investigate the occurrence of BRAF and cKIT impairments in patients with sinonasal melanoma in Southern Italy. Methods: Eleven sinonasal melanomas were screened for BRAF mutations and cKIT alterations by immunohistochemistry (CD117), fluorescence in situ hybridization and sequencing analyses. Results: A high prevalence (4/11; 36%) of BRAF mutations and lack of cKIT mutations were observed. Amplification of cKIT was found in 18% of cases; cKIT expression was detectable in 18% non-overlapping cases. No correlation between CD117 and cKIT alterations was observed. One (6%) cKIT and two (12%) BRAF mutations were detected in an additional series of 17 acral/mucosal melanomas from the same geographic areas. Conclusion: Mutations of cKIT are infrequent in sinonasal melanoma in Southern Italy. Copyright © 2013 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Abstract: The introduction of imatinib therapy for GISTs has represented a major breakthrough of oncology in the last decade, improving dramatically the prognosis of GIST patients. The discovery of an oncogenic event (kinase mutations) having a major predictive value underlines the need for a paradigm of classification combining thorough pathological examination and molecular analysis. The response to therapy with imatinib is indeed determined by the type of mutation in kinase genes. Genetic analysis should be therefore performed in all cases deserving such a therapy. The study of the resistance has enabled us to discover other important oncogenetic events (e.g. dysregulation of downstream pathways) that are important in the genesis and therapy of other tumours as well. Genetic studies have also allowed a molecular classification of GISTs, thus identifying subsets of GISTs (e.g. SDH-negative, paediatric) that do not behave as the “classical”, RTK-mutated tumours, probably representing different “entities”. [Copyright &y& Elsevier]

There is a spectrum of disorders that clinically manifest as a result of mast cell activation. A non-clonal form has emerged in the literature where many of the clinical features of systemic mastocytosis are shared despite having a distinct mast cell biology. In this review, we summarize key features of the science behind mast cell activation relevant to what is now known as non-clonal mast cell activation syndrome (nc-MCAS). We highlight the clinical manifestations of nc-MCAS with a focus on diagnosis and treatment. [ABSTRACT FROM AUTHOR]

Objective: 1) To further identify tumors other than GISTs; 2) To study cKIT gene mutation for proper Imatinib treatment. Methods: Fresh tumor tissue samples were obtained from: 2 tissue samples of GISTs; 1 parotid cancer; 1 soft tissue sarcoma; 1 chondrosarcoma; 1 liposarcoma and 1 pure seminoma. The fresh tissues were divided into two portions: one for histopathological diagnosis and cKIT protein study, the other for DNA extraction and cKIT gene mutation study. Results: The authors described the gastrointestinal stromal tumors (GISTs) and other malignancies associated with positive cKIT protein in Thailand. We also describe briefly the methodology and molecular techniques used in the study of the mutation of cKIT gene in our laboratory using DNA sequencing method. Examples of positive cKIT protein and deletion mutation as well as missense (point mutation) mutations from our own patients are shown. Mutation study of exons 9, 11, 13 and 17 of cKIT gene in all 7 patients was carefully done to assist us in treating those patients with Imatinib therapy. Conclusion: Mutation study of cKIT gene could be done routinely in our molecular laboratory in Thailand. We recommend cKIT gene mutation study be done in every case of GIST and tumor with positive cKIT protein other than GIST prior to initiation of Imatinib therapy.

Abstract: The Kit system, composed of Kit ligand (KL) and its tyrosine kinase receptor, cKit, has been well characterized in mammals. Studies have shown that it is involved in signaling between the oocyte and somatic cells during the process of follicle maturation. We characterized KL mRNA expression during follicle maturation in the domestic hen, examined regulation of KL and a possible function of the Kit system. KL mRNA expression was assessed using quantitative PCR (n =4 replicates) in follicles of various sizes (1, 3, 5, 6–12mm, F1). Expression of KL mRNA decreased significantly (p <0.01) with follicle development and was highest in <1mm follicles, which contained the theca as well as granulosa layers, with high levels also found in the granulosa layer of 3mm follicles and ovarian stroma. To study regulation of KL mRNA, granulosa cells from 6–8mm follicles (n =4 replicates) were plated in M199 plus 0.1% BSA in the presence of various treatments including: oocyte conditioned medium (OCM), Vitamin D3, FSH, estradiol, progesterone and testosterone. OCM caused a dose-related increase (p <0.05) in expression of KL mRNA; Vitamin D3 increased and FSH decreased expression of KL mRNA. cKit was detected (at the expected size) in the theca layer of 3–5mm follicles and in a lysate of whole <1mm follicles. Culture of granulosa cells in the presence of OCM resulted in a decrease of P4 secretion, an effect blocked by pre-incubation of OCM with cKit antibody. Although OCM caused a dose-related increase in E2 secretion from theca, this was not blocked by cKit antibody. [Copyright &y& Elsevier]

Cutaneous melanoma is a highly aggressive cancer with still limited, but increasingly efficacious, standard treatment options. Recent preclinical and clinical findings support the notion that cutaneous melanoma is not one malignant disorder but rather a family of distinct molecular diseases. Incorporation of genetic signatures into the conventional histopathological classification of melanoma already has great implications for the management of cutaneous melanoma. Herein, we review our rapidly growing understanding of the molecular biology of cutaneous melanoma, including the pathogenic roles of the mitogen-associated protein kinase (MAPK) pathway, the phosphatidylinositol 3 kinase [PI3K]/phosphatase and tensin homologue deleted on chromosome 10 [PTEN]/Akt/mammalian target of rapamycin [mTOR])PTEN (phosphatase and tensin homolog) pathway, MET (hepatocyte growth factor), Notch signaling, and other key molecules regulating cell cycle progression and apoptosis. The mutation Val600Glu in the BRAF oncogene (designated BRAF(V600E)) has been associated with clinical benefit from agents that inhibit BRAF(V600E) or MEK (a kinase in the MAPK pathway). Cutaneous melanomas arising from mucosal, acral, chronically sun-damaged surfaces sometimes have oncogenic mutations in KIT, against which several inhibitors have shown clinical efficacy. These findings suggest that prospective genotyping of patients with melanoma, combined with the growing availability of targeted agents, which can be used to rationally exploit these findings, should be used increasingly as we work to develop new and more effective treatments for this devastating disease. [ABSTRACT FROM AUTHOR]

Patient-specific therapeutic cells derived from induced pluripotent stem (iPS) cells may bypass the ethical issues associated with embryonic stem (ES) cells and avoid potential immunological reactions associated with allogenic transplantation. It is critical, for the ultimate clinical applicability of iPS cell-derived therapies, to establish feeder-free cultures that ensure efficient differentiation of iPS cells into therapeutic progenitors. It is also necessary to understand if iPS cell-derived progenitors differ from those derived from ES cells. In this study, we compared the efficiency of three different feeder-free cultures for differentiating mouse iPS cells into ckit+sca1+ hematopoietic progenitor cells (HPCs) and compared how differentiation and functionality varies between ES and iPS cells. Our results indicated that both iPS and ES cells can be efficiently differentiated into HPCs in suspension cultures supplemented with secretion factors from mouse bone marrow stromal cells (OP9-DL1 conditioned medium). The functionality of these cells was demonstrated by differentiation into CD11c+ dendritic cells (DCs). Both ES and iPS-derived DCs expressed activation molecules (CD86, CD80) in response to LPS stimulation and stimulated T cell proliferation in a mixed lymphocyte reaction (MLR). Extensive quantitative RT-PCR studies were used to study the differences in gene expression profiles of ckit+sca1+ cells generated from the various culture systems as well as differences between ES-derived and iPS-derived cells. We conclude that a feeder-free system using stromal conditioned medium can efficiently generate HPCs as well as functional DCs from iPS cells and the generated cells have similar gene expression profile as those from ES cells. [ABSTRACT FROM AUTHOR]

For targets belonging to the same family of receptors, inhibitors often act at more than one biological target and produce a synergistic effect. Separate pharmacophore-based comparative molecular field analysis (CoMFA) and comparative molecular similarity indices analysis (CoMSIA) models were developed from our data-set for the kinase insert domain-containing receptor (KDR), cKit and Flt1 inhibitors. These models showed excellent internal predictability and consistency; validation using test-set compounds yielded a good predictive power for the pIC50-value. The field contour maps (CoMFA and CoMSIA) corresponding to the KDR, cKit and Flt1 kinase subtypes reflected the characteristic similarities and differences between these types. These maps provided valuable information to facilitate structural modifications of the inhibitor to increase selectivity of the KDR over cKit and Flt1. [ABSTRACT FROM AUTHOR]

Abstract: Loss of cKit receptor in cutaneous melanomas was attributed to the down-regulation of AP2 transcription factor. Our analysis of 27 melanoma cell lines showed no correlation between AP2 and c-kit expression. Suggesting a post-transcriptional mechanism of cKit down-modulation, we performed genome-wide microRNA (miRNA) expression profiling and found that several miRNA species are commonly up-regulated in melanomas. Among them was mir-221, which can directly interact with c-kit 3′UTR and inhibit cKit protein translation. Observed inverse correlation of the c-kit and mir-221 expression in various melanocytic cells pointed to its involvement in regulation of cKit in melanoma. Moreover, a series of functional assays demonstrated that mir-221 could directly inhibit cKit, p27Kip1 and, possibly, other pivotal proteins in melanoma. Collectively, the studies presented here indicate that mir-221 could be a novel therapeutic target for the treatment of cutaneous melanoma. They also suggest that regulation of expression and functional activity of identified up-regulated miRNAs should be further studied in the context of malignant melanoma. [Copyright &y& Elsevier]

Aims: Small cell carcinoma of the ovary, hypercalcaemic-type (SCCOH) is morphologically similar to small cell carcinomas from other sites. The aims of this study were to (i) determine if a biomarker panel would distinguish small cell carcinomas of the ovary, cervix (SCCCx) and lung (SCCLu) and (ii) potentially determine the histogenesis of SCCOH. Methods and results: Nine ovarian small cell carcinomas (seven hypercalcaemic type; two pulmonary type), eight SCCCx and 22 SCCLu were immunostained for thyroid transcription factor (TTF)-1, WT-1, p16, cKIT and OCT3/4; a subset of cases were tested for human papillomavirus (HPV). WT-1 was diffusely positive in 6/7 SSCOH versus two of 33 other small cell carcinomas ( P ≤ 0.001). TTF-1 was diffusely positive in 20/22 SCCLu and 1/8 SCCCx, and negative in all SCCOH. p16 and cKIT demonstrated variable patterns of immunoreactivity in all cases. HPV was identified in 5/6 SCCCx; SCCOH and SCCLu were negative for HPV. Conclusions: Combined staining with WT-1 and TTF-1 will distinguish SCCOH from SCCLu and SCCCx with a sensitivity of 86% and specificity of 97%. HPV is specific for tumours of cervical origin, but p16 immunohistochemistry is not useful for this purpose. The presence of diffuse WT-1 supports a Müllerian origin for SCCOH, whereas the absence of cKIT and OCT3/4 argues against a germ cell origin. [ABSTRACT FROM AUTHOR]

Abstract: For targets belonging to the same family of receptors, inhibitors often act at more than one biological target and produce a synergistic effect. Separate CoMFA and CoMSIA models were developed from our data set for the KDR, cKit and Tie-2 inhibitors. These models showed excellent internal predictability and consistency, and validation using test-set compounds yielded a good predictive power for the pIC50 value. The field contour maps (CoMFA and CoMSIA) corresponding to the KDR, cKit and Tie-2 kinase subtypes reflected the characteristic similarities and differences between these types. These maps provided valuable information to facilitate structural modifications of the inhibitor to increase selectivity for the KDR over cKit and Tie-2. [Copyright &y& Elsevier]

We hypothesized that exposure to hyperbaric oxygen (HBO2) would mobilize stem/progenitor cells from the bone marrow by a nitric oxide (·NO) -dependent mechanism. The population of CD34+ cells in the peripheral circulation of humans doubled in response to a single exposure to 2.0 atmospheres absolute (ATA) O2 for 2 h. Over a course of 20 treatments, circulating CD34+ cells increased eightfold, although the overall circulating white cell count was not significantly increased. The number of colony-forming cells (CFCs) increased from 16 ± 2 to 26 ± 3 CFCs/100,000 monocytes plated. Elevations in CFCs were entirely due to the CD34+ subpopulation, but increased cell growth only occurred in samples obtained immediately posttreatment. A high proportion of progeny cells express receptors for vascular endothelial growth factor-2 and for stromal-derived growth factor. In mice, HBO2 increased circulating stem cell factor by 50%, increased the number of circulating cells expressing stem cell antigen-1 and CD34 by 3.4-fold, and doubled the number of CFCs. Bone marrow ·NO concentration increased by 1,008 ± 255 nM in association with HBO2. Stem cell mobilization did not occur in knockout mice lacking genes for endothelial ·NO synthase. Moreover, pretreatment of wild-type mice with a ·NO synthase inhibitor prevented the HBO2-induced elevation in stem cell factor and circulating stem cells. We conclude that HBO2 mobilizes stem/progenitor cells by stimulating ·NO synthesis. [ABSTRACT FROM AUTHOR]

Squamous cell carcinoma of the oesophagus (SCCO) is still a pathology of bad prognosis. Specific therapies are now developed against epidermal growth factor receptor (EGFR), human epidermal growth factor receptor 2, c-kit receptor (CD117), vascular endothelial growth factor (VEGF) and p53 protein. This study was aimed at assessing their expression in a large series of SCCO, as well as their potential therapeutic interest in this pathology. Immunohistochemical expression of these factors was assessed retrospectively in 107 cases of SCCO with primary surgery, as well as their relationships to recurrence, metastasis and overall survival on a long-term follow-up. Human epidermal growth factor receptor 2 and CD117 were expressed in less than 3% of the cases. Epidermal growth factor receptor and p53 were overexpressed in 68.2 and 66.4% of the cases, and VEGF in 38.3%. Epidermal growth factor receptor overexpression was significantly related to vascular invasion (P=0.023). Its diffuse positivity was significantly related in multivariate analysis to higher local recurrence (P=0.006) and lower overall survival (P=0.003), in a subgroup of patients of poor outcome who had received postoperative adjuvant treatment. These results highlight the great potential prognostic and therapeutic interest of evaluating EGFR diffuse positivity in locally advanced SCCO. [ABSTRACT FROM AUTHOR]

Stem cell factor (SCF) and its receptor, cKIT, are novel regulators of pathological neovascularization in the eye, which suggests that inhibition of SCF/cKIT signaling may be a novel pharmacological strategy for treating neovascular age-related macular degeneration (AMD). This study evaluated the therapeutic potential of a newly developed fully human monoclonal antibody targeting cKIT, NN2101, in a murine model of neovascular AMD. In hypoxic human endothelial cells, NN2101 substantially inhibited the SCF-induced increase in angiogenesis and activation of the cKIT signaling pathway. In a murine model of neovascular AMD, intravitreal injection of NN2101 substantially inhibited the SCF/cKIT-mediated choroidal neovascularization (CNV), with efficacy comparable to aflibercept, a vascular endothelial growth factor inhibitor. A combined intravitreal injection of NN2101 and aflibercept resulted in an additive therapeutic effect on CNV. NN2101 neither caused ocular toxicity nor interfered with the early retinal vascular development in mice. Ocular pharmacokinetic analysis in rabbits indicated that NN2101 demonstrated a pharmacokinetic profile suitable for intravitreal injection. These findings provide the first evidence of the potential use of the anti-cKIT blocking antibody, NN2101, as an alternative or additive therapeutic for the treatment of neovascular AMD. [ABSTRACT FROM AUTHOR]