Activating c-KIT Mutations Research Articles

Penile melanoma: a pathological report of two cases

BackgroundPenile melanoma (PM) is a rare tumor, accounting for less than 2% of all penile cancers. PM can occur on the surface of the glans, foreskin, and opening of the urethra. Furthermore, PM primarily affects older individuals and is not associated with sun exposure. Currently, there is no specific staging system for genitourinary tract melanomas, so these tumors are typically staged using the criteria for cutaneous melanoma. Limited data in the literature suggests that PM generally has a poor clinical prognosis.Case presentationHere, we describe two cases of PM. The first case affected a 62-year-old male who presented with hematuria and a painful tumor in the distal urethra, leading to a suspicion of penile cancer. The second case involved a 68-year-old male who noticed a rapidly evolving dark spot on his foreskin. Histological analysis confirmed the presence of melanoma in both patients. The tumors showed a diffuse and strong PRAME-positivity and lacked BRAF mutation in both cases. Additionally, the second tumor harbored an activating CKIT mutation. An enhanced PD-L1 expression was observed in both tumors.ConclusionsWe presented two rare forms of mucosal melanoma and highlighted the entities in the differential diagnosis. Based on our experience PRAME is a helpful marker for making the diagnosis of PM, and PD-L1 can predict the success of the immunotherapy. We also emphasize the need for an organ-specific staging system for PMs.

Abstract 1482: M4645 is a potent and highly selective inhibitor of disease relevant cKIT mutations for the treatment of metastatic or recurrent GIST

Abstract Oncogenic mutations in cKIT in exons 9 and 11 are the main driver of gastrointestinal stromal tumors (GIST). Imatinib is standard of care in 1L in unresectable metastatic or recurrent GIST, yet secondary resistance mutations in exons 13/14 and 17/18 of the cKIT kinase domain frequently arise limiting efficacy. Currently approved 2L and 3L therapies sunitinib and regorafenib target these mutations only to some extent and show limited clinical benefit. Even with the recent approval of ripretinib as 4L treatment, a high unmet medical need remains for patients with imatinib resistance mutations in exon13/14. M4645 was designed to particularly inhibit the frequent V654A resistance mutation in exon 13 of cKIT. Based on cKIT inhibitor scaffolds identified within the Merck compound collection, a 3,5,6-substituted indazole-lead was discovered. Substituent optimization in the 5- and 6-positions of the core scaffold resulted in highly selective kinase inhibitors. Through variation at the 3-position, potency and physicochemical properties were optimized, resulting in the candidate molecule M4645 which combined potency, kinase selectivity, and solubility with a good safety profile. The excellent kinase selectivity could be rationalized by the crystal structure of cKIT V654A mutant kinase domain in complex with M4645. M4645 showed potent inhibition of cKIT autophosphorylation and of downstream P-ERK1/2 and P-AKT signaling in cell lines bearing activating and resistance mutations of cKIT. Dose-dependent inhibition of cKIT activation was demonstrated in a GIST xenograft model expressing the cKIT resistance mutation V654A in exon 13. Strong antitumor activity was observed in cell line and patient-derived GIST xenograft models with this imatinib resistance mutation. Compared to imatinib, M4645 induced prolonged anti-tumor response in a GIST model with a primary activating mutation of cKIT. In summary, the favorable profile and strong antitumor activity, particularly in GIST models expressing the V654A resistance mutation, suggests superiority of M4645 compared to currently available > 2L treatment options for unresectable metastatic or recurrent GIST patients. Citation Format: Nina Linde, Andreas Blum, Dieter Dorsch, Ulrich Graedler, Michael Busch, Nina Glaser, Carl Petersson, Aaron Ruff, Eva Sherbetjian, Christina Esdar. M4645 is a potent and highly selective inhibitor of disease relevant cKIT mutations for the treatment of metastatic or recurrent GIST [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1482.

Correlation Between KIT Expression and c-Kit Mutations in 2 Subtypes of Canine Oral Melanocytic Neoplasms.

c-Kit mutations have been reported in 15% to 40% of certain human melanoma subtypes, including those histologically similar to canine oral malignant melanomas. Therapeutic response to tyrosine kinase inhibitors has been demonstrated in those human patients. As canine oral malignant melanomas tend to have a poor prognosis despite aggressive surgical removal, evaluation of KIT expression and identification of c-Kit mutations in canine oral melanocytic neoplasms was performed to determine if there is any indication that tyrosine kinase inhibitor drugs might effectively treat any of these cases. This study evaluated 27 canine oral malignant melanomas and 12 canine histologically well-differentiated oral melanocytic neoplasms for activating c-Kit mutations, determined differences in immunohistochemical expression of KIT and c-Kit mutation status, and determined if KIT expression could predict c-Kit mutation status. Among samples that contained intraepithelial nests of neoplastic melanocytes in the KIT-labeled sections, KIT was expressed within cells in these nests in 22/23 (96%) malignant melanomas and 5/7 histologically well-differentiated neoplasms. KIT was expressed in 10% to 30% of neoplastic melanocytes in the lamina propria in 3/24 (13%) malignant melanomas, but 0/9 (0%) histologically well-differentiated neoplasms. Next-generation sequencing identified 85 variants in c-Kit, including 9 nonsynonymous mutations that resulted in amino acid changes predicted to affect protein function. c-Kit mutations with predicted deleterious protein effects were more common in malignant melanomas (8/27 [30%] vs 1/12 [8%]). There was no apparent relationship between detected c-Kit mutations and KIT expression. These results do not support the use of therapies that target c-Kit.

C-KIT mutation role in oral malignant melanoma pathway: A narrative review

Aim: To investigate the role of the c-KIT mutation in oral malignant melanoma (OMM). Materials and Methods: The search strategy includes randomized control trials and clinical trials from the databases of PubMed Central, Cochrane, EBSCO, and MEDLINE from January 1999 to May 2019. A total of seven clinical trials that met all inclusion criteria were included in this systematic review. Results: A previous study found that c-KIT can initiate the activation variety of downstream pathways, including the MAPK/MEK and PI3K/AKT pathways, which play important roles in cancer development. The c-KIT mutation has resulted in a loss of heterozygosity at 12p13, and loss of p27KIP1 protein has been detected. Activating c-KIT mutations in the juxtamembrane and other domains has been considered as an oncogene and also a therapeutic target in various tumors. Conclusion: c-KIT is playing the role of an important target in the research of molecular therapy in patients with metastatic OMM with c-KIT mutations.

Identification of molecular genetic contributants to canine cutaneous mast cell tumour metastasis by global gene expression analysis.

Cutaneous mast cell tumours are one of the most common canine cancers. Approximately 25% of the tumours metastasise. Activating c-kit mutations are present in about 20% of tumours, but metastases occur in the absence of mutations. Tumour metastasis is associated with significantly diminished survival in spite of adjuvant chemotherapy. Available prognostic tests do not reliably predict whether a tumour will metastasise. In this study we compared the global expression profiles of 20 primary cutaneous mast cell tumours that metastasised with those of 20 primary tumours that did not metastasise. The objective was to identify genes associated with mast cell tumour metastatic progression that may represent targets for therapeutic intervention and biomarkers for prediction of tumour metastasis. Canine Gene 1.1 ST Arrays were employed for genome-wide expression analysis of formalin-fixed, paraffin-embedded biopsies of mast cell tumours borne by dogs that either died due to confirmed mast cell tumour metastasis, or were still alive more than 1000 days post-surgery. Decreased gene expression in the metastasising tumours appears to be associated with a loss of cell polarity, reduced cell-cell and cell-ECM adhesion, and increased cell deformability and motility. Dysregulated gene expression may also promote extracellular matrix and base membrane degradation, suppression of cell cycle arrest and apoptosis, and angiogenesis. Down-regulation of gene expression in the metastasising tumours may be achieved at least in part by small nucleolar RNA-derived RNA and microRNA-effected gene silencing. Employing cross-validation, a linear discriminant analysis-based classifier featuring 19 genes that displayed two-fold differences in expression between metastasising and non-metastasising tumours was estimated to classify metastasising and non-metastasising tumours with accuracies of 90–100% and 70–100%, respectively. The differential expression of 9 of the discriminator genes was confirmed by quantitative reverse transcription-PCR.

Comparison of lesional skin c-KIT mutations with clinical phenotype in patients with mastocytosis.

Activating c-KIT mutations cause abnormal mast cell growth and appear to play a role in mastocytosis. However, the correlation of c-KIT mutations with disease phenotypes is poorly characterized. To evaluate the correlation of c-KIT mutations with clinical presentations and laboratory findings. Total cellular RNA was isolated from the skin lesions of 43 adults and 7 children with mastocytosis, and PCR amplicons of cDNA were sequenced for c-KIT mutations. The most common activating mutation, KIT-D816V, was identified in 72% of adults and 57% of children. Additional activating mutations, namely, V560G and the internal tandem duplications (ITDs) 502-503dupAY, were detected in 12% of adults and 8% of children. V560G occurred more commonly in our patients than previously reported, and it appeared to be associated with more advanced disease. Otherwise, the presence or absence of activating mutations did not correlate with skin lesion morphology, disease extent or total serum tryptase levels. Four adults had expression only of wild-type KIT, while two others had expression of a truncated KIT lacking tyrosine kinase activity; yet these patients were clinically indistinguishable from those patients with activating c-KIT mutations. Activating c-KIT mutations exist in a significant portion of patients with mastocytosis, but not all patients showed expression of these mutations. Except for V560G, the presence or absence of activating c-KIT mutations did not predict the extent of disease. These observations suggest that although activating c-KIT mutations are associated with mast cell growth, other genes probably play a role in the cause of mastocytosis.

171 COMMD1 Restrains Tumor Promoting Activities of Myeloid Cells in Colon Cancer

G A A b st ra ct s primary resistance to imatinib. Amongst c-KIT/PDGFR wild type GIST, the most frequent driver mutation is BRAF-V600E, especially in small intestinal GIST. Thus, a mouse model of mutant Braf GIST might allow for development of more effective therapeutic strategies for these less common forms of GIST. Somatic activating mutations in c-Kit induce ICC hyperplasia and GIST in mouse models. However, whether GIST can arise from other cell types is unknown. In this study, we sought to generate a mouse model for Braf-mutant GIST from intestinal smooth muscle. Methods: Mice with a conditionally active (CA) mutant Braf allele, Braf-lox-stop-lox-V600E (Braf-CA), were crossed to mice carrying the tamoxifeninducible smooth muscle driver (Myh11-CreERT2). Adult mice received a single intraperitoneal injection of tamoxifen, ranging from 2.5-25mg/kg. The stomach, small intestine and colon were evaluated by H&E staining and immunohistochemistry. Results: Three weeks after tamoxifen injection, clusters of dysplastic cells with high nuclear-cytoplasmic ratio were observed in the intestinal muscular layer. The phenotype was most striking at the outer longitudinal layer of the greater curvature of stomach, but was also seen in the small intestinal and colonic muscle layer. The dysplastic cells were spindle-shaped and surrounded ganglion cells. CD34 immunoreactivity decorated, the dysplastic cells, a common finding in human GIST. Some of the clusters of dysplastic cells exhibited c-Kit-positivity and decreased a-SMA staining. c-Kit-positive cell clusters were also positive for anoctamin-1 (Ano1), an additional marker for human c-Kit-positive GIST. Conclusions: By conditionally activating mutant Braf in smooth muscle cells, we have developed a new mouse model for Braf-mutant GIST, as determined by characteristic histological features and markers. These results suggest that smooth muscle, rather than ICC, may be the cell-of-origin for Brafmutant GIST.

Molecular Mechanisms Underlying the Synergism of Erib and Hht in Treating t(8;21) AML with C-KIT mutations

AML1-ETO oncoprotein (AE) resulting from t(8;21)(q22;q22) translocation is known to play a pivotal role in leukemogenesis of t(8;21) AML, yet the activating mutations or over-expression of C-KIT occur in half of the AML patients with t(8;21). Animal models have proven that activating C-KIT mutations cooperate with AE in causing overt AML. The aberrant C-KIT expression was thus thought to be the major “second hit” in stepwise leukemogenesis of t(8;21) AML.Previous studies have demonstrated that Eriocalyxin B (EriB), a diterpenoid extracted from Isodon eriocalyx, induced apoptosis in t(8;21) leukemia cells through the NF-κB and MAPK signaling pathways and triggered degradation of AE dependent on caspase-3. By targeting AML1-ETO oncoprotein and activating apoptosis pathways simultaneously, EriB has potential to treat the t(8;21) leukemia.In this study, we observed the synergistic effect of EriB and the chemotherapy drug Homoharringtonine (HHT) on Kasumi-1, an AML cell line harboring t(8;21) and C-KIT mutations. Compared with using EriB or HHT alone, the combination of EriB and HHT (E+H) exhibited a higher efficacy to induce apoptosis and inhibit proliferation of Kasumi-1 cells. Data further showed that HHT enhanced the AE degradation caused by EriB in a dose/time-dependent manner. In addition, HHT significantly decreased the expression of C-KIT in Kasumi-1 cells, and the combination with EriB markedly enhanced this effect. Mechanism study revealed that E+H combination down-regulated the expression of transcriptional factor Sp1 through up-regulating miR-29b, separated Sp1 protein from Sp1/NF-κB complex, and released the Sp1/NF-κB complex from the C-KIT promoter. These data point to a mechanism involving Sp1/NF-κB/miR-29b regulatory network whereby E+H combination down-regulate C-KIT mRNA expression. In addition, HHT induced a proteasome-mediated degradation of C-KIT protein, which was further enhanced by EriB.From these observations, we conclude that the combined use of EriB and HHT might offer a potential therapeutic avenue for t(8;21) AML with C-KIT mutations. DisclosuresNo relevant conflicts of interest to declare.

Activating c-KIT mutations confer oncogenic cooperativity and rescue RUNX1/ETO-induced DNA damage and apoptosis in human primary CD34+ hematopoietic progenitors.

The RUNX1/ETO (RE) fusion protein, which originates from the t(8;21) chromosomal rearrangement, is one of the most frequent translocation products found in de novo acute myeloid leukemia (AML). In RE leukemias, activated forms of the c-KIT tyrosine kinase receptor are frequently found, thereby suggesting oncogenic cooperativity between these oncoproteins in the development and maintenance of t(8;21) malignancies. In this report, we show that activated c-KIT cooperates with a C-terminal truncated variant of RE, REtr, to expand human CD34+ hematopoietic progenitors ex vivo. CD34+ cells expressing both oncogenes resemble the AML-M2 myeloblastic cell phenotype, in contrast to REtr-expressing cells which largely undergo granulocytic differentiation. Oncogenic c-KIT amplifies REtr-depended clonogenic growth and protects cells from exhaustion. Activated c-KIT reverts REtr-induced DNA damage and apoptosis. In the presence of activated c-KIT, REtr-downregulated DNA-repair genes are re-expressed leading to an enhancement of DNA-repair efficiency via homologous recombination. Together, our results provide new mechanistic insight into REtr and c-KIT oncogenic cooperativity and suggest that augmented DNA repair accounts for the increased chemoresistance observed in t(8;21)-positive AML patients with activated c-KIT mutations. This cell-protective mechanism might represent a new therapeutic target, as REtr cells with activated c-KIT are highly sensitive to pharmacological inhibitors of DNA repair.

Gastrointestinal stromal tumor: analysis of outcome and correlation with c-kit status in Indian population.

The aim of the present study is to analyse the outcome and genotypic pattern of metastatic GIST patients which is largely unknown in India. The present study was a retrospective analysis of 24 patients of metastatic GIST. The case records were analysed for clinical profile, treatment response and prognostic factors. The archival samples were retrieved for c-kit mutation analysis in all but 5 patients for mutation analysis. The median age of the study population was 56 years. At a median follow up of 29 months, the PFS was 45% at 2 years. Activating c-kit mutations were detected in 10 cases (52.6%). 80% of the mutations were located in Exon 11. The outcome of metastatic GIST patients has definitely improved from a virtually incurable state to a disease where median OS has reached 60 months. The genotype of Indian patients with GIST may be different from the western population which needs to be confirmed in a larger study.

Secondary c‐Kit mutations confer acquired resistance to RTK inhibitors in c‐Kit mutant melanoma cells

Activation of the c-Kit receptor tyrosine kinase is rare in melanoma, but occurs in 20-40% of melanoma arising on mucosal membranes, acral skin and skin with chronic sun-induced damage. Many activating c-Kit mutations have been shown to be highly sensitive to imatinib mesylate, although the majority of patients with c-Kit mutant melanoma eventually progress on this inhibitor. We examined acquired resistance to imatinib and the newer generation inhibitor nilotinib in resistant c-kit mutant melanoma sublines. Four imatinib-resistant and six nilotinib-resistant sublines had acquired additional, secondary c-Kit mutations. The secondary A829P c-Kit mutation rendered cells resistant to imatinib, but did not suppress the activity of the tyrosine kinase inhibitors nilotinib and dasatinib. Sublines with an additional T670I c-Kit mutation showed resistance to imatinib, nilotinib and dasatinib, but responded to sunitinib. The concurrent inhibition of the MAPK and PI3K pathways was also effective at promoting apoptosis in the parent and derived resistant sublines. Our data provide a rationale for treating patients with melanoma progressing on imatinib or nilotinib with alternative RTK inhibitors or inhibitors targeting the MAPK and PI3K signalling cascades.

Core binding factor acute myeloid leukaemia and c-KIT mutations

Core binding factor (CBF) acute myeloid leukaemia (AML) represents 5-8% of all AMLs and has a relatively favourable prognosis. However, activating c-KIT mutations are reported to be associated with higher risk of relapse and shorter survival. To verify the incidence and prognostic value of c-KIT mutations in CBF AML, we retrospectively analysed bone marrow samples of 23 consecutive adult patients with de novo CBF AML [14 inv(16) and 9 t(8;21)] treated at a single institution from 2000 to 2011. All patients received standard induction chemotherapy with cytarabine, idarubicin and etoposide; 13 underwent allogeneic stem cell transplantation. c-KIT mutations in exons 8, 9, 10, 11, 13, 14 and 17 were assessed by PCR amplification in combination with direct sequencing. c-KIT mutations (3 in exon 10 and 4 in exon 17) were detected in 7/23 (30.4%) patients, 3 with t(8;21) and 4 with inv(16). No difference in c-KIT mutation status was observed between cases with inv(16) or t(8;21) alone and cases with additional cytogenetic abnormalities. No association between gender, age, white blood cell and platelet count, peripheral blood and bone marrow blast cells at diagnosis, achievement of complete remission, cytogenetic risk groups and Wilms tumour gene 1 (WT1) levels was found. On the contrary, lactate dehydrogenase (LDH) values were higher in mutated than in non-mutated patients (p=0.01). Overall survival (OS) rates were longer in CBF compared to the other types of AML and disease-free survival (DFS) was longer in inv(16) than in t(8;21) AML. OS and DFS were similar in mutated and non-mutated CBF AML patients. Our results confirm a better prognosis for CBF AML than all other AML categories, and for inv(16) than t(8;21) AML. However, no prognostic value for c-KIT mutational status was found in our series. The association between LDH levels and c-KIT mutation would indicate a more active proliferation for mutated CBF AML.

Evaluation of mesenchymal tumors of gastrointestinal tract with special reference to gastrointestinal stromal tumors – A tertiary care center experience

Background: Gastrointestinal tumors (GISTs) have attained significance in recent years because of their dramatic response to imatinib. Imatinib acts by inhibiting the proliferation and inducing apoptosis of GIST cells which express by activating c-kit mutation. GISTs with activating c-kit mutation show positivity for CD117 marker immunohistochemically. In this study, we analyzed the mesenchymal tumors of gastrointestinal tract morphologically and immunohistochemically. Materials And Methods: In this single-institutional retrospective study from January 1995 to April 2010, 52 mesenchymal tumors of gastrointestinal tract were included. Initially they were grouped under smooth muscle and stromal tumors based on their morphology and immunoreactivity for smooth muscle markers. Later on, they were subjected to additional immunostaining by CD117. Based on this, the mesenchymal tumors were reclassified. Results: In our study, the median age was 50 years (range 20-86 years). Stomach was the most common site ( n = 13). After CD117 immunostaining, 16 cases which were originally grouped under smooth muscle tumors were reclassified as GISTs. Conclusion: GISTs showing CD117 positivity are the largest group of mesenchymal tumors of gastrointestinal tract. Categorization of tumors as GIST helps to consider the patients for potential imatinib therapy.

Activating c-KIT mutations in a subset of thymic carcinoma and response to different c-KIT inhibitors

To analyze a multi-institutional series of type C thymic carcinomas (TCs) (including neuroendocrine tumors), focusing on the expression and mutations of c-KIT. Immunohistochemical expression of c-KIT/CD117, p63, CD5 and neuroendocrine markers, as well as mutational analysis of c-KIT exons 9, 11, 13, 14, 17 by direct sequencing of 48 cases of TCs. Immunohistochemical and molecular data were statistically crossed with clinicopathological features. Overall, 29 tumors (60%) expressed CD117, 69% were positive for CD5 and 85% (41 cases) for p63. Neuroendocrine markers stained all six atypical carcinoids and five poorly-differentiated thymic squamous cell carcinomas. Overall, six CD117-positive cases (12.5%) showed c-KIT mutation. No mutation was detected in CD117-negative tumors and carcinoids. All the mutations were found in poorly-differentiated thymic squamous cell carcinomas expressing CD117, CD5, p63 and lacking neuroendocrine markers (6 of 12 cases with these features). Mutations involved exon 11 (four cases: V559A, L576P, Y553N, W557R), exon 9 (E490K) and exon 17 (D820E). All TCs need an immunohistochemical screening with CD117, while c-KIT mutation analysis is mandatory only in CD117-positive cases, particularly when coexpressing CD5 and p63, lacking neuroendocrine differentiation. The finding of c-KIT mutation can predict efficacy with different c-KIT inhibitors.

Prevalence of c-KIT Mutations in Gonadoblastoma and Dysgerminomas of Patients with Disorders of Sex Development (DSD) and Ovarian Dysgerminomas

Activating c-KIT mutations (exons 11 and 17) are found in 10–40% of testicular seminomas, the majority being missense point mutations (codon 816). Malignant ovarian dysgerminomas represent ∼3% of all ovarian cancers in Western countries, resembling testicular seminomas, regarding chromosomal aberrations and c-KIT mutations. DSD patients with specific Y-sequences have an increased risk for Type II Germ Cell Tumor/Cancer, with gonadoblastoma as precursor progressing to dysgerminoma. Here we present analysis of c-KIT exon 8, 9, 11, 13 and 17, and PDGFRA exon 12, 14 and 18 by conventional sequencing together with mutational analysis of c-KIT codon 816 by a sensitive and specific LightCycler melting curve analysis, confirmed by sequencing. The results are combined with data on TSPY and OCT3/4 expression in a series of 16 DSD patients presenting with gonadoblastoma and dysgerminoma and 15 patients presenting pure ovarian dysgerminomas without DSD. c-KIT codon 816 mutations were detected in five out of the total of 31 cases (all found in pure ovarian dysgerminomas). A synonymous SNP (rs 5578615) was detected in two patients, one DSD patient (with bilateral disease) and one patient with dysgerminoma. Next to these, three codon N822K mutations were detected in the group of 15 pure ovarian dysgerminomas. In total activating c-KIT mutations were found in 53% of ovarian dysgerminomas without DSD. In the group of 16 DSD cases a N505I and D820E mutation was found in a single tumor of a patient with gonadoblastoma and dysgerminoma. No PDGFRA mutations were found. Positive OCT3/4 staining was present in all gonadoblastomas and dysgerminomas investigated, TSPY expression was only seen in the gonadoblastoma/dysgerminoma lesions of the 16 DSD patients. This data supports the existence of two distinct but parallel pathways in the development of dysgerminoma, in which mutational status of c-KIT might parallel the presence of TSPY.

Distinct classes of c-Kit–activating mutations differ in their ability to promote RUNX1-ETO–associated acute myeloid leukemia

AbstractThe t(8;21) RUNX1-ETO translocation is one of the most frequent cytogenetic abnormalities in acute myeloid leukemia (AML). In RUNX1-ETO+ patient samples, differing classes of activating c-KIT receptor tyrosine kinase mutations have been observed. The most common (12%-48%) involves mutations, such as D816V, which occur in the tyrosine kinase domain, whereas another involves mutations within exon 8 in a region mediating receptor dimerization (2%-13% of cases). To test whether distinct subtypes of activating c-KIT mutations differ in their leukemogenic potential in association with RUNX1-ETO, we used a retroviral transduction/transplantation model to coexpress RUNX1-ETO with either c-KitD814V or c-KitT417IΔ418-419 in murine hematopoietic stem/progenitor cells used to reconstitute lethally irradiated mice. Analysis of reconstituted animals showed that RUNX1-ETO;c-KitD814V coexpression resulted in 3 nonoverlapping phenotypes. In 45% of animals, a transplantable AML of relatively short latency and frequent granulocytic sarcoma was noted. Other mice exhibited a rapidly fatal myeloproliferative phenotype (35%) or a lethal, short-latency pre-B-cell leukemia (20%). In contrast, RUNX1-ETO;c-KitT417IΔ418-419 coexpression promoted exclusively AML in a fraction (51%) of reconstituted mice. These observations indicate that c-KitD814V promotes a more varied and aggressive leukemic phenotype than c-KitT417IΔ418-419, which may be the result of differing potencies of the activating c-Kit alleles.

C-kit gene mutation and CD117 expression in human anorectal melanomas

c-kit and BRAF mutations play an important role during the pathogenesis of melanoma. The subtypes of melanomas arising from different parts of the body have variable c-kit or BRAF mutation frequencies. Few studies in the literature have examined c-kit and BRAF mutation status in melanomas that occur in the anus and rectum. In this study, we analyzed 40 cases of anorectal melanoma for c-kit and BRAF mutations by DNA sequencing using paraffin-embedded tissues. c-kit Mutations were detected in exons 9, 11, 13, and 17. CD117 expression in tumor cells was analyzed by immunohistochemistry. Our study showed that a c-kit mutation was found in 7 of the 40 cases of anorectal melanoma. CD117 expression was detected in 16 of the 40 cases, and 3 of these 16 cases also had c-kit mutations. Mutations in BRAF were also identified in 2 patients. These results indicate that a subset of anorectal melanomas have activating c-kit mutations, which suggests that kinase inhibitors such as imatinib may be used to treat this subset of melanoma patients. In addition, our results show that c-kit mutations do not correlate with CD117 expression.

Update on Diagnosis and Treatment of Mastocytosis

Mastocytosis is a disorder characterized by increased numbers of mast cells in tissues. Recent clinical observations highlight the association of mastocytosis with an increased risk of anaphylaxis and underline the diversity of this disease. At the molecular level, recent studies have attempted to unravel specific gene expression profiles for activating c-kit mutations in the etiology of mastocytosis. The diagnosis may be facilitated by surrogate markers and detection of aberrant immunophenotypic surface markers. New therapeutic strategies are in development based on intracellular signal pathways, or on application of topical treatments, as are novel forms of cytoreductive therapy, including tyrosine kinase inhibitors.

KIT signaling regulates MITF expression through miRNAs in normal and malignant mast cell proliferation

AbstractActivating mutations in codon D816 of the tyrosine kinase receptor, KIT, are found in the majority of patients with systemic mastocytosis. We found that the transcription factor, microphthalmia-associated transcription factor (MITF), is highly expressed in bone marrow biopsies from 9 of 10 patients with systemic mastocytosis and activating c-KIT mutations. In primary and transformed mast cells, we show that KIT signaling markedly up-regulates MITF protein. We demonstrate that MITF is required for the proliferative phenotype by inhibiting colony-forming units with sh-RNA knockdown of MITF. Furthermore, constitutively active KIT does not restore growth of primary MITF-deficient mast cells. MITF mRNA levels do not change significantly with KIT signaling, suggesting posttranscriptional regulation. An array screen from mast cells identified candidate miRNAs regulated by KIT signaling. We found that miR-539 and miR-381 are down-regulated by KIT signaling and they repressed MITF expression through conserved miRNA binding sites in the MITF 3′-untranslated region. Forced expression of these miRNAs suppressed MITF protein and inhibited colony-forming capacity of mastocytosis cell lines. This work demonstrates a novel regulatory pathway between 2 critical mast cell factors, KIT and MITF, mediated by miRNAs; dysregulation of this pathway may contribute to abnormal mast cell proliferation and malignant mast cell diseases.

Pediatric Mastocytosis Is a Clonal Disease Associated with D816V and Other Activating c-KIT Mutations

Pediatric Mastocytosis Is a Clonal Disease Associated with D816V and Other Activating c-KIT Mutations