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Willmore-Payne C, Holden JA, Tripp S, Layfield LJHuman malignant melanoma: detection of BRAF and KIT activating mutations by high-resolution amplicon melting analysis. Hum Pathol 36:486-493
Abstract
Activating mutations in the BRAF kinase have been reported in a large number of cases of malignant melanoma. This suggests that therapy with specific RAF kinase inhibitors may find use in treating this disease. If the response to RAF kinase inhibition is dependent on the presence of an activated BRAF protein, it will be necessary to evaluate cases of malignant melanoma for the presence or absence of BRAF mutations. High-resolution amplicon melting analysis is able to detect single base-pair changes in DNA isolated from paraffin-embedded tissue sections and obviates the need for direct DNA sequencing. Results can be available within 48 hours. In this report, we used high-resolution amplicon melting analysis to evaluate 90 cases of malignant melanoma for BRAF mutations. Of these 90 cases, 74 were metastatic melanomas, 12 were primary cutaneous melanomas, and 4 were in situ melanomas. BRAF activation mutations were found in 43 cases (48%). Forty-one of these mutations were in exon 15. The mutations in exon 15 included V600E (34 cases), V600K (6 cases), and V600R (1 case). Two activating mutations were found in exon 11, G469V and G469R. The presence or absence of a BRAF mutation in the junctional component of an invasive melanoma was maintained in the invasive component. We also evaluated these 90 cases, as well as an additional 10 cases (total of 100) for the expression of c-kit. The majority of invasive and metastatic malignant melanomas did not express c-kit, although all in situ lesions and the junctional components of invasive lesions were strongly c-kit positive. Surprisingly, 2 cases of metastatic malignant melanoma (2%) showed strong and diffuse c-kit expression and contained a c-kit-activating mutation, L576P, as detected by high-resolution amplicon melting analysis and confirmed by direct DNA sequencing. These 2 c-kit mutation-positive cases did not contain BRAF mutations. The presence of a c-kit-activating mutation in metastatic malignant melanoma suggests that a small number of melanomas may progress by a somatic mutation of the c-kit gene. The presence of BRAF- and c-kit-activating mutations in malignant melanoma suggests new approaches to treating this disease involving specific tyrosine kinase inhibitors may prove worthwhile and that mutation analysis by high-resolution melting analysis might help guide therapy.
... KIT is pathologically activated in a variety of cancers. The majority of oncogenic KIT mutations are in exon 11, which encodes the regulatory juxtamembrane (JM) domain, or exon 17, which encodes the activation loop of the kinase domain (2)(3)(4)(5). Exon 11 mutations activate KIT by relieving the autoinhibition of the JM domain, while exon 17 mutations shift the conformational equilibrium of the kinase to the active state (6)(7)(8). ...
... Exon 11 mutations activate KIT by relieving the autoinhibition of the JM domain, while exon 17 mutations shift the conformational equilibrium of the kinase to the active state (6)(7)(8). For unclear reasons, exon 11 mutations predominate in gastrointestinal stromal tumor (GIST) and melanoma, whereas exon 17 mutations, exemplified by KIT D816V, predominate in systemic mastocytosis (SM), acute myeloid leukemia (AML), and germinomas (2)(3)(4)(5). ...
... Historically, exon 17-mutant KIT has been a challenging drug target, while exon 11-mutant KIT has been targetable with clinically available TKIs (1)(2)(3)(4)(5)(9)(10)(11). The first-generation of KIT inhibitors (imatinib, sunitinib, and regorafenib) transformed GIST driven by exon 11-mutant KIT from a lethal disease to a chronic condition (12). ...
KIT is a type-3 receptor tyrosine kinase that is frequently mutated at exon 11 or 17 in a variety of cancers. First-generation KIT tyrosine kinase inhibitors (TKI) are ineffective against KIT exon 17 mutations, which favor an active conformation that prevents these TKIs from binding. The ATP-competitive inhibitors, midostaurin and avapritinib, which target the active kinase conformation, were developed to inhibit exon 17–mutant KIT. Because secondary kinase domain mutations are a common mechanism of TKI resistance and guide ensuing TKI design, we sought to define problematic KIT kinase domain mutations for these emerging therapeutics. Midostaurin and avapritinib displayed different vulnerabilities to secondary kinase domain substitutions, with the T670I gatekeeper mutation being selectively problematic for avapritinib. Although gatekeeper mutations often directly disrupt inhibitor binding, we provide evidence that T670I confers avapritinib resistance indirectly by inducing distant conformational changes in the phosphate-binding loop. These findings suggest combining midostaurin and avapritinib may forestall acquired resistance mediated by secondary kinase domain mutations.
Significance
This study identifies potential problematic kinase domain mutations for next-generation KIT inhibitors midostaurin and avapritinib.
... Around 200 cancers were reported in different vital organs in human body [1]. Recent genetic screening showed that malignant tumors display a huge molecular events which can be used as a significant and potential therapeutic target for the treatment of cancer [5][6][7][8][9][10][11][12]. Mutation in BRAF have been reported as the major component in a number of human cancer types such as melanoma, thyroid and colon cancer [5,11,[13][14][15]. ...
... α-bromination of 3′-nitroacetophenone (5) have been accomplished using N-bromosuccinamide in DMF to give compound 6. 1 H NMR spectrum showed a singlet downfield signal at 5.06 ppm assigned for CH 2 Br. Condensation of compound 6 with 2-aminothiazole (7) in MeOH provided compound 8. 1 H NMR exhibited an additional signals at aromatic range at δ 8. 25,8.09 and 7.32 ppm attributed to the imidazothiazole protons. ...
BRAF mutation is commonly known in a number of human cancer types. It is counted as a potential component in treating cancer. In this study, based on structural optimization of previously reported inhibitors (3-fluro substituted derivatives of imidazo[2,1-b]thiazole-based scaffold), we designed and synthesized sixteen new imidazo[2,1-b]thiazole derivatives with m-nitrophenyl group at position 6. The electron withdrawing properties was reserved while the polarity was modified compared to previously synthesized compounds (-F). Furthermore, the new substituted group (–NO2) provided an additional H-bond acceptor(s) which may bind with the target enzyme through additional interaction(s). In vitro cytotoxicity evaluation was performed against human cancer cell line (A375). In addition, in vitro enzyme assay was performed against mutated B-Raf (B-Raf V600E). Compounds 13a, 13g and 13f showed highest activity on mutated B-Raf with IC50 0.021, 0.035 and 0.020 µM. All target compounds were tested for in vitro cytotoxicity against NCI 60 cell lines. Compounds 13a and 13g were selected for 5 doses test mode. Moreover, in silico molecular simulation was explored in order to explore the possible interactions between the designed compounds and the B-Raf V600E active site.
... KIT copy number was also most commonly detected in acral, with 27% and mucosal melanomas, with 26%. [14][15][16] There is also a strong correlation with KIT gene alterations are immunoreactivity with CD117, thus an emphasis should be put on the CD117 status. [13][14][15] Thus a routine application of CD117 immunohistochemistry in pathological diagnosis is indicative with a predictive purpose. ...
... [14][15][16] There is also a strong correlation with KIT gene alterations are immunoreactivity with CD117, thus an emphasis should be put on the CD117 status. [13][14][15] Thus a routine application of CD117 immunohistochemistry in pathological diagnosis is indicative with a predictive purpose. 7,13 BRAF V600E mutations are reportedly identified in 26.7% of conjunctival melanomas and 55.7% of cutaneous melanomas, but only in 1.1% of the mucosal melanomas. ...
Primary melanomas of the anus and rectum are rare neoplasms with aggressive
behavior, accounting for 0.1%-4.6% of anal canal tumors. Mucosal melanomas
account for approximately 1.2% of all melanomas, of which fewer than 25% are
anorectal. Histological evaluation with immunohistochemical stains like HMB-45,
S-100, vimentin and Melan A is required for definitive diagnosis. The 5-year survival
rate for anorectal melanomas (AM) was reported to be as low as < 20%, in contrast
to the value of approximately 80% for cutaneous melanomas. Furthermore,
up to 67% of patients are found to have distant metastases at the time of their
initial diagnosis with AM. Since the chemotherapy treatment possibilities are limited,
patients usually undergo mutation detection tests giving the opportunity of
targeted therapy. Herein we report a case of a patient with anorectal melanoma,
diagnosed in stage II and the pathomorphological and mutation status finding,
together with their correlation to tumor behavior and patient prognosis.
... La caracterización molecular de los melanomas vulvares puede ayudar a comprender mejor la carcinogénesis de esta patología (4). Las mutaciones del gen c-KIT son raras en los melanomas cutáneos, pero alrededor del 21,6 % de las mujeres con melanoma vulvar posee una mutación de este gen, lo cual lo hace una característica a tomar en cuenta (4,9,10). Se ha observado que c-KIT era el único marcador molecular de interés que variaba significativamente entre los melanomas vulvar y vaginal, con el 27 % en comparación con el 8 % de las muestras que albergaban la mutación, respectivamente (4,11). ...
os melanomas son neoplasias que surgen de la malignización de los melanocitos, generalmente relacionados con el daño solar acumulado por lo que la mayoría son cutáneos. Sin embargo, también puede aparecer en las superficies mucosas representando solo el 3%. Las mutaciones del gen c-KIT se encuentran en 21,6 % a diferencia de los melanomas cutáneos donde la mutación es rara. Por el contrario, las mutaciones NRAS y BRAF ocurren solo en el 10,2 % y 8,2 % de los melanomas vulvares, respectivamente, siendo más frecuente en los cutáneos. La mayoría de los melanomas vulvares se diagnostican en mujeres posmenopáusicas. Las manifestaciones clínicas incluyen: prurito, ulceración, sangrado o secreción vaginal, presentándose en etapas avanzadas.
... MITF expression is conserved in primary and metastatic malignant melanomas and is imperative in distinguishing cutaneous melanomas from nonmelanocytic lesions [56] [57]. Although raised from different cells, mastocytosis and melanoma share factors such as MITF, STAT3, and dependence on KIT signaling, suggesting some similarities in both pathologies [58,59]. Physiopathological implications connecting melanocytic tumors and abnormal proliferation of MCs have been reported [60], suggesting that patients with cutaneous mastocytosis undergo full-body screening for melanocytic lesions. ...
Activating mutations in KIT (CD117) have been associated with several diseases, including gastrointestinal stromal tumors and mastocytosis. Rapidly progressing pathologies or drug resistance highlight the need for alternative treatment strategies. Previously, we reported that the adaptor molecule SH3 binding protein 2 (SH3BP2 or 3BP2) regulates KIT expression at the transcriptional level and microphthalmia-associated transcription factor (MITF) expression at the post-transcriptional level in human mast cells and gastrointestinal stromal tumor (GIST) cell lines. Lately, we have found that the SH3BP2 pathway regulates MITF through miR-1246 and miR-5100 in GIST. In this study, miR-1246 and miR-5100 were validated by qPCR in the SH3BP2-silenced human mast cell leukemia cell line (HMC-1). MiRNA overexpression reduces MITF and MITF-dependent target expression in HMC-1. The same pattern was observed after MITF silencing. In addition, MITF inhibitor ML329 treatment reduces MITF expression and affects the viability and cell cycle progression in HMC-1. We also examine whether MITF downregulation affected IgE-dependent mast cell degranulation. MiRNA overexpression, MITF silencing, and ML329 treatment reduced IgE-dependent degranulation in LAD2- and CD34+-derived mast cells. These findings suggest MITF may be a potential therapeutic target for allergic reactions and deregulated KIT mast-cell-mediated disorders.
... KIT encodes a receptor tyrosine kinase (RTK) for the stem cell factor (SCF) ligand and functions as an upstream activator of the MAP kinase signaling pathway (Figure 1). The KIT rearranging in melanomas with enlargement of 4q12 showed a significant mutations [37,41], but advanced tumour sequencing has proven that they may contribute to only 2 to 5% of cases [42,43]. The KIT mutations are most predominant in CSD (8-20%), mucosal (16-25%), and acral (12-23%) melanomas [44,45]. ...
Melanoma is a severe skin cancer affecting thousands of people and a growing public
health concern worldwide. The potential hallmarks of melanoma are genetic instability and mutation
(GIAM), which are driving mechanisms for phenotypic variation and adaptation in melanoma. In
metastatic melanoma, DNA repair-associated genes are frequently expressed at higher levels than in
primary cancers, suggesting melanoma cells rely on genetic stability to spread distantly. The tumour
microenvironment is affected by genomic instability and melanoma mutation (GIMM), which plays
significant roles in developing GIMM and their contributions to the overall disease burden. The
GIAM is the crucial vulnerability of cancer cells, determining their sensitivity to harmful treatments,
including radiation and many chemotherapeutics. The high incidence of melanoma is typically
associated with genetic modifications, and several clinical and genetic interventions have been critical
in easing the burden.
... The genetic profiles of cutaneous melanoma and vulvar and vaginal melanoma are different in terms of mutations. The KIT gene mutation, which rarely appears in cutaneous melanoma patients [35,36], appears to be commonly occurring in vulvar melanoma (from 22 up to 31% of cases). It is more common in vulvar melanoma (27%) and less common in vaginal melanoma (8%) [37,38]. ...
Cutaneous melanoma is a relatively common neoplasm, with fairly well understood pathogenesis, risk factors, prognosis and therapeutic protocols. The incidence of this disease is increasing every year. The situation is different for rare malignancies such as vulvar melanomas and for the even rarer vaginal melanomas. The risk factors for vulvovaginal tumors are not fully understood. The basis of treatment in both cases is surgical resection; however, other types of treatments such as immunotherapy are available. This paper focuses on comparing the pathogenesis and risk factors associated with these neoplasms as well as the efficacy of two groups of drugs—anti-PD-L1 and anti-CTLA4 inhibitors—against both cutaneous melanoma and melanoma of the lower genital tract (vulva and vagina). In the case of cutaneous melanoma, the situation looks more optimistic than for vulvovaginal melanoma, which has a much worse prognosis and, as it turns out, shows a poorer response to immune therapy.
... In a clinical mTOR inhibitor trials, namely Temsirolimus or Everolimus are used in conjunction with a BRAF inhibitor (NCT01596140), also in another trial ASN003, an inhibitor of BRAF with enhanced selectivity against mTOR and PI3K kinases (NCT02961283). Combining low-dose inhibitors of mTOR with immunotherapy needs clinical validation because inhibition of mTOR can lead to immune activation or immune suppression depending on the mode of administration, timing, and dose [203] . ...
Melanoma is a relentless type of skin cancer which involves myriad signaling pathways which regulate many cellular processes. This makes melanoma difficult to treat, especially when identified late. At present, therapeutics include chemotherapy, surgical resection, biochemotherapy, immunotherapy, photodynamic and targeted approaches. These interventions are usually administered as either a single-drug or in combination, based on tumor location, stage, and patients' overall health condition. However, treatment efficacy generally decreases as patients develop treatment resistance. Genetic profiling of melanocytes and the discovery of novel molecular factors involved in the pathogenesis of melanoma have helped to identify new therapeutic targets. In this literature review, we examine several newly approved therapies, and briefly describe several therapies being assessed for melanoma. The goal is to provide a comprehensive overview of recent developments and to consider future directions in the field of melanoma.
... These included two melanomas, of which one had a transition mutation of leucine to proline in codon 576 (L576P) of exon 11. It was later found that L576P is the most common mutation in c-KIT in melanomas and that most mutations occur in exons 11 and 13 [47][48][49]. In addition, mutations of c-KIT seem to be more common than copy amplifications in melanomas and most of them occur in the kinase domain or in the juxta-membrane domain. ...
Melanomas exhibit the highest rate of somatic mutations among all different types of cancers (with the exception of BCC and SCC). The accumulation of a multimode of mutations in the driver oncogenes are responsible for the proliferative, invasive, and aggressive nature of melanomas. High-resolution and high-throughput technology has led to the identification of distinct mutational signatures and their downstream alterations in several key pathways that contribute to melanomagenesis. This has enabled the development of individualized treatments by targeting specific molecular alterations that are vital for cancer cell survival, which has resulted in improved outcomes in several cancers, including melanomas. To date, BRAF and MEK inhibitors remain the only approved targeted therapy with a high level of evidence in BRAFV600E/K mutant melanomas. The lack of approved precision drugs in melanomas, relative to other cancers, despite harboring one of the highest rates of somatic mutations, advocates for further research to unveil effective therapeutics. In this review, we will discuss potential druggable mutations and the ongoing research of novel individualized treatment approaches targeting non-BRAF mutations in melanomas.
... The c-KIT gene encodes KIT (CD117), which is a class III transmembrane receptor tyrosine kinase and is expressed in a variety of cells [19,20]. While c-KIT mutations are rare in cutaneous melanomas [21,22], 21.6% of women with VMs harbor a c-KIT mutation. The high rate of c-KIT mutations appears to be characteristic for VMs, [24]. ...
Ten percent of all women have pigmented vulvar lesions. Fortunately, most of these are benign but 1% of all melanomas in women affect the vulva. While the mortality rate of cutaneous melanoma has dropped by 7% annually during the last 5 years, the prognosis of vulvar melanoma remains dismal: the 5-year overall survival rate is 47% compared with 92% for cutaneous melanoma. The current evidence suggests that this likely results from a combination of delayed diagnosis and different tumor biology, treatment strategies, and treatment response. Although many landmark trials on checkpoint inhibitors included mucosal and vulvar melanomas, the results were often not reported separately. Post-hoc analyses indicate overall response rates between 19 and 37% for checkpoint inhibitors. A recently published retrospective study on vulvar melanomas suggests an objective response in 33.3% with a similar safety profile to cutaneous melanoma. Tyrosine kinase inhibitors may be considered in recurrent disease if a c-KIT mutation is present.
... KIT is a receptor tyrosine kinase that plays an important role in hematopoiesis, gametogenesis and melanogensis, and oncogenic KIT mutations have been identified in GISTs, mastocytosis, acute myeloid leukemia, melanoma and others [1][2][3]7,23,24]. Signaling studies of wild-type KIT and KIT mutants have showed that KIT mutants have different signaling properties in addition to the ligand independent activation which was considered as the main cause of cell transformation. ...
Activation of receptor tyrosine kinases needs tight control by tyrosine phosphatases to keep their normal function. In this study, we investigated the regulation of activation of the type III receptor tyrosine kinase KIT by protein tyrosine phosphatase receptor type E (PTPRE). We found that PTPRE can associate with wild-type KIT and inhibit KIT activation in a dose-dependent manner, although the activation of wild-type KIT is dramatically inhibited even when PTPRE is expressed at low level. The D816V mutation of KIT is the most frequently found oncogenic mutation in mastocytosis, and we found that PTPRE can associate and inhibit the activation of KIT/D816V in a dose dependent manner, but the inhibition is much weaker compared with wild-type KIT. Similar to mastocytosis, KIT mutations are the main oncogenic mutations in gastrointestinal stromal tumors (GISTs) although GISTs carry different types of KIT mutations. We further studied the regulation of the activation of GISTs-type KIT mutants and other mastocytosis-type KIT mutants by PTPRE. Indeed, PTPRE can almost block the activation of GISTs-type KIT mutants, while the activation of mastocytosis-type KIT mutants is more resistant to the inhibition of PTPRE. Taken together, our results suggest that PTPRE can associate with KIT, and inhibit the activation of both wild-type KIT and GISTs-type KIT mutants, while the activation of mastocytosis-type KIT mutants is more resistant to PTPRE.
... MM with oncogenic mutations in KIT have been reported in several studies that focused on the role of KIT in melanocyte transformation (19,34,41). Some cases of rare metastatic MM having KIT L576P activating mutation and strong and diffuse KIT expression (42) suggest that MM progression involves KIT activation and not loss of activity. It has been taken into consideration the use of tyrosine kinase inhibitors in these cases, which showed significant results in some studies (43,44). ...
The aim of the present study was to carried out a comparative immunohistochemical evaluation of CD117 (c-Kit), a biomarker that evaluates both tumor progression and prognosis, in different melanocytic lesions, to emphasize the significance of this biomarker in malignant melanoma (MM). The study was performed on 55 cases, represented by a control group, which included 5 cases of simple nevi and 5 cases of dysplastic nevi, as well as a study group consisting of 35 cases of primary MM and 10 metastases (one intestinal, 3 cutaneous - one satellite and two distant as well as 6 in the lymph nodes). The study group included 15 cases of superficial spreading melanoma (SSM), 10 cases of nodular melanoma (NM), 3 lentigo maligna melanoma (LMM), 3 cases of acral lentiginous melanoma (ALM) and 4 cases of amelanotic MM. CD117 was found to be massively involved in the process of tumorigenesis of cutaneous malignancies, being immunohistochemically undetectable in benign neural lesions, but densely expressed in dysplastic lesions and in situ melanoma areas. In invasive cutaneous MMs, CD117 expression tended to decrease with neoplasia progression proceding into the tumorigenic, vertical growth phase, being lower in the profound dermal component of tumors and in nodular MMs. To eliminate the epidermal barriers and gain a proliferative advantage to allow the transition to the vertical growth phase, it seems that MM should lose expression of c-Kit. Cutaneous metastases were found to express CD117 at a level comparable to their primary tumors, suggesting that other mechanisms interfere directly with the metastatic process and not loss of c-Kit expression by itself. CD117 overexpression in cutaneous melanocytic lesions correlates significantly with increased immunostaining intensity, suggesting that the immunohistochemical evaluation of CD117 may be a good method for screening patients, who could benefit from personalized therapy with tyrosine kinase inhibitors.
... CKIT mutation is the cause of 28% of cutaneous melanoma, leading to upregulation of MAPK and PI3K/AKT pathways, and is associated with induction of drug resistance. Imatinib, a CKIT inhibitor, led to 30% survival rate in melanoma patients with median survival of 3-4 months [28]. The high level of VEGF receptors in melanoma tumors has mediated neovasculature in tumor, immune suppression and poor progression, thereby blockade of VEGF has been considered as a promising approach [29]. ...
Melanoma is a poor immunogenic cancer and many treatment strategies have been used to enhance specific or nonspecific immunity against it. Dendritic cell (DC)-based cancer vaccine is the most effective therapies that have been used so far. Meanwhile, the efficacy of DC-based immunotherapy relies on critical factors relating to DCs such as the state of maturation and proper delivery of antigens. In this regard, the use of nanoparticulate delivery systems for effective delivery of antigen to ex vivo-generated DC-based vaccines that also poses adjuvanticity would be an ideal approach. In this review article, we attempt to summarize the role of different types of nanoparticulate antigen delivery systems used in the development of ex vivo-generated DC-based vaccines against melanoma and describe their adjuvanticity in mediation of DC maturation, cytoplasmic presentation of antigens to MHC class I molecules, which led to potent antigen-specific immune responses. As were represented, cationic liposomes were the most used approach, which suggest its potential applicability as delivery systems for further experiments in combination with either adjuvants or monoclonal antibodies.
... BRaf is constitutively activated in melanomas not only by mutation but also by upstream oncogenic molecular alterations of BRaf that can induce upregulation of BRaf and consequently of CCND1/cyclin D1. They include the following: autocrine production of growth factors (Satyamoorthy et al., 2003); mutational activation (although rare) of growth factor receptors, as reported for c-kit (Willmore-Payne, Holden, Tripp, & Layfield, 2005); and the presence of NRas mutations, one of the three isoforms of the Ras protein family (the other two are HRas and KRas), in which leucine in position 61 is substituted by glutamine (Q61L; Davies et al., 2002). NRas mutations appear in different papers between 15% and 20% (Muñoz-Couselo, Zamora Adelantado, Ortiz Vélez, Soberino-García, & Perez- Garcia, 2017) and 15% and 50% of melanomas (Ackermann et al., 2005;Ball et al., 1994;Davies et al., 2002;Gray-Schopfer et al., 2007) and are especially important because they constitutively activate both the MAPK pathway and the PI3K/Akt pathway, one of whose endpoints is an increase in CCND1/cyclin D1 transcription (Sekulic et al., 2009). ...
Cyclin D1 is a protein encoded by the CCND1 gene, located on 11q13 chromosome, which is a key component of the physiological regulation of the cell cycle. CCND1/cyclin D1 is upregulated in several types of human tumors including melanoma and is currently classified as an oncogene that promotes uncontrolled cell proliferation. Despite the demonstrated importance of CCND1/cyclin D1 as a central oncogene in several types of human tumors, its knowledge in melanoma is still limited. This review examines data published on upregulation of the CCND1 gene and cyclin D1 protein in the melanoma setting, focusing on the pathways and molecular mechanisms involved in the activation of the gene and on the clinical and therapeutic implications.
... Transforming mutations of KIT in AML are detected in about 5% of patients, and are typically located in exon 17, encoding the kinase domain activation loop, with D816V being the primary mutation. Interestingly, this same mutation is typical of systemic mastocytosis (SM), a rare disorder characterized by overproduction of mast cells that accumulate in the skin and organs (Nagata et al., 1995;Hirota et al., 1998;Kemmer et al., 2004;Willmore-Payne et al., 2005). Prognosis is generally poor for advanced SM patients, such as SM with an associated haematological neoplasm (SM-AHN), mast cell leukaemia (MCL) and aggressive SM (ASM), and treatment options are limited. ...
Mutations in two type‐3 receptor tyrosine kinases (RTKs), KIT and FLT3, are common in both acute myeloid leukaemia (AML) and systemic mastocytosis (SM) and lead to hyperactivation of key signalling pathways. A large number of tyrosine kinase inhibitors (TKIs) have been developed that target either FLT3 or KIT and significant clinical benefit has been demonstrated in multiple clinical trials. Given the structural similarity of FLT3 and KIT, it is not surprising that some of these TKIs inhibit both of these receptors. This is typified by midostaurin, which has been approved by the US Food and Drug Administration for mutant FLT3‐positive AML and for KIT D816V‐positive SM. Here, we compare the in vitro activities of the clinically available FLT3 and KIT inhibitors with those of midostaurin against a panel of cells expressing a variety of oncogenic FLT3 or KIT receptors, including wild‐type (wt) FLT3, FLT3‐internal tandem duplication (ITD), FLT3 D835Y, the resistance mutant FLT3‐ITD+ F691L, KIT D816V, and KIT N822K. We also examined the effects of these inhibitors in vitro and in vivo on cells expressing mutations in c‐CBL found in AML that result in hypersensitization of RTKs, such as FLT3 and KIT. The results show a wide spectrum of activity of these various mutations to these clinically available TKIs.
... The development of molecularly targeted therapies for melanoma was based on the knowledge that mutations in the BRAF gene are commonly seen in this disease. [7][8][9][10] This activating BRAF V600 mutation leads to aberrant activation of the mitogen-activated protein kinase (MAPK) pathway, a welldocumented cancer cell growth pathway. Multiple drugs targeting BRAF V600 mutations have been approved by the US Food and Drug Administration (FDA), including vemurafenib, dabrafenib, and encorafenib, based on their ability to improve survival outcomes for patients with BRAF-mutant melanoma. ...
Recently, the effectiveness of novel immune checkpoint inhibitors and BRAF-directed therapies has been demonstrated in advanced melanoma trial populations. Limited research, however, has evaluated the impact of these therapies in a real-world setting. The aim of this study was to evaluate treatment patterns and clinical outcomes among advanced melanoma patients treated with modern therapies within community oncology clinics. Adult patients with advanced melanoma who initiated treatment within the US Oncology Network between 1/1/14 and 12/31/16 were included. Data were sourced from electronic healthcare records. Patients were followed through 12/31/17. Descriptive analyses were performed to assess patient and treatment characteristics and Kaplan-Meier methods were used for time-to-event outcomes. In total, 484 patients met eligibility criteria (32.0% with brain metastasis, 12.6% with Eastern Cooperative Oncology Group performance status ≥2). In the first-line (1L) setting during the study period, 37.0% received anti-PD1 monotherapies, 26.4% ipilimumab monotherapy, 19.8% BRAF/MEK combination therapy, 6.4% BRAF or MEK monotherapy, 4.1% ipilimumab/nivolumab combination therapy and 6.2% other regimens. Differences in baseline demographic and clinical characteristics were observed across treatment groups. For the overall study population, the median (95% confidence interval) estimates for overall survival, time to next treatment and progression-free survival were 20.7 (16.0, 26.8), 5.8 (5.3, 6.5), and 4.9 (4.2, 5.7) months, respectively. The results of this study provide real-world insight into advanced melanoma treatment trends and clinical outcomes, including high utilization of immunotherapies and BRAF/MEK combination therapy. Future research can explore underlying differences in patient subpopulations and the sequence of therapies across lines of therapy.
... A number of somatic mutations, including NRAS, BRAF, KIT and PTEN, have been identified in melanoma pathogenesis, which has prompted the development of targeted therapies [11][12][13][14]. It was found that oncogenic mutations in the BRAF serine/threonine kinase gene of the mitogen-activated protein kinase (MAPK) pathway occurs in 41-55% of metastatic melanomas [15][16][17], and most commonly results in substitution of valine for glutamic acid at codon 600 (V600E, 74-95%), followed by valine for lysine (V600K, 5-20%) [17][18][19][20]. Inhibitors of BRAF V600E/K, such as vemurafenib, dabrafenib and encorafenib, are currently indicated in combination with inhibitors of the downstream MEK1/2 kinase, such as cobimetinib, trametinib and binimetinib for advanced melanoma. ...
Targeted therapies, based on identification of common oncogenic mutations such as BRAF V600E/K and monoclonal antibody immunotherapies, have transformed the treatment of melanoma. Dual mitogen-activated protein kinase (MAPK) pathway inhibition of BRAF V600E/K and MEK 1/2 kinases with BRAF–MEK inhibitors using dabrafenib–trametinib, vemurafenib–cobimetinib and encorafenib–binimetinib is now the standard of care for BRAF V600E/K tumours. Monoclonal antibodies, such as pembrolizumab and nivolumab, against programmed cell death protein (PD-1) on T cells, as well as ipilimumab against cytotoxic T lymphocyte antigen-4 (CTLA-4), enable restoration of suppressed T-cell antitumour response, and have also shown improved clinical benefit compared with traditional chemotherapy. Exploration of different combination therapies, sequence of treatment, and dosing strategies is ongoing, and the understanding of the pharmacokinetics (PK) and pharmacodynamics (PD) of these new agents is fundamental in devising the optimal regimen. Preclinical and clinical studies, as well as population PK modelling, provide essential data in terms of PK parameters, metabolism, interpatient variability, drug interactions and PD effects at the target. This review gathers the current evidence and understanding of the clinical PK and PD of drugs used in the modern treatment of melanoma, and the factors determining drug disposition, exposure and clinical response, and also highlighting areas of further research.
... Constitutively activating mutations in the BRAF serine-threonine kinase gene have been reported in 33-47% of primary melanomas [22][23][24] and 41-55% of metastatic melanomas [22,25,26], resulting in cancer cell growth and proliferation. The most common BRAF mutation is V600E (valine to glutamic acid at position 600) in 74-95% of patients, followed by V600K (valine to lysine) in 5-20% of patients [26][27][28][29]. ...
Purpose
The combination of a BRAF inhibitor dabrafenib and a MEK inhibitor trametinib (CombiDT) has improved outcomes compared with chemotherapy or BRAF inhibitor monotherapy in advanced BRAF V600E/K melanoma. However, CombiDT causes a high incidence of pyrexia and treatment interruptions. Pharmacokinetic analysis may provide an explanation for the pyrexia.
Methods
34 patients with Stage 3 BRAF V600 melanoma were treated with CombiDT on a clinical trial between August 2014 and June 2017. Plasma concentrations of drugs and metabolites were determined using validated LC–MS assays, in addition to analysis of a panel of cytokines.
Results
Pyrexia was experienced by 71% of the patients, with an additional 17% requiring dose interruption related to a pyrexia-like prodrome. Dabrafenib concentrations ranged from 4.0 to 4628 ng/ml and trametinib from 1.0 to 45 ng/ml in 34 patients. N-desmethyl-dabrafenib was the most prevalent metabolite, followed by carboxy- and hydroxy-dabrafenib. No definitive association between pyrexia and AUC or Cmin of the drugs, or metabolites could be observed. The level of IL-1B at the early during treatment (EDT) (as a % of pre-treatment) was higher in the pyrexia group (median 109% (range 32–681%) than in the no-incidence group [56% (26–79%)] (p = 0.029). Similarly, the level of IL-6 at EDT was higher in the pyrexia group [181% (34-3156%) vs 73% (57–101%)] (p = 0.028).
Conclusions
No apparent associations between pyrexia and exposure to the drugs or metabolites could be observed. Greater elevations in IL-1B and IL-6 were observed in patients with pyrexia during the first week of treatment compared to those without pyrexia.
... These mutations are associated with diseases characterized by neoplastic cell growth in cell lines expressing KIT, which include MCs, stromal cells, germline cells, melanocytes, hematopoietic progenitors in bone marrow, and a wide variety of neoplastic cells from various tumors. Thus, although KIT-driven disorders mostly include mastocytosis and gastrointestinal stromal tumors, where KIT mutations are detected in ~95% and ~75% of cases, respectively [101][102][103], KIT mutations have also been described in a subset of patients with seminoma [104], germinoma [65], melanoma [105], small cell lung cancer [106], colon cancer [107], neuroblastoma [108], and breast cancer [109], as well as in hematologic malignancies such as acute leukemia [110], myelodysplastic syndrome [111], and myeloproliferative neoplasm [112]. Most KIT mutations tend to cluster in small regions of the protein, especially at exons 11 and 17, although mutations involving other exons have been also reported. ...
Mast cells (MCs) are a key structural and functional component of both the innate and the adaptive immune systems. They are involved
in many different processes, but play a major role in the response to infections and in inflammatory reactions. In addition, MCs are the
main effector cells in allergy.
MC biology is far more complex than initially believed. Thus, MCs may act directly or indirectly against pathogens and show a wide variety
of membrane receptors with the ability to activate cells in response to various stimuli. Depending on where MCs complete the final stages
of maturation, the composition of their cytoplasmic granules may vary considerably, and the clinical symptoms associated with tissue MC
activation and degranulation may be also different. MCs are activated by complex signalling pathways characterized by multimolecular
activating and inhibitory interactions.
This article provides a comprehensive overview of MC biology, focusing predominantly on mechanisms of MC activation and the role of
MCs in the pathogenesis of allergic diseases.
... 4 In addition, mutations in c-KIT, which codes for CD117, a protein that binds to mast cell growth factor, stimulate the proliferation of mast cells and melanocytes. 2 Two case reports of association between MM and cutaneous mastocytosis occurred in adult females have been described in the literature, 3,5 both suggesting oestrogens as a possible pathogenic link. This is the third such case, and is the first in a male patient. ...
... Interestingly, mutations in the same genes can cause distinct malformations or cancers in NC-derived tissues. Loss-of-function RET mutations can cause HSCR but gain-of-function mutations can cause MEN2 (Takahashi et al., 1999); loss-offunction KIT mutations in melanocytes can cause piebaldism but gain-of-function mutations can cause melanoma (Willmore-Payne et al., 2005;Curtin et al., 2006). Gene mutations in the PHOX2B transcription factor cause life-threatening hindbrain malformations; as they can also affect vagal or sympatho-adrenal NC derivatives, these can be associated with either HSCR or neuroblastoma (Amiel et al., 2003). ...
This is a repeat preprint of a more recent upload - I've corrected the title and will soon remove the duplicate record.
... Interestingly, mutations in the same genes can cause distinct malformations or cancers in NC-derived tissues. Loss-of-function RET mutations can cause HSCR but gain-of-function mutations can cause MEN2 (Takahashi et al., 1999); loss-offunction KIT mutations in melanocytes can cause piebaldism but gain-of-function mutations can cause melanoma (Willmore-Payne et al., 2005;Curtin et al., 2006). Gene mutations in the PHOX2B transcription factor cause life-threatening hindbrain malformations; as they can also affect vagal or sympatho-adrenal NC derivatives, these can be associated with either HSCR or neuroblastoma (Amiel et al., 2003). ...
We review here some of the historical highlights in exploratory studies of the vertebrate embryonic structure known as the neural crest. The study of the molecular properties of the cells that it produces, their migratory capacities and plasticity, and the still-growing list of tissues that depend on their presence for form and function, continue to enrich our understanding of congenital malformations, pediatric cancers and evolutionary biology. Developmental biology has been key to our understanding of the neural crest, starting with the early days of experimental embryology and through to today, where increasingly powerful technologies contribute to further insight into this fascinating vertebrate cell population.
... In melanoma, CKIT mutations have been described in 39% of mucosal melanoma, 36% of acral lentiginous melanoma, 28% of cutaneous melanomas arising in areas of chronic sun-damaged skin, and none in melanomas of skin without chronic sun damage. 136,137 CKIT mutations or gene amplifications can lead to the constitutive ligand-independent activation of this receptor and upregulation of the MAPK and PI3K/AKT pathway. 122,138 CKIT mutations have been reported across several exons and were associated with the development of drug resistance. ...
Melanoma represents the most aggressive and the deadliest form of skin cancer. Current therapeutic approaches include surgical resection, chemotherapy, photodynamic therapy, immunotherapy, biochemotherapy, and targeted therapy. The therapeutic strategy can include single agents or combined therapies, depending on the patient's health, stage, and location of the tumor. The efficiency of these treatments can be decreased due to the development of diverse resistance mechanisms. New therapeutic targets have emerged from studies of the genetic profile of melanocytes and from the identification of molecular factors involved in the pathogenesis of the malignant transformation. In this review, we aim to survey therapies approved and under evaluation for melanoma treatment and relevant research on the molecular mechanisms underlying melanomagenesis.
... This suggests that KIT mutations have a role in the physiopathogeny and, therefore, are a potential therapeutic target in these subtypes of melanoma. In contrast, KIT mutations are rarely found in the largest subgroup of malignant melanomas, SM not associated with chronic exposure to sunlight: 1 in 100 cases studied by Willmore-Payne et al. (2005) [18]. Furthermore, phase II studies with imatinib in patients with SM, without KIT mutation analysis, were disappointing [19][20][21]. ...
Unlike their cutaneous counterparts, head and neck mucosal malignant melanomas (HNMM) are more aggressive, and their prognostic markers have not been fully elucidated. This study, comprising 28 patients with HNMM, aimed to establish the relationship between different mutations and outcome, define the incidence of KIT mutations in HNMM, and identify the correlation among therapeutic options, histopathological findings, demographic data, and clinical response. Clinical analysis included patient characteristics, staging, primary and palliative treatments, and disease-free survival and overall survival (OS). Progression-free survival and OS were analyzed. Paraffin blocks were selected following histologic analyses, enabling DNA extraction. PCR amplification of exons 9, 11, 13, and 17, with different DNA concentrations, was performed. Patients were predominantly females (57%) and aged 27-85 years. All patients underwent surgery; 17 received adjuvant radiotherapy, and recurrences occurred in 82% patients. Oncologic mutations in KIT were found in 7 of 7 tumors, 3 in exon 9, 3 in exon 11, and 1 in exon 13. Predictive factors for recurrence were mitotic rate, vascular invasion, and perineural spread. There were no significant differences in DFS and OS according to KIT mutation. Our study results suggest that some patients might benefit from appropriate targeted therapy with kinase inhibitors.
... Some mutations of the c-kit gene result in loss-of-function of KIT tyrosine kinase activity, while other mutations of the c-kit gene cause constitutive activation of KIT tyrosine kinase. These gain-of-function mutations of the c-kit gene are observed in GISTs (1), mastocytomas (3), seminomas (4) and melanomas (5). ...
Gastrointestinal stromal tumors (GISTs) are the most common mesenchymal tumors in the gastrointestinal (GI) tract. GISTs account for approximately 80% of the clinically relevant GI mesenchymal tumors. Although most GISTs show spindle cell morphology, 10-15% of GISTs show pure epithelioid configuration. Therefore, not only spindle cell tumors but also epithelioid cell ones developing in the GI tract are subject to the differential diagnoses of GISTs. GISTs are basically positive for KIT, a receptor tyrosine kinase (RTK) encoded by protooncogene c-kit, by immunohistochemistry, but approximately 5% of GISTs are only weakly or barely positive for KIT. Since almost all spindle cell type GISTs are strongly and diffusely positive for KIT regardless of different genetic subtypes, diagnosis of the spindle cell type GISTs is not difficult. On the other hand, epithelioid cell type GISTs show different staining patterns of KIT in different genetic backgrounds. Approximately half of the epithelioid cell type GISTs with platelet-derived growth factor receptor alpha (PDGFRA) gene mutation might show weak or undetectable staining of KIT. On the other hand, almost all GISTs are negative for desmin, which is a positive marker for mature smooth muscle cells, and S100 protein, which is a Schwann cell marker. Smooth muscle tumors such as leiomyomas and leiomyosarcomas, which usually show the spindle cell morphology, consist of approximately 10% of the clinically relevant GI mesenchymal tumors and are almost positive for desmin and negative for KIT and S100 protein. Schwannomas which nearly always show the spindle cell pattern, comprise up to 5% of the GI mesenchymal tumors, and almost all of them are positive for S100 protein and negative for KIT and desmin. Thus, most GI mesenchymal tumors are differentially diagnosed by immunohistochemistry (IHC) of KIT, desmin and S100 protein. However, mesenchymal tumors such as desmoids, solitary fibrous tumors (SFTs), inflammatory myofibroblastic tumors (IMTs), perivascular epithelioid cell tumors (PEComas), inflammatory fibroid polyps (IFPs), rarely develop in the GI tract, and have to be correctly diagnosed through detection of specific immunohistochemical markers and/or unique genetic aberrations.
... BRAF mutations and overexpression occur in about 40 to 70% of malignant melanoma. [4][5][6] Inhibiting the kinase BRAF results in rapid tumor regression and prolonged survival in patients. [10][11][12][13] Our previous studies have shown that silencing the BRAF gene can significantly inhibit the growth of melanoma. ...
... This genetic abnormality may be less likely in SNMM owing to the anatomic location of the disease [15]. In contrast, somatic mutations and/or increased copy numbers in the KIT gene, which have been identified in a small number of cutaneous melanomas [22], may be more frequent in mucosal melanomas [11]. Given the likely pathogenetic role the KIT gene has in mucosal melanomas, including those of the head and neck [23,24], screening for KIT aberrations may have potential diagnostic value, and the gene may present a therapeutic target. ...
Sinonasal mucosal melanoma (SNMM) is a rare oncological entity that comprises most head and neck mucosal melanomas. SNMM has distinctive genetic background, different from cutaneous melanoma. Survival outcomes among SNMM patients are poor; while there is no clear consensus on the optimal management of SNMM, the primary treatment modality is generally considered to be wide surgical excision, and radiation therapy (RT) is often used in the postoperative adjuvant setting to improve locoregional control. Systemic therapies have demonstrated little or no survival benefit, and most SNMM patients die of distant metastatic disease. Owing to the rarity of the disease, the literature describing treatment approaches for SNMM is lacking and largely limited to isolated case reports and retrospective series. Here, we describe contemporary diagnostic and therapeutic approaches to SNMM based on the most recent molecular and outcome data.
... The technique has already been employed to scan for somatic mutations in the KIT, BRAF, EGFR, ERBB2, TP53 and KRAS genes. [30][31][32][33][34][35] The causes of CHDs in the most cases are unknown, but the molecular evolution and gene analysis of heart have led to identification of some mutations associated with the CHDs. In our previous study in Kurdish patients with atrial and ventricular wall defects referred to Sanandaj and Kermanshah Hospitals, was detected no mutation in NKX2.5 gene and 3 mutations in TBX5 gene. ...
Background:
The mutations in GATA4 gene induce inherited atrial and ventricular septation defects, which is the most frequent forms of congenital heart defects (CHDs) constituting about half of all cases.
Method:
We have performed High resolution melting (HRM) mutation scanning of GATA4 coding exons of nonsyndrome 100 patients as a case group including 39 atrial septal defects (ASD), 57 ventricular septal defects (VSD) and four patients with both above defects and 50 healthy individuals as a control group. Our samples are categorized according to their HRM graph. The genome sequencing has been done for 15 control samples and 25 samples of patients whose HRM analysis were similar to healthy subjects for each exon. The PolyPhen-2 and MUpro have been used to determine the causative possibility and structural stability prediction of GATA4 sequence variation.
Results:
The HRM curve analysis exhibit that 21 patients and 3 normal samples have deviated curves for GATA4 coding exons. Sequencing analysis has revealed 12 nonsynonymous mutations while all of them resulted in stability structure of protein 10 of them are pathogenic and 2 of them are benign. Also we found two nucleotide deletions which one of them was novel and one new indel mutation resulting in frame shift mutation, and 4 synonymous variations or polymorphism in 6 of patients and 3 of normal individuals. Six or about 50% of these nonsynonymous mutations have not been previously reported.
Conclusion:
Our results show that there is a spectrum of GATA4 mutations resulting in septal defects.
... BRAF region comprised between position 140753211 and 140753460 on chromosome 7 (GRCh38) was amplified using Hot Star Taq Plus Kit, Qiagen, in a Mastercycler pro vapo protect, Eppendorf, with BRAF specific primers 5′-CTC TTC ATA ATG CTT GCT CTG ATA GG-3′and 5′-TAG TAA CTC AGC AGC ATC TCAGG-3′ [11] that allows to amplify a region of 250 base pairs encompassing the region that bears the BRAF c.1799T > A mutation. Afterwards, the amplicons were purified by QIAquick PCR Purification Kit, Qiagen, and then the purified amplicons were analyzed by agarose gel electrophoresis and quantified using QuBit dsDNA HS Assay kit in a Qubit 3.0 Fluorometer. ...
The MinION is a miniaturized high-throughput next generation sequencing platform of novel conception. The use of nucleic acids derived from formalin-fixed paraffin-embedded samples is highly desirable, but their adoption for molecular assays is hurdled by the high degree of fragmentation and by the chemical-induced mutations stemming from the fixation protocols. In order to investigate the suitability of MinION sequencing on formalin-fixed paraffin-embedded samples, the presence and frequency of BRAF c.1799T > A mutation was investigated in two archival tissue specimens of Hairy cell leukemia and Hairy cell leukemia Variant. Despite the poor quality of the starting DNA, BRAF mutation was successfully detected in the Hairy cell leukemia sample with around 50% of the reads obtained within 2 h of the sequencing start. Notably, the mutational burden of the Hairy cell leukemia sample as derived from nanopore sequencing proved to be comparable to a sensitive method for the detection of point mutations, namely the Digital PCR, using a validated assay. Nanopore sequencing can be adopted for targeted sequencing of genetic lesions on critical DNA samples such as those extracted from archival routine formalin-fixed paraffin-embedded samples. This result let speculating about the possibility that the nanopore sequencing could be trustably adopted for the real-time targeted sequencing of genetic lesions. Our report opens the window for the adoption of nanopore sequencing in molecular pathology for research and diagnostics.
... GIST with a mutation in either KIT or PDGFRA can be treated with selective small inhibitors such as Imatinib, Sunitinib or Regorafenib [30][31][32]. KIT copy number variations are described in melanoma, especially in acral (27.3%) and mucosal (26.3%) melanoma [33][34][35], in glioblastoma multiforme [36] and in intimal sarcoma [37]. They are detected together with BRAF V600E, NRAS or KIT mutations or even alone [38]. ...
Atypical fibroxanthomas (AFX) and pleomorphic dermal sarcomas (PDS) are frequent cutaneous sarcomas typically arising on sun-exposed skin in elderly patients. In contrast to AFX, which generally do not recur after complete excision, PDS locally recur in up to 50% and metastasize in up to 20%.
We recently detected characteristic UV-induced TP53 mutations as potential driver mutation in almost all PDS investigated as well as activating PIK3CA and RAS gene mutations in around one third of our tumors representing targets for personalized treatments in patients with unresectable or metastasized PDS.
In the present study, we identified amplifications and deletions in a small part of the PDS (6 of 27 cases) but not in AFX suggesting that copy number variations (CNV) might not be an initial event in tumor development but rather important during tumor progression. In addition to BRAF, KNSTRN, IDH1 and PDGFRA amplification, CNV analyses revealed deletions in the CDKN2A, KIT and PDGFRA genes. In cases where an appropriate FISH assay was established, the CNV results could be verified by FISH analysis.
Amplification of BRAF, KIT or PDGFRA and/or losses of CDKN2A might represent bad prognostic markers, although larger studies are needed to clarify their association with prognosis or progression in PDS.
... These observations indicate that parafibromin immunostaining is useful for diagnosis of PC, and that genetic testing for germline (Heinrich, Blanke, Druker, & Corless, 2002;Willmore-Payne, Holden, Tripp, & Layfield, 2005); or B-Raf proto-oncogene, serine/threonine kinase (BRAF) for melanoma and papillary thyroid cancer (Davies et al., 2002;Kimura et al., 2003). However, targeted therapies are not available for PC and at present genetic testing for somatic CDC73 mutations using parathyroid tumor DNA may not be clinically useful for establishing the diagnosis or staging, especially as such tumors may contain multiple mutations. ...
Parathyroid carcinoma (PC) may occur as part of a complex hereditary syndrome or an isolated (i.e. non-syndromic) non-hereditary (i.e. sporadic) endocrinopathy. Studies of hereditary, and syndromic forms of PC, which include the hyperparathyroidism-jaw tumour syndrome (HPT-JT), multiple endocrine neoplasia types 1 and 2 (MEN1 and MEN2), and familial isolated primary hyperparathyroidism (FIHP), have revealed some genetic mechanisms underlying PC. Thus, cell division cycle 73 (CDC73) germline mutations cause HPT-JT, and CDC73 mutations occur in 70% of sporadic PC, but in only ∼2% of parathyroid adenomas. Moreover, CDC73 germline mutations occur in 20-40% of patients with sporadic PC and may reveal unrecognized HPT-JT. This indicates that CDC73 mutations are major driver mutations in the aetiology of PCs. However, there is no genotype-phenotype correlation and some CDC73 mutations (e.g. c.679_680insAG) have been reported in patients with sporadic PC, HPT-JT, or FIHP. Other genes involved in sporadic PC include germline MEN1 and rearranged during transfection (RET) mutations and somatic alterations of the retinoblastoma 1 (RB1) and tumour protein P53 (TP53) genes, as well as epigenetic modifications including DNA methylation and histone modifications, and microRNA mis-regulation. This review summarizes the genetics and epigenetics of the familial syndromic and non-syndromic (sporadic) forms of PC. This article is protected by copyright. All rights reserved.
... Among BRAF mutant tumours, the frequency (24%) of BRAF V600K in our cohort was somewhat higher than expected. The frequency of BRAF V600K mutation has been reported to range between 6 and 30% (Willmore-Payne et al, 2005;Spittle et al, 2007;Ugurel et al, 2007;Halaban et al, 2010;Rubinstein et al, 2010;Long et al, 2011;Jewell et al, 2012;Lovly et al, 2012;Menzies et al, 2012;Bucheit et al, 2013;Greaves et al, 2013;Heinzerling et al, 2013). Of note, Long et al's Australian study determined that 20% of tumours had BRAF V600K oncogenic mutations (Long et al, 2011). ...
Background:
Cutaneous melanoma can metastasise haematogenously and/or lymphogenously to form satellite/in-transit, lymph node or distant metastasis. This study aimed to determine if BRAF and NRAS mutant and wild-type tumours differ in their site of first tumour metastasis and anatomical metastatic pathway.
Methods:
Prospective cohort of patients with a histologically confirmed primary cutaneous melanoma at three tertiary referral centres in Melbourne, Australia from 2010 to 2015. Multinomial regression determined clinical, histological and mutational factors associated with the site of first metastasis and metastatic pathway.
Results:
Of 1048 patients, 306 (29%) developed metastasis over a median 4.7 year follow-up period. 73 (24%), 192 (63%) and 41 (13%) developed distant, regional lymph node and satellite/in-transit metastasis as the first site of metastasis, respectively. BRAF mutation was associated with lymph node metastasis (adjusted RRR 2.46 95% CI 1.07-5.69, P=0.04) and sentinel lymph node positivity (adjusted odds ratio [aOR] OR 1.55, 95% CI 1.14-2.10, P=0.005). BRAF mutation and NRAS mutation were associated with increased odds of developing liver metastasis (aOR 3.09, 95% CI 1.49-6.42, P=0.003; aOR 3.17, 95% CI 1.32-7.58, P=0.01) and central nervous system (CNS) metastasis (aOR 4.65, 95% CI 2.23-9.69, P<0.001; aOR 4.03, 95% CI 1.72-9.44, P=0.001). NRAS mutation was associated with lung metastasis (aOR 2.44, 95% CI 1.21-4.93, P=0.01).
Conclusions:
BRAF mutation was found to be associated with lymph node metastasis as first metastasis and sentinel lymph node positivity. BRAF and NRAS mutations were associated with CNS and liver metastasis and NRAS mutation with lung metastasis. If these findings are validated in additional prospective studies, a role for heightened visceral organ surveillance may be warranted in patients with tumours harbouring these somatic mutations.
... Interestingly, besides BRAF V600E, 3 other BRAF variants were detected in our ATC series: G469V, G469A and D594A. These alterations are known oncogenes in malignant melanoma [23,24] and non-small-cell lung cancer (NSCLC) [25,26] but have so far not been reported in thyroid carcinoma. Since they were found in other human malignancies, we consider these alterations as likely oncogene also in the context of ATC. ...
Context:
Anaplastic thyroid carcinoma (ATC) represents one of the most aggressive carcinomas with no consistent survival benefit when treated with conventional radiochemotherapy. Approaches targeting "oncogene addiction" of ATC are increasingly explored and first promising results have been reported in single case studies.
Objective:
To determine the prevalence of mutations in known thyroid oncogenes and signalling pathways amendable to targeted therapy in a large cohort of ATC.
Results:
In 118 ATC (57 male/ 61 female) a total of 165 mutations were found. Genes involved in the MAPK/ERK and PI3K pathway (BRAF 11.0%, HRAS 4.2%, KRAS 7.6%, NRAS 7.6%, PI3KCA 11.8%) were altered in 33%. Targetable receptor tyrosine kinases were mutated in 11%. The most frequently altered genes were TERT in 86/118 (73%) and p53 in 65/118 (55%) cases. No mutations were found analysing ALK, KIT, MET and mTOR.
Materials and methods:
Next generation sequencing (NGS) was performed in FFPE samples from 118 ATC using MiSeq (Illumina) and CLC Cancer Research Workbench (CLCbio; Qiagen) for mutation analysis in: ALK, BRAF, CDKN2A, EGFR, ERBB2, HRAS, KIT, KRAS, MET, mTOR, NRAS, PDGFRA, PI3KCA, p53, RB1, RET and TSC2. Sanger sequencing was used to detect TERT promotor mutations.
Conclusions:
To our knowledge this is the largest study analysing mutations for targeted therapy of ATC. We found that 33% of ATC harbour mutations in pathways amendable to targeted therapy. Molecular screening in ATC is suggested for targeted therapies since current conventional treatment for ATC proved mainly futile.
... amplicon per sample was required to validate the run for a 1% sensitivity and confirmed as 1 previously described [16]. France) and LC480 thermocycler (Roche Diagnostics) as previously described [17,18] Diagnostics) were used as previously described to assess real-time allele specific 20 amplification [17,19]. All data and curves analysis were assessed using LightCycler 480 21 software v. ...
Background
Metastatic or unresectable melanoma is a serious and deadly disease. Anti-BRAF and immunotherapy improved overall survival in patients with metastatic disease. Thus, BRAF genotyping is important to choose the right therapy. Methods
In our study, we assessed and compared BRAF mutations in 59 formalin-fixed and paraffin-embedded tumor samples of patients with metastatic melanoma with next-generation sequencing (NGS), Cobas® 4800 BRAF V600 mutation test CE-IVD commercial kit, high-resolution melting PCR (HRM), multiplex real-time allele specific amplification (multiplexed RT-ASA) and immunohistochemistry (IHC). ResultsThirty-one samples were found bearing a BRAF mutation with NGS (52.5%), 28 with Cobas® test (47.5%), 28 with HRM (47.5%), 29 with multiplexed RT-ASA (49.2%) and 27 with IHC (45.8%). Based on NGS data, 26 (81.2%) were c.1799 T>A (p.Val600Glu), 3 (9.4%) were c. 1798-1799 GT>AA (p.Val600Lys), 1 was c.1789_1790 CT>TC (p.Leu597Ser) and 2 were complex mutations. Sensitivity was 90.3% for Cobas® test, 93.1% for multiplexed RT-ASA and 87.1% for IHC and HRM. Specificity was 100% for Cobas® test, IHC and multiplexed RT-ASA and 96.4% for HRM. The reference assay was NGS. Rare mutations were detected with NGS and HRM: c.1789_1790 CT>TC (p.Leu597Ser) mutation and the complex mutation c.1796 A>T; c.1797_1798 insACT (p.Thr599Thr; p.Thr599_Val600insThr). Our data suggest that multiplexed RT-ASA is the most sensitive assay but specific primers for each mutation are needed. HRM can detect all exon 15 mutations but has a lower sensitivity. Because of its specificity for Val600Glu mutation, IHC may be considered only as a screening tool and testing should be completed by a method able to detect other V600 mutations. BRAF Cobas® assay is Val600Glu-specific and has poor sensitivity for the other V600 mutations; thus, it looks important to use multiplex assays able to detect all V600 mutations because a false-negative result will deprive the patient of an important treatment option.
... [152,153] Although it also has been proposed recently that such mutations may complement the more common BRAF mutations, contributing to progression, [154,155] activation of CKIT may also occur in the absence of either NRAS or BRAF mutations. [152] Additionally, since CKIT and its ligand SCF are developmentally implicated in the proper development, melanocyte motility, and homing of melanocytes to the appropriate microenvironment [156,157]. ...
The tumor progression in melanocytic lesions is assumed to follow a multistep process where the genetic alterations and the morphological steps are closely related. In general terms, this process is usually identified pathologically as dysplasia, intraepithelial malignancy, and early neoplasm. However, melanocytic lesions with dysplasia are, on one hand, controversial in its diagnostic criteria and, on the other hand, difficult to grade and subcategorize due to its heterogeneity. The key condition is the atypical (dysplastic) melanocytic nevus (AMN), which requires both architectural and cytological features to be defined and always needs grading to provide a more accurate risk assessment for the development of malignant melanoma. At this point, it is essential to identify reliably and predict high-grade dysplasia/melanoma-in-situ (HGD-MIS) at clinical, dermatoscopic, biological, and pathological levels within these highly heterogeneous lesions.AMN-HGD and MIS share the same clinical and dermatoscopic features, which makes extremely hard to differentiate between them at this level. They are clearly different from atypical nevi with low-grade dysplasia, which usually present with soft clinical and dermatoscopic findings. Key dermatoscopic features for the low-grade vs. high-grade distinction are atypical pigment network, eccentric hyperpigmented blotches, and peripheral globules. Histologically, HGD-MIS is mainly defined by junctional asymmetry with both lentiginous and nested patterns, suprabasal melanocytes and at least three nuclear abnormalities (mainly nuclear enlargement, anisokaryosis and hyperchromatism as more predictive features). Biologically, these lesions reveal both cell kinetics and topographic genetic heterogeneity, which are mostly driven by abnormal TP53, dissociated from cyclin-dependent kinase inhibitors (p21WAF1-CDKN1A and p27KIP1-CDKN1B), especially for MIS. The differential diagnosis requires distinguishing them from atypical but not dysplastic melanocytic lesions (particularly in specific locations), and invasive malignant melanomas (mostly thin and non-tumorigenic status). The implications are also different for lentiginous and epithelioid dysplastic lesions, whose criteria are weighted in a slightly different way. Finally, the progression pathway in dysplastic melanocytic lesions is unlikely linear at morphological and genetic levels, and they show a distinct correlation with the molecular subtypes of malignant melanomas.
... [152,153] Although it also has been proposed recently that such mutations may complement the more common BRAF mutations, contributing to progression, [154,155] activation of CKIT may also occur in the absence of either NRAS or BRAF mutations. [152] Additionally, since CKIT and its ligand SCF are developmentally implicated in the proper development, melanocyte motility, and homing of melanocytes to the appropriate microenvironment [156,157]. ...
The tumor progression in melanocytic lesions is assumed to follow a multistep process where the genetic alterations and the morphological steps are closely related. In general terms, this process is usually identified pathologically as dysplasia, intraepithelial malignancy, and early neoplasm. However, melanocytic lesions with dysplasia are, on one hand, controversial in its diagnostic criteria and, on the other hand, difficult to grade and subcategorize due to its heterogeneity. The key condition is the atypical (dysplastic) melanocytic nevus (AMN), which requires both architectural and cytological features to be defined and always needs grading to provide a more accurate risk assessment for the development of malignant melanoma. At this point, it is essential to identify reliably and predict high-grade dysplasia/melanoma-in-situ (HGD-MIS) at clinical, dermatoscopic, biological, and pathological levels within these highly heterogeneous lesions.
AMN-HGD and MIS share the same clinical and dermatoscopic features, which makes extremely hard to differentiate between them at this level. They are clearly different from atypical nevi with low-grade dysplasia, which usually present with soft clinical and dermatoscopic findings. Key dermatoscopic features for the low-grade vs. high-grade distinction are atypical pigment network, eccentric hyperpigmented blotches, and peripheral globules. Histologically, HGD-MIS is mainly defined by junctional asymmetry with both lentiginous and nested patterns, suprabasal melanocytes and at least three nuclear abnormalities (mainly nuclear enlargement, anisokaryosis and hyperchromatism as more predictive features). Biologically, these lesions reveal both cell kinetics and topographic genetic heterogeneity, which are mostly driven by abnormal TP53, dissociated from cyclin-dependent kinase inhibitors (p21WAF1-CDKN1A and p27KIP1-CDKN1B), especially for MIS. The differential diagnosis requires distinguishing them from atypical but not dysplastic melanocytic lesions (particularly in specific locations), and invasive malignant melanomas (mostly thin and non-tumorigenic status). The implications are also different for lentiginous and epithelioid dysplastic lesions, whose criteria are weighted in a slightly different way. Finally, the progression pathway in dysplastic melanocytic lesions is unlikely linear at morphological and genetic levels, and they show a distinct correlation with the molecular subtypes of malignant melanomas.
Overview
Over 100 years ago, the Nobel Prize for Physiology and Medicine was given to Paul Ehrlich for postulating that “magic bullets” could specifically target and kill cells such as cancer cells based on their unique molecular features. The completed Human Genome Project and the cancer genomics revolution have now mapped many of the genetic changes underlying the unique features of common malignancies. Although cancer has long been recognized to be heterogeneous in its clinical presentation, course, pathology, and response to therapy, we now recognize that it is far more molecularly heterogeneous than anticipated and that such variability will require an individualized approach to patient care. Rather than a single magic bullet, we need an arsenal requiring precise delivery. While inter‐ and intratumoral genomic heterogeneity present significant challenges to cancer management and drug development, a number of developments foster hope for accelerated progress in the war on cancer. First, our catalogue of genomic targets underlying diverse cancers is rapidly growing. Second, we are beginning to understand how diverse mutations converge on a small number of druggable pathways. Third, we continue to develop drugs and biologic agents (e.g., immune checkpoint inhibitors) that target an increasingly diverse array of genomic subtypes of cancer. Finally, advances in “next‐generation” sequencing technologies now enable far earlier detection of disease and disease recurrence. This article focuses on these developments and how their integration is aiding in the precise delivery of “magic bullets.”
Objectives:
Neoplasms arising from the vulva are uncommon and comprise various subtypes. Given the recent advancements in the molecular aspects of oncologic pathology and how they have impacted cancer treatment, an understanding of recent innovations in the molecular features of vulvar lesions is important.
Materials and methods:
Systematic literature search was performed on PubMed, Google Scholar, and Scopus databases for molecular and genetic characteristics of vulvar neoplasms. Peer-reviewed literature published in English is included.
Results:
Squamous cell carcinoma (SCC) and its precursors are the predominant neoplasm at this site. Human papillomavirus (HPV) plays a crucial role in the pathogenesis of some of these lesions. Human papillomavirus-associated SCC follows the carcinogenic pathway driven by viral proteins E6 and E7 while HPV-independent SCC shows a high incidence of mutation of TP53 and CDKN2A genes. Mutations in the genes involving the PI3K-Akt pathway play an important role in the pathogenesis of both types of SCC. Among other vulvar malignancies, melanoma, and vulvar Paget disease (VPD) pose a significant clinical challenge and have unique molecular characteristics. Compared with dermal cutaneous melanoma, vulvar melanoma shows a higher rate of mutation of cKIT and NRAS genes and a lower rate of mutations in BRAF. Less than 20% of VPD shows amplification of ERBB2 and seldom shows mutation in genes involving the PI3K-Akt pathway.
Conclusions:
Several potentially targetable molecular pathways have emerged as they have been shown to be involved in the tumorigenesis of SCC, melanoma, and VPD.
BRAF mutation is commonly known in a number of human cancer types. It is counted as a potential component in treating cancer. In this study, based on structural optimization of previously reported inhibitors (3-fluro substituted derivatives of imidazo[2,1-b]thiazole-based scaffold), we designed and synthesized sixteen new imidazo[2,1-b]thiazole derivatives with m-nitrophenyl group at position 6. The electron withdrawing properties was reserved while the polarity was modified compared to previously synthesized compounds (-F). Furthermore, the new substituted group (-NO2) provided an additional H-bond acceptor(s) which may bind with the target enzyme through additional interaction(s). In vitro cytotoxicity evaluation was performed against human cancer cell line (A375). In addition, in vitro enzyme assay was performed against mutated B-Raf (B-Raf V600E). Compounds 13a, 13g and 13f showed highest activity on mutated B-Raf with IC50 0.021, 0.035 and 0.020 µM. All target compounds were tested for in vitro cytotoxicity against NCI 60 cell lines. Compounds 13a and 13g were selected for 5 doses test mode. Moreover, in silico molecular simulation was explored in order to explore the possible interactions between the designed compounds and the B-Raf V600E active site.
Melanoma is an aggressive skin malignancy, and the acral lentiginous melanoma (ALM) subtype affects non-sun-exposed sites such as the volar surface of the hands and feet and the subungual region and is most common in Asians, Hispanics, and Afro-descendants. The presence of different clones within the same tumor seems to influence the aggressiveness of tumors. Patients with mutations in the KIT gene have shown a good response to tyrosine kinase inhibitor therapy. We tested the hypothesis of intratumor heterogeneity through analysis of KIT gene mutations in ALM and determined the correlation between KIT mutations and demographic, clinical, and histopathological variables. Twenty-five ALM samples were examined. We selected up to four different regions per tumor for sequencing by the Sanger method for analysis of KIT gene exon 11 and exon 13 mutations. Advanced lesions were predominant, and the main histopathological characteristics of lesions were Breslow index >4.0 mm (17/25, 68%), Clark level IV/V (21/25, 84%), ulceration (16/25, 64%), and >3 mitoses/mm (8/25, 32%). KIT gene mutations were detected in 11/25 cases (44%), and all these 11 cases displayed intratumor heterogeneity, that is, at least 2 tumor regions had different mutational profiles. The predicted effect of most mutations detected was detrimental to protein function. No significant correlations between histopathological variables and either KIT mutations or intratumor heterogeneity were observed. The hypothesis of intratumor heterogeneity of KIT gene mutations in acral lentiginous melanoma was supported.
Cutaneous malignant melanoma is one of the few major cancers that continue to exhibit a positive rate of increase in the developed world. A wealth of epidemiological data has undisputedly implicated ultraviolet radiation (UVR) from sunlight and artificial sources as the major risk factor for melanomagenesis. However, the molecular mechanisms of this cause-and-effect relationship remain murky and understudied. Recent efforts on multiple fronts have brought unprecedented expansion of our knowledge base on this subject and it is now clear that melanoma is caused by a complex interaction between genetic predisposition and environmental exposure, primarily to UVR. Here we provide an overview of the effects of the macroenvironment (UVR) on the skin microenvironment and melanocyte-specific intrinsic (mostly genetic) landscape, which conspire to produce one of the deadliest malignancies.
We review here some of the historical highlights in exploratory studies of the vertebrate embryonic structure known as the neural crest. The study of the molecular properties of the cells that it produces, their migratory capacities and plasticity, and the still-growing list of tissues that depend on their presence for form and function, continue to enrich our understanding of congenital malformations, paediatric cancers and evolutionary biology. Developmental biology has been key to our understanding of the neural crest, starting with the early days of experimental embryology and through to today, when increasingly powerful technologies contribute to further insight into this fascinating vertebrate cell population.
Mutations in the BRAF gene have been described to occur in two-third of melanomas. The objective of the study was to establish the frequency of BRAF V600E/K/R mutation in a series of melanomas from Indian origin and to correlate mutation status with clinicopathological features. Seventy melanoma cases were evaluated for BRAF V600 mutation by pyrosequencing. Overall, BRAF mutations were detected in 30% of the patients. All mutations observed were missense type (GTG > GAG) resulting in p.V600E, while none showed V600K/R mutation. The frequency of BRAF V600E mutations were more in patients with onset age of 50 years. BRAF mutations were significantly associated with tumor site wherein more mutations were seen in tumors from head and neck and extremities region. Acral and mucosal tumor subtype showed a mutation frequency of 31% and 20%, respectively. Epithelial cell morphology tends to harbor frequent BRAF V600E mutation (36%) than other morphological subtypes. Tumors with ulceration and necrosis showed increased BRAF mutation rate (32.5% and 33%) respectively. In conclusion, this is the first study to report a mutation frequency of 30% in this cohort. Our results demonstrated that the BRAF V600E mutation is a frequent event in Indian melanomas, and represents an important molecular target for novel therapeutic approaches.
Breast cancer is the leading cause of cancer-related death in women worldwide. Mutations of the PIK3CA gene are found in approximately 25% of breast carcinomas and are reported as activators of the PI3K/AKT/mTOR pathway. This study aims to compare three assays for the somatic mutation detection of PIK3CA gene in FFPE tissues of patients with breast cancer. We compared Cobas® PIK3CA Mutation Test (Roche Diagnostics, Meylan, France), PCR amplification-refractory mutation system Scorpions® (ARMS) and High-Resolution Melting PCR assay (HRM) for the detection of PIK3CA mutations. Discrepant samples were assessed using Next Generation Sequencing (NGS). 46 FFPE breast carcinomas samples of patients treated for breast cancer have been assessed for PIK3CA mutations using the three PCR assays. Among the 46 samples, 17 (37.8%), 13 (28.36%) and 19 (41.3%) had a PIK3CA mutation, with Cobas®, ARMS and HRM assays respectively. Three different mutations of PIK3CA have been detected for one sample. Calculated kappa were 0.95[0.86;1] between Cobas® and HRM, 0.75[0.55;0.95] between Cobas® and ARMS and 0.72[0.51;0.92] between HRM and ARMS. Five samples were found with discrepant results. Our study shows that the Cobas® assay is suitable for PIK3CA mutation assessment in patients with breast cancer. HRM assay is also suitable for PIK3CA mutation assessment but requires a mutation characterization with a specific assay.
Overview
Over 100 years ago, the Nobel Prize in Physiology or Medicine was given to Paul Ehrlich for postulating that “magic bullets” could specifically target and kill cells such as cancer cells based on their unique molecular features. The completed Human Genome Project and the cancer genomics revolution have now mapped the specific genetic changes underlying unique features of many common malignancies. However, although cancer has long been recognized to be heterogeneous in its clinical presentation, course, and pathology, we now recognize that it is far more molecularly heterogeneous than anticipated and that such variability will require an individualized approach to patient care. Rather than a single magic bullet, we will need an arsenal and this arsenal will require precise delivery. While inter‐ and intratumoral genomic heterogeneity present significant challenges to cancer management and drug discovery, a number of developments foster hope for accelerated progress in the war on cancer. First, our catalog of genomic targets underlying diverse cancers is rapidly growing. Second, we are beginning to understand how diverse mutations converge on a small number of druggable pathways. Third, we continue to develop drugs and biologic agents (e.g., immune checkpoint inhibitors) that target an increasingly wide array of genomic subtypes of cancer. Finally, advances in next‐generation sequencing technologies now enable far earlier detection of disease recurrence than ever before. This chapter will focus on these developments and how their integration is aiding in the precise delivery of “magic bullets.”
Personalized medicine utilizes the molecular basis of disease to provide tools to stratify risk and predict treatment effect; and in doing so provides the opportunity for rational design of cancer therapeutics. In particular, the genetic events associated with rectal and anal malignancies have been translated into clinical applications that now guide screening efforts, serve as targets for therapeutic agents, and can function as predictive markers for treatment outcomes. For example, evaluation for defective cellular DNA-mismatch repair and for activating somatic mutations in the K-ras proto-oncogene have moved into the clinical arena and considered standard of care for screening and treatment decision-making. In addition, the epidermal growth factor receptor and vascular endothelial growth factor are both treatment-directed targets that can alter the life expectancy in individuals with these diseases. Thus by leveraging the science of genomics, the promise of personalized cancer medicine is delivery of highly effective and minimally toxic prevention, screening, and treatment modalities that are tailored to the genetic makeup of each patient and to the unique molecular makeup of that individual’s tumor.
Signal transduction pathways are central to all cellular biological processes, as they provide the link between extracellular or intracellular stimuli and an array of regulatory proteins, including protein kinases, ubiquitin ligases, and transcription factors. Given this, it is not surprising that signal transduction pathways are often deregulated in cancer. Indeed, melanoma is a paradigm for rewired signaling because most critical mutations discovered in this tumor type are centered around relatively few major signaling cues, the most significant of which are the mitogen-activated protein kinase (MAPK) and phosphatidylinositol-3-kinase (PI3K) pathways. Both pathways contain regulatory components with catalytic activities, making them the preferred targets for therapy. Here, we summarize our current understanding of the major deregulated signaling pathways in melanoma and the implications of such deregulation for tumor biology.
We discuss the biology of Ras signal transduction and the epidemiology of ras mutations in association with disease as a background for the development of a Raf kinase inhibitor, BAY 43-9006. Knowledge of Ras effector pathways has permitted genetic validation of numerous targets involved in the Ras signaling cascade. A key Ras effector pathway involves the kinase cascade RAF/MEK/ ERK (MEK: MAP/ERK kinase; ERK: extracellular signal related kinase). Indeed, we present studies of cell lines stably expressing mutant MEK constructs, which point to Raf kinase as a target for therapeutics with selective anti-tumor activity. Finally, a small molecule drug discovery program based on inhibition of Raf kinase activity is outlined and the initial pre-clinical development process of the Raf kinase inhibitor BAY 43-9006 is discussed.
In recent years, members of the protein kinase family have been discovered at an accelerated pace. Most were first described,
not through the traditional biochemical approach of protein purification and enzyme assay, but as putative protein kinase
amino acid sequences deduced from the nucleotide sequences of molecularly cloned genes or complementary DNAs. Phylogenetic
mapping of the conserved protein kinase catalytic domains can serve as a useful first step in the functional characterization
of these newly identified family members.
We discuss the biology of Ras signal transduction and the epidemiology of ras mutations in association with disease as a background for the development of a Raf kinase inhibitor, BAY 43-9006. Knowledge of Ras effector pathways has permitted genetic validation of numerous targets involved in the Ras signaling cascade. A key Ras effector pathway involves the kinase cascade RAF/MEK/ERK (MEK: MAP/ERK kinase; ERK: extracellular signal related kinase). Indeed, we present studies of cell lines stably expressing mutant MEK constructs, which point to Raf kinase as a target for therapeutics with selective anti-tumor activity. Finally, a small molecule drug discovery program based on inhibition of Raf kinase activity is outlined and the initial pre-clinical development process of the Raf kinase inhibitor BAY 43-9006 is discussed.
Gastrointestinal stromal tumors (GISTs) coexpress CD34 and the Kit tyrosine-kinase receptor (CD117). A subset of GISTs carry gain-of-function mutations of the c-kit proto-oncogene in its juxtamembrane domain. The relationship between the mutational status and histological as well as immunohistochemical features has not been assessed in detail. 36 GISTs and 14 other gastrointestinal mesenchymal tumors were investigated for their morphology and immunophenotype as well as for the presence of c-kit mutations. DNA was extracted from formalin-fixed, paraffin-embedded tissue. Exons 9, 11, 13, and 17 of c-kit were analyzed by SSCP. Bands with altered mobility were excised, reamplified, and sequenced. C-kit mutations in Exon 11 encoding the juxtamembrane domain were identified in 19 cases (52.8%), with deletions in 12 cases, insertions in 3 cases (2 of these as duplications), and point mutations in 4 cases. The mutations clustered between Codons 553 and 561, pinpointing the critical region for deregulated Kit receptor activation. In both Exons 9 and 13, single mutations could be identified, whereas no mutations were found in Exon 17. There were c-kit mutations in 66.6% of benign GISTs (14/21), 83.3% of the malignant (5/6), and 40% of the cases of intermediate malignancy (2/5). A low frequency of mutations in benign GISTs, as reported previously by other researchers, could not be observed in our panel. Interestingly, all GISTs with c-kit mutations displayed a spindle cell phenotype, whereas mutations were absent in all 7 tumors with an epithelioid component (P =.03). This finding suggests a relationship between c-kit mutation and histological subtype in GISTs.
Chronic myelogenous leukemia (CML) is caused by the BCR-ABL tyrosine kinase, the product of the Philadelphia chromosome. Imatinib mesylate, formerly STI571, is a selective inhibitor of this kinase.
A total of 532 patients with late--chronic-phase CML in whom previous therapy with interferon alfa had failed were treated with 400 mg of oral imatinib daily. Patients were evaluated for cytogenetic and hematologic responses. Time to progression, survival, and toxic effects were also evaluated.
Imatinib induced major cytogenetic responses in 60 percent of the 454 patients with confirmed chronic-phase CML and complete hematologic responses in 95 percent. After a median follow-up of 18 months, CML had not progressed to the accelerated or blast phases in an estimated 89 percent of patients, and 95 percent of the patients were alive. Grade 3 or 4 nonhematologic toxic effects were infrequent, and hematologic toxic effects were manageable. Only 2 percent of patients discontinued treatment because of drug-related adverse events, and no treatment-related deaths occurred.
Imatinib induced high rates of cytogenetic and hematologic responses in patients with chronic-phase CML in whom previous interferon therapy had failed.
Cancers arise owing to the accumulation of mutations in critical genes that alter normal programmes of cell proliferation, differentiation and death. As the first stage of a systematic genome-wide screen for these genes, we have prioritized for analysis signalling pathways in which at least one gene is mutated in human cancer. The RAS RAF MEK ERK MAP kinase pathway mediates cellular responses to growth signals. RAS is mutated to an oncogenic form in about 15% of human cancer. The three RAF genes code for cytoplasmic serine/threonine kinases that are regulated by binding RAS. Here we report BRAF somatic missense mutations in 66% of malignant melanomas and at lower frequency in a wide range of human cancers. All mutations are within the kinase domain, with a single substitution (V599E) accounting for 80%. Mutated BRAF proteins have elevated kinase activity and are transforming in NIH3T3 cells. Furthermore, RAS function is not required for the growth of cancer cell lines with the V599E mutation. As BRAF is a serine/threonine kinase that is commonly activated by somatic point mutation in human cancer, it may provide new therapeutic opportunities in malignant melanoma.
Activating mutations in the BRAF serine/threonine kinase are found in >70% of human melanomas, of which >90% are BRAF(V599E). We sought to investigate the role of the BRAF(V599E) allele in malignant melanoma. We here report that suppression of BRAF(V599E) expression by RNA interference in cultured human melanoma cells inhibits the mitogen-activated protein kinase cascade, causes growth arrest, and promotes apoptosis. Furthermore, knockdown of BRAF(V599E) expression completely abrogates the transformed phenotype as assessed by colony formation in soft agar. Similar targeting of BRAF(V599E) or wild-type BRAF in human fibrosarcoma cells that lack the BRAF(V599E) mutation does not recapitulate these effects. Moreover, these results are specific for BRAF, as targeted interference of CRAF in melanoma cells does not significantly alter their biological properties. Thus, when present, BRAF(V599E) appears to be essential for melanoma cell viability and transformation and, therefore, represents an attractive therapeutic target in the majority of melanomas that harbor the mutation.
Background
HER-2/neu and c-kit (CD117) onco-protein are increasingly being recognized as targets for therapy in solid tumors, but data on their role in malignant melanoma is currently limited. We studied the prevalence of overexpression of HER-2/neu and c-Kit in 202 patients with malignant melanoma to evaluate a possible prognostic value of these molecular targets in malignant melanoma.
Methods
Overexpression of HER-2/neu and c-Kit was evaluated using immunohistochemical assays in 202 archival tissue specimens.
Results
Between 1991 and 2001, 202 subjects (109 males; 54% and 93 females; 46%) with malignant melanoma were studied with a mean age of 57 years (age range: 15–101 years). The most common histologic type was amelanotic melanoma (n = 62; 30.7%) followed by superficial spreading melanoma (n = 54; 26.7%). The depth of penetration of melanoma (Breslow thickness, pT Stage) ranged from 0.4 mm (stage pT1) to 8.0 mm (stage pT4A). Mean thickness was 2.6 mm (stage pT3A). The ECOG performance scores ranged from 0 to 3. Only 2 patients (0.9%) revealed HER-2/neu overexpression, whereas 46 (22.8%) revealed c-Kit overexpression. Multivariate analysis performed did not show a significant difference in survival between c-Kit positive and negative groups (p = 0.36). Interestingly, not only was c-Kit more likely to be overexpressed in the superficial spreading type, a preliminary association between the presence or absence of c-Kit overexpression and the existence of another second primary tumor was also observed.
Conclusions
The results of our large study indicate that the HER-2/neu onco-protein neither has a role in melanogenesis nor is a potential target for clinical trials with monoclonal antibody therapy. This indicates there is no role for its testing in patients with malignant melanoma. Although c-Kit, expressed preferentially in the superficial spreading type, may not have prognostic value, it does have significant therapeutic implications as a molecular target warranting further investigation.
The RAS/mitogen-activated protein kinase pathway sends external growth-promoting signals to the nucleus. BRAF, a critical serine/threonine kinase in this pathway, is frequently activated by somatic mutation in melanoma. Using a cohort of 115 patients with primary invasive melanomas, we show that BRAF mutations are statistically significantly more common in melanomas occurring on skin subject to intermittent sun exposure than elsewhere (23 of 43 patients; P<.001, two-sided Fisher's exact test). By contrast, BRAF mutations in melanomas on chronically sun-damaged skin (1 of 12 patients) and melanomas on skin relatively or completely unexposed to sun, such as palms, soles, subungual sites (6 of 39 patients), and mucosal membranes (2 of 21 patients) are rare. We found no association of mutation status with clinical outcome or with the presence of an associated melanocytic nevus. The mutated BRAF allele was frequently found at an elevated copy number, implicating BRAF as one of the factors driving selection for the frequent copy number increases of chromosome 7q in melanoma. In summary, the uneven distribution of BRAF mutations strongly suggests distinct genetic pathways leading to melanoma. The high mutation frequency in melanomas arising on intermittently sun-exposed skin suggests a complex causative role of such exposure that mandates further evaluation.
Melanoma is a deadly disease affecting people worldwide. Genetic studies have identified different melanoma subtypes characterized by specific recurrently mutated genes and led to the successful clinical introduction of targeted therapies. Hotspot mutations in SF3B1 were recently reported in uveal melanoma. Our aim was to see if these mutations also occur in cutaneous melanoma.
We analyzed a cohort of 85 cutaneous melanoma including 22 superficial spreading, 24 acral-lentiginous, 36 nodular, and 3 lentigo-maligna melanomas. Exon 14 of SF3B1, containing the site of recurrent mutations described in uveal melanoma, was sequenced in all samples. Additionally, NRAS exon 1 and 2 and BRAF exon 15 were sequenced in all, KIT exons 9, 11, 13, 17, and 18 in 30 samples. High numbers of BRAF and NRAS mutations were identified with frequencies varying according to melanoma subtype. None of the samples were found to harbor a SF3B1 mutation.
We conclude that recurrent mutations in codon 625 of SF3B1 as reported in uveal melanoma are not present in most types of cutaneous melanoma. This highlights the genetic differences between cutaneous and uveal melanoma and the need for subtype specific therapeutic approaches.
Mutations in BRAF have recently been identified in a significant percentage of primary and metastatic cutaneous malignant melanomas. As ultraviolet (UV) exposure may play a role in the development of cutaneous melanoma lesions with BRAF mutations, BRAF mutation frequency in melanomas arising in sites protected from sun exposure may be lower than those from sun-exposed areas. Thus, we determined the BRAF mutation frequency in a panel of 13 mucosal melanomas and compared those data with data from all currently published series of cutaneous melanomas.
BRAF exon 15 DNA from 13 archival primary mucosal melanomas (eight vulvar, four anorectal, and one laryngeal) was sequenced using intron-based primers. As archival DNA occasionally produces poor-quality template, results were confirmed with a TspRI restriction fragment length polymorphism (RFLP) that distinguishes wild-type BRAF from the common mutant form V599E. A binomial test was used to compare the mutation frequency in the mucosal melanomas with the published mutation frequency in cutaneous melanomas.
None of the 13 mucosal melanomas in this series had an exon 15 BRAF mutation, as compared to 54/165 (33%) primary cutaneous melanomas with BRAF mutations in a compilation of all current published studies (p = 0.006).
These data suggest that UV exposure, plays a role in the genesis of BRAF mutations in cutaneous melanoma, despite the absence of the characteristic C>T or CC>TT mutation signature associated with UV exposure, and suggests mechanisms other than pyrimidine dimer formation are important in UV-induced mutagenesis.
Receptor tyrosine kinase genes were sequenced in non–small cell lung cancer (NSCLC) and matched normal tissue. Somatic mutations
of the epidermal growth factor receptor gene EGFR were found in 15of 58 unselected tumors from Japan and 1 of 61 from the United States. Treatment with the EGFR kinase inhibitor
gefitinib (Iressa) causes tumor regression in some patients with NSCLC, more frequently in Japan. EGFR mutations were found in additional lung cancer samples from U.S. patients who responded to gefitinib therapy and in a lung
adenocarcinoma cell line that was hypersensitive to growth inhibition by gefitinib, but not in gefitinib-insensitive tumors
or cell lines. These results suggest that EGFR mutations may predict sensitivity to gefitinib.
B-RAF is a serine/threonine-specific protein kinase that is mutated in approximately 70% of human melanomas. However, the role of this signalling molecule in cancer is unclear. Here, we show that ERK is constitutively activated in melanoma cells expressing oncogenic B-RAF and that this activity is required for proliferation. B-RAF depletion by siRNA blocks ERK activity, whereas A-RAF and C-RAF depletion do not affect ERK signalling. B-RAF depletion inhibits DNA synthesis and induces apoptosis in three melanoma cell lines and we show that the RAF inhibitor BAY43-9006 also blocks ERK activity, inhibits DNA synthesis and induces cell death in these cells. BAY43-9006 targets B-RAF signalling in vivo and induces a substantial growth delay in melanoma tumour xenografts. Our data demonstrate that oncogenic B-RAF activates ERK signalling, induces proliferation and protects cells from apoptosis, demonstrating that it is an important therapeutic target and thus provides novel strategies for clinical management of melanoma and other cancers.
Through careful clinicopathologic correlation, we identified 37 metastatic melanomas in the skin, all of which had intraepidermal components. These were compared with 43 microscopically similar primary melanomas with a predetermined panel of immunostains in general use in surgical pathology, including bcl-2 protein, mutant p53 protein, Ki-67 (MIB-1), proliferating cell nuclear antigen (PCNA), alpha-isoform actin, and CD117 (c-kit protein). There was no significant difference in bcl-2 or alpha-isoform actin staining patterns of primary vs secondary cutaneous melanomas. The expression of Ki-67 generally was higher in metastatic melanomas than in primary lesions, and the same was true of mutant p53 protein labeling; however, some overlap was observed. CD117 staining was retained in 65% of metastatic melanomas (24/37) when they originated from ocular primary tumors; nevertheless, that marker was lost in virtually all of the other metastatic melanocytic neoplasms, whereas primary melanomas demonstrated consistent reactivity for c-kit protein. Although they are not definitive, these trends in immunoreactivity could facilitate the process of distinguishing the multiple primary melanoma syndrome from melanomatous metastases to the skin. That undertaking is best approached with circumspection, because clinicopathologic discriminators for this diagnostic separation are still imperfect.
High-resolution amplicon melting analysis was used to scan for c-kit-activating mutations in exons 9, 11, 13, and 17 in 29 neoplasms diagnosed as gastrointestinal stromal tumors (GISTs). Immunohistochemically, 7 of 29 did not show strong CD 17 positivity and might represent true smooth muscle tumors or c-kit-negative GISTs. No c-kit-activating mutations were detected in the 7 CD117- cases by high-resolution amplicon melting analysis or direct DNA sequencing. Alterations in the remaining 22 CD117+ cases included 13 (59%) in exon 11, 2 (9%) in exon 9, 1 (5%) in exon 13, and none in exon 17. The genetic alterations consisted of point mutations and in-frame insertions, duplications, and deletions. In exon 11, 7 (54%) of 13 alterations have not been described previously. In 2 cases, the identical exon 11 mutation was observed in the primary tumor and a metastatic/recurrent lesion. In all cases, direct DNA sequencing confirmed that polymerase chain reaction products with an abnormal melting curve contained a mutation and products with a normal melting curve, a normal DNA sequence. High-resolution melting analysis can be used to scan DNA for potential c-kit-activating mutations and can aid in the diagnosis of GISTs.
Somatic mutations in the tyrosine kinase (TK) domain of the epidermal growth factor receptor (EGFR) gene are reportedly associated with sensitivity of lung cancers to gefitinib (Iressa), kinase inhibitor. In-frame deletions occur in exon 19, whereas point mutations occur frequently in codon 858 (exon 21). We found from sequencing the EGFR TK domain that 7 of 10 gefitinib-sensitive tumors had similar types of alterations; no mutations were found in eight gefitinib-refractory tumors (P = 0.004). Five of seven tumors sensitive to erlotinib (Tarceva), a related kinase inhibitor for which the clinically relevant target is undocumented, had analogous somatic mutations, as opposed to none of 10 erlotinib-refractory tumors (P = 0.003). Because most mutation-positive tumors were adenocarcinomas from patients who smoked <100 cigarettes in a lifetime ("never smokers"), we screened EGFR exons 2-28 in 15 adenocarcinomas resected from untreated never smokers. Seven tumors had TK domain mutations, in contrast to 4 of 81 non-small cell lung cancers resected from untreated former or current smokers (P = 0.0001). Immunoblotting of lysates from cells transiently transfected with various EGFR constructs demonstrated that, compared to wild-type protein, an exon 19 deletion mutant induced diminished levels of phosphotyrosine, whereas the phosphorylation at tyrosine 1092 of an exon 21 point mutant was inhibited at 10-fold lower concentrations of drug. Collectively, these data show that adenocarcinomas from never smokers comprise a distinct subset of lung cancers, frequently containing mutations within the TK domain of EGFR that are associated with gefitinib and erlotinib sensitivity.
Background
Mutations in BRAF have recently been identified in a significant percentage of primary and metastatic cutaneous malignant melanomas. As ultraviolet (UV) exposure may play a role in the development of cutaneous melanoma lesions with BRAF mutations, BRAF mutation frequency in melanomas arising in sites protected from sun exposure may be lower than those from sun-exposed areas. Thus, we determined the BRAF mutation frequency in a panel of 13 mucosal melanomas and compared those data with data from all currently published series of cutaneous melanomas.
Methods
BRAF exon 15 DNA from 13 archival primary mucosal melanomas (eight vulvar, four anorectal, and one laryngeal) was sequenced using intron-based primers. As archival DNA occasionally produces poor-quality template, results were confirmed with a TspRI restriction fragment length polymorphism (RFLP) that distinguishes wild-type BRAF from the common mutant form V599E. A binomial test was used to compare the mutation frequency in the mucosal melanomas with the published mutation frequency in cutaneous melanomas.
Results
None of the 13 mucosal melanomas in this series had an exon 15 BRAF mutation, as compared to 54/165 (33%) primary cutaneous melanomas with BRAF mutations in a compilation of all current published studies (p = 0.006).
Discussion
These data suggest that UV exposure, plays a role in the genesis of BRAF mutations in cutaneous melanoma, despite the absence of the characteristic C>T or CC>TT mutation signature associated with UV exposure, and suggests mechanisms other than pyrimidine dimer formation are important in UV-induced mutagenesis.
Human mastocytosis is characterized by increased mast cells.
It usually occurs as a sporadic disease that is often transient and
limited in children and persistent or progressive in adults. The
c-KIT protooncogene encodes KIT, a tyrosine kinase that is
the receptor for mast cell growth factor. Because mutated KIT can
transform cells, we examined c-KIT in skin lesions of 22
patients with sporadic mastocytosis and 3 patients with familial
mastocytosis. All patients with adult sporadic mastocytosis had somatic
c-KIT mutations in codon 816 causing substitution of valine
for aspartate and spontaneous activation of mast cell growth factor
receptor (P = 0.0001). A subset of four pediatric
onset cases with clinically unusual disease also had codon 816
activating mutations substituting valine, tyrosine, or phenylalanine
for aspartate. Typical pediatric patients lacked 816 mutations, but
limited sequencing showed three of six had a novel dominant
inactivating mutation substituting lysine for glutamic acid in position
839, the site of a potential salt bridge that is highly conserved in
receptor tyrosine kinases. No c-KIT mutations were found in
the entire coding region of three patients with familial mastocytosis.
We conclude that c-KIT somatic mutations substituting
valine in position 816 of KIT are characteristic of sporadic adult
mastocytosis and may cause this disease. Similar mutations causing
activation of the mast cell growth factor receptor are found in
children apparently at risk for extensive or persistent disease. In
contrast, typical pediatric mastocytosis patients lack these mutations
and may express inactivating c-KIT mutations. Familial
mastocytosis, however, may occur in the absence of c-KIT
coding mutations.
Mutations at the white spotting (w) locus in mice have deleterious effects on germ cells, melanocytes and hematopoietic stem cells. The w locus encodes the c-kit tyrosine-kinase receptor whose ligand is the product of the SI locus. Using monoclonal antibodies (MAb(s)) to the extracellular domain, we have evaluated the expression of c-kit in normal and transformed melanocytes. This cell lineage synthesizes a receptor with a mw of 145 kDa. The gene product is expressed in epidermal melanocytes and in a fraction of nevocytic and blue nevi. In primary melanomas, loss of the receptor is observed in more invasive lesions. Only 30% of the metastatic lesions express detectable levels of the receptor. These findings demonstrate that the c-kit product is down-regulated in melanocytes following malignant transformation. The functional relevance of this modulation remains to be evaluated.
The c-kit gene encodes a transmembrane receptor that has tyrosine kinase activity. c-kit plays a role in hematopoiesis, gametogenesis, and melanogenesis. c-kit is found in melanocytes, and there is evidence that expression is lost in melanoma. We studied 85 melanocytic lesions for c-kit by immunohistochemical techniques using a monoclonal antibody. The lesions included banal nevi, junctional and compound nevi with melanocytic dysplasia, nontumorigenic radial growth phase melanoma, tumorigenic vertical growth phase melanoma, and metastatic melanoma. We found intense membrane staining in normal melanocytes and mast cells. Staining in compound nevi was strongest in junctional and superficial dermal components, whereas dermal nevi showed weak reactivity. Dysplastic nevi stained strongly, particularly in junctional cells. In melanoma, strong reactivity was most prominent in radial growth phase disease, but there was little or no staining in vertical growth phase and metastatic melanomas. In summary, c-kit protein is expressed in normal melanocytes, benign nevi, dysplastic nevi and nontumorigenic melanoma, but expression is lost in tumorigenic primary melanomas and metastases. The role of c-kit loss in advanced melanoma requires additional investigation.
Tyrosine phosphorylation is one of the key covalent modifications that occurs in multicellular organisms as a result of intercellular communication during embryogenesis and maintenance of adult tissues. The enzymes that carry out this modification are the protein tyrosine kinases (PTKs), which catalyze the transfer of the phosphate of ATP to tyrosine residues on protein substrates. Phosphorylation of tyrosine residues modulates enzymatic activity and creates binding sites for the recruitment of downstream signaling proteins. Two classes of PTKs are present in cells: the transmembrane receptor PTKs and the nonreceptor PTKs. Because PTKs are critical components of cellular signaling pathways, their catalytic activity is strictly regulated. Over the past several years, high-resolution structural studies of PTKs have provided a molecular basis for understanding the mechanisms by which receptor and nonreceptor PTKs are regulated. This review will highlight the important results that have emerged from these structural studies.
The Ras/Raf/MEK (mitogen-activated protein kinase/ERK kinase)/ERK (extracellular-signal-regulated kinase) pathway is at the heart of signalling networks that govern proliferation, differentiation and cell survival. Although the basic regulatory steps have been elucidated, many features of this pathway are only beginning to emerge. This review focuses on the role of protein-protein interactions in the regulation of this pathway, and how they contribute to co-ordinate activation steps, subcellular redistribution, substrate phosphorylation and cross-talk with other signalling pathways.
CD 117 (KIT) is a transmembrane, tyrosine kinase growth factor receptor which is expressed on numerous diverse fetal and adult cells including hematopoietic cells, mast cells, melanocytes, germ cells, and the interstitial cells of Cajal. Its expression in tumors is also diverse, but with selective use and attention to specific staining patterns, this marker is useful in the identification of gastrointestinal stromal tumors, mast cell tumors, and seminomatous germ cell tumors.
Low-dose interferon alfa (IFNalpha) has been shown to have limited effects in the adjuvant treatment of patients with intermediate- and high-risk primary melanoma. We hypothesized that a combination regimen with low-dose interleukin-2 (IL-2) may improve survival prospects in these patients.
After wide excision of primary melanoma without clinically detectable lymph node metastasis (pT3 to 4, cN0, M0), 225 patients from 10 participating centers were randomly assigned to receive either subcutaneous low-dose IFNalpha2b (3 million international units [MU]/m2/d, days 1 to 7, week 1; three times weekly, weeks 3 to 6, repeated all 6 weeks) plus IL-2 (9 MU/m2/d, days 1 to 4, week 2 of each cycle) for 48 weeks, or observation alone. The primary end point was prolongation of a relapse-free interval.
Of the 225 enrolled patients, 223 were found to be eligible. Median follow-up time was 79 months. All evaluated prognostic factors were well balanced between the two arms of the study. Relapses were noticed in 36 of 113 patients treated with IFNalpha2b plus IL-2 and in 34 of 110 patients with observation alone. Five-year disease-free survival of those who had routine surgery supplemented by IFNalpha2b and IL-2 treatment was 70.1% (95% confidence interval [CI], 61.3% to 78.9%), compared with 69.9% in those receiving surgery and observation alone (95% CI, 60.7% to 79.1%) in the intention-to-treat analysis. Evaluation of the overall survival did not show any difference between treated and untreated melanoma patients (P =.93).
Adjuvant treatment of intermediate- and high-risk melanoma patients with low-dose IFNalpha2b and IL-2 is safe and well tolerated by most patients, but it does not improve disease-free or overall survival.
To further investigate the efficacy and safety of temozolomide plus thalidomide in patients with metastatic melanoma without brain metastases.
Patients with histologically confirmed advanced-stage metastatic melanoma were enrolled in an open-label, phase II study. The primary end point was response rate. Patients received temozolomide (75 mg/m2/d x 6 weeks with a 2-week rest between cycles) plus concomitant thalidomide (200 mg/d with dose escalation to 400 mg/d for patients < 70 years old, or 100 mg/d with dose escalation to 250 mg/d for patients >/= 70 years old). Treatment was continued until unacceptable toxicity or disease progression occurred.
Thirty-eight patients (median age, 62 years) with stage IV (three patients with M1a, eight with M1b, and 26 with M1c) or stage IIIc (one patient) melanoma and a median of four metastatic sites were enrolled, and received a median of two cycles of therapy. Twelve patients (32%) had an objective tumor response, including one with an ongoing complete response of 25+ months' duration and 11 with partial responses. Five patients achieving partial response with a more than 90% reduction of disease were converted to a complete response with surgery. Treatment was generally well tolerated. Median survival was 9.5 months (95% confidence interval, 6.05 to 19.38 months), with a median follow-up among survivors of 24.3 months.
The combination of temozolomide plus thalidomide seems to be a promising and well-tolerated oral regimen for metastatic melanoma that merits further study.
We apologize to our colleagues for our inability to cite multiple primary references due to space limitations. This work is supported in part by the McCabe Foundation, the Mary L. Smith Charitable Lead Trust, the Abramson Cancer Center of the University of Pennsylvania Pilot Projects Program, and Grant #IRG-78-002-26 from the American Cancer Society (all to D.A.T.), by the NIH grants CA-25874, CA-47159, CA-76674, and CA-10815 (to M.H.), and by funds from the Abramson Family Cancer Research Institute (D.A.T. and B.L.W.).
Most gastrointestinal stromal tumors (GISTs) express constitutively activated mutant isoforms of KIT or kinase platelet-derived growth factor receptor alpha (PDGFRA) that are potential therapeutic targets for imatinib mesylate. The relationship between mutations in these kinases and clinical response to imatinib was examined in a group of patients with advanced GIST.
GISTs from 127 patients enrolled onto a phase II clinical study of imatinib were examined for mutations of KIT or PDGFRA. Mutation types were correlated with clinical outcome.
Activating mutations of KIT or PDGFRA were found in 112 (88.2%) and six (4.7%) GISTs, respectively. Most KIT mutations involved exon 9 (n = 23) or exon 11 (n = 85). All KIT mutant isoforms, but only a subset of PDGFRA mutant isoforms, were sensitive to imatinib, in vitro. In patients with GISTs harboring exon 11 KIT mutations, the partial response rate (PR) was 83.5%, whereas patients with tumors containing an exon 9 KIT mutation or no detectable mutation of KIT or PDGFRA had PR rates of 47.8% (P =.0006) and 0.0% (P <.0001), respectively. Patients whose tumors contained exon 11 KIT mutations had a longer event-free and overall survival than those whose tumors expressed either exon 9 KIT mutations or had no detectable kinase mutation.
Activating mutations of KIT or PDGFRA are found in the vast majority of GISTs, and the mutational status of these oncoproteins is predictive of clinical response to imatinib. PDGFRA mutations can explain response and sensitivity to imatinib in some GISTs lacking KIT mutations.
Expression of KIT tyrosine kinase is critical for normal germ cell development and is observed in the majority of seminomas. Activating mutations in KIT are common in gastrointestinal stromal tumors and mastocytosis. In this study we examined the frequency and spectrum of KIT mutations in 54 testicular seminomas, 1 ovarian dysgerminoma and 37 non-seminomatous germ cell tumors (NSGCT). Fourteen seminomas (25.9%) contained exon 17 point mutations including D816V (6 cases), D816H (3 cases), Y823D (2 cases), and single examples of Y823C, N822K, and T801I. No KIT mutations were found in the ovarian dysgerminoma or the NSGCTs. In transient transfection assays, mutant isoforms D816V, D816H, Y823D, and N822K were constitutively phosphorylated in the absence of the natural ligand for KIT, stem cell factor (SCF). In contrast, activation of T801I and wild-type KIT required SCF. Mutants N822K and Y823D were inhibited by imatinib mesylate (Gleevec, previously STI571) whereas D816V and D816H were both resistant to imatinib mesylate. Biochemical evidence of KIT activation, as assessed by KIT phosphorylation and KIT association with phosphatidylinositol (PI) 3-kinase in tumor cell lysates, was largely confined to seminomas with a genomic KIT mutation. These findings suggest that activating KIT mutations may contribute to tumorigenesis in a subset of seminomas, but are not involved in NSGCT.
The rationale to target receptor protein tyrosine kinases (RPTKs) as an approach to cancer chemotherapy has continued to become more compelling with time. Preclinical and clinical data strongly support the involvement of specific RPTKs in the formation and progression of a subset of solid and liquid tumors. The advances in our understanding of the oncogenic activation of these receptors have been matched by the identification of new structural classes of kinase inhibitors that exhibit enormous improvements with regard to potency, specificity and efficacy. This article summarizes current knowledge of the most promising RPTK inhibitors in clinical trials or known to be in late stage preclinical development.
Over 30 mutations of the B-RAF gene associated with human cancers have been identified, the majority of which are located within the kinase domain. Here we show that of 22 B-RAF mutants analyzed, 18 have elevated kinase activity and signal to ERK in vivo. Surprisingly, three mutants have reduced kinase activity towards MEK in vitro but, by activating C-RAF in vivo, signal to ERK in cells. The structures of wild type and oncogenic V599EB-RAF kinase domains in complex with the RAF inhibitor BAY43-9006 show that the activation segment is held in an inactive conformation by association with the P loop. The clustering of most mutations to these two regions suggests that disruption of this interaction converts B-RAF into its active conformation. The high activity mutants signal to ERK by directly phosphorylating MEK, whereas the impaired activity mutants stimulate MEK by activating endogenous C-RAF, possibly via an allosteric or transphosphorylation mechanism.
Most patients with non-small-cell lung cancer have no response to the tyrosine kinase inhibitor gefitinib, which targets the epidermal growth factor receptor (EGFR). However, about 10 percent of patients have a rapid and often dramatic clinical response. The molecular mechanisms underlying sensitivity to gefitinib are unknown.
We searched for mutations in the EGFR gene in primary tumors from patients with non-small-cell lung cancer who had a response to gefitinib, those who did not have a response, and those who had not been exposed to gefitinib. The functional consequences of identified mutations were evaluated after the mutant proteins were expressed in cultured cells.
Somatic mutations were identified in the tyrosine kinase domain of the EGFR gene in eight of nine patients with gefitinib-responsive lung cancer, as compared with none of the seven patients with no response (P<0.001). Mutations were either small, in-frame deletions or amino acid substitutions clustered around the ATP-binding pocket of the tyrosine kinase domain. Similar mutations were detected in tumors from 2 of 25 patients with primary non-small-cell lung cancer who had not been exposed to gefitinib (8 percent). All mutations were heterozygous, and identical mutations were observed in multiple patients, suggesting an additive specific gain of function. In vitro, EGFR mutants demonstrated enhanced tyrosine kinase activity in response to epidermal growth factor and increased sensitivity to inhibition by gefitinib.
A subgroup of patients with non-small-cell lung cancer have specific mutations in the EGFR gene, which correlate with clinical responsiveness to the tyrosine kinase inhibitor gefitinib. These mutations lead to increased growth factor signaling and confer susceptibility to the inhibitor. Screening for such mutations in lung cancers may identify patients who will have a response to gefitinib.
Oncogenic mutations of molecules involved in the mitogen-activated protein kinase (MAPK) pathways provide signals mediating both tumor growth and invasion in various cancers including melanomas. BRAF somatic mutations, found in 66% of melanomas, have NIH3T3 transforming ability with the elevated kinase activity in vitro. We attempted to mediate RNA interference (RNAi) with HIV lentiviral vectors specific for either wild type or the most frequently mutated form of BRAF (V599E) in 10 melanoma cell lines, and found that RNAi inhibited the growth of most melanoma cell lines in vitro as well as in vivo, which was accompanied by decrease of both BRAF protein and ERK phosphorylation. Interestingly, the mutated BRAF (V599E)-specific siRNA inhibited the growth and MAPK activity of only melanoma cell lines with this mutation. Furthermore, BRAF RNAi inhibited matrigel invasion of melanoma cells accompanied with a decrease of matrix metalloproteinase activity and beta(1) integrin expression. These results clarify that the mutated BRAF (V599E) is essentially involved in malignant phenotype of melanoma cells through the MAPK activation and is an attractive molecular target for melanoma treatment. The lentivirus-mediated RNAi specific for oncogenic mutations may be a powerful technique for gene therapy of cancer.
Gefitinib (Iressa, Astra Zeneca Pharmaceuticals) is a tyrosine kinase inhibitor that targets the epidermal growth factor receptor
(EGFR) and induces dramatic clinical responses in nonsmall cell lung cancers (NSCLCs) with activating mutations within the
EGFR kinase domain. We report that these mutant EGFRs selectively activate Akt and signal transduction and activator of transcription
(STAT) signaling pathways, which promote cell survival, but have no effect on extracellular signal–regulated kinase signaling,
which induces proliferation. NSCLC cells expressing mutant EGFRs underwent extensive apoptosis after small interfering RNA–mediated
knockdown of the mutant EGFR or treatment with pharmacological inhibitors of Akt and STAT signaling and were relatively resistant to apoptosis induced
by conventional chemotherapeutic drugs. Thus, mutant EGFRs selectively transduce survival signals on which NSCLCs become dependent;
inhibition of those signals by gefitinib may contribute to the drug's efficacy.
The cytoplasmic serine/threonine kinase BRAF and receptor tyrosine kinases of the platelet-derived growth factor receptor (PDGFR) family are frequently activated in cancer by mutations of an equivalent amino acid. Structural studies have provided important insights into why these very different kinases share similar oncogenic hot spots and why the PDGFR juxtamembrane region is also a frequent oncogenic target. This research has implications for other kinases that are mutated in human tumours and for the treatment of cancer using kinase inhibitors.
Once a poorly defined pathologic oddity, in recent years, gastrointestinal stromal tumor (GIST) has emerged as a distinct oncogenetic entity that is now center stage in clinical trials of kinase-targeted therapies. This review charts the rapid progress that has established GIST as a model for understanding the role of oncogenic kinase mutations in human tumorigenesis. Approximately 80% to 85% of GISTs harbor activating mutations of the KIT tyrosine kinase. In a series of 322 GISTs (including 140 previously published cases) studied by the authors in detail, mutations in the KIT gene occurred with decreasing frequency in exons 11 (66.1%), 9 (13%), 13 (1.2%), and 17 (0.6%). In the same series, a subset of tumors had mutations in the KIT-related kinase gene PDGF receptor alpha (PDGFRA), which occurred in either exon 18 (5.6%) or 12 (1.5%). The remainder of GISTs (12%) were wild type for both KIT and PDGFRA. Comparative studies of KIT-mutant, PDGFRA-mutant, and wild-type GISTs indicate that there are many similarities between these groups of tumors but also important differences. In particular, the responsiveness of GISTs to treatment with the kinase inhibitor imatinib varies substantially depending on the exonic location of the KIT or PDGFRA mutation. Given these differences, which have implications both for the diagnosis and treatment of GISTs, we propose a molecular-based classification of GIST. Recent studies of familial GIST, pediatric GIST, and variant forms of GIST related to Carney's triad and neurofibromatosis type 1 are discussed in relationship to this molecular classification. In addition, the role of mutation screening in KIT and PDGFRA as a diagnostic and prognostic aid is emphasized in this review.
The molecular changes associated with the transition of melanoma cells from radial growth phase (RGP) to vertical growth phase (VGP, metastatic phenotype) are not yet well defined. We have demonstrated that the progression of human melanoma is associated with loss of expression of the transcription factor AP-2. In metastatic melanoma cells, this loss resulted in overexpression of MCAM/MUC18, MMP-2, the thrombin receptor (PAR-1), and lack of c-KIT expression. The transition from RGP to VGP is also associated with overexpression of the angiogenic factor IL-8. Additionally, the transition of melanoma cells from RGP to VGP is associated with overexpression of the transcription factors CREB and ATF-1, both of which may act as survival factors for human melanoma cells. Inactivation of CREB/ATF-1 activities in metastatic melanoma cells by dominant-negative CREB or by anti-ATF-1 single chain antibody fragment (ScFv), resulted in deregulation of MMP-2 and MCAM/MUC18, increased the sensitivity of melanoma cells to apoptosis, and inhibition of their tumorigenicity and metastatic potential in vivo. In this prospect article, we summarize our data on the role of AP-2 and CREB/ATF-1 in the progression of human melanoma and report on the development of new fully human antibodies anti-MCAM/MUC18 and anti-IL-8 which could serve as new modalities for the treatment of melanoma.
KIT is a target for imatinib mesylate (Gleevec; Novartis Pharma, Basel, Switzerland). Gastrointestinal stromal tumors (GISTs) express KIT and respond favorably to imatinib therapy. To determine other tumors in which such a molecular targeted therapy might be indicated, we investigated KIT expression in different human tumor types. Because recent studies in GISTs suggest that KIT-activating mutations predict response to imatinib therapy, we also sequenced a subset of positive tumors.
More than 3,000 tumors from more than 120 different tumor categories were analyzed by immunohistochemistry in a tissue microarray format. Seven commercially available anti-KIT antibodies were initially evaluated. The antibody A4502 (DAKO) was selected for analysis because of a high frequency of positivity in GIST and low staining background in other tissues. To determine the frequency of KIT mutations in various tumor types, the exons 2, 8, 9, 11, 13, and 17 (where mutations previously were reported) were sequenced in 36 tumors with strong KIT expression.
KIT positivity was detected in 28 of 28 GISTs (100%), 42 of 50 seminomas (84%), 34 of 52 adenoid-cystic carcinomas (65%), 14 of 39 malignant melanomas (35%), and eight of 47 large-cell carcinomas of the lung (17%), as well as in 47 additional tumor types. KIT mutations were found in six of 12 analyzed GISTs, but only in one of 24 other tumors.
The results suggest that KIT expression occurs infrequently in most tumor types and that, with the exception of GISTs, KIT gene mutations are rare in immunohistochemically KIT-positive tumors.
Recently, it was reported that BRAF mutations are frequent in melanoma. Previously, we analyzed a large series of paired primary and metastatic melanomas for NRAS codon 61 mutations and showed that they arise early and are preserved during tumor progression. Here, we have screened the same tumor samples for BRAF mutations.
Primary melanomas (n = 71) and corresponding metastases (n = 88) from 71 patients were screened for BRAF exon 11 and exon 15 mutations using single-strand conformational polymorphism and nucleotide sequence analysis
BRAF mutations were found in 42 of 71 patients (59%). Thirty-seven patients had mutations that lead to a Val599Glu change, whereas mutations resulting in Gly468Ser, Val599Arg, Val599Lys, and Lys600Glu changes were detected in one patient each. Furthermore, one patient had a 6-bp insertion between codons 598 and 599, encoding two threonine residues. In most cases, paired primary and metastatic lesions had the same BRAF genotype (i.e., mutations present in the primary tumors were preserved in the corresponding metastases, and mutations did not arise at the metastatic stage if they were not present in the primary lesion). Using laser-capture microdissection, BRAF mutations were found in the radial growth phase of the primary lesions. BRAF mutations occurred exclusively in tumors that were wild type for NRAS, and in total, 89% of the patients analyzed (63 of 71) had mutations in either of these two genes.
The Ras-Raf-mitogen-activated protein kinase/extracellular signal-regulated kinase-extracellular signal-regulated kinase signaling pathway is activated in the vast majority of melanomas. Activation occurs through either NRAS or BRAF mutations, both of which arise early during melanoma pathogenesis and are preserved throughout tumor progression.
EGF receptor gene mutations are common in lung cancers from bnever smokersQ and are associated with sensitivity of tumors to gefitinib and erlotinib
- W Pao
- V Miller
- M Zakowski
Pao W, Miller V, Zakowski M, et al. EGF receptor gene mutations are
common in lung cancers from bnever smokersQ and are associated
with sensitivity of tumors to gefitinib and erlotinib. Proc Natl Acad
Sci U S A 2004;101:13306 -11.
Proto-oncogene c-kit expression in malignant melanoma: protein loss with tumor progres-sion
- Montone
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Montone KT, van Bell P, Elenitsas R, Elder DE. Proto-oncogene c-kit expression in malignant melanoma: protein loss with tumor progres-sion. Mod Pathol 1997;10:939 -44.
Hematologic and cytogenetic responses to imatinib mesylate in chronic myelogenous leukemia
- Kantarjian
Kantarjian H, Sawyers C, Hochhaus A, et al. Hematologic and cytogenetic responses to imatinib mesylate in chronic myelogenous leukemia. New Engl J Med 2002;346:645 -52.
BRAF as a potential therapeutic target in melanoma and other malignancies
- Da Tuveson
- Bl Weber
- M Herlyn
Tuveson DA, Weber BL, Herlyn M. BRAF as a potential therapeutic
target in melanoma and other malignancies. Cancer Cell 2003;4:95 -8.
BRAF as a potential therapeutic target in melanoma and other malignancies
- Tuveson
EGF receptor gene mutations are common in lung cancers from “never smokers” and are associated with sensitivity of tumors to gefitinib and erlotinib
- Pao
Detection of c-kit–activating mutations in gastrointestinal stromal tumors by high resolution amplicon melting analysis
- Willmore
Progression of human cutaneous melanoma is associated with loss of expression of c-kit proto-oncogene receptor
- Natali