Expression of AID in malignant melanoma with BRAFV600E mutation

BRAF‐activating somatic mutations often exist in malignant melanoma. The underlying molecular mechanism of somatic BRAF mutation inductions remained to be clear. Activation‐induced cytidine deaminase (AID), a member of a cytidine deaminase family, and APOBEC3B induce somatic mutations and recently have been indicated to be involved in the pathomechanism of several kinds of cancers. The aim of this study was to explore the expression level of AID and APOBEC3B in BRAF‐mutation‐ containing malignant melanoma. Immunohistochemical study demonstrated that 9 of 10 malignant melanomas with high AID expression had BRAF^V600E mutation. Eight of them developed multiorgan metastases or multiple lymph node metastases afterwards. Although the size of the patient panel was small, the results indicate that there might be an association between AID expression and BRAF mutation in melanoma.     

Oncogenic BRAF signalling increases Mcl‐1 expression in cutaneous metastatic melanoma

The Bcl‐2 family member Mcl‐1 is essential for melanoma survival; however, the influence of oncogenic BRAF signalling remains elusive. In this study, Mcl‐1 splice variant expression was determined in a panel of melanoma cell lines in relation to BRAF mutational status. Mcl‐1L mRNA expression was increased in melanoma cells compared with primary melanocytes with significantly increased mRNA and protein expression observed in BRAF^V600E mutant melanoma cells. Although no change in Mcl‐1S mRNA was observed, Mcl‐1S protein expression also increased in BRAF mutant melanoma cells. Additionally, while over‐expression of mutant BRAF^V600E increased both Mcl‐1L and Mcl‐1S expression, inhibition of hyperactive BRAF signalling resulted in decreased Mcl‐1L expression. These studies suggest that the regulation of Mcl‐1 expression by BRAF signalling is increased by oncogenic activation of BRAF, revealing a mechanism of apoptotic resistance which may be overcome by the use of more specifically targeted Mcl‐1 inhibitors.     

Vemurafenib‐induced toxic epidermal necrolysis: possible cross‐reactivity with other sulfonamide compounds

Vemurafenib is a newly licensed target‐directed medication. It has been proven to improve the survival of patients with metastatic melanoma and the BRAF^V600E mutation; however, adverse cutaneous reactions are frequent. Few cases of life‐threatening severe cutaneous adverse reactions (SCARs) induced by vemurafenib have been reported. Dabrafenib, another selective BRAF inhibitor, has been licensed recently as an alternative drug with the same indications. From a molecular point of view, both vemurafenib and dabrafenib contain a sulfonamide group; cross‐reactivity to sulfonamide compounds has been reported in allergic patients. We report on a patient with vemurafenib‐induced toxic epidermal necrolysis (TEN). In vitro analysis of lymphocyte reactivity to vemurafenib showed positive results, confirming drug causality. In addition, lymphocytes from the patient reacted to dabrafenib and to the antibiotic sulfonamide drug sulfamethoxazole. Moreover, lymphocytes from two patients with cutaneous adverse reactions to sulfamethoxazole also reacted to vemurafenib and dabrafenib in vitro. These data strongly suggest that there might be clinical cross‐reactivity between BRAF inhibitors and sulfonamides in some patients. Thus, precautions should be taken to avoid sulfonamide drugs as much as possible in patients showing serious hypersensitivity reactions to vemurafenib and vice versa.     

MEK inhibitor cobimetinib rescues a dRaf mutant lethal phenotype in Drosophila melanogaster

Since Drosophila melanogaster has proven to be a useful model system to study phenotypes of oncogenic mutations and to identify new anti‐cancer drugs, we generated human BRAF^V600E homologous dRaf mutant (dRaf^A572E) Drosophila melanogaster strains to use these for characterisation of mutant phenotypes and exploit these phenotypes for drug testing. For mutant gene expression, the GAL4/UAS expression system was used. dRaf^A572E was expressed tissue‐specific in the eye, epidermis, heart, wings, secretory glands and in the whole animal. Expression of dRaf ^A572E under the control of an eye‐specific driver led to semi‐lethality and a rough eye phenotype. The vast majority of other tissue‐specific and ubiquitous drivers led to a lethal phenotype only. The rough eye phenotype was used to test BRAF inhibitor vemurafenib and MEK1/2 inhibitor cobimetinib. There was no phenotype rescue by this treatment. However, a significant rescue of the lethal phenotype was observed under a gut‐specific driver. Here, MEK1/2 inhibitor cobimetinib rescued Drosophila larvae to reach pupal stage in 37% of cases as compared to 1% in control experiments. Taken together, the BRAF^V600E homolog dRaf ^A572E exerts mostly lethal effects in Drosophila. Gut‐specific dRaf ^A572E expression might in future be developed further for drug testing.     

BRAF mutation testing algorithm for vemurafenib treatment in melanoma: recommendations from an expert panel

The incidence of melanoma has increased rapidly over the past 30 years, and the disease is now the sixth most common cancer among men and women in the U.K. Many patients are diagnosed with or develop metastatic disease, and survival is substantially reduced in these patients. Mutations in the BRAF gene have been identified as key drivers of melanoma cells and are found in around 50% of cutaneous melanomas. Vemurafenib (Zelboraf^®; Roche Molecular Systems Inc., Pleasanton, CA, U.S.A.) is the first licensed inhibitor of mutated BRAF, and offers a new first‐line option for patients with unresectable or metastatic melanoma who harbour BRAF mutations. Vemurafenib was developed in conjunction with a companion diagnostic, the cobas^® 4800 BRAF V600 Mutation Test. The purpose of this paper is to make evidence‐based recommendations to facilitate the implementation of BRAF mutation testing and targeted therapy in patients with metastatic melanoma in the U.K. The recommendations are the result of a meeting of an expert panel and have been reviewed by melanoma specialists and representatives of the National Cancer Research Network Clinical Study Group on behalf of the wider melanoma community. This article is intended to be a starting point for practical advice and recommendations, which will no doubt be updated as we gain further experience in personalizing therapy for patients with melanoma.     

BRAF V600E mutation as a prognostic factor in cutaneous melanoma patients

The prognostic significance of BRAF mutations in the natural course of melanoma is controversial. The aim of study was to assess the prognostic significance of BRAF V600E mutation in cutaneous melanoma patients. A total of 151 melanomas were included in the study. BRAF V600E mutation was detected using the real‐time PCR. BRAF V600E mutation rate was 51%. BRAF mutation rate was higher for young patients (61.4%) and upper limbs (63.2%), trunk (59.3%) and head and neck (59.2%) were the most frequently afflicted sites in BRAF‐mutant patients, whereas lower limbs were mostly affected in BRAF‐wild patients (77.8%). Likewise, acral melanomas rarely harbored BRAF mutation (17.1%). The disease‐free survivals regarding the entire and Stage III cohorts were longer in the BRAF‐mutant group than in the BRAF‐wild group (p = .006 and p = .004, respectively), whereas Stage I–II patients had no survival differences between BRAF statuses (p = .2). Likewise, BRAF‐mutant patients had better overall survival (OS) time compared to BRAF‐wild patients in all stages (p = .01), in Stage III (p = .01), and in Stage IV patients (p = .001). However, no differences between BRAF statuses were observed in Stage I–II melanomas (p = .3). In conclusion, BRAF V600E‐mutant melanomas show favorable prognostic impact on both disease‐free and OSs in all staged melanomas except local disease.     

BRAF inhibitor‐associated cutaneous squamous cell carcinoma: new mechanistic insight,emerging evidence for viral involvement and perspectives on clinical management

Mutations in the BRAF proto‐oncogene occur in the majority of cutaneous melanomas. The commonly detected valine (V) to glutamate (E) mutation (V600E) is known to drive melanomagenesis and has thus been the target of two highly selective chemotherapeutic agents: vemurafenib and dabrafenib. While BRAF inhibitor therapy has revolutionized the treatment of metastatic melanoma, unanticipated cutaneous toxicities, including the development of cutaneous squamous cell carcinomas (cSCCs), are frequently reported and hinder therapeutic durability. However, the mechanisms by which BRAF inhibitors induce cutaneous neoplasms are poorly understood, thus posing a challenge for specific therapies. In this review, we summarize the clinical and molecular profiles of BRAF inhibitor‐associated cSCCs, with a focus on factors that may contribute to disease pathogenesis. In particular, we discuss the emerging evidence pointing towards viral involvement in BRAF inhibitor‐induced cutaneous neoplasms and offer new perspectives on future therapeutic interventions. Continued clinical and mechanistic studies along this line will not only allow for better understanding of the pathogenic progression of BRAF inhibitor‐induced cSCCs, but will also lead to development of new therapeutic and preventative options for patients receiving targeted cancer therapy.     

Vemurafenib

Oral vemurafenib (Zelboraf^®) is a first-in-class, small molecule BRAF^V600E inhibitor indicated for the treatment of unresectable or metastatic melanoma in BRAF ^V600 mutation-positive patients (EU) or BRAF ^V600E mutation-positive patients (USA). Compared with intravenous dacarbazine, vemurafenib significantly improved overall survival and progression-free survival in patients with unresectable, previously untreated, BRAF ^V600E mutation-positive, stage IIIC or IV melanoma. Oral vemurafenib was generally well tolerated, with cutaneous adverse events among the most commonly occurring adverse events.     

A costly revolution for a subgroup of patients with metastatic melanoma

ORIGINAL ARTICLE: Hauschild A, Grob JJ, Demidov LV et al. Dabrafenib in BRAF‐mutated metastatic melanoma: a multicentre, open‐label, phase 3 randomised controlled trial. Lancet 2012; 380: 358–65. Background Dabrafenib, an inhibitor of mutated BRAF, has clinical activity with a manageable safety profile in phase I and II studies in patients with BRAF (V600)‐mutated metastatic melanoma. Hauschild et al. aimed to assess the efficacy of dabrafenib in a phase III trial of patients with BRAF (V600)‐mutated metastatic melanoma. Methods Patients were enrolled into a phase III trial between December 2010 and September 2011. This report is based on the cut‐off date of 19 December 2011. Patients with previously untreated stage IV or unresectable stage III BRAF (V600)‐mutated melanoma were randomly assigned (3:1) to receive dabrafenib (150 mg twice daily, orally) or dacarbazine (1000 mg m^?2 intravenously every 3 weeks). Patients were stratified according to American Joint Committee on Cancer stage. The primary endpoint was investigator‐assessed progression‐free survival (PFS) and was analysed by intention to treat. Safety was assessed per protocol. Findings Of the 733 patients screened, 250 were randomly assigned to receive either dabrafenib (187 patients) or dacarbazine (63 patients). Median PFS was 5·1 months for dabrafenib and 2·7 months for dacarbazine, with a hazard ratio of 0·30 (95% confidence interval 0·18–0·51, P < 0·0001). At cut‐off, 107 (57%) patients in the dabrafenib group and 14 (22%) in the dacarbazine group remained on randomized treatment. Treatment‐related adverse events (grade 2 or higher) occurred in 100 (53%) of the 187 patients who received dabrafenib and in 26 (44%) of the 59 patients who received dacarbazine. The most common adverse events with dabrafenib were skin‐related toxic effects, fever, fatigue, arthralgia and headache. The most common adverse events with dacarbazine were nausea, vomiting, neutropenia, fatigue and asthenia. Grade 3–4 adverse effects were uncommon in both groups. Interpretation Dabrafenib significantly improved PFS compared with dacarbazine.     

Vemurafenib‐induced hyperkeratosis of the areola treated with topical adapelene

We present a rare condition, hyperkeratosis of the areola, induced by vemurafenib. Only a few papers have described an association of BRAF inhibitors with hyperkeratosis of the areola and/or nipple. Vemurafenib is a selective BRAF inhibitor used in patients with unresectable or metastatic melanoma who are positive for the V600 mutation. This drug has been associated with numerous cutaneous side effects, both benign and malignant. We report a male patient with vemurafenib‐induced hyperkeratosis of the areola managed successfully with a topical retinoid, and describe for the first time a treatment for this side effect.     

Langerhans cell histiocytosis and Erdheim‐Chester disease,both with cutaneous presentations,and papillary thyroid carcinoma all harboring the BRAFV600E mutation

Langerhans cell histocytosis (LCH) and Erdheim‐Chester disease are two rare histiocytic disorders. Their occurrence in the same patient is more infrequent, but has been described. We report a case of a 38‐year‐old woman who presented with a diagnosis of single system cutaneous LCH. Subsequently, she developed multiple papules on her extremities consistent with a non‐LCH xanthogranuloma type lesion. BRAF^V600E mutation immunostain, VE1 was positive in the skin lesion, which was confirmed by molecular polymerase chain reaction (PCR) studies, initiating a complete systemic workup for Erdheim‐Chester disease. Systemic involvement was confirmed with bilateral sclerotic bone lesions and retroperitoneal and pelvic fibrosing disease. She was also found to have a BRAF^V600E mutation positive papillary thyroid carcinoma. New suspicious cutaneous lesions presenting in patients with a history of LCH need to be biopsied. A BRAF^V600E mutation in a non‐LCH histiocytic lesion with a xanthogranuloma phenotype (CD163/CD68/CD14/fascin/Factor 13a) should prompt an Erdheim‐Chester disease workup. This is a unique case of a woman with BRAF^V600E mutation positive Erdheim‐Chester disease and cutaneous LCH, while also being, to our knowledge, the first reported case in the English literature of it occurring in a patient with a BRAF^V600E mutation positive papillary thyroid carcinoma.     

Beyond the BRAFV600E hotspot: biology and clinical implications of rare BRAF gene mutations in melanoma patients

BRAF gene mutations can be found in approximately 50% of melanomas, but the most common BRAF mutation leads to substitution at residue 600 of the protein, from valine to glutamic acid. BRAF^V600E occurs in up to 95% of all melanoma cases and can be successfully blocked by using a combination of BRAF‐ and MEK inhibitors. The wider availability of next‐generation sequencing is revealing more non‐V600 BRAF mutations, and the clinical implications of these mutations are widely unknown. In this review, we will discuss the biology of the MAPK pathway and the different types of BRAF mutations as well as their effect on MEK activation. Current literature will be reviewed including in vitro data, case reports and case series.     

Aktuelle Fortschritte und Ausblicke in der Therapie des metastasierten Melanoms

Melanoma has long been considered as an extremely therapy‐resistant tumour. Recent developments in the area of immunotherapy as well as targeted therapy showed rapid development and excellent results. The anti‐CTLA‐4 antibody ipilimumab, which was approved in the USA and Europe in 2011, was the first substance in melanoma therapy to demonstrate an overall survival benefit. Another approval is expected in Europe for the specific BRAF‐inhibitor vemurafenib, which has shown a significant impact on progression‐free survival and overall survival in patients with the BRAF^V600E mutation. In this review the relevant agents in the substance classes of immunomodulatory drugs and small molecules are presented and discussed, and future prospects for combination therapies and developments in melanoma treatment are outlined.     

A comparative study of the cutaneous side effects between BRAF monotherapy and BRAF/MEK inhibitor combination therapy in patients with advanced melanoma: a single-centre experience

Background

Patients with advanced melanoma have a poor prognosis. Since the discovery of BRAF mutations in cutaneous melanoma, new pharmacological agents have been developed to inhibit this target. Although the survival of patients with advanced melanoma has improved with BRAF inhibitors, the emergence of drug resistance and the high incidence of cutaneous side effects represent important limitations.

Objectives

The aim of our study was to compare the incidence of cutaneous side effects between BRAF inhibitor monotherapy and BRAF and MEK inhibitor combination therapy in our cohort of patients.

Materials & methods

This studywas a longitudinal prospective observational study. The study population comprised 83 patients with advanced cutaneous melanoma presenting with BRAF V600 mutation. The inclusion criteria included: age above 18 years, metastatic cutaneous melanoma or melanoma with high risk of metastasis, the presence of BRAF V600 mutation, and treatment withBRAFinhibitors or a combination ofBRAF and MEK inhibitors.

Results

The majority of patients developed skin toxicity during treatment. The most common cutaneous side effects were localized hyperkeratosis and verrucous keratosis. Other cutaneous side effects observed were photosensitivity, squamous cell carcinoma, and keratoacanthoma.

Conclusion

Our results indicate that cutaneous side effects are generally observed during BRAF inhibitor monotherapy and are significantly different from those observed in patients treated with combination therapy.

     

There is a world beyond protein mutations: the role of non‐coding RNAs in melanomagenesis

Until recently, the general perception has been that mutations in protein‐coding genes are responsible for tumorigenesis. With the discovery of ^V600EBRAF in about 50% of cutaneous melanomas, there was an increased effort to find additional mutations. However, mutations characterized in melanoma to date cannot account for the development of all melanomas. With the discovery of microRNAs as important players in melanomagenesis, protein mutations are no longer considered the sole drivers of tumors. Recent research findings have expanded the view for tumor initiation and progression to additional non‐coding RNAs. The data suggest that tumorigenesis is likely an interplay between mutated proteins and deregulation of non‐coding RNAs in the cell with an additional role of the tumor environment. With the exception of microRNAs, our knowledge of the role of non‐coding RNAs in melanoma is in its infancy. Using few examples, we will summarize some of the roles of non‐coding RNAs in tumorigenesis. Thus, there is a whole world beyond protein‐coding sequences and microRNAs, which can cause melanoma.     

Congenital self‐healing reticulohistiocytosis with BRAF V600E mutation in an infant

Congenital self‐healing reticulohistiocytosis (CSHR) is a rare disorder characterized by benign skin lesions with a tendency to self‐heal. Multiple skin lesions are usually present in CSHR. It is very difficult to distinguish between CSHR and an invasive Langerhans cell histiocytosis. We present a case of a 5‐month‐old infant girl who had hypopigmented skin lesions distributed over her neck, thorax and torso. The skin lesions regressed spontaneously 2 months after the diagnosis of CSHR and the child has remained in complete remission without any sign of recurrence over a 2‐year follow‐up. BRAF V600E mutation was detected in lesional cells along with a low Ki‐67 proliferative activity of about 6%. BRAF oncogene‐induced senescence might contribute to a mechanism of self‐regression in CSHR; however, the exact role of the somatic BRAF V600E mutation in CSHR remains to be determined.     

Human papillomavirus evaluation of vemurafenib‐induced skin epithelial tumors: a case series

Erdheim–Chester disease (ECD) is a rare non‐Langerhans cell histiocytosis defined by heterogeneous multiorgan involvement. Due to the rarity of this disease strong evidence‐based therapies have yet to be established and prognosis has previously been considered to be poor, with more than half of patients dying within 3 years of initial presentation. We describe an 86‐year‐old woman with a 34‐year history of extensive cutaneous and internal nodules with typical pathological and immunophenotypical (CD68⁺/CD1a^?) features of ECD without evidence of the BRAF V600E mutation. The cosmetic appearance of cutaneous nodules and hoarse voice caused by vocal cord nodules has been managed surgically. More aggressive therapies reported for ECD were trialled for this patient, such as radiotherapy and interferon‐α, with no response. This case demonstrates a relatively good prognosis in ECD that has been managed conservatively.     

Diffuse melanosis cutis in the setting of BRAFV600E mutant melanoma and treatment with targeted therapies

Diffuse melanosis cutis (DMC) is a rare presentation of metastatic melanoma associated with a particularly guarded prognosis. We report a case of a 35‐year‐old man with BRAF^V600E metastatic melanoma treated with dabrafenib (as well as ipilimumab and whole brain radiotherapy), who is alive, 25 months after the onset of his DMC. This is significantly longer than the reported mean survival of 4 months, highlighting the importance of BRAF mutation testing and the promising survival advantage of using targeted therapies compared with conventional chemotherapeutic regimens.     

Cutaneous Adverse Events to Type I BRAF Inhibitors: An Analysis of Effects Associated with Each Inhibitor and Therapeutic Time Interval to Onset

The treatment of malignant melanoma with inhibitors targeting the BRAF V600E mutation has demonstrated dramatic clinical and radiographic response with improved progression-free and overall survival in the majority of patients receiving treatment. However, cutaneous adverse effects—from proliferative processes to more classic drug side effects—are increasingly being reported in patients on BRAF inhibitors. In this comprehensive literature review we provide (1) an all-inclusive list of cutaneous adverse effects associated with selective class I RAF inhibitors, (2) specific adverse effects associated with each inhibitor, and (3) the therapeutic time interval associated with the onset of all reported lesion types. Twenty-two studies reporting cutaneous adverse reactions with selective class I RAF inhibitor therapy were retrieved from PubMed and sourced from relevant articles referenced by other papers. We identified over 45 differently described lesion types, corresponding to close to 2,000 cases. The most commonly reported lesion types in order of decreasing frequency include inflammatory dermatoses, benign lesions, malignant lesions, and hair/nail-related abnormalities. For the most part, the terminologies used in the original studies were retained. Case totals and time-to-lesion onset are presented for every group, and where available, for individual lesion types, by associated BRAF inhibitor.     

BRAF,KIT and NRAS mutations and expression of c‐KIT,phosphorylated extracellular signal‐regulated kinase and phosphorylated AKT in Japanese melanoma patients

To clarify the status of gene mutation and activation of growth signal in melanoma of Japanese patients in vivo, we analyzed the mutation of BRAF exon 15, NRAS exon 2, and KIT exons 9, 11, 13, 17 and 18 in melanoma cells obtained by laser capture microdissection, and performed direct sequencing in 20 cases of acral lentiginous melanoma (ALM) and 17 cases of superficial spreading melanoma (SSM). In the study of the mutation of BRAF, pyrosequencing was also done. To examine the cell proliferation signaling, immunohistochemistry for phosphorylated extracellular signal‐regulated kinase (pERK), phosphorylated AKT (phosphorylated AKT) and c‐KIT was done. The mutation of BRAF p.V600E was detected in 13 cases of ALM (65.0%) and 12 cases of SSM (70.6%). No NRAS mutation was found in all cases. The mutation in exons 9, 11, and 18 of KIT was detected in nine cases. The mutation of BRAF and KIT showed no correlation with clinical stage, lymph node metastasis, tumor thickness, ulceration and histology. pERK and pAKT was observed in small population of melanoma cells and there was no correlation with gene mutation. Our results indicate that the mutations of BRAF and KIT exist in Japanese melanoma patients, however, the cell growth signaling may be regulated by not only these mutated genes, but by other unknown regulatory factors, which may affect the prognosis of melanoma.