Oncogenic KIT mutations in different exons lead to specific changes in melanocyte phospho-proteome.

UNLABELLED: Mutations in the proto-oncogene c-KIT (KIT) are found in several cancers, and the site of these mutations differs markedly between cancer types. We used site directed mutagenesis to induce KIT(559), KIT(642) and KIT(816) mutations in primary human melanocytes (PHM) and we investigated the impact of each mutation on KIT function. We studied canonical KIT-signaling pathways by immunoblotting, and we used stable isotope labeling by amino acids in cell culture (SILAC) and kinase prediction models to identify kinases differently activated in respective mutants. We validated our results with the analysis of phosphorylation levels of selected substrates for each kinase. We concluded that CK1 ε and δ are more active in cell clones harboring KIT(559) and KIT(642) mutations, whereas PAK4 is more active in clones with KIT(816) mutation. Our findings might help to develop further therapeutic options for tumors with specific KIT mutations in different domains. BIOLOGICAL SIGNIFICANCE: Different types of cancers harbor mutations in the oncogene KIT. The use of small molecules inhibitors directly targeting KIT had a limited success in the treatment of patients with KIT mutant cancers. Our study describes specific phospho-proteome changes due to different KIT mutations, and provides targets of further therapeutic options.

Combined Inhibition of MEK and Plk1 Has Synergistic Antitumor Activity in NRAS Mutant Melanoma.

About one-third of cancers harbor activating mutations in rat sarcoma viral oncogene homolog (RAS) oncogenes. In melanoma, aberrant neuroblastoma-RAS (NRAS) signaling fuels tumor progression in about 20% of patients. Current therapeutics for NRAS-driven malignancies barely affect overall survival. To date, pathway interference downstream of mutant NRAS seems to be the most promising approach. In this study, data revealed that mutant NRAS induced Polo-like kinase 1 (Plk1) expression, and pharmacologic inhibition of Plk1 stabilized the size of NRAS mutant melanoma xenografts. The combination of mitogen-activated protein kinase/extracellular signal-regulated kinase kinase (MEK) and Plk1 inhibitors resulted in a significant growth reduction of NRAS mutant melanoma cells in vitro, and regression of xenografted NRAS mutant melanoma in vivo. Independent cell cycle arrest and increased induction of apoptosis underlies the synergistic effect of this combination. Data further suggest that the p53 signaling pathway is of key importance to the observed therapeutic efficacy. This study provides in vitro, in vivo, and first mechanistic data that an MEK/Plk1 inhibitor combination might be a promising treatment approach for patients with NRAS-driven melanoma. As mutant NRAS signaling is similar across different malignancies, this inhibitor combination could also offer a previously unreported treatment modality for NRAS mutant tumors of other cell origins.

The combination of axitinib followed by paclitaxel/carboplatin yields extended survival in advanced BRAF wild-type melanoma: results of a clinical/correlative prospective phase II clinical trial.

BACKGROUND: Simultaneous chemotherapy with vascular endothelial growth factor (VEGF) inhibition has not shown additional benefit over chemotherapy alone in advanced melanoma. We tested administration of the potent VEGF inhibitor axitinib followed by paclitaxel/carboplatin to determine whether enhanced tumour proliferation during axitinib withdrawal leads to sustained chemosensitivity. METHODS: We conducted a prospective phase II trial in metastatic melanoma patients with ECOG performance status 0-1 and normal organ function. Axitinib 5 mg PO b.i.d. was taken on days 1-14 of each 21-day treatment cycle, and carboplatin (AUC=5) with paclitaxel (175 mg m(-2)) was administered on day 1 starting with cycle 2. 3'-Deoxy-3'-(18)F-fluorothymidine ((18)F-FLT)-PET scans were performed in five patients to assess tumour proliferation on days 1, 14, 17, and 20 of cycle 1. Molecular profiling for BRAF was performed for all patients with cutaneous, acral, or mucosal melanoma. RESULTS: The treatment was well tolerated. The most common grade 3 AEs were hypertension, neutropenia, and anaemia. Grade 4 non-haematologic AEs were not observed. Four of five patients completing (18)F-FLT-PET scans showed increases (23-92%) in SUV values during the axitinib holiday. Of 36 evaluable patients, there were 8 confirmed PRs by Response Evaluation Criteria in Solid Tumors. Overall, 20 patients had SD and 8 had PD as the best response. The median PFS was 8.7 months and the median overall survival was 14.0 months. Five BRAF(V600E/K) patients had significantly worse PFS than patients without these mutations. CONCLUSIONS: Axitinib followed by carboplatin and paclitaxel was well tolerated and effective in BRAF wild-type metastatic melanoma. 3'-Deoxy-3'-(18)F-fluorothymidine-PET scans showed increased proliferation during axitinib withdrawal.

Merkel cell carcinoma: mitoses, expression of Ki-67 and bcl-2 correlate with disease progression.

BACKGROUND: There are conflicting data on markers of disease progression and outcome of Merkel cell carcinoma. OBJECTIVE: We suggest to review histological and various immunohistochemical features of Merkel cell carcinoma specimens, in order to identify prognostic markers of clinical relevance. METHODS: We collected paraffin-embedded blocks from primary tumours from 26 patients diagnosed with Merkel cell carcinoma and determined the following: type and size of the tumour, number of mitoses, proliferation rate (Ki-67 antibody), (anti)-apoptosis rate (bcl-2, p53, p63 antibodies) and lymphatic vessel invasion (D2-40 antibody for podoplanin). Two authors blinded to clinical outcome, independently assessed and scored all samples. The findings were correlated with tumour progression, which was determined by local recurrence, lymph node- or distant metastases. RESULTS: During the average follow-up period of 63.4 months 12 (46%) patients had disease progression. Statistical analysis revealed Ki-67-staining (P = 0.005) as a marker of disease progression, high number of mitoses (P = 0.026) correlated with lymph node metastasis, while a tendency for increased Bcl-2 expression (P = 0.064) was found in patients with local recurrence. A higher number of invaded lymphatic capillaries showed a tendency in correlation with metastases (P = 0.072). CONCLUSION: The findings indicate that high numbers of mitoses, proliferation and survival of tumour cells as marked by Ki-67- and Bcl-2-staining, and infiltration of lymphatic vessels, might correlate with the biological behaviour of Merkel cell carcinoma.

Mycoplasma pneumoniae-associated mucositis--case report and systematic review of literature.

BACKGROUND: Mycoplasma pneumoniae, a bacterium known to be a common cause of pneumonia, has been documented to cause complications such as debilitating mucositis previously described as an atypical Stevens-Johnson syndrome without skin lesions. However, in the spectrum of epidermal dermatopathies, the condition is increasingly recognized as a separate entity, now termed M. pneumoniae-associated mucositis (MPAM). OBJECTIVES: We present a case of MPAM and systemically review the literature to discuss diagnostic and therapeutic options. METHODS: A systematic literature search was performed to find studies reporting MPAM in adults. We extracted and analysed patient demographics, disease symptomatology, diagnostic testing and treatment. RESULTS: Eleven articles, describing 12 patients and our own patient met the predefined criteria and were analysed. Respiratory, ocular and oral symptoms were present in all patients. Therapies predominantly included antibiotics (10 of 13) and immunosuppressive treatment (9 of 13) leading to complete resolution of symptoms in all patients. CONCLUSION: Our findings highlight that MPAM should be recognized as a distinct disease entity within the spectrum of epidermal dermatopathies. We discuss and show in our patient why M. pneumoniae IgA serum levels could prove to be more reliable diagnostic tools in the MPAM diagnosis than the widely used IgG and IgM titre levels.

Low-dose inhalation of interleukin-2 bio-chemotherapy for the treatment of pulmonary metastases in melanoma patients.

BACKGROUND: Interleukin-2 (IL-2) treatment for patients with metastatic melanoma has shown remarkable durable responses. Systemic administration of IL-2 may cause severe side effects, whereas local administration is considered to be a safe alternative. The lungs are common sites of metastases in melanoma patients causing considerable respiratory problems. We sought to evaluate the potential antitumoral effect of a low-dose inhalative IL-2 (lh-IL-2) regimen for patients with melanoma lung metastases. In addition, we explored the prophylactic potential of Ih-IL-2 after surgical removal of lung metastases in a study carried out in an outpatient setting. METHODS: Twenty patients with American Joint Committee on Cancer stage-IV (M1b and M1c) melanoma were enrolled in this study and treated with 3 × 3 million IU inhalative IL-2 q.d. together with monthly dacarbazine bolus injections. Five patients received lh-IL-2 after surgical resection of lung metastases to prevent recurrence of the disease (prophylaxis group, N=5). All other patients were enrolled in the treatment group (N=15). Clinical evaluations were carried out monthly and radiological follow-up was performed every third month. RESULTS: Nine patients in the treatment group had a clinical benefit with partial regression (27%) or stable disease (33%). Four patients had progression of lung metastases (26.7%) and two patients were not evaluable (13.3%). In the prophylaxis group, none of the patients developed new lung metastases during lh-IL-2 therapy. The median follow-up period was 7.8 months in the treatment group and 25.7 months in the prophylaxis group. In the majority of patients, treatment was well tolerated. CONCLUSIONS: Low-dose IL-2 inhalation might offer an effective and safe treatment option for lung metastases in melanoma patients. In addition, lh-IL-2 may have a prophylactic potential to prevent recurrence in the lungs after pulmonary melanoma metastasectomy. Administration can easily be performed in an outpatient setting, thus offering an attractive treatment option.

Sustainable correction of junctional epidermolysis bullosa via transposon-mediated nonviral gene transfer.

Sustainable correction of severe human genetic disorders of self-renewing tissues, such as the blistering skin disease junctional epidermolysis bullosa (JEB), is facilitated by stable genomic integration of therapeutic genes into somatic tissue stem cells. While integrating viral vectors can achieve this, they suffer from logistical and biosafety concerns. To circumvent these limitations, we used the Sleeping Beauty transposable element to integrate the LAMB3 cDNA into genomes of epidermal holoclones from six unrelated JEB patients. These cells regenerate human JEB skin that is normalized at the level of laminin 5 protein expression, hemidesmosome formation and blistering. Transposon-mediated gene delivery therefore affords an opportunity for stable gene delivery in JEB and other human diseases.

[New immunosuppressive agents for treating psoriasis]. / Neue Immunsuppressiva in der Therapie der Psoriasis.

Accumulative evidence suggests that psoriasis may be a genetically determined immunologenic inflammatory disorder based on an ongoing autoreactive Th-1 response. Various cytokines (e.g. IL-2, interferon-gamma etc.) are released and exert proliferative signals on to keratinocytes, which start proliferation that finally results in an incomplete differentiation. During this pathobiological process keratinocytes themselves express receptors that make them sensitive for growth inducing stimulation and also start the production of a set of cytokines that contribute to and maintain inflammation. Immunosuppressive agents, mostly by affecting T-cells may interfere with or even disrupt by rather unspecific mechanisms, this complex process of mutual stimulation of leucocytes and keratinocytes. In this manuscript we show mode of action, efficacy and side effects of Methotrexate, Ciclosporin A, Tacrolimus and Pimecrolimus, and discuss therapeutic options with mycophenolate mofetil and fumaric acid esters.

Pathogenesis of the permeability barrier abnormality in epidermolytic hyperkeratosis.

Epidermolytic hyperkeratosis is a dominantly inherited ichthyosis, frequently associated with mutations in keratin 1 or 10 that result in disruption of the keratin filament cytoskeleton leading to keratinocyte fragility. In addition to blistering and a severe disorder of cornification, patients typically display an abnormality in permeability barrier function. The nature and pathogenesis of the barrier abnormality in epidermolytic hyperkeratosis are unknown, however. We assessed here, first, baseline transepidermal water loss and barrier recovery kinetics in patients with epidermolytic hyperkeratosis. Whereas baseline transepidermal water loss rates were elevated by approximately 3-fold, recovery rates were faster in epidermolytic hyperkeratosis than in age-matched controls. Electron microscopy showed no defect in either the cornified envelope or the adjacent cornified-bound lipid envelope, i.e., a corneocyte scaffold abnormality does not explain the barrier abnormality. Using the water-soluble tracer, colloidal lanthanum, there was no evidence of tracer accumulation in corneocytes, despite the fragility of nucleated keratinocytes. Instead, tracer, which was excluded in normal skin, moved through the extracellular stratum corneum domains. Increasing intercellular permeability correlated with decreased quantities and defective organization of extracellular lamellar bilayers. The decreased lamellar material, in turn, could be attributed to incompletely secreted lamellar bodies within granular cells, demonstrable not only by several morphologic findings, but also by decreased delivery of a lamellar body content marker, acid lipase, to the stratum corneum interstices. Yet, after acute barrier disruption a rapid release of preformed lamellar body contents was observed together with increased organelle contents in the extracellular spaces, accounting for the accelerated recovery kinetics in epidermolytic hyperkeratosis. Accelerated recovery, in turn, correlated with a restoration in calcium in outer stratum granulosum cells in epidermolytic hyperkeratosis after barrier disruption. Thus, the baseline permeability barrier abnormality in epidermolytic hyperkeratosis can be attributed to abnormal lamellar body secretion, rather than to corneocyte fragility or an abnormal cornified envelope/cornified-bound lipid envelope scaffold, a defect that can be overcome by external applications of stimuli for barrier repair.

A localized variant of paraneoplastic pemphigus: acantholysis associated with malignant melanoma.

We report a 72-year-old male patient with a nodular malignant melanoma that was associated with focal suprabasal acantholysis (FSA). This phenomenon, which is regarded as an incidental finding by dermatopathologists, may be associated with inflammatory and also neoplastic skin diseases. Haematoxylin and eosin stained sections from an erythematous plaque surrounding the patient's tumour showed FSA, direct immunofluorescence (DIF) and indirect immunofluorescence (IIF) on normal human skin, monkey oesophagus and rat urinary bladder were negative. On electron microscopy few desmosomes could be detected in the basal cell layer of the acantholytic areas and there was a nearly complete loss of these structures in the spinous cell layer. Only remnants of cytoplasmic plaques and keratin filaments could be observed in those areas. In contrast, adherens junctions appeared to be well preserved. An enzyme-linked immunosorbent assay (ELISA) using recombinant fusion proteins as antigens did not show circulating autoantibodies against desmoglein 1 (Dsg1) or desmoglein 3 (Dsg3). In contrast, immunoblotting revealed autoantibodies directed against keratinocyte antigens with a molecular weight of 85 kDa and 250 kDa, the first band corresponding to the molecular weight of comigrating plakoglobin. Immunoprecipitation with patient serum also revealed a 85-kDa band. We conclude that these autoantibodies, probably in conjunction with cofactors produced by the tumour, could play a part in the pathogenesis of this variant of FSA, for which we propose the term 'localized paraneoplastic pemphigus.'