Peter Kurschat

Malignant Melanoma S3-Guideline "Diagnosis, Therapy and Follow-up of Melanoma"

This first German evidence-based guideline for cutaneous melanoma was developed under the auspices of the German Dermatological Society (DDG) and the Dermatologic Cooperative Oncology Group (DeCOG) and funded by the German Guideline Program in Oncology. The recommendations are based on a systematic literature search, and on the consensus of 32 medical societies, working groups and patient representatives. This guideline contains recommendations concerning diagnosis, therapy and follow-up of melanoma. The diagnosis of primary melanoma based on clinical features and dermoscopic criteria. It is confirmed by histopathologic examination after complete excision with a small margin. For the staging of melanoma, the AJCC classification of 2009 is used. The definitive excision margins are 0.5 cm for in situ melanomas, 1 cm for melanomas with up to 2 mm tumor thickness and 2 cm for thicker melanomas, they are reached in a secondary excision. From 1 mm tumor thickness, sentinel lymph node biopsy is recommended. For stages II and III, adjuvant therapy with interferon-alpha should be considered after careful analysis of the benefits and possible risks. In the stage of locoregional metastasis surgical treatment with complete lymphadenectomy is the treatment of choice. In the presence of distant metastasis mutational screening should be performed for BRAF mutation, and eventually for CKIT and NRAS mutations. In the presence of mutations in case of inoperable metastases targeted therapies should be applied. Furthermore, in addition to standard chemotherapies, new immunotherapies such as the CTLA-4 antibody ipilimumab are available. Regular follow-up examinations are recommended for a period of 10 years, with an intensified schedule for the first three years.

Identification of activated matrix metalloproteinase‐2 (MMP‐2) as the main gelatinolytic enzyme in malignant melanoma by in situ zymography

The involvement of extracellular matrix‐degrading enzymes, such as matrix metalloproteinases and serine proteases, during tumour progression and metastasis is well established. In particular, the activation of pro‐matrix metalloproteinase (MMP)‐2 on the surface of malignant cells by membrane‐bound MT1‐MMP has been shown to contribute to the invasive abilities of various tumours. This study presents evidence that in tissue of malignant melanomas, increased effective gelatinolytic activity is mainly located at sites where melanoma cells interact with the surrounding extracellular matrix. Forty‐one primary melanomas (30 superficial spreading and 11 nodular type) and six lymph node metastases were investigated by a modified technique of gelatin in situ zymography. This technique localizes areas of effective proteolytic activity within tissue sections. In 28/41 (68%) primary melanomas and in 6/6 (100%) metastases, considerable proteolysis was detected at the invading part of the tumour and especially at sites of tumour–stroma interactions, whereas no or only weak proteolytic activity was localized within the centres of solid nests of tumour cells. Zymographic analysis of extracts obtained from different areas of microdissected melanoma specimens identified activated MMP‐2 as the enzyme responsible for this activity. Immunohistochemical analysis detected strong staining for MMP‐2 and MT1‐MMP, even in areas in which no proteolytic activity was found by in situ zymography, emphasizing the importance of more functional techniques for the investigation of balanced proteolytic systems. This technology makes it possible to draw conclusions regarding the balance between activated proteases and inhibitors, which are frequently found to be present together in close proximity in vivo. Copyright

Mast cells play a protumorigenic role in primary cutaneous lymphoma

Primary cutaneous lymphomas (PCLs) are clonal T- or B-cell neoplasms, which originate in the skin. In recent years, mast cells were described as regulators of the tumor microenvironment in different human malignancies. Here, we investigated the role of mast cells in the tumor microenvironment of PCL. We found significantly increased numbers of mast cells in skin biopsies from patients with cutaneous T-cell lymphoma (CTCL) and cutaneous B-cell lymphoma (CBCL). Mast cell infiltration was particularly prominent in the periphery, at lymphoma rims. Interestingly, CTCL and CBCL patients with a progressive course showed higher mast cell counts than stable patients, and mast cell numbers in different stages of CTCL correlated positively with disease progression. In addition, mast cell numbers positively correlated with microvessel density. Incubating primary CTCL cells with mast cell supernatant, we observed enhanced proliferation and production of cytokines. In line with our in vitro experiments, in a mouse model of cutaneous lymphoma, tumor growth in mast cell-deficient transgenic mice was significantly decreased. Taken together, these experiments show that mast cells play a protumorigenic role in CTCL and CBCL. Our data provide a rationale for exploiting tumor-associated mast cells as a prognostic marker and therapeutic target in PCL.

Neuron Restrictive Silencer Factor NRSF/REST Is a Transcriptional Repressor of Neuropilin-1 and Diminishes the Ability of Semaphorin 3A to Inhibit Keratinocyte Migration

Neuropilin-1 (NRP1) is expressed by endothelial cells and neurons and serves as a receptor for both vascular endothelial growth factor (VEGF), an angiogenesis factor, and semaphorin 3A (Sema3A), a mediator of axonal guidance. We show here that NRP1 is also expressed in keratinocytes in vitro and in vivo. However, nothing has been reported about the regulation or function of keratinocyte NRP1. Using NRP1 promoter constructs in HaCaT cells, a keratinocyte cell line, we could demonstrate that a neuron restrictive silencer element (NRSE) was implicated in transcriptional repression of the NRP1 gene. Electrophoretic mobility shift assays demonstrated that the neuron restrictive silencer factor (NRSF) binds to NRSE. Overexpression of NRSF in HaCaT cells decreased NRP1 RNA and protein, whereas a dominant negative NRSF increased NRP1. Furthermore, the histone deacetylase inhibitor trichostatin A, an inhibitor of NRSF silencing activity, also increased NRP1 levels. NRP2 expression was not affected. Epidermal growth factor (EGF) and heparin-binding EGF-like growth factor (HB-EGF) strongly up-regulated NRP1 expression, concomitant with down-regulation of NRSF. Other keratinocyte mitogens such as keratinocyte growth factor (KGF) had no effect. To address function, HaCaT cells were exposed to two NRP1 ligands, VEGF165 and Sema3A. Neither had an effect on proliferation, whereas Sema3A, but not VEGF165, inhibited cell migration. Down-regulation of NRP1 by NRSF overexpression reduced Sema3A activity. It was concluded that NRSF is a transcription factor that silences NRP1 expression and thereby diminishes the Sema3A mediated inhibition of HaCaT keratinocyte migration.

Endothelial progenitor cell sprouting in spheroid cultures is resistant to inhibition by osteoblasts: A model for bone replacement grafts

Survival of tissue transplants generated in vitro is strongly limited by the slow process of graft vascularization in vivo. A method to enhance graft vascularization is to establish a primitive vascular plexus within the graft prior to transplantation. Endothelial cells (EC) cultured as multicellular spheroids within a collagen matrix form sprouts resembling angiogenesis in vitro. However, osteoblasts integrated into the graft suppress EC sprouting. This inhibition depends on direct cell–cell‐interactions and is characteristic of mature ECs isolated from preexisting vessels. In contrast, sprouting of human blood endothelial progenitor cells is not inhibited by osteoblasts, making these cells suitable for tissue engineering of pre‐vascularized bone grafts.

Site-directed mutagenesis in the B-neuropilin-2 domain selectively enhances its affinity to VEGF165, but not to semaphorin 3F.

Neuropilins (NRPs) are 130-kDa receptors that bind and respond to the class 3 semaphorin family of axon guidance molecules (SEMAs) and to members of the vascular endothelial growth factor (VEGF) family of angiogenic factors. Two NRPs have been reported so far, NRP1 and NRP2. Unlike NRP1, little is known about NRP2 interactions with its ligands, VEGF165 and SEMA3F. Cell binding studies reveal that VEGF165 and SEMA3F bind NRP2 with similar affinities, 5.2 and 3.9 nm, respectively, and are competitive NRP2 ligands. Immunoprecipitation studies show that the B (b1b2) extracellular domain of NRP2 is sufficient for VEGF165 binding, whereas SEMA3F requires both the A (a1a2) and B domains. To identify residues of B-NRP2 involved in VEGF165 binding, point mutations were introduced by site-directed mutagenesis. VEGF165 is a basic protein. Reduction of the electronegative potential of B-NRP2 by exchanging acidic residues for uncharged alanine (B-NRP2 E284A,E291A) in the 280–290 b1-NRP2 loop resulted in a 2-fold reduction in VEGF165 affinity. Conversely, enhancing the electronegative potential (B-NRP2 R287E,N290D and R287E,N290S) significantly increased VEGF165 affinity for B-NRP2 by 8- and 6.6-fold, respectively. The mutagenesis did not affect SEMA3F/B-NRP2 interactions. These results demonstrate that it is possible to alter VEGF165 affinity for NRP2 without affecting SEMA3F affinity. They also identify NRP2 residues involved in VEGF165 binding and suggest that modifications of B-NRP2 could lead to potentially high affinity selective inhibitors of VEGF165/NRP2 interactions.

Invasion of melanoma cells into dermal connective tissue in vitro: evidence for an important role of cysteine proteases.

Invasion of melanoma cells into the dermal connective tissue is a major characteristic in the complex process of metastasis. Proteases play an important role in tumor cell invasion as these enzymes are able to degrade most components of the extracellular matrix (ECM), and thus enable cells to penetrate interstitial connective tissues and basement membranes. We developed an improved culture model that allows the detailed study of melanoma cell invasion in vitro. In this model, high (BLM) or low (530) invasive melanoma cells were seeded on the dermal side of dead deepidermized dermis (DDD) and cultured for 14 days at the air/liquid interface. The high invasive cells invaded the tissue, leading to dermal tumor formation, whereas the low invasive cells did not. Analysis of the enzymatic activity of gelatinases by in situ gelatin zymography at neutral pH revealed proteolysis only in those composites cultured with high invasive melanoma cells. Interestingly, in situ zymograms performed at more acidic conditions, favoring the activity of cysteine proteases, exhibited markedly enhanced and widespread gelatinolysis compared to neutral pH. Cysteine protease inhibitors (E‐64 and leupeptin) significantly reduced invasion of melanoma cells into these composites. These results indicate an important role of cysteine proteases for tumor invasion.

Histopathological diagnostics of malignant melanoma in accordance with the recent AJCC classification 2009: Review of the literature and recommendations for general practice

Background: TNM classifications are the basis for diagnostic and therapeutic procedures in oncology. Histopathological reports have to enable a proper indexing of tumor specific findings into recent classifications.

RhoB controls coordination of adult angiogenesis and lymphangiogenesis following injury by regulating VEZF1-mediated transcription

Mechanisms governing the distinct temporal dynamics that characterize post-natal angiogenesis and lymphangiogenesis elicited by cutaneous wounds and inflammation remain unclear. RhoB, a stress-induced small GTPase, modulates cellular responses to growth factors, genotoxic stress and neoplastic transformation. Here we show, using RhoB null mice, that loss of RhoB decreases pathological angiogenesis in the ischaemic retina and reduces angiogenesis in response to cutaneous wounding, but enhances lymphangiogenesis following both dermal wounding and inflammatory challenge. We link these unique and opposing roles of RhoB in blood versus lymphatic vasculatures to the RhoB-mediated differential regulation of sprouting and proliferation in primary human blood versus lymphatic endothelial cells. We demonstrate that nuclear RhoB-GTP controls expression of distinct gene sets in each endothelial lineage by regulating VEZF1-mediated transcription. Finally, we identify a small-molecule inhibitor of VEZF1–DNA interaction that recapitulates RhoB loss in ischaemic retinopathy. Our findings establish the first intra-endothelial molecular pathway governing the phased response of angiogenesis and lymphangiogenesis following injury.

A decreased ratio between serum levels of the antagonistic angiopoietins 1 and 2 indicates tumour progression of malignant melanoma.

The growth of solid tumours like malignant melanoma depends on the ability of neoplastic cells to induce angiogenesis to ensure sufficient supply with nutrients and oxygen. The process of angiogenesis is tightly controlled by positive and negative regulators. Since many of these factors can be measured in the serum of patients, their use as tumour markers has been suggested. The angiopoietins 1 and 2 have been demonstrated to be secreted by various tumour cells. By binding to the Tie-2 receptor on endothelial cells, they regulate angiogenesis. Whereas angiopoietin-1 maintains quiescence of vessels, angiopoietin-2 increases angiogenesis by destabilising vessels and sensitising them to the effect of growth factors of the VEGF family. Since both angiopoietins compete for the same Tie-2 receptor and cause opposite effects concerning angiogenesis, the ratio between these two ligands is crucial. Therefore, we have measured serum levels of both angiopoietins in the serum of 148 melanoma patients at different stages of disease. Whereas angiopoietin-1 levels did not change during disease progression, angiopoietin-2 levels were significantly higher in advanced stage disease. Compared to the established tumour-marker S100B, angiopoietin-2 levels or the ratio between both angiopoietins did not show increased sensitivity for the early detection of advanced stages of malignant melanoma. In conclusion, the ratio between both angiopoietins is significantly altered in late stage melanoma patients, shifting the balance to favour angiogenesis.