Melanoma: Schuler G

Garbe C, Hauschild A, Volkenandt M, Schadendorf D, Stolz W, Reinhold U, Kortmann RD, Kettelhack C, Frerich B, Keilholz U, Dummer R, Sebastian G, Tilgen W, Schuler G, Mackensen A, Kaufmann R. · University Department of Dermatology, Tübingen, Germany. · Melanoma Res. · Pubmed #18337653 No free full text.

Abstract: Systemic medical treatment of melanoma is administered in the adjuvant and palliative setting. Adjuvant therapy may be considered in patients with primary melanoma with more than 1.5 mm tumor thickness and with regional node metastasis. Presently no indication for systemic adjuvant chemotherapy or for adjuvant therapy with nonspecific immune-stimulatory agents outside controlled studies is seen. Interferon-alpha is the first substance in the adjuvant therapy of melanoma, which has shown to present a significant advantage to the patients in some prospective randomized studies. Good arguments for using adjuvant interferon-alpha therapy in high-risk melanoma patients exist. Both high-dose and low-dose interferon-alpha show promise. The major indications for systemic chemotherapy and chemoimmunotherapy are inoperable recurrent tumors, inoperable regional metastases and distant metastases (stage IV). As treatment in such situations is primarily palliative, the effect of any regimen on the quality of life must be carefully weighed. As a first line treatment, single agent therapy is recommended, as polychemotherapy or biochemotherapy did not show significant advantages for prolongation of survival; hence they are more toxic. An urgent need for development of new treatment modalities is necessary and general principles of experimental immunotherapy are outlined.

Garbe C, Hauschild A, Volkenandt M, Schadendorf D, Stolz W, Reinhold U, Kortmann RD, Kettelhack C, Frerich B, Keilholz U, Dummer R, Sebastian G, Tilgen W, Schuler G, Mackensen A, Kaufmann R. · University Department of Dermatology, Tübingen, Germany. · Melanoma Res. · Pubmed #18227710 No free full text.

Abstract: The primary treatment of a melanoma is surgical excision. An excisional biopsy is preferred, and safety margins of 1 cm for tumor thickness up to 2 mm and 2 cm for higher tumor thickness should be applied either at primary excision or in a two-step procedure. When dealing with facial, acral or anogenital melanomas, micrographic control of the surgical margins may be preferable to allow reduced safety margins and conservation of tissue. The sentinel lymph node biopsy should be performed in patients whose primary melanoma is thicker than 1.0 mm and this operation should be performed in centers where both the operative and nuclear medicine teams are experienced. In clinically identified lymph node metastases, radical lymph node dissection is considered standard therapy. If distant metastases involve just one internal organ and operative removal is feasible, then surgery should be seen as therapy of choice. Radiation therapy for the primary treatment of melanoma is indicated only in those cases in which surgery is impossible or not reasonable. In regional lymph nodes, radiation therapy is usually recommended when excision is not complete (R1 resection) or if the nodes are inoperable. In distant metastases, radiation therapy is particularly indicated in bone metastases, brain metastases and soft tissue metastases.

Schultz ES, Schuler G. · Klinik für Dermatologie und Allergologie, Universitätsklinikum Giessen und Marburg GmbH, Deutschhausstrasse 9, 35037, Marburg, Deutschland. · Hautarzt. · Pubmed #18712326 No free full text.

Abstract: Malignant melanoma is a highly aggressive although immunogenic tumor which can be recognized and destroyed by the immune system. Therefore, immunotherapy has been represented an essential part of the therapeutic arsenal for decades. Besides non-specific immunotherapeutic approaches (whole tumor cell vaccine, cytokine therapy, toll-like receptor agonists), targeted immunotherapy has been made possible by the identification of tumor-associated antigens. Despite undisputable successes, the ultimate breakthrough has not yet been achieved. This overview deals with the fundamental aspects of antigen-specific immunotherapy and highlights future strategies to improve its clinical efficacy.

Enk AH, Becker JC, Schuler G. · Hautklinik der Ruprecht-Karls-Universität Heidelberg. · J Dtsch Dermatol Ges. · Pubmed #16895565 No free full text.

Abstract: Immunotherapy has assumed increasing importance in the therapy of malignant melanoma. The main reason is the high immunogenicity of the tumor itself, so that an immune response against the tumor often exists even without immune stimulation. The goal of modern immunotherapeutic approaches is to augment these anti-tumoral immune reactions to fight the tumor. Despite multiple successes, the ultimate breakthrough in the therapy of malignant melanoma has not yet been achieved. This overview summarizes the reasons for this lack of success and highlights future strategies for more successful therapy of malignant melanoma.

Garbe C, Hauschild A, Volkenandt M, Schadendorf D, Stolz W, Kortmann RD, Kettelhack C, Frerich B, Keilholz U, Dummer R, Sebastian G, Tilgen W, Schuler G, Mackensen A, Kaufmann R. · Universitäts-Hautklinik Tübingen. · J Dtsch Dermatol Ges. · Pubmed #16638065 No free full text.

This publication has no abstract.

Schultz ES, Schuler G. · Klinik für Dermatologie and Allergologie, Universitätsklinikum Giessen und Marburg, Standort Marburg. · HNO. · Pubmed #16167149 No free full text.

Abstract: In Germany, 6100 women and 5300 men contract a malignant melanoma of the skin every year. The chances of being cured are good in the early stages of the disease, but the average survival following distant metastasis is only 6 to 9 months. Therefore, early diagnosis is crucial, particularly as the skin is, by nature, a readily accessible organ. The introduction of epiluminescence microscopy has increased diagnostic accuracy significantly. In cases of doubt, complete excision of the suspect pigmented lesion is always advisable. A light skin type, presence of numerous naevi, genetic predisposition (familial history) and increased UV exposure are considered as risk factors. As a rule, adjuvant imunotherapy with interferon-alphais recommended in high-risk patients. A multidisciplinary approach consisting of surgery, radiotherapy, chemotherapy and immunotherapy has proved beneficial in advanced stages of metastasis.

Schuler-Thurner B, Schuler G. · Dermatologische Klinik mit Poliklink, Universitätsklinikum Erlangen. · J Dtsch Dermatol Ges. · Pubmed #16033483 No free full text.

This publication has no abstract.

Schadendorf D, Ugurel S, Schuler-Thurner B, Nestle FO, Enk A, Bröcker EB, Grabbe S, Rittgen W, Edler L, Sucker A, Zimpfer-Rechner C, Berger T, Kamarashev J, Burg G, Jonuleit H, Tüttenberg A, Becker JC, Keikavoussi P, Kämpgen E, Schuler G, Anonymous00068. · Skin Cancer Unit, German Cancer Research Center & University Hospital Mannheim, Mannheim. · Ann Oncol. · Pubmed #16418308 links to  free full text

Abstract: BACKGROUND: This randomized phase III trial was designed to demonstrate the superiority of autologous peptide-loaded dendritic cell (DC) vaccination over standard dacarbazine (DTIC) chemotherapy in stage IV melanoma patients. PATIENTS AND METHODS: DTIC 850 mg/m2 intravenously was applied in 4-week intervals. DC vaccines loaded with MHC class I and II-restricted peptides were applied subcutaneously at 2-week intervals for the first five vaccinations and every 4 weeks thereafter. The primary study end point was objective response (OR); secondary end points were toxicity, overall (OS) and progression-free survival (PFS). RESULTS: At the time of the first interim analysis 55 patients had been enrolled into the DTIC and 53 into the DC-arm (ITT). OR was low (DTIC: 5.5%, DC: 3.8%), but not significantly different in the two arms. The Data Safety & Monitoring Board recommended closure of the study. Unscheduled subset analyses revealed that patients with normal serum LDH and/or stage M1a/b survived longer in both arms than those with elevated serum LDH and/or stage M1c. Only in the DC-arm did those patients with (i) an initial unimpaired general health status (Karnofsky = 100) or (ii) an HLA-A2+/HLA-B44- haplotype survive significantly longer than patients with a Karnofsky index <100 (P = 0.007 versus P = 0.057 in the DTIC-arm) or other HLA haplotypes (P = 0.04 versus P = 0.57 in DTIC-treated patients). CONCLUSIONS: DC vaccination could not be demonstrated to be more effective than DTIC chemotherapy in stage IV melanoma patients. The observed association of overall performance status and HLA haplotype with overall survival for patients treated by DC vaccination should be tested in future trials employing DC vaccines.

Meyer RG, Britten CM, Siepmann U, Petzold B, Sagban TA, Lehr HA, Weigle B, Schmitz M, Mateo L, Schmidt B, Bernhard H, Jakob T, Hein R, Schuler G, Schuler-Thurner B, Wagner SN, Drexler I, Sutter G, Arndtz N, Chaplin P, Metz J, Enk A, Huber C, Wölfel T. · III. Medizinische Klinik, Johannes Gutenberg-Universitaet, Langenbeckstrasse 1, 55116 Mainz, Germany. · Cancer Immunol Immunother. · Pubmed #15627214 No free full text.

Abstract: A significant percentage of patients with stage II melanomas suffer a relapse after surgery and therefore need the development of adjuvant therapies. In the study reported here, safety and immunological response were analyzed after vaccination in an adjuvant setting with recombinant modified vaccinia virus Ankara carrying the cDNA for human tyrosinase (MVA-hTyr). A total of 20 patients were included and vaccinated three times at 4-week intervals with 5x10(8) IU of MVA-hTyr each time. The responses to the viral vector, to known HLA class I-restricted tyrosinase peptides, and to dendritic cells transfected with tyrosinase mRNA, were investigated by ELISpot assay on both ex vivo T cells and on T cells stimulated in vitro prior to testing. The delivery of MVA-hTyr was safe and did not cause any side effects above grade 2. A strong response to the viral vector was achieved, indicated by an increase in the frequency of MVA-specific CD4+ and CD8+ T cells and an increase in virus-specific antibody titers. However, no tyrosinase-specific T-cell or antibody response was observed with MVA-hTyr in any of the vaccinated patients. Although MVA-hTyr provides a safe and effective antigen-delivery system, it does not elicit a measurable immune response to its transgene product in patients with stage II melanoma after repeated combined intradermal and subcutaneous vaccination. We presume that modification of the antigen and/or prime-boost vaccination applying different approaches to antigen delivery may be required to induce an effective tyrosinase-specific immune response.

Schuler-Thurner B, Schultz ES, Berger TG, Weinlich G, Ebner S, Woerl P, Bender A, Feuerstein B, Fritsch PO, Romani N, Schuler G. · Department of Dermatology, University Hospital of Erlangen, D-91052 Erlangen, Germany. · J Exp Med. · Pubmed #12021308 links to  free full text

Abstract: There is consensus that an optimized cancer vaccine will have to induce not only CD8+ cytotoxic but also CD4+ T helper (Th) cells, particularly interferon (IFN)-gamma-producing, type 1 Th cells. The induction of strong, ex vivo detectable type 1 Th cell responses has not been reported to date. We demonstrate now that the subcutaneous injection of cryopreserved, mature, antigen-loaded, monocyte-derived dendritic cells (DCs) rapidly induces unequivocal Th1 responses (ex vivo detectable IFN-gamma-producing effectors as well as proliferating precursors) both to the control antigen KLH and to major histocompatibility complex (MHC) class II-restricted tumor peptides (melanoma-antigen [Mage]-3.DP4 and Mage-3.DR13) in the majority of 16 evaluable patients with metastatic melanoma. These Th1 cells recognized not only peptides, but also DCs loaded with Mage-3 protein, and in case of Mage-3DP4-specific Th1 cells IFN-gamma was released even after direct recognition of viable, Mage-3-expressing HLA-DP4+ melanoma cells. The capacity of DCs to rapidly induce Th1 cells should be valuable to evaluate whether Th1 cells are instrumental in targeting human cancer and chronic infections.

Atzpodien J, Neuber K, Kamanabrou D, Fluck M, Bröcker EB, Neumann C, Rünger TM, Schuler G, von den Driesch P, Müller I, Paul E, Patzelt T, Reitz M. · European Institute for Tumor Immunology and Prevention (EUTIP), Gotenstr. 152, 53175 Bonn, Germany. · Br J Cancer. · Pubmed #11870502 links to  free full text

Abstract: The purpose of this randomized trial was to evaluate the efficacy of combination chemoimmunotherapy compared with chemotherapy alone. A total of 124 patients were randomized to receive intravenous cisplatin (35 mg m(-2), days 1-3), carmustine (150 mg m(-2), day 1, cycles 1 and 3 only), dacarbacine (220 mg m(-2), days 1-3) and oral tamoxifen (20 mg m(-2), daily) in combination with (n=64) or without (n=60) sequential subcutaneous IL-2 and IFN-alpha. In those patients who received sequential immunotherapy, each cycle of chemotherapy was followed by outpatient s.c. IL-2 (10 x 10(6) IU m(-2), days 3-5, week 4; 5 x 10(6) IU m(-2), days 1, 3, 5, week 5) and s.c. IFN-alpha (5 x 10(6) IU m(-2), day 1, week 4; days 1, 3, 5, week 5). The overall response rate of patients treated with the combination of chemotherapy and IL-2/IFN-alpha was 34.3% with seven complete responses (10.9%) and 15 partial responses (23.4%). In patients treated with chemotherapy, only, the overall response rate was 29.9% with eight complete responses (13.3%) and 10 partial responses (16.6%). There was no significant difference in median progression free survival (0 months vs 4 months) and in median overall survival (12 months vs 13 months) for combined chemoimmunotherapy and for chemotherapy, respectively.

Schuler-Thurner B, Dieckmann D, Keikavoussi P, Bender A, Maczek C, Jonuleit H, Röder C, Haendle I, Leisgang W, Dunbar R, Cerundolo V, von Den Driesch P, Knop J, Bröcker EB, Enk A, Kämpgen E, Schuler G. · Department of Dermatology, University of Erlangen-Nuremberg, Erlangen, Germany. · J Immunol. · Pubmed #10975870 links to  free full text

Abstract: Dendritic cell (DC) vaccination, albeit still in an early stage, is a promising strategy to induce immunity to cancer. We explored whether DC can expand Ag-specific CD8+ T cells even in far-advanced stage IV melanoma patients. We found that three to five biweekly vaccinations of mature, monocyte-derived DC (three vaccinations of 6 x 106 s.c. followed by two i.v. ones of 6 and 12 x 106, respectively) pulsed with Mage-3A2.1 tumor and influenza matrix A2. 1-positive control peptides as well as the recall Ag tetanus toxoid (in three of eight patients) generated in all eight patients Ag-specific effector CD8+ T cells that were detectable in blood directly ex vivo. This is the first time that active, melanoma peptide-specific, IFN-gamma-producing, effector CD8+ T cells have been reliably observed in patients vaccinated with melanoma Ags. Therefore, our DC vaccination strategy performs an adjuvant role and encourages further optimization of this new immunization approach.

Thurner B, Haendle I, Röder C, Dieckmann D, Keikavoussi P, Jonuleit H, Bender A, Maczek C, Schreiner D, von den Driesch P, Bröcker EB, Steinman RM, Enk A, Kämpgen E, Schuler G. · Department of Dermatology, University of Erlangen-Nuremberg, D-91052 Erlangen, Germany. · J Exp Med. · Pubmed #10587357 links to  free full text

Abstract: Dendritic cells (DCs) are considered to be promising adjuvants for inducing immunity to cancer. We used mature, monocyte-derived DCs to elicit resistance to malignant melanoma. The DCs were pulsed with Mage-3A1 tumor peptide and a recall antigen, tetanus toxoid or tuberculin. 11 far advanced stage IV melanoma patients, who were progressive despite standard chemotherapy, received five DC vaccinations at 14-d intervals. The first three vaccinations were administered into the skin, 3 x 10(6) DCs each subcutaneously and intradermally, followed by two intravenous injections of 6 x 10(6) and 12 x 10(6) DCs, respectively. Only minor (less than or equal to grade II) side effects were observed. Immunity to the recall antigen was boosted. Significant expansions of Mage-3A1-specific CD8(+) cytotoxic T lymphocyte (CTL) precursors were induced in 8/11 patients. Curiously, these immune responses often declined after the intravenous vaccinations. Regressions of individual metastases (skin, lymph node, lung, and liver) were evident in 6/11 patients. Resolution of skin metastases in two of the patients was accompanied by erythema and CD8(+) T cell infiltration, whereas nonregressing lesions lacked CD8(+) T cells as well as Mage-3 mRNA expression. This study proves the principle that DC "vaccines" can frequently expand tumor-specific CTLs and elicit regressions even in advanced cancer and, in addition, provides evidence for an active CD8(+) CTL-tumor cell interaction in situ as well as escape by lack of tumor antigen expression.

Knippertz I, Hesse A, Schunder T, Kämpgen E, Brenner MK, Schuler G, Steinkasserer A, Nettelbeck DM. · Department of Dermatology, University Hospital Erlangen, Erlangen, Germany. · J Immunother. · Pubmed #19609245 No free full text.

Abstract: Dendritic cells (DCs) are professional antigen presenting cells and have key functions in the initiation of immune responses. Hence, antigen-loaded DCs have become important tools for active-specific immunotherapy. In addition to defining strategies for antigen loading, effective T-cell activation by DCs will depend on vaccination protocols that facilitate DC migration to secondary lymphoid tissues and expression of costimulatory molecules and cytokines. Adenoviral gene transfer has been successfully implemented for genetic antigen loading of DCs. In this study, we exploit an adenoviral vector encoding human CD40 ligand (CD40L), Ad5hCD40L, to establish DCs that feature both migration potential and prolonged secretion of the key T-helper 1 cytokine interleukin-12p70 (IL-12p70). Transduction of human monocyte-derived DCs with Ad5hCD40L resulted in efficient CD40L expression, which was detected only intracellularly, and in secretion of IL-12p70. Addition of recombinant interferon (IFN)-gamma shortly after DC transduction substantially increased IL-12p70 secretion. Maturation of DCs was achieved with a standard cytokine maturation cocktail (MC) containing prostaglandin E2 which, however, abolished IL-12p70 secretion by Ad5hCD40L-transduced cells in the absence of IFN-gamma. Only DCs treated with Ad5hCD40L, MC, and IFN-gamma migrated efficiently towards CCL19 and continued to secrete IL-12p70. Finally, DCs transduced with both Ad5hCD40L and an adenoviral vector encoding the melanoma antigen MelanA/MART-1 and treated with MC and IFN-gamma efficiently primed naive autologous CD8+ T cells into antigen-specific cytotoxic T lymphocyte. This strategy to generate DCs that exert both migration capacity and prolonged IL-12p70 secretion after intracellular CD40L expression and IFN-gamma treatment has the potential to further improve current DC vaccination protocols.

François V, Ottaviani S, Renkvist N, Stockis J, Schuler G, Thielemans K, Colau D, Marchand M, Boon T, Lucas S, van der Bruggen P. · Université Catholique de Louvain, Ludwig Institute for Cancer Research Ltd, Vrije Universiteit Brussel, Brussels, Belgium. · Cancer Res. · Pubmed #19435913 No free full text.

Abstract: Melanoma patients were injected with various vaccines containing a MAGE-A3 peptide presented by HLA-DP4. Anti-MAGE-A3.DP4 T cells were not detectable in the blood before vaccination, but their frequencies after vaccination ranged from 2 x 10(-6) to 2 x 10(-3) among the CD4(+) blood T lymphocytes of the patients. The CD4(+) blood T lymphocytes that stained ex vivo with HLA-DP4 tetramers folded with the MAGE-A3 peptide were selected by flow cytometry and amplified under clonal conditions. About 5% of the CD4(+) T-cell clones that recognized the MAGE-A3.DP4 antigen had a CD25(+) phenotype in the resting state. These CD25(+) clones had a high capacity to suppress the proliferation of another T-cell clone after peptide stimulation in vitro. Most of them had high FOXP3 expression in the resting state and an unmethylated FOXP3 intron 1. They produced active transforming growth factor-beta but none of cytokines IFN-gamma, interleukin-2 (IL-2), IL-4, IL-5, and IL-10. About 20% of CD25(-) clones had a significant but lower suppressive activity. Most of the CD25(-) clonal populations contained cells that expressed FOXP3 in the resting state, but FOXP3 demethylation was not observed. We conclude that MAGE-A3.DP4 vaccination can produce CD4(+) T cells that may exert regulatory T-cell function in vivo.

Erfurt C, Müller E, Emmerling S, Klotz C, Hertl M, Schuler G, Schultz ES. · Department of Dermatology, University Hospital Erlangen, Erlangen, Germany. · Int J Cancer. · Pubmed #19173283 No free full text.

Abstract: Melanoma-associated chondroitin sulfate proteoglycan (MCSP) (also known as high molecular weight-melanoma-associated antigen) represents an interesting target antigen for cancer immunotherapy which is expressed on human melanomas and other tumors such as breast carcinomas, gliomas, neuroblastomas and acute leukemias. MCSP seems to play an important functional role in melanoma as it is involved in tumor cell migration, invasion and angiogenesis. In this study, we isolated CD4(+) T helper cells from the blood of a healthy donor, recognizing a peptide from the MCSP core protein presented by HLA-DBR1*1101 molecules. T cell reactivity against the identified peptide could be detected in the blood of healthy donors and melanoma patients. MCSP specific T cells from the blood of a patient could be readily expanded by repeated peptide stimulation and recognized MCSP and HLA-DR expressing tumor cells. Our findings suggest that vaccination against MCSP helper T cell epitopes might be a promising approach to fight melanoma.

Garbe C, Schadendorf D, Stolz W, Volkenandt M, Reinhold U, Kortmann RD, Kettelhack C, Frerich B, Keilholz U, Dummer R, Sebastian G, Tilgen W, Schuler G, Mackensen A, Kaufmann R, Hauschild A. · Sektion Dermatologische Onkologie, Universitäts-Hautklinik, Liebermeister Strasse 25, D-72076 Tübingen, Germany. · J Dtsch Dermatol Ges. · Pubmed #18801142 No free full text.

This publication has no abstract.

Connerotte T, Van Pel A, Godelaine D, Tartour E, Schuler-Thurner B, Lucas S, Thielemans K, Schuler G, Coulie PG. · de Duve Institute, Université Catholique de Louvain, Department of Physiology and Immunology, Medical School of Vrije Universiteit Brussel, Brussels, Belgium. · Cancer Res. · Pubmed #18483279 links to  free full text

Abstract: Tumor regressions have been observed in a small proportion of melanoma patients vaccinated with a MAGE-A3 peptide presented by HLA-A1, administered as peptide, ALVAC canarypox virus containing a MAGE-A3 minigene, or peptide-pulsed dendritic cells (DC). There was a correlation between tumor regression and the detection of anti-MAGE-3.A1 CTL responses. These responses were monoclonal and often of a very low magnitude after vaccination with peptide or ALVAC, and usually polyclonal and of a higher magnitude after DC vaccination. These results suggested that, at least in some patients, surprisingly few anti-MAGE-3.A1 T-cells could initiate a tumor regression process. To understand the role of these T cells, we carried out a functional analysis of anti-MAGE-3.A1 CTL clones derived from vaccinated patients who displayed tumor regression. The functional avidities of these CTL clones, evaluated in lysis assays, were surprisingly low, suggesting that high avidity was not part of the putative capability of these CTL to trigger tumor rejection. Most anti-MAGE-3.A1 CTL clones obtained after DC vaccination, but not after peptide or ALVAC vaccination, produced interleukin 10. Transcript profiling confirmed these results and indicated that approximately 20 genes, including CD40L, prostaglandin D2 synthase, granzyme K, and granzyme H, were highly differentially expressed between the anti-MAGE-3.A1 CTL clones derived from patients vaccinated with either peptide-ALVAC or peptide-pulsed DC. These results indicate that the modality of vaccination with a tumor-specific antigen influences the differentiation pathway of the antivaccine CD8 T-cells, which may have an effect on their capacity to trigger a tumor rejection response.

Garbe C, Hauschild A, Volkenandt M, Schadendorf D, Stolz W, Reinhold U, Kortmann RD, Kettelhack C, Frerich B, Keilholz U, Dummer R, Sebastian G, Tilgen W, Schuler G, Mackensen A, Kaufmann R. · University Department of Dermatology, Tübingen, Germany. · Melanoma Res. · Pubmed #17992123 No free full text.

Abstract: Melanoma is a malignant tumor that arises from melanocytic cells and primarily involves the skin. The most important exogenous etiological factor is exposure to ultraviolet irradiation. Diagnosis of melanoma is based primarily on its clinical features, and the A-B-C-D rule is useful in identifying pigmented lesions, which are suspicious for melanoma (Asymmetry, Border irregular, Color inhomogeneous and Diameter more than 5 mm). Dermoscopy is very helpful in clarifying the differential diagnosis of pigmented lesions. About 90% of melanomas are diagnosed as primary tumors without any evidence for metastasis. The tumor-specific 10-year survival for all such tumors is about 75-85%. The most important prognostic factors for primary melanoma without metastases are vertical tumor thickness (Breslow depth) as measured on the histological specimen, presence of histopathologically recognized ulceration, invasion level (Clark level) and identification of micrometastases in the regional lymph nodes via sentinel lymph node biopsy. The current tumor node metastasis classification for the staging of primary melanoma is based on these factors. Melanomas can metastasize either by the lymphatic or by the hematogenous route. About two-thirds of metastases are originally confined to the drainage area of regional lymph nodes. A regional metastasis can appear as satellite metastases up to 2 cm from the primary tumor, as intransit metastases in the skin between the site of the primary tumor and the first lymph node and as regional lymph node metastases. In the stage of regional metastasis, the differentiation between micrometastasis and macrometastasis and the number of lymph nodes involved are crucial. As soon as distant metastasis develops, prognosis depends on the site of the metastasis and on the lactate dehydrogenase levels in the blood. The frequency and extent of follow-up examinations is based on the initial tumor parameters. In thin primary melanomas up to 1-mm tumor thickness, clinical examinations at 6-month intervals are sufficient and in thicker primary melanomas, at 3-month intervals. Lymph node sonography as well as determination of the tumor marker protein S100beta are recommended. Additionally, in the stage of regional metastasis, whole body imaging should be performed every 6 months; in the stage of distant metastasis, surveillance has to be scheduled individually.

Schierer S, Hesse A, Müller I, Kämpgen E, Curiel DT, Schuler G, Steinkasserer A, Nettelbeck DM. · Department of Dermatology, University Hospital Erlangen, Erlangen, Germany. · Int J Cancer. · Pubmed #17764070 No free full text.

Abstract: Adenoviral oncolysis is a promising new modality for treatment of cancer based on selective viral replication in tumor cells. However, tumor cell killing by adenoviral oncolysis needs to be improved to achieve therapeutic benefit in the clinic. Towards this end, the activation of anti-tumor immunity by adenoviral oncolysis might constitute a potent mechanism for systemic killing of uninfected tumor cells, thereby effectively complementing direct tumor cell killing by the virus. Knowledge of anti-tumor immune induction by adenoviral oncolysis, however, is lacking mostly due to species-specificity of adenovirus replication, which has hampered studies of human oncolytic adenoviruses in animals. We suggest the analysis of interactions of oncolytic adenoviruses with human immune cells as rational basis for the implementation of adenoviral oncolysis-induced anti-tumor immune activation. The goal of our study was to investigate how oncolytic adenoviruses affect human dendritic cells (DCs), key regulators of innate and adoptive immunity that are widely investigated as tumor vaccines. We report that melanoma-directed oncolytic adenoviruses, like replication-deficient adenoviruses but unlike adenoviruses with unrestricted replication potential, are not toxic to monocyte-derived immature DCs and do not block DC maturation by external stimuli. Of note, this is in contrast to reports for other viruses/viral vectors and represents a prerequisite for anti-tumor immune activation by adenoviral oncolysis. Furthermore, we show that these oncolytic adenoviruses alone do not or only partially induce DC maturation. Thus additional signals are required for optimal immune activation. These could be delivered, for example, by inserting immunoregulatory transgenes into the oncolytic adenovirus genome.

Held G, Wadle A, Dauth N, Stewart-Jones G, Sturm C, Thiel M, Zwick C, Dieckmann D, Schuler G, Hoogenboom HR, Lévy F, Cerundolo V, Pfreundschuh M, Renner C. · I Med Klinik, Saarland University Medical School, Homburg/Saar, Germany. · Eur J Immunol. · Pubmed #17559180 No free full text.

Abstract: MHC-peptide-specific Fab antibodies binding to HLA-A*0201 complexes presenting the wild-type EAAGIGILTV (EAA) or analogue Melan-A 10-mer ELAGIGILTV (ELA) peptide were generated to study efficacy of peptide processing and presentation. None of the selected Fab antibodies detected the naturally processed EAA/HLA-A*0201 complex on melanoma tumor cells, confirming the known low peptide number on the cell surface. To study the effect of peptide presentation and processing in more detail, genes coding for the A27L-mutated Melan-A protein or the processed ELA peptide were introduced into HLA-A*0201(+) B cells by infection with the respective recombinant vaccinia virus construct producing equimolar amounts of GFP-ubiquitin directly linked to the fragment of interest. Correlating GFP expression to actual numbers of peptide presented, 1100-2600 [corrected] ELA peptides had to be synthesized to be presented by a single MHC class I antigen-peptide complex. This number increased 10- to 20-fold when ELA peptide presentation from the A27L-mutated full length Melan-A protein was studied, since 16000-52000 [corrected] GFP molecules needed to be synthesized for the detection of one ELA peptide. Our results indicate that peptide processing rather than presentation is the rate-limiting step in our experimental setting and is much more ineffective for Melan-A than has been previously shown for other MHC class I-restricted epitopes.

Erfurt C, Sun Z, Haendle I, Schuler-Thurner B, Heirman C, Thielemans K, van der Bruggen P, Schuler G, Schultz ES. · Department of Dermatology, University Hospital of Erlangen, Hartmannstrasse 14, Erlangen, Germany. · J Immunol. · Pubmed #17548607 links to  free full text

Abstract: To avoid immune escape by down-regulation or loss of Ag by the tumor cells, target Ags are needed, which are important for the malignant phenotype and survival of the tumor. We could identify a CD4(+) T cell epitope derived from the human melanoma-associated chondroitin sulfate proteoglycan (MCSP) (also known as high m.w.-melanoma-associated Ag), which is strongly expressed on >90% of human melanoma lesions and is important for the motility and invasion of melanoma cells. However, MCSP is not strictly tumor specific, because it is also expressed in a variety of normal tissues. Therefore, self tolerance should prevent the induction of strong T cell responses against these Ags by vaccination strategies. In contrast, breaking self tolerance to this Ag by effectively manipulating the immune system might mediate antitumor responses, although it would bear the risk of autoimmunity. Surprisingly, we could readily isolate CD4(+) Th cells from the blood of a healthy donor-recognizing peptide MCSP(693-709) on HLA-DR11-expressing melanoma cells. Broad T cell reactivity against this Ag could be detected in the peripheral blood of both healthy donors and melanoma patients, without any apparent signs of autoimmune disease. In some patients, a decline of T cell reactivity was observed upon tumor progression. Our data indicate that CD4(+) T cells are capable of recognizing a membrane glycoprotein that is important in melanoma cell function, and it may be possible that the sizable reactivity to this Ag in most normal individuals contributes to immune surveillance against cancer.

Greiner S, Humrich JY, Thuman P, Sauter B, Schuler G, Jenne L. · Department of Dermatology, University Hospital Erlangen, Erlangen, Germany. · Clin Exp Immunol. · Pubmed #17034588 links to  free full text

Abstract: Vaccinia virus (VV) has been tested as oncolytic virus against malignant melanoma in clinical trials for more than 40 years. Until now, mainly strains comparable to viral strains used for smallpox vaccination have been probed for anti-tumoral therapy. We have shown recently that the wild-type strain Western Reserve (WR) can interfere with crucial functions of monocyte-derived dendritic cells (DCs). Our aim was to examine whether viral immune evasion mechanisms might be responsible for the ineffectiveness of WR-based vaccination strategies and whether the highly attenuated strain modified virus Ankara (MVA) differs from WR with respect to its possible immunostimulatory capacity after intratumoral injection. Using in vitro experiments, we compared the effect of both strains on melanoma cells and on local bystander DCs. We found that both VV-strains infected melanoma cells efficiently and caused disintegration of the actin cytoskeleton, as shown by fluorescence microscopy. In addition, both VV-strains caused apoptotic cell death in melanoma cells after infection. In contrast to MVA, WR underwent a complete viral replication cycle in melanoma cells. Bystander DCs were consecutively infected by newly generated WR virions and lost their capacity to induce allogeneic T cell proliferation. DCs in contact with MVA-infected melanoma cells retained their capacity to induce T cell proliferation. Immature DCs were capable of phagocytosing MVA-infected melanoma cells. Priming of autologous CD8(+) T cells by DCs that had phagocytosed MVA-infected, MelanA positive melanoma cells resulted in the induction of T cell clones specifically reactive against the model antigen MelanA as shown by enzyme-linked immunospot (ELISPOT) analysis. We conclude that the clinical trials with oncolytic wild-type VV failed probably because of suppression of bystander DCs and consecutive suppression of T cell-mediated anti-melanoma immunity. The attenuated VV-strain MVA facilitates the generation of tumour associated antigen (TAA)-specific T cell response as it is oncolytic for melanoma cells, but non-toxic for DC, and should be a promising candidate for intralesional metastatic melanoma therapy.

Dörrie J, Wellner V, Kämpgen E, Schuler G, Schaft N. · Department of Dermatology, University Hospital of Erlangen, Erlangen, Germany. · J Immunol Methods. · Pubmed #16780866 No free full text.

Abstract: Several kits are available to isolate RNA(1) from tissues. However, melanoma tissue is often rich in melanin that co-purifies with DNA/RNA and inhibits subsequent PCR reactions, hampering tumor antigen detection and amplification. This problem has not yet been addressed systematically. Here we generated a photometric protocol to determine both the melanin and RNA concentration by correcting the latter for the absorption coefficient of melanin at 260 nm. Subsequently, different combinations of silica-based RNA-binding, size-exclusion, and ion-exchange columns were used in 8 protocols for isolation of RNA from melanoma tissue to compare efficacy of melanin removal and yield of RNA. Furthermore, the capability of the different RNA preparations to function as template in RT-PCRs with products of different length, i.e. GAP-DH, tyrosinase, and gp100, was tested. We found that the combination of silica-based RNA-binding and size-exclusion columns was not sufficient to remove melanin from highly contaminated tumor samples, and subsequent RT-PCR failed to give larger products. However, protocols including ion-exchange columns resulted in efficient removal of melanin, while retaining reasonable RNA yields in samples from highly pigmented melanomas. Efficient RT-PCR of larger products turned out to be inversely correlated to the melanin contamination. This RNA-purification method will help scientists to isolate polynucleotides from melanin-containing tumor samples, which subsequently can be used in antigen detection assays and vaccination strategies using amplified total tumor RNA.

Römer W, Nömayr A, Greess H, Fiedler E, Platsch G, Schuler-Thurner B, Pfahlberg A, Hothorn T, Schuler G, Hornegger J, Bautz W, Kuwert T. · Nuklearmedizinische Klinik, Universität Erlangen-Nürnberg, Krankenhausstr. 12, 91054 Erlangen. · Nuklearmedizin. · Pubmed #16547570 No free full text.

Abstract: AIM: This study investigates whether interactive rigid fusion of routine PET and CT data improves localization, detection and characterization of lesions compared to separate reading. For this purpose, routine PET and CT scans of patients with metastases from malignant melanoma were used. PATIENTS, METHODS: In 34 patients with histologically confirmed malignant melanoma, FDG-PET and spiral CT were performed using clinical standard protocols. For all of these patients, gold standard was available. Clinical and radiological follow-up identified 82 lesions as definitely pathological. Two board-certified nuclear medicine physicians and two board-certified radiologists analyzed PET and CT images independently from each other. For each patient up to 32 anatomical regions (24 lymph node regions, 8 extranodular regions) were systematically classified. Discordant areas were interactively analyzed in manually and rigidly registered images using a commercially available fusion tool. No side-by-side reading was performed. RESULTS: Image fusion disclosed that the evaluation of the PET images alone led to a mislocalization in 26 of 91 focally FDG enhancing lesions. The overall sensitivities of PET, CT, and image fusion were 85, 88, and 94%, respectively; the overall specificities of PET, CT and image fusion were 98, 95 and 100%, respectively. Image fusion exhibited statistically significant higher specificity values as compared with CT. Ten definitely malignant sites were false-negative in CT, but could be detected by PET. On the other hand, twelve metastases were false-negative in PET, but could be detected by CT. These included two lesions, which had a clear correlate on the PET image when the fused images were evaluated. On the whole, registration of the PET and CT images yielded additional diagnostic information in 44% of the definitely malignant lesions. CONCLUSION: Retrospective image fusion of independently obtained PET and CT data is particularly valuable in exactly localizing foci of abnormal FDG uptake and improves the detection of metastases of malignant melanoma.