Annette Pflugfelder

Direct Injection of Protamine-protected mRNA: Results of a Phase 1/2 Vaccination Trial in Metastatic Melanoma Patients

In mice, injection of messenger RNA (mRNA) coding for tumor-associated antigens can induce antitumor immune responses and therefore offers a broadly applicable immunotherapy approach. We injected intradermally protamine-stabilized mRNAs coding for Melan-A, Tyrosinase, gp100, Mage-A1, Mage-A3, and Survivin in 21 metastatic melanoma patients. In 10 patients keyhole limpet hemocyanin (KLH) was added to the vaccine. Granulocyte macrophage colony-stimulating factor was applied as an adjuvant. Endpoints were toxicity and immune responses. No adverse events more than grade II have been observed. During treatment the frequency of Foxp3+/CD4+ regulatory T cells was significantly decreased upon mRNA vaccination in peripheral blood of the patients in the KLH arm, whereas myeloid suppressor cells (CD11b+HLA-DRlo monocytes) were reduced in the patients not receiving KLH. A reproducible increase of vaccine-directed T cells was observed in 2 of 4 immunologically evaluable patients. One of 7 patients with measurable disease showed a complete response. In conclusion, we show here that direct injection of protamine-protected mRNA is feasible and safe. The significant influence of the treatment on the frequency of immunosuppressive cells, the increase of vaccine-directed T cells upon treatment in a subset of patients together with the demonstration of a complete clinical response encourage further clinical investigation of the protamine-mRNA vaccine. This trial was registered at www.clinicaltrials.gov as ♯NCT00204607.

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.

Functional T Cells Targeting NY-ESO-1 or Melan-A Are Predictive for Survival of Patients With Distant Melanoma Metastasis

PURPOSE To analyze the prognostic relevance of circulating T cells responding to NY-ESO-1, Melan-A, MAGE-3, and survivin in patients with melanoma with distant metastasis. PATIENTS AND METHODS We examined 84 patients with follow-up after analysis (cohort A), 18 long-term survivors with an extraordinarily favorable course of disease before analysis (> 24 months survival after first occurrence of distant metastases; cohort B), and 14 healthy controls. Circulating antigen-reactive T cells were characterized by intracellular cytokine staining after in vitro stimulation. RESULTS In cohort A patients, the presence of T cells responding to peptides from NY-ESO-1, Melan-A, or MAGE-3 and the M category according to the American Joint Committee on Cancer classification were significantly associated with survival. T cells responding to NY-ESO-1 and Melan-A (hazard ratios, 0.29 and 0.18, respectively) remained independent prognostic factors in Cox regression analysis and were superior to the M category in predicting outcome. Median survival of patients possessing T cells responding to NY-ESO-1, Melan-A, or both was 21 months, compared with 6 months for all others. NY-ESO-1-responsive T cells were detected in 70% of cohort A patients surviving > 18 months and in 50% of cohort B patients. Melan-A responses were found in 42% and 47% of patients in cohorts A and B, respectively. In contrast, the proportion was only 22% for NY-ESO-1 and 23% for Melan-A in those who died within 6 months. CONCLUSION The presence of circulating T cells responding to Melan-A or NY-ESO-1 had strong independent prognostic impact on survival in advanced melanoma. Our findings support the therapeutic relevance of Melan-A and NY-ESO-1 as targets for immunotherapy.

A Dose-Escalation and Signal-Generating Study of the Immunocytokine L19-IL2 in Combination with Dacarbazine for the Therapy of Patients with Metastatic Melanoma

Purpose: L19-IL2 is an immunocytokine composed of an antibody fragment specific to the EDB domain of fibronectin, a tumor angiogenesis marker, and of human interleukin-2 (IL2). L19-IL2 delivers IL2 to the tumor site exploiting the selective expression of EDB on newly formed blood vessels. Previously, the recommended dose of L19-IL2 monotherapy was defined as 22.5 million international units (Mio IU) IL2 equivalents. In this study, safety and clinical activity of L19-IL2 in combination with dacarbazine were assessed in patients with metastatic melanoma. Experimental Design: The first 10 studied patients received escalating doses of L19-IL2 on days 1, 3, and 5 in combination with 1 g/m2 of dacarbazine on day 1 of a 3-weekly therapy cycle. Subsequently, 22 patients received L19-IL2 at recommended dose plus dacarbazine. Up to six treatment cycles were given, followed by a maintenance regimen with biweekly L19-IL2. Results: The recommended dose of L19-IL2 in combination with dacarbazine was defined as 22.5 Mio IU. Toxicity was manageable and reversible, with no treatment-related deaths. Twenty-nine patients were evaluable for efficacy according to Response Evaluation Criteria in Solid Tumors (RECIST). In a centralized radiology analysis, eight of 29 (28%) patients achieved a RECIST-confirmed objective response, including a complete response still ongoing 21 months after treatment beginning. The 12-month survival rate and median overall survival of the recommended dose–treated patients (n = 26) were 61.5% and 14.1 months, respectively. Conclusions: The repeated administration of L19-IL2 in combination with dacarbazine is safe and shows encouraging signs of clinical activity in patients with metastatic melanoma. This combination therapy is currently evaluated in a randomized phase II trial with patients with metastatic melanoma. Clin Cancer Res; 17(24); 7732–42. ©2011 AACR.

High Response Rate After Intratumoral Treatment With Interleukin-2: Results From a Phase 2 Study in 51 Patients With Metastasized Melanoma

Systemic high‐dose interleukin‐2 (IL‐2) achieved long‐term survival in a subset of patients with advanced melanoma. The authors reported previously that intratumorally applied IL‐2 induced complete local responses of all metastases in >60% of patients. The objectives of the current study were to confirm those results in a larger cohort and to identify patient or regimen characteristics associated with response.

Serum markers lactate dehydrogenase and S100B predict independently disease outcome in melanoma patients with distant metastasis

Background:Established prognostic factors are of limited value to predict long-term survival and benefit from metastasectomy in advanced melanoma. This study aimed to identify prognostic factors in patients with distant metastasis.Methods:We analysed overall survival of 855 institutional melanoma patients with distant metastasis by bivariate Kaplan–Meier survival probabilities and multivariate Cox hazard regression analysis.Results:Serum lactate dehydrogenases (LDH), S100B, the interval between initial diagnosis and occurrence of distant metastasis, the site of distant metastases, and the number of involved distant sites were significant independent prognostic factors in both bivariate and multivariate analyses. Visceral metastases other than lung (hazard ratio (HR) 1.8), elevated S100B (HR 1.7) and elevated LDH (HR 1.6) had the highest negative impact on survival. Complete metastasectomy was likewise an independent prognostic factor in multivariate analysis. This treatment was associated with favourable survival for patients with normal LDH and S100B values (5-year survival, 37.2%).Conclusion:The serum markers LDH and S100B were both found to be prognostic factors in melanoma patients with distant metastasis. Furthermore, complete metastasectomy had an independent favourable prognostic impact in particular for the patient subgroup with normal LDH and S100B values.

Incisional biopsy and melanoma prognosis: Facts and controversies

Facing the increasing number of melanoma patients is the controversial question of whether an incisional biopsy is associated with an unfavorable patient prognosis. Results of nine studies that occurred during the last four decades were reviewed. One of these studies was a large, prospective randomized controlled trial. Evidence from this trial and from most other studies is that incisional biopsies were not associated with an unfavorable prognosis for melanoma patients. Incisional biopsies are currently recommended for the histopathologic diagnosis of large tumors in facial, mucosal, and acral locations. Complete excisional biopsies are the generally recommended standard for melanoma surgery. Incisional biopsies of malignant melanoma do not negatively influence prognosis. Complete excision of primary melanoma is still the recommended standard of care and is a precondition for accurate histopathologic diagnosis.

Intralesional Treatment of Stage III Metastatic Melanoma Patients with L19-IL2 Results in Sustained Clinical and Systemic Immunologic Responses

Weide and colleagues show that intralesional therapy using the immunocytokine L19–IL2 elicited a high rate of clinical and systemic immune responses in patients with stage III melanoma and report their observation of favorable distant metastasis-free and overall survival in these patients after treatment. L19–IL2 is a recombinant protein comprising the cytokine IL2 fused to the single-chain monoclonal antibody L19. In previous studies, intralesional injection with IL2 has shown efficacy for the locoregional treatment of cutaneous/subcutaneous metastases in patients with advanced melanoma. The objectives of this study were to investigate whether (i) intralesional delivery of a targeted form of IL2 would yield similar results, with reduction of injection frequency and treatment duration; and (ii) systemic immune responses were induced by the local treatment. Patients with stage IIIB/IIIC melanoma and cutaneous/subcutaneous injectable metastases received weekly intratumoral injections of L19–IL2 at a maximum dose of 10 MIU/week for 4 consecutive weeks. Tumor response was evaluated 12 weeks after the first treatment. Twenty-four of 25 patients were evaluable for therapy-induced responses. A complete response (CR) by modified immune-related response criteria (irRC) of all treated metastases was achieved in 6 patients (25%), with long-lasting responses in most cases (5 patients for ≥24 months). Objective responses were documented in 53.9% of all index lesions [44.4% CR and 9.5% partial responses (by irRC)], and 36.5% of these remained stable, while 9.5% progressed. Toxicity was comparable with that of free IL2, and no serious adverse events were recorded. A significant temporary increase of peripheral regulatory T cells and natural killer cells, sustained increase of absolute CD4+ lymphocytes, and decrease of myeloid-derived suppressor cells were observed upon treatment. Finally, we recorded encouraging data about the progression time to distant metastases and overall survival. Cancer Immunol Res; 2(7); 668–78. ©2014 AACR.

Open label randomized study comparing 3 months vs. 6 months treatment of actinic keratoses with 3% diclofenac in 2.5% hyaluronic acid gel: a trial of the German Dermatologic Cooperative Oncology Group

Background Actinic keratoses (AK) are carcinomata in situ with the potential to develop into invasive carcinoma. Several studies have demonstrated that 3% diclofenac in 2.5% hyaluronic acid gel (HA) is effective and well tolerated in the treatment of AK. To date there are no large randomized multicentre trials with treatment durations longer than 90 days and histopathological control of treatment outcome.

Survival according to BRAF-V600 tumor mutations - An analysis of 437 patients with primary melanoma

The prognostic impact of BRAF-V600 tumor mutations in stage I/II melanoma patients has not yet been analyzed in detail. We investigated primary tumors of 437 patients diagnosed between 1989 and 2006 by Sanger sequencing. Mutations were detected in 38.7% of patients and were associated with age, histological subtype as well as mitotic rate. The mutational rate was 36.7% in patients with disease-free course and 51.7% in those with subsequent distant metastasis (p = 0.031). No difference in overall survival (p = 0.119) but a trend for worse distant-metastasis-free survival (p = 0.061) was observed in BRAF mutant compared to BRAF wild-type patients. Independent prognostic factors for overall survival were tumor thickness, mitotic rate and ulceration. An interesting significant prognostic impact was observed in patients with tumor thickness of 1 mm or less, with the mutation present in 6 of 7 patients dying from melanoma. In conclusion, no significant survival differences were found according to BRAF-V600 tumor mutations in patients with primary melanoma but an increasing impact of the mutational status was observed in the subgroup of patients with tumor thickness of 1 mm or less. A potential role of the mutational status as a prognostic factor especially in this subgroup needs to be investigated in larger studies.