Patrick Terheyden

Avelumab in patients with chemotherapy-refractory metastatic Merkel cell carcinoma: a multicentre, single-group, open-label, phase 2 trial

BACKGROUND Merkel cell carcinoma is a rare, aggressive skin cancer with poor prognosis in patients with advanced disease. Current standard care uses various cytotoxic chemotherapy regimens, but responses are seldom durable. Tumour oncogenesis is linked to Merkel cell polyomavirus integration and ultraviolet-radiation-induced mutations, providing rationale for treatment with immunotherapy antibodies that target the PD-L1/PD-1 pathway. We assessed treatment with avelumab, an anti-PD-L1 monoclonal antibody, in patients with stage IV Merkel cell carcinoma that had progressed after cytotoxic chemotherapy. METHODS In this multicentre, international, prospective, single-group, open-label, phase 2 trial, patients with stage IV chemotherapy-refractory, histologically confirmed Merkel cell carcinoma (aged ≥18 years) were enrolled from 35 cancer treatment centres and academic hospitals in North America, Europe, Australia, and Asia. Key eligibility criteria were an ECOG performance status of 0 or 1, measurable disease by Response Evaluation Criteria in Solid Tumors (RECIST) version 1.1, adequate haematological, hepatic, and renal function, and immune-competent status (patients with HIV, immunosuppression, haematological malignancies, and previous organ transplantation were excluded). Patient selection was not based on PD-L1 expression or Merkel cell polyomavirus status. Collection of biopsy material or use of archival tissue for these assessments was mandatory. Avelumab was given intravenously at a dose of 10 mg/kg every 2 weeks. The primary endpoint was confirmed objective response (complete response or partial response) assessed according to RECIST version 1.1 by an independent review committee. Safety and clinical activity were assessed in all patients who received at least one dose of study drug (the modified intention-to-treat population). This trial is registered with ClinicalTrials.gov as NCT02155647. FINDINGS Between July 25, 2014, and Sept 3, 2015, 88 patients were enrolled and received at least one dose of avelumab. Patients were followed up for a median of 10·4 months (IQR 8·6-13·1). The proportion of patients who achieved an objective response was 28 (31·8% [95·9% CI 21·9-43·1]) of 88 patients, including eight complete responses and 20 partial responses. Responses were ongoing in 23 (82%) of 28 patients at the time of analysis. Five grade 3 treatment-related adverse events occurred in four (5%) patients: lymphopenia in two patients, blood creatine phosphokinase increase in one patient, aminotransferase increase in one patient, and blood cholesterol increase in one patient; there were no treatment-related grade 4 adverse events or treatment-related deaths. Serious treatment-related adverse events were reported in five patients (6%): enterocolitis, infusion-related reaction, aminotransferases increased, chondrocalcinosis, synovitis, and interstitial nephritis (n=1 each). INTERPRETATION Avelumab was associated with durable responses, most of which are still ongoing, and was well tolerated; hence, avelumab represents a new therapeutic option for advanced Merkel cell carcinoma. FUNDING Merck KGaA, Darmstadt, Germany.

Constitutive activation of the Ras-Raf signaling pathway in metastatic melanoma is associated with poor prognosis.

Background Genes of the Raf family encode kinases that are regulated by Ras and mediate cellular responses to growth signals. Recently, it was shown that activating mutations of BRaf are found with high frequency in human melanomas. The Ras family member most often mutated in melanoma is NRas. Methods The constitutive activation of the Ras/Raf signaling pathway suggests an impact on the clinical course of the tumor. To address this notion, we analyzed tumor DNA from 114 primary cutaneous melanomas and of 86 metastatic lesions obtained from 174 patients for mutations in BRaf (exons 15 and 11) and NRas (exons 1 and 2) by direct sequencing of PCR products and correlated these results with the clinical course. Results In 57.5% of the tumors either BRaf or NRas were mutated with a higher incidence in metastatic (66.3%) than in primary lesions (50.9%). Although the majority of BRaf mutations affected codon 599, almost 15% of mutations at this position were different from the well-described exchange from valine to glutamic acid. These mutations (V599R and V599K) also displayed increased kinase and transforming activity. Surprisingly, the additional BRaf variants D593V, G465R and G465E showed a complete loss of activity in the in vitro kinase assay; however, cells overexpressing these mutants displayed increased Erk phosphorylation. The correlation of mutational status and clinical course revealed that the presence of BRaf/NRas mutations in primary tumors did not negatively impact progression free or overall survival. In contrast, however, for metastatic lesions the presence of BRAF/NRAS mutations was associated with a significantly poorer prognosis, i.e. a shortened survival. Conclusion We demonstrate a high – albeit lower than initially anticipated – frequency of activating BRaf mutations in melanoma in the largest series of directly analyzed tumors reported to date. Notably, the clinical course of patients harboring activating BRaf mutations in metastatic melanoma was significantly affected by the presence of a constitutive BRaf activation in these.

CD40-Ligated Dendritic Cells Effectively Expand Melanoma-Specific CD8+ CTLs and CD4+ IFN-γ-Producing T Cells from Tumor-Infiltrating Lymphocytes

Professional APC, notably dendritic cells (DC), are necessary for stimulation and expansion of naive T cells. By means of murine models, the interaction between CD40 on DC and its ligand CD154 has been recognized as an important element for conditioning of DC to prime and expand CTL. We translated these findings into the human system, scrutinizing the ability of DC to initiate clonal expansion of single T cells. DC generated under completely autologous conditions from peripheral blood monocytes were cocultured at a rate of 0.3 cell/well with melanoma-infiltrating T cells; this procedure guaranteed that either a CD4+ or a CD8+ cell interacted with the DC, thus avoiding the contact of more than one T cell to the DC. In the absence of further stimulation, this cloning protocol yielded almost exclusively CD4+ T cell clones that predominantly exhibited a Th2 phenotype. However, cross-linking of CD40 on DC resulted in the induction of IFN-γ-producing Th1 CD4+ T cell clones. In addition, CD40-activated DC were capable of expanding CD8+ CTL clones. The ratio of CD4 to CD8 T cell clones corresponded to the ratio present in the initial tumor-infiltrating lymphocyte preparation. The CTL clones efficiently lysed autologous tumor cells whereas autologous fibroblasts or MHC-mismatched melanoma cells were not killed. Our findings support the critical role of CD40/CD154 interactions for the induction of cellular immune responses.

Treatment of Melanoma

INTRODUCTION The incidence of melanoma has increased fivefold during the past three decades. Melanoma can no longer be classified as rare; rather, it is now one of the more frequent tumors. METHODS Recommendations for the diagnosis and treatment of melanoma are laid out in the interdisciplinary S2 guidelines of the German Cancer Society, upon which the present review is based. The goal of this article is to present the clinical core recommendations for treatment in all disease stages. RESULTS The operative management of primary melanoma usually takes place in two steps. A complete excisional biopsy with a safety margin of about 2 mm is performed in order to establish the histopathological diagnosis. Definitive surgical excision is performed with a safety margin of 1 cm in tumors up to 2 mm thick, 2 cm in thicker tumors. In tumors more than 1 mm thick, sentinel lymph node biopsy should be performed to aid in tumor staging. Radiotherapy is indicated in inoperable tumors of all stages. Adjuvant immunotherapy with interferon alpha is recommended in tumors of thickness >2 mm and in locoregional metastasis. If distant metastasis is present and R0 surgery is not an option, the treatment should primarily comprise monochemotherapy or alternatively the patient should be enrolled in a clinical trial. CONCLUSION The recommendations presented here are based predominantly on the results of prospective randomized trials.

Expression of the NKG2D ligand UL16 binding protein‐1 (ULBP‐1) on dendritic cells

Innate and adaptive immunity have not evolved separately. In this regard, the NKG2D molecule first identified on NK cells and classified as an activating NK cell receptor is also an important receptor for CD8+ T cells. Functional analyses of human NKG2D and its ligands, i.e. UL16 binding proteins (ULBP) and MHC class I chain‐related (MIC), have so far focused on immune cell‐target cell situations because of the expression of NKG2D ligands on infected, stressed or transformed cells. Here, however, we address a possible function of NKG2D/ULBP‐1 during the initiation of T cell responses. ULBP‐1 can be detected on mature dendritic cells both in situ in the T cell areas of lymph nodes as well as in vitro after artificial maturation. FCM analysis further demonstrated that although NKG2D is expressed to some degree on all analyzed T cell subsets from peripheral blood, in vitro stimulation of T cells results in up‐regulation of NKG2D on proliferating T cells. Using the sentinel lymph nodes of primary melanoma as a model for induction of defined T cell responses in vivo, we were able to demonstrate the expression of NKG2D on melanoma‐associated antigen‐specific T cells. Thus, our results suggest a role for NGK2D‐ULBP‐1 in the induction or reactivation of T cell responses.

Anti-vascular endothelial growth factor antibody bevacizumab in conjunction with chemotherapy in metastasising melanoma

PurposeThe combination of the antiangiogenic antibody bevacizumab with standard chemotherapy has improved the prognosis in patients with metastastic colorectal cancer and other advanced cancers. The role of combined anti-VEGF and chemotherapy in metastastic melanoma is just starting to be elucidated.MethodsWe tested this notion in three patients with advanced and therapy-refractory melanoma.ResultsInterestingly, two patients achieved objective regressions after three courses of therapy; the third patient, albeit progressing demonstrated a pronounced liquid necrosis in bulky lymphnode metastasis.ConclusionFurther studies are warranted to scrutinise these impressive therapeutic effects on the combination of bevacizumab and chemotherapy in melanoma.

[Immunochemotherapy of metastatic uveal melanoma with interferon alfa-2b, interleukin-2 and fotemustine. Case reports and review of the literature].

ZusammenfassungTrotz der primären Entwicklung in der Uvea werden Patienten mit metastasierendem Uveamelanom in der dermatologischen Onkologie vorgestellt. Diese Patienten werden häufig mit Therapieverfahren behandelt, die für Patienten mit metastasierenden kutanen Melanomen etabliert wurden. Allerdings divergiert sowohl die Biologie als auch das Metastasierungsverhalten des Uveamelanoms von dem des Hautmelanoms. Lymphogene Metastasen treten nie, hämatogene Metastasen treten oft spät auf, wobei die Leber – oft solitär – das am häufigsten befallene Organ ist. Die Überlebensprognose nach Auftreten hepatischer Metastasen ist mit 2 bis 5 Monaten sehr schlecht. Wir berichten über 3 Patienten mit metastasierendem Uveamelanom, die eine Immunochemotherapie mit Interferon-α2b, Interleukin-2 und Fotemustin erhalten haben. Mit dieser Therapie konnte bei einem der Patienten eine partielle Remission von mehr als 49 Monaten Dauer erzielt werden. Die beiden anderen Patienten erfuhren eine Stabilisierung der Erkrankung über 8 und 16 Monate. Dieser therapeutische Erfolg spiegelt sich in einem im Vergleich zu historischen Kontrollen deutlich verlängerten Überleben der Patienten von 14, 43+ und 59+ Monaten wider.SummaryIn spite of their tumor’s origin in the uveal tract, many patients suffering from advanced uveal melanoma are admitted to dermatological oncology units. Most patients with metastases from uveal melanoma receive treatments that were established for stage IV cutaneous melanoma. However, both the biology as well as the metastastic behaviour of this tumor is different from cutaneous melanoma. Lymphatic metastases do not occur, and hematogeneous metastases usually occur later and predominantly involve the liver. The prognosis is very bad ranging from 2 to 5 months.We describe three patients with advanced uveal melanoma who received immunochemotherapy containing interferon-α2b, interleukin-2, and fotemustine. This therapy induced a partial response of more than 49 months duration in one patient, whereas for the remaining patients the disease progression could be stabilized for eight and 16 months, respectively. This therapeutic success is reflected by a prolonged survival of 14, 43+, and 59+ months.

Longitudinal Analysis of MART‐1/HLA‐A2‐Reactive T Cells Over the Course of Melanoma Progression

An HLA‐A2‐positive patient with advanced stage IV melanoma was vaccinated with dendritic cells (DCs) pulsed with melanoma antigens, whereby the rapid progression of disease stalled for a period of 10 months. Monitoring of the cellular immune response against one of the vaccinated HLA‐A2‐restricted epitopes demonstrated both induction and subsequent decline in the number of interferon‐γ (IFN‐γ)‐producing MART‐1‐reactive cells present in the blood. Enumeration of reactive T cells by MART‐126‐35/HLA‐A2 tetramer staining revealed an induction of such cells after three vaccinations and a subsequent decline that most prominent at times of rapid disease progression. However, a substantial number of reactive cells were present even when no MART‐1 reactivity was detectable by functional assays. Isolation of such MART‐126−35‐reactive T cells by means of peptide/HLA‐A2‐coated magnetic beads demonstrated the persistence of a TCRVβ14+ T‐cell clone in this population over the whole observation period. Intracellular fluorescence‐activated cell sorter staining of such TCRVβ14+ T cells for IFN‐γ and interleukin‐2 after maximal stimulation with phorbol 12‐myristate 13‐acetate/ionomycin revealed an impairment in their capacity to produce cytokines at the end of the observation period. Thus, functional changes of individual T‐cell clones, e.g. clonal exhaustion, seem to be responsible for the known discrepancy between functional and phenotype assays for immune monitoring of tumour patients.

Sequential interferon-alpha2b, interleukin-2 and fotemustine for patients with metastatic melanoma.

The aim of this study was the evaluation of both the antitumour activity and toxicity of an immunochemotherapeutic regimen consisting of interferon-alpha2b and interleukin-2 in combination with fotemustine for patients with metastatic melanoma. To improve the penetration of fotemustine into the brain, it was given immediately after immunotherapy, when the blood-brain barrier is still disturbed. Of the 19 patients treated, three complete remissions (CRs) and one partial remission (PR) were induced, giving an objective response rate of 21% (95% confidence interval 6-46%). The durations of the CRs were 9, 19 and 44 months; the PR lasted for 59+ months. The overall survival times for the patients with CR were 21, 25 and 70+ months, and 59+ months for the PR. For nine patients (47%, 95% confidence interval 24-71%) disease was stabilized for a median period of 8 months (range 2-18 months), resulting in a median survival of 18 months (range 10-41+ months). No haematological toxicity of World Health Organization grade 3 or more was observed and in general toxicity was low. In summary, this immunochemotherapy regimen led to long-term survival in occasional patients, and about half of the patients achieved stable disease, with prolonged treatment- and progression-free survival compared with nonresponding patients. The occurrence of brain metastases, however, was not prevented, and in fact was the site of recurrence in those patients achieving a CR. Due to its low toxicity, this protocol can be applied at a community hospital level.

New developments in the biology and the treatment of metastatic Merkel cell carcinoma

Purpose of review Patients with stage IIIB und IV metastatic Merkel cell carcinoma (mMCC), who are not suitable candidates for surgery or radiotherapy, are unlikely to achieve lasting remission or tumor control by chemo or targeted therapy. In the majority of cases, the tumor arises from viral carcinogenesis associated with the Merkel cell polyomavirus (MCPyV). In MCPyV-negative tumors with a presumable ultraviolet carcinogenesis, a high mutational burden resulting in neoantigens was discovered. In two phase II clinical trials in either the first or second-line setting, a high response rate was observed for immunotherapies with antibodies blocking the programmed death-1 (PD-1) and programmed death ligand-1 (PD-L1) immune checkpoints. Recent findings The response rate was 56% with the anti-PD-1 inhibitor pembrolizumab as a first-line and 32% with the anti-PD-L1 antibody avelumab used as second-line therapy. Both treatments were well tolerated. Treatment response was rapid and in most cases maintained during follow-up, which, however, is still rather short. Whether the MCPyV or the PD-L1 status is predictive for treatment response and progression-free survival is still ambiguous. Additionally, clinical criteria for patient selection for immunotherapy of mMCC have not yet been defined. Summary PD-1/PD-L1 inhibition can be regarded as new first-line therapy for patients with mMCC not amendable by surgery and/or radiation.