Synchronous BRAF(V600E) and MEK inhibition leads to superior control of murine melanoma by limiting MEK inhibitor induced skin toxicity.

The BRAF inhibitor (BRAFi) treatment has led to impressive responses in BRAF(V600E) mutation-positive melanomas, but responses are not durable in many patients. As most of the BRAFi escape mechanisms involve ERK reactivation, combinations with MEK inhibitors (MEKi) are currently tested to improve BRAFi-mediated response durations. Additionally, such a combination is expected to reduce MEKi-induced skin toxicities, as these drugs are thought to have antagonistic effects on ERK activation in keratinocytes. However, preclinical in vivo data exploring the combination of BRAFi and MEKi to achieve improved tumor control in the absence of skin toxicities are limited. Using a murine Tyr::CreER(T2);Pten(LoxP/LoxP);Braf(CA/+) melanoma model, we have determined the effect of BRAFi and MEKi treatment and their combination on melanoma control and occurrence of adverse events. We found that the MEKi dosed beyond the maximum tolerable dose (MTD) led to stronger control of tumor growth than did the BRAFi, but mice had to be removed from treatment because of skin toxicity. The combination of BRAFi and MEKi reduced MEKi-associated skin toxicity. This allowed high and long-term dosing of the MEKi, resulting in long-term tumor control. In contrast to previous hypotheses, the addition of a BRAFi did not restore the MEKi-mediated downregulation of pERK1/2 in skin cells. Our data describe, for the first time, the alleviation of MEKi-mediated dose-limiting toxicity by addition of a BRAFi in a mouse melanoma model. Additional clinical Phase I studies should be implemented to explore MEKi dosing beyond the single drug MTD in combination with BRAFi.

Targeting BRAFV600E in an inducible murine model of melanoma.

The MAP kinase and PI3 kinase pathways have been identified as the most common pathways that mediate oncogenic transformation in melanoma, and the majority of compounds developed for melanoma treatment target one or the other of these pathways. In addition to such targeted therapies, immunotherapeutic approaches have shown promising results. A combination of these two treatment modalities could potentially result in further improvement of treatment outcome. To preclinically identify efficient treatment combinations and to optimize therapy protocols in terms of sequence and timing, mouse models will be required. We have crossed and characterized the Tyr::CreER(T2);PTEN(F-/-);BRAF(F-V600E/+) inducible melanoma model on a C57BL/6J background. Tumors from this model harbor the BRAF(V600E) mutation and are PTEN-deficient, making them highly suitable for the testing of targeted therapies. Furthermore, we crossed the model onto this specific background for use in immunotherapy studies, because most experiments in this field have been performed in C57BL/6J mice. Selective inhibition of BRAF(V600E) by PLX4720 treatment of melanoma-bearing mice resulted in a strong decrease of tumor outgrowth. Furthermore, the inducible melanomas had immune cell infiltrates similar to those found in human melanoma, and tumor-infiltrating lymphocytes could be cultured from these tumors. Our data indicate that the C57BL/6J Tyr::CreER(T2);PTEN(F-/-);BRAF(F-V600E/+) melanoma model could be used as a standard model in which targeted and immunotherapy combinations can be tested in a high-throughput manner.

Expression of the embryological morphogen Nodal in stage III/IV melanoma.

The characterization of factors involved in the etiology of melanoma may lead to the identification of new targets for therapy and prognostic markers. Recent data indicate that Nodal can be expressed by malignant melanoma cells and this expression seems to contribute to melanoma development and progression. The aim of this study was to investigate the potential of Nodal as a prognostic marker for stage III/IV patients and as a treatment target for melanoma immunotherapy. We analyzed, by means of immunohistochemistry, 63 patients with stage III/IV melanoma for expression of Nodal in their tumors taken during the course of their disease. Furthermore, to research potential safety issues when immunotherapeutically targeting Nodal, we assessed Nodal expression in normal human adult tissues. We found that Nodal can be expressed at all stages of melanoma progression and there is no significant increase in the frequency of Nodal expression in distant metastases. Furthermore, our data show that there is no correlation between the expression of Nodal during course of disease and survival of patients. Finally, when screening for Nodal expression in normal adult organ tissues, we found consistent expression in renal tissue. We conclude that Nodal expression cannot be used as a prognostic marker for survival of patients with stage III/IV melanoma and as this putative target is renally expressed, it is not well-suited for immunotherapeutic approaches. This study suggests that, despite strong previous suggestions, the role of Nodal in melanoma progression may be less prominent and immunotherapeutic targeting of Nodal could be potentially harmful.

Combination of targeted therapy and immunotherapy in melanoma.

The treatment of human melanoma has progressed markedly in recent years. Building on the observation that immune recognition is a frequent event in melanoma, a series of immunotherapeutic approaches have been evaluated in clinical trials, culminating in the first phase III study improving overall survival of melanoma patients since 20 years. However, the response rates seen upon immunotherapeutic interventions such as anti-CTLA4 treatment are often low. Furthermore, clinical responses can take several weeks to develop, during which time stage IV melanoma patients often deteriorate. Recent advances in our understanding of the genetic lesions in human melanoma now also allow the specific targeting of the signaling pathway alterations in this disease. Such targeted therapies can lead to high response rates, although the duration of these responses is thus far relatively short. We suggest that the combination of immuno and targeted therapy offers potential for synergy for both conceptual and practical reasons. In this review, we will discuss the potential and possible limitations for such combination therapy, and we describe the most promising combinations of targeted therapy and immunotherapy that can be tested in the clinic in the coming years. The concept of induction therapy by small molecule administration and consolidation by immunotherapeutics also has potential for the treatment of other human cancers.

Overall survival and PD-L1 expression in metastasized malignant melanoma.

BACKGROUND: Cancers are known to elude the immune system, for example, by MHC loss, FAS up-regulation, or increased secretion of TGF-beta. Recently, ligands of coinhibitory receptors like programmed cell death ligand-1 (PD-L1, B7-H1) have come to attention for their role in tumor immune escape. Various tumors have been tested for PD-L1 expression, and conflicting results were obtained regarding its correlative impact on patient survival. This study aimed to determine the prognostic relevance of PD-L1 expression for the survival of melanoma patients. METHODS: Paraffin-embedded nevi, primary melanoma, and in-transit, lymph node, and distant organ metastases from a set of 63 stages III-IV melanoma patients referred to the Netherlands Cancer Institute between 2000 and 2004 for a sentinel-node procedure or systemic therapy were studied. A large effort was invested in validating specific PD-L1 staining. In addition to immunological factors such as T-cell infiltration (CD8, CD4, and regulatory T cells), TGF-beta and MHC-I expression were assessed. RESULTS: Longitudinal analysis revealed no relevant PD-L1 expression on primary melanoma compared with metastatic disease. No significant correlations with prognosis were found regarding immunological factors, whereas known prognostic markers such as Breslow thickness and sex could be confirmed. Analyses of the overall survival of our patient cohort did not reveal a negative association with PD-L1 expression. CONCLUSIONS: Correlation of overall survival with PD-L1 expression by melanoma cells remains controversial, and future clinical studies should focus on antibody validation and time of analysis in respect to disease progression.

Lethal graft-versus-host disease in mouse models of T cell receptor gene therapy.

The transfer of T cell receptor (TCR) genes can be used to induce immune reactivity toward defined antigens to which endogenous T cells are insufficiently reactive. This approach, which is called TCR gene therapy, is being developed to target tumors and pathogens, and its clinical testing has commenced in patients with cancer. In this study we show that lethal cytokine-driven autoimmune pathology can occur in mouse models of TCR gene therapy under conditions that closely mimic the clinical setting. We show that the pairing of introduced and endogenous TCR chains in TCR gene-modified T cells leads to the formation of self-reactive TCRs that are responsible for the observed autoimmunity. Furthermore, we demonstrate that adjustments in the design of gene therapy vectors and target T cell populations can be used to reduce the risk of TCR gene therapy-induced autoimmune pathology.