Patient perception of the benefit of a BRAF inhibitor in metastatic melanoma: quality-of-life analyses of the BREAK-3 study comparing dabrafenib with dacarbazine.

BACKGROUND: In a randomized phase III study (BREAK-3), dabrafenib showed prolonged progression-free survival (PFS) (median 5.1 versus 2.7 months; hazard ratio = 0.30; 95% confidence interval 0.18-0.53; P < 0.0001) compared with dacarbazine (DTIC) in patients with BRAF V600E metastatic melanoma. Assessing how these results are transformed into a real health benefit for patients is crucial. METHODS: The EORTC QLQ-C30 questionnaire assessed quality of life (QoL) at baseline and follow-up visits. RESULTS: For DTIC, all functional dimensions except role dimension worsened from baseline at follow-up. For dabrafenib, all functionality dimensions remained stable relative to baseline or improved at week 6; mean change in seven symptom dimensions improved from baseline, with appetite loss, insomnia, nausea and vomiting, and pain showing the greatest improvement. In the DTIC arm, symptom dimensions were unchanged or worsened from baseline for all symptoms except pain (week 6), with the greatest exacerbations observed for fatigue and nausea and vomiting. Mixed-model-repeated measures analyses showed significant (P < 0.05) and/or clinically meaningful improvements from baseline in favor of dabrafenib for emotional and social functioning, nausea and vomiting, appetite loss, diarrhea, fatigue, dyspnea, and insomnia at weeks 6 and/or 12. After crossing over to dabrafenib upon progression (n = 35), improvements in all QoL dimensions were evident after receiving dabrafenib for 6 (n = 31) to 12 (n = 25) weeks. CONCLUSIONS: This first reported QoL analysis for a BRAF inhibitor in metastatic melanoma demonstrates that the high tumor response rates and PFS superiority of dabrafenib over DTIC is not only a theoretical advantage, but also transforms in a rapid functional and symptomatic benefit for the patient. ClinicalTrials.gov Identifier: NCT01227889.

Transfer of mRNA encoding recombinant immunoreceptors reprograms CD4+ and CD8+ T cells for use in the adoptive immunotherapy of cancer.

Human T lymphocytes can be redirected with a new defined specificity by expression of a chimeric T-cell receptor (immunoreceptor) for the use in adoptive immunotherapy of cancer. Whereas standard procedures use retroviral gene transduction to constitutively express immunoreceptors in T cells, we here explored for the first time mRNA electroporation to achieve transient immunoreceptor expression, and thereby minimizing the risk of persistence of potential autoaggression. CD4(+) and CD8(+) T cells were efficiently transfected with immunoreceptors specific for ErbB2 and CEA. The immunoreceptor expression was transient with half-maximal expression at day 2 and no detectable immunoreceptor expression at day 9 after electroporation. Immunoreceptor-transfected T cells were specifically activated upon coincubation with ErbB2(+) and CEA(+) tumor cells, respectively, resulting in secretion of interferon-gamma (IFNgamma), interleukin-2 (IL-2), and tumor necrosis factor-alpha (TNFalpha). Furthermore, immunoreceptor-transfected CD8(+) T cells specifically lysed ErbB2(+) and CEA(+) tumor cells, respectively. The RNA-transfected T cells retained their cytotoxic function after 2 days of activation and exhibited cytolytic activities like retrovirally transduced T cells. RNA electroporation of T cells thereby provides a versatile tool for transient immunoreceptor expression, which may be of advantage in avoiding the persistence of unintended autoaggression.

[Malignant melanoma of the lacrimal sac]. / Malignes Melanom des Tränensacks.

A 68-year-old woman presented with a 10-month history of right-sided epiphora, bloody tears, and medial canthal mass. Computed tomography revealed a soft tissue mass of the right lacrimal sac with widening of the bony nasolacrimal canal. External dacryocystorhinostomy with incisional biopsy confirmed the diagnosis of malignant melanoma. After staging, further therapy included orbital exenteration, lateral rhinotomy with en bloc resection of the lacrimal drainage apparatus, and adjuvant radioimmunotherapy. One year after surgery, no evidence of local recurrence or metastatic disease could be detected.

Dacarbazine (DTIC) versus vaccination with autologous peptide-pulsed dendritic cells (DC) in first-line treatment of patients with metastatic melanoma: a randomized phase III trial of the DC study group of the DeCOG.

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.

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-gamma (IFN-gamma)-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 TCRVbeta14+ T-cell clone in this population over the whole observation period. Intracellular fluorescence-activated cell sorter staining of such TCRVbeta14+ T cells for IFN-gamma 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.

Treatment of disseminated ocular melanoma with sequential fotemustine, interferon alpha, and interleukin 2.

Malignant melanoma of the uvea is remarkable for purely haematogenous dissemination and its tendency to metastasise to the liver. Although the liver is involved in up to 95% of patients, 50% of these also develop extrahepatic metastases, most often in the lungs, bone, skin, and brain. The only effective treatments reported to date relied on hepatic arterial chemoembolisation or -perfusion. The objective of this study was to establish a therapy protocol addressing patients with both sole liver involvement and systemic disease. Forty-eight patients with metastatic ocular melanoma received fotemustine 100 mg m(-2) either as 60-min infusion into the hepatic artery or as 15-min infusion via a peripheral vein, depending on the metastatic sites involved, i.e., restriction to the liver or hepatic together with extrahepatic disease. For the first treatment cycle this infusion was repeated after one week. For all cycles, subsequent to a three week resting period, patients received an immunotherapy consisting of subcutaneous interleukin 2 and interferon alpha(2). Although objective responses were more frequent within the cohort receiving intraarterial fotemustine (21.7 vs 8%), this difference did not translate into a significant benefit in overall survival, i.e., 369 and 349 days, respectively. Of note, this overall survival is much longer than that repeatedly reported for stage IV uveal melanoma not treated with fotemustine, suggesting a therapeutic activity of this cytostatic drug even after systemic administration.

Induction of systemic CTL responses in melanoma patients by dendritic cell vaccination: cessation of CTL responses is associated with disease progression.

Two HLA-A2-positive patients with advanced stage IV melanoma were treated with monocyte-derived dendritic cells (DC) pulsed with either tumor peptide antigens from gp100, MART-1 and MAGE-3 alone or in combination with autologous oncolysates. Clinically, the rapid progression of disease was substantially stalled and both patients were alive for more than 15 months after initiation of therapy. Specific CTL reactivity against several tumor antigens was detectable in peripheral blood, which declined just before reactivation of disease progression. Furthermore, CD3 zeta-chain expression detected by Western lotting was decreased in PBL at this time. In summary, our data confirm that DC-based vaccinations induce peptide-specific T cells in the peripheral blood of advanced-stage melanoma patients. Although successful induction of systemic tumor antigen-specific CTL may not lead to objective clinical tumor regression, their presence are indicative of a prolonged survival.

Interaction of Neisseria meningitidis with human dendritic cells.

Infection with Neisseria meningitidis serogroup B is responsible for fatal septicemia and meningococcal meningitis. The severity of disease directly correlates with the production of the proinflammatory cytokines tumor necrosis factor alpha (TNF-alpha), interleukin-1 (IL-1), IL-6, and IL-8. However, the source of these cytokines has not been clearly defined yet. Since bacterial infection involves the activation of dendritic cells (DCs), we analyzed the interaction of N. meningitidis with monocyte-derived DCs. Using N. meningitidis serogroup B wild-type and unencapsulated bacteria, we found that capsule expression significantly impaired neisserial adherence to DCs. In addition, phagocytic killing of the bacteria in the phagosome is reduced by at least 10- to 100-fold. However, all strains induced strong secretion of proinflammatory cytokines TNF-alpha, IL-6, and IL-8 by DCs (at least 1,000-fold at 20 h postinfection [p.i.]), with significantly increased cytokine levels being measurable by as early as 6 h p.i. Levels of IL-1beta, in contrast, were increased only 200- to 400-fold at 20 h p.i. with barely measurable induction at 6 h p.i. Moreover, comparable amounts of cytokines were induced by bacterium-free supernatants of Neisseria cultures containing neisserial lipooligosaccharide as the main factor. Our data suggest that activated DCs may be a significant source of high levels of proinflammatory cytokines in neisserial infection and thereby may contribute to the pathology of meningococcal disease.

[Nevus of Ota and choroid melanoma]. / Nävus Ota und Aderhautmelanom.

A 59-year-old patient presented with metastatic uveal melanoma that developed in a nevus of Ota. The nevus of Ota or oculodermal melanocytosis contains an increased number of dermal melanocytes in the distribution of the ophthalmic and maxillary divisions of the trigeminal nerve. Malignant transformation arising in a nevus of Ota may occur in all anatomical sites influenced by the nevus. Most often the choroid is involved. Although the nevus of Ota is rare in Caucasians, associated melanoma is more common than in Asians or black people. The dermatologist should be aware of this problem and aim at an interdisciplinary management of these patients.

Antibodies against listerial protein 60 act as an opsonin for phagocytosis of Listeria monocytogenes by human dendritic cells.

Human-monocyte-derived dendritic cells (MoDC) are very efficient in the uptake of Listeria monocytogenes, a gram-positive bacterium which is an important pathogen in humans and animals causing systemic infections with symptoms such as septicemia and meningitis. In this work, we analyzed the influence of blood plasma on the internalization of L. monocytogenes into human MoDC and compared the uptake of L. monocytogenes with that of Salmonella enterica serovar Typhimurium and Yersinia enterocolitica. While human plasma did not significantly influence the uptake of serovar Typhimurium and Y. enterocolitica by human MoDC, the efficiency of the uptake of L. monocytogenes by these phagocytes was strongly enhanced by human plasma. In plasma-free medium the internalization of L. monocytogenes was very low, whereas the addition of pooled human immunoglobulins resulted in the internalization of these bacteria to a degree comparable to the highly efficient uptake observed with human plasma. All human plasma tested contained antibodies against the 60-kDa extracellular protein of L. monocytogenes (p60), and anti-p60 antibodies were also found in the commercially available pooled immunoglobulins. Strikingly, in contrast to L. monocytogenes wild type, an iap deletion mutant (totally deficient in p60) showed only a minor difference in the uptake by human MoDC in the presence or the absence of human plasma. These results support the assumption that antibodies against the listerial p60 protein may play an important role in Fc-receptor-mediated uptake of L. monocytogenes by human MoDC via opsonization of the bacteria. This process may have a major impact in preventing systemic infection in L. monocytogenes in immunocompetent humans.

Isolation, enrichment, and culture of murine epidermal langerhans cells.

Langerhans cells are the epidermal variant of the dendritic cell system (1-5). They were-unknowingly, though-the first dendritic cell to be described: In 1868 Paul Langerhans published his observations of a dendritically shaped cell in the human epidermis (6). Until the early 1990s (i.e., the advent of methods for mass production of dendritic cells from blood or bone marrow) Langerhans cells served as a prototype dendritic cell. The basis of our current knowledge on the maturation of dendritic cells stems from experiments with epidermal Langerhans cells (7-9).

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.

Dendritic cell activation by danger and antigen-specific T-cell signalling.

Recent transplantation, animal and in vitro studies suggest a dependence of some immune reactions on tissue damage. Although many factors involved in enhancing immune responses through tissue damage have yet to be identified, recent data suggests that one of the targets of these cellular stress factors is the bone marrow derived dendritic cell (DC). DC are potent initiators of primary immune responses and hold the key to immune reactions through their ability to sense changes in their local environment and respond appropriately to induce T-cell immunity, or possibly tolerance. In the lymph node, DC are also influenced by antigen-specific signalling from T cells, which may extend and amplify DC antigen presenting capabilities, especially for the stimulation of cytotoxic responses. It now appears that both tissue damage and antigen-specific T-cell derived signals act together on the DC to promote the appropriate immune reaction to antigen. Thus DC antigen presenting behaviour is not only dependent on the context of antigen encounter in the periphery, but also on the availability of antigen-specific T cells and their T-cell receptor specificities.

Antigen presentation in extracellular matrix: interactions of T cells with dendritic cells are dynamic, short lived, and sequential.

Cognate interactions of naive T cells with antigen-presenting dendritic cells require physical cell-cell contacts leading to signal induction and T cell activation. Using a three-dimensional collagen matrix videomicroscopy model for ovalbumin peptide-specific activation of murine and oxidative mitogenesis of human T cells, we show that T cells maintain vigorous migration upon cognate interactions to DC (dendritic cell), continuously crawl across the DC surface, and rapidly detach (median within 6-12 min). These dynamic and short-lived encounters favor sequential contacts with the same or other DC and trigger calcium influx, upregulation of activation markers, T blast formation, and proliferation. We conclude that a tissue environment supports the accumulation of sequential signals, implicating a numeric or "digital" control mechanism for an ongoing primary immune response.

Classical chemotherapy for metastatic melanoma.

Despite a major effort in clinical research scrutinizing various treatment regimens for patients suffering from metastatic melanoma the prognosis for these patients still remains poor. The treatment options tested have ranged from monochemotherapy, polychemotherapeutic approaches including highly toxic regimens requiring autologous bone marrow rescue, immuno-modulatory therapies, e.g. defined cytokines such as interferon-alpha and interleukin-2 as well as vaccination therapy with dendritic cells or genetically modified tumour cells, and bio-chemotherapy. Although immuno-modulatory approaches are currently regarded as the most promising, to date no improved overall survival has been achieved by any of these measures especially if tested in large multicentre trials. The focus of this review will be on classical chemotherapy with emphasis on both cutaneous and uveal melanoma.

Mage-3 and influenza-matrix peptide-specific cytotoxic T cells are inducible in terminal stage HLA-A2.1+ melanoma patients by mature monocyte-derived dendritic cells.

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.

Maturation of dendritic cells leads to up-regulation of cellular FLICE-inhibitory protein and concomitant down-regulation of death ligand-mediated apoptosis.

Dendritic cells (DCs) disappear from lymph nodes 1 to 2 days after antigen presentation, presumably by apoptosis. To evaluate the role of death ligands in elimination of DCs, we analyzed the sensitivity of human DCs to CD95 ligand (CD95L) and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). We found mature DCs to be resistant to killing via CD95L or TRAIL, whereas only immature DCs were partially sensitive. However, all DC populations expressed CD95, TRAIL-R2, and TRAIL-R3 at comparable levels, suggesting that sensitivity to death ligand-induced DC apoptosis is not regulated at the receptor level. Interestingly, mature DCs highly expressed the caspase 8 inhibitory protein cFLIP, whereas only low levels were detected in immature DCs. Thus, death ligand sensitivity proved to be dependent on DC maturation and inversely correlated with expression levels of cFLIP. Induction of apoptosis by TRAIL or CD95L does not seem to play a role in the elimination of mature DCs, but instead might serve to regulate immature DC populations.

MHC class II and CD40 play opposing roles in dendritic cell survival.

In contrast to very immature dendritic cells (DC), mature DC are largely resistant to death by CD95 (CD95/APO-1) ligation. Investigation of other potential death-inducing ligands showed that mature DC were instead highly susceptible to apoptosis induced by cross-linking of MHC class II. Thus, increasing DC maturity correlates with increased resistance to CD95 killing, but an increased susceptibility to class II-mediated killing. Anti-I-A/I-E monoclonal antibodies (mAb) induced rapid (<2 h) apoptotic cell death in mature epidermal, spleen and bone marrow-derived DC, as determined by annexin/propidium iodide staining, morphological changes, decreased diploidy and loss in mitochondrial membrane potential. Although full class II-mediated killing required DC cytoskeletal motion, divalent cations and phosphatase activity, neither caspase activation, respiration, RNA or protein synthesis, NO production, nor CD95:CD95L interactions were required. Strikingly, DC pretreated by CD40 mAb cross-linking, but not by lipopolysaccharide or TNF-alpha, were completely resistant to class II-mediated killing. CD40-mediated protection was reduced in the presence of the SB202190 inhibitor of the mitogen-activated protein kinase p38 pathway, but appeared to be independent of p42/44 extracellular signal-related kinase or NF-KB activation. Our findings show that in addition to its role as an activator of antigen-presenting cell function, CD40 provides an important counter-signal against class II-induced apoptosis. Thus, these data point to an important role of the T cell in regulating DC survival.