Management of early melanoma recurrence despite adjuvant anti-PD-1 antibody therapy☆.

BACKGROUND: Anti-programmed cell death protein 1 (PD-1) antibodies (PD1) prolong recurrence-free survival in high-risk resected melanoma; however, approximately 25%-30% of patients recur within 1 year. This study describes the pattern of recurrence, management and outcomes of patients who recur with adjuvant PD1 therapy. PATIENTS AND METHODS: Consecutive patients from 16 centres who recurred having received adjuvant PD1 therapy for resected stage III/IV melanoma were studied. Recurrence characteristics, management and outcomes were examined; patients with mucosal melanoma were analysed separately. RESULTS: Melanoma recurrence occurred in 147 (17%) of ∼850 patients treated with adjuvant PD1. In those with cutaneous melanoma (n = 136), median time to recurrence was 4.6 months (range 0.3-35.7); 104 (76%) recurred during (ON) adjuvant PD1 after a median 3.2 months and 32 (24%) following (OFF) treatment cessation after a median 12.5 months, including in 21 (15%) who ceased early for toxicity. Fifty-nine (43%) recurred with locoregional disease only and 77 (57%) with distant disease. Of those who recurred locally, 22/59 (37%) subsequently recurred distantly. Eighty-nine (65%) patients received systemic therapy after recurrence. Of those who recurred ON adjuvant PD1, none (0/6) responded to PD1 alone; 8/33 assessable patients (24%) responded to ipilimumab (alone or in combination with PD1) and 18/23 (78%) responded to BRAF/MEK inhibitors. Of those who recurred OFF adjuvant PD1, two out of five (40%) responded to PD1 monotherapy, two out of five (40%) responded to ipilimumab-based therapy and 9/10 (90%) responded to BRAF/MEK inhibitors. CONCLUSIONS: Most patients who recur early despite adjuvant PD1 develop distant metastases. In those who recur ON adjuvant PD1, there is minimal activity of further PD1 monotherapy, but ipilimumab (alone or in combination with PD1) and BRAF/MEK inhibitors have clinical utility. Retreatment with PD1 may have activity in select patients who recur OFF PD1.

Five-year survival outcomes for patients with advanced melanoma treated with pembrolizumab in KEYNOTE-001.

BACKGROUND: Pembrolizumab demonstrated robust antitumor activity and safety in the phase Ib KEYNOTE-001 study (NCT01295827) of advanced melanoma. Five-year outcomes in all patients and treatment-naive patients are reported herein. Patients whose disease progressed following initial response and who received a second course of pembrolizumab were also analyzed. PATIENTS AND METHODS: Patients aged ≥18 years with previously treated or treatment-naive advanced/metastatic melanoma received pembrolizumab 2 mg/kg every 3 weeks, 10 mg/kg every 3 weeks, or 10 mg/kg every 2 weeks until disease progression, intolerable toxicity, or patient/investigator decision to withdraw. Kaplan-Meier estimates of overall survival (OS) and progression-free survival (PFS) were calculated. Objective response rate and PFS were based on immune-related response criteria by investigator assessment (data cut-off, September 1, 2017). RESULTS: KEYNOTE-001 enrolled 655 patients with melanoma; median follow-up was 55 months. Estimated 5-year OS was 34% in all patients and 41% in treatment-naive patients; median OS was 23.8 months (95% CI, 20.2-30.4) and 38.6 months (95% CI, 27.2-not reached), respectively. Estimated 5-year PFS rates were 21% in all patients and 29% in treatment-naive patients; median PFS was 8.3 months (95% CI, 5.8-11.1) and 16.9 months (95% CI, 9.3-35.5), respectively. Median response duration was not reached; 73% of all responses and 82% of treatment-naive responses were ongoing at data cut-off; the longest response was ongoing at 66 months. Four patients [all with prior response of complete response (CR)] whose disease progressed during observation subsequently received second-course pembrolizumab. One patient each achieved CR and partial response (after data cut-off). Treatment-related AEs (TRAEs) occurred in 86% of patients and resulted in study discontinuation in 7.8%; 17% experienced grade 3/4 TRAE. CONCLUSIONS: This 5-year analysis of KEYNOTE-001 represents the longest follow-up for pembrolizumab to date and confirms the durable antitumor activity and tolerability of pembrolizumab in advanced melanoma. CLINICAL TRIAL REGISTRY: ClinicalTrials.gov, NCT01295827.

Repression of microRNA-768-3p by MEK/ERK signalling contributes to enhanced mRNA translation in human melanoma.

Increased global protein synthesis and selective translation of mRNAs encoding proteins contributing to malignancy is common in cancer cells. This is often associated with elevated expression of eukaryotic translation initiation factor 4 (eIF4E), the rate-limiting factor of cap-dependent translation initiation. We report here that in human melanoma downregulation of miR-768-3p as a result of activation of the mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase (ERK) pathway has an important role in the upregulation of eIF4E and enhancement in protein synthesis. Melanoma cells displayed increased nascent protein production and elevated eIF4E expression, which was associated with the downregulation of miR-768-3p that was predicted to target the 3'-untranslated region of the eIF4E mRNA. Overexpression of miR-768-3p led to the downregulation of the endogenous eIF4E protein, reduction in nascent protein synthesis and inhibition of cell survival and proliferation. These effects were efficiently reversed when eIF4E was co-overexpressed in melanoma cells. On the other hand, introduction of anti-miR-768-3p into melanocytes upregulated endogenous eIF4E protein expression and increased global protein synthesis. Downregulation of miR-768-3p appeared to be mediated by activation of the MEK/ERK pathway, in that treatment of BRAF(V600E) melanoma cells with the mutant BRAF inhibitor PLX4720 or exposure of either BRAF(V600E) or wild-type BRAF melanoma cells to the MEK inhibitor U0126 resulted in the upregulation of miR-768-3p and inhibition of nascent protein synthesis. This inhibition was partially blocked in cells cointroduced with anti-miR-768-3p. Significantly, miR-768-3p was similarly downregulated, which was inversely associated with the expression levels of eIF4E in fresh melanoma isolates. Taken together, these results identify downregulation of miR-768-3p and subsequent upregulation of eIF4E as an important mechanism in addition to phosphorylation of eIF4E responsible for MEK/ERK-mediated enhancement of protein synthesis in melanoma.

Cotargeting histone deacetylases and oncogenic BRAF synergistically kills human melanoma cells by necrosis independently of RIPK1 and RIPK3.

Past studies have shown that histone deacetylase (HDAC) and mutant BRAF (v-Raf murine sarcoma viral oncogene homolog B1) inhibitors synergistically kill melanoma cells with activating mutations in BRAF. However, the mechanism(s) involved remains less understood. Here, we report that combinations of HDAC and BRAF inhibitors kill BRAF(V600E) melanoma cells by induction of necrosis. Cotreatment with the HDAC inhibitor suberoylanilide hydroxamic acid (SAHA) or panobinostat (LBH589) and the BRAF inhibitor PLX4720 activated the caspase cascade, but caspases appeared dispensable for killing, in that inhibition of caspases did not invariably block induction of cell death. The majority of dying cells acquired propidium iodide positivity instantly when they became positive for Annexin V, suggesting induction of necrosis. This was supported by caspase-independent release of high-mobility group protein B1, and further consolidated by rupture of the plasma membrane and loss of nuclear and cytoplasmic contents, as manifested by transmission electron microscopic analysis. Of note, neither the necrosis inhibitor necrostatin-1 nor the small interference RNA (siRNA) knockdown of receptor-interacting protein kinase 3 (RIPK3) inhibited cell death, suggesting that RIPK1 and RIPK3 do not contribute to induction of necrosis by combinations of HDAC and BRAF inhibitors in BRAF(V600E) melanoma cells. Significantly, SAHA and the clinically available BRAF inhibitor vemurafenib cooperatively inhibited BRAF(V600E) melanoma xenograft growth in a mouse model even when caspase-3 was inhibited. Taken together, these results indicate that cotreatment with HDAC and BRAF inhibitors can bypass canonical cell death pathways to kill melanoma cells, which may be of therapeutic advantage in the treatment of melanoma.

Suppression of PP2A is critical for protection of melanoma cells upon endoplasmic reticulum stress.

Endoplasmic reticulum (ER) stress triggers apoptosis by activating Bim in diverse types of cells, which involves dephosphorylation of Bim(EL) by protein phosphatase 2A (PP2A). However, melanoma cells are largely resistant to ER stress-induced apoptosis, suggesting that Bim activation is suppressed in melanoma cells undergoing ER stress. We show here that ER stress reduces PP2A activity leading to increased ERK activation and subsequent phosphorylation and proteasomal degradation of Bim(EL). Despite sustained upregulation of Bim at the transcriptional level, the Bim(EL) protein expression was downregulated after an initial increase in melanoma cells subjected to pharmacological ER stress. This was mediated by increased activity of ERK, whereas the phosphatase activity of PP2A was reduced by ER stress in melanoma cells. The increase in ERK activation was, at least in part, due to reduced dephosphorylation by PP2A, which was associated with downregulation of the PP2A catalytic C subunit. Notably, instead of direct dephosphorylation of Bim(EL), PP2A inhibited its phosphorylation indirectly through dephosphorylation of ERK in melanoma cells. Taken together, these results identify downregualtion of PP2A activity as an important protective mechanism of melanoma cells against ER stress-induced apoptosis.

Ets-1 mediates upregulation of Mcl-1 downstream of XBP-1 in human melanoma cells upon ER stress.

Past studies have shown that upregulation of the anti-apoptotic Bcl-2 family protein Mcl-1 is a major adaptive mechanism of melanoma cells to endoplasmic reticulum (ER) stress, and has an important role in resistance of the cells to apoptosis. In this study, we show that the increase in transcription of Mcl-1 in melanoma cells triggered by pharmacological ER stress inducers is mediated by the transcription factor Ets-1. By incremental deletion analysis of the Mcl-1 promoter, we identified a DNA fragment containing an Ets-1 binding site that is transcriptionally responsive to ER stress. Mutations in the Ets-1 binding site or knockdown of Ets-1 inhibited the increase in Mcl-1, indicating that Ets-1 has a critical role in transcriptional upregulation of Mcl-1. Similar to Mcl-1, Ets-1 was transcriptionally upregulated by ER stress. This was mediated by the IRE1α/XBP-1 branch of the unfolded protein response, as upregulation of Ets-1 was inhibited in melanoma cell lines deficient in IRE1α or XBP-1 established by short hairpin RNA knockdown. Activation of the PI3k/Akt pathway downstream of XBP-1 was also involved, in that inhibition of the pathway blocked upregulation of Ets-1. Inhibition of Ets-1 enhanced ER stress-induced apoptosis in melanoma cell lines and in fresh melanoma isolates, recapitulating the effect of inhibition of Mcl-1. These results reveal a key mechanism by which Mcl-1 is transcriptionally upregulated in melanoma cells by ER stress, and identify Ets-1 as a potential target for inhibition to sensitize melanoma cells to apoptosis.

Meeting report from the 7th International Melanoma Congress, Sydney, November, 2010.

The 2010 7th International Melanoma Congress sponsored by the Society for Melanoma Research and held in Sydney, Australia, was held together with the International Melanoma and Skin Cancer Centers group and the International Melanoma Pathology Study Group. As a consequence, there were over 900 registrants that included a wide range of clinicians (surgeons, medical oncologists, dermatologists) specialising in the management of melanoma as well as scientists and students carrying out laboratory-based research in melanoma. There was a general consensus that this grouping of clinicians, pathologists and scientists was mutually advantageous and plans are afoot to continue this grouping in future meetings. The meeting was dominated by the advances being made in treatment of melanoma with selective BRAF inhibitors but interest in epithelial mesenchymal transition and phenotypic changes in melanoma was apparent in many of the talks. The authors have attempted to capture many of the new developments in melanoma research but apologize to those speakers and poster presenters who had equally important findings not captured in these summaries.

Cystatin B inhibition of TRAIL-induced apoptosis is associated with the protection of FLIP(L) from degradation by the E3 ligase itch in human melanoma cells.

Past studies have identified a number of distinct mechanisms that contribute to the resistance of melanoma cells against apoptosis induced by TNF-related apoptosis-inducing ligand (TRAIL). In this report we show that cystatin B is another endogenous inhibitor of TRAIL-induced apoptosis. Cystatin B-deficient melanoma cell lines established by shRNA knockdown displayed increased apoptosis that was associated with enhanced activation of caspase-8 induced by TRAIL. This was not related to the inhibitory effect of cystatin B on the lysosomal cysteine proteases, cathepsin B and L, as they did not have a role in TRAIL-induced apoptosis in most melanoma cell lines even when cystatin B was inhibited. Instead, sensitization of melanoma cells to TRAIL-induced apoptosis by inhibition of cystatin B appeared associated with decreased stability of FLIP(L) as the levels of FLIP(L) were reduced because of shortened half-life time in melanoma cells deficient in cystatin B. In contrast, over-expression of cystatin B increased the levels of FLIP(L), decreased the amount of the E3 ligase Itch associated with FLIP(L), and reduced FLIP(L) ubiquitination. Inhibition of Itch by siRNA restored the levels of FLIP(L) and blocked sensitization to TRAIL-induced apoptosis associated with deficiency in cystatin B. Taken together, these results indicate that cystatin B regulates Itch-mediated degradation of FLIP(L) and thereby TRAIL-induced apoptosis in melanoma cells.

Apoptosis of human melanoma cells induced by inhibition of B-RAFV600E involves preferential splicing of bimS.

Bim is known to be critical in killing of melanoma cells by inhibition of the RAF/MEK/ERK pathway. However, the potential role of the most potent apoptosis-inducing isoform of Bim, Bim(S), remains largely unappreciated. Here, we show that inhibition of the mutant B-RAF(V600E) triggers preferential splicing to produce Bim(S), which is particularly important in induction of apoptosis in B-RAF(V600E) melanoma cells. Although the specific B-RAF(V600E) inhibitor PLX4720 upregulates all three major isoforms of Bim, Bim(EL), Bim(L), and Bim(S), at the protein and mRNA levels in B-RAF(V600E) melanoma cells, the increase in the ratios of Bim(S) mRNA to Bim(EL) and Bim(L) mRNA indicates that it favours Bim(S) splicing. Consistently, enforced expression of B-RAF(V600E) in wild-type B-RAF melanoma cells and melanocytes inhibits Bim(S) expression. The splicing factor SRp55 appears necessary for the increase in Bim(S) splicing, as SRp55 is upregulated, and its inhibition by small interfering RNA blocks induction of Bim(S) and apoptosis induced by PLX4720. The PLX4720-induced, SRp55-mediated increase in Bim(S) splicing is also mirrored in freshly isolated B-RAF(V600E) melanoma cells. These results identify a key mechanism for induction of apoptosis by PLX4720, and are instructive for sensitizing melanoma cells to B-RAF(V600E) inhibitors.

Utility of adjuvant systemic therapy in melanoma.

The lack of effective drugs in stage IV melanoma has impacted the effectiveness of adjuvant therapies in stage II/III disease. To date, chemotherapy, immunostimulants and vaccines have been used with minimal success. Interferon (IFN) has shown an effect on relapse-free survival (RFS) in several clinical trials; however, without a clinically significant effect on overall survival (OS). A recently conducted meta-analysis demonstrated prolongation of disease-free survival (DFS) in 7% and OS benefit in 3% of IFN-treated patients when compared with observation-only patients. There were no clear differences for the dose and duration of treatment observed. Observation is still an appropriate control arm in adjuvant clinical trials. Regional differences exist in Europe in the adjuvant use of IFN. In Northwest Europe, IFN is infrequently prescribed. In Central and Mediterranean Europe, dermatologists commonly prescribe low-dose IFN therapy for AJCC stage II and III disease. High-dose IFN regimens are not commonly used. The population of patients that may benefit from IFN needs to be further characterised, potentially by finding biomarkers that can predict response. Such studies are ongoing.

Small molecules and targeted therapies in distant metastatic disease.

Chemotherapy, biological agents or combinations of both have had little impact on survival of patients with metastatic melanoma. Advances in understanding the genetic changes associated with the development of melanoma resulted in availability of promising new agents that inhibit specific proteins up-regulated in signal cell pathways or inhibit anti-apoptotic proteins. Sorafenib, a multikinase inhibitor of the RAF/RAS/MEK pathway, elesclomol (STA-4783) and oblimersen (G3139), an antisense oligonucleotide targeting anti-apoptotic BCl-2, are in phase III clinical studies in combination with chemotherapy. Agents targeting mutant B-Raf (RAF265 and PLX4032), MEK (PD0325901, AZD6244), heat-shock protein 90 (tanespimycin), mTOR (everolimus, deforolimus, temsirolimus) and VEGFR (axitinib) showed some promise in earlier stages of clinical development. Receptor tyrosine-kinase inhibitors (imatinib, dasatinib, sunitinib) may have a role in treatment of patients with melanoma harbouring c-Kit mutations. Although often studied as single agents with disappointing results, new targeted drugs should be more thoroughly evaluated in combination therapies. The future of rational use of new targeted agents also depends on successful application of analytical techniques enabling molecular profiling of patients and leading to selection of likely therapy responders.

Immunotherapy of distant metastatic disease.

Immunotherapy of metastatic melanoma consists of various approaches leading to specific or non-specific immunomodulation. The use of FDA-approved interleukin (IL)-2 alone, in combination with interferon alpha, and/or with various chemotherapeutic agents (biochemotherapy) is associated with significant toxicity and poor efficacy that does not improve overall survival of 96% of patients. Many studies with allogeneic and autologous vaccines have demonstrated no clinical benefit, and some randomised trials even showed a detrimental effect in the vaccine arm. The ongoing effort to develop melanoma vaccines based on dendritic cells and peptides is driven by advances in understanding antigen presentation and processing, and by new techniques of vaccine preparation, stabilisation and delivery. Several agents that have shown promising activity in metastatic melanoma including IL-21 and monoclonal antibodies targeting cytotoxic T lymphocyte-associated antigen 4 (anti-CTLA-4) or CD137 are discussed. Recent advances of intratumour gene transfer technologies and adoptive immunotherapy, which represents a promising although technically challenging direction, are also discussed.

Biomarkers in melanoma.

Biomarkers are tumour- or host-related factors that correlate with tumour biological behaviour and patient prognosis. High-throughput analytical techniques--DNA and RNA microarrays--have identified numerous possible biomarkers, but their relevance to melanoma progression, clinical outcome and the selection of optimal treatment strategies still needs to be established. The review discusses a possible molecular basis for predictive tissue biomarkers such as melanoma thickness, ulceration and mitotic activity, and provides a list of promising new biomarkers identified from tissue microarrays that needs confirmation by independent, prospectively collected clinical data sets. In addition, common predictive serum biomarkers--lactate dehydrogenase, S100B and melanoma-inhibiting activity--as well as selected investigational serum biomarkers such as TA90IC and YKL-40 are also reviewed. A more accurate, therapeutically predictive classification of human melanomas and selection of patient populations that would profit from therapeutic interventions are among the major challenges expected to be addressed in the future.

Societal preference values for advanced melanoma health states in the United Kingdom and Australia.

BACKGROUND: No studies measure preference-based utilities in advanced melanoma that capture both intended clinical response and unintended toxicities associated with treatment. METHODS: Using standard gamble, utilities were elicited from 140 respondents in the United Kingdom and Australia for 13 health states. RESULTS: Preferences decreased with reduced treatment responsiveness and with increasing toxicity. CONCLUSIONS: These general population utilities can be incorporated into treatment-specific cost-effectiveness evaluations.

O(6)-methylguanine-DNA methyltransferase depletion and DNA damage in patients with melanoma treated with temozolomide alone or with lomeguatrib.

We evaluated the pharmacodynamic effects of the O(6)-methylguanine-DNA methyltransferase (MGMT) inactivator lomeguatrib (LM) on patients with melanoma in two clinical trials. Patients received temozolomide (TMZ) for 5 days either alone or with LM for 5, 10 or 14 days. Peripheral blood mononuclear cells (PBMCs) were isolated before treatment and during cycle 1. Where available, tumour biopsies were obtained after the last drug dose in cycle 1. Samples were assayed for MGMT activity, total MGMT protein, and O(6)-methylguanine (O(6)-meG) and N7-methylguanine levels in DNA. MGMT was completely inactivated in PBMC from patients receiving LM, but detectable in those on TMZ alone. Tumours biopsied on the last day of treatment showed complete inactivation of MGMT but there was recovery of activity in tumours sampled later. Significantly more O(6)-meG was present in the PBMC DNA of LM/TMZ patients than those on TMZ alone. LM/TMZ leads to greater MGMT inactivation, and higher levels of O(6)-meG than TMZ alone. Early recovery of MGMT activity in tumours suggested that more protracted dosing with LM is required. Extended dosing of LM completely inactivated PBMC MGMT, and resulted in persistent levels of O(6)-meG in PBMC DNA during treatment.

Phase I/II study of treatment with matured dendritic cells with or without low dose IL-2 in patients with disseminated melanoma.

BACKGROUND: In the present study, we have examined whether treatment of patients with metastatic melanoma with matured dendritic cell (DC) vaccines with or without low dose IL-2 may improve treatment outcomes. METHODS: Sixteen patients received DC vaccines (DCs) sensitized with autologous melanoma lysates and 18 patients received DCs sensitized with peptides from gp100, MART-1, tyrosinase, MAGE-3.A2, MAGE-A10 and NA17. IL-2 was given subcutaneously (sc) at 1 MU/m2 on the second day after each injection for 5-14 days in half of each group. DCs were given by intranodal injection. RESULTS: There were 2 partial responses (PR) and 3 with stable disease (SD) in the nine patients receiving DCs + peptides + IL-2, and 1 PR and 1 SD in nine patients treated with DCs + peptides without IL-2. There were only two patients with SD in the group receiving DCs + autologous lysates and no IL-2. Median overall survival for all patients was very good at 18.5 months but this was most probably due to selection of a favourable group of patients for the study. There was no significant difference in survival between the groups by log rank analysis. Treatment was not associated with significant side effects. The quality and yield of the DCs in the preparations were generally good. CONCLUSIONS: We conclude that mature DC preparations may be superior to immature DC preparations for presentation of melanoma peptides and that IL-2 may increase clinical responses to the DCs plus peptides. However, in our view the low response rates do not justify the cost and complexity of this treatment approach.

Temozolomide induces senescence but not apoptosis in human melanoma cells.

Temozolomide (TMZ), a DNA alkylating agent used in the treatment of melanoma, is believed to mediate its effect by addition of a methyl group to the O(6) position of guanine in DNA. Resistance to the agent may be in part due to the activity of O(6)-methylguanine-DNA methyl transferase (MGMT). In the present study, we show that sensitivity of melanoma cells to TMZ was dependent on their p53 status and levels of MGMT. Analysis of the mechanisms underlying reduced viability showed no evidence for induction of apoptosis even though marked levels of apoptosis was seen in TK6 lymphoma cells. Sensitivity of melanoma cells was associated with p53-dependent G2/M cell cycle arrest and induction of senescence. To verify the role of p53, the assays were repeated in presence of pifithrin-alpha, an inhibitor of p53. This resulted in increased viability of melanoma cells with wild-type p53 and reversed G2/M cell cycle arrest. Paradoxically, apoptosis was increased in melanoma but decreased as expected in TK6 lymphoma cells. These results are consistent with the view that TMZ is relatively ineffective against melanoma due to defective apoptotic signalling resulting from activation of p53. The nature of the defects in apoptotic signalling remains to be explored.