Targeting alternative splicing as a new cancer immunotherapy-phosphorylation of serine arginine-rich splicing factor (SRSF1) by SR protein kinase 1 (SRPK1) regulates alternative splicing of PD1 to generate a soluble antagonistic isoform that prevents T cell exhaustion.

BACKGROUND: Regulation of alternative splicing is a new therapeutic approach in cancer. The programmed cell death receptor 1 (PD-1) is an immunoinhibitory receptor expressed on immune cells that binds to its ligands, PD-L1 and PD-L2 expressed by cancer cells forming a dominant immune checkpoint pathway in the tumour microenvironment. Targeting this pathway using blocking antibodies (nivolumab and pembrolizumab) is the mainstay of anti-cancer immunotherapies, restoring the function of exhausted T cells. PD-1 is alternatively spliced to form isoforms that are either transmembrane signalling receptors (flPD1) that mediate T cell death by binding to the ligand, PD-L1 or an alternatively spliced, soluble, variant that lacks the transmembrane domain. METHODS: We used PCR and western blotting on primary peripheral blood mononuclear cells (PBMCs) and Jurkat T cells, IL-2 ELISA, flow cytometry, co-culture of melanoma and cholangiocarcinoma cells, and bioinformatics analysis and molecular cloning to examine the mechanism of splicing of PD1 and its consequence. RESULTS: The soluble form of PD-1, generated by skipping exon 3 (∆Ex3PD1), was endogenously expressed in PBMCs and T cells and prevents cancer cell-mediated T cell repression. Multiple binding sites of SRSF1 are adjacent to PD-1 exon 3 splicing sites. Overexpression of phosphomimic SRSF1 resulted in preferential expression of flPD1. Inhibition of SRSF1 phosphorylation both by SRPK1 shRNA knockdown and by a selective inhibitor, SPHINX31, resulted in a switch in splicing to ∆Ex3PD1. Cholangiocarcinoma cell-mediated repression of T cell IL-2 expression was reversed by SPHINX31 (equivalent to pembrolizumab). CONCLUSIONS: These results indicate that switching of the splicing decision from flPD1 to ∆Ex3PD1 by targeting SRPK1 could represent a potential novel mechanism of immune checkpoint inhibition in cancer.

Incidence, risk factors and outcomes of checkpoint inhibitor-induced liver injury: A 10-year real-world retrospective cohort study.

Background & Aims: Checkpoint inhibitors (CPI) account for increasing numbers of drug-induced liver injury (DILI) cases. We aimed to determine the incidence rate and risk factors associated with checkpoint inhibitor-induced liver injury (ChILI). Methods: Prescription event monitoring was performed on all melanoma and renal cancer patients who received CPI at a tertiary centre between 2011 and 2021. ChILI cases were identified using the definitions, grading, and causality assessment methods validated for DILI. We assessed risk factors associated with ChILI in CPI-naive patients using multivariable logistic regression model. Consecutive patients with suspected ChILI from two other tertiary centres were adjudicated and combined for case characterisation and outcomes of ChILI. Results: Out of 432 patients who received CPI over 10 years, ChILI occurred in 38 (8.8%) with an overall incidence rate of 11.5 per 1,000 person-months (95% CI 8.2-15.8). Probability of ChILI was highest in combination therapy (32%) and no new events occurred beyond 135 days of treatment. Risk factor analysis showed that combination therapy, female sex, higher baseline alanine transferase level and lower baseline alkaline phosphatase level were independently associated with higher risk of ChILI. In total, 99 patients were adjudicated to have ChILI from three centres. Although Common Terminology Criteria for Adverse Events classified 20 patients (20.2%) to have 'life-threatening' grade 4 hepatitis, ChILI severity was graded as mild in 45 (45.5%) and moderate in the remaining 54 (54.5%) cases. Conclusions: The real-world risk of ChILI is higher than previously reported. Among patients receiving dual CPI, this risk falls markedly after 4.5 months. As Common Terminology Criteria for Adverse Events overestimates its clinical severity, case-definition, evaluation and management of ChILI should be revised to harmonise care. Impact and implications: Using prescription event monitoring over a 10-year period, the incidence rate of checkpoint inhibitor induced liver injury (ChILI) based on established case definitions for drug-induced liver injury (DILI) is 11.5 per 1,000 person-months. Formal causality assessment identified an alternative cause in 19% of patients with suspected ChILI highlighting the importance of systematic evaluation by clinicians to minimise unnecessary immunosuppression. Intensity of monitoring in patients receiving combination therapy regime after 4.5 months of therapy can be reduced as the risk of new onset ChILI beyond this point is minimal. Current Common Terminology Criteria for Adverse Events (CTCAE) grading overestimates clinical severity of ChILI and hence contributes to avoidable hospitalisation.

Application of Genomic Sequencing to Refine Patient Stratification for Adjuvant Therapy in Renal Cell Carcinoma.

PURPOSE: Patients with resected localized clear-cell renal cell carcinoma (ccRCC) remain at variable risk of recurrence. Incorporation of biomarkers may refine risk prediction and inform adjuvant treatment decisions. We explored the role of tumor genomics in this setting, leveraging the largest cohort to date of localized ccRCC tissues subjected to targeted gene sequencing. EXPERIMENTAL DESIGN: The somatic mutation status of 12 genes was determined in 943 ccRCC cases from a multinational cohort of patients, and associations to outcomes were examined in a Discovery (n = 469) and Validation (n = 474) framework. RESULTS: Tumors containing a von-Hippel Lindau (VHL) mutation alone were associated with significantly improved outcomes in comparison with tumors containing a VHL plus additional mutations. Within the Discovery cohort, those with VHL+0, VHL+1, VHL+2, and VHL+≥3 tumors had disease-free survival (DFS) rates of 90.8%, 80.1%, 68.2%, and 50.7% respectively, at 5 years. This trend was replicated in the Validation cohort. Notably, these genomically defined groups were independent of tumor mutational burden. Amongst patients eligible for adjuvant therapy, those with a VHL+0 tumor (29%) had a 5-year DFS rate of 79.3% and could, therefore, potentially be spared further treatment. Conversely, patients with VHL+2 and VHL+≥3 tumors (32%) had equivalent DFS rates of 45.6% and 35.3%, respectively, and should be prioritized for adjuvant therapy. CONCLUSIONS: Genomic characterization of ccRCC identified biologically distinct groups of patients with divergent relapse rates. These groups account for the ∼80% of cases with VHL mutations and could be used to personalize adjuvant treatment discussions with patients as well as inform future adjuvant trial design.

Resistance Mechanisms and Barriers to Successful Immunotherapy for Treating Glioblastoma.

Glioblastoma (GBM) is inevitably refractory to surgery and chemoradiation. The hope for immunotherapy has yet to be realised in the treatment of GBM. Immune checkpoint blockade antibodies, particularly those targeting the Programme death 1 (PD-1)/PD-1 ligand (PD-L1) pathway, have improved the prognosis in a range of cancers. However, its use in combination with chemoradiation or as monotherapy has proved unsuccessful in treating GBM. This review focuses on our current knowledge of barriers to immunotherapy success in treating GBM, such as diminished pre-existing anti-tumour immunity represented by low levels of PD-L1 expression, low tumour mutational burden and a severely exhausted T-cell tumour infiltrate. Likewise, systemic T-cell immunosuppression is seen driven by tumoural factors and corticosteroid use. Furthermore, unique anatomical differences with primary intracranial tumours such as the blood-brain barrier, the type of antigen-presenting cells and lymphatic drainage contribute to differences in treatment success compared to extracranial tumours. There are, however, shared characteristics with those known in other tumours such as the immunosuppressive tumour microenvironment. We conclude with a summary of ongoing and future immune combination strategies in GBM, which are representative of the next wave in immuno-oncology therapeutics.

Vestibulotoxicity Associated With Platinum-Based Chemotherapy in Survivors of Cancer: A Scoping Review.

Background: Cochleotoxicity following the treatment with platinum-based chemotherapy is well documented. The potential for vestibulotoxicity is still unclear. This scoping review examined the extent of current research literature, summarized research findings and identified research gaps regarding vestibular-related adverse effects associated with platinum-based chemotherapy in survivors of cancer. Methods: Inclusion criteria followed the PICO principles: Participants, adult, and pediatric cancer patients of any cancer type; Intervention, platinum-based chemotherapy (such as cisplatin, carboplatin, and oxaliplatin); Control, none or any; Outcomes, vestibular-related adverse effects. English language articles published since 1978 were retrieved. Seventy-five eligible studies were identified from a systematic literature search, and relevant data were charted, collated, and summarized. Results: Testing for vestibulotoxicity predominately featured functional evaluation of the horizontal semicircular canal using the caloric and rotational tests. The rate of abnormal vestibular function test results after chemotherapy administration varied from 0 to 50%. The results of objective testing did not always correspond to patient symptoms. There is tentative support for patients with pre-existing loss of vestibular function to be more likely to experience vestibular toxicity after dosing with cisplatin. Conclusions: A number of studies reported significant evidence of vestibular toxicities associated with platinum-based chemotherapy, especially cisplatin. This scoping review emphasizes that vestibular toxicity needs more attention and comprehensive evaluation. Specifically, studies that analyse cumulative dose of platinum-based chemotherapy, affected sites of lesion in vestibular end organs, and the correlation and temporal patterns of cochlear and vestibular toxicity are needed.

Targeting gp100 and TRP-2 with a DNA vaccine: Incorporating T cell epitopes with a human IgG1 antibody induces potent T cell responses that are associated with favourable clinical outcome in a phase I/II trial.

A DNA vaccine, SCIB1, incorporating two CD8 and two CD4 epitopes from TRP-2/gp100 was evaluated in patients with metastatic melanoma. Each patient received SCIB1 via intramuscular injection with electroporation. The trial was designed to find the safest dose of SCIB1 which induced immune/clinical responses in patients with or without tumour. Fifteen patients with tumor received SCIB1 doses of 0.4-8 mg whilst 20 fully-resected patients received 2-8 mg doses. Twelve patients elected to continue immunization every 3 months for up to 39 months. SCIB1 induced dose-dependent T cell responses in 88% of patients with no serious adverse effects or dose limiting toxicities. The intensity of the T cell responses was significantly higher in patients receiving 4 mg doses without tumor when compared to those with tumor (p < 0.01). In contrast, patients with tumor showed a significantly higher response to the 8 mg dose than the 4 mg dose (p < 0.03) but there was no significant difference in the patients without tumor. One of 15 patients with measurable disease showed an objective tumor response and 7/15 showed stable disease. 5/20 fully-resected patients have experienced disease recurrence but all remained alive at the cut-off date with a median observation time of 37 months. A positive clinical outcome was associated with MHC-I and MHC-II expression on tumors prior to therapy (p = 0.027). We conclude that SCIB1 is well tolerated and stimulates potent T cell responses in melanoma patients. It deserves further evaluation as a single agent adjuvant therapy or in combination with checkpoint inhibitors in advanced disease.

Immuno-silent polymer capsules encapsulating nanoparticles for bioimaging applications.

PEGylated polymer capsules encapsulating LaVO4:Tb3+, GdVO4:Tb3+, Gd2O3:Tb3+, GdF3:Tb3+, YVO4:Tb3+ and iron oxide nanoparticles are promising new fluorescence, magnetic and magnetofluorescence imaging agents. Recently, we have reported their in vitro and in vivo level toxicity profiles which show the non-toxic nature of these polymer capsules encapsulating nanoparticles. However, prior to clinical use, it is essential to ensure that these agents are unlikely to activate immune responses. Herein, we investigated the immunocompatibility of polymer capsules with dendritic cells (DCs), macrophages (MOs), and major antigen presenting cell (APC) subsets required for the activation of innate and adaptive immunity. The capsules were efficiently internalized by both DCs and MOs in vitro. Importantly, despite the presence of intracellular capsules, there was no significant impact on the viability of the cells. We studied the impact of different capsules on the cytokine profiles of the DCs and MOs, which is known to be important for the polarization of T-cell immunity. None of the capsules elicited a change in cytokine secretion from the DCs. Furthermore, the capsules did not alter the polarization of either M1 or M2 MO subsets as determined by the balance of IL-12 and IL-10 secretion. These data support the notion that PEGylated polymer capsules loaded with nanoparticles have the potential to remain immunologically silent as they do not activate APCs nor do they hinder the response of DCs or MOs to pathogen activating signals. These systems, therefore, exhibit promising characteristics for bioimaging applications.

Stromal fibroblasts support dendritic cells to maintain IL-23/Th17 responses after exposure to ionizing radiation.

Dendritic cell function is modulated by stromal cells, including fibroblasts. Although poorly understood, the signals delivered through this crosstalk substantially alter dendritic cell biology. This is well illustrated with release of TNF-α/IL-1ß from activated dendritic cells, promoting PGE2 secretion from stromal fibroblasts. This instructs dendritic cells to up-regulate IL-23, a key Th17-polarizing cytokine. We previously showed that ionizing radiation inhibited IL-23 production by human dendritic cells in vitro. In the present study, we investigated the hypothesis that dendritic cell-fibroblast crosstalk overcomes the suppressive effect of ionizing radiation to support appropriately polarized Th17 responses. Radiation (1-6 Gy) markedly suppressed IL-23 secretion by activated dendritic cells (P < 0.0001) without adversely impacting their viability and consequently, inhibited the generation of Th17 responses. Cytokine suppression by ionizing radiation was selective, as there was no effect on IL-1ß, -6, -10, and -27 or TNF-α and only a modest (11%) decrease in IL-12p70 secretion. Coculture with fibroblasts augmented IL-23 secretion by irradiated dendritic cells and increased Th17 responses. Importantly, in contrast to dendritic cells, irradiated fibroblasts maintained their capacity to respond to TNF-α/IL-1ß and produce PGE2, thus providing the key intermediary signals for successful dendritic cell-fibroblasts crosstalk. In summary, stromal fibroblasts support Th17-polarizing cytokine production by dendritic cells that would otherwise be suppressed in an irradiated microenvironment. This has potential ramifications for understanding the immune response to local radiotherapy. These findings underscore the need to account for the impact of microenvironmental factors, including stromal cells, in understanding the control of immunity.

Biology of advanced uveal melanoma and next steps for clinical therapeutics.

Uveal melanoma is the most common intraocular malignancy although it is a rare subset of all melanomas. Uveal melanoma has distinct biology relative to cutaneous melanoma, with widely divergent patient outcomes. Patients diagnosed with a primary uveal melanoma can be stratified for risk of metastasis by cytogenetics or gene expression profiling, with approximately half of patients developing metastatic disease, predominately hepatic in location, over a 15-yr period. Historically, no systemic therapy has been associated with a clear clinical benefit for patients with advanced disease, and median survival remains poor. Here, as a joint effort between the Melanoma Research Foundation's ocular melanoma initiative, CURE OM and the National Cancer Institute, the current understanding of the molecular and immunobiology of uveal melanoma is reviewed, and on-going laboratory research into the disease is highlighted. Finally, recent investigations relevant to clinical management via targeted and immunotherapies are reviewed, and next steps in the development of clinical therapeutics are discussed.

Clinical trial designs for rare diseases: studies developed and discussed by the International Rare Cancers Initiative.

BACKGROUND: The past three decades have seen rapid improvements in the diagnosis and treatment of most cancers and the most important contributor has been research. Progress in rare cancers has been slower, not least because of the challenges of undertaking research. SETTINGS: The International Rare Cancers Initiative (IRCI) is a partnership which aims to stimulate and facilitate the development of international clinical trials for patients with rare cancers. It is focused on interventional--usually randomized--clinical trials with the clear goal of improving outcomes for patients. The key challenges are organisational and methodological. A multi-disciplinary workshop to review the methods used in ICRI portfolio trials was held in Amsterdam in September 2013. Other as-yet unrealised methods were also discussed. RESULTS: The IRCI trials are each presented to exemplify possible approaches to designing credible trials in rare cancers. Researchers may consider these for use in future trials and understand the choices made for each design. INTERPRETATION: Trials can be designed using a wide array of possibilities. There is no 'one size fits all' solution. In order to make progress in the rare diseases, decisions to change practice will have to be based on less direct evidence from clinical trials than in more common diseases.

Low calpain-9 is associated with adverse disease-specific survival following endocrine therapy in breast cancer.

BACKGROUND: The calpains are intracellular cysteine proteases that function in a variety of important cellular functions, including signalling, motility, apoptosis and survival. In breast cancer high calpain-1 and calpain-2 expression has been associated with adverse clinical outcome. Calpain-9 was thought to be exclusively expressed in the digestive tract; however recent studies have shown that this protein is also expressed in breast tissue. METHODS: We investigated the expression of calpain-9 in a large cohort of early stage breast cancer patients (n = 783) using immunohistochemistry on a tissue microarray. Patients had long-term follow-up information available for analysis. RESULTS: Low expression of calpain-9 was associated with patients over 40 years of age (P = 0.025), smaller tumour size (P = 0.001), lower tumour stage (P = 0.009), a more favourable Nottingham Prognostic Index value (P = 0.002) and positive oestrogen receptor status (P = 0.014). Calpain-9 expression was not associated with survival in the total patient cohort, however low calpain-9 expression was associated with adverse survival in patients who received endocrine therapy (P = 0.033), which remained significant in multivariate Cox regression analysis accounting for potential confounding factors (hazard ratio (HR) = 0.56, 95% confidence interval (95% CI) = 0.36-0.89, P = 0.013). Low calpain-9 expression was also associated with adverse survival in patients with an intermediate Nottingham Prognostic Index value (P = 0.009), and remained so in multivariate analysis (HR = 0.54, 95% CI = 0.36-0.82, P = 0.003). CONCLUSIONS: This study suggests that calpain-9 may play a role in breast cancer and that low expression is associated with poorer patient clinical outcome following endocrine therapy. Validation studies are warranted as determining expression of calpain-9 may provide important prognostic information.

Targeting human apurinic/apyrimidinic endonuclease 1 (APE1) in phosphatase and tensin homolog (PTEN) deficient melanoma cells for personalized therapy.

Phosphatase and tensin homolog (PTEN) loss is associated with genomic instability. APE1 is a key player in DNA base excision repair (BER) and an emerging drug target in cancer. We have developed small molecule inhibitors against APE1 repair nuclease activity. In the current study we explored a synthetic lethal relationship between PTEN and APE1 in melanoma. Clinicopathological significance of PTEN mRNA and APE1 mRNA expression was investigated in 191 human melanomas. Preclinically, PTEN-deficient BRAF-mutated (UACC62, HT144, and SKMel28), PTEN-proficient BRAF-wildtype (MeWo), and doxycycline-inducible PTEN-knockout BRAF-wildtype MeWo melanoma cells were DNA repair expression profiled and investigated for synthetic lethality using a panel of four prototypical APE1 inhibitors. In human tumours, low PTEN mRNA and high APE1 mRNA was significantly associated with reduced relapse free and overall survival. Pre-clinically, compared to PTEN-proficient cells, PTEN-deficient cells displayed impaired expression of genes involved in DNA double strand break (DSB) repair. Synthetic lethality in PTEN-deficient cells was evidenced by increased sensitivity, accumulation of DSBs and induction of apoptosis following treatment with APE1 inhibitors. We conclude that PTEN deficiency is not only a promising biomarker in melanoma, but can also be targeted by a synthetic lethality strategy using inhibitors of BER, such as those targeting APE1.

Surrogate endpoints for overall survival in metastatic melanoma: a meta-analysis of randomised controlled trials.

BACKGROUND: Recent phase 3 trials have shown an overall survival benefit in metastatic melanoma. We aimed to assess whether progression-free survival (PFS) could be regarded as a reliable surrogate for overall survival through a meta-analysis of randomised trials. METHODS: We systematically reviewed randomised trials comparing treatment regimens in metastatic melanoma that included dacarbazine as the control arm, and which reported both PFS and overall survival with a standard hazard ratio (HR). We correlated HRs for overall survival and PFS, weighted by sample size or by precision of the HR estimate, assuming fixed and random effects. We did sensitivity analyses according to presence of crossover, trial size, and dacarbazine dose. FINDINGS: After screening 1649 reports and meeting abstracts published before Sept 8, 2013, we identified 12 eligible randomised trials that enrolled 4416 patients with metastatic melanoma. Irrespective of weighting strategy, we noted a strong correlation between the treatment effects for PFS and overall survival, which seemed independent of treatment type. Pearson correlation coefficients were 0·71 (95% CI 0·29-0·90) with a random-effects assumption, 0·85 (0·59-0·95) with a fixed-effects assumption, and 0·89 (0·68-0·97) with sample-size weighting. For nine trials without crossover, the correlation coefficient was 0·96 (0·81-0·99), which decreased to 0·93 (0·74-0·98) when two additional trials with less than 50% crossover were included. Inclusion of mature follow-up data after at least 50% crossover (in vemurafenib and dabrafenib phase 3 trials) weakened the PFS to overall survival correlation (0·55, 0·03-0·84). Inclusion of trials with no or little crossover with the random-effects assumption yielded a conservative statement of the PFS to overall survival correlation of 0·85 (0·51-0·96). INTERPRETATION: PFS can be regarded as a robust surrogate for overall survival in dacarbazine-controlled randomised trials of metastatic melanoma; we postulate that this association will hold as treatment standards evolve and are adopted as the control arm in future trials. FUNDING: None.

Novel function for the p38-MK2 signaling pathway in circulating CD1c+ (BDCA-1+) myeloid dendritic cells from healthy donors and advanced cancer patients; inhibition of p38 enhances IL-12 whilst suppressing IL-10.

There is growing interest in myeloid (my) dendritic cells (DC) as an alternative to monocyte-derived DC (moDC) for immunotherapy. However, in contrast to moDC, little is known regarding the effect of malignancy on the function, abundance or use of intracellular signaling pathways in myDC. Understanding the molecular detail of circulating myDC is therefore important for future use in advanced cancer. Advanced cancer patients had similar numbers of circulating myDC to cancer-free patients and healthy individuals, and secreted similar levels of IL-1ß, IL-6, IL-10, IL-12 and IL-23. However, myDC from some patients failed to secrete the Th1-cytokine IL-12. Surprisingly, inhibiting p38 (p38i) signaling (using BIRB0796 or SB203580) markedly increased IL-12 secretion by myDC. This is in complete contrast to what is established for moDC where inhibiting p38 ablates IL-12. Interestingly, this was specific to IL-12, since IL-10 was suppressed by p38i in both DC types. The opposing effect of p38i on IL-12 was evident at the transcriptional level and in both DC types was mediated through the p38-MK2 pathway but did not involve differential phosphorylation of the distal Rsk kinase. Importantly, where patient myDC did not secrete IL-12 (or after treatment with suppressive melanoma lysate), p38i restored IL-12 to normal levels. In contrast to p38, inhibiting the other MAPK pathways had similar consequences in both DC types. We show for the first time the differential use of a major intracellular signaling pathway by myDC. Importantly, there are sufficient circulating myDC in advanced cancer patients to consider development of adoptive immunotherapy.

Adjuvant ganglioside GM2-KLH/QS-21 vaccination versus observation after resection of primary tumor > 1.5 mm in patients with stage II melanoma: results of the EORTC 18961 randomized phase III trial.

PURPOSE: The GM2 ganglioside is an antigen expressed in the majority of melanomas. The GM2-KLH/QS-21 vaccine induces high immunoglobulin M (IgM) and IgG antibody responses. The EORTC 18961 trial compared the efficacy of GM2-KLH/QS-21 vaccination versus observation. PATIENTS AND METHODS: A total of 1,314 patients with a primary tumor > 1.50 mm in thickness (T3-4N0M0; American Joint Committee on Cancer stage II) were randomly assigned to GM2-KLH/QS-21 vaccination (n = 657) or observation (n = 657). Treatment consisted of subcutaneous injections once per week from week 1 to 4, then every 3 months for the first 2 years and every 6 months during the third year. Primary end point was relapse-free survival (RFS). Secondary end points were distant metastasis-free survival (DMFS) and overall survival (OS). Analyses were by intent to treat. RESULTS: After a median follow-up of 1.8 years, the trial was stopped at the second interim analysis for futility regarding RFS (hazard ratio [HR], 1.00; P = .99) and detrimental outcome regarding OS (HR, 1.66; P = .02). After a median follow-up of 4.2 years, we had recorded 400 relapses, nine deaths without relapse, a total of 236 deaths. At 4 years, the vaccination arm showed a decreased RFS rate of 1.2% (HR, 1.03; 95% CI, 0.84 to 1.25) and OS rate of 2.1% (HR, 1.16; 95% CI, 0.90 to 1.51). Toxicity was acceptable, with 4.6% of patients ending study participation because of toxicity. CONCLUSION: GM2-KLH/QS-21 vaccination does not improve outcome for patients with stage II melanoma.

Sex is an independent prognostic indicator for survival and relapse/progression-free survival in metastasized stage III to IV melanoma: a pooled analysis of five European organisation for research and treatment of cancer randomized controlled trials.

PURPOSE: To study sex differences in survival and progression in patients with stage III or IV metastatic melanoma and to compare our results with published literature. PATIENTS AND METHODS: Data were retrieved from three large, randomized, controlled trials of the European Organisation for Research and Treatment of Cancer in patients with stage III and two trials in patients with stage IV melanoma. Cox proportional hazard models were used to calculate hazard ratios (HRs) and 95% CIs for females compared with males, adjusted for different sets of confounders for stage III and stage IV, respectively. RESULTS: In 2,734 stage III patients, females had a superior 5-year disease-specific survival (DSS) rate compared with males (51.5% v 43.3%), an adjusted HR for DSS of 0.85 (95% CI, 0.76 to 0.95), and an adjusted HR for relapse-free survival of 0.86 (95% CI, 0.77 to 0.95). In 1,306 stage IV patients, females also exhibited an advantage in DSS (2-year survival rate, 14.1% v 19.0%; adjusted HR, 0.81; 95% CI, 0.72 to 0.92) as well as for progression-free survival (adjusted HR, 0.79; 95% CI, 0.70 to 0.88). This female advantage was consistent across pre- and postmenopausal age categories and across different prognostic subgroups. However, the female advantage seems to become smaller in patients with higher metastatic tumor load. CONCLUSION: The persistent independent female advantage, even after metastasis to lymph nodes and distant sites, contradicts theories about sex behavioral differences as an explanation for this phenomenon. A biologic sex trait seems to profoundly influence melanoma progression and survival, even in advanced disease.

The ataxia telangiectasia mutated kinase pathway regulates IL-23 expression by human dendritic cells.

Little is known of the regulation of IL-23 secretion in dendritic cells (DC) despite its importance for human Th17 responses. In this study, we show for first time, to our knowledge, that the ataxia telangiectasia mutated (ATM) pathway, involved in DNA damage sensing, acts as an IL-23 repressor. Inhibition of ATM with the highly selective antagonist KU55933 markedly increased IL-23 secretion in human monocyte-derived DC and freshly isolated myeloid DC. In contrast, inhibiting the closely related mammalian target of rapamycin had no effect on IL-23. Priming naive CD4(+) T cells with ATM-inhibited DC increased Th17 responses over and above those obtained with mature DC. Although ATM blockade increased the abundance of p19, p35, and p40 mRNA, IL-12p70 secretion was unaffected. To further examine a role for ATM in IL-23 regulation, we exposed DC to low doses of ionizing radiation. Exposure of DC to x-rays resulted in ATM phosphorylation and a corresponding depression of IL-23. Importantly, ATM inhibition with KU55933 prevented radiation-induced ATM phosphorylation and abrogated the capacity of x-rays to suppress IL-23. To explore how ATM repressed IL-23, we examined a role for endoplasmic reticulum stress responses by measuring generation of the spliced form of X-box protein-1, a key endoplasmic reticulum stress transcription factor. Inhibition of ATM increased the abundance of X-box protein-1 mRNA, and this was followed 3 h later by increased peak p19 transcription and IL-23 release. In summary, ATM activation or inhibition, respectively, inhibited or augmented IL-23 release. This novel role of the ATM pathway represents a new therapeutic target in autoimmunity and vaccine development.

A comparative study of adhesion of melanoma and breast cancer cells to blood and lymphatic endothelium.

BACKGROUND: Lymphovascular invasion (LVI) is an important step in the metastatic cascade; tumor cell migration and adhesion to blood and lymphatic vessels is followed by invasion through the vessel wall and subsequent systemic spread. Although primary breast cancers and melanomas have rich blood vascular networks, LVI is predominately lymphatic in nature. Whilst the adhesion of tumor cells to blood endothelium has been extensively investigated, there is a paucity of information on tumor cell adhesion to lymphatic endothelium. METHODS AND RESULTS: Breast cancer (MDA-MB-231 and MCF7) and melanoma (MeWo and SKMEL-30) cell adhesion to lymphatic (hTERT-LEC and HMVEC dLy Neo) and blood (HUVEC and hMEC-1) endothelial cells were assessed using static adhesion assays. The effect of inflammatory conditions, tumor necrosis factor-α (TNF-α) stimulation of endothelial and tumor cells, on the adhesive process was also examined. In addition, the effects of TNF-α stimulation on tumor cell migration was investigated using haplotaxis (scratch wound) assays. Breast cancer and melanoma cells exhibited higher levels of adhesion to blood compared to lymphatic endothelial cells (p<0.001). TNF-α stimulation of endothelial cells, or of tumor cells alone, did not significantly alter tumor-endothelial cell adhesion or patterns. When both tumor and endothelial cells were stimulated with TNF-α, a significant increase in adhesion was observed (p<0.01), which was notably higher in the lymphatic cell models (p<0.001). TNF-α-stimulation of all tumor cell lines significantly increased their migration rate (p<0.01). CONCLUSIONS: Results suggest that metastasis resultant from lymphatic vessel-tumor cell adhesion may be modulated by cytokine stimulation, which could represent an important therapeutic target in breast cancer and melanoma.

Long-term results of the randomized phase III trial EORTC 18991 of adjuvant therapy with pegylated interferon alfa-2b versus observation in resected stage III melanoma.

PURPOSE: Adjuvant pegylated interferon alfa-2b (PEG-IFN-α-2b) was approved for treatment of resected stage III melanoma in 2011. Here, we present long-term follow-up results of this pivotal trial. PATIENTS AND METHODS: In all, 1,256 patients with resected stage III melanoma were randomly assigned to observation (n = 629) or PEG-IFN-α-2b (n = 627) for an intended duration of 5 years. Stratification factors were microscopic (N1) versus macroscopic (N2) nodal involvement, number of positive nodes, ulceration and tumor thickness, sex, and center. Recurrence-free survival (RFS; primary end point), distant metastasis-free survival (DMFS), and overall survival (OS) were analyzed for the intent-to-treat population. RESULTS: At 7.6 years median follow-up, 384 recurrences or deaths had occurred with PEG-IFN-α-2b versus 406 in the observation group (hazard ratio [HR], 0.87; 95% CI, 0.76 to 1.00; P = .055); 7-year RFS rate was 39.1% versus 34.6%. There was no difference in OS (P = .57). In stage III-N1 ulcerated melanoma, RFS (HR, 0.72; 99% CI, 0.46 to 1.13; P = .06), DMFS (HR, 0.65; 99% CI, 0.41 to 1.04; P = .02), and OS (HR, 0.59; 99% CI, 0.35 to 0.97; P = .006) were prolonged with PEG-IFN-α-2b. PEG-IFN-α-2b was discontinued for toxicity in 37% of patients. CONCLUSION: Adjuvant PEG-IFN-α-2b for stage III melanoma had a positive impact on RFS, which was marginally significant and slightly diminished versus the benefit seen at prior follow-up (median, 3.8 years). No significant increase in DMFS or OS was noted in the overall population. Patients with ulcerated melanoma and lower disease burden had the greatest benefit.

New anticancer immunotherapies.

The quest for immunotherapies against cancer has been ongoing for many years, and a greater understanding of the normal mechanisms involved in developing immune responses has now led to clinically effective therapies. With the licensing of Ipilimumab and Sipuleucel-T, immunotherapeutic strategies are taking their place alongside conventional treatments for cancer. This review will consider the different modalities of immunotherapy, highlighting clinical benefits observed and considering the immunological basis and evidence of their efficacy. Dendritic cell therapy, targeting activation and regulation of T cells, oncolytic virus vaccines and adoptive T cell therapies will all be considered, regarding the current situation and avenues for future exploration.