Temporary treatment cessation versus continuation of first-line tyrosine kinase inhibitor in patients with advanced clear cell renal cell carcinoma (STAR): an open-label, non-inferiority, randomised, controlled, phase 2/3 trial.

BACKGROUND: Temporary drug treatment cessation might alleviate toxicity without substantially compromising efficacy in patients with cancer. We aimed to determine if a tyrosine kinase inhibitor drug-free interval strategy was non-inferior to a conventional continuation strategy for first-line treatment of advanced clear cell renal cell carcinoma. METHODS: This open-label, non-inferiority, randomised, controlled, phase 2/3 trial was done at 60 hospital sites in the UK. Eligible patients (aged ≥18 years) had histologically confirmed clear cell renal cell carcinoma, inoperable loco-regional or metastatic disease, no previous systemic therapy for advanced disease, uni-dimensionally assessed Response Evaluation Criteria in Solid Tumours-defined measurable disease, and an Eastern Cooperative Oncology Group performance status of 0-1. Patients were randomly assigned (1:1) at baseline to a conventional continuation strategy or drug-free interval strategy using a central computer-generated minimisation programme incorporating a random element. Stratification factors were Memorial Sloan Kettering Cancer Center prognostic group risk factor, sex, trial site, age, disease status, tyrosine kinase inhibitor, and previous nephrectomy. All patients received standard dosing schedules of oral sunitinib (50 mg per day) or oral pazopanib (800 mg per day) for 24 weeks before moving into their randomly allocated group. Patients allocated to the drug-free interval strategy group then had a treatment break until disease progression, when treatment was re-instated. Patients in the conventional continuation strategy group continued treatment. Patients, treating clinicians, and the study team were aware of treatment allocation. The co-primary endpoints were overall survival and quality-adjusted life-years (QALYs); non-inferiority was shown if the lower limit of the two-sided 95% CI for the overall survival hazard ratio (HR) was 0·812 or higher and if the lower limit of the two-sided 95% CI of the marginal difference in mean QALYs was -0·156 or higher. The co-primary endpoints were assessed in the intention-to-treat (ITT) population, which included all randomly assigned patients, and the per-protocol population, which excluded patients in the ITT population with major protocol violations and who did not begin their randomisation allocation as per the protocol. Non-inferiority was to be concluded if it was met for both endpoints in both analysis populations. Safety was assessed in all participants who received a tyrosine kinase inhibitor. The trial was registered with ISRCTN, 06473203, and EudraCT, 2011-001098-16. FINDINGS: Between Jan 13, 2012, and Sept 12, 2017, 2197 patients were screened for eligibility, of whom 920 were randomly assigned to the conventional continuation strategy (n=461) or the drug-free interval strategy (n=459; 668 [73%] male and 251 [27%] female; 885 [96%] White and 23 [3%] non-White). The median follow-up time was 58 months (IQR 46-73 months) in the ITT population and 58 months (46-72) in the per-protocol population. 488 patients continued on the trial after week 24. For overall survival, non-inferiority was demonstrated in the ITT population only (adjusted HR 0·97 [95% CI 0·83 to 1·12] in the ITT population; 0·94 [0·80 to 1·09] in the per-protocol population). Non-inferiority was demonstrated for QALYs in the ITT population (n=919) and per-protocol (n=871) population (marginal effect difference 0·06 [95% CI -0·11 to 0·23] for the ITT population; 0·04 [-0·14 to 0·21] for the per-protocol population). The most common grade 3 or worse adverse events were hypertension (124 [26%] of 485 patients in the conventional continuation strategy group vs 127 [29%] of 431 patients in the drug-free interval strategy group); hepatotoxicity (55 [11%] vs 48 [11%]); and fatigue (39 [8%] vs 63 [15%]). 192 (21%) of 920 participants had a serious adverse reaction. 12 treatment-related deaths were reported (three patients in the conventional continuation strategy group; nine patients in the drug-free interval strategy group) due to vascular (n=3), cardiac (n=3), hepatobiliary (n=3), gastrointestinal (n=1), or nervous system (n=1) disorders, and from infections and infestations (n=1). INTERPRETATION: Overall, non-inferiority between groups could not be concluded. However, there seemed to be no clinically meaningful reduction in life expectancy between the drug-free interval strategy and conventional continuation strategy groups and treatment breaks might be a feasible and cost-effective option with lifestyle benefits for patients during tyrosine kinase inhibitor therapy in patients with renal cell carcinoma. FUNDING: UK National Institute for Health and Care Research.

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.

Surface polymer imprinted optical fibre sensor for dose detection of dabrafenib.

Dabrafenib is one of the most widely used of the new generation of targeted anti-cancer drugs. However, its therapeutic window varies for different patients and so there is an unmet need for methods to monitor the dose of drug which the patient receives and at the specific site where it acts. In the case of cancers, it is critical to measure the concentration of drug not just in the bloodstream overall, but in or near tumours, as these will not be the same over multiple time periods. A novel sensor based on an optical fibre long period grating (LPG) modified with a molecular imprinted polymer (MIP) has been developed with the ultimate aim of achieving minimally invasive measurements of Dabrafenib at the tumour site. A molecularly imprinted polymer specific for Dabrafenib was coated on a methacryloylalkoxysilane-functionalised optical fibre long period grating. In vitro experimental results demonstrate that the Dabrafenib sensitivity is 15.2 pm (µg mL-1)-1 (R2 = 0.993) with a limit of detection (LoD) of 74.4 µg mL-1 in serum solution. Moreover, the proposed sensor shows selective response to Dabrafenib over structurally similar 2-Aminoquinoline.

Cancer survivors treated with platinum-based chemotherapy affected by ototoxicity and the impact on quality of life: a narrative synthesis systematic review.

Objective: To identify any change in quality of life (QoL) caused by chemotherapy-induced toxicities, such as hearing loss and tinnitus, to provide information in order to improve services and aid clinicians in their decision-making. Design: This systematic review followed the preferred reporting items for systematic reviews and meta-analysis (PRISMA) checklist. The search terms were cancer, platinum-based chemotherapy, ototoxicity and "quality of life". Titles and abstracts, followed by full texts, were screened by two independent researchers. The relevant data were extracted and quality analysis was performed using the NIH Quality Assessment Tool. Study sample: About 308 titles and abstracts were screened, and 27 full-text articles were screened. Ten articles representing 11 studies were included in the review. Study design included cross-sectional studies, randomised control trials and longitudinal studies. Results: Diagnostic criteria consisted of audiograms, questionnaires and patient complaints. The study quality ranged from 21.43% to 85.71%. Overall results found that those treated with cisplatin had more hearing loss and tinnitus than those treated with other therapies. Furthermore, those with hearing loss and tinnitus were more likely to have a lower QoL. Conclusions: There is an urgent need to standardise diagnostics when investigating ototoxicity and its effect on QoL, particularly for research into risk factors, prevention and management.

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.

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.

Inherited variation in the PARP1 gene and survival from melanoma.

We report the association of an inherited variant located upstream of the poly(adenosine diphosphate-ribose) polymerase 1 (PARP1) gene (rs2249844), with survival in 11 BioGenoMEL melanoma cohorts. The gene encodes a protein involved in a number of cellular processes including single-strand DNA repair. Survival analysis was conducted for each cohort using proportional hazards regression adjusting for factors known to be associated with survival. Survival was measured as overall survival (OS) and, where available, melanoma-specific survival (MSS). Results were combined using random effects meta-analysis. Evidence for a role of the PARP1 protein in melanoma ulceration and survival was investigated by testing gene expression levels taken from formalin-fixed paraffin-embedded tumors. A significant association was seen for inheritance of the rarer variant of PARP1, rs2249844 with OS (hazard ratio (HR) = 1.16 per allele, 95% confidence interval (CI) 1.04-1.28, p = 0.005, eleven cohorts) and MSS (HR = 1.20 per allele, 95% CI 1.01-1.39, p = 0.03, eight cohorts). We report bioinformatic data supportive of a functional effect for rs2249844. Higher levels of PARP1 gene expression in tumors were shown to be associated with tumor ulceration and poorer OS.

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.

'Immunotherapeutic Strategies for Intra-cranial Metastatic Melanoma - a Meta-analysis and Systematic Review'.

Immune-activating anti-CTLA4 and anti-PD1 monoclonal antibodies (alone or in combination) are being used to treat advanced melanoma patients and can lead to durable remissions, and long-term overall survival may be achieved in between 50-60% of patients. Although intracranial metastases are very common in melanoma (about 50-75% of all patients with advanced disease), most of the pivotal prospective clinical trials exclude patients with intra-cranial metastases, certainly if their lesions are symptomatic and steroid-requiring and the degree of sensitivity of intra-cranial melanoma to immunotherapy remains uncertain, and requires further investigation especially in view of the demonstrable activity of RAF-MEK inhibitors in this clinical setting and the emergence of stereotactic radiotherapy. Our study aimed to evaluate the efficacy and toxicity of immunotherapy against advanced melanoma patients with brain metastases. In terms of comparative studies, only retrospective analyses could be identified. Based on 3 retrospective studies, treatment of patients with melanoma brain metastases with immunotherapeutic approaches improves overall survival substantially compared with supportive measures alone (no active anticancer treatment). The efficacy of targeted therapy appeared to be comparable to that of immune therapy in terms of overall survival, based on a small number of patients. The combination of concurrent radiation therapy to the brain and systemic immunotherapy led to improved overall survival compared to radiotherapy alone, suggesting potential synergism between the approaches, and combination treatment could be delivered safely. Our review supports the use of immunotherapeutic strategies for these patients although treatment efficacy appears to be lower for symptomatic lesions. In view of the extremely high efficacy of stereotactic radiotherapy approaches in the brain, understanding the interaction between radiotherapy and immunotherapy is vital and should be an area of active investigation.

A three-arm randomised phase II study of the MEK inhibitor selumetinib alone or in combination with paclitaxel in metastatic uveal melanoma.

AIMS: The MAPK pathway is constitutively activated in uveal melanoma (UM). Selumetinib (AZD6244, ARRY-142886), a MEK inhibitor, has shown limited activity as monotherapy in metastatic UM. Pre-clinical studies support synergistic cytotoxic activity for MEK inhibitors combined with taxanes, and here we sought to assess the clinical efficacy of combining selumetinib and paclitaxel. PATIENTS AND METHODS: Seventy-seven patients with metastatic UM who had not received prior chemotherapy were randomised to selumetinib alone, or combined with paclitaxel with or without interruption in selumetinib two days before paclitaxel. The primary endpoint was progression free survival (PFS). After amendment, the combination arms were combined for analysis and the sample size adjusted to detect a hazard ratio (HR): 0.55, 80% power at 1-sided 5% significance level. RESULTS: The median PFS in the combination arms was 4.8 months (95% CI: 3.8 - 5.6) compared with 3.4 months (2.0 - 3.9) in the selumetinib arm (HR 0.62 [90% CI 0.41 - 0.92], 1-sided p-value = 0.022). ORR was 14% and 4% in the combination and monotherapy arms respectively. Median OS was 9 months for the combination and was not significantly different from selumetinib alone (10 months) with HR of 0.98 [90% CI 0.58 - 1.66], 1-sided p-value = 0.469. Toxicity was in keeping with the known profiles of the agents involved. CONCLUSIONS: SelPac met its primary endpoint, demonstrating an improvement in PFS for combination selumetinib and paclitaxel. No improvement in OS was observed, and the modest improvement in PFS is not practice changing.

Standard Versus Modified Ipilimumab, in Combination With Nivolumab, in Advanced Renal Cell Carcinoma: A Randomized Phase II Trial (PRISM).

PURPOSE: Ipilimumab (IPI), in combination with nivolumab (NIVO), is an approved frontline treatment option for patients with intermediate- or poor-risk advanced renal cell carcinoma (aRCC). We conducted a randomized phase II trial to evaluate whether administering IPI once every 12 weeks (modified), instead of once every 3 weeks (standard), in combination with NIVO, is associated with a favorable toxicity profile. METHODS: Treatment-naïve patients with clear-cell aRCC were randomly assigned 2:1 to receive four doses of modified or standard IPI, 1 mg/kg, in combination with NIVO (3 mg/kg). The primary end point was the proportion of patients with a grade 3-5 treatment-related adverse event (trAE) among those who received at least one dose of therapy. The key secondary end point was 12-month progression-free survival (PFS) in the modified arm compared with historical sunitinib control. The study was not designed to formally compare arms for efficacy. RESULTS: Between March 2018 and January 2020, 192 patients (69.8% intermediate/poor-risk) were randomly assigned and received at least one dose of study drug. The incidence of grade 3-5 trAEs was significantly lower among participants receiving modified versus standard IPI (32.8% v 53.1%; odds ratio, 0.43 [90% CI, 0.25 to 0.72]; P = .0075). The 12-month PFS (90% CI) using modified IPI was 46.1% (38.6 to 53.2). At a median follow-up of 21 months, the overall response rate was 45.3% versus 35.9% and the median PFS was 10.8 months versus 9.8 months in the modified and standard IPI groups, respectively. CONCLUSION: Rates of grade 3-5 trAEs were significantly lower in patients receiving modified versus standard IPI. Although 12-month PFS did not meet the prespecified efficacy threshold compared with historical control, informal comparison of treatment groups did not suggest any reduction in efficacy with the modified schedule.

Phase II Study Investigating the Safety and Efficacy of Savolitinib and Durvalumab in Metastatic Papillary Renal Cancer (CALYPSO).

PURPOSE: Metastatic papillary renal cancer (PRC) has poor outcomes, and new treatments are required. There is a strong rationale for investigating mesenchymal epithelial transition receptor (MET) and programmed cell death ligand-1 (PD-L1) inhibition in this disease. In this study, the combination of savolitinib (MET inhibitor) and durvalumab (PD-L1 inhibitor) is investigated. METHODS: This single-arm phase II trial explored durvalumab (1,500 mg once every four weeks) and savolitinib (600 mg once daily; ClinicalTrials.gov identifier: NCT02819596). Treatment-naïve or previously treated patients with metastatic PRC were included. A confirmed response rate (cRR) of > 50% was the primary end point. Progression-free survival, tolerability, and overall survival were secondary end points. Biomarkers were explored from archived tissue (MET-driven status). RESULTS: Forty-one patients treated with advanced PRC were enrolled into this study and received at least one dose of study treatment. The majority of patients had Heng intermediate risk score (n = 26 [63%]). The cRR was 29% (n = 12; 95% CI, 16 to 46), and the trial therefore missed the primary end point. The cRR increased to 53% (95% CI, 28 to 77) in MET-driven patients (n/N = 9/27) and was 33% (95% CI, 17 to 54) in PD-L1-positive tumors (n/N = 9/27). The median progression-free survival was 4.9 months (95% CI, 2.5 to 10.0) in the treated population and 12.0 months (95% CI, 2.9 to 19.4) in MET-driven patients. The median overall survival was 14.1 months (95% CI, 7.3 to 30.7) in the treated population and 27.4 months (95% CI, 9.3 to not reached [NR]) in MET-driven patients. Grade 3 and above treatment related adverse events occurred in 17 (41%) patients. There was 1 grade 5 treatment-related adverse event (cerebral infarction). CONCLUSION: The combination of savolitinib and durvalumab was tolerable and associated with high cRRs in the exploratory MET-driven subset.

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.

Synthetic lethal targeting of DNA double-strand break repair deficient cells by human apurinic/apyrimidinic endonuclease inhibitors.

An apurinic/apyrimidinic (AP) site is an obligatory cytotoxic intermediate in DNA Base Excision Repair (BER) that is processed by human AP endonuclease 1 (APE1). APE1 is essential for BER and an emerging drug target in cancer. We have isolated novel small molecule inhibitors of APE1. In this study, we have investigated the ability of APE1 inhibitors to induce synthetic lethality (SL) in a panel of DNA double-strand break (DSB) repair deficient and proficient cells; i) Chinese hamster (CH) cells: BRCA2 deficient (V-C8), ATM deficient (V-E5), wild type (V79) and BRCA2 revertant [V-C8(Rev1)]. ii) Human cancer cells: BRCA1 deficient (MDA-MB-436), BRCA1 proficient (MCF-7), BRCA2 deficient (CAPAN-1 and HeLa SilenciX cells), BRCA2 proficient (PANC1 and control SilenciX cells). We also tested SL in CH ovary cells expressing a dominant-negative form of APE1 (E8 cells) using ATM inhibitors and DNA-PKcs inhibitors (DSB inhibitors). APE1 inhibitors are synthetically lethal in BRCA and ATM deficient cells. APE1 inhibition resulted in accumulation of DNA DSBs and G2/M cell cycle arrest. SL was also demonstrated in CH cells expressing a dominant-negative form of APE1 treated with ATM or DNA-PKcs inhibitors. We conclude that APE1 is a promising SL target in cancer.

Objective assessment of blood and lymphatic vessel invasion and association with macrophage infiltration in cutaneous melanoma.

The aims of this study were to investigate the role of vascular invasion (blood and lymphatic), vessel density and the presence of tumour-associated macrophages as prognostic markers in 202 cutaneous melanoma patients. Sections of primary melanoma were stained with lymphatic-specific antibody D2-40 to assess lymphatic vessel invasion and density in intratumoural and peritumoural areas; an antibody against endothelial marker CD34 was used to determine blood vessel invasion and density, and an antibody against CD68 was used to determine macrophage counts. Immunohistochemically determined vascular invasion (combined blood and lymphatic) was compared with that determined using haematoxylin and eosin (H&E) staining. The use of immunohistochemistry increased detection of vascular invasion from 8-30% of patients, and histological exam of H&E-stained tissue was associated with a false positive rate of 64%. Lymphatic vessel invasion occurred at a much higher frequency than blood vessel invasion (27 and 4% of patients, respectively). Although immunohistochemically detected vessel invasion was significantly associated with histological markers of adverse prognosis, such as increased Breslow thickness, ulceration and mitotic rate (all P<0.001), no associations with relapse-free or overall survival were observed. High macrophage counts were significantly associated with markers of aggressive disease, such as Breslow thickness, ulceration and mitotic rate (P<0.001, P<0.001, P=0.005, respectively), and lymphatic vessel invasion and high microvessel density (P=0.002 and P=0.003, respectively). These results suggest that vascular invasion is more accurately detected using immunohistochemistry and occurs predominantly via lymphatic vessels. The association of vessel characteristics with histological characteristics of the primary melanoma provides evidence for their biological importance in melanoma, but that they were not associated with clinical outcome attests to the value of existing histological prognostic biomarkers. We note that a high macrophage count may be associated with neovascularisation and primary tumour growth, and may also promote invasion through lymphatic vessels.

Ran GTPase is an independent prognostic marker in malignant melanoma which promotes tumour cell migration and invasion.

AIMS: Ran GTPase is involved in nucleocytoplasmic shuttling of proteins and is overexpressed in several cancers. The expression of Ran in malignant melanoma (MM) and its functional activity have not been described and were investigated in this study. METHODS: The prognostic value of Ran expression was tested in a series of 185 primary cutaneous MM cases using immunohistochemistry. The functional activity of Ran was investigated in the two melanoma cell lines. Ran expression was knocked down using two siRNAs and the effect on the expression of the c-Met oncogene, a potential downstream target of Ran, was tested. Functional effects of Ran knockdown on cell motility and cell proliferation were also assessed. RESULTS: Positive Ran expression was seen in 12.4% of MM and was associated with advanced clinical stage and greater Breslow thickness. Positive expression was an independent marker of shorter overall survival (p=0.023). Knockdown of Ran results in decreased expression of c-Met and the downstream c-met signalling targets ERK1/2. There was a significant reduction in cell migration (p<0.001) and cell invasion (p<0.001). c-Met knockdown decreased the expression of Ran through MAPK and PI3K-AKT in A375 cell line, inhibited the cell viability and migration of both A375 and G361 melanoma cell lines while invasion was enhanced. CONCLUSION: Ran is a poor prognostic marker in cutaneous MM. It upregulates expression of the oncogene c-Met and, possibly through this, it promotes cell motility which may in turn promote metastasis.

Novel approach for interleukin-23 up-regulation in human dendritic cells and the impact on T helper type 17 generation.

Interleukin-23 (IL-23) is important for T helper type 17 (Th17) responses and strategies to regulate IL-23 in human dendritic cells (DC) are limited. This study describes a novel means to control IL-23 secretion by conditioning DC with a phosphatidyl inositol 3-kinase inhibitor Wortmannin (WM). Treatment of monocyte-derived DC with WM increased Toll-like receptor (TLR) -dependent IL-23 secretion 10-fold and IL-12p70 twofold, but IL-27 was unaffected. The effect of WM was restricted to TLR3/4 pathways, did not occur through TLR2, TLR7/8 or Dectin-1, and was characterized by increased p19, p35 and p40 transcription. These responses were not solely dependent on phosphatidyl inositol 3-kinase as the alternative inhibitor LY294002 did not modulate IL-23 production. The normal patterns of activation of mitogen-activated protein kinase pathways were unaffected by WM-conditioning but IL-23 secretion required p38, ERK and JNK pathways. Importantly, this effect was manifest in populations of blood DC. Conditioning freshly isolated myeloid DC with WM before TLR3 or TLR4 triggering resulted in high levels of IL-23 secretion and an absence of IL-12p70. These WM-conditioned myeloid DC were highly effective at priming Th17 responses from naive CD4(+) T cells. Our findings provide a novel means to generate IL-23-rich environments and Th17 responses and suggest as yet unidentified regulatory factors, identification of which will provide new approaches to control IL-23-dependent immunity in infectious disease, autoimmunity and malignancy.

Interferon alfa-2a versus combination therapy with interferon alfa-2a, interleukin-2, and fluorouracil in patients with untreated metastatic renal cell carcinoma (MRC RE04/EORTC GU 30012): an open-label randomised trial.

BACKGROUND: In metastatic renal cell carcinoma combinations of interferon alfa-2a, interleukin-2, and fluorouracil produce higher response rates and longer progression-free survival than do single agents. We aimed to compare overall survival in patients receiving combination treatment or interferon alfa-2a. METHODS: RE04/30012 was an open-label randomised trial undertaken in 50 centres across eight countries. 1006 treatment-naive patients diagnosed with advanced metastatic renal cell carcinoma were randomly allocated (1 to 1) by minimisation to receive interferon alfa-2a alone or combination therapy with interferon alfa-2a, interleukin-2, and fluorouracil. Treatment was not masked. The primary endpoint was overall survival. Treatment groups were compared with a non-stratified log-rank test. Analysis was by intention to treat. This study is registered, number ISRCTN 46518965. FINDINGS: 502 patients were randomly assigned to receive interferon alfa-2a and 504 to receive combined treatment. Median follow-up was 37.2 months (24.8-52.3). Median overall survival was 18.8 months (17.0-23.2) for patients receiving interferon alfa-2a versus 18.6 months (16.5-20.6) for those receiving combination therapy. Overall survival did not differ between the two groups (hazard ratio 1.05 [95% CI 0.90-1.21], p=0.55; absolute difference 0.3% (-5.1 to 5.6) at 1 year and 2.7% (-8.2 to 2.9) at 3 years). Serious adverse events were reported in 113 (23%) patients receiving interferon alfa-2a and 131 (26%) of those receiving combined treatment. INTERPRETATION: Although combination therapy does not improve overall or progression-free survival compared with interferon alfa-2a alone, immunotherapy might still have a role because it can produce remissions that are of clinically relevant length in some patients. Identification of patients who will benefit from immunotherapy is crucial. FUNDING: UK Medical Research Council.