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Background : Early and accurate grading of renal cell carcinoma (RCC) improves patient risk stratification and has implications for clinical management and mortality. However, current diagnostic approaches using imaging and renal mass biopsy have limited specificity and may lead to undergrading. Methods : This study explored the use of hyperpolarised [1- 13 C]pyruvate MRI (HP 13 C-MRI) to identify the most aggressive areas within the tumour of patients with clear cell renal cell carcinoma (ccRCC) as a method to guide biopsy targeting and to reduce undergrading. Six patients with ccRCC underwent presurgical HP 13 C-MRI and conventional contrast-enhanced MRI. From the imaging data, three k-means clusters were computed by combining the k PL as a marker of metabolic activity, and the 13 C-pyruvate signal-to-noise ratio (SNR Pyr ) as a perfusion surrogate. The combined clusters were compared to those derived from individual parameters and to those derived from the percentage of enhancement on the nephrographic phase (%NG). The diagnostic performance of each cluster was assessed based on its ability to predict the highest histological tumour grade in postsurgical tissue samples. The postsurgical tissue samples underwent immunohistochemical staining for the pyruvate transporter (monocarboxylate transporter 1, MCT1), as well as RNA and whole-exome sequencing. Results: The clustering approach combining SNR Pyr and k PL demonstrated the best performance for predicting the highest tumour grade: specificity 85%; sensitivity 64%; positive predictive value 82%; and negative predictive value 68%. Epithelial MCT1 was identified as the major determinant of the HP 13 C-MRI signal. The perfusion/metabolism mismatch cluster showed an increased expression of metabolic genes and markers of aggressiveness. Conclusions: This study demonstrates the potential of using HP 13 C-MRI-derived metabolic clusters to identify intratumoral variations in tumour grade with high specificity. This work supports the use of metabolic imaging to guide biopsies to the most aggressive tumour regions and could potentially reduce sampling error.

Introduction: Localised renal masses are an increasing burden on healthcare due to the rising number of cases. However, conventional imaging cannot reliably distinguish between benign and malignant renal masses, and renal mass biopsies are unable to characterise the entirety of the tumour due to sampling error, which may lead to delayed treatment or overtreatment. There is an unmet clinical need to develop novel imaging techniques to characterise renal masses more accurately. Renal tumours demonstrate characteristic metabolic reprogramming, and novel MRI methods have the potential to detect these metabolic perturbations, which may therefore aid accurate characterisation. Here, we present our study protocol for the investigation of the differential biology of benign and malignant renal masses using advanced MRI techniques (IBM-Renal)., Methods and Analysis: IBM-Renal is a multiarm, single-centre, non-randomised, feasibility study with the aim to provide preliminary evidence for the potential role of the novel MRI techniques to phenotype localised renal lesions. 30 patients with localised renal masses will be recruited to three imaging arms, with 10 patients in each: (1) hyperpolarised [1- 13 C]-pyruvate MRI, (2) deuterium metabolic imaging (DMI) and (3) sodium MRI. The diagnosis will be made on samples acquired at biopsy or at surgery. The primary objective is the technical development of the novel MRI techniques, with the ultimate aim to understand whether these can identify differences between benign and malignant tumours, while the secondary objectives aim to assess how complementary the techniques are, and if they provide additional information. The exploratory objective is to link imaging findings with clinical data and molecular analyses for the biological validation of the novel MRI techniques., Ethics and Dissemination: This study was ethically approved (UK REC HRA: 22/EE/0136; current protocol version 2.1 dated 11 August 2022). The plans for dissemination include presentations at conferences, publications in scientific journals, a doctoral thesis and patient and public involvement., Trial Registration Number: NCT06016075., Competing Interests: Competing interests: GDS has received educational grants from Pfizer, AstraZeneca and Intuitive Surgical; consultancy fees from Pfizer, MSD, EUSA Pharma and CMR Surgical; Travel expenses from MSD and Pfizer; Speaker fees from Pfizer; Clinical lead (urology) National Kidney Cancer Audit and Topic Advisor for the NICE kidney cancer guideline. SJW is a founder and director of Pinto Medical Consultancy. FAG has research grants from GlaxoSmithKline and AstraZeneca, research support from GE Healthcare and has consulted for AstraZeneca on behalf of the University of Cambridge. All other authors have no competing interests., (© Author(s) (or their employer(s)) 2024. Re-use permitted under CC BY. Published by BMJ.)

Background: Clinical imaging tools to probe aggressiveness of renal masses are lacking, and T2-weighted imaging as an integral part of magnetic resonance imaging protocol only provides qualitative information. We developed high-resolution and accelerated T2 mapping methods based on echo merging and using k-t undersampling and reduced flip angles (TEMPURA) and tested their potential to quantify differences between renal tumour subtypes and grades., Methods: Twenty-four patients with treatment-naïve renal tumours were imaged: seven renal oncocytomas (RO); one eosinophilic/oncocytic renal cell carcinoma; two chromophobe RCCs (chRCC); three papillary RCCs (pRCC); and twelve clear cell RCCs (ccRCC). Median, kurtosis, and skewness of T2 were quantified in tumours and in the normal-adjacent kidney cortex and were compared across renal tumour subtypes and between ccRCC grades., Results: High-resolution TEMPURA depicted the tumour structure at improved resolution compared to conventional T2-weighted imaging. The lowest median T2 values were present in pRCC (high-resolution, 51 ms; accelerated, 45 ms), which was significantly lower than RO (high-resolution; accelerated, p = 0.012) and ccRCC (high-resolution, p = 0.019; accelerated, p = 0.008). ROs showed the lowest kurtosis (high-resolution, 3.4; accelerated, 4.0), suggestive of low intratumoural heterogeneity. Lower T2 values were observed in higher compared to lower grade ccRCCs (grades 2, 3 and 4 on high-resolution, 209 ms, 151 ms, and 106 ms; on accelerated, 172 ms, 160 ms, and 102 ms, respectively), with accelerated TEMPURA showing statistical significance in comparison (p = 0.037)., Conclusions: Both high-resolution and accelerated TEMPURA showed marked potential to quantify differences across renal tumour subtypes and between ccRCC grades., Trial Registration: ClinicalTrials.gov, NCT03741426 . Registered on 13 November 2018., Relevance Statement: The newly developed T 2 mapping methods have improved resolution, shorter acquisition times, and promising quantifiable readouts to characterise incidental renal masses., (© 2024. The Author(s).)

Multiple myeloma (MM) is a malignancy that is often driven by MYC and that is sustained by IRF4, which are upregulated by super-enhancers. IKZF1 and IKZF3 bind to super-enhancers and can be degraded using immunomodulatory imide drugs (IMiD). Successful IMiD responses downregulate MYC and IRF4; however, this fails in IMiD-resistant cells. MYC and IRF4 downregulation can also be achieved in IMiD-resistant tumors using inhibitors of BET and EP300 transcriptional coactivator proteins; however, in vivo these drugs have a narrow therapeutic window. By combining IMiDs with EP300 inhibition, we demonstrate greater downregulation of MYC and IRF4, synergistic killing of myeloma in vitro and in vivo, and an increased therapeutic window. Interestingly, this potent combination failed where MYC and IRF4 expression was maintained by high levels of the AP-1 factor BATF. Our results identify an effective drug combination and a previously unrecognized mechanism of IMiD resistance., Significance: These results highlight the dependence of MM on IKZF1-bound super-enhancers, which can be effectively targeted by a potent therapeutic combination pairing IMiD-mediated degradation of IKZF1 and IKZF3 with EP300 inhibition. They also identify AP-1 factors as an unrecognized mechanism of IMiD resistance in MM. See related article by Neri, Barwick, et al., p. 56. See related commentary by Yun and Cleveland, p. 5. This article is featured in Selected Articles from This Issue, p. 4., (©2023 The Authors; Published by the American Association for Cancer Research.)

Immunomodulatory drugs (IMiD) are a backbone therapy for multiple myeloma (MM). Despite their efficacy, most patients develop resistance, and the mechanisms are not fully defined. Here, we show that IMiD responses are directed by IMiD-dependent degradation of IKZF1 and IKZF3 that bind to enhancers necessary to sustain the expression of MYC and other myeloma oncogenes. IMiD treatment universally depleted chromatin-bound IKZF1, but eviction of P300 and BRD4 coactivators only occurred in IMiD-sensitive cells. IKZF1-bound enhancers overlapped other transcription factor binding motifs, including ETV4. Chromatin immunoprecipitation sequencing showed that ETV4 bound to the same enhancers as IKZF1, and ETV4 CRISPR/Cas9-mediated ablation resulted in sensitization of IMiD-resistant MM. ETV4 expression is associated with IMiD resistance in cell lines, poor prognosis in patients, and is upregulated at relapse. These data indicate that ETV4 alleviates IKZF1 and IKZF3 dependency in MM by maintaining oncogenic enhancer activity and identify transcriptional plasticity as a previously unrecognized mechanism of IMiD resistance., Significance: We show that IKZF1-bound enhancers are critical for IMiD efficacy and that the factor ETV4 can bind the same enhancers and substitute for IKZF1 and mediate IMiD resistance by maintaining MYC and other oncogenes. These data implicate transcription factor redundancy as a previously unrecognized mode of IMiD resistance in MM. See related article by Welsh, Barwick, et al., p. 34. See related commentary by Yun and Cleveland, p. 5. This article is featured in Selected Articles from This Issue, p. 4., (©2023 The Authors; Published by the American Association for Cancer Research.)

Renal cell carcinoma (RCC) is the 7th commonest cancer in the UK and the most lethal urological malignancy; 50% of all RCC patients will die from the condition. However, if identified early enough, small RCCs are usually cured by surgery or percutaneous procedures, with 95% 10 year survival. This study describes a newly developed non-invasive urine-based assay for the early detection of RCC. Our approach uses encoded magnetically controllable heterostructures as a substrate for immunoassays. These heterostructures have molecular recognition abilities and embedded patterned codes for a rapid identification of RCC biomarkers. The magnetic heterostructures developed for this study have a magnetic configuration designed for a remote multi axial control of their orientation by external magnetic fields, this control facilitates the code readout when the heterostructures are in liquid. Furthermore, the optical encoding of each set of heterostructures provides a multiplexed analyte capture platform, as different sets of heterostructures, specific to different biomarkers can be mixed together in a patient sample. Our results show a precise magnetic control of the heterostructures with an efficient code readout during liquid immunoassays. The use of functionalised magnetic heterostructures as a substrate for immunoassay is validated for urine specimen spiked with recombinant RCC biomarkers. Initial results of the newly proposed screening method on urine samples from RCC patients, and controls with no renal disorders are presented in this study. Comprehensive optimisation cycles are in progress to validate the robustness of this technology as a novel, non-invasive screening method for RCC., Competing Interests: Grant D. Stewart has received educational grants from Pfizer, AstraZeneca and Intuitive Surgical; consultancy fees from Pfizer, Merck, EUSA Pharma and CMR Surgical; travel expenses from Pfizer and Speaker fees from Pfizer. Jeroen Verheyen and Tarun Vemulkar are employees of Semarion. Russell P. Cowburn is non-executive director of Semarion. Russell P. Cowburn is employed by Durham Magneto Optics. All other authors have no financial disclosures., (This journal is © The Royal Society of Chemistry.)

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., Competing Interests: GPA has received consulting fees from Pfizer, GlaxoSmithKline, Clinicpace, Servier Pharmaceuticals, NuCANA Plc, AstraZeneca, and BenevolentAI paid to the University of Nottingham. All authors declare no conflicts of interest that relate to this work. Please refer to the accompanying ICMJE disclosure forms for further details., (© 2023 The Authors.)

Tumor behavior is intricately dependent on the oncogenic properties of cancer cells and their multi-cellular interactions. To understand these dependencies within the wider microenvironment, we studied over 270,000 single-cell transcriptomes and 100 microdissected whole exomes from 12 patients with kidney tumors, prior to validation using spatial transcriptomics. Tissues were sampled from multiple regions of the tumor core, the tumor-normal interface, normal surrounding tissues, and peripheral blood. We find that the tissue-type location of CD8 + T cell clonotypes largely defines their exhaustion state with intra-tumoral spatial heterogeneity that is not well explained by somatic heterogeneity. De novo mutation calling from single-cell RNA-sequencing data allows us to broadly infer the clonality of stromal cells and lineage-trace myeloid cell development. We report six conserved meta-programs that distinguish tumor cell function, and find an epithelial-mesenchymal transition meta-program highly enriched at the tumor-normal interface that co-localizes with IL1B-expressing macrophages, offering a potential therapeutic target., Competing Interests: Declaration of interests In the past 3 years, S.A.T. has consulted for Roche and Genentech and is a Scientific Advisory Board member of Qiagen, Foresite labs, Biogen, and GSK, as well as a consultant and equity holder as co-founder of Transition Bio., (Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved.)

Background: Surgery for renal cell carcinoma (RCC) with venous tumour thrombus (VTT) extension into the renal vein (RV) and/or inferior vena cava (IVC) has high peri-surgical morbidity/mortality. NAXIVA assessed the response of VTT to axitinib, a potent tyrosine kinase inhibitor., Methods: NAXIVA was a single-arm, multi-centre, Phase 2 study. In total, 20 patients with resectable clear cell RCC and VTT received upto 8 weeks of pre-surgical axitinib. The primary endpoint was percentage of evaluable patients with VTT improvement by Mayo level on MRI. Secondary endpoints were percentage change in surgical approach and VTT length, response rate (RECISTv1.1) and surgical morbidity., Results: In all, 35% (7/20) patients with VTT had a reduction in Mayo level with axitinib: 37.5% (6/16) with IVC VTT and 25% (1/4) with RV-only VTT. No patients had an increase in Mayo level. In total, 75% (15/20) of patients had a reduction in VTT length. Overall, 41.2% (7/17) of patients who underwent surgery had less invasive surgery than originally planned. Non-responders exhibited lower baseline microvessel density (CD31), higher Ki67 and exhausted or regulatory T-cell phenotype., Conclusions: NAXIVA provides the first Level II evidence that axitinib downstages VTT in a significant proportion of patients leading to reduction in the extent of surgery., Clinical Trial Registration: NCT03494816., (© 2022. The Author(s).)

Background: Immune checkpoint inhibitors (ICI) have emerged as active therapies in the management of advanced RCC. While multiple studies have shown clinical activity of ICIs in clear cell histologies, the evidence to support their use in non-clear cell (ncc) subtypes is based on smaller prospective trials and retrospective analyses., Objective: The objective of this review is to summarize the clinical outcomes of ICI-based therapies in ncc-subtypes and in tumors with sarcomatoid/rhabdoid features., Methods: We performed a systematic literature search using PubMed, Google Scholar and ASCO databases. The keywords "renal cell cancer" and "immune checkpoint inhibitors" and equivalents were used and all original publications between July 2016 and July 2021 were included., Results: We included a total of 14 publications, including two clinical trials and 12 case series. The most frequent histologies were papillary (up to 75-100%), unclassified (up to 34%) and chromophobe (up to 28%). ICI monotherapy showed some activity in both 1st and 2nd line with response rates up to 27%. ICI combination regimens yielded better activity than ICI monotherapy but, overall, a heterogeneous efficacy was noted across histologies. Overall, outcomes of ICIs were superior in tumors with sarcomatoid/rhabdoid features., Conclusion: The observed activity of ICI-based therapies was heterogeneous. Combination regimens, papillary subtype and sarcomatoid/rhabdoid features were associated with higher responses. These findings might help treatment decisions and require further validation., Competing Interests: Ana Filipa Palma dos Reis (has no conflict of interests to report), Diana Simão (has no conflict of interests to report), Thomas Odeny (has no conflict of interests to report), Chiara Rodrigues (has no conflict of interests to report), Mário Fontes (speaking role/advisory boards: Bristol Myers Squibb, Daiichi Sankyo, Gilead, Lilly, Merck Sharp & Dohme, Novartis, Pfizer, Roche, Servier), Ricardo da Luz (has no conflict of interests to report), Rajasree Pia Chowdry (has no conflict of interests to report), Sarah J Welsh (has no conflict of interests to report), Channing Paller (has no conflict of interests to report), Pedro C. Barata consultant (Institution): Astellas; Eisai; Janssen, EMD Serono; Dendreon; Pfizer, Seattle Genetics, BMS, Bayer, Guardant Health; contracted research (Institution): AstraZeneca, Merck; research grant (Institution): BlueEarth Diagnostics; speaker’s bureau (Institution): Bayer, Caris, Pfizer., (© 2022 – The authors. Published by IOS Press.)

Purpose: MEDI0680 is a humanized anti-programmed cell death-1 (PD-1) antibody, and durvalumab is an anti-PD-L1 antibody. Combining treatment using these antibodies may improve efficacy versus blockade of PD-1 alone. This phase II study evaluated antitumor activity and safety of MEDI0680 plus durvalumab versus nivolumab monotherapy in immunotherapy-naïve patients with advanced clear-cell renal cell carcinoma who received at least one prior line of antiangiogenic therapy., Patients and Methods: Patients received either MEDI0680 (20 mg/kg) with durvalumab (750 mg) or nivolumab (240 mg), all intravenous, every 2 weeks. The primary endpoint was investigator-assessed objective response rate (ORR). Secondary endpoints included best overall response, progression-free survival (PFS), safety, overall survival (OS), and immunogenicity. Exploratory endpoints included changes in circulating tumor DNA (ctDNA), baseline tumor mutational burden, and tumor-infiltrated immune cell profiles., Results: Sixty-three patients were randomized (combination, n = 42; nivolumab, n = 21). ORR was 16.7% [7/42; 95% confidence interval (CI), 7.0-31.4] with combination treatment and 23.8% (5/21; 95% CI, 8.2-47.2) with nivolumab. Median PFS was 3.6 months in both arms; median OS was not reached in either arm. Because of adverse events, 23.8% of patients discontinued MEDI0680 and durvalumab and 14.3% of patients discontinued nivolumab. In the combination arm, reduction in ctDNA fraction was associated with longer PFS. ctDNA mutational analysis did not demonstrate an association with response in either arm. Tumor-infiltrated immune profiles showed an association between immune cell activation and response in the combination arm., Conclusions: MEDI0680 combined with durvalumab was safe and tolerable; however, it did not improve efficacy versus nivolumab monotherapy., (©2022 The Authors; Published by the American Association for Cancer Research.)

Background: Efficient trial designs are required to prioritise promising drugs within Phase II trials. Adaptive designs are examples of such designs, but their efficiency is reduced if there is a delay in assessing patient responses to treatment., Methods: Motivated by the WIRE trial in renal cell carcinoma (NCT03741426), we compare three trial approaches to testing multiple treatment arms: (1) single-arm trials in sequence with interim analyses; (2) a parallel multi-arm multi-stage trial and (3) the design used in WIRE, which we call the Multi-Arm Sequential Trial with Efficient Recruitment (MASTER) design. The MASTER design recruits patients to one arm at a time, pausing recruitment to an arm when it has recruited the required number for an interim analysis. We conduct a simulation study to compare how long the three different trial designs take to evaluate a number of new treatment arms., Results: The parallel multi-arm multi-stage and the MASTER design are much more efficient than separate trials. The MASTER design provides extra efficiency when there is endpoint delay, or recruitment is very quick., Conclusions: We recommend the MASTER design as an efficient way of testing multiple promising cancer treatments in non-comparative Phase II trials., (© 2021. The Author(s).)

Background: The gut microbiome is implicated as a marker of response to  immune checkpoint inhibitors (ICI) based on preclinical mouse models and preliminary observations in limited patient series. Furthermore, early studies suggest faecal microbial transfer may have therapeutic potential, converting ICI non-responders into responders. So far, identification of specific responsible bacterial taxa has been inconsistent, which limits future application. The MITRE study will explore and validate a microbiome signature in a larger scale prospective study across several different cancer types., Methods: Melanoma, renal cancer and non-small cell lung cancer patients who are planned to receive standard immune checkpoint inhibitors are being recruited to the MITRE study. Longitudinal stool samples are collected prior to treatment, then at 6 weeks, 3, 6 and 12 months during treatment, or at disease progression/recurrence (whichever is sooner), as well as after a severe (≥grade 3 CTCAE v5.0) immune-related adverse event. Additionally, whole blood, plasma, buffy coat, RNA and peripheral blood mononuclear cells (PBMCs) is collected at similar time points and will be used for exploratory analyses. Archival tumour tissue, tumour biopsies at progression/relapse, as well as any biopsies from body organs collected after a severe toxicity are collected. The primary outcome measure is the ability of the microbiome signature to predict 1 year progression-free survival (PFS) in patients with advanced disease. Secondary outcomes include microbiome correlations with toxicity and other efficacy end-points. Biosamples will be used to explore immunological and genomic correlates. A sub-study will evaluate both COVID-19 antigen and antibody associations with the microbiome., Discussion: There is an urgent need to identify biomarkers that are predictive of treatment response, resistance and toxicity to immunotherapy. The data generated from this study will both help inform patient selection for these drugs and provide information that may allow therapeutic manipulation of the microbiome to improve future patient outcomes., Trial Registration: NCT04107168 , ClinicalTrials.gov, registered 09/27/2019. Protocol V3.2 (16/04/2021)., (© 2022. The Author(s).)

Differentiating aggressive clear cell renal cell carcinoma (ccRCC) from indolent lesions is challenging using conventional imaging. This work prospectively compared the metabolic imaging phenotype of renal tumors using carbon-13 MRI following injection of hyperpolarized [1- 13 C]pyruvate (HP- 13 C-MRI) and validated these findings with histopathology. Nine patients with treatment-naïve renal tumors (6 ccRCCs, 1 liposarcoma, 1 pheochromocytoma, 1 oncocytoma) underwent pre-operative HP- 13 C-MRI and conventional proton ( 1 H) MRI. Multi-regional tissue samples were collected using patient-specific 3D-printed tumor molds for spatial registration between imaging and molecular analysis. The apparent exchange rate constant ( k PL ) between 13 C-pyruvate and 13 C-lactate was calculated. Immunohistochemistry for the pyruvate transporter (MCT1) from 44 multi-regional samples, as well as associations between MCT1 expression and outcome in the TCGA-KIRC dataset, were investigated. Increasing k PL in ccRCC was correlated with increasing overall tumor grade (ρ = 0.92, p = 0.009) and MCT1 expression (r = 0.89, p = 0.016), with similar results acquired from the multi-regional analysis. Conventional 1 H-MRI parameters did not discriminate tumor grades. The correlation between MCT1 and ccRCC grade was confirmed within a TCGA dataset ( p < 0.001), where MCT1 expression was a predictor of overall and disease-free survival. In conclusion, metabolic imaging using HP- 13 C-MRI differentiates tumor aggressiveness in ccRCC and correlates with the expression of MCT1, a predictor of survival. HP- 13 C-MRI may non-invasively characterize metabolic phenotypes within renal cancer.

Background: Window-of-opportunity trials, evaluating the engagement of drugs with their biological target in the time period between diagnosis and standard-of-care treatment, can help prioritise promising new systemic treatments for later-phase clinical trials. Renal cell carcinoma (RCC), the 7 th commonest solid cancer in the UK, exhibits targets for multiple new systemic anti-cancer agents including DNA damage response inhibitors, agents targeting vascular pathways and immune checkpoint inhibitors. Here we present the trial protocol for the WIndow-of-opportunity clinical trial platform for evaluation of novel treatment strategies in REnal cell cancer (WIRE)., Methods: WIRE is a Phase II, multi-arm, multi-centre, non-randomised, proof-of-mechanism (single and combination investigational medicinal product [IMP]), platform trial using a Bayesian adaptive design. The Bayesian adaptive design leverages outcome information from initial participants during pre-specified interim analyses to determine and minimise the number of participants required to demonstrate efficacy or futility. Patients with biopsy-proven, surgically resectable, cT1b+, cN0-1, cM0-1 clear cell RCC and no contraindications to the IMPs are eligible to participate. Participants undergo diagnostic staging CT and renal mass biopsy followed by treatment in one of the treatment arms for at least 14 days. Initially, the trial includes five treatment arms with cediranib, cediranib + olaparib, olaparib, durvalumab and durvalumab + olaparib. Participants undergo a multiparametric MRI before and after treatment. Vascularised and de-vascularised tissue is collected at surgery. A ≥ 30% increase in CD8+ T-cells on immunohistochemistry between the screening and nephrectomy is the primary endpoint for durvalumab-containing arms. Meanwhile, a reduction in tumour vascular permeability measured by K trans on dynamic contrast-enhanced MRI by ≥30% is the primary endpoint for other arms. Secondary outcomes include adverse events and tumour size change. Exploratory outcomes include biomarkers of drug mechanism and treatment effects in blood, urine, tissue and imaging., Discussion: WIRE is the first trial using a window-of-opportunity design to demonstrate pharmacological activity of novel single and combination treatments in RCC in the pre-surgical space. It will provide rationale for prioritising promising treatments for later phase trials and support the development of new biomarkers of treatment effect with its extensive translational agenda., Trial Registration: ClinicalTrials.gov: NCT03741426 / EudraCT: 2018-003056-21 ., (© 2021. The Author(s).)

Purpose: To detect early response to sunitinib treatment in metastatic clear cell renal cancer (mRCC) using multiparametric MRI., Method: Participants with mRCC undergoing pre-surgical sunitinib therapy in the prospective NeoSun clinical trial (EudraCtNo: 2005-004502-82) were imaged before starting treatment, and after 12 days of sunitinib therapy using morphological MRI sequences, advanced diffusion-weighted imaging, measurements of R2* (related to hypoxia) and dynamic contrast-enhanced imaging. Following nephrectomy, participants continued treatment and were followed-up with contrast-enhanced CT. Changes in imaging parameters before and after sunitinib were assessed with the non-parametric Wilcoxon signed-rank test and the log-rank test was used to assess effects on survival., Results: 12 participants fulfilled the inclusion criteria. After 12 days, the solid and necrotic tumor volumes decreased by 28% and 17%, respectively (p = 0.04). However, tumor-volume reduction did not correlate with progression-free or overall survival (PFS/OS). Sunitinib therapy resulted in a reduction in median solid tumor diffusivity D from 1298x10-6 to 1200x10-6mm2/s (p = 0.03); a larger decrease was associated with a better RECIST response (p = 0.02) and longer PFS (p = 0.03) on the log-rank test. An increase in R2* from 19 to 28s-1 (p = 0.001) was observed, paralleled by a decrease in Ktrans from 0.415 to 0.305min-1 (p = 0.01) and a decrease in perfusion fraction from 0.34 to 0.19 (p<0.001)., Conclusions: Physiological imaging confirmed efficacy of the anti-angiogenic agent 12 days after initiating therapy and demonstrated response to treatment. The change in diffusivity shortly after starting pre-surgical sunitinib correlated to PFS in mRCC undergoing nephrectomy, however, no parameter predicted OS., Trial Registration: EudraCtNo: 2005-004502-82., Competing Interests: The authors have read the journal’s policy and have the following competing interests: ANP has received a speaker fee and travel expenses from GE Healthcare. SJW has received travel expenses from IPSEN. GDS has received educational grants from Pfizer, Astra Zeneca, and Intuitive Surgical; consultancy fees from Merck, Pfizer, EUSA Pharma and CMR Surgical; travel expenses and speaker fees from Pfizer. AW received a single Scientific Advisory Board fee from Roche. TE has received research support from Pfizer and AstraZeneca, and honoraria from Pfizer. TE was employed by AstraZeneca during part of the study duration and is now employed by Roche, and holds stock in AstraZeneca and Roche. Pfizer provided the study drug Sunitinib and an educational grant for the translational endpoint analysis, reported separately. There are no patents, products in development or marketed products associated with this research to declare. This does not alter our adherence to PLOS ONE policies on sharing data and materials.

Background: Immune-related adverse events (irAEs) typically occur within 4 months of starting anti-programmed cell death protein 1 (PD-1)-based therapy [anti-PD-1 ± anti-cytotoxic T-lymphocyte-associated protein 4 (CTLA4)], but delayed irAEs (onset >12 months after commencement) can also occur. This study describes the incidence, nature and management of delayed irAEs in patients receiving anti-PD-1-based immunotherapy., Patients and Methods: Patients with delayed irAEs from 20 centres were studied. The incidence of delayed irAEs was estimated as a proportion of melanoma patients treated with anti-PD-1-based therapy and surviving >1 year. Onset, clinical features, management and outcomes of irAEs were examined., Results: One hundred and eighteen patients developed a total of 140 delayed irAEs (20 after initial combination with anti-CTLA4), with an estimated incidence of 5.3% (95% confidence interval 4.0-6.9, 53/999 patients at sites with available data). The median onset of delayed irAE was 16 months (range 12-53 months). Eighty-seven patients (74%) were on anti-PD-1 at irAE onset, 15 patients (12%) were <3 months from the last dose and 16 patients (14%) were >3 months from the last dose of anti-PD-1. The most common delayed irAEs were colitis, rash and pneumonitis; 55 of all irAEs (39%) were ≥grade 3. Steroids were required in 80 patients (68%), as well as an additional immunosuppressive agent in 27 patients (23%). There were two irAE-related deaths: encephalitis with onset during anti-PD-1 and a multiple-organ irAE with onset 11 months after ceasing anti-PD-1. Early irAEs (<12 months) had also occurred in 69 patients (58%), affecting a different organ from the delayed irAE in 59 patients (86%)., Conclusions: Delayed irAEs occur in a small but relevant subset of patients. Delayed irAEs are often different from previous irAEs, may be high grade and can lead to death. They mostly occur in patients still receiving anti-PD-1. The risk of delayed irAE should be considered when deciding the duration of treatment in responding patients. However, patients who stop treatment may also rarely develop delayed irAE., Competing Interests: Disclosure CNO has received non-financial support from Merck Sharp & Dohme. XB receives a merit award supported by BMS. CA has received travel support from Amgen, BMS and Roche. PB has received sponsorship from MSD. WX has served on advisory boards for Merck Serono and Novartis and has received research funding from Merck Serono. BN has received personal financial compensation from Roche, Bristol-Myers Squibb, Merck Sharp & Dohme, Novartis and AstraZeneca, for public speaking, consultancy and participation in advisory board meetings. His institution (UZ Brussel) received research funding related to research projects conducted by his team from Pfizer, Novartis, Roche and Merck-Serono. CUB has an advisory role for BMS, MSD, Roche, Novartis, GSK, AZ, Pfizer, Lilly, GenMab, Pierre Fabre and Third Rock Ventures. He has received research funding from BMS, Novartis and NanoString. He has stock ownership in Uniti Cars and is co-founder of Immagene B.V. SJW has received travel support from Ipsen. SS has served on advisory boards for BMS, MSD, Merck, Novartis, Roche, Janssen, AstraZeneca and Amgen and has received research funding from Amgen, Endocyte, MSD and AstraZeneca. JM has intermittent project focused consultant or advisory relationships with Merck/Pfizer, Merck Sharp & Dohme, Amgen, Novartis, Bristol Myers Squibb, Sanofi and Pierre Fabre and has received travel support from Ultrasun, L'oreal, Merck Sharp & Dohme, Bristol Myers Squibb and Pierre Fabre outside of the submitted work. JLM is consultant advisor for BMS, Roche and Novartis. PAA has/had a consultant/advisory role for Bristol Myers Squibb, Roche-Genentech, Merck Sharp & Dohme, Array, Novartis, Merck Serono, Pierre Fabre, Incyte, Medimmune, AstraZeneca, Syndax, Sun Pharma, Sanofi, Idera, Ultimovacs, Sandoz, Immunocore, 4SC, Alkermes, Italfarmaco, Nektar, Boehringer-Ingelheim, Eisai and Regeneron. He also received research funds from Bristol Myers Squibb, Roche-Genentech and Array, and travel support from MSD. LZ has received honoraria from Roche, Bristol Myers Squibb, Merck Sharp & Dohme, Novartis and Pierre Fabre, research funding from Novartis and has served on advisory boards for Bristol Myers Squibb, Novartis, Pierre Fabre, Sun Pharma, Sanofi and Merck Sharp & Dohme. He has received travel support from Bristol Myers Squibb, Pierre Fabre, Sanofi, Amgen, Novartis and Sun Pharma. DBJ has served on advisory boards for Array Biopharma, BMS, Iovance, Jansen, Merck and Novartis, and received research funding from BMS and Incyte. CL has served on advisory boards for BMS, MSD, Novartis, Amgen, Roche, Merck, Serono and Sanofi. She has received honoraria from Roche, BMS, Novartis, Amgen, MSD, Pierre Fabre, Pfizer and Incyte, and speaker's bureau from Roche, BMS, Novartis, Amgen and MSD. She has received research funding from Roche and BMS. She has received travel support from BMS and MSD. She serves on the board of Avantis Medical Systems. MSC has served on advisory boards for Bristol Myers Squibb, MSD, Amgen, Novartis, Pierre Fabre, Roche, Sanofi, Merck, Ideaya, Regeneron, Nektar and Eisai and honoraria from Bristol Myers Squibb, MSD and Novartis. RJS has received research support from Amgen and Merck, as well as served as a paid consultant and/or on a scientific advisory board for Array, AstraZeneca, Asana Biosciences, Bristol Myers Squibb, Eisai, Iovance, Merck, Novartis, Oncosec, Pfizer and Replimune. GVL is consultant advisor for Aduro Biotech Inc, Amgen Inc, Array Biopharma inc, Boehringer Ingelheim International GmbH, Bristol-Myers Squibb, Highlight Therapeutics S.L., Merck Sharpe & Dohme, Novartis Pharma AG, Pierre Fabre, QBiotics Group Limited, Regeneron Pharmaceuticals Inc. AMM has served on advisory boards for BMS, MSD, Novartis, Roche, Pierre Fabre and QBiotics. All other authors have declared no conflicts of interest., (Copyright © 2021. Published by Elsevier Ltd.)

BCMA-CD3-targeting bispecific antibodies (BsAb) are a recently developed immunotherapy class which shows potent tumor killing activity in multiple myeloma (MM). Here, we investigated a murine BCMA-CD3-targeting BsAb in the immunocompetent Vk*MYC and its IMiD-sensitive derivative Vk*MYC hCRBN models of MM. The BCMA-CD3 BsAb was safe and efficacious in a subset of mice, but failed in those with high-tumor burden, consistent with clinical reports of BsAb in leukemia. The combination of BCMA-CD3 BsAb with pomalidomide expanded lytic T cells and improved activity even in IMiD resistant high-tumor burden cases. Yet, survival was only marginally extended due to acute toxicity and T cell exhaustion, which impaired T cell persistence. In contrast, the combination with cyclophosphamide was safe and allowed for a tempered pro-inflammatory response associated with long-lasting complete remission. Concurrent cytotoxic therapy with BsAb actually improved T cell persistence and function, offering a promising approach to patients with a large tumor burden.

Tumors consist of cancer cells and a network of non-cancerous stroma. Cancer-associated fibroblasts (CAF) are known to support tumorigenesis, and are emerging as immune modulators. Neutrophils release histone-bound nuclear DNA and cytotoxic granules as extracellular traps (NET). Here we show that CAFs induce NET formation within the tumor and systemically in the blood and bone marrow. These tumor-induced NETs (t-NETs) are driven by a ROS-mediated pathway dependent on CAF-derived Amyloid β, a peptide implicated in both neurodegenerative and inflammatory disorders. Inhibition of NETosis in murine tumors skews neutrophils to an anti-tumor phenotype, preventing tumor growth; reciprocally, t-NETs enhance CAF activation. Mirroring observations in mice, CAFs are detected juxtaposed to NETs in human melanoma and pancreatic adenocarcinoma, and show elevated amyloid and β-Secretase expression which correlates with poor prognosis. In summary, we report that CAFs drive NETosis to support cancer progression, identifying Amyloid β as the protagonist and potential therapeutic target.

Metastatic melanoma carries a poor prognosis despite modern systemic therapies. Understanding the evolution of the disease could help inform patient management. Through whole-genome sequencing of 13 melanoma metastases sampled at autopsy from a treatment naïve patient and by leveraging the analytical power of multi-sample analyses, we reveal evidence of diversification among metastatic lineages. UV-induced mutations dominate the trunk, whereas APOBEC-associated mutations are found in the branches of the evolutionary tree. Multi-sample analyses from a further seven patients confirmed that lineage diversification was pervasive, representing an important mode of melanoma dissemination. Our analyses demonstrate that joint analysis of cancer cell fraction estimates across multiple metastases can uncover previously unrecognised levels of tumour heterogeneity and highlight the limitations of inferring heterogeneity from a single biopsy.

The most common genetic abnormality in multiple myeloma (MM) is the deletion of chromosome 13, seen in almost half of newly diagnosed patients. Unlike chronic lymphocytic leukemia, where a recurrent minimally deleted region including MIR15A/MIR16-1 has been mapped, the deletions in MM predominantly involve the entire chromosome and no specific driver gene has been identified. Additional candidate loci include RB1 and DIS3 , but while biallelic deletion of RB1 is associated with disease progression, DIS3 is a common essential gene and complete inactivation is not observed. The Vk*MYC transgenic mouse model of MM spontaneously acquires del(14), syntenic to human chromosome 13, and Rb1 complete inactivation, but not Dis3 mutations. Taking advantage of this model, we explored the role in MM initiation and progression of two candidate loci on chromosome 13: RB1 and MIR15A/MIR16-1 . Monoallelic deletion of Mir15a/Mir16-1 but not Rb1 was sufficient to accelerate the development of monoclonal gammopathy in wildtype mice, and the progression of MM in Vk*MYC mice, resulting in increased expression of Mir15a/Mir16-1 target genes and plasma cell proliferation, which was similarly observed in patients with MM., Competing Interests: The authors declare no potential conflicts of interest

For all primary cutaneous squamous cell carcinomas (cSCCs), physical examination should include full skin examination, recording of tumour diameter and regional lymph-node-basin status. Surgery is the treatment of choice, with a minimal 5-mm margin. For elderly patients with well-differentiated tumours, other surgical modalities can be explored. Surgery for organ-transplant recipients should not be delayed. The issue with cSCC is identifying high-risk tumours with staging, as this may alter treatment and follow-up schedules. Adjuvant radiation therapy should be considered for incomplete resection, when re-excision is impossible or there are poor-prognosis histological findings. Recommendations are at least biannual dermatological surveillance for 2 years, but in elderly patients with small, well-differentiated tumours long-term follow-up is not always necessary. In case of positive lymph nodes, radical dissection is needed, with regional postoperative adjuvant radiation. Advanced cSCCs are defined as unresectable local, regional or distant disease requiring systemic treatment. Their only approved treat-ment is the PD-1 inhibitor, cemiplimab. Trials evaluating adjuvant or neo-adjuvant anti-PD-1 are ongoing. Platin-based chemo or anti-epidermal growth-factor-receptor therapies are possible second-line treatments. For transplant patients, minimizing immunosuppression and switching to sirolimus must be considered at first appearance of cSCC.

Anti-angiogenic agents, such as the multi-tyrosine kinase inhibitor sunitinib, are key first line therapies for metastatic clear cell renal cell carcinoma (ccRCC), but their mechanism of action is not fully understood. Here, we take steps towards validating a computational prediction based on differential transcriptome network analysis that phosphorylated adapter RNA export protein (PHAX) is associated with sunitinib drug treatment. The regulatory impact factor differential network algorithm run on patient tissue samples suggests PHAX is likely an important regulator through changes in genome-wide network connectivity. Immunofluorescence staining of patient tumours showed strong localisation of PHAX to the microvasculature consistent with the anti-angiogenic effect of sunitinib. In normal kidney tissue, PHAX protein abundance was low but increased with tumour grade (G1 vs. G3/4; p < 0.01), consistent with a possible role in cancer progression. In organ culture, ccRCC cells had higher levels of PHAX protein expression than normal kidney cells, and sunitinib increased PHAX protein expression in a dose dependent manner (untreated vs. 100 µM; p < 0.05). PHAX knockdown in a ccRCC organ culture model impacted the ability of sunitinib to cause cancer cell death ( p < 0.0001 untreated vs. treated), suggesting a role for PHAX in mediating the efficacy of sunitinib.

Background: Cell-free tumor-derived DNA (ctDNA) allows non-invasive monitoring of cancers, but its utility in renal cell cancer (RCC) has not been established., Methods: Here, a combination of untargeted and targeted sequencing methods, applied to two independent cohorts of patients (n = 91) with various renal tumor subtypes, were used to determine ctDNA content in plasma and urine., Results: Our data revealed lower plasma ctDNA levels in RCC relative to other cancers of similar size and stage, with untargeted detection in 27.5% of patients from both cohorts. A sensitive personalized approach, applied to plasma and urine from select patients (n = 22) improved detection to ~ 50%, including in patients with early-stage disease and even benign lesions. Detection in plasma, but not urine, was more frequent amongst patients with larger tumors and in those patients with venous tumor thrombus. With data from one extensively characterized patient, we observed that plasma and, for the first time, urine ctDNA may better represent tumor heterogeneity than a single tissue biopsy. Furthermore, in a subset of patients (n = 16), longitudinal sampling revealed that ctDNA can track disease course and may pre-empt radiological identification of minimal residual disease or disease progression on systemic therapy. Additional datasets will be required to validate these findings., Conclusions: These data highlight RCC as a ctDNA-low malignancy. The biological reasons for this are yet to be determined. Nonetheless, our findings indicate potential clinical utility in the management of patients with renal tumors, provided improvement in isolation and detection approaches.

The chimeric fusion oncogene early B-cell factor 1-platelet-derived growth factor receptor-β (EBF1-PDGFRB) is a recurrent lesion observed in Philadelphia-like B-acute lymphoblastic leukemia (B-ALL) and is associated with particularly poor prognosis. While it is understood that this fusion activates tyrosine kinase signaling, the mechanisms of transformation and importance of perturbation of EBF1 activity remain unknown. EBF1 is a nuclear transcription factor required for normal B-lineage specification, commitment and development. Conversely, PDGFRB is a receptor tyrosine kinase that is normally repressed in lymphocytes, yet PDGFRB remains a common fusion partner in leukemias. Here, we demonstrate that the EBF1-PDGFRB fusion results in loss of EBF1 function, multimerization and autophosphorylation of the fusion protein, activation of signal transducer and activator of transcription 5 (STAT5) signaling and gain of interleukin-7 (IL-7)-independent cell proliferation. Deregulation and loss of EBF1 function is critically dependent on the nuclear export activity of the transmembrane (TM) domain of PDGFRB. Deletion of the TM domain partially rescues EBF1 function and restores IL-7 dependence, without requiring kinase inhibition. Moreover, we demonstrate that EBF1-PDGFRB synergizes with loss of IKAROS function in a fully penetrant B-ALL in vivo. Thus, we establish that EBF1-PDGFRB is sufficient to drive leukemogenesis through TM-dependent loss of transcription factor function, increased proliferation and synergy with additional genetic insults including loss of IKAROS function.

Background: Selective kinase and immune checkpoint inhibitors, and their combinations, have significantly improved the survival of patients with advanced metastatic melanoma. Not all patients will respond to treatment however, and some patients will present with significant toxicities. Hence, the identification of biomarkers is critical for the selection and management of patients receiving treatment. Biomarker discovery often involves proteomic techniques that simultaneously profile multiple proteins but few studies have compared these platforms., Methods: In this study, we used the multiplex bead-based Eve Technologies Discovery assay and the aptamer-based SomaLogic SOMAscan assay to identify circulating proteins predictive of response to immunotherapy in melanoma patients treated with combination immune checkpoint inhibitors. Expression of four plasma proteins were further validated using the bead-based Millipore Milliplex assay., Results: Both the Discovery and the SOMAscan assays detected circulating plasma proteins in immunotherapy-treated melanoma patients. However, these widely used assays showed limited correlation in relative protein quantification, due to differences in specificity and the dynamic range of protein detection. Protein data derived from the Discovery and Milliplex bead-based assays were highly correlated., Conclusions: Our study highlights significant limitations imposed by inconsistent sensitivity and specificity due to differences in the detection antibodies or aptamers of these widespread biomarker discovery approaches. Our findings emphasize the need to improve these technologies for the accurate identification of biomarkers.

Early B cell factor 3 (EBF3) is an atypical transcription factor that is thought to influence the laminar formation of the cerebral cortex. Here, we report that de novo mutations in EBF3 cause a complex neurodevelopmental syndrome. The mutations were identified in two large-scale sequencing projects: the UK Deciphering Developmental Disorders (DDD) study and the Canadian Clinical Assessment of the Utility of Sequencing and Evaluation as a Service (CAUSES) study. The core phenotype includes moderate to severe intellectual disability, and many individuals exhibit cerebellar ataxia, subtle facial dysmorphism, strabismus, and vesicoureteric reflux, suggesting that EBF3 has a widespread developmental role. Pathogenic de novo variants identified in EBF3 include multiple loss-of-function and missense mutations. Structural modeling suggested that the missense mutations affect DNA binding. Functional analysis of mutant proteins with missense substitutions revealed reduced transcriptional activities and abilities to form heterodimers with wild-type EBF3. We conclude that EBF3, a transcription factor previously unknown to be associated with human disease, is important for brain and other organ development and warrants further investigation., (Copyright © 2017 American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.)

We examined aging-related differences in the contribution of visuomotor correction to force fluctuations during index finger abduction via the analysis of two datasets from similar subjects. Study (1) Young (N = 27, 23 ± 8 years) and older adults (N = 14, 72 ± 9 years) underwent assessment of maximum voluntary contraction force (MVC) and force steadiness during constant-force (CF) index finger abduction (2.5, 30, 65% MVC). For each trial, visual feedback of the force (VIS) was provided for 8-10 s and removed for 8-10 s (NOVIS). Visual gain of the force feedback at 2.5% MVC was high; 12- and 26-fold greater than the 30 and 65% MVC targets. Mean force, standard deviation (SD) of force, and coefficient of variation (CV) of force was calculated for detrended (<0.5 Hz drift removed) VIS and NOVIS data segments. Study (2) A similar group of 14 older adults performed discrete, randomly-ordered VIS or NOVIS trials at low target forces (1-3% MVC) and high visual gain. Study (1) For young adults the CV of force was similar between VIS and NOVIS for the 2.5% (4.8 vs. 4.3%), 30% (3.2 vs. 3.2%) and 65% (3.5 vs. 4.2%) target forces. In contrast, for older adults the CV of force was greater for VIS than NOVIS for 2.5% MVC (6.6 vs. 4.2%, p < 0.001), but not for the 30% (2.4 vs. 2.4%) and 65% (3.1 vs. 3.3%) target forces. At 2.5% MVC, the increase in CV of force for VIS compared with NOVIS was significantly greater (age × visual condition p = 0.008) for older than young adults. Study (2) Similarly, for older adults performing discrete, randomly ordered trials the CV of force was greater for VIS than NOVIS (6.04 vs. 3.81%, p = 0.01). When visual force feedback was a dominant source of information at low forces, normalized force variability was ~58% greater for older adults, but only 11% greater for young adults. The significant effect of visual feedback for older adults was not dependent on the order of presentation of visual conditions. The results indicate that impaired processing of visuomotor information underlies the greater motor variability observed in older adults during lab-based isometric contractions of a hand muscle.

Following the discovery that nearly half of all cutaneous melanomas harbour a mutation in the BRAF gene, molecular targeted kinase inhibitors have been developed for the treatment of metastatic melanoma and have dramatically improved outcomes for those patients with BRAF mutant disease, achieving high levels of objective response and prolonging survival. Since 2011, the specific BRAF targeted agents, vemurafenib and dabrafenib, and the MEK inhibitor, trametinib, have been licensed for the treatment of patients with unresectable or metastatic BRAF mutant melanoma. As with other biological targeted agents, these drugs are associated with predictable patterns of adverse events. Proactive toxicity management is important to ensure maximum treatment benefit and avoid unnecessary treatment discontinuation. We review the most common and serious adverse events associated with BRAF targeted agents and suggest management algorithms to guide practitioners in using these drugs effectively in the clinic.

The incidence and mortality from renal cell cancer (RCC) is increasing. RCC tumors are particularly vascular in nature as a result of disruption of the VHL gene and/or its upstream pathway leading to upregulation of the hypoxia-inducible factor transcription factor. The hypoxia-inducible factor pathway drives angiogenesis by upregulating VEGF and bFGF, amongst other proangiogenic downstream target genes. Therapies which target angiogenesis have been successful in treating metastatic RCC (mRCC) and the receptor tyrosine kinase inhibitor, pazopanib, is licensed for first line treatment of mRCC. This review details the past, current and future roles of pazopanib in the treatment of mRCC.

The hypoxia-inducible transcription factor (HIF) co-ordinates the response of tumours to low oxygen by stimulating genes involved in metabolism and angiogenesis. HIF pathway activation is associated with decreased progression-free survival and increased mortality; compounds that target this pathway are potential agents for the treatment of a range of solid tumour malignancies. Renal cancers are likely to be particularly sensitive to inhibition of the HIF pathway since ~80% show constitutive activation of HIF. We have previously described the di-substituted naphthalene derivative, CL67, which binds to a G-quadruplex higher-order structure in the HIF promoter sequence in vitro. We show here that CL67 blocks HIF expression leading to inhibition of HIF-transactivation and down-regulation of downstream target genes and proteins in renal carcinoma cell lines and in a mouse xenograft model of renal cancer. This inhibition is independent of pathways that control HIF abundance through oxygen-dependant degradation and oxygen dependant HIF sub-unit expression.

Metastatic tumors generally exhibit aerobic glycolysis (the Warburg effect). The advent of [18F]fluorodeoxyglucose positron emission tomography imaging, coupled with recent findings linking hypoxia-inducible factor (HIF-1alpha) overexpression to aggressive cancers, has rekindled an interest in this aspect of tumor metabolism. These studies explore the role of HIF-1alpha in human breast cancer lines and its relationship to glycolytic regulation. Here we demonstrate that, under normal oxygen conditions, nonmetastatic cells consume less glucose and express low HIF-1alpha, whereas metastatic cells constitutively express high glycolysis and HIF-1alpha, suggesting that dysregulation of HIF-1alpha may induce the Warburg effect. This hypothesis was tested by renormalizing HIF-1alpha levels in renal carcinoma cells, leading to inhibition of aerobic glycolysis.

Hypoxia-inducible factor-1 (HIF-1) is a transcription factor that plays a critical role in tumor growth by increasing resistance to apoptosis and the production of angiogenic factors such as vascular endothelial growth factor (VEGF). HIF-1 is a heterodimer comprised of oxygen-regulated HIF-1alpha and constitutively expressed HIF-1beta subunits. The redox protein thioredoxin-1 (Trx-1), which is found at high levels in many human cancers, increases both aerobic and hypoxia-induced HIF-1alpha protein in cells leading to increased expression of HIF-regulated genes. We have investigated whether two cancer drugs that inhibit Trx-1 signaling, PX-12 (1-methylpropyl 2-imidazolyl disulfide) and pleurotin, decrease HIF-1alpha protein levels and the expression of downstream target genes. Treatment of MCF-7 human breast cancer and HT-29 human colon carcinoma cells with PX-12 and pleurotin prevented the hypoxia (1% oxygen)-induced increase in HIF-1alpha protein. HIF-1-trans-activating activity, VEGF formation, and inducible nitric oxide synthase were also decreased by treatment with PX-12 and pleurotin under hypoxic conditions. PX-12 and pleurotin also decreased HIF-1alpha protein levels and HIF-1 trans-activation in RCC4 renal cell carcinoma cells that constitutively overexpress HIF-1alpha protein because of loss of the pVHL gene, indicating that HIF-1alpha is inhibited independently of the pVHL pathway. HIF-1alpha and VEGF protein levels in MCF-7 tumor xenografts in vivo were decreased by PX-12 treatment of mice. The results suggest that inhibition of HIF-1alpha by Trx-1 inhibitors may contribute to the growth inhibitory and antitumor activity of these agents.

Hypoxia-inducible factor 1 (HIF-1), a heterodimer of HIF-1alpha and HIF-1beta subunits, is a transcriptional activator central to the cellular response to low oxygen that includes metabolic adaptation, angiogenesis, metastasis, and inhibited apoptosis. Thioredoxin-1 (Trx-1) is a small redox protein overexpressed in a number of human primary tumors. We have examined the effects of Trx-1 on HIF activity and the activation of downstream genes. Stable transfection of human breast carcinoma MCF-7 cells with human Trx-1 caused a significant increase in HIF-1alpha protein levels under both normoxic (20% oxygen) and hypoxic (1% oxygen) conditions. Trx-1 increased hypoxia-induced HIF-1 transactivation activity measured using a luciferase reporter under the control of the hypoxia response element. Changes in HIF-1alpha mRNA levels did not account for the changes observed at the protein level, and HIF-1beta protein levels did not change. Trx-1 transfection also caused a significant increase in the protein products of hypoxia-responsive genes, including vascular endothelial growth factor (VEGF) and nitric oxide synthase 2 in a number of different cell lines (MCF-7 human breast and HT29 human colon carcinomas and WEHI7.2 mouse lymphoma cells) under both normoxic and hypoxic conditions. The pattern of expression of the different isoforms of VEGF was not changed by Trx-1. Transfection of a redox-inactive Trx-1 (C32S/C35S) markedly decreased levels of HIF-1alpha protein, HIF-1 transactivating activity, and VEGF protein in MCF-7 cells compared with empty vector controls. In vivo studies using WEHI7.2 cells transfected with Trx-1 showed significantly increased tumor VEGF and angiogenesis. The results suggest that Trx-1 increases HIF-1alpha protein levels in cancer cells and increases VEGF production and tumor angiogenesis.

Thymidylate synthase (TS) is an important enzyme catalysing the reductive methylation of dUMP to dTMP that is further metabolized to dTTP for DNA synthesis. Loss of viability following TS inhibition occurs as a consequence of depleted dTTP pools and at least in some cell lines, accumulation of dUTP and subsequent misincorporation of uracil into DNA. The expansion in dUTP pools is largely determined by the expression of the pyrophosphatase, dUTPase. Our previous work has shown that following TS inhibition the ability to accumulate dUTP was associated with an earlier growth inhibitory effect. 3 human lung tumour cell lines and HT29 human colon tumour cells transfected with dUTPase have been used to investigate the relationship between loss of viability following TS inhibition and dUTP accumulation. Cell cycle arrest typical of TS inhibition was an early event in all cell lines and occurred irrespective of the ability to accumulate dUTP or p53 function. However, a large expansion of dUTP pools was associated with mature DNA damage (4 h) and an earlier loss of viability following TS inhibition compared to cells in which dUTP pools were not expanded. In A549 cells damage to mature DNA may have been exacerbated by significantly higher activity of the excision repair enzyme, uracil-DNA glycosylase. Consistent with results using different inhibitors of TS, transfection of dUTPase into HT29 cells significantly reduced the cytotoxicity of a 24 h but not 48 h exposure to ZD9331. Although loss of viability can be mediated through dTTP deprivation alone, the uracil misincorporation pathway resulted in an earlier commitment to cell death. The relevance of this latter pathway in the clinical response to TS inhibitors deserves further investigation., (Copyright 2001 Cancer Research Campaign.)

Thymidylate synthase (TS) is an important target for cancer chemotherapy. However, several mechanisms of resistance to TS inhibitors have been described. One mechanism that may be relevant to short-term exposure to TS inhibitors occurs as a result of disruption of the autoregulatory loop, which allows TS to control its own translation. This disruption leads to up-regulation of TS protein and is generally thought to decrease efficacy. This study has investigated TS protein up-regulation using a range of TS inhibitors in both tumor and nonmalignant cell lines in vitro and in vivo. Up-regulation of TS protein showed a time-, dose-, and cell-type-specific response to treatment with ZD9331. This response was observed in W1L2 cells treated for 24 h at equitoxic doses of raltitrexed (6-fold), ZD9331 (10-fold), fluorouracil (5-fold), LY231514 (7-fold), AG337 (7-fold), and BW1843U89 (3-fold). Up-regulation was observed over a range of doses. Elevation of TS protein only persisted up to 12 h after removal of drug. The extent of induction does not depend on basal TS levels. Nontransformed human fibroblasts showed significantly greater up-regulation of TS protein than tumor cells exposed to an equitoxic dose of ZD9331. In vivo experiments using the L5178Y thymidine kinase -/- mouse lymphoma implanted into DBA2 mice also showed greater up-regulation of TS protein in normal intestinal epithelial cells compared with tumor cells. These results confirm that TS up-regulation is a common feature of TS inhibition in tumor cells and that it may occur to a greater extent in normal tissues, although the clinical implications of these findings remain to be determined.