A minimally invasive, lentiviral based method for the rapid and sustained genetic manipulation of renal tubules.

The accelerated discovery of disease-related genes emerging from genomic studies has strained the capacity of traditional genetically engineered mouse models (GEMMs) to provide in-vivo validation. Direct, somatic, genetic engineering approaches allow for accelerated and flexible genetic manipulation and represent an attractive alternative to GEMMs. In this study we investigated the feasibility, safety and efficiency of a minimally invasive, lentiviral based approach for the sustained in-vivo modification of renal tubular epithelial cells. Using ultrasound guidance, reporter vectors were directly injected into the mouse renal parenchyma. We observed transgene expression confined to the renal cortex (specifically proximal and distal tubules) and sustained beyond 2 months post injection. Furthermore, we demonstrate the ability of this methodology to induce long-term, in-vivo knockdown of candidate genes either through somatic recombination of floxed alleles or by direct delivery of specific shRNA sequences. This study demonstrates that ultrasound-guided injection of lentiviral vectors provides a safe and efficient method for the genetic manipulation of renal tubules, representing a quick and versatile alternative to GEMMs for the functional characterisation of disease-related genes.The authors wish to thank the core facilities (Biological Research Unit, Histopathology, Flow Cytometry and Microscopy) of the CRUK Cambridge Institute for advice and technical assistance. This work was funded by a CRUK Clinician Scientist Fellowship award (C37839/A12177).This is the final version. It was first published by NPG at http://www.nature.com/srep/2015/150605/srep11061/full/srep11061.html

Loss of PBRM1 rescues VHL dependent replication stress to promote renal carcinogenesis

AbstractInactivation of the VHL (Von Hippel Lindau) tumour suppressor has long been recognised as necessary for the pathogenesis of clear cell renal cancer (ccRCC); however, the molecular mechanisms underlying transformation and the requirement for additional genetic hits remain unclear. Here, we show that loss of VHL alone results in DNA replication stress and damage accumulation, effects that constrain cellular growth and transformation. By contrast, concomitant loss of the chromatin remodelling factor PBRM1 (mutated in 40% of ccRCC) rescues VHL-induced replication stress, maintaining cellular fitness and allowing proliferation. In line with these data we demonstrate that combined deletion of Vhl and Pbrm1 in the mouse kidney is sufficient for the development of fully-penetrant, multifocal carcinomas, closely mimicking human ccRCC. Our results illustrate how VHL and PBRM1 co-operate to drive renal transformation and uncover replication stress as an underlying vulnerability of all VHL mutated renal cancers that could be therapeutically exploited.</jats:p

Generation and Characterisation of a Pax8-CreERT2 Transgenic Line and a Slc22a6-CreERT2 Knock-In Line for Inducible and Specific Genetic Manipulation of Renal Tubular Epithelial Cells.

Genetically relevant mouse models need to recapitulate the hallmarks of human disease by permitting spatiotemporal gene targeting. This is especially important for replicating the biology of complex diseases like cancer, where genetic events occur in a sporadic fashion within developed somatic tissues. Though a number of renal tubule targeting mouse lines have been developed their utility for the study of renal disease is limited by lack of inducibility and specificity. In this study we describe the generation and characterisation of two novel mouse lines directing CreERT2 expression to renal tubular epithelia. The Pax8-CreERT2 transgenic line uses the mouse Pax8 promoter to direct expression of CreERT2 to all renal tubular compartments (proximal and distal tubules as well as collecting ducts) whilst the Slc22a6-CreERT2 knock-in line utilises the endogenous mouse Slc22a6 locus to specifically target the epithelium of proximal renal tubules. Both lines show high organ and tissue specificity with no extrarenal activity detected. To establish the utility of these lines for the study of renal cancer biology, Pax8-CreERT2 and Slc22a6-CreERT2 mice were crossed to conditional Vhl knockout mice to induce long-term renal tubule specific Vhl deletion. These models exhibited renal specific activation of the hypoxia inducible factor pathway (a VHL target). Our results establish Pax8-CreERT2 and Slc22a6-CreERT2 mice as valuable tools for the investigation and modelling of complex renal biology and disease.This work was supported by a Cancer Research UK Clinician Scientist Fellowship award (C37839/A12177) to AM. DA, BF, FY are funded by the Wellcome Trust Sanger Institute (grant number WT098051).This is the final version of the article. It first appeared from the Public Library of Science (PLOS) via https://doi.org/10.1371/journal.pone.014805

Prognostic significance of folate metabolism polymorphisms for lung cancer

Functional nonsynonymous single-nucleotide polymorphisms (nsSNPs) of folate metabolism genes can influence the methylation of tumour suppressor genes, thereby potentially impacting on tumour behaviour. To investigate whether such polymorphisms influence lung cancer survival, we genotyped 14 nsSNPs mapping to methylene-tetrahydrofolate reductase (MTHFR), methionine synthase (MTR), methionine synthase reductase (MTRR); DNA methyltransferase (DNMT2), methylenetetrahydrofolate dehydrogenase (MTHFD1) and methenyltetrahydrofolate synthetase (MTHFS) in 619 Caucasian women with incident disease, 465 with non-small cell (NSCLC) and 154 with small cell lung cancer (SCLC). The most significant association detected was with MTHFS Thr202Ala, with carriers of variant alleles having a worse prognosis (hazard ratio (HR)=1.49; 95% confidence interval: 1.14–1.94). Associations were also detected between overall survival (OS) in SCLC and homozygosity for MTHFR 222Val (HR=1.92; 1.03–3.58) and between OS from NSCLC and MTRR 175Leu carrier status (HR=1.36; 1.06–1.75). While there is evidence that variation in the folate metabolism genes may influence prognosis from lung cancer, current data are insufficiently robust to distinguish individual patient outcome

Prospective study evaluating the relative sensitivity of 18F-NaF PET/CT for detecting skeletal metastases from renal cell carcinoma in comparison to multidetector CT and 99mTc-MDP bone scintigraphy, using an adaptive trial design.

BACKGROUND: The detection of occult bone metastases is a key factor in determining the management of patients with renal cell carcinoma (RCC), especially when curative surgery is considered. This prospective study assessed the sensitivity of (18)F-labelled sodium fluoride in conjunction with positron emission tomography/computed tomography ((18)F-NaF PET/CT) for detecting RCC bone metastases, compared with conventional imaging by bone scintigraphy or CT. PATIENTS AND METHODS: An adaptive two-stage trial design was utilized, which was stopped after the first stage due to statistical efficacy. Ten patients with stage IV RCC and bone metastases were imaged with (18)F-NaF PET/CT and (99m)Tc-labelled methylene diphosphonate ((99m)Tc-MDP) bone scintigraphy including pelvic single photon emission computed tomography (SPECT). Images were reported independently by experienced radiologists and nuclear medicine physicians using a 5-point scoring system. RESULTS: Seventy-seven lesions were diagnosed as malignant: 100% were identified by (18)F-NaF PET/CT, 46% by CT and 29% by bone scintigraphy/SPECT. Standard-of-care imaging with CT and bone scintigraphy identified 65% of the metastases reported by (18)F-NaF PET/CT. On an individual patient basis, (18)F-NaF PET/CT detected more RCC metastases than (99m)Tc-MDP bone scintigraphy/SPECT or CT alone (P = 0.007). The metabolic volumes, mean and maximum standardized uptake values (SUV mean and SUV max) of the malignant lesions were significantly greater than those of the benign lesions (P < 0.001). CONCLUSIONS: (18)F-NaF PET/CT is significantly more sensitive at detecting RCC skeletal metastases than conventional bone scintigraphy or CT. The detection of occult bone metastases could greatly alter patient management, particularly in the context when standard-of-care imaging is negative for skeletal metastases.This work was supported by Cancer Research UK [grant number C19212/A16628]. The authors also received research support from the National Institute of Health Research Cambridge Biomedical Research Centre, Engineering and Physical Sciences Research Council Imaging Centre in Cambridge and Manchester, and the Cambridge Experimental Cancer Medicine Centre. The research has also been partly funded by a generous donation from the family and friends of a patient.This is the final version of the article. It first appeared from Oxford University Press via http://dx.doi.org/10.1093/annonc/mdv28

Treatment patterns and health outcomes in metastatic renal cell carcinoma patients treated with targeted systemic therapies in the UK

Funder: Bristol Myers Squibb Pharmaceuticals Ltd.Abstract: Background: Patients with metastatic renal cell carcinoma (mRCC) treated with targeted systemic therapies have demonstrated favourable outcomes in randomised controlled trials, however real-world evidence is limited. Thus, this study aimed to determine the effectiveness of targeted systemic therapies for patients with mRCC in routine clinical practice in the UK. Methods: A retrospective, observational, longitudinal study based on chart review of newly diagnosed adult mRCC patients treated at two UK hospitals from 2008 to 2015 was conducted. Targeted systemic therapies recommended for use in mRCC patients were evaluated across first to third lines of therapy (1LOT-3LOT). Important exclusions were treatment with cytokine therapy and within non-standard of care clinical trials. Primary outcome measure was overall survival (OS); data were analysed descriptively and using Kaplan-Meyer analysis. Results: 652 patients (65.3% male, 35.0% ≥70 years) were included. In 1LOT, 98.5% of patients received sunitinib or pazopanib. In 2LOT and 3LOT, 99.0 and 94.4% received axitinib or everolimus. Median OS was 12.9, 6.5 and 5.9 months at 1LOT, 2LOT and 3LOT respectively. Estimated OS at 1-year was 52.4% (95% CI: 48.6–56.4%) in 1LOT, 31.5% (25.2–39.5%) in 2LOT and 23.8% (10.1–55.9%) in 3LOT. Median OS from 1LOT in favourable, intermediate and poor MSKCC were 39.7, 15.8 and 6.1 months respectively. Conclusions: In this study, treatment was consistent with current National Institute for Health and Care Excellence (NICE) guidelines for mRCC patients. Although the study population favoured poorer prognosis patients, outcomes were more favourable than those for England at the same time. However, overall survival in this ‘real-world’ population remains poor and indicates significant unmet need for effective and safe treatment options to improve survival among mRCC patients

Integrated analyses of growth differentiation factor-15 concentration and cardiometabolic diseases in humans

Growth differentiation factor-15 (GDF15) is a stress response cytokine that is elevated in several cardiometabolic diseases and has attracted interest as a potential therapeutic target. To further explore the association of GDF15 with human disease, we conducted a broad study into the phenotypic and genetic correlates of GDF15 concentration in up to 14,099 individuals. Assessment of 772 traits across 6610 participants in FINRISK identified associations of GDF15 concentration with a range of phenotypes including all-cause mortality, cardiometabolic disease, respiratory diseases and psychiatric disorders, as well as inflammatory markers. A meta-analysis of genome-wide association studies (GWAS) of GDF15 concentration across three different assay platforms (n=14,099) confirmed significant heterogeneity due to a common missense variant (rs1058587; p.H202D) in GDF15, potentially due to epitope-binding artefacts. After conditioning on rs1058587, statistical fine mapping identified four independent putative causal signals at the locus. Mendelian randomisation (MR) analysis found evidence of a causal relationship between GDF15 concentration and high-density lipoprotein (HDL) but not body mass index (BMI). Using reverse MR, we identified a potential causal association of BMI on GDF15 (IVW p(FDR) = 0.0040). Taken together, our data derived from human population cohorts do not support a role for moderately elevated GDF15 concentrations as a causal factor in human cardiometabolic disease but support its role as a biomarker of metabolic stress.Peer reviewe

Prognostic effect of cytoreductive nephrectomy in synchronous metastatic renal cell carcinoma: a comparative study using inverse probability of treatment weighting.

PURPOSE: To test the hypothesis that cytoreductive nephrectomy (CN) improves overall survival (OS) of patients with synchronous metastatic renal cell carcinoma (mRCC), who subsequently receive targeted therapies (TT). METHODS: We identified 261 patients who received TT for synchronous mRCC with or without prior CN. To achieve balance in baseline characteristics between groups, we used the inverse probability of treatment weighting (IPTW) method. We conducted OS analyses, including IPTW-adjusted Kaplan-Meier curves, Cox regression models, interaction term, and landmark and sensitivity analyses. RESULTS: Of the 261 patients, 97 (37.2%) received CN and 164 (62.8%) did not. IPTW-adjusted analyses showed a statistically significant OS benefit for patients treated with CN (HR 0.63, 95% CI 0.46-0.83, P = 0.0015). While there was no statistically significant difference in OS at 3 months (P = 0.97), 6 months (P = 0.67), and 12 months (P = 0.11) from diagnosis, a benefit for the CN group was noted at 18 months (P = 0.005) and 24 months (P = 0.004). On interaction term analyses, the beneficial effect of CN increased with better performance status (P = 0.06), in women (P = 0.03), and in patients with thrombocytosis (P = 0.01). CONCLUSIONS: IPTW-adjusted analysis of our patient cohort suggests that CN improves OS of patients with synchronous mRCC treated with TT. On the whole, the survival difference appears after 12 months. Specific subgroups may particularly benefit from CN, and these subgroups warrant further investigation in prospective trials