Epstein-Barr Virus (EBV) detection and typing by PCR: a contribution to diagnostic screening of EBV-positive Burkitt's lymphoma

BACKGROUND: Epstein-Barr virus (EBV) is associated to the etio-pathogenesis of an increasing number of tumors. Detection of EBV in pathology samples is relevant since its high prevalence in some cancers makes the virus a promising target of specific therapies. RNA in situ hybridization (RISH) is the standard diagnostic procedure, while polymerase chain reaction (PCR)-based methods are used for strain (EBV type-1 or 2) distinction. We performed a systematic comparison between RISH and PCR for EBV detection, in a group of childhood B-cell Non-Hodgkin lymphomas (NHL), aiming to validate PCR as a first, rapid method for the diagnosis of EBV-associated B-cell NHL. METHODS: EBV infection was investigated in formalin fixed paraffin-embedded tumor samples of 41 children with B-cell NHL, including 35 Burkitt's lymphoma (BL), from Rio de Janeiro, Brazil, by in situ hybridization of EBV-encoded small RNA (EBER-RISH) and PCR assays based on EBNA2 amplification. RESULTS: EBV genomes were detected in 68% of all NHL. Type 1 and 2 accounted for 80% and 20% of EBV infection, respectively. PCR and RISH were highly concordant (95%), as well as single- and nested-PCR results, allowing the use of a single PCR round for diagnostic purposes. PCR assays showed a sensitivity and specificity of 96% and 100%, respectively, with a detection level of 1 EBV genome in 5,000–10,000 EBV-negative cells, excluding the possibility of detecting low-number EBV-bearing memory cells. CONCLUSION: We describe adequate PCR conditions with similar sensitivity and reliability to RISH, to be used for EBV diagnostic screening in high grade B-NHL, in "at risk" geographic regions

Epigenetic and oncogenic regulation of SLC16A7 (MCT2) results in protein over-expression, impacting on signalling and cellular phenotypes in prostate cancer

Felisbino S. received a fellowship from the Sao Paulo Research Foundation (FAPESP) ref. 2013/08830-2 and 2013/06802-1. Anne Y Warren research time funded by: Cambridge Biomedical Research Centre.Monocarboxylate Transporter 2 (MCT2) is a major pyruvate transporter encoded by the SLC16A7 gene. Recent studies pointed to a consistent overexpression of MCT2 in prostate cancer (PCa) suggesting MCT2 as a putative biomarker and molecular target. Despite the importance of this observation the mechanisms involved in MCT2 regulation are unknown. Through an integrative analysis we have discovered that selective demethylation of an internal SLC16A7/MCT2 promoter is a recurrent event in independent PCa cohorts. This demethylation is associated with expression of isoforms differing only in 5'-UTR translational control motifs, providing one contributing mechanism for MCT2 protein overexpression in PCa. Genes co-expressed with SLC16A7/MCT2 also clustered in oncogenic-related pathways and effectors of these signalling pathways were found to bind at the SLC16A7/MCT2 gene locus. Finally, MCT2 knock-down attenuated the growth of PCa cells. The present study unveils an unexpected epigenetic regulation of SLC16A7/MCT2 isoforms and identifies a link between SLC16A7/MCT2, Androgen Receptor (AR), ETS-related gene (ERG) and other oncogenic pathways in PCa. These results underscore the importance of combining data from epigenetic, transcriptomic and protein level changes to allow more comprehensive insights into the mechanisms underlying protein expression, that in our case provide additional weight to MCT2 as a candidate biomarker and molecular target in PCa.Publisher PDFPeer reviewe

A glycolytic phenotype is associated with prostate cancer progression and aggressiveness: a role for monocarboxylate transporters as metabolic targets for therapy.

Metabolic adaptation is considered an emerging hallmark of cancer, whereby cancer cells exhibit high rates of glucose consumption with consequent lactate production. To ensure rapid efflux of lactate, most cancer cells express high levels of monocarboxylate transporters (MCTs), which therefore may constitute suitable therapeutic targets. The impact of MCT inhibition, along with the clinical impact of altered cellular metabolism during prostate cancer (PCa) initiation and progression, has not been described. Using a large cohort of human prostate tissues of different grades, in silico data, in vitro and ex vivo studies, we demonstrate the metabolic heterogeneity of PCa and its clinical relevance. We show an increased glycolytic phenotype in advanced stages of PCa and its correlation with poor prognosis. Finally, we present evidence supporting MCTs as suitable targets in PCa, affecting not only cancer cell proliferation and survival but also the expression of a number of hypoxia-inducible factor target genes associated with poor prognosis. Herein, we suggest that patients with highly glycolytic tumours have poorer outcome, supporting the notion of targeting glycolytic tumour cells in prostate cancer through the use of MCT inhibitors.Pertega-Gomes N. and Sousa S. received fellowships from the Portuguese Foundation for Science and Technology (FCT), refs. SFRH/BD/61027/2009, and PTDC/SAU-MET/113415/2009, respectively. Felisbino S. received a fellowship from the Sao Paulo Research Foundation (FAPESP) ref. 2013/08830-2 and 2013/06802-1. We thank the core facilities at the Cancer Research UK Cambridge Institute led by James Hadfield (Genomics), Matt Eldridge (Bioinformatics) and Allen Hazelhurst (BRU). We also thank the support and critical advice on the project given by Christian Frezza and Marco Sciacovelli from The MRC Cancer Cell Unit and Professor Rui Henrique from Portuguese Institute of Oncology for providing samples from patients with metastatic prostate cancer.This is the final version of the article. It first appeared from Wiley via http://dx.doi.org/10.1002/path.454

Identification of potential therapeutic targets in prostate cancer through a cross-species approach.

Genetically engineered mouse models of cancer can be used to filter genome-wide expression datasets generated from human tumours and to identify gene expression alterations that are functionally important to cancer development and progression. In this study, we have generated RNAseq data from tumours arising in two established mouse models of prostate cancer, PB-Cre/PtenloxP/loxP and p53loxP/loxPRbloxP/loxP, and integrated this with published human prostate cancer expression data to pinpoint cancer-associated gene expression changes that are conserved between the two species. To identify potential therapeutic targets, we then filtered this information for genes that are either known or predicted to be druggable. Using this approach, we revealed a functional role for the kinase MELK as a driver and potential therapeutic target in prostate cancer. We found that MELK expression was required for cell survival, affected the expression of genes associated with prostate cancer progression and was associated with biochemical recurrence

Abstract A123: Preclinical evaluation of dual mTOR inhibitor, AZD2014, in prostate cancer

Abstract Background: An estimated 220,800 cases and 27,540 deaths from prostate cancer (PCa) will occur in the USA during 2015. Altered PI3K/AKT/mTOR signalling contributes to prostate cancer progression and transition to androgen-independent disease, for example one study reported 42% of primary and 100% of metastatic PCa tumours exhibited mutations, altered expression or copy number variations within this pathway. First generation mTOR inhibitors (preferentially inhibit mTORC1), have had limited anti-cancer effect in patients with PCa, possibly due to negative feedback activation of the AKT pathway via mTORC2. The dual mTORC1/2 inhibitor, AZD2014, may overcome this liability. Using a genetically engineered PTEN conditional mouse model (Ptenloxp/loxp;PB-Cre4), we have investigated the effects of AZD2014. The studies complement a clinical trial (NCT02064608) of AZD2014, given to men before radical prostatectomy and are timed for when invasive prostate carcinomas develop in the model around 10-14 months prior to onset of resistance to castration through AKT pathway activation. AZD2014, 15mg/kg daily, oral (with or without castration) or vehicle were administered for 14 days. Results: AZD2014 was well tolerated with no overt toxicity observed. Pharmacokinetic (PK) analysis revealed mean concentrations of 4.4±2.1μM of AZD2014 in the plasma samples collected 4 hours after day 14 dose. AZD2014 alone or combined with castration inhibited mTORC1 and mTORC2 measured by reductions in p4EBP1(Thr37/46) by approximately 48%±27% (p<0.001) and 37%±11% (p<0.001); pS6(Ser235/236) by 74%±43% (p<0.001) and 44%±13% (p<0.001) and pAKT(Ser473) by 36%±8% (p<0.001) and 20%±3% (p<0.01) as compared to vehicle-treated mice. AZD2014 treatment was anti-proliferative; Ki67 was significantly reduced in AZD2014-treated mice (70%±45%, p<0.001) or AZD2014 plus castration (42%±16%, p<0.001). Apoptosis was detected with cleaved caspase 3 and increased by 3.3-fold (p<0.001) in both AZD2014 or AZD2014 plus castration groups and 2-fold (p<0.001) in the castration only group, respectively. In all cases, 10 mice were used in each group and 80-120 randomly chosen images were analysed using Aperio automatic quantitative algorithms. Tumour volumes (ultrasound imaging) were reduced by 51% (p<0.05) comparing AZD2014 plus castration against control. HRMAS 1H NMR spectroscopy was used on tumour tissue to determine changes in metabolites following treatment and identified that the total choline to creatine ratio (t-Cho/Cr) was reduced by 40% in AZD2014-treated mice tumour samples (p<0.05) as compared to control-treated mice. Conclusions: Short term (14 days) treatment with AZD2014 with or without castration was associated with both pharmacodynamic and anti-tumour effects. The t-Cho/Cr ratio, previously reported as positively correlated with Gleason score in PCa patients, might be, in addition to our standard mTOR PD markers, utilised as a non-invasive biomarker of AZD2014 activity. The primary and phenotypic biomarker effects of monotherapy with AZD2014 in this relevant genetically engineered mouse model of prostate cancer will be compared with paired biopsies from the ongoing exploratory window study in the prostate cancer patients prior to prostatectomy, and may inform potential novel combination approaches that are translatable to the clinic. Citation Format: Chiranjeevi Sandi, Antonio Ramos-Montoya, Sergio L. Felisbino, Sarah Jurmeister, Basetti Madhu, Karan Wadhwa, John R. Griffiths, Frances M. Richards, Duncan I. Jodrell, David E. Neal, Sabina Cosulich, Barry Davies, Simon Pacey. Preclinical evaluation of dual mTOR inhibitor, AZD2014, in prostate cancer. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr A123.This is the accepted manuscript. The final version is available at http://mct.aacrjournals.org/content/14/12_Supplement_2/A123.short

PLEKHS1 drives PI3Ks and remodels pathway homeostasis in PTEN-null prostate

The PIP3/PI3K network is a central regulator of metabolism and is frequently activated in cancer, commonly by loss of the PIP3/PI(3,4)P2 phosphatase, PTEN. Despite huge research investment, the drivers of the PI3K network in normal tissues and how they adapt to overactivation are unclear. We find that in healthy mouse prostate PI3K activity is driven by RTK/IRS signaling and constrained by pathway feedback. In the absence of PTEN, the network is dramatically remodeled. A poorly understood YXXM- and PIP3/PI(3,4)P2-binding PH domain-containing adaptor, PLEKHS1, became the dominant activator and was required to sustain PIP3, AKT phosphorylation, and growth in PTEN-null prostate. This was because PLEKHS1 evaded pathway-feedback and experienced enhanced PI3K- and Src-family kinase-dependent phosphorylation of Y258XXM, eliciting PI3K activation. hPLEKHS1 mRNA and activating Y419 phosphorylation of hSrc correlated with PI3K pathway activity in human prostate cancers. We propose that in PTEN-null cells receptor-independent, Src-dependent tyrosine phosphorylation of PLEKHS1 creates positive feedback that escapes homeostasis, drives PIP3 signaling, and supports tumor progression

PLEKHS1 drives PI3Ks and remodels pathway homeostasis in PTEN-null prostate

The PIP3/PI3K network is a central regulator of metabolism and is frequently activated in cancer, commonly by loss of the PIP3/PI(3,4)P2 phosphatase, PTEN. Despite huge research investment, the drivers of the PI3K network in normal tissues and how they adapt to overactivation are unclear. We find that in healthy mouse prostate PI3K activity is driven by RTK/IRS signaling and constrained by pathway feedback. In the absence of PTEN, the network is dramatically remodeled. A poorly understood YXXM- and PIP3/PI(3,4)P2-binding PH domain-containing adaptor, PLEKHS1, became the dominant activator and was required to sustain PIP3, AKT phosphorylation, and growth in PTEN-null prostate. This was because PLEKHS1 evaded pathway-feedback and experienced enhanced PI3K- and Src-family kinase-dependent phosphorylation of Y258XXM, eliciting PI3K activation. hPLEKHS1 mRNA and activating Y419 phosphorylation of hSrc correlated with PI3K pathway activity in human prostate cancers. We propose that in PTEN-null cells receptor-independent, Src-dependent tyrosine phosphorylation of PLEKHS1 creates positive feedback that escapes homeostasis, drives PIP3 signaling, and supports tumor progression