Radiation-induced prodrug activation: extending combined modality therapy for some solid tumours

Combined chemoradiotherapy is the standard of care for locally advanced solid tumours. However, systemic toxicity may limit the delivery of planned chemotherapy. New approaches such as radiation-induced prodrug activation might diminish systemic toxicity, while retaining anticancer benefit. Organic azides have recently been shown to be reduced and activated under hypoxic conditions with clinically relevant doses of radiotherapy, uncaging pazopanib and doxorubicin in preclinical models with similar efficacy as the drug, but lower systemic toxicity. This approach may be relevant to the chemoradiation of glioblastoma and other solid tumours and offers potential for switching on drug delivery from implanted devices. The inclusion of reporters to confirm drug activation, avoidance of off-target effects and synchronisation of irradiation with optimal intratumoral drug concentration will be critical. Further preclinical validation studies of this approach should be encouraged

Detection and subtyping of Herpes simplex virus in clinical samples by LightCycler PCR, enzyme immunoassay and cell culture

BACKGROUND: Prompt laboratory diagnosis of Herpes simplex virus (HSV) infection facilitates patient management and possible initiation of antiviral therapy. In our laboratory, which receives various specimen types for detection of HSV, we use enzyme immunoassay (EIA) for rapid detection and culture of this virus. The culture of HSV has traditionally been accepted as the diagnostic 'gold standard'. In this study, we compared the use of real time PCR (LightCycler) for amplification, detection and subtyping of specific DNA with our in-house developed rapid and culture tests for HSV. RESULTS: The LightCycler PCR (LC-PCR) detected and subtyped HSV in 99% (66/67) of HSV positive specimens, compared to 81% (54/67) by rapid antigen EIA or 57% (36/63) by culture. A specimen was considered positive when two or more tests yielded HSV identifications or was culture positive. Discordant results were confirmed with an in-house developed PCR-ELISA or DNA sequence analysis. The typing results obtained with the LC-PCR and by culture amplified test were completely concordant. CONCLUSIONS: This study showed that the LC-PCR provided a highly sensitive test for simultaneous detection and subtyping of HSV in a single reaction tube. In addition to increased sensitivity, the LightCycler PCR provided reduced turn-around-times (2 hours) when compared to enzyme immunoassay (4 hours) or culture (4 days)

T-LAK cell-originated protein kinase (TOPK): an emerging target for cancer-specific therapeutics

‘Targeted’ or ‘biological’ cancer treatments rely on differential gene expression between normal tissue and cancer, and genetic changes that render tumour cells especially sensitive to the agent being applied. Problems exist with the application of many agents as a result of damage to local tissues, tumour evolution and treatment resistance, or through systemic toxicity. Hence, there is a therapeutic need to uncover specific clinical targets which enhance the efficacy of cancer treatment whilst minimising the risk to healthy tissues. T-LAK cell-originated protein kinase (TOPK) is a MAPKK-like kinase which plays a role in cell cycle regulation and mitotic progression. As a consequence, TOPK expression is minimal in differentiated cells, although its overexpression is a pathophysiological feature of many tumours. Hence, TOPK has garnered interest as a cancer-specific biomarker and biochemical target with the potential to enhance cancer therapy whilst causing minimal harm to normal tissues. Small molecule inhibitors of TOPK have produced encouraging results as a stand-alone treatment in vitro and in vivo, and are expected to advance into clinical trials in the near future. In this review, we present the current literature pertaining to TOPK as a potential clinical target and describe the progress made in uncovering its role in tumour development. Firstly, we describe the functional role of TOPK as a pro-oncogenic kinase, followed by a discussion of its potential as a target for the treatment of cancers with high-TOPK expression. Next, we provide an overview of the current preclinical progress in TOPK inhibitor discovery and development, with respect to future adaptation for clinical use

Overexpression of POLQ Confers a Poor Prognosis in Early Breast Cancer Patients

Depletion of POLQ (DNA polymerase theta) has recently been shown to render tumour cells more sensitive to radiotherapy whilst having little or no effect on normal tissues. This finding led us to investigate whether tumours that overexpress POLQ are associated with an adverse outcome. We therefore correlated the clinical outcomes of two retrospective series of patients with early breast cancer with the expression levels of POLQ, as determined by microarray gene expression analysis. We found that a significant number of tumours overexpressed POLQ and that overexpression was correlated with ER negative disease (p=0.047) and high tumour grade (p=0.004), both of which are associated with poor clinical outcomes. POLQ overexpression was associated with poor relapse free survival rates on both univariate (HR 5.80; 95% CI, 2.220 to 15.159; p<0.001) and multivariate analysis (HR 8.086; 95% CI 2.340 to 27.948 p=0.001). Analysis of other published clinical series confirmed that POLQ overexpression is associated with adverse clinical outcomes. The poor prognosis associated with POLQ is independent of other clinical or pathological features. The mechanism that causes this adverse outcome remains to be elucidated but may in part arise from resistance to adjuvant treatment. These findings, combined with the limited normal tissue expression of POLQ, make it a very appealing target for possible clinical exploitation

Application of Systematic Review Methodology to Food and Feed Safety Assessments to Support Decision Making

Systematic reviews are commonly used in human health research to provide overviews of existing evidence pertinent to clearly formulated specific questions, using pre-specified and standardised methods to identify and critically appraise relevant research, and to collect, report and analyse data from the studies that are included in the reviews. Formal systematic reviews have rarely been used in food and feed safety risk assessments and the existing systematic review methods in other disciplines may not be directly applicable in this field. This Guidance aims to assist the application of systematic reviews to food and feed safety risk assessments in support of decision making, by describing a framework for identifying the different types of question suitable for systematic review generated by the risk assessment process and for determining the need for systematic reviews when dealing with broad food and feed safety policy problems. The Guidance provides suggestions and examples for the conduct of eight key steps in the systematic review process (preparing a review, searching for studies, selecting studies for inclusion, collecting data from included studies, assessing the methodological quality of included studies, synthesising data from the studies, presenting data and results, and interpreting the results and drawing conclusions) for questions suitable for systematic reviews, taking into account issues that may be unique to food and feed safety. Due to its methodological rigor and its objective and transparent nature, systematic review methodology and its principles could provide additional value for answering well-formulated specific questions generated by the risk assessment process or other analytical frameworks in food and feed safety. Regular updates of this Guidance are foreseen in light of experience and new evidence both in food and feed safety and systematic review methodology

NVP-BEZ235 and NVP-BGT226, dual phosphatidylinositol 3-kinase/mammalian target of rapamycin inhibitors, enhance tumor and endothelial cell radiosensitivity

<p>Abstract</p> <p>Background</p> <p>The phosphatidylinositol 3-kinase (PI3K)/Akt pathway is activated in tumor cells and promotes tumor cell survival after radiation-induced DNA damage. Because the pathway may not be completely inhibited after blockade of PI3K itself, due to feedback through mammalian target of rapamycin (mTOR), more effective inhibition might be expected by targeting both PI3K and mTOR inhibition.</p> <p>Materials and methods</p> <p>We investigated the effect of two dual PI3K/mTOR (both mTORC1 and mTORC2) inhibitors, NVP-BEZ235 and NVP-BGT226, on SQ20B laryngeal and FaDu hypopharyngeal cancer cells characterised by EGFR overexpression, on T24 bladder tumor cell lines with H-Ras mutation and on endothelial cells. Analysis of target protein phosphorylation, clonogenic survival, number of residual γH2AX foci, cell cycle and apoptosis after radiation was performed in both tumor and endothelial cells. In vitro angiogenesis assays were conducted as well.</p> <p>Results</p> <p>Both compounds effectively inhibited phosphorylation of Akt, mTOR and S6 target proteins and reduced clonogenic survival in irradiated tumor cells. Persistence of DNA damage, as evidenced by increased number of γH2AX foci, was detected after irradiation in the presence of PI3K/mTOR inhibition, together with enhanced G2 cell cycle delay. Treatment with one of the inhibitors, NVP-BEZ235, also resulted in decreased clonogenicity after irradiation of tumor cells under hypoxic conditions. In addition, NVP-BEZ235 blocked VEGF- and IR-induced Akt phosphorylation and increased radiation killing in human umbilical venous endothelial cells (HUVEC) and human dermal microvascular dermal cells (HDMVC). NVP-BEZ235 inhibited VEGF-induced cell migration and capillary tube formation in vitro and enhanced the antivascular effect of irradiation. Treatment with NVP-BEZ235 moderately increased apoptosis in SQ20B and HUVEC cells but not in FaDu cells, and increased necrosis in both tumor and endothelial all cells tumor.</p> <p>Conclusions</p> <p>The results of this study demonstrate that PI3K/mTOR inhibitors can enhance radiation-induced killing in tumor and endothelial cells and may be of benefit when combined with radiotherapy.</p

Targeting TOPK sensitises tumour cells to radiation-induced damage by enhancing replication stress

T-LAK-originated protein kinase (TOPK) overexpression is a feature of multiple cancers, yet is absent from most phenotypically normal tissues. As such, TOPK expression profiling and the development of TOPK-targeting pharmaceutical agents have raised hopes for its future potential in the development of targeted therapeutics. Results presented in this paper confirm the value of TOPK as a potential target for the treatment of solid tumours, and demonstrate the efficacy of a TOPK inhibitor (OTS964) when used in combination with radiation treatment. Using H460 and Calu-6 lung cancer xenograft models, we show that pharmaceutical inhibition of TOPK potentiates the efficacy of fractionated irradiation. Furthermore, we provide in vitro evidence that TOPK plays a hitherto unknown role during S phase, showing that TOPK depletion increases fork stalling and collapse under conditions of replication stress and exogenous DNA damage. Transient knockdown of TOPK was shown to impair recovery from fork stalling and to increase the formation of replication-associated single-stranded DNA foci in H460 lung cancer cells. We also show that TOPK interacts directly with CHK1 and Cdc25c, two key players in the checkpoint signalling pathway activated after replication fork collapse. This study thus provides novel insights into the mechanism by which TOPK activity supports the survival of cancer cells, facilitating checkpoint signalling in response to replication stress and DNA damage

External beam radiation therapy and enadenotucirev: inhibition of the DDR and mechanisms of radiation-mediated virus increase

Ionising radiation causes cell death through the induction of DNA damage, particularly double-stranded DNA (dsDNA) breaks. Evidence suggests that adenoviruses inhibit proteins involved in the DNA damage response (DDR) to prevent recognition of double-stranded viral DNA genomes as cellular dsDNA breaks. We hypothesise that combining adenovirus treatment with radiotherapy has the potential for enhancing tumour-specific cytotoxicity through inhibition of the DDR and augmentation of virus production. We show that EnAd, an Ad3/Ad11p chimeric oncolytic adenovirus currently being trialled in colorectal and other cancers, targets the DDR pathway at a number of junctures. Infection is associated with a decrease in irradiation-induced 53BP1 and Rad51 foci formation, and in total DNA ligase IV levels. We also demonstrate a radiation-associated increase in EnAd production in vitro and in a pilot in vivo experiment. Given the current limitations of in vitro techniques in assessing for synergy between these treatments, we adapted the plaque assay to allow monitoring of viral plaque size and growth and utilised the xCELLigence cell adhesion assay to measure cytotoxicity. Our study provides further evidence on the interaction between adenovirus and radiation in vitro and in vivo and suggests these have at least an additive, and possibly a synergistic, impact on cytotoxicity

Whole tumor kinetics analysis of F-18-fluoromisonidazole dynamic PET scans of non-small cell lung cancer patients, and correlations with perfusion CT blood flow

Abstract Background To determine the relative abilities of compartment models to describe time-courses of 18F-fluoromisonidazole (FMISO) tumor uptake in patients with advanced stage non-small cell lung cancer (NSCLC) imaged using dynamic positron emission tomography (dPET), and study correlations between values of the blood flow-related parameter K 1 obtained from fits of the models and an independent blood flow measure obtained from perfusion CT (pCT). NSCLC patients had a 45-min dynamic FMISO PET/CT scan followed by two static PET/CT acquisitions at 2 and 4-h post-injection. Perfusion CT scanning was then performed consisting of a 45-s cine CT. Reversible and irreversible two-, three- and four-tissue compartment models were fitted to 30 time-activity-curves (TACs) obtained for 15 whole tumor structures in 9 patients, each imaged twice. Descriptions of the TACs provided by the models were compared using the Akaike and Bayesian information criteria (AIC and BIC) and leave-one-out cross-validation. The precision with which fitted model parameters estimated ground-truth uptake kinetics was determined using statistical simulation techniques. Blood flow from pCT was correlated with K 1 from PET kinetic models in addition to FMISO uptake levels. Results An irreversible three-tissue compartment model provided the best description of whole tumor FMISO uptake time-courses according to AIC, BIC, and cross-validation scores totaled across the TACs. The simulation study indicated that this model also provided more precise estimates of FMISO uptake kinetics than other two- and three-tissue models. The K 1 values obtained from fits of the irreversible three-tissue model correlated strongly with independent blood flow measurements obtained from pCT (Pearson r coefficient = 0.81). The correlation from the irreversible three-tissue model (r = 0.81) was stronger than that from than K 1 values obtained from fits of a two-tissue compartment model (r = 0.68), or FMISO uptake levels in static images taken at time-points from tracer injection through to 4 h later (maximum at 2 min, r = 0.70). Conclusions Time-courses of whole tumor FMISO uptake by advanced stage NSCLC are described best by an irreversible three-tissue compartment model. The K 1 values obtained from fits of the irreversible three-tissue model correlated strongly with independent blood flow measurements obtained from perfusion CT (r = 0.81)