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Mathematical modeling of tumor therapy with oncolytic viruses: Regimes with complete tumor elimination within the framework of deterministic models
Novozhilov, Artem S
Berezovskaya, Faina S
Koonin, Eugene V
Karev, Georgy P
Biology Direct
Oncolytic viruses that specifically target tumor cells are promising anti-cancer therapeutic agents. The interaction between an oncolytic virus and tumor cells is amenable to mathematical modeling using adaptations of techniques employed previously for modeling other types of virus-cell interaction.
A complete parametric analysis of dynamic regimes of a conceptual model of anti-tumor virus therapy is presented. The role and limitations of mass-action kinetics are discussed. A functional response, which is a function of the ratio of uninfected to infected tumor cells, is proposed to describe the spread of the virus infection in the tumor. One of the main mathematical features of ratio-dependent models is that the origin is a complicated equilibrium point whose characteristics determine the main properties of the model. It is shown that, in a certain area of parameter values, the trajectories of the model form a family of homoclinics to the origin (so-called elliptic sector). Biologically, this means that both infected and uninfected tumor cells can be eliminated with time, and complete recovery is possible as a result of the virus therapy within the framework of deterministic models.
Our model, in contrast to the previously published models of oncolytic virus-tumor interaction, exhibits all possible outcomes of oncolytic virus infection, i.e., no effect on the tumor, stabilization or reduction of the tumor load, and complete elimination of the tumor. The parameter values that result in tumor elimination, which is, obviously, the desired outcome, are compatible with some of the available experimental data.
This article was reviewed by Mikhail Blagosklonny, David Krakauer, Erik Van Nimwegen, and Ned Wingreen.
Related Articles
Targeting of Interferon-Beta to Produce a Specific, Multi-Mechanistic Oncolytic Vaccinia Virus
Kirn, David H
Wang, Yaohe
Le Boeuf, Fabrice
Bell, John
Thorne, Steve H
PLoS Medicine
Oncolytic viruses hold much promise for clinical treatment of many cancers, but a lack of systemic delivery and insufficient tumor cell killing have limited their usefulness. We have previously demonstrated that vaccinia virus strains are capable of systemic delivery to tumors in mouse models, but infection of normal tissues remains an issue. We hypothesized that interferon-beta (IFN-β) expression from an oncolytic vaccinia strain incapable of responding to this cytokine would have dual benefits as a cancer therapeutic: increased anticancer effects and enhanced virus inactivation in normal tissues. We report the construction and preclinical testing of this virus.
Methods and Findings
In vitro screening of viral strains by cytotoxicity and replication assay was coupled to cellular characterization by phospho-flow cytometry in order to select a novel oncolytic vaccinia virus. This virus was then examined in vivo in mouse models by non-invasive imaging techniques. A vaccinia B18R deletion mutant was selected as the backbone for IFN-β expression, because the B18R gene product neutralizes secreted type-I IFNs. The oncolytic B18R deletion mutant demonstrated IFN-dependent cancer selectivity and efficacy in vitro, and tumor targeting and efficacy in mouse models in vivo. Both tumor cells and tumor-associated vascular endothelial cells were targeted. Complete tumor responses in preclinical models were accompanied by immune-mediated protection against tumor rechallenge. Cancer selectivity was also demonstrated in primary human tumor explant tissues and adjacent normal tissues. The IFN-β gene was then cloned into the thymidine kinase (TK) region of this virus to create JX-795 (TK−/B18R−/IFN-β+). JX-795 had superior tumor selectivity and systemic intravenous efficacy when compared with the TK−/B18R− control or wild-type vaccinia in preclinical models.
By combining IFN-dependent cancer selectivity with IFN-β expression to optimize both anticancer effects and normal tissue antiviral effects, we were able to achieve, to our knowledge for the first time, tumor-specific replication, IFN-β gene expression, and efficacy following systemic delivery in preclinical models.
Stephen Thorne and colleagues describe, in a mouse model, an oncolytic vaccinia virus with interferon-dependent cancer selectivity that allows tumor-specific replication; it also expresses the IFN-β gene and hence has efficacy against tumors.
Editors' Summary
Normally, throughout life, cell division (which produces new cells) and cell death are carefully balanced to keep the body in good working order. But sometimes cells acquire changes (mutations) in their genetic material that allow them to divide uncontrollably to form cancers—disorganized masses of cells. Cancers can develop anywhere in the body and, as they develop, their cells acquire other genetic changes that enable them to move and start new tumors (metastases) elsewhere. Chemotherapy drugs kill rapidly dividing cancer cells but, because some normal cells are also sensitive to these drugs, it is hard to destroy the cancer without causing serious side effects. Consequently, researchers are trying to develop "targeted" therapies that attack the changes in cancer cells that allow them to divide uncontrollably but leave normal cells unscathed. One promising class of targeted therapies is oncolytic viruses. These viruses make numerous copies of themselves inside cancer cells (but not inside normal cells). Eventually the cancer cell bursts open (lyses), releases more of the therapeutic agent, and dies.
Why Was This Study Done?
Existing oncolytic viruses have two major disadvantages: they have to be injected directly into tumors, and therefore they can't destroy distant metastases; and they don't kill cancer cells particularly efficiently. In this study, the researchers have tried to adapt vaccinia virus (a virus that infects humans and which has recently been shown to kill tumor cells when injected into the bloodstream) in two ways: to both infect cancer cells selectively and then to kill them effectively.
They hypothesized that putting a gene that causes expression of a protein called interferon-beta (IFN-β) in a particular virus strain that is itself incapable of responding to IFN-β might achieve these aims. Human cells infected with viruses usually release IFNs, which induce an antiviral state in nearby cells. But vaccinia virus makes anti-IFN proteins that prevent IFN release. If the viral genes that encode these proteins are removed from the virus, the virus cannot spread through normal cells. However, many cancer cells have defective IFN signaling pathways so the virus can spread through them. IFN-β expression by the virus, however, should improve its innate anticancer effects because IFN-β stops cancer cells dividing, induces an antitumor immune response, and stops tumors developing good blood supplies.
What Did the Researchers Do and Find?
The researchers selected a vaccinia virus strain called WR-delB18R in which the B18R gene, which encodes an anti-IFN protein, had been removed from the virus. (WR is a wild-type virus.) In laboratory experiments, IFN treatment blocked the spread of WR-delB18R in normal human cells but not in human tumor cells. After being injected into the veins of tumor-bearing mice, WR-delB18R was rapidly cleared from normal tissues but persisted in the tumors. A single injection of WR-delB18R directly into the tumor killed most of the tumor cells. A similar dose injected into a vein was less effective but nevertheless increased the survival time of some of the mice by directly killing the tumor cells, by targeting the blood supply of the tumors, and by inducing antitumor immunity. Finally, when the researchers inserted the IFN-β gene into this WR-delB18R, the new virus—JX-795—was much better at killing tumors after intravenous injection than either WR or WR-delB18R.
What Do These Findings Mean?
These findings indicate that the vaccinia virus can be adapted so that it replicates only in tumor cells and kills these cells effectively after intravenous injection. In particular, they show that the strategy adopted by the researchers both optimizes the anticancer effects of the virus and minimizes viral replication in normal tissues. JX-795 is a promising oncolytic virus, therefore, particularly since vaccinia virus has been safely used for many years to vaccinate people against smallpox. Nevertheless, it will be some years before JX-795 can be used clinically. Vaccinia virus constructs like this need to be tested extensively in the laboratory and in animals before any attempt is made to test them in people and, even if they passes all these preclinical tests with flying colors, only clinical trials will reveal whether they can treat human cancer. Several related strains of vaccinia virus are currently undergoing clinical testing.
Additional Information.
Please access these Web sites via the online version of this summary at http://dx.doi.org/:10.1371/journal.pmed.0040353.
The US National Cancer Institute provides information on all aspects of cancer (in English and Spanish)
CancerQuest, from Emory University, provides information on all aspects of cancer (in several languages)
The UK charity Cancerbackup also provides information on all aspects of cancer
Wikipedia has a page on oncolytic viruses (note that Wikipedia is a free online encyclopedia that anyone can edit; available in several languages)
A short interview about oncolytic viruses with researcher Dr. John Bell is available on the Insidermedicine Web site
The Oncolytic virus Web page provides lists of oncolytic viruses classified by type
Related Articles
Towards Predictive Computational Models of Oncolytic Virus Therapy: Basis for Experimental Validation and Model Selection
Wodarz, Dominik
Komarova, Natalia
PLoS ONE
Oncolytic viruses are viruses that specifically infect cancer cells and kill them, while leaving healthy cells largely intact. Their ability to spread through the tumor makes them an attractive therapy approach. While promising results have been observed in clinical trials, solid success remains elusive since we lack understanding of the basic principles that govern the dynamical interactions between the virus and the cancer. In this respect, computational models can help experimental research at optimizing treatment regimes. Although preliminary mathematical work has been performed, this suffers from the fact that individual models are largely arbitrary and based on biologically uncertain assumptions. Here, we present a general framework to study the dynamics of oncolytic viruses that is independent of uncertain and arbitrary mathematical formulations. We find two categories of dynamics, depending on the assumptions about spatial constraints that govern that spread of the virus from cell to cell. If infected cells are mixed among uninfected cells, there exists a viral replication rate threshold beyond which tumor control is the only outcome. On the other hand, if infected cells are clustered together (e.g. in a solid tumor), then we observe more complicated dynamics in which the outcome of therapy might go either way, depending on the initial number of cells and viruses. We fit our models to previously published experimental data and discuss aspects of model validation, selection, and experimental design. This framework can be used as a basis for model selection and validation in the context of future, more detailed experimental studies. It can further serve as the basis for future, more complex models that take into account other clinically relevant factors such as immune responses.
Related Articles
ODE models for oncolytic virus dynamics
Komarova, Natalia L.
Wodarz, Dominik
Journal of theoretical biology
Replicating oncolytic viruses are able to infect and lyse cancer cells and spread through the tumor, while leaving normal cells largely unharmed. This makes them potentially useful in cancer therapy, and a variety of viruses have shown promising results in clinical trials. Nevertheless, consistent success remains elusive and the correlates of success have been the subject of investigation, both from an experimental and a mathematical point of view. Mathematical modeling of oncolytic virus therapy is often limited by the fact that the predicted dynamics depend strongly on particular mathematical terms in the model, the nature of which remain uncertain. We aim to address this issue in the context of ODE modeling, by formulating a general computational framework that is independent of particular mathematical expressions. By analyzing this framework, we find some new insights into the conditions for successful virus therapy. We find that depending on our assumptions about the virus spread, there can be two distinct types of dynamics. In models of the first type (the "fast spread" models), we predict that the viruses can eliminate the tumor if the viral replication rate is sufficiently high. The second type of models is characterized by a suboptimal spread (the "slow spread" models). For such models, the simulated treatment may fail, even for very high viral replication rates. Our methodology can be used to study the dynamics of many biological systems, and thus has implications beyond the study of virus therapy of cancers.
Related Articles
A Dynamical Systems Model for Combinatorial Cancer Therapy Enhances Oncolytic Adenovirus Efficacy by MEK-Inhibition
Bagheri, Neda
Shiina, Marisa
Lauffenburger, Douglas A.
Korn, W. Michael
PLoS Computational Biology
Oncolytic adenoviruses, such as ONYX-015, have been tested in clinical trials for currently untreatable tumors, but have yet to demonstrate adequate therapeutic efficacy. The extent to which viruses infect targeted cells determines the efficacy of this approach but many tumors down-regulate the Coxsackievirus and Adenovirus Receptor (CAR), rendering them less susceptible to infection. Disrupting MAPK pathway signaling by pharmacological inhibition of MEK up-regulates CAR expression, offering possible enhanced adenovirus infection. MEK inhibition, however, interferes with adenovirus replication due to resulting G1-phase cell cycle arrest. Therefore, enhanced efficacy will depend on treatment protocols that productively balance these competing effects. Predictive understanding of how to attain and enhance therapeutic efficacy of combinatorial treatment is difficult since the effects of MEK inhibitors, in conjunction with adenovirus/cell interactions, are complex nonlinear dynamic processes. We investigated combinatorial treatment strategies using a mathematical model that predicts the impact of MEK inhibition on tumor cell proliferation, ONYX-015 infection, and oncolysis. Specifically, we fit a nonlinear differential equation system to dedicated experimental data and analyzed the resulting simulations for favorable treatment strategies. Simulations predicted enhanced combinatorial therapy when both treatments were applied simultaneously; we successfully validated these predictions in an ensuing explicit test study. Further analysis revealed that a CAR-independent mechanism may be responsible for amplified virus production and cell death. We conclude that integrated computational and experimental analysis of combinatorial therapy provides a useful means to identify treatment/infection protocols that yield clinically significant oncolysis. Enhanced oncolytic therapy has the potential to dramatically improve non-surgical cancer treatment, especially in locally advanced or metastatic cases where treatment options remain limited.
Author Summary
Novel cancer treatment strategies are urgently needed since currently available non-surgical methods for most solid malignancies have limited impact on survival rates. We used conditionally replicating adenoviruses as cancer-fighting agents since they are designed to target and lyse cells with specific aberrations, leaving healthy cells undamaged. Highly malignant cells, however, down-regulate the adenovirus receptor, impairing infection and subsequent cell death. We demonstrated that disruption of the MEK pathway (which is frequently activated in cancer) up-regulated this receptor, resulting in enhanced adenovirus entry. Although receptor expression was restored, disruption of signaling interfered with adenovirus replication due to cell cycle arrest, presenting an opposing trade-off. We developed a dynamical systems model to characterize the response of cancer cells to oncolytic adenovirus infection and drug treatment, providing a means to enhance therapeutic efficacy of combination treatment strategies. Our simulations predicted improved therapeutic efficacy when drug treatment and infection occurred simultaneously. We successfully validated predictions and found that a CAR-independent mechanism may be responsible for regulating adenovirus production and cell death. This work demonstrates the utility of modeling for accurate prediction and optimization of combinatorial treatment strategies, serving as a paradigm for improved design of anti-cancer combination therapies.
Related Articles
Complex Spatial Dynamics of Oncolytic Viruses In Vitro: Mathematical and Experimental Approaches
Wodarz, Dominik
Hofacre, Andrew
Lau, John W.
Sun, Zhiying
Fan, Hung
Komarova, Natalia L.
PLoS Computational Biology
Oncolytic viruses replicate selectively in tumor cells and can serve as targeted treatment agents. While promising results have been observed in clinical trials, consistent success of therapy remains elusive. The dynamics of virus spread through tumor cell populations has been studied both experimentally and computationally. However, a basic understanding of the principles underlying virus spread in spatially structured target cell populations has yet to be obtained. This paper studies such dynamics, using a newly constructed recombinant adenovirus type-5 (Ad5) that expresses enhanced jellyfish green fluorescent protein (EGFP), AdEGFPuci, and grows on human 293 embryonic kidney epithelial cells, allowing us to track cell numbers and spatial patterns over time. The cells are arranged in a two-dimensional setting and allow virus spread to occur only to target cells within the local neighborhood. Despite the simplicity of the setup, complex dynamics are observed. Experiments gave rise to three spatial patterns that we call "hollow ring structure", "filled ring structure", and "disperse pattern". An agent-based, stochastic computational model is used to simulate and interpret the experiments. The model can reproduce the experimentally observed patterns, and identifies key parameters that determine which pattern of virus growth arises. The model is further used to study the long-term outcome of the dynamics for the different growth patterns, and to investigate conditions under which the virus population eliminates the target cells. We find that both the filled ring structure and disperse pattern of initial expansion are indicative of treatment failure, where target cells persist in the long run. The hollow ring structure is associated with either target cell extinction or low-level persistence, both of which can be viewed as treatment success. Interestingly, it is found that equilibrium properties of ordinary differential equations describing the dynamics in local neighborhoods in the agent-based model can predict the outcome of the spatial virus-cell dynamics, which has important practical implications. This analysis provides a first step towards understanding spatial oncolytic virus dynamics, upon which more detailed investigations and further complexity can be built.
Author Summary
Traditional chemotherapy of cancers is characterized by strong side effects, while showing a low success rate in the long term control of tumors. Besides small molecule inhibitors, which have shown great promise, oncolytic viruses present an emerging specific treatment approach. They are engineered viruses that spread from tumor cell to tumor cell, killing them in the process. Non-tumor cells are generally not infected. While clinical trials have given rise to promising results, reliable success remains elusive. Besides experiments, computational approaches provide a valuable tool to better understand the dynamics of virus spread through a growing population or tumor cells. Combining in vitro experimental approaches with computational models, we study the principles of virus spread through a spatially structured population of cells, which is of fundamental importance to understanding virus treatment of solid tumors. We describe different growth patterns that can occur, interpret them, and explore how they relate to the ability of the virus to induce tumor regression. We further define how these spatial dynamics relate to settings where cells and viruses mix more readily, such as in many cell culture experiments that are used to evaluate candidate viruses.
Related Articles
Intra-tumor Genetic Heterogeneity and Mortality in Head and Neck Cancer: Analysis of Data from The Cancer Genome Atlas
Mroz, Edmund A.
Tward, Aaron M.
Hammon, Rebecca J.
Ren, Yin
Rocco, James W.
PLoS Medicine
Although the involvement of intra-tumor genetic heterogeneity in tumor progression, treatment resistance, and metastasis is established, genetic heterogeneity is seldom examined in clinical trials or practice. Many studies of heterogeneity have had prespecified markers for tumor subpopulations, limiting their generalizability, or have involved massive efforts such as separate analysis of hundreds of individual cells, limiting their clinical use. We recently developed a general measure of intra-tumor genetic heterogeneity based on whole-exome sequencing (WES) of bulk tumor DNA, called mutant-allele tumor heterogeneity (MATH). Here, we examine data collected as part of a large, multi-institutional study to validate this measure and determine whether intra-tumor heterogeneity is itself related to mortality.
Methods and Findings
Clinical and WES data were obtained from The Cancer Genome Atlas in October 2013 for 305 patients with head and neck squamous cell carcinoma (HNSCC), from 14 institutions. Initial pathologic diagnoses were between 1992 and 2011 (median, 2008). Median time to death for 131 deceased patients was 14 mo; median follow-up of living patients was 22 mo. Tumor MATH values were calculated from WES results. Despite the multiple head and neck tumor subsites and the variety of treatments, we found in this retrospective analysis a substantial relation of high MATH values to decreased overall survival (Cox proportional hazards analysis: hazard ratio for high/low heterogeneity, 2.2; 95% CI 1.4 to 3.3). This relation of intra-tumor heterogeneity to survival was not due to intra-tumor heterogeneity's associations with other clinical or molecular characteristics, including age, human papillomavirus status, tumor grade and TP53 mutation, and N classification. MATH improved prognostication over that provided by traditional clinical and molecular characteristics, maintained a significant relation to survival in multivariate analyses, and distinguished outcomes among patients having oral-cavity or laryngeal cancers even when standard disease staging was taken into account. Prospective studies, however, will be required before MATH can be used prognostically in clinical trials or practice. Such studies will need to examine homogeneously treated HNSCC at specific head and neck subsites, and determine the influence of cancer therapy on MATH values. Analysis of MATH and outcome in human-papillomavirus-positive oropharyngeal squamous cell carcinoma is particularly needed.
To our knowledge this study is the first to combine data from hundreds of patients, treated at multiple institutions, to document a relation between intra-tumor heterogeneity and overall survival in any type of cancer. We suggest applying the simply calculated MATH metric of heterogeneity to prospective studies of HNSCC and other tumor types.
In this study, Rocco and colleagues examine data collected as part of a large, multi-institutional study, to validate a measure of tumor heterogeneity called MATH and determine whether intra-tumor heterogeneity is itself related to mortality.
Editors' Summary
Normally, the cells in human tissues and organs only reproduce (a process called cell division) when new cells are needed for growth or to repair damaged tissues. But sometimes a cell somewhere in the body acquires a genetic change (mutation) that disrupts the control of cell division and allows the cell to grow continuously. As the mutated cell grows and divides, it accumulates additional mutations that allow it to grow even faster and eventually from a lump, or tumor (cancer). Other mutations subsequently allow the tumor to spread around the body (metastasize) and destroy healthy tissues. Tumors can arise anywhere in the body—there are more than 200 different types of cancer—and about one in three people will develop some form of cancer during their lifetime. Many cancers can now be successfully treated, however, and people often survive for years after a diagnosis of cancer before, eventually, dying from another disease.
Why Was This Study Done?
The gradual acquisition of mutations by tumor cells leads to the formation of subpopulations of cells, each carrying a different set of mutations. This "intra-tumor heterogeneity" can produce tumor subclones that grow particularly quickly, that metastasize aggressively, or that are resistant to cancer treatments. Consequently, researchers have hypothesized that high intra-tumor heterogeneity leads to worse clinical outcomes and have suggested that a simple measure of this heterogeneity would be a useful addition to the cancer staging system currently used by clinicians for predicting the likely outcome (prognosis) of patients with cancer. Here, the researchers investigate whether a measure of intra-tumor heterogeneity called "mutant-allele tumor heterogeneity" (MATH) is related to mortality (death) among patients with head and neck squamous cell carcinoma (HNSCC)—cancers that begin in the cells that line the moist surfaces inside the head and neck, such as cancers of the mouth and the larynx (voice box). MATH is based on whole-exome sequencing (WES) of tumor and matched normal DNA. WES uses powerful DNA-sequencing systems to determine the variations of all the coding regions (exons) of the known genes in the human genome (genetic blueprint).
What Did the Researchers Do and Find?
The researchers obtained clinical and WES data for 305 patients who were treated in 14 institutions, primarily in the US, after diagnosis of HNSCC from The Cancer Genome Atlas, a catalog established by the US National Institutes of Health to map the key genomic changes in major types and subtypes of cancer. They calculated tumor MATH values for the patients from their WES results and retrospectively analyzed whether there was an association between the MATH values and patient survival. Despite the patients having tumors at various subsites and being given different treatments, every 10% increase in MATH value corresponded to an 8.8% increased risk (hazard) of death. Using a previously defined MATH-value cutoff to distinguish high- from low-heterogeneity tumors, compared to patients with low-heterogeneity tumors, patients with high-heterogeneity tumors were more than twice as likely to die (a hazard ratio of 2.2). Other statistical analyses indicated that MATH provided improved prognostic information compared to that provided by established clinical and molecular characteristics and human papillomavirus (HPV) status (HPV-positive HNSCC at some subsites has a better prognosis than HPV-negative HNSCC). In particular, MATH provided prognostic information beyond that provided by standard disease staging among patients with mouth or laryngeal cancers.
What Do These Findings Mean?
By using data from more than 300 patients treated at multiple institutions, these findings validate the use of MATH as a measure of intra-tumor heterogeneity in HNSCC. Moreover, they provide one of the first large-scale demonstrations that intra-tumor heterogeneity is clinically important in the prognosis of any type of cancer. Before the MATH metric can be used in clinical trials or in clinical practice as a prognostic tool, its ability to predict outcomes needs to be tested in prospective studies that examine the relation between MATH and the outcomes of patients with identically treated HNSCC at specific head and neck subsites, that evaluate the use of MATH for prognostication in other tumor types, and that determine the influence of cancer treatments on MATH values. Nevertheless, these findings suggest that MATH should be considered as a biomarker for survival in HNSCC and other tumor types, and raise the possibility that clinicians could use MATH values to decide on the best treatment for individual patients and to choose patients for inclusion in clinical trials.
Additional Information
Please access these websites via the online version of this summary at http://dx.doi.org/10.1371/journal.pmed.1001786.
The US National Cancer Institute (NCI) provides information about cancer and how it develops and about head and neck cancer (in English and Spanish)
Cancer Research UK, a not-for-profit organization, provides general information about cancer and how it develops, and detailed information about head and neck cancer; the Merseyside Regional Head and Neck Cancer Centre provides patient stories about HNSCC
Wikipedia provides information about tumor heterogeneity, and about whole-exome sequencing (note that Wikipedia is a free online encyclopedia that anyone can edit; available in several languages)
Information about The Cancer Genome Atlas is available
A PLOS Blog entry by Jessica Wapner explains more about MATH
Related Articles
Dual E1A Oncolytic Adenovirus: Targeting Tumor Heterogeneity With Two Independent Cancer-specific Promoter Elements, DF3/MUC1 and hTERT
Doloff, Joshua C.
Waxman, David J.
Cancer gene therapy
The therapeutic utility of oncolytic adenoviruses controlled by a single, tumor-specific regulatory element may be limited by the intra- and inter-tumoral heterogeneity that characterizes many cancers. To address this issue, we constructed an oncolytic adenovirus that uses two distinct tumor-specific promoters (DF3/Muc1 and hTERT) to drive separate E1A expression cassettes, in combination with deletion of the viral E1B region, which confers additional tumor selectivity and increased oncolytic activity. The resultant virus, Adeno-DF3-E1A/hTERT-E1A, induced higher levels of E1A oncoprotein, enhanced oncolysis, and an earlier and higher apoptotic index in infected tumor cells than following infection with Adeno-hTERT-E1A, which harbors a single hTERT promoter-driven E1A cassette. In isolated U251 human gliosarcoma cell holoclones (putative cancer stem cells), where DF3/Muc1 expression is substantially enriched and hTERT expression is decreased compared to the parental U251 cell population, E1A production and oncolysis were strongly decreased following infection with Adeno-hTERT-E1A but not Adeno-DF3-E1A/hTERT-E1A. The strong oncolytic activity of Adeno-DF3-E1A/hTERT-E1A translated into superior anti-tumor activity over Adeno-hTERT-E1A in vivo in a U251 solid tumor xenograft model, where hTERT levels were >90% suppressed and the DF3/Muc1 to hTERT expression ratio was substantially increased compared to cultured U251 cells. The enhanced anti-tumor activity of the dual-targeted Adeno-DF3-E1A/hTERT-E1A was achieved despite premature viral host cell death and decreased production of functional viral progeny, which limited tumor cell spread of the viral infection. These findings highlight the therapeutic benefit of targeting oncolytic viruses to heterogeneous tumor cell populations.
Cancer gene therapy; replication-conditional; oncolytic adenovirus; tumor heterogeneity; holoclone; cytochrome P450 prodrug activation
Related Articles
Choindroitinase ABC I-Mediated Enhancement of Oncolytic Virus Spread and Anti Tumor Efficacy: A Mathematical Model
Kim, Yangjin
Lee, Hyun Geun
Dmitrieva, Nina
Kim, Junseok
Kaur, Balveen
Friedman, Avner
PLoS ONE
Oncolytic viruses are genetically engineered viruses that are designed to kill cancer cells while doing minimal damage to normal healthy tissue. After being injected into a tumor, they infect cancer cells, multiply inside them, and when a cancer cell is killed they move on to spread and infect other cancer cells. Chondroitinase ABC (Chase-ABC) is a bacterial enzyme that can remove a major glioma ECM component, chondroitin sulfate glycosoamino glycans from proteoglycans without any deleterious effects in vivo. It has been shown that Chase-ABC treatment is able to promote the spread of the viruses, increasing the efficacy of the viral treatment. In this paper we develop a mathematical model to investigate the effect of the Chase-ABC on the treatment of glioma by oncolytic viruses (OV). We show that the model's predictions agree with experimental results for a spherical glioma. We then use the model to test various treatment options in the heterogeneous microenvironment of the brain. The model predicts that separate injections of OV, one into the center of the tumor and another outside the tumor will result in better outcome than if the total injection is outside the tumor. In particular, the injection of the ECM-degrading enzyme (Chase-ABC) on the periphery of the main tumor core need to be administered in an optimal strategy in order to infect and eradicate the infiltrating glioma cells outside the tumor core in addition to proliferative cells in the bulk of tumor core. The model also predicts that the size of tumor satellites and distance between the primary tumor and multifocal/satellite lesions may be an important factor for the efficacy of the viral therapy with Chase treatment.
Related Articles
The Utility of a Tissue Slice Model System to Determine Breast Cancer Infectivity by Oncolytic Adenoviruses
Pennington, Krista
Chu, Quyen D.
Curiel, David T.
Li, Benjamin D.L.
Mathis, J. Michael
The Journal of surgical research
Due to advances in viral design, oncolytic adenoviruses have emerged as a promising approach for treatment of breast cancer. Tumor tissue slices offer a stringent model system for preclinical evaluation of adenovirus therapies, since the slices retain a morphology and phenotype that more closely resembles the in vivo setting than cell line cultures, and it has been shown to have utility in the evaluation of viral infectivity and replication. In this study, we evaluated the efficacy of viral infection and replication using a tropism-modified oncolytic adenovirus.
Breast tumor tissue slices were infected with a tropism-modified oncolytic adenovirus, and a wild-type adenovirus for comparison. Efficiency of infection was evaluated using fluorescent microscopy, as the viruses used have been modified to express red fluorescent protein. Replication of the viruses was evaluated with quantitative real-time PCR to assay viral E4 genome copy number, a surrogate indicator for the number of virions. The breast tumor tissue slices were evaluated for the expression of CD46 expression by immunohistochemistry.
Infection and replication of our tropism modified oncolytic virus has been observed in breast cancer tissue slice model system and is comparative to wild-type virus. A qualitative increase in the number of cells showing RFP expression was observed correlating with increasing multiplicity of infection. Higher relative infectivity of the virus was observed in tumor tissue compared with normal breast tissue. Replication of the virus was demonstrated through increases in E4 copy number at 48 and 72 hours after infection in human breast tumor slices.
We have shown that a tropism modified oncolytic oncolytic adenovirus can infect and replicate in breast cancer tissue slices, which may be an important preclinical indicator for its therapeutic utility.
adenovirus; breast cancer; CD46; CAR; oncolytic; tissue slice model
Related Articles
Ex Vivo Infection of Live Tissue with Oncolytic Viruses
Diallo, Jean-Simon
Roy, Dominic
Abdelbary, Hesham
De Silva, Naomi
Bell, John C.
Journal of Visualized Experiments : JoVE
Oncolytic Viruses (OVs) are novel therapeutics that selectively replicate in and kill tumor cells1. Several clinical trials evaluating the effectiveness of a variety of oncolytic platforms including HSV, Reovirus, and Vaccinia OVs as treatment for cancer are currently underway2-5. One key characteristic of oncolytic viruses is that they can be genetically modified to express reporter transgenes which makes it possible to visualize the infection of tissues by microscopy or bio-luminescence imaging6,7. This offers a unique advantage since it is possible to infect tissues from patients ex vivo prior to therapy in order to ascertain the likelihood of successful oncolytic virotherapy8. To this end, it is critical to appropriately sample tissue to compensate for tissue heterogeneity and assess tissue viability, particularly prior to infection9. It is also important to follow viral replication using reporter transgenes if expressed by the oncolytic platform as well as by direct titration of tissues following homogenization in order to discriminate between abortive and productive infection. The object of this protocol is to address these issues and herein describes 1. The sampling and preparation of tumor tissue for cell culture 2. The assessment of tissue viability using the metabolic dye alamar blue 3. Ex vivo infection of cultured tissues with vaccinia virus expressing either GFP or firefly luciferase 4. Detection of transgene expression by fluorescence microscopy or using an In Vivo Imaging System (IVIS) 5. Quantification of virus by plaque assay. This comprehensive method presents several advantages including ease of tissue processing, compensation for tissue heterogeneity, control of tissue viability, and discrimination between abortive infection and bone fide viral replication.
Related Articles
Switching a Replication-Defective Adenoviral Vector into a Replication-Competent, Oncolytic Adenovirus
Nakashima, Hiroshi
Chiocca, E. Antonio
Journal of Virology
The adenovirus immediate early gene E1A initiates the program of viral gene transcription and reprograms multiple aspects of cell function and behavior. For adenoviral (Ad) vector-mediated gene transfer and therapy approaches, where replication-defective (RD) gene transfer is required, E1A has thus been the primary target for deletions. For oncolytic gene therapy for cancer, where replication-competent (RC) Ad viral gene expression is needed, E1A has been either mutated or placed under tumor-specific transcriptional control. A novel Ad vector that initially infected target tumor cells in an RD manner for transgene expression but that could be "switched" into an RC, oncolytic state when needed might represent an advance in vector technology. Here, we report that we designed such an Ad vector (proAdΔ24.GFP), where initial Ad replication is silenced by a green fluorescent protein (GFP) transgene that blocks cytomegalovirus (CMV)-mediated transcription of E1A. This vector functions as a bona fide E1A-deleted RD vector in infected tumor cells. However, because the silencing GFP transgene is flanked by FLP recombination target (FRT) sites, we show that it can be efficiently excised by Flp recombinase site-specific recombination, either when Flp is expressed constitutively in cells or when it is provided in trans by coinfection with a second RD herpes simplex virus (HSV) amplicon vector. This switches the RD Ad, proAdΔ24.GFP, into a fully RC, oncolytic Ad (rAdΔ24) that lyses tumor cells in culture and generates oncolytic progeny virions. In vivo, coinfection of established flank tumors with the RD proAdΔ24.GFP and the RD Flp-bearing HSV1 amplicon leads to generation of RC, oncolytic rAdΔ24. In an orthotopic human glioma xenograft tumor model, coinjection of the RD proAdΔ24.GFP and the RD Flp-bearing HSV1 amplicon also led to a significant increase in animal survival, compared to controls. Therefore, Flp-FRT site-specific recombination can be applied to switch RD Ad into fully oncolytic RC Ad for tumor therapy and is potentially applicable to a variety of gene therapy approaches.
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Oncolytic Virotherapy for Ovarian Carcinomatosis Using a Replication-Selective Vaccinia Virus Armed with a Yeast Cytosine Deaminase Gene*
Chalikonda, Sricharan
Kivlen, Maryann H.
O'Malley, Mark E.
Dong, Xiang Da (Eric)
McCart, J. Andrea
Gorry, Michael C.
Yin, Xiao-Yu
Brown, Charles K.
Zeh, Herbert J.
Guo, Z. Sheng
Bartlett, David L.
Cancer gene therapy
In this study, we assessed the ability of a highly tumor-selective oncolytic vaccinia virus armed with a yeast cytosine deaminase gene to infect and lyse human and murine ovarian tumors both in vitro and in vivo. The virus vvDD-CD could infect, replicate in and effectively lyse both human and mouse ovarian cancer cells in vitro. In two different treatment schedules involving either murine MOSEC or human A2780 ovarian carcinomatosis models, regional delivery of vvDD-CD selectively targeted tumor cells and ovarian tissue, effectively delaying the development of either tumor or ascites and leading to significant survival advantages. Oncolytic virotherapy using vvDD-CD in combination with the prodrug 5-fluorocytosine (5-FC) conferred an additional long-term survival advantage upon tumor-bearing immunocompetent mice. These findings demonstrate that a tumor-selective oncolytic vaccinia combined with gene-directed enzyme prodrug therapy (GDEPT) is a highly effective strategy for treating advanced ovarian cancers in both syngeneic mouse and human xenograft models. Given the biological safety, tumor selectivity and oncolytic potency of this armed oncolytic virus, this dual therapy merits further investigation as a promising new treatment for metastatic ovarian cancer.
poxvirus; ovarian cancer; peritoneal carcinomatosis; suicide gene; cytosine deaminase
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Virotherapy of Canine Tumors with Oncolytic Vaccinia Virus GLV-1h109 Expressing an Anti-VEGF Single-Chain Antibody
Patil, Sandeep S.
Gentschev, Ivaylo
Adelfinger, Marion
Donat, Ulrike
Hess, Michael
Weibel, Stephanie
Nolte, Ingo
Frentzen, Alexa
Szalay, Aladar A.
PLoS ONE
Virotherapy using oncolytic vaccinia virus (VACV) strains is one promising new strategy for cancer therapy. We have previously reported that oncolytic vaccinia virus strains expressing an anti-VEGF (Vascular Endothelial Growth Factor) single-chain antibody (scAb) GLAF-1 exhibited significant therapeutic efficacy for treatment of human tumor xenografts. Here, we describe the use of oncolytic vaccinia virus GLV-1h109 encoding GLAF-1 for canine cancer therapy. In this study we analyzed the virus-mediated delivery and production of scAb GLAF-1 and the oncolytic and immunological effects of the GLV-1h109 vaccinia virus strain against canine soft tissue sarcoma and canine prostate carcinoma in xenograft models. Cell culture data demonstrated that the GLV-1h109 virus efficiently infect, replicate in and destroy both tested canine cancer cell lines. In addition, successful expression of GLAF-1 was demonstrated in virus-infected canine cancer cells and the antibody specifically recognized canine VEGF. In two different xenograft models, the systemic administration of the GLV-1h109 virus was found to be safe and led to anti-tumor and immunological effects resulting in the significant reduction of tumor growth in comparison to untreated control mice. Furthermore, tumor-specific virus infection led to a continued production of functional scAb GLAF-1, resulting in inhibition of angiogenesis. Overall, the GLV-1h109-mediated cancer therapy and production of immunotherapeutic anti-VEGF scAb may open the way for combination therapy concept i.e. vaccinia virus mediated oncolysis and intratumoral production of therapeutic drugs in canine cancer patients.
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Immune Suppression during Oncolytic Virotherapy for High-Grade Glioma; Yes or No?
Koks, Carolien A.E.
De Vleeschouwer, Steven
Graf, Norbert
Van Gool, Stefaan W.
Journal of Cancer
Oncolytic viruses have been seriously considered for glioma therapy over the last 20 years. The oncolytic activity of several oncolytic strains has been demonstrated against human glioma cell lines and in in vivo xenotransplant models. So far, four of these stains have additionally completed the first phase I/II trials in relapsed glioma patients. Though safety and feasibility have been demonstrated, therapeutic efficacy in these initial trials, when described, was only minor. The role of the immune system in oncolytic virotherapy for glioma remained much less studied until recent years. When investigated, the immune system, adept at controlling viral infections, is often hypothesized to be a strong hurdle to successful oncolytic virotherapy. Several preclinical studies have therefore aimed to improve oncolytic virotherapy efficacy by combining it with immune suppression or evasion strategies. More recently however, a new paradigm has developed in the oncolytic virotherapy field stating that oncolytic virus-mediated tumor cell death can be accompanied by elicitation of potent activation of innate and adaptive anti-tumor immunity that greatly improves the efficacy of certain oncolytic strains. Therefore, it seems the three-way interaction between oncolytic virus, tumor and immune system is critical to the outcome of antitumor therapy. In this review we discuss the studies which have investigated how the immune system and oncolytic viruses interact in models of glioma. The novel insights generated here hold important implications for future research and should be incorporated into the design of novel clinical trials.
glioblastoma; oncolytic virotherapy; antitumor immunity
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Mathematical Modeling of Herpes Simplex Virus Distribution in Solid Tumors: Implications for Cancer Gene Therapy
Mok, Wilson
Stylianopoulos, Triantafyllos
Boucher, Yves
Jain, Rakesh K.
Clinical cancer research : an official journal of the American Association for Cancer Research
Although oncolytic viral vectors show promise for the treatment of various cancers, ineffective initial distribution and propagation throughout the tumor mass often limit the therapeutic response. A mathematical model is developed to describe the spread of herpes simplex virus from the initial injection site.
Experimental Design
The tumor is modeled as a sphere of radius R. The model incorporates reversible binding, interstitial diffusion, viral degradation, and internalization and physiologic parameters. Three species are considered as follows: free interstitial virus, virus bound to cell surfaces, and internalized virus.
This analysis reveals that both rapid binding and internalization as well as hindered diffusion contain the virus to the initial injection volume, with negligible spread to the surrounding tissue. Unfortunately, increasing the dose to saturate receptors and promote diffusion throughout the tumor is not a viable option: the concentration necessary would likely compromise safety. However, targeted modifications to the virus that decrease the binding affinity have the potential to increase the number of infected cells by 1.5-fold or more. An increase in the effective diffusion coefficient can result in similar gains.
This analysis suggests criteria by which the potential response of a tumor to oncolytic herpes simplex virus therapy can be assessed. Furthermore, it reveals the potential of modifications to the vector delivery method, physicochemical properties of the virus, and tumor extracellular matrix composition to enhance efficacy.
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Directed Evolution Generates a Novel Oncolytic Virus for the Treatment of Colon Cancer
Kuhn, Irene
Harden, Paul
Bauzon, Maxine
Chartier, Cecile
Nye, Julie
Thorne, Steve
Reid, Tony
Ni, Shaoheng
Lieber, Andre
Fisher, Kerry
Seymour, Len
Rubanyi, Gabor M.
Harkins, Richard N.
Hermiston, Terry W.
PLoS ONE
Viral-mediated oncolysis is a novel cancer therapeutic approach with the potential to be more effective and less toxic than current therapies due to the agents selective growth and amplification in tumor cells. To date, these agents have been highly safe in patients but have generally fallen short of their expected therapeutic value as monotherapies. Consequently, new approaches to generating highly potent oncolytic viruses are needed. To address this need, we developed a new method that we term "Directed Evolution" for creating highly potent oncolytic viruses.
Methodology/Principal Findings
Taking the "Directed Evolution" approach, viral diversity was increased by pooling an array of serotypes, then passaging the pools under conditions that invite recombination between serotypes. These highly diverse viral pools were then placed under stringent directed selection to generate and identify highly potent agents. ColoAd1, a complex Ad3/Ad11p chimeric virus, was the initial oncolytic virus derived by this novel methodology. ColoAd1, the first described non-Ad5-based oncolytic Ad, is 2–3 logs more potent and selective than the parent serotypes or the most clinically advanced oncolytic Ad, ONYX-015, in vitro. ColoAd1's efficacy was further tested in vivo in a colon cancer liver metastasis xenograft model following intravenous injection and its ex vivo selectivity was demonstrated on surgically-derived human colorectal tumor tissues. Lastly, we demonstrated the ability to arm ColoAd1 with an exogenous gene establishing the potential to impact the treatment of cancer on multiple levels from a single agent.
Using the "Directed Evolution" methodology, we have generated ColoAd1, a novel chimeric oncolytic virus. In vitro, this virus demonstrated a >2 log increase in both potency and selectivity when compared to ONYX-015 on colon cancer cells. These results were further supported by in vivo and ex vivo studies. Furthermore, these results have validated this methodology as a new general approach for deriving clinically-relevant, highly potent anti-cancer virotherapies.
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Explicit targeting of transformed cells by VSV in ovarian epithelial tumor-bearing Wv mouse models
Capo-chichi, Callinice D.
Yeasky, Toni M.
Heiber, Joshua F.
Wang, Ying
Barber, Glen N.
Xu, Xiang-Xi
Gynecologic oncology
Current treatment options for epithelial ovarian cancer are limited and therapeutic development for recurrent and drug-resistant ovarian cancer is an urgent agenda. We investigated the potential use of genetically engineered Vesicular Stomatitis Virus (VSV) to treat ovarian cancer patients who fail to respond to available therapies. Specifically, we examined the toxicity to hosts and specificity of targeting ovarian tumors using a Wv ovarian tumor model.
We first tested recombinant VSV for oncolytic activity in a panel of human ovarian epithelial cancer, immortalized, and primary ovarian surface epithelial cells in culture. Then, we test VSV oncolytic therapy using the immune competent Wv mice that develop tubular adenomas, benign tumor lesions derived from ovarian surface epithelial cells.
The expression of GFP encoded by the recombinant VSV genome was detected in about 5% of primary ovarian surface epithelial cells (3 lines) up to 30 days without significantly altering the growth pattern of the cells, suggesting the lack of toxicity to the normal ovarian surface epithelial cells. However, VSV-GFP was detected in the majority (around 90%) of cells that are either "immortalized" by SV40 antigen expression or cancer lines. Some variation in killing time courses was observed, but all the transformed cell lines were killed within 3 days.
We found that regardless of the inoculation route (intra bursal, IP, or IV), VSV specifically infected and replicated in the in situ ovarian tumors in the Wv mice without significant activity in any other organs and tissues, and showed no detectable toxicity. The epithelial tumor lesions were greatly reduced in VSV-targeted ovarian tumors in the Wv mice.
VSV oncolytic activity depends on a cell autonomous property distinguishing primary and transformed cells. The efficient oncolytic activity of VSV for the "immortalized" non-tumorigenic ovarian surface epithelial cells suggests that the selective specificity extends from pre-neoplastic to overt cancer cells. The results demonstrated the explicit targeting of ovarian epithelial tumors by VSV in immune com petent, ovarian tumor-bearing mouse models, and further support the utility of VSV as an effective and safe anti-cancer agent.
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Oncolytic Myxoma Virus: The path to clinic
Chan, Winnie M.
Rahman, Masmudur M.
McFadden, Grant
Many common neoplasms are still noncurative with current standards of cancer therapy. More therapeutic modalities need to be developed to significantly prolong the lives of patients and eventually cure a wider spectrum of cancers. Oncolytic virotherapy is one of the promising new additions to clinical cancer therapeutics. Successful oncolytic virotherapy in the clinic will be those strategies that best combine tumor cell oncolysis with enhanced immune responses against tumor antigens. The current candidate oncolytic viruses all share the common property that they are relatively nonpathogenic to humans, yet they have the ability to replicate selectively in human cancer cells and induce cancer regression by direct oncolysis and/or induction of improved anti-tumor immune responses. Many candidate oncolytic viruses are in various stages of clinical and preclinical development. One such preclinical candidate is myxoma virus (MYXV), a member of the Poxviridae family that, in its natural setting, exhibits a very restricted host range and is only pathogenic to European rabbits. Despite its narrow host range in nature, MYXV has been shown to productively infect various classes of human cancer cells. Several preclinical in vivo modeling studies have demonstrated that MYXV is an attractive and safe candidate oncolytic virus, and hence, MYXV is currently being developed as a potential therapeutic for several cancers, such as pancreatic cancer, glioblastoma, ovarian cancer, melanoma, and hematologic malignancies. This review highlights the preclinical cancer models that have shown the most promise for translation of MYXV into human clinical trials.
Myxoma Virus; Oncolytic Virotherapy; Hematological Malignancies; Pancreatic Cancer
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Armed and targeted measles virus for chemovirotherapy of pancreatic cancer
Bossow, S
Grossardt, C
Temme, A
Leber, MF
Sawall, S
Rieber, EP
Cattaneo, R
von Kalle, C
Ungerechts, G
Cancer gene therapy
No curative therapy is currently available for locally advanced or metastatic pancreatic cancer. Therefore, new therapeutic approaches must be considered. Measles virus (MV) vaccine strains have shown promising oncolytic activity against a variety of tumor entities. For specific therapy of pancreatic cancer, we generated a fully retargeted MV that enters cells exclusively through the prostate stem cell antigen (PSCA). Besides a high-membrane frequency on prostate cancer cells, this antigen is expressed on pancreatic adenocarcinoma, but not on non-neoplastic tissue. PSCA expression levels differ within heterogeneous tumor bulks and between human pancreatic cell lines, and we could show specific infection of pancreatic adenocarcinoma cell lines with both high- and low-level PSCA expression. Furthermore, we generated a fully retargeted and armed MV-PNP-anti-PSCA to express the prodrug convertase purine nucleoside phosphorylase (PNP). PNP, which activates the prodrug fludarabine effectively, enhanced the oncolytic efficacy of the virus on infected and bystander cells. Beneficial therapeutic effects were shown in a pancreatic cancer xenograft model. Moreover, in the treatment of gemcitabine-resistant pancreatic adenocarcinoma cells, no cross-resistance to both MV oncolysis and activated prodrug was detected.
oncolysis; measles virus; prodrug-converting enzyme; single-chain antibody; pancreatic cancer
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Imaging of Intratumoral Inflammation during Oncolytic Virotherapy of Tumors by 19F-Magnetic Resonance Imaging (MRI)
Weibel, Stephanie
Basse-Luesebrink, Thomas Christian
Hess, Michael
Hofmann, Elisabeth
Seubert, Carolin
Langbein-Laugwitz, Johanna
Gentschev, Ivaylo
Sturm, Volker Jörg Friedrich
Ye, Yuxiang
Kampf, Thomas
Jakob, Peter Michael
Szalay, Aladar A.
PLoS ONE
Oncolytic virotherapy of tumors is an up-coming, promising therapeutic modality of cancer therapy. Unfortunately, non-invasive techniques to evaluate the inflammatory host response to treatment are rare. Here, we evaluate 19F magnetic resonance imaging (MRI) which enables the non-invasive visualization of inflammatory processes in pathological conditions by the use of perfluorocarbon nanoemulsions (PFC) for monitoring of oncolytic virotherapy.
Methodology/Principal Findings
The Vaccinia virus strain GLV-1h68 was used as an oncolytic agent for the treatment of different tumor models. Systemic application of PFC emulsions followed by 1H/19F MRI of mock-infected and GLV-1h68-infected tumor-bearing mice revealed a significant accumulation of the 19F signal in the tumor rim of virus-treated mice. Histological examination of tumors confirmed a similar spatial distribution of the 19F signal hot spots and CD68+-macrophages. Thereby, the CD68+-macrophages encapsulate the GFP-positive viral infection foci. In multiple tumor models, we specifically visualized early inflammatory cell recruitment in Vaccinia virus colonized tumors. Furthermore, we documented that the 19F signal correlated with the extent of viral spreading within tumors.
These results suggest 19F MRI as a non-invasive methodology to document the tumor-associated host immune response as well as the extent of intratumoral viral replication. Thus, 19F MRI represents a new platform to non-invasively investigate the role of the host immune response for therapeutic outcome of oncolytic virotherapy and individual patient response.
Related Articles
Cancer Screening: A Mathematical Model Relating Secreted Blood Biomarker Levels to Tumor Sizes
Lutz, Amelie M
Willmann, Juergen K
Cochran, Frank V
Ray, Pritha
Gambhir, Sanjiv S
PLoS Medicine
Increasing efforts and financial resources are being invested in early cancer detection research. Blood assays detecting tumor biomarkers promise noninvasive and financially reasonable screening for early cancer with high potential of positive impact on patients' survival and quality of life. For novel tumor biomarkers, the actual tumor detection limits are usually unknown and there have been no studies exploring the tumor burden detection limits of blood tumor biomarkers using mathematical models. Therefore, the purpose of this study was to develop a mathematical model relating blood biomarker levels to tumor burden.
Methods and Findings
Using a linear one-compartment model, the steady state between tumor biomarker secretion into and removal out of the intravascular space was calculated. Two conditions were assumed: (1) the compartment (plasma) is well-mixed and kinetically homogenous; (2) the tumor biomarker consists of a protein that is secreted by tumor cells into the extracellular fluid compartment, and a certain percentage of the secreted protein enters the intravascular space at a continuous rate. The model was applied to two pathophysiologic conditions: tumor biomarker is secreted (1) exclusively by the tumor cells or (2) by both tumor cells and healthy normal cells. To test the model, a sensitivity analysis was performed assuming variable conditions of the model parameters. The model parameters were primed on the basis of literature data for two established and well-studied tumor biomarkers (CA125 and prostate-specific antigen [PSA]). Assuming biomarker secretion by tumor cells only and 10% of the secreted tumor biomarker reaching the plasma, the calculated minimally detectable tumor sizes ranged between 0.11 mm3 and 3,610.14 mm3 for CA125 and between 0.21 mm3 and 131.51 mm3 for PSA. When biomarker secretion by healthy cells and tumor cells was assumed, the calculated tumor sizes leading to positive test results ranged between 116.7 mm3 and 1.52 × 106 mm3 for CA125 and between 27 mm3 and 3.45 × 105 mm3 for PSA. One of the limitations of the study is the absence of quantitative data available in the literature on the secreted tumor biomarker amount per cancer cell in intact whole body animal tumor models or in cancer patients. Additionally, the fraction of secreted tumor biomarkers actually reaching the plasma is unknown. Therefore, we used data from published cell culture experiments to estimate tumor cell biomarker secretion rates and assumed a wide range of secretion rates to account for their potential changes due to field effects of the tumor environment.
This study introduced a linear one-compartment mathematical model that allows estimation of minimal detectable tumor sizes based on blood tumor biomarker assays. Assuming physiological data on CA125 and PSA from the literature, the model predicted detection limits of tumors that were in qualitative agreement with the actual clinical performance of both biomarkers. The model may be helpful in future estimation of minimal detectable tumor sizes for novel proteomic biomarker assays if sufficient physiologic data for the biomarker are available. The model may address the potential and limitations of tumor biomarkers, help prioritize biomarkers, and guide investments into early cancer detection research efforts.
Sanjiv Gambhir and colleagues describe a linear one-compartment mathematical model that allows estimation of minimal detectable tumor sizes based on blood tumor biomarker assays.
Editors' Summary
Cancers—disorganized masses of cells that can occur in any tissue—develop when cells acquire genetic changes that allow them to grow uncontrollably and to spread around the body (metastasize). If a cancer (tumor) is detected when it is small, surgery can often provide a cure. Unfortunately, many cancers (particularly those deep inside the body) are not detected until they are large enough to cause pain or other symptoms by pressing against surrounding tissue. By this time, it may be impossible to remove the original tumor surgically and there may be metastases scattered around the body. In such cases, radiotherapy and chemotherapy can sometimes help, but the outlook for patients whose cancers are detected late is often poor. Consequently, researchers are trying to develop early detection tests for different types of cancer. Many tumors release specific proteins—"cancer biomarkers"—into the blood and the hope is that it might be possible to find sets of blood biomarkers that detect cancers when they are still small and thus save many lives.
Why Was This Study Done?
For most biomarkers, it is not known how the amount of protein detected in the blood relates to tumor size or how sensitive the assays for biomarkers must be to improve patient survival. In this study, the researchers develop a "linear one-compartment" mathematical model to predict how large tumors need to be before blood biomarkers can be used to detect them and test this model using published data on two established cancer biomarkers—CA125 and prostate-specific antigen (PSA). CA125 is used to monitor the progress of patients with ovarian cancer after treatment; ovarian cancer is rarely diagnosed in its early stages and only one-fourth of women with advanced disease survive for 5 y after diagnosis. PSA is used to screen for prostate cancer and has increased the detection of this cancer in its early stages when it is curable.
What Did the Researchers Do and Find?
To develop a model that relates secreted blood biomarker levels to tumor sizes, the researchers assumed that biomarkers mix evenly throughout the patient's blood, that cancer cells secrete biomarkers into the fluid that surrounds them, that 0.1%–20% of these secreted proteins enter the blood at a continuous rate, and that biomarkers are continuously removed from the blood. The researchers then used their model to calculate the smallest tumor sizes that might be detectable with these biomarkers by feeding in existing data on CA125 and on PSA, including assay detection limits and the biomarker secretion rates of cancer cells growing in dishes. When only tumor cells secreted the biomarker and 10% of the secreted biomarker reach the blood, the model predicted that ovarian tumors between 0.11 mm3 (smaller than a grain of salt) and nearly 4,000 mm3 (about the size of a cherry) would be detectable by measuring CA125 blood levels (the range was determined by varying the amount of biomarker secreted by the tumor cells and the assay sensitivity); for prostate cancer, the detectable tumor sizes ranged from similar lower size to about 130 mm3 (pea-sized). However, healthy cells often also secrete small quantities of cancer biomarkers. With this condition incorporated into the model, the estimated detectable tumor sizes (or total tumor burden including metastases) ranged between grape-sized and melon-sized for ovarian cancers and between pea-sized to about grapefruit-sized for prostate cancers.
What Do These Findings Mean?
The accuracy of the calculated tumor sizes provided by the researchers' mathematical model is limited by the lack of data on how tumors behave in the human body and by the many assumptions incorporated into the model. Nevertheless, the model predicts detection limits for ovarian and prostate cancer that broadly mirror the clinical performance of both biomarkers. Somewhat worryingly, the model also indicates that a tumor may have to be very large for blood biomarkers to reveal its presence, a result that could limit the clinical usefulness of biomarkers, especially if they are secreted not only by tumor cells but also by healthy cells. Given this finding, as more information about how biomarkers behave in the human body becomes available, this model (and more complex versions of it) should help researchers decide which biomarkers are likely to improve early cancer detection and patient outcomes.
Additional Information.
Please access these Web sites via the online version of this summary at http://dx.doi.org/10.1371/journal.pmed.0050170.
The US National Cancer Institute provides a brief description of what cancer is and how it develops and a fact sheet on tumor markers; it also provides information on all aspects of ovarian and prostate cancer for patients and professionals, including information on screening and testing (in English and Spanish)
The UK charity Cancerbackup also provides general information about cancer and more specific information about ovarian and prostate cancer, including the use of CA125 and PSA for screening and follow-up
The American Society of Clinical Oncology offers a wide range of information on various cancer types, including online published articles on the current status of cancer diagnosis and management from the educational book developed by the annual meeting faculty and presenters. Registration is mandatory, but information is free
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Permissiveness of Human Cancer Cells to Oncolytic Bovine Herpesvirus 1 Is Mediated in Part by KRAS Activity
Cuddington, Breanne P.
Mossman, Karen L.
Journal of Virology
Oncolytic viruses (OVs) are attractive avenues of cancer therapy due to the absence of toxic side effects often seen with current treatment modalities. Bovine herpesvirus 1 (BHV-1) is a species-specific virus that does not induce cytotoxicity in normal primary human cells but can infect and kill various human immortalized and transformed cell lines. To gain a better understanding of the oncolytic breadth of BHV-1, the NCI panel of established human tumor cell lines was screened for sensitivity to the virus. Overall, 72% of the panel is permissive to BHV-1 infection, with corresponding decreases in cellular viability. This sensitivity is in comparison to a sensitivity of only 32% for a herpes simplex virus 1 (HSV-1)-based oncolytic vector. Strikingly, while 35% of the panel supports minimal or no BHV-1 replication, significant decreases in cellular viability still occur. These data suggest that BHV-1 is an OV with tropism for multiple tumor types and is able to induce cytotoxicity independent of significant virus replication. In contrast to other species-specific OVs, cellular sensitivity to BHV-1 does not correlate with type I interferon (IFN) signaling; however, mutations in KRAS were found to correlate with high levels of virus replication. The knockdown or overexpression of KRAS in human tumor cell lines yields modest changes in viral titers; however, overexpression of KRAS in normal primary cells elicits permissivity to BHV-1 infection. Together, these data suggest that BHV-1 is a broad-spectrum OV with a distinct mechanism of tumor targeting.
IMPORTANCE Cancer remains a significant health issue, and novel treatments are required, particularly for tumors that are refractory to conventional therapies. Oncolytic viruses are a novel platform given their ability to specifically target tumor cells while leaving healthy cells intact. For this strategy to be successful, a fundamental understanding of virus-host interactions is required. We previously identified bovine herpesvirus 1 as a novel oncolytic virus with many unique and clinically relevant features. Here, we show that BHV-1 can target a wide range of human cancer types, most potently lung cancer. In addition, we show that enhanced KRAS activity, a hallmark of many cancers, is one of the factors that increases BHV-1 oncolytic capacity. These findings hold potential for future treatments, particularly in the context of lung cancer, where KRAS mutations are a negative predictor of treatment efficacy.
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Oncolytic Measles Virus Encoding Thyroidal Sodium Iodide Symporter for Squamous Cell Cancer of the Head and Neck Radiovirotherapy
Li, Hongtao
Peng, Kah-Whye
Russell, Stephen J.
Human Gene Therapy
Oncolytic measles virus (MV) encoding the human thyroidal sodium iodide symporter (MV-NIS) has proved to be safe after intraperitoneal or intravenous administration in patients with ovarian cancer or multiple myeloma, respectively, but it has not yet been administered through intratumoral injection in humans. Squamous cell carcinoma (SCC) of the head and neck (SCCHN) usually is locally invasive and spreads to the cervical lymph nodes, which are suitable for the intratumoral administration of oncolytic viruses. To test whether oncolytic MV is an effective treatment for SCCHN, we used oncolytic MV-NIS to infect SCCHN in vitro and in vivo. The data show that SCCHN cells were infected and killed by MV-NIS in vitro. Permissiveness of the tumor cells to MV infection was not affected by irradiation after viral addition. Monitored noninvasively through radioiodine-based single-photon emission computed tomography/computed tomography, intratumorally virus-delivered NIS has concentrated the radioiodine in the MV-NIS–treated tumors in the FaDu mouse xenograft model of human SCCHN, and the antitumor effect could be boosted significantly (p<0.05) either with concomitant cyclophosphamide therapy or with appropriately timed administration of radioiodine 131I. MV-NIS could be a promising new anticancer agent that may substantially enhance the outcomes of standard therapy after intratumoral administration in patients with locally advanced SCCHN.
Li and colleagues investigate the use of oncolytic measles virus encoding human thyroidal sodium iodide symporter (MV-NIS) to treat squamous cell carcinoma of the head and neck (SCCHN) in vitro and in vivo. MV-NIS-treated tumors are able to concentrate administered radioiodine in a mouse xenograft model of human SCCHN, and the antitumor effect is significantly boosted by cyclophosphamide therapy.
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Modeling Evolutionary Dynamics of Epigenetic Mutations in Hierarchically Organized Tumors
Sottoriva, Andrea
Vermeulen, Louis
Tavaré, Simon
PLoS Computational Biology
The cancer stem cell (CSC) concept is a highly debated topic in cancer research. While experimental evidence in favor of the cancer stem cell theory is apparently abundant, the results are often criticized as being difficult to interpret. An important reason for this is that most experimental data that support this model rely on transplantation studies. In this study we use a novel cellular Potts model to elucidate the dynamics of established malignancies that are driven by a small subset of CSCs. Our results demonstrate that epigenetic mutations that occur during mitosis display highly altered dynamics in CSC-driven malignancies compared to a classical, non-hierarchical model of growth. In particular, the heterogeneity observed in CSC-driven tumors is considerably higher. We speculate that this feature could be used in combination with epigenetic (methylation) sequencing studies of human malignancies to prove or refute the CSC hypothesis in established tumors without the need for transplantation. Moreover our tumor growth simulations indicate that CSC-driven tumors display evolutionary features that can be considered beneficial during tumor progression. Besides an increased heterogeneity they also exhibit properties that allow the escape of clones from local fitness peaks. This leads to more aggressive phenotypes in the long run and makes the neoplasm more adaptable to stringent selective forces such as cancer treatment. Indeed when therapy is applied the clone landscape of the regrown tumor is more aggressive with respect to the primary tumor, whereas the classical model demonstrated similar patterns before and after therapy. Understanding these often counter-intuitive fundamental properties of (non-)hierarchically organized malignancies is a crucial step in validating the CSC concept as well as providing insight into the therapeutical consequences of this model.
Author Summary
Cancer is in essence a genetic disease that leads to uncontrolled cell proliferation, invasion and metastasis. The cancer stem cell (CSC) hypothesis states that tumors are not just a mass of uniform malignant cells but they are hierarchically organized, like normal tissues. At the top of such a hierarchy are cancer stem cells that fuel tumor growth in the long run, whereas the majority of other cells are able to divide only a few times. The experiments that support the CSC hypothesis are often criticized as being difficult to interpret. A novel approach to test the CSC paradigm is to integrate mathematical modeling with DNA variation data that carry the phylogenetic history of cells. We have developed a model that simulates the occurrence of such changes under both the CSC hypothesis and the classical, purely stochastic scenario. We found that although a CSC-driven tumor has a smaller number of tumorigenic cells, it triggers more malignant properties such as invasive growth, heterogeneity and evolutionary escape from peaks in the fitness landscape. These properties, that are unique to the CSC model, are enhanced even further when a treatment is applied to the tumor.
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Adult Industry Trade Association (AITA) to close – Emails to the Chair RE "Golden Eye International" involvement
It has been announced that the Adult Industry Trade Association (AITA) is to be closed down. The committee it seems feels that it is untenable and has been running at a loss since 2010.
After conducting research and feedback from the "Adult" Industry, that is Porn Industry to you and me, 64% of respondents felt that an adult trade association was needed but only 32% believed that AITA was an effective voice for the industry. These figures seem rather damning. Two Thirds believe their should be an association but that the AITA is rather useless actually acting as that association.
The Official AITA statement reads:
"With the limited resources available to AITA, it did not come as a surprise to learn that the majority of current members felt that AITA is underperforming in its provision of key services when considering their relative importance. It is clear that this cannot be addressed without a significant injection of cash. AITA has significantly reduced its running costs over the last three years, however with corresponding decreasing subscriptions it has been running at a significant loss since at least 2010. Due to the lack of ongoing support for AITA from the general UK adult industry, and after much deliberation, the committee believes that it is no longer tenable for AITA to continue after the end of the membership year (March 31st) and all committee members will resign on that date and the company closed down. The committee would like to thank all members for their support over the years. As individuals they will continue to work to support the UK adult industry where they can, offering ongoing advice in their particular area of expertise and continuing to network at adult industry events."
This is rather interesting as a statement made by the AITA December 12th reads
Trade body AITA has announced that it has appointed Jason Maskell of JCaz as its interim chairman following the resignation of Jerry Barnett. Maskell's tenure is scheduled to run until the end of March 2013.
A statement on the AITA website, posted 11th December, read: "The AITA committee would like to thank Jerry for all his time and work he has put in to AITA during his time as a committee member and chairman and wish him well for the future and all his does."
Now those following the Golden Eye International Legal action may not realise that Jason Maskell is also the head of Orchid MG, one of the litigants in the "Speculative Invoicing" action. Orchid MG were one of the Producers added to the action in Golden Eyes Appeal.
On the suggestion of one of the other Producers, I had written to the AITA before Maskell had taken over, and had received an email from the then head (Barnett), that whilst he did not agree with the action, he could understand why it happened, and that they should make the most of what they could do while they could do it. I thought an interesting response.
After Maskell took over in December 2012, and on realising he was one of the Producers added to the action, I thought, "Hmmm, this sounds like a conflict of interest", I was however misled into thinking that the AITA was a kind of self regulation group who might actually care for innocent people being targetted by Porn Barons and NOT a pressure group for the Porn Industry, my mistake.
I sent an email to the AITA detailing my concerns with the Golden Eye action and received, what to me was no more than a Golden Eye Press release from the AITA, I then sent a second email and got no response at all.
On Twitter this week Maskell attempted to engage me and offered me an interview on camera, using almost word for word, what Terence Stephens of "One Eyed Jack" had offered, I again politely declined, explaining again why I might not want to break rank, namely the fact I have already been threatened with legal action by Golden Eyes previous legal team. After failing to get me to do the interview, I pointed out to Maskell that he had not replied to my last email, and I would rather he did that, He responded by saying he wouldn't respond as I was "moaning about a court approved action" in my email, by Maskel of the AITA, I thought how odd (and unprofessional), far from moaning I thought I had laid out my concerns in a fairly cogent manner.
Anyhow, I include our email exchange here, just for the record, and would like to just add, that if the AITA are looking for a big cash injection, why on earth have they not asked Mr Ben Dover? Surely he is interested in the Industry that has made him a multi millionaire? No? Oh….
Was I really merely "moaning", about a legal action I didn't like?
The Speculative Invoicing Handbook – Part 2 :UPDATED
With news reaching us that the "Golden Eye International" launch of their Speculative Invoicing(Copyright Trolling) action is due to start in the New Year, we wanted to bring this latest tome to your attention. This quality guide was written by those fine folks at Being Threatened, in response to the number of innocent people targeted by previous companies, involved with these actions.
Download the PDF file here
Comments are always welcome.
We at ACS:BORE are not affiliated with the production of this book, although we endorse it as the best information to read with regard to receiving a letter from one of these companies. For further reading on this please see the excellent Torrent Freak post
My apologies, as pointed out I wrongfully referred to "Golden Eye International" as "Golden Eye Interactive", my apologies, it was an inadvertent mistake made in a moment of acute clarity of mind that a shell company based on an industrial estate in the UK with only two employees, and who operate only within the UK is NOT really an international company, my apologies again:-)
Open Rights Group appeals for funds, SO DOES "GOLDEN EYE INTERNATIONAL"!!
Open Rights Group (ORG) have stepped into the fracas of the Golden Eye International Court Case, that is threatening to bring more misery to innocent people in the UK. They have taken over from Consumer Focus and need some funding to continue to oppose the case, that the Pornographers have brought against thousands of O2 Customers.
For all those caught up in the Davenport Lyons/Acs:Law/Tilly Baily Irvine/Gallant Macmillan cases will now, there was NO talking to these people, it was a case of PAY UP OR ELSE.
Although Golden Eye International have been somewhat neutered by the High Court, they still use the SAME Software Monitor, that was shown to be corrupt and also the same "Expert Witness" that even ACS:LAW said was "Problematic"
To read what Open Rights want to do and to donate to their cause, please see here. http://www.openrightsgroup.org/blog/2012/org-goldeneye-intervene
In an amazing example of what can only be described as "Shameless Audacity" Golden Eye International are attempting the same thing.
GEILs Head Julian Becker who trades under the Optime Strategies moniker and whose business interests seem to cover a Security role at a London Synagogue and it is also the Parent company of Ben Dover Productions, has taken the Open Rights Group appeal for money as an insult.
Becker writes
"It would appear once again we have a difference of opinion on what constitutes a consumer — mine being someone who purchases goods or services theirs being someone who just takes them," Becker told XBIZ. "I'd like to think I have a decent grasp of the English language and my term to describe such a person would be thief rather than consumer."
Once again as is shown Becker is saying that anyone caught by his Software Monitor, is guilty of theft, now we already know that file sharing copyrighted material is "Copyright Infringement" NOT THEFT, however, Becker goes on
"They are appealing for funding for their court costs, which having checked our bank balance recently has given me the idea to do the same," he said. "The perception of pornographers outside the industry may well be filthy rich; however as anyone with an ability for rational thinking will appreciate any business who's core product has been decimated by piracy is experiencing a massive downturn in turnover and profitability."
Considering the material that Ben Dover Productions produce I would imagine the fanbase is dying off due to lack of quality material rather than "Copyright Infringement", it is very easy to say that it is because it is being copied and not brought, but I see it rather that even if it WAS being copied, those who copied it would never have bought it in the first place. I am NOT defending the downloading of copyright material here either, I just wonder if Golden Eye actually pay a license fee for ripping off other peoples titles, (XXX Factor complete with similar logo)
Regarding the "Outside perception of pornographers being "Filthy Rich"", I wonder if they might get that impression from Mr Beckers partner Mr Honey, AKA "Ben Dover" seen here in a BBC Interview. And here in a magazine article "I still have a very nice lifestyle. When I say I'm skint, people say, "No, you're not skint mate, it's just now you've only got three cars instead of five." So I wonder where these perceptions from people "outside the industry" come from? Note in the BBC interview there was NO talk about file sharing stealing business.
"Therefore any financial support, however small, that can be given will help the cause massively. If you can't give then supportive messages also are appreciated if not lost in the deluge of personal abuse and threats from the faceless keyboard warriors."
I wonder what he means? A man whose partner is a multi-millionaire is asking people of lesser means to contribute to a case of his own making, and that is RIGHT?
Whereby a Consumer group that KNOWS what happened with previous Legal actions, that were declared as "Legal Blackmail" in the "House of Lords", wants to raise money from those people affected BY those legal practices, and that is WRONG?
I think I have a pretty good grasp of the English language to, and I wouldn't like to say what I think about people like this, Pornographers attempting to squeeze the last pennies out of a dying beast, oh I better stop, I may being giving them a new film idea!
Golden Eye Interactive are due back in Court in December, to attempt to get 7000+names that they were declined at an earlier hearing.
I would urge anyone who feels the need to defend innocent people to contribute to the Open Rights appeal, at least that way there can be justice, Golden Eye are not interested in the justice, merely money to fill their coffers. £5000, is nothing to these people it is EVERYTHING to Open Rights Group (ORG)
Source for the quotes: http://newswire.xbiz.com/view.php?id=156690
Consumer Watchdog slams O2 in advice to ISPs on protecting customer details
In a damning briefing Consumer Focus the Consumer Watchdog who represented O2 Customers accused of copyright infringement by Pornographers Golden Eye International/Ben Dover Productions(GEIL/BDP), have lashed the ISP as irresponsible towards those very customers.
The briefing is advice to ISPs who are targeted by "Copyright Trolls", and how to balance the need for redress to those who believe their work has truly been infringed and the privacy of those they accuse. It is well worth reading, and can be found here.
After the past debacles involving the law firms, Davenport Lyons, ACS:LAW, Tilly Bailey Irvine ended in fines, suspensions and regulatory settlement agreements, most people thought the "Speculative Invoicing" schemes, or the "Pay up or else" letters were a thing of the past. With GEIL/BDP launching their attempt at an "Alternate revenue scheme" for their failing business, O2 should have stood in their way. They didn't.
Not only did O2 show complete disdain by capitulating to the pornographers at the expense of their customers, Consumer Focus highlights even more disgraceful behaviour on O2s part.
The High Court found the draft order and the draft letters to O2 customers which Golden Eye submitted as part of its application, and which O2 chose not to challenge, objectionable in a number of ways.
It is not feasible for a consumer watchdog to intervene in every single NorwichPharmacal order application, however we expect ISPs and online hosts to take responsibility for protecting their customers" rights. The draft order and draft letter before action proposed by Golden Eye were plainly problematic, yet O2 essentially only intervened to ensure that Golden Eye pays for the cost to O2 of matching the IP addresses with its customers" personal data.
O2"s refusal to ensure that its customers" rights are respected appears particularly odd in the light of the fact that Telefónica de España acted to defend its customers" data protection rights in Productores de Musica v Telefonica, which related to an application for a disclosure order.
Particularly odd indeed, one wonders why O2 capitulated to Pornographers when they didn't to the Music Producers. So why didn't O2 protect it's customers against predatory businesses like GEIL/BDP?
Why did they ignore the obvious problems with GEIL/BDPs approach?
Why did they not even turn up for the hearing?
Why did they only insist on their OWN protection and not of that of their customers?
Why indeed have they hung so many of their customers out to dry and left them vulnerable to a failing Pornography business desperate for cash?
Maybe because it is easy to take their customers for granted, maybe they feel they are so stupid they will stay with them rather than move ISP, one thing is for sure, when their customers start receiving GEIL/BDP letters maybe they will realise to late when their reputation suffers as it surely will.
O2 CANNOT simply use the tired excuse of "We have to abide by the rulings of a UK Court", THAT is NOT what happened here, O2 simply looked out for their OWN interests and betrayed their duty of care to their customers.
Golden Eye International /Ben Dover (GEIL) Neutered in the High Court
Golden Eye International/Ben Dover the latest group to try their hand at the Speculative Invoicing Scam, have been given permission to send their Court Approved letters out to O2 Customers.
The Letter, it would seem has been very much scrutinised and has been ordered by the Court to be toned down, after GEIL attempted to overstep their mark by using a letter that made some wild claims, regarding the amount they wanted and also what the ISP would do on their say so.
It remains to be seen the actual content of the letter, however early indications point to the fact that it is NOT allowed to contain a cash demand as GEIL's forerunners, Davenport Lyons, ACS:LAW, Tilly Baily Irvine, or Gallant Macmillan had done.
GEIL initially wanted to demand £700 per letter, this was quashed by the Judge overseeing the case.
More background on this can be found on this Blog or the always excellent Torrentfreak website.
What I wanted to highlight here, is some observations
Golden Eye International/Ben Dover may claim to have no connection with ACS:LAW but there is one, and one Julian Becker has already admitted to, the data gatherer, the person who will harvest the IP addresses of those suspected of uploading/downloading, is a man called Alireza Torabi of ng3 systems, Mr Torabi is the guy who got things spectacularly wrong with the IP addresses that led to ACS:LAW targeting innocent people. He is using the same software, so one can presume the same results.
Golden Eye International are also using the same "Expert Witness" Clem Vogler. Alireza Torabi was shown in the leaked emails to have a somewhat troublesome relationship with Andrew Crossley to the point that Crossleys cohort Terence Tsang, consulted and arranged for the monitoring to be done by Guardaley. One would have thought Torabi at least would have learnt his lesson!
Ben Dover aka Lindsay Honey, was interviewed in January 2009, with regard to the same action taken in the USA by Larry Flynt, The interview can be viewed here. as can be seen, Honey claims he KNEW that his sales were already going down the pan and that he had "Seen it coming" and had diversified into other areas, even attempting to become a serious actor…
The problem with this kind of "Legal Blackmail", as it was decribed in the House of Lords, is that it is VERY difficult to defend against. A person may have strong evidence that he has NOT downloaded one of their films, however to go to Court and have his name cleared costs a LOT of money. A person would more than likely pay up even if they are innocent through fear of not having the money to go to court to clear their name, and that is EXACTLY what people like GEIL are banking on. Of course being pornographers they have no shame, no understanding and no concern of what their actions have on the average person.
They want you to be frightened, they want you to fear going to Court, the reason being of course is that they have NO evidence whatsoever, against ANYONE. Don't believe me? See this quote from the ruling of Media C.A.T (ACS:LAW) by Judge Colin Birss
All the IP address identifies is an internet connection, which is likely today to be a wireless home broadband router. All Media CAT's monitoring can identify is the person who has the contract with their ISP to have internet access. Assuming a case in Media CAT's favour that the IP address is indeed linked to wholesale infringements of the copyright in question… Media CAT do not know who did it and know that they do not know who did it.
Julian Becker claims that GEIL are different, they all did, every Law Firm that has tried this, claimed to be different from the others, but they were not.
Becker says "In our first letter we seek to find out more information regarding evidence of an infringement of our copyright"
Andrew Crossley of ACS:LAW said "I make an enquiry of the recipient of my initial letter following receipt of evidence that their internet connection was utilized for the purposes of infringing copyright of our clients"
GEIL are not of course Lawyers, so it will be interesting to see what they will do when a letter comes through the door of some innocent subscriber of O2 and they say "Screw you, con merchants"
We will see.
One final point is that I wonder what O2 meant when they talk about their "Unique" position in this case?, the only unique position as I see it, is that they are the only ISP to have acquiesced to this form of racket since ACS:LAWs spectacular disintegration.
I am angry, pornography accusations can wreck lives and marriages, that is why I will fight for those wrongly accused.
I will leave the last word to Mr Honey…
"Fuck! Aren't I just the luckiest bastard in the World?
Not only do I get to film all of these fantasic looking girls stripping off for my camera, but I also get to slide my fingers knuckle-deep into their hot, wet snatches, then get my knob sucked into the bargain!! Have I got some awesome little jizz-junkies for you!"
And the comment:
"At the end of the day, if I can't make money out of porn, the only way I can make money is to get to the people who are not buying it"
O2 CENSORS "Golden Eye International" Debate from its Forums
This link is the only fragment left of a thread that was started on The O2 Forums. I joined the thread and posted a total of 5 posts (31/03_2012), engaging with the board users "Perksie" "Brownie" and "O2MACH"
I don't believe I was rude in any way. I pointed out that I believed that O2 did NOT fight for their customers and I used the published Judgement to prove the points that I made.
In a covering letter dated 20 September 2011 Golden Eye stated:
"We are the First Applicant and act for the Second – Fourteenth Applicants in this application.
It may be somewhat unusual for the Applicants to apply on their account. However, there has recently been a certain amount of publicity associated with this type of claim (ACS Law; Davenport Lyons). We therefore believe that we will be best served acting for ourselves."
The letter went on to request that the claim be considered on paper, and enclosed a copy of the skeleton argument which had prepared by counsel instructed on its behalf on the application against BT.
On 7 October 2011 Baker & McKenzie filed an acknowledgement of service on behalf of O2 stating that O2 did not intend to contest the claim.
I pointed out that it was strange that O2 were the only ISPs targeted by "Golden eye International" and further pointed out that PlusNet have been very robust since their experience with ACS:LAW and have come out on their own forums and said, they will resist a Norwich Pharmacal Hearing that would allow a company to have access to their Customers private information.
It seems O2 did not like this dose of truth. I have tried to contact O2 but have had NO REPLY
Their seems to be a media output on their behalf of "O2 had no choice" and "O2 FORCED to hand over customer details. Both of these IMHO are false.
O2 DID have a choice, they could have said NO.
"Golden Eye International" Commercial director Julian Becker has already stated in a rather laughable response to the Published Judgement that they use the SAME software that ACS:LAW used (Par 10),
This was blown out of the water at a previous hearing by Judge Birss who presiding over the ACS:LAW/Media C.A.T debacle (That used the same software to monitor) he stated.
"Assuming a case in MediaCATs favour that the IP Address is indeed limited to wholesale infringements of the copyright in question… MediaCAT do not know who did it, and know that they do not know who did it"
Further to my point of O2 HAVING a choice to say NO, I include the communication between O2 and ACS:LAW that quite clearly shows, they could have said no then, but merely asked ACS:LAW to change some of the legal arguments to suit them better.
If "Perksie", "Browni" or "O2MACH2" would like to continue our discussion, they are more than welcome to do so here..
For a look at how a TRUE ISP runs a forum see here…
UPDATE 1: From O2 ..
@acs_law_illegal Hi there, we've just moved to a new forum and only moved over the busiest topics
Hmmmm oh Reeeeaaaalllly

subset: commoncrawl


text:
The Orlando Magic have activated Hedo Turkoglu for tonight's game against the Toronto Raptors, and he will play for the first time since he suffered a broken left hand in the season-opener Nov. 2.
But Turkoglu won't have an easy transition.
Starting point guard Jameer Nelson has a sore hip and will not play tonight, team officials said. Nelson's absence likely will mean Turkoglu will bring the ball upcourt often tonight.
An MRI today revealed that Moore has a sprained ligament inside his left elbow and does not have any damage to the joint. Moore will not play tonight, and it's unclear how much time he will miss.
Ish Smith likely will start at point guard in Nelson's and Moore's absences.Josh Robbins covers the Orlando Magic and the NBA for the Orlando Sentinel. You can reach him via e-mail at email@example.com and connect with him on Facebook at facebook.com/JoshuaBRobbins. Follow him on Twitter at @JoshuaBRobbins.