Adjuvant sunitinib in patients with high-risk renal cell carcinoma: safety, therapy management, and patient-reported outcomes in the S-TRAC trial.

Background: Adjuvant sunitinib has significantly improved disease-free survival versus placebo in patients with renal cell carcinoma at high risk of recurrence post-nephrectomy (hazard ratio 0.76; 95% confidence interval, 0.59-0.98; two-sided P = 0.03). We report safety, therapy management, and patient-reported outcomes for patients receiving sunitinib and placebo in the S-TRAC trial. Patients and methods: Patients were stratified by the University of California, Los Angeles Integrated Staging System and Eastern Cooperative Oncology Group performance status score, and randomized (1 : 1) to receive sunitinib (50 mg/day) or placebo. Single dose reductions to 37.5 mg, dose delays, and dose interruptions were used to manage adverse events (AEs). Patients' health-related quality of life, including key symptoms typically associated with sunitinib, were evaluated with the European Organisation for Research and Treatment of Cancer Quality of Life Questionnaire (EORTC QLQ-C30). Results: Patients maintained treatment for 9.5 (mean, SD 4.4) and 10.3 (mean, SD 3.7) months in the sunitinib and placebo arms, respectively. In the sunitinib arm, key AEs occurred ∼1 month (median) after start of treatment and resolved within ∼3.5 weeks (median). Many (40.6%) AEs leading to permanent discontinuation were grade 1/2, and most (87.2%) resolved or were resolving by 28 days after last treatment. Patients taking sunitinib showed a significantly lower EORTC QLQ-C30 overall health status score versus placebo, although this reduction was not clinically meaningful. Patients reported symptoms typically related to sunitinib treatment with diarrhea and loss of appetite showing clinically meaningful increases. Conclusions: In S-TRAC, AEs were predictable, manageable, and reversible via dose interruptions, dose reductions, and/or standard supportive medical therapy. Patients on sunitinib did report increased symptoms and reduced HRQoL, but these changes were generally not clinically meaningful, apart from appetite loss and diarrhea, and were expected in the context of known sunitinib effects. Clinical trial registration: ClinicalTrials.gov, NCT00375674.

Prime-boost using separate oncolytic viruses in combination with checkpoint blockade improves anti-tumour therapy.

The anti-tumour effects associated with oncolytic virus therapy are mediated significantly through immune-mediated mechanisms, which depend both on the type of virus and the route of delivery. Here, we show that intra-tumoral oncolysis by Reovirus induced the priming of a CD8+, Th1-type anti-tumour response. By contrast, systemically delivered Vesicular Stomatitis Virus expressing a cDNA library of melanoma antigens (VSV-ASMEL) promoted a potent anti-tumour CD4+ Th17 response. Therefore, we hypothesised that combining the Reovirus-induced CD8+ T cell response, with the VSV-ASMEL CD4+ Th17 helper response, would produce enhanced anti-tumour activity. Consistent with this, priming with intra-tumoral Reovirus, followed by an intra-venous VSV-ASMEL Th17 boost, significantly improved survival of mice bearing established subcutaneous B16 melanoma tumours. We also show that combination of either therapy alone with anti-PD-1 immune checkpoint blockade augmented both the Th1 response induced by systemically delivered Reovirus in combination with GM-CSF, and also the Th17 response induced by VSV-ASMEL. Significantly, anti-PD-1 also uncovered an anti-tumour Th1 response following VSV-ASMEL treatment that was not seen in the absence of checkpoint blockade. Finally, the combination of all three treatments (priming with systemically delivered Reovirus, followed by double boosting with systemic VSV-ASMEL and anti-PD-1) significantly enhanced survival, with long-term cures, compared to any individual, or double, combination therapies, associated with strong Th1 and Th17 responses to tumour antigens. Our data show that it is possible to generate fully systemic, highly effective anti-tumour immunovirotherapy by combining oncolytic viruses, along with immune checkpoint blockade, to induce complementary mechanisms of anti-tumour immune responses.

Detection of candidate biomarkers of prostate cancer progression in serum: a depletion-free 3D LC/MS quantitative proteomics pilot study.

BACKGROUND: Prostate cancer (PCa) is the most common male cancer in the United Kingdom and we aimed to identify clinically relevant biomarkers corresponding to stage progression of the disease. METHODS: We used enhanced proteomic profiling of PCa progression using iTRAQ 3D LC mass spectrometry on high-quality serum samples to identify biomarkers of PCa. RESULTS: We identified >1000 proteins. Following specific inclusion/exclusion criteria we targeted seven proteins of which two were validated by ELISA and six potentially interacted forming an 'interactome' with only a single protein linking each marker. This network also includes accepted cancer markers, such as TNF, STAT3, NF-κB and IL6. CONCLUSIONS: Our linked and interrelated biomarker network highlights the potential utility of six of our seven markers as a panel for diagnosing PCa and, critically, in determining the stage of the disease. Our validation analysis of the MS-identified proteins found that SAA alongside KLK3 may improve categorisation of PCa than by KLK3 alone, and that TSR1, although not significant in this model, might also be a clinically relevant biomarker.

Oncolytic vaccinia virus as a vector for therapeutic sodium iodide symporter gene therapy in prostate cancer.

Oncolytic strains of vaccinia virus are currently in clinical development with clear evidence of safety and promising signs of efficacy. Addition of therapeutic genes to the viral genome may increase the therapeutic efficacy of vaccinia. We evaluated the therapeutic potential of vaccinia virus expressing the sodium iodide symporter (NIS) in prostate cancer models, combining oncolysis, external beam radiotherapy and NIS-mediated radioiodide therapy. The NIS-expressing vaccinia virus (VV-NIS), GLV-1h153, was tested in in vitro analyzes of viral cell killing, combination with radiotherapy, NIS expression, cellular radioiodide uptake and apoptotic cell death in PC3, DU145, LNCaP and WPMY-1 human prostate cell lines. In vivo experiments were carried out in PC3 xenografts in CD1 nude mice to assess NIS expression and tumor radioiodide uptake. In addition, the therapeutic benefit of radioiodide treatment in combination with viral oncolysis and external beam radiotherapy was measured. In vitro viral cell killing of prostate cancers was dose- and time-dependent and was through apoptotic mechanisms. Importantly, combined virus therapy and iodizing radiation did not adversely affect oncolysis. NIS gene expression in infected cells was functional and mediated uptake of radioiodide both in vitro and in vivo. Therapy experiments with both xenograft and immunocompetent Transgenic Adenocarcinoma of the Mouse Prostate (TRAMP) mouse models showed that the addition of radioiodide to VV-NIS-infected tumors was more effective than each single-agent therapy, restricting tumor growth and increasing survival. In conclusion, VV-NIS is effective in prostate cancer models. This treatment modality would be an attractive complement to existing clinical radiotherapy practice.

Spontaneous antibodies against Engrailed-2 (EN2) protein in patients with prostate cancer.

We reported the expression of the homeodomain-containing transcription factor Engrailed-2 (EN2) in prostate cancer and showed that the presence of EN2 protein in the urine was highly predictive of prostate cancer. This study aimed to determine whether patients with prostate cancer have EN2 autoantibodies, what the prevalence of these antibodies is and whether they are associated with disease stage. The spontaneous immunoglobulin (Ig)G immune response against EN2 and for comparison the tumour antigen New York Esophageal Squamous Cell Carcinoma 1 (NY-ESO-1), were tested by enzyme-linked immunosorbent assay (ELISA) in three different cohorts of prostate cancer patients as well as a group of men genetically predisposed to prostate cancer. Thirty-two of 353 (9·1%) of the SUN cohort representing all stages of prostate cancer demonstrated EN2 IgG responses, 12 of 107 patients (11·2%) in the advanced prostate cancer patients showed responses, while only four of 121 patients (3·3%) with castrate-resistant prostate cancer showed EN2 autoantibodies. No significant responses were found in the predisposed group. Anti-EN2 IgG responses were significantly higher in patients with prostate cancer compared to healthy control males and similarly prevalent to anti-NY-ESO-1 responses. While EN2 autoantibodies are not a useful diagnostic or monitoring tool, EN2 immunogenicity provides the rationale to pursue studies using EN2 as an immunotherapeutic target.

Synergistic cytotoxicity of radiation and oncolytic Lister strain vaccinia in (V600D/E)BRAF mutant melanoma depends on JNK and TNF-α signaling.

Melanoma is an aggressive skin cancer that carries an extremely poor prognosis when local invasion, nodal spread or systemic metastasis has occurred. Recent advances in melanoma biology have revealed that RAS-RAF-MEK-ERK signaling has a pivotal role in governing disease progression and treatment resistance. Proof-of-concept clinical studies have shown that direct BRAF inhibition yields impressive responses in advanced disease but these are short-lived as treatment resistance rapidly emerges. Therefore, there is a pressing need to develop new targeted strategies for BRAF mutant melanoma. As such, oncolytic viruses represent a promising cancer-specific approach with significant activity in melanoma. This study investigated interactions between genetically-modified vaccinia virus (GLV-1h68) and radiotherapy in melanoma cell lines with BRAF mutant, Ras mutant or wild-type genotype. Preclinical studies revealed that GLV-1h68 combined with radiotherapy significantly increased cytotoxicity and apoptosis relative to either single agent in (V600D)BRAF/(V600E)BRAF mutant melanoma in vitro and in vivo. The mechanism of enhanced cytotoxicity with GLV-1h68/radiation (RT) was independent of viral replication and due to attenuation of JNK, p38 and ERK MAPK phosphorylation specifically in BRAF mutant cells. Further studies showed that JNK pathway inhibition sensitized BRAF mutant cells to GLV-1h68-mediated cell death, mimicking the effect of RT. GLV-1h68 infection activated MAPK signaling in (V600D)BRAF/(V600E)BRAF mutant cell lines and this was associated with TNF-α secretion which, in turn, provided a prosurvival signal. Combination GLV-1h68/RT (or GLV-1h68/JNK inhibition) caused abrogation of TNF-α secretion. These data provide a strong rationale for combining GLV-1h68 with irradiation in (V600D/E)BRAF mutant tumors.

Lymphokine-activated killer and dendritic cell carriage enhances oncolytic reovirus therapy for ovarian cancer by overcoming antibody neutralization in ascites.

Reovirus is an oncolytic virus (OV), which acts by both direct tumor cell killing and priming of antitumor immunity. A major obstacle for effective oncolytic virotherapy is effective delivery of OV to tumor cells. Ovarian cancer is often confined to the peritoneal cavity and therefore i.p. delivery of reovirus may provide the ideal locoregional delivery, avoiding systemic dissemination. However, ovarian cancer is associated with an accumulation of ascitic fluid, which may interfere with oncolytic viral therapy. Here, we investigated the effect of ascites on reovirus-induced oncolysis against primary ovarian cancer cells and ovarian cancer cell lines. In the absence of ascites, reovirus was cytotoxic against ovarian cancer cells; however, cytotoxicity was abrogated in the presence of ascitic fluid. Neutralizing antibodies (NAb) were identified as the cause of this inhibition. Loading OV onto cell carriers may facilitate virus delivery in the presence of NAb and immune cells which have their own antitumor effector activity are particularly appealing. Immature dendritic cells (iDC), Lymphokine-activated killer (LAK) cells and LAKDC cocultures were tested as potential carriers for reovirus for tumor cell killing and immune cell priming. Reovirus-loaded LAKDC, and to a lesser degree iDC, were able to: (i) protect from NAb and hand-off reovirus for tumor cell killing; (ii) induce a proinflammatory cytokine milieu (IFNÉ£, IL-12, IFNα and TNFα) and (iii) generate an innate and specific antitumor adaptive immune response. Hence, LAKDC pulsed with reovirus represent a novel, clinically practical treatment for ovarian cancer to maximise both direct and innate/adaptive immune-mediated tumor cell killing.

Synergistic cytotoxicity of oncolytic reovirus in combination with cisplatin-paclitaxel doublet chemotherapy.

Oncolytic reovirus is currently under active investigation in a range of tumour types. Early phase studies have shown that this agent has modest monotherapy efficacy and its future development is likely to focus on combination regimens with cytotoxic chemotherapy. Indeed, phase I/II clinical trials have confirmed that reovirus can be safely combined with cytotoxic drugs, including a platin-taxane doublet regimen, which is currently being tested in a phase III clinical trial in patients with relapsed/metastatic head and neck cancer. Therefore, we have tested this triple (reovirus, cisplatin, paclitaxel) combination therapy in a panel of four head and neck cancer cell lines. Using the combination index (CI) method, the triple therapy demonstrated synergistic cytotoxicity in vitro in both malignant and non-malignant cell lines. In head and neck cancer cell lines, this was associated with enhanced caspase 3 and 7 cleavage, but no increase in viral replication. In vitro analyses confirmed colocalisation of markers of reovirus infection and caspase 3. Triple therapy was significantly more effective than reovirus or cisplatin-paclitaxel in athymic nude mice. These data suggest that the combination of reovirus plus platin-taxane doublet chemotherapy has significant activity in head and neck cancer and underpin the current phase III study in this indication.

Oncolytic Vaccinia virus and radiotherapy in head and neck cancer.

OBJECTIVE: Oncolytic forms of attenuated Vaccinia virus are now in clinical development, assessing the compatibility of this novel treatment with radiotherapy may reveal exploitable synergistic relationships. MATERIALS AND METHODS: In vitro analyses of cell killing, cell cycle effects and caspase activation were carried out on HN3, HN5, CAL27, Detroit, SIHN5B, and PJ41 cells. In vivo studies of the virus and X-radiation were performed on H&N xenografts in CD1 nude mice. RESULTS: Cell killing in vitro was demonstrated to be dose- and time-dependent. Infection causes an increase in S-phase and sub-G1 cells. A dose dependent increase in active caspase-3 indicated induction of apoptosis. Xenografts injected with Vaccinia stabilised and frequently completely regressed. Combination with radiation generated additional cell death, induction of caspase activity and in vivo further improved long term regression rates. CONCLUSIONS: These data support continued exploration of this therapy combination and indicates potential for clinical trials in head and neck cancer.

The role of HOX genes in normal hematopoiesis and acute leukemia.

The homeobox (HOX) genes are a highly conserved family of homeodomain-containing transcription factors that specify cell identity in early development and, subsequently, in a number of adult processes including hematopoiesis. The dysregulation of HOX genes is associated with a number of malignancies including acute myeloid leukemia (AML) and acute lymphoid leukemia (ALL), where they have been shown to support the immortalization of leukemic cells both as chimeric partners in fusion genes and when overexpressed in their wild-type form. This review covers our current understanding of the role of HOX genes in normal hematopoiesis, AML and ALL, with particular emphasis on the similarities and differences of HOX function in these contexts, their hematopoietic downstream gene targets and implications for therapy.

Measles virus causes immunogenic cell death in human melanoma.

Oncolytic viruses (OV) are promising treatments for cancer, with several currently undergoing testing in randomised clinical trials. Measles virus (MV) has not yet been tested in models of human melanoma. This study demonstrates the efficacy of MV against human melanoma. It is increasingly recognised that an essential component of therapy with OV is the recruitment of host antitumour immune responses, both innate and adaptive. MV-mediated melanoma cell death is an inflammatory process, causing the release of inflammatory cytokines including type-1 interferons and the potent danger signal HMGB1. Here, using human in vitro models, we demonstrate that MV enhances innate antitumour activity, and that MV-mediated melanoma cell death is capable of stimulating a melanoma-specific adaptive immune response.

Combination of a fusogenic glycoprotein, pro-drug activation and oncolytic HSV as an intravesical therapy for superficial bladder cancer.

BACKGROUND: There are still no effective treatments for superficial bladder cancer (SBC)/non-muscle invasive bladder cancer. Following treatment, 20% of patients still develop metastatic disease. Superficial bladder cancer is often multifocal, has high recurrences after surgical resection and recurs after intravesical live Bacillus Calmette-Guérin. Oncovex(GALV/CD), an oncolytic herpes simplex virus-1, has shown enhanced local tumour control by combining oncolysis with the expression of a highly potent pro-drug activating gene and the fusogenic glycoprotein. METHODS: In vitro fusion/prodrug/apoptotic cell-based assays. In vivo orthotopic bladder tumour model, visualised by computed microtomography. RESULTS: Treatment of seven human bladder carcinoma cell lines with the virus resulted in tumour cell killing through oncolysis, pro-drug activation and glycoprotein fusion. Oncovex(GALV/CD) and mitomycin C showed a synergistic effect, whereas the co-administration with cisplatin or gemcitabine showed an antagonistic effect in vitro. Transitional cell cancer (TCC) cells follow an apoptotic cell death pathway after infection with Oncovex(GALV/CD) with or without 5-FC. In vivo results showed that intravesical treatment with Oncovex(GALV/CD) + prodrug (5-FC) reduced the average tumour volume by over 95% compared with controls. DISCUSSION: Our in vitro and in vivo results indicate that Oncovex(GALV/CD) can improve local tumour control within the bladder, and potentially alter its natural history.

Recent clinical experience with oncolytic viruses.

There has been interest in using viruses to treat cancer for over a century. Recent clinical efforts, driven on by significant preclinical advances, have focussed on the safety of using replication-competent viruses. Recently published clinical trials of six oncolytic viruses (adenovirus, reovirus, measles, herpes simplex, Newcastle disease virus and vaccinia) have added to the accumulating data that endorse oncolytic viruses as a safe and well tolerated treatment approach. Conclusive evidence of efficacy remains to be demonstrated, but randomised clinical trials are now underway.

The roles of cell surface attachment molecules and coagulation Factor X in adenovirus 5-mediated gene transfer in pancreatic cancer cells.

Transduction of 11 pancreatic cancer cell lines with a replication-deficient adenovirus 5 expressing enhanced green fluorescent protein (Ad5EGFP) was analyzed and variable EGFP levels were observed, ranging from <1% to ∼40% of cells transduced, depending on the cell line. Efficient Ad5EGFP transduction was associated mainly with higher levels of cell surface Coxsackie and adenovirus receptor (CAR) but not with expression of α(v)ß(3) and α(v)ß(5) integrins and was fiber dependent. Reduction of CAR by RNA interference resulted in a corresponding decrease in Ad5EGFP transduction. Pre-treatment of Ad5EGFP with blood coagulation Factor X increased virus entry even in the presence of low CAR levels generated by RNA interference, suggesting a potential alternative route of Ad5 entry into pancreatic cancer cells. Immunohistochemistry carried out on 188 pancreatic ductal adenocarcinomas and 68 matched controls showed that CAR was absent in 102 (54%) of adenocarcinomas, whereas moderate and strong staining was observed in 58 (31%) and 28 (15%) cases, respectively. Weak or absent CAR immunolabeling correlated with poor histological differentiation of pancreatic cancer. In normal tissue, strong immunolabeling was detected in islet cells and in the majority of inter- and intralobular pancreatic ducts.

Disruption of HOX activity leads to cell death that can be enhanced by the interference of iron uptake in malignant B cells.

The HOX genes encode a family of transcription factors that are dysregulated in several malignancies and have been implicated in oncogenesis and cancer cell survival. Disruption of HOX protein function using the peptide HXR9 has shown anti-tumor effects against melanoma, lung cancer and renal cancer. In this report, we evaluated the expression of all 39 HOX genes in a panel of six malignant B-cell lines, including multiple myeloma cells and found different levels of expression of HOX family members suggesting that they also have a role in malignant B-cell survival. We show that disrupting HOX function using the peptide HXR9 induces significant cytotoxicity in the entire panel of cell lines. Importantly, we found that the cytotoxic effects of HXR9 can be enhanced by combining it with ch128.1Av, an antibody-avidin fusion protein specific for the human transferrin receptor 1 (CD71). Iron starvation induced by the fusion protein contributes to the enhanced effect and involves, at least in part, the induction of a caspase-independent pathway. These results show the relevance of HOX proteins in malignant B-cell survival and suggest that our therapeutic strategy may be effective in the treatment of incurable B-cell malignancies such as multiple myeloma.

Locoregional intravascular viral therapy of cancer: precision guidance for Paris's arrow?

Viral therapy of cancer includes strategies such as viral transduction of tumour cells with 'suicide genes', using viral infection to trigger immune-mediated tumour cell death and using oncolytic viruses for their direct anti-tumour action. However, problems still remain in terms of adequate viral delivery to tumours. A role is also emerging for single-organ isolation and perfusion. Having begun with the advent of isolated limb perfusion for extremity malignancy, experimental systems have been developed for the perfusion of other organs, particularly the liver, kidneys and lungs. These are beginning to be adopted into clinical treatment pathways. The combination of these two modalities is potentially significant. Locoregional perfusion increases the exposure of tumour cells to viral agents. In addition, the avoidance of systemic elimination through the immune and reticulo-endothelial systems should provide a mechanism for increased transduction/infection of target cells. The translation of laboratory research to clinical practice would occur within the context of perfusion programmes, which are already established in the clinic. Many of these programmes include the use of vasoactive cytokines such as tumour necrosis factor-alpha, which may have an effect on viral uptake. Evidence of activation of specific anti-tumour immunological responses by intratumoural and other existing methods of viral administration raises the intriguing possibility of a locoregional therapy, with the ability to affect distant sites of disease. In this review, we examined the state of the literature in this area and summarized current findings before indicating likely areas of continuing interest.

Clinical trials with oncolytic reovirus: moving beyond phase I into combinations with standard therapeutics.

It is time for those working on oncolytic viruses to take stock of the status of the field. We now have at our disposal an array of potential therapeutic agents, and are beginning to conduct early-phase clinical trials in patients with relapsed/metastatic cancers. By drawing on lessons learned during the development of other biological therapies, such as monoclonal antibodies and targeted small molecule inhibitors, we are now in a position to chart the course of the next wave of trials that will go beyond the phase I studies of safety and feasibility. In this article we review our approach to the development of oncolytic viruses as cancer therapeutics. In doing so, we emphasise the fact that this process is modular and involves multiple iterative steps between the laboratory and the clinic. Ultimately, at least in the medium term, the future of oncolytic virotherapy lies in combination regimens with standard anti-cancer agents such as radiation and chemotherapy.

Inhibition of the aquaporin 3 water channel increases the sensitivity of prostate cancer cells to cryotherapy.

Aquaporins (AQPs) are intrinsic membrane proteins that facilitate selective water and small solute movement across the plasma membrane. In this study, we investigate the role of inhibiting AQPs in sensitising prostate cancer cells to cryotherapy. PC-3 and DU145 prostate cancer cells were cooled to 0, -5 and -10 degrees C. The expression of AQP3 in response to freezing was determined using real-time quantitative polymerase chain reaction (RT-qPCR) and western blot analysis. Aquaporins were inhibited using mercuric chloride (HgCl(2)) and small interfering RNA (siRNA) duplex, and cell survival was assessed using a colorimetric assay. There was a significant increase in AQP3 expression in response to freezing. Cells treated with AQP3 siRNA were more sensitive to cryoinjury compared with control cells (P<0.001). Inhibition of the AQPs by HgCl(2) also increased the sensitivity of both cell lines to cryoinjury and there was a complete loss of cell viability at -10 degrees C (P<0.01). In conclusion, we have shown that AQP3 is involved directly in cryoinjury. Inhibition of AQP3 increases the sensitivity of prostate cancer cells to freezing. This strategy may be exploited in the clinic to improve the efficacy of prostate cryotherapy.