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.

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.

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.

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.

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.

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.

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.

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.

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.

Dendritic cells and T cells deliver oncolytic reovirus for tumour killing despite pre-existing anti-viral immunity.

Reovirus is a naturally occurring oncolytic virus currently in early clinical trials. However, the rapid induction of neutralizing antibodies represents a major obstacle to successful systemic delivery. This study addresses, for the first time, the ability of cellular carriers in the form of T cells and dendritic cells (DC) to protect reovirus from systemic neutralization. In addition, the ability of these cellular carriers to manipulate the subsequent balance of anti-viral versus anti-tumour immune response is explored. Reovirus, either neat or loaded onto DC or T cells, was delivered intravenously into reovirus-naive or reovirus-immune C57Bl/6 mice bearing lymph node B16tk melanoma metastases. Three and 10 days after treatment, reovirus delivery, carrier cell trafficking, metastatic clearance and priming of anti-tumour/anti-viral immunity were assessed. In naive mice, reovirus delivered either neat or through cell carriage was detectable in the tumour-draining lymph nodes 3 days after treatment, though complete clearance of metastases was only obtained when the virus was delivered on T cells or mature DC (mDC); neat reovirus or loaded immature DC (iDC) gave only partial early tumour clearance. Furthermore, only T cells carrying reovirus generated anti-tumour immune responses and long-term tumour clearance; reovirus-loaded DC, in contrast, generated only an anti-viral immune response. In reovirus-immune mice, however, the results were different. Neat reovirus was completely ineffective as a therapy, whereas mDC--though not iDC--as well as T cells, effectively delivered reovirus to melanoma in vivo for therapy and anti-tumour immune priming. Moreover, mDC were more effective than T cells over a range of viral loads. These data show that systemically administered neat reovirus is not optimal for therapy, and that DC may be an appropriate vehicle for carriage of significant levels of reovirus to tumours. The pre-existing immune status against the virus is critical in determining the balance between anti-viral and anti-tumour immunity elicited when reovirus is delivered by cell carriage, and the viral dose and mode of delivery, as well as the immune status of patients, may profoundly affect the success of any clinical anti-tumour viral therapy. These findings are therefore of direct translational relevance for the future design of clinical trials.

HOX transcription factors are potential therapeutic targets in non-small-cell lung cancer (targeting HOX genes in lung cancer).

The HOX genes are a family of homeodomain-containing transcription factors that determine the identity of cells and tissues during embryonic development. They are also known to behave as oncogenes in some haematological malignancies. In this study, we show that the expression of many of the HOX genes is highly elevated in primary non-small-cell lung cancers (NSCLCs) and in the derived cell lines A549 and H23. Furthermore, blocking the activity of HOX proteins by interfering with their binding to the PBX co-factor causes these cells to undergo apoptosis in vitro and reduces the growth of A549 tumours in vivo. These findings suggest that the interaction between HOX and PBX proteins is a potential therapeutic target in NSCLC.

Inflammatory tumour cell killing by oncolytic reovirus for the treatment of melanoma.

Reovirus is a promising unmodified double-stranded RNA (dsRNA) anti-cancer oncolytic virus, which is thought to specifically target cells with activated Ras. Although reovirus has been tested in a wide range of preclinical models and has entered early clinical trials, it has not previously been tested for the treatment of human melanoma. Here, we show that reovirus effectively kills and replicates in both human melanoma cell lines and freshly resected tumour; intratumoural injection also causes regression of melanoma in a xenograft in vivo model. Reovirus-induced melanoma death is blocked by caspase inhibition and is dependent on constituents of the Ras/RalGEF/p38 pathway. Reovirus melanoma killing is more potent than, and distinct from, chemotherapy or radiotherapy-induced cell death; a range of inflammatory cytokines and chemokines are released by infected tumour cells, while IL-10 secretion is abrogated. Furthermore, the inflammatory response generated by reovirus-infected tumour cells causes bystander toxicity against reovirus-resistant tumour cells and activates human myeloid dendritic cells (DC) in vitro. Hence, reovirus is suitable for clinical testing in melanoma, and may provide a useful danger signal to reverse the immunologically suppressive environment characteristic of this tumour.

Characterization of the adaptive and innate immune response to intravenous oncolytic reovirus (Dearing type 3) during a phase I clinical trial.

There is an emerging realization from animal models that the immune response may have both detrimental and beneficial therapeutic effects during cancer virotherapy. However, there is a dearth of clinical data on the immune response to viral agents in patients. During a recently completed phase I trial of intravenous reovirus type 3 Dearing (RT3D), heavily pretreated patients with advanced cancers received RT3D at doses escalating from 1 x 10(8) tissue culture infectious dose-50 (TCID(50)) on day 1 to 3 x 10(10) TCID(50) on 5 consecutive days of a 4 weekly cycle. A detailed analysis of the immune effects was conducted by collecting serial clinical samples for analysis of neutralizing anti-reoviral antibodies (NARA), peripheral blood mononuclear cells (PBMC) and cytokines. Significant increases in NARA were seen with peak endpoint titres >1/10 000 in all but one patient. The median fold increase was 250, with a range of 9-6437. PBMC subset analysis showed marked heterogeneity. At baseline, CD3+CD4+ T cells were reduced in most patients, but after RT3D therapy their numbers increased in 47.6% of patients. In contrast, most patients had high baseline CD3+CD8+ T-cell levels, with 33% showing incremental increases after therapy. In some patients, there was increased cytotoxic T-cell activation post-therapy, as shown by increased CD8+perforin/granzyme+ T-cell numbers. Most patients had high numbers of circulating CD3-CD56+ NK cells before therapy and in 28.6% this increased with treatment. Regulatory (CD3+CD4+CD25+) T cells were largely unaffected by the therapy. Combined Th1 and Th2 cytokine expression increased in 38% of patients. These data confirm that even heavily pretreated patients are capable of mounting dynamic immune responses during treatment with RT3D, although these responses are not clearly related to the administered virus dose. These data will provide the basis for future studies aiming to modulate the immune response during virotherapy.

Escherichia coli nitroreductase plus CB1954 enhances the effect of radiotherapy in vitro and in vivo.

Escherichia coli nitroreductase (NTR) converts the prodrug CB1954 (5-(aziridin-1-yl)-2,4-dinitrobenzamide) into a bifunctional alkylating agent that causes DNA crosslinks. In this study, the ability of NTR to enhance the combined effects of CB1954 and radiation has been tested in vitro and in vivo. Stably transduced ovarian cancer cells (SKOV3-NTR) that are sensitive to CB1954 (IC(50)=0.35 muM) demonstrated enhanced cytotoxicity when treated with CB1954 and single-fraction irradiation. The NTR-CB1954 system mediated a bystander effect in combination with radiation on transfer of conditioned medium from SKOV3-NTR, but not SKOV3, cells to SW480 target cells. The ability of CB1954 to enhance radiation-induced cytotoxicity in SKOV3-NTR (but not SKOV3) cells was also demonstrated by fluorescence-activated cell sorting (FACS) with dual staining for propidium iodide/fluorescein diacetate, 4',6-diamidino-2-phenylindole dichloride staining of apoptotic cells and measurement of double-stranded DNA breaks by FACS and confocal microscopy for gammaH2AX foci. Adenoviral delivery of NTR, under constitutive cytomegalovirus or tissue-specific CTP1 promoters, increased the in vitro cytotoxicity of CB1954 plus radiation in MTT and clonogenic assays. Finally, adenoviral delivery of NTR plus CB1954 enhanced the effect of fractionated radiotherapy (12 Gy in four fractions) in SW480 xenograft tumours in nude mice.

An interleukin-10 promoter polymorphism may influence tumor development in renal cell carcinoma.

PURPOSE: Polymorphisms in the promoter of the interleukin-10 (IL-10) gene may influence tumor development by altering the levels of IL-10 present in the serum or tumor microenvironment. In this study we looked for evidence of specific polymorphisms of the IL-10 promoter and whether lymphocyte expression of IL-10 correlates with specific genotypes. MATERIALS AND METHODS: Archival, paraffin embedded renal cell carcinoma tissue from 166 patients and 161 controls were genotyped for the IL-10-1082 single nucleotide polymorphism using real-time polymerase chain reaction. IL-10 protein expression in peripheral blood lymphocytes was assessed by standard enzyme-linked immunoassay in 32 patients with renal cancer. RESULTS: Patient-to-control comparisons identified the AA genotype to be significantly greater in patients with renal cell carcinoma (44% vs 30%, p <0.05). However, study of IL-10 protein expression in peripheral blood lymphocytes from patients with renal cancer showed no statistical difference in IL-10 expression among the GG, AA or AG genotypes. CONCLUSIONS: We found that there was a significantly larger proportion of patients with renal cell carcinoma with the AA homozygous genotype than in a normal population cohort. This result is in accordance with those in previous studies of prostate cancer and cutaneous malignant melanoma. In contrast to previous studies of other tumor types, no correlation could be established between IL-10-1082 polymorphism and serum IL-10.

Signet ring cell carcinoma in the prostate.

Metastases to the prostate gland are rare and often found in the context of widespread metastatic disease. We report an unusual case of primary gastric signet ring cell adenocarcinoma (SRCC) diagnosed over 1 year after treatment for metastatic disease in the prostate.

Coxsackie B and adenovirus receptor, integrin and major histocompatibility complex class I expression in human prostate cancer cell lines: implications for gene therapy strategies.

Gene therapy strategies based on modifying tumour cells using high efficiency adenoviral vectors have shown promise in the clinic. Recently the Coxsackie and adenovirus receptor (CAR) has been shown to mediate adenoviral entry into tumour cells, although previous studies also suggested a role for MHC class I heavy chain. Detailed evaluation of the expression of both CAR and MHC class I in prostate cancer cell lines would have important implications for therapeutic strategies. We have found that, unlike cell lines derived from other malignancies, in human and murine prostate cancer loss of CAR expression appears to be relatively infrequent and does not correlate with loss of MHC class I expression. These findings, together with the demonstration of appreciable levels of cell-surface expression of integrins, suggest that cancer vaccine strategies based on modifying whole prostate cancer cells should be feasible using the current generation of recombinant adenoviral vectors, without deleterious effects on either the virus vector or the target cell.