Despite advancement in therapies, overall survival rates for relapsed pediatric sarcomas are dismal. Newer therapies are needed to effectively salvage these patients. Oncolytic viruses (such as reovirus) and other genetically altered viruses (such as herpes simplex viruses and adenoviruses) have shown efficacy in a variety of solid tumors including sarcomas. Reolysin is an unmodified oncolytic reovirus that selectively replicates in Ras-activated cancer cells while not causing any significant human illness in its wild form.
By using a panel of pediatric sarcoma cell lines in vitro and flank xenografts in vivo, Reolysin was evaluated as a single agent and in combination with cisplatin and radiation therapy. Electron microscopy and immunohistochemistry was used to demonstrated a cytopathic effect in treated tumors.
Reolysin inhibited the proliferation and viability of sarcoma cell lines at a dose of 1 to 10 virus particles per cell. In vivo, 5 × 109 plaque-forming units (PFU) administered via the tail vein every other day for 3 doses every 21 days inhibited the growth of tumor xenografts with improvement in event-free survival. In the SKES1 Ewing sarcoma line, there was therapeutic enhancement when reovirus was administered in combination with radiation or cisplatin. In the RH30 line and the OS33 line, therapeutic enhancement was demonstrated with radiation and cisplatin, respectively.
Reovirus, or respiratory enteric orphan virus, was termed as such because it is not associated with a known disease state. The Dearing strain of reovirus (serotype 3) is a naturally occurring, ubiquitous, nonenveloped, human virus with a genome consisting of 10 segments of double-stranded RNA.1 Community-acquired reovirus infections are generally considered mild and are limited to the upper respiratory tract and gastrointestinal tract. The cytopathic effect of reovirus is restrained to cells transformed by an activated Ras signaling pathway.2-6 During naturally occurring infections in immunocompetent animals, the pathogenic effects appear to be minimal. The infectious cycle begins when the viral particles are converted in the gastrointestinal (GI) tract to intermediate subviral particles (ISVPs) because of the action of proteolytic enzymes. The reovirus initially infects the epithelial cells of the ileum and is thought to then cross through the intestinal M cells, moving to the Peyer's patches, and then the mesenteric lymph nodes through lymphatic dissemination.3, 4 The virus may eventually transit in the blood stream to extraintestinal organs and the central nervous system.7 Although it can infect many different organs, the reovirus produces very minor illness, if any. Seventy percent to 100% of adults will have antireovirus antibodies indicative of a prior exposure. The specific cytopathic effect of reovirus in Ras-transformed cells coupled with the absence of significant symptoms associated with human infection, make reovirus an attractive anticancer agent.
Reolysin is manufactured, clinical-grade, unmodified, Dearing strain reovirus capable of infecting and lysing a wide range of human tumors.7-12 There are 16 completed or ongoing phase 1 or phase 2 studies of Reolysin in Canada, the United Kingdom, and the United States. There are more than 290 patients receiving single or multiple doses intratumorally or intravenously either as a monotherapy or in combination with radiotherapy or chemotherapy. It is interesting to note that phase 1 dose-escalation studies using intravenous administration as monotherapy or combined with chemotherapies, including gemcitabine, docetaxel, and carboplatin/paclitaxel or combined with radiotherapy, have been completed with no maximum tolerated dose for Reolysin reached at doses up to 3 × 1010 TCID50. However, these results can be definitively addressed only in controlled trials.
Reovirus binds to ubiquitously expressed sialic acid on mammalian cells and is internalized. In reovirus-resistant cells, viral protein transcripts phosphorylate and activate the double-stranded RNA activated protein kinase (PKR). The activated PKR subsequently phosphorylates the α-subunit of the translation initiation factor (eIF-2), which in turn inhibits viral gene translation. The phosphorylation of eIF-2 by PKR protects normal cells from reovirus-mediated lysis. Viral transcripts are generated but not translated in normal cells.13 However, cells transfected with v-erbB, sos, or ras, all of which activate Ras-signaling pathways, are readily lysed by reovirus.14, 15 In cells transformed by activated Ras, or by some other component of the Ras signaling pathway, PKR phosphorylation is either inhibited or reversed. As a result, viral protein synthesis ensues, and the proliferating viral particles ultimately lyse the host cell.13-15
Activated signaling through the Ras pathway is common in pediatric sarcomas. In this article, we demonstrate that Reolysin inhibits tumor growth of a panel of human sarcomas xenografts after systemic administration. Viral particles are noted in tumor cells 24 hours and 48 hours after systemic injection by electron microscopy. Combination therapy with either cisplatin or external beam radiation may provide additional benefit and do not appear to limit the cytopathic effect.
MATERIALS AND METHODS
The tumor cell lines used included the RD and RH30 rhabdomyosarcoma lines (American Type Culture Collection, Manassas, Va); the Saos-2 (American Type Culture Collection, Manassas, Va), and OS187 (generous gift of Richard Gorlick, Albert Einstein College of Medicine, Bronx NY) osteosarcoma lines; SKPNDW and SK-ES1 Ewing sarcoma lines (generous gift of Mark Ladanyi, Memorial Sloan-Kettering Cancer Center, NY, NY); and HSSY-II and SYO-I synovial sarcoma lines (gift of Mark Ladanyi, Memorial Sloan Kettering Cancer Center). The cells were grown in monolayer at 37°C, 5% CO2 in media. Saos-2, OS187, SYO-1, and HSSY-II were grown in Eagle minimum essential medium (MEM), SK-PN-DW, RD, and SKES1 in Dulbecco modified Eagle medium (DME), and RH30 in Roswell Park Memorial Institute medium (RPMI) supplemented with 10% fetal bovine serum (Invitrogen, Carlsbad, Calif), 0.5% penicillin/streptomycin (Invitrogen), and 1% glutamine (Invitrogen). The OS2 and OS33 xenograft lines (a gift of Peter J. Houghton, St. Jude Children's Research Center, Memphis, TN) are tumor lines maintained by serial passage in mice as previously described.16
Reolysin was provided by Oncolytic Biotech (Calgary, Alberta, Canada). Cisplatin was purchased from Sigma Aldrich (St. Louis, Mo) and resuspended in phosphate-buffered saline.
Cell Proliferation Assay
Growth inhibition was determined by the microculture tetrazolium method. Briefly, cells were seeded in 96-well, flat-bottomed, microtiter plates at a density of 500 cells/well in 100 μL of media. After overnight incubation, 100 μL of media containing Reolysin was added to achieve specified final concentrations and a final volume of 200 μL/well. At 120 hours, the relative metabolic activities of treated and untreated cells were measured by mitochondrial conversion of 3-(4,5-dimethylthiazon-2-yl)-2,5-diphenyl tetrazolium bromide (MTT; Sigma, St. Louis, Mo) to formazine. At the completion of the drug treatment, 250 μg of MTT was added to each well and incubated at 37°C, 5% CO2 for 6 hours. Formazine crystals were dissolved in dimethyl sulfoxide and optical density at 595 nm measured on a VERSAmax spectrophotometer (Molecular Devices, Sunnyvale, Calif). Absorbance values were normalized to the values obtained for the vehicle-treated cells to determine the percentage of survival. The IC50 was defined as the concentration at which absorbance of the treated cells was 50% that of the controls. All experiments were performed in triplicate, and the data are the mean of 2 separate experiments.
Murine Xenograft Models
Athymic nu/nu mice (Harlan Sprague Dawley, Indianapolis, Ind) were implanted with subcutaneous flank tumors. For cell lines RH30, RD, SKES1, SK-PN-DW, HSSY-II, and SYO-1, cultured cells were harvested with trypsin/EDTA and resuspended in 50% Matrigel (Becton Dickinson, San Jose, Calif) at a concentration of 1 × 106 cells/0.1 mL. At 5 to 6 weeks of age, the mice were injected with 0.1 mL of cells suspended in Matrigel. The OS2 and OS33 tumor lines were established at St. Jude Children's Cancer Research Hospital and have been described previously.16 For transplantation with OS2 and OS33 tumor, mice were anesthetized with 4% isoflurane. A small incision was made in the flank of the mouse, and a 4-mm by 4-mm section of tumor implanted subcutaneously.
When the tumors were approximately 0.4 to 0.5 cm in diameter, tumor-bearing mice were randomized into groups of 8 mice with 1 treatment group and 1 control group. Treated mice received Reolysin 5 × 109 plaque-forming units (PFU) in 100 μL phosphate-buffer saline (PBS) or vehicle control every other day for 3 days starting on Day 1 and Day 22 as a tail vein injection. Assuming a spherical tumor, the volume was determined by the formula: mm3 = π × (D) × d2/6, where D is the maximal diameter and d is the diameter perpendicular to D. Volumes are expressed as relative tumor volumes (RTV), where the tumor volume at any given time point is divided by the starting tumor volume. The RTV for treated and control mice were measured a minimum of once per week. All experiments were conducted by using protocols and conditions approved by the Albert Einstein College of Medicine Institutional Animal Care and Use Committee.
Assessment of Tumor Response and Statistical Considerations
By criteria defined previously by Houghton et al.,16 progressive disease is defined as <50% regression from original tumor volume for the entire study period (RTV >0.5) and >25% increase in tumor volume at the end of the study period (RTV >1.25). Stable disease is defined as tumor regression that does not exceed 50% of the original tumor volume throughout the entire study period (RTV >0.5) and <25% increase in tumor volume at the end of the study period (RTV <1.25). A partial response is defined as greater than 50% regression in tumor volume (RTV <0.5) but with a measurable tumor mass of greater than 0.10cm.3 Loss of measurable tumor mass (<0.10cm3) at any point during the treatment period (6 weeks) was defined as a complete response (CR). A maintained CR was defined as a loss of measurable tumor mass (<0.10cm3) at any point after initiation of therapy without regrowth during the 6-week study period.
Statistical analysis was based on event-free survival (EFS). An event is defined as a relative tumor volume of 4× (ie, quadruple the starting tumor size) or death. EFS is defined as the time from the initiation of the study to an event. For those tumors not reaching an event by 6 weeks, the end of the study period, the time to event is defined as Day 43. For tumors reaching an event before Day 22, the RTV at Day 22 was calculated using the actual tumor volume at the time of the event. RTV values were compared with the nonparametric Wilcoxon rank-sum test with Bonferroni correction. The survival analysis for each treatment was determined by using the Kaplan-Meier method. Pairwise comparisons of the Kaplan-Meier curves were performed by a log-rank test with Bonferroni correction. The difference was considered to be significant when the P value was <.05. In combination testing, a treatment minus control (T-C) value is calculated from the median time to event for each group. The combination is considered to provide therapeutic enhancement when the T-C value for the combination is greater that the T-C for both single treatments evaluated and the EFS distributions for the single treatments are significantly better (P<0.05).17
Electron Microscopy Scanning
Electron microscopy was performed by JEOL (Jeol Ltd, Tokyo, Japan) 100CXII microscope on thin sections of tumor at specified time points. Greater than 100 high-powered fields (×54,000) for each tumor and vehicle controls were scanned for evidence of reovirus infection 24 and 48 hours after systemic administration of the virus.
Briefly, mice were anesthetized with 45 mg/kg pentobarbital sodium (Nembutal) then placed into a poly(methyl methacrylate) (Lucite) jig with 0.5 cm of lead body protection and individualized compartments through which a circular port was accessible for localized flank irradiation (20 Gy). A 40 MGC Philips (Best, Netherlands and Andover, Massachusetts) orthovoltage unit, operating at 320 kVp, 5 mA, and 0.5 mm copper filtration were used. All dose calculations are based on thermoluminescence dosimetry at a midline phantom within the jig.
Detection of apoptosis
Terminal uridine deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay was done on paraffin-embedded tissue sections using the apoptosis detection kit (Chemicon International, Temecula, Calif). Primary tumors from the control and the treated mice were harvested at the specified time points and fixed with formaldehyde. Paraffin-embedded tissue sections were deparaffinized with xylene and serial washes of ethanol. Endogenase peroxidase was quenched with 3% hydrogen peroxide. The slides were incubated with TdT enzyme for 1 hour at 37°C. The reaction was stopped with the stop buffer provided in the kit. Antidigoxigenin conjugate was added for 30 minutes at room temperature. Slides were then washed with PBS, and peroxidase substrate was added for 5 minutes at room temperature. Slides were counterstained with hematoxylin and then mounted with a coverslip. The number of brown-staining nuclei were visually analyzed under light microscopy, magnification ×400.
Reolysin Inhibited the Proliferation and Viability of Pediatric Solid Tumor Cell Lines
In the each of the rhabdomyosarcoma, Ewing sarcoma, osteosarcoma, and synovial sarcoma lines, Reolysin resulted in growth inhibition (Table 1). Cell viability in all cell lines approached 0 after 120 hours of continuous exposure to virus at a dose of 1 to 10 virus particles per cell (PFU of 1 to 10).
Table 1. Effect of REOLYSIN on Cells Cultured In Vitro
Plaque forming unit (pfu) is calculated at the time the cells are plated and refers to the number of virus particles per cells initially plated.
Systemic Administration of Reolysin Inhibits Growth of Heterotopic Human Tumor Implants in Severe Combined Immunodeficiency (SCID) Mice
In the rhabdomyosarcoma, Ewing sarcoma, synovial sarcoma, and osteosarcoma tumors, Reolysin demonstrated significant antitumor activity in xenograft models when compared with the untreated controls (Fig. 1). A partial response was seen in mice bearing the RH30 and SKES1 tumor implants. Stable disease was seen in OS33, and progressive disease in OS2 and HSSY-II. There was a statistically significant difference in the RTV at Day 22 (the first day of the second course of treatment) in all tumor lines. Furthermore, there was a statistically significant improvement in the EFS in all tumors. The response data are summarized in Table 2.
Table 2. Response Analysis for REOLYSIN® in Solid Tumor Xenografts
Kaplan-Meier Estimate of Median Time to Event
Day 22 Tumor Volume (treated/control)
EP: Experimental Period.
By Electron Microscopy (EM), Reovirus Is Identified in Cells 24 and 48 Hours After Systemic Administration
In the RH30 and OS33 lines, reovirus particles are visible by electron microscopy 24 hours (Fig. 2a) and 48 hours after systemic administration of Reolysin (Fig. 2b). These data confirm infection of tumor cells after intravenous administration of virus.
Reolysin Is Effective in Combination With DNA Damaging Agents
Given the finding that reovirus requires functional cellular transcription machinery to replicate, we evaluated the effect of Reolysin in combination with a DNA-damaging agent, cisplatin, and with radiation therapy. For these experiments, we used a reduced dose of Reolysin of 1 × 109 PFU administered via tail vein injection on Days 1, 3, and 5 only. In the SKES1 line (Fig. 3a), each Reolysin and cisplatin alone yielded progressive disease, whereas a complete response was seen with the combination. In the OS33 line, Reolysin alone yielded stable disease, cisplatin progressive disease, and the combination a partial response (Fig. 3b). Minimal or no effect was seen when Reolysin was used in combination with cisplatin in the HSSY-II line (Fig. 3c). The combination met criteria for therapeutic enhancement in both SKES1 and OS33 (Table 3).
Table 3. Response Analysis for Combination of REOLYSIN and Cisplatin in Solid Tumor Xenografts
K-M estimated Median Time to Event (T-C value)
p-value Combination vs Reovirus
p-value Combination vs Cisplatin
EP is Experimental Period. The median time to event is defined as 42 days if no event noted at the end of the experimental period.
Mice bearing RH30 rhabdomyosarcoma and SKES1 Ewing sarcoma xenografts were treated with Reolysin at a dose of 1 × 109 PFU on Days 1, 3, and 5. Five fractions of 4 Gy each were administered to the mice also starting on Day 1 for 4 consecutive days (total dose is 20 Gy). Progressive disease was seen in both tumor lines for Reolysin and radiation alone. However, in the RH30 xenograft, a complete response was seen after the combination therapy and a partial response with the combination in the SKES1 tumor (Fig. 4). The combination met criteria for therapeutic enhancement in both RH30 and SKES1 (Table 4).
Table 4. Response Analysis for Combination of REOLYSIN and Radiation in Solid Tumor Xenografts
K-M estimated Median Time to Event (T-C value)
p-value Combination vs Reovirus
p-value Combination vs Radiation
EP is Experimental Period. The median time to event is defined as 42 days if no event noted at the end of the experimental period.
Increased Degree of Apoptosis Is Seen in the Tumors Treated With the Combination of Reolysin and Radiation
RH30 and SKES1 tumors treated with the combination of Reolysin and radiation showed an increased number of apoptotic nuclei in comparison to the controls and to the individual treatment groups (Figs. 5a, 5b).
Viral oncolysis is defined as the killing of a cancer cell by infection and replication of a virus inside the cell. Exciting developments have been made in this field since its discovery in 1903. The major challenge in the development of these therapies in patients was the high rate of infectious complications resulting from the use of wild-type viruses. With the advent of recombinant DNA technology, it became possible to create attenuated virus mutants that were harmless to humans but still capable of infecting and killing tumor cells. Since then, several such viral mutants have been developed and are in clinical trials in cancer patients. A few of these include: human herpes virus (HSV1716), vaccinia virus (JX-594), Seneca Valley virus (SVV-001), and adenovirus (ONYX 015).18-21 Reovirus (Reolysin) is unique in the category of oncolytic viruses as being the only virus used in its wild form in patients.
Normally in cells infected with reovirus, the double-stranded RNA-activated protein kinase (PKR) is autophosphorylated in the presence of viral transcripts thus inhibiting viral protein synthesis and replication. Ras-activated cells inhibit the autophosphorylation of PKR, keeping it in an inactive state and allowing viral translation and eventual oncolysis to take place.6 The selective oncolysis of cells with increased signaling through Ras and the minimal effect on normal tissues makes Reolysin an interesting candidate for oncolytic viral therapy. We demonstrated that Reolysin has activity after systemic administration in a panel of pediatric sarcomas implanted in Athymic nu/nu mice and that this oncolytic effect is not inhibited when combined with DNA-damaging therapies (cisplatin and radiation). In fact, there is evidence of therapeutic enhancement of the combination in 3 of the 4 tumor lines evaluated.
Ras may be activated through mutations in the Ras proto-oncogene, upstream mitogenic signals such as tyrosine receptor kinases, or overexpression of growth-factor receptors. Regardless of the mechanism, 70% of cancers have activated Ras either by mutation or overexpression of upstream or downstream signaling regulators. However, it is not certain that Ras is the only intracellular signaling pathway capable of inhibiting autophosphorylation of PKR. For this reason, interrogation of the Ras pathway is not a focus of this article. In translating these results into the clinical setting, a valid biomarker for Reolysin activity has yet to be defined. Ras mutations are rare in pediatric malignancies, and down-stream proteins such as MAPK may be activated through multiple mechanisms. It will be an aim of a proposed pediatric phase 1 trial of Reolysin to help identify molecular determinants of Ras activity.
Previously, Reolysin was reported to have antitumor activity in metastatic human colon and ovarian xenografts in mice after systemic administration.9 In each of 7 sarcoma cell lines tested, Reolysin inhibited their proliferation and viability at a low number of virus particles per cell (1-10 PFU). Given the exponential growth of the tumor cells in culture, this implies efficient infection and replication of the virus in these pediatric tumors in vitro. In vivo, the RH30 rhabdomyosarcoma line and the SKES1 Ewing sarcoma line, treatment with Reolysin by systemic administration yielded partial responses. Stable disease is noted in the OS2 osteosarcoma xenograft. In all 5 lines evaluated, there was a statistically significant increase in the event-free survival in Reolysin-treated tumors. The capacity of the Reolysin to selectively infect tumor cells after systemic administration was confirmed by the demonstration of viral particles in tumor cells, by electron microscopy, 24 and 48 hours after tail vein injection (Fig. 2a, 2b). On the basis of these data, it is not known whether virus may spread cell to cell after oncolysis.
Enhanced in vitro and in vivo cytotoxic effects of combination of Reolysin with radiation therapy22 and Reolysin with chemotherapeutic agents23 has previously been reported in a variety of adult cancer cell lines and xenografts. It is easy to speculate that DNA-damaging agents commonly used in treatment of malignancies may alter viral replication and oncolysis. In the our study, the use of DNA-damaging agents in combination with Reolysin did not adversely affect the oncolytic capacity of the Reolysin. In 3 tumors lines treated with a single dose of cisplatin and 3 doses of Reolysin, there was therapeutic enhancement in the SKES1 Ewing sarcoma line. In the OS33 tumor line, there was a trend toward therapeutic enhancement, but Reolysin alone induced tumor growth delay beyond the 6-week experimental period. In the HSSY-II synovial sarcoma line, there was no therapeutic enhancement when Reolysin was combined with cisplatin. At a dose of 1 × 109 PFU for this combination study, Reolysin had little effect on the HSSY-II xenograft, and cisplatin provided no therapeutic enhancement. There was therapeutic enhancement and an increase in apoptotic nuclei in the RH30 rhabdomyosarcoma and the SKES1 Ewing sarcoma xenografts when 20 Gy radiation administered in 4 fractions was combined with 1 × 109 PFU Reolysin administered every other day for 3 doses.
In the multiple completed or ongoing phase 1 and phase 2 studies of Reolysin, in adult cancer patients worldwide, it has been used both intralesionally and via systemic administration, either alone or in combination with other cytotoxic agents. Intralesional delivery of Reolysin was evaluated in 3 phase 1 trials of advanced cancers in adults including prostate cancer and malignant gliomas.10 No dose-limiting toxicities were seen in these patients. Major adverse events (AEs) were grade 1 or 2 fever, myalgias, arthralgias, headaches, seizures (in patients with malignant gliomas), and elevation of alanine transaminase. Two recent phase 1 trials of systemic administration of Reolysin in adults with advanced cancers did not show any dose limiting toxicities. The major adverse events associated with Reolysin were, again, mild to moderate flu-like symptoms (such as fever, chills, headache, fatigue, rhinorrhea, and cough) as well as GI symptoms, including nausea, vomiting, and diarrhea. Moderate and transient alterations in hepatic function tests and hematology values were also observed.24, 25 A phase 2 study of intravenous Reolysin in adult patients with bone sarcomas metastatic to the lungs showed that 14 (42%) patients had stable disease for more than 2 months, 5 of whom had stable disease for more than 6 months.26 These results met the statistical endpoint for the trial of 3 or more patients having prolonged stabilization of disease for more than 6 months. However, these clinical data are less impressive than what would have been expected from the preclinical results. One of the reasons for this observation may be the finding that most humans have neutralizing antibodies against reovirus from past infections that might hinder the effect of reovirus by enhanced clearance of the virus by the host immune system. Although pre-existing antibodies have been reported not to interfere with the antitumor activity of reovirus in immunocompetent C3H mice when administered intratumorally,13 a recent report shows that systemic administration of reovirus in immunized mice was ineffective in inhibiting tumor growth.7 Combination of reovirus with immunosuppressive therapy in these mice completely restored the ability of the virus to inhibit tumor growth. Similarly, combination of reovirus with drugs such as cyclophosphamide is also shown to enhance antitumor activity likely because of suppression of the host immune system and decrease in neutralizing viral antibodies.27 Thus, this approach of immune suppression in combination with reoviral therapy may lead to better inhibition of tumor growth in patients and is currently being evaluated in adult and pediatric phase 1/2 trials. In addition, a double-blind, randomized, multicenter, phase 3 study (REO 018) is being initiated in North America and Europe.
In summary, in this article, we have demonstrated that Reolysin is able to infect and lyse pediatric sarcoma cells in vitro and to inhibit growth of sarcoma xenografts. These data suggest that Reolysin may be a promising target therapy in pediatric cancers and should be further evaluated in clinical trials. A phase 1 trial in pediatric patients with advanced malignancies is currently being planned.
CONFLICT OF INTEREST DISCLOSURES
This research work was supported in part by a grant from Oncolytics Biotech Inc, Calgary, Alberta, Canada.