A Phase I Trial of Intratumoral Administration of Reovirus in Patients With Histologically Confirmed Recurrent Malignant Gliomas
Authors: Peter Forsyth, Gloria Roldán, David George, Carla Wallace, Cheryl Ann Palmer, Don Morris, Gregory Cairncross, Maureen Vallee Matthews, James Markert, Yancey Gillespie, Matt Coffey, Brad Thompson, Mark Hamilton
Journal: Molecular Therapy (2008)

Reovirus is an oncolytic virus with activity in in vivo models of malignant gliomas (MGs). The primary aims were to determine the dose‐limiting toxicity (DLT) and maximum tolerated dose (MTD) of intratumoral administration of reovirus in patients with recurrent MGs. Response, survival, and time to progression (TTP) were secondary aims. Patients were adults, had Karnofsky Performance score ≥ 60, received prior radiotherapy with or without chemotherapy, and had up to the third recurrence of MG. Reovirus was administered intratumorally stereotactically at 1 × 107, 1 × 108, or 1 × 109 tissue culture infectious dose 50 (TCID50) in a volume of 0.9 ml. Twelve patients were treated at three dose levels (3, 6, and 3 patients, respectively). Seven were men, median Karnofsky Performance score was 80, and median age was 53.5 years. There were no grade III or IV adverse events (AEs) definitely or probably related to treatment. Ten patients had tumor progression, one had stabilization, and one was not evaluable for response. Median survival was 21 weeks (range, 6–234), and one is alive 54 months after treatment. Median TTP was 4.3 weeks (range, 2.6–39). An MTD was not reached. The intratumoral administration of the genetically unmodified reovirus was well tolerated using these doses and schedule, in patients with recurrent MG. Reovirus is an oncolytic virus with activity in in vivo models of malignant gliomas (MGs). The primary aims were to determine the dose‐limiting toxicity (DLT) and maximum tolerated dose (MTD) of intratumoral administration of reovirus in patients with recurrent MGs. Response, survival, and time to progression (TTP) were secondary aims. Patients were adults, had Karnofsky Performance score ≥ 60, received prior radiotherapy with or without chemotherapy, and had up to the third recurrence of MG. Reovirus was administered intratumorally stereotactically at 1 × 107, 1 × 108, or 1 × 109 tissue culture infectious dose 50 (TCID50) in a volume of 0.9 ml. Twelve patients were treated at three dose levels (3, 6, and 3 patients, respectively). Seven were men, median Karnofsky Performance score was 80, and median age was 53.5 years. There were no grade III or IV adverse events (AEs) definitely or probably related to treatment. Ten patients had tumor progression, one had stabilization, and one was not evaluable for response. Median survival was 21 weeks (range, 6–234), and one is alive 54 months after treatment. Median TTP was 4.3 weeks (range, 2.6–39). An MTD was not reached. The intratumoral administration of the genetically unmodified reovirus was well tolerated using these doses and schedule, in patients with recurrent MG. IntroductionThe current treatments for patients with malignant gliomas (MGs) are inadequate: The median survival for patients with glioblastoma multiforme (GBM), the most common MG, is only ∼1 year. Therefore, novel treatment strategies are needed. A number of replication‐competent oncolytic viruses have been selected or engineered as novel therapeutics for cancer.1Gromeier M Wimmer E Viruses for the treatment of malignant glioma.Curr Opin Mol Ther. 2001; 3: 503-508PubMed Google Scholar,2Parato KA Senger D Forsyth PA Bell JC Recent progress in the battle between oncolytic viruses and tumours.Nat Rev Cancer. 2005; 5: 965-976Crossref PubMed Scopus (449) Google Scholar These viruses selectively infect and kill tumor cells but leave normal cells unaffected (or relatively so) by exploiting genetic defects that are unique to tumor cells. For several oncolytic viruses (e.g., reovirus, herpes simplex virus, vesicular stomatitis virus) tumor cells have defects in a number of antiviral pathways, which render them susceptible to viral infection. Approximately 20 oncolytic viruses are being tested in clinical trials for a variety of cancers.2Parato KA Senger D Forsyth PA Bell JC Recent progress in the battle between oncolytic viruses and tumours.Nat Rev Cancer. 2005; 5: 965-976Crossref PubMed Scopus (449) Google Scholar For recurrent MGs in particular,3Shah AC Benos D Gillespie GY Markert JM Oncolytic viruses: clinical applications as vectors for the treatment of malignant gliomas.J Neurooncol. 2003; 65: 203-226Crossref PubMed Scopus (108) Google Scholar clinical trials using herpes simplex virus,4Markert JM Medlock MD Rabkin SD Gillespie GY Todo T Hunter WD et al.Conditionally replicating herpes simplex virus mutant, G207 for the treatment of malignant glioma: results of a phase I trial.Gene Ther. 2000; 7: 867-874Crossref PubMed Scopus (810) Google Scholar,5Rampling R Cruickshank G Papanastassiou V Nicoll J Hadley D Brennan D et al.Toxicity evaluation of replication‐competent herpes simplex virus (ICP null mutant 1716) in patients with recurrent malignant gliomas.Gene Ther. 2000; 7: 859-866Crossref PubMed Scopus (516) Google Scholar,6Papanastassiou V Rampling R Fraser M Petty R Hadley D Nicoll J et al.The potential for efficacy of the modified (ICP 34.5(–)) herpes simplex virus HSV1716 following intratumoural injection into human malignant glioma: a proof of principle study.Gene Ther. 2002; 9: 398-406Crossref PubMed Scopus (272) Google Scholar,7Harrow S Papanastassiou V Harland J Mabbs R Petty R Fraser M et al.HSV1716 injection into the brain adjacent to tumour following surgical resection of high‐grade glioma: safety data and long‐term survival.Gene Ther. 2004; 11: 1648-1658Crossref PubMed Scopus (278) Google Scholar Newcastle disease virus,8Csatary LK Gosztonyi G Szeberenyi J Fabian Z Liszka V Bodey B et al.MTH‐68/H oncolytic viral treatment in human high‐grade gliomas.J Neurooncol. 2004; 67: 83-93Crossref PubMed Scopus (152) Google Scholar,9Freeman AI Zakay‐Rones Z Gomori JM Linetsky E Rasooly L Greenbaum E et al.Phase I/II trial of intravenous NDV‐HUJ oncolytic virus in recurrent glioblastoma multiforme.Mol Ther. 2006; 13: 221-228Abstract Full Text Full Text PDF PubMed Scopus (283) Google Scholar and adenovirus10Chiocca EA Abbed KM Tatter S Lous DN Hochberg FH Barker F et al.A phase 1 open‐label, dose escalation, multi‐institutional trial of injection with an E1B‐attenuated adenovirus, ONYX‐015, into the peritumoral region of recurrent malignant gliomas, in the adjuvant setting.Mol Ther. 2004; 10: 958-966Abstract Full Text Full Text PDF PubMed Scopus (331) Google Scholar have been reported; trials using measles virus, poliovirus, and vaccinia virus are planned/in progress.We discovered that reovirus (respiratory enteric orphan), a double‐stranded RNA virus commonly isolated from the respiratory and gastrointestinal tracts of humans, infects and lyses tumors cells but not normal cells.11Strong J Coffey M Tang D Sabinin P Lee PW et al.The molecular basis of viral oncolysis: usurpation of the Ras signaling pathway by reovirus.EMBO J. 1998; 17: 3351-3362Crossref PubMed Scopus (449) Google Scholar,12Coffey MC Strong JE Forsyth PA Lee PW Reovirus therapy of tumors with activated Ras pathway.Science. 1998; 282: 1332-1334Crossref PubMed Scopus (637) Google Scholar,13Alain T Kim TS Lun X Liacini A Schiff LA Senger DL et al.Proteolytic disassembly is a critical determinant for reovirus oncolysis.Mol Ther. 2007; 15: 1512-1521Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar,14Wilcox ME Yang W Senger D Rewcastle NB Morris DG Brasher PM et al.Reovirus as an oncolytic agent against experimental human malignant gliomas.J Natl Cancer Inst. 2001; 93: 903-912Crossref PubMed Scopus (192) Google Scholar,15Hirasawa K Nishikawa SG Norman KL Alain T Kossakowska A Lee PW Oncolytic reovirus against ovarian and colon cancer.Cancer Res. 2002; 62: 1696-1701PubMed Google Scholar,16Hirasawa K Nishikawa SG Norman KL Coffey MC Thompson BG Yoon CS et al.Systemic reovirus therapy of metastatic cancer in immune‐competent mice.Cancer Res. 2003; 63: 348-353PubMed Google Scholar Reovirus does not cause disease in humans (hence its designation as "orphan") but can produce a lethal infection in neonatal17Tyler K Fields BN Reoviruses.in: Fields BN Knipe DM Howley PM Hanock RM Melnick JL Monath TP Virology. Lippincott‐Raven, Philadelphia, PA1996: 1597-1623Google Scholar and severe combined immunodeficiency nonobese diabetic18Sherry B Li XY Tyler KL Cullen JM Virgin HW IV Lymphocytes protect against and are not required for reovirus‐induced myocarditis.J Virol. 1993; 67: 6119-6124PubMed Google Scholar mice. Reovirus uses the activated ras signaling pathways (or its downstream elements) of host tumor cells to enhance proteolytic viral disassembly and therefore targets malignant tumor cells with activated ras.11Strong J Coffey M Tang D Sabinin P Lee PW et al.The molecular basis of viral oncolysis: usurpation of the Ras signaling pathway by reovirus.EMBO J. 1998; 17: 3351-3362Crossref PubMed Scopus (449) Google Scholar,12Coffey MC Strong JE Forsyth PA Lee PW Reovirus therapy of tumors with activated Ras pathway.Science. 1998; 282: 1332-1334Crossref PubMed Scopus (637) Google Scholar,13Alain T Kim TS Lun X Liacini A Schiff LA Senger DL et al.Proteolytic disassembly is a critical determinant for reovirus oncolysis.Mol Ther. 2007; 15: 1512-1521Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar We found promising efficacy animal models12Coffey MC Strong JE Forsyth PA Lee PW Reovirus therapy of tumors with activated Ras pathway.Science. 1998; 282: 1332-1334Crossref PubMed Scopus (637) Google Scholar,4Markert JM Medlock MD Rabkin SD Gillespie GY Todo T Hunter WD et al.Conditionally replicating herpes simplex virus mutant, G207 for the treatment of malignant glioma: results of a phase I trial.Gene Ther. 2000; 7: 867-874Crossref PubMed Scopus (810) Google Scholar,19Yang W Senger D Muzik H Shi ZQ Johnson D Brasher PMA et al.Reovirus prolongs survival and reduces the frequency of spinal and leptomeningeal metastases from medulloblastoma.Can Res. 2003; 63: 3162-3172PubMed Google Scholar,20Yang WQ Senger DL Lun XQ Muzik H Shi ZQ Dyck RH et al.Reovirus as an experimental therapeutic for brain and leptomeningeal metastases from breast cancer.Gene Ther. 2004; 11: 1579-1589Crossref PubMed Scopus (39) Google Scholar,21Yang WQ Lun X Palmer C Wilcox ME Muzik H Shi ZQ et al.Efficacy and safety evaluation of human reovirus type 3 in immunocompetent animals: racine and nonhuman primates.Clin Can Res. 2004; 10: 8561-8576Crossref PubMed Scopus (68) Google Scholar of brain tumors. Its intracerebral administration into the brains of nonhuman primates was well tolerated.21Yang WQ Lun X Palmer C Wilcox ME Muzik H Shi ZQ et al.Efficacy and safety evaluation of human reovirus type 3 in immunocompetent animals: racine and nonhuman primates.Clin Can Res. 2004; 10: 8561-8576Crossref PubMed Scopus (68) Google Scholar A number of phase I studies have been completed22Morris DG Forsyth PA Paterson AHG Fonseca K DiFrancesco LM Thompson BG et al.A phase 1 clinical trial evaluating intralesional Reolysin (reovirus) in histologically confirmed malignancies.Proc Am Soc Clin Oncol. 2002; 21 (24. Abstract 92)Google Scholar,23Yap TA Vidal L Pandha H Spicer J Digue L Coffey M et al.A phase I study of wild‐type reovirus, which selectively replicates in cells expressing activated Ras, administered intravenously to patients with advanced cancer.EJC Suppl. 2006; 4 (Abstract 348)Abstract Full Text PDF Google Scholar,24Vidal L Twigger K White CL Ahmed M Pandha HS Nutting CM et al.A phase I trial of intratumoral administration of reovirus type 3 (Reolysin) in combination with radiation in patients with advanced malignancies.Proc Amer Assoc Cancer Res. 2006; 47 (Abstract 3998)Google Scholar,25Gollamudi R Desai K Chaudhary I Ghalib MH Wong B Einstein M et al.Pharmacodynamic and safety study of REOLYSIN in patients with advanced solid tumors.Proc Am Soc Clin Oncol. 2007; 25 (Abstract 14004)Google Scholar examining the administration of reovirus intratumorally or in combination with radiation in patients with cutaneous metastases from systemic cancer, intratumorally in patients with T2 prostate cancer, and systemically (two studies) in patients with advanced malignancies. These studies found no significant toxicities in any of the treated patients, and a maximum tolerated dose (MTD) has never been reached.The objectives of this study were to determine the safety of the single intratumoral administration of escalating doses of reovirus. Although other clinical trials have directly administered herpes simplex virus4Markert JM Medlock MD Rabkin SD Gillespie GY Todo T Hunter WD et al.Conditionally replicating herpes simplex virus mutant, G207 for the treatment of malignant glioma: results of a phase I trial.Gene Ther. 2000; 7: 867-874Crossref PubMed Scopus (810) Google Scholar,5Rampling R Cruickshank G Papanastassiou V Nicoll J Hadley D Brennan D et al.Toxicity evaluation of replication‐competent herpes simplex virus (ICP null mutant 1716) in patients with recurrent malignant gliomas.Gene Ther. 2000; 7: 859-866Crossref PubMed Scopus (516) Google Scholar,6Papanastassiou V Rampling R Fraser M Petty R Hadley D Nicoll J et al.The potential for efficacy of the modified (ICP 34.5(–)) herpes simplex virus HSV1716 following intratumoural injection into human malignant glioma: a proof of principle study.Gene Ther. 2002; 9: 398-406Crossref PubMed Scopus (272) Google Scholar,7Harrow S Papanastassiou V Harland J Mabbs R Petty R Fraser M et al.HSV1716 injection into the brain adjacent to tumour following surgical resection of high‐grade glioma: safety data and long‐term survival.Gene Ther. 2004; 11: 1648-1658Crossref PubMed Scopus (278) Google Scholar or adenovirus10Chiocca EA Abbed KM Tatter S Lous DN Hochberg FH Barker F et al.A phase 1 open‐label, dose escalation, multi‐institutional trial of injection with an E1B‐attenuated adenovirus, ONYX‐015, into the peritumoral region of recurrent malignant gliomas, in the adjuvant setting.Mol Ther. 2004; 10: 958-966Abstract Full Text Full Text PDF PubMed Scopus (331) Google Scholar into the brains of patients with recurrent MGs, this is the first demonstration that the administration of a genetically unmodified (i.e., wild type) virus, using these doses, is safe and well tolerated.ResultsPatient characteristicsTwelve patients were enrolled from June 2002 to July 2005 (Table 1). The median age was 53.5 years (range: 40–61). Prior to the administration of reovirus nine patients had a diagnosis of GBM, two had anaplastic astrocytomas, and one had an anaplastic oligoastrocytoma. Median Karnofsky performance score was 80 (range: 60–90). Viral treatment was administered for the first, second, or third recurrence in six, five, and one patient, respectively. The median time interval between the diagnosis of an MG and reovirus administration was 12 months (range: 4–61). All patients received radiotherapy and 10 also received chemotherapy.Table 1Patient characteristics of 12 patients with recurrent malignant gliomas treated with Reovirus# PatientsaMay not add up to one hundred due to rounding.Gender Male758 Female542Performance status (KPS) 60325 70217 80217 90542Histology at first diagnosis of brain tumor Oligoastrocytoma grade II18 Anaplastic oligoastrocytoma18 Anaplastic astrocytoma325 Glioblastoma multiforme758Histology of recurrence prior to Reovirus administration Anaplastic astrocytoma217 Anaplastic oligoastrocytoma18 Glioblastoma multiforme975Prior therapy Radiotherapy (60 Gy)2100 Adjuvant chemotherapy1083Other medications at baseline Corticosteroids650 Anticonvulsants12100Dose (TCID50) 107325 108650 109325Surgery at Recurrence postadministration Resection650 None650Abbreviations: KPS, Karnofsky Performance score; TCID50, tissue culture infectious dose 50.a May not add up to one hundred due to rounding. Open table in a new tab Dose escalation and toxicityThe most common serious adverse event (AE) was a deterioration of motor strength related to tumor progression and not considered related to viral administration in any patient. Serious AEs (grade III or IV) and their relationship to viral administration are described in Table 2. The second patient in the first cohort [1 × 107 tissue culture infectious dose 50 (TCID50)] experienced recurrent seizures 2 weeks after reovirus administration related to noncompliance with anticonvulsants. The patient was also drowsy (grade III) related to bilateral hygromas unrelated to the study. With the addition of further antiseizure medications, the patient had grade III elevation of γ‐glutamyl transpeptidase and grade IV anorexia, also considered unlikely related to the viral treatment.Table 2Patients with serious adverse events (Grade III-IV)Relationship to Viral InjectionAdverse eventGradeUnrelatedUnlikelyPossibly Probably DefinitelyNeurological HeadacheIV1 SeizuresIII1 ConfusionIII21 DrowsinessIII21 AlteredIII2 visual fields SpeechIII2 Impairment SpeechIV1 Impairment Motor weaknessIII33 MotorIV1 Weakness AtaxiaIII1 InsomniaIII1 NeglectIII1Gastrointestinal AnorexiaIV1 VomitingIII1 NauseaIII1 ConstipationIII1Cardiovascular Pulmonary embolismIV1 TachycardiaIII1 HypoxiaIII3Others GGT elevationIII111 UrineIII11 Incontinence Apnea (sleep)III1 AstheniaIII1Abbreviations: GGT: y-glut amyl transpeptidase; CSF: cerebrospinal fluid; IC: intracranial. Open table in a new tab At the second dose level (1 × 108 TCID50), the second patient was admitted to the hospital for fever, headache, vomiting, anorexia, and weight loss 8 days after reovirus administration. The patient experienced a grade III glutamyl transpeptidase elevation, which was considered possibly related to viral infection. The third patient in this cohort experienced fever, nausea, and vomiting 4 days after reovirus administration of undetermined etiology. Four days later she was admitted to the hospital for nausea, dehydration, and anorexia. The patient then experienced a focal seizure and a grade III motor weakness attributed to cerebral edema. At the recommendation of the Data Safety Monitoring Board, three additional patients with a diagnosis of GBM were enrolled at that dose level, and none had a dose‐limiting toxicity (DLT). After escalating to the planned third dose level (1 × 109 TCID50) the MTD was not reached. None of the AEs at 1 × 109 TCID50 was definitely or probably related to the administration of reovirus.Six patients had tumor debulking at various times following their injection with reovirus. One patient experienced complications related to the neurosurgical procedure. Patient 6 had a postoperative cerebrospinal fluid leak with meningismus and a pulmonary embolism.Antibody responsesSamples of serum were collected at baseline and on days 0, 7, 14, 28, 35, 42, 56, 84, 112, and 168 (Table 3). Additional viral studies for the last six patients were required by Health Canada on Day 2 through Day 14. Ten patients (83%) became positive for serum‐specific antibodies from 1 to 4 weeks after viral administration. One patient had circulating antibodies detected for the first time on day 84.Table 3Viral dissemination and patient immune responsePatientDose (TC'D50)Serum antibodies increased above baselineRT-PCR serumRT-PCR salivaRT-Pcr urineRT-Pcr feces11 × 107D14NegNegNegNeg21 × 107D14NegNegNegNeg31 × 107neverNegNegNegNeg41 × 108D84aThis patient had not been evaluated for antibodies since day 7.NegNegNegNeg51 × 108D14NegNegNegNeg61 × 108D7NegNDNegNeg71 × 108D7NegNegNegNeg81 × 108neverNegPos D2 and 4NegPos D491 × 108D28NegPos at screeningNegNeg101 × 109D14NegNegNegNeg111 × 109D28NegNegNegPos D6 & 10 (neg D8)121 × 109D14NegNegNegNegAbbreviations: Ab, antibody; D, days after intratumoral viral administration; ND, not done; neg: persistent negative result through all the study; pos: positive;RT-PCR: reverse transcriptase-polymerase chain reaction.a This patient had not been evaluated for antibodies since day 7. Open table in a new tab Viral sheddingViral shedding was examined by specific reverse transcriptase‐polymerase chain reaction (RT‐PCR) in serum, sputum, urine, and stool using the same schedule described for antibody studies (Table 3). Viral RNA was not detected in the serum or urine of any patient. Viral shedding from the saliva was detected in one patient and in the feces from two patients. Saliva of patient 9 was positive at screening but remained negative after viral administration. In patient 8, viral RNA in saliva was found on days 2 and 4 and in feces on day 4. Patient 11 had viral RNA detected in feces on days 6 and 10.Clinical outcomeOne patient had stable disease, ten had progressive disease, and one was not evaluable for response (Table 4). This latter patient (number 4) had a small asymptomatic hemorrhage at week 4 and he had debulking surgery; he is alive 54 months after viral treatment (Figure 1). For the group as a whole the median time to progression (TTP) was 4.3 weeks (range: 2.6–39). The median overall survival was 21 weeks (range: 6–234 weeks). Six patients lived for >6 months, and three patients for >1 year (one of these had a recurrent GBM and two other types of recurrent MG); one is alive at 54 months after treatment with reovirus.Table 4Outcome of patients treated with reovirusPatientInitial diagnosisDiagnosis at injectiondose (TCID50)Best responseTTP (weeks)survival (weeks)1AAAA1 × 107PD11.6D, 42.12OA IIAOA1 × 107SD39D, 763AOAGBM1 × 107PD4.1D, 9.94AAAA1 × 108NENEA,2345AAGBM1 × 108PD7.4D, 63.46GBMGBM1 × 108PD3.4D, 14.37GBMGBM1 × 108PD8.7D,11.68GBMGBM1 × 108PD4.3D, 69GBMGBM1 × 108PD4.1D, 1310GBMGBM1 × 109PD3.6D, 35.611GBMGBM1 × 109PD7.4D, 28.112GBMGBM1 × 109PD2.6D, 11.7Abbreviations: A, alive; AA, anaplastic astrocytoma; AOA, anaplastic oligo-astrocytoma; D, deceased; GBM, glioblastoma multiforme; NE, not evaluable; OA II, oligo-astrocytoma grade II; PD, progressive disease; SD, stable disease; TTP, time to progression. Open table in a new tab Examination of tumor tissue after reovirus administrationSix of the twelve (50%) patients had surgery (ranging from 3 to 20 weeks) after the intratumoral viral treatment (patients 1, 4, 5, 6, 10, and 12). All had persistent or recurrent glioma and areas of necrosis. Of note, in three of the six patients (50%), focal collections of plasma cells were identified at the time of re‐resection that were not present in the tumors examined prior to the administration of reovirus. (Figure 2a and b). This unusual finding was considered as possibly related to reovirus treatment. In four patients, areas of normal brain were examined, and there was no evidence of encephalitis. Patient 12 had sarcomatous metaplasia consistent with the development of a gliosarcoma component that was present, but minor, in his original GBM.Figure 2Plasma cells identified at the time of re‐resection after reovirus administration.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Autopsy was performed in three patients. All had residual/recurrent tumor. Patient 8, who had been diagnosed with a GBM throughout his treatment, also had sarcomatous transformation of his tumor at autopsy, and was diagnosed with a gliosarcoma, WHO grade IV. There was no histologic evidence of focal/generalized encephalitis found in any patient.DiscussionThe major finding of this study is that the administration of reovirus directly into the tumors of patients with MGs was well tolerated and safe when given at the doses and schedule used. This is the first report of a live, replication competent, and genetically unmodified virus administered intracerebrally directly into the brain tumors of patients. Previous clinical trials of oncolytic viruses administered intracerebrally have all used viruses that are replication defective or attenuated by genetic manipulation.4Markert JM Medlock MD Rabkin SD Gillespie GY Todo T Hunter WD et al.Conditionally replicating herpes simplex virus mutant, G207 for the treatment of malignant glioma: results of a phase I trial.Gene Ther. 2000; 7: 867-874Crossref PubMed Scopus (810) Google Scholar,5Rampling R Cruickshank G Papanastassiou V Nicoll J Hadley D Brennan D et al.Toxicity evaluation of replication‐competent herpes simplex virus (ICP null mutant 1716) in patients with recurrent malignant gliomas.Gene Ther. 2000; 7: 859-866Crossref PubMed Scopus (516) Google Scholar,6Papanastassiou V Rampling R Fraser M Petty R Hadley D Nicoll J et al.The potential for efficacy of the modified (ICP 34.5(–)) herpes simplex virus HSV1716 following intratumoural injection into human malignant glioma: a proof of principle study.Gene Ther. 2002; 9: 398-406Crossref PubMed Scopus (272) Google Scholar,10Chiocca EA Abbed KM Tatter S Lous DN Hochberg FH Barker F et al.A phase 1 open‐label, dose escalation, multi‐institutional trial of injection with an E1B‐attenuated adenovirus, ONYX‐015, into the peritumoral region of recurrent malignant gliomas, in the adjuvant setting.Mol Ther. 2004; 10: 958-966Abstract Full Text Full Text PDF PubMed Scopus (331) Google Scholar,26Trask TW Trask RP Aguilar‐Cordova E Shine HD Wyde PR Goodman JC et al.Phase 1 study of adenoviral delivery of the HSV‐tk gene and ganciclovir administration in patients with recurrent malignant gliomas.Mol Ther. 2000; 1: 195-203Abstract Full Text Full Text PDF PubMed Scopus (235) Google Scholar,27Smitts PS Driesse M Wolbers J Kros M Avezaat C Treatment of relapsed malignant glioma with an adenoviral vector containing the herpes simplex thymidine kinase gene followed by ganciclovir.Mol Ther. 2003; 7: 851-858Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar,28Lang FF Bruner JM Fuller GN Aldape K Prados MD Chang S et al.Phase I trial of adenovirus‐mediated p53 gene therapy for recurrent glioma: biological and clinical results.J Clin Oncol. 2003; 21: 2508-2518Crossref PubMed Scopus (38) Google Scholar As in our study, these found no dose‐limiting toxicities at the dosages used (e.g., 3 × 109 plaque forming units for herpes simplex virus).4Markert JM Medlock MD Rabkin SD Gillespie GY Todo T Hunter WD et al.Conditionally replicating herpes simplex virus mutant, G207 for the treatment of malignant glioma: results of a phase I trial.Gene Ther. 2000; 7: 867-874Crossref PubMed Scopus (810) Google Scholar This was true even in trials in which a virus was administered into the brain itself adjacent to the tumor.7Harrow S Papanastassiou V Harland J Mabbs R Petty R Fraser M et al.HSV1716 injection into the brain adjacent to tumour following surgical resection of high‐grade glioma: safety data and long‐term survival.Gene Ther. 2004; 11: 1648-1658Crossref PubMed Scopus (278) Google Scholar,10Chiocca EA Abbed KM Tatter S Lous DN Hochberg FH Barker F et al.A phase 1 open‐label, dose escalation, multi‐institutional trial of injection with an E1B‐attenuated adenovirus, ONYX‐015, into the peritumoral region of recurrent malignant gliomas, in the adjuvant setting.Mol Ther. 2004; 10: 958-966Abstract Full Text Full Text PDF PubMed Scopus (331) Google Scholar The major concern of oncolytic viruses in brain tumors is viral infection in the brain. However, no evidence of focal or diffuse viral encephalitis was found histologically. The histological diagnosis of encephalitis can be subtle, but no patient had clinical evidence of encephalitis, which can be a far more sensitive indicator.This study was not designed to evaluate efficacy as a primary objective. Patients were not uniform with respect to number of recurrences, previous treatment regimes, or tumor type (Table 1), and magnetic resonance imaging may have limitations for the evaluation of agents such as viruses that cause immune infiltration of tumor beds after treatment. Several patients had longer‐than‐expected survivals following treatment with reovirus (three survived >1 year), and one remains a long‐term survivor. This might reflect patient selection rather than a treatment effect. Other trials using oncolytic viruses also report long‐term survivors,4Markert JM Medlock MD Rabkin SD Gillespie GY Todo T Hunter WD et al.Conditionally replicating herpes simplex virus mutant, G207 for the treatment of malignant glioma: results of a phase I trial.Gene Ther. 2000; 7: 867-874Crossref PubMed Scopus (810) Google Scholar,5Rampling R Cruickshank G Papanastassiou V Nicoll J Hadley D Brennan D et al.Toxicity evaluation of replication‐competent herpes simplex virus (ICP null mutant 1716) in patients with recurrent malignant gliomas.Gene Ther. 2000; 7: 859-866Crossref PubMed Scopus (516) Google Scholar or patients who have no histological evidence of recurrent glioma.4Markert JM Medlock MD Rabkin SD Gillespie GY Todo T Hunter WD et al.Conditionally replicating herpes simplex virus mutant, G207 for the treatment of malignant glioma: results of a phase I trial.Gene Ther. 2000; 7: 867-874Crossref PubMed Scopus (810) Google ScholarAntibody studies showed a rate of seroconversion (83%), consistent with other clinical studies using reovirus.23Yap TA Vidal L Pandha H Spicer J Digue L Coffey M et al.A phase I study of wild‐type reovirus, which selectively replicates in cells expressing activated Ras, administered intravenously to patients with advanced cancer.EJC Suppl. 2006; 4 (Abstract 348)Abstract Full Text PDF Google Scholar It is important in trials with oncolytic viruses to consider safety from occupational (i.e., exposure by health care staff) and public health perspectives. There was evidence of shedding (at least using RT‐PCR) in the saliva of one patient and in the feces from two patients, all of which was present within 10 days of viral inoculation but did not persist longer.A DLT was not reached over the dosage range used. A major limitation of our study is that it was stopped after using only three of the planned four dose levels because it took longer