- Email: [email protected]
II Trial of Intravenous NDV-HUJ Oncolytic Virus in Recurrent Glioblastoma Multiforme
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Phase I/II Trial of Intravenous NDV-HUJ Oncolytic Virus in Recurrent Glioblastoma Multiforme Arnold I. Freeman,1 Zichria Zakay-Rones,2 John M. Gomori,3 Eduard Linetsky,4,5 Linda Rasooly,1 Evgeniya Greenbaum,2 Shira Rozenman-Yair,6 Amos Panet,2 Eugene Libson,7 Charles S. Irving,6 Eithan Galun,1,* and Tali Siegal5 1
Goldyne Savad Institute of Gene Therapy, 3Department of Neuro-radiology, 4Department of Neurology, 7Department of Radiology, and 5 Gaffin Center for Neuro-Oncology, Hadassah University Hospital, Jerusalem 91120, Israel 2 Department of Virology, Hebrew University, Jerusalem 91120, Israel 6 Theravir Management LP, Jerusalem, Israel *To whom correspondence and reprint requests should be addressed. Fax: +972 2 642 9856. E-mail: [email protected].
Available online 28 October 2005
We undertook a Phase I/II trial in patients with apparent recurrent glioblastoma multiforme (GBM) based on imaging studies to determine the safety and tumor response of repetitive intravenous administration of NDV-HUJ, the oncolytic HUJ strain of Newcastle disease virus. The first part of the study utilized an accelerated intrapatient dose-escalation protocol with one-cycle dosage steps of 0.1, 0.32, 0.93, 5.9, and 11 billion infectious units (BIU) of NDV-HUJ (1 BIU = 1 109 EID50 50% egg infectious dose) followed by three cycles of 55 BIU. Virus was administered by intravenous infusion over 15 min. In the second part, patients received three cycles of 11 BIU. All patients without progressive disease were maintained with two doses of 11 BIU iv weekly. Eleven of the 14 enrolled patients (11–58 years, Karnofsky performance scale 50–90%) received treatment. Toxicity was minimal with Grade I/II constitutional fever being seen in 5 patients. Maximum tolerated dose was not achieved. Anti-NDV hemagglutinin antibodies appeared within 5–29 days. NDV-HUJ was recovered from blood, saliva, and urine samples and one tumor biopsy. One patient achieved a complete response. Intravenous NDV-HUJ is well tolerated. The findings of good tolerability and encouraging responses warrant the continued evaluation of NDV-HUJ in GBM, as well as other cancers. Key Words: Newcastle disease virus, NDV-HUJ, oncolytic virus, intravenous, glioblastoma, brain cancer, phase I/II trial, dose escalation, complete remission, apoptosis
INTRODUCTION Primary malignant gliomas are among the most lethal and difficult forms of cancer. Of these glioblastoma multiforme (GBM) is the most rapidly growing and most challenging [1]. Despite improvements, the outcome for GBM patients has not changed significantly over the past 30 years [2]. With maximal conventional therapy, survival is approximately 14 months from time of diagnosis and about 30 weeks from the time of recurrence [3]. Recent advances in the understanding of brain tumor biology have led to the development of novel therapeutic approaches [4,5], including oncolytic virotherapy [6–8]. Viruses, particularly adenovirus [9,10] and HSV [11,12], have been engineered to deliver therapeutic genes, while other viruses [13–15] have been utilized without engineering for their innate cytotoxic effects. Among the nonengineered oncolytic viruses, Newcastle disease virus (NDV) [16] has a long history as a
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broad-spectrum oncolytic agent that can destroy tumor cells and stimulate the immune system [17–25]. NDV is a single-stranded RNA virus, whose natural host is poultry. The 73-T [26], MTH68 [27], and PV701 (MK107) [28,29] strains of NDV have been the subject of several clinical studies. NDV-PV701 has recently been evaluated in a Phase I study of patients with advanced solid tumors; however, patients with CNS tumors were excluded from this study [29]. Anecdotal responses to MTH68 in malignant glioma have been reported [27]. NDV strains that are not pathogenic to poultry (lentogenic) have also been shown to kill cancer cells [24] but possibly by different mechanisms [17]. Infection of tumor cells by lentogenic NDV generates several innate danger signals leading to apoptosis. The lentogenic Ulster strain of NDV has been combined with various tumor cells as a tumor vaccine for different cancers, including GBM [30], but the use of a lentogenic NDV strain alone in oncolytic
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virotherapy has not been evaluated. HUJ is a new lentogenic strain of NDV (NDV-HUJ) that has a selective cytopathogenicity for human and animal cancer cell lines (L. Rasooly, E. Ovin, N. Hooshi, and E. Galun, unpublished results; Z. Zakay-Rones and A. Panet, unpublished results). We here present the first clinical evaluation of a lentogenic NDV strain administered systemically and the first evaluation of any NDV strain in GBM patients. We evaluated patients with recurrent GBM in a singlesite, open-label Phase I/II study of NDV-HUJ. The study was divided into two parts. The initial part of the study utilized a six-step intrapatient accelerated doseescalation scheme. In this scheme, patients advanced to the next higher dosage step after all patients had completed the lower step without any dose-limiting toxicities (DLT). Newly enrolled patients started at one dosage step higher than the one completed without DLT by all enrolled patients. The second part of the study utilized a constant dosing step, which was one lower than the highest step reached in the first part of the study.
RESULTS AND DISCUSSION Study Design, Patients, and Eligibility The intrapatient accelerated dose-escalation scheme provided each patient with the opportunity to receive a high and more likely potentially effective dose of NDV-HUJ. The lowest dosage step of 0.1 billion infectious units (BIU) corresponded to a previously reported NDV intravenous dosage [27]. The highest dosage step of 55 BIU was determined by preclinical toxicity testing. The step down from 55 to 11 BIU used in the second part of the study was intended to lower the risk of adverse events and was determined in part by the limited amount of
NDV-HUJ available for the study due to the high cost of initial production. We enrolled 14 patients (9 men and 5 women, ages 11 to 58 years) from December 2002 to December 2003 (Table 1, Fig. 1). All patients had been diagnosed with GBM based on histology and gadolinium-enhanced (Gd+) MRI, and all had a recurrence of GBM based on MRI evidence of mass effect associated with T1 gadolinium enhancement, T2 prolongation, elevated relative cerebral blood volume (rCBV) (3), and/or high choline on magnetic resonance spectroscopy (MRS). Three of the patients enrolled in Part II of the study were withdrawn during the screening period prior to starting NDV virotherapy as follows: Patient 08 died, 12 was found to have necrosis and not recurrent GBM upon repeat biopsy, and 14 had an abrupt deterioration in clinical condition. Prior therapy for the 11 assessable patients is listed in Table 1. Eight patients had at least one partial or full resection, all patients had received radiotherapy, and 9 had received temozolomide and/or intra-arterial carboplatin chemotherapy. Nine of the evaluable patients were diagnosed as GBM 17 to 49 weeks prior to virotherapy without prior history of low-grade gliomas, whereas 2 were diagnosed as GBM 5 to 6 weeks before virotherapy after having been previously diagnosed with lower grade glioma. The dosage steps reached by the patients are given in Table 2 and shown in Supplementary Fig. 1. Patients 02, 05, and 06 in Part I completed all three cycles of the 55 BIU maximum dose Step 6, while Patient 01 was withdrawn in the middle of the third cycle of Step 6 due to clinical and radiological disease progression. In Part II, Patients 07, 09, and 11 completed all three cycles of dosage Step 5 (11 BIU). Of the six patients that completed their last dosage step (Fig. 1), all except for Patient 07 continued with main-
TABLE 1: Patient characteristics Sex
Age (years)
Baseline KPS
Type GBM
Dx to VT (weeks)
Surgical procedures
Prior Rx
01 02 03
M M F
50 58 51
50 70 70
I I I
20 29 17
B B B
RT, TMZ RT, IAC RT
04 05 06 07 08 09 10 11 12 13 14
F F F F M M M M M M M
32 54 58 57 49 43 11 56 27 31 46
90 90 80 90 70 80 70 90 60 90 50
I I I I I I II I I II I
49 43 34 29 48 41 5 18 49 6 61
GTR GTR, GTR GTR, GTR GTR B PR B, PR GTR PR, GTR, GTR, GTR GTR, GTR, GTR PR
RT, IAC, TMZ RT, TMZ RT, IAC RT, IAC RT, IAC, TMZ RT, IAC, TMZ RT, TMZ RT RT, TMZ RT, TMZ RT, TMZ
Patient
Tumor location
Baseline tumor cross section (cm2)
R frontal L parietal R frontal + basal ganglia L temporal R parietal R frontal L frontal R temporal L parietal R parietal R frontal R parietal L frontal R parietal
31.5 34.8 16.0 12.5 2.4 11.5 20.5 14.4 9.5 38.4 12.9 16.3 45.0 22.3
Abbreviations: M, male; F, female; KPS, Karnofsky performance scale; Dx, diagnosis; GBM, glioblastoma multiforme; I, no previous diagnosis of a lower grade glioma; II, previous history of a lower grade glioma; VT, viral therapy; B, biopsy; PR, partial resection; GTR, gross total resection; Rx, treatment; RT, radiotherapy; IAC, intra-arterial carboplatin; TMZ, temozolomide; R, right; L, left.
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FIG. 1. Patient enrollment.
review of this patient’s MR imaging revealed brain stem involvement that was not appreciated at the time of enrollment. As seen in Table 3, neurological seizures occurred in 8 patients. Of the 15 seizures noted, 5 were classified as Grade 3 and 1 as Grade 4, according to the NDC Common Toxicity Criteria (CTC) version 2. Comparison of these seizures with the patients’ medical histories indicated that there were no changes in their frequency or quality following NDV administration. The stupor that occurred in Patient 03 (Table 3) was considered due to an enlarging brain tumor and is commonly seen in this situation. The patient’s MRI was consistent with progressive tumor and did not display new features suggestive of encephalopathy. None of the patients showed clinical findings indicative of inflammatory responses such as encephalitis/encephalopathy, meningitis, or other inflammatory responses; these are a concern for viral therapy in cancer patients. Toxicity of repeated intravenous NDV dosing was reported in Phase I studies of NDV PV701. They focused on acute adverse events (AEs) during infusion, general flulike AEs, and tumor-site-specific AEs. The AEs were reduced by priming dosing and following slower infusion
tenance dosing until radiological disease progression. Additional treatments that the patients received following withdrawal from maintenance dosing are given in Table 2. Time to progression (clinical and radiological) and overall survival are given for reference in Table 2, although we did not include them in the study as outcome measures. Survival ranged from 3 to 66 weeks from the start of virotherapy. Time to radiological progression ranged from 2 to 37 weeks. Although withdrawn from treatment at 8 weeks due to apparent radiological progression without clinical deterioration and subsequent treatment with temozolomide, Patient 11 continued to improve clinically and was free of disease progression out to 44 weeks. Toxicity We included 11 patients in the toxicity analysis (Table 3). Five patients developed fever, usually during their first cycle. Most patients also experienced adverse events that we determined to be unrelated or unlikely to be related to their virotherapy. One patient (No. 04) died on study due to disease progression and this was independently validated by the hospital’s safety board. A subsequent
TABLE 2: Summary of patient treatment outcomes Patient
Dosage steps started
01 02 03 04 05 06 07 09 10 11 13
1, 2, 3, 4, 5, 6, 6, 6 2, 3, 4, 5, 6, 6, 6, M 4, 5, 6 4, 5 5, 6, 6, 6, M 5, 6, 6, 6, M 5, 5, 5 5, 5, 5, M 5, 5 5, 5, 5, M 5, 5
Withdrawn during Step 5 or 6 X X X
X X
TTP (weeks) Imaging Clinical 15 23 CT CT 17 8 37 2 8 CT
12 23 4 3 53 18 9 37 2 44 4
OS from start of VT (weeks) 20 37 13 3 66 32 38 61 19 62 15
Additional therapies after withdrawal from VT
TMX, thyroid ablation TMZ IAC TMZ, IAC
Patients 08, 12, and 14 were withdrawn during baseline screening. Abbreviations: dosage steps in BIU, 1 = 0.1, 2 = 0.32, 3 = 0.93, 4 = 5.9, 5 = 11, 6 = 55; M, maintenance of dosage Step 5 twice weekly; TTP, time to progression; CT, tumor progression confirmed by CT following clinical progression; OS, overall survival; VT, viral therapy; TMX, tamoxifen; TMZ, temozolomide; IAC, intra-arterial carboplatin.
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TABLE 3: Adverse events in dosed patients Adverse event
1
No. of events by CTC grade 2 3
4
Total No. of patients with event of any grade (% pts)
Possibly or probably related to treatment Constitutional symptoms, fever
6
1
0
0
5 (45)
Unrelated or unlikely related to treatment Neurological, seizures Neurological, stupor Neurological, syncope Pain, headache Pain, abdominal Cardiovascular, hypertension Cardiovascular, thrombosis Cardiovascular, edema Infection, otitis media, without neutropenia, fever Gastrointestinal, vomiting Pulmonary, cough
0 0 0 1 1 1 0 1 1 0 0
9 0 0 4 0 0 0 0 4 0 1
5 1 2 1 0 0 2 0 0 1 0
1 0 0 0 0 0 0 0 0 0 0
8 1 1 3 1 1 2 1 3 1 1
(73) (9) (9) (27) (9) (9) (18) (9) (27) (9) (9)
Results are reported as independent events. More than one event may have occurred per patient or the same even may have occurred more than once in a given patient.
times [28]. In our study, the only AE related to NDV-HUJ administration was Grades I and II fever that occurred during the first cycle and resolved within several days. Other AEs, such as seizures, had occurred previously in the patients and were related to their underlying cancer. We determined them to be unrelated or unlikely to be related to treatment. The observed neurological AEs were not suggestive of increasing intracranial pressure or inflammatory response seen with viral encephalitis. No acute adverse events occurred during the infusion period, even though NDV-HUJ was administered rapidly over 15 min. The lack of AEs in our study compared to those seen in the NDV PV701 Phase I studies might be due to the lower doses administered in this study (11–55 BIU vs 24 to 96 billion PFU/m2), the inherent differences between the NDV strains, the high dose steroids that patients received as part of the GBM management, or a different spectrum of cancer patients. For these reasons, no priming doses were required to overcome the tachyphylactic-like reactions seen in studies using PV701. We observed no DLT and maximum tolerated dose is greater than the 55 BIU administered on 5 consecutive days for three cycles. Tumor Response All patients had obvious measurable disease (N2 cm2 contrast-enhanced tumor cross section) at treatment initiation (Table 1). As seen in Fig. 2, one patient (No. 09) had stable disease at first follow-up, a partial remission at second follow-up, and complete remission during maintenance dosing, at which time there was a significant improvement in neurological status and corticosteroid therapy was discontinued (tumor cross sections before, during, and after the study are given in Supplementary Table 1). The complete response did not prove durable (Table 2), and on routine imaging 3 months after complete disappearance of the tumor a lesion subsequently shown
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by histology to be GBM reappeared in the tumor bed. Three patients (Nos. 02, 05, and 06) had an increase in enhanced tumor cross section without a deterioration of their neurological status. As part of their clinical management, their tumors were biopsied. Perivascular lymphocytic infiltrates were found in the biopsied tumor of all three patients, 02, 05, and 06. The infiltrates were similar to those that have been described previously in the context of GBM and/or radiation-induced damage. Another patient (No. 11) did not have a change in neurological status and did not have an increase in enhanced tumor cross section, but was judged to have radiological disease progression based on increased enhancement within the resection cavity and changes in the FLAIR and rCBV MR images. As seen in Table 2, the time to clinical progression in Patients 05 and 11 occurred considerably later than their radiological progression, suggesting that patients can have radiological progressive disease that does not lead to immediate clinical deterioration. Of the three long-term survivors, two (Patients 05 and 11) had early radiological progression. All patients in the study eventually developed both clinical and radiological progression (Table 2). Molecular imaging has the potential of providing much-needed additional information on the response of glioma to novel therapies. In our study, postoperative changes in the tumoral bed made obtaining good rCBV and MRS difficult and increased the chances for misdiagnosis of radiation necrosis for recurrent glioblastoma at baseline. In the case of Patient 12 the baseline rCBV and MRS study was significantly questionable to warrant a biopsy prior to the start of dosing. The finding of radiation necrosis led to the screening withdrawal of the patient. In the few cases for which adequate data were available, rCBV and MRS confirmed the changes detected by conventioQ nal imaging, but did not add additional information.
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FIG. 2. Complete tumor response. (A) Patient 09 at baseline, (B) stable disease at first follow-up, (C) partial response (PR) at second follow-up, (D) PR at 20 weeks from start of virotherapy, and (E and F) complete response at 25 and 30 weeks from start of virotherapy.
[18F]Fluorodeoxyglucose positron emission tomography (FDG-PET) suffered from difficulty in distinguishing tumor from adjacent tissue and collateral reference sites. Response rates (partial response (PR) + complete response (CR)) in patients with recurrent GBM treated with the current gold standard, temozolomide, are low. Complete responses are particularly rare, being observed in 0/112 [31] and 2/128 [32] patients in multicenter studies of GBM at first relapse and in 1/375 patients [3] in the largest such study of recurrent GBM. We are unaware of any reports of spontaneous regression of GBM. The complete response seen in this study was accompanied by rapid neurological improvement and the gradual cessation of corticosteroids. At time of entry onto the NDV study this patient had measurable disease, which failed radiation therapy and two lines of chemotherapy. The possibility that the baseline Gd+ MR image represented tumor radiation necrosis and not recurrent GBM appears very unlikely on the basis of mass effect, elongated T2, a rCBV value of 3, and high choline on MRS, but cannot be completely ruled out in the absence of histologically proven recurrence. Diagnostically challenging malignant gliomas with nonclassical glioblastoma histologies can be mistaken in the absence of gene expression profiling for nonclassical anaplastic oligodendrogliomas [33], which have better survival and time to tumor progression. An explanation of the patient’s complete response due to misdiagnosis of necrosis or anaplastic oligodendroglioma
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is unlikely due to classical histology of glioblastoma found at the time of original diagnosis and upon recurrence following NDV-HUJ therapy. Since there is no other explanation for this complete response, we consider that it resulted from NDV-HUJ therapy. Antibody Response All 14 enrolled patients were tested at baseline for antiNDV hemagglutinin antibodies and were negative (Supplementary Table 2). Three patients (06, 09, and 10) developed antibodies during the first week of treatment. All patients developed antibodies by the third week, except for 2 patients (05 and 11), who developed antibodies only by the fifth week at the start of the third treatment cycle and by the eighth week at the start of maintenance dosing, respectively. In patients receiving long-term therapy, antibodies had either plateaued or started to decrease slightly by the eighth week. Antibody titers remained low throughout repetitive dosing. Virology We tested a total of 101 blood, saliva, and urine samples from five patients (01, 02, 03, 05, and 09) for infectious NDV by inoculation into the allantoic sac of embryonated eggs, a highly sensitive biological infectivity assay that indicates the presence or absence of infectious virus. All baseline samples were negative. For all five patients we recovered infectious NDV particles from blood, urine, and
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saliva samples taken during the first dosage cycle (Supplementary Table 3). We continued to recover infectious NDV particles from blood, saliva, and urine samples after the patients developed anti-hemagglutinin NDV antibodies. We also recovered infectious NDV from 9 of the 10 blood samples taken 9 days after the last dosage of NDV-HUJ from the previous dosage cycle. We recovered infectious NDV particles from a tumor biopsy sample of Patient 02, but not from the biopsy of Patient 05. Tumor cystic fluid samples obtained via an implanted Ommaya device from Patient 02 also tested positive for infectious NDV. Identity of the isolated virus was determined using anti-NDV antibodies. The recovery of infectious NDV particles from the tumor’s cyst fluid and the tumor tissue obtained by biopsy performed about 130 days after the start of virotherapy indicated that the NDV-HUJ reached the extracellular space of the tumor. It is likely that anti-NDV antibodies interfered with the potency of intravenously administered NDV, but not to the extent that all infectious particles were removed from circulation. Infectious NDV particles were found in the blood of patients after the patients’ serum anti-NDV antibody levels had reached plateau levels and were repeatedly detected 9 days following the last administration of virus. Further, Patient 09, who achieved a CR, also developed antibodies early. In future studies posttreatment tumor biopsies should be taken into consideration, when ethically justifiable. NDV-HUJ and Its Possible Mechanisms of Action Very little attention has been given to the safety and mechanisms of action of NDV strains, such as NDV-HUJ, whose pathogenicities for their natural avian hosts are attenuated to the extent of being nonpathogenic (lentogenic), which allows these strains to be used as poultry vaccines. The three major strains of NDV that have been evaluated in clinical studies, 73-T, MTH68, and PV701 (Massachusetts MK107) [34], are classified as mesogenic (moderately pathogenic) strains, whose use and importation are banned in many countries [35]. Mesogenic strains are able to replicate and produce viable progeny virus, which can infect adjacent cells. Their production of viable progeny virus is due in part to the conversion of the viral fusion surface protein from an inactive (F0) to an active (F1) form. This is made possible by a match between the proteases present in host tissues and the sequence of the cleavage site of the fusion protein that requires at least two basic (lysine or arginine) amino acids, between residues 113 and 116, and a phenylalanine at residue 117. The lentogenic HUJ strain has a 112G-R-Q-G-R-L117 sequence. In most tissues, lentogenic strains produce defective progeny, are monocyclic, and cannot easily spread between tissues. The replication of the HUJ strain in humans is probably quite limited, although the continued presence of circulating infectious particles for up to 9 days after dosing seen in
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this study suggests that limited replication in selected tissues, possibly in tumor tissue, occurs. Recent studies have suggested a number of mechanisms relevant to tumor virotherapy that do not involve the production of viable progeny virus particles. Infection of g-irradiated NCH 149 GBM cells by lentogenic NDV Ulster has been reported to lead to cytokine/chemokine induction, changes in cell surface molecules, and the induction of apoptosis [36]. Additional innate and adaptive immune responses have been associated with lentogenic NDV infection of tumor cells [37]. The release of NDV dsRNA into cytoplasm [38] and expression of HN on the cell surface [39] are two immune danger signals that increase IRF-3, IFN-a, TRAIL, TLR, and HSP levels [40,41]. Expression of HN on cell surfaces induces IFN-a in PBMCs [42]. The lentogenic LaSota and Ulster strains were shown to induce anti-tumor cytotoxicity in mouse macrophages and human monocytes [43]. These strains activated NF-nB in mouse macrophages and stimulated NO production [44]. We found that NDV-HUJ induces the inhibition of metabolism and cell death in C6 and RG2 rat malignant glioma cells and fibrosarcoma HT1080-positive control cells, whereas normal fibroblast MS5 cells were unaffected (L. Rasooly, E. Ovin, N. Hooshi, and E. Galun, unpublished results). The presence of apoptotic RG2 cells following incubation with NDV-HUJ suggested that the mechanism for the cytopathic effect of NDV-HUJ on malignant glioma cells is primarily apoptotic (L. Rasooly, E. Ovin, N. Hooshi, and E. Galun, unpublished results). The cytopathic effect of NDV-HUJ on Burkitt lymphoma Daudi cells has also been associated with apoptosis (D. Horesh, A. Panet, and Z. Zakay-Rones, unpublished results). Future Studies Further investigation of the mechanisms of action of the lentogenic strains of NDV is warranted in view of the results seen in this study, as well as the recent report of clinical benefit of lentogenic NDV-modified autologous tumor cell vaccine in GBM patients [30]. Future preclinical studies should be directed toward determining the role of NDV-HUJ selectivity and targeting for GBM cells in its anti-GBM tumor effects in vivo. The absence of major side effects and the complete tumor response seen in one patient provide initial evidence of a potential new concept of systemic virotherapy of GBM using lentogenic NDV and warrants further validation in a larger clinical trial. Future clinical studies may benefit from observations made in this study. In designing treatment schedules, advantage may be taken of the time prior to the appearance of anti-NDV antibodies. Further, the absence of major side effects at the maximum dosing indicates that significantly higher dosages might be explored, as well as a continuous daily dosing schedule. Molecular imaging techniques should be explored for their ability to provide additional information on tumor response. Inclusion criteria should include tumor biopsy
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and gene expression profiling, if possible. Withdrawal from treatment based solely on Gd+ MRI radiological progression should be reconsidered when designing future studies. Virotherapy using lentogenic NDV strains should also be evaluated in patients with lower grade gliomas.
PATIENTS AND METHODS Inclusion and exclusion criteria. Patients were required to have GBM histologically confirmed by the neuropathologist at Hadassah University Hospital and a tumor measurable by Gd+ MRI after failing conventional therapy. At least 4 weeks from completion of chemotherapy or radiotherapy was needed prior to entry into our study. Patients had to be between 3 and 70 years of age, be not pregnant, have an estimated life expectancy of greater than 2 months, have a Karnofsky performance status of 50% or greater, and be receiving a stable or decreasing dose of corticosteroids without an expected need for increase in dosing. Hepatic, renal, and bone marrow function requirements were Hb N 9 g%, WBC N 1000/mm3, platelet count N 30,000 mm3, creatinine b 2.5 mg%, liver function tests less than twice normal. Patients were not allowed to receive any investigational agents other than NDV-HUJ or any other anti-cancer agent during the study. A history of allergy to egg ovalbumin was a cause for exclusion. Other exclusion criteria included acute severe or life-threatening infection, severe depression or psychosis, or history of noncompliance with therapy. A written informed consent document approved by the Hadassah Hospital Institutional Review Board had to be understood and signed by the patient and by a spouse, parent, or guardian. We required both to sign due to alterations of psyche and understanding in some patients with GBM. NDV-HUJ. NDV-HUJ is a highly purified isolate originally derived from the naturally attenuated B1 NDV vaccine strain (ATCC, 1971), passaged 50 times in 10- to 11-day-old embryonated chicken eggs, and cloned twice by limiting dilution in specific-pathogen-free (SPF) embryonated hen’s eggs. The HUJ strain was classified as lentogenic and having no pathogenicity for its natural host, poultry, on the basis of its intracranial pathogenicity index of 0.0 determined by the Israel Ministry of Agriculture and a 112G-R-Q-G-RL117 cleavage site sequence in its surface fusion protein, as well as having a mean death time of N100 h. These certifications are required to allow importation of NDV-HUJ samples into countries where biosafety testing was performed [35]. Cultivation, concentration, and purification were carried out using routine methods [45]. Clinical lots of NDV-HUJ were prepared by growing the virus to high titers in SPF embryonated hen’s eggs (SPAFAS-Alpez, Mexico) and purifying the virus by pelleting and resuspension followed by ultracentrifugation in sucrose gradients. The purified virus was formulated as a suspension in PBS buffer, vialed as 1.1-ml aliquots, and stored at 708C until immediately before use. Clinical lots of NDV-HUJ met the release criteria approved by the Israel Ministry of Health. These criteria included European Pharmacopoeia 4, Section 2.6.16, Tests for Extraneous Agents in Viral Vaccines for Human Use adapted for Phase I/II clinical studies in recurrent GBM patients. Preclinical toxicity studies established the nonobserved adverse effect level to be the human equivalent dose of approximately 50 BIU by carrying out daily iv injections on 5 successive days/week for 3 weeks in rats.
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during validation runs indicated that the intended dose reached the end of the catheter. Treatment plan and dosages. The study protocol was approved by the Hadassah University Institutional Review Board. All patients were already receiving anti-convulsants because of their brain tumors and surgery. The first part of the study utilized a modified Simons Type 4B intrapatient, single-step, dose-accelerated dosage titration scheme [48]. Patients received one cycle of five consecutive daily doses at each dosage step. Dose escalation steps were 0.1, 0.32, 0.93, 5.9, and 11 BIU of NDV-HUJ. A dosage cycle was 7 days for the 0.1 and 0.32 BIU steps and 14 days for the higher dosages. A patient started accelerated dose escalation at the next highest dose above the one completed without DLT by previously enrolled patients. A patient advanced to the next accelerated dosage step after having completed the previous step without DLT. A patient completing the 11-BIU step received three additional cycles of 55 BIU. Patients with progressive disease were withdrawn from the study. The second constant dosing part of the study utilized three cycles at 11 BIU. Upon completion of the study dosing, patients without disease progression received maintenance dosing consisting of two iv doses of 11 BIU weekly. Follow-up continued for all patients after disease progression. Measurement of tolerability. Patients were extensively monitored before and after each treatment. Evaluations included physical examinations and clinical and neurological assessment. The National Cancer Institute CTC version 2.0 was used for grading the severity of AEs. DLT was defined as a study-related grade 3 or 4 AE. Symptoms clearly due to the underlying disease were not considered as DLTs. Measurement of effect. Efficacy was assessed by tumor response measured by Gd+ MRI. Patients were evaluated for response within 2 weeks following completion of the study dosing and again 10 weeks later. During maintenance dosing, patients were evaluated at 4- to 8week intervals. Tumor response was determined from the change in the tumor’s cross section (cm2) and was scored as CR, PR, stable disease, or progressive disease using standard criteria [49]. Clinical diagnosis of progressive disease was made by clinical/neurological deterioration in conjunction with increase in size of tumor determined by CT or MRI. Measurement of viral recovery. Blood, urine, and saliva samples were collected at various time intervals, stored at 708C, and later evaluated for NDV-HUJ viral recovery. If cerebral spinal fluid and biopsy samples were available these were also analyzed. Samples were screened for infectious virus by inoculation into the allantoic and yolk sacs of 10to 11-day-old embryonated eggs and checking the fluids for qualitative and quantitative hemagglutination (HA) 72–96 h postinoculation according to routine methods [46,50]. If sample was found to be negative in qualitative HA test, samples were further passaged three times to enable viral multiplication and further evaluated for qualitative/quantitative HA.
Viral assay. Virus titers were expressed as BIU measured by determination of egg infectious dose (1 BIU = 1 109 50% egg infectious dose (EID50) units). EID50 was determined by inoculation of serial dilutions into the allantoic sac of 10- to 11-day-old embryonated eggs and checking the fluids for hemagglutination 72–96 h postinoculation according to routinely used methods [46]. The EID50 value was calculated using the method of Reed and Muench [47].
Measurement of anti-NDV hemagglutinin antibodies. Serum samples were also collected at specific time intervals, stored at 708C, and tested for anti-NDV hemagglutinin antibodies by hemagglutination inhibition assay [46,50]. Sera were treated with receptor-destroying enzyme overnight and heat inactivated and serial twofold dilutions of inactivated sera were prepared, reacted with 4 hemagglutinating units of viral suspension, and incubated at room temperature for 30 min. Washed, 0.5% chicken RBCs were added and allowed to settle at room temperature. The endpoint used to designate the quantity of hemagglutination inhibition (HI) antibodies present in serum was the highest (last) dilution of serum that completely inhibits hemagglutination.
Intravenous administration of NDV-HUJ. One milliliter of a thawed and sonicated NDV-HUJ suspension was diluted into 20 ml of saline in a burrette (Voluset, Teva Medical) immediately before use and administered over 15 min through a peripheral or central venous line. Titer assays
Additional measurements. In conjunction with Gd+ MRI measurements, rCBV and single-voxel MRS measurements were performed. PET measurements using FDG-PET were carried out parallel to MRI measurements at baseline and at 2 and 10 weeks following study dosing.
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ACKNOWLEDGMENTS We thank nurse coordinators Sara Mizrachi and Yael Liberman and the Oncology Department of the Hadassah University Hospital for the clinical study and Gili Focht and Inbal Paz for study management. RECEIVED FOR PUBLICATION MAY 19, 2005; REVISED AUGUST 30, 2005; ACCEPTED AUGUST 30, 2005.
APPENDIX A. SUPPLEMENTARY DATA Supplementary data associated with this article can be found in the online version at doi:10.1016/j.ymthe. 2005.08.016. REFERENCES 1. Prados, M. D., and Levin, V. (2000). Biology and treatment of malignant glioma. Semin. Oncol. 27: 1 – 10. 2. Davis, F. G., et al. (1998). Survival rates in patients with primary malignant brain tumors stratified by patient age and tumor histological type: an analysis based on surveillance, epidemiology, and end results (SEER) data, 1973-1991. J. Neurosurg. 88: 1 – 10. 3. Wong, E. T., et al. (1999). Outcomes and prognostic factors in recurrent glioma patients enrolled onto phase II clinical trials. J. Clin. Oncol. 17: 2572. 4. Mischel, P. S., and Cloughesy, T. F. (2003). Targeted molecular therapy of GBM. Brain Pathol. 13: 52 – 61. 5. Rich, J. N., and Bigner, D. D. (2004). Development of novel targeted therapies in the treatment of malignant glioma. Nat. Rev. Drug Discovery 3: 430 – 446. 6. Fulci, G., and Chiocca, E. A. (2003). Oncolytic viruses for the therapy of brain tumors and other solid malignancies: a review. Front. Biosci. 8: e346 – e360. 7. Kirn, D., et al. (2001). Replication-selective virotherapy for cancer: biological principles, risk management and future directions. Nat. Med. 7: 781 – 787. 8. Shah, A. C., et al. (2003). Oncolytic viruses: clinical applications as vectors for the treatment of malignant gliomas. J. Neurooncol. 65: 203 – 226. 9. Vecil, G. G., and Lang, F. F. (2003). Clinical trials of adenoviruses in brain tumors: a review of Ad-p53 and oncolytic adenoviruses. J. Neurooncol. 65: 237 – 246. 10. Chiocca, E. A., et al. (2004). A phase I 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. 10: 958 – 966. 11. Markert, J. M., et al. (2000). Conditionally replicating herpes simplex virus mutant, G207, for the treatment of malignant glioma: results of a phase I trial. Gene Ther. 7: 867 – 874. 12. Todo, T., et al. (2000). Viral shedding and biodistribution of G207, a multimutated, conditionally replicating herpes simplex virus type 1, after intracerebral inoculation in aotus. Mol. Ther. 2: 588 – 595. 13. Phuong, L. K., et al. (2003). Use of a vaccine strain of measles virus genetically engineered to produce carcinoembryonic antigen as a novel therapeutic agent against glioblastoma multiforme. Cancer Res. 63: 2462 – 2469. 14. Thorne, S. H., and Kirn, D. H. (2004). Future directions for the field of oncolytic virotherapy: a perspective on the use of vaccinia virus. Expert Opin. Biol. Ther. 4: 1307 – 1321. 15. Wilcox, M. E., et al. (2001). Reovirus as an oncolytic agent against experimental human malignant gliomas. J. Natl. Cancer Inst. 93: 903 – 912. 16. Sinkovics, J. G., and Horvath, J. C. (2000). Newcastle disease virus (NDV): brief history of its oncolytic strains. J. Clin. Virol. 16: 1 – 15. 17. National Cancer Institute on Newcastle disease virus as a treatment for cancer, online at http://www.nci.nih.gov/cancerinfo/pdq/cam/NDV. 18. Bar-Eli, N., et al. (1996). Preferential cytotoxic effect of Newcastle disease virus on lymphoma cells. J. Cancer Res. Clin. Oncol. 122: 409 – 415. 19. Cassel, W. A., and Garrett, R. E. (1965). Newcastle disease virus as an antineoplastic agent. Cancer 18: 863 – 868. 20. Lorence, R. M., et al. (1994). Complete regression of human fibrosarcoma xenografts after local Newcastle disease virus therapy. Cancer Res. 54: 6017 – 6021. 21. Lorence, R. M., et al. (1994). Complete regression of human neuroblastoma xenografts in athymic mice after local Newcastle disease virus therapy. J. Natl. Cancer Inst. 86: 1228 – 1233.
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22. Phuangsab, A., et al. (2001). Newcastle disease virus therapy of human tumor xenografts: antitumor effects of local or systemic administration. Cancer Lett. 172: 27 – 36. 23. Reichard, K. W., et al. (1992). Newcastle disease virus selectively kills human tumor cells. J. Surg. Res. 52: 448 – 453. 24. Schirrmacher, V., et al. (2001). Antitumor effects of Newcastle disease virus in vivo: local versus systemic effects. Int. J. Oncol. 18: 945 – 952. 25. Tzadok-David, Y., et al. (1995). The effect of a mesogenic and a lentogenic Newcastle disease virus strain on Burkitt lymphoma Daudi cells. J. Cancer Res. Clin. Oncol. 121: 169 – 174. 26. Cassel, W. A., and Murray, D. R. (1992). A ten-year follow-up on stage II malignant melanoma patients treated postsurgically with Newcastle disease virus oncolysate. Med. Oncol. Tumor Pharmacother. 9: 169 – 171. 27. Csatary, L. K., et al. (2004). MTH-68/H oncolytic viral treatment in human high-grade gliomas. J. Neurooncol. 67: 83 – 93. 28. Lorence, R. M., et al. (2003). Overview of phase I studies of intravenous administration of PV701, an oncolytic virus. Curr. Opin. Mol. Ther. 5: 618 – 624. 29. Pecora, A. L., et al. (2002). Phase I trial of intravenous administration of PV701, an oncolytic virus, in patients with advanced solid cancers. J. Clin. Oncol. 20: 2251 – 2266. 30. Steiner, H. H., et al. (2004). Antitumor vaccination of patients with glioblastoma multiforme: a pilot study to assess feasibility, safety, and clinical benefit. J. Clin. Oncol. 22: 4272 – 4281. 31. Yung, W. K., et al. (2000). A phase II study of temozolomide vs. procarbazine in patients with glioblastoma multiforme at first relapse. Br. J. Cancer 83: 588 – 593. 32. Brada, M., et al. (2001). Multicenter phase II trial of temozolomide in patients with glioblastoma multiforme at first relapse. Ann. Oncol. 12: 259 – 266. 33. Nutt, C. L., et al. (2003). Gene expression-based classification of malignant gliomas correlates better with survival than histological classification. Cancer Res. 63: 1602 – 1607. 34. Hanson, R. P. (1964). Newcastle Disease Virus—An Evolving Pathogen, pp. 205–220. Univ. of Wisconsin Press, Madison/Milwaukee. 35. OIE (World Organisation for Animal Health) (2000). Manual of Standards for Diagnostic Tests and Vaccines; online at http://www.oie.int/eng/normes/mmanual/A_00036.htm. 36. Washburn, B., and Schirrmacher, V. (2002). Human tumor cell infection by Newcastle disease virus leads to upregulation of HLA and cell adhesion molecules and to induction of interferons, chemokines and finally apoptosis. Int. J. Oncol. 21: 85 – 93. 37. Schirrmacher, V. (2005). Clinical trials of antitumor vaccination with an autologous tumor cell vaccine modified by virus infection: improvement of patient survival based on improved antitumor immune memory. Cancer Immunol. Immunother. 54: 587 – 598. 38. Levy, D. E., and Marie, I. J. (2004). RIGging an antiviral defense—It’s in the CARDs. Nat. Immunol. 5: 699 – 701. 39. Zeng, J., et al. (2002). Induction of interferon-a and tumor necrosis factor-related apoptosis-inducing ligand in human blood mononuclear cells by hemagglutinin– neuraminidase but not F protein of Newcastle disease virus. Virology 297: 19 – 30. 40. Kadowaki, N., et al. (2000). Natural interferon alpha/beta-producing cells link innate and adaptive immunity. J. Exp. Med. 192: 219 – 226. 41. Zeng, J., et al. (2002). Stimulation of human natural interferon-alpha response via paramyxovirus hemagglutinin lectin–cell interaction. J. Mol. Med. 80: 443 – 451. 42. Fournier, P., et al. (2003). Two ways to induce innate immune responses in human PBMCs: paracrine stimulation of IFN-alpha responses by viral protein or dsRNA. Int. J. Oncol. 23: 673 – 680. 43. Schirrmacher, V., et al. (2000). Newcastle disease virus activates macrophages for antitumor activity. Int. J. Oncol. 16: 363 – 373. 44. Umansky, V., et al. (1996). Induction of NO synthesis in macrophages by Newcastle disease virus is associated with activation of nuclear factor-kappa B. Int. Immunol. 8: 491 – 498. 45. Slosaris, M., et al. (1989). Elevated virulence of Newcastle disease virus strains following serial passages in kidney cells in vitro. Avian Dis. 33: 248 – 253. 46. Sever, J. L. (1962). Application of a microtechnique to viral serological investigations. J. Immunol. 88: 320 – 329. 47. Reed, L., and Muench, J. A. (1938). A simple method of estimating 50 percent end points. Am. J. Hyg. 27: 493. 48. Simon, R., et al. (1997). Accelerated titration designs for phase I clinical trials in oncology. J. Natl. Cancer Inst. 89: 1138 – 1147. 49. Macdonald, D. R., et al. (1990). Response criteria for phase II studies of supratentorial malignant glioma. J. Clin. Oncol. 8: 1277 – 1280. 50. Allan, W., Lancaster, J. E., and Toth, B. (1978). Newcastle Disease Vaccines: Their Production and Use. Food and Agriculture Organization of the United Nations, Rome, 1978.
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Phase I/II Trial of Intravenous NDV-HUJ Oncolytic Virus in Recurrent Glioblastoma Multiforme Arnold I. Freeman,1 Zichria Zakay-Rones,2 John M. Gomori,3 Eduard Linetsky,4,5 Linda Rasooly,1 Evgeniya Greenbaum,2 Shira Rozenman-Yair,6 Amos Panet,2 Eugene Libson,7 Charles S. Irving,6 Eithan Galun,1,* and Tali Siegal5 1
Goldyne Savad Institute of Gene Therapy, 3Department of Neuro-radiology, 4Department of Neurology, 7Department of Radiology, and 5 Gaffin Center for Neuro-Oncology, Hadassah University Hospital, Jerusalem 91120, Israel 2 Department of Virology, Hebrew University, Jerusalem 91120, Israel 6 Theravir Management LP, Jerusalem, Israel *To whom correspondence and reprint requests should be addressed. Fax: +972 2 642 9856. E-mail: [email protected].
Available online 28 October 2005
We undertook a Phase I/II trial in patients with apparent recurrent glioblastoma multiforme (GBM) based on imaging studies to determine the safety and tumor response of repetitive intravenous administration of NDV-HUJ, the oncolytic HUJ strain of Newcastle disease virus. The first part of the study utilized an accelerated intrapatient dose-escalation protocol with one-cycle dosage steps of 0.1, 0.32, 0.93, 5.9, and 11 billion infectious units (BIU) of NDV-HUJ (1 BIU = 1 109 EID50 50% egg infectious dose) followed by three cycles of 55 BIU. Virus was administered by intravenous infusion over 15 min. In the second part, patients received three cycles of 11 BIU. All patients without progressive disease were maintained with two doses of 11 BIU iv weekly. Eleven of the 14 enrolled patients (11–58 years, Karnofsky performance scale 50–90%) received treatment. Toxicity was minimal with Grade I/II constitutional fever being seen in 5 patients. Maximum tolerated dose was not achieved. Anti-NDV hemagglutinin antibodies appeared within 5–29 days. NDV-HUJ was recovered from blood, saliva, and urine samples and one tumor biopsy. One patient achieved a complete response. Intravenous NDV-HUJ is well tolerated. The findings of good tolerability and encouraging responses warrant the continued evaluation of NDV-HUJ in GBM, as well as other cancers. Key Words: Newcastle disease virus, NDV-HUJ, oncolytic virus, intravenous, glioblastoma, brain cancer, phase I/II trial, dose escalation, complete remission, apoptosis
INTRODUCTION Primary malignant gliomas are among the most lethal and difficult forms of cancer. Of these glioblastoma multiforme (GBM) is the most rapidly growing and most challenging [1]. Despite improvements, the outcome for GBM patients has not changed significantly over the past 30 years [2]. With maximal conventional therapy, survival is approximately 14 months from time of diagnosis and about 30 weeks from the time of recurrence [3]. Recent advances in the understanding of brain tumor biology have led to the development of novel therapeutic approaches [4,5], including oncolytic virotherapy [6–8]. Viruses, particularly adenovirus [9,10] and HSV [11,12], have been engineered to deliver therapeutic genes, while other viruses [13–15] have been utilized without engineering for their innate cytotoxic effects. Among the nonengineered oncolytic viruses, Newcastle disease virus (NDV) [16] has a long history as a
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broad-spectrum oncolytic agent that can destroy tumor cells and stimulate the immune system [17–25]. NDV is a single-stranded RNA virus, whose natural host is poultry. The 73-T [26], MTH68 [27], and PV701 (MK107) [28,29] strains of NDV have been the subject of several clinical studies. NDV-PV701 has recently been evaluated in a Phase I study of patients with advanced solid tumors; however, patients with CNS tumors were excluded from this study [29]. Anecdotal responses to MTH68 in malignant glioma have been reported [27]. NDV strains that are not pathogenic to poultry (lentogenic) have also been shown to kill cancer cells [24] but possibly by different mechanisms [17]. Infection of tumor cells by lentogenic NDV generates several innate danger signals leading to apoptosis. The lentogenic Ulster strain of NDV has been combined with various tumor cells as a tumor vaccine for different cancers, including GBM [30], but the use of a lentogenic NDV strain alone in oncolytic
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virotherapy has not been evaluated. HUJ is a new lentogenic strain of NDV (NDV-HUJ) that has a selective cytopathogenicity for human and animal cancer cell lines (L. Rasooly, E. Ovin, N. Hooshi, and E. Galun, unpublished results; Z. Zakay-Rones and A. Panet, unpublished results). We here present the first clinical evaluation of a lentogenic NDV strain administered systemically and the first evaluation of any NDV strain in GBM patients. We evaluated patients with recurrent GBM in a singlesite, open-label Phase I/II study of NDV-HUJ. The study was divided into two parts. The initial part of the study utilized a six-step intrapatient accelerated doseescalation scheme. In this scheme, patients advanced to the next higher dosage step after all patients had completed the lower step without any dose-limiting toxicities (DLT). Newly enrolled patients started at one dosage step higher than the one completed without DLT by all enrolled patients. The second part of the study utilized a constant dosing step, which was one lower than the highest step reached in the first part of the study.
RESULTS AND DISCUSSION Study Design, Patients, and Eligibility The intrapatient accelerated dose-escalation scheme provided each patient with the opportunity to receive a high and more likely potentially effective dose of NDV-HUJ. The lowest dosage step of 0.1 billion infectious units (BIU) corresponded to a previously reported NDV intravenous dosage [27]. The highest dosage step of 55 BIU was determined by preclinical toxicity testing. The step down from 55 to 11 BIU used in the second part of the study was intended to lower the risk of adverse events and was determined in part by the limited amount of
NDV-HUJ available for the study due to the high cost of initial production. We enrolled 14 patients (9 men and 5 women, ages 11 to 58 years) from December 2002 to December 2003 (Table 1, Fig. 1). All patients had been diagnosed with GBM based on histology and gadolinium-enhanced (Gd+) MRI, and all had a recurrence of GBM based on MRI evidence of mass effect associated with T1 gadolinium enhancement, T2 prolongation, elevated relative cerebral blood volume (rCBV) (3), and/or high choline on magnetic resonance spectroscopy (MRS). Three of the patients enrolled in Part II of the study were withdrawn during the screening period prior to starting NDV virotherapy as follows: Patient 08 died, 12 was found to have necrosis and not recurrent GBM upon repeat biopsy, and 14 had an abrupt deterioration in clinical condition. Prior therapy for the 11 assessable patients is listed in Table 1. Eight patients had at least one partial or full resection, all patients had received radiotherapy, and 9 had received temozolomide and/or intra-arterial carboplatin chemotherapy. Nine of the evaluable patients were diagnosed as GBM 17 to 49 weeks prior to virotherapy without prior history of low-grade gliomas, whereas 2 were diagnosed as GBM 5 to 6 weeks before virotherapy after having been previously diagnosed with lower grade glioma. The dosage steps reached by the patients are given in Table 2 and shown in Supplementary Fig. 1. Patients 02, 05, and 06 in Part I completed all three cycles of the 55 BIU maximum dose Step 6, while Patient 01 was withdrawn in the middle of the third cycle of Step 6 due to clinical and radiological disease progression. In Part II, Patients 07, 09, and 11 completed all three cycles of dosage Step 5 (11 BIU). Of the six patients that completed their last dosage step (Fig. 1), all except for Patient 07 continued with main-
TABLE 1: Patient characteristics Sex
Age (years)
Baseline KPS
Type GBM
Dx to VT (weeks)
Surgical procedures
Prior Rx
01 02 03
M M F
50 58 51
50 70 70
I I I
20 29 17
B B B
RT, TMZ RT, IAC RT
04 05 06 07 08 09 10 11 12 13 14
F F F F M M M M M M M
32 54 58 57 49 43 11 56 27 31 46
90 90 80 90 70 80 70 90 60 90 50
I I I I I I II I I II I
49 43 34 29 48 41 5 18 49 6 61
GTR GTR, GTR GTR, GTR GTR B PR B, PR GTR PR, GTR, GTR, GTR GTR, GTR, GTR PR
RT, IAC, TMZ RT, TMZ RT, IAC RT, IAC RT, IAC, TMZ RT, IAC, TMZ RT, TMZ RT RT, TMZ RT, TMZ RT, TMZ
Patient
Tumor location
Baseline tumor cross section (cm2)
R frontal L parietal R frontal + basal ganglia L temporal R parietal R frontal L frontal R temporal L parietal R parietal R frontal R parietal L frontal R parietal
31.5 34.8 16.0 12.5 2.4 11.5 20.5 14.4 9.5 38.4 12.9 16.3 45.0 22.3
Abbreviations: M, male; F, female; KPS, Karnofsky performance scale; Dx, diagnosis; GBM, glioblastoma multiforme; I, no previous diagnosis of a lower grade glioma; II, previous history of a lower grade glioma; VT, viral therapy; B, biopsy; PR, partial resection; GTR, gross total resection; Rx, treatment; RT, radiotherapy; IAC, intra-arterial carboplatin; TMZ, temozolomide; R, right; L, left.
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FIG. 1. Patient enrollment.
review of this patient’s MR imaging revealed brain stem involvement that was not appreciated at the time of enrollment. As seen in Table 3, neurological seizures occurred in 8 patients. Of the 15 seizures noted, 5 were classified as Grade 3 and 1 as Grade 4, according to the NDC Common Toxicity Criteria (CTC) version 2. Comparison of these seizures with the patients’ medical histories indicated that there were no changes in their frequency or quality following NDV administration. The stupor that occurred in Patient 03 (Table 3) was considered due to an enlarging brain tumor and is commonly seen in this situation. The patient’s MRI was consistent with progressive tumor and did not display new features suggestive of encephalopathy. None of the patients showed clinical findings indicative of inflammatory responses such as encephalitis/encephalopathy, meningitis, or other inflammatory responses; these are a concern for viral therapy in cancer patients. Toxicity of repeated intravenous NDV dosing was reported in Phase I studies of NDV PV701. They focused on acute adverse events (AEs) during infusion, general flulike AEs, and tumor-site-specific AEs. The AEs were reduced by priming dosing and following slower infusion
tenance dosing until radiological disease progression. Additional treatments that the patients received following withdrawal from maintenance dosing are given in Table 2. Time to progression (clinical and radiological) and overall survival are given for reference in Table 2, although we did not include them in the study as outcome measures. Survival ranged from 3 to 66 weeks from the start of virotherapy. Time to radiological progression ranged from 2 to 37 weeks. Although withdrawn from treatment at 8 weeks due to apparent radiological progression without clinical deterioration and subsequent treatment with temozolomide, Patient 11 continued to improve clinically and was free of disease progression out to 44 weeks. Toxicity We included 11 patients in the toxicity analysis (Table 3). Five patients developed fever, usually during their first cycle. Most patients also experienced adverse events that we determined to be unrelated or unlikely to be related to their virotherapy. One patient (No. 04) died on study due to disease progression and this was independently validated by the hospital’s safety board. A subsequent
TABLE 2: Summary of patient treatment outcomes Patient
Dosage steps started
01 02 03 04 05 06 07 09 10 11 13
1, 2, 3, 4, 5, 6, 6, 6 2, 3, 4, 5, 6, 6, 6, M 4, 5, 6 4, 5 5, 6, 6, 6, M 5, 6, 6, 6, M 5, 5, 5 5, 5, 5, M 5, 5 5, 5, 5, M 5, 5
Withdrawn during Step 5 or 6 X X X
X X
TTP (weeks) Imaging Clinical 15 23 CT CT 17 8 37 2 8 CT
12 23 4 3 53 18 9 37 2 44 4
OS from start of VT (weeks) 20 37 13 3 66 32 38 61 19 62 15
Additional therapies after withdrawal from VT
TMX, thyroid ablation TMZ IAC TMZ, IAC
Patients 08, 12, and 14 were withdrawn during baseline screening. Abbreviations: dosage steps in BIU, 1 = 0.1, 2 = 0.32, 3 = 0.93, 4 = 5.9, 5 = 11, 6 = 55; M, maintenance of dosage Step 5 twice weekly; TTP, time to progression; CT, tumor progression confirmed by CT following clinical progression; OS, overall survival; VT, viral therapy; TMX, tamoxifen; TMZ, temozolomide; IAC, intra-arterial carboplatin.
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TABLE 3: Adverse events in dosed patients Adverse event
1
No. of events by CTC grade 2 3
4
Total No. of patients with event of any grade (% pts)
Possibly or probably related to treatment Constitutional symptoms, fever
6
1
0
0
5 (45)
Unrelated or unlikely related to treatment Neurological, seizures Neurological, stupor Neurological, syncope Pain, headache Pain, abdominal Cardiovascular, hypertension Cardiovascular, thrombosis Cardiovascular, edema Infection, otitis media, without neutropenia, fever Gastrointestinal, vomiting Pulmonary, cough
0 0 0 1 1 1 0 1 1 0 0
9 0 0 4 0 0 0 0 4 0 1
5 1 2 1 0 0 2 0 0 1 0
1 0 0 0 0 0 0 0 0 0 0
8 1 1 3 1 1 2 1 3 1 1
(73) (9) (9) (27) (9) (9) (18) (9) (27) (9) (9)
Results are reported as independent events. More than one event may have occurred per patient or the same even may have occurred more than once in a given patient.
times [28]. In our study, the only AE related to NDV-HUJ administration was Grades I and II fever that occurred during the first cycle and resolved within several days. Other AEs, such as seizures, had occurred previously in the patients and were related to their underlying cancer. We determined them to be unrelated or unlikely to be related to treatment. The observed neurological AEs were not suggestive of increasing intracranial pressure or inflammatory response seen with viral encephalitis. No acute adverse events occurred during the infusion period, even though NDV-HUJ was administered rapidly over 15 min. The lack of AEs in our study compared to those seen in the NDV PV701 Phase I studies might be due to the lower doses administered in this study (11–55 BIU vs 24 to 96 billion PFU/m2), the inherent differences between the NDV strains, the high dose steroids that patients received as part of the GBM management, or a different spectrum of cancer patients. For these reasons, no priming doses were required to overcome the tachyphylactic-like reactions seen in studies using PV701. We observed no DLT and maximum tolerated dose is greater than the 55 BIU administered on 5 consecutive days for three cycles. Tumor Response All patients had obvious measurable disease (N2 cm2 contrast-enhanced tumor cross section) at treatment initiation (Table 1). As seen in Fig. 2, one patient (No. 09) had stable disease at first follow-up, a partial remission at second follow-up, and complete remission during maintenance dosing, at which time there was a significant improvement in neurological status and corticosteroid therapy was discontinued (tumor cross sections before, during, and after the study are given in Supplementary Table 1). The complete response did not prove durable (Table 2), and on routine imaging 3 months after complete disappearance of the tumor a lesion subsequently shown
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by histology to be GBM reappeared in the tumor bed. Three patients (Nos. 02, 05, and 06) had an increase in enhanced tumor cross section without a deterioration of their neurological status. As part of their clinical management, their tumors were biopsied. Perivascular lymphocytic infiltrates were found in the biopsied tumor of all three patients, 02, 05, and 06. The infiltrates were similar to those that have been described previously in the context of GBM and/or radiation-induced damage. Another patient (No. 11) did not have a change in neurological status and did not have an increase in enhanced tumor cross section, but was judged to have radiological disease progression based on increased enhancement within the resection cavity and changes in the FLAIR and rCBV MR images. As seen in Table 2, the time to clinical progression in Patients 05 and 11 occurred considerably later than their radiological progression, suggesting that patients can have radiological progressive disease that does not lead to immediate clinical deterioration. Of the three long-term survivors, two (Patients 05 and 11) had early radiological progression. All patients in the study eventually developed both clinical and radiological progression (Table 2). Molecular imaging has the potential of providing much-needed additional information on the response of glioma to novel therapies. In our study, postoperative changes in the tumoral bed made obtaining good rCBV and MRS difficult and increased the chances for misdiagnosis of radiation necrosis for recurrent glioblastoma at baseline. In the case of Patient 12 the baseline rCBV and MRS study was significantly questionable to warrant a biopsy prior to the start of dosing. The finding of radiation necrosis led to the screening withdrawal of the patient. In the few cases for which adequate data were available, rCBV and MRS confirmed the changes detected by conventioQ nal imaging, but did not add additional information.
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FIG. 2. Complete tumor response. (A) Patient 09 at baseline, (B) stable disease at first follow-up, (C) partial response (PR) at second follow-up, (D) PR at 20 weeks from start of virotherapy, and (E and F) complete response at 25 and 30 weeks from start of virotherapy.
[18F]Fluorodeoxyglucose positron emission tomography (FDG-PET) suffered from difficulty in distinguishing tumor from adjacent tissue and collateral reference sites. Response rates (partial response (PR) + complete response (CR)) in patients with recurrent GBM treated with the current gold standard, temozolomide, are low. Complete responses are particularly rare, being observed in 0/112 [31] and 2/128 [32] patients in multicenter studies of GBM at first relapse and in 1/375 patients [3] in the largest such study of recurrent GBM. We are unaware of any reports of spontaneous regression of GBM. The complete response seen in this study was accompanied by rapid neurological improvement and the gradual cessation of corticosteroids. At time of entry onto the NDV study this patient had measurable disease, which failed radiation therapy and two lines of chemotherapy. The possibility that the baseline Gd+ MR image represented tumor radiation necrosis and not recurrent GBM appears very unlikely on the basis of mass effect, elongated T2, a rCBV value of 3, and high choline on MRS, but cannot be completely ruled out in the absence of histologically proven recurrence. Diagnostically challenging malignant gliomas with nonclassical glioblastoma histologies can be mistaken in the absence of gene expression profiling for nonclassical anaplastic oligodendrogliomas [33], which have better survival and time to tumor progression. An explanation of the patient’s complete response due to misdiagnosis of necrosis or anaplastic oligodendroglioma
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is unlikely due to classical histology of glioblastoma found at the time of original diagnosis and upon recurrence following NDV-HUJ therapy. Since there is no other explanation for this complete response, we consider that it resulted from NDV-HUJ therapy. Antibody Response All 14 enrolled patients were tested at baseline for antiNDV hemagglutinin antibodies and were negative (Supplementary Table 2). Three patients (06, 09, and 10) developed antibodies during the first week of treatment. All patients developed antibodies by the third week, except for 2 patients (05 and 11), who developed antibodies only by the fifth week at the start of the third treatment cycle and by the eighth week at the start of maintenance dosing, respectively. In patients receiving long-term therapy, antibodies had either plateaued or started to decrease slightly by the eighth week. Antibody titers remained low throughout repetitive dosing. Virology We tested a total of 101 blood, saliva, and urine samples from five patients (01, 02, 03, 05, and 09) for infectious NDV by inoculation into the allantoic sac of embryonated eggs, a highly sensitive biological infectivity assay that indicates the presence or absence of infectious virus. All baseline samples were negative. For all five patients we recovered infectious NDV particles from blood, urine, and
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saliva samples taken during the first dosage cycle (Supplementary Table 3). We continued to recover infectious NDV particles from blood, saliva, and urine samples after the patients developed anti-hemagglutinin NDV antibodies. We also recovered infectious NDV from 9 of the 10 blood samples taken 9 days after the last dosage of NDV-HUJ from the previous dosage cycle. We recovered infectious NDV particles from a tumor biopsy sample of Patient 02, but not from the biopsy of Patient 05. Tumor cystic fluid samples obtained via an implanted Ommaya device from Patient 02 also tested positive for infectious NDV. Identity of the isolated virus was determined using anti-NDV antibodies. The recovery of infectious NDV particles from the tumor’s cyst fluid and the tumor tissue obtained by biopsy performed about 130 days after the start of virotherapy indicated that the NDV-HUJ reached the extracellular space of the tumor. It is likely that anti-NDV antibodies interfered with the potency of intravenously administered NDV, but not to the extent that all infectious particles were removed from circulation. Infectious NDV particles were found in the blood of patients after the patients’ serum anti-NDV antibody levels had reached plateau levels and were repeatedly detected 9 days following the last administration of virus. Further, Patient 09, who achieved a CR, also developed antibodies early. In future studies posttreatment tumor biopsies should be taken into consideration, when ethically justifiable. NDV-HUJ and Its Possible Mechanisms of Action Very little attention has been given to the safety and mechanisms of action of NDV strains, such as NDV-HUJ, whose pathogenicities for their natural avian hosts are attenuated to the extent of being nonpathogenic (lentogenic), which allows these strains to be used as poultry vaccines. The three major strains of NDV that have been evaluated in clinical studies, 73-T, MTH68, and PV701 (Massachusetts MK107) [34], are classified as mesogenic (moderately pathogenic) strains, whose use and importation are banned in many countries [35]. Mesogenic strains are able to replicate and produce viable progeny virus, which can infect adjacent cells. Their production of viable progeny virus is due in part to the conversion of the viral fusion surface protein from an inactive (F0) to an active (F1) form. This is made possible by a match between the proteases present in host tissues and the sequence of the cleavage site of the fusion protein that requires at least two basic (lysine or arginine) amino acids, between residues 113 and 116, and a phenylalanine at residue 117. The lentogenic HUJ strain has a 112G-R-Q-G-R-L117 sequence. In most tissues, lentogenic strains produce defective progeny, are monocyclic, and cannot easily spread between tissues. The replication of the HUJ strain in humans is probably quite limited, although the continued presence of circulating infectious particles for up to 9 days after dosing seen in
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this study suggests that limited replication in selected tissues, possibly in tumor tissue, occurs. Recent studies have suggested a number of mechanisms relevant to tumor virotherapy that do not involve the production of viable progeny virus particles. Infection of g-irradiated NCH 149 GBM cells by lentogenic NDV Ulster has been reported to lead to cytokine/chemokine induction, changes in cell surface molecules, and the induction of apoptosis [36]. Additional innate and adaptive immune responses have been associated with lentogenic NDV infection of tumor cells [37]. The release of NDV dsRNA into cytoplasm [38] and expression of HN on the cell surface [39] are two immune danger signals that increase IRF-3, IFN-a, TRAIL, TLR, and HSP levels [40,41]. Expression of HN on cell surfaces induces IFN-a in PBMCs [42]. The lentogenic LaSota and Ulster strains were shown to induce anti-tumor cytotoxicity in mouse macrophages and human monocytes [43]. These strains activated NF-nB in mouse macrophages and stimulated NO production [44]. We found that NDV-HUJ induces the inhibition of metabolism and cell death in C6 and RG2 rat malignant glioma cells and fibrosarcoma HT1080-positive control cells, whereas normal fibroblast MS5 cells were unaffected (L. Rasooly, E. Ovin, N. Hooshi, and E. Galun, unpublished results). The presence of apoptotic RG2 cells following incubation with NDV-HUJ suggested that the mechanism for the cytopathic effect of NDV-HUJ on malignant glioma cells is primarily apoptotic (L. Rasooly, E. Ovin, N. Hooshi, and E. Galun, unpublished results). The cytopathic effect of NDV-HUJ on Burkitt lymphoma Daudi cells has also been associated with apoptosis (D. Horesh, A. Panet, and Z. Zakay-Rones, unpublished results). Future Studies Further investigation of the mechanisms of action of the lentogenic strains of NDV is warranted in view of the results seen in this study, as well as the recent report of clinical benefit of lentogenic NDV-modified autologous tumor cell vaccine in GBM patients [30]. Future preclinical studies should be directed toward determining the role of NDV-HUJ selectivity and targeting for GBM cells in its anti-GBM tumor effects in vivo. The absence of major side effects and the complete tumor response seen in one patient provide initial evidence of a potential new concept of systemic virotherapy of GBM using lentogenic NDV and warrants further validation in a larger clinical trial. Future clinical studies may benefit from observations made in this study. In designing treatment schedules, advantage may be taken of the time prior to the appearance of anti-NDV antibodies. Further, the absence of major side effects at the maximum dosing indicates that significantly higher dosages might be explored, as well as a continuous daily dosing schedule. Molecular imaging techniques should be explored for their ability to provide additional information on tumor response. Inclusion criteria should include tumor biopsy
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and gene expression profiling, if possible. Withdrawal from treatment based solely on Gd+ MRI radiological progression should be reconsidered when designing future studies. Virotherapy using lentogenic NDV strains should also be evaluated in patients with lower grade gliomas.
PATIENTS AND METHODS Inclusion and exclusion criteria. Patients were required to have GBM histologically confirmed by the neuropathologist at Hadassah University Hospital and a tumor measurable by Gd+ MRI after failing conventional therapy. At least 4 weeks from completion of chemotherapy or radiotherapy was needed prior to entry into our study. Patients had to be between 3 and 70 years of age, be not pregnant, have an estimated life expectancy of greater than 2 months, have a Karnofsky performance status of 50% or greater, and be receiving a stable or decreasing dose of corticosteroids without an expected need for increase in dosing. Hepatic, renal, and bone marrow function requirements were Hb N 9 g%, WBC N 1000/mm3, platelet count N 30,000 mm3, creatinine b 2.5 mg%, liver function tests less than twice normal. Patients were not allowed to receive any investigational agents other than NDV-HUJ or any other anti-cancer agent during the study. A history of allergy to egg ovalbumin was a cause for exclusion. Other exclusion criteria included acute severe or life-threatening infection, severe depression or psychosis, or history of noncompliance with therapy. A written informed consent document approved by the Hadassah Hospital Institutional Review Board had to be understood and signed by the patient and by a spouse, parent, or guardian. We required both to sign due to alterations of psyche and understanding in some patients with GBM. NDV-HUJ. NDV-HUJ is a highly purified isolate originally derived from the naturally attenuated B1 NDV vaccine strain (ATCC, 1971), passaged 50 times in 10- to 11-day-old embryonated chicken eggs, and cloned twice by limiting dilution in specific-pathogen-free (SPF) embryonated hen’s eggs. The HUJ strain was classified as lentogenic and having no pathogenicity for its natural host, poultry, on the basis of its intracranial pathogenicity index of 0.0 determined by the Israel Ministry of Agriculture and a 112G-R-Q-G-RL117 cleavage site sequence in its surface fusion protein, as well as having a mean death time of N100 h. These certifications are required to allow importation of NDV-HUJ samples into countries where biosafety testing was performed [35]. Cultivation, concentration, and purification were carried out using routine methods [45]. Clinical lots of NDV-HUJ were prepared by growing the virus to high titers in SPF embryonated hen’s eggs (SPAFAS-Alpez, Mexico) and purifying the virus by pelleting and resuspension followed by ultracentrifugation in sucrose gradients. The purified virus was formulated as a suspension in PBS buffer, vialed as 1.1-ml aliquots, and stored at 708C until immediately before use. Clinical lots of NDV-HUJ met the release criteria approved by the Israel Ministry of Health. These criteria included European Pharmacopoeia 4, Section 2.6.16, Tests for Extraneous Agents in Viral Vaccines for Human Use adapted for Phase I/II clinical studies in recurrent GBM patients. Preclinical toxicity studies established the nonobserved adverse effect level to be the human equivalent dose of approximately 50 BIU by carrying out daily iv injections on 5 successive days/week for 3 weeks in rats.
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during validation runs indicated that the intended dose reached the end of the catheter. Treatment plan and dosages. The study protocol was approved by the Hadassah University Institutional Review Board. All patients were already receiving anti-convulsants because of their brain tumors and surgery. The first part of the study utilized a modified Simons Type 4B intrapatient, single-step, dose-accelerated dosage titration scheme [48]. Patients received one cycle of five consecutive daily doses at each dosage step. Dose escalation steps were 0.1, 0.32, 0.93, 5.9, and 11 BIU of NDV-HUJ. A dosage cycle was 7 days for the 0.1 and 0.32 BIU steps and 14 days for the higher dosages. A patient started accelerated dose escalation at the next highest dose above the one completed without DLT by previously enrolled patients. A patient advanced to the next accelerated dosage step after having completed the previous step without DLT. A patient completing the 11-BIU step received three additional cycles of 55 BIU. Patients with progressive disease were withdrawn from the study. The second constant dosing part of the study utilized three cycles at 11 BIU. Upon completion of the study dosing, patients without disease progression received maintenance dosing consisting of two iv doses of 11 BIU weekly. Follow-up continued for all patients after disease progression. Measurement of tolerability. Patients were extensively monitored before and after each treatment. Evaluations included physical examinations and clinical and neurological assessment. The National Cancer Institute CTC version 2.0 was used for grading the severity of AEs. DLT was defined as a study-related grade 3 or 4 AE. Symptoms clearly due to the underlying disease were not considered as DLTs. Measurement of effect. Efficacy was assessed by tumor response measured by Gd+ MRI. Patients were evaluated for response within 2 weeks following completion of the study dosing and again 10 weeks later. During maintenance dosing, patients were evaluated at 4- to 8week intervals. Tumor response was determined from the change in the tumor’s cross section (cm2) and was scored as CR, PR, stable disease, or progressive disease using standard criteria [49]. Clinical diagnosis of progressive disease was made by clinical/neurological deterioration in conjunction with increase in size of tumor determined by CT or MRI. Measurement of viral recovery. Blood, urine, and saliva samples were collected at various time intervals, stored at 708C, and later evaluated for NDV-HUJ viral recovery. If cerebral spinal fluid and biopsy samples were available these were also analyzed. Samples were screened for infectious virus by inoculation into the allantoic and yolk sacs of 10to 11-day-old embryonated eggs and checking the fluids for qualitative and quantitative hemagglutination (HA) 72–96 h postinoculation according to routine methods [46,50]. If sample was found to be negative in qualitative HA test, samples were further passaged three times to enable viral multiplication and further evaluated for qualitative/quantitative HA.
Viral assay. Virus titers were expressed as BIU measured by determination of egg infectious dose (1 BIU = 1 109 50% egg infectious dose (EID50) units). EID50 was determined by inoculation of serial dilutions into the allantoic sac of 10- to 11-day-old embryonated eggs and checking the fluids for hemagglutination 72–96 h postinoculation according to routinely used methods [46]. The EID50 value was calculated using the method of Reed and Muench [47].
Measurement of anti-NDV hemagglutinin antibodies. Serum samples were also collected at specific time intervals, stored at 708C, and tested for anti-NDV hemagglutinin antibodies by hemagglutination inhibition assay [46,50]. Sera were treated with receptor-destroying enzyme overnight and heat inactivated and serial twofold dilutions of inactivated sera were prepared, reacted with 4 hemagglutinating units of viral suspension, and incubated at room temperature for 30 min. Washed, 0.5% chicken RBCs were added and allowed to settle at room temperature. The endpoint used to designate the quantity of hemagglutination inhibition (HI) antibodies present in serum was the highest (last) dilution of serum that completely inhibits hemagglutination.
Intravenous administration of NDV-HUJ. One milliliter of a thawed and sonicated NDV-HUJ suspension was diluted into 20 ml of saline in a burrette (Voluset, Teva Medical) immediately before use and administered over 15 min through a peripheral or central venous line. Titer assays
Additional measurements. In conjunction with Gd+ MRI measurements, rCBV and single-voxel MRS measurements were performed. PET measurements using FDG-PET were carried out parallel to MRI measurements at baseline and at 2 and 10 weeks following study dosing.
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ACKNOWLEDGMENTS We thank nurse coordinators Sara Mizrachi and Yael Liberman and the Oncology Department of the Hadassah University Hospital for the clinical study and Gili Focht and Inbal Paz for study management. RECEIVED FOR PUBLICATION MAY 19, 2005; REVISED AUGUST 30, 2005; ACCEPTED AUGUST 30, 2005.
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