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© 2003 American Society for Clinical Oncology Phase I Trial of Adenovirus-Mediated p53 Gene Therapy for Recurrent Glioma: Biological and Clinical Results
From the Departments of Neurosurgery, Pathology and Neuro-Oncology, The University of Texas M.D. Anderson Cancer Center, Houston, TX; Department of Neurosurgery, University of California, San Francisco, CA; Departments of Neuro-Oncology and Neurosurgery, University of Michigan, Ann Arbor, MI; North American Brain Tumor Consortium; Cancer Therapy Evaluation Program, Division of Cancer Therapy and Diagnosis, National Cancer Institute, Bethesda, MD; and Cancer Therapy Evaluation Program, National Cancer Institute, Bethesda, MD. Address reprint requests to Frederick F. Lang, MD, Department of Neurosurgery, The University of Texas M. D. Anderson Cancer Center, 1515 Holcombe Blvd., Unit 442, Houston, TX 77030-4009; email: flang{at}mdanderson.org.
Purpose: Advances in brain tumor biology indicate that transfer of p53 is an alternative therapy for human gliomas. Consequently, we undertook a phase I clinical trial of p53 gene therapy using an adenovirus vector (Ad-p53, INGN 201). Materials and Methods: To obtain molecular information regarding the transfer and distribution of exogenous p53 into gliomas after intratumoral injection and to determine the toxicity of intracerebrally injected Ad-p53, patients underwent a two-stage approach. In stage 1, Ad-p53 was stereotactically injected intratumorally via an implanted catheter. In stage 2, the tumor-catheter was resected en bloc, and the postresection cavity was treated with Ad-p53. This protocol provided intact Ad-p53–treated biologic specimens that could be analyzed for molecular end points, and because the resection cavity itself was injected with Ad-p53, patients could be observed for clinical toxicity. Results: Of fifteen patients enrolled, twelve underwent both treatment stages. In all patients, exogenous p53 protein was detected within the nuclei of astrocytic tumor cells. Exogenous p53 transactivated p21CIP/WAF and induced apoptosis. However, transfected cells resided on average within 5 mm of the injection site. Clinical toxicity was minimal and a maximum-tolerated dose was not reached. Although anti-adenovirus type 5 (Ad5) titers increased in most patients, there was no evidence of systemic viral dissemination. Conclusion: Intratumoral injection of Ad-p53 allowed for exogenous transfer of the p53 gene and expression of functional p53 protein. However, at the dose and schedule evaluated, transduced cells were only found within a short distance of the injection site. Although toxicity was minimal, widespread distribution of this agent remains a significant goal.
THE TREATMENT of malignant glioma remains a major therapeutic challenge. In patients with glioblastoma multiforme (GBM), the most common adult glioma, the median survival duration is 1 year, and only 15% of patients survive for 2 years after diagnosis despite the use of maximal conventional therapy.1,2 However, recent advances in the fundamental understanding of brain tumor biology have indicated that transfer of the tumor suppressor p53 using a gene therapy strategy is an alternative approach to brain tumor treatment.3–10 Transfection of p53 is a rational therapeutic strategy for human gliomas because p53 is frequently inactivated in astrocytic tumors either through mutation of the p53 gene,11–15 overexpression of murine double minute 2 (mdm-2; the primary negative regulator of p53), inactivation of p14ARF (an inhibitor of mdm-2),16,17 or interference with p53 posttranslational modifications (eg, phosphorylation).18 Furthermore, studies using a variety of approaches have shown that inactivation of p53 is a critical event in the formation and progression of gliomas.15,19–24 Lastly, wild-type p53 is a primary mediator of cell cycle arrest and apoptosis, parameters intimately involved in tumor growth and response to treatment.25 Transfer of p53 would be expected to restore or enhance these critical functions. Mercer et al3 initially demonstrated that plasmid-mediated transfection of the p53 gene is capable of suppressing cell growth in gliomas. Kock et al4 and Gomez-Manzano et al5,6 were among the first to demonstrate that delivering the p53 gene using an adenovirus vector (Ad-p53) results in dramatic apoptosis in glioma cell lines. In animal models, intratumoral injections of Ad-p53 have been shown to inhibit the subcutaneous growth of gliomas and to extend the survival of rodents harboring intracranially implanted gliomas.4,9,26 Moreover, Ad-p53 has been shown to restore the sensitivity of gliomas to radiotherapy and chemotherapy.8,18,27 Ad-p53 (INGN 201; ADVEXIN, Introgen Therapeutics, Inc, Houston, TX) is a type 5 replication-incompetent adenovirus in which the E1 region has been replaced with the cDNA of the wild-type p53 gene driven by the cytomegalovirus promoter.28,29 In addition to its anticancer effects in gliomas, Ad-p53 has been shown to be effective against a variety of other tumor types, including lung,30 colon,31 head and neck,32 ovarian,33 and breast cancers.34 Ad-p53 (INGN 201) has also been shown to produce minimal toxicity after direct intratumoral injection during phase I clinical trials of patients with lung30,35,36 or head and neck cancer.32,37,38 Despite the promising preclinical results in brain tumors and the encouraging clinical results in other tumor types, the clinical potential of Ad-p53 (INGN 201) in the treatment of human gliomas has not yet been demonstrated. Consequently, we undertook a phase I trial of Ad-p53 in the treatment of patients with recurrent malignant gliomas. The purpose of this trial was not only to determine the clinical toxicity of Ad-p53 but also to obtain molecular information regarding the expression and distribution of the p53 protein after intratumoral treatment of human gliomas with Ad-p53. To meet both of these objectives we used a two-stage surgical protocol, unique to brain tumor trials, which allowed treated patients to be observed for toxicity and provided brain tumor specimens that could be analyzed for the molecular effects of Ad-p53. We report here that intratumoral injection of Ad-p53 into gliomas was associated with minimal toxicity and resulted in transfer of the p53 gene and expression of a functionally active p53 protein. However, with the bolus injection method used in this trial, the distribution of Ad-p53 was limited to a short distance from the injection site.
Institutional Review The institutional review boards of all participating institutions approved this study.
Objectives
Study Design
Eligibility Criteria Patients ≥ 18 years old with a Karnofsky performance score ≥ 70 and with surgically accessible, histologically confirmed recurrent malignant glioma were enrolled in this study. Patients were required to have recovered from the toxic effects of prior therapy, and had to have adequate bone marrow (granulocyte count > 1,500/µL and platelet count > 100,000/µL), liver (ALT and alkaline phosphatase levels < two times institutional norms), and renal functions (creatinine < 1.5 mg/dL). Patients were excluded if they received radiotherapy during the 4 weeks before study entry, had an active uncontrolled infection, had evidence of bleeding diathesis, or were taking anticoagulants. Males and females were recruited with no sex preference, and there were no exclusions on the basis of race. Women who were pregnant, at risk for pregnancy, or breast-feeding were excluded. All of the patients were able to read and understand the informed consent form, and all signed it indicating that they were aware of the investigational nature of the study.
Adenoviral Vector Construct and Testing
Ad-p53 Dose
Stereotactic Biopsy Specimens Specimens obtained at the time of stereotactic biopsy as frozen sections were analyzed using hematoxylin and eosin staining for the presence of recurrent glioma. Formalin-fixed specimens were analyzed for baseline p53 expression using immunohistochemical detection with antibody PAb1801 (Oncogene Science, Cambridge, MA) via an avidin-biotin-peroxidase complex method as described previously.14,39 Frozen specimens were analyzed for p53 gene mutation using direct DNA sequencing to detect mutations in exons 5 to 8.
Surgical Specimens Sections immediately adjacent to those stained for p53 (10-µm separation) were analyzed by immunohistochemistry for the expression of p21CIP/WAF, a p53-inducible gene, using an anti-p21CIP/WAF monoclonal (Ab-3) antibody (Oncogene Science) as described previously.40 Adjacent sections were used in this analysis to determine whether the pattern of p21 staining overlapped the pattern of p53 staining. To more definitively determine whether individual p53-positive cells also expressed high levels of p21CIP/WAF, sequential double immunostaining for p53 and p21 was performed on the same section in two patient samples. Briefly, sections were first exposed to anti-p21CIP/WAF monoclonal antibody, followed by secondary antibody (chromogen was diaminobenzidine) and then placed in 70% alcohol and rinsed with phosphate-buffered saline. The section was then exposed to anti-p53 antibody PAb1801 followed by secondary antibody (chromogen was aminoethyl carbazole). The diaminobenzidine produces a brown stain, whereas the aminoethyl carbazole produces a red stain. To detect apoptotic cells, adjacent sections were also analyzed by terminal deoxynucleotidyl transferase–mediated deoxyuridine triphosphate-biotin nick end-labeling (TUNEL) using the DeadEnd Colorimetric TUNEL System (Promega, Madison, WI), according to the manufacturer’s instructions. The degree and distribution of apoptotic cells were analyzed relative to p53 immunostaining.
Clinical Patient Evaluation All patients underwent laboratory testing at baseline, at 24 hours after craniotomy, and on each on-study follow-up visit. Laboratory evaluations included hematology testing (complete blood count, differential, platelet count, prothrombin time, partial thromboplastin time), biochemistry (levels of standard electrolytes, calcium, magnesium, phosphate, blood urea nitrogen, creatinine, glucose, ALT, AST, total protein, albumin, alkaline phosphatase, bilirubin, total cholesterol, triglycerides, creatine phosphokinase, lactate dehydrogenase, and uric acid), and measurement of anticonvulsant levels. All patients underwent multiplanar magnetic resonance imaging (MRI) both with and without gadolinium contrast enhancement within 2 weeks of study entry to assess the location and size of the tumors. Noncontrast computed tomography was performed within 12 hours of catheter placement to assess the catheter position. Postresection MRI was performed within 48 hours of surgery (to assess the extent of resection), 4 weeks later, and then every 8 weeks after craniotomy during follow-up. Samples of plasma, sputum, urine, and stool were obtained for vector dissemination assays (see Cytopathic Effect Assay), and serum was collected for antiadenoviral-type 5 antibody assays (see Adenovirus Antibody Assay) at baseline and 24 hours after stereotactic injection of Ad-p53. After injection of Ad-p53 into the wall of the resection cavity (ie, after craniotomy), this sampling was repeated within 24 hours, after 2 weeks, and monthly.
Toxicity and Response Evaluation Because the major thrust of this study was to evaluate toxicity at each dose of Ad-p53, we made an attempt to evaluate its efficacy on the basis of clinical criteria. Because all of the patients underwent complete resection of the gadolinium-enhancing tumor mass, the primary outcome statistic was time to tumor progression as measured from the time of Ad-p53 injection into the wall of the resection cavity. Tumor recurrence was defined as the occurrence of a new region of MRI contrast enhancement that increases by 25% on sequential scans.
Adenovirus Antibody Assay
Cytopathic Effect (CPE) Assay
Statistical Methods
Patients and Treatment Fifteen patients received treatment in this study. Twelve patients completed both surgical stages of the protocol, whereas three patients completed stage 2 only (Table 1  ). The patients’ demographics, treatment dose and dose level, tumor type, prior treatments, and tumor features are listed in Table 1.
Molecular Analyses of Pretreatment Biopsies and Posttreatment Tumor Specimens
To assess the expression and distribution of exogenous p53, we performed immunohistochemical staining using an antibody specific to wild-type p53 (PAb-1801). Pretreatment biopsies were negative for p53 immunostaining in six specimens and demonstrated low levels or rare staining in two specimens (Table 2 ). In contrast, in all 10 assessable posttreatment patients, delivery of Ad-p53 resulted in robust p53 immunoreactivity in an inhomogeneous pattern around the catheter site (Table 2; Figs 2D and 3A). In areas of immunopositivity, 95% to 100% of the cells were intensely stained. Analysis of the sections at higher magnification revealed that p53 expression occurred within the nucleus of tumor cells, as would be expected of a transcription factor such as p53 (Fig 2E). However, we also observed cytoplasmic p53 expression in four specimens (Table 2; Fig 3B).
To more quantitatively determine the depth of spread of the vector, we measured the maximum distance from the injection site (defined by the catheter position) at which positive immunoreactivity was detectable in representative sections from each specimen (Table 2
In an effort to determine whether exogenous p53 was functional, we assayed specimens for increases in p21CIP/WAF, a known p53-inducible protein, by immunostaining with a p21-specific antibody. Analyses of sections adjacent to the p53-immunostained sections revealed that in seven of eight tested specimens, the distribution of p21 immunostaining was similar to that of exogenous p53, indicating that exogenous p53 was capable of inducing p21 (Figs 2F
To determine whether Ad-p53 was capable of inducing apoptosis, in situ TUNEL staining was undertaken. All four specimens analyzed demonstrated positive staining concentrated only around the injection site (Figs 2H
Clinical Studies: Adverse Events
Neurologic events potentially related to Ad-p53 occurred in two patients at dose level III (a grade 3 aphasia and a grade 2 aphasia). Because of the occurrence of the grade 3 toxicity, we evaluated three additional patients at this level (Table 1 ). None of these patients had significant neurologic toxicity, although one patient (patient 10) had an asymptomatic hematoma that was observed on MRI scans taken 3 months after treatment and was temporally related to trauma. Three patients were then treated at level IV with no significant toxicity. Thus, a maximum-tolerated dose was not defined in this study, and additional escalation was not undertaken because of difficulties with manufacturing and storing the agent at higher concentrations. It should be noted that three seizures were reported in two patients. However, these events occurred many months after treatment with Ad-p53 and were associated with tumor recurrence. Because of the common occurrence of seizures in this disease, we concluded that these events were not dose limiting.
Radiographic Evaluation
Immune Response to Adenovirus Type 5 To determine whether any systemic immune responses occurred after intracranial injection of Ad-p53 (INGN 201), we measured the titers of antiadenoviral type 5 immunoglobulin G antibodies both before Ad-p53 treatment and at fixed intervals after treatment. Ten (83%) of 12 assessable patients showed an antibody response (defined as > two-fold increase in antibody titer). Antibody titers increased 1 to 2 weeks after craniotomy, were maximal at 1 to 2 months, and decreased at 5 to 6 months (Table 4  ).
Adenovirus Vector Dissemination To determine whether intracranial injection of Ad-p53 (INGN 201) resulted in widespread systemic dissemination of the vector, we performed CPE assays at fixed intervals after treatment. We did not detect replication-deficient virus in patients’ plasma, urine, sputum, or rectal samples at any of the time points. There was no evidence for conversion of Ad-p53 to a replication-competent form.
Clinical Outcome
This phase I trial demonstrated that injection of Ad-p53 (INGN 201) into malignant brain tumors is safe and results in transfer of p53 gene to astrocytic tumor cells, leading to the expression of functionally active p53 protein that is capable of transducing other genes (eg, p21WAF/CIP) and inducing apoptosis in a subset of cells. Surgical specimens that preserved the anatomic integrity of the tissue showed limited distribution of Ad-p53 with the injection technique employed in this study. We showed that the two-stage surgical design used in this study is feasible for brain tumor patients. For new biologic agents, there has been an increasing desire to incorporate molecular analysis of tumor specimens into phase I trials.41,42 However, for brain tumors, in which tissue accessibility is a major problem and the potential for repeat biopsy is limited, such trials are particularly challenging.43 The incorporation of pretreatment biopsy and posttreatment craniotomy for tissue acquisition that was undertaken in this trial provided valuable biologic information that would otherwise not have been obtained if more standard phase I designs were followed. Importantly, the biologic evaluation did not interfere with the long-term clinical evaluation. However, the difficulties associated with obtaining adequate tissue in brain tumor patients limited our assessment of the biologic effects of Ad-p53 to only one time point after Ad-p53 delivery (3 days). Moreover, including cases of catheter implantation without Ad-p53 injection (as a negative control) may have been beneficial, but was not possible because of the complexity of the approach. Nevertheless, the design of this trial may be used as a model for future brain tumor studies.
The biologic analyses demonstrate robust p53 immunostaining within tumor cells, indicating expression of the p53 transgene. However, it must be recognized that the antibody used in this study could not distinguish between exogenous and endogenous p53. We interpreted the p53 immunostaining seen in the specimens to be from exogenously delivered p53 rather than endogenous p53 protein because in all but two patients, pretreatment biopsies did not demonstrate endogenous p53 immunoreactivity (consistent with wild-type p53 allele studies; Table 2 Although other clinical trials of head and neck32,38 and lung cancer30,35 have demonstrated p53 expression after intratumoral injection of Ad-p53 (INGN 201), to our knowledge, no other study has determined the spatial distribution of p53 relative to the injection site. We analyzed this parameter because preclinical studies indicated that adenoviral vectors are capable of extensive distribution throughout tumor-bearing rat brains.45 In addition, widespread delivery is particularly important in brain tumors, where tumor control requires transduction of unresectable tumor cells that infiltrate normal parenchyma several centimeters away from the solid tumor mass.46,47 Previous trials relied on molecular techniques that precluded anatomic analyses (eg, polymerase chain reaction) and therefore could not define spatial relationships. In contrast, by leaving the injection catheter in place and analyzing intact specimens with immunohistochemistry, we were able to demonstrate that with the bolus injection method used in this trial, the distribution of Ad-p53 in brain tumors was quite limited, reaching a zone of less than 1 cm around the injection site. Nevertheless, it seems that injection of Ad-p53 resulted in more widespread transgene expression than did instillation of producer cells, which was used in an earlier trial of herpes simplex virus–thymidine kinase (TK) gene therapy, in which only a few cells expressed the TK transgene.48 Thus, direct injection of adenovirus vectors may be more advantageous than the use of producer cells. Furthermore, although the narrow distribution could be overcome by multiple injections (as we did in the postresection stage of this trial), this technique is not ideal for brain tumors because of the potential for traumatic injury. The results in this trial provide useful baseline data for evaluating future methods aimed at improving the distribution of Ad-p53.
Our data indicate that the exogenously expressed p53 protein was functional. First, we showed that p53 expression correlated with p21CIP/WAF expression (Figs 2
The second piece of evidence indicating that p53 was functional was the correlation between zones of apoptotic (TUNEL-positive) cells and the area of tissue exposed to Ad-p53. Specifically, TUNEL-positive cells were essentially confined to the limited region around the catheter site where p53 expression was also high. Because the process of apoptosis results in degradation of cellular components, it was not surprising that p53-positive cells were not themselves TUNEL-positive. Instead, p53-positive cells were typically adjacent to regions of TUNEL-positive cells. It was of interest that the p53-positive cells were usually farther away from the catheter than were the TUNEL-positive cells. Although this observation may be related to nonspecific cell death from catheter manipulations, it may also relate to the timing of apoptosis induction relative to Ad-p53 exposure. In other words, as the Ad-p53 diffused from the site of injection, cells nearer the injection would have been exposed to Ad-p53 first and were thus more likely to have undergone apoptosis at the time of tissue sampling than were the more distant cells, which were still expressing p53; biopsies taken later than 72 hours after injection may have revealed more extensive apoptosis and less p53 staining. It was also of interest that some cells adjacent to the catheter site were p53-positive but TUNEL-negative. Although these cells should have had enough time to undergo apoptosis, it seemed that they were less sensitive to the apoptotic effects of Ad-p53. Indeed, in vitro studies have shown that glioma cells lines that do not harbor a mutation in their p53 gene are often less sensitive than mutant p53 gliomas to Ad-p53–mediated apoptosis,5,8,18 although wild-type p53 gliomas do undergo cell cycle arrest via p21WAF/CIP induction. Because most of the tumors in this study had normal p53 alleles (Table 2 The Ad-p53–associated toxicities identified in this study are similar to those reported in other cancer-related Ad-p53 (INGN 201) trials,30,32,35–38 and we interpret the occurrence of fever, fatigue, and headache in our study to be most consistent with a transient inflammatory response to the adenoviral vector. Previous trials in which p53 was injected into head and neck tumors that could be directly visualized demonstrated inflammatory changes at the injection site.32,37,38 Although the location within the intracranial compartment precluded direct observation of an inflammatory response, the pericavitary enhancement seen on MRI scans after Ad-p53 injection and its attenuation by administration of corticosteroids are indirect evidence of a local inflammatory response. Supporting this conclusion was the finding of a systemic increase in anti-Ad5 antibody titers in all but two of the patients within 2 months of Ad-p53 injection; similar increases in anti-Ad5 antibody have been reported in previous Ad-p53 trials.32,36,37 Lastly, MRI changes and an inflammatory response to Ad-p53 were also observed in animal studies. Smith et al49 reported studies of rats and monkeys in which injection of an adenoviral vector, albeit one containing the TK gene rather than p53, resulted in dose-dependent occurrence of fever; MRI of the animals showed increased enhancement around the injection tract, and histologic analysis of their brains demonstrated local astrogliosis that worsened as the dose increased. In preclinical studies from our laboratory,9 injection of Ad-p53 into Wistar rats resulted in local astrogliosis, which was most prominent on day 7 and had resolved by day 14. Thus, the potential of an immune response, although transient, may be an important component of the efficacy and toxicity of Ad-p53. In previous clinical studies, injection of Ad-p53 (INGN 201) into head and neck and non–small-cell lung cancer tumors resulted in dose-dependent shedding of the adenovirus in the urine and saliva or sputum.32,35 In contrast, using the same CPE assay, which can detect as few as 10 particle-forming units/mL, we were unable to identify any viral shedding in the urine or sputum both immediately and up to 2 months after Ad-p53 injection. We also did not detect the adenovirus in the plasma. However, our initial measurements were performed 24 hours after Ad-p53 injection, whereas previous trials detected the adenovirus within 30 minutes of treatment, and these titers became undetectable by 90 minutes. Thus, it is possible that in our study, some Ad-p53 was released into the plasma but was not detected because of our sampling schedule. Nevertheless, shedding of a virus after intracranial injection seems to be significantly less likely than that after injection into systemic tumors.
Although it was not intended to determine efficacy, the results of our trial indicate that Ad-p53 (INGN 201) warrants additional study as an anticancer agent (Table 5
We thank C. David James for performing mutational analysis of p53 specimens. We thank James Merritt, MD, and Deborah Wilson, PhD, from Therapeutics, Inc. We thank Ian Suk and Weiming Shi for their expert assistance with the figures. Special thanks go to Vivien Liu for her outstanding contribution as the primary clinical research nurse, and to MaryJo Gleason for her assistance with administration and oversight of the protocol. We thank David Wildrick for his editorial support and Sandra Flores for her diligent assistance in manuscript preparation.
Supported by National Cancer Institute grants CA62399 (to the North American Brain Tumor Consortium), CA62412, CA16672 (to The University of Texas M. D. Anderson Cancer Center), CA62422, MO1-RR00079 (to University of California, San Francisco), and MO1-RR00042 (to University of Michigan); and grants from the Anthony Bullock III Brain Tumor Research Fund and the Brian McCulloch Memorial Brain Tumor Fund (to F.F.L.).
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ABSTRACT
INTRODUCTION
2 method was used to determine the significant differences between dose levels. 
