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© 2002 American Society for Clinical Oncology Successful Adenovirus-Mediated Wild-Type p53 Gene Transfer in Patients With Bladder Cancer by Intravesical Vector InstillationByFrom the Departments of Medicine III and Urology, Johannes Gutenberg University, Mainz, and Department of Pathology, University Herdecke Witten, Germany; Canji Incorporated, San Diego, CA; and Schering-Plough Research Institute, Kenilworth, NJ. Address reprint requests to Martin Schuler, MD, Department of Medicine III, Johannes Gutenberg University, D-55101 Mainz, Germany; email: m.schuler{at}3-med.klinik.uni-mainz.de
PURPOSE: To study safety, feasibility, and biologic activity of adenovirus-mediated p53 gene transfer in patients with bladder cancer. PATIENTS AND METHODS: Twelve patients with histologically confirmed bladder cancer scheduled for cystectomy were treated on day 1 with a single intratumoral injection of SCH 58500 (rAd/p53) at cystoscopy at one dose level (7.5 x 1011 particles) or a single intravesical instillation of SCH 58500 with a transduction-enhancing agent (Big CHAP) at three dose levels (7.5 x 1011 to 7.5 x 1013 particles). Cystectomies were performed in 11 patients on day 3, and transgene expression, vector distribution, and biologic markers of transgene activity were assessed by molecular and immunohistochemical methods in tumors and normal bladder samples. RESULTS: Specific transgene expression was detected in tissues from seven of eight assessable patients treated with intravesical instillation of SCH 58500 but in none of three assessable patients treated with intratumoral injection of SCH 58500. Induction of RNA and protein expression of the p53 target gene p21/WAF1 was demonstrated in samples from patients treated with SCH 58500 instillation at higher dose levels. Distribution studies after intravesical instillation of SCH 58500 revealed both high transduction efficacy and vector penetration throughout the whole urothelium and into submucosal tumor cells. No dose-limiting toxicity was observed, and side effects were local and of transient nature. CONCLUSION: Intravesical instillation of SCH 58500 combined with a transduction-enhancing agent is safe, feasible, and biologically active in patients with bladder cancer. Studies to evaluate the clinical efficacy of this treatment in patients with localized high-risk bladder cancer are warranted.
AN ESTIMATED 261,000 new cases of bladder cancer are diagnosed worldwide per year. Bladder cancer is prevalent in the developed countries, where it affects mainly men and is frequently associated with a history of tobacco smoking or some occupational exposures, and in Northern Africa and Western Asia, where it is related to endemic infection with the parasite Schistosoma mansonii.1 In the Western world, 70% to 80% of patients present with superficial bladder tumors, which can be treated with transurethral resection.2,3 However, patients with less differentiated, large or multilocular bladder tumors as well as patients with carcinoma in situ or stage I bladder cancer are at high risk for tumor recurrence and development of muscle-invasive disease or distant metastases.4,5 Treatment strategies for such high-risk patients include local resection with close surveillance,2 local resection and intravesical therapy using bacillus Calmette-Guerin or cytotoxic agents,6-8 or radical cystectomy with urinary diversion or reconstructive surgery.9,10 Radical cystectomy provides optimal control of the bladder tumor, but at the price of organ loss. Intravesical and systemic medical therapies have substantial toxicities and bear the risk of local recurrence or tumor progression. Thus, new bladder-preserving treatment options for high-risk bladder cancer are required. Mutations of the p53 tumor suppressor gene are the most common genetic alteration in human cancers.11 The role of p53 in the prevention of oncogenic transformation, maintenance of genetic stability, and sensitivity to commonly used cancer treatments is well established.12,13 In some but not all studies, nuclear accumulation of p53 as an indicator for mutations in the p53 DNA binding domain was associated with an adverse prognosis in patients with bladder cancer.14-17 Hence, somatic gene transfer of the p53 tumor suppressor is an attractive new treatment modality for malignant bladder tumors. Preclinical cancer models have demonstrated that the expression of p53 by viral or nonviral gene transfer technology effectively induced apoptosis or sensitized cancer cells to drug- or radiation-induced cell death.18 These results have fostered the translation of p53 gene therapy into early clinical studies, which were conducted in patients with advanced lung, head and neck, ovarian, or liver cancers.19-23 Using intratumoral injection of adenoviral20-22,24 or retroviral19 p53 expression vectors, local transgene expression20,22 and evidence for local tumor regressions and induction of apoptosis19,24 were reported from several phase I and pilot studies. However, the only controlled phase II study in patients with newly diagnosed advanced non–small-cell lung cancer (NSCLC) failed to demonstrate a significant clinical benefit from local p53 gene transfer by intratumoral vector injection in combination with an effective first-line chemotherapy.25 One reason for this apparent clinical inactivity might be insufficient gene delivery and transduction after intratumoral injection of adenoviral p53 expression vectors. Systematic studies of these important parameters, however, are absent in cancer patients. One way to overcome the potential limitations of the intratumoral injection approach is the instillation of high-vector doses into cavitary organs, such as the pleural space,26 peritoneal cavity, or bladder. This should allow a homogeneous vector distribution along the tumor surfaces, as opposed to a vector distribution along the track of an injection needle. Preclinical studies have demonstrated the feasibility of this approach and have highlighted the importance of the addition of transduction-enhancing agents to maximize transgene expression in the bladder.27,28 To address this hypothesis, a study of safety, feasibility, and biologic activity of an intravesical instillation or an intratumoral injection of an adenoviral expression vector encoding wild-type p53 (SCH 58500) was conducted in patients with invasive bladder cancer. To allow assessment of vector distribution, transgene expression, and induction of p53 target genes or additional markers of biologic activity after the study treatment, only patients scheduled for radical cystectomy were enrolled onto this trial, enabling extensive tissue sampling for these analyses.
Patients Adult patients with histologically confirmed, muscle-invasive bladder cancer and indication for radical cystectomy were eligible for enrollment. Additional inclusion criteria were a life expectancy of at least 3 months, a Karnofsky performance score of at least 70%, and the absence of any clinical or laboratory evidence (WBC count  3,000/µL, absolute neutrophil count 1,000/µL, platelet count 100,000/µL, creatinine < 1.5 mg/dL, bilirubin < 1.5 mg/dL, AST and ALT < 1.5 times the upper limit of normal, and prothrombin and partial thromboplastin times within normal limits) for dysfunction of the hematopoietic, liver, renal, or coagulation systems. An interval of at least 4 weeks between prior chemotherapy, radiation, or major surgery was mandatory. Pregnant or nursing women, fertile women not practicing medically accepted contraception, patients with uncontrolled serious bacterial, fungal, or viral infections, human immunodeficiency virus–positive patients, and immunosuppressed patients were not eligible. Molecular or immunohistochemical evidence for an intratumoral p53 mutation was not required for eligibility. All patients provided written informed consent. After written informed consent, control tissue samples were obtained from patients with advanced bladder cancer or patients with nonmalignant bladder disease treated by cystectomy.
Study Design
Study Treatments
Study End Points
Clinical Monitoring
Virology Studies
Detection of SCH 58500 DNA and Expression of Transgenic p53, p21/WAF1, and the Coxsackie and Adenovirus Receptors
Analysis of Tissue Sections Localization of SCH 58500 was assessed using a direct in situ PCR method.36 Formalin-fixed paraffin-embedded tissues were cut into 5-µm sections, placed on in situ PCR slides, and baked for 2 to 3 hours at 60°C on a slide hot plate. The slides were washed in xylene to remove the paraffin, followed by an incubation with 0.02 N HCl and digestion with 2.5 µg/mL proteinase K (Qiagen) at 37°C for 30 minutes. The endogenous alkaline phosphatase activity was eliminated by incubating the slides in ice-cold 20% (vol/vol) acetic acid. Slides were dehydrated in graded alcohols and rehydrated in 45 µL of PCR master mix containing 1 µmol/L of each dinitro-phenyl (DNP)-labeled primer, 200 µmol/L of each dNTP, 2.5 mmol/L magnesium chloride, and 10 units of AmpliTaq DNA polymerase (Applied Biosystems). Primers were designed to amplify a SCH 58500-specific sequence located between the cytomegalovirus promoter (5'-CGTGTCACCGTCGTGGA-3') and the upstream p53 cDNA (5'-CCACTGCTTACTGGCTTATCGAAAT-3'). This primer selection prevents the amplification of genomic p53 DNA.29 Reactions were performed in a Perkin Elmer Gene Amp In Situ PCR System 1000 (Applied Biosystems) programmed for one cycle of denaturation at 95°C for 5 minutes and annealing at 55°C for 90 seconds, followed by 34 cycles of 94°C for 30 seconds and 55°C for 90 seconds. After completion of the PCR, slides were washed two times with standard saline citrate (0.3 mol/L NaCl and 0.03 mol/L sodium citrate) and blocked with casein solution (Vector, Burlingame, CA). For tissue sections, the DNP molecules incorporated into the PCR amplicons were detected using an anti-DNP antibody conjugated with alkaline phosphatase (Applied Biosystems). The sections were stained using the alkaline phosphatase substrate NBT/BCIP (nitro-blue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate) (Boehringer Mannheim, Germany) and then counterstained with Nuclear Fast Red (Vector). As a negative control, each section was processed, but the PCR reaction was performed without AmpliTaqDNA polymerase. Samples from rat bladders instilled with SCH 58500 or a ß-galactosidase–expressing adenoviral vector (Ad5.ß-gal) served as positive and negative controls. The protein expression of p53, p21/WAF1, apoptosis-related and cell cycle–related genes, and CAR was assessed by immunohistochemistry in formalin-fixed paraffin-embedded tissue sections. Primary antibodies against p53 (M7001, Dako Diagnostika, Hamburg, Germany), p21/WAF1 (M7202, Dako), Bcl-2 (M0887, Dako), Bak (AM04, Calbiochem, San Diego, CA), Bax (Ab-1/PC66, Calbiochem), MIB1 (dia 505, Dianova, Hamburg, Germany), and CAR (a gift from Dr Robert W. Finberg, Dana-Farber Cancer Institute, Boston, MA37,38) were used. Apoptotic cells were visualized by microscopy following the terminal deoxynucleotidyl transferase–mediated deoxyuridine triphosphate-biotin nick end-labeling (TUNEL) method39 and by means of laser scanning cytometry, as previously described.40 Normal bladder and tumor tissue samples from patients not treated with SCH 58500 served as controls.
Enrollment and Treatments Twelve patients from a single center were enrolled onto the study. Baseline characteristics and histologies of the study patients are listed in Table 2. Three patients were treated at dose level 1 in part A (intratumoral injection) of the study. No additional dose escalation was performed in part A. Nine patients were treated at three different dose levels in part B (intravesical instillation). Eleven patients underwent radical cystectomies after study treatment. In one patient, the tumor was determined to be unresectable with curative intent at laparotomy. Thus, tumor samples for assessment of the secondary end points were obtained from 11 patients treated with SCH 58500 at three dose levels.
Toxicity Postoperatively, one patient treated in part A suffered from WHO grade 1 fatigue. No toxicities were observed in the other two patients treated with intratumoral injection at cystoscopy. The predominant toxicities observed in patients treated in part B of the study were urethral and vesical burning, which reached WHO grade 2 in two patients and WHO grade 3 in another two patients. In addition, one patient each experienced WHO grade 2 and grade 3 abdominal pain. These symptoms were relieved in two patients treated at dose level 1 by a reduction of the contact time, for which the transurethral catheters were clamped. Hence, for patients treated at dose levels 2 and 3, the treatment was administered in two sequential 30-minute sessions. Additionally, patients treated at dose level 3 were premedicated with 50 mg of pethidine and 20 mg of butylscopolamine. Despite these modifications, the planned contact time had to be reduced by several minutes in three patients treated at dose level 2 and in one patient treated at dose level 3. All symptoms resolved immediately after release of the transurethral catheter and bladder irrigation with saline. No fever, chills, or other signs of systemic toxicity were observed in patients treated in part B. No alterations of laboratory parameters, including liver enzymes and bilirubin, were detected before surgery on day 3. Three patients were hospitalized because of fever of unknown origin within 4 to 6 weeks after surgery and quickly recovered under treatment with broad-spectrum antibiotics. In one of these patients, a methicillin-resistant Staphylococcus aureus was isolated from a catheter. Thus, even at the highest dose level of 7.5 x 1013 particles SCH 58500 administered by intravesical instillation, no dose-limiting toxicities were observed.
Transgene Expression and Biologic Activity To address whether the p53 transgene expression translated into biologic activity, we determined the quantitative expression of the p53 target gene p21/WAF1 by real-time RT-PCR analysis of tumor and normal bladder samples from patients treated with intravesical instillation of SCH 58500 or untreated control patients. The p21/WAF1 expression in tumor samples from untreated control patients was lower than in normal bladder samples (Table 3). Assaying nontumor bladder samples from patients treated with SCH 58500 instillation, moderate changes in p21/WAF1 expression were detected when compared with untreated controls (Table 3). However, in tumor samples from patients treated at the highest dose level of 7.5 x 1013 particles SCH 58500 p21/WAF1, expression was increased up to 40-fold compared with control tumor samples from patients not receiving gene therapy (Table 3). Immunohistochemical analyses revealed an increased p21/WAF1 protein expression after SCH 58500 treatment in tumor tissues but not in normal bladder samples from four patients with undetectable or low p21/WAF1 protein expression at baseline (Table 3). No significant correlation between transgene expression, p21/WAF1 induction, and CAR expression, as determined by RT-PCR analysis and immunohistochemistry, could be established. However, the CAR expression detected by immunohistochemistry exhibited a considerable heterogeneity among tumors from different patients as well as among different regions of the same tumor (not shown). Immunohistochemical analyses of p53 expression or expression of additional apoptosis-related or cell cycle–related genes revealed no consistent changes in relation to SCH 58500 treatment. Moreover, we failed to detect a significant induction of apoptosis as assessed by TUNEL staining and microscopy or laser scanning microscopy in samples taken at cystectomy approximately 48 hours after SCH 58500 treatment (not shown).
Taken together, these data demonstrate that a detectable p53 transgene expression in bladder tumors can be achieved by intravesical instillation of SCH 58500 in combination with a transduction-enhancing agent. At the highest dose level of 7.5 x 1013 particles SCH 58500, evidence for biologic activity in terms of RNA and protein expression of the p53 target gene p21/WAF1 was obtained.
Vector Distribution
Virologic Studies After SCH 58500 treatment, all patients in both study groups underwent extensive bladder irrigation with 6 L saline through a transurethral catheter over a period of 36 to 48 hours. Excretion of infectious adenoviruses was detected by a sensitive flow cytometry-based assay30 in samples taken from the first 2 to 4 L of void volume. No detectable urinary adenovirus excretion was found after 6 L of bladder irrigation (Fig 3). None of the urine samples taken 24 hours after study treatment gave a positive result in the qualitative on-site ELISA assay (not shown).
Long-Term Follow Up Nine of the 12 study patients were alive at a median follow-up of 30 months. In addition to SCH 58500 treatment and radical cystectomy, two patients received a platinum-based adjuvant chemotherapy regimen. In one patient treated in part A, fulminant liver metastases developed 4 weeks after surgery, which were not detectable on computed tomogram and ultrasound examinations performed at the preoperative staging. The patient was treated with palliative chemotherapy, but he died from progressive liver failure 7 weeks after cystectomy. One patient treated in part A developed a Mycoplasma pneumonia during adjuvant chemotherapy. In total, three patients died from disease progression, and one patient is being treated with palliative chemotherapy for recurrent disease.
A major challenge in the conservative management of localized bladder cancer is the frequent recurrence and progression to an advanced tumor stage in patients with high-risk tumors.2 To improve disease control, local tumor resection is combined with intravesical therapy with bacillus Calmette-Guerin or anticancer agents.8 However, these treatments have a substantial toxicity and may reduce the risk of recurrences but do not prevent disease progression.6,7 Thus, new treatment options for high-risk superficial bladder cancer are required. Mutations of the p53 tumor suppressor gene are frequently found in bladder cancer, are associated with an adverse prognosis in some studies, and may contribute to a more aggressive clinical course and resistance to anticancer treatment.14,15 In an ortopic injection model of bladder cancer, p53 gene transfer acted synergistically with cisplatin to prevent tumor growth and induce apoptosis in vivo.41 In the present phase I study, we tested whether adenoviral vector-mediated wild-type p53gene transfer is safe, feasible, and biologically active in patients with invasive bladder cancer. Taking advantage of the anatomy of the bladder, we planned to evaluate two different modes of vector administration: the intratumoral injection of vector solution, because it has been performed in several clinical studies of cancer gene therapy,19-21 and the intravesical vector instillation via a transurethral catheter. Because preclinical studies convincingly demonstrated that the transduction efficacy of adenoviral vectors instilled into the bladder can be dramatically enhanced by the addition of several compounds,27,28 here we administered intravesical SCH 58500 in combination with the transduction-enhancing agent Big CHAP.28 Both modes of administration of the study treatment, intratumoral injection at cystoscopy and transurethral intravesical instillation, were well tolerated and devoid of any detectable systemic toxicity. Successful gene transfer after intravesical instillation of SCH 58500 in combination with Big CHAP was detected by RT-PCR analysis in seven of eight assessable patients. Moreover, evidence for biologic activity of the transgene, as determined by quantitative RT-PCR analysis of RNA expression as well as by immunohistochemical analysis of protein expression of the p53 target gene p21/WAF1,32 was found in patients treated at higher dose levels. Transgene expression did not seem to correlate with the CAR expression status of the tumor samples as determined by RT-PCR analysis and immunohistochemistry. However, the relatively small number of patients enrolled onto this study and the detection methods for CAR expression might have influenced this result. Compared with the effective transduction achieved by intravesical vector instillation, no evidence for transgene expression was detected in the three patients treated by intratumoral injection of SCH 58500 at dose level 1, whereas SCH 58500 DNA sequences were detectable in two patients by PCR analysis. This was surprising, given that in a previous study in patients with NSCLC treated by intratumoral injection of SCH 58500, p53 transgene expression was detected in four of five assessable patients receiving the same vector dose of 7.5 x 1011 particles.20 Because intratumoral vector injection at cystoscopy is a relatively invasive procedure compared with transurethral vector instillation, it was decided not to proceed with the dose escalation in part A of this trial. Hence, we cannot exclude that at higher dose levels, a p53 transgene expression in bladder tumors would have been achieved by intratumoral injection of SCH 58500 at cystoscopy. Furthermore, the addition of Big CHAP or other transduction-enhancing agents28 might also be beneficial in the case of intratumoral vector injection in the bladder. However, in the light of the efficacy and ease of the intravesical instillation approach, intratumoral vector injection at cystoscopy clearly is the inferior approach for vector administration in bladder cancer. In contrast to the results obtained with the p53 target gene p21/WAF1, we found no consistent changes in the expression of p53, various cell cycle–related or apoptosis-related genes, or TUNEL staining in response to SCH 58500 administration. This observation might be limited by the small number of patients enrolled onto the trial and the availability of only a single time point for these examinations. Moreover, the activity of many genes regulating apoptosis is not controlled by their expression level but by conformational changes or changes in their subcellular localization,42 which cannot be detected by the methods applied in this study. Nevertheless, the p21/WAF1 response is a valid marker for biologic activity of transgenic p53, which has been confirmed in additional settings of clinical p53 gene therapy.35,43 In addition to molecular and immunohistochemical evidence for transgene expression and biologic activity, important information related to the vector distribution throughout the bladder and vector penetration into tumor tissues was gathered from this trial. We demonstrated by quantitative PCR analysis that administration of higher particle doses resulted in the recovery of higher copy numbers of SCH 58500-specific DNA from tissue samples (Fig 3). This was not unexpected; however, it suggests that together with the evidence for increased p21/WAF1 expression at high doses, a plateau of the biologic activity was not reached by the intravesical instillation of 7.5 x 1013 particles SCH 58500. Presently, technical limitations preclude the administration of a more concentrated adenovirus solution, leaving this issue unresolved. With respect to the vector distribution after intravesical instillation, we found a uniform distribution of SCH 58500 DNA throughout the normal urothelium and the luminal tumor tissues by in situ PCR analysis of bladder sections. Moreover, vector DNA could also be found in apparently submucosal tumor nodules as well as in cells in the Lamina propria. These results confirm the hypothesis that the instillation approach results in an improved vector distribution. In addition, they demonstrate that even submucosal tumor cells can be targeted by the luminal administration of an adenovirus in combination with a transduction-enhancing agent in the bladder. The optimal dosing schedule for intravesical SCH 58500 instillation remains to be established. Because of the procedure-associated discomfort observed in most patients treated in part B of this study, the contact times varied considerably. Yet SCH 58500 penetration and transgene expression analyses yielded promising results. It seems likely that even shorter contact times than the ones allowed in the course of this trial might result in sufficient transduction rates with lower local toxicity, a hypothesis supported by initial data from preclinical in vivo models. The intravesical instillation of SCH 58500 through a transurethral catheter also is environmentally safe, because infectious adenoviruses excreted in the urine after SCH 58500 treatment can easily be recovered in a contained system. The virologic studies performed in this trial suggest that if the bladder is sufficiently irrigated, infectious viral particles are only excreted with the first 4 L of irrigation fluid. This could minimize hospitalizations and could even allow outpatient treatment. The design of the present phase I study precluded the collection of data regarding the long-term effects of the intravesical administration of such high-vector doses as well as signs for clinical efficacy. However, important and unique data demonstrating effective vector distribution, transgene expression, and biologic activity after a clinically practicable and safe gene transfer procedure were obtained in patients with invasive bladder cancer. These results provide a strong rationale for future investigation of adenovirus therapy in bladder cancer and support trials addressing the clinical efficacy of intravesical SCH 58500 treatment in patients with superficial high-risk bladder cancer.
Supported by grants from Essex Pharma and Schering-Plough Research Institute. M.S. is supported by the Dr Mildred Scheel Stiftung für Krebsforschung. We thank Drs R. Uhlenbusch and H. Huberts, Essex Pharma, Dr J.A. Horowitz, SPRI, Drs M. Zepeda, J. Philopena, J. Shinoda, and S. Chang, Canji, Inc, and Dr R. Hohenfellner, Mainz, for support and contributions to this trial.
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Copyright © 2002 by the American Society of Clinical Oncology, Online ISSN: 1527-7755. Print ISSN: 0732-183X
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ABSTRACT
INTRODUCTION
