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Repeated Intravesical Instillations of an Adenoviral Vector in Patients With Locally Advanced Bladder Cancer: A Phase I Study of p53 Gene Therapy

Lance C. Pagliaro, Afsaneh Keyhani, Dallas Williams, Denise Woods, Baoshun Liu, Paul Perrotte, Joel W. Slaton, James A. Merritt, H. Barton Grossman, Colin P. Dinney

From the Department of Genitourinary Medical Oncology and the Department of Urology, The University of Texas M. D. Anderson Cancer Center; and Introgen Therapeutics, Inc, Houston, TX.

Address reprint requests to Lance C. Pagliaro, MD, Department of Genitourinary Medical Oncology, Box 427, The University of Texas M.D. Anderson Cancer Center, 1515 Holcombe Boulevard, Houston, TX 77030-4009; email: lpagliar{at}mdanderson.org.

	ABSTRACT

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION REFERENCES

 
Purpose: We investigated the feasibility, safety, and biologic activity of adenovirus-mediated p53 gene transfer in patients with locally advanced bladder cancer.

Patients and Methods: Patients with measurable, locally advanced transitional-cell carcinoma of the bladder who were not candidates for cystectomy were eligible. On a 28-day cycle, intravesical instillations of INGN 201 (Ad5CMV-p53) were administered on days 1 and 4 at three dose levels (10¹⁰ particles to 10¹² particles) or on either 4 or 8 consecutive days at a single dose level (10¹² particles).

Results: Thirteen patients received a total of 22 courses without dose-limiting toxicity. Specific transgene expression was detected by reverse transcriptase polymerase chain reaction in bladder biopsy tissue from two of seven assessable patients. There were no changes in p53, p21^waf1/cip1, or bax protein levels in bladder epithelium evident from immunohistochemical analysis of 11 assessable patients. Outpatient administration of multiple courses was feasible and well tolerated. A patient with advanced superficial bladder cancer showed evidence of tumor response.

Conclusion: Intravesical instillation of Ad5CMV-p53 is safe, feasible, and biologically active when administered in multiple doses to patients with bladder cancer. Observations from this study indicate that this treatment has an antitumor effect in superficial transitional-cell carcinoma. Improvements in the efficiency of gene transfer and the levels of gene expression are required to develop more effective gene therapy for bladder cancer.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION REFERENCES

 
APPROXIMATELY 80% of patients with bladder cancer are diagnosed with tumors confined to the bladder.¹ Endoscopic surgery is the standard treatment for superficial bladder cancer, but the majority of patients remain at risk for tumor recurrence or progression because of minimal residual disease and a urothelial field defect. The intravesical administration of bacille Calmette-Guérin (BCG) or other therapeutic agents as adjuvant therapy is effective and changes the natural history of the disease.² Unfortunately, 20% to 30% of patients still suffer recurrences despite BCG immunotherapy, and radical cystectomy is often necessary for continued local control and to prevent life-threatening progression and metastasis.

Therapeutic gene transfer is a new strategy for modifying the urothelium.³ In previous work, we used a mouse model to show that the luciferase reporter gene was highly expressed in bladder tissue after intravesical administration of an adenoviral vector, and in contrast to intravenous injection, there was nearly complete absence of transfection in other organs.⁴ Intravesical administration has potential advantages over intratumoral injection, which is the method commonly used for vector-mediated gene transfer in other tumor types.^3,5,6 In a recently reported study, Kuball et al⁷ looked for evidence of adenovirus-mediated wild-type p53 gene transfer in the cystectomy specimens of patients who received either intratumoral injection (7.5 x 10¹¹ particles in 1 mL) or intravesical instillation (7.5 x 10¹¹ to 7.5 x 10¹³ particles) of the vector preoperatively. They detected expression of the vector transgene in bladder tissue by reverse transcriptase polymerase chain reaction (RT-PCR) in seven of eight assessable patients after an intravesical instillation and in none of those who had received an intratumoral injection, though the sample size was small (three patients).

In the case of adenovirus-mediated gene therapy, some of the obstacles to achieving transfection of tumor cells include the difficulties in reaching a tissue target and the low transduction efficiency of the vector.⁸ The safety and convenience of intravesical administration offers an opportunity to overcome these problems, however, through repetitive dosing in the treatment of patients with bladder cancer. There have been no clinical studies to determine the safety, long-term effects, or biologic activity of multiple-dosing schedules or long-term intravesical treatment with an adenoviral vector for patients with bladder cancer. Intravesical gene therapy has potential future applications as a bladder-sparing intervention and for controlling minimal residual disease.³ Therefore, the feasibility of repetitive dosing with an adenoviral vector and long-term effects on the urothelium in vivo are important end points.

The p53 gene is frequently mutated in bladder cancer and is known to govern processes critical to cell proliferation and survival.⁹ Preclinical data indicate that adenovirus-mediated delivery of the wild-type p53 gene to human bladder cancer cell lines results in growth inhibition and apoptosis, including lines with and without p53 mutations.^10,11 Furthermore, animal studies and phase I data from a single study indicate that adenovirus-mediated p53 expression is cytotoxic to bladder cancer cells but not to normal urothelium.^7,12

To further investigate the safety, feasibility, and biologic activity of adenovirus-mediated wild-type p53 gene transfer in patients with bladder cancer, we conducted a phase I, dose-escalation study of INGN 201 (Ad5CMV-p53) administered intravesically to patients with measurable, mucosal lesions of the urinary bladder, who were not eligible for cystectomy. In this study, we also assessed the feasibility of endoscopic tissue sampling for monitoring the biologic effects of investigational therapy.

	PATIENTS AND METHODS

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION REFERENCES

 
Patient Eligibility
Adult patients with pathologically documented transitional-cell carcinoma (TCC) of the bladder were eligible for the study if they had refused or were not candidates for a cystectomy after failure of the primary therapy. The eligibility criteria were Zubrod performance status of 2 or lower; at least one bidimensionally measurable lesion in the bladder; at least one prior cisplatin-based cytotoxic treatment, unless contraindicated, for patients with muscle invasion or at least one prior course of BCG treatment for patients without muscle invasion; expected survival more than 12 weeks; urine viral culture negative for adenovirus; negative serology for human immunodeficiency virus type 1; no prior gene therapy; and written, informed consent. Patients with National Cancer Institute (NCI) grade 3 urinary incontinence, pregnant or lactating females, or patients with persistent toxicity of grade 3 or worse from prior chemotherapy were not eligible.

Study Design
This was a single-center, phase I, dose-escalation study of INGN 201 (Ad5CMV-p53) intravesical instillation. The study vector was supplied by the NCI Cancer Therapy Evaluation Program under a Cooperative Research and Development Agreement with Introgen Therapeutics, Inc (Houston, TX). Three patients were treated at each dose level, and dose escalation was permitted if no NCI grade 3 or worse toxicity was observed in each cohort. The protocol was approved by The University of Texas M.D. Anderson Cancer Center Institutional Review Board, NCI Cancer Therapy Evaluation Program, and the Recombinant DNA Advisory Committee of the National Institutes of Health Office of Biotechnology Activities. Informed consent was obtained from all participants.

Treatment Plan
The replication-defective adenoviral vector Ad5CMV-p53 contains the cytomegalovirus (CMV) promotor, wild-type human p53 cDNA, and an SV40 polyadenylation signal in a minigene cassette inserted into the E1-deleted region of modified adenovirus-5.¹³ Individual doses were 10¹⁰, 10¹¹, or 10¹² viral particles (total dose per instillation), and the first group of patients was to be treated on days 1 and 4 at each of these dose levels (Table 1). The second group of patients was to be treated on consecutive days (days 1 through 4) at 10¹² viral particles or the maximum-tolerated dose (MTD) defined in the first group. The third group of patients was to be treated on days 1 through 4 and days 8 through 11 at 10¹² viral particles unless a lower MTD was defined in the second group. The vector was suspended in phosphate-buffered saline, which was kept on ice until the time of administration, and a volume of 50 mL was used for each instillation with a dwell time of 20 minutes.

Clinical Monitoring
Patients were monitored for adverse effects for a minimum of 12 months. A follow-up cystoscopy was performed on day 4, 8, or 15 for patients in groups 1, 2, and 3, respectively, and on day 28 for all patients. Patients were seen for tumor assessment every month, including cystoscopy every 3 months, until there was evidence of progression or a cystectomy was performed. Patients who underwent cystectomy and were clinically disease-free were evaluated radiographically every 6 months thereafter. Hematology, serum chemistry (including electrolytes, ALT, AST, lactate dehydrogenase, total bilirubin, urea nitrogen, and creatinine), and urinalysis were performed before treatment and during follow-up visits. Urine was collected for cytology before treatment and on day 28.

Analysis of Bladder Biopsy Tissue
Tissue samples were either formalin-fixed or flash-frozen at the time of resection. For RT-PCR, RNA was extracted from thawed, homogenized, DNase-digested tissue. After reverse transcription, PCR was performed using primers specific to the Ad5CMV-p53 vector or to glyceraldehyde phosphate dehydrogenase (GAPDH). The bladder cancer cell line 5637 was transfected in vitro and used as a positive control. The vector-p53 transcript contains viral sequences from the CMV promotor, which serve to distinguish it from endogenous p53 mRNA.^5,6 For RT-PCR detection of vector-p53 gene expression, primers were constructed to bridge the viral and human transcribed sequences (Table 2; Fig 1). Only samples with a positive GAPDH reaction were considered assessable by RT-PCR. PCR products were resolved on 1% agarose gels and visualized by ethidium bromide staining.

View larger version (19K): [in this window] [in a new window]   Fig 1. Primers for reverse transcriptase polymerase chain reaction (PCR) of vector-p53 are designed to bridge the viral and human sequences, as shown. Abbreviations: bp, base pair; CMV, cytomegalovirus.

Statistical Methods
Three patients were to be treated at each dose level, with up to six patients at the final dose level. Toxicity was graded according to NCI common toxicity criteria (version 1.0). Toxicity not included in the toxicity scale was scored as grade 3 if hospitalization was required and grade 4 if toxicity was regarded as life-threatening. Overall survival was defined as the interval between the first treatment and death or last follow-up visit. Time to progression was defined as the interval between first treatment and appearance of a new metastasis, increased size of an established metastasis, increased size of an established bladder tumor, new hydronephrosis, or any progression in tumor stage (eg, from superficial to muscle-invasive). Positive urine cytology and recurrences of superficial TCC were not considered progression. Response was not an end point of the study, but any changes in tumor dimension, radiographic appearance, or cystoscopic appearance of an index lesion were noted.

	RESULTS

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION REFERENCES

 
Patient Characteristics
From December 1998 to June 2001, a total of 14 patients were registered, 13 of whom met eligibility criteria (Table 3). One patient was registered and subsequently found ineligible because of a positive urine adenoviral culture and lack of measurable disease on cystoscopy, and he was not treated with Ad5CMV-p53. All other patients had histologically confirmed TCC of the bladder. Nine patients were treated in the first group, three in the second group, and one in the third group, at which point we terminated the trial early because we had not observed significant toxicity and did not expect to reach the MTD. Of the patients treated, 10 had previously documented muscle-invasive bladder cancer, and three had extensive superficial disease that had recurred after BCG immunotherapy. Three patients who were treated with Ad5CMV-p53 subsequently underwent radical cystectomy for local control of residual TCC (patients 2, 11, and 14). Pretreatment tissue samples were obtained from all patients, and all but one patient had sufficient residual tumor for a second biopsy. Thus, tumor samples were successfully obtained from 12 patients, at all dose levels, before and after treatment with Ad5CMV-p53.

View larger version (111K): [in this window] [in a new window]   Fig 2. Patient 10, cystoscopic appearance day 1 (A) showing multiple superficial tumors; day 4 (B) showing superficial mucosal ulceration; day 28 (C) showing improvement; and p53 immunostain day 1 (D) and day 4 (E).

Detection of p53 Transgene
Tissue samples were obtained and flash-frozen in liquid nitrogen before and after treatment from 12 patients. Of these, seven were assessable for detection of vector-p53 expression by RT-PCR. The assessable posttreatment biopsies were obtained on day 4 (four patients), day 8 (two patients), or day 16 (one patient). We found vector-specific p53 expression in posttreatment bladder biopsies from two assessable patients who had received 10¹² viral particles per dose (Table 3; Fig 3). Transgene expression was detected in both tumor tissue and in random biopsies of normal-appearing mucosa.

View larger version (95K): [in this window] [in a new window]   Fig 3. Reverse transcriptase polymerase chain reaction detection of vector-p53 in tissue samples from patient 10. Glyceraldehyde phosphate dehydrogenase (GAPDH) detection day 28 is shown in lane 10, with positive control in lane 3. Size markers, lane 1; negative controls, lanes 5 and 8.

Long-Term Follow Up
Eleven of the 13 eligible patients have died. Two are alive at 15 and 44 months follow-up, respectively. Eleven patients have progressed, and the median time to progression was 2 months.

Patient 2 had muscle-invasive disease (T3b) previously treated with methotrexate, vinblastine, doxorubicin, and cisplatin chemotherapy. He received four courses at the lowest dose level (10¹⁰ viral particles), during which time there was stable, residual muscle-invasive TCC on bladder biopsy. There was no tumor progression, and a radical cystectomy was performed at 10 months. He remained disease-free at 44 months’ follow-up.

Patient 10 had a history of muscle-invasive TCC resected transurethrally, was not a candidate for cystectomy, and had only superficial (Ta/T1) lesions visible on day 1 (Fig 2A). Index lesions were reduced in size on days 4 and 28, after which the residual tumor tissue was resected entirely. He received a total of five courses, with 10¹² viral particles on days 1 and 4 of each course; during this time, he had a single recurrence of noninvasive TCC (Ta) that was resected. A second superficial recurrence was transurethrally resected at 12 months, during the follow-up period after treatment had been stopped. The patient died of a cerebral hemorrhage at 15 months. He did not experience progression after treatment with Ad5CMV-p53, and he was not known to have any residual or recurrent disease at the time of death.

Patient 14, the only patient treated at the highest dose level, demonstrated stable disease on day 16 (the cystoscopy was performed 1 day later than planned) and tumor progression on day 28. He tolerated eight instillations of Ad5CMV-p53 10¹² viral particles without any toxicity. He subsequently underwent radical cystectomy for local control and was alive with metastatic disease at 15 months of follow-up.

	DISCUSSION

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION REFERENCES

 
Our results demonstrate that daily outpatient administration of Ad5CMV-p53 intravesical instillation is feasible, safe, and well tolerated. Acute NCI grade 1 toxicities, including bladder spasm and superficial mucosal ulceration, were acceptable and provided further evidence of biologic activity from the treatment. The observation of acute inflammation on urine cytology obtained during long-term follow-up of three patients at the higher dose levels was an unexpected finding. Transduction of the vector-p53 gene into bladder tissue was confirmed by vector-specific RT-PCR analysis in two of seven assessable patients after as few as one dose, but in no patient did we see any significant change in the results of immunohistochemical staining for p53, p21^waf1/cip1, or bax. This analysis was confounded, in part, by abnormally high p53 staining in 11 of 12 assessable patients. Accumulation of immunoreactive p53 occurs in approximately 50% of TCC primary tumors and is usually related to the presence of mutations of the p53 gene and prolonged half-life of the abnormal gene product.¹⁴ The high frequency of increased p53 staining in this study was most likely a consequence of our patient selection, which targeted patients with advanced, treatment-refractory disease. We cannot exclude the possibility of p53 protein induction that was obscured by the high background.

The utility of endoscopic tissue sampling for the evaluation of biologic end points was also examined in this study. We successfully obtained bladder biopsies before and after treatment from 12 of 13 eligible patients, of which 11 were adequate for immunohistochemistry and seven contained RNA of sufficient quality for RT-PCR. We found that endoscopic retrieval of small tissue samples is an effective method for RT-PCR detection of therapeutic gene transduction, and that it can be accomplished in the course of routine cystoscopy with minimal additional discomfort to the patient and at multiple time points. Biopsies of normal-appearing mucosa were frequently found to be histologically abnormal, reflecting the well-described field effect that characterizes urothelial malignancy.¹⁵ In another recently published study, Kuball et al⁷ collected cystectomy specimens from 12 patients who received preoperative p53 gene therapy, of which 11 were assessable by RT-PCR and seven were positive for vector-p53 transgene expression. They also were unable to detect differences in immunostaining for p53, p21^waf1/cip1, and bax or for the TUNEL assay, despite having access to unlimited tissue.

Response was not an end point of the study, but all patients had measurable disease and were evaluated for evidence of antitumor activity. Eleven patients experienced progression within 3 months of starting treatment; nine patients had local progression, whereas two had progression of distant metastases. Patient 2 was treated at the lowest dose level of 10¹⁰ viral particles per dose and had stable TCC (grade 2) invading the muscularis propria throughout four courses of treatment. A radical cystectomy was performed at 10 months, and the patient was alive and disease-free at 44 months. This clinical course is unusual for muscle-invasive TCC that persists after neoadjuvant chemotherapy,¹⁶ as had occurred in this patient. Our data do not indicate that there is significant p53 transduction at this small dose, however, and it is likely that the favorable outcome reflected an indolent growth pattern rather than a treatment effect. Patient 10, conversely, received a 100-fold higher dose of 10¹² viral particles (days 1 and 4) and showed noticeable improvement in multiple superficial TCC tumors (grade 2 to 3). In addition, mucosal ulceration was noted on days 4 and 28, indicating a cytopathic effect of the treatment. Vector-specific p53 expression was detected by RT-PCR analysis of multiple biopsies obtained on day 4, and was negative on day 28 (Fig 3). On the basis of these observations, we suspect that there was an antitumor effect with Ad5CMV-p53 given at 10¹² viral particles on days 1 and 4.

Possible mechanisms of antitumor effect include p53 gene expression as well as a nonspecific inflammatory (or BCG-like) effect of the adenoviral vector. Patient 10 had histologically confirmed muscle-invasive TCC on a transurethral resection performed 5 months before study entry, so was not previously treated with BCG. We detected acute inflammation on the follow-up urine cytology, providing evidence of a cellular immune response. In light of the known sensitivity of superficial TCC to an inflammatory response,¹⁷ and considering the low level of vector-p53 expression observed, we favor the hypothesis that a BCG-like effect occurred in this patient.

Several factors can limit the efficiency of adenovirus-mediated gene transfer and may have contributed to the low levels of transduction observed in this study. In the specific case of the bladder mucosa, a protective glycosaminoglycan (GAG) layer forms a physiologic barrier to infection and could interfere with adenoviral gene transfer. In a rodent model of experimentally induced bladder tumors, however, the GAG layer was not present in tumor-bearing areas and seemed to block transfection of only the normal urothelium.¹⁸ Other factors that may interfere with adenoviral gene transfer are uneven distribution of the vector, which has been observed after intratumoral injection in animal models and results in focal rather than diffuse expression of the transgene;¹⁹ a neutralizing antibody response to the adenovirus; low levels of coxsackie and adenovirus receptor (CAR) expression on the surfaces of target cells, limiting the adhesion and internalization of viral particles;^20,21 and the genetic milieu of a particular cell, which may be more or less permissive for expression of the transgene after the vector has been internalized.²²

There is considerable interest in developing agents that enhance adenoviral gene transfer across the urothelial GAG layer.⁷ Connor et al²³ purified the polyamide Syn-3 for this purpose, but its effect in rats indicated interaction with CAR and increased adenoviral attachment, rather than GAG disruption alone, as the mechanism. Intravesical Syn-3 pretreatment also enabled efficient adenoviral gene transfer to human TCC cells growing superficially within the bladders of athymic nude mice, despite their relatively low CAR expression.²⁴ The CAR protein has been shown to facilitate intracellular adhesion in bladder cancer cell lines, indicating that its loss may not be a random event,²¹ and that the CAR status must be considered in the design of more effective bladder cancer gene therapy.

In conclusion, we have demonstrated that repeated daily administration of intravesical Ad5CMV-p53 is a feasible and well-tolerated method for therapeutic p53 gene transfer in patients with bladder cancer. RT-PCR analysis of tissue samples confirmed that bladder cancer cells were successfully transfected, but we found, as have other investigators,⁷ that there was not a detectable change in the immunostaining characteristics of bladder tissue after intravesical gene therapy. Clinical benefit was observed in a patient receiving long-term treatment with Ad5CMV-p53, although a nonspecific inflammatory response seems to have occurred rather than a p53-mediated antitumor effect. Recognition of this nonspecific mechanism will be important in future clinical trials of intravesical adenoviral gene therapy for bladder cancer, particularly because superficial bladder cancer and carcinoma-in-situ are logical targets for intravesical gene therapy.^3,9 Future work must address the need for more efficient gene delivery systems²⁵ and stronger transgene expression in the target tissue.

	ACKNOWLEDGMENTS

 
We thank Bogdan A. Czerniak, MD, PhD, William F. Benedict, MD, Richard E. Giles, PhD, and Christopher J. Logothetis, MD, for their guidance and support.

	NOTES

 
Supported, in part, by grant nos. CA76233 and CA91846, and in part by core grant no. CA16672 from the National Cancer Institute, Bethesda, MD.

	REFERENCES

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION REFERENCES

 
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9. Slaton JW, Benedict WF, Dinney CPN: p53 in bladder cancer: Mechanism of action, prognostic value, and target for therapy. Urology 57:852–859, 2001[CrossRef][Medline]

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16. Millikan R, Dinney C, Swanson D, et al: Integrated therapy for locally advanced bladder cancer: Final report of a randomized trial of cystectomy plus adjuvant M-VAC versus cystectomy with both preoperative and postoperative M-VAC. J Clin Oncol 19:4005–4013, 2001[Abstract/Free Full Text]

17. Ratliff TL: Role of the immune response in BCG for bladder cancer. Eur Urol 21:17–21, 1992 (suppl 2)

18. Shimizu H, Akasaka S, Suzuki S, et al: Preferential gene transfer to BBN-induced rat bladder tumor by simple instillation of adenoviral vector. Urology 57:579–584, 2001[CrossRef][Medline]

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21. Okegawa T, Pong RC, Li Y, et al: The mechanism of the growth-inhibitory effect of coxsackie and adenovirus receptor (CAR) on human bladder cancer: A functional analysis of CAR protein structure. Cancer Res 61:6592–6600, 2001[Abstract/Free Full Text]

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Submitted September 27, 2002; accepted January 9, 2003.

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