Introduction

Prostate cancer is the second leading cause of cancer death in men in the United States, and it is expected that some 27,000 men will die from it this year.¹ Treatment options for newly diagnosed, clinically localized prostate cancer include surgery and radiation therapy. Both treatments yield excellent long-term results (85% cure) in patients with organ-confined, low-risk disease (Stage T1/T2, Gleason score 6, and prostate-specific antigen (PSA) 10 ng/ml).^2,3 Unfortunately, radiation therapy is less effective against more aggressive forms of the disease (Gleason score 7 or PSA >10 ng/ml) as it fails to eradicate the cancer in 30–60% of patients, putting them at risk for future progression. Novel therapies that can improve the effectiveness of radiation therapy in these higher-risk patients need to be developed.

For the past 13 years, we have been developing a multimodal investigational cancer treatment that combines the oncolytic actions of human adenoviruses with the cytotoxic effects of chemo-radiosensitizing genes. The rationale for this investigational approach stems from our original observations that both suicide gene therapy and oncolytic virotherapy improved the effectiveness of radiation therapy in preclinical models of cancer.^4,5,6,7,8,9 On the basis of these results, we conducted two clinical trials targeting clinically localized prostate cancer using a first-generation oncolytic adenovirus (Ad5-CD/TKrep) armed with a bacterial cytosine deaminase (bCD)/wild-type herpes simplex virus thymidine kinase (HSV-1 TK) fusion gene. In two settings (locally recurrent and newly diagnosed) of prostate cancer, this investigational approach was associated with low toxicity and preliminary signs of efficacy were obtained.^10,11 At 5-year follow-up, the gene therapy alone was found to have had a significant impact on PSA doubling time, a non-validated, surrogate endpoint with significant prognostic power.¹² Other gene therapy strategies have also generated encouraging results in early-stage trials of prostate cancer.^{13,14,15,16,17,18}

Before proceeding to a randomized, controlled Phase III trial that will test the merit of combining replication-competent adenovirus-mediated suicide gene therapy with intensity-modulated radiotherapy (IMRT) versus IMRT alone, we decided to optimize our approach by making two improvements to our first-generation adenovirus. Relative to Ad5-CD/TKrep,⁶ the second-generation Ad5-yCD/mutTK_SR39rep-ADP adenovirus is armed with an improved yeast cytosine deaminase (yCD)/mutant SR39 herpes simplex virus thymidine kinase (HSV-1 TK_SR39) fusion gene (yCD/mutTK_SR39).¹⁹ Both enzyme products of the yCD/mutTK_SR39 fusion gene are more catalytically active than the prototype bacterial cytosine deaminase/HSV-1 TK gene contained in Ad5-CD/TKrep and generate a better chemotherapeutic and radiosensitization effect. Moreover, Ad5-yCD/mutTK_SR39rep-ADP also expresses high levels of the adenovirus death protein (ADP) gene, which enhances the oncolytic activity of replication-competent adenoviruses and improves local adenoviral spread.^{19,20,21,22,23,24} Here, we report the results of a Phase I trial with the second-generation Ad5-yCD/mutTK_SR39rep-ADP adenovirus in combination with IMRT in patients with newly diagnosed intermediate- to high-risk prostate cancer. We present our preliminary observations, which suggest that the gene therapy may have the potential to improve the outcome of conformal radiotherapy in certain patient groups.

Results

Patient baseline characteristics

Patient baseline characteristics are shown in Table 1. A total of nine patients in three cohorts were treated. The median age of patients was 63 years (range 53–81). The median PSA before any treatment (including hormone therapy) was 6.4 ng/ml (range 3.7–13.1). The mean Gleason score was 7 (range 6–9). Using accepted risk-group classification criteria (see Materials and Methods), five patients had intermediate-risk disease and four had high-risk disease. The median PSA follow-up period after the gene therapy is 12 months (range 2–16 months).

Table 1 - Patient baseline characteristics (Office of Biotechnology Activities protocol 0307-590).

Full table

Treatment schema

The treatment schema is depicted in Figure 1. Patients in Cohorts 1 and 2 received a single intraprostatic injection of Ad5-yCD/mutTK_SR39rep-ADP on Day 1 at 10¹¹ and 10¹² viral particles (vps), respectively. Three days later, patients were administered 2.6 weeks (13 days, weekdays only) of 5-fluorocytosine (5-FC) + valganciclovir (vGCV) prodrug therapy along with 74 Gy IMRT. To extend the radiosensitizing potential of the suicide gene therapy throughout the IMRT course, patients in Cohort 3 received two intraprostatic injections (10¹² vps per injection) of Ad5-yCD/mutTK_SR39rep-ADP on Days 1 and 22, with each injection being followed by a 2.6-week cycle of 5-FC + vGCV prodrug therapy and concomitant 74 Gy IMRT. Toxicity assessments were made once a week during the IMRT course and then at scheduled follow-up visits (see Materials and Methods). Prostate biopsies were obtained at 6, 12, and 24 months to assess the presence of adenocarcinoma.

Figure 1.

Treatment schema. Patients underwent a series of pre-treatment evaluations and had to meet all of the eligibility requirements to be enrolled in the trial. Patients in Cohorts 1 and 2 received a single intraprostatic injection of the Ad5-yCD/mutTK_SR39rep-ADP adenovirus on Day 1 at 10¹¹ and 10¹² viral particles (vps), respectively. Three days later, patients were administered 2.6 weeks (13 days, weekdays only) of 5-fluorocytosine (5-FC) + valganciclovir (vGCV) prodrug therapy and concomitant 74 Gy intensity-modulated radiotherapy (IMRT). Patients in Cohort 3 received two intraprostatic injections of Ad5-yCD/mutTK_SR39rep-ADP on Days 1 and 22 at 10¹² vps per injection. Each adenovirus injection was followed by a 2.6-week course (13 days, weekdays only) of 5-FC + vGCV prodrug therapy and concomitant 74 Gy IMRT. Toxicity assessments and serum prostate specific antigen (PSA) were obtained once a week until completion of the IMRT course and at scheduled follow-up visits thereafter. Prostate biopsies were obtained at 6, 12, and 24 months and analyzed for the presence of adenocarcinoma. The primary endpoint was toxicity through Day 90. Secondary endpoints were PSA response and local tumor control (prostate biopsy) at 6, 12, and 24 months. ADP, adenovirus death protein.

Full figure and legend (7K)

Acute toxicity

Adverse events through Day 90 (Day 1 being the day of the first adenovirus injection), regardless of their attribution, are reported in Table 2. The vast majority (92%) of adverse events were mild (grade 1) to moderate (grade 2), with only five events being grade 3 or above. Adverse events that occurred in two or more patients included lymphopenia (89%), gastrointestinal (GI) (diarrhea) and genitourinary (GU) (urinary frequency, nocturia, dysuria) events (67%), anemia (44%), elevation in hepatic transaminitis (44%), rigors/chills (33%), urinary retention (22%), neutropenia (22%), elevation in -glutamyl transpeptidase (22%), hyperglycemia (22%), hyponatremia (22%), fever (22%), hot flushes/sweats (22%), and abnormal prothrombin time/partial prothrombin time (22%). Most of these events were expected and could be attributed to dissemination of the Ad5-yCD/mutTK_SR39rep-ADP adenovirus to collateral tissues (elevation in hepatic transaminitis and -glutamyl transpeptidase, rigors/chills, fever), the hematological suppressive effects of the 5-FC + vGCV prodrug therapy (lymphopenia, anemia, neutropenia), and the well-known side effects of the radiation (GU and GI events, urinary retention) and hormone therapies (hot flushes/sweats). The grade 3 and 4 events of hyperglycemia, which occurred in diabetic patients, had an unknown basis and have been observed in two previous gene therapy trials.^10,11 Hyponatremia is often associated with hyperglycemia. There was no increase in acute toxicity with two (Cohort 3) rather than one (Cohorts 1 and 2) cycles of the gene therapy (data not shown).

Table 2 - Distribution of adverse events by severity for all cohorts through day 90 (Office of Biotechnology Activities protocol 0307-590).

Full table

There was one patient death (Patient 2) before the Day 90 toxicity endpoint. This patient completed the investigational therapy with minor toxicities (five grade 1 events, grade 3 lymphopenia) and died on Day 84 from complications (aspiration pneumonitis) that developed following elective abdominal surgery (ventral hernia). This event was reported to, and reviewed by, all institutional (Institutional Review Board, Institutional Biosafety Committe, Data and Safety Monitoring Board) and federal (Food and Drug Administration, Office of Biotechnology Activities)agencies and was judged to be unrelated to the gene therapy.

The toxicities observed in this Phase I trial (Office of Biotechnology Activities protocol 0307-590) were compared with those observed in our previous Phase I trial (Office of Biotechnology Activities protocol 0104-464) using the first-generation Ad5-CD/TKrep adenovirus.¹¹ The two studies enrolled similar types of patients, used the same mean adenovirus dose (10¹² vps) and injection procedures (transrectal ultrasound-guided), and used similar radiation doses (74 Gy versus 71 Gy). Therefore, any increase in toxicity observed here would likely be attributable to the activities of the yCD/mutTK_SR39 fusion and/or ADP genes. Despite the fact that Ad5-yCD/mutTK_SR39rep-ADP expresses more active suicide genes and high levels of ADP, there was no significant increase in hematologic, hepatic, GI, or GU toxicity relative to the previous trial conducted with the first-generation Ad5-CD/TKrep adenovirus (Table 3).

Table 3 - Comparison of adverse events in Office of Biotechnology Activities protocols 0307-590 and 0104-464.

Full table

Short-term efficacy

Post-treatment PSA and prostate biopsy results to date are provided in Table 4 As expected following definitive radiotherapy with or without hormone therapy, all patients experienced significant declines in PSA. Eight patients underwent at least one post-treatment prostate biopsy. Seven of eight patients (88%) were negative for adenocarcinoma at their last biopsy. The one positive patient (Patient 9) had extensive bilateral disease (10 of 12 cores positive) with a Gleason score of 9, representing the most advanced organ-confined prostate cancer that we have treated to date.

Table 4 - Post-treatment prostate specific antigen and prostate biopsy results (Office of Biotechnology Activities protocol 0307-590).

Full table

The post-treatment prostate biopsy results of our two clinical trials combining replication-competent adenovirus-mediated suicide gene therapy with conformal radiotherapy are summarized in Table 5 A total of 24 patients with intermediate- to high-risk prostate cancer have been treated to date. Fifteen patients were treated with the first-generation Ad5-CD/TKrep adenovirus and nine were treated with the second-generation Ad5-yCD/mutTK_SR39rep-ADP adenovirus. At least one post-treatment prostate biopsy has been obtained from 23 patients. Whereas 40% of these intermediate- to high-risk patients would have been expected to have a positive post-treatment biopsy had they been treated with radiotherapy alone (see Materials and Methods),^25,26,27 22% of evaluable patients treated with the gene therapy/radiotherapy combination were positive for adenocarcinoma at their last biopsy (odds ratio = 0.25, exact 95% confidence limits 0.07–0.95, P = 0.038). When the results were stratified by prognostic risk category, most of the treatment effect was observed in the intermediate-risk group. Specifically, 0 of 12 intermediate-risk patients (0%) were positive for adenocarcinoma at their last biopsy, which is better than expected (30%) for this subgroup of patients (P < 0.01, Fisher exact test). With a median PSA follow-up of 48 (protocol 0104-464) and 12 (protocol 0307-590) months, none of these 12 intermediate-risk patients (0%) have exhibited PSA relapse using accepted definitions of biochemical failure.^28,29 In contrast, 5 of 11 high-risk patients (45%) were positive for adenocarcinoma at their last biopsy, which is not significantly different from the expected result (56%) for this prognostic risk group (P = 0.72, Fisher exact test). Three high-risk patients (protocol 0104-464) have failed biochemically, all of whom were positive for cancer at their last biopsy.

Table 5 - Summary of post-treatment prostate biopsy results^a combining Office of Biotechnology Activities protocols 0104-464 and 0307-590.

Full table

Discussion

Using a second-generation oncolytic adenovirus armed with improved suicide genes and the ADP gene, we demonstrate here that replication-competent adenovirus-mediated suicide gene therapy can be combined safely with IMRT in men with newly diagnosed prostate cancer. When we combined the results of our three Phase I trials of 40 patients,^10,11,12 24 of whom also received conformal radiotherapy, 93% of the adverse events have been grade 2 or below. The only events of grade 3 or above that occurred in two or more men were lymphopenia (5 of 40; 13%) and hyperglycemia (7 of 40; 18%). Lymphopenia is an expected side effect of the prodrug therapy and resolves shortly after completion of the prodrug therapy course. The basis for the hyperglycemia is unknown, but it may be related to the fact that many of these elderly patients are known or borderline diabetics and/or to treatment-related stress. All other grade 3 or above events were isolated cases, and the vast majority were due to co-morbid conditions. There was no increase in toxicity when we compared two cycles versus one cycle of the gene therapy. Importantly, at 5-year follow-up, the gene therapy was not associated with any late side effects.¹² Together, these Phase I studies demonstrate the safety of this investigational approach in men with clinically localized prostate cancer.

Although it is easy to assess toxicity in clinical trials of prostate cancer, the assessment of efficacy is, paradoxically, more difficult. In the Phase III setting, survival remains the gold standard endpoint. However, prostate cancer has a long natural history, and the time from initial diagnosis to death can span one or more decades, making it difficult to assess the effectiveness of investigational therapies for newly diagnosed disease. Indeed, this is the primary reason most pharmaceutical companies do not undertake the development of new treatments for early-stage prostate cancer. Moreover, despite the fact that prostate cancer is among the few cancers in which disease progression can be monitored with a simple blood test (i.e., serum PSA), the absolute PSA level (i.e., nadir) after definitive radiotherapy does not have sufficient predictive power to make it a valid surrogate endpoint for clinical outcome. PSA doubling time is gaining acceptance and was recently shown to satisfy three of Prentice's four criteria required for validation of a surrogate endpoint.³⁰ However, PSA doubling time can be examined only in the context of a rising PSA. Biochemical (PSA) disease-free survival is the most common endpoint used in clinical trials of localized prostate cancer. However, it often requires long (5 years) follow-up and has not been proven to be prognostic for either prostate cancer–specific or overall survival. Moreover, the use of hormone therapy is becoming more common, even in intermediate-risk patients, which confounds and delays the analysis of post-treatment PSA results.

So how can the effectiveness of investigational treatments for newly diagnosed prostate cancer, such as gene therapy, be assessed in a timely manner before PSA relapse or death? Although the measure is not without its weaknesses, we believe that in the context of a randomized, controlled Phase III trial, prostate biopsy (12 cores) status at 2 years is a reasonable endpoint to assess the potential therapeutic effect of gene therapy when combined with loco-regional treatments such as definitive radiotherapy. One major drawback of using prostate biopsy as an endpoint, in addition to its invasiveness, is its high false negative rate, which is estimated to be 15–30% with the standard sextant biopsy.^31,32,33,34 This high error rate can be reduced, but not eliminated, by sampling more (e.g., 12) biopsy cores.³⁵ Another drawback is that the pathological determination of adenocarcinoma after radiotherapy is equivocal in 25% of cases at 2 years.^36,37,38,39 Staining for proliferation markers and high-molecular-weight cytokeratin can sometimes aid in the diagnosis of malignant versus atypical, non-malignant cells.

These weaknesses highlight the need to be cautious when assessing efficacy or making treatment decisions solely on the basis of prostate biopsy results. However, despite these drawbacks, at least five clinical studies have shown that men with a positive prostate biopsy at 2 years and later, signifying local treatment failure, have a significantly poorer prognosis and a much greater risk of developing biochemical/clinical recurrence and distant metastases than do men with a negative biopsy.^{25,26,27,36,37,38,39,40,41,42} Once distant metastases develop, most patients will die of prostate cancer despite the implementation of salvage therapies. Thus, prostate biopsy status at 2 years after radiotherapy is an independent predictor of eventual outcome. Here, we present our preliminary post-treatment biopsy observations, which suggest that replication-competent adenovirus-mediated suicide gene therapy may have the potential to improve local tumor control of conformal radiotherapy. Although we find these preliminary observations encouraging, we want to emphasize that the sample size (n = 23) is relatively small and the results were compared retrospectively with historical controls, for which there is a paucity of available data. Thus, no conclusion regarding the effect of the gene therapy can be drawn at this time.

We cannot conclude without commenting that our preliminary observations indicate that this adenovirus-based gene therapy approach might be showing a greater treatment effect in the intermediate- versus high-risk group. Specifically, whereas 45% of high-risk patients were positive for adenocarcinoma at their last biopsy, none of the intermediate-risk patients (0%) showed any evidence of disease based on their last prostate biopsy, serum PSA, and digital rectal examination. For the intermediate-risk subgroup, these preliminary biopsy results are better than expected relative to historical controls even when taking into consideration the possible effects of hormone therapy. Although hormone therapy is not curative (i.e., it does not eradicate the cancer), it does reduce the volume of cancer,⁴³ which may reduce the cancer detection rate via needle biopsy. As 3 of 12 intermediate-risk patients (25%) described here (Table 5) received short-term (6 months) androgen suppression therapy (AST), it is possible that the better than expected results are, in part, attributable to the use of hormone therapy. However, it should be noted that 7 of 11 high-risk patients (64%) received long-term (2 years) AST and the apparent effect of the gene therapy was much less pronounced in this subgroup. Nevertheless, we wish to emphasize again that caution should be used when interpreting these preliminary biopsy results and no conclusions regarding the effect of the gene therapy can be drawn at this time. Indeed, because AST obfuscates both prostate biopsy and PSA results, the use of AST will not be allowed in our follow-up randomized, controlled trial.

Assuming that these preliminary biopsy results are correct and that they will be confirmed in our follow-up trial, it is interesting to speculate on what may be underlying basis for the apparent differential effect between the intermediate- versus high-risk group. Adenovirus infection is a two-step process that involves the binding of the fiber knob to the coxsackie adenovirus receptor (CAR) followed by attachment of the penton base proteins to cell surface integrins.^44,45 The infectivity of human cells by adenoviruses correlates well with CAR levels, including prostate cancer cells.^46,47,48,49 More pertinent to the clinical observations presented here, it has been reported previously that CAR expression is reduced in high grade (Gleason grade of 4 and 5) prostate cancer.⁵⁰ Although there was significant overlap in CAR antibody reactivity among the different Gleason grades and these (and our) results await confirmation, they, nevertheless, might provide a molecular explanation for our preliminary findings. If these observations are indeed correct, it raises the possibility that high grade (Gleason score 8) prostate cancer may be beyond the reach of "conventional" adenovirus-based gene therapy approaches. Armed, oncolytic adenoviruses that can infect high grade prostate cancer independent of CAR may be worthy of investigation. It is also possible that the infiltrative nature of high grade prostate cancer and/or differences in tumor microenvironment could explain the apparent differential response of the intermediate- versus high-risk groups.

In summary, we have demonstrated in three clinical trials of prostate cancer the safety of replication-competent adenovirus-mediated suicide gene therapy without and with conformal radiotherapy. In both settings, encouraging signs of efficacy have emerged. On the basis of these results, we have designed a multicenter randomized, controlled Phase III trial that will test the hypothesis that replication-competent adenovirus-mediated suicide gene therapy combined with IMRT will improve clinical outcome compared with IMRT alone.

Materials and Methods

Study design. The primary objective was to determine the toxicity associated with intraprostatic injection of the second-generation, Ad5-yCD/mutTK_SR39rep-ADP adenovirus combined with 5-FC and vGCV prodrug therapy and IMRT. Secondary endpoints included post-treatment PSA response and presence of adenocarcinoma in needle biopsies of the prostate at 6, 12, and 24 months. All patients were treated in the Department of Radiation Oncology at the Henry Ford Health System. The protocol called for two cohorts of three patients, each of whom would receive a single intraprostatic injection of the Ad5-yCD/mutTK_SR39rep-ADP adenovirus on Day 1 at 10¹¹ vps (Cohort 1) and 10¹² vps (Cohort 2). Three days later, patients were administered 2.6 weeks (13 days, weekdays only) of 5-FC (150 mg/kg/day) and vGCV (1,800 mg/day) prodrug therapy concomitant with 74 Gy IMRT. As patients in Cohort 3 were scheduled to receive a second adenovirus injection on Day 22, 2.6 weeks (13 days) rather than 3 weeks (15 days) of prodrugs were used to preclude the possibility that the first prodrug therapy cycle would inhibit viral replication following the second adenovirus injection. Patients in Cohorts 1 and 2 were followed for 90 days before proceeding to the next cohort. After completion of Cohort 2 and review of the results by the Food and Drug Administration, Data and Safety Monitoring Board, and Institutional Review Board, a third cohort (Cohort 3) was enrolled, in which three patients received two intraprostatic injections of Ad5-yCD/mutTK_SR39rep-ADP on Days 1 and 22 at 10¹² vps, with each adenovirus injection being followed by a 2.6-week course of 5-FC + vGCV prodrug therapy concomitant with 74 Gy IMRT. The study was conducted under BB-IND 11253 (RAC protocol 0307-590) and complied with all federal and institutional regulations regarding human patient research. Informed consent was obtained from all research patients before their enrollment.

Patient selection. Patients had to meet all of the eligibility requirements of the protocol to be enrolled in the study. Accrual on the study was good. More than 50% of patients who met the initial inclusion criteria (Stage, Gleason score, PSA) expressed an interest and met all of the inclusion/exclusion criteria for the study. There were no protocol violations. Patients were required to have biopsy-proven evidence of prostate adenocarcinoma, Stage T1c–T4 and Gleason score 7 or serum PSA >10 ng/ml but <50 ng/ml. The following risk classifications were used: intermediate-risk: Stage T1/T2 and Gleason score = 7 or PSA >10 ng/ml but 20 ng/ml; high-risk: Stage T3/T4 or Gleason score 8 or PSA >20 ng/ml. Patients could not have any evidence of metastatic disease as evaluated by bone scan and computed tomography scan of the abdomen and pelvis. Patients were required to have adequate baseline organ function, as assessed by the following laboratory values: adequate renal function with serum creatinine 1.5 mg/dl or creatinine clearance 45 ml/min/m², platelet count 100,000/mm³, absolute neutrophil count 1,000/mm³, hemoglobin 10.0 mg/dl, normal partial prothrombin time and prothrombin time, bilirubin 1.5 mg/dl, and serum glutamic oxaloacetic transaminase (aspartate transaminase) and serum glutamic pyruvic transaminase (alanine aminotransferase) <2.5 times the upper limit of normal. Patients with acute infection, human immunodeficiency virus–positive tests, or a history of liver disease were excluded from the study.

Intraprostatic adenovirus injection. Adenovirus injections were performed transrectally under transrectal ultrasound guidance. A standardized adenovirus injection algorithm was developed based on pre-treatment prostate biopsy (6–14 cores). The algorithm used the following three criteria based on a total injection volume of 3 ml: (i) all sextants would be treated, (ii) each negative sextant would receive 0.25 ml, and (iii) each positive sextant would receive 0.25 ml + 1.5 ml/(the number of positive sextants). For example, if the pre-treatment biopsy showed adenocarcinoma in only two sextants, each of the four negative sextants would receive 0.25 ml (8.3%) of the prescribed dose, and each of the two positive sextants would receive 1.0 ml (33%) of the prescribed dose. In other words, all sextants were treated, but the adenovirus dose distribution was skewed to those sextants known to contain cancer. The adenovirus was deposited in each sextant in multiple aliquots using a 20-gauge needle.

Prodrug therapy. Patients in Cohorts 1 and 2 received 2.6 weeks (13 days, weekdays only) of prodrug therapy beginning on Day 4. Patients in Cohort 3 received 2 2.6-week cycles of prodrug therapy beginning on Days 4 and 25. For 5-FC (Ancobon; Roche Laboratories, Nutley, NJ), a total of 150 mg/kg/day was given orally in four equally divided doses. For vGCV (Valcyte; Roche Laboratories, Nutley, NJ), a total of 1,800 mg/day was given orally in two equally divided doses every 12 hours.

Intensity-modulated radiation therapy. From the initiation of prodrug therapy (Day 4), all patients received 74 Gy (37 fractions of 2 Gy per fraction) of IMRT using state-of-the-art planning and computed tomography simulation. Radiation was administered on an isocentrically mounted megavoltage equipment unit using photon energies 6 mV. The radiation dose was prescribed to the planning target volume.

Hormone therapy. AST was offered at the discretion of the treating physician after consultation with the patient. This consisted of luteinizing hormone–releasing hormone analogs without or with non-steroidal anti-androgen therapy. The gene therapy/IMRT was administered 2 months after initiation of AST.

Patient monitoring and follow-up evaluations. Toxicity assessments and serum PSA were performed once a week until completion of the IMRT course and then at 3, 6, 9, 12, 18, and 24 months and annually thereafter. Toxicity assessments were recorded on protocol-specific case report forms and were based on physical examinations by the treating physician, complete blood cell counts, and blood chemistries. GI and GU toxicities were graded by the treating physician using the Radiation Therapy Oncology Group morbidity scale. All other toxicities were graded using the National Cancer Institute's Common Terminology Criteria for Adverse Events (version 3). The presence of infectious adenovirus in serum and Ad5-yCD/mutTK_SR39rep-ADP viral DNA in blood was monitored at every blood draw until they were not detected in two consecutive measurements, as previously described.^10,11 No infectious adenovirus was detected in serum at any blood draw. Ad5-yCD/mutTK_SR39rep-ADP viral DNA was detectable by PCR in blood as late as Day 84 in Cohorts 1 and 2 and Day 118 in Cohort 3. As no infectious adenovirus was observed in patients' urine in two previous Phase I trials,^10,11 it was not monitored here. Infectious adenovirus was not monitored in stools.

Prostate biopsy. Prostate biopsies (6–14 cores) were obtained at 6, 12, and 24 months. In four cases, one of the three post-treatment biopsies was cancelled owing to concomitant medical issues. Biopsies were scored for adenocarcinoma using a two-tier classification system: negative or not-negative (positive). Treatment effects and immune reactions were noted but not used when scoring biopsies. Specimens containing suspicious or atypical cells were stained for Ki-67 and high-molecular-weight cytokeratin to ascertain the presence of proliferative cells and presence/absence of basal epithelial cells, respectively. Pre- and post-treatment biopsies were first graded and scored by several pathologists and then re-reviewed by a single GU pathologist (A.S.). There were no discrepancies among the pathologists regarding the assessment of adenocarcinoma in post-treatment biopsies. Thus, the post-treatment biopsies results reported in Table 5 are based on the determination of at least two pathologists with 100% concordance.

Based on the results of a randomized, controlled Phase III trial of men with low-, intermediate-, and high-risk prostate cancer,^25,26 it is expected that 30% of all patients will have a positive prostate biopsy at 2 years based on a sextant biopsy. However, for only intermediate- and high-risk patients, the biopsy positivity rate is 40% even with hormone therapy (A. Pollack, Fox Chase Cancer Center, Philadelphia, PA, personal communication, 17 July 2006). The expected biopsy positivity rate for high-risk patients treated with the radiation doses used here is 56%.²⁷

Manufacturing of Ad5-yCD/mutTK_SR39rep-ADP adenovirus. Clinical-grade Ad5-yCD/mutTK_SR39rep-ADP adenovirus was manufactured at the Baylor College of Medicine Vector Production Facility (Houston, TX). The adenovirus was supplied as a sterile frozen liquid in vials containing 1.25 ml at a concentration of 1 10¹² vps/ml. The infectious unit to vp ratio of the clinical lot was 13. The clinical lot was tested extensively for contaminants and met all Food and Drug Administration specifications. PCR revealed that the clinical lot contained <1 vp of recombinant adenovirus containing a wild-type E1A region per 3 10¹⁰ vps. The stability of the clinical lot was evaluated every 6 months, and no loss of titer or potency was observed during the course of this Phase I study.

Biostatistics. The Cochran–Mantel–Haenszel test was used to compare the overall proportion of negative prostate biopsies between the investigational therapy (gene therapy plus radiation therapy) and control therapy (radiation therapy), stratified by patient's risk category evaluated before treatment. Exact confidence limits for the common odds ratio (odds ratio of investigational versus control therapy) were calculated with the P-value for testing odds ratio = 1. The Fisher exact test was used to compare the proportional difference between the two groups for each risk category.

References

REFERENCES

1. American Cancer Society (2005). Cancer facts and figures 2005. American Cancer Society: Atlanta.
2. D'Amico, AV, Whittington, R, Malkowicz, SB, Schultz, D, Blank, K, Broderick, GA et al. (1998). Biochemical outcome after radical prostatectomy, external beam radiation therapy, or interstitial radiation therapy for clinically localized prostate cancer. JAMA 280: 969–974. | Article | PubMed | ChemPort |
3. Shipley, WU, Thames, HD, Sandler, HM, Hanks, GE, Zietman, AL, Perez, CA et al. (1999). Radiation therapy for clinically localized prostate cancer. JAMA 281: 1598–1604. | Article | PubMed | ISI | ChemPort |
4. Kim, JH, Kim, SH, Brown, SL and Freytag, SO (1994). Selective enhancement by an antiviral agent of the radiation-induced cell killing of human glioma cells transduced with HSV-tk gene. Cancer Res 54: 6053–6056. | PubMed | ISI | ChemPort |
5. Khil, MS, Kim, JH, Mullen, CA, Kim, SH and Freytag, SO (1995). Radiosensitization by 5-fluorocytosine of human colorectal carcinoma cells in culture transduced with cytosine deaminase gene. Clin Cancer Res 2: 53–57.
6. Freytag, SO, Rogulski, KR, Paielli, DL, Gilbert, JD and Kim, JH (1998). A novel three-pronged approach to selectively kill cancer cells: concomitant viral, double suicide gene, and radiotherapy. Hum Gene Ther 9: 1323–1333. | PubMed | ISI | ChemPort |
7. Rogulski, K, Wing, M, Paielli, D, Gilbert, J, Kim, JH, Freytag, S et al. (2000). Double suicide gene therapy augments the antitumor activity of a replication-competent lytic adenovirus through enhanced cytotoxicity and radiosensitization. Hum Gene Ther 11: 67–76. | Article | PubMed | ISI | ChemPort |
8. Rogulski, KR, Freytag, SO, Zhang, K, Gilbert, JD, Paielli, DL, Kim, JH et al. (2000). In vivo antitumor activity of ONYX-015 is influenced by p53 status and is augmented by radiotherapy.Cancer Res 60: 1193–1196. | PubMed | ISI | ChemPort |
9. Freytag, S, Paielli, D, Wing, M, Rogulski, K, Brown, S, Kolozsvary, A et al. (2002). Efficacy and toxicity of replication-competent adenovirus-mediated double suicide gene therapy in combination with radiation therapy in an orthotopic mouse prostate cancer model. Int J Radiat Oncol Biol Phys 54: 873–885. | Article | PubMed | ChemPort |
10. Freytag, SO, Khil, M, Stricker, H, Peabody, J, Menon, M, DePeralta-Venturina, M et al. (2002). Phase I study of replication-competent adenovirus-mediated double suicide gene therapy for the treatment of locally recurrent prostate cancer. Cancer Res 62: 4968–4976. | PubMed | ISI | ChemPort |
11. Freytag, SO, Stricker, H, Pegg, J, Paielli, D, Pradhan, DG, Peabody, J et al. (2003). Phase I study of replication-competent adenovirus-mediated double suicide gene therapy in combination with conventional-dose three-dimensional conformal radiation therapy for the treatment of newly-diagnosed, intermediate- to high-risk prostate cancer. Cancer Res 63: 7497–7506. | PubMed | ISI | ChemPort |
12. Freytag, SO, Stricker, H, Peabody, J, Pegg, J, Paielli, D, Movsas, B et al. (2007). Five-year follow-up of trial of replication-competent adenovirus-mediated suicide gene therapy for treatment of prostate cancer. Mol Ther 15: 636–642. | Article | PubMed |
13. Herman, JR, Adler, HL, Aguilar-Cordova, E, Rojas-Martinez, A, Woo, S, Timme, TL et al. (1999). In situ gene therapy for adenocarcinoma of the prostate: a phase I clinical trial. Hum Gene Ther 10: 1239–1249. | Article | PubMed | ISI | ChemPort |
14. Teh, BS, Aguilar-Cordova, E, Kernen, K, Chou, CC, Shaley, M, Vlachaki, MT et al. (2001). Phase I/II trial evaluating combined radiotherapy and in situ gene therapy with or without hormonal therapy in the treatment of prostate cancer—a preliminary report. Int J Radiat Oncol Biol Phys 51: 605–613. | Article | PubMed | ChemPort |
15. DeWeese, TL, van der Poel, H, Li, S, Mikhak, B, Drew, R, Goermann, M et al. (2001). A Phase I trial of CV706, a replication-competent, PSA selective oncolytic adenovirus, for the treatment of locally recurrent prostate cancer following radiation therapy. Cancer Res 61: 7464–7472. | PubMed | ISI | ChemPort |
16. Kubo, H, Gardner, TA, Wada, Y, Koeneman, KS, Gotoh, A, Yang, L et al. (2003). Phase I dose escalation clinical trial of adenovirus vector carrying osteocalcin promoter-driven herpes simplex virus thymidine kinase in localized and metastatic hormone-refractory prostate cancer. Hum Gene Ther 14: 227–241. | Article | PubMed | ISI | ChemPort |
17. Teh, BS, Ayala, G, Aguilar, L, Mai, WY, Timme, TL, Vlachaki, MT et al. (2004). Phase I-II trial evaluating combined intensity-modulated radiotherapy and in situ gene therapy with or without hormonal therapy in treatment of prostate cancer-interim report on PSA response and biopsy data. Int J Radiat Oncol Biol Phys 58: 1520–1529. | Article | PubMed | ISI | ChemPort |
18. Small, EJ, Carducci, MA, Burke, JM, Rodriguez, R, Fong, L, van Ummersen, L et al. (2006). A phase I trial of intravenous CG7870, a replication-selective, prostate-specific antigen-targeted oncolytic adenovirus, for the treatment of hormone-refractory, metastatic prostate cancer. Mol Ther 14: 107–117. | Article | PubMed | ChemPort |
19. Barton, KN, Paielli, D, Zhang, Y, Koul, S, Brown, SL, Lu, M et al. (2006). Second-generation replication-competent oncolytic adenovirus armed with improved suicide genes and the ADP gene demonstrates greater efficacy without increased toxicity. Mol Ther 13: 347–356. | Article | PubMed | ChemPort |
20. Tollefson, AE, Scaria, A, Hermiston, TW, Ryerse, JS, Wold, LJ and Wold, WS (1996). The adenovirus death protein (E3-11.6 K) is required at very late stages of infection for efficient cell lysis and release of adenovirus from infected cells. J Virol 70: 2296–2306. | PubMed | ISI | ChemPort |
21. Tollefson, AE, Ryerse, JS, Scaria, A, Hermiston, TW and Wold, WS (1996). The E3-11.6 kDa adenovirus death protein (ADP) is required for efficient cell death: characterization of cells infected with adp mutants. Virology 220: 152–162. | Article | PubMed | ISI | ChemPort |
22. Doronin, K, Toth, K, Kuppuswamy, M, Ward, P, Tollefson, AE and Wold, WS (2000). Tumor-specific, replication-competent adenovirus vectors overexpressing the adenovirus death protein. J Virol 74: 6147–6155. | Article | PubMed | ISI | ChemPort |
23. Doronin, K, Kuppuswamy, M, Toth, K, Tollelfson, AE, Krajcsi, P, Krougliak, V et al. (2001).Tissue-specific, tumor-selective, replication-competent adenovirus vector for cancer gene therapy. J Virol 75: 3314–3324. | Article | PubMed | ISI | ChemPort |
24. Toth, K, Tarakanova, V, Doronin, K, Ward, P, Kuppuswamy, M, Locke, JE et al. (2003). Radiation increases the activity of oncolytic adenovirus cancer gene therapy vectors that overexpress the ADP (E3-11.6K) protein. Cancer Gene Ther 10: 193–200. | Article | PubMed | ChemPort |
25. Pollack, A, Zagars, GK, Starkschall, G, Antolak, JA, Lee, JJ, Huang, E et al. (2002). Prostate cancer radiation dose response: results of the M.D. Anderson phase III randomized trial. Int J Radiat Oncol Biol Phys 53: 1097–1105. | Article | PubMed | ISI |
26. Pollack, A, Zagars, GK, Antolak, JA, Kuban, D and Rosen, II (2002). Prostate biopsy status and PSA nadir level as early surrogates for treatment failure: analysis of a prostate cancer randomized radiation dose escalation trial. Int J Radiat Oncol Biol Phys 54: 677–685. | Article | PubMed | ISI |
27. Zelefsky, MJ, Fuks, Z, Hunt, M, Lee, HJ, Lombardi, D, Ling, CC et al. (2001). High dose radiation delivered by intensity modulated conformal radiotherapy improves the outcome of localized prostate cancer. J Urol 166: 876–881. | Article | PubMed | ChemPort |
28. Cox, J and Kaplan, R. for the American Society for Therapeutic Radiology and Oncology Consensus Panel. (1997). Consensus statement: guidelines for PSA following radiation therapy. Int J Radiat Oncol Biol Phys 37: 1035–1041. | PubMed | ISI |
29. Buyounouski, MK, Hanlon, AL, Eisenberg, DF, Horwitz, EM, Feigenberg, SJ, Uzzo, RG et al. (2005). Defining biochemical failure after radiotherapy with and without androgen deprivation for prostate cancer. Int J Radiat Oncol Biol Phys 63: 1455–1462. | Article | PubMed | ISI |
30. Valicenti, RK, DeSilvio, M, Hanks, GE, Porter, A, Brereton, H, Rosenthal, SA et al. (2006). Posttreatment prostatic-specific antigen doubling time as a surrogate endpoint for prostate cancer specific survival: an analysis of radiation therapy oncology group protocol 92-02. Int J Rad Oncol Biol Phys 66: 1064–1071.
31. Fleshner, NE, O'Sullivan, M and Fair, WR (1997). Prevalence and predictors of a positive repeat transrectal ultrasound guided needle biopsy of the prostate. J Urol 158: 505–509. | Article | PubMed | ISI | ChemPort |
32. Epstein, JI, Walsh, PC, Sauvageot, J and Carter, HB (1997). Use of repeat sextant and transition zone biopsies for assessing extent of prostate cancer. J Urol 158: 1886–1890. | Article | PubMed | ChemPort |
33. Norberg, M, Egevad, L, Holmberg, L, Sparen, P, Norlen, BJ and Busch, C (1997). The sextant protocol for ultrasound-guided core biopsies of the prostate underestimates the presence of cancer. Urology 50: 562–566. | Article | PubMed | ChemPort |
34. Svetec, D, McCabe, K, Peretsman, S, Klein, E, Levin, H, Optenberg, S et al. (1998). Prostate rebiopsy is a poor surrogate of treatment efficacy in localized prostate cancer. J Urol 159: 1606–1608. | Article | PubMed | ChemPort |
35. Eskew, LA, Bare, RL and McCullough, DL (1997). Systematic 5 region prostate biopsy is superior to sextant method for diagnosing carcinoma of the prostate. J Urol 157: 199–203. | Article | PubMed | ChemPort |
36. Crook, JM, Perry, GA, Robertson, S and Esche, BA (1995). Routine prostate biopsies following radiotherapy for prostate cancer: results for 226 patients. Urology 45: 624–632. | Article | PubMed | ChemPort |
37. Crook, JM, Bahadur, YA, Robertson, SJ, Perry, GA and Esche, BA (1997). Evaluation of radiation effect, tumor differentiation, and prostate specific antigen staining in sequential prostate biopsies after external beam radiotherapy for patients with prostate carcinoma. Cancer 79: 81–89. | Article | PubMed | ChemPort |
38. Crook, JM, Bahadur, YA, Bociek, RG, Perry, GA, Robertson, SJ and Esche, BA (1997). Radiotherapy for localized prostate carcinoma: the correlation of pretreatment prostate specific antigen and nadir prostate specific antigen with outcome as assessed by systematic biopsy and serum prostate specific antigen. Cancer 79: 328–336. | Article | PubMed | ChemPort |
39. Crook, JM, Malone, S, Perry, G, Bahadur, Y, Robertson, S and Abdolell, M (2000). Postradiotherapy prostate biopsies: what do they really mean? results for 498 patients. Int J Radiat Oncol Biol Phys 48: 355–367. | Article | PubMed | ChemPort |
40. Scardino, PT and Wheeler, TM (1988). Local control of prostate cancer with radiotherapy. Frequency and prognostic significance of positive results of postirradiation prostate biopsy. Natl Cancer Inst Monogr 7: 95–103.
41. Kuban, DA, el-Mahdi, AM and Schellhammer, P (1992). The significance of post-irradiation prostate biopsy with long-term follow-up. Int J Radiat Oncol Biol Phys 24: 409–414. | PubMed | ChemPort |
42. Prestidge, BR, Kaplan, I, Cox, RS and Bagshaw, MA (1992). The clinical significance of a positive post-irradiation prostatic biopsy without metastases. Int J Radiat Oncol Biol Phys 24: 403–408. | PubMed | ChemPort |
43. Vaillancourt, L, Ttu, B, Fradet, Y, Dupont, A, Gomez, J, Cusan, L et al. (1996). Effect of neoadjuvant endocrine therapy (combined androgen blockade) on normal prostate and prostatic carcinoma: a randomized study. Am J Surg Pathol 20: 86–93. | Article | PubMed | ISI |
44. Bergelson, JM, Cunningham, JA, Drouguett, G, Kurt-Joned, EA, Krithivas, A, Hong, JS et al. (1997). Isolation of a common receptor for coxsackie B viruses and adenovirus 2 and 5. Science 275: 1320–1323. | Article | PubMed | ISI | ChemPort |
45. Wickham, TJ, Mathias, P, Cheresh, DA and Nemerow, GR (1993). Integrins alpha v beta 3 and alpha v beta 5 promote adenovirus internalization but not virus attachment. Cell 73: 309–319. | Article | PubMed | ISI | ChemPort |
46. Hemmi, S, Geertsen, R, Mezzacasa, A, Peter, I and Drummer, R (1998). The presence of human coxsackie and adenovirus receptor is associated with efficient adenovirus-mediated transgene expression in human melanoma cell cultures. Gene Ther 9: 2363–2373. | ChemPort |
47. Richardson, C, Brennan, P, Powell, M, Prince, S, Chen, YH, Spiller, OB et al. (2005). Susceptibility of B lymphocytes to adenovirus type 5 infection is dependent upon both coxsackie–adenovirus receptor and alpha v beta 5 integrin expression. J Gen Virol 86: 1669–1679. | Article | PubMed | ChemPort |
48. Li, Y, Pong, RC, Bergelson, JM, Hall, MC, Sagalowsky, AI, Tseng, CP et al. (1999). Loss of adenoviral receptor expression in human bladder cancer cells: a potential impact on the efficacy of gene therapy. Cancer Res 59: 325–330. | PubMed | ISI | ChemPort |
49. Okegawa, T, Li, Y, Pong, RC, Bergelson, JM, Zhou, J and Hsieh, JT (2000). The dual impact of coxsackie and adenovirus receptor expression in human prostate cancer gene therapy. Cancer Res 60: 5031–5036. | PubMed | ISI | ChemPort |
50. Rauen, K, Sudilovsky, D, Le, J, Chew, K, Hann, B, Weinberg, V et al. (2002). Expression of the coxsackie adenovirus receptor in normal prostate and in primary and metastatic prostate carcinoma: potential relevance to gene therapy. Cancer Res 62: 3812–3818. | PubMed | ISI | ChemPort |

Acknowledgements

This work was supported by grant P01 CA097012 (S.O.F.) from the National Cancer Institute. The authors declare that they have no competing financial interests.