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Departments of Radiation Oncology [S. O. F., J. P., D. P., D. G. P., X. X., S. B., J. H. K.], Urology [H. S., J. P.], Pathology [M. D-V.], and Biostatistics [M. L.], Henry Ford Health System, Detroit, Michigan 48202
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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0.5 ng/ml. The results demonstrate that replication-competent adenovirus-mediated double-suicide gene therapy can be combined safely with conventional-dose 3D-CRT in patients with intermediate- to high-risk prostate cancer. The shorter than expected PSA half-life in patients receiving more than 1 week of prodrug therapy may suggest a possible interaction between the oncolytic adenovirus and/or double-suicide gene therapies and radiation therapy. |
INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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6 and PSA 10 ng/ml), conventional-dose (70–75 Gy) EBRT results in excellent long-term cancer-free survival (
80%; Ref. 1
). In contrast, disease control in intermediate-risk (Stage T2b or Gleason score = 7 or PSA 10–20 ng/ml) and high-risk (Stage T2c or Gleason score 8 or PSA > 20 ng/ml) patients is considerably less (25–70%) based on the same criteria. This poorer outcome highlights the need for better radiation regimens for intermediate- to high-risk patients. The advent of 3D-CRT and IMRT has made it possible to deliver higher than conventional radiation doses to the prostate while sparing surrounding normal structures. Dose escalation has demonstrated that disease control in intermediate- to high-risk patients increases with radiation doses beyond 70 Gy (2, 3, 4, 5, 6, 7, 8) . With IMRT, prescription doses of 81–86.4 Gy have resulted in an estimated PSA failure-free survival at 3 years of 86% and 81% for intermediate- and high-risk patients, respectively (6 , 7) , which is markedly better than the results that can be achieved with conventional-dose EBRT. Such high radiation doses delivered by standard radiotherapy would result in unacceptable toxicity (9) . Remarkably, with IMRT, the 3-year actuarial likelihood of developing grade 2 or higher late rectal and urinary complications was only 4% and 15%, respectively (7) . The RTOG has reported similar low rates of urinary and GI toxicities with 3D-CRT to 79.2 Gy (10) .
Although the results of these sequential dose-escalation studies are very encouraging, they have not been validated in a randomized, prospective trial, and it has not been firmly established that PSA failure-free survival is a surrogate for either disease-specific or overall survival. Moreover, the results of such sequential dose studies are confounded by the fact that there has been significant stage migration in prostate cancer during the last decade (11, 12, 13) . There has been only one randomized, prospective, Phase III trial evaluating the merits of high-dose radiotherapy in patients with clinically localized (T1/T2) disease (14, 15, 16) . High-dose (78 Gy) 3D-CRT resulted in significantly better biochemical disease-free survival (62% versus 43% for 78 versus 70 Gy; P = 0.012) and marginally better freedom from distant metastases (98% versus 88% for 78 versus 70 Gy; P = 0.056) for intermediate- to high-risk patients (PSA > 10 ng/ml) at 6 years (15) . However, there was no improvement in local control as determined by sextant needle biopsy of the prostate at 2 years (14) , and no significant impact on overall survival at 6 years (90% versus 83% for 78 versus 70 Gy; P = 0.67; Ref. 15 ). Although it is likely that the gains observed in this landmark study represent a lower limit of the results that can be achieved with state-of-the-art conformal techniques (e.g., IMRT), until high-dose radiotherapy demonstrates a significant impact on cancer-specific survival in a randomized, multicenter trial, conventional-dose 3D-CRT is likely to remain a standard of practice, and we can expect that a significant fraction of intermediate- to high-risk patients will develop recurrent disease.
Biological approaches that increase the intrinsic radiosensitivity of tumor cells are an attractive alternative, or perhaps adjuvant, to dose escalation. Indeed, the gain achieved by biological enhancement may be many times greater than the gain made possible by increasing the prescription radiation dose. Androgen-deprivation therapy has demonstrated to be an effect adjuvant to radiotherapy in both preclinical models (17, 18, 19) and in the clinic (20, 21, 22, 23) . Although it is unclear whether androgen deprivation actually increases the intrinsic radiosensitivity of tumor cells, resulting in true radiosensitization (24) , its combined use with definitive radiotherapy has resulted in significantly better overall and disease-free survival, making it a standard of practice in some patient subsets (22 , 23) . Many issues remain regarding the optimal timing (immediate versus delayed), duration (short versus long term), and patient subsets that would benefit most from such treatment. The morbidity and costs associated with long-term androgen-deprivation therapy are not insignificant.
We have developed a biological, gene therapy-based approach designed to improve the effectiveness of radiation therapy. Our approach uses a replication-competent, oncolytic adenovirus to deliver a pair of therapeutic suicide genes to the tumor (25, 26, 27, 28) . One suicide gene, CD, converts the prodrug 5-FC into 5-fluorouracil, which on further conversion results in the inhibition of thymidylate synthase and the depletion of dTMP pools. This leads to an increase in DNA strand breaks and cell cycle redistribution, sensitizing cells to the lethal effects of radiation (29 , 30) . A second suicide gene, HSV-1 TK, converts thymidine analogues such as vGCV into their corresponding monophosphates, which ultimately get incorporated into DNA, resulting in termination of DNA synthesis and cell death (31) . We were the first to hypothesize that implementation of the HSV-1 TK system might sensitize tumor cells to ionizing radiation by inhibiting sublethal repair after radiation damage (32) . We and others have demonstrated in an extensive series of preclinical studies that both the CD and HSV-1 TK suicide gene systems are potent tumor cell radiosensitizers (25, 26, 27 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42) . We further hypothesized that the HSV-1 TK system may block the repair of damaged DNA induced by the CD system, resulting in synergistic cell kill. We therefore generated a CD/HSV-1 TK fusion gene that would allow for simultaneous use of the CD and HSV-1 TK systems (25 , 35) . In preclinical tumor models, the combination of CD and HSV-1 TK suicide gene therapies (double-suicide gene therapy) has demonstrated to be both an effective chemotherapeutic regimen and a potentiator of radiotherapy (26 , 27) . Moreover, we (26 , 27 , 43) and others (44) have demonstrated that the interaction between oncolytic adenoviral therapy and radiotherapy is at least additive in providing greater therapeutic efficacy without excessive toxicity. Our preclinical studies provided the scientific basis for this and other (45) Phase I studies in which the safety of adenovirus-mediated suicide gene therapy in combination with radiation therapy was evaluated in patients with newly diagnosed prostate cancer. The results support the thesis that replication-competent adenovirus-mediated double-suicide gene therapy can be combined safely with conventional-dose 3D-CRT and that, together, they may ultimately offer a new therapeutic option for patients with intermediate- to high-risk prostate cancer.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. If none of the patients experienced a DLT, as defined by any irreversible, treatment-related grade 3 or 4 toxicity, the study proceeded to the next cohort. Secondary objectives included determination of the persistence of transgene expression in the prostate and evaluation of therapeutic response as demonstrated by a decline in serum PSA and histopathological analysis for adenocarcinoma in sextant needle prostate biopsies. Because no patient in cohort 3 showed evidence of CD/HSV-1 TK transgene expression in the prostate at the 4-week time point, it was reasoned that 4 weeks of prodrug therapy could not be justified, and no patients were enrolled in cohort 6.
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7 or serum PSA > 10 but 50 ng/ml. Patients with Stage T1/T2 and Gleason score 7 and PSA < 20 ng/ml were considered intermediate risk, and patients with Stage T3/T4 or Gleason score 8–10 or PSA > 20 ng/ml were considered high risk. Patients had to possess the ability to give informed consent and express a willingness to meet all of the expected requirements of the protocol for the duration of the study. Patients could not have any evidence of metastatic disease as evaluated by bone scan and CT scan of the abdomen and pelvis. Suspicious abnormal areas on bone scans were evaluated by magnetic resonance imaging. Patients were required to have adequate baseline organ function, as assessed by the following laboratory values before initiating the protocol: (a) adequate renal function with serum creatinine 1.5 mg/dl or creatinine clearance 45 ml/min/m2; (b) platelet count 100,000/mm3; (c) absolute neutrophil count 1000/mm3; (d) hemoglobin 8.5 mg/dl; (e) normal PTT and PT; and (f) bilirubin 1.5 mg/dl and AST and ALT < 2.5 times the upper limit of normal. Patients with acute infection (any viral, bacterial, or fungal infection that required specific therapy), HIV-positive tests, or a history of liver disease were excluded from the study.
Pretreatment Planning and Injection of Ad5-CD/TKrep Adenovirus.
Pretreatment planning included transrectal ultrasound and sextant needle biopsy of the prostate. Transverse images of the prostate were obtained every 0.5 cm from base to apex. Ultrasound images were examined for hypoechoic regions and "areas of suspicion." Three-dimensional reconstruction of the prostate was performed with use of the treatment planning system. Pretreatment prostate biopsies were performed at least 5 weeks before the adenovirus injection (day 1) to allow for PSA normalization. Needle biopsies were obtained from the base, mid, and apex sextants of the left and right sides. If a patient’s biopsy showed adenocarcinoma in only one side, 67% (6.7 x 1011 vp) of the prescribed viral dose (1012 vp) was injected into that side, and the contralateral side received the remaining 33% (3.3 x 1011 vp). If a patient’s biopsy showed adenocarcinoma in both sides, then both sides were injected with 50% (5 x 1011 vp) of the prescribed viral dose.
Injection of the Ad5-CD/TKrep adenovirus was performed on an outpatient basis on day 1. The virus was diluted to the proper concentration with sterile saline in a final volume of 3 ml. The patient was placed in the left lateral decubitus position with the legs flexed and the knees brought up toward the chest. The virus was injected transrectally with transrectal ultrasound guidance to aid in the placement of the injection needle. The virus was deposited in multiple aliquots (6–12 deposits; 0.25–0.5 ml/deposit) divided from the original dose through two separate injection sites using a 20-gauge needle. After each deposit, the needle was withdrawn, the tip was positioned into the next injection area, and the next aliquot of virus was delivered. This was repeated until all of the virus was delivered.
Administration of Prodrugs.
Prodrugs were administered on an outpatient basis. 5-FC (Ancobon; Roche Laboratories) was administered orally beginning on day 3 and continued for 1 (cohorts 1–3), 2 (cohort 4), or 3 (cohort 5) weeks. A total of 150 mg/kg/day was given in four equally divided doses. vGCV (Valcyte; Roche Laboratories) was administered orally beginning on day 3 and continued for 1 (cohorts 1–3), 2 (cohort 4), or 3 (cohort 5) weeks. A total of 1800 mg/day were given in two equally divided doses every 12 h. The research nurse assigned to the trial counted the pills on a daily (weekday) basis to monitor patient compliance.
Administration of Radiation Therapy.
All patients received conventional-dose 3D-CRT using state-of-the-art treatment techniques, including 3D CT simulation (AcQsim CT simulator), 3D treatment planning (Pinnacle treatment planning system, version 5.2; ADAC Lab) and Varian 2100C/D accelerator. Typically, six conformal fields were used, and the daily fractional dose was 2 Gy. All patients received a dose of 50 Gy to the CTV, which included the prostate and seminal vesicles. A boost dose (20–24 Gy) was given to the prostate only. The PTV conformed to the CTV plus a 0.5–1.0-cm margin to account for subclinical extraprostatic tumor extension and variations in treatment set up and internal organ motion. Treatment was administered with use of an isocentrically mounted megavoltage unit with photon energy 6 MV. The source-to-axis distance was 100 cm.
Androgen-Deprivation Therapy.
Patients with high-risk disease were offered androgen-deprivation therapy consisting of Lupron (TAP Pharmaceuticals Products, Inc, Deerfield, IL) administered in six 4-month (30 mg) depots beginning 2 months before initiation of radiation therapy. Four high-risk patients in this trial (patients 4, 7, 14, and 15) refused such treatment. Patient 5, who had intermediate-risk disease, initially selected surgery for his primary treatment. However, the surgery was aborted because he was unable to be properly ventilated because of chronic obstructive pulmonary disease. As a result, his treatment was delayed several months. He was put on hormone therapy to help relieve his anxiety.
Patient Monitoring and Toxicity Grading.
Patient evaluations were conducted twice a week during the prodrug therapy course and then once a week thereafter until completion of the radiation course. The evaluations included a physical exam, serum PSA, complete blood counts and blood chemistries, PT and PTT, and the presence of infectious adenovirus in serum and Ad5-CD/TKrep viral DNA in blood until not detected in two consecutive measurements. Toxicities are reported through week 6 after completion of the radiation therapy course and were graded based on the NCI CTC, version 2. Acute urinary and lower GI toxicities were graded based on the NCI CTC to allow for comparison to our previous Phase I gene therapy trial without EBRT (BB-IND 8436; Ref. 28
), and using the RTOG acute morbidity scoring criteria4
to allow for comparison to RTOG 9406 (46)
. Because a high fraction of patients were impotent before treatment, erectile dysfunction was not graded. Serum testosterone levels and fibrinogen and fibrin degradation products were not monitored.
After treatment, patients received standard urological care. The following evaluations were performed every 3 months for the first year and then every 6 months thereafter: physical exam, serum PSA, complete blood counts and blood chemistries, PT and PTT, and presence of Ad5-CD/TKrep viral DNA in blood until it was not detected in two consecutive measurements. Sextant needle biopsy of the prostate was performed at 2 (cohort 1), 3 (cohort 2), and 4 (cohort 3) weeks for analysis of transgene expression in the prostate. Patients in cohorts 4 and 5 were biopsied at 12 weeks, and all patients were biopsied at 1 year for histopathological analysis of adenocarcinoma.
Manufacturing of Ad5-CD/TKrep Adenovirus.
Details regarding the construction and structure of the Ad5-CD/TKrep adenovirus have been published (25)
. Clinical-grade (GMP) Ad5-CD/TKrep adenovirus was manufactured at the Baylor College of Medicine Gene Vector Laboratory (Houston, TX). Descriptions of the safety testing performed on the Master Viral Bank and final product have been described previously (28)
. A semiquantitative PCR assay was developed to determine the level of wild-type adenovirus type 5 (Ad5) contamination in clinical batches, using primers that hybridize to the 55-kDa E1B region deleted in Ad5-CD/TKrep. Four of seven clinical batches produced contained <1 vp of wild-type Ad5 per 109 Ad5-CD/TKrep vp. These four batches were pooled, generating the clinical lot used here. The Ad5-CD/TKrep clinical lot was reevaluated every 3 months for titer with use of human embryonic kidney (HEK 293) cells and cytopathic potency on DU145 cells. The clinical lot showed no diminution in titer or potency over the study period.
Assays for Ad5-CD/TKrep Viral DNA in Blood, Shedding of Adenovirus, Titer of NABs, and Transgene Expression in Prostate Biopsies.
The presence of Ad5-CD/TKrep viral DNA in patient’s blood, shedding of infectious Ad5-CD/TKrep virus in patient’s serum, titer of NABs to adenovirus, and expression of the CD/HSV-1 TK transgene in prostate biopsies were measured as described previously (28)
. For analysis of transgene expression, prostate biopsy cores had to contain at least 10 highly fluorescent cells among a background of negative cells and no highly fluorescent cells in adjacent sections when the primary CD antibody was left out to be considered positive. The titers of NABs to adenovirus were determined before treatment and at 4 weeks after the adenovirus injection by the plaque reduction assay (28)
. All patients demonstrated an increase in NAB titer after the adenovirus injection (data not shown).
Calculation of PSA Half-Life and Statistical Analyses.
Patient PSA data were fitted to the first-order decay equation: PSA (t) = PSA (t = 0) *e-kt, where k is the first-order rate constant, and t is time in days. When the initial rise in PSA as a result of prostatic manipulation was ignored, there was excellent fit to an exponential decay curve (see Fig. 2
). The PSA half-life was calculated using the equation: ln(2)
/k. The mean PSA half-life in cohorts 1–3 (1 week of prodrug therapy) was compared with the half-lives in cohorts 4 and 5 (>1 week of prodrug therapy) by use of the Wilcoxon two-sample test.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. The median age of patients was 75 years, with a range of 54–82 years. Disease stage ranged from T1c to T3 (T1c, n = 5; T2a, n = 4; T2b, n = 1; T2c, n = 4; T3, n = 1). The mean Gleason score was 8, with a range of 6–9. The median pretreatment PSA level was 12.9 ng/ml, with a range of 2.5–30.9 ng/ml. The mean radiation dose was 7080 cGy. The median follow-up was 9 months, with complete toxicity assessment within 84 days after the initiation of the study therapy.
Toxicities.
Tables 2
and 3
summarize all AEs reported in two or more men. Because the timing of the early posttreatment prostate biopsy was not expected to affect toxicity, patients in cohorts 1–3, who received the same treatment, were grouped together. There were no DLTs or treatment-related serious AEs. The vast majority (94%) of AEs were mild (65% grade 1) to moderate (29% grade 2) in nature. The most frequent AEs were lymphopenia (93%), urinary frequency/urgency (87%), dysuria (73%), anemia (67%), leukopenia (67%), diarrhea (53%), hyperglycemia (47%), fatigue (47%), elevation in liver transaminases (40%), monocytosis (33%), GI discomfort (33%), and hypophosphatemia (33%). Most of these AEs were expected and attributable to the prodrug therapy (lymphopenia, anemia, diarrhea, GI discomfort), radiation therapy (urinary frequency/urgency, dysuria, diarrhea, fatigue), and probable dissemination of the Ad5-CD/TKrep adenovirus to collateral tissues (elevations in liver transaminases and monocytosis). A high incidence of hyperglycemia was also observed in our previous gene therapy trial without EBRT (28)
, suggesting that it may be related to the prodrug therapy and the fact the many of these elderly patients were known or borderline diabetics. The cause of the hypophosphatemia is unknown. The Cochran–Armitage test was used to examine the effect of increasing prodrug therapy duration on toxicity. Except for fever (P = 0.02), there was no significant association between the duration of prodrug therapy and any AE.
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There were no treatment-related serious AEs. There were 11 grade 3 events, including 1 event each of acute deep vein thrombosis, prolonged PT time, infection without neutropenia, hyperglycemia, hypophosphatemia, dyspnea, and urinary frequency/urgency. There were two grade 3 events each of lymphopenia and hypokalemia. Except for the events of lymphopenia, urinary frequency/urgency, and possibly dyspnea, none of the grade 3 events was treatment-related. One grade 3 event of lymphopenia occurred on day 30 in a patient (patient 2) who had grade 2 lymphopenia before treatment. It resolved 1 week later without alteration of the treatment course. The second grade 3 event of lymphopenia occurred on day 3 (patient 12) on initiation of 5-FC + vGCV prodrug therapy and resolved 3 days later. The grade 3 event of urinary frequency/urgency occurred on day 29 in a patient (patient 8) who had grade 2 urinary frequency/urgency and dysuria before treatment. The patient was administered Flomax, and the condition improved. The grade 3 event of dyspnea occurred on day 29 in a patient (patient 5) with a history of chronic obstructive pulmonary disease. The patient was administered Serevent and Proventil, and it resolved shortly thereafter.
Tables 4
and 5
compare the toxicities observed in this study (BB-IND 9852) with those in our previous Phase I gene therapy trial without EBRT (BB-IND 8436; Ref. 28
) and the RTOG 9406 dose escalation trial that used similar radiation doses and delivery methods (46)
. Relative to BB-IND 8436, there were significant increases in lymphopenia (P = 0.02) and leukopenia (P < 0.01), but not anemia, thrombocytopenia, or neutropenia (P > 0.39; Table 4
). All of these hematological events were transient, and none was clinically significant. Although the incidence of AST elevations was similar between the two trials (P = 0.7), for reasons unknown, the incidence of ALT elevations was increased significantly in the present study (P < 0.01). To compare acute bladder and bowel toxicities with those reported in RTOG 9406, urinary and lower GI events were graded with use of the RTOG morbidity scale.4
As expected, the addition of radiation therapy significantly increased acute bladder toxicity relative to BB-IND 8436 (P < 0.01). Although there was greater overall acute bladder toxicity in the present study relative to RTOG 9406 (P < 0.01), this difference became insignificant when considering grade 2 or higher events (P = 0.43). There was no significant increase in acute bowel toxicity relative to BB-IND 8436 or RTOG 9406 (P > 0.13).
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; Table 6
). Because of the high sampling error associated with the needle biopsy procedure, it is possible that this analysis underestimates the true persistence of transgene expression in the prostate.
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summarizes the posttreatment PSA and prostate biopsy results to date. As expected for patients receiving definitive radiation therapy, all have experienced significant declines in PSA. Except for possibly patient 1 (17 months out), it is unlikely that any have achieved a PSA nadir. With a median follow-up of only 9 months, 5 of 10 (50%) patients not treated with androgen-deprivation therapy have achieved a serum PSA 0.5 ng/ml. Two of six (33%) patients biopsied at 12 weeks (cohorts 4 and 5) were negative for adenocarcinoma. Only cohorts 1 and 2 have passed the 1-year time point. Four of six (67%) patients were negative for adenocarcinoma with patient 3 (positive) having only focal residual adenocarcinoma.
The posttreatment PSA kinetics of two patients from each cohort (cohorts 1–3 being grouped) are shown in Fig. 2
. As expected, most (12 of 15) patients experienced a rise (3–367%) in PSA immediately after the adenovirus injection that was likely attributable to prostatic manipulation. The PSA of patient 1 remained uncharacteristically elevated for 2 months and was accompanied by moderate acute/chronic inflammation of the prostate. After the initial rise, PSA in patients not treated with androgen-deprivation therapy declined, with first-order kinetics, yielding a mean half-life of 48 days (1.6 months) and a median of 39 days (1.3 months; Table 6
; Fig. 2
). The mean PSA half-life in patients administered >1 week of prodrug therapy (19 days; 0.6 months; n = 3) was significantly shorter than that of patients who received prodrugs for only 1 week (60 days; 2.0 months; n = 7; P < 0.02). It was also markedly shorter than that (2.4 months) reported previously for 841 men treated with 70 Gy of radiation therapy alone (47)
.
Presence of Infectious Adenovirus and Ad5-CD/TKrep Viral DNA in Blood.
The presence of infectious adenovirus and Ad5-CD/TKrep viral DNA in the patients’ blood was monitored twice a week during the prodrug therapy course and then once a week thereafter. Although Ad5-CD/TKrep viral DNA was detected in a patient’s blood as far out as day 45 (not shown), as was observed previously (28)
, no infectious adenovirus was detected in patient’s blood at any time point.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Normal tissue toxicity after exposure to ionizing radiation is commonly divided into two categories, acute and late, which reflect the radiosensitivity and turnover rate of the normal surrounding structures. For prostate cancer patients treated with radiation therapy, the most common acute (90 days) and late (>90 days) sequelae affect the genitourinary and GI systems. Damage to the bladder neck, urethra, and anterior rectal wall are the most serious, and although these events are rarely severe after conventional-dose 3D-CRT (grade 3, <5%), they can adversely affect a patient’s quality of life (48)
. One concern before initiating this study was the possibility of increased urinary and GI toxicities when combining oncolytic adenoviral and double-suicide gene therapies with radiation therapy (trimodal therapy). Because of their radiosensitizing effects, it was originally thought that the CD and HSV-1 TK suicide gene systems might exacerbate locoregional toxicities relative to radiation therapy alone. However, our preclinical toxicology studies in the mouse demonstrated that most of the locoregional toxicity associated with trimodal therapy was attributable to an interaction between the adenovirus, or more likely, an immune response to the adenovirus, and radiation and that the prodrug therapy contributes little to this effect (27)
. Indeed, in the dog, intraprostatic injection of a replication-competent adenovirus results in severe acute/chronic inflammation that is markedly greater than the inflammation observed with a replication-defective virus (49)
. The increased inflammation associated with replication-competent adenoviruses is likely attributable to increased expression of viral antigens, which is driven by E1A expressed from replication-competent vectors. As was observed here, 80% of patients developed acute/chronic inflammation of the prostate that was likely related to administration of adenovirus and/or radiation therapy. We are encouraged that despite the addition of oncolytic adenoviral and double-suicide gene therapies, the incidences of grade 2 or higher acute bladder and bowel toxicities were not significantly higher in the present study than would be expected for conventional-dose 3D-CRT alone (46)
. Although one patient developed grade 3 urinary frequency/urgency, the patient’s preexisting condition (grade 2 urinary frequency/urgency and dysuria) was likely a contributing factor. No patient required the use of a Foley catheter, and only one patient experienced minor (grade 1) rectal bleeding. It will be important to follow these patients long term to compare the development of late bladder and bowel toxicities to patients treated with conventional-dose 3D-CRT alone.
Another potential concern with adenovirus-mediated gene therapy is the dissemination of virus beyond the target organ, resulting in infection of critical organs such as the liver. The innate immune response to adenoviral gene therapy vectors and the cytotoxic effects of the CD and HSV-1 TK suicide gene systems can result in severe hepatotoxicity and death (50) . As was observed in our previous gene therapy trial without radiation (28) , a minority of patients exhibited elevations in liver transaminases, and all but one were grade 1. Moreover, when we combined the results of our two Phase I trials (Ref. 28 and the present study), only 1 of 31 (3%) patients experienced flu-like symptoms (fever, chills, and respiratory events) within 48 h after the adenovirus injection, and none demonstrated evidence of infectious adenovirus in their blood. A caveat is that although the presence of infectious adenovirus in the patients’ blood was monitored daily in our previous trial, it was monitored only twice a week beginning on day 4 in the present study. Thus, it is possible that had earlier time points been taken, infectious adenovirus would have been detected in the blood of the one patient who developed flu-like symptoms shortly after the adenovirus injection. This patient developed only minor sequelae that resolved the next day (flu-like symptoms) and shortly after completion of the prodrug therapy course (elevation in AST). Together our clinical results indicate that in most patients, little of the injected adenovirus disseminates beyond the prostate gland when either the transperineal (28) or the transrectal (present study) injection procedure is used. These results are consistent with our observations in the dog, in which we have demonstrated, using a replication-competent adenovirus that can be imaged noninvasively, that the vast majority of injected adenovirus remains in the prostate after direct intraprostatic injection (49) . Despite these positive results, dissemination of adenovirus beyond the prostate gland will continue to be monitored in our future Phase II and III trials.
A secondary study objective was to estimate the persistence of therapeutic transgene expression in the prostate. Such results will help define the optimal prodrug therapy course and will guide us in the design of future Phase II and III trials. Consistent with our previous gene therapy trial (28) , transgene expression was detected in the prostate up to 3 weeks after the adenovirus injection. These results are supported by our analyses of adenoviral DNA in blood, demonstrating that the virus can persist in patients as far out as day 76 (28) , although the latter assay is measuring neither transgene expression nor "active" virus. They contrast sharply with our observations in the dog, in which we observed transgene expression for only 5 or 6 days after intraprostatic injection of a replication-competent adenovirus (49) . We believe the short duration of transgene expression in the canine prostate is related to the very robust immune response to the adenovirus, although differences in the sensitivities of the methods used (invasive versus noninvasive) could be a contributing factor. Although most patients developed a demonstrable cellular immune response as well as circulating neutralizing antibodies to adenovirus (Ref. 28 ; not shown here), it appears that therapeutic transgene expression can persist in the human prostate for a greater than expected period of time. We consider these findings very encouraging because the longer the CD/HSV-1 TK transgene is expressed in the prostate, the greater the likelihood the suicide gene systems will exert chemotherapeutic and radiosensitizing effects.
Serum PSA typically declines with first-order kinetics with a half-life of 
2 months and reaches a nadir 18–36 months after radiation therapy (47
, 51, 52, 53, 54)
. Previous studies have demonstrated that the PSA half-life after radiotherapy is not prognostic for long-term outcome (47
, 54)
. Because the half-life of PSA is 3 days when the prostate is surgically removed (55)
, the PSA half-life after radiotherapy cannot simply reflect its clearance from the circulation but is likely to represent a complex function of the destruction and regrowth of PSA-secreting cells. That the PSA level declines at all after radiotherapy indicates that the destruction of PSA-secreting cells must exceed regrowth; it therefore follows that the rate of PSA decline should reflect the relative rate of destruction versus regrowth. In our previous gene therapy trial without radiation (28)
, PSA declined rapidly during the prodrug therapy course and was followed by a period of continued, but slower, decline in PSA as long as Ad5-CD/TKrep viral DNA was detected in blood. Once Ad5-CD/TKrep viral DNA disappeared from the circulation, patients began to fail biochemically. These results suggested that most of the observed decline in PSA was probably attributable to the cytotoxic effects of the CD and HSV-1 TK suicide gene systems. Because of the addition of radiation therapy in the present study, it is difficult to discern the relative contributions of the three therapeutic modalities (oncolytic adenoviral therapy, double-suicide gene therapy, and radiation therapy) to the observed decline in PSA. However, we are intrigued by the fact that the mean PSA half-life in patients administered >1 week of prodrug therapy (19 days; 0.6 months) was significantly shorter than that (60 days; 2.0 months) in patients receiving only 1 week of prodrug therapy and markedly shorter than the mean PSA half-life (2.4 months) reported previously for patients treated with 70 Gy of radiation therapy alone (47)
. Although the small sample size precludes any conclusions and PSA half-life is not prognostic for long-term outcome, the shorter than expected PSA half-life in patients receiving >1 week of prodrug therapy may suggest a possible interaction between the oncolytic adenoviral and/or double-suicide gene therapies and radiation therapy. Indeed, such interactions have been observed previously by us and others in preclinical tumor models (25, 26, 27
, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42)
. That therapeutic transgene expression can persist in the prostate for up to 3 weeks is consistent with this thesis.
Although PSA half-life after radiotherapy is not prognostic for long-term outcome, the predictive value of the PSA nadir continues to be controversial. Retrospective analyses have demonstrated that a PSA nadir 0.5 ng/ml predicts a favorable outcome when outcome is defined as freedom from biochemical failure (56, 57, 58, 59, 60, 61)
. Thus, the posttreatment PSA nadir might provide an early surrogate end point in investigational studies such as that described here. However, the PSA nadir that defines patients with a favorable versus unfavorable outcome has declined steadily over the years (from 1.0 to 0.5 to 0.2 ng/ml), and a recent Phase III study has called into question the prognostic value of the PSA nadir in patients who are failure free at 2 years (16)
. In contrast, posttreatment prostate biopsy status at 2 years has consistently been found to be a highly significant correlate of clinical outcome (7
, 16
, 62, 63, 64, 65, 66, 67, 68)
. Although more studies are needed to firmly establish a possible link between freedom from biochemical failure and disease-specific or overall survival (69)
, biochemical failure almost always precedes local failure and progression to distant metastases, the latter of which is a clinically meaningful end point. Thus, prostate biopsy status at 2 years has become the "gold standard" early end point for radiation therapy clinical trials. Earlier time points (<2 years) have less value because serial biopsy studies have shown that positive biopsy status decreases with longer follow-up, reaching a plateau approximately 2–3 years after radiotherapy (65
, 66)
. Moreover, a significant fraction of early posttreatment biopsies have an indeterminate histopathological status, making the interpretation of such results difficult (65)
. Both observations probably reflect the slow killing effect that radiation has on human prostate cancer cells in vivo. Because of the short follow-up in the present study (and small sample size), we cannot make any conclusions regarding the possible effect of combining oncolytic adenoviral and double-suicide gene therapies with radiotherapy on prostate biopsy status. Patients with residual adenocarcinoma and a declining PSA at 1 year will be reevaluated at 2 years. A randomized, prospective, two-arm Phase II trial with a much improved replication-competent adenovirus has been planned that will examine the effect of combining oncolytic adenoviral and double-suicide gene therapies with radiotherapy on prostate biopsy status at 2 years.
Prostate cancer has long been known to be a dose-responsive cancer (70, 71, 72, 73) . There are two ways to increase the effective radiation dose delivered to the tumor. One is to increase the prescription radiation dose by use of conformal techniques to limit the damage to normal surrounding tissues. The second is to increase the biological effect of the radiation dose delivered (i.e., radiosensitization). We believe that novel biological approaches that do not increase the toxicity of conformal radiotherapy, such as that described here, may ultimately provide an attractive alternative, or adjuvant, to high-dose radiotherapy. Although high dose conformal radiotherapy has produced very impressive results in nonrandomized (7 , 8) and randomized (14, 15, 16) single-institution trials, they need to be confirmed in a large, multicenter, randomized trial before high-dose conformal radiotherapy becomes a standard of practice. Moreover, the long-term complications associated with such high radiation doses are still largely unknown. We find two attractions of biological approaches. First, they may provide a greater therapeutic gain than is achievable with dose escalation. We have shown previously that the CD and HSV-1 TK suicide gene systems can generate in vitro sensitization enhancement ratios between 1.4 and 1.8 when implemented independently and on the order of 2 when combined (25 , 32, 33, 34, 35, 36) . Even if only a fraction of this radiosensitization could be obtained in the clinic, it would represent a greater therapeutic gain than is probably achievable with dose escalation using the very best conformal techniques (i.e., 75 Gy with a sensitization enhancement ratio of 1.3 would be equivalent to 97.5 Gy). Two key questions that remain unanswered are (a) what is the highest dose that can be delivered to the prostate with conformal techniques with acceptable long-term toxicity and (b) where on the sigmoidal dose–response curve does this dose fall? Even with IMRT, damage to the bladder neck and urethra may render this dose too low. A second attraction of biological approaches resides in their vast potential to be engineered to target the underlying molecular defects that drive the growth of malignant cells and their sensitivity to conventional cancer therapies. Thus it should be possible to develop biological approaches that specifically target the molecular defects that promote tumor cell radioresistance (e.g., overexpression of bcl-2; Ref. 74 ). The sequencing of the human genome and the explosion of knowledge that will ensue could provide the necessary fuel for the development of such novel biological approaches. We do not view dose escalation and biological approaches to be mutually exclusive; rather, we view them as complementary. Combining these two approaches is worth consideration once more convincing evidence regarding their overall merit becomes available.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 This work was supported by grants from the NIH (CA75456 and DK57833) and by an award from the RAID Program to S. O. F. 
2 To whom requests for reprints should be addressed, at Molecular Biology Research, Henry Ford Health System, One Ford Place, Wing 5D, Detroit, MI 48202-3450. Phone: (313) 876-1949; Fax: (313) 876-1950; E-mail: sfreyta1{at}hfhs.org
3 The abbreviations used are: EBRT, external beam radiation therapy; PSA, prostate-specific antigen; 3D-CRT, three-dimensional conformal radiotherapy; IMRT, intensity modulated radiotherapy; RTOG, Radiation Therapy Oncology Group; GI, gastrointestinal; CD, cytosine deaminase; 5-FC, fluorocytosine; vGCV, valganciclovir; HSV-1 TK, herpes simplex virus thymidine kinase; DLT, dose-limiting toxicity; vp, viral particle(s); CT, computed tomography; PTT, partial thromboplastin time; PT, prothrombin time; AST, aspartate aminotransferase; ALT, alanine aminotransferase; CTV, clinical target volume; PTV, planning target volume; CTC, Common Toxicity Criteria; NAB, neutralizing antibody; AE, adverse event.
4 http://www.rtog.org/members/toxicity/acute.html.
Received 7/ 2/03. Revised 8/14/03. Accepted 8/20/03.
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0.2 ng/mL defines disease freedom after radiotherapy for prostate cancer using modern techniques. Urology, 54: 968-971, 1999.[Medline]
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