Introduction

According to the Genetic Modification Clinical Research Information System (http://www4.od.nih.gov/oba/RAC/GeMCRIS/GeMCRIS.htm), more than 70 gene therapy protocols targeting prostate cancer have been reviewed by the Recombinant DNA Advisory Committee of the National Institutes of Health. Not surprisingly, only a fraction have progressed into the clinic, and even fewer have published results. The reasons for this meager progress are likely logistical (i.e., lack of resources, regulatory barriers, etc.) and should not reflect poorly on the gene therapy field. However, what our fledgling field needs now more than money or fewer regulations are winning strategies.

One question that every cancer gene therapist will face sooner or later is: What patient cohort should I test my approach in first? Similar to many cancers, prostate cancer is a complex disease, and different strategies will be required to demonstrate a benefit in a particular patient cohort. Most of the 27,000 men who succumb to prostate cancer each year in the United States die from metastatic disease, highlighting the need for better systemic therapies.¹ It is probably for this reason that gene-modified vaccine strategies were attempted first and have progressed furthest in the clinic. Not only are these strategies seductive because of their potential to affect metastatic disease, but they have scientific merit and have produced provocative results in phase 2 trials. However, it is important to keep in mind that the best available systemic therapies for prostate cancer, including hormone therapy and the recently approved Taxotere Sanofi-Aventis (Bridgewater, NJ) (docetaxel), are not curative but, instead, result in an extension of survival, which can be very modest. Moreover, both therapies are associated with significant morbidity and can have an adverse effect on the patient's quality of life. Although such agents have earned their place in the management of advanced prostate cancer, an extension of survival is not good enough, and we should not be satisfied with such gains. What are needed, in the long run, are therapies that will eradicate the disease at diagnosis, ultimately resulting in a reduction in the annual number of prostate cancer deaths.

The best way to manage prostate cancer, or any cancer for that matter, secondary to prevention, is to detect it early and apply effective treatments while it is still localized. Indeed, it is in the loco-regional setting that gene therapy may ultimately demonstrate its greatest benefit. Owing to a low efficiency of gene transfer, metastatic prostate cancer appears beyond the reach of current in vivo gene therapy technology. However, this limitation should not discourage gene therapists from moving forward. With the advent of prostate-specific antigen (PSA) screening, there has been a significant stage migration during the past two decades, and now only a small proportion (<10%) of prostate cancer patients has detectable metastatic disease at diagnosis.² Today, newly diagnosed prostate cancer is largely a loco-regional disease, making it within the reach of current in vivo gene therapy technology. The reason why 27,000 men die of prostate cancer every year is not necessarily because we detect it too late; rather, it is because our primary, loco-regional therapies fail too often. Except for patients with low-risk disease (Stage T2a, Gleason score 6, PSA < 10 ng/ml), loco-regional therapies such as surgery, radiotherapy, and hormone therapy fail in a significant proportion of cases, ultimately resulting in metastatic and, therefore, presently incurable disease; hence the need for better systemic therapies. If gene therapy could improve loco-regional tumor control and help eradicate the disease at diagnosis, the need for better systemic therapies would eventually diminish.

Here, we review the prostate cancer gene therapy trials that have been reported to date, including both in vivo and ex vivo approaches. Because different strategies will be required to affect localized versus metastatic disease, these two disease forms will be discussed separately. Several strategies have generated provocative results in early-stage trials. Although these results need to be confirmed in randomized, controlled phase 3 trials, for the time being they give us hope that gene therapy may someday earn a place in the management of prostate cancer.

Gene therapy strategies for the treatment of localized prostate cancer

Gene therapy for localized prostate cancer has progressed rapidly from replication-defective adenoviruses containing a single therapeutic gene, to replication-competent oncolytic adenoviruses lacking a therapeutic gene, to replication-competent oncolytic adenoviruses containing multiple therapeutic genes.^{3,4,5,6,7,8,9,10,11} These strategies have been evaluated in the setting of locally recurrent and newly diagnosed disease, and as single agents or in combination with radiation therapy (Table 1). All have demonstrated low toxicity in humans. The last approach, which was developed simultaneouslyand represents a combination of other approaches, has generated provocative results in two early-stage trials and is near to being tested in a randomized controlled phase 3 trial. Therefore, it will serve as a focal point for the following discussion. The results of other trials that have also been encouraging will be described and compared.

Table 1 - Prostate cancer gene therapy trials-localized disease.

Full table

Freytag et al. were the first to "arm" a replication-competent adenovirus with a therapeutic gene and propose combining gene therapy and oncolytic viral therapy with radiotherapy.^{12,13,14,15,16,17} Their rationale for using replication-competent, rather than replication-defective, adenoviruses as gene therapy vectors is based on the following points: (i) replication-competent adenoviruses are cytolytic and have demonstrated anti-tumor activity in preclinical tumor models and humans; (ii) replication-competent adenoviruses have the potential to replicate to high copy numbers, thereby resulting in significantly greater therapeutic gene expression in vivo; and (iii) replication-competent adenoviruses have greater potential to spread locally and therefore could infect a greater number of tumor cells. All these potential advantages have now been demonstrated in animal models and/or humans.

In their first phase 1 trial,⁷ 16 men with locally recurrent prostate cancer were administered a single intraprostatic injection of the replication-competent Ad5-CD/TKrep adenovirus (10¹⁰–10¹² viral particles, vp) followed by 1–2 weeks of 5-fluorocytosine (5-FC) and ganciclovir (GCV) prodrug therapy. Ad5-CD/TKrep contains a bacterial cytosine deaminase (CD)/wild-type herpes simplex virus thymidine kinase (HSV-1 TK) fusion gene in the E1 region in place of the 55 kd E1B gene, and lacks the adenoviral genes of the E3 region. Expression of the CD/TK fusion gene renders tumor cells sensitive to 5-FC and GCV. One strength of these suicide gene therapy strategies is that both demonstrate a bystander effect,^18,19,20 in which nearby tumor cells not expressing the genes can be killed owing to the local diffusion of toxic metabolites. This important attribute has the potential to extend the killing radius of the oncolytic adenovirus itself. Previous work by this group demonstrated that the CD and HSV-1 TK suicide gene therapies could augment the anti-tumor effects of replication-competent adenoviruses and, importantly, sensitize tumor cells to ionizing radiation.^{12,13,14,15,16,17}

Overall, the adenovirus-mediated gene therapy was well-tolerated. There were no dose-limiting toxicities, and 94% of the adverse events observed were mild (grade 1) to moderate (grade 2). The most frequent treatment-related side effects were pain/discomfort at the injection site (100% of patients), lymphopenia (56%), hematuria (56%), hyperglycemia (50%), anemia (44%), thrombocytopenia (38%), flu-like symptoms (38%), diarrhea (31%), and transaminitis (25%). All of these events were transient and self-limiting, and most could be attributed to the injection procedure (pain/discomfort at injection site, hematuria), dissemination of the Ad5-CD/TKrep adenovirus to collateral tissues (flu-like symptoms, transaminitis), and the well-known side effects of the 5-FC + GCV prodrug therapy (hematological events and diarrhea). The cause of the hyperglycemia is unknown, but it may be related to the fact that many of these elderly patients were known or borderline diabetics or it may have been the result of treatment-related stress. Although injection of adenovirus directly into the prostate gland results in local inflammation, no patient developed urinary retention requiring a Foley catheter.

Approximately half of the patients exhibited significant (>25%) declines in serum PSA and there were three (19%) objective responses (>50% PSA decline). However, all PSA responses were short-lived (<6 months). What was intriguing is that in all patients who responded, the rate of PSA decline was greatest during the prodrug therapy course, which was then followed by slower decline as long as the adenovirus persisted in patients (as measured by adenoviral DNA in blood). Once the adenovirus was eliminated, all patients exhibited PSA relapse. The inflection point immediately after completion of the prodrug therapy course suggested that the early rapid decline in PSA was, in part, due to the suicide gene systems. The slower PSA decline after the inflection point was likely due to the oncolytic actions of the Ad5-CD/TKrep adenovirus and/or resulting immune response. Together, these observations suggested two things: (i) the observed PSA responses were attributable to the investigational therapy (and resulting immune response), and (ii) the adenoviral DNA detected in blood likely reflected the presence of adenovirus back in the prostate (if not, then why did the PSA decline only when the adenovirus was present?). Importantly, histopathological evidence of tumor destruction was demonstrated in post-treatment prostate biopsies at 2 weeks. All post-treatment biopsies showed immune infiltrates comprised largely of polymorphonuclear cells, lymphocytes, and macrophages.

Encouraging results were also obtained in this same disease setting in a phase 1 trial conducted at Baylor College of Medicine with the replication-defective ADV/HSV-tk adenovirus, which contains the wild-type HSV-1 TK gene,³ and a phase 1 trial conducted at Johns Hopkins Medical Institute with the replication-competent CV706 adenovirus, which lacks a therapeutic gene.⁶ In the Baylor trial, the ADV/HSV-tk adenovirus dose was escalated to 2 10¹² vp and was followed by 2 weeks of GCV prodrug therapy. Only 1 of 18 (6%) patients experienced significant treatment-related toxicity (grade 3 transaminitis, grade 4 thrombocytopenia). This was an isolated case and was probably attributable to leakage (or injection) of the adenovirus into the circulation during the injection procedure. Notably, 3 of 18 (17%) subjects experienced a decline of more than 50% in PSA, in one case lasting for more than 1 year. The trial conducted at Johns Hopkins used the PSA-selective CV706 adenovirus, in which E1A expression, and therefore viral replication, is driven by a minimal PSA promoter. This feature was designed into the vector for safety reasons to restrict viral replication and cytolysis to the prostate.²¹ Subjects received a single intraprostatic injection (in 20–80 deposits) of CV706 up to a dose of 10¹³ vp, which is ten times the highest dose used in the trial with the Ad5-CD/TKrep adenovirus. Despite this high dose, the treatment was associated with low toxicity. Of the adverse events, 98% were mild to moderate. As observed in the trial with the Ad5-CD/TKrep adenovirus, a minority of subjects exhibited transaminitis and there was no grade 2 hepatotoxicity. Of 20 subjects, 5 (25%) subjects showed a decline of more than 50% in PSA, all of which occurred at the two highest dose levels (3 10¹² and 1 10¹³ vp), suggesting a possible dose effect. One objective response lasted 11 months. A secondary, or delayed, peak of CV706 DNA was detected in patients' circulation approximately 1 week after the adenovirus injection, providing suggestive evidence for viral replication in vivo.

The combined results of these three trials (54 patients) demonstrate clearly that adenovirus-mediated gene therapy, when administered intraprostatically, is associated with low toxicity in humans. Although encouraging PSA responses were observed in all three trials, the vast majority of responses were short-lived, indicating, at the time, that none of these approaches was efficacious enough to exert a sustained effect. However, a recent retrospective analysis of the trial conducted with the Ad5-CD/TKrep adenovirus yielded very provocative results.¹⁰ With a median PSA follow-up of 5 years, it was found that the gene therapy had a significant effect on PSA doubling time (PSADT), a non-validated surrogate endpoint that is highly predictive for the development of distant metastases and prostate cancer–specific mortality.^{22,23,24,25,26,27,28,29,30,31,32,33} When considering all evaluable subjects (i.e., those in whom the PSADT could be determined before and after the gene therapy, n = 14), the PSADT increased after the gene therapy from a mean of 17 to 31 months (median 16 to 22 months) (P = 0.014). Three of eight (38%) subjects at the lower-dose levels (10¹⁰ and 10¹¹ vp) and five of six (83%) subjects at the highest dose level (10¹² vp) exhibited a lengthening of PSADT after the gene therapy, suggesting a possible dose effect. Because the gene therapy slowed the rate of cancer growth in many patients, it delayed the point when salvage therapy was indicated by an average of 2 years. For two patients it is now more than 5 years since the gene therapy and salvage androgen suppression therapy (AST) has not been indicated. The results indicate that the adenovirus-mediated gene therapy may have provided a potential long-term benefit to patients, as demonstrated by a lengthening of the PSADT and delay in when salvage therapy was indicated.

What is intriguing is that the effect on PSADT persisted long after the Ad5-CD/TKrep adenovirus was eliminated from patients, begging the following question: How can adenovirus-mediated gene therapy slow the rate of cancer growth over a period of at least 6 years after the treatment course is completed? Although the answer to this question is unknown and under investigation, the most likely explanation is that it induced long-lasting anti-tumor immunity in some patients. This thesis is well supported by preclinical studies demonstrating that adenovirus-mediated suicide gene therapy can induce T-cell-dependent immunity in mice.^{34,35,36,37,38,39,40} If this hypothesis is correct, it raises the exciting possibility that adenovirus-mediated gene therapy may have the potential to impact both localized and metastatic disease.

An effect of gene therapy on PSADT was also reported by the Baylor group in an extended phase 1/2 trial conducted with the ADV/HSV-tk adenovirus.⁴ Although the median PSA follow-up for the entire patient cohort (n = 36) was not reported, at least 1 year's follow-up was available for 22 patients, some of whom received up to three adenovirus injections. The mean PSADT increased from 16 to 43 months (P = 0.027) after the gene therapy (median PSADT 12.2 versus 12.7 months). Patients who developed metastatic disease were censored from the analysis, which was not done in the 5-year follow-up analysis of the trial conducted with the Ad5-CD/TKrep adenovirus. If the observed lengthening of PSADT is attributable to long-lasting anti-tumor immunity, it could be argued that these patients should have been included in the primary analysis as they represent the group who could derive the greatest benefit from any systemic effects of the gene therapy.

Two of the gene therapy strategies described above have also been evaluated in the setting of newly diagnosed prostate cancer in combination with conformal radiotherapy. The scientific rationale for these trials draws on the original findings of Freytag and Kim, who demonstrated that both the CD and HSV-1 TK suicide gene systems had significant tumor radiosensitizing activity.^12,13 These findings were subsequently confirmed by several groups.^{41,42,43,44,45,46} In the trial conducted with the Ad5-CD/TKrep adenovirus,⁸ 15 subjects with intermediate- to high-risk prostate cancer (Gleason score 7 or PSA > 10 ng/ml) received a single intraprostatic injection of Ad5-CD/TKrep (10¹² vp) followed by an escalating duration of 5-FC + valganciclovir prodrug therapy and concomitant radiotherapy (70–74 Gy). The multi-modal treatment was associated with low toxicity. Except for the well-known side effects of prostate radiotherapy (gastrointestinal and genitourinary events), the adverse events resembled closely those observed in the previous trial without radiotherapy.⁷ Importantly, the toxicities associated with the three modalities (oncolytic adenoviral therapy, suicide gene therapy, radiotherapy) did not overlap, and the gene therapy did not exacerbate the most common side effects of prostate radiotherapy. Excellent safety results were also obtained by the Baylor group using the ADV/HSV-tk adenovirus in combination with 76 Gy radiotherapy.⁵

The phase 1 trial conducted with the Ad5-CD/TKrep adenovirus was followed by a similar study using an improved, second-generation adenovirus, Ad5-yCD/mutTK_SR39rep-ADP.¹¹ Ad5-yCD/mutTK_SR39rep-ADP was derived from Ad5-CD/TKrep and contains more catalytically active yeast CD^42,43 and mutant SR39 HSV-1 TK⁴⁷ genes in the E1 region, and the adenovirus death protein (ADP) gene in the E3 region. Both the yCD/mutTK_SR39 fusion and ADP genes are under the transcriptional control of the human cytomegalovirus promoter. The yCD/mutTK_SR39 fusion gene results in a greater chemo-radiosensitizing effect in vitro, and ADP expression enhances the local spread of replication-competent adenoviruses in vitro^{48,49,50,51,52} and in vivo.⁵³ In a preclinical model of human prostate cancer, Ad5-yCD/mutTK_SR39rep-ADP proved to be more efficacious than the parental Ad5-CD/TKrep adenovirus and did not increase toxicity.⁵³ Nine subjects, in three cohorts, with intermediate- to high-risk prostate cancer received an intraprostatic injection of Ad5-yCD/mutTK_SR39rep-ADP (10¹¹, 10¹², two injections of 10¹² vp), with each adenovirus injection followed by a 2.6-week course of 5-FC + valganciclovir prodrug therapy and concomitant 74 Gy intensity-modulated radiotherapy. Despite the use of a replication-competent adenovirus with a more catalytically active yCD/mutTK_SR39 fusion gene and the ADP gene, there was no significant increase in hematologic, hepatic, gastrointestinal, or genitourinary toxicity relative to the previous trial conducted with the Ad5-CD/TKrep adenovirus.

An advantage of targeting prostate cancer is that treatment effects can be assessed by serum PSA and prostate biopsy. Very provocative prostate biopsy results were obtained when the trials conducted with the Ad5-CD/TKrep and Ad5-yCD/mutTK_SR39rep-ADP adenoviruses were combined (24 patients).¹¹ Whereas 40% of these intermediate- to high-risk patients were expected to have a positive post-treatment biopsy had they been treated with radiotherapy alone,^54,55 22% of subjects treated with the gene therapy/radiotherapy combination were positive for adenocarcinoma at their last biopsy on the basis of 6–14 biopsy cores (mean of 9) (P = 0.038). What was even more interesting is that when the biopsy results were broken down by prognostic risk category, most of the treatment effect was observed in the intermediate-risk group. Specifically, 0 of 12 (0%) intermediate-risk subjects were positive for adenocarcinoma at their last biopsy, which is better than expected (30%) for this subgroup of patients (P < 0.01). In contrast, 5 of 11 (45%) high-risk subjects were positive for adenocarcinoma at their last biopsy, which is not significantly different from what is expected (56%) for this prognostic risk group (P = 0.72).⁵⁶

Although the assessment of treatment efficacy by prostate biopsy has weaknesses and these preliminary findings should be interpreted cautiously,¹¹ very similar to PSADT, prostate biopsy status at 2 years after radiotherapy is highly predictive for the development of distant metastases and prostate cancer–specific mortality (and biochemical relapse).^{57,58,59,60,61,62,63} With a median PSA follow-up of 3.3 years, 0 of 12 (0%) intermediate-risk subjects have exhibited clinical/biochemical (PSA) relapse. In contrast, 3 of 11 (27%) high-risk subjects have relapsed using contemporary definitions of biochemical failure. These preliminary results raise the possibility that replication-competent adenovirus–mediated suicide gene therapy may have the potential to improve local tumor control of conformal radiotherapy in select patient groups. This hypothesis will be tested in a follow-up randomized controlled phase 3 trial.

If these preliminary biopsy results are correct, much like the lengthening of PSADT observed in the locally recurrent setting, they beg an obvious question: What is the basis for the apparent differential effect observed between the intermediate- and high-risk groups? Although there are several possible explanations, one hypothesis is that it reflects the relative expression of the coxsackie adenovirus receptor (CAR). The infectivity of human cells, including prostate cancer cells, by adenoviruses in vitro correlates well with CAR levels.^64,65,66,67 It has been reported that CAR expression is reduced in high-grade (Gleason grades 4 and 5) prostate cancer.⁶⁸ Assuming these histopathological findings are correct, they could provide a simple explanation for the differential effect observed between the intermediate- and high-risk groups. It is also possible that fewer tumor cells in the high-risk group were infected with adenovirus owing to the infiltrative nature of high-grade prostate cancer.

In the parallel trial conducted at Baylor with the ADV/HSV-tk adenovirus,^5,9 all intermediate- and high-risk subjects also received hormone therapy, which obfuscates post-treatment PSA results. Rather dramatic (100% cancer-free) post-treatment prostate biopsy results were reported.⁹ It would seem, on the basis of this report, that combining adenovirus-mediated gene therapy with radiotherapy can impact all stages of localized prostate cancer, even the most aggressive forms. However, what was not disclosed in that report is that only 2 biopsy cores, rather than the standard 6–12, were taken at each post-treatment time point in most subjects. Although this suboptimal biopsy regimen was implemented for safety reasons (fear of fistula), no inferences regarding the possible effect of the gene therapy should be made owing to the excessively high sampling error (approximately 30% with 6 cores).^{69,70,71,72,73}

Liposome-mediated interleukin-2 (IL-2) gene therapy has been evaluated in men with locally advanced prostate cancer.⁷⁴ The rationale behind local administration of IL-2 gene therapy is that it has the potential to induce anti-tumor cytotoxic T-cell responses without systemic toxicity. Twenty-four subjects, in three cohorts, were given two intraprostatic injections of an IL-2 DNA–liposome complex over a dose range of 300–1,500 g. As expected, it proved to be safe, with the major side effects being pain/discomfort during the injection procedure and rectal bleeding. Although a significant proportion of patients showed PSA declines shortly after the gene therapy, all responses were short-lived and there were no durable PSA responses. An increased CD8/CD4 ratio was noted in post-treatment biopsies at 1 and 2 weeks.

Gene therapy strategies for the treatment of metastatic prostate cancer

Both in vivo and ex vivo gene therapy strategies targeting metastatic prostate cancer have been evaluated in the clinic (Table 2). Kubo and colleagues were among the first to evaluate an in vivo adenovirus-mediated gene therapy strategy targeting metastatic prostate cancer.⁷⁵ Their approach utilized a replication-defective adenovirus (Ad-OC-hsv-TK) containing the HSV-1 TK gene under the transcriptional control of the osteocalcin promoter.⁷⁶ The rationale for using the osteocalcin promoter to drive HSV-1 TK expression is that metastatic prostate cancer, which typically localizes to bone, assimilates to its new environment by taking on a bone stroma–like gene expression profile and expresses high levels of osteocalcin. Thus, HSV-1 TK gene expression is directed to the metastatic lesions. Eleven subjects were administered two intralesional injections of Ad-OC-hsv-TK at three dose levels (5 10⁹ to 5 10¹¹ vp/injection) followed by 3 weeks of valacyclovir prodrug therapy. Two subjects received localized injections in the prostatic fossa, four received injections in para-aortic lymph nodes, and five received injections in bony metastases. Overall, the treatment was well tolerated. Most of the adverse events were mild to moderate and could be attributed to either the prodrug therapy (lymphopenia, neutropenia) or dissemination of the adenoviral vector to collateral tissues (transaminitis, flu-like symptoms). Post-treatment biopsy of the injected lesions indicated that tumor cell apoptosis correlated with HSV-1 TK expression and CAR levels. Of 11 subjects, 1 (9%) exhibited a reduction of more than 50% in serum PSA after treatment; however, this event lasted less than 2 weeks. There were no objective radiological responses.

Table 2 - Prostate cancer gene therapy trials—metastatic disease.

Full table

Recently, results of a phase 1 study were reported in which a PSA-selective replication-competent adenovirus, CG7870, was administered intravenously to 23 patients with hormone-refractory metastatic prostate cancer.⁷⁷ CG7870 is similar to CV706^6,21 except that both the E1A and E1B genes are under the transcriptional control of prostate-specific (rat probasin and PSA, respectively) promoters. These features were designed into CG7870 to make the adenovirus even more prostate-specific than CV706. CG7870 also contains the immune-modulatory genes of the adenovirus E3 region. The treatment was well tolerated up to a dose of 3 10¹² vp. Treatment-related adverse events included mild to moderate flu-like symptoms, hypotension, lymphopenia, thrombocytopenia, and transaminitis. At a dose of 6 10¹² vp, a constellation of adverse events occurred in two patients, halting further dosing. These events included d-dimer formation, a hallmark of disseminated intravascular coagulation, transaminitis, and thrombocytopenia, all of which occurred in the young man who expired in the ornithine carbamoyltransferase gene therapy trial.⁷⁸ Although none of these events alone constituted a protocol-defined dose-limiting toxicity, accrual was, nevertheless, halted after two patients at this dose level.

Many laboratory findings were reported, including PSA, development of neutralizing antibodies to adenovirus, presence of infectious CG7870 adenovirus in body fluids (blood, urine, saliva), CG7870 viral DNA in blood, and various circulating cytokine levels (IL-1, IL-6, IL-10, and tumor necrosis factor-). Owing to the excessive demands that would be placed on patients, the presence of CG7870 adenovirus in metastatic lesions was not examined. However, it is intriguing that 5 of 23 (22%) patients exhibited a 25–49% decline in PSA, most of which occurred at the higher (>6 10¹¹ vp) adenovirus dose levels. As expected, all patients developed neutralizing antibodies to adenovirus. Shedding of infectious adenovirus was detected in the saliva of three patients but not in urine. After injection, CG7870 viral DNA was detected in blood through 90 minutes in all patients, through day 8 in approximately half (13 of 23, 57%) of patients, and through day 29 in a minority (3 of 23, 13%) of patients. The last group were all dosed at the highest dose level (6 10¹² vp). Secondary, or delayed, DNA peaks (defined as more than ten times above the DNA nadir) were observed in 70% of patients between days 2 and 8, which is suggestive of viral replication. These data should be interpreted cautiously as there is no direct evidence that the injected adenovirus actually reached the metastatic lesions, although the observed PSA declines would tend to support this contention. Moreover, the vast majority of adenovirus injected intravenously goes to liver, and liver has endogenous "E1A-like" activities (one of which is IL-6 inducible) that can support the replication of recombinant adenoviruses (even E1-deleted).^79,80,81,82 Such endogenous E1A-like activities could easily render a prostate-selective adenovirus such as CG7870 "not so prostate-selective." Along these lines, IL-6 (and IL-10) blood concentrations increased progressively with higher adenovirus doses, and IL-6 concentrations peaked shortly (6 hours) after infusion of the adenovirus. This could have set the stage for non-selective replication of CG7870 in liver.

Simon and colleagues were among the first to evaluate an ex vivo gene therapy strategy targeting metastatic prostate cancer.⁸³ Their strategy utilized an autologous granulocyte–monocyte colony stimulating factor (GM-CSF)–secreting cancer cell vaccine to treat men who were found to have metastatic prostate cancer at the time of surgery. The rationale for using GM-CSF as a cancer therapy is well founded scientifically and stems from the fact that it has demonstrated the greatest ability to induce durable tumoricidal anti-tumor immune responses in preclinical models.⁸⁴ GM-CSF recruits antigen-presenting cells, such as dendritic cells, to immunization sites, which, in turn, activate CD4⁺ and CD8⁺ T cells by priming them with oligopeptides derived from dying cancer cells. This ultimately results in the destruction of tumor cells by both T- and B-cell-mediated mechanisms.

Eight subjects were treated with autologous GM-CSF-secreting tumor vaccine that was generated ex vivo by retroviral transduction of surgically harvested cells. Subjects received up to six intradermal vaccinations every 3 weeks at two dose levels (1 10⁷ and 5 10⁷ cells per vaccination). Study endpoints included safety of the vaccination procedure, long-term toxicity, and induction of anti-tumor response to prostate adenocarcinoma measured by in vitro and in vivo assays.

The treatment was well tolerated. Most of the adverse events were expected, and they included skin reactions (injection site pain, erythema, swelling, pruritis) and flu-like symptoms (mild low-grade fevers, chills, malaise). All subjects demonstrated inflammatory cell infiltrates at the vaccination sites. Infiltrates were comprised largely of neutrophils and eosinophils, which increased significantly relative to pre-vaccination levels. Langerhans (skin dendritic cells) and macrophages were also noted in some biopsies. Whereas only two of eight (25%) subjects demonstrated positive delayed–type hypersensitivity tests to challenge with irradiated un-transduced autologous prostate tumor cells before vaccination, seven of eight (88%) exhibited positive delayed–type hypersensitivity tests after vaccination. Post-vaccination biopsies were characterized by ingress of macrophages, natural killer cells, T cells, and extensive eosinophilia. Importantly, 80% of CD3⁺ T cells expressed CD45RO, indicating T cell activation. An increase in antibody titers to prostate tumor antigens was observed in three of eight (38%) subjects. Although not a study endpoint, all subjects demonstrated significant declines in serum PSA, which is expected after prostate resection. However, all subjects exhibited disease progression.

Owing to the difficulty of obtaining a sufficient number of autologous tumor cells to generate patient-specific tumor vaccines, follow-up trials used "off-the-shelf" allogeneic tumor vaccines comprised of GM-CSF-secreting LNCaP and PC-3 human prostate adenocarcinoma cells (GVAX). The rationale for this approach stems from observations that prostate cancer from different subjects can express common tumor-associated antigens. In a phase 2 study, 34 subjects with hormone-refractory metastatic prostate cancer were vaccinated with a primer dose of 5 10⁸ GVAX cells.⁸⁵ Subjects were subsequently administered 12 booster vaccinations every 2 weeks at two dose levels (1 10⁸ or 3 10⁸ cells). At 2-year follow-up, 9 of 22 (41%, 2 were lost to follow-up) in the low-dose cohort, and 7 of 10 (70%) in the high-dose cohort, were alive, indicating a trend toward increased survival with the higher dose. There was also a trend toward a longer median time to disease progression (140 days versus 85 days) in the high-dose group as determined by bone scans. A second phase 2 study used a second-generation GVAX vaccine genetically engineered to express higher levels of GM-CSF.⁸⁶ Eighty subjects, in three cohorts, with hormone-refractory metastatic prostate cancer received an escalating dose of GVAX over a 24-week period. Of 19 subjects in the high-dose group, 6 (32%) exhibited PSA declines after repeat vaccinations. The proportion of subjects demonstrating an antibody response at 12 weeks correlated with the vaccine dose. The majority (62%) of subjects tested exhibited stable or reduced osteoclastic activity, which is a positive indicator in this setting. On the basis of these encouraging results, two randomized phase 3 trials are ongoing that will compare the efficacy of GVAX versus docetaxel and prednisone (Vital 1), and docetaxel plus GVAX versus docetaxel and prednisone (Vital 2), in men with metastatic hormone-refractory prostate cancer.

Other gene-modified vaccine strategies evaluated clinically used either PSA alone^{87,88,89,90,91} or PSA with three co-stimulatory molecules (B7-1, ICAM, LFA-3).⁹² On the basis of an excellent track record in other vaccination protocols, all of these strategies utilized poxviruses (vaccinia virus or fowlpox virus) as a vector. Poxviruses have several advantages, including a proven safety record, potent adjuvant activity, large genome size facilitating the insertion of therapeutic genes, and ease of manipulation.⁹³ Targeting PSA as a tumor antigen makes some sense given that malignant prostate cells, on a weight basis, express approximately ten times more PSA than the normal prostate epithelium, and PSA is (almost) a prostate-specific, but not prostate cancer–specific, antigen. However, in order for PSA-based approaches to work, the patient's immune system must break tolerance. As expected, all PSA-based vaccine strategies have demonstrated an excellent safety profile in the clinic. Although none has demonstrated objective PSA responses, several have shown PSA stabilization and PSA-specific T-cell responses in a significant proportion of patients. A phase 2 study conducted by the Eastern Cooperative Oncology Group reported that 45% of patients remained free of biochemical progression (defined as 50% increase in PSA), and 78% remained free of clinical progression, for 19 months.⁹¹ Approximately half of the patients showed PSA-specific T-cell responses.

Discussion

So what is the outlook for gene therapy in the treatment of prostate cancer? First, contrary to public perception, adenovirus-mediated gene therapy has an outstanding safety record in humans. All strategies evaluated to date, targeting either localized or metastatic disease, using either non-selective or prostate-selective approaches, administered as single agents or in combination with radiotherapy, have demonstrated low toxicity in more than ten clinical trials (>150 patients). Indeed, it could be argued that adenovirus-mediated gene therapy is associated with lower morbidity than currently approved treatments for prostate cancer, including surgery, conformal radiotherapy, hormone therapy, and chemotherapy. The argument still being made by some that current gene therapy strategies for prostate cancer are too toxic is simply false and is not supported by the clinical data. However, this excellent safety record does not mean that we can become complacent or take unjustifiable risks. On the contrary, patient safety should always be first and foremost in our minds, and we should treat every new patient as if he/she was our first.

So if excessive toxicity is not the problem, then why is gene therapy the Rodney Dangerfield of cancer therapy (i.e., it gets no respect)? Put simply, it is due to the fact that most, but not all, of the gene therapy strategies developed to date have not exhibited significant anti-tumor activity in the clinic. However, we are cautiously optimistic that the tide is about to turn. Because different strategies will be required for localized versus metastatic disease, each topic will be discussed separately.

Very provocative results have now been obtained, in two different clinical settings, indicating that adenovirus-mediated gene therapy may have the potential to improve outcome in clinically localized prostate cancer. Probably the most provocative data are from the trials conducted with the "armed" replication-competent Ad5-CD/TKrep and Ad5-yCD/mutTK_SR39rep-ADP adenoviruses.^10,11 When these were combined with conformal radiotherapy, better-than-expected post-treatment prostate biopsy results were obtained in the intermediate-risk subgroup (Stage T1/T2 and Gleason score = 7 or PSA > 10 ng/ml and 20 ng/ml). Although 25% of these patients also received short-term (6-month) AST, which can confound post-treatment biopsy results, a much greater percentage (64%) of high-risk patients received 2 years of AST, and there was no improvement over what was expected for this prognostic risk group. Thus, it would seem that these results cannot be explained by the occasional use of AST. Moreover, in the previous trial conducted without radiotherapy,¹⁰ histopathological evidence of tumor destruction was noted in several patients shortly after the gene therapy. These clinical observations, coupled with the fact that the suicide genes contained in these adenoviruses have demonstrated radiosensitizing activity in preclinical models, make it possible that the gene therapy augmented the tumoricidal effects of the radiotherapy in the intermediate-risk subgroup.

If confirmed in the follow-up randomized controlled Phase 3 trial, would the results be meaningful? We believe the answer is yes, for the following reasons. First, radiotherapy is a loco-regional cancer treatment. Thus, it makes sense to use local tumor control (i.e., prostate biopsy) to assess the efficacy of adjuvant therapies, such as gene therapy, that are designed to increase the effectiveness of radiotherapy. Second, at least five clinical studies have shown that men with a positive prostate biopsy at 2 years, 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.^{57,58,59,60,61,62,63} Once distant metastases develop, most men 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. Third, owing to the recent stage migration in prostate cancer, many patients walking into our clinics today fall into the intermediate-risk category. If these early-stage results can be confirmed in the phase 3 setting, adenovirus-mediated gene therapy could improve the outcome for the largest patient cohort that we will be treating in the future.

Moreover, the potentially good news may not end there. As demonstrated in the trial conducted with the Ad5-CD/TKrep adenovirus in the locally recurrent setting,¹⁰ at 5-year follow-up the gene therapy alone had a significant effect on PSADT, a surrogate endpoint with significant prognostic power.^22,33 Although PSADT is not quite ready to serve as an endpoint in lieu of survival, it was recently shown to satisfy three of Prentice's four criteria needed for validation, and narrowly missed the fourth.³³ So what is the potentially good news? These clinical observations raise the possibility that adenovirus-mediated gene therapy may have the potential to induce long-lasting anti-tumor immunity in patients. Whereas the Ad5-CD/TKrep adenovirus was eliminated from all patients by 3 months, the effect on PSADT has persisted for more than 6 years in some patients. Not only is this possibility well supported by preclinical studies, but the Baylor group has obtained evidence that adenovirus-mediated gene therapy can increase the levels of circulating activated CD4⁺ and CD8⁺ T cells.^94,95 Although these immune responses were short-lived (2 weeks) with the gene therapy only and were likely directed against the adenovirus, they were more robust and persistent (up to 1 year) when the gene therapy was combined with radiotherapy. We speculate that more robust, and possibly longer-lasting, T-cell responses would be observed with a replication-competent versus replication-defective adenovirus platform. Replication-competent adenoviruses are likely to result in greater tumor cell destruction, owing to their cytolytic activity, and unquestionably much greater expression of viral antigens, owing to the trans-activation activity of E1A as well as viral replication. Thus, not only could replication-competent adenoviruses result in greater tumor antigen presentation, but they might also provide a much greater "danger signal" to the immune system than replication-defective adenoviruses. This, in turn, might result in a much more pronounced cellular immune response, a hypothesis that has been confirmed in the dog prostate model (Freytag and Barton, unpublished results). Moreover, replication-competent adenoviruses can persist in patients for at least 4 months (as measured by adenoviral DNA in blood), raising the possibility that this "danger signal" could stimulate the immune system for a protracted period.

If the observed lengthening of PSADT can be linked to the induction of long-lasting anti-tumor immunity, it could have profound implications, for it would raise the possibility that adenovirus-mediated gene therapy could impact disseminated disease, even when administered locally (i.e., intratumorally). Although it is unlikely that these local strategies would induce significant tumor regression in patients with bulky metastatic disease, they might be potent enough to show meaningful anti-tumor activity against regional (i.e., seminal vesicle, lymph node) or distant micrometastatic disease. Administering the adenovirus locally has obvious safety advantages. However, it could also potentially improve efficacy against disseminated disease owing to a greater likelihood that tumor tissue would become infected, resulting in greater tumor antigen presentation and stimulation of the anti-tumor immune response. A drawback of delivering the adenovirus systemically is that most of it winds up in the liver, and there is no direct evidence in humans that the metastatic lesions actually become infected. Thus, in the localized setting, adenovirus-mediated gene therapy may be able to improve outcome through a variety of mechanisms, including (i) viral oncolytic effects, (ii) chemo-radiosensitizing effects of therapeutic genes, and (iii) immunological effects. Although the first two effects would likely impact only localized disease, the latter could impact both localized and metastatic disease.

In the metastatic setting, vaccine-based approaches are still the best strategy, and GVAX is currently the leading product. However, it is unlikely that vaccine strategies will be efficacious enough to eradicate the cancer in patients with advanced bulky disease and result in an actual cure. In this advanced setting, probably the best we can hope for is an extension of survival, docetaxel hopefully and it will be better than (2.5 months). Perhaps a better setting for gene-modified vaccine strategies is locally advanced disease, where the tumor burden is lower. In this setting, they could be combined with standard therapies to help eliminate micrometastases before they become a serious, and presently, incurable, problem. Although hormone therapy is currently used in the locally advanced setting to treat disseminated disease, hormone therapy is not curative (i.e., all patients eventually develop hormone-refractory disease), is associated with significant morbidity, and can adversely affect the patient's quality of life. Thus, better therapies in this setting are clearly needed. In terms of systemic administration of therapeutic adenoviruses, these approaches are unlikely to work until we develop ways to deliver them efficiently to the metastatic lesions while bypassing the liver.

So what are the next steps and how might we improve upon current approaches? First, the provocative early-stage results described above, obtained in both the localized and metastatic settings, need to be confirmed in randomized controlled phase 3 trials. Until this is done, gene therapy (rightfully) will never earn the respect of the clinical oncology community. Second, based on the preliminary biopsy findings of the trials conducted with the Ad5-CD/TKrep and Ad5-yCD/mutTK_SR39rep-ADP adenoviruses, it is possible that high-grade (Gleason score 8) prostate cancer may be beyond the reach of "conventional" adenovirus-based approaches. If the differential effect observed between the intermediate- and high-risk subgroups is in fact related to CAR levels, the development of therapeutic adenoviruses that can infect high-grade prostate cancer independent of CAR may be worthy of investigation. However, it is premature to draw this conclusion. The differential effect could also be easily explained by the infiltrative nature of high-grade disease (i.e., not all of the tumor present was infected by adenovirus). In this case, the development of injection algorithms designed to maximize the adenovirus distribution throughout the prostate gland is probably a better direction to take. Therapeutic adenoviruses that can be imaged non-invasively in vivo would aid greatly in this line of investigation for they would allow for quantification of local adenoviral spread (i.e., volume) after intraprostatic administration.⁹⁶ This is particularly relevant to localized prostate cancer, which is known to be a multi-focal disease.

In terms of enhancing the possible systemic effects of gene therapy, vectors armed with immune-stimulatory genes and prostate-specific antigens are currently under investigation and are probably the best strategy at the present time. Which vector (e.g., replication-competent adenovirus versus replication-defective adenovirus versus poxvirus), immune stimulatory gene (e.g., GM-CSF versus IL-12), or tumor antigen (e.g., PSA versus prostatic acid phosphatase) is the best to use is the topic of much debate. Possible advantages of replication-competent viruses (adenoviruses or poxviruses) is that they may result in greater expression of therapeutic genes (through their replicative ability), greater tumor antigen presentation (through their cytolytic activity), and a greater "danger signal" to the immune system (because of de novo expression of viral genes). These debates should be settled in the clinic, not in preclinical models or in the literature. Although it is unlikely that any of them will be efficacious enough to eradicate bulky metastatic disease, they may slow disease progression and result in an extension of survival. Indeed, the encouraging phase 3 results recently obtained with Sipuleucel-T (APC8015), a non-gene-therapy-based vaccine against prostatic acid phosphatase, demonstrate the potential of immunotherapies.⁹⁷ On the basis of the clinical data to date, it is likely that most will be associated with low toxicity, possibly making them an attractive alternative to hormone therapy and Taxotere.

Regardless of which gene therapy strategies ultimately prove to be winners, it is important that they be integrated into current standards of care for prostate cancer. Although the rationale for prostate-specific strategies was well founded 5 years ago, since the toxicity of "generic" (e.g., cytomegalovirus-driven) approaches was unknown at the time, they are much more difficult to justify today. The major reason for developing tissue/tumor-specific approaches is to limit normal tissue toxicity. However, all generic approaches evaluated in the clinic to date have resulted in low toxicity when administered intraprostatically, even when using an oncolytic adenovirus armed with two cytotoxic genes and combined with radiotherapy. Moreover, PSA-directed strategies cannot be used in combination with AST in hormone-sensitive disease. AST blocks the production of testosterone, which, in turn, abrogates PSA promoter activity. Indeed, this is the major reason why PSA levels drop so rapidly after implementation of AST (in contrast, tumor volume decreases by twofold (or by half)). AST has long been the standard of care for locally recurrent disease, and it is now part of the standard of care for newly diagnosed, locally advanced (Stage T3/T4) and high-risk (Gleason score 8, PSA > 20 ng/ml) disease. In the community, the use of AST has become widespread, and many physicians recommend hormone therapy to their patients if their PSA is above 10 ng/ml. Hence, the use of AST is now creeping into the intermediate-risk category. In contrast, "generic" gene therapy strategies can be combined with AST, meaning they can be integrated seamlessly into current standards of care. Moreover, from a product development standpoint, it makes more sense to develop a cancer therapeutic that can be applied to any human cancer, rather than just prostate cancer.

In summary, there is reason for cautious optimism that gene therapy may someday earn a place in the management of prostate cancer. If it does, one thing is certain, it will not be the result of the ideas or efforts of any one group. Many have contributed to this noble cause. And let us never forget one important thing, we are not doing this for us; we are doing this for them.

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Acknowledgments

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.