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Phase II Randomized Study of Vaccine Treatment of Advanced Prostate Cancer (E7897): A Trial of the Eastern Cooperative Oncology Group

From the Columbia University, New York, NY; Dana-Farber Cancer Institute; Beth Israel Deaconess Medical Center, Boston, MA; Cancer Institute of New Jersey, New Brunswick, NJ; Indiana University Medical Center, Indianapolis, IN; University of Pittsburgh, Pittsburgh, PA; National Cancer Institute, Bethesda, MD; University of Wisconsin, Madison, WI; and Fox Chase Cancer Center, Philadelphia, PA

Address reprint requests to Howard Kaufman, MD, Columbia University Medical Center, 177 Fort Washington Avenue, New York, NY 10032; e-mail: hlk2003{at}columbia.edu

	ABSTRACT

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION Authors' Disclosures of... REFERENCES

 
PURPOSE: A phase II clinical trial was conducted to evaluate the feasibility and tolerability of a prime/boost vaccine strategy using vaccinia virus and fowlpox virus expressing human prostate-specific antigen (PSA) in patients with biochemical progression after local therapy for prostate cancer. The induction of PSA-specific immunity was also evaluated.

PATIENTS AND METHODS: A randomized clinical trial was conducted by the Eastern Cooperative Oncology group and 64 eligible patients were randomly assigned to receive four vaccinations with fowlpox-PSA (rF-PSA), three rF-PSA vaccines followed by one vaccinia-PSA (rV-PSA) vaccine, or one rV-PSA vaccine followed by three rF-PSA vaccines. The major end point was PSA response at 6 months, and immune monitoring included measurements of anti-PSA and anti-vaccinia antibody titers and PSA-specific T-cell responses.

RESULTS: The prime/boost schedule was well tolerated with few adverse events. Of the eligible patients, 45.3% of men remained free of PSA progression at 19.1 months and 78.1% demonstrated clinical progression-free survival. There was a trend favoring the treatment group that received a priming dose of rV-PSA. Although no significant increases in anti-PSA antibody titers were detected, 46% of patients demonstrated an increase in PSA-reactive T-cells.

CONCLUSION: Therapy with poxviruses expressing PSA and delivered in a prime/boost regimen was feasible and associated with minimal toxicity in the cooperative group setting. A significant proportion of men remained free of PSA and clinical progression after 19 months follow-up, and nearly half demonstrated an increase in PSA-specific T-cell responses. Phase III studies are needed to define the role of vaccination in men with prostate cancer or those who are at risk for the disease.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION Authors' Disclosures of... REFERENCES

 
Prostate cancer is the most commonly diagnosed malignancy of men living in the United States. Despite local therapy, many patients will develop recurrent disease initially evident as prostate-specific antigen (PSA) progression followed by overt metastatic disease. Androgen ablation is only temporarily effective for recurrent disease after local therapy, secondary to the development of resistance.^1-5 Thus, alternative therapeutic strategies for patients with PSA progression are warranted. These patients demonstrate low tumor burdens and are ideal candidates for vaccine therapy.

Historically, the differential expression of antigens on malignant prostate tissue is the basis for prostate cancer vaccine development. PSA, a serine protease and a member of the glandular kallikrein family, is one such antigen that is expressed by normal and malignant prostatic tissue, and is found at very low levels only in normal breast and salivary gland tissue.^6-12 The use of PSA as a target for vaccine development is supported by the identification of several class I human leukocyte antigen (HLA-) restricted T-cell epitopes within the PSA coding sequences.^13-15 Two ten-mer peptides (designated PSA-1_aa41–150 and PSA-3_aa154–163) were isolated using peripheral blood T-cells derived from patients vaccinated against PSA and are restricted to the human HLA-A2 class I-motif.¹³ Recombinant poxviruses expressing these peptides or the full-length PSA sequence can be used to prime PSA-specific immune responses in animals and in some human patients.^16-20 Although approaches to induction of antigen-specific immunotherapy have included peptides, soluble proteins, dendritic cells pulsed with specific antigens, monoclonal antibodies, and recombinant plasmid DNA, viral vectors are attractive for immunotherapy, since they mimic natural infection, provide potent danger signals, and can induce potent immune responses. Vaccinia virus in particular offers an acceptable safety record and local dermal administration does not lead to systemic shedding of virus. In addition, benefits of vaccinia virus include the ability to express large eukaryotic genes, lack of nuclear integration, ease of construction without the need for complex packaging cell lines, and strong adjuvant properties of the virus. Vaccination with recombinant vaccinia virus expressing human PSA (rV-PSA) was safe and induced PSA-specific T-cell responses in animal models and in humans.^17–20 A study in 33 patients with advanced prostate cancer using monthly rV-PSA vaccinations demonstrated a specific T-cell response against HLA-A2-restricted PSA-3 peptide, but minimal clinical activity as a single agent.¹⁷

Efforts to improve vaccine effectiveness historically have included the use of heterologous prime boost approaches.^21-22 In fact, a heterologous prime/boost vaccine strategy using recombinant vaccinia and fowlpox viruses expressing human carcinoembryonic antigen (CEA) was recently evaluated and demonstrated superior immune responses in those patients receiving the prime/boost regimen.²¹ Fowlpox virus is replication defective in mammalian cells and have been used to express tumor antigens.²³ The lack of replication in host cells results in weak neutralizing antibody responses permitting subsequent boosting after initial exposure to these viruses. Furthermore, because these viruses express antigen for a longer period than vaccinia virus, the T-cell response may be significantly enhanced when using avipox vectors for immunization.²³ The avipoxviruses are nonpathogenic and offer substantial safety advantages over other viral agents.

This clinical trial evaluated a heterologous prime/boost vaccination schedule with vaccinia and fowlpox viruses expressing human PSA, in men with only biochemical progression after local therapy, to test the hypothesis that this approach is feasible in the cooperative group setting.

	PATIENTS AND METHODS

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Patient Characteristics
Eligible patients were required to have pathologically confirmed prostate cancer with tumor limited to the prostate. Staging was based on the 1997 American Joint Committee on Cancer system based on institutional pathology reports. Patients were enrolled through six Eastern Cooperative Oncology Group (ECOG) institutions. All patients needed to have completed local therapy at least 3 months before study entry. Patients must have had an increase in PSA with an absolute level greater than 2 ng/mL after prostatectomy by two measurements at least 1 month apart, or three consecutive increases in serum PSA after radiation therapy. Patients were also required to have a negative bone scan and computed tomography scan of the abdomen and pelvis within four weeks of study registration. Patients must have completed neoadjuvant chemotherapy or hormonal therapy at least 6 months before study entry, be fully recovered from such therapy, and have no evidence of PSA progression while on neoadjuvant treatment. The following criteria were also required: age > 18 years, life expectancy of at least 6 months, ECOG performance status of 0 or 1, adequate vital organ function (WBC > 3,500/µL; platelets > 100,000/µL; serum creatinine < 1.5 mg/dL or creatinine clearance > 50 mL/min; AST and ALT < 2 times the upper limit of the institution's normal range; and total bilirubin < 1.5 mg/dL), no active infections or known HIV infection, and no contraindications to receiving vaccinia virus. All patients were required to use a safe and effective method of contraception during the study to prevent virus transmission. All subjects provided written informed consent and the clinical protocol was approved by each institutional review board before patient enrollment.

Biochemical progression was defined as an increase in baseline PSA of greater than 50% documented by two different measurements (or three if patients had radiotherapy) taken at least 1 month apart. Patients were considered to have had a biochemical response if they experienced normalization or decrease in PSA. In patients with prior radiotherapy, a complete response consisted of PSA falling below the institution's upper limit of normal, and required three different measurements taken at least 1 month apart. In patients with prior prostatectomy, this was defined as an undetectable PSA on two successive occasions at least 1 month apart. A partial biochemical response was defined as a reduction of PSA by greater than 50% from baseline, and stable biochemical disease was defined as a reduction of less than 50% or an increase of less than 50%, documented by repeat measurements as previously described.

Clinical progression was defined as the appearance of new lesions or areas of malignant disease, or the development of symptoms consistent with metastatic disease, such as bone pain. If there was no clinical progression, the best overall response was defined as the biochemical response.

Treatment
Eligible patients were equally randomly assigned to one of three treatment arms using permuted blocks within strata, and dynamic balancing within institutions and HLA-A2 typing. Patients were randomly assigned to receive four rF-PSA vaccines (arm A), three rF-PSA vaccines followed by a single rV-PSA vaccine (arm B), or a single rV-PSA vaccine followed by three rF-PSA vaccinations (arm C). Vaccines were administered at 6-week intervals using 2.34 x 10⁸ plaque-forming units (PFU) of rV-PSA administered by intradermal injection or 1.5 x 10⁹ PFU of rF-PSA administered by intramuscular injection. Patients underwent history and physical examination every 6 weeks before vaccination and 3 weeks after the first vaccination. Blood was drawn for routine toxicity evaluation before the first vaccination (baseline sample), 3 weeks following the first immunization, and at the 6-, 12-, 18-, and 24-week time points. Additional blood was collected for immune monitoring at baseline, week 12, and week 24.

Vaccine Preparation
rV-PSA is a genetically engineered vaccinia virus containing a cDNA copy of the human PSA gene inserted into the thymidine kinase region of the Wyeth strain of vaccinia virus. The virus was cultured on embryonic epithelial cells and tested for bacterial, Mycoplasma, endotoxin, and adventitial viruses before vialing. Sterile vials containing 3.51 x 10⁸ PFU per 0.3 mL in phosphate-buffered saline (PBS) supplemented with 10% glycerol were stored at –70°C until just before administration. On the day of vaccination, vials were thawed to room temperature until completely warmed and a sterile syringe was used to withdraw 0.2 mL of virus from the vial, resulting in a final viral concentration of 2.34 x 10⁸ PFU, given by intradermal injection with a 25-gauge needle into the skin of the upper outer arm. The vaccination site was covered with a dry gauze and Tegaderm dressing (3M Health Care Ltd, St Paul, MN). All patients were instructed in proper vaccine site management, how to return and dispose of used dressings, and the importance of avoiding individuals at high risk for vaccinia-related complications for 7 to 10 days after vaccination.

rF-PSA was constructed in a similar manner using the human PSA cDNA for insertion into a nonessential region of wild-type fowlpox virus. The virus was passaged on embryonic epithelial cells, tested for infectious bacteria and viruses, and stored in sterile vials containing 1.782 x 10⁹ PFU of virus per 0.6 mL of PBS supplemented with 10% glycerol. The vials were stored at –70°C until the day of vaccination, when the vials were thawed to room temperature. A syringe was used to remove 0.5 mL of virus, containing 1.5 x 10⁹ PFU virus before injection. A 25-gauge needle was used to inject the virus into the muscle of the upper outer arm and the site was covered with a dry bandage.

These procedures were approved by each institutional biosafety committee and the National Institutes of Health Office of Biotechnology Activities.

Immunologic Monitoring
Antibody titers against vaccinia virus and PSA were tested by standard enzyme-linked immunosorbent assay as described elsewhere.²⁴ Briefly, serial dilutions of patient preimmune and postvaccination sera were applied to 96-well plates coated overnight with purified vaccinia virus lysate or PSA protein. Wells were incubated with human anti-immunoglobulin G conjugated to horseradish peroxidase and developed using a horseradish peroxidase-specific substrate and absorbance at 450 nm recorded. Postvaccination titers were considered significant if they were greater than three standard deviations above the mean preimmune titers for each patient.

Enzyme-linked immunosorbent spot (ELISPOT) assays were performed for all HLA-A2+ patients, using cryopreserved (using a rate-controlled cryomed freezer [Thermo Forma, Marietta, OH]) peripheral blood mononuclear cells (PBMC). PBMCs were phenotyped for expression of HLA-A2 molecules by flow cytometry using the anti-HLA-A2 mAb BB7.2 (American Type Culture Collection, Manassas, VA) and an immunoglobulin G isotype as a control. The verification of the A2.1 subtype was performed using polymerase chain reaction with sequence-specific primers as described previously.²⁵ ELISPOT assays for interferon-gamma (IFN-) production were performed in the ECOG reference laboratory, the Immunologic Monitoring and Cellular Products Laboratory at the University of Pittsburgh Cancer Institute (Pittsburgh, PA). Initially, attempts were made to detect the precursor frequency of PSA-specific T-cells in direct ELISPOT assays using a procedure previously described.²⁶ Briefly, 96-well nitrocellulose plates were coated overnight with 10 µg/mL of murine antihuman IFN- monoclonal antibody. After washing the plates, wells were blocked for 2 hours at 37°C with 200 µL complete medium containing Dulbecco’s Modified Eagle’s Medium, 10% human AB serum, 1% glutamine, and penicillin/streptomycin. Pre- and postvaccine PBMC specimens of each patient were thawed, cultured overnight in complete medium, and plated onto the precoated 96-well plates. In some cases, PBMCs were enriched in CD3+ T-cells by using immunobeads (Miltenyi Biotec, Auburn, CA) as per the manufacturer's instructions, before ELISPOT assays. Antigen-presenting cells (APCs) consisted of the HLA-A2 transporter associated with antigen processing–(TAP–)deficient T2 cells or autologous dendritic cells derived from PBMC samples. Responders were PBMC or CD3+ T cells added at 5 x 10⁴ cells per well. The ratio of responders to APC was 20:1, and APC were pulsed with 0, 20, or 100 µg/mL of the HLA-A2–restricted P3 PSA peptide (amino acids 154–163). An HLA-A2–restricted influenza and HIV peptide were used as controls. As positive assay controls, responder cells were also stimulated with 1 µg/mL OKT3 and plated at 5 x 10⁴ cells/well. After 24 hours incubation at 37°C and in 5% CO₂, plates were washed several times with PBS containing Tween-20 (0.05%) and incubated with 2 µg/mL biotinylated murine antihuman IFN- monoclonal antibody at 4°C overnight. Subsequently, wells were washed with PBS/Tween-20 and incubated with a 1:4000 dilution of avidin-linked alkaline phosphatase for 2 hours and washed with PBS. 100 µL of 5-bromo-4-chloro-3-indoyl phosphate/nitroblue/tetrazolium substrate was added for 1 hour and plates were washed and allowed to air dry. The number of individual spots/well was counted by a blinded observer using a Zeiss image analysis system. The precursor frequency was calculated as the number of spot-forming units from wells containing PBMC + APC + peptide after subtraction of the background (PBMC + APC alone) relative to the number of PBMC seeded per well. In most cases, direct ELISPOT assays were negative, and it was necessary to perform in vitro stimulation (IVS) with P3 peptide for 7 days before analysis by ELISPOT assay. In ELISPOT assays performed after IVS, a three-fold increase in the number of postvaccination T-cells responsive to the P3 peptide relative to the number of T-cells responsive to this peptide before vaccination (time 0) was considered significant.

Statistical Methods
The study was designed to distinguish a 30% progression-free rate at 6 months from a 5% rate. Assuming 20 eligible patients per arm, a specified arm would be considered worthy of further study if four or more patients remain free of biochemical progression at 6 months. Given this design, the study has 89% power with a two-sided alpha of less than 0.05. Descriptive statistics were used to characterize patients at study entry. Exact binomial confidence intervals were used to describe responses. Kruskal-Wallis tests were used to compare differences in age and baseline PSA level between treatment arms. The method of Kaplan and Meier was used to characterize time to progression. Cox proportional hazards regression model was used to study the association between progression-free survival time and some patient characteristic factors. Local polynomial smoothing methods were used to show a smoothed version of the population mean of PSA over time for each treatment arm. Random mixed-effects models were used to study changes in PSA over time and relationships between PSA and other study variables.

Changes in PSA slope before and after therapy were calculated because a short PSA doubling time, or a high log slope, has been strongly associated with clinical recurrence, and emerging data has suggested that PSA slope may be as a marker predicting prostate specific mortality.^27,28 It was assumed that log(PSA) changes linearly over time and fit a mixed-effects model to log(PSA) to compare the velocity of PSA change before and after treatment. Only those PSA values measured within 200 days before the randomization were included in the analysis for calculating the slope before treatment (due to the linearity assumption). The increase in number of PSA-reactive T-cells after IVS was explored among the HLA-A2 positive patients. A three-fold rise between baseline and week 24 was considered significant, and the proportion of patients with such a rise was computed for each treatment arm. All results use two-sided P values.

	RESULTS

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Patient Demographics
Seventy patients were enrolled and 64 were included in the final analysis. Table 1 lists patient characteristics at the start of the trial across the three arms. Six patients were deemed ineligible after registration, four patients had disease that was not limited to the prostate and two had skin conditions that precluded safe administration of vaccinia virus. Patients were stratified to assure that the proportion of HLA-A2 positive patients in each arm was balanced and overall 54% of the men were HLA-A2 positive. The median PSA level at study entry was 7.1 (range, 1.9 to 66.1) for all patients without statistically significant differences between treatment arms (P = .286). The median follow-up time was 19.1 months (range, 5.8 to 24.6 months).

View larger version (16K): [in this window] [in a new window]   Fig 1. Smoothed (bandwith = 100 days) group means for the natural log-transformed prostate-specific levels over time by treatment arms. PSA, prostate-specific antigen.

View larger version (17K): [in this window] [in a new window]   Fig 2. Prostate-specific antigen (PSA) progression-free survival for the randomly assigned patients on the three treatment arms. There is no significant difference between these treatment arms (P = .235). Prog, number of patients with PSA progression; PF, number of patients progression-free.

View larger version (18K): [in this window] [in a new window]   Fig 3. Clinical progression-free survival. There was no significant difference between these treatment arms (P = .704). Prog, number of patients with PSA progression; PF, number of patients progression-free.

View larger version (64K): [in this window] [in a new window]   Fig 4. Antivaccinia antibody titers before (PRE) and after (POST) vaccination in treatment arms A (A), B (B), and C (C).

View larger version (63K): [in this window] [in a new window]   Fig 5. Precursor frequency of prostate-specific antigen-specific T-cell responses in human leukocyte antigen- (HLA-) A2-expressing vaccine patients as measured by interferon- enzyme-linked immunosorbent spot assay before and after vaccination. Data is shown as the number of spots per 105 cells before and after vaccination for HLA-A2 positive patients in treatment arms A (A), B (B), and C (C).

	DISCUSSION

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The trial was designed to evaluate PSA responses at 6 months following a series of four vaccinations. Although we did not observe any objective biochemical responses, overall, 45.3% of the men treated on this study were free of PSA progression at 19.1 months of clinical follow-up. The median time to PSA progression has not yet been reached for the men in treatment arm C, and suggests a trend favoring the group that received a priming dose of vaccinia virus (see Fig 2). The trial was not designed to detect differences between the treatment arms, but rather to determine if more than 30% of the vaccinated patients would remain free of biochemical progression.

While the natural history of patients with PSA progression remains controversial, a 30% response rate was considered of interest for further study at the time the protocol was written in 1997.^4-5,29-31 A recent review of outcomes reporting for clinical trials involving patients with rising PSA have suggested that this population may not be ideal for most therapeutic interventions in prostate cancer.³² However, even this group recognized that immunologic strategies may be best suited for evaluation in these men since disease burden is low and the risk of recurrent disease may be significantly elevated. Nonetheless, the interpretation of PSA responses must be done cautiously since many variables affect PSA levels, including immune system manipulation (ie, development of anti-PSA antibody titers), patient demographics (ie, adverse characteristics, such as race), and the innate biology of individual tumors (ie, Gleason's score or disease stage). In our clinical trial we did not observe any significant differences in tumor biology or population demographics across treatment arms. Thus, while the observed rate of PSA progression was lower than we expected, especially for the vaccinia virus primed group, these results alone should not be used to determine the efficacy of the vaccine.

This clinical trial was not designed to evaluate clinical responses, yet it is interesting to note that with nearly 2 years of follow-up data, 78.1% of the men remain free of clinical progression with none of the treatment arms reaching median time to progression. The purpose of this trial was, however, to support future randomized clinical trials that are needed to better judge the clinical effectiveness of vaccines and other therapeutic agents in this group of patients. Future vaccine trials should take into consideration the patient population, biology of the underlying primary tumor, and status of the immune system, as these are all variables that can influence the response to therapeutic interventions.

Although the vaccines utilized in this trial expressed the full-length PSA gene and was theoretically capable of presenting the full range of HLA-restricted PSA epitopes in vivo, we focused our cellular immune monitoring on HLA-A2–expressing patients, since well-characterized HLA-A2–restricted PSA epitopes have been described, and the use of specific peptides improves the sensitivity of the ELISPOT assay. Our initial attempts to evaluate PSA-specific precursor frequency in PBMC were negative when a 24-hour direct ELISPOT assay with P3-pulsed APC was used. Consequently, we implemented several measures to improve the detection of PSA-reactive cells, including the use of autologous dendritic cells as APC, purification of CD3⁺ T-cells from PBMC, and IVS with the PSA-specific peptide, as previously described.³³ Although these maneuvers may have improved the ability to identify low-level precursors, we observed an increase in PSA-specific T-cells in 46% of the patients treated on this trial. Interestingly, some patients appeared to lose their PSA-specific T-cells after vaccination, which could be as a result of host immunosuppressive factors or active T-cell deletion.³⁴ There does not appear to be a significant difference in cell-mediated immune responses between the treatment arms, although more patients in the vaccinia treated arms (arms B and C) demonstrated an increase in PSA-reactive T-cells. The lack of humoral response to PSA is encouraging, since this would be one concern in using PSA as a marker for antitumor activity of such an approach.

In conclusion, we demonstrated the feasibility and tolerability of a prime/boost strategy using recombinant vaccinia and fowlpox viruses expressing PSA in men with PSA progression. Although we did not observe objective PSA responses, after almost 2 years of clinical follow-up, 45.3% of the men are free from PSA progression and 78.1% are free from clinical disease progression. None of the men tested exhibited evidence of an anti-PSA antibody response, but 46% had an increase in PSA-specific T-cell precursors following vaccination. The study was not powered to distinguish differences between treatment arms, but the trend favoring the group that received a priming dose of rV-PSA supports further study. Our results provide initial data to study the prime/boost regimen approach in larger clinical trials. One additional problem found in this study was the lack of PSA response. The addition of immune adjuvants, including cytokines and costimulatory molecules, may be important for improving the clinical and immune responses of this prime boost approach. Further randomized clinical trials of new generation vaccines using both the prime boost approach and immune adjuvants are needed to fully elucidate the benefit of this approach in men with prostate cancer or those who are at risk for developing overt metastatic disease.

	Authors' Disclosures of Potential Conflicts of Interest

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION Authors' Disclosures of... REFERENCES

 
The following authors or their immediate family members have indicated a financial interest. No conflict exists for drugs or devices used in a study if they are not being evaluated as part of the investigation. Acted as a consultant within the last 2 years: Howard L. Kaufman, Therion Biologics; Robert S. DiPaola, Therion Biologics.

	Acknowledgment

 
We thank Heidi Hoerig, James Gulley, and Phil Arlen for critical review of this manuscript.

	NOTES

 
This study was conducted by the Eastern Cooperative Oncology Group (Robert L. Comis, MD, Chair) and supported in part by Public Health Service Grants CA23318, CA66636, CA21115, CA14958, CA80775, CA49883, CA39229, CA21076, CA49957, and by the National Cancer Institute, National Institutes of Health and the Department of Health and Human Services. Its contents are solely the responsibility of the authors and do not necessarily represent the official views of the National Cancer Institute.

Preliminary results of this trial were presented at the 38th American Society of Clinical Oncology Annual Meeting, Orlando, FL, May 18–21, 2002.

Authors' disclosures of potential conflicts of interest are found at the end of this article.

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Submitted August 12, 2003; accepted March 3, 2004.

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