- Email: [email protected]
neu Peptide (E75) Vaccine for Prevention of Recurrence in High-Risk Prostate Cancer Patients
Longterm Followup Assessment of a HER2/neu Peptide (E75) Vaccine for Prevention of Recurrence in High-Risk Prostate Cancer Patients Jeremy D Gates, MD, Mark G Carmichael, MD, Linda C Benavides, MD, Jarrod P Holmes, MD, Matthew T Hueman, MD, Michael M Woll, MD, Constantine G Ioannides, PhD, Craig H Robson, MD, David G McLeod, MD, Sathibalan Ponniah, MD, George E Peoples, MD E75 is an immunogenic peptide from the HER2/neu protein that is expressed in prostate cancer. High-risk prostate cancer (HRPC) patients demonstrating varying levels of HER2/neu expression were vaccinated with E75 peptide plus granulocyte-macrophage colony-stimulating factor to prevent postprostatectomy PSA and clinical recurrences. STUDY DESIGN: Forty evaluable HRPC patients were prospectively identified using the validated Center for Prostate Disease Research/CaPSURE high-risk equation and enrolled. HLA-A2⫹ patients (n ⫽ 21) were vaccinated, and HLA-A2⫺ patients (n ⫽ 19) were followed as clinical controls. All patients were assessed for clinicopathologic factors, biochemical recurrence (consecutive PSA value ⱖ 0.2 ng/mL), clinical recurrence, and survival. RESULTS: Comparing the vaccinated and control groups, there were no statistical differences in clinicopathologic prognostic factors. At a median followup of 58.2 months (range 18.8 to 62.7 months), PSA recurrence rates were not different between vaccinated (29%) and control (26%) groups. Median time to recurrence from operation was 14.0 months (range 5.7 to 53.4 months) versus 8.5 months (range 4.7 to 34.1 months) (p ⫽ 0.7), respectively. Three vaccinated patients had PSA recurrences during the vaccine series. If these patients were excluded, median time to recurrence for the vaccinated group extends to 42.7 months (range 20.4 to 53.4 months) (p ⫽ 0.4). Study-wide, only one clinical recurrence and death occurred in a vaccinated patient that was early in the vaccine series. Subset analysis comparing vaccinated recurrent patients with control recurrences noted some statistical trends. CONCLUSIONS: The HER2/neu (E75) vaccine can prevent or delay recurrences in HRPC patients if completed before PSA recurrence. A larger randomized phase II trial in HLA-A2⫹ patients will be required to confirm these findings. (J Am Coll Surg 2009;208:193–201. © 2009 by the American College of Surgeons) BACKGROUND:
The prognostic and therapeutic significance of HER2/neu in breast cancer has stimulated investigation of the clinical
use of this receptor in other epithelial malignancies, such as prostate cancer.1 HER2/neu expression in prostate cancer is variable and appears to be involved in the progression of disease to androgen-independence.2 The role that HER2/
Disclosure Information: Nothing to disclose. Supported by the US Military Cancer Institute, Department of Surgery, Uniformed Services University of the Health Sciences, Center for Prostate Disease Research, and the Department of Clinical Investigation at Walter Reed Army Medical Center, Bethesda, MD. Supported by grants from the Center for Prostate Disease Research, a Congressionally funded program of the Henry M Jackson Foundation for the Advancement of Military Medicine. This article represents the personal viewpoint of the authors and cannot be construed as a statement of official Department of the Army, Department of the Navy, or Department of Defense policy.
Development Program, US Military Cancer Institute, Department of Surgery, Uniformed Services University of the Health Sciences, Bethesda, MD (Carmichael, Hueman, Woll, Ponniah, People); Department of Medicine, Hematology and Medical Oncology Service, Walter Reed Army Medical Center, Washington, DC (Carmichael); Department of Hematology and Medical Oncology, Naval Medical Center San Diego, San Diego, CA (Holmes); University of Texas MD Anderson Cancer Center, Houston, TX (Ioannides); Department of Urology, University of Texas Health Science Center at San Antonio, San Antonio, TX (Robson); and Center for Prostate Disease Research, Department of Urology, Walter Reed Army Medical Center, Washington, DC (McLeod). Correspondence address: COL George E Peoples, MD, FACS, Department of Surgery, General Surgery Service, Brooke Army Medical Center, 3851 Roger Brooke Dr, Ft Sam Houston, TX 78234. email: george. [email protected]
Received August 8, 2008; Revised September 24, 2008; Accepted October 23, 2008. From the Department of Surgery, General Surgery Service, Brooke Army Medical Center, Ft Sam Houston, TX (Gates, Benavides, People); Cancer Vaccine
© 2009 by the American College of Surgeons Published by Elsevier Inc.
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ISSN 1072-7515/09/$36.00 doi:10.1016/j.jamcollsurg.2008.10.018
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Abbreviations and Acronyms
CPDR ⫽ Center for Prostate Disease Research DTH ⫽ delayed-type hypersensitivity GM-CSF ⫽ granulocyte-macrophage colony-stimulating factor HRPC ⫽ high-risk prostate cancer IHC ⫽ immunohistochemistry
neu plays in prostate cancer has led to testing of targeted HER2/neu therapies, such as trastuzumab and HER2/neu peptide vaccination. The latter has been tested for prevention of recurrence in high-risk prostate cancer (HRPC) patients postprostatectomy by our Cancer Vaccine Development Program.3 HER2/neu is a proto-oncogene in the epidermal growth factor family of tyrosine kinases and encodes for a transmembrane glycoprotein highly expressed in many epithelialderived cancers.4 The HER2/neu protein has been shown to be an immune-recognized tumor-associated antigen.5-7 Its overexpression in prostate cancer has been variably reported from 0% to 87% by immunohistochemistry (IHC) and 0% to 53% by gene amplification.8,9 Although HER2/neu gene amplification is thought to exist in a small percentage of the prostate cancer population, HER2/neu overexpression appears to result from transcriptional activation.2,10 The Cancer Vaccine Development Program is involved with development and implementation of novel adjuvant vaccines targeting tumor-associated antigen, like HER2/ neu. Several immunogenic peptides within the HER2/neu sequence are recognized by cytotoxic T lymphocytes.11,12 E75, derived from the extracellular domain of the HER2/ neu protein (369 to 377: KIFGSLAFL),13 is the most studied HER2/neu-derived peptide.14-18 We have previously reported initial results in 27 patients from our phase Ib clinical trial of the E75 peptide vaccine in HRPC patients postprostatectomy. After completing the dose-escalation portion of the trial we found that the vaccine was safe and effective in eliciting an immune response, as reported.3 The trial completed enrollment with 46 patients and, although vaccine-induced E75-specific cytotoxic lymphocytes have been shown to lyse HER2/neu-expressing prostate cancer cells, the clinical significance of this vaccine-specific immune response has yet to be determined. To address the latter point, we have followed these vaccinated HRPC patients for 5 years and monitored their clinical course. In breast cancer, we have shown that the E75 vaccine can have a favorable impact on recurrence rates.19 The benefit appeared to be dependent on optimal dosing, and the HER2/neu-derived immunity waned without booster inoculations. Although, only 19% of prostate patients en-
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rolled in the phase Ib trial were optimally dosed and none received boosters, we present the 5-year clinical assessment of HRPC patients who were vaccinated with E75. These immunocompetent patients with prostate cancer were clinically and chemically disease-free after prostatectomy, but at high risk for PSA recurrence based on the Center for Prostate Disease Research (CPDR)/CaPSURE risk equation.20,21 By assessing these patients, we have attempted to determine if inducing E75-specific immunity conveys a clinical benefit by preventing PSA recurrence.
METHODS Patient characteristics and clinical protocols
The Institutional Review Board of the Department of Clinical Investigation, Walter Reed Army Medical Center, approved the clinical protocol. This clinical trial was conducted under an Investigational New Drug application (IND9187) approved by the US Food and Drug Administration. All patients had prostate cancers that expressed HER2/neu by immunohistochemistry (IHC 1 to 3⫹). Patients had undergone a prostatectomy and had been determined to be at increased risk for recurrence according to the CPDR/CaPSURE risk equation. Eligible HRPC patients were enrolled in the study and HLA-typed to determine their HLA-A2 status because E75 binds this specific HLA allele.13 HLA-A2 is found in ⬃40% to 50% of the general population.22 HLA-A2⫹ patients were vaccinated, and HLA-A2⫺ patients were followed as matched controls for clinical recurrence. Before vaccination, patients were skin-tested with a panel of recall antigens (Mantoux test ⫽ mumps, tetanus, and candida); patients must have had two of three positive (⬎ 5 mm) to be considered immunologically intact. HLA-A2 typing
Patients’ HLA-A2 status was confirmed by indirect staining with 10 L anti⫺HLA-A2 monoclonal antibodies, BB7.2 and MA2.1 (American Type Culture Collection; 1:10 dilution of culture supernatant) at 4°C for 30 minutes followed by a 30-minute incubation with goat-antimouse monoclonal antibodies conjugated with fluorescein isothiocyanate (Becton Dickinson) and analyzed on a BD FACSCalibur Analyzer (Becton Dickinson). Vaccine
The E75 peptide (KIFGSLAFL, HER2/neu, and 369 to 377) was produced commercially in good manufacturing practices grade by Multiple Peptide Systems, a US Food and Drug Administration⫺approved manufacturer. The purity of the peptide was verified by high-performance liquid chromatography and mass spectrometry, and the
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amino acid content determined by amino acid analysis. The peptide was purified to ⬎ 95%. Sterility and general safety testing were carried out by the manufacturer. Lyophilized peptide was reconstituted in sterile 0.9% NaCl solution at the following concentrations: 100 g in 0.5 mL, 500 g in 0.5 mL, and 1 mg in 0.5 mL. The peptide was mixed with granulocyte-macrophage colony-stimulating factor (GM-CSF; Immunex Corp) at 250 g in 0.5 mL, and the 1.0-mL inoculation was split and given intradermally at two sites within 5 cm of each other. Vaccination series
Patients in the treatment group received intradermal inoculations in the same extremity to the same draining lymph node basin and were assigned to one of four dose/schedule groups. Two patients received 100 g, six patients 500 g, and four patients 1,000 g E75 monthly for 6 months (100.6, 500.6, and 1,000.6, respectively). The variable dose helped determine the maximum tolerated and optimal biologic dose. A fourth group consisting of nine patients received 500 g peptide and 250 g GM-CSF, but with an alternate schedule omitting the fourth and fifth inoculations (500.4). Toxicity
Patients were observed 1-hour postvaccination for immediate hypersensitivity and returned 48 to 72 hours later to have their injection sites measured and questioned about toxicities. Toxicities were graded by the National Cancer Institute Common Terminology Criteria for Adverse Events, v3.0 and reported on a scale from 0 to 5. Progression from one dose group to the next occurred only if no substantial dose-limiting toxicity occurred in the lowerdose group. Patient-specific results are reported based on maximal local and systemic toxicity occurring during the series. Delayed-type hypersensitivity
A delayed-type hypersensitivity (DTH) reaction was assessed with 100 g E75 in 0.5 mL normal saline (without GM-CSF) injected intradermally with a parallel control (0.9% NaCl solution, same volume) at a site on the back or extremity (opposite side from the vaccination site) 1 month after completion of the vaccine series. DTH reaction was measured in two dimensions at 48 to 72 hours by using the sensitive ballpoint-pen method and reported as the orthogonal mean and compared with control.23 Clinical and chemical recurrences
Both vaccinated and control patients were followed for clinical or chemical, or both, recurrences through standard
Figure 1. Flow chart of patient enrollment and analysis. HRPC, high-risk prostate cancer.
cancer screening to include physical examination, laboratory, and radiographic studies. In general, patients were followed every 3 to 4 months for the first year, every 6 months for another 2 years, and then annually thereafter for an additional 2 years. Serum PSA level of ⬎ 0.2 ng/mL, confirmed by repeat serial laboratory examination, defined PSA recurrence. Statistical analysis
The p values for clinicopathologic factors were calculated using Wilcoxon, Fisher’s exact test, or chi-square as appropriate. The p values for immunologic response were calculated using Student’s t-test, paired or unpaired, as appropriate. Recurrences and mortality between the control and vaccinated groups were compared using chi-square test. Statistical significance is defined as p ⬍ 0.05.
RESULTS Patients
This longterm (5 years) followup study evaluating the E75 vaccine as a preventive strategy, enrolled 46 CPDR risk score⫺defined HRPC patients who were clinically diseasefree after standard therapies, but at high risk for recurrence (Fig. 1). On enrollment, patients were HLA-typed, and HLA-A2⫹ patients were vaccinated (E75 binds HLA-A2), and HLA-A2⫺ patients were followed prospectively as controls. At enrollment, HLA status was unknown and the most recent PSA level after operation was used to exclude patients with PSA recurrence. It was anticipated that the groups would be clinically similar. Six patients (n ⫽ 3 controls; n ⫽ 3 vaccinated) were not included in this study’s analysis because they withdrew from the study to enroll in an adjuvant chemotherapy trial (n ⫽ 1 HLAA2⫺), were anergic to the recall panel (n ⫽ 1 HLA-A2⫺), had a PSA recurrence before enrollment that was not initially recognized (n ⫽ 1 HLA-A2⫹), did not followup after enrollment (n ⫽ 2 HLA-A2⫺ and HLA-A2⫹), or did not
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Table 1. Prognostic Factors in High-Risk Prostate Cancer Patients Vaccinated (HLA-A2⫹) and Control (HLA-A2⫺) Groups
Age (y), median (range) Race (%) Caucasian African American Asian Tumor* (%) Organ confined (T1c⫺T2c) Extraprostatic (T3a⫺T3b) Postoperative Gleason score (%) Low (6⫺7) High (8⫺9) Positive margins (%) Updated CPDR (%) High risk (7.1⫺16.7) Very high risk (16.7⫺56.3) Her-2/neu mean† Overexpression (IHC ⱖ 3⫹) (%)
Vaccinated HLA-A2ⴙ (n ⴝ 21)
Control HLA-A2ⴚ (n ⴝ 19)
63 (48⫺75)
63 (53⫺72)
76.2 19 4.8
63.2 36.8 0
11.8 88.2
17.6 82.4
66.7 33.3 71.4
73.7 26.3 52.6
61.9 38.1 2.06 35.3
57.9 42.1 2.06 18.8
Wilcoxon/Fisher’s exact test/Chi-Square
p ⫽ 0.91 p ⫽ 0.3
p ⫽ 0.63
p ⫽ 0.74
p ⫽ 0.33 p⫽1
p ⫽ 0.44
*Seventeen patients per group. † No IHC data on four vaccinated and three control patients. CPDR, Center for Prostate Disease Research; IHC, immunohistochemistry.
complete vaccination and declined longterm followup (n ⫽ 1 HLA-A2⫹). Forty evaluable patients (n ⫽ 19 controls; n ⫽ 21 vaccinated) were available for analysis. To assess comparability (Table 1), median age, race, tumor invasion, postoperative Gleason score, and updated CPDR score were compared and found to be similar between the control and vaccinated groups. More vaccinated patients had a positive margin status (71.4% versus 52.6%; p ⫽ 0.3) and nearly twice the percentage of HER2/neu overexpression (IHC ⱖ 3⫹) compared with controls (35% versus 18%; p ⫽ 0.4). Overall, HLA-A2⫹ patients had consistently worse prognostic factors than their corresponding control group. Although these factors did not reach statistical significance individually, taken collectively they would suggest a higher expected recurrence rate among the HLAA2⫹ patients compared with the HLA-A2⫺ patients; there are too few patients given the number of prognostic variables to perform a multivariate analysis.
Immunologic response
DTH was used as a direct measure of the vaccine’s in vivo effectiveness and assessed at 1 month after completion of the vaccination series. The vaccinated group had a significant mean DTH reaction to E75 compared with the saline volume control (31.5 ⫾ 5.0 mm versus 9.7 ⫾ 1.6 mm; p ⬍ 0.001) as shown in Figure 3. Prevaccine DTH levels were not included in the initial trial design and were not available for comparison. This can be interpreted as an induction of an immunologic response to the vaccine, but might also reflect the presence or expansion of a preexisting E75specific response in HLA-A2⫹ patients.
Vaccine toxicity
Toxicity was monitored as described here. No grade 2 to 5 local or grade 3 to 5 systemic toxicities were observed or reported in the vaccinated group (Fig. 2). Only two (9.5%) grade 2 and eight (38.1%) grade 1 systemic toxicities were documented, and all patients had a grade 1 local reaction. Because most of the toxicity was believed to be a result of the direct effects of GM-CSF, the dose of this adjuvant was reduced to 125 g in five patients.
Figure 2. Maximum local and systemic toxicity in all vaccinated patients based on the NIH Common Terminology Criteria for Adverse Events.
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six recurrences in the vaccinated group occurring before completion of the vaccine series (range 54 to 139 days). Because the primary end point of the trial was disease prevention by the vaccine, if these recurrent patients were excluded from the analysis, then the median time to PSA recurrence extends to 42.7 months (range 20.4 to 53.4 months; p ⫽ 0.4) (Fig. 4B). Disease-free survival
Figure 3. Delayed-type hypersensitivity (DTH) reaction for all vaccinated patients (n ⫽ 21) was assessed with 100 g E75 (without granulocyte-macrophage colony-stimulating factor) in comparison with a control (0.9% NaCl solution) 1 month after completion of the vaccine series.
Clinical response
PSA recurrence and mortality from disease after operation were similar in the vaccinated versus control group (recurrence 28.6% versus 26.3%, respectively; p ⫽ 0.9; mortality 4.8% versus 0%, respectively; p ⫽ 0.3). Recurrences in the vaccinated (n ⫽ 6) and control (n ⫽ 5) groups were compared for prognostic factors. There was a larger percentage of the HLA-A2⫹-vaccinated patients, as shown in Table 2, who had higher postoperative Gleason scores, positive margins, very high-risk updated CPDR scores, and HER2/neu overexpression compared with HLA-A2⫺ controls. Overall, HLA-A2⫹ patients who recurred had consistently worse prognostic factors than their corresponding control recurrence group, although these factors did not reach statistical significance individually in this small cohort. After PSA recurrence, three patients within the control group started hormone therapy and one underwent radiation therapy. One control patient did not receive therapy after PSA recurrence. In the vaccinated group, four patients initiated hormone therapy after PSA recurrence and two patients had radiation therapy, one pre- and one post-PSA recurrence. One patient died in the vaccinated group from disease; this patient had a PSA recurrence during the middle of vaccination and died 57 months after enrollment. Time to recurrence
In Figure 4A, comparison of median time to PSA recurrence from operation for all vaccinated (n ⫽ 6) and control recurrences (n ⫽ 5) is shown (median 14.0 months, range 5.7 to 53.4 months versus median 8.5 months, range 4.7 to 34.1 months, respectively; p ⫽ 0.7). The 6-monthly inoculation series was conducted during 150 days with three of
Comparison of disease-free survival from the time of enrollment between the vaccinated and control groups was longer but not statistically significant (41.3 ⫾ 4.7 months versus 37.9 ⫾ 5.3 months, respectively; p ⫽ 0.6) (Fig. 5). Comparison of disease-free survival from the time of operation between the vaccinated and control groups was similar (47.7 ⫾ 5.0 months versus 48.6 ⫾ 5.5 months, respectively; p ⫽ 0.9) (Fig. 5). Surprisingly, time from operation to study enrollment between the vaccinated and control groups showed a difference of 4.2 months (6.5 ⫾ 1.0 months versus 10.7 ⫾ 1.9 months, respectively; p ⫽ 0.06) that trended toward significance (Fig. 5).
DISCUSSION Here we report the 5-year clinical followup of our completed phase Ib trial of the HER2/neu-derived E75 vaccine in disease-free high-risk prostate cancer patients. Previously, we reported on the dose-escalation stage of this trial.3 In this report, we confirm the safety and antigenicity of the vaccine in a larger cohort of patients and present findings suggestive of a potential clinical benefit. Although there was no difference in the PSA recurrence rate in these suboptimally vaccinated patients, there appears to be a delay in recurrence in those patients who completed the vaccination series before PSA recurrence. Although the proportion of recurrences between the vaccinated and control groups was similar, the time to recurrence was 65% longer in the vaccinated group. Despite the lack of a substantial difference in this small population of patients, the time to recurrence was accentuated (by 400%) when patients who recurred during the vaccination were excluded. These patients might not have received the full benefit of this preventive cancer vaccine. These findings must be tempered by the fact that the number of recurrences is small. In addition, based on our data with E75 in breast cancer, we have documented the optimal dose and need for boosters.24 In this trial, only 19% of these HRPC patients were optimally dosed and none received boosters. After enrollment into the study, subjects were HLAA2⫺typed. HLA-A2⫹ patients were vaccinated, and HLAA2⫺ patients were followed as clinical controls. The corre-
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Table 2. Prognostic Factors for High-Risk Prostate Cancer Patients Recurrences in Vaccinated (HLA-A2⫹) and Control (HLA-2⫺) Groups
Age (y), median (range) Race (%) Caucasian African American Tumor (%) Organ confined (T1c⫺T2c) Extraprostatic (T3a⫺T3b) Postoperative Gleason score (%) Low (6⫺7) High (8⫺9) Positive margins (%) Updated CPDR (%) High risk (7.1⫺16.7) Very high risk (16.7⫺56.3) Her-2/neu mean* Her-2/neu expression* Overexpression (IHC ⱖ 3⫹) (%)
Vaccinated recurrence HLA-A2ⴙ (n ⴝ 6)
Control recurrence HLA-A2ⴚ (n ⴝ 5)
Wilcoxon/Fisher’s exact test/Chi-Square
58.8 (51⫺74)
62.9 (54⫺67)
p ⫽ 0.74 p⫽1
66.7 33
80.0 20.0 p⫽1
0 100
0 100 p ⫽ 0.57
33.3 66.7 83.3
60.0 40.0 40.0
16.7 83.3 2.33
40.0 60.0 1.67
50
0
p ⫽ 0.24 p ⫽ 0.55
p ⫽ 0.46
*Two patients in control arm without Her-2/neu immunohistochemistry. CPDR, Center for Prostate Disease Research; IHC, immunohistochemistry.
lation between certain HLA molecules and prognosis has been established in melanoma and suggested in some other forms of cancer.25,26 De Petris and colleagues27 found that the HLA-A2 allele was overrepresented in prostate cancer patients when compared with the general population in Sweden. The HLA-A2 gene frequency also correlated with prostate cancer mortality in 19 European countries (p ⫽ 0.0056). In our trial, no statistical difference was seen between the vaccinated and control groups, virtually all clinicopathologic prognostic factors were worse in the HLAA2⫹-vaccinated group. In addition, there was a surprising finding of a longer time from operation to enrollment in the control group compared with the vaccinated group. This was unintentional, as investigators were unaware of the patients’ HLA type before enrollment. The only explanation for this finding is based on HLA type and might be additional evidence that HLA-A2⫹ status can be a poor prognostic factor in prostate cancer. The latter patients might have recurred sooner or more often after operation, thereby disqualifying them from study enrollment and resulting in overrepresentation in the control group of patients with longer disease-free survival at enrollment. Despite difficulties definitively determining the level of HER2/neu expression in prostate cancer, evidence would support the importance of HER2/neu in disease progression.2 An in vitro study showed that androgen-dependent LNCaP cells, when induced to express HER2/neu, converted into androgen-independent cells.28 Berger and col-
leagues29 demonstrated in vitro that HER2/neu levels are inversely related to androgen concentrations, with androgen stimulation causing an androgen-receptor⫺dependent decrease in HER2/neu mRNA and protein level. In vitro work in a murine model by this group then demonstrated that HER2/neu expression in orthotopic prostate tumors was increased after castration. After this, LNCaP cells were treated with an anti-androgen drug, bicalutamide, and were found to have increased HER2/neu expression. Clinical studies comparing HER2/neu expression showed an increasing percentage of patients with HER2/neu protein overexpression, with progression toward androgen-independent prostate cancer.2,30,31 In addition, the prognostic significance of HER2/neu overexpression in patients with bone metastatic prostate cancer who have undergone endocrine therapy is associated with decreased cause-specific survival and nonrecurrence rate.32 Overall, these findings are important in light of the role that HER2/neu plays in the stabilization of androgen-receptor protein levels and binding optimization of the androgen receptor to promoter/enhancer regions of androgen-regulated genes.33 HER2/neu can confer an increased malignant potential to prostate cancer cells through its ability to stimulate the androgen-receptor pathway in the absence of androgen as a patient’s clinical course evolves to the end point of androgenindependent prostate cancer. Collectively, this information suggests that HER2/neu might be a promising therapeutic target in prostate cancer patients.
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Figure 5. Time (months) from operation to enrollment and vaccination in the control (mean ⫾ SEM 10.7 ⫾ 1.9 months) and vaccinated group (mean ⫾ SEM 6.5 ⫾ 1.0 months) (p ⫽ 0.06). Diseasefree survival from operation in the control (mean ⫾ SEM 48.6 ⫾ 5.5 months) and vaccinated group (mean ⫾ SEM 47.7 ⫾ 5.0 months) (p ⫽ 0.91). Disease-free survival from enrollment in the control (mean ⫾ SEM 37.9 ⫾ 5.3 months) and vaccinated group (mean ⫾ SEM 41.3 ⫾ 4.7 months) (p ⫽ 0.64).
Figure 4. Time line from operation to recurrence. (A) Time to recurrence for all patients within the control (median 8.5 months; range 4.7 to 34.1 months) and vaccinated (median 14.0 months; range 5.7 to 53.4 months) group who had PSA or clinical recurrence during the trial (p ⫽ NS). (B) Excluding three patients from the vaccinated group (median 42.7 months, range 20.4 to 53.4 months) who recurred during the 150-day vaccination period (p ⫽ NS).
Use of a trastuzumab (Herceptin; Genentech), humanized monoclonal antibody targeting the extracellular domain of HER2/neu, has been studied in the context of prostate cancer. Morris and colleagues31 published a negative phase II trial in androgen-dependent and androgenindependent prostate cancer patients (n ⫽ 23) using trastuzumab alone or in combination with paclitaxel. Only 26% of the patients evaluated in this study were HER2/neu overexpressors (IHC 3⫹). The authors treated the patients
with trastuzumab as a single agent (until disease progression, when they added paclitaxel), in contrast to the breast cancer studies, which have shown a synergistic response to treatment with trastuzumab in combination with chemotherapy.1 Monotherapy, with trastuzumab in another phase II trial for hormone refractory prostate cancer patients (n ⫽ 18), who were chemotherapy-naïve, again showed poor efficacy based on PSA recurrence, causing trastuzumab administration to be stopped short of completion.34 Use of a different humanized monoclonal anti-HER2/neu antibody, pertuzumab, in castrated, chemotherapy-naïve, hormone refractory prostate cancer patients showed no PSA decline ⱖ 50%.35 Immunotherapy targeting prostate cancer continues to expand rapidly with development and refinement of techniques and the assortment of prostate-specific proteins (eg, PSA, prostatic acid phosphatase, prostate membrane-specific antigen, six transmembrane epithelial antigen of the prostate), which can be targeted.36 Several strategies have been undertaken to stimulate the immune system to bring about a favorable clinical response, such as protein or mRNA-loaded dendritic cell vaccines, autologous or allogenic tumor cell vaccines
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expressing GM-CSF, and protein-expressing virus vaccines with or without costimulatory molecules.37 Although the field of immunotherapy and its practical application in the clinical setting are growing, it is imperative that the timing and intent of such modalities be addressed. Immunotherapy can be used in both a therapeutic and a preventive role as recently described by Finn.38 Therapeutic immunotherapy can either be passive (ie, antibodies, T cells) or specific (ie, cancer vaccines), but because of a lack of clinical efficacy, as demonstrated in many trials, the trend is now toward using combination strategies with immune modulators (anti⫺ cytotoxic T lymphocyte antigen 4) or chemotherapy to obtain a cancer response. Preventive immunotherapy can be subdivided into prophylaxis in cancer-naïve patients versus prevention of recurrence in disease-free patients in the adjuvant setting. These latter immunotherapy strategies have not been studied extensively; the need for combination immunotherapy or chemotherapy, or both, has not been demonstrated, but might be required. In this study, the intent was to alter the recurrence pattern in high-risk prostate cancer patients who were deemed disease-free, thereby marking this as a preventive cancer vaccine trial in the adjuvant setting. At this time, it appears that we have conducted the only peptide cancer vaccine trial in humans targeting HER2/neu in prostate cancer. In our completed phase Ib trial with 5 years of clinical followup, we have shown that the HER2/neu (E75) vaccine is safe and effective in eliciting a peptide-specific immune response in HER2/neu-expressing HRPC postprostatectomy patients. Although, no difference was documented in the PSA recurrence rates between vaccinated and control patients, time to recurrence was substantially longer in the vaccinated group, especially among patients who completed the vaccination series before recurrence. Potential beneficial effects of the vaccine might also have been diluted by suboptimal dosing of the vaccine and the lack of booster inoculations. Finally, HLA-A2 can be an adverse prognostic factor in prostate cancer. Fully assessing the potential of this vaccine strategy will require a randomized phase II trial enrolling only HLA-A2⫹ patients and using optimal dosing and boosters of the E75 vaccine. Author Contributions Study conception and design: McLeod, Peoples Acquisition of data: Ponniah, Peoples Analysis and interpretation of data: Gates, Carmichael, Benavides, Holmes, Hueman, Ioannides, Robson, Ponniah, Peoples Drafting of manuscript: Gates, Peoples Critical revision: Gates, Ponniah, Peoples
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Acknowledgment: We thank Kathy Pangaro, Joyce Winters, and Diane Papay of the Cancer Vaccine Development Program for their excellent patient care and administration of the clinical trial, the staff of the Cancer Vaccine Development Program for their clinical and administrative assistance and Dr John Ward of the Department of Clinical Investigation at Brooke Army Medical Center for statistical input.
REFERENCES 1. Slamon DJ, Leyland-Jones B, Shak S, et al. Use of chemotherapy plus a monoclonal antibody against HER2 for metastatic breast cancer that overexpresses HER2. N Engl J Med 2001;344:783– 792. 2. Signoretti S, Montironi R, Manola J, et al. Her-2-neu expression and progression toward androgen independence in human prostate cancer. J Natl Cancer Inst 2000;92:1918–1925. 3. Hueman M, Dehqanzada Z, Novak T, et al. Phase I clinical trial of a HER-2/neu peptide (E75) vaccine for the prevention of prostate-specific antigen recurrence in high-risk prostate cancer patients. Clin Cancer Res 2005;11:7470–7479. 4. Anderson BW, Peoples GE, Murray JL, et al. Peptide priming of cytolytic activity to HER-2 epitope 369⫺377 in healthy individuals. Clin Cancer Res 2000;6:4192–4200. 5. Lustgarten J, Theobald M, Labadie C, et al. Identification of Her-2/Neu CTL epitopes using double transgenic mice expressing HLA-A2.1 and human CD. 8. Hum Immunol 1997;52: 109–118. 6. Zaks TZ, Rosenberg SA. Immunization with a peptide epitope (p369–377) from HER-2/neu leads to peptide-specific cytotoxic T lymphocytes that fail to recognize HER-2/neu⫹ tumors. Cancer Res 1998;58:4902–4908. 7. Knutson KL, Schiffman K, Cheever MA, et al. Immunization of cancer patients with HER-2/neu, HLA-A2 peptide, p369⫺377, results in shortlived peptide-specific immunity. Clin Cancer Res 2002;8:1014–1018. 8. Woll MM, Hueman MT, Ryan GB, et al. Preclinical testing of a peptide-based, HER2/neu vaccine for prostate cancer. Int J Oncol 2004;25:1769–1780. 9. Ross JS, Sheehan CE, Hayner-Buchan AM, et al. Prognostic significance of HER-2/neu gene amplification status by fluorescence in situ hybridization of prostate carcinoma. Cancer 1997; 79:2162–2170. 10. Skacel M, Ormsby AH, Pettay JD, et al. Aneusomy of chromosomes 7, 8, and 17 and amplification of HER-2/neu and epidermal growth factor receptor in Gleason score 7 prostate carcinoma: a differential fluorescent in situ hybridization study of Gleason pattern 3 and 4 using tissue microarray. Hum Pathol 2001;32:1392–1397. 11. Murray JL, Gillogly ME, Przepiorka D, et al. Toxicity, immunogenicity, and induction of E75-specific tumorlytic CTLs by HER-2 peptide E74 (369⫺377) combined with granulocyte macrophage colony-stimulating factor in HLA-A2⫹ patients with metastatic breast and ovarian cancer. Clin Cancer Res 2002;8:3407–3418. 12. Morrison C, Zanagnolo V, Ramirez N, et al. HER-2 is an independent prognositic factor in endometrial cancer: association with outcome in a large cohort of surgically stage patients. J Clin Oncol 2006;24:2376⫺2385.
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13. Fisk B, Blevins TL, Wharton JT, et al. Identification of an immunodominant peptide of HER-2/neu protooncogene recognized by ovarian tumor-specific cytotoxic T lymphocyte lines. J Exp Med 1995;181:2109–2117. 14. Garin-Chesa P, Campbell I, Saigo PE, et al. Trophoblast and ovarian cancer antigen LK26. Sensitivity and specificity in immunopathology and molecular identification as a folate-binding protein. Am J Pathol 1993;142:557–567. 15. Alberti S, Miotti S, Fornano M, et al. The CA-MOv18 molecule, a cell surface marker of human ovarian carcinomas, is anchored to the cell membrane by phosphatidylinositol. Biochem Biophys Res Commun 1990;171:1051–1055. 16. Jacob JA. Folate, DNA methylation, and gene expression: factors of nature and nurture. Am J Clin Nutr 2000;72:903–904. 17. Li PY, Del Vecchio S, Fonti R, et al. Local concentration of folate binding protein GP38 in sections of human ovarian carcinoma by in vitro quantitative autoradiography. J Nucl Med 1996;37: 665–672. 18. Weitman SD, Lark RH, Coney LR, et al. Distribution of the folate receptor GP38 in normal and malignant cell lines and tissues. Cancer Res 1992;52:3396–3401. 19. Amin A, Benavides LC, Holmes JP, et al. Assessment of immunologic response and recurrence patterns among patients with clinical recurrence after vaccination with a preventative HER2/ neu peptide vaccine: from US Military Cancer Institute Clinical Trials Group Study I-01 and I-02. Cancer Immunol Immunother 2008; Epub ahead of print. 20. Bauer JJ, Connelly RR, Sesterhenn IA, et al. Biostatistical modeling using traditional preoperative and pathological prognostic variables in the selection of men at high risk for disease recurrence after radical prostatectomy for prostate cancer. J Urol 1998;159:929–933. 21. Moul JW, Connelly RR, Lubeck DP, et al. Predicting risk of prostate specific antigen recurrence after radical prostatectomy with the Center for Prostate Disease Research and Cancer of the Prostate Strategic Urologic Research Endeavor databases. J Urol 2001;166:1322–1327. 22. Ellis JM, Henson V, Slack R, et al. Frequencies of HLA-A2 alleles in five U.S. population groups. Predominance of A*02011 and identification of HLA-A*0231. Hum Immunol 2000;61:334–340. 23. Sokal JE. Measurement of delayed skin-test responses. N Engl J Med 1975;293:501–502. 24. Holmes JP, Gates JD, Benavides LC, et al. Optimal dose and schedule of a HER2/neu (E75) peptide vaccine to prevent breast
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cancer recurrence: From US Military Cancer Institute Clinical Trials Group Study I-01 and I-02. Cancer 2008; Epub ahead of print. Lee JE. Factors associated with melanoma incidence and prognosis. Semin Surg Oncol 1996;12:379–385. Lee JE, Lowy AM, Thompson WA, et al. Association of gastric adenocarcinoma with the HLA class II gene DQB10301. Gastroenterology 1996;111:426–432. De Petris L, Bergfeldt K, Hising C, et al. Correlation between HLA-A2 gene frequency, latitude, ovarian and prostate cancer mortality rates. Med Oncol 2004;21:49–52. Craft N, Shostak Y, Carey M, et al. A mechanism for hormoneindependent prostate cancer through modulation of androgen receptor signaling by the HER-2/neu tyrosine kinase. Nat Med 1999;5:280–285. Berger R, Lin DI, Nieto M, et al. Androgen-dependent regulation of Her-2/neu in prostate cancer cells. Cancer Res 2006;66: 5723–5728. Osman I, Scher HI, Drobnjak M, et al. HER-2/neu (p185neu) protein expression in the natural or treated history of prostate cancer. Clin Cancer Res 2001;7:2643–2647. Morris MJ, Reuter VE, Kelly WK, et al. HER-2 profiling and targeting in prostate carcinoma. Cancer 2002;94:980–986. Nishio Y, Yamada Y, Kokubo H, et al. Prognostic significance of immunohistochemical expression of the HER-2/neu oncoprotein in bone metastatic prostate cancer. Urology 2006;68:110– 115. Mellinghoff IK, Vivanco I, Kwon A, et al. HER2/neu kinasedependent modulation of androgen receptor function through effects on DNA bind and stability. Cancer Cell 2004;6:517– 527. Ziada A, Barqawi A, Glode LM, et al. The use of trastuzumab in the treatment of hormone refractory prostate cancer; phase II trial. Prostate 2004;60:332–337. de Bono JS, Bellmunt J, Attard G, et al. Open-label phase II study evaluating the efficacy and safety of two doses of pertuzumab in castrate chemotherapy-naïve patients with hormonerefractory prostate cancer. J Clin Oncol 2007;25:257–262. Webster WS, Small EJ, Rini BI, et al. Prostate cancer immunology: biology, therapeutics, and challenges. J Clin Oncol 2005; 23:8262–8269. Sonpavde G, Spencer DM, Slawin KM, et al. Vaccine therapy for prostate cancer. Urologic Oncology 2007;25:451–459. Finn OJ. Molecular origins of cancer: cancer immunology. N Engl J Med 2008;358:2704–2715.
The prognostic and therapeutic significance of HER2/neu in breast cancer has stimulated investigation of the clinical
use of this receptor in other epithelial malignancies, such as prostate cancer.1 HER2/neu expression in prostate cancer is variable and appears to be involved in the progression of disease to androgen-independence.2 The role that HER2/
Disclosure Information: Nothing to disclose. Supported by the US Military Cancer Institute, Department of Surgery, Uniformed Services University of the Health Sciences, Center for Prostate Disease Research, and the Department of Clinical Investigation at Walter Reed Army Medical Center, Bethesda, MD. Supported by grants from the Center for Prostate Disease Research, a Congressionally funded program of the Henry M Jackson Foundation for the Advancement of Military Medicine. This article represents the personal viewpoint of the authors and cannot be construed as a statement of official Department of the Army, Department of the Navy, or Department of Defense policy.
Development Program, US Military Cancer Institute, Department of Surgery, Uniformed Services University of the Health Sciences, Bethesda, MD (Carmichael, Hueman, Woll, Ponniah, People); Department of Medicine, Hematology and Medical Oncology Service, Walter Reed Army Medical Center, Washington, DC (Carmichael); Department of Hematology and Medical Oncology, Naval Medical Center San Diego, San Diego, CA (Holmes); University of Texas MD Anderson Cancer Center, Houston, TX (Ioannides); Department of Urology, University of Texas Health Science Center at San Antonio, San Antonio, TX (Robson); and Center for Prostate Disease Research, Department of Urology, Walter Reed Army Medical Center, Washington, DC (McLeod). Correspondence address: COL George E Peoples, MD, FACS, Department of Surgery, General Surgery Service, Brooke Army Medical Center, 3851 Roger Brooke Dr, Ft Sam Houston, TX 78234. email: george. [email protected]
Received August 8, 2008; Revised September 24, 2008; Accepted October 23, 2008. From the Department of Surgery, General Surgery Service, Brooke Army Medical Center, Ft Sam Houston, TX (Gates, Benavides, People); Cancer Vaccine
© 2009 by the American College of Surgeons Published by Elsevier Inc.
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Abbreviations and Acronyms
CPDR ⫽ Center for Prostate Disease Research DTH ⫽ delayed-type hypersensitivity GM-CSF ⫽ granulocyte-macrophage colony-stimulating factor HRPC ⫽ high-risk prostate cancer IHC ⫽ immunohistochemistry
neu plays in prostate cancer has led to testing of targeted HER2/neu therapies, such as trastuzumab and HER2/neu peptide vaccination. The latter has been tested for prevention of recurrence in high-risk prostate cancer (HRPC) patients postprostatectomy by our Cancer Vaccine Development Program.3 HER2/neu is a proto-oncogene in the epidermal growth factor family of tyrosine kinases and encodes for a transmembrane glycoprotein highly expressed in many epithelialderived cancers.4 The HER2/neu protein has been shown to be an immune-recognized tumor-associated antigen.5-7 Its overexpression in prostate cancer has been variably reported from 0% to 87% by immunohistochemistry (IHC) and 0% to 53% by gene amplification.8,9 Although HER2/neu gene amplification is thought to exist in a small percentage of the prostate cancer population, HER2/neu overexpression appears to result from transcriptional activation.2,10 The Cancer Vaccine Development Program is involved with development and implementation of novel adjuvant vaccines targeting tumor-associated antigen, like HER2/ neu. Several immunogenic peptides within the HER2/neu sequence are recognized by cytotoxic T lymphocytes.11,12 E75, derived from the extracellular domain of the HER2/ neu protein (369 to 377: KIFGSLAFL),13 is the most studied HER2/neu-derived peptide.14-18 We have previously reported initial results in 27 patients from our phase Ib clinical trial of the E75 peptide vaccine in HRPC patients postprostatectomy. After completing the dose-escalation portion of the trial we found that the vaccine was safe and effective in eliciting an immune response, as reported.3 The trial completed enrollment with 46 patients and, although vaccine-induced E75-specific cytotoxic lymphocytes have been shown to lyse HER2/neu-expressing prostate cancer cells, the clinical significance of this vaccine-specific immune response has yet to be determined. To address the latter point, we have followed these vaccinated HRPC patients for 5 years and monitored their clinical course. In breast cancer, we have shown that the E75 vaccine can have a favorable impact on recurrence rates.19 The benefit appeared to be dependent on optimal dosing, and the HER2/neu-derived immunity waned without booster inoculations. Although, only 19% of prostate patients en-
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rolled in the phase Ib trial were optimally dosed and none received boosters, we present the 5-year clinical assessment of HRPC patients who were vaccinated with E75. These immunocompetent patients with prostate cancer were clinically and chemically disease-free after prostatectomy, but at high risk for PSA recurrence based on the Center for Prostate Disease Research (CPDR)/CaPSURE risk equation.20,21 By assessing these patients, we have attempted to determine if inducing E75-specific immunity conveys a clinical benefit by preventing PSA recurrence.
METHODS Patient characteristics and clinical protocols
The Institutional Review Board of the Department of Clinical Investigation, Walter Reed Army Medical Center, approved the clinical protocol. This clinical trial was conducted under an Investigational New Drug application (IND9187) approved by the US Food and Drug Administration. All patients had prostate cancers that expressed HER2/neu by immunohistochemistry (IHC 1 to 3⫹). Patients had undergone a prostatectomy and had been determined to be at increased risk for recurrence according to the CPDR/CaPSURE risk equation. Eligible HRPC patients were enrolled in the study and HLA-typed to determine their HLA-A2 status because E75 binds this specific HLA allele.13 HLA-A2 is found in ⬃40% to 50% of the general population.22 HLA-A2⫹ patients were vaccinated, and HLA-A2⫺ patients were followed as matched controls for clinical recurrence. Before vaccination, patients were skin-tested with a panel of recall antigens (Mantoux test ⫽ mumps, tetanus, and candida); patients must have had two of three positive (⬎ 5 mm) to be considered immunologically intact. HLA-A2 typing
Patients’ HLA-A2 status was confirmed by indirect staining with 10 L anti⫺HLA-A2 monoclonal antibodies, BB7.2 and MA2.1 (American Type Culture Collection; 1:10 dilution of culture supernatant) at 4°C for 30 minutes followed by a 30-minute incubation with goat-antimouse monoclonal antibodies conjugated with fluorescein isothiocyanate (Becton Dickinson) and analyzed on a BD FACSCalibur Analyzer (Becton Dickinson). Vaccine
The E75 peptide (KIFGSLAFL, HER2/neu, and 369 to 377) was produced commercially in good manufacturing practices grade by Multiple Peptide Systems, a US Food and Drug Administration⫺approved manufacturer. The purity of the peptide was verified by high-performance liquid chromatography and mass spectrometry, and the
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amino acid content determined by amino acid analysis. The peptide was purified to ⬎ 95%. Sterility and general safety testing were carried out by the manufacturer. Lyophilized peptide was reconstituted in sterile 0.9% NaCl solution at the following concentrations: 100 g in 0.5 mL, 500 g in 0.5 mL, and 1 mg in 0.5 mL. The peptide was mixed with granulocyte-macrophage colony-stimulating factor (GM-CSF; Immunex Corp) at 250 g in 0.5 mL, and the 1.0-mL inoculation was split and given intradermally at two sites within 5 cm of each other. Vaccination series
Patients in the treatment group received intradermal inoculations in the same extremity to the same draining lymph node basin and were assigned to one of four dose/schedule groups. Two patients received 100 g, six patients 500 g, and four patients 1,000 g E75 monthly for 6 months (100.6, 500.6, and 1,000.6, respectively). The variable dose helped determine the maximum tolerated and optimal biologic dose. A fourth group consisting of nine patients received 500 g peptide and 250 g GM-CSF, but with an alternate schedule omitting the fourth and fifth inoculations (500.4). Toxicity
Patients were observed 1-hour postvaccination for immediate hypersensitivity and returned 48 to 72 hours later to have their injection sites measured and questioned about toxicities. Toxicities were graded by the National Cancer Institute Common Terminology Criteria for Adverse Events, v3.0 and reported on a scale from 0 to 5. Progression from one dose group to the next occurred only if no substantial dose-limiting toxicity occurred in the lowerdose group. Patient-specific results are reported based on maximal local and systemic toxicity occurring during the series. Delayed-type hypersensitivity
A delayed-type hypersensitivity (DTH) reaction was assessed with 100 g E75 in 0.5 mL normal saline (without GM-CSF) injected intradermally with a parallel control (0.9% NaCl solution, same volume) at a site on the back or extremity (opposite side from the vaccination site) 1 month after completion of the vaccine series. DTH reaction was measured in two dimensions at 48 to 72 hours by using the sensitive ballpoint-pen method and reported as the orthogonal mean and compared with control.23 Clinical and chemical recurrences
Both vaccinated and control patients were followed for clinical or chemical, or both, recurrences through standard
Figure 1. Flow chart of patient enrollment and analysis. HRPC, high-risk prostate cancer.
cancer screening to include physical examination, laboratory, and radiographic studies. In general, patients were followed every 3 to 4 months for the first year, every 6 months for another 2 years, and then annually thereafter for an additional 2 years. Serum PSA level of ⬎ 0.2 ng/mL, confirmed by repeat serial laboratory examination, defined PSA recurrence. Statistical analysis
The p values for clinicopathologic factors were calculated using Wilcoxon, Fisher’s exact test, or chi-square as appropriate. The p values for immunologic response were calculated using Student’s t-test, paired or unpaired, as appropriate. Recurrences and mortality between the control and vaccinated groups were compared using chi-square test. Statistical significance is defined as p ⬍ 0.05.
RESULTS Patients
This longterm (5 years) followup study evaluating the E75 vaccine as a preventive strategy, enrolled 46 CPDR risk score⫺defined HRPC patients who were clinically diseasefree after standard therapies, but at high risk for recurrence (Fig. 1). On enrollment, patients were HLA-typed, and HLA-A2⫹ patients were vaccinated (E75 binds HLA-A2), and HLA-A2⫺ patients were followed prospectively as controls. At enrollment, HLA status was unknown and the most recent PSA level after operation was used to exclude patients with PSA recurrence. It was anticipated that the groups would be clinically similar. Six patients (n ⫽ 3 controls; n ⫽ 3 vaccinated) were not included in this study’s analysis because they withdrew from the study to enroll in an adjuvant chemotherapy trial (n ⫽ 1 HLAA2⫺), were anergic to the recall panel (n ⫽ 1 HLA-A2⫺), had a PSA recurrence before enrollment that was not initially recognized (n ⫽ 1 HLA-A2⫹), did not followup after enrollment (n ⫽ 2 HLA-A2⫺ and HLA-A2⫹), or did not
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Table 1. Prognostic Factors in High-Risk Prostate Cancer Patients Vaccinated (HLA-A2⫹) and Control (HLA-A2⫺) Groups
Age (y), median (range) Race (%) Caucasian African American Asian Tumor* (%) Organ confined (T1c⫺T2c) Extraprostatic (T3a⫺T3b) Postoperative Gleason score (%) Low (6⫺7) High (8⫺9) Positive margins (%) Updated CPDR (%) High risk (7.1⫺16.7) Very high risk (16.7⫺56.3) Her-2/neu mean† Overexpression (IHC ⱖ 3⫹) (%)
Vaccinated HLA-A2ⴙ (n ⴝ 21)
Control HLA-A2ⴚ (n ⴝ 19)
63 (48⫺75)
63 (53⫺72)
76.2 19 4.8
63.2 36.8 0
11.8 88.2
17.6 82.4
66.7 33.3 71.4
73.7 26.3 52.6
61.9 38.1 2.06 35.3
57.9 42.1 2.06 18.8
Wilcoxon/Fisher’s exact test/Chi-Square
p ⫽ 0.91 p ⫽ 0.3
p ⫽ 0.63
p ⫽ 0.74
p ⫽ 0.33 p⫽1
p ⫽ 0.44
*Seventeen patients per group. † No IHC data on four vaccinated and three control patients. CPDR, Center for Prostate Disease Research; IHC, immunohistochemistry.
complete vaccination and declined longterm followup (n ⫽ 1 HLA-A2⫹). Forty evaluable patients (n ⫽ 19 controls; n ⫽ 21 vaccinated) were available for analysis. To assess comparability (Table 1), median age, race, tumor invasion, postoperative Gleason score, and updated CPDR score were compared and found to be similar between the control and vaccinated groups. More vaccinated patients had a positive margin status (71.4% versus 52.6%; p ⫽ 0.3) and nearly twice the percentage of HER2/neu overexpression (IHC ⱖ 3⫹) compared with controls (35% versus 18%; p ⫽ 0.4). Overall, HLA-A2⫹ patients had consistently worse prognostic factors than their corresponding control group. Although these factors did not reach statistical significance individually, taken collectively they would suggest a higher expected recurrence rate among the HLAA2⫹ patients compared with the HLA-A2⫺ patients; there are too few patients given the number of prognostic variables to perform a multivariate analysis.
Immunologic response
DTH was used as a direct measure of the vaccine’s in vivo effectiveness and assessed at 1 month after completion of the vaccination series. The vaccinated group had a significant mean DTH reaction to E75 compared with the saline volume control (31.5 ⫾ 5.0 mm versus 9.7 ⫾ 1.6 mm; p ⬍ 0.001) as shown in Figure 3. Prevaccine DTH levels were not included in the initial trial design and were not available for comparison. This can be interpreted as an induction of an immunologic response to the vaccine, but might also reflect the presence or expansion of a preexisting E75specific response in HLA-A2⫹ patients.
Vaccine toxicity
Toxicity was monitored as described here. No grade 2 to 5 local or grade 3 to 5 systemic toxicities were observed or reported in the vaccinated group (Fig. 2). Only two (9.5%) grade 2 and eight (38.1%) grade 1 systemic toxicities were documented, and all patients had a grade 1 local reaction. Because most of the toxicity was believed to be a result of the direct effects of GM-CSF, the dose of this adjuvant was reduced to 125 g in five patients.
Figure 2. Maximum local and systemic toxicity in all vaccinated patients based on the NIH Common Terminology Criteria for Adverse Events.
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six recurrences in the vaccinated group occurring before completion of the vaccine series (range 54 to 139 days). Because the primary end point of the trial was disease prevention by the vaccine, if these recurrent patients were excluded from the analysis, then the median time to PSA recurrence extends to 42.7 months (range 20.4 to 53.4 months; p ⫽ 0.4) (Fig. 4B). Disease-free survival
Figure 3. Delayed-type hypersensitivity (DTH) reaction for all vaccinated patients (n ⫽ 21) was assessed with 100 g E75 (without granulocyte-macrophage colony-stimulating factor) in comparison with a control (0.9% NaCl solution) 1 month after completion of the vaccine series.
Clinical response
PSA recurrence and mortality from disease after operation were similar in the vaccinated versus control group (recurrence 28.6% versus 26.3%, respectively; p ⫽ 0.9; mortality 4.8% versus 0%, respectively; p ⫽ 0.3). Recurrences in the vaccinated (n ⫽ 6) and control (n ⫽ 5) groups were compared for prognostic factors. There was a larger percentage of the HLA-A2⫹-vaccinated patients, as shown in Table 2, who had higher postoperative Gleason scores, positive margins, very high-risk updated CPDR scores, and HER2/neu overexpression compared with HLA-A2⫺ controls. Overall, HLA-A2⫹ patients who recurred had consistently worse prognostic factors than their corresponding control recurrence group, although these factors did not reach statistical significance individually in this small cohort. After PSA recurrence, three patients within the control group started hormone therapy and one underwent radiation therapy. One control patient did not receive therapy after PSA recurrence. In the vaccinated group, four patients initiated hormone therapy after PSA recurrence and two patients had radiation therapy, one pre- and one post-PSA recurrence. One patient died in the vaccinated group from disease; this patient had a PSA recurrence during the middle of vaccination and died 57 months after enrollment. Time to recurrence
In Figure 4A, comparison of median time to PSA recurrence from operation for all vaccinated (n ⫽ 6) and control recurrences (n ⫽ 5) is shown (median 14.0 months, range 5.7 to 53.4 months versus median 8.5 months, range 4.7 to 34.1 months, respectively; p ⫽ 0.7). The 6-monthly inoculation series was conducted during 150 days with three of
Comparison of disease-free survival from the time of enrollment between the vaccinated and control groups was longer but not statistically significant (41.3 ⫾ 4.7 months versus 37.9 ⫾ 5.3 months, respectively; p ⫽ 0.6) (Fig. 5). Comparison of disease-free survival from the time of operation between the vaccinated and control groups was similar (47.7 ⫾ 5.0 months versus 48.6 ⫾ 5.5 months, respectively; p ⫽ 0.9) (Fig. 5). Surprisingly, time from operation to study enrollment between the vaccinated and control groups showed a difference of 4.2 months (6.5 ⫾ 1.0 months versus 10.7 ⫾ 1.9 months, respectively; p ⫽ 0.06) that trended toward significance (Fig. 5).
DISCUSSION Here we report the 5-year clinical followup of our completed phase Ib trial of the HER2/neu-derived E75 vaccine in disease-free high-risk prostate cancer patients. Previously, we reported on the dose-escalation stage of this trial.3 In this report, we confirm the safety and antigenicity of the vaccine in a larger cohort of patients and present findings suggestive of a potential clinical benefit. Although there was no difference in the PSA recurrence rate in these suboptimally vaccinated patients, there appears to be a delay in recurrence in those patients who completed the vaccination series before PSA recurrence. Although the proportion of recurrences between the vaccinated and control groups was similar, the time to recurrence was 65% longer in the vaccinated group. Despite the lack of a substantial difference in this small population of patients, the time to recurrence was accentuated (by 400%) when patients who recurred during the vaccination were excluded. These patients might not have received the full benefit of this preventive cancer vaccine. These findings must be tempered by the fact that the number of recurrences is small. In addition, based on our data with E75 in breast cancer, we have documented the optimal dose and need for boosters.24 In this trial, only 19% of these HRPC patients were optimally dosed and none received boosters. After enrollment into the study, subjects were HLAA2⫺typed. HLA-A2⫹ patients were vaccinated, and HLAA2⫺ patients were followed as clinical controls. The corre-
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Table 2. Prognostic Factors for High-Risk Prostate Cancer Patients Recurrences in Vaccinated (HLA-A2⫹) and Control (HLA-2⫺) Groups
Age (y), median (range) Race (%) Caucasian African American Tumor (%) Organ confined (T1c⫺T2c) Extraprostatic (T3a⫺T3b) Postoperative Gleason score (%) Low (6⫺7) High (8⫺9) Positive margins (%) Updated CPDR (%) High risk (7.1⫺16.7) Very high risk (16.7⫺56.3) Her-2/neu mean* Her-2/neu expression* Overexpression (IHC ⱖ 3⫹) (%)
Vaccinated recurrence HLA-A2ⴙ (n ⴝ 6)
Control recurrence HLA-A2ⴚ (n ⴝ 5)
Wilcoxon/Fisher’s exact test/Chi-Square
58.8 (51⫺74)
62.9 (54⫺67)
p ⫽ 0.74 p⫽1
66.7 33
80.0 20.0 p⫽1
0 100
0 100 p ⫽ 0.57
33.3 66.7 83.3
60.0 40.0 40.0
16.7 83.3 2.33
40.0 60.0 1.67
50
0
p ⫽ 0.24 p ⫽ 0.55
p ⫽ 0.46
*Two patients in control arm without Her-2/neu immunohistochemistry. CPDR, Center for Prostate Disease Research; IHC, immunohistochemistry.
lation between certain HLA molecules and prognosis has been established in melanoma and suggested in some other forms of cancer.25,26 De Petris and colleagues27 found that the HLA-A2 allele was overrepresented in prostate cancer patients when compared with the general population in Sweden. The HLA-A2 gene frequency also correlated with prostate cancer mortality in 19 European countries (p ⫽ 0.0056). In our trial, no statistical difference was seen between the vaccinated and control groups, virtually all clinicopathologic prognostic factors were worse in the HLAA2⫹-vaccinated group. In addition, there was a surprising finding of a longer time from operation to enrollment in the control group compared with the vaccinated group. This was unintentional, as investigators were unaware of the patients’ HLA type before enrollment. The only explanation for this finding is based on HLA type and might be additional evidence that HLA-A2⫹ status can be a poor prognostic factor in prostate cancer. The latter patients might have recurred sooner or more often after operation, thereby disqualifying them from study enrollment and resulting in overrepresentation in the control group of patients with longer disease-free survival at enrollment. Despite difficulties definitively determining the level of HER2/neu expression in prostate cancer, evidence would support the importance of HER2/neu in disease progression.2 An in vitro study showed that androgen-dependent LNCaP cells, when induced to express HER2/neu, converted into androgen-independent cells.28 Berger and col-
leagues29 demonstrated in vitro that HER2/neu levels are inversely related to androgen concentrations, with androgen stimulation causing an androgen-receptor⫺dependent decrease in HER2/neu mRNA and protein level. In vitro work in a murine model by this group then demonstrated that HER2/neu expression in orthotopic prostate tumors was increased after castration. After this, LNCaP cells were treated with an anti-androgen drug, bicalutamide, and were found to have increased HER2/neu expression. Clinical studies comparing HER2/neu expression showed an increasing percentage of patients with HER2/neu protein overexpression, with progression toward androgen-independent prostate cancer.2,30,31 In addition, the prognostic significance of HER2/neu overexpression in patients with bone metastatic prostate cancer who have undergone endocrine therapy is associated with decreased cause-specific survival and nonrecurrence rate.32 Overall, these findings are important in light of the role that HER2/neu plays in the stabilization of androgen-receptor protein levels and binding optimization of the androgen receptor to promoter/enhancer regions of androgen-regulated genes.33 HER2/neu can confer an increased malignant potential to prostate cancer cells through its ability to stimulate the androgen-receptor pathway in the absence of androgen as a patient’s clinical course evolves to the end point of androgenindependent prostate cancer. Collectively, this information suggests that HER2/neu might be a promising therapeutic target in prostate cancer patients.
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Figure 5. Time (months) from operation to enrollment and vaccination in the control (mean ⫾ SEM 10.7 ⫾ 1.9 months) and vaccinated group (mean ⫾ SEM 6.5 ⫾ 1.0 months) (p ⫽ 0.06). Diseasefree survival from operation in the control (mean ⫾ SEM 48.6 ⫾ 5.5 months) and vaccinated group (mean ⫾ SEM 47.7 ⫾ 5.0 months) (p ⫽ 0.91). Disease-free survival from enrollment in the control (mean ⫾ SEM 37.9 ⫾ 5.3 months) and vaccinated group (mean ⫾ SEM 41.3 ⫾ 4.7 months) (p ⫽ 0.64).
Figure 4. Time line from operation to recurrence. (A) Time to recurrence for all patients within the control (median 8.5 months; range 4.7 to 34.1 months) and vaccinated (median 14.0 months; range 5.7 to 53.4 months) group who had PSA or clinical recurrence during the trial (p ⫽ NS). (B) Excluding three patients from the vaccinated group (median 42.7 months, range 20.4 to 53.4 months) who recurred during the 150-day vaccination period (p ⫽ NS).
Use of a trastuzumab (Herceptin; Genentech), humanized monoclonal antibody targeting the extracellular domain of HER2/neu, has been studied in the context of prostate cancer. Morris and colleagues31 published a negative phase II trial in androgen-dependent and androgenindependent prostate cancer patients (n ⫽ 23) using trastuzumab alone or in combination with paclitaxel. Only 26% of the patients evaluated in this study were HER2/neu overexpressors (IHC 3⫹). The authors treated the patients
with trastuzumab as a single agent (until disease progression, when they added paclitaxel), in contrast to the breast cancer studies, which have shown a synergistic response to treatment with trastuzumab in combination with chemotherapy.1 Monotherapy, with trastuzumab in another phase II trial for hormone refractory prostate cancer patients (n ⫽ 18), who were chemotherapy-naïve, again showed poor efficacy based on PSA recurrence, causing trastuzumab administration to be stopped short of completion.34 Use of a different humanized monoclonal anti-HER2/neu antibody, pertuzumab, in castrated, chemotherapy-naïve, hormone refractory prostate cancer patients showed no PSA decline ⱖ 50%.35 Immunotherapy targeting prostate cancer continues to expand rapidly with development and refinement of techniques and the assortment of prostate-specific proteins (eg, PSA, prostatic acid phosphatase, prostate membrane-specific antigen, six transmembrane epithelial antigen of the prostate), which can be targeted.36 Several strategies have been undertaken to stimulate the immune system to bring about a favorable clinical response, such as protein or mRNA-loaded dendritic cell vaccines, autologous or allogenic tumor cell vaccines
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expressing GM-CSF, and protein-expressing virus vaccines with or without costimulatory molecules.37 Although the field of immunotherapy and its practical application in the clinical setting are growing, it is imperative that the timing and intent of such modalities be addressed. Immunotherapy can be used in both a therapeutic and a preventive role as recently described by Finn.38 Therapeutic immunotherapy can either be passive (ie, antibodies, T cells) or specific (ie, cancer vaccines), but because of a lack of clinical efficacy, as demonstrated in many trials, the trend is now toward using combination strategies with immune modulators (anti⫺ cytotoxic T lymphocyte antigen 4) or chemotherapy to obtain a cancer response. Preventive immunotherapy can be subdivided into prophylaxis in cancer-naïve patients versus prevention of recurrence in disease-free patients in the adjuvant setting. These latter immunotherapy strategies have not been studied extensively; the need for combination immunotherapy or chemotherapy, or both, has not been demonstrated, but might be required. In this study, the intent was to alter the recurrence pattern in high-risk prostate cancer patients who were deemed disease-free, thereby marking this as a preventive cancer vaccine trial in the adjuvant setting. At this time, it appears that we have conducted the only peptide cancer vaccine trial in humans targeting HER2/neu in prostate cancer. In our completed phase Ib trial with 5 years of clinical followup, we have shown that the HER2/neu (E75) vaccine is safe and effective in eliciting a peptide-specific immune response in HER2/neu-expressing HRPC postprostatectomy patients. Although, no difference was documented in the PSA recurrence rates between vaccinated and control patients, time to recurrence was substantially longer in the vaccinated group, especially among patients who completed the vaccination series before recurrence. Potential beneficial effects of the vaccine might also have been diluted by suboptimal dosing of the vaccine and the lack of booster inoculations. Finally, HLA-A2 can be an adverse prognostic factor in prostate cancer. Fully assessing the potential of this vaccine strategy will require a randomized phase II trial enrolling only HLA-A2⫹ patients and using optimal dosing and boosters of the E75 vaccine. Author Contributions Study conception and design: McLeod, Peoples Acquisition of data: Ponniah, Peoples Analysis and interpretation of data: Gates, Carmichael, Benavides, Holmes, Hueman, Ioannides, Robson, Ponniah, Peoples Drafting of manuscript: Gates, Peoples Critical revision: Gates, Ponniah, Peoples
J Am Coll Surg
Acknowledgment: We thank Kathy Pangaro, Joyce Winters, and Diane Papay of the Cancer Vaccine Development Program for their excellent patient care and administration of the clinical trial, the staff of the Cancer Vaccine Development Program for their clinical and administrative assistance and Dr John Ward of the Department of Clinical Investigation at Brooke Army Medical Center for statistical input.
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