Originally published as JCO Early Release 10.1200/JCO.2005.03.047 on September 12 2005

This Article Abstract Full Text (PDF) Purchase Article View Shopping Cart Alert me when this article is cited Alert me if a correction is posted Services Email this article to a colleague Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Save to my personal folders Download to citation manager Rights & Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Peoples, G. E. Articles by Ponniah, S. Search for Related Content PubMed PubMed Citation Articles by Peoples, G. E. Articles by Ponniah, S. Related Articles Related Editorial Social Bookmarking                            What's this?

Clinical Trial Results of a HER2/neu (E75) Vaccine to Prevent Recurrence in High-Risk Breast Cancer Patients

From the Clinical Breast Care Project, Walter Reed Army Medical Center, Washington, DC; Uniformed Services, University of the Health Sciences, Bethesda, MD; and the University of Texas M.D. Anderson Cancer Center, Houston, TX

Address reprint requests to George E. Peoples, MD, Department of Surgery, Walter Reed Army Medical Center, 6900 Georgia Ave, NW, Washington, DC 20307-5001; e-mail: george.peoples{at}na.amedd.army.mil

	ABSTRACT

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

 
PURPOSE: E75 is an immunogenic peptide from the HER2/neu protein that is highly expressed in breast cancer. We are conducting a clinical trial of an E75 + granulocyte-macrophage colony-stimulating factor vaccine to assess safety, immunologic response, and the prevention of clinical recurrences in patients with disease-free, node-positive breast cancer (NPBC).

PATIENTS AND METHODS: Fifty-three patients with NPBC were enrolled and HLA typed. HLA-A2⁺ patients (n = 24) were vaccinated, and HLA-A2^– patients (n = 29) are observed prospectively as clinical controls. Local/systemic toxicities, immunologic responses, and time to recurrence are being measured.

RESULTS: Only minor toxicities have occurred (one grade 3 [4%]). All patients have demonstrated clonal expansion of E75-specific CD8⁺T cells that lysed HER2/neu-expressing tumor cells. An optimal dosage and schedule have been established. Patients have developed delayed-type hypersensitivity reactions to E75 postvaccination compared with controls (33 v 7 mm; P < .01). HLA-A2⁺ patients have been found to have larger, more poorly differentiated, and more hormonally insensitive tumors compared to HLA-A2^– patients. Despite this, the only two deaths have occurred in the control group. The disease-free survival in the vaccinated group is 85.7% compared to 59.8% in the controls at 22 months' median follow-up with a recurrence rate of 8% compared to 21%, respectively (P < .19). Median time to recurrence in the vaccinated patients was prolonged (11 v 8 months), and recurrence correlated with a weak delayed-type hypersensitivity response.

CONCLUSION: This HER2/neu (E75) vaccine is safe and effective in eliciting a peptide-specific immune response in vivo. Induced HER2/neu immunity seems to reduce the recurrence rate in patients with NPBC.

	INTRODUCTION

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

 
HER2/neu is a proto-oncogene, and its protein product is highly expressed in many epithelial-derived cancers.¹ The HER2/neu protein has been found to be an immune-recognized tumor-associated antigen by several investigators.^2-4 Once identified as a tumor-associated antigen, several immunogenic peptides recognized by cytotoxic T lymphocytes (CTLs) were quickly described from the HER2/neu sequence.^5,6 E75 (KIFGSLAFL, HER2/neu, 369-377) was originally described by Fisk et al⁶ and has become the most studied HER2/neu-derived peptide both in vitro and in vivo.^7-11

The E75 peptide has been found in both in vitro and animal studies to be capable of inducing a peptide-specific, CTL-mediated immune response.^7,8 Preclinical studies led to three previous clinical trials of this peptide as an anticancer vaccine.^9-11 In these trials, the E75 peptide was used in combination with an immunoadjuvant (incomplete Freund's adjuvant [IFA], granulocyte-macrophage colony-stimulating factor [GM-CSF]) and injected as a simple vaccine without a delivery system. The trials were small (four, six, and 14 patients), targeted patients with metastatic cancer, and used many different immunologic monitoring assays. However, all three trials demonstrated that the E75 peptide is safe and capable of inducing a peptide-specific immune response in vivo. Unfortunately, little has been reported regarding the clinical impact of HER2/neu immunity, perhaps because of the late-stage disease of most of these patients.

Other trials have reinfused the E75 peptide (loaded autologous dendritic cells) and reinfused these cells back into the patient,^12,13 successfully inducing a peptide-specific CTL response in metastatic patients. Disis et al^14,15 have tested other HER2/neu-related vaccines consisting of combinations of longer peptides that are capable of binding HLA class II molecules including some with sequestered CTL epitopes (ie, E75). Although this strategy offers the potential benefit of stimulating CD4 helper T cells, it requires the host to process a longer peptide fragment to generate the E75 peptide for CTL stimulation. Neither approach produced better E75-specific responses than the E75 peptide alone mixed with an immunoadjuvant. Additionally, GM-CSF seems better than IFA as an immunoadjuvant for peptide vaccines.¹⁶

Therefore, our clinical trials are investigating E75 mixed with GM-CSF as a simple vaccine strategy that could be exported easily to the community. We are monitoring the safety while assessing the optimal dosing of this vaccine, which is required to produce a peptide-specific immunologic response. Most importantly, we are vaccinating immunocompetent patients with node-positive breast cancer (NPBC) who are disease-free after standard conventional therapies but at high risk for recurrence. We are also conducting a similar E75 vaccine trial in patients with prostate cancer who, after prostatectomy, are determined to be at high risk for recurrence based on the Centers for Prostate Disease Research risk-assessment score.^17,18 By studying these groups of patients, we are determining if induced E75-specific immunity conveys clinical benefit by preventing recurrence.

	PATIENTS AND METHODS

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

 
Patient Characteristics and Clinical Protocols
The Walter Reed Army Medical Center Department of Clinical Investigation approved the clinical protocol. This clinical trial is being conducted under an investigational new drug application (IND No. 9187) approved by the Food and Drug Administration. All patients had histologically confirmed NPBC that expressed HER2/neu by standard immunohistochemistry. All patients with NPBC had completed a standard course of surgery, chemotherapy, and radiation therapy (as required) before enrollment, and those patients on chemoprevention were continued on their specific regimen. After screening for eligibility criteria and proper counseling and consenting, patients with NPBC were enrolled into the studies and then HLA typed to determine their HLA-A2 status, because E75 binds this specific HLA allele found in approximately 40% to 50% of the general population.¹⁹ HLA-A2⁺ patients were vaccinated, and HLA-A2^– patients were observed prospectively 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 were considered immunocompetent if they reacted (>5 mm) to 2 antigens.

Vaccine
The E75 peptide was commercially produced in good manufacturing practices grade by Multiple Peptide Systems (San Diego, CA). Peptide purity was verified by high-performance liquid chromatography and mass spectrometry, and the amino acid content was determined by amino acid analysis. The peptide was purified to more than 95%. Sterility and general safety testing was carried out by the manufacturer. Lyophilized peptide was reconstituted in sterile saline at the following concentrations: 100 µg in 0.5 mL; 500 µg in 0.5 mL; and 1 mg in 0.5 mL. The peptide was mixed with GM-CSF (Berlex, Seattle, WA) at 250 µg in 0.5 mL, and the 1.0-mL inoculation was split and given intradermally at two sites within 5 cm of each other. All inoculations were given in the same extremity.

Vaccination Series
The study was performed as a two-stage safety trial (Table 1). In the first stage, three patients were assigned to each dose/schedule group receiving six monthly inoculations: 100 µg (100.6), 500 µg (500.6), or 1,000 µg (1,000.6) of E75 peptide + GM-CSF. A fourth group received 500 µg of peptide + GM-CSF but only four inoculations (500.4), omitting the fourth and fifth vaccination (Fig 1). In the second stage, four additional groups were vaccinated: 500.4, 500.6, 1,000.6, and 1,000.4.

View larger version (8K): [in this window] [in a new window]   Fig 1. Vaccination, phlebotomy, and clinical-visit schedule for patients with node-positive breast cancer. (A) Six-dose vaccination schedule; (B) Four-dose vaccination schedule. Blood samples were withdrawn prior to each vaccination and 1 and 6 months after series completion. LR, local reaction at 48 hours; DTH, delayed-type hypersensitivity.

Peripheral Blood Mononuclear Cell Isolation and Cultures
Blood was drawn from patients before receiving each inoculation and at 1 (postvaccination) and 6 months (long-term) after completing the series (Fig 1). Forty milliliters of peripheral blood was drawn into Vacutainer CPT tubes (Becton Dickinson, Franklin Lakes, NJ) and centrifuged for the isolation of peripheral blood mononuclear cell (PBMC) populations. PBMCs were washed in Hanks' balanced salt solution and resuspended in culture medium consisting of Iscove's modified Dulbecco's medium containing 10% human AB serum (Gemini Bio-Products, Woodland, CA) supplemented with 1x penicillin/L-glutamine/streptomycin, 1x sodium pyruvate, 1x nonessential amino acids, and 50 µmol/L of 2-mercaptoethanol (Life Technologies, Gaithersburg, MD). The PBMCs were used as a source of lymphocytes and for the preparation of dendritic cells (DCs). DCs were prepared by incubating the PBMCs in six-well plates for 1 hour followed by removal of the nonadherent cell population. Nonadherent PBMCs were grown in culture medium supplemented with 10 U of interleukin-2 per mL (R&D Systems, Minneapolis, MN) in a 48-well plate at 2 x 10⁶ cells per mL. Parallel cultures were stimulated with 25 µg/mL E75. Adherent cells were cultured in AIM-V and 5% human serum AB with 100 µg/mL of GM-CSF and 50 µg/mL of interleukin-4 per mL (R&D Systems) to obtain monocyte-derived DC populations. Recombinant human tumor necrosis factor alpha (R&D Systems) was added at 30 ng/mL on day 3 to induce maturation. DCs were then harvested at 6 days, incubated with E75 for 2 hours, and then used to restimulate the PBMC cultures that had previously been stimulated with E75 peptide. All cultures were maintained in a humidified incubator at 37°C in 5% CO₂. These peptide-stimulated PBMCs were used in cytotoxicity assays after 7 to 10 days in culture from DC stimulation.

HLA-A2:Immunoglobulin Dimer Assay
The presence of CD8⁺E75-specific cells in freshly isolated PBMCs from patients was directly assessed by using the dimer assay as previously described.²⁰ Briefly, the HLA-A2:immunoglobulin (Ig) dimer (Pharmingen, San Diego, CA) was loaded with the E75 or control peptide (E37) by incubating 1 µg of dimer with an excess (5 µg) of peptide and 0.5 µg of ß₂-microglobulin (Sigma Chemical Co, St Louis, MO) at 37°C overnight then stored at 4°C until used. Staining of freshly isolated PBMCs with the dimer preparations was carried out identically, and all incubations were performed at 4°C. Cells were plated at 5 x 10⁵ cells per well in round-bottom 96-well plates (Becton Dickinson, Mountain View, CA) and washed twice with stain buffer (Pharmingen). Human gamma-globulin (Sigma Chemical Co) was added, and the samples were incubated for 5 minutes before adding the dimer preparations. The cells were incubated with the peptide-loaded dimer (at 1 µg of dimer per well) for 45 minutes and washed once in phosphate-buffered saline. Cells were then stained with rat antimouse IgG1-phycoerythrin (clone A85-1), CD8-fluorescein isothiocyanate, and CD3-allophycocyanin (Pharmingen). Two-color fluorometric analysis was carried out on a Becton Dickinson FACScalibur analyzer (Becton Dickinson). The lymphocyte population was gated on forward and side scatter, and gated events were analyzed by using CellQuest software. The data are displayed as a dual-parameter density plot correlating CD8-fluorescein isothiocyanate and IgG1-phycoerythrin fluorescence. Quadrants were set on the basis of staining obtained by using irrelevant peptide (E37)-loaded dimers as a negative control. Results are expressed as the percent of E75-specific CTLs (control E37 dimer results subtracted) of the total CD8⁺ population.

Cytotoxicity Assays
E75-specific cytotoxicity in peptide-stimulated PBMC cultures was determined by standard 4-hour chromium-release assay. Briefly, targets were labeled with 100 to 150 µCi of sodium chromate (Perkin Elmer, Boston, MA) for 1.5 hours at 37°C and then washed twice and plated at 2,500 cells per well in 100 µL of media in 96-well U-bottom plates (Becton Dickinson). Effectors were added at an effector/target (E/T) ratio of 10:1 to 20:1 in 100 µL per well. After 4 hours of incubation, 100 µL of culture supernatant was collected, and radionuclide release was measured on a Microbeta Trilux counter (Perkin Elmer). All determinants were performed in triplicate. Results are expressed as percent-specific lysis as determined by: (experimental mean counts per minute [cpm] – spontaneous mean cpm)/(maximum mean cpm – spontaneous mean cpm) x 100. The target cells used in the cytotoxicity assays consisted of HER2/neu⁺, HLA-A2⁺ cell lines (SKOV3-A2, MCF-7, LNCaP) and HER2/neu⁺, HLA-A2^– cell lines (SKOV3, AU465, BT474, MB468). Not all tumors were used in every assay, but a minimum of two HLA-A2⁺ and two HLA-A2^– controls were used in each experiment. In some assays, the HER2/neu-transfected, HLA-A2⁺ B lymphoblastoid cell line J2R/H2N was used with its corresponding negative control, J2R (kindly provided by Keith Knutson, PhD, University of Washington, Seattle, WA).

Delayed-Type Hypersensitivity
One month after completion of the vaccination regimen, a delayed-type hypersensitivity (DTH) reaction was assessed with 100 µg of E75 (without GM-CSF), which was injected intradermally with a parallel control (sterile saline, same volume) at a site on the back or extremity (opposite side from the vaccination site). The DTH reaction was measured in two dimensions at 48 to 72 hours by using the sensitive ballpoint-pen method^21,22 and compared with control.

Clinical Recurrences
Both vaccinated and control NPBC patients were observed for clinical recurrence through standard cancer screening including physical exam, laboratory, and radiographic studies. All recurrences were confirmed by biopsy.

Statistical Analysis
Recurrence rates were compared between the vaccinated and control groups by using survival analysis by the Kaplan-Meier method, and the proportion of subjects who had recurrences were compared by using log-ranked analysis. P values for the demographic factors were calculated by using an independent sample t (not assuming equal variance), Mann-Whitney, or the ² test with Yates' correction as necessary. P values for comparisons of prevaccination and postvaccination DTH reactions, dimer assays, and cytotoxicity assays were calculated by using a paired two-sample t test.

	RESULTS

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

 
Patients
To investigate the E75 vaccine (specific for HLA-A2) in a preventive setting, we enrolled 53 patients with NPBC who were disease-free after standard therapies but at high risk for recurrence. Enrolled patients were HLA typed, and 24 HLA-A2⁺ patients were vaccinated, whereas 29 HLA-A2^– patients were observed prospectively as controls. Because HLA-A2 status was not known at enrollment, we anticipated that these groups would be clinically similar. However, Table 2 demonstrates that the patients with NPBC who were vaccinated compared to controls had tumors that were larger, more poorly differentiated, and less hormonally sensitive. Because of the latter, fewer vaccinated patients were on chemoprevention, which is known to reduce risk of recurrence.^23,24 In addition, fewer vaccinated patients received radiation therapy, which is known to reduce local recurrence and improve overall survival in both premenopausal and postmenopausal patients with NPBC.^25,26 Interestingly, a similar difference has been seen in the patients with prostate cancer who were enrolled in our trial.²⁷

Toxicity
After each vaccination, the patients were observed for 1 hour for signs of a hypersensitivity reaction; they then returned 48 to 72 hours later to be questioned regarding local and systemic reactions and have their injection sites checked. Both toxicities were graded per the National Institutes of Health Common Toxicity Criteria and reported on a scale of zero to five (Table 3). The trial was designed as a two-stage safety study with progression to the second stage only if the first stage was completed without dose-limiting toxicity (DLT; any hypersensitivity reaction or two patients developing grade 3 toxicity). No DLT was encountered. Overall, the vaccinated group reported no grade 4 or 5 systemic or local toxicities. One grade 3 (severe bone pain) (4%) and three grade 2 (12.5%) systemic toxicities were reported, and all patients had either a grade 1 or 2 local reaction (a desired effect). Because no DLT was encountered, no maximum-tolerated dose was achieved. Overall, 92% of the patients completed the vaccination series, and two patients withdrew by choice.

View larger version (9K): [in this window] [in a new window]   Fig 2. Clonal expansion of CD8+ E75-specific T cells over the vaccination series. E75-specific cytotoxic T lymphocytes in fresh unstimulated peripheral blood mononuclear cells were detected by using an HLA-A2:immunoglobulin dimer loaded with E75 or negative control peptide (E37) and anti-CD8 monoclonal antibody and analyzed by two-color flow cytometry. Results are presented as prevaccination (PRE), maximum (MAX), and postvaccination (POST; 1 month after series completion) response. FITC, fluorescein isothiocyanate; PE, phycoerythrin.

View larger version (8K): [in this window] [in a new window]   Fig 3. Individual E75-specific dimer responses to vaccination series in the first stage of the trial. Prevaccination (PRE), maximum (MAX), and postvaccination (POST; 1 month after series completion) levels of E75-specific cytotoxic T lymphocytes (control E37 dimer results subtracted) are shown as percentage of CD8+ cells. Responses are given for escalating-dose groups (100.6, 500.6, and 1,000.6) and schedule-reduction group (500.4).

View larger version (12K): [in this window] [in a new window]   Fig 4. Cumulative E75-specific dimer responses from both trial stages. CD8+E75-specific cytotoxic T lymphocytes (CTLs) in fresh unstimulated peripheral blood mononuclear cells from prevaccination (PRE), maximum (MAX), postvaccination (POST), and long-term (6 months postvaccination) time points. Results are means ± SE for all patients receiving six (A) or four (B) inoculations. Patients were categorized further as no pre-existing E75-specific CTLs receiving six (C) or four (D) inoculations or with pre-existing E75-specific CTLs receiving six (E) or four (F) inoculations.

Although the dimer assay provides phenotypic evidence of clonal expansion, it does not demonstrate the functional capacity of these cells. To address both aspects, dimer and cytotoxicity assays from all patients who have completed the vaccination series and 6 months of follow-up are presented in Figure 5. As mentioned previously, all patients responded to the vaccination series with clonal expansion of E75-specific CTLs in these experiments (0.56% ± 0.15% [prevaccination] to 1.66% ± 0.27% [maximum] E75-specific CD8⁺ T cells; P < .001) (Fig 5A). To assess the functional capability of these cells, we cocultured fresh bulk PBMCs with E75 peptide and stimulated them once with autologous DCs loaded with E75. We tested the ability of these peptide-stimulated PBMCs at 2 weeks to recognize and lyse HER2/neu-expressing tumors in chromium-release assays. The pattern of specific lysis of HER2/neu-expressing cancers varied over the course of the vaccination series and among different patients; however, similar to the dimer assays, the level of specific lysis tended to peak after the third inoculation. Figure 5B demonstrates the enhanced HER2/neu-specific cytotoxicity induced by the vaccination series (7.3% ± 1.4% [prevaccination] to 21.7% ± 2.2% [maximum] specific lysis at an E/T of 20:1; P < .001). Although the patterns of phenotypic and functional responses are similar, no direct correlation could be made between peak E75-specific dimer responses and levels of tumor-specific lysis.

View larger version (5K): [in this window] [in a new window]   Fig 5. Phenotypic (dimer) and functional (cytotoxicity) immunologic responses in each vaccination-complete patient. Prevaccination (PRE-VAC) and maximum (MAX) responses are given for clonal expansion of E75-specific cytotoxic T lymphocytes (A) and functional assessment (B) of short-term cultured, peptide-stimulated peripheral blood mononuclear cells in standard chromium-release assays against a panel of HLA-A2+, HER2/neu+ tumor targets with nonspecific lysis of HLA-A2–, HER2/neu+ tumor targets subtracted.

View larger version (3K): [in this window] [in a new window]   Fig 6. Delayed-type hypersensitivity reactions in vaccinated patients. After completion of the vaccination series, 100 µg of the E75 peptide without granulocyte-macrophage colony-stimulating factor and a saline control was placed intradermally at a site remote from the vaccination site. Induration was measured in two dimensions at 48 hours using the sensitive ballpoint-pen method and compared to control. Abbreviations: mm, millimeters; NS, normal saline.

In addition, the patients who did not complete the vaccination series were included in the analysis discussed above on the basis of intention to treat. Furthermore, it must be kept in mind that because this trial was of a dose-escalation design, not all patients were optimally dosed with the vaccine. For example, one of the two vaccinated patients who had recurrences received the lowest dose of the vaccine (100 µg).

	DISCUSSION

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

 
Our study uses a simple vaccine strategy consisting of the E75 HER2/neu peptide that was mixed with GM-CSF and administered intradermally to patients with NPBC who had no evidence of disease but are at high risk for recurrence. Results demonstrate that this vaccination strategy is safe and effective at raising E75-specific immunity, and this HER2/neu-specific immunity seems to reduce the clinical recurrence rate and improve DFS in vaccinated patients with NPBC compared to prospectively observed HLA-A2^– patients with NPBC.

Other studies have investigated the E75 peptide as a vaccine. Zaks and Rosenberg⁹ vaccinated four metastatic breast, ovarian, and colorectal cancer patients with E75 and IFA and found that three of four patients developed E75-specific CTLs; however, these CTLs could not recognize HER2/neu-expressing tumors in in vitro assays. Knutson et al¹⁰ vaccinated six stage III or IV breast or ovarian cancer patients with E75 mixed with GM-CSF; peptide-specific CTLs were demonstrated in two of four evaluable patients. Murray et al¹¹ vaccinated 14 metastatic breast and ovarian cancer patients with escalating doses of the E75 peptide mixed with GM-CSF, and in eight evaluable patients, seven developed a DTH response to the peptide, and four demonstrated E75-specific CTLs that could recognize HER2/neu-expressing tumors. These small trials demonstrate that the E75 vaccine is safe and that, when mixed with an immunoadjuvant, can be used to induce an in vivo peptide-specific immune response. Furthermore, they illustrate that a variety of HER2/neu-expressing cancers can be targeted by this vaccine. However, these studies did not address the question of whether E75-specific HER2/neu immunity provides a clinical benefit, most likely because they were performed in patients with metastatic disease, as have been the majority of cancer-vaccine trials to date.

One of the surprising findings of our study was the variability in the patient populations based on HLA-A2 status alone. Our study was specifically designed to enroll patients without knowing the HLA type initially. In the patients with breast cancer, the usual prognostic factors of tumor size, grade, and hormone receptor status were all worse in the HLA-A2⁺ group. Likewise, in a separate study with prostate cancer patients, the known prognostic factors of tumor size, postoperative Gleason score, margin status, and Centers for Prostate Disease Research risk score were all worse in the HLA-A2⁺ group. The link between HLA type and prognosis has been well established in melanoma and suggested in some other forms of cancer.^31,32 However, to determine if HLA-A2 status is an independent prognostic marker will require larger studies to be performed in these specific tumor types. For this study, the HLA-A2⁺ patients with NPBC should have had a higher recurrence rate based on all proven prognostic variables, yet after 2 years, the vaccinated HLA-A2⁺ patients have a 60% lower recurrence rate and a substantially better DFS.

In our study, toxicity seemed to be primarily associated with the GM-CSF, because the minor adverse effects follow the known profile for this cytokine drug. We chose this immunoadjuvant because it has been shown in preclinical studies to be an effective in vitro³³ and in vivo adjuvant.^10,11 In addition, in a study of a peptide-based melanoma vaccine, patients were randomly assigned to different immunoadjuvants, and GM-CSF was found to be superior to IFA,¹⁶ which may partially explain the results of Zaks and Rosenberg,⁹ who used IFA.

Our research provides additional evidence that the E75/GM-CSF vaccine is effective in producing both in vivo and in vitro peptide-specific immunity. Our primary in vitro monitoring assay has become the dimer assay, which is similar to the tetramer assay, because it allows for direct detection of peptide-specific clonal expansion but seems to be more sensitive and versatile.^20,27-30 The pattern of response demonstrated in these patients does not seem to be the result of clonal exhaustion but may be typical of a CTL response to a single CTL epitope seen repeatedly. The level of clonal expansion as well as the duration of E75-specific CTLs does seem to depend on pre-existing levels of peptide-specific CTLs, which suggests prior antigen exposure that may result in the alteration of T cell antigen receptor avidities and in levels of sensitization/tolerization of these CTLs. Vaccination dosages and/or schedules may need to be altered based on pre-existing levels of antigen-specific immunity. Furthermore, periodic booster inoculations may be required to maintain peptide-specific CTLs, or the addition of helper peptides may be required to maintain long-term immunologic memory. We are currently investigating the latter approach.

With our vaccine program, we are primarily interested in pursuing simple vaccination strategies that are exported easily to the community setting, targeting common antigens expressed on many different cancers, and testing these vaccines in well-designed trials that evaluate their clinical benefit to prevent, not treat, disease. We are pursuing the peptide strategy because it is straightforward, safe, inexpensive, and easily disseminated. Although the peptide approach is restricted by HLA type, peptides are now being routinely reported that bind other common non-HLA-A2 alleles. Although HER2/neu is overexpressed in only 30% of breast cancer,¹ it is also overexpressed in many other adenocarcinomas. Although not the ultimate target antigen, we pursued HER2/neu because it represents the "proof of principle" for the concept of a "universal" cancer vaccine for epithelial-derived cancers.

Finally, most cancer vaccines to date have been tested in patients with metastatic cancer. Although therapeutic vaccines are an admirable goal, we have pursued the avenue of prevention. Just as in countless animal studies, human clinical trials have shown that the immune system can be stimulated to prevent cancer. Specifically, in a large, well-designed, randomized trial, Vermoken et al³⁴ showed that an autologous tumor cell vaccine given after surgical extirpation of colon cancer prevented recurrence, and Doehn et al³⁵ demonstrated similar results for an autologous tumor cell lysate vaccine in resected, nonmetastatic renal cell carcinoma. Although not truly analogous, the prevention of primary cervical cancer has also been shown through a vaccination strategy.^36,37 Preventative cancer vaccine studies are finally becoming more common and are being conducted in a variety of tumor types but are probably most advanced in melanoma, as reviewed by Sabel and Sondak.³⁸

In conclusion, our study demonstrates that this simple HER2/neu (E75) vaccine is safe and effective in eliciting a peptide-specific immune response in HER2/neu-expressing patients with breast cancer. Induced HER2/neu immunity seems to reduce clinical recurrence rates and may improve DFS in patients with NPBC; however, the true clinical benefit of this vaccination strategy must be determined in a prospective phase III clinical trial in HLA-A2⁺ patients.

	Authors' Disclosures of Potential Conflicts of Interest

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

 
The authors indicated no potential conflicts of interest.

	NOTES

 
Supported by the Clinical Breast Care Project, a Congressionally funded program of the Henry M. Jackson Foundation for the Advancement of Military Medicine (Rockville, MD). Supported by the United States Army Medical Research and Materiel Command and the Department of Clinical Investigation at the Walter Reed Army Medical Center.

The opinions or assertions contained herein are the private views of the authors and are not to be construed as official or reflecting the views of the Department of the Army or the Department of Defense.

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

	REFERENCES

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

 
1. Slamon DJ, Godolphin W, Jones LA, et al: Studies of the HER-2/neu proto-oncogene in human breast and ovarian cancer. Science 244:707-712, 1989[Abstract/Free Full Text]

2. Ioannides CG, Fisk B, Fan D, et al: Cytotoxic T cells isolated from ovarian malignant ascites recognize a peptide derived from the HER2/neu proto-oncogene. Cell Immunol 151:225-234, 1993[CrossRef][Medline]

3. Yoshino I, Peoples GE, Goedegebuure PS, et al: HER2/neu-derived peptide(s) are shared antigens among human non-small cell lung cancer and ovarian cancer. Cancer Res 54:3387-3390, 1994[Abstract/Free Full Text]

4. Disis ML, Smith JW, Murphy AE, et al: In vitro generation of human cytolic T-cells specific for peptide derived from the HER-2/neu protooncogene protein. Cancer Res 54:1071-1076, 1994[Abstract/Free Full Text]

5. Peoples GE, Goedegeburre PS, Smith R, et al: Breast and ovarian cancer-specific cytotoxic T lymphocytes recognize the same HER2/neu-derived peptide. Proc Natl Acad Sci USA 92:432-436, 1995[Abstract/Free Full Text]

6. Fisk B, Blevins TL, Wharton JT, et al: Identification of an immunodominant peptide of the HER-2/neu proto-oncogene recognized by ovarian tumor-specific CTL lines. J Exp Med 181:2109-2117, 1995[Abstract/Free Full Text]

7. 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 6:4192-4200, 2000[Abstract/Free Full Text]

8. 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 52:109-118, 1997[CrossRef][Medline]

9. 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 58:4902-4908, 1998[Abstract/Free Full Text]

10. Knutson KL, Schiffman K, Cheever MA, et al: Immunization of cancer patients with HER-2/neu, HLA-A2 peptide, p369-377, results in short-lived peptide-specific immunity. Clin Cancer Res 8:1014-1018, 2002[Abstract/Free Full Text]

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 8:3407-3418, 2002[Abstract/Free Full Text]

12. Brossart P, Wirths S, Stuhler G, et al: Induction of cytotoxic T-lymphocyte responses in vivo after vaccinations with peptide-pulsed dendritic cells. Blood 96:3102-3108, 2000[Abstract/Free Full Text]

13. Kono K, Takahashi A, Sugai H, et al: Dendritic cells pulsed with HER-2/neu-derived peptides can induce specific T-cell responses in patients with gastric cancer. Clin Cancer Res 8:3394-3400, 2002[Abstract/Free Full Text]

14. Disis ML, Grabstein KH, Sleath PR, et al: Generation of immunity to the HER-2/neu oncogenic protein in patients with breast and ovarian cancer using a peptide-based vaccine. Clin Cancer Res 5:1289-1297, 1999[Abstract/Free Full Text]

15. Disis ML, Gooley TA, Rinn K, et al: Generation of T-cell immunity to the HER-2/neu protein after active immunization with HER-2/neu peptide-based vaccines. J Clin Oncol 20:2624-2632, 2002[Abstract/Free Full Text]

16. Schaed SG, Klimek VM, Panageas KS, et al: T-cell responses against tyrosinase 368-376(370D) peptide in HLA*A0201+ melanoma patients: Randomized trial comparing incomplete Freund's adjuvant, granulocyte macrophage colony-stimulating factor, and QS-21 as immunological adjuvants. Clin Cancer Res 8:967-972, 2002[Abstract/Free Full Text]

17. 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 159:929-933, 1998[CrossRef][Medline]

18. 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 166:1322-1327, 2001[CrossRef][Medline]

19. Lee TD: Distribution of HLA antigens in North American Caucasians, North American Blacks and Orientals, in J Lee (ed): The HLA System. New York, NY, Springer-Verlag, 1990, p. 141

20. Woll MM, Fisher CM, Ryan GB, et al: Direct measurement of peptide-specific CD8⁺ T cells using HLA-A2: Ig dimer for monitoring the in vivo immune response to a Her2/neu vaccine in breast and prostate cancer patients. J Clin Immunol 24:449-461, 2004[CrossRef][Medline]

21. Sokol JE: Measurement of delayed skin test responses. N Engl J Med 293:501-501, 1975[Medline]

22. Jordan TJ, Sunderam G, Thomas L, et al: Tuberculin reaction size measurements by the pen method compared to traditional palpation. Chest 92:234-236, 1987[Abstract/Free Full Text]

23. Fisher B, Bryant J, Dignam JJ, et al: National Surgical Adjuvant Breast and Bowel Project: Tamoxifen, radiation therapy, or both for prevention of ipsilateral breast tumor recurrence after lumpectomy in women with invasive breast cancers of one centimeter or less. J Clin Oncol 20:4141-4149, 2002[Abstract/Free Full Text]

24. Smith RE, Good BC: Chemoprevention of breast cancer and the trials of the National Surgical Adjuvant Breast and Bowel Project and others. Endocr Relat Cancer 10:347-357, 2003[Abstract]

25. Ragaz J, Jackson SM, Le N, et al: Adjuvant radiotherapy and chemotherapy in node-positive premenopausal women with breast cancer. N Engl J Med 337:956-962, 1997[Abstract/Free Full Text]

26. Overgaard M, Jensen MB, Overgaard J, et al: Postoperative radiotherapy in high-risk postmenopausal breast-cancer patients given adjuvant tamoxifen. Lancet 353:1641-1648, 1999[CrossRef][Medline]

27. Hueman MT, Gurney JM, Woll MM, et al: Clinical trial results of a HER2/neu (E75) vaccine to prevent recurrence in high-risk prostate cancer patients. Proc Am Assoc Cancer Res 45:163, 2004 (abstr 705)

28. Woll MM, Fisher CM, Ryan GB, et al: Evaluation of HLA-A2:Ig dimer for monitoring the in vivo immune response to a HER2/neu vaccine (E75) in prostate and breast cancer patients. Proc Am Assoc Cancer Res 43:142, 2002 (abstr 710)

29. Gurney JM, Woll MM, Storrer CE, et al: In vivo monitoring of the immune response in breast cancer patients receiving a HER2/neu peptide vaccine. J Am Coll Surg 187:S81-S82, 2003

30. Storrer CE, Hueman MT, Gurney JM, et al: Comparison of HLA-A2 dimer and tetramer molecules for the immunological monitoring of breast and prostate cancer vaccine clinical trials. Clin Invest Med 27:211D, 2004 (abstr 53.315)

31. Lee JE: Factors associated with melanoma incidence and prognosis. Semin Surg Oncol 12:379-385, 1996[CrossRef][Medline]

32. Lee JE, Lowy AM, Thompson WA, et al: Association of gastric adenocarcinoma with the HLA class II gene DQB10301. Gastroenterology 111:426-432, 1996[CrossRef][Medline]

33. Disis ML, Bernhard H, Shiota FM, et al: Granulocyte-macrophage colony-stimulating factor: An effective adjuvant for protein and peptide-based vaccines. Blood 88:202-210, 1996[Abstract/Free Full Text]

34. Vermoken JB, Claessen AME, van Tinteren H, et al: Active specific immunotherapy for stage II and III human colon cancer: A randomized trial. Lancet 353:345-350, 1999[CrossRef][Medline]

35. Doehn Ch, Richter A, Lehmacher W, et al: Adjuvant autologous tumour cell-lysate vaccine versus no adjuvant treatment in patients with M0 renal cell carcinoma after radical nephrectomy: 3-year interim analysis of a German multicentre phase-III trial. Folia Biol (Praha) 49:69-73, 2003

36. Koutsky LA, Ault KA, Wheeler CM, et al: A controlled trial of a human papillomavirus type 16 vaccine. N Engl J Med 347:1645-1651, 2002[Abstract/Free Full Text]

37. Jansen KU, Shaw AR: Human papillomavirus vaccines and prevention of cervical cancer. Annu Rev Med 55:319-331, 2004[CrossRef][Medline]

38. Sabel MS, Sondak VK: Tumor vaccines: A role in preventing recurrence in melanoma? Am J Clin Dermatol 3:609-616, 2002[CrossRef][Medline]

Submitted March 4, 2004; accepted June 1, 2005.

CiteULike    Complore    Connotea    Del.icio.us    Digg    Facebook    Reddit    Technorati    Twitter    What's this?

Related Editorial

• Cancer Vaccines: The Role of Tumor Burden in Tipping the Scale Toward Vaccine Efficacy
  Lupe G. Salazar and Mary L. Disis
  JCO 2005 23: 7397-7398 [Full Text]

This article has been cited by other articles:

				P. T.P. Kaumaya, K. C. Foy, J. Garrett, S. V. Rawale, D. Vicari, J. M. Thurmond, T. Lamb, A. Mani, Y. Kane, C. R. Balint, et al. Phase I Active Immunotherapy With Combination of Two Chimeric, Human Epidermal Growth Factor Receptor 2, B-Cell Epitopes Fused to a Promiscuous T-Cell Epitope in Patients With Metastatic and/or Recurrent Solid Tumors J. Clin. Oncol., November 1, 2009; 27(31): 5270 - 5277. [Abstract] [Full Text] [PDF]

				Y. Zhao, Q. J. Wang, S. Yang, J. N. Kochenderfer, Z. Zheng, X. Zhong, M. Sadelain, Z. Eshhar, S. A. Rosenberg, and R. A. Morgan A Herceptin-Based Chimeric Antigen Receptor with Modified Signaling Domains Leads to Enhanced Survival of Transduced T Lymphocytes and Antitumor Activity J. Immunol., November 1, 2009; 183(9): 5563 - 5574. [Abstract] [Full Text] [PDF]

				S. MATSUEDA, H. GAO, C. L. EFFERSON, N. TSUDA, S. ISHIYAMA, Y. LI, M. G. IOANNIDES, B. FISK, G. E. PEOPLES, and C. G. IOANNIDES N-Terminally LRMK-linked HER-2 peptides, AE-37 [p776(774-788)] and AE-47 [Ava-F7(776-788)], Aid Differentiation of E75-TCR+CD8+ Cells to Perforin-positive Cells Anticancer Res, July 1, 2009; 29(7): 2427 - 2435. [Abstract] [Full Text] [PDF]

				L. C. Benavides, J. D. Gates, M. G. Carmichael, R. Patel, J. P. Holmes, M. T. Hueman, E. A. Mittendorf, D. Craig, A. Stojadinovic, S. Ponniah, et al. The Impact of HER2/neu Expression Level on Response to the E75 Vaccine: From U.S. Military Cancer Institute Clinical Trials Group Study I-01 and I-02 Clin. Cancer Res., April 15, 2009; 15(8): 2895 - 2904. [Abstract] [Full Text] [PDF]

				L. Aurisicchio, D. Peruzzi, A. Conforti, S. Dharmapuri, A. Biondo, S. Giampaoli, A. Fridman, A. Bagchi, C. T. Winkelmann, R. Gibson, et al. Treatment of Mammary Carcinomas in HER-2 Transgenic Mice through Combination of Genetic Vaccine and an Agonist of Toll-Like Receptor 9 Clin. Cancer Res., March 1, 2009; 15(5): 1575 - 1584. [Abstract] [Full Text] [PDF]

				K. H. Park, E. Gad, V. Goodell, Y. Dang, T. Wild, D. Higgins, P. Fintak, J. Childs, C. dela Rosa, and M. L. Disis Insulin-like Growth Factor-Binding Protein-2 Is a Target for the Immunomodulation of Breast Cancer Cancer Res., October 15, 2008; 68(20): 8400 - 8409. [Abstract] [Full Text] [PDF]

				M. Barve, J. Bender, N. Senzer, C. Cunningham, F. A. Greco, D. McCune, R. Steis, H. Khong, D. Richards, J. Stephenson, et al. Induction of Immune Responses and Clinical Efficacy in a Phase II Trial of IDM-2101, a 10-Epitope Cytotoxic T-Lymphocyte Vaccine, in Metastatic Non-Small-Cell Lung Cancer J. Clin. Oncol., September 20, 2008; 26(27): 4418 - 4425. [Abstract] [Full Text] [PDF]

				J. P. Holmes, L. C. Benavides, J. D. Gates, M. G. Carmichael, M. T. Hueman, E. A. Mittendorf, J. L. Murray, A. Amin, D. Craig, E. von Hofe, et al. Results of the First Phase I Clinical Trial of the Novel Ii-Key Hybrid Preventive HER-2/neu Peptide (AE37) Vaccine J. Clin. Oncol., July 10, 2008; 26(20): 3426 - 3433. [Abstract] [Full Text] [PDF]

				A. D. Gritzapis, I. F. Voutsas, E. Lekka, N. Tsavaris, I. Missitzis, P. Sotiropoulou, S. Perez, M. Papamichail, and C. N. Baxevanis Identification of a Novel Immunogenic HLA-A*0201-Binding Epitope of HER-2/neu with Potent Antitumor Properties J. Immunol., July 1, 2008; 181(1): 146 - 154. [Abstract] [Full Text] [PDF]

				H. Conrad, K. Gebhard, H. Kronig, J. Neudorfer, D. H. Busch, C. Peschel, and H. Bernhard CTLs Directed against HER2 Specifically Cross-React with HER3 and HER4 J. Immunol., June 15, 2008; 180(12): 8135 - 8145. [Abstract] [Full Text] [PDF]

				M. Fasso, R. Waitz, Y. Hou, T. Rim, N. M. Greenberg, N. Shastri, L. Fong, and J. P. Allison SPAS-1 (stimulator of prostatic adenocarcinoma-specific T cells)/SH3GLB2: A prostate tumor antigen identified by CTLA-4 blockade PNAS, March 4, 2008; 105(9): 3509 - 3514. [Abstract] [Full Text] [PDF]

				G. E. Peoples, J. P. Holmes, M. T. Hueman, E. A. Mittendorf, A. Amin, S. Khoo, Z. A. Dehqanzada, J. M. Gurney, M. M. Woll, G. B. Ryan, et al. Combined Clinical Trial Results of a HER2/neu (E75) Vaccine for the Prevention of Recurrence in High-Risk Breast Cancer Patients: U.S. Military Cancer Institute Clinical Trials Group Study I-01 and I-02 Clin. Cancer Res., February 1, 2008; 14(3): 797 - 803. [Abstract] [Full Text] [PDF]

				S. M. Domchek, A. Recio, R. Mick, C. E. Clark, E. L. Carpenter, K. R. Fox, A. DeMichele, L. M. Schuchter, M. S. Leibowitz, M. H. Wexler, et al. Telomerase-Specific T-Cell Immunity in Breast Cancer: Effect of Vaccination on Tumor Immunosurveillance Cancer Res., November 1, 2007; 67(21): 10546 - 10555. [Abstract] [Full Text] [PDF]

				N. Tsuda, D. Z. Chang, T. Mine, C. Efferson, A. Garcia-Sastre, X. Wang, S. Ferrone, and C. G. Ioannides Taxol Increases the Amount and T Cell Activating Ability of Self-Immune Stimulatory Multimolecular Complexes Found in Ovarian Cancer Cells Cancer Res., September 1, 2007; 67(17): 8378 - 8387. [Abstract] [Full Text] [PDF]

				J. W. Park, M. E. Melisko, L. J. Esserman, L. A. Jones, J. B. Wollan, and R. Sims Treatment With Autologous Antigen-Presenting Cells Activated With the HER-2 Based Antigen Lapuleucel-T: Results of a Phase I Study in Immunologic and Clinical Activity in HER-2 Overexpressing Breast Cancer J. Clin. Oncol., August 20, 2007; 25(24): 3680 - 3687. [Abstract] [Full Text] [PDF]

				J. T. Garrett, S. Rawale, S. D. Allen, G. Phillips, G. Forni, J. C. Morris, and P. T. P. Kaumaya Novel Engineered Trastuzumab Conformational Epitopes Demonstrate In Vitro and In Vivo Antitumor Properties against HER-2/neu J. Immunol., June 1, 2007; 178(11): 7120 - 7131. [Abstract] [Full Text] [PDF]

				Y. Dang, K. L. Knutson, V. Goodell, C. dela Rosa, L. G. Salazar, D. Higgins, J. Childs, and M. L. Disis Tumor Antigen-Specific T-Cell Expansion Is Greatly Facilitated by In vivo Priming Clin. Cancer Res., March 15, 2007; 13(6): 1883 - 1891. [Abstract] [Full Text] [PDF]

				B. J. Czerniecki, G. K. Koski, U. Koldovsky, S. Xu, P. A. Cohen, R. Mick, H. Nisenbaum, T. Pasha, M. Xu, K. R. Fox, et al. Targeting HER-2/neu in Early Breast Cancer Development Using Dendritic Cells with Staged Interleukin-12 Burst Secretion Cancer Res., February 15, 2007; 67(4): 1842 - 1852. [Abstract] [Full Text] [PDF]

				S. Kitano, S. Kageyama, Y. Nagata, Y. Miyahara, A. Hiasa, H. Naota, S. Okumura, H. Imai, T. Shiraishi, M. Masuya, et al. HER2-Specific T-Cell Immune Responses in Patients Vaccinated with Truncated HER2 Protein Complexed with Nanogels of Cholesteryl Pullulan Clin. Cancer Res., December 15, 2006; 12(24): 7397 - 7405. [Abstract] [Full Text] [PDF]

				E. Ambrosino, M. Spadaro, M. Iezzi, C. Curcio, G. Forni, P. Musiani, W.-Z. Wei, and F. Cavallo Immunosurveillance of erbb2 carcinogenesis in transgenic mice is concealed by a dominant regulatory T-cell self-tolerance. Cancer Res., August 1, 2006; 66(15): 7734 - 7740. [Abstract] [Full Text] [PDF]

				H. Norell, M. Carlsten, T. Ohlum, K.-J. Malmberg, G. Masucci, K. Schedvins, W. Altermann, D. Handke, D. Atkins, B. Seliger, et al. Frequent Loss of HLA-A2 Expression in Metastasizing Ovarian Carcinomas Associated with Genomic Haplotype Loss and HLA-A2-Restricted HER-2/neu-Specific Immunity. Cancer Res., June 15, 2006; 66(12): 6387 - 6394. [Abstract] [Full Text] [PDF]

				Z. A. Dehqanzada, C. E. Storrer, M. T. Hueman, R. J. Foley, K. A. Harris, Y. H. Jama, T.-C. Kao, C. D. Shriver, S. Ponniah, and G. E. Peoples Correlations between Serum Monocyte Chemotactic Protein-1 Levels, Clinical Prognostic Factors, and HER-2/neu Vaccine-Related Immunity in Breast Cancer Patients Clin. Cancer Res., January 15, 2006; 12(2): 478 - 486. [Abstract] [Full Text] [PDF]

				M. L. Disis and H. K. Lyerly Global Role of the Immune System in Identifying Cancer Initiation and Limiting Disease Progression J. Clin. Oncol., December 10, 2005; 23(35): 8923 - 8925. [Full Text] [PDF]

This Article Abstract Full Text (PDF) Purchase Article View Shopping Cart Alert me when this article is cited Alert me if a correction is posted Services Email this article to a colleague Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Save to my personal folders Download to citation manager Rights & Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Peoples, G. E. Articles by Ponniah, S. Search for Related Content PubMed PubMed Citation Articles by Peoples, G. E. Articles by Ponniah, S. Related Articles Related Editorial Social Bookmarking                            What's this?