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Originally published as JCO Early Release 10.1200/JCO.2005.03.047 on September 12 2005 © 2005 American Society of Clinical Oncology. Clinical Trial Results of a HER2/neu (E75) Vaccine to Prevent Recurrence in High-Risk Breast Cancer PatientsFrom 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
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.
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 al14,15 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.
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
Vaccination Series
Toxicity The National Institutes of Health Common Toxicity Criteria (version 2, March 23, 1998) definitions of adverse events were applied. Both local toxicity at the injection sites as well as systemic toxicity were evaluated in all patients for all inoculations. By design, progression to the second stage occurred only if no significant toxicity occurred in the first stage.
Peripheral Blood Mononuclear Cell Isolation and Cultures
HLA-A2:Immunoglobulin Dimer Assay
Cytotoxicity Assays
Delayed-Type Hypersensitivity
Clinical Recurrences
Statistical Analysis
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
Vaccine and Vaccination Series The E75 peptide was mixed with GM-CSF (250 µg) and injected intradermally in the same extremity on a monthly basis. Table 1 provides the two-stage safety design and dose-escalation/schedule-reduction scheme. Patients were enrolled sequentially; however, if a patient failed to complete the series, a replacement patient was given the same dose and schedule until each group was complete.
Toxicity
Immunologic Response Blood was drawn from all patients before each vaccination and 1 (postvaccination) and 6 months (long-term) after completion of the series (Fig 1). Phenotypic and functional assays were performed on these samples, and they have been shown to correlate previously.20,27-29 E75-specific CTLs were assessed in fresh ex vivo PBMCs by the dimer assay, which has been shown to be more sensitive than the tetramer assay.30 A recurring pattern of clonal expansion has been observed among most patients with a growing percentage of CD8+E75-specific cells with successive vaccinations that peak during the series and then contract and plateau by the completion of the series.20 The peak clonal expansion most commonly occurs after the third inoculation, but this has varied among individual patients; therefore, we have reported the prevaccination, maximum, and postvaccination (1 month after completion) levels of CD8+E75-specific cells (Fig 2).
Figure 3 demonstrates the E75 dimer responses for the escalating doses of the vaccine from the first stage of the trial. There seems to be a correlation between dose and the induction of maximum E75-specific CTLs with the exception of the two patients receiving 100 µg, who had higher maximum responses than expected, which could be in part related to their high levels of pre-existing immunity (defined as > 0.3% CD8+E75-specific cells). The persistence of E75-specific CTLs seems to be related to the prevaccination levels of these cells. In the dose/schedule-reduction group (500.4), comparable E75-specific CTL induction was seen despite the reduced number of inoculations (Fig 3). Similarly, the persistence of peptide-specific CTLs were mostly seen in those patients with pre-existing levels.
To assess further the optimal vaccination schedule, the second-stage patients were vaccinated. Cumulative dimer responses from all doses are shown in Figure 4. When taken collectively, there was a statistically significant increase in CD8+E75-specific cells during (maximum, 2.20% ± 0.22%; P < .001) and after (postvaccination, 1.24% ± 0.29%; P < .02) vaccination compared to prevaccination levels (0.65% ± 0.15%). There was a strikingly similar clonal expansion response pattern between the six-inoculation group (Fig 4A) and the four-inoculation group (Fig 4B). The statistical difference persisted in these subset groups for the maximum levels of E75-specific CTLs (P < .002 and P < .001, respectively) but not for the postvaccination levels (P < .096 and P < .083, respectively) compared to the prevaccination levels. This finding suggests that the peaking and contracting pattern is not a result of overvaccination (clonal exhaustion), because the pattern remained the same even for the reduced schedule as well as among all dose groups. The number of E75-specific CTLs declines at 6 months but remains easily detectable, especially among patients without prevaccination levels of E75-specific cells (Figs 4C and 4D) (0.67% ± 0.21% [long-term] to 0.10% ± 0.04% [prevaccination]; P < .04) while returning to baseline levels for those patients with pre-existing E75 immunity (Figs 4E and 4F) in both the six-inoculation (Figs 4C and 4E) and four-inoculation (Figs 4D and 4F) groups. Paradoxically, the four-inoculation series seems to be better for those patients without pre-existing E75 immunity, whereas the six-inoculation series seems to be better for those patients with pre-existing immunity, suggesting an element of tolerance in these patients.
Overall, pre-existing E75 immunity (> 0.3% E75-specific CD8+ T cells) has been found in 45% of the patients. Long-term postvaccination persistence of E75-specific CTLs has been seen in 52% of the patients at 6 months, suggesting the potential need for periodic booster inoculations. There has been no correlation observed between the level of HER2/neu expression on the patients' tumors and their prevaccination, maximum, postvaccination, or long-term dimer response (data not shown). 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.
DTH Response A more direct measure of the vaccine's effectiveness in vivo is the DTH reaction. One month after series completion, 100 µg of E75 without GM-CSF was injected at a distant site with a saline control. The mean diameter of induration in the vaccinated NPBC patients was 33 mm (range, 17 to 53 mm) to E75 as compared to the saline controls of 7 mm (range, 0 to 17 mm) (P < .01) (Fig 6). DTH reactions did not significantly correlate with the dose-escalation/reduction scheme; however, there was a trend toward correlation between the postvaccination dimer response and the DTH reaction (data not shown).
Clinical Response Despite the greater preponderance of poor prognostic factors in the group of patients with NPBC who were vaccinated compared to their controls (Table 2), the only two deaths have occurred in the control group. Therefore, the overall survival is 100% for the vaccinated group compared to 93% for the control group. More importantly, only two of the 24 vaccinated NPBC patients (8%) have had recurrences, whereas six of the 29 HLA-A2– control patients (21%) have had recurrences to date at a median follow-up of 22 months (P < .19). The disease-free survival (DFS) rates in the vaccinated and control groups are 85.7% and 59.5%, respectively (Table 4).
The two patients with NPBC who were vaccinated and recurrences had the smallest DTH reactions, suggesting that they did not respond as well to the vaccine as other members of the vaccinated group. However, despite the weaker response, their median time to recurrence was substantially prolonged at 11 months, compared with 8 months for the patients in the control group who had recurrences. 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).
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.
The authors indicated no potential conflicts of interest.
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.
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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 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
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
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
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
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
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
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
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 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
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 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 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 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 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. Related Editorial
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ABSTRACT
INTRODUCTION
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. 
