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Introduction: Immunotherapy, including vaccines targeting the human EGFR2 (HER-2/neu) protein, is an active area of investigation in combatting breast cancer. Several vaccines are currently undergoing clinical trials, most of which are CD8+ T-cell-eliciting vaccines. AE37 is a promising primarily CD4+ T-cell-eliciting HER-2/neu breast cancer vaccine currently in clinical trials. Areas covered: This article reviews preclinical investigations as well as findings from completed and ongoing Phase I and Phase II clinical trials of the AE37 vaccine. Expert opinion: Clinical trials have shown the AE37 vaccine to be safe and capable of generating peptide-specific, durable immune responses. This has been shown in patients with any level of HER-2/neu expression. Early clinical findings suggest there may be benefit to AE37 vaccination in preventing breast cancer recurrence.

We have performed multiple adjuvant clinical trials using immunogenic peptides from the HER2/neu protein (AE37/E75/GP2) plus (GM-CSF) given intradermally to breast cancer patients. Four trials were performed with similar dose-escalation design with increasing doses of peptide (AE37/E75/GP2) and varying amounts of GM-CSF. Dose reductions (DRs) were made for significant local and/or systemic toxicity by decreasing GM-CSF for subsequent inoculations. Ex vivo and in vivo immunologic responses were used to compare groups. Of 132 patients, 39 required DR (30 for robust local reactions [DR-L]). DR patients, particularly DR-L, had greater immune responses both ex vivo and in vivo. Postvaccine delayed-type hypersensitivity in DR-L patients compared with all others was larger for E75 (p = 0.001), AE37 (p = 0.077) and GP2 (p = 0.076). All three peptide vaccines were safe and well-tolerated. These findings have led to a clinically relevant optimal vaccine dosing strategy, which may be applicable to other peptide-based cancer vaccines.

Background 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 ≥ 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.

PurposeHER-2/neu is overexpressed in breast cancer and is the source of immunogenic peptides. CD4+T-helper peptides for HER-2/neu are being evaluated in vaccine trials. The addition of Ii-Key, a four–amino-acid LRMK modification, increases vaccine potency when compared with unmodified class II epitopes. We present the results of the first human phase I trial of the Ii-Key hybrid HER-2/neu peptide (AE37) vaccine in disease-free, node-negative breast cancer patients.Patients and MethodsThe dose escalation trial included five dose groups, to determine safety and optimal dose of the hybrid peptide (100 μg, 500 μg, 1,000 μg) and granulocyte-macrophage colony-stimulating factor (GM-CSF; range, 0 to 250 μg). In the event of significant local toxicity, GM-CSF (or peptide in absence of GM-CSF) was reduced by 50%. Immunologic response was monitored by delayed-type hypersensitivity and [3H]thymidine proliferative assays for both the hybrid AE37 (LRMK-positive HER-2/neu:776-790) and AE36 (unmodified HER-2/neu:776-790).ResultsAll 15 patients completed the trial with no grade 3 to 5 toxicities. Dose reductions occurred in 47% of patients. In the second group (peptide, 500 μg; GM-CSF, 250 μg), all patients required dose reductions, prompting peptide-only inoculations in the third group. The vaccine induced dose-dependent immunologic responses in vitro and in vivo to AE37, as well as AE36.ConclusionThe hybrid AE37 vaccine seems safe and well tolerated with minimal toxicity if properly dosed. AE37 is capable of eliciting HER-2/neu–specific immune responses, even without the use of an adjuvant. This trial represents the first human experience with the Ii-Key modification, and to our knowledge, AE37 is the first peptide vaccine to show potency in the absence of an immunoadjuvant.

E75, a HER2/neu immunogenic peptide, is expressed in breast cancer (BCa). We have performed clinical trials of E75 + GM-CSF vaccine in disease-free, node-positive and node-negative BCa patients at high recurrence risk and recurrences were noted in both control and vaccine groups.Among the 186 BCa patients enrolled, 177 completed the study. Patients were HLA typed; the HLA-A2(+)/A3(+) patients were vaccinated; HLA-A2(-)/A3(-) patients were followed as controls. Standard clinicopathological factors, immunologic response to the vaccine, and recurrences were collected and assessed.The control group recurrence rate was 14.8 and 8.3% in the vaccinated group (P = 0.17). Comparing the 8 vaccinated recurrences (V-R) to the 88 vaccinated nonrecurrent patients (V-NR), the V-R group had higher nodal stage (or = N2: 75 vs. 5%, P = 0.0001) and higher grade tumors (%grade 3: 88 vs. 31%, P = 0.003). The V-R group did not fail to respond immunologically as noted by equivalent dimer responses and post-DTH responses. Compared to control recurrent patients (C-R), V-R patients trended toward higher-grade tumors and hormone-receptor negativity. C-R patients had 50% bone-only recurrences, compared to V-R patients with no bone-only recurrences (P = 0.05). Lastly, V-R mortality rate was 12.5% compared with 41.7% for the C-R group (P = 0.3).The vaccinated patients who recurred had more aggressive disease compared to V-NR patients. V-R patients had no difference in immune response to the vaccine either in vitro or in vivo. V-R patients, when compared to C-R patients, trended towards more aggressive disease, decreased recurrence rates, decreased mortality, and no bone-only recurrences.

Background: Primary hyperaparathyroidism (pHPT) is often accompanied by underlying thyroid pathology that can confound preoperative parathyroid localization studies and complicate intra-operative decision making. The aim of this study was to examine the utility of preoperative thyroid ultrasonography (US) in patients prior to undergoing parathyroidectomy for pHPT. Methods: An Institutional Review Board approved prospective study was undertaken from January 2005 through July 2008. All patients with pHPT meeting inclusion criteria (n=94) underwent preoperative thyroid ultrasound in addition to standard 99m Tc-sestamibi scintigraphy for parathyroid localization. Demographics, operative management and final pathology were examined in all cases. Results: Fifty-four of the 94 patients (57%) were noted to have a thyroid nodule on preoperative US, of which 30 (56%) underwent further examination with fine needle aspiration biopsy. Alteration of the operative plan attributable to underlying thyroid pathology occurred in 16 patients (17%), with patients undergoing either total thyroidectomy (n=9) or thyroid lobectomy (n=7). Thyroid cancer was noted in 33% of patients undergoing thyroid resection, and 6% of all patients with HPT. Conclusions: The routine utilization of preoperative thyroid ultrasound in patients prior to undergoing parathyroid surgery for pHPT is indicated. The added information from this non-invasive modality facilitates timely management of co-incidental, and sometimes malignant, thyroid pathology.

The authors conducted exploratory phase 1-2 clinical trials vaccinating breast cancer patients with E75, a human leukocyte antigen (HLA) A2/A3-restricted HER-2/neu (HER2) peptide, and granulocyte-macrophage colony-stimulating factor. The vaccine is given as adjuvant therapy to prevent disease recurrence. They previously reported that the vaccine is safe and effective in stimulating expansion of E75-specific cytotoxic T cells. Here, they report 24-month landmark analyses of disease-free survival (DFS).These dose escalation/schedule optimization trials enrolled lymph node-positive and high-risk lymph node-negative patients with HER2 (immunohistochemistry [IHC] 1-3(+) ) expressing tumors. HLA-A2/A3(+) patients were vaccinated; others were followed prospectively as controls for recurrence. DFS was analyzed by Kaplan-Meier curves; groups were compared using log-rank tests.Of 195 enrolled patients, 182 were evaluable: 106 (58.2%) in the vaccinated group and 76 (41.8%) in the control group. The 24-month landmark analysis DFS was 94.3% in the vaccinated group and 86.8% in the control group (P = .08). Importantly, because of trial design, 65% of patients received a lower than optimal vaccine dose. In subset analyses, patients who benefited most from vaccination (vaccinated group vs control group) had lymph node-positive (DFS, 90.2% vs 79.1%; P = .13), HER2 IHC 1+-2+ (DFS, 94.0% vs 79.4%; P = .04), or grade 1 or 2 (DFS, 98.4% vs 86.0%; P = .01) tumors and were optimally dosed (DFS, 97.3% vs 86.8%; P = .08). A booster program has been initiated; no patients receiving booster inoculations have recurred.The E75 vaccine has clinical efficacy that is more prominent in certain patients. A phase 3 trial enrolling lymph node-positive patients with HER2 low-expressing tumors is warranted.

BACKGROUND: The authors are conducting clinical trials of the HER-2/neu E75-peptide vaccine in clinically disease-free breast cancer (BC) patients. Their phase 1-2 trials revealed that the E75 + granulocyte-macrophage colony-stimulating factor (GM-CSF) vaccine is safe and effective in stimulating clonal expansion of E75-specific CD8+ T cells. They assessed the need for and response to a booster after completion of primary vaccination series. METHODS: BC patients enrolled in the E75 vaccine trials who were ≥6 months from completion of their primary vaccination series were offered boosters with E75 + GM-CSF. Patients were monitored for toxicity. E75-specific CD8+ T cells were quantified using the human leukocyte antigen-A2:immunoglobulin G dimer before and after boosting. RESULTS: Fifty-three patients received the vaccine booster. Median time from primary vaccination series was 9 months (range, 6-35 months), and median residual E75-specific immunity was 0.70% (range, 0-3.49%) CD8+ lymphocytes. Elevated residual immunity (ERI) (CD8+ E75-specific T cells >0.5%) was seen in 94.4% of patients at 6 months from primary vaccination series versus 48% of patients at >6 months (P = .002). The booster was well tolerated, with only grade 1 and 2 toxicity observed. Local reactions were more robust in patients receiving the booster at 6 months from primary vaccination series compared with those at >6 months (99.4 ± 6.1 mm vs 81.8 ± 4.1 mm, P = .01). In patients lacking ERI, 85% had increased ERI after vaccination (P = .0014). CONCLUSIONS: The HER-2/neu E75 peptide vaccine E75 stimulates specific immunity in disease-free BC patients. However, immunity wanes with time. A vaccine booster is safe and effective in stimulating E75-specific immunity in those patients without ERI. These results suggest that the booster may be most effective at 6 months after completion of the primary vaccination series. Cancer 2011. Published 2010 by the American Cancer Society.

Regulatory T cells (T Reg ), CD4 + CD25 + FOXP3 + , are implicated in suppressing tumor immune responses. We analyzed peripheral blood lymphocytes (PBL) from breast cancer patients receiving a modified HLA class II HER2/ neu peptide (AE37) vaccine for T Reg cells and correlated their levels with vaccine-specific immune responses. The mean CD4 + CD25 + FOXP3 + T Reg cells decreased in patients with vaccination with no significant difference in serum TGF-β levels. IFN-γ ELISPOT and DTH increased after vaccination with a good correlation between T Reg cell reduction and size of DTH to AE37. The T Reg cell reduction and associated immune response suggest that AE37 may be clinically useful.

BACKGROUND: HER-2/neu, overexpressed in breast cancer, is a source of immunogenic peptides that include GP2 and E75. Phase 2 testing of E75 as an adjuvant vaccine has suggested a clinical benefit. GP2, derived from the transmembrane portion of HER-2/neu, has differing binding characteristics and may be more immunogenic than E75. Results of the first phase 1 trial of GP2 peptide vaccine are presented. METHODS: Disease-free, lymph node-negative, human leukocyte antigen (HLA)-A2+ breast cancer patients were enrolled. This dose escalation trial included 4 groups to determine safety and optimal GP2 peptide/granulocyte-macrophage colony-stimulating factor (GM-CSF) dose. Toxicities were monitored. Immunologic response was assessed ex vivo via the HLA-A2:immunoglobulin dimer assay to detect GP2-specific CD8+ T cells (and E75-specific CD8+ T cells to assess epitope spreading) and in vivo via delayed type hypersensitivity (DTH) reaction (medians/ranges). RESULTS: Eighteen patients were enrolled. All toxicities were grade ≤2. Eight (88.9%) of 9 patients in the first 3 dose groups required GM-CSF dose reductions for local reactions ≥100 mm or grade ≥2 systemic toxicity. GM-CSF dose was reduced to 125 μg for the final dose group. All patients responded immunologically ex vivo (GP2-specific CD8+ T cells from prevaccination to maximum, 0.4% [0.0%-2.0%] to 1.1% [0.4%-3.6%], P < .001) and in vivo (GP2 pre- to postvaccination DTH, 0 mm [0.0-19.5 mm] to 27.5 mm [0.0-114.5 mm, P < .001). E75-specific CD8+ T cells also increased in response to GP2 from prevaccination to maximum (0.8% [0.0%-2.41%] to 1.6% [0.86%-3.72%], P < .001). CONCLUSIONS: The GP2 peptide vaccine appears safe and well tolerated with minimal local/systemic toxicity. GP2 elicited HER-2/neu–specific immune responses, including epitope spreading, in high-risk, lymph node-negative breast cancer patients. These findings support further investigation of the GP2 vaccine for the prevention of breast cancer recurrence. Cancer 2010. © 2010 American Cancer Society.

Background We have treated disease-free breast cancer patients with an HER2/ neu -derived peptide, E75, as an adjuvant vaccine. E75 was originally described as HLA-A2−restricted and has been previously tested in this population. Based on computer modeling, E75 is predicted to bind to HLA-A3, and preclinical data support this. We conducted a clinical trial of E75 in HLA-A3 + , A2 − (A3) patients. Study Design Disease-free breast cancer patients were enrolled after standard therapy in phase I/II trials. A3 patients were enrolled in parallel with A2 patients and vaccinated with E75 and granulocyte-macrophage colony-stimulating factor immunoadjuvant. A2 − , A3 − patients were followed as controls. Toxicities were graded. Immunologic responses were assessed by delayed-type hypersensitivity reactions and E75-specific interferon-γ enzyme-linked immunosorbent spot assay. Clinical recurrences were documented. Results Thirteen A3 patients completed the vaccine schedule. Clinicopathologic features were similar between A3, A2, and control patients, except for more HER2/ neu -overexpressing tumors in the A2 group and more estrogen-receptor/progesterone-receptor−negative tumors in A2 and A3 groups. Toxicity profiles and postvaccination delayed-type hypersensitivity were similar in A3 and A2 patients. Enzyme-linked immunosorbent spot assay results varied, but A3 patients' median spots increased pre- to postvaccination (p = 0.2). Recurrences were lower in the A3 group (7.7%) at 30-month median follow-up compared with published recurrence in A2-vaccinated (8.3%) and control groups (14.8%) at 26-month median follow-up. Conclusions HLA restriction limits potential use of peptide-based cancer vaccines. This trial demonstrates that HLA-A3 patients respond similarly to E75 vaccination as HLA-A2 patients, suggesting the potential use of the E75 vaccine in up to 76% of the population.

Background Coexisting thyroid nodules are the most common cause of false positive localization by radioscintigraphy in the preoperative evaluation for MIPS in patients with primary hyperparathyroidism (pHPT). This false positive finding can prompt full neck exploration in the setting of an unanticipated and incompletely evaluated thyroid nodule. Therefore, we are studying prospectively the routine use of preoperative thyroid US in patients with pHPT to determine the prevalence of concurrent thyroid disease and to assess how frequently this added information could alter the surgical plan. Materials and Methods Twenty-four patients with biochemically confirmed pHPT were evaluated with thyroid US after localizing 99mTc-sestamibi scintigraphy prior to parathyroid operation. Results Of the 24 patients, 38% (n = 9) had their operations altered from a planned MIPS or four-gland exploration due to coexisting thyroid nodule(s). Of these, 33% (n = 3) had underlying thyroid malignancy (all papillary thyroid cancer) requiring thyroidectomy in addition to parathyroidectomy. All but one patient had parathyroid adenoma as the cause of pHPT. Conclusion The routine use of preoperative thyroid US in patients with pHPT undergoing parathyroid surgery may aid in the timely diagnosis and treatment of coexisting thyroid disease. This added information secured before operation may avoid difficult intraoperative decision dilemmas and prevent the increased morbidity associated with a second neck exploration. A large scale prospective study is on-going.

Purpose: HER2/neu, a source of immunogenic peptides, is expressed in >75% of breast cancer patients. We have conducted clinical trials with the HER2/neu E75 peptide vaccine in breast cancer patients with varying levels of HER2/neu expression. Vaccine response based on HER2/neu expression level was analyzed.Experimental Design: Patients were stratified by HER2/neu expression. Low expressors (n = 100) were defined as HER2/neu immunohistochemistry (IHC) 1+ to 2+ or fluorescence in situ hybridization < 2.0. Overexpressors (n = 51) were defined as IHC 3+ or fluorescence in situ hybridization ≥ 2.0. Additional analyses were done stratifying by IHC status (0-3+). Standard clinocopathlogic factors, immunologic response (in vivo delayed-type hypersensitivity reactions; ex vivo human leukocyte antigen A2:immunoglobulin G dimer assay), and clinical responses (recurrence; mortality) were assessed.Results: Low-expressor (control, 44; vaccinated, 56) versus overexpressor patients (control, 22; vaccinated, 29) were assessed. Low expressors, overexpressors, and most IHC-status vaccinated groups responded immunologically. Vaccinated low-expressor patients had larger maximum immunologic responses compared with overexpressor patients (P = 0.04), and vaccinated IHC 1+ patients had increased long-term immune response (P = 0.08). More importantly, compared with controls, low-expressor patients had a mortality reduction (P = 0.08). The largest decrease in mortality was seen in IHC 1+ patients (P = 0.05). In addition, a subset of overexpressor patients (n = 7) received trastuzumab before vaccination, and this combination seems safe and immunologically beneficial.Conclusions: Most patients with various levels of HER2/neu expression responded immunologically and seemed to benefit from vaccination. The low expressors, specifically IHC 1+ patients, had more robust immunologic responses and may derive the greatest clinical benefit from the E75 vaccine.

BACKGROUND. E75, a HER-2/neu-derived peptide, was administered as a preventive vaccine with granulocyte-macrophage–colony-stimulating factor (GM-CSF) in disease-free lymph node-positive (NP) and lymph node-negative (NN) breast cancer (BCa) patients. The optimal biologic dose (OBD) was determined based on toxicity and immunologic response. METHODS. Patients were vaccinated over 6 months (3, 4, or 6 times) with different doses of E75 plus GM-CSF. Toxicities were graded per National Cancer Institute Common Terminology Criteria. GM-CSF was reduced for significant toxicity. Immunologic response was measured by delayed type hypersensitivity test (DTH), and E75-specific CD8+ T-cells were quantified with human leukocyte antigen-A2:immunoglobulin G dimer and flow cytometry. RESULTS. Ninety-nine patients (48 NP and 51 NN) were vaccinated in 7 dose groups. The OBD was 1000 μg E75 plus 250 μg GM-CSF monthly × 6. The optimal dose group (ODG, n = 29) experienced similar toxicities to the suboptimal dose group (SDG, n = 70), which was comprised of the remaining 6 groups. The ODG demonstrated a trend toward an increase in the average postvaccine dimer (0.87 ± 0.10% vs 0.67 ± 0.05%; P = .07), a significantly larger DTH response (21.5 ± 2.5 mm vs 11.3 ± 1.3 mm; P = .0002), and a trend toward decreased recurrences (3.4% vs 12.9%; P = .27). Compared with the SDG, the ODG had larger tumors (percentage ≥T2: 55% vs 23%; P = .004), more positive lymph nodes (percentage NP: 76% vs 37%; P = .001), and higher grade tumors (percentage grade 3: 52% vs 30%; P = .07), but a shorter median follow-up time (20 months vs 32 months; P < .001). CONCLUSIONS. Compared with suboptimally dosed patients, the optimally dosed E75 vaccine in disease-free BCa patients had similar toxicity but enhanced HER-2/neu-specific immunity that may lead to decreased recurrences with additional follow-up. Cancer 2008. Published 2008 by the American Cancer Society.

2565 Background: Regulatory T cells (Treg) inhibit effector T-cell function and prevent autoimmunity; they are recognized as obstacles to cancer immunotherapy. Tregs are predominantly CD4+, suggest...

BACKGROUND: HER2/neu-derived peptides that stimulate CD8+ T-lymphocytes are being evaluated in vaccine trials. We present results of a prospective, randomized, single-blinded phase II clinical trial of a HLA-A2/A3-restricted, HER2/neu peptide (GP2, HER2/neu 654-662) + GM-CSF immunoadjuvant vaccine versus GM-CSF alone in disease-free, high risk breast cancer (BCa) patients to prevent disease recurrence.METHODS: Disease-free, high risk BCa patients who have completed standard adjuvant therapy were enrolled and randomized to receive six monthly inoculations of either 500 mcg of GP2 with 125 mcg of GM-CSF (Peptide group; PG) or 125 mcg of GM-CSF alone (adjuvant group; AG). Toxicity was assessed after each inoculation. Immunologic response was monitored by measured delayed type hypersensitivity reactions (DTH) and an HLA-A2:Immunoglobulin dimer assay to detect GP2-specific CD8+ T-lymphocytes. Patients were monitored clinically, radiographically, and pathologically for recurrence.RESULTS: Thus far, 50 (27 PG, 23 AG) of the planned 200 patients have completed the primary series. The PG and AG have similar demographic/prognostic characteristics (Table 1). Toxicity profiles in the PG and AG were nearly identical with no grade 4-5 local toxicities and no grade 3-5 systemic toxicities in either arm. Median DTH reaction to GP2 increased significantly from pre-vaccination level after completion of the primary series (post-vaccination) in the PG group (1.0±0.8 cm to 18.0 ±3.1 cm; p= 2 cm66.7%52.2%0.45ER/PR negative40.7%43.5%0.92HER2 over-expressor59.3%47.8%0.60 Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 5110.

Abstract #3134 Background: CD4+CD25+FOXP3+ regulatory T cells (Tregs) have been implicated in the suppression of immune responses against various tumors. To monitor the potential induction of Tregs in breast cancer (BrCa) patients receiving a modified HLA Class II HER2/neu peptide (AE37) vaccine in a clinical trial, we have analyzed peripheral blood lymphocytes (PBL) from vaccinated patients for the presence of Tregs and correlated our findings with ex vivo immune assays and in vivo delayed type hypersensitivity (DTH) responses to the vaccine. Methods: Fifteen BrCa patients have completed 6 monthly injections of the AE37+GM-CSF vaccine in a dose escalation safety study. The AE37 peptide consists of a HER2/neu peptide (776-790) linked to the Ii-Key moiety of the HLA Class II-associated invariant chain, which enhances epitope interaction with the Class II molecule. PBL obtained pre- and post-vaccination were stained with anti-CD4/CD25 (n=15) and FOXP3 (n=9) antibodies (PCH101 and 236A/E7) and analyzed by flow cytometry. Cells were also stimulated ex vivo with AE37 peptide to measure IFN-γ ELISPOT, proliferation (3H-thymidine-cpm) and cytokine secretion (TGF-β). DTH responses to the AE37 peptide pre- and post-vaccination were also recorded. Results: The mean CD4+ and CD4+CD25+ T cell populations for all patients (n=15) did not change from pre- to post-vaccination (CD4+ = 52.3+3.3% vs. 50.5+3.9%, p=0.6; CD4+CD25+ = 1.9+0.2% vs. 2.4+0.5%, p=0.2). Tregs (CD4+CD25+FOXP3+) were reduced in all 9 patients tested pre- to post-vaccination for both FOXP3 antibodies (Ab) (FOXP3 Ab1 = 2.1+0.2% vs. 1.1+0.1%, p=0.002; FOXP3 Ab2 = 2.0+0.2% vs. 1.0+0.2%, p=0.0009). There was no difference in pre- to post-vaccination levels of TGF-β (2720+582 pg/ml vs. 3387+848 pg/ml; p=0.9). AE37-specific proliferative responses increased from pre- to post-vaccination (34+23cpm vs. 6427+1431 cpm; p Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 3134.

Abstract #3139 Background: Our Cancer Vaccine Development Program has performed phase I and II clinical trials using immunogenic peptides from the HER2/neu protein. AE37 (aa:776-790+Ii-Key) is HLA promiscuous whereas E75 (aa:369-377) and GP2 (aa:654-662) are HLA-A2/A3+ restricted. The peptides are located on the intracellular, extracellular, and transmembrane portions of the HER2/neu protein. We evaluate patients who required dose reductions and compare them to those who did not in our three peptide vaccine trials to determine the immunologic and clinical relevance of different responses to the peptide vaccines. Methods: Three vaccine trials were performed separately with similar dose escalation design by varying the amount of AE37, GP2 or E75 peptide and GM-CSF adjuvant. Dose reductions (DR) were made in event of significant toxicity (>100mm local or ≥grade 2 systemic) by decreasing GM-CSF (or peptide if no GM-CSF) by 50% for subsequent inoculations. We compared patients necessitating DR and those who did not. Immune response was measured ex vivo via 3H-thymidine proliferative assays or HLA-A2:Ig dimer assays and in vivo via DTH reactions pre- and post-vaccine. Recurrence and mortality data were available for E75 treated patients at 30-month median follow-up. Results: 132 patients underwent peptide vaccination and 39 patients required DR (Table 1). No patient had grade 3-5 local or systemic toxicities. DR patients, particularly those reduced for robust local reactions (DR-L), had greater immune responses ex vivo and in vivo. The post-vaccine DTH in DR-L patients compared to all other vaccinated patients was significantly larger in E75 (24.5±4.1mm vs. 12.7±1.3mm; p=0.001) and trended towards significant in AE37 (69.4±10.3mm vs. 35.8±14.9mm; p=0.08) and GP2 (48.9±14.1mm vs. 24.1±3.3mm; p=0.08). No recurrences or deaths were noted in the E75 DR patients; but for patients not requiring dose reductions there were 10 (12.5%) recurrences and 1 (1.25%) death.Discussion: All three peptide vaccines are safe and well-tolerated with minimal toxicity. The safety was partly due to reducing GM-CSF (or peptide if no GM-CSF) by 50% for patients with >100mm local reactions or >grade 2 systemic toxicity. DR patients, particularly DR-L patients, immunological responses were larger. This increased immune response (noted by DTH) suggests improved clinical response as there have been no recurrences or deaths among DR patients. Our analysis indicates that the optimal method of dosing HER2/neu peptide vaccines consists of constant peptide dose with a large enough dose of adjuvant to generate robust local reactions and that when local reactions are >100mm performing DR of adjuvant by 50% for subsequent inoculations. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 3139.

3016 Background: HER2/neu is overexpressed in breast cancer (BCa) and is the source of immunogenic peptides. CD4+ T helper peptides for HER2/neu are being evaluated in vaccine trials. The addition of Ii-Key, a 4-amino-acid LRMK modification, increases vaccine potency when compared to unmodified class II epitopes. We present the results of the first human Phase I trial of the Ii-Key hybrid HER2/neu peptide (AE37) vaccine in disease-free, node-negative BCa patients. Methods: The dose-escalation trial included 5 dose groups, to determine safety and optimal dose of the hybrid peptide (100mcg; 500 mcg; 1,000mcg) and GM-CSF (range 0–250mcg). In the event of significant toxicity, GM-CSF (or peptide in the absence of GM-CSF) was reduced by 50%. Immunologic response was monitored by DTH and 3H-thymidine proliferative assays for both the hybrid AE37 (LRMK + HER2/neu:776–790) and AE36 (unmodified HER2/neu:776–790). Results: All 15 patients completed the trial with no grade 3–5 local or systemic toxicities. Dose redu...

3067 Background: E75 is an immunogenic peptide from the HER2/neu protein that is expressed in prostate cancer. High-risk prostate cancer (HRPC) patients demonstrating various levels of HER2/neu expression were vaccinated with E75 peptide + GM-CSF to prevent post-prostatectomy prostate-specific antigen (PSA) and clinical recurrences. Methods: 40 HRPC patients were prospectively identified using the validated Center for Prostate Disease Research (CPDR)/CaPSURE high-risk equation and enrolled. HLA-A2+ patients (n=21) were vaccinated (V), and HLA-A2- patients (n=19) were followed as clinical controls (C). HER2/neu over-expressors (OE) were defined as IHC 3+ and low-expressors (LE) as IHC 0–2+. All patients were assessed for clinico-pathologic factors, PSA recurrence (≥0.2ng/ml), clinical recurrence and survival. Results: Comparing the V and C groups, there were no statistical differences in clinico-pathologic factors; however, the V group had larger tumors (≥T3b-T3c: 35% vs. 18%), higher postop Gleason scores...

22215 Background: HER2/neu is a tumor associated antigen that is over-expressed in several epithelial cancers. HER2/neu-specific immunity can prevent breast cancer in animal models. Immune surveillance against HER2/neu is a potential defense mechanism against cancer and is pursued as immunotherapy of breast cancer. Prior studies demonstrated that peptides from HER2/neu display varying binding affinities to different MHC Class II DR alleles. HLA-DR3 had the lowest affinity for HER2/neu peptides suggesting that DR3+ individuals may have reduced immunosurveillance against HER2/neu and thereby be susceptible to developing HER2/neu related cancers. We hypothesized that the presence of the DR3 allele may predispose patients to develop breast cancer, and if true, might expect to find higher numbers of DR3+ individuals among BrCa patients compared to the general population. Methods: Serologic and molecular testing for HLA-DR3 was performed on 52 HER2/neu+ (IHC 1–3+) breast cancer patients. We compared clinicopath...

Background: HER2/neu is a source of immunogenic peptides and is over-expressed in 30% of early stage breast cancer (BCa) patients. We have conducted clinical trials with the HER2/neu E75-peptide vaccine in node-positive and node-negative BCa patients demonstrating all levels of HER2/neu expression. Methods: A subset analysis review was performed of 165 BCa patients enrolled in our E75 vaccine trial based on the level of HER2/neu expression. Patients were HLA typed; HLA-A2+/A3+ patients were vaccinated (V); HLA-A2-/A3- patients served as controls (C). HER2/neu over-expressers (OE) were defined as FISH >2.0 and IHC 3+ HER2/neu tumors. Low-expressers (LE) included patients with IHC ranging from 0 to 2+. These 2 groups were assessed by clinicopathologic factors, immunologic response (in vivo DTH reactions and in vitro HLA-A2:IgG dimer assay) to the vaccine, and post-vaccine clinical responses (recurrence and survival). Results: Of 165 patients assessed; 94 were vaccinated (30 OE = 32%, 64 LE = 68%) and 71 were controls (22 OE = 31%, 49 LE = 69%). HER2/neu over-expressers were similar regarding prognostic and treatment factors, except a statistically larger number of V-OE patients were hormone receptor negative (ER/PR-; p=0.02) and node-negative (p=0.007). Clinicopathologic factors were not significantly different between control and V-LE patients. Immunologic responses were similar as measured by DTH reactions (post DTH V-OE=12.7+1.9 vs. V-LE=15.1+1.8; p=0.6), but V-OE demonstrated a decreased number of E75-specific CD8+ T cells when compared to V-LE post-vaccination (V-OE vs. V-LE Dimer: 0.6+0.06% vs. 0.8+0.07%; p=0.08; V-OE vs. V-LE Max Dimer: 1.5+0.1% 1.9+0.1%; p=0.04). At 30 months median follow-up, disease recurrence rates (RR) were similar between V-OE and C-OE patients (V-OE vs. C-OE RR: 4.3% vs. 5.6%; p=0.7); however there was a 50% reduction in mortality rate (MR) among patients that recurred (V-OE vs. C-OE MR: 25% vs. 50%; p=NS). RR was reduced for vaccinated patients with low HER2/neu expression (V-LE vs. C-LE RR: 6.4% vs. 11.3%; p=0.3). Importantly, MR among those that recurred was 0% vs. 38% (p=0.2) for V-LE and C-LE BCa patients, respectively. Conclusions: Patients with HER2/neu over-expressing breast cancer (FISH amplified (>2.0) and IHC 3+) respond well to the E75 vaccine immunologically and have a 50% absolute reduction in mortality compared to controls. Unexpectedly, patients with low-expressing (0 - 2+ on IHC) HER2/neu tumors show better response not only immunologically, but clinically with decreased breast cancer recurrence and 0% mortality following E75 peptide vaccination.