Ritesh Patil

Clinical trial results of the HER-2/neu (E75) vaccine to prevent breast cancer recurrence in high-risk patients: from US Military Cancer Institute Clinical Trials Group Study I-01 and I-02.

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).

Optimal dose and schedule of an HER-2/neu (E75) peptide vaccine to prevent breast cancer recurrence: from US Military Cancer Institute Clinical Trials Group Study I-01 and I-02.

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.

Use of booster inoculations to sustain the clinical effect of an adjuvant breast cancer vaccine

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.

Abstract P5-16-02: Final Results of the Phase I/II Trials of the E75 Adjuvant Breast Cancer Vaccine

Background: We have completed phase I/II clinical trials vaccinating breast cancer patients (pts) with E75, a HLA-A2/A3-restricted HER2/neu (HER2) peptide vaccine. The vaccine was administered in the adjuvant setting to prevent recurrences in high risk patients rendered disease-free with standard of care therapy. We have previously reported preliminary results indicating that the vaccine (including booster inoculations) is safe and effective in stimulating an anti-tumor immune response. Here, we report the final 5 year results from these trials. Methods: The phase I/II trials were performed as dose-escalation/schedule-optimization trials enrolling node positive and high-risk, node negative breast cancer patients with tumors expressing any level of HER2. HLA-A2/A3+ pts were enrolled into the vaccine group (VG) while HLA-A2/A3- pts were followed prospectively as the untreated control group (CG). The VG pts were given 4–6 monthly intradermal inoculations of E75 with GM-CSF during the primary vaccine series (PVS). In addition, a voluntary booster program was initiated during the trial, with booster inoculations being offered every 6 months after completion of the PVS. Patients were monitored for local and systemic toxicity (graded by NCI Common Terminology Criteria for Adverse Events). In vivo immune response was assessed in the VG by delayed type hypersensitivity (DTH) reactions to both E75 and saline, pre- and post-PVS. VG and CG pts were followed for 60 months (mo) and recurrences were documented. Demographic differences were compared with the Fisher9s exact test and disease-free survival was determined using the Kaplan-Meier method and compared by log-rank test. Results: 195 pts were enrolled, 6 withdrew (2 from VG, 4 from CG), 1 was lost to follow-up prior to vaccination, and 1 was found to be ineligible, leaving 187 evaluable pts; 108 in the VG and 79 in the CG. 53 pts volunteered for the booster program and received at least one booster inoculation. The VG and CG were well-matched with the only statistically significant difference being ER−/PR- status (31.1% in VG vs 17.7% in CG, p = 0.04). Vaccination was well tolerated (maximum local toxicity: 73.1% Grade 1, 26.9% Grade 2, 0% Grade 3; maximum systemic toxicity: 72.2% Grade 1, 15.7% Grade 2, and 2.8% Grade 3). In the VG, pre- to post-PVS E75 DTH significantly increased (mean 3.8 ±1.0 vs 14.8±1.4, p Conclusions: The E75 breast cancer vaccine is safe and well–tolerated. It elicits strong immune responses in vaccinated patients. At the end of the 5 year follow-up period, the E75 vaccine shows a strong trend toward preventing breast cancer recurrence in vaccinated patients. To investigate this vaccine (now known as NeuVax) further, the PRESENT trial, a prospective, randomized, double-blind, placebo-controlled, multi-center phase III registration trial has been initiated and is actively enrolling. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P5-16-02.

P1-13-02: Long-Term Clinical Benefit of Adjuvant Breast Cancer Vaccine: 5 Year Efficacy of E75 with Multiple Booster Inoculations.

Background: We are conducting phase I/II clinical trials vaccinating breast cancer patients with E75, an HLA-A2/A3 restricted HER2/neu (HER2) peptide mixed with GM-CSF. The vaccine has been studied in the adjuvant setting to prevent recurrences in clinically disease-free patients after completion of standard therapy. We have previously reported that the vaccine is safe, effectively stimulates HER2−specific immunity, and appears to improve disease-free survival at 24 months. Here, we report long-term data at a median follow-up of 60 months. Methods: The phase I/II trials were performed as dose escalation/schedule optimization trials enrolling node positive and high-risk, node-negative patients with tumors expressing any level of HER2. Vaccinated patients were given 4–6 monthly inoculations of E75 with GM-CSF immunoadjuvant. Due to waning immunity, a voluntary booster program was initiated, with inoculations every 6 months after completion of the primary vaccine series (PVS). Patients were monitored for local and systemic toxicities, which were graded by the NCI9s Common Terminology Criteria for Adverse Events. Vaccinated patients and controls were followed for 60 months and recurrences were documented. Demographic differences were compared with the Fisher9s exact test and survival was analyzed by the log-rank test. Results: 187 patients were enrolled; 108 in the vaccine group (VG) and 79 in the unvaccinated control group (CG). The vaccine and control groups were well-matched with the only statistically significant difference being ER-/PR- status (31.1% in VG vs 17.7% in CG, p=0.04). Vaccination was well tolerated with primarily grade 1 and grade 2 toxicity in the PVS (Local Toxicity: 85% Grade 1, 15% Grade 2, 0% Grade 3; systemic toxicity: 71% Grade 1, 14% Grade 2, and 3% Grade 3). Fifty-three of the VG patients received at least one booster, with 34 receiving a second booster, 25 a third, 22 a fourth, 12 a fifth, and 9 receiving at least six boosters. Booster inoculations were well-tolerated with only grade 1 and 2 local and systemic toxicities. There were delayed urticarial reactions in 7/53(13%) of the boosted patients occurring at a median of 9 days (5-21 days) after inoculation; these were grade 2 reactions and well-tolerated. After a median follow-up of 60 months, there has been a nonsignificant decrease in recurrences observed in the VG compared to the CG (10.6% vs 20.3%, p=0.098). The hazard ratio is 0.52 in the VG. In patients with immunity maintained with voluntary boosters, there have been even fewer (3.8%) recurrences (p=0.03). Conclusions: The E75 breast cancer vaccine is safe and well-tolerated. With long-term follow-up at 60 months, the E75 vaccine continues to show a strong trend toward preventing breast cancer recurrence in vaccinated patients particularly in patients whose immunity is maintained with booster inoculations. To investigate this further, a phase III trial with prospective boosting is being initiated. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P1-13-02.

Abstract LB-130: Immune reconstitution after chemotherapy correlates with increased in vitro immune response in breast cancer patients undergoing peptide vaccine therapy

Introduction: We are conducting a Phase II clinical trial of 2 HER2 peptide vaccines, GP2 (MHC Class I restricted) and AE37 (MHC Class II restricted), for the prevention of breast cancer (BCa) recurrence in disease-free, high risk patients (pts). We present analysis of T cell populations in trial patients at the time of enrollment and differences based on time since chemotherapy. We describe a “reconstitution” of the immune system after immunosuppressive chemotherapy. Methods: After completion of standard therapy, disease-free, BCa pts were enrolled. Demographic data was collected. Blood was collected prior to administration of their first vaccine. Peripheral blood mononuclear cells from 50 pts were isolated and evaluated for CD8+, CD4+CD8+, CD4-CD8- and CD4+ T cell populations. T cell proliferation responses were measured in patients of both arms of the trial; generically with FluM-specific CD8 cells (HLA-A2:Ig dimer assay) in the GP2 arm (n=58), and then with proliferation response to AE36 and AE37 in the AE37 arm (n=85). Linear regression analyses evaluated the relationship between time from chemotherapy and each T cell population. Immune responses of pts enrolled less than one year from chemotherapy ( 1yr group) using a t-test. Results: Chemotherapy regimens were determined by the treating oncologist and consisted primarily of anthracycline-based regimens with a taxane. Regression analysis revealed a significant correlation between time from chemotherapy and both CD4+ and CD8+ Tcell counts (R= .433, p=0.015 and R=.439, p=.014, respectively). Total T cell, CD4-CD8- and CD4+CD8+ populations, however, did not significantly correlate with increased time from chemotherapy (R=.28, p=0.128, R=.068, p=.715 and R=.058, p=.755, repectively). Comparison between the 1yr group in the GP2 arm (n=43 and n=15) for FluM-specific CD8 cells revealed a non-significant increase in immune response in the >1yr group (2.74 vs 3.57, p=0.15). A similar comparison in the AE37 arm (n=56 1yr) revealed increased proliferation in the >1yr group (AE36: 1110 vs 2167, p=0.034, AE37: 983 vs 2179, p=0.001). There were no significant differences between the >1yr and Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-130. doi:1538-7445.AM2012-LB-130

Monitoring of circulating tumor cell trends in a prospective, randomized, placebo-controlled HER2/neu peptide vaccine trial.

e11126 Background: Circulating tumor cells (CTC) may have a role as a prognostic marker and in monitoring therapeutic response in breast cancer (BrCa) patients. We are conducting a prospective, randomized phase II trial evaluating the HER2 peptide vaccines AE37 and GP2 in the adjuvant setting. AE37 is the Ii-Key hybrid of the AE36 peptide (HER2: 776-790) and GP2 is an HLA-A2+ -restricted peptide (HER2: 654-662). Both are capable of eliciting anti-HER2 tumor activity. We examined CTC trends in BrCa patients from our vaccine trial. METHODS Node positive or high-risk node negative BrCa patients with any level of HER2 expression disease-free after standard treatments were randomized to receive vaccine (either GP2 or AE37) + GM-CSF (immunoadjuvant) or GM-CSF alone. Vaccinations were given as six monthly intradermal inoculations. CTC were enumerated from 20 mL blood samples using the CellSearch System (Veridex, LLC Warren, NJ). CTC count was checked pre-vaccination and followed every 6 months for those with ≥1 CTC at baseline. p-values were calculated using Fishers exact test. RESULTS Baseline CTC levels were checked in 45 patients with 18 (40%) having ≥1 CTC. Follow-up CTC results were available for 12 patients. Eight of these patients had a subsequent CTC decrease while 4 patients had either increasing (2) or unchanged (2) CTC counts. In patients with a CTC decrease, 7 of 8 (87.5%) received vaccines compared to only 1 patient with a decrease after receiving GM-CSF alone. In those with no CTC decrease, only 1 of 4 (25%) received the vaccine while the remainder received GM-CSF only (p=0.067). In the 4 patients without CTC decrease, 2 developed BrCa recurrence with 1 death from metastatic disease. There were no events in patients with CTC decreases. CONCLUSIONS Clinically disease-free BrCa patients with detectable baseline CTC levels in our adjuvant vaccine trial who experience a CTC decrease were much more likely to have received the vaccine than the control. Monitoring CTC trends may be a promising clinically-relevant surrogate marker for BrCa recurrence. However, in the adjuvant setting, CTC were detectable in only 40% of patients suggesting the need for a more sensitive test for broader applicability.

Abstract LB-329: Defining rgulatory T-cells as CD4+CD25hiCD127- or as CD4+CD25hiFoxP3+ in immune monitoring of cancer vaccine clinical trials

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC Background: Regulatory T cells (Treg) are widely accepted to play a key role in the immune tolerance and escape of cancer cells. Originally Tregs were identified as being CD4+ T cells expressing high levels of CD25 (IL2 receptor). The identification of the transcription factor FoxP3 resulted in defining Tregs as CD4CD25hiFoxP3+ T cells by staining permeablized cells. Recently Tregs have been identified as CD4CD25hi T cells with low levels of CD127 (IL7 receptor) expressing a CD4+CD25hiCD127- cell surface phenotype. In an ongoing clinical trial with a MHC class II HER2/ neu pep tide vaccine we have evaluated these various definitions of Treg phenotypes for immunological monitoring. Methods: Tregs were quantified in disease-free, high risk BCa patients enrolled in adjuvant peptide-based breast cancer vaccine trials. Peripheral blood mononuclear cells (PBMCs) were both permeabilized and stained with CD4, CD25, and FoxP3 monoclonal antibodies and surface stained with CD4, CD25, and CD127. Samples were then analyzed by flow cytometry. The means are compared with t-test and correlation is determined with Pearsons correlation coefficient. Results: 196 PBMC samples from 72 patients were available for Treg quantification. There was a greater mean percentage of cells identified as Treg in permeabilized than nonpermeabilized cells by staining with CD4+CD25hi (5.9±0.1% vs 4.8 ±0.1%, p<0.01) and excellent correlation between the tests (r=0.78, p<0.01). There was a greater mean percentage of cells identified as Tregs by CD4+CD25hi than CD4+CD25hiFoxP3+ (5.9±0.1% vs 3.6±0.1%, p<0.01) with good correlation (r=0.68, p<0.01). There was a greater mean percentage of Tregs by CD4+CD25+ than CD4+CD25+CD127- (4.8±0.1 vs 4.0±0.1, p<0.01) with excellent correlation (r=0.89, p<0.01). There was a greater mean percentage of Tregs by CD4+CD25hiCD127- than Tregs by CD4+ CD25hiFoxP3+ although levels were similar (4.0±0.1 vs 3.6±0.1, p<0.01) and the two tests had good correlation (r=0.58, p<0.01). Conclusion: There was good correlation between various methods of defining Tregs with and without cell membrane permeabilization. Defining Treg cells as either CD4+CD25hiFoxP3+ or CD4+CD25+CD127- appear to be more specific than defining Tregs as CD4+CD25hi alone. Furthermore, defining Treg populations by surface staining as CD4+CD25hiCD127- yields similar levels with good correlation as CD4+CD25hiFoxP3+ and may be the preferred method for immune monitoring. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr LB-329.

Abstract LB-322: A modified HER2/neu peptide vaccine (2L9V-GP2) demonstrates enhanced CD8+ T cell recognition and stimulation in breast cancer patients at high risk for recurrence

Introduction : The affinity between tumor-associated antigens and MHC Class I complexes directly affects the degree of CD8+ T cell (CTL) response. GP2 (IISAVVGIL, p654-662) is a HER2 /neu -derived peptide, that has poor binding affinity to HLA-A2 (A2) and HLA-A3 (A3). We conducted a preclinical study to determine if amino acid substitutions in the GP2 peptide, predicted to increase binding affinity to A2 and A3, enhance CTL recognition and stimulation. Methods : Based on computer modeling algorithms (BIMAS, SYFPEITHI), two GP2 peptide variants were predicted to have higher binding affinity to A2: SPA-12(2L9V-GP2) and SPA-13(2L5L9V-GP2). Freshly isolated PBMCs were obtained from randomly chosen, disease-free, node positive (NP) and high risk node negative (NN) breast cancer (BCa) patients, who had completed standard adjuvant therapy and are enrolled in the E75 vaccine booster trial. Peptide-specific CD8+ T cells were measured by HLA-A2: IgG dimer (A2 pts) and by interferon-γ ELISPOT (A2, A3 pts) assays. Groups were compared by t-test (dimer) and Mann-Whitney U (ELISPOT) tests. Results: Thirty-five A2 (21 NP, 14 NN) and thirteen A3 (13 NP) samples were included in the study. Among A2 pts, the mean level of SPA-12-specific CTLs was greater than the GP2- (2.8±0.3% vs. 1.7±0.1%, p 5 cells) compared to GP2 (88, range 0-432 spots/10 5 cells, p=0.03) and SPA-13 (0, range 0-344 spots/10 5 cells, p Conclusion : The substituted sequences SPA-12 (2L9V-GP2) and SPA-13 (2L5L9V-GP2), both have higher predicted binding affinity to A2 and A3 than GP2. While SPA-12 showed significantly increased recognition and functional CTL responses compared to GP2, SPA-13 demonstrated lower responses. These data suggest that SPA-12 may be a more effective cancer vaccine than GP2, particularly in NP BCa patients. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr LB-322.