Kathy Schiffman

Factors that influence collection and engraftment of autologous peripheral-blood stem cells.

PURPOSE To analyze factors that affect the collection of peripheral-blood stem cells (PBSC) before transplant and the tempo of engraftment after transplant. PATIENTS AND METHODS A consecutive series of 243 patients with breast cancer (n = 87), malignant lymphoma (n = 90), multiple myeloma (n = 32), or other malignancies (n = 34) had PBSC collected following stimulation with colony-stimulating factors (CSFs) or after chemotherapy followed by CSF. Infusion of PBSC was performed following myeloablative chemotherapy with chemotherapy with or without total-body irradiation (TBI). Postinfusion CSFs were administered to 72 patients. An analysis of factors that influence CD34+ cell yield was performed by linear regression. Cox regression analysis was used to determine factors that affect the kinetics of granulocyte and platelet recovery following infusion of PBSC. RESULTS Mobilization with chemotherapy followed by CSF, a diagnosis of breast cancer, absence of marrow disease, no prior history of radiation therapy, and fewer cycles of conventional-dose chemotherapy were associated with a higher average daily yield of CD34+ cells. In the multivariate analysis, the CD34 content of infused cells and the use of a posttransplant CSF influenced neutrophil recovery after infusion of PBSC. CD34 content was also important for predicting platelet recovery. The use of postinfusion CSF was associated with a significant delay in platelet recovery in patients who received less than 5.0 x 10(6) CD34+ cells/kg, but there was no discernable effect in patients who received greater than 5.0 x 10(6) CD34+ cells/kg. CONCLUSION Disease status and prior treatment influence the ability to mobilize PBSC. CD34 cell dose is an important predictor of engraftment kinetics after PBSC transplant, regardless of disease or mobilization technique. The use of postinfusion CSF improves neutrophil recovery, but at low CD34 doses can delay platelet recovery.

Generation of T-Cell Immunity to the HER-2/neu Protein After Active Immunization With HER-2/neu Peptide–Based Vaccines

PURPOSE The HER-2/neu protein is a nonmutated tumor antigen that is overexpressed in a variety of human malignancies, including breast and ovarian cancer. Many tumor antigens, such as MAGE and gp100, are self-proteins; therefore, effective vaccine strategies must circumvent tolerance. We hypothesized that immunizing patients with subdominant peptide epitopes derived from HER-2/neu, using an adjuvant known to recruit professional antigen-presenting cells, granulocyte-macrophage colony-stimulating factor, would result in the generation of T-cell immunity specific for the HER-2/neu protein. PATIENTS AND METHODS Sixty-four patients with HER-2/neu-overexpressing breast, ovarian, or non-small-cell lung cancers were enrolled. Vaccines were composed of peptides derived from potential T-helper epitopes of the HER-2/neu protein admixed with granulocyte-macrophage colony-stimulating factor and administered intradermally. Peripheral-blood mononuclear cells were evaluated at baseline, before vaccination, and after vaccination for antigen-specific T-cell immunity. Immunologic response data are presented on the 38 subjects who completed six vaccinations. Toxicity data are presented on all 64 patients enrolled. RESULTS Ninety-two percent of patients developed T-cell immunity to HER-2/neu peptides (stimulation index, 2.1 to 59) and 68% to a HER-2/neu protein domain (stimulation index range, 2 to 31). Epitope spreading was observed in 84% of patients and significantly correlated with the generation of a HER-2/neu protein-specific T-cell immunity (P =.03). At 1-year follow-up, immunity to the HER-2/neu protein persisted in 38% of patients. CONCLUSION The majority of patients with HER-2/neu-overexpressing cancers can develop immunity to both HER-2/neu peptides and protein. In addition, the generation of protein-specific immunity, after peptide immunization, was associated with epitope spreading, reflecting the initiation of an endogenous immune response. Finally, immunity can persist after active immunizations have ended.

Pre-existent immunity to the HER-2/neu oncogenic protein in patients with HER-2/neu overexpressing breast and ovarian cancer

Immunomodulatory strategies, such as antibody therapy and cancer vaccines, are increasingly being considered as potential adjuvant therapies in patients with advanced stage breast cancer to either treat minimal residual disease or prevent relapse. However, little is known concerning the incidence and magnitude of the pre-existent breast cancer specific immune response in this patient population. Using the HER-2/neu oncogenic protein as a model, a well-defined tumor antigen in breast cancer, we questioned whether patients with advanced stage HER-2/neu overexpressing breast and ovarian cancers (III/IV) had evidence of pre-existent immunity to HER-2/neu. Forty-five patients with stage III or IV HER-2/neu overexpressing breast or ovarian cancer were evaluated for HER-2/neu specific T cell and antibody immunity. Patients enrolled had not received immunosuppressive chemotherapy for at least 30 days (median 5 months, range 1–75 months). All patients were documented to be immune competent prior to entry by DTH testing using a skin test anergy battery. Five of 45 patients (11%) were found to have a significant HER-2/neu specific T cell response as defined by a stimulation index ≥ 2.0 (range 2.0–7.9). None of eight patients who were HLA-A2 had a detectable IFNγ secreting T-cell precursor frequency to a well-defined HER-2/neu HLA-A2 T cell epitope, p369-377. Three of 45 patients (7%) had detectable HER-2/neu specific IgG antibodies, range 1.2–8.9 μg/ml. These findings suggest that patients with advanced stage HER-2/neu overexpressing breast and ovarian cancer can mount a T cell and/or antibody immune response to their tumor. However, in the case of the HER-2/neu antigen, the pre-existent tumor specific immune response is found only in a minority of patients.

Effect of dose on immune response in patients vaccinated with an HER-2/neu intracellular domain protein-based vaccine

PURPOSE To evaluate the safety of an HER-2/neu intracellular domain (ICD) protein vaccine and to estimate whether vaccine dose impacts immunogenicity. PATIENTS AND METHODS Twenty-nine patients with HER-2/neu-overexpressing breast or ovarian cancer and with no evidence of disease after standard therapy received a low- (25 microg), intermediate- (150 microg), or high-dose (900 microg) HER-2/neu ICD protein vaccine. The vaccine was administered intradermally, monthly for 6 months, with granulocyte-macrophage colony-stimulating factor as an adjuvant. Toxicity and both cellular and humoral HER-2/neu-specific immunity was evaluated. RESULTS The vaccine was well tolerated. The majority of patients (89%) developed HER-2/neu ICD-specific T-cell immunity. The dose of vaccine did not predict the magnitude of the T-cell response. The majority of patients (82%) also developed HER-2/neu-specific immunoglobulin G antibody immunity. Vaccine dose did not predict magnitude or avidity of the HER-2/neu-specific humoral immune response. Time to development of detectable HER-2/neu-specific immunity, however, was significantly earlier for the high- versus low-dose vaccine group (P =.003). Over half the patients retained HER-2/neu-specific T-cell immunity 9 to 12 months after immunizations had ended. CONCLUSION The HER-2/neu ICD protein vaccine was well tolerated and effective in eliciting HER-2/neu-specific T-cell and antibody immunity in the majority of breast and ovarian cancer patients who completed the vaccine regimen. Although the dose of vaccine did not impact the magnitude of T-cell or antibody immunity elicited, patients receiving the highest dose developed HER-2/neu-specific immunity more rapidly than those who received the lowest dose.

Humoral epitope-spreading following immunization with a HER-2/neu peptide based vaccine in cancer patients.

HER-2/neu is a tumor antigen in patients with breast and ovarian cancer. Multiple varieties of vaccine strategies are being developed to immunize patients against HER-2/neu. Studies in animal models have demonstrated both T cell and antibody immunity are needed to mediate an antitumor response. Thirty-five patients, immunized with HER-2/neu peptide based vaccines, were evaluated for the generation of HER-2/neu-specific antibody immunity. Sixty percent of patients developed HER-2/neu IgG specific antibody responses to at least one peptide included in their vaccine. Twenty-nine percent of patients developed IgG immunity to the native HER-2/neu protein after peptide immunization. Humoral intramolecular epitope-spreading within the HER-2/neu protein occurred in 49% of immunized patients. Intermolecular epitope-spreading to p53 was evident in 20% of vaccinated patients. Of those patients who developed new immunity to p53, 71% had demonstrated antibody epitope-spreading within HER-2/neu.

Pilot Study of an HLA-A2 Peptide Vaccine Using Flt3 Ligand as a Systemic Vaccine Adjuvant

A pilot vaccine study was conducted to test the safety and immunological efficacy of four monthly immunizations of an MHC class I peptide vaccine, the E75 HLA-A2 epitope from HER-2/neu, using flt3 ligand as a systemic vaccine adjuvant. Twenty HLA-A2-expressing subjects with advanced stage prostate cancer were randomly assigned to one of four immunization or treatment schedules: (a) Flt3 ligand (20 μ g/kg per day) administered subcutaneously daily for 14 days on a 28-day cycle, monthly for four months; (b) flt3 ligand course as above with the E75 peptide vaccine administered on day 7 of each flt3 ligand cycle; (c) flt3 ligand course as above with the E75 peptide vaccine administered on day 14 of each flt3 ligand cycle; or (d) E75 peptide admixed with granulocyte–macrophage colony-stimulating factor and administered intradermally once every 28 days, as has previously been reported. The primary endpoints of the study were the determination of safety and immunological efficacy in generating E75-specific T cells as determined by peptide-specific interferon-gamma ELIspot. Adverse events included one grade 3 skin reaction and the development of grade 2 autoimmune hypothyroidism in two subjects with preexisting subclinical autoimmune hypothyroidism. Dendritic cells were markedly increased in the peripheral blood of subjects receiving flt3 ligand with each repetitive cycle, but augmentation of antigen-presenting cells within the dermis was not observed. Apart from a single subject, no significant peptide-specific T-cell responses were detected by ELIspot, whereas delayed-type hypersensitivity responses were detectable in control subjects and in subjects receiving peptide vaccine early in the course of flt3 ligand administration. The absence of robust peripheral immune responses in the current study may be attributable to the small numbers of subjects or differences in the subject population. In addition, the inability of flt3 ligand to augment the number of peripheral skin antigen-presenting cells may have contributed to the absence of robust peptide-specific immunity detectable in the peripheral blood of immunized subjects treated with flt3 ligand.

Cancer vaccines targeting the HER2/neu oncogenic protein☆

Several advances in basic immunology over the last few years have forced a re-evaluation of cancer vaccine development. The most important finding has been that human tumors are immunogenic. The HER2/neu oncogenic protein is a tumor antigen. Existent antibody, helper T-cell, and cytotoxic T-cell immunity to HER2/neu have been detected in patients with cancer. The HER2/neu protein is an excellent therapeutic target for the immune system. Passive immunotherapy strategies, such as the infusion of monoclonal antibodies specific for HER2/neu, have been shown to be of clinical benefit in patients with HER2/neu-overexpressing malignancies. Inducing an active immune response by generating endogenous HER2/neu-specific antibodies and T cells may result in long-lived immunity and, hopefully, therapeutic benefit. In the majority of patients with pre-existent HER2/neu immunity, the antigen-specific antibodies and T cells detected are of low magnitude. Therefore, vaccine strategies aimed at boosting immunity already present may be effective in generating significant levels of HER2/neu-specific antibodies and T cells.

Immunotherapeutic approaches for the treatment of breast cancer.

The application of immunotherapeutic principlesto the treatment and prevention of breast cancer is arelatively new undertaking. Although cytokine infusions,cancer vaccines, and T cell therapy have been extensively studied in solid tumors such asmelanoma and renal cell carcinoma, the therapeuticefficacy of these approaches is not well explored inbreast cancer. The recent definition of tumor-specific immunity in breast cancer patients and theidentification of several breast cancer antigens hasgenerated enthusiasm for the application of immune-basedtherapies to the treatment of breast malignancies. In general, immunotherapies can be consideredeither non-specific, such as a general immunomodulator(e.g., a cytokine), or tumor-specific (e.g., a vaccinethat targets breast cancer tumor antigens). This review describes three major immunotherapeuticstrategies that have the potential to enhance orgenerate an anti-breast cancer T cell immune response:(i) cytokine therapy; (ii) cancer vaccines; and (iii) T cell therapy, and explores how each strategyhas been applied to the treatment of breastcancer.

Adoptive T-cell therapy for the treatment of solid tumours

Solid tumours can be eradicated by infusion of large amounts of tumour-specific T-cells in animal models. The successes seen in preclinical models, however, have not been adequately translated to human disease due, in part, to the inability to expand tumour antigen-specific T-cells ex vivo. Polyclonality and retention of antigen-specificity are two important properties of infused T-cells that are necessary for successful eradication of tumours. Investigators are beginning to evaluate the impact of attempting to reconstitute full T-cell immunity representing both major T-cell subsets, cytolytic T-cells and T-helper (Th) cells. One of the more important and often overlooked steps of successful adoptive T-cell therapy is the ex vivo expansion conditions, which can dramatically alter the phenotype of the T-cell. A number of cytokines and other soluble activation factors that have been characterised over the last decade are now available to supplement in vitro antigen presentation and IL-2. Newer molecular techniques have been developed and are aimed at genetically altering the characteristics of T-cells including their antigen-specificity and growth in vivo. In addition, advanced imaging techniques, such as positron emission tomography (PET), are being implemented in order to better define the in vivo function of ex vivo expanded tumour-specific T-cells.

Delayed type hypersensitivity response to recall antigens does not accurately reflect immune competence in advanced stage breast cancer patients

The development of delayed-type hypersensitivity (DTH) response to recall antigens has long been utilized as a measure of immune competence. It is assumed that because patients with advanced stage cancers exhibit multiple immune system defects they may not be responsive to immunization. We pre-selected patients with advanced HER-2/neu (HER2) overexpressing breast and ovarian cancers for enrolment into a phase I trial designed to evaluate the immunogenicity of a HER2 peptide vaccine based on the patients immune competence as assessed by DTH skin testing to common recall antigens (Multitest CMI, Institut Merieux, Lyon, France). At the time of a positive DTH response to tetanus toxoid (tt) peripheral blood was obtained to measure T cell responses to tt. Of 53 patients evaluated, 38 (72%) were not anergic. Among the 15 (28%) who were, seven patients with advanced stage breast cancer were re-tested a median of 26 days (range 12–150 days) after receiving a tt booster vaccination. Five of the seven had positive DTH responses when re-challenged with tt and six had peripheral blood tetanus specific T cell response with stimulation index > 2.0. Thus, the majority of patients studied with advanced stage breast or ovarian cancer were able to mount a DTH response to common recall antigens. Moreover, a negative response by DTH testing to a battery of common recall antigens was not a reflection of the breast cancer patients ability to mount a cell-mediated immune response to a vaccinated antigen, tt.