RESUMEN
Purpose: Triple-negative breast cancer (TNBC) represents a cancer stem cell-enriched phenotype. Hypoxia-inducible factor-1α (HIF-1α) induces the expression of proteins associated with stemness and is highly upregulated in TNBC. We questioned whether HIF-1α was immunogenic and whether vaccination targeting HIF-1α would impact the growth of basal-like mammary tumors in transgenic mice.Experimental Design: We evaluated HIF-1α-specific IgG in sera from controls and patients with breast cancer. Class II epitopes derived from the HIF-1α protein sequence were validated by ELISPOT. To assess therapeutic efficacy, we immunized Tg-MMTVneu and C3(1)Tag mice with HIF-1α Th1-inducing peptides. Stem cells were isolated via magnetic bead separation. Levels of HIF-1α and stem cells in the tumor were quantitated by Western blotting and flow cytometry.Results: The magnitude (P < 0.001) and incidence (P < 0.001) of HIF-1α-specific IgG were elevated in TNBC patients compared with controls. Both breast cancer patients and donors showed evidence of HIF-1α-specific Th1 and Th2 immunity. Three HIF-1α-specific Th1 class II restricted epitopes that were highly homologous between species elicited type I immunity in mice. After HIF-1α vaccination, mammary tumor growth was significantly inhibited in only C3(1)Tag (basal-like/stem cellhigh; P < 0.001) not TgMMTV-neu (luminal/neu/stem celllow; P = 0.859) murine models. Vaccination increased type I T cells in the tumor (P = 0.001) and decreased cells expressing the stem cell marker, Sca-1, compared with controls (P = 0.004).Conclusions: An HIF-1α vaccine may be uniquely effective in limiting tumor growth in TNBC. Inhibiting outgrowth of breast cancer stem cells via active immunization in the adjuvant setting may impact disease recurrence. Clin Cancer Res; 23(13); 3396-404. ©2016 AACR.
Asunto(s)
Vacunas contra el Cáncer/administración & dosificación , Subunidad alfa del Factor 1 Inducible por Hipoxia/inmunología , Neoplasias Mamarias Animales/terapia , Neoplasias de la Mama Triple Negativas/terapia , Animales , Vacunas contra el Cáncer/inmunología , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Femenino , Humanos , Subunidad alfa del Factor 1 Inducible por Hipoxia/sangre , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Inmunización , Inmunoglobulina G/sangre , Inmunoglobulina G/inmunología , Neoplasias Mamarias Animales/sangre , Neoplasias Mamarias Animales/inmunología , Neoplasias Mamarias Animales/patología , Ratones , Recurrencia Local de Neoplasia/sangre , Recurrencia Local de Neoplasia/inmunología , Recurrencia Local de Neoplasia/terapia , Células Madre Neoplásicas/inmunología , Neoplasias de la Mama Triple Negativas/sangre , Neoplasias de la Mama Triple Negativas/inmunología , Neoplasias de la Mama Triple Negativas/patologíaRESUMEN
Recent studies in patients with breast cancer suggest the immune microenvironment influences response to therapy. We aimed to evaluate the relationship between growth rates of tumors in common spontaneous mammary tumor models and immune biomarkers evaluated in the tumor and blood. TgMMTV-neu and C3(1)-Tag transgenic mice were followed longitudinally from birth, and MPA-DMBA-treated mice from the time of carcinogen administration, for the development of mammary tumors. Tumor-infiltrating CD4(+) and CD8(+) T-cells, FOXP3(+) T-regulatory cells, and myeloid-derived suppressor cells were assessed by flow cytometry. Serum cytokines were evaluated in subsets of mice. Fine needle aspirates of tumors were collected and RNA was isolated to determine levels of immune and proliferation markers. Age of tumor onset and kinetics of tumor growth were significantly different among the models. Mammary tumors from TgMMTV-neu contained a lower CD8/CD4 ratio than that of other models (p < 0.05). MPA-DMBA-induced tumors contained a higher percentage of FOXP3(+) CD4(+) T-cells (p < 0.01) and MDSC (p < 0.001) compared with the other models. Individuals with significantly slower tumor growth demonstrated higher levels of Type I serum cytokines prior to the development of lesions compared to those with rapid tumor growth. Moreover, the tumors of animals with more rapid tumor growth demonstrated a significant increase in the expression of genes associated with Type II immunity than those with slower-progressing tumors. These data provide a foundation for the development of in vivo models to explore the relationship between endogenous immunity and response to standard therapies for breast cancer.
Asunto(s)
Biomarcadores de Tumor/inmunología , Neoplasias de la Mama/inmunología , Inmunomodulación , Neoplasias Mamarias Animales/genética , Animales , Biomarcadores de Tumor/sangre , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/patología , Proliferación Celular/genética , Femenino , Factores de Transcripción Forkhead/genética , Humanos , Linfocitos Infiltrantes de Tumor/inmunología , Linfocitos Infiltrantes de Tumor/patología , Neoplasias Mamarias Animales/inmunología , Neoplasias Mamarias Animales/patología , RatonesRESUMEN
The enzyme-linked immunosorbent spot (ELISpot) assay is one of the most commonly used methods to measure antigen-specific T cells in both mice and humans. Some of the primary reasons for the popularity of the method are that ELISpot is highly quantitative, can measure a broad range of magnitudes of response and is capable of assessing critical cellular immune-related activities such as IFN-γ secretion and granzyme B release. Furthermore, ELISpot is adaptable not only to the evaluation of a variety of T-cell functions, but also to B cells and innate immune cells. It is no wonder that ELISpot has evolved from a research tool to a clinical assay. Recent Phase I and II studies of cancer vaccines, tested in a variety of malignancies, have suggested that ELISpot may be a useful biomarker assay to predict clinical benefit after therapeutic immune modulation. This article will discuss the most common applications of ELISpot, overview the efforts that have been undertaken to standardize the assay and apply the method in the analysis of human clinical trials, and describe some important steps in the process of developing a clinical-grade ELISpot.
Asunto(s)
Ensayo de Immunospot Ligado a Enzimas/métodos , Ensayo de Immunospot Ligado a Enzimas/normas , Linfocitos T/inmunología , Vacunas/inmunología , Antígenos/inmunología , Vacunas contra el Cáncer/inmunología , Vacunas contra el Cáncer/uso terapéutico , Ensayos Clínicos como Asunto/métodos , Granzimas/metabolismo , Humanos , Interferón gamma/metabolismoRESUMEN
PURPOSE: The primary objectives of this phase I/II study were to evaluate the safety and immunogenicity of combination therapy consisting of concurrent trastuzumab and human epidermal growth factor receptor 2 (HER2)/neu-specific vaccination in patients with HER2/neu-overexpressing metastatic breast cancer. PATIENTS AND METHODS: Twenty-two patients with stage IV HER2/neu-positive breast cancer receiving trastuzumab therapy were vaccinated with an HER2/neu T-helper peptide-based vaccine. Toxicity was graded according to National Cancer Institute criteria, and antigen specific T-cell immunity was assessed by interferon gamma enzyme-linked immunosorbent spot assay. Data on progression-free and overall survival were collected. RESULTS: Concurrent trastuzumab and HER2/neu vaccinations were well tolerated, with 15% of patients experiencing an asymptomatic decline in left ventricular ejection fraction below the normal range during combination therapy. Although many patients had pre-existing immunity specific for HER2/neu and other breast cancer antigens while treated with trastuzumab alone, that immunity could be significantly boosted and maintained with vaccination. Epitope spreading within HER2/neu and to additional tumor-related proteins was stimulated by immunization, and the magnitude of the T-cell response generated was significantly inversely correlated with serum transforming growth factor beta levels. At a median follow-up of 36 months from the first vaccine, the median overall survival in the study population has not been reached. CONCLUSION: Combination therapy with trastuzumab and a HER2/neu vaccine is associated with minimal toxicity and results in prolonged, robust, antigen-specific immune responses in treated patients.
Asunto(s)
Anticuerpos Monoclonales/uso terapéutico , Neoplasias de la Mama/terapia , Receptor ErbB-2/inmunología , Vacunación/métodos , Adulto , Anciano , Anticuerpos Monoclonales/efectos adversos , Anticuerpos Monoclonales Humanizados , Antineoplásicos/efectos adversos , Antineoplásicos/uso terapéutico , Neoplasias de la Mama/inmunología , Neoplasias de la Mama/patología , Vacunas contra el Cáncer/administración & dosificación , Vacunas contra el Cáncer/inmunología , Terapia Combinada , Fatiga/etiología , Femenino , Cefalea/etiología , Humanos , Estimación de Kaplan-Meier , Persona de Mediana Edad , Datos de Secuencia Molecular , Metástasis de la Neoplasia , Dolor/etiología , Péptidos/química , Péptidos/inmunología , Receptor ErbB-2/química , Linfocitos T/efectos de los fármacos , Linfocitos T/inmunología , Factor de Crecimiento Transformador beta/sangre , Trastuzumab , Resultado del Tratamiento , Vacunación/efectos adversosRESUMEN
BACKGROUND: Standardization of cell-based immunologic monitoring is becoming increasingly important as methods for measuring cellular immunity become more complex. We assessed the ability of two commonly used cell-based assays, tritiated thymidine incorporation (proliferation) and IFN-gamma ELISPOT, to predict T cell responses to HER-2/neu, tetanus toxoid (tt), and cytomegalovirus (CMV) antigens. These antigens were determined to be low (HER-2/neu), moderate (tt), and robustly (CMV) immunogenic proteins. Samples from 27 Stage II, III, and IV HER-2/neu positive breast cancer patients, vaccinated against the HER-2/neu protein and tt, were analyzed by tritiated thymidine incorporation and IFN-gamma ELISPOT for T cell response. RESULTS: Linear regression analysis indicates that both stimulation index (SI) (p = 0.011) and IFN-gamma secreting precursor frequency (p < 0.001) are significant indicators of antigen specific immunity. ROC curves plotted to assess the performance of tritiated thymidine incorporation and the ELISPOT assay indicate that SI is a significant indicator of low T cell response to the HER-2/neu vaccine (p = 0.05), and of moderate and robust responses to tt (p = 0.01) and CMV (p = 0.016), respectively. IFN-gamma precursor frequency is a significant indicator of a robust T cell response to CMV (p = 0.03), but not of moderate tt (p = 0.09), or low HER-2/neu (p = 0.09) T cell responses. CONCLUSION: These data underscore the importance of taking into consideration the performance characteristics of assays used to measure T cell immunity. This consideration is particularly necessary when determining which method to utilize for assessing responses to immunotherapeutic manipulations in cancer patients.