RESUMO
OBJECTIVES: Cancer-testis (CT) antigens are expressed in tumors but not normal tissues except the testis and could be targets for vaccine therapy in epithelial ovarian cancer (EOC). A-kinase anchoring protein 3 (AKAP-3) is a novel member of the CT antigen family. The aim of this study was to examine the expression of AKAP-3 in EOC and correlate with clinico-pathologic characteristics. METHODS: One step RT-PCR was performed with RNA from normal and ovarian cancer cell lines and 74 epithelial ovarian tumor tissues. AKAP-3-specific PCR product was amplified. The distribution of AKAP-3 expression and clinico-pathologic variables was analyzed. Survival distributions were estimated, and multivariate analyses were performed. RESULTS: AKAP-3 mRNA expression was demonstrated in 43/74 (58%) of the ovarian cancer specimens. AKAP-3 was expressed in normal testis, but not in other normal tissues. AKAP-3 expression significantly correlated with increased likelihood of residual tumor (P = 0.005), but no increase in the likelihood of recurrence or persistent disease (P = 0.06). Patients with AKAP-3 mRNA expression were found to have a significantly poorer overall survival (median 50 months) compared with patients without AKAP-3 expression (median not reached) (P = 0.007). Multivariate analysis of AKAP-3 expression, residual disease, and response to frontline chemotherapy found response to be the strongest predictor of overall survival (P = 0.012). CONCLUSIONS: Our data demonstrate that AKAP-3 is expressed at high frequency in patients with EOC. Since AKAP-3 demonstrates tumor-restricted expression and appears to be associated with worse overall survival, it could represent an attractive target for antigen-specific immunotherapy in EOC.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Neoplasias Ovarianas/genética , RNA Mensageiro/biossíntese , Proteínas de Ancoragem à Quinase A , Proteínas Adaptadoras de Transdução de Sinal/biossíntese , Adulto , Idoso , Idoso de 80 Anos ou mais , Células Epiteliais/patologia , Feminino , Humanos , Pessoa de Meia-Idade , Estadiamento de Neoplasias , Neoplasias Ovarianas/metabolismo , Neoplasias Ovarianas/patologia , RNA Mensageiro/genética , Taxa de SobrevidaRESUMO
NY-ESO-1 is one of the most immunogenic cancer antigens known to date, eliciting spontaneous immune responses in approximately 50% of patients with NY-ESO-1+ cancers. Spontaneous CD4+ and CD8+ T cell responses were found in patients with detectable NY-ESO-1 serum antibody, indicating an integrated type of immune response induced by NY-ESO-1+ malignancies. A close association between spontaneous NY-ESO-1 immunity and the HLA-DP4 allele was suggested in a recent study. To address these results, we assessed the NY-ESO-1 antibody and HLA-DP4 status of 102 patients with NY-ESO-1+ malignancies. However, no correlation between HLA-DP4 and NY-ESO-1 immunity was found. To explore the role of HLA-DP4-restricted CD4+ T cells in cancer immunity, we established HLA-DP4- restricted NY-ESO-1-specific CD4+ T cell clones by limiting dilution and repeated stimulation with NY-ESO-1 peptide p157-170 from NY-ESO-1 seropositive patients. A subset of CD4+ T cell clones was reactive with naturally processed NY-ESO-1 presented by autologous DCs that were pulsed with recombinant NY-ESO-1 protein, lysates of NY-ESO-1-expressing tumor cell lines, or transduced with recombinant NY-ESO-1 viral constructs in ELISPOT assays. Three different CD4+ T cell clones were used to mediate the specific lysis of allogeneic HLA-DP4+ Epstein-Barr virus-transformed B cells (EBV-B) pulsed with NY-ESO-1 p157-170. The Th1 phenotype and effector functions of the CD4+ T cell clones described here provide an important rationale for the activation of antigen-specific CD4+ T cells along with CD8+ T cells in cancer vaccination strategies.
Assuntos
Antígenos de Neoplasias/imunologia , Linfócitos T CD4-Positivos/química , Linfócitos T CD4-Positivos/fisiologia , Linfócitos T CD8-Positivos/química , Linfócitos T CD8-Positivos/fisiologia , Antígenos HLA-DP/biossíntese , Proteínas de Membrana/imunologia , Receptores de Interleucina-2/biossíntese , Linfócitos T Citotóxicos/química , Linfócitos T Citotóxicos/fisiologia , Células Apresentadoras de Antígenos/metabolismo , Antígenos de Neoplasias/sangue , Antígenos de Neoplasias/genética , Linfócitos T CD4-Positivos/metabolismo , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Linhagem Celular Tumoral , Células Clonais/química , Células Clonais/metabolismo , Células Clonais/fisiologia , Feminino , Regulação Neoplásica da Expressão Gênica/imunologia , Regulação Neoplásica da Expressão Gênica/fisiologia , Antígenos HLA-DP/genética , Cadeias beta de HLA-DP , Humanos , Imunofenotipagem/métodos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Melanoma/genética , Melanoma/metabolismo , Proteínas de Membrana/sangue , Proteínas de Membrana/genética , Neoplasias/genética , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/metabolismo , Subpopulações de Linfócitos T/química , Subpopulações de Linfócitos T/citologia , Subpopulações de Linfócitos T/metabolismoAssuntos
Academias e Institutos/organização & administração , Ensaios Clínicos como Assunto/métodos , Aprovação de Drogas/métodos , Imunoterapia/tendências , Neoplasias/terapia , Aprovação de Drogas/organização & administração , Humanos , Imunoterapia/métodos , Propriedade Intelectual , National Institutes of Health (U.S.) , Estados Unidos , United States Food and Drug AdministrationRESUMO
Using the recently developed ELISPOT cloning methodology, we obtained cDNA clone S35 coding for the Ag epitope recognized by a murine sarcoma Meth A-specific CTL clone AT-1. Analysis of truncated S35 constructs and overlapping peptides revealed that the peptide epitope was LGAEAIFRL. AT-1 CTL lysed peptide-pulsed CMS8 cells at a nanomolar concentration, and the peptide strongly stimulated IFN-gamma production in AT-1 CTL. Sequence homology indicated that the S35 was derived from a mouse homologue of human retinoic acid-regulated nuclear matrix-associated protein (ramp). The ramp gene consisted of 15 exons. The majority of the ramp mRNA was the transcript normally spliced between exons 14 and 15, but a minor population of mRNA with an extended exon 14 was also present in Meth A cells. The epitope was derived from the newly created open reading frame, which resulted from extension of exon 14 after splicing of the adjacent intronic sequence.
Assuntos
Epitopos de Linfócito T/biossíntese , Epitopos de Linfócito T/genética , Éxons/genética , Metilcolantreno , Sarcoma Experimental/imunologia , Linfócitos T Citotóxicos/imunologia , Sequência de Aminoácidos , Animais , Antígenos de Neoplasias/biossíntese , Antígenos de Neoplasias/genética , Antígenos de Neoplasias/isolamento & purificação , Antígenos de Neoplasias/metabolismo , Sequência de Bases , Proteínas de Transporte/biossíntese , Proteínas de Transporte/genética , Proteínas de Transporte/isolamento & purificação , Proteínas de Transporte/metabolismo , Células Clonais , Clonagem Molecular , Testes Imunológicos de Citotoxicidade , Proteínas de Ligação a DNA/biossíntese , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/isolamento & purificação , Proteínas de Ligação a DNA/metabolismo , Ensaio de Imunoadsorção Enzimática , Epitopos de Linfócito T/isolamento & purificação , Epitopos de Linfócito T/metabolismo , Éxons/imunologia , Biblioteca Gênica , Humanos , Interferon gama/análise , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos DBA , Dados de Sequência Molecular , Sarcoma Experimental/induzido quimicamente , Fatores de TranscriçãoRESUMO
NY-ESO-1 is one of the most immunogenic cancer antigens known to date, inducing humoral and cellular immune responses in a high proportion of patients with advanced NY-ESO-1-expressing cancers. The assessment of spontaneous and vaccine-induced CD8+ T cell responses has been limited to a small number of known NY-ESO-1 epitopes presented by MHC class I alleles. Recently, a new method to monitor NY-ESO-1-specific CD8+ T cell responses was introduced that does not depend on the individual MHC class I status and on predefined peptide epitopes. Antigen-presenting cells transduced with recombinant adenoviral vectors encoding NY-ESO-1 were used to stimulate CD8+ selected NY-ESO-1-specific T cells. Effector cells were tested for recognition of autologous B cell targets transfected with NY-ESO-1 using a recombinant vaccinia virus construct. Using a modified approach we identified the NY-ESO-1 p94-102 peptide as being recognized by CD8+ T cells in the context of HLA- B51. NY-ESO-1 p94-102 specific CD8+ T cells recognized naturally processed NY-ESO-1 presented by HLA-B51+ monocyte-derived dendritic and tumor cells. Transfection of target cells with NY-ESO-1 combined with different HLA class I alleles confirmed that the NY-ESO-1 peptide was naturally processed and recognized by HLA-B51-restricted CD8+ T cell lines and clones. Therefore, NY-ESO-1 p94-102 is a new candidate peptide antigen for cancer immunotherapy and for the monitoring of spontaneous and vaccine-induced NY-ESO-1-specific T cell responses in HLA- B51+ patients with NY-ESO-1 expressing malignancies.
