Purpose: NK cells exhibit cytotoxicity against neuroblastoma. Gene polymorphisms governing NK cell function, therefore, may influence prognosis. Two highly polymorphic genetic loci instrumental in determining NK cell responses encode the NK cell killer immunoglobulin-like receptors (KIR) and their class I human leukocyte antigen (HLA) ligands. We hypothesized that patients with a "missing ligand" KIR-HLA compound genotype may uniquely benefit from autologous hematopoietic stem cell transplantation (HSCT). Experimental Design: One hundred sixty-nine patients treated with autologous HSCT for stage IV neuroblastoma underwent KIR and HLA genotyping. Patients were segregated according to the presence or absence of HLA ligands for autologous inhibitory KIR. Univariate and multivariate analyses were done for overall and progression-free survival. Results: Sixty-four percent of patients lacked one or more HLA ligands for inhibitory KIR. Patients lacking a HLA ligand had a 46% lower risk of death [hazard ratio, 0.54; 95% confidence interval (95% CI), 0.35-0.85; P = 0.007] and a 34% lower risk of progression (hazard ratio, 0.66; 95% CI, 0.44-1.0; P = 0.047) at 3 years compared with patients who possessed all ligands for his/her inhibitory KIR. Among all KIR-HLA combinations, 16 patients lacking the HLA-C1 ligand for KIR2DL2/KIR2DL3 experienced the highest 3-year survival rate of 81% (95% CI, 64-100). Survival was more strongly associated with "missing ligand" than with tumor MYCN gene amplification. Conclusion: KIR-HLA immunogenetics represents a novel prognostic marker for patients undergoing autologous HSCT for high-risk neuroblastoma. (Clin Cancer Res 2009;15(23):7330-4) Neuroblastoma is the most frequently diagnosed cancer in infants and is the most common extracranial solid tumor in childhood (1). Prognosis varies, and risk assessment is based on several clinical and biological features, including age, stage at diagnosis, histopathology, and biomarkers of tumor aggressiveness (MYCN status, histology, and DNA ploidy; refs. 2,3). The risk of tumor progression likely depends not only on tumor biology but also on the host immune response. NK cells are capable of inhibiting colony formation of human neuroblastoma cells (4-7), and infusion of NK cells into nonobese diabetic/severe combined immunodeficient mice bearing human metastatic neuroblastoma leads to a significant improvement in overall survival (4). Based on recent advances linking NK cell function to NK cell genetics (8-11), we hypothesized that variations in genes responsible for regulating NK function may affect clinical outcomes for patients with neuroblastoma.The killer immunoglobulin-like receptor (KIR) gene cluster consists of 15 genes that encode both inhibitory and activating NK cell surface receptors instrumental in governing NK cell function. The similarly polymorphic human leukocyte antigen (HLA) class I gene loci encode three ligand groups for inhibitory KIR: HLA for KIR2DL2/KIR2DL3, HLA for KIR2DL1, and HLA for 12KIR3DL1. Ligation of inhi...

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Purpose: ESO is a tumor-specific antigen with wide expression in human tumors of different histologic types and remarkable spontaneous immunogenicity. We have previously shown that specific T H 1 and antibody responses can be elicited in patients with no detectable preexisting immune responses by vaccination with rESO administered with Montanide ISA-51 and CpG ODN 7909. The purpose of the present study was to characterize vaccine-induced ESO-specific CD4 + Tcell responses. Experimental Design: We generated CD4 + T cell clones from patient C2, who had the highest CD4 + Tcell response to the vaccine, and analyzed their fine specificity and HLA class II restriction to determine the recognized epitope.We then assessed the response to the identified epitope in all vaccinated patients expressing the corresponding HLA class II allele. Results: We found that ESO-specific CD4 + T cell clones from patient C2 recognize peptide ESO 119-143 (core region 123-137) presented by HLA-DR52b (HLA-DRB3*0202), a MHC class II allele expressed by about half of Caucasians. Importantly, following vaccination, all patients expressing DR52b developed significant responses to the identified epitope, accounting for, on average, half of the total CD4 + T cell responses to the 119-143 immunodominant region. In addition, analysis of ESO-specific DR52b-restricted CD4 + T cells at the clonal level revealed significant conservation of T cell receptor usage among different individuals. Conclusions: The identification of a DR52b-restricted epitope from ESO that is immunodominant in the context of vaccine-elicited immune responses is instrumental for the immunologic monitoring of vaccination trials targeting this important tumor antigen.

Purpose: Vaccination with full-length human tumor antigens aims at inducing or increasing antitumor immune responses, including CD8 CTL in cancer patients across the HLA barrier. We have recently reported that vaccination with a recombinant tumor-specific NY-ESO-1 (ESO) protein, administered with Montanide and CpG resulted in the induction of specific integrated antibody and CD4 T cell responses in all vaccinated patients examined, and significant CTL responses in half of them. Vaccine-induced CTL mostly recognized a single immunodominant region . The purpose of the present study was to identify genetic factor(s) distinguishing CTL responders from nonresponders. Experimental Design: We determined the HLA class I alleles expressed by CTL responders and nonresponders using high-resolution molecular typing. Using short overlapping peptides spanning the ESO immunodominant CTL region and HLA class I/ESO peptide tetramers, we determined the epitopes recognized by the majority of vaccine-induced CTL. Results: CTL induced by vaccination with ESO protein mostly recognized distinct but closely overlapping epitopes restricted by a few frequently expressed HLA-B35 and HLA-Cw3 alleles. All CTL responders expressed at least one of the identified alleles, whereas none of the nonresponders expressed them. Conclusions: Expression of HLA-B35 and HLA-Cw3 is associated with the induction of immunodominant CTL responses following vaccination with recombinant ESO protein. Because recombinant tumor-specific proteins are presently among the most promising candidate anticancer vaccines, our findings indicate that the monitoring of cancer vaccine trials should systematically include the assessment of HLA association with responsiveness.

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