title: Real-time quantitative polymerase chain reaction assessment of immune reactivity in melanoma patients after tumor peptide vaccination.
abstract: BACKGROUND
  Monitoring the immune response to epitope-specific vaccination in cancer patients is important for vaccine development. The traditional method, in which the in vitro sensitization of peripheral blood mononuclear cells (PBMCs) with epitope is compared before and after vaccination, is time-consuming and allows only a qualitative assessment of the response. We used a rapid, quantitative, real-time polymerase chain reaction (PCR) assay to directly measure the immune reactivity of patients' PBMCs to the vaccine epitope.
  
  
  METHODS
  PBMCs were obtained from melanoma patients before and after two rounds of vaccination with either g209-2M, a peptide derived from melanoma protein gp100 (n = 24), or ESg209-2M, a modified version of this peptide (n = 20). PBMCs were tested for immune reactivity by assaying interferon gamma (IFN gamma) protein release after in vitro sensitization with the epitope or for IFN gamma messenger RNA expression by real-time PCR. A twofold or more increase in IFN gamma protein release or a 1.5-fold or more increase in IFN gamma transcript accumulation in PBMCs after vaccination was considered to be evidence of a specific response. Correlation between the two methods was tested by use of the Spearman correlation coefficient after the results were ranked as positive or negative. All statistical tests were two-sided.
  
  
  RESULTS
  The results obtained with the two methods were strongly correlated (Spearman's rho = 0.72; P =.0006). The g209-2M and Esg209-2M peptides resulted in similar percentages of vaccine-specific reactivity in PBMCs after in vitro sensitization (63% and 65% of patients, respectively; Fisher's exact test P =.6 for comparison of the two groups). The PCR method could detect vaccine-specific reactivity in a subset of patients (38% and 35% of patients, respectively; Fisher's exact test P =.7 for comparison of the two groups).
  
  
  CONCLUSIONS
  Vaccination induces circulating antitumor lymphocytes, albeit in low frequencies, capable of directly reacting with tumor antigen. PBMCs of vaccinated individuals can respond to a vaccine-specific stimulus in a direct assay that does not require prolonged in vitro manipulations.
authors.affiliation: Surgery Branch, Division of Clinical Sciences, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892-1184, USA.
authors.forename: U S
authors.initials: US
authors.lastname: Kammula
authors.forename: F M
authors.initials: FM
authors.lastname: Marincola
authors.forename: S A
authors.initials: SA
authors.lastname: Rosenberg
mesh_terms: D016827:CD8 Antigens / Q000276:immunology; D019496:Cancer Vaccines / Q000008:administration & dosage*; D000939:Epitopes; D006801:Humans; D007371:Interferon-gamma / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism; D007963:Leukocytes, Mononuclear / Q000276:immunology*; D008545:Melanoma / Q000276:immunology* / Q000628:therapy*; D016133:Polymerase Chain Reaction / Q000379:methods*; D012333:RNA, Messenger / Q000032:analysis; D012878:Skin Neoplasms / Q000276:immunology* / Q000628:therapy*; D014407:Tumor Cells, Cultured
chemical_list: D016827:CD8 Antigens; D019496:Cancer Vaccines; D000939:Epitopes; D012333:RNA, Messenger; D007371:Interferon-gamma