WT1, PRAME, and PR3 mRNA Expression in Acute Myeloid Leukemia (AML).

Several tumor-associated antigens (TAAs) were recently identified, that could qualify as targets for immunotherapy, they could qualify (on RNA-level) for monitoring of tumor load. Here, we studied the expression levels of the immunogenic antigens PRAME (preferentially expressed antigen of melanoma), WT1 (Wilms' tumor gene), and PR3 (proteinase 3) on myeloid blasts by real-time quantitative polymerase chain reaction and correlated these data to the state and course of disease and to the defined subgroups of acute myeloid leukemia (AML). At first diagnoses, 41 of 47 patients tested showed overexpression of PRAME (87%), 38 of WT1 (81%), and 26 of PR3 (55%), with the highest expression levels for PRAME (2048-fold), followed by WT1 (486-fold) and PR3 (196-fold). Thereby, with 70%, the most frequent combination at first diagnoses was detected to be PRAME and WT1 (33/47 patients). Overall, 21 patients (45%) revealed overexpression for all 3 TAAs. Moreover, the highest expression levels of PRAME were found to be correlated with the FAB subtype M5, cytogenetic unfavorable risk groups, and AMLs arising from myelodysplasia (secondary AML; P=0.02). To compare TAA expression levels in the course of disease, expression data were calculatory adjusted to 100% blasts, revealing a relative increase in the PRAME expression levels during the course of persistent disease (3/4 cases). Independent of stage of disease, by trend, higher TAA expression levels were found on blasts derived from peripheral blood than those derived from the bone marrow. In conclusion, it is suggested that vaccine strategies for cancer immunotherapy should comprise different TAA peptides anticipating the diverse TAA expression levels on blasts evolving during the course of disease or treatment.

Significance of Frequencies, Compositions, and/or Antileukemic Activity of (DC-stimulated) Invariant NKT, NK and CIK Cells on the Outcome of Patients With AML, ALL and CLL.

Invariant natural killer T (iNKT)/natural killer (NK)/cytokine-induced killer (CIK) cells are important for immune surveillance. (I) Novel combinations of antibody 6B11 (targeting the Vα24-Jα18-invariant T-cell receptor) with CD4/CD8/CD1d/Vα24 for iNKT subset detection and "T/NK cell-like"-iNKT subsets were defined. Compared with healthy peripheral blood mononuclear cells (MNC) (significantly) lower proportions of iNKT cells (6B11/6B11CD3/6B11CD161), NK cells (CD3CD56/CD3CD161), and CIK cells (CD3CD56/CD3CD161) were found in peripheral blood MNC from acute myeloid (AML)/acute myeloid, lymphoid (ALL)/chronic lymphoid leukemia (CLL) patients in acute disease stages. Subtyping of iNKT cells revealed (significantly) higher proportions of CD3 T cells and CD161 NK cells in AML/ALL/CLL expressing 6B11 compared with healthy MNC. Prognostic evaluations showed higher proportions of iNKT/NK/CIK cells in favorable AML subgroups (younger age, primary, no extramedullary disease, achievement/maintenance of complete remission) or adult ALL and CLL patients. (II) iNKT/NK/CIK cell frequencies increased after (vs. before) mixed lymphocyte cultures of T-cell-enriched immune reactive cells stimulated with MNC/whole blood with or without pretreatment with "cocktails" (dendritic cells generating methods/kits inducing blasts' conversion to leukemia-derived dendritic cells from AML patients). Individual "cocktails" leading to "highest" iNKT cell frequencies could be defined. Antileukemic blast lytic activity correlated significantly with frequencies of iNKT/NK/CIK cells. In summary healthy MNC show significantly more iNKT/NK/CIK cells compared with AML/ALL/CLL MNC, a shift in the iNKT cell composition is seen in healthy versus leukemic samples and iNKT/NK/CIK cell-proportions in AML/ALL/CLL MNC samples correlate with prognosis. "Cocktail"-treated AML blasts lead to higher iNKT/NK/CIK cell frequencies and samples with antileukemic activity show significantly higher frequencies of iNKT/NK/CIK cells. Proportions of iNKT/NK/CIK cells should regularly be evaluated in AML/ALL/CLL diagnosis panels for quantitative/prognostic estimation of individual patients' antileukemic potential and their role in dendritic cells/leukemia-derived dendritic cells triggered immune surveillance.