RESUMO
The oncoantigen nucleophosmin-anaplastic lymphoma kinase (NPM-ALK) induces cellular and humoral immune responses in patients with NPM-ALK-positive anaplastic large cell lymphoma (ALCL). We characterize the NPM-ALK-specific T-cell responses in a cohort of pediatric and adolescent ALCL-patients in remission without Human Leucocyte Antigen (HLA)-preselection. First, we assessed NPM-ALK-reactive T-cell responses and their HLA-class I restriction in patients by using dendritic cells (DCs) transfected with in vitro transcribed (IVT) NPM-ALK-RNA for CD8 (n = 20) or CD3 (n = 9) T-cell stimulation. NPM-ALK-specific T-cells were detected in twelve of 29 patients (nine of 20 with CD8-selected and three of nine with CD3-selected cells). Recognition of NPM-ALK was restricted by HLA-C alleles in six of eight, and by HLA-B alleles in four of eight analyzed patients. No NPM-ALK-reactivity was detected in 20 healthy individuals. Second, in order to define possible immunogenic NPM-ALK-epitope regions, DCs pulsed with pools of overlapping long NPM-ALK-peptides were used to stimulate T-cells in further 22 patients and ten controls. Responsive T-cells were detected in 15 patients and in five controls. A peptide pool located in the middle of the kinase domain induced ALK-reactive T-cells in 14 of 15 responsive patients. We could narrow to single peptides between p327-p370 of NPM-ALK in four patients. In conclusion, using IVT-RNA, 40% of NPM-ALK-positive ALCL-patients in remission had detectable NPM-ALK-specific T-cell responses which were mainly restricted by HLA-B and -C alleles. Peptide stimulation of T-cells revealed responses in almost 70% of patients and allowed describing an immunogenic region located in the ALK-kinase domain.
RESUMO
Quantification of occult circulating tumour cells in blood or bone marrow (BM) enables the identification of patients with a high risk for relapse in nucleophosmin/anaplastic lymphoma kinase (NPM-ALK)-positive anaplastic large cell lymphoma (ALCL). We have developed a flow cytometric (FCM) assay to quantify the rare ALK- and CD30-positive ALCL cells. When ALCL cells were admixed with normal peripheral blood or BM, ALK- and CD30-positive cells could be detected above background level at an added concentration of 10(-5) for all three cell lines tested. Sensitivity and costs of the assay were compared with quantitative real-time polymerase chain reaction (PCR) for NPM-ALK. The results of the FCM assay and quantitative PCR for NPM-ALK correlated. The sensitivity of the PCR exceeded that of the FCM by at least one log. Quantitative PCR was more time-consuming and expensive than FCM. Both methods were compared on BM or blood samples from 11 ALCL patients. FCM using antibodies against ALK and CD30 can sensitively and specifically detect the circulating ALCL cells in BM or blood. This method needs to be tested in a larger cohort of patients to determine whether it has sufficient sensitivity to be used as a substitute for quantitative PCR.