The role of Wilms' tumor gene peptide-specific cytotoxic T lymphocytes in immunologic selection of a paroxysmal nocturnal hemoglobinuria clone.

OBJECTIVE: To clarify an expansion mechanism of a paroxysmal nocturnal hemoglobinuria (PNH) clone with the Wilms' tumor gene (WT1). MATERIALS AND METHODS: In PNH patients with the HLA-A*2402 allele, frequencies of peripheral blood (PB) WT1 peptide-specific and HLA-A*2402-restricted CD8+ cells and WT1 peptide-stimulated interferon-gamma-producing mononuclear cells (MNCs), cytotoxicity of WT1 peptide-specific and HLA-A*2402-restricted cytotoxic T lymphocyte (CTL) clone (TAK-1) cells on bone marrow (BM) MNCs, and after co-incubation with TAK-1 cells, changes in colony-forming unit granulocyte-macrophage colony formation of CD34+ cells and in CD59 expression in viable CD34+ cells were investigated. RESULTS: The frequencies of PB WT1 peptide-specific and HLA-A*2402-restricted CD8+ cells (p < 0.005) and WT1 peptide-stimulated interferon-gamma-producing MNCs (p < 0.02) were significantly higher in 5 PNH patients than 8 healthy volunteers (HV). In 5 PNH patients or 3 HV, TAK-1 cells significantly killed BMMNCs and suppressed colony formations of CD34+CD59+ and/or CD34+CD59- cells in the absence and presence of a WT1 peptide or only in the presence of the peptide, respectively, in an HLA-restricted manner. After co-incubation with TAK-1 cells, reduction rates of colony formation of CD34+CD59- cells were significantly less than those of CD34+CD59+ cells in 5 PNH patients (p < 0.002) and proportions of viable CD34+CD59- cells from 5 PNH patients significantly increased in the absence (p < 0.01) and presence (p < 0.01) of a WT1 peptide in an HLA-restricted manner. CONCLUSION: WT1 peptide-specific and HLA-restricted CTLs may play an important role in expansion of a PNH clone during immunologic selection and/or in the occurrence of BM failure via interferon-gamma in PNH.

Monitoring minimal residual disease in leukemia using real-time quantitative polymerase chain reaction for Wilms tumor gene (WT1).

We previously showed that Wilms tumor gene (WT1) expression level, measured by quantitative reverse transcriptase polymerase chain reaction (RT-PCR), was useful as an indicator of minimal residual disease (MRD) in leukemia and myelodysplastic syndrome. However, in conventional quantitative RT-PCR (CQ-PCR), RT-PCR must be performed for various numbers of cycles depending on WT1 expression level. In the present study, we developed a new real-time quantitative RT-PCR (RQ-PCR) method for quantitating WT1 transcripts. Results of intraassay and interassay variability tests demonstrated that the real-time WT1 assay had high reproducibility. WT1 expression levels measured by the RQ- and the CQ-PCR methods were strongly correlated (r = 0.998). Furthermore, a strong correlation was observed among WT1 transcript values normalized with 3 different control genes (beta-actin, ABL, and glyceraldehyde-3-phosphate dehydrogenase) and between relative WT1 transcript values with WT1 expression in K562 cells as the reference and absolute WT1 transcript copy numbers per microgram RNA. When WT1 expression and minor bcr-abl expression were concurrently monitored in 2 patients with bcr-abl-positive acute lymphoblastic leukemia, both MRDs changed mostly in parallel, indicating the reliability and validity of our RQ-PCR method. In conclusion, this RQ-PCR method is convenient and reliable for monitoring MRD and enables routine clinical use of a WT1 assay.

Localized relapse in bone marrow in a posttransplantation patient with t(6;9) acute myeloid leukemia.

We report a 38-year-old woman with t(6;9) acute myeloid leukemia who relapsed with localized leukemic cell growth in the bone marrow after she had undergone allogeneic bone marrow transplantation. The localized cell growth was first recognized by an apparent discrepancy in the DEK-CAN fusion transcript levels between the aspirates from the left and right iliac bone marrow. Magnetic resonance imaging of the iliac bone revealed localized cell accumulation in the left side. The nonhomogeneous and localized leukemic cell growth in this case may have been due to the graft-versus-leukemia effect following allogeneic transplantation with donor lymphocyte infusion. Serial monitoring of molecular markers for leukemia at different sites or magnetic resonance imaging of the bone marrow may be of value in detecting this type of relapse.

Enzyme immunoassay detection of platelet-derived microparticles and RANTES in acute coronary syndrome.

Platelet-derived microparticles (PDMPs) are produced by platelet activation or physical stimulation under various conditions. To evaluate changes in platelet and chemokine function in patients undergoing percutaneous transluminal coronary angioplasty (PTCA), we measured and compared levels of PDMPs and a C-C chemokine, regulated on activation normally T-cell express and secreted (RANTES), by ELISA. Levels of PDMP and RANTES in patients with acute coronary syndrome were significantly higher than those in the control groups (PDMP: 20.1 +/- 2.9 vs 80.4 +/- 7.3 U/ml, p < 0.001; RANTES: 18.6 +/- 3.7 vs 52.1 +/- 4.6 ng/ml, p < 0.01), but did not differ between the control groups and patients with stable angina. PDMP levels were higher in patients with acute myocardial infarction (AMI) than in patients with unstable angina (PDMP: 115.0 +/- 7.1 vs 63.9 +/- 6.2 U/ml, p < 0.001). There was no difference in the RANTES levels, however, between patients with AMI and patients with unstable angina. PDMP and RANTES levels were significantly decreased after PTCA (PDMP, p < 0.001; RANTES, p < 0.05), but without differences between the two groups. In addition, the level of PDMP was significantly correlated with that of RANTES or soluble CD40 ligand. These findings suggest that PTCA may prevent the development of AMI-associated complications in which activated platelets and RANTES play roles. Our ELISA method appears to be sufficient for monitoring PDMP and RANTES levels after PTCA in patients with acute coronary syndrome.

HLA class II haplotype and quantitation of WT1 RNA in Japanese patients with paroxysmal nocturnal hemoglobinuria.

It is unclear how a paroxysmal nocturnal hemoglobinuria (PNH) clone expands in bone marrow, although immune mechanisms involving cytotoxic T lymphocytes, autosomal proliferation, and apoptosis resistance have been hypothesized. To clarify aspects of immune mechanisms and proliferation of PNH cells, we investigated HLA-DRB1, -DQA1, and -DQB1 alleles by polymerase chain reaction (PCR)-based genotyping and expression of the Wilms' tumor gene, WT1, by real-time reverse transcriptase-PCR (RT-PCR) in 21 PNH and 21 aplastic anemia (AA) patients. HLA genotyping indicated that the frequency of DRB1*1501, DQA1*0102, and DQB1*0602 alleles in PNH patients and of DQB1*0602 allele in AA patients was significantly higher than in 916 Japanese controls, and that the HLA-DRB1*1501-DQA1*0102-DQB1*0602 haplotype, found in 13 of 21 PNH patients, 5 of 7 AA-PNH syndrome patients, and 7 of 21 AA patients showed significant differences compared with healthy individuals. RT-PCR analysis showed that the mean values of WT1 RNA were 3413, 712, and 334 copies/microg RNA in PNH, AA, and healthy individuals, respectively. The values for PNH patients were significantly higher than for AA patients and healthy volunteers and were correlated with the proportion of CD16b(-) granulocytes. The high frequency of HLA-DRB1*1501-DQA1*0102-DQB1*0602 haplotype in PNH, including AA-PNH syndrome, and AA patients suggests that linkage exists between the disorders and that immune mechanisms in an HLA-restricted manner play an important role in the pathogenesis of these disorders. In addition, high expression of WT1 RNA in PNH patients is related to a PNH clone, but it remains unclear whether this causes expansion of a PNH clone.

Rapid screening of leukemia fusion transcripts in acute leukemia by real-time PCR.

We established a real-time PCR method that can simultaneously detect 10 different fusion transcripts (major, minor and micro BCR/ABL, AML1/MTG8, PML/RARalpha, CBFbeta/MYH11, TEL/AML1, E2A/PBX1, MLL/AF4, and MLL/AF9) together with Wilms' tumor gene (WT1) transcripts. This screening method allowed the processing of six specimens concomitantly and required only one working day from RNA extraction to final results. Fifty-seven bone marrow (BM) samples from patients with acute leukemia were retrospectively screened for the presence of fusion and WT1 transcripts without knowledge of the cytogenetic data, and the fusion transcripts were detected in 20 of 57 samples (35.1%). The concordance between the present method and cytogenetic analysis was examined in 38 samples in which the cytogenetic data were available. In 12 of 38 samples, the PCR results agreed with the cytogenetic data, whereas in 4 of the remaining 26 samples, the translocations were detected by real-time PCR alone because of the insufficient number of metaphases obtained and presumably the submicroscopic or masked translocations. The WT1 levels ranged from 400 to 690,000 copies/microg RNA in BM from leukemia patients, whereas 0-470 copies/microg RNA were found in BM cells from BMT donors. This real-time PCR method enables rapid and efficient characterization of acute leukemia in addition to subsequent evaluation of minimal residual diseases.