Absence of gene mutation in TRAIL receptor 1 (TRAIL-R1) and TRAIL receptor 2 (TRAIL-R2) in chronic myelogenous leukemia and myelodysplastic syndrome, and analysis of mRNA Expressions of TRAIL and TRAIL-related genes in chronic myelogenous leukemia.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is an interferon (IFN)-induced molecule with apoptotic activity. We examined gene mutations in the death domains of TRAIL receptor 1 (TRAIL-R1) and TRAIL receptor 2 (TRAIL-R2), and in the TRAIL gene promoter in 46 chronic myelogenous leukemia (CML) patients. In 23 of the 46 patients, all the coding regions of TRAIL-R2 were also examined. However, no mutation or loss of heterozygosity was found. Furthermore, no mutation in the death domains of TRAIL-R1 and TRAIL-R2 genes, which causes amino acid change, was found in 18 myelodysplastic syndrome (MDS) patients. Ribonuclease protection assay (RPA) and real-time quantitative polymerase chain reaction using polymorphonuclear neutrophils of five new CML patients showed that the TRAIL mRNA expression was very low before in vitro IFN-alpha stimulation and markedly upregulated after IFN-alpha stimulation. FAS mRNA was also upregulated with IFN-alpha stimulation but the fold induction was far lower than that of TRAIL mRNA. In addition, RPA revealed that the ratio of (TRAIL-R1 plus TRAIL-R2) to TRAIL-R3 was also increased after IFN-alpha stimulation. Taken together, gene mutations of TRAIL-R1, TRAIL-R2 are infrequent in patients with CML and MDS. And so is the TRAIL promoter for CML. These mutations seem unrelated to tumorigenesis, disease progression, and response to IFN-alpha therapy in CML. A markedly high induction of TRAIL mRNA by IFN-alpha may have some relevance to IFN-alpha action in CML patients.

Effect of interferon-alpha on chromosome abnormalities in treated chronic myelogenous leukemia patients.

To investigate the relationship of chromosomal aberrations at blastic crisis (BC) in chronic myelogenous leukemia (CML), with previous therapies and with atomic bomb (AB) exposure, we studied 114 CML patients who developed BC, including 23 AB survivors in Hiroshima. In total, only 45.6% showed major-route abnormalities, which figure was far lower than those previously reported, implying possibility of geographical difference. Occurrence of major-route abnormality was not associated with either duration of chronic phase or survival time after BC. Patients treated with interferon-alpha (IFNalpha) showed lower frequency of major-route abnormalities and lower number of abnormal chromosomes than did patients treated with busulfan (Bu). The frequency of trisomy 8 was lower and monosomy 7 was higher in IFNalpha-treated than in Bu-treated patients. The frequency of unusual abnormalities at BC in IFNalpha-treated patients was indistinguishable from those in Bu-treated patients and, notably, a more common (40%) feature in IFNalpha-treated patients was no change in the cytogenetic picture. Thus, we conclude that IFNalpha action on chromosome aberration is basically quite neutral and that IFNalpha does not induce any specific aberrations, including unusual ones at BC, with an exception of deletion of chromosome 7. Atomic bomb exposure status did not make any difference in secondary abnormalities at BC.

Initial expression of interferon alpha receptor 2 (IFNAR2) on CD34-positive cells and its down-regulation correlate with clinical response to interferon therapy in chronic myelogenous leukemia.

In order to investigate the mechanism of interferon-alpha (IFNalpha) action in the treatment of chronic myelogenous leukemia (CML), we examined surface expressions of both type I interferon receptor 1 (IFNAR1) and 2 (IFNAR2) subunits on CD34-positive cells in bone marrow (BM) in a total of 57 CML patients. Initial cell-surface IFNAR2 expression at diagnosis assessed by flow cytometry widely distributed but showed overall significantly higher expression in CML patients when compared with normal controls. In 15 fresh patients who subsequently received IFNalpha therapy, IFNAR2 expression at diagnosis was significantly higher in cytogenetic good responders than in poor responders. Down-regulation of IFNAR2 expression during IFNalpha therapy was observed only in good responders but not in poor responders. In addition to protein level, both initial high IFNAR2c mRNA expression level and its down-regulation during IFNalpha therapy, in purified CD34-positive cells, were also observed only in good responders. In contrast to IFNAR2, cell-surface IFNAR1 expression was generally lower than IFNAR2, and correlation between either the pretreatment level or down-regulation of IFNAR1 and clinical response was not evident. With in vitro IFNalpha stimulation, CD34-positive cells showed down-regulations of cell-surface IFNAR2, and IFNAR1 to a lesser extent, in one good-responder patient, but not in one poor-responder patient. Serum soluble interferon receptor (sIFNR) was higher in untreated CML patients than in normal controls, without any correlation with clinical response to IFNalpha. Thus, the pretreatment protein and mRNA expression levels of IFNAR2 and their down-regulations during IFNalpha therapy correlate well with IFNalpha response in CML patients.

Chronic myelogenous leukemia with e13a3 (b2a3) type of BCR-ABL transcript having a DNA breakpoint between ABL exons a2 and a3.

We describe a patient with chronic myelogenous leukemia (CML), in whom the DNA breakpoint in the BCR-ABL fusion gene was determined to result in a rare e13a3 (b2a3) transcript. The breakpoint in BCR was intron 13, which was 30 bp downstream from exon 13, and the breakpoint in ABL was intron 2, and was 46 bp downstream from exon a2. This case conforms to the mechanism of DNA breakage occurring within ABL intron 2, but not at 5' to ABL exon a2. With our review of this case and the literature, it seems that CML with the BCR-a3 fusion product is associated with a low proportion of circulating immature cells, mild or lack of splenomegaly, slow progressiveness, rather resistance to IFN-alpha, and good response to imatinib mesylate. This is the first report of BCR-a3-type CML in which the exact DNA breakpoint was identified and located between exons a2 and a3 of the ABL gene.

Expression and functional analysis of granulocyte colony-stimulating factor receptors on CD34++ cells in patients with myelodysplastic syndrome (MDS) and MDS-acute myeloid leukaemia.

CD34++ cells from 45 patients with myelodysplastic syndrome (MDS) and MDS-acute myeloid leukaemia (MDS-AML) were observed by flow cytometry for the expression of granulocyte colony-stimulating factor receptor (G-CSFR). Ten patients had a significantly reduced expression of G-CSFR. Late stages of disease showed a higher proportion of either high or low G-CSFR expression than earlier stages. In MDS refractory anaemia (RA), G-CSFR was inversely related to CD33 expression. Most patients (9/10) with low G-CSFR expression had neutropenia of the peripheral blood. Neutropenia was less common in the normal group, but also occurred in the high expression group. No neutrophil response was observed following G-CSF administration to MDS-AML patients (6/6) with low G-CSFR expression. In the high expression group, patients (3/3) showed a response to G-CSF while, in the normal group (1/2), the response was minor. In the normal- or high-receptor-expressing groups, the receptors were functionally active in terms of apoptosis but not proliferation and clonogenic growth, although no clear correlation to receptor expression was observed. The G-CSFR signal transduction pathway in the normal and high group was not deficient of messenger RNA for either janus kinases (Jaks) or signal transducers and activators of transcription (Stats). These findings suggest that the lowered expression of G-CSFR may cause neutropenia in MDS and MDS-AML patients and, therefore, may partially explain the neutropenia in myelodysplastic patients.