Abstract
A substantial minority of patients with chronic myelogenous leukemia (CML) achieve a complete response (CR) to treatment with interferon- (IFN), defined as the disappearance of Philadelphia ...chromosome-positive metaphases. Currently it is unclear how long IFN treatment should be continued for such patients. We used a competitive reverse transcriptase-polymerase chain reaction (RT-PCR) to quantify levels of BCR-ABL transcripts in 297 peripheral blood specimens collected from 54 patients who had achieved CR with IFN. The median duration of observation was 1.9 years (range, 0.3-11.0 years). Total ABL transcripts were quantified as internal control and results were expressed as the ratio BCR-ABL/ABL. All 54 patients had molecular evidence of residual disease, although 3 patients were intermittently PCR negative. The median BCR-ABL/ABL ratio at the time of maximal response for each patient was 0.045% (range, 0%-3.6%). During the period of observation 14 patients relapsed, 11 cytogenetically to chronic phase disease and 3 directly to blastic phase. The median ratio of BCR-ABL/ABL at maximal response was significantly higher in patients who relapsed than in those who remained in CR (0.49% versus 0.021%,P < 0.0001). Our findings show that the level of residual disease falls with time in complete responders to IFN, but molecular evidence of disease is rarely if ever eliminated. The actual level of minimal residual disease correlates with the probability of relapse. We suggest that for patients who reach CR, IFN should be continued at least until relatively low levels of residual leukemia are achieved. (Blood. 2000;95:62-66)
Constitutive activation of the BCR-ABL tyrosine kinase is fundamental to the pathogenesis of chronic myeloid leukemia (CML). STI571 inhibits this activity and modulates the transcription of several ...genes. It was shown by differential display that the suppressor of cytokine signaling-2 (SOCS-2) gene was down-regulated by STI571 treatment in 14 of 16 BCR-ABL–positive cell lines and in 2 BCR-ABL–transfected murine lines, but not inBCR-ABL–negative counterparts. The effect was maximal at 2 hours and persisted for at least 24 hours after exposure to 1 µM STI571, whereas SOCS-1 and SOCS-3 expression were unaffected. Baseline levels of SOCS-2 were significantly higher in BCR-ABL–positive as compared withBCR-ABL–negative cell lines. It was similar in leukocytes and CD34+ cells from healthy persons (n = 44) and patients with CML in chronic phase (CP; n = 60) but significantly increased in patients with CML in blast crisis (BC; n = 20) (P < .0001). Mononuclear cells (MNCs) from 3 of 4 patients with CML in BC showed a 2-fold to 12-fold down-regulation ofSOCS-2 levels on in vitro exposure to STI571; moreover, a 2-fold to 11-fold decrease in SOCS-2 was observed in MNCs from 7 of 8 patients with CML in BC who responded to treatment with STI571. Refractoriness to STI571 or relapse after initial response was accompanied by augmentation of SOCS-2 expression. Ectopic overexpression of SOCS-2 in 32Dp210 cells slowed growth, inhibited clonogenicity, and increased their motility and sensitivity to STI571. Overall, the results suggest that SOCS-2 is a component of a negative feedback mechanism; it is induced by Bcr-Abl but cannot reverse its overall growth-promoting effects in blastic transformation.
We have tested whether peripheral blood mononuclear cells (PBMNCs) from interferon (IFN)-treated patients may lose residual BCR-ABL sequence-positive progenitor cells when long-term cultured for 35 ...days on allogeneic stromal cells. IFN-treated patients have low white blood cell counts and a fair number of BCR-ABL-negative colony-forming cells in the peripheral blood. Particularly, IFN responders show increased numbers of normal hematopoietic cells. We have quantitatively analyzed progenitor cells in PBMNCs of IFN-treated patients by combining the clonogenic assay in semisolid medium with interphase fluorescent in situ hybridization (FISH). Thus, the identification is possible of the BCR-ABL status of colony-forming progenitor cells. In IFN-treated patients, the number of BCR-ABL-positive CFCs is considerably decreased and BCR-ABL-negative CFCs appear in the peripheral blood. We could show that after LTC for 35 days of the same PBMNCs on irradiated allogeneic normal stromal cells residual BCR-ABL sequence-positive CFCs were still present. In some cases the relative number of BCR-ABL sequence-positive CFCs was found to be increased after LTC. A minor proportion of blood samples from IFN-treated patients did not give rise to CFCs after LTC on allogeneic stromal cells (three of 10 patients). Inter- and intraindividual variations can be found with regard to loss or gain of BCR-ABL sequence-positive colonies after LTC. We conclude that early CML progenitor cells persist in the peripheral blood of IFN-treated patients and that a certain proportion may survive long-term culture.
The BCR-ABL chimeric protein is thought to play a central role in the pathogenesis of Philadelphia (Ph) chromosome-positive leukemias, notably chronic myeloid leukemia (CML). There is compelling ...evidence that malignant transformation by BCR-ABL is critically dependent on its protein tyrosine kinase (PTK) activity. As a result, multiple signaling pathways are activated in a kinase-dependent manner, and thus the activation of such pathways may affect the expression of genes that confer the malignant phenotype. In this study, we used differential display to investigate the alterations of gene expression in BV173, a CML cell line derived from lymphoid blast crisis, after exposure to ST1571, which selectively inhibits ABL PTK activity. We show that the expression of a set of 12 genes is correlated with the kinase activity and that the profile of these genes reflects mechanisms implicated in the pathogenesis of CML. Several of the genes show a consistent pattern of altered regulation in all Ph-positive lymphoid cell lines, whereas others appear to be unique to BV173 cells. We conclude that BCR-ABL PTK activity drives the expression of specific target genes that contribute to the malignant transformation of Ph-positive cells. The identification of downstream molecules with a consistent regulation pattern may provide suitable targets for therapeutic intervention in the future.
The mechanism and target cell of the life-prolonging effect of interferon-alpha (IFN-alpha) in chronic myelogenous leukemia (CML) are controversial. We studied the influence of IFN-alpha treatment on ...the frequency of malignant hematopoietic precursor cells in the peripheral blood (PB) of CML patients during the course of the disease. PB 10-day colony-forming cells (PB-CFCs) were assessed with regard to their quantity, lineage distribution, and BCR-ABL status, as determined by fluorescence in situ hybridization (FISH). PB-CFC numbers were determined in 39 patients (29 in the chronic phase, 6 in an advanced stage, and 4 with progression to an advanced stage during follow-up). Thirty-one patients were evaluated either once or several times to determine the BCR-ABL status of the colonies. BCR-ABL negative PB-CFCs were detectable at diagnosis in 5 of 11 patients. A major reduction of BCR-ABL positive colonies to <25% of PB-CFCs was observed in 10/13 determinable IFN-alpha treated patients in early and late chronic phases, indicating a high proportion of BCR-ABL negativity at the clonogenic cell level. In contrast, only 3 of these patients had a cytogenetic response of <25% Philadelphia chromosome (Ph1)-positive metaphases in bone marrow cytogenetics. Treatment with IFN-alpha and/or hydroxyurea (HU) during chronic phase was accompanied by a reduction of PB-CFCs to subnormal levels (median 24 CFCs/ml) compared to controls (median 207 CFCs/ml), untreated patients in chronic phase (median 25,979 CFCs/ml), and patients with advanced disease (median 6,047 CFCs/ml). In blast crisis (6 patients), all colonies tested were BCR-ABL positive. Our results show that IFN-alpha treatment leads to a marked reduction of malignant myeloid precursor cells in the PB of CML patients, which exceeds the degree of cytogenetic remission. This offers an explanation for the good therapeutic efficacy and even life-prolonging effect of IFN-alpha, which is also observed in cytogenetic non-responders.
From the III. Medizinische Klinik, Medizinische Fakultät Mannheim, Universität Heidelberg, Mannheim, Germany (HK, NH, BS, MS, CL, RH, AH, PLR); Department of Hematology, Imperial College & ...Hammersmith Hospital London, United Kingdom (JVM)
Correspondence: Paul La Rosée, Klinikum Mannheim gGmbH, III. Medizinische Klinik Theodor-Kutzer-Ufer 7, D- 68167 Mannheim, Germany. E-mail: paul.larosee{at}med3.ma.uniheidelberg.de
The development of resistance to imatinib mesylate may partly depend on high Bcr-Abl-expression levels. Arsenic trioxide (ATO) has Bcr-Abl suppressing activity in vitro. Here we investigated means to improve ATO activity in CML by modulating cellular glutathione (GSH), a key regulator of ATO-activity in malignant disease. Our studies demonstrate that depletion of cellular glutathione using dl-buthionine-S,R-sulfoximine (BSO) enhances ATO activity against CML cells. GSH-depletion promotes enhanced Bcr-Abl specific activity of ATO through avid repression of Bcr-Abl protein levels and total cellular Bcr-Abl activity. These data provide a rationale for the clinical development of optimized ATO-based regimens through incorporation of GSH-modulators in CML treatment.
Key words: CML, Bcr-Abl, arsenic, glutathione, Imatinib, resistance.
PBMNC from patients with CML and healthy control persons were separated into plastic-adherent and nonadherent cell fractions. A colony assay in semi-solid medium was used to estimate the number and ...lineage commitment of CFC in each of the fractions. The CML blood-derived colonies were isolated and analyzed by FISH for BCR/ABL sequences. Thus, we were able to test the hypothesis whether a selective enrichment is possible of normal progenitor cells in the blood of CML patients in stable chronic phase after HU and/or IFN. Although the number of leukocytes differed considerably between patients at diagnosis and in stable chronic phase, the proportion of adherent and nonadherent cells was about the same in all preparations tested. There were also only minor differences of adherence between MNC of CML and normal origin. Furthermore, BCR/ABL-positive and negative colonies were equally distributed among unseparated, adherent, and nonadherent PBMNC fractions. In conclusion, an accumulation of BCR/ABL-negative CFC was not found in any of the PBMNC fractions. CFC from PBMNC of the same lineage commitment were simultaneously present in plastic-adherent and nonadherent cell fractions, indicating that their surface charges might be different and, on the other hand, that different lineage commitment precursors can be present in either of the fractions irrespective of CML or blood origin.
PDF
