Cases Of Chronic Myeloid Leukemia Research Articles

Gene expression changes associated with progression and response in chronic myeloid leukemia.

Chronic myeloid leukemia (CML) is a hematopoietic stem cell disease with distinct biological and clinical features. The biologic basis of the stereotypical progression from chronic phase through accelerated phase to blast crisis is poorly understood. We used DNA microarrays to compare gene expression in 91 cases of CML in chronic (42 cases), accelerated (17 cases), and blast phases (32 cases). Three thousand genes were found to be significantly (P < 10(-10)) associated with phase of disease. A comparison of the gene signatures of chronic, accelerated, and blast phases suggest that the progression of chronic phase CML to advanced phase (accelerated and blast crisis) CML is a two-step rather than a three-step process, with new gene expression changes occurring early in accelerated phase before the accumulation of increased numbers of leukemia blast cells. Especially noteworthy and potentially significant in the progression program were the deregulation of the WNT/beta-catenin pathway, the decreased expression of Jun B and Fos, alternative kinase deregulation, such as Arg (Abl2), and an increased expression of PRAME. Studies of CML patients who relapsed after initially successful treatment with imatinib demonstrated a gene expression pattern closely related to advanced phase disease. These studies point to specific gene pathways that might be exploited for both prognostic indicators as well as new targets for therapy.

Chronic myelomonocytic leukemia: the role of bone marrow biopsy immunohistology

The World Health Organization criteria for diagnosing chronic myelomonocytic leukemia (CMML) are largely based on findings observed in the peripheral blood and bone marrow aspirate. A specific diagnostic role for the bone marrow biopsy has not been adequately explored. We examined whether bone marrow biopsy supplemented by immunohistochemistry may be helpful in distinguishing CMML from cases of chronic myelogenous leukemia and atypical chronic myeloid leukemia (aCML). We immunostained 25 cases of CMML with paraffin reactive antibodies which included CD68 (KP1), CD68R (PG-M1), and CD163, and compared the results with those observed in six cases of chronic myelogenous leukemia and in three cases of atypical CML. In addition, we examined whether CD34 immunohistochemistry could be useful in separating cases of CMML with less than 10% blasts (type-1) from cases of CMML with blasts accounting for 10–19% (type-2), and cases of CMML in acute transformation to acute myeloid leukemia (blasts≥20%). The presence of nodules of plasmacytoid monocytes was investigated by CD123 staining. CD42b was used to highlight abnormal megakaryocytes. Our results demonstrate significant differences between the groups. CD34 analysis allowed separating CMML type-1 from type-2 and the former from CMML in acute transformation. CD123-positive plasmacytoid monocyte nodules were found only in CMML and not in the other two disease groups. Overlap between CMML and the other two groups were observed with CD68 immunostaining. CD68R was more restricted to bone marrow macrophages and monocytes than CD68, but the differences between CMML and chronic myelogenous leukemia or atypical CML were still not significant. Although CD42b immunostaining facilitated the detection of dwarf megakaryocytes often present in CMML, the distinction between those and the small forms seen in chronic myelogenous leukemia was still problematic.

Alteration in lipid profile in patients of chronic myeloid leukemia before and after chemotherapy

Background Chronic myeloid leukemia (CML) is a myeloproliferative disorder of pleuripotent hematopoietic progenitor cells characterized by excessive proliferation and accumulation of granulocytes and occasionally red blood cells and platelets. Methods We examined 30 newly diagnosed and proved cases of CML admitted in medical wards or attending a clinical hematology clinic. In addition to routine hematological investigations, lipid profile was done in all the patients at the time of presentation, 4–6 weeks after the start of chemotherapy and 6 months after the chemotherapy even if some of the patients were not in remission. Results Total serum cholesterol, HDL-cholesterol and LDL-cholesterol concentrations increased significantly after chemotherapy whereas serum triglyceride and VLDL-C cholesterol concentrations did not increase significantly. Also, lipid concentrations were correlated with disease activity. An inverse correlation was found with spleen size and total serum cholesterol, HDL-cholesterol and LDL-cholesterol. Serum triglyceride and VLDL-C concentrations were inversely related to the hemoglobin concentrations. There was no correlation of total cholesterol and LDL-cholesterol concentrations with hemoglobin. We found from the present study that low lipid concentrations are associated with poor prognosis of the disease. Conclusion We suggest that the estimation of lipid profile may be helpful in evaluating the response to chemotherapy in CML patients.

BCR/ABL rearrangement in two cases of Philadelphia chromosome negative chronic myeloid leukemia: deletion on the derivative chromosome 9 may or not be present

The BCR/ABL gene rearrangement is the causing factor in chronic myeloid leukemia (CML). In most cases, it is cytogenetically visualized as a translocation between chromosomes 9 and 22, known as the Philadelphia (Ph) translocation. About 5–10% of CML patients lack cytogenetic evidence of the Ph translocation but show BCR/ABL fusion by fluorescence in situ hybridization (FISH) or reverse transcriptase-polymerase chain reaction. Deletions around the breakpoints on the derivative 9 including ABL and or BCR sequences occur in 10–15% of Ph+ CML patients and are thought to have prognostic significance. We describe two patients with CML and normal karyotype in whom cryptic rearrangements involving chromosomes 9 and 22 resulted in the causative BCR/ABL gene. FISH with a three-color probe combination revealed BCR/ABL fusion on chromosome 9 without deletion in one patient; the other patient had BCR/ABL on chromosome 22 with an associated derivative 9 deletion. We discuss the proposed mechanisms in the formation of BCR/ABL in the setting of a normal karyotype. Some authors reported that patients with the chimeric gene located on the derivative 9 have a poor clinical course. We suggest that deletion rather than location of the chimeric gene alone is more likely to be associated with prognosis.

Myeloperoxidase cytochemical negativity: an unexpected but intrinsic property of blasts of all phases of chronic myeloid leukemia

Myeloperoxidase (MPO) cytochemical activity, recognized as a very important hallmark of myeloblasts, is generally negative in chronic myeloid leukemia (CML) blast crisis (BC). Because this finding is unexpected, being not in keeping with the myeloproliferative nature of CML, we tried to ascertain if MPO cytochemical negativity could be an intrinsic property of blasts of CML and hence present in the preblastic phases as well. Myeloperoxidase cytochemistry of peripheral blood blasts in 161 cases of CML, including 103 in chronic phase (CP) and 29 each in accelerated phase (AP) and BC, was assessed and compared with that of 30 cases of acute myeloid leukemia, AML-M2. Blasts of 97 (94.2%) of 103 cases of CP, 28 (96.6%) of 29 cases of AP, and 22 (75.9%) of 29 cases of BC were negative for MPO (<3% MPO-positive blasts). Compared with the strong MPO positivity, both in terms of intensity and proportion, in the AML-M2 cases, the positivity in the CML cases was generally weak and was seen in a small number of blasts (5-15%), except in one case of BC with 20% positive blasts. Absence or, at times, weak MPO cytochemical activity is an intrinsic property of blasts of all phases of CML, and use of the term myeloblast in CML should be understood to refer to a cell with this property. This also explains why MPO cytochemistry, despite its high reputation as a myeloid-lineage marker, generally does not help in CML BC. CML BC should therefore be considered as a possible diagnosis along with acute lymphoblastic leukemia, AML-M0, AML-M7, etc., in the setting of MPO-negative blasts. Similarity between MPO expression pattern in CML, i.e., negative in blasts and positive in the more mature cells, and that during maturation of normal myeloid series of cells shows the deranged myelopoiesis of CML to be undisturbed at least with respect to MPO expression. There is need for a more comprehensive study of blasts of preblastic phases.

Evidence for deletion of 9q as a two‐step process in chronic myeloid leukemia

Evidence for deletion of 9q as a two-step process in chronic myeloid leukemia. Chronic myeloid leukemia (CML) is characterized by the Philadelphia translocation t(9;22)(q34;q11) resulting in the BCR/ABL fusion gene. Submicroscopic deletion of the derivative chromosome 9 occurs in a subset of these patients and is associated with poor prognosis. In the current study, we present two unusual cases of CML selected from a series of 54 consecutive cases. A detailed study using classical cytogenetics and fluorescence in situ hybridization (FISH) analysis was done using dual color extra signal FISH and whole chromosome paint in order to elucidate the mechanism of 9q deletion. One case had two clones on interphase FISH, one with and one without chromosome 9q deletion. The other case had two clones on both cytogenetic and FISH analyses, one with and one without a marker chromosome carrying chromosome 9q sequences. In this latter case, the clone with deletion of the derivative chromosome 9 comprised 21.1% at diagnosis, increasing to 36.8% after 11 months, suggesting a growth advantage. We report here evidence that deletions on 9q in CML may occur through breakage and rearrangement of chromosomes resulting in derivative chromosomes and either a marker chromosome or fragment/episome, followed by loss of chromosome material from the cell.

Bcr/abl-Negative, Classic Myeloproliferative Disorders: Diagnosis and Treatment

Essential thrombocythemia, polycythemia vera, and myelofibrosis with myeloid metaplasia constitute the "classic" bcr/abl-negative myeloproliferative disorders (MPDs). Each of these MPDs represents a stem cell-derived clonal myeloproliferation with the respective features of thrombocytosis, erythrocytosis, and bone marrow fibrosis. Unlike with cases of chronic myeloid leukemia, in which the bcr/abl mutation is invariably detected, current diagnosis of essential thrombocythemia, polycythemia vera, and myelofibrosis with myeloid metaplasia is based on a consensus-driven set of clinical and laboratory criteria that have undergone substantial modification in recent times. The recent discovery of a recurrent activating Janus tyrosine kinase (JAK2) mutation (JAK2VG17F) in all 3 classic MPDs offers another opportunity for refining current diagnoses and disease classifications. In this article, we outline contemporary diagnostic algorithms for each of these disorders and provide an evidence-based approach to management.

Overexpression of Shp-2 is associated with the unlimited growth and apoptosis resistance of p210 bcr-abl-mediated chronic myeloid leukemia

To investigate the expression level of Shp-2 tyrosine phosphatase in chronic myeloid leukemia (CML) and its relationship with the unlimited growth and apoptosis resistance of p210 bcr/abl-induced malignant cells. In this study, p210 bcr/abl positive leukemia cell specimens were obtained from 25 CML cases, meanwhile, bone marrow and peripheral blood cell samples from 8 non-tumor individuals and 10 normal individuals were used as p210 bcr/abl negative controls. K562 and KU812 leukemia cells were used as p210 bcr/abl positive controls, and KG-1 leukemia cell line was used as Shp-2 positive control. Specimens of peripheral blood and bone marrow of 25 adult patients of chronic myelocytic leukemia, 15 males and 10 females, aged 28-64, were collected. Specimens of bone marrow of 8 basically healthy adult volunteers and specimens of peripheral blood of 10 healthy adult volunteers were used as controls. The total cell protein was collected and the expression of Shp-2 was examined by Western blotting. Human leukemia cells of the line K562 were cultured. Shp-2 specific sense and antisense oligonucleotides were added into the culture fluid respectively. The cell apoptosis was detected by flow cytometry (FCM). STI571, specific inhibitor of p210 bcr/abl was added into the cultured fluid of K562 cells, then Western blotting and FCM were used to detect the protein expression of Shp-2 and p210 bcr/abl, and cell apoptosis. Phosphorylated Shp-2 (pShp)-2 protein was overexpressed in 92% (23/25) of the CML cells, but lowly expressed or not expressed in the normal hematopoietic cells. The mean pShp-2 protein/beta-actin ratio of the primary CML leukemia cells was 0.91 +/- 0.62, significantly higher than those of the normal bone marrow cells and peripheral blood hematopoietic cells (0.16 +/- 0.09 and 0.03 +/- 0.05 respectively, both P < 0.01). The apoptotic rates of the CML cells treated by Shp-2 specific antisense oligonucleotide of the concentrations of 1 micromol/L and 4 micromol/L respectively for 72 h was 7.98% and 20.29% respectively, both significantly higher than that of the control group (4.06%, P < 0.01). The number of clone of CML cells treated by 0.25 micromol/L and 1.0 micromol/L Shp-2 specific antisense oligonucleotide for 7 days were 67% (37/60) and 11.9% (5/42) that of the control group. Twenty-four and 48 hours after the stimulation of STI571 the expression level of Shp-2 protein in the CML cells decreased time-dependently and the CML cell apoptotic rates were 31.15% and 38.69% respectively, both lower than that of the control group (33.6%). The number of clone of CML cells effected by 0.25 micromol/L and 1.0 micromol/L Shp-2 specific antisense oligonucleotide for 7 days was 67% (37/60) and 11.9% (5/42) that of the control group. Twenty-four and 48 hours after the stimulation of STI571 the expression level of Shp-2 protein in the CML cells decreased time-dependently and the CML cell apoptotic rates were 31.15% and 38.69% respectively, both lower than that of the control group (33.6%). (1) The pShp-2 protein is overexpressed in CML cells, which is associated with the unlimited growth and apoptosis resistance of malignant cells. (2) Shp-2 is upregulated by p210 bcr/abl oncoprotein in CML.

NUP98-LEDGF fusion and t(9;11) in transformed chronic myeloid leukemia

The molecular basis for disease progression in chronic myeloid leukaemia (CML) is poorly understood, but is believed to be a consequence of additional acquired genetic lesions. We describe here a case of CML who presented de novo in transformation with a t(9;11)(p21;p15) and NUP98-LEDGF fusion in addition to the t(9;22). The t(9;11) was present in only 2/45 (4%) of bone marrow metaphases, but 17/20 (85%) of metaphases from peripheral blood, suggesting an extramedullary or focal origin. This is the first description of NUP98-LEDGF in CML and strengthens the association between disease progression in and NUP98 abnormalities.

Breakpoints of variant 9;22 translocations in chronic myeloid leukemia locate preferentially in the CG‐richest regions of the genome

From 5% to 10% of 9;22 translocations in chronic myeloid leukemia (CML) are reported to occur in variant form, that is, with the involvement of other regions of the genome in 3-way or more rearrangements. The literature indicates that the alternative breakpoints are not distributed randomly in the genome but show hotspots. We present data on 289 unpublished cases of CML with variant 9;22 translocations having a total of 342 variant breakpoints, the largest independent series to date. We found that the distribution of breaks was in loose agreement with the literature but that some new hotspots were identified; furthermore, some published hotspots were not fully supported by our data. Moreover, when our 342 variant breakpoints were plotted against profiles of CG heterogeneity in the genome, a significant positive correlation between breakpoint locations and CG composition was observed. In an ancillary study, we compared the frequency of variant t(9;22) with that of variants of t(15;17) associated with acute promyelocytic leukemia (AML M3). We found that the frequency of the former, 9.3%, was significantly higher than that of the latter, 2.6%.

Chronic Myeloid Leukemia in a Man With Papillary Carcinoma of the Thyroid Treated With Radioactive Iodine

Leukemia as a second malignancy after treatment of thyroid cancer is rare. Most cases reported in the literature have occurred after cumulative doses higher than 800 mCi and it is most commonly acute leukemias. We report a case of chronic myeloid leukemia (CML) occurring in a 40-year-old man 14 years after treatment of papillary thyroid carcinoma. Our patient had the longest interval between the diagnosis of CML and administration of 131I.

Chromosome 17 abnormalities and mutation of the TP53 gene: correlation between cytogenetics, flow cytometry and molecular analysis in three cases of chronic myeloid leukemia

chronic myeloid leukemia (CML) have been described. This chromosomal region contains the tumor suppressor gene TP53 that may be an important factor in the evolution of this disease. In this study, we used flow cytometry and western blotting to assess p53 protein expression and single stranded conformational polymorphism to examine TP53 gene alterations in three patients with CML who showed alterations in 17p. Only the case with del(17)(p11) had p53 expression positive by flow cytometry and an abnormal migration pattern by SSCP analysis. The importance of the correlation between the results obtained with these techniques, as well as the clinical course of the patients, are discussed.

Loss of Response to Imatinib: Mechanisms and Management

The treatment of chronic myeloid leukemia (CML) has been revolutionized by the small molecule BCR-ABL-selective kinase inhibitor imatinib. Although imatinib is highly effective initially and generally well-tolerated, relapse is increasingly encountered clinically. Until recently, for the majority of CML patients with disease no longer responsive to imatinib, as well as for patients with imatinib intolerance, few effective therapeutic options existed. Our understanding of the major mechanisms of imatinib resistance has led to the clinical development of two novel BCR-ABL inhibitors that harbor significant therapeutic promise in early clinical trial experience. These agents, dasatinib (BMS-354825) and AMN107, are more potent inhibitors of BCR-ABL than imatinib, and moreover, harbor activity against nearly all imatinib-resistant BCR-ABL kinase domain mutant forms tested in vitro. Notably, neither of these compounds is effective against the imatinib-resistant BCR-ABL/T315I mutation. The potential availability of highly effective medications for the treatment of imatinib-resistant and intolerant cases of CML is expected to further complicate the timing of other effective therapies, such as allogeneic stem cell transplantation. Additionally, periodic genotyping of the BCR-ABL kinase domain to screen for drug-resistant mutations may play an increasingly important role in the future management of CML cases.

Qi therapy as a complementary therapy in chronic myeloid leukemia

We describe the successful treatment of a case of chronic myeloid leukemia with Qi therapy. The patient's disease was managed with conventional medical treatment and Qi therapy as a complementary therapy. Before Qi therapy, 95% of the patient's bone marrow showed evidence of disease. A second bone marrow sample five months after Qi therapy revealed that 38% of the bone marrow was normal; one year after Qi therapy the bone marrow was no longer producing any cancer cells. Although these results were obtained for a single case only and may not constitute conclusive evidence, the data suggest that Qi therapy given as a complementary therapy during conventional medical treatment may have beneficial effects on chronic myeloid leukemia.

Rational Approaches to the Design of Therapeutics Targeting Molecular Markers: The Case of Chronic Myelogenous Leukemia

Progress in understanding the molecular basis of signal transmission and transduction has contributed substantially to clarifying the mechanisms of leukemogenesis and of leukemia progression and has led to the identification of a number of specific molecular targets for treatment. Chronic myeloid leukemia (CML) has provided one of the best models, as the identification of a leukemia-specific hybrid tyrosine kinase (BCR-ABL, p210, p190) has led to the identification and the successful therapeutic application of a powerful tyrosine kinase inhibitor, imatinib. The BCR-ABL fusion gene is the result of a reciprocal translocation between the long arms of chromosomes 9 and 22, t(9;22)(q34;q11), which characterizes more than 95% of the cases of CML. The resulting chimeric proteins (P210 and P190), which retain a constitutively activated tyrosine kinase activity, have a causative role in the genesis of the leukemia process. In agreement with this observation, BCR-ABL tyrosine kinase inhibitors have recently emerged as powerful new therapeutic tools, obtaining extraordinary results in early chronic-phase CML as well as in more advanced phases of the disease. Although these results represent a remarkable breakthrough, there are still numerous issues, such as the emergence of resistance, that remain unsolved and that will need further investigation. In spite of its low incidence, CML remains a paradigmatic model for understanding the pathogenesis and therapeutic options of human leukemias.

Fluorescence in situ hybridization characterization of different cryptic BCR-ABL rearrangements in chronic myeloid leukemia

Using fluorescence in situ hybridization probes, obtained from bacterial artificial chromosome (BAC) libraries that relate to sequences either side of the BCR and ABL genes, this study characterized four chronic myeloid leukemia cases with cryptic BCR-ABL rearrangements. Each case showed evidence of a different underlying mechanism: one case showed a microinsertion of BCR into ABL, another a microinsertion of ABL into BCR, and the third showed a complex rearrangement including deletion of adjacent flanking sequences, consistent with the reverse translocation model of cryptic rearrangement. The fourth case also showed evidence of a more complex rearrangement involving chromosome 1.

Downregulation of Circadian Genes, PER1, PER2, and PER3, in Chronic Myeloid Leukemia.

Circadian rhythm is present in all eukaryotic and some prokaryotic life forms. This time-keeping system is organized in a hierarchical fashion and composed of a self-sustained central pacemaker in the suprachiasmatic nuclei of the anterior hypothalamus and peripheral oscillators in most body cells. A recent study demonstrated that mice deficient in the clock gene mPer2 are cancer prone and display a deregulated temporal expression genes involved in cell cycle regulation, such as c-Myc, Cyclin D1, Cyclin A, and Mdm-2. These mice display salivary gland hyperplasia, and a large portion of them develops lymphomas. The results revealed that Per2 is an essential circadian gene and it is associated with proliferation control in mammals. In a more recent study, it was demonstrated that an age-dependent decay of the circadian clock both at the behavioral and the molecular levels was observed in mPer1−/−mCry2−/− double-mutant mice. To gain further insights into the roles of circadian genes in chronic myeloid leukemia (CML), we analyzed peripheral blood from 21 healthy individuals and 35 CML patients (18 in blastic crisis and 17 in chronic phase) by real-time quantitative RT-PCR for the expression of circadian genes PER1, PER2, and PER3. In blastic crisis cases, the expression levels of all three PER genes were significantly impaired than in healthy individuals (PER1, 1:5.97, p < 0.005; PER2, 1:13.51, p < 0.0001; PER3, 1:2181.33; p < 0.0001); whereas, in chronic phase, only the expression levels of PER2 and PER3 were significantly impaired (PER1, 1:1.23, p > 0.5; PER2, 1:2.47, p < 0.05; PER3, 1:14.22; p < 0.0001). Mutational analysis of the whole gene and methylation analysis of CpGs sites at the promoter area were further performed to investigate the possible mechanisms. No mutation was found within the coding regions of the three PER genes in all CML cases. Methylation analysis using methylation-specific PCR and direct sequencing showed no methylation in the promoter areas of both PER1 and PER2 genes. In contrast, most of the CpG sites were methylated in the promoter area of PER3 gene in CML cases and none of these CpG sites were methylated in healthy individual cases. In addition, the methylated CpG frequencies of PER3 gene differed in patients at blastic crisis and at chronic phase (CpG, 8.24 ± 0.73 vs. 4.48 ± 0.48, p < 0.0001; T/CpG, 10.47 ± 0.67 vs. 14.67 ± 0.46, p < 0.0001; TG, 1.24 ± 0.32 vs. 0.81 ± 0.16, p > 0.05). Demethylation by treatment of hypermethylated K562 cells with 5′-aza-2′-deoxycytidine resulted in partial reactivation of PER3 expression. Our results suggest that the downregulated PER3 expression in CML was due to the inactivation of PER3 gene by methylation and the differential expression was correlated to the ratio of methylated CpG sites at the promoter region. We hope to explore the role of circadian genes in the tumorigenesis of leukemia and to establish circadian genes as novel and useful markers for diagnosis and references for therapies in leukemia.

Expression of Human Enhancer of Invasion 10 (HEI10) in Human Myeloid Leukemias and Downregulation during Differentiation of Hematopoietic Stem Cells.

Background: We previously investigated the gene expression profile of drug-resistant human leukemia K562 cells by the serial analysis of gene expression (SAGE) (Ichikawa et al. Int J Hematol, 2004; 79:276), and found that the HEI10 gene was overexpressed in K562 cells. With the same technology, we also observed that human CD34+ cells expressed high levels of HEI10 gene expression compared to erythroid progenitor cells (Fujishima et al. Int J Hematol, in press). HEI10 encodes a protein which belongs to a family of E3 ubiquitin ligase,and interacts with cyclin B1 leading to the regulation of cell cycle. In the present study, we examined the gene expression of HEI10 quantitatively in human leukemias in order to learn whether there would be any association between the expression levels and leukemia subtypes. We also investigated whether the expression levels of HEI10 would be related to differentiation of myeloid leukemia cell lines and normal hematopoietic stem cells.Methods: The quantitative real-time PCR method was employed to estimate the relative gene expression levels of HEI10. Total RNA was extracted from mononuclear cells of bone marrow that had been taken for diagnostic purposes. Patients include acute myeloid leukemia (AML) (n=21) and chronic myeloid leukemia (CML) (n=7; 2 in blastic crisis, 5 in chronic phase). Differentiation of human acute myeloid leukemia cell line HL60 cells was induced by PMA. Human normal CD34+ cells were purified from G-CSF mobilized blood stem cells and were induced to differentiate into erythroid, myeloid or dendritic cells by various cytokines.Results: Quantitative real-time PCR analysis revealed the high expression of HEI10 gene in patients with leukemia with undifferentiated phenotype (3 cases of AML M1 in FAB classification and 2 cases of CML in blast crisis) and low levels of expression in acute promyelocytic leukemia and CML in chronic phase. When HL60 cells were induced to differentiation with PMA, accumulation in the G0/G1 phase was observed and the expression of HEI10 was decreased. Differentiation of normal CD34+ cells also resulted in a decrease of HEI10 expression.Conclusions: The expression of HEI10 gene in hematopoietic cells appears to be upregulated when cells are immature. The role for HEI10 in hematopoietic stem cells remains to be elucidated. Biological effects of the HEI10 gene silencing in human hematopoietic cells are currently being investigated by RNAi technology. [Display omitted] [Display omitted]

STI571 Suppresses Proliferation by Restoring Nuclear Cyclin Dependent Kinase Inhibitors (CDKIs) while STI571+TRAIL Promotes Cell Death by Decreasing Cytoplasmic CDKIs.

Cyclin dependent kinase inhibitors (CDKIs), p27Kip1 and p21Cip1, function as cell cycle inhibitors when located in the nucleus. When localized to the cytoplasm, these CDKIs can function as anti-apoptotic molecules by sequestering/preventing the activation of pro-apoptotic proteins such as ASK1 and procaspase-3. Our lab has reported elevated cytoplasmic CDKIs and decreased nuclear CDKIs in hematopoietic cells expressing BCR/ABL, the oncogene found in more than 90% of cases of chronic myeloid leukemia. Within the past decade, STI571 has been shown highly promising for CML treatment. However, there is increasing evidence suggesting that the drug might function more as a suppressor of proliferation and less as a promoter of cell death. In the current studies, we differentiate the effect of STI571 on proliferation vs. survival by tracking the subcellular increase/decrease in CDKIs using MO7e cells engineered to express BCR/ABL. To determine if a correlation exists between STI571 resistance and levels of cytoplasmic anti-apoptotic CDKIs, we also investigated changes in levels of nuclear vs. cytoplasmic CDKIs in LAMA84 -S (sensitive to STI571) vs. LAMA84-R (resistant to STI571). And lastly, we tested whether activation of TRAIL would enhance cell death in STI571-resistant cells. STI571 treatment increases nuclear CDKIs, correlating directly with a decrease in proliferation of MO7e/p210 cells. However, the high levels of cytoplasmic CDKIs in MO7e/p210bcr/abl was not modulated following STI571 treatment and cell death was not prominent, unless growth factors were removed. Moreover, cytoplasmic p21Cip co-immunoprecipitated with ASK1 and procaspase 3. When compared with LAMA-S cells, LAMA-R cells expressed even higher levels of cytoplasmic CDKIs. Treatment with STI571 decreased cytoplasmic CDKIs in LAMA84-S cells and resulted in cell death. As hypothesized, LAMA84-R cells did not show reduction in cytoplasmic CDKIs and did not enter apoptosis. However, when treated with STI571 and TRAIL, LAMA84-R cells showed a decrease in cytoplasmic CDKIs, and increase in apoptosis. Based on these observations, we conclude that: 1. BCR/ABL expression reduces nuclear CDKIs but increases cytoplasmic CDKIs. 2. STI571 treatment restores nuclear CDKIs and reduces cell proliferation of BCR/ABL expressing cells under physiological conditions. 3. Treatment of STI571+TRAIL reduces cytoplasmic CDKIs and increases cell death of BCR/ABL expressing cells, most notably, the STI571-resistant cells. In conclusion, we show that the imbalance between nuclear (cell cycle inhibitor) and cytoplasmic (cell survival enhancer) CDKIs exist in BCR/ABL-hematopoietic cells.

Development of a Diagnostic Support System for Hematological Disorders Using a Self-Organizing Map.

Though crucial in determining treatment and prognosis, rapid and precise diagnosis of hematological disorders is often difficult. The fact that there is not always a hematologist in every medical institution is another reason why the development of a diagnostic system for hematological disorders is desirable. We used a self-organizing map (SOM) to establish a diagnostic support system for hematological disorders and investigated its usefulness. Data on peripheral blood (CBC, Ret, Diff), marrow differential cell count, immunophenotyping, and cytogenetic data from 402 patients diagnosed with hematological disorders between 1995 and 2003 were submitted into this system. These included 127 cases of acute myeloid leukemia (AML) 50 cases of acute lymphocytic leukemia (ALL), 17 cases of biphenotypic acute leukemia (BAL), one case of large-granular lymphocytic leukemia (LGLL), 55 cases of mature-B cell neoplasms (mature-B), 9 cases of mature-T cell neoplasms (mature-T), 43 cases of multiple myeloma (MM), and 100 other cases. The other cases consisted of 49 cases of myelodysplastic syndrome (MDS), 8 cases of myeloproliferative disorder (MPD), 26 cases of chronic myeloid leukemia (CML), 13 cases of idiopathic thrombocytopenia purpura (ITP), and 4 cases of anemia. The patients were classified into eight main groups (AML, B-ALL + BAL, T-ALL, LGLL, mature-B, mature-T, MM, and others) and a SOM of the main groups drawn. As SOMs express multi-dimensional data in a two-dimensional format, the map used was one with a toroid shape in which top and bottom and left and right are joined together. For subclassification, the multilayer map method was used to allow more detailed divisions. The method of display used was not dots; instead domains were established to include other related diseases in the surrounding area, and the probability of a specific blood disease was displayed, so that the classification made the relationship with the surrounding area visually comprehensible. Through a series of such modifications of SOM Ver.1.0, the handling of diseases, test results, and algorithms was also upgraded, and at present SOM Ver.4.0 is under development. For validation, an evaluation of the usefulness of Ver.4.0 was carried out using data from 65 fresh cases (AML, ALL, mature-B, MM and others). The same diagnosis as the clinical diagnosis was made in 17 of the 18 AML cases, both of the 2 ALL cases, 6 of the 8 mature-B cases, all 4 MM cases, and all 33 other cases by SOM Ver.4.0. The samples in which the initial SOM diagnosis did not agree with the clinical diagnosis were one AML:M6 classified as other, and two mature-B samples classified respectively as MM and other. Next we carried out validation of usefulness in diagnostic support in an actual medical treatment environment. Diagnoses of 10 newly admitted patients were evaluated by comparing diagnoses by residents, general physicians and hematologists with those by SOM Ver.4.0, resulting in predicted diagnoses of hematological disorders similar to the clinical diagnoses. All physicians who participated in evaluating the predicted diagnoses by SOM found the system to be a very useful tool for the diagnostic process and determining further appropriate action. The system thus appeared to constitute a useful diagnostic support tool for all physicians.