Ultrastructure of neutrophilic granulopoiesis in the bone marrow of patients with chronic myeloid leukemia (CML). A morphometric study with special emphasis on azurophil (primary) and specific (secondary) granules

In seven patients with chronic myeloid leukemia (CML) and ultrastructural and morphometric study was performed on neutrophilic granulopoiesis in bone marrow trephine biopsies. Bone marrow specimens from five patients without hematological abnormalities served as controls. In stable phases of CML, abnormalities of the maturing granulocytic lineage were most conspicuously expressed by an infrequently occurring nuclear disfiguration (blebs and disturbed bridging of segments). Morphometric evaluation included the numbers of azurphil (primary) and specific (secondary) granules, the cisternal length of the endoplasmic reticulum and the area of the mitochondrial profiles. These variables could be determined in early and late myeloblasts, promyelocytes, metamyelocytes, band cells and mature polymorphonuclear granulocytes. Statistical analysis with regard to control specimens demonstrated no significant differences in the total amount of neutrophil granules or of the other cell organelles.     

K562 chronic myeloid leukemia cells modify osteogenic differentiation and gene expression of bone marrow stromal cells

Bone marrow (BM) microenvironment plays an important role in normal and malignant hematopoiesis. As a consequence of interaction with the leukemic cells, the stromal cells of the bone marrow become deregulated in their normal function and gene expression. In our study, we found that mesenchymal stem cells (MSC) from BM of chronic myeloid leukemia (CML) patients have defective osteogenic differentiation and on interaction with K562 CML cells, the normal MSC showed reduced osteogenic differentiation. On interaction with K562 cells or its secreted factors, MSC acquired phenotypic abnormalities and secreted high levels of IL6 through NFκB activation. The MSC derived secreted factors provided a survival advantage to CML cells from imatinib induced apoptosis. Thus, a therapy targeting stromal cells in addition to leukemia cells might be more effective in eliminating CML cells.     

Evidence from molecular genetic and cytogenetic analyses that bone marrow histopathology is reliable in the diagnosis of chronic myeloproliferative disorders

The reliability of histopathological diagnosis in bone marrow specimens from patients with chronic myeloproliferative disorders (CMPD) was evaluated by correlating the histological findings with molecular genetic and cytogenetic analyses of the Ph¹-translocation. A rearrangement of m-bcr was detected only in patients (28/30) diagnosed histologically as chronic myeloid leukemia (CML). This finding was supported by the presence of a Ph¹-chromosome in 24/26 patients with CML examined. All the patients with other types of CMPD, including polycythemia vera (PV), primary thrombocythemia (PTH) and chronic megakaryocytic-granulocytic myelosis (CMGM), as well as those with unclassifiable CMPD (CMPD.UC) were Ph¹-negative (n = 38). The histopathological discrimination of CML from Ph¹-negative varieties of CMPD was also reliable for patients with myelofibrosis complicating CML, CMGM and CMPD.UC. The results demonstrate that bone marrow histopathology allows a reliable diagnosis of CML. This is in contrast with hematological data such as high platelet counts which show considerable overlapping in the various forms of CMPD.     

Vesicular monoamine transporter 2 (VMAT2) expression in hematopoietic cells and in patients with systemic mastocytosis.

Uptake of monoamines into secretory granules is mediated by the vesicular monoamine transporters VMAT1 and VMAT2. In this study, we analyzed their expression in inflammatory and hematopoietic cells and in patients suffering from systemic mastocytosis (SM) and chronic myelogenous leukemia (CML). Normal human and monkey tissue specimens and tissues from patients suffering from SM and CML were analyzed by means of immunohistochemistry, radioactive in situ hybridization, real time RT-PCR, double fluorescence confocal laser scanning microscopy, and immunoelectron microscopy. In normal tissue specimens, VMAT2, but not VMAT1, was expressed in mast cells, megakaryocytes, thrombocytes, basophil granulocytes, and cutaneous Langerhans cells. Further hematopoietic and lymphoid cells showed no expression of VMATs. VMAT2 was expressed in all types of SM, as indicated by coexpression with the mast cell marker tryptase. In CML, VMAT2 expression was retained in neoplastic megakaryocytes and basophil granulocytes. In conclusion, the identification of VMAT2 in mast cells, megakaryocytes, thrombocytes, basophil granulocytes, and cutaneous Langerhans cells provides evidence that these cells possess molecular mechanisms for monoamine storage and handling. VMAT2 identifies normal and neoplastic mast cells, megakaryocytes, and basophil granulocytes and may therefore become a valuable tool for the diagnosis of mastocytosis and malignant systemic diseases involving megakaryocytes and basophil granulocytes.     

Restoration of nonclonal hematopoiesis in chronic myelogenous leukemia with interferon alpha

Studies have shown that recombinant human alpha interferon (rIFN alpha) inhibits the growth of colonies of multipotential stem cells from human bone marrow. This report demonstrates that rIFN alpha inhibits the growth of such colonies from the bone marrow of patients with chronic myelogenous leukemia (CML) to a greater extent than from bone marrow of healthy individuals. It also shows that T lymphocyte colonies subcloned with interleukin 2 (IL-2) from CML mixed colonies were inhibited more by rIFN alpha than were similar colonies subcultured from normal mixed colonies. The report demonstrates that the Ph' chromosome is present in such T cell colonies subcultured from CML mixed colonies. When mixed colonies were grown from CML bone marrow in the presence of rIFN alpha, Ph' negative colonies were observed, whereas no such Ph' negative mixed colonies grew from a similar number of bone marrow cells incubated without rIFN alpha. These observations confirm that T lymphocytes derived from bone marrow stem cells are from the CML clone, and that the inhibition of growth of Ph' positive colonies, by rIFN alpha permits the growth of residual normal stem cells. The disappearance of the Ph-chromosome in subclones of T lymphocytes supports the notion of nonclonal hematopoiesis in patients with CML.     

An investigation of Ph1 chromosome in Chronic Myeloid Leukemia patients with different treatment modalities and hematological features

Chronic myeloid leukemia (CML) is characterized by a Ph¹ chromosome that derives through a translocation between chromosomes 9 and 22, i.e., t (9;22). Identifying the Ph¹ chromosome through cytogenetic analysis is an important aspect of CML diagnosis. The aim of this study was to determine the significance of cytogenetic analysis in the diagnosis of CML as well as to find out a relationship between chromosomal abnormalities and CML patients in different stages of treatment. Six CML patients were investigated for this study. The presence of Ph¹ chromosome was detected at different times of treatment using GTG banding on peripheral blood or bone marrow aspirations, and the results were analyzed using cytovision workstation. Hematological features were compared between newly diagnosed patients and patients under treatment. The Ph¹ chromosome was strongly associated with all cases of CML. The regression of Ph¹ chromosomes differed for each patient depending on the treatments and individual response to specific treatments.     

An overview of bone marrow transplantation for chronic myeloid leukemia.

The use of intensive therapy together with transplantation of marrow from a suitable donor is the only established curative treatment for patients with chronic myeloid leukemia (CML). However, marrow transplantation is hazardous, costly and applicable to relatively few patients. Therefore, we evaluated the results and limitations of marrow transplantation for CML and discussed new treatment strategies. We decided to select a limited number of papers that focused on the relevant issues rather than to undertake an exhaustive comparison of treatment results from different centres. Patients with CML in the chronic phase who receive marrow from a sibling with the same human leukocyte antigen type can expect to have a long-term disease-free survival rate of 50%. However, the procedure is associated with a mortality rate of 30%, mainly because of graft-versus-host disease (GVHD) and interstitial pneumonitis. Moreover, because of the requirements for age and histocompatibility only 10% of patients with chronic-phase CML are currently eligible. Transplantation earlier in the chronic phase (within 1 year after diagnosis), the use of marrow from matched, unrelated donors and the development of improved methods for reducing the incidence of GVHD all hold promise. In addition, the preliminary results of intensive therapy followed by transplantation with cultured autologous marrow have been encouraging. If further progress is to be made, continued optimism coupled with carefully developed and executed studies will be necessary.     

Multiple Chromosome Abnormalities Following Bone Marrow Transplant for Chronic Myelogenous Leukemia

A 44-year-old female was diagnosed in the chronic phase of chronic myelogenous leukemia (CML) and was confirmed to be Philadelphia chromosome positive by a bone marrow cytogenetic study. No additional cytogenetic abnormalities were found. The patient's cell counts were initially well controlled with hydrox-yurea. She then received an unrelated 6 of 6 HLA matched allo-geneic bone marrow transplant (BMT) from a male donor. The patient underwent myeloablative therapy with thiotepa and five fractions of total body radiation prior to the transplant. About four weeks after transplantation, the patient developed biopsy-proven graft-versus-host disease of the skin and GI tract. A blood sample was drawn at that time for cytogenetic analysis. Among 34 analyzed cells, 22 were normal male donor cells. The remaining 12 cells did not have the t(9;22), but had numerous structural abnormalities. While many cells were missing an X chromosome, other abnormalities, including deletions, rearrangements, dicentrics, acentric fragments, rings and marker chromosomes were non-clonal. No clinical evidence of progression from CML chronic phase was found, suggesting that the non-clonal abnor-malities were therapy related.     

Label-free MS(E) proteomic analysis of chronic myeloid leukemia bone marrow plasma: disclosing new insights from therapy resistance

Chronic myeloid leukemia (CML) is a pluripotent hematopoietic disorder that is currently considered incurable. The tyrosine kinase product of the Philadelphia chromosome, P210 BCR-ABL, provided a pathogenetic explanation for the initiation of the CML chronic phase and is the molecular therapeutic target for the disease. Imatinib mesylate, an orally available BCR-ABL kinase inhibitor, can induce haematologic and cytogenetic remission of CML. However, imatinib resistance occurs frequently, resulting in relapse. New treatment strategies are focusing on resistant CML stem cells and the bone marrow stroma. The identification of novel pathways and mechanisms in the bone marrow microenvironment could significantly contribute to the development of such strategies. In this work, we used a high-resolution label-free MS(E) proteomic approach to identify differential protein expression in the CML bone marrow plasma of responsive and resistant patients. Oxidative lipid metabolism and regulation of the switch from canonical to noncanonical WNT signaling may contribute to CML resistance in the bone marrow compartment.     

Blast colony-forming cell binding from CML bone marrow, or blood, on stromal layers pretreated with G-CSF or SCF

Blast colony-forming cells (CFU-BL) represent a specific subpopulation of special primitive progenitors characterized by colony formation only in close contact with a preformed stromal layer. CFU-BL derived from bone marrow of chronic myeloid leukaemia (CML) patients have been proved to adhere poorly to bone marrow derived stromal layers suggesting that the appearance of progenitors and precursors in the circulation is due to a defective adhesion of these cells to the bone marrow microenvironment. In the present experiments the effect of short-term incubation of preformed normal bone marrow stroma on the adherence of CML derived CFU-BL was studied. For stroma cultures bone marrow cells were cultured in microplates in the presence of hydrocortisone. Cultures were used when stromal layers became confluent and no sign of haemopoiesis could be observed. CFU-BL were studied by panning plastic non-adherent mononuclear (PNAMNC) bone marrow or blood cells. 8.9 +/- 2.4 colonies/103 PNAMNC (six experiments) were formed from normal bone marrow on stromal layers and 4.8 +/- 2.1 colonies/103 PNAMNC (five experiments) from CML bone marrow. Colony formation from normal bone marrow was not increased if stromal layers were incubated with 100 ng/mL granulocyte colony-stimulating factor (G-CSF) or stem cell factor (SCF). Incubation of stroma with G-CSF or SCF, however, increased the colony formation of PNAMNC from CML bone marrow or blood significantly. These findings suggest that local concentration of haemopoietic growth factors at the time of panning may influence the attachment of CML progenitors to the stroma.     

Response of granulocyte-committed progenitors from patients with chronic myeloid leukemia to humoral regulators release by macrophages in agar diffusion chamber culture

We investigated the responsiveness of granulocyte-committed progenitors (CFU-G/D) from patients with chronic myeloid leukemia (CML) and healthy subjects to stimulating and inhibiting activities released by murine macrophages in diffusion chamber culture. CFU-G/D from CML demonstrate a normal response to macrophage-derived stimulation. The responsiveness of CFU-G/D from patients with CML to indomethacin-sensitive inhibition was significantly suppressed. In this regard no difference between CFU-G/D from bone marrow and blood of patients with CML could be observed. Colonies formed both by CFU-G/D from healthy subjects and CML consisted exclusively of cells of granulocyte line: from myeloblasts up to polymorphonuclear granulocytes. Similar cellular composition of colonies could be noted during macrophage-derived stimulation and inhibition of CFU-G/D growth. In conclusion, we have demonstrated that CML CFU-G/D which proliferate and differentiate in diffusion chamber culture show a normal response to macrophage-derived stimulation but are less sensitive to indomethacin-dependent inhibition.     

Planimetric analysis of megakaryocytes in the four main groups of chronic myeloproliferative disorders.

Planimetry of megakaryocytes (MK) was performed in bone marrow biopsies (BMBs) from patients with chronic myeloproliferative disorders (CMPD) to substantiate cytomorphologic differences in this cell lineage between the four main groups of CMPD. The biopsy specimens were classified histologically prior to morphometry, according to the Hannover Classification of CMPD. Five histological groups were investigated, evaluating between 21 and 30 biopsies in each group. The five groups were as follows: (1) Chronic myelocytic leukemia (CML) of common type (CML.CT), (2) CML with megakaryocytic increase (CML.MI), (3) polycythemia vera (P. vera), (4) primary thrombocythemia (PTH), and (5) chronic megakaryocytic-granulocytic myelosis (CMGM). The results of five variables, i.e. the cellular and nuclear size, the cellular and nuclear form factor, and nuclear segmentation, were determined in at least 50 MK per BMB. The results reveal significant differences in MK nuclear and cellular size, as well as in nuclear segmentation between CML and the three other groups in that the nuclear and cellular size of the MK in CML are smaller than in P. vera, PTH, and CMGM. Moreover, the degree of nuclear segmentation or lobulation differs significantly between the three disorders characterized by large MK. Discriminant analysis permits 78-100% reliability of reclassification by morphometry compared with the histologic classification. A reduced reliability of the morphometric classification to around 80% was found between P. vera and PTH, as well as between P. vera and CMGM. In the design of this study, morphometry of MK lends added weight to the subjective classification of these disorders.     

Distribution of growth hormone and prolactin in secretory granules of the normal and neoplastic human adenohypophysis

Growth hormone [GH] and prolactin [PRL] can be demonstrated simultaneously in electron micrographs by means of the double immunocytochemical labeling technique using colloidal gold particles of two different sizes. This method was used to study biopsy specimens obtained from 15 patients suffering from acromegaly, 11 patients suffering from prolactinomas, and eight biopsy specimens obtained during adenomectomy from the normal, paraadenomatous pituitary tissue. Four granule populations with different immunoreactions were found: (1) granules containing GH only, (2) granules containing PRL only, (3) mixed granules containing GH and PRL, and (4) granules displaying no immunoreactivity. The existence of mixed granules indicated that the two hormones are synthesized by the same cell and in communicating compartments of the cells; i.e., the rough-surfaced endoplasmic reticulum. The number of GH-containing granules (pure GH granules and mixed GH-PRL granules) was greater than that of PRL-containing granules (pure PRL granules and mixed PRL-GH granules) in adenomas causing acromegaly and in the normal pituitary tissue, whereas the opposite was true for prolactinomas. The number of PRL-containing granules was larger in biopsy specimens from patients who had acromegaly and hyperprolactinemia than in patients with acromegaly and normal serum PRL levels.     

MYC in chronic myeloid leukemia: induction of aberrant DNA synthesis and association with poor response to imatinib

Untreated chronic myeloid leukemia (CML) progresses from chronic phase to blastic crisis (BC). Increased genomic instability, deregulated proliferation, and loss of differentiation appear associated to BC, but the molecular alterations underlying the progression of CML are poorly characterized. MYC oncogene is frequently deregulated in human cancer, often associated with tumor progression. Genomic instability and induction of aberrant DNA replication are described as effects of MYC. In this report, we studied MYC activities in CML cell lines with conditional MYC expression with and without exposure to imatinib, the front-line drug in CML therapy. In cells with conditional MYC expression, MYC did not rescue the proliferation arrest mediated by imatinib but provoked aberrant DNA synthesis and accumulation of cells with 4C content. We studied MYC mRNA expression in 66 CML patients at different phases of the disease, and we found that MYC expression was higher in CML patients at diagnosis than control bone marrows or in patients responding to imatinib. Further, high MYC levels at diagnosis correlated with a poor response to imatinib. MYC expression did not directly correlate with BCR-ABL levels in patients treated with imatinib. Overall our study suggests that, as in other tumor models, MYC-induced aberrant DNA synthesis in CML cells is consistent with MYC overexpression in untreated CML patients and nonresponding patients and supports a role for MYC in CML progression, possibly through promotion of genomic instability.     

Prognostic significance of the histomorphometric features of bone marrow trephine biopsies in patients with chronic myeloid leukemia

OBJECTIVE: To study the correlation of histomorphometric data of bone marrow trephine biopsies at the time of initial diagnosis in chronic myeloid leukemia (CML) patients with the patient prognosis. STUDY DESIGN: A total of 40 CML patients were divided equally in group I (developed accelerated phase or blast crisis within 18 months of initial diagnosis of chronic phase of CML) and group II (developed accelerated phase or blast crisis > 30 months after initial diagnosis of chronic phase of CML). The clinical, hematologic and histomorphometric data were compared in the 2 groups of CML patients. RESULTS: The percentage of bone marrow promyelocytes was significantly increased in group I. On morphometry, the number of blasts per square millimeter, the area of reticulin fibers per square millimeter and the percentage area occupied by reticulin fibers were statistically significant. CONCLUSION: There seems to be grounds for hope for predicting prognosis of CML patients at initial diagnosis based on histomorphometric findings. The percentage area of reticulin fibers and the number of blasts per square millimeter are important prognostic predictors in histomorphometry data.     

Increased accumulation of the glycoxidation product Nepsilon-(carboxymethyl)lysine in hearts of diabetic patients: generation and characterisation of a monoclonal anti-CML antibody

Heart failure is a condition closely linked to diabetes. Hyperglycaemia amplifies the generation of a major advanced glycation end product Nepsilon-(carboxymethyl)lysine (CML), which has been associated with the development of vascular and inflammatory complications. An increased accumulation of CML in hearts of diabetic patients may be one of the mechanisms related to the high risk of heart failure. Therefore, we investigated the localization of CML in diabetic hearts. To investigate the presence and accumulation of CML in tissues, a monoclonal anti-CML antibody was generated and characterised. With this novel monoclonal antibody against CML, the localization of CML was investigated by immunohistochemistry, in heart tissue of controls (n = 9) and heart tissue of diabetic patients (n = 8) without signs of inflammation or infarction. In addition, in the same subjects we studied the presence of CML in renal and lung tissues. CML staining was approximately sixfold higher in hearts from diabetic patients as compared to control hearts (2.0 +/- 0.3 and 0.3 +/- 0.2 A.U., respectively, P < 0.01). CML deposition was localized in the small intramyocardial arteries in endothelial cells and smooth muscle cells, but not in cardiomyocytes. These arteries did not show morphological abnormalities. The intensity of staining between arteries at the epicardial, midcardial and endocardial side did not vary significantly within patients. In renal tissues, CML staining was most prominent in tubules and in atherosclerotic vessels, without differences in intensity between controls and diabetic patients. In non-infected lungs, no CML was detected. In conclusion, CML adducts are abundantly present in small intramyocardial arteries in the heart tissue of diabetic patients. The accumulation of CML in diabetic hearts may contribute to the increased risk of heart failure in hyperglycaemia.     

Nature of the silver staining and association of acrocentric chromosomes in normal and leukemic human cells

Silver staining and acrocentric chromosome association (ACA) patterns were investigated in bone marrow cells as well as in peripheral blood cells (cultures with or without PHA) from 39 patients with chronic myelocytic leukemia (CML), including 20 cases being in blastic phase (BP CML), and 51 patients with acute leukemia (AL). Bone marrow cells and PHA-stimulated peripheral blood lymphocytes (from 10 and 17 healthy donors, respectively) were used as a control. The frequency of ACA in metaphases from bone marrow cells of all the above groups of patients was shown to be decreased compared to that in PHA-stimulated lymphocytes. Patients with BP CML and AL constituted the most heterogeneous groups although some of them demonstrated the highest ACA-frequency per cell. There is a pronounced correlation between Ag+-nucleolus organizer regions (NOR's) and the frequency of ACA. With the exception of CML, the correlation coefficients (0.83, 0.74, and 0.72) were highly significant for all the above groups (donors, BP CML, and AL patients, respectively). The distribution pattern of single chromosome pairs, according to their ACA frequency, differed with every individual studied, but it was similar in normal and leukemic cells of the same individual. From the above data a conclusion is made that the frequency of ACA may depend on the functional activity of the NOR's as well as on the cells type.     

Chronic myelogenous leukaemia exosomes modulate bone marrow microenvironment through activation of epidermal growth factor receptor

Chronic myelogenous leukaemia (CML) is a clonal myeloproliferative disorder. Recent evidence indicates that altered crosstalk between CML and mesenchymal stromal cells may affect leukaemia survival; moreover, vesicles released by both tumour and non‐tumour cells into the microenvironment provide a suitable niche for cancer cell growth and survival. We previously demonstrated that leukaemic and stromal cells establish an exosome‐mediated bidirectional crosstalk leading to the production of IL8 in stromal cells, thus sustaining the survival of CML cells. Human cell lines used are LAMA84 (CML cells), HS5 (stromal cells) and bone marrow primary stromal cells; gene expression and protein analysis were performed by real‐time PCR and Western blot. IL8 and MMP9 secretions were evaluated by ELISA. Exosomes were isolated from CML cells and blood samples of CML patients. Here, we show that LAMA84 and CML patients’ exosomes contain amphiregulin (AREG), thus activating epidermal growth factor receptor (EGFR) signalling in stromal cells. EGFR signalling increases the expression of SNAIL and its targets, MMP9 and IL8. We also demonstrated that pre‐treatment of HS5 with LAMA84 exosomes increases the expression of annexin A2 that promotes the adhesion of leukaemic cells to the stromal monolayer, finally supporting the growth and invasiveness of leukaemic cells. Leukaemic and stromal cells establish a bidirectional crosstalk: exosomes promote proliferation and survival of leukaemic cells, both in vitro and in vivo, by inducing IL8 secretion from stromal cells. We propose that this mechanism is activated by a ligand–receptor interaction between AREG, found in CML exosomes, and EGFR in bone marrow stromal cells.     

Clinical reagents of GM-CSF and IFN-α induce the generation of functional chronic myeloid leukemia dendritic cells in vitro

Dendritic cells (DCs) have been successfully induced in vitro from chronic myeloid leukemia (CML) cells, which may provide a promising immunotherapeutic protocol for CML. To facilitate the optimization of DCs-based vaccination protocols, we investigated the efficiency of in vitro generation of DCs from bone marrow mononuclear cells of CML patients by clinical reagents of GM-CSF and IFN-α. Bone marrow mononuclear cells were isolated from eight CML patients and CML-DCs were generated in the presence of different cytokines (Group A: GM-CSF for research and IL-4 for research; Group B: GM-CSF for injection and IFN-α for injection) in RMPI-1640 medium containing 10% human AB serum. After 8 days, the morphologic features of CML-DCs were observed and their immunophenotypes were analyzed by flow cytometry. The activity of CML-DCs was determined by evaluating their ability to stimulate allogeneic mixed lymphocyte reaction (allo-MLR) and anti-leukemic cytotoxic T lymphocytes (CTLs). The culture protocols were successful in generating functional CML-DCs from all the CML patients as evidenced by the significant upregulation of CD80, CD86, CD83 HLA-DR and CD1a compared to pre-cultured (p < 0.05), and increased allogeneic T cell stimulating proliferation capacity (p < 0.05). CML-DCs could stimulate a specific anti-leukemia response. In summary, we demonstrate that the combination of clinical reagents GM-CSF and IFN-α induced the generation of DCs that have the ability to stimulate a specific anti-leukemia CTLs response in vitro, indicating their feasibility for clinical vaccination protocols for CML patients.     

Osteopontin is upregulated by BCR-ABL

Chronic myelogenous leukemia (CML) is characterized by its hallmark oncogene BCR-ABL and the progression from a chronic phase toward an acute leukemia, with a differentiation arrest of the leukemic clone. In the present study, we conducted a microarray analysis using an inducible model of BCR-ABL expression based on the TET-OFF system, and we found that osteopontin (OPN), a component of stem cell niche, is overexpressed in BCR-ABL-expressing cells. Studies using mutant forms of BCR-ABL demonstrated that the BCR-ABL-induced OPN overexpression was a tyrosine kinase-dependent event. Furthermore, OPN concentration was significantly increased in the serum of leukemic mice generated by transplantation of BCR-ABL-expressing bone marrow cells. Most importantly, a significant increase of OPN concentration was observed in the serum of CML patients as compared to controls. Overall these results show that OPN is deregulated by BCR-ABL oncogene and suggest that OPN could be involved in CML stem cell biology.