Ultrastructure of neutrophilic granulopoiesis in the bone marrow of patients with chronic myeloid leukemia (CML)

In seven patients with chronic myeloid leukemia (CML) an 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. Partly supported by a grant from the Maria-Pesch Foundation, Cologne, Federal Republic of Germany     

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.     

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.     

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.     

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.     

Human AQP5 plays a role in the progression of chronic myelogenous leukemia (CML)

Aquaporins (AQPs) have previously been associated with increased expression in solid tumors. However, its expression in hematologic malignancies including CML has not been described yet. Here, we report the expression of AQP5 in CML cells by RT-PCR and immunohistochemistry. While normal bone marrow biopsy samples (n = 5) showed no expression of AQP5, 32% of CML patient samples (n = 41) demonstrated AQP5 expression. In addition, AQP5 expression level increased with the emergence of imatinib mesylate resistance in paired samples (p = 0.047). We have found that the overexpression of AQP5 in K562 cells resulted in increased cell proliferation. In addition, small interfering RNA (siRNA) targeting AQP5 reduced the cell proliferation rate in both K562 and LAMA84 CML cells. Moreover, by immunoblotting and flow cytometry, we show that phosphorylation of BCR-ABL1 is increased in AQP5-overexpressing CML cells and decreased in AQP5 siRNA-treated CML cells. Interestingly, caspase9 activity increased in AQP5 siRNA-treated cells. Finally, FISH showed no evidence of AQP5 gene amplification in CML from bone marrow. In summary, we report for the first time that AQP5 is overexpressed in CML cells and plays a role in promoting cell proliferation and inhibiting apoptosis. Furthermore, our findings may provide the basis for a novel CML therapy targeting AQP5.     

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.     

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.     

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.     

Effects of the tyrosine kinase inhibitor imatinib mesylate (STI571) on bone marrow features in patients with chronic myelogenous leukemia

Preliminary data are available about bone marrow (BM) changes in patients with chronic myeloid leukemia (CML) who received the molecularly targeted and highly effective tyrosine kinase inhibitor Imatinib mesylate (STI571). This review is focused on a systematic assessment of BM features detectable at different stages of CML (stable, accelerated, blastic) following long-term (more than 10 months) treatment. By applying enzyme- and immunohistochemistry including monoclonal antibodies visualizing proliferating cell nuclear antigen (PCNA) and apoptosis (anti-apostatin), a more elaborate insight into alterations affecting hematopoiesis and the stroma compartment was gained. In patients with stable-phase CML therapy resulted in a significant reduction in cellularity, neutrophil granulopoiesis and number of megakaryocytes, accompanied by a retrieval of erythroid precursors. In patients with Imatinib as the only treatment morphometric analysis of CD61+ megakaryopoiesis was in keeping with a significant decrease in maturation defects implying a lesser amount of atypical micromegakaryocytes almost consistent with normalization. Moreover, a reduction of the initially enhanced (CD34+) microvessel density was detectable associated with a decrease in luminal distension. Regression of marked to moderate myelofibrosis was recognizable in about 70% of patients especially in the accelerated and blastic phases. The amount of myeloblasts, CD34+ progenitor cells and lysozyme-expressing immature myelomonocytic cells declined with treatment, but recurred in about 19% of patients that developed a leukemic relapse after 21+/-6 months of therapy. Data on proliferative activity and apoptosis in general supported in vitro findings concerning the inhibitory effect of this agent on growth associated with a tendency for stimulated apoptosis, at least in responding patients.     

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.     

Effect of cytosine arabinoside on the differentiation of granulocyte-monocyte progenitors (CFU-GM) and myeloid leukemia blasts (CFU-L) in vitro

The serum concentrations of Ara-C are in the range from 10(-6) to 10(-8) M in LD-Ara-C treated patients. The growth of CFU-GM from bone marrow of healthy volunteers was depressed depending on Ara-C-concentration applied in vitro. The growth of CFU-L from peripheral blood of two patients with AML (M 2) and one patient with CML in blast crisis was differently influenced by Ara-C-application in vitro. An elevated proportion of mature cells was observed in smears of cultured cells with Ara-C from two patients. The usefulness of Ara-C for a differentiation inducing therapy is discussed.     

Increased fibroblast colony stimulating activity (F-CSA) in serum of myeloproliferative disorders

Sera of patients with primary myelofibrosis (PMF), primary thrombocythemia (PT), polycythaemia vera (PV) and chronic myeloid leukemia (CML) contained a significantly increased F-CSA (or F-CSAs) compared to those of normal subjects and patients with secondary thrombocytosis (ST). This F-CSA was heat sensitive and had the capacity to promote both proliferation and maturation of normal marrow fibroblast colony-forming cells (CFU-F). This F-CSA seemed to be different from human platelet derived growth factor (PDGF), tumor necrosis factor (TNF) and fibroblast growth factor (FGF) from bovine brain. This F-CSA might be of importance in the pathogenesis of bone marrow fibrosis in myeloproliferative disorders.     

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.     

Myeloid Derived Suppressor Cells (MDSCs) Are Increased and Exert Immunosuppressive Activity Together with Polymorphonuclear Leukocytes (PMNs) in Chronic Myeloid Leukemia Patients

Tumor immune tolerance can derive from the recruitment of suppressor cell population, including myeloid derived suppressor cells (MDSCs), able to inhibit T cells activity. We identified a significantly expanded MDSCs population in chronic myeloid leukemia (CML) patients at diagnosis that decreased to normal levels after imatinib therapy. In addition, expression of arginase 1 (Arg1) that depletes microenvironment of arginine, an essential aminoacid for T cell function, resulted in an increase in patients at diagnosis. Purified CML CD11b+CD33+CD14-HLADR- cells markedly suppressed normal donor T cell proliferation in vitro. Comparing CML Gr-MDSCs to autologous polymorphonuclear leukocytes (PMNs) we observed a higher Arg1 expression and activity in PMNs, together with an inhibitory effect on T cells in vitro. Our data indicate that CML cells create an immuno-tolerant environment associated to MDSCs expansion with immunosuppressive capacity mediated by Arg1. In addition, we demonstrated for the first time also an immunosuppressive activity of CML PMNs, suggesting a strong potential immune escape mechanism created by CML cells, which control the anti-tumor reactive T cells. MDSCs should be monitored in imatinib discontinuation trials to understand their importance in relapsing patients.     

Investigations on karyotype evolution in patients with chronic myeloid leukemia (CML)

In an attempt to relate karyotype evolution to clinical and hematological data serial chromosomal analyses were performed in 31 patients with chronic myeloid leukemia (CML), both in chronic and acute phases. Our results in Philadelphia chromosome (Ph1)-positive CML are in line with karyotype profiles described in the literature. In addition, we report on chromosomal findings in 4 cases of Ph1-negative disease, one presenting with an iso17q chromosome in the positive CML. The same chromosomal abnormality was observed in a small population of Ph1-negative cells present in one of two patients with mixed Ph1-positive/Ph1-negative CML. The first case of a female patient with the loss of a sex chromosome in Ph1-positive cells is reported. Two patients with unusually long and mild chronic phases despite the presence of trisomy 8 in their karyotypes are described. Our findings suggest that the order of appearance of additional chromosomal changes of CML is of prognostic significance for the progression and the clinical picture of the disease.     

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.     

Translocation of c-abl oncogene and PDGFB (c-sis) gene in a case of CML with 46,XY, t(22;22)

In a case of CML with a variant Philadelphia translocation (Ph1 or Ph) t(22;22) (q11;q13) in bone marrow cells and unstimulated peripheral blood cells, no cytogenetically detectable involvement of chromosome 9 was observed. Southern blot experiments using probes specific for bcr and c-sis however revealed rearrangement of the bcr, but not of PDGFB (c-sis) gene. Northern blot analysis of bone marrow RNA showed a very weak signal with the c-sis probe, while in a lymph-node biopsy PDGFB m-RNA could not be detected. Chromosomal in situ hybridization gave evidence for translocation of c-abl from chromosome 9 to Ph and of PDGFB from chromosome 22 to chromosome 9, as the result of a threefold translocation t(9;22;22).     

Prognostic significance of some clinical, morphological and cytogenetic findings in refractory anaemia (RA) and RA with sideroblasts

In 27 patients initially diagnosed as refractory anaemia (RA) or RA with sideroblasts (RA-S) according to the FAB-classification a number of clinical, morphological and cytogenetic parameters were correlated for prognostic significance. From these correlations it emerged that severe cytopenia is centrally positioned with regard to clinical course in RA and RA-S. Positive correlations were found to initial diagnosis, clonal cytogenetic abnormalities, progression to RA with an excess of blasts (RAEB) or acute myeloid leukaemia (AML), the percentage of bone marrow blast cells and prolonged half life for radioactively labeled iron. The degree of peripheral blood granulocytopenia, alone, was correlated to bone marrow hypoplasia. Moreover, the frequency of abnormal karyotypes was inversely correlated to bone marrow cellularity and proportional to the frequency of bone marrow blast cells. From these relationships it may be proposed that chromosome abnormalities are associated with prolonged blast cell generation times and inhibition of blast cell maturation resulting in reduced marrow cellularity and blast cell accumulation, and, in the peripheral blood, falling percentages of neutrophil granulocytes. With the blast cell accumulation the bone marrow cellularity again becomes hyperplastic and the preleukaemic condition is transformed into RAEB or AML.