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Chronic myeloid leukemia (CML) is a clonal myeloproliferative disorder of a stem cell, involving myeloid, erythroid, megacaryocyte, lymphoid B-cells and "natural killer" cells. The hallmark of CML is the Philadelphia (Ph) chromosome which is a shortened chromosome 22 (22q-) resulting from a reciprocal translocation involving chromosome 9 and chromosome 22, designed t (9;22) (q34;q11). This translocation juxtaposes parts of two genes; ABL on chromosome 9 and BCR (breakpoint cluster region) on chromosome 22. Transcription of the BCR/ABL fusion gene results in an hybrid mRNA that is translated into a 210 kDa or 190 kDa protein, depending on the location of the breakpoint in the bcr region. This protein plays a key role in CML: its tyrosine-kinase activity, that differs from the normal ABL product, may be involved in leukemic cell growth. Nonetheless, the loss of the negative cell growth regulation by c-ABL, or BCR/ABL fusion protein interaction with other cellular genes (such as RAS or c-MYC) could also be involved in CML pathophysiology. A better understanding of the molecular mecanisms of CML could lead to specific treatment, such as tyrosine-kinase inhibitors, synthetic oligodeoxynucleotides, or site-specific DNA-binding proteins designed against BCR/ABL oncogenic fusion sequence.

CD44 is a widely expressed, multifunctional, cell-surface glycoprotein that has been implicated in the regulation of normal hematopoiesis. In addition, expression of particular isoforms of CD44 has been associated with malignant transformation and/or the acquisition of metastatic potential. In this study, we used two recently developed monoclonal anti-CD44 antibodies, one reactive with an epitope shared by many CD44 isoforms and the other with an epitope unique to CD44 isoforms containing amino acids encoded by the alternatively spliced exon v10, to compare the expression of CD44 on primitive hematopoietic cells from the marrow of normal individuals and their neoplastic counterparts present in the peripheral blood of patients with chronic myeloid leukemia (CML). Multiparameter fluorescence-activated cell sorter (FACS) analysis and cell sorting studies showed that CD44 is normally expressed at high to very high levels on both long-term culture-initiating cells (LTC-IC) and granulopoietic colony-forming cells (granulocyte-macrophage colony-forming units [CFU-GM]). In contrast, primitive erythropoietic progenitors (burst-forming units-erythroid [BFU-E]) in normal marrow were more homogeneous in their expression of CD44, and very few (less than 5%) showed the very high levels of CD44 seen on 20% to 25% of LTC-IC and CFU-GM. Antibody staining showed the expression of exon v10-containing CD44 isoforms to be restricted to a small subpopulation (4% to 8%) of morphologically recognizable mature (CD34-) myeloid cells within the light-density fraction of normal marrow cells. Reverse transcription-polymerase chain reaction (RT-PCR) analysis showed the presence of two exon v10-containing mRNA species. In CML, a significantly greater proportion of the circulating neoplastic CFU-GM expressed very high levels of CD44, and these CFU-GM were accompanied by an increased number of light density v10+ cells, including some that coexpressed CD34. Nonmalignant hematopoietic progenitors mobilized by prior chemotherapy and growth factor treatment of patients with Hodgkin's disease or acute myeloid leukemia in remission showed no changes in CD44 expression relative to normal marrow progenitors. These results provide evidence of early differentiation-associated changes in CD44 expression during normal hematopoiesis in vivo that may be deregulated in the neoplastic clone of patients with CML.

The number of colony-forming units of granulocytes/monocytes (CFU-GM) in the peripheral blood of 7 patients with chronic myeloid leukemia (CML) in chronic phase (CP) receiving standard doses of busulfan (BSF, 0.1-0.2 mg/kg/day p.o.) was compared to that found in 8 patients with CML in CP not previously treated in order to establish if non-myeloablative chemotherapy mobilizes committed granulomonocytic progenitor cells into the circulation. The number (mean +/- SEM) of spontaneous CFU-GM in untreated patients was similar to that recorded in 10 sex- and age-matched controls, 2.6 +/- 1.9 and 3.5 +/- 2.1, respectively. BSF-treated patients showed significantly more spontaneous CFU-GM (13.9 +/- 7.5) than controls and untreated patients. Addition of recombinant human granulocyte/monocyte colony-stimulating factor to cultures promoted colony growth in controls but not in untreated and BSF-treated patients. These data seemingly indicate that: 1) administration of standard non-myeloablative doses of BSF to patients with CML in CP mobilizes CFU-GM into the circulation and 2) BSF therapy selects a granulomonocytic colony-forming progenitor cell population with increased autonomous growth potential. These findings may contribute to the understanding of the therapeutic role of BSF in CML.

The paper discusses certain distributive and clustering modes of stimulus P and of its receptors. When combined with topologic features peculiar to sources and stem cells, these parameters account not only for the cycling variations typical of normal stem cells but also for seemingly paradoxical shifts in proliferative activity of cells grafted into patients with chronic myeloid leukemias.

Four cases of endometrial extramedullary haemopoiesis are reported, all with associated haematological disease. The diagnoses of a myeloproliferative disorder and thalassaemia trait were made as a consequence of the histological observations and subsequent haematological investigations in two cases. The third case occurred in a patient with an established diagnosis of chronic myeloid leukaemia. The diagnosis of extramedullary haemopoiesis in the final case was made on autopsy material from a patient with multiple myeloma. The endometrium from five other women with known myelofibrosis was examined but extramedullary haemopoiesis was not found. Endometrium from 32 fetuses did not contain haemopoietic elements, excluding the likelihood of the endometrium being a common site for extramedullary haemopoiesis in development. Endometrial extramedullary haemopoiesis is an uncommon finding, but it is worthy of note, as it may herald the presence of an underlying haematological abnormality.

Macrophage inflammatory protein-1 alpha (MIP-1 alpha) is a negative regulator of normal haemopoietic stem cell proliferation. Insensitivity to MIP-1 alpha of progenitor cells in chronic myeloid leukaemia (CML) could, therefore, explain myeloid expansion in this disease. We compared the effects of MIP-1 alpha on progenitor cells in normal marrow and in the blood and marrow of patients with chronic phase CML. Plastic-adherent precursors of granulocyte-macrophage colony-forming cells (P delta progenitors) are very primitive progenitor cells and are detected by incubating them for 1 week in liquid culture and assaying the CFU-GM released into the supernatant. Direct CFU-GM assays were also used in this study. Daily addition of 300 ng/ml/day of MIP-1 alpha to P delta progenitor assays of normal marrow cells suppressed CFU-GM production by 50% and in CML bone marrow P delta cultures by 20-30%. The response of CML blood P delta progenitors was heterogeneous. In five of nine cases, CFU-GM production was doubled in the presence of MIP-1 alpha and in four of nine cases, it was reduced. Addition of 100-500 ng MIP-1 alpha to direct assays of CFU-GM stimulated colony formation by normal marrow and CML blood cells to a similar extent.

Available evidence suggests a double-pathway two-staged genetic alteration in the pathogenesis of Chronic Myeloid Leukaemia (CML). The regular Ph' defect results in BCR-ABL gene chimaerism on the one hand and suppressed synthesis of the protein responsible for Zn absorption on the other. The resulting Zn deficiency leads, through its metalloenzymes, to a low NAP activity and depressed DNA & RNA polymerase activities: the latter necessitates an adaptive mechanism to sustain cell division despite low zinc. This adaptation is in the form of another gene alteration; a point mutation in the BCR-ABL chimaeric gene, now an oncogene, whose onco-proteins are zinc-independent and stimulate cell division more efficiently (though abnormally also) than the polymerases while defying the usual mechanisms regulating DNA synthesis and cell division. Thus it seems possible that assisted transcellular zinc transport could prevent development of CML in Ph'-positive individuals and the enhanced (abnormal) cellular proliferation might be specifically inhibited.

INF-alpha therapy has been a major development in the treatment of CML. Maturing experience has confirmed its ability to induce durable major cytogenetic remissions, which translate into durable long-term disease control, and change in the natural history of the disease. Future studies aimed at improving the percentage and quality of cytogenetic remissions by combining INF-alpha therapy with other chemotherapeutic agents and/or biologicals are needed. The goal would be to identify strategies that can consistently achieve a major cytogenetic response rate of 40% to 50% in the general community setting with acceptable morbidity. Continued studies of the interactions between INF -alpha therapy and transplant strategies will permit the optimization of treatment for each individual patient with CML.

The growth of 66 long-term survivors, transplanted in two centres (Genoa S. Martino and Monza) is reported. Patients were all under age 15 at the time of bone marrow transplantation (mean 9.8 +/- 3.4 years; range 1.07-15 years) with a minimum follow-up of at least 12 months. They were divided into four groups. Group 1: eight patients with severe aplastic anemia (SAA): conditioning included cyclophosphamide (CY) 200 mg/kg only. Group 2: 32 patients with acute lymphoblastic leukemia (ALL), and acute myeloid leukemia (AML): conditioning included CY 120 mg/kg and 10-12 Gy fractionated total body irradiation (fTBI). Group 3: 20 patients with ALL, who had previously received cranial irradiation; conditioning included CY 120 mg/kg and fTBI with an additional testicular irradiation (4 Gy). Group 4: six patients with chronic myeloid leukemia (CML) and AML; conditioning included CY 200 mg/kg and busulfan (BU) 16 mg/kg. Group was impaired in all four groups, including the unirradiated groups (-0.2 +/- 0.7 and -0.5 +/- 0.6 delta-SDS in groups I and 4, respectively). Growth impairment-SDS was statistically significant in the two irradiated groups (-0.7 +/- 1.0 and -0.9 +/- 1.0 delta-SDS in groups 2 and 3, respectively), more marked in patients who had had previous cranial irradiation. Chronic graft-versus-host disease and its treatment were not found to have a major effect on growth.

The cardiac function, hemorheology and the lingual microcirculation of the 62 leukemic patients were investigated. The results showed that their left cardiac function was markedly damaged, and the blood specific viscosity and hematocrit lowered, the time of RBC electrophoresis extended, the ESR and the K value of ESR equation was increased, which suggested the blood agglutination was enhanced. The lingual microcirculation obviously displayed reducing the number of papilla fungiformis, of intrapapillar plexus of microvessel as well as vessel loops, it also revealed the increasing of the abnormal form of microvessel and of the dilating vessel loops. The colour of the blood flow showed pink and dark red, the condition around vessel loops showed exudation and bleeding in several cases.

We examined the hybrid bcr/abl mRNA present in 59 patients with chronic myeloid leukemia, using the reverse transcription method and polymerase chain reaction. Bcr/abl gene was found in 98% of patients. 60% of patients had b3a2 type of translocation, 40% type b2a2. Patients with b3a2 type had higher platelet count at diagnosis presentation than patients with b2a2. Other hematological data were similar in both groups.

We investigated the modulation of radio- and chemoresistance by caffeine and mechanisms of resistance in human leukemic cell lines and mononuclear cells from 18 leukemic patients. Caffeine synergistically potentiated cytotoxicity and apoptosis induced by ionizing radiation or carboplatin (CPt), but attenuated induction of apoptosis by daunorubicin (DNR) in KG-1a cells. Since caffeine released irradiated as well as DNR-treated KG-1a cells from G2M cell cycle arrest and CPt-treated cells from S-phase arrest, this release does not fully explain the different effects of caffeine. Caffeine synergistically reduced the level of the apoptosis inhibitor glutathione after irradiation or CPt treatment. In contrast, treatment with DNR plus caffeine diminished glutathione levels to a lesser extent than DNR alone. We conclude that the effect of caffeine on glutathione depletion represents a mechanism of action by which caffeine can modulate apoptosis. Caffeine increased CPt cytotoxicity in K562 cells and its doxorubicin-resistant subline (K562/ADM), but little effect was seen in HL-60 cells or mononuclear cells from leukemic patients. Multivariate cluster analysis revealed an association of CPt resistance with the expression of c-Fos, c-N-Ras, and p53 oncoproteins and with proliferative activity (S-phase of cell cycle), but not with Bcl-2 expression.

The clinical importance of CML lies in its poor responsiveness to chemotherapy which has proved highly effective in treating ALL. The scientific importance of CML resides in its role as a cancer prototype, permitting the identification of genes centrally involved in both neoplastic change and normal cellular differentiation. One of these genes, the fusion gene BCR/ABL resulting from the balanced translocation (9;22) has received wide attention owing to its intimate involvement in CML. Although a tremendous amount of data have been recently discovered about BCR/ABL, its exact role in leukemogenesis and normal hematopoiesis remains obscure. The study of CML cell lines has already been of considerable help in understanding the molecular events associated with the Ph chromosome [4]. Further advances are likely to be forthcoming, particularly at the molecular genetic, but also at the protein level. CML cell lines may offer an excellent means of addressing many issues as continuous cell lines represent an inexhaustible source of identical cell material that, in addition, can be made available to other researchers around the world. This overview on the thus far reported CML-derived cell lines supports the hypothesis that in some specimens of CML the target cells in which Ph translocation arises are not necessarily lineage-restricted committed progenitor cells, but are in fact in some (or all?) cases precommitted bipotential or multipotential progenitor or stem cells retaining the potential for differentiation in diverse hematopoietic directions [26]. In conclusion, established tumor cell lines with their unique phenotypic and karyotypic features have been extremely useful models for investigation of the molecular and biological characteristics of CML. Considerable progress in understanding the molecular and cell biology of CML has been achieved. Further advances in the knowledge of CML are expected to accrue with the productive use of these powerful research tools for many important unresolved issues. By so doing, these discoveries might open new avenues that promise to move clinicians closer to the goal of the prevention or cure of CML in all patients.

S9788 modulating resistance effect has been investigated on the activity of THP-DOX against multidrug-resistant K562R cells and compared to that of cyclosporin A and verapamil. Intracellular THP-DOX distribution and particulary its intranuclear concentration, with or without modulators, has been measured using confocal laser microspectrofluorometry. The kinetics of intranuclear accumulation of THP-DOX (1 microM in the medium), as a function of time, were rapid in K562S and K562R cells. Maximum accumulation of THP-DOX is reached in a few minutes (K562S, 400 microM; K562R, 40 microM). The addition of S9788, cyclosporin A and verapamil (5 microM) after one hour THP-DOX incubation, led to respectively 290, 250 and 114 microM. Uptake of THP-DOX was increased in K562R cells by a factor of 7 when S9788 was added. Results obtained on THP-DOX efflux from nuclei of K562S and K562R cells, after 3 hours of incubation without drug, showed a very short T1/2 (time corresponding to 50% decrease of intranuclear concentration of THP-DOX) in K562R cells (12 min) compared to that in K562S cells (150 min). The addition of S9788, cyclosporin A and verapamil (5 microM) led to a T1/2 of 90, 30 and 20 min respectively. The T1/2 of THP-DOX was increased in K562R cells by a factor of 7.5 when S9788 was added. We tried to correlate these results with those obtained in growth inhibition study. The IC50 (concentration which induces 50% growth inhibition) of THP-DOX, corresponding to one hour THP-DOX treatment and 3 days culture of K562S and K562R cells were respectively 230 and 7000 nM, and the RI (resistance index) value is 30. The addition of S9788, cyclosporin A and verapamil (5 microM), during the one hour treatment, led to an IC50 value of 350, 2000 and 5000 nM respectively. S9788 induced an IC50 value 20 times lower than without the modulator. Our study suggests that the higher activity of THP-DOX against K562R cells in the presence of S9788, compared to cyclosporin A and verapamil, is due to a higher intranuclear THP-DOX accumulation and to a strong decrease of drug efflux from K562R nuclei. This could be explained by a higher affinity of S9788 for membrane P-glycoprotein of K562R cells and/or an additional mechanism of action.

The leukemias can be divided into acute and chronic varieties, both of which have a myelocytic and lymphocytic type. When untreated, the acute leukemias are associated with a more rapid clinical course than are the chronic leukemias. Paradoxically, to the present time, the acute leukemias have been curable with chemotherapy, whereas the chronic leukemias have not. Until recently the treatment goal for patients with chronic leukemias has been to return the patient's life to normal for as long as possible. With recent advances, it may now be possible to cure some patients with chronic leukemia. Acute leukemias are treated with intensive chemotherapy, which is associated with severe and life-threatening side effects. Although this approach to treatment will cure some patients, the use of bone marrow transplantation has had an increasingly important role in the treatment of younger patients with these diseases.

Multidrug Resistant (MDR) plays a major role in chemoresistance. Alpha Interferon (IFN) and all trans retinoic acid (ATRA) have antiproliferative effect and IFN regulates several genes, some of them implicated in the regulation of MDR gene expression. We have studied the modulations of adriamycin cytotoxicity by IFN and/or ATRA on K 562 (MDR-) and resistant to adriamycin K 562 adri (MDR+) cell lines. We observed an important increase of adriamycin cytotoxicity on both K 562 and K 562 adri by low dose of IFN and ATRA. Studies of MDR gene expression shows an increase in K 562 adri after exposure to IFN or ATRA. So the observed effect is not due to a down regulation of MDR gene expression but probably to the own antiproliferative effect of IFN and ATRA in combination with the cytotoxicity of adriamycin.

We describe the results of clinical, cytogenetic, and molecular biological studies of a patient who, after two courses of treatment with Busulphan, has remained free of symptoms of Philadelphia chromosome-positive chronic myeloid leukemia for 27 years, except for the persistence of a Ph1 chromosome and the presence of its transcribed chimeric mRNA.

Serum ferritin (SF) was estimated using double antibody sandwich ELISA in 83 patients of acute and chronic leukemia at various stages of the disease. In 35 patients of acute lymphoblastic leukemia (ALL) in remission, the SF levels fell significantly from 550.63 ng/ml at presentation to 319.56 ng/ml but remained significantly higher than the control values of 46.14 ng/ml. In 28 patients of acute myeloid leukemia (AML), the SF values at 775.0 ng/ml were much higher than those in ALL patients and showed no decline with remission. This pattern was also seen in patients of chronic myeloid leukemia in blast crisis (CML-BC) with SF levels of 804.03 ng/ml at presentation and 717.43 ng/ml at partial remission. The values of SF were lowest in patients of CML in chronic phase ranging from 271.5 ng/ml to 332.12 ng/ml and showed no relationship with variation in total leucocyte count. No correlation was found between SF values and various clinical and laboratory parameters such as age, sex, fever, organomegaly, haemoglobin and total leucocyte count. Thus, while there appeared to be a correlation between remission and SF values in ALL, no such correlation existed between the activity of the disease and SF in other types of leukemia.

Chronic myeloproliferative disorders are clonal neoplasias that originate from a clonal pluripotent stem cell and may have a more or less pronounced tendency to acute progression. Except for chronic myelogenous leukemia marked by the Ph chromosome, the BCR/ABL rearrangement, or both, the other chronic myeloproliferative disorder subtypes show less specific, although recurrent, karyotypic and molecular abnormalities. We report here more recent advances in cytogenetics, molecular biology, and treatment of the most common chronic myeloproliferative disorders.

Three Saudi siblings presented with clinical features of chronic myelocytic leukaemia in early infancy. Their parents are first cousins. The mother was 22 years old during her first pregnancy. She had no history of abortion. The possibility of this familial disorder being congenital is discussed.

This study focuses on possible functional defects of circulating mature neutrophils in chronic phase chronic myelogenous leukemia (CML) and their modulation by interferon-alpha (IFN-alpha). Polymorphonuclear cells (PMN) of seven untreated and nine IFN-alpha-treated patients were evaluated for the following parameters by the following methods: generation of oxygen species, by luminol-dependent chemiluminescence; leukotriene B4 (LTB4) generation, by high-performance liquid chromatography (HPLC); expression of LTB4 and formylmethionyl-leucyl phenylalanine (FMLP) receptor sites, by 3H-binding assay; and GTPase activity, by 32P-gamma-GTP. Compared to normal controls, reduced values were obtained in treated and untreated CML for most parameters studied. Therapy with IFN-alpha resulted in significantly diminished values for oxygen species (NaF stimulation) and LTB4 (FMLP stimulation) generation, as well as FMLP receptor expression as compared to untreated CML. We conclude that alterations at the level of oxygen species production, mediator generation, receptor expression, and transmembrane signaling are involved in functional defects of circulating mature neutrophils from CML patients. IFN-alpha seems to enhance some of these functional defects, but the clinical relevance of these findings has be elucidated.

We investigated the expression of p53 in paraformaldehyde-lysine-periodate fixed normal and chronic myelogenous leukemia (CML) hemopoietic cells with flow cytometry and two monoclonal antibodies, PAb1801 and the mutant-conformation-associated PAb240. With both antibodies p53 proteins were detected in more than 50% of CD34+ cells and in more than 95% neutrophils but were undetectable in the CD34- myeloid precursors. The expression of a p53 protein reactive with PAb240 was closely associated with CD34+/HLA-DR+ cells and with cells in active cell cycle, while the p53 protein recognized by PAb1801 was mainly found in CD34+/HLA-DR- cells and in cells in the G0/G1 phases of the cell cycle. Treatment of chronic-phase CML cells with p53 antisense oligonucleotides resulted in significantly increased numbers of granulocyte-macrophage colony-forming unit colonies in 12 of 17 cases studied. Slightly reduced granulocyte-macrophage colony-forming unit colony numbers were observed in one case and no change in the four others. In eight samples of normal bone marrow cells, treatment with antisense oligonucleotides showed no consistent changes in granulocyte-macrophage colony-forming unit numbers. Our data suggest that the expression of the tumor suppressor p53 is involved in the regulation of both normal and CML hemopoiesis and that the inhibition of p53 expression could modulate the proliferation of CML hemopoietic cells and possibly of normal cells.

We found a K562 subclone (K562YO) that highly expressed the c-kit gene. K562YO had a higher capability of erythroid differentiation by hemin and cytosine arabinoside (Ara-C) than its parent K562 (KIT-). We obtained the transfectant expressing c-kit by introducing c-kit cDNA into K562 (KIT-). The differentiation of the transfectant was similar to that of the parent cell. Thus the difference described above was not due to the expression of c-kit. Next, we investigated the effects of stem cell factor (SCF) on the differentiation of the K562 cell expressing c-kit. SCF did not enhance the cell growth of K562YO. On the other hand, SCF suppressed induction of benzidine-positive cells when c-kit-positive cells were treated with hemin and Ara-C, especially at a low concentration. Furthermore, c-kit mRNA and protein were down-regulated during erythroid differentiation. SCF also downregulated the c-kit proteins. Our results suggest that the SCF/c-kit signals could act negatively for erythroid differentiation of the K562 cells expressing c-kit. K562YO is also useful for studying the mechanism that controls the expression of the c-kit gene because there is a K562 counterpart cell line that does not express this gene.

Most primitive hematopoietic cells appear to be normally quiescent in vivo, whereas their leukemic counterparts in patients with chronic myeloid leukemia (CML) are maintained in a state of rapid turnover. This difference is also seen in the long-term culture system, where control of primitive hematopoietic progenitor proliferation is mediated by interactions of these cells with marrow-derived mesenchymal cells of the fibroblast lineage. We now show that exogenous addition of macrophage inflammatory protein 1 alpha (MIP-1 alpha) to normal long-term cultures can reversibly and specifically block the activation of "primitive" (high proliferative potential), but not "mature" (lower proliferative potential), progenitors in the adherent layer of these cultures. Moreover, addition of MIP-1 beta after primitive-progenitor activation can prevent the subsequent return of these cells to a quiescent state a few days later as shown previously in similar experiments using antibodies to transforming growth factor beta. This suggests that the level of MIP-1 alpha (or a related MIP-1 alpha agonist) produced in LTCs, like the level of transforming growth factor beta, may be necessary, but is not on its own sufficient, to mediate the inhibitory activity of the regulatory cells in the adherent layer. Addition of MIP-1 alpha to similar long-term cultures containing normal marrow adherent layers but supporting exclusively neoplastic (CML) hematopoiesis did not block the cycling of primitive neoplastic progenitors. A defect in the responsiveness of CML cells to MIP-1 alpha (or a similarly acting chemokine) would explain their deregulated proliferative behavior in this model and, by extrapolation to the in vivo setting, suggests a molecular mechanism whereby the leukemic clone may become amplified at the stem-cell level. In addition, these findings suggest approaches to the therapy of CML, using inhibitors such as MIP-1 alpha for the protection of primitive normal cells.

Chronic myeloid leukemia (CML) is a clonal disorder arising from the hematopoietic stem cell, characterized by the Philadelphia chromosome (Ph) and, at the molecular level, by fusion of the BCR (breakpoint cluster region) gene and the c-ABL gene. The hallmark of CML is represented by a marked increase in the number of leukemic progenitors, as well as more mature cells, in the bone marrow (BM) and peripheral blood (PB). Despite expansion of the leukemic clone, normal Ph-negative stem cells have been demonstrated to survive in CML. Early observations of partial, but transient, restoration of Ph-negative hematopoiesis after high-dose chemotherapy have recently been extended by the use of myeloablative regimens followed by autografting with marrow or blood-derived stem cells. Moreover, treatment of early-stage CML patients with the biologic response modifier alpha-interferon (alpha-IFN) has led to the re-emergence of normal progenitor cells. Concurrently, "in-vitro" studies have reported that cultures of CML marrow in the presence of a stromal feeder-layer resulted in depletion of Ph-positive cells and predominance of Ph-negative hematopoietic precursors. Based on the assumption that normal and malignant stem cells may coexist in CML, several studies have recently been directed toward the characterization and "in-vitro" selection of benign progenitors within CML hematopoiesis. The results of those studies demonstrated that normal precursors can be phenotypically and functionally identified in the BM or PB of Ph-positive CML patients. These cells are included in the earliest identifiable hematopoietic cell compartment. Normal cells do not bear cell surface lymphoid or myeloid-lineage antigens, express high levels of the CD34 antigen, and fail to express the HLA-DR antigen. Furthermore, they possess a great capacity for adhering to marrow stroma. This cell population represents only a small minority of hematopoietic progenitors, but it retains many of the properties associated with putative hematopoietic stem cells. Thus, purification of a population of benign hematopoietic precursors that could be used for autologous bone marrow transplantation (ABMT) may be feasible in CML patients.

A chronic myelogenous leukemia (CML)-like disease can be induced in mice by infecting hematopoietic stem cells with a BCR/ABL-containing retrovirus; serial transplantation produces either normal or leukemic animals. In many patients with CML, autografting produces transient Philadelphia chromosome (Ph)-negativity, but Ph-negative hematopoiesis is prolonged in some cases. These and other observations suggest that at diagnosis, CML patients may have substantial numbers of normal stem cells in their marrow, which may in certain circumstances regain a proliferative advantage if leukemic hematopoiesis can be suppressed by intensive chemotherapy. Thus autografting may have the capacity to restore normal hematopoiesis for long periods in patients not eligible for treatment by allogeneic bone marrow transplantation.

We studied the effects of several cytokines on the development of granulocyte-macrophage (GM) progenitors using the serum-deprived culture. SCF plays an important role in the GM-CSF- or IL-3-dependent production of neutrophils and macrophages. In vitro colony assay also suggests an increase in sensitivity of GM progenitors to cytokines (GM-CSF, IL-3, G-CSF and/or SCF) in a patient with juvenile chronic myelogenous leukemia. A high level of serum IFN-gamma was associated with leukopenia and thrombocytopenia in a patient with hemophagocytic syndrome. Based on the evidence that IFN-gamma significantly inhibited the proliferation of GM progenitors, IFN-gamma-mediated suppression was suggested as one of the mechanisms causing cytopenia. In patients with aplastic anemia and neutropenia, an increase of serum G-CSF levels was observed when neutrophils decreased remarkably in number. However, the serum SCF levels were constant in these patients. A failure of SCF to enhance colony growth in some patients with aplastic anemia implies qualitative abnormalities of hematopoietic progenitors.

Fifteen patients with lymphoid blast crisis of chronic myelogenous leukemia (LyBC-CML) and five patients with acute lymphoblastic leukemia converting to Philadelphia-positive (Ph+) chronic myeloid leukemia (ALL Ph + CML) were followed. Seven of 15 (46.7%) LyBC-CML patients developed meningeal leukemia within a median period of 6 months (range 2-11 months), while there was no medullary relapse. Five of these responded well to triple intrathecal therapy. In the ALL Ph + CML patients, in spite of central nervous system (CNS) prophylaxis with IT MTX and 18 Gy cranial radiation, two of five patients (40%) experienced meningeal leukemia, one isolated and the other with medullary relapse. The data confirm that LyBC-CML patients experience a high incidence of meningeal leukemia. The role of CNS prophylaxis is not very clear, but its use may delay development and reduce morbidity due to CNS disease.

Marrow trepan biopsy was performed in 11 patients upon diagnosis of chronic myeloid leukemia and in clinicohematological remission of the disease. The patients were treated with alpha 2-interferon (reaferon) versus conventional myelosan (6 and 5 patients, respectively). The biopsies were studied histomorphometrically. In reaferon group the number of megakaryocytes and endosteal stromal cells diminished. This may be due to an indirect action of reaferon on hemopoietic tissue via endosteal cells of the marrow stroma. Reaferon is recommended for myeloid leukemia chronic with a granulocytic-megakaryocytic histological variant of the disease.

We report the clinical, hematologic, cytogenetic, and molecular characteristics of 13 patients with Philadelphia-negative (Ph-), bcr-negative atypical chronic myelogenous leukemia (CML). In the majority of cases, the phenotypic features at presentation resembled those of typical CML. However, these patients presented with a higher median age, lower median hemoglobin levels, and lower leukocyte and platelet counts than patients with Ph-positive CML. Cytogenetic analysis showed an abnormal karyotype in only one case. Southern blot investigation, using probes exploring the entire M-bcr region, demonstrated the absence of genomic bcr-abl rearrangements. The assessment of clonality in five patients (study of X-methylation patterns in females heterozygous at the DXS255 locus) indicated the proliferation of a monoclonal cell population. Disease evolution was mostly characterized by bone marrow failure, extramedullary infiltrates, and poor response to chemotherapy, without evidence of overt acute transformation. Our observations suggest that some hematologic and clinical features and the modalities of disease progression are presently the most helpful factors in distinguishing these bcr/abl-negative patients from those with typical bcr+CML. The differences existing also with chronic myelomonocytic leukemia (CMMoL), allow the consideration of ph-/bcr- CML as a separate entity, the nature of which remains to be elucidated.

We investigated the hemorheological, hematological and biochemical parameters in 30 cases of acute lymphocytic leukemia (ALL), 21 cases of acute myelogenous leukemia (AML) and 30 cases of chronic myelogenous leukemia (CML). The parameters studied include whole blood viscosity, plasma viscosity, erythrocyte sedimentation rate (ESR), red cell filterability, hematocrit, platelet count and aggregation, fibrinogen, hemoglobin, leucocyte count, bleeding time and lactate dehydrogenase activity (LDH). In the cases of ALL we observed significant decrease in whole blood viscosity, hemoglobin, hematocrit and platelet count but an increase in plasma viscosity, fibrinogen, bleeding time and LDH activity. In the cases of AML, we observed increase in whole blood viscosity, plasma viscosity, ESR, fibrinogen, leucocyte count, bleeding time and LDH activity but decrease in the hemoglobin, hematocrit and platelet count. In the cases of CML, we observed an increase of whole blood viscosity, plasma viscosity, ESR, fibrinogen elevation but decreases in bleeding time. In all cases, red cell filterability was unaffected.

The therapeutic efficacy and toxicity of alpha-interferon (alpha-IFN) (Roferon, Hoffmann-La Roche, Inc., Nutley, NJ) were determined in 15 children (age range, 6 to 20 years) with Philadelphia chromosome-positive chronic myelocytic leukemia (Ph+ CML). All patients had received cytoreductive therapy with either hydroxyurea (n = 13) or busulfan (n = 1) or both (n = 1) for 6 weeks to 46 months (median, 7 months) before beginning alpha-IFN therapy at a dose of 5 x 10(6) U/m2/d intramuscularly. This dose was escalated to 10 x 10(6) U/m2/d if leukemia was inadequately controlled. Ten children had a hematologic response, with nine showing a reduction in the percentage of Ph+ marrow cells, including four who had no detectable Ph+ cells in marrow samples collected 48 to 204 weeks after the initiation of therapy. Two of 15 patients remain free of Ph+ cells. Therapy was discontinued before week 104 in ten patients because of the following: (1) early hematologic responses without a decrease in Ph+ cells (three patients); (2) early resistant disease (one patient); (3) blast crisis (one patient); (4) progressive disease (two patients); (5) seizure attributed to high-dose alpha-IFN (one patient); or (6) an inadequate trial of alpha-IFN caused by aseptic necrosis or poor compliance (two patients). The most common side effects were mild and have included fever, malaise, headache, myalgias, and pain at the injection site. Adverse events causing interruption of therapy were seizures, aseptic necrosis, and myelofibrosis. alpha-IFN stabilizes the chronic phase of Ph+ CML in some children, is adequately tolerated when administered at a dose of 2.5 to 5 x 10(6) U/m2/d intramuscularly, and results in a significant decrease in the proportion of Ph+ metaphases in some patients. alpha-IFN in combination with an effective cytoreductive agent or agents appears worthy of further clinical testing in this disease.

Content of adenine nucleotides was studied in various compartments of blood platelets obtained from donors and patients with chronic myeloleukosis at various steps of the disease. Dissimilar biochemical defect was found in content of ATP and ADP, which varied in thrombocyte storage granules depending on severity of the disease. The data on adenine nucleotides content may be used for diagnosis of various steps of chronic myeloleukosis, for detection of thrombocytes anomaly and for evaluation of the state of medullary hemopoiesis.

All patients with chronic myelogenous leukemia (CML) undergo clinical transition from chronic to acute phase. This transition is often associated with deletion of the short arm of chromosome 17 in the form of the i(17q) aberration. Since the p53 gene is a suppressor gene and is located on 17p13, we examined the possibility that it is inactivated during progression of CML. Therefore, we studied the structure and expression of p53 in the leukemic cells of a large number of CML patients in acute phase. We found that although the gene is rarely rearranged, one p53 allele is completely deleted in patients with the i(17q) aberration as well as in some patients who do not show karyotypic changes. In all of these patients the remaining allele is inactivated through loss of expression, rearrangement, or point mutation. Detailed analysis of some patients who carry both p53 alleles indicated neither loss of expression nor structural alterations. It appears that p53 loss of function is associated with progression of around 25% of CML patients.

We studied the effect of recombinant interferon-alpha 2a (IFN alpha) on the interaction between stromal cells and granulocyte-macrophage colony-forming units (CFU-GM) from the marrow of normal individuals and chronic myeloid leukemia (CML) patients in chronic phase in long-term bone marrow cultures using preformed stromal layers. These stromal layers were established with marrow cells from normal allogeneic donors and grown to confluence in the presence or absence of IFN alpha at low concentration (100 U/ml). The number of CML CFU-GM localized within IFN alpha-treated stromal layers was significantly greater than the corresponding number localized within control stromal layers. Conversely, the distribution of normal CFU-GM between the adherent and nonadherent compartments was unaffected by IFN alpha treatment of the stromal layer. Preincubation of CML marrow cells with IFN alpha did not alter this distribution, so the observed effect of IFN alpha must be due to a primary action on stromal layers. Thus, in addition to its well known antiproliferative effect, IFN alpha enhances the capacity of marrow stromal cells to bind and/or retain CML progenitor cells, and the resulting restoration of normal regulatory control may be the basis for the selectivity of IFN alpha in CML.