Showing papers in "Leukemia in 1997"

Internal tandem duplication of the FLT3 gene is preferentially seen in acute myeloid leukemia and myelodysplastic syndrome among various hematological malignancies. A study on a large series of patients and cell lines

Abstract: In this study, we examined a large number of patients to clarify the distribution and frequency of a recently described FLT3 tandem duplication among hematopoietic malignancies, including 112 acute myelocytic leukemia (AML), 55 acute lymphoblastic leukemia (ALL), 37 myelodysplastic syndrome (MDS), 20 chronic myelogenous leukemia (CML), 30 non-Hodgkin's lymphoma (NHL), 14 adult T cell leukemia, 15 chronic lymphocytic leukemia (CLL) and 38 multiple myeloma (MM). We also evaluated 71 cell lines derived from 11 AML, 31 ALL, two hairy cell leukemia, three acute unclassified leukemia, 10 CML, 12 NHL including six Burkitt's lymphoma, and two MM. Using genomic PCR of exon 11 coding for the juxtamembrane (JM) domain and first amino acids of the 5'-tyrosine kinase (TK) domain, this length mutation was found only in AML (22/112, 20%) and MDS (1/37). According to the FAB subclassification, they were 5/18 (28%) of M1, 4/29 (14%) of M2, 3/17 (18%) of M3, 6/24 (25%) of M4, 4/20 (20%) of M5 and 1/9 of refractory anemia with excess of blast in transformation. In the various cell lines examined, this abnormality was determined in only one derived from AML and never found in other hematological malignancies. The sequence analysis of the abnormal PCR products revealed that 23 of 24 showed internal tandem duplication with or without insertion of nucleotides. In one AML, insertion and deletion without duplication was determined. All 24 lengthened sequences were in-frame. Duplication takes place in the sequence coding for the JM domain and leaves the TK domain intact. In conclusion, we emphasize that the length mutation of FLT3 at JM/TK-I domains were restricted to AML and MDS. Since all these mutations resulted in in-frame, this abnormality might function for the proliferation of leukemic cells.

Internal tandem duplication of FLT3 associated with leukocytosis in acute promyelocytic leukemia. Leukemia Study Group of the Ministry of Health and Welfare (Kohseisho).

Abstract: FLT3 is a member of receptor tyrosine kinases expressed in leukemia cells, as well as in hematopoietic stem cells. Recently, a somatic alteration of the FLT3 gene was found in acute myeloid leukemia, as an internal tandem duplication (FLT3/ITD) which caused elongation of the juxtamembrane (JM) domain of FLT3. Here we characterized the FLT3/ITD and investigated its clinical significance in acute promyelocytic leukemia (APL). Seventy-four newly diagnosed patients with APL, who were treated with the same protocol in a multi-institutional study, were studied for the FLT3/ITD. Genomic and message sequences of the FLT3 gene were amplified by means of polymerase chain reaction (PCR), and elongated PCR products were sequenced. Fifteen patients (20.3%) had FLT3/ITD, all of which were transcribed in frame. Location of the duplicated fragments (six to 30 amino acids) varied from patient to patient. However, they always contained either Y591 or Y599, but the tyrosine kinase domain was not significantly affected. This finding implied that signal transduction of FLT3 is amplified by the duplication. Clinically, the presence of FLT3/ITD was related to high peripheral white blood cell counts as well as peripheral leukemia cell counts (P < 0.0001), high LDH level (P = 0.04), and low fibrinogen concentration (P = 0.04). These data suggest that FLT3/ITD plays a significant role in progression of APL.

Anaplastic large cell lymphomas expressing the novel chimeric protein p80NPM/ALK: a distinct clinicopathologic entity.

Abstract: Some anaplastic large cell lymphomas (ALCLs) carry a specific chromosomal translocation, t(2;5)(p23;q35). Recently, we found a novel hyperphosphorylated 80-kDa protein tyrosine kinase, p80, in ALCLs with t(2;5). Subsequent cDNA cloning revealed p80 to be a fusion protein of two genes, the novel tyrosine kinase gene and the nucleophosmin gene, in accordance with the sequence of the NPM/ALK gene (Morris et al.). Meanwhile, the clinicopathologic features of p80-carrying ALCLs have remained unclear. Paraffin sections of 105 cases of ALCL were immunostained using anti-p80 antibody, and 30 of them were shown to express p80. Clinicopathological comparison between p80-positive and -negative ALCLs revealed that p80-positive cases occurred in a far younger patient age group and the patients showed a far better 5-year survival rate. These data showed that p80-positive ALCL is a distinct entity both clinically and pathogenetically, and should be differentiated from p80-negative ALCL.

Overexpression of tumor necrosis factor (TNF)-α and interferon (IFN)- γ by bone marrow cells from patients with myelodysplastic syndromes

Abstract: To clarify whether regulatory cytokines inhibit hematopoiesis in patients with myelodysplastic syndromes (MDS), malignancies characterized by the formation of cytopenias despite the presence of cellular bone marrow, expression of TNF-alpha and IFN-gamma by bone marrow cells was investigated using specific reverse transcriptase-polymerase chain reaction assays An enhanced expression of the mRNA for TNF-alpha was observed in most of the samples from MDS patients (11/14, 79%), whereas no enhancement was observed in bone marrow samples from AML (0/6), CML (0/2) or control cases (0/8) The expression of IFN-gamma was also enhanced in some of MDS cases (5/12, 42%) while AML (0/5), CML (0/2) and control cases (0/6) showed very low levels of IFN-gamma mRNA expression Immunohistochemical examination confirmed the scattered presence of TNF-alpha or IFN-gamma producing cells in the bone marrow of MDS patients The majority of these cells were CD68-positive macrophage lineage cells These results suggested that disruption of hematopoiesis in MDS might be caused by enhanced production of inhibitory regulatory cytokines especially TNF-alpha and occasionally IFN-gamma by bone marrow macrophages

Heteroduplex PCR analysis of rearranged T cell receptor genes for clonality assessment in suspect T cell proliferations

Abstract: Molecular analysis of T cell receptor (TCR) genes is frequently used to prove or exclude clonality and thereby support the diagnosis of suspect T cell proliferations. PCR techniques are more and more being used for molecular clonality studies. The main disadvantage of the PCR-based detection of clonal TCR gene rearrangements, is the risk of false-positive results due to 'background' amplification of similar rearrangements in polyclonal reactive T lymphocytes. Therefore, PCR-based clonality assessment should include analyses that discern between PCR products derived from monoclonal and polyclonal cell populations. One such method is heteroduplex analysis, in which homo- and heteroduplexes resulting from denaturation (at 94 degrees C) and renaturation (at lower temperatures) of PCR products, are separated in non-denaturing polyacrylamide gels based on their conformation. After denaturation/renaturation, PCR products of clonally rearranged TCR genes give rise to homoduplexes, whereas in case of polyclonal cells heteroduplexes with heterogeneous junctions are formed. We studied heteroduplex PCR analysis of TCR gene rearrangements with respect to the time and temperature of renaturation and the size of the PCR products. Variation in time did not have much influence, but higher renaturation temperatures (>30 degrees C) clearly showed better duplex formation. Nevertheless, distinction between monoclonal and polyclonal samples was found to be more reliable at a renaturation temperature of 4 degrees C, using relatively short PCR products. To determine the sensitivity of heteroduplex analysis with renaturation at 4 degrees C, (c)DNA of T cell malignancies with proven clonal rearrangements was serially diluted in (c)DNA of polyclonal mononuclear peripheral blood cells and amplified using V and C primers (TCRB genes) or V and J primers (TCRG and TCRD genes). Clonal TCRB and TCRD gene rearrangements could be detected with a sensitivity of at least 5%, whereas the sensitivity for TCRG genes was somewhat lower (10-15%). The latter could be improved by use of Vgamma member primers instead of Vgamma family primers. We conclude from our results that heteroduplex PCR analysis of TCR gene rearrangements is a simple, rapid and cheap alternative to Southern blot analysis for detection of clonally rearranged TCR genes.

Flow cytometry CD45 gating for immunophenotyping of acute myeloid leukemia

Abstract: A flow cytometry method has been introduced into the routine investigation of whole bone marrow samples following red blood cell lysis on the basis of a primary CD45/side scatter (SSC) gating procedure. Blast cells were first identified by CD45/SSC gating in 74 cases of acute myeloid leukemia (AML) and the results were compared to a conventional FSC/SSC gating procedure and to MGG-staining smears. The percentages of blast cells in these samples as defined by the morphological analysis of MGG smears correlated better with the values determined by CD45/SSC gating (r = 0.94) than with the blast cell counts recorded with FSC/SSC gating (r = 0.76). These findings were not surprising because while CD45 expression was regularly lower on leukemic blasts than on normal lymphoid and monocytic cells, the FCS/SSC characteristics of these populations were overlapping. In 53 samples, the blast cell populations were also analyzed with a panel of FITC-conjugated monoclonal antibodies that were utilized in double labeling with CD45-PE. We show that the CD45/SSC gating procedure improved phenotypic determination of the blast cells in three ways: (1) by discriminating between leukemic blast cells and residual normal cells; (2) by excluding normal cells from the phenotypic analysis of leukemic blast cells; and (3) by identifying blast cell heterogeneity in many cases of leukemia on the basis of different CD45 display. Moreover, this immunophenotyping procedure on whole bone marrow samples also allowed an efficient discrimination between the various cell lineages and facilitated the analysis of leukemic blasts present in low proportions.

Deficient activation of the CD95 (APO-1/Fas) system in drug-resistant cells.

Abstract: The molecular mechanisms for sensitivity and resistance of tumor cells towards chemotherapy are only partially understood. In chemosensitive leukemias and solid tumors, anticancer drugs have been shown to induce apoptosis. We previously identified activation of the CD95 (APO-1/Fas) receptor/CD95 ligand (CD95/CD95-L) system as a key mechanism for drug-induced apoptosis. Here, we show that therapeutic concentrations of doxorubicin, methotrexate and cytarabine also induce apoptosis via activation of the CD95 system in primary leukemia cells in vivo. CD95-resistant and doxorubicin-resistant leukemia and neuroblastoma cells display cross-resistance for induction of cell death. Down-regulation of CD95 expression was found in drug-resistant and CD95-resistant cell lines. Furthermore, up-regulation of CD95-L, previously shown to mediate drug-induced apoptosis in a variety of tumor cells, was completely blocked in doxorubicin-resistant cells. The prototype caspase (ICE/Ced-3 protease) substrate, poly(ADP-ribose)polymerase (PARP), was cleaved in sensitive, but not in resistant tumor cells following CD95 triggering or drug treatment. Since failure to activate CD95-L was not due to decreased drug uptake or increased drug efflux, non-multi-drug resistance (non-MDR) mechanisms are involved in this type of resistance. These findings suggested that an intact CD95 system plays a key role in determining sensitivity or resistance towards anticancer therapy.

Two acute monocytic leukemia (AML-M5a) cell lines (MOLM-13 and MOLM-14) with interclonal phenotypic heterogeneity showing MLL-AF9 fusion resulting from an occult chromosome insertion, ins(11;9)(q23;p22p23)

Abstract: We describe two new human leukemia cell lines, MOLM-13 and MOLM-14, established from the peripheral blood of a patient at relapse of acute monocytic leukemia, FAB M5a, which had evolved from myelodysplastic syndrome (MDS). Both cell lines express monocyte-specific esterase (MSE) and MLL-AF9 fusion mRNA. Gene fusion is associated with a minute chromosomal insertion, ins(11;9)(q23;p22p23). MOLM-13 and MOLM-14 are the first cell lines with, and represent the third reported case of, MLL gene rearrangement arising via chromosomal insertion. Both cell lines carry trisomy 8 which was also present during the MDS phase, as well as the most frequent trisomies associated with t(9;11), ie, +6, +13, +19 variously present in different subclones. Despite having these features in common, differences in antigen expression were noted between the two cell lines: that of MOLM-13 being CD34+, CD13-, CD14-, CD15+, CD33+; whereas MOLM-14 was CD4+, CD13+, CD14+, CD15+, CD33+. Differentiation to macrophage-like morphology could be induced in both cell lines after stimulation with INF-gamma alone, or in combination with TNF-alpha, which treatment also induced or upregulated, expression of certain myelomonocyte-associated antigens, including CD13, CD14, CD15, CD64, CD65 and CD87. Together, these data confirm that both cell lines are likely to be novel in vitro models for studying monocytic differentiation and leukemogenesis.

Tandem duplications of the FLT3 receptor gene are associated with leukemic transformation of myelodysplasia

Abstract: We recently reported an internal tandem duplication of the human flt3 receptor gene (FLT3) as a somatic mutation in 17% of acute myelogenous leukemia (AML). The present study revealed the duplication at the juxtamembrane and the first tyrosine kinase domains of FLT3 in seven of 92 (8%) patients with myelodysplastic syndrome (MDS) and AML with trilineage myelodysplasia (AML/TMDS), the diseases which may represent neoplastic changes of pluripotent stem cells. A tandem duplication of exon 11 of FLT3 was harbored by two of 58 (3%) patients with MDS and five of 34 (15%) with overt leukemia, including MDS-derived leukemia, AML/TMDS and therapy-related leukemia. Although the duplicated regions varied within exon 11 in each case, they occurred in-frame, and altered mRNA expressions were demonstrated by reverse-transcription polymerase chain reaction. Two cases of MDS with a FLT3 duplication transformed to overt leukemia within a few months. Longitudinal analyses in two other patients with leukemia revealed that the duplication was a late genetic event during the disease course; one of whom showed two independent duplications of FLT3 at the terminal therapy-resistant phase. Of seven patients with the FLT3 duplication, six had abnormal karyotypes, and four harbored a point mutation of the N-RAS and/or TP53 genes. Patients with FLT3 mutations have poor prognoses. This study uncovered the fact that the accumulation of genetic events, including FLT3 duplication, correlates with leukemic transformation from antecedent myelodysplasia and with subsequent disease progression.

Continuous infusion of low-dose 5-Aza-2'-deoxycytidine in elderly patients with high-risk myelodysplastic syndrome

Abstract: There is no standard therapy for elderly patients with high-risk myelodysplastic syndrome (MDS). The treatment options of low-dose Ara-C and haematopoietic growth factors are disappointing in regard to response rate or response duration. We tested the treatment with a 72-h continuous infusion of low-dose 5-Aza-2'-deoxycytidine (DAC) in a group of 29 elderly patients with high-risk MDS. In 15 patients (54%) we observed a response. Eight complete responses were reached, even among patients with bad prognostic cytogenetic findings. The actuarial median survival from the start of the therapy was 46 weeks. The only (and major) toxicity was myelosuppression, leading to a prolonged cytopenic period and thus leading to five toxic deaths (17%) in this high-risk patient group. We conclude that DAC is an effective drug in the treatment of MDS patients and that it probably works via its cytotoxic activity. Myelotoxicity is its major adverse effect.

Fas/Apo-1 (CD95) expression and apoptosis in patients with myelodysplastic syndromes.

Abstract: Apoptosis of hematopoietic progenitor cells is increased in myelodysplastic syndromes (MDS). We have studied Fas (CD95/Apo-1) antigen expression in 27 MDS patients (RARS 4, RA 3, RAEB 13; RAEB-t 3, CMML 4) and three AML secondary to MDS. We found that the Fas antigen was not expressed on normal bone marrow (BM) CD34+, CD14+, or glycophorin+ cells, and only slightly on CD33+ cells. Patients with MDS had upregulation of Fas expression on total bone marrow nuclear cells (BMMC) (t-test, P = 0.04), CD34+ (P = 0.013), CD33+ (P = 0.04), and glycophorin+ (P = 0.032) BM cells compared to controls. Fas expression did not correlate to the FAB subtype, the Bournemouth score, or to peripheral cytopenias. However, Fas expression intensity on CD34+ cells negatively correlated to the BM blasts number (Spearman, P = 0.01) suggesting that leukemic blasts cells lose Fas antigen expression with progression of myelodysplasia. Using both proliferation assays in liquid cultures and clonogenic progenitor assays in the presence of an agonist anti-Fas MoAb (CH11), we showed that the Fas protein was functional in some patients. Dose-dependent inhibition of DNA synthesis was observed in three out of seven patients studied. CFU-GM and BFU-E colonies suppression in some patients suggested that Fas can induce apoptosis in myeloid and erythroid BM progenitors of MDS patients. The TUNEL technique on BM smears gave a mean of 12.6% ± 2.5 of bone marrow apoptotic cells in five controls. Patients with MDS had increased bone marrow apoptosis (mean 39% ± 5.7, t-test, P = 0.012). Four out of 15 (26%) patients studied with a sensitive radiolabeled DNA ladder technique had typical DNA ladders indicative of advanced stages of apoptosis. Massive BM suicide was observed in patients with RA (2/2) and RAEB (8/11), whereas apoptosis rates were normal or low in patients with RAEB-t (3/3) or secondary AMLs (3/3). Moreover, high rates of apoptosis correlated to low Bournemouth score (Spearman, P = 0.01). No statistical correlation could be found between Fas expression and apoptosis rates. Our results confirm the importance of programmed cell death in MDS. The Fas antigen is clearly upregulated on BM cells, but its role in the pathophysiology of apoptosis in myelodysplasia is still unclear, indicating that many factors positively or negatively interfere with the Fas-mediated pathway of apoptosis in vivo and in vitro.

Urate oxidase in prevention and treatment of hyperuricemia associated with lymphoid malignancies.

Abstract: Standard prophylaxis and treatment of malignancy-associated hyperuricemia in the USA has been allopurinol with vigorous hydration, urinary alkalinization and osmotic diuresis. Urate oxidase, the enzyme that converts uric acid to allantoin (a readily excreted metabolite that has 5- to 10-fold higher solubility than uric acid), is an alternative therapy; however, few published findings support this practice. Between February 1994 and December 1996, we administered non-recombinant urate oxidase (Uricozyme) to 126 children with newly diagnosed non-B cell acute lymphoblastic leukemia (ALL) during the first 5 days of chemotherapy with methotrexate, 6-mercaptopurine or both. Their blood levels of uric acid and other indicators of tumor lysis were measured at diagnosis and during treatment and then compared with findings in 129 similarly treated historical controls who had received allopurinol to control hyperuricemia. Clinical responses to urate oxidase were also determined in eight patients with newly diagnosed B cell ALL or advanced-stage non-Hodgkin lymphoma. Patients treated with urate oxidase had rapid and significantly greater decreases in their blood uric acid levels than did the historical controls (median maximal level during treatment, 2.3 vs 3.9 mg/dl, P < 0.001). They also had lower creatinine (0.6 vs 0.7 mg/dl, P = 0.01) and blood urea nitrogen (11 vs 24 mg/dl, P < 0.001) levels. Similar findings were made in the eight cases of B cell ALL or non-Hodgkin lymphoma. None of the patients required dialysis for acute renal failure. Six (4.5%) of the 134 children given urate oxidase had allergic reactions, manifested primarily by urticaria, bronchospasm and hypoxemia. Thus, non-recombinant urate oxidase is a more effective uricolytic agent than allopurinol but is associated with acute hypersensitivity reactions, even in patients without a history of allergy.

Basic fibroblast growth factor (bFGF) upregulates the expression of bcl-2 in B cell chronic lymphocytic leukemia cell lines resulting in delaying apoptosis.

Abstract: To evaluate the function of HTLV-I env-pX gene in vivo, we developed two lines of transgenic rats (env-pX rats) that expressed env-pX gene products, under control of own LTR promotor. In various tissues of the rats, env and pX mRNAs were constitutively expressed, irrespective of age. At age 5 weeks, swelling of the bilateral ankle joints histologically showing synovial lining hyperplasia, severe chronic inflammation, erosion of the joint cartilage, and bone destruction with pannus formation began to develop in these env-pX rats. These histologic features resemble those of rheumatoid arthritis (RA) in man. High titered rheumatoid factors and low anti-dsDNA antibodies and hyper-gamma globulinemia were detected. Necrotizing arteritis resembling polyarteritis nodosa, polymyositis, myocarditis and Sjogren syndrome-like sialoadenitis developed, together with RA-like arthritis even in one individual animal. Thymic atrophy with low body weight was also observed. The evidence indicates that env-pX rats appear to be suitable animal models for elucidating pathogenetic mechanisms involved in not only HTLV-I related diseases but also various collegen vascular and autoimmune diseases of unknown etiology in man.

Mutations in the granulocyte colony-stimulating factor receptor gene in patients with severe congenital neutropenia.

Abstract: Previously, nonsense mutations in the gene encoding the granulocyte colony-stimulating factor receptor (G-CSF-R) have been described in three patients with severe congenital neutropenia (SCN) (Proc Natl Acad Sci USA 1994; 91: 4480; New Engl J Med 1995; 333: 487). The mutations resulted in the truncation of the carboxy-terminal region of G-CSF-R essential for transduction of maturation signals. Two of these patients developed acute myeloblastic leukemia (AML). We present the results of a search among 20 additional cases of congenital neutropenia (CN) and SCN for the presence of mutations in the cytoplasmic domain of G-CSF-R. This series includes patients with familial and nonfamilial forms of CN and SCN. Mutations in the G-CSF-R gene were found in two new SCN cases. These mutations were nonsense mutations, located in the same cytoplasmic region of G-CSF-R as those found earlier, resulting in the truncation of the C-terminus. Both of these patients developed AML. None of the other patients showed clinical symptoms or cytogenetic features indicative of AML or progression to leukemia. The analysis in this extended series of patients thus has revealed five SCN cases with G-CSF-R mutations, four of whom developed AML. These results add support to the notion that mutations in the G-CSF-R gene, affecting the maturation signaling function of the receptor, define a distinct subgroup of SCN with increased susceptibilty to AML.

Constitutive activation of mitogen-activated protein kinase pathway in acute leukemia cells

Abstract: Mitogen-activated protein (MAP) kinase appears to be one of the key regulators of cell proliferation and differentiation. Very little, however, has been revealed as to how MAP kinase is involved in leukemogenesis. We have studied the activation of the MAP kinase pathway in 100 human primary leukemia cells including 73 acute myelogenous leukemias (AMLs). Forty acute leukemia samples (40% of the total), including 37 AML samples (51% of AML), showed activation of MAP kinase as revealed by the mobility shift of the phosphorylated form of the protein and by in vitro kinase assay. This activation was correlated with MAP kinase kinase activity in these cells. In contrast, none of 14 chronic myelogenous leukemia samples showed the activation of MAP kinase. These results suggest that the MAP kinase pathway is constitutively activated in a subset of primary acute leukemias, and thus indicate the possible role of the constitutively activated MAP kinase in leukemogenesis.

Regulation of the cyclin-dependent kinase inhibitor p27 by degradation and phosphorylation

Abstract: The cell cycle has been the object of extensive studies for the past years. A complex network of molecular interactions has been identified. In particular, a class of cell cycle inhibitory proteins has been cloned and characterized but details of the molecular mechanism of their action have yet to be resolved. These inhibitors regulate the progression through G1 and the G1/S transition via the inhibition of the cyclin-dependent kinase (Cdk) activity. The potential function of these negative regulators as tumor suppressors provides new insights into the link between the cell cycle and oncogenesis. p27 is a potent inhibitor of Cdks. In quiescent cells p27 accumulates without an increase in mRNA or protein synthesis. Cell cycle regulation of p27 levels, both in normal and transformed human cells, occurs via the ubiquitin-proteasome pathway and, compared to proliferating cells, quiescent cells contain a far lower amount of p27 ubiquitinating activity. The specific proteolysis of p27 is probably involved in the pathway of activation of Cdks. p27 is a phosphoprotein and its phosphorylation is cell cycle regulated. Often phosphorylation is a signal for ubiquitination. p27 is phosphorylated exclusively on serine by Erk1 and almost exclusively on threonine by Cdk1 in in vitro experiments. This finding raises the question of whether and how phosphorylation by these kinases is involved in the process of p27 proteolysis.

DNA methylation changes in hematologic malignancies: biologic and clinical implications.

Abstract: DNA methylation changes are among the most common detectable abnormalities in human neoplasia. Hypermethylation within the promoters of selected genes appears to be especially common in all types of human hematopoietic neoplasms, and is usually associated with inactivation of the involved gene(s). Such hypermethylation-associated silencing of gene expression has been shown for several genes regulating the growth and differentiation of hematopoietic cells, including the estrogen receptor (ER) gene, P15, P16 and others. Hypermethylation within the promoters of some genes appear to be an early event in the pathogenesis of neoplasia (ER, P15), while other genes seem to become methylated during the progression of leukemias (HIC1, c-abl). The high prevalence of promoter methylation suggests that this molecular abnormality can be used to monitor disease activity during therapy. In addition, new technology allows the sensitive identification of gene hypermethylation in a background of normal cells, suggesting possible new strategies for the detection of minimal residual disease. Finally, reactivation of tumor-suppressor gene expression through pharmacologic inhibition of DNA methyltransferase and resultant DNA demethylation appears to be a promising new avenue of therapy in acute leukemia.

Treatment of neutropenia-related fungal infections with granulocyte colony-stimulating factor-elicited white blood cell transfusions: a pilot study.

Abstract: Neutropenia-related fungal infections can be life-threatening despite antifungal therapy. We evaluated the role of recombinant granulocyte colony-stimulating factor (rG-CSF)-elicited white blood cell (WBC) transfusions in patients with neutropenia-related fungal infections. Adult patients with hematologic malignancies, absolute neutrophil counts (ANC) <500/microl and fungal infections refractory to amphotericin B, received daily transfusions of rG-CSF-elicited and irradiated WBC transfusions from related donors. Donors received 5 microg/kg/day of rG-CSF subcutaneously. Donors achieved a mean ANC of 29.4 x 10(3) per microliter. The mean yield of neutrophils per transfusion was 41 x 10(9) (range, 10-116). Fifteen patients received a median of eight transfusions (range, 3-16). Fourteen patients had received rG-CSF for a median of 12 days. The median ANC baseline was 20/microl. Eleven patients had favorable responses and eight of them remained free of infection 3 weeks after therapy. Favorable responses occurred among patients with better Zubrod performance status (median, 3 vs 4) and shorter duration of both profound neutropenia (median, 15 vs 25 days) and active infection (median, 8 vs 17 days). The mean 1- and 24-h post-transfusion ANCs were 594/microl (range, 98-1472/microl) and 396/microl (range, 50-1475/microl), respectively. Adverse reactions were observed in nine of 35 donors and in the recipients of six of 130 transfusions. rG-CSF-elicited WBC transfusions may be a safe and promising approach for treating neutropenia-related fungal infections.

The EVI1 gene in myeloid leukemia

Abstract: Leukemia is an acquired genetic disease caused by the accumulation of chromosomal abnormalities which modify either the biochemical property or the level of expression of proteins. Frequent genetic abnormalities identified in human leukemia are chromosomal rearrangements such as chromosomal translocations and inversions. Chromosome band 3q26 is the site of the breakpoint of recurring translocations and inversions observed in patients with myeloid leukemias. Two genes located at 3q26 have been implicated in development or progression of myeloid leukemia. They are MDS1 and EVI1. MDS1, first identified as part of a fusion transcript resulting from the t(3;21)(q26;q22), encodes a small protein of unknown function. EVI1 encodes a zinc finger protein inappropriately overexpressed by chromosomal rearrangements (in man) or by retroviral insertion (in the mouse). Both genes are rearranged by the t(3;21)(q26;q22) and by the t(3;12)(p13;q22). As a result of the translocation, they are expressed as fusion genes either with AML1 or with TEL. EVI1 and MDS1 are unusual in that they can either encode separate proteins, or they can be expressed as one protein which we named MDS1/EVI1. EVI1 and MDS1/EVI1 have opposite functions as transcription factors. In this report, we review the current information on the two genes, and on their involvement in myeloid leukemia.

Results of decitabine therapy in the accelerated and blastic phases of chronic myelogenous leukemia

Abstract: The aim of the study was to evaluate the activity of decitabine, a hypomethylating agent, in the treatment of patients with chronic myelogenous leukemia (CML) in transformation. Thirty-seven patients with CML in blastic (20 patients) or accelerated phases (17 patients) were treated. Their median age was 52 years; 36 had Philadelphia chromosome-positive disease. Decitabine was given at 100 mg/m2 over 6 h every 12 h x 10 doses (1000 mg/m2) to 13 patients, and at 75 mg/m2 over 6 h every 12 h x 10 doses (750 mg/m2) to 24 patients. In blastic phase, two patients (10%) achieved a complete hematologic response (one with Ph suppression), and three (15%) had a hematologic improvement (marrow CR, platelets <100 x 10[3]/microl), for an overall response rate of 25%. In accelerated phase, six patients (35%) returned to a second chronic phase (two with Ph suppression), one (6%) had a hematologic improvement, and two (12%) had a partial hematologic response, for an overall response rate of 53%. Prolonged myelosuppression was the most significant side-effect. The median time to recovery of granulocytes above 500/microl was 48 days, and to recovery of platelets above 30 x 10(3)/microl, 31 days. Febrile episodes occurred in 25 patients (68%) including documented infections in 17 patients (46%). Decitabine has promising activity in CML. The most significant side-effect is prolonged myelosuppression. Decitabine may show activity in other myeloid disorders such as acute myeloid leukemia and myelodysplastic syndrome, as well as in other hematologic malignancies, alone or with other drug combinations. Its value in the context of stem cell support should also be investigated.

High bcl-2 expression in acute myeloid leukemia cells correlates with CD34 positivity and complete remission rate.

Abstract: Flow cytometric expression of bcl-2 protein was analyzed in 90 newly diagnosed acute myeloblastic leukemia (AML) patients using an anti-bcl-2 monoclonal antibody by direct immunofluorescence technique and results were correlated with FAB cytotype, CD34 expression and clinical outcome. Bcl-2 was expressed in all AML cases with different intensity. The mean fluorescence index (MFI), expressed as the ratio of sample mean channel:control mean channel, ranged from 3.0 to 39.5 with a median value of 14. The MFI was significantly higher (P = 0.01) in M0 (20.9) and M1 (18.3) than in M2 (11.7), M3 (12.4), M4 (11.8) and M5 (9.5) cytotypes. In addition, bcl-2 MFI significantly correlated both with CD34 positivity (P = 0.001) and with CD34 MFI (P = 0.01), being CD34 antigen expressed in 65% of patients with a bcl-2 MFI >14, and only in 35% of AML cases with a bcl-2 MFI >14. When bcl-2 intensity expression was correlated with complete remission (CR) rate, a higher MFI was associated with a low CR rate after standard intensive chemotherapy. In particular, CR was achieved in 86% of patients with a bcl-2 MFI 14 (P = 0.008). A further decrease of CR rate to 41% was observed in patients in whom a higher bcl-2 MFI was coupled with the presence of CD34 antigen on their blasts. By statistical analysis we also demonstrated that both bcl-2 high MFI (>14) and CD34 expression are independent prognostic factors for achieving CR in AML. These data raise the hypothesis that high values of bcl-2 may confer on myeloid blasts a higher resistance to standard chemotherapy. However, identification of patients with high expression of bcl-2 may be important for a different therapeutic approach.

Characteristic pattern of chromosomal gains and losses in marginal zone B cell lymphoma detected by comparative genomic hybridization

Abstract: Marginal zone B cell lymphoma (MZBCL) represents a distinct subtype of B cell non-Hodgkin's lymphoma, which has been recently recognized and defined as a disease entity. We investigated 25 cases (18 at primary diagnosis and seven during the course of disease) of MZBCL by comparative genomic hybridization (CGH) and compared these results with cytogenetic, fluorescence in situ hybridization (FISH), and Southern blot data. Twenty of the 25 cases (80%) showed gains (total 49) or losses (total 15) of genetic material. In extranodal, nodal, and splenic MZBCL, material of chromosomes 3 (52% of cases), 18 (32%), X (24%), and 1q (16%) was most frequently gained, whereas losses predominantly involved chromosomes 17 (16%) and 9 (12%). High-level amplifications involving the regions 18q21-23 and 18q21-22, respectively, were detected in two cases. Gains of chromosomes 1q and 8q and losses of chromosome 17 or 17p occurred more frequently in relapsed or progressive lymphomas. For all of the frequently affected chromosomes, CGH allowed narrowing of the relevant subregions including 3q21-23, 3q25-29 and 18q21-23. By Southern blot analysis, the BCL2, BCL6, and CMYC proto-oncogenes were found to be a part of the over-represented regions in two cases, one case, and two cases, respectively, with gains involving 18q, 3q or 8q. In 13 cases, CGH revealed chromosomal imbalances which were not detected by cytogenetic analysis but could be confirmed by FISH or Southern blot analysis in all cases investigated. On the other hand, CGH failed to detect trisomy 3, trisomy 18, and deletion 7q in three cases with a low proportion of tumor cells bearing these abnormalities, as shown by interphase FISH. The characteristic pattern of chromosomal gains and losses detected in this study confirms the distinct nature of MZBCL and may point to chromosomal regions involved in the pathogenesis of these neoplasms.

Cell cycle arrest and apoptosis of leukemia cells induced by L-asparaginase.

Abstract: Apoptotic cell death of murine leukemia cells induced by E. coli L-asparaginase was studied. Deprivation of L-asparagine from the culture of L5178Y cells by L-asparaginase caused the fragmentation of chromosomal DNA of the leukemia cells within 24 h. Prior to the degradation of DNA, cell cycles of L5178Y cells were found to be arrested in G1 phase, and evidence of the DNA strand breaks was initially observed in G1 phase cells as early as 8 h after the asparaginase treatment. Therefore, apoptosis of leukemia cells induced by L-asparaginase is an event that is associated with the cell cycle arrest in G1 phase.

Deletions at 11q identify a subset of patients with typical CLL who show consistent disease progression and reduced survival

Abstract: Eighty-four patients with typical chronic lymphocytic leukemia (CLL) (by morphological and immunophenotypic criteria) on whom karyotypes were available were studied. Binet stage at diagnosis and follow-up were defined. Survival was calculated from diagnosis. Fifty-one percent of patients had a karyotypic abnormality, the commonest being abnormalities at 13q14 (16%); these patients did not have significantly different survival from patients with normal karyotype. The second commonest abnormality was del(11q) (13%); these patients had significantly worse survival when compared both with patients with normal karyotype (P < 0.0001) and with other patients with karyotypic abnormality (P = 0.0012). All patients with del(11q) had progressed to stage C at follow-up while only 20% of the other patients had shown any disease progression (P < 0.0001). Del(11q) may identify a subset of patients with typical CLL who have worse survival and consistent disease progression and in future may help define a group of patients with CLL who could benefit from earlier or more intensive therapy.

Adoptive immunotherapy for relapsed multiple myeloma after allogeneic bone marrow transplantation (BMT): evidence for a graft-versus-myeloma effect.

Abstract: Adoptive immunotherapy with donor-derived buffy coat cells for relapsed hematological malignancies after allogeneic BMT is an established and highly effective treatment. We report a patient who relapsed on day +330 after allogeneic sibling BMT for multiple myeloma with multiple solid subcutaneous tumors consisting of plasma cells. Histology and immunocytology of the bone marrow did not show plasma cell infiltration. After cessation of the immunosuppression consisting of cyclosporine and methylprednisolone, a total of 6.2 x 10(7)/kg recipient body weight CD3+ T cells derived from the donor by leukapheresis were transfused on 4 consecutive days. To enhance the T cell effect six doses of 5 million units alpha interferon were given subcutaneously. Five days later the tumors started to shrink and have completely vanished since day x400 after BMT. The patient developed acute GVHD grade III of the liver and gut which was treated by reinduction of various immunosuppressive drugs. Up to now there is no evidence for relapse of the multiple myeloma, but the patient suffers from extensive chronic GVHD (gut and liver). This is the first report to demonstrate a graft-versus-myeloma effect for relapse with solid tumor manifestation after sibling BMT with donor-derived buffy coat cells as adoptive immunotherapy.

Functional features of neutrophils induced by G-CSF and GM-CSF treatment: differential effects and clinical implications

Abstract: G-CSF and GM-CSF are hematopoietic growth factors required for proliferation and differentiation of hematopoietic precursors. G-CSF is now widely used to overcome neutropenias of various origins. Beside the absolute number, the functional capacity of neutrophils at sites of inflammation is of major importance in host defense. This review summarizes major functional and phenotypical features of neutrophils induced by G-CSF treatment in patients with acquired and congenital neutropenias. Furthermore, we focus on the differential effect of G-CSF and GM-CSF on neutrophil function in vitro and in vivo. Some of the altered abilities of cytokine-induced neutrophils are important to understand side-effects of G-CSF therapy.

Cloning of the mouse BTG3 gene and definition of a new gene family (the BTG family) involved in the negative control of the cell cycle

Abstract: It is well known that loss of tumor suppressor genes and more generally of antiproliferative genes plays a key role in the development of most tumors. We report here the cloning of the mouse BTG3 gene and show that its human counterpart maps on chromosome 21. This evolutionarily conserved gene codes for a 30 kDa protein and is expressed in most adult murine and human tissues analyzed. However, we demonstrate that its expression is cell cycle dependent and peaks at the end of the G1 phase. This gene is homologous to the human BTG1, BTG2 and TOB genes which were demonstrated to act as inhibitors of cell proliferation. Its description allowed us to define better this seven gene family (the BTG gene family) at the structural level and to speculate about its physiological role in normal and tumoral cells. This family is mainly characterized by the presence of two conserved domains (BTG boxes A and B) of as yet undetermined function which are separated by a non-conserved 20-25 amino acid sequence.

Mechanisms of apoptotic cell death.

Abstract: Apoptosis is the mechanism by which cells are programmed to die under a wide range of physiological and developmental stimuli. Several mediators of programmed cell death have been identified and signals of apoptosis have been found to utilize common pathways, some of which have been elucidated. This review focuses on a number of apoptotic systems that have been widely studied and discuss recent progress and opinion in these areas. These include studies on Fas signaling, tumor suppressor genes, cell cycle interfaces, stress responses, genetic systems, and the Bcl-2 family. Understanding apoptosis from these perspectives sheds substantial light on processes of biological homeostasis. Furthermore, the ability to manipulate the apoptotic response may lead to novel therapeutic interventions in cancer and other diseases.

Potential role for concurrent abnormalities of the cyclin D1, p16CDKN2 and p15CDKN2B genes in certain B cell non-Hodgkin's lymphomas. Functional studies in a cell line (Granta 519).

Abstract: Abnormalities of several cell-cycle regulatory genes including cyclin D1, p16CDKN2 and p15CDKN2B have been described in B cell non-Hodgkin's lymphoma (B-NHL). We describe a new B-NHL cell line (Granta 519), with concurrent abnormalities of the cyclin D1, pl6CDKN2 and pl5CDKN2B genes. An independent clinical case of mantle cell NHL (Mc-NHL) with concomitant overexpression of cyclin D1, and deletion of the p16CDKN2 gene was also identified, suggesting that this combination of oncogenic aberration is a pathophysiologic contribution to a subset of NHL cases. More in-depth functional studies of this concept were facilitated by the availability of the cell line Granta 519 which was derived from a case of high-grade NHL and has a mature B cell immunophenotype. Cytogenetic analysis identified translocation t(11;14)(q13;q32) and complex rearrangements involving chromosomes 9p22, 13p21, 17pl1, and 18q21. Molecular analysis identified overexpression of cyclin D1 mRNA and biallelic deletion of the p16CDKN2 and p15CDKN2B genes. To elucidate the effect of these genetic abnormalities on the G1 control of Granta 519 cells, the level and function of the major components of the cyclinD/retinoblastoma (RB) pathway were investigated. Cyclin D1 was dominant among the D-type cyclins, formed abundant complexes with cyclin-dependent kinase (Cdk) Cdk4 rather than Cdk6, and the immunoprecipitated cyclin D1/Cdk4 holoenzyme was active as a pRB kinase. Electroporation of wild-type pl6CDKN2 arrested the Granta 519 cells in G1, consistent with the p16CDKN2 loss as a biologically relevant event during multistep evolution of the tumor, and with the expression of functional pRB. Direct cooperation of these distinct abnormalities to cell-cycle, deregulation in NHL cells was suggested by G1 acceleration upon inducible overexpression of cyclin D1 in a control breast cancer cell line lacking p16CDKN2, an effect which could be prevented by ectopic expression of p16CDKN2. Taken together, these data suggest that concurrent overexpression of cyclin D1 and functional elimination of p16CDKN2 and p15CDKN2B may characterize certain cases of mantle cell NHL, and that cooperation of the abnormalities is likely to provide a growth advantage of the tumour cells through more efficient inactivation of the RB tumor suppressor. Further clinicopathologic studies of this possibility are warranted.

Camptothecin-induced apoptosis in p53-null human leukemia HL60 cells and their isolated nuclei: effects of the protease inhibitors Z-VAD-fmk and dichloroisocoumarin suggest an involvement of both caspases and serine proteases.

Abstract: The human leukemia cell line, HL60 is very sensitive to various apoptotic stimuli and p53-null. The death-related cysteine proteases of the caspases family play a central role in the execution phase of apoptosis, and we recently reported the importance of serine protease activation in camptothecin-induced apoptotic endonuclease activation in HL60 cells. In the present study, we investigated the role of caspases (ICE/CED-3-related cysteine proteases) and serine proteases in cell death induced by the topoisomerase I inhibitor, camptothecin, in HL60 cells and in a cell-free system. We found that CPP32 is activated during camptothecin-induced apoptosis, and that N-benzyloxycarbony-Val-Ala-Asp (O-methyl) -fluoromethyketone (Z-VAD-fmk), a cell permeable caspase inhibitor blocks all features of apoptosis: morphological changes, cleavage of caspase 3 (CPP32/Yama/Apopain) and poly(ADP-ribose) polymerase, lamin B degradation and DNA fragmentation. However, Z-VAD-fmk and two other ICE/CED-3 inhibitors, YVAD-CHO and DEVD-CHO, were inactive in a cell-free system reconstituted from nuclei of untreated HL60 cells and cytosol from camptothecin-treated cells, suggesting that caspases are not required for endonuclease activation or lamin B cleavage in the cell-free system. By contrast, the serine protease inhibitors, 3,4-dichloroisocoumarin (DCI) and L-1-chloro-3-(4-tosylamido)-4-phenyl-2-butanone tosyl-L-phenylalanine chloromethyl ketone (TPCK), abolished the apoptosis-associated biochemical changes induced by camptothecin both in whole cells and in a cell-free system. DCI also inhibited CPP32 cleavage. Taken together, these results suggest that in HL60 cells, both CPP32 and serine proteases are activated in camptothecin-induced apoptosis.