Dysregulation of miRNA in Leukemia: Exploiting miRNA Expression Profiles as Biomarkers.

Micro RNAs (miRNAs) are a class of small non-coding RNAs that have a crucial role in cellular processes such as differentiation, proliferation, migration, and apoptosis. miRNAs may act as oncogenes or tumor suppressors; therefore, they prevent or promote tumorigenesis, and abnormal expression has been reported in many malignancies. The role of miRNA in leukemia pathogenesis is still emerging, but several studies have suggested using miRNA expression profiles as biomarkers for diagnosis, prognosis, and response to therapy in leukemia. In this review, the role of miRNAs most frequently involved in leukemia pathogenesis is discussed, focusing on the class of circulating miRNAs, consisting of cell-free RNA molecules detected in several body fluids. Circulating miRNAs could represent new potential non-invasive diagnostic and prognostic biomarkers of leukemia that are easy to isolate and characterize. The dysregulation of some miRNAs involved in both myeloid and lymphoid leukemia, such as miR-155, miR-29, let-7, and miR-15a/miR-16-1 clusters is discussed, showing their possible employment as therapeutic targets.

Clinical experience of granulocyte transfusion in the management of neutropenic patients with haematological malignancies and severe infection.

BACKGROUND: Prolonged chemotherapy-induced neutropenia is a major risk factor for the development of severe bacterial and fungal infections. Infectious manifestations may progress despite adequate anti-infectious treatment and lead to a very high short-term mortality. Granulocyte transfusion (GT) therapy is often considered. However, its efficacy is not well documented. METHODS: We retrospectively analyzed the clinical characteristics and outcome of a cohort of patients with haematological malignancies receiving GT during neutropenia and severe infection. RESULTS: A total of 30 patients with a median age of 46 y (range 3-82 y) who had received 1 or more GT were included. Acute leukaemia (80%) and non-Hodgkin lymphoma (17%) predominated as the underlying malignancy. All patients had severe and prolonged (median 16 days) neutropenia. The major indications for GT were persistent fever and clinical deterioration despite broad anti-infectious therapy, in combination with progressive pneumonia (n = 16), neutropenic enterocolitis (n = 6), and soft tissue infections (n = 3). GTs were given for a median of 3 transfusions (range 1-14). The median time to fever defervescence after GT was 14 days (range 6-33 days). For 11 patients, the resolution of fever and all signs of infection could directly be related to GT, and 3 of these patients became long-term survivors. Mortality at 30 days post-GT was 40% and at 6 months post-GT was 72%. GT was well tolerated. CONCLUSIONS: A substantial proportion of severely ill neutropenic patients appeared to benefit from GT. The results further underline the need for well- designed, randomized, prospective trials to determine the efficacy of this intervention in patients with life-threatening infectious complications.

Spontaneous and mitogen-induced cytokine production in lymphoproliferative diseases.

The levels of spontaneous and mitogen-induced production of pro- and anti-inflammatory cytokines were studied in patients with chronic lymphoid leukemia, non-Hodgkin's lymphocytic lymphomas, and multiple myeloma during the course of chemotherapy. Cytokine concentrations varied within a great range and did not conform to the normal distribution law. The levels of granulocyte and granulocyte-macrophage CSF were high during the debut, progress, and remission of the lymphoproliferative diseases. Imbalance of a wide spectrum of pro- and anti-inflammatory cytokines was observed during the debut and progress of the lymphoproliferative diseases, more often in chronic lymphoid leukemia and non-Hodgkin's lymphocytic lymphomas than in multiple myeloma.

Survival trends among 1,325 patients with chronic lymphocytic leukemia seen over the past 40 years in Israel.

In the light of recent data showing survival improvement of patients with chronic lymphocytic leukemia (CLL), we investigated clinical characteristics and survival patterns of patients with CLL over the last 40 years in Israel. Demographic and clinical data collected in the database of the Israeli CLL Study Group were analyzed. Of the 1,325 patients, 221 were diagnosed during the time period 1968-1989, 456 during 1990-1999, and 639 during 2000-2010. There was shift toward older age (median, 71 vs. 68 vs. 66 years) and a higher proportion of patients at Binet stage A at diagnosis (77.6% vs. 66.7% vs. 60.3%) in the more recent time periods. Median survival for the entire cohort was 10.9 years; 12.2 years for patients diagnosed at Binet stage A, 8.5 years for stage B, and 6.4 years for stage C patients. Older age, high-beta 2-microglobulin level, and expression of ZAP-70 predicted shorter survival. There were no apparent changes over time regarding gender, age or different clinical stages. Young patients with Binet stage A had lower life expectancy than the general population; but, in older ages, the survival rates were comparable. There were increased proportions of CLL patients diagnosed in early stages, and, at older age, during the last decades, however, survival rates according to sex, age, or stage remained stable. CLL continues to be an incurable disease affecting survival even in patients diagnosed at early stages. Survival benefit shown in recent trials using chemoimmunotherapy has still to be proven in wider general practice.

Effect of SLCO1B1 Polymorphisms on High-Dose Methotrexate Clearance in Children and Young Adults With Leukemia and Lymphoblastic Lymphoma.

High-dose (HD) methotrexate (MTX) is a critical component of treatment for hematologic malignancies in children and young adults. Therapeutic drug monitoring is necessary due to substantial interindividual variation in MTX clearance. Common function-altering polymorphisms in SLCO1B1 (encodes OATP1B1, which transports MTX) may contribute to clearance variability. We performed pharmacokinetic modeling using data for 106 children and young adults treated with HD MTX for hematologic malignancies; of 396 total courses of HD MTX, 360 consisted of 5 g/m2 over 24 hours. We evaluated the contribution of clinical covariates and SLCO1B1 genotype (388A>G and 521T>C) to MTX clearance variability. Of the clinical covariates studied, patient weight improved the pharmacokinetic model most significantly (P < 0.001). The addition of the SLCO1B1 variants individually further improved the model (P < 0.05 for each). An interaction between these variants was suggested when both were included (P = 0.017). SLCO1B1 genotype should be considered in efforts to personalize HD MTX dosing.

Diagnostic performance of the ClearLLab 10C B cell tube.

INTRODUCTION: Pre-analytical and analytical errors can threaten the reliability of flow cytometry (FC) results. A potential solution to some of these is the use of dry, pre-mixed antibodies, such as the ClearLLab 10C system. The purpose of the present study was to compare the diagnostic performance of the ClearLLab 10C B cell tube with that of our standard laboratory practice. METHODS: We compared the diagnoses made with the ClearLLab 10C B cell tube (experimental strategy) with those made with standard laboratory practice (standard strategy). Samples were selected aiming for representation of the full spectrum of B cell disorders, with an emphasis on mature B cell malignancies, as well as healthy controls. RESULTS: We included 116 samples (34 normal controls, 4 acute lymphoblastic leukemias, 54 mature lymphoproliferative disorders in peripheral blood and bone marrow, 3 myelomas, 6 bone marrow samples with involvement by lymphoma and 1 with elevated hematogone count, 14 lymph node samples, 1 cerebrospinal fluid, and 1 pleural effusion). There were two diagnostic errors (1.7%). The agreement between the two strategies in the percentage of CD19 cells and fluorescence intensity of CD5, CD19, CD20, CD200, and CD10 was very good. CONCLUSIONS: In this study, the ClearLLab 10C B cell tube performed similarly to our standard laboratory practice to diagnose and classify mature B cell malignancies.

The surface morphology of human B lymphocytes as revealed by immunoelectron microscopy.

Surface immunoglobulins (sIg) were detected on human lymphocytes by immunoelectron microscopy with peroxidase-conjugated antibodies. Blood, marrow, and thymus cells from normal individuals and patients with lymphoproliferative disorders were examined. Samples were fixed before exposure to specific reagents. Normal lymphocyts with detectable sIg, i.e. B lymphocytes, were characterized by a villous surface; nonlabeled blood lymphocytes and thymocytes were smooth cells. Intermediate cells were also found which in sections appeared moderately villous and labeled, thus identified as B lymphocytes. Further evidence for a relationship between villous surface and sIg was given by the finding of a few lymphocytes with polar concentration of labeled microvilli. In chronic lymphocytic leukemia patients, most cells exhibited a villous surface with parallel variations of the number of microvilli and of anti-immunoglobulin-binding capacity. However, some labeled smooth blastic cells were also observed. On the other hand, abnormal lymphocytes from Sézary's syndrome which could exhibit segments of villous membrane had no detectable sIg. This study confirms that in most cases human B lymphocytes have a characteristic surface appearance and that the detection of sIg in normal lymphocytes correlates with the presence of microvilli.

A glycoprotein associated with the membrane fraction of human B but not T lymphocytes.

A method is described which employs differential centrifugation and sucrose density gradient centrifugation to isolate a membrane fraction from human lymphocytes. Membrane preparations from long-term human cultured B- and T-lymphoid lines, peripheral blood lymphocytes, tonsillar lymphocytes, and thymocytes were analyzed on 0.5% sodium dodecyl sulfate-7.5% polyacrylamide gels stained for protein and carbohydrate. The most important finding was a major glycoprotein of approximately 30,000 daltons associated with the membrane preparations from B lymphocytes. T-lymphocyte preparations did not contain readily detectable amounts of this membrane-associated component. The T-cell lymphoid line MOLT-4 was unique in that it had a narrow protein band at approximately 30,000 daltons which did not contain carbohydrate.

Assessment of peripheral blood and bone marrow cells apoptosis caused by purine analogues in patients with chronic lymphocytic leukemia in correlation with parameters of disease progression.

Chronic lymphocytic leukemia (CLL) is a heterogeneous disease with variable clinical course and prognosis. Therefore, the role of prognostic factors is very important, especially for identifying the group of patients who require intensive treatment. The aim of this study was to assess whether the rate of apoptosis caused by purine analogues differs between patients with better or worse prognostic factors. The experiments were preformed in cultures of blood and bone marrow obtained from CLL patients. The cultures were supplemented with cladribine and fludarabine. We determined the percentage of caspase-3-positive cells and the BCL-2/BAX ratio, and subsequently these apoptosis markers were correlated with the expression of ZAP-70 and CD38, lymphocyte counts, lactate dehydrogenase and beta(2)-microglobulin levels and clinical stage according to the Rai classification. The results showed that bone marrow cells are more sensitive to apoptosis caused by purine analogues than cells derived from blood, supporting the idea that these two compartments have different proliferative statuses. The cells from ZAP-70-positive patients seem to enter apoptosis more readily than those from ZAP-70-negative patients; thus, ZAP-70-positive patients are more likely to benefit from treatment with purine analogues.

Mouse leukemia: depression of serum interferon production.

Production of circulating interferon is significantly impaired in AKR/J mice after development of lymphoblastic leukemia and in Balb/c mice with clinical signs of Friend erythroblastic leukemia. This alteration has been observed with three interferon inducers, each one known to elicit an interferon response in different cells.

Distinct alkaline phosphatase in serum of patients with lymphatic leukemia and infectious mononucleosis.

A distinct alkaline phosphatase (phosphatase N) was demonstrated in the serum of patients with acute lymphatic leukemia, chronic lymphatic leukemia, and infectious mononucleosis. This enzyme closely resembles that extracted from the thymus of mice with lymphoma or lymphatic leukemia, both in its electro-phoretic mobility and its substrate specificity. The phosphatase N activity was related to the clinical state of patients with lymphatic leukemia and disappeared with recovery from infectious mnononucleosis.

High dose methotrexate population pharmacokinetics and Bayesian estimation in patients with lymphoid malignancy.

The purpose of present study was to develop a population pharmacokinetic model of high dose methotrexate (HD-MTX) infusion in patients with lymphoid malignancy, to investigate the biological and clinical covariates related to the drug distribution and elimination. It is also the purpose to propose a limited sampling strategy (LSS) for the estimation of the time above the threshold (0.2 micromol.L(-1)). A total 82 patients with lymphoid malignancy were involved in the study. A pharmacokinetic model was developed using nonlinear mixed-effect model. The influence of demographic characteristics, biological factors, and concurrent administration were investigated. The final predictive performance was validated by bootstrap and cross-validation. Bayesian estimation was evaluated. The pharmacokinetics of HD-MTX was described by a two-compartment model. The pharmacokinetic parameters and the inter-individual variability were as follows: the clearance CL, 7.45 L.h(-1) (inter-individual variability 50.6%), the volume of the central and peripheral compartment V(1), 25.9 L (22.5%), V(2), 9.23 L (97.8%), respectively, and the intercompartmental clearance Q, 0.333 L.h(-1) (70.4%). The influence of serum creatinine on CL and weight on V(1) was retained in the final model. The protocol involved one sampling time at 44 h after the start of the infusion, allowing one to predict the time at which the MTX concentration reached the expected threshold (0.2 micromol.L(-1)). Serum creatinine and weight showed significant influence on methotrexate CL and V(1), respectively. Furthermore, a Bayesian estimation based on the covariates and 44 h sample was developed, allowing prediction of the individual methotrexate pharmacokinetic parameters and the time to 0.2 micromol.L(-1).

A C5a-Immunoglobulin complex in chronic lymphocytic leukemia patients is associated with decreased complement activity.

Chronic lymphocytic leukemia (CLL) is the most common adult leukemia in the Western world. The therapeutic approach to CLL includes chemotherapeutic regimens and immunotherapy. Complement-mediated cytotoxicity, which is one of the mechanisms activated by the therapeutic monoclonal antibodies, depends on the availability and activity of the complement (C) system. The aim was to study the structure of circulating C components and evaluate the importance of C5 structural integrity for C activity in CLL patients. Blood samples were collected from 40 naïve CLL patients and 15 normal controls (NC). The Western blot analysis showed abnormal C5 pattern in some CLL patients, while patterns of C3 and C4 were similar in all subjects. Levels of the C activation markers sC5b-9 and C5a were quantified before and after activation via the classical (CP) and alternative (AP) pathways. In patients with abnormal C5, basal levels of sC5b-9 and C5a were increased while activities of the CP and of the CP C5-convertase, the immediate C5-upstream complex, were decreased compared to NC and to patients with normal C5. The data indicate a link between CP activation and apparent C5 alterations in CLL. This provides a potential prognostic tool that may personalize therapy by identifying a sub-group of CLL patients who display an abnormal C5 pattern, high basal levels of sC5b-9 and C5a, and impaired CP activity, and are likely to be less responsive to immunotherapy due to compromised CP activity.

Current status of growth factors in the treatment of acute myeloid and lymphoblastic leukemia.

The safety of granulocyte colony-stimulating factor (G-CSF) and granulocyte-macrophage colony-stimulating factor (GM-CSF) in patients with acute leukemia has been well established in numerous clinical trials. The primary aim of these studies was to determine whether CSFs, when used as adjuncts to intensive chemotherapy, reduced the duration of neutropenia, prevented febrile neutropenia, infections, and hospitalization rates, and improved response and overall outcome in patients with acute myeloid leukemia (AML) or acute lymphoblastic leukemia (ALL). Despite considerable efforts in divers clinical settings, the potential advantages of hematopoietic growth factors in the management of these leukemias remain inconclusive. In general, individual published trials have shown declines in the incidence and/or duration of neutropenia but have not consistently demonstrated a reduction in the overall frequency of infectious complications or the duration of hospitalization. Most protocols also have failed to show a benefit in terms of disease-free or overall survival. Nevertheless, improvements in "soft" clinical end points, such as incidence of severe infections, may be clinically important and contribute, even if only incrementally, to the patient's quality of life. Selection of those patients likely to benefit from growth factors in a specific clinical setting is a worthwhile endeavour.

Human lymphocytes: 5'-nucleotidase-positive and -negative subpopulations.

The enzyme, 5'-nucleotidase (5'N) (E.C.-3.1.3.5) is present in lymphocytes isolated from the blood of normal subjects. This activity is markedly decreased or not detectable in the cells from three-quarters of patients with chronic lymphocytic leukemia (CLL), while supranormal levels are found in less than 10% of the cases. To determine whether the decreased 5'N value in CLL represents a lower activity per cell or fewer enzyme-containing cells than in the normal, conditions were established for the histochemical measurement of 5'N in human lymphocytes. It was found that the cells isolated from the blood of normal subjects or patients with CLL consist of 5'N-positive and 5'N-negative subpopulations. Normal subjects who had high 5'N specific activity were shown to have a greater percentage of 5'N-positive cells than individuals with low 5'N activity. Patients with CLL who had no activity by standard chemical assay had no 5'N-positive cells, while the exceptional patient with CLL with a higher than normal specific activity showed an percentage of 5'N-positive cells. It is suggested that the selective proliferation of 5'N-positive and 5'N-negative populations may account for the heterogeneity of 5'N in CLL.

Heterogeneity of 5'-nucleotidase activity in lymphocytes in chronic lymphocytic leukemia.

The specific activity of 5'-nucleotidase was determined in lymphocyte plasma membranes from 14 normal subjects and 10 patients with chronic lymphocytic leukemia (CLL). Whereas the enzyme was present in the preparation from normal lymphocytes, in 7 out of 10 CLL patients the membranes had markedly decreased or no detectable 5'-nucleotidase activity. The lack of this activity from the lymphocytes of most patients with CLL constitutes an alteration in a plasma membrane enzyme from the normal cell. The presence of the enzyme in the lymphocytes of some patients with CLL and its decrease in others provide further evidence for biochemical heterogeneity among patients with this disorder.

Synthesis of DNA and poly(adenosine diphosphate ribose) in normal and chronic lymphocytic leukemia lymphocytes.

Peripheral blood lymphocytes were isolated from 9 patients with chronic lymphocytic leukemia (CLL) and 12 normal control donors. The cells were assayed for synthesis of DNA and poly-(adenosine diphosphate ribose) (poly[ADPR]) immediately after isolation and on successive days following their treatment with phytohemagglutinin (PHA). Two different techniques were used to measure DNA synthesis. In the standard technique, DNA synthesis was measured by incubating intact cells with [(3)H]deoxythymidine. In the new technique, the lymphocytes were first rendered permeable to nucleotides, then DNA synthesis was measured by incubating them with [(3)H]deoxythymidine triphosphate in the presence of deoxyATP, deoxyGTP, deoxyCTP, ATP, and Mg(++). Both assays showed the anticipated rise in DNA synthesis after PHA stimulation of normal cells. PHA-stimulated lymphocytes from patients with CLL demonstrated low levels of DNA synthesis in both assay systems. The initial levels of poly(ADPR) synthesis were greater in CLL lymphocytes than in normal cells. Studies with a T-cell-depleted population of normal cells showed the same activity for poly(ADPR) synthesis that was demonstrated by the original population of normal cells. PHA stimulation produced an increase in poly(ADPR) synthesis in both the normal and CLL cells. The increase in poly(ADPR) synthesis in normal cells was coincident with the increase in DNA synthesis. The increase in poly(ADPR) synthesis in the CLL cells was dissociated from the delayed and diminished increase in DNA synthesis. Thus, CLL cells have higher than normal initial levels of poly(ADPR) synthesis. Poly(ADPR) synthesis is dissociated from DNA synthesis in CLL cells whereas it varies directly with DNA synthesis in normal lymphocytes.

Isolation and partial characterization of Fc gamma-binding proteins of human leukocytes.

We isolated and partially characterized three Fc-binding macromolecules from human leukocytes. Mononuclear cells from normal individuals and from five patients with chronic lymphocytic leukemia and neutrophils from normal donors were surface radiolabeled by using 125I and lactoperoxidase. After detergent solubilization of the cells, Fc gamma-binding macromolecules were purified by repetitive affinity chromatography under a variety of conditions and analyzed by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. Three radiolabeled macromolecules were isolated that retained specific ability to bind to Fc fragments. A 52,000-64,000-mol wt macromolecule was isolated from normal mononuclear and polymorphonuclear cells. A 43,000-mol wt band was characteristic of mononuclear cells, particularly from patients with chronic lymphocytic leukemia. A 33,000-mol wt molecule could be obtained from normal leukocytes under conditions that suggest it might be a proteolytic fragment.

Direct measurement of the rates of synthesis of plasma proteins in control subjects and patients with gastrointestinal protein loss.

The guanido carbon of hepatic arginine is the common precursor of urea and of the arginine of plasma proteins synthesized in the liver. It is possible to measure the momentary synthetic rates of plasma proteins by "pulse labeling" this arginine pool with bicarbonate-(14)C. In the current study, this method has been adapted in order to use urinary urea data and was applied to control subjects and patients with gastrointestinal protein loss. The assumptions required for this determination are discussed. There was close agreement between albumin synthetic rates measured by this method and albumin catabolic rates derived from simultaneous albumin-(131)I studies, supporting the validity of the method and suggesting that there is relatively little fluctuation in the rate of albumin synthesis from time to time. The albumin synthetic rates in six control subjects averaged 5.8 mg/kg per hr, while those of five patients with gastrointestinal protein loss averaged 7.2 mg/kg per hr. Thus in these patients, there was relatively little acceleration of albumin synthesis in response to continued loss of plasma proteins into the gastrointestinal tract. Fibrinogen synthetic rates averaged 1.9 mg/kg per hr in five control subjects and 3.2 mg/kg per hr in five patients with gastrointestinal protein loss. Transferrin synthetic rates exhibited considerable individual variation in both groups and averaged 0.24 mg/kg per hr in four control subjects and 0.31 mg/kg per hr in five patients with gastrointestinal protein loss. The method employed in this study offers several advantages in studying plasma protein metabolism. It provides a direct measurement of protein synthesis, applicable to several proteins simultaneously, does not require a long-term steady state in the metabolism of the proteins, and is capable of measuring short-term fluctuations in synthetic rates. Therefore, this approach is applicable to the investigation of the physiological factors controlling the rates of synthesis for plasma proteins.

Adenylate cyclase in thymus-derived and bone marrow-derived lymphocytes from normal donors and patients with chronic lymphocytic leukemia.

Lymphocytes were purified from peripheral blood of normal donors and patients with chronic lymphocytic leukemia (CLL) by Ficoll-Hypaque centrifugation. Adenylate cyclase activity, expressed as picomoles [(32)P]cyclic AMP generated per milligram protein per minute, was 57+/-4 in normals and 26+/-4 in CLL patients. Enzyme activity, expressed as picomoles [(32)P]cyclic AMP generated per 10(6) lymphocytes per minute, was 2.09+/-0.19 for normal lymphocytes and 1.10+/-0.16 for CLL lymphocytes. The differences between normal and CLL peripheral lymphocytes are highly significant (P < 0.001) with either method of calculating activity. Cyclic AMP levels (picomoles per 10(6) lymphocytes) also differed significantly: 1.38+/-0.29 for normals and 0.45+/-0.08 for CLL lymphocytes. Adenylate cyclase was assayed in lymphocytes enriched for bone marrow-derived (B) cells by removing E-rosetted thymus-derived (T) cells, and enriched for T cells by harvesting E-rosetted lymphocytes or by removing B cells with nylon wool absorption. Solutions to simultaneous equations gave the following calculated enzyme activities for pure B- and T-cell subpopulations (in picomoles [(32)P]cyclic AMP generated per milligram mg protein per minute): normal B, 196+/-22; normal T, 30+/-10; CLL B, 34+/-6; CLL T, 19+/-4. Thus. normal B-lymphocyte adenylate cyclase exceeds normal T-lymphocyte activity by more than sixfold, whereas in the case of CLL the enzyme activity in B lymphocytes is markedly reduced to levels comparable to T lymphocytes. The responses of lymphocytes to stimulation with the hormones prostaglandin E(1) and isoproterenol, and with NaF, were assessed. Compared with normal lymphocytes, enzyme activities were reduced in CLL lymphocytes incubated with these agents, but to a degree paralleling the reduced basal activities. Thus, the ratios between stimulated and basal adenylate cyclase levels in Ficoll-Hypaque-purified, normal lymphocytes were 2.3+/-0.1 after incubation with 10 muM isoproterenol, and 3.9+/-0.2 with 10 mM NaF, values which did not differ significantly from those obtained with CLL lymphocytes. When the enzyme activities calculated for purified T- and B-lymphocyte subpopulations were used to derive the stimulation ratios, the responses of normal and CLL T and B cells to these agents were also indistinguishable. The simplest explanation for these findings is a reduced number of normally responsive enzyme sites on the surface membranes of CLL lymphocytes, although alternative explanations are possible.