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.

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.

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.

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.

Plasma 25-hydroxyvitamin D concentration and lymphoma risk: results of the European Prospective Investigation into Cancer and Nutrition.

BACKGROUND: The relation between vitamin D status and lymphoma risk is inconclusive. OBJECTIVE: We examined the association between prediagnostic plasma 25-hydroxyvitamin D [25(OH)D] and lymphoid cancer risk. DESIGN: We conducted a study nested within the European Prospective Investigation into Cancer and Nutrition cohort of 1127 lymphoma cases and 1127 matched controls with a mean follow-up time of 7.1 y. Conditional logistic regression was used to estimate multivariable-adjusted incidence rate ratios of lymphoma risk in relation to plasma 25(OH)D. Season-standardized and season-specific 25(OH)D quartiles were used. We also analyzed 25(OH)D as a continuous variable and used predefined cutoffs. RESULTS: No statistically significant association between plasma 25(OH)D and overall lymphoid cancer risk was observed. A positive association for B-cell non-Hodgkin lymphoma was noted only in those with a diagnosis made during the first 2 y of follow-up (P-heterogeneity = 0.03), which suggests the possibility of reverse causality. Further analysis restricted to participants with ≥2 y of follow-up time showed a significant association between 25(OH)D and chronic lymphocytic leukemia (CLL) (n = 161): adjusted incidence rate ratios were 0.40 (95% CI: 0.18, 0.90; P-trend = 0.05) and 0.31 (95% CI: 0.13, 0.76; P-trend = 0.03) for the top compared with the bottom season-standardized and season-specific quartiles, respectively. Data on dietary vitamin D intake provided further support for the observed association (incidence rate ratio: 0.33; 95% CI = 0.12, 0.89; P-trend = 0.006). CONCLUSIONS: Our findings do not support a protective role of high 25(OH)D concentration in lymphoid cancers overall. However, they suggest that higher concentrations of 25(OH)D are associated with a reduced risk of CLL.

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.

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.

Elevated serum granulysin and its clinical relevance in mature NK-cell neoplasms.

Mature natural killer (NK)-cell neoplasms include extranodal NK/T cell lymphoma, nasal type (ENKL), aggressive NK-cell leukemia (ANKL) and chronic lymphoproliferative disorders of NK cells (CLPD-NK). Granulysin, a cytolytic granule protein, is expressed in cytotoxic T cells and NK cells, and is found in the sera as well, and functions as a cytotoxic and proinflammatory protein. Cytolytic proteins, such as granzyme B and perforin, have been shown to play crucial pathophysiological roles in NK/T cell neoplasms and have also been utilized for diagnostic purposes. Granulysin in NK-cell proliferative disorders, however, has yet to be fully analyzed. To elucidate the clinical relevance of granulysin in mature NK-cell neoplasms, we measured serum granulysin and analyzed cytolytic molecules immunohistologically. The median concentrations of serum granulysin were 39.0, 2.85, 2.8 and 1.35 ng/ml in ANKL, ENKL, CLPD-NK and healthy subjects, respectively (P < 0.01). Serum granulysin was significantly elevated in patients with ANKL compared with the levels in ENKL (P = 0.006) and CLPD-NK (P = 0.037). Furthermore, serum granulysin was correlated with whole-blood EBV viral load in ENKL and ANKL (P = 0.005) and was significantly reduced after treatment. Different expression patterns of cytolytic granule proteins were observed among the mature NK-cell neoplasms. Granulysin is closely associated with the characteristics of NK-cell neoplasms and serum granulysin may serve as a novel biomarker for these disorders.

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.

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.

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).

Release and clinical significance of soluble CD83 in chronic lymphocytic leukemia.

Soluble CD83 (sCD83), a potent immunosuppressive agent, circulates at elevated levels in some chronic lymphocytic leukemia (CLL) patients. We report that CLL patients with elevated plasma sCD83 levels had significantly shorter (P=0.038) treatment free survival. Culture of CLL cells with solid phase CD83 mAb+IL-4 significantly increases sCD83 release (23-117-fold, P=0.013) and ligation of normal donor PBMC with solid phase CD83 mAb alone induces similar significant increases in sCD83 release (P=0.003). RT-PCR analysis detected the presence of a transcript for sCD83 in 2/3 CLL samples. These results suggest sCD83 release may play a regulatory role in CLL progression.

Detection of anti-envoplakin and anti-periplakin autoantibodies by ELISA in patients with paraneoplastic pemphigus.

Paraneoplastic pemphigus patients (PNP) develop a group of autoantibodies, among which those against envoplakin and periplakin are almost always found. Epitope mapping has indicated that the linker subdomains of the proteins harbor the major antigenic sites recognized by PNP sera. In order to detect specific autoantibodies for the diagnosis of PNP, we expressed recombinant proteins containing linker subdomains of human periplakin and envoplakin in a human kidney cell line, and used them as the antigens for ELISAs. We found that all of the sera from 16 PNP patients recognized these two recombinant proteins by ELISA, and sera from 20 pemphigus vulgaris (PV), 12 pemphigus foliaceus (PF), 20 bullous pemphigoid (BP), 2 Castleman's tumor without PNP and 20 normal controls showed negative results. We also expressed the extracellular domain of desmoglein 3 (Dsg3) in the cell line, and used this recombinant Dsg3 as the ELISA antigen. Only 11 of our 16 PNP sera were positive, and most PV sera were positive. Our findings indicate that ELISAs using the recombinant proteins containing linker subdomains of envoplakin and periplakin expressed in a human cell line as the antigens are highly sensitive and specific for the diagnosis of PNP.

Detection of anti-asparaginase antibodies during therapy with E.coli asparaginase in children with newly diagnosed acute lymphoblastic leukemia and lymphoma.

BACKGROUND: Asparaginase is an effective antileukemic agent which is included in most front-line protocols for pediatric acute lymphoblastic leukemia (ALL) worldwide. Since asparaginase is a bacterial protein, it may induce formation of antibodies. The reported frequency of anti-asparaginase antibodies is highly variable: antibodies have been reported in as many as 79% of adults and as many as 70% of children after intravenous or intramuscular administration of E.coli asparaginase. PURPOSE: The aim of this study was to determine if the presence of antibodies during induction and continuation phases in newly diagnosed children with ALL and lymphoblastic lymphoma during therapy with E.coli asparaginase, had any correlation with various factors such as: age, gender, hypersensitivity reactions, response to therapy and Event Free Survival (EFS). PATIENTS AND METHODS: Between the period from March 2005 to May 2007, sixty-four children who attended the Menia outpatient pediatric oncology clinic, or were admitted to the inpatient department of the Menia oncology center, were enrolled in the study. Forty children had newly diagnosed ALL and 24 had lymphoblastic lymphoma. Patients were 48 males (75%) and 16 females (25%) with a male:female ratio 3:1. Their ages ranged from 3.5 to 17 years with mean age of 9.6 years. All patients received asparaginase therapy according to the St. Jude Total XIII protocol, in a dose of 10,000 IU/m(2)/dose, intramuscularly for 6-9 doses during the induction phase and another 6-9 doses during continuation phase according to disease status. RESULTS: Forty one patients achieved complete remission, 9 had partial remission, and 14 were lost to followup at different intervals of treatment. Antiasparaginase antibodies were detected in 36 patients (56%) out of 64 patients, and 37 patients (60%) out of 62 patients who were treated with asparaginase at day 8 and day 27 of induction phase respectively. Moreover, 33 patients (61%) out of 54 patients, and 41 patients (83%) out of 50 patients had positive antiasparaginase antibodies at week 10 and week 21 of continuation phase respectively. The 2-year EFS of the whole group was 50%. There was no statistically significant relation between positivity of antiasparaginase antibodies and the following: age, gender, hypersensitivity reaction, response to therapy and EFS. CONCLUSIONS: The presence of antiasparaginase antibodies was unrelated to age, gender, hypersensitivity reaction, response to therapy and event free survival of newly diagnosed children with acute lymphoblastic leukemia and lymphoblastic lymphoma.

A new high-throughput screening method for the detection of chronic lymphatic leukemia and myelodysplastic syndrome.

BACKGROUND: The VCS technology of Beckman Coulter differentiates white blood cells based on measures of their volume, conductivity and light scatter. The current study investigated the predictive value of index measures, known as research population data, for the detection of chronic lymphatic leukemia and myelodysplastic syndrome. METHODS: Blood cell counts were performed in samples from 44 patients with chronic lymphatic leukemia, 19 patients with myelodysplastic syndrome and 199 healthy blood donors using the Beckman Coulter LH750. Means and standard deviations of volume, conductivity and scatter of lymphocytes and neutrophils were evaluated as predictors for both diseases. Their specificity and selectivity were evaluated by logistic regression and receiver operating characteristic curve analysis. RESULTS: Research population data were significantly different among groups. For chronic lymphatic leukemia, standard deviations of lymphocytes scatter and volume showed most relevant differences in comparison to healthy blood donors (sensitivity 88.6%, specificity 84.4%). For myelodysplastic syndrome, standard deviations of neutrophils conductivity were most predictive (sensitivity 73.7%, specificity 93.0%). Areas under corresponding receiver operating characteristic curves were 0.941 and 0.951, respectively. CONCLUSIONS: Based on their high predictive value, research population data could be routinely used to screen for chronic lymphatic leukemia and myelodysplastic syndrome.

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.

Chemokine system and tissue infiltration in aggressive NK-cell leukemia.

NK cell-type lymphoproliferative disease of granular lymphocytes can be subdivided into aggressive NK-cell leukemia (ANKL) and chronic NK-cell lymphocytosis (CNKL). Hepatosplenomegaly is observed in ANKL patients, and hepatic failure is a common cause of death. Significant numbers of ANKL cells were pathologically observed in sinusoidal and interlobular regions of the liver, and in the splenic red pulp. In our previous study, ANKL cells were simultaneously positive for CXCR1 and CCR5. So, in order to elucidate the mechanism in the systemic migration of ANKL cells, we investigated the expression of the corresponding chemokines in ANKL compared with CNKL. The serum level of IL-8, MIP-1alpha and MIP-1beta was significantly elevated in ANKL patients, and ANKL cells were highly positive for IL-8, RANTES, MIP-1alpha and MIP-1beta according to intracellular staining and RT-PCR. These chemokines were also positively stained in hepatocytes. The interaction between Fas and Fas ligand (FasL) is supposed to be one of the mechanisms for liver dysfunction in ANKL. The serum concentration of soluble FasL was significantly high in ANKL patients, and ANKL cells expressed FasL protein in the cytoplasm. These results suggest that the chemokine system plays an important role in the transmigration of FasL-expressing ANKL cells.