Targeting the vulnerability to NAD+ depletion in B-cell acute lymphoblastic leukemia.

Although substantial progress has been made in the treatment of B-cell acute lymphoblastic leukemia (B-ALL), the prognosis of patients with either refractory or relapsed B-ALL remains dismal. Novel therapeutic strategies are needed to improve the outcome of these patients. KPT-9274 is a novel dual inhibitor of p21-activated kinase 4 (PAK4) and nicotinamide phosphoribosyltransferase (NAMPT). PAK4 is a serine/threonine kinase that regulates a variety of fundamental cellular processes. NAMPT is a rate-limiting enzyme in the salvage biosynthesis pathway of nicotinamide adenine dinucleotide (NAD) that plays a vital role in energy metabolism. Here, we show that KPT-9274 strongly inhibits B-ALL cell growth regardless of cytogenetic abnormalities. We also demonstrate the potent in vivo efficacy and tolerability of KPT-9274 in a patient-derived xenograft murine model of B-ALL. Interestingly, although KPT-9274 is a dual PAK4/NAMPT inhibitor, B-ALL cell growth inhibition by KPT-9274 was largely abolished with nicotinic acid supplementation, indicating that the inhibitory effects on B-ALL cells are mainly exerted by NAD+ depletion through NAMPT inhibition. Moreover, we have found that the extreme susceptibility of B-ALL cells to NAMPT inhibition is related to the reduced cellular NAD+ reserve. NAD+ depletion may be a promising alternative approach to treating patients with B-ALL.

Ordering of mutations in acute myeloid leukemia with partial tandem duplication of MLL (MLL-PTD).

Partial tandem duplication of MLL (MLL-PTD) characterizes acute myeloid leukemia (AML) patients often with a poor prognosis. To understand the order of occurrence of MLL-PTD in relation to other major AML mutations and to identify novel mutations that may be present in this unique AML molecular subtype, exome and targeted sequencing was performed on 85 MLL-PTD AML samples using HiSeq-2000. Genes involved in the cohesin complex (STAG2), a splicing factor (U2AF1) and a poorly studied gene, MGA were recurrently mutated, whereas NPM1, one of the most frequently mutated AML gene, was not mutated in MLL-PTD patients. Interestingly, clonality analysis suggests that IDH2/1, DNMT3A, U2AF1 and TET2 mutations are clonal and occur early, and MLL-PTD likely arises after these initial mutations. Conversely, proliferative mutations (FLT3, RAS), typically appear later, are largely subclonal and tend to be unstable. This study provides important insights for understanding the relative importance of different mutations for defining a targeted therapeutic strategy for MLL-PTD AML patients.

Dietary modulation of omega-3/omega-6 polyunsaturated fatty acid ratios in patients with breast cancer.

BACKGROUND: Polyunsaturated fatty acids of the omega-6 (omega-6) class, as found in corn and safflower oils, can act as precursors for intermediates involved in the growth of mammary tumors when fed to animals, whereas polyunsaturated fatty acids of the omega-3 (omega-3) class, as found in fish oil, can inhibit these effects. The effects of dietary intervention on the ratios of these fatty acids in breast and other adipose tissues have not previously been prospectively studied. PURPOSE: The present investigation was conducted to study the impact on the ratio of omega-3 and omega-6 polyunsaturated fatty acid in plasma and in adipose tissue of the breast and buttocks when women with breast cancer consume a low-fat diet and fish oil supplements. METHODS: Twenty-five women with high-risk localized breast cancer were enrolled in a dietary intervention program that required them to eat a low-fat diet and take a daily fish oil supplement throughout a 3-month period. Breast and gluteal fat biopsy specimens were obtained from each woman before and after dietary intervention. The fatty acid compositions of specimens of plasma, breast fat, and gluteal fat were determined by gas-liquid chromatography. Statistical analysis involved use of a two-sided paired t test. RESULTS: After dietary intervention, a reduction in the level of total omega-6 polyunsaturated fatty acids in the plasma was observed (P<.0003); moreover, total omega-3 polyunsaturated fatty acids increased approximately three-fold (P<.0001) and the omega-3/omega-6 polyunsaturated fatty acids ratio increased approximately fourfold (i.e., mean values increased from 0.09 to 0.41; P = .0001). An increase in total omega-3 polyunsaturated fatty acids in breast adipose tissue was observed following dietary intervention (P = .04); the omega-3/omega-6 polyunsaturated fatty acid ratio increased from a mean value of 0.05 to 0.07 (P = .0001). An increase in total omega-3 polyunsaturated fatty acids was observed in gluteal adipose tissue following the intervention (P = .05); however, the ratio of omega-3 to omega-6 polyunsaturated fatty acids (mean ratio values of 0.036-0.045; P = .06) was unchanged. CONCLUSION: Short-term dietary intervention can lead to statistically significant increases in omega-3/omega-6 polyunsaturated fatty acid ratios in plasma and breast adipose tissue. Breast adipose tissue changed more rapidly than gluteal adipose tissue in response to the dietary modification tested in this study. Therefore, gluteal adipose tissue may not be a useful surrogate to study the effect of diet on breast adipose tissue.

Does MIPO of fractures of the distal femur result in more rotational malalignment than ORIF? A retrospective study.

PURPOSE: Intraoperative control of rotational malalignment poses a big challenge for surgeons when using modern MIPO (minimally invasive plate osteosynthesis) techniques. We hypothesized that distal femoral fractures treated with MIPO technique are more often fixed in malrotation than those treated with open reduction internal fixation (ORIF). METHODS: In this retrospective study, we identified 20 patients who met the inclusion criteria and agreed to take part in the study. In ten patients MIPO was applied, in the other ten ORIF was used. Mean age was 44.8 (19-71 years). Functional status was assessed using clinical scores (Harris Hip Score, WOMAC Hip, KS Score, WOMAC Knee, Kujala Score). Rotational alignment was assessed with magnetic resonance imaging and compared to the opposite leg. RESULTS: We discovered a significant difference in the mean rotational difference between the MIPO group (14.3°) and the ORIF group (5.2°). Functionally, patients in the ORIF group outperformed patients in the MIPO group in all clinical scoring systems although no one proved to be statistically significant. MIPO technique was associated with significantly more rotational malalignment compared to ORIF in distal femur fracture fixation. However, implant failure and nonunion was more common in the ORIF group, with a revision rate of 3 versus 1 in the ORIF group. Clinical scoring did not significantly different between both groups. CONCLUSION: Taking into account the undisputable advantages of minimally invasive surgery, improved teaching of methods to avoid malrotation as well as regular postoperative investigations to detect any malrotation should be advocated.

A randomized study of melphalan 200 mg/m(2) vs 280 mg/m(2) as a preparative regimen for patients with multiple myeloma undergoing auto-SCT.

We aimed to examine whether doses of melphalan higher than 200 mg/m(2) improve response rates when used as conditioning before autologous transplant (ASCT) in multiple myeloma (MM) patients. Patients with MM, n=131, were randomized to 200 mg/m(2) (mel200) vs 280 mg/m(2) (mel280) using amifostine pretreatment. The primary end point was the proportion of patients achieving near complete response (⩾nCR). No treatment-related deaths occurred in this study. Responses following ASCT were for mel200 vs mel280, respectively, ⩾nCR 22 vs 39%, P=0.03, ⩾PR 57 vs 74%, P=0.04. The hazard of mortality was not statistically significantly different between groups (mel200 vs mel280; hazard ratio (HR)=1.15 (95% confidence interval (CI), 0.62-2.13, P=0.66)) nor was the rate of progression/mortality (HR=0.81 (0.52-1.27, P=0.36)). The estimated PFS at 1 and 3 years were 83 and 46%, respectively, for mel200 and 78 and 54%, respectively, for mel280. Amifostine and mel280 were well tolerated, with no grade 4 regimen-related toxicities and only one grade 3 mucositis (none with mel200) and three grade 3 gastrointestinal (GI) toxicities (two in mel200). Hospitalization rates were more frequent in the mel280 group (59 vs 43%, P=0.08). Mel280 resulted in a higher major response rate (CR+nCR) and should be evaluated in larger studies.

Comprehensive mutational analysis of primary and relapse acute promyelocytic leukemia.

Acute promyelocytic leukemia (APL) is a subtype of myeloid leukemia characterized by differentiation block at the promyelocyte stage. Besides the presence of chromosomal rearrangement t(15;17), leading to the formation of PML-RARA (promyelocytic leukemia-retinoic acid receptor alpha) fusion, other genetic alterations have also been implicated in APL. Here, we performed comprehensive mutational analysis of primary and relapse APL to identify somatic alterations, which cooperate with PML-RARA in the pathogenesis of APL. We explored the mutational landscape using whole-exome (n=12) and subsequent targeted sequencing of 398 genes in 153 primary and 69 relapse APL. Both primary and relapse APL harbored an average of eight non-silent somatic mutations per exome. We observed recurrent alterations of FLT3, WT1, NRAS and KRAS in the newly diagnosed APL, whereas mutations in other genes commonly mutated in myeloid leukemia were rarely detected. The molecular signature of APL relapse was characterized by emergence of frequent mutations in PML and RARA genes. Our sequencing data also demonstrates incidence of loss-of-function mutations in previously unidentified genes, ARID1B and ARID1A, both of which encode for key components of the SWI/SNF complex. We show that knockdown of ARID1B in APL cell line, NB4, results in large-scale activation of gene expression and reduced in vitro differentiation potential.

Transplantation of autologous peripheral blood progenitor cells procured after high-dose cytarabine-based consolidation chemotherapy for adults with acute myelogenous leukemia in first remission.

The purpose of the study was to evaluate the feasibility and efficacy of high-dose cytarabine-anthracycline consolidation chemotherapy followed by autologous transplantation of chemotherapy/rHuG-CSF-mobilized peripheral blood progenitor cells for adult patients with acute myelogenous leukemia in first remission. Fifty-nine consecutive patients (median age 45, range 18-69) with acute myelogenous leukemia in first remission were enrolled on a study of high-dose cytarabine-mitoxantrone consolidation chemotherapy used as a method of in vivo purging for the purpose of autologous peripheral blood progenitor cell transplantation. A median of 7 x 10(8) peripheral blood mononuclear cells/kg were infused 1 day after preparative conditioning with 11.25 Gy total body irradiation and cyclophosphamide (120 mg/kg). Forty-six patients received myeloablative chemo-radiotherapy followed by the infusion of chemotherapy/rHu-G-CSF-mobilized autologous peripheral blood progenitor cells. The median time to both neutrophil and platelet recovery from transplant was 15 days (range, 11-36 and 5-253+ days, respectively). After a median follow-up of 27 months, 31 patients remain alive with 27 in complete remission. Median remission duration for all eligible patients is 12 months, and actuarial leukemia-free survival at 3 years is 42 +/- 14%. The actuarial risk of relapse is 54 +/- 15%. Toxicity of autologous peripheral blood progenitor cell transplant included treatment-related death in two patients and grade III/IV organ toxicity in six. Advanced age was a negative prognostic factor for leukemia-free survival. Our results demonstrate that autologous transplantation of chemotherapy-mobilized peripheral blood progenitor cells is feasible in an unselected population of adult patients with acute myelogenous leukemia in first remission producing improved leukemia-free survival with minimal toxicity.

In silico prediction of receptor-mediated environmental toxic phenomena-application to endocrine disruption.

It is an objective of our institution to establish a virtual laboratory allowing for a reliable in silico estimation of the harmful effects triggered by drugs, chemicals and their metabolites. In the recent past, we have developed the underlying technology (Multi-dimensional QSAR: Quasar and Raptor) and compiled a pilot system including the 3D models of three receptors known to mediate endocrine-disrupting effects (the aryl hydrocarbon receptor, the estrogen receptor and the androgen receptor, respectively) and validated them against 310 compounds (drugs, chemicals, toxins). Within this set up we could demonstrate that our concepts are able to both recognize toxic compounds substantially different from those used in the training set as well as to classify harmless compounds clearly as being non-toxic. This suggests that our approach can be used for the prediction of adverse effects of drug molecules and chemicals.

Treatment of hairy-cell leukemia.

Hairy-cell leukemia is an unusual chronic lymphoid leukemia with distinctive clinical and pathological features. The management of this disorder has been revolutionized in the last decade with the discovery of the efficacy of alpha interferon and the inhibitors of adenosine metabolism, deoxycoformycin and chlorodeoxyadenosine. The best treatment protocol for hairy-cell leukemia has not yet been defined. Patients may still die from their disease, particularly in the early phases of treatment. Conversely, some patients appear not to require treatment and others respond well to splenectomy and need no further therapy. An individualized clinical approach is recommended, with a role for splenectomy in the patient with cytopenia and a relatively low number of hairy cells in the bone marrow. The first line drug treatment remains interferon alpha given for 12-18 months, following which the patient is observed for clinical relapse. Deoxycoformycin remains a useful experimental agent but cannot be recommended for routine clinical use until issues of long term toxicity are resolved. Chlorodeoxyadenosine is a very promising experimental drug, but confirmation of the early data in larger group trials is required. Similarly the adjunctive use of granulocyte colony stimulating factor appears useful, but will need further study in larger groups of patients. There is little or no role for alkylating agents or more intensive chemotherapy in the modern management of hairy-cell leukemia.

The biology of the cytokine sequence cascade.

Pretransplant and posttransplant use of hematopoietic growth factors in bone marrow transplantation (BMT) has shortened the time to engraftment. Severe neutropenia and thrombocytopenia have been the major clinical problems associated with autologous BMT. Efforts to maintain posttransplant levels of circulating neutrophils have focused on exploiting the synergistic action between various hematopoietic growth factor families. Ex vivo generation of distinct populations of expanded cells through simultaneous and sequential addition of hematopoietic growth factors was attempted. Cultures of CD34-selected cells with combinations of growth factors consisting of either recombinant human stem cell factor (rhSCF), recombinant human interleukin-6 (rhIL-6), and recombinant human interleukin-3 (rhIL-3) or rhSCF, rhIL-3, and recombinant human granulocyte-colony stimulating factor (rhG-CSF) generated two distinct but overlapping populations of cells. Delayed addition (on day 7) of rhIL-3 and rhIL-6 to cells cultured with rhSCF generated a population of cells significantly less mature than those cultured with continuous rhSCF, rhIL-3, and rhIL-6 alone. It appears that optimal generation of immediate and delayed cell populations can be achieved by simultaneous culture with rhSCF, rhIL-3, and rhG-CSF; and with rhSCF, rhIL-6, and rhIL-3. Questions remain regarding the cell populations most effective for generating and sustaining the required neutrophil numbers.

Pulmonary toxicity of high-dose chemotherapy for breast cancer: a non-invasive approach to diagnosis and treatment.

Drug-induced pulmonary toxicity is one of the most frequent non-hematologic toxicities in breast cancer patients receiving high-dose chemotherapy with cyclophosphamide, cisplatin and BCNU (CY/CDDP/BCNU). A non-invasive clinical scoring system was utilized in an attempt to diagnose and treat early lung toxicity in 64 consecutive breast cancer patients undergoing CY/CDDP/BCNU supported by peripheral blood progenitor cells. Following hospital discharge, patients who developed symptoms suggestive of lung toxicity were evaluated with physical examination, DLCO, 2-min walking oximetry and a chest radiograph. Clinically weighted scores were assigned as follows: crackles on lung exam, 2; decrease in corrected DLCO by > 10% from baseline, 3; decrease in O2 saturation by > or = 4% with a 2-min walk, 3; and interstitial infiltrates on chest radiograph, 3. Patients with scores > or = 6 were treated with prednisone (60 mg p.o. twice a day followed by a 2-month taper). Treatment was instituted in 37 patients (58%) a median of 56 days after high-dose chemotherapy. Steroid therapy was associated with rapid clinical improvement in most patients. No fatal complications or chronic pulmonary fibrosis was seen. This non-invasive clinical scoring system can be utilized as a model for the early diagnosis of lung toxicity. Further investigation is warranted for the development of preventative measures against this syndrome.

Allogeneic bone marrow transplantation for Hodgkin's and non-Hodgkin's lymphoma.

We studied 23 consecutive patients, median age 34 years, with relapsed or resistant aggressive lymphoma who underwent allogeneic BMT at the UCLA Medical Center Bone Marrow Transplantation Unit from 1 November 1984 to 30 March 1993. All patients were < 50 years of age and had sibling donors who were matched at the HLA-A, B and DR loci. Nine patients had Hodgkin's disease (HD) and 14 had non-Hodgkin's lymphoma (NHL); three of these had low grade histology and 11 had intermediate or high grade lymphoma histology. After a median follow-up of 34 months, eight patients are alive, seven without recurrent lymphoma. Five patients had early deaths. The disease-free survival for the entire group is 26% with an overall survival of 29%. There was no difference in survival rate on the basis of disease or histology. Comparing preparative regimens containing TBI to those without there was no difference in survival rate (P = 0.35). Neither age nor sex was a significant determinant of outcome (P = 0.63 and 0.36, respectively). Disease status at the time of transplantation proved to be the important determinant of outcome. Patients transplanted with chemotherapy sensitive disease (n = 9), defined as a partial or complete response to salvage chemotherapy, had a survival rate of 42%, which was significantly better than those who had refractory disease at transplantation (n = 14), who had a survival rate of 21% (P = 0.006). However, this small, but significant fraction of patients with refractory disease was curable. Thus, our data demonstrate that allogeneic bone marrow transplantation is an effective means of treatment for relapsed or aggressive Hodgkin's and non-Hodgkin's lymphoma.

Use of PEG-rHuMGDF in platelet engraftment after autologous stem cell transplantation.

This paper summarizes a pilot, sequential dose-escalation study of PEG-rHuMGDF in patients with advanced malignancies who had delayed platelet recovery after autologous stem cell transplantation (ASCT). Patients were randomized to receive either placebo (n = 11) or PEG-rHuMGDF at 5 (n = 9), 10 (n = 6), or 25 (n = 7) microg/kg/day by subcutaneous injection for 14 days and were monitored for 5 weeks. Across all treatment groups, eight patients had platelet recovery to > or = 20 x 10(9)/l by day 21. The proportion of patients achieving platelet recovery, the median number of days and units of platelet transfusions were similar for the placebo and the PEG-rHuMGDF groups. PEG-rHuMGDF was well tolerated at all dosages. The incidence rates of adverse events in all groups were similar. No deaths on study, no drug-related serious adverse events, and no development of neutralizing antibodies to MGDF occurred.

Autologous CD34-selected blood progenitor cell transplants for patients with advanced multiple myeloma.

Fifty-five patients with advanced multiple myeloma received purified CD34-selected peripheral blood progenitor cell transplants following myeloablative chemotherapy. A median of 4.1 x 10(6) CD34 cells/kg (range 1.2-30.7) were infused after busulfan (14 mg/kg) and cyclophosphamide (120 mg/kg); granulocyte-macrophage colony-stimulating factor was used until hematopoietic recovery. Median time to neutrophils >0.5 x 10(9)/l and platelets >20 x 10(9)/l were 12 days (range 10-16 and 8-184 days, respectively). Median follow-up of survivors from the time of transplantation is 33 months (range 7 to 44 months). Thirty-one patients are alive, 19 progression-free. Median progression-free survival is 14 months. Actuarial 3-year progression-free and overall survival are 29+/-14% and 47+/-17%. CD34-selection of peripheral blood progenitor cells provides effective hematopoietic support with significant progression-free and overall survival.

What has happened in norway after the ban of avoparcin? Consumption of antimicrobials by poultry.

When avoparcin was prohibited for use as feed additive in poultry in Norway on 31 May 1995, an increased incidence of Clostridium perfringens-associated necrotic enteritis (NE) and an increase in the use of antibacterial (AB) drug therapy in meat-type poultry was expected. The consumption of AB drugs for use against NE in poultry in the period 1990-2001 was investigated by use of sales statistics at the drug-wholesaler level. Defined daily dose (DDD) per kg live weight poultry was the unit of measurement for drug use (to correct for differences in the dosages). Sales figures of the AB drugs were converted to number of DDDpoultry sold for the numbers of broilers at risk (broilers were 97% of the slaughter poultry). Estimated annual percentages of the broilers treated against NE increased abruptly after the avoparcin ban--but in 1996, this figure declined to the same level as before the ban and has remained at that low level since then. In November 1995, narasin was approved temporarily as an ionophore feed additive (IFA) in broilers. The usage patterns of IFAs in broilers were measured as the weight of feed to which an IFA was added per broiler chicken produced. In 1996-2001, the IFAs used in broilers were predominantly narasin. We note that the temporary increase in NE after the avoparcin ban coincide with the period before narasin became available. The increase in the consumption of AB drugs for the treatment of NE in poultry following the avoparcin ban has been negligible.

Similar outcomes using myeloablative vs reduced-intensity allogeneic transplant preparative regimens for AML or MDS.

Although reduced-intensity conditioning (RIC) and non-myeloablative (NMA)-conditioning regimens have been used for over a decade, their relative efficacy vs myeloablative (MA) approaches to allogeneic hematopoietic cell transplantation in patients with AML and myelodysplasia (MDS) is unknown. We compared disease status, donor, graft and recipient characteristics with outcomes of 3731 MA with 1448 RIC/NMA procedures performed at 217 centers between 1997 and 2004. The 5-year univariate probabilities and multivariate relative risk outcomes of relapse, TRM, disease-free survival (DFS) and OS are reported. Adjusted OS at 5 years was 34, 33 and 26% for MA, RIC and NMA transplants, respectively. NMA conditioning resulted in inferior DFS and OS, but there was no difference in DFS and OS between RIC and MA regimens. Late TRM negates early decreases in toxicity with RIC and NMA regimens. Our data suggest that higher regimen intensity may contribute to optimal survival in patients with AML/MDS, suggesting roles for both regimen intensity and graft vs leukemia in these diseases. Prospective studies comparing regimens are needed to confirm this finding and determine the optimal approach to patients who are eligible for either MA or RIC/NMA conditioning.