ALBERT

Beschreibung: Key Points Application of capturing/sequencing, copy number, and RNA analysis technologies ensures comprehensive molecular diagnosis of Fanconi anemia.

Beschreibung: We investigated whether interleukin-1β (IL-1β) is differentially expressed in plasma cells from monoclonal gammopathy of undetermined significance (MGUS) and multiple myeloma (MM) patients because IL-1β appears to play a major role in the development of lytic bone lesions, the major clinical feature distinguishing MGUS from myeloma. In situ hybridization (ISH) for IL-1β was performed using bone marrow aspirates from 51 MM, 7 smoldering MM, 21 MGUS, and 5 normal control samples. Using the ISH technique IL-1β mRNA was detectable in the plasma cells from 49 of 51 patients with active myeloma and 7 of 7 patients with smoldering myeloma. In contrast, 5 of 21 patients with MGUS and 0 of 5 normal controls had detectable IL-1β message. Bone lesions were present in 40 of the 51 MM patients analyzed, and all 40 patients had IL-1β mRNA by ISH. These results show that greater than 95% of MM patients but less than 25% of MGUS patients are positive for IL-1β production. In the future, continued follow-up of IL-1β positive and negative MGUS patients should determine whether aberrant expression of plasma cell IL-1β is predictive of those MGUS patients that will eventually progress to active myeloma.

Beschreibung: Background: Deferitrin (GT56-252) is an orally active, tridentate, iron chelator under development to treat chronic iron overload conditions resulting from transfusion therapy. The compound is derived from desferrithiocin specifically modified to minimize toxicity associated with the parent compound (Bergeron et al., 1999). Deferitrin effects iron excretion with an efficiency of 13–18% when administered orally to Cebus apella monkeys, a species whose iron metabolism closely resembles that of humans. Iron excretion is predominantly by the fecal route (80–90%). Methods: This Phase 1 trial was an open-label, parallel-design, two-dose study. The study consisted of a screening period, a confinement period during which a single dose of deferitrin was administered on two consecutive days with pharmacokinetic measurements, and a follow-up visit. Twenty-six patients, divided in five panels, received at least one dose of deferitrin over two days, either fasted or fed, at five dose levels from 3 to 15 mg/kg. Deferitrin was administered as liquid (3 and 4.5 mg/kg), or as 50 or 250 mg capsules (4.5, 8, 11, and 15 mg/kg). Blood samples were taken at intervals up to 24 hours to characterize the pharmacokinetics of deferitrin, and 24-hour urine collections were conducted to measure deferitrin excretion. Safety laboratories included chemistry and hematology panels, coagulation parameters, urinalysis, and urine beta-2-microglobulin. Additional safety evaluations included physical examinations and electrocardiogrammes (ECG). Results: Deferitrin was well tolerated. There were no serious adverse events while patients were confined; however, one patient with pre-existing diabetes who received 4.5 mg/kg was hospitalized after an episode of hypoglycemic coma three days after the second dose. This had been a recurring phenomenon in this patient, and was determined by the investigator to be unrelated to administration of deferitrin. There were no clinically significant changes in safety laboratories, including chemistry, hematology, and coagulation studies, in urinalyses or urinary beta-2-microglobulin levels. ECGs performed before and after dosing were unremarkable. The AUC for the fed and fasted states were similar, both for the liquid and capsule, suggesting that bioavailability is not impaired by food. From 13 to 48% of the drug was present in serum as the 2:1 deferitrin: iron complex. The T½ (approximately 2 to 4 hours) was similar for all doses. Preliminary measurement of deferitrin levels in the urine showed recovery of approximately 75% at all doses. Approximately 2% of the deferitrin in the urine was complexed to iron. Conclusion: In this initial clinical study, deferitrin was safe, well-tolerated, and well-absorbed either with food and fasting in beta-thalassemia patients. Renal excretion of deferitrin uncomplexed with iron was similar to that seen in preclinical studies, which predict iron excretion to be via the fecal route. Further studies are ongoing to define the effect of deferitrin on iron balance.

Beschreibung: Abstract 501 Introduction & Purpose: Rivaroxaban is an oral Factor Xa inhibitor which has been licensed in Canada since 2008 and the United States since 2011 for the prevention of thromboembolic events following total hip and total knee arthroplasties. Multicentre research trials have shown clinical efficacy for prevention of deep venous thrombosis (DVT) and pulmonary embolism (PE). The aim of this study was to prospectively document the incidence and timing of thromboembolic and bleeding events in patients who received rivaroxaban as the primary prophylaxis in clinical practice. Methods: Prospective, observational study of patients given oral Factor Xa inhibitor (rivaroxaban) following primary and revision Total Hip Arthroplasty (THA) and Total Knee Arthroplasty (TKA). All patients were approached to participate and consent obtained. Patients treated with Rivaroxaban 10 mg po daily starting Post-Operative Day (POD) #1 and continued for 15 days. This protocol was approved for use at this institution and is NOT consistent with manufacturer's recommended dosing. All participants were followed up at 6 weeks and 3 months. Doppler ultrasound or venograms used to diagnose symptomatic proximal DVT at or above the popliteal vein. Spiral CT angiogram, angiogram or ventilation/perfusion V/Q scan were used to diagnose PE. All Doppler Ultrasound reports were reviewed by a radiologist to confirm findings and all spiral CT, CT angiogram or ventilation/perfusion scans or images were reviewed (where possible) to verify findings. Bleeding complications were documented as ‘on prophylaxis’ starting 2 hours after first dose of anticoagulant therapy until 24 hours after the 15thdose. All major and non-major bleeding events were reviewed by an internist to confirm severity of bleeding episode. Event rates are reported. Data reported on consented patients only. Research ethics approval was obtained for this study. Results: From June 2010 to Dec 2011, 2888 patients underwent total joint arthroplasty. Two thousand five hundred and thirty-five (88%) agreed to participate in the study. One hundred and fifty patients were treated with thromboprophylaxis other than rivaroxaban. Two thousand three hundred and forty-two were followed up at 3 months (98%). Forty-three patients were lost to follow-up. Complete data on 2342 patients is reported: 905 men, 1437 women with mean age 66 years. Total knees 1353 (primary 1229, revision 123, uni 1). Total hips 989 (primary 899, revision 90). DVT: Three DVT were reported at 6 weeks. Nine additional DVT's were reported at 3 months: 5 primary TKA and 6 Primary THAs 1 Rev THA. Total DVT= 12/2342= 0.5% (see Fig 1). PE: There were 7 confirmed PE during the first week post op: 6 primary TKA and 1 primary THA. Three additional PE's 6 weeks: 3 THA and 1 TKA. Five additional PE's reported at 3 months:4 THA and 1 TKA. Total PE 16/2342=0.7% (See Fig 2). Death: There have been 4 perioperative deaths. None were related to surgery, DVT, pulmonary embolism or bleeding. Bleeding: No major and 9 non-major surgical-site bleeds occurred. All but one were in primary THA. One major and 6 non-major non-surgical site bleeds occurred in patients who received rivaroxaban. Two additional major Non-surgical site bleeds occurred after having received rivaroxaban: one patient received only one dose of rivaroxaban and the other occurred 3–4 days after completion of treatment. Overall Major bleeds 0.04% and Non-Major bleeds 15/2342=0.6%. Transfusion: One hundred and fourteen patients (5%) received blood transfusions. Transfusion rates by procedure: unilateral THA 4%, Bilateral THA 40%, RTHA 27, unilateral PTKA 3%, bilateral TKA 16%,unicompartmental knee 0% and revision TKA 6%. No routine blood salvage or drains used for primary arthroplasties. Conclusions: The incidence of thromboembolic events within a period of 3 months was 12/2342=0.6% for DVT and 16/2342=0.7% for PE. The incidence of major bleeding was 0.04%. There were no deaths related to DVT, PE or bleeding. Preliminary results are surprising for the number of pulmonary emboli which occurred while patients were still in hospital and for the number of DVT's which occurred between 6 weeks and 3 months. The early PE's tended to occur in primary TKA. The late events tended to occur in primary THA and TKA. Disclosures: Murnaghan: Bayer Healthcare: Honoraria, Research Funding. Off Label Use: rivaroxaban is used for thromboprophylaxis after total joint replacement. our protocol gives first dose on day after surgery. Gollish:Bayer Healthcare: Honoraria, Research Funding.

Beschreibung: Tumor-related immunoglobulin heavy-chain (IgH) rearrangements are markers for polymerase chain reaction (PCR) detection of minimal residual disease (MRD) in B-cell malignancies. Nested PCR with patient IgH allele-specific oligonucleotide primers can detect 1 tumor cell in 104 to 106 normal cells. In childhood acute lymphoblastic leukemia (ALL), persistence of PCR-detectable disease is associated with increased risk of relapse. The clinical significance of qualitative PCR data can be limited, however, because patients can harbor detectable MRD for prolonged periods without relapse. Recent studies indicate that a quantitative rise in tumor burden identifies patients who are at high risk for relapse. Therefore, an efficient and reliable PCR method for MRD quantification is needed for ALL patients. We have developed a real-time PCR method to quantify MRD with IgH VH gene family consensus fluorogenically labeled probes. With this method, a small number of probes can be used to quantify MRD in a large number of different patients. The assay was found to be both accurate and reproducible over a wide range and capable of detecting approximately 1 tumor cell in 5 × 104 normal cells. We demonstrate that this methodology can discriminate between patients with persistence of MRD who relapse and those who do not. This technique is generally applicable to B-cell malignancies and is currently being used to quantify MRD in a number of prospective clinical studies at our institution.

Beschreibung: Human bone marrow (BM) cells contain low levels of the DNA repair protein, O6-alkylguanine-DNA alkyltransferase, which may explain their susceptibility to nitrosourea-induced cytotoxicity and the development of secondary leukemia after nitrosourea treatment. Isolated CD34+ myeloid progenitors were also found to have low levels of alkyltransferase activity. The level of alkyltransferase in CD34+ cells or in mononuclear BM cells did not increase after incubation with granulocyte-macrophage colony-stimulating factor, interleukin-3, stem cell factor, the combination, or 5637 conditioned medium. BCNU sensitivity remained unchanged as well. In addition, O6-benzylguanine depleted alkyltransferase activity in BM cells at concentrations as low as 1.5 mumol/L after a 1-hour exposure. O6-benzylguanine pretreatment markedly sensitized hematopoietic progenitor colony-forming cells to BCNU, resulting in a reduction in the dose of drug (termed the dose- modification factor) required to inhibit 50% of the colony formation (IC50) of threefold to fivefold. Since, unlike many other cell types, proliferating early (CD34+) hematopoietic precursors do not induce alkyltransferase, myelosuppression may be the dose-limiting toxicity of the combination of O6-benzylguanine plus BCNU in clinical trials.

Beschreibung: Stem cell factor (SCF) is expressed as an integral membrane growth factor that may be differentially processed to produce predominantly soluble (S) (SCF248) or membrane-associated (MA) (SCF220) protein. A critical role for membrane presentation of SCF in the hematopoietic microenvironment (HM) has been suggested from the phenotype of the Steel-dickie(Sld) mice, which lack MA SCF, and by studies performed in our laboratory (and by others) using long-term bone marrow cultures and transgenic mice expressing different SCF isoforms.Steel17H (Sl17H) is an SCF mutant that demonstrates melanocyte defects and sterility in males but not in females. The Sl17H allele contains a intronic mutation resulting in the substitution of 36 amino acids (aa’s) in the SCF cytoplasmic domain with 28 novel aa’s. This mutation, which affects virtually the entire cytoplasmic domain of SCF, could be expected to alter membrane SCF presentation. To investigate this possibility, we examined the biochemical and biologic properties of the Sl17H-encoded protein and its impact in vivo and in vitro on hematopoiesis and on c-Kit signaling. We demonstrate that compound heterozygous Sl/Sl17H mice manifest multiple hematopoietic abnormalities in vivo, including red blood cell deficiency, bone marrow hypoplasia, and defective thymopoiesis. In vitro, both S and MA Sl17H isoforms of SCF exhibit reduced cell surface expression on stromal cells and diminished biological activity in comparison to wild-type (wt) SCF isoforms. These alterations in presentation and biological activity are associated with a significant reduction in the proliferation of an SCF-responsive erythroid progenitor cell line and in the activation of phosphatidylinositol 3-Kinase/Akt and mitogen-activated protein-Kinase signaling pathways. In vivo, transgene expression of the membrane-restricted (MR) (SCFX9/D3) SCF in Sl/Sl17H mutants results in a significant improvement in peripheral red blood cell counts in comparison toSl/Sl17H mice.

Beschreibung: Iron accumulation and overload in beta thalassaemia patients are associated with significant morbidity and mortality. Iron chelators are used to manage iron accumulation but side effects and compliance issues restrict the use of available chelators. Deferitrin (Genzyme Corporation) is an orally available iron chelator intended for iron overload. Method: Patients were dosed in 4 cohorts, receiving 5, 10, 15 and 25 mg/kg/day of deferitrin. Deferitrin dosing in cohorts 1–3 was once daily for 12 weeks. Cohort 4 received deferitrin twice daily (BD) for 48 weeks (12.5mg/kg BD, 25 mg/kg/day). Pharmacokinetics (PK) were assessed in a subset of up to 5 patients in each cohort, pre-dose and 1, 2, 4 and 8 hours post dose. All patients had trough levels assessed at weeks 1, 6 and 12 (all Cohorts) and additionally at weeks 24, 36 and 48 for Cohort 4. PK parameters were determined by model independent (non-compartmental) analyses. Safety was assessed by collection of adverse events and laboratory assessments with renal parameters measured weekly due to observations of renal toxicity in preclinical testing. Efficacy (change in liver iron concentration (LIC)) was assessed by SQUID (superconducting quantum interference device) in Turin, Italy, between screening and end of study. Iron excretion and intake were estimated by calculation:Iron excretion due to deferitrin = Iron Intake (mg/kg/day) - TBI (mg/kg/day)Iron Intake (mg/kg/day) = [total mL pRBC (exclude last BT×) × 1.08] / [Weight (kg) × Days (Between 1st & last BT×)]TBI (mg/kg/day) = Change in LIC (mg Fe/g dry weight) × [10.6 (Angelucci Factor) / D (Days on deferitrin)] Key: pRBC = packed red blood cells, BT× = blood transfusion, TBI=Total Body Iron. Results: PK: PK for deferitrin dosed once daily was linear and dose proportional. The serum half-life was 1.3–1.8 hrs, clearance was 226–340 mL/min and mean residence time was 2.8–3.4 hrs for once daily dosing. PK data from BD dosing is not yet available. Safety: Deferitrin dosed once daily was generally well tolerated (Cohorts 1–3). Slight rises in transaminases were seen at 10 and 15 mg/kg/day. A large proportion of enrolled patients were hepatitis C positive (73%). When dosed BD (12.5 mg/kg BD in Cohort 4), 3 patients developed renal toxicity after 4–5 weeks of treatment. Two patients experienced increased proteinuria (max 3.73 g/L & 3.29 g/L) and one patient suffered acute renal failure (peak serum creatinine 4.1 mg/dL, lowest GFR 27 mmol/L). All patients recovered normal renal function after stopping treatment. No patients were re-challenged with deferitrin. Dosing was terminated in all patients because of safety concerns. Efficacy: Mean iron excretion in mg/kg/day (S.D) for Cohort 1 was 0.22 (0.22), Cohort 2 was 0.45 (0.14) and Cohort 3 was 0.33 (0.12). The reasons for the lack of dose proportionality in iron excretion are unclear. Efficacy could not be assessed in Cohort 4 due to early termination of the study. Conclusions: Deferitrin dosed once daily was generally well tolerated and associated with a mean iron excretion of 0.34 mg/kg/day. Deferitrin dosed BD (12.5mg/kg BD) was associated with unacceptable renal toxicity and led to study termination. Deferitrin does not appear to have an acceptable therapeutic margin to allow sufficient iron excretion for long-term administration.

Beschreibung: Follicular lymphomas (FLs) are neoplastic counterparts of normal germinal center (GC) B cells. FLs are characterized by t(14;18) with deregulation of the Bcl-2 (BCL2) gene. The presence of t(14;18) and overexpression of Bcl-2 is necessary, but not sufficient, to cause this disease. An array containing 588 complementary DNAs (cDNAs) was used to compare the gene expression between GC B cells and FL cells. To specifically monitor genes expressed in normal GC B and FL cells and not the entire tissue compartment, normal and malignant B cells were purified from tissues. Using the array, 37 genes were up-regulated and 28 were down-regulated in FL cells as compared to normal GC B cells. The expression level of each differentially expressed gene was verified by quantitative polymerase chain reaction. Following these studies 24 genes were up-regulated and 8 genes down-regulated with a P value less than .1. Included among the genes that were up-regulated in FLs were cell cycle regulator proteins CDK10, p120, p21CIP1, and p16INK4A; transcription factors/regulators Pax-5 and Id-2, which are involved in normal B-cell development; and genes involved in cell-cell interactions, tumor necrosis factor, interleukin-2Rγ (IL-2Rγ), and IL-4Rα. Among the genes that were down-regulated in FLs wereMRP8 and MRP14, which are involved in adhesion. Interestingly, several of these genes are localized within chromosomal regions already described to be altered in FLs. These findings provide a basis for future studies into the pathogenesis and pathophysiology of FL and may lead to the identification of potential therapeutic targets as well as antigens for immunotherapeutic strategies.

Beschreibung: During sickle cell-related pain episode, there is increased inflammation and decreased tissue perfusion as compared to baseline. Endothelin-1 may contribute to vasoconstriction. In addition, cell derived microparticles (C-MP) are membrane particles released during activation or apoptosis of cells such as platelets (PMP), leukocytes (LMP), red cells (RMP), and endothelium (EMP) and are emerging as biomarkers for inflammation and thrombosis. We investigated differences in blood counts, plasma endothelin-1, and cell-derived microparticles (C-MP) during steady state and pain crisis in 10 subjects with sickle cell disease (8 SS, 1 SC, and 1 S-beta thalassemia+; mean age 15.8 years± 4.6, range 6–20). C-MPs were measured by flow cytometry in platelet-poor plasma using fluorescent monoclonal antibodies: LMP by CD45, RMP by anti-glycophorin, PMP by CD31+/CD41+, and EMP by CD62E, or CD51, or CD31+/CD41−. Plasma endothelin-1 was measured by ELISA. White cell counts from hemoglobin SS individuals increased consistently during a vasoocclusive episode from a mean baseline WBC of 8817±2424 to 12,520 ± 4205 (p=0.03) during the pain episode, for a mean change of 4400±2525. There were no significant changes in hemoglobin or platelet count. PMP was normal at baseline, but there was a tendency to have lower levels during pain episodes. On the other hand, red cell microparticles (RMP) in 7 of 10 patients (those with total hemoglobin