1-year results from a prospective randomized trial comparing phlebotomy with deferasirox for the treatment of iron overload in pediatric patients with thalassemia major following curative stem cell transplantation

Background: As a result of previous transfusions, \u3b2-thalassemia major (TM) patients who have undergone curative hematopoietic stem cell transplantation (HSCT) are at increased risk of iron overload. There are, however, limited data on iron removal in such patients with either phlebotomy (PHL) or iron chelation. The aim of this study was to compare the efficacy, safety and convenience of the oral iron chelator deferasirox (DFX; Exjade\uae) with PHL for the treatment of iron overload in children with TM following HSCT, over a 1-year period. Methods: LB03T is a prospective, randomized trial enrolling children with TM aged 2-3 mg Fe/g dry weight [dw]). Eligible patients were randomized to PHL (6 mL/kg blood/2 weeks) or DFX (10 mg/kg/day starting dose; 5 mg adjustments up to 20 mg/kg/day were permitted). One of the primary endpoints was change in LIC assessed using magnetic resonance imaging techniques. Changes in serum ferritin levels, hemoglobin (Hb), total iron binding capacity (TIBC), non-transferrin-bound iron (NTBI), adverse events (AEs) and compliance with study treatment (PHL: ratio of performed:planned; DFX: tablet count) were also assessed. Convenience of treatment was evaluated using parents' responses to pre-prepared questions. Results: 27 patients were randomized to DFX or PHL; one patient randomized to PHL refused treatment, hence 12 patients received DFX and 14 received PHL. Mean age was 12.4 yrs and 53.8% were male. Patients were followed up for a mean of 12.0 months. 2 and 5 patients had DFX dose increases to 15 and 20 mg/kg/day, respectively. Mean DFX dose at last visit was 11.0 and 18.1 ng/mL in the LIC7 and LIC>7 groups, respectively. Median serum ferritin was significantly reduced from baseline over 12 months with DFX (\u2013497.5 ng/mL, P=0.004 vs baseline) and PHL (\u2013901.8 ng/mL, P<0.0001 vs baseline); there was no significant difference between groups (P=0.425). Mean LIC (for 20 patients with evaluable LIC following 1 yr of treatment) was significantly reduced with DFX (\u20135.78 mg Fe/g dw, P=0.0005 vs baseline) and PHL (\u20133.27 mg Fe/g dw, P=0.050 vs baseline); no significant difference between groups (P=0.270). In patients with serum ferritin levels 1000 ng/mL at baseline, DFX resulted in a significantly greater decrease in mean LIC than PHL (\u20138.1 vs \u20133.5 mg Fe/g dw, P=0.048; Table). For TIBC, the increase with DFX was significantly greater than with PHL (P=0.0003). NTBI decreased significantly from baseline by \u20131.91 \u3bcmol/L (P=0.014; n=9) with DFX (baseline 2.0 \u3bcmol/L) and \u20132.83 \u3bcmol/L (P=0.0015; n=11) with PHL (baseline 2.3 \u3bcmol/L); there was no significant difference between groups (P=0.362). NTBI and LIC were positively correlated (R=0.565; P=0.0026) at baseline and at last follow-up (R=0.881; P<0.0001). Baseline mean Hb was 12.5 and 12.6 g/dL in the DFX and PHL groups, respectively; levels were maintained with DFX (change \u20130.17 g/dL; P=0.426) (PHL: change \u20130.53 g/dL; P=0.033); no significant difference between groups (P=0.279). AEs reported for patients receiving DFX were skin rash [n=1], gastrointestinal upset [n=1], increased liver function tests [n=1]; for patients receiving PHL, difficulty with venous access [n=4] and distress during procedure [n=1] were reported. Compliance was excellent for 11 (91.7%) and 12 (85.7%); good for 1 (8.3%) and 1 (7.1%); and poor with 0 and 1 (7.1%) patients receiving DFX or PHL, respectively. Parents of 13/14 children randomized to PHL desired their children to receive DFX due to pain, risk of anemia and longer/more frequent hospital visits associated with PHL. Parents of 1/14 children were satisfied with PHL due to concerns over possible AEs with DFX. Conclusions: In pediatric post-HSCT patients with TM, both LIC and serum ferritin were reduced with DFX and PHL over 1 year. In patients with higher baseline iron burden, DFX decreased LIC to a greater extent than PHL. TIBC was also significantly increased with DFX compared with PHL. DFX dose adjustments from 10 to 20 mg/kg/day were required to achieve therapeutic goals in some patients, underscoring the need for appropriate and timely dose adjustments. DFX had a clinically manageable safety profile, compliance was high and the majority of parents with children receiving PHL stated a desire to switch to DF

TPP1 mutagenesis screens unravel shelterin interfaces and functions in hematopoiesis

Telomerase catalyzes chromosome end replication in stem cells and other long-lived cells. Mutations in telomerase or telomere-related genes result in diseases known as telomeropathies. Telomerase is recruited to chromosome ends by the ACD/TPP1 protein (TPP1 hereafter), a component of the shelterin complex that protects chromosome ends from unwanted end joining. TPP1 facilitates end protection by binding shelterin proteins POT1 and TIN2. TPP1 variants have been associated with telomeropathies but remain poorly characterized in vivo. Disease variants and mutagenesis scans provide efficient avenues to interrogate the distinct physiological roles of TPP1. Here, we conduct mutagenesis in the TIN2- and POT1-binding domains of TPP1 to discover mutations that dissect TPP1’s functions. Our results extend current structural data to reveal that the TPP1-TIN2 interface is more extensive than previously thought and highlight the robustness of the POT1-TPP1 interface. Introduction of separation-of-function mutants alongside known TPP1 telomeropathy mutations in mouse hematopoietic stem cells (mHSCs) lacking endogenous TPP1 demonstrated a clear phenotypic demarcation. TIN2- and POT1-binding mutants were unable to rescue mHSC failure resulting from end deprotection. In contrast, TPP1 telomeropathy mutations sustained mHSC viability, consistent with their selectively impacting end replication. These results highlight the power of scanning mutagenesis in revealing structural interfaces and dissecting multifunctional genes

Comparison of CBCs.

<p>Platelet counts are lower in <i>Nbeal2</i>^{<i>gps/gps</i>} mice compared to control mice in both set 1 (A) and set 2 (B) mice while hemoglobin levels are similar between the two groups (C). <i>Nbeal2</i>^{<i>gps/gps</i>} mice from set 1 exhibit significant neutropenia (D), which is not observed in set 2 by CBC (E) or flow cytometry (F). Mean platelet volume (G) and area (H) do not differ in set 1 mice, but show an increase in size for <i>Nbeal2</i>^{<i>gps/gps</i>} mice in set 2 (I).</p

Deficiency in platelet alpha granules.

<p><i>Nbeal2</i>^{<i>gps/gps</i>} platelets appear pale compared to wildtype (black arrows, A). Transmission electron microscopy (TEM) images show dark alpha granules in wildtype platelets (black arrows), which are missing in <i>Nbeal2</i>^{<i>gps/gps</i>} platelets. Red arrows indicate mitochondria (B).</p

<i>De novo</i> 8 bp deletion in the <i>Nbeal2</i> gene.

<p>The whole exome sequenced G6-ENU mouse inherited the <i>Nbeal2</i> deletion from a non-ENU parent 31925 (A). Sanger sequencing validates the heterozygous frameshift mutation in the suppressor pedigree (B). Western blot analysis of washed mouse platelets show a band at the expected size for NBEAL2 (~305kDa) in wildtype mice. This band is missing in <i>Nbeal2</i>^{<i>tm1Lex/tm1Lex</i>} mice as well as mice homozygous for the <i>Nbeal2</i>^<i>gps</i> allele (C). Schematic overview of the <i>Nbeal2</i> gene, the location of the deletion and the expected frameshift (D).</p

Expected and observed number of progeny in <i>Nbeal2</i>^<i>gps/+</i> crosses.

<p>Expected and observed number of progeny in <i>Nbeal2</i>^<i>gps/+</i> crosses.</p

Intensity of platelet staining and frequency of emperipolesis events in bone marrow megakaryocytes.

<p>Intensity of platelet staining and frequency of emperipolesis events in bone marrow megakaryocytes.</p

Emperipolesis of neutrophils in bone marrow and spleen of <i>NBEAL2</i> deficient mice.

<p>Increased emperiopolesis of neutrophils (black arrows) in <i>Nbeal2</i>^{<i>gps/gps</i>} mice compared to wildtype was observed in both histologic (A) and cytologic (B) preparations of bone marrow as well as spleen (B and D, respectively).</p