Iron Burden Research Articles

Restoring Iron Homeostasis in Pts Who Achieved Transfusion Independence after Treatment with Betibeglogene Autotemcel Gene Therapy: Results from up to 7 Years of Follow-up

Restoring Iron Homeostasis in Pts Who Achieved Transfusion Independence after Treatment with Betibeglogene Autotemcel Gene Therapy: Results from up to 7 Years of Follow-up

Blood Conservation Strategies in Adult Sickle Cell Patients on Chronic Transfusion Therapy: A Single Center Experience

▪Background: Adult sickle cell patients on chronic transfusion therapy (CTT) garnered special concern during the COVID-19 pandemic, when mass cancellation of blood drives threatened the national blood supply. In response, the American Society of Hematology proposed several strategies to decrease blood utilization, while maintaining adequate disease control for sickle cell patients on CTT. These included targeting a higher end hemoglobin S%, switching patients to simple transfusions when appropriate, and transitioning to alternative disease modifying therapies. There is little evidence to support the safety of altering exchange therapy regimens. Accordingly, at the Johns Hopkins Sickle Cell Center for Adults, a multidisciplinary team of clinicians and transfusion medicine specialists evaluated patients on a case by case basis to determine how their exchanges could be modified to accommodate for anticipated blood supply shortages. We describe our blood conservation efforts during the COVID-19 pandemic and resulting clinical outcomes for adult patients with sickle cell anemia (SCA).Methods: For inclusion in this IRB-approved retrospective study, patients received at least 7 monthly exchange transfusions between March 2019 and February 2020 and continued care through March 2021. Decisions regarding CTT were made prior to data collection. Modifications to chronic exchanges included increasing the fraction of cells remaining (FCR), decreasing the end hematocrit, or switching to a hemodilution method. Additionally, select patients transitioned to monthly simple transfusions if they were clinically stable, had a hemoglobin ≤ 7 g/dL, and had a persistently suppressed hemoglobin S% (≤ 30).We collected basic demographics, pre-exchange laboratory studies, and exchange parameters before and after each transfusion encounter in the year before (March 2019 - February 2020) and during (March 2020 - February 2021) the COVID-19 pandemic. Lastly, we recorded presentations to urgent care and the emergency department; and hospital admissions during each time period. We reported descriptive statistics for the cohort and compared outcomes using the Wilcoxon signed-rank test and Mann Whitney U test. We analyzed data using Stata/SE Version 16.1.Results: We identified 58 patients with SCA who qualified for inclusion (Table 1). Fifty-three patients remained on chronic exchange transfusions during the pandemic, and five were switched to simple transfusions. For patients who remained on chronic exchanges, most received conventional automated red cell exchange (RCE) prior to (85%) and during the pandemic (77%). Use of hemodilution increased (15% to 23% of patients). Forty-three patients experienced an increase in mean FCR (33.6 (SD 11.6) vs 37.7 (4.7), p = .00). Of those, 22 patients saw a concomitant decrease in mean end hematocrit (30.8 (1.7) vs 29.6 (1.1), p = .00). These changes resulted in a decline in the average number of units per procedure (8.6 (1.9) vs 7.3 (1.7), p = .00), which corresponded to 890 units conserved. Mean pre-transfusion hemoglobin values declined (9.4 (1.3) vs 9.2 (1.3), p = 0.01), but hemoglobin S%, reticulocyte count, and ferritin values were unchanged (p > 0.05). Acute care presentations and hospital admissions declined, which were likely spurred by concerns about COVID-19 infection (Table 2, p < 0.05).During the pandemic, three patients died, one of whom had been switched to simple transfusions. This patient experienced a consistent rise in hemoglobin S% until death. Two of the remaining patients on simple transfusions were switched back to automated RCE due to an increase in hemoglobin S% above goal (Figure 1).Conclusions: During the COVID-19 pandemic, we conserved red blood cell units through expanded use of hemodilution, higher FCR values, and switching some patients to simple transfusions. Patients who remained on exchanges maintained hemoglobin S% values near a goal of 30% without increasing iron burden. In contrast, the majority of patients who were switched to simple transfusions were unable to maintain goal hematologic parameters, and one patient died. Our data suggest that in a blood shortage crisis, changing the exchange procedure itself may be the safest means of conserving blood in a population of adult sickle cell patients; however longer follow-up is needed to ensure that these changes are safe. [Display omitted] DisclosuresLanzkron: GBT: Research Funding; Shire: Research Funding; Novo Nordisk: Consultancy; CSL Behring: Research Funding; Pfizer: Current holder of individual stocks in a privately-held company; Teva: Current holder of individual stocks in a privately-held company; Novartis: Research Funding; Imara: Research Funding; Bluebird Bio: Consultancy.

A simple clinical score to promote and enhance ferroportin disease screening

Ferroportin disease is a rare genetic iron overload disorder which may be underdiagnosed, with recent data suggesting it occurs at a higher prevalence than suspected. Costs and the lack of defined criteria to prompt genetic testing preclude large-scale molecular screening. Hence, we aimed to develop a readily available scoring system to promote and enhance ferroportin disease screening. Our derivation cohort included probands tested for ferroportin disease from 2008 to 2016 in our rare disease network. Data were prospectively recorded. Univariate and multivariate logistic regression were used to determine significant criteria, and odds ratios were used to build a weighted score. A cut-off value was defined using a ROC curve with a predefined aim of 90% sensitivity. An independent cohort was used for cross validation. Our derivation cohort included 1,306 patients. Mean age was 55±14 years, ferritin 1,351±1,357 μg/L, and liver iron concentration (LIC) 166±77 μmol/g. Pathogenic variants (n= 32) were identified in 71 patients. In multivariate analysis: female sex, younger age, higher ferritin, higher LIC and the absence of hypertension or diabetes were significantly associated with the diagnosis of ferroportin disease (AUROC in whole derivation cohort 0.83 [0.78-0.88]). The weighted score was based on sex, age, the presence of hypertension or diabetes, ferritin level and LIC. An AUROC of 0.83 (0.77-0.88) was obtained in the derivation cohort without missing values. Using 9.5 as a cut-off, sensitivity was 93.6 (91.7-98.3) %, specificity 49.5 (45.5-53.6) %, positive likelihood ratio 1.8 (1.6-2.0) and negative likelihood ratio 0.17 (0.04-0.37). We describe a readily available score with simple criteria and good diagnostic performance that could be used to screen patients for ferroportin disease in routine clinical practice. Increased iron burden associated with metabolic syndrome is a very common condition. Ferroportin disease is a dominant genetic iron overload disorder whose prevalence is higher than initially thought. They can be difficult to distinguish from each other, but the limited availability of genetic testing and the lack of definitive guidelines prevent adequate screening. We herein describe a simple and definitive clinical score to help clinicians decide whether to perform genetic testing.

Iron Overload in Patients With Heavily Transfused Sickle Cell Disease-Correlation of Serum Ferritin With Cardiac T2* MRI (CMRTools), Liver T2* MRI, and R2-MRI (Ferriscan®).

The treatment of sickle cell disease (SCD) is mainly supportive, except for a minority, who receive bone marrow transplantation (BMT). Serum ferritin (SF) is routinely available but is notoriously unreliable as a tool for iron-overload assessment since it is an acute-phase reactant. Although blood transfusion is one of the most effective ways to deal with specific acute and chronic complications of SCD, this strategy is often associated with alloimmunization, iron overload, and hemolytic reactions. This study, thus, aims to evaluate iron overload in patients with SCD on chronic blood transfusions and specifically, correlate SF with the current standard of care of iron-overload assessment using MRI-based imaging techniques. Amongst a historic cohort of 58 chronically transfused patients with SCD, we were able to evaluate 44 patients who are currently alive and had multiple follow-up testing. Their mean age (±SD) was 35 (9) years and comprised of 68.2% of women. The studied iron-overload parameters included cardiac T2* MRI, liver iron concentration (LIC) by Liver T2* MRI, and serial SF levels. Additionally, in a smaller cohort, we also studied LIC by FerriScan© R2-MRI. Chronic blood transfusions were necessary for severe vaso-occlusive crisis (VOC) (38.6%), severe symptomatic anemia (38.6%), past history of stroke (15.9%), and recurrent acute chest syndrome (6.9%). About 14 (24%) patients among the original cohort died following SCD-related complications. Among the patients currently receiving chelation, 26 (96%) are on Deferasirox (DFX) [Jadenu® (24) or Exjade® (2)], with good compliance and tolerance. However, one patient is still receiving IV deferoxamine (DFO), in view of the significantly high systemic iron burden. In this evaluable cohort of 44 patients, the mean SF (±SD) reduced marginally from 4,311 to 4,230 ng/ml, mean Liver T2* MRI dropped from 12 to 10.3 mg/gm dry weight, while the mean cardiac T2*MRI improved from 36.8 to 39.5 ms. There was a mild to moderate correlation between the baseline and final values of SF ng/ml, r = 0.33, p = 0.01; Cardiac T2* MRI ms, r = 0.3, p = 0.02 and Liver T2* MRI mg/kg dry weight, r = 0.6, p < 0.001. Overall, there was a positive correlation between SF and Liver T2* MRI (Pearson's r = 0.78, p < 0.001). Cardiac T2*MRI increased with the decreasing SF concentration, showing a negative correlation which was statistically significant (Pearson's r = −0.6, p < 0.001). Furthermore, there was an excellent correlation between SF ng/ml and LIC by FerriScan© R2-MRI mg/g or mmol/kg (Spearmen's rho = −0.723, p < 0.008) in a small subset of patients (n = 14) who underwent the procedure. In conclusion, our study demonstrated a good correlation between serial SF and LIC by either Liver MRI T2* or by FerriScan© R2-MRI, even though SF is an acute-phase reactant. It also confirms the cardiac sparing effect in patients with SCD, even with the significant transfusion-related iron burden. About 14 (24%) patients of the original cohort died over the past 15 years, indicative of a negative impact of iron overload on disease morbidity and mortality.

Guidelines for the monitoring and management of iron overload in patients with haemoglobinopathies and rare anaemias.

Guidelines for the monitoring and management of iron overload in patients with haemoglobinopathies and rare anaemias.

Reduced hepcidin expression enhances iron overload in patients with HbE/β-thalassemia: Α comparative cross-sectional study.

Iron homeostasis is regulated by hepcidin (HEPC) that controls the dietary iron absorption and iron recycling. HEPC deficiency contributes to iron overload in β-thalassemia patients. The present study aimed to investigate the correlation between HEPC concentration and serum iron status among hemoglobin E (HbE)/β-thalassemia patients and their parents (HbE trait and β-thalassemia trait) compared with healthy controls. This study is a comparative cross-sectional study in which iron profile and HEPC level were examined in 65 HbE/β-thalassemia patients (pretransfusion) and 65 parents at the Hospital Sultanah Nur Zahirah and in 130 students as healthy controls from Univesiti Sultan Zainal Abidin, Terengganu, Malaysia. Furthermore, six samples from each group (HbE/β-thalassemia patients, parents and healthy controls) were randomly selected for gene expression analysis of HEPC and ferroportin1 (FPN1) using reverse transcription quantitative PCR. The results demonstrated that serum HEPC level were significantly decreased in HbE/β-thalassemia patients and their parents (P<0.001) compared with healthy controls. In addition, the gene expression analysis showed a dramatically downregulated HEPC in HbE/β-thalassemia patients and their parents (P=0.001) compared with healthy controls. However, there was a marked upregulation of FPN1 in HbE/β-thalassemia patients and their parents (P=0.001) compared with healthy controls. Iron profiling results revealed a significantly increased serum ferritin in HbE/β-thalassemia patients and their parents compared with healthy controls (P<0.001). In summary, the present study demonstrated that HEPC expression level and serum level were significantly decreased in HbE/β-thalassemia patients and their parents, which was combined with a marked increased FPN1 expression level and serum ferritin level compared with healthy volunteers. These findings supported the hypothesis that downregulated HEPC could lose its function as a negative regulator of FPN1, resulting in iron overload in HbE/β-thalassemia patients. Subsequently, assessing HEPC and FPN1 gene expression may be a useful tool to determine the risk of iron toxicity in patients with HbE/β-thalassemia and their parents, and could therefore be considered as a therapeutic target in the management of iron burden in these patients.

LONG-TERM SAFETY AND EFFICACY OF DEFERIPRONE FOR THE TREATMENT OF CHRONICALLY TRANSFUSED, IRON-OVERLOADED PATIENTS WITH SICKLE CELL DISEASE OR OTHER ANEMIAS

Assess the safety and efficacy of deferiprone (DFP) in chronically transfused, iron-overloaded patients with sickle cell disease (SCD) or other anemias. FIRST was a multicenter, 1-year, noninferiority trial of DFP vs deferoxamine (DFO). Patients ≥2 years of age were randomly assigned 2:1 to receive DFP or DFO. Those who completed FIRST could enter FIRST-EXT, a 2-year extension in which all patients received DFP. Thus, the baseline was the start of FIRST for patients randomized to DFP (up to 3 years for the DFP-DFP group), and the start of FIRST-EXT for patients randomized to DFO in FIRST (up to 2 years for the DFO- DFP group). In both studies, efficacy endpoints were yearly changes from baseline in liver iron concentration (LIC), cardiac MRI T2*, and serum ferritin (SF) level. All adverse drug reactions (ADRs) for both studies were recorded. In FIRST, patients (N = 228) were 46.9% female, mean age was 16.9 (standard deviation [SD] 9.6; range 3–59) years, 84.2% had SCD and 15.8% had other anemias. At 1 year, there were no significant (p > 0.05) differences in change in LIC, cardiac MRI T2*, or SF measures between the DFP and DFO intent-to-treat groups. Common ADRs (≥5% of patients) in the DFP group were abdominal pain, vomiting, pyrexia, increased alanine transaminase, increased aspartate aminotransferase, neutropenia, nausea, and chromaturia; in the DFO group, they were pyrexia and injection site pain. Serious ADRs in the DFP group were neutropenia (2.6%), increased transaminases (1.3%), and agranulocytosis, sickle cell crisis, epididymitis, Propionibacterium infection, bacterial sinusitis (infections not associated with neutropenia), vascular device infection, and migraine (0.7% each); in the DFO group, they were abdominal pain, arthritis, and headache (1.3% each). 134 Patients continued to FIRST-EXT (n = 89 DFP-DFP; n = 45 DFO-DFP): 60.4% were male, mean age was 16.2 (SD 8.6; range 4–47) years, 85.8% had SCD and 14.2% had other anemias. At baseline, all patients had elevated LIC with a mean (SD) of 14.93 (7.61) mg/g dry weight (dw), all had normal cardiac iron with a mean (SD) MRI T2* of 32.69 (17.66) ms, and all but 1 had elevated SF with a mean (SD) of 3894 (2591) μg/L. LIC decreased with mean (SD) reductions of -2.64 (4.64), -3.91 (6.38), and -6.64 (7.72) mg/g dw after 1, 2, and 3 years, respectively (p < 0.01 for all years). Cardiac iron remains in the normal range for all patients. SF levels declined with mean (SD) changes from baseline to years 1, 2, and 3 of -1 (1986), -771 (2171), and -1016 (3617) μg/L, respectively (p < 0.05 for years 2 and 3). No new ADRs (observed in 30.6% of patients) were reported. One patient withdrew due to ADRs of thrombocytopenia and neutropenia, which resolved. Another patient withdrew due to generalized edema and died for reasons unknown 17 days after withdrawal from the study. DFP effectively controlled iron burden in chronically transfused patients with SCD and other anemias, and it was noninferior to DFO. DFP use was associated with reductions in LIC and SF levels. Cardiac MRI T2* remained normal. DFP was well tolerated in patients who received up to 3 years of treatment, with no new safety concerns. We thank Dr. M Badr, Dr. B Inusa, Dr. AAM Adly, Dr. Y Kilinc, C Fradette, and NT Temin for their contribution to the studies. Sponsored by Chiesi Global Rare Diseases; medical writing support provided by Oxford PharmaGenesis Inc., and funded by Chiesi.

Effects of B1+ Heterogeneity on Spin Echo-Based Liver Iron Estimates.

Liver iron concentration (LIC) measured by MRI has become the clinical reference standard for managing iron overload in chronically transfused patients. Transverse relaxivity (R2 or R2* ) measurements are converted to LIC units using empirically derived calibration curves. That flip angle (FA) error due to B1+ spatial heterogeneity causes significant LIC quantitation error. B1+ scale (b1 , [FAactual /FAspecified ]) variation is a major problem at 3 T which could reduce the accuracy of transverse relaxivity measurements. Prospective. Forty-seven subjects with chronic transfusional iron overload undergoing clinically indicated LIC assessment. 5 T/3 T dual-repetition time B1+ mapping sequence ASSESSMENT: We quantified the average/standard deviation b1 in the right and left lobes of the liver from B1+ maps acquired at 1.5 T and 3 T. The impact of b1 variation on spin echo LIC estimates was determined using a Monte Carlo model. Mean, median, and standard deviation in whole liver and right and left lobes; two-sided t-test between whole-liver b1 means. Average b1 within the liver was 99.3% ± 12.3% at 1.5 T versus 69.6% ± 14.6% at 3 T and was independent of iron burden (P< 0.05). Monte Carlo simulations demonstrated that b1 systematically increased R2 estimates at lower LIC (<~25 mg/g at 1.5 T, <~15 mg/g at 3 T) but flattened or even inverted the R2 -LIC relationship at higher LIC (≥~25 mg/g to 1.5 T, ≥~15 mg/g to 3 T); changes in the R2 -LIC relationship were symmetric with respect to over and under excitation and were similar at 1.5 T and 3 T (for the same R2 value). The R2* -LIC relationship was independent of b1 . Spin echo R2 measurement of LIC at 3 T is error-prone without correction for b1 errors. The impact of b1 error on current 1.5 T spin echo-based techniques for LIC quantification is large enough to introduce measurable intersubject variability but the in vivo effect size needs a dedicated validation study. 2.

A Comprehensive Analysis of the Erythropoietin-erythroferrone-hepcidin Pathway in Hereditary Hemolytic Anemias.

A Comprehensive Analysis of the Erythropoietin-erythroferrone-hepcidin Pathway in Hereditary Hemolytic Anemias.

Pancreatic MR imaging and endocrine complications in patients with beta-thalassemia: a single-center experience.

Iron deposition in various organs can cause endocrine complications in patients with transfusion-dependent beta-thalassemia. The aim was to investigate the relationship between endocrine complications and pancreatic iron overload using magnetic resonance imaging (MRI). Forty patients with transfusion-dependent thalassemia (TDT) were enrolled in the study. The magnetic resonance imagings of the patients were performed using a 1.5 Tesla Philips MRI scanner. Two out of three patients had at least one clinical endocrine complication. The rate of iron deposition was 62.5% in liver, and 45% in pancreas tissue, and was 12.5% in heart tissue. Pancreatic T2* and hepatic T2* values were significantly positively correlated (p = 0.006). Pancreatic T2* and ferritin were significantly negatively correlated (p = 0.03). Cardiac T2* values were negatively correlated with fasting blood glucose (p = 0.03). Patients with short stature had significantly higher cardiac iron burden (22.3 vs. 36.6 T2*ms; p 0.01), and patients with hypothyroidism had higher liver iron concentrations (9.9 vs. 6.4 LIC mg/g; p = 0.05). The ferritin level of 841ng/mL and liver iron concentration (LIC) value of 8.7mg/g were detected as the threshold level for severe pancreatic iron burden (AUC 70%, p:0.04, AUC 80%, p = 0.002, respectively). Moreover, males were found to have decreased pancreas T2* values compared with the values in females (T2* 19.3 vs. 29.9, p = 0.05). Patients with higher ferritin levels over than 840ng/mL should be closely monitored for pancreatic iron deposition, and patients with endocrine complications should be assessed in terms of cardiac iron burden.

Optimizing Iron Overload Screening in a Pediatric Center for Cancer and Blood Disorders

Background: Packed red blood cell (PRBC) transfusions are common in the management of pediatric hematology, oncology, and bone marrow transplant (HOB) patients, and can lead to accumulation of excess iron that the body is unable to excrete. Increased exposure can lead to the development of iron overload (IO), which results in toxic effects on the liver, heart, and endocrine organs. Treatment can reduce the body's iron burden and improve outcomes. Previous studies observed that patients who received >10 PRBC transfusions or had an elevated serum ferritin were at a higher risk of IO. Unfortunately, published guidelines for IO screening only exist for a minority of HOB patient subtypes (thalassemia, sickle cell, and bone marrow transplant). The Children's Hospital Colorado Center for Cancer and Blood Disorders (CCBD) lacked a system to track transfusion frequency and a standardized guideline to adequately screen for IO, which made this a prime process improvement opportunity.Objective: Improve screening for IO by increasing the percentage of CCBD patients with >10 PRBC transfusions who have received a serum ferritin to >75%.Methods: All patients seen in CCBD clinics in 2015 were evaluated for the total number of transfusions received prior to 2016 and whether additional workup for IO was obtained. With this baseline data, a multidisciplinary working group created 3 interventions to improve identification and screening of IO.First, initial screening recommendations were integrated into the electronic health record (EHR) to alert providers when a patient has had >10 PRBC transfusions, prompting them to order a screening serum ferritin. Second, after a review of existing literature and discussion with local content experts, a risk-based screening algorithm was created and linked to the EHR alert to guide further testing. The algorithm recommends obtaining a liver and cardiac MRI for iron content in patients with a ferritin persistently >500 ng/mL. It also provides recommendations for phlebotomy or chelation in patients with liver and/or cardiac IO based on MRI findings. Third, providers were educated on IO screening and treatment. Data on screening practices will be collected and analyzed on an ongoing basis to track the response to the interventions and modify our approach, if needed.Results: Analysis of preliminary data from 2015 demonstrates that 19% of patients seen in CCBD clinics had >10 transfusions (192/1006). The percentage of patients with >10 transfusions that had a serum ferritin drawn was 53% (101/192). However, only 45% of patients had a ferritin obtained after receiving 10 transfusions (86/192). The most common sub-group was patients with oncology diagnoses (94/192, 49%), but only 14% (13/94) of oncology patients had a screening ferritin after 10 transfusions. In contrast, 90% (26/29) of hematology and 68% (47/69) of bone marrow transplant (BMT) patients had a screening ferritin. The mean time to obtaining a ferritin after 10 transfusions was 392 days. When evaluating all patients who had a ferritin sent, the value was >500 ng/ml in 80% (81/101) and >1000 ng/ml in 60% (61/101). Despite having a ferritin >500 ng/ml, only 46% (37/81) of patients received additional testing with a cardiac and/or liver MRI for iron content. There is ongoing collection and analysis of data evaluating improvements in screening practices since the EHR alert, risk based screening algorithm, and staff education began.Conclusions: Relatively few CCBD patients at risk for IO received initial screening, and even fewer had additional testing to evaluate for organ based IO. One reason that fewer oncology patients were screened may be the lack of an available guideline for IO screening. In contrast, available screening recommendations for sickle cell, thalassemia, and BMT patients may explain the increased frequency of screening in hematology and BMT patients. However, even patients who were screened and had an elevated ferritin did not uniformly receive additional testing with a cardiac and/or liver MRI. The goal of this project is to establish a baseline percentage of high-risk patients screened for IO, and then increase this level using a CCBD-wide standardized approach of staff education and EHR lab test prompts according to a standardized algorithm. As a result of the proposed interventions patents may be diagnosed earlier and subsequently treated when the disease burden is lower. DisclosuresNo relevant conflicts of interest to declare.

Longer Term Follow-up on the First Patients with Severe Hemoglobinopathies Treated with Lentiglobin Gene Therapy

Background: LentiGlobin Drug Product (DP) contains autologous CD34+ cells transduced with the betibeglogene darolentivec (BB305) lentiviral vector, encoding a human β-globin gene with a point mutation (AT87Q) that confers anti-sickling properties similar to those with γ-globin. Human proof of concept for LentiGlobin treatment in transfusion-dependent β-thalassemia (TDT) and severe sickle cell disease (SCD) has been established in HGB-205 (NCT02151526), the original clinical study of gene therapy for hemoglobinopathies. Here, we provide an update on 5 previously-reported patients (4 TDT, 1 SCD) as well as early data from 2 additional treated patients with SCD.Methods: HGB-205 enrolled patients (5−35 years old) with TDT (≥100 mL/kg of packed red blood cells [pRBCs] per year) or severe SCD (e.g., failed hydroxyurea treatment and ≥2 acute chest syndrome episodes [ACS] or ≥2 vaso-occlusive crises [VOC] in the preceding year/year prior to regular transfusions). Following mobilization/apheresis for TDT or bone marrow harvest for SCD, autologous CD34+ cells were transduced with the BB305 lentiviral vector. Patients underwent myeloablative conditioning with busulfan, were infused with transduced cells and monitored for hematologic engraftment, vector copy number (VCN), transgenic hemoglobin (HbAT87Q) levels, and adverse events (AEs). Transfusion requirements and biological markers for TDT, and VOCs for SCD were also assessed.Results: As of June 2, 2017, 4 patients with TDT and 3 patients with severe SCD have received LentiGlobin DP; median follow-up is 23.4 (range, 3.4 - 42.2) months. Patient and DP characteristics and key outcomes for these 7 patients are shown in Table 1. All patients engrafted successfully. The toxicity profile was consistent with myeloablative conditioning with single-agent busulfan, with no DP-related ≥ grade 3 AEs or serious AEs and no evidence of clonal dominance reported to date. For patients with TDT, VCN, total Hb and HbAT87Q levels in peripheral blood remain stable, with no evidence of decreasing efficacy (Table 1). All patients with TDT remain free of RBC transfusions. In all 3 β0/βE patients, Hb levels are stable near or within the normal range. Importantly, iron burden and biological hallmarks of dyserythropoiesis have largely returned to normal. Two patients with TDT have discontinued iron chelation therapy and transitioned to therapeutic phlebotomy while the third patient (1202) has been able to discontinue both.All 3 patients with SCD showed increase in HbAT87Q production through 6 months or last follow-up. The first treated patient with severe SCD (1204) had no clinical symptoms or complications of SCD until, at about 30 months post-treatment, the patient developed a VOC following an episode of acute gastroenteritis with fever and dehydration, and was subsequently hospitalized. His total Hb levels (12.4 g/dL), HbAT87Q (6.1 g/dL) and peripheral blood VCN levels (2.3) have remained stable at 30 months of follow-up. The 2 recently treated patients with SCD (1207 and 1208) have 6 and 3 months of follow-up, have not undergone transfusions since Day 21 and 15 post-infusion, and have total Hb of 8.8 g/dL and 9.8 g/dL and HbAT87Q of 1.8 and 1.5 g/dL, respectively as of last follow-up. At approximately 6 months post-treatment, patient 1207 experienced an episode of ACS and was hospitalized. Total % anti-sickling Hb ([HbAT87Q + HbF + HbA2]/Total Hb * 100) in the 3 patients with SCD was 52%, 39% and 36% as of last follow-up. Levels of unconjugated bilirubin, lactate dehydrogenase and reticulocyte count had improved compared to screening in the 2 patients with SCD (1204 and 1207) who had ≥ 6 months follow-up.Summary: Data from this pioneering phase 1/2 clinical study continue to suggest that treatment with LentiGlobin DP elicits sustained HbAT87Q levels which may be sufficient to alleviate the clinical and biochemical effects of severe SCD and TDT, with an acceptable safety profile. In patients with TDT, sustained levels of HbAT87Q with longer term follow-up, abatement of dyserythropoeisis and discontinuation of iron chelation therapy confirm the results seen to date. For patients with SCD, the marked clinical improvement in 1 patient with nearly 2.5 years of follow-up and increasing HbAT87Q levels in recently treated patients suggest gene therapy may be a promising option. Follow-up data on the 3 patients with SCD and 2-year primary follow-up for patients with TDT will be presented. [Display omitted] DisclosuresPayen:bluebird bio: Honoraria, Research Funding. Brousse:Add Medica: Membership on an entity's Board of Directors or advisory committees. Bartolucci:Novartis: Consultancy; AddMedica: Consultancy, Research Funding. Beuzard:bluebird bio: Consultancy, Equity Ownership. Asmal:bluebird bio: Employment, Equity Ownership. Miller:bluebird bio: Employment, Equity Ownership. De Montalembert:Addmedica: Consultancy, Honoraria, Research Funding; Novartis: Consultancy, Honoraria, Research Funding. Leboulch:bluebird bio: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties. Ribeil:Addmedica: Membership on an entity's Board of Directors or advisory committees; Vitalaire: Other: Non-financial support; Novartis: Other: Non-financial support.

Pituitary Iron: Effect on Pituitary Volume and Function in Transfusion Dependent Thalassemia

The association of iron toxicity with vital organ dysfunction has been extensively investigated in transfusion-dependent-thalassemia (TDT). However, pituitary iron load and its effect on function have been infrequently addressed. Growth delay, hypogonadism and infertility resulting from iron-induced pituitary toxicity are still prevalent, impacting clinical care and patients' quality of life. Early recognition and timely intervention can potentially prevent irreversible damage and restore partial function.AimsWe aimed to determine pituitary iron load and anterior pituitary volume by MRI in various age groups of TDT and examine their association with systemic and organ-specific iron measures. Additionally, we attempted to stratify the occurrence of abnormal anterior pituitary function by these MRI findings. Identifying such risk thresholds could assist in establishing timing and effectiveness of treatment intervention.MRI (1.5T) measurements of anterior pituitary iron (R2) and volume (Vol) were compared to normal controls (Noetzli 2011) with resultant Z-scores. Mean annual ferritin was calculated and cardiac and liver iron concentration (LIC) were determined by MRI-T2* and superconducting quantum interference device (SQUID) biosusceptometry, respectively. Pituitary hormones (LH and FSH) and growth hormone (Z scores for IGF-1 and IGF-BP3) were determined.ResultsFifty-three TDT patients (51% females) age 25±8 years, (range 10-45 years) were enrolled. Thirty-three (62%) had a ZR2 value >2 (mean 5.4) indicating iron accumulation above 97.5th percentile. Thirty-two patients (60%) had a Z-score for anterior-volume (ZVOL) of less than -2 (mean -2.8) indicating gland shrinkage below the 2.5th percent. In 23 patients (43%) both ZR2 and ZVOL exceeded these thresholds. ZR2 inversely correlated with ZVOL; r =-0.32, p<0.02. Pituitary iron increased with age and differed between patients <18 yr (n=14) and those 18-45 yr (n=39) with a mean ZR2 at 0.9±0.6 and 4.3±0.5, respectively (p<0.001). Anterior pituitary volume declined in older patients, lagging after the iron accumulation (Figure).LH/FSH values and ZR2 showed inverse correlation: r = -0.27 (p<0.03) and -0.3 (p< 0.006) respectively. ZVOL and LH/FSH demonstrated a positive correlation: r = 0.40, p<0.05 and 0.37, p<0.003, respectively. Z(IGF-BP3) but not Z(IGF-1) had an inverse correlation with ZR2: r = -0.5 (p<0.001).ZR2 > 4 was found to be a significant predictor for gland shrinkage; Zvol < -2 was present in 17/33 patients with a ZR2>4 compared to 3/20 with a ZR2<4 (p<0.009). Receiver operator (ROC) analysis confirmed it with an AUC of 0.68. ZR2 >4 was also predictive of presence of clinically important cardiac (T2* <20ms, AUC=0.92, p<0.001) and hepatic iron (LIC >10mg/gd.w., AUC= 0.74, p<0.005). Mean ferritin significantly correlated with ZR2 (p<0.001) but not with the gland's volume.ConclusionsAnterior pituitary iron assessed by MRI, increases with age and correlates with total body iron burden in TDT, suggesting cumulative iron toxicity. It results in a decrease in gland size and impaired gonadotropin and growth hormone secretion. Pituitary iron exceeding ZR2 >4, appears to be a significant predictor for change in gland size, that also is associated with cardiac and liver iron-overload. Pituitary MRI and the concomitant liver and cardiac iron quantification can identify patients at high-risk for further pituitary damage, suggesting that early intervention can potentially prevent additional decline in hormonal secretion. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Iron Overload Decreases Engraftment of Mice Receiving Allogeneic or Syngeneic Bone Marrow Transplant

Background:Transfusional iron overload is a poor prognostic factor in patients undergoing hematopoietic stem cell transplantation (HSCT). In multiple clinical studies, elevated serum ferritin correlated with inferior transplant outcomes; however, ferritin is an imperfect marker of iron burden and may not correlate with other markers, such as liver iron content. These studies are also confounded because increased transfusional need often reflects more severe disease, an independent predictor of worse outcome. Thus, it remains unclear whether the correlation between iron status and transplant outcome is due to transfusional iron overload alone, or is more reflective of other comorbidities. We developed a mouse model of parenteral iron overload followed by high dose chemotherapy and allogeneic or syngeneic HSCT to study, in isolation, the effects of iron overload on engraftment and survival after transplant.Methods:Cohorts of female C57BL/6 (H-2Kb) recipients received 0, 5, 25, or 50mg iron dextran, in divided doses over 2 weeks. Mice were then conditioned with busulfan (20mg/kg daily x 5 days) and cyclophosphamide (100mg/kg daily for the last 3 days). Two days later, mice received 30 x 106 bone marrow cells from female BALB/c (H-2Kd) donors (allogeneic HSCT) or 5 x 106 bone marrow cells from female UBC-GFP (H-2Kb) donors (syngeneic HSCT). Mice received enrofloxacin (10mg/kg daily x 5 days) after transplant. Engraftment was evaluated by flow cytometry as the proportion of donor cells in blood or bone marrow. Iron-related parameters were measured at the time of euthanasia (Day +28 post-transplant). Statistical analysis was performed with GraphPad Prism.Results:Our model of iron overload using iron dextran at escalating doses showed a dose-dependent increase in markers of iron load, such as total liver iron (R2=0.86) and ferritin (R2=0.84)(Fig. 1A-B; both P<0.0001 for linear trend). After allogeneic HSCT, there was a statistically significant decrease in overall survival in all groups receiving iron dextran (range: 47-63%), as compared to the saline control (90%)(Fig 1C; P<0.05); this effect was not dose-dependent. Data from 3 pooled experiments (N=30 mice per group) showed an engraftment rate of 59% in the saline group, and 47%, 42%, and 21% in the 5mg, 25mg, and 50mg iron dextran groups, respectively, suggesting a dose-dependent effect of iron overload on engraftment (Fig. 1D). In the syngeneic HSCT model, iron overload prior to transplant similarly resulted in delayed RBC and platelet engraftment and decreased rates of overall engraftment (Fig. 1E,F,G).Conclusions:Iron overload had a dose-dependent negative effect on overall engraftment after allogeneic and syngeneic HSCT. For allogeneic HSCT, there was also a decrease in overall survival in all the groups receiving iron dextran. For syngeneic HSCT, in which graft rejection cannot occur, iron overload still negatively affected post-transplant engraftment, suggesting that this effect is not due solely to potential immunomodulatory effects of excess iron on graft rejection. The mechanism(s) underlying the effects of iron overload on survival and engraftment rates in allogeneic and syngeneic HSCT, along with the potential benefit of chelation therapy, remain to be determined.Figure 1: Effect of iron overload on survival and engraftment. Mean ± S.D. for (A) total liver iron and (B) ferritin for allogeneic HSCT. Kaplan-Meier survival estimates for (C) mortality and (D) percent of mice not engrafting >50% of nucleated cells for allogeneic HSCT (N=10 mice per group per replicate; 30 mice total per group). Mean ± S.D. for (E) percent red cell engraftment over time and (F) percent platelet engraftment of time in syngeneic HSCT. Kaplan-Meier survival estimates for (F) percent of mice not engrafting >50% of red blood cells (N=10 mice per group per replicate; 20 mice total per group). *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 as compared to the saline control or as specified. Arrow denotes day of transplant. [Display omitted] DisclosuresSpitalnik:New Health Sciences: Consultancy, Membership on an entity's Board of Directors or advisory committees.

Heme Regulates the Expression in Erythroblasts of Placenta Growth Factor, a Contributor to Pulmonary Hypertension in Sickle Cell Disease, through the Oxidant Response Transcription Factor NRF2

Patients with sickle cell disease (SCD) have elevated plasma levels of placenta growth factor (PGF), which promotes expression of the pulmonary vasoconstrictor endothelin-1 (ET-1) contributing to pulmonary hypertension, an important age-related and life-limiting complication of SCD. In SCD patients, markers of high iron burden are associated with the highest PGF levels, leading us to the investigation of a mechanism where excessive heme induces PGF expression. Our lab's published work has demonstrated a significant heme-bound iron (hemin) stimulation of the PGF promoter in human K562 cells and in primary human erythroid cells. Our initial experiments with N-acetylcysteine and the nonspecific oxidant hydrogen peroxide, suggested to us a role of oxidant stress in PGF regulation, hypothetically through the NRF2 antioxidant response mechanism.We conducted gene expression analysis by quantitative real time PCR for PGF in human K562 erythroblastoid cells, erythroid-differentiated human and mouse primary bone marrow cells before and after exposure to hemin. We tested the effect of Nrf2 agonists and an antagonist. We analyzed expression of heme oxygenase-1 as a positive control, and expression of several Nrf2 family members, as well as baseline genes. We performed siRNA gene knockdown experiments in cultured cells, chromatin immunoprecipitation experiments and intravenous injection of hemin (120 μmol/kg) in the tail veins of wild type and Nrf2 null mice, followed by assay of plasma PGF by ELISA and qPCR of bone marrow.We find that heme regulates the expression of several members of the Nrf2 family, which in turn may appear to affect PGF expression. More specifically, the antioxidant transcription factory family members NRF2, MafF, MafG and BACH1 are transcriptionally activated in a specific temporal pattern after heme exposure. NFE2 and MafK transcripts were simultaneously repressed by heme treatment. The well-characterized non-oxidative NRF2 agonist sulforaphane activated the endogenous PGF promoter and the NRF2 inhibitor brusatol inhibited heme induction of the endogenous PGF promoter. Specific gene knockdown results using siRNA supports additive roles for NRF2 and its heterodimeric partner MafG in activating the PGF promoter in response to heme, presumably as a heterodimer. We verified direct binding of NRF2 on the PGF promoter by chromatin immunoprecipitation experiments. While PGF regulation shows quite many common aspects to HO-1 regulation (heme and oxidative stress response), we find that protoporphyrin induces HO-1 (known to require BACH-1), but not PGF.In addition to the experimental data from our human K562 cell culture models, we have found corroborating results in primary erythroid-differentiated human and murine hematopoietic cells. In vivo, intravenous injection of heme in wild type mice produces robust secretion of PGF protein into plasma within 3 hours (192 ± 16 vs. 527 ± 62 pg/mL, P <0.0001, mean ± SEM, one-way ANOVA with Sidak's multiple comparison test) and this heme induction of plasma PGF protein is blunted in the Nrf2 null mice (527 ± 62 vs. 343 ± 21 pg/mL, P=0.004).Our results support a mechanism in which accelerated heme exposure in SCD promotes robust expression of PGF in erythroblasts during erythroid differentiation, through a pathway that involves EKLF, NRF2 and MafG, culminating in expression of the vascoconstrictor endothelin-1. This mechanism helps to explain the clinical observation that adults with SCD heavily exposed to red cell transfusion are more likely to develop high endothelin levels and pulmonary hypertension, as a potential consequence of excess heme trafficking from the turnover of transfused red cells. These results might inspire greater adherence to existing approved therapies to chelate iron in SCD. [Display omitted] DisclosuresOfori-Acquah:NuvOx Pharma: Patents & Royalties. Kato:Bayer: Research Funding; Global Blood Therapeutics: Consultancy; MAST Therapeutics: Research Funding; Novartis: Consultancy.

Role of Iron in the Molecular Pathogenesis of Diseases and Therapeutic Opportunities.

Iron is an essential mineral that serves as a prosthetic group for a variety of proteins involved in vital cellular processes. The iron economy within humans is highly conserved in that there is no proper iron excretion pathway. Therefore, iron homeostasis is highly evolved to coordinate iron acquisition, storage, transport, and recycling efficiently. A disturbance in this state can result in excess iron burden in which an ensuing iron-mediated generation of reactive oxygen species imparts widespread oxidative damage to proteins, lipids, and DNA. On the contrary, problems in iron deficiency either due to genetic or nutritional causes can lead to a number of iron deficiency disorders. Iron chelation strategies have been in the works since the early 1900s, and they still remain the most viable therapeutic approach to mitigate the toxic side effects of excess iron. Intense investigations on improving the efficacy of chelation strategies while being well tolerated and accepted by patients have been a particular focus for many researchers over the past 30 years. Moreover, recent advances in our understanding on the role of iron in the pathogenesis of different diseases (both in iron overload and iron deficiency conditions) motivate the need to develop new therapeutics. We summarized recent investigations into the role of iron in health and disease conditions, iron chelation, and iron delivery strategies. Information regarding small molecule as well as macromolecular approaches and how they are employed within different disease pathogenesis such as primary and secondary iron overload diseases, cancer, diabetes, neurodegenerative diseases, infections, and in iron deficiency is provided.

Oral Administration of Ginger-Derived Lipid Nanoparticles and Dmt1 siRNA Potentiates the Effect of Dietary Iron Restriction and Mitigates Pre-Existing Iron Overload in Hamp KO Mice.

Intestinal iron transport requires an iron importer (Dmt1) and an iron exporter (Fpn1). The hormone hepcidin regulates iron absorption by modulating Fpn1 protein levels on the basolateral surface of duodenal enterocytes. In the genetic, iron-loading disorder hereditary hemochromatosis (HH), hepcidin production is low and Fpn1 protein expression is elevated. High Fpn1-mediated iron export depletes intracellular iron, causing a paradoxical increase in Dmt1-mediated iron import. Increased activity of both transporters causes excessive iron absorption, thus initiating body iron loading. Logically then, silencing of intestinal Dmt1 or Fpn1 could be an effective therapeutic intervention in HH. It was previously established that Dmt1 knock down prevented iron-loading in weanling Hamp (encoding hepcidin) KO mice (modeling type 2B HH). Here, we tested the hypothesis that Dmt1 silencing combined with dietary iron restriction (which may be recommended for HH patients) will mitigate iron loading once already established. Accordingly, adult Hamp KO mice were switched to a low-iron (LFe) diet and (non-toxic) folic acid-coupled, ginger nanoparticle-derived lipid vectors (FA-GDLVs) were used to deliver negative-control (NC) or Dmt1 siRNA by oral, intragastric gavage daily for 21 days. The LFe diet reduced body iron burden, and experimental interventions potentiated iron losses. For example, Dmt1 siRNA treatment suppressed duodenal Dmt1 mRNA expression (by ~50%) and reduced serum and liver non-heme iron levels (by ~60% and >85%, respectively). Interestingly, some iron-related parameters were repressed similarly by FA-GDLVs carrying either siRNA, including 59Fe (as FeCl3) absorption (~20% lower), pancreatic non-heme iron (reduced by ~65%), and serum ferritin (decreased 40–50%). Ginger may thus contain bioactive lipids that also influence iron homeostasis. In conclusion, the combinatorial approach of FA-GDLV and Dmt1 siRNA treatment, with dietary iron restriction, mitigated pre-existing iron overload in a murine model of HH.

The effects of secondary iron overload and iron chelation on a radiation-induced acute myeloid leukemia mouse model

BackgroundPatients with myelodysplastic syndrome (MDS) require chronic red blood cell (RBC) transfusion due to anemia. Multiple RBC transfusions cause secondary iron overload and subsequent excessive generation of reactive oxygen species (ROS), which leads to mutations, cell death, organ failure, and inferior disease outcomes. We hypothesize that iron loading promotes AML development by increasing oxidative stress and disrupting important signaling pathways in the bone marrow cells (BMCs). Conversely, iron chelation therapy (ICT) may reduce AML risk by lowering iron burden in the iron-loaded animals.MethodsWe utilized a radiation-induced acute myeloid leukemia (RI-AML) animal model. Iron overload was introduced via intraperitoneal injection of iron dextran, and iron chelation via oral gavage of deferasirox. A total of 86 irradiated B6D2F1 mice with various levels of iron burden were monitored for leukemia development over a period of 70 weeks. The Kaplan-Meier estimator was utilized to assess AML free survival. In addition, a second cohort of 30 mice was assigned for early analysis at 5 and 7 months post-irradiation. The BMCs of the early cohort were assessed for alterations of signaling pathways, DNA damage response and gene expression. Statistical significance was established using Student’s t-test or ANOVA.ResultsIron loading in irradiated B6D2F1 mice accelerated RI-AML development. However, there was a progressive decrease in AML risk for irradiated mice with increase in iron burden from 7.5 to 15 to 30 mg. In addition, ICT decreased AML incidence in the 7.5 mg iron-loaded irradiated mice, while AML onset was earlier for the 30 mg iron-loaded irradiated mice that received ICT. Furthermore, analysis of BMCs from irradiated mice at earlier intervals revealed accelerated dysregulation of signaling pathways upon iron loading, while ICT partially mitigated the effects.ConclusionsWe concluded that iron is a promoter of leukemogenesis in vivo up to a peak iron dose, but further iron loading decreases AML risk by increasing cell death. ICT can partially mitigate the adverse effects of iron overload, and to maximize its benefit this intervention should be undertaken prior to the development of extreme iron overload.

Kidney Iron Burden Detection By T2 Star (T2*) Magnetic Resonance Imaging In Pediatric β Thalassemia Major Patients

Kidney Iron Burden Detection By T2 Star (T2*) Magnetic Resonance Imaging In Pediatric β Thalassemia Major Patients

A facile colorimetric method for the quantification of labile iron pool and total iron in cells and tissue specimens

Quantification of iron is an important step to assess the iron burden in patients suffering from iron overload diseases, as well as tremendous value in understanding the underlying role of iron in the pathophysiology of these diseases. Current iron determination of total or labile iron, requires extensive sample handling and specialized instruments, whilst being time consuming and laborious. Moreover, there is minimal to no overlap between total iron and labile iron quantification methodologies—i.e. requiring entirely separate protocols, techniques and instruments. Herein, we report a unified-ferene (u-ferene) assay that enables a 2-in-1 quantification of both labile and total iron from the same preparation of a biological specimen. We demonstrate that labile iron concentrations determined from the u-ferene assay is in agreement with confocal laser scanning microscopy techniques employed within the literature. Further, this assay offers the same sensitivity as the current gold standard, inductively coupled plasma mass spectrometry (ICP-MS), for total iron measurements. The new u-ferene assay will have tremendous value for the wider scientific community as it offers an economic and readily accessible method for convenient 2-in-1 measurement of total and labile iron from biological samples, whilst maintaining the precision and sensitivity, as compared to ICP-MS.