Lentiviral globin gene therapy with reduced-intensity conditioning in adults with ß-thalassemia: a phase 1 trial.

ß-Thalassemias are inherited anemias that are caused by the absent or insufficient production of the ß chain of hemoglobin. Here we report 6-8-year follow-up of four adult patients with transfusion-dependent ß-thalassemia who were infused with autologous CD34+ cells transduced with the TNS9.3.55 lentiviral globin vector after reduced-intensity conditioning (RIC) in a phase 1 clinical trial ( NCT01639690) . Patients were monitored for insertional mutagenesis and the generation of a replication-competent lentivirus (safety and tolerability of the infusion product after RIC-primary endpoint) and engraftment of genetically modified autologous CD34+ cells, expression of the transduced ß-globin gene and post-transplant transfusion requirements (efficacy-secondary endpoint). No unexpected safety issues occurred during conditioning and cell product infusion. Hematopoietic gene marking was very stable but low, reducing transfusion requirements in two patients, albeit not achieving transfusion independence. Our findings suggest that non-myeloablative conditioning can achieve durable stem cell engraftment but underscore a minimum CD34+ cell transduction requirement for effective therapy. Moderate clonal expansions were associated with integrations near cancer-related genes, suggestive of non-erythroid activity of globin vectors in stem/progenitor cells. These correlative findings highlight the necessity of cautiously monitoring patients harboring globin vectors.

Double Heterozygosity for Hb Durham-N.C. (HBB: c.344T>C) [ß114(G16)Leu→Pro] and the IVS-I-110 (HBB: c.93-21G>A) Causing a Severe ß-Thalassemia Phenotype.

The evaluation of a 10-month-old girl of Sicilian origin with a clinical phenotype of severe thalassemia led to the identification of two ß-globin gene defects, a ß-thalassemia (ß-thal), mutation at IVS-I-110 (HBB: c.93-21G>A) and a variant hemoglobin (Hb) mutation at codon 114 (HBB: c.344T>C) on the other allele, reported as Hb Durham-N.C. (also known as Hb Brescia) [ß114(G16)Leu→Pro] in the HbVar database. A very low Hb level (Hb 3.5 g/dL), microcytosis [mean corpuscular volume (MCV) 63.2 fL] and hypocromia [mean corpuscular Hb (MCH) 19.6 pg], increased red blood cell (RBC) distribution width (RDW) (36.0%), higher reticulocytes (6.2%), anisocytosis, poikilocytosis, hypocromia, basophilic stippling and inclusion body formation, were present in the affected subject. Analysis of other family components showed the presence of HBB: c.93-21G>A defect in the mother and in her brother, while Hb Durham-N.C. was absent in all other relatives, thus, this mutation has arisen as a de novo defect. This is the first case described as a severe thalassemic phenotype in a compound heterozygote carrier of this unstable Hb and a common ß-thalassemic allele. The important information gained from this case is that a rare dominant or recessive mutation may arise in every individual, even if this is a very rare event.

Longitudinal changes in LIC and other parameters in patients receiving different chelation regimens: Data from LICNET.

OBJECTIVES: The liver remains the primary site of iron storage, with liver iron concentration (LIC) being a strong surrogate of total body iron. MRI-R2 can accurately measure LIC. The LICNET (Liver Iron Cutino Network) was established to diagnostics of liver iron overload by MRI-R2 subjects with hemochromatosis in hematological disorders. The aims of the study were to look at variation in LIC measurements during time across different chelation regimens. METHODS: This was a cross-sectional study of 130 patients attending 9 Italian centers participating in the LICNET. LIC comparisons over time (T0 and T1 ) were made using t test and/or Wilcoxon test. RESULTS: LIC significantly decreased from MRI1 to MRI2 although at high variance (median change -0.8 mg Fe/g dw, range: -29.0 to 33.0; P = .011) and 7.7% of patients shifted from LIC values of high risk (>15 mg Fe/g dw) to an intermediate-risk category (7-15 mg Fe/g dw). Median change in LIC and correlation with serum ferritin levels (SF), during different chelation regimens, is reported. CONCLUSIONS: These findings suggest as longitudinal variation in the LIC is possible, across all chelation regimens. It confirms as SF levels not always can be used for estimating changes in LIC.

Prediction of cardiac complications for thalassemia major in the widespread cardiac magnetic resonance era: a prospective multicentre study by a multi-parametric approach.

Aims: Cardiovascular magnetic resonance (CMR) has dramatically changed the clinical practice in thalassemia major (TM), lowering cardiac complications. We prospectively reassessed the predictive value of CMR parameters for heart failure (HF) and arrhythmias in TM. Methods and results: We considered 481 white TM patients (29.48 ± 8.93 years, 263 females) enrolled in the Myocardial Iron Overload in Thalassemia (MIOT) network. Myocardial and liver iron overload were measured by T2* multiecho technique. Atrial dimensions and biventricular function were quantified by cine images. Late gadolinium enhancement images were acquired to detect myocardial fibrosis. Mean follow-up was 57.91 ± 18.23 months. After the first CMR scan 69.6% of the patients changed chelation regimen. We recorded 18 episodes of HF. In the multivariate analysis the independent predictive factors were myocardial fibrosis (HR = 10.94, 95% CI = 3.28-36.43, P < 0.0001), homogeneous MIO (compared with no MIO) (HR = 5.56, 95% CI = 1.37-22.51, P = 0.016), ventricular dysfunction (HR = 4.33, 95% CI = 1.39-13.43, P = 0.011). Arrhythmias occurred in 16 patients. Among the CMR parameters only the atrial dilation was identified as univariate prognosticator (HR = 4.26 95% CI=1.54-11.75, P = 0.005). Conclusions: CMR guided the change of chelation therapy in nearly 70% of patients, leading to a lower risk of iron-mediated HF and of arrhythmias than previously reported. Homogeneous MIO remained a risk factor for HF but also myocardial fibrosis and ventricular dysfunction identified patients at high risk. Arrhythmias were independent of MIO but increased with atrial dilatation. CMR by a multi-parametric approach dramatically improves cardiac outcomes and provides prognostic information beyond cardiac iron estimation.

The era of comparable life expectancy between thalassaemia major and intermedia: Is it time to revisit the major-intermedia dichotomy?

In the last few decades, the life expectancy of regularly transfused ß-thalassaemia major (TM) patients has dramatically improved following the introduction of safe transfusion practices, iron chelation therapy, aggressive treatment of infections and improved management of cardiac complications. How such changes, especially those attributed to the introduction of iron chelation therapy, improved the survival of TM patients to approach those with ß-thalassaemia intermedia (TI) remains unknown. Three hundred and seventy-nine patients with TM (n = 284, dead 40) and TI (n = 95, dead 13) were followed retrospectively since birth until 30 June 2015 or death. Kaplan-Meir curves showed statistically significant differences in TM and TI survival (P < 0·0001) before the introduction of iron chelation in 1965, which were no longer apparent after that date (P = 0·086), reducing the Hazard Ratio of death in TM compared to TI from 6·8 [95% confidence interval (CI) 2·6-17·5] before 1965 to 2·8 (95% CI 0·8-9·2). These findings suggest that, in the era of iron chelation therapy and improved survival for TM, the major-intermedia dichotomy needs to be revisited alongside future directions in general management and prevention for both conditions.

Multiparametric Cardiac Magnetic Resonance Survey in Children With Thalassemia Major: A Multicenter Study.

BACKGROUND: Cardiovascular magnetic resonance (CMR) plays a key role in the management of thalassemia major patients, but few data are available in pediatric population. This study aims at a retrospective multiparametric CMR assessment of myocardial iron overload, function, and fibrosis in a cohort of pediatric thalassemia major patients. METHODS AND RESULTS: We studied 107 pediatric thalassemia major patients (61 boys, median age 14.4 years). Myocardial and liver iron overload were measured by T2* multiecho technique. Atrial dimensions and biventricular function were quantified by cine images. Late gadolinium enhancement images were acquired to detect myocardial fibrosis. All scans were performed without sedation. The 21.4% of the patients showed a significant myocardial iron overload correlated with lower compliance to chelation therapy (P<0.013). Serum ferritin ≥2000 ng/mL and liver iron concentration ≥14 mg/g/dw were detected as the best threshold for predicting cardiac iron overload (P=0.001 and P<0.0001, respectively). A homogeneous pattern of myocardial iron overload was associated with a negative cardiac remodeling and significant higher liver iron concentration (P<0.0001). Myocardial fibrosis by late gadolinium enhancement was detected in 15.8% of the patients (youngest children 13 years old). It was correlated with significant lower heart T2* values (P=0.022) and negative cardiac remodeling indexes. A pathological magnetic resonance imaging liver iron concentration was found in the 77.6% of the patients. CONCLUSIONS: Cardiac damage detectable by a multiparametric CMR approach can occur early in thalassemia major patients. So, the first T2* CMR assessment should be performed as early as feasible without sedation to tailor the chelation treatment. Conversely, late gadolinium enhancement CMR should be postponed in the teenager age.

Deferiprone versus deferoxamine in thalassemia intermedia: Results from a 5-year long-term Italian multicenter randomized clinical trial.

In patients with thalassemia intermedia (TI), such as beta-TI, alpha-thalassemia (mainly HbH disease and mild/moderate forms of HbE/beta-thalassemia), iron overload is an important challenge in terms of diagnosis, monitoring, and treatment. Moreover, to date, the only possible chelators available are deferoxamine, deferasirox, and deferiprone. Here, we report the first 5-year long-term randomized clinical trial comparing the effectiveness of deferiprone versus deferoxamine in patients with TI. Body iron burden, which was determined by measuring serum ferritin levels in the same patient over 5 years and analyzed according to the generalized linear mixed model (GLMM), showed a linear decrease over time in the mean serum ferritin levels in both treatment groups (P-value = 0.035). The overall period of observation was 235.2 person-years for the deferiprone patients compared with 214.3 person-years for the deferoxamine patients. The results of the log-rank test suggested that the deferiprone treatment did not affect survival compared with the deferoxamine treatment (P-value = 0.360). The major adverse events observed included gastrointestinal symptoms and joint pain or arthralgia. Neutropenia and agranulocytosis were also detected, suggesting needing of strict hematological control. In conclusion, long-term iron chelation therapy with deferiprone is associated with an efficacy and safety similar to that of deferoxamine, suggesting that this drug is an alternative option in cases in which deferoxamine and deferasirox are contraindicated.

Long-term treatment with deferiprone enhances left ventricular ejection function when compared to deferoxamine in patients with thalassemia major.

Transfusion and iron chelation treatment have significantly reduced morbidity and improved survival of patients with thalassemia major. However, cardiac disease continues to be the most common cause of death. We report the left-ventricular ejection fraction, determined by echocardiography, in one hundred sixty-eight patients with thalassemia major followed for at least 5years who received continuous monotherapy with deferoxamine (N=108) or deferiprone (N=60). The statistical analysis, using the generalized estimating equations model, indicated that the group treated with deferiprone had a significantly better left-ventricular ejection fraction than did those treated with deferoxamine (coefficient 0.97; 95% CI 0.37; 1.6, p=0.002). The heart may be particularly sensitive to iron-induced mitochondrial damage because of the large number of mitochondria and its low level of antioxidants. Deferiprone, because of its lower molecular weight, might cross into heart mitochondria more efficiently, improving their activity and, thereby, myocardial cell function. Our findings indicate that the long-term administration of deferiprone significantly enhances left-ventricular function over time in comparison with deferoxamine treatment. However, because of limitations related to the design of this study, these findings should be confirmed in a prospective, randomized clinical trial.

Cardiac and hepatic iron and ejection fraction in thalassemia major: multicentre prospective comparison of combined deferiprone and deferoxamine therapy against deferiprone or deferoxamine monotherapy.

BACKGROUND: Due to the limited data available in literature, the aim of this multi-centre study was to prospectively compare in thalassemia major (TM) patients the efficacy of combined deferiprone (DFP) and deferoxamine (DFO) regimen versus either DFP and DFO in monotherapy by cardiovascular magnetic resonance (CMR) over a follow up of 18 months. METHODS: Among the first 1135 TM patients in the MIOT (Myocardial Iron Overload in Thalassemia) network, we evaluated those who had received either combined regimen (DFO + DFP, N=51) or DFP (N=39) and DFO (N=74) monotherapies between the two CMR scans. Iron overload was measured by T2* multiecho technique. Biventricular function parameters were quantitatively evaluated by cine images. RESULTS: The percentage of patients that maintained a normal global heart T2* value was comparable between DFP+DFO versus both monotherapy groups. Among the patients with myocardial iron overload at baseline, the changes in the global heart T2* and in biventricular function were not significantly different in DFP+DFO compared with the DFP group. The improvement in the global heart T2* was significantly higher in the DFP+DFO than the DFO group, without a difference in biventricular function. Among the patients with hepatic iron at baseline, the decrease in liver iron concentration values was significantly higher with combination therapy than with either monotherapy group. CONCLUSIONS: In TM patients at the dosages used in the real world, the combined DFP+DFO regimen was more effective in removing cardiac iron than DFO, and was superior in clearing hepatic iron than either DFO or DFP monotherapy. Combined therapy did not show an additional effect on heart function over DFP.

Serial echocardiographic left ventricular ejection fraction measurements: a tool for detecting thalassemia major patients at risk of cardiac death.

Cardiac damage remains a major cause of mortality among patients with thalassemia major. The detection of a lower cardiac magnetic resonance T2* (CMR-T2*) signal has been suggested as a powerful predictor of the subsequent development of heart failure. However, the lack of worldwide availability of CMR-T2* facilities prevents its widespread use for follow-up evaluations of cardiac function in thalassemia major patients, warranting the need to assess the utility of other possible procedures. In this setting, the determination of left ventricular ejection fraction (LVEF) offers an accurate and reproducible method for heart function evaluation. These findings suggest a reduction in LVEF≥7%, over time, determined by 2-D echocardiography, may be considered a strong predictive tool for the detection of thalassemia major patients with increased risk of cardiac death. The reduction of LVEF≥7% had higher (84.76%) predictive value. Finally, Kaplan-Meier survival curves of thalassemia major patients with LVEF≥7% showed a statistically significant decreased probability of survival for heart disease (p=0.0022). However, because of limitations related to the study design, such findings should be confirmed in a large long-term prospective clinical trial.

Long-term use of deferiprone significantly enhances left-ventricular ejection function in thalassemia major patients.

A multicenter randomized open-label long-term sequential deferiprone­deferoxamine (DFP-DFO) versus DFP alone trial (sequential DFP-DFO) performed in patients with thalassemia major (TM) was retrospectively reanalyzed to assess the variation in the left ventricular ejection fraction (LVEF) [1].

Neridronate improves bone mineral density and reduces back pain in ß-thalassaemia patients with osteoporosis: results from a phase 2, randomized, parallel-arm, open-label study.

Neridronate is a third generation bisphosphonate with established efficacy in metabolic bone disease. In this randomized, open-label study, 118 adults with ß-thalassaemia and bone mineral density (BMD) Z scores ≤-2·0 were randomized 1:1-500 mg calcium with 400 international unis (iu) vitamin D daily or 500 mg calcium with 400 iu vitamin D daily plus neridronate 100 mg intravenously every 90 d. Significant increases in BMD at the lumbar spine and total hip were noted in the neridronate group at 6 and 12 months from baseline (P < 0·001), and values were significantly higher than the control group at both time intervals. Neridronate also significantly decreased serum bone alkaline phosphatase and C-telopeptide of collagen type 1 levels from as early as 3 months (P = 0·04 and P < 0·001, respectively), reaching significantly lower values at 12 months compared with the control group (P < 0·05). Reductions in back pain and analgesic use were also evident, starting 3 months from commencing treatment. Treatment was well tolerated by all patients. In this largest randomized trial in thalassaemia-induced osteoporosis to date, neridronate was safe and effective in reducing bone resorption and increasing BMD. The associated reduction in back pain and improved quality of life will encourage adherence to therapy. (Clinicaltrials.gov identifier NCT01140321.).

IL28B polymorphisms influence stage of fibrosis and spontaneous or interferon-induced viral clearance in thalassemia patients with hepatitis C virus infection.

BACKGROUND: Polymorphisms in the interleukin-28B are important determinants in the spontaneous and drug-induced control of hepatitis C virus infection. DESIGN AND METHODS: We assessed the association of rs8099917 and rs12979860 polymorphisms with spontaneous viral clearance, severity of liver fibrosis, and response to interferon-monotherapy in 245 thalassemia major patients with hepatitis C virus infection. RESULTS: Ninety-eight patients (40%) had a spontaneous viral clearance while 147 patients (60%) developed a chronic infection. Spontaneous viral clearance was more frequent among patients with the T/T genotype of rs8099917 polymorphism (OR 2.130; P = 0.008) or C/C genotype of rs12979860 polymorphism (OR 2.425; P = 0.001). During observation, 131 patients with chronic infection underwent a liver biopsy; age (OR 1.058; P = 0.01) G/T or G/G genotypes of rs8099917 polymorphism (OR 3.962; P = 0.001), and C/T or T/T genotypes of rs12979860 polymorphism (OR 3.494; P = 0.005) were associated with severe liver fibrosis, independent of liver iron concentration. Finally, T/T genotype of rs8099917 polymorphism (OR 3.014; P = 0.03) or C/C genotype of rs12979860 polymorphism (OR 3.285; P = 0.01), age (OR 0.902; P = 0.001), female gender (OR 3.418; P = 0.01) and 2 or 3 virus C genotypes (OR 4.700; P=0.007) were independently associated with sustained virological response in 114 patients treated with alpha-interferon. Conclusions Polymorphisms in the interleukin-28B are associated with the control of hepatitis C virus infection in thalassemia major patients, and understanding allelic patterns has an important role in determining prognosis and therapeutic management.

Cardiac iron and cardiac disease in males and females with transfusion-dependent thalassemia major: a T2* magnetic resonance imaging study.

BACKGROUND: It has been repeatedly reported that female patients with thalassemia major survive longer than males and that the difference is due to a lower rate of cardiac disease in females. DESIGN AND METHODS: We compared the cardiac iron load as measured by T2* magnetic resonance imaging in 776 patients (370 males) examined at the National Research Council as part of an Italian cooperative study. We also established normal left ventricular ejection fraction values for our population. RESULTS: The prevalence of cardiac disease was higher in males than in females (105 males versus 69 females; P < 0.0001). Cardiac T2* was significantly lower in patients with heart dysfunction (P < 0.0001), but no difference was observed according to sex. Twenty males and five females had a history of cardiac arrhythmias. Their cardiac T2* was not significantly lower than that of patients without arrhythmias (24 ms versus 26 ms; P = 0.381), nor was there a difference between sexes. Liver T2* was significantly lower in males and females with heart dysfunction compared to those without. Ferritin levels were higher in patients of both sexes with heart dysfunction without significant differences between males and females. Conclusions Males and females are at the same risk of accumulating iron in their hearts, but females tolerate iron toxicity better, possibly as an effect of reduced sensitivity to chronic oxidative stress.

Deferasirox, deferiprone and desferrioxamine treatment in thalassemia major patients: cardiac iron and function comparison determined by quantitative magnetic resonance imaging.

BACKGROUND: Oral deferiprone was suggested to be more effective than subcutaneous desferrioxamine for removing heart iron. Oral once-daily chelator deferasirox has recently been made commercially available but its long-term efficacy on cardiac iron and function has not yet been established. Our study aimed to compare the effectiveness of deferasirox, deferiprone and desferrioxamine on myocardial and liver iron concentrations and bi-ventricular function in thalassemia major patients by means of quantitative magnetic resonance imaging. DESIGN AND METHODS: From the first 550 thalassemia subjects enrolled in the Myocardial Iron Overload in Thalassemia network, we retrospectively selected thalassemia major patients who had been receiving one chelator alone for longer than one year. We identified three groups of patients: 24 treated with deferasirox, 42 treated with deferiprone and 89 treated with desferrioxamine. Myocardial iron concentrations were measured by T2* multislice multiecho technique. Biventricular function parameters were quantitatively evaluated by cine images. Liver iron concentrations were measured by T2* multiecho technique. RESULTS: The global heart T2* value was significantly higher in the deferiprone (34 ± 11 ms) than in the deferasirox (21 ± 12 ms) and the desferrioxamine groups (27 ± 11 ms) (P = 0.0001). We found higher left ventricular ejection fractions in the deferiprone and the desferrioxamine versus the deferasirox group (P = 0.010). Liver iron concentration, measured as T2* signal, was significantly lower in the desferrioxamine versus the deferiprone and the deferasirox group (P = 0.004). CONCLUSIONS: The cohort of patients treated with oral deferiprone showed less myocardial iron burden and better global systolic ventricular function compared to the patients treated with oral deferasirox or subcutaneous desferrioxamine.

Detection of early cardiac dysfunction in patients with Beta thalassemia by tissue Doppler echocardiography.

BACKGROUNDS: In this study we tried to evaluate the prognostic significance of several echocardiographic parameters on the occurrence of heart failure or arrhythmias in patients with beta thalassemia. METHODS: We investigated possible differences in myocardial function between a population of 37 asymptomatic patients with beta thalassemia and 25 age-matched healthy controls, all of whom underwent an echocardiographic study, including tissue Doppler imaging (TDI), moreover plasmatic levels of N-terminal pro-BNP (NT-pro BNP) were measured in all patients. We followed the patients for 22 ± 8 months to evaluate adverse cardiac events. RESULTS: Conventional echocardiographic parameters of left ventricle were comparable in both groups. Whereas TDI peak systolic velocity (Sm) and diastolic parameter (E/Em ratio) were significantly abnormal in patients with thalassemia. Moreover eleven adverse cardiac events were observed during follow-up. Baseline systolic velocity (Sm) <7.9 cm/s was significantly associated with cardiac complications (P < 0.05). We also demonstrated that systolic velocity is inversely related to NT-proBNP plasmatic levels (P < 0.001). CONCLUSIONS: Our study suggests that mitral annular systolic velocity <7.9 cm/s is associated to the onset of adverse cardiac events.