Decrease in α-Globin and Increase in the Autophagy-Activating Kinase ULK1 mRNA in Erythroid Precursors from ß-Thalassemia Patients Treated with Sirolimus.

The ß-thalassemias are hereditary monogenic diseases characterized by a low or absent production of adult hemoglobin and excess in the content of α-globin. This excess is cytotoxic for the erythroid cells and responsible for the ß-thalassemia-associated ineffective erythropoiesis. Therefore, the decrease in excess α-globin is a relevant clinical effect for these patients and can be realized through the induction of fetal hemoglobin, autophagy, or both. The in vivo effects of sirolimus (rapamycin) and analogs on the induction of fetal hemoglobin (HbF) are of key importance for therapeutic protocols in a variety of hemoglobinopathies, including ß-thalassemias. In this research communication, we report data showing that a decrease in autophagy-associated p62 protein, increased expression of ULK-1, and reduction in excess α-globin are occurring in erythroid precursors (ErPCs) stimulated in vitro with low dosages of sirolimus. In addition, increased ULK-1 mRNA content and a decrease in α-globin content were found in ErPCs isolated from ß-thalassemia patients recruited for the NCT03877809 clinical trial and treated with 0.5-2 mg/day sirolimus. Our data support the concept that autophagy, ULK1 expression, and α-globin chain reduction should be considered important endpoints in sirolimus-based clinical trials for ß-thalassemias.

Outcomes in patients with chronic lymphocytic leukemia and TP53 aberration who received first-line ibrutinib: a nationwide registry study from the Italian Medicines Agency.

In this analysis we describe the effectiveness of first-line ibrutinib in 747 patients with chronic lymphocytic leukemia (CLL) and TP53 aberrations in a nationwide study with a 100% capture of patients who received the study drug. Median age was 71 years (range 32-95). An estimated treatment persistence rate of 63.4% (95% CI 60.0%-67.0%) and survival rate of 82.6% (95% CI 79.9-85.4%) were recorded at 24 months. Disease progression or death were the reasons for discontinuation in 182/397 patients (45.8%). A higher risk of treatment discontinuation was found to be associated with age, ECOG-PS and pre-existing heart disease, whereas ECOG ≥ 1, age ≥ 70 years and male sex were associated with an increased risk of death. Median post-progression overall survival (OS) was 12.2 months (95% CI 9.2-22.0). Post-discontinuation median OS in patients who discontinued ibrutinib for other reasons was not reached (95% CI 42.3 months - NA). Ibrutinib was an effective first-line treatment for CLL and TP53 aberrations in patients treated at large academic centers and community practice hospitals. Clinical characteristics at baseline may influence the effectiveness of ibrutinib, whereas the experience of prescribing centers and multi-hit or single-hit TP53 aberrations had no impact on outcome in this high-risk population.

Expression of γ-globin genes in ß-thalassemia patients treated with sirolimus: results from a pilot clinical trial (Sirthalaclin).

Introduction: ß-thalassemia is caused by autosomal mutations in the ß-globin gene, which induce the absence or low-level synthesis of ß-globin in erythroid cells. It is widely accepted that a high production of fetal hemoglobin (HbF) is beneficial for patients with ß-thalassemia. Sirolimus, also known as rapamycin, is a lipophilic macrolide isolated from a strain of Streptomyces hygroscopicus that serves as a strong HbF inducer in vitro and in vivo. In this study, we report biochemical, molecular, and clinical results of a sirolimus-based NCT03877809 clinical trial (a personalized medicine approach for ß-thalassemia transfusion-dependent patients: testing sirolimus in a first pilot clinical trial, Sirthalaclin). Methods: Accumulation of γ-globin mRNA was analyzed using reverse-transcription quantitative polymerase chain reaction (PCR), while the hemoglobin pattern was analyzed using high-performance liquid chromatography (HPLC). The immunophenotype was analyzed using a fluorescence-activated cell sorter (FACS), with antibodies against CD3, CD4, CD8, CD14, CD19, CD25 (for analysis of peripheral blood mononuclear cells), or CD71 and CD235a (for analysis of in vitro cultured erythroid precursors). Results: The results were obtained in eight patients with the ß+/ß+ and ß+/ß0 genotypes, who were treated with a starting dosage of 1 mg/day sirolimus for 24-48 weeks. The first finding of this study was that the expression of γ-globin mRNA increased in the blood and erythroid precursor cells isolated from ß-thalassemia patients treated with low-dose sirolimus. This trial also led to the important finding that sirolimus influences erythropoiesis and reduces biochemical markers associated with ineffective erythropoiesis (excess free α-globin chains, bilirubin, soluble transferrin receptor, and ferritin). A decrease in the transfusion demand index was observed in most (7/8) of the patients. The drug was well tolerated, with minor effects on the immunophenotype, and an only side effect of frequently occurring stomatitis. Conclusion: The data obtained indicate that low doses of sirolimus modify hematopoiesis and induce increased expression of γ-globin genes in a subset of patients with ß-thalassemia. Further clinical trials are warranted, possibly including testing of the drug in patients with less severe forms of the disease and exploring combination therapies.

Efficacy and Safety of Teriparatide in Beta-Thalassemia Major Associated Osteoporosis: A Real-Life Experience.

Osteoporosis represents a relevant cause of morbidity in adult Thalassemia Major (TM) population. Antiresorptive drugs such as bisphosphonates were demonstrated effective in preventing bone loss. Teriparatide (TP) is an anabolic agent approved for osteoporosis management in the general population, but its use has been very limited in TM patients so far. We evaluated TP efficacy and safety in TM-associated osteoporosis in real-life clinical practice. Retrospective evaluation of 11 TM patients (6 males, 5 females; mean age = 45 ± 4.38 years) with severe osteoporosis and multiple fractures under TP treatment. Mean TP treatment duration was 19 ± 7 months. TP withdrawal was due to poor compliance and side effects (fever and osteo-muscular pain) in two and three patients, respectively. After 12 and 24 months, BMD significantly increased at lumbar (+ 19% and 22%) and femoral sites (+ 13% and 13%). Osteocalcin and cross-laps levels increased after 12 and 24 months (+ 225 and + 54.2%; + 159 and 141%, respectively). No new fractures were detected during TP treatment. Baseline VAS score values (3 ± 3) did not significantly change after 12 and 24 months (3 ± 3 and 2 ± 3, respectively). Five out of eleven patients developed side effects. TP might be an effective treatment for TM-associated osteoporosis since it improves BMD, especially at the lumbar spine, and prevents fragility fractures. TM patients may have a higher frequency of side effects, especially muscle and bone pain under TP treatment, as compared to no TM population. Further studies are needed.

Thalassaemia is paradoxically associated with a reduced risk of in-hospital complications and mortality in COVID-19: Data from an international registry.

Although numerous patient-specific co-factors have been shown to be associated with worse outcomes in COVID-19, the prognostic value of thalassaemic syndromes in COVID-19 patients remains poorly understood. We studied the outcomes of 137 COVID-19 patients with a history of transfusion-dependent thalassaemia (TDT) and transfusion independent thalassaemia (TIT) extracted from a large international cohort and compared them with the outcomes from a matched cohort of COVID-19 patients with no history of thalassaemia. The mean age of thalassaemia patients included in our study was 41 ± 16 years (48.9% male). Almost 81% of these patients suffered from TDT requiring blood transfusions on a regular basis. 38.7% of patients were blood group O. Cardiac iron overload was documented in 6.8% of study patients, whereas liver iron overload was documented in 35% of study patients. 40% of thalassaemia patients had a history of splenectomy. 27.7% of study patients required hospitalization due to COVID-19 infection. Amongst the hospitalized patients, one patient died (0.7%) and one patient required intubation. Continuous positive airway pressure (CPAP) was required in almost 5% of study patients. After adjustment for age-, sex- and other known risk factors (cardiac disease, kidney disease and pulmonary disease), the rate of in-hospital complications (supplemental oxygen use, admission to an intensive care unit for CPAP therapy or intubation) and all-cause mortality was significantly lower in the thalassaemia group compared to the matched cohort with no history of thalassaemia. Amongst thalassaemia patients in general, the TIT group exhibited a higher rate of hospitalization compared to the TDT group (p = 0.001). In addition, the rate of complications such as acute kidney injury and need for supplemental oxygen was significantly higher in the TIT group compared to the TDT group. In the multivariable logistic regression analysis, age and history of heart or kidney disease were all found to be independent risk factors for increased in-hospital, all-cause mortality, whereas the presence of thalassaemia (either TDT or TIT) was found to be independently associated with reduced all-cause mortality. The presence of thalassaemia in COVID-19 patients was independently associated with lower in-hospital, all-cause mortality and few in-hospital complications in our study. The pathophysiology of this is unclear and needs to be studied in vitro and in animal models.

Impact of the preparation method of red cell concentrates on transfusion indices in thalassemia patients: A randomized crossover clinical trial.

BACKGROUND: The average hemoglobin content of red cell concentrates (RCC) varies depending on the method of preparation. Surprisingly less data are available concerning the clinical impact of those differences. STUDY DESIGN AND METHODS: The effects of two types of RCC (RCC-A, RCC-B) on transfusion regime were compared in a non-blinded, prospective, randomized, two-period, and crossover clinical trial. RCC-A was obtained by whole blood leukoreduction and subsequent plasma removal, RCC-B removing plasma and buffy coat first, followed by leukoreduction. Eligible patients were adult, with transfusion-dependent thalassemia (TDT). RESULTS: RCC-A contained 63.9 (60.3-67.8) grams of hemoglobin per unit (median with 1st and 3rd quartile), RCC-B 54.5 (51.0-58.2) g/unit. Fifty-one patients completed the study. With RCC-B, the average pre-transfusion hemoglobin concentration was 9.3 ± 0.5 g/dl (mean ± SD), the average transfusion interval 14.2 (13.7-16.3) days, the number of RCC units transfused per year 39.3 (35.4-47.3), and the transfusion power index (a composite index) 258 ± 49. With RCC-A, the average pre-transfusion hemoglobin concentration was 9.6 ± 0.5 g/dl (+2.7%, effect size 0.792), the average transfusion interval 14.8 (14.0-18.5) days (+4.1%, effect size 0.800), the number of RCC units transfused per year 34.8 (32.1-42.5) (-11.4%, effect size -1.609), and the transfusion power index 272 ± 61 (+14.1%, effect size 0.997). All differences were statistically highly significant (p < .00001). The frequency of transfusion reactions was 0.59% with RCC-A and 0.56% with RCC-B (p = 1.000). CONCLUSION: To reduce the number of RCC units consumed per year and the number of transfusion episodes, TDT patients should receive RCC with the highest average hemoglobin content.

Dual-energy X-ray absorptiometry pitfalls in Thalassemia Major.

BACKGROUND: Low mineral mass and reduced bone strength with increased fracture risk are the main causes of morbidity in Thalassemia Major (TM). The pathogenesis is multifactorial and includes ineffective erythropoiesis with medullary expansion, multiple endocrine dysfunctions, direct iron bone deposition, deferoxamine-induced bone dysplasia, and reduced physical activity associated with disease complications. Dual-energy X-ray absorptiometry (DXA) is the "gold standard" for bone mineral density (BMD) assessment and for bone strength and quality evaluation. This method identifies patients at greater risk of fragility fractures, guiding treatment and monitoring response to therapy. In TM, DXA shows limitations concerning BMD calculation accuracy and fracture risk prediction. One of the main challenges in the assessment of bone health in patients with TM is the accurate interpretation of densitometric results. PURPOSE: This review investigates the major pitfalls in DXA implementation and interpretation in TM. METHODS: Available literature has been assessed. CONCLUSIONS: DXA shows limitations in assessing bone mineral "status" in TM, especially in the paediatric population, due to the peculiar characteristics of bone architecture and deformities associated with the disease. A radiological technique adjustment in this population is mandatory.

Spermatozoal DNA damage in patients with B thalassaemia syndromes.

OBJECTIVE: to determine whether sperm DNA damage is increased in patients with beta-thalassaemia syndromes. DESIGN: prospective comparative assessment of sperm genomic integrity in thalassaemia patients and donor controls and correlation of sperm DNA damage with other measures of semen quality, reproductive hormones, ferritin, zinc and vitamin E levels. SETTING: Thalassaemia Centre of Paediatric and Adolescent Unit, Ferrara, Italy and Academic Research Institutions in the UK. SUBJECTS: twenty-eight thalassaemia major and thalassaemia intermedia patients attending the clinic for regular treatment. INTERVENTIONS: assessment of the degree of spermatozoa undergoing apoptosis by terminal deoxynucleotidyl transferase mediated assay (TUNEL) and measurement of the degree of those with compromised structural integrity as measured by the sperm chromatin structure assay (SCSA) using flow cytometry. MAIN OUTCOME MEASURES: the degree of spermatozoal DNA damage by TUNEL and SCSA and the correlation between these measures and sperm motility, concentration, morphology and serum FSH, LH, sex hormone binding globulin, free and total testosterone, ferritin, zinc and vitamin E using regression analysis and Student t-test. RESULTS: comparative analysis showed that beta-thalassaemia patients had significantly more sperm DNA damage (mean TUNEL=18.5%, SCSA=0.28) than controls (mean TUNEL=11.4%, mean SCSA=0.18) (p<0.001). Among thalassaemia patients there was a negative correlation between itchromatin structure damage and sperm concentration (r2=0.3, p<0.006). There was a significant negative correlation between serum ferritin levels and abnormal sperm morphology (r2=0.2, p<0.05). CONCLUSIONS: compared to controls there was a higher degree of DNA damage in spermatozoa of beta-thalassaemia patients. Thalassaemic patients with low sperm concentrations were more likely to have a higher degree of defective chromatin packaging. The negative association between ferritin levels and abnormal sperm morphology suggests a possible detrimental effect on spermatogenesis by the iron chelator desferrioxamine, which is used to reduce iron overload. Thalassaemic patients especially those being considered for assisted conception procedures should be counselled accordingly.

Impact of long-term iron chelation therapy on growth and endocrine functions in thalassaemia.

A large number of children treated from the time of diagnosis with modern transfusion and chelation therapy are now entering early adolescence or early adulthood, and only now can we evaluate how many complications, secondary to iron overload, can be prevented by daily s.c. desferrioxamine (DFX) therapy. In 1989, we planned a multi-centre study on growth and endocrine complications in patients who started chelation therapy with DFX early in life. Height, weight, endocrine complications, haematological variables and compliance with DFX were evaluated in a study group of 238 patients aged 2-17 years with beta-thalassaemia major regularly followed in 13 paediatric and haematological Italian centres. The LMS method by Cole and Green and the Mann-Whitney test were applied for statistical analysis. Twenty-six patients with thalassaemia (12.4%) had growth hormone insufficiency, five patients (2.1%) had primary hypothyroidism and four patients (1.7%) had hypoparathyroidism. Delayed puberty was present in 18.4% of boys and 17.7% of girls. At the beginning of chelation, standing height was in the normal range when compared to Swiss standards, while in the following years a progressive decline of growth was observed in both sexes. In conclusion, our study noted a positive effect of DFX therapy on sexual maturation and endocrine complications. Nevertheless, short stature has persisted despite major advances in treatment.

Chelation therapy and bone metabolism markers in thalassemia major.

The aim of our study was to investigate the effects of subcutaneous desferrioxamine (DFX) and oral deferiprone (L1) therapy on bone metabolism markers in patients with thalassemia major. We studied 17 patients with thalassemia receiving long-term treatment with desferrioxamine, 20 patients receiving long-term treatment with deferiprone, and 15 healthy age-matched controls. The following investigations were performed: a) intact parathyroid hormone (PTH), 25-hydroxyvitamin D [25(OH)D], 1,25-dihydroxyvitamin D [1,25(OH)2D] as endocrine parameters; b) alkaline phosphatase (ALP), bone alkaline phosphatase (BALP), osteocalcin (OC); c) bone resorption biochemical markers in serum and urine pyridinium crosslinks: hydroxylysyl-pyridinoline (HP) and lysyl-pyridinoline (LP); d) serum levels of cytokines and growth factors: transforming growth factor-beta1 (TGFbeta1), insulin-like growth factor-I (IGF-I), interleukin-1beta (IL-1beta), interleukin-6 (IL-6), tumor necrosis factor-a (TNFalpha); e) serum levels of IGF binding protein-3 (IGFBP-3). No significant differences among all studied variables were found in patients with thalassemia treated with desferrioxamine or deferiprone. In contrast, significant differences were found between patients with thalassemia and the control group: intact PTH was significantly lower in patients with thalassemia than in the controls (p < 0.0005), and a significant increase in ALP and BALP (p < 0.0005), but not in OC, was found in the patient group. With regard to bone resorption and remodeling markers, the urinary excretion of pyridinium crosslinks was higher in patients with thalassemia for HP fraction (p < 0.0005) and LP fraction (p = 0.002), as well as TGFbeta (p = 0.001). In contrast, IGF-I and IGFBP-3 were reduced when compared with controls. In conclusion, the study of bone metabolism markers in adult patients with thalassemia reveals a complex behavior with an increase in bone resorption indexes. Bone formation did not appear to be impaired. In particular, TGFbeta1 was higher in patients with thalassemia receiving L1 treatment.

HCV and HGV infection, iron overload and liver disease in multitransfused patients with thalassaemia and persistently normal or abnormal transaminase levels.

Prevalence and influence on liver disease of HCV and HGV infections, and HCV genotypes were studied in 28 HCV-Ab positive multitransfused thalassaemia patients with persistently normal ALT levels (group A) matched by sex and age with 28 patients with increased ALT levels (group B). Laboratory and virologic tests (all patients), liver biopsy (28 patients) and LIC by SQUID (30 patients) were performed. In group A, HCV-RNA was positive in 39%, genotype 2a was detected in 91%. In Group B, HCV-RNA was positive in 89%, prevalence of genotype 1b and 2a was 52% and 48% respectively; compared with group A, they had significantly increased values of gammaGT, AF, BA, TP, IgG, IgA, LIC (group B: 2,142 -/+ 1,524 microg/g liver; group A: 1,084 -/+ 610 microg/g liver). Overall prevalence of HGV-RNA was low (12.5%) and not significantly different between groups. Liver biopsies revealed no cirrhosis and severe fibrosis was found in 3 HCV viremic patients in group B. In 14 viremic patients examined both for LIC and liver histology, mild fibrosis was observed in 71%, in which iron overload was below 5 times the normal value. In conclusion, in patients with normal ALT levels, active HCV infection must be excluded by evaluation of HCV-RNA. Liver biopsy is indicated in HCV viremic patients, independent of ALT levels; in non-viremic patients, increased ALT levels may be due to iron overload and LIC measurement is indicated. Our data emphasise the crucial role of chelation therapy to maintain low LIC levels in order to prevent progression of fibrosis to cirrhosis in patients with HCV chronic hepatitis.

Diabetes mellitus and impaired glucose tolerance in thalassaemia major: incidence, prevalence, risk factors and survival in patients followed in the Ferrara Center.

Two hundred and seventy-three patients with thalassaemia major (TM) were followed in the Ferrara Thalassaemia Centre over a thirty-year period. Forty-two patients had insulin dependent diabetes mellitus (IDDM). The first case was diagnosed in 1973. The incidence of IDDM peaked in 1986 (3.9%), and it was 0.7% at the time of the study (March 1998). The prevalence of IDDM increased progressively over time, reaching 14.2% in 1998. Mean age at diagnosis of IDDM was 18.2 -/+ 3.6 years and this also rose significantly during the study period (p<0.01). Hypogonadism was present in 91% of patients with IDDM, hypothyroidism in 68%, hypoparathyroidism in 21%, and cardiopathy in 69%, all significantly more prevalent than in patients without IDDM. These complications appeared with the same frequency before and after the diagnosis of IDDM. Survival of patients with and without IDDM was similar and no difference in the primary cause of death was found between the two groups. Main risk factors associated with IDDM were poor compliance with desferioxamine (DFO) treatment (p<0.05%), advanced age at the start of intensive chelation therapy (p<0.001), liver cirrhosis or severe fibrosis (p<0.0001, odds ratio 9.5, CI 95% 2.8-32.6). Prevalence of impaired glucose tolerance (IGT) was highest in 1981, 1984, and 1985 when the incidence of IDDM was increasing; in 1995 the prevalence of IGT in patients aged 16-20 years was lower in comparison with that observed in 1975 (17% vs. 59%, p<0.01). Risk factors associated with IGT were: male sex (p<0.05), poor compliance with DFO therapy (p<0.05) and liver iron concentration 4 times above the normal value. In conclusion, our longitudinal study confirms that the incidence of IDDM and prevalence of IGT have been decreasing over the course of the last decade, appearing at a more advanced age, although some differences have not reached statistical significance. Iron overload and liver disease were the main associated risk factors, while positive family history for diabetes did not influence glucose metabolism in our patients.

Paraplegia due to spinal cord compression by extramedullary erythropoietic tissue in a thalassaemia intermedia patient with gynecomastia secondary to cirrhosis: successful treatment with hydroxyurea.

We report a 24 years old patient with thalassaemia intermedia and gynecomastia, complicated by paraplegia and urinary/fecal incontinence due to spinal cord compression by an extramedullary erythropoiesis (EE) mass, treated with long-term hydroxyurea (HU). Neurological improvement occurred during the first 6 weeks of HU therapy (1,000 mg/day) and magnetic resonance imaging showed a reduction in EE mass. HU dosage was reduced to 500 mg/day after 5 months, and treatment was discontinued after 25 months. Five months later there was a partial recurrence of neurological symptoms, which responded to radiotherapy. HU may have a role in the symptomatic treatment of spinal cord compression due to EE, particularly when radiotherapy is unavailable.

Healing of leg ulcers with hydroxyurea in thalassaemia intermedia patients with associated endocrine complications.

We report a preliminary clinical experience in the use of hydroxyurea (HU) for the treatment of leg ulcers in thalassaemia intermedia patients with associated endocrine complications. We administered HU 1 g/day to 6 adult patients with thalassaemia intermedia for 90 days. We observed an improvement (3 patients) or healing (3 patients) of chronic leg ulcers. Recurrence was observed in the absence of maintenance therapy (2 patients). Two patients discontinued HU because of adverse effects (stomach pain and fever). No significant increase in total Hb or HbF levels occurred. In conclusion, HU appears suitable for the treatment of leg ulcers unresponsive to conventional treatment in patients with thalassaemia intermedia.

Left ventricular remodeling, systolic function, and diastolic function in young adults with beta-thalassemia intermedia: a Doppler echocardiography study.

BACKGROUND: The aim of this study was to investigate the left ventricular (LV) remodeling and function in 24 asymptomatic young adults affected by beta-thalassemia intermedia (TI), in order to compare the obtained data with that of 80 patients affected by beta-thalassemia major (TM) and 65 healthy subjects. METHODS: LV volumes and shapes, mass index, mass/volume ratio, systolic and diastolic function, stroke volume, and cardiac index were determined by two-dimensional and M-mode echocardiography. RESULTS: In the TM and TI groups, LV volumes, diastolic and systolic shapes were significantly different from the control subjects, but the ejection fraction was slightly reduced only in the TM group. The TI group had larger LV volumes than did the TM group (mean [+/- SD] end-diastolic volume index, 99.4 +/- 21.9 vs 82.7 +/- 21.5 mL/m(2), respectively [p < 0.005]; mean end-systolic volume index, 42.8 +/- 12.2 vs 36.1 +/- 12.9 mL/m(2), respectively [p < 0.05]). Both groups showed an increase of the LV mass index, but the mass/volume ratio did not differ from the control subjects. The systolic volume index and the cardiac index were increased in both groups, but the increase was more pronounced in the TI group. Fractional shortening (FS) and the mean velocity of circumferential shortening (mVCFc) were decreased in the TM group (FS, 33.6 +/- 5.5% vs 36.9 +/- 4.1, respectively [p < 0.001]; mVCFc, 1.06 +/- 0.18 vs 1.17 +/- 0.12 circumference per second, respectively [p < 0.0001]). The LV contractile state was depressed only in the TM group, and the preload index was normal in both. LV filling showed an increase in the total flow velocity integral due to increases in the peak E wave (E) and peak A wave (A) velocities and integrals, with an increase of the E/A ratio in the TM group and a slight decrease in the TI group. The isovolumic relaxation time was prolonged in both groups. There was no major derangement in the pulmonary venous flow. CONCLUSIONS: Asymptomatic young adults with TI show significant increases in LV volumes, LV mass, and cardiac index that are more pronounced than those in TM patients. LV systolic function is preserved in the TI group but is slightly depressed in the TM group due to the increase of afterload and to reduced contractility. The hemodynamic and hematologic factors involved in the etiopathogenesis of these findings are discussed, such as the treatment strategy.