Anemia and iron overload as prognostic markers of outcomes in ß-thalassemia.

INTRODUCTION: Ineffective erythropoiesis and subsequent anemia as well as primary and secondary (transfusional) iron overload are key drivers for morbidity and mortality outcomes in patients with ß-thalassemia. AREAS COVERED: In this review, we highlight evidence from observational studies evaluating the association between measures of anemia and iron overload versus outcomes in both non-transfusion-dependent and transfusion-dependent forms of ß-thalassemia. EXPERT OPINION: Several prognostic thresholds have been identified with implications for patient management. These have also formed the basis for the design of novel therapy clinical trials by informing eligibility and target endpoints. Still, several data gaps persist in view of the challenge of assessing prospective long-term outcomes in a chronic disease. Pooling insights on the prognostic value of different measures of disease mechanism will be key to design future scoring systems that can help optimize patient management.

Prenatal Diagnosis of Cystic Fibrosis by Celocentesis.

Celocentesis is a new sampling tool for prenatal diagnosis available from 7 weeks in case of couples at risk for genetic diseases. In this study, we reported the feasibility of earlier prenatal diagnosis by celocentesis in four cases of cystic fibrosis and one case of cystic fibrosis and ß-thalassemia co-inherited in the same fetus. Celomic fluids were aspired from the celomic cavity between 8+2 and 9+3 weeks of gestation and fetal cells were picked up by micromanipulator. Maternal DNA contamination was tested and target regions of fetal DNA containing parental pathogenetic variants of CFTR and HBB genes were amplified and sequenced. Four of the five fetuses resulted as being affected by cystic fibrosis and, in all cases, the women decided to interrupt the pregnancy. In the other case, the fetus presented a healthy carrier of cystic fibrosis. The results were confirmed in three cases on placental tissue. In one case, no abortive tissue was obtained. In the last case, the woman refused the prenatal diagnosis to confirm the celocentesis data; the pregnancy is ongoing without complications. This procedure provides prenatal diagnosis of monogenic diseases at least four weeks earlier than traditional procedures, reducing the anxiety of patients and providing the option for medical termination of the affected fetus at 8-10 weeks of gestation, which is less traumatic and safer than surgical termination in the second trimester.

Iron chelation therapy for children with transfusion-dependent ß-thalassemia: How young is too young?

In this review, we provide a summary of evidence on iron overload in young children with transfusion-dependent ß-thalassemia (TDT) and explore the ideal timing for intervention. Key data from clinical trials and observational studies of the three available iron chelators deferoxamine, deferiprone, and deferasirox are also evaluated for inclusion of subsets of young children, especially those less than 6 years of age. Evidence on the efficacy and safety of iron chelation therapy for children ≥2 years of age with transfusional iron overload is widely available. New data exploring the risks and benefits of early-start iron chelation in younger patients with minimal iron overload are also emerging.

Prognostic Role of Multiparametric Cardiac Magnetic Resonance in Neo Transfusion-Dependent Thalassemia.

BACKGROUND: We prospectively evaluated the predictive value of multiparametric cardiac magnetic resonance (CMR) for cardiovascular complications in non-transfusion-dependent ß-thalassemia (ß-NTDT) patients who started regular transfusions in late childhood/adulthood (neo ß-TDT). METHODS: We considered 180 patients (38.25 ± 11.24 years; 106 females). CMR was used to quantify cardiac iron overload, biventricular function, and atrial dimensions, and to detect left ventricular (LV) replacement fibrosis. RESULTS: During a mean follow-up of 76.87 ± 41.60 months, 18 (10.0%) cardiovascular events were recorded: 2 heart failures, 13 arrhythmias (10 supraventricular), and 3 cases of pulmonary hypertension. Right ventricular (RV) end-diastolic volume index (EDVI), RV mass index (MI), LV replacement fibrosis, and right atrial (RA) area index emerged as significant univariate prognosticators of cardiovascular complications. The low number of events prevented us from performing a multivariable analysis including all univariable predictors simultaneously. Firstly, a multivariable analysis including the two RV size parameters (mass and volume) was carried out, and only the RV MI was proven to independently predict cardiovascular diseases. Then, a multivariable analysis, including RV MI, RA atrial area, and LV replacement fibrosis, was conducted. In this model, RV MI and LV replacement fibrosis emerged as independent predictors of cardiovascular outcomes (RV MI: hazard ratio (HR) = 1.18; LV replacement fibrosis: HR = 6.26). CONCLUSIONS: Our results highlight the importance of CMR in cardiovascular risk stratification.

RP1 Dominant p.Ser740* Pathogenic Variant in 20 Knowingly Unrelated Families Affected by Rod-Cone Dystrophy: Potential Founder Effect in Western Sicily.

Background and Objectives. Retinitis pigmentosa (RP) is the most common inherited rod-cone dystrophy (RCD), resulting in nyctalopia, progressive visual field, and visual acuity decay in the late stages. The autosomal dominant form (ADRP) accounts for about 20% of RPs. Among the over 30 genes found to date related to ADRP, RP1 pathogenic variants have been identified in 5-10% of cases. In a cohort of RCD patients from the Palermo province on the island of Sicily, we identified a prevalent nonsense variant in RP1, which was associated with ADRP. The objective of our study was to analyse the clinical and molecular data of this patient cohort and to evaluate the potential presence of a founder effect. Materials and Methods. From 2005 to January 2023, 84 probands originating from Western Sicily (Italy) with a diagnosis of RCD or RP and their relatives underwent deep phenotyping, which was performed in various Italian clinical institutions. Molecular characterisation of patients and familial segregation of pathogenic variants were carried out in different laboratories using Sanger and/or next-generation sequencing (NGS). Results. Among 84 probands with RCD/RP, we found 28 heterozygotes for the RP1 variant c.2219C>G, p.Ser740* ((NM_006269.2)*, which was therefore significantly prevalent in this patient cohort. After a careful interview process, we ascertained that some of these patients shared the same pedigree. Therefore, we were ultimately able to define 20 independent family groups with no traceable consanguinity. Lastly, analysis of clinical data showed, in our patients, that the p.Ser740* nonsense variant was often associated with a late-onset and relatively mild phenotype. Conclusions. The high prevalence of the p.Ser740* variant in ADRP patients from Western Sicily suggests the presence of a founder effect, which has useful implications for the molecular diagnosis of RCD in patients coming from this Italian region. This variant can be primarily searched for in RP-affected subjects displaying compatible modes of transmission and phenotypes, with an advantage in terms of the required costs and time for analysis. Moreover, given its high prevalence, the RP1 p.Ser740* variant could represent a potential candidate for the development of therapeutic strategies based on gene editing or translational read-through therapy for suppression of nonsense variants.

Nephrological Complications in Hemoglobinopathies: SITE Good Practice.

BACKGROUND: Hemoglobinopathies, among which thalassemic syndromes (transfusion-dependent and non-transfusion dependent thalassemias) and sickle cell disease (SCD), are the most widespread monogenic diseases worldwide. Hemoglobinopathies are endemic and spread-out all-over Italy, as result of internal and external migration flows. Nowadays, the increase therapeutic options associated to the general aging of patients with hemoglobinopathies related to the improvement in clinical management, contribute to the abnormalities in kidney function going from blood and urine test alterations to chronic kidney disease and end stage renal disease. METHODS: Here, we carried out a revision of the literature as panel of recognized experts in hemoglobinopathies with the consultancy and the revision of two nephrologists on kidney alteration and kidney disease in patients with TDT, NTDT and SCD. This is part of the action of the Italian society for the study of thalassemia and hemoglobinopties (SITE). The purpose of this "good practice (GP)" is to provide recommendations for follow-up and therapy for the management of kidney alterations in patients with TDT, NTDT and SCD. The literature review covers the period 1.1.2016 to 31.12.2022. In consideration of the rarity of these diseases, the analysis was extended from 5 to 7 years. Moreover, in the absence of relevant scientific papers in the identified time frame, we referred to pivotal or population studies, when available. Finally, in the absence of evidence-based data from prospective and randomized trials, the authors had to refer to expert opinion (expert consensus) for many topics. RESULTS: We generated question and answer boxes to offer a friendly consultation, using color code strategy and focused answers. CONCLUSIONS: The present GP will help in improving the clinical management, and the quality of care of patients with hemoglobinopathies.

An optimized procedure for preparation of conditioned medium from Wharton's jelly mesenchymal stromal cells isolated from umbilical cord.

Cell-free therapy based on conditioned medium derived from mesenchymal stromal cells (MSCs) has gained attention in the field of protective and regenerative medicine. However, the exact composition and properties of MSC-derived conditioned media can vary greatly depending on multiple parameters, which hamper standardization. In this study, we have optimized a procedure for preparation of conditioned medium starting from efficient isolation, propagation and characterization of MSCs from human umbilical cord, using a culture medium supplemented with human platelet lysate as an alternative source to fetal bovine serum. Our procedure successfully maximizes the yield of viable MSCs that maintain canonical key features. Importantly, under these conditions, the compositional profile and biological effects elicited by the conditioned medium preparations derived from these MSC populations do not depend on donor individuality. Moreover, approximately 120 L of conditioned medium could be obtained from a single umbilical cord, which provides a suitable framework to produce industrial amounts of toxic-free conditioned medium with predictable composition.

Antioxidant, Pro-Survival and Pro-Regenerative Effects of Conditioned Medium from Wharton's Jelly Mesenchymal Stem Cells on Developing Zebrafish Embryos.

Conditioned media harvested from stem cell culturing have the potential to be innovative therapeutic tools against various diseases, due to their high content of growth, trophic and protective factors. The evaluation in vivo of the effects and biosafety of these products is essential, and zebrafish provides an ideal platform for high-throughput toxicological analysis, concurrently allowing the minimization of the use of mammalian models without losing reliability. In this study, we assessed the biological effects elicited by the exposure of zebrafish embryos to a conditioned medium derived from Wharton's jelly mesenchymal stem cells. By a multiparametric investigation combining molecular, embryological, behavioural and in vivo imaging techniques, we found that exposure to a conditioned medium at a non-toxic/non-lethal dosage triggers antioxidant, anti-apoptotic and pro-regenerative effects, by upregulation of a set of genes involved in antioxidant defence (nrf2, brg1, sirt1, sirt6, foxO3a, sod2 and cat), glycolysis (ldha) and cell survival (bcl2l1, mcl1a and bim), coupled to downregulation of pro-apoptotic markers (baxa, caspase-3a and caspase-8). To our knowledge, this is the first study comprehensively addressing the effects of a conditioned medium on a whole organism from a developmental, molecular and behavioural perspective, and we are fairly confident that it will pave the way for future therapeutic application.

The Need for Translational Epidemiology in Beta Thalassemia Syndromes: A Thalassemia International Federation Perspective.

Epidemiology is the practical tool to provide information on which policy makers should base planning of services. Epidemiological data for thalassemia is based on inaccurate and often conflicting measurements. This study attempts to demonstrate with examples the sources of inaccuracy and confusion. The Thalassemia International Foundation (TIF) suggests that congenital disorders, for which increasing complications and premature death are avoidable through appropriate treatment and follow-up, should be given priority based on accurate data and patient registries. Moreover, only accurate information about this issue, especially for developing countries, will move national health resources in the right direction.

Development of a Thalassemia International Prognostic Scoring System (TIPSS).

A prognostic scoring system that can differentiate ß-thalassemia patients based on mortality risk is lacking. We analysed data from 3145 ß-thalassemia patients followed through a retrospective cohort design for the outcome of death. An a priori list of prognostic variables was collected. ß Coefficients from a multivariate cox regression model were used from a development dataset (n = 2516) to construct a formula for a Thalassemia International Prognostic Scoring System (TIPSS) which was subsequently applied to a validation dataset (n = 629). The median duration of observation was 10.0 years. The TIPSS score formula was constructed as exp (1.4 × heart disease + 0.9 × liver disease + 0.9 × diabetes + 0.9 × sepsis + 0.6 × alanine aminotransferase ≥42 IU/L + 0.6 × hemoglobin ≤9 g/dL + 0.4 × serum ferritin ≥1850 ng/mL). TIPSS score thresholds of greatest differentiation were assigned as <2.0 (low-risk), 2.0 to <5.0 (intermediate-risk), and ≥5.0 (high-risk). The TIPSS score was a good predictor for the outcome of death in the validation dataset (AUC: 0.722, 95%CI: 0.641-0.804) and survival was significantly different between patients in the three risk categories (P < 0.001). Compared to low-risk patients, the hazard ratio for death was 2.778 (95%CI: 1.335-5.780) in patients with intermediate-risk and 6.431 (95%CI: 3.151-13.128) in patients with high-risk. This study provides a novel tool to support mortality risk categorization for patients with ß-thalassemia that could help management and research decisions.

Incidence of cancer and related deaths in hemoglobinopathies: A follow-up of 4631 patients between 1970 and 2021.

BACKGROUND: The correlation between thalassemia and malignancies other than hepatocellular carcinoma (HCC) and the possible relationship between other hemoglobinopathies and tumor risk have been poorly evaluated. METHODS: Eight Italian specialized centers evaluated the incidence of malignant neoplasms in hemoglobinopathies as well as their sites and features. The study cohort included 4631 patients followed between 1970 and 2021 (transfusion-dependent ß-thalassemia, 55.6%; non-transfusion-dependent thalassemia, 17.7%; sickle cell disease, 17.6%; hemoglobin H disease, 8.3%). RESULTS: A total of 197 diagnoses of cancer were reported (incidence rate, 442 cases per 100,000 person-years). The liver was the most frequent site of tumors in both sexes, with a higher incidence (190 cases per 100,000 person-years) in comparison with the general population found in all types of hemoglobinopathies (except hemoglobin H disease). In recent years, tumors have become the second cause of death in patients with transfusion-dependent thalassemia. A lower risk of breast and prostate cancer was observed in the whole group of patients with hemoglobinopathies. The first cancer diagnoses dated back to the 1980s, and the incidence rate sharply increased after the 2000s. However, although the incidence rate of cancers of all sites but the liver continued to show an increasing trend, the incidence of HCC showed stability. CONCLUSIONS: These findings provide novel insights into the relationship between cancer and hemoglobinopathies and suggest that the overall risk is not increased in these patients. HCC has been confirmed as the most frequent tumor, but advances in chelation and the drugs that have led to the eradication of hepatitis C may explain the recent steadiness in the number of diagnoses that is reported here.

The place of allogeneic stem cell transplantation in aggressive B-cell non-Hodgkin lymphoma in the era of CAR-T-cell therapy.

Chimeric antigen receptor T (CAR-T) cells are a treatment option for patients with relapse/refractory (R/R) non-Hodgkin lymphoma (NHL), acute lymphoid leukemia and multiple myeloma. To date, diffuse large B-cell lymphoma (DLBCL), mantle cell lymphoma (MCL), follicular lymphoma (FL), and chronic lymphocytic leukemia (CLL) have been successfully treated with CAR-T cells directed against the CD19 antigen. However, when R/R disease persists after several treatment lines, patients with these diseases are often referred to transplantation centres to receive allogeneic stem cell transplantation (ALLO-SCT). ALLO-SCT and CAR-T cells share mechanism of actions, inducing immune effects of T-cells (and other cells after transplantation) against lymphoma cells, but they differ in several other characteristics. These differences justify unique positioning of each therapy within treatment algorithms. In this paper, we analyzed the results obtained after ALLO-SCT and CAR-T-cell therapy in patients with aggressive lymphomas (large B-cell lymphoma and MCL) to identify the ideal scenarios in which these 2 immunological therapies should be employed.

The use of hydroxyurea in the real life of MIOT network: an observational study.

BACKGROUND: Hydroxyurea (HU) has been widely used in clinical practice to manage patients with non-transfusion dependent thalassemia (NTDT). Few data are available about the effects of its administration in Italian patients. We assessed hematological and non-hematological outcomes following short- and long-term exposure to HU. RESEARCH DESIGN AND METHODS: We considered 71 NTDT patients (30 females) enrolled in the Myocardial Iron Overload in Thalassemia Network and treated for >12 months with HU. RESULTS: The mean duration of HU treatment was 8.23±5.79 years, starting at a mean age of 37.02±12.06 years. A significant increase in hemoglobin and mean corpuscular volume values and a down-regulation of all erythropoietic and/or hemolysis indices were detected after at least 12 months of treatment. In 28 patients the hemoglobin increase was ≥1.0 g/dl, associated with a higher HU dose. The hematological response dropped in long-term treatment. A favorable impact of HU treatment in limiting the progression of several complications typical of NTDT syndrome was observed. CONCLUSION: Our findings seemed to suggest that in several NTDT patients HU could be still a valid option to limit the advance in overall disease clinical burden without carrying significant adverse events and increase in mortality.

Early prenatal diagnosis of Hb Lepore Boston-Washington and ß-thalassemia on fetal celomatic DNA.

INTRODUCTION: Analysis of fetal DNA in at risk couples for thalassemia is performed from fetal trophoblast or amniotic fluid cells. Although these procedures are in common use, the main limitation is essentially due to the late gestation week in which diagnosis is performed. The celomic cavity develops around 4 weeks of pregnancy within the extraembryonic mesoderm and contains embryonic erythroid precursor cells as a source of fetal DNA that can be used to perform invasive prenatal diagnosis. METHODS: Celomatic fluids were obtained at 8 weeks of gestation in thirteen women with high-risk pregnancies. Twelve of these couples were at risk for Hb Lepore disease and ß-thalassemia and one couple represented a rare case in which both parents were carriers of Hb Lepore Boston-Washington. Fetal cells were isolated by micromanipulator and nested polymerase chain reactions were performed. RESULTS: The analysis was successfully performed in all examined cases. Two fetuses were found to have a compound heterozygosity for ß-thalassemia and Hb Lepore Boston-Washington, three fetuses were found to be carriers of ß-thalassemia, three fetuses of Hb Lepore, five were found without parental mutations. The genotypic analysis, carried out both by amniocentesis and on abortive tissue or after birth, showed concordance with results obtained on fetal celomic DNA. CONCLUSION: Our results unequivocally show that fetal DNA can be obtained by nucleated fetal cells present in celomatic fluid and demonstrate for the first time that prenatal diagnosis of ß-thalassemia and Hb Lepore may be feasible in an earlier time of pregnancy than other procedures.

Celomic Fluid: Laboratory Workflow for Prenatal Diagnosis of Monogenic Diseases.

BACKGROUND: Celomic fluid can be considered as an ultra-filtrate of maternal serum, containing a high protein concentration, urea, and many other molecules. It is an important transfer interface and a reservoir of nutrients for the embryo. Celomic fluid contains fetal cells that can be used for prenatal diagnosis of monogenic diseases in an earlier gestational period than villocentesis and amniocentesis. OBJECTIVE: The purpose of this study was to evaluate the characteristics of celomic fluid and to establish a workflow laboratory procedure for very early prenatal diagnosis of monogenic diseases. METHODS: Three hundred and eighty-five celomatic fluids were collected between the seventh and tenth week of gestation. We sampled 1 mL of celomic fluid in all cases. The embryo-fetal erythroid precursor cells were selected by the anti-CD71 microbead method or by a direct micromanipulator pick-up on the basis of their morphology. We amplified the extracted DNA using a nested polymerase chain reaction. Primers for short tandem repeat amplification were used to perform a quantitative fluorescent polymerase chain reaction evaluation to control maternal contamination. RESULTS: We observed maternal contamination in 95% of celomic fluids with a range between 5 and 100%. No fetal cells were observed in 0.78% of celomic fluids. The number of fetal cells ranged from a few units to several hundred. Isolation of embryo-fetal erythroblasts selected by the micromanipulator made diagnosis feasible in all cases. CONCLUSIONS: The selection of fetal cells by a micromanipulator and nested polymerase chain reaction analysis made celomatic fluid suitable for early prenatal diagnosis of monogenic disorders even in the presence of high maternal contamination and few fetal cells. The procedure reported in this study provides the opportunity for the use of celomic fluid sampled by celocentesis as an alternative to chorionic villi sampling and amniocentesis, to allow invasive prenatal diagnosis at a very early stage of pregnancy.