Haemoglobinopathies and other rare anemias in Spain: ten years of a nationwide registry (REHem-AR).

REHem-AR was created in 2013. The progressive implementation of neonatal screening for haemoglobinopathies in Spanish autonomous communities where the registry had not been implemented, as well as the addition of new centres during this period, has considerably increased the sample of patients covered. In this study, we update our previous publication in this area, after a follow-up of more than 5 years. An observational, descriptive, multicentre and ambispective study of adult and paediatric patients with haemoglobinopathies and rare anaemias registered in REHem was performed. The data are from a cross-sectional analysis performed on 1 June, 2023. The study population comprised 1,756 patients, of whom 1,317 had SCD, 214 had thalassaemia and 224 were diagnosed with another condition. Slightly more than one third of SCD patients (37%) were diagnosed based on neonatal bloodspot screening, and the mean age at diagnosis was 2.5 years; 71% of thalassaemia patients were diagnosed based on the presence of anaemia. Vaso-occlusive crisis and acute chest syndrome continue to be the most frequent complications in SCD. HSCT was performed in 83 patients with SCD and in 50 patients with thalassaemia. Since the previous publication, REHem-AR has grown in size by more than 500 cases. SCD and TM are less frequent in Spain than in other European countries, although the data show that rare anaemias are frequent within rare diseases. REHem-AR constitutes an important structure for following the natural history of rare anaemias and enables us to calculate investment needs for current and future treatments.

Does size matter? Two new deletions in the HBB gene cause ß0-thalassemia.

Most ß-thalassemias are caused by mutations involving one or a limited number of nucleotides within the gene or its adjacent regions. They can be substitutions or deletions; in these cases, the loss ranges from a single nucleotide to even the entire HBB gene, so we wonder if the phenotype is due to the size of the deletion or the location of the mutation. To clarify this, we present two new deletions in the ß-globin gene that cause ß0-thalassemia. The hematological parameters were determined with an automated cell counter; the Hb A2 and Hb F levels were measured by performance liquid chromatography. Hemoglobins were analyzed by capillary zone electrophoresis (Sebia Capillarys Flex system) and ion-exchange HPLC (BioRad Variant II ß-thalassemia Short Program). Molecular characterization was performed by automatic Sanger sequencing. The screening of common α-thalassemia point mutations and deletions in the world (21 in total) were carried out using multiplex PCR followed by reverse-hybridization with a commercial Alpha-Globin StripAssay kit. We have characterized two new mutations-(1) 1-bp deletion [CD61/62(-G)] [HBB:c.186_187delG], (2) 105-bp deletion [IVS-2-nt767-CD111] [HBB:c.316-84_333del]-and we have described, for first time in Spain, the 25-bp deletion [ß nts 252 - 276 deleted] [HBB:c.93-22_95del] mutation. These mutations were classified as pathogenic by UniProt Variants confirmed according to the American College of Medical Genetics and Genomics guidelines. These mutations present a phenotype compatible with ß0-thalassemia, supported by hematological parameters that correlate the degree of reduction in the synthesis of the ß-globin chain. Identification of this type of mutation is important for genetic counselling of partners where both are carriers, so that they are aware of the genetic risk of having affected children, allowing them to take an informed decision about their reproductive choices.

Genomic mutation profile in progressive chronic lymphocytic leukemia patients prior to first-line chemoimmunotherapy with FCR and rituximab maintenance (REM).

Chronic Lymphocytic Leukemia (CLL) is the most prevalent leukemia in Western countries and is notable for its variable clinical course. This variability is partly reflected by the mutational status of IGHV genes. Many CLL samples have been studied in recent years by next-generation sequencing. These studies have identified recurrent somatic mutations in NOTCH1, SF3B1, ATM, TP53, BIRC3 and others genes that play roles in cell cycle, DNA repair, RNA metabolism and splicing. In this study, we have taken a deep-targeted massive sequencing approach to analyze the impact of mutations in the most frequently mutated genes in patients with CLL enrolled in the REM (rituximab en mantenimiento) clinical trial. The mutational status of our patients with CLL, except for the TP53 gene, does not seem to affect the good results obtained with maintenance therapy with rituximab after front-line FCR treatment.

Sickle cell disease associated with thalassemia; description of a rare mutation.

Sickle cell disease (SCD) is a common hemoglobinopathy, secondary to alterations in the ß globin chain, resulting in an abnormal hemoglobin variant named as hemoglobin S. These disorders show a wide phenotypical spectrum, and the prevalence of these disorders has significantly changed over the time because of multiple factors such as migration. We report a case of a 17-year-old black male, born in Gambia, diagnosed with sickle cell disease, who presented an associated mutation only described in a Japanese family (Oshima et al., 1996).

Why is the novel Hb Miguel Servet visualised by CE-HPLC newborn and not by the CE-HPLC ß-thalassaemia programme?

Screening of haemoglobinopathies is indicated for the detection of sickle cell anaemia; thus, neonates can benefit from early and adequate treatment that prevents neurological damage, reduces morbidity and mortality associated with the disease. These types of programmes sometimes lead to unexpected findings. We present a new haemoglobin (Hb) variant (Hb Miguel Servet) detected by newborn screening. During neonatal screening of haemoglobinopathies by cation-exchange high-performance liquid chromatography (CE-HPLC) newborn, an Hb variant was detected. An analysis at 8 months of age using capillary zone electrophoresis (CZE) confirmed the presence of this new Hb. The molecular characterisation was performed by automatic sequencing of the α and ß globin genes in an ABI PRISM 3100 Genetic Analyzer. Hb analysis by CE-HPLC ß-thalassaemia short programmedid not indicate the presence of abnormal Hbs. By CZE showed a peak in the zone 12 zone comprising 3.3% of the total Hb. A new analysis by CE-HPLC on a Tosoh G8-2 (Horiba) shown a peak, in the region of HbA1b, did not interfere with the quantification of HbA1c. Sequencing of the ß gene revealed the substitution of a guanine for a thymine (GGT >TGT) in codon 69 of the second exon, resulting in substitution of cysteine for the amino acid glutamine (HBB:c.208G>T). Hb Miguel Servet is a ß-chain globin variant detected by CE-HPLC newborn (BioRad), by CZE and by CE-HPLC-CE Tosoh G8-2 (Horiba), but no by CE-HPLC-CE ß-thalassaemia short programme (BioRad). In fact, for all the techniques that are visualised, what would be detected would be the glutathione variant of Hb (Miguel Servet).

Efficacy and safety of high-dose intravenous iron as the first-choice therapy in outpatients with severe iron deficiency anemia.

BACKGROUND: Asymptomatic severe iron deficiency anemia is a common finding in subjects admitted to the outpatient anemia clinic. Although the condition can be easily be reversed with intravenous iron (IVI) therapy and several guidelines have suggested a restrictive threshold for using transfusion in hemodynamically stable patients, transfusion is often the rule in clinical practice. This study describes clinical practice results of IVI therapy without transfusion. STUDY DESIGN AND METHODS: In this multicenter retrospective observational study, data of severely anemic outpatients treated only with high-dose IVI with ferric carboxymaltose were collected. Inclusion criteria were hemoglobin (Hb) level of less than 7.0 g/dL and ferritin level of less than 30 ng/mL or mean corpuscular volume of less than 75 fL. RESULTS: Overall, 303 patients referred to the anemia clinic mainly from primary health care centers (46.2%) or the emergency department (28.7%) met the inclusion criteria. Median (interquartile range [IQR]) age was 47 (37-62) years and 84.5% were female. The median (IQR) Hb concentration at first visit was 6.5 (6.1-6.8) g/dL, 64 patients (21.1%) presented with a Hb level of less than 6.0 g/dL at diagnosis, and 11 of them (3.6%) had extreme anemia (Hb ≤ 5 g/dL). Gynecologic and gastroenteric bleeding were the main cause. After a mean IV administration of 1500 mg of iron, the Hb increased by a median of 5.7 g/dL. Thirteen patients experienced only mild side effects. CONCLUSIONS: In chronic very severe sideropenic anemias, third-generation IVI is effective and safe for quick correction and avoidance of red blood cell transfusion. These results suggest that more specific guidelines for this clinical setting are warranted.

Anemias hemolíticas autoinmunes: estudio retrospectivo de 93 pacientes / Autoimmune haemolytic anaemias: A retrospective study of 93 patients

INTRODUCCIÓN: Las anemias hemolíticas autoinmunes (AHAI) son enfermedades poco frecuentes y heterogéneas en su fisiopatología y comportamiento clínico, siendo el manejo de las mismas fundamentalmente empírico. PACIENTES Y MÉTODOS: Realizamos un estudio observacional, retrospectivo y multicéntrico de 93 pacientes diagnosticados de AHAI en 9 hospitales españoles entre 1987 y 2017, con una mediana de seguimiento de 28 meses. RESULTADOS: Mediana de edad de 67 años; un 85% de AHAI por anticuerpos calientes y un 64% AHAI primarias. Los valores de hemoglobina más bajos al diagnóstico se relacionaron con edad<45 años y el tipo serológico IgG+C. Un 92% recibieron tratamiento de primera línea, un 54% de segunda línea y un 27% de tercera línea. Las AHAI calientes fueron tratadas en primera línea con esteroides, con respuestas globales del 83% y completas del 58%. El rituximab en monoterapia o asociado a esteroides se administró a 34 pacientes con respuestas globales cercanas al 100% (respuestas completas 40-60%), relegando la esplenectomía a tercera línea. El tratamiento inmunosupresor se administró en pacientes con enfermedades autoinmunes o en dependientes de corticoides. DISCUSIÓN: Encontramos altas tasas de respuesta a esteroides, con tratamientos muy prolongados que provocan efectos secundarios y corticodependencia en un tercio de los pacientes. La asociación de esteroides con rituximab en primera línea podría estar indicada en pacientes con bajos niveles de hemoglobina y tipo serológico IgG+C. Las altas tasas de recaída hacen necesario el desarrollo de estudios aleatorizados con nuevos fármacos o la asociación con los ya existentes, que permitan mayor duración de las respuestas y con menores efectos secundarios

INTRODUCTION: Autoimmune haemolytic anaemia (AIHA) is an infrequent and heterogeneous disease in its pathophysiology and clinical behaviour, therefore it is generally managed empirically. PATIENTS AND METHODS: We conducted an observational, retrospective and multicentre study of 93 patients diagnosed with AHAI in 9 Spanish hospitals between 1987 and 2017, with a median follow-up of 28 months. RESULTS: Median age of 67 years; 85% AHAI for hot antibodies and 64% primary AHAI. The lowest haemoglobin values at diagnosis related to patients under 45 years of age and serological type IgG+C. Of the patients, 92% received first line treatment, 54% second line, and 27% third line. The warm AHAI were treated in first line with steroids, with overall responses of 83% and complete of 58%. Rituximab in monotherapy or in association with steroids was administered to 34 patients with overall responses close to 100% (complete responses 40-60%), relegating splenectomy to the third line. The immunosuppressive treatment was administered in patients with autoimmune diseases or in corticoid-dependent patients. DISCUSSION: We found high rates of response to steroids, with very prolonged treatments that cause side effects and corticoid dependence in a third of patients. The combination of steroids with rituximab in the first line, could be indicated in patients with low levels of haemoglobin and serological type IgG+C. The high relapse rates make necessary the development of randomised studies with new drugs or the combination with existing ones, which allow longer response times and with fewer side effects

Autoimmune haemolytic anaemias: A retrospective study of 93 patients. / Anemias hemolíticas autoinmunes: estudio retrospectivo de 93 pacientes.

INTRODUCTION: Autoimmune haemolytic anaemia (AIHA) is an infrequent and heterogeneous disease in its pathophysiology and clinical behaviour, therefore it is generally managed empirically. PATIENTS AND METHODS: We conducted an observational, retrospective and multicentre study of 93 patients diagnosed with AHAI in 9 Spanish hospitals between 1987 and 2017, with a median follow-up of 28 months. RESULTS: Median age of 67 years; 85% AHAI for hot antibodies and 64% primary AHAI. The lowest haemoglobin values at diagnosis related to patients under 45 years of age and serological type IgG+C. Of the patients, 92% received first line treatment, 54% second line, and 27% third line. The warm AHAI were treated in first line with steroids, with overall responses of 83% and complete of 58%. Rituximab in monotherapy or in association with steroids was administered to 34 patients with overall responses close to 100% (complete responses 40-60%), relegating splenectomy to the third line. The immunosuppressive treatment was administered in patients with autoimmune diseases or in corticoid-dependent patients. DISCUSSION: We found high rates of response to steroids, with very prolonged treatments that cause side effects and corticoid dependence in a third of patients. The combination of steroids with rituximab in the first line, could be indicated in patients with low levels of haemoglobin and serological type IgG+C. The high relapse rates make necessary the development of randomised studies with new drugs or the combination with existing ones, which allow longer response times and with fewer side effects.

C>A substitution in NT 46 of the 3' UTR region (the α complex protected region) of the alpha-1 globin gene: a non-deletional mutation or polymorphism?

AIMS: Untranslated regions (UTRs) play an important role in post-transcriptional regulation of gene expression, including by modulating messenger RNA (mRNA) transport out of the nucleus, translation efficiency, subcellular localisation and stability. Any mutation in this region could alter the stability of mRNA and thereby affect protein synthesis. We analysed if a mutation located in the α complex protected region of the α1 globin gene could cause non-deletional α-thalassaemia by affecting post-transcriptional stability (mRNA stability). METHODS: A total of 14 patients without anaemia, normal or slight microcytosis and hypochromia (medium concentration haemoglobin [MCH] <27 pg) were studied. Haemoglobin subtypes were screened using capillary zone electrophoresis and ion-exchange high-performance liquid chromatography (VARIANT II ß-Thalassaemia Short Program). The most common α-globin mutations were identified by multiplex PCR (Alpha-Globin StripAssay kit) and the molecular characterisation by automatic sequencing of alpha globin genes. RESULTS: All of them shown a novel transversion mutation in nt 778 (C>A), which is located in the 3' UTR in the α complex protected region [HBA1: c.*+46C>A]. CONCLUSIONS: This mutation is in the αRNAmin binding site, so a single nucleotide substitution in this region can decrease mRNA stability by potentially compromising the binding of α-complex protein to αRNAmin, favouring the decay of α-globin mRNA via erythroid cell-enriched endoribonuclease cleavage. In this case, it is a non-deletional α-thalassaemia. However, in silico and empirical studies predicted that it could be a silent polymorphism. Functional studies should be carried out to confirm whether it is a pathological mutation or a silent polymorphism.

Second cancers in MPN: Survival analysis from an international study.

One out of ten patients with Philadelphia-negative myeloproliferative neoplasms (MPN) develop a second cancer (SC): in such patients we aimed at assessing the survival impact of SC itself and of MPN-specific therapies. Data were therefore extracted from an international nested case-control study, recruiting 798 patients with SC diagnosed concurrently or after the MPN. Overall, 2995 person-years (PYs) were accumulated and mortality rate (MR) since SC diagnosis was 5.9 (5.1-6.9) deaths for every 100 PYs. A "poor prognosis" SC (stomach, esophagus, liver, pancreas, lung, ovary, head-and-neck or nervous system, osteosarcomas, multiple myeloma, aggressive lymphoma, acute leukemia) was reported in 26.3% of the patients and was the cause of death in 65% of them (MR 11.0/100 PYs). In contrast, patients with a "non-poor prognosis" SC (NPPSC) incurred a MR of 4.6/100 PYs: 31% of the deaths were attributed to SC and 15% to MPN evolution. At multivariable analysis, death after SC diagnosis was independently predicted (HR and 95% CI) by patient age greater than 70 years (2.68; 1.88-3.81), the SC prognostic group (2.57; 1.86-3.55), SC relapse (1.53; 10.6-2.21), MPN evolution (2.72; 1.84-4.02), anemia at SC diagnosis (2.32; 1.49-3.59), exposure to hydroxyurea (1.89; 1.26-2.85) and to ruxolitinib (3.63; 1.97-6.71). Aspirin was protective for patients with a NPPSC (0.60; 0.38-0.95). In conclusion, SC is a relevant cause of death competing with MPN evolution. Prospective data are awaited to confirm the role of cytoreductive and anti-platelet drugs in modulating patient survival after the occurrence of a SC.

Arterial thrombosis in Philadelphia-negative myeloproliferative neoplasms predicts second cancer: a case-control study.

Patients with Philadelphia-negative myeloproliferative neoplasm (MPN) are prone to the development of second cancers, but the factors associated with these events have been poorly explored. In an international nested case-control study, we recruited 647 patients with carcinoma, nonmelanoma skin cancer, hematological second cancer, and melanoma diagnosed concurrently or after MPN diagnosis. Up to 3 control patients without a history of cancer and matched with each case for center, sex, age at MPN diagnosis, date of diagnosis, and MPN disease duration were included (n = 1234). Cases were comparable to controls for MPN type, driver mutations and cardiovascular risk factors. The frequency of thrombosis preceding MPN was similar for cases and controls (P = .462). Thrombotic events after MPN and before second cancer were higher in cases than in controls (11.6% vs 8.1%; P = .013), because of a higher proportion of arterial thromboses (6.2% vs 3.7%; P = .015). After adjustment for confounders, the occurrence of arterial thrombosis remained independently associated with the risk of carcinoma (odds ratio, 1.97; 95% confidence interval, 1.14-3.41), suggesting that MPN patients experiencing arterial events after MPN diagnosis deserve careful clinical surveillance for early detection of carcinoma. This study was registered at www.clinicaltrials.gov as NCT03745378.

Second cancer in Philadelphia negative myeloproliferative neoplasms (MPN-K). A nested case-control study.

We conducted a large international nested case-control study including 1881 patients with Philadelphia-negative myeloproliferative neoplasms (MPN). Cases (n = 647) were patients with second cancer (SC: carcinoma, non-melanoma skin cancer, hematological second cancer, and melanoma) and controls (n = 1234) were patients without SC, matched with cases for sex, age at MPN diagnosis, date of MPN diagnosis, and MPN disease duration. The aim was to evaluate the risk of SC after exposure to cytoreductive drugs. Patients exposed to hydroxyurea (HU) (median: 3 years) had a risk of SC similar to unexposed patients (OR = 1.06, 95% CI 0.82-1.38). In contrast, in cancer-specific stratified multivariable analysis, HU had two-fold higher risk of non-melanoma (NM) skin cancer (OR = 2.28, 95% CI 1.15-4.51). A significantly higher risk of NM-skin cancer was also documented for pipobroman (OR = 3.74, 95% CI 1.00-14.01), ruxolitinib (OR = 3.87, 95% CI 1.18-12.75), and for drug combination (OR = 3.47, 95% CI 1.55-7.75). These three drugs did not show excess risk of carcinoma and hematological second cancer compared with unexposed patients. Exposure to interferon, busulfan, and anagrelide did not increase the risk. In summary, while it is reassuring that no excess of carcinoma was documented, a careful dermatologic active surveillance before and during the course of treatments is recommended.

High prognostic value of measurable residual disease detection by flow cytometry in chronic lymphocytic leukemia patients treated with front-line fludarabine, cyclophosphamide, and rituximab, followed by three years of rituximab maintenance.

It has been postulated that monitoring measurable residual disease (MRD) could be used as a surrogate marker of progression-free survival (PFS) in chronic lymphocytic leukemia (CLL) patients after treatment with immunochemotherapy regimens. In this study, we analyzed the outcome of 84 patients at 3 years of follow-up after first-line treatment with fludarabine, cyclophosphamide and rituximab (FCR) induction followed by 36 months of rituximab maintenance thearpy. MRD was assessed by a quantitative four-color flow cytometry panel with a sensitivity level of 10-4 Eighty out of 84 evaluable patients (95.2%) achieved at least a partial response or better at the end of induction. After clinical evaluation, 74 patients went into rituximab maintenance and the primary endpoint was assessed in the final analysis at 3 years of follow-up. Bone marrow (BM) MRD analysis was performed after the last planned induction course and every 6 months in cases with detectable residual disease during the 36 months of maintenance therapy. Thirty-seven patients (44%) did not have detectable residual disease in the BM prior to maintenance therapy. Interestingly, 29 patients with detectable residual disease in the BM after induction no longer had detectable disease in the BM following maintenance therapy. After a median followup of 6.30 years, the median overall survival (OS) and PFS had not been reached in patients with either undetectable or detectable residual disease in the BM, who had achieved a complete response at the time of starting maintenance therapy. Interestingly, univariate analysis showed that after rituximab maintenance OS was not affected by IGHV status (mutated vs unmutated OS: 85.7% alive at 7.2 years vs 79.6% alive at 7.3 years, respectively). As per protocol, 15 patients (17.8%), who achieved a complete response and undetectable peripheral blood and BM residual disease after four courses of induction, were allowed to stop fludarabine and cyclophosphamide and complete two additional courses of rituximab and continue with maintenance therapy for 18 cycles. Surprisingly, the outcome in this population was similar to that observed in patients who received the full six cycles of the induction regimen. These data show that, compared to historic controls, patients treated with FCR followed by rituximab maintenance have high-quality responses with fewer relapses and improved OS. The tolerability of this regime is favorable. Furthermore, attaining an early undetectable residual disease status could shorten the duration of chemoimmunotherapy, reducing toxicities and preventing long-term side effects. The analysis of BM MRD after fludarabine-based induction could be a powerful predictor of post-maintenance outcomes in patients with CLL undergoing rituximab maintenance and could be a valuable tool to identify patients at high risk of relapse, influencing further treatment strategies. This trial is registered with EudraCT n. 2007-002733-36 and ClinicalTrials.gov Identifier: NCT00545714.

Efficacy of repeated phlebotomies in hypertriglyceridemia and iron overload: A prospective, randomized, controlled trial.

BACKGROUND: High ferritin concentration is associated with hypertriglyceridemia, although it is not elucidated if iron overload has a causal role. OBJECTIVE: To evaluate the efficacy of repeated phlebotomies in patients with iron overload and hypertriglyceridemia. METHODS: Twelve weeks, 1:1 randomized, parallel-groups trial conducted at a University Hospital Lipid Clinic, including 86 subjects aged 18-70 years with serum ferritin >300 ng/mL in men or >200 ng/mL in women and triglycerides >200 mg/dL. Participants underwent: (1) three phlebotomies (every 3 weeks) and lipid-lowering dietary counseling or (2) lipid-lowering dietary counseling. The main outcome measured was the mean difference in percent change in triglyceride concentration between groups after the intervention. The mean differences in percent change of other clinical and biochemical variables (including cytokines and proinflammatory markers) after the intervention were also evaluated. RESULTS: Subjects who received phlebotomies showed a significant improvement in iron metabolism. The mean percent change in triglycerides between groups was -4.68 [-20.8, 11.4]%, P = .721. Retinol-binding protein 4 decreased by 9.98 ± 21.7% after phlebotomies, with a mean percent change between groups of -14.2 [-25.8, -2.73]%, P = .017, and correlated to gamma glutamyl transferase, alanine aminotransferase and aspartate aminotransferase change. Subjects with a large reduction in hepcidin showed a large improvement in liver enzymes and proinflammatory markers. CONCLUSIONS: A lipid-lowering diet plus a substantial reduction in iron deposits with repeated phlebotomies in subjects with hyperferritinemia and hypertriglyceridemia did not reduce triglyceride concentration in comparison with a lipid-lowering diet. Iron depletion for lipid management in these patients is not supported.