Sirolimus-Based Immunosuppression as GvHD Prophylaxis after Bone Marrow Transplantation for Severe Aplastic Anaemia: A Case Report and Review of the Literature

Congenital or acquired severe aplastic anaemia (SAA) is cured by bone marrow transplantation (BMT) from a histocompatible leukocyte antigen- (HLA-) identical sibling. The best conditioning regimen is cyclophosphamide (CTX) with or without antithymocyte globulin (ATG), followed by short-term methotrexate (MTX) and cyclosporine A (CsA) to prevent graft-versus-host disease (GvHD). In our pediatric oncology-hematology unit, a 5-year-old girl with SAA was treated with two BMT from the same HLA-identical sibling donor. Severe CsA-induced adverse events (severe hypertension and PRES) after the first BMT led necessarily to CSA withdrawal. Alternative immunosuppressive treatment for GvHD prevention as tacrolimus and mycophenolate were not tolerated by our patient because toxicity > grade II. For this reason we decided to administrate sirolimus alone as GvHD prophylaxis and to prevent disease relapse after the rescue BMT. Here we report the successful use of sirolimus alone for GvHD prophylaxis after the second transplant in a pediatric BMT setting for SAA

The Hereditary Hyperferritinemia-Cataract Syndrome in 2 Italian Families

Two 8- and 9-year-old brothers were referred to the Pediatric Oncology Unit, Perugia General Hospital, because of hyperferritinemia. Both had a history of bilateral cataract and epilepsy. Genetic investigation revealed two distinct mutations in iron haemostasis genes; homozygosity for the HFE gene H63D mutation in the younger and heterozygosity in the elder. Both displayed heterozygosity for C33T mutation in the ferritin light chain iron response element. A 7-year-old boy from another family was referred to our unit because of hyperferritinemia. Genetic analyses did not reveal HFE gene mutations. Family history showed that his mother was also affected by hyperferritinemia without HFE gene mutations. Magnetic resonance imaging in the mother was positive for iron overload in the spleen. Cataract was diagnosed in mother and child. Further genetic investigation revealed the C29G mutation of the ferritin light chain iron response element. C33T and C29G mutations in the ferritin light chain iron response element underlie the Hereditary Hyperferritinemia-Cataract Syndrome (HHCS). The HFE gene H63D mutation underlies Hereditary Haemochromatosis (HH), which needs treatment to prevent organ damages by iron overload. HHCS was definitively diagnosed in all three children. HHCS is an autosomal dominant disease characterized by increased L-ferritin production. L-Ferritin aggregates accumulate preferentially in the lens, provoking bilateral cataract since childhood, as unique known organ damage. Epilepsy in one case and the spleen iron overload in another could suggest the misleading diagnosis of HH. Consequently, the differential diagnosis between alterations of iron storage system was essential, particularly in children, and required further genetic investigation

Impact of Human Herpes Virus (HHV)-6 infection in manipulated haploidentical stem cell transplantation in children

Retrospective study of HHV-6 infection and disease after haploidentical stem cell transplan

High Incidence of Early Human Herpesvirus-6 Infection in Children Undergoing Haploidentical Manipulated Stem Cell Transplantation for Hematologic Malignancies

Human herpesvirus-6 (HHV-6) infection is increasingly recognized among allogeneic hematopoietic stem cell transplantation (HSCT) recipients, with 30% at risk of reactivation in the haploidentical setting. It has been associated with encephalitis, acute graft-versus-host disease, and graft failure. Here we report 2 cohorts of pediatric haploidentical manipulated HSCT in which, despite many differences, HHV-6 reactivation and disease occurred with very high incidence compared with data reported in the literature and represented the main early post-transplant infectious complication compared with other viral, bacterial, or fungal infections. The 2 cohorts were recruited at the pediatric transplant centers of Perugia (n\u202f=\u202f13), Barcelona (n\u202f=\u202f10), and Madrid (n\u202f=\u202f15). All patients received myeloablative conditioning regimens and 2 different types of ex vivo graft manipulation: CD34+ selection and regulatory T cell/conventional T cell infusion in 13 patients and CD45RA T cell depletion in 25 patients. Antiviral prophylaxis was acyclovir in 33 and foscarnet in 5 patients. HHV-6 DNAemia was checked by quantitative or qualitative PCR. In vitro experiments demonstrated that donor CD4+ T cells are the reservoir of HHV-6 and suggested a role of the graft composition in both transplant settings (rich in CD4+ T cells) in the high rate of HHV-6 infections. All patients presented very early HHV-6 DNAemia after transplantation, and although viremic, 9 patients (24%) developed symptomatic limbic encephalitis. All patients responded to antiviral treatment, and none died of infection, although 2 experienced long-term neurologic sequelae (22%). Moreover, 6 patients presented organ involvement in absence of other causes: 1 hepatitis, 1 pneumonia, 2 gastroenteritis, and 2 multiorgan involvement(1 encephalitis, pneumonia, and gastritis; 1 pneumonia and enteritis). Incidences of other viral, bacterial, and fungal diseases were lower in both cohorts. In vitro, HHV-6 was found to infect only CD4+ fraction of the graft. Co-culturing CD4+ T cells with CD56+ natural killer (NK) cells eliminated the virus, demonstrating the main role of NK cells in the antiviral immune response. All 38 pediatric patients undergoing these manipulated haploidentical HSCTs showed HHV-6 reactivation, and 14 of 38 developed HHV-6 disease with similar features in terms of timing, morbidity, response to treatment, and outcome. The graft composition in both transplant platforms, rich in CD4+ T cells and poor in NK cells, seems to play a key role. HHV-6 DNAemia surveillance was useful to diagnose and treat preemptively HHV-6 infection

Studio di una famiglia beta talassemica mediante NGS

Introduzione Variazioni geniche concomitanti alle mutazioni beta, (es. geni alfa globinici, geni regolatori BCL11A, HBG2, HBS1L-MYB), possono modulare il fenotipo clinico. L’analisi NGS offre la possibilità di identificare nuovi geni e nuove variazioni coinvolte nella modulazione del fenotipo. Materiali e Metodi Lo studio è stato condotto su 2 fratelli affetti da Beta Talassemia Major, diversa espressività fenotipica, identico genotipo beta -29 A>G /cod8 (-AA) e geni alfa normali. Abbiamo sviluppato un pannello NGS (SOPHiA GENETICS) per lo studio dei geni HBB, HBA1, HBA2, HBD, BMP6, BMP2, ERFE, FTL, HAMP, HFE, HJV, SLC11A2, SLC40A1, TFR2, TMPRSS6, PKLR, KFL1, BCL11A. Risultati Sono state identificate 44 varianti nel pt.1 e 45 del pt.2. Quattro varianti sono presenti solo nel pt.1 (1 in ERFE in eterozigosi, 1 in SLC40A1 in eterozigosi, 2 in TMPRSS6 in omozigosi) e due solo nel pt.2 (1 in SLC40A1 in eterozigosi e 1 in TMPRSS6 in eterozigosi). Inoltre 9 varianti comuni (1 in ERFE, 2 in BCL11A e 6 in TMPRSS6) presentavano differenze nell’assetto genotipico (omozigote/eterozigote). Il pt 1 presenta regime trasfusionale più intenso (2 GRC/settimana vs 1 GRC/settimana) senza sovraccarico marziale RMt2* (deferasirox). Il paziente 2 presenta moderato sovraccarico cardiaco ed epatico, ferritinemia elevata (deferasirox cpr rivestite, pregressa idiosincrasia epatica a deferasirox dispersibile e agranulocitosi con deferiprone). Conclusioni La caratterizzazione genomica individuale rappresenta sempre più una necessità per ottenere una correlazione tra genotipo/fenotipo e una potenziale stratificazione prognostica e terapeutica. I nostri dati, seppur preliminari, mostrano che 6 varianti segregano diversamente nei due fratelli

Emoglobinopatie e flussi migratori in Umbria, una regione non endemica

Le emoglobinopatie rappresentano un gruppo eterogeneo di disordini ereditari dell’emoglobina. L’Umbria è una regione considerata storicamente non endemica per tali patologie, tuttavia negli ultimi anni a causa dei flussi migratori la casistica di pazienti affetti seguiti presso il nostro Centro è in continua crescita. Dal 1988 al 2015 sono stati identificati 262 pazienti con alterazioni dei geni alfa o beta globinici (188 beta e 74 alfa), di cui 45 soggetti malati omozigoti o eterozigoti composti per le alterazioni a carico del gene beta globinico e 143 portatori, mentre 40 pazienti con alfa-2 talassemia, 27 con alfa-1 talassemia e 7 con triplicazione del gene alfa. 60% beta-talassemia, 85% drepanocitosi, 40% microdrepanocitosi e l’85% degli eterozigoti composti per varianti beta emoglobiniche sono stati diagnosticati negli ultimi tre anni. Inoltre, la maggior parte degli pazienti beta omozigoti o eterozigoti composti (84,5%) provenivano da paesi stranieri, mentre solo il 15,5% sono di origine italiana (Gorello et al, Hemoglobin). Tale fenomeno non si è arrestato infatti dal 2016 al 2018 sono stati identificati 152 pazienti (range età: 10 giorni-75 anni; rapporto M/F:76/76) aventi mutazioni a carico geni globinici. 104 pazienti con alterazioni a carico del gene beta globinico (2 talassemie, 6 drepanocitosi, 1 microdrepanocitosi, 1 HbE/HbE, 1 HbS/HbC, 93 portatori di varianti quantitative o qualitative); di cui 58 stranieri e 46 Italiani (36 Africani, 5 Americani, 4 Asiatici e 59 Europei). Mentre 48 pazienti con alterazioni a carico dei geni alfa globinici (10 alfa-1 talassemia, 29 alfa-2 talassemia, 2 malattia da HbH, 7 triplo alfa); di cui 30 stranieri e 18 Italiani (12 Africani, 6 Asiatici, 28 Europei e 2 i dati della provenienza non sono disponibili). Complessivamente dal 2012 ad oggi sono stati identificati 307 pazienti (74%) con alterazioni a carico dei geni globinici, di cui il 63% ha un’origine straniera (194 stranieri, 109 Italiani e 4 ND). Tale dato indica il forte incremento di pazienti malati o portatori che necessità quindi di un adeguamento sia in termini diagnostici che di gestione clinica del paziente anche in regioni storicamente non endemiche

Studio molecolare monocentrico di drepanocitosi e varianti falcemiche: valutazione di 6 loci polimorfi in geni regolatori

L’Hb S, variante emoglobinica più diffusa nel mondo, è dovuta a una mutazione a carico del gene beta globinico (HBB:c.20A>T). L’associazione tra alleli codificanti per HbS e alleli caratterizzati da differenti alterazioni possono dare origine a diversi fenotipi. Lo scopo del lavoro è quello di eseguire una caratterizzazione molecolare di pazienti presentanti agli esami di primo livello l’HbS, per effettuare una corretta stratificazione e una stretta correlazione genotipo/fenotipo. Dal 2000 al 2018 sono stati diagnosticati 37 pazienti (range età: 10 gg-60 anni, M/F: 26/10; 25 africani, 6 europei, 3 americani e 3 non disponibili). Lo studio del gene HBB ha permesso di identificare: 20 drepanocitosi, 10 microdrepanocitosi e 7 HbS/HbC. Inoltre sono stati studiati i geni alfa globinici e polimorfismi associati alla variazione di emoglobina fetale, essendo tali fattori possibili modulatori del fenotipo clinico. Nel gruppo dei 20 drepanocitosi (M/F: 17/3; 16 africani, 2 europei e 2 americani) sono stati identificati 5 pazienti con -3.7 in eterozigosi, mentre 1 con -3.7 in omozigosi. Nei 10 microdrepanocitosi (M/F: 7/3; 4 europei, 2 africani, 1 americano e 3 ND) sono stati evidenziati 4 diversi genotipi: HbS/cd39 (C>T), 3 pts; HbS /-29 (A>G), 3 pts; HbS /IVS-I-110 (G>A), 3 pts e HbS /IVS1-6 (T>C), 1 pt. L’analisi dei geni alfa globinici, eseguita in 9/10 pazienti, ha identificato in un paziente (Hb S/ cd 39(C>T)) una triplicazione del gene alfa. Nel gruppo dei 7 HbS/HbC (M/F:3/4; 7 africani) in 2 pazienti è stata riscontrata la mutazione -3.7 in eterozigosi. 33/37 pazienti sono stati analizzati per 5 loci polimorfi: HBG2:g.-158 C>T; BCL11A: rs1427407 G>T e rs10189857 A>G; HBS1L-MYB: rs28384513 A>C e rs9399137 T>C coinvolti nell’espressione della emoglobina fetale; 32/33 pazienti presentavano almeno un polimorfismo e 1/33 (HbS/HbS) è risultato negativo. BCL11A: rs10189857 A>G è il più polimorfo alterato in 19/33 pts (7 microdrepanocitosi, 7 drepanocitosi e 5 eterozigoti composti). HBS1L-MYB: rs9399137 T>C alterato in un paziente (HbS/cd39 (C>T)) risulta essere il meno polimorfo. Lo studio di C/EBPE: rs45496295 C>T è risultato negativo in tutti i pazienti analizzati. Lo studio di nuove alterazioni genetiche, sia nei suddetti locus genici che in altri, e l’incrocio con i dati clinici porterà sempre di più verso una precisa stratificazione dei pazienti, che risulta necessaria sia a fini diagnostici che per la gestione clinica del paziente

Clinical And Molecular Spectrum Of Glucose-6-Phosphate Isomerase Deficiency. Report Of 12 New Cases

Glucose-6-phosphate isomerase (GPI, EC 5.3.1.9) is a dimeric enzyme that catalyzes the reversible isomerization of glucose-6-phosphate to fructose-6-phosphate, the second reaction step of glycolysis. GPI deficiency, transmitted as an autosomal recessive trait, is considered the second most common erythro-enzymopathy of anaerobic glycolysis, after pyruvate kinase deficiency. Despite this, this defect may sometimes be misdiagnosed and only about 60 cases of GPI deficiency have been reported. GPI deficient patients are affected by chronic non-spherocytic hemolytic anemia of variable severity; in rare cases, intellectual disability or neuromuscular symptoms have also been reported. The gene locus encoding GPI is located on chromosome 19q13.1 and contains 18 exons. So far, about 40 causative mutations have been identified. We report the clinical, hematological and molecular characteristics of 12 GPI deficient cases (eight males, four females) from 11 families, with a median age at admission of 13 years (ranging from 1 to 51); eight of them were of Italian origin. Patients displayed moderate to severe anemia, that improves with aging. Splenectomy does not always result in the amelioration of anemia but may be considered in transfusion-dependent patients to reduce transfusion intervals. None of the patients described here displayed neurological impairment attributable to the enzyme defect. We identified 13 different mutations in the GPI gene, six of them have never been described before; the new mutations affect highly conserved residues and were not detected in 1000 Genomes and HGMD databases and were considered pathogenic by several mutation algorithms. This is the largest series of GPI deficient patients so far reported in a single study. The study confirms the great heterogeneity of the molecular defect and provides new insights on clinical and molecular aspects of this disease.PubMedWoSScopu

A prospective study on the efficacy of mobilization of autologous peripheral stem cells in pediatric oncohematology patients

Peripheral blood stem cells (PBSCs) are the preferred source in autologous transplantation. We assessed prospectively the efficacy of mobilization in pediatric patients and risk factors associated with its failure

Monitoring oral iron therapy in children with iron deficiency anemia: an observational, prospective, multicenter study of AIEOP patients (Associazione Italiana Emato-Oncologia Pediatrica)

Oral ferrous salts are standard treatment for children with iron deficiency anemia (IDA). The objective of our study was to monitor oral iron therapy in children, aged 3 months-12 years, with IDA. We prospectively collected clinical and hematological data of children with IDA, from 15 AIEOP (Associazione Italiana di Ematologia ed. Oncologia Pediatrica) centers. Response was measured by the increase of Hb from baseline. Of the 107 analyzed patients, 18 received ferrous gluconate/sulfate 2 mg/kg (ferrous 2), 7 ferrous gluconate/sulfate 4 mg/kg (ferrous 4), 7 ferric iron salts 2 mg/kg (ferric), 62 bis-glycinate iron 0.45 mg/kg (glycinate), and 13 liposomal iron 0.7-1.4 mg/kg (liposomal). Increase in reticulocytes was evident at 3 days, while Hb increase appeared at 2 weeks. Gain of Hb at 2 and 8 weeks revealed a higher median increase in both ferrous 2 and ferrous 4 groups. Gastro-intestinal side effects were reported in 16% (ferrous 2), 14% (ferrous 4), 6% (glycinate), and 0 (ferric and liposomal) patients. The reticulocyte counts significantly increased after 3 days from the start of oral iron supplementation. Bis-glycinate iron formulation had a good efficacy/safety profile and offers an acceptable alternative to ferrous iron preparations