Diagnosis and management of acquired aplastic anemia in childhood. Guidelines from the Marrow Failure Study Group of the Pediatric Haemato-Oncology Italian Association (AIEOP).

Acquired aplastic anemia (AA) is a rare heterogeneous disorder characterized by pancytopenia and hypoplastic bone marrow. The incidence is 2-3 per million population per year in the Western world, but 3 times higher in East Asia. Survival in severe aplastic anemia (SAA) has improved significantly due to advances in hematopoietic stem cell transplantation (HSCT), immunosuppressive therapy, biologic agents, and supportive care. In SAA, HSCT from a matched sibling donor (MSD) is the first-line treatment. If a MSD is not available, options include immunosuppressive therapy (IST), matched unrelated donor, or haploidentical HSCT. The purpose of this guideline is to provide health care professionals with clear guidance on the diagnosis and management of pediatric patients with AA. A preliminary evidence-based document prepared by a group of pediatric hematologists of the Bone Marrow Failure Study Group of the Italian Association of Pediatric Hemato-Oncology (AIEOP) was discussed, modified and approved during a series of consensus conferences that started online during COVID 19 and continued in the following years, according to procedures previously validated by the AIEOP Board of Directors.

Late-onset and long-lasting neutropenias in the young: A new entity anticipating immune-dysregulation disorders.

This study identifies a new chronic form of immune neutropenia in the young with or without detectable indirect anti-neutrophil antibodies, characterized by mild/moderate neutropenia low risk of severe infection (14%), tendency to develop autoimmune phenomena over the course of the disease (cumulative incidence of 58.6% after 20 years of disease duration), leukopenia, progressive reduction of absolute lymphocyte count and a T- and B-cell profile similar to autoimmune disorders like Sjogren syndrome, rheumatoid arthritis, and systemic lupus erythematosus (increased HLADR+ and CD3 + TCRγδ cells, reduced T regulatory cells, increased double-negative B and a tendency to reduced B memory cells). In a minority of patients, P/LP variants related to primary immuno-regulatory disorders were found. This new form may fit the group of "Likely acquired neutropenia," a provisional category included in the recent International Guidelines on Diagnosis and Management of Neutropenia of EHA and EUNET INNOCHRON ACTION 18233. The early recognition of this form of neutropenia would help clinicians to delineate better specific monitoring plans, genetic counseling, and potentially targeted therapies.

Hypomorphic RAG deficiency: impact of disease burden on survival and thymic recovery argues for early diagnosis and HSCT.

Patients with hypomorphic mutations in the RAG1 or RAG2 gene present with either Omenn syndrome or atypical combined immunodeficiency with a wide phenotypic range. Hematopoietic stem cell transplantation (HSCT) is potentially curative, but data are scarce. We report on a worldwide cohort of 60 patients with hypomorphic RAG variants who underwent HSCT, 78% of whom experienced infections (29% active at HSCT), 72% had autoimmunity, and 18% had granulomas pretransplant. These complications are frequently associated with organ damage. Eight individuals (13%) were diagnosed by newborn screening or family history. HSCT was performed at a median of 3.4 years (range 0.3-42.9 years) from matched unrelated donors, matched sibling or matched family donors, or mismatched donors in 48%, 22%, and 30% of the patients, respectively. Grafts were T-cell depleted in 15 cases (25%). Overall survival at 1 and 4 years was 77.5% and 67.5% (median follow-up of 39 months). Infection was the main cause of death. In univariable analysis, active infection, organ damage pre-HSCT, T-cell depletion of the graft, and transplant from a mismatched family donor were predictive of worse outcome, whereas organ damage and T-cell depletion remained significant in multivariable analysis (hazard ratio [HR] = 6.01, HR = 8.46, respectively). All patients diagnosed by newborn screening or family history survived. Cumulative incidences of acute and chronic graft-versus-host disease were 35% and 22%, respectively. Cumulative incidences of new-onset autoimmunity was 15%. Immune reconstitution, particularly recovery of naïve CD4+ T cells, was faster and more robust in patients transplanted before 3.5 years of age, and without organ damage. These findings support the indication for early transplantation.

New targets in pediatric Acute Myeloid Leukemia.

In the last few years the improvements of chemotherapy regimens and supportive care has progressively ameliorated the prognosis of children suffering from Acute Myeloid Leukemia (AML). However, a still high percentage of children do not respond to first line treatments or relapse and need to undergo further treatments. The need to explore new agents other than chemotherapy has been highlighted in the last years in order to overcome drug related resistance and toxicity. Recently, novel therapies have been studied within early phases pediatric trials and seem to show encouraging results. In fact, the knowledge of molecular abnormalities related to AML pathogenesis has permitted to identify selective drugs that may represent an important tool for the development of patient-tailored treatments. Nowadays, FLT3, Aurora Kinases, mTORS's and proteasome inhibitors represents the most promising drugs that are being used in pediatric AML studies.

Nuclear factor-kappa-B-inhibitor alpha (NFKBIA) is a developmental marker of NF-κB/p65 activation during in vitro oocyte maturation and early embryogenesis.

BACKGROUND: The oocyte-to-embryo transition (OET) requires a co-ordinated transcriptional programme acting through evolutionarily conserved events, and transcription factors (TFs) are known to control these processes. Here, we focus on nuclear factor (NF)-κB, a TF involved in several cellular processes, studying NFκB-inhibitor (NFKBIA) mRNA and its protein product, IκBα, during OET. NFKBIA and IκBα are part of a regulatory loop, as IκBα is the major down-regulator of NF-κB activation while NFKBIA transcription is activated by NF-κB. METHODS AND RESULTS: We found a dynamic correlation between NFKBIA transcript, expression of IκBα-protein and activation of NF-κB/p65 in bovine oocyte and embryo. During the transition from immature to in vitro matured bovine oocyte, we observed a decrease in maternal NFKBIA mRNA and a parallel increase of the IκBα-protein (both P < 0.05). In the embryo, NFKBIA neo-synthesis is activated as a consequence of embryo genome activation (EGA), and IκBα decreases. NF-κB/p65-binding activity was detectable at low levels in immature oocyte, disappeared in dormant metaphase II oocyte and was strong in the embryo, during embryonic NFKBIA synthesis. The level of NF-κB/p65 DNA binding correlates with the timing of meiotic silencing during bovine oocyte maturation and embryonic transcription reprogramming. CONCLUSIONS: The IκBα/NF-κB circuit appears to be a tightly stage-controlled mechanism that could govern OET, being activated at EGA. Our findings represent the first characterization of NFKBIA and IκBα as maternal effectors in both the bovine oocyte and embryo. We suggest a role for NFKBIA as a marker of NF-κB/p65 activation in the human oocyte and early embryo.

Daclizumab as useful treatment in refractory acute GVHD: a paediatric experience.

GVHD remains a serious complication after allogeneic SCT. We describe 13 paediatric patients treated with daclizumab for refractory acute GVHD (aGVHD). After 30 days of daclizumab administration, all patients with cutaneous aGVHD reached complete response. Among patients with gastrointestinal disease, 50 and 30% had complete and partial response, respectively, whereas 11 and 55% of patients with hepatic aGVHD achieved CR and PR, respectively. Overall, complete (46%) and partial (46%) responses were demonstrated in 92% of our patients, whereas the remaining patients (8%) were nonresponders. No life-threatening infectious episodes were recorded within 100 days from transplant in this selected group of paediatric patients. Overall 46% of patients were alive at a median of 461 days from SCT, but 50% of them developed chronic GVHD. In our experience, daclizumab proved to be a useful and safe treatment for refractory and steroid-resistant/dependent aGVHD, in particular for cutaneous and low-moderate intestinal involvement.

Haematopoietic stem cell transplantation in children in eastern European countries 1985-2004: development, recent activity and role of the EBMT/ESH Outreach Programme.

The paediatric population of 19 eastern European countries amounts to approximately 80 million children. Between 1985 and 2004, the number of centres performing haematopoietic stem cell transplantation (HSCT) in children increased from 1 in 1985 to 24 in 2004 and the yearly number of paediatric HSCTs rose from 1 in 1985 to 291 in 2004. Altogether, 2342 transplants were reported to the EBMT Registry during this time (Poland 953, Czech Republic 501, Hungary 269, Russia 217, Croatia 129, Slovakia 71, Bulgaria 45, Serbia and Montenegro 36, Slovenia 35, Belarus 33, Estonia 26, Lithuania 19 and Romania 8). Out of the 2342 transplants, 1487 (63.5%) transplants were performed in paediatric centres, 453 (19.3%) in centres for adults and 402 (17.2%) in combined centres. The number of children who underwent autologous HSCT (auto-HSCT) was 1053 (45%), whereas 1289 (55%) underwent allogeneic HSCT (allo-HSCT). Peripheral blood (PB) was the source of HSC in 751 (71.3%) out of 1053 auto-transplants, BM in 246 (23.4%) and PB+BM in 52 (4.9%) (missing data in 4, that is, 0.4%). Among the 1289 allo-transplants, BM was the source of HSC in 827 (64.3%), PB in 416 (32.3%), CB in 23 (1.8%) and BM+PB in 14 (1.1%) (missing data in 9, that is, 0.7%). Among them, 728 (57.4%) obtained HSC from MSD, 322 (25.4%) from UD, 195 (15.4%) from MMFD, 14 (1.1%) from CB family donor and 9 (0.7%) from CB unrelated donor (missing data in 21, that is, 1.6%). The number of children who underwent allo-HSCT for malignant diseases was 945 (73.4%), including ALL 376 (29.2%), AML 234 (18.2%), CML 177 (13.8%), MDS 97 (7.5%), NHL 35 (2.7%) and other malignancy 31 (2.4%), while 339 (26.9%) for non-malignant disorders, including SAA 202 (15.7%), immunodeficiencies 61 (4.7%), inborn errors of metabolism 40 (3.1%), Fanconi anaemia 19 (1.5%) and others 17 (1.3%). Out of 1053 recipients of auto-HSCT, 168 (16%) were transplanted for neuroblastoma, 129 (12.2%) for NHL, 124 (11.7%) for AML, 114 (10.8%) for ALL, 109 (10.4%) for Hodgkin's disease, 62 (5.9%) for Ewing's sarcoma, 16 (1.5%) for CNS tumour, 15 (1.4%) for Wilms tumour and 316 (30%) for other tumours. In 2001, the EBMT in collaboration with the European School of Haematology (ESH) developed the Outreach Programme, that is a programme supporting emerging HSCT projects and transplant centres in countries with limited resources and/or experience.

Early complications following haematopoietic SCT in children.

Early complications can be defined as those occurring within 100 days after transplant. Both epithelial and endothelial damage represent the pathogenetic basis for the onset of the most frequent complications. Clinical features related to endothelial damage depend on the involved district or on the grade and type of general distribution. Veno-occlusive disease (VOD) most often occurs within the first 20 days of haematopoietic SCT (HSCT) and is characterized by the obstruction of small intrahepatic venules and is caused by an initial injury of the sinusoid endothelial cells. The incidence in children ranges between 27 and 40%, and symptoms include hepatomegaly, portal hypertension and ascites. Early intervention with defibrotide (DF) proved to be effective for the treatment; however, overall mortality ranges between 20 and 50%. Thrombotic microangiopathy (TAM) incidence is 4-13%. It is often associated with the use of CYA or tacrolimus, and symptoms include haemolytic anaemia, thrombocytopenia and renal and/or central nervous system impairment. Treatment includes plasmapheresis and supportive care. The promising role of DF needs to be confirmed. The onset of engraftment syndrome may occur 1 or 2 days before the neutrophil count in peripheral blood increases. Clinical symptoms include fever not related to infection, respiratory involvement with pulmonary infiltrates or hypoxia and skin rash. Treatment consists of steroid administration for a few days. Haemorrhagic cystitis (HC) may occur early or later following transplant. Early-onset HC is related to mucosal damage caused by the catabolites of chemotherapy drugs, and late-onset HC is mostly caused by viral infections. The incidence ranges between 1 and 25%. Clinical symptoms include haematuria and dysuria without infections. Treatment includes hyperhydration and platelet support. In case of vescical clots, bladder irrigation is indicated. In advanced cases, hyperbaric oxygen administration or surgery may be useful. The use of cidofovir for BK virus-related HC seems encouraging, but further studies are needed to confirm its real efficacy.

A novel mutation in the PHF8 gene is associated with X-linked mental retardation with cleft lip/cleft palate.

Recently, two truncating mutations in the PHF8 (plant homeodomain finger protein 8) gene have been found to cause X-linked mental retardation associated with cleft lip/cleft palate (CL/P). One of the truncating mutations was found in the original family with Siderius-Hamel CL/P syndrome where only two of the three affected individuals had mental retardation (MR) with CL/P and one individual had mild MR. The second mutation was present in a family with four affected men, three of whom had MR and CL/P, while the fourth individual had mild MR without clefting. Here, we report a novel nonsense mutation (p.K177X) in a male patient who has MR associated with CL/P. The mutation results in a truncated PHF8 protein lacking the Jumonji-like C terminus domain and five nuclear localization signals. Our finding further supports the hypothesis that the PHF8 protein may play an important role in cognitive function and midline formation.

Haematopoietic stem cell transplantation trends in children over the last three decades: a survey by the paediatric diseases working party of the European Group for Blood and Marrow Transplantation.

This paper describes the trends in haematopoietic stem cell transplantation (HSCT) activity for children in Europe over the last three decades. We analysed 31,713 consecutive paediatric HSCTs reported by the European Group for Blood and Marrow Transplantation (EBMT) centres between 1970 and 2002. Data were taken from the EBMT registry and were compared according to period and centre category (paediatric or combined). Since 1996, there has been a significant increase in the number of HSCTs performed exclusively by paediatric centres, as well as in the number of alternative donor HSCTs, and in the use of peripheral blood stem cells (P<0.0001). The number of allogeneic HSCTs (allo-HSCTs) for acute lymphoblastic leukaemia, acute myeloblastic leukaemia and chronic myeloid leukaemia remained stable, whereas it increased for myelodysplastic syndromes and lymphomas, and decreased significantly for non-malignant diseases (P<0.0001). Multivariate analysis showed that younger age, human leukocyte antigen genoidentical donors, HSCT performed after 1996 and transplant centres performing more than 10 allo-HSCT/year were all associated with decreased transplant-related mortality (TRM) (P<0.0001). The number of autologus HSCTs (auto-HSCTs) for acute leukaemia decreased significantly, whereas it increased for solid tumours (P<0.0001). Multivariate analysis showed that both auto-HSCT performed before 1996 and paediatric solid tumours (P<0.0001) had higher TRM. Indications for paediatric HSCT have changed considerably during the last seven years. These changes provide tools for decision making in health-care planning and counselling.

A91V perforin variation in healthy subjects and FHLH patients.

Familial haemophagocytic lymphohistiocytosis (FHLH) is a heterogeneous autosomal recessive disorder characterized by hyperactivation of monocytes/macrophages. Perforin (PRF1) gene alterations have been documented in 40% of patients with FHLH. Although several mutations have been identified, a clear correlation between the individual molecular alteration and the phenotypic expression of the disease is still unclear. In particular, the role that the A91V substitution plays in the pathogenesis of the disease is still controversial. In the effort to make a conclusive remark to this issue, we here report on the frequency of the A91V mutation in a group of unrelated healthy families obtained from the "Centre d'Etude du Polymorphisme Humain" (CEPH), which are considered representative of the worldwide population. This frequency was compared to that observed in FHLH patients recruited through the Italian National Registry. The frequency in CEPH healthy subjects is 3.7%, thus indicating that the alteration represents a polymorphism. However, the frequency of this alteration in FHLH patients associated with PRF1 mutation is much higher than that observed in controls (26.2%, P = 0.0002), suggesting that the alteration is an important genetic susceptibility factor.

Survey on haematopoietic stem cell transplantation for children in Europe.

A recent report, prepared in March 2003, regarding the paediatric transplantation activity registered between 1970 and 2002 in the European Bone Marrow Transplantation (EBMT) database showed a decrease in the number of registrations in 2001 and in 2002. In order to validate this observation, the Paediatric Diseases Working Party (PDsWP) secretariat distributed a questionnaire to 395 institutions participating in the EBMT Registry. Each institution was requested to check the number of transplants they reported and to confirm or to correct the figures. As of 15 March 2004, replies had been received from 135 centres reporting a median of 48 transplants per centre over the study period, total 17 891 (58% of the total number). Among them, 55 confirmed their original figures, while 80 corrected the numbers. The overall number of autologous and allogeneic SCTs performed and not reported were 461 and 692, respectively. Most of the teams that corrected their figures stated that their data managers could provide missing data to the EBMT; 260 other teams, each reporting a median of 15 transplants during the study period, total 12 866 (42% of the total number) chose not to reply. A report prepared in March 2004, following the PDsWP survey, showed an increasing number of transplants performed on patients below 18 years of age between 1973 and 2002 and reported to the EBMT Registry (328 autologous and 628 allogeneic) as compared to the 2003 report. This first PDsWP survey, reaching more than 50% of activity in the field, illustrates that the decrease in activity we observed in the 2003 report does not correspond to a decrease in the number of transplants that were actually performed. It demonstrates the compliance of most major paediatric institutions and confirms the important role of cooperation between National Registries and EBMT Registries.

Home care for children following haematopoietic stem cell transplantation.

The quality of life of patients who undergo haematopoietic stem cell transplantation (HSCT) is affected by long periods of hospitalisation for the treatment of several complications. On this basis, 28 children who underwent 29 HSCTs were included in the Home Care (HC) programme of the Paediatric Haematology and Oncology Department of the Gaslini Children's Hospital to be discharged earlier. A total of 17 children were assisted for haematologic follow-up and support therapy administration. The remaining children were followed up for graft- versus-host disease and/or cytomegalovirus infection. Overall activity consisted of 1232 i.v. therapies, 501 blood tests, 58 red blood cell or platelet transfusions, 107 procedures on Central Venous Catheter. Median duration of the assistance per child was 25 days (range 1235) for a total of 1598 days. A total of 822 accesses at home replaced 459 and 363 out-patient and in-patient days of hospitalisation. The average cost per patient receiving HC (EUR 4,252) was significantly lower (P<0.01) when compared to the average cost per patient admitted to the hospital to undergo the same procedures (EUR 14,693). This report shows that HC is feasible for children following HSCT, that it reduces the discomfort of the patients and their families, and that it reduces costs.

Identification of novel RP2 mutations in a subset of X-linked retinitis pigmentosa families and prediction of new domains.

X-linked Retinitis Pigmentosa (XLRP) shows a huge genetic heterogeneity with almost five distinct loci on the X chromosome. So far, only two XLRP genes have been identified, RPGR (or RP3) and RP2, being mutated in approximately 70% and 10% of the XLRP patients. Clinically there is no clearly significative difference between RP3 and RP2 phenotypes. In the attempt to assess the degree of involvement of the RP2 gene, we performed a complete mutation analysis in a cohort of patients and we identified five novel mutations in five different XLRP families. These mutations include three missense mutations, a splice site mutation, and a single base insertion, which, because of frameshift, anticipates a stop codon. Four mutations fall in RP2 exon 2 and one in exon 3. Evidence that such mutations are different from the 21 RP2 mutations described thus far suggests that a high mutation rate occurs at the RP2 locus, and that most mutations arise independently, without a founder effect. Our mutation analysis confirms the percentage of RP2 mutations detected so far in populations of different ethnic origin. In addition to novel mutations, we report here that a deeper sequence analysis of the RP2 product predicts, in addition to cofactor C homology domain, further putative functional domains, and that some novel mutations identify RP2 amino acid residues which are evolutionary conserved, hence possibly crucial to the RP2 function.

Four year follow-up of a case of fucosidosis treated with unrelated donor bone marrow transplantation.

Fucosidosis is a rare autosomal recessive lysosomal disorder caused by alpha-fucosidase deficiency. We report a child with fucosidosis, second daughter of non-consanguineous parents, for whom biochemical diagnosis followed clinical evidence of the disease in her older sister. Based on previous experiences, the indication to transplant was considered. Since she lacked a matched sibling, an unrelated marrow donor was found. At pre-hematopoietic stem cell transplantation evaluation, first signs of neurological involvement were clinically detectable. MRI showed diffuse hypomyelination and auditory brainstem responses and somatic-sensorial evoked potentials were altered. Visual evoked potentials were normal, tortuosity in the retinal veins and peripapillary hemorrhages were detected. Bone marrow transplantation conditioning was with a regimen of busulphan, thiotepa and cyclophosphamide; in vivo Campath 1G, cyclosporin A and short course methotrexate were given to prevent graft-versus-host disease. The patient engrafted rapidly and her post-transplant course was complicated by moderate graft-versus-host disease, transient episodes of idiopathic thrombocytopenic purpura, repeated septic complications and recurrent episodes of Sweet's syndrome. Sequential short tandem repeat polymorphisms on peripheral blood and bone marrow cells documented the persistence of donor engraftment. Follow-up showed a progressive rise of enzymatic levels. Psychomotor development improved, as confirmed by evaluation of evoked potentials and by MRI scanning.

Megatherapy combining I(131) metaiodobenzylguanidine and high-dose chemotherapy with haematopoietic progenitor cell rescue for neuroblastoma.

Despite the use of aggressive chemotherapy, stage 4 high risk neuroblastoma still has very poor prognosis which is estimated at 25%. Metabolic radiotherapy with I(131) MIBG appears a feasible option to enhance the effects of chemotherapy. Seventeen patients having MIBG-positive residual disease received 4.1-11.1 mCi/kg of I(131) MIBG 7-10 days before initiating the high-dose chemotherapy cycle consisting of busulphan 16 mg/kg and melphalan 140 mg/m(2) followed by PBSC infusion. We compared the toxicity in these patients to that seen in 15 control subjects with neuroblastoma who underwent a PBSC transplant without MIBG therapy. We observed greater toxic involvement of the gastrointestinal system in children treated with I(131) MIBG: grade 2 or 3 mucositis developed in 13/17 patients treated with I(131) MIBG and in 9/15 treated without it. Grade 1-2 gastrointestinal toxicity occurred in 12/17 children given MIBG and in 5/15 of the controls. One child receiving I(131) MIBG developed transient interstitial pneumonia. Another child who also received I(131) MIBG after PBSC rescue developed fatal pneumonia after the third course of metabolic radiotherapy. Our experience indicates that MIBG can be included in the high-dose chemotherapy regimens followed by PBSC rescue for children with residual neuroblastoma taking up MIBG. Attention should be paid to avoiding lung complications. Prospective studies are needed to demonstrate the real efficacy of this treatment.

Stem cell transplantation from HLA-matched related donor for Fanconi's anaemia: a retrospective review of the multicentric Italian experience on behalf of AIEOP-GITMO.

Twenty-seven consecutive Italian patients with Fanconi's anaemia (FA) underwent stem cell transplantation (SCT) from an HLA-matched related donor in 10 Italian centres of the Associazione Italiana Ematologia ed Oncologia Pediatrica (AIEOP), Gruppo Italiano di Trapianto di Midollo Osseo (GITMO). Twenty-two patients (81.5%) were conditioned with low-dose (median 20 mg/kg) cyclophosphamide (Cy) and thoraco-abdominal or total body irradiation (median dose 500 cGy), five patients (18.5%) with high-dose Cy (median 120 mg/kg). Graft-vs.-host disease (GVHD) prophylaxis was carried out with cyclosporin A in 26 cases; methotrexate (MTX) was added in eight cases. One patient received MTX alone. The median follow-up was 36 months. Ninety-two percent of patients (25 out of 27) engrafted, grade II and III acute GVHD occurred in 28% and 8% of patients, respectively, with chronic GVHD in 12.5%. Conditioning-related toxicity was mild: 4% of patients had grade III mucositis, 7.4% had grade II haemorrhagic cystitis, 14.8% had grade III liver toxicity and 11.1% had grade III renal toxicity. Transplant-related mortality at 12 months was 19.2%, survival at 36 months was 81.5%, with a median Karnofsky score of 100%. No late tumours occurred after a mean follow-up of the survivors of 5 years. None of the studied variables significantly affected the survival, including conditioning regimen, acute GVHD and clinical non-haematological phenotype. Among the studied variables, only conditioning regimens containing high-dose Cy and the presence of genital abnormalities were significantly (P < 0.05) associated with an increased rate of acute GVHD. Our study demonstrates that the Italian FA patients undergoing SCT from an HLA-matched related donor have a very good outcome. These patients, when compared with others of different ethnic origin who underwent allogeneic bone marrow transplantation, showed a less severe non-haematological phenotype, raising the possibility that this milder phenotype may have, at least in part, contributed to the outcome. Our data may provide a useful tool for further studies aiming to correlate genotype with phenotype.