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.

A Case Series and Literature Review of Isavuconazole Use in Pediatric Patients with Hemato-oncologic Diseases and Hematopoietic Stem Cell Transplantation.

We analyzed the use of isavuconazole (ISA) as treatment or prophylaxis for invasive fungal disease (IFD) in children with hemato-oncologic diseases. A multicentric retrospective analysis was performed among centers belonging to the Italian Association for Pediatric Hematology and Oncology (AIEOP). Pharmacokinetic (PK) monitoring was applied by a high-performance liquid chromatography-tandem mass spectrometry (HLPC-MS/MS) assay. Twenty-nine patients were studied: 10 during chemotherapy and 19 after allogeneic hematopoietic stem cell transplantation (HSCT). The patients consisted of 20 males and 9 females with a median age of 14.5 years (age range, 3 to 18 years) and a median body weight of 47 kg (body weight range, 15 to 80 kg). ISA was used as prophylaxis in 5 patients and as treatment in 24 cases (20 after therapeutic failure, 4 as first-line therapy). According to European Organization for Research and Treatment of Cancer (EORTC) criteria, we registered 5 patients with proven IFD, 9 patients with probable IFD, and 10 patients with possible IFD. Patients with a body weight of <30 kg received half the ISA dose; the others received ISA on the adult schedule (a 200-mg loading dose every 8 h on days 1 and 2 and a 200-mg/day maintenance dose); for all but 10 patients, the route of administration switched from the intravenous route to the oral route during treatment. ISA was administered for a median of 75.5 days (range, 6 to 523 days). The overall response rate was 70.8%; 12 patients with IFD achieved complete remission, 5 achieved partial remission, 5 achieved progression, and 3 achieved stable IFD. No breakthrough infections were registered. PK monitoring of 17 patients revealed a median ISA steady-state trough concentration of 4.91 mg/liter (range, 2.15 to 8.54 mg/liter) and a concentration/dose (in kilograms) ratio of 1.13 (range, 0.47 to 3.42). Determination of the 12-h PK profile was performed in 6 cases. The median area under the concentration-time curve from 0 to 12 h was 153.16 mg·h/liter (range, 86.31 to 169.45 mg·h/liter). Common Terminology Criteria for Adverse Events grade 1 to 3 toxicity (increased transaminase and/or creatinine levels) was observed in 6 patients, with no drug-drug interactions being seen in patients receiving immunosuppressants. Isavuconazole may be useful and safe in children with hemato-oncologic diseases, even in the HSCT setting. Prospective studies are warranted.

Pharmacogenetics and induction/consolidation therapy toxicities in acute lymphoblastic leukemia patients treated with AIEOP-BFM ALL 2000 protocol.

Drug-related toxicities represent an important clinical concern in chemotherapy, genetic variants could help tailoring treatment to patient. A pharmacogenetic multicentric study was performed on 508 pediatric acute lymphoblastic leukemia patients treated with AIEOP-BFM 2000 protocol: 28 variants were genotyped by VeraCode and Taqman technologies, deletions of GST-M1 and GST-T1 by multiplex PCR. Toxicities were derived from a central database: 251 patients (49.4%) experienced at least one gastrointestinal (GI) or hepatic (HEP) or neurological (NEU) grade III/IV episode during the remission induction phase: GI occurred in 63 patients (12.4%); HEP in 204 (40.2%) and NEU in 44 (8.7%). Logistic regression model adjusted for sex, risk and treatment phase revealed that ITPA rs1127354 homozygous mutated patients showed an increased risk of severe GI and NEU. ABCC1 rs246240 and ADORA2A rs2236624 homozygous mutated genotypes were associated to NEU and HEP, respectively. These three variants could be putative predictive markers for chemotherapy-related toxicities in AIEOP-BFM protocols.

Hemopoietic stem cell transplantation in thalassemia: a report from the European Society for Blood and Bone Marrow Transplantation Hemoglobinopathy Registry, 2000-2010.

Allogeneic hemopoietic stem cell transplantation (HSCT) is the only method currently available to cure transfusion-dependent thalassemia major that has been widely used worldwide. To verify transplantation distribution, demography, activity, policies and outcomes inside the European Group for Blood and Marrow Transplantation (EBMT), we performed a retrospective non-interventional study, extracting data from the EBMT hemoglobinopathy prospective registry database. We included 1493 consecutive patients with thalassemia major transplanted between 1 January 2000 and 31 December 2010. In total, 1359 (91%) transplants were performed on patients <18 years old, 1061 were from a human leukocyte Ag-identical sibling donor. After a median observation time of 2 years, the 2-year overall survival (OS) and event-free survival (EFS; that is, thalassemia-free survival) were 88 ± 1% and 81 ± 1%, respectively. Transplantation from a human leukocyte Ag-identical sibling offered the best results, with OS and EFS of 91 ± 1% and 83 ± 1%, respectively. No significant differences in survival were reported between countries. The threshold age for optimal transplant outcomes was around 14 years, with an OS of 90-96% and an EFS of 83-93% when transplants were performed before this age. Allogeneic HSCT for thalassemia is a curative approach that is employed internationally and produces excellent results.

Outcome of children with high-risk acute myeloid leukemia given autologous or allogeneic hematopoietic cell transplantation in the aieop AML-2002/01 study.

We analyzed the outcome of 243 children with high-risk (HR) AML in first CR1 enrolled in the AIEOP-2002/01 protocol, who were given either allogeneic (ALLO; n=141) or autologous (AUTO; n=102) hematopoietic SCT (HSCT), depending on the availability of a HLA-compatible sibling. Infants, patients with AML-M7, or complex karyotype or those with FLT3-ITD, were eligible to be transplanted also from alternative donors. All patients received a myeloablative regimen combining busulfan, cyclophosphamide and melphalan; [corrected] AUTO-HSCT patients received BM cells in most cases, while in children given ALLO-HSCT stem cell source was BM in 96, peripheral blood in 19 and cord blood in 26. With a median follow-up of 57 months (range 12-130), the probability of disease-free survival (DFS) was 73% and 63% in patients given either ALLO- or AUTO-HSCT, respectively (P=NS). Although the cumulative incidence (CI) of relapse was lower in ALLO- than in AUTO-HSCT recipients (17% vs 28%, respectively; P=0.043), the CI of TRM was 7% in both groups. Patients transplanted with unrelated donor cord blood had a remarkable 92.3% 8-year DFS probability. Altogether, these data confirm that HSCT is a suitable option for preventing leukemia recurrence in HR children with CR1 AML.

Sirolimus-based graft-versus-host disease prophylaxis promotes the in vivo expansion of regulatory T cells and permits peripheral blood stem cell transplantation from haploidentical donors.

Hematopoietic stem cell transplantation (HSCT) from human leukocyte antigen (HLA) haploidentical family donors is a promising therapeutic option for high-risk hematologic malignancies. Here we explored in 121 patients, mostly with advanced stage diseases, a sirolimus-based, calcineurin-inhibitor-free prophylaxis of graft-versus-host disease (GvHD) to allow the infusion of unmanipulated peripheral blood stem cell (PBSC) grafts from partially HLA-matched family donors (TrRaMM study, Eudract 2007-5477-54). Conditioning regimen was based on treosulfan and fludarabine, and GvHD prophylaxis on antithymocyte globulin Fresenius (ATG-F), rituximab and oral administration of sirolimus and mycophenolate. Neutrophil and platelet engraftment occurred in median at 17 and 19 days after HSCT, respectively, and full donor chimerism was documented in patients' bone marrow since the first post-transplant evaluation. T-cell immune reconstitution was rapid, and high frequencies of circulating functional T-regulatory cells (Treg) were documented during sirolimus prophylaxis. Incidence of acute GvHD grade II-IV was 35%, and occurrence and severity correlated negatively with Treg frequency. Chronic GvHD incidence was 47%. At 3 years after HSCT, transpant-related mortality was 31%, relapse incidence 48% and overall survival 25%. In conclusion, GvHD prophylaxis with sirolimus-mycophenolate-ATG-F-rituximab promotes a rapid immune reconstitution skewed toward Tregs, allowing the infusion of unmanipulated haploidentical PBSC grafts.

T-cell receptor Vß skewing frequently occurs in refractory cytopenia of childhood and is associated with an expansion of effector cytotoxic T cells: a prospective study by EWOG-MDS.

Immunosuppressive therapy (IST), consisting of antithymocyte globulin and cyclosporine A, is effective in refractory cytopenia of childhood (RCC), suggesting that, similar to low-grade myelodysplastic syndromes in adult patients, T lymphocytes are involved in suppressing hematopoiesis in a subset of RCC patients. However, the potential role of a T-cell-mediated pathophysiology in RCC remains poorly explored. In a cohort of 92 RCC patients, we prospectively assessed the frequency of T-cell receptor (TCR) ß-chain variable (Vß) domain skewing in bone marrow and peripheral blood by heteroduplex PCR, and analyzed T-cell subsets in peripheral blood by flow cytometry. TCRVß skewing was present in 40% of RCC patients. TCRVß skewing did not correlate with bone marrow cellularity, karyotype, transfusion history, HLA-DR15 or the presence of a PNH clone. In 28 patients treated with IST, TCRVß skewing was not clearly related with treatment response. However, TCRVß skewing did correlate with a disturbed CD4(+)/CD8(+) T-cell ratio, a reduction in naive CD8(+) T cells, an expansion of effector CD8(+) T cells and an increase in activated CD8(+) T cells (defined as HLA-DR(+), CD57(+) or CD56(+)). These data suggest that T lymphocytes contribute to RCC pathogenesis in a proportion of patients, and provide a rationale for treatment with IST in selected patients with RCC.

Gonadal and uterine function in female survivors treated by chemotherapy, radiotherapy, and/or bone marrow transplantation for childhood malignant and non-malignant diseases.

OBJECTIVE: To evaluate gonadal function and uterine volume in a cohort of female survivors treated by chemotherapy, radiotherapy, and/or stem cell transplantation (SCT) for childhood malignant and non-malignant diseases. DESIGN: An observational study. SETTING: S. Matteo Hospital, Pavia, Italy. POPULATION: A cohort of 135 female survivors. METHODS: A clinical, hormonal, and ultrasonographic evaluation. Thirty-three patients (24%) had non-malignant haematologic diseases (thalassaemia or sickle cell anaemia), 68 (50%) had leukaemia, 23 (17%) had lymphomas, and 11 (8%) had solid tumours. In total, 106 patients had received SCT, preceded by a conditioning regimen. MAIN OUTCOME MEASURES: Anti-Müllerian hormone (AMH) and Inhibin-B, and uterine volume. RESULTS: The median concentrations of AMH and Inhibin-B in the entire cohort were 0.12 ng/ml (interquartile range, IQR, 0.1-0.5 ng/ml) and 3.5 pg/ml (IQR 0.1-13.2 pg/ml), respectively. In a stepwise ordered logistic regression analysis, conventional chemotherapy for the treatment of malignancies, as opposed to total body irradiation (TBI), was the only oncologically significant predictor of increased AMH levels (OR 4.8, 95% CI 1.9-12, P < 0.001). Conditioning treatment before or after menarche did not influence AMH concentrations (P = 0.24). The best predictor of reduced uterine volume was TBI during the preparation for the allograft (OR 3.5, 95% CI 1.4-8.4, P = 0.006). Increasing age at treatment (OR 0.86, 95% CI 0.77-0.95, P = 0.04), chemotherapy, as opposed to other treatments (OR 0.09, 95% CI 0.03-0.28, P < 0.001), and solid tumours as opposed to either leukaemia/lymphomas or non-malignant diseases (OR 0.2, 95% CI 0.07-0.56, P = 0.002) were associated with larger uterine volumes. CONCLUSIONS: Conditioning therapies for SCT, including TBI, had the worst effects on uterine volume and gonadal reserve. Increasing age at treatment and conventional chemotherapy were associated with less detrimental effects on uterine volume.

Polymer-hematite nanocomposites: templating effect of commercial ion-exchangers in the growth of size-controlled iron oxide nanoparticles.

The commercially available ion-exchange resin Amberlite IR120 has been used as the stabilizing agent for the preparation of a size controlled nanostructured hematite phase. The employed synthetic approach, based on the loading of Fe3+ by ion exchange and the subsequent treatment with an aqueous base solution, produces a nanocomposite material with a remarkably high content of oxide nanoparticles (iron content = 18.8% w/w, corresponding to 24.9% w/w of Fe2O3). Scanning electron microscopy and energy dispersive X-ray spectroscopy investigation show that the iron distribution is egg-like and parallels the distribution of the sulfonic groups of the ion-exchanger. Transmission electron microscopy characterization reveals that the size of the ferric oxide nanoparticles in the nanocomposites is narrowly distributed in the 4-6 nm range and that it is the same after the first and the fifth ion-exchange-precipitation cycle. Selected area Electron Diffraction (SAED) analysis of the nanostructured oxide after five ion-exchange-precipitation cycles indicates that it is hematite with a distorted structure.

Biomechanical evaluation of the phases during simulated endotracheal intubation (ETI): pilot study on the effect of different laryngoscopes.

Endotracheal Intubation (ETI) is a common airway procedure used to connect the larynx and the lungs through a windpipe in patients under emergency situations. The process is carried out by a laryngoscope inserted into the mouth, used to help doctors in visualizing the glottis and inserting the tube. Currently, very few studies on objective evaluation of the biomechanics of the doctors during the procedure have been done. Additionally, these studies have been concentrated only on the overall performance analysis, without any segmentation, with a consequent loss of important information. In this paper, the authors present a preliminary study on a methodology to objectively evaluate and segment the biomechanical performance of doctors during the ETI, using surface electromyography and inertial measurement units. In particular, the validation has been performed by comparing three kinds of laryngoscopes involving an expert doctor. Finally, results are presented and commented.

T-cell therapy for EBV-associated nasopharyngeal carcinoma: preparative lymphodepleting chemotherapy does not improve clinical results.

BACKGROUND: We and others have demonstrated that adoptive cell therapy with Epstein-Barr virus (EBV)-specific autologous cytotoxic T lymphocytes (CTLs) may control disease progression in patients with EBV-associated nasopharyngeal carcinoma (NPC). With the aim of favoring in vivo T-cell expansion, we optimized our cell therapy approach by administering higher doses of EBV-specific CTLs, following lymphodepleting chemotherapy. PATIENTS AND METHODS: Eleven patients with EBV-related NPC in whom conventional treatment failed have been enrolled. Patients received nonmyeloablative lymphodepleting chemotherapy consisting of cyclophosphamide and fludarabine. Two doses of autologous EBV-specific CTLs were subsequently infused, 2 weeks apart. Study end points were feasibility and clinical outcome. RESULTS: All patients enrolled completed the treatment and were assessable for analysis. The median dose of CTLs per infusion was 3.7 × 10(8). Therapy was well tolerated, with no severe adverse events ascribable to either chemotherapy or cell therapy. Disease control (defined as either tumor regression or disease stabilization lasting >4 months) was obtained in 6 of 11 patients, in keeping with previously published results. CONCLUSIONS: Our data confirm that EBV-specific CTL therapy is safe and associated with antitumor activity in patients with advanced NPC. The use of lymphodepleting chemotherapy before high-dose CTL infusion did not enhance the clinical benefit observed in our previous series.

T cell therapy for nasopharyngeal carcinoma.

Among the novel biologic therapeutics that will increase our ability to cure human cancer in the years to come, T cell therapy is one of the most promising approaches. However, with the possible exception of tumor-infiltrating lymphocytes therapy for melanoma, clinical trials of adoptive T-cell therapy for solid tumors have so far provided only clear proofs-of-principle to build on with further development. Epstein-Barr virus (EBV)-associated malignancies offer a unique model to develop T cell-based immune therapies, targeting viral antigens expressed on tumor cells. In the last two decades, EBV-specific cytotoxic T-lymphocytes (CTL) have been successfully employed for the prophylaxis and treatment of EBV-related lymphoproliferative disorders in immunocompromised hosts. More recently, this therapeutic approach has been applied to the setting of EBV-related solid tumors, such as nasopharyngeal carcinoma. The results are encouraging, although further improvements to the clinical protocols are clearly necessary to increase anti-tumor activity. Promising implementations are underway, including harnessing the therapeutic potential of CTLs specific for subdominant EBV latent cycle epitopes, and delineating strategies aimed at targeting immune evasion mechanisms exerted by tumor cells.

Hematopoietic stem cell transplantation for advanced myelodysplastic syndrome in children: results of the EWOG-MDS 98 study.

We report on the outcome of children with advanced primary myelodysplastic syndrome (MDS) transplanted from an HLA-matched sibling (MSD) or an unrelated donor (UD) following a preparative regimen with busulfan, cyclophosphamide and melphalan. Ninety-seven patients with refractory anemia with excess blasts (RAEB, n=53), RAEB in transformation (RAEB-T, n=29) and myelodysplasia-related acute myeloid leukemia (MDR-AML, n=15) enrolled in the European Working Group of MDS in Childhood (EWOG-MDS) 98 study and given hematopoietic stem cell transplantation (HSCT) were analyzed. Median age at HSCT was 11.1 years (range 1.4-19.0). Thirty-nine children were transplanted from an MSD, whereas 58 were given the allograft from a UD (n=57) or alternative family donor (n=1). Stem cell source was bone marrow (n=69) or peripheral blood (n=28). With a median follow-up of 3.9 years (range 0.1-10.9), the 5-year probability of overall survival is 63%, while the 5-year cumulative incidence of transplantation-related mortality (TRM) and relapse is 21% each. Age at HSCT greater than 12 years, interval between diagnosis and HSCT longer than 4 months, and occurrence of acute or extensive chronic graft-versus-host disease were associated with increased TRM. The risk of relapse increased with more advanced disease. This study indicates that HSCT following a myeloablative preparative regimen offers a high probability of survival for children with advanced MDS.