Maria Giuseppina Cefalo

Mobilization of healthy donors with plerixafor affects the cellular composition of T-cell receptor (TCR)-αβ/CD19-depleted haploidentical stem cell grafts

BackgroundHLA-haploidentical hematopoietic stem cell transplantation (HSCT) is suitable for patients lacking related or unrelated HLA-matched donors. Herein, we investigated whether plerixafor (MZ), as an adjunct to G-CSF, facilitated the collection of mega-doses of hematopoietic stem cells (HSC) for TCR-αβ/CD19-depleted haploidentical HSCT, and how this agent affects the cellular graft composition.MethodsNinety healthy donors were evaluated. Single-dose MZ was given to 30 ‘poor mobilizers’ (PM) failing to attain ≥40 CD34+ HSCs/μL after 4 daily G-CSF doses and/or with predicted apheresis yields ≤12.0x106 CD34+ cells/kg recipient’s body weight.ResultsMZ significantly increased CD34+ counts in PM. Naïve/memory T and B cells, as well as natural killer (NK) cells, myeloid/plasmacytoid dendritic cells (DCs), were unchanged compared with baseline. MZ did not further promote the G-CSF-induced mobilization of CD16+ monocytes and the down-regulation of IFN-γ production by T cells. HSC grafts harvested after G-CSF + MZ were enriched in myeloid and plasmacytoid DCs, but contained low numbers of pro-inflammatory 6-sulfo-LacNAc+ (Slan)-DCs. Finally, children transplanted with G-CSF + MZ-mobilized grafts received greater numbers of monocytes, myeloid and plasmacytoid DCs, but lower numbers of NK cells, NK-like T cells and Slan-DCs.ConclusionsMZ facilitates the collection of mega-doses of CD34+ HSCs for haploidentical HSCT, while affecting graft composition.

Human iPSC for Therapeutic Approaches to the Nervous System: Present and Future Applications

Many central nervous system (CNS) diseases including stroke, spinal cord injury (SCI), and brain tumors are a significant cause of worldwide morbidity/mortality and yet do not have satisfying treatments. Cell-based therapy to restore lost function or to carry new therapeutic genes is a promising new therapeutic approach, particularly after human iPSCs became available. However, efficient generation of footprint-free and xeno-free human iPSC is a prerequisite for their clinical use. In this paper, we will first summarize the current methodology to obtain footprint- and xeno-free human iPSC. We will then review the current iPSC applications in therapeutic approaches for CNS regeneration and their use as vectors to carry proapoptotic genes for brain tumors and review their applications for modelling of neurological diseases and formulating new therapeutic approaches. Available results will be summarized and compared. Finally, we will discuss current limitations precluding iPSC from being used on large scale for clinical applications and provide an overview of future areas of improvement. In conclusion, significant progress has occurred in deriving iPSC suitable for clinical use in the field of neurological diseases. Current efforts to overcome technical challenges, including reducing labour and cost, will hopefully expedite the integration of this technology in the clinical setting.

The combination of bortezomib with chemotherapy to treat relapsed/refractory acute lymphoblastic leukaemia of childhood

Achieving complete remission (CR) in childhood relapsed/refractory acute lymphoblastic leukaemia (ALL) is a difficult task. Bortezomib, a proteasome inhibitor, has in vitro activity against ALL blasts. A phase I‐II trial, reported by the Therapeutic Advances in Childhood Leukaemia and Lymphoma (TACL) consortium, demonstrated that bortezomib with chemotherapy has acceptable toxicity and remarkable activity in patients with relapsed ALL failing 2–3 previous regimens. We evaluated bortezomib in combination with chemotherapy in 30 and 7 children with B‐cell precursor (BCP) and T‐cell ALL, respectively. Bortezomib (1·3 mg/m2/dose) was administered intravenously on days 1, 4, 8, and 11. Chemotherapy agents were the same as those used in the TACL trial, consisting of dexamethasone, doxorubicin, vincristine and pegylated asparaginase. Three patients (8·1%) died due to infections. Twenty‐seven patients (72·9%) achieved CR or CR with incomplete platelet recovery (CRp). Fourteen had minimal residual disease (MRD) lower than 0·1%. Twenty‐two of 30 BCP‐ALL patients (73·3%) and 5/7 patients (71%) with T‐cell ALL achieved CR/CRp. The 2‐year overall survival (OS) is 31·3%; CR/CRp patients with an MRD response had a remarkable 2‐year OS of 68·4%. These data confirm that the combination of bortezomib with chemotherapy is a suitable/effective option for childhood relapsed/refractory ALL.

Wernicke Encephalopathy in Pediatric Neuro-oncology: Presentation of 2 Cases and Review of Literature

Wernicke encephalopathy represents a well-known entity characterized by a set of cognitive and neurologic alterations. Wernicke encephalopathy is rare and under-recognized in childhood and may be fatal. Few cases have been documented in pediatric oncology. We report on 2 Wernicke encephalopathy cases that occurred in children having a brain tumor. The diagnosis of Wernicke encephalopathy was suggested by clinical manifestations associated with the typical radiologic findings and a laboratory evidence of thiamine deficiency. No large series have been published to support the evidence that pediatric malignancies represent a demonstrated factor of increased risk to develop a Wernicke encephalopathy. Moreover, the diagnosis may be even more difficult in brain tumors, considering the overlapping symptoms and the risk of encephalopathy related to both the disease and the treatment. Wernicke encephalopathy should be considered in all children with cancer presenting a neurologic deterioration, mainly in brain tumors. An early diagnosis is imperative for a prompt therapy that might prevent or minimize the irreversible brain damage related to this condition.

Behavioral disorders as unusual presentation of pediatric extraventricular neurocytoma: report on two cases and review of the literature

BackgroundExtraventricular neurocytomas (EVNs) are rare parenchymal brain tumors, distinct from central neurocytomas that are typically located within the supratentorial ventricular system. Seizures and headache represent the most common symptoms of extraventricular neurocytomas in the cerebral hemisphere both in adult and pediatric population.Case presentationWe describe two cases of pediatric EVN with clinical onset characterized by behavioral and attention deficit/ hyperactivity disorders. The association between behavioral/attention disorders in childhood and the presence of a frontal neurocytoma has never been described before. Furthermore, inappropriate levels of inattention, hyperactivity and impulsivity are common among the neurobehavioral and developmental disorders in childhood. We reviewed 43 pediatric cases of extraventricular neurocytoma included in the PubMed database and their clinical presentation, and we never found this unusual relationship.ConclusionIn childhood, the attention/hyperactivity disorders seem to be often over-diagnosed. When these deficits are more subtle and do not well-fit in a specific neurocognitive disorder, the clinicians should have a suspicion that they might mask the clinical features of a frontal lesion. This paper is focused on the clinical presentation of the extraventricular neurocytoma and the possible organic etiology of an attention and hyperactivity deficit.

Lethal sepsis and malignant transformation in severe congenital neutropenia: Report from the Italian Neutropenia Registry

To the Editor: The occurrence of lethal sepsis and myelodysplasia/acute myeloid leukemia (MDS/AML) may affect patients diagnosed with severe congenital neutropenia (SCN) at variable rates depending upon the cohorts studied [1]. The Severe Chronic Neutropenia International Registry (SCNIR) reported a cumulative incidence (CI) of sepsis/death of 10% after 15 years from the start of therapy with granulocyte colony-stimulating factor (G-CSF) [2]. The French Severe Chronic Neutropenia Registry (SCNFR) reported 6 septic deaths out of 101 SCN cases, while in the Swedish cohort nobody died of sepsis [3,4]. As for MDS/AML, the CIs after 15 years from the beginning of G-CSF therapy were 22%, 11% and 31%, respectively, in the SCNIR, SCNFR and Swedish cohorts [2,3,4]. In the present study, we evaluated the incidence of lethal sepsis and MDS/AML in patients registered in the Italian Neutropenia Registry (INR). Starting from 2003, the INR identified 350 patients affected with various type of neutropenia from 35 centers belonging to Associazione Italiana Ematologia-Oncologia Pediatrica (AIEOP). Among them, 23 had SCN and considered eligible for the study. Median age at diagnosis of SCN was 8.17 months (IQR: 2.83– 32.15) and median duration of follow up was 6.44 years (IQR; 2.99–12.05) (Table I). During the course of the study, two patients died of non-transplant related sepsis, after voluntary decision by the family to stop G-CSF treatment. The lethal sepsis CI was 10% after three years from the beginning of G-CSF treatment. One patient out of 23 developed MDS, while no leukemia cases were reported. The MDS CI was 6% (95% IC 1–35) at four years after the start of G-CSF. In our study, the incidence of MDS was far lower than that described in the Swedish cohort and in SCNIR patients treated with G-CSF doses greater than 8mcg/kg. On the contrary, it is closer to the French cohort and to the SCNIR group treated with G-CSF dosage lower than 8mcg/kg (15%). The reason for the low MDS incidence, could be the small size and the young age of the cohort; also, in some cases, the choice of “early” transplantion might have prevented the malignant transformation. Moreover, SCN cases (i.e., GATA2 gene defects) with a high transforming potential were not included in our cohort, possibly reducing the incidence of malignant events. On the other hand, the lethal sepsis risk in our cohort was similar to those described in SCNIR and in the French cohort. Deaths were mainly related to voluntary interruption G-CSF treatment, probably because underestimation of its vital importance and general lack of adherence to daily subcutaneous infusions. Even with the limitations of the small sample size and relatively short follow up, due to the young age of patients in the Registry and to the ongoing SCN cases registration, we emphasize that the management and follow up of patients with SCN remains a matter of concern. The education of patients and their families and strict follow up could minimize the risk of improper administration and help to perform early diagnosis of possible malignant evolution.

Cord blood transplantation in children with hemoglobinopathies

Introduction: Current challenges in the field of hematopoietic stem cell transplantation (HSCT) for hemoglobinopathies include the possibility of using alternative donors to expand the number of candidates to an allograft. Umbilical cord blood transplantation (UCBT) have extended the access to HSCT in patients lacking matched related or unrelated adult donors, and appears particularly appealing in non-malignant diseases by virtue of the associated low incidence of graft-versus-host disease (GVHD). Areas covered: Outcomes of patients with thalassemia or sickle cell disease given related UCBT is at least as good as that of patients receiving bone marrow (BM) allograft from human leucocyte antigen (HLA)-identical siblings, provided the UCB unit contains adequate cell dose. The experience with unrelated UCBT has been less encouraging, primarily due to low engraftment rates and delayed hematopoietic recovery. New promising approaches of ex vivo graft manipulation aimed at overcoming the drawback of cell dose limitation are under investigation. Expert opinion: We suggest that HSCT should be pursued whenever an HLA-identical CB unit is available, employing it either alone or in combination with BM from the same donor. Based on currently reported data, unrelated UCBT appears to be a suboptimal strategy for patients with hemoglobinopathies, unless it is performed in the context of clinical trials aimed at exploring specific treatment platforms of ex vivo UCB-graft manipulation.

Streptococcus pneumoniae pharyngeal colonization in school-age children and adolescents with cancer.

Patients with cancer, particularly those with hematologic malignancies, are at an increased risk of invasive pneumococcal disease (IPD) and they are included in the list of subjects for whom pneumococcal vaccination is recommended. The main aim of this study was to evaluate Streptococcus pneumoniae colonization in school-aged children and adolescents with cancer to determine the potential protective efficacy of 13-valent pneumococcal conjugate vaccine (PCV13). An oropharyngeal swab was obtained from 277 patients (age range 6-17 years) with cancer during routine clinical visits and analyzed for S. pneumoniae using real-time polymerase chain reaction. S. pneumoniae was identified in 52 patients (18.8%), including 47/235 (20.0%) with hematologic malignancies and 5/42 (11.9%) with solid tumors. Colonization declined significantly with an increase in age (odds ratio [OR] 0.34, 95% confidence interval [CI] 0.16-0.71, and OR 0.30, 95% CI 0.11-0.82 in children aged 10-14 and ≥15 years, respectively, as compared to those <10 years). Carriage was more common among patients with leukemia or lymphoma than in children with solid tumors. Co-trimoxazole prophylaxis was significantly associated with reduced pneumococcal carriage (OR 0.41, 95% CI 0.19–0.89). A total of 15/58 (25.9%) and 26/216 (12.0%) children were colonized by PCV13 serotypes among cancer patients previously vaccinated and not vaccinated with 7-valent pneumococcal conjugate vaccine (PCV7), respectively. In conclusion, this study indicates that children and adolescents with cancer are frequently colonized by S. pneumoniae. Because most of the carried serotypes are included in PCV13, this vaccine is presently the best solution to reduce the risk of IPD in these patients.

Whole Genome MBD-seq reveals different CpG methylation patterns in Azacytidine-treated Juvenile Myelomonocytic Leukaemia (JMML) patients

Juvenile myelomonocytic leukaemia (JMML) is a rare and severe form of early childhood leukaemia characterized by clonal proliferation of myelomonocytic cells, progressive anaemia, thrombocytopenia, hepatosplenomegaly and increased fetal haemoglobin (HbF) (Chan et al, 2009). The only available curative treatment is allogeneic haematopoietic stem cell transplantation (HSCT), but prognosis remains poor (Niemeyer & Kratz, 2008; Locatelli & Niemeyer, 2015). It has previously been shown that aberrant DNA hypermethylation of distinct target genes defines more aggressive variants of JMML (Flotho et al, 2007; Furlan et al, 2009; Olk-Batz et al, 2011; Poetsch et al, 2014). Azacytidine (AZA) is a DNA hypomethylating agent recently employed for JMML treatment (Locatelli & Niemeyer, 2015). To evaluate AZA treatment impact on aberrant methylation in JMML, we screened global DNA methylation in 3 JMML patients before and after AZA treatment. We found global DNA hypermethylation in both preand post-AZA treated patient samples compared to healthy controls, and a significant, although patient-specific, AZA-induced hypomethylation effect. Furthermore, we identified several differentially methylated coding and non-coding species of RNA, depicting a complex deregulation at different levels of transcription and translation in JMML. CD34 cells were isolated from frozen mononuclear cell suspensions. Three JMML patients at both diagnosis (t0 group) and after the third cycle of AZA (t1 group) (Furlan et al, 2009) were studied (Table SI); three additional healthy donor (HD) control samples were also included (HD group). Informed consent was obtained from either parents or legal guardians according to the Declaration of Helsinki. Approval for this study was obtained from the Institutional Review Board of the “Bambino Ges u” Children’s Hospital, Rome. JMML patients were treated with AZA as a compassionate use. Methods for CD34 cells isolation, their percentage assessment and genomic DNA extraction and sequence analysis are detailed in Data S1. The complete DNA-seq datasets are available at the European Bioinformatics Institute European Nucleotide Archive (EBI ENA) database (http://www. ebi.ac.uk/ena; accession number: PRJEB19377). Gene lists and ontologies are available in supplemental files. After Methyl Binding Domain (MBD) sequencing (EBI ENA database; http://www.ebi.ac.uk/ena) we compared t0 and HD groups (File S1), detecting 987 different transcriptional units corresponding to 714 coding and 273 non-coding sequences, mostly hypermethylated in t0 (Fig 1A Panel i, ii). A functional protein association network (STRING; http://string-db.org/) (Szklarczyk et al, 2015) identified 18 different pathways possibly impaired by deregulated DNA-methylation (Figure S1; Table I; false discovery rate [FDR] < 0 05). The same analysis on t1 and HD groups identified 643 unique transcriptional units, with 468 coding and 175 non-coding sequences, indicating a strong tendency towards hypermethylation in JMML samples (File S2; Fig 1A Panel i, ii). Again, the functional protein association network identified 10 putative impaired pathways, similarly to t0 vs. HD analysis (Figure S2; Table I; FDR < 0 05), even if DNA hypermethylation after AZA treatment was reduced. Involvement of membrane elements, such as adhesion molecules, developmental processes and cell cycle-related genes was recurrent in our analysis, meaning that identifying insights of these pathways could help to further enlighten pathogenic mechanisms and novel targets for therapy in JMML. Unexpectedly, direct comparative methylation analysis of samples obtained at onset of JMML and after AZA treatment (t0 vs. t1) did not show any significant difference (File S3), suggesting a probable unspecific and unique patient-related pharmacological effect, which was also evident when considering single-case analysis (Figure S3; File S4, S5, S6). Notably, 453 differentially methylated coding regions were shared between newly diagnosed JMML and AZA-treated samples compared to HD (Table SII). Additionally, 261 and 15 coding regions with different methylation status were specific for t0 and t1 respectively (Fig 1B Panel i; Table SII). Among non-coding regions, we also found 165 sequences shared between the two groups (Table SII), while 107 and 10 were unique in the t0 vs. HD and t1 vs. HD, respectively (Fig 1B Panel ii; Table SII). Interestingly, 439 coding and 161 non-coding specific genomic regions preserved their hypermethylated status, describing an apparent resistance to AZA treatment (Fig 1B Panel iii, iv; Table SII). We found different non-coding RNA species, such as microRNAs, splicing RNAs, long noncoding RNAs/antisense transcripts (AS) and other nondescript RNA species (Table SII; Supplemental files), identifying a complex deregulation of preand post-transcriptional processes in JMML pathogenesis possibly acting in a cooperative way. correspondence

T Cell Immunotherapy for Immune Reconstitution and GVHD Prevention After Allogeneic Hematopoietic Stem Cell Transplantation

Many different studies have demonstrated that early recovery of the adaptive immune system after allogeneic hematopoietic stem cell transplantation (HSCT) is predominantly sustained by peripheral expansion of donor-derived, mature lymphocytes transferred with the graft. Different approaches based on the infusion of donor T cells after HSCT have been developed mainly to accelerate immune recovery and to treat/prevent (a) malignancy recurrence, (b) life-threatening infections, and (c) immune-mediated disorders, such as graft-versus-host disease (GVHD). For many years, donor lymphocyte infusion (DLI) has been a widely used approach to prevent and to treat leukemia recurrence, to convert mixed chimerism into complete donor chimerism, and to accelerate immune reconstitution of patients after HSCT. More sophisticated strategies of adoptive infusion of T cell lines/clones capable of mediating a graft-versus-leukemia (GVL) response, while avoiding GVHD occurrence, or specific for the most life-threatening pathogens (e.g., cytomegalovirus, Epstein-Barr virus, and adenovirus) have been envisaged and successfully tested in pilot trials in the early post-transplantation period. Also, ex vivo expanded regulatory T (Treg) cells have been shown to be beneficial for preventing GVHD post-HSCT. In this review, we will focus on DLI as well as more complex cellular therapies that require extensive cell manipulation.