ABSTRACT Purpose: Oncological diseases, especially if experienced in the first 3 years of life, expose young children to conditions of serious physical and emotional stress causing delays in their physical development, cognitive acquisitioning and issues related to their emotional and social functioning. This study examines the effects of the participation in an early childcare intervention to support the children's development, which was proposed to children from 0 to 36 months, at the moment of cancer diagnosis in their hospital setting. Method: A prospective multicenter programme was offered to young children (<3 years) newly diagnosed with cancer. These children were treated in three pediatric oncology centers affiliated to the Italian Association of Pediatric Hematology-Oncology. The children's strengths and weaknesses, in different developmental areas, were examined pre-programme and 6 months post-programme. Results: Fifty-three young children were enrolled in the study. The data showed that at the moment of diagnosis the majority of the children demonstrated low scores across all the abilities in the subscales examined, highlighting a mental age below their chronological age (ΔT0 = 3.5 months). However, post-programme, the children were shown to have significantly reduced the gap between their chronological and mental age (ΔT1 = 1 month), demonstrating improvements in all their developmental abilities. Conclusion: The study confirms significant implications for early childcare programmes, delivered in hospitals for young children with cancer, in terms of support for their ongoing physical, cognitive and social development. Therefore, the study demonstrated the necessity, for all pediatric oncology centers, to invest in early childhood education programmes.

Natural killer (NK) cells are indicated as favorite candidates for innovative therapeutic treatment and are divided into two subclasses: immature regulatory NK CD56bright and mature cytotoxic NK CD56dim. Therefore, the ability to discriminate CD56dim from CD56bright could be very useful because of their higher cytotoxicity. Nowadays, NK cell classification is routinely performed by cytometric analysis based on surface receptor expression. Here, we present an in-flow, label-free and non-invasive biophysical analysis of NK cells through a combination of light scattering and machine learning (ML) for NK cell subclass classification. In this respect, to identify relevant biophysical cell features, we stimulated NK cells with interleukine-15 inducing a subclass transition from CD56bright to CD56dim. We trained our ML algorithm with sorted NK cell subclasses (≥86% accuracy). Next, we applied our NK cell classification algorithm to cells stimulated over time, to investigate the transition of CD56bright to CD56dim and their biophysical feature changes. Finally, we tested our approach on several proband samples, highlighting the potential of our measurement approach. We show a label-free way for the robust identification of NK cell subclasses based on biophysical features, which can be applied in both cell biology and cell therapy.

Leukocytes are major cellular components of the inflammatory and immune response systems. After their generation in the bone marrow from hematopoietic stem cells, they maturate as granulocytes (neutrophils, eosinophils, and basophils), monocytes, and lymphocytes. The abnormal accumulation and proliferation of immature blood cells (blasts) lead to severe and widespread diseases such as leukemia. We have recently shown that KCTD15, a member of the potassium channel tetramerization domain containing protein family (KCTD), is remarkably upregulated in leukemic B-cells. Here, we extend our investigation by monitoring the KCTD15 expression levels in circulating lymphocytes, monocytes, and granulocytes, as well as in leukemia cells. Significant differences in the expression level of KCTD15 were detected in normal lymphocytes, monocytes, and granulocytes. Interestingly, we also found overexpression of the protein following leukemic transformation in the case of myeloid cell lineage. Indeed, KCTD15 was found to be upregulated in K562 and NB4 cells, as well as in HL-60 cell lines. This in vitro finding was corroborated by the analysis of KCTD15 mRNA of acute myeloid leukemia (AML) patients reported in the Microarray Innovations in Leukemia (MILE) dataset. Collectively, the present data open interesting perspectives for understanding the maturation process of leukocytes and for the diagnosis/therapy of acute leukemias.

Long non–coding RNAs (lncRNAs) are emerging as key gene regulators in the pathogenesis and development of various cancers including B lymphoblastic leukaemia (B–ALL). In this pilot study, we used RNA–Seq transcriptomic data for identifying novel lncRNA–mRNA cooperative pairs involved in childhood B–ALL pathogenesis. We conceived a bioinformatic pipeline based on unsupervised PCA feature extraction approach and stringent statistical criteria to extract potential childhood B–ALL lncRNA signatures. We then constructed a co–expression network of the aberrantly expressed lncRNAs (30) and protein–coding genes (754). We cross–validated our in–silico findings on an independent dataset and assessed the expression levels of the most differentially expressed lncRNAs and their co–expressed mRNAs through ex vivo experiments. Using the guilt–by–association approach, we predicted lncRNA functions based on their perfectly co–expressed mRNAs (Spearman’s correlation) that resulted closely disease–associated. We shed light on 24 key lncRNAs and their co–expressed mRNAs which may play an important role in B–ALL pathogenesis. Our results may be of clinical utility for diagnostic and/or prognostic purposes in paediatric B–ALL management.

Leukemic cells originate from the malignant transformation of undifferentiated myeloid/lymphoid hematopoietic progenitors normally residing in bone marrow. As the precise molecular mechanisms underlying this heterogeneous disease are yet to be disclosed, the identification and the validation of novel actors in leukemia is of extreme importance. Here, we show that KCTD15, a member of the emerging class of KCTD ((K)potassium Channel Tetramerization Domain containing) proteins, is strongly upregulated in patients affected by B-cell type acute lymphoblastic leukemia (B-ALL) and in continuous cell lines (RS4;11, REH, TOM-1, SEM) derived from this form of childhood leukemia. Interestingly, KCTD15 downregulation induces apoptosis and cell death suggesting that it has a role in cellular homeostasis and proliferation. In addition, stimulation of normal lymphocytes with the pokeweed mitogen leads to increased KCTD15 levels in a fashion comparable to those observed in proliferating leukemic cells. In this way, the role of KCTD15 is likely not confined to the B-ALL pathological state and extends to activation and proliferation of normal lymphocytes. Collectively, data here presented indicate that KCTD15 is an important and hitherto unidentified player in childhood lymphoid leukemia, and its study could open a new scenario for the identification of altered and still unknown molecular pathways in leukemia.

Traditionally, hematopoietic stem cell transplantation (HSCT) from both HLA-matched related and unrelated donors (UD) has been used for treating children with acute leukemia (AL) in need of an allograft. Recently, HLA-haploidentical HSCT after αβ T-cell/B-cell depletion (αβhaplo-HSCT) was shown to be effective in single-center studies. Here, we report the first multicenter retrospective analysis of 127 matched UD (MUD), 118 mismatched UD (MMUD), and 98 αβhaplo-HSCT recipients, transplanted between 2010 and 2015, in 13 Italian centers. All these AL children were transplanted in morphological remission after a myeloablative conditioning regimen. Graft failure occurred in 2% each of UD-HSCT and αβhaplo-HSCT groups. In MUD vs MMUD-HSCT recipients, the cumulative incidence of grade II to IV and grade III to IV acute graft-versus-host disease (GVHD) was 35% vs 44% and 6% vs 18%, respectively, compared with 16% and 0% in αβhaplo-HSCT recipients (P < .001). Children treated with αβhaplo-HSCT also had a significantly lower incidence of overall and extensive chronic GVHD (P < .01). Eight (6%) MUD, 32 (28%) MMUD, and 9 (9%) αβhaplo-HSCT patients died of transplant-related complications. With a median follow-up of 3.3 years, the 5-year probability of leukemia-free survival in the 3 groups was 67%, 55%, and 62%, respectively. In the 3 groups, chronic GVHD-free/relapse-free (GRFS) probability of survival was 61%, 34%, and 58%, respectively (P < .001). When compared with patients given MMUD-HSCT, αβhaplo-HSCT recipients had a lower cumulative incidence of nonrelapse mortality and a better GRFS (P < .001). These data indicate that αβhaplo-HSCT is a suitable therapeutic option for children with AL in need of transplantation, especially when an allele-matched UD is not available.

Relapse of acute lymphoblastic leukemia (ALL) may occur in extramedullary sites, mainly central nervous system (CNS) and testis. Optimal post-remissional treatment for isolated extramedullary relapse (IEMR) is still controversial. We collected data of children treated with hematopoietic stem cell transplantation (HSCT) for ALL IEMR from 1990 to 2015 in Italy. Among 281 patients, 167 had a relapse confined to CNS, 73 to testis, 14 to mediastinum, and 27 to other organs. Ninety-seven patients underwent autologous HSCT, 79 received allogeneic HSCT from a matched family donor, 75 from a matched unrelated donor, and 30 from an HLA-haploidentical donor. The 10-year overall survival was 56% and was not influenced by gender, ALL blast immune-phenotype, age, site of relapse, duration of first remission, and type of HSCT. In multivariable analysis, the only prognostic factors were disease status at HSCT and year of transplantation. Patients transplanted in third or subsequent complete remission (CR) had a risk of death 2.3 times greater than those in CR2. Children treated after 2000 had half the risk of death than those treated before that year. Our results suggest that both autologous and allogeneic HSCT may be considered for the treatment of pediatric ALL IEMR after the achievement of CR2.

Introduction. Severe acute graft-versus-host disease (GVHD) remains the most relevant complication after allogeneic HSCT. Although its incidence in the pediatric population is lower than in adults, children with severe acute GVHD and who do not respond to first-line treatment with systemic steroids still have a poor prognosis. The exact incidence of steroid-refractory acute GVHD in children is still not precisely defined, as well as the risk of non-relapse mortality (NRM) due to steroid-refractory acute GVHD. Aim of our study was to analyze the frequency of acute GVHD unresponsive to first-line steroid treatment in children and adolescents given allogenic HSCT, to describe the second line treatment employed, and the outcome of patient with this complication.

We report on 109 patients with hemophagocytic lymphohistiocytosis (HLH) undergoing 126 procedures of allogeneic hematopoietic stem cell transplantation (HSCT) between 2000 and 2014 in centers associated with the Italian Pediatric Hematology Oncology Association. Genetic diagnosis was FHL2 (32%), FHL3 (33%), or other defined disorders known to cause HLH (15%); in the remaining patients no genetic abnormality was found. Donor for first transplant was an HLA-matched sibling for 25 patients (23%), an unrelated donor for 73 (67%), and an HLA-partially matched family donor for 11 children (10%). Conditioning regimen was busulfan-based for 61 patients (56%), treosulfan-based for 21 (20%), and fludarabine-based for 26 children (24%). The 5-year probabilities of overall survival (OS) and event-free survival (EFS) were 71% and 60%, respectively. Twenty-six patients (24%) died due to transplant-related causes, whereas 14 (13%) and 10 (9%) patients experienced graft rejection and/or relapse, respectively. Twelve of 14 children given a second HSCT after graft failure/relapse are alive and disease-free. Use of HLA-partially matched family donors was associated with higher risk of graft failure and thus with lower EFS (but not with lower OS) in multivariable analysis. Active disease at transplantation did not significantly affect prognosis. These data confirm that HSCT can cure most HLH patients, active disease not precluding successful transplantation. Because in HLH patients HLA-haploidentical HSCT performed through CD34+ cell positive selection was found to be associated with poor sustained engraftment of donor cells, innovative approaches able to guarantee a more robust engraftment are warranted in patients given this type of allograft.

Allogeneic stem cell transplantation (HSCT) is largely adopted as post-remissional therapy in children with acute myeloid leukemia (AML) in first complete remission (CR1) but with high-risk characteristics (including high-risk cytogenetics or high levels of minimal residual disease at the end of induction therapy) or to rescue patients reaching CR2 after a previous relapse.

a Center for Advanced Biomaterials for Healthcare@CRIB, Istituto Italiano di Tecnologia (IIT), Largo Barsanti e Matteucci 53, 80125 Naples, Italy. BMT Unit, Department of Pediatric Hemato-Oncology, Santobono-Pausilipon Hospital, Naples, Italy. Interdisciplinary Research Centre on Biomaterials (CRIB) and Dipartimento di Ingegneria Chimica, dei Materiali e della Produzione Industriale, Università degli Studi di Napoli "Federico II", Piazzale Tecchio 80, 80125 Naples, Italy.

Histology and histopathology are based on the morphometric observations of quiescent cells. Their diagnostic potential could largely benefit from a simultaneous screening of intrinsic biophysical properties at single-cell level. For such a purpose, we analyzed light scattering signatures of individual mononuclear blood cells in microfluidic flow. In particular, we extracted a set of biophysical properties including morphometric (dimension, shape and nucleus-to-cytosol ratio) and optical (optical density) ones to clearly discriminate different cell types and stages. By considering distinctive ranges of biophysical properties along with the obtained relative cell frequencies, we can identify unique cell classes corresponding to specific clinical conditions (p < 0.01). Based on such a straightforward approach, we are able to discriminate T-, B-lymphocytes, monocytes and beyond that first results on different stages of lymphoid and myeloid leukemia cells are presented. This work shows that the simultaneous screening of only three biophysical properties enables a clear distinction between pathological and physiological mononuclear blood stream cells. We believe our approach could represent a useful tool for a label-free analysis of biophysical single-cell signatures.

This study report clinical course, etiology, management, and long-term outcome of children who developed toxic epidermal necrolysis-like reaction (TEN-LR) after allogeneic hematopoietic stem cell transplantation (allo-HSCT). We retrospectively collected children with TEN-LR occurring after allo-HSCT performed in 2 pediatric bone marrow units between 2005 and 2014. We identified 6 cases of TEN-LR of 322 patients (1.8%). Possible triggers of TEN included antibiotics, antiepileptics, antimycotics, and Mycoplasma infection. In 3 patients TEN-LR occurred concurrently with severe multiorgan acute graft versus host disease. The management of TEN included administration of high doses of intravenous immunoglobulins and steroids (n=6), anti-tumor necrosis factor (n=3), and plasmapheresis (n=3) and whenever possible, discontinuation of the potentially causative drugs. Four patients (66%) reached a complete clinical response of TEN-LR after a median of 11.2 days. Two children (34%) are presently alive, 1 with long-term ocular sequelae. TEN-LR is a potentially lethal complication that may occur after HSCT also in pediatric patients. In our experience, TEN-LR and acute graft versus host disease probably coexisted and an overlap between the 2 forms is suggested. The multidisciplinary approaches involving specialized nurses, hematologists, dermatologists, burn surgeons, and infectious disease specialists is crucial to treat these patients.