Rita Maria Pinto

Clinical, genetic, and cellular analysis of 49 osteopetrotic patients: implications for diagnosis and treatment

Background: Osteopetrosis, a genetic disease characterised by osteoclast failure, is classified into three forms: infantile malignant autosomal recessive osteopetrosis (ARO), intermediate autosomal recessive osteopetrosis (IRO), and autosomal dominant osteopetrosis (ADO). Methods: We studied 49 patients, 21 with ARO, one with IRO, and 27 with type II ADO (ADO II). Results: Most ARO patients bore known or novel (one case) ATP6i (TCIRG1) gene mutations. Six ADO II patients had no mutations in ClCN7, the only so far recognised gene implicated, suggesting involvement of yet unknown genes. Identical ClCN7 mutations produced differing phenotypes with variable degrees of severity. In ADO II, serum tartrate resistant acid phosphatase was always elevated. Bone alkaline phosphatase (BALP) was generally low, but osteocalcin was high, suggesting perturbed osteoblast differentiation or function. In contrast, BALP was high in ARO patients. Elevated osteoclast surface/bone surface was noted in biopsies from most ARO patients. Cases with high osteoclasts also showed increased osteoblast surface/bone surface. ARO osteoclasts were morphologically normal, with unaltered formation rates, intracellular pH handling, and response to acidification. Their resorption activity was greatly reduced, but not abolished. In control osteoclasts, all resorption activity was abolished by combined inhibition of proton pumping and sodium/proton antiport. Conclusions: These findings provide a rationale for novel therapies targeting pH handling mechanisms in osteoclasts and their microenvironment.

Allogeneic hematopoietic stem cell transplantation in thalassemia major: results of a reduced-toxicity conditioning regimen based on the use of treosulfan

Sixty thalassemia patients (median age, 7 years; range, 1-37) underwent allogeneic hematopoietic stem cell transplantation (HSCT) after a preparation combining thiotepa, treosulfan, and fludarabine. Before HSCT, 27 children were assigned to risk class 1 of the Pesaro classification, 17 to class 2, and 4 to class 3; 12 patients were adults. Twenty patients were transplanted from an HLA-identical sibling and 40 from an unrelated donor. The cumulative incidence of graft failure and transplantation-related mortality was 9% and 7%, respectively. Eight patients experienced grade II-IV acute GVHD, the cumulative incidence being 14%. Among 56 patients at risk, 1 developed limited chronic GVHD. With a median follow-up of 36 months (range, 4-72), the 5-year probability of survival and thalassemia-free survival are 93% and 84%, respectively. Neither the class of risk nor the donor used influenced outcome. This treosulfan-based preparation proved to be safe and effective for thalassemia patients given allogeneic HSCT.

Genotype-Phenotype Relationship in Human ATP6i-Dependent Autosomal Recessive Osteopetrosis

Autosomal-recessive osteopetrosis is a severe genetic disease caused by osteoclast failure. Approximately 50% of the patients harbor mutations of the ATP6i gene, encoding for the osteoclast-specific a3 subunit of V-ATPase. We found inactivating ATP6i mutations in four patients, and three of these were novel. Patients shared macrocephaly, growth retardation and optic nerve alteration, osteosclerotic and endobone patterns, and high alkaline phosphatase and parathyroid hormone levels. Bone biopsies revealed primary spongiosa lined with active osteoblasts and high numbers of tartrate-resistant acid phosphatase (TRAP)-positive, a3 subunit-negative, morphologically unremarkable osteoclasts, some of which located in shallow Howship lacunae. Scarce hematopoietic cells and abundant fibrous tissue containing TRAP-positive putative osteoclast precursors were noted. In vitro osteoclasts were a3-negative, morphologically normal, with prominent clear zones and actin rings, and TRAP activity more elevated than in control patients. Podosomes, alphaVbeta3 receptor, c-Src, and PYK2 were unremarkable. Consistent with the finding in the bone biopsies, these cells excavated pits faintly stained with toluidine blue, indicating inefficient bone resorption. Bone marrow transplantation was successful in all patients, and posttransplant osteoclasts showed rescue of a3 subunit immunoreactivity.

Outcome of children with acute leukemia given HLA-haploidentical HSCT after αβ T-cell and B-cell depletion

Allogeneic hematopoietic stem cell transplantation (HSCT) from an HLA-haploidentical relative (haplo-HSCT) is a suitable option for children with acute leukemia (AL) either relapsed or at high-risk of treatment failure. We developed a novel method of graft manipulation based on negative depletion of αβ T and B cells and conducted a prospective trial evaluating the outcome of children with AL transplanted with this approach. Eighty AL children, transplanted between September 2011 and September 2014, were enrolled in the trial. All children were given a fully myeloablative preparative regimen. Anti-T-lymphocyte globulin from day -5 to -3 was used for preventing graft rejection and graft-versus-host disease (GVHD); no patient received any posttransplantation GVHD prophylaxis. Two children experienced primary graft failure. The cumulative incidence of skin-only, grade 1-2 acute GVHD was 30%; no patient developed extensive chronic GVHD. Four patients died, the cumulative incidence of nonrelapse mortality being 5%, whereas 19 relapsed, resulting in a 24% cumulative incidence of relapse. With a median follow-up of 46 months for surviving patients, the 5-year probability of chronic GVHD-free, relapse-free survival (GRFS) is 71%. Total body irradiation-containing preparative regimen was the only variable favorably influencing relapse incidence and GRFS. The outcomes of these 80 patients are comparable to those of 41 and 51 children given transplantation from an HLA-identical sibling or a 10/10 allelic-matched unrelated donor in the same period. These data indicate that haplo-HSCT after αβ T- and B-cell depletion represents a competitive alternative for children with AL in need of urgent allograft. This trial was registered at www.clinicaltrials.gov as #NCT01810120.

Adhesion Molecule Expression, Clinical Features and Therapy Outcome in Childhood Acute Lymphoblastic Leukemia

In view of the relevance of adhesion molecule expression for the mechanisms of homing, trafficking and spreading of malignant cells, we have investigated the expression of surface adhesion molecules in lymphoblasts from 57 acute lymphoblastic leukemia (ALL) cases and tried to correlate the adhesive phenotype with immunological typing, prognostic factors at diagnosis and clinical follow-up. Blasts from all cases expressed adhesion molecules at high rates. β1 integrin chain (CD18) was consistently found on blasts from most ALL cases; among integrins of the β2 family, LFA-1 was detected in 58% of cases, in the virtual absence of other α chains. CD54 and CD58 were expressed in variable proportions by ALL blasts and CD44 was detected in the majority of the malignant cells, whereas the CD62L selectin was only present in 24% of cases. B-lineage ALLs displayed similar adhesion molecule phenotypes irrespective of maturational stages of the leukemic cells. We found a significantly reduced expression of β2 αL integrins in the hybrid ALL cases (CD13 and/or CD33 positive). However, these cases did not show differences in clinical presentation and behaviour in comparison with patients of other groups. We did not find a significant correlation between adhesion molecule expression and well established risk factors (age, white blood cell count, central nervous system involvement, chromosomal abnormalities), with the exception of splenomegaly, that was significantly associated with CD18 expression. In the follow-up, no evidence of significant correlation between adhesive phenotype and adverse events such as leukemic relapse and death was found. In conclusion, although expression of adhesion molecules on lymphoblasts confirms the phenotypic heterogeneity of ALL, it appears that this is not relevant for the clinical aspects of the disease and for prognosis.

Early and progressive insulin resistance in young, non-obese cancer survivors treated with hematopoietic stem cell transplantation.

It is unclear whether there is a causative relationship between the development of metabolic syndrome (MS) and increased risk of early cardiovascular morbidity in patients receiving hematopoietic stem cell transplantation (HSCT) during childhood. Early identification of risk factors associated with insulin resistance, MS, and abnormal glucose tolerance during childhood or adolescence in these patients could represent a useful tool for preventing cardiovascular disorders.

RABGAP1L gene rearrangement resulting from a der(Y)t(Y;1)(q12;q25) in acute myeloid leukemia arising in a child with Klinefelter syndrome

In this study, we report the molecular cytogenetic characterization of an acute myeloid leukemia with a der(Y)t(Y;1)(q12;q25) in bone marrow cells in a child with Klinefelter syndrome. Conventional cytogenetics demonstrated the unbalanced translocation, i.e., a trisomic 1q25-qter juxtaposed to Yq12 replaced the terminal segment of chromosome Y was acquired and present only on bone marrow cells. Fluorescence in situ hybridization showed that the breakpoint at 1q25 disrupted RABGAP1L, a strongly expressed gene in CFU-GEMM, erythroid cells, and megakaryocytes, while the Yq12 breakpoint fell within the heterochromatic region. As der(Y)t(Y;1)(q12;q25) was an isolated cytogenetic change, RABGAP1L rearrangement as well as gene(s) dosage effects correlated to 1q25-qter trisomy, and Yq12-qter loss may make a major contribution to leukemogenesis and/or disease progression.

Rapid T-cell receptor CD4+ repertoire reconstitution and immune recovery in unrelated umbilical cord blood transplanted pediatric leukemia patients

Umbilical cord blood transplantation has been successfully employed for treatment of many immune and hematologic disorders. The aim of this study was to evaluate the quality of immune reconstitution after umbilical cord blood transplantation in 6 leukemia children. T-cell receptor Vβ third complementary region spectratyping was used for monitoring the contribution of the thymic pathway in patients’ immune reconstitution. Absolute numbers of lymphocyte subsets (T, B, and natural killer), and lymphoproliferative in vitro response to mitogens, recovered within 12 months after transplantation. Furthermore, an overall diversification of T-cell receptor complexity in the repopulating T cells, with a polyclonal Gaussian profiles in most (74%) of total families was observed. Noteworthy, we showed a wider and more rapid reconstitution of T-cell receptor CD4+ T cell families compared with T-cell receptor CD8+ T ones still exhibiting some perturbations at 24 months. These data show that umbilical cord blood transplantation allows immune reconstitution already within 12 months with generation of newly diversified CD4+ T lymphocyte subsets.

Pearson Syndrome: A Retrospective Cohort Study from the Marrow Failure Study Group of A.I.E.O.P. (Associazione Italiana Emato-Oncologia Pediatrica)

Pearson syndrome (PS) is a very rare and often fatal multisystemic mitochondrial disorder involving the liver, kidney, pancreas, and hematopoietic and central nervous system. It is characterized principally by a transfusion-dependent anemia that usually improves over time, a tendency to develop severe infections, and a high mortality rate. We describe a group of 11 PS patients diagnosed in Italy in the period 1993-2014. The analysis of this reasonably sized cohort of patients contributes to the clinical profile of the disease and highlights a rough incidence of 1 case/million newborns. Furthermore, it seems that some biochemical parameters like increased serum alanine and urinary fumaric acid can help to address an early diagnosis.

Pre-transplant manipulation processing of umbilical cord blood units: Efficacy of Rubinstein's thawing technique used in 40 transplantation procedures.

BACKGROUND Umbilical cord blood (UCB) is a valid alternative to be used in transplanted patients. Limitations of the use of stem cells depends on the small number of cells available; this is the reason why UCB can be used only in very low-weight patients. In this study we have evaluated the efficacy of cellular manipulation before transplant and in particular, before thawing the units through the Rubinstein method. METHODS We have evaluated the results obtained after thawing 40 UCB to be used for as many patients affected by several pathologies (21 ALL, 6 AML, 3 MDS, 2 LNH, 2 histiocytosis, 2 β-thalassemia, 1 Chédiak-Higashi syndrome, 1 Fanconi anemia, 1 Wiskott-Aldrich syndrome and 1 Omenn syndrome). RESULTS After thawing, nucleated cells (NC) mean recovery was 76.81% (SD±15.41). The quantity of NC obtained was 124.29×107 (SD±43.18) and in only 5 cases the number of NC after the procedure was lower than the requested graft dose. Among the last ones, in two cases only we did not achieve the target after manipulation. The post-manipulation cellular viability was 83.48% (SD±10.6). For all the units shipment complied with all the necessary procedures; in fact the temperature never rose above -120°C. CONCLUSION In our study we highlighted the efficacy of UCB thawing technique, with the same method defined in 1995 at the New York Blood Centre that guarantees an excellent NC recovery and maintains a high level of cell viability.