Letizia Pomponia Brescia

HLA-haploidentical stem cell transplantation after removal of αβ+ T and B cells in children with nonmalignant disorders

Twenty-three children with nonmalignant disorders received HLA-haploidentical hematopoietic stem cell transplantation (haplo-HSCT) after ex vivo elimination of αβ(+) T cells and CD19(+) B cells. The median number of CD34(+), αβ(+)CD3(+), and B cells infused was 16.8 × 10(6), 40 × 10(3), and 40 × 10(3) cells/kg, respectively. No patient received any posttransplantation pharmacologic prophylaxis for graft-versus-host disease (GVHD). All but 4 patients engrafted, these latter being rescued by a second allograft. Three patients experienced skin-only grade 1 to 2 acute GVHD. No patient developed visceral acute or chronic GVHD. Cumulative incidence of transplantation-related mortality was 9.3%. With a median follow-up of 18 months, 21 of 23 children are alive and disease-free, the 2-year probability of disease-free survival being 91.1%. Recovery of γδ(+) T cells was prompt, but αβ(+) T cells progressively ensued over time. Our data suggest that this novel graft manipulation strategy is safe and effective for haplo-HSCT. This trial was registered at www.clinicaltrials.gov as #NCT01810120.

Natural killer cells in the treatment of high-risk acute leukaemia

Several studies have shown that in patients with acute leukaemia given allogeneic haematopoietic stem cell transplantation (allo-HSCT) large part of the therapeutic effect lies on the anti-tumour effect displayed by cells of both adaptive and innate immunity. This evidence has also opened new scenarios for the treatment of patients with other haematological malignancies/solid tumours. In particular, donor-derived natural killer (NK) cells play a crucial role in the eradication of cancer cells in patients given an allograft from an HLA-haploidentical relative, especially when there is a killer inhibitory-receptor (KIR)-KIR ligand mismatched in the donor-recipient direction. Alloreactive donor-derived NK cells have been also demonstrated to kill recipient antigen-presenting cells and cytotoxic T lymphocytes, thus preventing graft-versus-host disease (GvHD) and graft rejection and to largely contribute to the defence against cytomegalovirus infection in the early post-transplant period. Several clinical studies have recently focused also on the influence of NK-cell activating receptors on the outcome of allo-HSCT recipients; in particular, B/x haplotype donors offer clinical advantages compared with A/A donors, even when the donor is an HLA-identical volunteer. Altogether, these data have provided the rationale for implementing phase I/II clinical trials based on adoptive infusion of either selected or ex vivo activated NK cells from an HLA-mismatched donor. This review summarizes the biological and clinical data on the role played by NK cells in patients with high-risk acute leukaemia, focusing also on the still unsolved issues and the future perspectives related to the approaches of adoptive NK cell therapy.

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.

HLA-haploidentical T cell-depleted allogeneic hematopoietic stem cell transplantation in children with fanconi anemia

We report the outcome of 12 consecutive pediatric patients with Fanconi anemia (FA) who had neither an HLA-identical sibling nor an HLA-matched unrelated donor and who were given T cell-depleted, CD34(+) positively selected cells from a haploidentical related donor after a reduced-intensity, fludarabine-based conditioning regimen. Engraftment was achieved in 9 of 12 patients (75%), and the cumulative incidence of graft rejection was 17% (95% confidence interval [CI], 5% to 59%). Cumulative incidences of grades II to IV acute and chronic graft-versus-host disease were 17% (95% CI, 5% to 59%) and 35% (95% CI, 14% to 89%), respectively. The conditioning regimen was well tolerated, with no fatal regimen-related toxicity and 3 cases of grade III regimen-related toxicity. The cumulative incidence of transplant-related mortality was 17% (95% CI, 5% to 59%). The 5-year overall survival, event-free survival, and disease-free survival were 83% (95% CI, 62% to 100%), 67% (95% CI, 40% to 93%), and 83% (95% CI, 62% to 100%), respectively. These data demonstrate that a fludarabine-based conditioning regimen, followed by infusion of high doses of T cell-depleted stem cells, is able to ensure engraftment with good overall survival and disease-free survival, confirming the feasibility of haploidentical hematopoietic stem cell transplantation in FA. To the best of our knowledge, this is the largest series of hematopoietic stem cell transplantation from a haploidentical related donor in FA patients reported to date.

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.

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.

Adoptive immunotherapy with antigen-specific T cells during extracorporeal membrane oxygenation (ECMO) for adenovirus-related respiratory failure in a child given haploidentical stem cell transplantation.

We report on the successful infusion of human adenovirus (HAdV)‐specific T cells in a child with congenital amegakaryocytic thrombocytopenia, given T‐cell‐depleted hematopoietic stem cell transplantation (HSCT) from the HLA‐haploidentical mother during extracorporeal membrane oxygenation (ECMO) for severe HAdV‐related respiratory failure. Donor‐derived, interferon (IFN)‐γ‐secreting HAdV‐specific T cells were enriched using the cytokine capture assay, after in vitro stimulation with overlapping peptides from the immunodominant HAdV5 hexon protein. Two weeks after T‐cell transfer, viral load decreased and ECMO was discontinued. T‐cell responses to HAdV antigens were documented after four weeks and were associated with viral clearance, immune reconstitution and clinical amelioration. Pediatr Blood Cancer 2014;61:376–379.

Extracorporeal photopheresis for paediatric patients experiencing graft-versus-host disease (GVHD).

Extracorporeal photopheresis (ECP) is broadly used in adults with cutaneous T-cell lymphoma, acute or chronic graft-versus-host disease (GVHD), rejection of solid organ transplants, and a variety of autoimmune, cell-mediated diseases. The predominant use of ECP in children and adolescents is for treating GVHD. Children pose specific challenges to ECP, due to their unique physiology and to patients size. Herein, we will focus on current clinical trials with ECP in children with GVHD, with an emphasis on technical and clinical issues that are peculiar to the paediatric setting.

Zoledronic acid boosts γδ T-cell activity in children receiving αβ+ T and CD19+ cell-depleted grafts from an HLA-haplo-identical donor

ABSTRACT We demonstrated that γδ T cells of patients given HLA-haploidentical HSCT after removal of αβ+ T cells and CD19+ B cells are endowed with the capacity of killing leukemia cells after ex vivo treatment with zoledronic acid (ZOL). Thus, we tested the hypothesis that infusion of ZOL in patients receiving this type of graft may enhance γδ T-cell cytotoxic activity against leukemia cells. ZOL was infused every 28 d in 43 patients; most were treated at least twice. γδ T cells before and after ZOL treatments were studied in 33 of these 43 patients, till at least 7 mo after HSCT by high-resolution mass spectrometry, flow-cytometry, and degranulation assay. An induction of Vδ2-cell differentiation, paralleled by increased cytotoxicity of both Vδ1 and Vδ2 cells against primary leukemia blasts was associated with ZOL treatment. Cytotoxic activity was further increased in Vδ2 cells, but not in Vδ1 lymphocytes in those patients given more than one treatment. Proteomic analysis of γδ T cells purified from patients showed upregulation of proteins involved in activation processes and immune response, paralleled by downregulation of proteins involved in proliferation. Moreover, a proteomic signature was identified for each ZOL treatment. Patients given three or more ZOL infusions had a better probability of survival in comparison to those given one or two treatments (86% vs. 54%, respectively, p = 0.008). Our data indicate that ZOL infusion in pediatric recipients of αβ T- and B-cell-depleted HLA-haploidentical HSCT promotes γδ T-cell differentiation and cytotoxicity and may influence the outcome of patients.

Structural variation in SBDS gene, with loss of exon 3, in two Shwachman-Diamond patients.

Shwachman-Diamond Syndrome (SDS) is an autosomal recessive disease (#260400) characterized by pancreatic exocrine insufficiency, hematological abnormalities, bone marrow failure with increased risk for acute myelodysplasia and acute myeloid leukaemia and skeletal alterations. As suggested by international guidelines [1], the SDS clinical diagnosis is confirmed by detection of bi-allelic pathogenic variants in SBDS gene, localized at 7q11.21 [2,3]. Targeted mutation analysis of exon 2 by PCR-RFLP discloses in at least 90% of SDS patients one of the three common mutations, (c.185_184TANCT, c.258+2TNC and the combination of [c.183_184TANCT:258+2TNC] on one allele). Two of the commonmutations are observed concomitantly in approximately 62% of SDS patients [3]. In our laboratories, since 2003, we performed SBDSmolecular analysis confirming the clinical diagnosis in 115 patients, and all of them harboured at least one of the common mutations in exon 2. We recently found two families, each of themwith two affected children, identified with their Unique Patient Number (UPN) as UPN 42, UPN 43 and UPN 61, UPN 76, carrying only the [c.258+2TNC]mutation; no other DNA changes was found after complete sequencing analysis of all five exons of SBDS and their flanking intronic regions. In both families, the clinical picture of the patients completely fits the diagnostic criteria: in all of them, at clinical diagnosis, both hematological manifestations (neutropenia, thrombocytopenia and hypocellular bone marrow) and exocrine pancreas dysfunction (with reduced levels of fecal elastase and serum lipase) were present. No patient presented skeletal defects but a growth delay was a common symptom. UPN 61 underwent bone marrow transplantation at age 2 following an EBV infection requiring blood transfusions. Western-blot analysis using SBDS antibody did not demonstrate any SBDS protein in the three buffy coats available, suggesting the presence of a more complex change on the second allele (Fig. 1). On genomicDNAof the four patientswe performed a deletion/duplication analysis by long-range PCRwith the specific primers designed by Boocock et al. [2], but matching them differently to obtain longer amplicons containing more exons. When using the primers [forward for exon 2 and reverse for exon 4], that amplify the region from exon 2 to exon 4, we observed in all patients the expected band of 3756bp and the presence of a second smaller band of approximately 3000bp (Fig. 2). We then extracted the DNA from the smaller band and used it as template in a new long-range PCR experiment, with the same primers.