Valentina Bertaina

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.

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.

Strategies to accelerate immune recovery after allogeneic hematopoietic stem cell transplantation

ABSTRACT The interplay existing between immune reconstitution and patient outcome has been extensively demonstrated in allogeneic hematopoietic stem cell transplantation. One of the leading causes of infection-related mortality is the slow recovery of T-cell immunity due to the conditioning regimen and/or age-related thymus damage, poor naïve T-cell output, and restricted T-cell receptor (TCR) repertoires. With the aim of improving posttransplantation immune reconstitution, several immunotherapy approaches have been explored. Donor leukocyte infusions are widely used to accelerate immune recovery, but they carry the risk of provoking graft-versus-host disease. This review will focus on sophisticated strategies of thymus function-recovery, adoptive infusion of donor-derived, allodepleted T cells, T-cell lines/clones specific for life-threatening pathogens, regulatory T cells, and of T cells transduced with suicide genes.

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.

IDO1 involvement in mTOR pathway: a molecular mechanism of resistance to mTOR targeting in medulloblastoma

Medulloblastoma (MB) is the most common malignant brain tumor in children. Despite therapeutic advancements, high-risk groups still present significant mortality. A deeper knowledge of the signaling pathways contributing to MB formation and aggressiveness would help develop new successful therapies. The target of rapamycin, mTOR signaling, is known to be involved in MB and is already targetable in the clinical setting. Furthermore, mTOR is a master metabolic regulator able to control cell growth versus autophagy decisions in conditions of amino-acid deprivation that can be due to IDO1 enzymatic activity. IDO1 has been also implicated in the regulation of inflammation, as well as of T cell-mediated immune responses, in a variety of pathological conditions, including brain tumors. In particular, IDO1 induces expansion of regulatory T-cells (Treg), preventing immune response against tumor cells. Analysis of 27 MB tissue specimens for the expression of both mTOR and IDO1 showed their widespread expression in all samples. Testing their cooperation in vitro, a significant involvement of IDO1 in mTOR immunogenic pathway was found, able to counteract the aim of rapamycin treatment. In MB cell lines, inhibition of mTOR strongly induced IDO1 expression and activity, corroborating its ability to recruit Treg cells in the tumor microenvironment. The mTOR/IDO1 cross talk was found to be strictly specific of MB cells. We demonstrated that mTOR pathway cross talks with IDO1 pathway to promote MB immune escape, possibly contributing to failure of mTOR- targeted therapy.

Cytomegalovirus in hematopoietic stem cell transplant recipients - management of infection

ABSTRACT Introduction: Cytomegalovirus (CMV) still causes significant morbidity and mortality in patients given allogeneic hematopoietic stem cell transplantation (HSCT). Despite effective pharmacotherapy, potentially life-threatening CMV disease occurs nowadays in up to 10% of HSCT recipients; moreover, routinely used anti-CMV agents have been shown to be associated with morbidity. Areas covered: This review examines different issues related to diagnosis and management of CMV infection in HSCT recipients, paying particular attention to the monitoring of CMV-specific immune recovery, approaches of adoptive cell therapy and new antiviral drugs. Expert commentary: Despite advances in diagnostic tests and treatment, there is still room for refining management of CMV in HSCT recipients. Immunological monitoring should be associated in the future to virological monitoring. The safety profile and efficacy of new anti-CMV agents should be compared with that of standard-of-care drugs. Donor-derived, pathogen-specific T cells adoptively transferred after transplantation could contribute to reduce the impact of CMV infection on patient’s outcome.

Che‐1 is targeted by c‐Myc to sustain proliferation in pre‐B‐cell acute lymphoblastic leukemia

Despite progress in treating B‐cell precursor acute lymphoblastic leukemia (BCP‐ALL), disease recurrence remains the main cause of treatment failure. New strategies to improve therapeutic outcomes are needed, particularly in high‐risk relapsed patients. Che‐1/AATF (Che‐1) is an RNA polymerase II‐binding protein involved in proliferation and tumor survival, but its role in hematological malignancies has not been clarified. Here, we show that Che‐1 is overexpressed in pediatric BCP‐ALL during disease onset and at relapse, and that its depletion inhibits the proliferation of BCP‐ALL cells. Furthermore, we report that c‐Myc regulates Che‐1 expression by direct binding to its promoter and describe a strict correlation between Che‐1 expression and c‐Myc expression. RNA‐seq analyses upon Che‐1 or c‐Myc depletion reveal a strong overlap of the respective controlled pathways. Genomewide ChIP‐seq experiments suggest that Che‐1 acts as a downstream effector of c‐Myc. These results identify the pivotal role of Che‐1 in the control of BCP‐ALL proliferation and present the protein as a possible therapeutic target in children with relapsed BCP‐ALL.

Unrelated donor vs HLA-haploidentical alpha/beta T-cell and B-cell depleted HSCT in children with acute 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.

Preservation of antigen-specific functions of αβ T cells and B cells removed from hematopoietic stem cell transplants suggests their use as an alternative cell source for advanced manipulation and adoptive immunotherapy

Hematopoietic stem cell transplantation is standard therapy for numerous hematological diseases. The use of haploidentical donors, sharing half of the HLA alleles with the recipient, has facilitated the use of this procedure as patients can rely on availability of a haploidentical donor within their family. Since HLA disparity increases the risk of graft-versus-host disease, T-cell depletion has been used to remove alloreactive lymphocytes from the graft. Selective removal of αβ T cells, which encompass the alloreactive repertoire, combined with removal of B cells to prevent EBV-related lymphoproliferative disease, proved safe and effective in clinical studies. Depleted αβ T cells and B cells are generally discarded as by-products. Considering the possible use of donor T cells for donor lymphocyte infusions or for generation of pathogen-specific T cells as mediators of graft-versus-infection effect, we tested whether cells in the discarded fractions were functionally intact. Response to alloantigens and to viral antigens comparable to that of unmanipulated cells indicated a functional integrity of αβ T cells, in spite of the manipulation used for their depletion. Furthermore, B cells proved to be efficient antigen-presenting cells, indicating that antigen uptake, processing, and presentation were fully preserved. Therefore, we propose that separated αβ T lymphocytes could be employed for obtaining pathogen-specific T cells, applying available methods for positive selection, which eventually leads to indirect allodepletion. In addition, these functional T cells could undergo additional manipulation, such as direct allodepletion or genetic modification.

Indoleamine 2,3-dioxygenase-1 (IDO1) expression by childhood acute myeloid leukemias inhibits T-cell production of IFN-γ and confers an unfavorable prognosis

Indoleamine 2,3-dioxygenase 1 (IDO1) degrades tryptophan into kynurenine (KYN) and other immune suppressive molecules that inhibit effector T cells and promote regulatory T-cell differentiation. We have previously shown that IDO1 mRNA and protein are detectable in blast cells from 52% of adults with newly diagnosed acute myeloid leukemia (AML). Herein, we investigated IDO1 expression and function in 41 children with AML (median age=10 years, range 1-17). In 20/41 cases, leukemia blast cells up-regulated IDO1 after in vitro challenge with IFN-γ. Of interest, microenvironmental IFN-γ was higher in IDO(pos) compared with IDO(neg) patients. In line with these results, bone marrow (BM)-resident T cells produced more IFN-γ, but not IL-4 or IL-17, compared with T cells from normal BM samples. KYN levels significantly increased in supernatants of IFN-γ-stimulated AML cells (21.0 μM/L, range 6.1-36.0) compared with unstimulated cultures (0.85 μM/L, range 0.4-1.7; p=0.0022), in parallel with tryptophan consumption (2.95 μM/L, range 1.0-37.0, after challenge with IFN-γ compared with 38.1 μM, range 18.2-50.0, in unstimulated cultures; p<0.0001). In a mixed tumor cell lymphocyte culture, AML blasts primed with IFN-γ inhibited Th1 cytokine production by allogeneic CD8+ and, to a lesser extent, CD4+ T cells, while enhancing Th2 cytokine release. The provision of D,L-1-methyl-tryptophan (1MT), an IDO inhibitor, to T-cell/AML co-cultures partially restored IFN-γ production by both CD4+ and CD8+ T cells. Furthermore, IDO-expressing AML blasts inhibited NK-cell degranulation, as measured through CD107a expression. Finally, 5-year overall survival was significantly better for IDO(neg) patients (34 months) compared with IDO(pos) ones (64.7 months; p=0.0438; Figure ​Figure1).1). In conclusion, IDO suppresses Th1 responses/NK activity and may portend an unfavorable prognosis in childhood AML. Figure 1 The 5-year overall survival of IDO-positive and IDO-negative patients with AML is shown.