Publications by authors named "Barbara Buldini"

50 Publications

Targeting the plasticity of mesenchymal stromal cells to reroute the course of acute myeloid leukemia.

Blood 2021 Aug;138(7):557-570

Women's and Children's Health Department, Haematology-Oncology Clinic and Laboratory, University of Padova, Padova, Italy.

Bone marrow (BM) microenvironment contributes to the regulation of normal hematopoiesis through a finely tuned balance of self-renewal and differentiation processes, cell-cell interaction, and secretion of cytokines that during leukemogenesis are altered and favor tumor cell growth. In pediatric acute myeloid leukemia (AML), chemotherapy is the standard of care, but >30% of patients still relapse. The need to accelerate the evaluation of innovative medicines prompted us to investigate the role of mesenchymal stromal cells (MSCs) in the leukemic niche to define its contribution to the mechanism of leukemia drug escape. We generated a humanized 3-dimensional (3D) niche with AML cells and MSCs derived from either patients (AML-MSCs) or healthy donors. We observed that AML cells establish physical connections with MSCs, mediating a reprogrammed transcriptome inducing aberrant cell proliferation and differentiation and severely compromising their immunomodulatory capability. We confirmed that AML cells modulate h-MSCs transcriptional profile promoting functions similar to the AML-MSCs when cocultured in vitro, thus facilitating leukemia progression. Conversely, MSCs derived from BM of patients at time of disease remission showed recovered healthy features at transcriptional and functional levels, including the secretome. We proved that AML blasts alter MSCs activities in the BM niche, favoring disease development and progression. We discovered that a novel AML-MSC selective CaV1.2 channel blocker drug, lercanidipine, is able to impair leukemia progression in 3D both in vitro and when implanted in vivo if used in combination with chemotherapy, supporting the hypothesis that synergistic effects can be obtained by dual targeting approaches.
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http://dx.doi.org/10.1182/blood.2020009845DOI Listing
August 2021

Low-Blood Lymphocyte Number and Lymphocyte Decline as Key Factors in COPD Outcomes: A Longitudinal Cohort Study.

Respiration 2021;100(7):618-630. Epub 2021 Apr 26.

Department of Cardiac, Thoracic, Vascular Sciences and Public Health, University of Padova, Padova, Italy.

Background: Smokers with and without chronic obstructive pulmonary disease (COPD) are at risk of severe outcomes like exacerbations, cancer, respiratory failure, and decreased survival. The mechanisms for these outcomes are unclear; however, there is evidence that blood lymphocytes (BL) number might play a role.

Objective: The objective of this study is to investigate the relationship between BL and their possible decline over time with long-term outcomes in smokers with and without COPD.

Methods: In 511 smokers, 302 with COPD (COPD) and 209 without COPD (noCOPD), followed long term, we investigated whether BL number and BL decline over time might be associated with long-term outcomes. Smokers were divided according to BL number in high-BL (≥1,800 cells/µL) and low-BL (<1,800 cells/µL). Clinical features, cancer incidence, and mortality were recorded during follow-up. BL count in multiple samples and BL decline over time were calculated and related to outcomes.

Results: BL count was lower in COPD (1,880 cells/µL) than noCOPD (2,300 cells/µL; p < 0.001). 43% of COPD and 23% of noCOPD had low-BL count (p < 0.001). BL decline over time was higher in COPD than noCOPD (p = 0.040). 22.5% of the whole cohort developed cancer which incidence was higher in low-BL subjects and in BL decliners than high-BL (31 vs. 18%; p = 0.001) and no decliners (32 vs. 19%; p = 0.002). 26% in the cohort died during follow-up. Furthermore, low-BL count, BL decline, and age were independent risk factors for mortality by Cox regression analysis.

Conclusion: BL count and BL decline are related to worse outcomes in smokers with and without COPD, which suggests that BL count and decline might play a mechanistic role in outcomes deterioration. Insights into mechanisms inducing the fall in BL count could improve the understanding of COPD pathogenesis and point toward new therapeutic measures.
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http://dx.doi.org/10.1159/000515180DOI Listing
April 2021

Childhood cancer in Italy: background, goals, and achievements of the Italian Paediatric Hematology Oncology Association (AIEOP).

Tumori 2021 Apr 20:3008916211007934. Epub 2021 Apr 20.

Pediatric Onco-Hematology, Stem Cell Transplantation and Cellular Therapy Division, Regina Margherita Children's Hospital, Azienda Ospedaliera-Universitaria Città della Salute e della Scienza, University of Torino, Piemonte, Italy.

This article reviews the primary goals and achievements of the Italian Association for Pediatric Hematology-Oncology (Associazione Italiana Ematologia Oncologia Pediatrica [AIEOP]), a national cooperative group that has been working for children and adolescents with cancer in Italy since 1975.
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http://dx.doi.org/10.1177/03008916211007934DOI Listing
April 2021

CD56, HLA-DR, and CD45 recognize a subtype of childhood AML harboring CBFA2T3-GLIS2 fusion transcript.

Cytometry A 2021 Aug 2;99(8):844-850. Epub 2021 Apr 2.

Pediatric Hemato Oncology, Maternal and Child Health Department, University of Padua, Padua, Italy.

The presence of CBFA2T3-GLIS2 fusion gene has been identified in childhood Acute Myeloid Leukemia (AML). In view of the genomic studies indicating a distinct gene expression profile, we evaluated the role of immunophenotyping in characterizing a rare subtype of AML-CBFA2T3-GLIS2 rearranged. Immunophenotypic data were obtained by studying a cohort of 20 pediatric CBFA2T3-GLIS2-AML and 77 AML patients not carrying the fusion transcript. Enrolled cases were included in the Associazione Italiana di Ematologia Oncologia Pediatrica (AIEOP) AML trials and immunophenotypes were compared using different statistical approaches. By multiple computational procedures, we identified two main core antigens responsible for the identification of the CBFA2T3-GLIS2-AML. CD56 showed the highest performance in single marker evaluation (AUC = 0.89) and granted the most accurate prediction when used in combination with HLA-DR (AUC = 0.97) displaying a 93% sensitivity and 99% specificity. We also observed a weak-to-negative CD45 expression, being exceptional in AML. We here provide evidence that the combination of HLA-DR negativity and intense bright CD56 expression detects a rare and aggressive pediatric AML genetic lesion improving the diagnosis performance.
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http://dx.doi.org/10.1002/cyto.a.24339DOI Listing
August 2021

Relative expansion of CD19-negative very-early normal B-cell precursors in children with acute lymphoblastic leukaemia after CD19 targeting by blinatumomab and CAR-T cell therapy: implications for flow cytometric detection of minimal residual disease.

Br J Haematol 2021 05 14;193(3):602-612. Epub 2021 Mar 14.

National Research and Clinical Center for Pediatric Hematology, Oncology and Immunology, Moscow, Russian Federation.

CD19-directed treatment in B-cell precursor acute lymphoblastic leukaemia (BCP-ALL) frequently leads to the downmodulation of targeted antigens. As multicolour flow cytometry (MFC) application for minimal/measurable residual disease (MRD) assessment in BCP-ALL is based on B-cell compartment study, CD19 loss could hamper MFC-MRD monitoring after blinatumomab or chimeric antigen receptor T-cell (CAR-T) therapy. The use of other antigens (CD22, CD10, CD79a, etc.) as B-lineage gating markers allows the identification of CD19-negative leukaemia, but it could also lead to misidentification of normal very-early CD19-negative BCPs as tumour blasts. In the current study, we summarized the results of the investigation of CD19-negative normal BCPs in 106 children with BCP-ALL who underwent CD19 targeting (blinatumomab, n = 64; CAR-T, n = 25; or both, n = 17). It was found that normal CD19-negative BCPs could be found in bone marrow after CD19-directed treatment more frequently than in healthy donors and children with BCP-ALL during chemotherapy or after stem cell transplantation. Analysis of the antigen expression profile revealed that normal CD19-negative BCPs could be mixed up with residual leukaemic blasts, even in bioinformatic analyses of MFC data. The results of our study should help to investigate MFC-MRD more accurately in patients who have undergone CD19-targeted therapy, even in cases with normal CD19-negative BCP expansion.
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http://dx.doi.org/10.1111/bjh.17382DOI Listing
May 2021

Thioridazine requires calcium influx to induce MLL-AF6-rearranged AML cell death.

Blood Adv 2020 09;4(18):4417-4429

Haematology-Oncology Clinic and Laboratory, Department of Woman and Child Health, and.

In pediatric acute myeloid leukemia (AML), intensive chemotherapy and allogeneic hematopoietic stem cell transplantation are the cornerstones of treatment in high-risk cases, with severe late effects and a still high risk of disease recurrence as the main drawbacks. The identification of targeted, more effective, safer drugs is thus desirable. We performed a high-throughput drug-screening assay of 1280 compounds and identified thioridazine (TDZ), a drug that was highly selective for the t(6;11)(q27;q23) MLL-AF6 (6;11)AML rearrangement, which mediates a dramatically poor (below 20%) survival rate. TDZ induced cell death and irreversible progress toward the loss of leukemia cell clonogenic capacity in vitro. Thus, we explored its mechanism of action and found a profound cytoskeletal remodeling of blast cells that led to Ca2+ influx, triggering apoptosis through mitochondrial depolarization, confirming that this latter phenomenon occurs selectively in t(6;11)AML, for which AF6 does not work as a cytoskeletal regulator, because it is sequestered into the nucleus by the fusion gene. We confirmed TDZ-mediated t(6;11)AML toxicity in vivo and enhanced the drug's safety by developing novel TDZ analogues that exerted the same effect on leukemia reduction, but with lowered neuroleptic effects in vivo. Overall, these results refine the MLL-AF6 AML leukemogenic mechanism and suggest that the benefits of targeting it be corroborated in further clinical trials.
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http://dx.doi.org/10.1182/bloodadvances.2020002001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7509857PMC
September 2020

The hematopoietic stem cell marker VNN2 is associated with chemoresistance in pediatric B-cell precursor ALL.

Blood Adv 2020 09;4(17):4052-4064

Department of Stem Cell Transplantation, University Children's Hospital Zurich, Zurich, Switzerland; and.

Most relapses of acute lymphoblastic leukemia (ALL) occur in patients with a medium risk (MR) for relapse on the Associazione Italiana di Ematologia e Oncologia Pediatrica and Berlin-Frankfurt-Münster (AIEOP-BFM) ALL protocol, based on persistence of minimal residual disease (MRD). New insights into biological features that are associated with MRD are needed. Here, we identify the glycosylphosphatidylinositol-anchored cell surface protein vanin-2 (VNN2; GPI-80) by charting the cell surface proteome of MRD very high-risk (HR) B-cell precursor (BCP) ALL using a chemoproteomics strategy. The correlation between VNN2 transcript and surface protein expression enabled a retrospective analysis (ALL-BFM 2000; N = 770 cases) using quantitative polymerase chain reaction to confirm the association of VNN2 with MRD and independent prediction of worse outcome. Using flow cytometry, we detected VNN2 expression in 2 waves, in human adult bone marrow stem and progenitor cells and in the mature myeloid compartment, in line with proposed roles for fetal hematopoietic stem cells and inflammation. Prospective validation by flow cytometry in the ongoing clinical trial (AIEOP-BFM 2009) identified 10% (103/1069) of VNN2+ BCP ALL patients at first diagnosis, primarily in the MRD MR (48/103, 47%) and HR (37/103, 36%) groups, across various cytogenetic subtypes. We also detected frequent mutations in epigenetic regulators in VNN2+ ALLs, including histone H3 methyltransferases MLL2, SETD2, and EZH2 and demethylase KDM6A. Inactivation of the VNN2 gene did not impair leukemia repopulation capacity in xenografts. Taken together, VNN2 marks a cellular state of increased resistance to chemotherapy that warrants further investigations. Therefore, this marker should be included in diagnostic flow cytometry panels.
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http://dx.doi.org/10.1182/bloodadvances.2019000938DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7479947PMC
September 2020

Flash survey on severe acute respiratory syndrome coronavirus-2 infections in paediatric patients on anticancer treatment.

Eur J Cancer 2020 06 7;132:11-16. Epub 2020 Apr 7.

The Cancer Centre for Children, The Children's Hospital at Westmead, Australia.

Introduction: Since the beginning of COVID-19 pandemic, it is known that the severe course of the disease occurs mostly among the elderly, whereas it is rare among children and young adults. Comorbidities, in particular, diabetes and hypertension, clearly associated with age, besides obesity and smoke, are strongly associated with the need for intensive treatment and a dismal outcome. A weaker immunity of the elderly has been proposed as a possible explanation of this uneven age distribution. Thus, there is concern that children treated for cancer may allso be at risk for an unfavourable course of infection. Along the same line, anecdotal information from Wuhan, China, mentioned a severe course of COVID-19 in a child treated for leukaemia.

Aim And Methods: We made a flash survey on COVID-19 incidence and severity among children on anticancer treatment. Respondents were asked by email to fill in a short Web-based survey.

Results: We received reports from 25 countries, where approximately 10,000 patients at risk are followed up. At the time of the survey, more than 200 of these children were tested, nine of whom were positive for COVID-19. Eight of the nine cases had asymptomatic to mild disease, and one was just diagnosed with COVID-19. We also discuss preventive measures that are in place or should be taken and treatment options in immunocompromised children with COVID-19.

Conclusion: Thus, even children receiving anticancer chemotherapy may have a mild or asymptomatic course of COVID-19. While we should not underestimate the risk of developing a more severe course of COVID-19 than that observed here, the intensity of preventive measures should not cause delays or obstructions in oncological treatment.
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http://dx.doi.org/10.1016/j.ejca.2020.03.021DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7141482PMC
June 2020

Health technology assessment-based approach to flow cytometric immunophenotyping of acute leukemias: a literature classification.

Tumori 2020 Feb 14:300891620904412. Epub 2020 Feb 14.

Fondazione IRCCS Istituto Nazionale dei Tumori, Direzione Scientifica, Milan, Italy.

Objective: Acute leukemia (AL) is a broad, heterogeneous group of malignant diseases. The diagnostic workup of AL is based on several clinical and laboratory findings, including flow cytometric immunophenotyping. However, the role of this assay in the diagnosis of AL has not been systematically investigated. The aim of this study was to determine the accuracy and utility of flow cytometric immunophenotyping in the identification, characterization, and staging of AL.

Methods: We performed a systematic selection and classification of the literature since 1980, focused on flow cytometric immunophenotyping of AL. We applied a 6-variables model to cover both the technical capabilities and the clinical value of flow cytometric immunophenotyping in the diagnosis of AL.

Results: Using 3 key words (acute leukemia, immunophenotyping, flow cytometry), we screened the literature from January 1985 to April 2015 in PubMed and Embase databases and found 1010 articles. A total of 363 were selected and submitted to the expert panel, which selected a final data set of 248 articles to be analyzed. Of these, 160 were focused on clinical and biological issues, 55 were technical articles, and 31 were reviews. These 248 articles were then analyzed according to the 6-variables model and definitively classified.

Conclusions: We assessed the literature on flow cytometric immunophenotyping of AL over 3 decades as the first step toward an evidence-based analysis of the impact of this technology on the clinical management of patients with AL.
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http://dx.doi.org/10.1177/0300891620904412DOI Listing
February 2020

Flow-Cytometric Monitoring of Minimal Residual Disease in Pediatric Patients With Acute Myeloid Leukemia: Recent Advances and Future Strategies.

Front Pediatr 2019 11;7:412. Epub 2019 Oct 11.

Children's Cancer Research Institute (CCRI), St. Anna Kinderkrebsforschung, Vienna, Austria.

Minimal residual disease (MRD) by multiparametric flow cytometry (MFC) has been recently shown as a strong and independent prognostic marker of relapse in pediatric AML (pedAML) when measured at specific time points during Induction and/or Consolidation therapy. Hence, MFC-MRD has the potential to refine the current strategies of pedAML risk stratification, traditionally based on the cytogenetic and molecular genetic aberrations at diagnosis. Consequently, it may guide the modulation of therapy intensity and clinical decision making. However, the use of non-standardized protocols, including different staining panels, analysis, and gating strategies, may hamper a broad implementation of MFC-MRD monitoring in clinical routine. Besides, the thresholds of MRD positivity still need to be validated in large, prospective and multi-center clinical studies, as well as optimal time points of MRD assessment during therapy, to better discriminate patients with different prognosis. In the present review, we summarize the most relevant findings on MFC-MRD testing in pedAML. We examine the clinical significance of MFC-MRD and the recent advances in its standardization, including innovative approaches with an automated analysis of MFC-MRD data. We also touch upon other technologies for MRD assessment in AML, such as quantitative genomic breakpoint PCR, current challenges and future strategies to enable full incorporation of MFC-MRD into clinical practice.
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http://dx.doi.org/10.3389/fped.2019.00412DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6798174PMC
October 2019

Relapses and treatment-related events contributed equally to poor prognosis in children with ABL-class fusion positive B-cell acute lymphoblastic leukemia treated according to AIEOP-BFM protocols.

Haematologica 2020 07 10;105(7):1887-1894. Epub 2019 Oct 10.

Pediatric Hematology-Oncology Department, Ruth Rappaport Children's Hospital, Rambam Health Care Campus, Rappaport Faculty of Medicine, Technion-Israel Institute of Technology, Haifa, Israel.

ABL-class fusions other than characterize around 2-3% of precursor B-cell acute lymphoblastic leukemia. Case series indicated that patients suffering from these subtypes have a dismal outcome and may benefit from the introduction of tyrosine kinase inhibitors. We analyzed clinical characteristics and outcome of 46 ABL-class fusion positive cases other than treated according to AIEOP-BFM (Associazione Italiana di Ematologia-Oncologia Pediatrica-Berlin-Frankfurt-Münster) ALL 2000 and 2009 protocols; 13 of them received a tyrosine kinase inhibitor (TKI) during different phases of treatment. ABL-class fusion positive cases had a poor early treatment response: minimal residual disease levels of ≥5×10 were observed in 71.4% of patients after induction treatment and in 51.2% after consolidation phase. For the entire cohort of 46 cases, the 5-year probability of event-free survival was 49.1+8.9% and that of overall survival 69.6+7.8%; the cumulative incidence of relapse was 25.6+8.2% and treatment-related mortality (TRM) 20.8+6.8%. One out of 13 cases with TKI added to chemotherapy relapsed while eight of 33 cases without TKI treatment suffered from relapse, including six in 17 patients who had not received hematopoietic stem cell transplantation. Stem cell transplantation seems to be effective in preventing relapses (only three relapses in 25 patients), but was associated with a very high TRM (6 patients). These data indicate a major need for an early identification of ABL-class fusion positive acute lymphoblastic leukemia cases and to establish a properly designed, controlled study aimed at investigating the use of TKI, the appropriate chemotherapy backbone and the role of hematopoietic stem cell transplantation. (Registered at: .
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http://dx.doi.org/10.3324/haematol.2019.231720DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7327633PMC
July 2020

Next-generation sequencing of PTEN mutations for monitoring minimal residual disease in T-cell acute lymphoblastic leukemia.

Pediatr Blood Cancer 2020 01 1;67(1):e28025. Epub 2019 Oct 1.

Department of Woman's and Child's Health, University-Hospital of Padua, Padua, Italy.

Minimal residual disease (MRD) analysis has become a powerful indicator to refine therapy in acute lymphoblastic leukemia (ALL). Here, we present an MRD detection based on the next-generation sequencing of PTEN exon 7 mutations (NGS-PTEN) in 30 pediatric T-cell ALL patients. By comparing the NGS-PTEN results with current quantitative PCR of antigen receptor gene rearrangements (qPCR-Ig/TR), an overall concordance of 80% was found between the two methods. However, the NGS-PTEN qualified a lower number of high-risk patients than qPCR-Ig/TR. These findings suggest that NGS-PTEN is a promising tool that could potentially be used to support current MRD methodologies for T-ALL patients.
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http://dx.doi.org/10.1002/pbc.28025DOI Listing
January 2020

Cerebrospinal fluid analysis by 8-color flow cytometry in children with acute lymphoblastic leukemia.

Leuk Lymphoma 2019 11 25;60(11):2825-2828. Epub 2019 Apr 25.

Pediatric Onco-Hematology Unit, Department of Women's and Children's Health, University of Padova, Padova, Italy.

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http://dx.doi.org/10.1080/10428194.2019.1602269DOI Listing
November 2019

Flow-cytometric minimal residual disease monitoring in blood predicts relapse risk in pediatric B-cell precursor acute lymphoblastic leukemia in trial AIEOP-BFM-ALL 2000.

Pediatr Blood Cancer 2019 05 18;66(5):e27590. Epub 2018 Dec 18.

Children's Cancer Research Institute, Vienna, Austria.

Background: Flow-cytometric monitoring of minimal residual disease (MRD) in bone marrow (BM) during induction of pediatric patients with acute lymphoblastic leukemia (ALL) is widely used for outcome prognostication and treatment stratification. Utilizing peripheral blood (PB) instead of BM might be favorable, but data on its usefulness are scarce.

Procedure: We investigated 1303 PB samples (days 0, 8, 15, 33, and 52) and 285 BMs (day 15) from 288 pediatric ALL patients treated in trial AIEOP-BFM ALL 2000. MRD was assessed by four-color flow cytometry and evaluated as relative, absolute, and kinetic result.

Results: In B-ALL only, PB measures from early time points correlated with relapse incidence (CIR). Best separation occurred at threshold <1 blast/μL at day 8 (5-year CIR 0.02 ± 0.02 vs 0.12 ± 0.03; P = 0.044). Patients with highest relapse risk were not distinguishable, but PB-MRD at days 33 and 52 correlated with prednisone response and postinduction BM-MRD by PCR (P < 0.001). Kinetic assessment did not convey any advantage. In multivariate analysis including day 15 BM-MRD, PB-MRD measures lost statistical power.

Conclusions: In summary, PB-MRD in pediatric B-ALL correlates with outcome and risk parameters, but its prognostic significance is not strong enough to substitute for BM assessment in AIEOP-BFM trials. It might, however, be valuable in treatment environments not using multifaceted risk stratification with other MRD measures.
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http://dx.doi.org/10.1002/pbc.27590DOI Listing
May 2019

Unrelated donor vs HLA-haploidentical α/β T-cell- and B-cell-depleted HSCT in children with acute leukemia.

Blood 2018 12 22;132(24):2594-2607. Epub 2018 Oct 22.

Department of Pediatric Hematology/Oncology and Cell and Gene Therapy, Istituto di Ricovero e Cura a Carattere Scientifico (IRCCS), Ospedale Bambino Gesù, Rome, Italy.

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 ( < .001). Children treated with αβhaplo-HSCT also had a significantly lower incidence of overall and extensive chronic GVHD ( < .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 ( < .001). When compared with patients given MMUD-HSCT, αβhaplo-HSCT recipients had a lower cumulative incidence of nonrelapse mortality and a better GRFS ( < .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.
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http://dx.doi.org/10.1182/blood-2018-07-861575DOI Listing
December 2018

A Novel t(8;14)(q24;q11) Rearranged Human Cell Line as a Model for Mechanistic and Drug Discovery Studies of NOTCH1-Independent Human T-Cell Leukemia.

Cells 2018 Oct 9;7(10). Epub 2018 Oct 9.

UOC Immunologia e Diagnostica Molecolare Oncologica, Istituto Oncologico Veneto-IRCCS, Padova 35128, Italy.

-translocated T-lineage acute lymphoblastic leukemia (T-ALL) is a rare subgroup of T-ALL associated with deletions, inactivation, and absence of or mutations. This subtype of T-ALL has been associated with induction failure and aggressive disease. Identification of drug targets and mechanistic insights for this disease are still limited. Here, we established a human NOTCH1-independent -translocated T-ALL cell line that maintains the genetic and phenotypic characteristics of the parental leukemic clone at diagnosis. The University of Padua T-cell acute lymphoblastic leukemia 13 (UP-ALL13) cell line has all the main features of the above described -translocated T-ALL. Interestingly, UP-ALL13 was found to harbor a heterozygous R882H mutation typically found in myeloid leukemia. Chromatin immunoprecipitation coupled with high-throughput sequencing for histone H3 lysine 27 (H3K27) acetylation revealed numerous putative super-enhancers near key transcription factors, including MYC, MYB, and LEF1. Marked cytotoxicity was found following bromodomain-containing protein 4 (BRD4) inhibition with AZD5153, suggesting a strict dependency of this particular subtype of T-ALL on the activity of super-enhancers. Altogether, this cell line may be a useful model system for dissecting the signaling pathways implicated in NOTCH1-independent T-ALL and for the screening of targeted anti-leukemia agents specific for this T-ALL subgroup.
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http://dx.doi.org/10.3390/cells7100160DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6209910PMC
October 2018

The genetic basis and cell of origin of mixed phenotype acute leukaemia.

Nature 2018 10 12;562(7727):373-379. Epub 2018 Sep 12.

Johns Hopkins Medical Institutions, Baltimore, MD, USA.

Mixed phenotype acute leukaemia (MPAL) is a high-risk subtype of leukaemia with myeloid and lymphoid features, limited genetic characterization, and a lack of consensus regarding appropriate therapy. Here we show that the two principal subtypes of MPAL, T/myeloid (T/M) and B/myeloid (B/M), are genetically distinct. Rearrangement of ZNF384 is common in B/M MPAL, and biallelic WT1 alterations are common in T/M MPAL, which shares genomic features with early T-cell precursor acute lymphoblastic leukaemia. We show that the intratumoral immunophenotypic heterogeneity characteristic of MPAL is independent of somatic genetic variation, that founding lesions arise in primitive haematopoietic progenitors, and that individual phenotypic subpopulations can reconstitute the immunophenotypic diversity in vivo. These findings indicate that the cell of origin and founding lesions, rather than an accumulation of distinct genomic alterations, prime tumour cells for lineage promiscuity. Moreover, these findings position MPAL in the spectrum of immature leukaemias and provide a genetically informed framework for future clinical trials of potential treatments for MPAL.
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http://dx.doi.org/10.1038/s41586-018-0436-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6195459PMC
October 2018

Genetic risk factors for VIPN in childhood acute lymphoblastic leukemia patients identified using whole-exome sequencing.

Pharmacogenomics 2018 10 7;19(15):1181-1193. Epub 2018 Sep 7.

Charles-Bruneau Cancer Center, CHU Sainte-Justine Research Center, Montreal, QC, H3T1C5, Canada.

Aim: To identify genetic markers associated with vincristine-induced peripheral neuropathy (VIPN) in childhood acute lymphoblastic leukemia.

Patients & Methods: Whole-exome sequencing data were combined with exome-wide association study to identify predicted-functional germline variants associated with high-grade VIPN. Genotyping was then performed for top-ranked signals (n = 237), followed by validation in independent replication group (n = 405).

Results: Minor alleles of rs2781377/SYNE2 (p = 0.01) and rs10513762/MRPL47 (p = 0.01) showed increased risk, whereas that of rs3803357/BAHD1 had a protective effect (p = 0.007). Using a genetic model based on weighted genetic risk scores, an additive effect of combining these loci was observed (p = 0.003). The addition of rs1135989/ACTG1 further enhanced model performance (p = 0.0001).

Conclusion: Variants in SYNE2, MRPL47 and BAHD1 genes are putative new risk factors for VIPN in childhood acute lymphoblastic leukemia.
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http://dx.doi.org/10.2217/pgs-2018-0093DOI Listing
October 2018

The presence of mutated and deleted PTEN is associated with an increased risk of relapse in childhood T cell acute lymphoblastic leukaemia treated with AIEOP-BFM ALL protocols.

Br J Haematol 2018 09 25;182(5):705-711. Epub 2018 Jun 25.

Department of Women's and Children's Health, Paediatric Hematology and Oncology, University of Padova, Padova, Italy.

Notwithstanding the improvement in treatment results for paediatric T cell acute lymphoblastic leukaemia (T-ALL) it remains important to understand if genetic aberrations influence therapy response. PTEN tumour suppressor gene inactivation is a frequent event in T-ALL but its effect on patient therapy response is debatable. We analysed the effect of the presence of mutated PTEN on outcome in 257 children with T-ALL treated with Associazione Italiana di Ematologia e Oncologia Pediatrica (AIEOP)-Berlin-Frankfürt-Münster (BFM) protocols. PTEN mutations were present in 31 (12·1%) patients and were significantly associated with increased risk of relapse. PTEN mutations also indicate a poor prognosis in T-ALL patients in the absence of NOTCH1 mutations or in the group of patients with co-presence of PTEN mutation and deletions. These results indicate that PTEN genomic aberrations and the biologically consequential PTEN inactivation contribute to adverse therapy response in T-ALL patients; PTEN status as a biomarker may contribute to the development of new molecularly-defined stratification algorithms.
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http://dx.doi.org/10.1111/bjh.15449DOI Listing
September 2018

Hematopoietic stem cell transplantation for isolated extramedullary relapse of acute lymphoblastic leukemia in children.

Bone Marrow Transplant 2019 02 13;54(2):275-283. Epub 2018 Jun 13.

Oncoematologia Pediatrica, I.R.C.C.S. Ospedale Pediatrico Bambino Gesù, Roma, Italy.

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.
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http://dx.doi.org/10.1038/s41409-018-0259-5DOI Listing
February 2019

International cooperative study identifies treatment strategy in childhood ambiguous lineage leukemia.

Blood 2018 07 2;132(3):264-276. Epub 2018 May 2.

National Program for Antineoplastic Drugs for Children (PINDA), Chilean National Pediatric Oncology Group, Hospital Roberto del Rio/Universidad de Chile, Santiago, Chile.

Despite attempts to improve the definitions of ambiguous lineage leukemia (ALAL) during the last 2 decades, general therapy recommendations are missing. Herein, we report a large cohort of children with ALAL and propose a treatment strategy. A retrospective multinational study (International Berlin-Frankfurt-Münster Study of Leukemias of Ambiguous Lineage [iBFM-AMBI2012]) of 233 cases of pediatric ALAL patients is presented. Survival statistics were used to compare the prognosis of subsets and types of treatment. Five-year event-free survival (EFS) of patients with acute lymphoblastic leukemia (ALL)-type primary therapy (80% ± 4%) was superior to that of children who received acute myeloid leukemia (AML)-type or combined-type treatment (36% ± 7.2% and 50% ± 12%, respectively). When ALL- or AML-specific gene fusions were excluded, 5-year EFS of CD19 leukemia was 83% ± 5.3% on ALL-type primary treatment compared with 0% ± 0% and 28% ± 14% on AML-type and combined-type primary treatment, respectively. Superiority of ALL-type treatment was documented in single-population mixed phenotype ALAL (using World Health Organization and/or European Group for Immunophenotyping of Leukemia definitions) and bilineal ALAL. Treatment with ALL-type protocols is recommended for the majority of pediatric patients with ALAL, including cases with CD19 ALAL. AML-type treatment is preferred in a minority of ALAL cases with CD19 and no other lymphoid features. No overall benefit of transplantation was documented, and it could be introduced in some patients with a poor response to treatment. As no clear indicator was found for a change in treatment type, this is to be considered only in cases with ≥5% blasts after remission induction. The results provide a basis for a prospective trial.
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http://dx.doi.org/10.1182/blood-2017-12-821363DOI Listing
July 2018

Epigenetic heterogeneity affects the risk of relapse in children with t(8;21)RUNX1-RUNX1T1-rearranged AML.

Leukemia 2018 05 2;32(5):1124-1134. Epub 2018 Feb 2.

Women and Child Health Department, Haematology-Oncology Clinic and Lab, University of Padova, Padova, Italy.

The somatic translocation t(8;21)(q22;q22)/RUNX1-RUNX1T1 is one of the most frequent rearrangements found in children with standard-risk acute myeloid leukemia (AML). Despite the favorable prognostic role of this aberration, we recently observed a higher than expected frequency of relapse. Here, we employed an integrated high-throughput approach aimed at identifying new biological features predicting relapse among 34 t(8;21)-rearranged patients. We found that the DNA methylation status of patients who suffered from relapse was peculiarly different from that of children maintaining complete remission. The epigenetic signature, made up of 337 differentially methylated regions, was then integrated with gene and protein expression profiles, leading to a network, where cell-to-cell adhesion and cell-motility pathways were found to be aberrantly activated in relapsed patients. We identified most of these factors as RUNX1-RUNX1T1 targets, with Ras Homolog Family Member (RHOB) overexpression being the core of this network. We documented how RHOB re-organized the actin cytoskeleton through its downstream ROCK-LIMK-COFILIN axis: this increases blast adhesion by stress fiber formation, and reduces mitochondrial apoptotic cell death after chemotherapy treatment. Altogether, our data show an epigenetic heterogeneity within t(8;21)-rearranged AML patients at diagnosis able to influence the program of the chimeric transcript, promoting blast re-emergence and progression to relapse.
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http://dx.doi.org/10.1038/s41375-017-0003-yDOI Listing
May 2018

Pre- and post-transplant minimal residual disease predicts relapse occurrence in children with acute lymphoblastic leukaemia.

Br J Haematol 2018 03 23;180(5):680-693. Epub 2018 Jan 23.

Clinic of Paediatric Haemato-Oncology, Department of Women's and Children's Health, University of Padua, Padua, Italy.

Relapse remains the leading cause of treatment failure in children with acute lymphoblastic leukaemia (ALL) undergoing allogeneic haematopoietic stem cell transplantation (HSCT). We retrospectively investigated the prognostic role of minimal residual disease (MRD) before and after HSCT in 119 children transplanted in complete remission (CR). MRD was measured by polymerase chain reaction in bone marrow samples collected pre-HSCT and during the first and third trimesters after HSCT (post-HSCT1 and post-HSCT3). The overall event-free survival (EFS) was 50%. The cumulative incidence of relapse and non-relapse mortality was 41% and 9%. Any degree of detectable pre-HSCT MRD was associated with poor outcome: EFS was 39% and 18% in patients with MRD positivity <1 × 10 and ≥1 × 10 , respectively, versus 73% in MRD-negative patients (P < 0·001). This effect was maintained in different disease remissions, but low-level MRD had a very strong negative impact only in patients transplanted in second or further CR. Also, MRD after HSCT enabled patients to be stratified, with increasing MRD between post-HSCT1 and post-HSCT3 clearly defining cohorts with a different outcome. MRD is an important prognostic factor both before and after transplantation. Given that MRD persistence after HSCT is associated with dismal outcome, these patients could benefit from early discontinuation of immunosuppression, or pre-emptive immuno-therapy.
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http://dx.doi.org/10.1111/bjh.15086DOI Listing
March 2018

DNA variants in DHFR gene and response to treatment in children with childhood B ALL: revisited in AIEOP-BFM protocol.

Pharmacogenomics 2018 Jan 6;19(2):105-112. Epub 2017 Dec 6.

Charles-Bruneau Cancer Center, CHU Sainte-Justine Research Center, Montreal, QC, H3T1C5, Canada.

Aim: We have previously reported an association of dihydrofolate reductase promoter polymorphisms with reduced event-free survival in childhood acute lymphoblastic leukemia (ALL) patients treated with Dana Farber Cancer Institute protocol. Here, we assessed whether these associations are applicable to other protocol, based on different methotrexate doses.

Methods: Genotypes for six tag polymorphisms and resulting haplotypes were analyzed for an association with ALL outcome.

Results: The association was found with the polymorphisms A-680C, A-317G and C-35T in high-risk group patients. Carriers of haplotype *1 had a remarkably higher risk of events compared with noncarriers and a lower probability of event-free survival (21.4 vs 81.3%).

Conclusion: The role of DHFR variants in predicting the outcome of childhood ALL extends beyond single-treatment protocol and can be useful biomarker in personalizing treatment.
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http://dx.doi.org/10.2217/pgs-2017-0153DOI Listing
January 2018

Prognostic significance of flow-cytometry evaluation of minimal residual disease in children with acute myeloid leukaemia treated according to the AIEOP-AML 2002/01 study protocol.

Br J Haematol 2017 04 27;177(1):116-126. Epub 2017 Feb 27.

Department of Woman and Child Health, Laboratory of Haematology-Oncology, University of Padova, Padova, Italy.

In children with acute myeloid leukaemia (AML), assessment of initial treatment response is an essential prognostic factor; methods more sensitive than morphology are still under evaluation. We report on the measurement of minimal residual disease (MRD), by multicolour flow-cytometry in one centralized laboratory, in 142 children with newly diagnosed AML enrolled in the Associazione Italiana di EmatoOncologia Pediatrica-AML 2002/01 trial. At the end of the first induction course, MRD was <0·1% in 69, 0·1-1% in 16 and >1% in 51 patients. The 8-year disease-free survival (DFS) of 125 children in morphological complete remission and with MRD <0·1%, 0·1-1% and ≥1% was 73·1 ± 5·6%, 37·8 ± 12·1% and 34·1 ± 8·8%, respectively (P < 0·01). MRD was also available after the second induction course in 92/142 patients. MRD was ≥0·1% at the end of the first induction course in 36 patients; 13 reached an MRD <0·1% after the second one and their DFS was 45·4 ± 16·7% vs. 22·8 ± 8·9% in patients with persisting MRD ≥0·1% (P = 0·037). Multivariate analysis demonstrated that MRD ≥0·1% after first induction course was, together with a monosomal karyotype, an independent adverse prognostic factor for DFS. Our results show that MRD detected by flow-cytometry after induction therapy predicts outcome in patients with childhood AML and can help stratifying post-remission treatment.
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http://dx.doi.org/10.1111/bjh.14523DOI Listing
April 2017

AIEOP-BFM consensus guidelines 2016 for flow cytometric immunophenotyping of Pediatric acute lymphoblastic leukemia.

Cytometry B Clin Cytom 2018 01 21;94(1):82-93. Epub 2017 Feb 21.

Laboratory of Pediatric Onco-Hematology, Women and Child Department, University of Padova, Padova, Italy.

Immunophenotyping by flow cytometry (FCM) is a worldwide mainstay in leukemia diagnostics. For concordant multicentric application, however, a gap exists between available classification systems, technologic standardization, and clinical needs. The AIEOP-BFM consortium induced an extensive standardization and validation effort between its nine national reference laboratories collaborating in immunophenotyping of pediatric acute lymphoblastic leukemia (ALL). We elaborated common guidelines which take advantage of the possibilities of multi-color FCM: marker panel requirements, immunological blast gating, in-sample controls, tri-partite antigen expression rating (negative vs. weak or strong positive) with capturing of blast cell heterogeneities and subclone formation, refined ALL subclassification, and a dominant lineage assignment algorithm able to distinguish "simple" from bilineal/"complex" mixed phenotype acute leukemia (MPAL) cases, which is essential for choice of treatment. These guidelines are a first step toward necessary inter-laboratory standardization of pediatric leukemia immunophenotyping for a concordant multicentric application. © 2017 International Clinical Cytometry Society.
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http://dx.doi.org/10.1002/cyto.b.21518DOI Listing
January 2018
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