Publications by authors named "Andrea Assanelli"

44 Publications

Ruxolitinib for chronic steroid-refractory graft versus host disease: a single center experience.

Leuk Res 2021 Jun 11;109:106642. Epub 2021 Jun 11.

Haematology and Bone Marrow Transplant Unit, IRCSS San Raffaele Scientific Institute, Italy. Electronic address:

Background: Chronic Graft versus Host Disease (GvHD) is a serious complication of allogeneic hematopoietic stem cell transplant that severely impacts quality of life and long-term survival. About 50-to-60 % of patients treated with steroids require a further line of therapy due to lack of sustained response. Ruxolitinib, a JAK1/2 inhibitor, has recently been approved for the treatment of acute GvHD.

Methods: We aimed to retrospectively evaluate ruxolitinib efficacy and safety in a cohort of patients diagnosed with moderate (25 %) or severe (75 %) steroid-refractory or steroid-dependent chronic GvHD. Response evaluation was performed at three and six months.

Results: Thirty-six patients received ruxolitinib after a median of three previous lines (range, r 1-11) for a median of 8.6 months (r 1-51.6). Cutaneous GvHD was the most frequent presentation. We observed an overall response of 59 % (CR 9%, PR 50 %) at three months and 62 % (CR 15 %, PR 46 %) at six months. Two patients had hematologic disease recurrence and were censored at relapse; no other permanent discontinuation due to adverse events were documented. Cutaneous, oral, genital and ocular GvHD significantly improved after treatment. 2-year overall survival and 2-year transplant related mortality were 74 % and 19 % respectively. Ruxolitinib was associated with a significant reduction of steroid dose.

Conclusion: Ruxolitinib was confirmed to be a safe and effective option as salvage treatment also for advanced stages of chronic GvHD. Longer follow up is needed to evaluate durability of response. Prospective analyses on larger cohorts are ongoing.
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http://dx.doi.org/10.1016/j.leukres.2021.106642DOI Listing
June 2021

Posttransplantation Cyclophosphamide- and Sirolimus-Based Graft-Versus-Host-Disease Prophylaxis in Allogeneic Stem Cell Transplant.

Transplant Cell Ther 2021 Jun 1. Epub 2021 Jun 1.

Hematology and Bone Marrow Transplantation Unit, IRCCS San Raffaele Scientific Institute, Milan, Italy.

Post-transplantation cyclophosphamide (PTCy) has emerged as a promising graft-versus-host-disease (GVHD) prophylaxis in the setting of allogeneic hematopoietic stem cell transplantation (HSCT) from haploidentical donors and more recently in matched donor transplants. Herein, we describe our real-life experience on 249 adult patients undergoing allogeneic HSCT, from HLA-matched related (MRD), HLA-matched unrelated (MUD), or mismatched related donors (MMRD). Patients received unmanipulated peripheral blood stem cells (PBSCs), using a GVHD prophylaxis with PTCy and sirolimus. Mycophenolate mofetil was added in MUD or MMRD. In the HLA-matched donor group (MRD, n = 48, MUD, n = 50), the cumulative incidence of grades II-IV and III-IV acute GvHD was 23% and 9% at 100 days, respectively. The cumulative incidence of chronic GvHD was 25% at 2 years, severe only for 5% of the patients. The cumulative incidences of relapse and transplant-related mortality (TRM) were 31% and 9% at 2 years, respectively. The 2-year overall survival (OS) was 72% and progression-free survival (PFS) 60%; the composite endpoint of GvHD/relapse-free survival (GRFS) was 52% at 2 years. In the haploidentical donor group (n = 151), we documented a cumulative incidence of grades II-IV and III-IV acute GVHD of 35% and 20% at 100 days, respectively, and a cumulative incidence of chronic GvHD of 39% at 2 years. We observed severe chronic GVHD in 15% of the patients. The cumulative incidence of relapse and TRM was 32% and 25% at 2 years, respectively. The 2-year OS was 48%, whereas PFS was 43%; GRFS was 28% at 2 years. However, more patients in the haploidentical group presented high/very high disease risk index (DRI) and higher HCT-comorbidity index. In patients classified in the low-intermediate DRI, 2-year GRFS was 53% in MRD, 65% in MUD, and 46% in haploidentical HSCT (P = .33). Sirolimus-PTCy platform deserves further investigation as an alternative to calcineurin-inhibitor-based GVHD prophylaxis for all donor sources. In patients presenting a low-intermediate DRI, this strategy translates in relevant survival independently from the transplant source.
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http://dx.doi.org/10.1016/j.jtct.2021.05.023DOI Listing
June 2021

Continuous positive airway pressure and pronation outside the intensive care unit in COVID 19 ARDS.

Minerva Med 2020 Sep 30. Epub 2020 Sep 30.

Unit of General Medicine and Advanced Care, IRCCS San Raffaele Scientific Institute, Milan, Italy.

Background: The efficacy and safety of continuous positive airway pressure and respiratory physiotherapy outside the ntensive care unit during a pandemic.

Methods: In this cohort study performed in February-May 2020 in a large teaching hospital in Milan, COVID-19 patients with adult respiratory distress syndrome receiving continuous positive airway pressure (positive end-expiratory pressure = 10 cm H2O, FiO2 = 0.6, daily treatment duration: 4x3hcycles) and respiratory physiotherapy including pronation outside the intensive care unit were followed up.

Results: Of 90 ARDS patients treated with continuous positive airway pressure (45/90, 50% pronated at least once) outside the intensive care unit and with a median (interquartile) follow up of 37 (11-46) days, 45 (50%) were discharged at home, 28 (31%) were still hospitalized, and 17 (19%) died. Continuous positive airway pressure failure was recorded for 35 (39%) patients. Patient mobilization was associated with reduced failure rates (p=0.033). No safety issues were observed.

Conclusions: Continuous positive airway pressure with patient mobilization (including pronation) was effective and safe in patients with ARDS due to COVID-19 managed outside the intensive care unit setting during the pandemic.
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http://dx.doi.org/10.23736/S0026-4806.20.06952-9DOI Listing
September 2020

The first case of COVID-19 treated with the complement C3 inhibitor AMY-101.

Clin Immunol 2020 06 29;215:108450. Epub 2020 Apr 29.

Hematology and Bone Marrow Transplantation Unit, IRCCS San Raffaele Scientific Institute, Milan, Italy; University Vita Salute San Raffaele, Milan, Italy. Electronic address:

Acute respiratory distress syndrome (ARDS) is a devastating clinical manifestation of COVID-19 pneumonia and is mainly based on an immune-driven pathology. Mounting evidence suggests that COVID-19 is fueled by a maladaptive host inflammatory response that involves excessive activation of innate immune pathways. While a "cytokine storm" involving IL-6 and other cytokines has been documented, complement C3 activation has been implicated as an initial effector mechanism that exacerbates lung injury in preclinical models of SARS-CoV infection. C3-targeted intervention may provide broader therapeutic control of complement-mediated inflammatory damage in COVID-19 patients. Herein, we report the clinical course of a patient with severe ARDS due to COVID-19 pneumonia who was safely and successfully treated with the compstatin-based complement C3 inhibitor AMY-101.
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http://dx.doi.org/10.1016/j.clim.2020.108450DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7189192PMC
June 2020

Fast reshaping of intensive care unit facilities in a large metropolitan hospital in Milan, Italy: facing the COVID-19 pandemic emergency

Crit Care Resusc 2020 04 1;22(2):91-94. Epub 2020 Apr 1.

IRCCS San Raffaele Scientific Institute, Milan, Italy.

At the end of 2019, the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) outbreak spread from China all around the world, causing thousands of deaths. In Italy, the hardest hit region was Lombardy, with the first reported case on 20 February 2020. San Raffaele Scientific Institute — a large tertiary hospital and research centre in Milan, Italy — was immediately involved in the management of the public health emergency. Since the beginning of the outbreak, the elective surgical activity of the hospital was rapidly reduced and large areas of the hospital were simultaneously reorganised to admit and assist patients with coronavirus disease 2019 (COVID-19). In addition, the hospital became the regional referral hub for cardiovascular emergencies in order to keep ensuring a high level of health care to non-COVID-19 patients in northern Italy. In a few days, a COVID-19 emergency department was created, improving the general ward capacity to a total number of 279 beds dedicated to patients with COVID-19. Moreover, the number of intensive care unit (ICU) beds was increased from 28 to 72 (54 of them dedicated to patients with COVID-19, and 18 to cardiology and cardiac surgery hub emergencies), both converting pre-existing areas and creating new high technology spaces. All the involved health care personnel were rapidly trained to use personal protection equipment and to manage this particular category of patients both in general wards and ICUs. Furthermore, besides clinical activities, continuously important research projects were carried out in order to find new strategies and more effective therapies to better face an unprecedented health emergency in Italy.
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April 2020

Lung Ultrasound to Evaluate Invasive Fungal Diseases after Allogeneic Hematopoietic Stem Cell Transplantation.

Infect Chemother 2019 Dec;51(4):386-392

Hematology and Bone Marrow Transplant Unit, IRCCS San Raffaele Scientific Institute, Milano, Italy.

Invasive fungal diseases (IFDs) are a leading cause of infection-related-mortality after allogeneic hematopoietic stem cell transplantation (HSCT). In this prospective pilot study, we investigated the use of bedside lung ultrasound (US) in IFD management. Ten consecutive hematological patients, who developed pulmonary IFD after HSCT, were included in the study. Standard computed tomography scan and lung US were performed at IFD diagnosis and 10 days after antifungal treatment. The lung US demonstrated a high sensitivity in the detection of lung lesions at IFD diagnosis and in the follow-up examinations. It is of potential clinical relevance for IFD management in hematological patients.
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http://dx.doi.org/10.3947/ic.2019.51.4.386DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6940370PMC
December 2019

Clinical implementation of low-dose total body irradiation using topotherapy technique.

Phys Imaging Radiat Oncol 2019 Oct 16;12:74-79. Epub 2019 Dec 16.

Medical Physics, San Raffaele Scientific Institute, Milan, Italy.

Background And Purpose: The topotherapy technique was recently suggested as a robust alternative to helical radiation delivery for total body irradiation (TBI). It allows to deliver a discrete number of beams with fixed gantry. A Topotherapy-based low-dose TBI technique was optimized and clinically implemented.

Materials And Methods: TBI delivery was split in two parts: the first treating from the head to half thigh and the second the remaining legs. An in-silico investigation aimed to optimize plan parameters was first carried out on four patients. For the upper plan, field width and pitch were fixed to 5 cm and 0.5: the combined impact of five modulation factor (MF) values and different field configurations (6/8/12 fields) was investigated. For the lower plan, two anterior/posterior beams (field width: 5 cm; pitch: 0.5; MF:1.5) were used. After assessing the optimal technique, set-up/quality assurance/image-guidance procedures were defined and the technique clinically implemented: 23 patients were treated up to now.

Results: The best compromise between treatment time and planning target volume (PTV) coverage/homogeneity was found for MF = 1.5 and 8 fields. All clinical plans were automatically optimized using an "ad-hoc" plan template: excellent PTV coverage (PTV95%>98.5%) and homogeneity (median SD:4%) were found with a median beam-on time of 17/9 min for the upper/lower plan. All patients were successfully treated and transplanted.

Conclusions: TBI delivered with the topotherapy approach robustly guarantees adequate coverage and dose homogeneity. Semi-automatic clinical plans can be quickly generated and efficiently delivered.
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http://dx.doi.org/10.1016/j.phro.2019.11.009DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7807637PMC
October 2019

Interleukin-6 as Biomarker for Acute GvHD and Survival After Allogeneic Transplant With Post-transplant Cyclophosphamide.

Front Immunol 2019 1;10:2319. Epub 2019 Oct 1.

Haematology and Bone Marrow Transplant Unit, IRCCS San Raffaele Scientific Institute, Milan, Italy.

Although the outcome of allogeneic hematopoietic stem cell transplantation (allo-HSCT) has dramatically improved in the past decade, it is still compromised by transplant-related mortality (TRM), mainly caused by Graft-vs. -Host Disease (GvHD). We conducted a prospective observational study to ascertain the potential of serum interleukin-6 (IL6) levels, measured before conditioning and 7 days after allo-HSCT, in predicting acute GvHD, TRM and survival after allo-HSCT with Post-Transplant Cyclophosphamide (PT-Cy) based GvHD prophylaxis. Between April 2014 and June 2017, we collected samples from 166 consecutive allo-HSCT patients. By ROC analysis, we identified a threshold of 2.5 pg/ml for pre-transplant IL6 and 16.5 pg/ml for post-transplant IL6. Both univariate and multivariate analyses confirmed the ability of high baseline IL6 levels to predict worse OS (HR 4.3; < 0.01) and grade II-IV acute GvHD (HR 1.8; = 0.04), and of high post-transplant IL6 to identify patients with worse OS (HR 3.3; < 0.01) and higher risk of grade II-IV (HR 5; < 0.01) and grade III-IV acute GvHD (HR 10.2; < 0.01). In multivariate analysis, both baseline (HR 6.7; < 0.01) and post-transplant high IL6 levels (HR 3.5; = 0.02) predicted higher TRM. IL6 may contribute to the risk stratification of patients at major risk for aGvHD and TRM, potentially providing a window for additional prophylactic or preemptive strategies to improve the quality of life in the early post-transplant phase and the outcome of allo-HSCT.
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http://dx.doi.org/10.3389/fimmu.2019.02319DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6779849PMC
October 2020

Clofarabine and Treosulfan as Conditioning for Matched Related and Unrelated Hematopoietic Stem Cell Transplantation: Results from the Clo3o Phase II Trial.

Biol Blood Marrow Transplant 2020 02 9;26(2):316-322. Epub 2019 Oct 9.

Hematology and Bone Marrow Transplant Unit, IRCCS San Raffaele Scientific Institute, Milan, Italy. Electronic address:

Allogeneic hematopoietic stem cell transplantation (allo-HSCT) can be curative for patients with hematologic malignancies. The ideal conditioning regimen before allo-HSCT has not been established. We conducted a Phase II study to evaluate the tolerability and efficacy of clofarabine and treosulfan as conditioning regimen before allo-HSCT. The primary objective was to evaluate the cumulative incidence of nonrelapse mortality (NRM) on day +100. Forty-four patients (36 with acute myelogenous leukemia, 5 with acute lymphoblastic leukemia, 3 with myelodysplastic syndromes) were enrolled. The median patient age was 47 years, and the median duration of follow-up was 27 months. The conditioning regimen was based on clofarabine 40 mg/m (days -6 to -2) and treosulfan 14 g/m (days -6 to -4). Allogeneic hematopoietic stem cells were derived from a sibling (n = 22) or a well-matched unrelated donor (n = 22). Graft-versus-host disease (GVHD) prophylaxis consisted of antithymocyte globulin, rituximab, cyclosporine, and a short-course of methotrexate. The regimen allowed for rapid engraftment and a 100-day NRM of 18%, due mainly to bacterial infections. The incidences of grade II-IV acute GVHD and chronic GVHD were 16% and 19%, respectively. The rates of overall survival (OS), progression-free survival, and relapse at 2 years were 51%, 31%, and 50%, respectively. Significantly different outcomes were observed between patients with low-intermediate and patients with high-very high Disease Risk Index (DRI) scores (1-year OS, 78% and 24%, respectively). Our findings show that the use of treosulfan and clofarabine as a conditioning regimen for allo-HSCT is feasible, with a 78% 1-year OS in patients with a low-intermediate DRI score. However, 1-year NRM was 18%, and despite the intensified conditioning regimen, relapse incidence remains a major issue in patients with poor prognostic risk factors.
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http://dx.doi.org/10.1016/j.bbmt.2019.09.032DOI Listing
February 2020

Lentiviral haemopoietic stem/progenitor cell gene therapy for treatment of Wiskott-Aldrich syndrome: interim results of a non-randomised, open-label, phase 1/2 clinical study.

Lancet Haematol 2019 May 10;6(5):e239-e253. Epub 2019 Apr 10.

CSD Pharma Consulting, Redhill, UK; Orchard Therapeutics, London, UK.

Background: Wiskott-Aldrich syndrome is a rare, life-threatening, X-linked primary immunodeficiency characterised by microthrombocytopenia, infections, eczema, autoimmunity, and malignant disease. Lentiviral vector-mediated haemopoietic stem/progenitor cell (HSPC) gene therapy is a potentially curative treatment that represents an alternative to allogeneic HSPC transplantation. Here, we report safety and efficacy data from an interim analysis of patients with severe Wiskott-Aldrich syndrome who received lentiviral vector-derived gene therapy.

Methods: We did a non-randomised, open-label, phase 1/2 clinical study in paediatric patients with severe Wiskott-Aldrich syndrome, defined by either WAS gene mutation or absent Wiskott-Aldrich syndrome protein (WASP) expression or a Zhu clinical score of 3 or higher. We included patients who had no HLA-identical sibling donor available or, for children younger than 5 years of age, no suitable 10/10 matched unrelated donor or 6/6 unrelated cord blood donor. After treatment with rituximab and a reduced-intensity conditioning regimen of busulfan and fludarabine, patients received one intravenous infusion of autologous CD34+ cells genetically modified with a lentiviral vector encoding for human WAS cDNA. The primary safety endpoints were safety of the conditioning regimen and safety of lentiviral gene transfer into HSPCs. The primary efficacy endpoints were overall survival, sustained engraftment of genetically corrected HSPCs, expression of vector-derived WASP, improved T-cell function, antigen-specific responses to vaccinations, and improved platelet count and mean platelet volume normalisation. This interim analysis was done when the first six patients treated had completed at least 3 years of follow-up. The planned analyses are presented for the intention-to-treat population. This trial is registered with ClinicalTrials.gov (number NCT01515462) and EudraCT (number 2009-017346-32).

Findings: Between April 20, 2010, and Feb 26, 2015, nine patients (all male) were enrolled of whom one was excluded after screening; the age range of the eight treated children was 1·1-12·4 years. At the time of the interim analysis (data cutoff April 29, 2016), median follow-up was 3·6 years (range 0·5-5·6). Overall survival was 100%. Engraftment of genetically corrected HSPCs was successful and sustained in all patients. The fraction of WASP-positive lymphocytes increased from a median of 3·9% (range 1·8-35·6) before gene therapy to 66·7% (55·7-98·6) at 12 months after gene therapy, whereas WASP-positive platelets increased from 19·1% (range 4·1-31·0) to 76·6% (53·1-98·4). Improvement of immune function was shown by normalisation of in-vitro T-cell function and successful discontinuation of immunoglobulin supplementation in seven patients with follow-up longer than 1 year, followed by positive antigen-specific response to vaccination. Severe infections fell from 2·38 (95% CI 1·44-3·72) per patient-year of observation (PYO) in the year before gene therapy to 0·31 (0·04-1·11) per PYO in the second year after gene therapy and 0·17 (0·00-0·93) per PYO in the third year after gene therapy. Before gene therapy, platelet counts were lower than 20 × 10 per L in seven of eight patients. At the last follow-up visit, the platelet count had increased to 20-50 × 10 per L in one patient, 50-100 × 10 per L in five patients, and more than 100 × 10 per L in two patients, which resulted in independence from platelet transfusions and absence of severe bleeding events. 27 serious adverse events in six patients occurred after gene therapy, 23 (85%) of which were infectious (pyrexia [five events in three patients], device-related infections, including one case of sepsis [four events in three patients], and gastroenteritis, including one case due to rotavirus [three events in two patients]); these occurred mainly in the first 6 months of follow-up. No adverse reactions to the investigational drug product and no abnormal clonal proliferation or leukaemia were reported after gene therapy.

Interpretation: Data from this study show that gene therapy provides a valuable treatment option for patients with severe Wiskott-Aldrich syndrome, particularly for those who do not have a suitable HSPC donor available.

Funding: Italian Telethon Foundation, GlaxoSmithKline, and Orchard Therapeutics.
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http://dx.doi.org/10.1016/S2352-3026(19)30021-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6494976PMC
May 2019

Intrabone hematopoietic stem cell gene therapy for adult and pediatric patients affected by transfusion-dependent ß-thalassemia.

Nat Med 2019 02 21;25(2):234-241. Epub 2019 Jan 21.

Rare Disease Center, Fondazione IRCCS Ca' Granda, University of Milan, Milan, Italy.

ß-thalassemia is caused by ß-globin gene mutations resulting in reduced (β) or absent (β) hemoglobin production. Patient life expectancy has recently increased, but the need for chronic transfusions in transfusion-dependent thalassemia (TDT) and iron chelation impairs quality of life. Allogeneic hematopoietic stem cell (HSC) transplantation represents the curative treatment, with thalassemia-free survival exceeding 80%. However, it is available to a minority of patients and is associated with morbidity, rejection and graft-versus-host disease. Gene therapy with autologous HSCs modified to express ß-globin represents a potential therapeutic option. We treated three adults and six children with ß or severe ß mutations in a phase 1/2 trial ( NCT02453477 ) with an intrabone administration of HSCs transduced with the lentiviral vector GLOBE. Rapid hematopoietic recovery with polyclonal multilineage engraftment of vector-marked cells was achieved, with a median of 37.5% (range 12.6-76.4%) in hematopoietic progenitors and a vector copy number per cell (VCN) of 0.58 (range 0.10-1.97) in erythroid precursors at 1 year, in absence of clonal dominance. Transfusion requirement was reduced in the adults. Three out of four evaluable pediatric participants discontinued transfusions after gene therapy and were transfusion independent at the last follow-up. Younger age and persistence of higher VCN in the repopulating hematopoietic cells are associated with better outcome.
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http://dx.doi.org/10.1038/s41591-018-0301-6DOI Listing
February 2019

First Occurrence of Plasmablastic Lymphoma in Adenosine Deaminase-Deficient Severe Combined Immunodeficiency Disease Patient and Review of the Literature.

Front Immunol 2018 2;9:113. Epub 2018 Feb 2.

San Raffaele Telethon Institute for Gene Therapy (SR-TIGET), Pediatric Immunohematology and Bone Marrow Transplantation Unit, Scientific Institute San Raffaele (IRCCS), Milan, Italy.

Adenosine deaminase-deficient severe combined immunodeficiency disease (ADA-SCID) is a primary immune deficiency characterized by mutations in the ADA gene resulting in accumulation of toxic compounds affecting multiple districts. Hematopoietic stem cell transplantation (HSCT) from a matched donor and hematopoietic stem cell gene therapy are the preferred options for definitive treatment. Enzyme replacement therapy (ERT) is used to manage the disease in the short term, while a decreased efficacy is reported in the medium-long term. To date, eight cases of lymphomas have been described in ADA-SCID patients. Here we report the first case of plasmablastic lymphoma occurring in a young adult with ADA-SCID on long-term ERT, which turned out to be Epstein-Barr virus associated. The patient previously received infusions of genetically modified T cells. A cumulative analysis of the eight published cases of lymphoma from 1992 to date, and the case here described, reveals a high mortality (89%). The most common form is diffuse large B-cell lymphoma, which predominantly occurs in extra nodal sites. Seven cases occurred in patients on ERT and two after haploidentical HSCT. The significant incidence of immunodeficiency-associated lymphoproliferative disorders and poor survival of patients developing this complication highlight the priority in finding a prompt curative treatment for ADA-SCID.
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http://dx.doi.org/10.3389/fimmu.2018.00113DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5801298PMC
March 2019

Adjuvant role of SeptiFast to improve the diagnosis of sepsis in a large cohort of hematological patients.

Bone Marrow Transplant 2018 04 12;53(4):410-416. Epub 2018 Jan 12.

Hematology and BMT Unit, IRCCS San Raffaele Scientific Institute, via Olgettina 60, 20132, Milano, Italy.

Febrile neutropenia and sepsis are common and life-threatening complications in hematological diseases. This study was performed retrospectively in 514 patients treated for febrile neutropenia at our institute, to investigate the clinical usefulness of a molecular tool, LightCycler® SeptiFast test (SF), to promptly recognize pathogens causing sepsis in hematological patients. We collected 1837 blood samples of 514 consecutive hematological patients. The time of processing is short. Overall, 757 microorganisms in 663 episodes were detected by molecular test and standard blood cultures (BC): 73.6% Gram-positive bacteria, 23.9% Gram-negative bacteria, and 2.5% fungal species. This large analysis demonstrated a significant episode-to episode agreement (71.9%) between the two methods, higher in negative samples (89.14%), and a specificity of 75.89%. Clinical variables that gave a statistically significant contribution to their concordance were absolute neutrophil count, ongoing antimicrobial therapy, timing of test execution, and organ localization of infection. The large analysis highlights the potential of molecular-based assays directly performed on blood samples, especially if implementing the detection of antibiotic resistance genes, which was lacking in the used study.
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http://dx.doi.org/10.1038/s41409-017-0039-7DOI Listing
April 2018

Enteric Microbiome Markers as Early Predictors of Clinical Outcome in Allogeneic Hematopoietic Stem Cell Transplant: Results of a Prospective Study in Adult Patients.

Open Forum Infect Dis 2017 6;4(4):ofx215. Epub 2017 Oct 6.

Laboratory of Microbiology and Virology, San Raffaele Scientific Institute, Milan, Italy.

Background: Infections and graft-vs-host disease (GvHD) still represent major, not easily predictable complications in allogeneic hematopoietic stem cell transplant (allo-HSCT). Both conditions have been correlated to altered enteric microbiome profiles during the peritransplant period. The main objective of this study was to identify possible early microbiome-based markers useful in pretransplant risk stratification.

Methods: Stool samples were collected from 96 consecutive patients at the beginning of the pretransplant conditioning regimen (T) and at 10 (T) and 30 (T) days following transplant. When significant in univariate analysis, the identified microbiome markers were used in multivariate regression analyses, together with other significant clinical variables for allo-HSCT-related risk stratification. Four main outcomes were addressed: (1) septic complications, (2) GvHD, (3) relapse of the underlying disease, and (4) mortality.

Results: The presence of >5% proinflammatory Enterobacteriaceae at T was the only significant marker for the risk of microbiologically confirmed sepsis. Moreover, ≤10% Lachnospiraceae at T was the only significant factor for increased risk of overall mortality, including death from both infectious and noninfectious causes.Finally, a low bacterial alpha-diversity (Shannon index ≤ 1.3) at T was the only variable significantly correlating with an increased risk of GvHD within 30 days.

Conclusions: Microbiome markers can be useful in the very early identification of patients at risk for major transplant-related complications, offering new tools for individualized preemptive or therapeutic strategies to improve allo-HSCT outcomes.
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http://dx.doi.org/10.1093/ofid/ofx215DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5714175PMC
October 2017

NK cell recovery after haploidentical HSCT with posttransplant cyclophosphamide: dynamics and clinical implications.

Blood 2018 01 6;131(2):247-262. Epub 2017 Oct 6.

Unit of Immunogenetics, Leukemia Genomics and Immunobiology.

The use of posttransplant cyclophosphamide (PT-Cy) as graft-versus-host disease (GVHD) prophylaxis has revolutionized haploidentical hematopoietic stem cell transplantation (HSCT), allowing safe infusion of unmanipulated T cell-replete grafts. PT-Cy selectively eliminates proliferating alloreactive T cells, but whether and how it affects natural killer (NK) cells and their alloreactivity is largely unknown. Here we characterized NK cell dynamics in 17 patients who received unmanipulated haploidentical grafts, containing high numbers of mature NK cells, according to PT-Cy-based protocols in 2 independent centers. In both series, we documented robust proliferation of donor-derived NK cells immediately after HSCT. After infusion of Cy, a marked reduction of proliferating NK cells was evident, suggesting selective purging of dividing cells. Supporting this hypothesis, proliferating NK cells did not express aldehyde dehydrogenase and were killed by Cy in vitro. After ablation of mature NK cells, starting from day 15 after HSCT and favored by the high levels of interleukin-15 present in patients' sera, immature NK cells (CD62LNKG2AKIR) became highly prevalent, possibly directly stemming from infused hematopoietic stem cells. Importantly, also putatively alloreactive single KIR NK cells were eliminated by PT-Cy and were thus decreased in numbers and antileukemic potential at day 30 after HSCT. As a consequence, in an extended series of 99 haplo-HSCT with PT-Cy, we found no significant difference in progression-free survival between patients with or without predicted NK alloreactivity (42% vs 52% at 1 year, = NS). Our data suggest that the majority of mature NK cells infused with unmanipulated grafts are lost upon PT-Cy administration, blunting NK cell alloreactivity in this transplantation setting.
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http://dx.doi.org/10.1182/blood-2017-05-780668DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5757695PMC
January 2018

Human Herpesvirus 6 Infection Following Haploidentical Transplantation: Immune Recovery and Outcome.

Biol Blood Marrow Transplant 2016 12 30;22(12):2250-2255. Epub 2016 Sep 30.

Unit of Hematology and Bone Marrow Transplantation, IRCCS San Raffaele Scientific Institute, Milan, Italy; Vita-Salute San Raffaele University, Milan, Italy. Electronic address:

Human herpesvirus 6 (HHV-6) is increasingly recognized as a potentially life-threatening pathogen in allogeneic hematopoietic stem cell transplantation (alloSCT). We retrospectively evaluated 54 adult patients who developed positivity to HHV-6 after alloSCT. The median time from alloSCT to HHV-6 reactivation was 34 days. HHV-6 was present in plasma samples from 31 patients, in bone marrow (BM) of 9 patients, in bronchoalveolar lavage fluid and liver or gut biopsy specimens from 33 patients, and in cerebrospinal fluid of 7 patients. Twenty-nine patients developed acute graft-versus-host disease (GVHD), mainly grade III-IV, and 15 had concomitant cytomegalovirus reactivation. The median absolute CD3 lymphocyte count was 207 cells/µL. We reported the following clinical manifestations: fever in 43 patients, skin rash in 22, hepatitis in 19, diarrhea in 24, encephalitis in 10, BM suppression in 18, and delayed engraftment in 11. Antiviral pharmacologic treatment was administered to 37 patients; nonetheless, the mortality rate was relatively high in this population (overall survival [OS] at 1 year, 38% ± 7%). A better OS was significantly associated with a CD3 cell count ≥200/µL at the time of HHV-6 reactivation (P = .0002). OS was also positively affected by the absence of acute GVHD grade III-IV (P = .03) and by complete disease remission (P = .03), but was not significantly influenced by steroid administration, time after alloSCT, type of antiviral prophylaxis, plasma viral load, or organ involvement. Although HHV-6 detection typically occurred early after alloSCT, better T cell immune reconstitution seems to have the potential to improve clinical outcomes. Our findings provide new insight into the interplay between HHV-6 and the transplanted immune system.
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http://dx.doi.org/10.1016/j.bbmt.2016.09.018DOI Listing
December 2016

Lentiviral haemopoietic stem-cell gene therapy in early-onset metachromatic leukodystrophy: an ad-hoc analysis of a non-randomised, open-label, phase 1/2 trial.

Lancet 2016 Jul 8;388(10043):476-87. Epub 2016 Jun 8.

San Raffaele Telethon Institute for Gene Therapy (SR-TIGET), Division of Regenerative Medicine, Stem Cells and Gene Therapy, IRCCS San Raffaele Scientific Institute, Milan, Italy; Pediatric Immunohematology Unit, IRCCS San Raffaele Scientific Institute, Milan, Italy; Bone Marrow Transplantation Program, IRCCS San Raffaele Scientific Institute, Milan, Italy; Vita Salute San Raffaele University, Milan, Italy. Electronic address:

Background: Metachromatic leukodystrophy (a deficiency of arylsulfatase A [ARSA]) is a fatal demyelinating lysosomal disease with no approved treatment. We aimed to assess the long-term outcomes in a cohort of patients with early-onset metachromatic leukodystrophy who underwent haemopoietic stem-cell gene therapy (HSC-GT).

Methods: This is an ad-hoc analysis of data from an ongoing, non-randomised, open-label, single-arm phase 1/2 trial, in which we enrolled patients with a molecular and biochemical diagnosis of metachromatic leukodystrophy (presymptomatic late-infantile or early-juvenile disease or early-symptomatic early-juvenile disease) at the Paediatric Clinical Research Unit, Ospedale San Raffaele, in Milan. Trial participants received HSC-GT, which consisted of the infusion of autologous HSCs transduced with a lentiviral vector encoding ARSA cDNA, after exposure-targeted busulfan conditioning. The primary endpoints of the trial are safety (toxicity, absence of engraftment failure or delayed haematological reconstitution, and safety of lentiviral vector-tranduced cell infusion) and efficacy (improvement in Gross Motor Function Measure [GMFM] score relative to untreated historical controls, and ARSA activity, 24 months post-treatment) of HSC-GT. For this ad-hoc analysis, we assessed safety and efficacy outcomes in all patients who had received treatment and been followed up for at least 18 months post-treatment on June 1, 2015. This trial is registered with ClinicalTrials.gov, number NCT01560182.

Findings: Between April, 2010, and February, 2013, we had enrolled nine children with a diagnosis of early-onset disease (six had late-infantile disease, two had early-juvenile disease, and one had early-onset disease that could not be definitively classified). At the time of analysis all children had survived, with a median follow-up of 36 months (range 18-54). The most commonly reported adverse events were cytopenia (reported in all patients) and mucositis of different grades of severity (in five of nine patients [grade 3 in four of five patients]). No serious adverse events related to the medicinal product were reported. Stable, sustained engraftment of gene-corrected HSCs was observed (a median of 60·4% [range 14·0-95·6] lentiviral vector-positive colony-forming cells across follow-up) and the engraftment level was stable during follow-up; engraftment determinants included the duration of absolute neutropenia and the vector copy number of the medicinal product. A progressive reconstitution of ARSA activity in circulating haemopoietic cells and in the cerebrospinal fluid was documented in all patients in association with a reduction of the storage material in peripheral nerve samples in six of seven patients. Eight patients, seven of whom received treatment when presymptomatic, had prevention of disease onset or halted disease progression as per clinical and instrumental assessment, compared with historical untreated control patients with early-onset disease. GMFM scores for six patients up to the last follow-up showed that gross motor performance was similar to that of normally developing children. The extent of benefit appeared to be influenced by the interval between HSC-GT and the expected time of disease onset. Treatment resulted in protection from CNS demyelination in eight patients and, in at least three patients, amelioration of peripheral nervous system abnormalities, with signs of remyelination at both sites.

Interpretation: Our ad-hoc findings provide preliminary evidence of safety and therapeutic benefit of HSC-GT in patients with early-onset metachromatic leukodystrophy who received treatment in the presymptomatic or very early-symptomatic stage. The results of this trial will be reported when all 20 patients have achieved 3 years of follow-up.

Funding: Italian Telethon Foundation and GlaxoSmithKline.
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http://dx.doi.org/10.1016/S0140-6736(16)30374-9DOI Listing
July 2016

Pioglitazone as a novel therapeutic approach in chronic granulomatous disease.

J Allergy Clin Immunol 2016 06 4;137(6):1913-1915.e2. Epub 2016 Apr 4.

San Raffaele Telethon Institute for Gene Therapy (TIGET), Pediatric Immunohematology and Bone Marrow Transplantation Unit, San Raffaele Scientific Institute, Milan, Italy.

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http://dx.doi.org/10.1016/j.jaci.2016.01.033DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4889778PMC
June 2016

High Doses of Antimetabolites Followed by High-Dose Sequential Chemoimmunotherapy and Autologous Stem-Cell Transplantation in Patients With Systemic B-Cell Lymphoma and Secondary CNS Involvement: Final Results of a Multicenter Phase II Trial.

J Clin Oncol 2015 Nov 17;33(33):3903-10. Epub 2015 Aug 17.

Andrés J.M. Ferreri, Giovanni Donadoni, Marta Bruno-Ventre, Andrea Assanelli, Marco Foppoli, Giovanni Citterio, Federico Caligaris-Cappio, and Fabio Ciceri, Istituto di Ricovero e Cura a Carattere Scientifico, San Raffaele Scientific Institute; Massimo Di Nicola, Istituto Nazionale dei Tumori, Milano; Maria Giuseppina Cabras and Alessandro Fanni, Hospital Businco, Cagliari; Caterina Patti and Antonino Mulè, Hospital "V. Cervello," Palermo; Michael Mian, Ospedale di Bolzano, Bolzano; Renato Zambello, Azienda Ospedaliera di Padua, University of Padua, Padua; Corrado Tarella, Azienda Ospedaliera Ordine Mauriziano-Umberto I, University of Turín, Turín; Alfonso M. D'Arco, Polo Oncologico Nocera-Pagani, Nocera Inferiore; and Gianluca Doa, Azienda Ospedaliera di Nuoro, Nuoro, Italy.

Purpose: Treatment of secondary CNS dissemination in patients with aggressive lymphomas remains an important, unmet clinical need. Herein, we report the final results of a multicenter phase II trial addressing a new treatment for secondary CNS lymphoma based on encouraging experiences with high doses of antimetabolites in primary CNS lymphoma and with rituximab plus high-dose sequential chemoimmunotherapy (R-HDS) in relapsed aggressive lymphoma.

Patients And Methods: HIV-negative patients with aggressive B-cell lymphoma and secondary CNS involvement at diagnosis or relapse, age 18 to 70 years, and Eastern Cooperative Oncology Group performance status ≤ 3 were enrolled and treated with high-doses of methotrexate and cytarabine, followed by R-HDS (cyclophosphamide, cytarabine, and etoposide) supported by autologous stem-cell transplantation (ASCT). Treatment included eight doses of rituximab and four doses of intrathecal liposomal cytarabine. The primary end point was 2-year event-free survival; the planned accrual was 38 patients.

Results: Thirty-eight patients were enrolled; CNS disease was detected at presentation in 16 patients. Toxicity was usually hematologic and manageable, with grade 4 febrile neutropenia in 3% of delivered courses and grade 4 nonhematologic toxicity in 2% of delivered courses. Four patients died because of toxicity. Autologous stem cells were successfully collected in 24 (89%) of 27 patients (median, 10 × 10(6)/kg); 20 patients underwent ASCT. Complete response was achieved in 24 patients (complete response rate, 63%; 95% CI, 48% to 78%). At a median follow-up of 48 months, 17 patients remained relapse free, with a 2-year event-free survival rate of 50% ± 8%. At 5 years, 16 patients were alive, with a 5-year overall survival rate of 41% ± 8% for the whole series and 68% ± 11% for patients who received transplantation. Systemic (extra-CNS) and/or meningeal disease did not affect outcome.

Conclusion: The combination of high doses of antimetabolites, R-HDS, and ASCT is feasible and effective in patients age 18 to 70 years old with secondary CNS lymphoma, and we propose it as a new standard therapeutic option.
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http://dx.doi.org/10.1200/JCO.2015.61.1236DOI Listing
November 2015

High rate of hematological responses to sorafenib in FLT3-ITD acute myeloid leukemia relapsed after allogeneic hematopoietic stem cell transplantation.

Eur J Haematol 2016 Jun 21;96(6):629-36. Epub 2015 Sep 21.

Unit of Hematology and Bone Marrow Transplantation, IRCCS San Raffaele Scientific Institute, Milan, Italy.

Relapse represents the most significant cause of failure of allogeneic hematopoietic stem cell transplantation (HSCT) for FLT3-ITD-positive acute myeloid leukemia (AML), and available therapies are largely unsatisfactory. In this study, we retrospectively collected data on the off-label use of the tyrosine kinase inhibitor sorafenib, either alone or in association with hypomethylating agents and adoptive immunotherapy, in 13 patients with post-transplantation FLT3-ITD-positive AML relapses. Hematological response was documented in 12 of 13 patients (92%), and five of 13 (38%) achieved complete bone marrow remission. Treatment was overall manageable in the outpatient setting, although all patients experienced significant adverse events, especially severe cytopenias (requiring a donor stem cell boost in five patients) and typical hand-foot syndrome. None of the patients developed graft-vs.-host disease following sorafenib alone, whereas this was frequently observed when this was given in association with donor T-cell infusions. Six patients are alive and in remission at the last follow-up, and four could be bridged to a second allogeneic HSCT, configuring a 65 ± 14% overall survival at 100 d from relapse. Taken together, our data suggest that sorafenib might represent a valid treatment option for patients with FLT3-ITD-positive post-transplantation relapses, manageable also in combination with other therapeutic strategies.
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http://dx.doi.org/10.1111/ejh.12647DOI Listing
June 2016

Post-transplantation Cyclophosphamide and Sirolimus after Haploidentical Hematopoietic Stem Cell Transplantation Using a Treosulfan-based Myeloablative Conditioning and Peripheral Blood Stem Cells.

Biol Blood Marrow Transplant 2015 Aug 19;21(8):1506-14. Epub 2015 May 19.

Division of Regenerative Medicine, Stem Cells and Gene Therapy, Hematology and Bone Marrow Transplantation Unit, San Raffaele Scientific Institute, Milan, Italy.

Haploidentical hematopoietic stem cell transplantation (HSCT) performed using bone marrow (BM) grafts and post-transplantation cyclophosphamide (PTCy) has gained much interest for the excellent toxicity profile after both reduced-intensity and myeloablative conditioning. We investigated, in a cohort of 40 high-risk hematological patients, the feasibility of peripheral blood stem cells grafts after a treosulfan-melphalan myeloablative conditioning, followed by a PTCy and sirolimus-based graft-versus-host disease (GVHD) prophylaxis (Sir-PTCy). Donor engraftment occurred in all patients, with full donor chimerism achieved by day 30. Post-HSCT recovery of lymphocyte subsets was broad and fast, with a median time to CD4 > 200/μL of 41 days. Cumulative incidences of grade II to IV and III-IV acute GVHD were 15% and 7.5%, respectively, and were associated with a significant early increase in circulating regulatory T cells at day 15 after HSCT, with values < 5% being predictive of subsequent GVHD occurrence. The 1-year cumulative incidence of chronic GVHD was 20%. Nonrelapse mortality (NRM) at 100 days and 1 year were 12% and 17%, respectively. With a median follow-up for living patients of 15 months, the estimated 1-year overall and disease-free survival (DFS) was 56% and 48%, respectively. Outcomes were more favorable in patients who underwent transplantation in complete remission (1-year DFS 71%) versus patients who underwent transplantation with active disease (DFS, 34%; P = .01). Overall, myeloablative haploidentical HSCT with peripheral blood stem cells (PBSC) and Sir-PTCy is a feasible treatment option: the low rates of GVHD and NRM as well as the favorable immune reconstitution profile pave the way for a prospective comparative trial comparing BM and PBSC in this specific transplantation setting.
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http://dx.doi.org/10.1016/j.bbmt.2015.04.025DOI Listing
August 2015

Plerixafor on demand in ten healthy family donors as a rescue strategy to achieve an adequate graft for stem cell transplantation.

Transfusion 2015 Aug 26;55(8):1993-2000. Epub 2015 Feb 26.

Blood Transfusion Service, IRCCS San Raffaele Scientific Institute, Milan, Italy.

Background: In allogeneic hematopoietic stem cell (HSC) transplantation, the collection of an appropriate number of HSCs while maintaining a high level of safety for healthy donors is fundamental. Inadequate HSC mobilization can be seen with the standard use of granulocyte-colony-stimulating (G-CSF). Plerixafor (PL) is a chemokine receptor CXC Type 4-stromal-derived factor 1 inhibitor; its HSC-mobilizing properties are synergistic with G-CSF in poor mobilizing patients. The use of PL as adjuvant or alternative to G-CSF in healthy donors has shown a good safety profile but is so far off-label.

Study Design And Methods: We report 10 healthy HSC donors treated with PL because of insufficient response to G-CSF alone or contraindication to G-CSF. Eight donors did not mobilize enough CD34+ cells with G-CSF alone because poor mobilizers or because insufficient HSCs were harvested according to the clinical need of the patient; in two cases G-CSF administration and marrow harvest were unfeasible or contraindicated in the donor.

Results: The use of PL for mobilization increased the number of circulating CD34+ cells by 2.8-fold and the CD34+/kg collection by 3.0-fold. Only mild adverse events were reported (bone pain or discomfort) and not univocally attributable to PL. Rate of engraftment and graft-versus-host disease were similar to those seen in recipients of grafts from G-CSF only-mobilized donors.

Conclusion: We exposed 10 allogeneic donors to mobilization with PL. PL was well tolerated in all cases and ensured procurement of an adequate graft for transplantation resulting in a normal hematopoietic engraftment.
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http://dx.doi.org/10.1111/trf.13059DOI Listing
August 2015

Treosulfan based reduced toxicity conditioning followed by allogeneic stem cell transplantation in patients with myelofibrosis.

Hematol Oncol 2016 Sep 3;34(3):154-60. Epub 2014 Dec 3.

Hematology and Bone Marrow Transplantation Unit, IRCCS San Raffaele Scientific Institute, Milano, Italy.

Allogeneic transplantation is the only potentially curative strategy for myelofibrosis, even in the era of new drugs that so far only mitigate symptoms. The choice to proceed to allogeneic transplantation is based on several variables including age, disease phase, degree of splenomegaly, donor availability, comorbidities and iron overload. These factors, along with conditioning regimen and time to transplantation, may influence the outcome of ASCT. We report 14 patients affected by myelofibrosis with a median age of 57 years (range, 41-76) receiving a treosulfan-fludarabine based reduced toxicity conditioning. Patients (pts) received a stem cell transplantation from an HLA identical (n = 10) or matched unrelated donor (n = 4). All pts had a complete myeloablation followed by engraftment and in 12 out of 13 evaluated pts donor chimerism was 100% at 1 month. In most cases a reduction of splenomegaly and a reduction (or resolution) of bone marrow fibrosis was observed. After a median follow-up of 39 months (range, 3-106), the 3-year probability of overall survival and disease free survival was 54 +/- 14% and 46 +/- 14%, respectively. The cumulative incidence of non-relapse mortality at 2 years was 39 +/- 15%. Causes of non-relapse mortality were: infection (n = 2), GvHD (n = 2) and haemorrhage (n = 1). We can conclude that a treosulfan and fludarabine based conditioning has a potent myeloablative and anti-disease activity although non-relapse mortality remains high in this challenging clinical setting. Copyright © 2014 John Wiley & Sons, Ltd.
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http://dx.doi.org/10.1002/hon.2183DOI Listing
September 2016