Publications by authors named "Alice Bertaina"

106 Publications

Impact of treosulfan exposure on early and long-term clinical outcome in pediatric allogeneic HSCT recipients: a prospective multicenter study.

Transplant Cell Ther 2021 Oct 1. Epub 2021 Oct 1.

Department of Pediatrics, Leiden University Medical Center, Leiden, The Netherlands. Electronic address:

Background: Treosulfan-based conditioning has gained popularity in pediatric allogeneic hematopoietic stem cell transplantation (HSCT) because of its presumed favorable efficacy and toxicity profile. Treosulfan is used in standardized dosing regimens based on body surface area. The relationship between systemic treosulfan exposure, early and long term clinical outcome in pediatric patients undergoing allogeneic hematopoietic stem cell transplantation (HSCT) for non-malignant diseases is as yet unresolved.

Objective: In this study we assessed the association between treosulfan exposure and early, and in particular, long term clinical outcomes.

Study Design: We conducted a multicenter, prospective observational study and included 110 pediatric patients with non-malignant diseases transplanted between 2011 and 2019 in Leiden, The Netherlands and Rome, Italy. Blood samples were collected and treosulfan area under the curve (AUC) was estimated as a measure of exposure. Cox proportional hazard survival analyses were performed to assess the relationship between treosulfan exposure, OS and EFS. The predictive value of systemic treosulfan exposure for the occurrence of toxicity within 28 days is evaluated using a multivariable logistic regression analysis.

Results: In the overall cohort, overall survival (OS) and event-free survival (EFS) at 2 years were 89.0% and 75.3%, respectively, with an excellent OS of 97% in children under the age of 2 years. The occurrence of grade II-IV aGvHD, the level of 1-year whole blood chimerism, and 2-year OS and EFS were not correlated with treosulfan exposure. The occurrence of skin toxicity (odds ratio (OR) 3.97, 95% confidence interval (CI) 1.26-13.68, p=0.02) and all grade mucositis (OR 4.43, 95%CI 1.43-15.50, p=0.02), but not ≥ grade 2 mucositis (OR 1.51, 95%CI 0.52-4.58, p=0.46) was related to high treosulfan exposure (>1750 mg*h/L).

Conclusion: Our study demonstrates that standardized treosulfan-based conditioning results in a favorable OS and EFS in infants and children with non-malignant diseases, independent of interindividual variation in treosulfan exposure. These outcomes can be achieved without the need for therapeutic drug monitoring (TDM), thereby emphasizing the advantage of treosulfan use in this category of patients. Although higher treosulfan exposure increases the risk of skin toxicity, there is no absolute necessity for therapeutic drug monitoring if proper preventive skin measures are taken. More research is needed to assess whether de-escalation of treosulfan doses is possible in order to minimize early and long term toxicity without compromising efficacy.
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http://dx.doi.org/10.1016/j.jtct.2021.09.018DOI Listing
October 2021

TCRαβ/CD19 depleted HSCT from an HLA-haploidentical relative to treat children with different non-malignant disorders.

Blood Adv 2021 Sep 30. Epub 2021 Sep 30.

Sapienza, University of Rome, Italy.

Several non-malignant disorders (NMDs), either inherited or acquired, can be cured by allogeneic hematopoietic stem cell transplantation (HSCT). Between January 2012 and April 2020, 70 consecutive children affected by primary immunodeficiencies, inherited/acquired bone marrow failure syndromes, red blood cell disorders or metabolic diseases, lacking a fully-matched donor or requiring urgent transplantation, underwent TCRαβ/CD19-depleted haploidentical HSCT from an HLA-partially matched relative as part of a prospective study (#NCT01810120). Median age at transplant was 3.5 years (range 0.3-16.1); median time from diagnosis to transplant was 10.5 months (2.7 for SCID patients). Primary engraftment was obtained in 51 patients, while 19 and 2 patients experienced either primary or secondary graft failure (GF), the overall incidence of this complication being 30.4%. Most GFs were observed in children with disease at risk for this complication (e.g., aplastic anemia, thalassemia). All but 5 patients experiencing GF were successfully retransplanted. Six patients died of infectious complications (4 had active/recent infections at time of HSCT), the cumulative incidence of transplant-related mortality (TRM) being 8.5%. Cumulative incidence of grade I-II acute GvHD was 14.4% (no patient developed grade III-IV acute GVHD). Only one patient at risk developed mild chronic GvHD. With a median follow-up of 3.5 years, the 5-year probability of overall and disease-free survival was 91.4% and 86.8%, respectively. In conclusion, TCRαβ/CD19-depleted haploidentical HSCT from an HLA-partially matched relative is confirmed to be an effective treatment for children with NMDs. Prompt donor availability, low incidence of GvHD and TRM make this strategy an attractive option in NMDs patients.
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http://dx.doi.org/10.1182/bloodadvances.2021005628DOI Listing
September 2021

Outcomes of pediatric patients with therapy-related myeloid neoplasms.

Bone Marrow Transplant 2021 Sep 3. Epub 2021 Sep 3.

Department of Pediatrics, University of Colorado School of Medicine, Aurora, CO, USA.

Long-term outcomes after allogeneic hematopoietic cell transplantation (HCT) for therapy-related myeloid neoplasms (tMNs) are dismal. There are few multicenter studies defining prognostic factors in pediatric patients with tMNs. We have accumulated the largest cohort of pediatric patients who have undergone HCT for a tMN to perform a multivariate analysis defining factors predictive of long-term survival. Sixty-eight percent of the 401 patients underwent HCT using a myeloablative conditioning (MAC) regimen, but there were no statistically significant differences in the overall survival (OS), event-free survival (EFS), or cumulative incidence of relapse and non-relapse mortality based on the conditioning intensity. Among the recipients of MAC regimens, 38.4% of deaths were from treatment-related causes, especially acute graft versus host disease (GVHD) and end-organ failure, as compared to only 20.9% of deaths in the reduced-intensity conditioning (RIC) cohort. Exposure to total body irradiation (TBI) during conditioning and experiencing grade III/IV acute GVHD was associated with worse OS. In addition, a diagnosis of therapy-related myelodysplastic syndrome and having a structurally complex karyotype at tMN diagnosis were associated with worse EFS. Reduced-toxicity (but not reduced-intensity) regimens might help to decrease relapse while limiting mortality associated with TBI-based HCT conditioning in pediatric patients with tMNs.
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http://dx.doi.org/10.1038/s41409-021-01448-xDOI Listing
September 2021

Supportive Care During Pediatric Hematopoietic Stem Cell Transplantation: Prevention of Infections. A Report From Workshops on Supportive Care of the Paediatric Diseases Working Party (PDWP) of the European Society for Blood and Marrow Transplantation (EBMT).

Front Pediatr 2021 29;9:705179. Epub 2021 Jul 29.

Department of Pediatrics, University Hospital Center Zagreb, Zagreb, Croatia.

Specific protocols define eligibility, conditioning, donor selection, graft composition and prophylaxis of graft vs. host disease for children and young adults undergoing hematopoietic stem cell transplant (HSCT). However, international protocols rarely, if ever, detail supportive care, including pharmaceutical infection prophylaxis, physical protection with face masks and cohort isolation or food restrictions. Supportive care suffers from a lack of scientific evidence and implementation of practices in the transplant centers brings extensive restrictions to the child's and family's daily life after HSCT. Therefore, the Board of the Pediatric Diseases Working Party (PDWP) of the European Society for Blood and Marrow Transplantation (EBMT) held a series of dedicated workshops since 2017 with the aim of initiating the production of a set of minimal recommendations. The present paper describes the consensus reached within the field of infection prophylaxis.
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http://dx.doi.org/10.3389/fped.2021.705179DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8358428PMC
July 2021

Standardizing Definitions of Hematopoietic Recovery, Graft Rejection, Graft Failure, Poor Graft Function, and Donor Chimerism in Allogeneic Hematopoietic Cell Transplantation: A Report on Behalf of the American Society for Transplantation and Cellular Therapy.

Transplant Cell Ther 2021 08;27(8):642-649

West Virginia University, Morgantown, West Virginia.

Allogeneic hematopoietic cell transplantation (allo-HCT) is potentially curative for certain hematologic malignancies and nonmalignant diseases. The field of allo-HCT has witnessed significant advances, including broadening indications for transplantation, availability of alternative donor sources, less toxic preparative regimens, new cell manipulation techniques, and novel GVHD prevention methods, all of which have expanded the applicability of the procedure. These advances have led to clinical practice conundrums when applying traditional definitions of hematopoietic recovery, graft rejection, graft failure, poor graft function, and donor chimerism, because these may vary based on donor type, cell source, cell dose, primary disease, graft-versus-host disease (GVHD) prophylaxis, and conditioning intensity, among other variables. To address these contemporary challenges, we surveyed a panel of allo-HCT experts in an attempt to standardize these definitions. We analyzed survey responses from adult and pediatric transplantation physicians separately. Consensus was achieved for definitions of neutrophil and platelet recovery, graft rejection, graft failure, poor graft function, and donor chimerism, but not for delayed engraftment. Here we highlight the complexities associated with the management of mixed donor chimerism in malignant and nonmalignant hematologic diseases, which remains an area for future research. We recognize that there are multiple other specific, and at times complex, clinical scenarios for which clinical management must be individualized.
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http://dx.doi.org/10.1016/j.jtct.2021.04.007DOI Listing
August 2021

Harnessing Mechanisms of Immune Tolerance to Improve Outcomes in Solid Organ Transplantation: A Review.

Front Immunol 2021 10;12:688460. Epub 2021 Jun 10.

Division of Hematology, Oncology and Stem Cell Transplantation and Regenerative Medicine, Department of Pediatrics, Stanford University School of Medicine, Stanford, CA, United States.

Survival after solid organ transplantation (SOT) is limited by chronic rejection as well as the need for lifelong immunosuppression and its associated toxicities. Several preclinical and clinical studies have tested methods designed to induce transplantation tolerance without lifelong immune suppression. The limited success of these strategies has led to the development of clinical protocols that combine SOT with other approaches, such as allogeneic hematopoietic stem cell transplantation (HSCT). HSCT prior to SOT facilitates engraftment of donor cells that can drive immune tolerance. Recent innovations in graft manipulation strategies and post-HSCT immune therapy provide further advances in promoting tolerance and improving clinical outcomes. In this review, we discuss conventional and unconventional immunological mechanisms underlying the development of immune tolerance in SOT recipients and how they can inform clinical advances. Specifically, we review the most recent mechanistic studies elucidating which immune regulatory cells dampen cytotoxic immune reactivity while fostering a tolerogenic environment. We further discuss how this understanding of regulatory cells can shape graft engineering and other therapeutic strategies to improve long-term outcomes for patients receiving HSCT and SOT.
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http://dx.doi.org/10.3389/fimmu.2021.688460DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8222735PMC
June 2021

Toxoplasmosis in Pediatric Hematopoietic Stem Cell Transplantation Patients.

Transplant Cell Ther 2021 04 10;27(4):292-300. Epub 2020 Dec 10.

Lucile Packard Children's Hospital Stanford, Palo Alto, California; Division of Infectious Diseases, Department of Pediatrics, Stanford University School of Medicine, Stanford, California; Dr Jack S. Remington Laboratory for Specialty Diagnostics, Palo Alto Medical Foundation, Palo Alto, California.

Infection due to the protozoa Toxoplasma gondii can be life-threatening in hematopoietic stem cell transplantation (HSCT) recipients. Most cases of toxoplasmosis in HSCT recipients result from reactivation of latent infection in individuals who were Toxoplasma-seropositive before transplantation and did not receive appropriate prophylaxis. Pretransplantation screening with Toxoplasma IgG and IgM antibodies is suggested for all allogeneic HSCT recipients and their donors and all autologous HSCT recipients. Prevention of toxoplasmosis in T. gondii-seropositive HSCT recipients requires primary prophylaxis, preemptive screening, or both. Trimethoprim-sulfamethoxazole (TMP-SMX) is the preferred agent for Toxoplasma prophylaxis and should be continued for 6 months or until the patient is no longer receiving immunosuppression, whichever is longer, assuming that immune reconstitution has occurred. Preemptive weekly screening with whole blood Toxoplasma PCR should be considered for seropositive HSCT recipients if prophylaxis cannot be given or if prophylaxis other than TMP-SMX is used. The signs, symptoms, and radiographic findings of toxoplasmosis in HSCT recipients can be nonspecific, and the diagnosis requires a high degree of suspicion. Common presentations include fever, encephalopathy with mental status changes or seizures, and pneumonia. A Toxoplasma PCR analysis from whole blood (and other body fluids/tissues according to clinical symptoms) should be obtained in patients in whom there is a concern for toxoplasmosis. Treatment with oral pyrimethamine, sulfadiazine, and leucovorin for at least 6 weeks is the first-line therapy and should be followed by secondary prophylaxis. In this article, we review the published literature regarding the epidemiology, clinical presentation, treatment, and prevention of toxoplasmosis in HSCT recipients.
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http://dx.doi.org/10.1016/j.jtct.2020.11.003DOI Listing
April 2021

Response to Trimethoprim-Sulfamethoxazole in a Pediatric Hematopoietic Stem Cell Transplant Recipient With Disseminated Toxoplasmosis: A Case Report.

J Pediatric Infect Dis Soc 2021 Aug;10(6):745-748

Lucile Packard Children's Hospital Stanford, Palo Alto, California, USA.

We describe the presentation and treatment of a patient who developed ongoing fever and diagnosed with disseminated toxoplasmosis post-hematopoietic stem cell transplantation. He was initially treated with trimethoprim-sulfamethoxazole (TMP-SMX) and there was dramatic improvement in his fever curve. He successfully completed a modified course of therapy.
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http://dx.doi.org/10.1093/jpids/piab006DOI Listing
August 2021

Toxoplasmosis Among 38,751 Hematopoietic Stem Cell Transplant Recipients: A Systematic Review of Disease Prevalence and a Compilation of Imaging and Autopsy Findings.

Transplantation 2021 Feb 4. Epub 2021 Feb 4.

Division of Infectious Diseases, Department of Pediatrics, Stanford University School of Medicine, Stanford, CA, USA Dr. Jack S. Remington Laboratory for Specialty Diagnostics, Palo Alto Medical Foundation, Palo Alto, CA, USA Stanford University School of Medicine, Stanford Prevention Research Center, Stanford, CA, USA Division of Hematology Oncology and Stem Cell Transplantation and Regenerative Medicine, Department of Pediatrics, Stanford University School of Medicine, Stanford, CA, USA Department of Radiology, Thoracic Imaging, Stanford University School of Medicine, Stanford, CA, USA Department of Radiology, Neuroimaging and Neuro-intervention, Stanford University School of Medicine, Stanford, CA, USA.

Background: Toxoplasmosis in hematopoietic stem cell transplant-recipients (HSCT) can be life threatening if not promptly diagnosed and treated.

Methods: We performed a systematic review (PubMed last search 03/29/2020) of toxoplasmosis among HSCT-recipients and calculated the toxoplasmosis prevalence across studies. We also created a compilation list of brain imaging, chest imaging and autopsy findings of toxoplasmosis among HSCT-recipients.

Results: We identified 46 eligible studies (47 datasets) with 399 toxoplasmosis cases among 38751 HSCT-recipients. There was large heterogeneity in the reported toxoplasmosis prevalence across studies, thus formal meta-analysis was not attempted. The median toxoplasmosis prevalence among 38751 HSCT-recipients was 2.14% (range 0-66.67%). Data on toxoplasmosis among at-risk R+HSCT-recipients were more limited (25 studies; 2404 R+HSCT-recipients [6.2% of all HSCT-recipients]) although the median number of R+HSCT-recipients was 56.79% across all HSCT-recipients. Median toxoplasmosis prevalence across studies among 2404 R+HSCT was 7.51% (range 0-80%) vs 0% (range 0-1.23%) among 7438 R-HSCT. There were limited data to allow meaningful analyses of toxoplasmosis prevalence according to prophylaxis-status of R+HSCT-recipients.

Conclusion: Toxoplasmosis prevalence among HSCT-recipients is underestimated. The majority of studies report toxoplasmosis prevalence among all HSCT-recipients rather than only among the at-risk R+HSCT-recipients. In fact, the median toxoplasmosis prevalence among all R+/R- HSCT-recipients is 3.5-fold lower compared to the prevalence among only the at-risk R+HSCT-recipients and the median prevalence among R+HSCT-recipients is 7.51-fold higher than among R-HSCT-recipients. The imaging findings of toxoplasmosis among HSCT-recipients can be atypical. High-index of suspicion is needed in R+HSCT-recipients with fever, pneumonia or encephalitis.
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http://dx.doi.org/10.1097/TP.0000000000003662DOI Listing
February 2021

Human-engineered Treg-like cells suppress FOXP3-deficient T cells but preserve adaptive immune responses .

Clin Transl Immunology 2020 25;9(11):e1214. Epub 2020 Nov 25.

Department of Pediatrics Division of Hematology, Oncology Stem Cell Transplantation and Regenerative Medicine Stanford University School of Medicine Stanford CA USA.

Objectives: Genetic or acquired defects in FOXP3 regulatory T cells (Tregs) play a key role in many immune-mediated diseases including immune dysregulation polyendocrinopathy, enteropathy, X-linked (IPEX) syndrome. Previously, we demonstrated CD4 T cells from healthy donors and IPEX patients can be converted into functional Treg-like cells by lentiviral transfer of (CD4). These CD4 cells have potent regulatory function, suggesting their potential as an innovative therapeutic. Here, we present molecular and preclinical data supporting CD4 cell clinical progression.

Methods: The molecular characterisation of CD4 cells included flow cytometry, qPCR, RNA-seq and TCR-seq. The suppressive function of CD4 cells was assessed in xenograft-versus-host disease (xeno-GvHD) and FOXP3-deficient IPEX-like humanised mouse models. The safety of CD4 cells was evaluated using peripheral blood (PB) humanised (hu)- mice testing their impact on immune response against pathogens, and immune surveillance against tumor antigens.

Results: We demonstrate that the conversion of CD4 T cells to CD4 cells leads to specific transcriptional changes as compared to CD4 T-cell transduction in the absence of FOXP3, including upregulation of Treg-related genes. Furthermore, we observe specific preservation of a polyclonal TCR repertoire during cell production. Both allogeneic and autologous CD4 cells protect from xeno-GvHD after two sequential infusions of effector T cells. CD4 cells prevent hyper-proliferation of CD4 memory T cells in the FOXP3-deficient IPEX-like hu-mice. CD4 cells do not impede expansion of antigen-primed T cells or tumor clearance in the PB hu-mice.

Conclusion: These data support the clinical readiness of CD4 cells to treat IPEX syndrome and other immune-mediated diseases caused by insufficient or dysfunctional FOXP3 Tregs.
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http://dx.doi.org/10.1002/cti2.1214DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7688376PMC
November 2020

Veno-occlusive Disease in HSCT Patients: Consensus-based Recommendations for Risk Assessment, Diagnosis, and Management by the GITMO Group.

Transplantation 2021 04;105(4):686-694

Clinica Medica, Ospedali Riuniti di Ancona, Ancona, Italy.

Variation in clinical practice affects veno-occlusive disease management, mainly in patients who undergo allogeneic hematopoietic stem cell transplantation. Disputes about diagnostic criteria, treatment, and prophylaxis, due to the lack of high-quality data, are at the base of this variability. With the aim of limiting inconsistency in clinical care, thus improving both patient outcomes and data collection reliability, the Italian Society of Stem cell transplant (Gruppo Italiano Trapianto Midollo Osseo e Terapia Cellulare) launched a collaborative effort to formulate recommendations based on integration of available evidence and expert's consensus. A systematic method, according to US National Institute of Health guidelines and Italian National System for Guidelines, was used. Twenty-nine recommendations were approved with a strong (20) or weak (9) level of agreement, while 26 were rejected. In particular, the panel pointed out the need to achieve an early diagnosis, encouraging the adoption of European Society for Blood and Marrow Transplantation criteria and the prompt use of ultrasonography. Moreover, our experts strongly recommended in favor of prophylactic use of ursodeoxycholic acid. As soon as a veno-occlusive disease diagnosis is established, treatment with defibrotide should be started for at least 21 days. A number of areas of uncertainty, particularly concerning risk stratification and use of diagnostic tools such as elastography has been identified and discussed.
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http://dx.doi.org/10.1097/TP.0000000000003569DOI Listing
April 2021

HLA-haplotype loss after TCRαβ/CD19-depleted haploidentical HSCT.

Bone Marrow Transplant 2021 03 17;56(3):733-737. Epub 2020 Oct 17.

Department of Pediatrics, Division of Stem Cell Transplant and Regenerative Medicine, Stanford University, Stanford, CA, USA.

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http://dx.doi.org/10.1038/s41409-020-01081-0DOI Listing
March 2021

Engineered type 1 regulatory T cells designed for clinical use kill primary pediatric acute myeloid leukemia cells.

Haematologica 2021 10 1;106(10):2588-2597. Epub 2021 Oct 1.

Department of Pediatrics, Division of Stem Cell Biology and Regenerative Medicine, Stanford School of Medicine, Stanford, CA; Stanford Institute for Stem Cell Biology and Regenerative Medicine, Stanford School of Medicine, Stanford.

Type 1 regulatory (Tr1) T cells induced by enforced expression of IL-10 (LV-10) are being developed as a novel treatment for chemotherapy-resistant myeloid leukemias. In vivo, LV-10 cells do not cause graft vs host disease while mediating graft vs leukemia (GvL) effect against adult acute myeloid leukemia (AML). Since pediatric AML (pAML) and adult AML are different on a genetic and epigenetic level, we investigate herein whether LV-10 cells also efficiently kill pAML cells. We show that the majority of primary pAML are killed by LV-10 cells, with different levels of sensitivity to killing. Transcriptionally, pAML sensitive to LV-10 killing expressed a myeloid maturation signature. Overlaying the signatures of sensitive and resistant pAML onto the public NCI TARGET pAML dataset revealed that sensitive pAML clustered with M5 monocytic pAML and pAML with MLL rearrangement. Resistant pAML clustered with myelomonocytic leukemias and those bearing the core binding factor translocations inv(16) or t(8;21)(RUNX1-RUNX1T1). Furthermore, resistant pAML upregulated the membrane glycoprotein CD200, which binds to the inhibitory receptor CD200R1 on LV-10 cells. To examine if CD200 expression on target cells can impair LV-10 cell function, we overexpressed CD200 in myeloid leukemia cell lines ordinarily sensitive to LV-10 killing. Indeed, LV-10 cells degranulated less and killed fewer CD200-overexpressing cells compared to controls, indicating that pAML can utilize CD200 expression for immune evasion. Altogether, the majority of pAML are killed by LV-10 cells in vitro, supporting further LV-10 cell development as an innovative cell therapy for pAML.
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http://dx.doi.org/10.3324/haematol.2020.263129DOI Listing
October 2021

Making a case for using γδ T cells against SARS-CoV-2.

Crit Rev Microbiol 2020 Nov 7;46(6):689-702. Epub 2020 Oct 7.

Stem Cell Transplantation and Regenerative Medicine, Department of Pediatrics, Stanford University School of Medicine, Palo Alto, CA, USA.

Intensive worldwide efforts are underway to determine both the pathogenesis of SARS-CoV-2 infection and the immune responses in COVID-19 patients in order to develop effective therapeutics and vaccines. One type of cell that may contribute to these immune responses is the γδ T lymphocyte, which plays a key role in immunosurveillance of the mucosal and epithelial barriers by rapidly responding to pathogens. Although found in low numbers in blood, γδ T cells consist the majority of tissue-resident T cells and participate in the front line of the host immune defense. Previous studies have demonstrated the critical protective role of γδ T cells in immune responses to other respiratory viruses, including SARS-CoV-1. However, no studies have profoundly investigated these cells in COVID-19 patients to date. γδ T cells can be safely expanded using existing inexpensive FDA-approved drugs such as bisphosphonate, in order to test its protective immune response to SARS-CoV-2. To support this line of research, we review insights gained from previous coronavirus research, along with recent findings, discussing the potential role of γδ T cells in controlling SARS-CoV-2. We conclude by proposing several strategies to enhance γδ T cell's antiviral function, which may be used in developing therapies for COVID-19.
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http://dx.doi.org/10.1080/1040841X.2020.1822279DOI Listing
November 2020

Identification of dual positive CD19+/CD3+ T cells in a leukapheresis product undergoing CAR transduction: a case report.

J Immunother Cancer 2020 09;8(2)

Stanford University, Stanford Cancer Institute, Stanford California, USA.

Background: Chimeric antigen receptor (CAR) therapy and hematopoietic stem cell transplantation (HSCT) are therapeutics for relapsed acute lymphocytic leukemia (ALL) that are increasingly being used in tandem. We identified a non-physiologic CD19+/CD3+ T-cell population in the leukapheresis product of a patient undergoing CAR T-cell manufacturing who previously received a haploidentical HSCT, followed by infusion of a genetically engineered T-cell addback product. We confirm and report the origin of these CD19+/CD3+ T cells that have not previously been described in context of CAR T-cell manufacturing. We additionally interrogate the fate of these CD19-expressing cells as they undergo transduction to express CD19-specific CARs.

Main Body: We describe the case of a preteen male with multiply relapsed B-ALL who was treated with sequential cellular therapies. He received an αβ T-cell depleted haploidentical HSCT followed by addback of donor-derived T cells genetically modified with a suicide gene for iCaspase9 and truncated CD19 for cell tracking (RivoCel). He relapsed 6 months following HSCT and underwent leukapheresis and CAR T-cell manufacturing. During manufacturing, we identified an aberrant T-cell population dually expressing CD19 and CD3. We hypothesized that these cells were RivoCel cells and confirmed using flow cytometry and PCR that the identified cells were in fact RivoCel cells and were eliminated with iCaspase9 activation. We additionally tracked these cells through CD19-specific CAR transduction and notably did not detect T cells dually positive for CD19 and CD19-directed CARs. The most likely rationale for this is in vitro fratricide of the CD19+ 'artificial' T-cell population by the CD19-specific CAR+ T cells in culture.

Conclusions: We report the identification of CD19+/CD3+ cells in an apheresis product undergoing CAR transduction derived from a patient previously treated with a haploidentical transplant followed by RivoCel addback. We aim to bring attention to this cell phenotype that may be recognized with greater frequency as CAR therapy and engineered αβhaplo-HSCT are increasingly coupled. We additionally suggest consideration towards using alternative markers to CD19 as a synthetic identifier for post-transplant addback products, as CD19-expression on effector T cells may complicate subsequent treatment using CD19-directed therapy.
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http://dx.doi.org/10.1136/jitc-2020-001073DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7490929PMC
September 2020

Sustained fetal hematopoiesis causes juvenile death from leukemia: evidence from a dual-age-specific mouse model.

Blood Adv 2020 08;4(15):3728-3740

Winthrop P. Rockefeller Cancer Institute, College of Medicine, University of Arkansas for Medical Sciences, Little Rock, AR.

It is not clear whether disrupted age-specific hematopoiesis contributes to the complex manifestations in leukemia patients who carry identical mutations, particularly in pediatric and adult patients with similar clinical characteristics. By studying a dual-age-specific mouse model, we demonstrate that (1) loss of Pten during the fetal-to-adult hematopoiesis switch (hematopoiesis switch) causes sustained fetal hematopoiesis, resulting in death in juvenile leukemia; (2) myeloid-biased hematopoiesis in juvenile mice is associated with the sustained fetal properties of hematopoietic stem cells (HSCs); (3) the age specificity of juvenile myelomonocytic leukemia depends on the copy number of Pten and Nf1; (4) single-allelic Pten deletion during the hematopoiesis switch causes constitutive activation of MAPK in juvenile mice with Nf1 loss of heterozygosity (LOH); and (5) Nf1 LOH causes monocytosis in juvenile mice with Pten haploinsufficiency but does not cause lethality until adulthood. Our data suggest that 1 copy of Pten is sufficient to maintain an intact negative-feedback loop of the Akt pathway and HSC function in reconstitution, despite MAPK being constitutively activated in juvenile Pten+/ΔNf1LOH mice. However, 2 copies of Pten are required to maintain the integrity of the MAPK pathway in juvenile mice with Nf1 haploinsufficiency. Our data indicate that previous investigations of Pten function in wild-type mice may not reflect the impact of Pten loss in mice with Nf1 mutations or other genetic defects. We provide a proof of concept that disassociated age-specific hematopoiesis contributes to leukemogenesis and pediatric demise.
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http://dx.doi.org/10.1182/bloodadvances.2020002326DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7422126PMC
August 2020

A study assessing the feasibility of randomization of pediatric and young adult patients between matched unrelated donor bone marrow transplantation and immune-suppressive therapy for newly diagnosed severe aplastic anemia: A joint pilot trial of the North American Pediatric Aplastic Anemia Consortium and the Pediatric Transplantation and Cellular Therapy Consortium.

Pediatr Blood Cancer 2020 10 9;67(10):e28444. Epub 2020 Aug 9.

Boston Children's Hospital and Harvard Medical School, Boston, Massachusetts.

Background: Recent data show survival after matched unrelated donor (MUD) bone marrow transplantation (BMT) is similar to matched sibling procedures for young patients with severe aplastic anemia (SAA). Donor delays, risk of transplant-related mortality (TRM), and concern about chronic graft versus host disease raise questions about whether MUD BMT or immune suppression therapy (IST) should be preferred initial therapy for young patients lacking matched sibling donors.

Procedure: We performed a pilot trial to assess the feasibility of randomizing patients under age 26 with newly diagnosed SAA to receive IST versus MUD BMT. Primary aims assessed the acceptability of randomization and timing of BMT. Secondary aims measured toxicities, response, and survival.

Results: Sixty-seven patients with possible SAA were screened at nine centers. Of 57 with confirmed SAA, 23 underwent randomization and received therapy with a median follow-up of 18 months. Of 12 randomized to BMT, 10 started BMT as initial therapy at a median of 36 days after randomization. One BMT recipient experienced secondary graft failure, requiring a second procedure. Six of 11 randomized to IST responded, whereas five with refractory disease underwent successful salvage BMT. One patient achieving complete response relapsed after discontinuation of immune suppression and died of infection after salvage BMT.

Conclusions: This feasibility study showed that a high percentage of patients underwent randomization and received up-front MUD BMT. Our study lays the groundwork for a larger randomized trial that will define best initial therapy for young patients with SAA who have an available MUD.
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http://dx.doi.org/10.1002/pbc.28444DOI Listing
October 2020

Phenotypic and Functional Characterization of NK Cells in αβT-Cell and B-Cell Depleted Haplo-HSCT to Cure Pediatric Patients with Acute Leukemia.

Cancers (Basel) 2020 Aug 5;12(8). Epub 2020 Aug 5.

Laboratory of Immunology, Istituto di Ricovero e Cura a Carattere Scientifico (IRCCS) Ospedale Policlinico San Martino, 16132 Genoa, Italy.

NK cells can exert remarkable graft-versus-leukemia (GvL) effect in HLA-haploidentical hematopoietic stem cell transplantation (haplo-HSCT). Here, we dissected the NK-cell repertoire of 80 pediatric acute leukemia patients previously reported to have an excellent clinical outcome after αβT/B-depleted haplo-HSCT. This graft manipulation strategy allows the co-infusion of mature immune cells, mainly NK and γδT cells, and hematopoietic stem cells (HSCs). To promote NK-cell based antileukemia activity, 36/80 patients were transplanted with an NK alloreactive donor, defined according to the KIR/KIR-Ligand mismatch in the graft-versus-host direction. The analysis of the reconstituted NK-cell repertoire in these patients showed relatively high proportions of mature and functional KIRNKG2ACD57 NK cells, including the alloreactive NK cell subset, one month after HSCT. Thus, the NK cells adoptively transfused with the graft persist as a mature source of effector cells while new NK cells differentiate from the donor HSCs. Notably, the alloreactive NK cell subset was endowed with the highest anti-leukemia activity and its size in the reconstituted repertoire could be influenced by human cytomegalovirus (HCMV) reactivation. While the phenotypic pattern of donor NK cells did not impact on post-transplant HCMV reactivation, in the recipients, HCMV infection/reactivation fostered a more differentiated NK-cell phenotype. In this cohort, no significant correlation between differentiated NK cells and relapse-free survival was observed.
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http://dx.doi.org/10.3390/cancers12082187DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7463940PMC
August 2020

Engineering the Bridge between Innate and Adaptive Immunity for Cancer Immunotherapy: Focus on γδ T and NK Cells.

Cells 2020 07 22;9(8). Epub 2020 Jul 22.

Stem Cell Laboratory and Cell Therapy Center, IRCCS Istituto Giannina Gaslini, Via G. Gaslini, 516147 Genova, Italy.

Most studies on genetic engineering technologies for cancer immunotherapy based on allogeneic donors have focused on adaptive immunity. However, the main limitation of such approaches is that they can lead to severe graft-versus-host disease (GvHD). An alternative approach would bolster innate immunity by relying on the natural tropism of some subsets of the innate immune system, such as γδ T and natural killer (NK) cells, for the tumor microenvironment and their ability to kill in a major histocompatibility complex (MHC)-independent manner. γδ T and NK cells have the unique ability to bridge innate and adaptive immunity while responding to a broad range of tumors. Considering these properties, γδ T and NK cells represent ideal sources for developing allogeneic cell therapies. Recently, significant efforts have been made to exploit the intrinsic anti-tumor capacity of these cells for treating hematologic and solid malignancies using genetic engineering approaches such as chimeric antigen receptor (CAR) and T cell receptor (TCR). Here, we review over 30 studies on these two approaches that use γδ T and NK cells in adoptive cell therapy (ACT) for treating cancer. Based on those studies, we propose several promising strategies to optimize the clinical translation of these approaches.
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http://dx.doi.org/10.3390/cells9081757DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7464083PMC
July 2020

Transplantation Induces Profound Changes in the Transcriptional Asset of Hematopoietic Stem Cells: Identification of Specific Signatures Using Machine Learning Techniques.

J Clin Med 2020 Jun 1;9(6). Epub 2020 Jun 1.

Department of Clinical and Biological Sciences, University of Turin, 10043 Turin, Italy.

During the phase of proliferation needed for hematopoietic reconstitution following transplantation, hematopoietic stem/progenitor cells (HSPC) must express genes involved in stem cell self-renewal. We investigated the expression of genes relevant for self-renewal and expansion of HSPC (operationally defined as CD34+ cells) in steady state and after transplantation. Specifically, we evaluated the expression of ninety-one genes that were analyzed by real-time PCR in CD34+ cells isolated from (i) 12 samples from umbilical cord blood (UCB); (ii) 15 samples from bone marrow healthy donors; (iii) 13 samples from bone marrow after umbilical cord blood transplant (UCBT); and (iv) 29 samples from patients after transplantation with adult hematopoietic cells. The results show that transplanted CD34+ cells from adult cells acquire an asset very different from transplanted CD34+ cells from cord blood. Multivariate machine learning analysis (MMLA) showed that four specific gene signatures can be obtained by comparing the four types of CD34+ cells. In several, but not all cases, transplanted HSPC from UCB overexpress reprogramming genes. However, these remarkable changes do not alter the commitment to hematopoietic lineage. Overall, these results reveal undisclosed aspects of transplantation biology.
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http://dx.doi.org/10.3390/jcm9061670DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7355574PMC
June 2020

γδ T Cells: The Ideal Tool for Cancer Immunotherapy.

Cells 2020 05 24;9(5). Epub 2020 May 24.

Laboratorio Cellule Staminali post-natali e Terapie Cellulari, IRCCS G. Gaslini, Via G. Gaslini 5, 16147 Genova, Italy.

γδ T cells have recently gained considerable attention as an attractive tool for cancer adoptive immunotherapy due to their potent anti-tumor activity and unique role in immunosurveillance. The remarkable success of engineered T cells for the treatment of hematological malignancies has revolutionized the field of adoptive cell immunotherapy. Accordingly, major efforts are underway to translate this exciting technology to the treatment of solid tumors and the development of allogeneic therapies. The unique features of γδ T cells, including their major histocompatibility complex (MHC)-independent anti-cancer activity, tissue tropism, and multivalent response against a broad spectrum of the tumors, render them ideal for designing universal 'third-party' cell products, with the potential to overcome the challenges of allogeneic cell therapy. In this review, we describe the crucial role of γδ T cells in anti-tumor immunosurveillance and we summarize the different approaches used for the ex vivo and in vivo expansion of γδ T cells suitable for the development of novel strategies for cancer therapy. We further discuss the different transduction strategies aiming at redirecting or improving the function of γδ T cells, as well as, the considerations for the clinical applications.
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http://dx.doi.org/10.3390/cells9051305DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7290982PMC
May 2020

Genome editing of donor-derived T-cells to generate allogenic chimeric antigen receptor-modified T cells: Optimizing αβ T cell-depleted haploidentical hematopoietic stem cell transplantation.

Haematologica 2021 03 1;106(3):847-858. Epub 2021 Mar 1.

Department of Pediatrics, Stanford University School of Medicine, Stanford, CA, USA

Allogeneic hematopoietic stem cell transplantation is an effective therapy for high-risk leukemias. In children, graft manipulation based on the selective removal of aβ T cells and B cells has been shown to reduce the risk of acute and chronic graft-versus-host disease, thus allowing the use of haploidentical donors which expands the population of recipients in whom allogeneic hematopoietic stem cell transplantation can be used. Leukemic relapse, however, remains a challenge. T cells expressing chimeric antigen receptors can potently eliminate leukemia, including those in the central nervous system. We hypothesized that by engineering the donor aβ T cells that are removed from the graft by genome editing to express a CD19-specific chimeric antigen receptor, while simultaneously inactivating the T-cell receptor, we could create a therapy that enhances the anti-leukemic efficacy of the stem cell transplant without increasing the risk of graft-versus-host disease. Using genome editing with Cas9 ribonucleoprotein and adeno-associated virus serotype 6, we integrated a CD19-specific chimeric antigen receptor inframe into the TRAC locus. More than 90% of cells lost T-cell receptor expression, while >75% expressed the chimeric antigen receptor. The initial product was further purified with less than 0.05% T-cell receptorpositive cells remaining. In vitro, the chimeric antigen receptor T cells efficiently eliminated target cells and produced high cytokine levels when challenged with CD19+ leukemia cells. In vivo, the gene-modified T cells eliminated leukemia without causing graft-versus-host disease in a xenograft model. Gene editing was highly specific with no evidence of off-target effects. These data support the concept that the addition of aβ T-cell-derived, genome-edited T cells expressing CD19-specific chimeric antigen receptors could enhance the anti-leukemic efficacy of aβ T-celldepleted haploidentical hematopoietic stem cell transplantation without increasing the risk of graft-versus-host disease.
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http://dx.doi.org/10.3324/haematol.2019.233882DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7928014PMC
March 2021

Myeloablative conditioning for allo-HSCT in pediatric ALL: FTBI or chemotherapy?-A multicenter EBMT-PDWP study.

Bone Marrow Transplant 2020 08 17;55(8):1540-1551. Epub 2020 Mar 17.

Paediatric Onco-Haematology, City of Science and Health of Turino, Regina Margherita Children's Hospital, Torino, Italy.

Although most children with acute lymphoblastic leukemia (ALL) receive fractionated total body irradiation (FTBI) as myeloablative conditioning (MAC) for allogeneic hematopoietic stem cell transplantation (allo-HSCT), it is an important matter of debate if chemotherapy can effectively replace FTBI. To compare outcomes after FTBI versus chemotherapy-based conditioning (CC), we performed a retrospective EBMT registry study. Children aged 2-18 years after MAC for first allo-HSCT of bone marrow (BM) or peripheral blood stem cells (PBSC) from matched-related (MRD) or unrelated donors (UD) in first (CR1) or second remission (CR2) between 2000 and 2012 were included. Propensity score weighting was used to control pretreatment imbalances of the observed variables. 3.054 patients were analyzed. CR1 (1.498): median follow-up (FU) after FTBI (1.285) and CC (213) was 6.8 and 6.1 years. Survivals were not significantly different. CR2 (1.556): median FU after FTBI (1.345) and CC (211) was 6.2 years. Outcomes after FTBI were superior as compared with CC with regard to overall survival (OS), leukemia-free survival (LFS), relapse incidence (RI), and nonrelapse mortality (NRM). However, we must emphasize the preliminary character of the results of this retrospective "real-world-practice" study. These findings will be prospectively assessed in the ALL SCTped 2012 FORUM trial.
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http://dx.doi.org/10.1038/s41409-020-0854-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8376634PMC
August 2020

Treatment of steroid resistant acute graft versus host disease with an anti-CD26 monoclonal antibody-Begelomab.

Bone Marrow Transplant 2020 08 13;55(8):1580-1587. Epub 2020 Mar 13.

Dipartimento di Oncoematologia Pediatrica, IRCCS, Ospedale Pediatrico Bambino Gesu', Sapienza, Università di Roma, Roma, Italy.

We have treated 69 patients with steroid refractory acute graft versus host disease (SR-aGvHD), with an anti-CD26 monoclonal antibody (Begelomab): 28 patients in two prospective studies (EudraCT No. 2007-005809-21; EudraCT No. 2012-001353-19), and 41 patients on a compassionate use study. The median age of patients was 42 and 44 years; the severity of GvHD was as follows: grade II in 8 patients, grade III in 33, and grade IV in 28 patients. There were no adverse events directly attributable to the antibody. Day 28 response was 75% in the prospective studies and 61% in the compassionate use patients, with complete response rates of 11 and 12%. Response for grade III GvHD was 83 and 73% in the two groups; response in grade IV GvHD was 66 and 56% in the two groups. Non relapse mortality (NRM) at 6 months was 28 and 38%. Overall there were 64, 56, 68% responses for skin, liver, and gut stage 3-4 GvHD. The overall survival at 1 year was 50% for the prospective studies and 33% for the compassionate use patients. In conclusion, Begelomab induces over 60% responses in SR-aGvHD, including patients with severe gut and liver GvHD, having failed one or more lines of treatment.
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http://dx.doi.org/10.1038/s41409-020-0855-zDOI Listing
August 2020

Supportive care during pediatric hematopoietic stem cell transplantation: beyond infectious diseases. A report from workshops on supportive care of the Pediatric Diseases Working Party (PDWP) of the European Society for Blood and Marrow Transplantation (EBMT).

Bone Marrow Transplant 2020 06 6;55(6):1126-1136. Epub 2020 Feb 6.

Division of Pediatric Hematoloy/Oncology, Schneider Children's Medical Center of Israel, Petach Tikva, Israel.

Hematopoietic stem cell transplantation (HSCT) is currently the standard of care for many malignant and nonmalignant blood diseases. As several treatment-emerging acute toxicities are expected, optimal supportive measurements critically affect HSCT outcomes. The paucity of good clinical studies in supportive practices gives rise to the establishment of heterogeneous guidelines across the different centers, which hampers direct clinical comparison in multicentric studies. Aiming to harmonize the supportive care provided during the pediatric HSCT in Europe, the Pediatric Diseases Working Party (PDWP) of the European Society for Blood and Marrow Transplantation (EBMT) promoted dedicated workshops during the years 2017 and 2018. The present paper describes the resulting consensus on the management of sinusoidal obstructive syndrome, mucositis, enteral and parenteral nutrition, iron overload, and emesis during HSCT.
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http://dx.doi.org/10.1038/s41409-020-0818-4DOI Listing
June 2020

Outcome of children relapsing after first allogeneic haematopoietic stem cell transplantation for acute myeloid leukaemia: a retrospective I-BFM analysis of 333 children.

Br J Haematol 2020 05 3;189(4):745-750. Epub 2020 Feb 3.

Division of Pediatric Hematology and Oncology, Department of Pediatrics, Universities of Medical University Hannover, Hannover, Germany.

Outcome of 333 children with acute myeloid leukaemia relapsing after a first allogeneic haematopoietic stem cell transplantation was analyzed. Four-year probability of overall survival (4y-pOS) was 14%. 4y-pOS for 122 children receiving a second haematopoietic stem cell transplantation was 31% and 3% for those that did not (P = <0·0001). Achievement of a subsequent remission impacted survival (P = <0·0001). For patients receiving a second transplant survival with or without achieving a subsequent remission was comparable. Graft source (bone marrow vs. peripheral blood stem cells, P = 0·046) and donor choice (matched family vs. matched unrelated donor, P = 0·029) positively impacted survival after relapse. Disease recurrence and non-relapse mortality at four years reached 45% and 22%.
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http://dx.doi.org/10.1111/bjh.16441DOI Listing
May 2020

Health-Related Quality of Life and Emotional Difficulties in Chronic Granulomatous Disease: Data on Adult and Pediatric Patients from Italian Network for Primary Immunodeficiency (IPINet).

J Clin Immunol 2020 02 20;40(2):289-298. Epub 2019 Dec 20.

Unit of Pediatric Haemato-Oncology, Policlinico Giovanni XXIII Hospital, University of Bari, Piazza Giulio Cesare, 11, 70124, Bari, Italy.

Chronic granulomatous disease (CGD) is a primary immunodeficiency characterized by life-threatening infections, inflammation, and autoimmunity with an impact on health-related quality of life (HRQoL). Few data are available for children, whereas no study has been conducted in adults. Here, we investigated HRQoL and emotional functioning of 19 children and 28 adults enrolled in Italian registry for CGD. PEDsQL and SDQ were used for children and their caregivers, and adults completed the SF-12 questionnaire. Mean scores were compared with norms and with patients affected by chronic diseases. Comparisons were made for CGD patients who underwent or not hematopoietic stem cell transplantation (HSCT). When compared with norms, CGD children exhibited higher difficulties in social/school areas, peer relationship, and conduct/emotional problems (< 5 years of age), as scored by proxies. Differently, CGD adults reported higher difficulties both in mental and physical area than norms. Only for children, clinical status had a damaging effect on psychosocial and school dimensions, whereas age had a negative impact on social areas. No significant difference was observed between patients treated or not with HSCT. When compared with patients affected by chronic diseases, CGD children and adults both displayed fewer physical disabilities. Differently, in mental scale adults scored lower than those with rheumatology diseases and had similar impairment in comparison with patients with diabetes mellitus and cancer. This study emphasized the impact of CGD on HRQoL since infancy and its decline in adulthood, with emotional difficulties occurring early. HRQoL impairment should be considered in clinical picture of CGD and pro-actively assessed and managed by clinicians.
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http://dx.doi.org/10.1007/s10875-019-00725-1DOI Listing
February 2020
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