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To analyze and compare the tear immunological profile in ocular GVHD (oGVHD) patients with that in non-oGVHD patients and to correlate them with ocular surface parameters based on the International Chronic Ocular GVHD Consensus Group (ICCGVHD) diagnostic criteria.Tear samples from 20 individuals who underwent allo-hematopoietic stem cell transplantation and were grouped according the presence or absence of oGVHD were analyzed using Bio-Plex assay.IL-8 and MIP-1α levels were significantly higher in tears from oGVHD patients compared with those in tears from non-oGVHD patients (p0.001 and p=0.001, respectively). Tear IL-8 levels correlated significantly with OSDI criteria (ρ=0.5159, p=0.001), ocular hyperemia (ρ=0.469, p=0.002), and corneal staining (ρ=0.339, p=0.032), whereas tear Mip-1α levels correlated with OSDI score (ρ=0.358, p=0.023).We demonstrated higher tear levels of IL-8 and MIP-1α in oGVHD patients and significant correlations between theses cytokines and ocular surface parameters based on the ICCGVHDCG criteria.

Malignant extracranial germ cell tumors (GCTs) are rare in pediatric patients and are usually extremely sensitive to chemotherapy. Relapsed or refractory tumors, although rare, established the need for second-line therapies, including high-dose chemotherapy with autologous stem cell transplantation (HDCT/ASCT). However, there are few data on its use in children with GCTs. We present a retrospective analysis of all patients diagnosed with extracranial GCTs who received HDCT/ASCT at two Brazilian pediatric cancer centers from May 1999 to December 2019. We identified a total of 34 patients with a median age at diagnosis of 2.8 years (range, 0 to 18.8), who received HDCT/ASCT. Most patients (73%) received carboplatin, etoposide and melphalan (CEM) as a HDCT regimen. Fourteen patients received a second-line conventional dose chemotherapy (CDCT), 14 received a third-line CDCT and five received even a fourth-line CDCT prior to HDCT/ASCT. After a median follow-up of 22.7 months (range, 0.3 to 198.1), 16 patients had died after tumor relapse/progression and 2 patients died from HDCT/ASCT toxicity. We observed a 5-year OS of 47.1% and 5-year EFS of 44.1%. The 5-year OS for patients referred for HDCT/ASCT with progressive disease was 10% compared to 62.5% for those who achieved disease control before HDCT/ASCT (p = 0.001). In our experience, heavily pretreated children and adolescents with extracranial GCTs achieved considerable survival rates with HDCT/ASCT since, at least, partial control of their disease was possible before starting HDCT/ASCT. The role of HDCT/ASCT in pediatric patients with GCTs should be investigated in prospective trials.

Haploidentical hematopoietic-cell transplantation using post-transplant cyclophosphamide(Haplo-PTCy) is a feasible procedure in children with haematologic malignancies. However, data of a large series of children with acute leukaemia(AL) in this setting is missing. We analysed 144 AL Haplo-PTCy paediatric recipients; median age was 10 years. Patients had acute lymphoblastic(ALL; n = 86) or myeloblastic leukaemia(AML; n = 58) and were transplanted in remission(CR1: n = 40; CR2: n = 57; CR3+: n = 27) or relapse (n = 20). Bone marrow was the graft source in 57%; donors were father (54%), mother (35%), or sibling (11%). Myeloablative conditioning was used in 87%. Median follow-up was 31 months. At day +100, cumulative incidence (CI) of neutrophil recovery and acute GVHD (II-IV) were 94% and 40%, respectively. At 2-years, CI of chronic GVHD and relapse, were 31%, 40%, and estimated 2-year overall survival (OS), leukaemia-free survival (LFS) and graft-versus-host-relapse-free survival (GRFS) were 52%, 44% and 34% respectively. For patients transplanted in remission, positive measurable residual disease (MRD) prior to transplant was associated with decreased LFS (p = 0.05) and GRFS (p = 0.003) and increased risk of relapse (p = 0.02). Mother donor was associated with increased risk of chronic GVHD (p = 0.001), decreased OS (p = 0.03) and GRFS (p = 0.004). Use of PBSC was associated with increased risk of chronic GVHD (p = 0.04). In conclusion, achieving MRD negativity pre-transplant, avoiding use of mother donors and PBSC as graft source may improve outcomes of Haplo-PTCy in children with AL.

THE BRAZILIAN SOCIETY FOR BLOOD AND MARROW TRANSPLANTATION (SBTMO) PRESENTS THE BRAZILIAN GUIDELINES ON HEMATOPOIETIC STEM CELL TRANSPLANTATION

To better understand the outcomes of HSCT in Brazil, we conducted a multicenter study using the CIBMTR database. Seven participating centers extracted their own data through the Data Back to Center tool. Main indications for HSCT-auto were MM(51%), NHL(18%) and HL(17%); Allogeneic, AML(24%), ALL(23%) and SAA(15%). For acute leukemias, risk of death was higher in the 18-40 years group (HR=1.18,p=0.022), 40-60(HR=1.19,p

This is an observational and cross-sectional study, carried out in May 2020, targeting adult individuals of both sexes who are members of multiprofessional teams working in Brazilian HSCT units in the current period of the pandemic by completing and analyzing a questionnaire. pre-formulated. HSCT units that cannot access the questionnaire were excluded from the study. The analysis of the operation profile of HSCT units in Brazil, through the application of a pre-structured questionnaire, is not an accurate tool, since it assumes some premises that may prove to be wrong, especially in this current scenario in Brazil. However, the data reveal the vulnerability of patients with onco-hematological diseases to infection by COVID-19, especially during HSCT procedures, in relation to the general population. Despite its limitations, it can be valuable to plan policies.

The choice of alternative donors for HCT for patients without an HLA-matched related donor depends on several factors. We compared major HCT outcomes in 212 consecutive children transplanted at 11 centers in Brazil for acute leukemia or MDS from an HLA-matched unrelated donor (MUD, n = 95), mismatched unrelated donor (MMUD, n = 47) or unrelated umbilical cord blood (UCB, n = 70). Most had ALL (61%), bone marrow (57%) as the graft source and 95% received a MAC regimen. The 3-year OS probability were 57, 55, and 37% after HCT from MUD, MMUD, and UCB, respectively (HR 1.68, 95%CI 1.07-2.63; P = .02). In comparison with MUD, OS was similar after transplantation of a ≥ 6/8 HLA-matched or a high cell dose (>5 × 107 TNC/kg) CB unit (HR 1.41, 95%CI 0.88-2.27; P = .15). NRM was higher for UCB (HR 3.90, 95%CI 1.43-10.7; P = .01) but not for MMUD (HR 1.03, 95%CI 0.53-2.00; P > .20). Advanced disease (HR 2.05, 95%CI 1.26-3.33; P < .001) and UCB with high probability of being < 6/8 HLA-matched (HR 5.34, 95%CI 2.0-13.9; P < .001) were associated with higher mortality. Relapse and acute GVHD were similar among groups, while PGF was higher among UCB transplants (P = .002) and chronic GVHD among MMUD group (HR 2.88, 95% CI 1.05-7.88; P = .04). Our results suggest that in Brazil HCT outcomes performed with MMUD and MUD donors were comparable, while with UCB units < 6/8 HLA-matched were associated with higher NRM for children with acute leukemia or MDS.

The objective of this study is research produced by the multi-professional group of Education and Health in a center for the care of children and adolescents with cancer in a Brazilian city. We selected an investigation that aims to study and determine the school specificities of pupils submitted to hematopoietic cell transplantation (HCT). We studied the process of school intervention with 200 student-patients, in the interval of 2000-2015. Four annotations were made for the articulation of knowledge focused on the social practices of language of the current school year. The students submitted to the HCT present a process of irregular schooling necessitating orientation to the specific curricular demands. RESUMO O objeto deste estudo sao pesquisas produzidas pelo grupo multiprofissional de Educacao e Saude em um centro de atendimento infanto-juvenil ao paciente com câncer de uma cidade brasileira. Selecionamos uma investigacao que tem como objetivo estudar e determinar as especificidades escolares dos alunos submetidos ao transplante de celulas hematopoieticas (TCH). Estudamos o processo de intervencao escolar com 200 alunos-pacientes, no intervalo de 2000-2015. Realizou-se quatro anotacoes para a articulacao de conhecimentos voltados as praticas sociais de linguagem do ano escolar em curso. Os alunos submetidos ao TCH apresentam processo de escolarizacao irregular necessitando de orientacao as demandas especificas curriculares.

Background: Peripheral blood stem cell concentrations are traditionally adjusted to 20–40 × 106 leukocytes/mL prior to freezing. This low cell concentration at cryopreservation implies larger volumes with more dimethyl sulfoxide being used, and higher cost and toxicity at the time of transplant. Higher cell concentrations have been reported but this is not widely accepted. Moreover, the influence of cell concentration on engraftment has not been well documented. Therefore, this study retrospectively analyzed the influence of peripheral blood stem cell concentration at freezing on engraftment after autologous hematopoietic stem cell transplantation. Method: Leukapheresis products were plasma-depleted and cryopreserved with 5% dimethyl sulfoxide, 6% hydroxyethylamide solution and 4% albumin in a −80 °C freezer. Individual patient data from hospital records were reviewed. Results: Fifty consecutive patients with oncological diseases underwent 88 leukaphereses. Median age was six years (range: 1–32 years) and median weight was 19 kg (range: 8–94 kg). Median leukocyte concentration was 109 × 106/mL at collection and 359 × 106 (range: 58–676 × 106) at freezing with 78% viability (range: 53–95%); leukocyte recovery after thawing was 95% (range: 70–100%). In multivariate analysis, cell concentration (p-value = 0.001) had a negative impact on engraftment. Patients infused with bags frozen with 600 × 106 leukocytes/mL, engraftment was after 14 days (range: 13–22 days). Conclusion: In patients with adequate CD34 cell collections, total leukocyte concentrations of 282 × 106/mL, freezing with 5% dimethyl sulfoxide and 6% hydroxyethylamide solution without a controlled-rate freezer, and storing cells at −80 °C yielded excellent engraftment. Further increases in cell concentration may delay engraftment, without affecting safety. Keywords: Cryopreservation, Stem cell transplantation, peripheral, Autografts, Pediatrics, Dimethyl sulfoxide

Langerhans cell histiocytosis (LCH) is a rare disease, with an estimated incidence of 0.5 per 100,000 children in the United States of America1. HCL occurs due to differentiation of myeloid precursors into CD1a+ / CD207+ cells and is characterized by constitutional activation of the MAPK2 signaling pathway, leading to a spectrum of organ involvement and dysfunction. Treatment of HCL is risk-adjusted: single lesions may respond to local treatment whereas multisystem disease requires systemic therapy. Although survival for patients without organ dysfunction is excellent3, mortality in those with compromised organs at risk (hematopoietic system, liver, and/or spleen) reaches 20%2,4. Despite the progress made in the treatment of HCL, disease reactivation rates remain above 30% and the best second-line treatment has not yet been established. Treatment failure is associated with increased morbidity and mortality, including an association with neurodegeneration2. As it is a rare disease and generally has a good prognosis, few scientific studies are evaluating the role of allogeneic hematopoietic stem cell transplantation (HSCT) in the treatment of this disease. In 2015 Veys et al5 published retrospective results of 87 high-risk patients transplanted between 1990 and 2013. Myeloablative conditioning regimens (MAC) based on total body irradiation or busulfan6 were the most used until the 2000s, and reduced-intensity conditioning regimens (RIC) – predominantly a combination of Melphalan and Fludarabine – were most used between 2000 and 2013. Transplant-associated mortality rates in 3 years were similar between RIC and MAC conditioning regimens (21% versus 15%, respectively). Recurrence was higher in the RIC group compared to the MAC group (28% versus 8%, respectively), however, the 3-year overall survival (OS) was similar (77% versus 71%, respectively), since the patients who relapsed after RIC transplantation could be rescued with chemotherapy. More recently, Kudo et al7 published a retrospective study with 30 patients with refractory LCH who underwent HSCT between 1996 and 2014. Eleven patients received myeloablative conditioning regimen based on total body radiotherapy (RCT) with a dose equal to or greater than 8 Gy or busulfan, and 19 of reduced intensity based on Fludarabine and Melphalan, associated or not with low dose of RCT. There was no significant difference between the conditioning regimen modalities, with OS of 56.8% for the RIC group and 63.6% for the MAC group. Disease status was the main prognostic factor, with a 5-year OS of 100% for patients who arrived at HSCT with disease in remission or with partial remission, versus 54.5% for those who had active disease at the time of the procedure. Regarding the type of donor used and the source of stem cells, there is great variation, with greater frequency for unrelated and extensive use of bone marrow and umbilical cord, and apparently, there is no impact on survival rates.5, 6 There are few case reports and extremely restricted performance of autologous HSCT in HCL.

In this study, we report on major MRD or URD BMT outcomes in pediatric patients with SAA in Brazil. This was a retrospective study, which included 106 patients ≤18 years old who received a first BMT for SAA. All patients received bone marrow as graft source from an MRD (n = 69) or a URD (n = 37). Conditioning regimen was non-myeloablative in 73.6% of cases, and GVHD prophylaxis comprised a calcineurin inhibitor plus methotrexate in 89.6% of patients. After a median follow-up of 4.5 years after BMT, 81 patients are alive, with a 4-year OS of 77% and no statistically significant difference between the MRD and URD groups (82% vs. 69%, respectively; P = .08). Grade III-IV aGVHD at 6 months and cGVHD at 2 years were observed in 8% and 14% of cases, respectively, and were not statistically different between the groups. Twenty-five (23%) patients died at a median of 2.9 months after BMT. Our study showed that 4-year OS after BMT was not statistically different between MRD and URD recipients. This study shows that the outcomes of pediatric patients transplanted for SAA with a URD in Brazil are approaching those of MRD transplants. In contrast, OS after MRD BMT was lower than we would expect based on previous reports. The wide range of preparatory regimens used by the study centers highlights the need for standardized protocols for these children. Our findings provide a benchmark for future studies focused on improving BMT outcomes in this setting in Brazil.

Background:Natural Killer Cells are innate immune system cells important in host defenses against viruses and tumor cells. Two subpopulations are well described: NK CD56bright CD16neg (NK56++16-) which is less frequent in peripheral blood (PB) and associated with high cytokine production, and NK56dim16pos (NK56+16+), more frequent in PB and associated with high cytotoxic activity. In allogeneic hematopoietic stem cell transplantation (allo-HSCT), NK cells are important for infection control, and also for the disease control. The mismatch between donor NK cell receptors and host HLA may lead to a stronger graft versus leukemia (GVL), possibly modulating graft versus host disease (GVHD). However, only a few studies evaluated NK subpopulations recovery after HSCT and the impact on transplant outcomes. Objectives: The aim of the study was to analyze NK cells subpopulations reconstitution after HSCT and their possible impact on HSCT endpoints: relapse, GVHD (acute and chronic), transplantation related mortality and overall survival. Patients and Methods: Overall, 77 patients from 4 HSCT centers (60 % male, median age 17 years, range 1-74), receiving bone marrow (BM, 47%), umbilical cord (UCB, 30%) or peripheral blood (PB, 23%) from unrelated (80%) or related donors (20%) were studied. The most common diagnosis was acute leukemia (AML 38%, ALL 34%). Most patients received myeloablative conditioning (MAC) regimens (61%). Antithymocyte globulin (ATG) was used in 27 patients (35%) and total body irradiation (TBI) in 40 (52%). Median follow up time was 82 months (22-107). NK subpopulations were quantified by multiparametric flow cytometry at engraftment and days 3, 7, 14, 21 and 60 after engraftment. We have then elaborated graphs plotting cells counts over time (until 60 days) and calculated the area over the curve (AOC) for each subpopulation [NK56++16- (NK56-index) and NK56+16+ cells (NK16-index)], considering 0.5x10e9/L as cut-off value for both subpopulations. A higher AOC represents a more profound NK deficiency. Using receiver operating characteristic (ROC) curves we determined cut offs for the AOC that could provide the best correlation with survival. Results:The overall survival (OS) in 80 months was significantly worse in patients with NK56 deficiency, defined by a NK56-index > 14592.31 cells/mm3.days (29% x 73% for non- deficient, p=0.0001, Figure 1). NK56 deficient patients also had a higher cumulative incidence (CI) of non-relapse related mortality (NRM: 35% x 15%, p=0.03, Figure 2). GVHD (acute or chronic) was similar between low NK56-index and high NK-56 index patients. In multivariate analysis, high NK56index remained significantly associated with worse OS [hazard ratio (HR) 3.5; 95% confidence interval (95%CI) 1.6-7.6], and higher NRM (HR 2.9; 95%CI 1.0-8.0). There were no significant correlations between NK16-index with any of the analyzed HSCT outcomes. Conclusion: A higher NK56-index is significantly associated with worse outcome in long term survival and non relapse related mortality in patients submitted to allogeneic hematopoietic stem cell transplantation. This strategy might be helpful in identifying risk groups and possible earlier therapeutic interventions. Disclosures No relevant conflicts of interest to declare.

Background: Most studies in immune reconstitution after allogeneic hematologic stem cell transplantation (alloHSCT) focus on adaptive immunity, particularly in lymphocyte recovery. Classical monocytes (cMo) are part of the innate immunity, playing an important role in the defense against fungal and bacterial infections. The innate and adaptive immunity are closely related, and non-classical monocytes (ncMo) participate in this connection, regulating T cell response. However, the role of these cells in the graft versus tumor effect and graft versus host disease (GVHD) is still unclear. Objectives: The aim of the study was to investigate cMo and ncMo reconstitution after alloHSCT and a possible impact of these cells subsets on acute and chronic GVHD, relapse, non-relapse related mortality (NRM) and overall survival (OS). Patients and Methods: This is a retrospective analysis of 77 patients who underwent alloHSCT in 4 different transplant centers in Brazil. Peripheral blood samples were collected from patients at neutrophil engraftment (NE) and 3, 7, 14, 21, and 42 days after NE. The cMo (DR+, CD11c+, CD14+, CD16-) and ncMo (DR+, CD11c+, CD14-, CD16+) were classified and analyzed by 8-color multiparametric flow cytometry (FACSCanto II). In order to analyze the possible impact of duration and severity of monocytopenia after transplant, we calculated then the 'mono-index' and the 'ncMo-index', based on the area over the curve (AOC) of absolute cMO counts (AMC) and absolute ncMo counts, respectively, considering a horizontal line of AMC 0.5x10e9/L as cut-off value for both subpopulations. Results: The medium follow up was 82 months. Out of 77 patients, 46 (60%) were male, and the median age was 17 years (range: 1-74). The stem cell source was bone marrow in 36 (47%), cord blood in 23 (30%), and peripheral blood in 18 (23%); and donors were matched unrelated in 61 (79%), and matched related in 16 (21%) cases. Most patients received myeloablative conditioning (n=47, 61%); and acute leukemia was the most common diagnosis (72%). A total of 40 (52%) patients received total body irradiation (TBI), and 27 (35%) patients received antithymocyte globulin (ATG). We calculated the possible impact of cMo at all timepoints and found no differences in the main outcomes between patients with lower or higher counts. We then analyzed the AOC of all cMo counts (the mono-index). A higher index represents a more severe deficiency of cMo. We then used the receiver operating characteristic (ROC) curve to define the cut-off value of 14234.4 cells/days x mm3 to discriminate patients as cMo deficient (n=31, 40%) or non-deficient (n=46, 60%). Non relapse mortality (NRM) at 6 years was higher in cMo deficient patients calculated by the mono-index (40% vs. 16% for non-deficient, p=0.02, Figure 1). Deficient patients also had worse OS at 6 years (58% vs. 37% for non-deficient, p=0.03, Figure 2). In multivariate analyses, cMo deficiency calculated by the mono-index remained significantly associated with NRM (hazard ratio 3.07, 95% confidence interval) and OS (HR 2.05, 95%CI 1.20-7.85). We observed no impact of mono-index on acute or chronic GVHD or relapse. We observed no impact of the ncMo counts at all timepoints or the ncMo-index in any of the analyzed outcomes. Conclusion: cMo deficiency calculated by the mono-index can predict mortality in alloHSCT. Mono-Index is an inexpensive, accessible and easy to perform tool that might help clinicians in predicting unfavorable outcomes. Further studies are needed to confirm these findings and to test how different transplant strategies or therapeutic changes might interfere with monocytes recovery in alloHSCT. Disclosures No relevant conflicts of interest to declare.

Introduction: As well as best allogeneic donor choice is imperative to bone marrow transplant (BMT), best conditioning regimen is one of the major cornerstones of the treatment success. Although high-dose busulfan have been employed along with cyclophoshamide (BuCy) for more than two decades as one of the best options for allogeneic BMT in acute myeloid leukemia patients (AML), we were unable to attain the best published outcomes in our center, which led us to pursue new treatment strategies, including alternative conditioning regimens. We chose to use a combination of Busulfan and Melphalan (BuMel) together with Busulfan plasma concentration monitoring. Objective: to compare outcomes of children and young adults who underwent allogeneic BMT for AML using two different myeloablative conditioning regimens without radiotherapy. Methods: retrospective analysis of data. Results: from September, 1999, to July, 2016, 40 patients (30 male), median age 4,6 yo (1,2 to 20,7) underwent allogeneic BMT at Instituto de Oncologia Pediatrica (GRAACC - IOP - UNIFESP). Third-four patients had matched related donors. Median follow-up was 472 days. BuCy (oral busulfan 16 mg/kg or intravenous equivalente dose and cyclophosphamide 200 mg/kg) was the conditioning regimen for 15 patients, and BuMel (oral busulfan 16 mg/kg or intravenous equivalent dose and melphalan 140 mg/m2) for the other 25. Graft versus host disease (GVHD) prophylaxis included rabbit antithymocyte globulin for six matched unrelated transplants. Half of the patients were in first, 19 in second and one in third remission. Bone marrow stem cells were used in the majority of the procedures (38). Only two out of 15 patients who received BuCy as conditioning regimen are alive (OS 13%), versus 15 of the 25 who were transplanted using BuMel (OS 60%). Leukemia relapse was cause of death in 70% for BuCy, followed by transplant-related mortality (TRM) in 23% and GVHD in 7% of the cases. In BuMel group, main cause of death was GVHD (50%), followed by leukemia relapse (20%), TRM (20%) and one disseminated fusariosis. Busulphan pharmacokinetics was available for 18 procedures (17 BuMel and one BuCy). Death rate for those patients was 44%, against 68% for those who had not their busulphan plasmatic levels monitored. However, BuCy was the chosen conditioning regimen for 14 of the 22 patients in the latter group. Conclusion: in view of the unsatisfactory results obtained with BuCy for allogeneic BMT in AML patients in our center urgent measures were required. The choice of a different conditioning regimen as well as busulphan pharmacokinetics monitoring from May, 2012 on, have led us to attain overall survival rates comparable to Center for International Blood and Marrow Transplant Research (CIBMTR) data. Longer follow up will be required to confirm our current data Disclosures No relevant conflicts of interest to declare.