ALBERT

Description: Aberrant methylation in promoter-associated CpG islands has been recognized as a major mechanism for silencing tumor suppressor genes in many malignant diseases. In multiple myeloma (MM), a limited number of studies of gene methylation have been reported, with conflicting results. We determined the methylation status of 9 suppressor tumor genes in 68 newly diagnosed MM patients by methylation specific PCR (MSP). Ten DNA of normal healthy donors were used as controls. The target genes chosen are involved in many cellular pathways as DNA repair (MGMT), cell cycle regulation (p15INK4b and p16INK4a), cell-cell adherence (E-cadherin), apoptosis (DAP-k and BNIP3), hormonal response (RARb and ER) and Jak/STAT3 signaling pathway (SHP1). An association between hypermethylation and loss of expression of these genes has been demonstrated. The correlation between methylation status and risk factors was assessed by Fisher exact test or Chi-square test (categorical variables) or Student’s t-test (continuous variables). Overall survival (OS) was estimated using the Kaplan-Meier method. Differences between survival curves were estimated by the log-rank test. Multivariate analysis of factors associated with OS was performed by Cox regression. With a median follow-up of 15.5 months, 16 patients died (23%). Healthy donor samples were negative for methylation in all 9 genes tested. Hypermethylation was detected in 50% of patients for E-cadherin, 43% for p16, 16% for p15, 15% for SHP1, 13% for ER and BNIP3, 12% for RARb, 6% for DAP-k, and 0% for MGMT. Overall, 54 patients (79%) presented at least one hypermethylated gene (1 in 19 patients, 2 in 17 patients, 3 in 12 patients, 4 in 5 patients, and 5 in 1 patient). By univariate analysis, hypermethylation of DAP-k (p

Description: 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.

Description: Hepatic sinusoidal obstruction syndrome (SOS) is a serious complication in hematopoietic stem cell transplant (HSCT) recipients. To determine the impact of pretransplantation hyperferritinemia on the risk of SOS after HSC transplantation, we retrospectively studied 427 HSCT recipients (179 autologous and 248 allogeneic). Serum ferritin levels were measured before transplantation. Patients with and without a diagnosis of SOS were compared regarding demographics; underlying disease; transplant characteristics; receipt of imatinib, busulfan, total body irradiation, gemtuzumab, vancomycin, acyclovir, or methotrexate; and baseline serum ferritin. Univariate and multivariate (stepwise logistic regression) analyses were performed. SOS was diagnosed in 88 patients (21%) at a median of 10 days (range, 2-29 days) after transplantation. By multivariate analysis, allogeneic HSC transplantation (odds ratio [OR] = 8.25; 95% confidence interval [95% CI], 3.31-20.57), receipt of imatinib (OR = 2.60; 95% CI, 1.16-5.84), receipt of busulfan (OR = 2.18; 95% CI, 1.25-3.80), and ferritin serum level higher than 1000 ng/dL (OR = 1.78; 95% CI, 1.02-3.08) were risk factors for SOS. A ferritin serum level higher than 1000 ng/dL in the pretransplantation period is an independent risk factor for SOS. The results suggest the need for prospective studies addressing the use of iron chelation in the pretransplantation period.

Description: Invasive fungal diseases (IFDs) represent an important cause of treatment failure in adults with acute leukemia. Because of leukemia’s heterogeneity, the risk for IFDs is highly variable. We therefore apply a risk-adapted antifungal strategy with strong emphasis on pretreatment and day-15 posttreatment to allow earlier and more individualized interventions. We determine pretreatment risks for IFDs based on 4 factors: (1) host fitness for standard therapy (ie, fit, unfit, or frail); (2) leukemia resistance (high vs low probability of achieving complete remission [CR]); (3) anticipated treatment-related toxicity such as neutropenia, mucositis, and steroid-induced immunosuppression; and (4) patient exposure to opportunistic fungi. Accordingly, we stratify patients as high, intermediate, or low risk for IFDs and apply risk-adapted antifungal strategies, including primary or secondary prophylaxis and diagnostic-based preemptive or empiric therapy. Prevention of IFDs also relies on optimizing organ function, decreasing exposure to opportunistic fungi, and improving net state of immunosuppression with use of better-tolerated and investigational agents for unfit patients and those with adverse leukemia biology. Novel targeted and safe therapies that can achieve higher rates of sustained CR among patients with adverse genetics offer the best promise for reducing the burden of IFDs in these patients.

Description: Background and Objective: Benign constitutional neutropenia (BCN) or benign ethnic neutropenia (BEN) is an asymptomatic condition characterized by mild chronic neutropenia in patients with no history of recurrent infections. In most of these patients neutropenia is discovered incidentally in a complete blood count ordered for routine screening. This is particularly concerning for general practitioners because neutropenia is associated with infections and hematologic malignancies. Thus, many patients are commonly referred to a specialist for evaluation, which leads to further testing and increased costs of care without evidence of improved personal or public health. To ensure that individuals with BCN are healthy, some diagnostic methods can be used to assess neutrophil mobilization from reserve compartments, including epinephrine, corticosteroids, endotoxins and vigorous exercise. We herein present an alternative form of assessment for individuals with BCN based on neutrophil circadian variation. Neutrophils in healthy individuals have a physiological increase of between 10 and 20% by early afternoon. In this study, we hypothesized that because patients with BCN have higher G-CSF basal levels and a greater physiological response to this cytokine, a larger increase in neutrophils can be detected in a blood test performed in the afternoon compared to routine tests performed in the morning. A physiological increase in neutrophil levels in individuals with BCN would be of great practical interest. This could reduce costs of laboratory tests and reduce medical and patient concerns. Methods: We studied patients who had neutropenia that was detected in routine blood count and with suspected BCN. Patients with clinical symptoms, laboratory test result changes and history of chronic, inflammatory, autoimmune or neoplastic diseases were excluded. Patients 〉60 years were excluded because they have a higher prevalence of myelodysplasia and other concomitant diseases. Neutrophil counts were evaluated in patients with BCN using paired blood counts performed in the early morning (between 7:00 and 9:00 am) and in the early afternoon (between 1:00 and 3:00 pm). The neutrophil counts were stratified into the following four groups: 〈 500, 500-1000, 1000-1500 and 1500-2000/µL. Results: We evaluated 157 patients with neutropenia and 118 individuals met the entry criteria. Their median age was 36 years (range, 3-60 years), and 41 (35%) were male. Median blood count results were as follows: white blood count, 2880/µL; hemoglobin, 12.9 g/dL; and platelets, 223,000/µL. The difference between the median neutrophil counts performed in the morning (1164/µL) and afternoon (2070/µL) was 904/µL (95% CI=770-1040; p

Description: Introduction: The prompt initiation of a betalactam antibiotic in febrile neutropenia (FN) is standard of care in patients with hematologic malignancies and hematopoietic cell transplant (HCT) recipients. The use of a glycopeptide is recommended by guidelines in situations such as suspected catheter-related, skin and soft tissue infection, pneumonia or hemodynamic instability, but the level of these recommendations is weak. The aim of this study was to evaluate the use of vancomycin in febrile neutropenia (FN) in four Brazilian centers in which more restrictive criteria for using vancomycin have been applied. Methods: We retrospectively reviewed all episodes of FN from 2013-2019 in four hospitals: 2 teaching public, 1 public and 1 private hospital, and looked at reasons for giving vancomycin and outcomes. Criteria for using vancomycin was infection by methicillin-resistant Gram-positive bacteria, skin or soft tissue infection and shock. Results: We recorded 461 episodes of FN in 312 patients. Median age was 50 years (17 - 86) and 61% were male. FN occurred after chemotherapy in 70% of cases and after HCT in 30% (allogeneic in 11.5%). The most frequent underlying diseases were acute myeloid leukemia (28%), non-Hodgkin's lymphoma (25%), acute lymphoid leukemia (16%) and multiple myeloma (13.4%). Colonization by methicillin-resistant Staphylococcus aureus (MRSA) was present in only 3%, and quinolone prophylaxis was given in 32% of episodes. According to international guidelines, vancomycin should have been started at first fever in 120 episodes (26%) but it was given in only 28 (6%). Reasons for use at first fever were skin or soft tissue infection (n=10), hypotension or shock (n=8), catheter-related infection (n=3), mucositis (n=1), and no apparent reason (n=6). Vancomycin was added after day 1 in 69 episodes (15%), for the following reasons: Gram-positive bacteremia (n=36), skin or soft tissue infection (n=7), mucositis (n=7), hypotension or shock (n=6), pneumonia (n=3) and no apparent reason (n=10). Bacteremia was documented in 143 episodes (31%): 81 by Gram-negative and 62 by Gram-positive bacteria. Bacteremia due to Staphylococcus aureus occurred in only 4 episodes (1 MRSA). The 15- and 30-day mortality was 5.4% and 9.8%, respectively, with no significant differences among private (7.6%) and public (11.3%) hospitals. Conclusions: Despite the lack of benefit of empiric vancomycin in FN either in terms of fever duration or mortality, vancomycin is still widely used in to manage FN in hematologic patients and HCT recipients, potentially increasing toxicity. In our hospitals vancomycin was given in a very low proportion of episodes, despite having classic indications for its use as per guidelines. Our findings highlight the importance of caution in adopting recommendations from guidelines without looking at local epidemiology especially if the level recommendation is weak. These measures have the potential to decrease coast and toxicity. Disclosures No relevant conflicts of interest to declare.

Description: 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.

Description: This is a retrospective analysis of 1084 patients who received an allogeneic HSCT in 10 Brazilian Centers, from February 1983 to March 2003, aiming to validate the EBMT risk score. Data from transplanted patients and donors regarding patients age, disease stage at transplantation, HLA full-match sibling donor or full-match unrelated donors, donor-recipient gender match and the interval from diagnosis to transplantation, were used as variables to calculate the EBMT risk score. This score was analyzed using Cox Proportional Hazards Model. The OS, DFS, TRM and relapse were analyzed using Kaplan-Meier and cumulative incidence, whenever appropriate. In all there were 647 (60%) males and 437 (40%) females, the median age was 32 years old (range 1 – 59); 898 (83%) were in chronic phase, 146 (13%) were in accelerated phase and 40 (4%) were in blast crisis; 151 (14%) were younger than 20 years old, 620 (57%) were between 20 and 40 and 313 (29%) were older than 40; 1025 (95%) received HLA full-match sibling transplant and only 59 (5%) received an unrelated transplant. Female donor to male recipient occurred in 283 (26%) transplants. The interval from diagnosis to transplantation was less than 12 months in 223 (21%) cases and greater in 861 (79%). The OS, DFS, TRM and, relapse were 49%, 50%, 45%, 25%, respectively. The risk score 0–1 occurred in 179 (17%), score 2 in 397 (37%), score 3 in 345 (32%), score 4 in 135 (12%), and score 5–6 in 28 (2%). The risk scores 0–1 and 2 did not show any difference in terms of OS (58% and 55%, respectively) but they were significantly better than scores 3 or more (score 3 – 44%, 4 – 36 % and, 5-6 - 27%, respectively) (P