Fanconi anemia is a rare hereditary disease showing genetic heterogeneity due to a variety of mutations in genes involved in DNA repair pathways, which may lead to different clinical manifestations. ...Phenotypic variability makes diagnosis difficult based only on clinical manifestations, therefore laboratory tests are necessary. New advances in molecular pathogenesis of this disease led researchers to develop a diagnostic test based on Western blot for FANCD2. The objective of the present study was to determine the efficacy of this method for the diagnosis of 84 Brazilian patients with Fanconi anemia, all of whom tested positive for the diepoxybutane test, and 98 healthy controls. The FANCD2 monoubiquitinated isoform (FANCDS+/FANCD2L-) was not detected in 77 patients (91.7%). In 2 patients (2.4%), there was an absence of both the monoubiquitinated and the non-ubiquitinated proteins (FANCD2S-/FANCD2L-) and 5 patients (5.9%) had both isoforms (FANCD2S+/FANCD2L+). This last phenotype suggests downstream subtypes or mosaicism. All controls were diepoxybutane negative and were also negative on the FANCD2 Western blot. The Western blot for FANCD2 presented a sensitivity of 94% (79/84) and specificity of 100% (98/98). This method was confirmed as an efficient approach to screen Brazilian patients with deleterious mutations on FANCD2 (FANCD2S-/FANCD2L-) or other upstream genes of the FA/BRCA pathway (FANCDS+/FANCD2L-), to confirm the chromosome breakage test and to classify patients according to the level of FA/BRCA pathway defects. However, patients showing both FANCD2 isoforms (FANCD2S+/FANCD2L+) require additional studies to confirm mutations on downstream Fanconi anemia genes or the presence of mosaicism.
We transplanted 47 patients with Fanconi anemia using an alternative source of hematopoietic cells. The patients were assigned to the following groups: group 1, unrelated bone marrow (N = 15); group ...2, unrelated cord blood (N = 17), and group 3, related non-sibling bone marrow (N = 15). Twenty-four patients (51%) had complete engraftment, which was not influenced by gender (P = 0.87), age (P = 0.45), dose of cyclophosphamide (P = 0.80), nucleated cell dose infused (P = 0.60), or use of anti-T serotherapy (P = 0.20). Favorable factors for superior engraftment were full HLA compatibility (independent of the source of cells; P = 0.007) and use of a fludarabine-based conditioning regimen (P = 0.046). Unfavorable factors were > or = 25 transfusions pre-transplant (P = 0.011) and degree of HLA disparity (P = 0.007). Intensity of mucositis (P = 0.50) and use of androgen prior to transplant had no influence on survival (P = 0.80). Acute graft-versus-host disease (GVHD) grade II-IV and chronic GVHD were diagnosed in 47 and 23% of available patients, respectively, and infections prevailed as the main cause of death, associated or not with GVHD. Eighteen patients are alive, the Kaplan-Meyer overall survival is 38% at approximately 8 years, and the best results were obtained with related non-sibling bone marrow patients. Three recommendations emerged from the present study: fludarabine as part of conditioning, transplant in patients with < 25 transfusions and avoidance of HLA disparity. In addition, an extended family search (even when consanguinity is not present) seeking for a related non-sibling donor is highly recommended.
Terbutryn is a widely used preemergence and postemergence
s-triazine herbicide. This pesticide is used in agriculture as a control agent for most grasses and many annual broadleaf weeds in cereal and ...legume fields, and under fruit trees. Unexpectedly, this compound was found to persist in the environment (240 and 180 days in pond and river sediment, respectively) and to have the tendency to move from treated soils to water compartments through water runoff and leaching. However, only scant information is available about the genotoxic properties of terbutryn. In the present in vitro study, we investigated the relationship between cytogenetic damage, as evaluated in the sister-chromatid exchange (SCE) assay and the micronucleus (MN) test, and primary DNA damage (as evaluated by the “comet” assay). Cytogenetic and primary DNA damage were recorded in vitro in freshly isolated human peripheral blood leukocytes. Our results showed that the tested compound failed to produce any significant increases in SCE or MN, neither in the absence nor in the presence of S9-mix. However, terbutryn was found to induce primary DNA damage, more pronounced without S9 mix, even though in the absence of a clear trend for dose-dependence and in the presence of a concomitant mild cytotoxic effect.
: Toxoplasma infection following bone marrow transplantation (BMT) is infrequently reported. We report 9 cases of disseminated Toxoplasma gondii infection in BMT recipients documented during an ...11‐year period at our institution. The incidence of T. gondii infection in our institution (1.14 per 100 allogeneic BMT) is higher than previously reported. The most frequently affected sites were the brain, lungs, and heart. Findings common to most patients who developed toxoplasmosis were positive pre‐transplant serology, allogeneic transplant and graft‐versus‐host disease and its treatment, as well as BMT from matched unrelated donors. All 9 patients died and 8 were diagnosed only after autopsy. Heightened awareness of the occurrence of toxoplasmosis in marrow recipients, especially in highly endemic areas, and early diagnosis and therapy are needed for a better outcome.
Background
Telomere biology diseases (TBD) result from defective telomere maintenance, leading to bone marrow failure. The only curative treatment for aplastic anemia related to TBD is a ...hematopoietic cell transplant (HCT). Although reduced‐intensity conditioning (RIC) regimens decrease transplant‐related mortality, non‐hematological phenotypes represent a major challenge and are associated with poor long‐term follow‐up outcomes.
Objective
To describe the outcome of TBD patients transplanted for marrow failure.
Study Design
This is a retrospective, single‐center study describing the outcomes of 32 consecutive transplants on 29 patients between 1993 and 2019.
Results
The median age at transplantation was 14 years (range, 3–30 years). Most patients received a RIC regimen (n = 28) and bone marrow (BM) from an unrelated donor (n = 16). Four patients received a haploidentical transplant. Chimerism was available for 27 patients with a median time to neutrophil recovery of 20 days (13–36 days). Primary graft failure occurred in one patient, whereas second graft failure occurred in two. Acute GVHD grade II‐IV and moderate to severe chronic GVHD occurred in 22% of patients at risk. Fourteen patients were alive after HCT at the last follow‐up (median, 6 years; 1.4–19 years). The 5‐year overall survival was better after matched sibling donor (MSD) transplantation compared to other hematopoietic stem cell sources (88.9% vs. 47.7%; p = .05; CI = 95%). Overall, 15 patients died after HCT, most of them (n = 11) after the first year of transplant, due to non‐hematological disease progression or complication of chronic GVHD.
Conclusions
Hematopoietic cell transplantation is a potentially curative treatment option for TBD, nonetheless the poor outcome reflects the progression of non‐hematologic disease manifestations, which should be considered when transplantation is indicated.
The generation, transmission (e.g. power lines, transformers, service wires, and electrical panels), and use (e.g. home appliances, such as electric blankets, shavers, and televisions) of electrical ...energy is associated with the production of weak electric and magnetic fields (EMF) which oscillate 50 (Europe) or 60 (USA) times per second (power-line frequency), falling in the extremely-low frequency (ELF) region of the electromagnetic spectrum. Epidemiological reports suggest a possible association between exposure to ELF-EMF and an increased risk of cancer (e.g. childhood acute leukaemia). Benzene is an established human leukomogen. This xenobiotic, which is unlikely to be the ultimate carcinogen, is metabolized in the liver to its primary metabolite phenol, which is hydroxylated to hydroquinone (1,4-benzenediol) and 1,2,4-benzenetriol. In this in vitro approach, to test the genotoxic and / or co-genotoxic potency of ELF-EMF, the cytokinesis block micronucleus (MN) method with Jurkat cells has been used. A 50 Hz magnetic field (MF) of 5 mT field strength was applied for different length of time (from 1 to 24 h), either alone or with benzene, 1,4-benzenediol, or 1,2,4-benzenetriol. Our preliminary results show that, after 24 h exposure, the frequency of micronucleated cells in MF-exposed cultures is 1.9 fold higher than in sham-exposed (control) cultures. Benzene exposure does not show any cytogenetic activity, whereas 1,4-benzenediol or 1,2,4-benzenetriol alone significantly affect the number of MN in Jurkat cells, as compared to untreated cultures. Moreover, co-exposure to ELF-MF does not seem to affect the frequency of micronuclei induced by benzene, 1,4-benzenediol, or 1,2,4-benzenetriol.
Fanconi anemia (FA) is an autosomal recessive genetic disease characterized by progressive bone marrow failure, susceptibility to cancer and multiple congenital anomalies. There is important clinical ...variability among patients and the knowledge of factors which might predict outcome would greatly help the decision making regarding the choices of treatment and the appropriate time to start it. Future studies of the possible correlation between specific mutations with specific clinical presentations will provide the answer to one of these factors. At our Center we standardized a rapid and precise screening test using a mismatch PCR assay for a specific mutation (3788-3790del in exon 38 of gene FANCA) in Brazilian FA patients. We present the results obtained after screening 80 non-consanguineous FA patients referred from all regions of Brazil with a clinical diagnosis of FA supported by cellular hypersensitivity to diepoxybutane. We were able to detect the 3788-3790del allele in 24 of the 80 (30%) FA patients studied. Thirteen of the 80 (16.25%) were homozygotes and 11 of the 80 (13.75%) were compound heterozygotes, thus confirming the high frequency of the FANCA 3788-3790del mutation in Brazilian FA patients. The identification of patients with specific mutations in the FA genes may lead to a better clinical description of this condition, also providing data for genotype-phenotype correlations, to a better understanding of the interaction of this specific mutation with other mutations in compound heterozygote patients, and ultimately to the right choices of treatment for each patient with improvement of the prognosis on future studies.
Minimal residual disease (MRD) measurement in B-cell precursor acute lymphoblastic leukemia (BCP-ALL) has been consolidated in the literature as the strongest adverse prognostic factor for overall ...and disease-free survival in both child and adult studies. However, only recently has multiparametric flow cytometry (MFC) been fully standardized with the addition of new markers and acquisition of a higher number of cellular events, which can improve the objectivity and sensitivity of the test.
This prospective study aims to test a single 10-color flow cytometric assay compared to standardized 8-color two tubes in patients with BCP-ALL before bone marrow transplantation, follow by analysis of the kinetic of medullary B-cell recovery after transplant.
Prospective analysis of 75 consecutive patients (pcts) transplanted between 2019/January and 2022/May, with MRD evaluation in the month before HSCT, day30, day100 and day360. Flow cytometry was performed on bone marrow samples after bulk-lysis with ammonium chloride when necessary, and subsequent staining with antibody panels. A 10-color tube included the backbone markers CD19, CD45, CD34, CD10, and CD20, in addition to CD38 and CD81 to improve separation between normal/reactive and malignant BCP, and four discriminatory markers placed in the same tube (CD66c and CD123 in PE, CD73 in BV605, and CD304 in APC-R700). This tube was compared to the standardized EuroFlow®method, which was composed of two 8-color tubes previously described. FACSCantoII™and FACSLyric™flow cytometry's were used. Data were analyzed with Infinicyt. The achieved sensitivity and percentages of lymphoblasts, normal mature and precursor B-cells, plasmatic cells and stromal cells were compared before and after transplantation. Statistical analyses were performed in SPSS Statistics v.20.0 software.
34 ALL (42.6%), being 26 BCP-ALL and 8 high-risk T-ALL, and 41 AML (57.4%), median age 31.6 (4.1-62.5 years) for ALL and 42.9 (0.6-74.2 years) for AML; 45(60%) in first complete remission (CR1), 21 CR2 (28%), one CR3 and 7 active diseases. Among 64 patients in morphologic remission, we found 38(59.4%) MRD negative and 26(40.6%) MRD positive before transplant. Pos-transplantation MRD and maturation analysis was possible in 26 pcts at d30, 24 at d60, 57 at d100 and 18 at d360. All but two samples had more than 9.000.000 cell events acquired, median lower limit of detection (LoD) was 0,0002% and lower limit of quantification (LoQ) was 0,0005% in both assays. All patients had the same percentages of lymphoblasts, normal mature and precursor B-cells, plasmatic cells, in the two different assays. The posttransplant B-cell recovery kinetic was similar between BCP-ALL, T-cell ALL and myeloid leukemia recovery after transplantation, and different between adults and children at day100 and day360, but not in day30.
This study showed that this 10-color single tube strategy achieves the same sensitivity as the 8-color assay and allows correct identification of MRD, achieving maximum sensitivities and specificities in a single tube, and still maintaining the best discrimination between pathological B cells and normal B cell precursors. B-cell maturation kinetics were similar between leukemia types, and slightly different between adults and children. Additional studies and a larger sample size are needed to confirm these findings.
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