Allogenic peripheral stem cell transplantation from HLA-matched related donors for adult sickle cell disease: remarkable outcomes from a single-center trial.

Adult patients with sickle cell disease (SCD) are highly susceptible to stem cell transplant complications, including drug toxicity, graft versus host disease (GVHD), and graft rejection due to SCD-related tissue damage, endothelial activation, and inflammation. The scarcity of compatible stem cells for transplantation further limits treatment options, with only 43 cases of adult allogeneic peripheral blood stem cell transplantation (allo-PSCT) from human leukocyte antigen (HLA)-identical sibling donors reported in the international registry for the period 1986-2013. Herein we report remarkable outcomes in a cohort of adult SCD patients who underwent allo-PSCT using a fludarabine (Flu), busulfan (Bu), and anti-T-cell lymphocyte globulin (ATG)-based conditioning regimen in combination with very low dose total body irradiation (TBI), followed by post-transplant cyclophosphamide (Cy) and sirolimus as GVHD prophylaxis. We performed a single-center, retrospective study consisting of 20 consecutive patients (mean age 33.4 years) who underwent allo-PSCT from HLA-matched related donors with a conditioning regimen of Flu 150/Bu 3.2/Cy 29/ATG 30 (Fresenius)/TBI 200 between September 2013 and September 2017. Data were validated by an independent data audit group of the affiliated JACIE-accredited transplantation center. All patients experienced a sustained donor cell engraftment. Full donor chimerism (total cell) occurred within 180 days in all patients. Mean duration of follow-up was 13.8 months (range: 0.3-50 months), with 12 (60%) patients completing 12 months. No non-relapse mortality or graft rejection occurred. Successful treatment was achieved without the presence of graft loss, grade III-IV acute GVHD, extensive chronic GVHD, or other major complications. Allo-PSCT in combination with Flu 150/Bu 3.2/Cy 29/ATG 30(Fresenius)/TBI 200- Cy/Sirolimus therapy yielded encouraging outcomes with no mortality and low incidence of GVHD. Further controlled studies will be necessary to compare transplant protocols and long-term outcomes.

Effectiveness of fludarabine- and busulfan-based conditioning regimens in patients with acute myeloblastic leukemia: 8-year experience in a single center.

OBJECTIVE: Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is a curative treatment for acute myeloblastic leukemia (AML). Because the conditioning regimen of busulfan plus cyclophosphamide carries significant risks of toxicity, we evaluated the factors affecting survival after fludarabine replacement instead of cyclophosphamide. METHODS: The study included 55 patients who underwent allo-HSCT for AML and received busulfan, fludarabine, and antithymocyte globulin (ATG). RESULTS: Forty-eight patients received a myeloablative regimen; 7 patients received a reduced-intensity conditioning regimen. The neutrophil and platelet engraftment times were 12 days (range 9 to 20) and 12 days (range 7 to 19), respectively. Graft-vs-host disease (GvHD) developed in 10% and 50% of the patients, respectively. Seven patients received donor lymphocyte infusion. Of them, 5 patients developed grade I or II GvHD, one grade IV GvHD. The median follow-up period was 20.6 months. The predicted progression-free survival (PFS) at 1 and 3 years after transplantation was 78% and 74%, respectively. The overall survival (OS) at 1, 3, and 5 years was 76%, 74%, and 62%, respectively. Treatment-related mortality (infection in 1 patient, GvHD in 2 patients) occurred in 3 patients (5.5%). Multivariate analysis revealed that OS and PFS were not influenced by age, dose of busulfan or ATG, or presence of cytomegalovirus antigenemia. Acute GvHD and pretransplantation minimal residual disease positivity negatively affected the transplant outcome. The presence of active disease at the time of transplantation was found as an independent risk factor for AML. CONCLUSIONS: Busulfan- and fludarabine-based conditioning regimens are effective for AML, and have acceptable toxicity, morbidity, and mortality.

Alterations of circulating endothelial cells after apheresis in patients with sickle cell disease: a potential clue for restoration of pathophysiology.

OBJECTIVES: The potential influence of automated red cell exchange (ARCE) on endothelial activation is not well established. This study was intended to assess whether ARCE influences circulating endothelial cells (CECs) in patients with sickle cell disease. BACKGROUND: Automated red cell exchange (ARCE) has been used to protect the patient from complications of sickle cell disease. However, the expected benefits vary in different patients. CECs reflect endothelial activation. We hypothesize that suppression of endothelial activation may be an important mechanism of ARCE. METHODS: The study included 20 patients with sickle cell disease who underwent 30 apheresis procedures. We used flow cytometry to directly compare pre- and post-apheresis CEC number (prior to ARCE and 5 days after ARCE) during the steady state and painful crisis. We also determined if independent variables (the level of plasma nitrite concentration, the percentage of circulating hemoglobin S, and painful crisis) significantly contributed to the CEC level. RESULTS: The mean CEC number decreased (P = 0.04), while progenitor CECs did not change in patients with sickle cell disease after ARCE compared with pre-ARCE values (P>0.05). Clinical factors such as the volume of replacement fluid and the citrate infusion rate did not correlate with post-apheresis CECsand progenitor CEC numbers. The independent variables were not significantly associated with CEC and progenitor CEC numbers. CONCLUSIONS: ARCE can alter the CEC number, suggesting the possibility of suppression of endothelial activation. This may highlight the efficacy of ARCE for prevention or management of sickle cell vaso-occlusive crisis.

Detecting methylation patterns of p16, MGMT, DAPK and E-cadherin genes in multiple myeloma patients.

Multiple myeloma (MM) is a B-cell neoplasia characterized by the clonal proliferation of plasma cells. Besides known genetic abnormalities, epigenetic changes are also known to effect MM pathogenesis. DNA methylation is an epigenetic mechanism that silences genes by adding methyl groups to cytosine-guanine dinucleotides at the promoter regions. In this study, the methylation status of four genes; p16, O6-methyl guanine DNA methyl transferase (MGMT), death-associated protein kinase (DAPK) and E-cadherin (ECAD); at the time of diagnosis was investigated using methylation-specific polymerase chain reaction (MS-PCR). In the 20 cases studied; methylation of the promoter regions of p16, MGMT, DAPK and ECAD genes was detected in 10%, 40%, 10% and 45% of the cases, respectively. In 65% (13/20) of cases, at least one of the genes studied had promoter methylation; while 35% of cases (7/20) had methylated promoters of more than one gene. There was a significant correlation between promoter hypermethylation of MGMT and the presence of extramedullary involvement; but for the other genes no correlation was found regarding disease properties like age, disease stage, clinical course and the presence of lytic bone lesions. Determining the methylation profiles of genes in MM, could lead to a new understanding of the disease pathogenesis and guide the assessment of treatment options.

A detachment technique based on the thermophysiologic responses of cultured mesenchymal cells exposed to cold.

Trypsinization has generally been used as a technique to detach adherent mesenchymal stromal cells (MSC). However, this technique involves chemical manipulation. This study was designed to identify whether detachment of MSC can be induced by cold without using trypsin. MSC isolated from bone marrow were detached via trypsin or exposed to -20 degrees C for 1, 5 or 10 min at all passages. Compared with trypsinization, exposing MSC to -20 degrees C for 10 min resulted in a significant decrease in MSC number and viability. In conclusion, although detachment of adhered MSC on culture dishes via exposure to cold may allow structurally and functionally intact detached cells, the technique requires improvement of the thermotolerance of MSC.

Cytogenetic findings and clinical outcomes of adult acute myeloid leukaemia patients.

The role of cytogenetic findings in determining the diagnosis, therapy and prognosis of acute myeloid leukaemia (AML) has become more valuable by the day. In this study, the results of conventional and molecular cytogenetic analyses and clinical outcomes of 66 AML patients of different subgroups aged between 16 and 82 were evaluated. Chromosomal abnormalities were detected in 17 (25.7%) patients cytogenetically at the time of diagnosis, whereas molecular cytogenetic abnormalities were detected in 21 (31.8%) patients by fluorescence in situ hybridisation (FISH). Thirty-eight (57.6%) patients had a normal karyotype. In 8 patients, we did not obtain suitable chromosomes for karyotype analysis and in 3 patients conventional cytogenetics were not requested by the physician. During clinical follow-up, 21 patients (31.8%) achieved complete remission (CR), 2 had partial remission (PR) (3.0%) and 4 patients had progressive disease (6.06%). Twenty-eight (42.4%) patients died during treatment and no follow-up data were available in 7 cases. Among patients with chromosome abnormalities detected by either one of the two cytogenetic methods (n=28), 12 had achieved CR, 9 of whom were already categorised in the good prognostic group with t(15;17), inv16 or t(8;21). As for the normal karyotype, each patient displayed a different clinical course, which is probably due to the molecular changes in leukaemia-related genes. Here we report our findings, which correlate with previous reports and conclude that cytogenetics is a crucial marker in leukaemia diagnosis and conventional and molecular cytogenetics should be performed as well as molecular genetic diagnostic methods.

Plasma exchange in critically ill patients with sickle cell disease.

Red cell exchange transfusion is the recommended therapy for patients with sickle cell disease who have complicated vaso-occlusive episodes. However, the role of the therapeutic plasma exchange in the management of the potentially life-threatening complications in patients with sickle cell disease is not well known. To determine whether plasma exchange had a cumulative effect on the red cell exchange in patients with sickle cell disease who developed multi-organ failure during the post red cell exchange period, we performed plasma exchange in the nine episodes of multi-organ failure of 7 patients with sickle cell anemia. The median age of those patients was 21 years (range, 9-50 years). The criterion of the multi-organ failure was defined as organ failure of two or more organs i.e. lung, liver, or renal, established according to Acute Physiological and Chronic Health Evaluation-II (APACHE-II) criteria. The average total plasma exchange volume was 1.0 times the patient's plasma volume. The patients had a good outcome, with a survival rate at 86% after one year of follow-up. Plasma exchange may have cumulative benefits in the treatment of severe illness in patients with sickle cell disease who underwent automatic red cell exchange therapy.

The first 2 years of clinical experience with peripheral blood stem cell transplantation for various hematological malignancies: results from a single Baskent University Center.

Autologous stem cell transplantation is the current standard approach for patients with multiple myeloma and relapsed or refractory lymphoma. Nonmyeloablative allogeneic stem cell transplantation has been applied worldwide. We analyzed the results of transplantation activity from 2004 to 2006. Seven evaluable patients younger than 65 years old with stage II/III multiple myeloma were treated with high-dose melphalan therapy (140 mg/m(2)) plus autologous peripheral blood stem cell transplantation. Complete responses or tumor reductions of more than 75% were obtained in all patients. At a median follow-up of 10 months, all patients remained disease-free. Four patients with acute myeloblastic leukemia underwent nonmyeloablative allogeneic peripheral stem cell transplantation. Their median age was 30 years. One patient was refractory and the others were in hematological remission. The patients received fludarabine-based preparative regimens. All patients received fully matched blood from a related donor 2 days after chemotherapy in conjunction with graft-versus-host disease prophylaxis. One refractory patient with >90% engraftment had late autologous reconstitution at 3 months with evidence of relapse. All other patients in remission remained with >90% donor cell engraftment. These patients are disease-free at 13, 10, and 2 months. Toxicity was minimal. These results showed promise due to the minimal toxicity observed with the conditioning regimens which indicated the feasibility of these procedures.

Thrombotic Thrombocytopenic Purpura with Reversible Neurological Features: Brain Diffusion MRI with ADC Map, Spect and EEG Findings. A Case Report.

Although nervous system involvement is common in thrombotic thrombocytopenic purpura (TTP), abnormalities on computerized tomography, magnetic resonance imaging and electroencephalography are not encountered so frequently and if present, these abnormalities are often reversible. We describe a 39-year-old woman with recurring transient focal neurological findings found to have laboratory findings consistent with TTP. In cerebral diffusion weighted images (DWI), diffuse cortical hyperintensity was noted in right frontal lobe, but the ADC (apparent diffusion coefficient) map was normal. Electroencephalography demonstrated lateralized slowing and repeated DWI showed diffuse cortical hyperintensity in the right hemisphere. SPECT showed luxury perfusion in the right hemisphere areas. The patient's condition resolved with plasmapheresis. Our patient illustrates that diffuse hemispheric involvement can be seen in DWI and EEG, and SPECT may show luxury perfusion after resolution of neurological findings in TTP cases. To our knowledge, this is the first TTP case in which the ADC map was normal.

Conventional and molecular cytogenetic findings of myelodysplastic syndrome patients.

Myelodysplastic syndrome (MDS) involves myeloid cells of the bone marrow, which is important in progressive bone marrow insufficiency. Of all MDS patients, 40%-50% have at least one chromosomal rearrangement. Loss of specific chromosomal regions like 5q- and 7q- are usually the secondary cytogenetic abnormalities associated with MDS. In order to detect chromosome abnormalities associated with MDS, bone marrow samples from 26 patients diagnosed as MDS were obtained prior to chemotherapy. Both conventional cytogenetic analyses and fluorescence in situ hybridisation (FISH) methods were performed and locus-specific probes for 5q and 7q were used. Results obtained were compared. Twenty-one patients had normal karyotypes and four patients had abnormal karyotypes, while in one patient we could not obtain metaphases from cultures. Three patients with normal karyotypes revealed del (5q), two patients had del (7q) and one patient had monosomy (7). A total of 10 of 26 patients had chromosome changes visualised by either conventional or molecular cytogenetics (approximately 38.5%). Our results show that both methods are important in diagnosis and follow up of MDS patients. When used together, conventional cytogenetics and FISH detect clinically significant chromosome abnormalities in MDS patients.

Erythrocyte membrane ATPase activity of G6PD-deficient individuals and the effect of primaquine metabolite(s) on membrane ATPase enzymes.

Erythrocyte membrane Na+/K+, Ca2+/Mg2+ and Mg2+ ATPase activities in addition to the calmodulin-activated Ca2+/Mg2+ ATPase enzyme were measured in both G6PD-deficient and normal individuals. Although all three membrane ATPase activities were somewhat higher in the G6PD-deficient erythrocytes, only activated Ca2+/Mg2+ ATPase activity was significantly increased. The effect of primaquine on the membrane ATPases was also compared with other ATPase inhibitors. Primaquine was ineffective on erythrocyte membrane ATPase in-vitro. However, sera containing primaquine metabolite(s) were inhibitory to Ca2+/Mg2+ and Mg2+ ATPase systems of only G6PD-deficient erythrocytes. Other ATPase inhibitors showed a similar inhibitory effect in G6PD-deficient and normal erythrocytes, indicating a specific influence of primaquine on ATPase system in G6PD deficiency. It is suggested that this effect of primaquine may be an additional factor for haemolysis observed in the people with GdB- type of G6PD deficiency among the Mediterranean populations.