Chromosomal aberrations in ascetic fluid of metastatic gastric cancer patients: A clustering analysis.

To date, no specific pattern of chromosomal abnormalities has been established in gastric cancer (GC). Cytogenetic analysis was performed using G-banding and fluorescence in situ hybridization (FISH) in 9 ascetic fluids from GC patients, and the clustering patterns of chromosomal abnormalities were studied. Twenty-six different types of chromosomal abnormalities were identified. In contrast to structural abnormalities, the gain or loss of chromosomes was infrequent. Moreover, five main clusters of chromosomal abnormalities were identified by clustering analysis. Extensive cytogenetic complexity, specific chromosomal abnormalities and karyotype heterogeneity are the main characterizations of GC. Some of the recurrent and novel chromosomal abnormalities with distinct clustering patterns identified in this study may play important roles for GC initiation and progression and could serve as promising diagnostic and prognostic markers in GC patients.

Comprehensive mutational analysis of primary and relapse acute promyelocytic leukemia.

Acute promyelocytic leukemia (APL) is a subtype of myeloid leukemia characterized by differentiation block at the promyelocyte stage. Besides the presence of chromosomal rearrangement t(15;17), leading to the formation of PML-RARA (promyelocytic leukemia-retinoic acid receptor alpha) fusion, other genetic alterations have also been implicated in APL. Here, we performed comprehensive mutational analysis of primary and relapse APL to identify somatic alterations, which cooperate with PML-RARA in the pathogenesis of APL. We explored the mutational landscape using whole-exome (n=12) and subsequent targeted sequencing of 398 genes in 153 primary and 69 relapse APL. Both primary and relapse APL harbored an average of eight non-silent somatic mutations per exome. We observed recurrent alterations of FLT3, WT1, NRAS and KRAS in the newly diagnosed APL, whereas mutations in other genes commonly mutated in myeloid leukemia were rarely detected. The molecular signature of APL relapse was characterized by emergence of frequent mutations in PML and RARA genes. Our sequencing data also demonstrates incidence of loss-of-function mutations in previously unidentified genes, ARID1B and ARID1A, both of which encode for key components of the SWI/SNF complex. We show that knockdown of ARID1B in APL cell line, NB4, results in large-scale activation of gene expression and reduced in vitro differentiation potential.

Autologous transplant remains the preferred therapy for relapsed APL in CR2.

Despite their favorable prognosis, 10-20% of acute promyelocytic leukemia (APL) patients relapse. Reinduction therapy is often followed by autologous hematopoietic cell transplantation (auto-HCT). Arsenic trioxide (ATO) has become part of standard reinduction and is often followed by auto-HCT. Data on patients in CR2 were collected from two large transplant registries (Center for International Blood and Marrow Transplant Research (CIBMTR) and European Group for Blood and Marrow Transplant (EBMT)) and two specialty referral centers. The outcome of patients in CR2 who received only ATO-based therapy as reinduction was retrospectively compared with those who got an auto-HCT, with or without ATO. Prognostic factors included age, disease risk, extramedullary disease and duration of CR1. Of 207 evaluable patients, the median age was 31.5 years, 15.3% had extramedullary disease and median WBC at diagnosis was 4.8 × 10(9)/L. Sixty-seven patients received ATO alone and 140 underwent auto-HCT. The groups were comparable for age, gender, extramedullary disease, risk group and duration of CR1. At 5 years, overall survival (OS) was 42% and 78% for the ATO-only and auto-HCT groups, respectively (P<0.001). In addition, OS was associated with longer duration of CR1 (P=0.002), but not with disease risk at diagnosis. These data suggest that auto-HCT for APL patients in CR2 results in better OS than ATO-based therapy alone.

Expression analysis of microRNA-125 in patients with polycythemia vera and essential thrombocythemia and correlation with JAK2 allele burden and laboratory findings.

INTRODUCTION: The JAK2V617F mutation has emerged in recent years as a diagnostic as well as a treatment target in patients with polycythemia vera (PV) and essential thrombocythemia (ET). The disease phenotype is also influenced by other factors such as microRNA (miRNA) deregulation. The aim of this study was to investigate miR-125 expression level in these patients with those obtained from healthy control subjects and its correlation with JAK2 allele burden and laboratory findings. METHODS: In total, forty patients with a clinical diagnosis of PV and ET were examined at the time of diagnosis. Ten healthy subjects were checked as controls. We performed JAK2 V617F allele burdens measurement and expression analysis of miR-125b-5p, miR-125b-3p, miR-125a-5p, and miR-125a-3p in leukocytes isolated from peripheral blood by quantitative real-time polymerase chain reaction. RESULTS: MiR-125b-5p and miR-125a-5p were upregulated in both patients with PV (P = 0.00 and P = 0.003, respectively) and ET (P = 0.02 and P = 0.002, respectively). In PV group, a significant correlation was observed between miR-125a-5p and platelet counts (P = 0.01, r = 0.531). The correlation between miRNA and JAk2 allele burden was not significant. CONCLUSION: In conclusion, our data indicate that other factors such as aberrant miR-125 expression may influence on the disease phenotype in patients with PV and ET.

Low frequency of c-MPL gene mutations in Iranian patients with Philadelphia-negative myeloproliferative disorders.

BACKGROUND: Myeloproliferative disorders are a group of diseases characterized by increased proliferation of myeloid lineage. In addition to JAK2V617F mutation, several mutations in the c-MPL gene have been reported in patients with philadelphia-negative chronic myeloproliferative disorders that could be important in the pathogenesis of diseases. The aim of the present study was to investigate the frequency of c-MPL and JAK2V617F mutations in Iranian patients with Philadelphia-negativemyeloproliferative disorders. MATERIAL AND METHODS: Peripheral blood samples were collected from 60 patients with Philadelphia-negative MPD) Subgroups ET and PMF) and 25 healthy subjects as control group. The mutation status of c-MPL and Jak2V617F were investigated by using Amplification-refractory mutation system (ARMS) and Allele-Specific PCR (AS-PCR), respectively. The results were confirmed by sequencing. RESULTS: Among 60 patients, 34 (56.6%) and 1(1.7%) had Jak2V617F and c-MPL mutation, respectively. Patients with Jak2V617F mutation had higher WBC counts and hemoglobin concentration than those without the mutation (p= 0.005, p=0.003). In addition, for all healthy subjects in control group, mutations were negative. CONCLUSIONS: The present study revealed that the c-MPL mutations unlike the Jak2V617F mutations are rare in Iranian patients with Ph-negative MPNs and the low mutation rate should be considered in the design of screening strategies of MPD patients.

Hematopoietic SCT in Iranian children 1991-2012.

This study presents the pediatric hematopoietic SCT (HSCT) activity in Iran between 1991 and 2012. Overall, 1105 fifteen-year-old or younger patients have undergone HSCT (975 allogeneic and 130 autologous). Annual HSCTs have been increasing steadily since 2007. HLA-matched siblings and other related donors were the main source of HSCs, although since 2008 a national HLA registry has been established to fill the gap for patients lacking a related donor. Inherited abnormalities of RBCs (45.88%), leukemias (27.6%) and BM failure syndromes (11.94%) constituted the majority of HSCTs during this period. Two-year overall survival and disease-free survival rates for all patients were 74.2% (95% confidence interval (CI): 71.6-77) and 66.3% (95% CI: 63.5-69.3), respectively. Leading cause of death in allogeneic group was TRM (165 deaths) and relapse caused the majority of deaths in the autologous group (39 deaths). All HSCTs from the beginning have been performed exclusively with TBI-free-conditioning regimens, which provides unique data for comparison with activities of other centers. Encouraging survival rates provide a basis for future studies on the extensive applicability of TBI-free-conditioning regimens in pediatric HSCT.

Modified protocol for improvement of differentiation potential of menstrual blood-derived stem cells into adipogenic lineage.

OBJECTIVES: To characterize potency of menstrual blood-derived stem cells (MenSCs) for future cell therapies, we examined differentiation potential of MenSCs into adipocytes. MATERIALS AND METHODS: Differentiation potential of MenSCs in comparison to bone marrow stem cells (BMSCs) was assessed in conventional culture medium. Differentiation potential of MenSCs into adipocytes was improved using different combinations of growth factors and hormones. RESULTS: First, we demonstrated that MenSCs preserve their appearance and karyotypic stability during passages. Although these cells express mesenchymal stem cells markers, they cannot simply be classified as mesenchymal stem cells due to expression of embryonic stem cells marker, OCT-4. Oil red O staining showed that differentiated MenSCs in conventional medium with/without retinoic acid (protocols 1 and 2) did not attain adipocyte characteristics, whereas differentiated BMSCs in conventional medium accumulated oil vacuoles typically. Nevertheless, real-time RT-PCR results showed that LPL gene expression was up-regulated in both protocols 1 and 2, whereas LEPR was up-regulated only in protocol 2 (fortified with retinoic acid). Surprisingly, protocol 3 (including rosiglitazone) had odd influence on mRNA expression of all genes (LEPR, LPL and PPAR-γ). Oil red O staining confirmed fat-producing ability of MenSCs under protocol 3. CONCLUSIONS: Presented data suggest an efficient differentiation protocol for in vitro production of MenSC-derived adipocytes. These cells are suggested to be an apt alternative to BMSCs for future stem cell therapy of soft tissue injuries.

Reduced intensity conditioning is effective for hematopoietic SCT in dyskeratosis congenita-related BM failure.

BM failure (BMF) is a major and frequent complication of dyskeratosis congenita (DKC). Allogeneic hematopoietic SCT (allo-HSCT) represents the only curative treatment for BMF associated with this condition. Transplant-related morbidity/mortality is common especially after myeloablative conditioning regimens. Herein, we report nine cases of patients with DKC who received an allo-SCT at five different member centers within the Eastern Mediterranean Blood and Marrow Transplantation Registry. Between October 1992 and February 2011, nine DKC patients (male, 7 and female, 2), with a median age at transplantation of 19.1 (4.9-31.1) years, underwent an allo-HSCT from HLA-matched, morphologically normal-related donors (100%). Preparative regimens varied according to different centers, but was reduced intensity conditioning (RIC) in eight patients. Graft source was unstimulated BM in five cases (56%) and G-CSF-mobilized PBSCs in four (44%) cases. The median stem cell dose was 6.79 (2.06-12.4) × 10(6) cells/kg body weight. GVHD prophylaxis consisted of CsA in all nine cases; MTX or mycophenolate mofetil were added in five (56%) and two (22%) cases, respectively. Anti-thymocyte globulin was administered at various doses and scheduled in four (44%) cases. Median time-to-neutrophil engraftment was 21 (17-27) days. In one case, late graft failure was noted at 10.4 months post allo-HSCT. Only one patient developed grade II acute GVHD (11%). Extensive chronic GVHD was reported in one case, whereas limited chronic GVHD occurred in another four cases. At a median follow-up of 61 (0.8-212) months, seven (78%) patients were still alive and transfusion independent. One patient died of metastatic gastric adenocarcinoma and graft failure was the cause of death in another patient. This study suggests that RIC preparative regimens are successful in inducing hematopoietic cell engraftment in patients with BMF from DKC. Owing to the limited sample size, the use of registry data and heterogeneity of preparative as well as GVHD prophylaxis regimens reported in this series, we are unable to recommend a particular regimen to be considered as the standard for patients with this disease.

Therapeutic outcomes of transplanting autologous granulocyte colony-stimulating factor-mobilised peripheral mononuclear cells in diabetic patients with critical limb ischaemia.

The efficacy and safety of transplanting autologous mesenchymal stem cells (MSCs), from granulocyte-colony-stimulating factor (G-CSF)-mobilised peripheral blood, was investigated in diabetic patients with critical limb ischaemia (CLI). After 3 months, the transplanted group of patients (n=7) showed a significant improvement in ischaemia manifestations, including pain and neurological signs, wound healing and the rate of lower-limb amputation, compared to the control group of patients (n=14). Pain was significantly reduced in the transplanted group compared to controls (P=0.014). The ankle-brachial index (ABI) and the pulse strength within ischaemic tissues of the transplanted group were significantly improved (P=0.035 and P=0.01, respectively). Importantly, 50% of the control group (7/14 patients) faced major amputation of a limb at the study's conclusion, compared to none of 7 patients in the transplanted group (P=0.047). The safety of transplantation was confirmed by observing no adverse reactions among the transplanted group, including infection and immunological rejection. Hence, this study provides further evidence that transplantation of autologous peripheral blood MSCs, mobilised by G-CSF, induces angiogenesis and improves the wound healing process in diabetic patients with CLI.

Allogeneic matched-sibling hematopoietic cell transplantation for AML: comparable outcomes between Eastern Mediterranean (EMBMT) and European (EBMT) centers.

Allogeneic hematopoietic cell transplantation (HCT) activity significantly increased in the Eastern Mediterranean area over the past decade. However, comparative outcomes with longer established centers, especially European Blood and Marrow Transplantation (EBMT) centers, have not been reported. We compared outcomes of matched-sibling allogeneic HCT between East Mediterranean Blood and Marrow Transplantation (EMBMT) and EBMT centers for adult patients with AML in first CR using myeloablative conditioning. We matched 431 patients from EMBMT with 431 patients from EBMT centers according to patient, disease and transplant characteristics. EMBMT recipients and donors were more likely to be CMV seropositive. There were no significant differences in the incidence of acute or chronic GVHD, or the 3-year cumulative incidence of non-relapse mortality (NRM) and relapse incidence (RI) between the two groups (NRM: EMBMT=16% vs EBMT=11), (RI: EMBMT=13% vs EBMT=19%). Notably, the 3-year leukemia-free survival (LFS) and OS were similar between the groups (LFS: EMBMT=70±2% vs EBMT=69±3%), (OS: EMBMT=74±2% vs EBMT=73±2%). Despite differences in socioeconomics, health resources and transplant experience, matched-sibling allogeneic HCT outcomes in emerging centers in the EMBMT region appear similar to EBMT centers.

Direct short-term cytotoxic effects of BIBR 1532 on acute promyelocytic leukemia cells through induction of p21 coupled with downregulation of c-Myc and hTERT transcription.

Acute promyelocytic leukemia (APL) is characterized by specific t(15;17), distinct morphologic picture, and clinical coagulopathy that contribute to the morbidity and mortality of the disease. This study aims to investigate the effects of antitelomerase compound BIBR1532 on APL cells (NB4). BIBR 1532 exerts a direct short-term growth suppressive effect in a concentration-dependent manner probably through downregulation of c-Myc and hTERT expression. Our results also suggest that induction of p21 and subsequent disturbance of Bax/Bcl-2 balanced ratio as well as decreased telomerase activity may be rational mechanisms for the potent/direct short-term cytotoxicity of high doses of BIBR1532 against NB4 cells.

Reduced-intensity conditioning hematopoietic SCT for pediatric patients with LAD-1: clinical efficacy and importance of chimerism.

Pediatric patients with leukocyte adhesion deficiency type-I (LAD-I) experience severe and recurrent life-threatening bacterial infections with failure of pus formation and delayed wound healing. LAD-I is a rare inherited disease caused by mutation in the leukocyte CD18 integrin expression, resulting in defective adherence and migration of leukocytes, in particular neutrophilic granulocytes through the intravascular space. Hematopoietic SCT is the only curative treatment option available to patients with LAD-I. Since 2007, in a prospective trial, reduced-intensity conditioning regimen have been developed for 10 consecutive patients with LAD-I who were referred to our center. Based upon available data, it is the first time that such a number of patients affected by LAD-I have been treated with this regimen. This study attempts to show that reduced-intensity regimen leads to a favorable result in LAD-I patients even in those who have experienced comorbid complications. Following transplantation, some patients develop mixed chimerism, however, in our study mixed chimerism was not followed by transplant rejection.

High expression of Fas ligand on cord blood dendritic cells: a possible immunoregulatory mechanism after cord blood transplantation.

BACKGROUND: Allogeneic cord blood transplantation is associated with less severe graft-versus-host disease (GVHD). Dendritic cells (DCs), as the most potent antigen-presenting cells of the immune system, play a central role in the development of GVHD. Because apoptosis induction is one of the known mechanisms that DCs use to regulate T-cell responses, we studied the immunostimulatory and apoptosis induction capacities of cord blood dendritic cells (CBDCs) and peripheral blood dendritic cells (PBDCs) to evaluate the mechanisms underlying the lower incidence of GVHD after cord blood transplantation. Presence of apoptosis-related markers Fas, Fas ligand (FasL), and CD40 and costimulatory molecules, along with the proportion of myeloid and lymphoid DCs subsets, were also measured on CBDCs and PBDCs. METHODS: Fresh CBDCs and PBDCs were isolated from cord and peripheral mononuclear cells as lineage-negative cells by using monoclonal antibodies against CD3, CD11b, CD14, CD16, CD19, CD56, CD34, and CD66b. DCs were cocultured with allogeneic T cells, and the effect of CBDCs and PBDCs on T-cell apoptosis and proliferation were determined through flow cytometric analysis and 3H-thymidine incorporation. RESULTS: Our findings showed that CBDCs markedly augment apoptosis of CD3+ T-cells. FasL expression on CBDCs was significantly higher than on PBDCs. However, there was no difference between Fas expression on CBDCs and PBDCs. Moreover, CBDCs were poor stimulators of allogenic T cells in mixed leukocyte reaction compared with adult peripheral blood DCs. They also displayed decreased expression of HLA-DR and CD86 molecules. The ratio of lymphoid DCs (CD11c-, CD123+) to myeloid DCs (CD11c+, CD123-) was also significantly higher in CBDCs compared with PBDCs. CONCLUSIONS: It seems that less severe GVHD after cord blood transplantation is due not only to a higher degree of immaturity of CBDCs, but also to delivery of apoptotic signals to the host T cells that recognize allo-MHC molecules on CBDCs in the early phase of immune response.

The detection of disseminated tumor cells in bone marrow and peripheral blood of gastric cancer patients by multimarker (CEA, CK20, TFF1 and MUC2) quantitative real-time PCR.

OBJECTIVE: To investigate the suitability of multimarker detection of DTCs in PB and BM of GC patients. DESIGN AND METHOD: A qRT-PCR assay was developed to estimate the number of CEA, CK20, TFF1 and MUC2 transcripts in PB and BM samples of 35 GC patients prior to the initiation of therapy. PB samples from healthy volunteers and BM from patients with hematological malignancies were used as negative controls. RESULTS: In PB analysis; 22.9%, 37.1%, 31.4%, and 22.9% of GC patients and in BM analysis; 20%, 28.6%, 45.7%, and 22.9% of GC patients were positive for CEA, CK20, TFF1 and MUC2 mRNAs, respectively. Samples from the control group were negative for the expression of all the markers tested in this study. A higher positive ratio was obtained with the multimarker detection in comparison to the single marker detection. There was a significant correlation between the PB and BM samples for DTC detection. CONCLUSION: Multimarker detection assay is a reliable and powerful tool for the early detection of DTCs in GC patients.

Population pharmacokinetics of oral high-dose busulfan in adult patients undergoing hematopoietic stem cell transplantation.

BACKGROUND AND THE PURPOSE OF THE STUDY: Many factors have been reported that contribute to the wide intra- and inter-patient variability of Busulfan (Bu) disposition. The purpose of this study was to develop a population pharmacokinetic model and to determine the covariates affecting the pharmacokinetics (PK) of Bu in Iranian adult patients who received oral high-dose as a conditioning regimen before Hematopoietic Stem Cell Transplantation (HSCT). METHODS: A population PK analysis was performed in 30 patients who received an oral Bu and cyclophosphamide regimen before HSCT. Bu was given orally according to the protocol of the institution. In order to prevent seizures caused by Bu, phenytoin was administered orally one hour before each dose of Bu.A total of 180 blood samples were analyzed by HPLC and PK parameters were estimated by the non-linear mixed effect model by MONOLIX 3.1 program. A one-compartment model with an additive error model was used to describe the concentration-time profile of Bu. RESULTS: Patients' disease and weight was found to be the determinant factors for clearance (CL) and the volume of distribution (Vd) according to Monolix analysis. The covariate entered in final model followed by these equations:[Formula: see text][Formula: see text] In this limited study, the age (15-43 years) had no significant effect. For a patient weighting 60 kg, the typical CL and Vd were estimated to be 13.4 l/hr and 42.6 L, respectively. The interindividual variability of CL and Vd were 13.6 and 6.3%, respectively. There was no significant metabolic induction in these four days as is evident by comparing the trough levels of Bu. However it should be mentioned that, one tailed t-test p-values of the days of two and three, two and four and three and four were 0.083, 0.069 and 0.388, respectively. MAJOR CONCLUSIONS: Results of this study showed that the type of disease was a determinant of CL and the weight of patient was a determinant of Vd for Bu population PK parameters. A reliable PK parameters and Css, estimated from only one plasma concentrations (5 hrs after the first dose), were validated. Since these methods require few sampling and are easy to be used, the limited sampling methods might be advantageous in the routine clinical practice.

Hematopoietic SCT activity in Asia: a report from the Asia-Pacific Blood and Marrow Transplantation Group.

The hematopoietic SCT (HSCT) activity in nine Asian countries/regions was surveyed to overview the current situation. Data of 58 113 HSCTs (allogeneic: 63% vs autologous: 37%) performed between 1986 and 2006 by 432 transplant teams were collected. The number of HSCTs has been increasing in the past two decades in most countries/regions. The increase in allogeneic HSCTs is greater than in autologous HSCTs. The proportion of unrelated donors among allogeneic HSCTs in 2006 varied widely from <1% (Iran and Vietnam) to 62% (Japan). The use of each stem cell source, that is, BM, PBSC, cord blood and others (including co-infusion of BM and PBSC), also varied widely (36, 58, 0.1 and 6% in HSCT from related donors, respectively, and 53, 11, 35 and 1% in HSCT from unrelated donors, respectively). HSCTs have been continuously increasing for all indications except for chronic myelogenous leukemia and solid tumors. Hemoglobinopathy is a common indication among non-malignant diseases in many Asian countries/regions except for China, Japan and Korea. This survey clearly shows the recent progress of HSCTs in Asia and also some differences in donor and stem cell selection and disease application among countries/regions.