bluuebunny/crossref_metadata_2025 huggingface dataset:
High Incidence of Posttransplant Lymphoproliferative Disorder after Unrelated Cord Blood Transplantation.
Authors: Yoo Keon Hee , Lee Soo Hyun , Sung Ki Woong , Jung Hye Lim , Ki Chang Seok , Ko Young Hyeh , Koo Hong-Hoe
Published: October 2019

Though posttransplant lymphoproliferative disorder (PTLD) is a well-documented complication arising in recipients of solid organ or hematopoietic stem cell transplantation, data about PTLD following cord blood transplantation (CBT) are limited. Currently less than 20 cases of PTLD have been described after CBT in the literature world-wide. However, we hypothesized that several factors such as relatively lower number and naïve nature of cord blood T-lymphocytes, frequent donor-recipient HLA disparity and in vivo T-cell depletion by ATG might lead unrelated cord blood recipients more susceptible to PTLD. We retrospectively reviewed the medical records of 45 pediatric recipients who received unrelated CBT at our institution from Jan 2002 to Jan 2006. At least 4/6 matched cord blood units were selected for transplant and some patients (n=10) received double cord blood units. ATG was included in the preparative regimen in all but one case. Cyclosporine with either methyl-prednisolone or mycophenolate mofetil were used for GvHD prophylaxis. We assessed the cumulative incidence, clinical features, pathologic findings, and treatment results of PTLD after unrelated CBT. Only pathologically proven cases were included for analysis. Five patients developed PTLD at 3 to 8 mo posttransplant (median, 4 mo). The cumulative incidence of PTLD was 14.1% at 2 year estimated by Kaplan-Meier method. Four of the 5 patients were positive for EBV serology before transplant. The involved sites were variable such as lymph nodes, brain, lung, liver, kidney, parotid gland, and colon. Three patients had multiple sites of PTLD involvement. Biopsied samples of 5 patients revealed diffuse CD20 positivity (n=4), positive for EBV in situ hybridization and/or EBNA/LMP (n=4), monomorphic (n=2), and polymorphic (n=3) histology. One patient had lesions showing diffuse CD3 positivity instead of CD20 suggesting T-PTLD. Two patients with monomorphic disease were cured with rituximab therapy and 2 of 3 with polymorphic PTLD became free of disease after immune withdrawal. The other one with polymorphic B-PTLD underwent progressive course despite immune withdrawal and rituximab therapy. In conclusion, the incidence of PTLD after unrelated CBT in this study was much higher than previously reported. Clinical suspicion and thorough diagnostic work-up followed by early therapeutic intervention should be given not to miss the potentially curable disease.
Low Risk of Relapse in Pediatric Patients after Double Unit Cord Blood Transplantation.
Authors: Rosenthal Joseph , Bolotin Ellen , Pawlowska Anna B. , Falk Peter , Forman Stephen
Published: October 2019

Unrelated umbilical cord blood transplantation (UCBT) has become a standard therapeutic option for pediatric patients who may benefit from hematopoietic stem cell transplantation but lack an adequate HLA-identical related donor. UCB has the advantages of rapid availability and presumably lower risk of severe, acute graft-versus-host disease (GVHD) despite donor-recipient human leukocyte antigen (HLA) disparity. Double-unit UCBT (DUCBT) extends access to transplantation to patients who were previously disqualified on the basis of the available cell dose in a single unit. Recent studies report high engraftment rate, low incidence of severe acute GVHD and a relatively low transplantation-related mortality in DUCBT. (Barker Blood.2005;105:1343–1347). It is unknown, however, whether patients with advanced hematological malignancies and patients with the severe co-morbidities would benefit from DUCBT. Seven patients, 5 males and 2 females, age 5.2–15 yrs (median 14 yr), 5 with advanced hematological malignancies; were given DUCBT. The diagnoses were AML (n=3; refractory n=1, 2CR n=1, 3CR n=1)), ALL (n=4; 1CR Ph+ I n =1, 2CR Ph+ n=1, both MRD+, 2CR n=2). Five of seven were non-Caucasian (71%). Co-morbidities included recent invasive fungal infection (n=3), acute pancreatitis (n=1), and bulbar paralysis (n=1). Conditioning regimes were FTBI and melphalan d (n=3) and fludarabine and melphalan (n=4). Myeloid engraftment occurred in all patients. The median time to an absolute neutrophil count >500 was 34 days (range 26–74 days). Three patient remained platelet transfusion dependent after day 100. In the remaining 4 patients the median time to a platelet count >20,000 unsupported were 51 days (range 44–69 days). All patients (100%) experienced Grades II-III acute GVHD. There were no patients with Grade IV GVHD and no deaths from acute GVHD. Four patients developed extensive chronic GVHD of the skin only. The 100 day transplant related mortality was 0%. One patient died on day + 200 from respiratory failure. None of the patient suffered relapse. The disease-free survival is 80% with a median day +348 post transplant (range 116–1069). The observation that DUCBT may be associated with a reduced risk of relapse in patients with high-risk leukemia deserves further evaluation. Larger studies will be needed to investigate potential mechanisms by which DUCBT could mediate an anti-leukemic effect and to confirm the clinical experience.
Therapeutic Angiogenesis in Arterial Limb Ischaemia by Autologous Bone Marrow Transplantation.
Authors: Novoa J.E. , Medina M.A. , Portillo F. , Salgado M. , Sequeira T. , Nuñez N. , Olivet C.
Published: October 2019

Background: therapeutic angiogenesis has recently been developed as a new method of treatment for several ischaemic diseases; both experimentally and clinically there is preliminary data suggesting that implantation of bone marrow-mononuclear cells into ischaemic limbs increases collateral vessels formation.

Aims: to evaluate viability of the therapeutic angiogenesis using hematopoyetic bone marrow progenitors mobilized by G-CSF (granulocyte colony stimulating factor) and saffety of the procedure.

Methods: 28 patients developing critical arterial limb ischaemia (candidates to amputation) were included in this study (International Working Party). 15 men and 13 women. Median age 65 years old (58 – 74). Mobilized by filgrastim (Neupogen ®) 5 μg/kg weight daily (5 days). Bone marrow harvest at 5th day. Local anaesthesia was employed in all the patients. Unmanipulated cells were injected in the affected limb in 2 ml aliquots into the gastrocnemius muscle. All the patients received injections unless in one limb. 1 patient received treatment in both. Each patient were evaluated regularly for rest pain, amount of required analgesia, healing of the ulcers, peack walking time, Doppler and angiographic findings. The mean number of injected mononuclear cells was 1,5 ×109/kg. All the patients received low molecular weight heparin (nadroparin, Fraxiparine ®) 3800 – 5600 IU anti-Xa subcutaneously, aspirin 81 mg and pentoxifiline 400 mg daily, as medical treatment after the procedure for at least 60 to 90 days. A control population of 27 vascular patients affected by critical arterial limb isquemia was considered. They don’t received angiogenic treatment, only the same medical antithrombotic schedule described.

Results: there were no secondary effects for the mobilization or injection of cells in the 28 treated patients. There were no dead patients secondary to the procedure. Moreover, in 22 patients with a median follow up of 18 months, 20 patients showed an improvement of all parameters, mainly pain at rest, peack walking time and skin trophic lesions. One patient suffered amputation of the affected extremity because obstruction of an old by-pass (3,6%). On the control population, amputation of the affected extremity was necessary in 25 of the 27 patients evaluated with medical treatment only (90,7%). The statistical differences between the two groups were highly significant in favor of the angiogenic group. They were evaluated by the chi square test and log rank test (p<0,05).

Conclusions: our preliminary data suggest that autologous bone marrow transplantation can be performed safely and appears to be a benefical therapy for selected patients with severe peripheral arterial disease.

(drnovoa51@yahoo.es or drnovoa@netgate.com.uy).
Different Outcome for Angioimmunoblastic T-Cell Lymphoma (AIL) and Peripheral T-Cell Lymphoma Unspecified (PTCL-U) Following Upfront Autologous Stem Cell Transplantation.
Authors: Reimer Peter , Ruediger Thomas , Weissinger Florian , Mueller-Hermelink Hans Konrad , Engert Andreas , Einsele Hermann , Wilhelm Martin
Published: October 2019

Introduction: Peripheral T-cell lymphomas (PTCL) are rare diseases and optimal treatment strategies still remain to be defined. With the exception of the ALK-positive anaplastic large cell lymphoma (ALCL) that shows a favourable outcome following conventional chemotherapy, PTCL are known for their poorer prognosis compared to aggressive B-cell lymphomas. However, the impact of the different PTCL-subtypes on treatment outcome has not been clearly demonstrated in prospective studies. PTCL unspecified (PTCL-U) and angioimmunoblastic T-cell lymphoma (AIL) represent the most common subtypes of PTCL in Western countries, accounting for approximately 70% of PTCL. We therefore analysed the data of our study on myeloablative radiochemotherapy followed by autologous stem cell transplantation (ASCT) in primary diagnosed PTCL with regard to the main histologic subtypes.

Material and Methods: From 06/00 to 06/06 92 patients with confirmed diagnosis of PTCL entered the study. Primary cutaneous PTCL and ALK+ ALCL were excluded from the trial. Main subtypes were PTCL-U (n= 37) and AIL (n= 28) accounting for 65 of the 92 patients (71%). 0f these patients 53 (PTCL, n= 31; AIL, n= 22) were evaluable for the analysis (82%).

Results: Median age was 50 years in the PTCL-U and 47.5 years in the AIL group, respectively. The International Prognostic Index (IPI) did not differ in both groups. In the PTCL-U and the AIL group a low/intermediate-low risk was found in 35% and 36%, respectively and a high/intermediate-high risk in 65% and 64%, respectively. There were slightly more patients in stage IV in the AIL group compared to the PTCL-U group (64% versus 53%). In addition, more patients in the AIL group complained of B-symptoms and had bone marrow involvement compared to the PTCL-U group (86% versus 66% and 48% versus 39%, respectively). However, in an intent-to treat analysis only 58% in the PTCL-U group compared to 82% in the AIL group underwent ASCT mainly due to a higher rate of patients with progressive disease in the PTCL-U group. The median overall survival (OS) was 11 months in the PTCL-NOS and 20 months in the AIL group. Regarding only patients undergoing ASCT, the median OS was 13.5 months in the PTCL-U and 25.5 months in the AIL group.

Conclusion: Our analysis suggests that patients with AIL, although showing a slightly more unfavourable risk profile at diagnosis, benefit more from upfront autotransplantation than patients with PTCL-U in our study.
Curable Potential with TBI Containing Regimen (L-PAM/TBI) Followed by In Vivo Purged Auto-PBSCT for Patients with Relapsed or Refractory Follicular Lymphoma.
Authors: Tsujimura Akane , Taji Hirofumi , Kato Harumi , Kagami Yoshitoyo , Yamamoto Kazuhito , Ogura Michinori , Morishima Yasuo
Published: October 2019

Background: High-dose therapy (HDT) followed by autologous stem cell transplantation (ASCT) is one of the treatment options for relapsed or refractory follicular lymphoma (FL). However, whether ASCT is able to bring cure for relapsed FL, or what optimal high-dose regimen is remaining to be established.

Patients and Methods: Chemotherapy to obtain response and in vivo purging effect was CHASER regimen reported previously: rituximab 375 mg/m2 on day 1; cyclophosphamide 1200mg/m2 on day 3; cytarabine 2g/m2 on days 4–5; etoposide 100mg/m2 on days 3–5. Patients who achieved partial or complete response by CHASER were treated with L-PAM/TBI regimen comprised four fractionated TBI (8–12Gy) on days -6 to -5 or -5 to -4 and high-dose L-PAM 60 mg/m2 on days -3 to -1 followed by autologous peripheral blood stem cell transplantation (auto-PBSCT) on day 0.

Results: Seventeen patients (median age 45 yrs; range, 26–59 yrs) were treated by this regimen. A median value of infused CD34 positive cells was 3.1×106/kg (range, 1.7–10.1). All patients experienced grade 4 hematological toxicities and febrile neutropenia (FN) were observed in most of patients. Protracted thrombocytopenia was observed in some patients. No grade 4 non-hematologic toxicity was observed. Major grade 3 non-hematologic toxicities were FN and gastrointestinal. Two patients experienced grade 3 vomiting (11.8%), and nine patients experienced grade 3 diarrhea (52.9%), and two patients experienced grade 3 mucositis (11.8%). Two patients developed interstitial pneumonitis on days 130 and 229. They improved by steroid treatment immediately. The median times to reach a white blood cell count greater than 1.0×109/L and platelet count 50×109/L were 11days and 17days, respectively. Engraftment was obtained in all patients. There was no treatment related mortality within days 28 after auto-PBSCT. At a median follow-up of 2.6 years (range, 0.3–4.5 years), all patients were alive without relapse. No second malignancies was observed until now.

Conclusions: L-PAM/TBI regimen followed by in vivo-purged auto-PBSCT collected by CHASER might bring high %CR and have curable potential for patients with relapsed or refractory advanced FL, although large-scaled study is needed.
Early Harvest Followed by Melphalan-Dexamethasone and Second-Line Autologous Stem Cell Transplantation in AL Amyloidosis.
Authors: Palladini Giovanni , Perfetti Vittorio , Russo Paola , Lavatelli Francesca , Nuvolone Mario , Obici Laura , Merlini Giampaolo
Published: October 2019

Autologous stem cell transplantation (ASCT) is considered the most effective therapy in AL, but treatment related mortality (TRM) is significant. Melphalan-dexamethasone (MDex) is well tolerated and grants a high response rate (Blood2004; 103: 2936-8). In order to reduce TRM, after stem cell harvest, we treated AL patients eligible for ASCT with first-line MDex, offering second-line ASCT to non-responders (NR). Eligibility to ASCT was determined according to Comenzo and Gertz (Blood2002; 99: 4276-82), including good and intermediate risk patients. Response to MDex was evaluated every 3 cycles. MDex was continued until either complete remission (CR) or partial hematologic response (PR) associated with organ response (PR+OR) was reached. MDex was discontinued after 3 cycles in NR or after 9 cycles in patients in PR without organ response (PRNOR). Patients in PRNOR or NR received second-line therapy: ASCT if they were still eligible, or thalidomide-dexamethasone (TDex, Blood2002; 105: 2949-51) if they became ineligible for ASCT. Forty patients (median age 55 years, range 32–67 years, 20 males, 15 good-risk) were enrolled between March 2002 and June 2005. Twenty-five (63%) responded to MDex (11 CR, 14 PR). Hematologic response was accompanied by OR in 23 cases. There was no TRM. Seventeen patients (42%), 15 NR and 2 in PRNOR received second-line therapy. Seven were still eligible for ASCT. Although 2 of them were enrolled as good-risk, at the time of ASCT all were intermediate-risk and received conditioning with melphalan 140 mg/m2. Two patients died within 100 days and 2 (29%) responded. The 2 responders had obtained PRNOR after MDex. Ten patients received second-line TDex. One died within 100 days and none responded. Overall, 6 patients (15%), none of whom responded to MDex, died. Median follow-up of living patients is 27 months. Median survival was not reached. Response to MDex improved survival (p<.001). The efficacy and tolerability of MDex is confirmed. Approximately 60% of NR to MDex lose eligibility for ASCT. After MDex, second-line ASCT is associated with apparently increased TRM. Response to second-line ASCT was observed only in patients who obtained PR with MDex.
Results of Autotransplantation in Follicular Large-Cell Lymphoma.
Authors: Copelan Edward A. , Crilley Pamela Ann , Pohlman Brad , Elder Patrick , Pant Shubham , Marathe Pallavi , Brodsky Isadore , Lin Thomas S. , Bolwell Brian J.
Published: October 2019

The role of autotransplantation in the treatment of large-cell follicular lymphoma is uncertain. We analyzed results of autotransplantation performed from 1/91 through 7/06 in follicular large-cell non-Hodgkin’s lymphoma (NHL) at 3 institutions using preparation with busulfan, cyclophosphamide and VP-16 and compared results to those of patients with diffuse large-cell NHL (DLCL) receiving identical preparation over the same period. Fifty-nine patients with follicular large-cell NHL (FLCL) were compared to 430 patients with DLCL. There were no significant differences between the 2 groups in regards to age (DLCL: median 51 years; range 18–74 years; follicular: median 55 years; range 33–77years), sex, remission status at transplantation, bulky disease at diagnosis, or follow-up(median 4 years). Bone marrow involvement at diagnosis occurred more frequently in FLCL patients (P=0.03). Regimen-related mortality at 100 days for patients with FLCL is 4% (95% CI: 0-14%); estimated relapse-free survival at 4 years is 53% (95% CI: 20-83%) and relapse rate at 4 years is 43% (95% CI: 13%-73%). Relapses beyond 4 years occurred in 2 patients with FLCL and 4 with DLCL. Relapse-free survival was adversely affected in both groups by chemotherapy resistant disease or transplantation beyond second remission (P=0.02). There were no significant differences between the two groups in the incidence of early mortality, 4 year lymphoma-free survival, relapse rate at 4 years, or relapse beyond 4 years. In conclusion, the results of autotransplantation in FLCL are similar to those in DLCL. Patients who undergo transplantation in chemosensitive first relapse or earlier have favorable outcomes. Late relapse occurs infrequently.
Lentiviral MDR1 Gene Transfer Confers Radioprotection to Human CD34(+) Haematopoietic Progenitor Cells.
Authors: Maier Patrick , Herskind Carsten , Katharina Fleckenstein , Laufs Stephanie , Zeller Jens W. , Wenz Frederik , Fruehauf Stefan
Published: October 2019

Normal haematopoietic stem cells are particularly sensitive to radiation-induced apoptosis. Overexpression of the multidrug resistance 1 (MDR1) gene product, P-glycoprotein (P-gp), leads to suppression of apoptosis and is radioprotective in the human lymphoblastoid cell line TK6. Therefore, gene therapy with MDR1 might protect bone marrow cells during tumour-radiotherapy. The aim of this study was to test if human CD34(+) blood stem cells can be protected from the effect of radiation by MDR1 gene transduction. MDR1 cDNA was cloned into the lentiviral SIN-vector pHR’SINcPPT-SEW to replace the eGFP cDNA. CD34(+) cells isolated from four individual donors were exposed to lentiviral supernatants with an MOI of 10 of either HR’SIN-MDR1 or HR’SINcPPT-SEW as a control. After lentiviral transduction, 0.8×105 cells of transduced and non-transduced CD34(+) control cells, respectively, were irradiated with 0–8 Gy and held in liquid culture under differentiation conditions. Ten days after irradiation, the amount of MDR1 expressing cells was determined by the Rhodamine-(Rh−)123 efflux assay and the MDR1-expression rate was monitored by real-time PCR. Additionally, the differentiation status was tested with FACS-analyses for CD11b (myeloid and natural killer cells), CD15 (neutrophils, eosinophils, monocytes), CD33 (myeloid progenitor cells, monocytes), and CD34 (hematopoietic precursors) expression. To test the potential of MDR1 to protect differentiated cells, unirradiated cells were irradiated after 10 days in liquid culture. Apoptotic cells were detected 48 hours later by staining with Annexin V. The transduction efficiency for the MDR1-virus was 3–18%, for the GFP-virus 12–60%. The proportion of RH-123-negative (=MDR1-positive) cells of all four donors increased with escalating radiation doses (e.g. 18–54% from 0–8 Gy). Irradiation of the differentiated cells after ten days of liquid culture also led to an increase of RH-123-negative cells with escalating radiation doses (e.g. 12.5–17% from 0–8 Gy). We found a correlation between radiation dose and differentiation status: independent on transduction the amount of CD11b-cells increased at 2–4 Gy (e.g from 23–45%) and decreased to 9% with 8 Gy; a similar course was also seen for CD15 expression. Our results clearly indicate the radioprotective effect of human blood stem cells by lentiviral MDR1-overexpression. Thus, enhancing repopulation by surviving stem cells may increase the irradiation tolerance of hematopoietic cells and thus contribute to widening the therapeutic range in radiotherapy.
Economics of Intravenous Fludarabine Phosphate and Oral Fludarabine Phosphate for the Treatment of B-Cell Chronic Lymphocytic Leukemia in a Developing Country - Brazil.
Authors: Bigni Ricardo , Araújo Gabriela T.B. , Fonseca Marcelo C.M. , Dubin Jane , Coelho Arthur M. , Bello Reinaldo Dal
Published: October 2019

B-cell chronic lymphocytic leukemia (B-CLL) is the commonest leukemia occurring in Western countries, and arises predominantly in older age groups. Intravenous fludarabine phosphate offers the possibility of a prolonged progression-free interval, an increased response rate and a trend toward better tolerability in comparison with standard polychemotherapy regimens. A more convenient oral formulation of fludarabine phosphate, has been developed. We undertook a literature review that has demonstrated that oral fludarabine phosphate has a similar clinical efficacy and safety profile to the IV formulation and does not adversely affect quality of life. Also we performed a retrospective analysis of patients receiving IV fludarabine for (B-CLL) at our institution to determine the management and resource use of these patients. A cost minimization analysis was then conducted. The per patient costs for the treatment of drug-related adverse events were in local currence R$ 1.476,87 for oral fludarabine and R$ 2.119,64 for IV fludarabine. When costs of a full course of each treatment were compared, the costs per patient for oral fludarabine and IV fludarabine were R$ 22.152,43 and R$ 23.160,31 respectively. In this preliminary analysis, considering that oral fludarabine has a similar efficacy rate and tolerability profile to IV fludarabine, the oral formulation appeared to have a lower total cost per patient. These data require confirmation with formal assessment of cost-effectiveness.
Cost Effectiveness of Post-Remission Intensive Therapy in Patients with Acute Leukemia.
Authors: Hsu Hui-Chi , Gau Jyh-Pyng , Chern Hsiouh-Hsiang , Chau Wing-Keung , Tzeng Cheng-Hwai , Ho Chao-Hung
Published: October 2019

Background: We assessed the cost-effectiveness of high dose arabinoside (HiDAC)-based and allogeneic stem cell transplantation (alloSCT)-based therapy in patients with acute leukemia.

Patients and Methods: We analyzed the outcome, cost and cost-effectiveness of 106 patients treated between 01/94 and 01/02 (94 AML/12 ALL). Forty-two young patients at either intermediate or unknown cytogenetic risk received post-remission intensive therapy (24 HiDAC-based / 18 alloSCT-based therapy).

Results: After a median follow-up of 50 months, the estimated 7 year overall survival for the HiDAC-based group showed a tendency to be higher than the alloSCT-based group (48% versus 28%; p=0.1452). HiDAC-base group spent a significantly lower total cost (USD 51,857 versus 75,474; p=0.004) than the alloSCT-based group. Cost-effectiveness analysis showed that the mean cost per year of life saved for the HiDAC-based group is considerably less expensive than the alloSCT-based group (USD 11,224 versus 21,564). The reduced total cost for the HiDAC-based group originated from lower cost in room fees, medication, laboratory and procedure, but not in blood transfusion and professional man-power fees.

Conclusion: HiDAC therapy as initial post-remission intensive therapy is a cost-effective approach in AML patients at either intermediate or unknown cytogenetic risk, which deserves further prospective clinical study to address this issue.
Trisomy 21 Expands the Megakaryocyte-Erythroid Progenitor Compartment in Human Fetal Liver-Implications for Down Syndrome AMKL.
Authors: Tunstall-Pedoe Oliver , de la Fuente Josu , Bennett Phillip R. , Fisk Nicholas M. , Vyas Paresh , Roberts Irene A.G.
Published: October 2019

Children with Down syndrome (DS) have a uniquely high frequency of acute megakaryoblastic leukemia (AMKL)- ~500-fold increased compared to children without trisomy 21 (T21). At least two genetic events are required but are not sufficient for DS-AMKL: T21 and N-terminal truncating mutations in the key megakaryocytic transcription factor GATA1. This tight association of T21 with GATA1 mutations and the development of AMKL in a narrow temporal window (fetal life-5yrs) makes DS-AMKL a highly informative model of multi-hit leukemogenesis in which the first steps occur in utero. However, the individual contributions of T21 and mutant GATA1 in the leukemogenesis are unclear. To specifically investigate the role of T21 in DS-AMKL and why leukemia-initiation is confined to fetal (or early post-natal) life we have studied fetal hemopoiesis in DS during the second and third trimester in 16 fetuses (gestational age 15–37 weeks) where an antenatal diagnosis of DS with T21 was made by amniotic fluid fetal cell karyotyping. Samples of fetal blood (n=13), fetal liver (n=9) and fetal bone marrow (n=8) were screened for mutations in the GATA1 gene genomic DNA by DHPLC or direct sequencing (sensitivity of detecting a GATA1 mutation is 1–5% by DHPLC). No GATA1 mutations were detected. This allowed us to study the impact of T21 independent of GATA1 mutation on fetal hemopoiesis. DS fetuses showed marked qualitative and quantitative abnormalities in hemopoiesis. While the total number of CD34+ cells in DS and normal fetal liver were comparable, DS fetuses had a striking increase in bi-potential megakaryocyte-erythroid progenitors (MEP; CD34+CD38+FcgloCD45RA+− 74.4% vs 27.0% of fetal liver CD34+/CD38+ cells. Peripheral blood from all DS fetuses studied compared to normal fetal blood samples showed dysmegakaryopoiesis (abnormally shaped and/or giant platelets and MK fragments), dyserythropoiesis (macrocytes, poikilocytes, basophilic stippling), increased numbers of blast cells and also had an increased percentage of MEPs − 40.3% vs 26.9%. By contrast, there was no difference in the number of MEP nor erythroid or MK lineage morphology in DS fetal bone marrow compared to normal fetal bone marrow. CD34+ cells from DS fetal liver and fetal blood expressed both fl GATA1 and GATA1s mRNA indicating that dysmegakaryopoiesis and erythropoiesis were not due to lack of expression of fl GATA1. These data indicate, for the first time, that T21 by itself profoundly disturbs megakaryopoiesis and erythropoiesis and leads to an increased of frequency of MEP. This has important implications since it provides a testable hypothesis for the role of T21 in the initiating step of AMKL, namely that T21 expands a fetal liver-derived progenitor compartment which forms a substrate upon which GATA1 mutations then confer a further selective advantage.
Insufficient Inhibition by Aspirin of Platelet TXA2 Synthesis and Platelet Recruitment Is Associated with Myonecrosis in Patients with ST-Segment Elevation Myocardial Infarction.
Authors: Santos Maria-Teresa , Fuset Maria P. , Moscardo Antonio , Ruano Miguel , Perez Francisca , Breña Silvia , Piñon Marta , Valles Juana
Published: October 2019

Heterogeneity in response to aspirin (ASA) treatment or “aspirin resistance” could be of importance in patients with ST-segment elevation myocardial infarction (STEMI). Reduced effect of ASA in platelets could be due to partial inhibition of cyclooxygenase-1 (COX-1) or to COX-1-independent mechanisms. We evaluated the effect of ASA treatment in patients with STEMI for: a) platelet TXA2 synthesis; b) platelet recruitment elicited by TXA2-dependent and -independent mechanisms and c) the possible association of these aspects of platelet reactivity with serum markers of myonecrosis. We studied 62 consecutive ASA-treated patients shortly after the onset of the acute event. Analytical determinations were performed within 48 h of the onset of the acute event (mean ± SD, 23.12 ± 11.61 h). Fifty-two of the patients were treated with fibrin-specific fibrinolytic agents and 56 were also anticoagulated with enoxaparin. A group of 62 ASA-free and 10 ASA-treated controls were also evaluated. TXA2 synthesis and platelet recruitment (fluid-phase pro-aggregatory activity of cell-free releasates) were assessed after collagen stimulation (1 μg/mL) of whole blood in patients and control subjects. Optimal inhibition of TXA2 and platelet recruitment was assumed if these were greater than 95% and 80%, respectively, compared with the ASA-free population of normal donors. Comparisons between categorical data were performed using chi-squared tests. The Kruskal-Wallis non-parametric test was used for comparisons between groups. A two-sided P value < 0.05 was considered significant. Partial inhibition of TXA2 by ASA was found in 21 patients (34%). This was associated with significant elevations of Troponin-T, CK-MB mass, CK and recruiting activity vs. 41(66%) patients with blocked TXA2 production. This was independent of fibrinolysis and platelet COX-2 expression was not augmented. TXA2 blockade was achieved after subsequent daily treatments or platelet incubation with ASA in vitro, suggesting reduced sensitivity of COX-1 to ASA. In addition, 28 patients (45%) had an abnormally elevated recruiting activity, despite TXA2 blockade, which was also associated with increased myonecrosis. In conclusion, aspirin resistance, elicited by both TXA2-dependent and TXA2-independent mechanisms, was prevalent in patients with STEMI. This study describes for the first time the association of partial platelet TXA2 inhibition with myonecrosis.
Identification and Partial Characterization of Fanconi Anemia Associated Polypeptides (FAAPs) Using a Versatile Multiprotein-Complex Purification Method.
Authors: Ali Abdullah M. , Singh Thiyam R. , Meetei Ruhikanta A.
Published: October 2019

Fanconi Anemia (FA) is an autosomal recessive and X-linked disorder characterized by congenital abnormalities, progressive bone marrow failure, and a high incidence of hematological (acute leukemia) and non-hematological malignancies (squamous cell carcinomas of the head and neck or gynecologic system). FA is genetically heterogeneous disease and to date 12 complementation groups are known of which 11 gene products have been identified (FANC- A, B, C, D1, D2, E, F, G, J, L, M). Eight of the FA gene products, FANCA, FANCB, FANC, FANCE, FANCF, FANCG, FANCL and FANCM form a multiprotein FA core complex. This complex is required for the monoubiquitination of FANCD2 upon DNA damage by various genotoxic agents. The other two FA proteins; FANCD1/BRCA2 and FANCJ are believed to act “downstream” of FANCD2. In order to understand the role of FA proteins in DNA repair pathway it is necessary to find all the FA genes and their interacting partners. We have established a two-step purification method using 6XHis and FLAG tags for the biochemical and functional characterization of the FA core complex proteins. In an attempt to isolate interacting partners of FANCM and FANCL proteins; we have established two different HeLa cell lines; HeLa-HF-FANCM and HeLa-HF-FANCL, stably expressing HF-FANCM and HF-FANCL recombinant proteins respectively. Two step affinity purification was carried out to isolate the complexes from the extracts prepared from stable cell lines. Two polypeptides, namely, FAAP16 and FAAP100 were identified by mass-spectrometry as major interacting partners of FANCM and FANCL respectively. The interaction of FAAP16 and FAAP100 with other FA core complex proteins was confirmed by reciprocal affinity purification coupled mass-spectrometry using HeLa cells stably expressing HF-FAAP16 and HF-FAAP100 proteins. Furthermore, suppression of FAAP16 and FAAP100 in HeLa cells using siRNA resulted in a reduced MMC-induced FANCD2 monoubiquitination. Studies are being carried out to understand the precise role of these proteins in the FA core complex. These data suggest additional proteins interact with FA core complex members and demonstrate the utility of the purification method in delineating interacting proteins involved in FA.
Proteasome Inhibitor/Imatinib Combinations Exert Potentiated Antileukemia Efficacy but Not Cardiotoxicity in Chronic Myeloid Leukemia.
Authors: Zhou Guangbiao , Hu Zheng , Pan Xiaofen , Liu Jinglei , Ma Liyuan , Liu Xiaoli , Meng Fanyi , Li Juan , Chen Zhu , Chen Saijuan
Published: October 2019

Whether imatinib mesylate (IM)-based combinatory regimens could be an option for overcoming limitations of IM in treating chronic myeloid leukemia (CML), e.g., resistance developed by leukemic cells, unsatisfactory efficacy on patients at accelerated and blastic phases, and potential cardiotoxity, warrants further investigation. Here we report that the proteasome inhibitors, Bortezomib (BOR) and PSI, significantly potentiate proliferation/growth inhibition and enhance apoptotic effects of IM on K562 cells, CD34+ cells from CML patients, and BCR-ABL-expressing murine hematopoietic progenitor cells. BOR and PSI potentiate IM-triggered activation of caspases and inactivation of BCR-ABL oncoprotein, while caspase inhibitor blocks the apoptotic effect and reverse BCR-ABL inactivation. BOR/IM and PSI/IM combinations also attenuate DNA binding activity of NFκB. Intriguingly, low dose BOR/IM combination yields potent effects on inhibition of tumor growth, induction of apoptosis in leukemic cells, and inactivation of BCR-ABL oncoprotein, and does not cause weight loss or cardiotoxicity in null mice injected with K562 cells as compared to animals treated with IM at high doses. These results suggest a clinical trial to test the efficacy of IM/BOR combination in treating CML.
Methylprednisolone-Treatment Induces Dephosphorylation of Syk in B-CLL Cells In Vitro.
Authors: Boelens Jerina , Lust Sofie , Van Gele Mireille , Derycke Lara , Janssens Ann , Van Hoecke Barbara , Philippé Jan , Bracke Marc , Poelman Martine , Offner Fritz
Published: October 2019

Glucocorticoids (GCs) are often implemented in the treatment regimens of B-cell Chronic Lymphocytic Leukemia (B-CLL). Apart from caspase-involvement and downregulation of Lyn, the exact mechanisms of action in apoptosis induction in B-CLL remain uncertain. We studied methylprednisolone (MP)-induced apoptosis in B-CLL and found a greater sensitivity of ZAP−70+ B-CLL cells for MP-induced cell death, which was accompanied by a glucocorticoid receptor-dependent decrease of ZAP-70 expression. Micro-array data correlated this downregulation of ZAP-70 with an upregulation of the phosphatase PTP1B (Protein Tyrosine Phosphatase 1B). This observation was confirmed on genomic and protein level by RT-qPCR and Western blotting. Next, functional assays with the phosphatase-inhibitor ortho sodium vanadate and with the PTP1B-inhibitor 3-(3,5-Dibromo-4-hydroxy-benzoyl)-2-ethyl-benzofuran-6-sulfonicacid-(−4-(thiazol-2-ylsulfamyl)-phenyl)-amide were performed. Both drugs were able to inhibit the GC-induced ZAP-70 downregulation, which is suggestive for the role of PTP1B. In order to explore this further, we studied the effect of MP on the phosphorylation of Syk by Western blotting and flow cytometry. We did not take phosphorylated ZAP-70 into account as phosphorylation of ZAP-70 is very unclear in CLL cells. We observed GC-induced dephosphorylation of Syk (tyrosine residues 352 and 526), associated with a decrease of total Syk. The kinetics were completely in accordance with those of the ZAP-70 downregulation, occurring only after 12 hours after the start of treatment. Next, we performed immunoprecipitation experiments to investigate the reciprocal influence of Syk and ZAP-70 and found co-immunoprecipitation of Syk, ZAP-70 and hsp90, suggesting a close relation of ZAP-70 and Syk in performing their tasks. In spite of the unclear status of ZAP-70 phosphorylation, ZAP-70 positive B-CLL cells allow for more effective IgM-signaling than their ZAP-70 negative counterparts, probably by blocking inhibitors of signalling through Syk. Moreover, ZAP-70 positive B-CLL cells undergo stronger and prolonged BCR-induced phosphorylation of tyrosine residues 352 and 526 of Syk, which are already prominent in unstimulated conditions. The kinetics and concentration overlapping downregulation of ZAP-70 and dephosphorylation of Syk after MP treatment suggests a relation between these two protein tyrosine kinases in performing their tasks. This was further strengthened by the observation of a co-immunoprecipitation of ZAP-70, Syk and hsp90. In conclusion, in vitro GC treatment of ZAP-70+ B-CLL cells is associated with a downregulation of ZAP-70 and a dephosphorylation of Syk tyrosines 352 and 526 via a GR-dependent way. The upregulation of PTP1B at both genomic and protein level, and the results of the functional assays, are suggestive for a role of this phosphatase during treatment with GCs.
Identification of Genes with Prognostic and Therapeutic Relevance in Multiple Myeloma Using Serial Analysis of Gene Expression Technique.
Authors: Felix Roberta S. , Colleoni Gisele W.B. , Vettore Andre L. , Yamamoto Mihoko , Almeida Manuella S.S. , Andrade Valeria C.C. , Chauffaille Maria de Lourdes L.F. , da Silva Wilson A. , Caballero Otavia L. , Simpson Andrew J. , Zago Marco Antonio
Published: October 2019

Serial analysis of gene expression (SAGE) allows a comprehensive profiling of gene expression within a given tissue and also an assessment of transcript abundance.

Objectives: We generated SAGE libraries from normal and neoplastic plasma cells with the aim of identifying genes differentially expressed in MM that could be useful as new potential diagnostic, prognostic markers or therapeutic targets.

Material and methods: Normal plasma cells were obtained from palatine tonsils of 20 children who underwent tonsillectomy. MM SAGE library was obtained from bone marrow plasma cells of two IgGk newly diagnosed MM patients. Purified normal and neoplastic plasma cells were isolated after magnetic sorting of CD-138-positive cells.

Results: We obtained 29,918 SAGE tags from normal and 10,340 tags from tumor libraries. Computer-generated genomic analysis identified 50 overexpressed genes in MM library. After comparison of unique tags expression levels, we selected 12 overexpressed (at least 10 times) genes in the MM library (ZFHX1B, XBP1, PIM2, LGALS1, RANBP2, P53CSV, DDX5, LSM5, JUND, MAPKAPK2, SP140 and NTN1) for further investigation. Expression of 10 out of 12 genes was validated by quantitative real-time PCR in purified plasma cells of 31 MM patients and three controls. XBP1, RANBP2, P53CSV, DDX5, MAPKAPK2 were overexpressed in at least 50% of MM cases. Comparative analyses of relative expression (2-D D CT) of the 10 genes in 31 MM cases and 3 controls showed statistically significant difference between cases and controls for all genes but PIM2 and ZHFX1B, confirming the SAGE data. Also, we could identify that RANBP2 and ZHFX1B have prognostic impact in MM OS (p = 0.040 and p = 0.0502, respectively). Multivariate analysis confirmed RANBP2 expression as a good independent prognostic marker.

Conclusions: SAGE technique allowed the identification of genes differentially expressed in normal and neoplastic plasma cells. These genes may have important role in tumorigenesis or may be possible therapeutic targets for MM control, particularly P53CSV and MAPKAPK2, or prognostic impact, as RANBP2.
Impaired S Phase Arrest in AML Cells with a FLT3-ITD Treated with Clofarabine Allows Prolonged Drug exposure to Reverse the Chemoresistant Phenotype.
Authors: Seedhouse Claire , Grundy Martin , Shang Shili , Ronan John , Pimblett Heather , Russell Nigel H. , Pallis Monica
Published: October 2019

Abstract 1000

Poster Board I-22

We have previously reported that AML cells with a FLT3-ITD have enhanced DNA repair mechanisms following exposure to DNA-damaging drugs which may be a mechanism of chemoresistance. Clofarabine is a novel nucleoside analogue, active in S-phase, with efficacy in AML and is incorporated into DNA as clofarabine triphosphate. Here we show that in FLT3-ITD cells enhanced repair, and therefore resistance to clofarabine-induced DNA damage and toxicity, can be reversed by prolonged drug incubation. When treated with clofarabine, FLT3-ITD-harbouring MOLM13 and MV4.11 cells undergo similar levels of DNA damage (γH2A.X foci) to FLT3 wildtype (WT) cells (HL60 and KG1). After a short pulse of drug the FLT3-ITD cells have a superior repair capability than WT cells; following a 2 hour washout period γH2A.X positivity found immediately after treatment had almost completely disappeared in the FLT3-ITD cells (<10% γH2A.X remaining), whereas in the FLT3-WT cells significant damage (γH2A.X) remained (>40%). Furthermore, after a 1 hour pulse of clofarabine, whereas the FLT3-WT cells under go rapid S phase arrest the S-phase checkpoint fails in the FLT3-ITD cells: reduction in the proportion of cells synthesising DNA is >80% in FLT3-WT cells and <10% in FLT3-ITD cells. Cell cycle arrest in response to DNA damage in S phase is affected via loss of the transcriptional regulator cdc25A. This loss of expression of cdc25A fails to take place in clofarabine-treated FLT3 mutant cells compared to WT cells. In addition, cdc25A mRNA levels are maintained by the FLT3-ITD as demonstrated by siRNA to FLT3 which reduced cdc25A mRNA levels in MV4.11 cells by 87.5%. Primary FLT3 mutant samples from AML patients(n=3) also display impaired cell cycle arrest upon treatment with clofarabine and show enhanced sensitivity on prolonged treatment (24 hours) compared to wildtype samples (n=2). We conclude that there is a reversal of phenotype in mutant FLT3 cells dependant on the length of exposure to clofarabine. Efficient DNA repair renders the cells resistant to a short pulse of the drug, but a failure of cell cycle checkpoint(s) in S phase, mediated by cdc25A, renders the cells sensitive to prolonged exposure. These results may have implications for the scheduling of clofarabine in clinical studies.

Disclosures:

No relevant conflicts of interest to declare.
Prognostic Value of Monosomal Karyotype in Patients with Primary Acute Myeloid Leukemia On Behalf of Spanish CETLAM Group.
Authors: Granada Isabel , Brunet Salut , Hoyos Montserrat , Costa Dolors , Aventín Anna , Marugán Isabel , Vallespí Teresa , Barnues Marta , Duarte JJ , Solé Francesc , Teixidó Montserrat , Mascaró M. , Llorente Andreu , Guardia Ramon , Tormo Mar , Duarte Rafael F. , Millá Fuensanta , Esteve Jordi , Ribera Josep-Maria , Sierra Jordi
Published: October 2019

Abstract 1003

Poster Board I-25

Introduction:

Recently, the cooperative group HOVON-SAKK has refined the prognostic impact of cytogenetic abnormalities in acute myeloid leukemia (AML) by introducing the concept of monosomal karyotype (MK). This consists of ≥ 2 autosomal monosomies or one autosomal monosomy in addition to a structural alteration. In their experience, MK would explain the poor prognosis of AML with a complex karyotype.

Objective:

To investigate the prognostic impact of MK in patients with primary (de novo) AML enrolled in the Spanish CETLAM group protocols (AML 94/99/03). Also, to determine whether considering MK added predictive value to the cytogenetic classification of the Medical Research Council (MRC).

Methods:

Retrospective analysis of data from 1149 AML patients. Chromosomal formula was centrally reviewed with karyotypes being classified by the presence of MK and allocated into the MRC risk categories. Complete remission (CR) rate, disease-free survival (DFS) and overall survival (OS) were calculated.

Results:

The karyotype was assessable in 904 (79%) of the 1149 cases. In 145 of the 904 cases (16%), abnormalities involving CBF gene were detected and in 437 (48%) the karyotype was normal (NK). In 253 (28%) additional patients the karyotype was not monosomal; of them, 61 (24%) belonged to the unfavorable MRC with 17 cases harboring a complex karyotype ≥ 5 abnormalities, 7 cases with rearrangements 3q, 13 cases with -7, 9 cases with 5q abnormalities and 16 cases with t(6;9)). The remaining 69 (7.7%) patients had a MK; of them, 59 (85.5%) were from the unfavorable MRC category and included 43 cases with complex karyotype ≥ 5 abnormalities, 6 cases with rearrangements 3q, 5 cases with -7, 5 cases with alterations of 5q). The following table summarizes the results in terms of CR rate, DFS and OS:

Conclusions:

The addition of MK to the MRC cytogenetic classification refines the prognostic prediction. In our series, the dismal outcome of patients with MK is confirmed; these patients had worse prognosis than those with adverse cytogenetics without MK. Alternative treatment strategies are mandatory for MK+ patients.

Supported in part by grants: GR1-01075, ECO07/90065, PI080672 and RD06/0020/0101.

Disclosures:

No relevant conflicts of interest to declare.
Arsenic Trioxide as First Line Therapy for New Case of APL, Results and Long Term Follow up.
Authors: Ghavamzadeh Ardeshir , Alimoghaddam Kamran , Ghaffari Seyed Hamidolah , Rostami Shahrbano , Jahani Mohamad , Mossavi Seyed Asadollah , Iravani Massoud , Khodabandeh Ali , Baybordi Eissa , Bahar Babak , Jalili Mahdi
Published: October 2019

Abstract 1023

Poster Board I-45

Arsenic Trioxide approved for treatment of APL patients who relapsed after first line treatment failure by ATRA and chemotherapy. Its efficacy as first line treatment studied in at least three trials (including our center reports) previously. Here we are studying its efficacy, safety and long term follow up of new cases of APLwho treated by Arsenic trioxide without ATRA and/or Chemotherapy.

Material and methods:

between 1999 and 2009 we treated 190 new cases of APL by Arsenic Trioxide ( 0.15 mg/kg/day till CR ). Presence of t(15,17) confirmed in all cases by cytogenetic study and/or RT-PCR. In patients who achieved to CR we applied 4 more courses of daily ATO with the same dose for 28 days. Patients followed every 3 months by physical exam, hemogram and RT-PCR on peripheral blood for detection of relapse.

Results:

Median age of our patients was 29 years. Remission rate was 84.7% and median time to achieve to CR was 30 days. Median follow up was 37 months (maximum105 months) and for patients in remission, DFS was 73.4%. Also chance of 3 and 5 years DFS and OS were 71.3%, 83% and 67.6%, 72% respectively. Also Although early mortality was higher in patients with WBC more than 10000 at presentation, we couldn't find any significant correlation between DFS or OS and at presentation WBC count.

Conclusion:

Arsenic trioxide is effective as first line treatment of new cases of APL. Its long term results is comparable with conventional treatment of APL. optimization of treatment needs better supportive care to reduce early mortality due to APL differentiation syndrome and hemorrhagic episodes.

Disclosures:

No relevant conflicts of interest to declare.