bluuebunny/crossref_metadata_2025 huggingface dataset:
Preliminary Study of the Autoantigens on the Membrane of Erythropoietic Cells of the Patients with BMMNC- Coomb's Test(+) Hemocytopenia
Authors: Fu Rong , Liu Hui , Shao Zonghong , Wang Jun , Li Lijuan
Published: October 2019

Abstract 4435

Objective

To observe the relationship between EPO receptor(EPOR) and autoantibodies-IgG/IgM (auto-Ab) on the membrane of erythropoietic cells of the patients with bone marrow mononuclear cell Coomb's (BMMNC-Coomb's) test(+) immuno-related pancytopenia(IRP), and then explore the probable autoantigens of auto-Ab in IRP.

Methods

46 newly diagnosed IRP patients (15 with auto-Ab on erythropoietic cells and 31 without auto-Ab on erythropoietic cells) and 18 healthy donors as controls were enrolled in this study. EPOR expression on their nuclear erythrocytes were tested with flow cytometry to observe the relationship between EPOR and auto-Ab; After sorting erythropoietic cells in bone marrow, EPOR mRNA and protein Stat5,P-Stat5 were investigated by RT-PCR and Western blot to observe the production of EPOR and EPO/EPOR signal transduction; Finally, EPOR expression on the membrane were tested again after stripping auto-Ab with glycine buffer.

Results

(1) EPOR of auto-Ab(+) arm(1.59±0.87)% was significantly lower than that of auto-Ab (-) arm(4.58±4.09)%(P<0.01), and the latter was significantly higher than that of normal controls (2.27±1.76)%(P<0.05); EPOR of IRP patients was negatively correlated with their auto-Ab (r=-0.543,P=0.000) and its regression equation was Y(EPOR)=0.040-0.335X(auto-Ab);(2)EPOR mRNA of auto-Ab(+) arm(0.685±0.136)was significantly higher than that of auto-Ab (-) arm(0.554±0.116)(P<0.01)and normal controls (0.580±0.119)(P<0.05);(3)Protein Stat5 of auto-Ab(+) arm(1.45±0.94) was significantly higher than that of normal controls (0.54±0.36)(P<0.05); While P-Stat5 of auto-Ab(+) arm(0.42±0.18) was significantly lower than that of normal controls (0.85±0.38)(P<0.05); (4) EPOR expression became higher while auto-Ab became lower after stripping with glycine buffer.

Conclusion

The auto-Ab of some IRP patients might block or competitively inhibit the EPOR on the membrane of erythropoietic cells. EPOR was one of autoantigens in IRP.

Disclosures:

No relevant conflicts of interest to declare.
Characterization of CDR3 Structure of Vβ21 T Cell Clones In Patients with P210BCR-ABL Positive CML and B-ALL
Authors: Zha Xianfeng , Chen Shaohua , Yang Lijian , Li Bo , Chen Yu , Yan Xiaojuan , Li Yangqiu
Published: October 2019

Abstract 4455

The clonally expanded T cells identified in most cancer patients which were considered to respond to tumour association antigen (TAA) have definitely specific anti-tumor cytotoxicity. P210BCR-ABL protein regards as the hallmark of CML and known to be causative of the disease, and also expresses in partial acute lymphoblastic leukaemia (ALL) can induce to yield tumor-specific cytotoxic T lymphocytes (CTL) which can kill bar-abl+ tumor cells in many trials. In our previous study, we found the oligoclonal expansion of TCR Vβ21 subfamily in the peripheral blood (PB) of 8 cases with P210BCR-ABL positive CML and 3 cases with P210BCR-ABL positive B-ALL patients and 1 case with P210BCR-ABL negative B-ALL patients using the genescan technology. To further elucidate the oligoclonal TCR Vβ21 features, we carried out sequencing analysis of CDR3 region of each oligoclonal TCR Vβ21 and determined the primary structure of the CDR3 region. The 12 TCR Vβ21 clones used different Jβ segments, Jβ2.3 segment was used in 4 clones, both Jβ2.1 and Jβ1.1 segments were used in 2 clones respectively, Jβ1.2, Jβ1.3, Jβ2.2 and Jβ2.7 segments were used in 1 clone. Only a conserved amino acid motif (SLxxV) was found within the CDR3 region from 3 patients with CML. Then we constructed the three-dimensional structures of the CDR3 sequences from all 12 clones by homology modeling methods and through pairwise comparing with the three-dimensional structures of all CDR3 sequences, the results showed that the conformation of the CDR3 region either containing the conserved amino acid motif or using the same Jβ segment displayed low similarity. Interesting, the conformation among some clones whose CDR3 region contain different amino acid motif and did not use the same Jβ segment were high resemble. In conclusions, our findings described that the high frequency of TCR Vβ21 subfamily expansion emerged in p210BCR-ABL positive CML and B-ALL patients, and according to perplexing features of CDR3 region, it could not easy to confirmed that the oligoclonal expanded TCR Vβ21 subfamily must recognize to p210BCR-ABL protein, however, primary structure characterization of CDR3 region, in combination with spatial structure features, ultimately might help to identify the tumor-associated antigen immune attack on tumor cell in p210BCR-ABL positive CML or B-ALL patients.

Disclosures:

Li: The study was supported by grants from National “863” project (No. 2006AA02Z114) and Key project of Natural Science Foundation of Guangdong province, China (No. 05103293, 9251063201000001).: Research Funding.
ABL Mutations In Early Chronic Phase Chronic Myeloid Leukemia (CP-CML) Are Associated with a Greater Likelihood of Progression and Shorter Survival
Authors: Carella A. M. , Garuti A. , Cirmena G. , Catania G. , Pica G. , Beltrami G. , Patrone F.
Published: October 2019

Abstract 4458

Acquired resistance to Imatinib in advanced phase of CML has been associated with mutations in the kinase domain (KD) of BCR-ABL. We have recently reviewed the status of the mutations in 52 patients with early CP-CML on the samples collected at diagnosis. Mutations were identified by direct sequency (DS) with BidDye Terminator V1.1. cycle sequencing kit and analyzed with a 3130 AB capillary electrophoresis system. Twenty-eight patients had low risk, 10 intermediate risk and 14 high risk, according to Sokal/Euro. Ten out of 14 high Sokal risk patients showed the following mutations: Y253C, S265R, E255K, F359Y, N374S, E255V, E255V, E255V, R332L, E334G. Three of these patients progressed during Imatinib and second-line TKIs and died of blastic phase CML at 23, 33 and 69 months. Curiously, S265R and N374S mutations disappeared during Imatinib treatment but were substituted during follow-up by other two mutations: E255L and H396R. The patient carrying E255L mutation died in blastic phase at 33 months and the one with mutation H396R was well controlled by Nilotinib and he is now alive in CMR 26 months after. Only one out of the 10 intermediate Sokal risk carried KD mutations at diagnosis (D363G). This patient is alive in MMR at 26 months after diagnosis under Imatinib. None of the 28 low Sokal risk patients carried KD mutations at diagnosis and no patients developed cytogenetic evolution while on treatment. In conclusion, the fact that KD mutations were more present in patients with high Sokal risk supports the hypothesis that the probability developing a mutation is related to the basic biology of the disease rather than being merely a random event.

Disclosures:

No relevant conflicts of interest to declare.
High Levels of Nm23-H2 Protein Are a Specific and Invariable Feature of CML-CP Cells Due to Post Transcriptional Control Via a Bcr-Abl Dependent Pathway
Authors: Tschiedel Sabine , Cross Michael , Müller Enrica , Jilo Annette , Wang Song-Yau , ucinic Vladan , Lange Thoralf , Al-Ali Haifa Kathrin , Niederwieser Dietger W
Published: October 2019

Abstract 4461

Introduction:

We have previously identified Nm23-H2 as a tumor antigen in a case of chronic myeloid leukemia. In the following project we investigated the molecular background for the increased expression of Nm23-H2 in CML cells from a large number of patients.

Material and Methods:

Clinical samples from 30 patients with CML in chronic phase at diagnosis [Bcr-Abl/Abl transcript levels from 69.5 to 93% IS (international scale)], in addition to patients with molecular response to imatinib [n=10; median 2 (range 0.1–11) % IS Bcr-Abl/Abl transcript level] and to imatinib resistant patients [n=10; median 66 (range 37–100) % IS Bcr-Abl/Abl transcript level] were investigated. Subpopulations were isolated from the blood (CD3+, CD14+) or bone marrow (CD34+) by FACS sorting. AML samples were obtained from 12 patients with de novo AML and 3 patients with secondary AML at diagnosis. Ba/F3 cells expressing either wild-type Bcr-Abl (Bcr-Ablwt Ba/F3), the T315I mutant of Bcr-Abl (Bcr-AblT315I Ba/F3) or the E255K mutant of Bcr-Abl (Bcr-AblE255K Ba/F3) were used in the cell line model.

Messenger RNA levels of Nm23-H2 were measured using the QuantiTect Primer Assay (Qiagen). Results were normalized to mRNA for the ribosomal protein RPLP0. Western blotting for Nm23-H2 was performed by standard techniques using chemiluminescence and immunocytochemistry was performed using Nm23-H2 and ß-Actin antibodies. Mean fluorescence intensity was determined using the LSM Image Browser software.

Results:

Nm23-H2 protein is over-expressed in the cytoplasm of mononuclear cells from 30/30 CML patients at diagnosis and 10/10 patients with resistance to imatinib. In contrast, patients responding to Imatinib had markedly reduced levels of Nm23-H2 protein. Over-expression is limited to Bcr-Abl+ progenitor and myeloid populations and is absent from (Bcr-Abl-) T lymphoid cells, normal donors and 14/15 acute myeloid leukemia samples. The increase in Nm23-H2 protein is due to post-transcriptional regulation, with no detectable increase in mRNA levels. The increase in Nm23-H2 can be reversed by imatinib or nilotinib treatment of Ba/F3 cells expressing wild-type Bcr-Abl, but not of cells expressing the respective resistant Bcr-Abl mutants.

Conclusions:

The post-transcriptional up-regulation of Nm23-H2 is therefore a common and specific property of CML closely associated with Bcr-Abl activity, making Nm23-H2 a potential target for the development of novel immunotherapeutic or pharmaceutical therapies for CML.

Disclosures:

Al-Ali: Novartis: Consultancy, Honoraria. Niederwieser:Bristol-Myers Squibb: Speakers Bureau; Novartis: Speakers Bureau.
Treatment with TKIs Overcomes Imatinib Resistance through the PLCgamma-1 Signaling Pathway In Imatinib Resistant Human CML Cell Lines
Authors: Romano Alessandra , Vigneri Paolo , Romano Chiara , Vetro Calogero , Stella Stefania , Massimino Michele , Stagno Fabio , Raimondo Francesco Di , Liotta Lance , Espina Virginia
Published: October 2019

Abstract 4468

Suppression of BCR-ABL1 catalytic activity by the tyrosine kinase inhibitor (TKI) Imatinib Mesylate (IM) has dramatically improved the natural history of Chronic Myeloid Leukemia (CML) and - to date - represents the first and most successful example of effective anti-cancer targeted therapy {Druker, 2009}. Despite the success, patients can become resistant. Since IM-resistance in CML patients occurs despite adequate suppression of BCR-ABL activity, it is likely due to the activation of other pathways, and for this reason we need to discover novel Bcr-Abl independent pathways than can become the targets of resistant cells.

Therefore we used immortalized CML human cell lines K562 and LAM84, both sensitive and resistant to Imatinib, to study the signaling in sensitive/resistant cell lines following treatment with 4 different compounds. Reverse-Phase protein microarrays were used to quantitatively map 35 cell signaling pathway endpoints, including survival, proliferation, drug resistance, apoptosis, and autophagy. Cells were incubated with IM 1uM, Dasatinib 1uM, Nilotinib 1uM or LY-29400210 uM and after 2 or 12 hours were placed in a preservative that suppresses fluctuations in kinase pathway proteins (Espina, 2008).

5/35 protein endpoints were linked together and suppressed by Dasatinib, even in the resistant cell line: PLC-y-1-Tyr783, and its upstream (ShCTyr317 SrcTyr416) and downstream targets (mTORSer2448, STAT5Tyr694, ERKThr202/Tyr204) without interfering with AKT activation status on Ser473 compared to Imatinib (p=0.0031 for K562, p= 0.042 for LAM84), Nilotinib (p=0.0034 for K562, p= 0.043 for LAM84), LY-294002 (p=0.0009 for K562, p= 0.015 for LAM84).

In Imatinib-sensitive cell line LAM84 there were no differences between Dasatinib and Imatinib in the modulation of the pathway, compared to IM-sensitive K562 cell line (p=0.005), thus confirming the different profile among these two CML models.

Dasatinib showed a greater suppression of the PLC-y-1 pathway compared to LY-294002 in both resistant cell lines (K562, p=0.0009 and LAM84, p=0.015).

These data confirm and extend the conclusions of Markova et al. (Oncogene 2010) showing PLC-y-1 as a mechanism of death in sensitive cells. Our data showed the mechanism by which second generation TKIs (dasatinib) can overcome Imatinib resistance by suppressing the PLC-y-1 pathway.

Disclosures:

No relevant conflicts of interest to declare.
Validation of a New Flow Cytometry-Based Method to Discriminate Malignant From Normal Stem Cells In CML
Authors: del Castillo Santiago , García Regina , Hernandez Agustin M , Entrena Laura , Llano Ma Paz Queipo De , Campos Arturo , Rosell Ana , Cerda Maria , Ramirez Gemma
Published: October 2019

Abstract 4474

Introdution:

Previously, leukemic stem cells in Chronic Myelogenous Leukemia could only be identified indirectly by using culture techniques. Jeroen J.W et al have developed a new flow cytometric approach that allows a direct discrimination of CML stem cells from their normal counterparts within single patient samples. CD34+CD38- leukemic stem cells could be discriminated from normal stem cells by high CD90 expression, which is a specific feature of CML stem cells, while CD90 expression is low on their normal counterparts.

Material: A total of 8 patients and 3 health controls were included in this study.

Result:

Health controls did not present CD90 molecular expression in stem cells. 100% of the patients presented different levels of CD90 in CD34+/CD38- cells. Our patients seemed to show an asociation between CD34+/CD38- stem cells which express CD90+ and bcr-abl levels. We have evolutive studies of 4 patients who are under treatment with imatinib; in those patients we apreciate decrease in the level of CD90 related to good response.

Conclusion:

Our results confirm Jeroen's data; we think that this new technique will expand our possibilities to identify CML stem cell specific targets and may improve efficacy assessment of CML treatment as well.

Disclosures:

García: Celgene: Research Funding.
Phase II Study of Dasatinib In Patients with Relapsed Chronic Lymphocytic Leukemia.
Authors: Al-Ameri Ali M , Badoux Xavier , Ferrajoli Alessandra , Wierda William G. , Fayad Luis , Estrov Zeev , Bickel Susan , Keating Michael J , O'Brien Susan
Published: October 2019

Abstract 4488

Src-family kinases (SFK) are described to be over expressed in CLL resulting in increased BCR signaling. Aberrant activation of the SFK, Lyn, leads to defective apoptosis of CLL cells in-vitro. C-Abl kinase is also overexpressed in CLL cells compared to normal B-lymphocytes. Since dasatinib is a dual Src and c-abl kinase inhibitor with in-vitro pro-apoptotic properties in CLL cells, we investigated the activity of dasatinib in patients (pts) with CLL. We designed a phase II study of dasatinib in pts with relapsed CLL/SLL and T-PLL. Pts were eligible if they were previously treated and had indication for therapy according to NCI-working group criteria. All pts had adequate performance status, renal and liver function prior to therapy. Treatment consisted of dasatinib 50mg given orally twice daily. In case of suboptimal response the dose of dasatinib could be increased up to a maximum dose of 70mg twice daily. Dose reductions to 20mg twice daily were permitted for toxicity. Pts were assessed for treatment response according to 1996 NCI-WG criteria. Seventeen pts have been enrolled in this study. The median age was 67 years (42-83 years), 9 (52%) had Rai stage III-IV, median beta-2 microglobulin levels was 5.9 (3.0 – 11.8) mg/L. The median number of prior treatments was 4 (1 – 8). An objective response (PR) was observed in 1 patient, 13 pts had no objective response and 3 pts were not evaluable for response due to early discontinuation of therapy (0-3 days). Fourteen pts remained on therapy for a median of 2 (0-19) months with 4 pts discontinuing due to disease progression and 9 pts discontinuing due to adverse events and lack of response. Hematological toxicities consisted of grade 3–4 neutropenia in 76% of the pts, grade 3–4 thrombocytopenia in 44% of pts and grade 1–2 anemia in 80%. Non-hematological toxicity consisted of grade 3–4 fatigue in 1 patient and grade 3 pleural effusions in another patient. Grade 1 and 2, toxicities included flushing 38%, headache 38%, fatigue 46% anorexia and nausea 46%, and diarrhea 23%. Several pts showed evidence of biological activity. Treatment with dasatinib lacks efficacy in pts with heavily pretreated CLL. Responses occurred in only 6% of pts and dasatinib administration was associated with a high incidence of neutropenia.

Disclosures:

O'Brien: Bristol-Myers Squibb: Research Funding.
Dasatinib for Chronic Phase Chronic Myeloid Leukemia After Failure of Imatinib In Korean Patients.
Authors: Lee Ha Yeon , Chang Myung-Hee , Jung Chul Won , Kim Dong Hwan (Dennis) , Kim Won Seog , Kim Seok Jin , Kim Kihyun , Jang Jun Ho , Yoon Sung-Soo , Lee Kyoo-Hyung , Kim Hyeoung Joon , Chung Joo-Seop , Sohn Sang Kyun
Published: October 2019

Abstract 4494

Background:

Although imatinib could induce efficacious and stable responses in patients with chronic myeloid leukemia (CML), resistance is increasingly problematic. Dasatinib, a BCR-ABL inhibitor with 325-fold greater potency in vitro than imatinib, induced MCyR 59%, CCyR 48%, PFS 90% and OS 96% for patients who can not tolerate or are resistant to imatinib in START-C.

Method:

We assessed retrospectively the efficacy and safety of dasatinib in patients with chronic phase (CP)-CML, resistant or intolerant to imatinib who received dasatinib 70 mg to 140 mg per day. Dose was adjusted according to toxicity.

Result:

Between 21 June 2005 and 31 March 2010, medical records of 47 patients from 6 centers in Korea were reviewed: 22 with imatinib-resistant and 1 with imatinib-intolerant CP-CML. 8 Imatinib–resistant ABL kinase domain mutations were found including E255K, T315I, F317L. Median duration of dasatinib therapy was 20.6 months. CHR, MCyR, CCyR and MMR was attained in 91%, 79%, 56% and 44% of patients, respectively. 18 month MMR rate was 71%, 67% in imatinib-intolerant and 53% in imatinib-resistant group. There was no difference in PFS according to the Sokal score, to the best response of imatinib to the type of mutation. 3-year PFS was 71% and OS was 79% with a median follow up of 20.6 months. There was no disease progression or death in imatinib intolerant group. Grade 3/4 anemia, neutropenia and thrombocytopenia were reported in 36%, 49% and 45% of patients, respectively. Non-hematologic toxicity (grade 3/4) consisted of infection(15%), dyspnea(4%), pleural effusion(6%), abdominal pain(2%) and skin rash(2%).

Conclusion:

Dasatinib showed promising efficacy and tolerability in imatinib-resistant or -intolerant CP-CML in Korean patients.

Disclosures:

No relevant conflicts of interest to declare.
Extramedullary Relapse of Acute Adult Leukemia Following Allogeneic Stem Cell Transplantation:10 Cases Analysis.
Authors: Shi Jimin , Luo Yi , Tan Yamin , Zhu Xiaoli , He Jingsong , Zheng Weiyan , Xie Wanzhuo , Li Li , Ye Xiujin , Zhang Jie , Cai Zhen , Lin Maofang , Huang He
Published: October 2019

Abstract 4513

Background:

Up to date, the knowledge concerning the predisposing factors, pathogenesis and mechanisms of isolated extramedullary relapse (EMR) of acute leuekmia (AL) has not been well elucidated.

Method:

We retrospectively analyzed 215 patients who underwent allogeneic hematopoietic stem cell transplantation (allo-HSCT) at our center from 1998 to 2006. All of them were adult AL patients (110 acute myeloid leukemia (AML), 102 acute lymphocytic leukemia (ALL) and 3 acute mixed lineage leukemia). 10 cases (4.7%) experienced relapse of extramedullary sites. Among these 10 patients, there were four male and six female patients with a median age of 25 years (range, 22–38 years). Six patients were AML and four patients were ALL. The median interval from diagnosis to transplantation was 9 months (range, 6–15 months). Five patients had cytogenetics or molecular abnormalities. All the patients were in first CR. All the donors were HLA-identical matched (5 siblings and 5 unrelated donor). Conditioning regimen included BU and CY without total body irradiation (TBI). Graft-versus-host disease (GVHD) prophylaxis is consisted of cyclosporin A, a short-term methotrexate and mycophenolate mofetil.

Result:

Ten patients experienced isolated EMR at a median of 10 months (range, 3–28months) after allo-HSCT. Sites of EMR varied widely and included CNS (leptomeninges, brain, retro-orbital tissue and paraspinal tissue), skin, bone, pelvis and breasts. Five patients experienced two or more sites of EMR. The most frequently involved in relapse sites were CNS (4 patients) and skin (3 patients). Three patients subsequently developed bone marrow relapse, two patients in 2 and 3 months after the initial EMR and the third patient in 3 months after the second onset of EMR. The remaining seven patients did not develop bone marrow involvement at a follow-up ranged from 3 to 8 months after the initial EMR. Three patients received donor lymphocytes infusion (DLI) combined with chemotherapy and (or) irradiation, two patients achieved CR and remain free of disease for 25 and 32 months after transplant whereas one died due to the second EMR and the following marrow relapse. Two patients received local surgery in combination with radiotherapy or chemotherapy and both remain free of disease for 7 and 18 months post transplant. Among five patients who were only treated with local irradiation or chemotherapy or supportive care, four died from progressive disease.

Conclusion:

The prognosis of EMR is generally considered to be unfavorable. An intensified regimen combined of local interventions with systemic chemotherapy may be benefit for patients.

Disclosures:

No relevant conflicts of interest to declare.
The Value of Donor Lymphocyte Infusions In Unstable Mixed Chimerism In Patients with Beta Thalassemia.
Authors: Karasu Gülsün Tezcan , Ye¸silipek Akif , Karaüzüm Sibel , Uygun Vedat , Akcan Mediha , Manguoğlu Esra , Küpesiz Alphan , Hazar Volkan
Published: October 2019

Abstract 4521

The only curative option for ß talasemia remains allogeneic hematopoietic stem cell transplantation (HSCT) to correct the genetic defect and provide a normal hemoglobin level in the recipient. Engraftment of donor derived cells is necessary for a success of transplantation. However, it has been shown that complete donor hematopoesis is not essential for sustained engraftment in thalassemia. Donor and recipient cells may coexist and produce a functional graft commonly referred to as donor/recipient mixed chimerism (MC) even with a low amount of engrafted donor cells. We documented our experience in a cohort of 19 beta thalassemia patients receiving DLI (donor lymphocyte infusions) following 21 transplantations with unstable mixed chimerism (MC). Seven patients received DLI with the indication of having more than 25% residual host cells (RHC) (MClevel3(residual host cells>25%)) early after transplantation and assigned as early-DLI group. Four patients although had MClevel3 in the early period, received deferred DLI and assigned as late-DLI group. The difference between these two groups, in respect to preserving functional graft, was not found statistically significant. However the chimerism kinetics of these early and late-DLI groups were also different and a slower decline in percentage of donor cells were observed in patients preserving grafts. Ten patients received DLI in the later period and although a significant increase in percentage of donor cells was not detected, increase in hemoglobin levels was provided. The infusions were safe with acceptable risk for GVHD. We believe that patients with more than 25% of RHC within two months early intervention with DLI seems feasible. Deferring DLI to a later period especially if the decrease in percentage of donor cells is so prompt, seems less effective. In the later period, we propose to consider the hemoglobin level as the main determinative factor for DLI application rather than the percentage of donor cells in line with expectance of better long-term tolerance. A better understanding of the mechanisms underlying the occurrence of persistent MC and strategies for induction and maintenance of tolerance will provide better interventions and outcomes for these patients.

Disclosures:

No relevant conflicts of interest to declare.
Successful Haploidentical Stemcell Transplantation In a Patient with Fanconi Anemia and AML.
Authors: Duerken Matthias , Schneider Henriette , Soerensen Jan , Bader Peter
Published: October 2019

Abstract 4586

Allogeneic hematopoetic transplantation is the only curative treatment in patients with fanconi anemia indicated as soon transfusion dependency occurs and a matched family or unrelated donor is available. Overall survival for matched or nearly (5/6) matched unrelated donor transplantation improved since the introduction of fludarabine-based conditioning regimens from 30% to 53%. Transplantation after malignant transformation, in heavily pretransfused patients or from alternative donors is associated with a dismal outcome. We report the case of a 7 year old girl with fanconi anemia who developed AML-M4 (monosomy 7 and gain on chromosome 3) with 24% peripheral blasts during donor search for HCT. Remission was acchieved with a mild chemotherapeutic regimen consisting of a single dose of intravenous (300mg/m2) and intrathecal (40mg) cytarabine and daily oral thioguanine (cumulative dose: 930mg/m2). 4 weeks after diagnosis of AML, the girl was transferred for allogeneic PBSCT (CD3/19 depleted) from an unrelated mismatched (7/10) donor. Conditioning regimen consisted of Fludarabine, Busulfex, ATG and OKT-3. Unfortunately, mixed chimerism was observed and on day +21 graft rejection was diagnosed. The patient underwent a second PBSCT from the same donor after conditioning with Fludarabine, Alemtuzumab and Cyclophosphamide and 12 days later received a stem-cell-boost due to poor hematological regeneration. Prolonged aplasia under treatment with G-CSF was suspicious for non-engraftment. The indication for a third PBSCT with her haploidentical father as donor was set. Conditioning in the aplastic patient with elevated autologous T-Lymphocytes consisted of ATG only for 3 days. Full engraftment was achieved on day +11. CMV-reactivation was effectively treated with a combination therapy of valganciclovir and foscavir. One year after the third PBSCT, the patient is in continuing morphologic remission with normal Cytogenetics and complete donor chimerism. Mild Graft vs. Host Disease (Grade I, Skin) dissolved after short term therapy with Prednison. Still she suffers under minor restrictions in her every day life. We demonstrate here the case of a CMV-positive patient with fanconi anemia and AML, polytransfused (27 red-cell-packs and 94 platelet-transfusions) who survived mismatched PBSCT of her CMV-positive father after two failed PBSCT from an alternative 7/10-mismatched donor. To our knowledge this is the first patient with fanconi anemia and AML, successfully transplanted from an haploidentical donor.

Disclosures:

No relevant conflicts of interest to declare.
ICOS Gene Polymorphisms In B-Cell Chronic Lymphocytic Leukemia In a Polish Population.
Authors: Frydecka Irena , Karabon Lidia , Jedynak Anna , Tomkiewicz Anna , Pawlak Edyta , Wolowiec Dariusz , Kielbinski Marek , Kuliczkowski Kazimierz , Woszczyk Dariusz , Wrobel Tomasz
Published: October 2019

Abstract 4614

Introduction:

There is strong evidence that altered immunological function entails an increased risk of B-cell chronic lymphocytic leukemia (B-CLL). The main mechanism of an antitumor response depends on T-cell activation. Unlike the constitutively expressed CD28, inducible costimulatory molecule (ICOS) is expressed on the T-cell surface after activation. ICOS enhances all the basic T-cell responses to a foreign antigen, namely proliferation, secretion of lymphokines, the up-regulation of molecules that mediate cell-cell interaction, and effective help for antibody secretion by B cells. ICOS is essential for both efficient interaction between T and B cells and normal antibody responses to T cell-dependent antigens. It does not up-regulate the production of interleukin-2, but superinduces the synthesis of interleukin-10. Our previous results indicated a role of ICOS gene as a susceptibility locus to B-CLL. Therefore an extended study was undertaken to evaluate the association between four ICOS polymorphisms (which were recently described as functional ones) and susceptibility to B-CLL in a Polish population.

Methods:

A case-control study of 296 individuals including 146 B-CLL patients was conducted on four polymorphisms in the ICOS gene. Genotyping of the polymorphisms ICOSISV1+173T>C (rs10932029), ICOSc.1624C>T (rs10932037), ICOSc.2373G>C (rs4675379), and ICOSc.602A>C (rs10183087) was done using allelic discrimination methods with the TaqManÒ SNP Genotyping Assay.

Results:

There were no statistically significant differences in the allele, genotype, and haplotype distributions between B-CLL patients and healthy controls for any of the investigated polymorphic markers in the ICOS gene. However, we noted that patients carrying genotype ISV1+173T>C [TT], ICOSc.602A>C [AA], ICOSc.1624C>T [CC], and ICOSc.2373G>C [GG] have a decreased frequency of progression to a higher Rai stage during 60-month follow-up (21.35 vs. 40.8%, p=0.013) compared with other individuals.

Conclusion:

This study showed that the investigated polymorphisms do not modulate the risk of B-CLL in the Polish population, but are associated with disease dynamics in particular with the time to Rai stage progression.

Disclosures:

No relevant conflicts of interest to declare.
Cyclin D1 mRNA Expression In CLL: a Pilot Study.
Authors: Mocikova Heidi , Pekova Sona , Zejskova Lenka , Spacek Martin , Kozak Tomas
Published: October 2019

Abstract 4619

Background.

Expression of cyclin D1 demonstrated by immunohistochemistry is seen in 95% of mantle cell lymphomas and in other lymphoproliferative diseases including 10% of chronic lymphocytic leukemias (CLL). This study analyzed the impact of cyclin D1 positivity on time to treatment (TTT) and overall survival (OS) measured by RT PCR in newly diagnosed CLL patients and correlation with reported prognostic factors.

Patients and methods.

Level of cyclin D1 (quantitative real-time RT-PCR with a specific TaqMan flurescent hybridization probe) mRNA expression was analysed in 72 samples (57 peripheral blood and 15 bone marrow) from patients with newly diagnosed CLL. Cyclin D1 expression (cut-off according to ROC curve >3 over the threshold expression in healthy donors) was reported as positive. Fisher's exact test was used to analyze the relationship of cyclin D1 positivity and prognostic factors: del17p, del11q, unmutated IgVH, trisomy 12, ZAP 70 and CD38 positivity, elevated B2microglobulin, elevated LDH and lymphocyte doubling time (LDT) <6 months. The comparison of time to treatment (TTT) and prognostic factors with cyclin D1 positivity was calculated via Spearman correlation coefficient. Survival curves were calculated by Kaplan-Meier survival analysis and comparison between subgroups was performed by the log-rank test.

Results.

Cyclin D1 was positive in 29 (40%) CLL patients. Although cyclin D1 was not statistically significant for TTT (P=0,145), a trend was observed, suggesting a negative prognostic impact of cyclin D1 overexpression in CLL. Following variables correlated significantly with TTT: del17p (P=0.037), del11q (P=0.003), unmutated IgVH (P=0.004), trisomy 12(P=0.024), positive CD38 (P=0.014), elevated B2microglobulin (P<0.001), elevated LDH (P<0.001) and LDT <6 months (P<0.001). Del 17p, del 11q, trisomy 12 and elevated B2microglobulin were independent factors for TTT in the multivariate analysis. None of these factors were significant for overall survival due to the short follow-up.

Conclusion.

Cyclin D1 measured by RT-PCR from peripheral blood or bone marrow has no statistically singificant impact on TTT or OS, though a trend pointing to cyclin D1 overexpression in CLL as a negative prognostic marker can be suggested. This data should be confirmed on a larger cohort of patients with a longer follow-up.

Disclosures:

No relevant conflicts of interest to declare.
Left Atrium Enlargement Is An Independent Predictor of Overall Mortality In Patients with Cardiac AL Amyloidosis.
Authors: Mohty Dania , Pibarot Philippe , Darodes Nicole , Tanguy Benedict , Lavergne David , Echahidi Najmeddine , Virot Patrice , Bordessoule Dominique , Jaccard Arnaud
Published: October 2019

Abstract 4621

Background:

Primary systemic amyloidosis (AL) is a severe plasma cell disorder characterized by amyloid fibrils extracellular deposition in different organs. Myocardial involvement is frequent and has major impact on prognosis. Echocardiography (TTE) is the most common test performed when cardiac involvement is suspected. We hypothesized that a simple measurement of left atrium enlargement (LA) by TTE may provide an important risk marker for this disease.

Methods and results:

Between 1997 and 2010, 114 patients were diagnosed with systemic AL. Sixty one patients of the cohort (54%) had cardiac involvement on the basis of standard criteria defined as: mean LV wall thickness >12mm with no other cardiac causes. They had first TTE within 16 days of diagnosis. Patients were mainly treated with conventional chemotherapy (M-Dex) or with new agents for refractory or relapsing patients We retrospectively collected demographic baseline characteristics along with biological and echo data of these patients. Mean age was 61±11 years; 60% were male; 19% had hypertension. Mean left ventricular ejection fraction and mean LV wall thickness were respectively 58±13% and 15±3 mm. Mean follow up time was 2.1±2 years. None had significant valvular heart disease. LA enlargement was defined by M mode as > 40 mm in male and > 36 mm in female. Patients with enlarged LA had significantly more hypertension and lower ejection fraction and more hypertrophied LV walls (All P< 0.05). At 3 years, survival rate was markedly reduced in patients with enlarged LA vs. those with normal LA: 34±9% vs.75 ±9% (P =0.02). By multivariate analysis, after adjusting for age, gender, and presence of hypertension, LA enlargement remained an independent predictor of overall mortality at 3 years (HR=2.88; CI (1.12-8.84); P=0.03).

Conclusion:

In patients with systemic AL amyloidosis and cardiac involvement, LA enlargement, a surrogate marker of diastolic dysfunction and elevated LV filling pressure, is a powerful independent predictor of long-term mortality. Therefore LA enlargement may help to enhance risk stratification in patients presenting with this disease.

Disclosures:

No relevant conflicts of interest to declare.
Differential Gene Expression Involved In Angiogenesis, Metabolism, Cell Proliferation and Self-Renewal and Pluripotency In Myelodysplastic Syndromes (MDS) and Acute Myeloid Leukemia (AML)
Authors: Falantes Jose F , Calderón Cristina , Trujillo Pablo , Martin-Antonio Beatriz , González Jose , Urbano Alvaro , Carmona Magdalena , Espigado Ildefonso
Published: October 2019

Abstract 4646

Background

Myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML) are clonal disorders characterized by clinical and prognostic heterogeneity, mainly explained by different genetic abnormalities among other factors. The role of some key genes involved in disease pathogenesis in both entities remain unclear.

Methods and patients

Study objective was to analyse differences in gene expression related to angiogenesis, metabolism and cell proliferation, self-renewal and pluripotency in patients (pts) with MDS and AML. Thirty-three bone marrow (BM) samples at diagnosis were analysed and distributed in 4 different groups: control group (n=8), low-risk MDS (LR-MDS:<10% BM blasts; n=15), high-risk MDS (HR-MDS:>10% BM blasts; n=4) and AML (n=6). Total RNA was isolated from BM samples. Genes analysed were: vascular endothelial growth factor (VEGF) for angiogenesis, MYC, macrophage migration inhibitory factor (MIF) and glycogen synthase (GYS) for metabolism and cell-proliferation and Oct4 as transcription factor required to maintain an undifferentiated state for self-renewal and pluripotency. Gene expression was quantified by qRT-PCR in triplicate using ß-actin gene as control. SPSS software (v.16) and Mann-Whitney U-test were applied for statistical analysis (P value ≤.05 was considered significant in all cases).

Results

After analysis, only mRNA levels of VEGF and MYC showed significant difference between LR-MDS and the control group. However, higher expression of all genes were observed in HR-MDS vs LR-MDS (p≤.05) as shown in table 1. When compared HR-MDS to AML, no difference was observed in VEGF, MYC, MIF and GYS, but significant difference was noticed in mRNA expression of Oct4 in AML samples (p=.032) vs HR-MDS. Globally, gene expression in MDS (pts with LR and HR-MDS) was significant lower than in AML pts in all genes studied as expected.

Conclusions

Increased expression of VEGF, cMYC, MIF, GYS and OCT4 in HR-MDS vs LR-MDS and in AML vs MDS (global) suggests that these factors may play a relevant role in pathogenesis of both entities. These results point towards a different biological behaviour in less proliferative disease respect advanced stages and AML in different cellular pathways involved in disease progression. They might also justify clinical heterogeneity among patients with MDS and in patients with AML vs MDS as a sole group, and also being responsible for different response to treatment options. Analysis of protein expression is ongoing.

Disclosures:

No relevant conflicts of interest to declare.
Identification of JAK1 Mutation In 40 Cases of Adult Acute T Lymphoblastic Leukemia
Authors: Sun Aining , Wang Wenjuan , Chen Guanghua , Depei Wu , Chen Suning , Chang Huirong , Ding Zixuan
Published: October 2019

Abstract 4652

Objective

This study was purposed to analyze the mutations of JAK1 in bone marrow cells from adult patients with newly-diagnosed T cell acute lymphoblastic leukemia (T-ALL).

Methods

The entire coding sequence of JAK1 in bone marrow mononuclear cells (MNCs) from 40 adult T cell ALL patients were screened by polymerase chain reaction (PCR) and direct sequencing, then the clinical features of JAK1 mutation positive patients were analyzed.

Results

JAK1 mutations were identified in 5%(2/40) patients, one is point mutation, the other is insertion mutation, resulting in changes in amino acid sequences, and the two types of mutations are different from those mutations found before.

Conclusion

The JAK1 mutations were found in about 5% newly-diagnosed T cell acute lymphoblastic leukemia, which was lower than those report abroad.

Disclosures:

No relevant conflicts of interest to declare.
Preclinical Safety of Baxter's Recombinant Factor IX
Authors: Dietrich Barbara , Kubik Susan , Auer Wilfried , Wuersch Kuno , Reipert Birgit , Horling Frank , Wolfsegger Martin , Ehrlich Hartmut J. , Scheiflinger Friedrich , Schwarz Hans Peter , Muchitsch Eva-Maria
Published: October 2019

Abstract 4654

Baxter has developed a rFIX product which is produced by a genetically engineered Chinese hamster ovary (CHO) cell line in a cell culture medium free from any animal or human proteins. The objective of this preclinical study-program was to evaluate the safety of Baxter's rFIX in different species.

The preclinical program included studies on established models of general safety pharmacology (conscious, telemetered cynomolgus monkey), thrombogenicity (rabbit, Wessler test), single and repeated dose toxicity (mouse, rat, cynomolgus monkey), local tolerance (rabbit), and comparative immunogenicity (mouse, rat, cynomolgus monkey). Commercially available licensed rFIX and pdFIX served as active reference items.

Thrombogenicity studies showed no thrombogenic potential of Baxter's rFIX. General safety pharmacology revealed no adverse effects on clinical signs, cardiovascular or respiratory variables. Single dose toxicity studies in mice, administered at doses of up to 7500 IU/kg BW, and repeated administration of Baxter's rFIX in rats and cynomolgus monkeys, administered doses of up to 750 IU/kg, confirmed the safety of the new product. Furthermore, Baxter's rFIX was well tolerated at the injection site after intravenous injection. No differences in immunogenicity between Baxter's rFIX and the reference items were revealed by comparative immunology studies.

This good safety profile of Baxter's rFIX was the basis for proceeding with human trials, which have recently been initiated.

Disclosures:

Dietrich: Baxter Innovations GmbH: Employment. Kubik:Baxter Innovations GmbH: Employment. Auer:Baxter Innovations GmbH: Employment. Wuersch:Baxter Innovations GmbH: Employment. Reipert:Baxter Innovations GmbH: Employment. Horling:Baxter Innovations GmbH: Employment. Wolfsegger:Baxter Innovations GmbH: Employment. Ehrlich:Baxter Innovations GmbH: Employment. Scheiflinger:Baxter Innovations GmbH: Employment. Schwarz:Baxter Innovations GmbH: Employment. Muchitsch:Baxter Innovations GmbH: Employment.
The cytokines of helper T cells in Patients with immune Thrombocytopenic Purpura
Authors: Li Feng , Wu Boting , Zhan Yanxia , Cheng Yunfeng
Published: October 2019

Abstract 4663

Immune thrombocytopenic purpura (ITP) is traditionally considered an auto-antibody mediated disease. Recently the role of T cell immunity in ITP has been recognized and discussed by various researchers. In order to investigate changes in T cell subgrouping of ITP patients, we enrolled 20 previously untreated ITP patients (8 males, 12 females) with a median age of 41 (range: 20 to 81) and 20 normal controls. After standard dose of prednisone treatments (1mg·kg-1·d-1), 12 patients achieved complete remission and 7 partial remission. Using real-time RT-PCR methods, we measured the expression of 7 T cell associated genes including T-bet, IFN-γ, GATA-3, TGF-β, Foxp3, IL-2, IL-4 in PBMC of ITP patients before and after treatment and of normal controls. The results showed that T-bet, IFN-γ and IL-2 were significantly over-expressed in ITP patients (p<0.001), which represented a Th1 up-regulation. After treatment, T-bet, IFN-γ, IL-2 and IL-4 were markedly down-regulated, while Foxp3 were up-regulated (p<0.05). Interestingly, TGF-β showed a different pattern between old and young patients. It was down-regulated (p<0.05) among patients younger than 60, while up-regulated in older patients. In conclusion, we found significant change of T cell associated gene expression in ITP patients. And standard dose of prednisone treatment could effectively tune T cell subgroups to a suppressive pattern.

Disclosures:

No relevant conflicts of interest to declare.
Romiplostim Treatment Mimicking Cyclic Thrombocytopenia In 3 Patients with Chronic Idiopatic Thrombocytopenic Purpura
Authors: Iuliano Francesco , Mingrone Tecla , Infusino Stefania , Perricelli Alessia , Pomillo Angelo , Luci Maria , Maio Massimo Di
Published: October 2019

Abstract 4665

Introduction

The thrombopoietic growth factors (TGFs) are a novel class of compounds for the treatment of chronic immune thrombocytopenia (ITP). The first of these agents to receive regulatory approval, romiplostim and eltrombopag, have demonstrated impressive efficacy and tolerability in randomized controlled trials and open-label extension studies of several years duration and stand poised to revolutionize the management of ITP. Nonetheless, critical questions regarding the safety of these agents, remain partially unanswered. The aim of this report is to focus on the incidence of thrombocytosis and rebound thrombocytopenia mimicking cyclic thrombocytopenia in a series of 15 patients treated with romiplostim in a single institution.

Methods

15 patients (8 M and 7 F, median age 45 years range 21–76) with chronic ITP (median onset 6 years, range 1–11), 1/15 splenectomized, plts median 2 ×109/L, (range 18–24) were treated with Romiplostim initial dose of 1 mg/kg, weekly checks, with only weekly increase of 1 mg/kg to reach a platelet count ≥ 50 × 109/L. The target platelet count range was 50 to 250 × 109/L. Twenty-two percent of patients (3/15) were receiving concurrent treatment for ITP (corticosteroids, danazol) at the time of first romiplostim dose. These medications were discontinued after platelet counts reached 50 × 109/L.

Results

A platelet response had been achieved by 30% of patients after the first dose and by 51% of patients after the third dose. Treatment response was observed in 100% of pts treated after 6 weeks. In three patients after the eighth week, with average values plts 120×109/l, the subsequent administration of the drug led to an increase in platelet count > 1000×109/l, with rapid fall to <30 ×109/l at the third week suspension in 2/3, while the third patient is in CR at six months follow-up despite treatment was stopped.

Conclusion

According to an analysis of all patients with ITP treated with romiplostim in 4 controlled trials have been reported 3 thrombocytosis events in 271 pts (1.1%). In our series the percentage was 20% (3/15), much higher than that reported in the literature, with no thrombotic complications related. Interesting in a case CR was still maintained at 8 months of follow-up despite cessation of treatment. It would be very useful to create a network of information between users of romiplostim to evaluate on a larger series, the true incidence of thrombocytosis and any related thrombotic phenomena associated in a day by day clinical practice.

Disclosures:

No relevant conflicts of interest to declare.
Clinical Characteristics of Thrombotic Thrombocytopenic Purpura with Severe ADAMTS13 Deficiency
Authors: Jang Moon , Chong So Young , Kim Inho , Jung Chul W. , Oh Doyeun
Published: October 2019

Abstract 4666

The clinical significance of ADAMTS13 activity for response to treatment, mortality rate, recurrence, and prognosis is unclear. Therefore, we investigated the characteristics of severe ADAMTS13 deficiency and evaluated its clinical significance in Thrombotic thrombocytopenic purpura (TTP). The Korean TTP Registry includes 66 patients from 13 teaching hospitals in Korea who received the diagnosis of TTP from January 2005 to December 2008. Blood samples obtained upon admission were sent for ADAMTS13 analysis (multimer analysis by sodium dodecyl sulfate electrophoresis and/or ELISA) to a central laboratory along with patient clinical information. After 6 months, patient data regarding treatment, response, and prognosis were collected on standardized report forms. Patients with severe ADAMTS13 deficiency had lower serum creatinine levels (P=0.001) and WBC counts (P=0.050) than patients with non-severe ADAMTS13 deficiency. Although severe ADAMTS13 deficiency was associated with better response rate (75% vs 53%, P=0.145), remission rate (81% vs 61%, P=0.209), and mortality rate (19% vs 31%, P=0.508) than non-severe ADAMTS13 deficiency, treatment outcomes did not differ significantly between groups. After adjusting for clinical and laboratory features, multivariate analysis did not reveal any independent risk factors for TTP-associated mortality. Patients with severe ADAMTS13 deficient had lower serum creatinine levels and WBC counts at presentation but Severe ADAMTS13 activity deficiency at TTP diagnosis does not appear to have prognostic significance.

Disclosures:

No relevant conflicts of interest to declare.