bluuebunny/crossref_metadata_2025 huggingface dataset:
Plerixafor and G-CSF Versus Cyclophosphamide and G-CSF for Stem Cell Mobilization in Patients with Multiple Myeloma.
Authors: Nazha Aziz , Cook Rachel , Vogl Dan T. , Mangan Patricia A. , Hummel Kimberly , Cunningham Kathleen , Luger Selina , Porter David , Schuster Stephen J. , O'Doherty Una , Sell Mary , Siegel Don L. , Stadtmauer Edward A.
Published: October 2019

Abstract 2146

Poster Board II-123

Introduction:

High dose melphalan and autologus stem cell transplant remains an effective treatment for patients with either early or refractory multiple myeloma (MM). Collection of sufficient numbers of stem cells for more than one transplant is optimal. G-CSF with chemotherapy, particularly cyclophosphamide (CY/G-CSF), has been a widely used and effective regimen for stem cell collection in MM. Plerixafor, a CXCR4 antagonist, when combined with G-CSF has been shown in a large randomized clinical trial to be superior to G-CSF alone. A comparison of plerixifor/G-CSF to CY/G-CSF is presented here.

Materials and Methods:

We performed a single institution retrospective analysis of 365 patients with MM who underwent stem cell mobilization and harvest at the University of Pennsylvania Abramson Cancer Center from January 2002 to December 2007. All patients were harvested early in the course of their disease. 76 patients were excluded from this analysis (23 had incomplete data on induction regimen, 19 had incomplete data on stem cell collection, 16 had incomplete data on mobilization regimen, 10 underwent allogeneic transplants, 2 had bone marrow rather than peripheral blood harvests, 2 had stem cells collected at an outside institution, 2 had chemotherapy mobilization other than CY and 2 had medical complications prior to harvest and after mobilization). Therefore, 289 patients were included in the analysis; 16 received plerixafor/G-CSF, 198 received CY/G-CSF, and 75 received G-CSF alone.

Results:

The median number of collected stem cells was 7.95 × 106 CD34+/kg in plerixafor/G-CSF group, 7.7 × 106 CD34+/kg in Cy/G-CSF group and 4.5 × 106 CD34+/kg in G-CSF alone group. The median number of apheresis days was 2 days, 2 days and 4 days respectively. The percentage of the patients who collected ≥ 6 × 106 CD34+/kg in < 3 apheresis was 63% (10/16), 62% (123/198) and 19% (14/75) respectively. The percentage of the patients who collected ≥ 6 × 106CD34+/kg <5 apheresis was 81% (13/16), 69% (136/198) and 23% (17/75) respectively. The mean CD34+/kg collected erither after CY/G-CSF or plerixafor/G-CSF was higher than G-CSF alone (p<0.0001 for each analysis).

Conclusion:

This analysis suggests that plerixafor/G-CSF and CY/G-CSF mobilization result in similar and adequate stem cell harvest numbers for autologous stem cell transplantation for MM. Both approaches are superior to G-CSF alone. The choice of plerixafor/G-CSF vs CY/G-CSF for stem cell mobilization will therefore depend on further analysis of the relative costs, toxicities and long term outcome of these regimens.

Disclosures:

Stadtmauer: genzyme: Consultancy.
Wnt-Pathway Directed Compound Targets Blast Crisis and Chronic Phase CML Leukemia Stem Progenitors.
Authors: Peterson Luke F , Turbiak Anjanette J , Giannola Diane M , Donato Nicholas , Showalter Hollis H.D. , Fearon Eric R , Talpaz Moshe
Published: October 2019

Abstract 2168

Poster Board II-145

The deregulated tyrosine kinase associated with t(9;22) fusion protein BCR-ABL is efficiently targeted by tyrosine kinase inhibitors (Imatinib, Nilotinib and Dasatinib). It is generally believed that most patients have residual disease. The inability to eliminate the refractory leukemia stem cell in chronic myeloid leukemia (CML) is not well understood. The refractory stem cell is present despite effective inhibition of the BCR-ABL kinase in these cells, and dissociation between kinase inhibition and cell death implies lack of dependency on BCR-ABL aberrant kinase activity, unlike the one seen in more mature CML cells. Thus the oncogene addictive dependency of these leukemia stem cells may be overcome by intrinsic and/or extrinsic factors/pathways. One such pathway involved in hematopoeitic stem cell maintenance is the Wnt/β-catenin signaling pathway. BCR-ABL signaling is known to directly activate the Wnt/β-catenin pathway. The loss of function of a negative regulator of the Wnt/β-catenin pathway, known as GSK3β is associated with CML progression from chronic phase to the blast phase. Lastly, the loss of β-catenin in murine models impairs the self renewal capacity of both the normal and the BCR-ABL+ leukemia stem cell. Thus this pathway is important during various stages of the disease.

We explored the effect of Wnt inhibition using a novel Wnt pathway inhibitor (AG-214, University of Michigan). AG-214 is structurally related to a previously reported Wnt-inhibitor. AG-214 is able to antagonize β-catenin/TCF in luciferase reporter assays, and expression of Wnt targets in colon cancer cell lines. We utilized a serum free culture system with 5 added cytokines and treated both CML blast crisis CD34+ as well as chronic phase CD34+ cells with AG-214. Our in vitro experiments show that primary blast crisis CD34+ cells are induced to undergo apoptosis at an IC-50 of approximately 2 μM. Furthermore, combination of 1.25 μM of AG-214 with 2 μM Imatinib achieved greater than 50% apoptosis. Analysis of chronic phase CML CD34+ cells showed that targeting of Wnt/β-catenin pathway requires higher concentration of the Wnt-inhibitor (>2.5 μM), and that the addition of Imatinib can cooperate to enhance the apoptotic response. Furthermore, chronic phase progenitors (Lin-CD38+/CD34+) are more sensitive to lower concentrations of AG-214 whereas 5 μM is required to induce significant apoptosis of ∼70% in the primitive leukemia stem cell (Lin-/CD38-/CD34+) population, and addition of 2 μM Imatinib increased their apoptotic response to ∼84%. Normal CD34+ cells do not undergo significant apoptosis with 5 μM of AG-214 (∼10%) even when combined with 2 μM Imatinib (∼20%). Targeting of the Wnt/β-catenin pathway enhances apoptosis in both blast crisis and chronic phase CML progenitors and leukemia stem cells.

Disclosures:

No relevant conflicts of interest to declare.
A Common but Overlooked Mechanism of BCR-ABL1 Kinase Inhibitor Resistance in Chronic Myeloid Leukemia.
Authors: Bruey Jean-Marie , Kantarjian Hagop M. , Ma Wanlong , Yeh Chen-Hsiung , Lee Tai-Sung , O'Brien Susan , Giles Francis , Estrov Zeev , Cortes-Franco Jorge , Albitar Maher
Published: October 2019

Abstract 2179

Poster Board II-156

Point mutations in the BCR-ABL1 protein are the most commonly reported mechanism of resistance to imatinib mesylate (Gleevec or STI-571) treatment in patients with chronic myelogenous leukemia (CML). We have reported that some patients with imatinib-resistant CML express an alternatively spliced BCR-ABL1 mRNA with a 35-bp insertion (BCR-ABL135INS), but the prevalence and effect of this mutation have not been well delineated. We sought to improve understanding of this mutation in order to provide insight into mechanisms of resistance to tyrosine kinase inhibitors. Using a sensitive PCR method, we determined the prevalence of the alternatively spliced BCR-ABL135INS mRNA in a group of 168 patients with chronic-phase CML resistant to imatinib. Expression of truncated protein was confirmed by Western blot in patient samples. The effects of various kinase inhibitors on human K562 CML cells transfected with BCR-ABL135INS cDNA were tested by measuring cell survival, caspase-3 activation, and the BIM activation pathway. In our analysis of BCR-ABL1 genotypes in 168 imatinib-resistant chronic-phase CML patients, (56%) of patients expressed various levels of BCR-ABL135INS whereas only (25%) had detectable point mutations in BCR-ABL1. Expression of this truncated BCR-ABL1 fusion protein was also confirmed by immunoprecipitation at levels proportional to those predicted by mRNA quantification. In vitro, BCR-ABL135INS expression conferred resistance to the tyrosine kinase inhibitors imatinib, dasatinib, and nilotinib in a concentration-dependent fashion, but had no effect on the protein-synthesis inhibitor homoharringtonine (HHT or omacetaxine) or the aurora kinase inhibitor MK-0457 (VX-680). The combination of imatinib with nilotinib or HHT was synergistic in overcoming BCR-ABL135INS-induced resistance. Moreover, the resistance to imatinib induced by BCR-ABL135INS was associated with loss of BIM expression. This loss of BIM was overcome by pretreatment with HHT. In conclusion, these findings emphasize the importance of the overlooked alternatively spliced BCR-ABL135INS found in imatinib-resistant CML patients. Optimal patient management in the future will likely require periodic testing for the expression of BCR-ABL-135INS mRNA during therapy. This may help in predicting various levels of resistance or the potential for eliminating the disease by adjusting the treatment strategy.

Disclosures:

No relevant conflicts of interest to declare.
Alefacept Treatment for Graft Vs. Host Disease May Not Suppress the Graft Vs. Leukemia Effect.
Authors: Dray Liliane , Matusevich Aliza , Resnick Igor , Aker Memet , Stepansky Polina , Or Reuven , Shapira Michael Y.
Published: October 2019

Abstract 2223

Poster Board II-200

Introduction:

In general it is expected that strong immune suppression that alleviates GVHD, will increase the risk of relapse. Alefacept (Amevive®) is an immunosuppressive dimeric fusion protein that is used for psoriasis control. We showed its remarkable effect in acute steroid resistant/dependent and chronic extensive GVHD.

Methods:

To date, 42 patients with a median age of 31.5 years (range 3-66) were treated by us with alefacept due to acute (n=28) or chronic (n=14) steroid resistant/dependent GVHD (27 males, 15 females). Twenty were transplanted from HLA matched family members, 14 from HLA matched unrelated donors, 8 from mismatched donors and 2 from unrelated cord blood units. Pretreatment acute GVHD grade ranged 2-4 (median 3) and involved the skin (30), gut (19) and liver (7). All the patients with chronic GVHD had extensive involvement prior to therapy.

Results:

The median time from transplantation to alefacept was 42.5 days and 13 months in acute and chronic GVHD respectively (range 18-110d and 3-47.5m) and a median of 9 (range 1-25) injections that were given per patient. Thirty-four out of the 41 evaluable patients (83%) responded to the treatment (23/28 and 11/13 in the acute and chronic group respectively). Despite this high response rate, demonstrating the deep immunosuppressive and immunomodulative effect of alefacept, only 5/41 evaluable patients (figure 1) have relapsed (with a median follow-up of 30.8 months on the 17 survivors). Other than the 5 patients that relapsed (all treated with calcineurin inhibitor and steroids), full-donor peripheral blood chimerism (100% donor cells and no residual host-type DNA) was stable throughout the treatment period and later in all but one patient, that developed mixed chimerism under alefacept treatment. His chimerism returned to full-donor chimerism with taper-down of immune suppression. Currently, 17/42 (40.5%) patients are alive (figure 1), most with improved or stable GVHD. Twenty-six patients died due to either: GVHD progression (n=14), progression of the basic disease (n=4), infections (n=5) or other causes (n=3).

Conclusions:

Alefacept is effective and safe for the treatment of acute or chronic steroid resistant/dependent GVHD and may discriminate between GVHD and GVL.

Disclosures:

No relevant conflicts of interest to declare.
High Levels of B-Cell Activating Factor During the Peri-Transplant Period Are Associated with the Reduced Incidence of Acute Graft-Versus-Host Disease Following the Myeloablative Allogeneic Stem Cell Transplantation.
Authors: Cho Byung Sik , Min Chang Ki , Kim Hee Je , Lee Seok , Kim Yoo Jin , Jeong Dae-Chul , Cho Bin , Kim Hack Ki , Eom Ki-Seong , Cho Seok Goo , Kim Dong Wook , Lee Jong Wook , Min Woo Sung , Park Jong Won , Kim Chun Choo , Chung Nak Gyun
Published: October 2019

Abstract 2229

Poster Board II-206

To determine whether serum B-cell activating factor (BAFF) levels that were previously found to be elevated in patients with chronic graft-versus-host disease (GVHD), and C-reactive protein (CRP) levels known as a predictor for aGVHD as well as a marker for systemic inflammation during the peri-allogeneic stem cell transplantation (SCT) period, could be used to predict the occurrence of acute GVHD (aGVHD), 45 consecutive patients who had undergone myeloablative allogeneic SCT for hematologic malignancies were assessed. Serum BAFF and CRP levels were measured using ELISA (R&D Systems, Minneapolis, MN, USA) before conditioning and on day 0, day +7, and day +14 after transplant. Thirty-three of 45 patients (cumulative incidence, 73=) developed aGVHD between 16 days and 98 days after transplant. Analyses using repeated measures of ANOVA revealed that the serum BAFF levels were significantly lower in patients with aGVHD than in those without aGVHD (P=0.001), whereas no association was detected between CRP levels and aGVHD (P=0.508). Receiver operating characteristic curve (ROC) analysis showed that serum BAFF levels at every time point were available for the prediction of development of aGVHD (pre-conditioning; P=0.005, day 0; P=0.002, day +7; P=0.004, and day +14; P=0.005). Using ROC curve analysis, the identical cutoff value of 43 pg/ml at every time point that divides patients into two groups, high (> 43 pg/ml) or low (≤ 43 pg/ml) BAFF group, was determined, which could assure 75= sensitivity and 73-82= specificity for the prediction of aGVHD at every time point. The analyses of the cumulative incidence of aGVHD at each time point by BAFF groups (high vs. low) showed that serum BAFF level at every time point plays a significant predictive role for the occurrence of aGVHD (pre-conditioning; P=0.040, day 0; P=0.023, day +7; P=0.003, and day +14; P=0.026). This study is, to the best of our knowledge, the first to show that high serum BAFF levels during the peri-transplant period may play a protective role against aGVHD in humans. The results of this study also show that the BAFF levels during peri-transplant period may be considered to be a predictor for aGVHD. Further trials with a larger cohort will be necessary to determine a definite cutoff value of high BAFF level as well as to construct an index with previously known factors for the prediction of aGVHD.

Disclosures:

No relevant conflicts of interest to declare.
MicroRNAs Are Differentially Expressed Among Cytogenetic Subgroups in Multiple Myeloma.
Authors: Corthals Sophie L , Jongen-Lavrencic Mojca , de Knegt Yvonne , Goldschmidt Hartmut , Lokhorst Henk , Sonneveld Pieter
Published: October 2019

Abstract 2833

Poster Board II-809

Micro-RNAs (miRNAs) are a class of small non-coding single stranded RNAs of 20-22 nucleotides in length. MiRNAs regulate gene expression by binding to the 3' UTR of target mRNAs, which leads to either mRNA degradation or translational inhibition of this target mRNA. MiRNAs play a critical role in cell growth, survival and differentiation and have been suggested to function as tumor suppressor and oncogenes, and/or play a critical role in the pathogenesis of Multiple Myeloma (MM). We investigated miRNA expression in 41 newly diagnosed patients with MM who were included in a clinical trial. MiRNA expression profiling was performed in BM derived CD138 selected plasma cells (PC) obtained from newly diagnosed MM patients with a minimum monoclonal PC purity of > 80%. A quantitative PCR-method (TaqMan low density arrays-microRNA assay', Applied Biosystems) was used for miRNA quantification. The relative expression levels of miRNAs were calculated using the 2-DDCt method and the data was normalized using the endogenous control RNU48. 176/365 miRNAs (48.2%) were expressed in these MM patients. Unsupervised hierarchical clustering based on the average-linkage method and principal component analysis (PCA) was performed in Partek software (Partek Genomics Suite), showing a differential hierarchy for the chromosomal translocation or gain subgroups. When patients with or without chromosome 13 deletions were compared, no correlation was found between expression levels of miRNAs located on chromosome 13 and the deletion status of this chromosome as determined by FISH. Three miRNAs, i.e. miRNA-126, miRNA-145 and miR-517c were identified that were different between MM patients with or without chromosome 13 deletion. These miRNAs were not located on chromosome 13. Likewise, when comparing the miRNA expression in MM patients with or without chromosome 1q gain, the three most differentially expressed miRNAs were miRNA-23a, miRNA-200a# and miRNA-145, which are not on chromosome 1. In addition, miR-18a, located on chromosome 1 was upregulated in MM patients with 1q gain. MM patients with t(4;14) showed differential expression of miRNA-520g, miRNA-28 and miRNA-502. Currently we evaluate potential association of microRNA expression with the clinical response of MM patients to Bortezomib.

In conclusion, our data indicate that miRNAs are differentially expressed in subgroups of MM patients characterized by common cytogenetic abnormalities.

Disclosures:

Goldschmidt: Johnson and Johnson: Research Funding, Speakers Bureau. Sonneveld:Johnson and Johnson: Research Funding, Speakers Bureau.
SIGNIFICANCE of ABSOLUTE LYMPHOCYTE COUNT at RELAPSE as a PROGNOSTIC FACTOR in PATIENTS with T-CELL NON-HODGKIN'S LYMPHOMA.
Authors: Choi Dae Ro , Yoon Dok Hyun , Ahn Heui June , Cho Yoojin , Kim Eun Kyoung , Kim Shin , Huh Jooryung , Lee Dae Ho , Kim Sang We , Lee Jung Shin , Suh Cheolwon
Published: October 2019

Abstract 2939

Poster Board II-915

Introduction:

Peripheral blood absolute lymphocyte count (ALC) at the time of diagnosis is a prognostic indicator in hematologic malignancies. However, no reports have addressed whether ALC at the time of first relapse (ALC-R) has a prognostic significance in the patients with relapsed T-cell Non-Hodgkin's lymphoma (NHL). We retrospectively studied the prognostic significance of ALC-R in these patients.

Patients and Methods:

We identified 63 patients who had a documented relapsed disease after having reached a complete response, including at least an unconfirmed complete response between 1993 and 2007 and we analyzed their ALC-R and following variables at the time of first relapse; age, gender, the number of extranodal sites, lactate dehydrogenase, ECOG performance status, stage and international prognostic index.

Results:

Out of the 63 patients, 23 (36.5%) had a peripheral T-cell lymphoma, not otherwise characterized, while 15 (23.8%) had an extranodal NK/T-cell lymphoma, nasal type, 6 (9.5%) anaplastic large-cell lymphoma, 5 (7.9%) angioimmunoblastic T-cell lymphoma, 2 (3.2%) primary cutaneous T cell lymphoma and 12 (19%) other types. Among them, 32 (50.8%) had an ALC-R ≥ 1.25 × 109/L, 47 (74.6%) had an ECOG PS 0 or 1 and by IPI at relapse, 0, 1, 2, 3, 4, and 5 were 20.6%, 25.4%, 23.8%, 23.8%, 4.8%, and 1.6%, respectively. Univariate analyses showed that good ECOG PS (HR, 0.408; 95% CI 0.190-0.876; p=0.022) and high ALC at relapse (HR, 0.394; 95% CI 0.210-0.740; p=0.004) were associated with longer survival from relapse (Table 1). Multivariate analysis also showed that high ALC at relapse (HR, 0.369; 95% CI 0.187-0.726; p=0.004) and good ECOG PS (HR, 0.295; 95% CI 0.131-0.666; p=0.003) were still associated with longer survival outcome (Table 2).

Conclusions:

The high ALC-R predicted a better prognosis in patients with relapsed T-cell NHL, suggesting that the host immune system might have a crucial role.

Disclosures:

No relevant conflicts of interest to declare.
PTEN Regulates SYK-Directed AKT Activation in MCL.
Authors: Rudelius Martina , Pittaluga Stefania , Sebasigari Denise , Davies-Hill Theresa , Klier Margit , Quintanilla-Martinez Leticia , Fend Falko , Tabe Yoko , Raffeld Mark
Published: October 2019

Abstract 2941

Poster Board II-917

Mantle cell lymphoma (MCL) can be divided into two clinicopathologic subtypes, a common or “typical” form, and an aggressive “blastoid” variant. A subset of MCL, including all of the aggressive blastoid variants, shows constitutive activation of the PI3K/AKT pathway. Since the BCR-associated SYK tyrosine kinase can activate the PI3K/AKT pathway during normal BCR signaling, we wished to assess its role in MCL.

Five MCL cell lines (Granta 519, Rec-1, Jeko, Z138C and JVM-2) and 32 primary cases were studied for SYK activation and phosphorylation of several SYK targets, including blnk and PLC-gamma. The functional role of SYK was assessed by pharmacologic inhibition with R406, Piceatannol and SYK II inhibitor (2-(2-Aminoethylamino)-4-(3-trifluoromethyl-anilino)-pyrimidine-5-carboxamide, Dihydrochloride, Dihydrate). To rule out off target effects functional data was validated by siRNA experiments. Subsequently we analysed the effects of PTEN and constitutively active (S380A) PTEN in 2 cell lines (Granta 519 and Z138C).

All cell lines were found to have constitutively activated SYK accompanied by phosphorylation of its downstream targets blnk and PLC-gamma. Inhibition of SYK with pharmacologic inhibitors or by specific siRNA resulted in AKT inactivation, suggesting a causal relationship between SYK activation and AKT activation in the cell lines. However, all primary MCL cases demonstrated constitutive activation of SYK, regardless of the activation state of AKT. Interestingly, p-AKT expression correlated with PTEN inactivation, as assessed by p-PTEN expression, in both primary cases and MCL cell lines. Increasing PTEN phosphatase activity, by introducing a constitutively active PTEN, abrogated AKT phosphorylation, without affecting SYK activity. These data suggest that SYK activity in MCL is necessary, but not sufficient to activate the PI3K/AKT pathway, and that an additional critical step is the inactivation of PTEN. This study reveals new complexity in the dysregulation of survival pathways in MCL that may be relevant to pathogenesis.

Disclosures:

No relevant conflicts of interest to declare.
Constitutive Activation of Rac GTPases Is Induced by Cooperating Mutations in a Human Model of MLL-AF9 Leukemia.
Authors: Wunderlich Mark , Link Kevin A , Chou Fu-Sheng , Mulloy James C.
Published: October 2019

Abstract 2959

Poster Board II-935

Retroviral transduction of primary human umbilical cord blood CD34+ cells with a gene encoding MLL-AF9 (MA9) efficiently promotes immortalization in vitro and development of leukemia in a xenotransplant model. MA9 cells remain cytokine dependent with a strict requirement for Flt3 signaling. Additionally, the cells are hypersensitive to inhibition of the RAC GTPases. In this current study, we have examined a potential link between these pathways. We found that MA9 cells proliferate in culture media containing Flt3 Ligand (FL) as the sole cytokine, indicating that Flt3 signaling is sufficient for growth and survival. Furthermore, introduction of a mutant Flt3-ITD gene in these cells (MA9-ITD cells) not only promotes cytokine independent growth, but also significantly decreases leukemia latency in xenotransplant experiments. Consistent with the persistent requirement for active Rac signaling, we found that MA9 cells stimulated with FL induce the activated form of Rac (Rac-GTP) while MA9-ITD cells contain constitutively high levels of Rac-GTP, even after serum starvation and in the absence of all cytokines. Interestingly, we found that expression of the activated mutant NRas-G12D in MA9 cells (MA9-NRas cells) also leads to constitutive Rac activation, cytokine independent growth, and decreased leukemia latency upon transplantation into immunodeficient mice. Taken together, these results indicate that RAC activation may be a common integrating pathway by which secondary mutations cooperate in the progression towards leukemia. Consistent with this notion, both MA9-ITD and MA9-NRas cells remain sensitive to Rac inhibition with a small molecule Rac inhibitor or shRNA knockdown of Rac1 or Rac2. These data indicate that Rac signaling remains critical for MA9 survival and proliferation even in the context of additional mutations and highlights the therapeutic potential for targeting Rac.

Disclosures:

No relevant conflicts of interest to declare.
Recurrent Miscarriage in Women with and without Antiphospholipid Syndrome: Prognosis and Predictors of a Subsequent Successful Delivery.
Authors: Cohn Danny M , Middeldorp Saskia , Korevaar Johanna C , Dawood Feroza , Büller Harry R , Goddijn Mariëtte , Farquharson Roy
Published: October 2019

Abstract 2971

Poster Board II-947

Introduction:

Women with antiphospholipid syndrome (APS) are at increased risk of recurrent miscarriage. Remarkably, the outcome of a successive pregnancy in women with APS and recurrent miscarriage is merely unknown.

Patients and methods:

We performed a cohort study including all women with 2 or more consecutive miscarriages who attended the recurrent miscarriage clinic of Liverpool Women's Hospital, Liverpool, UK between 1988 and 2006. All women underwent a systematic diagnostic work up including testing for APS. Women with proven APS (according to the SAPPORO criteria) were studied, and outcomes were compared to women with unexplained recurrent miscarriage. We excluded all women from couples with other reasons for recurrent miscarriage.

Results:

737 women were included, of whom 220 (30%) had APS. Mean age (32 years) and obstetric history (mean number of prior losses: 3) did not differ between women with and without APS. Of women with APS, 148 (67%) had a successful pregnancy outcome as compared to 324 women without APS (63%), OR=1.2 (95%CI 0.9 to 1.7). No differences were found for birth weight, gestational age, and intra-uterine growth retardation. Women with APS who received aspirin and heparin more often had a successful pregnancy 53/67 (79%) as compared to women without treatment 26/44 (59%); OR=2.6 (95%CI 1.0 to 6.6). Combined use of aspirin and heparin was not associated with an increased success rate in women without APS: 25/43 (58%) vs 204/305 (67%); OR=0.7 (95%CI 0.3-1.4).

Conclusions:

The prognosis of a successive pregnancy in women with recurrent miscarriage and APS was comparable to women with unexplained recurrent miscarriage. However, the combined use of heparin and aspirin was associated with a significantly increased chance of a successful pregnancy in women with APS.

Disclosures:

No relevant conflicts of interest to declare.
Elevated Cholinesterase Activity in Patients with TIA and Other Thrombotic Disorders.
Authors: Horstman Larry Lawrence , Ahn Yeon-Soong , Esquenazi Jacob , Jy Wenche , Bidot Carlos J , Ortega Camile
Published: October 2019

Abstract 2991

Poster Board II-967

Background.

The natural function of ACTH in blood is not well defined. However elevated plasma level was reported in patients with chronic inflammation, suggesting as one of inflammatory markers. Erythrocytes possess membrane-bound acetylcholinesterase (AChE) while plasma contains a less specific cholinesterase (ChE). We reported at a previous meeting [Blood 2008, 112(11) Abst #3849] that AChE activity of RBC-derived microparticles (RMP) is 6 times higher than those in PMP. In this study we measured plasma ACTH levels in several groups and encountered unexpectedly high plasma ChE activities in patients with TIA and other thrombosis.

Methods.

(i) Patient population.

A series of n=108 consenting patients were recruited sequentially having various disorders For purpose of analysis, they were divided in 2 main groups: n=53 with thrombosis (TBS), F/M 27/26; and n=55 non-TBS, F/M 24/31. Thrombosis group consists of venous and arterial thrombosis including those with TIA. Non TBS group consists of anemias, thrombocytopenias including ITP, MDS.

(ii) Assay

was essentially by Ellman's method. In our system, milli-absorbance units/min (mA/min) x 0.065 = umols substrate cleaved/min. Values reported here are in unitsof mA/min per mL plasma. Normal controls (NC, n=14) had a cutoff of 3000 mA/min per mL plasma, = mean +2SD.

(iii) Sample handling.

Platelet-poor plasma (PPP) was prepared by centrifuging 10 min at 1800 xg, then frozen in aliquots. For assay, it was diluted 1:20 with saline, then 5 uL and 10 uL were used in 96-well microtiter plate.

Results:

(i) Significantly higher ChE activity was observed in thrombosis (TBS) patients compared to non-TBS. Mean value ±SD in TBS was 2928 ±773, and in Non-TBS was 1897 ±713 (units as above). This difference was significant, p<0.001. (ii) When analyzed elevated levels of ACTH above 2SD of normal controls (n=26) between 2 groups, the TBS group had elevated ChE (>3000) in 30% of patients, while the Non-TBS had elevated ChE in 7.8%. (iii) Further analysis among thrombosis group revealed that the subgroup with neurological TBS (TIA, minor strokes) had the most consistent elevations of ACTH; 10 of15 with TIA group (66%) had ChE activities >3000.

Summary/ Discussion.

Although ChE and AChE in blood has long been studied, to our knowledge, this is the first report to show a relation between plasma ChE and thrombosis. Its elevation is very pronounce in thrombosis of the CNS, manifesting as TIA. Further study is warranted to elucidate how ChE is related to AChE of the CNS and on red cells.

Disclosures:

No relevant conflicts of interest to declare.
Pharmacokinetics of Intravenous Uricase-PEG 20, a Drug for Managing Hyperuricemia in Tumor Lysis Syndrome.
Authors: Zhang Zhihua , Fiorino Anthony
Published: October 2019

Abstract 3086

Poster Board III-23

Background

Tumor lysis syndrome can result in hyperuricemia and the precipitation of monosodium urate crystals in the renal tubules, leading to renal failure and dialysis. The enzyme uricase, found in most mammals but not humans, metabolizes uric acid into the highly soluble allantoin. Rasburicase (Elitek/Fasturtec, Sanofi Aventis), an unmodified recombinant uricase, is used to treat and prevent hyperuricemia associated with tumor lysis syndrome. However, it requires multiple doses and is highly immunogenic. Uricase-PEG 20 is a pegylated recombinant uricase, designed to have an extended half life and reduced immunogenicity compared with unmodified uricase, that has been administered intramuscularly to gout patients in clinical studies. However, it has not previously been studied as an intravenous agent.

Methods

Intravenous toxicology studies, including pharmacokinetic studies, were performed with Uricase-PEG 20 in rats and cynomolgus monkeys. Uricase-PEG 20 was administered to rats at 1, 4 and 10 mg/kg and uricase enzyme activity was assessed at 1, 6, 24, 72 and 144 hrs after dosing. In monkeys, Uricase-PEG 20 was administered at 0.5, 1 and 5 mg/kg and uricase activity was assessed at 15 min and 1, 4, 24, 72 and 144 hrs after administration.

Results

In rats, the half-life for Uricase-PEG 20 ranged from 44.2 to 104.5 hrs (median, 70.7 hrs); the volume of distribution varied from 38.8 to 129.0 mL/kg (median 94.6), and the clearance varied from 0.70 to 1.48 mL/hr/kg. In monkeys, the half-life ranged from 184 to 214 hrs (median 194.5 hrs), the volume of distribution ranged from 17.2 to 25.2 mL/kg (median 19.9 mL/kg), and the clearance ranged from 0.063 to 0.102 mL/hr/kg. In both species, measures of exposure including Cmax and AUClast increased in a dose-proportional manner. The median half-lives of 70.7 hrs in rats and 194.5 hrs in cynomolgus monkeys is significantly longer than the half-life reported for rasburicase in similar models (<2 hrs in rats and <4 hrs in baboon monkeys).

Conclusions

The long half-life of Uricase-PEG 20 compared to that of unmodified uricase suggests that Uricase-PEG 20 may require less frequent dosing and thus have better clinical utility than unmodified uricase. Uricase-PEG20 will be used at intravenous doses ranging from 0.05 to 0.4 mg/kg in initial human studies.

Disclosures

Zhang: EnzymeRx: Employment. Fiorino:EnzymeRx: Employment.
Sall4, a Stem Cell Factor, Promotes Chemoresistance by Regulates the Side Population Cell Phenotype.
Authors: Jeong Ha-Won , Cui Wei , Guo Ye , Yang Youyang , Lu Jiayun , Gao Chong , Chai Li
Published: October 2019

Abstract 3093

Poster Board III-30

The side population (SP) phenotype, defined as the verapamil-blockable ability to efflux the nucleic acid dye Hoechst 33342, has been used to enrich for stem cells in several human normal tissues, cancers, and multiple cell lines including blood cells, and thus may be useful for the identification and isolation of cancer stem cells, which has emerged as a new therapeutic target for cancer treatment. However, the molecular mechanism(s) operating in SP cells remain unclear. Previous studies show that the stem cell factor, SALL4 plays a central role in the self-renewal of embryonic and leukemic stem cells. In this study, we determined the role of SALL4 in side population (SP). Using FACS sorting, we found the presence of SP fractions in four leukemic cell lines: HL-60, NB-4, RPMI8226 and KG1a. SALL4 expression was more abundant in SP cells compared with non-SP cells by 2- 4 fold in these leukemic cell lines. Knockdown of SALL4 in purified SP cells resulted in a reduction of SP population, indicating that SALL4 is required for the self-renewal of SP cells in this culture system. Since the side population phenotype is known to be mediated by the ATP-binding cassette proteins, we further investigated whether SALL4 could regulate the ATP transporters in SP cells. Chromatin immunoprecipitation (ChIP), luciferase assays and real-time PCR analysis demonstrated that SALL4 could bind to the promoter region of ABCA3, one of the ABC transporters, and activated its expression. In addition, while overexpression of SALL4 led to drug resistance in cell lines, SALL4-knockdown cells displayed more sensitive to drug treatments than the control cells. Further more, SALL4 expression was associated with primary AML treatment status (N= 64 cases), with higher SALL4 mRNA expression seen in drug resistance and partial remission patients than that from complete remission cases. Taken together, our results suggest that SALL4 plays an important in the sensitivity to drug treatment, at least in part, through the maintenance of SP cells, and is responsible for drug-resistance in leukemia.

Disclosures

No relevant conflicts of interest to declare.
Comorbidity Is An Independent Prognostic Factor in AML: Comparison of Two Comorbidity Scores.
Authors: Novotny Juergen , Aisenbrey Achim , Nückel Holger , Dührsen Ulrich
Published: October 2019

Abstract 3106

Poster Board III-43

Objective

Many factors have been studied to predict outcome and allocate treatment in AML. The best established prognostic factors are karyotype and age. However, comorbidity may play an important role in the outcome of AML. We applied two comorbidity scores at the time of first admission in order to test this hypothesis.

Methods

We retrospectively analysed 198 consecutive patients with AML. All patients were included irrespective of the applied therapy. Karyotype risk group was 11.6 % good, 68.0 intermediate and 20.4 % poor risk, respectively. The median survival was 382 days. The median age was 62 years (range 20 – 81), 95 were male, 103 were female. The criteria of the Charlson Comorbidity Index (CCI) and the recently developed Hematopoietic Cell Transplantation-Specific Comorbidity Index (HCT-CI) were applied. However, the cut-offs were set lower than in the original publication in order to detect the possible impact of minor comorbidity.

Results

In our study, the HCT-CI (left figure) separated clearly between patients with a score of more than one (dotted line), one (dashed line) and zero (continous line) (median survival: 100 vs 328 vs 718 days; log rank test p<0.0001). Similarly, the CCI separated the patients with more than one (dotted line), one (dashed line) and none (continous line) (median survival: 94 vs 293 vs 515 days; log rank test p<0.0001). Karyotype (p < 0.001), HCT-CI (p = 0.003) and age (cut-off 60 years; p = 0.006) we shown as independent risk factors by multivariate analysis. The CCI (right figure) separated only between patients with a score of zero (continous line) and those with at least one score point. Patients with one (dashed line) and patients more than one (dotted line) were not separated.

Conclusions

Our findings show that HCT-CI is an additional prognostic factor in AML, at least in our cohort. We extend previous findings severalfold. Firstly, our analysis includes 90 patients younger than 60 years and was not restricted to patients older than 60 years.

Secondly, we directly compared two comorbidity indices, showing that the HCT-CI discriminaates between three risk groups. Thirdly, we showed that comorbidity is an independent predictor for survival.

In conclusion comorbidity seems to be an independent prognostic factor and should be studied prospectively to clarify its use for stratification in therapeutic studies.

Disclosures

No relevant conflicts of interest to declare.
Lactadherin as a Probe for Phosphatidylserine Exposure and as An Anticoagulant for the Procoagulant Activity in the Study of Stored Platelets.
Authors: Zhou Jin , Hou Jinxiao , Li Wen , Zhang Xiaoqian , Fu Yueyue , Li Huibo , Xie Rui , Zhang Zhuo , Gilbert Gary E. , Shi Jialan
Published: October 2019

Abstract 3150

Poster Board III-87

Background

Phosphatidylserine (PS) can support coagulant reactions. However, it is uncertain how the location and extent of PS exposure to the membranes of stored platelets affect such reactions. We compared annexin V with lactadherin as a way of detecting how of PS exposure influences the procoagulant properties of stored platelets in platelet concentrates (PCs).

Method

PS exposure and the relevant procoagulant activity (PCA) of platelets in 5 different PCs were investigated by flow cytometry, confocal microscopy, coagulation time analysis and enzymatic assays. PS exposure was separately measured using annexin V and lactadherin, respectively.

Results

Exposure of PS to stored platelets promoted thrombin formation. A progressive increase in PS exposure was detected by flow cytometry. Moreover, using lactadherin, we identifed higher levels of PS exposure on the platelets and platelet-derived microparticles (PMPs) compared to detection using annexin V. The percentage of PS-positive cells was 0.02 % by annexin V versus 0.3 % by lactadherin on day 0, 7.5 % by annexin V versus 12.3 % by lactadherin on day 5, and 29 % by annexin V versus 44.3 % by lactadherin on day 9. Rare microparticles (MPs) were released from fresh platelets, and, the number of PMPs increased approximately 2-fold on day 5 and further progressively increased. Using lactadherin and platelets in the earlier stage of storage, confocal microscopy revealed earlier and localized PS exposure based on plasma membrane staining. For later storage platelets, increased levels of PS-positive platelets and PMPs were clearly detected by both annexin V and lactadherin. Thirty-two nM lactadherin or annexin V prolonged coagulation time 2.4 fold versus 2 fold. The productions of thrombin and intrinsic/extrinsic factor Xase were approximately inhibited 85 % and 60 % by lactadherin and annexin V, respectively.

Conclusion

PS exposure was localized to the cellular rims, blebbing vesicles and thin elongated filopodia-like areas on banked platelets. Furthermore, lactadherin provides a more accurate measurement of PS exposure and the relevant with PCA, which is an important factor to consider for transfusion medicine. Our findings of elevated PS-positive platelets and PMPs indicate that platelets should not be stored for extended periods of time.

Disclosures

No relevant conflicts of interest to declare.
Phage-Displayed Peptide-Q Dot Nanocrystal Combo for High-Sensitivity Bacterial Detection in Plasma.
Authors: Sainath Rao Shilpakala , Ketha Krishna Mohan V. , Atreya Chintamani D
Published: October 2019

Abstract 3153

Poster Board III-90

Introduction

Despite improved phlebotomy practices, refrigeration of red cells, freezing of plasma and improved materials for transfusion product collection and storage, bacterial contamination of transfusion products is still a longstanding problem. Current bacterial detection tests relevant to transfusion medicine, especially for stored platelets have limitations with regard to time, specificity and sensitivity. There is a need for new and improved cost-effective high-affinity detection probes to fill this gap. In this study, using plasma spiked with Bacillus cereus 4342 and B. anthracis-Sterne as an experimental system, we identified peptides from a bacteriophage-displayed random peptide library that selectively bind and detect the Bacillus strains. By labeling the peptides with Q dot-liquid nanocrystals, the detection sensitivity of the peptides was further enhanced.

Methods

A commercially available bacteriophage-displayed random peptide library was screened using B. cereus 4342 as bait, using appropriate controls under stringent conditions. The screening and subsequent sequencing of the phage DNA identified two phages each containing a coding sequence for 12-amino acid peptide that are selectively capable of binding to the Bacillus. Based on the nucleic acid sequence, the two synthetic peptides with biotin tag were prepared for detection assays and to enhance the detection sensitivity further, the peptides were labeled with streptavidin-conjugated fluorescent quantum-dots (Q dots). Fluorescence was measured either by a plate reader or using a fluorescence microscope.

Results

The two synthetic peptides selectively bound to the Bacillus strains in both dot blot and ELISA assays. The membrane-based dot blot assay demonstrated an assay sensitivity of 103 colony forming units/ml (CFU/ml), whereas ELISA demonstrated a sensitivity of 102 CFU/ml detection limit. Fluorometry analysis of spiked plasma samples revealed that the two peptides were able to bind to B. cereus 4342 and B. anthracis Sterne and detect these bacteria in plasma at 102 CFU/ml concentrations. The peptide-Qdot combo even detected a single bacterium in fluorescence microscopy.

Conclusion

Overall, the results reported here validate the usefulness of affinity-selected recombinant filamentous phage-derived peptides in combination with Qdot-liquid nanocrystals as high sensitivity detection probes for bacteria in various platforms and settings relevant to the blood safety and transfusion medicine.

The findings and conclusions in this abstract have not been formally disseminated by the Food and Drug Administration and should not be construed to represent any Agency determination or policy.

Disclosures

No relevant conflicts of interest to declare.
Screening of Telomere Length in Peripheral Blood Lymphocytes From Patients with Aplastic Anemia by Flow-Fluorescence in Situ Hybridization.
Authors: Nishio Nobuhiro , Takahashi Yoshiyuki , Muramatsu Hideki , Hama Asahito , Nagata Toshihito , Kojima Seiji
Published: October 2019

Abstract 3205

Poster Board III-142

Telomeres in peripheral blood cells from patients with aplastic anemia (AA) are shorter than those from normal individuals. One possible explanation is that telomere shortening reflects the extent and duration of damage to hematopoietic stem cells and the number of compensatory stem cell divisions. Patients with such stressed hematopoiesis probably do not respond to immunosuppressive therapy (IST). Cryptic forms of dyskeratosis congenita (DC) have been recognized in patients with AA who do not have physical abnormalities, but who harbor the gene mutation associated with telomere maintenance. To identify such patients is important because those who have these mutations might respond poorly to IST. The present study evaluates the usefulness of screening telomere length in peripheral blood lymphocytes from patients at the time of diagnosis with AA by flow-fluorescence in situ hybridization (flow-FISH) using Telomere PNA kits (Dako Cytomation, Glostrup, Denmark). After gating diploid cells based on propidium iodine staining, lymphocytes were isolated according to size and granularity. Relative telomere length (RTL) was calculated as the ratio between the telomere signal of each sample and the telomere signal of the control cell line. Because telomeres shorten with age, we obtained age-adjusted measurements for comparison. We used flow-FISH to measure the length of telomeres in peripheral blood lymphocytes from five and two patients with classical and cryptic DC who had the TERT mutation, respectively, to determine whether these two forms of DC can be screened. Significant telomere shortening was detected in all seven patients compared with age-matched normal individuals. To determine the association between telomere length and response to IST, we compared telomere lengths in peripheral blood lymphocytes from 61 normal controls and 27 patients with AA (very severe, severe and non-severe, n = 9 each) at diagnosis who received IST with antithymocyte globulin (ATG) and cyclosporine. The median age of the patients was 9.0 years (range, 1.5 to 14.8). The causes of AA were unknown etiology in 25 patients and hepatitis in 2. In total, 17 of the 27 patients (63%) responded to IST at 6 months after administration of ATG. Age-adjusted delta RTLs did not significantly differ between normal controls and responders (p = 0.181), but those in non-responders were significantly lower than those in normal individuals (p = 0.000). Our findings suggest that screening telomere length in patients with AA at diagnosis is useful for detecting cryptic DC and for predicting responses to IST.

Disclosures

No relevant conflicts of interest to declare.