bluuebunny/crossref_metadata_2025 huggingface dataset:
Comparative Efficacy of First Line Treatment of Chronic Myeloid Leukaemia (CML): A Systematic Review and Meta-Analysis.
Authors: Mealing Stuart , Barcena Leticia , Hawkins Neil , Clark James , Davis Catherine
Published: October 2019

Abstract 3436

Introduction:

Historically, interferon-alpha, hydroxyurea or chemotherapy were used as first line therapy for individuals newly diagnosed with CML. Current practice, however, recommends that tyrosine-kinase inhibitors (TKIs) be used in this patient group, with imatinib (1st generation TKI) being the current standard of care. Recent publications provide data from randomized clinical trials on 2nd generation TKIs (dasatinib and nilotinib) in the first-line setting. In the absence of randomized head to head evidence for all three TKI's, a Bayesian mixed treatment comparison meta-analysis provides a means of indirectly estimating the treatment effect of one intervention relative to another.

Methods:

A systematic review identified RCTs that reported or summarized efficacy data for any of the treatments of interest in treatment-naive CML patients. In order to maximise the evidence network, RCT's of non-TKI's were also included in the review. Where reported, data was extracted for molecular, hematological and cytogenic response at three montly intervals. In addition, safety and progression free and overall survival data was extracted for all reported timepoints. Extracted data was analyzed in a Bayesian mixed treatment comparison using a fixed-effects model. The outputs from all analyses were expressed as odds ratios with Imatinib 400mg SID used as the baseline intervention in all analyses.

Results:

Overall, 44 articles arising from 19 distinct clinical trials were included in the review. The results for complete cytogenetic response (CCyR) at twelve months (expressed as both response probabilities and odds ratios) are presented in table 1.

High dose imatinib performed no better than standard dose imatinib. However, response with both 2nd generation TKIs was significantly better than with standard dose imatinib with the effect size being doubled on all endpoints. Head to head results for nilotinib and dasatinib showed no statistically significant differences (p=0.05) between the two products on all endpoints (see table 2).

Conclusions:

Compared to Imatinib, the use of 2nd generation TKI's for the treatment of newly diagnosed CML results in a significant improvement in complete cytogenic response. However, the current evidence base is not rich enough to distinguish between the two 2nd generation TKIs either in terms of response or progression free survival. The analyses presented will be strengthened by the inclusion of additional RCT data that may become available in the future.

Disclosures:

Mealing: Oxford Outcomes: Employment. Barcena:Oxford Outcomes: Employment. Hawkins:Oxford Outcomes: Employment. Clark:Oxford Outcomes: Employment. Davis:Bristol Myers-Squibb: Employment.
The Potential Role of Circulating IL-17 Levels During the Peri-Transplant Period as a Predictor for Early Leukemia Relapse After Allogeneic Stem Cell Transplantation.
Authors: Cho Byung Sik , Lim Ji-Young , Yahng Seung-Ah , Lee Sung-Eun , Eom Ki-Seong , Kim Yoo-Jin , Chung Nak-Gyun , Jeong Dae-Chul , Lee Seok , Kim Heeje , Cho Seok-Goo , Kim Dong-Wook , Lee Jong-Wook , Min Woo-Sung , Park Chong-Won , Min Chang-Ki
Published: October 2019

Abstract 3454

IL-17 is involved in inducing and mediating proinflammatory responses. The association of IL-17 with tumor growth or graft-versus-host disease (GVHD) has become the object of controversy. To determine the role of IL-17 levels during the peri-transplant period on alloreactive responses, serum IL-17 (sIL-17) levels of 95 patients with leukemia (52 AML, 38 ALL, 5 CML) who had undergone myeloablative allogeneic stem cell transplantation were measured using ELISA before conditioning and on day 0, day +7, and day +14 after transplantation. With the median follow-up of 17 months (range, 9–26), overall survival and disease-free survival rates were 70.9%±9.2% and 66.3±6.2%, respectively, and cumulative incidence of relapse and non-relapse mortality were 23.4%±5.7% and 10.3%±3.3%, respectively. Among 18 patients relapsed, 10 patients were relapsed within 180 days (range, 76–172) after transplants, and cumulative incidence of the early relapse was 10.5%±3.2%. The cumulative incidences of acute GVHD with grade II-IV and chronic GVHD were 55.8%±5.1%and 69%±5%, respectively. Analyses using repeated measures of ANOVA and mean values of sIL-17 revealed that patients relapsed within 180 days had higher sIL-17 levels during peri-transplant period, whereas no association was detected between sIL-17 levels and other clinical outcomes including acute GVHD. Receiver operating characteristic curve analysis also showed that sIL-17 levels at every time point were available for the prediction of the early relapse. Patients with advanced disease status at transplantation, one of factors associated with early relapse in univariate analyses, had higher sIL-17 levels at pre-conditioning and day 0. However, the result of multivariate analyses showing that sIL-17 levels at each time points were the only factor associated with early relapse suggests the importance of sIL-17 level itself than advanced disease status at transplantation. This study represents the first one demonstrating the early change of sIL-17 during the peri-transplant period and its association with early leukemic relapse in human. The predictive role of sIL-17 during the peri-transplant period for early leukemic relapse may be explained by the association of sIL-17 with tumor itself or tumor growth as well as possible inhibition of the function of graft-versus-leukemia effectors, such as natural killer cell, by sIL-17. Further studies are needed to precisely define the potential roles of IL-17.

Disclosures:

No relevant conflicts of interest to declare.
Influence of Glutathione S-Transferase A1 and P1 Polymorphisms on the Outcome of Hematopoietic Stem Cell Transplantation with Busulfan Based Conditioning Regimen In Children.
Authors: Lee Ji Won , Kang Hyoung Jin , Yuk Yen Ju , Jang Mi Kyoung , Han Eun Jong , Kim Nam Hee , Kim Hyery , Park Kyung Duk , Shin Hee Young , Ahn Hyo Seop
Published: October 2019

Abstract 3492

Introduction:

Busulfan has narrow therapeutic range. High exposure is associated with systemic toxicity such as veno-occlusive disease (VOD) while underexposure results in graft failure or relapse. In children, pharmacokinetic variability was found to be high even after the introduction of intravenous busulfan. Busulfan is metabolized via liver through conjugation with glutathione S-transferase (GST) family, and inter-individual variability may be explained by GST polymorphisms. Thus, we investigated the influence of GST polymorphisms on the clinical outcomes of hematopoietic stem cell transplantation (HSCT) with busulfan based conditioning regimen in children.

Patients and methods:

We studied patients who underwent HSCT at Seoul National University Children's Hospital from November, 2001 to November, 2008. IV busulfan (0.8 mg/kg/dose for patients≥10 kg and 1.1 mg/kg/dose for patients <10 kg, q 6hr for 4 days) was used in combination with cyclophosphamide or fludarabine as conditioning regimen. Etoposide was added for acute lymphocytic leukemia (ALL). GST polymorphisms (GSTA1 375 A > G, -52 G > A, -567 G > T, -631 G > T, -69 C > T, GSTM1 deletion, GSTT1 deletion, and GSTP1 313 A > G) were analyzed by multiplex PCR amplification and SNP genotyping.

Results:

A total of 70 patients (48 male, 22 female) were analyzed. The diagnoses were ALL in 32, AML in 26, and other diseases in 12 patients. Median age at HSCT was 8.8 years (range 1.0–19.0 years). Bone marrow or peripheral blood stem cell transplantations (BMT/PBSCT) were conducted in 42 patients, and cord blood transplantations (CBT) in 28 patients. Graft failure occurred in 11 (15.7%) patients (1 (2.4%) in BMT/PBSCT, 10 (35.7%) in CBT) and relapse occurred in 15 (21.4%) patients (12 (28.6%) in BMT/PBSCT, 3 (10.7%) in CBT) after HSCT. Patients having GSTA1 *A/*A and GSTP1 313 A/A genotype (N=33) showed higher incidence of graft failure than the others (N=37) (27.3% vs 5.4%, P =.01). Conversely, the incidence of hyperbilirubinemia over grade 3 was significantly lower in the patients with GSTA1 *A/*A and GSTP1 313 A/A genotype than the other patients (6.1% vs 24.3%, P =.05). Event free survival (EFS) of patients with GSTA1 *A/*A and GSTP1 313 A/A genotype was significantly lower than the EFS of the other patients in both BMT/PBSCT and CBT, and the main causes of event were graft failure in CBT and relapse in BMT/PBSCT.

Conclusions:

In children undergoing HSCT with busulfan based conditioning regimen, GST A1 and P1 polymorphisms seem to have influence on the graft failure, relapse and complications. To confirm our results, further studies about the influence of GST A1 and P1 polymorphisms on the pharmacokinetics of busulfan are needed.

Disclosures:

No relevant conflicts of interest to declare.
Similar Overall Survival and Toxicity After CB Transplanation with Co-Infusion of CD34+ Cells From a Third Party Donor Compared to Myeloablative MUD Stem Cell Transplantation In High Risk Patients
Authors: Kwon Mi , Infante Maria , Balsalobre Pascual , Muñoz Cristina , Buno Ismael , Gayoso Jorge , Serrano David , Diez-Martin Jose L.
Published: October 2019

Abstract 3532

In the absence of an HLA matched sibling donor, an unrelated adult donor matched (MUD) at 8 of 8 alleles for HLA A, B, C, DRB1 is considered as first alternative. However, only 50% of patients in Europe have an available suitable donor in a median time of 4 months. For those patients without a suitable MUD, we have adopted CB (cord blood) stem cell transplantation with the co-infusion of CD34+ cells from a third party non HLA-identical donor (Dual transplantation). The objective of this study was to analyze toxicity and overall survival (OS) rates of patients who underwent this procedure and compare them with a cohort of patients who received a myeloablative MUD transplantation in a single centre and during a similar period of time.

Patients:

Between 2005 and 2010, 16 consecutive patients with high risk disease underwent a total of 18 dual transplants and 17 patients, with similar pre-SCT characteristics recieved a myeloablative MUD transplantation with 12/12 HLA identity in a single centre (Table 1). Transplants performed before 2005, those with manipulated grafts and with HLA mismatches were excluded.

Results:

There were no significant differences in age, gender, pre-SCT disease status and previous therapy lines between both groups. Three cases among the dual group showed primary graft failure (2 of them showing third party donor cells engraftment) who were rescued by a second CB transplant in two cases and a MUD in one case (succesfull in 2). There were no graft failures in the MUD group. GVHD rates are shown in table 1. OS was 50% in the dual group and 53% in the MUD group with a median follow-up of 33 (1-67) y 13 months (1,1-48,5) respectively (Figure 1). The cumulative relapse incidence at 2 years was 54% in the dual group and 53% in the MUD group. There were no relpses after 2 years in both groups. All the transplant related deaths (TRM) took place within the first year after transplantation with no significant differences in cumulative incidence between both groups (p=0,69).

Conclusions:

In our experience, CB transplantion together with the co-infusion of CD34+ cells from a third party HLA-mismatched donor offers OS and DFS rates comparable to those from myeloablative HLA 12/12 MUD trasnplantation with an acceptable TRM. Therefore, dual transplantion is still our first choice for patients without an available MUD or for whom trasplant is urgent.

Disclosures:

No relevant conflicts of interest to declare.
Minimal Residual Disease Status at Day +100 Post Allogeneic Hematopoietic Stem Cell Transplantation Is a Powerful Predictor for Post-Transplant Outcome In Patients with High Risk Acute Leukemia
Authors: Tang XiaoWen , Sun Xingwei , Xue Shengli , Shi Xiaolan , Zhu Mingqing , Shen Wenhong , Chen Li , Sun Aining , Depei Wu
Published: October 2019

Abstract 3550

Background and Objectives Relapse after allogeneic hematopoietic stem cell transplantation (allo-HSCT) is still a major cause for the failure in treatment. It has been shown that there was a closely relationship between the level of minimal residual disease (MRD) and relapse in acute leukemia (AL) patients; However, the application of multiparameter flow cytometry (MFC) for MRD assessment in high risk patients with AL who undergoing allo-HSCT is little concerned. We retrospectively analysed the serial results of MRD of 52 high risk patients with AL to evaluates the prognostic value of MRD pre and post transplantation.

Methods

52 patients with a median age of 29 (13–55) years have been enrolled on this study in our hospital from January 2003 to September 2008.Diagnoses included AML (n=27) and ALL (n=25). The patients had been analyzed retrospectively the level of MRD pre-(day-30)and post-HSCT(day+30 and +100)using three color FCM with CD45/SSC gating and a comprehensive panel of monoclonal antibodies, at least one leukemia associated aberrant immunophenotype (LAIP) at diagnosis. According to the cutoff value 0.1%, two groups were defined based on the level of patient's MRD level< (low level group) or >= (high level group) 0.1%.

Results

The median follow up were 23 (range 1–60) months. 1.MRD level declines significantly (P=0.03) post transplant. 2. There were significantly difference between low level and high level group at day -30 before transplant with 3 years event free survival(EFS) and relapse free survival (RFS)(77.4% and 88.4% vs. 22.3% and 25.7%, p=0.007and p=0.001 respectively). 3. Concerning about MRD at day +100 after transplant, outcome was significantly better among patients with low level MRD group versus high group including 3 years OS,EFS and RFS(84.2%, 79.5% and 89.5% versus 22.9%, 9.5% and 11.2%).4. The median time from high level MRD detected first time to clinical relapse was 2.5 (range from 1 to 33) months in relapsed patients. 5. The patients with cGVHD had better 3 years OS and EFS than that without cGVHD(86.3% vs 12.1%, p<0.001 and 65.3% vs.14.8%, p< 0.001 respectively). 6. Multivariate Cox regression analysis revealed that MRD on day +100 as well as chronic GVHD were independent parameters predictive for OS and EFS.

Conclusions

MRD monitoring pre- and post-transplant is an important tool to predict the outcome of transplantation for patients with high risk AL. The MRD check point at day +100 should be considered crucial for subsequent therapeutic decisions after allogeneic transplantation.

Disclosures:

No relevant conflicts of interest to declare.
Intensive Induction Chemotherapy Followed by Autologous Stem Cell Transplantation (ASCT) In Patients with Enteropathy-Associated T-Cell Lymphoma: a Prospective Study by the Nordic Lymphoma Group (NLG-T-01)
Authors: Jantunen Esa , Relander Thomas , Lauritzsen Grete Fossum , Hagberg Hans , Anderson Harald , Cavallin-Ståhl Eva , Holte Harald , Østerborg Anders , Merup Mats , Brown Peter De Nully , Kuittinen Outi , Erlanson Martin , Østenstad Bj∅rn , Fagerli Unn-Merete , Gadeberg Ole , Sundstrom Christer , Delabie Jan , Ralfkiaer Elisabeth , Vornanen Martine , d’Amore Francesco
Published: October 2019

Abstract 3565

Enteropathy-associated T-cell lymphoma (ETCL) is a rare lymphoma often, but not always, associated with celiac disease and characterized by poor prognosis when treated with conventional chemotherapy. In previous studies long-term survival has been achieved in only 10–20% of the patients. Limited data is available on the feasibility and efficacy of intensive induction chemotherapy followed by autologous stem transplantation (ASCT) in this rare lymphoma entity. We therefore specifically analysed the outcome of ETCL patients included in a large prospective phase II study (NLG-T-01) performed by the Nordic Lymphoma Group. The NLG-T-01 study included 160 patients with systemic alk-negative peripheral T-cell lymphoma over the period 2002–2007. The patients received CHOEP-14 × 6 followed by ASCT after BEAM or BEAC in responsive patients. The study included altogether 21 patients (13 %) with ETCL. There were 16 males and 5 females with a median age of 55 years (32-65) at diagnosis. Eighteen patients (86 %) had advanced disease, three patients (14 %) had a bulky tumour, nine patients (43 %) presented with B symptoms and four (19%) with elevated serum lactate dehydrogenase. Response status after three and six courses was CR or CRu in 67 % patients. Fourteen patients (67 %) received BEAM or BEAC supported by blood stem cell graft (median number of stem cells infused 5.4 × 106/kg). Of these, 6 patients relapsed with a median of 219 days from ASCT. Of the 7 patients (33%), who did not reach ASCT because of refractory/progressive disease, 5 died early due to lymphoma. At a median follow-up of 45 months, 10 patients (45 %) are alive. The progression-free survival is 40 %. One patient (5%) died due to early transplant-related cause (disseminated candidiasis). In this prospective study, intensive induction chemotherapy followed by ASCT was feasible in the majority of younger patients with EATL. In a subset of patients, who should clinically and biologically be further characterized, long-term outcome seems promising when compared to historical controls. Whether addition of other chemotherapeutic agents, antibodies such as alemtuzumab or other biologicals may further improve long-term outcome remains to be studied.

Disclosures:

No relevant conflicts of interest to declare.
C-Abl Regulates Mcl-1 Gene Expression In Chronic Lymphocytic Leukaemia Cells
Authors: Allen John C , Zuzel Mirko , Lin Ke , Slupsky Joseph R
Published: October 2019

Abstract 3579

Chronic lymphocytic leukaemia (CLL) is a prevalent malignancy characterised by the clonal expansion of mature B cells that are resistant to apoptosis. This resistance to apoptosis will partly be due to Mcl-1 expression because high levels of this protein in CLL cells correlate with poor disease prognosis and resistance to chemotherapy. Thus, understanding the mechanism(s) regulating Mcl-1 expression in CLL cells may be useful in the development of new therapies for this incurable disease. In the present study we show a strong relationship between c-Abl and Mcl-1 expression in CLL cells. We show that treatment of CLL cells with Abl-specific siRNA or with imatinib, to inhibit c-Abl activity, results in the downregulation of Mcl-1 protein and mRNA. A major regulator of Mcl-1 gene expression is STAT3. Our data show that CLL cells expressing high levels of c-Abl also show elevated levels of phospho-STAT3, and that STAT3 phosphorylation in CLL cells is dependent on c-Abl activity. However, STAT3 phosphorylation by c-Abl is not direct, and requires activation of NFkB, secretion of autocrine IL6 and active PKC. Taken together, our results provide clearer definition of the pathobiological role of c-Abl in CLL cells. Given that high NFkB activation, plasma IL6 levels and Mcl-1 all correlate with poor disease prognosis in CLL, our work potentially connects several features of CLL cells that are important to their pathophysiology by suggesting a central role of c-Abl in their regulation. Since CLL cells expressing high levels of c-Abl and Mcl-1 belong to the unmutated, poor prognostic group of CLL, c-Abl inhibition may have therapeutic application in the treatment of this disease, especially for those patients who are resistant to conventional chemotherapeutic agents.

Disclosures:

No relevant conflicts of interest to declare.
Epigenetic Silencing of Mir-218 Regulates CDK6 Expression In Pre-B Acute Lymphoblastic Leukemia Cells
Authors: Li Xiaoqing , Liu Jun , Zhou Rui , Zheng Jine , Yao Junxia , He Yanli , Chen Xiangjun , Ding Jie , Huang Shiang , Chen Xian-Ming
Published: October 2019

Abstract 3621

Increasing evidence suggests that dysregulation of miRNAs plays an important pathological role in various malignant diseases including acute leukemia. To reveal the contributions of aberrant epigenetic modifications to the deregulated miRNA expression in precursor B-cell acute lymphoblastic leukemia, we examined the miRNA expression profile in NALM-6 cells after treatment with the combination of 5-AZA-2'-deoxycytidine (AZA) and trichostatin A (TSA). We found that the treatment significantly increased expression of 34 miRNAs and decreased the expression of 10 miRNAs. One of the most significantly upregulated miRNAs is miR-218, an intronic miRNA that can be transcribed from either pri-miR-218-1 or pri-miR-218-2, residing in the intron of the SLIT2 gene or SLIT3 gene respectively. Interestingly, we detected that pri-miR-218-1 and its host gene SLIT2, but not pri-miR-218-2 and SLIT3, were induced by AZA plus TSA treatment. Consistent with this observation, we showed that the CpG islands in SLIT2 promoter was highly methylated in NALM-6 cells and AZA plus TSA treatment significantly decreased DNA methylaiton in this region. We found that targeting of the 3'untranslated region of CDK6, a bona fide oncogenic factor, by miR-218 resulting in translational repression. Overexpression of miR-218 expression in NALM-6 cells by transfection of miR-218 precursor decreased cellular expression of CDK6 at the protein level, but not at the message level. AZA and TSA treatment decreased CDK6 expression in NALM-6 cells, presumably through upregulating miR-218. Our results indicate that epigenetic regulation plays an important role in controlling miRNA expression in human acute lymphoblastic leukemia cells. Epigenetic silencing of miR-218 may contribute to the overexpression of CDK6 in NALM-6 cells.

Disclosures:

No relevant conflicts of interest to declare.
Genome-Wide Epigenetic Analysis of Cancer Stem Cells (CSCs) In Acute Myeloid Leukemia.
Authors: Yamazaki Jumpei , Estecio Marcos R , Jelinek Jaroslav , Graber David , Lu Yue , Ramagli Louis , Liang Shoudan , Kornblau Steven M. , Issa Jean-Pierre
Published: October 2019

Abstract 3640

Background & Aims:

The hypothesis that cancer is driven by Cancer Stem Cells (CSCs or Cancer-Initiating Cell) has recently attracted a great deal of attention. Epigenetic mechanism such as DNA methylation and histone modification play an important role in cancer cells and also in normal stem cells. However, their role remains unclear in CSCs. We sought to determine if CSCs have distinct epigenetic patterns in acute myeloid leukemia (AML).

Methods:

Peripheral blood samples in AML patients were separated to obtain stem cells (CD34+CD38-) and progenitor cells (CD34+CD38+) by magnetic cell sorting (MACS®, Myltenyi biotec). To study DNA methylation in CSCs in AML, we performed genome wide screening using methylated CpG island microarray (MCAM), which detects 7202 promoter CpG islands, 1348 non-promoter CpG islands, and 632 non-CpG island promoter methylation. MCAM was performed on 4 AML patient samples Next, we evaluated the methylation status of 7 genes which showed apparent higher DNA methylation in stem cells or progenitor cells in MCAM analysis, using a quantitative bisulfite-pyrosequencing for each population of stem cell, progenitor cell, and mature cells (CD34-) from peripheral blood samples in 6 AML patients. For histone modification analysis, we used Chromation immuprecipitation followed by massively parallel sequencing (ChIP-Seq) for stem cell and progenitor cell populations for H3K4me3 which is known to be a marker for activated genes.

Results:

By MCAM, we found minimal differences between stem cells and progenitor cells present in 2 out of 4 AML patients. Those few genes (<1%) which were shown to have higher DNA methylation in stem or progenitor cells by MCAM analysis were likely false positives, as no significant difference was found when analyzed by quantitative bisulfite-pyrosequencing. DNA methylation status for stem cell-related gene (OCT4, SOX2, MYC, HOXB4, and KLF4) also showed no significant difference. By ChIP-seq analysis, we found differences in 2362 genes between stem cells and progenitor cells. In stem cells, H3K4me3 was enriched in genes (Bmi1, Notch1, Wnt1, and etc) which are known to be important for stem cell function, but they were not enriched in the progenitor cell population. In pathway analysis of the H3K4me3 data, Hypoxia-Inducible Factor signaling, NFkB signaling, and p53 signaling are found to be enriched specifically in the stem cell population whereas Cellular Growth and Cell Cycle, and DNA Damage Response signaling are found in the progenitor cell population.

Conclusions:

There is no significant difference in DNA methylation between stem cell, progenitor cell or mature cell populations in AML. DNA methylation of promoter CpG islands is unlikely to explain tumor hierarchy in AML. Rather, histone modifications seem to have a greater significance in this regard.

Disclosures:

No relevant conflicts of interest to declare.
High Throughput Sequencing of Acute Lymphoblastic Leukemia Small RNAs.
Authors: Wei Michael C , Osborn Elizabeth O , Cleary Michael
Published: October 2019

Abstract 3650

MicroRNAs are small, non-coding RNAs that regulate gene expression and play key roles in cancer by modulating oncogene and tumor suppressor pathways. We are investigating the clinical and prognostic roles of miRNA expression in pediatric leukemias using high-throughput sequencing as a profiling tool. To establish the methodology, we have utilized high-throughput sequencing to quantify small RNA expression from eight acute lymphoblastic leukemia cell lines and one MLL-rearranged infant ALL patient sample. We generated sequencing libraries from these cells, conducted high-throughput sequencing using the Illumina platform, and established a custom bioinformatics pipeline for data analysis. Over 50 million individual sequence reads were analyzed. These sequences were mapped against a database of human miRNAs, and the frequency of miRNA expression among samples was enumerated. Expression of hematopoietic-specific miR-142 and miR-181 cluster miRs was found in these leukemia samples, while the liver-specific miR-122 was not expressed. miR-196b, previously reported to be over-expressed in MLL-rearranged leukemias, was expressed in 3/3 MLL-rearranged leukemia cell lines and 1/5 non-MLL cell lines. Expression of individual miRNAs was validated by quantitative PCR. Additional analysis of MLL-associated miRNAs and novel small RNAs will be presented. Our results demonstrate the feasibility and potential of high-throughput sequencing to profile the expression of small RNAs from leukemia cells, and we plan to apply these methods to additional primary patient samples to examine prognostic and clinical correlations with small RNA expression patterns.

Disclosures:

No relevant conflicts of interest to declare.
Differential Effect of An Autoantibody to Thrombin on Fibrinogen Cleavage and Protein C Activation.
Authors: Shastri Aditi , Baker Kelty R. , Thiagarajan Perumal
Published: October 2019

Abstract 3652

A 76 year old male experienced an unexpected blood loss of 1300 ml during an elective left shoulder replacement for degenerative joint disease. His post-operative course was uncomplicated. Further laboratory testing revealed a prolonged thrombin time of 33 seconds (control of 17.7–20.3 seconds). The reptilase time was within normal limits. He also had a normal factor V, × and fibrinogen level. The prolonged thrombin time failed to correct upon addition of normal plasma. The patient's IgG fraction was isolated by protein G-Sepharose chromatography and tested for its effect in coagulation assays. The patient's purified IgG (2.1 mg/ml) prolonged the thrombin time in normal plasma when tested with a commercial bovine thrombin time reagent. Under similar conditions, normal IgG had no effect. These laboratory tests suggest an acquired antibody to thrombin.

The antibody was further characterized by testing its effect on purified human thrombin. The antibody prolonged the clotting time of human thrombin in normal plasma in a concentration dependent manner. However, the antibody had no effect on the esterolytic activity of human or bovine thrombin as measured by the proteolysis of the chromogenic thrombin substrate, S-2238. These results suggest that the epitope for the antibody overlaps the fibrinogen binding site on thrombin but does not include thrombin's active enzymatic site.

We also tested the effect of the purified antibody on thrombomodulin-dependent thrombin activation of protein C. The patient's antibody was incubated with protein C, rabbit thrombomodulin and calcium containing buffer. The activation of protein C was measured using the chromogenic protein C substrate S2366. The antibody had no effect on protein C activation by thrombin.

Acquired antibodies to thrombin develop after exposure to bovine thrombin, commonly used for surgical hemostasis. These antibodies are also associated with antibodies to factor V and a low factor V level. This patient's clinical and laboratory profile is intriguing, given the lack of previous exposure to bovine or human thrombin and the presence of normal Factor V levels. This novel antibody may represent formation of a spontaneous autoantibody to thrombin due to failure of immunological surveillance mechanisms.

Disclosures:

No relevant conflicts of interest to declare.
Intracranial Hemorrhage In Patients with Inherited Bleeding Disorder In Children.
Authors: Park Ji Kyoung , Lee Soon Yong , Park Young Sil
Published: October 2019

Abstract 3657

Intracranial hemorrhage (ICH) is known to be a severe although uncommon complication of inherited bleeding disorder. A 12-year retrospective study was conducted to investigated the episodes of ICH in children with an inherited bleeding disorder who where followed by a regional hemophilia comprehensive center in South Korea. We review cases of ICH events in patients with inherited bleeding disorders in children and investigate characteristics, treatment, and prognosis. Between January 1998 and August 2010, a total of 13 ICHs in 9 patients were identified.

ICHs were occurred for 7 patients with hemophilia A, one patient with hemophilia B, and one patient with factor VII deficiency. All patients were male and severe hemophilia A(2 patients); moderate hemophilia A(5 patients); moderate hemophilia B(1 patients). One of the patients with severe hemophilia A had inhibitors. The median age of patients was 6years 11months old. (range, 7 days≂f15 years old). Types of ICHs were 8 SDH, 2 EDH, 1 intracerebral with IVH, 1 intracerebral with SDH and 1 SDH with SAH. 8 cases were occurred spontaneously and 5 cases were occurred after traumatic events. Presenting symptoms were headache (6 events), seizure (2 events), vomiting (1 event), stuporous mentality (1 event), lethargy (1 event), irritability (1 event) but one patient with traumatic SDH had no symptom. Initial diagnostic modality was CT scan. One patient with severe hemophilia A who had inhibitors was initially treated with FEIBA® and controlled well. The remaining patients with hemophilia were treated with factor eight/nine concentrate. The patients with factor VII deficiency was treated with recombinant factor VIIa (Novoseven®). The patient with moderate hemophilia B required surgical evacuation. The mean duration of hospitalization was 12.8 days (range, 3–25 days). ICH was the first significant bleeding event in 3 patients – one with moderate hemophilia A, aged 7 days, and one with moderate hemophilia A, aged 5months, and one with factor VII deficiency, aged 1months. The median follow-up duration was 73 months (range, 4–149 months). Two younger patients had complication with long term sequale like hydrocephalus, cerebral palsy, and epilepsy. For one patient, antibody to factor VIII was developed due to factor administration.

ICH can occur in patient with a severe inherited bleeding disorder and also in patients with moderate hemophilia. Younger patients with ICH may have poor prognosis and neurologic sequale more frequently. Symptoms of younger patients was atypical so their parental recognition and medical diagnosis delayed. The median duration between onset of symptom and administration of factor is 2.6 days(range, 0–8 days). Delayed administration of factor caused the patients to have long term sequale. Rapid and appropriate factor administration is important to treatment course and prognosis.

Disclosures:

No relevant conflicts of interest to declare.
Suicide Gene (HSVtk) Ablation of Hematopoietic Stem Cells and Their Progeny In the Rhesus Macaque Model: An Approach to Deletion of Dangerous Clones.
Authors: Barese Cecilia , King Connor , Sellers Stephanie , Krouse Allen E , Metzger Mark E , Traversari Catia , Bordignon Claudio , Kornblau Steven M. , Marini Frank , Donahue Robert E , Dunbar Cynthia E.
Published: October 2019

Abstract 3759

For genetic blood diseases, such as primary immunodeficiencies, gene therapy targeted to hematopoietic stem cells (HSCs) is a feasible and now proven effective therapeutic option for patients who lack a histocompatible HSC. However, the risk of adverse outcomes resulting from insertional oncogenesis is a major concern. We are investigating whether inclusion of the herpes simplex virus thymidine kinase (HSVtk) gene into integrating vectors into rhesus macaque HSCs confers ganciclovir (GCV) sensitivity allowing ablation of vector-containing cells from the blood and other hematopoietic compartments, as an approach to increasing safety of gene therapy procedures. HSVtk suicide genes have been studied in detail in transduced mature T cells, but never in stem and progenitor cells. We infused autologous CD34+ cells transduced ex vivo with gammaretrovirus vectors encoding the HSVtk as suicide gene along with marker genes into 4 rhesus macaques, following myeloablative irradiation. In the first animal, a vector consisting of the MND backbone driving the sr39 high affinity tk mutant, and IRES and a truncated NGFR marker gene was used. A stable marking level of 5% NGFR+ circulating cells was observed for 6 months following transplantation, confirmed by q-PCR. The drug GCV was infused at 5 mg/Kg BID for 21 days. This animal had complete elimination of vector-containing cells in all peripheral blood lineages as assessed by flow cytometry and qPCR, and remains negative now 4 months after GCV discontinuation. Three additional animals were transplanted with autologous CD34+ cells transduced with a vector containing a standard HSVtk gene and GFP as a marker. These animals had lower stable marking levels of approximately 1% at 4 months post-transplant, and after 21 days of GCV, had a clear decrease in the level of GFP+ cells, but not complete ablation, likely due to lower drug-sensitivity of the tk protein expressed by this vector. Cells with a lower level of GFP expression were not eliminated, supporting this hypothesis. Additional animals receiving cells transduced with the sr39 tk retroviral vector and with a lentiviral vector containing a codon-optimized HSVtk are in progress. These data suggest that inclusion of a suicide gene in integrating vectors may be an effective way to address genotoxicity concerns, should clonal outgrowth occur, and increase safety of HSC-targeted gene therapy.

Disclosures:

No relevant conflicts of interest to declare.
Feline Leukocyte Adhesion Deficiency.
Authors: Giger Urs , Pratt Suzanne M. , Palena Christina , Kennedy Doug R , Bauer Thomas R.
Published: October 2019

Abstract 3779

Leukocyte adhesion deficiency (LAD) caused by CD18 deficiency is an autosomal recessive immunodeficiency which has been described in humans, Irish Setter dogs, Holstein cattle, and transgenic mice. We report here on the characterization of the clinical features and leukocyte function abnormalities of a novel feline model of leukocyte adhesion deficiency.

The proband was a 5 year old male domestic longhair cat with a life-long history of infections, inflammation and granulomas. Severe gingivitis was recognized at 8 weeks of age which progressed to peridontitis and loss of all deciduous and adult teeth before 1 year of age. Over the next years this cat experienced recurrent severe upper respiratory infections, gastroenteritis, anterior uveitis, various abscesses, and epidermal sloughing with sepsis secondary to small grooming lacerations. The various illnesses seemed to be responsive to various long-term antibiotic as well as glucocorticoid treatments. Complete blood cell counts revealed a persistent severe leukocytosis (50-91,000/μl) due to mature neutrophilia with few toxic changes, and mild lymphocytosis, monocytosis and eosinophilia. Serum immunoglobulin concentrations were normal.

Compared to feline control cells, neutrophils of the proband did not express any CD18 on the cell surface as determined with two monoclonal anti-CD18 antibodies but normal CD45 expression. Adhesion of affected neutrophils with and without PMA activation was severally impaired; the adhesion of normal feline neutrophils could be completely blocked by the anti-CD18 antibody. In a proliferation assay the proband's T-cells responded weakly at 1 pg SEA but near normally at 100 pg SEA, suggesting a CD18-independent T-cell response in cats. Sequencing of the feline CD18 β-integrin gene is being completed.

In conclusion, this naturally-occurring feline model of CD18 deficiency exhibits similar features to LAD in other species. However, feline LAD seems clinically milder allowing for a longer life expectancy possibly due to T-cell independent activities. This LAD model may be helpful in developing and assessing novel therapies.

Disclosures:

No relevant conflicts of interest to declare.
Human Interleukin-3/Granulocyte Macrophage-Colony Stimulating Factor Knock-In Mice Support Human Myeloid Cell Reconstitution and Human Immune Responses In the Lung.
Authors: Willinger Tim , Rongvaux Anthony , Takizawa Hitoshi , Eynon Elizabeth E. , Stevens Sean , Manz Markus G , Flavell Richard A.
Published: October 2019

Abstract 3789

Humanized mice, i.e. mice with a functional human immune system, have great potential to study human immunology in vivo and to allow vaccine testing. To this end, mice need to fully support engraftment with human immune cells, allow infection with human pathogens, and mount effective human immune responses to pathogens. A major limitation of current humanized mice is the poor development and function of human myeloid cells. Here we report a novel strategy to overcome this limitation by generating human interleukin-3/granulocyte macrophage colony stimulating factor knock-in (hIL-3/GM-CSF KI) mice to create a better environment for human myeloid cells. These mice faithfully expressed human GM-CSF and IL-3 and developed pulmonary alveolar proteinosis (PAP) due to elimination of mouse GM-CSF. We demonstrate that hIL-3/GM-CSF KI mice engrafted with human CD34+ hematopoietic cells had improved human myeloid cell reconstitution in the lung. In particular, hIL-3/GM-CSF KI mice supported the development of human alveolar macrophages that partially rescued the PAP syndrome. In addition, these mice showed an enhanced systemic inflammatory response to LPS. Finally, humanization of IL-3 and GM-CSF lead to a stronger innate immune response against influenza virus infection. In summary, hIL-3/GM-CSF KI mice represent a new mouse model to study human immune responses in the lung and against pathogens such as influenza.

Disclosures:

Stevens: Regeneron Pharmaceuticals: Employment; AnaptysBio Inc: Employment.
Toll Receptors and Langerhans Cell Histiocytosis.
Authors: Very Gayane badalian , Vergilio Jo-Anne , Calicchio Monica , Fleming Mark D. , Rollins Barrett
Published: October 2019

Abstract 3793

Introduction:

Langerhans cell histiocytosis is a clonal proliferative disease of antigen presenting cells. Clinically, it is associated with aberrant secretion of cytokines from both Langerhans cells (LCs) themselves and immune cells associated with the disease. Toll-like receptors (TLRs) are membranous and cytoplasmic proteins involved in pattern-based recognition of bacterial and viral products. Activation of TLRs results in production of pro-inflammatory cytokines, modulation of the immune response, and maturation of LCs. TLRs activate signal transduction pathways involving IRFs (interferon regulatory factors), NFkB (Nuclear Factor-k-B) and MAPK (mitogen activated protein kinases).

In this study, we have examined LCH samples for their expression of TLRs and downstream effector proteins.

Methods:

We examined archived samples from 42 LCH patients. Paraffin embedded samples were sectioned and stained for immunohistochemistry (IHC) using antibodies directed against TLR1-9; IRF3, 7 and 8; MEK1/2 and ERK1/2. RNA was isolated from the same samples using laser-capture microdissection, and the expression of TLR receptors, effectors and downstream targets was analyzed using qRT-PCR. These data were compared with gene expression data published in Gene Expression Omnibus using data set GSE16395.

Results:

IHC results showed upregulation in pathologic LCs compared to normal LCs of TLR 1, 3, 5, 7, 8 and 9, but not TLR 4 and 6. Moreover, upregulation of downstream pathways involving MEK1/2 and ERK1/2 and IRF8 in was also observed in pathologic LCs. Gene expression analysis showed up-regulation of the effectors of TLRs including CASP8, MAP3K7, and PPARA as well as genes in the NFKB pathway (CCL2, NFKb1, NFKB2, NFKBIL1, NFRKB, and REL), the JNK/p-38 pathway (ELK1, c-FOS, MAP3K1 and MAP3K8), and the interferon regulatory factor pathway (CXCL10, IRF7, IRF8 and TBK1). Comparison with the gene expression data base confirmed the results obtained from qRT-PCR for the gene expression of TLRs, their effectors, and downstream.

Conclusion:

Upregulation of TLRs and the pathways they control occurs commonly in LCH. Therapies directed toward inhibition of TLRs and their downstream targets may provide a means of modulating the adverse inflammatory aspects of LCH.

Disclosures:

No relevant conflicts of interest to declare.
Mesenchymal Stromal Cells Accelerate Hematopoietic Reconstitution and Mediate An IL6-Dependent Regeneration of the Intestinal Epithelium of Lethally Irradiated Mice.
Authors: Francois Moira , Birman Elena , Coutu Daniel L. , Forner Kathy-Ann , Galipeau Jacques
Published: October 2019

Abstract 3846

Apoptosis of radiation-sensitive stem cells in the bone marrow and the gut are the main complications arising from radiation exposure, whether therapeutic or accidental. Bone marrow-derived mesenchymal stromal cells (MSC) were shown to exert regenerative properties through the secretion of factors which act in a paracrine manner. In this study, we demonstrated that a single intra-peritoneal injection of C57Bl/6 MSC rescued lethally irradiated Balb/c mice. Histological analysis and Ki67 immunostaining of jejunum sections collected 48 hours and 5 days post MSC injection indicated that MSC protected the gastro-intestinal epithelium from severe radiation-induced damages and prompted the regeneration of the gut by stimulating proliferation of the crypt stem cell pool. Using interleukin-6 null MSC, we demonstrated that IL-6 derived from the MSC played a role accelerating gastro-intestinal epithelium regeneration. We also observed that MSC injection prevented severe anemia and restored granulocyte and platelet counts to normal values faster than the control group. Accelerated regeneration of the bone marrow and extra-medullar hematopoiesis in the spleen of MSC transplanted mice corroborated this observation. Our results suggest that IL-6 derived from MSC or used on its own may be exploited to alleviate radiation induced injuries to patients undergoing radiotherapy or subject to accidental radiation exposure

Disclosures:

No relevant conflicts of interest to declare.
Microenvironment-Mediated Regulation of Micrornas In B-Lymphocytes as a Novel Mechanism for Terminal B-Cell Differentiation and Lymphomagenesis.
Authors: Lin Jianhong , Lwin Tint , Tam Wayne , Zhao Jian-Jun , Crespo Luis , Choi Yong Sung , Zhang Xinwei , Tannenbaum Victoria , Sotomayor Eduardo M. , Moscinski Lynn C. , Dalton William S. , Wright Ken , Tao Jianguo
Published: October 2019

Abstract 3853

B-cell differentiation is tightly regulated by synchronized suppression and/or induction of specific transcription factors. Among them, B-cell lymphoma 6 (BCL6) and PRDM1 are considered to be master regulators for germinal center formation and terminal B-cell differentiation. Dysregulation of BCL6 and PRDM1 also have been associated with lymphomagenesis. Their regulation still need further study especially at the posttranscriptional level. Here, by using co-culture system and whole genomic microRNA microarray profiling, we show for the first time that direct B lymphoma cell-stroma cell contact between follicular dendritic cells and B-lymphocytes could induce upregulation of miR-30 family and downregulation of miR-9 and let-7 family. In silico analysis showed that miR-30s can target genes BCL6 and miR-9/let7 can target PRDM1 with direct binding sites in 3`UTR region of their mRNAs. The microarray data can be proved by microRNA specific Q-RT-PCR. Specifically, by both gain of function and loss of function studies, we functionally verified that FDCs Regulate Expression of BCL6 and PRDM1 via Cell-Cell Direct Contact induced correlated microRNA dysregulation. To further validate the direct interaction between BCL6 and miR-30, we constructed luciferase reporters containing the BCL6 3`-UTR that included miR-30 binding sites and a mutant 3`-UTR harboring mutations in the “seed pairing” sequences of the miR-30 binding site. Co-transfection of miR-30 and reporter construct into cells significantly decreased luciferase activity in wild-type but not in mutant BCL6-3`-UTR transfected cells, supporting the role of miR-30 family in the regulation of BCL6 expression. BCL6 and PRDM1 and their regulation miRNAs, let-7 and miR-30, also can be validated in primary normal B-lymphocytes and lymphoma cells by using our co-culture system. Dysregulation of BCL6 and PRDM1 is often associated with lymphomagenesis. We firstly identified that BCL6 is the direct target of miR-30 family and also verified PRDM1 is the target of miR-9, and let-7 in our system. Our studies provide a novel mechanism of post-transcriptional regulation of BCL6 and PRDM1 by several microRNAs. In the context of micro-environment, it provides a clue for germinal center B-cell differentiation as well as B-cell lymphomas progression regulated by lymphocyte cell-stroma cell contact through microRNAs.

Disclosures:

No relevant conflicts of interest to declare.