bluuebunny/crossref_metadata_2025 huggingface dataset:
Gamma Prime Fibrinogen Does Not Cause Arterial Thrombosis
Authors: Walton Bethany L , Getz Todd M , Bergmeier Wolfgang , Uitte de Willige Shirley , Wolberg Alisa S.
Published: October 2019

Elevated levels of the acute phase plasma protein fibrinogen are associated with increased risk of thrombosis. Using a mouse model of arterial thrombosis, we have previously shown that elevated total fibrinogen levels increase the rate of fibrin formation, increase thrombus fibrin content, and directly promote arterial thrombosis (Machlus et al. 2011 Blood 117:4953-63). Fibrinogen is composed of two copies each of three polypeptide chains: Aα, Bβ, and γ. The fibrinogen γ chain undergoes alternative splicing, resulting in a dominant form (γA/γA) that comprises ∼90% of circulating fibrinogen and a minor species (γA/γ’) that comprises 8-15% of circulating fibrinogen. Epidemiologic studies have detected elevated levels of γA/γ’ fibrinogen in patients with a history of arterial thrombosis, indicating that this isoform associates with thrombotic risk. However, in vitro data show that γA/γ’ fibrinogen has anticoagulant properties due to its ability to bind and sequester thrombin, and suggest that its expression is upregulated in response to a proinflammatory/procoagulant process. To determine whether γA/γ’ fibrinogen is pro- or antithrombotic in vivo, we purified γA/γA and γA/γ’ fibrinogen from human plasma by anion exchange chromatography, raised circulating levels in mice by intravenous infusion, and determined the effects of increased levels of these proteins on arterial thrombus formation in vivo. We also determined the effects of γA/γA and γA/γ’ fibrinogen on whole plasma clot formation, platelet aggregation, and endogenous procoagulant activity. Compared to buffer controls (7.3 [4.6-40.0] minutes, median [range]), γA/γA fibrinogen shortened the time to carotid artery occlusion (5.5 [4.0-8.0] minutes, P<0.05), whereas γA/γ’ fibrinogen did not (7.6 [4.9-40.0] minutes, P=0.9). Both γA/γA and γA/γ’ fibrinogen could be cleaved by murine and human thrombin, were incorporated into murine and human clots, and supported murine and human thrombin-induced platelet aggregation. When γA/γA or γA/γ’ fibrinogen was spiked into normal human or murine plasma, both γA/γA and γA/γ’ fibrinogen accelerated the fibrin formation rate (P<0.02). However, γA/γA fibrinogen increased the rate to a greater extent than γA/γ’ fibrinogen (P<0.02). Interestingly, compared to buffer controls (18.9 [9.8-126.2] ng/mL), mice infused with γA/γ’ fibrinogen had lower levels of circulating plasma thrombin-antithrombin complexes (6.2 [0.9-89.4] ng/mL, P<0.01) following arterial injury, whereas mice infused with γA/γA fibrinogen did not (13.2 [10.7-80.1] ng/mL, P=0.7). These data suggest γA/γ’ fibrinogen exhibits “antithrombin I” activity in vivo by binding thrombin and decreasing circulating prothrombotic activity. Together, these findings indicate that elevated levels of the γA/γA fibrinogen isoform promote arterial thrombosis in vivo, whereas the γA/γ’ isoform does not.

Disclosures:

No relevant conflicts of interest to declare.
Anti-Factor VIII A2 Epitopes In a Murine Bleeding Model
Authors: Eubanks Joshua , Baldwin Wallace Hunter , Markovitz Rebecca , Parker Ernest T , Meeks Shannon L.
Published: October 2019

Up to 30% of patients with severe hemophilia A will develop inhibitory antibodies to factor VIII (fVIII inhibitors). In addition, autoimmune antibodies to fVIII can develop in non-hemophiliacs, producing acquired hemophilia A, which frequently produces life- or limb-threatening bleeding. Patients with congenital hemophilia who develop inhibitors usually have a polyclonal antibody response directed against the A2 and C2 domains of fVIII. Patients with acquired hemophilia typically have a more limited B-cell epitope response with antibodies directed against the A2 or C2 domain but not both. We have shown that within the C2 domain of fVIII antibody epitope is more important than inhibitory titer in predicting pathogenicity in a murine in vivo bleeding model. In this project we investigated the pathogenicity of a diverse panel of anti-A2 monoclonal antibodies (Mabs) in the murine in vivo bleeding model. We have previously characterized anti-A2 antibodies into groups A, AB, B, BCD, C, D, DE, and E based on the pattern of overlap on the B-cell epitopes in a competition ELISA. Table 1 shows the characteristics of the anti-A2 Mabs. Mabs were injected retro-orbitally into Exon 16 hemophilic (E16) mice at a dose of 0.5 umg per g body weight (∼ 65nM plasma concentration). Fifteen minutes later, mice were injected with B-domain deleted human fVIII at a dose of 180U/kg (∼ 2.5nM plasma concentration). In addition, a subset of Mabs has also been tested at a high dose of 360U/kg (∼5 nM). Two hours after fVIII injection, the mice were anesthetized and a 4mm tail snip was performed. Blood was collected in a tube of normal saline over 40 minutes and measured. 4A4, 2-76, and 1D4 are all high inhibitory titer, type I Mabs that produced significant bleeding with 180 U/kg fVIII when compared to control. In addition, 2-76 and 4A4 were tested at the higher dose of fVIII and significant bleeding was again seen. In comparison, the high titer type II Mab 2-54 had a similar inhibitory titer but no significant bleeding at either dose of fVIII. B94 is a type II inhibitor with a similar inhibitory profile to 2-54, but maximum inhibition is 45% as compared to 82% for 2-54. B94 also was not pathogenic at either fVIII dose tested. Both 4C7—a non-inhibitory Mab—and B25—a very low titer Mab that would be predicted to have residual fVIII activity at the Mab concentration tested—did not produce significant bleeding. The inhibitory titer alone did not predict bleeding phenotype within a diverse panel of anti-A2 Mabs. This discrepancy combined with similar findings in the C2 domain stress the importance of inhibitor properties not detected in the standard Bethesda assay in predicting response to fVIII therapy.

Disclosures:

No relevant conflicts of interest to declare.
Venous Thromboembolism (VTE) Risk Assessment & Prophylaxis In Irish Hospitalized Obstetric Patients: A Nationwide Cross-Sectional Study
Authors: Noone Caroline M , O'Shea Susan , Crowley Maeve P , Higgins John
Published: October 2019

Background

Pulmonary embolism continues to be the second leading cause of mortality in pregnancy and the puerperium. VTE complicates 1 - 2/1000 pregnancies, and the risk increases with age, mode of delivery, and presence of co-morbidities. Literature has shown that the use of low molecular weight heparin (LMWH) is safe in this patient cohort.

Aims

Assessment of the prevalence of VTE risk in hospitalised women in both the antenatal and postnatal groups to determine the proportion of at-risk patients who receive LMWH prophylaxis appropriately.

Methods

The study period was September 2011 to November 2012. All inpatients in the participating hospitals on the day of investigation were assessed for risk of VTE on the basis of hospital chart review. Risk profile was assessed in accordance with the 2009 Royal College of Obstetricians and Gynaecologist Guidelines. Patients undergoing procedures or on the labour ward at the time of review were excluded. Ethical approval was obtained from the ethics committees governing all centres.

Results

610 pregnancies were reviewed across 19 centres. The average age of was 31+/- 5.65yrs (Range 16-47), with 21.87% aged over 35. 22% had a parity of 3 or more. The average weight was 71.51kg (Range 42-134kg, SD 14.482kg). Data on BMI was available for 77% - 34% were overweight and 21% were obese. 1% had a BMI>40. 31% were antenatal and 69% were postnatal.

63% of antenatal patients were low risk (<2 risk factors), 35% were intermediate risk (2 or more risk factors, prophylaxis should be considered) and 2% were high risk. All the high risk patients were on prophylaxis at an appropriate dose. 4% of the low risk patients were on prophylaxis unnecessarily. Only 7% of the intermediate risk patients were on correctly dosed prophylaxis.

Among postnatal patients, 41% were low risk (<2 risk factors), 58% were intermediate risk (2 or more risk factors, require prophylaxis) and <1% were high risk. 80% were appropriately risk stratified and put on LMWH if necessary. 59% of patients should have been on LMWH but only 42% were (92% Tinzaparin and 8% Enoxoparin). This included 8 patients who were on LMWH unnecessarily. 38% were on too low a dose.

Conclusion

VTE prophylaxis remains a central issue in obstetric care given its prominent role in maternal morbidity and mortality and the increasing prevalence of risk factors such as obesity and increasing maternal age. It is clear that while there is good awareness of the risk in the postnatal period, there is less emphasis on risk assessment in antenatal patients where prophylaxis is rarely used. Those on prophylaxis are also likely to be on an incorrect dose. There is a clear role for a national guideline to standardize care for all pregnant women. On the basis of these findings, we have authored a guideline and this is now in use as a reference in all Irish maternity units.

Disclosures:

No relevant conflicts of interest to declare.
The RNA Environment Of Hematopoietic Stem Cells
Authors: Wendt George R , Oshima Motohiko , Iwama Atsushi
Published: October 2019

Recent reports in RNA biology have suggested that long non-coding RNAs, edited RNAs, and alternatively spliced RNAs (henceforth referred to as non-canonical RNAs) play a relatively unclear but critical role in numerous cells, tissues, and diseases. Of interest to hematology, several recent studies have also shown that these non-canonical RNAs all play various roles in both normal and malignant hematopoiesis, but no study yet has comprehensively examined these non-canonical RNAs in hematopoietic stem cells (HSCs).

To this end, we conducted microarray and RNAseq analyses of mouse hematopoietic cells at various stages of differentiation. Microarray analysis detected several thousand possible lincRNAs expressed in hematopoietic stem and progenitor cells (HSPCs), several hundred of which were significantly enriched in CD34-LSKs compared to more differentiated populations. RNAseq analysis provided a more detailed look at these non-coding RNAs, as well as a slightly more conservative estimate of the abundance and structure of the long non-coding RNAs expressed and significantly enriched in HSPCs. In addition to examining lincRNAs, RNAseq data allowed us to profile alternative splicing events and RNA editing events in HSPCs. Interestingly, several hundred potentially significant HSC-specific alternative splicing events and RNA editing events were also discovered.

In order to test whether these non-canonical RNAs play a functional role in HSPCs we performed shRNA mediated knockdown of several interesting HSC-specific lincRNAs and alternatively-spliced transcripts. We found that knockdown of several of these candidates was incompatible with hematopoiesis, both in vitro and in vivo. To better understand how these particular non-canonical RNAs function in HSCs, we are currently carrying out RNA FISH studies.

In this study, we explored the RNA environment of HSCs and compared it to their downstream progenitors. We discovered several hundred novel non-coding transcripts, hundreds of alternative splicing events and hundreds of RNA editing events unique to HSCs. Functional investigation of some of these events revealed that these non-canonical RNAs do indeed play a critical role in HSC maintenance. The data presented here should eventually be an asset to other HSC researchers interested in the RNA biology of HSCs.

Disclosures:

No relevant conflicts of interest to declare.
G-CSF Treatment Induces Toll-Like Receptor Signaling and Regulates Hematopoietic Stem Cell Function
Authors: Schuettpelz Laura G. , Borgerding Joshua N. , Gopalan Priya , Christopher Matt , Romine Molly , Woloszynek Jill , Greenbaum Adam , Link Daniel C.
Published: October 2019

Recent studies demonstrate that inflammatory signals regulate hematopoietic stem cells (HSCs). Granulocyte-colony stimulating factor (G-CSF) is often induced with infection and plays a key role in the stress granulopoiesis response. However, its effects on HSCs are unclear. Herein, we show that treatment with G-CSF induces expansion and increased quiescence of phenotypic HSCs, but causes a marked, cell-autonomous HSC repopulating defect. RNA profiling and flow cytometry studies of HSCs from G-CSF treated mice show that multiple toll- like receptors (TLRs) are upregulated in HSCs upon G-CSF treatment, and gene set enrichment analysis shows enhancement of TLR signaling in G-CSF-treated HSCs. G-CSF-induced expansion of phenotypic HSCs is reduced in mice lacking the TLR signaling adaptors MyD88 or Trif, and the induction of quiescence is abrogated in mice lacking these adaptors. Furthermore, loss of TLR4 mitigates the G-CSF-mediated HSC repopulating defect. Interestingly, baseline HSC function is also dependent on TLR signaling. We show that HSC long-term repopulating activity is enhanced in Tlr4-/- and MyD88-/- mice, but not Trif-/- mice. One potential source of TLR ligands affecting HSC function in the bone marrow is the gut microbiota. Indeed, we show that in mice treated with antibiotics to suppress intestinal flora, G-CSF induced HSC quiescence and hematopoietic progenitor mobilization are attenuated. Moreover, in germ free mice, HSC long-term repopulating activity is enhanced. Collectively these data suggest that low level TLR agonist production by commensal flora contributes to the regulation of HSC function and that G-CSF negatively regulates HSCs, in part, by enhancing TLR signaling. Our finding of enhanced TLR signaling upon G-CSF treatment, and the mitigation of G-CSF’s effects in mice deficient for TLR signaling or commensal organisms, suggest that TLR antagonists and/or agonists may ultimately be used clinically to enhance engraftment following bone marrow transplantation or applied toward the treatment of patients with bone marrow failure.

Disclosures:

No relevant conflicts of interest to declare.
MiR-29a Maintains Hematopoietic Stem Cell Self-Renewal and Is Required For Myeloid Leukemogenesis
Authors: Hu Wenhuo , Dooley James , Chung Stephen S. , Yalcin Safak , Shin Yu Sup , De Strooper Bart , Liston Adrian , Park Christopher Y.
Published: October 2019

microRNAs (miRNAs) are important regulators of both embryonic and adult tissue stem cell self-renewal. We previously showed that ectopic expression of miR-29a, a miRNA highly expressed in HSCs as well as in human acute myeloid leukemia (AML) stem cells, in immature mouse hematopoietic cells is sufficient to induce a myeloproliferative disorder that progresses to AML. During the early phase of this disease, miR-29a induces aberrant self-renewal of committed myeloid progenitors, strongly suggesting a role for miR-29a in regulating HSC self-renewal. In order to determine the role of miR-29a in HSC function, we have evaluated our recently described miR-29a/b1 null mouse. Homozygous deletion of miR-29a/b1 resulted in reduced bone marrow cellularity and reduced colony forming capacity of hematopoietic stem and progenitor cells (HSPCs). The phenotype was mediated specifically by miR-29a since miR-29b expression was not significantly altered in HSCs and reconstitution of miR-29a/b1 null HSPCs with miR-29a, but not miR-29b, rescued in vitro colony formation defects. Self-renewal defects were observed in miR-29a deficient HSCs in both competitive and non-competitive transplantation assays, and these deficits were associated with increased HSC cell cycling and apoptosis. Gene expression studies of miR-29a deficient HSCs demonstrated widespread gene dysregulation including a number of up-regulated miR-29a target genes including DNA methylation enzymes (Dnmt3a, -3b) and cell cycle regulators (e.g. Cdk6, Tcl1, Hbp1, Pten). Knockdown of one of these targets, Dnmt3a, in miR-29a deficient HSCs resulted in partial restoration of colony formation, providing functional validation that Dnmt3a mediates part of miR-29a null HSPCs functional defects. miR-29a loss also abrogated leukemogenesis in the MLL-AF9 retroviral AML model. Together, our results demonstrate that miR-29a positively regulates HSC self-renewal and is required for myeloid leukemogenesis.

Disclosures:

No relevant conflicts of interest to declare.
Comparative Analysis Of Cytogenetic Evolution Patterns During Relapse In The Hematopoietic Stem Cell Transplantation and Chemotherapy Settings Of Patients With Acute Leukemia
Authors: Cho Young-Uk , Chi Hyun-Sook , Park Sang Hyuk , Kim Young Jin , Jang Seongsoo , Seo Eul-Ju , Park Chan-Jeoung
Published: October 2019

Background

Despite significant therapeutic progress, many patients with acute leukemia die from deteriorating disease after relapse. Relapse of acute leukemia has repeatedly been associated with cytogenetic clonal evolution. However, only a few studies focused on the direct comparison of cytogenetic evolution patterns during relapse of leukemia after hematopoietic stem cell transplantation (HSCT) and conventional chemotherapy. Thus, we performed comparisons of the cytogenetic patterns of patients with acute leukemia both at diagnosis and relapse after HSCT or standard chemotherapy (SC). Cytogenetic patterns with FLT3 mutational instability were also compared.

Methods

This retrospective analysis was based on a total of 516 patients. Among them, 349 were diagnosed with acute myeloid leukemia (AML) who developed relapse after HSCT (n = 125) or SC (n = 224); 167 were diagnosed with acute lymphoblastic leukemia (ALL) and developed relapse after HSCT (n = 63) or SC (n = 104). Cytogenetic analysis and FLT3 mutation detection were performed according to standard methods.

Results

In patients with AML as shown in Table 1, differences in the karyotypes between diagnosis and relapse were more frequent in the HSCT cohort than in the SC cohort (53.2% vs. 40.1%, respectively; P = 0.035). Development of more than three new cytogenetic changes was also more frequent in the HSCT cohort than in the SC cohort (31.4% vs. 15.3%, respectively; P = 0.046). Overall, FLT3 mutation instability did not correlate to the clonal cytogenetic changes. However, restricted to patients showing FLT3 mutation instability, tyrosine kinase domain (TKD) mutation was more frequently involved in patients with cytogenetic changes compared to those without (46.2% vs. 5.0%, respectively; P = 0.008) (Table 2). Conversely, in patients with ALL, frequency and pattern of the cytogenetic alterations between diagnosis and relapse did not differ between HSCT and SC cohorts. For both types of acute leukemia, the median number of cytogenetic alterations increased from 1.0 at diagnosis to 2.0 at relapse (P < 0.001) in the HSCT cohort. The increasing number of cytogenetic alterations was also seen in the SC cohort (P = 0.011).

Conclusions

Higher frequencies of clonal cytogenetic changes and more complex cytogenetic patterns were observed in the HSCT cohort compared to the SC cohort. This finding may be associated with an adverse outcome for relapse patients after HSCT. FLT3 TKD mutation could have a certain role in promoting cytogenetic progression in a subset of AML patients. In addition, our data extends the findings of recent previous studies, which described patterns of clonal evolution in acute leukemia patients.

Disclosures:

No relevant conflicts of interest to declare.
Exosomes as Markers Of Therapeutic Response In Patients With Acute Myeloid Leukemia
Authors: Boyiadzis Michael , Hong Chang-Sook , Whiteside Theresa L
Published: October 2019

Objective

Exosomes are virus-size (50-100 nm) double membrane-bound microvescicles formed within the endocytic compartments and released into the extracellular space via the fusion of multivescicles bodies with the cell membrane. While all cells release exosomes under physiologic conditions, tumor cells are avid exosome producers. In this study, we isolated exosomes from plasma of acute myeloid leukemia (AML) patients at diagnosis, during and after chemotherapy and studied their protein and TGF-β1 content. The hypothesis was tested that these exosomal components may serve as biomarkers of response to chemotherapy in AML.

Methods

Exosome fractions were isolated from plasma by exclusion chromatography on Sephadex G50 columns followed by ultracentrifugation. Exosome protein content was determined and expressed in μg protein/mL plasma. Exosome-associated TGF-β1 was measured by ELISA following exosome sonication/acidification and expressed in pg/exosome protein/mL plasma.

Results

Exosomes isolated from AML patients’ plasma (n=16) at diagnosis and prior to any treatment had a higher mean protein content (55.2 ± 13.8 μg protein/mL) than those isolated from sera of normal controls (n=7), (13.1 ± 2.4 μg protein/mL, p<0.009). Following a course of induction chemotherapy, a reduction of the exosome protein level (23.8 ± 4.4 μg protein/mL, p<0.05) was observed in AML patients’ plasma (n=9). The exosome protein levels in the patients who at 14 days after the initiation of induction chemotherapy had a significant reduction of their blasts (to <5%) were lower compared to those in the patients with residual leukemia (18.5 ± 5.8 vs 30.8 ± 7.5 μg protein/mL, p=0.2). The exosome protein levels during consolidation therapy with high dose cytarabine in patients who had achieved complete remission (n=10) was higher (52.4 ± 8.9 μg protein/mL) compared to levels measured immediately after induction chemotherapy (p<0.02) and approaching those seen at diagnosis. Protein levels of exosomes isolated from plasma of patients (n=5) in long-term remission (>2 years after completing consolidation therapy) normalized to the levels which were not different from those of normal controls. Levels of active TGF-β1 carried by exosomes obtained at AML diagnosis were high and then decreased (p<0.005) following induction chemotherapy. Similar to protein levels, TGF-β1 levels in plasma-derived exosomes were elevated (p<0.004) during consolidation chemotherapy. In AML patients in long-term remission, TGF-β1 levels carried by exosomes normalized, approaching levels seen in exosomes of normal controls.

Conclusions

Our results indicate that exosomal protein and TGF-β1 levels in AML plasma could serve as biomarkers of response to chemotherapy and might reflect the presence/absence of residual disease after therapy.

Disclosures:

No relevant conflicts of interest to declare.
FLT3-ITD Internal Tandem Duplication Confers Poor Prognosis In Patients With Acute Promyelocytic Leukemia Treated With The AIDA Protocols. Long-Term Follow-Up Analysis
Authors: Breccia Massimo , Loglisci Giuseppina , Loglisci Maria Giovanna , Ricci Roberto , Diverio Daniela , Latagliata Roberto , Foà Robin , Lo Coco Francesco
Published: October 2019

Although previous studies in APL have revealed that the FlT3-ITD mutation is associated with an elevated presenting WBC count, hypogranular variant (M3v) morphology and the short (bcr3) isoform of PML-RARA, the prognostic significance of FlT3 mutations in APL has not been firmly established. We report an update of these patients with a median follow-up of 9 years in which we observed that the presence at baseline of the FlT3-ITD mutation confers a very poor overall survival (OS). One hundred and forty-seven patients with newly diagnosed APL were observed and treated with the AIDA (73 patients) and AIDA2000 protocols (74 patients) at the Sapienza University of Rome during the period April 1993-October 2010. Diagnosis was initially established morphologically and subsequently confirmed in all cases by RT-PCR. The following clinical characteristics at diagnosis were analyzed according to the FlT3 status: age, sex, FAB classification, peripheral WBC and platelet count, hemoglobin, karyotype, PML/RAR isoform and relapse risk. For statistical analysis, the Wilcoxon-Mann-Whitney test was used to compare non-parametric series and the Fisher’s exact test to compare categories. OS was estimated using the Kaplan-Meier method, whereas relapse-free considered as events relapse and death in CR. Thirty-three patients were identified as FlT3+, 19 were males (57%) and 14 were females; 27% had a variant type according to the FAB classification and 36% were classified as high-risk. Twenty-one FlT3+ patients (63%) presented the bcr3 transcript compared to 37/114 (32%) FlT3- patients (p=0.002). Eight FlT3+ patients (24%) experienced a differentiation syndrome compared to 14 (12%) in the negative cohort (p=0.02). After a median follow-up of 9 years (range 5-19), we could document a significant worse long-term outcome for FlT3+ patients: OS was 96% in the FlT3- cohort compared to 39% in the FlT3+ cohort (p=0.0001), relapse-free survival (RFS) 90% in FlT3- patients vs 30% in the FlT3+ ones (p=0.017), CIR at 9 years was 4% (95% CI, 1,279-6,238) for the FLT3-negative cohort and 60% (95% CI,22.632-81.236) for the FLT3-positive subset (p=0.0001). While this study confirms that there is no difference in response to induction with the AIDA schedule in FlT3+ APL patients, the longest follow-up so fare reported has allowed to demonstrate the significantly worse long-term outcome for this subset of APL, in terms of OS, RFS and DFS. Further studies aimed at investigating whether other clonal mutations may play a role in this unfavorable subset of patients are needed.

Disclosures:

No relevant conflicts of interest to declare.
Prognostic Significance Of The Cytogenetic Evolution After The Hematopoietic Stem Cell Transplantation In Adult Acute Myeloid Leukemia
Authors: Yuasa Mitsuhiro , Uchida Naoyuki , Kaji Daisuke , Ota Hikari , Ishiwata Kazuya , Tsuji Masanori , Yamamoto Hisashi , Yamamoto Go , Asano-Mori Yuki , Makino Shigeyoshi , Yoneyama Akiko , Wake Atsushi , Izutsu Koji , Taniguchi Shuichi
Published: October 2019

Background

Cytogenetic abnormalities at diagnosis have significant impacts on disease outcome and clonal evolution at cytogenetic level is considered to be associated with relapse and refractoriness to chemotherapy. Relapse of AML was reported to be associated with cytogenetic clonal evolution in 40% of patients who have received chemotherapy. However, little is known about the association of cytogenetic clonal evolution and relapse after allogeneic hematopoietic stem cell transplantation (allo-HSCT). This study was conducted to see the karyotypic changes of the AML relapsed after allo-HSCT and to see whether the presence of the cytogenetic evolution has a prognostic impact.

Patients and Methods

We retrospectively reviewed patients diagnosed as AML who underwent allo-HSCT for the first time at our institute from Jan. 2006 to Dec. 2012 consecutively and analyzed the cytogenetic evolution patterns in patient at relapse of AML after HSCT.

Results

Three hundred and thirty patients were included in this study. We identified fifty-seven (17%) relapsed in bone marrow after HSCT. We excluded two patients, because of that one patient was not performed bone marrow aspiration after relapse and the other received auto-PBSCT after allo-HSCT due to an engraftment failure. So, we analyzed the bone marrow karyotypic changes before and after transplantation of 55 patients. Thirty-eight (69%) were male, and median age at allo-HSCT was 56 years (range, 21-82). Eight (14%) are still alive. Thirty-four (61%) received umbilical cord blood transplantation (UCBT), ten (18%) did related HSCT, and eleven (20%) did unrelated HSCT. Eleven (20%) had normal karyotype, fourteen (25%) had one chromosomal abnormality, eleven (20%) had two abnormalities, and nineteen (35%) had more than three abnormalities before HSCT. Median observation time after HSCT was 16 months (1-75). Thirty (55%) showed gain or loss of chromosomal abnormalities from original one (group1), sixteen (29%) showed no karyotypic change between HSCT, and nine (16%) showed totally different karyotype. We defined the cytogenetic evolution group as group 1, and the others as group 2. Median day of relapse after HSCT was not different between group 1 and 2 (4 months (range 1-73), 6 months (range 1-26), P=0.29). Shorter survival time after relapse was observed in group 1 (5.9% vs. 16.8% at 2 year post-relapse, P= 0.04) (figure. 1).

Conclusion

Although durations from HSCT to relapse were comparable between group 1 and 2, survival time after relapse was significantly shorter in group 1 than in group 2, which indicates that cytogenetic clonal evolution may confer resistant characters against treatments, and needs novel therapeutic applications.

Disclosures:

No relevant conflicts of interest to declare.
Trends In Survival Outcome Of Elderly Patients With B-Lineage Acute Lymphoblastic Leukemia : Analysis Of SEER Database
Authors: Guru Murthy Guru Subramanian , Mehta Paulette , Venkitachalam Raji
Published: October 2019

Background

B-lineage Acute Lymphoblastic Leukemia (B-ALL) is the commonest form of ALL in elderly age. Prognosis in this group is generally considered poor due to higher incidence of t(9,22) and other mutations which increase with age and presence of higher comorbidities. It is unclear if the survival has improved over time in this high risk group with addition of newer agents like tyrosine kinase inhibitors and monoclonal antibodies along with chemotherapy.

Methods

Patients were selected from the 13 SEER registry sites (1992 onwards) in the Nov 2012 submission data. Patients included were 60 years and above, with microscopy confirmed B-ALL as the first primary malignancy diagnosed between 1992-2010. We excluded patients diagnosed only at autopsy/death certificate and without survival or demographic data. Patients were evaluated by period of diagnosis (1992-2001 and 2002-2010) and age at diagnosis (60-69 years, 70 years and above). Baseline characteristics were noted (age, sex, race including white, black, Native American, Asian/Pacific Islander). Descriptive statistics were used for comparison of baseline characteristics. One-year and 5-year overall survival (OS) were estimated by Kaplan-Meier method and compared by log rank test. Determinants of OS were analyzed with Cox-regression method. Statistical analyses were done with significance level of p < 0.05.

Results

Among 646 patients who were ≥60 years with B-ALL, 239 patients were in 1992-2001 group and 407 patients were in 2002-2010 group. Mean age was 71.7 years for patients in 1992-2001 group and 70.8 years for patients in 2002-2010 group (p=0.12). Both groups had similarly more males than females, more whites than blacks and other races (Table 1). Patients between 60-69 years had 1-year OS of 50.3 % in 2002-2010 group compared to 32.3 % in the 1992-2001 group (p=0.01). Five-year OS in 2002-2010 group was 20.2% compared to 8.1% in 1992-2001 (p<0.01)(Figure 1). Patients 70 years or older had no significant improvement in 1 year OS or 5 year OS over this period of time (5 year OS 5.5 % in 1992-2001 vs. 12.2 % in 2002-2010, p-NS) (Figure 2). In multivariate analysis, 2002-2010 group was associated with lower mortality (HR 0.77, P=0.004) and age >70 was associated with higher mortality (HR 1.56, P=0.001).

Conclusion

Patients between the ages of 60 and 69 years with B-lineage ALL treated after 2001 had improved 1-year OS and 5-year OS compared to those treated prior to 2001 reflecting efficacy of new therapies in this age group. However, patients above 70 years of age need more effective and safe therapeutic options to improve the survival outcome.

Disclosures:

No relevant conflicts of interest to declare.
Elimination Of Minimal Residual Disease (MRD) In AML Patients With a Novel Fc-Optimized FLT3 Antibody (4G8-SDIEM)
Authors: Salih Helmut R. , Hofmann Martin , Grosse-Hovest Ludger , Nuebling Tina , Bamberg Maximilian , Aulwurm Steffen , Buehring Hans-Joerg , Bethge Wolfgang A. , Kanz Lothar , Jung Gundram
Published: October 2019

Chemotherapeutic treatment induces a morphological complete remission (CR) in the majority of leukemia patients, but minimal residual disease (MRD) remains detectable by sensitive methods and constitutes the basis for relapse. Immunotherapeutic strategies are rarely sufficient to eliminate large tumor burden, but can successfully eliminate MRD. This is exemplified by the efficacy of donor lymphocyte infusion (DLI) and allogeneic stem cell transplantation (SCT) in leukemia. Here we report the first clinical experience with a novel Fc-optimized antibody directed to the receptor tyrosine kinase FLT3 expressed on the surface of leukemia cells. This antibody may potently eliminate MRD after conventional therapy and achieve this goal with less toxicity (i.e. GvHD) than DLI and SCT. After in vitro characterization of the antibody, which revealed its markedly increased ability to mediate antibody dependent cytotoxicity (ADCC), in particular of NK cells, against leukemic cells (Hofmann et al, Leukemia 2012), the antibody (4G8SDIEM) was produced in a university owned unit and applied on a compassionate need basis. Upon application in AML patients with active disease, potent NK cell activation and anti-leukemia activity were observed, but effects were rather short lived, most likely due to unfavorable NK:leukemia cell ratios in the bone marrow. Consecutively we applied 4G8SDIEM to AML patients in morphological CR after SCT which displayed increasing MRD levels (mutNPM1) indicative of imminent relapse and for which no other treatment options were available. In 3 of the 4 patients treated so far, a single application of 4G8SDIEM (in 2 cases followed by treatment with one dose of DLI) resulted in rapid elimination of MRD. The non-responsive patient displayed high titers of preexistent anti-Fab antibodies capable of binding 4G8SDIEM, which provides a potential explanation for the lacking response. One patient that had received subsequent DLI died of complications due to GvHD after reaching and maintaining a molecular CR. The other two patients (1 with, 1 without DLI) remained in molecular CR for at present 17 and 12 months after 4G8-SDIEM application without further specific treatment. No stem cell toxicity was observed. Our results imply that 4G8SDIEM is capable to reduce or eliminate MRD after conventional therapy. Based on these observations, a phase I/II trial enrolling AML patients in CR with detectable MRD is in preparation.

Disclosures:

Hofmann: Synimmune, GmbH, Tuebingen, Germany: Employment. Grosse-Hovest:Synimmune, GmbH, Tuebingen, Germany: Employment. Aulwurm:Synimmune, GmbH, Tuebingen, Germany: Employment. Jung:Synimmune GmbH, Tuebingen, Germany: Equity Ownership, Honoraria.
Parenteral CoQ10 Formulation (BPM31510) Significantly Improves Survival In Animal Model Of Leukemia Including The Resolution Of Paraplegia Due To Brain Metastasis
Authors: Narain Niven R , Mauro Lucia M , Akmaev Viatcheslav R , Benaim Ely , Sarangarajan Rangaprasad , Jimenez Joaquin J
Published: October 2019

Leukemia cells exhibit alterations in intermediary metabolism similar to other cancers, wherein ATP sourcing is shunted from mitochondrial oxphos towards glycolytic preponderance (Warburg Effect) to meet oncogenic proliferative demands. A consequence of this metabolic switch is the simultaneous short-circuit of the programmed death pathways, leading to a immortalization program in cancer cells including leukemia. Delivery of high levels of CoQ10 in a lipid nanodispersion mixture (BPM31510) has been demonstrated to preferentially shift metabolic networks from glycolysis towards mitochondrial-centric oxphos and recapitulation of apoptotic pathways in various cancers in vitro and in vivo models. Given the centrality of the Bcl-2 involvement in the etiology of leukemia, this study focused on investigation of the effectiveness of BPM31510 in animal models of erythroid and myeloid leukemia. Human acute erythro-leukemia (K562) and acute myeloid leukemia (KG1) models were developed in immune-compromised mice. The mice (total n=120 for each model respectively) were randomized into four (n=30/group) treatment groups: Untreated (control); BPM31510 (75 mg/kg, once/day); chemotherapy (Adriamycin [5mg/kg; once/wk]+AraC [25mg/kg; 5 days] and BPM31510+chemotherapy. All dosing were intravenous and followed a protocol of 3wk treatment followed by 1wk rest. In both leukemia models, combination of BPM31510 with chemotherapy was associated with significant increase in survival compared to other groups. BPM31510 alone improved survival compared to chemotherapy in myeloid leukemia, not in erythroleukemia model. In a separate study, a rat (Fisher 344) chloroleukemia (MIA C51) model of CNS leukemia was developed that demonstrated paraplegia and urinary retention as a result of brain metastasis. Administration of BPM31510 (50 mg/kg/day, IP) was associated with complete resolution of limb paralysis demonstrating the ability of BPM31510 in penetrating into the CNS. Moreover, BPM31510 (50 mg/kg/day, IP) administration was associated with significant increase in survival in animals with metastasis to the lungs and liver. The data provides encouraging evidence of the potential translational use of CoQ10 containing BPM31510 in the treatment of leukemia. A Phase 1 study in relapse acute leukemia is to be started shortly.

Disclosures:

Narain: Berg: Employment. Akmaev:Berg: Employment. Benaim:Berg: Employment. Sarangarajan:Berg: Employment. Jimenez:Berg: Membership on an entity’s Board of Directors or advisory committees.
Arginine Deprivation With Pegylated Arginine Deiminase Induces Death Of Acute Myeloid Leukaemia Cells In Vivo
Authors: Miraki-Moud Farideh , Ariza-McNaughton Linda , Ghazaly Essam A , Hodby Katharine A , Luong Phuong , Clear Andrew , Sohrabi Fareeda , Bomalaski John , Gribben John , Bonnet Dominique , Szlosarek Peter , Taussig David C
Published: October 2019

Introduction

Malignant cells require amino acids for a wide range of core functions. Amino acid deprivation using enzymatic degradation has been used to induce remission in acute lymphoblastic leukemia for decades. Amino acid deprivation may also benefit patients with acute myeloid leukemia (AML). We have previously shown that AML cells lack of argininosuccinate synthetase 1(ASS1), a key enzyme in the pathway that produces arginine (1). Here we tested the effect of an arginine depleting agent, pegylated arginine deiminase (ADI-PEG 20) on primary AML cells in a xenograft model of AML.

Methods

NOD/SCID/interleukin 2 gamma chain null (NSG) mice were transplanted with 6 primary AML samples. 12 weeks after transplantation of AML mice received either ADI-PEG 20, cytarabine, ADI-PEG 20 plus cytarabine or vehicle. ADI was administered weekly for 4 doses and cytarabine was given for 10 consecutive days (0.2mg per day, roughly equivalent to 40mg in humans). Five weeks after treatment started, mice were killed and the percentage of AML in the bone marrow was determined by flow cytometry. Blood was collected to quantify plasma arginine using liquid chromatography-mass spectrometry/mass spectrometry.

Results

Plasma arginine levels were depressed following ADI-PEG 20 administration confirming that arginine depletion was achieved in vivo. In all six experiments the combination of ADI-PEG 20 and cytarabine induced a significant reduction in levels of AML compared to control (Figure 1). Critically the combination of ADI-PEG 20 and cytarabine was significantly better than cytarabine alone in three of six experiments.

Conclusion

Our experiments show that arginine deprivation by ADI-PEG 20 can decrease the leukemic burden in mice transplanted with primary AML cells. The combination of ADI with cytarabine had a greater effect than cytarabine alone in half the experiments. These results provide the rationale to test ADI-PEG 20 with cytarabine in clinical trials.

1. Peter W. Szlosarek, Fiona Luong, Andrew Clear, David Taussig, Simon Joel, Maria Calaminici, Silvana Debernardi, Jude Fitzgibbon, John S. Bomalaski, Arthur E. Frankel, and Dominique Bonnet. Pegylated arginine deiminase (ADI-PEG 20) as a potential novel therapy for argininosuccinate synthetase-deficient acute myeloid leukemia. Cancer Research: April 15, 2011. AACR 102nd Annual Meeting 2011, (AACR Abstract # 467)

F. M-M and L. A-M contributed equally

D.B., P.W.S. and D.C.T contributed equally

Disclosures:

Bomalaski: Polaris Group: Employment, Equity Ownership. Szlosarek:Polaris Group: Research Funding.
GADD45a Is a Tumor Suppressor In BCR-ABL-Driven Leukemogenesis
Authors: Mukherjee Kaushiki , Sha Xiaojin , Bhatia Ravi , Skorski Tomasz , Hoffman Barbara , Liebermann Dan
Published: October 2019

The BCR-ABL fusion oncogene which encodes a fused deregulated tyrosine kinase causes chronic myelogenous leukemia (CML) in humans. Imatinib, a small molecule ABL kinase inhibitor has been highly effective in treating chronic phase (CP) CML patients. However, a substantial number of patients undergo relapse due to development of resistance to imatinib therapy that leads to blast crisis (BC-CML), which is invariably fatal within weeks to months. Additional genetic aberrations assist in progression and identification of key players that are responsible for transformation is of utmost importance from a therapeutic point of view. Growth arrest DNA damage 45a (Gadd45a) gene, a member in the gadd45 family of genes including Gadd45b & Gadd45g, was identified as a myeloid differentiation primary response gene. There is evidence consistent with it’s involvement in G2/M cell cycle arrest and apoptosis in response to multiple stressors, including genotoxic and oncogenic stress. To investigate the effect of Gadd45a in the development of CML, adaptive bone marrow transplantation experiments with either wild type or Gadd45a null myeloid progenitors expressing 210-kD BCR-ABL fusion oncoprotein revealed that loss of Gadd45a accelerated BCR-ABL driven CML resulting in the development of a more aggressive AML/BC like disease. Recent newly obtained data indicate that number of Gadd45a deficient Leukemic stem cells (LSC) harboring BCR-ABL increased as disease progressed confirming Gadd45a as a crucial tumor suppressor in CML. Recent data also indicate, that transformed Gadd45a deficient progenitors exhibit increased proliferation and decreased apoptosis, associated with enhanced PI3K-AKT-mTOR-4E-BP1 signaling and upregulated oncogenic p30C/EBPα. More importantly, newly obtained data indicate that Gadd45a transcript levels in peripheral blood of human blast crisis (BC-CML) samples was found to be reduced compared to accelerated phase (AP-CML), chronic phase (CP-CML) and normal controls, assessed by Quantitative real time PCR analysis. Collectivly these data strongly suggest that Gadd45a expression is a novel prognostic indicator of CML progression, implicating Gadd45a as a downregulated target of BCR-ABL associated with progression to more aggressive stages. To conclude, our findings provide novel evidence that Gadd45a functions as a suppressor of BCR/ABL driven myeloid leukemogenesis, & that suppresion of Gadd45a is associated with CML progression. These data provide the impetus to further elucidate the role Gadd45a plays in suppressing the development of CML, and explore how its loss contributes to the progression of CML to more aggressive leukemic phenotypes.

Disclosures:

No relevant conflicts of interest to declare.
Protein-Coding and Non-Coding RNA Expression Profiles Of Mesenchymal Stem Cells In Patients With Myelodysplastic Syndromes
Authors: Zhao Youshan , Chang Chunkang , Li Xiao
Published: October 2019

Background

A few reports have suggested chromosomal and gene expression abnormalities in mesenchymal stem cells in patients with MDS. Recently years, there is growing evidence for a role of the non-coding RNA in the transcriptional control of gene expression. Characterization of protein-coding and non-coding RNA expression in mesenchymal stem cells of MDS patients could be strategic for understanding gene expression regulation and role of marrow microenvironment in the pathophysiology of MDS.

Methods

In this study, gene expression profiles of MSC in 10 patients with MDS were compared with 5 healthy individuals using human transcriptome array. This array allows comprehensive examination of gene expression and noncoding transcripts, as well as detection of coding SNPs and genome-wide identification of alternative splicing. Real-time RT-PCR was performed to confirm the expression levels of selected transcripts.

Results

In MSC of MDS patients, 2628 genes were significantly differentially expressed (p≤0.01) in comparison to healthy individuals, of which 945(36%) were protein-coding transcripts. Gene ontology and pathway analysis were performed on those protein-coding genes. The significant significant ontology themes for up-regulated gene in MDS include cell division, mitosis, DNA repair and regulation of transcription, and for down-regulated genes include cytokine-mediated signaling pathway, inflammatory response and positive regulation of mitosis. Pathway analysis identified the deregulated gene pathway were main associated with purine metabolism, cytokine-cytokine receptor interaction, toll-like receptor signaling pathway, and so on. In non-coding RNA, 79 were long noncoding RNA (LncRNA), which were reported in database. We used gene coexpression networks to cluster thousands of transcripts into phenotypically relevant coexpression modules. The coexpression patterns of lncRNAs and protein-coding RNAs in MDS and controls were different. In the MDS coexpression network, differentially expressed lncRNAs were mainly focused on gene function related to ribosome biogenesis, tRNA processing, and so on.

Conclusions

These results demonstrated the differential RNA expression profile between MDS and healthy individuals, suggesting that lncRNAs may play an important role in dysfunctional microenvironment in MDS.

Disclosures:

No relevant conflicts of interest to declare.
Cytogenetic Response Based On Revised IPSS Cytogenetic Risk Stratification and Minimal Residual Disease Monitoringby FISH In MDS Patients Treated By Low-Dose Decitabine
Authors: Li Xiao , Chang Chunkang , He Qi , Xu Feng , Zhang Qingxia , Wu Lingyun , Su Jiying , Zhang Xi , Zhou Liyu , Wu Dong , Zhang Zheng
Published: October 2019

Objective

To observe and analyze the response characteristics based on revised IPSS (IPSS-R) cytogenetic risk stratification in patients with myelodysplastic syndromes (MDS) who received low-dose decitabine treatment.

Patients and methods

Eighty-seven MDS patients received decitabine treatment (15-20mg/M2/d×5/per course) with a median of 4 courses (range, 1-10). The clinical and cytogenetic response after treatment were observed to evaluate whether the IPSS-R cytogenetic risk stratification could predict treatment response. For those patients with abnormal karyotypes who achieved clinical complete response (CR), percentage of clonal cells in bone marrow (BM) was calculated by G-banding together with fluorescence in situ hybridization (FISH) to determine the minimal residual disease (MRD). In addition, the effect of cytogenetic response on overall survival (OS) was also analyzed.

Results

Sixty of 87 patients (69.0%) achieved treatment response including 25 cases (28.7%) with CR. For the 44 patients with abnormal chromosome, 27 cases (61.3%) achieved cytogenetic response including 18 with complete cytogenetic response (cCR). The patients carrying poor or very poor karyotypes presented better clinical and cytogenetic response than those with intermediate or good karyotypes. Among the patients with poor or very poor karyotypes, the ones carrying chromosome 7 aberrance showed better response than the others. Among 18 patients with abnormal karyotypes who achieved clinical CR, 4 cases presented cytogenetic PR or NR through G-banding analysis. FISH analysis revealed an over 5 % of clonal cells in BM in two patients who presented G-banding defined cytogenetic CR. In addition, longer median OS (24 months) were observed for patients who achieved cytogenetic response than those who did not (12 months) (P=0.007)

Conclusion

IPSS-R cytogenetic risk stratification could be used to predict the clinical and cytogenetic response to decitabine treatment in MDS patients. The predicting effect may be related to the chromosome 7 involvement rather than the number of abnormal karyotypes. FISH together with G-banding analysis should be available in determining MRD in MDS patients after treatment.

Disclosures:

No relevant conflicts of interest to declare.