bluuebunny/crossref_metadata_2025 huggingface dataset:
High Prevalence Of Aspirin-Resistance In Myeloproliferative Neoplasms
Authors: Demory Jean-Loup , Bruge Judith , Cambier Nathalie , Rose Christian , Charpentier Agnès
Published: October 2019

Aspirin is recommended and widely prescribed to MPN patients without any evaluation of its efficacy. We report on 46 patients followed in our institution for a Myeloproliferative Neoplasm and treated by low-dose aspirin (75 to 160 mg p. day) alone or aside a cytoreductive therapy, mainly hydroxy-urea ; on the occasion of a routine visit we measured the response of their platelets to arachidonic acid in order to assess their sensibility to aspirin. Most patients (25) had ET, 15 had PV, 4 had CML and 2 Myelofibrosis. Response to arachidonic acid was measured on a citrated whole blood sample using an impedance-based semi-automatic aggregometer (Multiplate®) ; TRAP (Thrombin Receptor Activating Peptide) aggregation was performed systematically as a positive control. In 20 patients out of 46 (43,5%) there was persistence of a significant aggregability to arachidonic acid defining the resistance to aspirin according to our reference values : 10/25 ET (38%), 9/15 PV (62%)and 1/4 LMC. These proportions are much higher than those we note in heart or vascular diseases as well as those reported in published series. The observance of aspirin treatment could be considered doubtful only in a minority of patients (5/46) ; as the resistance appeared more frequent in PV we could assume a biais linked to the utilization of whole blood, but most PV patients were well controlled and did not have an elevated hematocrit. In untreated or refractory ET patients, the persistence of an increased platelet count suggests that aspirin dosage was not adequate although there was no statistical significance between daily dose and resistance. We observed an ischemic complication (transient stroke) in a single patient whose platelet count was not controlled despite hydroxy-urea but remained sensible to aspirin.

We conclude that aspirin resistance is frequent in MPN treated with low-dose aspirin and that a monitoring is worth to increase the dose or change for another inhibitor.

Disclosures:

No relevant conflicts of interest to declare.
Clinical Spectrum and Molecular Pathophysiology of Shwachman-Diamond Syndrome
Authors: Shimamura Akiko
Published: October 2019

Shwachman-Diamond Syndrome (SDS) is an inherited marrow failure syndrome associated with exocrine pancreatic dysfunction and leukemia predisposition. SDS patients may also manifest additional non-hematologic abnormalities. Autosomal recessive mutations in the SBDS gene are found in over 90 percent of patients fitting the classical clinical phenotype of SDS. The advent of genetic testing has revealed an unexpectedly broad range of SDS phenotypes. Through the Shwachman-Diamond Syndrome Registry, we found that diagnosis may be obscured by cryptic or non-classical presentations of SDS. The timely diagnosis of SDS carries profound ramifications for medical management and treatment. We are developing assays utilizing massively parallel next generation sequencing to address this challenging diagnostic problem. Clinical applications of next generation sequencing to the diagnostic algorithm for marrow failure or myelodysplastic syndrome and implications for medical treatment will be explored.

Disclosures:

No relevant conflicts of interest to declare.
Day 30 Platelet Count As a Prognostic Marker of Survival in Acute Myeloid Leukemia
Authors: Mangaonkar Abhishek , Mohsin Jamal , Mansour Joshua , Mondal Ashis , Kolhe Ravindra B. , Jillella Anand , Kota Vamsi , Puttagunta Haarika
Published: October 2019

Introduction:

Acute myeloid leukemia (AML) affects about 3.5 per 100,000 people per year in the western world. High dose chemotherapy with anthracycline and cytarabine combination has resulted in complete remission rates of around 60% even in elderly patients. Complete remission (CR) is commonly defined as bone marrow blast count of less than 5% with recovery of all three cell lines in the peripheral blood. CR with incomplete count recovery is where there is no evidence of persistent disease but there is an incomplete recovery of blood counts, is also seen frequently and it has been shown to have poor prognosis as compared to patients with normal count recovery. Above normal platelet count is seen in small percentage of patients and the significance of this has not been well described.. We hypothesized that patients with a high platelet count at recovery from induction therapy have an increased survival as compared to those with normal or below normal platelet counts.

Methods:

We did a retrospective chart review of 123 consecutive patients with a diagnosis AML at Georgia Regents University Cancer Center that were admitted for induction treatment between 2005 and 2012. Highest platelet count between days 25 to 35 from the date of initiation of induction chemotherapy was taken for this analysis. Patients were divided into three groups: platelet count <50,000 (n=21); 50,000-400,000 (n=80) & >400,000 (n=22). Stratified survival with Kaplan-Meir curves were calculated for each of the groups.

Results:

Mean follow-up period was 94.122 months and median survival was 33.917 months. Mean survival (in months) for the first group was 23.157; 57.801 for the second group & 187.388 for the third group. Difference between survival curves was statistically significant (p=0.003).

Conclusion:

Patients with complete remission appear to have significant survival advantage as compared to those with incomplete platelet recovery. Review of patients across various large clinical trials from US showed that the patients with CR have 3-5 year survival advantage as compared to those with incomplete platelet recovery. Platelet count at the end of one month after initiation of induction chemotherapy can also be used to predict long-term prognosis. This observation would serve as a valuable addition to the already established prognostic markers such as cytogenetics and molecular studies. Our results need to be validated in a larger set of patients.

Disclosures

Kolhe: Georgia Regents University: LLS grant Other, Research Funding. Jillella:Emory University: Research Funding. Kota:Georgia Regents University: LLS grant Other, Research Funding; Emory University: Advisory board, Ponatinib, Advisory board, Ponatinib Other; Teva: Speakers Bureau.
G17V Rhoa Mutation in Circulating DNA Is a Useful Marker for Diagnosis of AITL and AITL-Related Lymphoma
Authors: Nakamoto-Matsubara Rie , Sakata-Yanagimoto Mamiko , Nguyen Tran , Furuta Junichi , Ito Takayoshi , Komeno Takuya , Ogawa Seishi , Chiba Shigeru
Published: October 2019

[Background] Angioimmunoblastic T-cell lymphoma (AITL) is a subtype of peripheral T-cell lymphoma. We and others reported that the G17V RHOA mutation is frequent and highly specific to AITL and AITL-like peripheral T-cell lymphoma, not otherwise specified (PTCL-NOS). Thus, detection of this mutation serves as a novel diagnostic strategy for AITL and AITL-like PTCL-NOS.

[Aim] Analysis of disease-specific mutations in serum DNA from AITL and PTCL-NOS patients.

[Method] Genomic DNA was collected from tumors and sera from 16 AITL and PTCL-NOS patients and 11 B-cell malignancy patients. Seven out of 16 T-cell lymphoma and all B-cell malignancy patients did not receive prior treatment. Amplicon-based sequencing by Ion Torrent PGM was performed to analyze the G17V RHOA mutation as well as TET2 and DNMT3A mutations in these samples.

[Result] Three out of 7 tumor samples of T-cell lymphoma patients whose serum was preserved prior to treatment were positive for the G17V RHOA mutation. In the serum of these 3 patients, allele frequencies of the G17V RHOA mutation were 0.006 - 0.072 (mean 0.015, SDV 0.026), while 0 (mean 0, SDV 0) in the serum of the remaining 4 T-cell lymphoma patients and all the 11 B-cell malignancy patients whose tumor samples did not show RHOA mutation. Because we set cut off value as 0.002 (0.2%) based on the ROC curve, RHOA mutation was segregated into positive and negative faithfully according to whether the mutation was identified in the tumor samples. Then, we analyzed TET2 and DNMT3A mutations in sera of 5 patients whose tumor samples were positive for TET2 (n=2; a single mutation in each sample) and DNMT3A (n=3; 5 mutations all together), respectively. TET2 and DNMT3A mutations were detected in all the 5 sera, although 2 out of the 5 DNMT3A mutations were undetectable. In the clinical courses, RHOA mutations became undetectable after the treatment in sera of all the 3 patients whose pre-treatment sera were positive, while TET2 mutations were detected even after the treatment in sera of both patients whose pre-treatment sera were positive.

[Conclusion] Biopsy is sometimes difficult in lymphoma patients with progressed diseases. Identification of the disease-specific G17V RHOA mutation in serum may provide a non-invasive method to diagnose AITL and AITL-like PTCL-NOS, although inapplicable to detect minimum residual disease. Stable detection of TET2 mutations regardless of chemotherapy might indicate the presence of the mutations in the 'pre-cancerous cells' which are resistant to the chemotherapy.

Disclosures

No relevant conflicts of interest to declare.
The Prognostic Value of Molecular Bone Marrow Involvement in Aggressive Lymphoma
Authors: Goldschmidt Neta , Darawshy Fares , Slyusarevsky Elena , Pogrebijski Galina , Ben Yehuda Dina , Gatt Moshe E.
Published: October 2019

Introduction: Morphological bone marrow (BM) involvement in diffuse Large B-cell lymphoma (DLBCL) and primary T-cell lymphoma (PTCL) has prognostic and therapeutic implications. The prognostic role of molecular BM involvement (B and T cell gene rearrangement (GR) studies), in patients with no evidence for involvement is uncertain.

Objective: To evaluate the prognostic role of molecular BM involvement in patients with DLBCL and PTCL, and its significance in the rituximab era.

Methods: This is a retrospective study of 416 patients with DLBCL (358) or PTCL (58) treated 1995-2010. All patients underwent BM biopsy for morphology and GR studies on BM aspirates at diagnosis and/or at end of treatment (EOT). Most patients were treated with CHOP or R-CHOP based protocols after diagnosis. Medical records were reviewed for details of initial disease characteristics, type and response to treatment, relapse if occurred and the date of death or last follow-up. Patients were classified into one of three groups according to BM results: morphology positive (MorphPos); morphology negative/GR positive (MorphNeg/GRpos); morphology negative/GR negative (MorphNeg/GRneg).

Main outcome measures: Overall survival (OS) and progression free survival (PFS) among the groups of patients at diagnosis and at EOT.

Results: At diagnosis there were 89 MorphPos, 49 MorphNeg/GRpos and 278 MorphNeg/GRneg patients. GRpos patients had lower complete remission rate (73.9%) than GRneg patients (84.8%; p=0.087). The median OS differed significantly among groups: MorphPos patients 66.4 months (95%CI: 50.7-82); MorphNeg/GRpos patients 89 months (95%CI: 66-111); and MorphNeg/GRneg patients 125 months (95%CI: 113-136), p<0.001. MorphNeg status improved PFS (p<0.001), however GRneg status did not (p=0.09). The hazard ratio for GRpos at diagnosis in MorphNeg patients was 1.52 (95% CI: 0.97-2.4; p=0.068). Among the subgroup of DLBCL patients, the median OS was significantly different: MorphPos patients 75.6 months (95%CI: 57.7-94.1); MorphNeg/GRpos patients 95.5 months (95%CI: 66.9-124.1); and MorphNeg/GRneg patients 129.6 months (95%CI: 118.2-141.1), p=0.036. 73% of DLBCL patients received rituximab. Rituximab improved OS (p<0.001) in all DLBCL patients, yet did not overcome the GR-positivity negative effect on OS. For PTCL patients, BM morphology and/or GR status did not affect survival. 72 patients were tested at the EOT, 5 were MorphPos and 17 GRpos. EOT MorphPos patients had marginally worse OS (p=0.07). EOT GR status in MorphNeg patients did not affect survival (p=0.5)

Conclusions: BM GR positivity at diagnosis, independent of BM morphological involvement, predicts worse OS in DLBCL even in the rituximab era, but not in PTCL. EOT morphologic but not molecular involvement, had marginally negative effect on survival.

Disclosures

No relevant conflicts of interest to declare.
Cooperation Between the CD20 Receptor and hIFN-Alpha Receptor in Overriding the Blocked CD20 Cell-Signaling in Rituximab-Resistant B-NHL By the Fusion Protein Anti-CD20-hIFN-Alpha
Authors: Vega Gabriel G , Franco-Cea Luz A , Huerta-Yepez Sara , Mayani Hector , Martinez-Maza Otoniel , Bonavida Benjamin , Vega Mario I.
Published: October 2019

Introduction: The standard treatment of B-NHL consists of rituximab in combination with CHOP (RCHOP) and results in a significant clinical response. Rituximab inhibits cell-proliferation and inhibits cell survival/anti-apoptic signaling pathways. A subset of patients does not initially respond and a subset of responding patients develops resistance to RCHOP. The genetic engineering of a fusion protein, α-CD20-hIFN-α, was found to be active in the rituximab-resistant B-NHL cell lines.

Objective: To investigate the underlying mechanism by which α-CD20-hIFN-α signals in the resistant lines.

Hypothesis: We hypothesized that the treatment with the α-CD20-hIFN-α may result in the cooperation of both α-CD20 and hIFN-α and their interactions with corresponding receptors that will result in overriding α-CD20 blocked cell signaling.

Methods: Rituximab-resistant cell lines, R-2F7 and R-Ramos, were used as models. Cell signaling was determined by western. Sensitivity to drug-induced apoptosis was done by activation of caspase 3 by flow cytometry.

Results: Treatment of the R lines with α-CD20-hIFN-α resulted in the inhibition of cell growth and sensitization to doxorubicin-induced apoptosis. Treatment with single agents alone or combination was not effective. Treatment with the α-CD20-hIFN-α resulted in the inhibition of the NFκB and the p38 MAPK pathways. In addition, the hIFN-mediated signaling pathway, namely, PKC-d, was also inhibited by the α-CD20-hIFN-α.The role of PKC-d in drug sensitization was corroborated by the use of the specific inhibitor, Rotterin, which reversed the drug sensitization by α-CD20-hIFN-α and doxorubicin

Conclusion: The ability of the α-CD20-hIFN-α to inhibit cell survival and anti-apoptotic pathways, that was not achieved with single agents or combination, suggested that there may be a crosslinking of the CD20 and hIFN-α receptors by α-CD20-hIFN-α and results in triggering the cells via both receptors and inhibiting intracellular survival pathways and sensitization to drug apoptosis.

Clinical Implication: The findings also suggest the potential therapeutic application of the combination of α-CD20-hIFN-α and drugs for the treatment of patients resistant to RCHOP.

Disclosures

No relevant conflicts of interest to declare.
Management and Outcomes of Plasmablastic Lymphoma: A Single-Center Experience
Authors: Gupta Arjun , Naina Harris V.
Published: October 2019

Background: Plasmablastic lymphoma (PBL) is a rare aggressive B-cell neoplasm characteristically occurring in patients with HIV infection. Given their rarity, most of the existing literature is from case reports or heterogenous pooled data. Optimal therapy is not clearly defined and outcomes are traditionally poor with median survival in the order of months. We describe the epidemiology, management and outcomes of this rare malignancy at a single center.

Methods: We identified patients with PBL diagnosed between 2000-2015 at Parkland Memorial Hospital. Patients were followed till June 2015. Structured forms were used to interview patients and to abstract relevant (demographics, diagnostics, pathology, therapy and outcomes) clinical data. Logistic regression was used to assess variables for association with the odds of treatment failure. Log-rank test was used to assess differences in survival by treatment regimen. Statistical analysis was performed using SAS software version 9.0 (SAS Institute).

Results: Twenty-nine patients with PBL were diagnosed in the study period. Median age at presentation was 42 years; 24 (83%) were male. Twenty-sex (90%) occurred in patients with HIV infection; median CD4+ count at diagnosis was 93/mm3; median viral load was 97,823 copies/mL. Only 5 patients (17%) were actively taking antiretroviral therapy at the time of diagnosis. Three patients were immunocompetent. The most common primary site of disease was the gastrointestinal tract (13 patients) followed by oral/maxillofacial sites (10 patients). Twenty patients (69%) presented with adanced stage disease (stage III or IV disease). EBV was detected in all 26 cases where it was tested; HHV-8 was negative in all patients. CD20 positivity was seen in only 4 patients (14%). MYC rearrangements were described in 70% patients. EPOCH (etoposide, prednisolone, vincristine, cyclophosphamide and hydroxydaunorubicin), was the most common treatment regimen used in 16 patients (56%). Radiation therapy was administered in 28% of patients. Overall survival across the cohort was 9.9 months. Twenty patients (69%) died during the follow-up period; their median survival was 2.6 months. Nine patients were alive after a median follow-up of 47.8 months. Those who were treated with EPOCH demonstrated a better median overall survival compared to those who were not (26.3 months vs 1.2 months, p value 0.01).

Conclusions: PBL is a rare AIDS-related lymphoma most commonly affecting the oral cavity and gastrointestinal tract. It mostly occurs in HIV patients not on HAART. Patients usually present with advanced stage disease. OS continues to be poor but EPOCH regimens can improve survival compared to non-EPOCH regimens, and should be further explored in this setting.

Disclosures

No relevant conflicts of interest to declare.
Fishing with a Transgenic Line: Using Zebrafish to Elucidate Mechanisms and Therapeutics in NUP98-NSD1 AML
Authors: Filiaggi Corey , Deveau Adam P , Prykhozhij Sergey , Dellaire Graham , Berman Jason N.
Published: October 2019

The NUP98-NSD1 (NND1) translocation is a fusion oncogene recently identified in pediatric acute myeloid leukemia (AML), where it occurs in approximately 16% of patients. NND1 predicts a dismal prognosis, with a 4-year event-free survival <10%. The mechanism of action of NND1 may be through the activation of the posterior homeobox gene, HOXA9. NND1 patients often harbour an internal tandem duplication of fms-like tyrosine kinase 3 (FLT3-ITD), another genetic lesion associated with poor prognosis. Co-expression of NND1 and FLT3-ITD results in worse survival than either aberration in isolation. NND1 may be sufficient to produce a myeloproliferative phenotype, but the interaction with FLT3-ITD activates essential downstream signaling pathways necessary for AML pathogenesis. A better understanding of the mechanisms by which NND1 dysregulates hematopoiesis and interacts with FLT3-ITD is fundamental to developing targeted therapies to improve the outcome in this disease.

The zebrafish has been established as a robust and reliable model of hematologic malignancies, with conserved genetics and ease of genetic interrogation. Our group previously generated a transgenic zebrafish model expressing the related fusion oncogene, NUP98-HOXA9, in which embryos had anemia and expansion of myeloid cells, and adult fish exhibited a myeloproliferative neoplasm (MPN). Using this model, we discovered novel downstream epigenetic regulators that could be targeted therapeutically and restore normal embryonic hematopoiesis. Moreover, the up-regulated genes that we identified correlated with features of high-risk AML in human datasets, highlighting the translational relevance of this human disease model and justifying the employment of this approach to investigate NND1-driven AML (Deveau et al, Leukemia 2015).

Plasmid constructs have been generated that incorporate human NND1 into the zebrafish using the Tol2 system, with detection by green fluorescent protein (GFP) expression. Injection of CMV-NND1-sGFP revealed strong GFP expression from 24-48 hours post fertilization (hpf) ubiquitously and in hematopoietic cells. Whole-mount in situ hybridization experiments of plasmid-injected embryos have shown that, similar to the NUP98-HOXA9 model, embryos expressing NND1 develop a pre-leukemic state, with a decrease in red blood cell marker expression (gata1) and an increase in myeloid marker expression (l-plastin). Currently no animal models exist for NND1 AML. Our initial studies have revealed a myeloproliferative phenotype in zebrafish embryos, providing an in vivo tool for further genetic and epigenetic interrogation, as well as a preclinical platform for novel drug discovery in this disease.

Disclosures

No relevant conflicts of interest to declare.
SLP76 Is Ectopically Expressed in Chronic Lymphocytic Leukemia Cells and Involves in B-Cell Receptor Signaling
Authors: Herishanu Yair , Dorozella Nili , Shapiro Mika , Perry Chava , Katz Ben-Zion
Published: October 2019

In the last decade, the B-cell receptor (BCR) has emerged as a pivotal stimulus in CLL pathogenesis. The BCR responsiveness in CLL cells is heterogeneous among patients and correlates with disease aggressiveness. Here we show for the first time, that SLP76 a key scaffold protein in T-cell receptor (TCR) signaling, is ectopically expressed in CD19+ purified CLL cells (purity >95%) in the majority of patients, and is co-expressed with other components of the TCR pathway, including LCK and ZAP70. SLP76 mRNA levels correlated with its protein expression and SLP76 protein levels were higher in unmutated IGHV and ZAP70+ CLL cells. SLP76 was found to be functionally active in CLL cells, as it becomes phosphorylated in response to BCR engagement in a time dependent manner. Activation with anti-IgM antibody results in phosphorylation of SLP76 on the positive regulatory tyrosine residue Y128 residue with increased physical association with Btk, peaking after 15 minutes. The negative regulatory residue S376 became phosphorylated only after 45', concomitantly with downregulation of the tyrosine phosphorylation. SLP76 phosphorylation in response to BCR engagement did not correlate with total ZAP70 expression. Pre-incubation of CLL cells with the LCK kinase inhibitor LCKi and with the SYK inhibitor R406, inhibited SLP76 phosphorylation in response to BCR activation, while the BTK inhibitor-ibrutinib had no effect. These suggest that LCK and SYK, but not ZAP70, play a central role in the upstream signaling involved in SLP76 activation in CLL cells. Knockdown of SLP76 in CLL cells resulted in decreased induction of BTK and PLCγ2 phosphorylation after BCR activation with anti-IgM. Consistent with our findings that SLP76 in involved in BCR signaling in CLL cells, we found that high total SLP76 expression was associated with a shorter time to first progression or need for treatment. In conclusion, SLP76 is ectopically expressed in CLL cells and plays a role in BCR signaling in those cells.

Disclosures

No relevant conflicts of interest to declare.
Netrin-1 and Its Receptors Unc5b and DCC May be Useful Targets for Preventing Multiple Myeloma Bone Lesions
Authors: Mediero Aránzazu , Mazumder Amitabha , Cronstein Bruce
Published: October 2019

Purpose: Multiple Myeloma is a hematologic malignancy that involves the malignant proliferation of plasma cells. This type of malignancy is particularly trophic to bone where it induces osteoclastic destruction of bone. We have recently demonstrated that osteoclast differentiation is dependent upon the autocrine/paracrine expression of the laminin-like matrix protein and chemorepulsant netrin1 and binding of netrin1 to the receptor unc5b. We therefore asked whether targeting netrin1 and its receptors unc5b and DCC could regulate myeloma spread in a murine model of myeloma.

Methods: 106 5TGM1-GFP myeloma cells (GFP expressing) were inoculated through the tail vein in 9 week old female C57Bl/KaLwRijHsd mice. Two weeks after inoculation animals were injected with monoclonal antibodies against Netrin-1, Unc5b and DCC weekly for 4 weeks (n=each). IVIS was performed for in vivo localization of myeloma bone lesions at the end of the experiment. Bone mineral density was measured by DEXA scanner and vertebrae and long bones were collected and prepare for microCT and histology analysis.

Results: IVIS imaging revealed a marked decrease in bone lesions in the anti-Netrin-1 and anti-Unc5b treated groups (n=6 and 9 mice respectively) when compared to control mice (n=6), whereas treatment results with anti-DCC antibody were more heterogeneous (n=7). There was an increase in total bone mineral density in anti-Unc5b and anti-DCC treated mice (0.63±0.09 g/cm3 and 0.8±0.09 g/cm3 respectively vs. 0.42±0.02 g/cm3, p<0.5). microCT analysis revealed no changes in cortical bone parameters (Bone volume/total volume (BV/TV), bone volume (BV), total volume (TV) and Bone Mineral Density (BMD)) for any treatment group, but there was increased in these parameters in Netrin-1 and Unc5b antibody treated groups when trabecular bone was analyzed, consistent with the decrease in myeloma lesions. There was also an increase in Trabecular thickness (Tb.Th), trabecular number (Tb.N.) and a decrease in trabecular separation (Tb.Sp.). TRAP staining revealed decreased osteoclasts in both anti-Netrin-1 and -Unc5b treated mice (7±1 and 6±1 cells/hpf respectively vs. 15±1 cells/hpf for control, p<0.001, n=5) but not for anti-DCC treated mice (13±1 cell/hpf vs. 15±1 cells/hpf for control, p=ns, n=5), and immunofluorescence analysis reveal a decrease in Cathepsin K positive that correlated with the decrease in TRAP-positive osteoclasts.

Conclusions: Netrin-1 and Unc5b treatment decreases osteoclast formation in a murine model of myeloma and decreases myeloma bone lesions. Targeting Netrin-1 or its receptor Unc5b may be a novel therapeutic approach for multiple myeloma.

Disclosures

No relevant conflicts of interest to declare.
Thrombin Generation in Children after Acute Venous Thromboembolism
Authors: Zia Ayesha , Journeycake Janna M. , Sarode Ravi
Published: October 2019

Introduction: Venous thromboembolism (VTE) is a major and growing problem in children. The annual rate of VTE has increased by 70% in the U.S hospitalized children in the past decade. Accompanying this increase in VTE is the attendant increase in chronic, post-thrombotic sequeale of VTE. Strategies to identify children at high risk of developing poor outcomes after initial management of VTE are unclear. We hypothesized that 'global coagulation assessment' may identify children likely to suffer from poor thrombosis outcomes following an initial event. In this pilot study, we therefore, evaluated thrombin generation (TG) after an acute episode of provoked VTE in children.

Methods: Baseline patient, thrombus characteristics and thrombophilia testing were prospectively collected for 50 consecutive children with acute, provoked VTE after informed consent. Subjects with cancer, protein C, protein S and antithrombin deficiency, persistent antiphospholipid positivity and inflammatory disorders were excluded. Thrombin generation, using corn trypsin inhibited treated plasma by calibrated automated thrombogram was measured in the presence and absence of thrombomodulin (TM), 4 weeks after completion of anticoagulation therapy, and compared to 50 healthy controls. All cases were treated according to the 9th edition of American College of Chest Physicians Clinical Practice Guidelines.

Results: The mean age of VTE cases was 14 years (range: 6 weeks to 18 years). Two thirds (33/50) of VTE cases consisted of deep venous thrombosis of the limbs and/or pulmonary embolism, and nearly 20% (10/50) were cerebral sinovenous thrombosis. Thrombin generation of subjects showed significantly higher endogenous thrombin potential (ETP) compared to healthy controls (1150 ± 281 vs. 763 ± 274 nM.min; p <0.001). There were no significant differences in the other TG parameters (lag time and thrombin peak) between VTE cases and healthy controls. Inhibition by TM was attenuated in patients compared to controls (22% vs. 45.5% p<0.001). A significant negative correlation was found between those with elevated FVIII levels (>150%) at end of therapy and TM induced reduction of ETP (r=0.297 vs. 0.171; p<0.01).

In conclusion, a novel finding in children with provoked VTE was persistent elevation of TG after successful completion of anticoagulation therapy. A role for FVIII as an important cause of hypercoagulability after anticoagulation therapy is reflected by the reduced inhibitory effect of TM at high FVIII levels. Further studies are ongoing to determine the utility of TG as a biomarker for predicting poor outcomes after VTE in children.

Disclosures

Journeycake: CSL, Baxalta, NovoNordisk: Consultancy; ATHN: Research Funding; Biogen: Speakers Bureau; ATHN: Membership on an entity's Board of Directors or advisory committees.
Development of Multi-Lineage Hematopoiesis in Two Novel Zebrafish runx1 mutants
Authors: Bresciani Erica , Carrington Blake , Kwon Erika Mijin , Jones Marypat , Wincovitch Stephen , Sood Raman B. , Liu Paul P.
Published: October 2019

Long term hematopoietic stem cells are essential for the life-long maintenance of the hematopoietic system of an organism. The transcription factor RUNX1 is required for the emergence of definitive hematopoietic stem cells (HSCs) from the hemogenic endothelium during the embryo development. Runx1 knockout mouse embryos lack all definitive blood lineages and cannot survive past embryonic day 13. However, we previously showed that zebrafish homozygous for an ENU-induced nonsense mutation in runx1 (runx1W84X/W84X) were able to recover from a larval "bloodless" phase and develop to fertile adults with multi-lineage hematopoiesis, suggesting the formation of runx1-independent adult HSCs. However, our finding was based on a single zebrafish model, which requires verification in additional, independent models.

In order to further investigate if a RUNX1-independent pathway exists for the formation of adult HSCs, we generated two new runx1 mutants, a deletion of 8 bp (runx1del8/del8) and a deletion of 25 bp (runx1del25/del25) within exon 4 of runx1, respectively, using the Transcription activator-like effector nucleases (TALENs) technology. These mutations cause frameshifts and premature terminations within the runt-homology domain,, resulting in loss of function of runx1 (runx1-/-). Both runx1del8/del8 and runx1del25/del25 mutant embryos had normal primitive hematopoiesis but failed to develop definitive hematopoiesis. Time-lapse recordings with confocal microscopy revealed that, indeed, there was no emergence of HSCs from the ventral wall of dorsal aorta in the runx1-/- embryos. The runx1-/- larvae gradually lost circulating primitive blood cells and became bloodless between 8 and 14 days post fertilization (dpf). However they gradually regained circulating blood cells between 15 and 20 dpf. Eventually, about 40% of runx1del8/del8 and runx1del25/del25 mutants developed to fertile adults with circulating blood cells of multi-lineages. Taken together, our data is consistent with the previously described runx1W84X/W84X phenotype and supports the possibility of a runx1-independent mechanism for HSC formation and definitive hematopoiesis.

Disclosures

No relevant conflicts of interest to declare.
Stem Cell-like Megakaryocyte Progenitors As Driving Forces of IFN-Induced Emergency Megakaryopooesis
Authors: Haas Simon , Hansson Jenny , Klimmeck Daniel , Loeffler Dirk , Velten Lars , Uckelmann Hannah , Wurzer Stephan , Prendergast Áine M. , Schnell Alexandra , Hexel Klaus , Santarella-Mellwig Rachel , Blaszkiewicz Sandra , Kuck Andrea , Geiger Hartmut , Milsom Michael D. , Steinmetz Lars M , Schroeder Timm , Trumpp Andreas , Krijgsveld Jeroen , Essers Marieke A. G.
Published: October 2019

Recent reports have highlighted the importance of platelets as inflammatory and immune cells in addition to their well-known function in thrombosis. Systemic inflammation associated with acute infection triggers the release of immune-modulatory agents and the interaction of platelets with neutrophils to facilitate the formation of neutrophil extracellular traps (NETs). This provokes a rapid consumption of platelets resulting in a transient thrombocytopenia. During acute inflammation, low platelet levels are associated with a loss of vascular integrity and hemorrhage, as well as septic shock resulting in increased mortality. Therefore, a fast recovery of platelet levels is essential. However, the exact mechanism by which platelet levels are rapidly regenerated after acute inflammation remains unknown.

Here, we show that inflammatory signaling instructs a rapid Mk maturation program at distinct levels of megakaryopoiesis to regenerate the lost platelet pool. While megakaryocyte progenitors (MkPs) are efficiently driven into endomitosis leading to a strong increase in mature Mks, a small pool of potent stem-like megakaryocyte-committed progenitors (SL-MkPs) within the phenotypic HSC compartment replaces the lost MkPs. Type I Interferon (IFN) signaling triggers FoxO3a inactivation, resulting in cell cycle activation of these normally quiescent SL-MkPs. Moreover, IFN signaling mediates a rapid functional and cellular maturation program of SL-MkPs, associated with mTOR and STAT1 dependent increase in cell size, enhanced Mk protein production and appearance of alpha granular precursors.

Interestingly, while acute inflammatory signaling drives rapid maturation of Mk progenitors resulting in increased platelet production, repeated cycles of type-I IFN mediated inflammation triggers a constant push of maturation and thus leads to a partial exhaustion of Mk progenitors. These findings reveal that depending on the context and timing, the same IFN stimulus can be associated with opposing effects on megakaryopoiesis. This may explain why some inflammatory states are associated with thrombocytosis, while others provoke thrombocytopenia.

Disclosures

No relevant conflicts of interest to declare.
Targeting Bone Marrow Mesenchymal Stromal Cells Using Cre-Recombinase Transgenes
Authors: Zhang Jingzhu , Link Daniel C.
Published: October 2019

The bone marrow microenvironment contains hematopoietic niches that regulate the proliferation, differentiation, and trafficking of hematopoietic stem/progenitors cells (HSPCs). These hematopoietic niches are comprised of a heterogeneous population of stromal cells that include, endothelial cells, osteoblasts, CXCL12-abundant reticular (CAR) cells, mesenchymal stem cells (MSCs), arteriolar pericytes, and sympathetic nerves. Emerging data suggest that specific stromal populations may regulate distinct types of HPSCs. Thus, it is important to have validated approaches to interrogate and target specific stromal cell populations. Prior studies have shown that Prx1-Cre, Osx-Cre, Lepr-Cre, and Nes-Cre broadly target mesenchymal stromal cells in the bone marrow. Here, we rigorously define the stromal cell populations targeted by two Cre-transgenes that are commonly used to target osteolineage cells (Ocn-Cre, and Dmp1-Cre) and introduce a new Cre-transgene (Tagln-Cre) that efficiently targets bone marrow pericytes. For each Cre-transgene, we performed lineage mapping using ROSA26Ai9/Ai9 mice, in which cells that have undergone Cre-mediated recombination express tdTomato. In some cases, we further crossed these mice to introduce the Cxcl12gfp transgene, which can be used to define GFP-bright CAR cells. Immunostaining of bone sections and flow cytometry were used to define the target stromal cell population(s) in these mice.

Osteocalcin (Bglap, Ocn) is primarily expressed in mature osteoblasts. Accordingly, Ocn-Cre is widely used to specifically target osteoblasts. However, our lineage mapping studies show that Ocn-Cre targets not only all osteoblasts, but also 72 ± 4.0% of CAR cells. Ocn-Cre also targets a subset of NG2+ arteriolar pericytes. Dentin matrix acidic phosphoprotein 1 (Dmp1) is expressed primarily in osteocytes, and Dmp1-Cre has been widely used to specifically target osteocytes. However, we show that Dmp1-Cre also efficiently targets endosteal osteoblasts and approximately 40% of CAR cells. To target bone marrow pericytes, we tested several Cre-transgenes, ultimately focusing on Tagln-Cre. Transgelin (Tagln, SM22a) is broadly expressed in pericytes, smooth muscle cells, and cardiomyocytes. Lineage-mapping studies show that Tagln-Cre targets all arteriolar and venous sinusoidal pericytes in the bone marrow. It also targets osteoblasts and 75 ± 5.2% of CAR cells.

There are several recent studies that have ascribed specific functions to osteoblasts or osteocytes based on targeting using Ocn-Cre or Dmp1-Cre, respectively. In light of our data, these conclusions need to be re-evaluated. Ocn-Cre, Dmp1-Cre, and Tagln-Cre each target a subset of CAR cells. Studies are underway to determine whether these CAR subsets have unique expression profiles and functions. Finally, Talgn-Cre represents a new tool for investigators in the field to efficiently target bone marrow pericytes.

Disclosures

No relevant conflicts of interest to declare.
Evidence for a Common Immune Pathophysiology in Acquired Aplastic Anemia and Ulcerative Colitis
Authors: Nakagawa Noriharu , Maruyama Hiroyuki , Zaimoku Yoshitaka , Maruyama Kana , Saito Chizuru , Katagiri Takamasa , Hosokawa Kohei , Ishiyama Ken , Yamazaki Hirohito , Nakao Shinji
Published: October 2019

Background: Acquired aplastic anemia (AA) is occasionally preceded or complicated by the other autoimmune diseases such as rheumatoid arthritis and ulcerative colitis (UC), but the pathogenetic links between these diseases remain unknown. A number of case reports attributed the development of AA in UC patients to mesalazine that had been administered before the onset of AA. However, some UC patients who were referred to our clinical because of "mesalazine-induced AA" had increased percentages of glycosylphosphatidylinositol-anchored protein-deficient (PNH-type) cells that serve as a marker for immune-mediated bone marrow (BM) failure. This suggested an immune pathophysiology of UC-associated AA similar to that of idiopathic AA.

Methods: To address this issue, we analyzed clinical and laboratory data including HLA-DRB1 alleles and PNH-type cells of 14 AA patients (six males and eight females) associated with UC who were referred to our clinic from 2001 through 2015.

Results: The median age of the patients was 55 (range, 22-76). Diagnoses of the 14 patients were severe AA in eight, non-severe AA in five, and hypomegakaryocytic thrombocytopenia in one. UC preceded AA in 11 patients and nine of them received mesalazine at the time of AA diagnosis, while three AA patients developed UC 12, 72, and 36 months after diagnosis of AA. Increased percentages of PNH-type granulocytes (0.35% to 3.2%) were detected in 11 (79%) patients. Of 9 PNH(+) patients with AA preceded by UC, seven had received mesalazine while two had not been treated with mesalazine. The prevalence of increased PNH-type cells in AA patients who developed UC was 67% (2/3). Six patients were positive for DRB1*15:02, a well-known allele associated with susceptibility both to UC and AA in Japanese patients (Table). Another major DR15 allele, DRB1*15:01, was possessed by four patients. Seven patients received immunosuppressive therapy with either cyclosporine (CsA) alone (two patient) or CsA+antithymocyte globulin (five patients) and six of them achieved a remission of AA.

Conclusions: The high prevalence of increased PNH-type cells indicates that AA complicated by UC is a legitimate immune-mediated BM failure and that mesalazine may be irrelevant to the development of AA. The high frequency of DRB1*15:02 suggests the two diseases share an immune pathophysiology. Genome wide association studies on UC patients may help to identify genes associated with a susceptibility to AA.

Figure 1. Figure 1.

Disclosures

No relevant conflicts of interest to declare.
In Vivo Selection Unmasks a Dormant Pool of Repopulating Hematopoietic Clones
Authors: Adair Jennifer E , Schefter Lauren E , Humphrys Daniel R , Haworth Kevin G , Hocum Jonah D , Trobridge Grant D. , Kiem Hans-Peter
Published: October 2019

Long-term clonal tracking studies utilizing hematopoietic stem and progenitor cells (HSPCs) in nonhuman primates receiving myeloablative transplantation demonstrate a successive pattern of repopulation: short-term repopulating cells are succeeded by long-term clones. However, the duration of short-term repopulation and the numbers of clones contributing to either short or long-term repopulation are unclear. Here, we tracked >11,000 unique clones in 8 pigtail macaques for up to 9 years following myeloablative transplantation with autologous, lentivirus gene-modified CD34+ HSPCs. Seven of these animals received cells expressing the P140K mutant methylguanine methyltransferase transgene, which is resistant to the combination of O6-benzylguanine (O6BG) and bis-chloroethylnitrosourea (BCNU) chemotherapy, thus conferring a selective advantage to gene-modified cells in vivo. After transplantation and before in vivo selection with O6BG/BCNU, we observed a successive pattern of hematopoietic reconstitution, with short-term clones declining within 100 days after transplantation. Within the first year after transplant, the percent of persistent clones varied from animal-to-animal, ranging from 8% to 54% of clones detected at a >1% frequency, and remained stable in the absence of selective pressure. Importantly, when animals engrafted with P140K-expressing cells were administered O6BG/BCNU we observed novel clonal patterns, which directly correlated with transplanted cell dose and time of chemotherapy administration after transplant. In all animals, chemotherapy induced emergence of previously undetected clones. In animals receiving ≤12x106 CD34+ cells/kg at the time of transplant (n = 4), chemotherapy also induced a re-emergence of previously declined short-term repopulating clones or a stabilization (i.e. decreased fluctuation) of repopulating clones identified between 100 days and 1 year after transplant. However, in animals receiving robust cell doses, ≥35x106 CD34+ cells/kg (n = 2), chemotherapy more than 1 year after transplant induced a completely novel clonal repertoire. In one animal receiving 22x106 CD34+ cells/kg at transplant, chemotherapy administration beginning <1 year (253 days) after transplant induced clonal stability, which was maintained through two additional chemotherapy treatments. These data suggest that some short-term repopulating clones may have long-term repopulation ability, but revert to a dormant phase within the first year after transplant. Additionally, these data indicate that transplant of excess repopulating cells results in early dormancy of a large proportion of repopulating clones. Together, these findings suggest that previous estimates of HSPC frequency based on clone tracking are an underestimate of true graft repopulation potential.

Disclosures

No relevant conflicts of interest to declare.
Co-Opting the Genetic Redundancy of the Pax Family to Treat PAX5 Mutated Pre-B ALL
Authors: Horwitz Marshall S , Hart Matthew R
Published: October 2019

Pre-B cell ALL is the most common pediatric malignancy. More than a third of cases acquire loss-of-function mutations of PAX5, a regulatory transcription factor which is required for the differentiation of B lymphocytes. Additionally, germline loss of function PAX5 mutation is a cause of familial pre-B cell ALL. These observations indicate that loss of PAX5 activity drives leukemogenesis. We have seen that PAX5 re-expression in PAX5 -mutated pre-B cell ALL restores differentiation capacity and promotes apoptosis. However, because the gene is deleted or otherwise mutated it may not be possible to easily repair or restore PAX5 expression in leukemic cells. Nevertheless, PAX5 possesses two closely related paralogs, PAX2 and PAX8, which are structurally and functionally similar to PAX5, yet are neither expressed in lymphocytes nor mutated in ALL. Toward the goal of exploiting PAX family genomic redundancy for therapeutic purposes, we have tested the ability of PAX2 and PAX8 to replace PAX5 in pre-B ALL cell lines. Preliminary results indicate that both PAX2 and PAX8 rescue B cell differentiation and function similarly to PAX5 in this context. These results include modulation of B cell developmental markers, CD10, CD19 and CD22, as well as induction of PAX5 target genes, CD79a and BACH2. Cells also display a reduction in size which may be indicative of the large to small B cell transition and importantly show increased levels of cell death following PAX2 and PAX8 expression. We have further investigated the epigenetic status of PAX2 and PAX8 promoters in B cells and find that both loci are hypermethylated, suggesting that demethylation with agents such as methotrexate, may represent a therapeutic entry point for activating expression of PAX5 paralogs. Finally, based on observations that electrical polarization during early embryogenesis regulates developmental transcription factor expression, including for PAX genes, we have tested the ability of approved drugs targeting ion channels for their ability to induce PAX factor expression and thereby complement for loss of PAX5 in pre-B cell ALL. The anti-parasitic drug ivermectin, which activates chloride channels, is one of several compounds that appear promising in early studies.

Disclosures

No relevant conflicts of interest to declare.
Induction of NK and T Cell Immune Responses Against Leukemia Cells By Bispecific NKG2D-CD16 and -CD3 Fusion Proteins
Authors: Koerner Samuel , Kanz Lothar , Grosse-Hovest Ludger , Jung Gundram , Salih Helmut R
Published: October 2019

Monoclonal antibodies (mAbs) have by now become an established tool in therapy of many malignancies. The interaction of a mAb's Fc portion with Fcγ receptors (FcγR) on immune effector cells is important for its efficacy, but often insufficient to potently induce antitumor immunity e.g. due to FcγR polymorphisms. Moreover, Fc parts of mAbs may bind to FcγRs expressed on non-cytotoxic cells (e.g., platelets and B cells) and interact with FcγRs that do not trigger cytotoxicity (e.g. CD16b on granulocytes). These shortcomings can be overcome by novel antibody formats like bispecific antibodies allowing for improved activation of a specific pool of effector cells. Here we report on the development and preclinical characterization of two bispecific fusion proteins that target ligands of the immunoreceptor NKG2D (NKG2DL) which are widely expressed on malignant cells but generally absent on healthy tissue. Our fusion proteins consist of the extracellular domain of NKG2D as targeting moiety for tumor-expressed NKG2DL fused to Fab-fragments of either an agonistic CD3 or CD16 antibody. Specific binding of these NKG2D-CD3 and NKG2D-CD16 constructs was confirmed using NKG2DL-transfectants with regard to their target arm and either NK cells or T cells with regard to their different effector parts. Dose titration assays revealed an increased affinity of NKG2D-CD16 to the FcγR on NK cells as compared to our previously described (e.g., Steinbacher et al, 2014) Fc-optimized NKG2D-IgG1 fusion protein, which was mirrored by a potently increased ability to induce lysis of NKG2DL-tranfectants and NKG2DL-positive primary acute myeloid leukemia (AML) cells by allogeneic NK cells. The novel NKG2D-CD3 construct in turn was found to potently activate allogeneic and autologous CD4+ and CD8+ T cells. Next we comparatively analyzed the efficacy of T cells and NK cells to lyse autologous leukemia cells upon treatment with NKG2D-CD3 and NKG2D-CD16, respectively, by using PBMC of AML patients (blast counts 30-70%) directly ex vivo in long term cytotoxicity assays. NKG2D-CD16 potently induced AML cell lysis, and this was, in line with their proliferative and higher effector potential, by far exceeded upon stimulation of T cells with NKG2D-CD3. Taken together, we here introduce novel "antibody-like" bispecific constructs that take advantage of the highly tumor-restricted expression of NKG2DL and potently activate the reactivity of NK or T cells for immunotherapy of leukemia.

Disclosures

No relevant conflicts of interest to declare.