bluuebunny/crossref_metadata_2025 huggingface dataset:
The Cell Loss after Thawing in Cord Blood Units
Authors: Karasu Gulsun , Uygun Vedat , Kilic Suar Caki , Ozturk Zeynep , Dincer Zeynep , Yesilipek M. Akif
Published: October 2019

Umbilical cord blood (CB) are increasingly used for allogeneic transplantation as an alternative source to bone marrow or peripheral stem cell for patients who lack a human leucocyte antigen (HLA)-matched donor. Cell dose is one of the main determinant factor for selection of the unit and it has been shown to correlate with time and probability of engraftment. Cell doses available for selection of the unit are the ones evaluated before cryopreservation. Processing of the graft either with cryopreservation and/or thawing inevitably lead to cell loss but the data showing to what extent is very limited although this has great importance especially for the units with critical cell counts. To evaluate the extent of the cell loss after thawing and the effect of preservation duration on cell loss and the viability, we retrospectively analyzed our single center experience in a cohort of patients receivingunrelated CBT.

Sixty cord blood units used for unrelated CBT in fifty-eight patients in our Pediatric Stem Cell Transplantation Unit were included in the study. One of the patient was transplanted with double cord blood unit and one had recurrent CBT because of previous graft failure.

Median number of declared TNC per kilogram of recipient body weight was 16x107/kg (range: 3,16-59,1). After thawing the median TNC dose decreased to 11,1x107/kg (range:1,5-34), with median cell viability at 80%. The median TNC recovery was found as 70%. Median CD 34+ cell count declared by the banks per kilogram body weight of recipient was 6,0x105/kg (range: 0,5-37). The CD 34 cell count of one of the units used in a patient having double cord blood infusion was not available. The CD34 cell count of the other complementary unit was 0,48x105/kg. Since the total cell counts received bu the recipient was given, the CD34 cell count of the patient having double CB infusion was not included in statistical analysis. The median postthawed CD 34 cell count was 3,4 x105/kg (range: 0-21)and median CD34 cell recovery was %58.

The storage duration of the CB units in liquid nitrogen was available for 59 units and that was 52±30 months (range:3-130 months). Correlation analysis did not show any significant correlation between CB unit storage duration and viability (r=-0,2 p=0.07). Also any correlation between storage time and also TNC and CD34 cell recovery were not identified.

Our study indicated that approximately 30% of cell loss is expectable in cord blood transplantation because of either cryopreservation or thawing. It should be kept in mind while assessing acceptibility of cord blood units with limited cell counts

Disclosures

No relevant conflicts of interest to declare.
Recombinant scAAV2 Vector-Mediated Ex Vivo Transduction of Primary Human Hematopoietic Stem Cells from a β-Thalassemia Patient and Human β-Globin Gene Expression in a Murine Xenograft Model in Vivo
Authors: Song Liu-Jiang , Zhang Xin-Hua , Zhu Jun , Wu Jue-Lian , Yin Xiao-Ling , Tan Meng-Qun
Published: October 2019

Background: The most severe form of β-thalassemia, β°-thalassemia major, is characterized by the complete absence of normal β-globin chain, and is often lethal. Autologous transplantation of genetically-modified hematopoietic stem cells (HSCs) using lentiviral vectors have been used successfully to achieve clinical efficacy in one patient, although clonal expansion of a myeloid cell population also occurred in this patient which was associated with the activation of a cellular proto-oncogene, HMGA2. We reasoned that recombinant vectors based on a non-pathogenic human parvovirus, the adeno-associated virus (AAV), might offer a safer alternative. We have previously documented that although the conventional single-stranded (ss) AAV2 vectors mediated β-globin gene transfer and expression in primary human fetal liver cells and in human HSCs from patients with β-thalassemia patients in vitro, the level of transgene expression was sub-optimal. In the present study, we investigated whether double-stranded self-complementary (sc) AAV2 vectors could overcome this limitation.

Methods: Human HSCs, obtained from a β-thalassemia homozygous patient, were mock-transduced or transduced with recombinant scAAV2-β-globin vectors at 5×104 vgs/cell, followed by i.v. injection into sub-lethally irradiated NOD/SCID mice (2.65 cGy total body irradiation), which were also pre-treated with 200 µg purified anti-IL2RB/CD122 monoclonal antibody. Recipient mice were sacrificed 12 weeks post-transplantation. Bone marrow cells from recipient mice were analyzed by BFU-E assays. Human β-globin gene expression in human erythroid progenitor cells from transplanted mice was evaluated by RT-PCR.

Results: Pre-treatment of NOD/SCID mice with anti-CD122 antibody improves engraftment of human HSCs in bone marrow of receipt mice. Human β-actin (538-bp) and β-globin (272-bp) transcripts were detected by RT-PCR in bone marrow cells from all recipient mice, indicating that recombinant scAAV2-β-globin–transduced HSCs from a patient with β-thalassemia were successfully transduced and transplanted in these mice and that human β-globin gene was transcriptionally active 12 weeks post-transplantation.

Conclusion: Our results indicate that human HSCs from β-thalassemia patients can be efficiently transduced by recombinant scAAV2-β-globin vectors followed by expression of normal human β-globin gene. These studies provide the proof-of-concept that scAAV2 vector-mediated gene transfer into human HSCs might be a potentially safer alternative approach for gene therapy of β-thalassemia.

Disclosures

No relevant conflicts of interest to declare.
Risk of Clostridium Difficile Infection in Leukemia and Lymphoma Patients
Authors: Abdelfatah Mohamed , Mohamed Amr , Rabih Nayfe , Kathleen Enriquez , Ala Nijim , Richard Watkins
Published: October 2019

Background: Clostridium difficile is the most important cause of nosocomial infectious diarrhea. The prevelance of C. difficile infection (CDI) has been poorly investigated in patients with hematologic malignancies such as leukemia and lymphoma.

Purpose: To evaluate the clostridium difficile infection (CDI) incidence, severity, recurrence, developing sepsis and 30-days mortality among hospitalized patients with Leukemia and lymphoma.

Methods: IRB approved retrospective case control study. 2020 patients were admitted for >48 hours in our institution between July 2007 and December 2013 with CDI, and had adequate follow up. CDI was defined as more than 3 episodes of loose stools in less than 24 hrs with a positive CD stool toxin assay. Recurrent CDI (RCDI) defined as readmission for recurrent diarrhea with 2nd positive stool test 15 days following complete resolution of previous diarrheal episode and cessation of CDI treatment.

Computerized medical records to investigate demographics, laboratory blood work, Carlson Co-morbidity index, and medication used. Patients were divided into leukemia lymphoma group and control group. Group-1 included all patients with Leukemia lymphoma; AML, CML, ALL CML.

Univariate predictors of CDI and multiple logistic regressions were used to assess whether patients with leukemia lymphoma had higher incidence of severity, recurrence and mortality. Controlling for Age, Serum Albumin, modified Carlson Co-morbidity index (CCI), PPI, antibiotics, and CDI treatment.

Results: 2020 patients had been identified; in group-1 19 out of 95 patients (25%) had RCDI. In the control group, 364 out of 1655 patients (18%) had RCDI (p=0.614). 30-days mortality 7 out of 95 patients died within group-1 versus 157 out of 1924 in control group (p=0.799). Criteria for sepsis was fulfilled in 37 out of 95 in group-1 versus 482 out of 1924 in control group (p=0.003)

Conclusion: We conclude that hospitalized patient with Leukemia and lymphoma had higher incidence of developing sepsis from CDI. There was no difference in the incidence of recurrence and 30-days mortality between patients with Leukemia lymphoma than control group. Physicians should have high suspicious for CDI in Leukemia lymphoma patients. Depend on this study we suggest early diagnosis and starting second line stronger treatment may help in decrease the incidence of CDI related sepsis.

Disclosures

No relevant conflicts of interest to declare.
Parental, in Utero, and Early-Life exposure to Benzene and Risk of Childhood Leukemia: A Meta-Analysis
Authors: Smith Martyn T. , Carlos-Wallace Frolayne M , Zhang Luoping , Rader Gabriella , Steinmaus Craig
Published: October 2019

Benzene is an established cause of adult leukemia but its association with childhood leukemia remains unclear. We performed a systematic review of the epidemiologic literature on benzene and childhood leukemia, and used meta-analysis to explore causal inference, evaluate bias, and identify sources of heterogeneity. The summary relative risk estimate (RR) for childhood leukemia for all studies combined was 1.84 (95% confidence interval [CI]: 1.43, 2.37; n=23). In studies of acute myeloid leukemia (AML), the RR was 2.22 (95%CI: 1.65, 2.99; n=8). For studies of acute lymphocytic leukemia (ALL), the RR was lower (RR=1.62; 95%CI: 1.28, 2.04; n=16). Summary relative risks were higher for studies examining maternal (RR=1.96; 95%CI: 1.39, 2.78; n=13) versus paternal exposure (RR=1.23; 95%CI: 1.07, 1.41; n=14), and in studies assessing benzene exposure based on maternal occupation (RR=1.71; 95%CI: 0.91, 3.24; n=6) compared to paternal occupation (RR=1.18; 95%CI: 0.94, 1.50; n=6) or traffic density (RR=0.97; 95%CI: 0.75, 1.25; n=6). Summary relative risks were also higher for exposure during gestation (RR=1.91; 95%CI: 1.32, 2.75; n=12) compared to other periods, and remained elevated in studies adjusted for smoking or socioeconomic status. Overall, these findings suggest that benzene is associated with increased risk of childhood leukemia.

Disclosures

Smith: University of California: Consultancy.
Complement Activation May Trigger the Onset of Thrombotic Thrombocytopenic Purpura in Patients with Severe ADAMTS13 Deficiency
Authors: Hu Xiao-Hui , Bao Jialing , Ueda Yoshiyasu , Miwa Takashi , Song Wenchao , Zheng X. Long
Published: October 2019

Thrombotic thrombocytopenic purpura (TTP), a potential fatal syndrome, is often associated with severe deficiency of plasma ADAMTS13 activity, either resulting from ADAMTS13 mutations or acquired anti-ADAMTS13 autoantibodies that inhibit plasma ADAMTS13 activity. Patients with severe ADAMTS13 do not always have TTP signs and symptoms, which often occur following infections or inflammatory responses. The mechanism of TTP flare is not fully understood. In the present study, complement activation markers (iC3b, C5b, Bb, and C4b) were determined by enzyme-linked absorbent assays (ELISA) in the initial plasmas (prior to plasma exchange) of 20 patients with acquired TTP with severe ADAMTS13 deficiency (less than 20% of normal) and plasmas from 20 healthy controls. Of 20 TTP patients, 19 exhibited positive inhibitor in the 50:50 mixing study. Plasma levels of iC3b (1,000 ± 1,062 ng/ml), sC5b-9 (1,342±867 ng/ml), and Bb (38.2±47.7 ng/ml), as well as C4b (74.3±49.5 ng/ml) in acquired TTP patients were significantly higher than those in healthy controls (p value less than 0.01) These results indicate that complement activation in both classic and alternative pathways is a common phenomenon in patients with acquired autoimmune TTP. To demonstrate the causative effect of complement activation in TTP, we turned to our Adamts13 null mice. C57BL/6 (Adamts13-/-) mice are resistant to the development of spontaneous and Shigatoxin-induced TTP syndrome. When injected with a murine specific monoclonal antibody against complement factor H (CFH) (800 micro grams/mouse), which inhibits binding of circulating CFH to endothelial cells and C3b, Adamts13-/- mice (C57BL/6) developed more severe thrombocytopenia and anemia than wild type mice did within 6 days without additional challenge. However, renal insufficiency manifested by the increase of plasma BUN concentration was similar in both groups (Fig. 1). These results indicate that complement activation through an alternative pathway, following antibody-mediated inhibition of CFH or other complement regulatory components, may trigger the onset of TTP in light of severe ADAMTS13 deficiency.

Figure 1 Figure 1.

Disclosures

No relevant conflicts of interest to declare.
Granulocyte-Colony Stimulating Factor (G-CSF) in Secondary Prophylaxis for Advanced-Stage Hodgkin Lymphoma Treated with ABVD Chemotherapy: A Cost-Effectiveness Analysis
Authors: Cheung Matthew C , Prica Anca , Graczyk Joanna , Buckstein Rena , Chan Kelvin KW
Published: October 2019

Background: G-CSF is commonly administered to patients with advanced-stage Hodgkin Lymphoma (HL) with severe neutropenia during therapy, although such an approach does not appear to provide a survival benefit. Although the therapy may reduce episodes of febrile neutropenia, any benefit might be offset by increased costs and the potential for increased bleomycin lung toxicity with G-CSF exposure, suggested in prior studies (Martin et al., JCO 2005). As an alternative to secondary prophylaxis, single institution studies have suggested that ABVD chemotherapy can be administered without G-CSF support, treatment delays, or dose reductions. The relative costs and benefits of such an approach compared to routine use of G-CSF is unknown.

Methods: We constructed a Markov decision-analytic model to compare the strategy of secondary prophylaxis with G-CSF to a strategy of "no G-CSF" in response to therapy-related severe neutropenia for a cohort of 40 year-old patients with clinical stage IIB to IV HL treated with 8 cycles of ABVD. A 2-year time horizon was simulated. Baseline probability estimates and utilities were derived from a systematic review of relevant published studies. Direct medical costs were obtained from publicly available administrative databases or from the literature and applied to the health states. All costs and benefits were discounted by 3%. A Canadian public health payer's perspective was considered and costs were presented in 2013 Canadian dollars. Key variables were subjected to sensitivity analyses.

Results: The quality-adjusted life years (QALYs) attained with the G-CSF and "no G-CSF" strategies were 1.403 and 1.416, respectively, for a net expected benefit of 0.013 QALYs associated with omitting G-CSF. Costs for the strategies with and without G-CSF were $38,971 and $33,982, respectively, with a cost savings of $4,989 when G-CSF is omitted. In the base case analysis, the "no G-CSF" strategy was associated with both cost savings and improved quality-adjusted outcomes compared to secondary prophylaxis; therefore, the "no G-CSF" approach was dominant. This analysis was robust to one-way sensitivity analyses involving all key variables; the "no-GCSF" strategy remained dominant even when the cost of G-CSF was zero (Figure 1). In probabilistic sensitivity analysis (1000 simulations), the "no-G-CSF" strategy remained dominant in all pairwise simulations compared to an approach with G-CSF for secondary prophylaxis (Figure 2).

Conclusions: For patients with severe neutropenia during ABVD chemotherapy for advanced-stage HL, a strategy without G-CSF support, treatment delay or dose reduction is associated with improved quality-adjusted outcomes and cost savings and is the preferred approach.

Figure 1 Figure 1.

Figure 2 Figure 2.

Disclosures

No relevant conflicts of interest to declare.
Profilin2 Is Controlled By the Iron Regulatory Proteins and Modulates Iron Homeostasis
Authors: Luscieti Sara , Pilo Boyl Pietro , Galy Bruno , Gutierrez Lucia , Shvartsman Maya , Morales M. Puertos , Hentze Matthias W. , Witke Walter , Sanchez Mayka
Published: October 2019

Objective: The IRPs/IRE regulatory network plays a central role in the control of cellular iron homeostasis. Using a high throughput approach, we have previously identified novel IRP1 and IRP2 interacting mRNAs. Among the identified mRNAs, we studied more in depth Profilin2 (Pfn2), a protein involved in endocytosis and neurotransmitters release. The aim of this work is to characterize Pfn2 as a novel IRPs target mRNA and study its role in iron homeostasis.

Materials and Methods: Mouse and human Pfn2 mRNAs were tested by non-radioactive competitive electrophoretic mobility shift assays (EMSA) for the binding to IRP1 and IRP2. To test the responsiveness of Pfn2 to IRP activity, Pfn2 mRNA levels were analyzed in mice with intestinal IRP1 and IRP2 deficiency. The labile iron pool (LIP) was measured in HeLa and Hepa1-6 cell lines with transient or stable overexpression of Pfn2. Tissues derived from Pfn2 knock-out mice were analyzed for iron content, measured by atomic absorption or colorimetric assay, and for mRNA and protein levels of iron-related genes.

Results: Combination of EMSA experiments and bioinformatic analyses allowed the identification of a novel and conserved 3’UTR iron responsive element in Pfn2 mRNA with an atypical hexanucleotide apical loop (AAGUGG). Pfn2 mRNA levels were significantly reduced (~20-25%) in duodenal samples from mice with IRP1 and IRP2 intestinal specific ablation, suggesting that IRPs exert a positive effect on Pfn2 mRNA expression in vivo. Overexpression of Pfn2 cDNA in HeLa and Hepa1-6 cells reduces LIP levels compared to control cells. Finally, analysis of Pfn2 KO mice showed iron accumulation in discrete areas of the brain (olfactory bulb, hippocampus and midbrain) together with an hepatic iron deficiency with ferritin reduction.

Conclusion: Our results indicate that Pfn2 is controlled by the IRP regulatory system in vivo and that Pfn2 modulates iron homeostasis in cell lines and mice.

Funding: Work supported by grant SAF2012-40106 from Spanish Secretary of Research, Development and Innovation (MINECO) and grant CIVP16A1857 “Ayudas a proyectos de Investigación en Ciéncias de la Vida - Fundación Ramón Areces” to M.S. M.S. held a research contract under the Ramón y Cajal program from the Spanish Ministry of Science and Innovation (RYC-2008-02352).

Disclosures

No relevant conflicts of interest to declare.
Targeting Meningeoma-1 Driven AML through Epigenetic Modulation of the Cell of Origin
Authors: Riedel Simone , Bernt Kathrin M , Haladyna Jessica , Bezzant Matthew , Stevens Brett , Pollyea Daniel A. , Sinha Amit U. , Armstrong Scott A. , Wei Qi , Jordan Craig T. , Neff Tobias
Published: October 2019

Meningeoma-1 (MN1) overexpression in AML is common and predicts a poor prognosis. Forced expression of MN1 in early murine hematopoietic progenitors (CMP and LSK) but not hematopoietic stem cells (HSC) or committed progenitors (GMP) induces an aggressive myeloid leukemia as a single hit. This leukemia is strictly dependent on the high-level expression of a defined gene expression program in the cell of origin, which includes the key component HoxA9. This “susceptibility program” has been proposed as a therapeutic target in MN1high AML, but means to modulate this program have so far remained elusive.

We previously showed that the pharmacologically targetable histone methyltransferase Dot1l is critically required for HoxA9 expression in MLL-rearranged leukemias. We now report dependence on Dot1l of the MN1 “susceptibility-program” in murine MN1-driven leukemia using a genetic loss of function model. Key genes including HoxA9 depend on Dot1l in normal LSK-cells, and in MN1 transduced leukemogenic early progenitors. Inactivation of Dot1l prevents leukemia establishment and abrogates leukemia maintenance in vivo. Phenotypically, deletion of Dot1l in MN1-driven leukemias induces differentiation, cell cycle arrest and apoptosis. RNA-Seq data document that deletion of Dot1l antagonizes the cell of origin-derived susceptibility program in MN1 leukemias, including HoxA9 expression. We also confirm previous reports that MN1-transduced HSC can expand ex vivo but fail to cause leukemia in vivo. This has been linked to a lower expression level of HoxA9 in normal and MN1-transformed HSCs compared to early progenitors. We confirm that HoxA9 expression levels in MN1-transduced HSCs are lower than in MN1 transduced progenitors. Importantly, in contrast to MN1-transduced progenitors, this lower, “HSC-level” expression of HoxA9 is independent of Dot1l. This suggests a developmental switch at the HSC-to-progenitor transition that involves increased expression of HoxA9 and a change from Dot1l-independent to Dot1l-dependent regulation. The high HoxA9 expression necessary to cooperate with MN1 to cause AML is strictly Dot1l-dependent.

HOXA9 is co-expressed with MN1 in a subset of clinical MN1high AML. Exposure of MN1highHOXA9high AML primary patient samples to a pharmacologic inhibitor of DOT1L (EPZ4777) demonstrated sensitivity to DOT1L inhibition as expected from the murine model. Taken together, these data point to DOT1L as a potential therapeutic target in MN1high AML. The targeted epigenetic modulation of the interplay between an oncogenic lesion and its cooperating normal developmental program could represent a new paradigm for the treatment of AML.

Disclosures

Pollyea: Celgene: Membership on an entity's Board of Directors or advisory committees. Armstrong:Epizyme : Consultancy.
The Mir-17-92 Cluster Contributes to MLL Leukemia through the Repression of PKNOX1, a MEIS1 Competitor
Authors: Mian Yousaf A , Zeleznik-Le Nancy J.
Published: October 2019

Leukemias which arise as a result of translocations between the MLL gene and one of more than 80 different partner genes have a relatively poor prognosis. As a result of chromosome translocation, the carboxy-terminal portion of MLL containing the H3K4 methyltransferase domain is replaced by partner protein domains. In most cases this causes constitutive recruitment of transcriptional elongation machinery to MLL target genes. Downstream targets of MLL are aberrantly upregulated and include developmentally important HOX genes and MEIS1, as well as multiple microRNAs. Here we examine the contribution of specific miRNAs in the miR-17-92 cluster to MLL leukemia through the use of custom antagomiR oligonucleotides. Combinatorial treatment with antagomiRs against miR-17 and miR-19a dramatically reduce colony forming ability of MLL-fusion containing cells, but not non-MLL AML controls. To determine the mechanism by which these miRNAs contribute to leukemia, we validated PKNOX1 as a target of both miR-17 and miR-19a. MEIS1 and PKNOX1 are TALE domain proteins that participate in ternary complexes with HOX and PBX proteins. Here we establish the competitive relationship between PKNOX1 and MEIS1 in PBX-containing complex formation and determine the antagonistic role of Pknox1 to leukemia in a murine MLL-AF9 model. Collectively, these data implicate the miR-17-92 cluster as part of a regulatory mechanism necessary to maintain MEIS1/HOXA9 -mediated transformation in MLL leukemia. This approach represents a paradigm where targeting multiple non-homologous miRNAs may be utilized as a novel therapeutic regimen.

Disclosures

No relevant conflicts of interest to declare.
PAX5-PML Induces Pro B Acute Lymphoblastic Leukemia in Mice
Authors: Imoto Naoto , Kurahashi Shingo , Hayakawa Fumihiko , Yasuda Takahiko , Sugimoto Keiki , Kojima Yuki , Morishita Takanobu , Inagaki Yuichiro , Tsuzuki Shinobu , Naoe Tomoki , Kiyoi Hitoshi
Published: October 2019

PAX5 is a transcription factor required for B-cell development and maintenance. We previously showed that PAX5-PML, a fusion gene found in acute lymphoblastic leukemia (ALL), dominant negatively inhibited PAX5 transcriptional activity. Reported data including ours revealed that PAX5 fusion proteins had possible oncogenic ability; however, leukemogenicity of PAX5 fusion genes and other PAX5 mutations in mice model has not been clarified, yet. Here we demonstrated leukemia development in mice by introducing PAX5-PML. Pro B cells derived from mouse fetal liver were transfected with PAX5-PML expression vector and transplanted into mice. All 8 transplanted mice died with pro B ALL from day 63 to 158. Leukemic cells could be serially transplanted and mice died more rapidly with repetition (Figure A). Among the target genes transcriptionally activated by PAX5, expressions of BLNK, Fcer2a, and CD72 were significantly repressed in leukemia cells but repression of CD19 and CD79a were mild, suggesting the importance of down regulation of these genes for differentiation block. We compared mRNA expression profile between leukemia cells and normal pro B cells and gene set enrichement analysis (GSEA) identified candidates for second hits for development of leukemia. We analyzed the mechanism of the selective repression of CD19, Fcer2a, and BLNK and the significance of the second hit candidates, using a cell line established from leukemia cells of the third transplanted mouse. The results will show the meeting.

Figure 1 Figure 1.

Disclosures

Sugimoto: Otsuka Pharmaceutical Co., Ltd: Employment. Naoe:Zenyaku Kogyo: Research Funding; Dainippon Sumitomo Pharma: Research Funding; Kyowa Hakko Kirin Co. LTD: Research Funding; Chugai Pharmaceutical Co. LTD: Research Funding; Novartis Pharma,: Research Funding; Bristol-Myers Squibb: Research Funding; Otsuka Pharmaceutical Co. LTD: Research Funding; FUJIFILM Corporation: Research Funding. Kiyoi:Zenyaku Kogyo: Research Funding; Dainippon Sumitomo Pharma: Research Funding; Kyowa Hakko Kirin Co. LTD.: Research Funding; Chugai Pharmaceutical Co. LTD: Research Funding; Bristol-Myers Squibb: Research Funding; FUJIFILM Corporation: Research Funding.
Loss of Wild-Type Kras is Associated with Enhanced Fitness and Increased Mitogen-Activated Protein Kinase Pathway Addiction in K-RasG12D Acute Myeloid Leukemia
Authors: Burgess Michael R. , Hwang Eugene , Wandler Anica M. , Asthana Saurabh , Davis Elizabeth M. , Le Beau Michelle M. , Taylor Barry , Shannon Kevin
Published: October 2019

Functional analysis of therapeutic outlier responses in cancer therapy can identify unexpected synthetic lethal interactions and uncover biomarkers that predict enhanced sensitivity to targeted agents. We performed preclinical trials of the MEK inhibitor PD0325901 (PD901) in primary murine acute myeloid leukemias (AMLs) generated using retroviral insertional mutagenesis in KrasG12D “knock-in” mice. Similar to previous observations Nf1 and Nras mutant leukemias, treatment with PD901 prolonged survival in six independent leukemias harboring diverse retroviral integrations. Intriguingly, one outlier leukemia, clone Kras.101, demonstrated a greater than 4-fold improvement in median overall survival when treated with the MEK inhibitor. Importantly, this AML demonstrated a novel retroviral integration at relapse, indicating clonal evolution during treatment. The relapsed clone displayed phenotypic resistance upon transplantation into secondary recipients and retreatment with PD901. We performed a detailed analysis of the paired PD901-sensitive/resistant Kras.101 clones to identify the mediators of mitogen-activated protein kinase pathway (MAPK) addiction. Functional studies demonstrated that the novel dominant retroviral integration in the PD901 resistant clone did not directly alter drug response. However, genome-wide analysis of copy number alterations revealed a gain of chromosome 6 involving the Kras locus in the resistant clone, which was confirmed by spectral karyotyping and fluorescence in situ hybridization. Surprisingly, the PD901-sensitive leukemia harbored two copies of oncogenic KrasG12D due to somatic uniparental disomy, while the resistant clone harbored two mutant and one wild-type Kras alleles. This observation and paired single nucleotide polymorphism analysis indicated that the PD901-resistant leukemia was evolutionarily ancestral to the drug sensitive clone. Consistent with this hypothesis, competitive repopulation experiments with fluorescently labeled leukemia cells demonstrated increased fitness of the drug sensitive leukemia in the absence of the MEK inhibitor and over-expression of wild type K-Ras in the drug-sensitive leukemia reduced fitness in vivo. Finally, utilizing a tetracycline-inducible system, we found that wild-type Kras expression enhances the fitness of the Kras.101 clone only in the presence of the MEK inhibitor. Together, these data suggest that loss of wild-type Kras imparts a fitness advantage at the expense of increased MAPK pathway addiction, which results in enhanced sensitivity to MEK inhibition in leukemias driven by oncogenic K-Ras.

Disclosures

No relevant conflicts of interest to declare.
Premature Platelet Activation and Resistance to P2Y12 Inhibitors in Rasa3 Mutant Mice
Authors: Bergmeier Wolfgang , Paul David S , Stefanini Lucia , Robledo Raymond F. , Chan E. Ricky , Getz Todd M , Piatt Raymond , Boulaftali Yacine , Adams Mark D , Peters Luanne L.
Published: October 2019

The small GTPase RAP1 is critical for platelet activation and thrombus formation. RAP1 activity in platelets is controlled by the guanine nucleotide exchange factor CalDAG-GEFI and an unknown regulator operating downstream of the ADP receptor, P2Y12, the target of antithrombotic therapy. Here we provide evidence that the GTPase-activating protein, RASA3, is a critical inhibitor of platelet activation and the missing link in the P2Y12/RAP1 signaling pathway. Genetic inactivation of Rasa3 led to premature activation and markedly reduced lifespan of circulating platelets in mice (t1/2=14 hrs vs. 55 hrs in controls). The increased platelet turnover and the resulting thrombocytopenia were reversed by concomitant deletion of CalDAG-GEFI. Rasa3 mutant platelets were hyperresponsive to agonist stimulation, both in vitro and in vivo. Importantly, activation of Rasa3 mutant platelets occurred independently of ADP feedback signaling and was insensitive to inhibitors of P2Y12 or PI3 kinase. Thus, constitutively active RASA3 ensures that circulating platelets remain quiescent by restraining CalDAG-GEFI/RAP1 signaling. At sites of vascular injury, P2Y12 signaling is required to inhibit RASA3 and enable sustained RAP1-dependent platelet activation and thrombus formation. Our findings provide critical mechanistic insights for the antithrombotic effect of P2Y12 inhibitors and may lead to improved diagnosis and treatment of platelet-related disorders.

Disclosures

No relevant conflicts of interest to declare.
Epithelial (E)-Cadherin Is a Novel Regulator of Platelet Function
Authors: Krause Diane , Halene Stephanie , Booth Carmen Jane , Jin Huiyan , Zou Siying , Teixeira Alexandra M , Zhang Ping-Xia
Published: October 2019

Epithelial (E-) cadherin is an adhesion molecule that mediates cell-cell interactions, and is important in pluripotent stem cell reprogramming. We are investigating the role of E-cadherin in megakaryocyte differentiation and platelet function, and propose that E-cadherin mediates interactions that facilitate the essential roles of platelets. We have evidence that mature megakaryocytes and platelets express E-cadherin, and therefore generated a megakaryocyte-specific E-cadherin knockout mouse using the PF4-Cre system. E-cadherin deleted mice are viable and fertile. Despite having normal platelet counts and mean platelet volume, platelet function is abnormal. E-cadherin deletion in platelets significantly increases bleeding time in adult mice, with wild type (Ecadw/w) bleeding times of 225±52 secs, and homozygous deletion (Ecadf/f) times of 880±134 secs (p=0.005). In vivo platelet depletion using systemic administration of anti-CD42b antibody in the Ecadf/f mice causes death, likely due to hemorrhage and failure of hemostasis, which is not observed in Ecadw/w with similar levels of platelet depletion, suggesting a platelet function defect that becomes more evident under stress. We performed immunofluorescence to probe platelet structure as a potential explanation for the phenotypes, and observed disrupted b-tubulin architecture in the E-cadherin null platelets. Also, static platelet adhesion assays revealed that E-cadherin deficient platelets have impaired adhesion on fibrinogen, relative to a BSA substrate (p=0.0005). We have extended our studies to the human system, and initial studies using an E-cadherin blocking antibody in human platelets show impairment of aggregation in response to ADP, epinephrine, and thrombin. Taken together, the results demonstrate that E-cadherin contributes to the delicate balance between bleeding, hemostasis, and thrombosis. Future studies will focus on identifying how E-cadherin regulates hemostasis, with an emphasis on the interactions mediated by E-cadherin, whether between platelets, or with other cells in the blood.

Disclosures

No relevant conflicts of interest to declare.
Basic Mechanisms of Arterial and Venous Thrombosis
Authors: Renné Thomas
Published: October 2019

Combinations of proinflammatory and procoagulant reactions are the unifying principle for a variety of disorders affecting the cardiovascular system. Factor XII (FXII, Hageman factor) is a plasma protease that initiates the contact system. This system starts a cascade of procoagulant and proinflammatory reactions via the intrinsic pathway of coagulation, and the bradykinin producing kallikrein-kinin system, respectively. The biochemistry of the contact system in vitro is well understood, however its in vivo functions are just beginning to emerge. This presentation will summarize roles of the FXII-driven contact system in vivo. Genetically altered mice and large animal models have shown that FXII is essential for thrombus formation while being dispensable for hemostatic processes that terminate blood loss. Challenging the dogma of a coagulation balance, targeting FXII protected from cerebral ischemia without interfering with hemostasis. In contrast, excess FXII activity is associated with a life threatening inflammatory disorder, hereditary angioedema. Platelet polyphosphate (an inorganic polymer), neutrophil extracellular traps (NETs) and mast cell heparin activate FXII with implications on the initiation of thrombosis and edema. A key aspect of the talk will be the analysis of common principles, interactions and cross-talk between coagulation and inflammation, and the use of the novel FXII blocking antibody 3F7 in cardiopulmonary bypass system. Elucidating the FXII-driven contact system offers the exciting opportunity to develop strategies for safe interference with both thrombotic and inflammatory diseases.

Disclosures

No relevant conflicts of interest to declare.
How Do We Define the Genomic Landscape?
Authors: Dave Sandeep S.
Published: October 2019

High throughput sequencing is a revolutionary technology for the definition of the genomic features of tumors. This talk will provide a review of the relevant methodologies for non-experts in the field. The presentation will include a discussion of how high throughput sequencing is performed, its relative strengths and weaknesses, and how it is applicable to formalin-fixed and fresh/frozen tissue samples. The talk will also describe future directions in the genomic analysis of tumors.

Disclosures

No relevant conflicts of interest to declare.
Clinical Implications of the New Molecular Knowledge for Treatment of Diffuse Large B-Cell Lymphoma
Authors: Friedberg Jonathan W.
Published: October 2019

For more than 35 years, the standard induction therapy for patients with newly diagnosed diffuse large B-cell lymphoma (DLBCL) consists of cyclophosphamide, doxorubicin, vincristine, and prednisone (CHOP). Until recently, efforts to improve upon this regimen were empiric and without success, and consisted of adding additional cytotoxic drugs, intensifying doses and schedules of drugs, and consolidating initial responses with high dose therapy and autologous stem cell support. The addition of rituximab to standard CHOP therapy (RCHOP) more than a decade ago improved progression-free and overall survival, and now the majority of patients presenting with DLBCL are cured with induction therapy. Biological insights from gene expression profile (GEP) studies indicate substantial heterogeneity of DLBCL, which is largely responsible for treatment failure. We are now at the cusp of being able to utilize rational targeted therapies in biologically-defined subgroups of patients of DLBCL. In addition to cell-of-origin defined subgroups, GEP has defined stromal signatures and pathway-specific signatures in DLBCL which also predict outcome and potentially define a group of patients for a biologically-targeted therapy approach. At present, clinical development strategies include combining novel agents with standard RCHOP treatment and/or evaluating novel agents as maintenance or consolidation after RCHOP treatment. Two recent failures illustrate the challenges inherent in these approaches: RCHOP + bevacizumab (targeting angiogenesis) and R-CHOP followed by enzastaurin (targeting protein kinase C-b). Ongoing randomized trials are evaluating R-CHOP + bortezomib (proteasome inhibitor targeting NFkB), R-CHOP + ibrutinib (Bruton’s tyrosine kinase inhibitor targeting B-cell receptor signaling pathway), and R-CHOP + lenalidomide (immunomodulator with pleotropic effects) in specific GEP (or immunohistochemistry surrogates for GEP)-defined groups of patients. Finally, the majority of treatment failures in DLBCL appear to occur in patients with overexpression of c-MYC oncogene (often in combination with BCL-2 protein), which represents up to one third of patients when defined using immunohistochemistry. This unique subgroup occurs across cell-of-origin designations, and represents a substantial opportunity to improve outcome in DLBCL with the addition of novel therapeutics. This talk will review the considerable challenges in translating the exciting biology defined by GEP into the clinic for patients with DLBCL, provide an update on current and future trial designs, and conclude with a forward looking time to when GEP, or an accurate surrogate, is a component of the standard diagnostic algorithm to define optimal therapeutic approach to DLBCL.

Disclosures

Off Label Use: Novel agents for DLBCL.
Platelets Induce Neutrophil Extracellular Traps in Transfusion-Related Acute Lung Injury
Authors: Looney Mark R.
Published: October 2019

Transfusion-related acute lung injury (TRALI) is the leading cause of transfusion-related mortality in the U.S. and a major cause of transfusion-associated morbidity including increased time on mechanical ventilation and length of stay in the intensive care unit and the hospital. Neutrophils have been identified as critical cellular mediators in the pathogenesis of TRALI in both clinical studies and in experimental settings using a variety of injury models. Platelets have been implicated as a blood product that can trigger TRALI, and endogenous platelet activation contributes to lung injury. Platelets bind to the surface of neutrophils to form heterotypic aggregates, and activated platelets can trigger the formation of neutrophil extracellular traps (NETs), which is a new mode of neutrophil death that is distinct from apoptosis and necrosis. NETs are produced in experimental TRALI and are increased in post-transfusion plasma from patients who develop TRALI. Blocking platelet activation reduces the production of NETs and lung injury, and inhibiting NETs by blocking extracellular histones or dismantling the NET structure with DNase1 are strongly protective in TRALI. In conclusion, TRALI is an immune-mediated event in which activated platelets, neutrophils, and NETs, contribute to injury and are therefore targets for therapeutic intervention.

Disclosures

No relevant conflicts of interest to declare.
Inherited Predisposition to Myelodysplastic Syndrome and Acute Leukemia
Authors: Churpek Jane E.
Published: October 2019

Although generally thought of as pediatric conditions, inherited forms of myelodysplastic syndrome (MDS) and acute leukemia (AL) are increasingly recognized among adult patients. At present, at least nine genes, including ANKRD26, CEBPA, GATA2, PAX5, RUNX1, SRP72, TERC, TERT, and TP53, are known to cause familial MDS and/or AL syndromes. Several other promising candidate genes are emerging from ongoing research on the many pedigrees identified without a mutation in one of these already recognized genes. Clinical recognition of individuals with these syndromes is essential for optimal care of the patient and his/her at-risk family members and requires familiarity with the subtle clinical features of each syndrome and a high index of suspicion by the treating physician. Once recognized, genetic testing should be performed to identify the specific syndrome present as each can have unique aspects to their clinical care. For example, individuals with familial MDS/AL due to TERT or TERC abnormalities require monitoring of lung function and screening for head and neck and anogenital cancers, whereas individuals with platelet dysfunction due to familial platelet disorder/RUNX1 or ANKRD26 mutations require careful planning prior to surgical procedures to prevent bleeding complications. Due to significant overlap in the clinical presentation, often a multigene-based approach to genetic testing is necessary. Unique aspects of genetic testing in this population include: 1) tissue type selection as many of the genes that cause the familial MDS/AL syndromes are also somatically mutated in hematologic malignancies so results from DNA derived from peripheral blood or bone marrow in an individual with MDS or AL are difficult to interpret; and 2) urgency as allogeneic hematopoietic stem cell transplantation may be pursued quickly and requires knowledge of the specific mutation in the family to identify the optimal stem cell donor. The management of affected individuals who have not yet developed hematologic malignancies can be challenging as many may show morphologic signs of dysplasia in the bone marrow that may not truly represent overt malignancy. The decision of when to pursue allogeneic hematopoietic stem cell transplantation with curative intent is especially difficult. Ongoing research to define the specific events that trigger malignant transformation and how to optimally detect these events is underway. Practical algorithms for the clinical recognition, genetic testing, and management of individuals with these syndromes based on currently available knowledge as well as research seeking to improve the clinical care of these patients will be explored. A summary of the yield of next generation sequencing-based genetic testing strategies for familial presentations of MDS/AL will also be provided.

Disclosures

No relevant conflicts of interest to declare.
Predisposition to Myeloproliferative Neoplasms
Authors: Skoda Radek C.
Published: October 2019

Familial forms of myeloproliferative neoplasms (MPN) and genetic contribution to sporadic cases of MPN have long been recognized. In the majority of cases, familial MPN is inherited as an autosomal dominant trait. The penetrance varies from around 20% to up to 100% in some pedigrees. We can distinguish two types of familial MPN. Type 1 has high penetrance, polyclonal hematopoiesis and hyperproliferation of a single hematopoietic lineage, caused by mutations in a single gene and usually manifesting at birth or early childhood. Examples are mutations in the genes for the erythropoietin receptor, thrombopoietin or its receptor, MPL. The type 2 familial MPNs are characterized by clonal hematopoiesis, low penetrance and manifestation beginning in most cases later in adult life. These type 2 familial MPNs are classical examples of inherited predisposition to a clonal malignant disease, in which acquired somatic mutations in hematopoietic cells are required for disease manifestation. Affected family members typically display the same acquired driver mutations in the genes for JAK2 (JAK2-V617F or JAK2-exon12), MPL (MPL-W515), or calreticulin (CALR) as patients with sporadic MPN. The mutated genes and the mechanism of how these inherited germline mutations predispose to MPN have not yet been elucidated. The search for these germline mutations has been hampered by the low penetrance of MPN manifestation and the rare occurrence of pedigrees that are large enough for genetic studies. Furthermore, the few candidate gene mutations that have been identified to date do not map to one gene locus and the function of the candidate genes does not fall into a common category. Two models of how the germline predisposition interacts with acquired driver mutations can be considered. First, the germline mutation may increase the mutation rate for gene mutations in JAK2, MPL, and CALR. Second, the germline mutation functionally synergizes with mutations in JAK2, MPL, and CALR and promotes disease initiation. The current state of our studies and studies in other laboratories will be discussed.

Disclosures

Skoda: Novartis: Consultancy; Sanofi: Consultancy.
Regulated RNA Expression and Normal Erythropoiesis
Authors: Higgs Douglas
Published: October 2019

Although a small number of the vast array of long non-coding RNAs (lncRNAs) have known effects on cellular processes, in general their contribution to development, differentiation and disease remains unknown. We have shown that some intragenic enhancers, when active, behave as alternative promoters producing lncRNA transcripts that are processed using the canonical signals of their host gene. More recently we have also analyzed intergenic lncRNAs to determine the extent to which they too might originate from intergenic enhancers. We find that intergenic lncRNAs in erythroid cells are almost evenly divided between those arising from enhancer-associated or promoter-associated elements and that these RNAs differ with respect to their conservation and tissue specificity. Of considerable interest we find that expression of lncRNAs arising from enhancers is associated with enhanced expression of neighboring protein coding genes during erythropoiesis.

Disclosures

No relevant conflicts of interest to declare.
Stem Cell Paradigms in Tissue Regeneration and Cancer
Authors: Fuchs Elaine
Published: October 2019

Stem cells (SCs) have the ability to self-renew long term and differentiate into one or more tissues. Typically, SCs are used sparingly to replenish cells during normal homeostasis. However, even SCs that are quiescent must be able to respond quickly to injury in order to fuel rapid tissue regeneration. How SCs balance self-renewal and differentiation is of fundamental importance to our understanding of normal tissue maintenance and wound repair. Increasing evidence suggests that the regulatory circuitry governing this balancing act is at the root of some types of tumors both in mice and in humans. The skin is an excellent model system to understand how SCs function in normal tissue generation and how this process goes awry in cancer. Many of the principles we have unearthed by studying skin stem cells have parallels to the hematopoietic system. We have identified quiescent and “primed”/active SCs within the skin and shown that primed SCs have a lower threshold for activation to enter tissue regeneration. We have learned that during normal homeostasis, SC behavior is controlled not only through cues received from their microenvironment but also through signals emanating from their differentiating lineages. We have been dissecting how extrinsic signaling to SCs trigger a cascade of transcriptional changes that govern SC activation during tissue development, homeostasis and hair regeneration. Our findings provide new insights into our understanding of the process of SC activation, and in so doing have revealed mechanisms which are also deregulated in a variety of different human cancers. Our goal is to understand how SCs start and stop making tissue, and how this changes in cancer. Our recent discoveries on this topic have led us to the realm of identifying and characterizing cancer SCs (tumor-initiating cells) of squamous cell carcinomas (SCCs) of the skin. We developed a new method to knockdown genes specifically in skin and oral progenitors, enabling us to screen not only the differences between cancer and normal SCs, but also the myriad of gene alterations surfacing from the Human Cancer Sequencing project. Our screens have illuminated new oncogenes and tumor suppressors for SCCs, among the most prevalent and life-threating cancers world-wide that include cancers of lung, esophagus, breast, cervix, prostate, throat and oral tissues. Our findings are unearthing new targets for cancer therapeutics.

Disclosures

No relevant conflicts of interest to declare.