Serial Bleeding Research Articles

Retrotransposons are co-opted to activate hematopoietic stem cells and erythropoiesis.

Hematopoietic stem cells (HSCs) and erythropoiesis are activated during pregnancy and after bleeding by the derepression of retrotransposons, including endogenous retroviruses and long interspersed nuclear elements. Retrotransposon transcription activates the innate immune sensors cyclic guanosine 3',5'-monophosphate-adenosine 5'-monophosphate synthase (cGAS) and stimulator of interferon (IFN) genes (STING), which induce IFN and IFN-regulated genes in HSCs, increasing HSC division and erythropoiesis. Inhibition of reverse transcriptase or deficiency for cGAS or STING had little or no effect on hematopoiesis in nonpregnant mice but depleted HSCs and erythroid progenitors in pregnant mice, reducing red blood cell counts. Retrotransposons and IFN-regulated genes were also induced in mouse HSCs after serial bleeding and, in human HSCs, during pregnancy. Reverse transcriptase inhibitor use was associated with anemia in pregnant but not in nonpregnant people, suggesting conservation of these mechanisms from mice to humans.

Deletion of tissue factor pathway inhibitor isoform beta or gamma, but not alpha, improves clotting in hemophilic mice

BackgroundTissue factor pathway inhibitor (TFPI) regulates tissue factor–triggered coagulation. Humans and mice express transcripts encoding for multidistributed (endothelial, platelet, and plasma) 3-Kunitz domain TFPIα and endothelial membrane–anchored 2-Kunitz TFPIβ. Mice express a third transcript, γ, that encodes plasma lipoprotein–associated 2-Kunitz TFPI. In humans, proteolysis of α and/or β produces plasma lipoprotein–associated 2-Kunitz TFPI at lower levels. In clinical trials, monoclonal antibodies that target all TFPI isoforms extend coagulation and correct bleeding in hemophilic patients but with some thrombosis risks. ObjectivesTo determine the impact of TFPI isoform-specific deletions on promoting clotting in hemophilic mice. MethodsEngineered TFPI isoform–specific, hemophilic (factor VIII-null) mice were evaluated for clotting. ResultsMice expressing any single TFPI isoform were healthy. Thrombin generation assays identified TFPIγ as the dominant anticoagulation isoform in mouse plasma. Hemostasis was assessed by serial bleeding times from a tail vein laceration. Repeatedly, after a clot forms, it was manually disrupted; the number of clots/disruptions occurring over a 15-minute period were reported. C57BL/6 and hemophilic mice clot on average 25.6 vs 5.4 times, respectively. On a hemophilia background, TFPIβ or TFPIγ-specific deletion improved clotting to 14.6 and 15.2 times, respectively (P < .0001). TFPIα-specific deletion was without impact, clotting 5.1 times. Heterozygous deletion of TFPIβ was effective, clotting 11.8 times (P < .0001). Heterozygous deletion of TFPIα or TFPIγ alone was ineffective, clotting 3.0 and 6.1 times, respectively, but heterozygous TFPIαγ deletion improved clotting to 11.2 times (P < .001). ConclusionIn hemophilic mice, endothelial TFPIβ and plasma γ-derived 2-Kunitz TFPI individually contribute more to bleeding than total TFPIα.

Deciphering the regulatory landscape of murine splenic response to anemic stress at single-cell resolution

AbstractStress erythropoiesis can be influenced by multiple mediators through both intrinsic and extrinsic mechanisms in early erythroid precursors. Single-cell RNA sequencing was conducted on spleen tissue isolated from mice subjected to phenylhydrazine and serial bleeding to explore novel molecular mechanisms of stress erythropoiesis. Our results showed prominent emergence of early erythroblast populations under both modes of anemic stress. Analysis of gene expression revealed distinct phases during the development of emerging erythroid cells. Interestingly, we observed the presence of a "hiatus" subpopulation characterized by relatively low level of transcriptional activities that transitions between early stages of emerging erythroid cells, with moderate protein synthesis activities. Moreover, single-cell analysis conducted on macrophage populations revealed distinct transcriptional programs in Vcam1+ macrophages under stress. Notably, a novel marker, CD81, was identified for labeling central macrophages in erythroblastic islands (EBIs), which is functionally required for EBIs to combat anemic stress. These findings offer fresh insights into the intrinsic and extrinsic pathways of early erythroblasts' response to stress, potentially informing the development of innovative therapeutic approaches for addressing anemic-related conditions.

Development of a novel ddPCR assay for nonclinical and clinical pharmacokinetic characterization of ICVB-1042.

e14693 Background: ICVB-1042 is a novel chimeric oncolytic Adenovirus (Ad) with potential for intravenous administration. A key component of the clinical assessment of ICVB-1042 will be pharmacokinetic (PK) viral genome analysis by Polymerase Chain Reaction (PCR) to understand plasma viral kinetics and tumor replication. Droplet Digital PCR (ddPCR) is more sensitive and more accurately quantifies a lower template concentration compared to traditional quantitative PCR. Our ddPCR assay targets ICVB-1042 and will not amplify other Ad5 or Ad34 viruses. The aim is to show that this assay specifically measures ICVB-1042 and may be used as a clinical biomarker. Methods: PCR primers and probe were designed to target the Ad5/Ad34 chimeric fiber of ICVB-1042. Template ICVB-1042 was spiked into representative human and murine sample matrices. Preclinical studies tested assay performance in vitro using patient derived disassociated tumor cells (DTCs) and in vivo using tumor bearing and non-tumor bearing mouse models. Viral replication and PK were assessed in vitro by analyzing supernatant of infected cells at multiple timepoints and in vivo with serial bleeds from different animals. Viral distribution was assessed by isolating viral DNA from homogenized tissue samples taken at multiple timepoints. Results: Analysis of ICVB-1042 by ddPCR showed linear quantification over a wide range of viral concentrations (5E5-5E12 viral genomes/mL) with sensitivity to low concentrations of virus (lower limit of quantification = 1.11E2 total viral genomes). Viral genomes were detected and quantified in human plasma samples spiked with ICVB-1042. Non-spiked samples were negative for signal. ICVB-1042 viral genomes were also detectible in tumor sections from ICVB-1042-treated NSG mice. Increasing viral genomes could be detected in vitro at days 6 and 9 post ICVB-1042 infection in the supernatants of several DTCs (2 donors each). The measured increase in viral genomes was accompanied by viral mediated cell death. ICVB-1042 could be detected in vivo in the plasma of non-tumor bearing CD-1 mice 1 and 24 hours post ICVB-1042 dosing but was cleared and not detected out to 90 days post dose. ICVB-1042 was detected post dose in the plasma of tumor bearing NSG mice and was again cleared. ICVB-1042 was detectable in plasma samples and accompanied by anti-tumor efficacy at later timepoints. Studies directly comparing tumor bearing to non-tumor bearing NSG mice have shown that the virus present in the plasma is due to tumor specific replication. Tissue viral genome distribution differences could also be detected in C57BL/6 mice expressing huCD46, the viral tropism of ICVB-1042. Conclusions: ICVB-1042 viral genomes can be detected using ddPCR. This assay can be used to assess viral replication in vitro and PK and distribution in vivo. Use of ddPCR to detect ICVB-1042 in patient samples will be key to the characterization of clinical efficacy.

Acute Anemic Stress Is Modulated By Intercellular Mitochondria Transport from Erythroblastic Island Macrophages to Early Erythroblasts Via CD47-Sirpα Interactions

Stress erythropoiesis can be modulated by multiple cellular and molecular mediators from early stages of erythroblasts and precursors via cell intrinsic and extrinsic mechanisms. To investigate novel cell autonomous and non-autonomous mechanisms to overcome erythroid stress, we adopted phenylhydrazine (PHZ) induced acute anemic stress in mice and analyzed bulk RNA-seq on splenic early erythroblasts (Ter119+CD44high), and found significant enrichment of the early erythroid progenitor populations, as well as up-regulation of cell-cycle, DNA replication, proliferation and mitochondrial metabolism related genes (Gata1, Tal1, Sox6, E2Fs, Lin54, Foxo3 etc), suggesting that the most prominent erythroblastic cell-autonomous responses to stress include proliferation and energy metabolism. Next, we aim to explore the cell extrinsic pathways to mediate stress erythropoiesis from the perspective of the erythroid niche, i.e., erythroblastic islands (EBIs) macrophages. Intercellular mitochondria transfer is an emerging topic with implications in multiple biological processes, while it remains elusive whether mitochondria can be transported intercellularly within the EBIs, and if such a phenomenon is functionally relevant. In our study, we infused EBI macrophages into PHZ or serial bleeding induced anemic mice, and demonstrated that exogenous EBI macrophage infusion accelerated the erythroid regeneration in both modes of acute anemic stressors in a dynamic manner. More interestingly, we infused EBI macrophage isolated from mito-Dendra2 mice that express trackable mitochondria, and observed that mitochondria are transferred from EBI macrophages to early stages of erythroblasts. We conducted in vitro co-culture of EBI macrophages isolated from mito-Dendra2 mice and early erythroblasts isolated from PHZ treated tdTomato-Vav-Cre mice, and observed that mitochondria transfer mainly occurs via cell-cell contact mediated direct uptake during erythroid stress. Single cell RNA-sequencing (scRNA-seq) revealed that early erythroblasts receiving mitochondria exhibit up-regulation of a myriad of of proliferation signatures that are enriched in RNA binding (Rps2, Rpl28, Rpl10a, etc.), translation/protein synthesis (Eif5a, Eef1g, Ybx1, etc.), ATP metabolism (Atp5o, Atp5d, Atp6v0e, etc.) related molecular functions. The result pointed to the functional importance of intercellular mitochondria transfer to mediate the bioenergetic profiles of recipient erythroblasts by enhancing their protein/ATP synthesis and proliferation. Furthermore, scRNA-seq analysis revealed a subset of metabolically active erythroid populations marked by CD47 that receive higher abundance of mitochondria, as well as exhibiting enrichment in proliferation signatures, suggesting a potential role of the CD47 labelled erythroblasts in mitochondria transfer mediated erythroid regeneration. Notably, CD47 or Sirpα blockade in vivo leads to remarkable decline in both mitochondria transfer events as well as their mediated erythroid recovery. Thus, we postulate that there is a clear association between CD47 and mitochondria transfer mediated stress erythropoiesis. Collectively, our study explored the cell intrinsic and extrinsic pathways of early erythroblasts' responses to overcome stress, and shed light on the regulatory mechanisms of mitochondria transfer through CD47-SIRPα mediated cell-cell contacts and/or their downstream signaling in the context of stress erythropoiesis.

Column Agglutination Assay Using Polystyrene Microbeads for Rapid Detection of Antibodies against SARS-CoV-2.

Rapid serology platforms are essential in disease pandemics for a variety of applications, including epidemiological surveillance, contact tracing, vaccination monitoring, and primary diagnosis in resource-limited areas. Laboratory-based enzyme-linked immunosorbent assay (ELISA) platforms are inherently multistep processes that require trained personnel and are of relatively limited throughput. As an alternative, agglutination-based systems have been developed; however, they rely on donor red blood cells and are not yet available for high-throughput screening. Column agglutination tests are a mainstay of pretransfusion blood typing and can be performed at a range of scales, ranging from manual through to fully automated testing. Here, we describe a column agglutination test using colored microbeads coated with recombinant SARS-CoV-2 spike protein that agglutinates when incubated with serum samples collected from patients recently infected with SARS-CoV-2. After confirming specific agglutination, we optimized centrifugal force and time to distinguish samples from uninfected vs SARS-CoV-2-infected individuals and then showed concordant results against ELISA for 22 clinical samples, and also a set of serial bleeds from one donor at days 6-10 postinfection. Our study demonstrates the use of a simple, scalable, and rapid diagnostic platform that can be tailored to detect antibodies raised against SARS-CoV-2 and can be easily integrated with established laboratory frameworks worldwide.

In vivo study for the hemostatic efficacy and foreign body reaction of a new powder-type polysaccharide hemostatic agent.

PurposeVarious hemostatic agents have been introduced in therapy as postoperative bleeding is a poor prognostic factor for postoperative outcomes. These products can be divided into those that directly promote the hemostatic cascade and those that physically form a barrier by absorbing blood. The latter, powder-type hemostatic agents have the advantages of being inexpensive and more absorbable with less foreign body reactions (FBRs) and are applicable to a relatively wide area. This study was conducted to verify the safety and efficacy of a newly invented polysaccharide product (OOZFIX, Theracion Biomedical), which improves blood absorption and hemostatic effects.MethodsTwo separate animal experiments were performed. The first evaluated FBRs histologically at 3 days, 2 weeks, and 4 weeks, after implantation of OOZFIX in rats, and the second compared hemostatic performance of OOZFIX and Arista AH (Bard) in the porcine liver punch biopsy model.ResultsWe found minimal FBRs in the 3-day group and no reactions in both the 2-week and 4-week groups after implantation of hemostatic agents. The time to hemostasis of OOZFIX was not significantly different from that of Arista AH (median [interquartile range]: 9 [6–10] minutes vs. 8 [6–10] minutes, respectively; P = 0.522). When comparing the serial bleeding grade tendency, there was no statistical difference between OOZFIX and Arista AH (P = 0.656).ConclusionOOZFIX caused a minimal FBR that disappeared within 2 weeks in vivo, and its hemostatic performance was comparable with that of an existing agent, Arista AH. Further clinical studies are required in the future.

Anti-CD21 Chimeric Antigen Receptor (CAR)-T Cells for T Cell Acute Lymphoblastic Leukaemia (T-ALL)

CAR-T cell therapy against CD19 has changed the treatment landscape in relapsed/refractory (r/r) B-ALL. R/r T-ALL has a dismal prognosis, with an unmet need for effective targeted therapies. Several unique challenges mean that CAR-T cell therapy has yet to be successfully translated to T-ALL. Most strategies have targeted pan-T cell antigens (CD7, CD5) but these are limited by T cell aplasia and fratricide, requiring elimination of CAR-T antigen surface expression during manufacture. An ideal target would be exclusively or largely confined to the malignant T cell component but published examples of these (CD1a and TRBC1) are expressed in only minor T-ALL subsets.We previously showed that CD21 is expressed in a NOTCH-dependent manner in T-ALL (Leukemia. 2013, 27:650) and have developed it as a potential immunotherapy target, being primarily expressed on normal B cells, with minimal expression on mature T cells.70% of human T-ALL cell lines (9/16) expressed surface CD21 by flow cytometry (FACS), with a median antigen density in positive lines of 2545/cell. In primary T-ALL, 57% of presentation samples (n=58) expressed CD21 (median antigen density 1168/cell). 45% of relapse (n=11) and 20% of primary refractory cases (n=30) expressed CD21, with a similar antigen density to presentation samples. CD21 positivity varied by maturational stage, with highest expression in cortical T-ALL (80% of cases) followed by pre-T (72%), mature (67%), ETP (25%) and pro-T (17%).Healthy donor blood (n=14) showed CD21 expression limited to B cells and a low proportion (11%) of T cells (10-fold lower intensity v B cells, 316 antigens/cell). T cell CD21 expression was not up-regulated upon activation with CD3/CD28 antibodies (n=6) and was not associated with markers of differentiation/exhaustion.To target CD21, DNA gene-gun vaccination of rats with a plasmid encoding full-length CD21, followed by phage display was performed and multiple anti-CD21 scFvs isolated. These were cloned into 4-1BBζ CARs and expressed in primary T cells but failed to kill or secrete cytokines in response to CD21+ SupT1 cells.CD21 is a bulky molecule, with 15/16 sushi repeats in the extracellular domain. All isolated scFvs were found to bind membrane-distal domains. We hypothesized that ineffective signalling due to inadequate synapse formation was responsible for poor performance of anti-CD21 CAR-T, and that binders to membrane-proximal epitopes would signal more efficiently. We re-vaccinated rats with the first 5 sushi repeats of CD21 and generated a library of binders which bound CD21 at this membrane-proximal region.Multiple candidate binders expressed as CARs were functional, with cytotoxicity and interferon-γ secretion in response to CD21+ target cells. However, non-specific background cytokine secretion was seen against CD21 negative cells, and no IL-2 secretion was seen. Re-cloning binders into a fragment antigen binding (Fab)-CAR architecture yielded constructs capable of specific cytotoxicity, IFN-γ and IL2 secretion against a CD21+ cell line but not its CD21 negative counterpart (n=6). Our lead anti-CD21 candidate CAR specifically proliferated in vitro, without fratricide or premature exhaustion/ differentiation, and was active against low-density CD21-positive cell lines (n=3) and primary cells from 2 T-ALL patients. Improved functionality of Fab v scFv-based CAR was not driven by higher affinity binding or CAR surface expression.We tested anti-CD21 CAR in murine models of T-ALL. NSG mice were injected with SupT1-luciferase cells and treated with aCD19 or aCD21 CAR-T on day +5. At 2 weeks post treatment, markedly lower disease burden was seen in CD21 CAR-T v CD19 recipients by bioluminescence imaging (median radiance 71700 v 790000 p=0.0079). Further, we injected primary T-ALL blasts in another cohort, treating with aCD19 or aCD21 CAR-T on D+20. Serial bleeds from day 27 post CAR-T showed tumour control in aCD21 CAR treated mice (p=0.024) with an overall survival advantage (median OS 44 days vs undefined, HR = 19.8, p = 0.0069, n=4/group).In summary, we propose CD21 as a novel target for CAR-T cell therapy in T-ALL. Its expression is largely restricted to the malignant T cell compartment, overcoming issues with fratricide and on-target off-tumour effects seen in many T-ALL CAR-T strategies to date. Despite the complexity of the target, we have successfully generated an aCD21 CAR that is functional both in vitro and in vivo. DisclosuresMaciocia: Autolus: Current equity holder in publicly-traded company. Onuoha: Autolus: Ended employment in the past 24 months. Khwaja: Pfizer: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Novartis: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Jazz Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Astellas: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Abbvie: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Maciocia: Autolus: Current equity holder in publicly-traded company, Research Funding. Pule: Autolus: Current Employment, Current equity holder in publicly-traded company.

Blockade of the Neonatal Fc Receptor (FcRn) Represents an Effective Mechanism for the Removal of Pathogenic Autoantibodies in Primary Immune Thrombocytopenia

Introduction: The neonatal Fc receptor, FcRn is ubiquitously expressed, & is responsible for maintaining the half-life of IgG & albumin, by rescuing these proteins from intracellular lysosomal degradation. Blockade of the interaction of FcRn with IgG would be expected to prevent salvage of both non-pathogenic and pathogenic IgG. Patients with primary immune thrombocytopenia (ITP) have autoantibodies against platelet membrane proteins such as the cell adhesion receptor CD61/CD41 (GPIIb/IIIa). Rozanolixizumab (a high affinity IgG4 monoclonal antibody (mAb) that specifically inhibits IgG binding to FcRn) is currently being evaluated in phase 2 clinical studies in patients with ITP. The aim of the current study was to understand the in vitro properties of rozanolixizumab & to explore the in vivo effects of a surrogate anti-mouse FcRn mAb (murinized “4464”) in a mouse model of ITP.Methods: The affinity of rozanolixizumab for FcRn was tested using surface plasmon resonance technology (Biacore), with the antibody captured by an anti-Fc immobilized on the sensor chip & soluble FcRn in solution, whilst the affinity of fluorescently labelled rozanolixizumab for human FcRn expressed on the surface of Madin-Darby canine kidney (MDCK) cells was measured by flow cytometry. Recycling & transcytosis of human IgG (hIgG) was also assessed in FcRn-transfected MDCK cells. For IgG tracking experiments, endothelial & epithelial cell lines were incubated with an AF647-conjugated IgG for 24 hours in the presence or absence of rozanolixizumab. Prior to imaging, nuclei were labelled using Hoechst at 37°C & cells were then visualized by fluorescence microscopy. Phagocytosis of anti-human MHC I (W6/32)-coated platelets by THP-1 cells was assessed by flow cytometry at 1 hour. The effect of rozanolixizumab on IgG recycling in vivo was assessed in the human FcRn-transgenic (hFcRn-Tg) mouse: hIgG at 500mg/kg was administered IV to mice on day 1 & dosed with antibodies on day 2; serial bleeds were then taken at multiple time points post antibody dosing & levels of hIgG, albumin & rozanolixizumab were quantified using LC-MS/MS. For the induction of ITP in mice, animals received an anti-CD41 antibody (MWReg30), delivered by osmotic minipump (~1µg of antibody delivered every 24 hours) & the anti-mouse FcRn mAb (4464) was administered at 30mg/kg either on days 0, 2 & 4 (prophylactic dosing) or on days 3 & 5 (therapeutic dosing); IVIg (1g/kg) was also evaluated & control mice received either PBS or an isotype-matched control mAb (101.4). Daily blood samples were taken to assess platelet numbers.Results: Rozanolixizumab has a high affinity for FcRn in a protein-protein (Biacore) assay with a KD of ~25pM at both pH6.0 & pH7.4; in the cell-based assay, the affinity was 1nM at both pH's. Rozanolixizumab efficiently inhibited the recycling & transcytosis of IgG in human FcRn-transfected MDCK cells (IC50 0.4nM & 1.1nM, respectively). Confocal microscopy experiments showed that there was an increased accumulation of IgG in cells in the presence of rozanolixizumab, relative to an isotype control mAb, & that the IgG was co-localized in lysosomes where it appeared to be degraded. FcRn has been postulated to play a direct role in phagocytosis, but the phagocytosis of opsonized platelets by THP-1 cells was unaffected by blockade with rozanolixizumab. Treatment with rozanolixizumab resulted in a dose-dependent accelerated clearance of hIgG in the hFcRn-Tg mouse; hIgG levels in all treated groups were significantly lower vs PBS controls from 24 hours post-dosing until the end of the experiment 8 days post dosing (p<0.01). No effect on albumin levels occurred in these mice. In the mouse model of ITP, both prophylactic & therapeutic treatment regimens with 4464 resulted in an increased number of platelets compared to control animals that received either PBS or 101.4. Animals that received 4464 from day 0 had significantly greater platelet numbers than control animals on days 4 & 5 (p<0.05); therapeutic dosing gave a significantly greater platelet number than control animals on day 5 (p<0.05).Conclusion: Rozanolixizumab is a high affinity mAb that inhibits the IgG recycling function of FcRn in vitro &in vivo . An anti-mouse FcRn mAb demonstrated efficacy in a mouse model of ITP. These data support the evaluation of rozanolixizumab in patients with ITP, & a phase 2 clinical study is currently ongoing in this patient population. DisclosuresEddleston:UCB Pharma: Employment. Greenslade:UCB Pharma: Employment. Qureshi:UCB Pharma: Employment. West:UCB Pharma: Employment. Atherfold:UCB Pharma: Employment. Amirkhosravi:UCB Pharma: Research Funding. Desai:UCB Pharma: Research Funding. Rivera-Amaya:UCB Pharma: Research Funding. Smith:UCB Pharma: Consultancy, Equity Ownership. Shock:UCB Pharma: Employment.

Abstract PR19: Monitoring of melanoma progression utilizing multiplatform biomarkers of blood cell-free DNA

Abstract Blood biopsies utilizing cell-free DNA (cfDNA) represent an alternative, minimally invasive source that permits repeated sampling to enable melanoma patient monitoring during treatment and follow-up. However, the sensitivity of cfDNA biomarker detection is limited to tumor sampling availability, which can vary significantly. This encourages the use of multiplatform biomarkers to improve sensitivity in assessing melanoma progression. Here we explored the detection of different cfDNA biomarker types, DNA mutations and copy number amplification (CNA), in blood of melanoma patients. To assess the utility of these cfDNA biomarker types, we utilized a highly sensitive multigene (54-gene cancer panel) digital next-generation sequencing (NGS) assay containing 0.1% sensitivity or multiplex droplet digital PCR ddPCR. cfDNA mutations were assessed by a multigene digital NGS assay in a cohort of preoperative stage III/IV patients rendered disease-free (n=44), whereby 75% of patients had detectable cfDNA genomic aberrations. Patients with high tumor mutation burden (TMB) (≥3 mutations) were associated with worse overall survival (OS) (p=0.048) compared to those with &amp;lt;3 mutations. Next, we assessed 98 serial bleeds obtained from a cohort of preoperative stage IIIA-C melanoma patients rendered disease-free followed by adjuvant immunotherapy with follow-up until stage IV distant metastasis (n=12), where cfDNA mutation burden (CMB) levels correlated with TMB (p=0.019). This enabled earlier detection of stage IV disease recurrence compared to imaging (p&amp;lt;0.01), and captured tumor heterogeneity, and rising CMB levels prerecurrence clinical detection. An expanded 70-gene digital NGS panel was used to assess melanoma brain metastasis (MBM) in 26 preoperative MBM patients who had paired resected MBM tumors-cfDNA. Interestingly, tumor-identified MBM mutations were detected in the paired cfDNA at a concordance of 48%, 54%, and 58% at mutant allele frequency cut-offs of 0%, 0.5%, and 1%, respectively. Encouragingly, cfDNA-tumor SNV concordance was 88% in known melanoma driver genes (BRAF/NRAS). We then assessed a novel 1q21.3 CNA during melanoma progression in patients receiving checkpoint inhibitor immunotherapy (CII). Using a multiplex ddPCR assay targeting four 1q21.3 gene targets, we identified 77% (n=31) of patients at the time of progressive disease while on CII had 1q21.3 CNA in plasma whereby patients with stable disease, partial response, or disease free had low or no 1q21.3 CNA cfDNA. These results demonstrate the ability to use different multiplatform cfDNA biomarkers to monitor melanoma disease progression for recurrence and treatment response. The study demonstrates that cfDNA as mutation detection can be improved in melanoma using other genomic aberrations in monitoring immunotherapy in melanoma patients. Citation Format: David S. Hoon, Selena Y. Lin, Rebecca Gentry, Steve O’Day. Monitoring of melanoma progression utilizing multiplatform biomarkers of blood cell-free DNA [abstract]. In: Proceedings of the AACR Special Conference on Melanoma: From Biology to Target; 2019 Jan 15-18; Houston, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(19 Suppl):Abstract nr PR19.

A Factorial Analysis of Drug and Bleeding Effects in Toxicokinetic Studies.

The ICH revised the S3A guidance allowing blood to be microsampled for toxicokinetic analysis from the main study cohorts of rats in general toxicology studies. The resulting changes in the hemogram have been examined in healthy animals but the ability to read through the data when there are toxicological changes has not been thoroughly examined in the literature. To address this, a toxicology study in Sprague Dawley rats was conducted where animals received repeated doses of saline or valproic acid by IP injection daily for 7 days. Animals in both treatment groups were unbled, serially bled (6 bleeds/animal at 0.1 ml/bleed) or compositely bled (2 bleeds/animal at 0.6 ml/bleed) on days 1 and 7 for TK analysis. No statistically significant changes in the clinical pathology were observed for either the serial bleed or composite bleed animals when compared with their respective unbled control; however, a 4%-7% decrease in erythrocyte counts following serial bleeding and a 5%-19% decrease following composite bleeding was observed. When all the clinical pathology and organ weight data were equivalence tested, both the serial bleed and composite bleed results were equivalent to their unbled controls except for the erythroid parameters in the composite bleed group. Toxicokinetic analysis of the blood samples resulted in comparable concentration-time curves, regardless of the method of blood collection. Under these study conditions, the results show blood microsamples can be collected from the core or recovery cohort of animals in a toxicology study without impacting the toxicological interpretation in rats.

Cell-intrinsic regulation of peripheral memory-phenotype T cell frequencies.

Memory T and B lymphocyte numbers are thought to be regulated by recent and cumulative microbial exposures. We report here that memory-phenotype lymphocyte frequencies in B, CD4 and CD8 T-cells in 3-monthly serial bleeds from healthy young adult humans were relatively stable over a 1-year period, while Plasmablast frequencies were not, suggesting that recent environmental exposures affected steady state levels of recently activated but not of memory lymphocyte subsets. Frequencies of memory B and CD4 T cells were not correlated, suggesting that variation in them was unlikely to be determined by cumulative antigenic exposures. Immunophenotyping of adult siblings showed high concordance in memory, but not of recently activated lymphocyte subsets. To explore the possibility of cell-intrinsic regulation of T cell memory, we screened effector memory-phenotype T cell (TEM) frequencies in common independent inbred mice strains. Using two pairs from these strains that differed predominantly in either CD4 TEM and/or CD8 TEM frequencies, we constructed bi-parental bone marrow chimeras in F1 recipient mice, and found that memory T cell frequencies in recipient mice were determined by donor genotypes. Together, these data suggest cell-autonomous determination of memory T niche size, and suggest mechanisms maintaining immune variability.

Stress-Induced Hyperglycemia in White-Throated and White-Crowned Sparrows: A New Technique for Rapid Glucose Measurement in the Field.

Organisms experience stressors, and the physiological response to these stressors is highly conserved. Acute stress activates both the sympathetic nervous system and the hypothalamic-pituitary-adrenal axis, increasing epinephrine, norepinephrine, and glucocorticoids, collectively promoting glucose mobilization. While this is well characterized in mammals, the hyperglycemic response to stress in avian and nonavian reptiles has received less attention. A number of factors, ranging from time of day to blood loss, are reported to influence the extent to which acute stress leads to hyperglycemia in birds. Here we characterized the glycemic response to acute handling stress in two species of free-living sparrows: white-throated sparrows (WTSPs: Zonotrichia albicollis) in St. Mary's County, Maryland, and white-crowned sparrows (WCSPs: Zonotrichia leucophrys) in Tioga Pass Meadow, California. We validated a novel technique for rapid field measurement of glucose using a human blood glucose meter, FreeStyle Lite. As expected, acute handling stress elevated blood glucose at both 15 and 30 min postcapture as compared to baseline for both WTSPs and WCSPs. In addition, handling for 30 min without bleeding had the same hyperglycemic effect as handling with serial bleeds in WCSPs. Finally, body condition that was measured as abdominal fat score predicted stress-induced blood glucose in WTSPs but not in WCSPs. Our results are consistent with the mammalian literature on acute stress and energy mobilization, and we introduce a new field technique for avian field biologists.

Murine Pharmacokinetic Studies.

Murine pharmacokinetics (PK) represents the absorption, distribution, metabolism, and elimination of drugs from the body, which helps to guide clinical studies, ultimately resulting in more effective drug treatment. The purpose of this protocol is to describe a serial bleeding protocol, obtaining blood samples at six time points from single mouse to yield a complete PK profile. This protocol has proved to be rapid, highly repeatable, and relatively easy to acquire. Comparing with the conventional PK studies, this method not only dramatically reduces animal usage, but also decreases sample variation obtained from different animals.

Monitoring bottlenose dolphin leukocyte cytokine mRNA responsiveness by qPCR.

Both veterinarians caring for dolphins in managed populations and researchers monitoring wild populations use blood-based diagnostics to monitor bottlenose dolphin (Tursiops truncatus) health. Quantitative PCR (qPCR) can be used to assess cytokine transcription patterns of peripheral blood mononuclear cells (PBMC). This can supplement currently available blood tests with information on immune status. Full realization of this potential requires establishment of normal ranges of cytokine gene transcription levels in bottlenose dolphins. We surveyed four dolphins over the span of seven months by serial bleeds. PBMC were stimulated with phytohaemagglutinin (1, 5, and 10 μg/mL) and concanavalin A (1 μg/mL) for 48 H in vitro. RNA from these cultures was probed by qPCR using Tursiops truncatus-specific primers (IL-1α, IL-1β, IL-1RA, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12p40, IL-13, IL-18, IFN-γ and TNF-α). Two blood samples from an additional bottlenose dolphin diagnosed with acute pulmonary disease add further perspective to the data. We observed that mitogen choice made a significant difference in the magnitude of gene transcription observed. On the other hand, most cytokines tested exhibited limited intra-animal variation. However, IL-6 and IL-12p40 differed between older and younger dolphins. Furthermore, the magnitude of mitogenic response clusters the tested cytokines into three groups. The data provide a reference for the selection of target cytokine mRNAs and their expected range of mitogen-stimulated cytokine gene transcription for future studies.

Abstract 3788: Monitoring of multimodality immune checkpoint inhibitor treatment efficacy in metastatic melanoma patients through molecular analysis of circulating tumor cells

Abstract There is a dire need for molecular tools that can monitor therapy efficacy and prognosis in melanoma patients who suffer from distant metastasis. Blood biopsies offer a minimally invasive platform for clinicians to monitor melanoma patients. In particular, the molecular analysis of circulating tumor cells (CTCs) would be advantageous as they reflect both mutation and transcriptome profile of the tumor. However, the isolation and assessment of a minimal number of CTCs present in 10 ml of blood still remains challenging. To address the needs for more informative molecular CTC assays, our group used well-established blood-based melanoma mRNA CTC biomarkers to demonstrate utility in several multicenter phase II/III clinical trials through a direct quantitative RT-PCR approach. Here, we employed the microfluidics-based ClearCell® FX system (Clearbridge BioMedics) system, non-antibody based approach to develop a dual molecular assay to characterize CTCs for our previously established melanoma mRNA biomarkers (MART1, TRP2, MAGEA3, PAX, and GALNCT) and the clinically-actionable BRAF V600E mutation (mt). The assay was optimized for CTC isolation from whole blood using three BRAF V600E (+) melanoma cell lines and demonstrated sensitivity for 25 melanoma cells in 10 ml (107 cells) of blood. Immunofluorescence (IF) staining of the melanoma associated antigens verified CTCs were of melanoma origin post-isolation in stage IV patients. Next, the assay was subjected to 100 blood samples collected from 56 melanoma stage III/IV patients. We identified CTCs in 94% of pre-operative (pre-op) patients (n=15/16) as determined by detection of ≥2 mRNA markers. BRAF V600E mt was detected in 100% of pre-op patient (n=5/5) CTCs with BRAF V600E (+) tumors using a sensitive droplet digital PCR (ddPCR) assay. Bloods from patients undergoing active mono/combination immunotherapy (ipilimumab, nivolumab, pembrolizumab) were also evaluated by our CTC biomarker panel. Serial bleeds (2-5 bleeds) from 22 patients were profiled and correlated with clinicopathological factors. To facilitate further biomarker identification predictive of therapy response, CTCs isolated from stage IV patients undergoing immunotherapy were cultured to develop spheres and eventually primary cell lines, and further analyzed by deep RNA sequencing. This study developed a unique platform to analyze patient CTCs for melanoma biomarkers, and provides a potential platform to identify novel CTC-derived melanoma biomarkers indicative of multimodality treatment efficacy. Citation Format: Selena Lin, Stella Lam, Shuichi Ohe, Kevin Tran, Irene Ramos, Ali Asgar Bhagat, Chwee Teck Lim, Steven J. O'Day, Leland Foshag, Dave Hoon. Monitoring of multimodality immune checkpoint inhibitor treatment efficacy in metastatic melanoma patients through molecular analysis of circulating tumor cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3788. doi:10.1158/1538-7445.AM2017-3788

Cohort study of the incidence of spontaneous preterm birth and septic abortion referred by pathological examination in Gifu prefecture in Japan

AimsTo clarify the incidence of spontaneous preterm birth (PB) and septic abortion (sab) in Gifu prefecture in Japan. Study designThis prospective, population-based cohort study was approved by our hospital's Institutional Review Board. All 36 hospitals (100%) in Gifu prefecture offering obstetrical services participated in the study. Patient enrollment criteria were: sab and PB from 22 to <37weeks gestation (WG), excluding for maternal and fetal indications. Pathological examinations before 36 WG and associated factors for both PB and chorioamnionitis (CAM) stage 3 were analyzed by multiple logistic regression analysis judging from minimum daily clinical information in Gifu prefecture. ResultsThe sab rate per all deliveries was 29/16871 (0.17%) at 16.9±2.9 WG. The total spontaneous PB rate was 615/16871 (3.65%) at 34.5±2.7 WG, with birth weight (BW) 2267±557g. There were 26 (0.15%) PBs from 22+0 to 27+6 WG (weeks+days) at 25.2±1.5 WG, with BW 745±199g. Among 214 pathological examinations, CAM was detected in 80% (sab) and 63% (PB<36 WG), respectively. Funisitis were 14% and 17% respectively. Episodes of serial genital bleeding and/or hematoma at <12 WG were more frequent in sab and earlier PB (<28 WG) associated with CAM stage 3 (odds 1.9, P<0.0001). Combined factors such as bleeding and past history of CAM correlated with earlier delivery at 23.4±5.9 WG (P=0.0032). ConclusionIn Gifu prefecture, the incidence of sab was 0.17% (per all deliveries) and 3.65% of spontaneous PB. The combined risk of past CAM history and bleeding was associated with earlier delivery among total preterm birth.

Evaluation and Optimization of Blood Micro-Sampling Methods: Serial Sampling in a Cross-Over Design from an Individual Mouse.

Current practices applied to mouse pharmacokinetic (PK) studies often use large numbers of animals with sporadic or composite sampling that inadequately describe PK profiles. The purpose of this work was to evaluate and optimize blood microsampling techniques coupled with dried blood spot (DBS) and LC-MS/MS analysis to generate reliable PK data in mice. In addition, the feasibility of cross-over designs was assessed and recommendations are presented. The work describes a comprehensive evaluation of five blood microsampling techniques (tail clip, tail vein with needle hub, submandibular, retro-orbital, and saphenous bleeding) in CD-1 mice. The feasibility of blood sampling was evaluated based on animal observations, ease of bleeding, and ability to collect serial samples. Methotrexate, gemfibrozil and glipizide were used as test compounds and were dosed either orally or intravenously, followed by DBS collection and LC-MS/MS analysis to compare PK with various bleeding methods. Submandibular and retro-orbital methods that required non-serial blood collections did not allow for inter-animal variability assessments and resulted in poorly described absorption and distribution kinetics. The submandibular and tail vein with needle-hub methods were the least favorable from a technical feasibility perspective. Serial bleeding was possible with cannulated animals or saphenous bleeding in non-cannulated animals. Of the methods that allowed serial sampling, the saphenous method when executed as described in this report, was most practical, reproducible and provided for assessment of inter-animal variability. It enabled the collection of complete exposure profiles from a single mouse and the conduct of an intravenous/oral cross-over study design. This methodology can be used routinely, it promotes the 3Rs principles by achieving reductions in the number of animals used, decreased restraints and animal stress, and improved the quality of data obtained in mouse PK studies.This article is open to POST-PUBLICATION REVIEW. Registered readers (see "For Readers") may comment by clicking on ABSTRACT on the issue's contents page.

HB9 Represses Hematopoietic Stem Cell Proliferation and Induces Senescence

Introduction: HB9 is a transcription factor encoded by homeobox gene B9 (HLXB9). It is physiologically expressed during early embryonic development as well as in pancreatic beta- and motor neuronal cell development. Ectopic HB9 expression is found in infant acute myeloid leukemia with translocation t(7;12), accounting for up to one third of infant AML cases with a poor 3-year EFS of 0% irrespective of the treatment approach. We previously showed that HB9 regulates cell-cell interaction/adhesion (Wildenhain et al. Leukemia, 2010) in hematopoietic cells and influences the prostaglandin signalling pathway (Wildenhain & Ingenhag et al. JBC, 2012). In this study we focussed on the oncogenic potential of HB9 in hematopoiesis.Methods: To investigate the oncogenic influence of HB9 expression on hematopoiesis, we developed an in vivo murine transplantation model. HB9-transduced lineage negative (Lin-) murine HSCs were transplanted into lethally irradiated wild-type mice and we monitored hematopoietic reconstitution and leukemia emergence by serial retroorbital bleedings for up to one year. Final analysis included comprehensive flow cytometric analysis of all hematopoietic compartments, with respect to dissemination of blast cells and cellular distribution. In vitro studies included proliferation as well as cell cycle analysis. Senescent phenotype was characterized by senescence-associated beta-galactosidase staining and cellular morphology. Knockdown of p53 was obtained via transfection of siRNA.Results: Transplantation of HB9- or mock-transduced murine Lin- cells into lethally irradiated wild-type recipient mice (n=10) showed >80% donor chimerism and HB9-transduced Lin- cells gave rise to all hematopoietic lineages (B-lineage: CD19+, T-lineage: CD3+, NK-lineage: Nk1.1+, granulocytic lineage: Gr-1+, Monocytic lineage: CD11b+) in the peripheral blood, indicating no lineage-related preference of HB9-expressing HSCs. Reconstitution of peripheral blood cell compartments in HB9 transplanted mice, however, was significantly decreased in all three lineages (CD3+: 9.5-fold, CD19+: 34.7-fold , Gr+: 1.8-fold) compared to the control group with respect to copy number, mRNA and protein expression. We did not observe an accumulation of hematopoietic stem (LT-HSC, ST-HSC, MPP) and precursor cells subsets (CLP, MEP, CMP, GMP) in the bone marrow of mice transplanted with HB9-positive Lin- cells. Finally, mice transplanted with HB9-transduced Lin- cells did not develop leukemia after 12 months follow-up. The decreased reconstitution capacity of HB9 expressing HSCs led us to the assumption that HB9 represses cellular proliferation in vivo.Thus we performed proliferation studies in vitro. Ectopic expression of HB9 in the murine NIH3T3 cell line revealed a complete inhibition of cell proliferation compared to mock control (n=3). The same effect was observed in human HT1080 cell line. Cell cycle analysis revealed a significant decrease of the S-phase (2-fold, p<0.05), stalling the cells in G1 and G2 phase of the cell cycle. In both cell line models HB9-transduced cells developed a senescent phenotype being multinuclear, flattened and enlarged. Staining for senescence-associated β-galactosidase activity was positive in HB9-transduced cells in contrast to complete absence in mock-transduced cells. Immunoblot analysis revealed that the HB9 dependent cell cycle arrest was mediated via p53-induced upregulation of p21. Knockdown experiments using p53-targeting siRNAs confirmed that the p53-signalling is responsible for the growth arrest because p53-knockdown was able to reverse the effect.Conclusion:In our study HB9 represses hematopoietic stem cell proliferation in vivo and induces a senescent phenotype in vitro. Senescence is an evasion mechanism in response to aberrant oncogene expression and induction of senescence is the first evidence for an oncogenic potential of HB9. Future studies elucidating the signal pattern of HB9-induced senescence will shed new light on the pathomechanism and potential therapeutic targets in the treatment of translocation t(7;12) positive AML. DisclosuresNo relevant conflicts of interest to declare.

Overcoming the Immunosuppressive Tumor Microenvironment of Hodgkin Lymphoma Using Chimeric Antigen Receptor T Cells

Patients with Hodgkin lymphoma (HL) who relapse or are refractory to first-line therapy have a poor prognosis and some of these patients require innovative therapeutic approaches. Our group and others have demonstrated the clinical efficacy of anti-CD19 chimeric antigen receptor T cells (CART19) for B cell malignancies. However, B cell antigens such as CD19 are typically absent from Hodgkin Reed-Sternberg (HRS) cells. Targeting the CD30 antigen using CART cells has met with limited clinical success to date. In HL the neoplastic HRS cells often represent ~1% of the tumor mass and the remainder is comprised of a highly immunosuppressive tumor microenvironment (TME). Within the TME, tumor-associated macrophages (TAM) have a key role in promoting tumor growth and inhibiting the anti-tumor immune response, and the presence of TAM in HL is associated with a poor prognosis. HL therefore represents an excellent model of tumor-induced immunosuppression that can potentially represent a barrier to successful CART therapy. The objective of this study was therefore to develop an anti-HL CAR T cell immunotherapy that would also overcome the immunosuppression of the HL microenvironment.We sought to define a tumor antigen that was co-expressed on HRS and on TAM. Using immunohistochemistry and flow cytometry we confirmed previous reports that CD123 (the IL-3Rα chain) is expressed on HRS in 50-60% of HL patients and in 100% HL cell lines (HDLM-2, KMH2, SUPHD1, and L428). Importantly, CD123 was also seen on several HL-associated immune cells of the TME, including TAM. Exposure of macrophages to HDLM-2 cells polarized them to an M2-like phenotype (Figure 1 A) with expression of CD123. Luminex analysis of HDLM-2/TAM co-culture supernatants revealed high levels of IL-13, and blockade of IL-13 partially reversed the immunosuppressive phenotype. We then developed a model of TAM-mediated CAR T cell inhibition, using immunosuppressive M2-like macrophages derived by exposure to IL-4 or to HDLM-2 cells. These macrophages could suppress “third party” CART19 proliferation in response to B-acute lymphoblastic leukemia. We then used anti-CD123 CART cells (CART123) to target TAM. Importantly, CART123 actively recognized and killed TAM, thereby resisting TAM-mediated inhibition (as opposed to control CART19 cells) (Figure 1 B).We therefore sought to investigate the utility of anti-CD123 CART for the treatment of HL (initial results for this part presented at ASH 2014). In vitro, CART123 specifically degranulate, proliferate, produce cytokines and kill HL cells. A clinically-relevant xenograft model of HL was generated using luciferase+ HDLM-2 cells in NSG mice. In this model mice showed progressive increase in tumor burden over approximately 6 weeks, reproducing the indolent nature of the human disease. At day ~43 mice were randomized to receive 1.5 million CART123 cells or control T cells. CART123 induced complete and durable eradication of disseminated tumor within 14 days, leading to 100% relapse-free and 100% overall survival at 8 months while mice treated with control T cells had a median survival of 128 days (Figure 2 A). Tumor elimination was associated with extensive CAR T cell expansion as detected by flow cytometry in serial peripheral blood bleedings. In order to prove the formation of an immunological memory, at day 250 CART123 treated mice were re-challenged with the HDLM-2 cell line. The tumor was promptly rejected, associated with a re-expansion of CART123 cells in the peripheral blood (~10 months after T cell injection) leading to an overall survival advantage as compared to controls (Figure 2 B).In summary, we showed that human CD123-redirected T cells display potent therapeutic activity against disseminated HL. In Hodgkin Lymphoma (HL) CD123 is expressed on malignant Reed-Sternberg cells as well as in M2-polarized tumor associated macrophages (TAMs): importantly CART123 can target simultaneously both the HRS and the TAM without being resistant to immunosuppression. We have previously demonstrated that CART123 can lead to myelosuppression in preclinical models, suggesting that our findings could be translated to treat patients with refractory HL in the context of stem cell transplantation using “short-acting” RNA-CAR123 T cells or depletable lentivirally-transduced CART123. [Display omitted] [Display omitted] DisclosuresRuella:Novartis: Patents & Royalties. Kenderian:Novartis: Patents & Royalties, Research Funding. Wasik:Gilead Sciences: Equity Ownership; Seattle Genetics: Honoraria; Novartis: Research Funding; University of Pennsylvania: Patents & Royalties: NPM-ALK as an omncogene; University of Pennsylvania: Patents & Royalties: CAR T-cells; Gilead Sciences: Research Funding; Pharmacyclics: Research Funding. June:Novartis: Honoraria, Patents & Royalties, Research Funding; Johnson & Johnson: Honoraria; Pfizer: Honoraria; Celldex: Consultancy, Equity Ownership; Immune Design: Consultancy, Equity Ownership; Tmunity Therapeutics: Equity Ownership; Novartis: Honoraria, Patents & Royalties, Research Funding. Gill:Novartis: Patents & Royalties, Research Funding.