Transcriptomic Systems Biology Analyses with Buparlisib (BKM120) Pan PI3K Inhibition in Canine B Cell Lymphoma (BCL): Leveraging Comparative Oncology for Cancer Therapeutics and Biomarker Discovery

Abstract Background: The canine is a highly appealing model for cancer research and discovery in part due to comparable histopathological features with humans, a fully intact immune system, similar clinicopathologic features, a more comparable body size and pharmacokinetic properties than the mouse, varied breed-specific incidence rates as well as a shared environment with humans. We and others have shown prominent transcriptomic overlap of human and canine NHL (cNHL) (McDonald T et al. Onctogarget, 2018). PI3K/Akt signaling plays an important role in lymphomagenesis, which is also a promising therapeutic target. However, identification of predictive genetic aberrations of therapeutic efficacy remains elusive. We evaluated the clinical activity of the pan-PI3K inhibitor, buparlisib, in a pilot clinical study in cNHL. Methods :We enrolled and treated 10 dogs with buparlisibwho were diagnosed with BCL in an IRB and IACUC approved clinical study. Cases included 2 treatment naïve and 8 dogs with relapsed disease that had relapsed s/p CHOP (6), L' asparaginase (1) and VELCAP (1) treatment. Pet owners were consented and the study subjects received buparlisib9mg/kg orally for 28 consecutive days. Analysis for tumor response were evaluated on weekly basis through direct tumor measurement or use of x-rays. Post-therapy fine needle aspirates (FNA) were collected on Days 0, 7 and 21 to examine predictors of response to BKM120. RNA from fine need aspirate cells were isolated and the transcriptomic changes were evaluated using Canine Genome 2.0 Affymetrix Array, followed by unbiased systems biology assessment for biological pathways using Gene Set Enrichment Analysis (GSEA) and Ingenuity Pathway Analysis (IPA). We performed unbiased assessment to determine pertinent biological pathways associated with treatment response. The overall impact was used to determine the global effect on tumor progression and cancer risk based on the specific regulation of each gene. A Carcinogenic Risk Score (CRS) was calculated based on these values to determine if there is a promoted risk for cancer (positive value) or inhibitory risk for cancer (negative value) by summing the log2 fold-change values of key genes and subtracting this from the sum of log2 fold-change values of the tumor suppressors when comparing pre-treated to BKM120 treated dogs. Results: Following four weeks of BKM120 treatment, the overall response rate was 30% with 1 complete response lasting 42 days; 2 partial responses lasting 55 and 72 days; 3 stable disease; and 4 progressive disease. Mild treatment related toxicities such elevated blood glucose, thrombocytopenia and anemia, fever, nausea and lethargic symptoms, with no treatment related toxicities in 2 cases were noted. Principal Component Analysis (PCA) and hierrachical clustering analysis of differentially expressed genes show that differentially expressed genes to cluster together in all dogs during post 2 week, indicating a consistent biological activity by BKM120 in all dogs regardless of breed, prior treatment or disease status. Pathway network analysis based on differentially expressed genes predicted activation of upstream regulators associated with tumor suppression including SOX1, SOX3 and GMNN (Week 1) and CEBPA (Week 2). Analysis of "key genes" involved in multiple biological processes appeared to be associated with response of PI3K inhibitortreatment. This included down regulation of CREBBP with a Cancer Risk Score (CRS) of -0.97 and downregulation of VIM, CDH3, WNT3, WNT5B and FGFR2 with a CRS of -2.98 (Fig 1). Conclusion: Results from our pilot study in cNHL showed encouraging clinical responses with a pan-PI3K inhibitor in 3 of 10 dogs. Furthermore, our unbiased characterization of biological pathways revealed that the observed GEP changes associated with tumor suppression and they reduced the risk for cancer progression. Overall, the canine model appears to be particularly attractive model that may be leveraged for the study of clinical and biological responses to novel therapeutic oncologic agents. Disclosures Evens: Bayer: Consultancy; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees; Abbvie: Consultancy; Novartis: Consultancy; Acerta: Consultancy; Seattle Genetics, Inc.: Membership on an entity's Board of Directors or advisory committees, Research Funding; Pharmacyclics International DMC: Membership on an entity's Board of Directors or advisory committees; Tesaro: Research Funding; Janssen: Consultancy; Affimed: Consultancy.

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Functional Effects of Low-Dose IL-2 in Patients with Chronic Graft Versus Host Disease

Blood ◽

10.1182/blood.v128.22.667.667 ◽

2016 ◽

Vol 128 (22) ◽

pp. 667-667

Author(s):

Jennifer Whangbo ◽

Bryn Falahee ◽

John Koreth ◽

Haesook T. Kim ◽

Sarah Nikiforow ◽

...

Keyword(s):

T Cells ◽

Clinical Response ◽

Board Of Directors ◽

Differentially Expressed Genes ◽

Research Funding ◽

Low Dose ◽

Differentially Expressed ◽

Advisory Committees ◽

Response Groups ◽

Pre Treatment

Abstract Introduction: CD4+CD25+FoxP3+ regulatory T cells (Treg) have broad suppressive activity and play a central role in the maintenance of immune tolerance and prevention of chronic graft-versus-host disease (cGVHD) after allogeneic hematopoietic stem cell transplantation (HSCT). Interleukin-2 (IL-2) is a key growth factor for Treg and we previously reported that daily administration of low-dose IL-2 in patients with refractory cGVHD induces selective expansion of Treg and NK cells. No expansion of CD4 conventional T cells (Tcon) or CD8 T cells was noted. In a Phase 2 study, 64% of patients had clinical responses. Predictors of clinical response included younger age, early IL-2 initiation and a higher Treg:Tcon ratio at study baseline and at week 1 of IL-2 therapy. However, there were no significant differences in overall Treg expansion or plasma IL-2 levels between responders and non-responders during the initial 12 week treatment period. We therefore asked whether functional or gene expression differences in Treg or effector T cells could distinguish clinical response to IL-2. Methods: In vitro Treg suppression assays were performed using cryopreserved patient samples obtained at study baseline and week 6 of IL-2 therapy. CD4+CD25+CD127- Treg were cultured with CellTrace Violet-labeled CD4+CD25- Tcon in the presence of stimulating antibody-coated beads. Proliferation was measured as CellTrace Violet dilution by flow cytometry after 4 days of incubation. All patient samples were tested against the same healthy donor (HD) Treg and Tcon. Differential gene expression between clinical responders and non-responders was examined by RNA Seq analysis of sorted Treg, Tcon, CD8 and NK cells at study baseline and week 4 of IL-2 therapy. Results: To compare Treg cell function between response groups, we tested the ability of patient Treg to suppress the proliferation of HD Tcon. To determine whether there were qualitative differences in effector T cells between the response groups, we measured suppression of patient Tcon by HD Treg. At study baseline, clinical responders appear to have higher Treg suppressive function although the differences between the response groups were not significant due to small sample size (Figure 1A). Patient Tcon in responders and non-responders were similarly suppressed by HD Treg. At week 6 of IL-2 therapy, Treg from non-responders showed significant improvement in suppressive activity against HD Tcon, whereas there was no significant change in suppressive function of Treg from responders (Figure 1B). Thus, despite the lack of clinical improvement in cGVHD symptoms, non-responder Treg show both numeric increase and functional improvement in response to IL-2 therapy. To identify other cell-intrinsic differences that distinguish clinical response to IL-2, we performed RNA Seq on sorted Treg, Tcon, CD8 and NK cells in 3 responders and 3 non-responders. Differentially expressed genes were identified using the DEseq package. There were very few differentially expressed genes between response groups within the pre-treatment Treg, CD8 and NK cell samples (Figure 2). In contrast, 93 genes (92 upregulated, 1 downregulated) with greater than 4-fold difference in expression levels were identified in pre-treatment Tcon samples between responders and non-responders. The top 10 gene ontogeny terms associated with these differentially expressed genes include cell-cell adhesion and ion transport processes. By week 4 of IL-2 therapy, the gene expression profiles of responder and non-responder cells were very similar. There were no differentially expressed genes within Treg, and only 5 within Tcon. Conclusions: In addition to increasing absolute Treg numbers, daily low-dose IL-2 improves Treg suppressive function in patients who do not exhibit clinical cGVHD improvement. Interestingly, RNA Seq results suggest that the determinants of clinical response do not lie within the Treg. Instead, the greatest differences between response groups were detected in pre-treatment CD4 Tcon, implying that lack of clinical response reflects resistance of CD4 effector cells to suppression mediated by Treg in vivo. Further analysis of differentially expressed Tcon genes may help elucidate the mechanisms of resistance to IL-2 therapy in patients with cGVHD. Disclosures Koreth: LLS: Research Funding; prometheus labs inc: Research Funding; kadmon corp: Membership on an entity's Board of Directors or advisory committees; amgen inc: Consultancy; millennium pharmaceuticals: Research Funding; takeda pharmaceuticals: Membership on an entity's Board of Directors or advisory committees. Armand:Otsuka: Research Funding; Roche: Research Funding; BMS: Consultancy, Research Funding; Infinity: Consultancy; Merck & Co., Inc.: Consultancy, Research Funding; Sequenta: Research Funding; Sigma Tau: Research Funding; Tensha: Research Funding. Soiffer:Kiadis: Membership on an entity's Board of Directors or advisory committees; Juno: Membership on an entity's Board of Directors or advisory committees; Jazz Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees. Ritz:Kiadis: Membership on an entity's Board of Directors or advisory committees.

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Integrated Transcriptomic and Proteomic Analyses of Inflammasome in Myelodysplastic Syndromes and Chronic Myelomonocytic Leukemia

Blood ◽

10.1182/blood-2019-128094 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 2991-2991

Author(s):

María Zurdo ◽

Ana M Hurtado López ◽

Tzu Hua Chen-Liang ◽

Helios Martínez-Banaclocha ◽

Laura Palomo ◽

...

Keyword(s):

Board Of Directors ◽

Differentially Expressed Genes ◽

Myelodysplastic Syndromes ◽

Peripheral Blood ◽

Research Funding ◽

Chronic Myelomonocytic Leukemia ◽

Differentially Expressed ◽

Advisory Committees ◽

Myelomonocytic Leukemia

Background and aim: Inflammasome and pyroptosis overactivation have recently been associated as fundamental mechanisms in the ineffective hematopoiesis of myelodysplastic syndromes (MDS). Chronic myelomonocytic leukemia (CMML) shares histological and clinical characteristics with MDS but, within clinical differences, It stands out a high association with inflammatory/autoimmune diseases in which a disproportionate activation of inflamasome has been implicated. Our hypothesis is that CMML cases show a higher inflammasome activation with respect to the MDS subset, a relevant difference both in terms of potential therapeutic targets and pathogenic clues. The main objective is to confirm, describe and quantify these differences using high-performance and multi-gene/protein methods. Methods: We performed enhanced RNA-seq in bone marrow mononucleated cells of 27 CMML at diagnosis, 10 MDS and 9 controls (103 million average readings). We selected 116 genes related to the inflammasome and reviewed the differential expression between cases and controls. We evaluated by multiplex immunoassay the profile of 28 cytokines in peripheral blood in 35 CMML patients, 37 MDS and 8 controls. Subsequently, we studied whether these differentially expressed genes / cytokines showed differences in CMML depending on the mutational state of TET2, SRSF2 and ASXL1. Finally, we compared in vitro the degree of activation of inflamasome in the monocytoid component of 8 CMML patients versus 7 controls. Results: In the transcriptomic analysis of the inflamasome genes in patients with CMML, we found 30 of 116 differentially expressed genes compared with healthy controls. Of those 30 genes, 26 showed a pro-inflammatory function and, of them, 18 were up-regulated. Of the 4 differentially expressed genes with an anti-inflammatory function, 3 were significantly under-expressed in CMML patients. We highlight, due to the quantitative difference, the overexpression of two genes coding for monocyte chemotactic proteins, CCL7 and CCL2 (FC = 269.21, p = 0.032; FC = 11.79, p = 0.03) That pro-inflammatory transcriptional profile was not so evident in the cases of MDS: of the 29 differentially expressed genes with pro-inflammatory function, 18 were down-regulated. Subsequently, we designed a customized panel for proteomic analysis including 9 of the 30 differentially expressed genes in CMML. We found that, in a relevant percentage of cases, also proinflammatory cytokines derived from these differentially expressed genes were elevated (62.5%) in peripheral blood of patients, compared to healthy donors; pointing towards the key role of gene transcription in the definition of the pro-inflammatory sense of the proteomic dimension of inflammasome in CMML. Next, we found that those patients with CMML and somatic mutations of TET2 had a higher expression of CCL7 and CCL2 compared to patients with CMML wild type, with a tendency to significance in the first case and significant in the second (FC 11.9, p = 0.15; FC 7.8, p = 0.03). Finally, we conducted in vitro stimulation studies at diagnosis in patients with CMML confirming that the canonical activation of the NLRP3 inflammasome (increased production of IL-1β) is significantly enhanced with respect to control individuals. Conclusion: We describe for the first time, a hyperactivation in CMML, compared with MDS, of the components of the inflammasome. Hyperactivation associated in CMML to a gene transcriptional mechanism and related, in the case of the two most over-expressed genes, CCL2 and CCL7, to the presence of mutations in TET2. Our findings point to new therapeutic targets whose modulation could restore inefficient hemopoiesis and potential diagnostic and prognostic biomarkers in CMML. Disclosures Díez-Campelo: Celgene Corporation: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding. Jerez:Novartis: Honoraria; Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees.

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HIF-Mediated and Non-HIF-Mediated Differential Gene Expressions in Sickle Cell Reticulocyte and Their Impact on Clinical Manifestations

Blood ◽

10.1182/blood-2021-153079 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 950-950

Author(s):

Xu Zhang ◽

Jihyun Song ◽

Binal N. Shah ◽

Jin Han ◽

Taif Hassan ◽

...

Keyword(s):

Gene Expression ◽

Differentially Expressed Genes ◽

Research Funding ◽

Clinical Manifestations ◽

Hemoglobin Concentration ◽

Differentially Expressed ◽

Erythroid Progenitors ◽

Advisory Committees ◽

Deconvolution Analysis ◽

Stress Erythropoiesis

Abstract Reticulocytosis in sickle cell disease (SCD) is driven by tissue hypoxia from hemolytic anemia and vascular occlusion. Gene expression changes caused by hypoxia and other factors during reticulocytosis may impact SCD outcomes. We detected 1226 differentially expressed genes in SCD reticulocyte transcriptome compared to normal Black controls. To assess the role of hypoxia-mediating HIFs from other regulation of changes of the SCD reticulocyte transcriptome, we compared differential expression in SCD to that in Chuvash erythrocytosis (CE), a disorder characterized by constitutive upregulation of HIFs in normoxia. Of the SCD differentially expressed genes, 28% were shared between CE and SCD and thus classified as HIF-mediated. The HIF-mediated changes were generally in genes promoting erythroid maturation. We found that genes encoding the response to endoplasmic reticulum stress generally lacked HIF mediation. We then investigated the clinical correlation of erythroid gene expression for the 1226 differentially expressed genes detected in SCD reticulocytes, using clinical measures and gene expression data previously profiled in peripheral blood mononuclear cells (PBMCs) of 157 SCD patients at the University of Illinois at Chicago (UIC). Normal PBMCs contain only a small number of erythroid progenitors, but in SCD or CE PBMCs the erythroid transcriptome is enriched due to elevated circulating erythroid progenitors from heightened erythropoiesis (PMID: 32399971). We applied deconvolution analysis to assess the clinical correlation of erythroid gene expression, using a 16-gene expression signature of erythroid progenitors previously identified in SCD PBMCs. Deconvolution analysis uses the proportion of cell/tissue or specific marker genes (here the erythroid specific 16-gene signature) to dissect gene expression variation in biological samples with cell/tissue type heterogeneity. We correlated, in the 157 UIC patients, erythroid gene expression with i) degree of anemia as indicated by hemoglobin concentration, ii) vaso-occlusive severe pain episodes per year, and iii) degree of hemolysis measured by a hemolysis index. The analysis identified 231 genes associated with at least one of the complications. Increased expression of 40 erythroid specific genes, including 15 HIF-mediated genes, was associated with all three complications. These 40 genes are all upregulated in SCD reticulocytes and correlated with low hemoglobin concentration, frequent severe pain episodes, and high hemolysis index, suggesting that these manifestations may share a relationship to stress erythropoiesis-driven transcriptional activity. Expression quantitative trait loci (eQTL) contain genetic polymorphisms that associate with gene expression level, which can be viewed as a natural experiment to investigate the causal relations between gene expression change and phenotypic outcomes. To assess the causal effect of erythroid gene expression, we tested association between erythroid eQTL and the clinical manifestations in 906 SCD patients from the Walk-PHaSST and PUSH cohorts. We first mapped erythroid eQTL in the 157 UIC patients, who were previously genotyped by array, applying deconvolution algorithm on the same PBMC data for the 1226 differential genes in SCD reticulocytes, and detected 54 distinct eQTL for 30 genes at 5% false discovery rate. After adjusting for multiple comparisons, we found that the C allele of rs16911905, located in the β-globin cluster and associated with increased erythroid expression of HBD (encodes δ-globin of hemoglobin A 2), significantly correlated with lower hemoglobin concentration (β=-0.064, 95% CI -0.092 - -0.036, P=6.7×10 -6). The C allele was also associated with higher hemolytic rate (P=0.031), less frequent pain episodes (P=0.045), and increased erythroid expression of HBB here encoding sickle β-globin (P=5.1x10 -5). The association of the C allele with lower hemoglobin concentration was then validated in 242 patients from the UIC cohort (β=-0.071, 95% CI -0.13 - -0.011, P=0.023), as was the trend of association with higher hemolytic rate (P=0.0031) and less pain episodes (P=0.034). Our findings reveal HIF- and non-HIF-mediated genes in SCD stress erythropoiesis, and identify novel clinical associations for a HBD eQTL. Our study highlights the correlation of altered erythroid gene expression with SCD hemolytic and vaso-occlusive manifestations. Disclosures Saraf: Global Blood Therapeutics: Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding; Pfizer: Research Funding. Gordeuk: Modus Therapeutics: Consultancy; Novartis: Research Funding; Incyte: Research Funding; Emmaus: Consultancy, Research Funding; Global Blood Therapeutics: Consultancy, Research Funding; CSL Behring: Consultancy.

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Personalized Transcriptomic Analyses Identify Unique Signatures That Correlate with Genomic Subtypes in Acute Myeloid Leukemia (AML) Using Explainable Artificial Intelligence

Blood ◽

10.1182/blood-2020-139522 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 33-34 ◽

Cited By ~ 1

Author(s):

Yazan Rouphail ◽

Nathan Radakovich ◽

Jacob Shreve ◽

Sudipto Mukherjee ◽

Babal K. Jha ◽

...

Keyword(s):

Machine Learning ◽

Board Of Directors ◽

Research Funding ◽

State Of The Art ◽

Data Monitoring Committee ◽

Biological Pathways ◽

Machine Learning Algorithms ◽

Advisory Committees ◽

Altered Expression ◽

Individual Level

Background Multi-omic analysis can identify unique signatures that correlate with cancer subtypes. While clinically meaningful molecular subtypes of AML have been defined based on the status of single genes such as NPM1 and FLT3, such categories remain heterogeneous and further work is needed to characterize their genetic and transcriptomic diversity on a truly individualized basis. Further, patients (pts) with NPM1+/FLT3-ITD- AML have a better overall survival compared to patients with NPM1-/FLT3-ITD+, suggesting that these pts could have different transcriptomic signature that impact phenotype, pathophysiology, and outcomes. Many current transcriptome analytic techniques use clustering analysis to aggregate samples and look at relationships on a cohort-wide basis to build transcriptomic signatures that correlate with phenotype or outcome. Such approaches can undermine the heterogeneity of the gene expression in pts with the same signatures. In this study, we took advantage of state of the art machine learning algorithms to identify unique transcriptomic signatures that correlate with AML genomic phenotype. Methods Genomic (whole exome sequencing and targeted deep sequencing) and transcriptomic data from 451 AML pts included in the Beat AML study (publicly available data) were used to build transcriptomic signatures that are specific for AML patients with NPM1+/FLT3-ITD+ compared to NPM1+/FLT3-ITD, and NPM1-/FLT3-ITD-. We chose these AML phenotypes as they have been described extensively and they correlate with clinical outcomes. Results A total of 242 patients (54%) had NPM1-/FLT3-, 35 (8%) were NPM1+/FLT3-, and 47 (10%) were NPM1+/FLT3+. Our algorithm identified 20 genes that are highly specific for NPM1/FLT3ITD phenotype: HOXB-AS3, SCRN1, LMX1B, PCBD1, DNAJC15, HOXA3, NPTXq, RP11-1055B8, ABDH128, HOXB8, SOCS2, HOXB3, HOXB9, MIR503HG, FAM221B, NRP1, NDUFAF3, MEG3, CCDC136, and HIST1H2BC. Interestingly, several of those genes were overexpressed or underexpressed in specific phenotypes. For example, SCRN1, LMX1B, RP11-1055B8, ABDH128, HOXB8, MIR503HG, NRP1 are only overexpressed or underexpressed in patients with NPM1-/FLT3-, while PCBD1, NDUFAF3, FAM221B are overexpressed or underexpressed in pts with NPM1+/FLT3+. These genes affect several important pathways that regulate cell differentiation, proliferation, mitochondrial oxidative phosphorylation, histone modification and lipid metabolism. All these genes had previously been reported as having altered expression in genomic studies of AML, confirming our approach's ability to identify biologically meaningful relationships. Further, our algorithm can provide a personalized explanation of overexpressed and underexpressed genes specific for a given patient, thus identifying targetable pathways for each pt. Figure 1 below shows three pts with the same genotype (NPM1+/FLT3-ITD+) but demonstrate different transcriptomic patterns of overexpression or underexpression that affect different biological pathways. Conclusions We describe the use of a state of the art explainable machine learning approach to define transcriptomic signatures that are specific for individual pts. In addition to correctly distinguishing AML subtype based on specific transcriptomic signatures, our model was able to accurately identify upregulated and downregulated genes that affecte several important biological pathways in AML and can summarize these pathways at an individual level. Such an approach can be used to provide personalized treatment options that can target the activated pathways at an individual level. Disclosures Mukherjee: Partnership for Health Analytic Research, LLC (PHAR, LLC): Honoraria; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; EUSA Pharma: Consultancy; Celgene/Acceleron: Membership on an entity's Board of Directors or advisory committees; Bristol Myers Squib: Honoraria; Aplastic Anemia and MDS International Foundation: Honoraria; Celgene: Consultancy, Honoraria, Research Funding. Maciejewski:Alexion, BMS: Speakers Bureau; Novartis, Roche: Consultancy, Honoraria. Sekeres:BMS: Consultancy; Takeda/Millenium: Consultancy; Pfizer: Consultancy. Nazha:Jazz: Research Funding; Incyte: Speakers Bureau; Novartis: Speakers Bureau; MEI: Other: Data monitoring Committee.

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Single-Cell Pathway Enrichment and Regulatory Profiling of Multiple Myeloma across Disease Stages

Blood ◽

10.1182/blood-2019-131361 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 364-364

Author(s):

Tianjiao Wang ◽

Hua Sun ◽

Daniel Cui Zhou ◽

Ruiyang Liu ◽

Lijun Yao ◽

...

Keyword(s):

Cell Cycle ◽

Multiple Myeloma ◽

Board Of Directors ◽

Research Funding ◽

Plasma Cells ◽

Cell Types ◽

Differentially Expressed ◽

P Value ◽

Advisory Committees ◽

Pathway Enrichment

Multiple myeloma (MM) is a hematological malignancy, defined by aberrant monoclonal proliferation of plasma cells in the bone marrow, that to date remains an incurable disease despite advances in treatment. Key genetic and epigenetic alterations that drive MM pathogenesis have been identified, but a comprehensive profile of affected cellular pathways has yet to be fully characterized. In this study, we integrate whole-genome and whole-exome sequencing data with single-cell RNA sequencing (scRNA-seq) data from 13 patients across multiple treatment stages to 1) assess differential pathway enrichment between tumor subpopulations, 2) trace the clonal evolution of dominant disease mechanisms, and 3) investigate signaling interactions between surrounding cell types. We also analyzed bulk genomic and transcriptomic data from 662 additional Multiple Myeloma Research Foundation (MMRF) tumor samples as a large reference cohort for highly prevalent pathway disturbances. To assess whether tumor subpopulations rely on different oncogenic programs for proliferation, we analyzed the differential expression of key genes (FDR-adjusted p-value <0.05) in 12 canonical oncogenic pathways. Cell cycle, Hippo, RTK/RAS, and NFkB pathways contain the highest numbers of differentially expressed genes, with certain subclusters upregulating as many as 25% of annotated cell cycle genes and over 90% of annotated Hippo genes, whereas p53, Notch, Nrf2, and DNA repair genes tend to be uniformly expressed across subpopulations. Next, we evaluated changes in pathway enrichment across different disease timepoints, with the goal of capturing the reorganization of functional profiles through successive treatment and relapse cycles. We assessed statistical enrichment of pathways containing differentially expressed genes (DEGs) unique to Smoldering Multiple Myeloma (SMM), primary, and relapse stages using the KEGG pathway database (n = 2, 17, and 7 pathways, respectively; FDR-adjusted p-value of enrichment < 0.05). SMM is the only stage where hematopoietic differentiation and the PI3K-Akt pathways are variably expressed between plasma cell subpopulations, suggesting that these pathways may play a role in initiating events. Only primary tumor samples show significant intra-tumor variability of p53 regulation, which is lost in the relapsed tumor and may reflect selection due to treatment. Relative to SMM, primary and relapse samples are enriched for changes in the MAPK, NFkB, RAP1, and cell cycle pathways, indicating potential sources of tumor resistance. We then analyzed pathway enrichment within the tumor microenvironment to enhance our understanding of tumor development in the context of surrounding tissues. We see frequent changes in many immune cell types in TLR signaling as the disease progresses, driven by differential expression of NFkB1A, JUN, and FOS, all of which are key upstream regulators of the AP-1 pathway and responders to the MAPK and PI3K signaling cascades. These microenvironment changes may be complementary to the PI3K and MAPK dysregulation observed in tumor plasma cells. Proteasome and ubiquitin genes, which affect secretion, autophagy, and apoptosis pathways that may be relevant to MM pathogenesis are also frequently differentially expressed in immune cells between disease stages. Finally, we integrate bulk whole-exome and whole-genome sequencing analysis (from both the 13-patient cohort and MMRF) to obtain a more complete understanding of how pathways become dysregulated in MM. Our findings advance the understanding of how MM tumor subpopulations differentially regulate cellular pathways and interact within the tumor microenvironment. Disclosures O'Neal: Wugen: Patents & Royalties: Patent Pending; WashU: Patents & Royalties: Patent Pending. Rettig:WashU: Patents & Royalties: Patent Application 16/401,950. Oh:Incyte: Membership on an entity's Board of Directors or advisory committees; Blueprint Medicines: Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy. Vij:Bristol-Myers Squibb: Honoraria, Research Funding; Celgene: Honoraria, Research Funding; Genentech: Honoraria; Janssen: Honoraria; Karyopharm: Honoraria; Sanofi: Honoraria; Takeda: Honoraria, Research Funding. DiPersio:Amphivena Therapeutics: Consultancy, Research Funding; Magenta Therapeutics: Equity Ownership; Karyopharm Therapeutics: Consultancy; Incyte: Consultancy, Research Funding; RiverVest Venture Partners Arch Oncology: Consultancy, Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy; Bioline Rx: Research Funding, Speakers Bureau; Macrogenics: Research Funding, Speakers Bureau; WUGEN: Equity Ownership, Patents & Royalties, Research Funding; NeoImmune Tech: Research Funding; Cellworks Group, Inc.: Membership on an entity's Board of Directors or advisory committees.

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Transcriptome Profiling of Pediatric Myeloproliferative Neoplasms Demonstrates Dysregulation of Platelet-Relevant Genes and Enrichment of Inflammatory and JAK2 Mediated Complement Pathways

Blood ◽

10.1182/blood-2019-125103 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 4203-4203

Author(s):

Nicole Kucine ◽

Amanda R. Leonti ◽

Aishwarya Krishnan ◽

Rhonda E. Ries ◽

Ross L. Levine ◽

...

Keyword(s):

Bone Marrow ◽

Board Of Directors ◽

Platelet Activation ◽

Research Funding ◽

Mononuclear Cells ◽

Myeloproliferative Neoplasms ◽

Differentially Expressed ◽

Rna Seq ◽

Advisory Committees ◽

Normal Controls

Introduction : Myeloproliferative neoplasms (MPNs) are rare clonal bone marrow disorders in children characterized by high blood counts, predisposition to clotting events, and the potential to transform to myelofibrosis or acute myeloid leukemia (AML). Children with MPNs have lower rates of the known driver mutations (in JAK2, MPL, and CALR) than adult patients, and the underlying pathways and molecular derangements in young patients remain unknown. Given the lack of knowledge about pediatric MPNs, it is critical that we gain a better understanding of the dysregulated pathways in these diseases, which is necessary for improving disease understanding and broadening treatment options in children. Therefore, the objective of this work was to identify differentially expressed genes and pathways between children with MPNs and healthy controls, as well as children with AML, to guide further study. Methods : Mononuclear cells were extracted from peripheral blood of pediatric MPN patients (n=20) and pediatric and young adult AML patients (n=1410), and bone marrow of normal controls (NC, n=68). AML patient samples were being evaluated as part of a Children's Oncology Group planned analysis. To identify an expression profile unique to MPNs, transcriptome data from MPN patients was contrasted against NC and AML patients. All samples were ribodepleted and underwent Illumina RNA-Seq to generate transcriptome expression data. All analyses were performed in R. Differentially expressed genes were identified using the voom function from the limma package (v. 3.38.3), and enriched pathways were identified using the pathfindR package (v. 1.3.1). Unsupervised hierarchical clustering and heatmap generation was performed using the ComplexHeatmap package (v. 1.20.0). Results : MPN patient samples showed a unique expression signature, distinct from both AML patients and normal controls. Unsupervised PCA plot (Figure 1A) and heatmaps (Figure 1B) show that MPN samples cluster together. There were 4,012 differentially expressed (DE) genes in MPNs compared to NC and 6,743 DE genes in MPNs compared to AML patients. There were 2,493 shared genes between the 2 groups (Figure 1C.) Significantly DE genes between MPNs and other groups included multiple platelet-relevant genes including PF4 (CXCL4), PF4V1, P2RY12, and PPBP (CXCL7). Interestingly, PF4V1 was the most DE gene in MPNs compared to AML, and third highest versus NC. Dysregulation of some of these genes has been seen in adult MPNs, as well as thrombosis. Further comparison of transcriptome profiles between children with (n=13) and without (n=7)JAK2 mutations showed upregulation of three genes, CFB, C2, and SERPING1, which are all known complement genes, implicating complement activation in JAK2-mutated MPN patients. Complement activation has previously been reported in adult MPNs. Pathway enrichment analysis shows a number of immune and inflammatory pathways as enriched in MPN patients compared to both AML and NC. There were 179 enriched pathways in MPNs compared to AML and 142 compared to NC, with 134 common pathways (Figure 1D.) The systemic lupus erythematosus pathway was the most heavily enriched pathway in MPNs compared to both AML and NC. Additional pathways with significant enrichment include hematopoietic cell lineage, cytokine-cytokine interactions, DNA replication, and various infection-relevant pathways. The JAK-STAT signaling pathway was also enriched in MPNs compared to both AML and NC, as was the platelet activation pathway. Conclusion: Transcriptome evaluation of childhood MPNs shows enrichment of numerous inflammatory and immune pathways, highlighting that, as in adult MPNs, inflammation is implicated in pediatric MPNs. Furthermore, specific complement genes were upregulated in JAK2-mutant MPN. Upregulation of platelet-specific genes implies potential insights into disease mechanisms and warrants more study. Variations in the cell populations may account for some of the differences seen, however all samples were largely mononuclear cells, making their comparisons reasonable. Further analysis of this early data is needed to better assess inflammatory changes and platelet activation in pediatric MPNs, as are larger sample sizes. Individual cells may have differential expression of various genes, and future experiments with single-cell RNA-seq would be helpful to further elucidate differences. Disclosures Levine: Novartis: Consultancy; Loxo: Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy, Research Funding; Gilead: Consultancy; Roche: Consultancy, Research Funding; Lilly: Honoraria; Amgen: Honoraria; Qiagen: Membership on an entity's Board of Directors or advisory committees; Imago Biosciences: Membership on an entity's Board of Directors or advisory committees; C4 Therapeutics: Membership on an entity's Board of Directors or advisory committees; Prelude Therapeutics: Research Funding; Isoplexis: Membership on an entity's Board of Directors or advisory committees.

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Short-Term Risk for Progression in Patients with Smoldering Multiple Myeloma: The Impact of CD56 Expression

Blood ◽

10.1182/blood-2020-139214 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 11-11

Author(s):

Laura Notarfranchi ◽

Rosanna Vescovini ◽

Roberta Segreto ◽

Sabrina Bonomini ◽

Paola Storti ◽

...

Keyword(s):

Multiple Myeloma ◽

Clinical Study ◽

Board Of Directors ◽

Research Funding ◽

Prognostic Score ◽

Short Term ◽

Advisory Committees ◽

Risk Of Progression ◽

Cd56 Expression

The identification of risk factors for progression is critical in the clinical management and appropriate follow up of patients with Smoldering Multiple Myeloma (SMM). The early identification of patients with possible short-term progression to Multiple Myeloma (MM) could lead to anticipate the treatment. Several prognostic score identify in SMM patients the main risk factors for progression to MM. The two most used risk stratification models in SMM are the Mayo Clinic model, based on the tumor burden and the free light chains ratio, and the Spanish PETHEMA group model based on the immunophenotyped to identify abnormal plasma cells (PCs) and the reduction of the unevolved immunoglobulins. However, significant discrepancies between these two clinical models currently used in clinical practice has been recently underlined. For this reason, new parameters to identify possible new parameters for progression in SMM need to be defined. The aim of this study was to validate the main prognostic score and to investigate the possible role of the immunphenotype as risk factor for progression in a monocentric cohort of patients with SMM. We retrospectively evaluated a cohort of SMM patients admitted to a single haematological center (Hematology and BMT Unit, University Hospital of Parma) between 2014 and 2018. We analyzed a total cohort of 80 patients diagnosed with SMM according to the IMWG recently updated diagnostic criteria. All patients analysed underwent to Bone Marrow (BM) examination and imaging evaluation was performed in order to exclude the presence of bone disease and/or focal lesions. Both immunophenotypic and FISH analysis were performed of BMPCs. The median age of the SMM patients analysed was 68 years (range 36-93 years). Median percentage of BMPCs was 15% (range 10-40%) in the entire population. Median serum M-protein was 2 g/dL (range: 0.17-4.5). FLC ratio value was available in 66 patients: in 47 (71%) the ratio was unbalanced, 26 (39%) had a FLC ratio ≤ 0.125 or ≥ 8 and in 6 (9%) it was > 20. The presence of a reduction of one or two uninvolved immunoglobulins occurred in 61% of the entire population. The median follow up time was 27 months (range 0 - 76 months) for whole population. Overall 22 patients of the entire cohort progressed to MM with a median the time to progression (TTP) of 22 months. Firstly, we validated the currently score of progression in our cohort of SMM patients. By univariate analysis we found that percentage of BMPCs, abnormal FLC ratio and presence of immunoparesis were significantly correlated with progression to active MM (p<0.005 for each variable). Any significant correlation was not observed with age, sex, Ig isotype and light chain's type (p=NS). Afterwards, we study and confirm the significance of the risk stratification models. "Pethema" (p=0.0002), "20-2-20" Mayo score (p=0.0005) and also the "Danish score" (p= 0.0173) turned out statistically significant. Then, we investigate the possible role of immunophenotype in the risk of progression. Dividing the population-based on CD56 expression, we found that the median TTP in CD56- SMM patients was 21 months as compared to 34 months in CD 56+ SMM patients (p= 0.08). Moreover CD56- patients progressed without a significant increase of the monoclonal component (p=0.48) as compared to those CD56+ SMM patients (p=0.023). Finally, a relationship between CD56 expression and the hyperdiploidy was wound finding that CD56- SMM patients had a significant lower presence of hyperdiploidy as compared to those with CD56+ BMPCs (p=0.024) In conclusion, our data indicate that in SMM patients the factors, which mostly impact on the short-term risk of progression to active MM, are the entity of the PCs infiltrate, the immunoparesis and abnormal FLC ratio. Therefore, we identified the absence of CD56 expression by BMPCs as a possible factor for a more aggressive disease regardless to the tumoral burden. Disclosures Giuliani: Celgene: Membership on an entity's Board of Directors or advisory committees, Other: Participation in congresses, Research Funding; Janssen Pharmaceutical: Membership on an entity's Board of Directors or advisory committees, Other: Clinical study sponsorship; participation in congresses, Research Funding; Millennium Pharmaceutical: Other: Clinical study sponsorship, Research Funding; GSK: Other: Clinical study sponsorship, Research Funding; Takeda: Membership on an entity's Board of Directors or advisory committees; Bristol-Myers Squibb: Other: Participation in congresses.

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Totality of Scientific Evidence in the Development of ABP 798, a Biosimilar to Rituximab

Blood ◽

10.1182/blood-2020-142723 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 35-36

Author(s):

Dietger Niederwieser ◽

Caroline Hamm ◽

Patrick Cobb ◽

Theingi Thway ◽

Cecily Forsyth ◽

...

Keyword(s):

Sensitivity Analysis ◽

Clinical Study ◽

Board Of Directors ◽

Research Funding ◽

Percent Reduction ◽

Advisory Committees ◽

Comparative Clinical Study ◽

Full Analysis

Background: ABP 798 is a proposed biosimilar to rituximab reference product (RP). Regulatory approval of biosimilars is based on a totality of evidence (TOE) approach which includes similarity in analytical characteristics (structural and functional), in mechanism of action (MoA) -clinical pharmacology, pharmacokinetics (PK), pharmacodynamics (PD), and in comparative clinical studies. Evidence from analytical, functional, clinical PK/PD, and efficacy and safety evaluations indicate that ABP 798 is similar to rituximab RP. Similarity of efficacy and safety in patients with non-Hodgkin lymphoma (NHL) was demonstrated in a randomized, double-blind, comparative clinical study in which the primary analysis was based on data from central, independent, and blinded assessments of disease and the sensitivity analysis was based on data using investigator assessments of disease. Here we present in vitro chemo synergy, preclinical in-vivo tumor efficacy data and show how the results of the sensitivity analysis using investigator assessments of the response rate from the NHL clinical study further add to the TOE supporting similarity of ABP 798 with the RP. Methods: In vitro synergy with chemotherapeutic agents was investigated by assessing the effect of docetaxel, gemcitabine, or doxorubicin with ABP 798 and rituximab RP in SU-DHL-6 cell lines and measured using the ATP-based cell viability endpoint (ATPLite). In vivo efficacy was assessed by evaluating the anti-tumor activity of ABP 798 and rituximab RP in NHL xenograft models in NOD/SCID mice using CD20-expressing RL and Raji cell lines. A sensitivity analysis was conducted to assess the robustness of the results of the primary efficacy endpoint of the proportion of patients with the best overall response of complete response, partial response, or unconfirmed complete response (ORR) in the NHL comparative clinical study using the investigator's assessment of disease on the full analysis set. Waterfall plots of the maximum percent reduction in the sum of the products of the greatest diameters (SPD) for index nodal lesions based on both the central, independent, blinded assessment of disease and the investigator's assessment of disease were used. Results: In vitro study results demonstrated synergistic activity of the combination of docetaxel and ABP 798 or the RP (synergy score = 6.35±0.69 [with ABP 798]; 6.79±0.12 [with RP]). In contrast, combinations with gemcitabine (synergy score = 2.68±0.20 [with ABP 798] and 2.54±0.29 [with RP]) or doxorubicin (synergy score = 1.95±0.56 [with ABP 79]) and 3.23±0.53 [with RP]) were additive. In RL cell lines, ABP 798 inhibited tumor growth similarly to rituximab RP, with no statistical difference between dose groups at 3 and 30 mg/kg (p = 0.951 and p = 0.996, respectively). In Raji cells, ABP 798 and rituximab RP significantly and similarly inhibited tumor growth when compared to an IgG1 isotype control (p < 0.001 for 3 and 30 µg/kg doses). ABP 798, at both dose levels, inhibited tumor growth similarly to rituximab RP by 27-41%, with no statistical difference. In the NHL comparative clinical study, risk difference (RD) of ORR with 1-sided 95% lower and upper confidence limits from the adjusted linear model was 7.7% (-3.2%, 18.6%) using central, blinded, independent assessments of the modified full analysis set based on randomized treatment assignment. The sensitivity analysis using investigator assessments of disease of the full analysis set of all randomized subjects based on the randomized treatment assignment received was RD = 0.5% (-7.8%, 8.8%). Waterfall plots of the maximum percent reduction in the SPD for index nodal lesions further demonstrate similarity of efficacy with central or local assessments of disease. Conclusions: In vitro and in vivo preclinical studies demonstrated chemo synergy and in vivo tumor efficacy. The sensitivity analysis of ORR using local assessments in the NHL comparative clinical study was consistent with results from the primary efficacy analysis; the maximum percent reduction in index nodal lesions was similar for central and local assessments of disease. These results further support that ABP 798 is similar to the RP across a range of efficacy evaluations as well as primary and secondary mechanisms of action and add additional supporting data to the TOE. Disclosures Niederwieser: Novartis: Speakers Bureau; Cellectis: Membership on an entity's Board of Directors or advisory committees; Daiichi: Research Funding; Amgen: Speakers Bureau. Hamm:Amgen: Consultancy. Cobb:Amgen: Consultancy. Thway:Amgen: Current Employment, Current equity holder in publicly-traded company. Forsyth:Amgen: Consultancy; Janssen Pharmaceuticals Australia: Honoraria, Membership on an entity's Board of Directors or advisory committees. Tucci:Amgen: Consultancy. Delwail:Amgen: Consultancy. Hajek:Janssen: Consultancy, Honoraria, Research Funding; Amgen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Consultancy, Honoraria, Research Funding; BMS: Consultancy, Honoraria, Research Funding; Novartis: Consultancy, Research Funding; Takeda: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Abbvie: Consultancy, Honoraria; PharmaMar: Consultancy, Honoraria; Oncopeptides: Consultancy. Hanes:Amgen: Current Employment, Current equity holder in publicly-traded company.

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Gas1 and Kif27 Genes Are Strongly up-Regulated Biomarkers of Hedgehog Inhibition (PF-04449913) on Leukemia Stem Cells in Phase I Acute Myeloid Leukemia and Chronic Myeloid Leukemia Treated Patients

Blood ◽

10.1182/blood.v118.21.1535.1535 ◽

2011 ◽

Vol 118 (21) ◽

pp. 1535-1535

Author(s):

Viviana Guadagnuolo ◽

Cristina Papayannidis ◽

Ilaria Iacobucci ◽

Sandra Durante ◽

Carolina Terragna ◽

...

Keyword(s):

Stem Cell ◽

Phase I ◽

Board Of Directors ◽

Myeloid Leukemia ◽

Research Funding ◽

Fold Change ◽

Differentially Expressed ◽

Advisory Committees ◽

Before And After ◽

Hh Signaling

Abstract Abstract 1535 Recent studies show that uncontrolled Hedgehog (Hh) pathway activation contributes to leukemia development and growth, and that targeted pathway inhibition is likely to offer an efficient therapeutic opportunity. PF-04449913, a Hh pathway inhibitor, is a new selective and potent inhibitor of leukemia self-renewal and is currently being evaluated in phase I clinical trials. In order to identify new potential clinical biomarkers for the PF-04449913 (Hh inhibitor), we studied leukemia stem cell population (CD34+ subpopulation) collected before and after 28 days treatment in a phase I dose escalation protocol (Clinical Trial Gov. NTC00953758) enrolling selected hematological malignancies. This experimental clinical trial enrolled Myelofibrosis (MF), MDS, blastic phases Chronic Myeloid Leukemia (CML), chronic myelomonocytic leukemia (CMML) and Acute Myeloid Leukemia (AML) patients. We were able to collect and separate highly purified (98%) bone marrow hematopoietic progenitor cells (CD34+ populations) from 5 AML, 1 MF and 2 CML patients by immunomagnetic separation, and analysed them for gene expression profile (GEP) using Affimetrix HG-U133 Plus 2.0 platform. We have observed that 1197 genes were differentially expressed between CD34+ cells collected before and after 28 days of PF-04449913 dose finding oral therapy. Clustering of their expression profiles showed that mostly genes differentially expressed are mainly related to Hh signaling,this providing further evidences that PF-04449913 really therapeutically targets the Hh pathway. Regarding genes involved in Hh signaling pathway, Gas1 and Kif27 were strongly upregulated (fold change 1.0947 and 1.12757 respectively; p-value 0.01 and 0.02 respectively) in CD34+ leukemia stem cells after 28 days exposure to PF-04449913 as compared to baseline, suggesting these two genes have potential as new biomarkers of activity. GAS-1 (growth arrest specific 1 gene) is a Sonic Hedgehog (Shh)-binding protein; it acts to sequester Shh and inhibit the Shh signalling pathway. Kif27 (kinesin family member 27) mainly acts as a negative regulator in the Hh signaling pathway, and inhibits the transcriptional activator activity of Gli1 by inhibiting its nuclear translocation. Other genes were differentially expressed after ‘ex- vivo’ treatment with PF-04449913 as compared to baseline: we observed a down regulation of Bcl2 (fold change −1.03004), ABCA2 (fold change −1.08966), LEF1 (fold change −1.28457), Gli1 (fold change −1.0775), Smo (fold change −1.07702), and an upregulation of Gli2 (fold change 1.08191). Conclusions: This data demonstrates thatPF-04449913 specifically targets the Hh Pathway in CD34+ cells, suggesting that Hh inhibition may impair leukemia stem cell maintenance. In addition, we identify several new potential biomarkers (e.g. Gas1 and KIF27). Taken together, these data may be useful for patient selection strategies and subsequent eradication of the leukaemia stem cell. Acknowledgments. Work supported by Pfizer, European LeukemiaNet, FIRB 2008, PRIN 2009, AIRC, AIL, COFIN, University of Bologna and BolognAIL. Disclosures: Soverini: Novartis: Consultancy; ARIAD: Consultancy; Bristol-Myers Squibb: Consultancy. Levin:Pfizer Oncology: Employment; Pfizer Oncology: Equity Ownership. Courtney:Pfizer Oncology: Employment; Pfizer Oncology: Equity Ownership. Baccarani:Pfizer Oncology: Consultancy; Novartis: Consultancy; BMS: Consultancy; Ariad: Consultancy; Novartis: Research Funding; Pfizer Oncology: Honoraria; Novartis: Honoraria; BMS: Honoraria; Ariad: Honoraria; Novartis: Membership on an entity's Board of Directors or advisory committees; BMS: Membership on an entity's Board of Directors or advisory committees; Ariad: Membership on an entity's Board of Directors or advisory committees. Cortes:Novartis: Consultancy; Novartis: Research Funding; BMS: Consultancy, Research Funding; Ariad: Consultancy, Research Funding; Pfizer: Consultancy, Research Funding. Oehler:Pfizer Oncology: Research Funding. McLachlan:Pfizer: Employment. Van Arsdale:Pfizer: Employment. Martinelli:Novartis: Consultancy, Honoraria; BMS: Consultancy, Honoraria.

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Enhanced Global Hemostatic Potentials with a Bispecific Antibody to Factors IXa and X (ACE910) in the Whole Blood By Rotation Thromboelastometry (ROTEM)

Blood ◽

10.1182/blood.v126.23.3503.3503 ◽

2015 ◽

Vol 126 (23) ◽

pp. 3503-3503 ◽

Cited By ~ 4

Author(s):

Koji Yada ◽

Keiji Nogami ◽

Yasuaki Shida ◽

Masahiro Takeyama ◽

Ryu Kasai ◽

...

Keyword(s):

Clinical Study ◽

Board Of Directors ◽

Whole Blood ◽

Research Funding ◽

Bispecific Antibody ◽

Hemophilia A ◽

Advisory Committees ◽

Group A ◽

Group B ◽

Group D

Abstract Background: ACE910, a humanized bispecific antibody to factor (F) IXa and FX mimicking the functions of FVIII, exerts tenase activities without FVIII (a) (Kitazawa et al. Nature Medicine. 2012;18, 1570). In primate hemophilia A (HA) models, the hemostatic enhancing effect of ACE910 has been reported and the clinical study investigating the effect and safety of ACE910 for human HA patients is on-going. However, the hemostatic effect of ACE910 remains unquantified and difficult to be evaluated. Objectives: In this study, we evaluated the viscoelastmetric parameters in the whole blood obtained from HA patients under long-term treatment of ACE910 in phase 1 and its extension studies, utilizing rotation thromboelastometry (ROTEM), in order to investigate global hemostatic function of HA patients under treatment with ACE910. Methods: Ca2+-triggered hemostatic functions were assessed by ROTEM in the citrated whole blood samples obtained and stabilized at the indicated points from five severe HA cases with inhibitors (Case 1 : 39 BU/ml, 2 : 41 BU/ml, 3 : 17 BU/ml, 4 : 11 BU/ml and 5 : 111 BU/ml ) and two severe cases without inhibitors (Case 6 and 7) under subcutaneous administration of ACE910, with the different types of dosing regimen (group A with 0.3 mg/kg/week subcutaneous injection (loading dose of 1 mg/kg) for Case 1, 2, group B with 1 mg/kg/week (loading dose of 3 mg/kg) for Case 3, 4, group C with 3 mg/kg/week for Case 6 and group D changing from group A to group C for Case 5, 7) in a course of clinical study. The samples from the subjects prior to administration of ACE910 were spiked ex vivo with ACE910 and the hemostatic functions of them were also evaluated by ROTEM. The parameters of clot time (CT), clot formation time (CFT), maximum clot formation (MCF) and alpha angle were evaluated. The hemostatic potentials in the samples from twenty healthy volunteers and other ten HA patients with the various FVIII:C were evaluated by ROTEM as controls and the correlation between FVIII:C and CT was obtained. Annualized bleeding rates (ABR) of each case were calculated. The studies were approved by local ethics committee and the informed consent was obtained from each patient. Results: Addition of ACE910 (f.c. 10 or 30 μg/ml) into the sample from each case prior to administration of ACE910 shortened CT from 5,562 ± 374 sec (median 5,924 sec) to 1,475 ± 138 or 1,131 ± 82 sec, equivalent to FVIII:C 3.3 or 12.2 IU/dL, respectively, in an ACE910 concentration dependent manner. In group A, the plasma concentration of ACE910 got to 12 ± 5 μg/ml at 12 week. CT was shortened to 1,443 ± 24 sec, equivalent to FVIII:C 3.7 IU/dL maximally at 47 week consistent with the result of the spiked result. ABR decreased from 38.6 ± 25.8 to 1.1 ± 0.8, showing 97% decrease. As for Case 3 in group B (ACE910: 31 μg/ml at 12 week), CT was shortened to 1,164 sec, equivalent to FVIII:C 11.9 IU/dL maximally at 47 week consistent with the spiked data. ABR apparently decreased from 38.6 to 3.6. In Case 4 who dropped out the clinical study at 4 week, CT was shortened to 977 sec, equivalent to FVIII:C 22 IU/dL, maximally at 3 week, and continued to be shortened till 29 week (1,758 sec, equivalent to FVIII:C 1.1 IU/dL). In group C, the shortening of CT from 1,594 ± 103 sec, equivalent to FVIII:C 2.0 ± 1.0 (median 3.6) IU/dL (before administration of ACE910), to 1,226 ± 71 sec (8.5 ± 2.5 (median 10.4) IU/dL) (at steady state) was observed in consistent with the decrease of ABR from 8.1 to 1.1. In group D, CT was shortened from 3,405 ± 386 sec to 1,409 ± 85 sec, equivalent to FVIII:C 4.2 IU/dL after middle to high dose escalation in consistent with decrease of ABR from 22.3 ± 9.6 to 3.8 ± 2.6. Among all cases except Case 4, CT and ABR were of clinically relevant difference between before and after administration of ACE910. Conclusions: The comprehensive hemostatic function evaluated by ROTEM in hemophilia A patients irrespective of presence of inhibitors was improved after administration of ACE910 in a dose dependent fashion, resulting in the reduction of ABR, which suggested the hemostatic effectiveness of ACE910 for HA patients. Disclosures Yada: Chugai Pharmaceutical Co., Ltd: Research Funding. Nogami:Bayer, Novo Nordisk, Baxalta. Biogen: Research Funding; Chugai: Membership on an entity's Board of Directors or advisory committees; Bayer, NovoNordisk, Baxalta, Chugai, Kaketsuken, Pfizer, Biogen: Honoraria. Shida:Chugai Pharmacoceutical Co. Ltd.: Research Funding. Takeyama:Chugai Pharmaceutical Co., Ltd.: Research Funding. Kasai:Chugai Pharmaceutical Co., Ltd.: Employment. Shima:Chugai Pharmaceutical Co., Ltd: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Biogen: Honoraria, Research Funding; Pfizer: Honoraria, Research Funding; Baxalta: Honoraria, Research Funding; Novo Nordisk: Honoraria, Research Funding; Kaketsuken: Honoraria; Bayer: Honoraria, Research Funding.

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