bluuebunny/crossref_metadata_2025 huggingface dataset:
Herpes Simplex Virus Initiates the Intrinsic Pathway of Coagulation.
Authors: Gershom Edwin S , Sutherland Michael R , Lollar Pete , Pryzdial Ed L.
Published: October 2019

Abstract 3173

Poster Board III-113

A hemostatic response to vascular injury is initiated by the extrinsic pathway of coagulation and amplified by the intrinsic pathway. We previously reported that purified herpes simplex virus type-1 (HSV1) has constitutive extrinsic pathway tissue factor (TF) and anionic phospholipid on its surface derived from the host cell, and can consequently bypass strict cellular control of coagulation. The current work addresses the hypothesis that HSV1-induced plasma coagulation also involves intrinsic pathway, factor VIII (FVIII), and upstream contact activation pathway, factor XII (FXII). HSV1-initiated clotting was accelerated when purified FVIII was added to FVIII-deficient plasma. The involvement of FVIII was confirmed by an inhibitory anti-FVIII antibody (Ab), which attenuated HSV1-initiated clotting of normal plasma. High HSV1 concentrations predictably reduced the effect of FVIII due to the availability of excess viral TF. To further define clotting mechanisms initiated by HSV1, the extrinsic pathway was disabled using factor VII-deficient plasma. The intrinsic pathway may be triggered by contact activation of FXII by kallikrein when bound to a suitable surface. Here we found that an inhibitor of activated FXII, corn trypsin inhibitor, and anti-FXII Ab attenuated HSV1-initiated clotting. Direct activation of purified FXII by the virus was not observed. Supporting the involvement of kallikrein in FXII activation due to HSV1, anti-kallikrein Ab was also inhibitory. The current work shows that HSV1 can trigger and amplify coagulation through the intrinsic and contact activation pathways, and suggests an additional mechanism that may explain prior reports linking virus infection to vascular pathology.

Disclosures

No relevant conflicts of interest to declare.
Immunogenicity of FVIII: a Role for Thrombin.
Authors: Skupsky Jonathan , Zhang Ai-Hong Allan , Scott David W.
Published: October 2019

Abstract 3175

Poster Board III-115

Administration of human Factor VIII to hemophilia A (FVIII-/-) mice is a useful small animal model to study the immune response in patients given therapeutic FVIII. These mice manifest a robust, T-cell dependent, antibody response to exogenous FVIII treatment even when encountered through traditionally tolerogenic routes, whereas a different foreign protein like ovalbumin (OVA) is much less immunogenic by these routes. Interestingly, administration of FVIII with OVA led to an immune response to both proteins, suggesting that the function of FVIII in the clotting cascade led to additional “danger” signals to co-administered proteins. Thus, we propose that FVIII is particularly immunogenic because of its function in the coagulation cascade that leads to thrombin formation. We showed that native FVIII is poorly immunogenic when it cannot activate downstream coagulation factors. Thus, heat-inactivated FVIII is poorly immunogenic despite containing normal T-cell epitopes. Importantly, native FVIII is less immunogenic in mice treated with warfarin (which blocks vitamin K-dependent enzymes) or with the direct thrombin-inhibitor, hirudin. Based on the hypothesis that thrombin generation is necessary for the immunogenicity and formation of inhibitors to FVIII, we wished to uncouple this immune response from the disease. When hemostatically normal BALB/c mice were injected directly with thrombin and OVA, they formed increased immune responses to OVA compared to mice given OVA alone. Thus, a likely mechanism is that FVIII treatment initiates a thrombin burst, which we propose is an immunogenic “danger” signal, leading indirectly to dendritic cell activation and promotion of an immune response. (Supported by NIH RO1 HL061883, NIH T32 HL007698, and AHA Fellowship 0815219E)

Disclosures

No relevant conflicts of interest to declare.
Children with NCI Standard Risk Acute Lymphoblastic Leukemia (ALL) and TEL-AML1 or Favorable Chromosome Trisomies Are Almost Certain to Be Cured with Graduated Intensity Therapy: Results of the CCG - 1991 Study.
Authors: Matloub Yousif , Bostrom Bruce C. , Angiolillo Anne L , Devidas Meenakshi , Heerema Nyla A. , Gastier-Foster Julie M. , Hunger Stephen P , Sather Harland , Linda Stephen , Carroll William L. , Nachman James B , Gaynon Paul S.
Published: October 2019

Abstract 320

Three thousand fifty four children with NCI standard-risk (SR) ALL were enrolled on CCG-1991; 2075 non-T ALL eligible patients were randomized and began treatment with IT cytarabine, vincristine, dexamethasone, and pegylated asparaginase. Bone marrow status was assessed at Day 7, 14, and 28 of Induction. Slow early responders (Day 7/14 M3-M3 and M3-M2; or M2 at Day 28 Induction) and patients with unfavorable cytogenetics were non-randomly assigned to a COG augmented BFM therapy regimen (N Engl J Med 1998; 338:1663).Thirteen hundred and thirty patients were analyzed for the presence of trisomy 4 and trisomy 10 (DT); and 1041 patients were evaluated for the presence of TEL/AML1 (ETV6/RUNX1) (TEL) fusion transcript. Twenty four percent of patients were positive for DT, and 41% were positive for TEL. Among the randomized subset, 23.8% had DT, and 40.8% had TEL. Six year event-free survival (EFS) and overall survival (OS) rates are given in the tables below for all patients and also separately for the randomized patients, by DT and TEL status. There was a significant different in EFS and OS between patients that were DT+ vs. DT- and also between patients that were TEL+ vs. TEL-.The overall 6-year EFS and OS for all patients with B-lineage ALL are 88.1±1.1% and 94.7±0.8%; and for the randomized patients they are 89.4±2.4% and 95.9±0.8%. We conclude that COG risk-adapted therapy is curative for patients with NCI-SR criteria and DT and/or TEL.

Disclosures:

No relevant conflicts of interest to declare.
In Vitro Large-Scale Expansion of Unfractionated Mononucleated Cells of Human Umbilical Cord Blood: Investigation for Clinical-Grade Generation of Cord Blood Hematopoietic Cells.
Authors: Mao Ping , Duan Huaxin , Wang Caixia , Deng Tingfen , Luo Changru , Xu Yanli , Zhang Yuping
Published: October 2019

Abstract 3222

Poster Board III-159

Previous studies of expansion for hematopoietic stem/progenitor cells showed progenitor cells could reach a great expansion but were mainly performed at small culture volume. The poor cell number of expanded cells even great folds of expansion in a no more than 10 ml culture system limited the application for clinic transplantation. Thereof, we expanded umbilical cord blood (UCB) hematopoietic cells at a large scale in 250 ml bioreactor culture system. The mononuclear cell isolated form fresh UCB samples were ex vivo expanded in 250 ml bioreactor with serum-free medium supplied with SCF, FL3 and TPO at concentration 20 ng/ml. Cell number, surface markers and colony forming potential after 7 days of expansion have been evaluated. The sub-lethally irradiated animal models were used to analyze engraftment capability of HSC by transplant of expanded cells in severe combined immunodeficient and nonobese diabetic (NOD/SCID) mice. The detection of human hematopoietic cells in the bone marrow of the mice was performed at 6 wk after the transplant. We have successfully expanded hematopoietic progenitor cells 5 times using the modified bioreactor. The cell viability showed no obvious variations during culture. Total cells increased from 1.76±0.50 ×108 (range 1.2-2.5) to 4.14±0.83×108 (range 3.7-5.6) after expansion, CD34+ cells from 1.25±0.31 ×106 (range 0.96-1.75) to 3.96±0.78×106 (range 2.88-5.04) and CD133+ cells from 1.55±0.69×106 (range 0.96-2.75) to 4.77±0.88×106 (range 4.00-5.04). The colonies per 105 cells of CFU-E /BFU-E, CFU-GM and CFU-Mix increased from 369.6±71.5 to 1648.5±504.3, 42.8±81.4 to 146.4±54.5, 39.1±10.3 to 144.7±38.8, respectively. The positive expression of CD34+ was 0.7%±0.28% at day 0 and 0.9%±0.34% after culture in bioreactor at day 7 and CD133+ cells was 0.8%±0.24% and 1.1%±0.35%. There was no significant survive rate difference between expanded and nonexpanded cells transplantation group. The analysis of multilineage hematopoiesis showed that transplanted human hematopoietic cells is represented in murine bone marrow cells by detection the percentage of human cells identified with human specific anti CD3/19/33/45/61/71 MoAb by FACS and the human specific gene Alu-1 and Cat-1 by PCR.

Disclosures

No relevant conflicts of interest to declare.
Combined High Resolution Genomic and Expression Profiles Microarray Analysis in Sezary Syndrome.
Authors: Caprini Elisabetta , Cristofoletti Cristina , Arcelli Diego , Fadda Paolo , Citterich Mauro Helmer , Sampogna Francesca , Magrelli Armando , Torreri Paola , Censi Federica , Frontani Marina , Scala Enrico , Picchio Maria Cristina , Monopoli Alessandro , Lombardo Giuseppe Alfonso , Taruscio Domenica , Narducci Maria Grazia , Russo Giandomenico
Published: October 2019

Abstract 3238

Poster Board III-175

We have used single nucleotide polymorphism (SNP) and comparative genomic hybridization array (aCGH) to study DNA copy number (CN) changes and loss of heterozygosity (LOH) in a series of 28 patients affected by Sézary syndrome (SS), a rare form of cutaneous-T-cell lymphoma (CTCL). Our data identified, further confirming previous studies, recurrent losses of 17p13.2-p11.2 and 10p12.1-q26.3 occurring in 71 and 68% of cases respectively; common gains were detected for 17p11.2-q25.3 (64%) and chromosome 8/8q (50%). Moreover, we identified novel genomic lesions recurring in more than 30% of tumours: loss of 9q13-q21.33 and gain of 10p15.3-10p12.2. Individual chromosomal aberrations did not show a significant correlation with prognosis, however when more than three recurrent chromosomal alterations (gain or loss) were considered, a statistical association was observed using Kaplan-Meier survival analysis. Crossing genomic data mapping with those obtained from gene expression profiles of matching samples we were able to identify a total of 113 deregulated transcripts in aberrant chromosomal regions that included cancer related genes such as members of the NF-kB pathway (NKIRAS2, PSMD3, BAG4, BTRC, TRAF2) that might explain its constitutive activation in CTCL. Matching this list of genes with those discriminating patients with different survival times we identify several common candidates that might exert critical roles in SS, like PIP5K1B and BUB3. Taken together this study confirms and maps more precisely the regions of gain and loss and, combined to transcriptional profiles, suggest a novel set of genes of potential interest in SS.

Disclosures

No relevant conflicts of interest to declare.
Pediatric Precursor-B ALL with BTG1 Deletions Show a Distinct Genomic Profile.
Authors: Waanders Esmee , Leeuwen Frank N. van , Verwiel Eugene , Reijmersdal Simon V van , Levers Marloes R , Galen Joost C van , Sonneveld Edwin , Hoogerbrugge Peter M. , Kessel Ad Geurts van , Kuiper Roland P.
Published: October 2019

Abstract 3244

Poster Board III-181

Recent genome-wide profiling studies have revealed that childhood acute lymphoblastic leukemia (ALL) is characterized by recurrent microdeletions, including the cell cycle regulator CDKN2A, the B-cell differentiation genes PAX5, EBF1 and IKZF1 (Ikaros) and the anti-proliferative gene B-cell translocation gene 1 (BTG1). In a previous study, we have shown that BTG1 is an important determinant of glucocorticoid sensitivity (Van Galen et al. Blood/ ASH Annual Meeting Abstracts, 2008). In the present study we have characterized these cases in more detail and elucidated the frequency of recurrent lesions in BTG1 deletion cases. Using locus-specific MLPA screening of an unselected cohort of 305 precursor B-ALL cases, we identified 26 microdeletions (8.5%). All deletions encompassed BTG1 only. We were able to genomically profile 22 diagnosis samples using Affimetrix SNP6.0 arrays. Of these, 12 did not develop a relapse during a minimal of 4,5 years of follow up. The mean number of CNVs was 29.6 of which 10.3 gains and 22.5 losses (median size 512 kb and 115 kb respectively). BTG1 deletions were generally focal, varying in size from 104 kb to 1,4 Mb. In all but one patient the breakpoints at the 5' end of the deletion tightly clustered and subsequent fine-mapping using qPCR revealed that this breakpoint cluster was located within intron 1 of the BTG1 gene. At the 3'end of the deletion, four breakpoint clusters could be identified. Analysis of the copy number variation (CNV) profiles showed that patients with a BTG1 deletion more often harbored a deletion in IKZF1 compared to an unselected cohort of pre-B ALL cases (27% vs 7%, chi-square p=0.042). In contrast, recurrent CNVs like PAX5, EBF1 and CDKN2A/B occur in similar frequencies (23%, 9% and 32% vs 17%, 0% and 23% respectively). In addition, the BTG1 deletion cases that developed into a relapse showed significantly more often a deletion in CDKN2A/B compared to the BTG1 deletion cases that did not develop a relapse (60% vs 8%, p=0.02). Together, these data indicate that pediatric precursor-B ALL carrying BTG1 deletions have distinct genomic profiles, showing increased frequencies of deletions in IKZF1 and CDKN2A.

Disclosures

No relevant conflicts of interest to declare.
12 Years Experience in Peru with Related Allogeneic Stem Cell Transplantation (allo-sct) in Children with High Risk ALL.
Authors: Navarro Juan Ramon , Murillo Sergio , Rodriguez Jackeline , Moreno Mariela , Aranda Lourdes , Chumpitaz Gloria
Published: October 2019

Abstract 3356

Poster Board III-244

Background:

ALL accounts for approximately 70-83% of all childhood leukemia. High risk ALL is a big problem in our country and allogeneic related HSCT has proven to be a curative treatment for high risk patients with ALL.

Patients and Methods:

We analyzed the clinical data of 51 children (27 boys and 24 girls) with ALL undergoing related BMT/PSCT from Edgardo Rebagliati Hospital between 1996 and 2008. Survival probabilities were analyzed by Kaplan and Meier method. Outcome of different subgroups was compared by the log-rank test.

Results:

The recipients median age at transplantation was 10.7 years (range, 1 - 18 years). The disease status at conditioning were 1st CR (n=33), 2nd CR or more (n=18). The median interval from diagnosis to BMT was 14 months (range, 4 - 93 months). We have used two conditioning regimen: TBI/CTX/VP16 (TBI-1200Gy/CTX-120 mg/kg/ VP16-40mg/kg) in 34 patients (66.7%) or TBI/CTX 16 (31.4%) and 1 patient (2%) standard BuCy conditioning regimen. Graft-versus-host disease (GVHD) prophylaxis was methotrexate and cyclosporin. The median infused CD34 cell number was 6.21×106/kg (range 1.61-14.9×106/kg). The resource of hematopoietic cell was bone marrow 33.3% (17 patients), peripheral blood, 64.7% (33 patients) and both 2% (1 patient). Acute GVHD occurred in 10 (19.6%) patients, and chronic GVHD occurred in 15 (29.4%) patients.

Twelve year overall survival (OS) is 59.8% ± 8.7% (n= 51) and leukemia-free survival probabilities (DFS) were 62.9% ± 8.2%. Patients with 1st CR and 2nd CR had a 5 year EFS of 60.1% and 67.3% (log-rank test, P 0.92), respectively.

The mortality non related to transplant was 29.5%; 10 patients (19.6%) due to progressive disease and five patients (9.9%) by sepsis.

Conclusions:

Allo-SCT is well tolerated in high risk ALL children with prolonged OS and DFS. Not found statistical significance on OS and DFS between the conditioning regimen used, between status pre-transplant (1st CR vs 2nd CR) and we found statistical significance in favor to PB as a source of SC on DFS.

Disclosures:

No relevant conflicts of interest to declare.
Allogeneic Hematopoietic Cell Transplantation (Allo-HCT) for Treatment of Pediatric Philadelphia Chromosome-Positive Acute Lymphoblastic Leukemia (ALL).
Authors: Burke Michael , Trotz Barb , Cao Qing , Weigel Brenda , Kumar Ashish , Smith Angela , Verneris Michael
Published: October 2019

Abstract 3377

Poster Board III-265

Introduction:

Allo-HCT with best available donor for children with Philadelphia positive (Ph+) ALL has previously been considered standard practice. Since the introduction of imatinib into the treatment of this disease, the role of allo-HCT is more uncertain.

Patients and Methods:

We investigated the impact of remission status, graft source, and imatinib use on transplant outcomes for thirty-seven children with Ph+ ALL who received an allo-HCT at the University of Minnesota between 1990 and 2006. The median age at HCT was 7.47 (range; 1.4 -16.4) years. Thirteen patients received imatinib therapy pre and/or post-HCT (imatinib group) and 24 patients, received either no imatinib (n=23) or only after post-HCT relapse (n=1) (non-imatinib group).

Results:

There was no difference in disease-free survival (DFS) or relapse between the imatinib and non-imatinib groups at 3 years (62% / 15% vs. 53% / 26%; p=0.99; 0.81, respectively). There was no significant difference in transplant outcomes between matched related donor or unrelated donor (umbilical cord blood or matched unrelated marrow) recipients whereas patients receiving allo-HCT in first remission (CR1) had superior DFS and less relapse compared to patients transplanted in ≥CR2 (71% / 16% vs. 29% / 36%; p=0.01; p=0.05).

Conclusions:

Based on this retrospective analysis at a single institution, the use of imatinib either pre and/or post-transplant does not appear to significantly impact outcomes for children with Ph+ ALL and allo-HCT with the best available donor should be encouraged in CR1.

Disclosures:

No relevant conflicts of interest to declare.
Comparative Analysis of Outcomes of Allogeneic Peripheral Blood Stem Cell Transplantation From Related and Unrelated Donors.
Authors: Kang Byung Woog , Kim Shi Nae , Moon Joon Ho , Jung Joo-Seop , Cho Goon-Jae , Jo Deog Yeon , Kim Yeo-Kyeoung , Kim Hyeoung Joon , Ryoo Hun-Mo , Eom Hyeon Seok , Lee Sang Min , Joo Young-Don , Won Jong Ho , Park Moo-Rim , Kim Min Kyoung , Hyun Myung Soo , Sohn Sang Kyun
Published: October 2019

Abstract 3381

Poster Board III-269

This study compared the results of allogeneic peripheral blood stem cell transplantation (PBSCT) from unrelated and related donors, and involved 235 consecutive patients from 10 centers who received an allogeneic PBSCT for hematological malignancies between Jan 2004 and Dec 2008. Among these patients, 160 (68.1%) received an HLA-matched related PBSCT and 75 (31.9%) a matched unrelated PBSCT. Sixty-five patients (27.7%) had a high-risk disease status at transplantation. The cumulative incidence of acute graft-versus-host disease (GVHD) was 43.9% for the related PBSCT group and 59.3% for the unrelated PBSCT group (P-value:0.011). Although the cumulative incidence of chronic GVHD was no different between the related (54.2%) and unrelated (64.9%; P-value:0.199) PBSCT groups, the cumulative incidence of extensive chronic GVHD was higher among the unrelated PBSCT group (34.9%) than among the related PBSCT group (17.0%; P-value:0.015). Plus, the unrelated PBSCT group showed a higher cumulative incidence of CMV infection (44.6%) than the related PBSCT group (26.8%; P-value:0.002). The overall survival rate at four-year was 58.2% versus 49.1% (p=0.698) and the cumulative incidence of relapse 28.4% versus 25.0% (P-value:0.289) for the related and unrelated PBSCT groups, respectively. Among the factors examined, unrelated PBSCT (P-value:0.024), the CD34-positive cell count (>6 × 106/kg; P-value:0.041), and CMV infection (P-value:0.066) were all related with a higher incidence of extensive chronic GVHD. However, in a multivariate analysis, only unrelated PBSCT was identified as a risk factor for the development of extensive chronic GVHD (hazard ratio=2.012, 95% confidence interval=1.006-4.023; P-value:0.048). In conclusion, the overall survival and relapse incidence were not significantly different between the related and unrelated PBSCT groups. However, a higher incidence of CMV infection and extensive chronic GVHD was observed in the unrelated PBSCT group.

Disclosures:

No relevant conflicts of interest to declare.
Increased Prevalence of Inhibitors in Mexican-Hispanic Patients with Severe Hemophilia A Enrolled in the Universal Data Collection Project.
Authors: Carpenter Shannon , Soucie J. Michael , Sterner Sophia , Presley Rodney J
Published: October 2019

Abstract 3488

Poster Board III-425

Neutralizing inhibitor formation occurs in up to 20-30% of patients with severe factor VIII deficiency, leading to significantly increased morbidity in affected individuals. It has been well-established that patients of African descent have a higher prevalence of inhibitor development. [Oldenburg, J et al. Semin Hematol, 2004] The Hispanic population also has been assumed to have an increase in inhibitor development when compared with Caucasians. The study presented here is the first to definitively demonstrate an increased prevalence of inhibitors in the Hispanic population. We compared inhibitor prevalence among various racial and ethnic groups in a cross-sectional analysis of 6198 males with severe hemophilia A that participated in the Universal Data Collection project sponsored by the Centers for Disease Control and Prevention. We used logistic regression analysis to control for potential confounding variables including age, insurance type (as a proxy for access to care and socio-economic status), age at first bleed, age at diagnosis and use of prophylaxis. The included table shows those variables that were determined to be independently predictive of inhibitors. We assigned Mexican derivation to participants who labeled themselves as Hispanic and who were born either in Mexico, in states bordering Mexico or in states with large Mexican populations as established by Census data. The prevalence of high titer inhibitors in the Mexican-Hispanic population was 26.3% compared to 16.4% for Caucasian patients [OR 1.5, 95% CI 1.1, 1.9], and 26.8% for African-Americans. The underlying cause of increased inhibitor prevalence in these populations is still unknown, though a recent study in African-Americans demonstrated wild-type factors unique from commercially available product. [Viel KR, et al. Inhibitor of Factor VIII in Black Patients with Hemophilia. N Engl J Med, 2009] Further investigation of this phenomenon in the Mexican-Hispanic population, as well as the potential impact of differing immune responses, is warranted.

Multivariate analysis of ethnicity and other variables found to be independently predictive of a prevalent inhibitor Characteristic Odds Ratio 95% CI Race/Ethnicity African-American 1.5 1.2 - 1.9 Mexican Hispanic 1.5 1.1 - 1.9 Hispanic 1.2 0.9 - 1.7 Other 1.2 0.9 - 1.6 White Ref Age* (years) <2 4.2 3.0 - 5.9 2-5 6.4 5.1 - 8.0 6-10 2.8 2.2 - 3.5 11-18 1.7 1.4 – 2.1 >18 Ref Insurance type Medicare 1.8 1.4 - 2.3 Medicaid 1.3 1.1 - 1.5 State program 1.1 0.6 - 1.9 TRICARE 1.0 0.4 - 2.1 Other 0.8 0.6 - 1.2 Uninsured 1.6 1.0 - 2.4 Commercial Ref Prophylaxis Yes 0.6 0.5 - 0.7 No Ref * Age with inhibitor or last UDC visit if no inhibitor

The authors wish to acknowledge the contributions of the Hemophilia Treatment Center Network Investigators in the completion of this study.

Disclosures:

No relevant conflicts of interest to declare.
Population Pharmacokinetic Model of ADVATE in Pediatric and Adult Patients with Hemophilia A.
Authors: Oh Myungshin , Björkman Sven , Schroth Phillip , Fritsch Sandor , Collins Peter , Fischer Kathelijn , Blanchette Victor S. , Casey Kathleen M. , Spotts Gerald , Ewenstein Bruce M.
Published: October 2019

Abstract 3492

Poster Board III-429

Introduction

The objective of this analysis was to characterize the population pharmacokinetic (PK) model of ADVATE® (Antihemophilic Factor (Recombinant), Plasma/Albumin-Free Method) in hemophilia A patients. This included estimation of typical population pharmacokinetic parameters and inter-individual and residual variability and identification of covariates that are significant predictors of variability in a pooled population of children and adults.

Patients and Methods

Plasma FVIII activity PK data were collected for 3 ADVATE® clinical trials in previously treated patients: 184 full PK data sets for 100 adults/adolescents, aged 10 to 65 years, and from 52 reduced sample PK data sets for 52 children, aged 1 to 6 years. Population PK analysis was conducted using non-linear mixed effects modeling with the first-order integral approximation method in SAS® software (NLMIXED procedure). A two-compartment model was used as the base model and the influence of age and weight were explored.

Results

Two-compartment PK models with additive plus proportional residual variability model and exponential inter-individual variability model adequately described the data. Clearance (CL) is significantly correlated with age and body weight and central volume of distribution (V1) is also related with body weight. The estimated population PK parameters were (mean parameter, (inter-individual variability %)): CL (2.92 mL/kg·h, 22%), V1 (0.46 dL/kg, 5.2%), peripheral volume V2 (0.09 dL/kg) and inter-compartmental clearance Q (2.07 mL/ kg·h).

Conclusions

A population PK model that describes the combined PK data from adults and pediatric studies has been constructed. A significant portion of inter-individual variability in both volume and clearance can be explained by subject weight. An additional smaller effect of age on clearance but not volume was observed. A population PK model for Factor VIII could provide the clinician with advantages in designing a patient specific treatment regimen. It could provide more relevant guidance in individual patient pharmacokinetics than just incremental recovery without the burden of a full PK assessment of the patient.

Disclosures:

Oh: Baxter: Employment. Off Label Use: Prophylaxis is not indicated in the US. Björkman:Baxter: Consultancy; Octapharma: Consultancy. Schroth:Baxter: Employment. Fritsch:Baxter: Employment. Collins:Bayer: Consultancy; Novo Nordisk: Consultancy; Baxter: Consultancy. Fischer:Bayer: Consultancy; Wyeth: Consultancy; Baxter: Consultancy. Blanchette:Bayer: Consultancy; Baxter: Consultancy. Casey:Baxter: Employment. Spotts:Baxter: Employment. Ewenstein:Baxter: Employment.
Impaired Interaction Between Regulatory T Cells and Dendritic Cells in Immune Thrombocytopenia.
Authors: Catani Lucia , Sollazzo Daria , Curti Antonio , Trabanelli Sara , Palandri Francesca , Polverelli Nicola , Baccarani Michele , Vianelli Nicola , Lemoli Roberto
Published: October 2019

Abstract 3511

Poster Board III-448

CD4+CD25+ regulatory T cells (Tregs) are critical in maintaining self-tolerance and preventing organ-specific autoimmune diseases. However, the role of Tregs in the pathogenesis of immune thrombocytopenia (ITP), an immune disorder in which increased platelet clearance is caused by antiplatelet autoantibodies, has not yet been clarified. The purpose of the present study was to investigate whether the interaction between dendritic cells (DCs) and regulatory T cells (Tregs) may play a pathogenetic role in ITP. Forty patients with active disease and 35 healthy subjects were enrolled into the study. We firstly characterized the number (by flow cytometry) and the suppressive activity (by modified allogeneic mixed leukocyte reaction) of Tregs in ITP. We found that the absolute number of Tregs was significantly decreased in ITP patients in comparison to healthy subjects (CD4+CD25highFoxp3+ T cells (5.5±4.3 vs 11.6±6.9 cells/microL; p below 0.01) and CD4+CD25highCD127low-negative T cells (50.9±27.3 vs 80.5±37.7 cells/microL; p below 0.02)). We documented also that in ITP suppressive activity of Tregs was defective as compared to healthy subjects. In parallel experiments we studied the in vitro conversion of CD4+CD25- T cells, either from ITP patients or healthy subjects, into CD4+CD25+Foxp3+ Tregs by co-coltures with mature autologous/allogeneic DCs. The flow cytometry analysis showed that in ITP the low number of circulating Tregs may be partly due to the reduced ability of DCs to convert non-Treg cells into Tregs. We then explored the in vitro capability of CD4+CD25+ Tregs, either from ITP patients or healthy subjects, to inhibit allogeneic DCs maturation by flow cytometry evaluation of the expression of the costimulatory molecules CD80 and CD86. We demonstrated that in ITP patients CD4+CD25+ Tregs show lower ability to inhibit DCs maturation because they do not affect the expression of CD80 and CD86 molecules. This finding may be related to the lower level of Interleukin-10 and Interleukin-6 in the cocoltures. Taken together, these findings document that the interaction between DCs and Tregs is altered in ITP and suggest that this dysfunction may play a pathogenetic role.

Supported in part by BolognaAIL (Italian association against Leukemia, Bologna section)

Disclosures:

No relevant conflicts of interest to declare.
Irradiated Third Party Peripheral Blood Mononuclear Cells Enhance Umbilical Cord Blood Engraftment.
Authors: Budak-Alpdogan Tulin , Jeganathan Gajan , Lee Kuo-Chieh , Mrowiec Zbigniew Roger , Medina Daniel , Todd Dennis , Moore Dirk , Bertino Joseph , Strair Roger
Published: October 2019

Abstract 3533

Poster Board III-470

Umbilical Cord Blood (UCB) is a readily available, alternate donor source for allogeneic Hematopoietic Stem Cell (HSC) transplantation and can be used with lower stringency HLA-matching between donor and host. However, only limited numbers of cells are available and non-engraftment or delayed engraftment occurs often when recipients receive single UCB units that contain < 2.5 × 107 TNC/kg recipient body weight. This excludes single UCB as a donor source for many adult patients who might otherwise benefit from an UCB transplant. Both immunologic and non-immunologic mechanisms may contribute to UCB HSC engraftment failure. UCB T-cells are phenotypically and functionally immature with a high percentage of CD45RA+ T cells and T cells are known to facilitate engraftment in human HSC transplantation. Co-transplantation of irradiated T-cells supported engraftment in an immunocompetent mouse transplant model without increasing GvHD (Waller et al. Blood94: 3222,1999).

The working hypothesis for his study is that the irradiated third party peripheral blood mononuclear cells (PBMNCs) induce a cytokine or cellular response that facilitates hematopoietic engraftment. Long-term culture initiating cell (LTC-IC) frequencies determined with limiting dilution assays were statistically higher when either purified UCB derived CD34+ cells or CB MNCs cells were plated with irradiated PBMNCs (Figure 1).

Figure 1 Figure 1.

To assess the impact of irradiated third party PBMCs on UCB engraftment we utilized sub-lethally irradiated NOD/SCID-IL2Rgnull mice in the presence and absence of irradiated third-party human PBMNCs. After sublethal irradiation (240 cGy) female 8 week-old animals were transplanted with either UCB MNCs corresponding to 8×103 (n=5) or 2.66×103 (n=9), or 8.88×102 CD34+ cells (n=9) with or without co-transplantation of 1×106 irradiated (2500 cGy) PBMNCs, and 4 animals received only irradiated PBMNCs as the control group. Engraftment levels are compared by calculating SCID repopulating cell (SRC) frequency. Co-transplantation of PB MNCs with UCB MNCs provided a steady, statistically significant increase in both short-term and long term hematopoietic engraftment (Figure 2).

Figure 2 Figure 2.

In these pre-clinical studies, we have confirmed in vitro effects on LTIC formation and in vivo UCB graft-enhancing effects of irradiated PBMNCs. If applied in clinic, this cellular therapy option might overcome a major barrier to successful allogeneic UCB HSC transplantation: engraftment failure, making the procedure more applicable and safer.

Disclosures:

No relevant conflicts of interest to declare.
Clonogenic MM Progenitor Cells Reside In CD19-CD38++ Plasma Cells
Authors: Hosen Naoki , Sugiyama Haruo
Published: October 2019

Abstract 1897

For effective treatment of multiple myeloma (MM), it is important to identify clonogenic MM progenitor cells and target them. However, it is still controversial where MM progenitor cells reside. It was reported that CD19+ B cells from MM patients generated MM upon transplantation to NOD/SCID mice, while CD19-CD38++ plasma cells generated MM disease in SCID-hu or rab model. In this study, we performed both of those two xenograft models with a series of MM patient samples. To increase engraftment efficiencies, we used highly immuno-deficient NOD/Scid, IL-2Rγnull (NOG) mice as recipients, and cells were injected directly into bone marrow (BM) or intravenously to new born pups. CD19+ cells from 10 MM samples were transplanted, but none of them engrafted in NOG mice. Thus, the significance of CD19+ B cells was unclear in the MM patients examined in this study. On the other hand, in SCID-rab model, CD19-CD38++ plasma cells, but not CD19+ B cells, generate MM disease in 5 out of 13 MM patient samples. In addition, in vitro MM colony-forming cells were highly enriched in CD19-CD38++ plasma cells. Taken together, our results indicate that clonogenic MM progenitors reside in CD19-CD38++ cells and are essential targets to eradicate MM clones.

Disclosures:

No relevant conflicts of interest to declare.
Haploinsufficiency of Ribosomal Protein S6 In Mice Mimics Bone Marrow Failure Syndromes In Humans
Authors: Park Christopher Y. , McGowan Kelly A , Glader Bertil , Barsh Gregory S , Weissman Irving L.
Published: October 2019

Abstract 194

Haploinsufficiency of ribosomal proteins has been implicated in the pathogenesis of Diamond Blackfan anemia and 5q- myelodysplastic syndrome, two diseases that share phenotypic features including anemia and thrombocytosis. The molecular and cellular basis of the specific cytopenias observed in affected individuals has remained enigmatic. Here, we examined the effect of bone marrow-specific hemizygosity for a ribosomal protein, Rps6, in the mouse, and find that the model recapitulates many aspects of human bone marrow failure syndromes. Rps6lox/+;Tg.MxCre animals exhibit a robust macrocytic anemia that worsens over time with a 36% reduction in red blood cell count and a 25% increase in mean corpuscular volume (MCV) at 16-weeks post induction of Cre recombinase. Mutant mice also exhibit marked leukopenia and a progressive thrombocytosis. Morphologic examination of Rps6lox/+;Tg.MxCre/+ bone marrow reveals a prominent megakaryocytosis (2.7-fold increase) composed of small, dysplastic megakaryocytes and a 50% reduction in cellularity in compared to control animals. There is also a significant increase in the number of immature erythroid precursors (basophilic erythroblasts, P=0.004), and a significant decrease in mature erythroid cells (poly- or orthochromatic erythroblasts, P=0.027). We also measured HSC and committed progenitors in Rps6lox/+; Tg.MxCre/+ mutant and nonmutant (Tg.MxCre/+) controls. Flow cytometric evaluation revealed that mutant animals exhibited a significant decrease in HSCs (P=0.027), accompanied by significant increases of most downstream committed progenitor populations (MPP, P=0.002; PreMegE, P=0.004; Pre CFU-E, P=8.5×10−6; MpK, P=0.00007) (Pronk et al., Cell Stem Cell 2007). Mutant animals also exhibited a significant (P=0.017) reduction of more mature erythroid progenitor cells (CFU-E/ProEry). Based on prior work implicating p53 in the pathogenesis of bone marrow failure, Rps6lox/+;Tg.MxCre mice were crossed with p53 knockout (Trp53ko/ko) animals and evaluated. In Rps6lox/+;Tg.MxCre/+ animals, deficiency for p53 completely mitigated the hematopoietic alterations in the peripheral blood and the bone marrow. Together, these data indicate that Rps6 haploinsufficiency mimics many aspects of human bone marrow failure syndromes and does so through a p53-dependent pathway. This mouse model will serve as a useful tool to investigate the molecular and cellular mechanisms underlying bone marrow failure syndromes mediated by RPS haploinsufficiency.

Disclosures:

Weissman: Amgen, Systemix, Stem cells Inc, Cellerant: Consultancy, Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees.
Pacmed Salvage Therapy for Advanced High-Risk Multiple Myeloma (AHRMM)
Authors: Alsayed Yazan , Waheed Sarah , Szymonifka Jackie , Nair Bijay , Usmani Saad , van Rhee Frits , Anaissie Elias J. , Grazziutti Monica , Hoering Antje , Barlogie Bart
Published: October 2019

Abstract 1969

Background:

High-risk MM remains a very difficult clinical challenge despite advances in therapy for the majority of patients who have benefited from the use of high-dose therapies and novel agents. Recognizing that MM from the outset represents a genomically highly complex malignancy with even further accelerated acquisition of mutations with every further relapse, we have tried to develop multi-agent combinations employing drugs with efficacy in other high-grade tumors such as large cell lymphomas and incorporated novel agents as well.

Patients and Methods:

Eighty-four patients with AHRMM were given PACMED comprising cisplatin (15-25mg/m2 CI × 3d), cytarabine (1.0-1.5g/m2/d × 3), cyclophosphamide (1.0-1.5g/m2/d CI × 3), mesna (1.0-1.5g/m2/d CI × 3), etoposide (0.3-0.5g/m2/d × 3) and DEX (40-100mg/d × 3); additional agents included bortezomib (1.0-1.6mg/m2 on days 1 + 4), thalidomide (100-200mg/d × 4d) or lenalidomide (25-100mg/d × 4) and rapamycin (3mg d 1, 1mg d 2–4) with or without HPC boost. Statistical methods included Cox regression modeling for OS and EFS, along with Kaplan-Meier methodology for survival and cumulative incidence plots. Survival comparisons were made using the logrank test.

Results:

Baseline characteristics included age >=65 in 18%, B2M >=3.5mg/L in 68% and >5.5mg/L in 38%, CRP >=8mg/L in 60%, LDH >=ULN in 57%, and cytogenetic abnormalities (CA) in 62%. Gene expression profiling (GEP)-defined high-risk (70 genes, R70; 80 genes, R80) was present in 80% and 71%; PR (Proliferation), MF and MS subtypes were present in 44%, 27% and 14%. Prior transplants (Tx) had been given to 96%, including 40% who received 2Tx, 25% with 3Tx and 14% with >3Tx. PR was achieved by 29%, including 14% n-CR and 8% CR. 1-year estimates of OS and EFS were low at 13% and 8%, and median durations were 5 and 3 months. Increased age and high LDH were the only baseline characteristics adversely affecting both OS and EFS. The 29 patients with neither of these risk factors experienced 1-yr OS/EFS rates of 31%/17%; the corresponding values with 1 risk factor (n=47) were 5%/4% and with both risk factors (n=8) 0%/0%.

Conclusion:

Single cycle PACMED provides only transient tumor control in this heavily pretreated population with 80% displaying GEP-defined high-risk and 62% CA, as a manifestation of end-stage MM. We are currently evaluating repeated cycles of PACMED earlier in the disease course in high-risk MM, in the context of a Super-BEAM transplant regimen.

Disclosures:

No relevant conflicts of interest to declare.
Convergent Mechanisms of Somatic Mutations In Polycythemia Vera
Authors: Wang Kai , Swierczek Sabina , Hickman Kimberly , Hakonarson Hakon , Prchal Josef T.
Published: October 2019

Abstract 1979

Polycythemia vera (PV) is an acquired clonal blood disorder characterized by variable increased number of clonal myeloid cells (erythrocytes, granulocytes and platelets) and mostly normal polyclonal T lymphocytes. Most patients carry a gain-of function JAK2 V617F mutation in 2 copies acquired by a uniparenteral disomy; i.e. a copy-neutral loss of heterozygosity (LOH) in chromosome 9p in PV pathogenesis. Yet, JAK2 V617F mutation is not a PV-initiating event and the family clustering of PV suggests a contribution of inherited genetic events to its pathogenesis. Interestingly, majority of PV subjects acquire the JAK2 V617F mutation on a distinct a germline SNP 9p24.1 haplotype. Using whole-genome SNP arrays, we assayed 34 T-cells and 66 granulocytes from PV patients (including 32 pairs from the same patient). Comparison to a control population identified multiple germline SNPs around JAK2 as being associated with PV susceptibility (rs11999802, P=1.8×10-8, OR=4.4). Analysis of somatic genomic changes in granulocytes revealed strong genetic heterogeneity: we detected 9p LOH in 36% of the patients, 9p chromosomal gain in 1 patient, as well as “fractional LOH” in 14% of the patients where a small fraction of granulocytes have LOH. Interestingly, the magnitude of somatic LOH in 9p was strongly associated with somatic V617F allelic dosage (r=0.74, P=4.8×10-12), suggesting that LOH preferentially increase V617 PV subclone without altering genomic copy number. Adjusting for the fractional LOH, we did not have sufficient power to detect association between rs11999802 germline genotypes and increased dosage of somatic V617F mutations. Analysis of granulocytes with heterozygous rs11999802 genotypes demonstrated that LOH increases the relative fraction of germline risk alleles versus non-risk alleles. In summary, germline risk variants surrounding JAK2 predispose to somatic SNVs within JAK2, whose allelic dosage can be further increased by a serial subclonal expansion of allele-specific copy-neutral LOH or chromosomal duplications. Our study illustrates how convergent mechanisms of somatic mutations increase the genetic load of gain-of-function mutations contributing to the clonal expansion and disease pathogenesis.

Figure 1 Box plot of the percentage of somatic V617F mutation in JAK2 over each of the fractional LOH measures in granulocytes. Strong correlation between these two measures was detected (r=0.74, P=4.8×10-12). Figure 1. Box plot of the percentage of somatic V617F mutation in JAK2 over each of the fractional LOH measures in granulocytes. Strong correlation between these two measures was detected (r=0.74, P=4.8×10-12).

Disclosures:

No relevant conflicts of interest to declare.
Transgene Dose- and Age-Dependent Development of Myeloproliferative Neoplasm Phenotypes In JAK2V617F Transgene Mice
Authors: Ho Wanting , Zhao Wanming , Zhao Zhizhuang Joe
Published: October 2019

Abstract 1980

Myeloproliferative neoplasms (MPNs) are heterogeneous hematologic disorders represented by three main phenotypes: polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF). The major molecular lesion in these diseases is JAK2V617F, which occurs in over 95% patients with PV and in over 50% of patients with ET or PMF. The pathogenic effects of JAK2V617F have been demonstrated by retrovirus-mediated gene transfer, transgenic, and knock-in mouse models, but the precise mode of JAK2V617F action is not clear. Interestingly, in the knock-in model, expression of JAK2V617F causes severe PV-like disease but not ET-like phenotype as seen in patients. To verify the pathogenic role of JAK2V617F, we further characterized the phenotypes of three lines of JAK2V617F transgenic mice generated by using the vav gene promoter which drives expression of transgenes in the hematopoietic system. These mice developed MPN-like phenotypes in a transgene dose- and age-dependent manner. Line A mice have a JAK2V617F gene copy number of 13; they develop MPN phenotype with marked increases in blood counts and enlarged spleens as early as 4–6 weeks after birth. In contrast, lines B and D mice have a transgene copy number of 2 and 1, respectively, and it takes nearly 70 weeks for these mice to show MPN-like phenotypes. The phenotype of line A mice is particularly noteworthy. Essentially all the hemizygous line A mice displayed an ET-like phenotype with marked elevations in platelet counts (usually over 4000×109/L by the age of 15 weeks), but only a slight increase in red cell and white cell counts. In contrast, all the homozygous mice exhibited a clear PV-like phenotype with elevations in all three types of blood cells, although their platelets hardly ever went over 4000×109/L. The hemizygous mice developed myelofibrosis after 30 weeks while the homozygous mice showed the symptom within only 10 weeks. As expected, the increased blood cell counts and formation of myelofibrosis are associated with mobilization of hematopoietic stem/progenitor cells to peripheral hematopoietic tissues (blood, spleen, and liver). By conducting stem cell transplant experiments, we further proved that JAK2V617F-induced ET and PV-like phenotypes are transplantable. Our study demonstrates that transgenic expression of JAK2V617F is capable of producing all three phenotypes of MPNs in a transgense dose- and age-dependent manner. Our transgenic mice thus represent an excellent model system to study MPNs.

Disclosures:

No relevant conflicts of interest to declare.
Protein Kinase C Isoform ε Negatively Regulates ADP-Induced Thromboxane Generation by Regulating cPLA2 Activation and Calcium Mobilization In Platelets
Authors: Bynagari Yamini , Lakhani Parth , Bhavaraju Kamala , Jin Jianguo , Rico Mario C , Kim Soochong , Woulfe Donna S , Kunapuli Satya P.
Published: October 2019

Abstract 2020

Positive regulatory role of Protein Kinase C (PKC) isoforms in platelets have been extensively studied. However, negative regulatory roles of PKCs in platelets are poorly understood. In this study we investigated the mechanism by which PKCs negatively regulate ADP-induced thromboxane generation and identified PKC isoforms involved in this process. Pan PKC inhibition with GF109203X potentiated ADP-induced cPLA2 phosphorylation suggesting that PKCs negatively regulate thromboxane generation by regulating cPLA2 activation. Inhibition of PKCs potentiated ADP-induced ERK activation and intracellular calcium mobilization, two upstream signaling molecules of cPLA2.These data suggest that PKCs negatively regulate thromboxane by regulating ERK activation and calcium mobilization, which inturn regulate cPLA2 activation. Pan-PKC inhibition potentiated ADP-induced, P2Y1 receptor-mediated calcium mobilization in platelets independent of P2Y12-receptor. Pretreatment of platelets with GF109203X, a Pan PKC inhibitor, but not Go-6976, a classical PKC isoform inhibitor, potentiated ADP-induced thromboxane generation. Thus, we investigated the role of various novel class of PKC isoforms (nPKCs) in platelets. We have previously demonstrated that nPKC η, θ, δ positively regulates agonist-induced thromboxane generation in platelets. Thus, we investigated if the role of nPKC ε in ADP-induced thromboxane generation using PKC ε knockout mice (PKCε KO). ADP-induced thromboxane generation in PKC ε KO murine platelets was ten-fold higher than that of wild type platelets. Furthermore, PKC ε KO mice exhibited shorter times to occlusion in FeCl3-induced arterial injury model and shorter bleeding times in Tail bleeding experiments. We conclude that PKCε negatively regulates ADP-induced thromboxane generation in platelets and thereby offers protection against thrombosis.

Disclosures:

No relevant conflicts of interest to declare.