bluuebunny/crossref_metadata_2025 huggingface dataset:
The Influence of Polymorphisms of Interleukin-17F Genes On the Susceptibility and Clinical Outcome of Multiple Myeloma.
Authors: Saitoh Takayuki , Kasamatsu Tetsuhiro , Yokohama Akihiko , Handa Hiroshi , Tsukamoto Norifumi , Karasawa Masamitsu , Ogawara Hatsue , Nojima Yoshihisa , Murakami Hirokazu
Published: October 2019

Abstract 1784

Poster Board I-810

Introduction

Recent several cytokine studies have shown dysfunctional T cell responses in multiple myeloma (MM) patients. Regulatory T (Treg) cells, is essential for dominant immunologic tolerance. This decreased number and function of Treg cells in MM has been reported, leading to dysfunctional T cell responses. Interleukin-17 F(IL-17F) is a relatively new cytokine that regulates the adaptive and innate immune systems. In vivo studies in murine disease indicate that the Th17 lineage plays a pathogenic role in autoimmune disease. IL-17 polymorphism has been implicated in autoimmunity, including ulcerative colitis and asthma. Polymorphisms were studied, including the coding-region sequence variant single nucleotide polymorphism rs763780 (7488T/C), which causes a His-to-Arg substitution at amino acid 161 (H161R). We examined the single nucleotide polymorphisms (SNPs) in the promoter regions of the IL-17 genes in patients with MM, and analyzed the relationship between IL-17 SNPs and clinical features.

Methods

Eighty one patients with MM [age range, 40-83 years; stage I (n=9), stage II (n=21), stage III (n=51); IgA(n=13), IgG(n=48), IgD(n=1), non-secretary (n=3), Bence Jones(n=16)] and 100 healthy controls were included. Genomic DNA was isolated from peripheral blood using the DNA Kit (QIAGEN, Hilden, Germany). Genotyping in IL-17F was determined by PCR based technique. Genotype and allele frequencies were compared between the study groups using χ2-test. The characteristics and laboratory features of the MM patients with each IL-10 promoter polymorphism were compared using χ2-tests and student t-tests. Probability values <0.05 were considered statistically significant.

Results

The frequencies of the genotypes in MM patinets were as follows: TT (76%), TC (18%), and CC (6%). Patients with MM had a higher frequency of The IL-17F CC genotype compared with healthy controls (P<0.05). No significant difference in the clinical presentation at diagnosis was indicated according to IL-17 polymorphism considering sex, Ig type, Durie-Salmon staging system, and international staging system.

Conclusion

Homozygosity of the H161R variant was associated with susceptibility of MM. These observations suggest that IL-17 may affect dysfunctional T cell responses in MM.

Disclosures

No relevant conflicts of interest to declare.
Gadolinium Containing Contrast Agent Promotes Multiple Myeloma Cell Growth: Implication for Clinical Use of MRI in Myeloma.
Authors: Fulciniti Mariateresa , Sundararaman Swaminathan , Nanjappa Puru , Amin Samir B , Chevireddy Prajwal , Gokden Neriman , Hiatt Kim , High Whitney , Shammas Masood A , Prabhala Rao , Hideshima Teru , Tassone Pierfrancesco , Anderson Kenneth C. , Shah Sudhir V. , Munshi Nikhil C.
Published: October 2019

Abstract 1809

Poster Board I-835

Bone marrow infiltration by myeloma cells and osteolytic bone lesions are the major features of Multiple Myeloma. Magnetic Resonance Imaging (MRI) has been used in MM not only to image bone marrow (BM) and to identify lytic bone disease but to also evaluate therapeutic response and prognosis. Gadolinium (Gd)-based contrast agents are frequently used to enhance MRI resolution. We evaluated effect of the most common Gd-containing agent, Omniscan, on myeloma cells. We observed that Omniscan induced both time and dose dependent MM cell growth in vitro (8-20 fold increase relative to control). Importantly, the presence of BMSC enhanced the effect of Omniscan on growth of both MM cell lines and primary MM cells. However, Omniscan was not able to overcome cytotoxic effects of conventional and novel agents in MM. This growth promoting effects were not observed on normal BM stromal cells. Evaluating the molecular mechanism of action of Omniscan on MM cells, we observed time dependent ERK1/2 phosphorylation as well as reversal of growth promoting effects of Omniscan by specific inhibition of ERK signaling; however, Omniscan had no effect on STAT3 and AKT signaling pathways. Next, we investigated in vivo effect of Omniscan in a murine xenograft model of MM. Following detection of tumor, mice were treated with either iv Omniscan or PBS. Treatment with Omniscan significantly induced MM tumor growth compared to control mice (1042 ±243 mm3 vs 502 ±137 mm3 respectively; p=0.0001). Finally in autopsies in 8 MM patients with repeated exposure to Omniscan, we quantified gadolinium in various tissues using Inductively-coupled mass spectrometry. We observed massive quantities of gadolinium accumulation in tissues of these MM patients regardless of their renal function. These results, confirming both in vitro and in vivo growth promoting effects of Gd-containing contrast agent on MM, suggest the need for further analysis of the mechanism of its action on myeloma cells and careful analysis of its clinical impact in MM patients undergoing MRI evaluation.

Disclosures

No relevant conflicts of interest to declare.
A Clinically Usable Gene Expression Report for Risk Assessment and Identification of Possible Therapeutic Targets in Multiple Myeloma.
Authors: Meiβner Tobias , Seckinger Anja , Hielscher Thomas , Rème Thierry , Moreaux Jerome , Benner Axel , Bertsch Uta , Jauch Anna , Neben Kai , Möhler Thomas , Klein Bernard , Goldschmidt Hartmut , Hose Dirk
Published: October 2019

Abstract 1829

Poster Board I-855

Introduction

In addition to current clinical and cytogenetic risk factors, several highly predictive gene expression based risk stratifications have been proposed in multiple myeloma. At the same time, putative drugable targets have been identified which are only expressed in a subpopulation of myeloma patients (e.g. AURKA). Whereas assessment of both works well within a clinical trial or an experimental setting, they can currently not readily be applied to clinical routine.

Methods

As reference a group of 300 Affymetrix U133 Plus 2.0 DNA microarrays from patients with multiple myeloma is preprocessed using GC-RMA. Quality control of the DNA microarrays is implemented according to the MACQ-Project. Gene expression based prediction of sex, immunoglobulin- and light chain type is used as sample identity-test within a multicenter-setting. Gene expression based risk stratification (IFM-score, 70-gene high risk score, gene expression based proliferation index) and molecular classifications are assessed as published, as are individual target genes e.g. AURKA. To classify a patient within a prospective clinical routine setting, the documentation by value strategy (Kostka & Spang, 2008) was adapted for GC-RMA preprocessing and is used for documenting the quantitative preprocessing information of the reference group. The gene expression based report is developed in the open source language R, containing a GUI based on Gtk2, and the final report is created as a PDF-file.

Results

We present here our publicly available (http://code.google.com/p/gep-r) open source software-framework (GEP-R) that allows creating a gene expression based report from Affymetrix raw-data. The risk stratification of an individual patient is assessed and based on saved preprocessing information of a reference cohort by treating the individual patient's expression data as being part of this group, assuring comparable risk stratification. Results can be interpreted and commented within the report and a PDF based document be created. The generation of the report can be performed within short time on a standard computer.

Conclusion

Gene expression reporting allows validated assessment of risk and of individual therapeutic targets in myeloma patients within a clinical routine setting.

Disclosures

No relevant conflicts of interest to declare.
Significant Impact of IL-17A Gene Polymorphism On Transplant Outcomes After HLA-Fully-Matched Unrelated Bone Marrow Transplantation.
Authors: Takami Akiyoshi , Espinoza J. Luis , Onizuka Makoto , Kawase Takakazu , Katagiri Takamasa , Ohtake Shigeki , Morishima Yasuo , Fukuda Takahiro , Kodera Yoshihisa , Nakao Shinji
Published: October 2019

Abstract 2305

Poster Board II-282

IL-17 plays an active role in autoimmune diseases and organ transplant rejection through its mediating proinflammatory responses and inducing inflammatory cytokine and chemokine production. This study examined the impact of donor and recipient polymorphism in the IL-17A gene on the clinical outcomes in unrelated HLA-fully-matched myeloablative bone marrow transplantation (BMT) through the Japan Marrow Donor Program. The IL-17A polymorphism (rs2275913, G197A) was retrospectively analyzed in a total 145 recipients with hematologic malignancies and their unrelated donors. In patients with standard risk disease, the donor IL-17A-197A genotype was associated with significantly improved OS (adjusted HR, 0.32; 95% CI, 0.13-0.79; p=0.01) and reduced transplant related mortality (TRM) (adjusted HR, 0.27; 95% CI, 0.11-0.67; p=0.005), but no impact on disease relapse or the development of grades II-IV acute GVHD or chronic GVHD. Patients with standard risk disease receiving transplants from donors with the 197A genotype had better 5-year OS (71% vs. 40%, p=0.02; Figure) and lower 5-year TRM rate (27% vs. 60%, p=0.02). The IL-17A polymorphism did not significantly influence the transplant outcomes in patients with high risk disease. These results suggest transplantation from IL-17A-197A positive donor is advantageous in improving OS and reducing TRM after HLA-matched unrelated BMT for standard risk hematologic malignancies.

Disclosures:

No relevant conflicts of interest to declare.
Autologous Transplantation for Nodular Lymphocyte-Predominant Hodgkin Lymphoma (NLPHL).
Authors: Popat Uday , Hosing Chitra , Fanale Michelle , Medeiros L. Jeffrey , Alousi Amin M. , Giralt Sergio A. , de Lima Marcos J. , Qazilbash Muzaffar H. , Kebriaei Partow , Younes Anas , Khouri Issa , Andersson Borje , Champlin Richard , Anderlini Paolo
Published: October 2019

Abstract 2310

Poster Board II-287

Background:

NLPHL is an uncommon type of Hodgkin lymphoma characterized by nodules composed of large neoplastic B-cells (CD20+), lymphocyte predominant (LP) cells (previously known as L& H cells), associated with many small reactive B-cells. Patients with NLPHL typically have an indolent clinical course, but the disease can recur, often with histologic evidence of progression or transformation. Because of its rarity, limited information is available, mainly from retrospective studies. Very little information is available about salvage therapy particularly about the role of autologous transplantation in this disease. The purpose of this study was to evaluate outcomes of autologous transplantation in patients with relapsed or refractory NLPHL.

Patients and Methods: All patients with NLPHL who underwent autologous transplantation between January 1990 and December 2008 were included in this study. Pathology specimens were reviewed. The diagnosis of NLPHL was confirmed in all cases and, at time of transplant, many patients had histologic evidence of T-cell/histiocyte rich large B-cell lymphoma. During these 19 years, 26 patients (24 males) underwent autologous transplantation. Median age at transplantation was 35 years (range 13-51). At transplantation, 9 (35%) were in complete remission (CR); 13 (50%) were in partial remission (PR); and 4 (15%) had stable (SD) or progressive disease (PD). Median number of prior chemotherapy regimens was 3 (1-4). LDH was elevated in 10 (38%). Peripheral blood stem cells were used as stem cell source in 23 (86%) patients. Conditioning regimen were BEAM(10), BEAM-R(5), CBV(6), and other regimens in 5 patients. Peripheral blood stem cells were used as stem cell source in 23 (86%) patients.

Results:

Following transplant, 22 patients achieved complete remission. No treatment related mortality or treatment related MDS/AML occurred. Seven patients have relapsed. With a median follow up of 50 months (range, 2-138 months), the 5-year overall (OS) and event-free survival (EFS) were 76% (SE 9%) and 69%(SE 10%), respectively. For chemosensitive patients, The 5-year overall (OS) and event-free survival (EFS) were 82% (SE 10%) and 79% (SE 10%), respectively. Three out of 4 patients (75%) with chermoresistant disease relapsed compared to 4 out of 22 (18%) of patients with chemosensitive disease. Five patients died, all of recurrent disease.

Conclusion:

High-dose chemotherapy and autologous transplantation is highly effective in patients with refractory NLPHL or relapsed NLPHL including patients with histologic evidence of transformation to T-cell/histiocyte rich large B-cell lymphoma, and results in long term remission in most of patients.

Disclosures:

No relevant conflicts of interest to declare.
A Pathobiological Role of the Insulin Receptor in Chronic Lymphocytic Leukemia.
Authors: Malek Sami , Parkin Brian , Ouillette Peter , Kujawski Lisa , Erba Harry Paul , Campagnaro Erica L. , Kaminski Mark S. , Shedden Kerby , Saiya-Cork Kamlai
Published: October 2019

Abstract 2324

Poster Board II-301

The presence of genomic aberrations in CLL, including del17p and del11q, affects CLL patient survival, and thus an in-depth understanding of the genes influenced through these deletions is important. Further, genes affecting CLL disease progression that is observed in patients with recurrent genomic deletions, including del11q, remain incompletely characterized. Focusing therefore on del11q in CLL, we employed comparative expression array profiling (Affymetrix 133 2.0 plus arrays) of RNA from sorted CD19+ cells from 10 cases with and nine cases without del11q to identify stable transcriptome differences associated with del11q. Through these efforts we have identified for the first time differential expression of the insulin receptor (INSR) in CLL and predominant INSR expression in CLL with del11q. We have validated this finding in a cohort of 219 CLL cases. Our aggregate data suggest biologically and clinically meaningful INSR expression in a substantial subset of all CLL cases. INSR stimulation by insulin in CLL cells expressing the INSR resulted in the activation of canonical INSR signaling pathways, including the AKT-mTOR and Ras/Raf/Erk pathways, and INSR activation partially abrogated CLL cell apoptosis ex vivo. Clinically, CLL cases with INSR expression displayed larger lymphnodes and higher Rai stage at study enrollment and the disease progressed faster requiring repeat therapy earlier than CLL cases with low or absent INSR expression. This novel data provides the first description of a pathobiological role of the insulin receptor in CLL biology and disease progression, thus identifying a potential mediator and mechanism for del11q-associated CLL phenotypes. The identification of a transmembrane tyrosine kinase receptor with direct effects on CLL biology and clinical behavior offers opportunities for CLL prognostication and targeted therapy development and further strengthens the case for involvement of the INSR in cancer biology.

Disclosures:

Malek: Cephalon: Honoraria, Speakers Bureau; Celgene: Honoraria, Speakers Bureau; Affymetrix: Research Funding. Erba:Lilly: Research Funding; Antisoma: Research Funding; Wyeth: Research Funding; Cephalon: Honoraria, Research Funding; MGI Pharma: Honoraria; Pharmion: Honoraria; Celgene: Honoraria; BMS: Honoraria; Novartis: Honoraria, Research Funding; Genzyme: Consultancy, Honoraria, Research Funding; Gemin-X: Research Funding; Kanisa: Research Funding.
Chronic Lymphocytic Leukaemia Drives the Production of T Regulatory Cells From the CD4+ CD25- Compartment with a CD27high/CD127 Low Phenotype.
Authors: Piper Karen , Karanth Mamtha , McLarnon Andrew , Kalk Emma , Khan Naeem , Murray Jim A , Pratt Guy , Moss Paul
Published: October 2019

Abstract 2330

Poster Board II-307

Patients with chronic lymphocytic leukaemia (CLL) have defects in both cellular and humoral immunity including changes in the numbers and function of T regulatory cells (Tregs). The identification of Tregs is an ever-evolving field and in this study we readdressed the phenotype using the markers CD25, FoxP3 and CD127-/lo and confirmed function by classical suppressor assays in CLL patients on and off treatment. Using the combination CD4 and FoxP3 we observed increased Treg frequencies in CLL patients, in particular with advanced disease, supporting previous studies showing an increase in Tregs in CLL. However in contrast to previous studies, there was no increase in the CD25+ FoxP3+ population in CLL patients rather the increase in FoxP3 expression occurred in the CD25- compartment of CLL patients. Interestingly CLL induced a 7-fold increase in the expression of FoxP3 in CD4+CD25- T cells following short-term co-culture. The T regulatory cells in CLL patients had a significantly higher expression of CD27 compared to healthy controls and although CD127 expression was low in both healthy and CLL patients it was significantly lower in CLL patients.

Fludarabine treatment initially induced increased expression of FoxP3 in the CD4+ T cell compartment but this declined gradually to reach levels below that pre-treatment. Here we propose that CLL drives the production of Tregs from the CD4+CD25- compartment as has been shown recently in Non-Hodgkins Lymphoma (NHL) and the mechanism of induction could provide alternative avenues for treatment.

Disclosures:

No relevant conflicts of interest to declare.
SHIP1 and PTEN Co-Inactivation and Akt Pathway Targeting in ALL.
Authors: Diccianni Mitchell B. , Barnhill Lisa M. , Lo Tony , Yu Alice L.
Published: October 2019

Abstract 2386

Poster Board II-363

Despite success in the treatment of Acute lymphoblastic leukemia (ALL), relapse and death continue to occur and in survivors, secondary malignancies due to the impact of aggressive chemotherapy negatively impact the quality and duration of life. Akt plays a central role in signal transduction and is negatively regulated by PTEN and, in cells of hematopoietic lineage, SHIP1; it is positively regulated by PIK3CA. Despite multiple levels of regulation, Akt is aberrantly activated in many cancers including ALL. To understand the mechanism behind Akt activation in ALL, PIK3CA, PTEN and SHIP1 genes were assessed in leukemia cell lines and primary samples. No sample harbored PIK3CA mutation. PTEN was expressed in just one-third of the cell lines analyzed, but in two-thirds of the primary ALL of both T- and B-lineage, though all were in the phosphorylated (inactivated) form. SHIP1 was expressed in all leukemia cell lines, except for Jurkat and K562 cell lines. In general, expression of SHIP1 protein was much higher in cell lines of the B-lineage than of the T-lineage. The Jurkat T-ALL cell line, long observed to lack SHIP1 protein, harbored biallelic inactivation by null mutation and a frame-shift deletion; no SHIP1 mutations were identified in the K562 cell line. In contrast to leukemia cell lines, no full length SHIP1 was detected in primary T-ALL, and only ∼20% of the primary B-precursor ALL expressed full length SHIP despite the expression of full length transcript. Both ALL lineages expressed truncated isoforms of SHIP, which cloning and sequence analysis revealed was due to premature termination resulting from the frame-shift and other translationally-inactivating alternative splicing. The co-inactivation of SHIP and PTEN may create therapeutic opportunities. The PI3KCA inhibitor LY294002 has been shown to sensitize glioblastoma and colon carcinoma to doxorubicin. To determine if these agents acted synergistically in T-ALL, cell lines Molt16 (SHIP1+/PTEN-), Jurkat (SHIP1-/PTEN-) and normal MNCs (SHIP1+/PTEN+) were subjected to various combinations of the PIK3CA inhibitor LY294002 and doxorubicin and cell proliferation and viability assessed. Normal cells were generally insensitive to both agents except at high concentrations. LY294002 and doxorubicin inhibited viability in Molt16 with IC50s of 10 uM and 0.1 ug/ml, respectively, with no evidence of synergy. Jurkat cells exhibited a similar IC50 to LY294002, but were more sensitive to doxorubicin (IC50s of 10 uM and 0.025 ug/ml, respectively), with the two agents demonstrating some synergy when added simultaneously at low concentrations. In summary, we present evidence of Akt pathway deregulation in T-ALL due to SHIP1/PTEN inactivation and suggest that agents targeting this pathway may provide new directions into the therapeutic treatment of T-ALL.

Disclosures:

No relevant conflicts of interest to declare.
Pseudonormokalemia in Patients with Thrombocytosis.
Authors: Goldman Raimonda , Steinberg Amir , Carreiro Jennifer , Panagopoulos Georgia , Cooper Marvin , Levine Randy L.
Published: October 2019

Abstract 2420

Poster Board II-397

Introduction:

Pseudohyperkalemia represents an artificial elevation in serum potassium concentration. It is well described that patients with thrombocytosis may have elevated serum but normal plasma potassium. The difference between serum and plasma potassium is felt to be due to potassium release from platelets during clotting. We propose that a similar mechanism will lead to “pseudonormokalemia,” where serum potassium appears to be in the normal range (3.5-5.0 MEq/L) despite below-normal levels in the plasma(<3.5 MEq/L).

Method:

The interim analysis of this study was sent for consideration to ASH Annual Meeting on November 2006 (108: 3964) We now present the final analysis by comparing 69 thrombocytosis patients (platelets>500,000/uL) to 68 control patients (platelets<500,000/uL). Patients were identified from a list of lab results generated by a computer search and serum and plasma potassium and CBC were then drawn simultaneously.

Results:

There was no statistically significant difference in sex distribution or age between the two groups. The average platelet count was 667,960/uL in the thrombocytosis group (SD 151,1 uL) and 286,790/uL (SD 103,9 uL) in the control group (p value <0.001). In both groups, serum potassium was higher than plasma potassium. The analysis comparing serum potassium levels between the two groups showed that serum potassium was significantly higher in the thrombocytosis group than in the control. In fact, we found a strong positive correlation (spearman's rho=0.61, p<.001) between platelet count and the difference in values in serum potassium minus plasma potassium. As the platelet count increases, so does the difference between the serum and plasma potassium. The thrombocytosis group was noted to have a difference between serum and plasma potassium of .51 MEq/L (SD .30 MEq/L) while the control group had a difference of .21 MEq/L (SD .20 MEq/L)

Conclusion:

Patients with thrombocytosis and normal serum potassium may actually be hypokalemic as this study demonstrates.

Disclosures:

No relevant conflicts of interest to declare.
The Quality and Accuracy of Hemophilia Information on the Internet.
Authors: Breakey Vicky R , Ignas Danial M , Denburg Avram E , Blanchette Victor S.
Published: October 2019

Abstract 244

The Internet has become a major source of health information for our patients, especially those with chronic illnesses. Hemophilia is an inherited bleeding disorder that is due to a deficiency of coagulation factor VIII (hemophilia A) or Factor IX (hemophilia B). There are many websites devoted to hemophilia, but currently there are no data on the quality of the information provided on the Internet. To assess the quality, accuracy and readability of information related to hemophilia that is available on the Internet, three reviewers (two physicians and one lay person with severe hemophilia A) reviewed the first three pages of hits for the search term “hemophilia” on the four most popular Internet search engines. The validated DISCERN rating instrument for written patient information was applied to assess the general quality of the information (Charnock et al. 1999). Content was assessed for breadth and accuracy using an adapted Checklist Rating System Instrument (Bichakjian et al. 2002). The World Federation of Hemophilia's “Guidelines for the Management of Hemophilia” were referenced as the gold standard of hemophilia information. Readability was assessed using the Flesch Reading Score and the Flesch-Kinkaid Grade level score. After omission of duplicates and inaccessible sites, a total of 31 websites were reviewed. The sites were 42% commercial, 29% institutional and 29% charitable. Reviewers showed good inter-rater reliability when assessing overall quality with the DISCERN tool, with a quadratic weighted kappa >0.6. Four websites (13%) contained a total of 15 inaccuracies. Readability of the five most highly-rated websites suggests that content is written at the equivalent of a grade 10/11 level, much higher than the grade 8 level suggested for patient education materials. In conclusion, the 31 websites containing hemophilia-related information varied widely in the content and quality of information with 13% of sites citing inaccuracies. Hemophilia treatment centers should be aware of the hemophilia resources available on the Internet and must guide patients in finding the most comprehensive and accurate information. Health care providers should be actively involved in the development of thorough and accurate web-based hemophilia education that is written at an appropriate level to meet the needs of our patients.

Disclosures:

Breakey: Baxter: Educational Grant for Hemophilia Patient Education. Blanchette:Baxter Healthcare Corporation: Educational Grant for Work in Hemophilia, Honoraria.
Targeting PKCtheta Inhibits Alloreactivity and Graft-Versus-Host Disease While Preserving Graft-Versus-Leukemia and Anti-Infection Activities.
Authors: Yu Xue-Zhong , Valenzuela Javier , Iclozan Cristina , Hossain Mohammad , Hopewell Emily , Bronk Crystina , Wang Junmei , Celis Esteban , Engelman Robert , Waller Edmund K. , Blazar Bruce R. , Beg Amer
Published: October 2019

Abstract 2458

Poster Board II-435

Introduction:

When used as therapy for hematopoietic malignances, allogeneic hematopoietic stem cell transplantation (HCT) relies on the graft-versus-leukemia (GVL) effect to eradicate residual tumor cells through immunologic mechanisms. However, graft-versus-host-disease (GVHD) is a potentially lethal complication of allogeneic BMT. Thus, inhibition of GVHD, while preserving GVL and protective responses against infectious agents, can enhance the therapeutic potential of BMT. GVHD is initiated by alloreactive donor T cells that recognize mismatched major and/or minor histocompatibility antigens and cause severe damage to hematopoietic and epithelial tissues. Protein kinase C theta isoform (PKCθ) is crucial in TCR signaling and regulates the nature of lymphocyte-specific effector responses. The aim of this study was to investigate the significance of PKCθ in donor T-cell-mediated GVHD, anti-leukemia, and antiviral infection.

Methods:

Our results were validated using clinically relevant murine bone marrow transplantation (BMT) models. GVHD was measured by recipient survival and clinical signs such as body weight loss. Leukemia/lymphoma was induced by intravenous injection of a luciferase expressing A20 clone (a B lymphoma cell line derived from BALB/c mice). Tumor invasion was quantitated using the Xenogen IVIS-200 bioluminescence imaging system. Murine cytomegalovirus (MCMV) infection was induced by intraperitoneally injecting MCMV in the recipients after GVHD was established.

Results:

Using 3 distinct mouse models of allogeneic BMT, we found that T cells lacking PKCθ are unable to undergo robust expansion and cause damage to recipient hematopoietic or epithelial tissues, demonstrating that an essential requirement for PKCθ in alloreactivity and GVHD development. In contrast, PKCθ-/- T cells retain the ability to respond to virus infection post-BMT. Mechanistically, absence of PKCθ raises the threshold for T cell activation that selectively impacts alloresponses, but T cell responses triggered by infection or following administration of antigen plus microbial adjuvant are relatively well preserved in the absence of PKCθ Furthermore, we evaluated the role of PKCθ in GVHD and GVL responses, and found that PKCθ-/- T cells have at least a 10-fold reduction in the ability to induce GVHD but only a ∼2.5-fold reduction in GVL compared to WT T cells. Thus, absence of PKCθ impacts GVHD responses more significantly than GVL responses.

Conclusion:

These findings validate PKCθ as a potentially unique therapeutic target that is required for GVHD induction but not for GVL or protective responses to infectious agents in allogeneic BMT.

Disclosures:

No relevant conflicts of interest to declare.
Niche Induced Myelodysplasia and Secondary Hematopoietic Neoplasia Caused by Deletion of Dicer1 in Osteoprogenitor Cells.
Authors: Raaijmakers Marc HGP , Mukherjee Siddhartha , Guo Shangqin , Kobayashi Tatsuya , Schoonmaker Jesse , Aung Zinmar , Ebert Benjamin L. , Al-Shahrour Fatima , Hasserjian Robert P , Vallet Sonia , Scadden Edward , Lin Charles P , Scadden David T.
Published: October 2019

Abstract 247

Introduction:

Mesenchymal cells are a part of virtually every tissue in metazoans and are thought to participate in organ formation and homeostasis. In the hematopoietic system, mesenchymal cells of the osteoblast lineage have revealed their role as regulators of normal stem cell and hematopoietic physiology. Whether these cells, which have been relegated a relatively non-descript role of ‘stroma', participate in processes that result in disease is relatively understudied.

Methods:

To explore this, we conditionally deleted Dicer1, the endonuclease essential for miRNA biogenesis, from osteoprogenitor cells by intercrossing transgenic mice expressing a GFP-Cre recombinase under the transcriptional control of the osteoblastic lineage specific osterix promoter to mice containing conditional (floxed) Dicer1 alleles.

Results:

Deletion of Dicer1 from osteoprogenitor cells resulted in markedly disordered hematopoiesis, affecting multiple lineages and recapitulating key features of human myelodysplastic syndrome (MDS). These features included ineffective hematopoiesis with cytopenia, multilineage dysplasia, increased proliferation and intramedullary apoptosis of primitive hematopoietic cells, decreased B-cell progenitors, increased bone marrow vascularity and the propensity to develop hematopoietic neoplasms (myeloid sarcoma and acute monocytic leukemia-like disease). Comparative genomic hybridization of tumor and germline tissues revealed multiple genetic aberrancies in myeloid sarcomas induced by the Dicer1 deleted environment. The hematopoietic abnormalities were entirely microenvironment dependent with intact Dicer1 in hematopoietic cells. Transplantation of wild-type hematopoietic cells into the mutant environment recapitulated these abnormalities whereas, conversely, transplantation of hematopoietic cells from mutant mice into a wild-type environment resulted in complete normalization of hematopoiesis. In addition, hematopoietic abnormalities were not observed when Dicer1 was deleted from mature osteoblasts indicating a central role of osteoprogenitor cells in the regulation of hematopoiesis. Finally, gene expression profiling and cytokine arrays from osteoprogenitor cells identified candidate molecular effectors of the observed hematopoietic abnormalities.

Conclusions:

The data demonstrate that a distinctive, differentiation stage specific, stromal subset of osteolineage cells can induce a highly dysfunctional hematopoietic system, recapitulating key characteristics of human myelodysplastic syndrome. Further, they reveal that primary changes in a tissue microenvironment can initiate neoplastic disease.

Disclosures:

Scadden: Fate Therapeutics: Consultancy.
Adjudicating Bleeding Outcomes in a Large Thromboprophylaxis Trial in Critical Illness.
Authors: Arnold Donald M. , Rabbat Christian , Lauzier Francois , Zytaruk Nicole , Heels-Ansdell Diane , Cook Deborah
Published: October 2019

Abstract 2471

Poster Board II-448

Background:

Adjudication in clinical trials can confirm or refute eligibility, describe cointerventions, judge appropriateness of care, or assess the severity of morbidity outcomes.

Objective:

To refine the adjudication process, calibrate 4 adjudicators, and measure agreement on bleeding severity in an international trial of heparin thromboprophylaxis (PROTECT).

Methods:

Independently and blinded to each others' ratings and study drug, 4 adjudicators used web-based methods to examine 40 charts of patients assessed by local research coordinators to have either major (20 patients) or minor (20 patients) bleeding. We discussed reasons for disagreement after the first 20 charts to remediate and recalibrate. We calculated crude agreement, chance-corrected agreement (kappa), and chance-independent agreement (phi).

Results:

For 45 events in 40 patients, pair-wise crude agreement ranged from 86.7-93.3% (average 82.2%). Overall kappa was 0.81. Phi (which can only analyze pair-wise values) ranged from 0.75-0.87. We resolved all disagreements. During adjudication discussions, we 1) addressed methodological issues (e.g., handling recurrent bleeds), 2) added a category (e.g., no bleed), 3) expanded a category (e.g., a major bleed did not have to be overt if other criteria were fulfilled), and 4) divorced procedure-grounded definitions (such as the need for transfusion or therapeutic interventions) from bleeding severity criteria (e.g., the patient could still be classified as having no bleed or a minor bleed if 2 units of PRBCs were transfused for anemia).

Conclusions:

After independent quadruplicate review of 45 bleeding events, we documented satisfactory agreement for bleeding outcomes, refined the adjudication process, and calibrated adjudicators for the remainder of the trial. Henceforth, charts will be randomly allocated to pairs of adjudicators for blinded review.

Disclosures:

No relevant conflicts of interest to declare.
Cost-Effectiveness of Filgrastim and Pegfilgrastim as Primary Prophylaxis against Febrile Neutropenia in Lymphoma Patients Receiving R-CHOP Chemotherapy.
Authors: Lathia Nina , Isogai Pierre K , Walker Scott , Cheung Matthew C , Krahn Murray , DeAngelis Carlo , Mittmann Nicole
Published: October 2019

Abstract 2475

Poster Board II-452

Introduction:

Non-Hodgkin Lymphoma (NHL) is the fifth most common type of malignancy in Canada. The most common subtype of NHL is diffuse large B-cell lymphoma (DLBCL). Initial standard treatment for DLBCL includes combination immuno-chemotherapy with rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP). This regimen is typically administered every 21 days for a total of 6 cycles. Febrile neutropenia (FN) is a serious toxicity of lymphoma chemotherapy and patients with this condition must be treated aggressively as it could lead to complications such as prolonged hospitalization and death. Granulocyte-colony stimulating factors such as filgrastim and pegfilgrastim are efficacious in preventing FN, yet their cost-effectiveness has not been evaluated in the publicly funded Canadian healthcare system.

Methods:

A Markov model was constructed to evaluate the cost-effectiveness (cost-utility) of filgrastim and pegfilgrastim as primary prophylaxis versus no primary prophylaxis against FN in DLBCL patients receiving induction chemotherapy. Health states included in the model were hospitalization for FN, and receiving chemotherapy with no FN. It was assumed that patients in the no primary prophylaxis arm of the model who experienced a FN episode would receive secondary prophylaxis with filgrastim for all subsequent chemotherapy cycles. The time horizon of the model was 18 weeks, the time period over which the six cycles of chemotherapy are administered. The analysis was conducted from the hospital perspective. Costs are reported in 2009 Canadian dollars and outcomes in quality-adjusted life years (QALY). One-way sensitivity analyses were done on model parameters. A probabilistic sensitivity analysis was done to evaluate overall uncertainty in the model.

Results:

In the base case analysis costs associated with the no primary prophylaxis, filgrastim and pegfilgrastim interventions were $6044, $9450 and $15899, respectively. Quality-adjusted life years associated with the three interventions were 0.198, 0.200, and 0.202 respectively (over the 18-week model time horizon). The incremental cost-effectiveness ratio (ICER) of filgrastim compared to no primary prophylaxis was estimated to be $1.7 million (M)/QALY [95% confidence interval: -14M/QALY (dominated) to $15M/QALY]; for pegfilgrastim compared to filgrastim the ICER was estimated to be $4.4M/QALY [95% confidence interval: -25M/QALY (dominated) to $22.7M/QALY]. All one-way sensitivity analyses yielded ICERs of greater than $1M/QALY.

Conclusions:

The ICERs for filgrastim and pegfilgrastim when used as primary prophylaxis against FN in DLBCL patients receiving induction chemotherapy are well above the usually accepted cost-effectiveness threshold of $50,000/QALY.

Disclosures:

Mittmann: Amgen Canada: Unrestriced educational grant.
PI3 Kinase, Phospholipase C (PLC)-γ, and RasGRPs Act Cooperatively to Activate the Ras-Extracellular-Related Kinase (ERK) Pathway in Response to Cytokines in Normal and Kras Mutant Myeloid Cells.
Authors: Diaz-Flores Ernesto , Goldschmidt Hana Leah , Depeille Philippe , Ng Victor , Roose Jeroen , Shokat Kevan , Braun Benjamin S. , Shannon Kevin
Published: October 2019

Abstract 2512

Poster Board II-489

Introduction: KRAS is the most common gain-of-function mutation in human cancer and occur in some myeloid malignancies. Despite extensive data implicating aberrant responsiveness to cytokines in the pathogenesis of myeloid leukemias with hyperactive Ras, the underlying mechanisms are largely unknown. We investigated the biochemical basis of hypersensitivity to granulocyte macrophage colony stimulating factor (GM-CSF), IL-3, and stem cell factor (SCF) in primary cells from Mx1-Cre, KrasG12D mice. Methods: We utilized conventional biochemical techniques, phospho-specific flow cytometry, and small molecule inhibitors to interrogate signaling downstream of the activated GM-CSF and c-Kit receptors. Results: We found that endogenous KrasG12D expression deregulates multiple effectors in primary myeloid lineage cells. Although Ras-GTP levels are markedly elevated in bone marrow cells from KrasG12D mice, these cells only fully activate the Raf/MEK/ERK cascade upon cytokine stimulation. We used a “chemical genetic” strategy to explore the biochemical mechanism linking the activated GM-CSF receptor to oncogenic K-RasG12D and ERK. Surprisingly, inhibitors of type I PI3 kinases (PI-90, PI-103) as well as a p110 γ/α isoform inhibitor (AS605240) interfered with GM-CSF and SCF induced Ras/MAPK activation in wild-type and KrasG12D myeloid cells. These effects of PI3 kinase are mediated through activation of PLC-γ and the RasGRP guanine nucleotide exchange factors. Functional studies confirmed that inhibiting PI3 kinase or PLC-γ blocked KrasG12D-mediated myeloid progenitor colony growth. This PI3 kinase/PLC-γ module also functions downstream of the activated c-kit receptor in lineage- cKit+ Sca1+ (LSK) cells in SCF-induced ERK activation. Conclusions: PI3 kinase, PLC-γ, and RasGRPs function upstream of normal and oncogenic K-Ras to efficiently activate the Raf/MEK/ERK cascade in primary hematopoietic cells. These data infer that PI3 kinase is both an important upstream regulator as well as a major downstream effector of Ras-GTP. Significance: Targeting PI3 kinase in hematologic cancers characterized by hyperactive Ras may have unanticipated biochemical and functional consequences in malignant and normal hematopoietic cells.

Disclosures:

No relevant conflicts of interest to declare.
NIPA Phosphorylation and Inactivation at G2/M Is Mediated by ERK2.
Authors: Zech Michael , Illert Anna Lena , Albers Corinna , Bassermann Florian , Peschel Christian , Duyster Justus
Published: October 2019

Abstract 2513

Poster Board II-490

Regulated oscillation of protein expression is an essential mechanism of cell cycle control. The SCF class of E3 ubiquitin ligases is involved in this process by targeting cell cycle regulatory proteins for degradation by the proteasome. We previously reported cloning of NIPA (Nuclear Interaction Partner of ALK) in complex with constitutively active oncogenic fusions of ALK, contributing to the development of lymphomas and sarcomas. Subsequently we characterized NIPA as a F-Box protein that defines an oscillating ubiquitin E3-ligase. The SCF-NIPA complex targets nuclear cyclin B1 for ubiquitination in interphase while phosphorylation of NIPA in late G2 phase and mitosis inactivates the complex to allow for accumulation of cyclin B1, a critical event for proper G2/M transition. Thus, SCF-NIPA executes an important G2/M checkpoint control. We recently specified three serine residues Ser 354, 359 and 395 implicated in NIPA phosphorylation at G2/M. These data suggest a sequential NIPA phosphorylation, where initial Ser 354 and 359 phosphorylation is most crucial for SCF-NIPA inactivation by dissociating the SCF-NIPA complex. Here we aimed to find the kinase responsible in this initial most important phosphorylation step. Using in vitro kinase assays we identified both ERK1 and ERK2 to phosphorylate NIPA with high efficiency. Mutation of either Ser 354 or Ser 359 abolished ERK-dependent NIPA phosphorylation. Inhibition of ERK1/2 activity in cell lines by specific inhibitors resulted in decreased NIPA phosphorylation at G2/M. To differentiate between phosphorylation by ERK1 and ERK2, we combined cell cycle analysis with stable expression of microRNA's targeting both isoforms. To this end NIH/3T3 cells were retrovirally transduced with microRNAs targeting ERK1 and 2 and cell cycle progression was analysed by BRDU/PI labeling. Using this approach, we are able to show that ERK2 but not ERK1 mediates NIPA phosphorylation at G2/M. Furthermore, ERK2 silencing leads to a distinct phenotype in cell cycle progression with a delay of ERK2 knockdown cells at the G2/M transition. Thus, our data indicate, that the recently described divergent functions of ERK1 and ERK2 in cell cycle regulation could be in part due to the differential ability of these kinases to phosphorylate and inactivate NIPA at G2/M. Since checkpoint proteins such as NIPA are constitutively inactivated in tumor cells ERK2 might represent an interesting target to reconstitute important cell cycle checkpoint controls in malignant cells.

Disclosures:

No relevant conflicts of interest to declare.
A Sensitized Screen Uncovers a Novel Platelet Phenotype in a Loss-of-Function Jak2 Allele.
Authors: Anderson Nicole , Javed Mojib Javadi , Bailey Monica L , Huang Kai , Berberovic Zorana , Flenniken Ann , Wang Chen , Minden Mark D. , Paulson Robert , McNagny Kelly M. , Stanford William L. , Barber Dwayne L
Published: October 2019

Abstract 2516

Poster Board II-493

Our group has undertaken a concerted effort to develop novel heritable mouse strains via ethylnitrosourea (ENU)-based random mutagenesis. We have complemented this strategy to perform a dominant sensitized screen to identify mouse mutants with altered recovery following treatment with 5-fluoruracil (5-FU). We isolated a heritable line with elevated platelets as well as an overshoot in platelet numbers when exposed to 5-FU. Affected mice also displayed megakaryocytosis and centrilobular hepatic lipidosis.

The mutation was mapped to a 8.7Mb region on mouse Chromosome 19, which contained the candidate gene Jak2. The entire coding region of Jak2 was sequenced and a mutation in exon 19 was found. The causative mutation was located at nucleotide 2740 and leads to an A to T transversion in exon 19 of JAK2 and causes a substitution of a Lys to a premature stop codon at amino acid 914. The resulting protein product terminates within the JH1 kinase domain of JAK2.

JAK2 914 is distinct from the JAK2 null allele characterized by the Ihle and Pfeffer labs. For example, JAK2−/− embryos die at E12.5, whereas JAK2914/914 embryos survive until E13.5, suggesting that the truncated protein product may serve as a scaffold. Differences were also observed in clonogenic potential of both strains. For example, JAK2+/− mice had increased CFU-C whereas JAK2914/+ were normal. Splenic BFU-E and CFU-C were increased in JAK2+/− mice while both cell types were decreased in JAK2914/+. In contrast, bone marrow CFU-E were increased in both strains of mice and splenic CFU-E were normal in JAK2+/− and JAK2914/+. In addition, both mouse strains display elevated platelets when animals are housed under resting conditions.

The hepatic phenotype is phenocopied by Leptin Receptor null mice that are hyperphagic, poorly cold-adapted, sterile and have enhanced fat conversion resulting in lipidosis.

In conclusion, a sensitized screen has shown that JAK2 regulates the response to the chemotherapeutic drug, 5-fluorouracil. These findings have significance to potential myelosuppressive effects of the clinical use of 5-FU in colorectal and pancreatic cancer.

Disclosures:

No relevant conflicts of interest to declare.
Exo1 Independent DNA Mismatch Repair Pathway in Hematopoiesis.
Authors: Desai Amar , Qing Yulan , Liu Min , Kenyon Jonathan , Nwafor Uchenna , Gerson Stanton L.
Published: October 2019

Abstract 2526

Poster Board II-503

The DNA mismatch repair pathway (MMR) is a fundamental process in cells that functions to correct mispaired bases and insertion/deletion loops caused by errors in replication. Failure in MMR can lead to the accumulation of mutations and carcinogenesis, notably hereditary nonpolyposis colorectal cancer (HNPCC). In hematopoiesis, loss of MMR results in methylating agent resistance and an HSC repopulation defect. Our research focused on the significance of Exonuclease 1, the enzyme responsible for excising mispaired bases in MMR. Interestingly Exo1−/− mice display a much milder pathogenic phenotype when compared to the MMR deficient MSH2−/− mice; characterized by lower mutation rates, lower levels of microsatellite instability, and in humans no association with HNPCC. These findings led us to hypothesize that the limited phenotype observed in Exo1−/− mice is due to (a) complementation through another exonuclease occurs which restores the proficiency of the MMR pathway or (b) an alternative pathway exists which allows for exonuclease independent repair.

To test this hypothesis we derived primary MEFs from WT, Exo1−/− and MSH2−/− mice and performed temozolomide sensitivity assays. We observed that Exo1−/− MEFs are similarly sensitive as WT cells when treated with the drug, confirming proficient MMR, while MSH2−/− MEFs display a decreased sensitivity. Additionally, comet assays with the same cell populations show a persistence of DNA single strand breaks in the Exo1−/− and WT cells 24 hours post temozolomide treatment, consistent with proficient MMR activity, while MSH2−/− MEFs show no such damage. To confirm functional MMR in hematopoiesis of Exo1−/− mice we conducted a competitive repopulation study. We found that Exo1−/− and WT marrow engraft at similar levels 8 and 16 weeks post transplantation. We subsequently treated these mice with 80 mg/kg temozolomide to determine if Exo1−/− marrow progenitors would confer a competitive survival advantage post treatment. Consistent with our hypothesis we found that the ratio of marrow cells remained approximately 50:50 (Exo1:WT) in contrast to data obtained from MSH2−/− mice in which the MSH2−/− cells display a strong 95:5 (MSH2:WT) advantage post drug treatment. This strongly suggests functional MMR in the Exo1 deficient mice. We next coimmunoprecipitated the MMR complex by pulling down Mlh1 in Exo1−/− and WT MEFs. Exo1, a key exonuclease in mismatch repair, does not modulate hematopoietic function as do the other MMR proteins and appears to be complemented by an as yet undefined exonuclease.

Disclosures:

No relevant conflicts of interest to declare.