bluuebunny/crossref_metadata_2025 huggingface dataset:
Transcriptional and Translational Regulation of the Cancer-Testis Antigen PRAME Mediated by Pathogen-Associated Molecular Patterns
Authors: Wadelin Frances R , Spriggs Keith A , Falcone Franco H , Heery David M
Published: October 2019

Abstract 2475

PRAME (preferentially expressed antigen in melanoma) is a germinal tissue-specific gene that is expressed at high levels in a number of haematological malignancies. In AML, PRAME is a positive prognostic indicator, being associated with a favourable response to chemotherapy and increased overall survival, even in the presence of unfavourable karyotypes. In contrast, over-expression of PRAME mRNA is associated with poor prognosis in CML, NHL, HD, CLL and myeloma, being associated with progressive disease and chemo-resistance. Despite proposals to implement PRAME as a tool for monitoring minimal residual disease and as an immunotherapeutic target, the physiological functions of PRAME in normal and tumour cells are unclear. A role in the regulation of retinoic acid signalling has been proposed, although interactions of PRAME with retinoic acid receptors are weak compared to other coregulator proteins. Sequence homology and structural predictions suggest that PRAME is a member of the family of leucine-rich repeat (LRR) proteins that function in cellular immunity, bearing most resemblance to the LRR domains of Toll-like receptors (in particular TLR3 and TLR4) and internalin proteins. LRR proteins can bind pathogen-associated molecular patterns (PAMPs) including proteins, nucleic acids and sugars, and consistent with this PRAME has been reported to interact with OPA proteins from Neisseriae gonorrhoeae. Here we show that expression of PRAME in HL60 leukaemic cells is strongly induced by lipopolysaccharide (LPS) or peptidoglycan in the presence of interferon (IFN)-gamma, whereas no induction was observed after exposure to other PAMPs such as zymosan A. This burst of PRAME expression peaked between 1 and 4 hrs post treatment, but was not mediated by the proximal promoter region. Moreover, we show that treatment of HL60 with LPS and IFN-gamma increases the association of PRAME transcripts with polysomes indicating PRAME is also translationally regulated, and we provide evidence that PRAME 5'UTR contains an internal ribosome entry sequence for CAP-independent translation. Finally, we show that the subcellular localisation of PRAME protein in HL60 and U937 cells is modulated by treatment with LPS and IFN-gamma. In summary, our studies indicate that the LRR protein PRAME is subject to transcriptional and translational regulation following cellular exposure to bacterial PAMPs and interferon, suggesting it may have a role in innate immunity.

Disclosures:

No relevant conflicts of interest to declare.
Gene Expression Profiling Using the Illumina ‘DASL’ Platform on RNA Extracted From Formalin Fixed Paraffin Embedded (FFPE) Tissue Identifies Distinct Prognostic Groups In CHOP-R Treated DLBCL
Authors: Barrans Sharon , Worrillow Lisa , Care Matthew , Crouch Simon , Smith Alex , Patmore Russell , Davies Andrew , Tooze Reuben , Roman Eve , Jack Andrew
Published: October 2019

Abstract 2485

Diffuse large B cell lymphoma (DLBCL) is a heterogenous disease, which has been subclassified into germinal centre (GCB) and activated B-cell (ABC) type using gene expression profiling. This has been shown to separate DLBCL into distinct prognostic sub-groups in patients treated with either CHOP or CHOP-R therapy. Previous studies have required the use of fresh or frozen samples for the extraction of RNA of sufficient quality to permit whole genome expression analysis. The Illumina ‘DASL' platform allows for highly reproducible gene expression data to be generated from FFPE material, which opens up large series' of retrospective data for detailed expression studies.

The aim of this study was therefore to determine whether the Illumina DASL platform could yield reproducible results on formalin fixed paraffin embedded (FFPE) biopsies from a large series of archival CHOP-R treated DLBCL samples.

RNA was extracted from paraffin sections using the Ambion Recoverall extraction kit, with 179/206 (87%) of cases yielding >200ng of RNA sufficient for DASL analysis. The DASL assay was performed according to Illumina protocols. Using stringent exclusion criteria, 157/179 (88%) cases yielding results that were considered to be of sufficiently high quality to be included in the analysis. To fully assess the reproducibility of the assay, 35 cases were analysed on 2–8 occasions across multiple experimental days.

Using Pearson's correlation, with full-linkage clustering, four discrete clusters were identified (n=28, 40, 46 and 43). Of important note, 95% of the samples were seen to cluster more tightly with their repeats than with any other sample, with all duplicated samples being called in the same cluster with 100% accuracy, suggesting that the technique is highly reproducible.

Univariate Kaplan-Meier survival analysis showed that the clusters identified patients with very different outcomes. Two of the clusters showed identical survival curves and therefore these clusters were merged to give 3 clusters with 2-year overall survivals (OS) of 51% (n=71), 65% (n=46) and 77% (n=40), log rank p=0.03, with a 3.7 year follow-up.

This data supports the use of gene expression profiling to classify DLBCL patients into clinically relevant prognostic groups. The Illumina DASL assay allows for highly reproducible gene expression data to be produced in valuable, archival data series, and also in the context of clinical trials, where the majority of the tissue available for study is FFPE. The patients identified in this study as having a sub-optimal response to CHOP-R should be considered for alternative therapies, which should be validated in the context of a clinical trial.

Disclosures:

No relevant conflicts of interest to declare.
Expression of the Neonatal Fc Receptor FcRn Is Not Required for the Amelioration of Murine ITP by IVIg or a Monoclonal Antibody
Authors: Crow Andrew R. , Suppa Sara J , Lazarus Alan H
Published: October 2019

Abstract 2529

There are several theories as to the mechanism of intravenous immunoglobulin (IVIg) in the treatment of autoimmune diseases such as immune thrombocytopenia (ITP). One prominent theory involves accelerated pathogenic autoantibody clearance by saturation of the neonatal Fc Receptor (FcRn). FcRn is an IgG receptor, and FcRn within endosomes binds endocytosed IgG and diverts IgG from degradation. In the treatment of ITP, it has been theorized that high concentrations of IVIg saturate FcRn, reducing the ability of the pathogenic anti-platelet antibodies to bind FcRn, increasing their catabolism, and thus rapidly decreasing their serum concentration which results in a decrease in their ability to induce platelet clearance. Mice lacking FcRn have only 20–30% of the level of endogenous IgG as compared with wild-type mice and have an accelerated clearance rate of both endogenous and injected IgG. We have utilized these mice in a murine model of ITP to help understand the role, if any, that FcRn plays in IVIg's therapeutic activity.

Both IVIg (2 g/kg body weight) and monoclonal antibodies to CD44 (2 mg/kg body weight; please see submitted abstract ID# 29504, “Amelioration of murine immune thrombocytopenia by CD44 antibodies: a potential therapy for ITP?”) successfully ameliorate murine ITP. To definitively determine if FcRn is required for the acute amelioration of ITP by these two therapeutics, we employed FcRn deficient mice in the murine ITP model. Here, we demonstrate that FcRn deficient mice (B6.129X1-Fcgrttm1Dcr/DcrJ) injected with an anti-platelet antibody exhibit a slightly more profound degree of thrombocytopenia than wild-type mice. FcRn deficient mice treated with IVIg or the anti-CD44 antibody KM114 (at a 3 log fold lower dosage than IVIg) were protected from ITP to the same extent as wild-type mice.

FcRn has an absolute requirement for the protein β2 microglobulin (β2M) to be functionally expressed. Specifically, β2M deficient mice do not possess functional FcRn and also show low endogenous IgG levels and increased clearance of IgG. To verify and substantiate the results found with FcRn deficient mice, we next employed β2M deficient mice in the murine ITP model and found that β2M deficient mice (B6.129P2-B2mtm1Unc/J) treated with IVIg or KM114 were also protected from ITP to the same extent as wild-type mice.

These data suggest that for both high dose IVIg as well as low dose monoclonal CD44 antibody treatment in an acute ITP model, FcRn expression is dispensable.

Disclosures:

No relevant conflicts of interest to declare.
Resveratrol, a Natural Antioxidant From Grapes and Red Wines, Prevents EBV-Associated Lymphoproliferation and Transformation through Inducing Apoptosis
Authors: Espinoza Luis J. , Takami Akiyoshi , Nakata Katsuya , Kato Shunichi , Kasahara Yoshihito , Nakao Shinji
Published: October 2019

Abstract 2543

Epstein-Barr virus (EBV) infection and reactivation is an increasing complication in immune deficient patients, particularly after allogeneic hematopoietic stem cell transplantation (HSCT) or solid organ transplantation (SOT). Whereas tapering of immunosuppression, donor lymphocyte infusion, and rituximab may be effective for this complication after HSCT, severe adverse events such as fatal GVHD and infections could develop thereafter. Resveratrol, a natural antioxidant compound found in grapes and red wines, possesses various biological activities including antitumor, anti-inflammatory, and antimicrobial efficacies “(Nature Reviews Drug Discovery 5, 493–506, 2006). We report here anti-EBV effects of resveratrol in eliminating EBV infected cells and preventing EBV-associated B-cell transformation. Resveratrol, at nontoxic concentrations to normal cells, effectively prevented the transformation of primary B cells derived from a panel of 16 healthy donors as well as cord blood mononuclear cells derived from 10 different donors. In addition, resveratrol induced dose-dependent apoptosis, assessed by FACS-based detection of Annexin V, in 7 different established EBV-transformed lymphoblastoid cell lines (LCLs) and in the Burkitt lymphoma EBV+ Raji and Daudi cell lines. These effects were consistently associated with the activation of caspase-8 and a 60 % down-regulation of the anti-apoptotic protein BCL-2. The addition of the caspase-8 inhibitor c-FLIPL to the cell culture vigorously inhibited the resveratrol induced apoptosis. Moreover, the resveratrol treatment decreased by 80% the EBV nuclear antigen 1 (EBNA1) expression, a critical protein required for the persistency of EBV infection. These results suggest that resveratrol may have potential roles in preventing EBV-associated lymphoproliferative diseases in immune compromised patients. Since resveratrol is a nontoxic, inexpensive and clinically available agent, it may be useful in preventing EBV- infection and reactivation after HSCT and SOT.

Disclosures:

No relevant conflicts of interest to declare.
New Instrument for Comprehensive Symptom Profile Assessment In Patients with Malignant Lymphomas
Authors: Novik Andrei A , Ionova Tatyana I , Kalyadina Svetlana A , Fedorenko Denis A , Mochkin Nikita E , Kurbatova Kira A , Nikitina Tatyana P , Gorodokin Gary I
Published: October 2019

Abstract 2561

Comprehensive symptom assessment before and during treatment in lymphoma patients is worthwhile. We aimed to develop a new symptom assessment tool — Comprehensive Symptom Profile in Lymphoma Patients (CSP-Lym) and test its applicability to this patient population.

The CSP-Lym is a self-reported tool which allows the assessment of the severity of 45 symptoms in lymphoma patients. It consists of numerical rating scales, scored from “0” (no symptom) to “10” (most expressed symptom). Thirteen symptom clusters have been identified, which were clinically relevant and increased the practicability of the tool. Utility of the CSP-Lym was demonstrated by means of interviewing both patients and physicians. Applicability of the tool with preliminary analysis of psychometric properties was tested in a pilot study. 75 patients with different types of malignant lymphomas (Stage – I-IV) were included in the study: Non-Hodgkin's lymphoma – 26; Hodgkin's lymphoma – 49. Mean age was 33.5 years old; male/female distribution –28/47. Patients filled out the CSP-Lym before and at different time-points of treatment.

The utility of the CSP-Lym was demonstrated. The patients acknowledged the comprehensiveness of the tool. All of the items were easy for the patients to read and understand. Completion of the CSP-Lym in paper and pencil format took 5–7 min. The data produced by the tool were clear for interpretation by physicians and were used by them in day-to-day decision making.

The construct validity of the CSP-Lym was proven by factor analysis and “known-group” comparison. A six factor structure was found for the core symptom items, which explained 65% of the total variance. The analysis of symptom severity in the groups with and without B-symptoms revealed differences between the groups: all of the symptoms were more severe in patients with B-symptoms. Statistically significant differences between the groups (p<0.05) were found for the majority of symptoms. Reliability of the CSP-Lym was satisfactory: Chronbach's alpha coefficient varied from 0.63 to 0.92. Sensitivity to changes was demonstrated by the comparison of symptom severity before and after treatment. The majority of symptoms after treatment were less severe than at base-line.

Thus, the CSP-Lym is an appropriate and practical tool to assess the symptom severity in lymphoma patients. The utility of the questionnaire was shown; preliminary psychometric properties appeared to be satisfactory. Further studies are needed before the wide-spread use of the CSP-Lym in clinical practice and clinical trials.

Disclosures:

No relevant conflicts of interest to declare.
Clinical Consequences of Disseminating the Bortezomib Postmarketing Safety Alerts In Japan
Authors: Tanimoto Tetsuya , Murashige Naoko , Oshima Yasuo , Hori Akiko , Kodama Yuko , Matsumura Tomoko , Kami Masahiro
Published: October 2019

Abstract 2572

Backgroud:

In Japan, bortezomib (Velcade; Janssen Pharmaceutical Corporation, Tokyo, Japan) was approved on October 2006 for relapsed or refractory multiple myeloma, and sales began on the open market in December 2006. The approval process clarified potential risk for interstitial lung disease (ILD) among bortezomib-treated Japanese patients as we reported before (Blood 2006; 107:3492–4, Lancet Oncol 2008; 9:414–5). By 2007, various safety alerts were widely disseminated through the responsible pharmaceutical company, the media, the academic societies and the regulatory authority of Japan (Pharmaceuticals and Medical Devices Agency, PMDA) concerning the potential ILD risk of bortezomib (J Clin Oncol 2008; 26:5820–3). In this study, we investigated whether notification of patients and providers about these safety alerts influenced the clinical outcomes after bortezomib therapy in Japan.

Methods:

We reviewed all bortezomib-related adverse drug reaction (ADR) reports available from open source database of PMDA from December 2006 to December 2009. The indication of bortezomib remained unchanged in this period. We compared the number of reported cases and their outcomes between the early postmarketing period (from December 2006 to December 2007) and the late period after the dissemination of the safety alerts concerning ILD (from January 2008 to December 2009).

Results:

In the study period, 419 ADR cases were identified after bortezomib therapy. The proportion of ILD to any ADR was 25.0% (9 of the 36 cases) in December 2006, 8.3% (13 of the 156 cases) in 2007, 12.0% (17 of the 142 cases) in 2008 and 7.1% (6 of the 85 cases) in 2009 (Table 1). When we compared the early postmarketing period before the safety alerts and the late period after the safety alerts, the death proportion of the cases with any ADRs seemed similar. Forty of the 192 cases (20.8%) died in the early period and 47 of the 227 cases (20.7%) in the late period. On the other hand, the death proportion among ILD cases appeared to decrease remarkably (Table 2). Nine of the 22 ILD cases (40.9%) died in the early period and 1 of the 23 ILD cases (4.3%) died in the late period (p=0.004, Fisher's exact test).

Conclusions:

Notification of patients and providers about the safety alerts appeared to influence the clinical practice and to improve proportion of fatal outcomes for bortezomib-treated ILD patients. This study illusrates the importance of communicating drug safety information to the public.

Disclosure:

Oshima: Sanofi-Aventis: Employment.
Readiness for Transition From Pediatric Sickle Cell Care: Exploratory Analyses of Domains of Readiness and Total Scores
Authors: Smith Wally R. , Smith Virginia , Godder Kamar , Sisler India , Casper Robbie , Johnson Shirley , Brice Wanda , Bitsko Matthew , Engram April , Morgan Alma
Published: October 2019

Abstract 2576

Background:

In just one generation, the average survival of patients with sickle cell anemia has increased from 14 years to nearly 50 years, necessitating transition programs from pediatric to adult care. We developed a transition program to provide continuity of care from pediatric to adult settings. In order to provide education and skills via an individualized plan for transition of care, we assessed confidence/self-efficacy, knowledge, skills, and attitudes surrounding transition of 34 African-American individuals aged 15–21 (mean 17). We hypothesized that there would be correlation between each of these domains, and that such correlation would suggest that each domain predicts initial readiness/sophistication for transition.

Methods:

We administered a survey battery both of items we constructed and of previously used survey items assessing medical, Educational/Vocational, Health Benefits, Social, Independent Living, Feelings, Sickle Cell Stress, and Self-Efficacy domains. We assigned/mapped these content items, using clinical judgment and consensus, to four domains of “readiness for transition”: confidence/self-efficacy, knowledge, skills, and attitudes. Readiness scores for each domain were calculated such that a summary score of 100 would indicate perfect readiness in each domain. The complete question battery is available.

Results are shown in the table below.

Conclusions:

Attitudinal readiness, knowledge readiness, confidence readiness, and skill readiness for transition were low initially in sickle cell adolescents enrolled in a transition program. Percentage of maximum possible scores was lowest for attitude, and highest for skill. Both attitude and knowledge scores showed very high correlation with total readiness score. However, several component scores were moderately correlated with each other. Transition programs for SCD should consider assessing and enhancing not only knowledge, but also attitudes, confidence, and skill.

Disclosure:

No relevant conflicts of interest to declare.
Coordinated Regulation of Embryonic and Adult Hematopoietic Stem Cell Activity by PTPN11/Shp2.
Authors: Zhu Helen He , Ji Kaihong , Alderson Nazilla , He Zhao , Li Shuangwei , Li Linheng , Feng Gen-Sheng
Published: October 2019

Abstract 2630

Shp2, a tyrosine phosphatase with two SH2 domains, is implicated in malignant blood diseases. Germline dominant active mutations in PTPN11 that codes for Shp2 are found in approximately 50% of Noonan syndrome (NS) patients who have high risk of juvenile myelomonocytic leukemia (JMML). Somatic gain-of-function mutations in PTPN11 have also been detected in nonsyndromic JMML, pediatric AML, B-cell precursor ALL and myelodysplastic syndromes. The majority of leukemia-associated PTPN11 mutations disrupt the auto-inhibitory interactions between its N-terminal SH2 and the phosphatase domain, resulting in elevated catalytic activity. However, the physiological function of Shp2 in normal hematopoiesis in adults remains to be elucidated, although a Shp2 function in embryonic hematopoiesis was documented previously. Mouse embryonic stem cells (mESCs) homozygous for targeted exon 3 deletion are defective in differentiation to all blood cell lineages, suggesting its role in commitment/specification of hematopoietic stem cells (HSCs) during embryogenesis. Indeed, our previous experiments suggested a developmental blockage from mESC to mesoderm and hemangioblast differentiation by the exon 3 deletion. Notably, distinct mechanisms are involved in embryonic and adult hematopoiesis. Despite an essential role for specification of HSC fate from embryonic mesoderm, SCL/tal-1 was found dispensable for HSC maintenance and differentiation in adults. Similar observation was made for transcription factor Runx. We show here that inducible ablation of PTPN11/Shp2 in the hematopoietic compartment leads to severe cytopenia in bone marrow, spleen and periphery. Shp2 removal suppressed the number and function of HSCs and myeloid progenitors. Shp2-deficient HSCs failed to reconstitute irradiated recipient mice, due to combined defects in homing, self-renewal and survival. Therefore, PTPN11/Shp2 indeed plays an essential role in maintenance of a functional HSC pool in adult mammals. This study reveals a critical signaling component conserved in embryonic and adult hematopoiesis. Given the essential role of Shp2 in normal HSCs, developing a chemical that distinguishes wild-type and mutant Shp2 enzymes may be a therapeutic approach for elimination of leukemic stem cells (LSCs) harboring mutant PTPN11/Shp2.

Disclosures:

No relevant conflicts of interest to declare.
A Meta-Analysis of Nodular Sclerosis Hodgkin Lymphoma Identifies Risk Loci at 6p21.32
Authors: Cozen Wendy , Li Dalin , Best Timothy , Berg David J. Van Den , Skol Andrew , Cortessis Victoria K. , Mack Thomas M , Glaser Sally L. , Schumacher Fred , Weiss Lawrence , Nathwani Bharat N. , Hwang Amie , Bhatia Smita , Strong Louise C , Robison Leslie L. , V Conti David , Onel Kenan
Published: October 2019

Abstract 2686

Nodular sclerosis Hodgkin lymphoma (NSHL) is epidemiologically and pathologically distinct from other Hodgkin lymphoma subtypes. It is highly heritable and is the most common form of HL in young adults. In a combined analysis of results from two genome-wide association scans (one undertaken at the University of Southern California using the Illumina Human610-Quad beadchip and the other at the University of Chicago using the Affymetrix Genome-Wide Human SNP Array 6.0) of 395 European-origin NSHL patients and 3,245 controls, we identified two single nucleotide polymorphisms (SNPs) at chromosome 6p21.32 directly genotyped in both scans associated with disease risk at genome-wide levels of significance (rs9268542: p=8.2×10−10, odds ratio (OR)= 1.63, 95% Confidence Interval (CI) = 1.40–1.91; and rs204999: p=2.3×10-9, OR= 0.54, 95% CI: 0.44=0.66). These SNPs are located in distinct haplotype blocks and are in only weak linkage disequilibrium (LD) (r2=0.06). Joint analysis revealed that each SNP was independently associated with disease risk, with the global likelihood ratio test remaining statistically significant for each group (p-value for 2 df test USC = 7.46 × 10-9 and UC=.3.51 × 10-6). Neither SNP is in a known gene or microRNA. rs9268542 is in LD with SNPs in the HLA-DRA gene (r2=0.92-0.93) and is a cis-acting expression QTL (eQTL) for several HLA Class II genes (HLA-DRB1-HLA-DRB5; HLA-DQB1-HLA-DQB2; all p=1×10-5). Of note, rs6903608, an imputed SNP in the same region also surpassing genome-wide significance for association with NSHL, is an eQTL for these same HLA Class II genes at p=1×10-20. rs204999 is in LD with SNPs in the nearby PRRT1 (r2=0.82) and EGFL8 (r2=0.81) genes, for which function remains poorly defined. Thus, we identified one SNP on chromosome 6p21.32 associated with NSHL that, by eQTL analysis, may be implicated in the transcriptional regulation of HLA-class II genes, a known susceptibly region for NSHL, and one SNP in the genomic region that appears to be independently-associated with NSHL, but which does not appear to be linked to the HLA locus.

Disclosures:

No relevant conflicts of interest to declare.
High Incidence of Peripheral Blood Plasmacytosis In Patients with Dengue Virus Infection: a Prospective Study
Authors: Thai Khao T.D. , Wismeijer Josta A. , Zumpolle Catrien M. , de Jong Menno D. , Vde ries Peter J. , Kersten Marie Jose
Published: October 2019

Abstract 2772

Introduction:

One of the characteristic features of dengue virus (DENV) infection is the occurrence of leukopenia and thrombocytopenia, probably resulting from virus induced bone marrow suppression. Despite the general bone marrow suppression, polyclonal peripheral blood plasmacytosis has occasionally been described in DENV infected patients. The frequency of peripheral blood (PB) plasmacytosis in patients with dengue infection, the origin of these plasma cells (PCs) and the mechanisms by which they appear in the blood are not known. We initiated this prospective observational study to quantify and describe the kinetics and phenotype of PB plasmacells (PCs) in these patients.

Methods:

Morphological examination of the peripheral blood smear was performed in 35 sequential returned travelers suspected of DENV infection, with a history of less than 14 days of fever. Flow cytometric (FC) analysis for the characterization and immunophenotyping of lymphocyte subsets and PCs was performed in 31 patients. Follow-up samples were available for 8 patients.

Results:

Our results show that PB plasmacytosis is a very common hematological finding in DENV infection, with extreme values of up to 36% of total white blood cells in some patients. Depending on the number of days since the onset of fever at presentation, PB plasmacytosis was observed in 64% to 73% of 28 patients with confirmed DENV infection, and in none of 7 patients with other febrile illnesses. PB plasmacytosis was the most pronounced before 7 days after onset of illness and declined rapidly thereafter, to completely disappear after 14 days of illness. The median percentage of PCs at day 7 was 2.5% (range 0–36%; 25–75 interquartile range: 0–8%). The median percentage of PCs was significantly higher in patients with secondary DENV infection than in patients with primary infection (4.5% versus 1.0%; p=0.05). Viral RNA was detectable in 18 of 28 DENV infected patients with a highly variable viral load, but there was no correlation between viral load and percentage of PCs. We found an excellent correlation between percentage of PCs as assessed by morphology and by flow cytometry (r2= 0.85). The majority of CD138+ PCs (89%) had a shared immunophenotype (CD45+/CD19−/CD56−), which differed from normal plasmacells which are generally CD19+. In all cases the PCs were polyclonal.

Conclusion:

PB plasmacytosis, characterized by a transient presence of polyclonal PCs in the circulation, is a common event in DENV infection and is probably the result of a vigorous humoral immune response to dengue. With an increasing number of travelers to areas where dengue virus is endemic, it is important also for hematologists to recognize this benign cause of sometimes extreme plasmacytosis, for which no invasive procedures such as bone marrow examinations are needed.

Disclosures:

No relevant conflicts of interest to declare.
Cross Laboratory Validation of An In Vitro Screen for Cytokine Release Syndrome
Authors: Bannish Gregory , Makropoulos Dorie , Perpetua Meredith , Thomas Gareth , Bate Simon , Walker Mindi , Bugelski Peter
Published: October 2019

Abstract 2778

Monoclonal antibodies (mAb) are widely used in anti-inflammatory and tumor therapy. They are highly effective in certain diseases, but can cause a range of adverse effects. Toxicity may result from the expected pharmacological effects of the antibody and from interactions with antigen expressed on tissues other than the intended target. The results range from mild infusion reactions to cytokine release syndrome (CRS). A number of mAbs are associated with CRS on the first dose in humans (including: anti-CD3, anti-CD52; anti-CD20, anti-CD25 and anti-CD28 superagonist) that were not predicted by preclinical studies in animals. To address this issue, we have developed an in vitro screen using cultured human whole blood that assess the potential for mAb to cause CRS. The purpose of these experiments was to determine if the assay can be transferred to different laboratory and conducted using a different analytical platform and different donors. mAbs are bound to Protein A coated polystyrene beads (Spherotech) and incubated with whole blood from 6 normal human volunteers diluted in media (1:10) in 96 well plates. PBS (no beads) and Autologous Plasma are negative controls, while LPS (1 ug/mL); LPS and superagonist anti-CD28 (ANC28.1/5D10, Ancell) serve the positive controls. After incubation for 48 hr, the supernatants are collected and cytokines (IFN-γ, TNFα, IL-1, IL-2, IL-6, IL-8, IL-10 and IL-12) assayed using SearchLight® (Aushon Biosystems) or MSD® (Meso Scale Discovery) platforms. The response to superagonist anti-CD28 was distinctly different from the response to LPS, anti-CD20 and anti-CD52. Although the absolute values for the cytokines differed across the analysis platforms, Spearman Rank Order analysis showed a statistically significant correlation for most cytokines. Moreover, when the average values were normalized to the response to LPS, hierarchical cluster analysis classified the responses measured by the two platforms in an identical manner. In conclusion, this study has shown that an in vitro whole blood based assay can classify mAb according to the potential to cause CRS in humans and suggest that the assay can be transferred successfully across laboratories and analysis platforms.

Disclosures:

No relevant conflicts of interest to declare.
Exploring the Biological Role of Kruppel-Like Factor 2 In Cytotoxic T Lymphocytes
Authors: Preston Gavin Charles , Cantrell Doreen A
Published: October 2019

Abstract 2783

Kruppel-like factor 2 (KLF2) is a transcription factor which has been shown to be a critical regulator of T lymphocyte quiescence and trafficking. KLF2 is highly expressed in naïve CD8 T cells but its expression is transcriptionally downregulated in effector cytotoxic T lymphocytes (CTL). To understand how KLF2 might co-ordinate biological processes in CTL, we have determined the impact of preventing KLF2 downregulation on the transcriptional program of antigen receptor triggered CD8 T cells. Our data show that CTL which fail to downregulate KLF2 have a strikingly different transcriptional program to normal CTL. Immune activated CD8 T cells that sustain KLF2 expression thus show increased expression of 672 genes and decreased expression of 205 genes compared to normal CTL that have lost KLF2. Previous studies have indicated that high levels of KLF2 correlate with long term T cell survival and the development of memory CD8 T cells. We therefore questioned whether the KLF2 regulated genes identified in our experiments gave any insight as to why increasing levels of KLF2 in immune activated T cells might control T cell memory. In this context, we noted that KLF2 downregulation is necessary for TCR triggering of the CTL mitotic pathway. The molecular basis for this effect includes that KLF2 drives expression of intracellular cell cycle inhibitors. It was equally striking, however, that KLF2 could induce expression of the inhibitory receptor Carcinoembryonic Antigen-related Cell Adhesion Molecule 1 (Ceacam1, CD66a), which can suppress the T cell proliferative response. The present report confirms that KLF2 controls the repertoire of chemokine receptors and adhesion molecules expressed by CTL. Previous studies have identified the adhesion molecule CD62L and the G protein coupled receptor S1P1 as direct gene targets for KLF2. The present data support that KLF2 positively regulates expression of these molecules but also describes a cell autonomous role for KLF2 to negatively regulate the expression of the inflammatory chemokine receptor CXCR3 in antigen primed CTL. The loss of KLF2 is thus essential to allow CTL to traffic to CXCR3 ligands. One other striking observation was that KLF2 expression in CTL upregulates expression of the IL-6 receptor and Serine Protease Inhibitor 6 (Spi6). The latter molecule has a key role in protecting CTL from self injury inflicted by granzymes and is critical for the generation of T cell memory. IL-6 receptor expression is similarly important for memory T cell survival. Collectively, these data identify new KLF2 regulated genes and biological functions in CD8 T cells and provide important insights as to how this transcription factor controls T cell immune responses and might determine the effector/memory fate of a CTL.

Disclosures:

No relevant conflicts of interest to declare.
The Combination of Azacitidine (AZA) and Recombinant Erythropoietin (rEPO) Can Induce Rapidly Hematological Response In Intermediate-2 and High-risk MDS (including RAEB-t)
Authors: Kartsios Haris , Loukidis Kostas , Papadopoulos Vassilios , Effraimidou Smaragda , Spyrou Anastasia , Kokoviadou Kyriaki , Papadakis Emmanouil , Kioumi Anna , Korantzis Ioannis
Published: October 2019

Abstract 2914

Background:

AZA, a DNA hypomethylating agent, provides 50–60% responses in higher-risk MDS after administration of 6 courses of treatment. Recent laboratory data suggests that demethylation with AZA upregulates EPO-receptor mRNA (Wallach, 2009). AZA might also affect several genes involved in cell cycle, metabolism and signal transduction which are down-regulated in bone marrow erythroid cells in MDS patients non-responsive to rEPO. There is currently insufficient data to combine AZA and rEPO in MDS patients.

Patients-Methods:

We explored the safety and the efficacy of the AZA-rEPO combination in a cohort of 10 (M/F: 5/5) patients (pts) with a median age of 75(67-83) years. Diagnosis (WHO classification) was: RAEB-2: 5, CMML: 2 and RAEB-t: 3; IPSS was: int-2 in 8/10 and high in 2/10 pts. Median time from diagnosis was 6(1-31) months. 9/10 pts were transfusion dependent, 8/10 were refractory to previous rEPO administration while 2/10 pts were not treated with rEPO but their diagnostic serum EPO levels were >200 U/L. Patients were given AZA at FDA/EMEA-approved schedule (75 mg/m2/d x7d/4-weekly) initially for 5 courses and continued if response was obtained. rEPO (40,000IU/week) was given until achievement of steady Hb level >10.5 g/dL or until AZA discontinuation.

Results:

Median follow-up was 6.5(1-14) months. Patients received a median of 5 cycles (range 2–13) of AZA; 9/10 pts were treated with ≥5 courses of AZA. The median time of rEPO administration was 82(76-142) days. Best response (IWG 2006 criteria) was CR in 1/10 pts (RAEB-2: 1), marrow CR in 1/10 pts (RAEB-t: 1), and stable disease with hematological improvement (HI) in 4/10 pts (RAEB-t: 1, RAEB-2: 2, CMML: 1) leading to an overall response rate of 60%. As soon as 2 courses of AZA-rEPO were given, 5/6 responders experienced HI-erythroid response, 3/6 obtained HI-platelet response and 2/6 achieved HI-neutrophil response. Adverse events included 2 episodes of febrile neutropenia, nausea (2/10 pts), reversible renal impairment (2/10 pts) and hemorrhagic complications (3/10 patients). Currently, 9/10 patients remain alive, 1 patient experienced progression to AML and the estimated probability of 1 year-Progression Free Survival is 75%.

Conclusions:

This study provides clinical evidence that the AZA-rEPO combination is safe and rapidly effective in higher risk MDS pts. Our results emphasize the necessity for randomized trials in order to further evaluate the AZA-rEPO combination in MDS.

Disclosures:

No relevant conflicts of interest to declare.
Decitabine Is Effective In Patients with Myelodysplastic Syndromes Who Failed Prior Intensive Regimen: No Negative Impact of Prior Therapy
Authors: Jabbour Elias , Kantarjian Hagop , Kadia Tapan , Borthakur Gautam , Cortes Jorge E. , Cornelison A. Megan , Ravandi Farhad , Garcia-Manero Guillermo
Published: October 2019

Abstract 2936

Background:

Decitabine is a hypomethylating agent (HMA) with demonstrated activity and an acceptable safety profile for the treatment of all FAB subtypes of myelodysplastic syndromes (MDS). A retrospective analysis was conducted to determine the effect of prior MDS treatment on overall response rate (ORR), duration of response, time to response, and overall survival (OS).

Methods:

Of 188 patients, 54 had prior MDS treatment consisting of cytotoxic chemotherapy (n=31), immunomodulators (n=21), stem cell transplant (n=4), monoclonal antibodies (n=2), oblimersen (n=1), or HMAs (n=1) were identified from two clinical trials with decitabine (Phase III, 3-day and Phase II, 5-day dosing studies). Patients were stratified on the basis of those who had received prior treatment (PT, n=54) for MDS and those who had not (NPT, n=134). Descriptive statistics were used to quantify the differences between the two groups for baseline characteristics, response rates (based on IWG 2006 criteria), safety and survival data.

Results:

Baseline characteristics showed the PT group had a longer time from diagnosis; the NPT group had a greater percentage of patients with High risk and Int-2 IPSS scores. There were differences noted in the baseline NPT group (5-day dosing only), with more patients with FAB classification of RAEB and RAEB-t (n=45), whereas the baseline PT group had a greater number of CMML patients (n=8). Objective response rate (ORR), overall improvement rate (OIR) and median survival were greater in NPT patients with no significant difference observed between the groups (Table 1). Patients with PT had a shorter duration of improvement (median 208 days) vs. the NPT group (median 296 days, p=NS) for the 5-day dosing study.

Conclusions:

Decitabine is active in patients with MDS with low rate of side effects. Prior therapy with intensive regimens have no impact on response to decitabine and such patients should be considered for hypomethylating therapy.

Disclosures:

Jabbour: Eisai Inc.: Editorial and statistical support, Honoraria. Kantarjian:Novartis: Research Funding; Pfizer: Research Funding; Bristol Myers Squibb: Research Funding; Novartis: Consultancy. Borthakur:Eisai Inc.: Research Funding. Cortes:Novartis: Research Funding; Pfizer: Consultancy, Research Funding; Bristol Myers Squibb: Research Funding. Ravandi:Eisai Inc.: Research Funding; Eisai Inc.: Honoraria.
Incidence of Plasma Cell Myeloma In Ethnic Chinese In British Columbia, a 30-Year Survey From 1975–2004
Authors: Chan Vicky W. , Song Kevin , Mang Oscar , Ip Dennis , Au Wing Y
Published: October 2019

Abstract 2956

Introduction:

The incidence of plasma cell myeloma (PCM) in East Asian population is known to be 3–4 times lower than that in Caucasians. This may be due to genetic or environmental factors. Migrant studies may separate these factors to some extent by determining whether the disease incidence follows the parent or destination prevalence. British Columbia (BC) has a large population of Chinese migrants and therefore, it is an ideal population for migrant studies. Hong Kong (HK) is chosen as the comparison population because it has meticulous census data and a population-based cancer registry.

Methods:

Computerized records of all PCM cases diagnosed from 1975 to 2004 were retrieved from the British Columbia Cancer Agency (BCCA) and the Hong Kong Cancer Registry (HKCR). Cases with Chinese ethnicity in BC were identified by surname using a previously validated methodology and verified from electronic charts. Age distribution of HK, BC and BC Chinese populations for consecutive 5-secular year (yr.) periods were obtained from census departments from the respective governments. The crude incidence rates of PCM were calculated and 95% CI were determined using normal approximation of Poisson distribution. Projected PCM incidence rates in BC Chinese using HK and BC models were calculated and standard incidence ratios (SIR) estimated.

Results:

There was a steady increase in PCM from 1975 to 2004: from 42 to 159 cases/yr. in HK and from 86 to 207 cases/yr. in BC. This is likely due to population growth and aging. The proportion of population with age >65 grew from 5.4% to 11.2% in HK and from 9.7% to 13.2% in BC. From 1975–2004, a total of 106 cases of BC Chinese PCM were identified, increasing from 0.4 to 8 cases/yr. The BC Chinese population grew from 0.07 to 0.37 million, while the elderly subgroup (age >65) grew from 7.9% to 12.9%. Age specific mean 30-year crude incidence/100,000 person-years and 95% CI in the 3 cohorts were determined (Table 1). The standardized incidence ratio (SIR) of PCM in BC Chinese from 1975–2004 was 0.83 (95% CI 0.68–1.00) when compared to HK and 0.44 (0.36-0.53) when compared to the BC population. Cumulative SIRs of PCM in BC Chinese when compared to BC population remained low for incremental 5-year from 1975–2004 (Table 2).

Conclusion:

The incidence of PCM in ethnic Chinese in British Columbia is lower compared to the Caucasian counterparts, but similar to the incidence seen in HK. The trend is apparent in all age groups over 45 years old. This observation suggests a strong genetic predisposition in the development of PCM.

Disclosures:

No relevant conflicts of interest to declare.
High Resolution Genome-Wide Analyses Revealed That Bortezomib Selects a Prediagnosis Clone In Relapsed Patients with Multiple Myeloma
Authors: Magrangeas Florence , Avet-Loiseau Hervé , Decaux Olivier , Gouraud Wilfried , Harousseau jean Luc , Anderson Kenneth C. , Moreau Philippe , Munshi Nikhil C. , Minvielle Stephane
Published: October 2019

Abstract 2960

Despite advances and significant improvement in survival, multiple myeloma (MM) remains incurable and nearly all patients relapse after treatment. In order to gain insights into the genomic lesions associated with acquisition of drug resistance and progression of disease, we performed high resolution genome-wide single nucleotide polymorphism and copy number analyses on matched diagnostic and relapse samples from 24 MM patients either treated with proteasome inhibitor (bortezomib)-based induction regimen (n=12) or conventional chemotherapeutic agents (n= 12). All relapse samples have a clear relationship to the diagnosis clone. The vast majority of patients (92%) acquired additional copy number abnormalities (CNAs) or uniparental disomy (UPD) at relapse or exhibited change in the pattern of lesions present at diagnosis. Of these, 45% acquired new lesions and 41% both acquired new lesions and lost lesions present at diagnosis. Remarkably, loss of lesions at relapse was significantly associated with initial bortezomib treatment (8 out of 12 versus 1 out of 12; P = 0.009). Moreover, in 75% of the bortezomib-treated MM, the lesions lost at relapse included either UPD or deletion providing direct evidences that the relapse clone arose from a common prediagnosis clone present as a minor population at diagnosis that acquired additional abnormalities before emerging as the relapse clone. These results suggest that resistance to novel therapeutic agent known to target MM cells in the bone marrow milieu preferentially goes through with selection of minor prediagnosis clone while escape to conventional chemotherapeutic agents is almost exclusively associated with clonal evolution from diagnosis clone. These data support the proposal to combine several anti-myeloma drug upfront in order to obtain long-term remissions.

Disclosures:

Anderson: Millennium Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau. Munshi:Millennium Pharmaceuticals: Honoraria, Speakers Bureau.
Improved Myeloma Targeting by Combination of the Human Anti-CD38 Antibody Daratumumab with Lenalidomide and Bortezomib
Authors: van der Veer Michael S. , de Weers Michel , van Kessel Berris , Bakker Joost M , Wittebol Shulamiet , Parren Paul W.H.I. , Lokhorst Henk M , Mutis Tuna
Published: October 2019

Abstract 3030

Although multiple myeloma (MM) is considered an incurable malignancy of antibody-producing clonal plasma cells, over the past decade significant progress has been made in MM treatment by the availability of novel immunomodulating agents such as lenalidomide (LEN) and bortezomib (BORT). We are currently exploring the possibility to further improve MM therapy by combining LEN and BORT with novel agents, such as daratumumab (DARA), a human antibody directed against CD38 which mediates MM cell lysis via ADCC (antibody dependent cellular cytotoxicity), CDC (complement dependent cytotoxicity) and apoptosis. Our initial in vitro work show significantly improved MM cell killing by combined DARA with LEN treatment. We now investigated whether further improvement of this combination therapy is possible by addition of BORT. In ex vivo assays, which allow us to address killing directly in BM-MNC isolated from MM patients, we demonstrate that the DARA+LEN+BORT combination significantly exceeds the effectiveness of LEN+BORT treatment (P<0.001). Remarkably, triple therapy was also very effective, even when there was little or no response against LEN+BORT (P=0.0001). In other assays we further mimicked the in vivo conditions by incubating BM-MNC not only with LEN, BORT and DARA for 48 hours but also by adding complement. Addition of complement resulted in substantial enhancement of DARA-mediated MM tumor cell killing. This killing could not be further improved by addition of LEN and BORT. In conclusion, we demonstrate that addition of DARA to a LEN+BORT treatment regime increases overall clearance of MM cells ex vivo, suggesting that patients, even when not responding to LEN+BORT treatment, might benefit from a DARA+LEN+BORT combination therapy.

Disclosures:

Veer: GenMab, Utrecht, NL: Research Funding. Weers:Genmab: Employment. Bakker:GenMab, Utrecht, Netherlands: Employment. Parren:Genmab: Employment. Lokhorst:GenMab, Utrecht,NL: Research Funding. Mutis:GenMab, Utrecht, NL: Research Funding.
Microenvironment-Mediated Bim Down-Regulation through Induction of Microrna-181a Protects Cells From Apoptosis In Mantle-Cell and Other Non-Hodgkin B-Cell Lymphomas
Authors: Lwin Tint , Lin Jian-Hong , Choi Yong Sung , Zhang Xinwei , Moscinski Lynn , Wright Kenneth , Sotomayor Eduardo M. , Dalton William , Tao Jianguo
Published: October 2019

Abstract 3118

Follicular dendritic cells (FDCs), essential components of the lymph node microenvironment, regulate and support B lymphocyte differentiation, survival, and lymphoma progression. Here, we demonstrated that adhesion of mantle cell and other non-Hodgkin lymphoma cells to FDCs reduced cell apoptosis, associated with decreased levels of Bim, a pro-apoptotic protein. Bim down-regulation was epigenetically regulated via up-regulation of microRNA-181a (miR-181a). miR-181a overexpression decreased, whereas miR-181a inhibition increased Bim levels by directly targeting Bim. Furthermore, we found that cell adhesion-up-regulated miR-181a contributes to FDC-mediated cell survival through Bim down-regulation, implicating miR-181a as an upstream effector of the Bim-apoptosis signaling pathway. miR-181a inhibition and Bim up-regulation significantly suppressed FDC-mediated protection against apoptosis in lymphoma cell lines and primary lymphoma cells. Thus, FDCs protect B-cell lymphoma cells against apoptosis, in part through activation of a miR-181a-dependent mechanism involving down-regulation of Bim expression. We demonstrate, for the first time, that cell-cell contact controls tumor cell survival and apoptosis via microRNA in mantle cell and other non-Hodgkin lymphomas. Regulation of microRNAs by B-cell-FDC interaction may support B-cell survival, representing a novel molecular mechanism for cell-adhesion mediated drug resistance (CAM-DR) and a potential therapeutic target in B-cell lymphomas.

Disclosures:

No relevant conflicts of interest to declare.