bluuebunny/crossref_metadata_2025 huggingface dataset:
Distinct CD8+/CD57+ Large Granular Lymphocytic Populations Seen by Flow Cytometry Are Associated with Monoclonal T-Cell Populations, a Retrospective Case-Control Study of Large Granular Lymphocytic Leukemia
Authors: Ohgami Robert S. , Zehnder James L. , Arber Daniel A.
Published: October 2019

Abstract 2707

Large granular lymphocytic (LGL) leukemia is a disease with chronic elevations of large granular lymphocytes for > 6 months associated with a T-cell clone and often cytopenias. While a defined classification has been established in the 2008 WHO, the clinical and pathologic diagnosis often remains elusive, as patients can exhibit significant polyclonal or oligoclonal proliferations of LGLs in reactive conditions. Therefore predicting and defining patients with increased LGLs and significant monoclonal proliferations remains difficult. In an attempt to identify flow cytometry patterns which might be associated with monoclonality, we performed a retrospective review of patients at our institute between 2003–2010 with significant populations of peripheral blood LGLs in which T-cell clonality tests were also performed. 66 independent cases were identified and were divided impartially by time into a study set (cases from 2008–2010) and an experimental set (cases from 2003–2007). In the study set (2008-2010), 31 flow cytometry cases were identified, which stratified into 16 cases with monoclonal T-cell populations and 15 with oligoclonal or polyclonal populations. Comparing flow cytometry scatter-plots, we identified what appeared to be a discrete flow cytometry pattern. Specifically, those cases with distinct CD8+(dim)/CD57+ populations were significantly associated with T-cell monoclonality (p =< 0.0001) as well as lower neutrophil counts (average ANC= 1.78, p < 0.001) (See Figure 1). Such a preliminary analysis suggested that identification of patients with specific CD8+(dim)/CD57+ populations might define patients with not only monoclonal proliferations, but also with clinically and pathologically significant disease. To further evaluate this hypothesis, we used these criteria (discrete CD8+(dim)/CD57+ populations) to examine, in a blinded analysis, our experimental set of 35 consecutive cases of large granular lymphocytosis from 2003–2007. 11 cases were identified which demonstrated significant CD8+(dim)/CD57+ populations. Yet again, we observed that distinct populations of CD8+(dim)/CD57+ populations were associated with T-cell monoclonality (p =0.037), as well as neutropenia (average ANC=1.49; p=0.046). These findings demonstrate and define a major subset of clinically and pathologically significant patients through the use of flow cytometry immunophenotyping.

Disclosures:

No relevant conflicts of interest to declare.
The Kinase Inhibitor TKI258 Is Active Against the Novel CUX1-FGFR1 Fusion Detected In a Patient with T-Lymphoblastic Leukemia/Lymphoma and t(7;8)(q22;p11)
Authors: Wasag Bartosz , Lierman Els , Meeus Peter , Cools Jan , Vandenberghe Peter
Published: October 2019

Abstract 2715

The 8p11 myeloproliferative syndrome (EMS) is an aggressive, atypical stem cell disorder associated with chromosome translocations that constitutively activate FGFR1 by fusion to diverse partner genes. Here, we describe a case with a clinical and hematological diagnosis of T-lymphoblastic leukemia/lymphoma and with a t(7;8)(q22;p11) on cytogenetic analysis. We identified the fusion partner involved and characterized this translocation functionally in vitro using the interleukin 3 (IL3) dependent Ba/F3 cell line.

The translocation was analyzed in more detail by FISH using FGFR1 flanking probes. We could confirm the 8p11 breakpoint and 7q as the partner chromosome. Using 5'-RACE CUX1 (7q22) was identified as the fusion partner of FGFR1 in this patient with T-lymphoblastic leukemia/lymphoma. CUX1 is a homeobox family DNA binding protein not previously described as a fusion partner in hematological malignancies. To evaluate the transforming potential of this novel fusion, the CUX1-FGFR1 fusion was cloned and used to transform Ba/F3 cells. CUX1-FGFR1 expressing Ba/F3 cells displayed IL3 independent proliferation thus demonstrating the oncogenic character of this fusion protein. Western blotting of the transformed Ba/F3 cells showed activation of FGFR1 as well as its downstream target STAT5. Treatment of the CUX1-FGFR1 expressing Ba/F3 cells with the kinase inhibitors PKC412 and TKI258 significantly inhibited cell growth with an IC50 of 483 and 489 nM respectively. With western blotting a direct effect of both inhibitors on FGFR1 kinase activity as well as on different downstream effectors was proven. Furthermore using an annexinV/propidium iodide-based apoptosis assay, we could show that PKC412 and TKI258 both induced apoptosis followed by cell death in inhibitor treated CUX1-FGFR1 transformed Ba/F3 cells. The antiproliferative effect of the inhibitors could be rescued by addition of IL3 for the TKI258 treated but not for PKC412 treated CUX1-FGFR1 expressing cells. This observation indicates a selective action of TKI258 on FGFR1 signaling at the concentrations used. In contrast, for PKC412 non-specific cytotoxicity is also contributing to the antiproliferative effect. In summary, we identified a novel CUX1-FGFR1 fusion in a case with EMS and a novel t(7;8)(q22;p11), and demonstrated the oncogenic potential of CUX1-FGFR1 in the Ba/F3 cell line. This new fusion partner CUX1 contains a potential coiled coil domain that can explain the observed constitutive FGFR1 activation, as has been elaborately demonstrated for other oncogenic kinase fusions. The in vitro data presented here using the inhibitor TKI258 support the use of this compound for the treatment of the novel CUX1-FGFR1 fusion as well as other constitutively active FGFR1 fusion proteins.

Disclosures:

No relevant conflicts of interest to declare.
Detection, Comprehensive Analysis and Clinical Validation of Chromosomal Aberrations by Multiplex Ligation-Dependent Probe Amplification In Chronic Lymphocytic Leukemia
Authors: Abdool Adam , Donahue Amber C. , Wohlgemuth Jay G , Yeh Chen-Hsiung
Published: October 2019

Abstract 2716

Background:

Current strategies based on karyotyping or fluorescent in situ hybridization (FISH) for detection of chromosomal abnormalities in chronic lymphocytic leukemia (CLL) are laborious, time-consuming, and costly and have limitations in resolution. Multiplex ligation-dependent probe amplification (MLPA) can simultaneously detect copy number changes of multiple loci in a single PCR, making it an attractive alternative. We developed and validated an MLPA protocol for comprehensive, automated data analysis and interpretation of chromosome abnormalities associated with CLL.

Patients and Methods:

Reference ranges for individual MLPA probes were established from a group of 50 healthy control subjects. Using these ranges we built an automated spreadsheet-based analysis system that includes multiple quality checks; samples that fail these checks are flagged and not reported. Each target was given a call of “deletion,” “normal,” or “amplification,” depending on whether the normalized ratio fell within or outside of the established normal range (mean ± 2SD or mean ± 3SD). After establishing the normal references ranges for each probe, we used the MLPA assay to characterize chromosome abnormalities in blood samples from 100 patients with suspected CLL that had been previously tested with FISH.

Results:

The maximum normal ranges were distributed between 0.82 and 1.18 for the mean ± 2SD values (ie, 95% CI, P = 0.05), and between 0.73 and 1.27 for the mean ± 3SD values (ie, 99.7% CI, P = 0.01). MLPA showed good concordance with FISH results in the 100 clinically suspected CLL cases. In 6 of these cases, abnormalities detected by MLPA were in regions not covered by FISH, including additional copy number gains on chromosomes 18q21.1 and 19, and novel micro-deletions at 19q13.43 and 19p13.2 loci. Excluding these cases, MLPA showed 94% sensitivity, 94% concordance, and 93% specificity relative to FISH. MLPA detected abnormalities in 3 FISH-negative cases and failed to detect abnormalities in three 13q- cases with low percentages of leukemic cells (7%, 12% and 19%). The limit of detection of the CLL MLPA assay was about 20% leukemic clones.

Conclusions:

This MLPA-based assay for chromosome abnormalities in CLL showed excellent concordance with FISH. This multiplex assay represents a fast (roughly 2–3 days total process to report time vs 7–10 days for FISH), high-throughput, accurate, and user-friendly process for that has potential for use as a first-line screening test for detection of chromosome abnormalities associated with CLL in the clinical laboratory.

Disclosures:

No relevant conflicts of interest to declare.
High-Resolution Comparative Genomic Hybridization of Mirna Genes In Therapy-Related AML Identifies a Somatic Deletion of MiR-223
Authors: Ramsingh Giridharan , Matson Angela , Walter Matthew J. , Link Daniel C.
Published: October 2019

Abstract 2759

MicroRNAs (miRNAs) are small non-coding RNAs that regulate gene expression and diverse cellular processes. Expression profiling of miRNAs has identified dysregulated miRNAs in many cancers, including acute myeloid leukemia (AML). However, the mechanism for altered miRNA gene expression and the frequency of miRNA gene mutations in AML is largely unknown. We performed next-generation sequencing and high-resolution comparative genomic hybridization (CGH) to determine the frequency of miRNA gene mutations in 30 patients with therapy-related AML (t-AML). The sequencing is completed, and final analysis is underway. Herein, we report the results of the CGH. We designed custom CGH arrays for all 835 miRNA genes in miRBase (version 14) and 44 genes involved in miRNA processing. Average probe spacing was 30 bp for miRNA genes and 80 bp for miRNA processing genes and 10 kb flanking regions were interrogated for all genes. In each case, genomic DNA from leukemic blasts was compared with DNA from a skin biopsy from that patient. The median age of the 30 patients with t-AML was 49.2 years (25-77) with M:F ratio 13:17. The mean blast % was 81% (31-100). Consistent with previous reports, many of these patients had an abnormal karyotype with abnormalities of chromosome 5 and 7, 13% and 17% respectively. As expected, CGH analysis confirmed copy number alterations already identified by cytogenetics. In addition, we identified a single t-AML sample (from a male patient) that carried a small (435 kb) hemizygous deletion of miR-223 on chromosome × that was not apparent by cytogenetics. Quantitative PCR of genomic DNA confirmed the loss of the miR-223 gene, and real time RT-PCR demonstrated loss of miR-223 expression. Of note, miR-223 has been implicated in the regulation of granulopoiesis, and mice lacking miR-223 display a myeloproliferative phenotype (Johnnidis, Nature 2008). We screened an additional 27 AML patients for miR-223 expression and identified 3 other samples with miR-223 expression 2 standard deviations below the normal (based on CD34+ cells from healthy donors). No copy number alterations in miR-223 were detected in these patients, suggesting epigenetic silencing of miR-223. Consistent with this possibility, one of these patients carried a t(8;21), which has been shown to epigenetically silence miR-223 (Fazi, Cancer Cell 2007). The mechanism by which miR-223 is silenced in the other two AML samples is under investigation. In summary, cytogenetically silent deletions of miRNA genes are uncommon in t-AML. Loss of miR-223 expression can occur through somatic mutation or epigenetic silencing and is likely to contribute to leukemic transformation in a subset of patients with AML.

Disclosures:

No relevant conflicts of interest to declare.
Lack of IL-7 Receptor Alpha Chain (CD127) Expression In T Cells Is a Hallmark of T-Cell Immunodeficiency In Schimke Immuno-Osseous Dysplasia (SIOD)
Authors: Sanyal Mrinmoy , Dionis Kira Y , Baradaran-Heravi Alireza , Dekel Benjamin , Bökenkamp Arend , Boerkoel Cornelius F. , Lewis David B.
Published: October 2019

Abstract 2767

Schimke immuno-osseous dysplasia (SIOD) is an autosomal recessive, incompletely penetrant, childhood disorder associated with biallelic loss-of-function mutations of SMARCAL1 (swi/snf-related matrix-associated actin-dependent regulator of chromatin, subfamily-a-like-1) gene. The SMARCAL1 gene encodes for the DNA annealing helicase although it is unknown why this impairment results in skeletal dysplasia, renal dysfunction, and T-cell lymphopenia. The representation of T-cell subsets in SIOD patients is further characterized by a high proportion of memory (CD45RA−CD45RO+) T cells, however, the etiology of T-cell immunodeficiency has not been elucidated. Here, we demonstrate that T cells in individuals affected by SIOD express markedly reduced surface levels of IL-7 receptor alpha chain (CD127) as compared to their unaffected sibling or normal individuals. In contrast, the alpha-chain component of the IL-2 receptor (CD25) was expressed normally. This reduction in IL-7 receptor alpha chain was observed in all T-cell subsets including naïve T cells. This suggests that the origin of the IL-7 receptor alpha chain deficiency is likely intrathymic, rather than occurring after emigration to the periphery. In addition, T cells from SIOD patients were less responsive to stimulation with IL-7, indicating a loss of functional receptor. From these observations, we propose the lack of functional IL-7 receptor expression in T cells, and possibly their earlier progenitors, may have restricted T-cell development in SIOD patients. The mechanism for reduced IL-7 receptor expression is currently under investigation.

Disclosures:

No relevant conflicts of interest to declare.
Characterization of the Immunoregulatory Function of Human TCRαβ+ CD4-CD8- Double-Negative T Cells
Authors: Voelkl Simon , Gary Regina , Mackensen Andreas
Published: October 2019

Abstract 2776

Regulatory T (Treg) cells have been shown to be involved in downregulating immune responses in autoimmunity, transplant rejection, and graft-versus-host disease. Moreover, the important role of Treg cells in tumor progression has also been extensively investigated and Treg-mediated immune suppression has emerged as a crucial mechanism of tumor evasion. Recently, a novel subset of TCRαβ+ CD4−CD8− (double-negative, DN) T cells has been characterized to specifically suppress immune responses in mice. Here we demonstrate that human DN T cells belong to the family of inducible Treg cells with potent suppressor activity towards CD4+ and CD8+ T-cell responses. Resting DN T cells failed to suppress responder cells, whereas antigen presenting cell (APC)-stimulated DN T-cells and freshly isolated CD4+CD25+ Treg cells revealed a strong suppressive activity. Of importance, when more potent stimulators such as allogeneic dendritic cells were used for activation of responder T cells, CD4+CD25+ Treg cells were unable to mediate any suppressor function, while APC-primed DN T cells were still able to suppress. The suppressive activity of DN T cells is neither mediated indirectly by modulation of APCs nor by competition for T-cell growth factors. Furthermore, DN T-cell mediated suppression towards responder T cells requires cell-cell contact and is TCR dependent. Based on the importance of Treg cells in tumor immunity, we determined the frequency of circulating DN T cells in patients with malignant diseases. Of interest, the DN T-cell pool revealed an increased frequency in cancer patients compared to healthy controls, indicating that these suppressor cells might limit tumor-specific T-cell responses and thereby impair tumor immunosurveillance. Taken together, our results demonstrate that human DN T cells are a new subset of inducible Treg cells exerting a very potent suppressive activity towards cellular immune responses. Further understanding of the mechanisms involved in human DN T-cell suppression may have important implications for novel immunotherapies.

Disclosures:

No relevant conflicts of interest to declare.
Resveratrol Induces Cell Growth Arrest and Cell Death of Adult T-Cell Leukemia Cells Via Deacetylation of STAT3 by SIRT1
Authors: Ito Shigeki , Oyake Tatsuo , Murai Kazunori , Ishida Yoji
Published: October 2019

Abstract 2844

Adult T-cell leukemia (ATL) is an aggressive malignancy of peripheral T cells infected with human T-cell leukemia virus type 1 (HTLV-1). The prognosis of aggressive ATL patients remains poor because of its resistance to conventional chemotherapy. Therefore, new therapeutic targets should be explored. We recently reported that signal transducers, and activators of transcription 3 (STAT3)/survivin pathway is critical for cell proliferation and survival of ATL cells (Ito S. et al. Leuk Res. 2010. 34:352-357). Thus, STAT3 may be one of the therapeutic targets in ATL. Recent report suggested that acetylation in the SH2 domain of STAT3 is an important regulatory modification that influences protein-protein interaction and transcriptional regulation. Although STAT3 has been shown to interact with p300 acetyltransferase, the mechanism of deacetylation of STAT3 remains unknown. It has been recently shown that fasting-activated longevity protein sirtuin 1 (SIRT1) deacetylase/STAT3 pathway in the regulation of gluconeogenesis. We hypothesized that STAT3 deacetylation by SIRT1 may influence the cell growth and survival of ATL cells. In this study, we investigated the effects of resveratrol, an activator of SIRT1 deacetylase, on HTLV-1 infected T-cell lines, KUT1 and MT2 cells. We found that resveratrol significantly suppressed cell proliferation in a dose- and time-dependent manner by trypan blue exclusion method (P<0.01, ED50=50mM). Resveratrol also induced apoptosis in these cells by flow cytometric analysis with AnnexinV/propidium iodide staining (P<0.01). We next examined intracellular signaling in resveratrol-treated cells. Immunoblot analysis showed the suppression of constitutive phosphorylation of STAT3 at Tyr705 and IkBα at Ser32 but not JAK3 and STAT5 of both cells 24 hours after the treatment of resveratrol. Interestingly, resveratrol reduced acetylation of STAT3 at Lys685. We also observed the cleavage of caspase-3 and poly(ADP-ribose) polymerase (PARP) in resveratrol-treated cells, indicating that resveratrol induces caspase-dependent apoptosis in these cells. To better understand the mechanism by which resveratrol induces STAT3 deacetylation, we examined the relationship between SIRT1 and STAT3. SIRT1 protein expression level was elevated after the treatment of resveratrol. Immunoprecipitation assay revealed the physical interaction of STAT3 with SIRT1 in MT2 cells, suggesting that the mechanism of action of resveratrol involves the deacetylation of STAT3 through the interaction of STAT3 with SIRT1.

Our data indicate that resveratrol presents a potent anti-leukemia effect in part via the deacetylation of STAT3 in HTLV-1 infected cells. These mechanistic findings suggest that resveratrol may have a potential in the treatment of ATL.

Disclosures:

No relevant conflicts of interest to declare.
The Novel Bispecific Antibody [(CD20)2xCD16] Efficiently Triggers Lysis of Neoplastic B Cells
Authors: Glorius Pia , Baerenwaldt Anne , Kellner Christian , Staudinger Matthias , Parren Paul W.H.I. , van De Winkel Jan G. J. , Humpe Andreas , Valerius Thomas , Repp Roland , Nimmerjahn Falk , Gramatzki Martin , Peipp Matthias
Published: October 2019

Abstract 2846

Although the CD20 antibody rituximab considerably improved the treatment of B-cell neoplasias, novel optimized CD20-targeting strategies may still offer therapeutic gains. Analysis of Fc receptor (FcR) polymorphisms of rituximab-treated patients and animal studies demonstrated the crucial role for activating FcRs and effector cell recruitment. In the current study a novel CD20-directed bispecific antibody (bsab) [(CD20)2×CD16] in the tribody format was evaluated to enhance antibody dependent cellular cytotoxicity (ADCC). The heterodimeric molecule forms a CD16-directed F(ab) fragment with a CD20-directed scFv c-terminally fused to both peptide chains. [(CD20)2×CD16] was expressed in HEK-293T cells and purified using affinity chromatography. Specific binding to CD20- or FcγRIIIa- (CD16a) positive cells was demonstrated by flow cytometry. No significant difference in binding to both CD16a alloforms (CD16a-F158 and CD16a-V158) was observed. In chromium release assays [(CD20)2×CD16] mediated specific lysis of different burkitt's lymphoma cell lines in a dose-dependent manner even at low picomolar concentrations. Potency of target cell lysis was improved compared to rituximab (10-fold) irrespective of the effector cell donors' CD16a V/F-polymorphism status. In addition, the bsab triggered lysis of primary tumor cells from 12/12 lymphoma patients (CLL; MCL; MZL). [(CD20)2×CD16] triggered significantly stronger target cell lysis than rituximab or the parental CD20 antibody. Importantly, specific lysis of primary tumor cells was also observed using autologous NK cells as effector cells, suggesting that [(CD20)2×CD16] is capable in triggering tumor cell lysis in the presence of a strong MHC I inhibitory signal. Interestingly, when B-cell depletion was analyzed in whole blood, significantly higher depletion rates were obtained with the bsab compared to rituximab (75% vs 25%). Depletion of B cells was accompanied by activation of NK cells, as evidenced by CD69 expression on NK cells. Finally, NOD-SCID mice were transplanted with CD34-positive cord blood cells to reconstitute a humanized hematopoietic system. Distribution and numbers of engrafted human B cells and human CD16-positive effector cells were analyzed by flow cytometry. CD16 was expressed on splenic monocytes and NK cells. After daily injection of [(CD20)2×CD16] for three days a significant reduction of B cells was observed. Thus, the CD16-directed bispecific antibody [(CD20)2×CD16] may extend the therapeutic range and represents a promising candidate for further clinical development.

Disclosures:

Parren: Genmab: Employment. van De Winkel:Genmab: Employment.
Bortezomib Induces Caspase-9-Mediated Apoptosis through Activation of the BH3-Only Proteins Noxa, Bik and Puma In Both ALK-Positive and ALK-Negative Anaplastic Large Cell Lymphoma Cells
Authors: Cillessen Saskia AGM , Hijmering Nathalie J , Moesbergen Laura M , Ossenkoppele Gert J. , Oudejans Joost J , Meijer Chris JLM
Published: October 2019

Abstract 2847

Anaplastic large cell lymphoma (ALCL) is a CD30 positive T-cell lymphoma that can be divided into a systemic and a primary cutaneous type. Systemic ALCL can be further divided into an anaplastic lymphoma kinase (ALK) expressing type and an ALK-negative type. Despite intensive treatment regimens, the disease will be fatal in 20–30% of the systemic ALK-positive and 50–70% of the systemic ALK-negative ALCL patients. A recent study in primary ALCL samples has demonstrated an increased expression of a fraction of NF-κB target genes, suggesting upregulation of NF-κB activity in ALCL tumor cells. NF-κB activity can be inhibited by the proteasome inhibitor bortezomib resulting in induction of apoptosis. In this study, we therefore investigated if bortezomib can induce apoptosis of cultured lymphoma cells of three systemic ALK-positive and three ALK-negative ALCL patients and seven ALCL cell lines and we examined the mechanisms by which bortezomib induced cytotoxicity in these ALCL cells.

Treatment with bortezomib resulted in induction of apoptosis in all ALK-positive and ALK-negative ALCL patient samples and ALCL cell lines tested, when we compared the percentage cell death with the non-neoplastic CD4- and CD8-positive PBMC and tonsil T-cells from healthy donors. The lethal dose (LD50) varied between 54nM and more than 100nM after 24 hours and varied between 21nM and 52nM after 48 hours of exposure. ALK-negative ALCL cases were more sensitive to bortezomib and showed significant lower LD50 values than ALK-positive ALCL cells. We show that bortezomib-induced cell death in ALK-positive and ALK-negative ALCL is dependent on caspase-9 and/or caspase-8 mediated apoptosis and that bortezomib induces depolarization of the mitochondrial membrane. mRNA-expression and protein analysis revealed clearly upregulation of the BH3-only proteins Noxa, Bik and Puma, resulting in Bak and Bax release from the anti-apoptotic proteins Mcl-1 and Bcl-2. We also demonstrated that ALCL cells relatively resistant to bortezomib were characterized by high expression of Bcl-2A1, suggesting the possibility of pre-defining patients most likely to benefit from bortezomib therapy.

Our preclinical data support the therapeutic application of bortezomib as potential drug in the treatment of ALCL, especially ALK-negative ALCL patients to improve their prognosis.

Disclosures:

No relevant conflicts of interest to declare.
Preclinical Evaluation of A Potent 2nd Generation Small-Molecule Hsp90 Inhibitor STA-9090 In Hematological Cell Lines
Authors: Ying Weiwen , Proia David , He Suqin , Sang Jim , Foley Kevin , Du Zhenjian , Blackman Ronald k , Wada Yumiko , Sun Lijun , Koya Keizo
Published: October 2019

Abstract 2899

STA-9090 is a potent, second generation, small-molecule Hsp90 inhibitor, with a chemical structure unrelated to the first-generation, ansamycin family of Hsp90 inhibitors. In preclinical in vitro and in vivo studies, STA-9090 has shown potency up to 100 times greater than the first-generation Hsp90 inhibitors against a wide range of solid and hematological cancer types including those resistant to imatinib, sunitinib, erlotinib, and dasatinib. STA-9090 is currently being evaluated two Phase 1 and four Phase 2 trials (non-small cell lung, GIST, colon, and gastric) in solid tumor cancers; and two trials in hematologic cancers. Additional Phase 2 trials in several other indications are planned for 2H 2010.

Inhibition of Hsp90 by STA-9090 results in the destabilization of a broad range of oncogenic kinases often overexpressed or mutated in hematological cancers. For example, the nucleophosmin-anaplastic lymphoma kinase (NPM-ALK) expressed in the anaplastic large cell lymphoma (ALCL) cell line Karpas 299, is degraded rapidly in the presence of STA-9090 in vitro, resulting in the loss of viability. Similar results were shown in other NPM-ALK driven ALCL cells including SU-DHL-1 and SR-786 with IC50 less than 20 nM. Stability of other kinases common to hematological malignancies, such as Bcr-Abl, FLT3 and c-Kit, were also shown to be highly sensitive to STA-9090, resulting in potent cell death of cell lines addicted to signaling by these kinases.

In vivo, STA-9090 was highly effective in a subcutaneous xenograft model of diffuse large B-cell lymphoma SU-DHL-4 with resulting %T/C values of 26, 4, -90 and -93 when dosed at 25, 50, 75 and 100 mg twice per week, respectively. Importantly, 75 and 100 mg/kg STA-9090 dosed 2 times per week for a total of 3 weeks (150 and 200 mg/kg weekly) resulted in 25% and 50% of the animals in each group being free of tumors by the end of the study, respectively. MV4-11, an AML (FLT3ITD) cell line, turned out to be one of the most sensitive xenograft models to STA-9090 treatment. STA-9090 at 100 mg/kg or125mg/kg once weekly was highly efficacious with 37.5% of mice achieving tumor free with acceptable toxicity at the end of the 3-week treatment period.

In conclusion, STA-9090 exhibits preferable biological profiles both in vitro and in vivo in treating hematological malignances. Clinical studies for using STA-9090 both once weekly and twice weekly are ongoing.

Disclosures:

Ying: Synta Pharmaceuticals: Employment. Proia:Synta Pharmaceuticals: Employment. He:Synta Pharmaceur: Employment. Sang:Synta Pharmaceuticals: Employment. Foley:Synta Pharmaceuticals: former employee. Du:Synta Pharmaceuticals: former employee. Blackman:Synta Pharmaceuticals: Employment. Wada:Synta Pharmaceuticals: Employment. Sun:Synta Pharmaceuticals: Employment. Koya:Synta Pharmaceuticals: Employment.
PDE4 Inhibitors Enhance DNA Damage-Induced Apoptosis In CLL
Authors: Meyers John , Lerner Adam
Published: October 2019

Abstract 2902

Treatment of primary chronic lymphocytic leukemia (CLL) cells with PDE4 inhibitors induces apoptosis while comparable treatment of primary human T and B cells from healthy donors does not. PDE4 inhibitor treatment also augments glucocorticoid-induced apoptosis in CLL cells. We now report that PDE4 inhibitor treatment enhances CLL apoptosis induced by DNA damage. Using drug combination analysis, treatment with etoposide and rolipram resulted in a greater than additive apoptotic effect than predicted by a Loewe additivity model at many dose combinations. In order to directly address the role of DNA damage and rule out effects on drug uptake and efflux, we next examined the ability of PDE4 inhibitors to modulate gamma-irradiation-induced apoptosis in CLL. While most patients demonstrated in vitro sensitivity to rolipram and gamma irradiation, a small subset of patients, including those known to be p53 mutant, that lacked a substantial response to ionizing radiation also lacked sensitivity to rolipram (p < 0.01). Those patients with intact sensitivity to radiation, demonstrated at least an additive, or greater-than-additive at some doses, effect on apoptosis when PDE4 inhibitor treatment was combined with gamma irradiation treatment (p < 0.05 at 1 Gy and 20 mM rolipram). As judged by comet assays, gamma radiation-induced DNA double stranded breaks were repaired within one hour of induction regardless of exposure to rolipram. As judged by Western analysis and flow cytometry, the kinetics of gamma irradiation-induced H2AX phosphorylation, a marker for DNA damage, were not altered by addition of rolipram in a manner that correlated with apoptosis. Finally, rolipram treatment did not further augment levels of p53, p21cip, or phospho-Chk2. These results suggest that PDE4 inhibitors did not alter double strand DNA break repair nor alter upstream radiation-induced p53-mediated signaling events. As PDE4 inhibitors are known to induce CLL apoptosis through a mitochondrial pathway, our current studies are focused on identifying the mechanism by which DNA damage and PDE4 inhibitor signaling pathways converge at the mitochondrion to enhance apoptosis. Regardless of the precise mechanism by which these supra-additive apoptotic effects occur, our study adds credence to the concept that PDE4 inhibitors may prove to be important sensitizing adjuvants in the treatment of human B cell malignancies.

Disclosures:

No relevant conflicts of interest to declare.
Study on the Membrane Hemopoietic Cytokine Receptor Expression on CD34+ Bone Marrow Cells of the Patients with Myelodysplastic Syndromes
Authors: Shao Zonghong , Li Lijuan , Fu Rong , Wang Huaquan , Yue Lanzhu , Liu Hui , Wang Jun , Wang Honglei , Ruan Erbao , Qu Wen , Liang Yong , Wang Guojin , Wang Xiaoming , Liu Hong , Song Jia , Wu Yuhong , Xing Limin , Guan Jing
Published: October 2019

Abstract 2908

Objectives

This study was to detect if there were abnormalities of membrane hemopoietic cytokine receptor expression on CD34+ bone marrow cells in MDS.

Methods

34 newly diagnosed MDS(12 in low-risk and 22 in high-risk) cases and 32 normal controls were enrolled in this study. Their CD34+CD38+ and CD34+CD38- bone marrow cells and the expressions of stem cell factor receptor(SCF-R),erythropoietin receptor (EpoR), granulocyte colony-stimulating factor receptor (G-CSFR) and thrombopoietin receptor (TpoR) on those cells were measured by flow cytometry.

Results

The mean percentage of CD34+ BMMNCs of MDS cases in high risk[(2.94±4.79)%)] was significantly higher than that of control group[(0.95±1.06)%](P<0.05). The mean percentages of CD34+CD38+ cells were significantly lower in low risk and high risk groups[(86.98±6.83)% and (83.57±9.86)% respectively] than that in control group [(92.41±3.43)%], thus the percentage of CD34+CD38- cells was significantly higher in either low-risk or high-risk group[(13.03±6.84)% and (16.42±9.85)% respectively]than that in control group[(7.59±3.43)%](P<0.05). In control group, the mean percentage of antigen expression of EpoR was significantly lower in CD34+CD38+ cells [(17.72±20.24) %] than that in CD34+CD38- cells [(64.65±21.02)%](P<0.01), The expressions of SCF-R,G-CSFR and TpoR on CD34+CD38- cells were not significantly different from these on CD34+CD38+ cells. The expression of EpoR on CD34+CD38+ cells of low-risk and high-risk MDS groups[(7.01±6.82)% and (7.16±9.45)% respectively] were significantly lower than that of control group[(17.72±20. 24) %] (P<0.05), The expression of G-CSFR on CD34+CD38+ cells of low-risk and high-risk MDS groups[(22.65±12.14)% and (26.50±19.65)% respectively] were significantly lower than that of control group[(45.13±23.41)%](P<0.01). The amount of EpoR on CD34+CD38-cells of low-risk and high-risk MDS groups[(40.18±20.38)% and (28.58±17.00)% respectively] were significantly lower than that of control group[(64.65±21.02)%](P<0.01), The expression of TpoR on CD34+CD38- cells of low-risk and high-risk MDS groups[(4.46±7.45)% and (3.23±4.55)% respectively] were significantly lower than that of control group[(15.33±14.95)%](P<0.01). The incidence of cytopenia of MDS cases with low expression rates of hemopoietic cytokine receptors on CD34+cells were higher than that of MDS with high expression rates of hemopoietic cytokine receptors.

Conclusions

There were abnormalities of differentiation and membrane hemopoietic cytokine receptors expression of CD34+ bone marrow cells in MDS, which were associated with MDS cytopenia and might be useful for MDS diagnosis.

Disclosures:

No relevant conflicts of interest to declare.
A Favorable Outcome In Children with Juvenile Myelomonocytic Leukemia (JMML) with RAS Mutations
Authors: Doisaki Sayoko , Muramatsu Hideki , Hama Asahito , Shimada Akira , Takahashi Yoshiyuki , Kojima Seiji
Published: October 2019

Abstract 2909

Introduction:

Juvenile myelomonocytic leukemia (JMML) is a rare clonal myeloproliferative disorder that affects young children. It is characterized by a specific hypersensitivity of JMML cells to granulocyte-macrophage colony-stimulating factor (GM-CSF) in vitro. The pathogenesis of JMML involves disruption of GM-CSF signal transduction resulting from mutations of the components of the RAS signaling pathway, including NF1, PTPN11, NRAS, and KRAS. Somatic point mutations of the RAS genes at codons 12, 13, and 61 are found in approximately 20% of patients. Although most patients with JMML die due to progressive disease within 12 months unless treated with hematopoietic stem cell transplantation (HSCT), Matsuda et al reported that JMML patients with NRAS or KRAS glycine to serine substitution improved spontaneously. Other groups in Europe did not confirm this observation, and treatment for patients with JMML and RAS mutations is controversial. Therefore, in the present study, we analyze the association between the mutational status of RAS and prognoses of patients with JMML.

Patients and Methods:

Eighty children diagnosed with JMML between 1988 and 2010 were studied retrospectively. We performed a mutational analysis of NRAS, KRAS, PTPN11, and C-CBL genes.

Results:

Seventeen patients (21%) had RAS mutations [NRAS (n = 13) and KRAS (n = 4)], while PTPN11 and C-CBL mutations were found in 28 patients (35%) and 5 patients (6.3%), respectively (Four patients were included in the previous report; Matsuda et al, Blood, 2007). Five children had clinical evidence of NF1 mutations. Among NRAS mutations, G12D and G13D were the most common (n = 6 and n = 5, respectively). Only one patient carried a G12S substitution, which was reported as a favorable mutation. Three patients with KRAS mutations had G13D substitutions. Compared to patients with other mutations or without any aberrations, patients with RAS mutations were significantly younger at diagnosis (median age: 12 months vs. 24 months, p = 0.011), while other known predictive factors such as HbF level and platelet count were not significantly different at diagnosis (median HbF level: 9.1 % vs. 22.2 %, p = 0.295; median platelet count: 27.5 × 109/L vs. 49.0 × 109/L, p = 0.390). Monosomy 7 was observed in seven patients without RAS mutations, and all patients with RAS mutations had normal karyotypes. Among untransplanted patients with RAS mutations, three achieved long-term survival (20, 84, and 209 months after diagnosis). The probability of 5-year overall survival estimated by the Kaplan-Meier method was significantly higher for patients with RAS mutations than for those without (85.7% vs. 30.4%, p = 0.033).

Conclusion:

These results suggest that JMML patients with RAS mutations may be a distinct subgroup with favorable outcomes in spite of other than G12S.

Disclosures:

No relevant conflicts of interest to declare.
Cytogenetic Evolution (CE) In Patients (pts) with Low and Intermediate Risk Myelodysplastic Syndromes (MDS) Is Associated with Poor Prognosis
Authors: Li Liunan , Jabbour Elias , Borthakur Gautam , Faderl Stefan , Kadia Tapan , Ravandi Farhad , Pierce Sherry A. , Wierda William G. , Abruzzo Lynne V. , Kantarjian Hagop , Garcia-Manero Guillermo
Published: October 2019

Abstract 2941

Introduction:

MDS is a spectrum of abnormalities in the proliferation and differentiation of hematopoietic stem cells that result in peripheral cytopenias, bone marrow dysplasia and increased risk of transformation to acute myelogenous leukemia (AML). Cytogenetic abnormalities occur in more than 50% of patients (pts) and have an impact on survival and risk of transformation to AML. CE, or acquisition of additional clonal chromosomal abnormalities, has been reported to occur in 30 to 50% of primary MDS pts. Their impact on prognosis and transformation into AML among pts with low and intermediate risk MDS is not known. In this study, we analyzed the impact of CE on prognosis in lower risk MDS.

Methods:

we reviewed 722 pts clinic records of low and intermediate risk MDS pts at MD Anderson Cancer Center (MDACC) from 2000–2010 and conducted a retrospective analysis of all MDS pts with at least two consecutive cytogenetic analysis (365 patients, 50.6%) and compared the cytogenetic evolution group (CE group) with the group without cytogenetic changes (no CE group). Cytogenetic analysis was performed in the Cytogenetics Laboratory at MDACC.

Results:

CE was detected in 200 pts (55%). Characteristics of patients with CE are: median age 65 years (23-91), IPSS int-1 79%, diploid CG 42%, excess blasts 25%. Pts with CE were more frequently female (p=0.005), and had more frequently abnormalities of chromosome 5 and 7 (p<0.001) at baseline. There were no statistically significant difference between these two groups (p>0.05) regarding age, WBC, platelet, hgb, ANC, BM blasts percent, diagnosis (RA or RAEB), and IPSS score. There were more chr.-5/-7, insufficient metaphases, and other abnormalities, but less diploid cases in CE group compared with no CE group (p<0.001). History of malignancy (p=0.001) and prior chemotherapy exposure were also associated with CE (p=0.001), but this was not as strong for radiation exposure (p=0.066). Also, more CE patients required therapy for MDS compared to no CE patients (p=0.039). Progression free survival was significantly extended in no CE patients (p=0.02). Overall survival was a longer in no CE (34.1months), compared with CE group (26.2 months), although this was not statistically significant.

Conclusion:

CE is more commonly observed among pts with high-risk features, and is usually associated with disease progression and resistance. Also, prior malignancy and chemotherapy exposure were associated with CE in this study. This data indicates that genomic instability has a role in disease progression in MDS. Further analysis of CE in MDS is needed.

Disclosures:

No relevant conflicts of interest to declare.
Identification of Prognostic Markers by Gene Expression Profiling In Myelodysplastic Syndrome Hematopoietic Stem Cells
Authors: Pellagatti Andrea , Cazzola Mario , Giagounidis Aristoteles , Perry Janet , Malcovati Luca , Porta Matteo G. Della , Jädersten Martin , Killick Sally , Vyas Paresh , Hellström-Lindberg Eva , Boultwood Jacqueline , Wainscoat James S.
Published: October 2019

Abstract 298

The myelodysplastic syndromes (MDS) are a heterogeneous group of clonal hematopoietic stem cell malignancies that are characterized by ineffective hematopoiesis resulting in peripheral cytopenias and a hypercellular bone marrow. Approximately 40% of patients with MDS will develop an acute myeloid leukemia. It is important to establish prognosis of MDS patients since the treatment options vary from supportive care to bone marrow transplantation. In order to determine the relationship of gene expression levels to prognosis and so identify new molecular markers, we have used gene expression profiling to study the transcriptome of the hematopoietic stem cells of 125 MDS patients with a minimum 12 month follow up. The CD34+ cells obtained from MDS patients and healthy individuals were analyzed using Affymetrix U133 Plus2.0 arrays. The patients were split randomly in a training set (n=84) and a test set (n=41). Supervised principal components analysis was used to identify genes correlated with survival. Using the 84 patients in the training set, the Cox scores were computed for each gene, and the principal components calculated on the genes with the highest Cox scores. The first of the principal components was then used to generate a regression model to predict the survival in the test set. Finally, for each probe set an importance score was calculated equal to its correlation with the supervised principal component predictor. This approach returned a list of 150 top ranked probe sets correlated with survival. Patients in the training set were split into tertiles based on the predictor (low, medium and high score) and patients in the test set were assigned to their predicted class, and Kaplan-Meier plots were generated for both training and test set. The differences in survival for both training and test set were statistically significant (Figure 1). Top ranked genes showing lower expression levels in patients with shorter survival include CDH1, LEF1 and AKAP12/Gravin. Top ranked genes showing higher expression levels in patients with shorter survival include IL23A, WT1 and PTHR2. Figure 2 shows survival of patients divided into tertiles of expression for the individual genes CDH1, LEF1 and WT1. It is probable that the genes identified in this study will become the first validated molecular markers for MDS prognosis. Multivariate analysis is currently being performed.

Figure 1 Figure 1.

Figure 2 Figure 2.

Disclosure:

No relevant conflicts of interest to declare.
Towards Effective Immunotherapy of Multiple Myeloma: Enhanced Elimination of Myeloma Cells by Combination of Lenalidomide with the Human CD38 Monoclonal Antibody Daratumumab
Authors: van der Veer Michael S. , de Weers Michel , van Kessel Berris , Wittebol Shulamiet , Bakker Joost M , Parren Paul W.H.I. , Lokhorst Henk M , Mutis Tuna
Published: October 2019

Abstract 3059

Since its approval by the FDA, lenalidomide (LEN) demonstrates impressive results in patients with newly-diagnosed and relapsed multiple myeloma (MM). Despite these improvements, MM remains incurable, and new innovative treatments need to be developed in efforts to improve long term outcomes. Recently we have developed a new human CD38 antibody, daratumumab (DARA) which mediates MM cell killing via complement dependent cytotoxicity (CDC), antibody dependent cellular cytotoxicity (ADCC) and apoptosis. In the current study we evaluated the potential benefits of combining lenalidomide (LEN) with daratumumab (DARA). We demonstrate that DARA-dependent ADCC of purified primary MM cells, as well as of CD38+ UM 9 MM cells was significantly augmented by pre-treatment of PBMC effector cells with LEN. Importantly, a synergism between LEN and DARA was observed in ex-vivo assays, which allowed us to investigate ADCC directly in whole BM-MNC, thus without isolating MM cells from their natural environment. Interestingly, DARA-induced ADCC was significantly upregulated in PBMC derived from three MM patients who were undergoing LEN treatment. In conclusion, these results support the hypothesis that powerful and complementary effects may be achieved by combining LEN and DARA in clinical MM management.

Disclosures:

Veer: GenMab, Utrecht: Research Funding. Weers:Genmab: Employment. Bakker:GenMab, Utrecht, Netherlands: Employment. Parren:Genmab: Employment. Lokhorst:GenMab: Research Funding. Mutis:GenMab: Research Funding.
Comparison of Conventional Cytology Vs. High Sensitivity Flow Cytometry for the Diagnosis of Leptomeningeal Involvement by Hematological Lymphoid Malignancies
Authors: Lam Selay , Chin-Yee Ian H. , Keeney Michael , Popma Janice , Rizkalla Kamilia , Mangel Joy
Published: October 2019

Abstract 3112

Objective:

To compare the utility of high sensitivity (5-colour) flow cytometry (FCM) vs. conventional cytology (CC) for detecting cerebrospinal fluid (CSF) involvement in patients with hematological lymphoid malignancies.

Methods:

The results of diagnostic evaluations on all CSF samples analyzed for involvement by neoplastic lymphoid cells between January 2005 and February 2010 were reviewed retrospectively. Cases were identified by reviewing logs of all FCM procedures performed during that time period. FCM was performed on the CSF using a 5-antibody panel (“high sensitivity”).

Result:

108 patients (62M/46F) diagnosed with non-Hodgkin lymphoma or acute lymphoblastic leukemia underwent a total of 609 lumbar punctures (LP). The 359 samples that were sent for both CC and FCM form the basis of this analysis. The majority of the LP's (312/359, 87%) were negative for malignant cells by both FCM and CC (FCM-/CC-). 47 samples showed infiltration by tumor cells; of these, 25 (7%) were FCM+/CC+, and 22 (6%) were FCM+/CC-. No cases (0%) were FCM-/CC+. Using FCM as the gold standard, CC had a specificity of 100%, but a sensitivity of only 53%.

Conclusion:

High-sensitivity FCM has superior sensitivity to CC for diagnosing leptomeningeal involvement by lymphoid malignancies. CC failed to identify any additional cases that were not seen on FCM. This raises the question of whether performing CC on CSF samples to search for neoplastic lymphoid cells is of any additional diagnostic value.

Disclosures:

No relevant conflicts of interest to declare.