bluuebunny/crossref_metadata_2025 huggingface dataset:
The Medicinal and Antitumor Mushroom, Agaricus Blazei Murill, Activates NF-κB Via TLR2 but Not TLR4 In Monocytic Cells, and Stimulates Monocyte-Derived Dendritic Cells (MDDC) to Increased Cell Surface Marker Expression and Cytokine Production, and May Thus Have Adjuvant Effect In MDDC Cancer Vaccines
Authors: Hetland Geir , Tryggestad Anne Merete Aaland , Espevik Terje , Ryan Liv , Tangen Jon-Magnus , Johnson Egil , Kvalheim Gunnar
Published: October 2019

Abstract 3904

Agaricus bM (AbM) is an edible Basidiomycetes mushroom from Brazilian rain forest that is used in traditional medicine against cancer and other diseases. It is rich in immunomodulating substances like β-glucans and is shown to inhibit tumors and adjuvate hepatitis B virus DNA vaccine in mouse models. Previously, we have found that an AbM-based (82%) extract, AndoSan™, additionally containing Basidiomycetes mushrooms Hericium erinaceum and Grifola frondosa, also protected against gram positive and gram-negative sepsis and allergy in mice. Now, to examine the mechanisms behind AbM's biological effects and to see whether it is beneficial as adjuvant in DC cancer vaccines, cellular surface markers on stimulated MDDC were studied by flow cytometry and cytokine production by a 17-plex Luminex system. Transcription factor NF-kB activation, which is linked to cytokine production, cell cyclus regulation and more, was then determined by a luciferase assay system and western blotting as translocation of NF-kB from cytosol to nucleus in AbM stimulated promonocytic THP-1 cells and in HEK293 cells, transfected with CD14/MD2 and TLR2 or TLR4. In both cell lines, AbM induced NF-kB activation via TLR2, but inhibited such activation induced via TLR4. After 24h incubation of MDDC with AndoSan™, there was down-regulated CD11c, de novo CD69 and enhanced CD86 expression on the cells, as well as increased levels in the culture supernatants of cytokines TNFα, IL-1β, IL-2, IL-6, IL-8, MIP-1β, IFNγ and G-SCF. Moreover, when this mixed mushroom extract was added as adjuvant to a DC melanoma vaccine, there was increased proliferation (H3-thymidine incorporation) of cancer-peptide-specific T cells. We conclude that in monocytes and probably also MDDC, AbM activates transcription factor NF-kB via TLR2 stimulation but inhibits such activation induced via the LPS receptor, TLR4. In MDDC the AbM-based extract AndoSan™ induced differential expression of cell surface markers for cell adhesion, activation and antigen presentation, and increased production of a leukocyte growth factor and proinflammatory, chemotactic and some Th1-type but not Th2-type cytokines in vitro. This may explain some of the known antitumor properties of AbM and its effect against infection and allergy. Since preliminary data suggest that the AbM-based extract, AndoSan™, may also have positive adjuvant effect in MDDC-based cancer vaccine, it will be tested further ex vivo and in clinical trials with cancer patients subjected to such DC vaccines.

Disclosures:

Hetland: Immunpharma AS: Patents & Royalties, Research Funding. Tangen:Immunopharma AS: Research Funding. Johnson:Immunopharma AS: Patents & Royalties.
Incompletely Activated CD4+ T Cells Induce Granzyme B+ Regulatory B Cells In An Interleukin 21-Dependent Manner
Authors: Jahrsdoerfer Bernd , Dahlke Karen , Hagn Magdalena , Sontheimer Kai , Beyer Thamara , Lunov Oleg , Fabricius Dorit , Tron Kyrylo , Nienhaus G. Ulrich , Simmet Thomas
Published: October 2019

Abstract 3905

Immune regulation is central for the development of an efficient cellular immune response. Both Treg cells and plasmacytoid DC can suppress T cell proliferation in a granzyme B (GzmB)-dependent and perforin-independent manner. In the present study we found that, depending on stimulation with interleukin (IL-) 21, B cells (BC) can also express GzmB and effectively suppress T cell proliferation. GzmB expression in BC is enhanced by BC receptor engagement, and is suppressed by CD40 ligation. Since CD4+ T cells are a main source of IL-21, we tested whether they can induce GzmB in BC. We found that incompletely activated CD4+ T cells, but not fully activated T cells induce GzmB in co-cultured BC. Using confocal microscopy, we showed that BC-derived GzmB is enzymatically active and that GzmB+ BC transfer GzmB to CD4+ T cells. Furthermore, GzmB+ BC decreased CD4+ T cell expression of the TCR-zeta chain, a GzmB target, which is required for T cell proliferation. Our results suggest BC may regulate cellular adaptive immune responses by Treg cell-like mechanisms. Inhibition of BC-derived GzmB may represent a novel strategy to induce more effective and comprehensive cellular immune responses.

Disclosures:

No relevant conflicts of interest to declare.
Activation of Acute Lymphocytic Leukemia Blasts with CpG ODN, IL-4 and CD40 Ligand Facilitates Enhanced Anti-Leukemic CTL Responses
Authors: Fabricius Dorit , Breckerbohm Lucia , Queudeville Manon , Vollmer Angelika , Eckhoff Sarah M. , Pfeil Johanna , Strauss Gudrun , Fulda Simone , Jahrsdoerfer Bernd , Meyer Lueder H , Debatin Klaus-Michael
Published: October 2019

Abstract 3907

Acute lymphoblastic leukemia (ALL) is the most common pediatric malignancy. Although the vast majority of patients initially respond to up-front chemotherapy, relapses occur in approximately 20% of cases and have a poor prognosis. Thus, novel therapeutic strategies are required to treat minimal residual disease and improve long-term survival. B cell precursor (BCP)-ALL cells express low levels of costimulatory and antigen-presenting molecules and therefore are poorly recognized by the immune system. Previous reports show that CpG oligodeoxynucleotides (CpG) can induce immunogenicity of non-Hodgkin's lymphomas including B-CLL, and that CpG may decrease BCP-ALL tumor burden in a murine xenograft model. In the present study we investigated the effect of various combinations of known potent B cell stimulators including CpG, Interleukin (IL)-2 family cytokines and CD40 ligand (CD40L) on the immunogenicity of both primary BCP-ALL cells and a series of established BCP-ALL cell lines. Using combinations of CpG, IL-4 and CD40L we were able to enhance expression of CD40, CD54, CD86 and MHC class II on BCP-ALL cells, resulting in an increased capacity of BCP-ALL cells to induce proliferative T cell responses and to generate cytotoxic T cells (CTLs). Of note, these CTLs exhibited significantly enhanced anti-leukemic cytotoxicity not only towards treated but also towards untreated BCP-ALL cells, with similar effects found in both allogeneic and syngeneic settings. The strongest effects were observed after exposure of BCP-ALL cells to a combination of CpG, IL-4 and CD40L, whereas untreated control BCP-ALL cells induced minimal T-cell proliferation and cytotoxicity only. Importantly, CpG in combination with IL-4 and CD40L was significantly more effective than CpG alone. Our results demonstrate that combined treatment with CpG, IL-2 family cytokines and CD40L is more efficient than CpG alone in inducing an immunogenic phenotype in BCP-ALL cells. Further studies using a humanized leukemia mouse model may open novel therapeutic approaches in the management of BCP-ALL, for example by using adoptive T cell transfer after in-vitro stimulation of autologous T cells with activated BCP-ALL cells.

Disclosures:

No relevant conflicts of interest to declare.
AT9283, a Novel Pan-Aurora/JAK-2 Kinase Inhibitor Suppresses Tumor Growth In Aggressive B-Cell Non-Hodgkin's Lymphoma
Authors: Mahadevan Daruka , Liu Xiaobing , Persky Daniel Oscar , Miller Thomas P. , Squires Matthew S , Qi Wenqing
Published: October 2019

Abstract 3930

Aurora kinases (A and B) are oncogenic serine/threonine kinases that play a central role in regulating the mitotic phase of the eukaryotic cell cycle. Auroras are over-expressed in numerous tumors including aggressive B-cell non-Hodgkin's lymphomas (B-NHL) and are considered validated oncology targets. AT9283 a pan-Aurora/JAK-2 inhibitor has undergone early phase trials in acute and chronic myeloid leukemia with promising anti-tumor activity. Hence, we hypothesized that 1) targeting mitosis with AT9283 would be effective in promoting apoptosis in aggressive B-NHL cell lines, and 2) addition of a microtubule targeting agent activating the spindle assembly checkpoint would lead to enhanced anti-tumor activity in a mouse xenograft model of mantle cell lymphoma (MCL).

AT9283 inhibited Aurora A and B activity as demonstrated by reducing phosphorylation of Aurora A and histone H3 as well as inhibition of cell proliferation (IC50 of 0.02–1.6 μM) in a variety of aggressive B-NHL cell lines (diffuse large B-cell lymphoma, MCL and transformed follicular lymphoma). B-NHL cells treated with AT9283 exhibited endoreduplication confirming the mechanism of action of an Aurora kinase inhibitor. Treatment of B-NHL cells with AT9283 induced apoptosis demonstrated by flow cytometry and PARP-cleavage in a dose and time dependent manner. Aurora A over-expression has been demonstrated to override the spindle assembly checkpoint (SAC) and result in resistance to microtubule targeting agent (e.g. taxols and vinca alkaloids) induced apoptosis. At very low doses (5-50 nM) apoptosis was almost doubled in the combination (30%) compared to AT9283 or docetaxel alone (15%) compared to controls. A mouse xenograft MCL model (Granta-519) demonstrated that AT9283 (10 and 15 mg/kg) or docetaxel (10 mg/kg) alone had modest anti-tumor activity (10-20% tumor growth inhibition). However, AT9283 plus docetaxel demonstrated a statistically significant tumor growth inhibition of >60% over control. The body weights of all mice in all cohorts did not change significantly (within 10%) during the study and mice appeared to tolerate treatment(s) well. Together, our results suggest that AT9283 plus docetaxel may represent a novel therapeutic strategy in aggressive B-cell NHL and warrant early phase clinical trial evaluation [Funded by the Lymphoma SPORE, P50 CA130805501A1].

Disclosures:

No relevant conflicts of interest to declare.
Identification of AML and MDS Patients Who Could Potentially Benefit From Temozolomide Therapy: Proportion of Patients with Low Methyl Guanine Methyltransferase (MGMT) Expression Level
Authors: Brandwein Joseph , Kassis Jeannine , Leber Brian , Howson-Jan Kang , Minden Mark D. , Greaves Kim , Pouliot Jean-Francois
Published: October 2019

Abstract 3985

Introduction:

Temozolomide is an imidazotetrazine derivative of dacarbazine that functions as an alkylating agent. Temozolomide anti-tumor activity is attributed to methylation of DNA, leading to the formation of O6-methylguanine that eventually leads to apoptosis. Resistance to temozolomide may largely be conferred by O6-methylguanine DNA methyltransferase (MGMT) over-expression. The level of MGMT expression will determine the ability of a cell to withstand damage from an alkylating chemotherapeutic agent. Brandwein et al. (Leukemia 2007;21:821-4) previously showed that the complete response rate to temozolomide therapy in patients with acute myeloid leukemia (AML) was higher when MGMT expression was low.

Methods:

In this prospective multicenter study we evaluated MGMT expression by Western blot in bone marrow or peripheral blood from older patients with previously untreated AML and high risk myelodysplastic syndrome (MDS). Expression was characterized by comparison with b-actin and results were expressed as an MGMT:b-actin ratio: Negative: < 0.2; Weak: 0.2 – 0.25; and Strong:> 0.25 MGMT promoter methylation was also evaluated using methylation-specific PCR. Both protein expression and promoter methylation were tested in a central lab.

Results:

Data were obtained for 152 patients; median age was 76 years old (range 46–94 years). A majority (63%) were male. Primary diagnosis was AML (84%) and MDS (16%). MGMT expression level could be determined in 128 of the 152 patients tested (84%). Expression ratios were: Negative (41%), Weak (11%), and Strong (48%). MGMT promoter methylation could be detected in 16% of cases in patients with either Negative or Weak expression ratio and in 2% of patients with Strong expression ratio. A majority of patients with promoter methylation (82%) had low MGMT expression.

Discussion:

One-half of patients with AML/MDS had either a Negative or Weak MGMT expression level, and would therefore be potential candidates for temozolomide therapy. These low levels could be explained by promoter methylation in a minority of cases. Thus, low level of MGMT expression in AML/MDS may be due to direct alteration of the MGMT gene or modulation of expression at the post-transcriptional level.

Disclosures:

Brandwein: Merck: Honoraria, Research Funding. Off Label Use: temozolomide for acute myeloid leukemia. Kassis:Merck: Research Funding. Leber:Merck: Research Funding. Howson-Jan:Merck: Research Funding. Minden:Merck: Research Funding. Greaves:Schering-Plough/Merck: Employment. Pouliot:Schering-Plough/Merck: Employment.
Biomarkers In Cell Death of Imatinib-Resistant Ph-Leukemia Cells During Treatment with mTOR Inhibitor, Everolimus
Authors: Minami Miho , Minami Yosuke , Kuwatsuka Yachiyo , Kiyoi Hitoshi , Naoe Tomoki
Published: October 2019

Abstract 3988

Aberrant activation of mammalian target of rapamycin (mTOR) signaling pathway has been reported in hematological malignancies including leukemia initiating cells. Although rapamycin and its analogs have proven effective as anticancer agents, the mechanism of action and the solid biomarkers of response have not been fully elucidated. We investigated detailed biomarkers during the cell death of imatinib (IM)-resistant Ph-positive (Ph+) leukemia cells due to quiescence or mutations at the ABL-kinase domain after treatment with mTOR inhibitor, everolimus (Eve, RAD001). Ph+ leukemic NOD/SCID/IL2rγnull (NOG) mice cells were long co-cultured with S17 stromal cells and treated with IM and Eve. While slow-cycling (Hoechst 33342low/Pyronin Ylow) CD34+ cells were insensitive to IM in spite of BCR-ABL-dephosphorylation, combination treatment with IM and Eve induced substantial cell death including the CD34+ population. In Baf3/p210T315I cells, IM-resistant Ph+ leukemia cell line harboring T315I-mutation, Eve also induced cell death with low IC50 values in PI-exclusion assays. In murine model cutaneously injected with Baf3/p210T315I cells, in vivo-treatment with Eve decreased tumor formation. In these systems during treatment with Eve, we did not observe evident dephosphorylations of BCR-ABL, mTOR itself and 4EBP1, but rapid S6K-dephosphorylation with lower doses and decreased expression of MCL-1. Furthermore, the feedback-loop effects such as reversely increased phosphorylations of AKT (Ser473) and FOXO1/3a were also detected during the cell death. We are now investigating more efficient strategies using inhibitors screening kit and also planning to examine new generation of mTOR inhibitors to overcome the IM-resistance due to quiescence or T315I-mutation.

Disclosures:

Naoe: Kyowa-Kirin: Research Funding; Novartis: Research Funding; Janssen: Research Funding.
Assessing Proteostasis and Proteasome Stress In Light Chain Amyloidosis
Authors: Oliva Laura , Palladini Giovanni , Cerruti Fulvia , Pengo Niccolò , Cascio Paolo , Merlini Giampaolo , Sitia Roberto , Cenci Simone
Published: October 2019

Abstract 3992

Recently, proteasome inhibitors (PI) proved powerful against multiple myeloma (MM), the neoplastic transformation of plasma cells. The balance between proteasome expression and degradative workload (mainly contributed by protein synthesis) proved a crucial determinant of apoptotic sensitivity of MM cells to proteasome inhibition (Bianchi et al, Blood 2009). Light chain amyloidosis (AL) is a plasma cell dyscrasia caused by a bone marrow plasma cell clone synthesizing structurally unstable, misfolded, monoclonal immunoglobulin (Ig) light chains, which polymerize into amyloid fibrils. Interestingly, AL is proving even more sensitive than MM to PI in clinical trials with unprecedented response rates (>80%) rapidly achieved in previously untreated patients (Kastritis et al, J Clin Oncol 2010), raising the question as to whether, and if so why, AL cells are intrinsically more sensitive than MM to PI. We hypothesized that AL cells suffer from intense proteasome stress linked to the synthesis of the misfolded Ig light chain, thereby facing constitutive proteotoxicity.

To test this hypothesis, we set out to optimize purification of primary bone marrow plasma cells from AL patients, and determine: intrinsic sensitivity to the PI bortezomib (by FACS); proteasome activity (by fluorogenic assays); accumulation of ubiquitinated (Ub) proteins and Ig light chain (by immunofluorescence).

Our ex vivo studies demonstrated twofold higher PI sensitivity in AL plasma cells as compared to primary MM cells (EC50 in 24 hr apoptosis assays: AL, 8.3 ± 2.2 nM; MM, 15.1 ± 3.0 nM). We also found that, similar to MM cells, proteasome activity of primary AL plasma cells varies greatly among different patients (5.2 ± 3.6 nM substrate specifically cleaved by the chymotryptic β-peptidase activity per cell per min). Furthermore, accumulation of Ub proteins strongly correlates with light chain content, suggesting a crucial role for paraprotein synthesis and/or retention on proteasome stress. Interestingly, unlike MM cells, we failed to detect a clear correlation between proteasome activity and ex vivo assessed PI sensitivity, possibly due to intracellular toxicity of the misfolded light chain. The resulting hypothesis that different mutations could result in different intrinsic proteotoxicity in AL cells is currently being tested.

In conclusion, our integrated approach indicates that AL cells are intrinsically more sensitive to PI than MM cells, providing a potential explanation for the excellent clinical responses. Moreover, we established a technological platform to investigate proteostasis and proteotoxic stress in primary AL cells. This strategy may help investigate the efficacy of proteostasis regulators on plasma cell dyscrasias, including MM, and identify molecular markers of clinical use to predict disease severity and response to therapy.

Disclosures:

No relevant conflicts of interest to declare.
Myeloid-Derived Suppressive Cells Belonging to the Leukemic Clone Account for Immunosuppression In CMML
Authors: Droin Nathalie , Itzykson Raphael , Rameau Philippe , Morabito Margot , Braun Thorsten , Louache Fawzia , Zhang Yanyan , Botton Stephane De , Cordonnier Catherine , Quesnel Bruno , Fenaux Pierre , Vainchenker William , Solary Eric
Published: October 2019

Abstract 3997

We have shown previously that cells identified as monocytes in the peripheral blood of patients with chronic myelomonocytic leukemia (CMLL) included a variable proportion of CD14-negative, CD24-positive immature granulocytes. These cells synthesize and secrete alpha-defensin 1–3 that inhibit M-CSF-driven monocyte differentiation into macrophages through interaction with the P2Y6 purinergic receptor (Droin N et al, Blood 2010).

In the present study, we show that these CD14-,CD24+ immature granulocytes also inhibit the proliferation of autologous lymphocytes activated with anti-CD3 and anti-CD28 antibodies through cell-cell contact. This functional property suggested that these cells could be “myeloid-derived suppressive cells” (MDSC), which was supported by their phenotype that included expression of CD15 marker at their surface and survivin, S100A8 and S100A9, Cyclin D2 and Cyclin D3 at the mRNA level. STAT3 and STAT6 were found constitutively phosphorylated in these immature granulocytes that responded to Toll-like receptor agonists such as LPS or Pam-6. CD14-positive monocytes of the leukemic clone activated these MDSC through production of IL-13 and induction of arginase 1 mRNA, which could be reproduced by recombinant IL-13. On the other hand, activation of these MDSC did not require induction of the nitric oxide synthase mRNA, in agreement with their granulocytic origin.

We were able to generate immature myeloid cells expressing CD24 with morphology similar to that of peripheral blood MDSC by in vitro culture of various subpopulations of bone marrow CD34-positive cells obtained from CMML patients, including the most immature CD34+/CD38-/CD90+ cells. Furthermore, generation of these cells could be recapitulated in vivo by xenotransplantation of CMML CD34+ cells in NOG mice, albeit with lower efficacy than CD14+ cells. In patients with high grade CMML included in a phase II clinical trial, decitabine was observed to decrease both CD14+,CD24- monocytes and CD14-,CD24+ immature granulocytes.

Altogether, these data suggest that CMML initiating cells generate CD14-positive monocytes and, in most patients, an additional population of CD14-negative immature granulocytes with suppressive properties towards innate and acquired immune response. Generation of these cells may account for the high sensitivity of CMML patients to autoimmune and infectious diseases.

Disclosures:

Fenaux: Celgene: Honoraria, Research Funding; Novartis: Honoraria, Research Funding; Janssen Cilag: Honoraria, Research Funding; ROCHE: Honoraria, Research Funding; AMGEN: Honoraria, Research Funding; GSK: Honoraria, Research Funding; Merck: Honoraria, Research Funding; Cephalon: Honoraria, Research Funding.
Efficacy of Azacitidine In the Treatment of Chronic Myelomonocytic Leukemia
Authors: Teichman Melissa L. , Wetzstein Gene A. , Ho Viet Q. , Lancet Jeffrey E. , List Alan F. , Komrokji Rami S.
Published: October 2019

Abstract 4017

Background:

Chronic myelomonocytic leukemia (CMML) is a heterogeneous disease sharing features of myelodysplastic syndromes (MDS) and myeloproliferative neoplasms (MPN). FDA approved indications for azacitidine and decitabine include CMML as subset of MDS. Fewer than 10 patients with CMML, however, were treated in each of the original studies. In this study we report our institutional experience of azacitidine treatment of CMML patients.

Methods:

This was a retrospective review of CMML patients who received azacitidine at Moffitt Cancer Center. The primary endpoint was determining response rate to azacitidine utilizing International Working Group 2006 criteria (IWG 2006). Secondary objectives were to assess treatment tolerance and overall survival. Descriptive statistics were used for baseline characteristics and response rates. Kaplan-Meier estimates were used for evaluation of overall survival.

Results:

Between July 2004 and December 2009, 35 CMML patients treated with azacitidine were identified. Table-1 summarizes baseline characteristics of those patients. Based on Dusseldorf CMML risk criteria one patient (2.9%) was low risk, 17 (48.7%), intermediate, 7 (20%) high risk and 10 (28.6%) were unknown. According to MD Anderson CMML risk model, 11 (31.4%) were low risk, 12 (34.3%) int-1, 2 (5.7%) int-2, 1 (2.9%) high risk and 9(25.7%) unknown. The median number of azacitidine cycles was 6.0 (1-34)

The best response rates by IWG 2006 criteria were complete response (CR) 5 (14.3%), marrow CR 4 (11.4%), partial response (PR) 1 (2.9%), and hematological improvement (HI) 7 (20%). The overall response rate was 48.6%. The median OS was 25 month (95%CI 13.8–36.1 mo).

Conclusions:

In this retrospective analysis, response to azacitidine in CMML was similar to response rates reported in other MDS patients on azacitidine studies. The median overall survival is comparable to AZA-001 randomized clinical study.

Disclosures:

Lancet: Celgene: Research Funding. List:Celgene: Research Funding. Komrokji:Celgene: Research Funding, Speakers Bureau.
Thrombocytopenia Predicts for Poor Survival In Patients with Lower Risk Myelodysplastic Syndromes (MDS)
Authors: Cruz Raymond , Galili Naomi , Khan Ghulam Sajjad , Raza Azra
Published: October 2019

Abstract 4021

The incidence of thrombocytopenia in MDS is reported to be as high as 40–60% in various reports and is more commonly seen in patients presenting with higher risk disease (∼80%) by International Prognostic Scoring System (IPSS) compared to Int-1 (∼50%) or low risk MDS (12%). Thrombocytopenic MDS patients are at an increased risk of bleeding and hemorrhagic complications have been reported in 3– 50% patients with fatal hemorrhage occurring in 14–20% cases. The prognostic significance of thrombocytopenia in lower risk MDS patients (low+Int-1 risk) has not been clearly defined. To determine whether thrombocytopenia in lower risk MDS patients affects survival, we performed a retrospective study of 1,402 patients diagnosed with MDS between 1987 and 2010. Of these, 687 patients were classified by IPSS as follows: 22%(151/687) were identified as having low risk, 44% (301/687) had Int-1 risk, 4% 26/387) had Int-2, and 30% (209/687) had high risk disease. The overall incidence of thrombocytopenia (platelets <100,000/ul) across all groups was 54% (373/687) with the highest incidence being in the higher risk groups (46%), followed by Int-1 (43%) and low risk (7.5%). Of the 452 lower risk patients, 41% were thrombocytopenic (187/452). Overall, 256 lower risk patients had platelets of <150,000 and these were further divided into three subgroups based on platelet counts ranging between: 1) 100–149,000/ul; 2) 50–99,000/ul; 3) less than 50,000/ul. Median overall survival (OS) was obtained for each group. Statistical significance was calculated using Kruskal-Wallis rank sum test of equality of population. Non-thrombocytopenic lower risk patients showed a statistically significant higher median OS compared to thrombocytopenic patients (3.2 vs. 1.3 yrs p < 0.0005). Median OS progressively decreased with declining platelet range (2.4, 1.6 and 1.2 years) below 150,000/ul. Patients whose platelet counts were less than 50,000/ul had survival rates that were comparable to Int -2 patients (1.2 yrs). We conclude that the presence of thrombocytopenia predicts for poor survival in lower risk MDS patients and this information may be helpful in making therapeutic decisions.

Disclosures:

No relevant conflicts of interest to declare.
Prognostic Relevance of Genomic Aberrations In Light Chain Only Multiple Myeloma
Authors: Jiang Nan , Qi Connie , Trieu Young , Reece Donna E. , Chang Hong
Published: October 2019

Abstract 4036

Background:

Multiple myeloma (MM) is characterized by an expansion of clonal plasma cells with production of monoclonal immunoglobulin. The majority of MM patients produce an intact immunoglobulin, but in a subset of patients (≂f15%), the tumor produces monoclonal light chains only (LCO). Although specific genomic aberrations have emerged as a major prognostic factor in MM, the genomic changes and their prognostic impact on LCO myeloma patients are not clear.

Methods:

A total of 86 patients with LCO MM identified by urine and serum immunoelectrophoresis were included in this study. They were all uniformly treated with high dose chemotherapy followed by autologous stem cell transplant (ASCT). The genomic risk factors including del(13q), del(17p), t(4;14), 1q21 gain and 1p loss– were evaluated by cytoplasmic fluorescence in situ hybridization (cIg-FISH) in clonal plasma cells and correlated with patients clinical outcomes.

Results:

cIg-FISH detected del(13q) in 41%, t(4;14) in 12%, del(17p) in18%, 1q21 gain in 25%, and 1p loss in 19% of the evaluable cases. In our entire cohort, the median post-transplant follow-up was 36.5 months with a median progression free survival (PFS) of 24.8 months [95% confidence interval (CI): 18.4–31.3] and overall survival (OS) of 68.8 months (95% CI: 50.2–87.5). Patients with del(13q) and 1q21 gains had a significantly shorter PFS (median 15.8 vs. 33.4 months, p=0.002; median 19.1 vs. 33.4 months, p=0.011, respectively), and shorter OS (median 56.2 vs. 80.4 months, p=0.021; median 26.9 vs. 77.9 months, p=0.006, respectively), than those without such genetic abnormalities. In addition, 1p loss was associated with a significantly shorter PFS (median 18.2 vs. 37.9 months, p=0.001). However, there was no significant difference in PFS or OS in patients with or without the high-risk genetic factors t(4;14) or del(17p). On multivariate analysis adjusting for all 5 genetic risk factors, del(13q) was an independent prognostic factor for PFS (p=0.011) and OS (p=0.045).

Conclusion:

Although LCO MM had a similar incidence of genetic abnormalities to common MM, only del(13q) and chromosome 1 abnormalities appear to adversely affect the survival in our cohort. Further larger, prospective studies are warranted to verify the role of these genomic aberrations in the genetic risk stratification of LCO MM.

Disclosures:

Reece: Celgene: Honoraria, Research Funding.
Molecular Profiling of Extramedullary and Medullary Plasmacytomas Compared to Multiple Myeloma
Authors: Mahindra Anuj , Amin Samir B. , Sohani Aliyah R. , Motyckova Gabriela , Patel Kishan , Vallet Sonia , Pozzi Samantha , Santo Loredana , Cirstea Diana , Toomey Christiana E. , Michaelson James S. , Hochberg Ephraim P. , Cin Paola Dal , Rodig Scott J , Hideshima Teru , Anderson Kenneth C. , Munshi Nikhil C. , Raje Noopur
Published: October 2019

Abstract 4042

Plasmacytomas are rare clonal proliferations of plasma cells that though cytologically identical to plasma cell myeloma, present with osseous or extraosseous growth pattern. Understanding their molecular characteristics can provide crucial insights into their pathogenesis and risk of progression to multiple myeloma (MM). To investigate the differences between extramedullary (EMP) and medullary plasmacytomas (MP) and MM without plasmacytomas, we sought to molecularly profile these tumors by tissue microarrays, gene expression, microRNA, and FISH.

We identified 85 patients from our data base with a pathological diagnosis of plasmacytoma. Of the 85 patients, 13 patients presented with EMP, and 72 had MP. Among the patients with EMP (n=13), 2 patients presented with multiple lesions. Three of 13 (23%) patients progressed to develop MM at a median of 12 months. 72 patients presented with MP, of which 21 had solitary lesions and 27 (37%) progressed to MM at a median of 20.5months. There was a male preponderance (67% vs 33%) and the median age at diagnosis was 60.5 years (range 27.7–87.6). The mean overall survival for patients with EMP was 121 months (95% confidence interval[CI] 97–144 months) and for patients with MP was 102 months (95% CI 93–128 months) {p=0.025}.

MicroRNA (miRNAs) profiling was performed on MP (n=19), EMP (n=7) and MM samples (n=66). Data was normalized using U6 endogenous control. Gene expression profiling was performed and correlated with the miRNA data to identify genes and transcripts of interest. miRNA 127, which regulates SET D8, was upregulated four fold in both MP and EMP compared to MM. miRNA 493, which regulates cadherin 11 and PTCH 1, both of which have been associated with metastatic potential in solid tumors, was similarly downregulated four fold in both MP and EMP compared to MM. A tissue microarray was created on 52 patients (8: EMP, 44: MP,) in whom paraffin-embedded tissue was available. Additional evaluation using SET 8, cadherin 11 antibodies and validation of additional functional targets is ongoing and will be reported.

Differential expression patterns of factors involved in proliferation, survival, adhesion, and stroma-tumor cell interactions may help explain plasmacytoma biology and identify factors responsible for progression to MM. These insights may help identify new therapeutic approaches and targets in the treatment of these plasma cell disorders.

Disclosures:

No relevant conflicts of interest to declare.
Targeting Syk (spleen tyrosine kinase) Inhibits the Proliferation, Migration and Viability of Multiple Myeloma Cells
Authors: Körber Ruth-Miriam , Held Stefanie AE , Daecke Solveig , von Lilienfeld-Toal Marie , Bringmann Anita , Brossart Peter
Published: October 2019

Abstract 4071

Introduction:

Multiple myeloma (MM) is a malignant plasma cell disorder characterized by clonal expansion of single plasma cells in the bone marrow. Despite high-dose melphalan therapy with autologous stem cell transplantation (ASCT) and the introduction of active drugs like bortezomib or lenalidomide that have been associated with improved survival MM is still incurable. Thus, the identification of novel molecular targets and additional treatment options are warranted. In B-cell malignancies such as chronic lymphocytic leukaemia (CLL) or diffuse large B cell lymphoma, the inhibition of the tyrosine kinase Syk gave promising preclinical and first clinical results.In our study, we analyzed the potential of Syk as a target in MM.

Methods:

The MM cell lines AMO-1, U266, MMS-1 and RPMI8226 and primary MM cells were treated with the Syk-inhibitors BAY61-3606 or piceatannol and proliferation, migration and apoptosis induction were analyzed. Effects on involved intracellular signaling cascades were determined by Western blotting.

Results:

Incubation of MM cell lines with Syk-inhibitors resulted in a reduced proliferation and and SDF-1 induced migration, that was accompanied by a concentration dependent inhibition of the MAP kinase signaling characterized by reduced phosphorylation of ERK an p38 molecules. Furthermore, the nuclear localized expression of the NF-kB members RelA and RelB was inhibited in treated cells. In addition, Syk inhibition induced apoptosis in MM cell lines and primary MM cells in a dose-dependent manner as demonstrated by flow cytometry, caspase-3 activity and PARP-1 cleavage. While there was no effect on the expression of xIAP, survivin or MCL-1, Syk inhibition reduced the expression of pro-apoptotic Bcl-2 and Bcl-xl molecules and increased the release of cytochrome c, indicating that the apoptotic cell death is mediated via the internal mitochondrial pathway. Combination of piceatannol with lenalidomide and orally bioavailable dual phosphatidylinositol 3-kinase/mammalian target of rapamycin inhibitor NVP-BEZ235but not with bortezomib or dexamethasone enhanced the cytotoxic effects of the compound. In line with the results from previous experiments the addition of MAPK inhibitors PD98059, SP600125, U0126, SB202190 and SB203580 to piceatannol further increased the efficacy of Syk inhibition.

Conclusions:

Our results show that Syk inhibition might represent a promissing new treatment option in MM with an increased efficacy when combined with lenalidomide or MAP kinase inhibitors.

Disclosures:

No relevant conflicts of interest to declare.
Functional Role of BAALC In Leukemogenesis
Authors: Berg Tobias , Heuser Michael , Kuchenbauer Florian , Park Gyeongsin , Fung Stephen , Lin Grace , Leung Malina , Lai Courteney , Krauter Jürgen , Ganser Arnold , Humphries R. Keith
Published: October 2019

Abstract 4194

Cytogenetically normal acute myeloid leukemia (CN-AML) patients with high BAALC or MN1 expression have a poor prognosis. Whereas the oncogenic function of MN1 is well established, the functional role of BAALC in hematopoiesis is not known.

We therefore compared the expression of BAALC and MN1 in 140 CN-AML patients by quantitative PCR. To further assess the impact of BAALC on leukemogenesis we used retroviral gene transfer into primary murine bone marrow cells and cells immortalized with NUP98-HOXD13 (ND13) and HOXA9. Transduced cells were assessed in vitro by colony forming assays and for their sensitivity to treatment with all-trans retinoic acid (ATRA). They were also evaluated by in vivo transplantation into lethally-irradiated mice.

In the 140 CN-AML patients analyzed, the expression of BAALC and MN1 was highly correlated (R=0.71). Retroviral overexpression of MN1 or BAALC in the Hox gene-immortalized bone marrow cells did not cause upregulation of the other gene, suggesting that these genes do not regulate each other. In murine bone marrow cells BAALC did not immortalize the cells in vitro as assessed by serial replating of transduced cells in methylcellulose assays. Transplantation of transduced cells resulted in negligible engraftment of approximately 1 percent at 4 weeks after transplantation. However, co-transduction of BAALC into NUP98-HOXD13 cells (which are very sensitive to the treatment with all-trans retinoic acid) increased the 50 percent inhibitory concentration (IC50) of ATRA by 4.3-fold, suggesting a negative impact of BAALC on myeloid differentiation. We next evaluated whether the differentiation inhibiting effects of BAALC may cooperate with the self renewal-promoting effects of HOXA9 to induce leukemia in mice. Mice receiving transplants of murine bone marrow cells transduced with BAALC and HOXA9 developed myeloid leukemias with a median latency of 139.5 days that were characterized by leukocytosis, massively enlarged spleens (up to 1.02 g), anemia and thrombocytopenia. Infiltrations of myeloid cells were also found in liver, spleen, and kidney. The disease was transplantable into secondary animals. By Southern blot analysis we found one to two BAALC viral integrations per mouse, suggesting that clonal disease had developed from BAALC-transduced cells.

We demonstrate for the first time that BAALC blocks myeloid differentiation and promotes leukemogenesis when combined with the self-renewal promoting oncogene HOXA9. Due to its prognostic and functional effects BAALC may become a valuable therapeutic target in leukemia patients.

Disclosures:

No relevant conflicts of interest to declare.
Clinical & Molecular Analysis of Patients with Type 2 (Qualitative) Hereditary Antithrombin (AT) Deficiency
Authors: Patnaik Mrinal M. , Guenther Jennifer , Pruthi Rajiv , Heit John
Published: October 2019

Abstract 4201

Background:

Hereditary AT deficiency is classified as type 1 (quantitative) or type 2 (qualitative). Type 2 deficiency can be further subdivided into type 2a (reactive center loop [RCL]), type 2b (heparin binding domain), and type 2c (pleiotropic) based on functional and molecular AT analysis. Patients with heterozygous type 2b AT deficiency are thought to be at a lower risk for venous thromboembolism (VTE).

Objectives:

1) To estimate the frequency of type 2 hereditary AT deficiency. 2) To utilize molecular analysis to accurately sub type patients with type 2 defects. 3) To correlate thrombotic and obstetric complications with AT deficiency sub types.

Methods:

Apparently unrelated Mayo Clinic AT-deficient patients (n=20) were categorized as type 1 or 2 based on plasma AT activity and antigen, or by molecular analysis for previously reported type 2a-c mutations. Demographic and clinical characteristics were abstracted from patient medical records. The SERPINC1 putative promoter region, all exons (n=7) and splice junctions, and the 3′UTR were PCR amplified from leukocyte genomic DNA, and sequenced with nested forward and reverse primers. For patients without identifiable mutations, multiplex ligand-dependent probe amplification (MLPA) was performed.

Results:

Out of 20 probands tested 7 (35%) had type 2 AT deficiency. The mean patient age at diagnosis was 36 years (range 18–65) and 5 (69%) were women. There were 2 patients with type 2a, 4 with type 2b (including 1 with a homozygous defect), and 1 with type 2c AT deficiency. The mean plasma AT (range) activity/antigen for these patients were 50% (37-67%)/93% (78-103%) [AT activity & antigen normal range=80-130%]. 6 patients had a previously described mutation, whereas 1 had a novel mutation [S380R] affecting the RCL (Table1). All patients with type 2a and type 2c AT deficiency had unprovoked VTE occurring at a young age. All 3 patients with heterozygous type 2b AT deficiency had no VTE or obstetric complications. One patient with a homozygous type 2b defect (AT Vienna) presented with an unprovoked DVT at age 15. One family with AT Toyama (type 2b AT deficiency) had 5 asymptomatic adult family members with the mutation.

Background:

Molecular testing is important for an accurate subtyping of patients with type 2 AT deficiency. Type 2 defects have a diverse clinical spectrum. Patients with heterozygous type 2b AT deficiency have a low rate of VTE and obstetric complications. Homozygous type 2b AT deficiency can be compatible with adult life.

Disclosures:

No relevant conflicts of interest to declare.
Patients with Cancer Who Develop a First Venous Thromboembolic Event After Surgery Are at High Risk of Venous Thromboembolism Recurrence During the Anticoagulation Period
Authors: Louzada Martha L , Lazo-Langner Alejandro , Carrier Marc , Dao Vi , Zhang Jerry , Rodger Marc A. , Kovacs Michael J. , Wells Philip
Published: October 2019

Abstract 4202

Background:

It is unknown whether patients with cancer who develop VTE after a surgical procedure have the same risk of recurrent VTE as clinical patients cancer-associated thrombosis. VTE recurrence risk in non-cancer patients with VTE after surgery is approximately 1% in the 3 months following completion of anticoagulation. It is unknown whether surgical patients with cancer follow the low risk of recurrence as other provoked VTEs or whether they have the high recurrence risk typical of cancer patients.

Methods:

We performed a post-hoc analysis of a single centre retrospective cohort study conducted at the Thrombosis Unit of the Ottawa Hospital. The charts of patients with cancer and VTE followed from 2002 to 2004 and from 2007 to 2008 were reviewed. We sought to compare the risk of recurrent VTE between patients with cancer who developed a first VTE after major surgery with all other patients with cancer-associated thrombosis. We included patients > or = 18 years of age with active malignancy and objectively diagnosed index VTE [pulmonary embolism (PE), proximal deep venous thrombosis (DVT) of the legs or arms, PE + DVT; unusual site thrombosis]. After the first VTE, all patients received a minimum of 6 months of anticoagulation. In the surgery group, index VTE was considered associated with the intervention if it occurred within the first 3 months after the procedure.

Results:

543 patients were included. 121 patients had VTE after surgery and 17 (13.1%) developed a recurrence during therapeutic anticoagulation. Of 422 clinical patients, 61 (14.7%) had a recurrent VTE (Table). The relative risk of recurrent VTE comparing patients who had and who did not have surgery was non-significant (RR= 0.97 (95%CI: 0.587 – 1.574; p= 1.000) suggesting that patients with cancer who undergo surgery have similar risk of developing a recurrent VTE during anticoagulation as patients with cancer-associated VTE who do not undergo surgery. VTE recurrence occurred predominantly within the first 6 months of anticoagulation [Surgery: 9 of 17 patients (52.9 %); no surgery: 45 of 61 (73.7%) patients (p=0.1377)] (Figure). There was no significant difference in VTE recurrence risk according to anticoagulant strategy, tumor site, histology, TNM stage, age or gender between surgery and no surgery groups.

Conclusion:

Patients with cancer who develop VTE after surgery have similar risk of developing a recurrent VTE during the anticoagulation period as clinical patients with cancer-associated VTE.

Disclosures:

Rodger: Pfizer: Research Funding; Leo Pharma: Research Funding; Sanofi Aventis: Membership on an entity's Board of Directors or advisory committees, Research Funding; Canadian Institutes of Health Research: Research Funding; Heart and Stroke Foundation: Research Funding.
Treatment with the Small Molecule Genistein Increases Hepcidin Expression In Zebrafish Embryos and Human Hepatocytes
Authors: Gibert Yann , Zhen Aileen W. , Nguyen Nancy H. , Fraenkel Paula G.
Published: October 2019

Abstract 4245

Hepcidin, a transcriptionally regulated peptide hormone, produced by hepatocytes in response to iron overload, decreases intestinal iron absorption and macrophage iron release via effects on ferroportin. Hepcidin is a potential drug target for patients with iron overload syndromes, because its levels are inappropriately low in these patients. Among patients with hereditary hemochromatosis, premenopausal women exhibit less severe iron overload than their male counterparts. While this has been attributed to menstrual blood loss, we hypothesized that estrogens may play a role in regulating Hepcidin expression. We tested estradiol and the phytoestrogens, genistein, apigenin, daizdein, and genistin, for their effects on hepcidin expression in zebrafish embryos. In addition to its estrogenic effects, genistein is known to inhibit multiple protein tyrosine kinases. We found that genistein treatment from 28–52 hours post-fertilization in zebrafish embryos enhanced hepcidin transcript levels relative to liver size, as assessed by whole mount in situ hybridization, while estradiol and the other phytoestrogens did not produce a similar effect. To evaluate whether these effects were conserved in human hepatocytes, we treated HepG2 cells with genistein for 24 hours. We found that genistein treatment was associated with a significant increase in Hepcidin transcript levels, which was abrogated by co-treatment with the bone morphogenic protein (BMP) signaling antagonist dorsomorphin, but not by co-treatment with the estrogen receptor antagonist ICI 182,780. In conclusion, genistein promotes Hepcidin expression in hepatocytes in a BMP-dependent, but estrogen receptor independent manner. Future experiments will address the signaling pathway by which genistein exerts its effect on Hepcidin expression.

Disclosures:

No relevant conflicts of interest to declare.
Nitrogen Monoxide Inhibits Heme Synthesis In Reticulocytes
Authors: Mikhael Marc , Apte Sameer , Soe-Lin Shan , Ponka Prem
Published: October 2019

Abstract 4252

Anemia of chronic disease (ACD) is a condition that often manifests in patients with chronic immune activation due to chronic infections, autoimmune disorders, cancer and other diseases. The pathogenesis of ACD is complex and involves inefficient erythropoietin production, immune-mediated inhibition of erythropoiesis, and retention of iron in hemoglobin-processing macrophages. During their development, erythroid cells are closely associated with macrophages. In inflammatory conditions, activated macrophages generate large quantities of the gaseous molecule, nitric oxide (NO), which has numerous effects on iron metabolism. In this study, we explored the possibility that NO affects iron metabolism in erythroid cells. We treated reticulocytes with the NO donors, sodium nitroprusside (SNP) and S-Nitroso-N-acetyl-D,L-penicillamine (SNAP). We show that NO inhibits 59Fe incorporation from 59Fe-transferrin into reticulocytes and their heme. Significantly, 5-aminolevulinic acid (ALA, the product of ALA synthase, which catalyzes the first step of heme synthesis) reversed the SNP-mediated decrease in 59Fe incorporation into heme but not the cellular 59Fe uptake. In addition, SNP treatment led to an increase in eIF2α phosphorylation (which is known to occur in heme-deficient cells) and decreased globin translation. Importantly, the addition of ALA to SNP-treated reticulocytes prevented the effect of SNP on eIF2α phosphorylation and reversed globin synthesis inhibition. This indicates that in SNP treated reticulocytes, the phosphorylation of eIF2α and inhibition of globin synthesis occur indirectly, via NO's effect on erythroid-specific ALA synthase (ALA-S2). These results led us to conclude that NO has two distinct effects on reticulocytes, namely: a decrease in ALA-S2 activity and a decrease in transferrin-mediated iron uptake. The profound impact of NO on heme synthesis, iron uptake and globin translation in reticulocytes raises the possibility that NO production by macrophages could also contribute to ACD.

Disclosures:

No relevant conflicts of interest to declare.