bluuebunny/crossref_metadata_2025 huggingface dataset:
Resveratrol Induces Cell Cycle Arrest and Apoptosis in Malignant NK Cells Via JAK2/STAT3 Pathway Inhibition
Authors: Trung Ly Quoc , Espinoza Luis Jorge , Takami Akiyoshi , Yoshida Akiyo , Nakao Shinji
Published: October 2019

Abstract 1343

Natural killer (NK)-cell malignancies, particularly aggressive NK-cell leukemias/lymphomas, have poor prognoses. Although recent regimens that include L-asparaginase substantially improve outcomes, novel therapeutic approaches are needed to improve clinical responses. Recent reports have shown that the signal transducer and activator of transcription 3 (STAT3) pathway is critical for proliferation and survival of malignant NK cells. Resveratrol is a naturally-occurring polyphenol that has been extensively studied for its anti-inflammatory, cardioprotective, and anti-cancer activities. In this study, we investigated the potential anti-tumor activities of resveratrol against the NK cell lines KHYG-1, NKL, NK-92, and NK-YS. Resveratrol significantly suppressed cell proliferation in a dose- and time-dependent manner in these four cell lines. Flow cytometry analysis with annexin V/propidium iodide staining showed a variable but consistent induction of apoptosis in the four cell lines treated with resveratrol for 48 hours, ranging from 57.1±6.9% of apoptosis in the L-asparaginase resistant cell line KHYG-1 to 53.4±9.7%, 28.8±3.3%, and 51.7±6.7% in NKL, NK-92, and NK-YS cells respectively (Fig. 1a). Notably, the anti-tumor activity of resveratrol against NK cell lines was p53 independent as demonstrated by equal efficacy of resveratrol against NK cell lines pretreated with the p53 inhibitor Pifithrin-α. Immunoblot analysis to study intracellular signaling in resveratrol-treated cells showed suppression of constitutively active STAT3 in all four cell lines 24 hours after treatment. Remarkably, resveratrol inhibited JAK2 phosphorylation, but had no effect on other known upstream mediators of STAT3 activation such as PTEN and Tyk2 (Fig. 1b). Resveratrol induced robust G1 cell cycle arrest and down-regulation of two anti-apoptotic proteins, Mcl-1 and survivin, both of which are downstream effectors of the STAT-3 pathway. Furthermore, resveratrol enhanced the pro-apoptotic and anti-proliferative activities of L-asparaginase against NKL and NK-92 cells by 32% and 126% respectively.

These data indicate that resveratrol possesses a potent anti-tumor effect via inactivation of the STAT3 pathway in malignant NK cells. These mechanistic findings suggest that resveratrol may have therapeutic potential against NK cell malignancies. Our finding that resveratrol is a bonafide JAK2 inhibitor extends its therapeutic potential to other diseases with deregulated JAK2 signaling.

Disclosures:

No relevant conflicts of interest to declare.
AML Exosomes Regulate Stromal Bystander Cells and Provide a Novel RNA Biomarker Platform
Authors: Huan Jianya , Hornick Noah , Lenz Kyle , Goloviznina Natalya A , Skinner Amy M. , Tyner Jeffrey W , Roberts Charles T , Kurre Peter
Published: October 2019

Abstract 1378

Cell membrane-derived vesicles carrying protein and RNA cargo have recently emerged as potent mediators of cell-cell communication. These vesicles traffic from their tissue of origin into the blood stream, providing a serum-based and minimally invasive biomarker platform for cancer-specific RNA and protein signatures. We were interested in evaluating vesicle production by acute myeloid leukemia (AML) cells, with an emphasis on RNA content and cell-cell trafficking. Here we demonstrate for the first time that cell lines and primary cells from AML patients release exosome-sized vesicles (30–100nm) capable of transfer to bystander cells. Analysis of their non-protein content revealed the enrichment of many coding- and non-coding RNAs with relevance to AML biology, including prognostically important transcripts encoding FLT3, NPM-1, IGF-IR, CXCR4 and MMP9. In addition, we demonstrated the presence of a diverse group of transcription factor mRNAs (MYC, MEF2C, GATA1, SHIP1, ID1, E2F1, CEPBA CEPBB) in exosomes. PCR array studies also confirmed the broad representation of cellular miRNAs. Among the 234 miRNAs represented, the levels of several (miR −9, −155 and −223 and LET-7A) were enriched over the levels seen in donor cells (U6 RNA control). When examining cell-cell trafficking of AML exosomes, we noted rapid entry into co-cultured stromal cells, with maximum uptake occurring between 2 and 3 hours. AML-derived mRNA transcripts were transferred to bystander cells without direct cell-cell contact after Transwell co-culture or direct exposure to purified exosome preparations, or via vesicle-rich media. Results show that exosome trafficking induced changes in gene expression (OPN, ANGPT, SCF, SDF-1A) and altered the secretion of cytokines and angiopoietic growth factors by bone marrow stromal cells. Additional proof-of-concept studies provided evidence for the canonical function of transferred RNA. Specifically, we found that transfer of IGF-IR mRNA led to proliferative responses in co-cultured OP9 stromal cells that were abrogated by the IGF-IR specific inhibitor picropodophyllin. Further, exosomes derived from AML cell lines (HL60 and Molm14) altered c-FOS and VEGF expression in OP9 cells as well as improving viability and promoting migration toward SDF-1A by a hematopoietic progenitor cell line (Baf3), respectively. Our observations support the concept that AML exosomes exhibit regulatory potential in the leukemic bone marrow niche via direct cytoplasmic cell-cell transfer of transcripts encoding tyrosine kinase receptors and transcription factor as well as micro- RNAs. We propose that AML exosomes contribute to the extrinsic signaling cues in the microenvironment that promote disease resistance in AML and that they are rich in biomarkers to guide diagnosis and treatment.

Disclosures:

No relevant conflicts of interest to declare.
Oral Realgar-Indigo Naturalis Formula Achieved Comparably High Rate of Remission and Survival to Intravenous Arsenic Trioxide As a Front-Line Treatment in Newly Diagnosed Acute Promyelocytic Leukemia: A Multi-Center Randomized Trial APL07
Authors: Zhu Zhu-Hong , Wu De-Pei , Jin Jie , Li Jian-Yong , Ma Jun , Wang Jiangxiang , Jiang Hao , Chen Sai-Juan , Huang Xiao-Jun
Published: October 2019

Abstract 140

Background

Arsenic trioxide increases the rate of complete remission (CR) and prolongs long-term overall survival in patients with acute promyelocytic leukemia (APL). We demonstrated that an oral arsenic-containing Realgar-Indigo naturalis formula (RIF) could yield clear synergistic effects in an in vivo murine APL model as well as an in vitro APL cell lines. The efficacy and safety of this formula were confirmed by a CR rate of 96.7% and reasonable safety profile in a multicenter Phase II clinical trial in China. To find out whether oral RIF could achieve similar remission and survival with ATO, the Chinese APL Cooperative Group conducted a phase 3 randomized study (APL07) that compared oral RIF with ATO in both the induction and maintenance therapies.

Methods

In this phase 3 randomized trial, 242 newly diagnosed APL patients were randomly assigned to receive oral RIF (n=121, daily 60 mg per kg body weight) or ATO (n=121, daily 0.15 mg per kg body weight) as a part of induction therapy. All patients also received all-trans-retinoic acid (ATRA; daily 25 mg per square meter) as induction therapy, and were between the ages of 15 and 60 years. After achieving CR, all patients were given three courses of consolidation chemotherapy with anthracycline and cytosine arabinoside. The maintenance treatment included eight cycles of sequential use of ATRA and RIF or ATO for two years. The primary endpoint was disease-free survival (DFS). The secondary endpoints were the rate of CR and overall survival (OS) and safety.

Results

At a median follow-up of 28 months, no significant differences were noted between RIF group and ATO group with respect to CR rate (99.1% vs.97.2%, P=0.62), DFS (98.9% vs.97.64%, P=0.6) (Figure 1A) and OS at 3 years (99.1% vs.96.6%, P=0.18) (Figure 1B). The rates of adverse events were similar in the two groups. The rate of treatment-related grade 3/4 liver toxic effects was 9.6% in the RIF group and 12.0% in the ATO group(p=0.67). When on the arsenic-containing treatment, the plasma arsenic content was higher in the ATO group than in the RIF group (56.3 vs. 23.6 μg/L, p=0.0002). The median levels of arsenics concentration of plasm, urine, hair and nail samples after completing therapy 12 months were below the lower limit of the normal control.

Conclusions

Oral Realgar-Indigo naturalis formula yielded a comparable high remission and long-term survival with arsenic trioxide as front-line treatment of newly diagnosed APL.

Disclosures:

No relevant conflicts of interest to declare.
Reduced Intensity Conditioning Allogeneic Stem cell Transplantation (RIC-alloSCT) for Elderly Acute Myeloid Leukaemia (AML) Patients Over 60 Years: A “donor” Versus “no donor” Comparison
Authors: Peterlin Pierre , Raffoux Emmanuel , Chevallier Patrice , Guillaume Thierry , Gastinne Thomas , Le Gouill Steven , Moreau Philippe , Dombret Hervé , Mohty Mohamad , Delaunay Jacques
Published: October 2019

Abstract 2037

Acute Myeloid Leukemia(AML) in elderly patients is associated with a poor outcome even for patients able to receive intensive chemotherapy. Relapse rate remain elevated around 60 to 80% at 3 years especially in case of unfavourable cytogenetics. From more than 10 years, the approach of RIC-alloSCT has permitted to extend the age limit of transplant in this population. However, feasibility remains controversial for patient aged of 60 years or more because of more frequent co-morbidities translating into an higher risk of treatment related mortality (TRM). To address the question of feasibility and results of RIC-alloSCT in AML patients of 60 years or more in a intend to treat manner approach, we have conducted a retrospective bi-centric analysis based on a comparison “donor” versus “no donor” for patient in first complete remission (CR1).

Methods:

96 AML were included, all have received a 3+7 or equivalent induction regimen, between 2001–2009 in two different French centers and have benefited of an HLA typing in CR1. All patients of the “donor” group are considered for a RIC-alloSCT after one or two courses of consolidation.

Results:

The “donor” group is constitute of 30 patients with a median age of 65.5 years and with an HLA identical donor identified. Eighteen 60%) have been finally transplanted with a median period of 188 days from diagnosis to graft. The “no donor” group is constituted by 66 patients with a median age of 68.5. The two groups are similar considering cytogenetics risk and haematological features of the disease.

Median follow up is 48 months for alive patients. No difference of overall survival (OS) or Leukemia free survival (LFS) has been observed between the ”donor” and “no donor group” (respectively 51% vs 44%, p=0.7 and 35%vs 32%, p=0.27). When restricting analysis to the patient who could actually received the RIC allo-SCT, the transplant group (n=18) has a significant better outcome for both OS and LFS as compared to the “non transplant” (n=78) group (69% vs 32% and 52% vs 29% respectively). In the transplant group, no patient died from transplant-related mortality. Acute and extensive chronic graft versus host disease (aGVHD and cGVHD) occurred respectively in 5/18 and in 8/18 patients of the “transplant” group.

Conclusion:

In a intend to treat manner, RIC-alloSCT provides similar outcome as compared to chemotherapy alone in this series of 96 patients. However, both OS and LFS are significantly improved for patients who have actually received RIC-alloSCT. Toxicity appears to be limited in the “transplant” group. Recruiting patients in this study is still ongoing and final results will be presented at the congress.

Disclosures:

No relevant conflicts of interest to declare.
A Novel EGLN1/PHD2 High-Frequency Variant in Tibetans Protects Against Hypoxia-Induced Polycythemia.
Authors: V Felipe R Lorenzo , Huff Chad Daniel , Myllymäki Mikko , Swierczek Sabina , Salama Mohamed E , Semenza Gregg , Gordeuk Victor R. , Jorde Lynn , Koivunen Peppi , Prchal Josef T.
Published: October 2019

Abstract 2079

The hypoxic response, mediated by hypoxia inducible transcription factors (HIFs), is central to the control and development of many essential biological functions, including erythropoiesis. As a high altitude population, Tibetans are genetically adapted to the environmental stress of high altitude, having normal hemoglobin concentration both at sea level and at high altitude. Several haplotypes have undergone positive selection in Tibetans, including variations at or near the EGLN locus, which encodes prolyl 4-hydroxylase (PHD2). PHD2 triggers the degradation of hypoxia-inducible factors (HIFs) that mediate many physiological responses to hypoxia, including erythropoiesis.

We now describe two high-frequency Tibetan variants, PHD2D4E and PHD2C127S, that originated on the same haplotype about ∼6,000 years ago and contribute functionally to the Tibetan high-altitude phenotype. The PHD2D4E, C127S variant exhibits a lower Km value for O2, suggesting that it promotes HIF degradation under hypoxia. Because HIF directly stimulates erythropoiesis, the measured kinetic PHD2D4E, C127S properties were experimentally reproduced in native PHD2D4E, C127S erythroid progenitors that had decreased proliferation under hypoxic conditions, whereas wild-type progenitors have increased proliferation. Our results demonstrate that the Tibetan selected PHD2D4E, C127S variant contributes to protection from polycythemia at high altitude.

We describe the first high-altitude adaptive mutations in Tibetans and demonstrate that they abrogate hypoxia-induced HIF-mediated augmentation of erythropoiesis, explaining Tibetan protection from polycythemia at high altitude.

Disclosures:

No relevant conflicts of interest to declare.
Impact of Factor IXa Content On Function, Safety and Efficacy of Recombinant Factor IX Products.
Authors: Auer Wilfried , Rottensteiner Hanspeter , Schrenk Gerald , Hoellriegl Werner , Schiviz Alexandra , Scheiflinger Friedrich , Schwarz Hans Peter , Turecek Peter L , Muchitsch Eva-Maria
Published: October 2019

Abstract 2233

Baxter has developed a new recombinant factor IX (rFIX) product (BAX 326) for treating patients with hemophilia B. The aim of the presented studies was to evaluate the function, safety and efficacy of BAX 326 compared to a commercially available rFIX regarding differences in activated FIX (FIXa) content.

The FIXa concentration of BAX 326 was about 10 fold less than that of a commercial rFIX product. To address the influence of FIXa on functional FIX in vitro assays, the FIXa concentration of BAX 326 was increased. Samples were analyzed by one-stage clotting assay, non-activated partial thromboplastin time assay and thrombin generation assay. In all three assays, FIXa affected the potency determination for FIX, indicating that rFIX products should preferentially contain a low FIXa content.

The thrombogenic potential of BAX 326 was assessed in rabbits using a modified Wessler Test at 750 IU/kg (10-fold human clinical dose). No thrombogenic potential was observed with BAX 326 (individual scores of 0), whereas the mean score for the commercially available rFIX was 0.5. After increasing the FIXa concentration of BAX 326 to equalize it with the commercially available rFIX, a mean score of 0.42 (individual scores 0 – 0.5) was determined. These data strongly suggest that the differences in preclinical thrombogenicity were caused by the higher FIXa content of the commercially available rFIX, thereby confirming earlier findings [1].

Efficacy of the rFIX products was studied in hemophilia B (FIX ko) mice that received prophylactic treatment with 75 IU/kg of BAX 326 or the commercially available rFIX. Treated animals were analyzed in two primary pharmacodynamic models: a carotid occlusion model and one using thrombelastography. In both models, results for the two groups were similar, demonstrating that despite its lower FIXa content, BAX 326 was as efficacious as a commercially available rFIX at 75 IU/kg (p<0.0076).

Disclosures:

Auer: Baxter Innovations GmbH: Employment. Rottensteiner:Baxter Innovations GmbH: Employment. Schrenk:Baxter Innovations GmbH: Employment. Hoellriegl:Baxter Innovations GmbH: Employment. Schiviz:Baxter Innovations GmbH: Employment. Scheiflinger:Baxter Innovations GmbH: Employment. Schwarz:Baxter Innovations GmbH: Employment. Turecek:Baxter Innovations GmbH: Employment. Muchitsch:Baxter Innovations GmbH: Employment.
Factor V Leiden Mutation Increases the Risk of Venous Thromboembolism in Cancer Patients – Results From the Vienna Cancer and Thrombosis Study (CATS).
Authors: Pabinger Ingrid , Ay Cihan , Dunkler Daniela , Thaler Johannes , Reitter Eva-Maria , Marosi Christine , Zielinski Christoph , Mannhalter Christine
Published: October 2019

Abstract 2253

Background:

Patients with cancer are at an increased risk of venous thromboembolism (VTE). The risk varies markedly in different patient populations and improvement of the prediction of VTE would be of advantage for tailoring thrombosis prophylaxis. Factor V Leiden is the most common genetic risk factor for VTE and the impact of factor V Leiden on cancer-associated thrombosis is not yet fully elucidated.

Objective:

To study the impact of factor V Leiden on the risk of VTE in cancer patients.

Patients and Methods:

Nine-hundred-eighty-two patients with newly diagnosed cancer (n=745) or progression of disease after complete or partial remission (n=237) were included in the cancer and thrombosis study (CATS), a prospective observational single centre cohort study at the Medical University Vienna. Patients were followed for a maximum period of 2 years. Blood samples were collected at inclusion and factor V Leiden was determined by genotyping. The main outcome measure was symptomatic or lethal objectively confirmed VTE. All VTE events were adjudicated independently.

Results:

Of the 982 patients (median age 62 years, interquartile range (IQR) 52–68, 537 men, 445 women) factor V-Leiden was found in 72 (7.3%), 70 had a heterozygous and two a homozygous genotype. Ten of 72 (14%) patients with factor V-Leiden developed VTE, whereas this was the case in 69 of 910 (7.6%) patients without factor V-Leiden. Interestingly, both patients with homozygous factor V Leiden developed VTE. In multivariable analysis that included age, sex, different tumour types, newly diagnosed versus recurrence of disease and the treatment modalities (chemotherapy, radiotherapy and surgery) the hazard ratio (HR) for factor V Leiden was 2.04 (95% confidence interval (CI) 1.04–3.97)). In patients with newly diagnosed tumours the HR for factor V Leiden was 3.7 (95% CI 1.2–12.2) after 30 days. In Kaplan Meier analysis the probability for development of VTE after 6 months was 5.7% in those without and 13% in those with factor V Leiden, after one year the corresponding rates were 7.3% and 15%.

Conclusions:

Factor V Leiden is a genetically determined and thus disease-independent parameter, which is associated with VTE in cancer patients, especially shortly after cancer diagnosis, and could therefore be used for individual risk assignment.

Disclosures:

No relevant conflicts of interest to declare.
Immunogenicity of Blood Group Antigens in Female and Male Transfusion Recipients: Is It Possible to Avoid Immunization to RBC Antigens?
Authors: Szymanski Irma O.
Published: October 2019

Abstract 4377

In men blood group incompatibilities between a donor and blood transfusion recipient may stimulate RBC antibody production, whereas in women both transfusion and pregnancies play a role. Immunogenicities of various RBC antigens correlate with their ability to induce a specific antibody response. These parameters were calculated on data obtained previously in 711 patients found to be immunized in pre-transfusion tests using the method of Giblett (Transfusion 1961), and corrected for the fraction of antibody decline in time by the method of Torney and Stack (Blood 2009).

Immunogenicities of RBC antigens expressed as a fraction of that of the K antigen (Potency = 1) are shown in columns IV & VII (method of Giblett) and in V & VIII (method of Torney & Stack) of the following table.

I Antigen X II Anti-X, N III Anti-X, Females, N IV Females Potency vs. K V Females, Corrected Potency VI Males, Anti-X, N VII Males, Potency Vs. K VIII Males, Corrected Potency C 71 51 0.227 0.156 20 0.108 0.075 c 37 27 0.164 0.141 10 0.074 0.063 E 220 117 0.554 0.565 103 0.594 0.606 e 9 5 0.244 4 0.238 Fya 44 28 0.122 0.086 16 0.085 0.060 Fyb 3 1 0.007 0.004 2 0.017 0.012 Jka 60 41 0.225 1.083 19 0.127 0.611 Jkb 5 3 0.015 2 0.012 K 153 84 1 1 69 1 1 k 1 1 0.488 0 S 18 13 0.051 0.027 5 0.024 0.013 s 1 0 1 M 21 10 0.056 0.069 11 0.075 0.093

In males the corrected potencies of E and Jka were about 60% of that of K, but in females the corrected potencies of Jka and K were equal. The antigens C, c, Fya,Jka and S had higher corrected

potencies in females than in males, probably reflecting the additional role of pregnancies in immunization. Note the big difference in antibody response between allelic non-Rh antigens (e.g. Jka vs. Jkb).

Immunogenicity of the Rh D antigen was not calculated since Rh negative patients receive Rh negative blood and Rh negative pregnant women are given Rh immune prophylaxis. However, in this study 29.6 % of the patients were Rh negative. Over 70% of the susceptible patients had been immunized to the Rh D.

In conclusion, RBC antibodies interfere with the benefits of RBC transfusions and may also cause severe adverse effects. Introduction of molecular methods into Blood Bank setting could eventually minimize exposure and immunization of transfusion recipients to incompatible RBC antigens. Information about immunogenicity of various blood group antigens may point out those antigens that should not be given to patients who are lacking them. It is also critical to understand why immunization to the D antigen is still prevalent in order to pave the way to future multiple antigen matching.

Disclosures:

No relevant conflicts of interest to declare.
Greater Need for FVIII Replacement in Patients of Type Non-O Blood Groups with Moderate Hemophilia A
Authors: Lima Magaly BPLV , Siqueira Lúcia Helena , Saraiva Ana Suely , Montalvão Silmara Aparecida , Machado Tânia de Fátima Gomes Siegl , Trento Thais , Oliveira-Filho Aldemir Branco , Ozelo Margareth C , Lemos José Alexandre Rodrigues
Published: October 2019

Abstract 4390

Hemophilia A is a bleeding disorder of variable expressivity caused by mutations in clotting factor 8 gene located on Xq28 chromosome. In Brazil, treatment of such patients consists of replacement therapy with FVIII concentrate derived from human plasma, which is administered either to control an episode of hemorrhage or prophylactically in order to prevent bleeding and its consequences, such as arthropathy. Recent studies have demonstrated that the half-life of FVIII administered to patients of the type O blood group with severe hemophilia A is shorter than that in patients of Type non-O groups. A likely explanation for this fact would be the greater plasma concentration of von Willebrand factor (vWF) in individuals with type non-O blood. In this study, was investigated the association between the blood group and the annual consumption of FVIII concentrate (UI/Kg/year) in the last three years among 99 patients (68 with moderate and 31 with mild hemophilia A) treated at the two blood centers in Brazil (Hemocentro do Pará and Hemocentro/UNICAMP). Most cases were treated for episodes of hemorrhage. Determinations of antigen levels of von Willebrand factor (vWF:Ag), activity of ristocetin cofactor (vWF:RiCof) and FVIII clotting activity (FVIII:C) were performed to investigate the correlation between these variables and immunophenotypes of the ABO blood system. The Mann-Whitney test was used for the comparison of FVIII consumption (UI/Kg/year) between O type and non-O type hemophiliacs, with the level of significance set to 5% (p<0.05). Unexpectedly, significantly greater FVIII consumption was found among patients of the non-O type group (p=0.017). In the evaluation of patients with moderate hemophilia A, plasma levels of vWF:RiCof were significantly higher in non-O type patients in comparison to those with type O blood (p=0.045). No significant differences were found between blood groups in the evaluation of patients with mild hemophilia A. The present findings lead one to conclude that the higher level of plasma vWF:RiCof (which could exert an influence on the increased half-life of FVIII) in patients of the non-O type group with moderate hemophilia A was not enough to impede the need for greater FVIII replacement and lessen the frequency of bleeding in this group. The authors hypothesize that other variables, such as mutations in moderate hemophilia A, affect the stability of the FVIII molecule, which has antigen A and B determinants in individuals of non-O type groups. Further studies involving a larger sample are needed to corroborate the findings of the present investigation.

Disclosures:

No relevant conflicts of interest to declare.
The Average Baseline BCR-ABL Levels Are Significantly Higher in Patients with Resistance to Dasatinib As First-Line Treatment for Early Chronic Phase Chronic Myeloid Leukemia
Authors: Tomasetti Cristian , Kantarjian Hagop M. , Ramchandani Ritesh , Jabbour Elias , Quintás-Cardama Alfonso , Parmigiani Giovanni , Cortes Jorge E.
Published: October 2019

Abstract 4436

Background:

The development of drug resistance remains a problem for some chronic myeloid leukemia (CML) patients treated with tyrosine kinase. Point mutations are the main mechanism behind drug resistance, and they can occur prior to therapy. Thus, there may be a positive correlation between the baseline BCR-ABL levels and the occurrence of drug resistance.

Methods:

We define a “relapse/no-response” as a BCR-ABL/ABL transcript ratio of >10% (international scale) at 6 months or later during continuous dasatinib treatment. Statistical analysis is performed an all early chronic phase CML treated with dasatinib as first-line therapy (n=102) in a prospective clinical trial at the M.D. Anderson Cancer Center. The probability of having drug resistant leukemic cells already present at diagnosis is also estimated.

Results:

Seven of the 90 (7.8%) evaluable patients had a “relapse/no-response”. The mean (median) BCR-ABL/ABL transcript ratio at baseline for the “relapse/no-response” group was 23.3% (20.7%), while for the rest of the patients was 14.1% (9.9%), a statistically significant difference. The incidence rates of “relapse/no-response” for patients with a BCR-ABL/ABL transcript ratio at baseline equal to or larger than 20% was 13.8%, versus 7.8% overall for the entire dataset. Our estimates suggest that if the therapy were to be started on average one month earlier, the probability of a “relapse/no-response” would decrease from 7.8% to 6.9%.

Conclusions:

The BCR-ABL transcript levels at baseline are significantly higher for those patients later incurring in a “relapse/no-response”. Moreover, our estimates suggest that starting the treatment as soon as possible after diagnosis may be critical for reducing the number of patients developing drug resistance.

Disclosures:

Kantarjian: Novartis: Research Funding; Pfizer: Research Funding; Ariad: Research Funding; Bristol-Myers Squibb: Research Funding. Jabbour:Pfizer: Honoraria; Novartis: Honoraria; BMS: Honoraria. Cortes:Novartis: Consultancy, Research Funding; Bristol-Myers Squibb: Consultancy, Research Funding; Pfizer: Consultancy, Research Funding; Ariad: Consultancy, Research Funding.
Prognostic Significance of Immunocytometric Count of Medullar Blasts in Myelodysplastic Syndromes
Authors: Molteni Alfredo , Speziale Valentina , Riva Marta , Nichelatti Michele , Greco Rosa , Cesana Clara , Scarpati Barbara , Morra Enrica
Published: October 2019

Abstract 4945

Background:

bone marrow blast count is a crucial parameter to determinate the prognostic score according to IPSS in myelodysplastic syndromes. However even among the major experts the agreement about detection of a blast cells by optical microscopy is less than 90% (Mufti et al: Haematologica 2008). The aim of this study is to evaluate prognostic significance of immunocytometric count of medullar blasts compared to cytological count.

Method:

in a retrospective analysis of 104 patients with a minimum follow-up of three years, IPSS was calculated replacing cytological count of blasts with the flow cytometry one. Blasts were expressed as percentages of total cellularity, and identified by the combination of CD45/SSC with/without CD33, CD34 and CD117 antigens. The monoclonal antibodies used were CD34 FITC, CD117 PE, CD45 PerCp, CD33 APC. Acquisition of information on 1×105 stained cells corresponding to the whole bone marrow cellularity was assessed on a dual-laser FACSCalibur flow cytometer using the CellQUEST software (Becton Dickinson, San José CA USA). Survival of “low + intermediate-1 risk” (“IPSS1+2”) patients was compared to “intermediate-2 + high risk” ones (“IPSS3+4”) using Kaplan Meier method followed by logrank test. The comparison of ability among all blast count models to distinguish the probability of survival of the two groups (“IPSS 1+2” vs “IPSS3+4”) was based on χ2 value obtained by the logrank test.

Results:

considering IPSS obtained from any of the immunocytometric markers considered (CD33, CD34, CD117 and CD45dim/SSC), “IPSS1+2” group had always an extremely better survival than “IPSS 3+4” group with a level of statistical significance similar to survival study of “standard IPSS”. Of all markers, CD45dim/SSC was the most capable to distinguish survival of “IPSS1+2” group versus “IPSS3+4” group (χ2 = 42. 5). Among others, CD117 and CD33 had a better discriminating power than cytological count (respectively χ2 = 38, 1 and χ2 = 22, 3 versus χ2 18, 2); only CD34 was worse than cytological count (χ2 =14, 2).

Conclusion:

the immunocytometric markers considered are demonstrated to be even better than cytological count as parameter in IPSS score. Their use has to be encouraged, especially where there is little experience in optical microscopy.

Disclosures:

No relevant conflicts of interest to declare.
Targeting Mir-548m-HDAC6 Axis Circumvents Stroma- Mediated Drug Resistance (CAM-DR) and Clonogenicity in Non-Hodgkin B-Cell Lymphomas
Authors: Lwin Tint , Zhao Xiaohong , Cheng Fengdong , Zhang Xinwei , Zhang Yizhuo , Huang Andy , Moscinski Lynn , Dalton William S. , Sotomayor Eduardo M. , Tao Jianguo
Published: October 2019

Abstract 5094

A dynamic interaction occurs between the lymphoma cell and its microenvironment, with each profoundly influencing the behavior of the other. Here, we demonstrate that adhesion of mantle cell lymphoma (MCL) and other non-Hodgkin lymphoma cells to lymph node stromal cells, follicular dendritic cells (FDCs), confers drug resistance, enhances lymphoma cell clonogenicity, and is associated with induction of histone deacetylase 6 (HDAC6). Furthermore, stroma down-regulated miR-548m contributes to HDAC6 up-regulation, and HDAC6 is a key determinant for FDC-mediated lymphoma cell survival and colony formation. We further showed that stroma-mediated drug resistance and clonogenic growth are reversed by enforced expression of miR-548m and inhibition of HDAC6. The HDAC6-selective inhibitor tubastatin significantly enhances cell death, abolishes cell adhesion-mediated drug resistance, and suppresses clonogenicity and lymphoma growth suppression ex vivo and in vivo. Together, these data suggest that the lymphoma–stroma interaction in lymph node microenvironment directly impacts the biology of lymphoma through epigenetic regulation of miRNA and HDAC, with HDAC6 as a potential therapeutic target to overcome drug resistance in MCL and other B-cell lymphomas.

Disclosures:

No relevant conflicts of interest to declare.
Molecular Diagnosis of One Pedigree with Protein S Deficiency and Antithrombin Deficiency
Authors: Zhou Rong-Fu , Tao Hong , Ouyang Jian , Zhang Xian , Yang Yonggong , Zhang QiGuo , Xu Jingyan , Chen Bing
Published: October 2019

Abstract 5140

Objective

To identify gene mutations for one patient and his family members with protein S and antithrombin deficiency.

Methods

ELISA were used to detect protein S (PS), protein C (PC) and antithrombin (AT) activities for the proband and family members, respectively. The genomic DNA was extracted from the peripheral blood of proband and family members. All exons and their flanks of protein S gene and antithrombin gene were amplified by polymerase chain reaction (PCR). The PCR products were sequenced directly. The mutation-related exons of his famliy members were amplified by PCR and sequenced directly.

Results

The proband was a 49-year-old male. He presented with sudden left lower extremity swelling and pain without casues. Regular examination revealed that his APTT, PT, and TT were all in normal levels, but D- dimmer was 5. 62mg/L, Color doppler ultrasonography showed thrombosis in his left femoral vein. The activity of PS for his family members was ‡1 0%, ‡2 0%, ‡3 0%, ‡4 130. 8%, ‡5 8. 4%, ‡1 0%, ‡2 0%, and that of AT was ‡1 129. 1%, ‡2 51. 9%, ‡3 73.2%, ‡4 119. 1%, ‡5 136. 2%, ‡1 65. 5% and ‡2 60. 1%, respectively. The sequencing analysis showed that a heterozygous missense mutation G68395T (NG_009813. 1) was detected in Exon 4 of PS gene leading to the substitution of Arg90 by Leu (NP_000304. 2) for the propositus. The heterozygous mutation (Arg90Leu) was also found in other family members. A heterozygous (nonsense) mutation G12444A (NG_012462. 1) was detected in Exon 4 of AT gene leading to Trp257Ter (NP_000479. 1) for the propositus. The mutation (Trp257Ter) was found in other family members with reduced activity of AT. These two mutations (G68395T in PS gene and G12444A in AT gene) were not reported before and were thus novel ones.

Conclusion

The novel mutation G68395T in PS gene and G12444A in AT gene might be the causes of deficiency of PS and AT for the family.

Disclosures:

No relevant conflicts of interest to declare.
Noncanonical Wnt Signaling Maintains Hematopoietic Stem Cells in Different Zones
Authors: Sugimura Ryohichi , Li Linheng
Published: October 2019

Abstract 639

Hematopoietic stem cells (HSCs) are maintained in a balance between quiescent state and proliferating state. While proliferating HSCs are critical for supporting the routine blood production, quiescent HSCs are essential for long term maintenance and can also be activated to replenish lost proliferating or active HSCs. How the different states of HSCs are regulated is a fundamental question. Accumulated evidence supports a model that quiescent HSCs are located in the endosteal zone and active HSCs are in the perivascular zone. The underlying signaling to regulate the quiescence and activation in different niches remains largely unknown. To address this question, we have analyzed the expression profile of Wnt receptors, Frizzleds, in HSCs. We found that noncanonical Wnt signaling via receptor Frizzled8 (Fz8) and co-receptor Flamingo presents in and functionally maintains quiescent HSCs in the endosteal zone (Sugimura, et al., Cell 2012). However, it has not been clear whether and how active HSCs in the perivascular zone are regulated by Wnt signaling.

Recently, we detected that another noncanonical Wnt receptor, Frizzled5 (Fz5), is expressed in metabolically active (indicated by Mitotracker) HSCs and also in Nestin-GFP+ mesenchymal stem cells (MSCs) in the perivascular zone of central marrow. Fz5 expresses neither in H2B-GFP label-retaining quiescent HSCs nor in endosteal cells and nor in sinusoidal cells as well. Using an Mx1-Cre:Fz5 knockout mouse model, we found a 60% decrease of HSCs isolated from central marrow, but no change in the number of HSCs isolated from endosteum. Functionally, hematopoietic reconstitution was not affected in the primary transplantation, but was substantially decreased (by 80%) in the secondary transplantation compared to the control.

This indicates that Fz5 maintains HSCs in the perivascular zone. To examine the role of Fz5 in Nestin+ MSCs for HSC maintenance, we examined the Nestin-Cre:Fz5 model. We observed a large loss of CD49hiHSCs that were reported to represent intermediate (IT) HSCs. We further found a correlation of the quiescent vs. active HSCs respectively to long term (LT) HSCs vs. IT-HSCs with the latter population sensitive to 5FU treatment. Mechanistically we observed that Fz5 inactivation also led to a loss of Cdc42 polarity in the HSCs residing in the perivascular niche. The results suggest that Fz5-mediated noncanonical Wnt signaling regulates polarity of active HSCs in the perivascular zones. Future study is required to see whether the Fz5-Cdc42 mediated polarity in HSCs is associated with symmetric vs. asymmetric division.

We propose that noncanonical Wnt signaling maintains quiescent and active HSCs reside respectively in the endosteal zone and the perivascular zone. In these zones, Fz8 and Fz5 are differentially expressed and mediate noncanonical Wnt signaling for HSC maintenance in the endosteal niche and to regulate active HSC action in the perivascular niche.

Disclosures:

No relevant conflicts of interest to declare.
Low RPS14 Expression in MDS without 5q- Aberration Is Associated with Increased Apoptosis of Erythrocytes and Predicts Prolonged Survival and Possible Response to Lenalidomide in Lower-Risk Patients
Authors: Wu Lingyun , Li Xiao , Xu Feng , Zhang Yan , He Qi , Chang Chunkang
Published: October 2019

Abstract 698

Purpose:

Haploinsufficiency of the ribosomal protein S14 (RPS14) has been identified as a causal factor in 5q- syndrome. This study was carried out to investigate RPS14 expression in myelodysplastic syndrome (MDS) without 5q deletion and the role under-expressed RPS14 plays in pathogenesis and the prognosis and lenalidomide-response predicting of non-5q-MDS.

Patients and Methods:

The expression level of RPS14 transcription was detected in 156 MDS patients without 5q-. The apoptosis index of bone marrow (BM) erythrocytes was also analyzed. Furthermore, patient prognosis with low or normal RPS14 expression was analyzed and the role of RPS14 expression in lenalidomide-response prediction was elevated.

Results:

The reduced expression of RPS14 occurred in 83 of 156 (53.2%) patients. Patients with RPS14 under-expression presented higher platelet counts in the peripheral blood compared to RPS14 normal patients (p=0.012). RPS14 expression status was inversely corrected with the apoptosis index of erythrocytes from BM (r=-0.54, p=0.013). Patients with low RPS14 expression have a higher two-year survival probability than normal RPS14 cases in the international prognosis scoring system (IPSS) lower-risk group (90.8% vs. 71.7%; p = 0.018). A multivariate analysis showed RPS14 expression status was an independent predictor for survival in lower-risk MDS patients without 5q deletion. Twelve patients were treated with lenalidomide. Five of seven patients achieved an erythroid response in the low RPS14 expression group (5/7, 71.4%), compared to zero responses in the five normal RPS14 patients (p=0.018).

Conclusion:

Low RPS14 expression is common in MDS patients without 5q deletion and is associated with increased apoptosis of erythrocytes in lower-risk MDS. Additionally, low RPS14 predicts prolonged survival and possible response to lenalidomide in lower-risk MDS patients.

Disclosures:

No relevant conflicts of interest to declare.
TAS-117, a Novel Selective Akt Inhibitor Demonstrates Significant Growth Inhibition in Multiple Myeloma Cells in Vitro and in Vivo
Authors: Mimura Naoya , Ohguchi Hiroto , Cirstea Diana , Cottini Francesca , Gorgun Gullu Topal , Minami Jiro , Suzuki Rikio , Shimomura Toshiyasu , Utsugi Teruhiro , Hideshima Teru , Anderson Kenneth C.
Published: October 2019

Abstract 942

The PI3K/Akt pathway mediates multiple myeloma (MM) cell growth and drug resistance, and targeting this molecule is a promising therapeutic option. In this study, we examined anti-MM activities of TAS-117 (TAIHO PHARMACEUTICAL CO., LTD., JAPAN), a selective potent Akt inhibitor in MM cell lines including MM.1S, MM.1R, OPM1 and H929 cells with high level of baseline Akt phosphorylation. TAS-117 induced significant growth inhibition in these cell lines, associated with downregulation of phosphorylation (Ser473 and Thr308) of Akt and downstream molecule FKHR/FKHRL1, without cytotoxicity in normal peripheral blood mononuclear cells. TAS-117 triggered G0/G1 arrest followed by apoptosis, evidenced by increased annexin V-positive cells, in both MM.1S and H929 cell lines. Apoptosis was further confirmed by cleavage of caspase-8, -3 and PARP. Interestingly, TAS-117 also induced: autophagy, evidenced by increased LC3-II; as well as endoplasmic reticulum (ER) stress, confirmed by induction of phospho-eIF2α, phospho-IRE1α and a molecular chaperone BiP/GRP78. Since the bone marrow (BM) microenvironment plays a crucial role in MM cell pathogenesis including drug resistance, we further examined the effect of TAS-117 in the presence of BM stromal cells (BMSCs). TAS-117 induced significant cytotoxicity in MM cells even in the presence of BMSCs, associated with downregulation of phospho-Akt. Importantly, TAS-117 inhibited secretion of IL-6 from BMSCs, and exogenous IL-6 and IGF-1 did not block cytotoxicity induced by this agent. We have previously shown the bortezomib activates Akt, and that Akt inhibition with bortezomib triggers synergistic MM cell cytotoxicity. TAS-117 enhanced bortezomib-induced cytotoxicity in MM.1S cells, associated with increased CHOP followed by PARP cleavage, suggesting that TAS-117 augments bortezomib-induced ER stress and apoptotic signaling. TAS-117 also enhanced cytotoxicity induced by other therapeutic agents (ie, rapamycin, dexamethasone, 17-AAG) in MM.1S cells. Finally, we examined anti-MM activities of TAS-117 in a xenograft murine model. Oral administration of TAS-117 for 14 days significantly inhibited growth of H929 plasmacytoma and was well tolerated. Taken together, the novel and selective Akt inhibitor TAS-117 blocks MM cell growth in vitro and in vivo, providing the preclinical framework for clinical evaluation of this agent to improve patient outcome in MM.

Disclosures:

Shimomura: TAIHO PHARMACEUTICAL CO., LTD.: Employment. Utsugi:TAIHO PHARMACEUTICAL CO., LTD.: Membership on an entity's Board of Directors or advisory committees. Anderson:Celgene, Millennium, BMS, Onyx: Membership on an entity's Board of Directors or advisory committees; Acetylon, Oncopep: Scientific Founder, Scientific Founder Other.
A Novel ENU-Induced Intronic Mutation of Ank1 Causing Quantitative Defect of Ankyrin-1 Models Hemolytic Hereditary Spherocytosis in Mouse
Authors: Du Xin , Huang Hua , Krieg Lara
Published: October 2019

Abstract 989

The ability of red blood cells (RBCs) to maintain the surface area and deformability is vital for their survival. The maintenance of membrane surface is dependent upon the strong cohesion between the lipid bilayer and the skeletal network, achieved by vertical linkages between transmembrane proteins and spectrin tetramers. Mutations causing functional deficiencies in these proteins have been identified in various hemolytic anemias. Here we reported a mild hereditary spherocytosis (HS) and hemolytic anemia phenotype in mouse, named hema6, induced by N-ethyl-N-nitrosourea (ENU) mutagenesis. Hema6 phenotype is transmitted as a semidominant trait as heterozygous mice are less severely affected than homozygotes. The causal mutation was traced to a single nucleotide transition in the deep intronic region of intron 13 of gene Ank1, encoding the anchorage protein ankyrin-1 in RBC. In vitro minigene assay revealed two abnormally spliced transcripts in addition to wild-type mRNA. The wild-type Ank1 transcript was detected in the homozygous mutant mouse with 30% reduction in expression level compared to that in wild type mouse. The aberrant transcripts presumably encoded a 509 amino acids protein, which lacks beta-spectrin binding domain and C-terminal regulatory region. The truncated protein was not detected by western blotting using currently available antibodies against full-length ankyrin-1 in homozygous hema6 erythrocyte ghosts, whereas the wild-type Ankyrin-1 are present with reduced quantity. Employing biochemical and cell biology assays, we characterized the mechanism by which Ank1hema6 mutation causes hemolytic hereditary spherocytosis in mouse. Hema6 strain provides a novel tool to study ankyrin-1 and its pathogenesis role in HS.

Disclosures:

No relevant conflicts of interest to declare.
Erythrocyte Advanced Glycation End-Products as Novel Mediators of Endothelial Dysfunction Following Transfusion
Authors: Mangalmurti Nilam S. , Friedman Jessica , Siegel Don L. , Lee Janet , Albelda Steven M.
Published: October 2019

Abstract SCI-47

Red cell transfusions are associated with the development of acute lung injury in critically ill patients, yet the mechanisms for this association remain unknown. We have previously shown that stored red blood cells (RBCs) express the advanced glycation end-product Nε-carboxymethyl lysine (Nε-CML) (1). Advanced glycation end-products (AGEs), a heterogeneous group of adducts formed during states of increased oxidative stress or hyperglycemia, are thought to exert their effects by binding to membrane receptors, including RAGE (the receptor for advanced glycation-end products). RAGE is a multiligand pattern-recognition receptor implicated in vascular disease whose function in lung endothelium remains unknown. Our previous data demonstrate that RAGE ligands on stored erythrocytes can induce reactive oxygen species in lung endothelial cells. We therefore hypothesized that RAGE ligands on the surface of stored RBCs exacerbate lung inflammation by interacting with the pulmonary endothelium, thus possibly participating in the pathogenesis of lung injury following transfusion. We first examined lung endothelial cells at baseline using multiple techniques and observed low basal expression of RAGE. Because RAGE ligands can induce the expression of RAGE, and stored red blood cells (RBCs) express the RAGE ligand Nε-carboxymethyl lysine (Nε-CML), we investigated whether RBC transfusion would augment RAGE expression on lung endothelium utilizing a syngeneic model of RBC transfusion. Increased lung endothelial RAGE expression and enhanced lung inflammation and endothelial activation was observed, as evidenced by increases in lung high mobility group box 1 (HMGB1) and vascular cell adhesion molecule (VCAM-1) expression following transfusion. These effects were mediated by RAGE, as endothelial activation was absent in RBC-transfused Rage knockout mice. Therefore, functional consequences of RBC transfusion are augmented RAGE expression, increased RAGE ligation, and endothelial activation, thus linking RAGE activation to lung inflammation following RBC transfusion.

Disclosures:

No relevant conflicts of interest to declare.