bluuebunny/crossref_metadata_2025 huggingface dataset:
Human FcγRIII (CD16) Polymorphism Screening Is Enhanced with Pyrosequencing Analysis
Authors: Matlawska-Wasowska Ksenia , Gale James , Khalili Parisa , Wilson Bridget S , Vasef Mohammad , Winter Stuart S.
Published: October 2019

Abstract 4911

Surface specific antigens expressed on the cell membrane of hematopoietic cells are attractive target for antibody mediated cancer cells therapy. Monoclonal antibodies involve various mechanisms to eliminate cancer cells, including antibody dependent cellular cytotoxicity (ADCC) and phagocytosis (ADCP) mediated by immune effector cells such as NK cells and macrophages bearing FcγRIIIA (CD16) receptor. Previous studies reported that clinical efficacy of monoclonal antibodies can be linked to the single nucleotide polymorphism found at position 559 in cDNA of the gene encoding FcγRIIIA. This allelic polymorphism generateses the following allotypes V/V, F/V or F/F at amino acid position 158 and can affect binding of mAbs and immune cell effector function. CD16-mediated binding is most efficient with the V/V genotype, and least efficient with the F/F genotype, leading to a range of efficacy in mAb-mediated targeted therapies. Currently, many patients are not screened for CD16 heterozygosity. Nevertheless there is a clear need for a diagnostic assay that will allow estimating the allelic polymorphism of a patient undergoing treatment with monoclonal antibodies utilizing ADCC/ADCP. Here we hypothesized that the pyrosequencing might improve a screening for polymorphisms of human FcγRIIIA-158 receptor. We studied 42 normal human subjects for the incidence of V/V, F/V and F/F CD16 polymorphisms using pyrosequencing technologies and compared to nested PCR-based allele-specific restriction assay. Compared to pyrosequencing, the nested PCR-based allele-specific restriction assay was 0. 33 sensitive and 1. 0 specific in discriminating the V/V and F/F genotypes, and 0. 33 sensitive and 0. 7 specific for the V/V and F/V genotypes (Table 1). Compared to pyrosequencing, the nested PCR-based allele-specific restriction assay was relatively insensitive and not specific in distinguishing the V/V genotype from other genotypes. Since the efficacy of the mAb-based targeted immunotherapy may be highly dependent upon the CD16 polymorphism in any given individual, we propose that pyrosequencing of the CD16 receptor be routinely evaluated in all patients. Such practices might prevent patients from randomizing to receive targeted therapies to that hematological malignancies for that have little or no therapeutic potential.

Table 1 V/V F/V F/F pyrosequencing 6 16 20 allele-specific restriction assay 2 23 17 sensitivity - 0.33 0.33 specificity - 0.7 1.0

Disclosures:

No relevant conflicts of interest to declare.
Activity of Lenalidomide in a Phase II Single Institution Study in Non-Del(5q) Transfusion Dependent, Patients with Low and Intermediate-1 Risklower-Risk, Non-Del(5q) Myelodysplastic Syndromes (MDS))): Results From a Single-Institution Phase II Study
Authors: Raza Azra , Falkenstern John , Yeh Fiona , Castagna Ginevra , De Los Reyes Antonietta , Galili Naomi
Published: October 2019

Abstract 4929

In a previous multi-center Phase II study, lenalidomide resulted in a 43% overall hematologic response with 26% of patients achieving transfusion independence (TI) among 214 non-del(5q) red blood cell (RBC) transfusion-dependent patients with International Prognostic Scoring System (IPSS) lower-risk MDS (Raza et al Blood. 2008). A single institution follow-up trial was conducted in patients with low or Int-1 risk non-del(5q) MDS, a platelet count of >50, 000 and requiredat least 2 units of blood required in 8 weeks. Fifty-six patients received lenalidomide at a starting dose of 10 mg qday. There were 41 males and 15 females, and the median age was 71 years. By IPSS, 27 had low-risk disease and 22 had Int-1-risk disease, while IPSS could not be assigned for 7 patients. By cytogenetics, 38 patients had a normal karyotype, 12 showed abnormalities, while data could not be obtained for 4 patients due to culture failure. Morphologic classification consisted of: RA=17, RCMD=16, RARS=4, RCMD-RS=7, RARS-T=1 and RAEB-1=11. Median duration of MDS was 12 months (range 1–120 months). Median transfusion requirement was 2 units in 8 weeks prior to starting therapy. Median duration of lenalidomide therapy was 4 months (range, 1–36+ months). Treatment was generally well tolerated. The most common Grade 3/4 toxicity was myelosuppression, with 17 (30%) experiencing thrombocytopenia, 12/(21%) neutropenia and 1 leukopenia (2%). Other Grade 3/4 toxicities included diarrhea (7%) rash (2%), and fatigue (2%). At least one dose reduction was required for 33 (59%) patients. Intent-to-treat analysis showed hematologic response (HI) by the IWG-R criteria in 23 (41%) patients, no response in 25(44%) patients, and other failure in 8 (14%) patients. Among the 23 responders, 18 (32%) achieved TI. Median time to response was 2 cycles (6 weeks), with 6 patients responding in cycle 1, 9 in cycle 2, 5 in cycle 3 and 1 each in cycles 4, 5, 6, respectively. Median duration of response was 294 days (approximately 10 months), with durations ranging from 44+ days to 936+ days (+ indicates continuing response). We confirm in this 56-patient, single institution phase II study that lenalidomide has substantial clinical activity in transfusion-dependent, lower-risk non-del(5q) MDS, with results comparable to those previously reported in the multi-center phase II study.

Disclosures:

No relevant conflicts of interest to declare.
Autoimmunity in Patients (pts) with Chronic Myelomonocytic Leukemia (CMML): A Frequent Finding
Authors: Swedeh Amer , Patnaik Mrinal , Alfakara Dima , Ashrani Aneel A , Pruthi Rajiv , Elliott Michelle , Litzow Mark , Al-Kali Aref
Published: October 2019

Abstract 4930

Background:

Autoimmune disorders have been observed in pts with myeloid neoplasms but their frequency and impact on the natural history of the myeloid neoplasms remains to be defined.

Aim:

To describe the frequency and prognostic impact of autoimmunity in pts with CMML.

Methods:

After IRB approval, a retrospective chart review of all pts diagnosed with CMML at Mayo Clinic between 1994 – 2011. Autoimmunity (AI) was defined as positive serologic tests (Anti-nuclear antibodies (ANA), rheumatoid factor (RF), anti –cyclic citrullinate peptide (CCP), and antineutrophil cytoplasmic antibodies (ANCA)), diagnosis of connective tissue disease (Rheumatoid arthritis, RA), vasculitis, or auto-immune thrombocytopenia (ITP). Survival estimates were calculated using Kaplan-Meier estimates and Cox regression model for multivariate analysis for via JMP software v. 9.

Results:

During the study period, 288 pts with CMML were evaluated at Mayo Clinic, 35% (n=100) were female. Median age at diagnosis was 71yr (20–95). Median hemoglobin was 11 g/dL(6–16), median white blood cell (WBC) 12 x109/L (1–302), and median platelet count 86 x109/L (1–1110). Median peripheral blood (PB) and bone marrow (BM) blasts were 0 and 5 respectively, while median PB and BM monocytes were 20% and 8% respectively. Eight-nine percent of pts had CMML1, while 10% had CMML2 and only 2 pts had CMML transforming into acute myeloid leukemia (-t). Karyotype analysis performed in demonstrated normal (diploid) karyotype in 180 (63%) pts and complex karyotype in 31 pts (11%). JAK2 mutation analysis was performed in 76 pts, 13% (10/76) of which were positive for JAK2V617F mutation.

Autoimmunity was found in 46 (16%) of 288 pts. When tested, positive ANA, RF, CCP, and ANCA were found in 28/89 (31%), 8/61 (13%), 1/20 (5%), and 7/25 (28%), respectively. However, RA was reported positive in 6/288 (2%), and vasculitis was reported in only 4 pts. Median overall survival of the 288 patients was 496 days. Pts with evidence of autoimmunity had a median survival of 400 days compared to 527 days to the rest of the group (p 0. 73). On multivariate analysis, autoimmunity was not an independent risk factor for survival (p 0. 15), but age, platelets, WBC and body mass index (BMI) were independent factors.

Conclusion:

Autoimmunity is a frequent finding in pts with CMML. When tested, autoimmune antibodies were very frequent up to 31% of pts with CMML, but this did not confer into higher frequency of autoimmune diseases. Presence of positive autoimmune antibodies did not affect overall survival adversely.

Disclosures:

No relevant conflicts of interest to declare.
Outcome of 5-Azacitidine and Decitabine Treatment in Elderly Patients with Myelodysplastic Syndrome
Authors: Nam Seung-Hyun , Lim Taekyu , Kim Bong-Seog
Published: October 2019

Abstract 4959

Background:

Myelodysplastic syndrome (MDS) are heterogenous group of hematologic disorders broadly characterized by cytopenias associated with a dysmorphic and usually cellular bone marrow, and by consequent ineffective blood cell production. For patients with MDS an epigenetic therapy with hypomethylating agents is considered standard of care. We evaluated the outcome of 5-Azacitidine and Decitabine treatment in elderly patients with myelodysplastic syndrome in our veterans hospital.

Method:

For analysis, patients were included if they were > 60 years, had morphological evidence of MDS per bone marrow examination at the time of diagnosis and were treated with either hypomethylating agent, 5-azacitidine and decitabine. The patients received 5-azacitidine 75mg/m2, d1–7 or decitabine 20mg/m2, d1–5 at four weeks interval. The primary end point was overall response rate.

Results:

A total of 25 patients were analyzed. Median age was 68 years (range 61–81 years). 9(36%) patients had received 5-azacitidine and 16(64%) patients had received decitabine. The International Prognostic Scoring System risk category was Intermediate-2/High in 44%. A median of 5 courses (range 1–16) were delivered and 60%(15/25) patients were treated 4 cycles over. The overall response rate was 59%. Patients who showed response (CR+PR+HI) had significantly longer overall survival than those who did not (30 months vs 5months, p=0. 01). With a median follow-up duration of 12 months, median overall survival was 14. 7 months. The difference in overall survival was evident in the Intermediate-2/High risk group but not in the Intermediate-1 risk group. However, the difference between 5-azacitidine and decitabine group was not stastically significant.

Conclusion:

In our study, both 5-azacitidine and decitabine were feasible and effective in elderly patients with myelodysplastic syndrome. The overall survival was significantly longer in patients showing response. There were needed additional large-scale prospective studies.

Disclosures:

No relevant conflicts of interest to declare.
Adenosine Regulates Bone Metabolism Via A1, A2A and A2B Receptors in Bone Marrow Cells From Normal and Patients with Multiple Myeloma
Authors: He Wenjie , Park Jeesun , Mazumder Amitabha , Cronstein Bruce
Published: October 2019

Abstract 4977

Background:

Multiple myeloma is characterized by osteolytic bone lesions, wherein coupled bone remodeling is disrupted with increased osteoclast activation and decreased osteoblast differentiation. We have previously demonstrated that adenosine, acting via A2A receptors, diminishes human and murine osteoclast formation and others have reported that adenosine, acting at A2Breceptors, promotes osteoblast differentiation in murine osteoblast precursors and cell lines. In this study, we examined the effect of adenosine on osteoblast and osteoclast differentiation derived from multiple myeloma (MM) patients.

Methods:

Human bone marrow was collected from multiple myeloma patients. Bone marrow stromal cells (BMSCs) and bone marrow derived mononuclear (BMMs) cells were isolated and osteoblasts and osteoclastswere cultured, respectively. Adenosine A1 receptor agonist CHA and antagonist Rolofylline, A2A receptor agonist CGS and antagonist ZM, and A2b receptor agonist BAY and antagonist MRS 1754, A3receptor agonist IB-MECA and antagonist MRS 1191; and dipyridamole, a nucleoside transport inhibitor, were added to the culture media. Alkaline phosphatase (ALP) activity assay was used to quantitate the osteoblast differentiation. In vitro osteoblast calcification was determined by alizarin red staining. TRAP+ staining was used to examine the osteoclast differentiation and bone resorption assay was used to study the osteoclast activity.

Results:

We found that A1R blockade by rolofyllineand A2aR ligation by CGS21680 inhibited differentiation of both normal and MM BMMs into TRAP+ multinucleated cells (IC50= 1nM for A1R, IC50= 10μM for A2AR;p<0. 001, n=3 for both). The inhibition of osteoclast differentiation by Rolofylline was also seen in bone resorption assay (Pit formation assay). The A2A receptor antagonist completely reversed the effects of CGS21680 on osteoclast differentiation. Moreover, enhanced adenosine accumulation in the presence of dipyridamole (0. 5μM) and A2BR activation promoted the differentiation of BMSCs from myeloma patients into osteoblasts shown byArlizarin red staining and ALP activity assay (by 1. 8 ± 0. 41 and 1. 57 ± 0. 26 fold, respectively, p<0. 05, compared with osteogenic media only, n=3 for both).

Conclusions:

These results indicate that adenosine A2 receptors may be useful targets for the treatment and prevention of MM-induced bone disease.

Disclosures:

No relevant conflicts of interest to declare.
Hypophosphorylated Retinoic Acid Receptor Alpha Inhibits the Proliferation of Multiple Myeloma Cells
Authors: Zheng Dong , Li Juan , Xiaojuan Guo , Chaoxu Zheng , Longjuan Zhang
Published: October 2019

Abstract 4990

Background:

Previous studies have shown that the effects of all-trans retinoic acid (ATRA) are mediated mainly through retinoic acid receptor alpha (RARα) and that the phosphorylation of a serine residue at amino acid 77 in the AF-1 domain of RARα was required for the basal activation function of RARα. In the present study, the effect of hypophosphorylated RARα(RARαS77A) in the proliferation of multiple myeloma(MM) was investigated.

Methods:

To determine whether RARαS77A would inhibit the proliferation of MM cells, RPMI8226 cells were transfected with constructing lentivirus-RARαS77A. The proliferation inhibition of ATRA and RARαS77A on MM cells were detectd by CCK-8. The molecular mechanisms of ATRA and RARαS77A on MM cells were explored by flow cytometer and western blotting.

Results:

The proliferation of RPMI8226 cells was inhibited by ATRA in a dose- and time-dependent manner. Compared with the control group, ATRA significantly arrested the cell cycle at the G1 phase (P<0. 05). The proliferation of MM cells transfected with lentivirus-RARαS77A was significantly inhibited compared with cells transfected with the control vector (p<0. 05). In addition, G1 arrest was significantly observed in MM cells transfected with lentivirus-RARαS77A (p<0. 05). The expression levels of retinoblastoma protein (Rb) and caspase-3 in the ATRA-treated and lentivirus-RARαS77A-transfected groups were increased compared with the control group.

Conclusion:

RARαS77A could mimic the proliferation inhibition and apoptosis promotion effects of ATRA on human RPMI8226 myeloma cells. Similar to ATRA, RARαS77A could induce G1 arrest by increasing the expression levels of Rb and caspase-3 in the anti-proliferation of MM cells.

Disclosures:

No relevant conflicts of interest to declare.
Identification and Characterization of Inhibitors That Specifically Target TORC2 in Multiple Myeloma
Authors: Hoang Bao , Gera Joseph , Lichtenstein Alan
Published: October 2019

Abstract 5026

Recent data indicate that TORC2 is necessary for survival of multiple myeloma (MM) cells. Currently, drugs targeting mTOR either inhibit TORC1 alone or both TORC1 and TORC2. To identify drugs that specifically target TORC2, we performed a drug screen in a yeast-two hybrid system to identify compounds that prevented an interaction between rictor and mTOR. We identified several potential compounds and have begun to characterize their molecular activity. These compounds induced significant MM cell apoptosis when used at concentrations below 4 uM. This cytotoxic effect was accomplished by a down regulation of TOR activity which was specific for TORC2 (ie., decreased S473 AKT phosphorylation and NDRG T346 phosphorylation). Co-immunoprecipitation experiments confirmed that at least some of the compounds prevented binding of rictor to mTOR within MM cells while having no effect on binding of raptor to mTOR. In addition, myeloma cells expressing TORC2 phosphomimetic protein substrates (AKTS473D or SGKS422D) were significantly less sensitive to apoptosis as compared to the empty vector control when treated with these compounds. These data suggest that the drug-induced cytotoxicity was mediated specifically through the inhibition of TORC2 kinase activity. These results are the initial characterization of TORC2-specific drugs and support a rationale for targeting TORC2 in multiple myeloma.

Disclosures:

No relevant conflicts of interest to declare.
Heparin Does Not Increase Live Births in Pregnant Women with Previous Unexplained Recurrent Miscarriages: A Systematic Review and Meta-Analysis
Authors: Al-Khabori Murtadha K. , Al-Hosni Zainab S. , Ghaithi Hajer M. Al , Maamari Altaf M. Al , Bessiso Wafa S. , Farsi Khalil Al , Al-Huneini Mohammed , Al-Riyami Arwa Z. , Daar Shahina
Published: October 2019

Abstract 504

Background:

The impact of heparin in pregnant women with previous unexplained recurrent miscarriages remains uncertain. The use of heparin in this patient population has been increasing with the hope of improving the rate of live births. We performed a systematic review of the randomized clinical trials addressing this question.

Methods:

We searched MEDLINE (searched July 1st 2012), CENTRAL (Issue 7 of 12, July 2012), American Society of Hematology (searched July 1st 2012), clinical trials registries (http://clinicaltrials.gov/, searched July 1st 2012), and bibliographies of relevant studies for randomized clinical trials comparing heparin (unfractionated or low molecular weight) to other best care approaches. Use of aspirin was allowed in either study arms. Included patients were pregnant women over 18 years of age with previous recurrent unexplained miscarriages (at least two at any trimester). We performed a meta-analysis using random effects models to estimate the pooled odds ratio. Statistical heterogeneity was calculated by using the I2 statistic.

Results:

Eight trials proved eligible, five of which provided data from 1141 patients that could be included in the meta-analysis and three of which remain unpublished. Only the subgroup of patients with no antiphospholipid antibodies or inherited thrombophilia was included. There was a total of 839 live births observed. Enoxaparin was used in four trials and nadroparin was used in one trial. All trials randomized patients before the 8th week of gestation. There were 430 live births (among a total of 561 pregnancies) and 409 live births (among a total of 580 pregnancies) in the heparin and other best care treatment arms respectively. The difference between the two treatment arms was not statistically significant with a pooled odds ratio of 1.49 (95% confidence interval: 0.84, 2.66; P = 0.17). There was a substantial heterogeneity among the results of different trials (Chi2 = 14.91, P = 0.005; I2 = 73%).

Conclusions:

Available evidence suggests that heparin does not increase live births in pregnant women with previous unexplained recurrent miscarriages with no evidence of antiphospholipid antibodies or inherited thrombophilia. Future trials should explore new treatment strategies.

Disclosures:

No relevant conflicts of interest to declare.
The Significance of CD44ICD in Difuse Large B- Cell Lymphoma Cell Line SUDHL2 and the Preliminary Study On the Mechanism
Authors: Feng Ru , Duan Wenbing , Chen Dayan , Wei Xiaolei , Wei Yongqiang , Huang Fen , Yu J. Jessica , Ye B. Hilda
Published: October 2019

Abstract 5087

Objective:

Diffuse large B cell lymphoma (the diffuse large B-cell lymphoma, DLBCL), is one of the most common type of non-Hodhkin's lymphomawith high heterogeneity. γ-secretase can induce the hydrolysis of CD44, which play a key important in DLBCL, producing the CD44 intracellular domain. According to our research, the inhibition of CD44ICD can decrease the expression of NF-„KB and Stat-3 in SUDHL2 cell line from ABC-DLBCL cell line. Therefore the aim of this study was put important on the change of proliferation, apoptosis and main surface markers, ERK1/2 and phosphor-ERK1/2 in CD44 positive cell line from ABC-DLBCL after inhibiting the production of CD44ICD.

Method:

The surface expression of CD44 in SUDHL2 and OCI-ly3 cell lines was tested by flow cytometry, and then chose the CD44 positive cell line. Using 0. 1, 1. 0, 5. 0, 10, 25, 50, 75 and 100μM DAPT inhibited the production of CD44ICD in CD44 positive cell line respectively, and then tested the cell proliferation, apoptosis and main surface markers(CD44, CD19, CD20) by MTT analysis, Annexin V-FITC/PI and flow cytomety separately after 24h. mean while western blot was used to detected whether ERK1/2 and phosphor-ERK1/2 expressed in the chosen cell line, if they expressed in it, tested their change after blocking the release of CD44ICD for 1h.

Results

Conclusion

The ERK1/2 signal path participated in the regulation of SUDHL2 cell line, moreover after inhibited the release of CD44ICD, both the ERK1/2 and phosphor-ERK1/2 could be regulated up, however further study must go on.

Disclosures:

No relevant conflicts of interest to declare.
A Modified Glasgow Prognostic Score (mGPS) Based On Systemic Inflammatory Response Is a Useful Indicator to Predict Response and Survival in Patients with Newly Diagnosed Diffuse Large B-Cell Lymphoma (DLBCL)
Authors: Jung Sung-Hoon , Yang Deok-Hwan , Ahn Jae-Sook , Kim Yeo-Kyeoung , Kim Hyeoung-Joon , Lee Je-Jung
Published: October 2019

Abstract 5089

Systemic inflammatory responses as evidenced by CRP and albumin have reported as a useful indicator of prognosis in patients with advanced cancers. We retrospectively evaluated the relationship between the systemic inflammation score and survival in 213 patients with DLBCL receiving the first-line R-CHOP chemotherapy between October 2003 and December 2011 at Chonnam National University Hwasun Hospital. Patients were assorted into three groups based on initial serum CRP, albumin and LDH level at diagnosis, as named by ¡°modified Glasgow prognostic score (mGPS)¡±. Patients with elevated CRP (> 1. 0 mg/dL), hypoalbuminemia (< 3. 5 g/dL) and elevated LDH (> 1¢¥ upper normal value) were assigned a score of 2. Patients in whom one or two of these biochemical abnormalities was present were assigned of 1. Patients in whom neither of these abnormalities was present were assigned a score of 0. In Cox multivariate analysis, the independent poor prognostic factors for progression free survival (PFS) were mGPS 2 (HR 5. 415, 95% CI 2. 028–14. 454, P = 0. 001), ECOG PS ¡Ã 2 (HR 3. 504, 95% CI 1. 655–7. 419, P = 0. 001), and bulky lesion ¡Ã 10cm (HR 2. 030, 95% CI 1. 037–3. 972, P = 0. 039). In addition, the independent poor prognostic factors for overall survival (OS) were mGPS 2 (HR 5. 898, 95% CI 2. 028–14. 454, P = 0. 001) and ECOG PS ¡Ã 2 (HR 3. 525, 95% CI 1. 642–7. 567, P = 0. 001). The overall response rate, including complete response and partial response, for the R-CHOP chemotherapy was decreased according to the mGPS; 96. 7% at mGPS 0, 87% at mGPS 1, and 75% at mGPS 2 (P = 0. 009). This study suggest that the modified Glasgow prognostic score (mGPS) based on systemic inflammatory responses (CRP, albumin and LDH) was a useful prognostic indicator for OS and PFS and also a reliable method to predict the response for R-CHOP chemotherapy in patients with newly diagnosed DLBCL.

Disclosures:

No relevant conflicts of interest to declare.
Significance of Clinical Factors As Prognostic Indicators for Mature T-Cell Non-Hodgkin Lymphoma Patients: A Retrospective Analysis of Chinese Cases
Authors: Xie Wanzhuo , Hu Keyue , Xu Fan , Zhou De , Huang Weijia , Li Li , He Jingsong , Shi Jimin , Luo Yi , Zhu Jingjing , Wei Guoqing , Zhang Jie , Lin Maofang , Ye Xiujin , Cai Zhen , Huang He
Published: October 2019

Abstract 5093

Objectives:

To retrospectively analyze the significance of different clinical factors for predicting the prognosis of mature T-cell non-Hodgkin lymphoma (MTCL) patients.

Methods:

Two hundred and fifty-two MTCL patients admitted from 2005 to 2011 into the First Affiliated Hospital of the Zhejiang University were retrospectively reviewed. All diagnoses were confirmed by histopathological hematoxylin and eosin (HE) staining and prognostic values of β2-microglobulin (β2-MG) levels, lactate dehydrogenase (LDH) levels, B symptoms, Ann Arbor stages, international prognostic index (IPI), Eastern Cooperative Oncology Group (ECOG) performance status, bone marrow involvement (BMI) and extra nodal involvement (ENI) for the overall survival (OS) were evaluated. Additionally, we compared the OS of patients treated with CHOP (cyclophosphamide, doxorubicin, vincristine, and prednisone) and patients treated with an alternative intensive chemotherapy.

Results:

At a median follow-up of 23 months, the OS rate was 23. 8%. Our results revealed that the presence of B symptoms (P <0. 001), ECOG score≥2 (P <0. 001), BMI (P <0. 001), elevated LDH levels (P <0. 001), elevated β2-MG levels (P<0. 001), Ann Arbor stages III/IV (P=0. 007) and IPI ≥3 (P=0. 001) were factors for a poor OS prognosis and intensive chemotherapy showed a better OS outcome than CHOP treatment.

Conclusion:

Elevated LDH and β2-MG levels, B symptoms, Ann Arbor stage III/IV, BMI, high IPI indexes and ECOG scores predict an unfavorable prognosis of MTCL patients. When compared with the classical CHOP regimen, the intensive chemotherapy treatment can improve the prognosis of patients with MTCL.

Disclosures:

No relevant conflicts of interest to declare.
The Potential Role of STAT's in Anti-Leukemic Therapy with Different Drugs
Authors: Kiper Hatice Demet , Kaymaz Burcin Tezcanli , Purclutepe Ozlem , Dalgic Ceyda Tunakan , Selvi Nur , Dalmizrak Aysegul , Avci Cigir Biray , Kosova Buket , Sahin Fahri , Aktan Cagdas , Saydam Guray
Published: October 2019

Abstract 5120

STAT pathways play a pivotal role in oncogenesis and leukemogenesis, thus targeting STAT signalling appears to be an effective anticancer treatment strategy. It has been described that constitutive activation of STAT3 and STAT5 plays a pro-oncogenic role both in acute and chronic myeloid neoplasms. In this study, we aimed to clarify the potential relationship between drug-induced apoptosis with different agents and STAT pathway. A third-generation bisphosphonate; zoledronate, an angiotensin-converting enzyme inhibitor (ACE-I); enalapril, a proteasome inhibitor which is used for treatment of multiple myeloma; bortezomib and a second-generation tyrosine kinase inhibitor; dasatinib were examined in this goal.

Cell viability and cytotoxicity tests were conducted by using Trypan blue dye exclusion and XTT assays, respectively. Apoptotic analyses were performed by AnnexinV-EGFP staining method and fluorescence microscopy. Expression levels of STAT3, −5A and −5B genes were analysed in myeloid cell lines by qRT-PCR.

The results showed that zoledronate; bortezomib and dasatinib decreased viability and proliferation and induced apoptosis in CML cell line K562 in a dose- and time-dependent manner which is associated by prominent decrease of STAT3, STAT5A and STAT5B mRNA expressions. Enalapril was also found to be cytotoxic and induced apoptosis in APL cell line HL60 in a dose- and time-dependent manner and the expression levels of STAT5A gene have significantly reduced in enalapril-treated HL60 cells as compared to untreated controls. Treatments of cell lines with other drugs were also associated with significant apoptosis in certain time points. The results and changes in expression of STAT's in mRNA level at 72nd hours are summarized in table.

Taken together all these data showed that targeting STAT pathways by different drugs may be an appropriate approach in anti-leukemic therapy. This finding is important to propose that discovery or identification of novel agents targeted STATs may open new windows to the other hematological and solid malignancies which are associated with aberrant STAT expression.

Table: The changes in STAT expressions after drug exposure in time-dependent manner with the dose of IC50. DRUGS CELL LINE IC50 APOPTOSIS (%) STAT3 mRNA Down Regulation (%) STAT5A mRNA Down Regulation (%) STAT5B mRNA Down Regulation (%) ENALAPRIL HL-60 7 μM 20 20* 76 5* ZOLEDRONATE K562 60 μM 34 63 31 57 BORTEZOMIB K562 177 μM 37 98 100 99 DASATINIB K562 3,314 nM 75 NA 33 78 * : Not significant NA: not applied

Disclosures:

No relevant conflicts of interest to declare.
Characterization of T-Cell Lymphoblastic Leukemia Genetic Etiology
Authors: Drury Luke , Brett Benjamin , Sheetz Todd , Dupuy Adam J
Published: October 2019

Abstract 5128

Introduction.

T-cell lymphoblastic leukemia (T-ALL) is inflict nearly 1500 children annually in the US. We recently reported using Sleeping Beauty mutagenesis as a forward genetics approach to model two major subtypes of T-ALL, typical and early T-cell precursor (ETP). The genetic mutations that define these subtypes is incomplete. Moreover, identifying driver from passenger mutations within these tumors is paramount for developing targeted therapeutics to specific genetic lesions.

Method.

T-ALL tumors were generated using a Cre-inducible Sleeping Beauty system to drive mutagenesis at either early (Vav-iCre) or late (CD4-Cre) stage of T-cell development. Synthetic gene standards were spiked into samples at known concentrations to permit quantitation of transposon insertion events within individual tumors. Samples were prepared by ligation mediated PCR and sequenced using an Illumina HiSeq. Using the standard as a barometer, common insertion sites (CIS) were defined as initiating, transition, or progression based on their abundance. We further developed a network analysis pathway to identify CISs within similar signaling axes.

Results.

Spiked standards were reproducibly seen at expected abundances in several replicated tumors. Further, we are able to distinguish insertions found at high abundances in tumors from lower occurring or background events. Together this allowed us to identify the order of mutations in each tumor and characterize mutations as initiators or progression events. Using our network analysis pathway we found Myc and Stat5b signaling axes driving the majority of ETP-ALL and Notch1 signaling driving typical T-ALL.

Conclusions.

Our data are the first to utilize NextGen sequencing techniques to quantitate CIS events to understand the genetic etiology of T-ALL. Coupled with our network analysis software, we are able to more accurately identify driving pathways in individual T-ALL tumors induced by Sleeping Beauty mutagenesis.

Disclosures:

No relevant conflicts of interest to declare.
Platelet Activation and Inhibition in Sickle Cell Disease (PAINS) Study
Authors: Frelinger Andrew L. , Jakubowski Joseph A. , Brooks Julie K. , Zayas Sabrina L. , Berny-Lang Michelle A. , Barnard Marc R. , Heeney Matthew M. , Michelson Alan D.
Published: October 2019

Abstract 5147

Platelet activation/aggregation in sickle cell disease (SCD) may promote tissue ischemia, suggesting antiplatelet therapy may be useful. However, assessing platelet function and the effect of antiplatelet therapy in blood from SCD patients may be confounded by hemolysis with release of ADP. Here we evaluate levels of platelet activation markers in SCD adolescents vs. normal controls and compare, by multiple methods, the effect of in vitro blockade of the platelet ADP receptor P2Y12 by prasugrel's active metabolite, R-138727. Platelet activation markers in blood from SCD adolescents (n=15) and healthy adults (n=10), and the effect of R-138727 (0. 1 – 10 μM) added in vitro, were evaluated with and without ADP stimulation. Circulating levels of platelet-monocyte and platelet-neutrophil aggregates were significantly higher (p <0. 01) in SCD patients than in healthy controls. R-138727, in a concentration-dependent manner, inhibited platelet function in both SCD patients and healthy subjects as judged by ADP-stimulated light transmission aggregation, VerifyNow P2Y12 assay, multiple electrode aggregometry, and flow cytometric analysis of platelet vasodilator-stimulated phosphoprotein, activated GPIIb-IIIa and P-selectin. The R-138727 IC50s for each assay were not significantly different in SCD vs. healthy subjects. In summary: 1) The high circulating levels of platelet-monocyte and platelet-neutrophil aggregates demonstrate in vivo platelet activation in SCD and may be useful as markers of the in vivo pharmacodynamic efficacy of antiplatelet therapy in SCD. 2) The similar in vitro R-138727 IC50s in SCD and healthy subjects suggest that the prasugrel dose-dependence for platelet inhibition in SCD patients will be similar to that previously observed in healthy subjects.

Disclosures:

Frelinger: Eli Lilly: Consultancy, Research Funding; Daiichi Sankyo: Research Funding; GLSynthesis: Research Funding. Jakubowski:Eli Lilly: Employment. Heeney:Novartis: Consultancy, Research Funding; Eli Lilly and Company: Research Funding; Pfizer: Consultancy. Michelson:Eli Lilly: Data monitoring committee and idependent external monitor of clinical trials, Research Funding; Takeda: Research Funding; Oxygen Biotherapeutics: Research Funding; Alexion: Research Funding; Omthera: Research Funding.
Hereditary Hemolytic Anemia in Korea From 1997 to 2011: A Study by the Korean Hereditary Hemolytic Anemia Working Party of the Korean Society of Hematology
Authors: Park Eun Sil , Jung Hye Lim , Cho Hee Soon , Hah Jeong-Ok , Park Sung Sup , Koo Hong Hoe , Kim Moon Kyu , Kim Yeo-Kyeoung , Kim Young Dae , Kim Hee Jin , Kim Heung Sik , Kim Soon Ki , Seo Jong Jin , Seong Moon-Woo , Song Sang Hoon , Shin Hee Young , Ahn Hyo Seop , Yoo Eun Sun , Lyu Chuhl Joo , Yoon Hoi Soo , Lee Kun Soo , Lee Mee Jeong , Lim Young Tak , Im Ho Joon , Jeong Dae Chul , Choi Eun Jin , Choi Hyoung Soo , Han Dong kyun , Chueh Hee Won
Published: October 2019

Abstract 5157

Background:

With the development of diagnostic technique, an accurate diagnosis of hereditary hemolytic anemia (HHA)- red blood cell (RBC) membranopathy, hemoglobinopathy, RBC enzymopahty – have been made. Therefore, we surveyed the prevalence and characteristics of patients diagnosed as HHA during recent five years in Korea.

Methods:

Through the use of questionnaires, information on the clinical and laboratory findings of HHA diagnosed from 2007 to 2011 in Korea was collected. The globin gene analysis (direct sequencing) and RBC enzyme analysis was performed at the representative laboratories. A total of 203 cases were collected in this study by the Korean Hereditary Hemolytic Anemia Working Party of the Korean Society of Hematology.

Results:

Patients number of RBC membranopathy, hemoglobinopathy, and RBC enzymopahty was 125, 47, and 31, respectively. Percentage of patients with dominant family history was 57% in patients with hereditary spherocytosis (n=116) and dominant symptoms were anemia, jaundice, splenomegaly and gallstones. Osmotic fragility test and flow cytometric method for detection of RBC membrane defect were performed about 60% of patients. RBC membrane protein analysis using sodium dodecyl sulfate polyacrylamide gel electrophoresis was performed on 59 patients. Of the 47 cases of hemoglobinopathies, 36 cases (77%) were β-thalassemia minor, 10 cases (21%) were α-thalassemia minor and one case (2%) was unstable Hb, Hb M-Saskatoon (beta 64 His-→Tyr). Median age at diagnosis was 7 years (range: 6 months–58 years). Eleven of 47 cases (23%) had family history of HHA. As all thalassemia patients were thalassemia minor, they presented with mild jaundice or pallor. Of the 31 patients were diagnosed as RBC membranopathy, pyruvate kinase deficiency was 3 cases and glucose-6 phosphate dehydrogenase deficiency was 2, and other various forms were reported.

Conclusions:

We could confirm that accurate diagnosis has been made in more patients using elegant diagnostic technique. However, more defined diagnostic approaches were needed in this rare disease and further systematic supporting systems for patients and their families were warranted in public health aspect.

Disclosures:

No relevant conflicts of interest to declare.
The Ischemic Disease: A Joke Where There's Nothing to Be Gained
Authors: Corinaldesi Giorgio
Published: October 2019

Abstract 5198

This study explored the hypothesis that the ischemic disease (ID) is associated with systemic alterations of the blood coagulation system: platelet activation (sCD40L: determined by ELISA) and release of various proteins (CRP: determined by nephelometry, IL-6: determined by ELISA).

Plasma concentration of these factors were higher in patients with ischemic disease: sCD40L (ID: 724. 2 +/− 58. 4 vs controls 212 +/− 8. 6 pg/ml; p<0001), CRP (ID: 5. 76+/−1. 8 vs controls 2. 54 +/− 0. 3g/l; p<001), IL-6 (ID: 6. 36 +/− 1. 32 vs controls 2. 02 +/− 064 pg/ml; p<001).

Platelet activation predicts the risk of ischemic events, in fact CD40L binds and activates alfa IIb/beta 3, and increases PMC (platelet – monocyte complexes), and PDMP (platelet derived microparticle), it also promotes the release of several chemokines (FP4, RANTES, CxCL, ENA 78, NAP-2) that may play an important role in plaque destabilization; high levels of CD40L predict cardiovascular risk of death or myocardial infarction, or stroke within 8 to 12 month in patients with asyntomatic low grade stenosis (carotid or coronary).

Several data suggest that CRP has a prognostic value in patients with negative troponin and it is predictive for the development of CAD and refractory ischemic disease, plaque destabilization, and future adverse events, or for the development of severe disease.

IL-6 was strongly associated with markers of inflammation (CRP, fibrinogen, white cells count), plasma viscosity, elevated markers of coagulation (fibrin, D-Dimer, FVIII, FIX); moreover IL-6 is associated with insulin resistance, and its levels are high in metabolic syndrome; it is significantly correlated with age, BMI, white cell count; IL-6 increases dendritic platelet forms and acts synergistically with thrombopoietin and stem-cell factor in stimulating megakaryocytopoiesis.

Although it has been controversial, IL-6, PCR, and CD40L appear now to be very promising for quantifying the degree of ischemic disease and to monitor or to identify the efficacy and the response to the therapy.

Disclosures:

No relevant conflicts of interest to declare.
The G-CSF Produces Long-Term Changes in Gene and Mirnas Expression Profiles in CD34+ From Healthy Donors
Authors: Báez Alicia , Prats-Martín Concepción , Álvarez-Laderas Isabel , Garcia Estefania , Sanchez-Abarca Luis Ignacio , Martin-Antonio Beatriz , Urbano-Ispizua Alvaro , Simón Jose Antonio Pérez
Published: October 2019

Abstract 588

Introduction:

The G-CSF (granulocyte colony-stimulating factor) is the cytokine most commonly used for the mobilization of hematopoietic progenitor cells (HPCs) from healthy donors used in allogenic transplantation (allo-HSCT). Although the administration of G-CSF is considered safe, the knowledge about its long-term effects, especially in HPCs, is limited. The aim of this study was to analyze whether G-CSF induces changes in gene and miRNAs expression profiles in HPCs from healthy donors, and determine whether or not these changes persist at the long term.

Materials and Method:

CD34 + cells were isolated by immunomagnetic separation and sorting from 5 healthy donors before mobilization with G-CSF and at afterwards at 5, 30 and 365 days after mobilization. A pool of samples from PB not mobilized was used as reference group. We analyzed the expression of 375 miRNAs using TaqMan MicroRNA Arrays Human v2.0 (Applied Biosystems), and the gene expression profile using Whole Human Genome Oligo microarray kit 4×44K (Agilent). The expression levels of genes and miRNAs were obtained by the 2-ΔΔCTmethod. From expression data hierarchical clustering was performed using the Euclidean distance. To identify genes and miRNAs differentially expressed due to the effect of G-CSF at different time-points a cut-off value of level expression 2,5 above or below the control values was used and non-parametric Mann-Whitney test was applied. All analysis were performed using the Multi-experiment Viewer 4.7.1. The function of the miRNAs and genes of interest was determined from the various databases available online (TAM database, Gene Ontology, TargetScan Human).

Results:

Seven miRNAs were differentially expressed in HPCs at 5, 30 and 365 days after mobilization with G-CSF, as compared to HPCs obtained from not mobilized PB. In addition, 67 genes were also differentially expressed after administration of G-CSF, whose expression profiles remained abnormal 1 year after mobilization.

These genes are involved in biological processes such as hematopoietic cell proliferation, ribosomal protein synthesis, cell metabolism and transmembrane transportation. Interestingly, 8 of these genes are target of the miRNAs also identified in the current study, which suggests that their expression might be regulated by these miRNAs.

Conclusion:

The G-CSF modifies gene expression profiles and miRNAs in HPCs from healthy donors. Remarkably, these changes were observed from early time-points and persisted at least 1 year after exposure to the drug. This effect of G-CSF on HPCs have not been previously reported and might be clinically relevant.

Disclosures:

No relevant conflicts of interest to declare.
Experience with 2 Dose Schedules of Inotuzumab Ozogamicin, Single Dose, and Weekly, in Refractory-Relapsed Acute Lymphocytic Leukemia (ALL)
Authors: O'Brien Susan , Thomas Deborah A. , Jorgensen Jeffrey L. , Kebriaei Partow , Ravandi Farhad , Kwari Monica , Faderl Stefan , Cortes Jorge E. , Jabbour Elias J. , York Sergernne , Garris Rebecca , Kantarjian Hagop M.
Published: October 2019

Abstract 671

Background:

CD22 expression occurs in more than 90% of patients with ALL. Inotuzumab ozogamicin, a CD22 monoclonal antibody bound to calicheamicin, is active in ALL.

Study aims:

Evaluate the efficacy and toxicity profiles of two schedules of inotuzumab, single-dose and weekly, in refractory-relapsed ALL.

Patients and methods:

Patients with refractory-relapsed ALL were eligible. Pediatric patients were allowed on therapy after 10 adults were treated. Eligibility criteria were standard including normal organ functions (renal, hepatic). The original single dose schedule gave inotuzumab at 1.3–1.8 mg/m2 IVq 3–4 weeks. After the initial 49 patients were treated, the schedule was modified to inotuzumab weekly, 0.8 mg/m2 D1, 0.5 mg/m2 D8 and 15, q 3–4 weeks. The latter schedule was based on higher invitro efficacy with more frequent exposure, and possible lower clinical toxicity.

Results:

83 patients were treated, 49 with single dose, and 34 with weekly dose. 29% were ≥ 60 yrs; 71% were in salvage 2 or beyond; only 8 (10%) were in salvage 1 with CRD1>12 months; 28% had Ph+ or t (4; 11); 10 (12%) had prior allogeneic SCT. Overall 14 patients (17%) achieved CR, 23 (28%) had CRP (no platelet recovery), and 9 (11%) had marrow CR (no recovery of counts), for an overall response rate of 46/83=55%. Response rate was 28/49 (57%) with single dose and 18/34 (53%) with weekly dose. Only 4 patients died in the first 4 weeks of therapy. Among 28 patients with cytogenetic abnormalities who achieved response, 25 (89%) achieved completed cytogenetic response. Among 44 patients who achieved response and had MRD studies by multi-parameter flow cytometry, 28 (64%) became MRD negative. The median survival was 5.4 months, 5.0 months with the single dose and 6.3 months with the weekly dose. Reversible grade 1–2 and 3–4 bilirubin elevations were observed in 24% and 4% respectively on single-dose and in 3% and 0% on weekly dose. Reversible grade 1–2 and 3–4 liver enzyme elevations were observed in 55% and 2% respectively on single-dose and in 21% and 6% respectively on weekly dose. Adverse factors for response included: salvage 2 or later vs. salvage 1 (49% vs. 71%); Ph+ and t(4; 11) abnormalities vs. others (39% vs. 62%). Allogeneic SCT was performed 22/49 patients (49%) on single-dose and in 9/34 (26%) so far (shorter follow-up) on weekly schedule: veno-occlusive disease was observed in 5/22 with single-dose (23%) and in 1/9 with weekly dose (11%).

Conclusions:

Inotuzumab is highly active in refractory relapsed ALL. Weekly dose appears to be equally effective and less toxic than single dose.

Disclosures:

Cortes: Pfizer: Consultancy, Research Funding. Jabbour:Pfizer: Honoraria.
Loss of Wild-Type Jak2 Allele Enhances Myeloid Cell Expansion and Accelerates Myelofibrosis in Jak2V617F Knock-in Mice
Authors: Akada Hajime , Akada Saeko , Yan Dongqing , Hutchison Robert , Mohi Golam
Published: October 2019

Abstract 809

The activating JAK2V617F mutation is the most common mutation found in Philadelphia chromosome (Ph)-negative myeloproliferative neoplasms (MPNs), which include polycythemia vera (PV), essential thrombocythemia (ET) and primary myelofibrosis (PMF). Although a majority of MPN patients carry heterozygous JAK2V617F mutation, loss of heterozygosity (LOH) on chromosome 9p involving JAK2 has been observed in ∼30% of patients with MPNs particularly in PV and PMF. JAK2V617F homozygosity through 9pLOH has been linked to more severe MPN phenotype. However, the contribution of 9pLOH in the pathogenesis of MPNs remains unclear. To investigate the role of wild-type JAK2 in MPNs induced by JAK2V617F, we have utilized conditional Jak2 knock-out and Jak2V617F knock-in alleles and generated heterozygous, hemizygous and homozygous Jak2V617F mice. Whereas heterozygous Jak2V617F expression results in a polycythemia vera-like disease in mice, loss of wild-type Jak2 allele in hemizygous or homozygous Jak2V617F mice results in a significantly greater increase in reticulocytes, white blood cells, neutrophils and platelets in the peripheral blood and larger spleen size. We also have found that hemizygous or homozygous Jak2V617F expression significantly increased megakaryocyte-erythroid progenitors in the bone marrow and spleens and marked infiltration of neutrophils in the liver compared with heterozygous Jak2V617F. More importantly, hemizygous or homozygous Jak2V617F mice show accelerated myelofibrosis compared with heterozygous Jak2V617F-expressing mice. Thus, loss of wild type Jak2 allele increases myeloid cell expansion and enhances the severity of the MPN. Together, these results suggest that wild-type Jak2 serves as a negative regulator of MPN induced by Jak2V617F.

Disclosures:

No relevant conflicts of interest to declare.