bluuebunny/crossref_metadata_2025 huggingface dataset:
Mesenchymal Stromal Cells Expressing Interferon α Limit the Development of Acute Myeloid Leukemia, Inducing Apoptosis In Vitro and In Vivo
Authors: Desbourdes Laura M , Guess Adam J , Hasgur Suheyla , Overholt Kathleen M , Yu Minjun , Otsuru Satoru , Horwitz Edwin M
Published: October 2019

Introduction

The 5-year survival for patients with acute myeloid leukemia (AML) has stagnated for over two decades at about 60% for children, 40% for young adults, and <15% for elderly patients. While most patients achieve remission, approximately 50% will relapse which is generally attributed to the persistence of leukemic stem cells. Interferon α (IFNα) is an effective therapy for patients with AML due to multiple mechanisms of action. However, high serum levels are associated with many adverse effects. In this proof-of-concept study, we used engineered mesenchymal stem/stromal cells (MSC) to deliver high concentrations of IFNα locally to an AML chloroma, potentially diminishing the poorly tolerated systemic side-effects.

Methods

Bone marrow MSCs from C57BL/6 mouse were isolated and transduced with a lentiviral vector expressing murine IFNα (IFNα-MSCs) and/or GFP (GFP MSCs). After measuring IFNα secretion by ELISA and confirming activity by the induction of the MHC I expression on the transduced cells, the anti-AML activity of these transduced MSCs was assessed by co-culture with the C57BL/6 AML cell line c1498 which expresses DsRed and firefly luciferase (FFluc). Apoptotic cell frequencies and cell cycle phase distributions of leukemia cells with or without MSCs were assessed by flow cytometry. The in vivo validation has been performed by subcutaneous injection of c1498 cells (chloroma) with or without GFP MSCs or IFNα MSCs in C57BL/6 mice. Tumor growth was monitored by bioluminescence imaging every 3 or 4 days.

Results

Flow cytometric analysis and ELISA confirmed the secretion of bio-active of IFNα by transduced MSCs (41.5 ng/1E06 MSCs/24h). In co-cultures, the presence of IFNα MSCs at the ratio 100:1 (c1498: MSC) significantly decreased the population of c1498 cells mainly by inducing apoptosis compared to MSC-free or GFP MSC co-cultures while no effect was observed on cell cycle distribution. The pro-apoptotic effect of IFNα MSCs was then investigated in vivo by subcutaneous injection of c1498 cells with or without MSCs (ratio 10:1) in C57BL/6 mice.The presence of IFNα MSCs significantly decreased leukemic cell mass as shown by the bioluminescence of the DsRed+ FFLuc+ c1498 cells. This result was confirmed by flow cytometric analysis of the percentage of DsRed + cells in the chloroma.

Conclusions

This study shows that IFNα MSCs present a strong anti-leukemic effect in vitro and in vivo promoting apoptosis and thus decreasing the leukemic burden. Further experiments will focus on a potential synergetic effect with Cytarabine treatment and a preclinical study using human IFNα MSCs in a xenograft murine model.

Disclosures

No relevant conflicts of interest to declare.
DNMT3A Mutation inChinese Childhood Acute Myeloid Leukemia
Authors: Li Weijing , Gao Chao , Cui Lei , Liu Shuguang , Zhao Xiaoxi , Zhang Ruidong , Zheng Huyong , WU Minyuan , Li Zhigang
Published: October 2019

Purpose DNMT3A mutation has been found in approximately 20% of adult acute myeloid leukemia (AML) patients and in 0~1.4% of children with AML, and the hotspots of mutations are mainly located in the catalytic methyltransferase domain, thereinto, mutation R882 accounts for 60%. Although the prognosis of adult patients with DNMT3A mutations is dismal, the prognosis of DNMT3A mutation in childhood AML is still unclear. Here, we tried to determine the incidence and prognostic significance of DNMT3A mutations in Chinese childhood AML.

Methods We detected the mutations in DNMT3A exon 23 by PCR and direct sequencing in 342 children with AML (0~16 years old) from January in 2005 to June in 2013, treated on BCH-2003 AML protocol. The correlation of DNMT3A mutations with clinical characteristics, fusion genes, other molecular anomalies (FLT3-ITD, NPM1, C-KIT and WT1 mutations), and treatment outcome were analyzed.

Results DNMT3A mutations were detected in 4 out of 342 (1.2%) patients. The mutations in coding sequences included S892S, V912A, R885G, and Q886R. Furthermore, there were two intronic mutations (c.2598-15C>T and c.2739+55A>C) found in one patient. No association of DNMT3A mutations with common clinical features was found. Two patients with DNMT3A mutations died of relapse or complications during treatment, one patient gave up treatment due to introduction failure in day33. Only one patient achieved continuous complete remission.

Conclusions DNMT3A mutations were rare in Chinese children with AML. The mutation positions were different from the hotspots reported in adult AML. DNMT3A mutations may have adverse impact on prognosis of children with AML.

Disclosures

No relevant conflicts of interest to declare.
Dynamics of Molecular Response in AML Patients with NPM1 and FLT3 Mutations Undergoing Allogeneic Stem Cell Transplant
Authors: Salem Rana , Massoud Radwan , Mahfouz Rami , Bazarbachi Ali , El Cheikh Jean
Published: October 2019

BACKGROUND: Concomitant NPM1 and FLT3 mutation occurs in 20% of AML patients. Molecular response and achievement of negative minimal residual disease (MRD) are strong predictors of long-term outcome. However, little is known about the dynamics of molecular response in NPM-1 and FLT-3 double positive mutations.

OBJECTIVE: To assess the dynamics of molecular response to treatment in AML adult patients with concomitant FLT3 and NPM1 mutations.

DESIGN: Retrospective single center study.

SETTING: This study was approved by the institutional review board of American University of Beirut Medical Center.

PATIENTS OR OTHER PARTICIPANTS:

Twelve consecutive newly diagnosed (n=11) or relapsed (n=1) AML patientsreceived Idarubicin/cytarabineinduction and one or two consolidation(s) (Table 1). Ten patients received allogeneic stem cell transplant (allo-SCT) followed by post-transplantsorafenibmaintenance. Median follow-up was 11.5 (6-27) months. All transplanted patients remain alive and disease free.

INTERVENTIONS: FLT3 mutation was tested on DNA using a qualitative method with a sensitivity of0.01%. NPM-1 mutation was tested on cDNA using a qualitative or a quantitative RT-PCR with a sensitivity of 0.01% and0.008 NCN respectively. Patients were tested at diagnosis, after induction, after each consolidation, before and at days 30, 60 and 100 afterallo-SCT.

MAIN OUTCOMES MEASURES: Kinetics of NPM1 and FLT3 molecular response.

RESULTS:

After induction, FLT3 became negative in all tested patients (n=10). After first consolidation, FLT-3 remained or became positive in 10/11 tested patients whereas a molecular relapse was noted in one patient who developed a hematological relapse and rapidly died. No molecular positivity for FLT-3 was noted later on, whether after second consolidation or post-transplant. Conversely, NPM-1 mutation became negative in 2 out of 12 tested patients after induction, in 1 additional patient after first consolidation and in 6 additional patients afterallo-SCT, mostly after startingsorafenib. NPM-1 MRD value remained elevated in 3 out of 4 patients with quantitative assessment at diagnosis and post induction.

CONCLUSION: FLT3 become negative early after induction while NPM1 negativity lags behind. Persistent NPM-1 MRD does not seem to predict post-transplant outcome and may indeed become negative after sorafenib. These results need confirmation in larger studies.

Figure Dynamics of NPM1 and FLT3 Molecular Response Figure. Dynamics of NPM1 and FLT3 Molecular Response

Disclosures

No relevant conflicts of interest to declare.
Leukemia Stem-like KG-1a Cells Escape the Synergistic Cytotoxic Effect of Arsenic Trioxide and Aclacinomycin: Regulated By Survivin
Authors: Ye Yongbin , Xu Xiaojun , Liu Qifa
Published: October 2019

Leukemia stem-like KG-1a cells escape the synergistic cytotoxic effect of arsenic trioxide and aclacinomycin: regulated by survivin

Yongbin YE1 , Xiaojun.XU1 , Liu Qifa2

Correspondence to: Xiaojun XU. E-mail: doctorxu@163.com

1Department of Hematology, Zhongshan Hospital of Sun Yat-sen University & Zhongshan City People Hospital, Zongshan 528403

2Department of Hematology, Nanfang Hospital of Southern Medical University, Guangzhou,510515

Abstract AIM OF STUDY: Arsenic trioxide combined with aclacinomycin has a synergistic cytotoxic effect on leukemia stem cell-like cells KG-1a in our pervious study, however, there are still a part of KG-1a cells escaped the synergistic cytotoxicity, survivin may plays in a key role in this process. In this study, we have studied the interaction and mechanism of survivin in regulating leukemia stem-like KG-1a cell escape the synergistic cytotoxic effect of arsenic trioxide and aclacinomycin. MATERIALS AND METHODS: The anti-proliferation effect was detected by CCK-8 and colony-forming assay, protein-protein chip assay was used to analysis the expression level of survivin before and after combination treatment. The induction of apoptosis and cell cycle arrest in KG-1a cell line were detected by FACS, the expression of related signal pathway protein was detected by western blot. RESULTS: The anti-proliferation of KG-1a cells caused by arsenic trioxide or aclacinomycin showed a time- and dose-independent manner. However, protein-protein chip assay showed that the expression of survivin had a significant increase after the combination treatment (p<0.05), but when survivin was suppressed, the apoptosis rate had a more significant increase than the single drug treatment. Meanwhile, more prominent cell cycle arrest was observed in the combination treatment, further study found that suppression of survivin combined with chemotherapy may activate the related apoptosis pathway protein but suppress the PI3K/AKT signal pathway. CONCLUSION: survivin play an important role in regulating leukemia stem cell escape the synergistic cytotoxic effect of arsenic trioxide and aclacinomycin. The mechanism of regulation process may through activate the related apoptosis pathway protein and supperss the PI3K/AKT signal pathway. This study may provide a further benefit for reversing chemotherapy resistant for acute leukemia

Key words: arsenic trioxide,aclacinomycin, Acute leukemia; survivin; chemotherapy resistant

Disclosures

No relevant conflicts of interest to declare.
Comparative Analysis Between RQ-PCR and Digital Droplet PCR of BCL2/IGH Rearrangement in the Peripheral Blood and Bone Marrow of Early Stage Follicular Lymphoma
Authors: Cavalli Marzia , De Novi Lucia Anna , Della Starza Irene , Cappelli Luca , Nunes Vittorio , Pulsoni Alessandro , Del Giudice Ilaria , Guarini Anna , Foa Robin
Published: October 2019

Figure Figure.

Disclosures

No relevant conflicts of interest to declare.
The Addition of Novel Agents to Front-Line Therapy Significantly Improve the Short-Term and Long-Term Therapeutic Effects of High Risk Waldenstrom's Macroglobulinemia Patients in China
Authors: Xiong Wenjie , Yi Shuhua , Li Heng , Li Zengjun , Lv Rui , Liu Wei , Xu Yan , Zou Dehui , Hao Mu , Qiu Lugui
Published: October 2019

Background: Waldenstrom's Macroglobulinemia (WM) is a B-cell proliferative malignancy characterized by immunoglobulin M monoclonal gammopathy and bone marrow infiltrated by B cell, lymphoplasmacytic cells and plasma cells. Some studies have shown that novel agents such as Rituximab and Bortezomib combined chemotherapy could improve WM patients' survival. However, few studies had focused on the efficiency of these agents on ISSWM intermediate or high risk group and no systematical investigations had been published for the Eastern WM patients.

Methods: We systematically reviewed the records of 91 WM patients with intermediate or high risk ISSWM group, who had been diagnosed at Institute of Hematology & Blood Disease Hospital, Chinese Academy of Medical Sciences& Peking Union Medical College.

Results: There was male predominant (75.8%) for the entire population, with a median age of 62 years (range32-87) at diagnosis. There were 36(39.6%) and 55(60.4%) patients in the intermediate or high risk ISSWM group, respectively. The percentage of hepatomegaly, splenomegaly, B symptomand elevated LDH were 16.9%, 34.1%, 33.3% and 18.6%, respectively. Among them, 36 patients received novel agents combination first-line chemotherapy and 55 patients received conventional chemotherapy. The complete remission (CR) rate and overall response rate(ORR) were 26.5% and 76.5% in the novel agent group respectively, which were higher than the conventional group( 26.5% v.s. 10%p=0.047; 76.5% v.s.56%, p=0.055). With a median follow-up of 35 months, The novel agent group induced a significantly longer progression-free survival (PFS) compared with the conventional group (77months v.s.45 months, p=0.04).The mediate overall survival (OS) in the novel agent group was not reached, which was also significantly longer than the conventional group(NR v.s. 62months,p=0.045).

Conclusion:Compared with the conventional therapy, the novel agent combination significantly improved the short-term and long-term therapeutic effects in intermediate/high risk patients of WM.

Disclosures

No relevant conflicts of interest to declare.
Post Transplant Brentuximab Maintenance Appears More Effective Than Post Transplant Salvage Brentuximab for Relapsed/Refractory Hogkin's Lymphoma
Authors: Chidiac Amanda , Massoud Radwan , Haidar Mohamad , Fares Elie , Bazarbachi Ali , El Cheikh Jean
Published: October 2019

POST TRANSPLANT BRENTUXIMAB MAINTENANCE APPEARS MORE EFFECTIVE THAN POST TRANSPLANT SALVAGE BRENTUXIMAB FOR RELAPSED /REFRACTORY HODGKIN LYMPHOMA

Amanda Chidiac1, Radwan Massoud 1, Mohamad Haidar1, Elie Fares1, Ali Bazarbachi1 and Jean El Cheikh1

1Bone Marrow Transplantation Program, American University of Beirut Medical Center, Beirut, Lebanon

Keywords: Hodgkin Lymphoma, Relapsed/Refractory, Brentuximab.

Context: Brentuximab Vedotin (BV) is a chimeric anti CD30 IgG1 antibody, conjugated to synthetic antitubulin momomethyl auristatin. BV is approved for the treatment of classical HL in relapse either after autologous stem cell transplantation (ASCT) or after two lines of combination chemotherapy in transplant ineligible patients. The AETHERA trial revealed increased PFS when BV is used as maintenance therapy after ASCT.

Objective: Compare the effectiveness of BV as maintenance or salvage therapy after ASCT for relapsed/refractory (R/R) HL.

Design: A retrospective analysis conducted on patients with R/R HL treated with BV after ASCT either as maintenance or salvage therapy. Analysis after a median follow up of 12 months is reported.

Setting: The study was conducted in a single institution; American University of Beirut Medical Center (AUBMC) after IRB approval.

Patients: The study included 15 adult patients with R/R HL treated with BV with the following indications:

á Maintenance therapy after ASCT in high risk patients

á Salvage therapy for relapse after ASCT or allo-SCT

Intervention: BV at 1.8mg/kg IV every 4 weeks for the above indications.

Main outcome measures: response rate (RR), overall survival (OS) and progression free survival (PFS).

Results: Nine high-risk patients (60%) received 4 cycles of BV as maintenance therapy post ASCT. Six additional patients (40%) in relapse after ASCT (n=5) or after allo-SCT (n=1) received BV as salvage therapy for an average of 4 cycles (range 3-21). Patients characteristics are listed on Table 1. RR was 33% in 6 patients who received BV as salvage post SCT. Two patients proceeded with allo-SCT in the salvage group. After a median follow up of 9 months, only 2 patients (22%) in the maintenance group progressed and all patients are still alive. Conversely, in the salvage group, 4 patients (67%) progressed and 1 patient developed secondary AML and died.

Conclusion: BV therapy showed an efficacy only as maintenance treatment after auto-SCT but was less successful when used as post-transplant salvage in R/R HL. Four cycles of maintenance may suffice. Prospective trials with larger samples are warranted to support these findings.

Disclosures

No relevant conflicts of interest to declare.
Treatment of Drug-Resistant Forms of Mycosis Fungoides/Sezary Syndrome
Authors: Gorenkova Liliya , Penskaya Ekaterina , Kravchenko Sergey , Kovrigina Alla , Savchenko Valeriy G.
Published: October 2019

Mycosis Fungoides (MF) and leukemic variant - Sezary Syndrome (SS) are the most frequent diseases among cutaneous T-cell lymphomas. These tumors have a chronic progradient nature. Up to present time there is no evident of effective therapy, particularly late-stage of diseases.

About 30% of patients have no effect from interferon, radiotherapy and combined treatment regimens. Older age (median age - ...) of such patients limits therapy options. Several lines of previous treatment lead to tumor resistance and higher accumulated toxicity. Thus, use of potentially effective chemotherapeutic agents in lower doses will reduce myelotoxicity and increase the cohort of patients. Gemcitabine is an effective medication in treatment of MF/SS. Its efficacy is preserved in small doses with prolonged duration of infusion and allows decreasing myelotoxicity. It was firstly discovered in therapy of solid tumors.

In current study were involved 14 patients with resistant forms of MF/SS (10 patients - GM 3 - SS, 1 case GM transformation into large cell lymphoma). We used 6-hour infusion of gemcitabine at dosage of 250 mg/m2. 86% of patients achieved a general response: 29% complete remission, 50% - partial remission, 21% - stabilization of disease, 14% (2 patients)-progression.

Present study showed promising results in treatment of patients with advanced stages of MF/SS which had at least two preceding lines of therapy without an effect. Common response (PR/CR) was received in high percent of cases. This treatment protocol is reproducible in elderly patients. For long-term conclusions further studies are needed.

Disclosures

No relevant conflicts of interest to declare.
Mixed Phenotype Blast Phase of Chronic Myeloid Leukemia in the Era of Tyrosine Kinase Inhibitor Therapy
Authors: Gong Zimu , Bai Shi , Hu Shimin
Published: October 2019

Background

Chronic myeloid leukemia is a myeloproliferative neoplasm that is characterized by BCR-ABL1fusion gene and typically exhibits three distinct stages: chronic phase, accelerated phase and blast phase (BP). With tyrosine kinase inhibitors (TKIs) becoming standard front-line therapy, the progression of CML to BP has been dramatically decreased. In pre-TKI era, the blasts were of myeloid lineage in approximately 70% of patients with CML-BP, lymphoid lineage in approximately 30% of patients, and rarely, mixed phenotype. In the era of TKI era, cases of CML-BP with mixed phenotype are rarely reported to date.

Methods

Cases of CML diagnosed since year 1998 when TKI therapy was implemented in our institution to present were reviewed for blastic transformation. Clinical characteristics and laboratory data were collected. Lineage was assigned based on flow cytometry, cytochemistry or immunohistochemistry according to the 2008 WHO classification.

Results

A total of 430 patients were diagnosed with CML-BP: 278 (64.7%) had myeloid BP, 138 (32.1%) lymphoid BP, and 14 (3.3%) mixed phenotype BP (Table 1). Of these 14 cases of mixed phenotype BP, 10 were of B/myeloid and 4 T/myeloid. Molecular assay showed that 4 patients had p190 subtypeBCR-ABL1transcript and the remaining 10 patients had p210 subtype. There are 8 men and 6 women. The median age at diagnosis of CML-BP was 52 (range 23-70). Five patients presented with CML-BP initially, and the remaining 9 patients developed BP later during treatment with a median interval of 17.4 months from initial diagnosis of CML to blast transformation.

Twelve of the 14 patients received TKIs, 11 patients received multi-agent chemotherapy (including hyper-CVAD regimen), and 7 patients underwent allogeneic stem cell transplantation (allo-SCT). In 12 patients with known treatment response, 11 achieved complete cytogenetic response, and 7 achieved major molecular response. Of the 7 cases treated with allo-SCT, all achieved complete cytogenetic response, and 6 achieved major molecular response. Patients with mixed phenotype BP had a slightly better prognosis than patients with myeloid or lymphoid BP. The overall survival after blastic transformation was 17.8 months (range 0.7-73.2 months) in patients with mixed phenotype BP, comparing to 9.6 month in patients with myeloid BP and 15.9 months in patients with lymphoid BP. However, limited by the small sample size, no statistical significance was reached.

Conclusion

Blastic transformation of CML with mixed phenotype is rare. The prognosis of patients with mixed BP is poor. Aggressive chemotherapy in combination with TKIs might be effective, and allo-SCT may offer a chance of deep response and probably longer overall survival.

Disclosures

No relevant conflicts of interest to declare.
Nilotinib As Second or Third-Line Therapy for Myeloid Chronic Leukemia Chronic-Phase in Mexican Patients
Authors: Ayala Manuel , Domínguez Jacqueline , Chavez Antonieta
Published: October 2019

Background.- Nilotinib has 30-fold higher potency and increased selectivity against Bcr-Abl.Fast and deep responses can be achieved with nilotinib after failure to imatinib. In this study, we report the response rates and long-term results of using nilotinib after failure to imatinib or beyond. At the Specialty Hospital CMN "La Raza" nilotinibexperience launching this late 2006, has developed institutional experience.

Objective.- To evaluate the cytogenetic and molecular response to treatment with nilotinib in the second line treatment of patients with CML-CP and its correlation with early molecular-response.

Methods- During the experience with nilotinib as second-line therapy or beyond on CML-CP, 110 CML patients have been treated; established dose of nilotinib for all phase was 400 mg BID. Dose adjustments due toxicity were performed in accordance to previous publications, hematological and no hematological toxicity were scored in conformance to CTCAE v3. Patients were evaluated for hematologic, cytogenetic and molecular responses. Of all patients with CP-CML diagnosed treated with nilotinib, sixty-nine patients were included in the analysis, we selected those who had undergone a molecular evaluation at 3 months. In this study by BCR-ABL mutation status was analyzed. Twenty-seven percent (30 of 110) of the imatinib-resistant patients had BCR-ABL mutation(s) at baseline. One imatinib-resistant patients had the T315I mutation at baseline. The data from 69 patients with CP-CML were analyzed. Distribution of Sokal risk groups were as follows low-16/intermediate-14/high-57/NA-13%.

Results.- Overall, 57 of the 69 patients (83%) of them had a transcript of ≤ 10% at 3 months. Forty-five out of 69 patients (65%) with transcript of ≤10% at 3 months achieved MMR at 12 months, while only 2 of 12 patients (17%) with ratio >10% had MMR, and 83% did not reach the MMR. The EFS by level of transcripts ≤10% at 3 months in 57 patients with 2 events was 96% while in >10% in 12 patients with 4 events was 67%, with an average estimate of time of EFS of 62 vs 43 months respectively. The EFS by MMR at 12 months in 32 patients without events was 100%, while without MMR in 37 patients with 6 events was 84% P=0.022. Nilotinib induced complete cytogenetic responses in 60 percent and 48% molecular responses in the 110 patients treated, 58% of which were major molecular responses, and deep molecular response 42%. To date, 96 of the treated patients remain alive (87%) and continue on treatment.

Conclusions.- Nilotinib (Tasigna) is efficacy and well-tolerated treatment for patients with CML as second-line or beyond in our patients, The 48-month follow-up results show that nilotinib therapy continued to be effective, is comparable to diverse results of other international reports in developed countries.

Disclosures

No relevant conflicts of interest to declare.
Managing BCR-ABL 1 Negative Myeloproliferative Neoplasms in an Emerging Economic Environment
Authors: Benneh Amma A. , Asare Eugenia Vicky Naa Kwarley , Olayemi Edeghonghon
Published: October 2019

Background:

Myeloproliferative neoplasms (MPN) are a group of diseases characterized by clonal disorders of the pluripotent stem cell. The annual incidence of all subtypes of MPN is 6-10/100,000 population. MPN without Philadelphia chromosome or BCR-ABL1 fusion gene such as Polycythaemia Vera(PV), Essential Thrombocythaemia (ET) and Myelofibrosis (MF) are known as BCR-ABL1 negative MPN. Diagnosis and management of these conditions in a limited resource environment can be challenging. Some successes have however been chalked when diagnostic and therapeutic difficulties have been overcome.

Methodology: A retrospective patient chart review of patients who attended Hematology clinic at the Department of Hematology, Korle-Bu Teaching Hospital was conducted between January 2005 and December 2015.This review included all adult patients diagnosed with PV, ET and MF over the period. Diagnosis of MPN was made mainly by film comment, bone marrow aspirate, trephine biopsy and JAK 2 mutation analysis if patient could afford. Patients records were analyzed for demographic characteristics, clinical signs, hematological parameters, treatment options and outcomes.

Results:

The annual incidence of MPN over the period under study was 2.4 per year. The median age was 46 (range 24 - 75) years. There was male predominance of 1.9: 1. Splenomegaly was the commonest organomegaly seen. Polycythaemia Vera was the commonest (10; 38.5%) BCR-ABL1 negative MPN to be diagnosed at the hospital within the period. Treatment options available for patients with PV was venesection and hydroxycarbamide. For ET and MF it was hydroxycarbamide and supportive treatment. The longest survivor within the period of study has lived with Polycythaemia Vera for the past 10 years.

Conclusion:

The diagnosis and management of BCR-ABL1 negative MPN in emerging economic environments can be challenging as resources are limited. Certain conditions such as Hyperreactive malarial splenomegaly (HMS) can also mimic MPN and as such a high index of suspicion coupled with appropriate diagnostic facilities are required for early diagnosis and prompt management. Despite these odds, the disease is controlled reasonably well in our environment giving the patients more years of quality life than they would have had without any intervention.

Disclosures

Asare: Intramural University of Ghana Research fund: Research Funding; Vanderbilt University Medical Center Gift Funds: Research Funding. Olayemi:Intramural University of Ghana Research fund: Research Funding; Vanderbilt University Medical Center Gift Funds: Research Funding.
Extra Medullary Hematopoiesis in Patients with Polycythemia Vera Rare and Unusual Presentations
Authors: Yassin Mohamed A. , Mohamed Shehab , Nashwan Abdulqadir
Published: October 2019

Extramedullary Hematopoisis refers to the presence of hemopoietic elements in locations other than the bone marrow medullary space, it may be seen in many conditions, including chronic hemolytic anemias like thalassemia Intermedia or major. The common sites of involvement are the liver, spleen and the lymph nodes. Less common sites include the central nervous system, adrenal gland, kidney, perirenal soft tissues, breast, peritoneum and gastrointestinal tract the paraspinal region is a relatively uncommon location for these hemopoietic deposits. There are a few case reports of EMH occurring in the spineMost cases have been reported in connection with thalassemia, although it has also been described in patients with sickle cell anemia,myelodysplastic syndrome and myelofibrosis. To the best of our knowledge, there are very few documented case reports in the literature of patients with polycythemia vera and Extra medullary Hemayopoiesis.The reported cases are limited in this regard only 11 cases were reported from 1979 till 2015, Paraprasesis is the most common presenting symptom, Age between 30 to 75 year with Male to female 6:1,Thoracic region is most affected site Most cases treated by surgery ,radiotherapy or both Poorer outcome compared to MF cases

Overall the mortality in EMH with polycythemia vera is higher than MF and thalassemia, some of them due to fatal location of cord compression (cervical)

No definite guidelines have as yet been formulated for the treatment of such patients with EMH. Excision and decompression, radiotherapy, hypertransfusion and hydroxyurea alone or in combination have been proposed as well as lack of consensus about what is complete remission is it clinical or clinical and radiological and what is partial response is it any reduction of the mass lesion or any degree of clinical improvement in conclusion there is necessity to establish consus for treatment and definition of CR ,PR or No response in EMH with polycythemia vera.

Disclosures

No relevant conflicts of interest to declare.
Application of Next Generation Sequencing for Prognostic Stratification in Myelodysplastic Syndromes
Authors: Xu Yuanyuan , Li Yan , Hou Guangyuan , Chen Chongjian , Yu Li
Published: October 2019

Myelodysplastic syndromes (MDS) are a heterogeneous group of hematological malignancies characterized by a deregulation of blood cell formation with cytopenia in varying degrees and frequently develop into acute myeloid leukemia (AML). Next generation sequencing (NGS) has significantly contributed to diagnosis and prognostication in patients with MDS. To explore the role of acquired mutations in MDS biology and clinical features, we performed mutational profiling of 111 genes in 125 patients with MDS based on target-sequencing. The results showed that 89% of patients had one or more carcinogenic mutations. Mutation frequencies of several genes, including ASXL1 (16.8%), RUNX1 (14.4%) and TET2 (12%), were above 10% in MDS patients. According to univariate analysis in 108 patients with survival data, mutations of GATA1/2, TP53 and DNMT3A were identified as adverse prognostic factors for overall survival (OS), while RUNX1, KRAS/NRAS, SRSF2 and TET2 mutations were determined as unfavorable factors for progression free survival (PFS). By multivariate COX regression analysis, mutations of KRAS/NRAS, GATA1/2 and DNMT3A were independent risk factors for OS, whereas IDH1/2 gene mutations were favorable factors for PFS. By evaluation of the clinical benefit of hypomethylating agents (HMAs), it showed that for patients harboring mutations associated DNA methylation or not and receiving HMAs treatment or not, the order of the outcome from good to bad was geneWT with non-HMAs, geneWT with HMAs, genemutwith HMAs and genemutwith non-HMAs. Therefore, target-sequencing of mutational spectrum is a feasible and highly promising prediction method for prognostic evaluation in patients with MDS, which would contribute to personalized therapeutic decisions.

Disclosures

No relevant conflicts of interest to declare.
Sustained Remissions after Discontinuation of Ibrutinib in Relapsed/Refractory CLL: A Basis for Reducing Drug Toxicity and Treatment Costs?
Authors: Houranieh Jenna , Fleischman Roger A
Published: October 2019

In contrast to traditional chemotherapy, patients responding to biological or targeted therapies often are treated indefinitely until progression or toxicity. This therapeutic model, however, increases treatment costs, may induce greater toxicity and theoretically could select for earlier emergence of drug resistance. Moreover, little data is available regarding the outcomes of patients who discontinue targeted therapies after achieving remission. In this regard, we report two patients with relapsed/refractory CLL who chose to stop therapy unrelated to toxicity or disease status after the induction of remission by the BTK inhibitor ibrutinib. Patient A started ibrutinib for progressive CLL at an absolute lymphocyte count (ALC) of 137,000mm3 and recurrent hemolytic anemia. After 5 months, the hemolysis had resolved (Hgb 15.6 g/dl) while the ALC had declined to 9200mm3. Treatment was then interrupted due to patient preference. One month after drug discontinuation, the ALC was in the normal range at 1400mm3 and remained within or near the normal range for a total of 12 months. Two months later, the ALC was again markedly elevated at 68,000mm3and anemia recurred. The patient then agreed to restart ibrutinib. Thus far, after 5 months of re-treatment, he has had prompt resolution of the anemia and achieved a partial remission with a slowly decreasing lymphocytosis. Patient B was started on ibrutinib for a rising ALC (26,000mm3) and severe hemolytic anemia. After 9 months of treatment, the hemoglobin was 13 g/dl and the ALC was in the normal range at 3300mm3. Due to unrelated medical problems, ibrutinib therapy was stopped. Currently, 6 months since drug discontinuation, the ALC remains in the normal range and no other signs of CLL are present. These clinical observations suggest that interruption of ibrutinib may be feasible in at least some CLL patients who achieve remission. Even if flow cytometry were performed at monthly intervals to detect early recurrence and ensure prompt re-institution of therapy, the cost savings would still be considerable. Of course, clinical trials will be necessary to confirm equivalent long-term efficacy and overall survival for intermittent versus continuous ibrutinib therapy in CLL.

Disclosures

No relevant conflicts of interest to declare.
12-Month Full Donor Chimerism Is Associated with Low-Grade Acute Graft Versus Host Disease (aGVHD) and Predicts Superior Outcomes in Reduced Intensity (RIC) Alemtuzumab-Based Transplants for MDS and AML
Authors: Kanellopoulos Alex George , Paneesha Shankaranarayana , Xenou Evgenia , Qureshi Iman , Eden Dewi Tomos , Sutton David John , Pryor Rebecca , Cain Lorna , Arbuthnot Carolina , Lovell Richard , Borg Anton , Randall Katie , Holder Kathy , Suhr Julie , Baker Lynda , Ryan Lynn , Kaparou Maria , Kishore Bhuvan , Nikolousis Emmanouil
Published: October 2019

Introduction

Reduced intensity (RIC) Alemtuzumab conditioned allogeneic stem cell transplants have enabled a successful graft versus leukaemia effect while GvHD rates were low. The impact of chimerism on outcome has scarcely been studied in RIC, Alemtuzumab-based transplants.

Patient characteristics

Retrospective analysis of 50 patients with high risk MDS (n=14) and AML (n=36) undergoing RIC-Alemtuzumab transplant in our center since 2006. Median age was 62 years (range 51-72) and there were 33 male and 17 female recipients. All donors were fully (10/10) matched in HLA-loci. 11 patients had a sibling donor while 39 had a matched unrelated donor. 29 patients were conditioned with fludarabine, melphalan and alemtuzumab while 21 patients received fludarabine, busulphan and alemtuzumab.

Results

The median follow-up of patients was 18.2 months (range 15-90). Median overall survival was 27.4 months and median progression free survival 33 months. At 3 months post-HSCT 72% (n=36) and 18% (n=9) of patients were full and mixed chimeras respectively. At 12 months post-HSCT n=19 and n=10 of patients were full and mixed donor respectively. 14 patients (28%) developed grade 1-2 GvHD while only 1 (2%) grade 3-4 GvHD. Patients who attained 12-month full donor chimerism in whole blood (WB), had a mean PFS of 85 months as compared to 81.5 months for mixed chimeras (p= 0.04). In 3-months, those who were full donor in WB had a mean PFS 95 months compared to 33 for mixed chimeras in WB (p= 0.016). 3, 12 -month T-cell chimerism did not influence OS and PFS. The presence of grade I-II GVHD was significantly associated with 12-month full donor chimerism in both WB and T-cells (p<0.05).

Conclusion

In our series of AML/MDS patients who received RIC Campath-based allografts, full donor chimerism is a surrogate marker for progression free and overall survival. All grade I/II aGVHD patients achieved full donor chimerism in whole blood.

Disclosures

Paneesha: Abvie: Honoraria. Kishore:celgene: Other: travel grant.
Haploidentical Stem Cell Transplantation for Rare Pediatric Diseases at Peking University People's Hospital
Authors: Chen Yao , Chen Huan , Xu Lan-Ping , Chen Yu-Hong , Wang Jing-Zhi , Wang Feng-Rong , Zhang Xiao-Hui , Liu Kai-Yan , Huang Xiao-Jun
Published: October 2019

Background:Rare diseases requiring hematopoietic stem cell transplantation (SCT) present a challenge for BMT centers. The patients present at a low frequency, and there are often no established conditioning regimens for these disorders, especially for children without HLA matched sibling donors. We report the conditioning regimen using haploidentical related donors, and outcomes for the following rare disorders transplanted at our center from January 2013 through December 2015. During the past three years we had patients presented with the following: Fanconi anemia (FA), dyskeratosis congenital (DC), adrenal leukodystrophy (ALD) and Wiskott-Aldrich syndrome (WAS).

Methods:We treated these children with rare disorders that required SCT at our center, and are reporting the transplant outcomes for these rare diseases.

Results: All children were transplanted with haploidentical related donors using G-CSF mobilized bone marrow and peripheral blood stem cell. Conditioning regimens and treatment plans were mainly following Beijing protocol and varied with type of disease. Patient characteristics and outcomes are presented in Table 1. No graft failure occurred and three of 5 patients (60%) are alive and well with no evidence of disease.

Conclusions: Our program has treated a variety of rare disorders with an event-free-survival rate of 60%. Our data suggests haploidentical SCT for rare pediatric diseases seems to be challenging and promising.

Disclosures

No relevant conflicts of interest to declare.
Potent Anti-Platelet Metabolite, 12-HETrE, Inhibits Platelet Activation and Thrombosis In Vivo Via Activation of the IP Receptor
Authors: Tourdot Benjamin , Isingizwe Zitha , Yeung Jennifer , Adili Reheman , Holinstat Michael
Published: October 2019

Platelet activation is a critical component of clot formation. Under pathophysiological conditions platelet-mediated clot formation can lead to an occlusive thrombotic event resulting in myocardial infarction and stroke. Regulation of platelet reactivity relies on an understanding of the complex intra-platelet signaling that takes place following activation of various receptors on the platelet surface. Our lab recently identified a novel oxidized metabolite known as 12-HETrE that is formed from 12-lipoxygenase (12-LOX) oxidation of the ω-6 fatty acid DGLA in the platelet. Since DGLA constitutes one of the predominant fatty acids contained in the phospholipids on the platelet membrane and is also a rich source of dietary fatty acid intake through a variety of plant oils, we had originally sought to determine if this metabolite contained inherent regulatory properties on the human platelet. We successfully showed not only that 12-HETrE potently regulates hemostasis and thrombosis in vivo, but additionally identified that the metabolite signals inhibition of platelet activity through a GαS-linked GPCR resulting in formation of cAMP and activation of PKA and VASP. In the current study, we identified for the first time the receptor activated by 12-HETrE in the human platelet. To identify which GPCR is activated by 12-HETrE, we utilized both pharmacologic as well as genetic approaches. Our ex vivo pharmacological data suggested 12-HETrE predominantly signals through the IP receptor in the human platelet. This finding was confirmed using an IP receptor knockout mouse as well as human cell lines expressing or absent IP receptor on their surface. Finally, these findings were confirmed in vivo with wild type or IP knockout mice and assessing the ability of 12-HETrE to inhibit laser-induced thrombosis. Hence, these findings show for the first time that 12-HETrE does in fact regulate inhibition of platelets through the IP receptor. Since 12-HETrE has already been shown to inhibit thrombosis without a significant increase in bleeding diathesis, the current study has elucidated a new target for IP receptor activation and an inhibition of platelet activity and occlusive thrombus formation and should be evaluated for its potential as a new class of anti-thrombotic therapeutics.

Disclosures

No relevant conflicts of interest to declare.
New Functional Assays to Selectively Quantify APC- and TFPI-Cofactor Activities of Protein S in Plasma
Authors: Alshaikh Nahla , Rosing Jan , Thomassen Stella , Castoldi Elisabetta , Simioni Paolo , Hackeng Tilman M.
Published: October 2019

Background: Protein S is an important anticoagulant in the down-regulation of blood coagulation by acting as cofactor for two main regulators of coagulation. Protein S acts as a cofactor for activated protein C (APC) in the inactivation of the procoagulants FVa and FVIIIa and for TFPI in the formation of the TFPI-FXa encounter complex that subsequently inactivates TF-FVIIa. Several studies have indicated that protein S deficiency is associated with increased risks of venous thrombosis.

Aims: Current protein S functional assays are influenced by plasma determinants such as FVLeiden. The aim of the study was to develop thrombin generation based assays that enable quantification of both the APC- and TFPI-cofactor activities of protein S in plasma whereas the outcome of the assays should not be affected by the presence of FVLeiden or other plasma variables.

Methods: APC- and TFPI-cofactor activities of protein S in plasma were measured using calibrated automated thrombography (CAT) in protein S-depleted plasma supplemented with a small amount of sample plasma either in the presence of anti-TFPI antibodies and APC (APC-cofactor activity) or at excess full-length TFPI without APC (TFPI-cofactor activity). Total and free protein S levels in plasma were measured by ELISA's.

Results: Average APC-cofactor activities of protein S were 113%, 122%, and 87% in plasma from normal individuals (n=10), FV Leiden heterozygotes (n=4), and FV Leiden homozygotes (n=2), respectively, while the average APC-cofactor activity of protein S in plasma from heterozygous protein S-deficient individuals (n=21) was significantly lower (55%). Similar trends were observed for the TFPI-cofactor activity of protein S, with averages of 108%, 112%, and 97% in plasma from individuals with normal protein S levels and different FV Leiden genotypes, and 64% in plasma from protein S-deficient patients. APC-cofactor activities of protein S correlated significantly with free and total protein S antigen levels, while TFPI-cofactor activities correlated less with protein S antigen levels.

Summary/Conclusion: We have developed functional protein S assays that measure both the TFPI- and APC-cofactor activities of protein S in plasma and that are not affected by the FV Leiden mutation or other plasma influences.

Disclosures

No relevant conflicts of interest to declare.
PAR1 Antagonists Development and Clinical Utility
Authors: Coughlin Shaun
Published: October 2019

Thrombin is a potent activator of platelets and other cells. The mechanism by which thrombin, a protease, regulates cellular behaviors like a hormone was revealed by expression cloning of a G protein-coupled receptor now known as protease-activated receptor-1 (PAR1). Thrombin activates PAR1 by cleaving its N-terminal ectodomain to reveal a new N-terminus that then serves as a tethered peptide ligand, binding to the heptahelical domain to trigger G protein activation. A synthetic hexapeptide mimicking this new N-terminus activates PAR1 without receptor cleavage. Point mutations that prevent receptor cleavage render PAR1 unactivatable by thrombin without altering activation by exogenous tethered ligand peptide. Replacement of the thrombin cleavage site with sites for other proteases allows PAR1 to signal in response to those other proteases. Removal of sequence N-terminal to the cleavage site and creation of the new protonated amino group at N-terminal of the tethered ligand sequence is necessary for its function, explaining how the tethered ligand is silent in the intact receptor and activated by receptor cleavage. Thus, PARs are, in essence, peptide receptors that carry their own ligands, which can be unmasked by receptor cleavage. Mammalian cells utilize four PARs to respond to coagulation proteases and other proteases with trypsin-like specificity. Three, PAR1, PAR3 and PAR4 can mediate responses to thrombin, with PAR3 and PAR4 mediating platelet activation by thrombin in mice and PAR1 and PAR4 platelet activation in humans. Inhibition of thrombin signaling via PARs in platelets decreases thrombus formation in animal models of platelet-dependent thrombosis. These and other studies led to development of the PAR1 antagonist vorapaxar (Zontivity), a first-in-class antiplatelet agent approved for secondary prevention of atherothrombotic events in selected patients with prior myocardial infarction and peripheral arterial disease. What was and was not learned from animal and human studies and remaining questions related to clinical utility of PAR inhibition will be discussed. The structure-function studies outlined above support the tethered ligand model, but a structure that reveals how the tethered ligand binds and how binding leads to transmembrane signaling and G protein activation is needed. Vorapaxar provided a tool for stabilizing PAR1 protein during solubilization, purification and crystallization. In collaboration with Brian Kobilka and colleagues, we solved a crystal structure of off-state PAR1 in complex with its antagonist vorapaxar, which explains pharmacological properties of vorapaxar and efforts to solve a complementary on-state structure are ongoing.

Disclosures

Coughlin: Merck: Research Funding; Novartis: Consultancy; Portola Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees.
The Impact of 3D Chromosomal Topology in Acute Leukemia
Authors: Aifantis Iannis
Published: October 2019

Acute myeloid leukemia (AML) is the most common adult leukemia characterized by excessive proliferation of abnormal myeloid progenitors. AML continues to have a dismal survival rate amongst all subtypes of leukemia (<50% five-year overall survival rate), which can largely be attributed to limited advances in treatment regimens that, for the last decades, have relied on the use of two non-targeted cytotoxic drugs: cytarabine and anthracycline. Large-scale sequencing efforts have shed new light on genetic and epigenetic determinants of AML. Interestingly, these studies identified a frequent co-occurrence of somatic mutation between genes encoding cohesin complex subunits (such as STAG2, SMC1A, RAD21 and SMC3) and well-characterized AML oncogenic triggers, such as FLT3-ITD, TET2, and NPM1. Recent work has demonstrated an important role for the cohesin complex in normal stem/progenitor self-renewal and differentiation, gene regulation, and suppression of myeloproliferative neoplasms and AML, despite the precise mechanisms underlying these functions remaining poorly understood. It is believed that cohesin may suppress tumor formation by regulating chromatin looping at loci critical for self-renewal and myeloid progenitor differentiation. Utilizing established models of murine and human AML, this we will focus on the molecular mechanisms of cohesin-dependent myeloid tumor-suppression, with an emphasis on understanding novel treatment approaches that can exploit these functions. Using established protocols for identifying genome-wide changes in chromatin topology and gene expression, we propose undertaking an extensive characterization of cohesin-regulated chromatin changes driving AML. Furthermore, we investigate the application of targeted agents in cohesin-deficient AML whilst extensively mapping the mechanisms-of-action underlying these specific treatments. Ultimately, our work aims to generate novel, pre-clinical disease models of cohesin-mutated AML with strong mechanistic insights into the tumor-suppressive function of this complex.

Disclosures

No relevant conflicts of interest to declare.