bluuebunny/crossref_metadata_2025 huggingface dataset:
Pregnancy and Primary Mediastinal B-Cell Lymphoma
Authors: Mangasarova Jana Konstantinovna , Kravchenko Sergey Kirillovich , Magomedova Aminat Umaraskhabovna , Baryakh Elena , Vorobiev Vladimir Ivanovich , Savchenko Valeri G.
Published: November 2019

Introduction. Favorable therapeutic regimen for pregnant women with primary mediastinal B-cell

Lymphoma (PMBCL) is determined by tumor mass, somatic status, period of pregnancy and potential risk of teratogenic effects of medications which penetrate through placenta.

Aim. To estimate chemotherapy efficacy of PMBCL in case of pregnancy and its toxicity for fetus.

Patients and Methods. Since 2004 to 2015 years in National Research Center for Hematology were treated 106 patients with primary mediastinal B-cell lymphoma. In 8 (7,5%) from them disease debuted during 2-3 trimester of pregnancy, one women had two fetuses simultaneously. Before delivery women underwent induction chemotherapy with VACOPB- regimen in 5/8 cases and with R+DAEPOCH in 3/8 cases.

Results. After induction treatment had been performed 6/8 women achieved a partial remission, 1/8 had progression, 1/8 - complete remission. High dose chemotherapy was performed in 7/8 women after 1 month following delivery. It was born 9 children (4 boys and 5 girls). Median body weight was 2000 g. (1300 - 3600 g.). Median growth was 47 cm. (40 - 53 cm). In two cases when rituximab had been administered to women before delivery, children were diagnosed with intrauterine pneumonia. One child, born from woman after VACOPB chemotherapy had thrombosis of superior vena cava. All women continue to be in complete remission of PMBCL, children are practically healthy without malformations. The median follow-up of patients was 40 months (15 - 78 months).

Conclusions. Pregnancy is not a contradiction for induction chemotherapy of PMBCL in 2-3 trimesters. Rituximab shouldn't be administered before delivery because of a high risk of infectious in fetus.

Disclosures

No relevant conflicts of interest to declare.
Retrospective Study of the Efficacy and Safety of Treatment with Subcutaneous Injection of Bortezomib in De Novo Patients with MM
Authors: Fu Chengcheng , Ma Ling , Yan Shuang , Liu Hui , Xu Yun , Depei Wu
Published: November 2019

[Objective]

The induction therapy of multiple myeloma (MM) has dramatically changed due to the availability of new drugs such as bortezomib.To explore the efficacy and safety of subcutaneous injection of bortezomib in the treatment of de novo MM patients, a total of 56 patients treated with PAD form June 2008 to January 2015 were retrospective analyzed.

[Methods]

29 patients in PAD group received intravenous injection of bortezomib 1.3mg/m2, d1,4,8,11, Adr iv 40mg/m2, d1, Dex iv 20mg, d1,2,4,5,8,9,11,12; another 28 in improved PAD group received subcutaneous injection of bortezomib1.3mg/m2, d1,4,8,11, Adr iv 40mg/m2, d1, Dex iv 20mg, d1,2,4,5,8,9,11,12. The efficacy and safety of two groups were analyzed.

[Results]

The response rate >= VGPR after 4 cycles of PAD or improved PAD were 51.7% and 51.9% respectively.With a median follow up of 24 months,the median TTP , PFS and OS of the PAD group was 47 months, 42 months, 63 months respectively, but the time of the improved PAD group wasn't shown up yet.In the improved PAD group,2y- TTP ,PFS and OS was 94.4%, 94.4%, 100%. With a follow-up to 60 months,the OS of standard-risk group[without FISH 1q21+, or t(4;14), or p53-]was 61.9%,but the survival was only 31.9% in FISH high-risk group. OS, PFS of the patients with 1q21 amplification was 18.7-56.4 months and 9.7-53.5 months, showed statistical difference (P=0.029), (P=0.037), compared to FISH negative patients,which was 51.5-71.3 months and 39.4-68.4 months. The study found that the hemoglobin < 100 g/L or 1q21+ were the poor prognosis factors for patients' OS, while subcutaneous injection of bortezomib could improve OS(P=0.042), mainly because compared to PAD group,Grade 3 or worse adverse events were significantly reduced in improved PAD group. the most common were peripheral neuropathy (P=0.049), infection(0.004). When α=0.1, leukopenia(P=0.086), diarrhea(P=0.093) also showed statistically significant difference.

[Conclusions]

The improved PAD regimen by changing bortezomib from intravenous administration to subcutaneous injection significantly reduced adverse events, improved the safety of clinical application of bortezomib without affecting curative effect, and had greatly improved the OS.1q21 amplification was the poor prognostic factors in PAD induction treatment of MM.

Disclosures

No relevant conflicts of interest to declare.
Improved Outcome of Relapsed or Refractory Pediatric B-Cell Non-Hodgkin Lymphoma in Japan
Authors: Osumi Tomoo , Mori Tetsuya , Fujita Naoto , Saito Akiko M. , Nakazawa Atsuko , Tsurusawa Masahito , Kobayashi Ryoji
Published: November 2019

Introduction

Relapsed or refractory pediatric B-cell non-Hodgkin lymphoma (B-NHL) has been reported to be extremely difficult to cure. We previously conducted a retrospective study of pediatric relapsed or refractory B-NHL in Japan between 1996 and 2004 and found that the four-year overall survival rate was only 20.5% (Fujita N. e al. PBC. 2008). Rituximab, a chimeric anti-CD20 monoclonal antibody, is expected to be effective in improving the prognosis of pediatric relapsed or refractory B-NHL and was approved for use in 2003 in Japan. Here, we retrospectively assessed the treatment and prognosis of pediatric relapsed or refractory pediatric B-NHL in the rituximab era in Japan.

Methods

We collected relapsed pediatric B-NHL cases from patients enrolled in our current study, JPLSG B-NHL03 study, which was the first nationwide multicenter trial for newly diagnosed pediatric B-NHL. We collected information on treatment and outcome after induction failure or relapse.

Results

In 33 patients enrolled this study, the median age at diagnosis was 9.7 years and male predominance was observed. Nine cases were pathologically subclassified initially as diffuse large B-cell lymphoma, 11 as Burkitt lymphoma, 11 as Burkitt Leukemia and two as others. According to initial Murphy staging, most cases were in the advanced stage. The most common site of relapse was abdomen (8 cases), followed by bone marrow (8 cases) and central nervous system (7 cases). Twenty-three cases relapsed in one site while 10 did so in multiple sites. Among them, 20 relapsed within six months after initial diagnosis.

Among them, 28 patients received some rituximab combined treatment as salvage therapy. R-ICE (rituximab, ifosfamide, carboplatin and etoposide) regimen was the most popular and used in 22 patients as first-line salvage therapy. As a result, 22 patients (66.7%) achieved complete remission (CR) by some salvage therapy. 23 received hematopoietic stem cell transplantation (HSCT). Their 5-year overall survival rate was 48.5%, which was far more superior to both our previous study (Figure 1) and another on relapsed or refractory B-NHL in childhood.

In risk factor analysis for survival, rituximab combined treatment and HSCT did not influence the outcome, but achievement of CR after salvage treatment resulted in significantly better survival (68.2% vs 9.1%, p=0.001). These results suggest that the advent of rituximab induced an improvement of CR rate for relapsed paediatric B-NHL, which resulted in an improvement of the survival rate.

Conclusion

In conclusion, the prognosis of the pediatric relapsed or refractory B-NHL in a Japanese cohort remained poor but is showing improvement in the rituximab era.

Disclosures

No relevant conflicts of interest to declare.
R-EPOCH Is Superior to R-CHOP As a First-Line Regimen in De Novo DLBCL Patients with High Ki-67 Expression
Authors: Huang Jia-Jia , Jiang Wenqi , Li Zhi-Ming
Published: November 2019

Diffuse large B-cell lymphoma (DLBCL) patients with high Ki-67 expression receive limited benefits from R-CHOP (rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone) therapy. This study aims to compare the R-EPOCH (etoposide, prednisone, vincristine, cyclophosphamide, and doxorubicin) and R-CHOP regimens as first-line therapy in DLBCL patients with high Ki-67 expression. Data from 44 untreated DLBCL patients with high Ki-67 expression receiving R-EPOCH therapy were matched with those from 132 untreated DLBCL patients with high Ki-67 expression receiving R-CHOP therapy based on the International Prognostic Index (IPI: age, Ann Arbor stage, performance status, LDH level, number of extranodal sites), gender, and Ki-67 expression. In the R-EPOCH group, 42/44 patients were eligible for response evaluation. A total of 35 patients (83.3%) achieved complete remission (CR); 6 patients (14.3%) achieved partial remission (PR); and one patient (2.4%) exhibited progressive disease (PD) after 2 cycles of therapy. Patients in the R-EPOCH group presented better survival outcomes than those in the R-CHOP group (3-year overall survival [OS]: 89.9% vs. 70.2%, p=0.041; 3-year progression-free survival [PFS]: 86.6% vs. 59.7%, p=0.024). The survival superiority of the R-EPOCH over the R-CHOP regimen persisted when considering only patients of low-to-intermediate IPI risk, but it was not observed in those of high IPI risk. Our data suggest that R-EPOCH is superior to R-CHOP as a first-line regimen in DLBCL patients with high Ki-67 expression, especially in those of low-to-intermediate IPI risk.

Disclosures

No relevant conflicts of interest to declare.
Fc Gamma Rll (CD32b) Expression Affects Anti-CD20 Monoclonal Antibody Cellular Responses and Is Down Regulated in Rituximab Resistant Pre-Clinical Models
Authors: Mavis Cory , Stewart Kayle , Gu Juan J , Wallace Paul K. , Hernandez-Ilizaliturri Francisco J.
Published: November 2019

Rituximab's primary mechanisms-of-action include: antibody-dependent cellular cytotoxicity (ADCC), complement-dependent cytotoxicity (CMC), induction of apoptosis and anti-proliferation. The acquirement of phenotypic changes in cancer cells or host immune cells over time may affect rituximab responsiveness and underscores the complexity of the potential mechanisms-of-resistance to anti-CD20 monoclonal antibodies (mAbs). Laboratory studies had demonstrated the importance of FcγRIIa and FcγRIII activating receptors in the biological activity of rituximab and other monoclonal antibodies (mAbs) in cancer medicine. Recently, investigators had focused their efforts in the study of FcγRIIb function expressed primarily on normal and malignant B-cells and its role in inflammatory conditions or hematological malignancies. FcγRIIb is an inhibitory low affinity Fcg receptor capable to induce potent inhibitory (apoptotic) signals via immune receptor tyrosine-based inhibition motif (ITIM). Previously, we generated several rituximab-resistance lymphoma cell lines (RRCL) and demonstrated a global down-regulation of CD20. On the other hand, forced expression of CD20 in RRCL did not restore rituximab sensitivity suggesting the existence of additional mechanisms of resistance to mAbs targeting CD20 (Tsai et al Clin Cancer Res. 2012;18:1039-50). To this end, we evaluated the expression and significance of FcγRIIb (CD32b) in RRCL. FcγRIIb gene and protein expression levels were determined in a panel of rituximab sensitive (RSCL) or RRCL by quantitative polymerase chain reaction (qPCR) and western blotting respectively. Subsequently, we silenced FcγRIIb in RSCL using siRNA interference and evaluated changes in the capacity of several mAbs targeting CD20 (rituximab, ofatumumab, and obinotuzumab) of inducing CMC or ADCC in vitro. RRCL were found to have almost a complete loss in FcgRIIb mRNA and protein levels when compared to RSCL. In addition, silencing of FcγRIIb in RSCL decreased the capacity of rituximab (P=0.04 [ADCC] and P=0.15 [CMC]), ofatumumab (P=0.02 [ADCC] and P =0.10 [CMC]), and obinituzumab (P=0.02 [ADCC] and P<0.01 [CMC]) in inducing effective ADCC and CMC when compared to scramble transfected cells. Together our data suggests that the chronic exposure of lymphoma cells to rituximab results in the decreased expression of FcγRIIb. In addition, FcγRIIb low affinity inhibitory receptors appear to contribute to rituximab anti-tumor activity in vitro. Additional in vivo experiments are ongoing to further define the role of FcγRII in the anti-tumor activity of mAbs targeting CD20 and how it is down regulated in the context of acquired resistance to rituximab-based therapies. (Research, in part, supported by The Eugene and Connie Corasanti Lymphoma Research Fund)

Disclosures

No relevant conflicts of interest to declare.
The Efficacy and Tolerability of Generic of Imatinib in a Very Specific Group of Chronic Myeloid Leukemia (CML)
Authors: Conchon Monika
Published: November 2019

Introduction:

Most of the patients with CML started to receive a generic formulation of imatinib (IM) in May 2013, available in the Public Health in Brazil.

The lack of information about the tolerability and efficacy of generics was a concern to patients and doctors who have been treating with Glivec, the original IM from Novartis since 2002.

Objectives:

To evaluate the efficacy and tolerability of generic of IM in CML chronic phase (CP) patients who had achieved a stable major molecular response (MMR) with Glivec before May 2013.

Methods:

The absolute level of BCR-ABL transcript from peripheral blood was serially measured every 3 to 6 months by RT-qPCR. Only level variation > 0,1% expressed on the International Scale and adverse events were noted.

Results:

Forty CML-CP patients in MMR defined as BCR-ABL (IS) £ 0,1% in at least to consecutive times, who started the generic IM in May 2013 were evaluated. All of them received 400 mg of generic IM during 24 months.

Of 40 patients, 24 (60%) had no variation on sequential analysis and 13 (32,5%) had one or two variation of BCR-ABL/BCR between 0,1% to 1,16%, but re-achieved MMR.

Two patients, due to compliance issue, lost MMR. One re-achieved MMR with generic imatinib, the other one with nilotinib.

One patient presented hepatic toxicity grade 3, and was switched to dasatinib, without lack of MMR.

Overall, MMR was maintained in all but one patient (98%).

Conclusion:

In this specific MMR group, the use of generic IM was found to be safe and highly effective in maintaining MMR.

Disclosures

No relevant conflicts of interest to declare.
Clinical Relevance of Failure to Achieve Early Molecular Response in Chronic Myeloid Leukemia in Chronic Phase
Authors: Lee Ji Yun , Lim Sung Hee , Kim Hae Su , Yoo Kwai Han , Song Haa-Na , Cho Jinhyun , Park Silvia , Kim Seok Jin , Kim Won Seog , Kim Kihyun , Jang Jun Ho , Jung Chul Won
Published: November 2019

Purpose

The early molecular response (EMR, ≤ 10% BCR-ABL1 at 3 months) of tyrosine kinase inhibitor (TKI) treatment for patients with chronic myeloid leukemia (CML) in chronic phase (CP) has been reported to have strong correlation with long-term outcome. We aim to investigate the prognostic interaction of the EMR and major molecular response (MMR).

Methods

We retrospectively reviewed data for a total of 165 patients with newly diagnosed CML-CP who received TKIs (imatinib, nilotinib, or dasatinib) as first-line treatment between January 2003 and April 2013. Of the total 128 patients who were regularly monitored by peripheral blood molecular analysis, 85 had a BCR-ABL1 assessment at 3 months and were finally included in the analysis.

Results

The median age of all patients was 49 years and 87.1% received imatinib as first line treatment. High risk group by Sokal and EUTOS were 29.4% and 14.1%, respectively. Patients with EMR (n = 56, 65.9%) had a tendency to have low risk disease and to be treated with 2nd generation of TKIs. With a median follow-up duration of 53.6 months (range, 5.4-131.3), the 5-year OS, 5-year FFS, and 5-year EFS were 92.5%, 74.8%, and 68.0%, respectively. Median time to achieve MMR was 11.1 months (95%CI, 8.4 - 13.8). The outcomes at 5 year comparing patients whose BCR-ABL1 transcript levels ≤ 10% vs >10% at 3 months were as follows: OS, 92.2% (95% CI 84.9-99.1) vs 92.8% (95% CI 83.7-102.3), p = 0.819; FFS, 84.7% (95% CI, 75.6-94.4) vs 57.4% (95% CI, 39.0-75.0), p < 0.001; and EFS, 73.6% (95% CI 62.5-85.5) vs 57.8% (95% CI 40.0-76.0), p = 0.050. Six (10.7%) of 56 patients with BCR-ABL1 transcript levels ≤ 10% at 3 months failed to achieved an MMR and 18 (62.1%) of 29 patients with > 10% at 3 months achieved an MMR. Based on these heterogeneous clinical outcomes, we further explored subgroup analysis according to the achievement of MMR for refined discrimination of survival outcomes. There was no significant difference of clinical outcomes between ≤ 10% vs > 10% at 3 months among the patients who achieved MMR (OS, p = 0.376; FFS, p = 0.793; and EFS, p = 0.266). In patients who did not achieved MMR, only FFS was significantly difference between ≤ 10% vs > 10% at 3 months (OS, p = 0.489; FFS, p = 0.014; and EFS, p = 0.199).

Conclusion

Patients who failed to achieve EMR but finally reached MMR have excellent prognosis that whether we have to change TKI for EMR failure is to be addressed by ongoing prospective clinical trials.

Disclosures

Jang: Alexion Pharmaceuticals: Research Funding.
Leukocytosis Can Predict Thrombotic Events in Myelofibrosis
Authors: Vassalli Laura Coutinho , Malafaia Emilia Carolina , Chauffaille Maria L. , Kerbauy Daniella
Published: November 2019

Thrombotic events are the main complication of Philadelphia-negative chronic myeloproliferative neoplasms (MPN). In polycythemia vera (PV) and essential thrombocythemia (ET), risk factors for thrombosis are well established, such as age greater than 60 years and previous thrombosis. However, the role of JAK2 V617F mutation and leukocytosis at diagnosis as risk factor for thrombosis is still controversial. Our aim was to identify factors related to the risk for thrombotic events in the studied population.This study is a retrospective non-interventional cohort. All of the analyses were performed using the database of 142 patients with MPN regularly followed at the Hematology Division (at UNIFESP-SP) from 1992 to 2014. Diagnosis was established according to WHO criteria. We analyzed the JAK2 V617F mutation, hemoglobin (g/dL), hematocrit (%), white blood cells (x109/L) and platelets (x109/L) at the diagnosis and DIPSS-Plus risk score (International Working Group for Myelofibrosis Research and Treatment, 2009). These variables were associated with thrombotic event at any time.Of the 142 patients, 54 had diagnosis of PMF, 28 of PV, 33 of ET and 27 of post-essential thrombocythaemic myelofibrosis (post ET MF) or post-polycythaemic myelofibrosis (post-PV MF). This last group was included in myelofibrosis group for statistical purposes.

Thrombotic events were more frequent in PV patients (39.2%), followed by ET (33.3%), and PMF (20.9%).

From those which JAK2 mutation was obtained, it was positive in 92.4% of PV patients, 62% of PMF and 50% of ET. In none of the three groups, the presence of JAK2 V617F mutation was related to increased risk of thrombosis.

In myelofibrosis, leukocytosis was higher among thrombotic patients (median of 13.7 in thrombotic group versus 9.7x 109/L; p 0.0379). None of the other parameters, hemoglobin, hematocrit, platelets and DIPSS-Plus were statistically significant. In ET, the hemoglobin level at diagnosis was significantly higher in the presence of thrombosis (mean of 14.57 in thrombotic group against 13.03 g/dL in the non-thrombotic one, p 0.0428). The other parameters, hematocrit, white blood cells and platelets were not relevant. The median WBC in the thrombotic group was 9.4 and in the non-thrombotic one 9.3 x109/L. Finally, in polycythemia vera, none of the variables were related to thrombosis. Among the studied population, leukocytosis was increased in patients with thrombotic event in MF. Thus, monitoring leukocyte count in MF is essential to predict thrombosis risk and should be further studied in order to define therapeutic goals in these patients.

Disclosures

No relevant conflicts of interest to declare.
Patient-Reported Outcomes in Myelofibrosis Patients Help to Identify Patients' Needs
Authors: Ionova Tatyana , Nikitina Tatyana , Lomaia Elza , Myasnikov Alexandr , Pospelova Tatyana , Evseev Andrey , Gritsenko Taras , Lyamkina Anna , Machulaytene Elena , Usacheva Elena , Rodionova Anna , Kurbatova Kira
Published: November 2019

Myelofibrosis (MF) is the most symptomatic of the myeloproliferative neoplasms and is associated with the greatest symptom burden and poorest prognosis. Patient-reported outcomes is an effective way to identify patients' needs and risks/benefits of MF treatment. We aimed to study quality of life (QoL) and symptom burden in MF patients in a real-world setting.

44 MF patients - 27 primary MF, 8 post-essential thrombocytopenia, 9 post-polycythemia vera - were enrolled in the multicenter real-world QoL study. Mean age - 60.8±13.3; male/female - 14/30. All the patients received the best available treatment (BAT, n=28) or novel treatment modality ruxolitinib (n=16) for at least 6 months (range 6-160 mths). A high proportion of patients (80%) had intermediate to high prognostic risk scores according to International Prognostic Scoring System. All the patients completed the QoL questionnaire SF-36, symptom assessment questionnaire CSP-MF and Patient Global Impression of Change (PGIC) tool. Integral QoL Index (IQoLI) in MF patients was calculated on the basis of SF-36 and QoL impairment grade was assessed in comparing with QoL population norms (PN). Comparison t-test for independent samples or Mann-Whitney test was applied.

The heterogeneity of MF patients population in terms of QoL impairment was shown: 55% of patients had mild QoL impairment (IQoLI≤25% from PN), 7% - moderate (IQoLI≤25-50% from PN), 38% - severe or critical QoL impairment (IQoLI≤50% from PN). Patients receiving BAT exhibited more pronounced QoL impairment as compared to patients receiving ruxolitinib (p<0.05); they had worse physical functioning, general health, vitality, social functioning, and mental health (p<0.05). All the patients experienced multiple symptoms; the most severe symptoms were fatigue, inactivity and pain in bones/muscles. The symptoms were more expressed in patients on BAT as compared to patients on ruxolitinib (p<0.005). Patient's impression of health changes was better in patients treated with ruxolitinib: the mean PGIC score was higher in patients on BAT on ruxolitinib - 4.4 vs 2.3 (p=0.001).

Quality of life and perceived change in health condition are better and symptom severity is less in MF patients on ruxolitinib therapy than those on BAT. Results of this real-world study demonstrate benefits of ruxolitinib therapy from patient perspective. Patient-reported outcomes are of help to better identify the needs of MF patients.

Disclosures

No relevant conflicts of interest to declare.
The Expression of the Co-Inhibitory Molecule PD-1 and Its Ligand PD-L1 in Patients with MDS
Authors: Geng Suxia , Weng Jianyu , Huang Xin , Wu Ping , Deng Chengxin , Lai Peilong , Du Xin
Published: November 2019

Programmed death receptor 1 (PD-1) is an important immunosuppressive molecule and expresses on activated T cells, B cells and myeloid cells. PD-L1, a primary ligand PD-1, is mainly expressed on T cells and primary B cell surfaces and plays a role in the differentiation and apoptosis of these cells, induces a coinhibitory signal in activated T cells, promotes T cells apoptosis, incompetence and functional exhaustion. The expression of PD-1 is high in patients with hematologic malignancies and a high expression of PD-L1 is found on hematologic malignancies cells.

To further know the characteristics of PD-1 and PD-L1 in patients with MDS, we detected theexpression of PD-1 and PD-L1 in peripheral blood (PB) and bone marrow (BM) samples from 25 RAEB and 10 RARS patients using Real-time PCR. The PD1 and PD-L1 expression levels of 13 PB and 8 BM samples from normal individuals were as controls. The PD-1 levels of PB samples from 25 RAEB patients [42.40(4.5-173.96)%] were significantly higher than that from normal controls [32.32(19.45-41.38)%, P=0.026]. While the level of PD-1 in 10 RARS patients was comparable to that of normal controls and RAEB patients (P=0.401 and P=0.352). Compared to normal controls [23.72(3.23-39.2)%], the median PD-1 level of BM from 10 RAEB patients[36.81(12.14-151.52)%] showed an increasing tendency, but the difference was not statistically significant (P=0.062). PD-L1 expression levels of PB samples from RARS patients were significantly lower than that of normal controls (P=0.009). There were no significant difference between RAEB patients and normal controls about the PD-L1 expression level in PB and BM samples (P=0.248 and P=0.181) and between RAEB and RARS patients about PD-L1 expression level in PB samples (P=0.243). PD-1 level in BM samples from 11 RAEB patients with remission was (31.32±15.75)% and it was lower significantly than that before treatment [(59.94±47.44)%, P=0.034]. After progression or transformation, the expression level of PD1 (54.72±37.27)% increased again and was higher than that in remission (P=0.028).There were also no significant difference on PD-L1 expression among before treatment, bone marrow remission and progression or transformation (P>0.05). In 8 RAEB patients transformed to AML after treatment, PD-1 level has a decreasing tendency(P =0.05)and the change of PD-L1 has no significant difference (P>0.05).

In conclusion, PD1 mRNA level increased significantly in patients with RAEB and the changes of PD-1 was associated with the evolution of the disease after treatment with demethylating agents. The level of PD1 may be used as an indicator to determine the efficacy, but the changes of PD-L1 was not found regularity.

Disclosures

No relevant conflicts of interest to declare.
A Case Report on Coexisting JAK2 V617F and Calr exon 9 Mutation in Essential Thrombocythemia
Authors: Rashid Munazza , Ahmed Rifat Zubair , Ahmed Shariq , Nadeem Muhammad , Ahmed Nuzhat , Shamsi Tahir
Published: November 2019

Myeloproliferative Neoplasms (MPNs) are a heterogeneous group of clonal disorders derived from multipotent hematopoietic myeloid progenitors. Classic "BCR-ABL1-negative" MPNs is an operational sub-category of MPNs that includes polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF). These three disorders are characterized by stem cell-derived clonal myeloproliferation. The most common mutation in the MPNs PV, ET and PMF is JAK2 V617F.

JAK2 V617F can be detected in about 95% of patients with PV while remaining 5% of PV patients carry a somatic mutation of JAK2 exon 12. Approximately one third of patients with ET or PMF do not carryany mutation in JAK2 or MPL. In December 2013 mutations were described in calreticulin (CALR) gene in 67-71% and 56-88% of JAK2 V617F and MPL negative patients with ET and PMF, respectively. Since this discovery, CALR mutations have not only been recommended to be included in the diagnostic algorithm for MPNs, but also CALR exon 9 mutations have been recognised to have clinical utility as mutated patients have a better outcome than JAK2 V617F positive patients.CALR mutations have also been reported to be mutually exclusive with JAK2 V617F or MPL mutations. According to our knowledge so farthere have been only six reports published,which described patients harbouring concurrent JAK2 V617F and CALR exon 9 mutations; seven ET, three PMF, one PV and one MPN-U.

In the present study we are reporting ET patient with coexisting JAK2 V617F and CALR exon 9 mutations from our center. In July 2011, 55-years-old female patient was referred to our hospital with a history of gradual elevation of platelet counts accompanied with pain in right hypochondriac region and feet. Bone Marrow aspirate consisted of 'Stag-horn' appearance Megakarocytes. Multiple platelets aggregates and islands were seen throughout the aspirate smear. ARMS-PCR for JAK2 V617F mutation was positive whereas bidirectional Sanger sequencing for CALR exon 9 exhibited c.1214_1225del12 (p.E405_D408del) mutation pattern.

Disclosures

No relevant conflicts of interest to declare.
Effects of Decitabine and Homoharringtonine in Combination in Leukemia Cell Lines
Authors: Geng Suxia , Yao Han , Weng Jianyu , Huang Xin , Wu Ping , Deng Chengxin , Lu Zesheng , Du Xin
Published: November 2019

Background: The methylation inhibitor decitabine (DAC) has great therapeutic value for myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML). However, DAC monotherapy is associated with relatively low rates for overall response and complete remission. We aimed to investigate the effects of the combination of DAC and homoharringtonine (HHT) in the SKM-1 and Kg-1a cell lines and explore their associated mechanisms.

Methods: Cell viability was estimated by MTS assay. Cell apoptosis were detected by flow cytometry analysis and PI/Annexin V staining. Western blot and quantitative reverse transcription-polymerase chain reaction assays were performed to detect the expression of apoptosis-related genes including caspase-3, caspase-9, BCL-2 and BCL-XL and DNA methyltransferases(DNMT) including DNMT1, DNMT3A and DNMT3B. Changes in LINE-1 gene methylation were assessed by quantitative polymerase chain reaction after bisulfite conversion. The effects of the combinations were estimated using CalcuSyn software.

Results: The combination of DAC and HHT showed synergistic effects for inhibiting cell viability in SKM-1 and KG-1a cell lines. This combination can enhance inhibition of colony formation and apoptosis induction of DAC alone in SKM-1 cell line. However, in Kg-1a cells, this combination only enhanced the apoptosis induction of DAC alone. For SKM-1 cells, further study found that different doses of DAC and HHT alone have different effects on the expression of the apoptosis-related genes caspase-3, caspase-9, BCL-XL and BCL-2. The combination of 0.4 μM DAC and 5 nM HHT treatment significantly increased the mRNA expression of caspase-3 (P=0.0005) and caspase-9 (P=0.0075) and decreased the expression of BCL-2 (P=0.0331), but has no significantly effect on the BCL-XL (P=0.3436) compared with 0.4 μM DAC alone. However, 4 μM DAC plus 50 nM HHT had no significant effects on the mRNA and protein expression of the apoptosis-related genes examined compared with 4 μM DAC alone (P>0.05). Low-dose DAC induced greater hypomethylation than higher doses of the drug. Whereas HHT had no demethylation effects, and also had no enhancement effects with DAC on the hypomethylation and mRNA and protein expression of DNMT1, DNMT3A and DNMT3B in SKM-1 cells.

Conclusion: The combination of DAC and HHT has synergistic effects on cell viability inhibition and enhancement effects on cell apoptosis in SKM-1 and KG-1a cell lines. But this combination did not enhance the hypomethylation effect of DAC.These data suggest that DAC used in combination with HHT has clinical potential in the treatment of MDS and AML.

Disclosures

No relevant conflicts of interest to declare.
Chidamide, a Novel Histone Deacetylase Inhibitor, Displays Potent Antitumor Activity Against MDS Cells Mainly through JAK2/STAT3 Signaling Inhibition
Authors: Chang Chunkang , Zhao Sida , Guo Juan , Zhao Youshan , Fei Chengming , Gu Shucheng , Li Xiao
Published: November 2019

Background:

Many studies have indicated that histone deacetylases (HDAC) activity is always increased in a lot of human tumors, and inhibition of HDAC activity are promising new strategies in the treatment of cancers. Chidamide (CS055/HBI-8000), a novel histone deacetylases inhibitor (HDACi) of the benzamide class, is currently under clinical trials. Despite emerging information on the effect of chidamide in multiple cancers, little is yet known about mechanism of action, and there were few reports about this drug's effects on myelodysplastic syndromes (MDS). In this study, we aimed to investigate the antitumor activities of chidamide on MDS cell line SKM-1 and to explore the possible mechanism.

Methods:

We treated MDS cells with different concentrations of chidamide. The effect of chidamide on HDAC activity of MDS cells was studied by using a HDAC Fluorometric Activity Assay Kit. The induction of histone acetylation was confirmed by detecting acetylated histone H3 and H4 using Western blot analysis. The effect of chidamide on the proliferation of MDS cells was analyzed by Cell Counting Kit (CCK-8) assay. Apoptosis were detected by Annexin V/propidium iodide (PI) double-labeled cytometry. Cell cycle was analyzed by a PI method. The acetylation levels of genes promoterassociated histone H3 and H4 were examined by chromatin immunoprecipitation (ChIP) and quantitative real-time PCR. Quantitativereal-time PCR and Western blot were used to detect the expression of signaling pathway factors (SOCS3, JAK2, STAT3,Bcl-XL,Bcl-2 and Mcl-1).

Results:

Our results demonstrated chidamide suppressed MDS cells growth in a time- and dose-dependent manner, and induced cell apoptosis and cell cycle arrest at G0/G1phase. Chidamide was able to significantly inhibit the HDAC activity and increase the acetylated histoneH3 and H4 of MDS cells. ChIP analysis further indicated that chidamide induced acetylated histones accumulation in the promoter of SOCS3.Moreover, chidamide upregulated the mRNA and protein expression ofSOCS3, and also significantly downregulated JAK2/STAT3 signaling. Further, we identified reduced expression of STAT3 downstream targets with treatment of chidamide, including Bcl-XL, Bcl-2 and Mcl-1, which may explain the cytotoxic effects of chidamide on MDS cells.

Conclusion:

These results demonstrate that chidamide possesses significant cytotoxic effects on MDS cells mainly through histone modifications of SOCS3 and consequently JAK2/STAT3 signaling inhibition. Therefore, Our data provide rationale for clinical investigation of chidamide in MDS.

Disclosures

No relevant conflicts of interest to declare.
Interlaboratory Variation in CD34+ Cell Quantification in Low Risk Myelodysplastic Syndromes
Authors: Font Patricia , Subira Dolores , Matarraz Sergio , Benavente Celina , Cedena Teresa , Morado Marta , Perez Corral Ana , Bellon Jose Maria , Diez-Martin Jose Luis
Published: November 2019

Background: Accurate bone marrow (BM) blast count is essential for establishing prognosis in lower-risk myelodysplastic syndromes (MDS). Flow cytometry immunophenotyping (FCI) helps to evaluate the most immature BM cell compartment, but it is not known whether the CD34+ myeloid cell count in MDS are comparable among different observers. We investigated the FCI approaches used for enumeration of CD34 + myeloid cells in MDS in the routine practice in 6 clinical Spanish centers, and then explored if data were reproducible among participants.

Methods: In this prospective study, participants exchanged 47 FCI files from MDS BM samples with <5% blasts, and quantified the CD34+ myeloid cells in a blind manner. No agreement regarding cytometers, number, reagents, fluorochromes and events recorded were previously established.

Results: The mAb combinations selected to identify BM myeloid CD34+ cells was highly variable: CD45 was included in 45/47 (95.7%) files, CD34/CD45/CD117 in 37 (78.7%), and HLA-DR in 19 files (40.4%). The FCI assays used in every centre were quite different, but a very good concordance among the 6 FCI observers for CD34+ cell enumeration was observed with the intraclass correlation coefficient (0.720). Considering the specific analysis of each sample, 2 groups were distinguished. Twenty-five cases (53.2%) exhibited the highest concordance, with an interlaboratory coefficient of variation (CV) <30%, or a difference among observers <0.5%. In the remaining 22 cases, the concordance among participants was low. Comparison of the characteristics of high and low agreement assays showed no statistical differences. In order to determine whether the lower concordance obtained among observers after the analysis of these 22 samples could be improved, we designed a protocol with specific guidelines with gating strategies for identification of the CD34+ myeloid cells in several consecutive steps. The strategy was designed for samples with the CD34/CD45/CD117 mAb combination (n=15), with or without HLADR. Using this strategy, the interlaboratory CV improved in 7/15 cases (46.66%), but no improvement was obtained in the remaining 8 cases. Finally, the percentage of CD34+ myeloid cells was reproducible in 32/47 cases (68%) diagnosed with low risk MDS. Conclusions: Despite different methodology, accuracy among observers in CD34 cell quantification is feasible. Selection of appropriate protocols and targeted training would help to enhance the reproducibility of the technique among different observers. However, the intrinsic heterogeneity of these cells in MDS might justify the difficulty for their identification in some cases.

Disclosures

No relevant conflicts of interest to declare.
Cytogenetics of Patients with Myelodysplasic Syndrome in Venezuela
Authors: Vizcaino Jennifer , De Salvo Cardullo Luigi , Salas-Auvert Rodofo J , Hassanhi Manzur , De Salvo Dalila del V , Herrera-Merchan Humberto C
Published: November 2019

The myelodysplastic syndromes are clonal hematological disorders of the stem Cell, and clinically characterized by ineffective hematopoiesis and morphological dispoyesis. The affected patients have a high risk of leukemic transformation. In a total of (n=416) patients studied, (n=247) patients (59.3%) corresponded with a female gender, with an average age of = 59 years (limits 15-99 yrs), and (n=169) male patients (40.6%), with average age of =62 years (limits 09-89 yrs). In (n = 295) studied patients, 71% had normal karyotype 46, XX or 46, XY, where in 16% (n = 66) of the samples cell growth was not attained. In (n = 54) of patient samples, 13% showed karyotype changes, including those frequently reported for MDS; and 11.1% (n = 6) with -Y, 11q 1.8% (n = 1), del20q 3.7% (n = 2), +8 3.7% (n = 2), - 21 1.8% (n = 1), del 7q 1.8% (n = 1), inv3 1.8% (n = 1), complex karyotypes 12.9 % (n = 7) cases. Other alterations include, simple anomalies: +2 1.8% (n = 1), +13 5.5% (n = 3), del17q 1.8% (n = 1), del4p 1.8 %(n=1), del9q 3.7% (n = 2), der21q 3.7 %(n = 2), del17p3 3.7% (n = 2), t (1; 7) 1.8% (n = 1), t (4; 12) 1.8% (n = 1), rob13 1.8% (n = 1); 14, r2 1.8%(n = 1); double anomalies, delXq and del10q (n = 1), del18p and del13q (n = 1). Among other findings, polymorphisms in (n=15) patients (27.7%), such as 1qh + (n = 2), 9qh + (n = 4), 16qh + (n = 3), 14PS + (n = 1), 15ps + (n = 2) , 22ps + (n = 1), inv9 (n = 2). According with IPSS-R karyotypes, samples were classified as very favorable 12.9% (n = 7), pro 3.7% (n = 4), Intermediate 40.7% (n = 22), 1.8% unfavorable ( n = 1) and very favorable 18.5% (n = 12.9).

Disclosures

No relevant conflicts of interest to declare.
Phenotypic Characteristics of Tumor Cells in Patients with Chronic Lymphocytic Leukemia into Different Prognostic Groups
Authors: Kuvshinov Aleksei , Voloshin Sergei , Martynkevich Irina , Martynenko Ludmila , Garifullin Andrei , Kleina Elizaveta , Abdulkadyrov Kudrat
Published: November 2019

Background. The presence or absence of certain cluster of differentiation on the tumor cells of chronic lymphocytic leukemia may affect the course of the disease. Influence of genetic abnormalities on the prognosis of the disease was also proved.

Aim. To determine the relationship of the phenotype of tumor cells with genetic prognostic groups of patients with chronic lymphocytic leukemia (CLL).

Methods. Thirty-five adult pts (median age 61 year, range 44 - 82; male 24, female 11) with diagnosed CLL were included in the study. The CLL was diagnosed according to the standard basic examination (complete blood count with differential, multicolor flow cytometry (MFC) of blood and bone marrow (BM), lymph node and BM immunohistochemistry (IHC), computered tomography). Cytogenetic studies were performed on blood samples using standard GTG-method. Interphase FISH analyses were performed according to the manufacturer's protocol using DNA probes: LSI 13(RB1)13q14, LSI ATM (11q22), CEP12, LSI TP53 (17p13.1) (Abbott). Immunophenotype (IFT) of CLL cells assessed with combinations: CD3/CD19, CD19/CD5, CD19/CD11c, CD19/CD20, CD19/CD22, CD19/CD23, CD19/CD25, CD19/CD38, CD19/CD43, CD19/CD81, CD19/HLA-DR, and CD19/CD5/CD23.

Results. Stratification of patients into prognostic groups was performed based on identified GA. Favorable prognosis - patients with del(13q) (n = 9); neutral prognosis - normal karyotype (n = 14) or trisomy of chromosome 12 (n = 4); unfavorable prognosis - del(17p) (n = 3), del(11q) (n = 3) and the complex karyotype (n = 2).

Expression of CD20 was lower, and CD38 - higher in adverse group (51.0±16.31 % and 36.02±10.35 %, respectively) versus neutral or favorable groups (CD20+ - 83.17±5.52 % and 84.41±4.7 %; CD38 - 10.46±4.8 %, and 12.44±4.1 %, respectively, p <0.05). The expression level of CD20 and CD38 did not differ between the neutral and favorable groups.

The number of patients with CD38 expression more than 10% was higher in the unfavorable group (7/8) versus favorable (4/8) (p <0.05). At the same time overexpression of CD38 was observed more frequently in patients with a lack of expression of CD23 on CD5 tumor cells (CD5+CD23-) (p<0.05).

Expression of HLA-DR was higher in patients with MRD-negative remissions (3/4) versus patients with MRD-positive remissions (1/8) (p<0.05).

Conclusions. The study of the influence of various factors on the prognosis and course of CLL requires a comprehensive approach. Further researches are needed to determine the relationship between CLL affecting factors like genetic abnormalities, phenotypic characteristics of the tumor cells and MRD.

Disclosures

No relevant conflicts of interest to declare.
The BET Protein BRD2 Cooperates with CTCF to Enforce a Transcriptional Boundary in Erythroid Cells
Authors: Hsu Sarah C. , Bartman Caroline , Stonestrom Aaron J , Edwards Christopher R , Gilgenast Thomas , Emerson Daniel , Huang Peng , Werner Michael T. , Jahn Kristen , Keller Cheryl A. , Giardine Belinda , Hardison Ross C. , Raj Arjun , Phillips-Cremins Jennifer E. , Blobel Gerd A.
Published: November 2019

Pharmacologic inhibitors of the bromodomain and extraterminal motif (BET) family of proteins have shown promise in the treatment of hematologic and other malignancies and are being developed for clinical use. However, BET inhibitors do not discriminate between the family members BRD2, BRD3, and BRD4, and thus the mechanistic basis for their therapeutic efficacy is not well understood. In addition, BRD2 and BRD4 are individually required for the activation of genes driven by the erythroid transcription factor GATA1 (Stonestrom et al., Blood 2015), yet how BRD2 in particular contributes to this process has not been studied. We examined BRD2 occupancy genome-wide in erythroid cells and find that BRD2 colocalizes extensively with the architectural/insulator protein CCCTC-binding factor (CTCF). We define a functional hierarchy whereby CTCF is required for BRD2 to occupy co-bound sites, while CTCF binding is BRD2-independent. Using CRISPR/Cas9-based genome editing we identify a boundary element occupied by CTCF and BRD2 that is adjacent to a GATA1-driven enhancer and ensures appropriate transcriptional regulation at the locus. Employing single-molecule RNA FISH we show that either site-specific CTCF disruption or BRD2 depletion leads to increased correlation in mature mRNA levels between the genes flanking this boundary, suggesting that they become inappropriately coregulated. Taken together these findings indicate that BRD2 collaborates with CTCF to constrain the activity of an erythroid enhancer and reveal a potential new role for BET proteins in chromatin domain boundary function.

Disclosures

No relevant conflicts of interest to declare.
FANCA Fine-Tunes Chromosome Segregation By Controlling BUBR1K250 Acetylation at the Kinetochores
Authors: Sater Zahi Abdul , Sharma Richa , Sierra Potchanant Elizabeth , He Ying , Nalepa Grzegorz
Published: November 2019

Fanconi anemia (FA) is an inherited bone marrow failure syndrome associated with genomic instability, high risk of acute myeloid leukemia (AML) and other malignancies. Somatic mutations within the FA/BRCA signaling network occur in AML in the general population, reflecting the importance of FA genes in tumor suppression. While the role of FA signaling in DNA damage repair and replication is well-established, we and others found that the FA network is essential for error-free chromosome segregation during cell division. Both interphase and mitotic errors contribute to the evolution of genomic instability during FA-/- human and murine hematopoiesis in vivo. However, the molecular mechanisms of FA pathway-dependent genome housekeeping during mitosis are incompletely understood.

Through a synthetic lethal kinome-wide shRNA screen in FANCA patient cells, we discovered interphase and mitotic phosphosignaling networks that FANCA-/- cells depend on for survival, including the BUB1-BUBR1 axis of the spindle assembly checkpoint (SAC). BUB1 and BUBR1 are essential SAC kinases that prevent premature anaphase onset and chromosome mis-segregation by inhibiting the APC (anaphase-promoting complex) ubiquitin ligase at the centromeres until all kinetochores achieve correct attachment to the spindle microtubules. Our super-resolution microscopy and biochemistry experiments revealed that FANCA shuttles to kinetochores upon mitotic entry and physically interacts with BUB1 and BUBR1 at the kinetochore-microtubule attachment sites in attachment- and tension-dependent manner. Consistent with impaired SAC, we found that that anaphase onset as well as APC-mediated degradation of cyclin B1, BUBR1 and CDC20 all occur prematurely in FANCA-/- cells. We found that FANCA is essential for BUBR1 lysine 250 (K-250) acetylation at prometaphase kinetochores, and we confirmed that endogenous BUBR1K250 acetylation is disrupted in FANCA-/- primary patient cells using a validated acetyl-specific antibody. BUBR1K250 acetylation event works as a molecular switch in which BUBR1 is converted from a degradation target to a potent inhibitor of the APC ligase. Further, we observed that loss of FANCA disrupts kinetochore recruitment of the BUBR1K250 acetyltransferase PCAF and its upstream regulator, FANCD1/BRCA2.

Our findings establish the first mechanistic connection between FANCA, the canonical SAC tumor suppressor cascade and the FA effector FANCD1/BRCA2. These findings further our understanding of the mechanisms of genomic instability and carcinogenesis resulting from loss of FA signaling. Since impaired BUBR1K250 acetylation causes chromosomal instability and cancer in vivo, our results have a direct translational relevance.

Figure. Figure.

Disclosures

No relevant conflicts of interest to declare.