bluuebunny/crossref_metadata_2025 huggingface dataset:
Feasibility of a Cell-Based Oncolytic Virotherapy Targeting Bone Marrow Lesions of Multiple Myeloma
Authors: Sato Kota , Futami Muneyoshi , Chit Ung Weng , Nakamura Takafumi , Tojo Arinobu
Published: October 2019

Virotherapy is a potentially promising modality to treat intractable malignant tumors that are resistant to conventional chemotherapy and molecularly targeted therapy. From a viewpoint of clinical application, vaccinia virus (VV) has several advantages over other oncolytic viruses, such as a short life cycle, potent tissue penetration and robust lytic activity. We showed that intravenous injection of miRNA (let-7a)-regulated and thymidine kinase-deleted VV (MDVV) efficiently infects and kills multiple myeloma cells with much less adverse effects on normal cells and tissues in SCID mice carrying a subcutaneous xenograft (Blood 2014 124:2082). However, in another xenograft model carrying bone marrow (BM) myeloma lesions, MDVV failed to reduce tumor burden or prolong survival probably due to its poor penetrance to BM. Then, for an alternative strategy, we tested the feasibility of a cell-based virus delivery which targets BM lesions using myeloma cells infected ex vivo with MDVV.

A human myeloma cell line KPMM2 is absolutely dependent on interleukin-6 or a murine stromal cell line such as HESS-5 for its continuous growth and survival (Blood 1997 90:2437). It recapitulates BM myeloma lesions and IgG paraproteinemia when transplanted into SCID mice intravenously. Furthermore, KPMM2 cells are highly susceptible to and efficiently killed by MDVV. First, we tested whether KPMM2 cells can serve as MDVV carriers to cause secondary infection. MDVV was originally labeled by F-luc, and KPMM2 cells were transduced with R-luc (target: T), then seeded onto a HESS-5 monolayer. Twenty-four hrs later, MDVV-infected KPMM2 cells (effector: E) were added at various E/T ratios. Dual F/R-luc assay showed that target cell viability was significantly reduced at E/T ratio ≥ 1 along MDVV propagation. This lytic effect was dependent on time and a multiplicity of infection (MOI), but not perturbed by the co-culture with cord blood mononuclear cells (bystander). Next, we validated the possibility that MDVV-infected KPMM2 cells can deliver MDVV to BM after intravenous injection into SCID mice. Most of KPMM2 cells show high level expression of CXCR4, a critical regulator of myeloma cell homing to BM, and retain comparable CXCR4 expression even after MDVV infection. Actually, 48 to 72 hrs after 1x107 MDVV-infected KPMM2 cells (labeled by F-luc) were injected into SCID mice, in vivo imaging analysis detected the accumulation of prominent F-luc signal at the BM following early phase trap in the lung (Figure). Taken together, the present cell-based strategy is a unique way to enable targeted delivery of MDVV to BM and application of oncolytic virotherapy to hematological malignancies, especially multiple myeloma. The therapeutic efficacy of MDVV-infected KPMM2 cells on BM myeloma lesions in SCID mice is currently under investigation.

Figure Figure.

Disclosures

No relevant conflicts of interest to declare.
A 5-Day Cytoreductive Chemotherapy Followed By Haplo-Identical HSCT (FA5-Bucy) As a Tumor-Ablative Regimen Improved the Survival of Patients with Advanced Hematological Malignancies
Authors: Yang Ting , Ren Jinhua , Lin Qiaoxian , Yuan Xiaohong , Luo Xiaofeng , Liu Tingbo , Zheng Xiaoyun , Chen Zhizhe , Hu Jianda
Published: October 2019

Background: Haploidentical hematopoietic stem cell transplantation (haplo-HSCT) has been used when HLA-matched siblings are not available. Conditioning regimens aim to reduce tumor burden prior to HSCT and provide sufficient immunoablation.

Patients and Interventions: We report the outcome of haplo-HSCT in 63 consecutive patients (19 females/44 males) with high-risk or relapsed/refractory hematological malignancies (n=29-AML; 8-sAML; 19-ALL; 5-advanced-MDS; 2-CML-BC), followed at our Center from 2/2013 to 12/2015. Median age was 20 years (range: 1.1-49). Twenty-one patients achieved remission prior to transplant, while 42 did not (7%-98% BM blasts). No patient had severe infection or organ failure before HSCT. Patients received FA5-BUCY registered on http:// ClinicalTrials.gov (NCT02328950), i.e., 5-day salvage chemotherapy (Fludarabine/Ara-C) and conditioning (Busulfan/Cyclophosphamide). GvHD prophylaxis included ATG, CsA, MMF and short-term MTX.

Results: All patients received stem cells from bone marrow and peripheral blood, and achieved successful engraftment, except two who died before. With a median follow-up of 269 days (120-1081), 42/63 patients are still alive and disease-free. Two-year OS and RFS were similar in patients not in remission and in those in complete remission (61.3% vs 56.3%, p=0.88; 58.3% vs 56.3%, p=0.991). Non-relapse mortality and relapse incidence were 22.2% and 11.1%, respectively. Severe acute-GvHD occurred in 4/63 patients. Transplant-related mortality was low at day+100 (17.5%) and for the entire study period (20.6%). Unexpectedly, few patients experienced mild-to-moderate toxicity, and main causes of death were infection and GvHD. BM blast counts, age,and donor-recipient gender-pairs did not affect the outcome. Less chemotherapy cycles prior to HSCT might result in more favorable outcome.

Conclusions: Thus, haplo-HSCT with FA5-BUCY appears promising for advanced disease, especially when TBI and amsacrine, used for FLAMSA, are not available and in pediatric patients for whom TBI is not recommended.

Two-year OS and RFS in patients not in remission prior to HSCT compared to those in complete remission. The two-year OS and RFS in patients not in remission were similar to those from patients in complete remission (61.3% vs 56.3%, p=0.88; 58.3% vs 56.3%, p=0.991). Two-year OS and RFS in patients not in remission prior to HSCT compared to those in complete remission. / The two-year OS and RFS in patients not in remission were similar to those from patients in complete remission (61.3% vs 56.3%, p=0.88; 58.3% vs 56.3%, p=0.991).

Disclosures

No relevant conflicts of interest to declare.
Preclinical Characterization of TTI-281, a Novel, Orally Bioavailable BET Inhibitor: Predictive Biomarker Identification and Combination Treatment Analysis
Authors: Choi Jaehyun , Wang Chunlei , Wang Zezhou , Schimmer Aaron D. , Petrova Penka S. , Slassi Malik , Uger Robert A.
Published: October 2019

Small molecule inhibitors of the bromodomain and extraterminal domain (BET) proteins are being actively tested in clinical trials for many cancers, but patient selection strategies remain poorly defined. We have previously demonstrated TTI-281 is an efficacious BET inhibitor in multiple hematological malignancies models. Here, we present candidate biomarker genes that predict TTI-281 activity in vitro. Furthermore, we assessed the preclinical activity of TTI-281 in combination with 17 other drugs and found five compounds that improve TTI-281 potency.

We first determined the GI50 values for TTI-281 in 19 hematological cell lines. These GI50 values were then correlated with publicly available basal mRNA expression data for 58 candidate genes. Two novel candidates that predict the sensitivity of cancer cells to TTI-281 were identified: BCLAF1 (prediction accuracy: 83.3%) and HAPLN2 (prediction accuracy: 88.9%). Restricting the analysis to leukemia cells and including a third biomarker candidate gene (BCL2L1) improved the prediction accuracy to 100%.

Next, 17 drugs that are either approved or in development for the treatment of hematological malignancies were tested in combination with TTI-281. Five drugs were identified that significantly improved the potency (1.5-3.5 fold decrease in GI50) or maximum inhibitory effect (1.7-3.1 fold increase in maximum efficacy) of TTI-281 on multiple leukemia cells: azacitidine, everolimus, idelalisib, nilotinib, and vorinostat. However, the effects of combining TTI-281 with these five drugs were not observed in all hematological tumor cells, thus further studies are required to investigate the molecular mechanisms by which TTI-281 synergizes with other drugs.

In summary, the BET inhibitor TTI-281 is a promising anti-tumor agent with novel predictive biomarker candidates, capable of synergizing with azacitidine, everolimus, idelalisib, nilotinib, and vorinostat. Our preclinical data provide a strong rationale for further validating the current predictive biomarkers for TTI-281 in a clinical setting and probing the mechanism by which TTI-281 achieves synergy when combined with other anti-cancer agents.

Disclosures

Choi: Trillium Therapeutics: Employment. Wang:Trillium Therapeutics: Employment. Wang:Trillium Therapeutics: Employment, Patents & Royalties. Schimmer:Novartis: Honoraria. Petrova:Trillium Therapeutics Inc: Employment, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties. Slassi:Trillium Therapeutics: Employment, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties. Uger:Trillium Therapeutics: Employment, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties.
Liposomal Delivery of a Phosphodiesterase 5 Inhibitor Enhances Prostacyclin-Mediated Adenosine Triphosphate Release from Human Red Blood Cells
Authors: Bowles Elizabeth , Sprague Randy , Ercal Nuran
Published: October 2019

Pulmonary arterial hypertension (PAH) is characterized by high pulmonary vascular resistance (PVR) and right heart failure. Red blood cells (RBCs) of PAH patients fail to release the vasodilator, adenosine triphosphate (ATP), when deformed as would occur when traversing the lung. Such a defect could contribute to increased PVR. However, RBCs of PAH patients do release ATP in response to prostacyclin (PGI2) analogs and this is augmented by phosphodiesterase 5 (PDE5) inhibitors. Current PAH treatment includes PGI2 analogs and PDE5 inhibitors alone or in combination. Unfortunately, these drugs can have untoward side effects. Encapsulation of drugs within liposomes (small lipid-membraned vesicles) that can be targeted to RBCs has been shown to increase effectiveness and tolerability of some medications.

The objective of this study was to determine if encapsulation of the PDE5 inhibitor zaprinast (ZAP) within liposomes is an effective means to deliver this class of drugs to human RBCs and if this approach would augment ATP release stimulated by the PGI2 analog UT-15c. Human RBCs were isolated and incubated with either blank liposomes (n=10), liposomes containing ZAP (n=9) or liposomes containing the PDE3 inhibitor, cilosatzol (CILO, n=10). RBCs (20% hematocrit) were then treated with UT-15c (1 µM). ATP release was measured before and 5, 10, and 15 min after the addition of UT-15c. In the presence of empty liposomes, the dose of UT-15c used did not stimulate ATP release. However, UT-15c did stimulate ATP release from RBCs pretreated with ZAP encapsulated by liposomes (P<0.01). The average time for maximal release was 9 ± 1 min. In contrast, when exposed to liposomes containing CILO, there was no ATP release following UT-15c administration.

These studies demonstrate that the selective delivery of a PDE5 inhibitor to human RBCs potentiates UT-15c induced ATP release. Moreover the findings are consistent with the hypothesis that directed delivery of this class of drugs to PAH RBCs could be a new and important method to augment PGI2 analog-induced ATP release from these cells. Such an approach could significantly limit side effects of both drugs without compromise of their therapeutic effectiveness in PAH.

Disclosures

No relevant conflicts of interest to declare.
A Prospective Study of Thyroid Function Status in Patients of Haemoglobin E Beta Thalassemia and Correlation with Serum Ferritin Level
Authors: Dolai Tuphan Kanti , Baul Shuvra Neel , Mandal Prakas Kumar , De Rajib , Chakrabarti Prantar
Published: October 2019

Introduction

Haemoglobin E-beta-thalassaemia (EBT) represent approximately 50 per cent of those affected with severe beta thalassemia.The highest frequencies are observed in India, Bangladesh and throughout Southeast Asia. Endocrinopathies are now amongst the common complications of thalassaemia and it is multifactorial in origin. Iron overload in EBT is also multifactorial. This study was undertaken to evaluate the thyroid dysfunction in patients of EBT and its correlation with serum ferritin levels.

Methods

EBT patients were evaluated prospectively to assess thyroid dysfunction status and correlate it with serum ferritin levels. High performance liquid chromatography was performed with Bio-rad beta thalassaemia short program variant II. Serum ferritin estimation was done with microplate immunoenzymometric assay and thyroid assay for TSH, free T4 and T3 were done by access 2 Immunoassay System, Beckman Coulter.

Results

50 patients with EBT were evaluated. The mean age of patients were 19.7 years (range: 12-47). There were 28 males and 22 females. There were 41(82%) and 9(18%) transfusion dependent and transfusion independent patients respectively and 40(80%) were on chelation therapy. In this cohort 22(44%) patients had thyroid dysfunction. Six (12%) and 16(32%) of patients were having hypothyroidism and subclinical hypothyroidism respectively. Mean ± Standard deviation (S.D) of serum ferritin level with hypothyroidism, subclinical hypothyroidism and euthyroidism was 1077 ± 371.8 ng/ml ,1422 ± 1361.0 ng/ml and 1252 ± 664.4 ng/ml respectively with correlation coefficient =0 [Fig 1 and Fig 2]. Serum ferritin levels do not predict thyroid dysfunction in patients of EBT. Male and female have equal preponderance for thyroid dysfunction. Patients with subclinical hypothyroidism were having few symptoms compared to frank hypothyroidism and majority of symptoms in either scenario were masked by thalassemia itself. Earlier age of onset of EBT is associated more with thyroid dysfunction however the association is not statistically significant (p=0.2). There was no association between spleen size and thyroid dysfunction (p=0.7).

Conclusions

Thyroid dysfunction was seen in 42% of EBT patients. Prevalence of hypothyroidism was found to be higher in EBT patients compared to general population but a definite correlation with the serum ferritin levels could not be established.

Figure 1 Mean serum ferritin level and thyroid status Figure 1. Mean serum ferritin level and thyroid status Figure 2 Correlation of serum TSH and ferritin level (correlation coefficient =0) Figure 2. Correlation of serum TSH and ferritin level (correlation coefficient =0)

Disclosures

No relevant conflicts of interest to declare.
Average Chronic Pain Medication Needed in Adult Patient with Sickle Cell Disease and Its Relation to Vitamin D Level
Authors: Master Samip , Patan Shajadi , Cingam Shashank , Shi Runhua , Mansour Richard Preston
Published: October 2019

Introduction: The chronic pain in sickle cell disease (SCD) arises from chronic bone damage as a consequence of bone marrow infraction during vaso-occulsive events. There are certain barriers to adequate pain management in adult patients with SCD, namely, limited knowledge among the clinicians, inadequate assessment, concerns about addiction, and biases against opioid use. We did retrospective analysis to investigate the average pain medication needed by adult patient with SCD. We also did analysis to see if there was a relation between plasma vitamin D level and amount of pain medication needed.

Methods:

We take care of approximately 300 active adult SCD at Hematology clinic at our institute. We did a retrospective analysis of 458 adult patients with SCD seen at our clinic between 2001 and 2016. We collected data on type of SCD, plasma 25 -hydroxyvitamin d level and amount of opioid pain medication in mg. To get uniform units of opiates, we converted all the different opiates into morphine.

Results:

The average morphine dose in a 24 hours period needed to manage chronic pain in an adult patient with SS type was 84 mg with standard deviation of 72, for SC type was 60 mg with standard deviation of 72 and for sickle beta thal type was 72 mg with standard deviation of 71. There were 4 patients with SS with hereditary persistence of hemoglobin F and average opiate dose in them was 84 mg.

We obtained vitamin d level on 223 patients and out of them, 47 had vitamin d level of <4.2 ng/ml (lowest level reportable by our lab). We also found negative correlation between amount of pain medication and vitamin D level. The spearman correlation coefficient was -0.2 and p value was <0.01.

Conclusion:

Because we were unable to find any previous reports of the correlation of vitamin D levels and opiate use in an adult population with SCD, we believe that this is the first study to report this correlation. It also provides a rough estimate regarding average amount of opiates that an adult patient with SCD needs. This correlation between Vitamin D levels and opiate requirement supports our current practice of screening all patients for Vitamin D deficiency using 25hydroxy vitamin D levels and treating all patients who are found to be deficient. We do not know if this Vitamin D replacement will reduce pain, bone health or the amount of opiate medication needed in the future.

Disclosures

No relevant conflicts of interest to declare.
Tumor Metastasis in VWF Deficient Mice: The Contributions of PGE2 and VEGF
Authors: Sosa Iberia Romina , Chava Kankana , Guillory Bobby , Cohen Daniel N
Published: October 2019

Dysregulation of the hemostatic system in malignancy leads to a hypercoagulable state and thrombotic complications. In addition to thrombosis, growing evidence from both human and animal studies, suggests that constituents of the hemostatic system also play an important role in tumor progression and metastasis. Platelets contribute to metastasis by protecting tumor cells from immune detection and inducing epithelial-mesenchymal transition, thereby facilitating the extravasation of circulating tumor cells out of the vasculature. Moreover, platelet-tumor cell interactions have been shown to promote prostaglandin E2 (PGE2) synthesis, an important modulator of angiogenesis and proliferation. The role of von willebrand factor (VWF) in malignancy has been of particular interest as it serves as an adhesive link between platelets and endothelium, and could possibly facilitate platelet interactions with tumor cells. Yet, the absence of VWF is associated with increased metastatic potential in a B16 melanoma mouse model, suggesting that the primary contribution of VWF to the metastatic model may not be platelet mediated. We employed a combined approach using cell lines and mouse models to explore the interaction of platelets and VWF in metastasis.

In vitro studies using lung and melanoma cells lines show that the interaction of resting and activated platelets with tumor cells leads to upregulation of COX-2 expression and PGE2 biosynthesis but does stimulate VEGF-A production. In an experimental metastasis model of C57BL/6 wild type and VWF null mice, the injection of B16 melanoma cells by tail vein yielded a significant increase in the number of pulmonary metastatic foci. PGE2 measurements from mouse serum showed reduced levels of prostaglandin in VWF deficient mice injected with tumor, suggesting that the absence of VWF precludes an important tumor cell-platelet interaction that facilities PGE2 production. Moreover, preliminary results show increased VEGF-A in VWF deficient mice, suggesting that increased metastasis in this model may be the result of dysregulated angiogenesis.

In conclusion, platelet-tumor cell interaction yield a pro-metastatic effect, in part mediated by PGE2 production. Despite its unique role in platelet adhesion and thrombus formation, VWF does not facilitate the interaction of platelets and tumor cells to produce PGE2. Its protective effect may lie in its ability to modulate angiogenesis via VEGF-specific pathways.

Disclosures

No relevant conflicts of interest to declare.
Dynamics of Hemorragic Events and Primary Hemostasis Deffects during Treatment with Ibrutinib. Real-Life Experience
Authors: Acedo Natalia , Loscertales Javier , Aguado Beatriz , Paz Yizel , Jimenez-Barral Elena , Arriero Alvaro , Alegre Adrian
Published: October 2019

INTRODUCTION: Ibrutinib is a first-in-class Bruton kinase inhibitor (BTK) registered for the treatment of Chronic Lymphocytic Leukemia (CLL), mantle cell linphoma (MCL) and Waldenström Macroglobulinemia (WM).

Increased risk of bleeding events is asociated with ibrutinib, mostly grade ≤2. Alleged mechanism for this risk could be the role of BTK in platelet activation via GPIb, GPVI and FVW.

We here present our experience regarding hemorragic events and hemostasis tests alterations in CLL patients (pts) treated with ibrutinib.

PATIENTS AND METHODS: 15 pts with relapsed CLL were started on ibrutinib between december/2012 and july/2016. Median age was 69 years (range: 42-83 years). 12 of them were male. Median exposure to the drug was 17 month (range 1- 38).

After 9 to 36 months from initiation of treatment (median 22 months) 6/15 were thouroughly interviewed about personal history of bleeding events and the following laboratory work was performed: factor VIII level, FVW antigen and activity, platelet function analysis (PFA-100TM) with ADP and epinephrine, and aggregation studies with colagen, ADP and arachidonic acid. Along these tests all patients had blood counts with platelets more than 100 x 10e9/L.

RESULTS: 3 out of 15 pts had bleeding events (grade 1 or 2), 2 consisting in spontaneus bruising and 1 in upper grastrointestinal bleeding, in the first weeks of treatment. 4 pts underwent minor invasive procedures with no complications, witholding the drug at least 3 days before and after. 2 pts were on concomitant antiplatelet (1) or anticoagulant (1, LMWH, due to AF) and had no bleeding events.

All pts tested (6/6) had normal or slightly elevated leves of F VIII, FVW:Ag and FVW:Ac. 2 pts had abnormal PFA-100TM and abnormal aggregations with any agonist. One case had normal PFA-100 TM while aggregations with colagen and AA were mildly altered.

COMMENTS: Our limited experience shows a moderate risk of minor bleedings consisting with a platelet disfunction drug-driven effect. Despite the fact that we have no baseline tests done, alterations in primary hemostasis test, including PFA-100TM and specially aggregometry could be expected early on after exposure to ibrutinib.

Disclosures

Loscertales: Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Gilead Sciences: Honoraria, Membership on an entity's Board of Directors or advisory committees; Roche: Honoraria, Speakers Bureau. Alegre:Janssen: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees.
Outcomes of Venous Thromboembolism (VTE) Treatment in Thrombocytopenic Leukemic Patients
Authors: Khan Maliha , Nassif Mohammed , Alzubaidi Mohanad , Cox Travis , Oo Thein H. , Garg Naveen , Rojas-Hernandez Cristhiam Mauricio
Published: October 2019

BACKGROUND

The current standard of care for the treatment of cancer-related VTE is low molecular heparin. Treatment of VTE in leukemia is complicated as patients suffer severe thrombocytopenia during the different phases of their malignant disease. The optimal treatment of cancer-related VTE in this population has still not been widely studied. Therefore, there is a dire need for research to provide evidence-based guidance on the optimal management of cancer related-VTE in leukemia population.

OBJECTIVES

The primary objective of the study was to estimate the recurrence rate of VTE (proximal lower extremity, pulmonary embolism or both) at 6 months from the institution of VTE-related treatment. Secondary objectives included overall survival, recurrent VTE event-free survival, and rate of complications attributed to therapies for VTE.

METHODS

A retrospective chart review of adult patients with thrombocytopenic leukemia patients who suffered VTE and were treated at MD Anderson Cancer Center between January 2002 and March 2016. Demographic, clinical, and VTE treatment-related data were collected. Primary and secondary outcomes were compared among the different VTE treatment-modalities using non-parametric methods. Kaplan-Meier curves were calculated and compared between the groups of VTE-treatment modalities using Log-Rank test. All comparisons were performed with a test significance of 0.05.

RESULTS:

At the time of abstract submission, a total of 101 patient-data have been analyzed. The most common type of leukemia was AML (2/3 of all cases). Study outcomes were compared between three groups: anticoagulation only, inferior vena cava filter only and no VTE-treatment intervention. We found no statistically significant differences in the demographic and clinical characteristics between the three groups, except for a lower median platelet count in the patients in whom no VTE-treatment intervention was performed (p<0.05). The VTE recurrence rate was low (<5%) and not different between the intervention groups. The median overall survival of the general population was poor (< 6 months) and significantly worse in patients who did not receive anticoagulation therapy for the management of VTE (p<0.01).

CONCLUSION:

Our study is the largest cohort reported to date on management of VTE in thrombocytopenic leukemia patients. Our preliminary data indicated that anticoagulation therapy and non-pharmacologic interventions for the management of VTE seemed to be safe and equivalent for the secondary prevention of recurrent VTE in that population. Future prospective cohort studies will likely confirm these findings.

Disclosures

No relevant conflicts of interest to declare.
Markers of Coagulation and Angiogenesis Can Serve As Surrogate Biomarkers for Disease Activity and Survival in Cancer Patients
Authors: Beleva Elina Angelova , Deneva Tanya , Popov Veselin , Grudeva Janet G.
Published: October 2019

Based on observations of the link between hemostatic system and malignancy it is plausible to consider that plasma coagulation factors could serve as easily accessible markers for detection of malignant state and monitoring of cancer patients. Therefore, we hypothesized that tissue factor (TF), angiopoietin-2 (Ang-2) and soluble urokinase plasminogen activator receptor (suPAR) might perform as diagnostic and predictive biomarkers for the presence of malignancy as well as for the development of cancer-related events: metastatic progression and death.

To analyze their performance TF-associated microparticle activity (MPTF), tissue factor antigen (TFAg), Ang-2 and suPAR were determined in 128 cancer patients with various cancer types receiving chemotherapy by sequential measurements (up to four times) at prespecified time points incorporated into interim and end-of-treatment analysis. MPTF activity was measured with Zymuphen MPTF kit, TFAg, suPAR and ANG-2 were quantitated by ELISA. Patients were followed up for progression free survival (PFS) from therapy initiation until occurrence of a combined event defined as either metastatic progression or death. In order to determine the predictive ability of hemostatic biomarkers for a PFS event Log-rank (Mantel-Cox) test was applied. ROC analysis was used to determine the cut off values for detection of an event, cut off values were transformed into binary variables and Kaplan-Meier curves were constructed. Cox regression analysis was applied to explore quantitatively the effect on the risk of progression.

Results: Cut off values for the occurrence of an event were MPTF < 0.5 pg/ml (AUC 0.591), TFAg (√ pg/ml) > 8.28 (AUC 0.603), Ang-2 > 302.3 pg/ml (AUC 0.615) and suPAR (√ pg/ml) > 2.83. Shortened PFS was associated with decreased MPTF procoagulant activity and elevated plasma levels of TFAg, Ang-2, suPAR. As significant predictors for PFS were determined only TFAg (RR 4.57, p=0.038), Ang-2 (RR 2.138, p=0.031) and suPAR (RR 2.238, p=0.049).

Conclusion: TFAg, Ang-2 and suPAR might be identified as significant adverse predictors of metastatic progression or death in cancer patients and risk is increased with elevated plasma levels.

Disclosures

No relevant conflicts of interest to declare.
Mechanism of Action of Hydroxychloroquine in the Antiphospholipid Syndrome
Authors: Müller-Calleja Nadine , Manukyan Davit , Ruf Wolfram , Lackner Karl
Published: October 2019

Objective. The antiphospholipid syndrome (APS) is characterized by thromboembolic events and/or recurrent abortions in the presence of pathogenic antiphospholipid antibodies (aPL). Hydroxychloroquine (HCQ) is recommended for thromboprophylaxis in patients with systemic lupus erythematosus and aPL. We aimed at identifying mechanisms responsible for the beneficial effects of HCQ in the APS.

Methods. Monocytic cells were stimulated with human aPL in vitro and expression of tissue factor (TF) and tumor necrosis factor (TNFα) was quantified by qRT-PCR. For intracellular ROS detection, cells were stained with OxyBurst and analyzed by flow cytometry. Live cell imaging was performed by confocal laser scanning microscopy to show translocation of NADPH oxidase 2 (NOX2) and TLR8 on stimulation. Finally, the effect of HCQ was tested in an in vivo model of venous thrombosis.

Results. HCQ prevents the induction of TF and TNFα by aPL in MonoMac1 cells, by blocking the activation of endosomal NOX2. This latter effect is not mediated by direct interference with the previously described uptake of aPL into the endosome but by inhibiting the recruitment of the catalytic subunit of NOX2 (gp91phox) into the endosome. Furthermore, HCQ protects mice from aPL induced and NOX2 mediated thrombus formation in vivo.

Conclusion. We propose that interference with the assembly of endosomal NOX2 is a major effect of HCQ responsible for its antithrombotic effect in the APS. Since most if not all APS patients harbor aPL that activate endosomal NOX2 this probably explains the well established efficacy of HCQ in APS patients.

Disclosures

No relevant conflicts of interest to declare.
Insight into Donor Deferral Pattern Based on Peripheral Blood Counts: An Experience from Southern Pakistan
Authors: Irfan Syed Mohammed , Sultan Sadia , Abbas Syeda Alia , Ashar Sana
Published: October 2019

Introduction:

American association of blood banking (AABB) recommends screening of all potential donors by a copper sulphate technique, but this method has chances of false acceptance as well as false deferral. Additionally others valuable blood parameters remain undetermined. Yet this is practically applicable in developed countries with low prevalence of anemia and community infections. In Pakistan anemia's secondary to nutritional deficiency or infectious diseases are much common. It makes the application of this crude method questionable. With this background we determined the whole peripheral blood counts by automated hematology analyzers in all prospective blood donors.

This is to provide the pre-donation deferral rate of the healthy blood donors based on peripheral blood counts. The basic knowledge about frequency, types and severity of anemia among donors will help to plan a strategy to promote donor recruitment and overall national health.

Methods:

Prospective records of all the reported donors were collected from January-2014 to December-2015 at Liaquat National Hospital, Karachi.

Results:

Overall 36954 potential donors reported to the blood bank, out of which 33853 were selected and 3101 were deferred, which makes the deferral rate of 8.39%. Of total, 264 (0.71%) donors were excluded based on history whereas 174 (0.47%) donors were excluded due to examination findings while majority [n=2663 (7.20%)] of donor were deferred based on complete blood counts.

Based on peripheral blood counts; anemia (91.8%) represents the major cause of deferral, followed by raised total leukocytic count (3.7%) and polycythemia (3.3%) respectively and thrombocytopenia (1.0%) was the least potential cause. Microcytic-hypochromic anemia was found in 58.5% donors followed by normocytic and macrocytic anemias in 38.9% and 2.4% respectively. Mild anemia was seen in 78.2% followed by moderate and severe anemias in 20.5% and 1.18% respectively.

Conclusion:

In view of frequently anemic donors in the present study, it is strongly recommended to have a full blood counts rather to rely on semi-quantitative methods which led to acceptance of anemic donors.

High prevalence of anemia among Pakistani blood donors signifies deteriorating health status not only in donor population but also in general population as well. Astonishingly high TLC (sub clinical infections) turned out as second important cause of deferral. This situation recalls for strenuous efforts to overcome as it entails that we will likely to face more dearth of donors in future.

Disclosures

No relevant conflicts of interest to declare.
Prognostic Significance of IDH1 and IDH2 mutations in Older Patients with Primary Cytogenetically Normal Acute Myeloid Leukemia
Authors: Wang Jinghan , Ma Zhixin , Wang Qinrong , Yu Mengxia , Yin Xiufeng , Li Xia , Suo Shanshan , Shen Dan , Wang Yungui , Huang Xin , Pan Hanzhang , Wang Huanping , Chen Zhimei , Huang Jiansong , Mao Liping , Yu Wenjuan , Wei Juying , Qian Wenbin , Mai Wenyuan , Meng Haitao , Tong Hongyan , Chen Suning , Marcucci Guido , Chen Saijuan , Jin Jie
Published: October 2019

IDH1 and IDH2 mutations are frequently identified in older patients with AML.With the development and application of IDH inhibitor, the prognostic significance of these mutations in old patients is of primary importance. To assess the impact on clinic outcome, we analyzed 224 older (> 60 years) patients with primary cytogenetically normal acute myeloid leukemia (CN-AML) similarly treated with intensive induction chemotherapy. Our analysis identified IDH1 and/or IDH2 (IDH1/2), IDH1 or IDH2 mutations in 63(28%), 29(12.9%) and 35(15.6%) patients, respectively. In general, IDH1 and IDH2 mutations were mutually exclusive, with only one patient having both IDH1 R132C and IDH2 R140Q mutations. IDH2 R172 mutations were exclusive with all other mutations analyzed (NPM1, CEBPA, DNMT3A, IDH1 and FLT3-ITD). There was a strong interaction between IDH2 mutation and NPM1 mutational status. Concurrent presence of IDH2 R140 and NPM1 mutations was independently associated with longer overall survival (OS; P=0.038, HR=0.42) and event free survival (EFS; P=0.034, HR=0.46) compared to the wild type counterpart in multivariable analyses. In contrast, IDH1 mutations were independently associated with shorter OS (P=0.004, HR=1.98) and EFS (P=0.014, HR=1.72) in multivariable analyses. Notably, IDH1 R132 mutations harbored higher levels of total 2-hydroxyglutarate compared to IDH2 R140Q mutations. Patients with IDH1 mutations or IDH2 R140 and NPM1 mutations harbored also distinct miRNA-expression signatures. Future studies will establish whether these difference between IDH1 and IDH2 mutations will be relevant with respect to response of the respective subsets of patients to the emerging therapies with IDH1 and IDH2 inhibitors.

Disclosures

No relevant conflicts of interest to declare.
Early Deaths in Pediatric Acute Leukemia; A Challenge in Developing Countries
Authors: Hafez Hanafy Ahmed , Solaiman Rawaa , Bilal Dalia , Shalaby Lobna m
Published: October 2019

Background and objectives: The survival rates of children with acute leukemia is consistently improving, due to the lower relapse rates in addition to reducing treatment-related mortality, which is mainly a result of infectious causes. The aim of the study is to describe the incidence and risk factors associated with early deaths (first 42 days in treatment) among children with acute leukemia. Methods: This is a retrospective study included newly diagnosed patients with acute leukemia who presented to the National Cancer Institute, Cairo University between Jan. 2011 to Dec. 2013. Patients' data were collected from their files and analyzed for the total and early death rates and proposed causes of death. Results: The study included 370 patients, 253 with acute lymphoblastic leukemia (ALL), 100 with acute myeloid leukemia (AML) and 17 with mixed phenotypic acute leukemia (MPAL). The total death rate among the whole group was 40.5% (n=150) and induction death rate was 19.2% (n=71). AML was accompanied with higher rates of total and induction deaths as they were 58% and 25% respectively, compared to 33.6% and 17.4% in ALL. These early deaths were attributed mostly to infection 64.7% (n=46) and cerebrovascular accidents 18.3% (n=13). Early deaths were significantly higher in patients with age below 2 years old (p. value=0.008), and in those with poor response to therapy (p. value= 0.001). Using enhanced supportive care measures as better infection control, appropriate antibiotic guidelines and available intensive care unit during 2013 had significantly reduced the overall and induction mortality rates (27.8% and 13.8% respectively in 2013 versus 45% and 20.3% in 2011 and 49% and 25% in 2012). Conclusion: Induction deaths in pediatric acute leukemia remain a major challenge in developing countries and constitute an increasing fraction of all deaths. Accordingly, using a well equipped cancer centers with better supportive care guidelines is essential to further improve the survival in these group of patients.

Key words: Acute Leukemia- Pediatrics- Early Death- Infection

Disclosures

No relevant conflicts of interest to declare.
Clinical Significance of Myeloblasts Percentage in the De Novo Acute Myeloid Leukemia with Erythroid Hyperplasia
Authors: Qiu Shaowei , Wei Hui , Lin Dong , Zhang Guangji , Wei Shuning , Zhou Chunlin , Liu Kaiqi , Wang Ying , Liu Bingcheng , Liu Yuntao , Gong Benfa , Gong Xiaoyuan , Feng Sizhou , Mi Yingchang , Han Mingzhe , Wang Jianxiang
Published: October 2019

Objects: Investigate the clinical significance of myeloblasts percentage in the de novo acute myeloid leukemia(AML) with erythroid hyperplasia from a single center of China

Methods: We retrospectively collected information regarding clinical characteristics and survival data from de novo101 patients with newly diagnosed AML with erythroid hyperplasia (> 50% erythroid cells) from 2002-2016. The total patients were divided into two groups according to the myeloblasts percentage in total marrow cells. Cases with<20% total myeloblast was defined as low-blast group while cases with>20% total myeloblast was defined as high-blast group. Median follow-up of patients was 10.03 months.

Results:1.A total of 101 patients were diagnosed as de novo acute myeloid leukemia with erythorid hyperplasia according to WHO2008 criteria, 70 patients was classified as low-blast group, 31 patients was classified as high-blast group. Compared with high-blast group, in the low-blast group, the median age was older (39.8 vs 33.3, p=0.044), male was predominant (71.4% vs 48.4%, p=0.01). But complete remission (CR) after induction chemotherapy was lower in high-blast group(44.4% vs 57.4%, p=0.019).There were no difference in the white blood count(WBC), chromosome karyotype and incidence of various gene mutations.

2. The median survival time in the low-blast group and high-blast group was 39.19 months and 23.43 months respectively. 3-year overall survival(OS) was 53.1% and 43.4%(p=0.668), 3-year disease free survival(DFS) was 71.4% and 47.8% (p=0.297).

3. Univariable analysis showed that OS of 101 AML patients was correlated with prognosis subtype, remission of induction chemotherapy, transplant and relapse, and not related with myeloblast percentage exceed 20% of total morrow cells or not, WBC, age, gender and induction regimens. DFS of the all patients was correlated with the remission of induction chemotherapy and prognosis subtype. Multivariable analysis showed that OS was only correlated with remission of induction chemotherapy and transplant, DFS was only correlated with remission of induction chemotherapy.

Conclusions: In the AML patients with erythroid hyperplasia with<20% total myeloblast, the age was older and CR was higher than that with >20% total myeloblast, but the survival was similar in both patients regardless of myeloblast percentage. The patients who attained CR after induction chemotherapy and received transplantation would get better outcome.

Disclosures

No relevant conflicts of interest to declare.
Monitoring AML Disease Status with Next Generation Sequencing
Authors: Liu Zach , Dimov Nikolay
Published: October 2019

Recent advance in next generation sequencing (NGS) have confirmed that AML is a heterogeneous malignancy harboring may many genetic mutations. These mutations have been studied for leukemia genesis, diagnosis and therapeutic targets. Monitoring minimal residual diseases has also been studied recently. We summarized our experience with NGS in morning AML disease status.

NGS data during 2014 and 2016 from patient with newly diagnosed or AML and/or AML follow-up patients along with bone marrow biopsy, FISH/cytogenetics, flow cytometric results were reviewed. Targeted sequencing was performed with customized panel (34 genes) on Ion PGM platform from Life Technology Inc.

41 AML patients with complete bone marrow work-up with bone marrow morphology, flow cytometry, FISH/cytogenetics (MFFC) and NGS were collected. At least one sample with complete work-up for each patient was included. Majority of the patients had several studies (2-8 samples). 15 out of 41 (36.6%) has complete remission based on bone marrow morphology, flow cytometry, FISH/cytogenetic studies. No mutations were detected among these 15 patients. 17 patients (41%) showed concordant result with other technologies, i.e. when the patient was in remission based on MFFC, No mutations were detected. When patient had recurrent AML or residual disease, mutations were detected. It worth to point out that 2 patients showed positive mutation without detectable increase in myeloblasts. These 2 patients had relapsed AML within 3 months. Different subclones were detected at different intervals in 1 patient. 2 (0.5%) patients (1 with newly diagnosed AML and 1 with early recurrent AML) showed no detectable mutations. Mutations were detected in 5 patients (12%) with AML remission by MFFC, additional follow-up is need for these patients. The most common mutations included TET2, ASXL1, DNMT3A, RUNX1, IDH1 and TP53.

NGS is valuable to assess the AML status despite of heterogeneous genetic abnormalities. Although the NGS results were concordant with bone marrow morphology, FISH/cytogenetics and flow cytometry in most of the cases (87.5%), persistent mutations may be detectable in cases without detectable residual AML by other modalities, which may be associated with minimal residual disease or early relapse, and need further evaluation. Clonal evaluation may occur at molecular level occasionally.

Disclosures

No relevant conflicts of interest to declare.
Fusion Protein Ompc-CD154 Inhibits Proliferation and Induces Apoptosis in B-NHL Cell Lines By a Mechanism That Involves MAPK P38 and TRAF2 Activation
Authors: Pantoja-Escobar Gerardo , Romo-Dorantes Laura , Morales-Martinez Mario , Vega Gabriel G , Martinez-Maza Otoniel , Huerta-Yepez Sara , Vega Mario I.
Published: October 2019

CD40 is a member of the TNF super family, which is expressed mainly in B cells, dendritic cells and macrophages, and the interaction with its ligand CD154 is essential for the development of the immune response. The activation or selective inhibition of this pathway is part of various therapeutic strategies for the treatment of diseases with inflammatory or immune origin, as well as various types of tumors. The fusion protein OmpC-CD154 expresses 10 amino acids of CD154 (Ser149-Trp140) and is capable of binding to CD40, inducing apoptosis and inhibiting proliferation in NHL cell lines. In this study we demonstrate that the fusion protein OmpC-CD154 induced apoptosis and inhibition of proliferation in several B-NHL cell lines. In addition, we analyzed the mechanisms involved in the induction of apoptosis and the inhibition of proliferation. Our results by Western blot demonstrate that OmpC-CD154 induced degradation of Bcl-6 by a MAPK p38 activation dependent mechanism. Pre-treatment of Ramos and Raji cell lines with a specific chemical p38 inhibitor reverted the effect of OmpC-CD154 in proliferation. Our results showed that the fusion protein can also inhibit the constitutive activation of both the canonical and non-canonical NF-kB pathways, and this inhibition was associated with the activation of the TRAFs pathway, as it promotes TRAF2 phosphorylation, and the decreasing of the expression of TRAF3. All these effects were greater than those observed with recombinant human CD154 on B-NHL cell lines. The present findings establish that a CD154 peptide fused with a protein exhibits significant cytotoxicity on B-NHL cells. Current studies are evaluating fusion proteins with CD154 peptide that are not antigenic in vivo and that have significant anti-tumor effects, both direct and on the host immune response, with minimal toxicity.

Disclosures

No relevant conflicts of interest to declare.
Prognostic Significance of Serum LDH in B Cell Chronic Lymphoproliferative Disorders: A Single-Institution Study of 829 Cases in China
Authors: Liu Huimin , Xiong Wenjie , Li Heng , Lv Rui , Liu Wei , Yi Shuhua , Li Zengjun , Qiu Lugui
Published: October 2019

Background: B-cell chronic lymphoproliferative disorders (B-CLPD) comprise several indolent lymphoma subtypes with similar characteristics but also difference and heterogeneity. Several prognostic systems had been established for separate subtypes, such as Rai/Binet stage system for CLL, FLIPI for follicular lymphoma. Here, we want to explore the prognostic role of some common clinical characteristics in each lymphoma subtype, such as serum LDH.

Materials and methods: The clinical data of 829 patients with B-CLPD including clinical features, LDH expression level, cytogenetic abnormalities and therapeutic outcome were analyzed retrospectively in department of lymphoma & myeloma, Institute of hematology & Blood disease hospital from April 1990 to August 2013, and the prognosis of B-CLPD patients with different expression levels of LDH was compared, including overall survival (OS) and progression-free survival (PFS) rates.

Results: 829 patients were divided into three groups, including 426 (51.4%) patients with chronic lymphocytic lymphoma (CLL), 307 (37.0%) patients with non-CLL B-CLPD and 96 (11.6%) patients with BLPD-U. Elevated serum LDH at diagnosis was detected in 112 (26.3%), 85 (27.7%) and 23 (24.9%) of CLL, non-CLL B-CLPD and BLPD-U group, respectively. Patients with elevated LDH B-CLPD had poorer 5-year overall survival rate (CLL: 63.8% vs 83.0%; non-CLL B-CLPD: 62.2% vs 78.1%; BLPD-U: 47.4% vs 88.6%) and 5-year progression-free survival rate (CLL: 52.0% vs 78.2%; non-CLL B-CLPD: 40.8% vs 75.6%; BLPD-U: 48.9% vs 89.5%) than LDH normal patients. Univariate analysis showed that LDH expression, hemoglobin level, platelet count, B symptoms, P53 deletion were prognostic factors for CLL patient survival, while LDH expression, platelet count, albumin level affected non-CLL B-CLPD patient survival significantly and LDH expression, albumin level, ECOG PS scores for BLPD-U patient survival. Multivariate analysis showed that elevated LDH was an independent significant prognostic factor for overall survival (P<.001) and progression-free survival (P<.001) in all three groups.

Conclusion s: Based on these data, we conclude that elevated serum LDH at diagnosis is an unfavorable prognostic factor in patients with B-CLPD.

Disclosures

No relevant conflicts of interest to declare.