bluuebunny/crossref_metadata_2025 huggingface dataset:
Bio-Inspired Artificial Red Blood Cell: Design, Pre-Clinical Results and Novel Indications
Authors: Doctor Allan
Published: November 2019

In blood, the primary role of RBCs is to transport oxygen and to regulate regional blood flow distribution, via highly regulated mechanisms. In the setting of exsanguinating haemorrhage or severe anemia, RBC loss may critically restrict tissue oxygenation and result in morbidity and mortality. In such cases, transfusion of whole blood or RBCs can significantly improve survival. However, blood products including RBCs have limited availability and portability and present additional challenges related to type matching, pathogenic contamination risks, and short shelf-life. These issues lead to substantial logistical barriers to their pre-hospital use or in austere battlefield and remote civilian conditions. To date, efforts to develop hemoglobin (Hb) based oxygen carriers (HBOCs) have failed, because of design flaws which do not preserve physiologic interactions of Hb with: O2 (they capture O2 in lungs, but do not release O2 effectively to tissue) and nitric oxide (NO) (they trap NO, causing vasoconstriction). While robust efforts are underway to resolve these issues, recent research breakthroughs have led to bio-inspired engineering of RBC surrogates, using various cross-linked, polymeric and encapsulated Hb in the form of 'bio-synthetic RBC surrogates'. These 'next-generation' Hb-based oxygen carriers (HBOCs) can potentially provide therapeutic oxygenation when whole blood or RBC are not available. This talk will comprehensively review both historical and new HBOC designs, including current state-of-the-art and novel bio-inspired artificial RBC designs in development, along with a critical discussion of successes and challenges in this field.

Disclosures

Doctor: Biogen: Membership on an entity's Board of Directors or advisory committees; Fresenius Kabi: Honoraria; KaloCyte, Inc.: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties; Etiometry: Membership on an entity's Board of Directors or advisory committees.
Three-D Codes in the Human Genome
Authors: Aiden Erez Lieberman
Published: November 2019

Stretched out from end-to-end, the human genome -a sequence of 3 billion chemical letters inscribed in a molecule called DNA -is over 2 meters long. Famously, short stretches of DNA fold into a double helix, which wind around histone proteins to form the 10nm fiber. But what about longer pieces? Does the genome's fold influence function? How does the information contained in such an ultra-dense packing even remain accessible?In this talk, I describe our work developing 'Hi-C' (Lieberman-Aiden et al., Science, 2009; Aiden, Science, 2011) and more recently 'in-situHi-C' (Rao & Huntley et al., Cell, 2014), which use proximity ligation to transform pairs of physically adjacent DNA loci into chimeric DNA sequences. Sequencing a library of such chimeras makes it possible to create genome-wide maps of physical contacts between pairs of loci, revealing features ofgenome folding in 3D. Next, I will describe recent work using in situ Hi-C to construct haploid and diploid maps of nine cell types. The densest, in human lymphoblastoid cells, contains 4.9 billion contacts, achieving 1 kb resolution. We find that genomes are partitioned into contact domains (median length, 185 kb), which are associated with distinct patterns of histone marks and segregate into six subcompartments. We identify ∼10,000 loops. These loops frequently link promoters and enhancers, correlate with gene activation, and show conservation across cell types and species. Loop anchors typically occur at domain boundaries and bind the protein CTCF. The CTCF motifs at loop anchors occur predominantly (>90%) in a convergent orientation, with the asymmetric motifs "facing" one another. Next, I will discuss the biophysical mechanism that underlies chromatin looping. Specifically, our data is consistent with the formation of loops by extrusion (Sanborn & Rao et al., PNAS, 2015). In fact, in many cases, the local structure of Hi-C maps may be predicted in silicobased on patterns of CTCF binding and an extrusion-based model. Finally, I will show that by modifying CTCF motifs using CRISPR, we can reliably add, move, and delete loops and domains. Thus, it possible not only to "read" the genome's 3D architecture, but also to write it.

Disclosures

No relevant conflicts of interest to declare.
Ex Vivo Drug Sensitivity Screens Identify Personalized Treatment Options for CLL Patients
Authors: Skånland Sigrid Strand , Henrik Bendiksen , Cremaschi Andrea , Thimiri Govinda Raj Deepak Balaji , Munthe Ludvig Andre , Tjønnfjord Geir Erland , Taskén Kjetil
Published: November 2019

In the era of targeted therapies, it remains a challenge to determine the optimal treatment for the individual cancer patient. By screening the tumor cells for drug sensitivity ex vivo it is possible to predict to which drugs and drug combinations the cancer will respond. We have developed an ex vivo culture protocol that mimics the chronic lymphocytic leukemia (CLL) tumor microenvironment and allows cell proliferation. Using our unique method, we performed drug sensitivity screens on B cells from 22 CLL patients and 5 matched healthy donors. The drug library consisted of 70 single drugs and 39 drug combinations that are clinically relevant for treatment of CLL, including venetoclax (Bcl-2 antagonist), ibrutinib (Btk inhibitor) and idelalisib (PI3K inhibitor). B cells were cultured in 384-well plates in the presence of drugs covering a concentration range of 5 log10 increments. Drug sensitivity was assessed after three days using the CellTiter-Glo luminescent cell viability assay. The Drug Sensitivity Score (DSS) was then calculated for each treatment based on area under the concentration-response curve. We observed specificity towards CLL cells for drugs including TAK-659 (SYK inhibitor), TGR-1202 and duvelisib (PI3K inhibitors), trametinib (MEK1/2 inhibitor) and venetoclax. Ten combinations with venetoclax were investigated, and all showed high drug sensitivity and specificity towards CLL cells. When ranking the drug combinations from high to low DSS, CLL selectivity and obtained synergy, combinations of venetoclax with trametinib, ibrutinib or duvelisib showed the highest potency. Of interest, similar effects were not observed for venetoclax in combination with other Btk or PI3K inhibitors. In order to understand the mechanisms underlying these differences, signaling analyses by use of phospho flow is currently ongoing. In summary, by applying drug sensitivity screens, we have identified drugs and drug combinations that kill CLL cells. These findings should be investigated further in animal models and in precision medicine clinical trials.

Disclosures

Tjønnfjord: Mundipharma: Research Funding.
Predictive and Prognostic Factors Associated with the Incidence and Mortality of Hematologic Malignancies
Authors: Deloughery Emma P. , Shatzel Joseph J
Published: November 2019

Background:

Identifying individual and systemic risk factors are key to reducing cancer incidence and improving mortality. Unfortunately for hematologic malignancies, few clear-cut common cause-and-effect relationships exist. Multiple myeloma has been associated with Black race and obesity, and the lymphomas and leukemias differ in incidence with age. In order to identify potential risk factors, in this study we compared incidence and mortality rates among U.S. states for myeloma, all leukemias and lymphomas, and correlated these rates with the prevalence of several lifestyle and demographic characteristics.

Methods:

Data came from the following sources: cancer incidence and mortality data from 1999-2015 came from the CDC WONDER database; race and poverty prevalence from the 2015 American Community Survey; gross domestic product (GDP) data from the Bureau of Economic Analysis; median household income from the Census Bureau; and weight and physical activity data from the Behavioral Risk Factor Surveillance System survey. Spearman's rank correlation coefficient was used to compare cancer incidence/mortality and potential risk factors.

Results:

Myeloma incidence and mortality were more common in obese individuals (incidence, rho = 0.27, P = 0.05; mortality, 0.38, P = 0.006) and individuals identifying as Black (0.65, P < 0.001; 0.42, P = 0.002). Incidence was lower in physically active individuals. (-0.51, P < 0.001).

Leukemia incidence and mortality were more common in individuals identifying as white (0.45, P = 0.001; 0.36, P = 0.009). Mortality was positively associated with obesity (0.39, P = 0.005), and negatively associated with physical activity (-0.36, P = 0.009) and median household income (-0.39, P = 0.004).

Lymphoma incidence and mortality were more common in white individuals (0.38, P = 0.006; 0.40, P = 0.004). Incidence was negatively associated with state poverty percentage (-0.40, P = 0.003). Mortality was positively associated with obesity (0.35, P = 0.01), and negatively associated with a healthy weight (-0.35, P = 0.01).

Discussion:

This data suggests that weight and activity are modifiable risk factors for a variety of hematologic cancers, while race may be a non-modifiable risk factor. Individual and state wealth do not appear to play a large role in incidence or mortality. Although further study is needed, these results underscore the importance of maintaining a healthy weight and regular physical activity in cancer prevention. This data also confirms the higher incidence of myeloma in Black individuals and suggests lymphoma and leukemias may be more common among those identifying as white.

Disclosures

Shatzel: Aronora, Inc.: Consultancy.
Modified Conditioning Regimen with Idarubicin Prior to Autologous Hematopoietic Stem Cell Transplantation in B-Cell Non-Hodgkin Lymphoma
Authors: Tian Chen , Li Yueyang , You M. James , Zhao Zhigang , Zhang Yizhuo , WANG Yafei
Published: November 2019

High-dose chemotherapy followed by autologous hematopoietic stem cell transplantation (auto-HSCT) has become a standard consolidation treatment for patients with invasive lymphoma. The combination of cyclophosphamide, carmustine, and etoposide (CBV) is a commonly used conditioning regimen prior to auto-HSCT for patients with invasive lymphoma. Many modifications of the CBV regimen, including the addition of idarubicin, have been employed, with varying results. We retrospectively analyzed the clinical characteristics and treatment outcomes of 36 patients with invasive B-cell non-Hodgkin lymphoma treated with a conditioning regimen prior to auto-HSCT between January 2015 and June 2018. For our analysis, the patients were divided into two groups: one group received the classic CBV conditioning regimen and the other group received idarubicin at a dose of 8 mg/m2 for 3 days in addition to the CBV regimen. Overall survival (OS), median progression-free survival (PFS), adverse reactions, and hematopoietic reconstitution time were compared between the two groups. OS and PFS were analyzed using the Kaplan-Meier method, and the log-rank test was used for comparison between groups. Cox regression was used for multivariable analysis of other clinical factors. The median follow-up time was 29 months. Among the 36 patients in the cohort, 19 were male and 17 were female, with a male-to-female ratio of 1.12 to 1. The median age of onset was 39.5 years; 18 patients were older than 40 years and 18 were younger than 40 years. Twenty-six patients (72%) had an international prognostic index score of 0-2 and 10 patients (28%) had a score of 3-5. According to Ann Arbor staging criteria, 9 patients (25%) had stage I-II disease and 27 patients (75%) had stage III-IV disease. A total of 21 patients achieved complete remission and 15 patients achieved partial remission before transplantation. There were no significant differences between the groups in terms of neutrophil counts (P = 0.795) or platelet reconstitution time (P = 0.551). Also, there was no difference in adverse reactions between the two groups, suggesting that the addition of idarubicin to the conditioning regimen did not aggravate adverse reactions. OS and PFS were significantly longer in the idarubicin group than among patients who did not receive idarubicin. In the multivariable analysis, the use of idarubicin and complete remission state before auto-HSCT were associated with improved prognosis. These results indicate that the use of idarubicin with the CBV conditioning regimen prior to auto-HSCT is a safe and effective choice for patients with invasive B-cell non-Hodgkin lymphoma.

Keywords: B-cell non-Hodgkin lymphoma; conditioning regimen; autologous hematopoietic stem cell transplantation; idarubicin

Disclosures

No relevant conflicts of interest to declare.
Management of Primary Gastrointestinal Non-Hodgkin Lymphoma with Lenalidomide
Authors: Tian Chen , Li Yueyang , Wang Yafei , You M. James
Published: November 2019

Primary gastrointestinal non-Hodgkin lymphoma is a heterogeneous malignancy with limited data to guide management. We analyzed the clinical characteristics and treatment outcomes of patients with primary gastrointestinal non-Hodgkin lymphoma treated with or without lenalidomide in a single-center patient cohort. The Kaplan-Meier method was used for survival analysis. The log-rank test was used to perform univariate survival analysis, and Cox multivariable analysis was carried out to analyze significant (P < 0.05) factors from the univariate analysis. The cohort included 219 patients, of whom 140 (64%) had primary gastric lymphoma and 79 (36%) had primary intestinal lymphoma. Most tumors (205; 93%) were composed of predominantly B-cell lymphoma; diffuse large B-cell lymphoma was the most common pathologic type for both primary gastric lymphoma and primary intestinal lymphoma. Univariate analysis showed that lesion location, age, clinical stage, and sex were not associated with overall survival, but MYC rearrangement, tumor cell type, international prognostic index score, and malignant degree were associated with prognosis. Multivariable analysis showed that MYC rearrangement and tumor cell type were independent prognostic factors. The OS and PFS of patients with MYC rearrangement were shorter than that of patients without MYC rearrangement indicating that MYC rearrangement was associated with decreased survival. Neither overall survival nor progression-free survival differed between patients who received chemotherapy with surgery and those who received chemotherapy without surgery, but patients who received surgery alone had a poor prognosis. Surgery was mainly conducted to relieve tumor-related complications or to make the diagnosis. For patients with invasive B-cell lymphoma with MYC rearrangement, the addition of lenalidomide (25 mg daily, days 1-21) to chemotherapy were given. Both of the OS and PFS of patients with lenalidomide were prolonged compared with patients without lenalidomide indicating that lenalidomide could improve the prognosis of patients with invasive B-cell lymphoma with MYC rearrangement.

Keywords: primary gastrointestinal lymphomas, lenalidomide, prognosis

Disclosures

No relevant conflicts of interest to declare.
Evaluating Thrombocytopenia and Other Biomarkers in Multi-Organ Failure during Sickle Cell Crisis
Authors: Shanmugasundaram Krithika , Higgins Ixavier , El Rassi Fuad A , McLemore Morgan L.
Published: November 2019

Background: In sickle cell anemia, very few markers of disease have been shown to correlate with activity and severity of crisis. We aimed to identify any patterns amongst biomarkers and co-morbidities that correlate with death from multi-organ failure when admitted for sickle cell crisis.

Methods: We identified 20 patients with sickle cell anemia who died of multi-organ failure at Grady Memorial Hospital from 2010-2016. We described baseline characteristics, such as hemoglobin phenotype, age, gender as well as features at presentation such as vital signs, laboratory data, clinical signs and symptoms (I.e. worsening pain), and co-morbidities. We examined bivariate associations between clinical characteristics and the length of survival after multi-organ failure. In a secondary analysis, we investigated clinical and demographic features that most accurately predict infection with gram-negative bacteria. To achieve this, we fit a multivariate logistic regression model and perform leave-one-out cross validation to assess the predictive performance of the fitted model on unseen data.

Results: At baseline, the average age of a patient is 43.8 years (sd=14.7 years). A majority of our cohort are women (60%), do not smoke (70%), and were not on hydroxyurea (80%). Time to death from multi-organ failure was 3.05 days (95% C.I.: 0.20, 5.90), and only 43% identified having "worsening pain." Among complications, 23% had a stroke and 25% had an infection with gram-negative bacteria. With regards to laboratory data, average WBC was 13.4 (95% C.I.: 11.04, 15.70), Platelets 155 (95% C.I.: 102.22, 208.38), d-dimer 12,892 (95% C.I.: -2498.47, 33437.87) and ferritin 3108 (95% C.I.: 795.07, 5421.63). When using a univariate analysis, we identified that stroke (p=0.008), age (p=0.022) and platelet count at the time of presentation (p=0.039) correlated with time to death. We also identified a statistically significant decrease in the platelet count from baseline to time of multi-organ failure (-105.6, p<0.001). Body mass index (BMI) and worsening pain jointly predict infection with gram-negative bacteria with small error (0.133).

Conclusion: We have described a cohort of patients with sickle cell anemia who died of multi-organ failure and found that some factors may have correlated with time to death. The most poignant factors include age, platelet count, and drop in platelet count from baseline. It is also interesting that a proportion of these patients developed a gram-negative bacterial infection, which is a common cause of mortality in sepsis. Although our cohort is small, these data may help guide future studies with larger cohorts of patients to identify what puts these patients at risk of death from multi-organ failure.

Disclosures

No relevant conflicts of interest to declare.
Anti-Leukemic Efficacy of Talazoparib and APE1 Inhibitor III Combined with Decitabine in Myeloid Malignancies
Authors: Kohl Vanessa , Brendel Susanne , Flach Johanna , Kleiner Helga , Weiss Christel , Seifarth Wolfgang , Nowak Daniel , Hofmann Wolf-Karsten , Fabarius Alice , Popp Henning D.
Published: November 2019

Malignant hematopoietic cells of myelodysplastic syndromes (MDS)/chronic myelomonocytic leukemias (CMML) and acute myeloid leukemias (AML) may be particularly vulnerable to inhibition of poly(ADP ribose) polymerase 1/2 (PARP1/2) and apurinic/apyrimidinic endonuclease 1 (APE1). PARP1/2 and APE1 are critical enzymes involved in single-strand break repair and base excision repair, respectively. Here, we investigated the cytotoxic efficacy of talazoparib and APE1 inhibitor III, inhibitors of PARP1/2 and APE1, as single-agents, combined with decitabine and combined with each other in CD34+ MDS/CMML cells and in CD34+ or CD34- AML cells in comparison to healthy CD34+ donor cells.

The surviving fraction of CD34+ MDS/CMML cells (n = 8; 4 MDS and 4 CMML), CD34+ or CD34- AML cells (n = 18) and healthy CD34+ donor cells (n = 8) was analyzed using the CellTiter-Glo luminescent cell viability assay (Promega, Southampton, UK). Cell proliferation of untreated MDS/CMML and AML cells was determined by trypan blue exclusion assay (Merck, Darmstadt, Germany). PARP1/APE1 mRNA expression was evaluated using validated primer sets for PARP1 (Hs_PARP1_1_SG QuantiTect Primer Assay, NM_001618) and APE1 (Hs_APEX1_1_SG QuantiTect Primer Assay, ENST00000216714) (Qiagen, Hilden, Germany). Immunofluorescence microscopy of γH2AX foci was performed using a JBW301-derived mouse monoclonal anti-γH2AX antibody (Merck).

Talazoparib and APE1 inhibitor III demonstrated critical anti-leukemic efficacy as single-agents in about 19-25% of MDS/CMML/AML cell samples (Figure 1A and B). Low doses of talazoparib and APE1 inhibitor III further increased the cytotoxic efficacy of decitabine in about 78-86% of MDS/CMML/AML cell samples. Moreover, low doses of APE1 inhibitor III increased the cytotoxic efficacy of talazoparib in about 68% of MDS/CMML/AML cell samples.

In summary, talazoparib and APE1 inhibitor III demonstrated substantial anti-leukemic efficacy as single-agents, in combination with decitabine and combined with each other. Hence, our findings support further investigation of these agents in sophisticated clinical trials.

Figure 1 Cytotoxic efficacy of talazoparib and APE1 inhibitor III in healthy CD34+ donor cells, in CD34+ myelodysplastic syndrome (MDS)/chronic myelomonocytic leukemia (CMML) cells and in CD34+ or CD34- acute myeloid leukemia (AML) cells after 3 days of treatment. (A) The mean IC50 of talazoparib was significantly lower (*p = 0.016) in 1 MDS (MDS#2), 1 CMML (CMML#2) and 3 AML cell samples (AML#1, AML#2, AML#3) as compared to 8 healthy donor cell samples. (B) The mean IC50 of APE1 inhibitor III was substantially lower (p = 0.059) in 1 MDS (MDS#2) and 5 AML cell samples (AML#1, AML#2, AML#3, AML#6, AML#12) as compared to 8 healthy donor cell samples.

Figure 1

Disclosures

Fabarius: Novartis: Research Funding.
Clinical Study on Part Factors Affecting the Effect of Platelet Transfusion during Hematopoietic Stem Cell Transplantation
Authors: Yang Jie , Li Xiuwen , Chen Bao-An
Published: November 2019

Objective :At present, patients with hematopoietic stem cell transplantation (HSCT) are gradually increasing due to hematological diseases. However, there are no further studies on the factors affecting the effect of platelet transfusion during HSCT. This study analyzed the possible factors that affect the platelet transfusion efficiency.

Methods: The clinical data of patients with HSCT were collected from the Department of Hematology, Zhongda Hospital Affiliated to Southeast University from March 2015 to March 2018. According to the inclusion and exclusion criteria, a retrospective statistical analysis on the factors affecting the infusion effect was taken in all patients from +1 d to the day that the peripheral blood granulocyte count was more than 0.5×109/L after transplantation. The factors are including gender, age, types of disease, whether initial treatment, platelet inventory days in the effective period, types of transplantation, duration of disease and transfusion times. Furthermore, non-conditional logistic regression analysis was performed on the mentioned factors with statistical significance.

Results: A total of 52 patients, 299 platelet transfusions were included in this study. The patients involve 28 men and 24 women, and there were 233 effective infusion and 66 invalid infusion. After chi-square test, the effects of gender, age, disease types, whether initial treatment, transplantation types, duration of disease and transfusion times on platelet transfusion were statistically significant(P<0.05), while platelet inventory days within the validity period had no statistically significant effect on platelet transfusion (P≥0.05). Furthermore, the results of non-conditional logistic regression analysis showed that gender, disease type and whether initial treatment had a statistically significant impact on the effect of platelet transfusion (P<0.05).

Conclusion: There are many factors related to poor platelet transfusion during HSCT, in which the gender, disease types and whether initial treatment are the main influencing factors.

Disclosures

No relevant conflicts of interest to declare.
Impact of Minimal Residual Disease Detected By Multiparameter Flow Cytometry on Outcomes of Allogeneic Hematopoietic Stem Cell Transplantation for Acute Myeloid Leukemia Patients in First Complete Remission
Authors: Konova Zoya V. , Parovichnikova Elena N. , Galtseva Irina V. , Kapranov Nikolay M. , Davydova Julia O. , Drokov Mikhail Yu. , Nareyko Maria V. , Popova Natalia N. , Dmitrova Anna A. , Maslikova Ulyana V. , Starikova Olga S. , Usikova Ekaterina I. , Mikhaltsova Ekaterina D. , Koroleva Olga M. , Vasilyeva Vera A. , Lobanova Tatiana I. , Kuzmina Larisa A. , Savchenko Valeriy G.
Published: November 2019

Introduction.

Allogeneic hematopoietic stem cell transplantation (allo-HSCT) could be curative for acute myeloid leukemia (AML) patients. However, the disease relapse after allo-HSCT lead to poor outcomes almost in all cases. Minimal residual disease (MRD) is a strong prognostic marker of increased risk of relapse and reflects overall (OS) and relapse-free survival (RFS) in patients with AML. Immunophenotypic evaluation using multiparameter flow cytometry (MFC) is an important strategy for detecting MRD, because of its simplicity and wide availability.

Aim. Determine the impact of MRD detected by MFC before allo-HSCT on clinical outcomes.

Patients and methods.

To assess the impact of MRD status before transplantation we include 56 AML patients who underwent allo-HSCT in National Research Center for Hematology between July 2016 and June 2019. Patient's characteristics are shown in table 1. All these patients were in first compete morphologic remission at the time of allo-HSCT. MRD was evaluated by MFC. Bone marrow samples were obtained before allo-HSCT and were analyzed by 6-color MFC (BD FACS Canto II, USA). Positive MRD was identified as a cell population deviating from the normal patterns of antigen expression on specific cell lineages at specific stages of maturation for all patients. In addition, for patients with LAIP at diagnosis, positive MRD was also defined as a cell population carrying LAIP markers at diagnosis.

The probabilities of OS and RFS were estimated using the Kaplan-Meier method.

Results.

Among 56 AML patients before allo-HSCT (all were in CR at the time of MRD analysis). Median of follow-up was 9.3 months.11 patients (19,6%) were identified as MRD+ (0,011%-5,08%, mean 1%). And 5 of them relapsed (45,5%). Out of 45 MRD negative patients before HSCT relapse was registered in one (relapse rate 2,2%). Statistical analysis revealed significant differences between MRD- and MRD+ patients: MRD+ had much worse OS (22,9% vs. 85.5%, p=0.0047, Fig.1A) and RFS (26% vs. 97.4%, p<0,0001, Fig.1B).

Conclusions.

Despite the presence of morphological CR before allo-HSCT, MRD persistence can lead to worse probabilities of OS and RFS, which is comparable to patients who were transplanted in relapse. Because of the association between MRD and relapse risk, it can be assumed, that testing for MRD before allo-HSCT may help to identify a subgroup of patients at high risk of relapse who might benefit from preemptive posttransplantation treatment.

Disclosures

No relevant conflicts of interest to declare.
Prevalence of the Antiphospholipid Antibodies in Young Adults with Ischemic Stroke
Authors: Vaccarino Antonella , Pagliaro Maria , Rollone Clara , Melis Fabio , De Marco Federica , Savio Katia , Guido Marilena , Bazzan Mario , Imperiale Daniele , Pini Massimo
Published: November 2019

Background: Ischemic stroke is rare in young adults, with an incidence of about 10-15% of all the ischemic strokes. In about one third of these patients a cause is missing. Among patients with antiphospholipid antibodies syndrome (APS), stroke is the first thrombotic event in about 13% of cases.

Aims of our project were: to evaluate the prevalence of antiphosfolipid antibodies (aPL),to investigate on the prevalence of conventional risk factors and to define the radiological characteristics of the ischemic lesion.

Materials and methods: this is a no profit, observational multicenter prospective study. Inclusion criteria were: age older than 18 and younger than 55 years, informed written consent, a clinical and radiological diagnosis of stroke.

Patient's data were collected at diagnosis and after 30 days from stroke. If any aPL positivity was found the patient was referred to our service to further/eventually confirm the diagnosis of APS. For each patient these data were collected: age, sex, body mass index, personal and familial history, concomitant co morbidities and therapies, cardiovascular risk factors, drug abuse.

CT scan or angioCT or MRI was always performed at diagnosis, aPL profile was determined at diagnosis and eventually confirmed after 12 weeks according to the Sapporo criteria. None of the patients had a previous diagnosis of APS.

Results: enrolled patients from January 2017 to December2018 were 46 out of 425 ischemic stroke (10.8%). We found 11/46 aPL positivity patients. Among these patients, 7 were confirmed at 12 weeks (15%). Baselines characteristics of the study population are detailed in table 1. We found a high prevalence of associated conventional cardiovascular risk factors: hypertension (56%), dyslipidemia ( 50%), obesity (55%), smoke ( 52%). We didn't find any correlation between APS and a clear radiological pattern on MRI and CT scan.

Conclusions: Prevalence of APS was 15% in our cohort of young patients with stroke, 85% of which had an high risk aPL profile. The detection frequency is similar to the recent APS-ACTION and literature findings. In our cohort stroke was a relapse of a previous ischemic event in 24% of the patients, while in 15% there was a stroke's relapse. Even if these data should be confirmed with a wider number of patients, it seems to be useful to evaluate the presence of aPL in a young patient with ischemic stroke .

Disclosures

No relevant conflicts of interest to declare.
Prognostic Value of Circulating Plasma Cells in Newly Diagnosed Multiple Myeloma
Authors: Chen Lijuan , Li Jianyong
Published: November 2019

Multiple myeloma (MM) is biologically diverse and there is a significant variation in survival time from a few months to several years. The presence of circulating plasma cells (CPCs) is associated with a worse prognosis in patients with MM. This study retrospectively analyzed CPCs by 8-color multi-parameter flow cytometry in 108 cases of newly diagnosed MM patients to investigate its value for outcome prediction. Among them, 58 (53.7%) patients were CPCs positive expression. The optimum cutoff predicting for overall survival was determined as 0.29% by using a ROC analysis. Compared with patients with CPCs < 0.29% (n = 75, 69.4%), those with CPCs ≥0.29% (n = 33, 30.6%) showed lower Erythrocyte sedimentation rate(ESR) (P = 0.0032), but higher lactate dehydrogenase (LDH), ferritin (FER) , BM PCs and P53 deletion in BM by FISH (P = 0.001, 0.003, 0.014, and 0.001,respectively). With the median follow-up time 17 months (range, 2.0-37.0), the median PFS in the subgroups with CPCs<0.29% and ≥0.29% was not reached and17.0 months (95% confidence interval (CI):14.85-19.15), respectively, and the median OS was not reached and 12.5months (95% CI: 6.35-18.65), respectively. On multivariate analysis for OS, factors independently predictive of mortality were CPCs≥0.29% (hazard ratio (HR) 4.172; 95% CI, 1.61-10.79; P=0.003), Deletion P53(HR 11.54; 95% CI, 4.06-32.84; P<0.001). We further developed a convenient two-factor risk stratification based on CPCs and p53 deletion according to the results of log-rank test, univariate and multivariate analysis. The high-risk group was defined as both CPCs ≥ 0.29% and P53 deletion, accounting for 10% of the population, have a dire prognosis (median PFS = 5 months; OS = 10 months) despite modern therapies .These results identified CPCs as an unfavorable prediction for the outcome of MM. A combination of p53 deletion may screen out a high-risk subgroup which should be considered for novel therapeutic approaches.

Disclosures

No relevant conflicts of interest to declare.
Diplotype Analysis of NUDT15 Variants By Targeted Sequencing in Pediatric Acute Lymphoblastic Leukemia
Authors: Yu Chih-Hsiang , Chang Ya-Hsuan , Chou Wen-Chien , Wang Der-Shiun , Lin Shu-Wha , Chen Hsuan-Yu , Yang Yung-Li
Published: November 2019

Background: The NUDT15 polymorphisms, V18_V19insGV and R139C, located on exon 1 and exon 3 were associated with intolerance of 6-mercaptopurine (6MP), which were frequently used during the maintenance therapy of childhood acute lymphoblastic leukemia (ALL). However, these two NUDT15 diplotypes with significant different biological activity, mono-allelic *1/*2 and complex homozygosity *3/*6, could not be distinguished from each other by direct Sanger sequencing of exon 1 and exon 3. We applied targeted sequencing of NUDT15 cDNA to clarify the diplotypes of complex homozygous of NUDT15. Methods: Thirty-five ALL patients who were diagnosed in National Taiwan University Hospital with both V18_V19insGV and R139C of NUDT15 were included in this study. A single amplicon (562 bp) covers all the coding regions of NUDT15 was amplified from cDNA and then sequenced by paired-end sequencing using MiSeq reagent kit v3 (600 cycles). For validation, the amplicons from complex homozygous NUDT5 were cloned into pCR-Blunt II-TOPO vector. Result: By targeted sequencing, thirty-three patients were identified as mono-allelic *1/*2. Two complex homozygous NUDT15, *3/*6 and *2/R34T, were identified in patient No.27 and No.37. We also cloned amplicons from these two patients into vector and the results were the same as targeted sequencing. These two patients with complex homozygous NUDT15 variants tolerated only 1~2 mg/m2 daily mercaptopurine doses. Conclusions: Targeted sequencing of NUDT15 cDNA is a simple method to determine the NUDT15 diplotype and helpful for the identifications of complex homozygous NUDT15, which should be identified before the administration of 6MP to avoid excessive myelosuppression caused by improper of 6MP dosage. Figure: (A) NUDT15 haplotypes. (B) The primers is used for direct Sanger sequencing and cDNA targeted sequencing. (C) The NUDT15 diplotype of patient No.27 is analyzed using cDNA cloning followed by Sanger sequencing.

Figure

Disclosures

No relevant conflicts of interest to declare.
Disease Risk Comorbidity Index for Patients Receiving Haploidentical Allogeneic Hematopoietic Transplantation
Authors: Mo Xiaodong , Zhang Xiaohui , Xu Lanping , Wang Yu , Yan Chenhua , Chen Huan , Chen Yuhong , Han Wei , Wang Fengrong , Wang Jingzhi , Liu Sining , Liu Kaiyan , Huang Xiaojun
Published: November 2019

Purpose or Background:

We aimed to develop a disease risk comorbidity index (DRCI) based on Disease Risk Index (DRI) and Hematopoietic Cell Transplantation-Specific Comorbidity Index (HCT-CI) in patients receiving haploidentical hematopoietic stem cell transplantation (haplo-HSCT).

Method or Case:

We examined 889 patients undergoing haplo-HSCT from 2015 to 2016. We used a Cox multivariable model to identify factors prognostic of disease-free survival (DFS) in a training subset (n = 593). A weighted score using these factors was assigned to the remaining patients (validation cohort; n = 296). This work was supported by the Capital's Funds for Health Improvement and Research (grant number 2018-4-4089).

Results or Progress:

The multivariable model identified two independent predictors of DFS: DRI and HCT-CI before HSCT. A weighted score of 2 was assigned to very high risk DRI, and a weighted score of 1 was assigned to high-risk DRI and intermediate- and high-risk HCT-CI in the scoring system (i.e., haplo-HSCT). In the validation cohort, the 3-year DFS was 65.2% (95% CI, 58.2-72.2%), 55.8% (95% CI, 44.9-66.7%), and 32.0% (95% CI, 5.8-58.2%) for the low-, intermediate- and high-risk group, respectively (P=0.005). Haplo-DRCI can predict relapse (P<0.001), non-relapse mortality (NRM, P<0.001), DFS (P<0.001), and overall survival (OS, P<0.001) in total population and in disease-specific subgroups, particularly in acute leukemia patients. Increasing score was also significantly predictive of increased relapse (P<0.001), increased NRM (P=0.001), decreased DFS (P<0.001), and decreased OS (P<0.001) in an independent historical cohort (n=526).

Conclusion or Discussion:

These data confirmed that haplo-DRCI can effectively risk stratifies haplo-HSCT recipients and provide the tool to better predict who will best benefit from haplo-HSCT.

Disclosures

No relevant conflicts of interest to declare.
Gfi1 Upregulates c-Myc Expression and Promotes c-Myc-Driven Cell Proliferation
Authors: Zhang Yangyang , Dong Fan
Published: November 2019

Gfi1 is a zinc-finger transcriptional repressor that plays an important role in hematopoiesis. When aberrantly activated, Gfi1 may function as a weak oncoprotein in the lymphoid system, but collaborate strongly with c-Myc in lymphomagenesis. c-Myc is a transcription factor that is frequently activated in human cancers including leukemia and lymphoma mainly due to its overexpression as a result of gene amplifications and chromosomal translocations. c-Myc overexpression may also result from stabilization of c-Myc protein, which is highly unstable and rapidly degraded through the ubiquitin-proteasome pathway. The mechanism by which Gfi1 collaborates with c-Myc in lymphomagenesis is incompletely understood. c-Myc activates gene expression by forming a heterodimeric complex with the partner protein Max, but may also repress target genes through interaction with transcription factor Miz-1. We previously showed that Gfi1 indirectly interacts with c-Myc through Miz-1 and collaborates with c-Myc to repress CDK inhibitors p21Cip1 and p15Ink4B. In this study, we show that Gfi1 augmented the level of c-Myc protein transiently expressed in Hela cells and the levels of MycER fusion protein stably expressed in the mouse pro-B Ba/F3 and myeloid 32D cells. The C-terminal ZF domains of Gfi1, but not its transcriptional repression and DNA binding activities, were required for c-Myc upregulation. Notably, although Miz-1 has been shown to stabilize c-Myc protein, the expression of c-Myc V394D mutant, which is defective in Miz-1 interaction, was still upregulated by Gfi1, suggesting that Gfi1-mediated c-Myc upregulation was independent of Miz-1 interaction. We further show that Gfi1 overexpression led to reduced polyubiquitination and increased stability of c-Myc protein. Interestingly, the levels of endogenous c-Myc mRNA and protein were augmented upon induction of Gfi1 expression in Ba/F3 and Burkitt lymphoma Ramos cells transduced with the doxycycline-inducible Gfi1 lentiviral construct, but reduced in Gfi1-knocked down human leukemic HL60 and U937 cells. Additionally, targeted deletion of Gfi1 resulted in reduced c-Myc expression in mouse lineage negative bone marrow cells, which was associated with a decline in the expression of c-Myc-activated target genes. The oncogenic potential of Myc derives from its ability to stimulate cell proliferation. Our results demonstrate that inducible expression of Gfi1 in Ba/F3 cells expressing MycER promoted Myc-driven cell cycle progression and proliferation. Thus, in addition to its role in c-Myc-mediated transcriptional repression, Gfi1 upregulates c-Myc expression at both mRNA and protein levels, leading to enhanced expression of c-Myc-activated genes and augmented cell proliferation driven by c-Myc. Together, these data may reveal a novel mechanism by which Gfi1 collaborates with c-Myc in lymphomagenesis.

Disclosures

No relevant conflicts of interest to declare.
The Effect of Chronic Viral Hepatitis on Serum Ferritin Level in Thalassemia Patients
Authors: Rujeerapaiboon Natthapat , Tantiworawit Adisak , Piriyakhuntorn Pokpong , Rattanathammethee Thanawat , Hantrakool Sasinee , Chai-Adisaksopha Chatree , Rattarittamrong Ekarat , Norasetthada Lalita , Charoenkwan Pimlak
Published: November 2019

Background: Serum ferritin is widely used as a marker of iron overload in thalassemia patients. However, the ferritin level is affected by active infections or inflammation. The association between viral hepatitis and serum ferritin level in thalassemia patients is still unclear. This study aimed to determine the effect of chronic viral hepatitis on serum ferritin level in thalassemia patients.

Methods: This was a cross-sectional study in thalassemia patients aged ≥15 years-old at Chiang Mai University hospital. We expected that thalassemic patients in our clinic have a mean serum ferritin of 767 ng/mL with a standard deviation of 210 ng/mL. As a result, we have to enroll a total of 28 patients to demonstrate 30% difference of mean serum ferritin when the power was set at 80% with alpha level of 0.05. Information on chronic viral hepatitis, mean serum ferritin and liver iron concentration (LIC) as measured by T2* MRI were collected. Chronic viral hepatitis status was confirmed by either HBV DNA or HCV RNA testing. Patients were categorized to hepatitis and non-hepatitis group. Serum ferritin levels were compared between two groups. LIC measurement was used as a gold standard for iron overload. Subgroup analysis was performed according to iron overload and transfusion requirement status. Categorical and continuous variables were compared using the Chi-squared test and T-test, respectively. The correlation between viral loads and mean serum ferritin levels was analyzed by Pearson's correlation.

Result: Of 32 thalassemia patients (25 non-transfusion dependent [NTDT] and 7 transfusion dependents [TDT]), 13 patients had chronic viral hepatitis (7 with hepatitis B and 6 with hepatitis C infections). The LIC between hepatitis and non-hepatitis groups were not significantly different (7.28 [SD 4.7] vs 9.08 [SD 5.2] mg Fe/g, p=0.19). In the higher LIC group (≥ 5 mg Fe/g), the mean serum ferritin level was higher in the hepatitis group than non-hepatitis group (1,776 [SD 488] vs 967 [SD 860] ng/mL, p=0.03). For the lower LIC group (<5 mg Fe/g), the mean ferritin levels were not significantly different between the hepatitis and non-hepatitis groups (646 [SD 224] vs 459 [SD 205] ng/mL, p=0.22). The correlation between the viral load and mean ferritin level in NTDT group showed a significant linear correlation with R=0.7 (p=0.04).

Conclusions: We observe a higher serum ferritin level among thalassemia patients who concurrently have chronic viral hepatitis. Chronic viral hepatitis is a possible cause of a falsely high ferritin level in these patient population. Furthermore, the viral load is positively correlated with serum ferritin level.

Disclosures

No relevant conflicts of interest to declare.
C-Myc-Driven and PRMT5-Dependent Regulation of Multiple Myeloma Cell Proliferation through TNRC6B Gene
Authors: Xu Peipei , Dan Guo , Chen Bing , Zhao Quan , Zhou Rong-Fu , Ouyang Jian
Published: November 2019

Objective: The protein arginine methyltransferase 5 (PRMT5) catalyzes the symmetrical bimethylation of arginine residues, which plays an important regulatory role in the life process. Transcription factor c-Myc is widely proved involved in the alteration and development of tumors, while the regulatory mechanism of c-Myc-mediated gene expression has not been fully understood. In this article we try to investigate the functional role of PRMT5 and c-Myc in regulating multiple myeloma cell proliferation.

Methods: The protein and mRNA expression levels of PRMT5 in myeloma cells and normal cells were detected by Western Blot and qPCR. Lentivirally transduced shRNAs targeting PRMT5 (sh-PRMT5) was constructed using lentivirus mediated RNAi technology, and was packaged to infect MM cells to select positive colonies. The effect of PRMT5 on the proliferation of MM cells was detected by cck-8 assay. Annexin V/7-AAD double staining flow cytometry was used to detect cell survival and apoptosis rate. The expression of apoptotic suppressor gene c-Myc in sh-PRMT5 cell lines was detected by Western Blot and qPCR. Finally, double-knockdown of PRMT5 and c-myc was performed and gene expression differentiation were identified by RNA sequencing (RNA-seq).

Results: PRMT5 expression in MM cell lines (RPMI8226 and U266) was relatively high compared with peripheral blood mononuclear cells (PBMCs), which was correlated with progression-free survival (PFS) and overall survival (OS) in MM patients. After sh-PRMT5 infection to MM cell lines, puromycin selection was performed and efficient gene knockdown was evaluated by Western Blot and qPCR. After targeted silencing of PRMT5 expression, the proliferation of MM cells was decreased and apoptosis was significantly increased (P < 0.05). C-Myc protein and mRNA levels were significantly downregulated after PRMT5 gene silencing. The results showed that PRMT5 promoted the proliferation of MM cells and inhibited the apoptosis of MM cells by regulating the expression of apoptotic suppressor gene c-Myc. Subsequently, RNA-seq confirmed SMCHD1 as the common candidate target gene of c-myc and PRMT5. Therefore, we speculated that PRMT5 and c-Myc may jointly regulate TNRC6B gene to promote the proliferation of MM cells.

Conclusion: Altogether, our study not only proposed the mechanism PRMT5 and c-myc in regulating multiple myeloma related gene expression, but also provided a new strategy and theoretical basis for clinical treatment of multiple myeloma.

Disclosures

No relevant conflicts of interest to declare.
Combined Targeting of Distinct c-Myc and JunB Transcriptional Programs for Multiple Myelioma Therapy
Authors: Lind Judith , Czernilofsky Felix , Vallet Sonia , Fan Fengjuan , Sattler Martin , Bakiri Latifa , Wagner Erwin F , Percherstorfer Martin , Podar Klaus
Published: November 2019

c-Myc plays a pivotal role in multiple myeloma (MM) pathogenesis; and the BET protein BRD4 is a key regulator of c-Myc transcription. Recently, a pathophysiologic role in MM has also been attributed to the AP-1 family member JunB. Approaches to target transcription factors (TFs), such as c-Myc and JunB, currently emerge among the most promising novel anti-MM strategies, with a potentially high therapeutic index. However, redundancy phenomena represent a major challenge for targeting c-Myc- or JunB-mediated tumor proliferation programs.

MZ-1 is a novel proteolysis-targeting chimera combining the recognition sequence for the E3-ligase Von-Hippel-Lindau with JQ1, a moiety that targets BRD4. Indeed, beyond direct inhibition by JQ1, MZ-1 significantly decreased BRD4 as well as c-Myc protein levels in MM cell lines and primary cells. Consequently, MZ-1 inhibited MM cell growth through G0/G1 arrest; and induced tumor cell death. Moreover, patient-derived BMSC- or exogenous IL-6- induced BRD4/c-Myc upregulation in MM cells was inhibited by MZ-1 treatment, indicating that targeting BRD4, at least in part, overcomes the protective effect of the microenvironment. Of note, MZ-1 did not have an impact on BMSCs/IL-6- induced upregulation of JunB RNA or protein levels. Conversely, dox-induced knockdown of BMSC/IL-6-triggered JunB upregulation in TetshJunB/MM.1S cells did not decrease BRD4/c-Myc RNA or protein levels. Similar data were obtained in other MM cell lines and primary cells. RNAseq, unbiased GSEA, and luciferase reporter assays of representative target genes further supported the co-existence of c-Myc- and JunB-mediated proliferative programs, which are initiated by the same stimuli. Importantly, MZ-1 in combination with dox-induced knockdown of BMSC/IL-6-triggered JunB upregulation in TetshJunB/MM.1S cells significantly decreased both BRD4/c-Myc as well as JunB protein levels; synergistically inhibited tumor cell proliferation and induced cell death. Similar results were obtained in other MM cell line and primary cells upon treatment with MZ-1 and transient siJunB. Finally, our preliminary results show the synergistic in vivo activity of MZ-1 in BMSC:TetshJunB/MM.1S versus BMSC:TetshSCR/MM.1S-carrying doxycyclin- treated NSG mice. Taken together, these data for the first time delineate the existence of distinct c-Myc and JunB transcriptional programs that contribute to MM proliferation. They thereby provide the preclinical rationale for the development of therapeutic strategies that combine targeting of distinct transcriptional programs to improve outcome in MM.

Disclosures

Vallet: Roche Pharmaceuticals: Consultancy; Pfizer: Honoraria; MSD: Honoraria. Podar:Celgene: Consultancy, Honoraria; Amgen Inc.: Honoraria; Roche Pharmaceuticals: Research Funding; Takeda: Consultancy; Janssen Pharmaceuticals: Consultancy, Honoraria.