bluuebunny/crossref_metadata_2025 huggingface dataset:
Bone Marrow Derived Mesenchymal Stem Cells and Radiation Response: a Kinetic Study of Gene Response Using Microarray Analysis.
Authors: Garrigou Philippe , Mayol Jean-Francois , Mouret Catherine , Delaunay Christophe , Drouet Michel , Herodin Francis , Peinnequin Andre , Pugnière Pascal
Published: October 2019

Abstract 4575

Mesenchymal Stem cells (MSC) are an important radiosensitive component of the so called hematopoietic stem cell niche. Importantly this supportive microenvironment influences the stem cell repopulation capacity as well as the quiescent/non proliferative state of hematopoietic cells. Senescence is considered as a major process in MSC response following irradiation. However, other studies have reported in mice the reduction of the pool of bone marrow mesenchymal stem/progenitor cells following TBI independently of senescence. An altered osteoblastic differentiation was pointed out in these studies. Furthermore, MSC have been shown to be involved in the repair of ionizing radiation damage of distant epithelial sites which requires adherence genes mitigation. The aim of this study was to clarify some of these points using an in vitro model of irradiation and short term culture.

Briefly, confluent human BM-MSC were irradiated at the dose of 2.5 Gy (dose rate: 95 cGy.min-1) and immediately put into culture (Minimum essential medium supplemented with 10% FCS and 10 μg/ml of ciprofloxacin, penicillin and streptomycin). Six, 12, 24, 48 and 72 hours after irradiation, cells were harvested and lysed. Total RNAs were purified using the automatic Qiacube system (Qiagen,Courtaboeuf, France) and processed on DNA microarray scanner (Agilent technologies Inc.) according to supplier's recommendations. Data were analyzed with GeneSpring GX Expresion Analysis software version 10.0 (Agilent) in order to identify the transduction pathways involved.

No apoptosis was observed during this short term incubation. Among other genes we identified plasminogen activator inhibitor 1 (PAI-1) as a factor highly upregulated after irradiation, in addition to CD151. This is in accordance with MSC response to nutrient-poor, hypoxic stress environment (Copland et al, Stem cells 2009). As MSC are radiosensitive cells, this may indicate that PAI impacts MSC survival through the mitigation of their adhesiveness to surrounding matrices. As PAI-1 is an important factor involved in the balance of blood coagulation and fibrinolysis as well as in the regulation of angiogenesis, one may speculate the consequences of PAI-1 release from MSC on blood homeostasis. Work is going on to describe the main response target genes. This could allow us to identify therapeutic strategies based on ex-vivo or in vivo manipulation of MSC in a purpose of tissue remodelling.

Disclosures:

No relevant conflicts of interest to declare.
Expression of the Transcription Factor Lymphoid Enhancer Binding Factor-1 (LEF-1) Is Correlated to Aberrantly Increased Fibromodulin Transcripts in Chronic Lymphocytic Leukemia (CLL).
Authors: Erdfelder Felix , Gehrke Iris , Gandhirajan Rajesh Kumar , Hertweck Magdalena , Razavi Regina , Paesler Julian , Filipovich Alexandra , Uhrmacher Sabrina , Poll-Wolbeck Simon Jonas , Hallek Michael , Kreuzer Karl Anton
Published: October 2019

Abstract 4589

Chronic lymphocytic leukemia (CLL) is characterized by accumulation of monoclonal CD5+ B lymphocytes. Fibromodulin is a secreted extracellular matrix protein usually found in articular cartilage, bones, connective tissue and collagen rich tissues but has been shown to be excessively expressed in CLL. Moreover, we and others could show that WNT signaling is highly activated in CLL. The aim of our study was to investigate a possible connection between fibromodulin overexpression and the WNT pathway. Moreover, we wanted to explore possible relations between these parameters and the prognosis of CLL. Fibromodulin mRNA transcripts were correlated with the mRNA expression of the WNT pathway transcription factors lymphoid enhancer binding factor-1 (LEF-1) and T cell factor-4 (TCF-4) in primary CLL cells. Furthermore, we assessed correlations between the mRNA expression levels with ZAP-70 and CD38 protein expression. These parameters have been shown to be associated with a poor prognosis. Real-time reverse transcriptase-polymerase chain reaction (RT-PCR) was used and calculated PCR-efficiency corrected relative expression values of Fibromodulin, LEF-1 and TCF-4 mRNA in primary CLL-cells determined. ZAP-70 and CD38 expression was determined by flow cytometry analysis. Based on 20 primary CLL samples we found a significant positive correlation between Fibromodulin and LEF-1 mRNA levels (Spearman's rho = 0.65, p = 0.009). Also, LEF-1 and TCF-4 were found to be strongly correlated (Spearman's rho = 0.61, p = 0.027). In contrast, fibromodulin and TCF-4 mRNA levels were only weakly correlated (Spearman's rho = 0.33, p = 0.271). CD38 and ZAP-70 did not correlate significantly to any of the other values. Both parameters did also not exhibit significant correlations with fibromodulin, LEF-1, and TCF-4 mRNA. The positive correlation of TCF-4 and LEF-1 was expected as LEF-1 has been shown to be a target gene of TCF transcription factors. The strong positive correlation of fibromodulin and LEF-1 indicates that fibromodulin might be a target gene of LEF-1 or part of the same (TCF-4 independent) transcription regulation pathway. Studies investigating theses functional relationships are underway.

Disclosures:

Hallek: BayerScheringAG: Honoraria, Research Funding.
Human Embryonic Stem Cell-Derived and Fetal Macrophages Exhibit Mainly Features of Alternative (M2) Polarization.
Authors: Klimchenko Olena , Di Stephano Antonio , Solary Eric , Routier Melanie , Vainchenker William , Norol Françoise
Published: October 2019

Abstract 4598

Monocyte heterogeneity has long been recognized and 2 functional subsets of human monocytes that exert specific roles in homeostasis and inflammation in vivo, M1 and M2 monocytes, has been described. The different monocyte subsets seem to reflect developmental stages with distinct physiological roles but few is known whether the macrophage diversity arises in early ontogeny.

Human embryonic stem cells (hESC) provide an unique model for in vitro studies of the early ontogeny of the hematopoietic system. Human embryonic monocytes were obtained from embryoid bodies cultured for 3 weeks in the presence of BMP4, VEGF and a mixture of hematopoietic cytokines. The sorted CD14+CD45+ cells were further cultured for 7 -10 days in the presence of GM- and M-CSF. Embryonic stem cells derived monocytes and macrophages were identified by several criteria including morphology, cytochemical staining, ultrastructural and functional features. In contrast to early mouse hematopoiesis that bypass the series of monoblaste/monocyte precursors human embryonic monocyte lineage developped by process similar to definitive monopoiesis. Two distinct subpopulations of monocytes, first CD14+CD16- then CD14+CD16+ could be successively identified. Transcriptional and functional assays suggest that CD14+CD16- population is not an embryonic counterpart of CD14+CD16- “inflammatory” adult monocytes but a precursor for CD14+CD16+ macrophages and dendritic cells. Moreover, both tested populations possess phenotypical markers of M2 polarization and were positive for a expression of scavenger receptors CD163+ and CD36+ of CCR5 and negative for CD62L. Additional transcriptional studies and chemokine pattern confirmed their IL10 high IL12low phenotype, clearly indicating their M2 polarization state. M2 polarization was confirmed in fetal macrophages derived from CD34+ cells issued from livers of first-trimester fetuses. Involvment of embryonic and foetal monocytes in homeostasis are under investigation.

Collectively, our data provide insight into alternative macrophage polarization during early embryogenesis and adds further data to the growing body of evidence that establishment of macrophage heterogeneity is related to early ontogeny.

Disclosures:

No relevant conflicts of interest to declare.
Effect of Sickle Cell Disease Type, Age and Gender On the Prevalence of Chronic Organ Damage in Adults with Sickle Cell Disease.
Authors: Osarogiagbon Raymond U. , Haider Syed N , Tang Jun
Published: October 2019

Abstract 4607

Introduction

The high mortality risk that sickle cell disease (SCD) patients experience from infancy is cumulative through adulthood, largely because of the effect of cumulative end organ damage, which is a more powerful predictor of early mortality than frequency of painful episodes. The latter, though, gets more attention from patients and caregivers. Any vascular territory is susceptible to damage. The most common target organs are the brain, lungs, kidneys, retina and joints. We examined the prevalence of the full spectrum of end organ damage in a cohort upon entry into our adult SCD program and compared the clinical and laboratory characteristics of patients based on age, gender and SCD type.

Patients and Methods

Retrospective review of prospectively collected data on 118 adults upon entry into our program between February 2005 and October 2008. All patients underwent a standardized battery of tests to evaluate hematological and biochemical parameters at entry. Historical presence of episodes of acute chest syndrome, pneumonia, stroke, avascular necrosis, osteomyelitis, leg ulcers, priapism, and cholecystectomy and hydroxurea therapy was quantified. Pulmonary hypertension (PHT) was defined as a tricuspid regurgitant jet velocity (TRJV) ≥2.5 m/s on Doppler echocardiography; sickle cell nephropathy (SCN) as glomerular filtration rate, (GFR) < 90ml/min. and/or 24-hour protein>300mg and/or urine protein/creatinine ratio>0.3); cerebrovascular disease (CVD) as evidence of previous ischemic and/or hemorrhagic infarct and/or aneurysm formation on brain MRI/MR angiography; and sickle cell retinopathy (SCR) as background to proliferative retinopathy) on fluorescent retinal angiography. Characteristics of patients were evaluated with t-test for continuous variables and chi-square test for the categorical variables.

Results

The relevant statistically significant correlative variables in these 3 comparisons are shown in the following tables.

Conclusions

Our study highlights the various differences in the prevalence of sickle cell disease-related end organ damage and morbidity among different age, gender and two major sickle cell disease categories. It shows the significant progression of organ damage with advancing age and the more severe nature of SS/Sβ0 phenotype. Further expansion of this assessment may help identify specific high risk groups that can be targeted as candidates for more intensive preventive interventions.

Disclosures:

No relevant conflicts of interest to declare.
TDC Imaging and Atoxic Contrast and Cerebrovascular Lesions in SCD Adults Patients.
Authors: Leite Ana Claudia C , Veloso Orlando CG , Lobo Clarisse Lopes de Castro , Oliveira Raquel VC , Bahia Paulo RV
Published: October 2019

Abstract 4612

Silent infarcts have been reported in approximately 20% of children with SCD. The prevalence in SCD adult patients has not been studied previously.

METHODS

We studied 187 patients (mean age 29 years) Each of these patients underwent a neurological examination, duplex scan of carotids and TCD image with atoxic contrast (micro bubbles). Eighty of these patients were sorted to perform MRI and MRA to study lacunar infarction, encephalomalacia, leukoencephalopathy, Moya Moya syndrome and aneurysm (measured by number and size).

RESULTS

The overall prevalence of silent infarcts was 52%. Comparing the two methods regarding sensitivity and specificity, obtained respectively 88 and 85%. The positive predictive value of the TCD image with contrast was 80%. There was only one case of intracranial aneurysm, observed by both imaging methods. The other asymptomatic patients with abnormal TCD (low definition of image contrast or absence of flow to TCD image) presented diagnostic criteria of vascular occlusion with secondary collateral (Moya Moya Syndrome).

CONCLUSIONS

Use of atoxic contrast (micro bubbles) increases the sensitivity of TCD image for diagnosis of intracranial lesions in SCD adults patients, even in asymptomatic individuals, enabling the diagnosis of silent infarcts.

Disclosures:

No relevant conflicts of interest to declare.
Changes in Serum Protein Profiles During Allogeneic Hematopoietic Stem Cell Transplantation (allo-HSCT): Clinical Significance of Serum C-Reactive Protein and Haptoglobin Levels.
Authors: Lee Se Ryeon , Ryu Joohyun , Ahn Jae-Sook , Kim Yeo-Kyeoung , Yang Deok Hwan , Lee Je-Jung , Sung Hwa Jung , Choi Chul Won , Kim Byung Soo , Park Byoung Chul , Kim Hyeoung-Joon
Published: October 2019

Abstract 4658

Background

Successful hematopoietic stem cell transplantation involves the restoration of hematopoietic function after engraftment, arising from the differentiation and proliferation of hematopoietic stem cells. Several factors influence the course of allo-HSCT. In particular, knowledge of serum proteome changes during the allo-HSCT period might increase the efficacy of diagnosis and disease prevention efforts.

Method

The sera from five representative patients who underwent allo-HSCT were analyzed by 2-DE and LC-MS/MS. To verify the clinical significance of identified serum proteome, we assessed the representative APPs, C-reactive protein (CRP) and haptoglobin in 78 patients who underwent allo-HSCT at Chonnam National University Hospital (CNUH) who were included between June 2002 and April 2008. Serum level of CRP and haptoglobin were measured weekly from D-7 or D-0 to D+21 of allo-HSCT.

Result

Fourteen proteins showing significant changes in expression were further examined, and most were identified as APPs. Studies of 78 additional patients confirmed that CRP and haptoglobin undergo expression changes during allo-HSCT and thus have the potential to serve as representative markers of various hematologic diseases. Maximal CRP level affected the development of MTCs and other problems, FUO. In particular, an increase in CRP level 21 days after allo-HSCT was found to be an independent risk factor for MTC. Maximal haptoglobin and haptoglobin level 14 days after allo-HSCT were predictive of relapses in underlying hematologic disease.

Conclusion

Our results reveal the usefulness of a proteomic approach in the clinical follow-up of patients after allo-HSCT, and suggest that CRP and haptoglobin can be monitored after allo-HSCT to assess risks of early transplant-related complications and relapse.

Disclosures:

No relevant conflicts of interest to declare.
Post-Thaw Viability of Cryopreserved Hematopoietic Progenitor Cell Grafts Influences Patients' Predisposition towards Infectious Complications in the Post-Transplant Period.
Authors: Vrhovac Radovan , Peric Zinaida , Jurenec Silvana , Kardum-Skelin Ika , Jelic-Puskaric Biljana , Siftar Zoran , Mesaric Jasna , Minigo Hrvoje , Jaksic Branimir
Published: October 2019

Abstract 4666

Cell viability in peripheral blood progenitor cell (PBPC) grafts and its influence on the clinical course following transplantation was evaluated in 81 consecutive transplantations (72 autologous, 9 allogeneic) performed in patients with hematological diseases. Viability of cells in PBPC grafts, assessed by trypan blue dye exclusion test immediately upon collection was 98.6±3.5%, after addition of dimethyl sulfoxide (DMSO) 73.3±21.8%, and post-thaw 65.2±16.1%. It did not differ significantly between patients with different diagnoses, gender, age, type of priming used, dose of G-CSF administered or number of CD34+ cells collected. However, grafts stored for more than 60 days showed lower post-thaw viability compared to the ones thawed in the 60 days following cryopreservation (56.6±15.2% vs. 67.6±15.5%, p=0.04). A strong, statistically significant correlation was found between pre-cryopreserved CD34+ cell dose and hematopoietic recovery in patients, expressed as time to reach ANC>1×109/l and platelets >20×109/l (R2=0.14 p=0.001, R2=0.12 p=0.005, respectively). Occurrence of febrile neutropenia and total number of febrile days did not differ significantly between patients depending on the dose of CD34+ cells infused. On the contrary, post-thaw graft viability did not influence engraftment time, but there was a predisposition towards infectious complications in the post-transplant period in patients receiving grafts with lower percentage of viable cells. These patients developed febrile neutropenia more often (72.2% vs. 50% of patients, p=0.05) and had more febrile days (2.4±2.6 vs. 1.5±2.3, p=0.05) following transplantation. Our results demonstrate that PBPC grafts are capable of long term engraftment regardless of the graft storage time or percentage of viable cells post-thaw, which confirms the robustness of CD34+ cells during the freeze/thaw procedures carried out in daily clinical practice. Granulocyte concentration in PBPC grafts could have an influence on infectious complications following transplantation. This is an intriguing finding that deserves further investigation on a larger number of patients, less heterogeneous in terms of underlying disease, type of transplantation or conditioning regimen used.

Disclosures:

No relevant conflicts of interest to declare.
HHV-6 Encephalitis in Pediatric Unrelated Umbilical Cord Blood Transplantation: Role for Ganciclovir Prophylaxis?
Authors: Cheng Frankie Wai Tsoi , Lee Vincent , Leung Wing Kwan , Chan Paul Kay Sheung , Leung Ting Fan , Shing Matthew MK , Li Chi Kong
Published: October 2019

Abstract 4667

Background

The role of ganciclovir as HHV-6 prophylaxis in unrelated hematopoietic stem cell transplant (HSCT) setting remains controversial.

Methods

We performed a 8-year retrospective review of patients received unrelated HSCT from January 2000 to September 2008. From January 2002, ganciclovir prophylaxis 5mg/kg twice daily for 7 days for all unrelated HSCT before transplant was adopted. The other transplant policies including antibacterial, antifungal, antiviral and graft-versus-host disease control policies remained unchange in that period. The prevalence of HHV-6 encephalitis was studied before and after the change in policy.

Result

Fifty-four unrelated HSCT were performed from January, 2000 to September, 2008. Total four cases (7.4%) of HHV-6 encephalitis were diagnosed. Two cases out of 16 cases (12.5%) diagnosed before adoption of the policy; 2 cases out of 38 cases (5.3%) diagnosed afterward. All of them were unrelated umbilical cord blood (UCB) transplant recipients. Two cases had significant residual neurological deficit and refractory seizure. The other two cases died of other transplant-related mortalities.

Conclusion

We conclude that HHV-6 encephalitis is still a rare complication of unrelated HSCT and may be more common in unrelated UCB transplantation. Routine use of ganciclovir as HHV-6 prophylaxis in all unrelated HSCT recipients may not be justified.

Disclosures:

No relevant conflicts of interest to declare.
Pulmonary Infection After Allo-Transplantation: Fungus or Tuberculosis? 2 Case Report.
Authors: Yin Songmei , Xie Shuangfeng , Li Yiqing , Nie Danian , Wang Xiuju , Ma Liping , Xiao Jie
Published: October 2019

Abstract 4671

Introduction

Infection after allo-Stem cell transplantation (SCT) is a major cause for post allo-SCT deaths. The pathogens involved are diverse and sometimes difficult to be distinguished. Treatment is primarily based on clinical history, blood analysis, chest X-ray, computerized tomography (CT) examination and pathogen culture of sputum or bronchoaleolar lavage fluid. Empirical treatment is often used before the results of pathogen culture are available. Here, we reported 2 cases of allo-SCT with similar clinical background but infections due to different pathogens.

Clinical information

At the onset of the pulmonary diseases, anti-GVHD drugs (Including prednisone, CsA) were already withdrawn for both cases. There was no GVHD progression after withdrawal of the anti-GVHD drugs. Both cases had fever, ranging from 38 to 39 centigrade. Patients were short of breath. Oxygen inhalation was needed. Case 1 was in severe hypoxia. The oxygen saturation was down to 70%∼80% at the peak severity. Neither of the 2 cases was in need of mechanical ventilation. CT images showed that these 2 cases had similar lung injuries (Fig 1 and Fig 2). Tissue biopsy revealed that different pathogens were involved in these 2 cases, although they had identical disease history, similar duration after transplantation, same clinical symptoms and signs, even similar CT images. The histological findings of lung biopsies showed that the pathogen for case 1 was aspergillums, whereas that of case 2 was tubercle bacillus. Both cases responded well to the specific treatment, respectively. Patients are now in disease-free state.

Conclusions

Both tuberculosis and fungus infection could show diffused foci on CT image. The diffused lesions of tuberculosis tend to fuse, whereas little cavities were more frequently seen in fungus infections. After treatment, the lesions of fungus were much easier to be clear than tuberculosis. Diagnostic treatment might be used to distinguish these 2 diseases if biopsy cannot be performed or pathogen culture is negative.

Disclosures:

No relevant conflicts of interest to declare.
CHILDHOOD Biphenotypic ACUTE LEUKEMIA with High Incidence of Basophilic Differentiation: The CONTRIBUTION of Ultrastructural Methods to Diagnosis.
Authors: Resnitzky Peretz , Luria Drorit , Shaft Dina , Avrahami Gali , Jeison Marta , Stark Batia , Yaniv Isaac
Published: October 2019

Abstract 4698

The diagnosis of Biphenotypic Acute Leukemia (BAL) is still a challenge in clinical hematology. In spite of the use of the score formulation of the European Group for the Immunological Classification of Leukemias (EGIL) there are still cases difficult to define. We assessed the contribution of ultrastructural morphology and cytochemistry to the diagnosis of BAL in pediatric leukemic patients. Twenty six patients diagnosed at Schneider Children's Medical Center of Israel between the years 1989 to 2004 have been classified as BAL in a retrospective analysis evaluated by various combinations of light-microscopy (LM) morphology, immunophenotype flow-cytometry, cytogenetics and electron-microscopy (EM) including myeloperoxidase (MPO) and platelet peroxidase (PPO) reaction. The identification of myeloid features by EM was based on the presence of 1) MPO positive granules and MPO reactivity in the nuclear membrane, profiles of endoplasmic reticulum (ER) and in the membranes of the Golgi system; 2) PPO reaction in the nuclear membrane and profiles of ER, and 3) the presence of primary basophilic granules in the cytoplasm of the blasts. The EGIL scoring system has been used with the addition of the ultrastructural findings for the definition of BAL. In 24 cases the morphologic appearance and cytochemistry by LM of the leukemic cells were that of undifferentiated blasts. By cytogenetic evaluation abnormal karyotypes were detected in 16 patients, normal in 4 and unknown in 6 patients. Out of 26 patients 15 had the combination of T-ALL with myeloid phenotype and 11 had the B-lineage with myeloid features. Unexpectedly the blasts of 9 of the 26 BAL patients had basophilic differentiation as indicated by the presence of typical primary basophilic granules with MPO reactivity in a scattered pattern as previously reported in basophylic leukemia. Interestingly 7 of these 9 cases had a T lymphoid component. By contrast among 15 patients who had FAB M0, 9 had the B lineage markers. The remaining 2 patients had M1 and M7 features with T lymphoid phenotype. The contribution of EM studies enabled to establish the diagnosis of BAL in 11 out of 26 patients, in other 6 patients it allowed for the refinement of the AML subgroup such as basophilic leukemia and in the remaining 9 patients EM confirmed the diagnosis of BAL as defined by flow cytometry.

Conclusions

Our study identified a subgroup of children with acute leukemia in whom BAL could be suggested only by the addition of ultrastructural studies. EM could further refine and confirm the diagnosis of BAL in all the other cases. Of interest is the high incidence of basophylic differentiation among BAL pediatric patients, and its association with T lymphoid phenotype.

Disclosures:

No relevant conflicts of interest to declare.
Molecular Profiling of Cancer Testis Antigens in Chronic Lymphocytic Leukemia.
Authors: Dubovsky Jason A , Berchmans Emmanuel , Powers John J. , Hui-Yi Lin , Gao Yang , Sotomayor Eduardo , Pinilla Javier
Published: October 2019

Abstract 4701

Background

Chronic Lymphocytic Leukemia (CLL) is characterized by the progressive accretion of long-lived mature B-lymphocytes. Although the classical Rai and Binet staging is still commonly used, a new molecular understanding has identified specific signatures which could help predict disease progression and survival. Given the recent success of immunotherapeutic strategies and immunomodulatory drugs in the treatment of CLL we sought to identify potentially immunologically relevant targets and their relation to well known disease criterion.

Methods

Our study characterized the mRNA expression of 29 known cancer-testis antigens (CTAs) in 66 patients with CLL at varying stages of disease using a RT-PCR based expression panel. Relevant clinical criterion such as RAI stage, B2m, ZAP-70, IGVH mutational status, CD38, cytogenetics by FISH analysis, WBC count, age, gender, and treatment among others were then taken into account. The binary RNA expression data associated with the clinical and demographic factors were evaluated using chi-square or Wilcoxon rank sum analysis.

Results

Of the cancer-testis antigens tested, the MAGE family of CTAs revealed statistically significant correlations with multiple clinical criteria. Our analysis reveals a correlation between previous chemo-immunotherapy treatment and MAGE-A1, B2, E1, MAD-CT-2, SPA-17, and PAGE-5 expression. Beyond treatment, total white blood cell count was shown to have a significant association with MAGE family members A1, A3, and B2 expression. In addition, MAD-CT-2 and MAGE-B2 were significantly correlated with the expression of FMC-7 and SSX-4 and LAGE-1 correlated with the presence of B-cell symptomatology.

Conclusions

Preliminary RT-PCR based CTA phenotyping has unveiled interesting correlations to clinical criteria, opening multiple avenues for future immunotherapeutic interventions as well as possible prognostic value in CLL. Further investigation to better understand the biological value of this information in warranted.

Disclosures:

Pinilla: Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Bristol-Myers Squibb: Research Funding; exelixis: Research Funding.
Application of SENP-1, HIF-1α and VEGF Gene Expressions in Prognostic Prediction for Normal Karyotypic Acute Leukemia Patients with NPM1 + and FLT3-ITD-.
Authors: Huang Binbin , Xiu Bing , Liang Aibin
Published: October 2019

Abstract 4723

In our study, 26 BM species from 23 normal karyotypic acute leukemia patients (non-APL) with NPM+ and FLT3-ITD- gene mutation were collected in our department. Compared to healthy controls, SENP1, HIF-1alpha and VEGF mRNA expression in ALL and AML patients were elevated to 10.0, 13.2, 16.6 and 43.1, 97.75, 240.0 folds, respectively, by RT-real time PCR. The levels of SENP1, HIF-1αand VEGF mRNA in AML patients were greatly higher than those in ALL (p<0.05). SENP1 and HIF-1alpha expression levels were positively and significantly correlated (linear regression analysis, r = 0.88, p<0.05) both in ALL and AML. The correlation between HIF-1alpha and VEGF mRNA level was statistically significant (linear regression analysis, r = 0.34, p<0.05). Among AML patients, SENP1, HIF-1alpha and VEGF gene expressive level in non-response (NR) patients (n=7) were higher than patients with CR (n=9), p<0.01. For ALL patients, the expressive level (‘X±S) of SENP1, HIF-1alpha, VEGF gene in NR(n=3)were slightly higher than those in CR (n=4): 10.5±2.7, 6.8±3.4; 13.2±16.6, 3.9±3.0; 13.1±10.1,11.1±12.1. Elevated gene expression of SENP1, HIF-1alpha and VEGF indicate shorter survival time of patients, p<0.01.The levels of SENP1, HIF-1alpha and VEGF gene decreased after treatment in 3 AML patients. Thus, higher SENP1, HIF-1alpha and VEGF gene levels might be associated with shortened survival and worse clinical process. Therefore, although higher expression of SENP1, HIF-1alpha and VEGF gene might throw an impact on the prognostic prediction in acute leukemia patients, confirmation of this hypothesis need to be done in more large population.

Disclosures:

No relevant conflicts of interest to declare.
Viral Antigens Trigger Granzyme B Secretion by Human CD5+ B1 Cells in the Presence of Interleukin 21.
Authors: Sontheimer Kai , Blackwell Sue , Hagn Magdalena , Ebel Verena , Beyer Thamara , Simmet Thomas , Jahrsdoerfer Bernd
Published: October 2019

Abstract 4729

CD5+ B1 cells are thought to play a key role in innate immune responses by producing and secreting natural immunoglobulins, which bind to a wide range of bacterial, viral antigens as well as auto-antigens. Here we demonstrate that B1 cells from human cord blood are able to express the pro-apoptotic serine protease granzyme B (GrB) in response to viruses including tick-borne encephalitis virus, rabies virus, and hepatitis B virus. Using pharmacological tools we reveal that this response depends on B cell receptor stimulation, toll-like receptor 9 engagement and interleukin 21 (IL-21), a cytokine primarily found in the acute phase of viral infections. While up to 35% of freshly isolated B1 cells directly respond with GrB expression to stimulation with inactivated viruses in the presence of IL-21, similar, though lower reactions in CD5- B2 cells are only found after, but not before vaccination of the donor against the respective virus. Of note, GrB-expressing B1 cells feature a homogeneous CD19+CD5+GrB+CD20+CD27-CD38-IgD- phenotype and B1 cell-derived GrB is secreted in an enzymatically active state, reaching levels comparable to those secreted by activated cytotoxic cells. GrB induction requires activation of similar signaling pathways as in CTL and NK cells including members of the JAK/STAT pathway. Our findings suggest GrB secretion by B1 cells represents a novel innate immune response mechanism. GrB-secreting B1 cells may play a role in early anti-viral immune defense, and may contribute to elevated serum GrB levels found in various viral diseases. Further studies will elucidate whether CD5+ B1 cells, possibly in co-operation with NK cells, exhibit cytotoxicity towards virus-infected as well as tumor cells.

Disclosures:

No relevant conflicts of interest to declare.
Prognostic Implications of Procalcitonin and NT-ProBNP in Neutropenic Patients.
Authors: Young Kim Sun , Han Hye Young , Jeong Dae-Chul
Published: October 2019

Abstract 4743

Background

Procalcitonin (PCT) has been increasingly used as an inflammatory marker to identify patients with systemic infection. N-terminal pro brain natriuretic peptide (NT-proBNP) elevation can be used to predict future cardiac events and survival. The aim of this study was to evaluate the role of PCT and NT-proBNP measurements in febrile neutropenic patients in terms predicting outcomes in these patients.

Methods

Neutropenia was defined as neutrophil count of 500 cells/mm3 or less or a count of < 1,000 cells/mm3 with a predicted decrease to < 500 cells/mm3. Fever in a neutropenic patient is defined as a single measurement of oral temperature of more than 38.3 °C or a temperature of 38.0 °C for 1 hr. From July 2008 and May 2009, immunocompromized patients with neutropenia were screened for PCT, NT-proBNP at the time of diagnosis, 3 days later and 3 days after fever subsided.

Results

A total of 32 patients aged between 2.5 and 13.5 years (17 boys and 15 girls) were admitted because of neutropenic fever at the Chungnam National University Hospital. PCT levels were not significantly different among 3 different times of measurements, but NT-proBNP was significantly higher at 3 days after admission compared with those of at diagnosis and after fever was subsided. PCT and NT-proBNP levels at the time of diagnosis were significantly correlated with each other. Three patients with higher NT-proBNP showed symptoms of heart failure and arrhythmia but PCT was not significantly elevated in these patients.

Conclusion

PCT and NT-proBNP at the time of diagnosis were correlated with each other. PCT levels were not significantly changed among 3 different times of measurements but NT-proBNP was significant index of heart failure especially 3 days after fever developed.

Disclosures:

No relevant conflicts of interest to declare.
Mathematical Modeling in Optimizing Methotrexate-Based Chemotherapy.
Authors: Kasenda Benjamin , Rehberg Marcel , Franzem Melanie , Fritsch Kristina , Markert Anna , Finke Jürgen , Lebiedz Dirk , Illerhaus Gerald
Published: October 2019

Abstract 4766

Introduction

Optimization of doses and administration schedules for anticancer drugs is crucial for effective and safe treatment. Since chemotherapy (CT) remains based predominately on empiric data, the creation of a mathematical model describing the complex interplay between dosing, tumor-response, agents' clearance, and toxicity, presents the opportunity to optimize treatment strategies of established but also new drugs. Primary central nervous system lymphoma (PCNSL) is a rare disease whose incidence is rising. Standard treatment of methotrexat (MTX) based CT has been approved in several studies. Here we report the most recent development of a real-life mathematical model describing the relationship between MTX serum levels and myelosuppression to optimize current treatment protocols.

Materials and Methods

We retrospectively analyzed 38 patients (pts) diagnosed and treated for PCNSL from 2005-2009. Pts <65 yrs of age were treated according to the high-dose MTX induction protocol (MTX 8g/m2/4hrs followed by leucovorin rescue), whereas treatment of pts >65 yrs of age followed the elderly protocol (MTX 3g/m2/4hrs followed by leucovorin rescue). Leukocyte and MTX serum levels were documented. We used an extended version of the mathematical model by Friberg et al. (J Clin Oncol 2002) as basis for our computations. The model was fitted to the data by using a least-squares-error approach.

Results

First order elimination proves sufficient for describing the pharmacokinetics of MTX. The average elimination rate is 0.25 h-1 with similar inter-patient and inter-therapy variation: the STD is 0.03 h-1 among patients and 0.02 h-1 among treatments. Similar analysis of all present parameters highlights the overall robustness of our approach.

Conclusion

Our model makes it possible to predict myelosuppression after treatment with MTX following leucovorin rescue based on clinical data. The parameters estimated are in accordance with those provided by Friberg et al. and thus confirm the model's general relevance. Extensions of the model describing tumor size reduction by MTX will be implemented to describe the complex interplay between MTX serum levels, clearance and anti-tumor effect. Additionally, mathematical optimal control theory methods will be employed to compute optimized schedules, i.e. doses and timing for the drugs that minimize tumor size and do not violate toxicity constraints such as leukocyte count.

Disclosures:

No relevant conflicts of interest to declare.
LATE EFFECTS IN Hodgkin's POPULATION Treated BETWEEN 1980-2005 IN A SINGLE INSTITUTION.
Authors: Gini Guido , Caraffa Patrizia , Offidani Massimo , Trappolini Silvia , Bocci Caterina , Stronati Andrea , Corvatta Laura , Polloni Claudia , Gentili Silvia , Savini Agnese , Montanari Mauro , Capelli Debora , Poloni Antonella , Olivieri Attilio , Leoni Pietro
Published: October 2019

Abstract 4769

From 1980 to 2005 in our institution we've diagnosed and treated 361 patients (pts) affected by Hodgkin's disease. At the moment of diagnosis the median age of the patients was 29 years (range 13-69).About 72% of patients were in the stage I-II and 28% were in the stage III-IV. More than 54% of all patients received ABVD, 17% received MOOP, 10% MOOP/ABVD, 3% VBM and 1% received Stanford V scheme. Over 70% of all patients received radiotherapy (RT) with median dose of 4500 cGy (range 1000-16210). Over 94% of all patients obtained a Complete remission, however 11% relapsed. 130 patients among 361 are at this moment evaluable for late effects of chemo-radiotherapy.26 pts were treated in the period 1980-84 (25%) ; 6 pts treated 1985-89 (4,6%);21 pts treated in 1990-94 (16,1%); 24 pts treated in 1995-99 (19%) and 35 pts in the period 2000-2005 (27%). 50 pts among 130 (38,5%) present late effects related to the therapy. 24 pts (48%) present heart disease: 13 pts (40%) Valvulopathy,8 pts (25%) Ischemic heart disease, 7 pts (21%) Congestive heart failure, 4 pts (14%) Pericardic disease. Therapies of these patients were performed : In 1980-89 (11 pts), 6 pts (25%) received MOOP + RT,2 pts (8,3%) received M/A and 3 pts only RT. In 1990-99 (7 pts), 3 pts(12,5%) received ABVD +RT, 2 pts(8,3%) received MA+RT, 1 pt(4,1%) received ABVD and 1 pt(4,1%) received M/A. In 2000-05 (6 pts), 2 pts (8,3%) received ABVD+RT and 4pts(16,6%) received only ABVD. 18 pts present Endocrinological-disease, in all cases, represented by hypothyroidism; 16 pts among 18 had done Chemoteraphy and Radiotheraphy, while 2 pts received only Chemotheraphy. Besides 8 pts show both endocrinological and cardiological disease; in these cases 3 pts (37,5) had done ABVD+RT, 4 pts (50%) MOOP+RT and 1 patient (12,5%) only RT. Furthermore 26/130 (20%) developed a second neoplasm.

Conclusions

In our population 38% of patients had developed secondary late effects and we assess that about 15% of our Hodgkin's population die for a secondary late effect of therapies Although there are many variables for the development of a late effect we think that this analysis could lead to try to reduce the late toxicities of treatments.

Disclosures:

Offidani: Celgene: Honoraria; Janssen Cilag: Honoraria.
Single Agent Metronomic Cyclophosphamide in Refractory,Non-Hodgkin's Lymphoma.
Authors: Hamm Caroline , Kanjeekal Sindu M. , Mathews John , Alam Yasmin , Yoshida Sam , Elfiki Tarek
Published: October 2019

Abstract 4776

n this retrospective review, we report a case series of eight patients with refractory, heavily pre-treated non-Hodgkin's lymphoma(NHL), treated with low dose cyclophosphamide. Median number of prior treatment regimens was 3. The median disease free survival was 15.5 months with three patients in ongoing CR at the time of reporting with 15 - 36 months of followup. In a comparative group of 6 patients with other malignancies beside NHL (3 chronic lymphocytic leukemia, 2 breast cancer, 1 thymoma, and one small cell carcinoma) the median progression free survival was significantly worse at 3 months. Interestingly the thymoma had a progression free survival of 36 months.

Toxicity was minimal. One patient stopped because of thrombocytopenia. One of the patients with ongoing CR at 36 months stopped after 11 months of treatment. One patient died in CR of anal cancer after 15 months of treatment.

These results demonstrate an easily tolerable and surprisingly effective treatment option in patients with non-Hodgkin's lymphoma who are not candidates for bone marrow transplantation.

The mechanism of action of metronomic chemotherapy has been proposed as anti-angiogenic. We suggest an alternate mechanism of action: that of immunomodulation. We are suggesting this alternate explanation in view of the significant difference in the response rate between those disease that are known to immunogenic and those that may be less so, as well as the well-known immunogenic affect of cyclophosphamide. Further studies are ongoing to investigate this hypothesis.

Disclosures:

No relevant conflicts of interest to declare.
Combination of Enzastaurin and HDAC Inhibitors Synergically Induce Apoptosis in Lymphoma Cells.
Authors: Bodo Juraj , Sedlak Jan , Maciejewski Jaroslaw P. , Hsi Eric D.
Published: October 2019

Abstract 4783

Introduction

Histone deacetylase inhibitors (HDACis) are approved for use in the setting of cutaneous T-cell lymphoma with modest benefit. Enzastaurin is an investigational PKCβ inhibitor that has growth inhibitory and pro-apoptotic effects in both B and T-cell lymphoma. Specifically, enzastaurin-induced inhibition of PKC leads to rapid accumulation of β-catenin that triggers c-Jun dependent induction of p73, followed by apoptosis. We investigated the cytotoxicity and mechanisms of cell death of combination enzastaurin and low concentrations of HDACis in B-cell lymphoma and T-cell lymphoma cell lines and primary lymphoma/leukemia cells.

Experimental design

Apoptosis was measured by flow cytometry and PARP cleavage. Phospho-GSK3β (S9), pS6, phospho-c-jun (S73) and β-catenin were analyzed by Western blot or quantum-dot immunoflourescence as measures of PKCβ inhibition. Cytotoxicity was determined by WST-1 proliferation assay and colony forming cell (CFC) assays.

Results

As expected, enzastaurin induced dephosphorylation of GSK3β and S6RP associated with increased β-catenin expression followed by phosphorylation of c-jun (S73) and PARP cleavage in SU-DHL-6 (diffuse large B-cell lymphoma line) cells. Treatment with low concentrations of suberoylanilide hydroxamic acid (SAHA) showed slight or no changes in studied proteins. Combined enzastaurin/SAHA treatment resulted in strong synergistic apoptosis in two treated germinal center B-cell-like and two activated B-cell-like lymphoma cell lines, two T-cell lymphoma cell lines and four different primary lymphoma/leukemia samples. Similarly, combined enzastaurin/ valproic acid treatment induced synergistic apoptosis in SU-DHL-6 cell line, suggesting the synergy is generalizable to other HDACis. In comparison to the single agent treatment, combined enzastaurin/ SAHA treatment resulted in activation of proapoptotic MAPK, c-jun N-terminal kinase, further increase of phospho c-jun (S73) levels, increased FasL levels, and amplification of PARP cleavage. Quantitative immunofluorescence assay showed a more rapid increase of β-catenin levels with the combination than either agent alone. Furthermore, compared to the low dose SAHA treatment alone, hyperacetylation of histone H3 was detected in samples when enzastaurin was added in combination with low dose SAHA, likely the consequence of displacement of HDAC by β-catenin. In addition, no change in CFC output in normal bone marrow exposed to this combination was observed.

Conclusion

Enzastaurin/ HDACi therapy can synergistically inhibit growth and induce apoptosis in lymphoid malignancy through increased biochemical effects attributed to each agent. These data support further investigation of addition of PKCβ inhibitors to HDACi in order to increase their anti-lymphoma effects.

Disclosures:

No relevant conflicts of interest to declare.
Hepatitis B Virus Reactivation in Malignant Lymphoma with Occult HBV Carrier.
Authors: Fukushima Noriyasu , Mizuta Toshihiko , Urata Chisako , Tanaka Mariko , Yokoo Masako , Ide Masaru , Hisatomi Takashi , Sueoka Eisaburo , Kimura Shinya
Published: October 2019

Abstract 4794

Background

In surface antigen of hepatitis B virus (HBsAg)-positive carrier for anticancer treatment of malignant lymphoma, it is well recognized that reactivation of hepatitis B virus (HBV) occasionally occurs. However, there have been only a few studies of HBV reactivation in serum HBsAg-negative and hepatitis B core antigen (HBcAb)-positive occult HBV carriers. We looked at both retrospective and prospective studies to determine the prevalence, clinical course and risk factor of HBV reactivation during chemotherapy in lymphoma patients.

Patients and methods

Forty-nine of 128 (38.3%) lymphoma patients were HBsAg negative and HBcAb positive, and 25 of these patients were then given liver function tests and HBsAg tests monthly and serum HBV DNA every 3 months.

Results

HBV reactivation was observed in two patients (4.0%) who had received intensive chemotherapy including steroid and rituximab. Immediate administration of entecavir therapy after elevation of HBV DNA level was conducted, and this resulted in reduction of it and improvement of liver function test.

Conclusions

Rituximab plus steroid-containing regimens may increase the risk of HBV reactivation in HBsAgnegative and HBcAb-positive lymphoma patients. More ambitious prospective studies are required to establish clinically useful or cost-effective follow-up methods for control of HBV reactivation in lymphoma patients with occult HBV infection.

Disclosures:

No relevant conflicts of interest to declare.
Population Pharmacokinetic of Native Escherichia Coli Asparaginase.
Authors: Borghorst Stephan , Pieters Rob , Kuehnel Hans Juergen , Boos Joachim , Hempel Georg
Published: October 2019

Abstract 4803

Introduction

Native Escherichia Coli Asparaginase (ASNase) is an integral component in the therapy of acute lymphoblastic leukemia (ALL) and non-Hodgkin's Lymphoma (NHL). There is a great interindividual variability in treatment intensity in patients treated with the same dose of ASNase. Population pharmacokinetics (PopPK) provides the possibility to divide the overall variability of a population in an inter- and intraindividual element and to develop more precise dosing recommendations. Furthermore, pharmacokinetic parameters can be estimated as well as possible covariates that may influence the pharmacokinetics of the drug can be identified.

Patients and Methods

The model building dataset consisted of 16 patients (233 samples) receiving 5000 U/m2 ASNase (Asparaginase Medac®) 8 times according to the DCOG-ALL 10 treatment protocol. Asparaginase activity was measured in a randomized clinical Phase 2 study comparing the pharmacokinetic and pharmacodynamic of a newly developed recombinant ASNase with that of the established ASNase (Asparaginase Medac®)[R. Pieters et al. Blood. 2008 Dec 15. 112(13):4832-8]. The PopPK-model was developed using NONMEM (version VI) with First Order Conditional Estimation (FOCE) method and INTERACTION option.

Results

A linear 2-compartmental model with a combined proportional (0.9%) and additive (48.1U/l) error model described the data adequately. The pharmacokinetic parameters estimated were: Total systemic clearance 0.135 ± 12.8% l/h/70kg, volume of distribution of the central compartment 4.27 ± 13.1% l/70kg, volume of distribution in the peripheral compartment 0.83 ± 80.4% l/70kg and intercompartmental clearance 0.058 l/h/70kg (mean ± interindividual variability). Body weight was identified as the most important covariate. Validity of the model was verified by simulating different dosages of ASNase (2500U/m2 and 10000U/m2) in induction and reinduction of the ALL-BFM treatment protocol. The median and mean ASNase activity was compared with published data [E. Ahlke et al. Br J Haematol. 1997 Mar. 96(4):675-81 and Boos et al. Eur J Cancer. 1996 Aug. 32A(9):1544-50]. Furthermore pharmacokinetic data obtained by a noncompartmental analysis [R. Pieters et al. Blood. 2008 Dec 15.112(13):4832-8] were compared with the pharmacokinetic data estimated by the PopPK model. Both procedures indicated on face validity of the PopPK model.

Conclusion

This PopPK analysis provides the first step in the development of a PopPK model for ASNase. Face validity of the PopPK model could be demonstrated and will be confirmed with an independent dataset.

Disclosures:

Pieters: Medac GmbH: Research Funding. Kuehnel:Medac GmbH: Employment. Boos:Medac GmbH: Honoraria. Hempel:Medac GmbH: Honoraria.