bluuebunny/crossref_metadata_2025 huggingface dataset:
Upregulation of HLA-G Gene Expression in Malignant Hematopoietic Cells Treated with 5-Aza-2′- Ddeoxycytidine.
Authors: Alsabty Firas , Mistrik Martin , Polakova Katarina
Published: October 2019

Abstract 4567

Introduction

Human leukocyte antigen G (HLA-G) is a nonclassic HLA class I antigen with restricted distribution in normal tissues. It exerts multiple immunregulatory functions that have been suggested to contribute to the immune evasion of tumour cells. Ectopic HLA-G expression observed in some pathological conditions such as malignant transformation may be triggered by epigenetic modifications such as DNA demethylation or histone acetylation.

Materials and Methods

Mononuclear cells were isolated from peripheral blood of newly diagnosed previously untreated patients with acute myeloblastic leukemia (AML) (n=9) and chronic lymphocytic leukemia (CLL) (n=5) by standard Ficoll-Hypaque density gradient centrifugation. Isolated cells were resuspended in RPMI 1640 medium supplemented with 2mM L-glutamine, 200 μg/ml gentamicin, 0.125 μg/ml amphotericin B and 10% heat-inactivated fetal bovine serum. Demethylating treatment of cells was carried out with100 μM 5-aza-2x- deoxycytidine (AdC) (Sigma) for 3 days. Human choriocarcinoma cell lines JEG3 and JAR (ATCC, Rockville, MD) were used as HLA-G positive and negative controls, respectively. Real time polymerase chain reaction (RT-PCR) and semiquantitative RT-PCR were performed using the ABI Prism 7000 Sequence Detection System and AmpliTaq Gold DNA polymerase to detect HLA-G mRNAs transcriptions. The HLA-G protein expression was examined by western blot analysis using mAb 4H84.

Results

HLA-G transcripts in AdC untreated leukemia samples were demonstrated in 3 out of 5 patients (60%) with B-CLL and in 5 out of 9 patients (56%) with AML. Treatment with demethylating agent AdC resulted in up-regulation of HLA-G transcription and expression of HLA-G protein in 5 out of 8 (63%) examined leukemia cell lines (Table 1).

Conclusions

we conclude that DNA methylation is an important control mechanism of HLA-G gene expression, and treatment of human leukemia with demethylating agent AdC may up-regulate HLA-G gene expression and induce HLA-G protein synthesis in some patients that may allow leukemic cells to escape recognition and destruction by cytotoxic T-cells or NK cells. Therefore patients should be monitored for HLA-G expression in order to follow risk of AdC therapy.

Disclosures:

No relevant conflicts of interest to declare.
Mesenchymal Stem Cells From Patients with Acute Myeloid Leukemia (AML) Can Differentiate Into B-Cells in NOD/SCID/IL-2Rγ-/- Mice.
Authors: Wang Rui-Yu , Shi Yue-Xi , Zeng Zhihong , Schober Wendy D. , McQueen Teresa J. , Hopwood Vicki L. , Korchin Borys , Konopleva Marina , Andreeff Michael
Published: October 2019

Abstract 4573

Human mesenchymal stem cells (MSCs) derived from bone marrows are characterized by high proliferative potential and pluripotentiality to differentiate into multiple lineages such as osteo-, chondro-, and adipogenic cells. MSC express CD105, CD73 and CD90, but not CD45, CD34, CD14 or CD11b, CD79alpha or CD19 and HLA-DR surface molecules. In this study, we observed that MSC derived from the bone marrow of four AML patients differentiated into B-cell lymphoblasts with NOD/SCID/IL-2Rg-/- engraftment potential.

MSC cell lines were established by culturing adherent cells from newly diagnosed AML (n=4) age 20 to 74 years in alpha-DMEM medium supplement with 20% fetal bovine serum. Surface antigen phenotype analysis and G-banding karyotype analysis were performed in passage 2 to 4. FACS-sorted CD90 positive cells were then intravenously (I.V.) injected into NOD/SCID/IL-2Rg-/- (NOG) mice via tail vein (n=9) or into the bone marrow (n=3). Circulating cells were analyzed for CD19, CD33, CD34, and CD90 expression on day 36, 45, 60, 75 after injection of MSC.

Results

1) G-banding showed normal karyotype in all MSC; 2) Injected MSC engrafted and differentiated in NOG mice. Surprisingly, CD19 positive cells were found in all samples starting on day 36 (table) and increased on day 60 and 75 (from d36: 6.9±3.5%, d45:0.7±0.1%, d60:2.6 ± 1.6% and d75: 9.3 ± 1.0%); 3) CD90 positive cells were found on day 45 (range from 0.07-3.96% and decreased to 0.1-0.5% on day 75). Low percentage of CD33 (day 45: 0.19-0.78% and day 60: 0.12-2.53%) and CD34 positive cells (day 45: 0.32-1.9% and day 60: 0.21-2.39%) were observed before day 60 and were undetectable by day 75.

Table shows the percentages of CD19+ cells found in circulation in NOD/SCID/IL-2Rg-/- (NOG) mice after MSC I.V. or intra-bone marrow injection. (* Mice died after phlebotomy.)

Conclusion

Human MSC derived from AML bone marrows have the capacity to differentiate into CD19 positive B lymphocyte in NOG mice in vivo. It has previously been reported that AML can be propagated by a leukemic stem cell with lymphoid characteristics (Cancer Cell 2006, 10, 363-74). Data reported here suggest the possibility that AML-derived MSC give rise to lymphoid cells that engraft in NOG mice. This unexpected finding could shed light on the role of stroma cells in the pathogenesis and propagation of leukemias.

Disclosures:

No relevant conflicts of interest to declare.
Homing and Engraftment of Bone Marrow Derived Cells in Irradiated Mouse Lungs.
Authors: Salimi Umar , Rajagopalan Malolan , Rwigema Jean-Claude , Dixon Tracy , Shen Hongmei , Epperly Michael W. , Stripp Barry , Greenberger Joel
Published: October 2019

Abstract 4600

To determine the degree to which murine bone marrow migrates to and can repopulate damaged lung, we studied lung stem cell expression of the Clara Cell Specific Protein (CCSP). Several different models have been used to deplete the lung stem cells. We used CCtk mice which expresses the HSV-tk under control of the CCSP promoter. Subcutaneous administration of gancicylovir (50 mg/ml) over a 7 day period using Alzet osmotic pumps results in the reduction of CCSP RNA and CCSP positive cells. To determine if a reduced dose of gancicylovir reduced CCSP cells to a nonfatal degree and opened a stem cell niche for homing of hematopoietic cells, CCtk mice had osmotic pumps placed subcutaneously containing a lower dose of 25 mg/ml of gancicylovir. The lungs were explanted at serial times up to 7 days after pump placement. RNA was extracted and real time polymerase chain reaction (RT-PCR) using primers specific for CCSP was used to quantitate CCSP expression. CCtk mice treated with gancicylovir showed a 70% reduction of lung CCSP expression. CCtk mice implanted with osmotic pumps containing 25 mg/ml gangcicylovir and injected intravenously with bone marrow from sex-mismatched GFP+ transgenic FVB male mouse 2 days later were followed for thirty days. The mice were sacrificed, lungs removed, single cell suspensions prepared, stained with a PE labeled anti-CD45 monoclonal antibody and analyzed by flow cytometry for GFP positive, CD45 negative cells. No GFP+, CCSP+ cells cells were detected. Injection of FVB mice with naphthalene (which kills differentiated but not stem CCSP+ cells) at 200 mg/kg intraperitoneally resulted in an 87% decrease in CCSP expression. When injected with bone marrow cells from GFP+ transgenic mice at serial times to 7 days and sacrificed at day 30, again no GFP+ CCSP+ cells were identified. In a third attempt to remove additional CCSP cells and prepare sites for homing of marrow cells, FVB mice were treated with naphthalene (200 mg/kg) followed by 20 Gy irradiation to the lungs 48 hr later, then injected with GFP+ bone marrow, 24 hr later. The mice were sacrificed 30 days later, and examined for GFP+, CD45- cells. Low numbers of GFP+ CCSP+ cells were detected. The data indicates a low baseline level of hematopoietic stem cell potential to home and differentiate to CCSP positive lung cells.

Supported by NIH-2R01CA119927-08A1.

Disclosures:

No relevant conflicts of interest to declare.
The Role of Periodic Limb Movement in Sleep of Patients with Sickle Cell Disease.
Authors: Roizenblatt Marina , Roizenblatt Suely S , Vicari Perla , Arruda Martha M A S , Chediek Felipe , Abrantes Fabiano , Morais Antonio V , Tufik Sergio , Figueiredo Maria Stella
Published: October 2019

Abstract 4609

Background

In last decade, there has been growing research in sleep disorders associated with sickle cell disease (SCD), particularly focusing on oxyhemoglobin desaturation. The coexistence of periodic limb movement (PLM) and anemia is well known, however it remains controversial to which extent dopamine deficiency, lower serum ferritin levels or iron deficiency contribute to the pathophysiology of PLM in anemia.

Objective

The aim of this study is to evaluate the relationship between PLM and other manifestations of SCD

Methods

Eight male patients with SCD were age and BMI-matched to 16 healthy controls (1:2) for sleep evaluation by clinical inventory and polysomnography (PSG). PLMS were defined as limbs movements lasting 0.5–5 seconds that recur every 5 to 90 seconds in a series of ≥ 4. Serum levels of ferritin and hemoglobin were assessed.

Results

Comparing to controls (aged 28.6 ±5.9 yrs, BMI of 22.1 ±3.8 kg/m2), SCD patients (aged 28.0 ±7.3 yrs, BMI of 21.4 ±4.6 kg/m2 p>0.05, both) scored their sleep satisfaction as moderate (75% vs. 12.5%, p=0.005), whereas 50% of controls scored it as very satisfactory (vs. none of the SCD patients, p=0.02). Awakenings in the middle of the night were frequently reported by SCD patients (87.5% vs. 43.7%, p= 0.05) as well as pain complaints (87.5% vs. 37.5%, p=0.03). PSG data revealed that, compared to controls, SCD patients exhibited decrease in sleep efficiency (76.7% ±12.6 vs. 87.9% ±4.8, p=0.004), in REM sleep percentage (10.6 % ±6.0 vs. 20.3% ±5.8, p=0.001), and increase in wake after sleep onset (65.3%±38.9 vs. 32.4% ±20.2, p=0.012), arousal index (16.3/hour ±8.5 vs. 9.5/hour ±4.9, p=0.021), PLM index (13.1 ±10.1 vs. 2.8 ±3.3, p= 0.001), apnea hypopnea index (8.8/hour ±5.6 vs. 3.4/hour ±5.0, p=0.025). Lower mean oxyhemoglobin saturation (88.0 mmHg ±3.4 vs. 92.4 mmHg ±5.4, p=0.049), and hemoglobin levels (8.7g/dl ±1.1 vs. 15.8g/dl ±0.7, p<0.001) were also verified in SCD patients. Interestingly, ferritin levels did not differ between groups (209.6±132.8 in SCD patients vs. 214.8 ±142.2, p=0.949). Linear regression showed that SCD condition represented an independent risk feature for PLM (R2 = 0.51, p<0.001). Correlation between PLM and the number of comorbities related to SCD was substantial (r=0.79, p=0.02)

Conclusions

Subjective sleep complaints reported by patiens with SCD were supported by PSG findings of sleep disruption and lower mean oxyhemoglobin saturation. Increase in PLM in SCD patients correlated the number of comorbities of the disease. In contrast with the literature, ferritin did not play a role in the pathophysiology of PLM in these patients.

This study was supported by FAPESP CEPID 98/14303-3 and AFIP

Disclosures:

No relevant conflicts of interest to declare.
Pregnancy as a Risk Factor for the Patients with Hodgkin Disease: Preliminary Report.
Authors: Grudeva Janet G.
Published: October 2019

Abstract 4636

Pregnancy as a risk factor for the patients with Hodgkin disease: preliminary report

Grudeva-Popova J1, Nenova I1, Spasova M2

1Department of Oncology and Hematology, 2Department of Pediatrics and Medical Genetic, University Hospital “St George” – Plovdiv, Bulgaria

Hodgkin disease is a potentially curable lymphoid malignancy, affecting predominantly young age. However, subsequent pregnancy in women in child-bearing age, previously diagnosed with Hodgkin disease, may raise some medical and ethical considerations regarding the prognosis of both mother and child. There are few published reports with controversial results about the influence of pregnancy on the duration of remission and overall survival of this subgroup of patients. The objective of the study is to analyze the long-term sequelae of subsequent pregnancy for women previously diagnosed with Hodgkin disease.

Patients and methods

We studied retrospectively 224 patients with Hodgkin disease, diagnosed, treated and followed-up during a 15-year period (1994-2009) at the Department of Hematology and the Department of Oncology and Hematology, University Hospital. Women in child-bearing age numbered 51, and pregnancy after the diagnosis of Hodgkin disease occurred in 5 of them. We analyzed the prognostic factors and possible influence of pregnancy on the duration of remission, and overall survival by logistic regression model and κaplan-Meier survival estimation.

Results

Relapse occurred in 3 out of 5 women with Hodgkin disease. Subsequent pregnancy with fatal outcome occurred in 2 of the 3 soon after delivery. We identified the following prognostically unfavorable factors in this subgroup of patients: advanced clinical stage, presence of B symptoms at the diagnosis of Hodgkin disease, lymphocyte depletion histology, and short time interval between the end of therapy and pregnancy. For the other 2 women pregnancy ended successfully for the mother and child, possibly because of a time interval exceeding 3 years between occurrence of remission and the pregnancy. Median survival time of the observed group was 73 months (95% CI: 10-137) and was shorter, compared to the survival time of the patients in the control group: 212 months (95%CI: 136-288).

Conclusion

The preliminary results of the study of this small subgroup of patients with Hodgkin disease show that women in child-bearing age previously diagnosed with Hodgkin disease, particularly with unfavorable prognosis, have increased relapse risk in case of subsequent pregnancy. The relapse risk appears to be higher in the first 3 years after remission induction.

Disclosures:

No relevant conflicts of interest to declare.
Optimal Post-Dose Levels of Cyclosporine A at a Higher Peak Concentration Are Correlated with the Area Under the Concentration-Time Curve of Twice-Daily Infusion and Oral Administration in Allogeneic Hematopoietic Stem Cell Transplantation.
Authors: Inoue Yasuyuki , Saito Tasuku , Ogawa Kohei , Nishio Yuji , Kosugi Seiju , Suzuki Yoshinori , Shibuya Yasushi , Kato Masayuki , Takahashi Masatomo , Miura Ikuo
Published: October 2019

Abstract 4656

Introduction

Cyclosporine A (CyA), a calcineurin inhibitor, is widely used as an immunosuppressor for the prevention of acute graft-versus-host disease (aGVHD) in allogeneic hematopoietic stem cell transplantation (alloHSCT). However, the optimal regimen of CyA has not yet been unequivocally established. Among liver transplant patients, it is important to maintain a peak CyA concentration and to monitor area under the concentration-time curve (AUC) and 2 h after CyA dosing (C2) for prophylaxis against graft rejection. We investigated the pharmacokinetics of CyA delivered by twice-daily infusion and oral administration maintained with a peak level above 1000 ng/ml to keep AUC 0-24 higher than 10000 ng-h/ml in 10 patients with hematological malignancy who underwent alloHSCT.

Methods

CyA was started as a twice-daily infusion at 1.5 mg/kg and then orally administered at twice the infusion dose in order to maintain the trough blood concentration between 200 and 500 ng/ml and with a peak level above 1000 ng/ml. Serial blood samples were collected at 0, 1, 2, 3, 5, 8, and 12 h after CyA dosing (C0,C1,C2,C3,C5,C8,C12) on days 14-21 after transplantation and on days 7-14 after switching to oral administration, and the AUC was calculated.

Results

The mean AUC0-12 for twice-daily infusion and oral administration was 7443±998.5 ng-h/ml and 7185±1714 ng-h/ml, respectively. Among all the patients, the AUC0-24 for both CyA twice-daily infusion and oral administration was higher than 10000 ng-h/ml. Two close relationships were observed between AUC0-12 and the C3 level for infusion and between AUC0-12 and the C8 level for oral administration. Engraftment was achieved in all cases, and two patients suffered from grade 2 aGVHD. None of the patients had grades 3-4 aGVHD or other serious complications.

Conclusion

This strategy was well tolerated, and the C3 level for twice-daily infusion and the C8 level for oral administration were the optimal points for monitoring of CyA concentration in the early phase of alloHSCT.

Disclosures:

No relevant conflicts of interest to declare.
Lymphoproliferative Response to Herpes Simplex Virus, Cytomegalovirus, Epstein-Barr Virus, Varicella Zoster Virus, Human Herpes Virus 6, 7 and 8 Antigen Stimulation in Pediatric Allogeneic Stem Cell Transplant Recipients.
Authors: Cheng Frankie Wai Tsoi , Chan Paul Kay Sheung , Leung Wing Kwan , Lee Vincent , Shing Matthew MK , Li Chi Kong , Leung Ting Fan
Published: October 2019

Abstract 4663

Introduction

Delayed immune reconstitution is a major cause of transplant-related mortality in allogeneic stem cell transplant setting. Reactivation of herpes viruses is a common post-transplant complication. In this study, we evaluate the recovery of cell-mediated immunity (CMI) to various herpes viruses by measuring lymphoproliferative response (LPR) to specific recall antigens.

Materials and Methods

Cell-mediated immunity was evaluated by the in-vitro lymphoproliferative response of peripheral blood mononuclear cells (PBMC) to specific purified HSV, VZV, CMV, EBV, HHV-6, 7, 8 antigens. Results were expressed as stimulation index (SI). SI≥a3 was regarded as positive lymphoproliferative response (LPR). Serial measurements were made at before transplant, and monthly during the initial 6 months post-transplant then quarterly till 12 months post-transplant.

Results

From 2001-2004, 36 patients (M=19; F=17) with median age 10.5 years old were recruited. Hematological malignancies accounted for 58.3% of cases. Most transplants were from matched siblings (MSD) with peripheral blood stem cells (PBSC) as the source of stem cells. Altogether 50% of subjects showed positive LPR to HSV, CMV and VZV antigens at 2-4 months post-transplant; A significant upsurge of LPR were observed at 4-6 months post-transplant. However, no positive LPR to HSV, HHV-6,7 and 8 antigens were observed. The antibody status of donor and recipient for HSV, CMV and VZV were associated with the timing of recovery of CMI. Donor choice and stem cell source were important determinants of eventual LPR at day 100 post-transplant.

Conclusion

More than half of transplant recipients developed satisfactory LPR to HSV, CMV and VZV at 2-4 months after transplant. Pre-transplant serostatus of donor and recipient, donor choice and stem cell are important determinants of LPR to herpes viruses.

Disclosures:

No relevant conflicts of interest to declare.
Despite Tolerance of Imatinib Prior to Transplant, Imatinib Induces Prolonged Exanthematous Pustulosis in a Patient with Sclerodermatous Graft-Versus-Host Disease: A Case Report.
Authors: Bishara Michael , Hamm Caroline
Published: October 2019

Abstract 4670

Treatment of sclerodermatous chronic graft versus host disease (cGVHD) remains a daunting challenge. Despite developments in prophylaxis for cGVHD, supportive care measures remain as the primary mode of therapy, with little evidence of treatments that reverse the process. Steroids and immunosuppressants are often ineffective, and extracorporeal photopheresis has been used with varying success. Recently, case reports have reported on the successful use of imatinib in treating sclerodermatous GVHD (Moreno-Romero et al, 2008; Leonardo Magro et al., 2009). We report a case of a 40-year-old male patient with extensive sclerodermatous cGVHD. In 2005 he underwent an unrelated stem cell transplant (SCT) for chronic myeloid leukemia. His transplant course was complicated by thrombotic thrombocytopenic purpura (TTP) associated with all standard immunosuppressive drugs (cyclosporine, tacrolimus and sirolimus). Two years later, the patient developed sclerodermatous GVHD and was given plaquenil, steroids, and photopheresis to manage this complication. Unfortunately, after months of treatment, he showed progressive disease, including increased contractures, and difficulties with inspiration. Based on case reports, and the patient's tolerance of imatinib prior to SCT, he was started on imatinib for his sclerodermatous GVHD. Within 5 days he developed generalized erythema, pustular rash, and skin sloughing (Figure 1). He was hospitalized for this severe reaction and was treated with high dose steroids after which, his symptoms began to improve. Resolution of erythema did not occur until months following initial exposure. Histological differential diagnosis included pustular psoariasis and Sneedon-Wilkinson syndrome. The skin biopsy revealed subcorneal pustular dermatosis (Figure 2). In view of this history, it is suggested that the exanthematous pustulosis was induced by imatinib in this patient. We recommend that close attention is paid to patients with cGHVD who are treated with imatinib and to intervene early in those who develop severe cutaneous reactions by stopping imatinib and starting appropriate therapy.

Figure 1 SEQ – Imatinib induced generalized erythema, pustular rash, and skin sloughing developed five days into treatment. Figure 1. SEQ – Imatinib induced generalized erythema, pustular rash, and skin sloughing developed five days into treatment. Figure 2 SEQ - skin biopsy revealing subcorneal pustular dermatosis Figure 2. SEQ - skin biopsy revealing subcorneal pustular dermatosis

Disclosures:

No relevant conflicts of interest to declare.
Prognostic Significance of Beta-2 Microglobulin (B2M) in Adult Acute Lymphoblastic Leukemia (ALL).
Authors: Thomas Deborah A. , O'Brien Susan , Faderl Stefan , Cortes Jorge , Borthakur Gautam , Burger Jan A. , Garris Rebecca , Wierda William G. , Keating Michael J. , Kantarjian Hagop M.
Published: October 2019

Abstract 4679

Beta-2 microglobulin (B2M) is 12-kDa light chain protein that binds to the alpha chain of class 1 MHC molecules, dissociated into extracellular fluids after degradation of HLA. Elevated levels of serum B2M have historically been associated with a poor prognosis in several lymphoproliferative disorders. The prognostic relevance of B2M in myeloid disorders such as acute myelogenous leukemia and myelodysplastic syndrome has also been noted, in some cases providing further refinement of existing prognostic models. The significance of pretreatment serum B2M levels has not been extensively studied in adult ALL. In our historical experience with the conventional VAD (vincristine, doxorubicin, dexamethasone) regimen, elevated levels of B2M were associated with a lower complete remission (CR) rate, worse survival (OS), and higher propensity for development of CNS leukemia, after adjusting for renal dysfunction and older age [Kantarjian, Am J Med 93:599, 1992]. The analysis was somewhat limited by the strong negative impact of the mature B-cell ALL subset were the significantly high levels of B2M were associated with poor prognosis in the era prior to intensive multi-agent chemotherapy regimens. We assessed the associations of pretreatment B2M levels with outcome in 320 adolescents and adults with de novo ALL (Burkitt leukemia/lymphoma, Philadelphia chromosome positive ALL, and lymphoblastic lymphoma subtypes were excluded from the analysis) treated with the more intensive hyper-CVAD regimen (fractionated cyclophosphamide, vincristine, doxorubicin, dexamethasone alternating with methotrexate and cytarabine) and its variants (modified hyper-CVAD with or without anthracycline intensification and with or without rituximab). Median serum B2M level was 3 mg/L (range 0-14.7). As observed previously, the CR rate was lower for patients (pts) with elevated levels of B2M, an effect which persisted after excluding the elderly subset and accounting for renal dysfunction (93% versus 98%, p=.02). The 3-yr rates for CRD and OS were also worse if B2M was elevated above the median, 55% vs 70% (p=0.09) and 60% vs 70% (p=.01), respectively. These findings confirm the prior observations in a larger more homogenous population of de novo ALL treated with intensive chemotherapy. A limited multivariate analysis identified the independent prognostic influence of B2M on survival. Additional analyses will be conducted to determine how these observations can be best incorporated into clinical practice.

Disclosures:

No relevant conflicts of interest to declare.
Utility of Gene Scan for Detection of MRD in Precursor B-Cell Neoplasm.
Authors: Abdel-ghaffar Hasan Ahmed , Salem Dalia A. , Evans Paul , El-deen Omar Sharaf , Shamaa Sameh S.
Published: October 2019

Abstract 4720

Reliable Techniques are needed for detection of MRD to evaluate the effectiveness of the treatment in patients with precursor B-cell leukemia/lymphoma and to predict impending relapse prior to clinical manifestations. Currently, PCR analysis of Ag receptor gene rearrangements and flowcytometry (FC) represent the most clinically useful techniques for MRD analysis in ALL. In this study, we assessed the applicability of a non–sequence based PCR technique for the detection of MRD namely gene scan (GS) as compared to multicolor FC and real time quantitative PCR (RQ PCR). As a pilot study, results of MRD using the three techniques were assessed in 8 follow-up bone marrow samples of patients with precursor B-Cell leukemia/lymphoma. MRD detection using fluorescent GS was positive in 87.5% of samples compared to 37.5% using four colour FC. RQ-PCR was applied on 4 samples using patient-specific allele-specific oligonucleotide and revealed the presence of MRD in all of them. Only 3 of those sample gave positive result for MRD using both 4-colour FC and GS. Fluorescent GS technique as a known simple, rapid, and cost effective method might be a reliable technique for detection of MRD in precursor B-Cell leukemia/lymphoma as it showed a considerable sensitivity compared to four colour FC. However, a large-scale study for accurate assessment of sensitivity and specificity of Fluorescent GS in comparison with RQ-PCR as a gold standard technique is now undergoing.

Disclosures:

No relevant conflicts of interest to declare.
Distribution of Cytoskeleton Protein Nestin in Acute Leukemia.
Authors: Du Xin , Yang Xuhui , Ling Wei , Tian Xia , Zou Maoxian , Chen Zhengguang , Wang Tao , Weng Jianyu , Wu Suijing , Guo Rong , Zhang Xiuming , Lahn Bruce T. , Xiang Andy Peng
Published: October 2019

Abstract 4721

Objective

Nestin was detected in some tumor cells and nestin expression was positive relative with malignancy of glioma, angioma and melanoma. Nestin maybe was considered as marker of tumor malignancy and cell proliferation. There was no report about nestin expression in acute myeloid leukemia (AML) and acute lymphoblast leukemia (ALL). Herein, we describe the clinicopathological and immunohistochemical features of 48 patients with acute leukemia, introducing nestin as an additional marker that identifies these tumors .Meanwhile, Nestin and ki67 antigen expression were done to identify the relation of nestin and cell proliferation in AML, and the correlation between histoscore of nestin expression on leukemic blasts and patients' complete remission rate (CR) and overall survival (OS) was analyzed in AML.

Methods

Nestin and ki67 antigen expression in a series of 37 AML samples, 11 ALL samples, 2 multiple myeloma samples (MM) and 2 chronically myeloid leukemia samples (CML) were done with immunohistochemistry. Histoscore of nestin expression on leukemic blasts with AML and ALL was done and analyzed with independent t-test. The correlation of histoscore of nestin expression with patients' CR rate in AML was analyzed by the method of chi-square test. Patients' OS analysis was determined by the method of Kaplan and Meier, with log-rank analysis to estimate statistical significance in AML. P values of <0.05 were considered significant.

Results

Among 37 cases with AML, Nestin expression was observed in most leukemic blasts, and matural granulocytic cell also expressed nestin in 30 cases .The ratio of tumor cell expressing nestin and total tumor cells was over fifty percents in AML in 14 patients. The ratio of tumor cell expressing nestin and total tumor cells was over ten percents in AML in 7 patients. The ratio of tumor cell expressing nestin and total tumor cells was less ten percents in AML in 9 patients. Nestin expression was not observed in 8 patients. Where ki67 expressed in AML, nestin expressed intensely in 7 cases with AML. But other cases did not express ki67. However, histoscore of nestin expression on leukemic blasts in AML was not correlated with patients' CR (p=0.701) and OS (p=0.245). Nestin immune-reactivity in ALL mainly focused on matural granulocytic cells, matural lymphocytic cells and tumor cells did not express nestin. Compared with AML, there was significant statistical significance (p=0.007).

Conclusions

These findings constitute the first conclusive evidence that stem cell marker nestin expression in leukemic blasts with AML, CML, MM, but nestin did not express in leukemic blasts with ALL. Nestin may be a useful immunohistochemical marker for identifying AML and ALL. Nestin expression was not correlated with cell proliferation, patients' CR and OS in AML.

Disclosures:

No relevant conflicts of interest to declare.
Leukemic Presentation Is Predictive Indicator for Relapse for Patients with Follicular Lymphoma Treated with Rituximab Containing Initial Therapy.
Authors: Kodaira Makoto , Takeuchi Kengo , Nara Eriko , Nakano Kenji , Ueda Kyoko , Yamada Shuhei , Mishima Yuko , Yokoyama Masahiro , Terui Yasuhito , Hatake Kiyohiko
Published: October 2019

Abstract 4763

INTRODUCTION

Peripheral blood involvement (leukemic presentation) is considered as a part of bone marrow involvement and detected about 18% of bone marrow (BM) involvement of follicular lymphoma (FL). It is not known whether leukemic presentation is an adverse prognostic factor in rituximab era.

METHOD

We retrospectively evaluate prognostic value of peripheral blood involvement in patients with follicular lymphoma received rituximab containing regimen as an initial therapy from October 2000 to January 2009. Leukemic presentation was defined by morphologic identification of an abnormal lymphoid population in the peripheral blood.

RESULT

Total 129 patients were treated with rituximab containing initial therapy. Bone marrow involvement was detected in 39 /108 (36.1%) patients and leukemic presentation was identified in 8 / 39 (20.5%) of patients with BM involvement. Leukemic presentation shows significant poorer progression free survival than BM involvement without peripheral blood involvement. (2yr PFS 23.4% vs 73.3% p=0.015) Multivariate analysis conducted by Cox proportional hazard analysis including five variables of FLIPI revealed Hb <12g/dl (HR 2.769 95% CI: 1.111-6.902 p=0.029) and leukemic presentation (HR 5.127 95% CI: 1.898-13.853 p=0.001) were significant independent adverse prognostic factor for PFS.

CONCLUSION

Leukemic presentation is a significant adverse prognostic factor of FL in rituximab era. Prognostic value of leukemic presentation should be validated in further study.

Disclosures:

No relevant conflicts of interest to declare.
Phase I Trial Examining Addition of Gemcitabine to CHOP in Intermediate Grade NHL.
Authors: Reagan John L. , Butera James N. , Rosmarin Alan G. , Nadeem Ahmed , Schiffman Fred J. , Sikov William , Winer Eric , Mega Anthony
Published: October 2019

Abstract 4765

BACKGROUND

Gemcitabine induces a 20% response as single agent therapy in patients with relapsed or refractory NHL. We report phase I findings of gemcitabine in combination with standard CHOP chemotherapy with G-CSF support for intermediate grade NHL. The protocol was modified during enrollment to include rituximab in CD 20+ lymphomas.

METHODS

Patients received CHOP plus gemcitabine at 500 mg/m2 (Cohort 1) or 750 mg/m2 (Cohort 2) on days 1,4 of each 21 day cycle. Accrual was suspended once each cohort was filled. Dose escalation occurred after all patients in the cohort were determined to not have a dose limiting toxicity.

RESULTS

Between 4/02 and 5/04 10 patients were enrolled and completed the study treatment (6 in cohort 1, 4 in cohort 2). In Cohort 1, grade 3 toxicities included neutropenia, anemia, neuropathy, and constipation. Grade 4 toxicities were febrile neutropenia, and thrombocytopenia. In Cohort 2, grade 3 toxicities included neutropenia, thrombocytopenia, mucositis, anemia and intestinal obstruction. Grade 4 toxicities included febrile neutropenia, neutropenia, and thrombocytopenia. One patient developed MDS 36 months after chemotherapy. Three of four patients in Cohort 2 developed dose limiting toxicities (mucositis and thrombocytopenia) requiring dose reduction of gemcitabine after cycle 1. Overall, the survival rate at 2.5 years was 71%.

CONCLUSIONS

This Phase I trial concludes that gemcitabine 500mg/m2 on days 1 and 4 of each 21 day cycle is the maximum tolerated dose when combined with standard CHOP chemotherapy with G-CSF support for intermediate grade NHL. Response rates are encouraging for this novel chemotherapeutic regimen.

Disclosures:

Off Label Use: Gemcitabine was added to standard CHOP chemotherapy in this trial.. Sikov:Eli Lilly: Honoraria.
Extranodal NK/T-Cell Lymphoma, Nasal Type: Experience of Management in a Referral Hospital in a Development Country.
Authors: Castellanos-Sinco Humberto , Rivas-Vera Silvia , Argote Victoria E. Ferrer , Peñafiel Christian Omar Ramos , Salinas-Torres Ana Carmen , Romo-Jiménez Angélica , Hernández-Sánchez Merit , Martínez-Murillo Carlos
Published: October 2019

Abstract 4767

Introduction

Extranodal NK/T-cell lymphoma, nasal type is an entity of difficult diagnosis. It is characterized by damage and vascular destruction, necrosis, cytotoxic phenotype and Epstein Barr virus association. It is prominent in Asia, Mexico, Central and South America, predominantly in men.

Material and methods

It is an observational, descriptive and retrospective study, based on medical records of NK/T-cell lymphoma patients of the General Hospital of Mexico between 1992-2009.

Results

Of 632 non-Hodgkin lymphomas (NHL), 113 had a T cell lymphoma. Only 32 cases (28.3%) were eligible for the study. The age range was 19-56 years with a media of 42. The male:female ratio was 2.2:1. The most frequent clinical presentation symptoms were nasal obstruction, facial swelling and pain. Central nervous system involvement was present only in two cases and bone marrow infiltration in one. Seventy eight percent were on clinical stages I or II and 56% had B symptoms. In 7/10 patients the Epstein Barr association was documented by hystopathological study. The treatment was completed in 31/32 individuals. Fifty five percent showed some response, regardless of the therapy used. The CHOP regimen was the first line treatment in 80.6% of cases and only 13% received fractionated radiotherapy as initial treatment. The CHOP arm had an overall response rate of 60% (complete remissions, unconfirmed complete remissions, partial remissions), and 100% of the patients in the radiotherapy arm presented complete response. Most patients of the radiotherapy arm were on stage IE. The overall survival rate was 63% at 36 months. The average follow-up was 40 months. There were no differences in overall survival rates between the two arms, but the radiotherapy arm had a higher rate of complete remissions.

Conclusions

In this study we report the clinical features and response to treatment of the patient population treated at the General Hospital of Mexico. The results are similar to other international reports. Although the incidence of NK/T-cell lymphoma is low, it is necessary to standardize its treatment and these preliminary data warrant further trial of radiotherapy as the initial treatment and chemotherapy as consolidation for advanced stages of the disease.

Disclosures:

No relevant conflicts of interest to declare.
Inhibition of Fatty Acid Synthase Suppresses c-Met Receptor Kinase and Induces Apoptosis in Diffuse Large B Cell Lymphoma.
Authors: Uddin Shahab , Hussain Azhar , Bavi Prashant , Al-Kuraya Khawla
Published: October 2019

Abstract 4787

Targeted approaches are expected to revolutionize cancer treatment in near future. Fatty acid synthase (FASN), the enzyme responsible for de novo synthesis of fatty acids has emerged as a potential therapeutic target for several cancers however its role in diffuse large B-cell lymphoma (DLBCL) has not been fully elucidated.. Therefore, we investigated the expression of FASN in tissue micro array cohort of 301 DLBCL patients. FASN was found to be expressed in 62.6% (162/259) DLBCL samples and was seen in highly proliferative tumors manifested by high Ki67 (p<0.0001). Significant association was found between tumors expressing high FASN and c-Met tyrosine kinase (p<0.0002) as well as p-AKT (p=0.0309). In vitro, pharmacological FASN inhibition and SiRNA targeted against FASN triggered caspase dependent apoptosis and suppressed expression of c-Met kinase in DLBCL cell lines which further highlighted the molecular link between FASN and c-Met kinase. Finally, simultaneous targeting of FASN and c-Met with specific chemical inhibitors induced a synergistically stimulated apoptotic response in DLBCL cell lines. These findings provide evidence of an active role of FASN in DLBCL evolution by specifically regulating tyrosine kinases related to malignant transformation strongly suggest that targeting FASN may have therapeutic value in treatment of DLBCL.

Disclosures:

No relevant conflicts of interest to declare.
Transient Laboratory Findings Upon First Dosing with T-Cell Engaging BiTE® Antibody Blinatumomab in Non-Hodgkin Lymphoma Patients.
Authors: Nagorsen Dirk , Zugmaier Gerhard , Kufer Peter , Klinger Matthias , Viardot Andreas , Goebeler Mariele , Schmidt Margit , Klappers Petra , Wolf Andreas , Brandl Christian , Baeuerle Patrick A , Bargou Ralf C
Published: October 2019

Abstract 4798

Blinatumomab is a single-chain bispecific antibody construct with specificity for CD19 and CD3 belonging to the class of bispecific T cell engager (BiTE®). It has shown high response rates as single agent after treatment of patients with indolent and mantle cell lymphoma and B-precursor acute lymphocytic leukemia. We here report from an ongoing phase 1 trial in non-Hodgkin lymphoma patients on transient laboratory findings seen upon start of continuous i.v. infusion of blinatumomab. The vast majority of adverse events (AEs) during 4-8 weeks of infusion occurred within the first two days of treatment. Lymphopenia (up to CTCAE grade 4) was the most frequent early laboratory AE, and can be explained by rapid B cell depletion and initial redistribution of T cells. Except for B cell decline, all laboratory parameters were transient and normalized within days during continued treatment. Dose-dependent increases were seen for AST and ALT (maximally grade 1/2), CRP, and D-dimer during the first days of treatment. Dose-dependent declines during the first days of treatment were observed for platelets and hemoglobin, which normalized after a few days. Of note, no drug-related thromboembolic events were observed. Serum levels of cytokines IL-6, IL-2, IFNγ, and IL-10 showed small dose-dependent increases, which reversed to baseline within hours. No association between cytokine levels and thrombocytopenia was found. Moreover, there was no evident correlation between any laboratory abnormality and clinical adverse event observed during the first days of treatment. The laboratory AEs induced by blinatumomab appear to reflect consequences of the onset of redirected target cell lysis, T cell activation and bystander effects. In conclusion, blinatumomab causes characteristic laboratory findings during the first days of treatment, which are transient, not associated with clinically relevant AEs and do not require treatment interruption. Compared to AEs caused by chemotherapy regimens, the laboratory findings observed after start of blinatumomab treatment are considered rather mild.

Disclosures:

Nagorsen: Micromet: Employment, Equity Ownership. Zugmaier:Micromet: Employment, Equity Ownership. Kufer:Micromet: Employment, Equity Ownership, Patents & Royalties. Klinger:Micromet: Employment, Equity Ownership. Schmidt:Micromet: Employment, Equity Ownership. Klappers:Micromat: Employment, Equity Ownership. Wolf:Micromet: Employment, Equity Ownership. Brandl:Micromet: Employment, Equity Ownership. Baeuerle:Micromet: Employment, Equity Ownership. Bargou:Micromet: Consultancy, Patents & Royalties.
Selective HDAC6 Inhibition and Antileukemic Activity of the Novel HDAC Inhibitor ST80 in Myeloid Leukemia Cell Lines.
Authors: Hackanson Bjorn W. , Rimmele Leander , Jung Manfred , Lübbert Michael
Published: October 2019

Abstract 4808

The antileukemic activity of histone deacetylase inhibitors (HDACi) has driven the search for epigenetic drugs with higher substrate specificity. Most of the currently used HDACi target class I, II and IV, with some bearing a class preference but only very few being selective in inhibiting specific HDACs, such as the HDAC6-selective inhibitor tubacin. HDAC6 inhibition leads to acetylation of non-histone proteins such as Hsp90 and alpha-tubulin. As it was recently demonstrated that HDAC6 is overexpressed in AML cells, we sought to investigate the effect of selective HDAC6 inhibition by the novel hydroxamic acid derivate ST80 in myeloid cell lines.

Methods

ST80 (selective HDAC6 inhibitor, 30-fold higher enzyme inhibition as compared to HDAC1) and the non-selective novel hydroxamic acid derivates ST13 (pan-HDACi) and ST34 (pan-HDACi with preference for class I) had previously been tested in enzymatic assays for their HDAC inhibitory potential (Scott et al. Mol Cancer Res. 6:1250-8, 2008). Cell lines HL60, Kasumi-1, NB4, THP1, U937 and K562 were treated with 10 nM to 30 μM of these three drugs. Viability and growth inhibition were determined using trypan blue staining. Acetylation of histone H3, H4 and alpha-tubulin and HDAC6 expression were determined by Western blot and quantified by densitometry. Tubulin-selective acetylation was calculated as the ratio of tubulin acetylation vs. H4 acetylation (ac-tubulin:ac-H4 quotient).

Results

At 1 μm, ST80, ST13 and ST34 all acetylated tubulin (8-, 8- and 2-fold in NB4, 11-, 14- and 3.4-fold in HL60, respectively, after a 12 h treatment). However, the calculated ac-tubulin:ac-H4 ratio of ST80 was 15- and 8-fold higher in NB4 and 9-fold higher in HL60 when compared to ST13 and ST34. The inhibitory concentration (IC) 50 (cell growth) of ST80 in the six myeloid cell lines ranged from 2.8 μM (NB4) to 5.1 μM (Kasumi-1) after 48 h treatment. Median cell viability of all 6 cell lines at 48 h was 93.7 % (range: 87.0 - 96.8 %) at 1 μm and 90.3 % (65.7 - 95.7 %) at 5 μm of ST80. HDAC6 protein levels were strongly variable between cell lines; however, growth inhibition by ST80 was independent of HDAC6 expression.

Conclusion

The novel hydroxamic acid derivate ST80 shows antileukemic activity in myeloid cell lines at low micromolar concentrations, which affect cell viability only modestly. The degree of relative tubulin acetylation by ST80 indicates a selective HDAC6 inhibitory activity in myeloid leukemias. The favorable ratio of ST80 growth inhibition vs. cytotoxicity warrants combination studies of this drug with other compounds.

Disclosures:

No relevant conflicts of interest to declare.
Recipient T and B Lymphocytes Dictate the Severity of Antibody-Mediated Transfusion-Related Acute Lung Injury (TRALI).
Authors: Fung Yoke Lin , Kim Michael , Speck Edwin R , Freedman John , Semple John W.
Published: October 2019

Abstract 641

Transfusion-related acute lung injury (TRALI) is a serious complication of transfusion and has been ranked as the leading cause of transfusion-related fatalities. The majority (approx. 80%) of TRALI reactions are associated with and probably initiated by donor alloantibodies recognizing recipient granulocytes and/or human leukocytes antigens (HLA). Nonetheless, many details of the immunopathogenesis of TRALI are unknown. Previous studies have shown that a murine anti-MHC (H-2Kd) class I antibody (clone 34-1-2s) can induce TRALI in mice (Looney et al J Clin Invest. 116:1615,2006) and we utilized this model in an attempt to understand the role that recipient lymphocytes might play in TRALI reactions. BALB/c (H-2d) mice were injected iv with titrations of 34-1-2s and body temperature, morbidity/mortality, pulmonary granulocyte accumulation and serum levels of MIP-2 (the murine analog of human neutrophil chemokine IL8) were measured at various time points. Results showed that when BALB/c mice were administered 34-1-2s, a significant drop (N=20) in rectal body temperature indicating shock occurred within 30 min post-infusion, with evidence of recovery beginning at 1 hour post-infusion. Visible signs of breathing difficulty were apparent but there was no mortality observed. A significant granulocyte accumulation (N=20) within the lungs was also observed by 30 min post-infusion, which continued on to the end of the experiment (2 hours post-infusion). Serum MIP-2 levels were also significantly elevated concurrently with the granulocyte accumulation. To determine the role of recipient lymphocytes on these responses, BALB/c mice with severe combined immunodeficiency (SCID; lacking T and B lymphocytes) were infused with 34-1-2s. Compared with the BALB/c recipients, the decreases in rectal temperatures in the SCID mice were significantly greater (N=18) and there was a 66% mortality rate (N=18) with symptoms of severe respiratory distress and tracheal edema with 30 minutes after infusion of 34-1-2s. In addition, there was a significantly greater accumulation of pulmonary granulocytes in the SCID mice at lower doses of 34-1-2s and the antibody stimulated the production of significantly higher serum levels of MIP-2. These findings were also seen in 34-1-2s-infused SCID mice that were first depleted of natural killer cells suggesting that NK cells play no role in the enhanced severity of the antibody-mediated TRALI reaction. Taken together, these results suggest that recipient T and B lymphocytes have a protective role in suppressing antibody-mediated TRALI reactions perhaps by modulating recipient chemokine production. They identify a potentially new recipient mechanism that controls the severity of antibody-mediated TRALI.

Disclosures:

No relevant conflicts of interest to declare.