bluuebunny/crossref_metadata_2025 huggingface dataset:
Comparison of Array Comparative Genomic Hybridization (aCGH) to FISH and Cytogenetics in Prognostic Evaluation of CLL.
Authors: O'Malley Dennis P. , Giudice Christina , Chang Averee S , Chang Dorothy , Barry Todd S , Hibbard Michele K , Chen Rebecca , Chen Sing-Tsung
Published: October 2019

Abstract 4393

INTRODUCTION

Chronic lymphocytic leukemia (CLL) is a commonly encountered hematologic neoplasm. Evaluation of prognosis in CLL is strongly based on genetic findings and the most commonly used studies are classical cytogenetics and targeted interphase fluorescence in situ hybridization (FISH). High resolution array comparative genomic hybridization (aCGH) is a relatively new and robust method of evaluating chromosomal alterations over the entire genome. We compared aCGH with routine cytogenetics and FISH in detecting genetic alterations in newly-diagnosed CLL cases.

MATERIALS AND METHODS

aCGH testing was performed on 55 cases of CLL in addition to a standard panel of FISH probes (ATM on 11q22, trisomy 12, 13q14, p53 on 17p13 using a standard cutoff for positivity of 10%). These results were compared to a control group of 100 CLL cases upon which routine cytogenetics and FISH were performed. The frequency of detecting abnormalities was compared between the groups and discordant results between methodologies were compared.

RESULTS

In the control group (n=100), the mean age was 71 (52-86) with a male to female ratio of 1.6:1. Genetic abnormalities were detected by classical cytogenetics in 19% (19/100) of cases as compared to FISH which detected abnormalities in 66% (66/100) of cases (Table 1). An additional group of 55 CLL cases [male to female ratio of 2.2:1 and a mean age of 71 (52-90)] was analyzed by both aCGH and FISH. This additional group of CLL cases showed a similar frequency of genetic abnormalities by FISH (60%; 27/45). In contrast to FISH, aCGH detected genetic abnormalities in 82% (45/55) of CLL cases (Table 1).

aCGH identified genetic abnormalities not detected by FISH studies in 16% (7/45) of cases whereas FISH identified abnormalities not detected by aCGH in only 7% (3/45) of cases. Rare recurring genetic alterations were detected by aCGH, which would not have been detected by a standard FISH panel, and included losses in 6q, 8p, 10q, 14q32, and 18q, and gains in 10q.

DISCUSSION

Classical cytogenetics is often performed in cases of CLL, but is not particularly useful as CLL cells are often difficult to grow in culture and because of the low rate of detecting common genetic alterations. Our findings suggest that aCGH is an effective and robust technique for evaluating recurring genetic abnormalities in CLL and is better than classical cytogenetics and standard FISH in detecting common genetic abnormalities in CLL.

Disclosures:

No relevant conflicts of interest to declare.
Mechanism Research of Magnetic Nanoparticle Fe3O4 and 5-Bromotetrandrine On Reversal of Multidrug Resistance in K562/A02 Leukemic Cells.
Authors: Chen Bao-An , Wu Wei-Wei , Cheng Jian , Gao Feng , Xu Wen-Lin , Gao Chong , Ding Jia-Hua , Sun Xin-Chen , Xu Cui-Rong , Chen Ning-Na , Xia Guo-Hua , Wang Xue-Mei , Chen Wen-Ji , Liu Li-Jie , Li Xiao-Mao
Published: October 2019

Abstract 4827

Purpose

The present study aimed to research the machenism of magnetic nanoparticles Fe3O4 (Fe3O4-MNPs) and 5-bromotetrandrine (BrTet) on multidrug resistance cell line K562/A02 solitarily or symphysially.

Mothed

The proliferation of K562 and K562/A02 cells, and the cytotoxicity of PMBCs which were cultured with daunomycin (DNR) alone or in combination with Fe3O4-MNPs (0.1, V/V), BrTet (0.5μM) or both for 48 h, were evaluated by MTT assay. DNR accumulation of K562, K562/A02 cells and PMBCs were analyzed by fluorospectrophotometry after incubated with 2μM DNR in the absence or presence with Fe3O4-MNPs (0.1 V/V), BrTet (0.5μM) or both for 48 h. Real time–PCR analyses and western blotting were performed to examine the mRNA and proteins level, respectively.

Result

The results showed that the combination of Fe3O4-MNPs and BrTet with effective concentration could exaggerate cytotoxicity against MDR cell line K562/A02 significantly. Flow cytometry assay showed that 0.5μM BrTet in combination with Fe3O4-MNPs (0.1, V/V) significantly enhanced the intracellular accumulation of DNR in K562/A02 cells and its potency was greater than that of BrTet or Fe3O4-MNPs alone at the same concentrations. While both Fe3O4-MNPs and BrTet in company with DNR did not increase the cytotoxicity to PMBCs. Both BrTet and Fe3O4-MNPs inhibited the overexpression of MRP1, MRP2, MRP4 and MRP5, and down regulated, mrps mRNA expression in K562/A02 cells to some extent.

Conclusion

We propose that Fe3O4-MNPs loaded with DNR and BrTet probably have synergetic effect on reversal in multidrug resistance. However, this combination shows less cytotoxicity to normal cells and reveals better target. The results may provide evidence for clinic application of them as reversal agents of drug resistance.

Disclosures

No relevant conflicts of interest to declare.
Diagnostic Utility of Flow Cytometry in Myelodysplastic Syndrome: A Retrospective Validation From a Single Centre.
Authors: Topcuoglu Pervin , Dalva Klara , Bakanay Sule Mine , Bozdag Sinem Civriz , Arslan Onder , Ozcan Muhit , Arat Mutlu , Beksac Meral , Gurman Gunhan
Published: October 2019

Abstract 4846

Myelodysplastic syndrome (MDS) is heterogeneous clonal hematopoietic stem cell disorder characterized by cytopenia(s) and dysplasia in one or more cell lineage. Though flow cytometry (FCM) is an important diagnostic tool in hematopoietic cell disorders, a prominent immunophenotyping feature in MDS may not be determined. In this study, we retrospectively evaluated flow cytometric features of bone marrow samples diagnosed as MDS with clinical and hematological findings.

Patients-Method

Between Feb 2004 and March 2009, flow cytometric parameters of 73 patients (M/F: 50/23) with MDS were re-analyzed. Median age was 59 years (17-89 ys). Our general principles are to evaluate quality of bone marrow samples, to determine proportion of the cells and features of their light scatter, and to give percentage of the blast. When detected a finding of dysplasia in the first analysis, the second step includes the determination of the maturation of the cells and the presence of the aberrant antigen expression.

Results

The samples were interpreted as MDS in % 76.7, MDS-RAEB-1 or RAEB-2 in %16.4, myeloproliferative disorder in %1.4 and non-diagnostic in %6.8 of the cases by flow cytometric examination. We detected variable degrees of hypogranulation in myeloid lineage in %82.2 of the samples by the light scatter features of the cells: 85% of severe and 15% of moderate or mild hypogranulation. The ratio of myeloid and lymphoid was changing from 0.3 to 17.5 (median 2). The decreasing of this ratio (<1) was observed in 19.4% of the samples. We detected altered expression of mature granulocyte. These included decreasing or lack of expression in CD15 45/73 (61.4%), CD13 38/70 (54.3%), CD16 53/67 (79.1%), CD11b 51/71 (71.8%), CD24 44/69 (65.2%), CD10 23/72 (31.9%) and MPO 14/72 (19.4%). Besides, bright expression of CD33 in 53.5% of the samples was observed. CD36 and CD56 in myeloid lineage were co-expressed in about 50 % of the samples. In 80.8 % of the samples dysplasia in erythroid compartment could be evaluated: Expression of CD71 according to glycophorin A (ratio <1) was decreased in 23.7 %. When we made similar analysis in the samples without RAEB-1 and -2 as pathological examination of bone marrow, 13.4 % of the samples could not be evaluated in favor of dysplasia. Of the samples with dysplasia hypogranulation, aberrant antigen expression of myeloid lineage and eryhtroid dysplasia were observed in 92.1%, 34.1% and 31.5%, respectively. In conclusion, FCM events may help to the differantial diagnosis of MDS especially when combining with clinical events. Improving of the analysis by focusing on the blast characteristics may be a standard approach to evaluate for low risk MDS.

Disclosures

No relevant conflicts of interest to declare.
Differential Diagnosis of Myelodisplastic Syndrome, with Flow Cytometry.
Authors: De Salvo Cardullo Luigi , Duerto Mirian C , De Salvo Dalila DV , Salas-A Rodolfo J , Weir-M Jesus E , Nuñez Ana M , Nuñez Alonso A , Plumacher Zaida A , Fernandez Olga Ch , Avila Estela BEATRIZ , Rios Mariela J , Paz Luis S , Salas Dalida DJ , Fuenmayor Alba M
Published: October 2019

Abstract 4860

Our flow cytometry laboratory processed 38 bone marrow (BM) samples (24 males and 14 females) from patients with Myelodisplasic Syndrome diagnosis, from January 2008 to May 2009. Upon examination, most BM smears corresponded with an hypercellular bone marrow and increased intermediate and immature cells. All patients classified according to OMS for Myelodisplasic Syndromes. Twenty-four (n=24) patients (65%) presented RA, five (n=5) patients (13%) RAEB-1, one (n=1) 1% presented RARS, and eight (n=8) patients (21%) classified as RAEB-II. Cytogenetic result analyses for all patients classified according with the Index Prognostic Standard Score (IPSS). Twenty-five (n=25) patients or 66% corresponded with a good cytogenetic risk; seven (n=7) or 20% corresponded with an intermediate risk and six (n=6) or 14% with poor risk. Cell cytometry results corresponded with increased immature cells. In 20 patients, the DR/CD34 average 27,29% (range 0-67); in 24 patients, the DR/CD33 average 31,80% (range 6-100); in 29 patients, the DR/CD117 average 18,89% (range 0-43); in 7 patients, the CD33/CD34 average 22,14% (range 0-50); in 5 patients, the CD38 average 47,60% (range 32-61); in 4 patients, the BCL2 average 21,75% (14-26 and in 3 patients, the DR/CD64 average 44,33% (range 30-60). All patients with high average of DR/CD64 classified as CMML. Patients with RAEB-II diagnosis classified as MDS in transformation. Most patients displayed 10% CD34+ values higher than normal human average. We conclude the cytometry is an important tool in the differential diagnosis of MDS.

Disclosures

No relevant conflicts of interest to declare.
Arsenic Trioxide-Mediated Growth Inhibition of Myeloma Cells Is Associated with An Extrinsic Signaling Pathway Involved in Caspase 3 and Caspase 8.
Authors: Shi Jumei , Wu Yi , Wang Siqing , Meng Xiuqin , Wei Rong , Hu Xiaojing , Zangari Maurizio , Tricot Guido J. , Zhan Fenghuang
Published: October 2019

Abstract 4899

Arsenic trioxide (ATO) is a well-known inhibitor of cell proliferation in certain forms of malignancy and has been successfully used in the treatment of acute promyelocytic leukemia. Preclinical and clinical studies showed that ATO has anti-myeloma effects both as a single agent and in the combination therapy; however, the underlying molecular mechanism remains elusive. This study was performed to evaluate the molecular mechanism underlying its anti-myeloma activities. Cells from OPM2, U266, RPMI8226 myeloma cell lines and patients diagnosed with myeloma (n=6) were cultured with various concentrations of ATO for 4 days. Cell growth and viability were assayed by trypan blue dye exclusion. Cell cycle and apoptosis were analyzed by flow cytometry using CellQuest software and Vybrant Apoptosis Assay Kit. Alterations of the signaling pathways induced by ATO were tested by real-time PCR and western blot. ATO induced potent inhibition of myeloma cell growth compared with untreated control cells. Further investigation showed that ATO down-regulated c-Myc and phosphorylated (p)-Rb, while it up-regulated p53, p21Clip1, and p27Kip1 proteins, resulting in G2/M cell cycle arrest and cell growth inhibition. ATO treatment increased mRNA levels of interferon regulatory factor-1 (IRF-1) and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), as well as protein levels of caspase 8 and cleaved caspase 3, indicating involvement of the extrinsic apoptotic pathway. No significant change was detected in the expression levels of Bax, Bcl-xL caspase 9 and Bcl-2, indicating that the intrinsic signaling pathway was not involved. A pan-caspase inhibitor abrogated ATO-induced apoptosis of myeloma cells. Our data suggest that ATO induces apoptosis in MM cells most likely through an extracellular signaling pathway.

Disclosures

No relevant conflicts of interest to declare.
Compassionate Use of Combined Anti-Inflammatory and Angiostatic Treatment as Third-Line Therapy in Eight Patients with Multiple Myeloma – a Combined Treatment Setting of Lenalidomide with Pioglitazone, Dexamethasone and Low-Dose Treosulfan.
Authors: Reichle Albrecht , Vogelhuber Martin , Holler Ernst , Hahn Joachim , Andreesen Reinhard
Published: October 2019

Abstract 4958

Introduction

Therapeutic options for patients with refractory and multifold pretreated multiple myeloma (MM) are limited, as patients have already received the novel targeted therapy approaches at this stage. Continuous production and release of pro-inflammatory cytokines may account for its resistance towards cytotoxic agents. Therefore, a multi-targeted approach consisting of anti-angiogenic, anti-inflammatory and anti-tumor components was implemented for response induction prior to and after allogeneic transplantation.

Methods

Within a compassionate-use program eight patients suffering from refractory or relapsed osteolytic multiple myeloma (MM) or plasma cell leukemia (PCL) and proven progression of serum paraprotein levels were treated in third-line continuously with daily lenalidomide 10 mg, pioglitazone 60 mg, treosulfan 250 mg once daily, and dexamethasone 1 mg.

Results

Here we report results of eight patients (48 to 63 years old) treated for refractory (n= 7) or relapsed (n= 1) multiple myeloma (n= 7) or PCL (n= 1). Prior therapy consisted of double autologous transplantation with high-dose treosulfan/melphalan conditioning, lenalidomide and/or bortezomib. One patient suffered from progressive disease following allogeneic stem cell transplantation (allo-SCT). No side effects > WHO 2 occurred on treatment. In one patient, bortezomib-induced motoric polyneuropathia partially resolved on study medication. Biomodulatory therapy induced clinically meaningful and very rapid responses, stable disease, n= 2, partial remission (PR), n= 3, and very good PR (VPR, n= 3). Therapy-induced PR and VPR facilitated allo-SCT in 2 refractory patients. A third patient with refractory disease after allo-SCT received donor lymphocytes. Two patients with allo-SCT survived in VPR for 10+ and 3+ months, respectively. One patient remained in ongoing VPR since 9 months after donor lymphocyte infusion. Patients not proceeding to allo-SCT had SD for 3 (PLC) and 4 months, VPR and PR for 3+, 4+ and 5+ months.

Conclusions

Together, biomodulatory therapy has remarkable clinical activity in pretreated and refractory patients with MM. The eight cases show comparatively high efficacy at low toxicity rates of a biomodulatory acting therapy approach: Although the substances have limited or no efficacy at the respective dose levels, and were already administered previously at higher dose-levels (i.e. treosulfan and/or lenalidomide), the combined treatment setting is promising since it targets the myeloma cells itself as well as the surrounding stroma. Therefore, this treatment schedule deserves further investigation and discharges now in a novel multi-center phase II trial for third-line therapy in multiple myeloma.

Disclosures

No relevant conflicts of interest to declare.
Prospective Exploratory Phase II Studies of A Rotating Regime of Nilotinib and Imatinib for Frontline Treatment of Philadelphia POSITIVE (Ph+) Chronic Myeloid Leukemia (CML) and Acute Lymphoblastic Leukemia (ALL).
Authors: Saglio Giuseppe , Abruzzese Elisabetta , Alimena Giuliana , Bocchia Monica , Carella Angelo Michele , Fanin Renato , Martinelli Giovanni , Rosti Gianantonio , Russo Domenico , Specchia Giorgina , Baccarani Michele
Published: October 2019

Abstract 4972

The introduction of Imatinib (IM) and subsequently of Dasatinib and Nilotinib (NI) has made a substantial contribution to the therapy of all Ph+ leukemias. With IM alone, a complete cytogenetic response (CCgR) is achieved in about 75% of CML patients who are treated frontline, but about 15% of them loose the response in the first 3 years. A complete hematologic response (CHR) is achieved in almost all ALL patients, but about 50% of them relapse with BCR-ABL mutated clones within one year. With NI alone, a complete cytogenetic response is obtained in about 40% of Ph+ leukemia patients who are resistant to IM, and in more than 90% of early chronic phase, previously untreated, CML patients. NI is a derivative of IM, but has different pharmacokinetic and pharmacodynamic properties, and inhibits most of BCR-ABL mutants which are resistant to IM. Moreover, the toxicity profile of the two drugs is different. The administration of two tyrosine kinase inhibitors (TKIs) may increase the strength of initial therapy, so increasing the rate and the solidity of the response, and reducing the rate of failures. To test the feasibility and the validity of this hypothesis the GIMEMA CML WP designed a rotating regime of NI and IM to be tested front-line in patients with Ph+ CML, and in the patients with Ph+ ALL who are more than 60 years old. The first course is with NI 400 mg twice daily. The second course is with IM 400 mg once daily. A course lasts 3 months in CML, and 6 weeks in ALL. Treatment duration (study core) is 2 years in CML (8 courses), and 36 weeks in ALL (6 courses). The primary endpoint is event-free survival at 24 months in CML, and disease-free survival at 6 months in ALL. CML patients enrollment (n = 120) has been completed between February and August 2009. The results of the first interim analysis at 3 and 6 months will be presented. The enrollment of ALL patients will begin September 2009.

Disclosures

No relevant conflicts of interest to declare.
Effect of CD20 Expression Levels On B-Cell Lymphoma Patients' Background and Prognosis.
Authors: Suzuki Yuhko , Yoshida Tsutomu , Nakamura Naoya , Togano Tomiteru , Miyazaki Koji , Ishii Ryuuji , Ohsaka Manabu , Danbara Mikio , Horie Ryouichi , Nakayama Meijin , Okayasu Isao , Higashihara Masaaki
Published: October 2019

Abstract 5000

AIM

The anti-CD20 antibody Rituximab has improved the disease-free survival and overall survival of patients with several types of B-cell lymphoma, in which CD20 is expressed on the cell surface and in the cytoplasm. Flow cytometry indicated that CD20 expression varies from null, weak to strong. Despite its clinical importance, the influence of CD20 expression levels on lymphoma therapy has not been well elucidated. Patients, Material and

Methods

214 cases were analyzed, all newly diagnosed with B-cell lymphoma at our institute from January in 2002 to April in 2009. All were biopsied and analyzed by flow cytometry. The biopsy specimens were fixed in formalin and stained with Hematoxylin-Eosin; they were also immunostained. Histological subtypes were defined according to the World Health Organization Classification Ver. 3. The mean florescence intensities of CD20 and CD19 were determined by flow cytometry, and cytoplasmic expression of CD20 was determined by immunohistochemistry. 1) The cases were categorized as follows: A: CD20-negative, B: CD20/19 less than 20%, C: CD20/19 20-50% and D: CD20/19 more than 50%. Patients' backgrounds, pathological diagnoses, primary lesions, etc. were also analyzed. 2) Among DLBCL cases, 76 treated with R-chemo were selected and analyzed with respect to treatment response and overall survival.

Results

1) Diagnoses: DLBCL 128, Follicular lymphoma 58, MALT 7, CLL 4 and so on. Among the DLBCL cases, immunohistochemistry indicated six (4%) were CD20-negative and three were CD20-positive; the ages in the CD20-negative DLBCL group tended to be high (74.28 vs 64.36, p=0.06, t-test). Weak CD20 expression was observed in 15 cases (B: 4, C:11). No statistically significant differences were seen with respect to gender, IPI, clinical stage, biopsy lesion, CD5 expression or Karyotype. No FL cases were CD20-negative, but two ( 3.4%) showed low CD20 expression. 2) Kaplan-Meier analysis of 76 cases of DLBCL treated with R-Chemo showed a significantly lower overall survival for Group A, (CD20-negative) while there was no significant difference in overall survival of Groups B+C(CD20-weak) and D(CD20-normal). Our results indicate that even patients with lower levels of CD20 expression in B-cell lymphoma may respond favorably to anti-CD20 therapy. Further studies will be necessary to explore this possibility.

Disclosures

No relevant conflicts of interest to declare.
“Lymphomatous and Leukemic Meningitis – Patterns of Cytomorphology and Value of MRI and Protein Analysis”.
Authors: Strik Herwig Matthias , Perske Christina , Proemmel Peter , Nagel Ingelore , Nagel Holger
Published: October 2019

Abstract 5011

Introduction

Lymphomatous and leukemic meningitis (LM), although a well known and relatively frequent complication of aggressive lymphoma and leukemia, are still difficult to detect. With cytomorphology, neoplastic lyphocytes are difficult to distinguish from inflammatory lymphocytes. We evaluated here if specific morphological criteria can improve this differentiation. Moreover, we assessed the sensitivity of MRI and protein analysis for the detection of LM in comparison with CSF-cytology.

Patients and Methods

To establish cytomorphological criteria, 42 cytospin preparations of CSF from patients with confirmed CSF involvement by aggressive lymphoma or acute leukemia were compared with 26 samples of inflammatory diseases. CSF cytology was analyzed morphologically for pre-selected parameters of cell, cytoplasm and nucleic appearance and the presence of mitoses or apoptoses. For the comparison of cytology and MRI, 38 patients with leukemic or lymphomatous meningitis were evaluated retrospectively for MRI-signs of neoplastic meningitis and for CSF-protein abnormalities (total protein, oligoclonal bands, lactate, ferritine).

Results

as expected, none of the cytomorphological parameters sharply discerns neoplastic and inflammatory transformation. However, neoplastic cells were significantly larger than inflammatory lymphocytes with a mean of 3.0 as opposed to 1.8 times the size of normal small lymphocytes (p=0.0001). Moreover, irregular shape, pointed borders of the cytoplasm, and deep notches in the nucleus were significantly more often found with neoplastic than with inflammatory lymphocytes.

The total cell count was elevated in 68% of cases of lymphomatous meningitis. While cytomorphology could achieve approx. 90% sensitivity for the detection of LM, spinal and/or cranial MRI only detected 71% of cases with normal and 52% with elevated cell counts. Total protein was elevated in 77% of cases, lactate in 55% and ferritine in 48%. Oligoclonal IgG was found in 11% isolated in the CSF and in 18% in CSF and serum identically. Only 3/38 patients (8%) had completely normal CSF cell count and proteins.

Conclusions

CSF cytology is more sensitive than MRI for the detection of LM, but application of both methods clearly enhances the sensitivity by approx. 10%. No single cytomorphological parameter is sufficient to detect neoplastic lymphocytes. Considering a combination of cell size and irregular shape of cell and nucleus may improve the diagnostic accuracy of CSF dissemination by aggressive hematological malignancies.

Disclosures

Strik: Mundipharma : Consultancy, Honoraria, Research Funding, Speakers Bureau; Essex/Shering-Plough/Merck: Consultancy, Honoraria, Speakers Bureau; Medac: Research Funding.
FDG-PET for Staging and Follow-up of HIV-Infected Patients with Non Hodgkin Lymphoma and Hodgkin Lymphoma: A Single Center Experience.
Authors: Cantoni Silvia , Ravelli Erika , Moioli Maria Cristina , Rusconi Chiara , Zilioli Vittorio Ruggero , Basilico Claudia Maria , Gargantini Livio , Nosari Annamaria , Volonterio Alberto , Rossetti Claudio , Morra Enrica
Published: October 2019

Abstract 5014

Introduction

Risk of developing non Hodgkin lymphomas (NHL) is known to be increased among patients (pts) infected by HIV-1 and sporadic non-HIV related cases of Hodgking lymphomas (HL) are also described. HIV-positive pts also feature a virus-related systemic lymphoadenopathy which makes nodal involvement by lymphoma difficult to evaluate using conventional imaging. The role of positron-emission tomography using 18F-fluorodeoxyglucose (FDG-PET) in the staging and evaluation of response to treatment of pts with NHL and HL is well established in immunocompetent pts, while only limited information is available on lymphomas arising in the setting of HIV infection. We report on a retrospective analysis of FDG-PET results in a group of HIV pts with lymphoma, both NHL and HL, followed-up at our hospital.

Patients and methods

Twelve HIV-positive male pts with a diagnosis of lymphoma (7 high grade and 1 follicular NHL, 4 HL) underwent a total of 22 PET scans. PET results were compared with those obtained by computed tomography (CT). Nine PET were performed at staging, 7 at restaging following chemotherapy, and 6 at follow-up. At diagnosis of lymphoma: median CD4 count was 200 cells/mcL (range 98-451); HIV genome was undetectable in 5/9 pts, in the remaining 4 pts for whom the data was available viral loads ranged from 103 to 1,452,720 copies/mL; 10/12 pts were already receiving HAART, while the remaining 2 pts started treatment upon diagnosis of lymphoma.

Results

In the majority of cases (17/22 = 77%) results of PET, CT scan and clinical status were concordant, being diagnostic of either lymphoma presence (11/17) or absence (6/17). No false positive results were recorded at follow-up. In 5/22 cases (23%) PET and CT scan were discordant. In 4 cases PET yielded a false negative result; in 2 cases at diagnosis (2 pts with HL) and in 2 cases at follow-up (both in the pt with follicular lymphoma). In the remaining case, a PET-negative CT-positive adenopathy was demonstrated which proved to be reactive in nature at biopsy.

Discussion

Our results suggest that FDG-PET is useful in the evaluation of lymphoma in the setting of HIV infection, similarly to what has been observed among immunocompetent pts. Although the number of patients is limited, in our experience PET proved to be able to discriminate between reactive and lymphomatous involvement of lymphoid tissue as demonstrated by absence of false positive results in pts evaluated at follow-up. These results need to be confirmed by larger clinical trials.

Disclosures

No relevant conflicts of interest to declare.
Ex-Vivo Expanded Peripheral Blood Stem Cells (EVEC) Compared with Un Manipulated Peripheral Blood Stem Cells (PBSC) Autologous Transplantation for Multiple Myeloma: A Pair Match Analysis.
Authors: Milpied Noel-Jean , Marit Gerald , Dazey Bernard , Boiron Jean-Michel , Ivanovic Zoran , Foucaud Carine , Bijou Fontanet , Bouabdallah Krimo , Schmitt Anna , Sauvezie Mathieu , Cailliot Christian , Reiffers Josy
Published: October 2019

Abstract 502

Autologous stem cell transplantation with PBSC after high-dose chemotherapy remains standard therapy for patients with symptomatic Multiple Myeloma (MM). Strategies to minimize complications could significantly reduce the morbidity of that procedure. One possibility could be to shorten the duration of induced neutropenia through the injection of an ex-vivo expanded graft. Nineteen patients (pts) received EVEC after high-dose Melphalan (HDM) (200 mg/m2) as the only graft. The ex-vivo expanded procedure has been described elsewhere (Boiron et al. Transfusion 2006 and Ivanovic et al. Transfusion 2006). Briefly, thawed peripheral blood CD 34+ cells collected after G-CSF mobilisation and selected with immunomagnetic devices were incubated for 10 days in a serum free medium (Maco Biotech HP01) with Stem Cell Factor (Amgen), G-CSF (Amgen) and TPO (Amgen: 7 pts; Cellgenix:12 pts). The expanded cells were then thoroughly washed and injected 48h after the HDM injection. The ex-vivo expansion lead to a median fold of 5,4 for CD34+ cells (1,3-11,8); 118 for CD33+ (1-703880); 3386 for CD14+ (4-101075); 28,5 for CD13+ (10-703880) and 13 for CFUs (6-21). The median N° of CD34+ cells injected was 14×10e6/kg (5,3-48). The results of these transplants were compared to those achieved in 38 pts who received unmanipulated PBSC after HDM. Pts and controls were matched for age, sex, stage of the disease, first line chemotherapy ( VAD or VD) status of the disease at time of transplant, year of transplant, time between diagnosis and transplant, CD34+ mobilisation technique (HD cytoxan + G-CSF or G-CSF alone) and the median N° of total nucleated cells and of CD34+ collected. The results are summarized on the table:

There was no secondary neutropenia in the patients who received EVEC. With a median FU of the entire cohort of 30 m, the median OS for pts who received their first transplant with EVEC and with PBSC is 69 m and not reached respectively (p=NS), the median PFS is 18 m and 27 m (p = NS) and the median time to progression is 14 m and 15 m (p=NS).

Conclusion:

EVEC is feasible, safe and reduce significantly the morbidity of autologous stem cell transplantation after HDM for multiple myeloma.

Disclosures:

Milpied: Amgen France: Honoraria.
Adaptor Protein Lnk Binds to JAK3 and Negatively Regulates a Mutant Activating Allele Associated with Megakaryocytic Leukemia.
Authors: Koren-Michowitz Maya , Gery Sigal , Nowak Daniel , Koeffler Phillip H
Published: October 2019

Abstract 5044

The adaptor protein Lnk is known to associate with hematopoietic cytokine receptors such as cKIT, MPL and PDGFR, as well as, non-receptor tyrosine kinases such as JAK2, and is considered to have an inhibitory effect on these signaling pathways. JAK3 is expressed mainly in the hematopoietic system and its absence is associated with autosomal recessive severe combined immunodeficiency (SCID). Recently, activating mutations of JAK3 were described in transient myeloproliferative disorder (TMD) and acute megakaryocytic leukemia (AMKL) in Down syndrome (DS) patients as well as adult non-DS AMKL. JAK3 mutations were also rarely described in solid tumors and B-ALL. The 50% homology between JAK3 and JAK2 has led us to study the association between Lnk and JAK3.293T cells were co-transfected with cDNAs encoding either wild-type (WT) JAK3 or JAK3 harboring an activating A572V mutation (JAK3 A572V), as well as the WT V5-tagged Lnk. Whole cell lysates were used for immunoprecipitation with either V5-tag or JAK3 antibodies. Binding of Lnk and JAK3 was detected by Western blot probed with JAK3 or V5-tag antibodies. To determine which domain of Lnk is responsible for the binding, we constructed a series of V5-tagged Lnk mutants including a mutation in the SH2 domain (R392E), deletion of the SH2 domain (del SH2) and deletion of the PH and SH2 (del SH2/PH) domains. Our results show that WT Lnk binds to WT JAK3, as well as JAK3 A572V. The R392E and del SH2 Lnk mutants retained JAK3 binding capacity while deletion of both SH2 and PH domains of Lnk abolished JAK3 binding. In order to study the biological effect of Lnk binding to JAK3, we infected CMK cells, a megakaryocytic leukemia cell line harboring JAK3 A572V, with a bicistronic retroviral MSCV-IRES-GFP (MIG) WT Lnk vector. Effect on growth was assessed in GFP positive sorted cells by cell count and colony formation in methylcellulose. CMK cells infected with MIG WT Lnk grew slower in liquid culture and had decreased clonogenic growth in soft agar culture compared to cells infected with MIG vector alone.

In summary, we show for the first time that Lnk can bind to WT and mutant JAK3 and slow the growth of leukemic cells harboring an activating JAK3 mutation. Developing a small molecule mimetic of Lnk may have a therapeutic role in the treatment of hematopoietic malignancies associated with a variety of activated tyrosine kinase receptors and non-receptor tyrosine kinases including JAK3, as well as secondary signaling proteins.

Disclosures

No relevant conflicts of interest to declare.
The Effect of siRNA Down-Regulating XIAP On the Apoptosis and Chemo-Sensitivity of K562 Cells in Vitro.
Authors: Xie Shuangfeng , Yin Songmei , Li Dan , Nie Danian , Li Yiqing , Ma Liping , Wang Xiuju , Xiao Jie
Published: October 2019

Abstract 5053

Introduction

Failure for chemotherapy in leukemia patients is often due to innate or acquired multi-drug resistance of leukemia cells. The X-linked inhibitor of apoptosis protein (XIAP) is a potent inhibitor of apoptosis and is also involved in drug resistance. The relationship between XIAP and drug resistance is not fully clarified. Here, we manipulated the expression of XIAP in K562 human chronic myelogenous leukemia cells and explore the changes in cell apoptosis and sensitivity to cytarabin.

Methods

Small interfering RNA (siRNA) targeting XIAP was created, K562 cells were either transfected with XIAP siRNA or control siRNA by Nucleofector transfection. The siRNAs were labelled with Cy3. Real-time RT-PCR and Western blot were used to determine the expression of XIAP in K562 cells. Flow cytometry was used to detect apoptotic cells and 50% inhibiting concentration (IC50) of cytarabin was also determined.

Results

The transfection rate was 87% in general. The transfection was also confirmed by fluorescence microscopy. After 48h, the expression levels of XIAP mRNA and protein were much lower in XIAP siRNA-transfected cells than in control siRNA-transfected cells (mRNA, 0.37±0.10 vs. 1.41±0.13, P<0.05 ; protein, 0.39±0.03 vs. 0.99±0.08, P<0.05). There was no significant difference in the fractions of apoptotic cells between these two groups at 48h after transfection (37.04%±1.77% vs. 47.43%±1.13%, P>0.05). Cytarabin was added in different concentrations right after Nucleotransfection. After 48h, the IC50 of Cytarabin in the XIAP siRNA-transfected cells was 2.28-fold lower than that in the control siRNA-transfected cells (82.60±21.56μg/ml vs. 188.67±44.48μg/ml, P<0.05). The percentage of apoptotic cells was much higher in the XIAP knockdown cells than in the control cells (47.43%±1.13% vs. 37.04%±1.77% P<0.05).

Conclusions

XIAP siRNA can specifically down-regulate the expressions of XIAP mRNA and protein, promote the drug-sensitivity to cytarabin and increase the cell apoptosis in K562 cells. This might be a novel choice for leukemia treatment.

Disclosures

No relevant conflicts of interest to declare.
Pediatric Venous Thromboembolism: Redefining Epidemiology.
Authors: Setty Bhuvana A , O'Brien Sarah H. , Kerlin Bryce A.
Published: October 2019

Abstract 5055

Introduction

Pediatric venous thromboembolism (VTE) is a multifactorial disease most commonly seen in children with complex medical conditions. The data regarding associated conditions in children is sparse. The most comprehensive source regarding these associations is derived from the Canadian registry of 137 children with VTE collected from 1990-1992.

Objective

To refine the spectrum of pediatric VTE associated illnesses, utilizing a large, comprehensive claims database.

Methods

The Healthcare Cost and Utilization Project (HCUP) Kids' Inpatient Database (KID) 2006 was utilized to identify children <18 years old with in-hospital VTE. These children were identified by the presence of at least one of the following ICD-9-CM diagnosis or procedure codes: 325, 452, 453(.0, .2-.42), 453 (.8-.9), 415 (.0-.11), 38 (.05, .07, .09), or 99.10. The remaining diagnostic ICD-9-CM codes were then utilized to assign a preliminary Complex Chronic Condition (CCC) category for each patient, using previously defined criteria. This categorization was further refined by a manual translation of the diagnostic codes by one of the investigators (BAS or BAK). The incidence of in-hospital VTE by geographic region, hospital type, age, gender, ethnicity, and median household income was estimated.

Results

4,731 children met the inclusion criteria (1.76/1,000 discharges). The major underlying illnesses were cardiovascular (18%), malignancy (17%), neuromuscular disease (11%), gastrointestinal (8%), hematology/immunology (7%), and metabolic disease (6%). Trauma and surgical interventions were present in 5 % while 5% of VTE was idiopathic. Renal, respiratory, and autoimmune diseases were less commonly associated with VTE (<5%). Interestingly, the incidence of VTE was highest in the Midwest (2.12/1,000 discharges) and lowest in the South (1.60/1,000 discharges). As seen in previous studies, VTE was more commonly seen in infants and adolescents. There was a slight predominance of males with VTE (1.22:1). 4.2% of the VTE were associated with in-hospital death.

Conclusion

Pediatric VTE is rarely an idiopathic illness. More commonly it is associated with a chronic underlying medical condition. These data refine our understanding of the spectrum of underlying health conditions in children with VTE. This is limited by the inability to take into account the use of various medications and outpatient interventions that may have contributed to the occurrence of the VTE. Future studies should focus on the epidemiology of VTE within each associated disease category.

Disclosures

No relevant conflicts of interest to declare.
Evaluation of the Gene Encoding Calcium Diacylglycerol Guanine Nucleotide Exchange Factor I (CalDAG-GEFI) in Human Patients with Congenital Qualitative Platelet Disorders.
Authors: Puetz John , Boudreaux Mary
Published: October 2019

Abstract 5078

Normal platelet function is dependent on an orchestrated series of interactions resulting in primary hemostasis. Dysfunction in any step of platelet activation and aggregation results in abnormal platelet function and abnormal mucocutaneous bleeding. Defects in agonist/receptor interactions, membrane phospholipid and cytoskeleton structure, signal transduction, storage pool content and release have all been described. While some congenital qualitative platelet function disorders such as Bernard-Soulier syndrome or Glanzmann thrombasthenia are well characterized at the molecular and genetic level, the majority of congenital platelet function disorders are not. Recently, insights into the molecular and genetic causes of platelet signal transduction and secretion pathway disorders have been found in animals. Dogs and cattle with recurrent abnormal mucocutaneous bleeding symptoms and abnormal in vitro platelet aggregation have been found to be caused by a mutation in the calcium-diacylglycerol guanine nucleotide exchange factor I (CalDAG-GEFI) gene. Genetic ablation of CalDAG-GEFI in mice has resulted in abnormal platelet function and bleeding. Polymorphisms in the human CalDAG-GEFI gene have been linked to Kindlin-3/ FERMT3 mutations resulting in a leukocyte adhesions defect associated with platelet dysfunction (LAD-III or LAD-1/variant syndrome). To date, mutations in the CalDAG-GEFI gene in humans associated with abnormal platelet function and bleeding have not been described. To determine if mutations in the human CalDAG-GEFI gene are associated with abnormal mucocutaneous bleeding and platelet aggregation dysfunction, we have begun sequencing the CalDAG-GEFI gene in human patients with a congenital qualitative platelet function disorder of unknown etiology. As control groups, we will also evaluate the CalDAG-GEFI gene sequence of unaffected family members and unrelated blood donors known to have normal platelet aggregation. Preliminary results of our analysis will be presented.

Disclosures

No relevant conflicts of interest to declare.
Association of Red Blood Cell Distribution Width (RDW) with Laboratory Parameters in Anemic Adults.
Authors: Means Robert T.
Published: October 2019

Abstract 5083

RDW, a quantitative measure of anisocytosis, has been utilized for many years in the differential diagnosis of anemia. More recently, studies have reported that RDW varies with renal function and markers of inflammation, and that RDW is a predictor of mortality (Scand. J. Clin. Lab. Invest. 2009; 68:745-8; Arch. Pathol. Lab. Med. 2009; 133:628-32; Arch. Intern. Med. 2009; 169:588-94). However, these reports were based on studies of large populations of unselected outpatients, many of whom were hematologically normal. Utilizing a de-identified database of 32 anemic adult inpatients (17 male/15 female, 27 Caucasian) who had undergone diagnostic bone marrow examination as part of an earlier study (J. Clin. Lab. Haematol. 1999; 21:161-7), associations between RDW and clinical laboratory parameters in a more restricted patient subset were examined.

RDW values for 26/32 patients were in the highest quintile observed in the National Health and Nutrition Examination Survey (RDW > 13.8%). Significant differences between patients in the highest quintile and other patients were observed for hemoglobin concentration (9.9 g/dL vs. 11.6 g/dL, p = 0.01), serum soluble transferrin receptor (sTfR) (30.3 nM vs. 16.7 nM, p = 0.02), and serum interleukin (IL)-1 (1.6 pg/mL vs. 0.3 pg/mL, p =0.03). No other significant differences were observed in other automated hematologic markers, in biochemical indicators of iron status, renal function, or inflammation, or in marrow aspirate iron score. When the correlation between RDW and the other parameters studied was evaluated, the only statistically significant correlations found were with C-reactive protein (CRP) (r = -0.49, p = 0.009) and sTfR (r = 0.53, p = 0.002). The significance of the correlation between sTfR and RDW was independent of other indicators of iron status, CRP, and serum IL-1. In contrast, the significance of the correlation between CRP and RDW was dependent on variation in sTfR and serum ferritin concentrations. In this group of patients with who generally had either high-normal or high RDW values, CRP and RDW were inversely correlated.

In summary, associations between RDW and other laboratory or clinical parameters observed in unselected patient populations may not be applicable to complex anemic inpatients. The association between sTfR concentration and RDW appears to be independent of the relationship between sTfR concentration and patient iron status.

Disclosures

Means: Beckman Coulter, Inc.: Consultancy, Honoraria.
Haploidentical Hematopoietic Stem Cell Transplantation for Non-Hodgkin's Lymphoma with Bone Marrow Involvement.
Authors: Wang HengXiang , Xue Mei , Liu Jing , Yan Hong-Min , Duan Lian-Ning , Zhu Ling , Wang Zhi-Dong
Published: October 2019

Abstract 5124

Here, we report the preliminary results of haploidentical hematopoietic stem cell transplantation (Haplo-HSCT) with G-CSF mobilized bone marrow grafts without T cell depletion for 10 cases with refractory non-Hodgkin's lymphoma accompanied by bone marrow involvement. The patients were aged ranging 7 to 38 years and six were of T- and four were B-cell origin at diagnosis. Eight patients received a conditioning regimen consisting of high-doses of cytarabine and cyclophosphamide with total body irradiation, while two cases busulfan, thiotepa and cyclophosphamide. All patients had rapid engraftment with the mean time for neutrophil and platelet recovery was 16.6 days and 19.2 days, respectively. Three cases died within six months after transplantation from severe acute graft-versus-host disease, fungal infection or relapse. The others are currently alive at a median follow-up of 60.7 months (range: 44-81months). Therefore, haplo-HSCT might provide an opportunity of myeloablative therapy for refractory lymphoma with bone marrow involvement.

Disclosures

No relevant conflicts of interest to declare.
PU.1 Relieves Its GATA-1-Mediated Repression near Cebpa and Cbfb During Transdifferentiation of Murine Erythroleukemia - Tool of Inducing Leukemic Blasts to Differentiate.
Authors: Burda Pavel , Curik Nikola , Kokavec Juraj , Mikulenkova Dana , Skoultchi Arthur , Zavadil Jiri , Stopka Tomas
Published: October 2019

Abstract 547

Transcription factors GATA-1 and PU.1 interact on DNA to block transcriptional programs of undesired lineage during hematopoietic commitment. Murine erythroleukemia (MEL) cells that co-express GATA-1 and PU.1 are blocked at the blast stage but respond to down-regulation of PU.1 or up-regulation of GATA-1 by inducing terminal erythroid differentiation. To test whether GATA-1 blocks PU.1 in MEL cells we have conditionally activated a transgenic PU.1 protein fused with the estrogen receptor ligand-binding domain (PUER), resulting in activation of a myeloid transcriptional program. Gene expression arrays identified components of the PU.1-dependent transcriptome negatively regulated by GATA-1 in MEL cells, including CCAAT/enhancer binding protein (C/EBP) alpha (Cebpa) and Core-binding factor, beta subunit (Cbfb) that encode two key hematopoietic transcription factors. Inhibition of GATA-1 by siRNA resulted in derepression of PU.1 target genes. Chromatin immunoprecipitation and reporter assays identified PU.1 motif sequences near Cebpa and Cbfb co-occupied by PU.1 and GATA-1 in the leukemic blasts. Substantial derepression of Cebpa and Cbfb is achieved in MEL cells by either activation of PU.1 or knockdown of GATA-1. Furthermore, transcriptional regulation of these loci by manipulating the levels of PU.1 and GATA-1 involves quantitative increases in a transcriptionally active chromatin mark: acetylation of histone H3K9. Collectively, we demonstrate that either activation of PU.1 or inhibition of GATA-1 efficiently reverse the transcriptional block imposed by GATA-1 and lead to activation of a myeloid transcriptional program directed by PU.1.

The mechanism of PU.1 and GATA-1 in leukemic state and upon leukemia differentiation involves the following putative steps: at myeloid genes such as Cebpa, PU.1 binds directly to DNA but is repressed by GATA1 that binds directly to PU.1 molecules on DNA. Activation of PU.1-ER and stable levels of GATA-1 create excess of availabel PU.1, which is not paired by availabel GATA-1 on DNA, allowing thus gene activation. Similarly, on erythroid genes such as Nfe2, GATA1 is bound to DNA, but is repressed by PU.1 that binds to this GATA-1 molecule. Activation of GATA1-ER creates an excess of availabel GATA-1 which is not paired on DNA by availabel PU.1, also allowing gene activation. Our mechanistic study implicates that transcription factor manipulation, such as inhibition of GATA-1 or activation of PU.1 in erythroleukemias, may represent an efficient tool of inducing leukemic blasts to differentiate. (Grants NR9021-4, 10310-3, 2B06077)

Disclosures:

No relevant conflicts of interest to declare.