bluuebunny/crossref_metadata_2025 huggingface dataset:
X-Reactivation Impacts Human iPSC Differentiation Potential Towards Blood
Authors: Moraghebi Roksana , Kirkeby Agnete , Larsson Marcus , Herbst Andreas , Parmar Malin , Soneji Shamit , Woods Niels-Bjarne
Published: October 2019

To determine novel key regulators that direct ES/iPS cell differentiation to hematopoietic lineages, we compared the gene expression profiles of multiple iPS cell lines with defferential blood forming capacity. We generated multiple iPS cell lines from amniotic fluid derived mesenchymal stromal cells (AF-iPS) which differentiated towards hematopoietic lineages using our standardized and highly reproducible differentiation protocol. Of the 9 AF-iPS cell lines derived from an individual female patient, the average efficiency of CD45+ hematopoietic cells was 14.2 +/- 9% (range 1.6 to 26.3%).

To elucidate the possible reasons for this diversity in efficiency, we grouped the AF-iPS cell lines on the basis of lowest and highest blood differentiation capacity and compared their gene expression profiles by microarray. We found very few changes above 1.5-fold, but interestingly, among the 11 genes that were over-expressed in the AF-iPSC lines with poor blood differentiation efficiency 10 were located on X chromosome, and the remaining one reported to be involved in Notch signalling.

A combination of cumulative sum analysis and the location of differentially expressed genes on the X chromosome identified putative regions of reactivation at multiple, but distinct locations. The possibility of X-reactivation in these female lines was reinforced further where lower levels of XIST were seen in AF-iPSC lines shown to have low blood forming potential, however only half of the iPS cell lines with high blood differentiation capacity showed normal XIST expression when compared to the amniotic fluid mesenchymal starting cell material.

To determine whether the block in differentiation was tissue specific we tested the differentiation capacity of the AF-iPSC lines towards neuronal lineages. Intriguingly we found neural cell differentiation was not hampered within all lines with poor blood potential suggesting that the over-expression of genes as a consequence of X-reactivation can impart a specific negative effect on differentiation towards the blood lineages from pluripotency stage, while not having an effect on neuronal cell development.

To further define the source of this block, we have begun working knocking down the overexpressed genes on X chromosome in lines with poor blood differentiation potential to determine whether the efficiency can be increased (or fully rescued) with one, or a combination of these 11 candidate genes. These results have implications for the identification and selection of female iPS lines suitable for therapeutic purposes.

Disclosures:

No relevant conflicts of interest to declare.
In Vitro Generation Of Osteoclasts From Interleukin-3 (IL-3)-Dependent Mouse Hematopoietic Cells
Authors: Wang Shunqing , Hong Huixian
Published: October 2019

Objective

This work aims to develop a new strategy to generate murine osteoclasts in vitro using IL-3-dependent cells prepared by 6-day IL-3 treatment of murine bone marrow cells

Methods

1. Here, we describe an alternative method for in vitro generation of osteoclasts, which involves the use of interleukin (IL)-3-dependent murine bone marrow cells. Bone marrow cells, isolated from 6- to 8-week old C57BL/6 were cultured in α-MEM containing 10% FBS in tissue culture dishes overnight to remove stromal cells. Then, non-adherent bone marrow cells were harvested and continued in α-MEM containing 10% FBS without (control) or with IL-3 (1 ng/ml) for 6 days. While no cells survived in the control culture after the 6-day culturing, the IL-3-treated culture gave rise to a significant number of surviving cells. These IL-3-depedent cells were capable of differentiating to osteoclasts in response to M-CSF and RANKL stimulation. Moreover, these IL-3-dependent cells can be further expanded by plating them in non-treated plastic dishes followed with M-CSF treatment; they continued to survive and proliferate in non-treated plastic dishes in the presence of M-CSF for up to 4 days. After 4-day M-CSF treatment, these cells can be lifted by EDTA, and they were still able to differentiate into osteoclasts upon subsequent stimulation of M-CSF and RANKL.

2. We performed the in vitro bone resorption assay, Semiquantitative Reverse Transcription (RT)-PCR, Western Analysis, Infection of Murine Bone Marrow Cells (BMCs) to test whether the osteoclasts generated from IL-3-dependent murine bone marrow cells are different from the osteoclasts generated from traditional method.

Results

1. IL-3 can maintain the survival of murine bone marrow cells for up to 6 days and these cells still keep their capacity to generate osteoclasts. The capacity of IL-3-dependent cells to form osteoclasts decreases with time of IL-3 treatment and IL-3 dependent cells can be further expanded by M-CSF without significant loss of the osteoclastogenic potential.

2. IL-3-dependent cells can form functional osteoclasts. RANKL induces the expression of osteoclast genes in IL-3-dependent cells. RANKL activates some of RANK signaling pathways in IL-3-dependent cells. Importantly, we found that IL-3 dependent murine bone marrow cells can be infected by retrovirus encoding GFP.

Conclusions

1) We have developed a new strategy to generate murine osteoclasts in vitro using IL-3-dependent cells prepared by 6-day IL-3 treatment of murine bone marrow cells.

2) IL-3-dependent cells can be infected by retrovirus, permitting further experimental manipulations to express or knock down genes in IL-3-dependent cells for studying the molecular mechanism controlling differentiation and proliferation of osteoclast precursors or delineating molecular events in early osteoclastogenesis.

Disclosures:

No relevant conflicts of interest to declare.
Methylene Blue Selectively Inhibits B Cell Development and E2A Activity
Authors: Soans Eroica , Choi John K
Published: October 2019

Introduction

E2A, a basic helix loop helix transcription factor, is essential for B lymphocyte proliferation, survival and differentiation. Mutations in E2A are associated with B cell leukemia and lymphomas. Our preliminary data suggests that E2A activity is altered by HSP70 and since HSP70 ATPase activity is inhibited by methylene blue (MB), we hypothesized that MB will alter E2A activity and thus alter B cell development.

Methods

We studied the effects of MB on normal and leukemic B cells compared to its effect on myeloid cells. The cell lines used were REH, NALM-6 and BAF3-p185 cells compared to myeloid cell lines, CCRF and CMK for the proliferation assay. To study MB effect on normal B and myeloid progenitors, methylcellulose colony formation assays with murine bone marrow cells (strain 129S1/SVImJ, 8-12 weeks old mice) were performed. We also treated mice with MB in their drinking water (1000 μM) up to 16 weeks to study the in-vivo effect on B cells as compared to granulocyte, macrophages, T cells and NK cells in the peripheral blood by flow cytometry analysis. To understand the mechanisms by which MB alters B cell proliferation, we examined the effects of MB on cell cycle progression, apoptosis and E2A activity in B cells.  For cell progression NALM-6 cells were treated with 4 μM MB for 0, 6, 12 and 24 hrs and assayed using BRDU staining by flow cytometry analysis. Further, apoptosis was measured using Annexin V staining by flow cytometry analysis in NALM-6 cells up to 48hrs post treatment with MB. Lastly, E2A activity was measured using an E2A-specific luciferase reporter assay in 293T cells.

Results

MB decreased the proliferation of precursor B cell lines, REH, NALM-6 and BAF3-p185 cells compared to myeloid cell lines, CCRF and CMK. Similar effects of MB were also seen with normal B and myeloid progenitors as assayed by methylcellulose colony formation assays with murine bone marrow cells. Similarly, there is a significant decrease in peripheral B cells as compared to granulocytes, macrophages, T cells and NK cells when the mice were treated with 1000 μM of methylene blue in their drinking water as compared to those that remained untreated. MB caused apoptosis and decreased the S phase fraction of treated NALM-6 cells. In addition, we found that MB inhibited E2A activity using an E2A-specific luciferase reporter assay.

Conclusion

Our findings suggest that MB is a specific inhibitor of B cell proliferation, survival and development. This effect may be mediated by the inhibition of E2A activity by methylene blue.

Disclosures:

No relevant conflicts of interest to declare.
Genetic Modifiers Of Beta-Thalassaemia Associated With Management Without Blood Transfusion: Ten Years Experience
Authors: Ansari Saqib Hussain , Shamsi Tahir S , Khan Mohammed Tahir , Munzir Saima , Erum Sajida , Perveen Kousar , Parveen Sadia , Farzana Tasneem , Ashraf Mushtaq
Published: October 2019

Background

Long survival with improve quality life in treatment of thalassaemia patients can be procure by blood transfusion and sedulous chelation but, it is cumbrous. Therapeutic manoeuvers designed to stabilize or increase hemoglobin without transfusion and to reduce the blood transfusion. Efforts have been undertaken to find an alternative approach to transfusion in β-thalassaemia.

Methods

On the basis of our previous studies evaluating the safety & efficacy of Hydroxyurea (HU) in beta thalassemia patients which is an oral Hb-F augmentation agent, at 10-15 mg/kg/day was used on 473 patients for Mean duration of 3 years under the guidelines of Helsinki’s declaration. Response was measured by using transfusion requirement prior to 6 months period of enrollment in the study as control. Patients were finally divided into Complete Responders (CR), partial responders (PR) and non-responders(NR).Patients’ genetic profile was investigated. Chi square test was applied to check association between variables, p-value ≤0.05 considered significant.

Results

Complete responders were 176, PR were 194 while 103 were NR.  Most common genetic mutations observed were IVS1-5 (46%) either homozygous or heterozygous, Fr 8-9 (14.3%), IVS1-1 (11%), Cd 30 (5.5%), Fr 41-42 (5.5%), Del-619 (5%) &Cap+1 (1.9%). Astounding results were found when association of these mutations with response of HU was observed. Response was found to be closely related with gene mutations Cd-30 (61.5 % Responders), IVS1-1 (58 % Responders), Cap+1 (44.4 % Responders), IVS1-5 (33.6 % Responders). Homozygous Xmn-I polymorphism was observed in 21% & heterozygous in 24% of the patients, while 55 % had no Xmn-I polymorphism. Xmn-I polymorphism was found to be significantly associated with Response 67 % (p-value 0.00).

Conclusion

Hydroxyurea showed promising results in this long term follow up of large cohort of patients. We suggest screening all newly diagnosed thalassaemic children for Genetic characteristic in order to offer Hb-F Augmenting agent as an alternative to blood transfusion.

Disclosures:

No relevant conflicts of interest to declare.
Distinct Chemokine Receptor Profile In Chronic Lymphocytic Leukaemia and Richter Transformed Diffuse Large B Cell Lymphomas Compared To Germinal Center B Cells and De Novo Diffuse Large B Cell Lymphomas
Authors: Wenzl Kerstin , Troppan Katharina , Deutsch Alexander JA , Linkesch Werner , Neumeister Peter , Beham-Schmid Christine
Published: October 2019

Chemokine receptors are G-protein-coupled cell surface receptors, which dissociate upon activation by their ligands and cause downstream signaling. Recently, several studies have revealed the crucial contribution of chemokine receptors and their ligands in normal B-cell differentiation and development of hematopoietic malignancies. The Richter syndrome (RS) represents the clinico-pathologic transformation of chronic lymphocytic leukaemia (CLL) to an aggressive lymphoma, most commonly diffuse large B-cell lymphoma (DLBCL). Due to the lack of knowledge on the chemokine receptors in RS, we aimed to investigate their expression profile in Richter syndrome patients. Therefore, we investigated the mRNA expression levels of 18 known chemokine receptors (CCR1-CCR9, CXCR1-CXCR7, XCR1, CX3CR1) by using semi-quantitative real time PCR on seven samples of paired (CLL and transformed DLBCL) RS samples, and six samples of de novo DLBCL, additionally four CLL samples –all of them transformed into DLBCL-, and eight transformed DLBCL samples –all of them originated from CLL- were included. Four samples of peripheral B cells and four samples of germinal center B cells (GCB) served as non-neoplastic controls.

11 out of 18 chemokine receptors were differentially expressed in CLL (RL) and transformed DLBCL originated from CLL (RH) compared to GCBs. RL exhibited an at least 15-fold higher expression of CCR2, CCR4, CCR7, CXCR3, and XCR1, as well as a de novo expression of CCR3, CCR8, CXCR1, CXCR2, and CX3CR1 compared to GCB. Additionally to these chemokine receptors, CCR1 also showed significant higher expression levels comparing GCB and RH samples. Only one chemokine receptor was found to be differentially expressed in our seven paired RS samples: CCR6 showed a trend of up-regulation in CLL components. Interestingly, the transformed DLBCL originated from CLL were characterized by a significant up-regulation of six chemokine receptors (CCR3, CCR7, CXCR1, CXCR3, CXCR4, and CX3CR1) and a down-regulation of CCR5 compared to DLBCL.

Our data indicate that the chemokine receptor expression profile of CLL and transformed DLBCL, originated from CLL samples, differs substantially from those of non neoplastic germinal center B cells and de novo DLBCL, suggesting other cellular origin and an impact on more aggressive clinical course of Richter syndrome patients. Hence, these multiple deregulated CC and CXC receptor might serve as useful prognostic tool to separate high malignant Richter syndrome from de novo DLBCL.

Disclosures:

No relevant conflicts of interest to declare.
Elevated Nuclease Activity Correlates With Clinical Spectrum Of Plasma Cell Dyscrasias
Authors: Patel Jaymin M. , Vahia Ankit V , Nanjappa Puru , Bajwa Ashun , Shah Sima , Yu-Tzu Tai , Anderson Kenneth C. , Shammas Masood , Munshi Nikhil C.
Published: October 2019

Over the last decade, there has been increasing interest in mechanisms that underlie genomic instability which is implicated in tumor progression, development of drug resistance and possibly even oncogenesis. We have observed that dysregulated homologous recombination (HR) is an important contributor to genomic instability. In this study, we aim to broaden our understanding of HR by exploring alternate pathways that may affect genomic instability such as nuclease activity.

Methods

We have developed an assay to measure total cellular nuclease activity and compare activity levels across a spectrum of plasma cell dyscrasias. Total nuclease activity in cell lines and patient samples was assessed using a novel plasmid degradation assay. Briefly, multiple myeloma (MM) cell lines and primary MM patient cells were lysed at equal cell concentration. The lysates were mixed with plasmid DNA in triplicate/quadruplicate and incubated at 37°C for fixed time points ranging from 0 to 30 minutes. Following forced cessation of enzymatic activity, samples were fractionated on agarose gel and resultant image analyzed for band intensity by Kodak 1D Image analysis software. The ratio of degraded plasmid over time was analyzed via nonlinear regression to calculate a half-life and k-constant.

Results

Non-linear regression analysis of data derived from assays involving 6 MM cell lines (RPMI, OPM1, H292, U266, INA6 & MM1S) with three pellets each of equal size taken from non-confluent cell culture with > 85% viability showed statistically significant reproducibility of best fit curve with an average R-squared of 0.93. Similar regression analysis could not be performed on data derived from normal PBMC pellets because they showed minimal degradation with linear regression slope of (-0.62). Lastly, non-linear regression analysis on 22 patient samples with a plasma cell dyscrasia diagnosis, revealed a broader range of activity by half-life. Further differentiation of plasma cell dyscrasia patients into 2 groups revealed a significantly higher half-life in MGUS & smouldering (9499.8 minutes) versus newly diagnosed and refractory or relapsed multiple myeloma patients (9.4,19.3 minutes respectively).

Conclusions

In conclusion, we show that nuclease activity, implicated in elevated recombination and genomic instability, can be measured in a statistically reproducible fashion by our in vitro assay and may be used to differentiate plasma cell dyscrasias across a clinical spectrum. Further analysis of nuclease activity in clinically annotated samples for correlation with clinical features, genomic data and survival outcomes is ongoing.

Disclosures:

Anderson: celgene: Consultancy; onyx: Consultancy; gilead: Consultancy; sanofi aventis: Consultancy; oncopep: Equity Ownership; acetylon: Equity Ownership.
KLF4 induces apoptosis in T-ALL through the BCL2/BCLXL pathway
Authors: Xu Bing , Wang Xiangmeng , Li Peng , Li Wei , Dong Huijuan , Zhou Yong , Li Yanyan
Published: October 2019

KLF4, also known as GKLF (gut KLF), is a member of the KLF zinc finger-containing transcription factor family. Klf4 together with Oct4, Sox2, and c-Mycare widely referred to as ‘Yamanaka factors’ because mouse somatic cells can be reprogrammed into pluripotent stem cells following their ectopic expression. The transcription factor Kruppel-like factor 4 (KLF4) may induce tumorigenesis or suppress tumor growth in a tissue-dependent manner. In T cell leukemia and pre-B cell lymphoma cells, KLF4 acts as a tumor suppressor. We found that over expression of KLF4 induced human acute T cell lymphoblastic leukemia (T-ALL) cell lines to undergo apoptosis through the BCL2/BCLXL pathway, and we confirmed KLF4-induced apoptosis in primary samples from T-ALL patients. We further characterized KLF4 function in human early and mature T cells. Our analysis uncovered that KLF4 suppressed the transcription of other T cell-associated genes in T-ALL.

Disclosures:

No relevant conflicts of interest to declare.
In Vitro Effect Of VPA Together With ATRA and Ara-C On Proliferation and Viability Of Pediatric AML Cell Line THP-1
Authors: Anak Sema , Kanbay Ayca Gul , Gulec Cagri
Published: October 2019

In addition to its HDAC inhibitory property, Valproic acid is also known as anticonvulsant agent and mood stabilizer in the treatment of bipolar disorders. Due to its HDAC inhibitory activity and its safety in long-term usage, VPA is presumed to be a good candidate for cancer treatment. It is known that VPA induces apoptosis in leukemic cells, while not in normal cells. VPA is reported as an effective agent in treatment of pediatric AML in clinical studies and  is also well tolerated in children.

In this study, the in vitro effect of the combination of HDAC inhibitor VPA with Ara-C and ATRA which are used in AML therapy, is investigated on AML cells. For this purpose, the effect of VPA, Ara-C and ATRA on proliferation of AML cell line THP-1 is tested in cell culture condition. To assess the effect on cell proliferation, p21 expression was measured by RT-PCR method. The use of VPA alone, did not affect the cell viability, while increasing the expression of the p21 gene. VPA in combination with Ara-C, increased the expression of p21 gene more than the other combinations. Thus it is determined that the p21 gene expression is higher as a result of known cell cycle stops.

In this study, the understanding of how effective is VPA together with ATRA and/or Ara-C on AML cells, might be a good start for animal studies and clinical trials as a remarkable data for the development of new chemotherapeutic protocols.

Disclosures:

No relevant conflicts of interest to declare.
Reduction Of Immune Repertoire Diversity Through DNA Sequence Constraints At VDJ Junctions
Authors: Howie Bryan , Robins Harlan , Carlson Christopher S
Published: October 2019

B and T lymphocytes are effector cells of the adaptive immune system. These cells express surface receptors that bind a huge variety of antigens, and together they comprise a person’s immune repertoire. A diverse repertoire is essential for mounting robust immune responses against a wide range of pathogens, and repertoire diversity affects the probability that DNA sequencing can uniquely tag a clonally expanded population of cells for the detection of minimum residual disease (MRD) during cancer treatment.

Immune repertoire diversity arises partly through the combinatorial splicing of gene segments from the variable (V), diversity (D), and joining (J) regions of a B or T cell receptor locus. Much additional diversity is created through the stochastic insertion and deletion of nucleotides at the splice junctions, and by somatic hypermutation (SHM) in maturing lymphocytes. The generation of junctional diversity is an important part of this process, but it may be constrained by the underlying biological mechanisms.

To explore the landscape of junctional diversity among immune receptor loci, we developed a likelihood model that can annotate VDJ junctions in the presence of SHM and compute the probability that a given receptor sequence was generated only once in a person’s repertoire, which is essential for tracking MRD. Using high-throughput sequencing data from several individuals and a range of receptor loci, we identify mechanistic constraints that limit B and T cell receptor diversity. For example, we show that the usual variability in CDR3 length is reduced at the immunoglobulin kappa (IgK) locus, and we connect this finding to sequence motifs that constrain nucleotide deletion at the ends of IgK gene segments. Our findings will inform future genetic studies of the adaptive immune system, and they provide quantitative guidance for deciding which cancer clones can be tracked for reliable MRD detection.

Disclosures:

Howie: Adaptive Biotechnologies: Employment, Equity Ownership. Robins:Adaptive Biotechnologies: Consultancy, Equity Ownership, Patents & Royalties. Carlson:Adaptive Biotechnologies: Consultancy, Equity Ownership, Patents & Royalties.
Mutaome Profiling and Retrospective Mutaome Profiling Using Archived Bone Marrow Smear In AML
Authors: Liu Hongxing , Wang Fang , Teng Wen , Lin Yuehui , Yang Junfang , Zhang Xian , Yin Qing , Chen Xue , Zhu Ping , Tong Chunrong
Published: October 2019

Background

Acute myeloid leukemia (AML) is a heterogeneous disease with respect to presentation and clinical outcome. In recent years, more and more somatic mutations and their clinical significance were identified in AML. Most AML patients carry multiple gene mutations and the repertoire of mutations changing during the disease process, which determine the patient's unique clinical manifestations. Herein we recommend using the novel word ”mutaome¡± for representing the repertoire of somatic gene mutations in a specific tumor tissue, and aimed to establish a panel of mutation profiling protocol and retrospective profiling using archived bone marrow smear for clinical use in AML.

Methods and Cases

Mutation profiling protocol for CEBPA, DNMT3A, FLT3-ITD/TKD, IDH1, IDH2, KIT, NPM1, PHF6 and TET2 by PCR and Sanger sequencing was established, with the detection sensitivity about 15% to 20%. Bone Marrow (BM) or peripheral blood (PB) was collected form patients with newly diagnosed or relapsed. Archived BM smear on glass slides at the time of newly diagnosed were used for retrospective mutation analysis.

Results

1) Totally 61 archived smear, 106 fresh BM or PB and 2 paraffin-embedded pathological specimens from 157 patients were analyzed. Age ranged from 1 to 77 years old, with the median age of 27 years old.

2) 60.4% (102/169) samples carrying at least one mutation, 51.0% (53/102) of them carrying 2 or more mutations, 30.4% (31/102) carrying mutations within 2 or more different genes. The number of mutated sample for each gene is 33 for CEBPA¡¢9 DNMT3A¡¢35 FLT3¡¢6 IDH1¡¢7 IDH2¡¢7 KIT¡¢19 NPM1¡¢8 PHF6¡¢and 21 TET2.

3) Paired archived smear and relapsed samples were analyzed for 11 cases, 9 of them showing difference, detailed results shown in Table 1.

4) Totally 53 patients come to our hospital after certain treatment, with the tumor cells below 15% and without AML gene mutation results. For these patients, archived BM smear samples were used for retrospective mutation profiling and then to guide targeted therapy and stratified evaluation.

Conclusions

Panel testing for gene mutations is effective method for detection of AML molecular markers. For the patients came with partial remission and without gene mutation result, archived bone marrow smear sample can be used for retrospective mutation analysis and then help guiding targeted therapy and stratified evaluation.

Disclosures:

No relevant conflicts of interest to declare.
MicroRNA Dysregulation in Newly Diagnosed Chronic Myeloid Leukaemia Patients
Authors: Gordon Jane E. A. , Bailey Charles G , Rasko John , Ritchie William , Watkins Dale B. , White Deborah L. , Leclercq Tamara , Hughes Timothy P
Published: October 2019

Introduction

Heterogeneity within the CML patient population is evidenced by variable response dynamics and tolerability between patients treated with tyrosine kinase inhibitors (TKIs). Clinical risk scores have utility however do not inform upon the biology underpinning differential response. OCT1 activity has been established as a predictor of imatinib response but is not predictive for second generation TKIs nilotinib or dasatinib.

In CML, microRNAs (miRNAs) have been implicated in BCR-ABL mediated signalling pathways and alter rapidly after TKI treatment. Indeed, we have previously established that miR-142-3p decreases within 14 days of TKI initiation and correlates with the SOKAL score.

Method

We performed miRNA profiling on 75 newly diagnosed, clinical trial CML patients (56% male, 44% female, median age 51 years) with greater than 24 months follow-up. RNA was extracted from peripheral blood mononuclear cells. The expression of 800 mature miRNA was analysed using the nCounter Human v2 miRNA Expression Assay (NanoString Technologies). In addition, IC50 values for imatinib and nilotinib were determined on all samples as previously described.

Results

The miRNA expression levels obtained on this platform showed a wide dynamic range and substantial interpatient variation. This included miRNA previously reported in CML in vitro and patient studies: interquartile ranges were 52414 to 121632 for miR-142-3p, 3120 to 15599 for miR-451a, 2658 to 12023 for miR-150, 828 to 1461 for miR-181a-5p. In addition, we were able to identify miRNAs that were associated with nilotinib IC50 and imatinib IC50.

Discussion

The current study highlights the variability evident at the miRNA level between newly diagnosed CML patients. It provides a rationale to further examine the role of miRNA in CML pathogenesis, TKI response and define patient heterogeneity from diagnosis.

Disclosures:

White: Novartis: Research Funding; BMS: Research Funding, Speakers Bureau; Ariad: Research Funding; CSL: Research Funding. Hughes:Novartis: Honoraria, Research Funding; BMS: Honoraria, Research Funding; ARIAD: Honoraria, Research Funding.
Therapy Related Acute Lymphoblastic Leukemia: Pethema Experience
Authors: Kelleher Nicholas John , Gallardo David , Brunet Salut , Montesinos Pau , Ribera Josep-Maria , Barba Pere , Garcia Olga
Published: October 2019

Background

Therapy related acute lymphoblastic leukemia, a subset of secondary acute lymphoblastic leukemia has been estimated as accounting for between 1.2 and 6.9% of all adult acute lymphoblastic leukemia cases. It has been associated with an increased frequency of high risk cytogenetic alterations and with worse clinical outcomes. It has been suggested these patients should be included in high risk treatment protocols.

Method

In order to evaluate these characteristics in a group of similar patients we contacted centres working within the PETHEMA group to request data on patients diagnosed with ALL asking for clinical information including the presence or absence of previous neoplasia and of previous cytotoxic therapy along with treatment responses and survival data.

Results

We received information on 429 patients of whom 22 had received cytotoxic therapy for a prior neoplasm.Patients were divided into group 1 with prior cytotoxic therapy, group 2 with prior neoplasia without cytotoxic therapy and group 3 de novo ALL. We found patients in group 3 to be younger than the other two groups Group 1( 55 years) Group 2 (65 years) Group 3 (34 years) (p=0.001). No statistically significant difference was shown for white cell count, cytopenias, CNS involvement, LDH or for B versus T immunophenotype. Nor did our series show a significant difference in the frequencies of high risk cytogenetics between the groups. Figures for complete remission [Group 1- 13 (93%); Group 2- 6 (75%); Group 3-346 (85%) p=0.477] were higher in group 1 therapy related ALL compared with de novo patients without reaching clinical significance. Nor was a statistically significant difference shown for 3 year overall survival [Group 1 (80%); Group 2 (38%); Group 3 (47%) p=0.151] , 3 year event free survival [Group 1 (67%); Group 2 (38%); Group 3 (42%) p=0.24] or for complete remission duration [Group 1 (75%);Group 2 (50%); Group 3 (60%) p=0.462]

Conclusion

Apart from age, our series did not show an increase in poor risk clinical or cytogenetic features in therapy related ALL patients compared with de novo disease cases and nor was clinical outcome demonstrated to be worse. This would suggest that risk stratification should be carried out using currently recognized parameters without specifically taking into account the status of therapy related disease.

Disclosures:

No relevant conflicts of interest to declare.
Pathological Confirmation Of Apparent Metastatic Disease Is Essential: Incidentally Diagnosed Low Grade B Cell Lymphomas In Patients With Solid Tumors
Authors: Gaballa Sameh , Abdelmalek Cherif , Alpdogan Onder , Axelrod Rita S. , Campling Barbara , Kelly William kevin , Rodriguez Joanna , Gitelson Elena
Published: October 2019

Background

The detection of skeletal metastasis, enlarged lymph nodes or parenchymal lesions in patients (pts) with established solid tumors most commonly denotes advanced stage disease. If not confirmed histologically, a subset of pts might be over staged and mismanaged palliatively.

Methods

We report 7 cases of low-grade B cell lymphomas diagnosed in a bone, lymph node or lung biopsy during staging workup in patients with suspected metastatic solid tumors.

Results

All pts were men aged 41 to 80 yo diagnosed with: non-small cell lung (NSCL), prostate, lip squamous cell, bladder, renal cell cancer (CA) and nasal adenoid cystic carcinoma. Imaging studies done during initial workup or follow up after resection of the primary tumor revealed bone metastasis, lymphadenopathy or lung nodules suggesting advanced stage of the primary CA. Invasive workup of the lesions in question revealed incidental low-grade B cell lymphomas (2 low grade lymphomas of bone, 2 CLL/SLL, 1 BALT, 1 marginal zone and 1 nodular lymphocyte predominant Hodgkin lymphoma). In all cases, this led to down staging and changed the management of the solid CA. Surgical resection of the tumor was done after it was down staged from non-resectable to resectable in 2 pts with head/neck CA; 1 pt with NSCL was down staged from stage IV to IIIA and received chemo/radiotherapy; 1 pt with prostate CA was down staged from stage IV to I; 3 pts were down staged from stage IV and were in remission from previously resected solid tumors. Only 2 pts required therapy for the newly diagnosed lymphoma. Avidity of the lesions revealed SUV ranging from 1.28 to 14.11 in the bone and from 2.17 to 6.98 in the lymph nodes.

Conclusions

Accurate staging of pts with solid tumors is critical in defining optimal goals of therapy. The growing use of PET/CT scans results in a higher rate of incidentally detected bone lesions or lymphadenopathy. Whereas a number of solid tumors readily spread to bone and lymph nodes, a spectrum of indolent lymphoid disorders may coexist in pts with established solid tumors. These lymphomas may remain asymptomatic for years. This might be misinterpreted as advanced stage solid tumor unless confirmed histologically.

Disclosures:

No relevant conflicts of interest to declare.
Can Clinical Parameters Predict Bone Marrow Involvement In DLBCL?
Authors: Baillargeon Paul , Nistala Puja , Doll Donald
Published: October 2019

Diffuse large B-cell lymphoma (DLBCL) is the most common non-Hodgkin lymphoma, accounting for about 1/3rd cases of NHL. At diagnosis, DLBCL involves the bone marrow in approximately 15-25% of cases. However, the bone marrow may reveal discordant morphology from the lymph node, usually an indolent lymphoma.

Bone marrow biopsy is routinely used in the staging evaluation of patients with DLBCL. The aim of this study was to determine if clinical variables could predict bone marrow involvement by DLBCL.

Methods

Retrospective chart review of all patients with a diagnosis of DLBCL at University of Missouri-Columbia/Ellis Fischel Cancer Center from 1993-2013. The following parameters were reviewed: bone marrow biopsy reports, anemia (Hb<10g/dl), leukopenia (wbc<4000), thrombocytopenia (<150,000), lactate dehydrogenase level and clinical stage. Statistical analysis was performed by Fischer Exact test and logistic regression analysis.

Results

80 charts were available for review and 7 patients had bone marrow involvement by DLBCL. Univariate analysis for cytopenias (p=0.459), elevated LDH (p=0.442), anemia (p=0.436), leukopenia (p=1.0) and thrombocytopenia (p=0.434) and bone marrow involvement by DLBCL showed no statistical significance (Fischer Exact test). Multivariate analysis comparing anemia, leukopenia, thrombocytopenia, elevated LDH and clinical stage with respect to bone marrow involvement revealed p=0.789, p=0.134, p=0.062, p=0.827 and p=0.521 respectively, and disclosed no statistical significance (logistic regression analysis). Univariate (Fisher exact tests and Cochran-Mantel-Haenszel statistics) and multivariate (logistic regression) analyses were used to test for the relationship of these parameters with bone marrow involvement.

Disclosures:

No relevant conflicts of interest to declare.
Breast Implant Related Anaplastic Large Cell Lymphoma ALK- (ALCL ALK-) – Case Report
Authors: Arbelaez Alejandro , Catley Laurence , Pool Louis
Published: October 2019

Case presentation

A 29 underwent bilateral cosmetic breast augmentation 10 years previously (McGhan Textured Round 400 mL implants). Six months before, she developed slowly progressive right breast pain and inflammatory signs associated with fluid collection around the right breast that was drained. A yellow cloudy fluid was examined and showed atypical large lymphoid cells. The cell block prepared in another institution showed numerous lymphoid cells including large atypical cells with lobated nuclei. PET CT scan was negative, same as bone marrow aspirate and trephine for lymphoma infiltration. Following bilateral removal of the breast implants, further histopathology studies showed no infiltration by lymphoma of the breast capsules or scar tissue. However, right breast peri prosthetic fluid microscopy showed a population of single malignant cells with scanty cytoplasm, numerous mitosis, and nuclei showing single and multiple nucleoli. Some cells showing horseshoe nuclei. The malignant cells were positive for CD30 and LCA and negative for CD20, CD68, AE1-3, and ALK 1. FISH for ALK was not possible (Fig 1)

Discussion

Primary breast lymphomas are very rare conditions; they represent less than 1% of all NHL and less than 0.7% of all breast malignancies. There have been some cases reported in the medical literature of ALCL ALK- associated with breast implants. All the cases have been described in patients with textured implants, such as in this case and the reason is unknown. There are two main types of ALCL of the breast based on published case reports: a mass and an effusion. Primary breast effusion associated ALCL portends a good prognosis despite the fact that they are ALK-. The development of ALCL proximal to breast implants suggests that they are the result of an immune reaction to the silicone. Whether they represent true malignancy or a localised reactive phenomenon is not entirely clear yet. In previous case series, the condition has been described as indolent. However, given the low incidence of this condition and the limited literature available; it is difficult to know what the best treatment approach is. Following confirmation of the diagnosis, treatment options were discussed with the patient and the preferred option was active treatment with local radiation after removal of breast implants.

Disclosures:

No relevant conflicts of interest to declare.
The Combination Of Arsenic, Interferon-Alpha, and Zidovudine Restores An “Immunocompetent-like” Micro-Environment In Patients With Adult T-Cell Leukemia/Lymphoma
Authors: Kchour Ghada , Rezaee S. A. Rahim , Farid Reza , Ghantous Akram , Rafatpana Houshang , Tarhini Mahdi , Kooshyar Mohamad-Mehdi , El Hajj Hiba , Berry Fadwa , Nasser Roudaina , Shirdel Abbas , Dassouki Zeina , Ezzedine Mohamad , Rahimi Hossein , Ghavamzadeh Ardeshir , de Thé Hugues , Hermine Olivier , Mahmoudi Mahmoud , Bazarbachi Ali
Published: October 2019

Background

HTLV-I associated adult T-cell leukemia/lymphoma (ATL) carries a dismal prognosis due to chemo-resistance and immuno-compromised micro-environment. The combination of zidovudine and interferon-alpha (IFN) significantly improved survival in ATL. Promising results were reported by adding arsenic trioxide to zidovudine and IFN.

Methods

Here we assessed Th1/Th2/Treg cytokine gene expression profiles in 16 ATL patients before and 30 days after treatment with arsenic/IFN/zidovudine, in comparison with HTLV-I healthy carriers and sero-negative blood donors.

Results

ATL patients at diagnosis displayed a Treg/Th2 cytokine profile with significantly elevated transcript levels of Foxp3, interleukin-10 (IL-10), and IL-4 and had a reduced Th1 profile evidenced by decreased transcript levels of interferon-γ (IFN-γ) and IL-2. Most patients (15/16) responded, with CD4+CD25+ cells significantly decreasing after therapy, paralleled by decreases in Foxp3 transcript. Importantly, arsenic/IFN/zidovudine therapy sharply diminished IL-10 transcript and serum levels concomittant with decrease in IL-4 and increases in IFN-γ  and IL-2 mRNA, whether or not values were adjusted to the percentage of CD4+CD25+ cells.

Conclusions

The observed shift from a Treg/Th2 phenotype before treatment toward a Th1 phenotype after treatment with arsenic/IFN/zidovudine may play an important role in restoring an immuno-competent micro-environment, which enhances the eradication of ATL cells and the prevention of opportunistic infections.

Disclosures:

Off Label Use: Off label use of arsenic trioxide, zidovudine and interferon-alpha for the treatment of adult T cell leukemia/lymphoma.
Birinapant Enhances Bendamustine-Induced Apoptosis In Activated B Cell-Diffuse Large Cell Lymphoma Cells
Authors: Joshi Indira D , Smith Mitchell R
Published: October 2019

Birinapant (TL32711), a Smac mimetic in clinical testing, potently targets Inhibitor of Apoptosis Proteins (IAPs, including cIAPs and XIAP) to unblock intrinsic and extrinsic pathways, enabling caspase-dependent apoptosis via multiple signals. Birinapant also inactivates canonical NF-kB signaling through cIAPs. We investigated the pro-apoptotic effects of birinapant, alone and in combination with bendamustine (BDM), an active lymphoma therapeutic agent, in a panel of B cell lymphoma cell lines representing germinal center/follicular (GC) vs. activated B cell (ABC) subtypes. We hypothesized that the efficacy of this potential combination therapeutic strategy might differ between GC and ABC lymphoma types, as ABC are reported to be NF-kB-dependent. We used the following EBV negative cell lines: WSU-FSCCL t(14:18)+ follicular lymphoma (FL), FC-TxFL2 t(14:18)+ transformed FL, and SU-DHL4 GC-type diffuse large B cell lymphoma (DLBCL) as examples of GC origin lymphomas. U2932 and TMD8 cell lines represent ABC-type DLBCL.  Apoptosis was determined by annexin V staining and confirmed by caspase-3 activation, each assessed by flow cytometric methods following 48 h incubation. Birinapant had little effect (<5% annexin V+ cells) as a single agent on any of these B cell lymphoma cell lines at ≤ 100 nM, though a low level of apoptosis (7-12% annexin V+ cells) was detectable at 10-20 µM in GC types. Addition of birinapant 30-60 minutes prior to BDM did not further enhance the already high level (>50% annexin V+) of apoptosis induced by 10 uM BDM in WSU-FSCCL and FC-TxFL2,  and only slightly enhanced the low level of BDM-induced apoptosis in the GC DLBCL cell line DHL-4 (to 10-15%). In the ABC DLBCL cell lines, however, whereas 10uM BDM induced <5% annexin V+ cells for U2932 and 10-15% for TMD8, addition  of 100 nM birinapant 30-60 minutes prior to 10 uM BDM induced 35-40% annexin V+ cells in each of these ABC-DLBCL cell lines. This enhancement was schedule-dependent, not observed when birinapant was added after BDM. Thus, the cell lines representing FL and transformed FL are sensitive to BDM at clinically-achievable concentrations, without further enhancement by birinapant. The 3 DLBCL lines were relatively insensitive to BDM compared with FL cells, but BDM-induced apoptosis was markedly enhanced when birinapant was added before (but not after) BDM in the 2 ABC type DLBCL lines. Further explorations into the mechanism of birinapant sensitization of ABC-DLBCL to BDM, issues of dose and schedule, and role of NF-kB-dependency are ongoing. These data suggest that therapeutic trials of BDM plus birinapant would be of interest in ABC type DLBCL.

Disclosures:

No relevant conflicts of interest to declare.
Long Term Outcomes Of Imatinib In Chronic Myeloid Leukemia In Saudi Population, Single Center Study
Authors: Alsobhi Enaam Mohammed , Abrar Mohammed m , Abdelaal Mohammed A , Alsaeed Ahmad S. , Al-Absi Ahmed , Zahrani Zaid A , Warsi Ashraf A , Al-Amri Abdulfattah , El-Hemaidi Ihab
Published: October 2019

Background

The introduction of Imatinib therapy has significantly changed the treatment of patients with newly diagnosed chronic myeloid leukemia (CML) and improved survival. Since the International Randomized Study of Interferon (IRIS), a number of studies were conducted involving diverse populations and showed significant variations in the treatment outcome. To date, there has been no published study on the effectiveness of imatinib in adult CML patients in Saudi Arabia. The aim of the present study was to present a single-institution experience in the treatment with imatinib of newly diagnosed patients with CML and compare it with results from international studies.

Methods

A total of 101 medical records of consecutive adult CML patients treated with imatinib as first line therapy at King Abdulaziz Medical City, Jeddah, Saudi Arabia between 2001 and 2012 were retrospectively reviewed. Survival and response rates were evaluated.

Results

The estimated overall survival (OS) rates at 5 and 10 years were 95%±2.3% when patients were stratified by cytogenetic type (stander vs.variant Ph positive chromosome) at presentations, significant difference in OS, EFS, and PFS were noted (P=0.001). Complete haematological response was achieved in 94 (93.1%) of our patients, cytogenetic response (CR) in 84 (83.2%) while complete and major cytogenetic response (MCR) were observed in 70 (69.3%) and 6 (5.9%) of the patients respectively. (MR), 62 patients (61.4%) achieved major molecular response (MMR) and 34 (33.7%) complete molecular response.

Conclusion

compared to other studies among different population, our results confirm the previously noted variation in the response to imatinib. Our study has shown that Ph variant has an impact on the outcome. Further study may be indicated. However second TKI generations as first line in treatment CML with Ph variants should be consider!

Disclosures:

No relevant conflicts of interest to declare.
Chromosomal Instability: A Probable Unfavorable Prognostic Factor For Patients Of Myeloidysplastic Syndromes
Authors: Xiao Yajuan , Huang Yuanlu , Xu Na , Lin Rong , Zhou Xuan , Xiao Xiaozhen , Gao Guanlun , Xiaoli, MD Liu
Published: October 2019

Objective

Myelodysplastic syndromes (MDS) are a group of heterogeneous hematopoetic stem cell clonal disorders with a high frequency of karyotypic abnormalities (40-60%). Among karyotypic abnormalities, abnormal chromosome numbers (aneuploidy) occurs frequently. In aneuploidy, chromosomal instability (CIN) is defined as persistent mis-segregation of whole chromosomes and is caused by defects during mitosis with an odd number of chromosomes. CIN is associated with tumor heterogenesis, multidrug resistance and aggressiveness in solid tumor. Hence, we performed a one-center study on MDS patients to uncover the role of CIN in MDS clinical development.

Method

A total of 104 cases , 62 male and 42 female, aged from 15 years to 89 years, were tested by fluorescent in situ hybridization (FISH) and karyotypic analysis before any therapeutic intervention. According to the cytogenetic analysis of those two technology they were separated into 5 groups including: CIN, normal karyotype, complex karyotype excluding CIN, deletion chromosome 7 abnormality and other chromosomal abnormalities. All cases were followed up for a median of 19.5 months.

Results

Karyotyping and FISH identified 70 (67.3%) patients with abnormal karyotypes containing 32 cases of CIN, 9 cases of deletion chromosome 7 abnormality and 5 cases of complex karyotype excluding CIN. The median survival for CIN group was 13 months (incredible interval:6-20 months) compared with 23 months (incredible interval :20-27 months) in all cases, 44months in normal karyotype, 23 months in deletion chromosome 7 abnormality and 13 months in complex karyotype excluding CIN group (P=0.001 for log rank method). In CIN group, 11 cases transformed into acute leukemia with a incidence of 34% with no significant difference with total cases. And the length of time for leukemia transformation shows no significant difference between CIN group and total cases.

Conclusion

Chromosomal instability in MDS patients of the study revealed worst prognosis compared with other groups. This may suggest that chromosomal instability in MDS chromosomal abnormality confer a significant independent adverse impact on patients survival. However this effect might have no relation to leukemia transformation.

Disclosures:

No relevant conflicts of interest to declare.
Evaluation Of The JAK2-V617F Gene Mutation In Egyptian Patients With Essential Thrombocythemia and Polycythemia Vera
Authors: El Zaafarany Maha Ibrahim , Abdel-Ghaffar A. Hasan , Elkhodary Tawfik R. , Salem Dalia A. , Soliman Eman A. , Shamaa Sameh S.
Published: October 2019

An activating mutation of Janus kinase 2 (JAK2-V617F) was previously described in chronic myeloproliferative disorders (MPD). In previously published studies, the frequency of the JAK2-V617F mutation was determined to be 80–90 % for patients with polycythemia vera (PV) and 40–70 % for essential thrombocythemia (ET). In this study, we analyzed the relationship between the JAK2-V617F mutation and clinical-hematological parameters in Egyptian patients with MPD and compared these findings with published studies from other geographic regions and previous studies in EGYPT. A total of 56 patients were studied; of which, 32 were diagnosed with PV and 24 with ET. The mutation status of JAK2 was determined using allele-specific oligonucleotide (ASO) PCR assay. We found that 53% of the PV group and 79% of the ET group were positive for the JAK2-V617F mutation. When all patients were analyzed; patient age, levels of WBCs, levels of hemoglobin, levels of platelets and splenomegaly were significantly different in patients with the JAK2-V617F mutation (p < 0.05). The JAK2-V617F mutation is frequently detected in the Egyptian patients with MPD, and especially in patients with ET. Hence, it would be useful to include JAK2 mutation screening in the initial evaluation of patients suspected to have MPD especially ET.

Disclosures:

No relevant conflicts of interest to declare.