bluuebunny/crossref_metadata_2025_split huggingface dataset:
Selection For Brentuximab Vedotin Resistant Lymphoma Cell Lines Leads To Downregulation Of Surface CD30 Expression
Authors: Chen Robert , Hou Jessie , Reddy Megan , Newman Edward , Forman Stephen J. , Kane Susan 
Published: October 2019

Abstract

Background
Brentuximab vedotin (BV) is an antibody-drug conjugate that delivers a potent cytotoxic agent, monomethyl auristatin E (MMAE), specifically to cells expressing surface CD30. In pivotal phase II trials BV demonstrated a complete response rate (CR) of 34% in patients with relapsed/refractory Hodgkin lymphoma (HL) and CR of 50% in patients with relapsed/refractory anaplastic large cell lymphoma (ALCL). However, patients who do not attain CR will eventually develop progressive disease despite active treatment with BV. We aim to select for BV resistant lymphoma cell lines and examine for alterations in surface CD30 expression in cell lines.

Methods
In vitro experiments used two HL cell lines (L428, KM-H2) and one ALCL cell line (Karpas 299). The selection of BV resistant cell lines was done in two different approaches (pulsatile and constant exposure). Confirmation of BV resistance was done by MTS assays and cell counting assays. Measurement of surface CD30 was performed by flow cytometry using monoclonal mouse anti-human CD 30, clone Ber-H2.

Results
MTS assay showed the IC50 of parental cell lines to be: L428 (24 ug/ml), KM-H2 (9 ug/ml), and Karpas (16 ng/ml). The cell lines were incubated at supra-IC50 dosage until no proliferation was seen and then rescued with BV free media in the pulsatile approach. Only L428 was able to be rescued and kept in BV free media until consistent proliferation was seen (defined as incremental growth for 3 consecutive weeks). L428 was then incubated again in 50 mg/ml of BV until consistent proliferation was seen. For the constant exposure approach, the cell lines were incubated at sub-IC50 dosages and dosages of BV were adjusted up depending on cell proliferation rates. Only KM-H2 and Karpas 299 showed consistent proliferation in the constant exposure approach. We were able to select for 1 resistant line per cell type. Cell counting assay showed that all three resistant cell lines were able to proliferate in the presence of BV at dosages twice their respective IC50s (Figure 1, only L428 is shown). MTS assays showed that all resistant cell lines had increased IC50s (Table 1). Flow cytometry showed downregulation of surface CD30 expression in Karpas 299 and KM-H2 but not L428 (Table1). For Karpas 299, both the percentage of CD30+ cells and the median CD30 intensity decreased in the resistant line. For KM-H2, only the median CD30 intensity decreased in the resistant line.

Conclusion
Prolonged exposure of HL and ALCL cell lines to BV led to resistant cell lines. When compared to their parental cell lines, two out of three resistant lines showed downregulation in surface CD30 expression.

Disclosures:
Chen: Seattle Genetics, Inc.: Consultancy, Research Funding, Speakers Bureau, Trave expenses Other.
CRM1 As a New Therapeutic Target For Non-Hodgkin Lymphoma
Authors: Shi Yuankai , Wang Jianfei , Han Xiaohong , Zhang Ningning , Wang Shuai , Yao Jiarui 
Published: October 2019

Abstract

Background
The nuclear export protein chromosomal region maintenance 1 (CRM1) may play a role in human neoplasia and serve as a novel target for cancer treatment. Investigators recently developed orally bioavailable selective inhibitors of nuclear export (SINEs) that irreversibly bind to CRM1 and block its function. Our objective was to evaluate the therapeutic efficacy of the novel SINEs KPT-185 and KPT-276 against NHL in vitro and in vivo and elucidate the mechanism of CRM1 inhibitor-mediated antitumor activity.

Methods
Cell viability, apoptosis and cell cycle were evaluated in 8 B and T cell lymphoma cell lines (Jeko-1, Mino, Granta519, Sp53, RL, Hut102, Hut78 and Jurkat) which were treated with KPT-185; Primary tumor cells from 5 patients and normal human lymphocytes from 2 healthy volunteers were treated directly with KPT-185. Tumor suppressor proteins were detected by western blot to explore the possible mechanisms of KPT-185 inducing lymphoma cells growth inhibition and apoptosis. BALB/c nude mice bearing Jeko-1 tumors were treated orally with KPT-276 (similar structure to KPT-185, but improved animal pharmacokinetics) to examine the efficacy and side-effects of KPT-276.

Results
KPT-185 displayed potent antiproliferative properties at submicromolar concentrations (half-maximal inhibitory concentrations, 60-120 nM) and induced cell-cycle arrest and apoptosis in NHL cell lines and normal lymphocytes. The antitumor activity mainly consisted of regulating cell growth and apoptosis mechanisms by inducing caspase cleavage and downregulating the expression of antiapoptotic proteins such as CRM1, nuclear factor-kB, and survivin. Furthermore, oral administration of KPT-276 significantly suppressed tumor growth and prolonged survival in mice with NHL xenografts without any major toxic effects (P < 0.001). Analysis of tumor remnants in the mice demonstrated that KPT-276 trapped the antiapoptoic protein survivin within the nuclei of NHL cells.

Conclusions
We observed the biologic and pharmacologic activity of CRM1-inhibiting SINEs in NHL cells, primary NHL tumor samples, and a murine NHL xenograft model. SINE CRM1 inhibitors inhibited growth of lymphoma cells both in vitro and in vivo. The antitumor activity of the SINEs resulted primarily from induction of caspase activity and downregulation of expression of antiapoptoic proteins such as survivin and NF-kB. The preclinical in vitro and in vivo results reported herein support further study of CRM1-inhibiting SINEs as novel therapeutics for NHL.

Disclosures:
No relevant conflicts of interest to declare.
A Combined Molecular and Clinical Prognostic Index For Relapse and Survival In Cytogenetically Normal AML (PINA)
Authors: Pastore Friederike , Dufour Annika , Benthaus Tobias , Metzeler Klaus H. , Maharry Kati , Schneider Stephanie , Ksienzyk Bianka , Mellert Gudrun , Zellmeier Evelyn , Kakadia Purvi M. , Unterhalt Michael , Feuring-Buske Michaela , Buske Christian , Braess Jan , Sauerland Cristina , Heinecke Achim , Krug Utz , Berdel Wolfgang E. , Büchner Thomas , Woermann Bernhard J. , Hiddemann Wolfgang , Bohlander Stefan K. , Marcucci Guido , Spiekermann Karsten , Bloomfield Clara D. , Hoster Eva 
Published: October 2019

Abstract

Background
Cytogenetically normal acute myeloid leukemia (CN-AML) is the largest and most heterogeneous cytogenetic AML subgroup. For the practicing clinician it is difficult to know how to use the prognostic information of the growing number of clinical and molecular markers. Our purpose was to develop a widely applicable prognostic model by combining well-established pre-treatment patient and molecular characteristics.

Patients and methods
Two prognostic indices for CN-AML, one with regard to overall survival (PINAOS) and the other regarding relapse-free survival (PINARFS) were derived based on a cohort of 669 CN-AML patients treated within the AML Cooperative Group 99 (AMLCG99) study.

Results
Based on age (median: 60 years [range: 17-85 years]), performance status, white blood count, and presence or absence of NPM1 mutation, biallelic CEBPA mutation, and FLT3-ITD, patients were classified into three risk groups according to PINAOS and PINARFS: 29% of all and 32% of responding patients had low risk (5-year OS 72%; 5-year RFS 55%), 56% and 39% intermediate risk (5-year OS 28%; 5-year RFS 27%), and 15% and 29% high risk disease (5-year OS 3%; 5-year RFS 8%) (Figure 1). PINAOS and PINARFS further subdivided the European LeukemiaNet (ELN) favorable-genetic group as well as the ELN intermediate-I-genetic group. Both, PINAOS and PINARFS were confirmed in a large, independent, and comparable CN-AML cohort of 529 patients from the Cancer and Leukemia Group B (CALGB/Alliance) trials (Figure 2).

Conclusions
We have developed and validated the first prognostic indices specifically designed for CN-AML patients of all ages combining well-established molecular and clinical variables easily applicable in routine clinical care. The integration of both clinical and molecular markers could provide a basis for individualized patient care by risk-adapted therapy of CN-AML.

Disclosures:
No relevant conflicts of interest to declare.
Subclonal IKZF1 Deletions Indicate a Multiclonal Evolution In BCR-ABL1-Positive B-Cell Precursor ALL
Authors: Boehm Verena , Lebenatus Annett , Bartels Marius , Nagel Inga , Trautmann Heiko , Gökbuget Nicola , Hoelzer Dieter , Pfeifer Heike , Siebert Reiner , Kneba Michael , Ottmann Oliver , Brüggemann Monika 
Published: October 2019

Abstract
The transcription factor Ikaros, encoded by the IKZF1 gene, has multiple functions in different steps of hematopoiesis. Loss of function mutations in IKZF1 are frequently associated with BCR-ABL1-positive ALL. IKZF1 deletions comprise whole gene deletions as well as smaller intragenic deletions. All IKZF1 aberrations are associated with a poor prognosis in terms of overall survival and frequency of relapse.
IKZF1 deletions were evaluated in a cohort of 180 adult BCR-ABL1-positive B-cell precursor ALL cases. MLPA analysis was used to assess both whole gene and intragenic deletions, whereas breakpoint-specific consensus multiplex PCR [Caye et al., Haematologica, 2013] and real-time quantitative (RQ)-PCR were used to specifically detect the recurrent intragenic deletions Δ2-7, Δ2-8, Δ4-7 and Δ4-8. Intragenic IKZF1 deletions were detected in 129 patients (71.7%). More than one intragenic deletion was detected in a subset of 44 patients. Unexpectedly, in the majority of these patients (41/180 patients; 22.8%), significant differences in relative frequencies of single intragenic deletions were detected by RQ-PCR analysis. Together with the finding of more than two distinct intragenic deletions in 19/44 patients this clearly indicates the presence of subclonal or oligoclonal intragenic IKZF1 deletions. In the remaining 3/44 patients, number and frequencies of detected intragenic deletions rather suggested the presence of bi-allelic deletions within the whole leukemic bulk. Specificity of individual (sub-) clonal deletions was validated by Sanger sequencing. These data indicate that IKZF1 deletions arise independently in different subclones more frequently than it was estimated in earlier reports.
The quantitative breakpoint-specific PCR approach was additionally used to correlate the detection of intragenic IKZF1 mutations with established Ig/TCR-based monitoring of minimal residual disease (MRD) during/after treatment in a subset of 17 patients. Comparative IKZF1- and Ig/TCR-based MRD monitoring was performed on a total of 207 samples of initial diagnosis and follow-up. Whereas good correlation of Ig/TCR-MRD results and detection of IKZF1 deletions was seen in 9 out of 17 patients, different kinetics of Ig/TCR-MRD results and IKZF1-based leukemia monitoring in the remaining 8 patients were additionally indicative for the existence of IKZF1 deletions in leukemic subclones besides the major leukemic clone with their own kinetics of clearance and persistence.
Illegitimate RAG-mediated recombination has been proposed as responsible mechanism for recurrent intragenic deletions in IKZF1. Our data suggest that these lesions occur repeatedly during leukemogenesis and support a model of multiclonal evolution in BCR-ABL1-positive B-cell precursor ALL.

Disclosures:
No relevant conflicts of interest to declare.
Activation Of EphrinB2/EphB4 Influences Myeloid Leukemia Cell Migration and Invasion
Authors: Zhou Xuan , Xiaoli Liu , Xu Na , Xiao Yajuan , Zhang Jinfang , Huang Bintao , Du Qingfeng 
Published: October 2019

Abstract
Eph receptors and ephrin ligands are cell-surface molecules capable of bidirectional signaling that control cell-cell interactions, migration and invasion. However, their role and regulation in myeloid leukemia cells remain to be defined. To address the hypothesis that Ephrin/EphB is an important regulator of myeloid leukemia cell migration and invasion, we first screened the mRNA levels of 23 eph and ligand ephrin RTK family members in myeloid leukemia cells (K562, HL-60, THP-1) and mononuclear cells from healthy donors, then found that EphB4, EphA5, EfnA1 highly expressed in most myeloid leukemia cells compared to healthy donors(P<0.05). Both the mRNA and protein levels of EphB4 and EphA5 were detected in 13 primary myeloid leukemia cells (5 from patients with extramedullary leukemia among 13 cases) and 10 mononuclear cells from healthy donors by real-time RT-PCR and Immunoblot analysis. The results showed that both the mRNA and protein levels of EphB4 and EphA5 were higher in 13 primary myeloid leukemia cells relative to the 10 healthy donors (P=0.046). Moreover, the EphB4 were highly expressed in 5 patients with extramedullary leukemia compared with 8 patients without extramedullary leukemia. These findings indicated that EphB4 and EphA5 expression were correlated with the development of myeloid leukemia cells, moreover, EphB4 may be closely related with myeloid leukemia cell migration or invasion. To further clarified the question, migration were determined in leukemia cell lines (K562 cells) which were treated with clustered ephrinA1–Fc proteins, ephrinB2–Fc proteins and Fc proteins by transwell migration assay. Invasion were also determined by matrigel invasion assay. The results showed that, after ephrinB2–Fc stimulation, the numbers of K562 cells migrating through transwell chamber were signiﬁcantly enhanced compared to Fc proteins stimulation (1.8 to 2.5-fold, P<0.05), meanwhile, the numbers of K562 cells invading the matrigel also enhanced (1.2 to 1.8-fold, P<0.05). However, after ephrinA1–Fc stimulation, the numbers of K562 cells migrating through transwell chamber didn’t signiﬁcantly increase compared to Fc proteins stimulation (P>0.05), and the numbers of K562 cells invading the matrigel also didn’t enhanced (P>0.05). These findings indicated that ephrinB2–Fc could activate EphB4, leading to the change of myeloid leukemia cell migration and invasion. Further study may help to assess a promising potential of this protein to be used as a prognostic marker or as a target for a molecular therapy.

Disclosures:
No relevant conflicts of interest to declare.
Copy Number Variations and IKZF1 Mutations In Pediatric CML
Authors: Krumbholz Manuela , Tauer Josephine , Kronnie Geertruy te , Ekici Arif , Suttorp Meinolf , Metzler Markus 
Published: October 2019

Abstract
Chronic myeloid leukemia (CML) occurring in children and adolescents is associated with a number of clinical and molecular features that differ from the correspondent disease in patients in older age. Detailed molecular analyses of the genomic BCR-ABL1 breakpoints in a cohort of 60 pediatric individuals from the German CML-paed I and CML-paed II trial revealed a significantly different breakpoints distribution compared to adult CML. Especially the observed bimodal breakpoint distribution pattern in the BCR gene and a higher proportion of breakpoints within Alu repeat regions vary between pediatric CML and adult CML and resembles the pattern observed in adult BCR-ABL1-postitive acute lymphoblastic leukemia.
To identify additional genetic variations, in addition to the BCR-ABL1 fusion gene, high resolution whole-genome microarray analyses using Affymetrix Cytogenetics Whole-Genome 2.7M or CytoScan® HD arrays were performed in a selected sub-cohort of pediatric CML patients from the German CML-paed II trial; twenty individuals diagnosed in chronic phase (CP) and two individuals diagnosed in blast crisis (BC) were screened. In contrast to adult CML patients in whom about 25% of individuals in CP exhibit detectable CNVs, nearly 60% of pediatric patients have CNVs (1.9 CNVs per case) in this initial stage of CML. All identified CNVs were private and no recurrent genetic aberration was associated with early disease manifestation in children. Two patients in BC showed an increased number of CNVs (6.5 CNVs per case) which is in accordance with data from adult patients, indicating that additional secondary genetic events are associated with disease progression. One of the patients in lymphoid CML-BC harbored a deletion involving the IKZF1 gene, an aberration frequently observed in advanced disease stages in adult CML and ALL.
To further evaluate the incidence of IKZF1 mutations and their impact on the disease progression in pediatric CML patients, deep sequencing of IKZF1 was performed in 55 patients; 52 individuals in CP and 3 individuals in BC. Previously performed whole genome array analyses showed a deletion of exon 4 to 7 in the IKZF1 gene in one of the three analyzed patients in BC. Deep sequencing identified no additional mutation within IKZF1 in both remaining individuals. Interestingly, two patients in CP showed a single nucleotide insertion resulting in a premature stop codon, E35 and Y348 respectively. One patient developed imatinib resistantance 1.5 years after treatment onset. The second patient underwent stem cell transplantation 1 year after initial diagnosis. To date, with a follow-up of 7 and 6 years respectively, no patient progressed to advanced disease. These findings are in line with findings in adult CML, where IKZF1 aberrations are preferentially observed in advanced disease stages or imatinib resistant patients.

Disclosures:
No relevant conflicts of interest to declare.
Impact Of Myelofibrosis (MF) In MDS Treated With Azacitidine (AZA). A Single Center Study
Authors: Legoupil Clemence , Sebert Marie , Braun Thorsten , Gardin Claude , Martin Antoine , Brechignac Sabine , Harel Stephanie , Fenaux Pierre , Ades Lionel 
Published: October 2019

Abstract

Background
Bone marrow fibrosis is observed in 10-20% of MD, and is a poor prognostic factor, both in lower and higher risk MDS (Della Porta, JCO 2009). AZA, the current reference treatment for higher risk MDS, approved in EU for patients with up to 30% bone marrow blasts not candidates to intensive chemotherapy (IC) or allogeneic SCT, gives 50-60% responses and improves OS in higher-risk MDS but its role in MDS with myelofibrosis remains unknown.

Methods
Between 2004 and 2012, we treated at our center 172 consecutive MDS patients (pts) including FAB RAEB-T / WHO AML with 20-30% blasts, with AZA (75 mg/m2/d x7 d every 4 weeks, for a median of 6 cycles). We assessed in those patients the impact of myelofibrosis (MF), evaluated on bone marrow trephine biopsy and graded according to the European consensus on grading bone marrow fibrosis.

Results
Median age of the 172 pts was 73 years and 67% were males. According to WHO classification, 1 had del(5q) syndrome, 4 RARS/RCMD-RS, 24 RCMD, 37 RAEB-1, 54 RAEB-2, 29 AML (20-30% blasts), 17 CMML and 6 MDS unclassified. Median Hb level, WBC, platelet count and marrow blast count were 9.4 g/dl (range 3.5-15), 1.6 G/L (0-58), 75G/l (8-1080) and 12%(1-29), respectively. IPSS was low, Int-1, Int-2, High and a failure in 1(>1%), 29(17%), 65 (38%), 59 (34%) and 18 (11%) patients, respectively. Twenty-three pts (13%) had grade 2-3 myelofibrosis (MF). Patients with MF were younger (median 68 vs. 74 years, p= 0.04), but had similar hematological characteristics: hemoglobin (median 9 vs. 9.45g/dl, p= 0.69); WBC (2.2 vs. 1.6 G/l, p= 0.48) Platelet (47 vs 77 G/l,p= 0.43) and bone marrow blast (10 vs. 12%, p=0.67). IPSS was int 1, int 2, high and a failure in 4, 7, 6 and 6 patients respectively without difference compared to patients without fibrosis. Cytogenetics was complex in 8, del(20q) +/- 1 additional abn in 5 patients, normal in 4 patients, failed in 6 patients (but with trisomy 8 and monosomy 7, resp, detected in 2 patients by FISH analysis). Overall, 73 (42%) patients achieved a response according to IWG2006 criteria, including 31 (18%) CR. The response rate and CR rate were 52% and 17% respectively in pts with MF, compared to 44% and 20% in pts without MF (p= 0.507 and p=1.00, respectively). With a median follow-up of 6.5 yr, median OS was 12 months in pts with MF, compared to 16 months in pts without MF (p=0.45).

Conclusion
In this MDS series, presence of MF was not associated with specific features (but blasts were often counted on marrow aspirates, with a possible underestimate in case of MF). Response to AZA and survival after AZA onset did not significantly differ based on the presence of MF.

Disclosures:
No relevant conflicts of interest to declare.
Aberrant Overexpression Of CD14 In Granulocytes Sensitizes Innate Immune Response In mDia1 Heterozygous Myelodysplastic Syndromes
Authors: Keerthivasan Ganesan , Zhao Baobing , Harris Chad E , Zhang Ling , Gao Juehua , McElherne James , Yang Jing , Basiorka Ashley A , Schipma Matthew , Boultwood Jacqueline , Verma Amit , List Alan , Williams David A. , Ji Peng 
Published: October 2019

Abstract
The myelodysplastic syndromes (MDS) are a group of pre-leukemic diseases characterized by increased risk of acute myeloid leukemia (AML). Heterozygous loss of chromosome 5q (5q-) is the most common cytogenetic abnormality in MDS. DIAPH1 is localized to 5q31 and encodes one of the formin family proteins, mDia1, involved in the regulation of linear actin polymerization. Mice with mDia1 deficiency develop hematologic features mimicking human myeloproliferative neoplasm, but its role in the pathogenesis of MDS is unclear. Here we report that mDia1 is largely dispensable for normal hematopoiesis. However, the committed or mature granulocytes in mDia1 heterozygous and knockout mice were activated and showed increased actin polarization. Strikingly, CD14 was aberrantly overexpressed in the bone marrow and peripheral granulocytes of mDia1 heterozygous and knockout mice in a cell-autonomous manner, leading to a hypersensitivity of the innate immune response to lipopolysaccharide (LPS) stimuli through CD14-Toll like receptor 4 (TLR4) signaling. Chronic stimulation with LPS accelerated the occurrence of MDS in mDia1 heterozygous and knockout mice. Similar findings of CD14 overexpression were observed in the bone marrow granulocytes of 5q- MDS patients, but not normal patients or MDS patients without 5q deletion. These patients exhibited relatively frequent infections with long duration of disease. Mechanistically, mDia1 was required for the endocytosis of CD14 upon LPS stimulation. Heterozygosity and loss of mDia1 led to diminished interferon expression that is dependent on CD14 endocytosis. Thus, our study revealed an essential role of the innate immune signaling in the pathogenesis of 5q- MDS. Specifically, heterozygosity or loss of mDia1 leads to a cell autonomous hypersensitivity of innate immune system in the granulocytes that causes myeloid dysplasia. Together with previous reported evidence of dyserythropoiesis due to loss of RPS14, and dysmegakaryopoiesis due to loss of mir-145/146a, these data fully recapitulate the pathogenesis of tri-lineage dysplasia in 5q- MDS. In addition, our study highlighted the significance of the activated CD14/TLR signaling pathway in the pathogenesis of MDS, which could serve as a novel target for therapeutic management.

Disclosures:
No relevant conflicts of interest to declare.