bluuebunny/crossref_metadata_2025_split huggingface dataset:
The Histone Deacetylase Inhibitor Vorinostat (SAHA) Induces Apoptosis and Cell Cycle Arrest in Hodgkin Lymphoma (HL) Cell Lines by Altering Several Survival Signaling Pathways and Synergizes with Doxorubicin, Gemcitabine and Bortezomib.
Authors: Georgakis Georgios V. , Yazbeck Victor Y. , Li Yang , Younes Anas 
Published: October 2019

Abstract
Several epigenetic changes have been recently implicated in the pathogenesis of several human malignancies, including HL. This pathway is currently explored for cancer therapy using hypomethylating agents and histone deacetylase (HDAC) inhibitors. The purpose of this study was to examine the potential clinical value for HDAC inhibition in HL. Therefore, we examined the single agent activity of vorinostat (suberoylanilide hydroxamic acid, SAHA) in a panel of three well defined HL-derived cell lines (HD-LM2, L-428, and KM-H2). Furthermore, we determined the molecular mechanisms of vorinostst activity in these cell lines. Vorinostat showed antiproliferative effect in all HL cell lines in a time- and dose-dependent manner, as determined with the MTS assay. The most sensitive cell line was the HD-LM2 with an IC50 of 2 micromolar, but antiproliferative activity was observed at concentrations between 0.1 and 0.5 micromolar. This antiproliferative effect was due to cell cycle arrest at the G2/M phase, as determined by Propidium Iodide DNA staining and FACS analysis. This effect was most prominent in the HD-LM2 cells, as the G2M fraction increased by 6 fold within 24 hours of incubation with 10 micromolar of vorinostat, and by 4 fold when 5 micromolar was used. Cell cycle arrest was followed with an increase in Annexin-V binding indicative of induction of apoptosis. This apoptotic effect was associated with activation of the caspase pathway, which resulted in caspase 3 and PARP cleavage. Furthermore, the antiproliferative activity was associated with de-phosphorylation of three important survival signals that are known to promote HL survival: STAT6, Akt and ERK. To determine the potential synergy of vorinostat with conventional chemotherapy and modern targeted small molecules, we treated HL cell lines with the combination of vorinostat and doxorubicin, gemcitabine, and the proteasome inhibitor bortezomib. In all the combinations SAHA significantly enhanced the effect of the other drugs, as determined with the MTS assay and the calculation of the Combination Index. Moreover, the combination of vorinostat and bortezomib arrested the cells in G2/M phase more efficiently and showed increased rate of apoptosis than each drug alone. In contrast, there was no added benefit when vorinostat was combined with the heat shock protein-90 inhibitor 17-AAG or with the mTOR inhibitor CCI-779. Our data indicate that HDAC inhibition by vorinostat may have a clinical value in HL, especially when combined with bortezomib or with chemotherapy.
The Significance of BAALC Expression and CEBPa Mutations as New Prognostic Factors in Pediatric Acute Myeloid Leukemia with Normal Karyotype.
Authors: Mizushima Yasuhiro , Adachi Souichi , Taki Tomohiko , Shimada Akira , Hayashi Yasuhide , Tsukimoto Ichiro , Yui Masahiro , Kamitsuji Yuri , Hiraumi Yoshimi , Matsubara Hiroshi , Watanabe Motonobu , Watanabe Ken-ichiro , Nakahata Tatsutoshi 
Published: October 2019

Abstract
Approximately half of children with acute myeloid leukemia (AML) have no chromosomal or genetic abnormalities that can predict outcome. To improve the prognostic stratification on this heterogeneous group of patients, novel markers need to be investigated. Recently, CEBPa (CCAAT/enhancer binding protein alpha) mutations have been described as a favorable prognostic factor and the high expression level of BAALC (brain and acute leukemia, cytoplasmic) was found to be a poor prognostic marker, in adult AML with normal karyotype. To explore their significance in childhood, we studied the prognostic impact of the high expression level of BAALC and CEBPa mutations in pediatric AML with normal karyotype. Samples were available from pediatric patients 3 months to 15 years of age, treated on Japanese Childhood AML Cooperative Study Group Protocol, AML99. BAALC expression was determined by comparative real-time RT-PCR assay in 29 patients. The relative BAALC expression was determined using the comparative cycle threshold method. The median value of healthy volunteers was used as cutoff. The mutational analysis of CEBPa was performed in 49 patients with RT-PCR followed by direct sequencing. For samples carrying mutations, additional subcloning analysis were carried out. 72.4% (21 of 29) of patients had high BAALC expression and 27.6% (8 of 29) had low. High BAALC expression was associated with the FAB subtypes M0, M1, and M2, whereas low BAALC expression correlated with M4 and M5. Overall survival and event-free survival was 52.3%; 49.5% (high BAALC expression group) and 75.0%; 62.5% (low BAALC expression group), respectively. CEBPa mutations were detected in four patients (8.2 %, 4 of 49; two each in M1 and M2). N-terminal frameshift mutations and inframe insertions in the basic-leucine zipper (bZIP) domain were detected. Novel mutations (212 insertion (ins) C, 214–224 deletion CCCCGCACGCG, 720 ins CGCACC, 1074 ins AGA, 1092 ins CAC) were identified. One patient had mutations occurred in both N-terminal part and bZIP domain. Moreover, above patients with CEBPa mutations had not cooperating mutations with FLT3-ITD (fms-like tyrosine kinase 3 internal tandem duplication) and maintain in complete remission for at least 21 months duration. In conclusion, BAALC expression and CEBPa mutations may be prognostic factors in pediatric AML with normal karyotype.
Improved Measurement of Minimal Residual Disease (MRD).
Authors: Morley Alexander A. , Latham Sue , Brisco Michael J. , Sykes Pamela J. , Kuss Bryone , Waters Keith 
Published: October 2019

Abstract
A new and improved method for measurement of minimal residual disease (MRD) was developed.
Method
the total repertoire of leukaemic rearrangements of the immunoglobulin heavy chain (IgH) gene.was determined by performing multiple parallel Q-PCRs in microplates to determine usage of individual V and J segments. This enabled detection and quantification of clones ranging in size from 100% to approximately 0.03% of the leukemic population. MRD measurement involved nested Q-PCR using the specific V and J primers and internal primers based on the sequence of the rearrangement of interest.
Following informed parental consent, 25 children with B-ALL were studied at the end of induction therapy. Under general anesthesia, 4 aspirations, 2 from each iliac spine, were performed and on each sample MRD was measured on 2 different days.
This enabled definition of the laboratory and sampling factors important in measurement of MRD.
Results
Repertoire analysis was very effective in identifying rearrangements of the IgH gene suitable for use as molecular markers, being superior to the commonly-used BIOMED-2 protocol for identification of both large and small clones. Two or more rearrangements marking large clones were detected in 20 of the 25 patients.
The median MRD level at the end of induction was 2.1 x 10−5. A level of > 10−3 was seen in 4 patients and < 10−7 in 4. Sensitivity of detection in a single sample was approximately 2 x 10−6, which is 1–2 orders of magnitude better than current techniques.
Figure Figure
The SD of measurement depended on the number of target rearrangements present in the sample, reflecting the Poisson statistical uncertainty inherent in measurement of small numbers of events. For > 50 rearrangements SD was 0.23 log units, but below this level the SD rose steeply. 50 targets in 1 μg of DNA corresponds to an MRD of approximately 3 x 10−4.
Figure Figure
There was significant sampling error. In 1 patient there was 1000-fold MRD difference between the 2 iliac spines.
Conclusions
We recommend that, for MRD measurement,
-an aspiration should be performed from each iliac spine, with each sample being quantified separately and an average obtained -each measurement should involve at least 10 μg of good-quality DNA
The MRD value so obtained should have sufficient accuracy, sensitivity and precision for clinical decisions based upon the value to be made with confidence.
The described methods for MRD measurement should also be applicable to monitoring of all B-lymphocyte neoplasms, in addition to ALL.
Notch1 Mutations in Adult T Lymphoblastic Lymphoma and T-ALL.
Authors: Baleydier Frederic , Simon Arnauld , Bergeron Julie , Millien Corinne , Beldjord Kheira , Lepretre Stephane , Dombret Herve , Asnafi Vahid , Macintyre Elizabeth A. 
Published: October 2019

Abstract
Notch1 is recognised to be involved in human T-ALL oncogenesis, initially by implication of its intracellular domain in the rare translocation t(7;9) and more recently with the description of mutations in NOTCH1 exons coding the heterodimerisation (HD) +/− PEST domains in 50% of T-ALL. Favorable prognosis impact of Notch1 mutations has been shown in pediatric patients. Little data is available regarding Notch mutations in adult T-ALL, and even less in adult T Lymphoblastic lymphoma (T-LBL). We have therefore searched for Notch1 mutations in adult T-ALL and T-LBL, all of which have undergone comparable, TCR and oncogene based classification (TLX1/3, SIL-TAL1, CALM-AF10, LMO1/2, TAL1 and LYL1). We searched for mutations in exons 26, 27 and 34 by direct sequencing in 46 T-ALL (median age 27,5y. range 16–54; 6 < 18y) and 32 T-LL (median 31y., range 16–70y.; 3 < 18y.). Notch1 mutations were identified in 22/46 (48%) of T-ALL and 16/32 (50%) of T-LBL. The type of mutations differed, insofar as HD only mutations were seen in 16/22 (73%) of mutated T-ALL but only 4/16 (25%) of mutated T-LL whereas PEST only mutations were seen in 2/22 (9%) T-ALL compared to 8/16 (50%) of T-LBL. The incidence of HD and PEST mutations was identical in T-ALL (4/16; 25%) and T-LBL (6/22; 27%). Combining T-ALL and T-LBL, TLX1hi or TLX3 expression was seen in 7/71 (10%) and 5/71 (7%) cases respectively. Notch1 mutations were seen in 7/7 TLX1+ cases but only 1/5 TLX3 (p=0.004). With respect to stage of maturation arrest, Notch mutations in T-ALL were relatively rare in mature TCR+ cases (2/15 vs. 20/31 p=0.001), whereas they were found at all stages of maturation arrest in T-LBL.
These data demonstrate that Notch 1 mutations are as frequent in T-LBL as T-ALL, but more frequently involve the PEST domain. They are in favour of preferential occurrence of NOTCH1 mutations in TLX1 relative to TLX3 proliferations, suggesting that evaluation of prognostic impact of Notch1 should not be performed independently of evaluation of TLX1/3 status, since TLX1 generally identifies good prognosis and TLX3 poor prognosis T-ALL. Within the 46 LALA94 adult T-ALLs, Notch1 mutated cases were of marginally superior prognosis (p=0.50); this is currently being evaluated within the ongoing adult GRAALL03 T-ALL trial.
FLT-3 Activity and Its Response to Drugs Can Be Determined in AML Blast Cells by FLT-3 Phosphorylation Status Using Flow Cytometry.
Authors: Grafone Tiziana , Palmisano Michela , Ottaviani Emanuela , Martelli Alberto Maria , Cappellini Alessandra , Follo Matilde , Testoni Nicoletta , Paolini Stefania , Baccarani Michele , Martinelli Giovanni 
Published: October 2019

Abstract
One of the most common molecular defects identified in acute myeloid leukemia (AML) patients is an activating mutation of FLT3 tyrosine kinase. The identification of activated FLT3 as a contributor to the cause and progression of much leukemia has led to its consideration as a potential target for therapy. Since small molecule FLT3 kinase inhibitors are actually in clinical trials; a robust and standardized method for screening of FLT3 receptor activation is necessary.
We evaluated the expression level of FLT3 receptor (CD135) by Facs analysis. We developed a flow cytometry method to measure FLT3 phosphorylation (P-FLT3) in samples with <10 (e)5 cells. The amount of P-FLT3 in the samples was determined as the mean fluorescence intensity (MFI). The P-FLT3 status of the treated samples was expressed as a percentage of the untreated control (100%). The method was first validated in FLT3 wild-type (HL-60) and mutant (MV4-11/ITD+) as well as FLT3 negative (K562) cell lines. The method also provides to be reproducible with samples AML from patients. Analysis was performed after exposure to drugs, in vitro and in vivo. In response to increasing drugs concentration (CEP-701 and SU11657) there was a linear reduction in P-FLT3. The results validate a rapid method to detect P-FLT3 protein at the single cell level by flow cytometry, and enable an accurate assessment of FLT3 kinase activity in blast cells in response to novel tyrosine kinase inhibitors.
Specific Tumor Targeting and Potent Bystander Killing with SGN-35, an Anti-CD30 Antibody Drug Conjugate.
Authors: Okeley Nicole M. , Alley Stephen C. , Cerveny Charles G. , Andreyka Jamie , Sanderson Russell J. , Anderson Martha , Benjamin Dennis R. , Law Che-Leung , Sievers Eric , Ihle Nathan C. 
Published: October 2019

Abstract
CD30 is expressed on the surface of Reed Sternberg cells in Hodgkin’s Lymphoma (HL) and on anaplastic large cell lymphoma (ALCL) cells. The anti-mitotic agent monomethyl auristatin E (MMAE) has been conjugated to cAC10, the anti-CD30 monoclonal antibody SGN-30, to yield the antibody-drug conjugate SGN-35. SGN-35 is internalized into lysosomes of CD30-positive cells and MMAE is released intracellularly when the protease-sensitive linker is cleaved by lysosomal proteases such as cathepsin B. In mouse xenograft models of HL and ALCL, complete remissions have been observed at well tolerated doses of SGN-35. To characterize further the mode of action in vivo, mice with established CD30-positive xenografts were treated with SGN-35, and the concentration of released MMAE was measured in the tumor and serum. The tumor concentrations of released MMAE achieved were several hundred-fold above the IC50 for MMAE in tumor cell cultures. Peak tumor to serum ratios for released MMAE reached more than 1000:1. In tumors and serum, the peak released MMAE concentrations were achieved 3 days and 1 hour post dose, respectively. As a considerable portion of the tumor volume in HL consists of a CD30-negative cellular infiltrate, the ability of SGN-35 to kill CD30-negative cells in mixed cell populations was investigated. First, cell growth medium from the SGN-35-treated HL cell line L540cy (CD30 positive) was shown to be cytotoxic to the SGN-35-insensitive Ramos cells (CD30 negative). Measurements with mass spectrometry confirmed that MMAE was released into the growth medium of SGN-35-treated L540cy cells, and that the observed concentration of MMAE was consistent with the cytotoxic effect on the Ramos cells. Second, a mixed culture of Karpas 299 cells (ALCL, CD30 positive) and Ramos cells was treated with SGN-35 and examined by FACS. The viability of both cell populations was reduced or eliminated, in contrast to proliferation observed in single Ramos cultures treated with SGN-35 or untreated mixed cultures. These results demonstrate that SGN-35 effectively targets the potent cytotoxic agent MMAE to CD30-positive tumor cells, while free MMAE released by these tumor cells is also available for killing CD30-negative infiltrating bystander cells. SGN-35 is a promising treatment for tumors with homogeneous (ALCL) and heterogeneous (HL) expression of CD30. Clinical trials with SGN-35 are expected to begin by the end of 2006.
Characterization of T-Cell Lymphoma Cell Lines with a Novel t(9;14)(q34;q11.2) Rearrangement: Does gamma-Secretase Inhibitor Sensitivity Depend on Intragenic NOTCH1 Breakpoint?.
Authors: Suzuki Shinsuke , Nagel Stefan , Schneider Bjoern , Kaufmann Maren , Anders Dorothea , Tsubouchi Hirohito , Uozumi Kimiharu , Arima Naomichi , Drexler Hans G. , MacLeod Roderick A.F. 
Published: October 2019

Abstract
Activating mutations and deletions affecting specific NOTCH1 protein domains have been recently shown to occur widely in T-cell neoplasia, e.g. in T-acute lymphoblastic leukemia (T-ALL). However, knowledge of NOTCH1 chromosomal alterations is largely based on a single cell line model (SUP-T1) with t(7;9)(q35;q34) in which NOTCH1 truncated at exon 24 is juxtaposed with TCRB. We describe the characterization of a novel rearrangement, t(9;14)(q34.3;q11) in two T-cell lymphoma cell lines, HD-MAR and HT-1. FISH analysis using fosmid clones and sequencing of fragments identified by long distance inverse PCR showed that in both cases t(9;14) effected tail-to-tail juxtaposition of intron 27 of NOTCH1 with TCRA genes, namely 5′-TRAV40 in HD-MAR, and intron 2 of TRAV5 in HT-1. Thus, in both cell lines t(9;14) places NOTCH1, truncated immediately 3′ of the HD-domain, under transcriptional control of TCRA. The 14q11.2 breakpoints in HD-MAR and HT-1 lie, respectively, near the proximal E-delta enhancer and amid a cryptic enhancer region represented by a cluster of T-cell specific DNase-I hypersensitive sites. Western blotting revealed prominent expression of truncated activated NOTCH1 polypeptides, ranging in size from 100 to 115 kDa in both cell lines. Antibodies recognizing ANK and TAD domains, believed essential for inducing T-ALL, detected the aberrant polypeptides. Moreover, treatment with gamma-secretase inhibitor (GSI) altered expression patterns of NOTCH1 polypeptides and induced growth inhibition due to G0/G1 cell cycle arrest in both t(9;14) cell lines, in stark contrast to GSI-resistant SUP-T1 cells wherein truncation occurs before the heterodimerization (HD) domain. (Another recently described t(7;9) cell line (CUTLL1) which is GSI-sensitive also carries a NOTCH1 breakpoint at intron 27.) The same protein species were not detectable by antibodies recognizing the transmembrane domain of NOTCH1 which requires GS for exposure suggesting nuclear access requires GS-cleavage. Immunostaining confirmed extranuclear blocking of NOTCH1 in response to GSI in HD-MAR/HT-1 but not in SUP-T1. In contrast, repression of HES1 occurred in response to GSI irrespective of NOTCH1 breakpoint location, suggesting its non-involvement in growth signaling. In addition to providing cell line models for a new NOTCH1 disease translocation, these data suggest that the sensitivities of T-cell neoplasias bearing NOTCH1 translocations may critically depend on whether 9q34 breakpoints lie upstream or downstream of the HD domain.
Gene Expression Profiling Comparison between the Major Mature B-Cell Neoplasms and Their Normal Cellular and Anatomic Counterparts: Identification of Candidate Genes Potentially Involved in Lymphomagenesis.
Authors: Blin Nicolas , Bossard Celine , Harousseau Jean-Luc , Charbonnel Catherine , Gouraud Wilfried , Campion Loic , Magrangeas Florence , Minvielle Stephane , Loiseau Herve Avet 
Published: October 2019

Abstract
Gene expression profiling has provided new insights into the understanding of mature B cell neoplasms by relating each one to its normal counterpart, so that they can be to some extent classified according to the corresponding normal B-cell stage. Thus, diffuse large B cell (DLBCL) and follicular lymphoma (FL) have been related to a germinal center precursor whereas mantle cell lymphoma (MCL) or marginal zone lymphoma (MZL) are more likely to derive from naïve and memory B cell, respectively. However, little is still known about the physiopathology of B-cell lymphomas and particularly the deregulated pathways involved in their oncogenesis. To further investigate that point, we performed laser capture microdissection (LCM) of the three anatomic lymphoid compartments (i.e germinal center, mantle zone and marginal zone) taken from nine normal spleens and lymph nodes and magnetic cell separation of the four normal B cell subpopulations (i.e naïve B cells, centroblasts, centrocytes and memory B cells) purified from twelve normal tonsils for gene expression profiling by cDNA microarray. These molecular profiles have been compared to those of the four most frequent mature B cell neoplasms in adult (i.e DLBCL, FL, MZL and MCL), each one isolated from five previously untreated patients. Unsupervised analysis by hierarchical clustering of the normal anatomic and cellular populations could discriminate the germinal from the extra-germinal populations by genes involved in cell proliferation (e.g. E2F5, CCNB2, BUB1B and AURKB), DNA repair (e.g. PCNA and EXO1), cytokine secretion (e.g. IL8, IL10RB, IL4R and TGFBI) and apoptosis (e.g. CASP8, CASP10, BCL2 and FAS). Supervised analysis of the comparison between each B-cell lymphoma and its anatomic and cellular physiologic equivalent identified molecular deregulations concerning several genes’families characterizing the different histologic subtypes. Genes associated with cellular adhesion and ubiquitin cycle were significantly up-regulated in MCL (FCGBP, ITGAE, USP7, VCAM1) and MZL (CTGF, CDH1, ITGAE) whereas germinal center derived lymphomas (i.e. DLBCL and FL) mainly showed up-regulation of genes involved in cell proliferation (TNFRSF17, SEPT8) and immune response (FCER1G, XBP1, IL1RN). Few deregulated genes were common to the four subtypes, principally associated with cell proliferation (CYR61, GPNMB), cytosqueleton organization (EPB41L3) and carbohydrates metabolism (GNPDA1), suggesting potential similar oncogenic pathways. Those preliminary results are compatible with both subtype-specific and overall mechanisms of lympomagenesis and should be verified in a wider range of samples to confirm the oncogenic events involved in this heterogeneous disease.
Alternative Splicing Generates a BCL6 Isoform Encoding a Compact Repressor.
Authors: Shen Yulei , Ge Baosheng , Ramachandrareddy Himabindu , McKeithan Timothy , Chan Wing-Chung 
Published: October 2019

Abstract
BCL6 encodes a BTB/POZ zinc finger (ZF) transcriptional repressor which is essential for normal germinal center (GC) reactions. Its dysregulated expression contributes to the pathogenesis of B cell non-Hodgkin lymphoma (NHL). The BCL6 gene consists of ten exons, BCL6 translation start site is located in exon 3, and the six-ZF DNA binding domain is encoded by exons 7 to 10. BCL6S, was cloned from the cDNA of a BCL-6 positive cell line, DHL-16. DNA sequencing showed that BCL6S was a normal splicing isoform that excludes the entire exon 7 of the BCL6 gene and encodes a 650 amino acid (aa) protein. BCL6S was detected by RT-PCR in all human cell lines and tissues expressing BCL6, but was not detected in the mouse B cell lymphoma cell line A20. BCL6S accounts for 1/8 to 1/10 of total BCL6 transcripts. Luciferase reporter assays demonstrated that BCL6S could repress typical BCL6 target genes ( BCL6, CD23b, Blimp1, MIP1α ) as effectively as the full length BCL6. BCL6S retains the last four ZFs of BCL6 and localizes in the cell nucleus. Protein-protein interaction assays confirmed that BCL6 and BCL6S could form homodimers and heterodimers. DNA binding assays showed no affinity differences between BCL6 and BCL6S; however, a single mutation at the N-terminus of the POZ domain (L19 to H) not only disrupted BCL6S dimerization but also significantly decreased the DNA binding affinity, indicating that BCL6 dimerization may stabilize the ZF-DNA binding. In conclusion, we have identified a novel BCL6 splicing isoform. BCL6S, a compact repressor as potent as BCL6. POZ dimerization plays an important role in stabilization of BCL6 DNA binding whereas the first two ZFs are not absolutely required. The biological role of BCL6S in GC B cells needs to be further investigated.