bluuebunny/crossref_metadata_2025_split huggingface dataset:
Identification of High-Level DNA Amplifications in AML with Complex Karyotype Using Array-CGH.
Authors: Rücker Frank G. , Bullinger Lars , Miller Simone , Kestler Hans A. , Lichter Peter , Dohner Konstanze , Dohner Hartmut 
Published: October 2019

Abstract
Complex karyotype acute myeloid leukemia (AML), commonly defined as the presence of three or more chromosome abnormalities without specific fusion transcripts, is seen in approximately 10–15% of all AML cases. In this subset of cases, genomic losses and gains are much more frequent than balanced translocations, indicating other mechanisms of leukemogenesis. One possible mechanism is the activation of oncogenes through high-level DNA amplifications. To detect high-level DNA amplifications and to identify corresponding candidate genes, we applied comparative genomic hybridization to microarrays (array-CGH) in 100 cases of complex karyotype AML and correlated the findings with gene expression profiling (GEP) data. For array-CGH a custom-made 2.8k-microarray was used consisting of 2799 different BAC- or PAC-vectors with an average resolution of approximately 2 Mb. Hybridization experiments were performed in a dye-swap setting; significant aberrations were defined as mean plus/minus three times the standard deviation of a set of balanced clones for each individual experiment. In selected cases correlation with global gene expression studies was performed to further delineate candidate genes.
We identified 50 high-level DNA amplifications in 20 different genomic regions. Amplifications occurring in at least two cases mapped to (candidate genes in the amplicon) 11q23.3-q24.1 (n=10; ETS, FLI1, APLP2); 11q23.3 (n=8;MLL, DDX6, LARG, SC5DL); 21q22 (n=5; ERG, ETS2); 9p24 (n=4; JAK2); 13q12 (n=4; CDX2, FLT3, PAN3); 8q24 (n=3; C8FW, MYC); 12p13 (n=2; FGF6, CCND2); 20q11 (n=2; ID1, BCL2L1); and 11q13 (n=2; STARD10, GARP, RAD30, DLG2). To better characterize the genomic architecture of the amplicons, we applied array-CGH using an 8.0k-microarray with an average resolution of approximately 1 Mb. Using this approach highly complex amplicon structures with several distinct amplicon peaks were identified for e.g. the amplified regions in 8q24, 11q23, and 13q12. In addition, parallel analysis of GEP in a subset of 43 of 100 cases displayed overexpressed candidate genes in the critical amplified regions; for some of the genes an oncogenic role has been implicated e.g. C8FW and MYC in 8q24, ETS1, FLI1 and APLP2 in 11q24.1, as well as FLT3 and CDX2 in 13q12. In conclusion, using high-resolution genome-wide screening tools such as array-CGH, a large number of high-level DNA amplifications were identified in AML with complex karyotype suggesting a more general role for protooncogene activation in this AML subset. This high-resolution technique allows the detection of complex amplicon structures with several distinct amplicon peaks pinpointing to selective candidate genes. In addition, correlation with GEP studies facilitates the delineation of overexpressed candidate genes within the amplified regions.
Commonly Dysregulated Genes in Murine Acute Promyelocytic Leukemia (APL) Cells.
Authors: Yuan Wenlin , Payton Jacqueline E. , Holt Matthew S. , Link Daniel C. , Watson Mark A. , DiPersio John F. , James Ley Timothy 
Published: October 2019

Abstract
To identify genes that are commonly dysregulated in a murine model of acute promyelocytic leukemia (mice expressing PML-RARa under control of the murine cathepsin G locus), we first defined gene expression patterns during normal murine myeloid development; serial gene expression profiling studies were performed with primary murine hematopoietic progenitors that were induced to undergo myeloid maturation in vitro with G-CSF. This system provided a method for obtaining highly enriched populations of murine myeloid cells at well-defined stages of differentiation: stem and progenitor cells were enriched on Day 0, promyelocytes were the predominant cell in culture on Day 2–3, mid-myeloid cells were most abundant on Day 4–5, and terminally differentiated myeloid cells were enriched on Day 6–7. These stages were validated using well-characterized “marker” genes for each developmental stage. Many genes were reproducibly expressed in restricted developmental “windows”, suggesting a structured hierarchy of expression that is relevant for the induction of developmental fates and/or differentiated cell functions. Some genes relevant for the pathogenesis of acute myelogenous leukemia (AML) also showed dynamic changes during myeloid differentiation.
We compared the normal myeloid developmental transcriptome to that of APL cells derived from the mCG-PML-RARa knock-in mice. While many promyelocyte-specific genes were highly expressed in all APL samples, 116 genes were reproducibly dysregulated in many independent APL samples. These included 4 classes:genes that were highly expressed in murine APL cells but not in normal myeloid cells at any stage of development,genes normally expressed only in early hematopoietic progenitors (d0) but abnormally expressed in APL cells,genes normally expressed only in late myeloid cells (d7) but also expressed in APL cells, andgenes normally expressed in promyelocytes (d2) but not in APL cells.
Many of these dysregulated genes had previously been linked to oncogenic transformation, including Fos, Jun, Egr-1, TNF, and VCAM-1. However, we found that this set of commonly dysregulated genes was expressed normally in preleukemic promyelocytes from the same mouse model, suggesting that dysregulation occurs as a “downstream” event during disease progression. These studies suggest that the genetic events that lead to APL progression may converge on common pathways that are important for leukemia pathogenesis.
Complex Interaction of the MoeA-N and MoeA-C Domains of Gephyrin Is Required for the Immortalization Activity of MLL-Gephyrin.
Authors: Imamura Toshihiko , Luo Roger T. , Thirman Michael J. 
Published: October 2019

Abstract
More than 40 proteins have been identified as partners of MLL in acute leukemia. The partner proteins are categorized based on their subcellular localization into either the nucleus or cytoplasm. Recent studies have proposed that the mechanism of transformation of MLL-cytoplasmic protein fusion products is mediated by the oligomerization ability of partner proteins. Gephyrin, which is involved in synaptic anchoring of glycine receptor and certain GABAA receptor subtypes, is a rare partner of MLL in patients with AML. Using an in vivo cross linking assay, we confirmed the dimerization activity of a domain within the MoeA-N domain of Gephyrin, which is located at amino acids 456–476. However, we found that this domain was dispensable for immortalization in the methylcellulose colony-forming assay, indicating that the presence of a dimerization domain was not sufficient for transformation. We also observed that the Tubulin binding domain, which is located at amino acids 322–376, was also dispensable. To evaluate which domain of Gephyrin is critical for the immortalization activity of MLL-Gephyrin, we performed a colony-forming assay in methylcellulose and found that both the MoeA-N (amino acids 354–528) and the MoeA-C (amino acids 691–769) domains of Gephyrin are essential for immortalization. In addition, we observed that a MLL- Gephyrin (444–476) deletion mutant formed dimers using the in vivo cross linking assay. To explore the possibility that both MoeA-N and MoeA-C domains are associated with dimer formation of MLL- Gephyrin, we performed co-immunoprecipitation experiments with three fragments of Gephyrin, MoeA-N, MoCF (amino acids 532–690) and MoeA-C. Our data showed that both the MoeA-N and MoeA-C domains interacted with the MoCF domain of Gephyrin. Taken together, these findings show that Gephyrin has multiple dimerization domains, and that the interaction that links the MoeA-N, MoeA-C, and MoCF domains is critical for the immortalization activity of the MLL- Gephyrin fusion protein. Our data indicate that complex patterns of dimerization exist among MLL partners and suggest that specific types of dimerization domains might be critical for MLL-associated transforming activity.
Survivin Is Negatively Regulated by Early Growth Response (Egr)-1 Transcription Factor in Acute Myeloid Leukemia.
Authors: Tamm Ingo , Schmelz Karin , Dörken Bernd , Wagner Mandy 
Published: October 2019

Abstract
Survivin, a member of the inhibitor of apoptosis protein family, is involved in both, inhibition of apoptosis and regulation of cell division. It is expressed in embryonic and fetal tissues as well as in the majority of human leukemias, but is undetectable in normal differentiated adult tissue in vivo. The molecular mechanisms involved in the cancer-specific re-expression of survivin are unclear. In this study, we describe a novel mechanism for over-expression of survivin in AML.
Using electrophoretic mobility shift assays (EMSA), we show that the early growth response (Egr)-1 transcription factor binds to the sequence 5′ GAGGGGGCG 3′ within the human survivin promoter after induction by phorbol 12-myristate-13-acetate (PMA) in vitro. Furthermore, chromatin immunoprecipitation (ChIP) analysis confirmed the specific binding of Egr-1 to the proximal survivin promoter in PMA treated entire leukemia cells.
To further analyze the functionality of the Egr-1 site within the survivin promoter in entire cells, transient transfections of p53 wildtype and mutated cell lines with wildtype Egr-1 expression vector were performed. In these overexpression experiments, mRNA and protein levels of survivin but not of control protein were downregulated after exogenous expression of wildtype Egr-1. Using reporter-gene assays, basal survivin promoter activity was decreased significantly by wildtype Egr-1 transfection, whereas mutant Egr-1 did not change activity of the survivin promoter, implying that Egr-1 specifically blocks survivin expression at the transcriptional level. In addition, Egr-1 over-expression sensitized cells to TRAIL-induced apoptosis.
To investigate the expression pattern of Egr-1 in different AML cell lines and control samples from healthy donors, we analyzed the expression of Egr-1 by RT-PCR. Fitting with the above shown data, Egr-1 was expressed at significantly lower levels in AML cell lines expressing high survivin levels than in healthy control samples with low survivin levels.
Taken together, we show that the transcription factor Egr-1 binds to the human survivin promoter in vitro and in entire cells, downregulates survivin expression independently of p53 and sensitizes cells to TRAIL-induced apoptosis. Since Survivin is often overexpressed in AML, downregulating Survivin expression by activating Egr-1 may be an interesting therapeutic option.
Identification of Meis1 as a Target of CREB in Myeloid Leukemogenesis.
Authors: Esparza Samuel D. , Shankar Deepa , Nelson Stanley , Sakamoto Kathleen 
Published: October 2019

Abstract
The cyclic AMP response element binding protein, CREB, is a leucine zipper transcription factor that induces the expression of genes that regulate proliferation and survival. We previously demonstrated that CREB expression was increased in bone marrow cells from patients with acute leukemia. Transgenic mice that overexpress CREB in myeloid cells develop a myeloproliferative/myelodysplastic syndrome after one year. Although CREB has been implicated in the development of leukemia, the mechanisms by which CREB overexpression leads to a malignant phenotype remain elusive. To identify CREB target genes, we performed microarray analysis with RNA from two CREB overexpressing cell lines, K562 and NFS60. Both murine (NFS60) and human (K562) myeloid leukemia cell lines were stably transfected with CREB under the control of the myeloid-specific HMRP-8 promoter. Meis1, an important transcription factor in hematopoiesis, was found to be upregulated 40-fold in CREB overexpressing cells compared to controls. In the development of granulocytes, Meis1 expression decreases as cells differentiate. Similar to CREB, Meis1 is expressed in CD34+ hematopoietic cells but not in CD34- cells. Meis1 expression has been reported to promote self-renewal of stem cells and inhibit G-CSF-dependent differentiation into granulocytes. We hypothesized that CREB overexpression may lead to persistent expression of Meis1, increased self-renewal, and a block in differentiation of primitive hematopoietic progenitor cells, thereby contributing to leukemogenesis. We first examined Meis1 expression in CREB overexpressing K562 and NFS60 cells. Significantly higher levels of Meis1 were detected in Western blot analysis with lysates from CREB overexpressing K562 cells compared to parental cells. In addition, the degree of CREB expression correlated with the degree of Meis1 expression when evaluated at the protein level. Experiments performed using the NFS60 cell line showed similar results. To investigate Meis1 expression in primary myeloid leukemia cells, bone marrow from patients with acute myeloid leukemia (AML) were analyzed for CREB and Meis1 expression. Samples from three of the four patients had elevated CREB expression. Patients whose cells had the highest levels of CREB also had highest expression of Meis1. The one patient with minimal CREB expression showed no detectible levels of Meis1. Evaluation of cell lines and primary cells using quantitative real time PCR is underway to determine potential differences in CREB and Meis1 expression at the mRNA level. Chromatin immunoprecipitation will also be performed to determine whether Meis1 is a direct target of CREB. Our data demonstrate that CREB overexpression correlates with overexpression of Meis1 in both cell lines and primary AML cells. These results suggest that CREB and Meis1 are co-regulated in AML cells and may contribute to the leukemia phenotype.
Dose-Dense Induction (Sequential-HAM) in Primary Acute Myeloid Leukemia - A Pilot Study of the AML-CG.
Authors: Braess Jan , Staib Peter , Ludwig Wolf D. , Wormann Bernhard J. , Reichle Andreas , Berdel Wolfgang , Buchner Thomas , Gruneisen Andreas , Schmid Christoph , Schmid Christian , Kern Wolfgang , Spiekermann Karsten , Fiegl Michael , Feuring-Buske Michaela , Bohlander Stefan , Heinicke Achim , Sauerland Christ , Unterhalt Michael , Hiddemann Wolfgang 
Published: October 2019

Abstract
Background: Dose intensity is considered one of the prime determinants of antileukemic efficacy in induction treatment of acute myeloid leukemia (AML) - as demonstrated by the superior long-term results of double induction versus conventional induction. In an attempt to further pursue this historically successful strategy an ongoing phase II study of the AML-CG pilots the feasibility of the S-HAM regimen (HD-AraC 3g/m2/12h d1,2,8,9; Mitoxantrone 10mg/m2 d3,4,10,11) in first-line treatment of de-novo AML. In this regimen the interval between the two induction cycles is reduced from 17 days (double induction) to a minimum of 3 days (S-HAM) - thereby increasing dose-intensity more than 2-fold in the critical early phase of treatment.
Results: In the past 18 months 99 patients have been recruited into the trial with a median age of 52 years (range 18 – 78). Of 93 patients evaluable for response the following results were achieved: CR 62%, CRp 24%, PL 6%, ED 8% - resulting in an overall response rate (ORR) of 86%. The early death rate (ED) of 8% and the toxicity profile compared favourably with a historical control group within the AML-CG 1999 study (subgroup: de-novo AML, age less than 60 years, HAM-HAM double induction) with an ED rate of 14% (ORR 68%, persistent leukemia (PL) 18%). The high antileukemic efficacy of S-HAM was also demonstrated by the fact that 88% of patients had a bone marrow blast count of < 10% one week after therapy as compared to less than 48% of patients of the HAM-HAM double induction group. If patients had an adequate blast clearance on day 18 pegylated G-CSF was applied every 10 days until neutrophil recovery. The median time to neutrophil recovery was 30 days after start of treatment with S-HAM which was substantially shorter than following either TAD-HAM or HAM-HAM double induction in the AML-CG 1999 trial (both with a median of 45 days). Since the S-HAM regimen has proven feasible at the present dose level a dose escalation was performed with an additional day of HD-AraC and Mitoxantrone in the first cycle of the sequential regimen.
Conclusion: In the future the appropriate dose level of the S-HAM regimen will then constitute the experimental arm for a randomized comparison of a dose-intensified regimen S-HAM - combining a promising antileukemic activity with significantly reduced duration of critical neutropenia - versus standard double induction for patients younger than 60–70 years in the next generation of the AML-CG studies.
Bone Marow (BM) Involvement in Diffuse Large B Cell Lymphoma (DLBCL): Clinical Impact of Discordant Disease.
Authors: Chhanabhai Mukesh , Connors Joseph , Seville Wayne , Matso Dan , Gascoyne Randy 
Published: October 2019

Abstract
Background and Methods: Most published studies have suggested that survival of de novo DLBCL with discordant BM involvement by small B cell lymphoma is indistinguishable from patients having a negative staging BM. The aim of the study was to investigate the incidence and clinical impact of BM involvement by concordant and discordant B cell lymphoma in patients with DLBCL seen in a single institution over a 5 year period (1\1\2000 – 31\12\2004). The cases were identified from pathology records and BCCA Lymphoid Cancer Database.
Results: The group of interest for this study comprised 652 patients with de novo DLBCL with staging marrow available for review. 60 of 652 (9.2%) of patients with DLBCL had concordant large B-cell lymphoma in their bone marrow. 523 (80.2%) were negative, 16 patients showed what we considered to be atypical lymphoid (ALH) infiltrates lacking definitive features of malignancy. In 50 patients (7.7%) the bone marrow showed discordant histology with predominantly small B-cells, some showing paratrabecular localization. In total there were five cases of TCRBCL in the study, three with marrow involvement. Furthermore, all 54 cases of primary mediastinal B-cell lymphoma (PMBCL) had a negative BM. Of the 652 cases with DLBCL with staging bone marrows available, merging the pathology and clinical databases resulted in 599 patients with complete clinical records. Of these, bone marrows were either not done or deemed inadequate in 101 cases. Therefore, there were 488 patients with DLBCL with an interpretable marrow of which 344 had advanced stage disease. Staging marrows in these patients were negative = 264, positive 41, discordant 28, and ALH 11. The overall survival and progression free survival were strongly affected by the IPI score for these 344 cases (P<0.00001) (see figure). Compared to those without BM involvement patients with concordant large B-cell lymphoma in their BM (n=41) had the worst outcome and those with discordant small B-cell lymphoma in the bone marrow (n=28) had an inferior but intermediate outcome (median survivals (months) = not reached;12 and 20, respectively).
Conclusion: Diffuse large B-cell lymphoma is a heterogenous group of lymphomas as demonstrated by gene expression profiling. Our data suggests that discordant low grade B-cell lymphoma in patients who have coincident DLBCL has a poorer prognosis and the presence or absence of BM disease has clinical significance. Though we had very few cases, TCRBCL show a higher incidence of BM involvement in keeping with the reported literature. The absence of marrow disease in PMBCL is consistent with recent data indicating it is a biologically distinct form of DLBCL.
Figure Figure
Primary Mediastinal Large B-Cell Lymphoma (PMBL) Outcome May Be Significantly Improved by the Addition of Rituximab to Dose-Adjusted (DA)-EPOCH and Obviates the Need for Radiation: Results from a Prospective Study of 44 Patients.
Authors: Dunleavy Kieron , Pittaluga Stefania , Janik John , Grant Nicole , Shovlin Margaret , Steinberg Seth , Raffeld Mark , Staudt Louis , Jaffe Elaine S. , Wilson Wyndham H. 
Published: October 2019

Abstract
PMBL is a distinct clinicopathologic entity characterized by young age, female preponderance, localized disease, prominent sclerosis and CD30+. Gene expression profiling reveals a unique molecular signature, distinct from other DLBCL subtypes, with similarity to classical Hodgkin lymphoma (HL) (J Exp Med 198: 851, 2003). HL is typically CD20 negative whereas PMBL has robust CD20 staining. As with HL, the risk of local failure after anthracycline-based therapy in PMBL has led to routine mediastinal radiation. Given the young median age, female predominance and high cure rates, long-term toxicities from secondary malignancies and coronary artery disease can be life threatening. We prospectively evaluated the role of DA-EPOCH± R without routine radiation in 44 patients with untreated PMBL. The first 18 patients received DA-EPOCH alone and the subsequent 26 received DA-EPOCH+R. DA-EPOCH was administered for 6–8 cycles as described (Blood99: 2685, 2002). Most patients had adverse prognostic features with bulky disease, elevated LDH and extranodal sites, which were balanced among the 2 groups.
Patient Characteristics Characteristics All Patients DA-EPOCH DA-EPOCH-R Total Patients 44 18 26 Gender (F/M) 26:18 (1.44) 10:8 (1.25) 19:9 (1.88) Median age, y (range) 34 (12–70) 34 (20–62) 34 (12–70) Median Mass cm (range) 9.8 (3–19.7) 8.4 (5.1–15.7) 10.2 (3–19.7) Bulky mass > 6 cm 34 (83%) 13 (87%) 21 (81%) ECOG PS > 1 4 (9%) 2 (11%) 2 (8%) Stage III or IV 19 (43%) 9 (50%) 10 (38%) LDH > Normal 32 (73%) 14 (78%) 18 (69%) Extranodal sites 25 (57%) 9 (50%) 16 (63%) Pleural effusion 15 (34%) 4 (22%) 11(42%)
IHC profiling was similar in both groups and consistent with gene expression profiling of PMBL. Analysis of 40 cases showed CD20+ 40/40 (100%), CD10+ 2/30 (7%), BCL-6+ 21/26 (81%), MUM-1+ 10/24 (42%) and high MIB-1 with median (range) of 82% (54–98). At a median potential follow-up of 9.5 and 4.2 years, EFS and OS are shown below for DA-EPOCH and DA-EPOCH-R, respectively. Rituximab was associated with a significantly improved EFS (p=.038) and OS (p=0.023) by 2-tailed exact log-rank test with caveats associated with any non-randomized comparison. Three patients on DA-EPOCH-R had positive PET and biopsy after treatment. One received radiation (event), one recieved salvage chemotherapy and radiation (event), and one no further treatment after biopsy. DA-EPOCH-R is highly effective in PMBL with OS of 100% and obviated the need for radiation/surgery in 23/26 (88%) patients. Rituximab may significantly improve EFS and OS with DA-EPOCH-based treatment. Accrual continues.
Figure Figure