bluuebunny/crossref_metadata_2025_split huggingface dataset:
The Combination of 2-CDA and Rituximab in Patients with Chronic Lymphocytic Leukemia (CLL): A Prospective Multicenter Phase II Trial (SAKK 34/02).
Authors: Leupin Nicolas , Schuller Jan C. , Solenthaler Max , Tichelli Andre , Gratwohl Alois , Beretta Kurt , Bargetzi Mario J. , Himmelmann Andreas , Hanselmann Susanne , Zenhausern Reinhard 
Published: April 2020

Abstract
Introduction: This trial aimed to determine the efficacy and toxicity of an induction immunochemotherapy consisting of rituximab and cladribine (2-chlorodeoxyadenosine, 2-CDA) in patients (pts) suffering from chronic lymphocytic leukemia (CLL).
Methods: Inclusion criteria were CLL at first diagnosis or after one treatment with alkylating agents. The regimen consisted of four remission induction cycles. In cycle 1, 2-CDA (0.1 mg/kg/day) was administered for 5 days. In cycles 2-4, Rituximab (375 mg/m^2) was given on day 1 followed by 2-CDA (0.1 mg/kg body weight), in intervals of 28 days. Responding pts (complete remission (CR), very good partial remission (VGPR) or nodular partial remission (NPR)) underwent stem cell mobilization chemotherapy with Cyclophosphamide (4g/m^2 on day 2) G-CSF (10 microgram/kg s.c. daily, from day 4 on), and Rituximab (375 mg/m^2) on day 1 and 8 as in vivo purging. If no CR, VGPR or NPR was achieved, up to 4 cycles CHOP were administered. Primary endpoint was CR, secondary endpoints were VGPR, NPR and toxicity after induction and feasibility of stem cell mobilization. For response evaluation, staging procedures included clinical examination as well as bone marrow biopsies and CT-scans. A total of 41 pts was planned using Simon’s two-stage design with 5% significance and 80% power for the null hypothesis of CR rate &lt; 25% and the alternative hypothesis of CR rate &gt; 45%.
Results: 42 pts were included, median age 53.8 y (range 38 – 65), WHO performance status 0 in 33 pts and 1 in 9 pts, stage Binet B in 20 pts, Binet C in 8 pts and progressive A in 14 pts. 2 pts were not evaluable for response. 9 pts reached CR (22.5%, 95% CI: 11–38%). Overall response rate including 15 VGPR and 2 NPR was 65% (CI: 48–79%). Of the 14 non-responders, 8 underwent CHOP treatment of which 2 achieved VGPR. 20 patients underwent mobilization and 8 pts refused further protocol treatment. 14 pts had leucapheresis. Stem cell harvest was feasible in 7 pts, all with &#8805; 2×10^6 cells/kg. Fever and infection were reported respectively in 13 and 9 pts. Infusion related adverse events of Rituximab were moderate and occurred mainly after the first infusion. 42 of 158 cycles (27%) were associated with grade 3 or 4 neutropenia and 6 of 158 cycles (4%) with grade 3 thrombocytopenia.
Conclusions: Although the expected CR rate was not achieved, a combination of Rituximab and 2-CDA is an effective and well tolerated treatment.
Identification of Driver and Passenger Mutations of FLT3 by High-Throughput DNA Sequence Analysis and Functional Assessment of Candidate Alleles.
Authors: Frohling Stefan , Scholl Claudia , Levine Ross L. , Loriaux Marc , Boggon Titus J. , Bernard Olivier A. , Berger Roland , Dohner Hartmut , Dohner Konstanze , Ebert Benjamin L. , Teckie Sewit , Golub Todd R. , Schittenhelm Marcus M. , Lee Benjamin H. , Griffin James D. , Stone Richard M. , Heinrich Michael C. , Deininger Michael W.N. , Druker Brian J. , Gilliland D. Gary 
Published: April 2020

Abstract
Activating mutations in the juxtamembrane domain or the activation loop of the receptor tyrosine kinase FLT3 occur in many cases of acute myeloid leukemia (AML), but it is not known whether genomic alterations outside these regions contribute to leukemogenesis. High-throughput DNA sequence analysis has provided insights into the mutational profiles of various cancers and represents a promising strategy for the identification of novel therapeutic targets. However, recognizing the subset of genomic alterations that are functionally relevant has proven difficult. We used a high-throughput platform to interrogate the entire FLT3 coding sequence in a cohort of 222 adult AML patients without known FLT3 mutations and experimentally tested the functional consequences of each candidate leukemogenic allele by exogenous expression in BaF3 cells. DNA sequencing detected nine non-synonymous sequence variants in six exons that were not known single-nucleotide polymorphisms. Functional assessment of these alleles identified four novel activating mutations in the extracellular domain, the juxtamembrane domain, and the activation loop that induced constitutive kinase activity, differentially activated downstream signaling pathways, and conferred varying sensitivity to pharmacologic FLT3 inhibition. In contrast, the remaining five alleles, including mutations in highly conserved, key functional domains of FLT3, were not associated with increased kinase activity and aberrant signal transduction. These findings support the concept that acquired mutations in cancer may not contribute to malignant transformation, and underscore the importance of functional validation of candidate alleles discovered using high-throughput genomic screens, to distinguish between ‘driver’ mutations underlying cancer development, and biologically neutral ‘passenger’ alterations.
Dual Epigenetic Control of CCAAT/Enhancer Binding Protein α (C/EBPα) Expression in Acute Myeloid Leukemia.
Authors: Hackanson Bjoern , Bennett Kristi L. , Brena Romulo M. , Jiang Jinmai , Maharry Katie , Whitman Susan P. , Schmittgen Thomas D. , Luebbert Michael , Marcucci Guido , Bloomfield Clara D. , Plass Christoph 
Published: April 2020

Abstract
CCAAT/enhancer binding protein α (C/EBPα) is a master regulator for myeloid differentiation. As a potential tumor suppressor, its loss of function through mutations and posttranslational mechanisms has been thoroughly investigated in acute myeloid leukemia (AML). Recently, we have demonstrated the importance of epigenetic alterations in deregulating C/EBPα expression patterns in lung cancer and head and neck squamous cell carcinoma (HNSCC). In this study we investigated the role of DNA methylation and other epigenetic factors in the regulation of C/EBPα in AML using 94 patient samples and 7 leukemia cell lines. A comprehensive and quantitative DNA methylation analysis of C/EBPα’s large CpG island using MassARRAY (Sequenom©) technology and BioCOBRA identified a distinct and densely methylated upstream promoter region (−1423 bp to −1121 bp in relation to the transcription start site) in 20% (19 of 94) of AML patient samples and in five of seven leukemia cell lines, while the core promoter remained unmethylated. This aberrant DNA methylation pattern was associated with two generally prognostically favorable cytogenetic subgroups: inv(16)(p13q22) and t(15;17)(q22;q21). While DNA methylation levels in normal bone marrow samples (NBM) were very low (median: 0%, range: 0%–5%) when compared to NBM, we observed significantly higher DNA methylation levels in the inv(16) and t(15;17) cytogenetic subgroups, with median DNA methylation levels of 29% (range: 1% to 81%) and 5% (range: 1% to 94%), respectively (p&lt;0.05). However, using semi-quantitative RT-PCR, we could not detect a significant correlation between DNA methylation of C/EBPα and its expression in the inv(16) subgroup. Interestingly, while epigenetic-targeting compounds induced up-regulation of C/EBPα mRNA in vitro, C/EBPα protein was diminished. This unexpected scenario was postulated to be compatible with the presence of a microRNA targeting C/EBPα mRNA. Using a computational microRNA prediction approach and functional studies, we show that C/EBPα mRNA is a target for microRNA-124a. We additionally demonstrate that microRNA-124a is frequently silenced by epigenetic mechanisms in AML, becomes upregulated following epigenetic treatment and targets the C/EBPα 3′-untranslated region (3′UTR). In this way, C/EBPα protein expression is reduced in a posttranscriptional manner. Our results indicate that epigenetic alterations of C/EBPα are a frequent event in AML and that epigenetic treatment can result in down-regulation of a key hematopoietic transcription factor.
Genome-Wide Identification of Aberrant Promoter Associated CpG Island Methylation in Acute Lymphoblastic Leukemia.
Authors: Kuang Shao-qing , Tong Weigang , Yang Hui , Lee Mathew K. , Fang Zhi-Hong , Jelinek Jeroslav , Issa Jean-Pierre , Garcia-Manero Guillermo 
Published: April 2020

Abstract
Aberrant DNA methylation is a common molecular feature of both pediatric and adult ALL. Specific methylation patterns predict for poor prognosis (Shen et al Blood 2004), and reactivation of epigenetically inactivated molecular pathways results in induction of leukemia cell death (Kuang et al. Oncogene 2007). Until now most studies of methylation in ALL have been based on arbitrary gene selection methods. To overcome this limitation and to study hundreds of promoter CpG islands simultaneously, we have developed a method that combines MCA (Methylated CpG Island Amplification) with either RDA (Representational Difference Analysis) or the Agilent Promoter Microarray platform. With these methods differentially methylated DNA treated with bisulfite is generated after mixing tester DNA (in our case DNA from de novo refractory Ph negative and MLL negative ALL patients) with driver DNA (normal B cell controls) and using specific restriction enzymes and several rounds of PCR. DNA fragments thus generated are either cloned (RDA) or labeled and spotted on the Agilent Array. Using this technology, that can potentially interrogate up to 17K promoters, we have identified 932 promoters targets of aberrant DNA methylation in poor risk ALL from patients that cannot be currently identified by standard molecular methods (Ph and MLL negative). The genes associated with these promoters are distributed through the human genome but an overrepresentation of methylated promoters located in chromosomes 3, 9, 11 and 19 was detected. Using molecular pathway clustering analysis, 404 of these genes are grouped together in 29 specific functional pathways. We have validated the methylation of 31 of these 923 genes by bisulfite pyrosequencing. Of these, 27 (87%) were confirmed to be hypermethylated in 23 human leukemia cell lines but not in normal controls (N=15). Methylation status analysis of these 27 genes allowed for the segregation of T cell versus B cell leukemia cell lines. Fifteen of these genes (GIPC2, RSPO1, MAGI1, CAST1, ADCY5, HSPA4L, OCLN, EFNA5, MSX2, GFPT2, GNA14, SALL1, MYO5B, ZNF382 and MN1) were also frequently hypermethylated in primary ALL samples. Expression analysis of 6 of these genes (GIPC2, MAGI1, ADCY5, HSPA4L, OCLN and GNA14) in leukemia cell lines further confirmed methylation associated gene silencing. Treatment of methylated/silenced cell lines with 5′-aza-2′-deoxycytidine and trichostatin A resulted in gene re-expression, further confirming the role of DNA methylation in their silencing. In summary, we have identified in excess of 900 targets of aberrant DNA methylation in ALL. The study of the epigenetically suppressed pathways represented by these genes should allow us to further understand the molecular pathogenesis of ALL and develop new prognostic biomarkers for patients with Ph and MLL negative disease.
Epigenetic Inactivation of the Secreted Frizzled-Related Protein Gene Family in Multiple Myeloma.
Authors: Jost Edgar , Gezer Deniz , Wilop Stefan , Herman James G. , Osieka Rainhardt , Galm Oliver 
Published: April 2020

Abstract
Multiple myeloma (MM) is a B-cell neoplasm that is characterized by the accumulation of malignant plasma cells in the bone marrow. Previous molecular studies have largely focused on acquired genetic aberrations in MM. There is increasing evidence that in addition to genetic aberrations epigenetic processes play a major role in carcinogenesis. Aberrant methylation of CpG islands near gene promoter regions is the most widely studied epigenetic abnormality in human malignancies and is associated with loss of gene function. This epigenetic event acts as an alternative to mutations and deletions to disrupt tumor suppressor gene function. The Wnt pathway has been recognized to be essential for normal organ development, and a role for Wnt signal transduction at several stages of lymphocyte differentiation and in the self-renewal of hematopoietic stem cells could be demonstrated. Alterations in the Wnt pathway have been shown to contribute to the pathogenesis of various human malignancies. Epigenetic silencing of the family of secreted frizzled-related proteins (SFRPs), which act as Wnt antagonists, was recently reported in several solid tumors and in acute lymphoblastic leukemia. In order to investigate the potential role of abnormal Wnt signaling in MM, we determined the methylation status of the promoter-associated CpG islands of SFRP-1, 2, 4 and 5 in the MM cell lines U266, LP-1, RPMI-8226 and OPM-2. Methylation-specific polymerase chain reaction (MSP) analysis revealed that promoter hypermethylation of the SFRP genes was a frequent event in MM cell lines (SFRP-1: 2/4, SFRP-2: 2/4, SFRP-4: 1/4 and SFRP-5: 3/4). Aberrant methylation of SFRP-1 and SFRP-2 in MM cell lines was associated with transcriptional silencing, as determined by real-time reverse transcriptase polymerase chain reaction. Treatment of cell lines that carry a hypermethylated SFRP-1 and SFRP-2 gene, respectively, with the demethylating agent 5-aza-2′-deoxycytidine resulted in gene reexpression. We then analyzed by MSP the methylation status of SFRP-1, 2, 4 and 5 in 76 specimens obtained from MM patients. The frequency of aberrant methylation among the primary patient samples was 38.2% (29/76) for SFRP-1, 59.2% (45/76) for SFRP-2, 2.6% (2/76) for SFRP-4 and 7.6% (6/76) for SFRP-5. There was a high incidence of concomitant hypermethylation of SFRP-1 and SRFP-2. We conclude that promoter hypermethylation of the SFRP genes is a novel epigenetic event in MM that may contribute to aberrant activation of the Wnt pathway. Further studies are warranted to elucidate the functional consequences of aberrant Wnt signaling by downregulation of the SFRP genes in the pathogenesis of MM. Additionally, the increasing evidence for the important role of DNA methylation changes in malignant plasma cell disorders may serve as a basis for the use of epigenetically targeted therapeutic approaches in MM in the future.
Host Plasmacytoid or Conventional Dendritic Cells Alone Are Sufficient To Initiate Graft-Versus-Host Disease.
Authors: Koyama Motoko , Hashimoto Daigo , Aoyama Kazutoshi , Matsuoka Ken-ichi , Karube Kennosuke , Takeda Kiyoshi , Ohshima Koichi , Akashi Koichi , Tanimoto Mitsune , Harada Mine , Teshima Takanori 
Published: April 2020

Abstract
Graft-versus-host disease (GVHD) is a major complication after allogeneic hematopoietic stem cell transplantation. Alloantigen expression on host dendritic cells (DCs) is critical to initiate GVHD. DCs can be divided into two main subpopulations; conventional DCs (cDCs) and plasmacytoid DCs (pDCs), however, the contribution of each DC subset to elicit GVHD remains unclear. We examined the ability of cDCs and pDCs to initiate GVHD. pDCs, cDCs and B cells were isolated from C57BL/6 (B6: H–2b) mice treated with Flt3 ligand in order to expand DCs. pDCs were enriched from bone marrow by depleting CD3+, CD19+, CD11b+, and CD49b+ cells, followed by a FACS sorting of CD11cint B220+ cells. cDCs and B cells were sorted from splenocytes as CD11chi B220− cells and CD11c− B220+ cells, respectively. Isolated pDCs showed plasmacytoid morphology, produced IFN-α in response to CpG oligonucleotide. Although pDCs stimulated allogeneic T cells far less potently than cDCs, stimulation with CpG enhanced their allostimulatory capacity as potent as cDCs. We compared the ability of each DC subset to initiate GVHD by an add-back study of MHC class II-expressing DCs into MHC class II-deficient (II−/−) mice that were resistant to CD4-dependent GVHD. Lethally irradiated II−/− B6 mice were injected with 2 × 106 pDCs, cDCs or B cells from wild-type (II+/+) B6 mice on day -1 and injected with 2 × 106 CD4+ T cell from BALB/c (H–2d) mice on day 0. A flow cytometric analysis of the mesenteric lymph nodes on day +5 demonstrated significantly greater expansion of donor CD4+ T cells in recipients of pDCs or cDCs than those of B cells (Table). While injection of B cells did not cause any sign of GVHD, injection of pDCs or cDCs alone was sufficient to produce clinical and pathological GVHD (Table), thus breaking GVHD resistance of II−/− mice. We next examined the ability of pDCs to induce CD8-dependent GVHD in MHC-matched transplant using mice deficient in functional MHC class I expression (β2m−/−). Again, injection of pDCs or cDCs alone was sufficient to cause expansion of donor CD8+ T cells (p&lt;0.05). We next asked whether signaling through Toll-like receptors (TLRs) could be required for pDCs to initiate GVHD. However, injection of pDCs isolated from MyD88/TRIF-double deficient mice was able to initiate GVHD as potent as wild-type pDCs, thus demonstrating that pDCs initiate GVHD in a TLR signaling independent manner. These results provide important information for developing strategies aimed at inactivating host DCs to prevent GVHD.
Impact of each APC subpopulation on GVHD APC Donor CD4 expansion (×103±SE) Clinical GVHD score (mean±SE) Pathological GVHD score (mean±SE) *p&lt;0.05 compared with B cells B cell 0.1 ± 0.0 2.1 ± 0.2 2.1 ± 0.2 pDC 5.3 ± 2.4* 4.3 ± 0.3* 7.4 ± 0.5* cDC 9.7 ± 3.8 * 3.8 ± 0.5 * 7.2 ± 0.7*
A Shift in the Intestinal Microflora towards Pro-Inflammatory Bacteria and Signaling Via Toll-Like-Receptor 2 Triggers Early Onset of Graft-Versus-Host Disease.
Authors: Nogai Axel , Heimesaat Markus M. , Bereswill Stefan , Thiel Eckhard , Goebel Ulf B. , Uharek Lutz 
Published: April 2020

Abstract
Pattern recognition receptors such as Toll-like-receptors (TLR) and NOD proteins bind specifically to microbial components and activate innate immune responses. Reducing the bacterial load by antibiotic treatment has been shown to ameliorate the severity of graft-versus-host-disease (GvHD) after allogenic stem cell transplantation. The impact of bacteria and TLRs on the induction of T-cell-alloreactivity and GvHD was investigated in a murine transplantation model. C57B/10 wild-type (wt) mice served as recipients of MHC-mismatched Balb/c grafts after conditioning with treosulfan and cyclophosphamide. Analyses of the gut microbiota in fecal samples showed a pronounced increase of luminal load of both gram-positive and gram-negative bacteria within 11 days after stem cell transplantation. The increase in the Escherichia coli load was significantly correlated with the severity of graft-versus-host-disease and with decreased survival rates. Analysis of the kinetics of fecal E. coli load and the histologial and clinical GvHD scores revealed that the increase of the fecal E. coli load is detected simultanously to the onset of clinical overt GvHD. Because TLR 2 and 4 represent the major receptors for gram-positive and gram-negative bacteria, respectively, the incidence and severity of GvHD was investigated in TLR2, TLR4 and TLR2/4 deficient mice on a C57B/10 background. The onset of GvHD was delayed in recipient mice lacking TLR2 and both TLR2 and 4 (p&lt;0.05) whereas a single TLR4 deficiency had no influence. Interestingly, there was no correlation of E. coli load and GvHD in TLR2-deficient mice.Taken together, these results support potential roles of TLR2-dependent signaling and gut bacterial components in the induction and early onset of intestinal GvHD. In expansion of these findings, our data elucidate the role of the innate immune system and its receptors for the induction of alloreactive T-cell responses, providing evidence that modulation of the intestinal gut flora might prevent or reduce GvHD in humans.
Lymphopenia Promotes the Development of Graft-Versus-Host Disease upon Donor Leukocyte Infusion.
Authors: Sachs Jessica , Sykes Megan 
Published: April 2020

Abstract
In murine models of hematopoeitic cell transplantation (HCT), a graft-versus-leukemia (GVL) effect can be effectively separated from graft-versus-host disease (GVHD) by giving delayed donor-leukocyte infusions (DLI) to established mixed hematopoietic chimeras (MC) (Mapara, Transplantation 2003). In these models, the absence of conditioning-induced inflammation at the time of DLI prevents trafficking to GVHD target tissues, limiting the GVH response to the lymphohematopoeitic system (Chakraverty, J Exp Med 2006). While clinical trials have shown that the same outcome is possible in humans, some patients develop severe GVHD following delayed DLI (Dey, Blood Biol Marrow Transplant 2003). In contrast to mice, who recover T cell numbers quickly after HCT through de novo thymopoiesis, humans remain lymphopenic for many months. The current study investigates the role of recipient lymphopenia in the development of GVHD following delayed DLI to MC. Allogeneic (B10.A) or syngeneic (B6) lymphocytes (30 × 106 whole splenocytes) and bone marrow cells (10 × 106) were administered to unconditioned RAG–1 knockout (KO) B6 recipients or to established MC, and animals were followed for development of GVHD. By day 7 after injection, the RAG KO recipients of allogeneic lymphocytes (allogeneic group) had lost significantly more weight (p&lt;0.001) and had significantly higher clinical GVHD scores (p&lt;0.0001) than RAG KO recipients of syngeneic lymphocytes (syngeneic group) or MC recipients of allogeneic lymphocytes (MC group). Histologic evaluation on day 7 showed significantly increased GVHD in liver and colon of the allogeneic group. Chimerism analysis on day 7 showed the presence of an ongoing lymphohematopoietic GVH response (LGVHR) in both the allogeneic and MC groups, though the response was slower in the MC group. When followed over time, clinical GVHD in the allogeneic group resolved by day 56, weight differences disappeared by day 65, but mild histologic GVHD persisted as late as day 239. Importantly, the same dose of allogeneic T cells given to freshly conditioned animals results in rapid, lethal GVHD, whereas these unconditioned, lymphopenic animals developed only a relatively mild, largely self-limited GVHD. These results demonstrate that in the absence of conditioning-induced inflammation, lymphopenic recipients of allogeneic T cells do develop mild GVHD, consistent with the possibility that recipient lymphopenia contributes to the GVHD seen in human MC recipients of DLI. The mechanisms by which lymphopenia promotes GVHD in non-conditioned MC recipients of DLI require further investigation, but may include the absence of regulatory T cells, the potential for lymphopenia-induced proliferation of T cells, and the presence of infection-induced inflammation.