bluuebunny/crossref_metadata_2025 huggingface dataset:
Importance of Transplantation Variables in Marrow to Muscle Conversion.
Authors: Abedi Mehrdad , Greer Deborah A. , Foster Bethany A. , Colvin Gerald A. , Harpel Jashua A. , Dooner Mark S. , Quesenberry Peter J.
Published: April 2020

We have previously shown that conversion of marrow to skeletal muscle is increased by cardiotoxin injury and we have demonstrated levels of up to 12% conversion. We have now used the same model of cardiotoxin injury to determine variables that affects this conversion. Muscle fibers were defined by their morphology and presence of desmin and dystrophin staining and absence of CD45 staining. In a GFP to B6 transplant model, there was a significant increase in the incidence of muscle chimersim with increasing the dose of marrow donor cells unrelated to the level of hematopoietic chimersim. Radiation was a critical factor and a combination of 500cGy of whole body and 500cGy of lower extremity radiation was found as the optimal model. However radiation impact was mostly due to its effect on hematopoietic chimerism and there was very little local effect on muscle. Higher doses of radiation were detrimental and resulted in significant muscle atrophy. Transplantation of marrow cells to NOD/Scid mice without radiation resulted in muscle engraftment although this was a rare event. Furthermore, timing of the cardiotoxin injury relative to the transplantation was an important factor since injection of cardiotoxin before or right after transplant resulted in minimal conversion while injury seven days or more after transplant resulted in significant conversion events. When sublethal doses of cardiotoxin were injected systemically after marrow transplant GFP positive muscle fibers were seen but the incidence was much lower than direct intramuscular injections of cardiotoxin. Multiple cycles of mobilization and injury also improved the incidence of GFP positive fibers in the regenerated muscle. We conclude that in addition to injury, cell mobilization and cell selection, improvements in the efficiency of this process can be achieved by applying specific doses and distribution of radiation (i.e. local and systemic), by giving the appropriate doses of marrow cells and by selecting the appropriate time for injury. Combining these models gives quantitatively significant fiber conversions approaching levels that would be therapeutically effective in the treatment of muscular dystrophies.
Variation in Transfusion Rates amongst Canadian Academic and Community Medical Centers.
Authors: Hutton Brian , Fergusson Dean , Hebert Paul C. , Tinmouth Alan , McIntyre Lauralyn , Kmetic Andrew
Published: April 2020

Purpose: Few recent studies have examined approaches to transfusion practice in high-risk patients. We therefore documented practice variation following repair of hip fracture or cardiac surgery, as well as those requiring intensive care following a surgical intervention or multiple trauma.

Methods: We documented rates of red cell transfusion in 41,568 patients admitted to 11 hospitals across Canada between August 1998 and August 2000 as part of a retrospective observational cohort study. Adjusted odds of transfusion, median number of transfusions per patient and median number of transfusions per patient day were compared across sites. We also compared mean nadir hemoglobin concentrations from center to center in the subgroup of 7,552 patients that received red cells.

Results: The overall rate of red cell transfusion was 38.7 %, and ranged from 23.8% to 51.9% across centers among the 41,568 peri-operative and critically ill patients. Women were more likely to be transfused (43.7% versus 35.3%, p<0.0001), with higher rates of transfusion in 8 of 11 centers. Following adjustment for the effects of age, gender, severity of illness and the presence of twelve different comorbidities, the odds of transfusion ranged from 0.44 to 1.53 overall, from 0.42 to 1.22 in patients undergoing a hip fracture repair, from 0.72 to 3.17 in cardiac surgical patients undergoing cardiac surgery, and from 0.27 to 1.11 in critically ill and trauma patients (figure 1). Examination of both median number of units transfused and median number of units transfused per patient day also revealed between-site variation (figure 2). In the 7,552 transfused patients, the mean adjusted nadir hemoglobin was 74.0±4.83g/L overall, and ranged from 66.9±1.7g/L to 84.5±1.6g/L across centers. Similar differences among centers were observed amongst hip fracture patients (71.2±2.9g/L to 82.8±1.7g/L), cardiac surgical patients (65.7±1.1g/L to 77.3±1.0g/L) and critically ill and trauma patients (66.1±3.04g/L to 87.5±2.5g/L).

Conclusions: We noted significant differences in the rates of red cell transfusion and nadir hemoglobin concentrations in various surgical and critical care settings. Such variation provides the impetus for investigators to explore its potential causes, and furthermore to attempt to define optimal transfusion practice in a variety of clinical settings by conducting randomized controlled trials.
Mutation Status of Imatinib Mesylate-Resistants CML Patients and Clinical Outcomes: A French Multicenter Retrospective Study for the fiLMC Group.
Authors: Corm Selim , Nicollini Franck , Borie Dominique , Sorel Nathalie , Leguay Thibault , Hayette Sandrine , Facon Thierry , Roy Laurence , Giraudier Stephane , Marit Gerald , Preudhomme Claude , Michalet Mauricette , Mahon Francois-Xavier , Thuilliez Michel , Guilhot Francois , Roche-Lestienne Catherine
Published: April 2020

Imatinib mesylate (IM) is highly effective in newly diagnosed CML. Unfortunately, acquired abl-kinase domain mutations are commonly identified to be associated to resistance or relapse in patients with advanced disease. Distinct mutation exhibit varying degrees of resistance, and mutation occurring in the ATP-binding loop (P loop) or in positions in direct contact with IM (i.e. T315) may be correlated with subsequent disease progression.

In this study, we investigated the bcr-abl mutation status of imatinib-resistant CML patients and correlated to the disease outcome.

Patients and methods: From CML patients diagnosed in 5 French centers, 67 IM-resistants patients presenting mutations by direct sequencing of the abl kinase domain at time of resistance or relapse were included in this study. When starting IM monotherapy (J1) at 400 to 600 mg per day, 45 patients were in chronic phase of CML, 12 patients were in accelerated phase of the disease and 7 patients were in blast crisis. The median duration of IM is 29 months (range 3–54) and the median delay between diagnosis and J1 is 34 months (range 0–168). At the end of the study, 23 patients were dead. According to the mutation status, a Kaplan-Meier curve was construct for time to death to J1, and survival were compared using log rank test.

Results: By direct sequencing, 17 patients presented mutations in the P-loop (residues 252 to 255), 10 patients presented the T315M mutation alone, 1 patient had the T315 and the M351 mutations, and 39 patients presented mutations in other kinase domains. According to the mutation status, survival difference between patients presenting P-loop mutations or mutations in an other abl-kinase (including T315), no significant difference was observed (p=0.07). However, survival difference between patients presenting P-loop mutations, T315 mutation and mutations in an other abl-kinase domains was highly significant (p=0.0031, figure). The median survival for the P-loop and T315 mutated patients was of 39 months and 29 months respectively, and median survival not reached for others.

Conclusion: By this study we confirm that either the T315 mutation or mutations in the P-loop had a poor prognosis. Detailed correlation analysis of mutation status according to clinical features and response to increased IM-dose will be reported.

Figure Figure
Divergent Point of Developmental Cell Fate of Common Precursor for Cardiomyocyte and Hemangioblast Is Accompanied by the Presence or Absence of Expression of PDGFRalpha and Flk-1.
Authors: Hirata Hirokazu , Murakami Yoshinobu , Tanaka Yosuke , Inoue Kayoko , Ishikawa Masakazu , Sasada Masataka , Asahara Takayuki , Kawamata Shin
Published: April 2020

Pluripotency of embryonic stem (ES) cells provides a powerful tool for dissecting the cellular and molecular mechanisms regulating germ layer fate determination and tissue formation. Both of hemangioblasts and cardiomyocytes are thought to be derived from lateral plate mesoderm of early embryo, but little is known about the phenotypic specification in these two lines of differentiation at the divergent point through development. Utilizing the in vitro differentiation system of murine ES cells in the presence or absence of stromal cell layer, and gene expression data in the microarray analysis, we determined a set of cell-surface molecules as specific markers, which enabled efficient enrichment of hemangioblasts or cardiac myocytes. Furthermore, we represent the evidences that the sorted fraction with hemangioblast-specific markers markedly produced both of blood island and endotherial colonies in stromal-free culture system.

Materials and Methods

Murine embryonic stem cell line, EB5 was cultured on gelatin-coated plates for mesodermal differentiation after withdrawal of LIF and Blastcidin. The expression of surface molecules related to mesodermal differentiation was evaluated with flowcytometry or quantitative RT-PCR techniques. The cell population defined by combination of the candidate surface molecules was sorted at day 5 and seeded on PA-6 or OP-9 stromal cell layers, or in conditioned medium of PA-6 cells for stroma-free assay until day 12 of differentiation. Differentiational direction of each culture sample was evaluated by the expression of genes or membrane molecules specific for tissue lineages with quantitative RT-PCR or flowcytometric techniques. The cardiogenic potential was also assessed by scoring beating colonies co-cultured on stromal cells.

Results and Discussion

In combination with cell surface expression of Flk-1 (VEGFR2), which is known to be a restricted marker for mesodermal tissues during early embryonic development, upregulated expression of Platelet-Derived Growth Factor (PDGF) receptor alpha on cell surface was detected more frequently in the fraction containing pre-cardiomyocytic cells. In contrast, negative fraction for PDGFRalpha contributed to efficient formation of hematopoietic burst colonies growing together with endotherial cells engulfing DiI- acetylated LDL. We also cofirmed that these suspended hematopoietic cells were spurting out of hemangioblast-like compartment in time-lapse imaging analysis. Our findings may provide a new experimantal window to analyze the common mesodermal precursors for hemangioblasts and cardiomyocytes in vitro. We will search for tissue stem cells bearing the corresponding surface markers in human cell sources, and evaluate their differentiation potentials and therapeutic effect on ischemic diseases.
Specificity of Smad Signaling during Primitive Erythropoiesis.
Authors: Zafonte Brian T. , Huber Tara L. , Keller Gordon , Evans Todd
Published: April 2020

Bone morphogenetic proteins (BMPs) comprise a sub-family of TGF-beta-like molecules that exert a wide range of biological activities during development, and are essential for normal hematopoiesis. However, the precise stage in development that BMP signaling regulates hematopoiesis is not defined. Three proteins, Smad1, Smad5, and Smad8 transmit BMP signals to the nucleus to activate the expression of hematopoietic-specific transcription factors. These Smads are homologous in their sequences, and appear to be regulated similarly, however their specificity in regulating hematopoiesis remains undefined. Although Smad proteins are regulated post-translationally, their expression is also under transcriptional control during development. We examined the specificity of Smad1/5/8 activity in the context of primitive erythropoiesis, using the mouse embryonic stem cell /embryoid body (ES/EB) system. We exploited ES cells with GFP targeted to the brachyury locus, in order to identify specific sub-sets of progenitors. Smad1 transcript levels are initially upregulated as ES cells become fated to mesoderm and hematopoietic progenitors, but the levels are significantly decreased in cells derived from differentiating primitive erythroid colonies. In contrast, Smad5 transcript levels show the opposite profile, being more correlated with erythroid differentiation. To directly assess the role of these Smads during erythropoiesis, their activity is being manipulated in ES cells during the commitment phases of embryonic hematopoiesis. For this purpose, inducible ES cell lines were generated capable of forcing the expression of wildtype Smad1 or Smad5, or a dominant-negative isoform of Smad5, at any stage of ES/EB development. Colony assays were used to analyze quantitatively the hematopoietic potential of these cells. Forced expression of Smad1 results in a marked increase in primitive red blood cell colony formation as compared to control ES cells. Maintenance of Smad1 expression does not appear to inhibit terminal differentiation. Based on a time-study of the induction, the effect on erythoid colonies could be due to expansion of earlier progenitors. Current experiments using the in vitro blast assay are examining the direct effect of Smad1 expression on earlier (hemangioblast) development. This data, and analogous analyses of cells induced to express Smad5 or the dominant-negative Smad isoform are in progress and will be presented. These studies should facilitate our understanding of the specificity of BMP-regulated Smads during commitment and differentiation of embryonic stem cells and hematopoietic progenitors.
HLA-G Antigen Expression Is Associated with a More Aggressive, Unfavorable Outcome and Immunosuppression in Chronic Lymphocytic Leukemia.
Authors: Nueckel Holger , Rebmann Vera , Jan Duerig , Ulrich Duehrsen , Hans Grosse-Wilde
Published: April 2020

Background: The human leukocyte antigen (HLA)-G molecule exhibits limited tissue distribution and exerts multiple immunoregulatory functions. Recent studies indicate an ectopic up-regulation in tumor cells which may favor their escape from antitumor immune responses. The role of HLA-G in B cell chronic lymphocytic leukemia (B-CLL) has not been defined.

Experimental design: HLA-G expression was studied retrospectively in circulating B-CLL cells from 47 patients by flow cytometry using the MEM/G9 monoclonal antibody.

Results: The proportion of leukemic cells expressing HLA-G varied from 1% to 54%. Patients with < 23% HLA-G positive cells (according to ROC analysis; designated as the HLA-G negative group) had a significantly longer progression-free survival (PFS) time than patients with > 23% of positive cells (median PFS 120 versus 23 months, p=0.0001). Multivariate analysis revealed that HLA-G expression (hazard ratio 4.81; p=0.002) was next to the initial staging according to Binet (hazard ratio 8.6; p=0.0001) the best independent prognostic factor compared to other known prognostic factors like ZAP-70 status (hazard ratio 3.6; p=0.029) or CD38 status (hazard ratio 1.83; p=0.37). Humoral and cellular immunosuppression was significantly more prominent in HLA-G positive as compared to HLA-G negative patients group (median immunglobuline G (g/l) 4.3 versus 7.3; median total T-cells (per μl) 824 versus 2540).

Conclusions: In B-CLL the level of HLA-G expression is correlated with the degree of immunosuppression and prognosis. To our knowledge this is the first report that describes an association of HLA-G antigen expression and the course of the disease in B-CLL patients. Thus, HLA-G may contribute to the impairment of immune responses against tumor cells and infections. These findings need to be confirmed in a prospective study.
Potential Value of N-Desulfated Heparin without Anticoagulant Activity in Treatment of Complement-Mediated Lysis of Paroxysmal Nocturnal Hemoglobinuria Erythrocytes.
Authors: Ninomiya Haruhiko , Nagasawa Toshiro
Published: April 2020

Paroxysmal nocturnal hemoglobinuria (PNH) is clinically characterized by the complement (C’)-mediated intravascular lysis of PNH-affected erythrocytes and thrombotic diathesis. Treatment methods of intravascular hemolysis, especially on the hemolytic crisis, have not been established yet. Although several reagents have been used for the treatment of PNH hemolysis, the value of prednisone/prednisolone in treating the hemolysis of PNH is controversial; anti-C5 antibody is under clinical study. We previously demonstrated that heparin and low-molecular weight heparin (LMWH) inhibit C’-mediated hemolysis of PNH erythrocytes in vitro, by inhibiting insertion of C5b-7 into the erythrocyte membranes (Ninomiya et al: Inhibition of complement-mediated haemolysis in paroxysmal nocturnal haemoglobinuria by heparin or low-molecular weight heparon. Brit J Haematol109,875–881,2000). However, that the concentration of heparin (or even that of LMWH) required to inhibit C’-mediated lysis of PNH erythrocytes efficiently induces a profound prolongation of APTT (activated partial thromboplastin time) of plasma unables their clinical application. In the current study, we examined the inhibitory effects of heparin, LMWH (dalteparin), and N-desulfated heparin on C’-mediated lysis of PNH erythrocytes (sucrose hemolytic assay) and their anticoagulant effects on normal plasma coagulation system (APTT, TT (thrombin time), and AT-III (antithrombin)-enhancing activity). N-desulfated heparin inhibited C’-mediated lysis of PNH erythrocytes, equivalently to heparin or LMWH, on the basis of their uronic acid (UA) contents; IC50 values of heparin, LMWH, and N-desulfated heparin, was 0.8 U/ml (UA 2.9 μg/ml), 0.5 IU/ml (UA 2.3μg/ml), and 3.0μg/ml (UA 0.9μg/ml), respectively. Whereas the concentrations of heparin and LMWH, that exerted their anti-C’ activity efficiently, induced prolongations of APTT profoundly to clinically unapplicable extents, N-desulfated heparin (up to 50 μg/ml) did not show any anticoagulant activity. These data show a potential value of N-desulfated heparin for the treatment of C’-mediated intravascular lysis of PNH erythrocytes without bleeding complications due to its administration. Because anticoagulant therapy may be beneficial and indicated for the treatment of hemolysis in PNH, especially in hemolytic crisis increasing the risk of thrombotic complications, cocktail therapy of heparin (or LMWH) with N-desulfated heparin may be proposed.
Functional Characterization of Telomerase RNA Variants Found in Patients with Hematological Disorders.
Authors: Ly Hinh , Calado Rodrigo T. , Allard Paulette A. , Baerlocher Gabriela M. , Lansdorp Peter M. , Young Neal S. , Parslow Tristram G.
Published: April 2020

Human telomerase uses a portion of its integral RNA component (hTER) as the template to synthesize telomeres at chromosome ends. hTER sequence polymorphisms have been observed in some patients with bone marrow failure syndromes such as aplastic anemia, but the functional significance of most such variants is unknown. Here, we report the functional characteristics of ten previously-described and two newly discovered hTER disease-associated polymorphisms. Most of these hTER variants adversely affected telomerase enzymatic function as measured in the telomerase reconstituted human cells. Similar loss-of-function effects were also seen directly in primary lymphocytes collected from two of the patients. The majority of the functional deficits stemmed from perturbations of the predicted hTER RNA secondary structure, and corresponded well with the degrees of telomere shortening observed in patients. In contrast, hTER variants anticipated to be inconsequential polymorphisms, which were also found in healthy subjects, did not interfere with telomerase function. Loss of telomerase activity and of telomere maintenance resulting from inherited hTER mutations may predispose some patients to aplastic anemia and other marrow failure disorders.
FLT3 Is Fused to ETV6 in a Myeloproliferative Disorder with a t(12;13)(p13;q12) Translocation.
Authors: Vu Hoang Anh , Xinh Phan Thi , Shimizu Seiko , Masuda Michihiko , Motoji Toshiko , Tokunaga Katsushi , Sato Yuko
Published: April 2020

The FLT3, located at band 13q12 and encoding a receptor tyrosine kinase (RTK), is one of the most frequently mutated genes in hematologic malignancies including ALL, MDS, and AML. The most common mutation of the FLT3 is an internal tandem duplication in exons 14 and 15, whereas other mutations have also been found at and around codon 835 of exon 20. These activating mutations promote constitutive RTK activity in the absence of ligand, proposing FLT3 as an attractive therapeutic target for directed inhibition. However, many questions with regards to the biology of FLT3 and its role in leukemogenesis remain to be clarified. Despite its highly frequent mutations, FLT3 has never been reported to fuse to any other genes, a phenomenon usually observed in other RTKs. Here, we report a case of a novel fusion gene between FLT3 and ETV6 at 12p13, a well-known target for a number of translocations. The patient, a 68-year-old female, was diagnosed as myeloproliferative disorder with hypereosinophilia in May 2002. Peripheral blood showed WBC 33.6x106/L (3% myelocytes, 33.5% neutrophils, 54% eosinophils, 1.5% basophils, 1.0% monocytes and 7% lymphocytes), Hb 119g/L and platelet counts 5,450x106/L. The bone marrow (BM) was marked hypercellular with 0.9% blasts, 6.0% promyelocytes, 15.6% myelocytes, 8.1% immature eosinophils and 19.2% mature eosinophils. Karyotype of BM cells was 46, XX, t(12;13)(p13.1;q12.3–13)[28]/46, XX[2]. Under the suspicion of Ph-negative CML, she was treated with IFNα with no response. Then, HU was started and her WBC decreased to 30x106/L. FISH analysis showed that the breakpoint at 12p13 occurred within ETV6, while the breakpoints at 13q12 occurred at two locations, within FLT3 or CDX2. To identify the fusion partner of ETV6, 3′-RACE PCR was performed. Sequence analysis of PCR-products revealed 4 types of ETV6/FLT3 transcripts. These fusion transcripts were confirmed by Northern blot analysis. Each ETV6/FLT3 transcript contained the entire helix-loop-helix domain of ETV6 (exons 1 to 4 or 5) and almost all of the functional domains of FLT3 including the tyrosine kinase domain (from exons 14, 16 or 17), suggesting that the resultant chimeric protein would be constitutively activated FLT3 kinase. Of them, three are in-frame fusion, presumably encoding for the approximately 58, 62, and 83 kD fusion proteins. However, Western blot analysis showed only expression of the 58 and 83 kD proteins. RT-PCR detected the reciprocal FLT3/ETV6 transcript, comprising the FLT3 exons 1 to 13 frameshifly fused to the ETV6 exons 6 to 8, within which a stop codon appeared at codon 33- downstream from the fusion point. Functional studies to assess the oncogenic properties of these fusion proteins are now in progress. Our findings provide an evidence that FLT3 is also involved in hematologic malignancies as a fusion gene.
A “Single” Dose of G-CSF after Autologous Stem Cell Transplantation in Patients with Malignant Lymphoma - Third Interim Analysis of Multicentre Randomized Trial Comparing Standard Schedule with Delayed Application and Placebo.
Authors: Faber Edgar , Pytlik Robert , Trneny Marek , Slaby Jiri , Kozak Tomas , Raida Ludek , Papajik Tomas , Zikesova Eva , Zapletalova Jana , Maresova Ivana , Hamouzova Michaela , Indrak Karel
Published: April 2020

The optimal schedule of G-CSF given after autologous peripheral stem cell transplantation (ASCT) has not been defined yet. Here we present results of the third interim analysis of randomized multicentre trial comparing standard application of G-CSF from day +5 (arm A) with delayed dosing (G-CSF was started when WBC reached 0,5x109/l and neutrophil count 0,1x109/l; arm B) and placebo (arm C). In order to eliminate the influence of diagnosis and conditioning and to minimize the impact of graft quality only patients with malignant lymphoma conditioned by BEAM and with graft harvested in maximum 3 aphereses containing at least 5,0x106 CD 34+cells/kg were included. Patients signed informed consent before registration into the study that had been approved by local ethical and national health care authorities. 115 patients at median age 45 (range 21 – 64) were randomized by July 15th, 2004. The data of 97 patients were analysed. There was no difference in age, lymphoma bone marrow involvement at diagnosis, number of chemotherapy cycles and the use of radiotherapy before transplantation and CD 34+ cells content in graft between any two of the three arms. Duration of neutropenia below 0,5x109/l and 1,0x109/l and number of days to neutrophil engraftment over 0,5x109/l and 1,0x109/l are listed in the table. A significant difference in duration of neutropenia and time to neutrophil engraftment was found between the arms A and B (p=0,002 – 0,005), the arms A and C (p<0,0001) and also between the arms B and C (p=0,04–0,0001). A trend for delayed platelet engraftment was observed in both arms with G-CSF (A and B). Duration of hospitalisation was significantly longer in placebo group (arm C), however, there was no prolongation of fever or antibiotic use. The delayed „single” application of G-CSF (usually with one or two doses sufficient for full neutrophil recovery) appears to be safe and cost effective. Comparing with standard G-CSF schedule only one day delay in median neutrophil engraftment was observed. We hope to finish the study within the year 2004. Supported by the grant MSN 15110004.

Arm A / G-CSF from day +5 (n=33) Arm B / Tailored initiation of G-CSF (n=32) Arm C / Placebo (n=32) * median(range) Days with neutropenia <0,5x109/l (days) 7 (4–10)* 8 (6–11)* 10 (6–30)* Days with neutropenia <1,0x109/l (days) 8 (5–15)* 9 (7–11)* 11,5 (6–30)* Neutrophil engraftment >0,5x109/l (day +) 10 (9–12)* 11 (9–15)* 14 (9–30)* Neutrophil engraftment >1,0x109/l (day +) 11 (9–22)* 12 (9–15)* 15 (10–30)* Platelet engraftment >20x109/l (day +) 12 (2–33)* 12 (7–30)* 11 (7–24)* Platelet engraftment >50x109/l (day +) 17,5 (8–100)* 16,5 (9–115)* 13 (8–32)*
Insertion Sites of Retroviral Gene-Marking Vectors Influence the Contribution of Individual, Non-Malignant Cell Clones to Long-Term Hematopoiesis.
Authors: Kustikova Olga , Li Zhixiong , Baum Christopher , Fehse Boris
Published: April 2020

Replication-defective gammaretroviral vectors have successfully been used for gene marking and therapy studies. Recently, we and others have shown that proto-oncogene upregulation due to retroviral vector insertion may trigger malignant transformation of engineered cells. We have now investigated whether insertional mutagenesis may also influence the contribution of individual cell clones to normal long-term hematopoiesis. Therefore, using LM-PCR we retrieved retroviral insertion sites from hematopoietic cells of mice exhibiting progression to oligoclonal or monoclonal hematopoiesis upon serial bone marrow transplantation. From 28 mice analyzed (12 primary, 16 secondary) we obtained a total of 67 insertions that matched the murine genome. In samples harvested from primary recipients seven months after transplantation of gene-marked cells a number of intriguing hits (HoxB4, Evi1, Ly78, Ccnd3, Pip5k2a) was found obviously already reflecting selection for long-term repopulating ability. Strikingly, almost all clones analyzed five months after serial transplantation revealed hits in genes with an established or potential role in the self-renewal process of hematopoietic stem cells (e.g., Vegfa, HoxB4, CyclinD3, Evi1) or other genes involved in the regulation of cell survival (Ly78, Pip5k2a, Irf2bp, Cflar). Two (Evi1, Ccnd3) among the 13 insertions associated with serial repopulation activity were already established common integration sites (CIS) in the context of mouse leukemias elicited by replication-competent retroviruses (RTCGD database). Using RT-PCR transcriptional activation involving fusion transcripts originating from the retroviral LTR into exons could be detected for the two genes (HoxB4, Vegfa) tested. Interestingly, Evi1 was the only gene targeted several times - with distinct insertions in 5 different clones. However, despite the fact that Evi1 was also the only established proto-oncogene found to be marked, four of the clones detected in primary recipients were lost upon serial transplantation. This indicates that insertion into the Evi1 locus, although having the potential to promote engraftment and/or survival of a given clone, was not sufficient for malignant transformation. Thus our data demonstrate a delicate regulatory balance following insertional mutagenesis by replication-deficient retroviral vectors. These findings have major implications for interpreting results from diagnostic gene marking experiments, for gene therapy, and the discovery of genes regulating stem cell turnover.

(The first two authors contributed equally to this work)
Breakpoint-Specific Targeting of p210BCR-ABL mRNA by siRNA Alters Progenitor Cell Proliferative Rate in CML.
Authors: Marley Stephen B. , Withey Jane , Harvey Amanda , Crompton Mark , Gordon Myrtle
Published: April 2020

Abrogating expression of p210BCR-ABL or of associated cell-signalling proteins in CML through siRNA-targeted destruction of mRNA has enormous potential in CML, both as a research tool and as a potential therapy, especially for imatinib resistant disease. We have investigated a p210BCR-ABL junction-specific (b3a2) siRNA. We used an Oligofectamine transfection protocol which gives high transfection rates with minimal toxicity. In the Bcr-Abl-positive cell line, K562, this siRNA reduced p210BCR-ABL expression by up to 80% compared with actin controls and induced significant levels of apoptosis. Silencing of p210BCR-ABL has been reported by Scherr et al (Blood 2003, 101:1566–69) not to induce apoptosis in primary Ph+ cells. Similarly, we showed that the initial CFU-GM plating efficiency from b3a2 CML peripheral bloods treated with breakpoint-specific siRNA was not significantly reduced (100+/−5% of untreated controls). Replating large numbers of individual CFU-GM and analysing the distribution of secondary colonies can quantify the proliferative capacity of the myeloid progenitor pool. Using this assay, we showed that siRNA transfection significantly reduced the proliferative capacity of CML CFU-GM in a breakpoint-specific fashion (56+/−18% of untreated controls). This reduction in proliferative capacity is very similar to the CML-specific reduction in proliferative capacity induced by Interferon-alpha (50+/−40% of untreated controls) or by imatinib mesylate (50+/−30% of untreated controls). To our knowledge, this is the first demonstration of a functional consequence of siRNA treatment of primary CML haemopoietic cells. Our findings support the view that siRNA-based therapies may ultimately be of benefit, particularly in the management of CML
Centrosome Aberrations in Chronic Myeloid Leukemia (CML) Are Correlated with Disease Progression and Chromosome Instability.
Authors: Giehl Michelle , Fabarius Alice , Zheng Chun , Frank Oliver , Hochhaus Andreas , Hafner Mathias , Hehlmann Rudiger , Seifarth Wolfgang
Published: April 2020

Purpose: Numerical and structural centrosome abnormalities are hallmarks of a variety of cancers and have been implicated in chromosome missegregation, chromosomal instability, and aneuploidy. These phenomena already occur in preneoplastic lesions like oral leukoplakia, early cervical neoplasias, and small benign tumors of colon and breast. Moreover, deviations from normal karyotype seem to increase as tumors enlarge and become malignant. Genetic instability is a common feature in chronic myeloid leukemia (CML). We sought to establish a relationship between centrosome abnormalities and cytogenetic aberrations in CD34+ cells from CML patients at diagnosis (chronic phase - CP) and in blast crisis (BC).

Methods: Diagnosis of CML was established by hematologic, cytogenetic and molecular parameters. Treatment was performed according to the protocols of the German CML study group (www.kompetenznetz-leukaemie.de). CD34+ cells from ten umbilical cord blood specimens served as negative controls. Centrosome number and morphology were analyzed by immunofluorescence microscopy. In brief, CD34+ cells from ficollized peripheral blood samples were concentrated by magnetic cell sorting (MACS) and cytospun onto coated slides. After methanol fixation cells were incubated with antibodies directed to centrosomal proteins Pericentrin and gamma-Tubulin. Antibody-antigen complexes were stained by incubation with FITC- and Cy3-conjugated secondary antibodies.

Results: CML CP samples tested at initial diagnosis (n=20) already displayed numerical and structural centrosome aberrations (30.0% +/−2.3) as compared with corresponding normal control cells (n=10) (2.3% +/−1.1). In BC samples (n=10) an increase of centrosome aberrations was observed (58.0% +/−2.0).

Conclusion: The findings suggest that centrosome defects in CML occur early and are already present at primary diagnosis. Centrosome defects may contribute to disease progression by generation of further chromosome instability leading to accumulation of alleles carrying pro-oncogenic mutations and loss of alleles containing normal tumor suppressor genes and thus accelerating complex genomic changes associated with CML BC.
Loss of IRF-4 Exacerbates the CML-Like Phenotype of IRF-8 Knockout Mice.
Authors: Schatz Jonathan , Ren Ruibao , Singh Harinder
Published: April 2020

IRF-4 and IRF-8 are related interferon regulatory factors that are required for the development and function of myeloid and lymphoid cells. Mice deficient in IRF-8 develop a myeloproliferative disorder with many similarities to human chronic myeloid leukemia (CML). Our group has shown that in B-lymphocyte development, IRF-4 and 8 function redundantly to control the pre-B to B transition. Though expression of IRF-4 has been demonstrated in myeloid cells, its role in these lineages has not been defined. We hypothesized that, similar to their role in B-cells, IRF-4 and 8 may to some extent function redundantly in myeloid cells. To test this hypothesis, myelopoiesis was analyzed in IRF-4/8 double knockout (DKO) mice. We found that from seven weeks of age, the DKO animals have a more aggressive leukemic phenotype than the IRF-8 KO mice. The white blood cell (WBC) counts of DKO animals were typically in the range of 40,000 to 80,000 cells per microliter, campared to 15,000 to 20,000 for age-matched IRF-8 KO animals. FACS analysis showed that the increased WBCs in the DKO animals was due to a massive expansion of mature granulcytes. The absolute granulocyte counts of DKO animals were typically 7-fold to 10-fold higher than those of IRF-8 KO animals. In addition, histologic preparations showed that by 15 weeks of age the spleens, lymph nodes, and bone marrow of DKO animals were invaded by large numbers of mature granulocytes and pseudo-Gaucher cells, with complete effacement of the normal micro-architecture. Age-matched IRF-8 KO animals showed invasion to a lesser degree, with preservation of many of the normal architectural features. In conclusion, IRF-4 is an important myeloid tumor suppressor, whose loss augments the myeloproliferative disease manifested in IRF-8 deficient mice. This suggests the two transcription factors, similar to their role in B-cell development, function redundantly to control the normal maturation of myeloid-lineage cells.
Intrinsic Regulation of the Interactions between the SH3 Domain of p85 Subunit of Phosphatidylinositol-3 Kinase and BCR/ABL Oncogenic Tyrosine Kinase.
Authors: Ren Shuyue , Xue Fan , Feng Jan , Skorski Tomasz
Published: April 2020

BCR/ABL fusion tyrosine kinase is responsible for the initiation and maintenance of the Philadelphia chromosome (Ph1)-positive chronic myelogenous leukemia (CML) and a cohort of acute lymphocytic leukemias (ALL). Our previous studies showed that a signaling protein phosphatidylinositol-3 kinase (PI-3k) is essential for the growth of CML cells, but not of normal hematopoietic cells, and that p85 subunit of PI-3k co-immunoprecipitates with BCR/ABL (Skorski et al., (1995) Blood 86, 726–36; Skorski et al., (1997) Embo J 16, 6151–61; Klejman et al., (2002) Oncogene 21, 5868–76). Therefore, we made an attempt to better characterize the p85 - BCR/ABL interactions. Here we show that SH3 domain of p85 (p85-SH3) pulls-down the p210BCR/ABL kinase from hematopoietic cell lysates. In addition, we characterize the p85-SH3 mutants, which abrogate or enhance this interaction. The results of pull-down assays of the p85-SH3 mutants seem to support the assumption that p85-SH3 interacts with the BCR/ABL protein network via the proline-rich (PxxP) region. One of the surprising findings was the enhanced binding affinity of the tyrosine to phenylalanine p85-SH3 domain mutants in comparison to the wild-type p85-SH3. Based on these results we speculate on the capability of p85-SH3 to interact with BCR/ABL and on the p85-SH3 conformational requirements necessary for this reaction. PxxP - binding appears to be required for the interaction of p85-SH3 with BCR/ABL protein complex and activation of the catalytic activity of PI-3k, whereas subsequent BCR/ABL-dependent phosphorylation of the tyrosines may facilitate the release of activated PI-3k from the complex.
Cooperative Anti-Leukemic Effects of Imatinib and Mcl-1 Antisense: Identification of Mcl-1 as a Novel Target in CML.
Authors: Aichberger Karl J. , Mayerhofer Matthias , Krauth Maria-Theresa , Skvara Hans , Florian Stefan , Sonneck Karoline , Akgul Cahit , Derdak Sophia , Pickl Winfried F. , Wacheck Volker , Selzer Edgar , Monia Brett P. , Moriggl Richard , Valent Peter , Sillaber Christian
Published: April 2020

The BCR-ABL tyrosine kinase inhibitor STI571 (Imatinib) has successfully been introduced in the treatment of patients with CML. However, despite encouraging initial data and high expectations long term results are not available yet, and recent data suggest that resistance against STI571 can occur. Therefore, current studies focus on novel potential drug targets in CML cells. Recently, several anti-apoptotic members of the Bcl-2-family including Mcl-1 have been implicated in the regulation of survival of BCR/ABL+ cell lines and therefore proposed as potential targets. We have examined expression of Mcl-1 in primary CML cells and various BCR/ABL-transformed cell lines. Independent of the phase of disease, isolated primary CML cells expressed Mcl-1 mRNA and the Mcl-1 protein in a constitutive manner. The BCR/ABL-inhibitor STI571 decreased the expression of Mcl-1 in these cells. Correspondingly, BCR/ABL enhanced Mcl-1 promoter activity, Mcl-1 mRNA, and Mcl-1 protein in Ba/F3 cells. BCR/ABL-dependent expression of Mcl-1 in Ba/F3 cells was counteracted by the MEK-inhibitor PD98059, but not by the PI3-kinase inhibitor LY294002. Identical results were obtained for constitutive expression of Mcl-1 in primary CML cells and the CML-derived cell lines K562 and KU812. To investigate the role of Mcl-1 as a survival-related target gene in CML cells, Mcl-1 siRNA or an Mcl-1 antisense oligonucleotide (ASO) were applied. Both the ASO and the siRNA-induced downregulation of Mcl-1 were found to be associated with a substantial decrease in viability of K562 cells. Moreover, the Mcl-1 ASO was found to cooperate with STI571 in producing growth-inhibition in both STI571-sensitive and STI571-resistant K562 cells. Together, our data identify Mcl-1 as a survival factor and novel target in CML. Whether this concept is of clinical significance remains to be determined in forthcoming clinical trials.
CEBPα Mutations in Childhood Acute Myeloid Leukemia.
Authors: Liang Der-Cherng , Shih Lee-Yung , Huang Chein-Fuang , Hung Iou-Jih , Yang Chao-Ping , Liu Hsi-Che , Jaing Tang-Her , Wang Lin-Yen , Chang Wan-Hui
Published: April 2020

Transcription factor CCAAT/enhancer binding protein alpha(C/EBPα) is essential for granulocyte differentiation. CEBPα mutations have been described in 7–10% of adult patients with acute myeloid leukemia (AML) at initial diagnosis and conferred a favorable prognosis. CEBPα mutations in childhood AML has not been reported yet. In this study, we sought to assess the frequency of CEBPα mutration, its clinicohematologic correlation and to analyze the cooperating mutations, including FLT3 and N-ras mutations in childhood AML. CEBPα mutations were analyzed in 117 children (age one day to 17 years, median 5 years) with de novo AML. CEBPα mutation status was examined by performing DNA polymerase chain reaction (PCR) followed by direct sequencing for each PCR product. CEBPα mutations were detected in 7 (6.0%) of 117 patients. Of the 7 patients with CEBPα mutations, 4 had FAB subtype of M2, 2 M1 and 1 M4. All had intermediate cytogenetics and comprised 11.5% (7/61) of the intermediate cytogenetic risk group. Five of the 7 mutations occurred in both N-terminal part and bZIP domain, one had N-terminal frameshift mutation and the remaining one had an inframe insertion in the bZIP domain. Clonal analysis using expand long template PCR assay with a primer pair covering entire coding sequence was performed in all 5 patients carrying two different mutations. The results demonstrated that one had homozygous combined mutation and 4 had heterozygous biallelic mutations with the cloned alleles carrying single mutations at N-terminal or bZIP domain in the different alleles or harboring combined mutations in the same alleles. None of the remission marrow samples obtained from 5 patients who had CEBPα mutations at diagnosis carried the mutations. FLT3-ITD mutations were detected in 2 of the 7 patients with CEBPα mutations compared to 15 of the 109 patients without CEBPα mutations. None of the patients carrying CEBPα mutations had FLT3-TKD mutations. Two of the 7 CEBPα(+) patients also harbored N-ras mutations compared to 10 of the 110 patients without CEBPα mutations. Taken together, 4 of 7 CEBPα(+) patients had cooperating mutations including FLT3-ITD or N-ras as compared to 27 in 109 CEBPα(−) patients (P=0.081). There were no statistical differences in age, WBC count, LDH level, 5-year overall survival and event-free survival between patients with and without CEBPα mutations; similar results were obtained if we only analyzed patients with intermediate cytogenetics. The present data showed that the frequency of CEBPα mutations in our series of childhood AML was similar to that in adults. CEBPα mutations were frequently associated with FLT3-ITD or N-ras mutations supporting the two-hit hypothesis for the pathogenesis of AML.
Internal Tandem Duplication and Kinase Domain Mutation of FLT3 in Pediatric Acute Myeloid Leukemia Treated on Japan Cooperative AML 99 Protocol.
Authors: Shimada Akira , Taketani Takeshi , Taki Tomohiko , Tabuchi Ken , Hanada Ryoji , Tawa Akio , Tsuchida Masahiro , Horibe Keizo , Tsukimoto Ichiro , Hayashi Yasuhide
Published: April 2020

FLT3 is a type III receptor tyrosine kinase and this kinase mutation is most frequently observed in cases of acute myeloid leukemia (AML). Approximately 30% of adult AML patients have internal tandem duplication (ITD) of the juxtamembrane domain or point mutation of kinase domain (D835Mt) leading to the constitutive activation of downstream signaling pathways and aberrant cell growth. We examined FLT3-ITD and D835Mt in 160 pediatric AML patients treated on Japan pediatric AML cooperative treatment protocol, AML 99. This protocol is consisted of intensification of multi-drug chemotherapy and early application of stem cell transplantation (SCT) for the high risk patients (monosomy 7, Ph1, or delayed remission etc.). FLT3-ITD was found in 22 of 160 (13.8%) patients, including 5 of 15 (33.3%) patients with FAB-M3, 5 of 25 (20.0%) with M5, and 4 of 24 (16.7%) with M1. No specific chromosomal abnormalities were associated with ITD; 9 of 17 (52.9%) patients with ITD showed normal karyotype except for t(15;17). The D835Mt was found in 12 of 160 (7.5%) patients, including 4 of 15 (26.7%) patients with M3. The initial WBC count and the age at diagnosis in ITD or D835Mt patients were significantly higher than those in patients with wild type (WT) of FLT3 (p< 0.05). Except for M3, 12 of 17 (70.6%) patients with ITD received SCT (3 patients in 1st CR, 1 patient in 2nd CR, 3 patients in 1st relapse, although 4 patients in non-CR). Eight patients showed early relapse after SCT and died. No patients with D835Mt showed the induction failure or relapse. In M3, only one patient with FLT3-WT died in early induction phase. The overall survivals (OS) for patients with ITD, D835Mt and WT by Kaplan-Meier method were 33%, 100% and 80%, respectively (p<0.05). These results suggest that patients with ITD are associated with poor outcome despite of receiving SCT, while those with D835Mt are associated with good outcome.
MLL/AF9 Fusion Transcript Quantification in t(9;11)-Positive AML Identify Patients with High Risk of Relapse.
Authors: Scholl Claudia , Eiwen Karina , Frohling Stefan , Schlenk Richard F. , Dohner Hartmut , Dohner Konstanze
Published: April 2020

Detection of minimal residual disease (MRD) in acute myeloid leukemia (AML) with specific gene fusions is an important tool for the assessment of response to treatment and the individual risk of relapse. We evaluated the predictive value of MLL/AF9 fusion-transcript quantification using real-time RT-PCR for disease relapse and verified the results with nested RT-PCR. A t(9;11) was identified in 27 younger AML patients (16–60 years) entered into the multicenter trials AML HD93 and AML HD98-A of the AML Study Group Ulm. One hundred seventy-eight samples (bone marrow [BM], n=84; peripheral blood [PB], n=79; leukapheresis product [LP], n=15) from 22 patients were available for real-time RT-PCR analysis. In 6 of these 22 patients, only samples from diagnosis were obtained, the remaining 16 patients were analyzed during and after therapy. The sensitivity of the real-time RT-PCR was 10−3 and of the nested RT-PCR 10−4 to 10−5. The MLL/AF9 copy number at diagnosis was not predictive of subsequent clinical outcome. Eight of the 16 evaluable patients became real-time RT-PCR-negative after double induction therapy. Six of these 8 patients are in continuous complete remission (CR), 1 died in CR from graft-versus-host disease, and 1 relapsed 11 months after diagnosis. Eight of 16 patients had detectable fusion-transcript levels after achieving complete hematologic remission. Six of these 8 patients relapsed after a median of 8.6 months (range, 3.6 to 13.5) from diagnosis, one patient died in CR 3 weeks after autologous BM transplantation, and one patient underwent allogeneic BM transplantation and is in continuous CR 16 months after diagnosis. Of 3 patients, who relapsed, we were able to analyze bone marrow 1 months before hematologic relapse occurred. Two of these 3 patients were real-time RT-PCR-negative after therapy and became positive 1 month before relapse, 1 patient was still real-time RT-PCR-positive after therapy and had increased MLL/AF9 copy numbers 1 month before relapse occurred. Five Patients, who were in continuous CR, were real-time RT-PCR-negative in all analyzed samples during follow up (median, 56 months; range, 13.8 to 68.9 months). One hundred seventy-two patient samples were examined by nested RT-PCR. Real-time RT-PCR-negative samples were also negative with nested RT-PCR, therefore nested RT-PCR did not enhance sensitivity of real-time PCR. We conclude that real-time RT-PCR is convenient for the detection of MRD in t(9;11)-positive AML and real-time RT-PCR positivity in at least one sample during therapy correlates with a high risk of relapse.