bluuebunny/crossref_metadata_2025 huggingface dataset:
Tyrosine Kinase Inhibitors Dasatinib, Nilotinib and Imatinib Have an Impact on Both CD8+ T Lymphocytes and CD4+CD25+FoxP3+ Regulatory T Cells by Downregulation of the NF-κB Pathway.
Authors: Fei Fei , Yu Yingzhe , Schmitt Anita , Chen Jinfei , Chen Baoan , Rojewski Markus , Ringhoffer Mark , von Harsdorf Stefanie , Greiner Jochen , Goetz Marlies , Guillaume Philippe , Dohner Hartmut , Bunjes Donald , Schmitt Michael
Published: April 2020

Background: For CML/ALL patients with refractory disease or at relapse after allogeneic stem cell transplantation (allo-PBSCT), one might administer tyrosine kinase inhibitors (TKIs) like imatinib (Glivec), nilotinib (Tasigna) and dasatinib (Sprycel), or donor lymphocyte infusions (DLIs). TKIs will inhibit the proliferation of CML progenitor cells, but might also hamper the graft-versus-leukemia (GVL) effect considered to be crucial for the eradication of the disease. Moreover, CD8+ T cells specific for cytomegalovirus (CMVpp65) might be impaired.

Methods: Imatinib, nilotinib and dasatinib were added at concentrations of 0–20 μM, 0–4 μM and 0–50 nM respectively to proliferation assays of Tregs and CD8+ T cells. Mixed lymphocyte peptide cultures (MLPCs) were performed with peptides derived from

influenza matrix protein (IMP), CMVpp65 as a viral antigen and the receptor for hyaluronic acid mediated motility (RHAMM-R3) as a leukemia-associated antigen.

CD8+ T cells from these MLPCs from healthy donors and patients with CML after allo-PSCT by tetramer staining/multi-color flow cytometry and enzyme linked immunosorbent spot (ELISPOT) assays, as well as Tregs after 3 days of culture with anti-CD3 and anti-CD28. Activity of T cells from the peripheral blood of patients under dasatinib and after withdrawal of dasatinib. Western blots (WBs) for T cell receptor (TCR) related molecules and NFkB were performed.

Results: The release of interferon gamma and granzyme B by CD8+ HLA-A2/tetramer+effector T cells specific for IMP, CMVpp65 and RHAMM-R3 was inhibited by all TKIs in a dose-dependent fashion and correlating to the time of TKI exposure. The inhibition was reversible after removal of the drugs from the MLPC. The proliferation and function of Tregs were also significantly inhibited by TKIs in a dose-related fashion. In WBs, TKIs decreased the expression of ZAP70, Lck and Akt, as well as NFκB p65/p100/p105 and c-Rel. T cell activity was impaired when TKIs were clinically administered. The potency of T cell inhibition was imatinib: nilotinib: dasatinib = 1:2:40 under therapeutical serum respectively culture medium levels (2 μM:1 μM: 25 nM).

Conclusion: When administering TKIs to patients after allo-PBSCT, the inhibition of both CD8+ and Tregs must be taken into consideration with respect to GVL and anti-viral T cell response. Inhibition of CD8+ T lymphocytes was partially reversible after removal of TKIs from the cultures. Using western blotting analysis, we found that these effects are mediated at least in part by down-regulating the levels of phosphorylation of TCR and NF-κB family members.
Frequent CD7 Antigen Loss in Aggressive Natural Killer-Cell Leukemia Can Be a Useful Diagnostic Marker.
Authors: Kim Sun-Hee , Yoo Eun-Hyung , Kim Hee-Jin , Kim Won Seog
Published: April 2020

Aggressive natural killer-cell leukemia (ANKL) is a rare neoplasm characterized by systemic proliferation of NK cells with rapidly progressive clinical course and fatal outcome. Because of the aggressive clinical course, rapid and accurate diagnosis of ANKL is critical. However, the differential diagnosis of NK cell lymphoproliferative disorders including hemophagocytic lymphohistiocytosis is still challenging in the absence of a distinct diagnostic hallmark. Furthermore, cases with a low burden of malignant cell polpuation makes it more difficult. To find any diagnostic markers in ANKL, we analyzed clinical data and laboratory findings from bone marrow studies in Korean patients with bone marrow involvement of ANKL. From January 2000 to July 2007, a total of 20 cases were diagnosed with ANKL based on morphologic and immunophenotypic findings from bone marrow studies. The leukemic cells were surface CD3–CD16/56+ large granular lymphocytes with pale or lightly basophilic cytoplasm containing azurophilic granules. We retrospectively analyzed clinical features and laboratory findings including complete blood count (CBC), Epstein-Barr virus (EBV) status, serum lactate dehydrogenase (LDH) level, immunophenotype, and cytogenetic results from medical records. There were 6 (30%) women and 14 (70%) men with a median age of 44 years (range, 2–70 years). Hepatomegaly (70%), splenomegaly (60%), and lymphadenopathy (30%) were frequently observed. Peripheral blood counts were variable; anemia (hemoglobin <10g/dL) was predominant in 14 patients and thrombocytopenia (platelet <100×109/L) in 16. The proportion of leukemic NK cells ranged 3∼70%. EBV was detected in 15 of 18 cases (83%) by EBV in situ hybridization or EBV quantitative PCR. Cytogenetic studies were performed in 18 cases, and karyotypic abnormalities were observed in 50% (9/18). There were no recurrent cytogenetic abnormalities, except 6q abnormalities observed in 4 cases (4/18, 22%). The immunophenotype of the leukemic NK cells by flow cytometry was cytoplasmic CD3+, surface CD3−, CD16/56+, CD2+, and CD5−. Most cases were CD4− (13/16, 81%) and CD8− (11/14, 79%). Of note, loss of CD7 antigen was observed in 10 patients (10/20; 50%) (normal NK cells: CD2+, CD7+, and CD5−). There were no significant differences in clinical or laboratory parameters between the CD7+ and CD7− groups. All three cases with deletion of 6q revealed absent expression of CD7. When the CD7 loss was combined with cytogenetic abnormalities, clonal markers could be identified in 75% of ANKL cases. We observed frequent CD7 antigen loss in our series of Korean patients with ANKL. This characteristic immunophenotypic finding can provide a reliable and timely information as a diagnostic marker in ANKL along with cytogenetic findings. Therefore, immunophenotypic analysis of the expression of CD7 should be included in the diagnostic workup of NK cell neoplasms.
The AML1/ETO Target LAT2 Membrane Adaptor Molecule Is Regulated during Normal Monocytic Differentiation.
Authors: Duque-Afonso Jesus , Solari Leticia , Luebbert Michael
Published: April 2020

LAT2 (NTAL/LAB/WBSCR5) is a 28 KDa membrane protein which acts as adaptor molecule in the signalling pathways of FcεR I, c-Kit, B cell and T cell receptor. Bone marrow-derived mast cells from knock-out (KO) mice are hyperresponsive to stimulation via FcεR I. Although LAT2 is highly expressed in B cells, no major changes were found in function or development of B cells from LAT2 KO mice. An autoimmunity syndrome in LAT2 KO mice is caused, at least in part, by hyperreactivity and higher proliferation of T cells. Previously, we showed that LAT2 mRNA is repressed in vivo by AML1/ETO which was confirmed by others in several large series of primary AML blasts. We wished to elucidate the possible role of LAT2 during the myelopoiesis. AML1/ETO was induced by Ponasterone A in an ecdysone-inducible system in U937 cells (9/14/18 cell line). AML bone marrow samples from 43 patients (pts) were analyzed for LAT2 expression. Several myeloid cell lines were treated either with ATRA, DMSO or PMA for 3 days. Normal CD34+ cells were differentiated ex vivo by G-CSF towards granulocytes and by GM-CSF plus IL-4 towards monocytes and dendritic cells. LAT2 expression was determined by Northern and Western blot. LAT2 protein was repressed not only in AML1/ETO positive primary AML blasts (6/6), but also in blasts from patients with deletions of chromosome 7 (3/4) and the t(15;17) (4/4); expression was moderate to high in AML blasts with normal karyotype (14/15). LAT2 was expressed in normal monocytes and even higher in alveolar macrophages but not in granulocytes of healthy donors. It was downregulated after ATRA-induced granulocytic differentiation of NB4, HL60 and U937 cells but upregulated after DMSO-induced granulocytic differentiation of HL60 cells and PMA-induced monocytic-macrophage differentiation of HL60, U937 and Kasumi-1 cells. In normal CD34+ cells, LAT2 was strongly induced 7 days after the addition of G-CSF and GM-CSF+IL4 respectively, but after 14 days it was downregulated (0.7 +/− 0.4-fold) by G-CSF-induced granulocytic differentiation and upregulated (5.8 +/− 2.8-fold) by GM-CSF+IL4-induced monocytic-DC differentiation. Conditional expression of AML1/ETO in 9/14/18-U937 cells partially inhibited the PMA- and vitamin D3-induced monocytic differentiation of these cells, as determined by FACS for CD11b and CD11c. In conclusion, LAT2 protein is strongly repressed by AML1/ETO in primary leukemias and is upregulated during the monocytic differentiation in several cell lines and normal CD34+ cells. Further studies in a LAT2 knock-down by shRNAs in U937 cells are warranted to functionally address its possible role in monocytic differentiation.
The Identification and Characterization of Zebrafish Mast Cells.
Authors: Berman Jason N. , Seibert Jake , Dobson Tristan , Teh Eve , Hrytsenko Olga , Pohajdak Bill , Fraser Robert , Paw Barry , Lin Tong-jun
Published: April 2020

Mast cells (MCs), tissue counterparts to mammalian basophils, are known for their role in allergic reactions, inflammation and cancer progression, yet their developmental origin remains controversial due to limitations studying these cells in traditional animal models. The zebrafish provides a highly efficient system for studying vertebrate MC development. All other major hematopoietic lineages have zebrafish counterparts and the fundamental genetic mechanisms controlling hematopoiesis are well conserved. We are the first to identify zebrafish MCs in the gill and intestine. These cells demonstrate classic MC phenotypes including prominent metachromatic granules following staining with toluidine blue and positive immunohistochemical reactions to antibodies against human tryptase and C-KIT. Electron microscopy demonstrates a striking morphologic resemblance to mammalian MCs. Functional studies using the stimulating agent, Compound 48/80 or formalin-killed Aeromonas result in MC degranulation and increased blood levels of key mediators, such as tryptase. These cells also express carboxypeptidase A5 (cpa5), a zebrafish homologue of the human mast cell-specific CPA enzymes. Cpa5 expression in zebrafish embryonic blood cells begins at 24 hours post fertilization and co-localizes with a number of established granulocytic and monocytic markers suggesting that MCs arise from a common granulocyte/monocyte progenitor. Morpholino knockdown studies have demonstrated that the transcription factors gata-2 and pu.1, but not gata-1 are necessary for early MC development. Interestingly, friend of gata-1 (fog-1) may also be required, but in a gata-1 dependent manner. Ongoing morpholino and mutant rescue studies will further establish in vivo the contribution that these transcription factors make to vertebrate MC development. Finally, we have cloned a cpa5 promoter element and shown it can drive expression of the green fluorescent protein in early zebrafish MCs providing a means for generating transgenic zebrafish lines to model human MC diseases for use in high throughput small molecule modifier screens.
Kinase Suppressor of Ras Plays a Critical Role in Modulating Inflammatory Mast Cell Functions.
Authors: Chen Meng , Horn Whitney , Li Xiaohong , Knowles Scott , Ingram David , Chen Shi , Yang Feng-Chun , Clapp D. Wade
Published: April 2020

The Raf/MEK/extracellular signal-regulated kinase (ERK) kinase cascade and the Ras-PI3-K-Akt pathways are intricately regulated and evolutionarily conserved pathways that have been implicated in specialized cellular functions including proliferation, differentiation, survival and degranulation. Recent data suggest that the strength and duration of these signals is maintained by extracellular growth factors and integrin stimuli as well as intracellular protein scaffolds. In the present study, we investigated the role of Kinase suppressor of Ras (KSR), a scaffold that appears to regulate both Ras-Erk and Ras-PI3-K activity in influencing mast cell function. In vivo, KSR−/− mice have a 2–3 fold reduction of resident mast cells in multiple organs including the peritoneum and the skin as evaluated by scoring Alcian blue positive cells. To evaluate the mechanistic underpinnings of these in vivo observations, bone marrow derived mast cells (BMMCs) were generated and proliferation, survival, degranulation, and migration was examined. A 3–4 fold reduction in kit-ligand mediated proliferation as measured by [3H]thymidine incorporation was observed in KSR−/− BMMCs as compared to WT BMMCs. In addition, a 50% increase in apoptosis was observed in KSR−/− mast cells as compared to that in WT cells as measured by flow cytometeric analysis using Annexin/PI staining. Given that Erk and Akt are established molecular targets control proliferation and survival, respectively; we next performed western blots to evaluate if the changes in biological activity was associated with these signaling pathways. Importantly, a reduction in phosphorylation of ERK and phosphorylation of AKT was observed in the KSR −/− BMMCs as compared to that in WT BMMCs. Given the role of PI3-K signals in mediating cytoskeletal organization in mast cells, we next tested whether the reduction in PI3-K signals was associated with a reduction in degranulation and migration. Following stimulation with kit-ligand and cross-linking of the IgE receptor, KSR−/− mast cells were found to have a 30–50% decrease in b-hexosaminidase release. Moreover, KSR−/− mast cells have up to a 5 fold reduction in migration to kit-ligand as measured over a range of kit-ligand concentrations. Collectively, the in vivo and in vitro studies suggest that KSR is an important regulatory kinase that may be a viable molecular target for modulating inflammatory mast cell functions.
Flt3 Receptor Inhibition Abolishes Constitutive NFκB Activation in High-Risk Myelodysplastic Syndrome and Acute Myeloid Leukemia.
Authors: Grosjean Jennifer , Ades Lionel , Bohrer Simone , Fenaux Pierre , Kroemer Guido
Published: April 2020

High-risk myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML) are characterized by the constitutive activation of the anti-apoptotic transcription factor NF-kappaB, via the activation of the IKK complex. We show that constitutive activation of the receptor tyrosine kinase Flt3 is responsible for IKK activation and this activation of the NF-kappaB pathway was found to involve a not yet described phosphorylation of the IKK and IkBa complex involving tyrosine residues compared to serine residues in the classical NF-kappaB pathway. Chemical inhibition or knockdown of Flt3 with small interfering RNAs abolished NF-kappaB activation in MDS and AML cell lines, as well as in primary CD34+ bone marrow cells from patients, causing mitochondrial apoptosis. Epistatic analysis involving the simultaneous inhibition of Flt3 and IKK indicated that both kinases act via the same anti-apoptotic pathway. An IKK2 mutant with a constitutive kinase activity and a plasma membrane-tethered mutant of NEMO that activates IKK1/2 prevented the cytocidal action of Flt3 inhibition. IKK2 and Flt3 physically associated in MDS and AML cells and Flt3 inhibition caused the release of IKK2 from a preferential association with the plasma membrane. Flt3 inhibition only killed CD34+ bone marrow cells from high-risk MDS and AML patients, in correlation with the blast numbers and the NF-kappaB activity, yet had no lethal effect on healthy CD34+ cells or cells from low-risk MDS. These results suggest that Flt3 inhibitors might exert an anti-neoplastic effect in high-risk MDS and AML through inhibition of constitutive NF kappaB activation.
Polymorphisms in Detoxification and DNA Repair Genes in Myelodysplastic Syndromes.
Authors: Seedhouse Claire H. , Fischer Stephanie , Germing Ulrich , Russell Nigel , Haase Detlef
Published: April 2020

The maintenance of genetic stability within haematopoietic stem cells is essential for normal haematopoiesis and this is emphasised by the association of leukemias and myelodysplastic syndromes (MDS) with genetic instability. DNA is normally protected from damage via a number of complex pathways including detoxification and DNA repair pathways. Inefficient processing of DNA damage may result in an increased susceptibility to leukemia and MDS. Genetic polymorphisms exist in many genes within the DNA damage processing pathways, some of which affect the cells ability to maintain genetic stability. We have studied polymorphisms in the homologous DNA repair genes RAD51 (RAD51-g135c) and XRCC3 (XRCC3-Thr241Met) and the detoxification gene GSTM1 (deletion polymorphism) in 384 MDS samples. The GSTM1 polymorphism was studied using PCR and the RAD51 and XRCC3 genotypes were assayed simultaneously using a SNaPshot technique. The genotype distributions of RAD51-g135c and GSTM1 did not differ significantly from those reported in the literature. However the distribution of the XRCC3-Thr241Met polymorphism was found to be significantly different, with an over-representation of the variant Met allele, when compared to previously published frequencies in both control and AML populations1 (Pearson Chi-square p=0.001 and 0.016 respectively). The MDS samples were further stratified with respect to FAB groups and cytogenetics. No associations were found between any of the three genetic polymorphisms and FAB type. Interestingly the XRCC3-Met variant genotype was significantly more common in MDS samples with normal karyotypes compared to aberrant karyotypes (Pearson Chi-square p=0.01). Whilst the presence of a single polymorphic variant may display only a subtle effect, polymorphic variants of more than one gene involved in the same pathway are likely to be biologically important with respect to the cellular ability to maintain genetic integrity and hence may play a role in MDS pathogenesis. RAD51, XRCC3 and GSTM1 genotypes were therefore studied in combined analyses. The triple variant genotype (RAD51-135c/XRCC3-241Met/GSTM1-null) occurred in 4.5% of MDS patients compared to 0.6% of healthy controls1 (p=0.01) implicating this genotype in MDS. Collectively these results suggest that polymorphisms in genes which process DNA damage play an important role in MDS pathogenesis, a role which may be more significant in the absence of competing karyotype alterations. 1 Seedhouse C, Faulkner R, Ashraf N, Das-Gupta E, Russell N. Polymorphisms in genes involved in homologous recombination repair interact to increase the risk of developing acute myeloid leukemia. Clin Cancer Res.2004;10:2675–2680.
PI-PLCbeta1 Expression in Patients with High-Risk Myelodysplastic Syndromes Is Affected by Azacitidine Treatment.
Authors: Finelli Carlo , Follo Matilde Y. , Bosi Costanza , Mongiorgi Sara , Clissa Cristina , Martinelli Giovanni , Baccarani Michele , Martelli Alberto M. , Cocco Lucio
Published: April 2020

Phosphoinositide-specific phospholipase C (PI-PLC) beta1 is a key enzyme in nuclear signal transduction, and it is involved in many cellular processes, such as proliferation and differentiation (Manzoli L et al, Prog Lipid Res, 2005). In particular, the involvement of the PI-PLCbeta1 gene in erythroid differentiation lead us to investigate this gene in patients affected by high-risk Myelodysplastic Syndromes (MDS). It is still unclear what is the pathogenesis of the evolution of MDS into Acute Myeloid Leukemia (AML), even if the presence of a mono-allelic and cryptic deletion of the PI-PLCbeta1 gene, as well as an impaired regulation of the PI3K/Akt/mTOR axis, have been recently hypothesized to be implicated in mechanisms related to the disease progression (Lo Vasco VR et al, Leukemia, 2004;Follo MY et al, Cancer Res, 2007). In the present study, we performed a relative quantification, by Real-Time Polymerase Chain Reaction (PCR) analysis, on high-risk MDS patients, at baseline and during treatment with azacitidine. Furthermore, we evaluated the expression of the PI-PLCbeta1 gene on healthy donors and the HL60 cell line, which is useful for testing the accuracy of the technology because of its low expression of PI-PLCbeta1. To analyze and quantify the levels of the two different splicing variants of the PI-PLCbeta1 gene (a and b), we used a TaqMan isoform specific approach. We studied 8 patients with high-risk MDS (IPSS risk high or intermediate-II) treated with azaciditine, 5 of them showing a favourable response to treatment (1 patient: complete remission; 2 patients: partial remission; 2 patients: haematologic improvement). During the treatment with azacitidine, the non responsive patients (3/8) did not show any significant change in the levels of PI-PLCbeta1 mRNAs, whilst all the responders showed a marked increase of the PI-PLCbeta1 mRNA as compared with their baseline amount. Interestingly, the responsive patients showed fluctuations of PI-PLCbeta1 levels that could be related to their clinical status.Our data show a correlation between azacitidine treatment and PI-PLCbeta1 signalling in high-risk MDS, hinting at the likelihood that azacitidine could affect the transcriptional activity of PI-PLCbeta1, which is indeed a key player in the control of cell cycle.
T-Cell Receptor Vß CDR3 Oligoclonality Frequently Occurs in Childhood Refractory Cytopenia and Severe Aplastic Anemia.
Authors: de Vries Andrica C.H. , Langerak Anton W. , Verhaaf Brenda , Niemeyer Charlotte Marie , Stary Jan , Schmiegelow Kjeld , van Wering Elisabeth , Beishuizen Auke , Pieters Rob , Van Den Heuvel M.M.
Published: April 2020

(Very) Severe acquired aplastic anemia ((v)SAA) and myelodysplastic syndrome (MDS) are rare diseases in childhood. (V)SAA is a bone marrow failure syndrome characterized by immune mediated destruction of hematopoietic progenitors. MDS is a malignant clonal stem cell disorder, in which the hypoplastic variant is, in case of absence of a cytogenetic clone, difficult to separate from (v)SAA. Recently, studies provided a “molecular signature” of autoimmunity in adult (v)SAA, by showing oligoclonality of TCR Vß CDR3 region, which is refered to as TCR Vß skewing. We investigated the value of TCR Vß repertoire analysis in pediatric MDS-RC and (v)SAA patients. Peripheral blood and/or bone marrow mononuclear samples of patients with (v)SAA (n=38), MDS-RC (n=28) and 18 controls were analysed with Vß heteroduplex analysis of extracted RNA. Skewing was found in 21/38 (55%) of the (v)SAA patients and in 17/28 (61%) of the RC patients. Seventeen patients with clinical (v)SAA showed no oligoclonality. A significant difference in TCR skewing was found between the (v)SAA + MDS-RC patients as compared to the controls (χ2 analysis, p=0.001), but not between MDS-RC and (v)SAA (χ2 analysis, p=0.8). In this study paired samples (PB and BM) of 25 cases showed a high correlation between the skewing results in both compartments (Pearson correlation, rr 0.98). In this study TCR Vß repertoire analysis did not discriminate between MDS-RC and (v)SAA. Prospective studies will be necessary to investigate whether there is a role for this molecular tool in pediatric MDS-RC for the identification of a subset of patients that is associated with auto-immunity and therfore could be treated with IST up-front, and whether it can be used for molecular response monitoring.
A Complex Karyotype but Not Monosomy 7 Is an Independent Prognostic Factor in Advanced Childhood MDS.
Authors: Gohring Gudrun , Michalova Kyra , Beverloo Berna , Betts David , Harbott Jochen , Haas Oskar , Kerndrup Gitte , Sainati Laura , Bergstraesser Eva , Hasle Henrik , Starý Jan , Trebo Monika , van den Heuvel-Eibrink Marry , Zecca Marco , Fischer Alexandra , Noellke Peter , Locatelli Franco , Schlegelberger Brigitte , Niemeyer Charlotte M.
Published: April 2020

Disclosure: No relevant conflicts of interest to declare.

To study the clinical significance of recurrent chromosome aberrations in childhood MDS, cytogenetic data of 394 consecutive children with refractory cytopenia (RC) (N=215), RAEB (N=141) and RAEB-T (N=38) analyzed in the regional cytogenetic reference centers and registered in the prospective study EWOG-MDS 98 between 1998 and 2005 were evaluated. At diagnosis, a karyotype could be defined in 279/394 patients (pts) (71%). No karyotype was obtained in 16% of pts with RC compared to 8% pts with RAEB and RAEB-t (p<0.001). Clonal chromosome aberrations were more common in pts with advanced MDS (RAEB and RAEB-T, 61%) compared to RC (29%), and in pts with secondary (69%) compared to primary MDS (36%) (p<0.001). Monosomy 7 was the most frequent aberration occurring with similar frequency in RC (47% of abnormal karyotypes) compared to advanced MDS (49%) and in primary (53%) compared to secondary (41%) MDS. In addition, aberrations typical for de novo AML such as aberrations involving 11q23 or 3q, t(6;9) and del(9q) were noted in morphologically and clinically unequivocal MDS cases. Recurrent aberrations of adult MDS like isolated del(5q), del(20q) and -Y were very uncommon indicating a different pathogenesis of these cases. In pts with advanced MDS, there was no significant difference in overall survival (OS) of pts with normal karyotype (44% ± 18) compared to pts with monosomy 7 (58% ± 19) and patients with other karyotypes (61% ± 22). However, pts with advanced MDS and a complex karyotype (defined by ≥ 3 chromosome aberrations, presence of structural aberrations and excluding clonal evolution of monosomy 7) had a shorter OS (16% ±15, p<0.01). OS and event-free survival after hematopoietic stem cell transplantation (HSCT) in pts with complex karyotypes was inferior compared to that of pts with other cytogenetic aberrations (p=0.012 and 0.039, respectively). Within the group of pts with secondary MDS, complex karyotypes were found in MDS evolving from inherited bone marrow failure disorders or after radio-/ chemotherapy, but absent in familial MDS and cases evolving from acquired aplastic anemia. As shown in a multivariate Cox analysis, advanced MDS, secondary MDS, the presence of a complex karyotype and HSCT were identified as independent prognostic factors for OS. Thus, this study demonstrates the prognostic significance of cytogenetic findings in advanced childhood MDS independent of HSCT.
Myelodysplastic Syndrome: A Study of VA Population.
Authors: Matacia-Murphy Gina M. , Al Ali Najla H. , Schrager Jeffrey A. , Beg M.S. , Safa Malek M. , Komrokji Rami S.
Published: April 2020

Background: Epidemiology and outcome of myelodysplastic syndromes (MDS) in the United States is not well recognized. MDS became reportable to the Surveillance, Epidemiology, and End Results program (SEER) in 2001. Only one study is published examining the SEER data between 2001–2003 on MDS incidence, epidemiology and outcome. We report first study of MDS among large population in the Veteran Affair system.

Methods: There are approximately 127 VA Medical Centers diagnosing and/or treating Cancer patients. The data collected by the medical centers cancer registries is aggregated as the VA Central Cancer Registry (VACCR) and is maintained by the Chief Program Office for Oncology at VA in Washington DC. We used the VACCR to analyze VA patients with MDS diagnosed between 1995 and 2005. The data was downloaded from the database using ICD-03 histology codes for MDS. Data was entered and analyzed using bio-statistical software SPSS. Kaplan-Meier curves were used for estimates of overall survival, chi square for comparison of categorical variables and t-test for continuous variables.

Results: Between 1995 and 2005, 1411 MDS cases were registered in the VACCR database. The median age was 75 years. Among those 1379 (97.7%) were males and 1196 (84.4%) were white patients, 171 (12.1%) black, and 44 (3.5%) other race. Majority of cases were reported as MDS, NOS 935 (66.3%), Refractory anemia (RA) 180 (12.8%), Refractory anemia with ring sideroblasts (RARS) 105 (7.4%), Refractory anemia with multilineage dysplasia (RCMD) 22 (1.6%), 5 q syndrome 13 (0.9%), Refractory anemia with excess blasts (RAEB) 105 (7.4%), Refractory anemia with excess blast in transformation 17 (1.2%), and therapy related MDS (t-MDS) 34 (2.4%). No IPSS data were available. The median overall survival (OS) was 1.8 year (95% CI 1.5–2.1). The 3 year and 5 year overall survival was 30% and 22% respectively. The median OS for RA patients was 3.7 year, RARS 4.9 year and for RAEB was 0.8 year. No difference was observed in OS between whites and black (median OS 1.73 versus 2.65, p value 0.28). Their was a non statistically significant trend for improvement of OS in patients diagnosed in 2004–2005 compared to 1995–2003 (time where azacitidine and lenalidomide were approved) median OS of 3.2 versus 1.8 (p value 0.64).

Conclusion: This is the first report of MDS case series in the VA system. Outcome is similar to what is described in literature and SEER data (3 Year OS was 30% in our VA population study compared to 35% in recently published SEER data). No racial disparities are observed in outcome. A trend towards better overall survival is noted in the last 2 years since approval of new treatment options. Coding for WHO subtypes, IPSS and treatment options should be considered in all large MDS registries.
Angiogenic Signature of Multiple Myeloma Determined by Gene Expression Profiling.
Authors: Hose Dirk , Moreaux Jerome , Meissner Tobias , Rossi Jean-Francois , Benner Axel , Mahtouk Karene , Seckinger Anja , Heiss Christiane , Hundemer Michael , Reme Thierry , Hillengass Jens , Herde Katja , Bertsch Uta , DeVos John , Wenisch Sabine , Pantesco Veronique , Jauch Anna , Jourdan Eric , Goldschmidt Hartmut , Klein Bernard , Moehler Thomas
Published: April 2020

BACKGROUND. Angiogenesis is a hallmark of active multiple myeloma and a therapeutic target, e.g. by thalidomide. Others and we have shown this to be due to a differential and novo expression of pro/antiangiogenic genes in MM as well as an effect of increased plasma cell number. AIM of this study was to investigate based on the expression of (anti)angiogenic genes alone (consensus list determined by medline search),whether for respective samples being PPC, BMPC or MMC can be predicted,what expression differences between these entities exist,if differences between the expression pattern of MMC from early stage vs. therapy requiring MM can be used for prediction, andwhether the survival of MM–patients undergoing HDT is different in two groups distinguished by their angiogenic signature.

PATIENTS AND METHODS. 187 newly diagnosed MM/MGUS–patients (65 training (TG) / 122 independent validation group (VG)), 14 normal donors (ND) and 12 in vitro generates PPC samples were included. Bone marrow aspirates were CD138-purified by activated magnetic cell sorting. RNA was in-vitro transcribed and hybridised to Affymetrix HG U133 A+B GeneChip (TG) and HG U133 2.0 plus arrays (VG). Expression data were gcrma-normalised and the empirical Bayes algorithm used. P-Values were adjusted using the Benjamini-Hochberg method (Bioconductor). The PANP-algorithm was used to identify expressed genes. iFISH was performed on purified MM-cells using probesets for chromosomes 1q21, 9q34, 11q23, 11q13, 13q14, 15q22, 17p13, 19q13, 22q11 and the translocations t(4;14) and t(11;14). Selected expression data were verified by real time RT-PCR and western blotting.

RESULTS.

On TG and VG, being BMPC or PPC is predicted by a 17 gene predictor (31 probe sets) without error. Being MMC is predicted with an error rate of 6.9/4.7% with an overall-error rate of 5.8/4.3% in TG/VG, respectively. 5 proangiogenic genes/7 probe sets (HGF [hepatocyte growth factor], ADM [adrenomedullin], CXCL2 [chemokine (C-X-C-motif)-ligand 2], IGF1 [insulin-like growth factor 2], Met [HGF-receptor]) and 1 antiangiogenic/1 probe set (SerpinF1, [Serpin peptidase inhibitor F1]) are differentially expressed between these entities concordantly in TG and VG. Attribution of MMC to early or therapy requiring can not be predicted. Most patients with MGUS and MM display 1 or several over expressed angiogenic gene. Survival analysis (EFS/OAS) will now be performed based on these findings.

CONCLUSION. Our analysis reveals novel targets for antiangiogenic therapy of multiple myeloma as anti-HGF. There is evidence that angiogenesis related genes are activated early during disease progression as rationale for early intervention using antiangiogenic compounds.
Molecular and Transcriptional Characterization of the Novel 17p11.2-p12 Chromosome Amplification in Multiple Myeloma.
Authors: Fabris Sonia , Todoerti Katia , Mosca Laura , Agnelli Luca , Ronchetti Domenica , Intini Daniela , Lionetti Marta , Ciceri Gabriella , Deliliers Giorgio Lambertenghi , Bertoni Francesco , Neri Antonino
Published: April 2020

Multiple myeloma (MM) is a fatal malignancy of clonal bone marrow plasma cells characterized by a high genomic instability increasing with disease progression. We describe here a genomic amplification at 17p11.2–p12, an unstable chromosomal region characterized by a large number of low copy repeats which have been proven to mediate deletion and duplication in several genomic disorders and amplifications in solid tumors. An approximately 5 Mb 17p11.2–p12 amplified region was detected in KMS-26 myeloma cell line by SNP microarray analysis. Further FISH mapping showed two unidentified amplified chromosomes as well as a complex pattern of rearranged chromosomes 17. The analysis of transcriptional profiles in a proprietary data base of myeloma cell lines, identified 12 significantly overexpressed genes in KMS–26 amplified region, including TNFRSF13B/TACI, COPS3, and NCOR1. The evaluation of their expression levels in a data base including 11 monoclonal gammopathy of uncertain significance, 121 MM and 9 plasma cell leukaemia, showed a significant overexpression of at least one gene in 13 patients. FISH analyses of these patients identified one MM carrying a 3.8 Mb amplified region and two MMs with gains specifically involving the TACI locus. Interestingly, the complete inactivation of TP53 at 17p13.1 was found in KMS–26 whereas a monoallelic loss was identifiable in two of the three patients carrying gain/amplification. Our data suggest that, as in the case of solid tumors, amplification/gain of the 17p11.2–p12 in MM could be mediated by the presence of repeats located in this region and may provide insights for defining novel candidate myeloma-associated genes.
Evaluation of the Cytogenetic Aberration Pattern in AL Amyloidosis Compared to Monoclonal Gammopathies Not Requiring Treatment: Translocation t(11;14) Is More Frequent in AL Amyloidosis.
Authors: Bochtler Tilmann , Schonland Stefan O. , Jauch Anna , Heiss Christiane , Benner Axel , Cremer Friedrich W. , Hose Dirk , Moos Marion , Bartram Claus R. , Ho Anthony D. , Goldschmidt Hartmut , Hegenbart Ute
Published: April 2020

Introduction: Cytogenetic aberrations (CA) have emerged as important pathogenetic and prognostic factors in plasma cell disorders. However, in AL amyloidosis (AL) only a few reports with small numbers of patients have been published.

Methods: Using interphase FISH analysis in CD138+ cells, we evaluated the role of CA in a series of 85 AL patients as compared to 146 patients with a monoclonal gammopathy without treatment requirement in a prospective manner. Our panel included IgH translocations t(11;14), t(4;14), t(14;16) and translocation of 14q32 with an unidentified partner, gains of 1q21, 11q23 and 19q13 as well as deletions of 8p21, 13q14 and 17p13. Using these probes we could detect at least one of these aberrations in 95% of AL and in 88% of the control group. Age, gender and plasma cell content were statistically equally distributed among both groups.

Results: The most frequent aberration in AL was t(11;14), which was detected in 45% of AL patients as compared to 26% of the control group (p=0,056). It was strongly associated with the lack of an intact immunoglobulin (p<0,001), thus accounting for the frequent light chain only subtype in AL. Markedly, t(11;14) was more frequently found in combination with gain 11q23 in AL than in the control group (20% versus 6%, p = 0.005). Other frequent aberrations in AL included deletion 13q14 (32%) and gain 1q21 (21%), which were observed in the control group at comparable frequencies (34% and 20%). The overlapping character of the underlying plasma cell disorder in both disease entities was also emphasized by the similarities of branching patterns of the five major CA in cluster analysis applied in 169 patients (figure 1). The relation of clinical parameters and chromosomal aberrations was also evaluated. The analyzed CA had no impact on the organ involvement pattern in AL patients.

Conclusions: We observed a high frequency for t(11;14) in AL. Apart from this finding, the cytogenetic patterns known in monoclonal gammopathy of unknown significance and multiple myeloma were widely shared by AL amyloidosis.

Figure Figure
Cathepsin G, a Potent Platelet Aggregation Activator, Is Upregulated in Multiple Myeloma Patients during Treatment with Lenalidomide (Revlimid).
Authors: Pal Rekha , Kennedy Ryan , Mansour L. Renee , Stirling David , Mapara Markus Y. , Roodman G. David , Lentzsch Suzanne
Published: April 2020

Lenalidomide induces impressive response rates in patients with multiple myeloma. Unfortunately, one of the major side effects of this therapy is the increased risk of thromboembolic events (TEE) up to 17%. The reason for a higher risk of TEE associated with Lenalidomide is not known, but it has been found that aspirin is very effective in preventing TEEs induced by thalidomide and lenalidomide (Rajkumar, 2005). Cathepsin G is a serine proteinase present in the azurophilic granules of polymorphonuclear neutrophils (PMNs) and is a strong platelet aggregation agonist with potency similar to thrombin. In the current study we investigated the effect of the immunomodulatory derivatives of thalidomide (IMiDs) including Actimid and lenalidomide on cathepsin G in vitro and in vivo. To investigate the effect of IMiDs in vitro CD34+ hematopoietic progenitors were cultured in the presence of IL-3, IL-6 and SCF supporting the development of granulocytes and treated with Actimid or DMSO (control). RNA and protein were extracted for the analysis of cathepsin G. A significant up-regulation of cathepsin G (7.7 fold) was detected in oligonucleotide gene array analysis after 3 days of treatment with Actimid as compared to control group. These results were confirmed by RT-PCR, which showed a 3.7 and 7.6 fold mRNA-increase on day 6 and day 10 of culture compared to control. Next we analyzed the cathepsin G levels of patients (n=10) treated with lenalidomide before treatment, on day 15 of each cycle (1–4) as well as 1, 2, and 3 month after stop of lenalidomide treatment. We observed a continuous significant increase of the mRNA levels of PMNs over the course of treatment (baseline: 1 fold, cycle 2: 1.7 fold, cycle 3: 4.8 fold, cycle 4: 20.7 fold). These data were confirmed by measuring cathepsin G levels in patient serum by ELISA. Cathepsin G significantly increased from a baseline mean of 53 ng/ml to 77.5 ng/ml (cycle 2), 129.2 ng/ml (cycle 3) and 145.5 ng/ml (cycle 4). After stop of the lenalidomide treatment we observed a time dependent normalization to baseline levels of the cathepsin G with 61 ng/mL (1 month post treatment), 42.5 ng/mL (2 months post treatment), and 49.2 ng/mL (3 months post treatment). Our data show that IMiDs up-regulate the potent platelet aggregation activator cathepsin G in hematopoietic cells and thereby might contribute to the development of TEE in patients receiving IMiD treatment. Based on our data TEE prophylaxis should be continued for at least 1 more month after stop of therapy. For the first time our results provide an explanation why aspirin is effective in preventing TEE in patients receiving IMiDs. Further studies are needed to determine whether cathepsin G helps to predict TEE. Inhibition of cathepsin G might be a potential therapeutic target for preventing the hypercoaguable state induced by IMiD treatment.
Autophagy as a Target Pathway in Multiple Myeloma: A Forward Chemical Genetic Approach.
Authors: Shen John P. , Divakaran Sanjay , Kuai Letian , Wang Xiang , Ong Shao-En , Amaravadi Ravi , Wood John , Carr Steven , Schreiber Stuart , Haggarty Stephen , Bradner James
Published: April 2020

Autophagy is an evolutionarily conserved process by which cellular structures may be degraded to support ongoing biogenesis. Recent evidence has identified the induction of autophagy following growth factor pathway inhibition, rapid tumor expansion and proteasome inhibition. Thus, autophagy is a highly desirable pathway for the development of targeted therapeutics in cancer, in particular in tumors characterized by misfolded protein stress such as multiple myeloma (MM). Unfortunately, there are only several known classes of autophagy inhibitors and the emerging biology of autophagy has identified few validated targets. We therefore performed a forward chemical genetic study of autophagy using novel techniques in high-content imaging, high-throughput screening and multidimensional data analysis. Using LN229 glioblastoma cells stably transfected with an LC3-EGFP construct, we created a robust screening platform capable of quantifying the number, size and fluorescence intensity of autophagosomes. Assay validation was performed with the known autophagy inhibitor chloroquine, which induces a characteristic shift from diffuse to punctate fluorescence. Initially, we profiled a library of approximately 3,500 structurally diverse bioactive small molecules to leverage possible downstream mechanistic insights. Assay positives and controls were re-tested in the adherent H1299 adenocarcinoma cell line likewise stably expressing LC3-EGFP. Known autophagy inhibitors were enriched with this approach: nocodazole, bafilomycin A1, colchicine and monensin. Structure-activity relationship analysis of the primary screening data and 34 novel autophagy modulators revealed a compelling activity of bis-indolyl maleimides. We confirmed sub-micromolar inhibition of autophagy by three such compounds using a quantitative, radiolabeled protein degradation assay (tritiated tyrosine pulse-chase), LC3-II immunoblotting and quantitative measurement of autophagosomes across three cell lines. From these studies we selected a lead compound, K252a, for further characterization and tested twenty-two closely related analogues. Detailed SAR analysis has identified features of the molecule required for inhibition of autophagy and sites permissive for further functionalization in ongoing target identification studies. Using MM as a translational model system of misfolded protein stress, we found dose-dependent, selective cytotoxicity in MM of K252a compared to analogues lacking autophagy inhibitory activity. We report here the results of a forward chemical genetic study of autophagy which identifies and characterizes novel small molecule inhibitors of autophagy and establishes a rationale for clinical studies of autophagy inhibitors in the treatment of MM.
Wnt3/RhoA/ROCK Signaling Pathway Is Involved in VLA6-Dependent Adhesion-Mediated Drug Resistance of Multiple Myeloma.
Authors: Kobune Masayoshi , Kawano Yutaka , Takimoto Rishu , Matsunaga Takuya , Kato Junji , Hamada Hirofumi , Niitsu Yoshiro
Published: April 2020

Adhesion of myeloma cells to BM stromal cells is now considered to play a critical role in chemo-resistance. However, little is known about the molecular mechanism of cell adhesion mediated drug resistance (CAM-DR) in multiple myeloma. In this study, we focused on relationship between drug resistance and expression of Wnts, the factor regulating the cell adhesion and proliferation, in myeloma cells. To gain insight into involvement of Wnt signaling in CAM-DR, we first screened the expression of Wnt family in myeloma cell lines (RPMI8226, ARH77, KMS-5 and MM1S) by reverse transcription-polymerase chain reaction analysis. Although the mRNAs of Wnt2b, Wnt7a and Wnt10b were variably expressed in some of myeloma cell lines, Wnt3 mRNA was detected in all the myeloma cells examined. KMS-5 and ARH77, which highly expressed Wnt3 protein, tightly adhered to human BM stromal cells and accumulation of β-catenin and GTP-bounded RhoA was observed in these myeloma cell lines. Conversely, RPMI8226 and MM1S, which modestly expressed Wnt3 protein, rather weakly adhered to human BM stromal cells. We then examined the relevance of Wnt3 expression to adhesive property to stromal cells and to CAM-DR of myeloma cells. KMS-5 and ARH-77 exhibited apparent CAM-DR against Doxorubicin. This CAM-DR was significantly reduced by anti-integrinβ1 antibody, anti- integrinα6 antibody and a Wnt-receptor competitor, secreted Frizzled related protein-1 and Rho kinase inhibitor (Y27632 and OH-fasudil), but not by the specific inhibitor of canonical signaling (DKK-1), indicating that Wnt-mediated CAM-DR which is dependent on integrinα6/β1 (VLA-6)-mediated attachment to stromal cells is induced by Wnt/RhoA-Rho kinase (ROCK) pathway signal. This CAM-DR for doxorubicin was also significantly reduced by Wnt3 siRNA transfer to KMS-5 and further augmented by addition of Wnt3 conditioned medium. These results indicate that Wnt3 contributes to VLA-6-mediated CAM-DR via the Wnt/RhoA/ROCK pathway of myeloma cells. Thus, the Wnt3/RhoA/ROCK signaling pathway could be a promising molecular target to overcome CAM-DR.
KD7150, a Novel Mercaptoketone-Based HDAC Inhibitor, Exerts Potent Anti-Multiple Myeloma Effects In Vitro and In Vivo by Induction of DNA Damage and Mitochondrial Signaling.
Authors: Feng Rentian , Ma Huihui , Hassig Christian A. , Payne Joseph E. , Smith Nicholas D. , Mapara Markus Y. , Hager Jeffrey H. , Lentzsch Suzanne
Published: April 2020

Histone deacetylase (HDAC) inhibitors have emerged as a class of novel and promising anti-cancer agents and their activities are currently being investigated in both the pre-clinical and clinical settings. Using an unbiased, ultrahigh throughput screening system a novel mercaptoketone-based HDAC inhibitor KD5170 was identified. This novel non-hydroxamic acid non-benzamide compound was profiled for its anti-myeloma activity in vitro and in vivo. KD5170 inhibited the proliferation of multiple myeloma (MM) cell lines and the viability of CD138+ primary MM cells by induction of apoptosis. This was accompanied by an increase of acetylation of histones and activation of caspase-3, -8 and -9. Treatment with KD5170 caused a loss of mitochondrial membrane potential measured by JC-1 staining and resulted in release of apoptogenic factors such as cytochrome c, smac, and AIF from mitochondria. Furthermore, KD5170 induced oxidative stress and oxidative DNA damage in myeloma cells, as evidenced by the upregulation of heme oxygenase-1 and H2A.X phosphorylation, respectively. Combination of KD5170 with the proteasome inhibitor bortezomib or TNF-related apoptosis-inducing ligand synergistically enhanced the anti-myeloma activity. Resistance of MM cells to KD5170 was associated with activation of the ERK/MAPK pathway under treatment with KD5170. Pretreatment with the MAPK-inhibitor restored the sensitivity to KD5170, suggesting that the combination of KD5170 with a MAPK inhibitor (U0126) could overcome drug resistance. Growth of myeloma tumor xenografts in KD5170-treated nude mice was significantly inhibited (1401 mm3 ± 596 versus 442 mm3 ± 221; P = .009) and survival was prolonged (9 days and 17.5 days; P= .0095) compared to vehicle-treated mice. Accordingly, our in vivo data showed that histone acetylation was markedly upregulated in tela in spleen or tumor tissues of the animals treated with KD5170 as early as 2 hours. In conclusion, our data show that KD5170 has anti-myeloma activity in vitro and in vivo, mediated by the induction of apoptosis due to DNA damage and mitochondrial signaling.