A randomized inter-group trial comparing more intensive treatment strategies to a common standard arm 3 + 7 (CSA) was conducted in patients with non-M3 AML. Untreated patients ≥ 60 years were allocated to the CSA (n = 132) or to the study group arms (n = 1154) of the AMLCG (TAD/HAM versus HAM/HAM ± G-CSF followed by TAD and maintenance) and the OSHO (intermediate-dose ara-C/mitoxantrone followed by ara-C/mitoxantrone). Median age of the 1147 eligible patients was 69 (range 60–87) years. CR/CRi status at 90 days was not significantly different between the CSA (54

Oncogene-induced senescence, e.g., in melanocytic nevi, terminates the expansion of pre-malignant cells via transcriptional silencing of proliferation-related genes due to decoration of their promoters with repressive trimethylated histone H3 lysine 9 (H3K9) marks. We show here that structurally distinct H3K9-active demethylases-the lysine-specific demethylase-1 (LSD1) and several Jumonji C domain-containing moieties (such as JMJD2C)-disable senescence and permit Ras/Braf-evoked transformation. In mouse and zebrafish models, enforced LSD1 or JMJD2C expression promoted Braf-V600E-driven melanomagenesis. A large subset of established melanoma cell lines and primary human melanoma samples presented with a collective upregulation of related and unrelated H3K9 demethylase activities, whose targeted inhibition restored senescence, even in Braf inhibitor-resistant melanomas, evoked secondary immune effects and controlled tumor growth in vivo.

While plasma cells are well-established bone marrow (BM) long-term residents with unique features,1Manz R.A. Thiel A. Radbruch A. Lifetime of plasma cells in the bone marrow.Nature. 1997; 388: 133-134Crossref PubMed Scopus (654) Google Scholar only a few studies indicate memory B (memB) cells as persisting in BM and other lymphoid organs. Little is known about phenotypic and functional differences between circulating and tissue-resident memB cells. No major differences were seen for memB cells from spleen, tonsils, BM, and peripheral blood (PB) regarding maturation and activation status, migratory traits, adhesion marker, or the death receptor CD95.2Giesecke C. Frölich D. Reiter K. Mei H.E. Wirries I. Kuhly R. et al.Tissue distribution and dependence of responsiveness of human antigen-specific memory B cells.J Immunol. 2014; 192: 3091-3100Crossref PubMed Scopus (61) Google Scholar Thus, it was deduced that phenotypically similar memB cells recirculate between the different organs. However, differences were seen for ICOS-L, CD62L, ITGβ7, and CD31 in this study, suggesting tissue-specific functions, which have not been analyzed in detail yet. In another study, BM-resident memory T cells have been characterized with distinct functional features compared with their circulating counterparts, particularly exhibiting a more resting state.3Okhrimenko A. Grün J.R. Westendorf K. Fang Z. Reinke S. von Roth P. et al.Human memory T cells from the bone marrow are resting and maintain long-lasting systemic memory.Proc Natl Acad Sci U S A. 2014; 111: 9229-9234Crossref PubMed Scopus (128) Google Scholar To elucidate any differences between human BM-resident memB cells and their circulating counterparts, we now performed a comprehensive comparative analysis using paired BM and PB samples. Detailed methodology is provided in the Methods section in this article's Online Repository at www.jacionline.org. Briefly, paired BM diaphysis/femur head and PB samples were obtained from 58 patients (mean age, 62 years; 50% men, 50% women) undergoing hip joint replacement surgery and allocated to different analyses including microarray, cytometric analyses, and cell culture experiments. The study was approved by the Charité Berlin local ethics committee (EA1/269/14). After isolation of PBMCs and bone marrow mononuclear cells (BMMCs), cells were cultured in 1.5 mL Iscove Modified Dulbecco Medium (IMDM)/10% FCS/1% penicillin/streptomycin in 12-well plates at 37°C and 5% CO2 for 5 hours for cytokine detection (TNF-α and IL-6) or 6 days for Annexin-V and Ki-67 assays. Stimulations were performed by either heat-killed formalin-fixed Staphylococcus aureus Cowan strain I (1:10,000 diluted; Merck Millipore, Darmstadt, Germany), 6 μg/mL CpG (ODN 2006; InvivoGen, San Diego, Calif), and 1 μg/mL pokeweed mitogen (Sigma-Aldrich, München, Germany) or 1 μg/mL human recombinant CD40L (Biolegend, Fell, Germany) and 10 μg/mL F(ab')2 antihuman IgM + IgG (Affymetrix eBioscience, Frankfurt am Main, Germany). After 1 hour of stimulation, 7.5 μg/mL brefeldin A (Sigma-Aldrich) was added to the culture for the remaining 4 hours to facilitate detection of intracellular TNF-α and IL-6. Intracellular α-defensin 1-3 (DEFA1-3) expression was measured immediately after cell isolation. For inducing DEFA1-3 expression, lymphocytes were cultured with PB- or BM-derived serum and CD45hiCD15+ cells for 18 hours at 37°C, 5% CO2. Flow cytometric analyses were carried out using the LSRII Fortessa Flow Cytometer (Becton Dickinson, Franklin Lakes, NJ) (see Table E1 and Fig E1 in this article's Online Repository at www.jacionline.org). Switched and nonswitched memory B cells from 3 paired PBMC and BMMC (diaphysis plus femur head) samples were sorted. After RNA isolation, microarray hybridization and bioinformatic analysis were performed (see Table E2 in this article's Online Repository at www.jacionline.org). An altered B cell subset distribution was apparent among mature B cells with reduced percentages of CD27+IgD+ nonswitched memory B cells as well as increased percentages of CD27−IgD− double negative B cells and CD38hiCD27hi plasma cells in BM when compared to PB (Fig 1, A). Our data provide evidence that BM memB cells are more resting compared with their circulating counterparts. This was indicated by reduced HLA-DR and CXCR3, but increased Fas-ligand expression on BM cells (Fig 1, B and C). Moreover, upon reactivation, BM memB cells displayed less proliferation and apoptosis, as well as reduced production of the inflammatory cytokines TNF-α and IL-6 (Fig 1, D). Transcriptome analyses revealed a significantly higher amount of α-defensin (DEFA) transcripts in BM memB cells (Fig 2, A) and accordingly an increased DEFA1-3 protein expression (Fig 2, B). This molecule class belongs to the antimicrobial peptides functioning as part of innate immunity.4Ganz T. Defensins: antimicrobial peptides of innate immunity.Nat Rev Immunol. 2003; 3: 710-720Crossref PubMed Scopus (2341) Google Scholar Few studies have indicated DEFA1-3 expression by adaptive immune cells. An upregulation was found in PB T cells of patients with drug-induced subcutaneous diseases,5Morel E. Alvarez L. Cabañas R. Fiandor A. Díaz R. Escamochero S. et al.Expression of alpha-defensin 1-3 in T cells from severe cutaneous drug-induced hypersensitivity reactions.Allergy. 2011; 66: 360-367Crossref PubMed Scopus (34) Google Scholar as well as in CD8+ T cells from long-term HIV-infected nonprogressors whose cells secrete DEFA1-3 upon stimulation.6Zhang L. Yu W. He T. Yu J. Caffrey R.E. Dalmasso E.A. et al.Contribution of human alpha-defensin 1, 2, and 3 to the anti-HIV-1 activity of CD8 antiviral factor.Science. 2002; 298: 995-1000Crossref PubMed Scopus (445) Google Scholar This state of affairs contributes to the anti–HIV-1 activity of CD8 antiviral factor. Low DEFA1-3 expression in PB memB cells could be induced by addition of BM serum (see Fig E2, A, in this article's Online Repository at www.jacionline.org), or by BM CD45hiCD15+ cells, highly expressing DEFA (Fig E2, B). We proposed that DEFA1-3 RNA- and protein-rich exosomes are released from these CD15+DEFA1-3high cells into the serum and subsequently taken up by BM memB cells. Exosomes are small vesicles released from the surface of many cell types into body fluids carrying mRNA, microRNA, or DNA as well as proteins.7Lawson C. Vicencio J.M. Yellon D.M. Davidson S.M. Microvesicles and exosomes: new players in metabolic and cardiovascular disease.J Endocrinol. 2016; 228: R57-R71Crossref PubMed Scopus (234) Google Scholar Our hypothesis was supported by the finding that significant amounts of DEFA1 and DEFA3 mRNA as well as protein were detectable in exosomes isolated from BM but not in those from PB serum (Fig E2, C and D). Importantly, the addition of the exosome uptake inhibitor cytochalasin-D to the coculture assay with CD15+DEFA1-3high cells profoundly reduced the DEFA1-3 expression in PB-derived memB cells (Fig E2, E). Cytochalasin-D disrupts the actin polymerization and thereby prevents exosome uptake by target cells.8Svensson K.J. Christianson H.C. Wittrup A. Bourseau-Guilmain E. Lindqvist E. Svensson L.M. et al.Exosome uptake depends on ERK1/2-heat shock protein 27 signaling and lipid Raft-mediated endocytosis negatively regulated by caveolin-1.J Biol Chem. 2013; 288: 17713-17724Crossref PubMed Scopus (459) Google Scholar This suggests that the induction of DEFA1-3 expression in memB cells is indeed exosome-dependent and provides first evidence for an exosome-related communication between cells of the innate and adaptive immune system in the BM by mediating DEFA1-3 transfer. Whether or not BM memB cells are inherently capable of DEFA mRNA transcription and translation without the help of exosomes cannot be determined from our data and needs further investigation.Fig 2RNA of switched (sw) and nonswitched (non sw) memB cells from paired human PBMC and BMMC samples of 3 different donors (D1-D3) was used for microarray analysis. A, The gene expression pattern for indicated differentially expressed genes as heatmap based on the Linear Model for Microarray Analysis (LIMMA) algorithm. B, The DEFA1-3 protein expression in CD27+ memB cells (n = 20) as percentage and mean fluorescence intensity. PE, Phycoerythrin. Mean ± SEM values are given.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Functionally, supernatants from BMMCs showed a higher bactericidal activity against Streptococcus pneumoniae than did those from PB (see Fig E3, A, in this article's Online Repository at www.jacionline.org), which is potentially attributable to higher numbers of CD15+DEFA1-3high cells and DEFA1-3high BM memB cells. Inactivation of DEFA with the α1-proteinase inhibitor9Panyutich A.V. Hiemstra P.S. van Wetering S. Ganz T. Human neutrophil defensin and serpins form complexes and inactivate each other.Am J Respir Cell Mol Biol. 1995; 12: 351-357Crossref PubMed Scopus (120) Google Scholar resulted in reduced killing activity in almost 40% of functionally positive BMMC supernatants (Fig E3, B). BMMC supernatants were defined as functionally positive if bacterial growth was inhibited by at least 1 log. Because of a lack of specific neutralizing antibodies targeting DEFA1-3, we could not exclusively inhibit these antimicrobial peptides in the bacterial killing assay. In consequence, our data do not prove that DEFA is responsible for bactericidal activity in the BM, but rather indicate their significant contribution, along with other antimicrobial molecules, to bacterial killing. Preliminary data also suggest a higher DEFA expression in splenic memB cells, which could hint at a similar role in antimicrobial activity as proposed for the BM (data not shown). In summary, our study provides evidence that BM-residing memB cells differ phenotypically and functionally from those in PB. We show evidence that the BM contains niches for resting memB cells and suggest a novel, essentially innate mechanism by which memB cells could contribute to BM immunity. Before isolation of PBMCs/BMMCs, bone pieces were incubated for 10 minutes in IMDM medium/10% FCS/1% penicillin/streptomycin (Merck Millipore) at room temperature under constant agitation followed by cell filtration with a 100-μm cell strainer. PBMCs/BMMCs were isolated from heparin PB samples and bone diaphysis/femur heads with density gradient centrifugation (Biocoll separating solution; Merck Millipore). Flow cytometric analysis was performed at the LSRII Fortessa Flow Cytometer (Becton Dickinson) configured with a blue (488 nm, 100 mW), yellow-green (561 nm, 100 mW), red (639 nm, 40 mW), violet laser (404 nm, 50 mW) and ultraviolet laser (355 nm, 60 mW), enabling the detection of up to 18 colors simultaneously. Sorted cells from PB or BM were measured at 4°C at the Aria II-SORP Flow Cytometer (BD Biosciences) equipped with a blue (488 nm, 100 mW), yellow-green (561 nm, 50 mW), red (639 nm, 40 mW), violet laser (408 nm, 50 mW) and 15 fluorescence channels. Both instruments allow precise laser focusing with a cuvette flow cell gel coupled to the collection optics. Details about detection optics/filter configurations are depicted in Fig E4. Data were evaluated with the FlowJo-9.8.5 software (TreeStar, Ashland, Ore). Absolute cell numbers were determined with differential PB counts. Dead cells were excluded by propidium iodide (Biolegend) or LD Pacific Blue/Orange (Thermo Fisher Scientific, Darmstadt, Germany) staining. For intracellular staining, the fixation/permeabilization buffer set (DEFA1-3, IL-6, TNF-α) and the Foxp3/Transcription Factor Staining Buffer Set (Ki-67) were used according to manufacturer's instructions (Affymetrix eBioscience). Apoptosis was assessed with the Annexin-V FITC Kit (BD Biosciences, Heidelberg, Germany). RNA was extracted with the RNeasy Micro Kit with prior addition of β-mercaptoethanol and additional DNase I digestion according to manufacturer's instructions (Qiagen, Hilden, Germany). RNA integrity was checked by 2100 Bioanalyzer (Agilent Technologies, Santa Clara, Calif). RNA (2 ng) was used for coupled cDNA synthesis and amplification process with the RNA Ovation Pico WTA System V2 (NuGEN, San Carlos, Calif). To generate biotin-labeled targets, 5.5 μg cDNA per reaction was used in the Encore Biotin Module (NuGEN) and added to a hybridization cocktail for hybridization to the Human Gene 2.0 ST Array according to the manufacturer's GeneChip Hybridization Wash and Stain Kit containing eukaryotic control cRNA and a control oligonucleotide B2 (Affymetrix). Hybridization, staining, and washing processes were achieved at 45°C and 60 rpm for 16 hours in the GeneChipHybridization Oven 640 and the GeneChip Fluidics Station 450 (Affymetrix). The scanning was carried out with the GeneChip Scanner 3000 7G (Affymetrix). All Affymetrics HGU-133plus2 mRNA-array samples passed an initial quality control based on the R-package “arrayQualityMetrics,” version 3.24.0.E1Kauffmann A. Gentleman R. Huber W. arrayQualityMetrics–a bioconductor package for quality assessment of microarray data.Bioinformatics. 2009; 25: 415-416Crossref PubMed Scopus (635) Google Scholar Raw intensity expression values were background corrected, log2-transformed, and quantile normalized with the GCRMA algorithm of the R “oligo” package version 1.32.0.E2Carvalho B.S. Irizarry R.A. A framework for oligonucleotide microarray preprocessing.Bioinformatics. 2010; 26: 2363-2367Crossref PubMed Scopus (979) Google Scholar A linear contrast model was fit to the normalized data using the R package “limma” version 3.24.15E3Ritchie M.E. Phipson B. Wu D. Hu Y. Law C.W. Shi W. et al.limma powers differential expression analyses for RNA-sequencing and microarray studies.Nucleic Acids Res. 2015; 43: e47Crossref PubMed Scopus (15342) Google Scholar to identify differentially expressed transcripts. R version was 3.2.2 and bioconductor version was 3.1. In addition, a gene set enrichment analysis (GSEA) showed comparable results to LIMMA, based on the R-package GSEABase (Morgan M, Falcon S, Gentleman R, 2016, GSEABase: Gene set enrichment data structures and methods. R package version 1.36.0., data not shown). In Table E2, the arithmetic mean of combined log differences (nonswitched and switched memB cells of all 3 patients per gene) is displayed. BM and PB serum was freshly prepared by centrifugation. Exosomes were isolated from BM- and PB-derived serum samples with the Total Exosome Isolation Reagent, followed by RNA extraction with the Total Exosome RNA and Protein Isolation Kit according to manufacturer's instructions (Thermo Fisher Scientific). cDNA was generated with the high-capacity cDNA reverse transcription kit (Applied Biosystems). The following thermal cycler program at a GeneAmp PCR System 9700 (Applied Biosystems) was applied: 10 minutes 25°C, 120 minutes 37°C, 5 minutes 85°C. PCR reactions were performed in 96-well MicroAmpOptical plates (Applied Biosystems) in a final volume of 11 μL composed of 2 μL cDNA, 4.25 μL Diethyl pyrocarbonate (DEPC)-treated water, 6.25 μL TaqManUniversal PCR Master Mix (Applied Biosystems), and 0.5 μL 6-carboxyfluorescein (FAM)-labeled DEFA1 (Hs00414018_m1) and DEFA3 (Hs00234383_m1) primer pair/probe sets (Thermo Fisher Scientific). 18S rRNA was used as normalizer gene (primer pair/probe sets: Thermo Fisher Scientific). The following PCR program was used: 1× (2 minutes 50°C), 1× (10 minutes 95°C), 40× to 50× (15 seconds 95°C, 1 minute 60°C). PCR was performed at a 7500 Real-Time PCR System (Applied Biosystems). PBMCs and BMMCs were cultured for 1 day at 37°C, 5% CO2, in IMDM medium supplemented with ITS-G (5 μg/mL Insulin, 5 μg/mL Transferrin, 5 ng/mL Sodium-Selenite; Thermo Fisher Scientific) followed by freezing of supernatants. 1 × 102 colony-forming units of wild-type serotype 2 Streptococcus pneumoniae (strain D39/NCTC 7466) were incubated with IMDM + 0.1% FCS medium only, or with 5 μg/mL cathelicidin antimicrobial peptide LL-37 (Innovagen AB, Lund, Sweden) as positive control, or with respective PBMC and BMMC supernatants for 0, 3, and 6 hours in a total volume of 100 μL, 96-well plates (Sarstedt, Nümbrecht, Germany) at 37°C, 5% CO2. For α-defensin inhibition, supernatants of PBMCs and BMMCs were preincubated with 1 mg/mL alpha 1-proteinase inhibitor (Sigma-Aldrich) for 1 hour at 37°C, 5% CO2, before addition to the bacteria. Subsequently bacteria were plated on Columbia Agar plates with 5% sheep PB (BD Biosciences), cultured for 1 day at 37°C, and then quantified for colony-forming units. Supernatants of Staphylococcus aureus Cowan strain I/CpG/pokeweed mitogen–stimulated PBMCs and BMMCs were freezed at day 6 after stimulation. DEFA1-3 concentration was determined with a human DEFA1-3 ELISA kit including a monoclonal biotinylated antibody and a streptavidin-peroxidase conjugate (Hycult Biotech, Beutelsbach, Germany) according to manufacturer's instructions. The measurement was done at a Tecan Sunrise microplate reader with Magellan-Data Analysis Software 6.4 (Tecan Group, Männedorf, Switzerland). The 2-sided Mann-Whitney U and the Wilcoxon matched-pairs signed-ranks test were applied. Values were expressed as mean ± SEM. Statistical analysis was performed with the GraphPad Prism 6 software (La Jolla, Calif). P values of .05 or less were considered statistically significant.Fig E2BM-derived serum and CD45hiCD15+ cells induce a high DEFA1-3 expression in memB cells. A, cDNA expression levels of DEFA1 and DEFA3 were determined in exosome samples isolated from PB (black bars, n = 5) or BM (gray bars, n = 5) with quantitative RT-PCR. The relative expression to 18S rRNA is given. B, Exosome preparations from PB (upper graphs) and BM (lower graphs) were analyzed with flow cytometry. Exosomes were identified as Live-Dead−CD63+CD9+ particles (left) and analyzed for intracellular expression of DEFA1-3 (right). Shown is a representative experiment out of 4. C and D, (1 to 2) × 105 sorted CD19+/CD3+ lymphocytes from PB (black bars, n = 6) and BM (gray bars, n = 3-6) were cultured in 300 μL IMDM medium supplemented either with 20% FCS and (Fig E2, B) with 5% FCS + 15% BM- or PB-derived serum or (Fig E2, C) with 5% FCS + BM- or PB-derived (1 to 2) × 105-sorted CD45hiCD15+ cells. After 18 hours of incubation at 37°C, 5%CO2, the DEFA1-3 expression in CD19+CD27+ memB cells was determined by flow cytometry. Mean values of MFI ± SEM are given. E, For exosome transfer inhibition, sorted BM CD45hiCD15+ granulocytes and PB CD19+/CD3+ lymphocytes were separately preincubated with 20 μM cytochalasin-D (Sigma-Aldrich) for 40 minutes at 37°C, 5% CO2. Afterwards, CD19+/CD3+ lymphocytes from PB were cultured for 18 hours with 20% FCS or 20% FCS + BM-derived CD45hiCD15+ cells in the presence or absence of cytochalasin-D (Cyt.D; n = 3). The histograms show the DEFA1-3 expression as % of maximum for memB cells after culture with FCS (dashed line), with FCS + CD15+ cells (gray filled), and with FCS + CD15+ + Cyt.D (white filled). The graph on the right depicts the MFI values obtained from the 3 experiments for the DEFA1-3 expression by memB cells after incubation with FCS + CD15+ cells (circles) or with FCS + CD15+ cells + Cyt.D (squares). Differences between PBMCs and BMMCs or between 20% FCS control and other stimulations were statistically evaluated with the Wilcoxon matched-pair signed-rank test. APC, Allophycocyanin; FITC, fluorescein isothiocyanate; MFI, mean fluorescence intensity. *P ≤ .05.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig E3Higher antimicrobial activity by BMMCs than PBMCs supernatants. A, Growth of Streptococcus pneumoniae (S.pn.) was measured for indicated time points in the presence of supernatants (sup.) from cultured BMMCs (circles) and PBMCs (rhombuses) compared with mock. Antimicrobial LL-37 (5 μg/mL) served as positive control (triangle). B, BMMC responders (circles), who mitigated the growth of S.pn. by at least 1 log compared with control, from Fig E3, A (n = 13 of 17), were mock treated (triangle) or incubated with 1 mg/mL alpha 1-proteinase inhibitor (α1-PI, squares; n = 8) and growth of S.pn. was analyzed against control showing a neutralization of the antimicrobial activity in 3 of 8 BMMC supernatants. Shown is the number of colony-forming units (CFU/mL) as mean ± SEM values (PB: n = 13, BM: n = 17, controls: n = 5). Differences between PBMC or BMMC supernatants were statistically evaluated with the Wilcoxon matched-pair signed-rank test (Fig E3, A) and with the unpaired Mann-Whitney test (Fig E3, B). **P ≤ .01, ***P ≤ .001.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig E4Filter configurations for flow cytometry measurements. A, Filter arrangement shown for LSRII Fortessa Flow Cytometer configured with a blue, yellow-green, red, violet laser, and ultraviolet laser. B, Filter arrangement depicted for Aria II-SORP Flow Cytometer cell sorter equipped with a blue, yellow-green, red, and violet laser.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Table E1Fluorochrome-conjugated monoclonal antihuman antibodies for flow cytometryAntibodyCloneCompanyB-cell subsets CD19 V500HIB19BD Biosciences CD19 FITCHIB19Biolegend CD27 BV650O323Biolegend CD27 PEM-T271BD Biosciences IgD-APC H7IA6-2BD Biosciences CD38 APCHIT2Biolegend CD10 PE-Cy7HI10aBiolegend CD24 PerCP Cy5.5ML5BD Biosciences IgM PerCP Cy5.5MHM-88BiolegendT cells CD3 Pacific BlueUCHT1BD Biosciences CD3 PerCP Cy5.5OKT3BiolegendGranulocytes, NK cells CD15 FITCHI98Biolegend CD16 PE-Cy7, Pacific Blue3G8Biolegend CD45 AF700HI30Biolegend CD56 PEHCD56BiolegendFunctional marker HLA-DR PerCP-Cy5.5L243Biolegend BAFF-R FITC11C1Biolegend TACI PE1A1Biolegend CXCR3 FITC49801R&D Systems CXCR4 PE12G5Biolegend CCR7 FITC150503R&D Systems CD95 FITCDX2Biolegend Fas-L FITCSB93aAbcam DEFA1-3 BiotinD21Hycult Biotech Ki-67 FITCREA183Miltenyi Biotec TNF-α FITCMAb11Affymetrix eBioscience IL-6 PEMQ2-6A3BD BiosciencesExosomes CD9 FITCHI9aBiolegend CD63 APCH5C6BiolegendIsotype controls IgG1κ BiotinMOPC-21Hycult Biotech IgG1κ FITCMOPC-21Biolegend IgG1κ PEMOPC-21BD Biosciences IgG2a/κ PEMOPC-173Biolegend IgG2a/κ PerCP-Cy5.5MOPC-173Biolegend IgG2b/κ FITCMPC-11BiolegendStreptavidin conjugates Streptavidin PEBD Biosciences Streptavidin Pacific BlueThermo Fisher ScientificCD, Cluster of differentiation; V500, violet 500; BV650, brilliant violet 650; APC-H7, allophycocyanin-H7; PE-Cy7, phycoerythrin-cyanin7; PerCP Cy 5.5, peridinin chlorophyll protein-cyanin 5.5; NK, natural killer cells; AF700, Alexa Fluor 700; HLA-DR, human leukocyte antigen-DR; BAFF-R, B cell activating factor receptor; TACI, transmembrane activator and CAML interactor; CXCR3/4, C-X-C chemokine receptor type 3/4; CCR7, C-C chemokine receptor type 7; Fas-L, Fas ligand. Open table in a new tab Table E2Differentially expressed genes (log-fold change) of memB cells from BM compared to PBUpregulated genesDownregulated genesDEFA37.18EGR11.84AZU11.16FASLG−0.01NDUFB6−1DEFA1B6.21MPO1.82ZNF3311.15IPW−0.01ADPRM−1.01FOSB4.24IL-61.81BEX11.15SORD−0.01PRICKLE2-A S3−1.01CTSG3.89SIK11.7IER51.14NSUN6−0.02CEP19−1.01CD693.37NR4A11.68MMP81.13ZNF280D−0.02SORD−1.02NR4A23.37NFKBIA1.68PPP1R15B1.12HLA-DRB3−0.02GRK4−1.03FOS3.34SCARNA101.67HBB1.12HLA-DRB4−0.03WEE1−1.04DEFA43DUSP101.62ARL4A1.11LTB−0.04LTB−1.04RNASE22.74LTF1.58PTGR21.1PTGR2−0.05MORC3−1.06S100A92.59IPW1.57MS4A31.08HLA-DRA−0.06CCT6P1−1.07RNASE32.56KLF61.43RASGEF1B1.07SPDYA−0.06ZNF280D−1.1RGS22.52CSRNP11.41IFRD11.07HLA-DRA−0.06DLEU2L−1.11RGS12.5DUSP51.4DDX3X1.07ZNF280D−0.07ANXA2P2−1.11S100A82.49TMEM881.39SDCBP1.06SMAD4−0.12RAP2A−1.13GRASP2.4OR2L1P1.37DDIT31.05HBG2−0.16IQGAP2−1.15CEACAM82.34KDM6B1.33TSPYL21.05KLHL42−0.17SMAD4−1.16DUSP12.27ZNF1651.33CXCR41.04IPW−0.17PDIA3P1−1.17AREG2.24SRGN1.32ECRP1.03CCT6P1−0.19HGB2−1.17CEACAM62.22SPDYA1.31CRISP31.03SORD−0.21FAR2P2−1.17CD832.14TRAJ581.3TNFRSF13C0.81MORC3−0.23RBM12B-AS1−1.19JUNB2.07CLC1.3HLA-DRB10.45HLA-DRA−0.27IGHD−1.21JUN2.04NSUN61.28HLA-DRB50.34FAS−0.29CXorf31−1.25SLC2A32.04AHSP1.26SMAD40.33IPW−0.29KLHL42−1.42AREG2.01CAMP1.25CCR70.27TNFRSF13B−0.32RAD51D−1.44NR4A32.01PER11.24TNF0.24ZNF280D−0.33SNHG19−1.73S100A122CSTA1.24IGHD0.21MORC3−0.47FAR2P2−2.14RGCC1.99HBD1.22HLA-DRB10.13CXCR3−0.56PPBP−2.3YPEL51.99PTGER41.22HLA-DRB40.11MORC3−0.57ELANE1.97GADD45B1.2HLA-DRB30.06SORD−0.62PRTN31.9KMT2E-A S11.2GRK40.01CD1C−0.68PPP1R15A1.85SCARNA51.18CD80−0.73 Open table in a new tab CD, Cluster of differentiation; V500, violet 500; BV650, brilliant violet 650; APC-H7, allophycocyanin-H7; PE-Cy7, phycoerythrin-cyanin7; PerCP Cy 5.5, peridinin chlorophyll protein-cyanin 5.5; NK, natural killer cells; AF700, Alexa Fluor 700; HLA-DR, human leukocyte antigen-DR; BAFF-R, B cell activating factor receptor; TACI, transmembrane activator and CAML interactor; CXCR3/4, C-X-C chemokine receptor type 3/4; CCR7, C-C chemokine receptor type 7; Fas-L, Fas ligand.

Transcription factor AP-1 is constitutively activated and IRF4 drives growth and survival in ALK+ and ALK– anaplastic large cell lymphoma (ALCL). Here we demonstrate high-level BATF and BATF3 expression in ALCL. Both BATFs bind classical AP-1 motifs and interact with in ALCL deregulated AP-1 factors. Together with IRF4, they co-occupy AP-1-IRF composite elements, differentiating ALCL from non-ALCL. Gene-specific inactivation of BATFs, or global AP-1 inhibition results in ALCL growth retardation and/or cell death in vitro and in vivo. Furthermore, the AP-1-BATF module establishes TH17/group 3 innate lymphoid cells (ILC3)-associated gene expression in ALCL cells, including marker genes such as AHR, IL17F, IL22, IL26, IL23R and RORγt. Elevated IL-17A and IL-17F levels were detected in a subset of children and adolescents with ALK+ ALCL. Furthermore, a comprehensive analysis of primary lymphoma data confirms TH17–, and in particular ILC3-skewing in ALCL compared with PTCL. Finally, pharmacological inhibition of RORC as single treatment leads to cell death in ALCL cell lines and, in combination with the ALK inhibitor crizotinib, enforces death induction in ALK+ ALCL. Our data highlight the crucial role of AP-1/BATFs in ALCL and lead to the concept that some ALCL might originate from ILC3.

Cellular senescence induced by chemotherapy leads to the acquisition of stemness in cancer cells, which results in enhanced tumour-promoting capacity after forced release or spontaneous escape from the senescent cell-cycle arrest. Cells can go into senescence under stressed conditions to prevent the spread of potentially cancerous cells. Clemens Schmitt and colleagues show that cellular senescence induced by chemotherapy leads to the acquisition of 'stemness' in cancer cells, which allows them to escape senescence and promote tumour growth. Interestingly, such cells gain elevated tumour-initiating capacity compared with cells that have never undergone senescence. Cellular senescence is a stress-responsive cell-cycle arrest program that terminates the further expansion of (pre-)malignant cells1,2. Key signalling components of the senescence machinery, such as p16INK4a, p21CIP1 and p53, as well as trimethylation of lysine 9 at histone H3 (H3K9me3), also operate as critical regulators of stem-cell functions (which are collectively termed ‘stemness’)3. In cancer cells, a gain of stemness may have profound implications for tumour aggressiveness and clinical outcome. Here we investigated whether chemotherapy-induced senescence could change stem-cell-related properties of malignant cells. Gene expression and functional analyses comparing senescent and non-senescent B-cell lymphomas from Eμ-Myc transgenic mice revealed substantial upregulation of an adult tissue stem-cell signature, activated Wnt signalling, and distinct stem-cell markers in senescence. Using genetically switchable models of senescence targeting H3K9me3 or p53 to mimic spontaneous escape from the arrested condition, we found that cells released from senescence re-entered the cell cycle with strongly enhanced and Wnt-dependent clonogenic growth potential compared to virtually identical populations that had been equally exposed to chemotherapy but had never been senescent. In vivo, these previously senescent cells presented with a much higher tumour initiation potential. Notably, the temporary enforcement of senescence in p53-regulatable models of acute lymphoblastic leukaemia and acute myeloid leukaemia was found to reprogram non-stem bulk leukaemia cells into self-renewing, leukaemia-initiating stem cells. Our data, which are further supported by consistent results in human cancer cell lines and primary samples of human haematological malignancies, reveal that senescence-associated stemness is an unexpected, cell-autonomous feature that exerts its detrimental, highly aggressive growth potential upon escape from cell-cycle blockade, and is enriched in relapse tumours. These findings have profound implications for cancer therapy, and provide new mechanistic insights into the plasticity of cancer cells.

Clonal hematopoiesis of indeterminate potential (CHIP) occurs in an age-related manner and associates with an increased risk of hematologic cancer, atherosclerotic disease, and shorter overall survival. Little is known about the cell of origin, repartition patterns of clonal mutations within the hematopoietic differentiation tree, and its dynamics under evolutionary pressure. Using targeted sequencing, CHIP was identified in 121 out of 437 elderly individuals (27.7%). Variant allele frequencies (VAFs) of 91 mutations were studied in six peripheral blood cell fractions. VAFs were significantly higher in monocytes, granulocytes, and NK-cells compared to B- or T cells. In all cases with available bone marrow material, mutations could be identified in Lin−CD34+CD38− HSCs with subsequent expansion to myeloid primed progenitors. In 22 patients with solid cancer receiving (radio-)chemotherapy, longitudinal study of 32 mutations at 121 time points identified relative VAF changes of at least 50% in 13/32 mutations. VAFs of DNMT3A, were stable in 12/13 cases (P < .001). Cancer patients with a clonal mutation other than DNMT3A required more often red blood cell transfusions and dose reductions. Our results provide novel insights into cellular distribution of clonal mutations, their dynamics under chemotherapy, and advocate for systematic analyses for CHIP in cancer patients.

Increased serum ferritin (SF) is common in hematologic malignancies; however, its prognostic role in acute myeloid leukemia (AML) is not clearly established. We examined the impact of baseline SF on long-term survival in 137 intensively treated AML patients. Patients and baseline characteristics were retrieved from an AML database at Charité University Medical Center Berlin, Campus Virchow Clinic. After c-reactive protein (CRP)-based adjustment for inflammation, patients were grouped according to their baseline SF level. Survival analysis was performed accordingly. A significant decline in overall survival and relapse-free survival was observed in patients with high SF as compared to those with low SF. Furthermore, elevated baseline SF remained an independent poor prognostic factor within the multivariate analysis and was associated with a significant higher risk of relapse and non-relapse mortality (NRM). In conclusion, our data show that elevated baseline SF has a negative impact on long-term survival in intensively treated AML patients.

The MabThera and Involved field Radiotherapy study investigated efficacy and safety of involved field (IF) radiotherapy in combination with the anti-CD20 antibody Rituximab for early-stage follicular lymphoma (FL) in a prospective, single-arm multicenter phase 2 design. Eighty-five stage I-II FL patients received 8 cycles of Rituximab (375mg/m(2)) and IF irradiation (30/40 Gy). The primary endpoint was progression-free survival (PFS) 2 years from treatment start. Secondary endpoints were overall survival (OS), complete response rates, toxicity, quality of life, and minimal residual disease (MRD) response with protocol defined visits up to month 30. For the primary endpoint, PFS at 2 years was 85% for the intention-to-treat set. Long-term data were captured in selected sites and evaluated as post hoc analysis in the per protocol (PP) set: PFS and OS were 78% and 96% at 5 years with a median follow-up of 66 or 78 months, respectively. There were 17/76 recurrences in the PP set, of which 14 were outside the radiation volume only. MRD analyses revealed a clonal marker in 36% of patients at diagnosis. All but 1 marker positive patients experienced a molecular treatment response. There were 13 serious adverse events (4 related to the therapy) during the first 30 months. IF radiotherapy combined with Rituximab is well tolerated and highly efficient with low rates of recurrence in the first years in early-stage FL. The efficacy is comparable with more aggressive therapy approaches without compromising the quality of life and maintains for an extended follow-up of more than 5 years.

Abstract Adoptive T-cell therapy (ATT) efficacy is limited when targeting large solid tumors. The evaluation of ATT outcomes using accessory treatment would greatly benefit from an in vivo monitoring tool, allowing the detection of functional parameters of transferred T cells. Here, we generated transgenic bioluminescence imaging of T cells (BLITC) mice expressing an NFAT-dependent click-beetle luciferase and a constitutive Renilla luciferase, which supports concomitant in vivo analysis of migration and activation of T cells. Rapid transferability of our system to preestablished tumor models was demonstrated in the SV40-large T antigen model via both crossbreeding of BLITC mice into a T-cell receptor (TCR)-transgenic background and TCR transduction of BLITC T cells. We observed rapid tumor infiltration of BLITC CD8+ T cells followed by a burst-like activation that mirrored rejection kinetics. Using the BLITC reporter in the clinically relevant H-Y model, we performed female to male transfers and detected H-Y-specific alloreactivity (graft-versus-host disease) in vivo. In an H-Y solid tumor model, we found migration of adoptively transferred H-Y TCR-transgenic CD4+ T cells into the tumor, marked by transient activation. This suggests a rapid inactivation of infiltrating T cells by the tumor microenvironment, as confirmed by their expression of inhibitory receptors. In summary, the BLITC reporter system facilitates analysis of therapeutic parameters for ATT, is rapidly transferable to models of interest not restricted to tumor research, and is suitable for rapid screening of TCR clones for tumor rejection kinetics, as well as off-target effects. Cancer Immunol Res; 6(1); 110–20. ©2018 AACR.

To analyze the prognostic impact of distinct combinations of molecular aberrations, a retrospective analysis of 620 acute myeloid leukemia (AML) was performed. The mean patient's age was 57 (16-85) years. 47% of patients were characterized by abnormal karyotype: favorable (11%), intermediate (67%), and adverse (22%) risk. We analyzed NPM1, FLT3, DNMT3A, IDH1/2, and c-KIT mutations in diagnostic samples. Molecular genetics and cytogenetics considered adverse risk for 28% of patients, favorable for 26%, and intermediate in 46% of patients. More common mutations in whole cohort of patients and in patients with normal karyotype (NK) were NPM1 (22% and 36%), FLT3- ITD (23% and 31%). In addition, DNMT3A and IDH1 mutations were found in 18% and 17% of NK-AML patients, respectively. IDH2 mutations were associated with older patients age (p=.0001). DNMT3A and NPM1 mutations were more frequently detected in patients with monocyte/myelomonocytic differentiation of leukemic blasts (p=.0001). C-kit mutations were more common in Core Binding Factor AML.

Purpose Gemcitabine is standard of care in the adjuvant treatment of resectable pancreatic ductal adenocarcinoma (PDAC). The epidermal growth factor receptor tyrosine kinase inhibitor erlotinib in combination with gemcitabine has shown efficacy in the treatment of advanced PDAC and was considered to improve survival in patients with primarily resectable PDAC after R0 resection. Patients and Methods In an open-label, multicenter trial, patients were randomly assigned to one of two study arms: gemcitabine 1,000 mg/m2 days 1, 8, 15, every 4 weeks plus erlotinib 100 mg once per day (GemErlo) or gemcitabine (Gem) alone for six cycles. The primary end point of the study was to improve disease-free survival (DFS) from 14 to 18 months by adding erlotinib to gemcitabine. Results In all, 436 patients were randomly assigned at 57 study centers between April 2008 and July 2013. A total of 361 instances (83%) of disease recurrence were observed after a median follow-up of 54 months. Median treatment duration was 22 weeks in both arms. There was no difference in median DFS (GemErlo 11.4 months; Gem 11.4 months) or median overall survival (GemErlo 24.5 months; Gem 26.5 months). There was a trend toward long-term survival in favor of GemErlo (estimated survival after 1, 2, and 5 years for GemErlo was 77%, 53%, and 25% v 79%, 54%, and 20% for Gem, respectively). The occurrence or the grade of rash was not associated with a better survival in the GemErlo arm. Conclusion To the best of our knowledge, CONKO-005 is the first study to investigate the combination of chemotherapy and a targeted therapy in the adjuvant treatment of PDAC. GemErlo for 24 weeks did not improve DFS or overall survival over Gem.

Introduction: Irrespective of its genomic B-cell origin, classical Hodgkin's lymphoma (cHL) is characterized by the virtual lack of gene products whose expression constitutes the B-cell phenotype. Epigenetic repression of B-cell-specific genes was previously postulated to contribute to the lost B-cell phenotype in cHL. Restoration of the B-cell phenotype may not only revert a hallmark of cHL but provide a new Achilles' heel by sensitizing cHL to clinically established antibody therapies targeting B-cell surface receptors as well as small compounds interfering with B-cell receptor (BCR) signaling. Methods: We engineered cHL cell lines to carry a CD19 reporter, and conducted a high-throughput pharmacological screening with more than 28,000 compounds to identify drugs that promote re-expression of the B-cell phenotype. Results: We found three chemicals to robustly enhance CD19 transcription. Since two of them reportedly interfere with epigenetic regulators, we performed chromatin immunoprecipitation assays, showing that these compounds lowered transcriptionally repressive lysine 9-trimethylated histone H3 (H3K9me3) levels at the CD19 promoter. Inhibition of the H3K9-methyltransferase EHMT2, a possible target structure of these two compounds, by BIX-01294 or shRNA-mediated knockdown resulted in increased CD19 transcript levels, suggesting that EHMT2 might be involved in repression of the B-cell phenotype in cHL. Furthermore, the anti-leukemic and differentiation-promoting agents arsenic trioxide (ATO) and all-trans retinoic acid (ATRA), both not part of the screened library, were found to reconstitute the silenced B-cell transcriptional program and impair viability of cHL cell lines. In combination with a screening-identified chemical, ATO evoked re-expression of the CD20 surface receptor, which could be therapeutically exploited by enabling CD20 antibody-mediated direct apoptosis and antibody-dependent cellular cytotoxicity of Hodgkin cells. Even more strikingly, restoration of the B-cell phenotype profoundly sensitized an expanded panel of eight cHL cells towards the B-cell Non-Hodgkin's lymphoma-tailored small compound inhibitors of BCR signaling, ibrutinib and idelalisib, where dramatic death rates were observed after priming with two restore agents. Further investigations, including pre-clinical mouse trials, are currently in progress and will be reported at the meeting. Conclusions: In essence, we present here a novel “Restore & Target” strategy that builds on the re-expression of a lost tumor cell phenotype in the first place followed by it specific exploration as druggable vulnerability in a subsequent step. Such a strategy would expand the arsenal of treatment options for cHL by a “chemo-free” combination, and might not only be of interest for relapsed or refractory patients. Keywords: B-cell receptor (BCR); CD20; Hodgkin lymphoma (HL).

Background: Clonal hematopoiesis of indeterminate potential (CHIP) is defined by the presence of hematologic cancer associated mutations in the peripheral blood (PB) of at least 10% of elderly people without history of hematologic disorders (Genovese et al ., NEJM, 2014; Jaiswal et al ., NEJM, 2014). At present, caution is needed when predicting clinical consequences from CHIP in healthy people. An essential step towards a better understanding of CHIP requires identification of the cell of origin, clonal expansion patterns within the hematopoietic differentiation tree, and its dynamic behavior under stress scenarios (e.g. chemotherapy).

BCR-ABL-positive acute myeloid leukemia (AML) is a rare subtype of AML that is now included as a provisional entity in the 2016 revised WHO classification of myeloid malignancies. Since a clear distinction between de novo BCR-ABL+ AML and chronic myeloid leukemia (CML) blast crisis is challenging in many cases, the existence of de novo BCR-ABL+ AML has been a matter of debate for a long time. However, there is increasing evidence suggesting that BCR-ABL+ AML is in fact a distinct subgroup of AML. In this study, we analyzed all published cases since 1975 as well as cases from our institution in order to present common clinical and molecular features of this rare disease. Our analysis shows that BCR-ABL predominantly occurs in AML-NOS, CBF leukemia, and AML with myelodysplasia-related changes. The most common BCR-ABL transcripts (p190 and p210) are nearly equally distributed. Based on the analysis of published data, we provide a clinical algorithm for the initial differential diagnosis of BCR-ABL+ AML. The prognosis of BCR-ABL+ AML seems to depend on the cytogenetic and/or molecular background rather than on BCR-ABL itself. A therapy with tyrosine kinase inhibitors (TKIs) such as imatinib, dasatinib, or nilotinib is reasonable, but-due to a lack of systematic clinical data-their use cannot be routinely recommended in first-line therapy. Beyond first-line treatment of AML, the use of TKI remains an individual decision, both in combination with intensive chemotherapy and/or as a bridge to allogeneic stem cell transplantation. In each single case, potential benefits have to be weighed against potential risks.

e15717Background: The combination of nabpaclitaxel/gemcitabine (AG) is one standard of care treatment for patients (pts) with advanced pancreatic adenocarcinoma (APC). Only insufficient data accord...

In Chronic Myeloid Leukemia (CML), standard treatment consists of modern tyrosine-kinase inhibitors (TKI). Nevertheless, there is evidence that immune responses against leukemia-associated antigens (LAA) may play an important role in disease control. Dendritic cell (DC)- based immunotherapy is able to induce T cell responses against LAA and might therefore pose an interesting therapeutic option in CML, especially in the setting of minimal residual disease (MRD). GMP production of DC for clinical vaccination remains a time- and cost- intensive procedure and standardized DC generation is warranted. We asked whether maturation-induction with IFN-γ and IFN-α has an influence on functional properties of DC derived from peripheral blood mononuclear cells (PBMC) in CML patients. Monocyte-derived DC from healthy donors and from patients with CML were analyzed after maturation-induction with our TNF-α-containing standard cytokine cocktail with or without addition of IFN-α and/or IFN-γ. Our results confirm that the addition of IFN-γ leads to enhanced IL-12 secretion in healthy donors. In contrast, in CML patients, IFN-γ was not able to increase IL-12 secretion, possibly due to a higher degree of cell adherence and lower cell yield during the cell culture. Our data suggest, that- in contrast to healthy donors-, additional interferons are not beneficial for maturation induction during large-scale DC production in patients with CML.

ObjectivesWith increasing numbers of therapeutic options in inoperable pancreatic cancer (PAC), patients tend to receive more than just a first line (FL) therapy.MethodsAll patients who started FL for PAC at our institution (1997-2012) were retrospectively studied to identify patient's and treatment characteristics. Significant parameters in regard to second-line (SL) related survival were looked for as the basis for a prognostic model. This score was validated in a patient cohort from the CONKO-003 study.ResultsTwo hundred eighty of 521 (53.7%) patients received SL therapy, median overall survival (OS) from the beginning of SL (OS2) was 5.1 months. Significant more SL patients had undergone surgery, a higher Karnofsky performance state (KPS) and a duration of FL longer than 4 months.Prognostic factors impacting OS2 were KPS, carbohydrate antigen 19-9 levels at start of SL and the duration of FL. These 3 factors establish a prognostic score-validated in CONKO-003-for SL patients with 3 subgroups: "good" (median OS2, 9.3 months), "intermediate" (median OS2, 7.1 months), "poor" prognosis (median OS2, 3.8 months; P < 0.001).ConclusionsAmong patients with PAC, more than 50% receive SL therapy. Our prognostic model identifies 3 subgroups and can identify patients with a maximum benefit of SL therapy.

Flow cytometric immunophenotyping represents an indispensable tool in hematological and immunological diagnostics. The most frequent indications include lymphocyte phenotyping and the diagnosis and monitoring of benign and malignant hematologic diseases. The role of immunophenotyping in clinical practice is evolving rapidly. This review provides an overview of its current applications and limitations.

by undertaking a retrospective longitudinal study in CML patients in BC. We identified CML patients in BC from our previous study for whom we had 5 or more serial samples in total prior to and/or following progression to the blastic disease phase [1] . The 5 CML patients (2 female, 3 male) had a median age of 62 years (range 46–76), and 4 were treated with one or more of the tyrosine kinase inhibitors (TKIs) ( table 1 ). The other had undergone stem cell transplant (SCT). The study was performed using previously described standard molecular biology methods [1] . The MSI2 expression in 4 of the 5 patients appeared to mirror the BCR-ABL1 kinetics ( fig. 1 ). With the exception of the post-SCT samples of patient 5, the MSI2 expressions were lower than the BCR-ABL1 transcript numbers. BCR-ABL1 was undetectable in the post-SCT samples. The notion that regular and close monitoring of MSI2 mRNA levels in serial samples might identify patients at risk of progressing to BC was not supported by this study. Moreover, the data presented here show that an increase in MSI2 transcripts does not precede an increase in BCRABL1 mRNA levels. More importantly, this raises the question as to whether increased MSI2 expression in BC is the cause or effect. The early identification of chronic myeloid leukemia (CML) patients in the chronic phase (CP) who are at risk of progression to blast crisis (BC) would enhance the clinical management of these individuals. Recently, we verified reported data showing increased levels of Musashi 2 (MSI2) transcripts in CML patients in BC compared to those in CP, implying a role for MSI2 in CML transformation [1–3] . MSI2 , a posttranscriptional regulator, is reported to control hematopoietic stem cell self-renewal and differentiation [2, 3] . MSI2 achieves this control by binding to mRNA and thereby impeding translation of the target RNA [2, 3] . Interestingly, we found the MSI2 transcript numbers were significantly lower (p < 0.0001) in CML patients in CP at diagnosis than normal adult blood control samples [1] . Based on these observations and knowing that MSI2 expression is restricted to early hematopoietic progenitors, we reasoned that regular close monitoring of its mRNA levels might identify patients at risk of transformation prior to detecting increasing levels of BCR-ABL1 . If confirmed, it would provide a window in which to intervene early with alternative therapy with a view to reversing the progression, before the patient becomes refractory to further treatment. We addressed this notion Received: January 14, 2016 Accepted: March 1, 2016 Published online: May 10, 2016