EZH2 Mutations Are Related to Low Blast Percentage in Bone Marrow and -7/del(7q) in De Novo Acute Myeloid Leukemia

<div><p>The purpose of the present work was to determine the incidence and clinical implications of somatic EZH2 mutations in 714 patients with de novo acute myelogenous leukemia by sequencing the entire coding region. EZH2 mutations were identified in 13/714 (1.8%) of AML patients were found to be more common in males (<i>P</i> = 0.033). The presence of EZH2 mutations was significantly associated with lower blast percentage (21–30%) in bone marrow (<i>P</i><0.0001) and -7/del(7q) (<i>P</i> = 0.025). There were no differences in the incidence of mutation in 13 genes, ASXL1, CBL, c-KIT, DNMT3A, FLT3, IDH1, IDH2, MLL, NPM1, NRAS, RUNX1, TET2, and WT1, between patients with and without EZH2 mutations. No difference in complete remission, event-free survival, or overall survival was observed between patients with and without EZH2 mutation (<i>P</i>>0.05). Overall, these results showed EZH2 mutation in de novo acute myeloid leukemia as a recurrent genetic abnormality to be associated with lower blast percentage in BM and -7/del(7q).</p></div

Comparison of clinical and laboratory features between AML patients with and without EZH2 mutation.

ψ<p>Risk status: Better-risk: inv(16)/t(16;16), t(8;21),t(15;17); Intermediate-risk: normal, +8, t(9;11), other undefined risk; Poor-risk: complex, −5, 5q−, −7, 7q−, 11q23(non t(9;11)), inv(3), t(3;3), t(6;9), t(9;22).</p><p>(A) Structure of the EZH2 protein and location of EZH2 mutations. (B) DNA sequencing chromatograms of AML genomic DNA samples showing 14 mutations in 13 AML patients.</p

Kaplan-Meier survival curves according to EZH2 mutation status.

<p>(A) EFS in de novo AML patients according to EZH2 mutations. The green line represents patients with mutated EZH2 (n = 12); and magenta line, patients with unmutated EZH2 (n = 277; <i>P</i> = 0.2283); (B) OS in de novo AML patients according to EZH2 mutations. The green line represents patients with mutated EZH2 (n = 12); and the magenta line represents patients with unmutated EZH2 (n = 277; P = 0.5001).</p

Additional file 1 of CD7-directed CAR T-cell therapy: a potential immunotherapy strategy for relapsed/refractory acute myeloid leukemia

Additional file 1: Figure S1. Cytotoxicity and cytokines analysis of the CD7 CAR T-cells. Figure S2. Diagrammatic sketch of the treatments and response. Figure S3. Infusion-related hepatic toxicities. Figure S4. Flow cytometry analysis of the fraction of T-cells and NK cells in the PB after infusion. Figure S5. Flow cytometry of the T-cell fractions in the PB after infusion of CART cells. Table S1. The result of SNP array (Cytoscan 750K/HD) at diagnosis. Table S2. A panel of 222 genes detected by next-generation sequencing. Table S3. A panel of targeted transcriptome RNA sequencing (RNA-seq)

Genomic array data.

<p>A: The respective breakpoints and FISH clones at ACTR2 and inside RAF1 of der(2)t(2;3)(p14;p25) in KARPAS-1106P as revealed by genomic arrays expands the FISH data. B: Color coded plots of FARAGE (purple), KARPAS-1106P (pink), MEDB-1 (blue), U-2940 (green)—show genomic copy number (solid) and LOH (barred) at 12 loci (1p12, 2p15, 2p16, 6q23, 7p22, 7q31, 8q24, 9p21, 9p24, 16p13, 15q23, 19q13, together with OMIM genes below. Copy number polymorphic regions (<a href="http://dgv.tcag.ca/dgv/app/home" target="_blank">http://dgv.tcag.ca/dgv/app/home</a>) are shown between, listing gains (blue), losses (red), and copy number neutral alterations, such as inversions (gray).</p

Spectral Karyotyping (SKY): FARAGE (A), KARPAS-1106P (B), MEDB-1 (C) and U-2940 (D).

<p>Inverse DAPI G-banding shown left of corresponding pseudocolored SKY. Arrows show deletions (white), duplications (red), and translocations (ochre). After trypsin G-banding (not shown) analyses were extended to prepare consensus karyotypes. Individual metaphases sometimes departed from consensus karyotypes, e.g. note loss of Y-chromosome in MEDB-1. Note absence of key cytogenetic rearrangements and relative lack of rearrangement when compared to cHL cell lines. Thus the salient cytogenetic features of PMBL are essentially negative with respect to neighboring entities, cHL and PMBL.</p

Fluorescence in situ hybridization (FISH) Analysis.

<p>A-D: Shows the cytogenetic configuration of focal deletions affecting 16p13 including the SOCS1 locus (arrows) in three PMBL cell lines: monoallelic in FARAGE, biallelic in KARPAS1106P and U-2940 effected by t(16;16) rearrangement, but absent in MEDB-1. E/F: Shows deletions affecting the PTPN1 locus in KARPAS-1106P (E) and MEDB-1 (F). Twin partial chromosome 20 long-arm deletions in KARPAS-1106P effect PTPN1 monosomy, while in MEDB-1 where the gene is mutated this locus escapes deletion despite proximity to a translocation breakpoint therein. G-I: Shows analysis of der(3)t(2;3)(p14;p25) in KARPAS-1106P. The respective breakpoints at 2p14 and 3p25 were placed close to ACTR2 (within clone RP11-441L10) and RAF1 (within RP11-148M13). Coordinates and labelling scheme are shown below. Coordinates (MBp) are from HG19. FISH was performed using tilepath BAC clones.</p

Gene expression at genomically rearranged loci.

<p>A: Microarray expression data for genes implicated at CNV (mainly deletions). Note, for example gene silencing accompanying focal biallelic deletions at multiple loci: including, CD58 at 1p12 in KARPAS-1106P; MSH6 and FBXO11 at 2p26 in FARAGE; TNFAIP3, AIMP2 at 6q23 in U2940; EIF2AK1 at 7p22 in U-2940; CDKN2A at 9p21 in KARPAS-1106P and U-2940; CD274 at 9p24 in U-2940; SOCS1 at 16p13 in KARPAS-1106P and U-2940; and KIAA0355 at 19q13 in U-2940. B: Shows qPCR expression of select target genes at recurrent PMBL amplicons (2p15, 9p24) and a deletion (16p13) set against TBP reference in cell lines FARAGE, KARPAS-1106P, MEDB-1, and U-2940 (PMBL) shown blue, alongside reference SU-DHL-8 (DLBCL) shown red. Diamonds indicate undetectable expression. Quantitative data were verified by twofold or more biological replication.</p