Glycosylation pattern and cell surface expression of FLT3 receptor differ in FLT3 mutants.

<p>(A) FLT3 mutants were cultured without IL-3 for 24 hours prior to lysis. One out of three independently representative experiments is shown. (B) To illustrate the expression of the 150/130 kDa forms of FLT3 semi-quantitive analyses of western blot band intensity were performed. Depicted is the ratio of the 150 kDa to the 130 kDa FLT3 form. Values are expressed as mean +/− S.D. of three independent experiments. Ratio of 150 kDa to 130 kDa form of FLT3 was significantly different for each FLT3-TKD (p≤0.009) and each FLT3-ITD (p≤0.002) cell line, but not for FLT3-V592A and -I867S expressing cells compared to FLT3-WT expressing cells. FLT3-WT expressing cells showed a significant differnence to the groups of FLT3-WT-like mutants (p = 0.039), FLT3-TKD mutants (≤0.001) and FLT3-ITD mutants (p≤0.001). Further FLT3-WT like mutants were significantly different compared to the FLT3-TKD group (p≤0.001) and FLT3-TKD mutants were significantly different compared to FLT3-ITD cell lines (p = 0.004). (*) indicates significance of grouped analyses and individually compared to FLT3-WT expressing cells. (C) Ba/F3 cell lines stably expressing the indicated FLT3 constructs were stained with CD-135 antibody and analyzed by flow cytometry. Expression is depicted as difference of geometric mean to isotype control (ΔGmean). Values are expressed as means +/− S.D. of four independent experiments. (*) indicates significance compared to FLT3-WT expressing cells.</p

FLT3 mutants display a wide range of activating phenotypes in Ba/F3 cells.

<p>Ba/F3 cells stably transduced with indicated constructs were seeded at a density of 4×10⁴ cells/ml and cultured in presence and absence of 10 ng/ml IL-3. (A) Viable cells were counted after 72 h using trypan blue exclusion. Control indicates viable Ba/F3 MIY cells treated with 10 ng/ml IL-3. Values are expressed as mean +/− S.D. of nine independent experiments. (*) indicates significant higher growth compared to FLT3-WT expressing cells. (B) After incubation for 48 hours cells were stained with Annexin-V and 7-AAD. The percentage of apoptotic cells was determined using flow cytometry. Values are expressed as mean +/− S.D. of at least three independent experiments. (*) indicates significant higher apoptosis compared to FLT3-WT expressing cells. (C) Ba/F3 cells expressing FLT3 mutations were cultured without IL-3 for 24 hours. Cells were treated with 100 ng FLT3-ligand for 10 minutes prior to lysis to analyze phosphorylation of FLT3 receptor at Tyr591 in a chemiluminescent ELISA assay. The differences of FLT3-ligand stimulated to untreated results are shown. Values are expressed as mean +/− S.D. of three independent experiments. For further statistical analyses FLT3 mutants were divided into groups of FLT3-WT-like (FLT3-I867S/-V592A), FLT3-TKD (FLT3-D839G/-D835V/-D835Y) and FLT3-ITD (FLT3-ITD1/-ITD2/-ITD3) cell lines. In grouped analysis FLT3-WT expressing cells were significantly different compared to the FLT3-TKD (p = 0.019) but not to FLT3-WT-like cell lines. Groups of FLT3-WT-like, FLT3-TKD and FLT3-ITD cell lines were significant among each other (p≤0.030). (*) indicates significance of grouped analyses and individually compared to FLT3-WT expressing cells. RLU: Relative light units.</p

Prognostic impact and genetic stability of FLT3-ITD and FLT3-TKD mutations in AML.

<p>(A) OS in 672 patients was significantly different between <i>FLT3</i>-mutated patients compared to patients with <i>FLT3</i>-WT*. OS in patients with <i>FLT3</i>-TKD mutations was not different compared to <i>FLT3</i>-WT* expressing patients (HR: 0.8, 95% CI: 0.7–1.7). Patients with a <i>FLT3</i>-ITD showed a significantly worse OS compared to those with <i>FLT3</i>-WT* (HR: 1.4, 95% CI: 1.1–1.8). There was no significant difference in the OS of patients with both <i>FLT3</i>-ITD and <i>FLT3</i>-TKD mutation compared to those with <i>FLT3</i>-ITD (HR 1.1, 95% CI: 0.3–3.4). (B) RFS in 443 patients in first complete remission was significantly different between <i>FLT3</i>-mutated patients compared to patients with <i>FLT3</i>-WT*. Patients with <i>FLT3</i>-TKD mutation had no significant superior RFS compared to <i>FLT3</i>-WT* (HR: 0.8, 95% CI: 0.4–1.4). Patients with a <i>FLT3</i>-ITD showed a significantly worse RFS compared to those with <i>FLT3</i>-WT* (HR: 1.7, 95% CI: 1.3–2.2). There was no significant difference in the RFS of patients with both <i>FLT3</i>-ITD and <i>FLT3</i>-TKD mutation compared to those with <i>FLT3</i>-ITD (HR 1.2, 95% CI: 0.3–5.0). (C) 10 of 113 patients positive for <i>FLT3</i>-WT* at diagnosis acquired a <i>FLT3</i> mutation at relapse. 27 of 35 (77%) patients with a <i>FLT3</i>-ITD at initial diagnosis displayed a <i>FLT3</i>-ITD at relapse, whereas the majority of patients with a <i>FLT3</i>-TKD mutation (7/11; 63%) lost this mutation at relapse. (D) <i>FLT3</i>-ITD mRNA levels in 20 patients with a <i>FLT3</i>-ITD at diagnosis and at relapse were calculated. <i>FLT3</i>-ITD mRNA levels were calculated as following: (<i>FLT3</i>-ITD/<i>FLT3</i>-WT)/(<i>FLT3</i>-ITD/<i>FLT3</i>-WT+1). Median <i>FLT3</i>-ITD mRNA level was significantly higher at the time of relapse compared to first diagnosis (0.54 [range 0.37–1.00] versus 0.40 [range 0.08–0.88]; p&lt;0.001, Wilcoxon test).</p

FLT3-ITD mutants induce STAT5 and Tyr 591 phosphorylation whereas FLT3 point mutations rely on AKT and MAPK activation.

<p>(A) Ba/F3 cells expressing FLT3 mutations were cultured without IL-3 for 24 hours prior to lysis. Blots were probed against STAT5 pTyr694, AKT pSer473 and MAPK pThr202/pTyr204 stripped and subsequently reprobed against total STAT5, AKT and MAPK. One out of three independent representative experiments is shown. (B) Semi-quantitive analysis of (p)STAT5 band intensity was performed to calculate the ratio between pSTAT5 and total STAT5. Values are expressed as mean +/− S.D. of three independent experiments. (*) indicates significance to FLT3-WT expressing cells. The signal intensity of Ba/F3 MIY expressing cells has been subtracted from FLT3-WT and FLT3 mutant values. (C) Ba/F3 cells expressing FLT3 mutations were cultured without IL-3 for 24 hours prior to lysis. Equal amount of whole cell lysates were used in a chemiluminescent ELISA assay detecting phosphorylation of FLT3 receptor at Tyr591. Values are expressed as mean +/− S.D. of three independent experiments. Grouped analysis of FLT3 mutants revealed significant differences between FLT3-WT expressing cells and FLT3-WT-like cell lines (p = 0.045) as well as FLT3-WT expressing cells and FLT3-ITD cell lines (p = 0.005). Further the group of FLT3-ITD cell lines showed significant differences compared to FLT3-WT-like mutants (p = 0.008) and FLT3-TKD cell lines (p≤0.001). (*) indicates significance of grouped analyses and individually compared to FLT3-WT expressing cells. The signal intensity of Ba/F3 MIY expressing cells has been subtracted from FLT3-WT and FLT3 mutant values. RLU: Relative light units. (D) 4×10⁴ cells/ml stably transduced with indicated constructs, were cultured in presence or absence of 2 µM MK 2206 and/or 10 ng/ml IL-3. Cells were counted after 72 hours by trypan blue exclusion. (*) indicates significant growth reduction by MK 2206 compared to untreated cells. Proliferation with MK 2206 is shown in relation to untreated cells. Control indicates for Ba/F3 MIY expressing cells treated with 2 µM MK 2206 in the presence of IL-3. Values are expressed as mean +/− S.D. of three independent experiments.</p

Multivariable analysis of prognostic factors for OS, RFS and CR.

<p>To evaluate the independent prognostic impact of FLT3-ITD and FLT3-TKD on OS, RFS and CR, all candidate prognostic factors were included in the Cox regression model without selection of variables. The analyses were performed using 535 complete datasets of patients with regard to OS for the candidate prognostic factors. HR: Hazard Ratio; CI: confidence interval; CL: confidence limit.</p

Heatmap of FLT3 signaling and gene expression profiles in AML blast cells.

<p>(A) Graphic illustration of FLT3 mutants with respect to biological characteristics according to performed experiments. FLT3 mutants were clustered hierarchically based on similarity and displayed in columns. Experiments were ordered in rows as indicated. (B) Heatmap of probe sets differentially expressed in <i>FLT3</i>-ITD, -TKD and -WT CN-AML. The 27 probe sets were selected according to FLT3 and NPM1 mutation status (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0089560#pone.0089560.s005" target="_blank">Table S3</a>) with following characteristics: Log fold change &gt; or &lt;1.5. If no probe set met this criterion, the ten most significant probe sets were selected. Rows: probe sets (n = 27); Columns: patients (n = 213).</p

Overall Survival (OS) and Relapse-Free Survival (RFS) in 349 patients with cytogenetically normal acute myeloid leukemia and <i>NPM1</i> mutation treated in the AMLCG99 study.

<p>(A) OS in patients with <i>NPM1</i> type A mutation versus <i>NPM1</i> rare type mutation. (B) OS in patients with <i>NPM1</i> type A mutation versus <i>NPM1</i> rare type mutation with or without an additional <i>FLT3-ITD</i>. (C) RFS in patients with <i>NPM1</i> type A mutation versus <i>NPM1</i> rare type mutation. (D) RFS in patients with <i>NPM1</i> type A mutation versus <i>NPM1</i> rare type mutation with or without an additional <i>FLT3-ITD</i>. <b>Abbreviations</b>: CR, complete remission; <i>FLT3-ITD</i>+, presence of an internal tandem duplication in the fms-related tyrosine 3 gene; <i>FLT3-ITD</i>-, absence of an internal tandem duplication in the fms-related tyrosine 3 gene; <i>NPM1-A</i>, mutation in the nucleophosmin gene consisting of an insertion of the tetranucleotide TCTG; <i>NPM1-RA</i>, mutation in the nucleophosmin gene other than type A.</p

Multivariable Cox regression models for OS and RFS in 349 <i>NPM1</i>-mutated patients.

<p>White blood cell count, platelet count, hemoglobin level, lactase dehydrogenase level, bone marrow blasts, de novo AML versus non de novo AML, performance status, sex, age, type A versus rare type <i>NPM1</i> mutation, <i>FLT3-ITD</i>, monoallelic <i>CEBPA</i> mutations, biallelic <i>CEBPA</i> mutations were included in the Cox regression models for OS and RFS with backward elimination. The analyses were performed using 313 patients for OS and 227 RFS who had data for all these variables. A p-value of <0.05 was considered as indicating significant differences. All parameters that did not have a significant impact on OS or RFS are not shown in the table, except for the <i>NPM1</i> mutation type.</p><p><b>Abbreviations</b>: <i>CEBPA</i>, CCAAT/enhancer-binding protein alpha gene; CI, confidence interval; <i>FLT3-ITD</i>, internal tandem duplication of the <i>FLT3</i> gene; HR, hazard ratio; negative, absence of <i>FLT3-ITD</i>; <i>NPM1</i>, nucleophosmin gene; <i>NPM1-A</i>, mutation in the nucleophosmin gene leading to the insertion of the tetranucleotide TCTG; <i>NPM1-RA</i>, mutation in the nucleophosmin gene other than type A; OS, Overall survival; p, p value; positive, presence of <i>FLT3-ITD</i>; RFS, Relapse-free survival; WBC, white blood cell count.</p><p>Multivariable Cox regression models for OS and RFS in 349 <i>NPM1</i>-mutated patients.</p

PDX AML cells allow genetic engineering without altering molecular sample characteristics.

<p><b>(A)</b> Scheme of the process of generating transgenic PDX (t-PDX) AML cells. PDX cells were transduced after first or second retransplantation cycle. <b>(B)</b> Scheme of the vector constructs. <b>(C)</b> Transduction rate in t-PDX AML cells after lentiviral transduction and cell amplification in mice was measured by FACS analysis of fluorochrome or NGFR expression. Each mark visualizes data obtained from a single transduction. Open mark: no transgenic cells were detectable. <b>(D)</b> Enrichment of transgenic cells using flow cytometry was measured using mCherry expression after cell amplification in mice. <b>(E)</b> Genetic engineering does not alter immunophenotype; primary cells, untransduced PDX cells after fourth retransplantation and enriched transgenic t-PDX cells were analyzed by multicolor flow cytometry; specific fluorescence intensity is depicted. See also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0120925#pone.0120925.s003" target="_blank">S3C Fig.</a> for exemplary FACS plots of AML-372. Raw data is depicted in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0120925#pone.0120925.s011" target="_blank">S3 Table</a>. <b>(F)</b> Genetic engineering does not markedly alter AML-specific mutations; genomic DNA was isolated out of primary cells, untransduced PDX cells and enriched transgenic t-PDX cells; VAF of mutations was profiled by targeted resequencing. <i>BCOR</i> (BCL-6 corepressor); <i>KRAS</i> (Kirsten rat sarcoma viral oncogene homolog); <i>NRAS</i> (neuroblastoma RAS viral oncogene homolog); <i>TP53</i> (tumor protein p53). Raw data is depicted in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0120925#pone.0120925.s010" target="_blank">S2 Table</a>.</p

BLI is highly sensitive and reliable in single mice.

<p><b>(A)</b> 1x10⁵ t-PDX AML-372 cells were injected into two mice. At indicated days after cell injection, mice were monitored by BLI. Images of one representative mouse are shown. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0120925#pone.0120925.s005" target="_blank">S5A Fig.</a> for further images. <b>(B)</b> BLI signals from the kinetic in <b>A</b> were quantified in both animals (diamonds); cells positive for both hCD45 and hCD33 in PB were analyzed in parallel (circles). <b>(C)</b> t-PDX AML-372 cells were injected into three mice per group at absolute numbers indicated; 1 and 8 days after cell injection, mice were monitored by BLI; images are shown of one representative mouse per group.</p