High Expression of <i>c-kit</i> mRNA Predicts Unfavorable Outcome in Adult Patients with t(8;21) Acute Myeloid Leukemia

<div><p>The reason that a certain subgroup of acute myeloid leukemia (AML) patients with t(8;21) translocation (generating the <i>AML1/ETO</i> fusion gene) displays a poor survival remains elusive. The proto-oncogene <i>c-kit</i> is expressed in approximately 80% of AML cases. The kinase domain mutation of the <i>c-kit</i> gene, one of the most common gain-of-function mutations associated with t(8;21) AML, predicts higher relapse risk and poor prognosis. However, the role of <i>c-kit</i> high expression in t(8;21) AML remains poorly understood. Here we evaluated the prognostic significance of <i>c-kit</i> expression levels in AML patients. The mRNA expression of <i>c-kit</i> was determined by real-time quantitative reverse transcription PCR in 132 adult AML patients. Patients were grouped into quartiles according to <i>c-kit</i> expression levels (Q1–Q4, each quartile containing 25% of patients) and divided into <i>c-kit</i> high (Q4; n = 33) and <i>c-kit</i> low (Q1–Q3; n = 99). High <i>c-kit</i> expression was associated with <i>AML1/ETO-</i>positive and with <i>c-kit</i> mutation. Of note, 35.8% of the <i>AML1/ETO-</i>positive AML patients carrying wild-type <i>c-kit</i> expressed high levels of <i>c-kit</i>, suggesting that other factors are involved in <i>c-kit</i> overexpression. High <i>c-kit</i> expression was associated with inferior overall and event-free survival in <i>AML1/ETO-</i>positive patients and was independently predictive for overall and event-free survival in multivariate analyses in a <i>c-kit</i> mutation-independent manner. Thus, high <i>c-kit</i> expression serves as a reliable molecular marker for poor prognosis, supporting a pathogenetic role of <i>c-kit</i> signaling in <i>AML1/ETO-</i>positive AML. <i>AML1/ETO-</i>positive patients with high <i>c-kit</i> expression might benefit from early treatment modifications and molecular target therapies.</p></div

Selective high expression of <i>c-kit</i> in <i>AML1/ETO</i>-positive AML cell lines and patients.

<p><b>(A)</b> qPCR showing <i>c-kit</i> expression in myeloid leukemia cell lines. CML-BC: chronic myeloid leukemia in blast crisis. Bars indicate the mean±SEM from three independent experiments. <i>ABL1</i> levels were measured for normalization. <b>(B)</b> Normalized <i>c-kit</i> expression in pretreatment samples of 461 patients with <i>de novo</i> AML (GEO database, GSE6891). The gene expression was determined using gene-expression arrays (Affymetrix HGU133 Plus 2.0 GeneChips), which reflected by the intensity of hybridization of labeled mRNA to the gene chip. Median values are depicted by the horizontal lines. The Mann-Whitney U test was used to compare expression levels between groups. <b>(C)</b> qPCR showing <i>c-kit</i> expression level in bone marrow samples from untreated AML patients at diagnosis and healthy donors. <i>ABL1</i> levels were measured for normalization. Median values are depicted by the horizontal lines. The Mann-Whitney U test was used to compare expression levels between groups.</p

The GFPuv-labeled <i>Psa</i> colonization and migration in kiwifruit leaf veins.

<p>The inoculated leaves were observed under a fluorescence stereomicroscope (Leica MZ12) at different days after inoculation. Bar = 2 mm.</p

Transmission electron micrographs of cross sections of kiwifruit twigs infected with the GFPuv-labeled <i>Psa</i> 5 DAI.

<p>(a), Bacterial cells in intercellular spaces of the parenchyma. Bar = 2 μm; (b), bacterial colonization in parenchyma cells where the cell walls are ruptured and organelles have disintegrated. Bar = 2 μm; (c), bacterial colonization in intercellular spaces where the middle lamella tended to be ruptured. Bar = 0.5 μm.</p

Higher expression of <i>c-kit</i> in <i>AML1/ETO-</i>positive AML predicts more inferior prognosis.

<p><b>(A-C)</b> Kaplan-Meier estimate for OS and EFS in indicated patient subgroups. Survival curves were compared using log-rank test.</p

Positive correlation between <i>AML1/ETO</i> and <i>c-kit</i> expression levels in <i>AML1/ETO</i>-positive AML.

<p><b>(A)</b> The patients described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0124241#pone.0124241.g001" target="_blank">Fig 1C</a> were divided into high and low <i>c-kit</i> expression groups, which described in detail in statistical analysis. The threshold is depicted as a dashed line. Patient characteristics are described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0124241#pone.0124241.t001" target="_blank">Table 1</a>. <b>(B)</b> Stratification of <i>AML1/ETO</i>-positive AML patients with high and low <i>c-kit</i> expression according to <i>c-kit</i> mutation status. <b>(C)</b> Correlation between <i>c-kit</i> and <i>AML1/ETO</i> levels in <i>AML1/ETO</i>-positive AML patients was assessed by the Spearman rank correlation coefficient. Gene expression was detected by qPCR. <i>ABL1</i> levels were measured for normalization. Black circles indicate AML patients carrying wt<i>c-kit</i>.</p

The migration of the GFPuv-labeled <i>Psa</i> in kiwifruit twigs in different temperature treatments by cutting inoculation.

<p>Samples were taken 2 cm from the inoculation site. The GFPuv-labeled <i>Psa</i> were isolated and cultured in BPA plates supplemented with KAN (50 μg·mL⁻¹) and colonies were counted under the fluorescence microscope.</p

Light and transmission electron micrographs of a cross-section of kiwifruit leaf infected by the GFPuv-labeled <i>Psa</i>.

<p>(a), Intercellular bacteria in the mesophyll cells at 5 dai. Bar = 50 μm; (b), bacterial colonization in the palisade parenchyma where the cells were abnormal. Bar = 10 μm. (c), bacteria that have colonized the intercellular space. Bar = 1 μm. Abbreviations: upper epidermis (Ue); palisade parenchyma (Pp); spongy mesophyll (Sm); lower epidermis (Le); bacteria (ba).</p

Formation and spread of the leaf lesions by spraying the GFPuv-labeled <i>Psa</i>.

<p>The inoculated leaves were observed under a fluorescence stereomicroscope (Leica MZ12) at different days after inoculation. Bar = 2 mm</p

Population size of the GFPuv-labeled <i>Psa</i> in kiwifruit twigs after inoculation of different wound types.

<p>The GFPuv-labeled <i>Psa</i> were isolated and cultured in BPA plates supplemented with KAN (50 μg·mL⁻¹) and colonies were counted under the fluorescence microscope. Values in columns followed by different letters were significantly different according to Fisher’s protected LSD test among lenticel, natural rubbing, leaf scar and cutting (P = 0.05).</p