Xing-xing Chen

U2AF1 Mutations in Chinese Patients with Acute Myeloid Leukemia and Myelodysplastic Syndrome

Somatic mutations of U2AF1 gene have recently been identified in myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML). In this study, we analyzed the frequency and clinical impact of U2AF1 mutations in a cohort of 452 Chinese patients with myeloid neoplasms. Mutations in U2AF1 were found in 2.5% (7/275) of AML and 6.3% (6/96) of MDS patients, but in none of 81 CML. All mutations were heterozygous missense mutations affecting codon S34 or Q157. There was no significant association of U2AF1 mutation with blood parameters, FAB subtypes, karyotypes and other gene mutations in AML. The overall survival (OS) of AML patients with U2AF1 mutation (median 3 months) was shorter than those without mutation (median 7 months) (P = 0.035). No difference in the OS was observed between MDS patients with and without U2AF1 mutations. Our data show that U2AF1 mutation is a recurrent event at a low frequency in AML and MDS.

Overexpressed let-7a-3 is associated with poor outcome in acute myeloid leukemia.

Dysregulation of microRNA let-7a-3 has been identified in several solid tumors and is associated with prognosis of patients. However, the pattern of let-7a-3 expression and the impact on prognosis has not yet been studied in acute myeloid leukemia (AML). The purpose of this study is to investigate the expression status of let-7a-3 and its clinical significance in AML patients using real-time quantitative PCR. Overexpression of let-7a-3 was identified in 25 of 102 (25%) de novo AML. There was no significant difference in age, blood parameters, FAB/WHO subtypes, karyotype risks and nine gene mutations (FLT3-ITD, NPM1, C-KIT, IDH1/IDH2, DNMT3A, C/EBPA and N/K-RAS) between patients with and without let-7a-3 overexpression (P>0.05). The patients with let-7a-3 overexpression had similar rates of complete remission (CR) as those without let-7a-3 overexpression (50% vs. 56%, P=0.693). Although the overall survival (OS) of AML patients with let-7a-3 overexpression (median 12 months,) was shorter than those without overexpression (median 25 months), the difference was not statistically significant (P=0.228). However, among those 51 obtained CR, patients with let-7a-3 overexpression had significantly shorter OS than those without let-7a-3 overexpression (P=0.029). The difference in relapse-free survival (RFS) was also significant between two groups (P=0.005). These findings suggest that let-7a-3 overexpression is a common event and is associated with poor clinical outcome in AML.

Overexpression of miR-378 is frequent and may affect treatment outcomes in patients with acute myeloid leukemia

MicroRNA miR-378 plays important roles in tumorigenesis by enhancing cell survival, reducing apoptosis, promoting tumor growth, angiogenesis and promoting cell migration and invasion. Abnormal expression of miR-378 has been observed in various types of cancers. The aim of this study was to investigate the expression status of miR-378 and its clinical significance in patients with acute myeloid leukemia (AML) using real-time quantitative PCR. miR-378 overexpression was identified in 26 of 84 (31%) AML patients. The patients with miR-378 overexpression had lower hemoglobin level than those without miR-378 overexpression (66 versus 78 g/L, respectively, P=0.010). The frequency of miR-378 overexpression in FAB-M2 subtype was higher than other subtypes (44% versus 20%, P=0.032). Moreover, the frequency of miR-378 overexpression was higher in patients with t(8;21) than in others (64% versus 24%, P=0.012). The status of miR-378 expression was not correlated with the mutations of eight genes (FLT3-ITD, NPM1, C-KIT, IDH1/IDH2, DNMT3A, C/EBPA and U2AF1). The difference in relapse-free survival was observed between patients with and without miR-378 overexpression (P=0.049). These findings suggest that miR-378 up-regulation is a common event and might have an adverse impact on prognosis in AML.

MiR-378 Promotes the Migration of Liver Cancer Cells by Down-Regulating Fus Expression

Background: miR-378 regulates osteoblast differentiation and participates in tumor cell self-renewal and chemo-resistance. However, the function of miR-378 in liver cancer cell migration has not been reported to date. Methods: miR-378 expression was examined using real-time quantitative PCR. HepG2 cell migration and liver cell invasion were examined using wound-healing and cell invasion assays. Additionally, HepG2 cell metastasis was analyzed in nude mice. Results: miR-378 over-expression enhances HepG2 cell proliferation, migration and liver cell invasion. Typical metastatic lesions were found in the livers of mice injected with miR-378-transfected cells, and high levels of the CMV promoter were detected in the nodules, indicating that miR-378 promoted the metastasis of the tumor cells to the liver. We also demonstrated that miR-378 down-regulated Fus expression. Conclusions: Our results suggested that miR-378 enhanced cell migration and metastasis by down-regulating Fus expression.

Dysregulation of miR-124-1 predicts favorable prognosis in acute myeloid leukemia

OBJECTIVE MicroRNA miR-124 has been suggested as a tumor suppressor for its role in inhibiting cell growth, inducing differentiation and promoting apoptosis. The present study was aimed to investigate the expression status of miR-124-1 and its clinical relevance in patients with acute myeloid leukemia (AML). DESIGNS AND METHODS Real-time quantitative PCR was performed to detect the expression level of miR-124-1 in AML patients. The clinical significance of miR-124-1 expression in AML was investigated. RESULTS miR-124-1 underexpression was identified in 30 (36%) of 83 AML patients. No significant difference could be observed in sex, age and blood parameters between the patients with and without miR-124-1 underexpression. The frequency of miR-124-1 underexpression was higher in the patients with t(15;17) than in others (62% versus 30%, P = 0.040). The status of miR-124-1 expression was not correlated with the mutations of nine genes (FLT3-ITD, NPM1, C-KIT, IDH1/IDH2, DNMT3A, N/K-RAS and C/EBPA). The patients with miR-124-1 underexpression had borderline longer overall survival and relapse-free survival than those without miR-124-1 underexpression (P = 0.052 and 0.045, respectively). CONCLUSIONS These findings suggest that miR-124-1 underexpression is a common event and might have a favorable impact on prognosis in AML.

Development of high-resolution melting analysis for the detection of the MYD88 L265P mutation

OBJECTIVE Recurrent L265P mutation of myeloid differentiation primary response gene 88 (MYD88) has been identified in a proportion of diffuse large B-cell lymphoma (DLBCL) and chronic lymphocytic leukemia. The present study aimed to establish a rapid, sensitive, and reliable method using high-resolution melting analysis (HRMA) to detect MYD88 L265P mutation in DLBCL. DESIGNS AND METHODS The sensitivity of HRMA in the detection of MYD88 L265P mutation was evaluated. MYD88 L265P mutation was further screened in 120 patients with DLBCL. The results of HRMA were validated by direct DNA sequencing. RESULTS For the detection of MYD88 L265P mutation, the reproducible maximal sensitivity of HRMA was 5% higher than that obtained by direct DNA sequencing (25%). Heterozygous MYD88 L265P mutations were identified in 11 (9.2%) DLBCL cases, all of which were diagnosed as non-germinal-center B cell (non-GCB) DLBCL. CONCLUSIONS The HRMA assay is a rapid, sensitive, reliable, and high-throughput method to screen MYD88 L265P mutation and could be used in clinical diagnostic laboratories.

Development of a high-resolution melting analysis for the detection of the SF3B1 mutations.

SF3B1, located on chromosome 2q33.1, encodes a core component of RNA-splicing machinery, and its mutation has been described in myelodysplastic syndromes (MDS) characterized with ring sideroblasts (RS). To explore the reliability and sensitivity of the high-resolution melting analysis (HRMA) technique for the identification of the SF3B1 mutations, mutations in 92 patients with MDS were detected in this study. The sensitivity could reach 5%, obviously higher than the 25% of direct DNA sequencing. A low frequency (5.4%) of SF3B1 mutations were identified in patients with MDS, including three cases of K700E, one case of H662Q, and one case of K666M. Further, SF3B1 mutations were more frequently recurrent in the 33% of patients with MDS characterized with RS, whereas in other subtypes of MDS, only 2.3% of patients were detected with SF3B1 mutations (p=0.006). In conclusion, a rapid, reproducible, sensitive, and high-throughput HRMA assay has been established for the scanning of SF3B1 mutations.

The Methylation Status of the DDX43 Promoter in Chinese Patients with Chronic Myeloid Leukemia

Aberrant DNA methylation is a common epigenetic alteration and an important feature in human cancers. The DEAD box polypeptide 43 (DDX43) has been found to be overexpressed in various solid tumors and some hematologic malignancies. In the present study, we investigated the methylation status of the DDX43 promoter in 87 Chinese patients with chronic myeloid leukemia (CML) using real-time quantitative methylation-specific polymerase chain reaction and examined the DDX43 transcript in 35 patients using real-time quantitative polymerase chain reaction. DDX43 promoter hypomethylation was observed in 22 (25.3%) CML patients. No significant correlation was found between the hypomethylation of the DDX43 promoter with the age, sex, white blood cell counts, hemoglobin concentration, platelet counts, and chromosomal abnormalities of CML patients (p>0.05). The frequency of DDX43 hypomethylation in patients in the chronic phase, in the accelerated phase, and in blast crisis was 23.4% (15/64), 25.0% (2/8), and 33.3% (5/15), respectively (p>0.05). There was a significant correlation between DDX43 hypomethylation and DDX43 transcript (r=0.469, p=0.004). Our data suggest that hypomethylation of the DDX43 promoter may be an early and frequent molecular event in the development of CML in Chinese patients.

Methylation of CTNNA1 promoter: Frequent but not an adverse prognostic factor in acute myeloid leukemia

The reduced expression of CTNNA1 gene, a putative tumor suppressor gene, has been found in several cancers including acute myeloid leukemia (AML). CTNNA1 expression is regulated by methylation and histone deacetylation. However, the clinical significance of CTNNA1 methylation in AML is rarely known. The present study was aimed to investigate the methylation status of CTNNA1 promoter region using methylation-specific PCR (MSP) and its clinical relevance in Chinese AML patients. Patients with CTNNA1 hypermethylation had significantly lower level of CTNNA1 transcript than those without CTNNA1 hypermethylation (P=0.031). The relationship of CTNNA1 methylation with clinical parameters was evaluated. Aberrant hypermethylation of CTNNA1 gene was found in 23.9% (37/155) AML cases. The status of CTNNA1 methylation was not correlated with the mutations of seven genes (FLT3-ITD, NPM1, C-KIT, IDH1/IDH2, DNMT3A, N/K-RAS and C/EBPA). There was no significant difference in the rates of complete remission (CR) between patients with and without CTNNA1 methylation. Although the overall survival (OS) time of the CTNNA1-methylated AML was shorter than that of CTNNA1-unmethylated group (6 months vs 9 months), the difference was not statistically significant (P=0.681). Our data suggest that CTNNA1 methylation is a recurrent event but has no influence on prognosis in AML.

SF3B1 mutation is a rare event in Chinese patients with acute and chronic myeloid leukemia.

OBJECTIVE Somatic mutations of SF3B1 gene have recently been identified in myelodysplastic syndrome and chronic lymphocytic leukemia. The frequency and clinical relevance of SF3B1 mutations have been rarely studied in acute myeloid leukemia (AML) and chronic myeloid leukemia (CML). The present study was aimed to analyze the frequency of SF3B1 mutations in AML and CML. DESIGNS AND METHODS High-resolution melting analysis (HRMA) was established to detect the mutation hotspots (codon E622, H662, K666, and K700) of SF3B1 gene in 275 AML and 81 CML patients. RESULTS Heterozygous SF3B1 mutations were detected in three AML patients by HRMA. Direct DNA sequencing identified one K666T, one K666N and one K700E mutations. All three AML patients had normal karyotypes. One case also had NPM1 and DNMT3A mutations, one had FLT3 internal tandem duplication and DNMT3A mutations, and the other had NPM1 mutation. No SF3B1 mutations were detected in CML patients. CONCLUSIONS SF3B1 mutation is a rare molecular event in Chinese AML and CML patients.