Detection of NPM1 Mutations in Acute Myeloid Leukemia by using Drop-Off Droplet Digital PCR and its Clinical Application.

BACKGROUND: Nucleophosmin 1 (NPM1) mutations, which occur in 25 - 30% of acute myeloid leukemia (AML) and 50 - 60% of AML with normal karyotype, have been identified as an important marker for stratification of prog-nosis in AML. This study aimed to establish a new quantitative polymerase chain reaction (PCR) technique, the drop-off droplet digital PCR (ddPCR), for rapid and sensitive detection of NPM1 mutations in AML. METHODS: We established the drop-off ddPCR system and verified its performance. NPM1 mutations were screened in 130 AML patients by drop-off ddPCR and were validated by Sanger sequencing and next-generation sequencing (NGS). Then, the NPM1 mutation burden was dynamically monitored in five patients. RESULTS: The limit of blank (LOB) of drop-off ddPCR established for NPM1 mutation was 3.36 copies/µL, and the limit of detection (LOD) was 5.00 - 5.37 copies/µL in 50 ng DNA, and the sensitivity was about 0.05%, which had good linearity. Drop-off ddPCR identified 33/130 (25.4%) NPM1 mutated cases, consistent with Sanger sequencing. In 18 NPM1 positive cases selected randomly, NGS identified fourteen with type A mutation, two with type D mutation, and two with rare type mutations. The mutation burden of NPM1 mutation analyzed by NGS was consistent with the drop-off ddPCR. The sequential samples were detected for measurable residual disease (MRD) monitoring in 5 patients showed that the NPM1 mutation burden was consistent with clinical remission and recurrence. Compared with traditional ddPCR, drop-off ddPCR was also suitable for MRD monitoring. CONCLUSIONS: In this study, we established a drop-off ddPCR method for detecting three common mutations in AML with good sensitivity and repeatability, which can be used to screen mutations in newly diagnosed AML patients and for MRD monitoring after remission to guide treatment.

Reduced protocadherin17 expression in leukemia stem cells: the clinical and biological effect in acute myeloid leukemia.

BACKGROUND: Leukemia stem cell (LSC)-enriched genes have been shown to be highly prognostic in acute myeloid leukemia (AML). However, the prognostic value of tumor suppressor genes (TSGs) that are repressed early in LSC remains largely unknown. METHODS: We compared the public available expression/methylation profiling data of LSCs with that of hematopoietic stem cells (HSCs), in order to identify potential tumor suppressor genes in LSC. The prognostic relevance of PCDH17 was analyzed on a cohort of 173 AML patients from The Cancer Genome Atlas (TCGA), and further validated in three independent cohorts (n = 339). RESULTS: We identified protocadherin17 (PCDH17) and demonstrated that it was significantly down-regulated and hypermethylated in LSCs compared with HSCs. Our analyses of primary AML patient samples also confirmed these deregulations. Clinically, low PCDH17 expression was associated with female sex (P = 0.01), higher WBC (P < 0.0001), higher percentages of blasts in bone marrow (BM) and peripheral blood (PB) (P = 0.04 and < 0.001, respectively), presence of FLT3-internal tandem duplications (P = 0.002), mutated NPM1 (P = 0.02), and wild-type TP53 (P = 0.005). Moreover, low PCDH17 expression predicted worse overall survival (OS) in four independent cohorts as well as in the molecularly defined subgroups of AML patients. In multivariable analyses, low PCDH17 expression retained independent prognostic value for OS. Biologically, PCDH17 expression-associated gene signatures were characterized by deregulations of EMT- and Wnt pathway-related genes. CONCLUSIONS: PCDH17 gene was silenced by DNA methylation in AML. Low PCDH17 expression is associated with distinct clinical and biological features and improves risk stratification in patients with AML.

Down-regulation of miR-29c is a prognostic biomarker in acute myeloid leukemia and can reduce the sensitivity of leukemic cells to decitabine.

BACKGROUND: MicroRNA-29c (miR-29c) is abnormally expressed in several cancers and serves as an important predictor of tumor prognosis. Herein, we investigate the effects of abnormal miR-29c expression and analyze its clinical significance in acute myeloid leukemia (AML) patients. In addition, decitabine (DAC) has made great progress in the treatment of AML in recent years, but DAC resistance is still common phenomenon and the mechanism of resistance is still unclear. We further analyze the influences of miR-29c to leukemic cells treated with DAC. METHODS: Real-time quantitative PCR (RQ-PCR) was carried out to detect miR-29c transcript level in 102 de novo AML patients and 25 normal controls. miR-29c/shRNA-29c were respectively transfected into K562 cells and HEL cells. Cell viability after transfection was detected by cell counting Kit-8 assays. Flow cytometry was used to detect apoptosis. RESULTS: MiR-29c was significantly down-regulated in AML (P < 0.001). Low miR-29c expression was frequently observed in patients with poor karyotype and high risk (P = 0.006 and 0.013, respectively). Patients with low miR-29c expression had a markedly shorter overall survival (OS) than those with high miR-29c expression (P < 0.001). Multivariate analysis confirmed the independent prognostic value of low miR-29c expression in both the whole cohort as well as the cytogenetically normal AML (CN-AML) subset. Over-expression of miR-29c in K562 treated with DAC inhibited growth, while silencing of miR-29c in HEL promoted growth and inhibited apoptosis. MiR-29c overexpression decreased the half maximal inhibitory concentration (IC50) of DAC in K562, while miR-29c silencing increased the IC50 of DAC in HEL. The demethylation of the miR-29c promoter was associated with its up-regulated expression. Although miR-29c demethylation was also observed in DAC-resistant K562 (K562/DAC), miR-29c expression was down-regulated. MiR-29c transfection also promoted apoptosis and decreased the IC50 of DAC in K562/DAC cells. CONCLUSIONS: Our results suggest that miR-29c down-regulation may act as an independent prognostic biomarker in AML patients, and miR-29c over-expression can increase the sensitivity of both non-resistant and resistant of leukemic cells to DAC.

Methylation-independent CHFR expression is a potential biomarker affecting prognosis in acute myeloid leukemia.

CHFR acts as a tumor suppressor gene, which is frequently inactivated caused by its promoter hypermethylation in various solid tumors. Although a recent study showed that CHFR hypermethylation was a frequent event in acute myeloid leukemia (AML) and correlated with adverse clinical outcome, herein, we found that CHFR methylation was a rare event in patients with myeloid malignancies (including AML, chronic myeloid leukemia, and myelodysplastic syndromes), but its expression may serve as an independent prognostic biomarker in AML. CHFR expression was assessed by real-time quantitative PCR, whereas CHFR methylation was detected by methylation-specific PCR and bisulfite sequencing PCR. In AML patients, lower CHFR expression was associated with lower complete remission (CR) rate, and CHFR expression was significantly increased in CR after chemotherapy. Moreover, patients with lower CHFR expression showed shorter overall survival and leukemia-free survival, and multivariate analysis confirmed that lower CHFR expression was an independent risk factor in AML. Importantly, the prognostic value of CHFR expression was validated using the published Gene Expression Omnibus datasets. Notably, CHFR promoter was nearly unmethylated in patients with myeloid malignancies. Our findings revealed that lower CHFR expression was independently associated with unfavorable prognosis in AML. Moreover, aberrant CHFR promoter methylation was a rare event in myeloid malignances.

Intragenic hypomethylation of DNMT3A in patients with myelodysplastic syndrome.

BACKGROUND: DNMT3A is a DNA methyltransferase that acts in de novo methylation. Aberrant expression of DNMT3A has been reported in several human diseases, including myelodysplastic syndrome (MDS). However, the pattern of DNMT3A methylation remains unknown in MDS. METHODS: The present study was aimed to investigate the methylation status of DNMT3A intragenic differentially methylated region 2 (DMR2) using real-time quantitative methylation-specific PCR and analyze its clinical significance in MDS. RESULTS: Aberrant hypomethylation of DNMT3A was found in 57% (51/90) MDS cases. There were no significant differences in age, sex, white blood cell counts, platelet counts, hemoglobin counts and World Health Organization, International Prognostic Scoring System and karyotype classifications between DNMT3A hypomethylated and DNMT3A hypermethylated groups. However, the patients with DNMT3A hypomethylation had shorter overall survival time than those without DNMT3A hypomethylation (11 months vs. 36 months, p=0.033). Multivariate analysis confirmed the independent adverse impact of DNMT3A hypomethylation in MDS. CONCLUSIONS: Our data suggest that DNMT3A DMR2 hypomethylation may be a negative prognostic hallmark in MDS.

Low NKD1 expression predicts adverse prognosis in cytogenetically normal acute myeloid leukemia.

Dysregulation of NKD1 has been identified in several solid tumors. However, the status of NKD1 expression and its clinical implication in acute myeloid leukemia remain largely elusive. NKD1 transcript level in bone marrow mononuclear cells was detected by real-time quantitative polymerase chain reaction in 126 de novo acute myeloid leukemia patients and 30 controls. Clinical significance of NKD1 expression was obtained by the comparison between the patients with low and high NKD1 expression. NKD1 messenger RNA level was significantly decreased in acute myeloid leukemia patients compared with controls ( p = 0.019). There were no significant differences between patients with low and high NKD1 expression in sex, age, peripheral blood cells, bone marrow blasts, French-American-British/World Health Organization subtypes, and karyotypes/karyotypic classifications ( p > 0.05). Although no significant difference was observed in complete remission rate between NKD1low and NKD1high patients ( p > 0.05), Kaplan-Meier analysis revealed that NKD1low patients showed shorter overall survival time than NKD1high patients in whole-cohort acute myeloid leukemia, non-M3 acute myeloid leukemia, and cytogenetically normal acute myeloid leukemia ( p = 0.014, 0.063, and 0.020). Multivariate analyses disclosed the low NKD1 expression was an independent risk factor in cytogenetically normal acute myeloid leukemia patients (hazard ratio = 0.397, p = 0.017). Moreover, the prognostic value of NKD1 expression was confirmed by gene expression profile data in cytogenetically normal acute myeloid leukemia patients ( p = 0.028 and 0.011). NKD1 showed significantly increased level after induction chemotherapy achieved complete remission in follow-up paired acute myeloid leukemia patients ( p < 0.001). These findings indicated that reduced NKD1 expression is associated with unfavorable clinical outcome in cytogenetically normal acute myeloid leukemia.

CDH1 (E-cadherin) expression independently affects clinical outcome in acute myeloid leukemia with normal cytogenetics.

BACKGROUND: Epithelial-mesenchymal transition (EMT) is a critical process which involves in tumor metastasis. As an important EMT marker gene, CDH1 (E-cadherin) expression and its clinical implication in acute myeloid leukemia (AML) remain largely elusive. METHODS: Real-time quantitative PCR (RQ-PCR) was carried out to examine CDH1 transcript level in 123 de novo AML patients and 34 controls. RESULTS: Compared with controls, CDH1 was significantly downregulated in AML (p<0.001). The median level of CDH1 expression divided total AML patients into CDH1 low-expressed (CDH11ow) and CDH1 high-expressed (CDH1high) groups. There were no significant differences between the two groups in age, peripheral blood cell counts, complete remission (CR) rate, and the distribution of FAB/WHO subtypes as well as karyotypes/karyotypic classifications (p>0.05). However, CDH11ow group tended to have a higher bone marrow (BM) blasts (p=0.093). The spearman correlation analysis further illustrated a trend towards a negative correlation between CDH1 expression level and BM blasts (r=-0.214, p=0.052). CDH1low group had a tendency towards a lower frequency of N/K-RAS mutations (p=0.094). Furthermore, CDH1low patients had markedly shorter overall survival (OS) time in cytogenetic normal AML (CN-AML) (p=0.019). Both univariate and multivariate analyses confirmed the prognostic value of CDH1 expression in CN-AML patients (p=0.027 and 0.033, respectively). CONCLUSIONS: CDH1 downregulation acted as an independent prognostic biomarker in CN-AML patients.

The prognostic implication of SRSF2 mutations in Chinese patients with acute myeloid leukemia.

Recently, somatic mutations in SRSF2 gene have been discovered in a proportion of hematologic malignancies including acute myeloid leukemia (AML). This study was aimed to investigate SRSF2 mutations in Chinese AML patients. High-resolution melting analysis (HRMA) was developed to screen SRSF2 mutations in 249 cases with AML, and then direct DNA sequencing was used to verify the results of HRMA. In this study, 3.6 % (9/249) of Chinese AML patients were found with heterozygous SRSF2 mutations. Patients with SRSF2 mutations were older than those with wild-type SRSF2 (P = 0.014). No differences in the sex, blood parameters, French-American-British classification (FAB) subtypes, and karyotypes were observed between AML patients with and without SRSF2 mutations. Although the overall survival (OS) of SRSF2-mutated patients was inferior to those without mutations in both whole AML patients (median 4 vs. 11 months, respectively; P = 0.006) and cytogenetically normal patients (median 2 vs. 12 months, respectively; P = 0.008), multiple analysis disclosed that SRSF2 mutation was not an independent prognostic factor in AML patients. These results suggest that SRSF2 mutation occurs at a low frequency in aged AML patients and might not be associated with adverse prognosis in Chinese AML patients.

Let-7a-3 hypomethylation is associated with favorable/intermediate karyotypes but not with survival in acute myeloid leukemia.

Aberrant methylation of let-7a-3 promoter has been observed in various malignancies. However, the clinical relevance of let-7a-3 methylation remains poorly known in acute myeloid leukemia (AML). This study was to investigate the let-7a-3 methylation status and to explore its clinical significance in AML. let-7a-3 promoter was significantly hypomethylated in AML patients compared to controls (median 4.51 vs 0.49) (P = 0.0003). Receiver operating characteristic curve (ROC) analysis discriminated all patients or cytogenetically normal patients from controls with an areas under the ROC curve (AUC) of 0.737 or 0.783, respectively (P < 0.001). Patients with favorable/intermediate karyotypes had significantly higher let-7a-3 unmethylation than controls. Patients with DNMT3A mutations had a trend of high level of let-7a-3 unmethylation than did those with wild-type DNMT3A (median 6.76 vs 3.66, P = 0.096). There was no significant difference in overall survival between patients with and without hypomethylated let-7a-3 (median 12 vs 5 months, P = 0.103). No correlation was observed between the level of let-7a-3 expression and let-7a-3 unmethylation in AML samples (R = 0.197, P = 0.150). However, the level of let-7a-3 expression was increased in a dose-dependent manner in THP-1 line treated with 5-aza-dC, while the methylation density of let-7a-3 promoter decreased with 5-aza-dC dose. Our findings suggest that let-7a-3 hypomethylation is associated with favorable and intermediate karyotypes but not a prognostic predictor for AML patients. Let-7a-3 expression may be partially regulated by promoter methylation.

Hypermethylation of DLX4 predicts poor clinical outcome in patients with myelodysplastic syndrome.

BACKGROUND: Hypermethylation of DLX4 (distal-less homeobox 4) has been disclosed in a variety of cancers. Our work was aimed to examine the pattern of DLX4 methylation and further investigate its clinical relevance in patients with myelodysplastic syndrome (MDS). METHODS: Real-time quantitative methylation-specific PCR and bisulfite sequencing PCR were carried out to detect the level of DLX4 methylation. Clinical significance of DLX4 methylation was analyzed between the DLX4 hypermethylated and non-hypermethylated patients. RESULTS: DLX4 was significantly hypermethylated in MDS patients than controls (p<0.001). No significant differences were observed between the hypermethylated and non-hypermethylated MDS patients in white blood cells, platelets, age, WHO classifications, FAB classifications, IPSS risks, and common gene mutations (p>0.05). However, DLX4 hypermethylated patients tended to have higher hemoglobin (HB) than DLX4 non-hypermethylated patients (p=0.079). Moreover, there was a trend that male patients, poor karyotype patients, and IPSS Int-2/High patients had a higher frequency of DLX4 hypermethylation (p=0.067, 0.065, and 0.068). DLX4 hypermethylated patients had significantly shorter overall survival than DLX4 non-hypermethylated patients (p=0.004). Multivariate analysis confirmed the prognostic value of DLX4 methylation in MDS patients (p<0.001). CONCLUSIONS: Our study indicated that DLX4 hypermethylation was a frequent event and acted as an independent prognostic biomarker in de novo MDS patients.

Reduced miR-215 expression predicts poor prognosis in patients with acute myeloid leukemia.

OBJECTIVE: Abnormal expression of microRNA-215 has been identified in a variety of solid cancers. However, little is known about the expression pattern of microRNA-215 in acute myeloid leukemia. This study was to investigate the status of microRNA-215 expression and further analyze its clinical significance in acute myeloid leukemia. METHODS: Real-time quantitative polymerase chain reaction assay was performed to evaluate the expression level of microRNA-215 in 113 patients with acute myeloid leukemia. Besides, the relationship between microRNA-215 levels and clinical and pathological factors was explored. RESULTS: Compared with the healthy individuals, microRNA-215 expression in acute myeloid leukemia patients was significantly down-regulated (P= 0.001). MicroRNA-215 low-expressed patients had higher white blood cells than microRNA-215 high-expressed patients (P= 0.014). The incidence of FLT3/ITD mutation in the patients with low microRNA-215 expression was significantly higher than those with high microRNA-215 expression (P= 0.025). MicroRNA-215 low-expressed patients had significantly shorter overall survival than microRNA-215 high-expressed patients in both non-M3 acute myeloid leukemia patients and cytogenetically normal patients (P= 0.017 and P= 0.044, respectively). Meanwhile, multivariate analysis confirmed the adverse prognostic value of microRNA-215 expression in acute myeloid leukemia patients with non-M3 subtypes. CONCLUSIONS: Our study demonstrates that reduced microRNA-215 expression is a common event and is associated with poor clinical outcome in acute myeloid leukemia.

Down-Regulation of miR-186 Correlates with Poor Survival in de novo Acute Myeloid Leukemia.

BACKGROUND: MicroRNA-186 (miR-186) plays an important role in the pathogenesis of several cancers. Our study was intended to investigate the expression status and the prognostic implication of miR-186 in acute myeloid leukemia (AML). METHODS: Real-time quantitative PCR was carried out in 112 de novo AML patients and 28 controls. RESULTS: The level of miR-186 expression in AML was significantly down-regulated compared to normal controls (p < 0.001). Patients with low miR-186 expression presented significantly older age than those with high miR-186 expression (p = 0.004). MiR-186high patients had a significantly higher frequency of CEBPA mutation than miR-186low patients (20% and 4%, respectively, p = 0.022). In addition, miR-186low patients had a significantly lower complete remission (CR) rate (30% vs. 53%, respectively, p = 0.028) than miR-186high patients. Moreover, miR-186low patients showed significantly shorter overall survival (OS) time than miR-186high patients in both whole AML and non-M3 patients (p = 0.023 and 0.026, respectively). Additionally, the adverse prognostic impact of miR-186 down-regulation was also shown in both whole AML and non-M3 patients without CEBPA mutation (p = 0.017 and 0.023, respectively). CONCLUSIONS: Our study suggests that miR-186 down-regulation is a frequent event and predicts poor prognosis in de novo AML patients.

Epigenetic inactivation of DLX4 is associated with disease progression in chronic myeloid leukemia.

Aberrant DNA methylation of various genes has been identified to be associated with disease progression in chronic myeloid leukemia (CML). Our study was intended to investigate DLX4 methylation pattern in different clinical stages of CML and further determine its role in regulating DLX4 expression. Real-time quantitative methylation-specific PCR and bisulfite sequencing PCR were applied to detect DLX4 methylation. 5-aza-2'-deoxycytidine (5-aza-dC) was used for demethylation studies. DLX4 was significantly hypermethylated in CML patients (P = 0.002) especially in blastic phase (BC) stage (P < 0.001) as compared with controls. Moreover, DLX4 methylation level in BC stage was significantly higher than in chronic phase (CP) stage (P < 0.001). DLX4 methylation density was significantly increased during the progression of CML among the tested two patients (P < 0.001). DLX4 hypermethylation occurred with the highest incidence in BC stage (83%), lower incidence in acute phase (AP) stage (43%), and the lowest incidence in CP stage (26%) (P = 0.001). Moreover, t(9; 22) with additional alteration cases had significantly higher frequency of DLX4 hypermethylation compared with the other cytogenetics (P = 0.010). Significantly negative correlation was observed between DLX4 methylation and DLX4-TV2 (the shorter DLX4 isoform) expression (R = -0.382, P = 0.001, n = 78) but not between DLX4 methylation and BP1 (the longer DLX4 isoform) expression (R = 0.134, P = 0.244, n = 78) in CML patients. Both DLX4-TV2 and BP1 mRNA were significantly increased after 5-aza-dC treatment in K562 cell line (P < 0.001). Our study indicated that hypermethylation of DLX4 correlated with disease progression of CML. Moreover, DLX4 expression was regulated by its methylation in CML.

IDH1 and IDH2 mutation analysis in Chinese patients with acute myeloid leukemia and myelodysplastic syndrome.

The somatic mutations of isocitrate dehydrogenase genes (IDH1 and IDH2) have been identified in a proportion of hematologic malignancies. We examined IDH1 R132 and IDH2 R140/R172 mutations by high resolution melting analysis and direct sequencing in Chinese patients with different myeloid malignancies including 198 acute myeloid leukemia (AML), 82 myelodysplastic syndrome (MDS), 85 chronic myeloid leukemia, and 57 myeloproliferative neoplasms. IDH1 and IDH2 mutations were found in four (2.0%) and ten (5.0%) AML and in two (2.4%) and three (3.6%) MDS cases, but not in other patients. IDH1 and IDH2 mutations were heterozygous and mutually exclusive. IDH1/2 mutations were significantly more frequently observed in cytogenetically normal AML or MDS compared to those without mutations. There was no difference in overall survival of both AML and MDS patients with or without IDH1/2 mutations (P = 0.177 and 0.407, respectively). In conclusion, IDH1/2 mutations are recurrent but rare molecular aberrations in Chinese AML and MDS.

Dysregulation of LINC00324 associated with methylation facilitates leukemogenesis in de novo acute myeloid leukemia.

INTRODUCTION: LINC00324 was overexpressed and facilitated carcinogenesis in various solid malignant tumors. However, the role of LINC00324 in leukemogenesis remains to be elucidated. METHODS: The relative expression and unmethylation levels of LINC00324 were detected by real-time quantitative PCR (RT-qPCR) and real-time quantitative methylation-specific PCR (RT-qMSP). Cell proliferation experimental and flow cytometer (FCM) was used to detect the change of proliferation and apoptosis in leukemia cell lines after overexpression of LINC00324. RESULTS: The results showed that the expression of LINC00324 and the methylation level of the promoter region were significantly negatively correlated in AML patients. Moreover, patients with lower LINC00324 expression showed more prolonged overall survival (OS). Remarkably, overexpression of LINC00324 in leukemia cell lines promoted the proliferation of target cells and inhibited their apoptosis. CONCLUSION: Our findings firstly identified that the hypomethylation of LINC00324 was a common molecular event in de novo AML patients. The abnormally upregulated LINC00324 promotes proliferation and inhibits apoptosis in leukemia cells.

Reduced expression of lncRNA DLEU7-AS1 is a novel favorable prognostic factor in acute myeloid leukemia.

The objective of our study was to measure DLEU7-AS1 expression in de novo acute myeloid leukemia (AML) whilst also analyzing its clinical relevance. We used gene expression data from The Cancer Genome Atlas (TCGA), Gene Expression Omnibus (GEO), Cancer Cell Line Encyclopedia (CCLE) and Genotype-Tissue Expression project (GTEx) to assess the expression profile of DLEU7-AS1 in pan-cancers, cancer cell lines and normal tissues. Reverse transcription-quantitative PCR was used to measure DLEU7-AS1 expression in bone marrow from 30 normal individuals and 110 patients with de novo AML. DLEU7-AS1 expression was found to be markedly reduced in the AML samples of the TCGA pan-cancer datasets. In our PCR validation, DLEU7-AS1 expression was significantly decreased in the AML samples compared with that in controls (P<0.001). Low DLEU7-AS1 expression (DLEU7-AS1low) correlated positively with lower blood platelet counts (P=0.029). In addition, low DLEU7-AS1 expression was more frequently observed in the intermediate (58%; 44/76) and favorable karyotypes (65%; 15/23) compared with that in the poor karyotype (10%; 1/10; P=0.005). In particular, patients with high expression levels of DLEU7-AS1 (DLEU7-AS1high) showed lower complete remission rates (P=0.002) than patients with DLEU7-AS1low. Survival analysis revealed that patients with DLEU7-AS1low had longer overall survival (OS) than patients with DLEU7-AS1high (P<0.05). Multivariate Cox analysis demonstrated that in patients with non-acute promyelocytic leukemia (non-M3) who were ≤60 years old, DLEU7-AS1 expression was an independent prognostic factor for OS. Furthermore, we found distinct correlations among the expression of DLEU7-AS1, infiltration by immune cells and immune checkpoint genes in AML.

Detection of the JAK2 mutation in myeloproliferative neoplasms by asymmetric PCR with unlabeled probe and high-resolution melt analysis.

BACKGROUND: Several methods have been established to detect the JAK2 V617F mutation, a frequent event involved in the pathogenesis of myeloproliferative neoplasms (MPNs). High-resolution melt (HRM) analysis is a newly established technique without the requirement of any gel-based post-PCR handling. METHODS: An asymmetric PCR with unlabeled specific probe was developed and combined to HRM analysis o screen for JAK2 V617F mutation. RESULTS: Heterozygous mutation was easily distinguished from homozygous JAK2 for the obvious shape change. Homozygous JAK2 mutant can be also well separated from wild-type JAK2 in the presence of internal temperature calibrators. The easily recognizable and maximal sensitivity of HRM analysis was 5% for the detection of JAK2 V617F mutation, higher than 25% of direct sequencing. In the test of blind screening of 223 samples (111 Ph- MPNs, 60 Ph+ chronic myeloid leukemia, and 52 acute myeloid leukemia), JAK2 V617F mutations were found in 78 (70%) patients with MPNs, but in none with chronic and acute myeloid leukemia. HRM analysis of all cases was fully concordant with the results of PCR-RFLP and direct sequencing. CONCLUSIONS: The HRM method with unlabeled probe could be used as convenient, sensitive and reliable diagnostic test for detection of JAK2 V617F mutation.

[Overexpression of LncRNA ITGB2-AS1 Predicts Adverse Prognosis in Acute Myeloid Leukemia].

OBJECTIVE: LncRNA ITGB2-AS1 has been found to play important roles in the occurrence and development of human solid tumors. However, its role in hematological diseases, especially acute myeloid leukemia (AML), remains unclear. The aim of this study was to identify the expression pattern of ITGB2-AS1 in AML patients and to further explore its clinical significance. METHODS: ITGB2-AS1 expression was analyzed in public datasets (including TCGA and GSE63270) and further validated in a cohort of 109 AML patients by real-time quantitative PCR (RT-qPCR). RESULTS: The level of ITGB2-AS1 was up-regulated among two independent cohorts (TCGA, P<0.05; GSE63270, P<0.05), which was confirmed by the data from 109 AML patients enrolled in this study (P<0.05). Clinically, high ITGB2-AS1 expression was associated with older age (P=0.023) and lower complete remission (CR) rate (P=0.005). Multivariate analysis identified that high ITGB2-AS1 expression was an independent prognostic factor not only for CR rate (P=0.027) but also for overall survival (OS) time (P=0.011), and ITGB2-AS1 was positively correlated with ITGB2 expression in both TCGA (r=0.74, P<0.001) and clinical data detected in this study (r=0.881, P<0.001). High ITGB2 expression was also associated with older age (P=0.02) and lower CR rate (P=0.020). Moreover, high ITGB2 expression predicted worse OS (P=0.028). CONCLUSION: ITGB2-AS1 is overexpressed in AML and predicts poor prognosis in AML patients.

[Quantification of GRAF gene expression in patients with acute myeloid leukemia using EvaGreen real time quantitative PCR].

OBJECTIVE: To quantify the expression level of GRAF gene in acute myeloid leukemia (AML) and analyze its clinical significance. METHODS: The EvaGreen real-time quantitative polymerase chain reaction (RQ-PCR) assay was established and performed to measure the GRAF gene transcripts in 71 cases with AML and 21 with nonmalignant hematological diseases. The clinical correlation of GRAF expression was analyzed. RESULTS: The established EvaGreen RQ-PCR assay had good specificity, reproducibility and sensitivity. The GRAF expression level was significantly lower in AML (0.01%-169.75%, median 3.82%) than that in controls (14.49%-126.85%, median 56.04%) (P<0.05). There was no correlation between the level of GRAF transcript and the sex, age, hematologic parameters, FAB subtypes and karyotypic groups (P>0.05). CONCLUSION: The GRAF gene was down-regulated in AML, which might play a role in the leukemogenesis.

[Quantification of the PRAME transcripts in patients with acute myeloid leukemia].

OBJECTIVE: To analyze the expression level and clinical significance of the preferentially expressed antigen of melanoma (PRAME) transcripts in patients with acute myeloid leukemia (AML). METHODS: Real-time quantitative polymerase chain reaction with EvaGreen dye was established to detect the expression level of PRAME transcripts in the bone marrow mononuclear cells of 56 AML cases and 20 controls. The clinical association of PRAME transcripts was analyzed. RESULTS: The PRAME transcripts were 0-1.46% (median 0.18%) and 0-21 618.09% (median 9.79%) in controls and AML cases, respectively (P< 0.01). Among the FAB subtypes, those with M1, M2, M3 and M4 had significantly higher level of PRAME transcripts than controls. However, those with M5 had similar level of PRAME transcripts as controls. There was a significantly negative correlation between the PRAME transcripts and cytogenetic risk groups (r= -0.438, P= 0.001). Cases in low risk had significantly higher level of PRAME transcripts than those in intermediate and high risk. Among cases with AML-M2, those with t(8;21) had significantly higher level of PRAME transcripts (135.06% -21 618.09%, median 2201.88%) than those without t(8;21)(0.14% -1696.30%, median 17.97%)(P= 0.002). In a patient with sequential samples, PRAME transcripts significantly decreased after induction therapy and significantly increased after relapse. CONCLUSION: The PRAME transcript was highly expressed in AML patients and was a favorable marker of prognosis. Quantification of PRAME transcript can be used in monitoring disease status of AML.