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 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.

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.

[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.

Hypomethylation of MIR-378 5'-flanking region predicts poor survival in young patients with myelodysplastic syndrome.

BACKGROUND: Previous studies have disclosed up-regulation of MIR-378 in acute myeloid leukemia (AML), and might consequently affect the outcome of the patients. Correspondingly, hypomethylation of MIR-378 was also identified in AML, particularly for FAB-M2 subtype with t(8;21) chromosomal translocation. Nevertheless, the methylation status of MIR-378 has not been illustrated in myelodysplastic syndrome (MDS). Herein we designed to understand the methylation pattern of MIR-378 involved in MDS and clinical interrelation thereof. METHODS: Real-time quantitative methylation-specific PCR (RQ-MSP) was performed to evaluate the methylation degree of MIR-378 5'-flanking region on bone marrow mononuclear cells collected from 95 de novo MDS patients. Five gene mutations (IDH1, IDH2, DNMT3A, U2AF1, and SF3B1) were detected by high-resolution melting analysis to further evaluate the clinical relevance of hypomethylation of MIR-378. RESULTS: Unmethylated level of MIR-378 5'-flanking region was significantly higher in MDS patients than that in controls (p = .034). Hypomethylated MIR-378 was identified in 20 of 95 (21%) cases with MDS. Male patients appeared to be more frequent to harbor MIR-378 hypomethylation compared to female patients (15/55, 27.3% vs. 4/40, 10.0%, p = .04). There was no significant difference in age, white blood cell counts, platelet counts, hemoglobin concentration, and karyotypes between the patients with and without MIR-378-hypomethylation. Distinct distribution of five gene mutations was not observed in the two groups as well. However, MIR-378-hypomethylated patients had significantly shorter overall survival than those without MIR-378 hypomethylation (p = .036). Moreover, among patients <60 years, hypomethylation of MIR-378 was confirmed to be an independent adverse prognostic factor by both Kaplan-Meier and Multivariate Cox analyses. CONCLUSION: Hypomethylation of MIR-378 5'-flanking region is an adverse prognosticator in MDS, particularly in patients <60 years.

DOK6 promoter methylation serves as a potential biomarker affecting prognosis in de novo acute myeloid leukemia.

BACKGROUND: Downstream of tyrosine kinase 6 (DOK6), which is specifically expressed in the nervous system, was previously recognized as an adapter only in neurite outgrowth. Recent studies also demonstrated the potential role of DOK6 in solid tumors such as gastric cancer and breast cancer. However, previous studies of DOK6 have not dealt with its roles in myeloid malignancies. Herein, we verified the promoter methylation status of DOK6 and further explored its clinical implication in de novo acute myeloid leukemia (AML). METHODS: A total of 100 newly diagnosed adult AML patients were involved in the current study. DOK6 expression and methylation were detected by real-time qPCR and methylation-specific PCR (MSP), respectively. Bisulfite sequencing PCR (BSP) was performed to assess the methylation density of the DOK6 promoter. RESULTS: Downstream of tyrosine kinase 6 promoter methylation was significantly increased in AML patients compared to controls (P = .037), whereas DOK6 expression significantly decreased in AML patients (P < .001). The expression of DOK6 was markedly up-regulated after treated by 5-aza-2'-deoxycytidine (5-aza-dC) in THP-1 cell lines. The methylation status of the DOK6 promoter was associated with French-American-British classifications (P = .037). There was no significant correlation existed between DOK6 expression and its promoter methylation (R = .077, P = .635). Interestingly, of whole-AML and non-APL AML patients, both have a tendency pertaining to the DOK6 methylation group and a significantly longer overall survival (OS) than the DOK6 unmethylation group (P = .042 and .036, respectively). CONCLUSION: Our study suggested that DOK6 promoter hypermethylation was a common molecular event in de novo AML patients. Remarkably, DOK6 promoter methylation could serve as an independent and integrated prognostic biomarker not only in non-APL AML patients but also in AML patients who are less than 60 years old.

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.

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.

Overexpression of lncRNA PANDAR predicts adverse prognosis in acute myeloid leukemia.

BACKGROUND AND PURPOSE: Abundant studies have shown that lncRNA PANDAR plays an oncogenic role in human solid tumors. Although abnormal expression of PANDAR has been well investigated in solid tumors, it was rarely studied in hematologic diseases. Hence, the aim of this study was to determine the PANDAR expression level and its clinical significance in patients with acute myeloid leukemia (AML). MATERIALS AND METHODS: For detecting the expression level of PANDAR in 119 AML patients and 26 controls, real-time quantitative PCR was used in this study. The prognostic values were evaluated by using Kaplan-Meier analysis, Cox regression analyses, and logistic regression analysis. RESULTS: PANDAR was significantly overexpressed in AML and might be a promising biomarker which could distinguish AML from normal samples (P<0.001). Patients with high expression of PANDAR (PANDAR high) were older and showed higher bone marrow blasts than patients in PANDAR low group (P=0.029 and 0.032, respectively). Significant differences between these groups were also detected regarding risk group and karyotype finding (P=0.009 and 0.041, respectively). Importantly, PANDAR high patients presented a significant lower complete remission rate compared to PANDAR low patients (P<0.001). Furthermore, Kaplan-Meier analysis showed that PANDAR high patients had shorter overall survival compared to PANDAR low patients observing the whole AML cohort, and also in the non-M3 group of patients (P<0.001 and P=0.005, respectively). Multivariate analysis of Cox and logistic regression analysis confirmed that high PANDAR expression was an independent unfavorable risk factor for overall survival and complete remission in both observed patient groups. CONCLUSION: These results revealed that PANDAR was overexpressed in AML, and that higher PANDAR expression was associated with poor clinical outcome. Our study therefore suggests that PANDAR expression is a promising biomarker for prognostic prediction for AML.

SETBP1 mutations in Chinese patients with acute myeloid leukemia and myelodysplastic syndrome.

BACKGROUND: Somatic mutations in SETBP1 gene have recently been detected in hematologic malignancies. The present study aimed to explore the frequency and clinical correlations of SETBP1 mutations in patients with acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS). METHODS: In this study, we used high-resolution melting analysis (HRMA) to detect the SETBP1 mutations in a cohort of 363 patients with AML or MDS. RESULTS: A total of 1.2% (3/249) of AML and 1.8% (2/114) of MDS patients were found with heterozygous SETBP1 mutations. In AML, patients with SETBP1 mutations showed higher hemoglobin (P = 0.004) and were more frequently recurrent in AML-M4 subtype (P = 0.034). All five SETBP1 mutated patients had normal karyotypes. The patients with SETBP1 mutations had significantly higher incidences of concurrent SRSF2 mutations (P = 0.002). HRMA could detect SETBP1 mutations with 5% sensitivity, obviously higher than 25% of Sanger sequencing. CONCLUSIONS: We established a rapid, inexpensive, high-throughput and sensitive method to screen SETBP1 mutations. SETBP1 mutations were a rare molecular event in AML and MDS patients.

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.

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.

KRAS overexpression independent of RAS mutations confers an adverse prognosis in cytogenetically normal acute myeloid leukemia.

The prognostic value of RAS mutations has been systematically investigated in acute myeloid leukemia (AML). However, clinical significance of RAS expressions in AML remains poorly determined. To explore the clinical significance, we analyzed KRAS and NRAS expressions in 143 de novo AML patients by real-time quantitative PCR. KRAS and NRAS expressions were significantly up-regulated in AML patients. KRAS and NRAS mutations were identified in 4% (6/143) and 8% (12/143) of these patients, respectively. However, no significant association was observed between RAS mutations and expressions. High KRAS expression was associated with older age, higher white blood cells, and a tendency of higher platelets, whereas high NRAS expression was only correlated with older age. Complete remission (CR) rate and overall survival of AML patients were adversely affected by KRAS overexpression, but not NRAS overexpression. Multivariate analysis revealed that KRAS acted as an independent prognostic predictor in cytogenetically normal AML (CN-AML). Moreover, the prognostic value of KRAS expression was validated using the published data from Gene Expression Omnibus datasets. In the follow-up patients, KRAS expression rather than NRAS expression in CR time tended to decrease compared to newly diagnosis time, and both KRAS and NRAS expressions were significantly increased when in relapse time. Our findings revealed that RAS overexpression and mutations were common events in AML with potential therapeutic target value. KRAS overexpression independent of RAS mutations conferred an adverse prognosis in CN-AML.

Efficacy and safety of decitabine in treatment of elderly patients with acute myeloid leukemia: A systematic review and meta-analysis.

Elderly patients with acute myeloid leukemia (AML) have limited treatment options concerned about their overall fitness and potential treatment related mortality. Although a number of clinical trials demonstrated benefits of decitabine treatment in elderly AML patients, the results remains controversial. A meta-analysis was performed to evaluate efficacy and safety of decitabine in treatment of elderly AML patients. Eligible studies were identified from PubMed, Web of Science, Embase and Cochrane Library. Nine published studies were included in the meta-analysis, enrolling 718 elderly AML patients. The efficacy outcomes were complete remission (CR), overall response rate (ORR) and overall survival (OS). Safety was evaluated based on treatment related grades 3-4 adverse events (AEs) and early death (ED) rate. Pooled estimates with 95% confidence interval (CI) for CR, ORR and OS were 27% (95% CI 19%-36%), 37% (95% CI 28%-47%) and 8.09 months (95% CI 5.77-10.41), respectively. The estimated treatment related early death (ED) incidences were within 30-days 7% (95% CI 2%-11%) and 60-days 17% (95% CI 11%-22%), respectively. Thrombocytopenia was the most common grades 3-4 AEs. Subgroup analyses of age, cytogenetics risk, AML type and bone marrow blast percentage showed no significant differences of treatment response to decitabine. In conclusion, decitabine is an effective and well-tolerated therapeutic alternative with acceptable side effects in elderly AML patients.

Reduced intensity conditioning of allogeneic hematopoietic stem cell transplantation for myelodysplastic syndrome and acute myeloid leukemia in patients older than 50 years of age: a systematic review and meta-analysis.

PURPOSE: A systematic review and meta-analysis were performed to explore the efficacy and safety of allogeneic hematopoietic stem cell transplantation with a reduced intensity conditioning regimen in elderly patients with myelodysplastic syndrome (MDS) or acute myeloid leukemia (AML). METHODS: Overall survival (OS) and event-free survival (EFS) were established as the primary endpoints for directly assessing the efficacy, and non-relapse mortality (NRM) for safety. The eligible patients were at or above 50 years of age, and the outcomes of the typical elderly patients (≥60 years) were analyzed individually. RESULTS: The pooled estimates (95% confidence interval (CI)) for 1-year OS, EFS and NRM were 65 (55-74)  %, 50 (44-55)  % and 26 (21-30)  %, respectively; as for the patients ≥60 years of age, these were 63 (53-72) %, 46 (41-50)  % and 28 (23-32) %, respectively. No significantly statistical difference achieved between MDS and AML patients in 1-year EFS and NRM [relative risk (RR) 0.91, 95% CI 0.80-1.04; P = 0.172 and RR 1.18, 95% CI 0.82-1.69; P = 0.365]. The patients with lower diseases risk had the possibility of higher OS rate at ≥ 3 years than those with higher diseases risk (RR 1.37, 95% CI 0.95-1.97; P = 0.088). The patients had significantly higher 2-year OS and EFS rates in complete remission (CR, CR1 and CR2) at transplantation compared to those with advanced diseases (P < 0.05). CONCLUSIONS: RIC-alloHSCT is a feasible treatment option for the patients older than 50 year of age with MDS and AML. Advanced diseases status and higher diseases risk may be the poor factors for prognosis.

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.

Hypomethylation of let-7a-3 is associated with poor prognosis in myelodysplastic syndrome.

Abnormal methylation of let-7a-3 has been found in various cancers and may consequently affect their survival. In this study, real-time quantitative methylation specific PCR (RQ-MSP) was used to determine the unmethylation level of let-7a-3 in 95 patients with myelodysplastic syndrome (MDS). The hypomethylation of let-7a-3 promoter was detected in 22 of 95 (23.2%) patients with MDS compared to 4.2% (1/24) of controls (p= 0.0419). Moreover, the frequency of let-7a-3 hypomethylation was higher in older patients (≥70 years) than in younger patients (<70 years). No significant difference was observed in distribution of WHO, IPSS, and cytogenetic classification. However, hypomethylated patients had significantly shorter overall survival than those without hypomethylation (p= 0.007). Moreover both Kaplan-Meier and Multivariate Cox analyses confirmed that let-7a-3 hypomethylation was an independent prognostic risk factor in cohorts of MDS patients with lower-risk disease. Our study suggested that let-7a-3 hypomethylation may predict poor outcome in MDS.

Efficacy and Safety of Lenalidomide for Treatment of Low-/Intermediate-1-Risk Myelodysplastic Syndromes with or without 5q Deletion: A Systematic Review and Meta-Analysis.

BACKGROUND: Lenalidomide could effectively induce red blood cell (RBC) transfusion independence (TI) in patients with lower-risk (Low/Intermediate-1) myelodysplastic syndrome (MDS) with or without 5q deletion. However whether lenalidomide ultimately improves the overall survival (OS) of lower-risk MDS patients and reduces the progression to AML remains controversial. METHOD: A meta-analysis was conducted to examine the efficacy and safety of lenalidomide in the treatment of lower-risk MDS. Efficacy was assessed according to erythroid hematologic response (HI-E), cytogenetic response (CyR), OS and AML progression. Safety was evaluated based on the occurrence rates of grades 3-4 adverse events (AEs). RESULTS: Seventeen studies were included consisting of a total of 2160 patients. The analysis indicated that the overall rate of HI-E was 58% with 95% confidence interval (CI) of 43-74%. The pooled estimates for the rates of CyR, complete CyR, and partial CyR were 44% (95% CI 19-68%), 21% (95% CI 13-30%) and 23% (95% CI 15-32%), respectively. The patients with 5q deletion had significantly higher rate of HI-E and CyR than those without 5q deletion (P = 0.002 and 0.001, respectively). The incidences of grades 3-4 neutropenia, thrombocytopenia, leukopenia, anemia, deep vein thrombosis, diarrhea, fatigue and rash were 51% (95% CI 30-73%), 31% (95% CI 20-42%), 9% (95% CI 5-13%), 7% (95% CI 2-12%), 3% (95% CI 2-5%), 3% (95% CI 1-5%), 2% (95% CI 1-4%) and 2% (95% CI 1-3%), respectively. Lenalidomide significantly improved OS (HR: 0.62, 95% CI 0.47-0.83, P = 0.001) and lowered the risk of AML progression in del(5q) patients (RR: 0.61, 95% CI 0.41-0.91, P = 0.014). CONCLUSIONS: In spite of the AEs, lenalidomide could be effectively and safely used for the treatment of lower-risk MDS patients with or without 5q deletion.

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.