TET2 expression is a potential prognostic and predictive biomarker in cytogenetically normal acute myeloid leukemia.

TET2 (Ten-Eleven Translocation 2) gene is a member of TET family that can modify DNA through catalyzing the conversion of 5-methylcytosine (5-mC) into 5-hydroxymethylcytosine (5-hmC). Although TET2 mutation has been disclosed in a variety of hematopoietic malignancies, the prognostic implication of TET2 expression in acute myeloid leukemia (AML) remains largely elusive. In this study, real-time quantitative PCR was carried out to detect the level of TET2 transcript in 134 de novo AML patients and 35 healthy donors. TET2 mRNA level was significantly down-regulated in AML patients compared with controls (p = 0.010). Among the French-American-British (FAB) subtypes, the incidence of TET2 under-expression in M0/M1 subtypes was significantly higher than in the other subtypes M2/M3/M4/M5/M6 (p = 0.017), and also markedly higher than in the other granulocyte subtypes M2/M3 (p = 0.005). TET2 low-expressed patients showed a significantly higher frequency of NPM1 mutations than TET2 high-expressed patients. Although there was no significant difference in complete remission rate between two groups (low and high TET2 expression), patients with low TET2 expression had markedly shorter overall survival (OS) in both non-M3 and cytogenetically normal AML (CN-AML) (p = 0.016 and 0.044, respectively). Furthermore, multivariate analysis confirmed the prognostic value of TET2 expression on OS among CN-AML patients (p = 0.049). Importantly, TET2 expression in complete remission (CR) time was significantly higher than newly diagnosis time (p = 0.001), and was returned to lower level when in relapse time (p < 0.001). These findings indicated that down-regulation of TET2 expression was a common event and acted as a prognostic and predictive biomarker in CN-AML 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.

DLX4 hypermethylation is a prognostically adverse indicator in de novo acute myeloid leukemia.

Hypermethylation of distal-less homeobox 4 (DLX4) has been increasingly identified in several cancers. Our study was aimed to determine the role of DLX4 methylation in regulating DLX4 expression and further analyze its clinical significance in de novo acute myeloid leukemia (AML) patients. DLX4 methylation level was detected by real-time quantitative methylation-specific PCR and bisulfite sequencing PCR. Treatment with 5-aza-2'-deoxycytidine (5-aza-dC) was used for demethylation studies. Clinical significance of DLX4 methylation was obtained by the comparison between the patients with and without DLX4 methylation. DLX4 was significantly methylated in AML patients compared with controls (P < 0.001). DLX4 methylation was negatively associated with DLX7 (the shorter DLX4 isoform) (R = -0.202, P = 0.021) but not BP1 (the longer DLX4 isoform) (R = -0.049, P = 0.582) expression in AML patients. DLX7 and BP1 messenger RNA (mRNA) were significantly increased after 5-aza-dC treatment in leukemic cell lines THP1 and Kasumi-1. DLX4 methylated patients showed significantly higher frequency of U2AF1 mutation compared with DLX4 unmethylated patients (P = 0.043). Both all AML and non-M3 patients with DLX4 methylation presented significantly lower complete remission rate than those with DLX4 unmethylation (P = 0.001 and <0.001, respectively). DLX4 methylated cases had significantly shorter overall survival than DLX4 unmethylated cases among both all AML (P = 0.003), non-M3 AML (P = 0.001), and cytogenetically normal AML (P = 0.032). Multivariate analysis confirmed that DLX4 methylation was independent risk factor in both all AML and non-M3 patients. Our study indicates that DLX4 hypermethylation is negatively associated with DLX7 expression and predicts poor clinical outcome in de novo AML patients.

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.

The Antisenescence Effect of Exosomes from Human Adipose-Derived Stem Cells on Skin Fibroblasts.

Human adipose-derived stem cells (ADSCs) have become a promising therapeutic approach against skin aging. Recent studies confirm that exosomes partially mediate the therapeutic effect of stem cells. This study successfully isolated exosomes from the ADSC culture medium and discovered that ADSC-derived exosomes (ADSC-Exos) could alleviate human dermal fibroblast (HDF) senescence and stimulate HDF migration. Moreover, ADSC-Exos increased the type I collagen expression level and reduced the reactive oxygen species (ROS) and senescence-associated ß-galactosidase (SA-ß-Gal) activity in HDFs. In addition, we demonstrated that ADSC-Exos significantly inhibited senescence-related protein expression levels of p53, p21, and p16. In conclusion, our results have revealed the antisenescence effects of ADSC-Exos on HDFs and ADSC-Exos may be a novel cell-free therapeutic tool for antiaging.

[Research Progress in the Role of Tim3/galectin-9 in Hematological Malignancy--Review].

The incidence of hematological malignant tumor is increasing year by year, and seriously affecting the human health. In addition to the traditional radiation and chemotherapy, immunotherapy has achieved a certain effect in the treatment of blood tumor, but it is limited by exhaustion of CD8+ T cell. The exhaustion of CD8+ T cells is mainly related to the activation of some immune checkpoint inhibitors, such as (Tim3), programmed death 1 (PD-1), cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), etc. Among them, Tim3 is getting more and more attention. The combination of Tim3 and its ligand galectin-9 produced a strong cellular immunosuppressive effect. Tim3 has been proved to be associated with CD8+ T cell exhaustion in many tumors. In this review, the application of Tim3/galectin-9 in hematologic tumors is briefly summarized so as to provide theoretical basis for clinical diagnosis and treatment of these diseases.

Micronucleus assay for monitoring the genotoxic effects of arsenic in human populations: A systematic review of the literature and meta-analysis.

The micronucleus (MN) assay has been used to determine the potential genotoxic effects in human populations exposed to arsenic. Some of these studies found an increase in MN frequency among exposed individuals, but others found no increase or inconclusive results. Thus, the main purpose of this current study was to investigate whether MN can be used as a biomarker for the arsenic exposure, as well as whether or not the different cell types that have been used to monitor MN frequency differ in their sensitivity to upon arsenic exposure. A systematic literature review was conducted followed by a meta-analysis. The review identified 25 useful studies with data from 3232 exposed individuals (15 studies assaying lymphocytes, 16 assaying buccal cells, and 9 assaying urothelial cells), with 18 studies measuring drinking water exposure, 5 measuring occupational exposure, one measuring coal burning, and one measuring dietary exposure. The meta-analysis indicated that the overall estimates of Mean Ratio (MR, defined as the mean value of the response in the exposed group divided by the mean value of the response in the reference group) were 2.95 (95% confidence interval (CI): 2.00 to 4.35), 2.36 (95% CI: 1.77 to 3.15), and 2.82 (95% CI: 1.86 to 4.28) for MN assays conducted with lymphocytes, buccal cells, and urothelial cells in the MN assay, respectively. Subgroup analysis showed that when the exposure method was drinking water, the MN frequencies increased significantly in lymphocytes (MR = 3.59, 95% CI: 2.30 to 5.60), in buccal cells (MR = 2.35, 95% CI: 1.76 to 3.15), and in urothelial cells (MR = 3.16, 95% CI: 2.02 to 4.97). However, when the exposure method was the occupational setting or others, the MN detection using the three types of cells did not find significant differences between groups. Subgroup analysis also showed that lymphocyte MN frequencies increased significantly in both routine-culture MN assays (MR = 2.88, 95% CI: 1.15 to 7.24) and cytokinesis-block MN assays (MR = 2.89, 95% CI: 1.84 to 4.55). The performance of the MN assay with different types of cells was also compared, but no significant differences were found. Therefore, our analysis indicates that MN can be used as an effective biomarker for monitoring arsenic-exposed populations, and that MN assays conducted with lymphocytes, buccal cells, and urothelial cells do not differ in their ability to detect the genetic damage from arsenic.

Over-expression of miR-675 in formalin-fixed paraffin-embedded (FFPE) tissues of breast cancer patients.

BACKGROUND: Dysregulation of miR-675 has been found in a variety of solid tumors. MiR-675 has been suggested as having both oncogenic and tumor suppression properties in cancer. However, there is no evidence whether miR-675 is involved in breast cancer. The objective of this study was to evaluate the expression status of miR-675 and its clinical relevance in breast cancer patients. METHODS: The expression level of miR-675 was detected in 100 breast cancer patients and 38 cancer-free controls using real-time quantitative PCR. The clinicopathological characteristics of miR-675 in breast cancer were also investigated. All statistical analyses were performed using SPSS 20.0. RESULTS: The study showed that miR-675 was significantly up-regulated in breast cancer patients compared with controls (P < 0.01). There was no significant difference in age, lymph nodes stage, ER status and PR status between patients with and without miR-675 over-expression (P > 0.05). The frequency of miR-675 over-expression was higher in the patients of histological grade I-II than in others (50% versus 9%, P = 0.011). The expression level of miR-675 had a high correlation with miR-24/93/98/378 in breast cancer patients. CONCLUSIONS: Taken together, our study demonstrated that miR-675 in formalin-fixed paraffin-embedded (FFPE) tissues might serve as a good source for biomarker discovery and breast cancer validation.

High expression of dual-specificity phosphatase 5 pseudogene 1 (DUSP5P1) is associated with poor prognosis in acute myeloid leukemia.

The purpose of this study was to investigate the expression status of Dual-Specificity Phosphatase 5 Pseudogene 1 (DUSP5P1) and its clinical relevance in patients with acute myeloid leukemia (AML). Real-time quantitative PCR (RQ-PCR) was performed to detect the status of DUSP5P1 expression in 89 patients with de novo AML and 24 normal controls. The level of DUSP5P1 expression was significantly up-regulated in AML compared to controls (P=0.031). The patients with high expression of DUSP5P1 had higher percentage of blasts in bone marrow (BM) than those without high expression (P=0.027). The occurrence rate of DUSP5P1 high expression was significantly higher in M1 (2/8, 25%) and M2 subtypes (9/33, 27%) than in M3 subtype (0/17, 0%) (P=0.034). At the same time, the frequency of DUSP5P1 high expression in patients with intermediate (13/53, 24%) and poor karyotypes (5/11, 45%) was significantly higher than that in patients with favorable karyotype (0/21, 0%) (P=0.003). Meanwhile, DUSP5P1 high-expressed patients had significantly shorter overall survival (OS) than those with low expression (median 4.5 vs. 10.5 months, respectively, P=0.038). Our findings indicated that high expression of DUSP5P1 may identify high-risk AML patients and is associated with poor prognosis in AML.

STAT3 activation in tumor cell-free lymph nodes predicts a poor prognosis for gastric cancer.

STAT3 is constitutively activated in many human cancers including gastric cancer and plays crucial roles in modulating cancer cell proliferation, survival, metastasis as well as the microenvironment of pre-metastatic niches. Accumulating evidence has implicated STAT3 as a promising target for cancer therapy and it has been well established that tumor cell metastasized to lymph node is associated with poor prognosis. However, little is known about the relation between STAT3 activation in tumor cell-free lymph nodes and patient clinical outcomes. The objective of the current study was to investigate the role of STAT3 activity in tumor cell-free lymph nodes in tumor progression and prognosis for gastric cancer patients. Immunohistochemical analyses for p-STAT3, Ki-67, CD68 and Bcl-xL were performed in tumor cell-free lymph nodes from 60 gastric cancer patients. Survival analysis was conducted by using the Kaplan-Meier method. Immunohistochemical analyses showed that hyperactivity of STAT3 in tumor cell-free lymph nodes was significantly associated with tumor recurrence, and STAT3 activation pattern coincides with expression Ki-67, CD68, Bcl-xL. Survival analysis revealed that persistent STAT3 activation in uninvolved lymph nodes was positively associated with poor overall survival (P<0.05). These findings suggest that STAT3 activation in tumor-free lymph nodes is involved in the pathogenesis and metastasis of gastric cancer and that elevated STAT3 activity in lymph nodes prior to tumor cell arrival may indicate a poorer prognosis. These clinical studies support our findings in mouse tumor models showing that STAT3 activation is crucial for pre-metastatic niche formation and metastasis.

Prostate cancer-specific and potent antitumor effect of a DD3-controlled oncolytic virus harboring the PTEN gene.

Prostate cancer is a major health problem for men in Western societies. Here we report a Prostate Cancer-Specific Targeting Gene-Viro-Therapy (CTGVT-PCa), in which PTEN was inserted into a DD3-controlled oncolytic viral vector (OV) to form Ad.DD3.E1A.E1B(Δ55)-(PTEN) or, briefly, Ad.DD3.D55-PTEN. The woodchuck post-transcriptional element (WPRE) was also introduced at the downstream of the E1A coding sequence, resulting in much higher expression of the E1A gene. DD3 is one of the most prostate cancer-specific genes and has been used as a clinical bio-diagnostic marker. PTEN is frequently inactivated in primary prostate cancers, which is crucial for prostate cancer progression. Therefore, the Ad.DD3.D55-PTEN has prostate cancer specific and potent antitumor effect. The tumor growth rate was almost completely inhibited with the final tumor volume after Ad.DD3.D55-PTEN treatment less than the initial volume at the beginning of Ad.DD3.D55-PTEN treatment, which shows the powerful antitumor effect of Ad.DD3.D55-PTEN on prostate cancer tumor growth. The CTGVT-PCa construct reported here killed all of the prostate cancer cell lines tested, such as DU145, 22RV1 and CL1, but had a reduced or no killing effect on all the non-prostate cancer cell lines tested. The mechanism of action of Ad.DD3.D55-PTEN was due to the induction of apoptosis, as detected by TUNEL assays and flow cytometry. The apoptosis was mediated by mitochondria-dependent and -independent pathways, as determined by caspase assays and mitochondrial membrane potential.

Identification of a novel protein, LYRIC, localized to tight junctions of polarized epithelial cells.

Tight junctions (TJ) are multiprotein complexes that function to regulate paracellular transport of molecules through epithelial and endothelial cell layers. Many new tight junction-associated proteins have been identified in the past few years, and their functional roles and interactions have just begun to be elucidated. In this paper, we describe a novel protein LYsine-RIch CEACAM1 co-isolated (LYRIC) that is widely expressed and highly conserved between species. LYRIC has no conserved domains that would indicate function and does not appear to be a member of a larger protein family. Data from analysis of rat and human tissue sections and cell lines show that LYRIC colocalizes with tight junction proteins ZO-1 and occludin in polarized epithelial cells, suggesting that LYRIC is part of the tight junction complex. LYRIC dissociates from ZO-1 when junctional complexes are disrupted, and as tight junctions reform, ZO-1 relocalizes before LYRIC. These results suggest that LYRIC is most likely not a structural component required for TJ formation, but rather is recruited during the maturation of the tight junction complex.