Eiko Yamamoto

Fusobacterium species are part of the gut microbiome in humans. Recent studies have identified overrepresentation of Fusobacterium in colorectal cancer tissues, but it is not yet clear whether this is pathogenic or simply an epiphenomenon. In this study, we evaluated the relationship between Fusobacterium status and molecular features in colorectal cancers through quantitative real-time PCR in 149 colorectal cancer tissues, 89 adjacent normal appearing mucosae and 72 colonic mucosae from cancer-free individuals. Results were correlated with CpG island methylator phenotype (CIMP) status, microsatellite instability (MSI), and mutations in BRAF, KRAS, TP53, CHD7, and CHD8. Whole-exome capture sequencing data were also available in 11 cases. Fusobacterium was detectable in 111 of 149 (74%) colorectal cancer tissues and heavily enriched in 9% (14/149) of the cases. As expected, Fusobacterium was also detected in normal appearing mucosae from both cancer and cancer-free individuals, but the amount of bacteria was much lower compared with colorectal cancer tissues (a mean of 250-fold lower for Pan-fusobacterium). We found the Fusobacterium-high colorectal cancer group (FB-high) to be associated with CIMP positivity (P = 0.001), TP53 wild-type (P = 0.015), hMLH1 methylation positivity (P = 0.0028), MSI (P = 0.018), and CHD7/8 mutation positivity (P = 0.002). Among the 11 cases where whole-exome sequencing data were available, two that were FB-high cases also had the highest number of somatic mutations (a mean of 736 per case in FB-high vs. 225 per case in all others). Taken together, our findings show that Fusobacterium enrichment is associated with specific molecular subsets of colorectal cancers, offering support for a pathogenic role in colorectal cancer for this gut microbiome component.

Abstract Large intergenic noncoding RNAs (lincRNA) have been less studied than miRNAs in cancer, although both offer considerable theranostic potential. In this study, we identified frequent upregulation of miR-196a and lincRNA HOTAIR in high-risk gastrointestinal stromal tumors (GIST). Overexpression of miR-196a was associated with high-risk grade, metastasis and poor survival among GIST specimens. miR-196a genes are located within the HOX gene clusters and microarray expression analysis revealed that the HOXC and HOTAIR gene were also coordinately upregulated in GISTs which overexpress miR-196a. In like manner, overexpression of HOTAIR was also strongly associated with high-risk grade and metastasis among GIST specimens. RNA interference–mediated knockdown of HOTAIR altered the expression of reported HOTAIR target genes and suppressed GIST cell invasiveness. These findings reveal concurrent overexpression of HOX genes with noncoding RNAs in human cancer in this setting, revealing miR-196a and HOTAIR as potentially useful biomarkers and therapeutic targets in malignant GISTs. Cancer Res; 72(5); 1126–36. ©2012 AACR.

DNA methylation plays a key role in the silencing of numerous cancer‐related genes, thereby affecting a number of vital cellular processes, including the cell cycle checkpoint, apoptosis, signal transduction, cell adhesion and angiogenesis. Also widely altered in human malignancies is the expression of microRNAs (miRNAs), a class of small noncoding RNAs that act as posttranscriptional regulators of gene expression. Furthermore, emerging evidence now supports the idea that DNA methylation is crucially involved in the dysregulation of miRNAs in cancer. This is in part the result of technological advances that enable more comprehensive analysis of miRNA expression profiles and the epigenome in cancer cells, which has led to the identification of a number of epigenetically regulated miRNAs. As with protein‐coding genes, it appears that miRNA genes involved in regulating cancer‐related pathways are silenced in association with CpG island hypermethylation. In addition, methylation in CpG island shore regions and DNA hypomethylation also appear to contribute to miRNA dysregulation in cancer. Aberrant DNA methylation of miRNA genes is a potentially useful biomarker for detecting cancer and predicting its outcome. Moreover, re‐expression of miRNAs and the replacement of tumor suppressive miRNAs using miRNA mimics or expression vectors could be effective approaches to cancer therapy.

To clarify the cooperative roles of recurrently identified mutations and to establish a more precise risk classification system in acute myeloid leukemia (AML), we comprehensively analyzed mutations in 51 genes, as well as cytogenetics and 11 chimeric transcripts, in 197 adult patients with de novo AML who were registered in the Japan Adult Leukemia Study Group AML201 study. We identified a total of 505 mutations in 44 genes, while only five genes, FLT3, NPM1, CEBPA, DNMT3A and KIT, were mutated in more than 10% of the patients. Although several cooperative and exclusive mutation patterns were observed, the accumulated mutation number was higher in cytogenetically normal AML and lower in AML with RUNX1-RUNX1T1 and CBFB-MYH11, indicating a strong potential of these translocations for the initiation of AML. Furthermore, we evaluated the prognostic impacts of each sole mutation and the combinations of mutations and/or cytogenetics, and demonstrated that AML patients could be clearly stratified into five risk groups for overall survival by including the mutation status of DNMT3A, MLL-PTD and TP53 genes in the risk classification system of the European LeukemiaNet. These results indicate that the prognosis of AML could be stratified by the major mutation status in combination with cytogenetics.

Indoleamine 2,3-dioxygenase (IDO) is a tryptophan-catabolising enzyme inducing immune tolerance. The present study aimed to investigate IDO expression and its prognostic significance in endometrial cancer. Indoleamine 2,3-dioxygenase expression in endometrial cancer tissues (n=80) was immunohistochemically scored as four groups (IDO−, 1+, 2+, and 3+). The high IDO expression (IDO2+ or 3+) in tumour cells was found in 37 (46.3%) of the 80 cases, and was positively correlated with surgical stage, myometrial invasion, lymph-vascular space involvement, and lymph node metastasis, but not with the histological grade. Patients with high IDO expression had significantly impaired overall survival and progression-free survival (PFS) (P=0.002 and P=0.001, respectively) compared to patients with no or weak expression of IDO (IDO− or 1+). The 5-year PFS for IDO−/1+, 2+, and 3+ were 97.7, 72.9, and 36.4%, respectively. Even in patients with early-stage disease (International Federation of Gynecology and Obstetrics I/II, n=64), the PFS for IDO2+/3+ was significantly poor (P=0.001) compared to that for IDO−/1+. On multivariate analysis, IDO expression was an independent prognostic factor for PFS (P=0.020). These results indicated that the high IDO expression was involved in the progression of endometrial cancer and correlated with the impaired clinical outcome, suggesting that IDO is a novel and reliable prognostic indicator for endometrial cancer.

Abstract Purpose: Tumor escape from host immune systems is a crucial mechanism for disease progression. We recently showed that the immunosuppressive enzyme indoleamine 2,3-dioxygenase (IDO) is a prognostic indicator for endometrial cancer. The purpose of the present study was to investigate the relationship between IDO expression and tumor-infiltrating lymphocytes (TIL) or natural killer (NK) cells and to clarify their prognostic effect in endometrial cancer. Experimental Design: Immunohistochemical staining for IDO expression in endometrial cancer tissues (n = 65) was done. Tumor-infiltrating CD3+ and CD8+ lymphocytes, as well as CD57+ NK cells, were counted in serial tissue sections. Results: High IDO expression in tumor cells was found in 32 of 65 cases and was positively correlated with myometrial invasion, nodal metastasis, and lymph-vascular space involvement. We also found a significant correlation between high IDO expression and reduced numbers of CD3+, CD8+, and CD57+ cells infiltrating into both the tumor epithelium and stroma. Patients with high IDO expression, a low number of stromal CD3 (<60), low intraepithelial CD8 (<25), or low stromal CD8 (<40) had significantly impaired progression-free survival. On multivariate analysis, IDO expression and the number of stromal CD3+ TILs were independent prognostic factors for impaired progression-free survival. Conclusions: Tumoral IDO expression correlated with a reduced number of TILs and NK cells in endometrial cancer, possibly contributing to disease progression and impaired clinical outcome. These findings suggest that targeting IDO to restore host antitumor immunity may be a therapeutic strategy for endometrial cancer.

Indoleamine 2,3-dioxygenase (IDO) is a tryptophan-catabolizing enzyme that induces immune tolerance. The purpose of the present study is to investigate IDO expression and its functional role in ovarian cancer cells in vitro and in vivo.IDO expression was immunohistochemically scored in surgically-resected ovarian cancer tissues (n=60), and its association with tumor-infiltrating lymphocyte (TIL) count or patient survival was analyzed. Next, IDO cDNA was transfected into the human ovarian carcinoma cell line SKOV3, establishing stable clones of IDO-overexpressing cells (SK-IDO). SK-IDO cells were characterized in vitro as well as in vivo using a nude mouse xenograft model.High IDO expression in tumor cells was found in 34 (56.7%) cases and was correlated with a reduced number of CD8+ TIL. Patients with high IDO expression had significantly impaired overall and progression-free survival compared to patients with no or low IDO expression. There were no significant differences in in vitro cell proliferation, migration, invasion, or chemosensitivity to paclitaxel between the SK-IDO and control vector-transfected (SK-pcDNA) cells. However, tumor peritoneal dissemination was significantly increased in SK-IDO-xenografted mice compared to SK-pcDNA-xenografted mice. This tumor-progressive effect in SK-IDO-xenografted mice was abrogated by oral administration of the IDO inhibitor 1-methyl-tryptophan (1-MT). Finally, treatment with weekly i.p. paclitaxel combined with daily administration of 1-MT significantly prolonged the survival of the SK-IDO-xenografted mice compared to treatment with paclitaxel alone.These results suggest that IDO is involved in ovarian cancer progression in vivo and may be a promising therapeutic target for advanced ovarian cancer.

Altered expression of microRNA (miRNA) is strongly implicated in cancer, and recent studies have shown that the silencing of some miRNAs is associated with CpG island hypermethylation. To identify epigenetically silenced miRNAs in gastric cancer (GC), we screened for miRNAs induced by treatment with 5-aza-2'-deoxycytidine and 4-phenylbutyrate. We found that miR-34b and miR-34c are epigenetically silenced in GC and that their downregulation is associated with hypermethylation of the neighboring CpG island. Methylation of the miR-34b/c CpG island was frequently observed in GC cell lines (13/13, 100%) but not in normal gastric mucosa from Helicobacter pylori-negative healthy individuals. Transfection of a precursor of miR-34b and miR-34c into GC cells induced growth suppression and dramatically changed the gene expression profile. Methylation of miR-34b/c was found in a majority of primary GC specimens (83/118, 70%). Notably, analysis of non-cancerous gastric mucosae from GC patients (n = 109) and healthy individuals (n = 85) revealed that methylation levels are higher in gastric mucosae from patients with multiple GC than in mucosae from patients with single GC (27.3 versus 20.8%; P < 0.001) or mucosae from H. pylori-positive healthy individuals (27.3 versus 20.7%; P < 0.001). These results suggest that miR-34b and miR-34c are novel tumor suppressors frequently silenced by DNA methylation in GC, that methylation of miR-34b/c is involved in an epigenetic field defect and that the methylation might be a predictive marker of GC risk.

Epigenetic gene inactivation plays a key role in the development of various types of cancer. Using methylated CpG island amplification coupled with representational difference analysis to identify genes inactivated by DNA methylation in gastric cancer, we identified seven DNA fragments corresponding to the 5' CpG islands of the affected genes. One of the clones recovered was identical to the 5' flanking region of DFNA5, a gene previously shown to be associated with deafness and induced by DNA damage. Further analysis revealed that DFNA5 is expressed in normal tissues but is down-regulated in gastric cancer cell lines due to methylation of the region around its transcription start site. Treating gastric cancer cells that lacked DFNA5 expression with a methyltransferase inhibitor, 5-aza-2'-deoxycytidine, restored the gene's expression. Methylation of DFNA5 was detected in 50% of primary gastric tumors, and was correlated with positivity for Epstein-Barr virus and the absence of metastasis. Moreover, introduction of exogenous DFNA5 into silenced cells suppressed colony formation. Taken together, these data suggest that the silencing of DFNA5 occurs frequently in gastric cancer and may play a key role in development and progression of the disease.

OBJECTIVES: Sessile serrated adenomas (SSAs) are known to be precursors of sporadic colorectal cancers (CRCs) with microsatellite instability (MSI), and to be tightly associated withBRAFmutation and the CpG island methylator phenotype (CIMP). Consequently, colonoscopic identification of SSAs has important implications for preventing CRCs, but accurate endoscopic diagnosis is often difficult. Our aim was to clarify which endoscopic findings are specific to SSAs. METHODS: The morphological, histological and molecular features of 261 specimens from 226 colorectal tumors were analyzed. Surface microstructures were analyzed using magnifying endoscopy. Mutation inBRAFandKRASwas examined by pyrosequencing. Methylation ofp16,IGFBP7,MLH1andMINT1, -2, -12and -31was analyzed using bisulfite pyrosequencing. RESULTS: Through retrospective analysis of a training set (n=145), we identified a novel surface microstructure, the Type II open-shape pit pattern (Type II-O), which was specific to SSAs withBRAFmutation and CIMP. Subsequent prospective analysis of an independent validation set (n=116) confirmed that the Type II-O pattern is highly predictive of SSAs (sensitivity, 65.5%; specificity, 97.3%).BRAFmutation and CIMP occurred with significant frequency in Type II-O-positive serrated lesions. Progression of SSAs to more advanced lesions was associated with further accumulation of aberrant DNA methylation and additional morphological changes, including the Type III, IV and V pit patterns. CONCLUSIONS: Our results suggest the Type II-O pit pattern is a useful hallmark of the premalignant stage of CRCs with MSI and CIMP, which could serve to improve the efficacy of colonoscopic surveillance.

Abstract Altered expression of microRNAs (miRNA) occurs commonly in human cancer, but the mechanisms are generally poorly understood. In this study, we examined the contribution of epigenetic mechanisms to miRNA dysregulation in colorectal cancer by carrying out high-resolution ChIP-seq. Specifically, we conducted genome-wide profiling of trimethylated histone H3 lysine 4 (H3K4me3), trimethylated histone H3 lysine 27 (H3K27me3), and dimethylated histone H3 lysine 79 (H3K79me2) in colorectal cancer cell lines. Combining miRNA expression profiles with chromatin signatures enabled us to predict the active promoters of 233 miRNAs encoded in 174 putative primary transcription units. By then comparing miRNA expression and histone modification before and after DNA demethylation, we identified 47 miRNAs encoded in 37 primary transcription units as potential targets of epigenetic silencing. The promoters of 22 transcription units were associated with CpG islands (CGI), all of which were hypermethylated in colorectal cancer cells. DNA demethylation led to increased H3K4me3 marking at silenced miRNA genes, whereas no restoration of H3K79me2 was detected in CGI-methylated miRNA genes. DNA demethylation also led to upregulation of H3K4me3 and H3K27me3 in a number of CGI-methylated miRNA genes. Among the miRNAs we found to be dysregulated, many of which are implicated in human cancer, miR-1-1 was methylated frequently in early and advanced colorectal cancer in which it may act as a tumor suppressor. Our findings offer insight into the association between chromatin signatures and miRNA dysregulation in cancer, and they also suggest that miRNA reexpression may contribute to the effects of epigenetic therapy. Cancer Res; 71(17); 5646–58. ©2011 AACR.

MicroRNAs (miRNAs) play pivotal roles in numerous biological processes, and their dysregulation is a common feature of human cancer. Thanks to recent advances in the analysis of the cancer epigenome, we now know that epigenetic alterations, including aberrant DNA methylation and histone modifications, are major causes of miRNA dysregulation in cancer. Moreover, the list of miRNA genes silenced in association with CpG island hypermethylation is rapidly growing, and various oncogenic miRNAs are now known to be upregulated via DNA hypomethylation. Histone modifications also play important roles in the dysregulation of miRNAs, and histone deacetylation and gain of repressive histone marks are strongly associated with miRNA gene silencing. Conversely, miRNA dysregulation is causally related to epigenetic alterations in cancer. Thus aberrant methylation of miRNA genes is a potentially useful biomarker for detecting cancer and predicting its outcome. Given that many of the silenced miRNAs appear to act as tumor suppressors through the targeting of oncogenes, re-expression of the miRNAs could be an effective approach to cancer therapy, and unraveling the relationship between epigenetic alteration and miRNA dysregulation may lead to the discovery of new therapeutic targets.

BRAF is an important gene in colorectal cancers (CRCs) that is associated with molecular characterization and resistance to targeted therapy. Although microRNAs (miRNAs) are useful biomarkers of various cancers, the association between miRNA and BRAF in CRCs is undefined. Therefore, this study was conducted to identify a relationship between specific miRNA molecules and BRAF mutation in CRCs and serrated lesions. miRNA array was used for the measurement of 760 miRNAs in 29 CRCs. To assess the identified miRNAs, quantitative reverse transcription–PCR was performed on 721 CRCs, 381 serrated lesions and 251 non-serrated adenomas. Moreover, proliferation and invasion assays were conducted using cell lines. miRNA array analysis revealed that microRNA-31 (miR-31)-5p was the most up-regulated miRNA in CRCs with mutated BRAF (V600E) compared with CRCs possessing wild-type BRAF (including cases with KRAS mutation). High miR-31 expression was associated with BRAF and KRAS mutations and proximal location (P < 0.0001). High miR-31 expression was related to cancer-specific mortality [multivariate hazard ratio = 2.06, 95% confidence interval: 1.36–3.09, P = 0.0008]. Functional analysis demonstrated that miR-31 inhibitor decreased cell invasion and proliferation. With regard to serrated lesions, high miR-31 expression was less frequently detected in hyperplastic polyps compared with other serrated lesions. In conclusion, associations were identified between miR-31, BRAF and prognosis in CRC. Transfection of miR-31 inhibitor had an antitumour effect. Thus, miR-31 may be a promising diagnostic biomarker and therapeutic target in colon cancers. Moreover, high miR-31 expression in serrated lesions suggested that miR-31 may be a key molecule in serrated pathway.

Cancers exhibiting the CpG island methylator phenotype (CIMP) are found among a wide variety of human malignancies and represent a subclass of tumors showing concurrent hypermethylation of multiple CpG islands. These CIMP-positive tumors often exhibit characteristic molecular and clinicopathological features, suggesting CIMP represents a distinct carcinogenic pathway. However, marker genes to define CIMP have been largely inconsistent among studies, which has caused results to vary. Nonetheless, recent advances in genome-wide methylation analysis have enabled the existence of CIMP to be confirmed, and large-scale cancer genome analyses have begun to unravel the previously unknown molecular basis of CIMP tumors. CIMP is strongly associated with clinical outcome, suggesting it may be a predictive biomarker.

Abstract Epigenetic mechanisms such as histone modification play key roles in the pathogenesis of multiple myeloma (MM). We previously showed that EZH2, a histone H3 lysine 27 (H3K27) methyltransferase, and G9, a H3K9 methyltransferase, are potential therapeutic targets in MM. Moreover, recent studies suggest EZH2 and G9a cooperate to regulate gene expression. We therefore evaluated the antitumor effect of dual EZH2 and G9a inhibition in MM. A combination of an EZH2 inhibitor and a G9a inhibitor strongly suppressed MM cell proliferation in vitro by inducing cell cycle arrest and apoptosis. Dual EZH2/G9a inhibition also suppressed xenograft formation by MM cells in vivo. In datasets from the Gene Expression Omnibus, higher EZH2 and EHMT2 (encoding G9a) expression was significantly associated with poorer prognoses in MM patients. Microarray analysis revealed that EZH2/G9a inhibition significantly upregulated interferon (IFN)-stimulated genes and suppressed IRF4-MYC axis genes in MM cells. Notably, dual EZH2/G9a inhibition reduced H3K27/H3K9 methylation levels in MM cells and increased expression of endogenous retrovirus (ERV) genes, which suggests that activation of ERV genes may induce the IFN response. These results suggest that dual targeting of EZH2 and G9a may be an effective therapeutic strategy for MM.

Angiotensin II, a main effector peptide in the renin-angiotensin system, acts as a growth-promoting and angiogenic factor via type 1 angiotensin II receptors (AT(1)R). We have recently demonstrated that angiotensin II enhanced tumour cell invasion and vascular endothelial growth factor (VEGF) secretion via AT1R in ovarian cancer cell lines in vitro. The aim of the present study was to determine whether AT1R expression in ovarian cancer is correlated with clinicopathological parameters, angiogenic factors and patient survival. Immunohistochemical staining for AT1R, VEGF, CD34 and proliferating cell nuclear antigen (PCNA) were analysed in ovarian cancer tissues (n = 67). Intratumour microvessel density (MVD) was analysed by counting the CD34-positive endothelial cells. Type 1 angiotensin II receptors were expressed in 85% of the cases examined, of which 55% were strongly positive. Type 1 angiotensin II receptors expression was positively correlated with VEGF expression intensity and MVD, but not with histological subtype, grade, FIGO stage or PCNA labelling index. In patients who had positive staining for AT1R, the overall survival and progression-free survival were significantly poor (P = 0.041 and 0.017, respectively) as compared to those in patients who had negative staining for AT1R, although VEGF, but not AT1R, was an independent prognostic factor on multivariate analysis. These results demonstrated that AT1R correlated with tumour angiogenesis and poor patient outcome in ovarian cancer, suggesting its clinical potential for a novel molecular target in strategies for ovarian cancer treatment.

A subset of colorectal cancers (CRCs) show simultaneous methylation of multiple genes; these tumors have the CpG island methylator phenotype (CIMP). CRCs with CIMP show a specific pattern of genetic alterations, including a high frequency of BRAF mutations and a low frequency of p53 mutations. We therefore hypothesized that genes inactivated by DNA methylation are involved in the BRAF- and p53-signaling pathways. Among those, we examined the epigenetic inactivation of insulin-like growth factor-binding protein 7 ( IGFBP7 ) expression in CRCs. We found that in CRC cell lines, the silencing of IGFBP7 expression was correlated with high levels of DNA methylation and low levels of histone H3K4 methylation. Luciferase and chromatin immunoprecipitation assays in unmethylated cells revealed that p53 induces expression of IGFBP7 upon binding to a p53 response element within intron 1 of the gene. Treating methylated CRC cell lines with 5-aza-2′-deoxycytidine restored p53-induced IGFBP7 expression. Levels of IGFBP7 methylation were also significantly higher in primary CRC specimens than in normal colonic tissue ( P < 0.001). Methylation of IGFBP7 was correlated with BRAF mutations, an absence of p53 mutations and the presence of CIMP. Thus, epigenetic inactivation of IGFBP7 appears to play a key role in tumorigenesis of CRCs with CIMP by enabling escape from p53-induced senescence.

Dysregulation of microRNAs (miRNAs) has been implicated in bladder cancer (BCa), although the mechanism is not fully understood.We aimed to explore the involvement of epigenetic alteration of miRNA expression in BCa.Two BCa cell lines (T24 and UM-UC-3) were treated with 5-aza-2'-deoxycytidine (5-aza-dC) and 4-phenylbutyric acid (PBA), after which their miRNA expression profiles were analyzed using a TaqMan array (Life Technologies, Carlsbad, CA, USA). Bisulfite pyrosequencing was used to assess miRNA gene methylation in 5 cancer cell lines, 83 primary tumors, and 120 preoperative and 47 postoperative urine samples.Receiver operating characteristic (ROC) curve analysis was used to assess the diagnostic performance of the miRNA gene panel.Of 664 miRNAs examined, 146 were upregulated by 5-aza-dC plus PBA. CpG islands were identified in the proximal upstream of 23 miRNA genes, and 12 of those were hypermethylated in cell lines. Among them, miR-137, miR-124-2, miR-124-3, and miR-9-3 were frequently and tumor-specifically methylated in primary cancers (miR-137: 68.7%; miR-124-2: 50.6%; miR-124-3: 65.1%; miR-9-3: 45.8%). Methylation of the same four miRNAs in urine specimens enabled BCa detection with 81% sensitivity and 89% specificity; the area under the ROC curve was 0.916. Ectopic expression of silenced miRNAs in BCa cells suppressed growth and cell invasion.Our results indicate that epigenetic silencing of miRNA genes may be involved in the development of BCa and that methylation of miRNA genes could be a useful biomarker for cancer detection.

Gastrointestinal stromal tumors (GIST) are the most important mesenchymal tumors of the gastrointestinal tract. The vast majority of GISTs exhibit activating mutations of KIT or PDGFRA, but epigenetic alteration of GISTs is largely unknown. In this study, we aimed to clarify the involvement of DNA methylation in GIST malignancy.A total of 106 GIST specimens were studied. Levels of LINE-1 methylation were analyzed using bisulfite pyrosequencing. In addition, methylation of three other repetitive sequences (Alu Yb8, Satellite-α, and NBL2) was similarly analyzed, and CpG island hypermethylation was analyzed using MethyLight. Array-based comparative genomic hybridization (array CGH) was carried out in 25 GIST specimens.LINE-1 hypomethylation was significantly correlated with risk, and high-risk GISTs exhibited significantly lower levels of LINE-1 methylation than low-risk (61.3% versus 53.2%; P = 0.001) or intermediate-risk GISTs (60.8% versus 53.2%; P = 0.002). Hypomethylation of Satellite-α and NBL2 was also observed in high-risk GISTs. By contrast, promoter hypermethylation was relatively infrequent (CDH1, 11.2%; MLH1, 9.8%; SFRP1, 1.2%; SFRP2, 11.0%; CHFR, 9.8%; APC, 6.1%; CDKN2A, 0%; RASSF1A, 0%; RASSF2, 0%) and did not correlate with LINE-1 methylation or risk. Array CGH analysis revealed a significant correlation between LINE-1 hypomethylation and chromosomal aberrations.Our data suggest that LINE-1 hypomethylation correlates significantly with the aggressiveness of GISTs and that LINE-1 methylation could be a useful marker for risk assessment. Hypomethylation may increase the malignant potential of GISTs by inducing accumulation of chromosomal aberrations.

Subgroups of colorectal carcinomas (CRCs) characterized by DNA methylation anomalies are termed CpG island methylator phenotype (CIMP)1, CIMP2, or CIMP-negative. The pathogenesis of CIMP1 colorectal carcinomas, and their effects on patients' prognoses and responses to treatment, differ from those of other CRCs. We sought to identify genetic somatic alterations associated with CIMP1 CRCs.We examined genomic DNA samples from 100 primary CRCs, 10 adenomas, and adjacent normal-appearing mucosae from patients undergoing surgery or colonoscopy at 3 tertiary medical centers. We performed exome sequencing of 16 colorectal tumors and their adjacent normal tissues. Extensive comparison with known somatic alterations in CRCs allowed segregation of CIMP1-exclusive alterations. The prevalence of mutations in selected genes was determined from an independent cohort.We found that genes that regulate chromatin were mutated in CIMP1 CRCs; the highest rates of mutation were observed in CHD7 and CHD8, which encode members of the chromodomain helicase/adenosine triphosphate-dependent chromatin remodeling family. Somatic mutations in these 2 genes were detected in 5 of 9 CIMP1 CRCs. A prevalence screen showed that nonsilencing mutations in CHD7 and CHD8 occurred significantly more frequently in CIMP1 tumors (18 of 42 [43%]) than in CIMP2 (3 of 34 [9%]; P < .01) or CIMP-negative tumors (2 of 34 [6%]; P < .001). CIMP1 markers had increased binding by CHD7, compared with all genes. Genes altered in patients with CHARGE syndrome (congenital malformations involving the central nervous system, eye, ear, nose, and mediastinal organs) who had CHD7 mutations were also altered in CRCs with mutations in CHD7.Aberrations in chromatin remodeling could contribute to the development of CIMP1 CRCs. A better understanding of the biological determinants of CRCs can be achieved when these tumors are categorized according to their epigenetic status.

The concept of the CpG island methylator phenotype (CIMP) in colorectal cancer (CRC) is widely accepted, although the timing of its occurrence and its interaction with other genetic defects are not fully understood. Our aim in this study was to unravel the molecular development of CIMP cancers by dissecting their genetic and epigenetic signatures in precancerous and malignant colorectal lesions. We characterized the methylation profile and BRAF/KRAS mutation status in 368 colorectal tissue samples, including precancerous and malignant lesions. In addition, genome-wide copy number aberrations, methylation profiles, and mutations of BRAF, KRAS, TP53, and PIK3CA pathway genes were examined in 84 colorectal lesions. Genome-wide methylation analysis of CpG islands and selected marker genes revealed that CRC precursor lesions are in three methylation subgroups: CIMP-high, CIMP-low, and CIMP-negative. Interestingly, a subset of CIMP-positive malignant lesions exhibited frequent copy number gains on chromosomes 7 and 19 and genetic defects in the AKT/PIK3CA pathway genes. Analysis of mixed lesions containing both precancerous and malignant components revealed that most aberrant methylation is acquired at the precursor stage, whereas copy number aberrations are acquired during the progression from precursor to malignant lesion. Our integrative genomic and epigenetic analysis suggests early onset of CIMP during CRC development and indicates a previously unknown CRC development pathway in which epigenetic instability associates with genomic alterations. The concept of the CpG island methylator phenotype (CIMP) in colorectal cancer (CRC) is widely accepted, although the timing of its occurrence and its interaction with other genetic defects are not fully understood. Our aim in this study was to unravel the molecular development of CIMP cancers by dissecting their genetic and epigenetic signatures in precancerous and malignant colorectal lesions. We characterized the methylation profile and BRAF/KRAS mutation status in 368 colorectal tissue samples, including precancerous and malignant lesions. In addition, genome-wide copy number aberrations, methylation profiles, and mutations of BRAF, KRAS, TP53, and PIK3CA pathway genes were examined in 84 colorectal lesions. Genome-wide methylation analysis of CpG islands and selected marker genes revealed that CRC precursor lesions are in three methylation subgroups: CIMP-high, CIMP-low, and CIMP-negative. Interestingly, a subset of CIMP-positive malignant lesions exhibited frequent copy number gains on chromosomes 7 and 19 and genetic defects in the AKT/PIK3CA pathway genes. Analysis of mixed lesions containing both precancerous and malignant components revealed that most aberrant methylation is acquired at the precursor stage, whereas copy number aberrations are acquired during the progression from precursor to malignant lesion. Our integrative genomic and epigenetic analysis suggests early onset of CIMP during CRC development and indicates a previously unknown CRC development pathway in which epigenetic instability associates with genomic alterations. Colorectal cancer (CRC) is a leading cause of cancer mortality worldwide, but the incidence of CRC can be reduced through detection and removal of colorectal adenomas. Notably, however, most small colorectal polyps do not progress to malignancy; thus, the identification of precursor lesions likely to become cancerous is also extremely important for reducing CRC mortality. CRCs are thought to arise, in part, through the accumulation of genetic changes, including mutations of oncogenes and tumor suppressor genes.1Vogelstein B. Fearon E.R. Hamilton S.R. Kern S.E. Preisinger A.C. Leppert M. Nakamura Y. White R. Smits A.M. Bos J.L. Genetic alterations during colorectal-tumor development.N Engl J Med. 1988; 319: 525-532Crossref PubMed Scopus (6128) Google Scholar It is also generally accepted that CRCs can exhibit either of two genetic instabilities: chromosomal instability (CIN) or microsatellite instability (MSI).2Grady W. Carethers J. Genomic and epigenetic instability in colorectal cancer pathogenesis.Gastroenterology. 2008; 135: 1079-1099Abstract Full Text Full Text PDF PubMed Scopus (718) Google Scholar In addition to these genetic changes, epigenetic alterations, including DNA methylation and histone modification, play critical roles in the development of CRC; in addition, an increasing number of genes involved in cell cycle control, DNA repair, tumor invasiveness, and the response to growth factors have been identified as targets of hypermethylation in CRC.3Jones P.A. Baylin S.B. The epigenomics of cancer.Cell. 2007; 128: 683-692Abstract Full Text Full Text PDF PubMed Scopus (3780) Google Scholar, 4Suzuki H. Tokino T. Shinomura Y. Imai K. Toyota M. DNA methylation and cancer pathways in gastrointestinal tumors.Pharmacogenomics. 2008; 9: 1917-1928Crossref PubMed Scopus (43) Google Scholar, 5Lao V.V. Grady W.M. Epigenetics and colorectal cancer.Nat Rev Gastroenterol Hepatol. 2011; 8: 686-700Crossref PubMed Scopus (523) Google Scholar, 6Curtin K. Slattery M.L. Samowitz W.S. CpG island methylation in colorectal cancer: past, present and future.Patholog Res Int. 2011; 2011: 902674Crossref PubMed Google Scholar These epigenetic alterations are thought to be the main driving force in a subset of CRCs exhibiting concurrent hypermethylation of multiple loci, which is termed the CpG island methylator phenotype (CIMP).7Toyota M. Ahuja N. Ohe-Toyota M. Herman J.G. Baylin S.B. Issa J.P. CpG island methylator phenotype in colorectal cancer.Proc Natl Acad Sci U S A. 1999; 96: 8681-8686Crossref PubMed Scopus (2178) Google Scholar CIMP-positive CRCs show characteristic clinicopathological and molecular features, including proximal tumor location, female sex, older age, high tumor grade, wild-type TP53, frequent BRAF and KRAS mutations, and MSI. In addition, several studies support the hypothesis that CRCs can be categorized into three subclasses based on aberrant CpG island methylation: CIMP-high (CIMP-H; also known as CIMP1), CIMP-low (CIMP-L; also known as CIMP2), and CIMP-negative (CIMP-N). CIMP-H CRCs are significantly associated with a BRAF mutation, MLH1 methylation, and subsequent MSI.7Toyota M. Ahuja N. Ohe-Toyota M. Herman J.G. Baylin S.B. Issa J.P. CpG island methylator phenotype in colorectal cancer.Proc Natl Acad Sci U S A. 1999; 96: 8681-8686Crossref PubMed Scopus (2178) Google Scholar, 8Toyota M. Ohe-Toyota M. Ahuja N. Issa J.P. Distinct genetic profiles in colorectal tumors with or without the CpG island methylator phenotype.Proc Natl Acad Sci U S A. 2000; 97: 710-715Crossref PubMed Scopus (410) Google Scholar, 9Weisenberger D. Siegmund K. Campan M. Young J. Long T. Faasse M. Kang G. Widschwendter M. Weener D. Buchanan D. Koh H. Simms L. Barker M. Leggett B. Levine J. Kim M. French A. Thibodeau S. Jass J. Haile R. Laird P. CpG island methylator phenotype underlies sporadic microsatellite instability and is tightly associated with BRAF mutation in colorectal cancer.Nat Genet. 2006; 38: 787Crossref PubMed Scopus (1582) Google Scholar, 10Shen L. Toyota M. Kondo Y. Lin E. Zhang L. Guo Y. Hernandez N. Chen X. Ahmed S. Konishi K. Hamilton S. Issa J. Integrated genetic and epigenetic analysis identifies three different subclasses of colon cancer.Proc Natl Acad Sci U S A. 2007; 104: 18654Crossref PubMed Scopus (467) Google Scholar, 11Samowitz W. Albertsen H. Herrick J. Levin T. Sweeney C. Murtaugh M. Wolff R. Slattery M. Evaluation of a large, population-based sample supports a CpG island methylator phenotype in colon cancer.Gastroenterology. 2005; 129: 837-845Abstract Full Text Full Text PDF PubMed Scopus (495) Google Scholar A link between CIMP-L CRCs and KRAS mutations was first reported by Ogino et al,12Ogino S. Kawasaki T. Kirkner G.J. Loda M. Fuchs C.S. CpG island methylator phenotype-low (CIMP-low) in colorectal cancer: possible associations with male sex and KRAS mutations.J Mol Diagn. 2006; 8: 582-588Abstract Full Text Full Text PDF PubMed Scopus (257) Google Scholar and it was subsequently confirmed by other groups,10Shen L. Toyota M. Kondo Y. Lin E. Zhang L. Guo Y. Hernandez N. Chen X. Ahmed S. Konishi K. Hamilton S. Issa J. Integrated genetic and epigenetic analysis identifies three different subclasses of colon cancer.Proc Natl Acad Sci U S A. 2007; 104: 18654Crossref PubMed Scopus (467) Google Scholar, 13Hinoue T. Weisenberger D.J. Lange C.P. Shen H. Byun H.M. Van Den Berg D. Malik S. Pan F. Noushmehr H. van Dijk C.M. Tollenaar R.A. Laird P.W. Genome-scale analysis of aberrant DNA methylation in colorectal cancer.Genome Res. 2012; 22: 271-282Crossref PubMed Scopus (510) Google Scholar but much remains unknown about their respective molecular and clinicopathological features. Reducing the incidence of cancers, such as CRC, will require a better understanding of the mechanisms underlying carcinogenesis and the molecular alterations occurring in premalignant lesions. For example, although experimental evidence has confirmed the presence of CIMP in CRCs, the role of CIMP in the progression of precancerous lesions toward cancer is not yet fully understood. In recent years, sessile serrated adenomas (SSAs) have been the origin of MSI-positive/CIMP-H cancers, which account for approximately 10% to 15% of sporadic CRCs.14Spring K. Zhao Z. Karamatic R. Walsh M. Whitehall V.L.J. Pike T. Simms L. Young J. James M. Montgomery G. Appleyard M. Hewett D. Togashi K. Jass J. Leggett B. High prevalence of sessile serrated adenomas with BRAF mutations: a prospective study of patients undergoing colonoscopy.Gastroenterology. 2006; 131: 1400Abstract Full Text Full Text PDF PubMed Scopus (477) Google Scholar, 15Leggett B. Whitehall V. Role of the serrated pathway in colorectal cancer pathogenesis.Gastroenterology. 2010; 138: 2088-2100Abstract Full Text Full Text PDF PubMed Scopus (759) Google Scholar In addition, studies have also shown that CIMP is frequently observed among MSI-negative CRCs.10Shen L. Toyota M. Kondo Y. Lin E. Zhang L. Guo Y. Hernandez N. Chen X. Ahmed S. Konishi K. Hamilton S. Issa J. Integrated genetic and epigenetic analysis identifies three different subclasses of colon cancer.Proc Natl Acad Sci U S A. 2007; 104: 18654Crossref PubMed Scopus (467) Google Scholar, 16Nagasaka T. Koi M. Kloor M. Gebert J. Vilkin A. Nishida N. Shin S. Sasamoto H. Tanaka N. Matsubara N. Boland C.R. Goel A. Mutations in both KRAS and BRAF may contribute to the methylator phenotype in colon cancer.Gastroenterology. 2008; 134: 1950Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar From these and other studies of many tumors, a model was suggested in which CIN and epigenetic instability (CIMP) represent the two major pathways of CRC development, with up to 50% of CRCs being characterized as CIMP.16Nagasaka T. Koi M. Kloor M. Gebert J. Vilkin A. Nishida N. Shin S. Sasamoto H. Tanaka N. Matsubara N. Boland C.R. Goel A. Mutations in both KRAS and BRAF may contribute to the methylator phenotype in colon cancer.Gastroenterology. 2008; 134: 1950Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar, 17Goel A. Nagasaka T. Arnold C. Inoue T. Hamilton C. Niedzwiecki D. Compton C. Mayer R. Goldberg R. Bertagnolli M. Boland C.R. The CpG island methylator phenotype and chromosomal instability are inversely correlated in sporadic colorectal cancer.Gastroenterology. 2007; 132: 127Abstract Full Text Full Text PDF PubMed Scopus (241) Google Scholar This means that the origin of a large fraction of CIMP cancers remains unclear. High-resolution magnifying colonoscopy is a powerful diagnostic tool for detecting premalignant lesions. According to Kudo's classification, the pit patterns of nonneoplastic lesions are classified as type I (normal colon) or type II [hyperplastic polyp (HP)], whereas the pit patterns of neoplastic lesions are classified as types III, IV, and V.18Kudo S. Tamura S. Nakajima T. Yamano H. Kusaka H. Watanabe H. Diagnosis of colorectal tumorous lesions by magnifying endoscopy.Gastrointest Endosc. 1996; 44: 8-14Abstract Full Text Full Text PDF PubMed Scopus (913) Google Scholar, 19Kudo S. Lambert R. Allen J. Fujii H. Fujii T. Kashida H. Matsuda T. Mori M. Saito H. Shimoda T. Tanaka S. Watanabe H. Sung J. Feld A. Inadomi J. O'Brien M. Lieberman D. Ransohoff D. Soetikno R. Triadafilopoulos G. Zauber A. Teixeira C. Rey J. Jaramillo E. Rubio C. Van Gossum A. Jung M. Vieth M. Jass J. Hurlstone P. Nonpolypoid neoplastic lesions of the colorectal mucosa.Gastrointest Endosc. 2008; 68: S3-S47Abstract Full Text Full Text PDF PubMed Scopus (437) Google Scholar Recently, we performed an integrative analysis of the morphological, pathological, and molecular signatures in colorectal precancerous lesions and identified a novel pit pattern (type II, open pits) that was specific to SSAs.20Kimura T. Yamamoto E. Yamano H.O. Suzuki H. Kamimae S. Nojima M. Sawada T. Ashida M. Yoshikawa K. Takagi R. Kato R. Harada T. Suzuki R. Maruyama R. Kai M. Imai K. Shinomura Y. Sugai T. Toyota M. A novel pit pattern identifies the precursor of colorectal cancer derived from sessile serrated adenoma.Am J Gastroenterol. 2012; 107: 460-469Crossref PubMed Scopus (152) Google Scholar Those results depict an important relationship between morphological characteristics and molecular alterations that will significantly improve our ability to detect premalignant lesions. In the present study, our aim was to uncover the molecular evolution of CIMP cancers through an integrative analysis of many precursor and malignant colorectal lesions. Based on their genetic and epigenetic signatures, we propose a model in which CRCs develop via four distinct pathways. We also provide evidence that CIMP and CIN are not completely mutually exclusive, so that chromosomal aberrations may play important roles in a subset of CIMP cancers. These findings will improve our understanding of the pathogenesis of CRCs and could potentially contribute to better clinical management of premalignant lesions. Colorectal tumor tissues were collected from Japanese patients who underwent endoscopic or surgical resection of a colorectal tumor at Akita Red Cross Hospital (Akita, Japan). A total of 368 specimens from 192 precursor lesions, 38 noninvasive carcinomas [carcinoma in situ (CIS)], 100 CRCs, and 38 samples of adjacent normal tissue were analyzed in this study. Informed consent was obtained from all patients before collection of the specimens. Approval of this study was obtained from the Institutional Review Board of Akita Red Cross Hospital and Sapporo Medical University (Sapporo, Japan). By using the standard phenol/chloroform procedure, genomic DNA was extracted from biopsy specimens obtained before endoscopic or surgical resection. CRC cell lines were maintained and treated with 5-aza-2′-deoxycytidine, as previously described.21Akino K. Toyota M. Suzuki H. Mita H. Sasaki Y. Ohe-Toyota M. Issa J.P. Hinoda Y. Imai K. Tokino T. The Ras effector RASSF2 is a novel tumor-suppressor gene in human colorectal cancer.Gastroenterology. 2005; 129: 156-169Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar High-resolution magnifying endoscopes (CF260AZI; Olympus, Tokyo, Japan) were used for all colonoscopic analyses. Colorectal subsite locations were defined as right side colon proximal to the splenic flexure (cecum, ascending colon, hepatic flexure, and transverse colon), left side colon distal to the splenic flexure (splenic flexure, descending colon, and sigmoid colon), and rectum (rectosigmoid and rectum). All detected colorectal tumors were observed at high magnification after staining with indigo carmine dye and 0.05% crystal violet. Surface microstructures were classified according to Kudo's pit pattern classification system.18Kudo S. Tamura S. Nakajima T. Yamano H. Kusaka H. Watanabe H. Diagnosis of colorectal tumorous lesions by magnifying endoscopy.Gastrointest Endosc. 1996; 44: 8-14Abstract Full Text Full Text PDF PubMed Scopus (913) Google Scholar, 19Kudo S. Lambert R. Allen J. Fujii H. Fujii T. Kashida H. Matsuda T. Mori M. Saito H. Shimoda T. Tanaka S. Watanabe H. Sung J. Feld A. Inadomi J. O'Brien M. Lieberman D. Ransohoff D. Soetikno R. Triadafilopoulos G. Zauber A. Teixeira C. Rey J. Jaramillo E. Rubio C. Van Gossum A. Jung M. Vieth M. Jass J. Hurlstone P. Nonpolypoid neoplastic lesions of the colorectal mucosa.Gastrointest Endosc. 2008; 68: S3-S47Abstract Full Text Full Text PDF PubMed Scopus (437) Google Scholar Most often, one biopsy specimen was collected from each lesion for the extraction of genomic DNA. However, when two or more pit patterns were found in a single lesion (eg, adenoma to carcinoma transition), biopsy specimens were obtained for each respective pit pattern (see Supplemental Figure S1 at http://ajp.amjpathol.org). Thereafter, the lesions underwent endoscopic mucosal resection, endoscopic submucosal dissection, or surgical resection, after which histological analyses were performed (see Supplemental Figure S1 at http://ajp.amjpathol.org). Conventional adenomas, such as tubular adenoma and tubulovillous adenoma, were diagnosed using standard criteria. Serrated lesions, including HP, traditional serrated adenoma (TSA), and SSA, were classified based on criteria previously described by Torlakovic et al.22Torlakovic E. Skovlund E. Snover D. Torlakovic G. Nesland J. Morphologic reappraisal of serrated colorectal polyps.Am J Surg Pathol. 2003; 27: 65-81Crossref PubMed Scopus (550) Google Scholar Serrated lesions that did not satisfy the criteria for SSA or TSA were defined as HP. Tumors were classified into three categories: precursor lesions (HP, tubular adenoma, tubulovillous adenoma, TSA, and SSA), CIS, and CRCs. Methylated CpG island amplification microarray (MCAM) analysis was performed as previously described.23Goto Y. Shinjo K. Kondo Y. Shen L. Toyota M. Suzuki H. Gao W. An B. Fujii M. Murakami H. Osada H. Taniguchi T. Usami N. Kondo M. Hasegawa Y. Shimokata K. Matsuo K. Hida T. Fujimoto N. Kishimoto T. Issa J.P. Sekido Y. Epigenetic profiles distinguish malignant pleural mesothelioma from lung adenocarcinoma.Cancer Res. 2009; 69: 9073-9082Crossref PubMed Scopus (112) Google Scholar A BioPrime Plus Array CGH Genomic Labeling System (Life Technologies, Carlsbad, CA) was used to label MCA amplicons from tumor samples with Alexa Fluor 647 (Life Technologies, Carlsbad, CA), and those from a pooled mixture of normal colonic tissue were labeled with Alexa Fluor 555 (Life Technologies). Labeled MCA amplicons were then hybridized to a custom human CpG island microarray (G4497A; Agilent Technologies, Santa Clara, CA), which included 15,134 probes covering 6157 unique genes. After washing, the array was scanned using an Agilent DNA Microarray Scanner (Agilent Technologies), and the data were processed using Feature Extraction software version 10.7 (Agilent Technologies), and analyzed using GeneSpring GX version 11 (Agilent Technologies). Unsupervised hierarchical clustering and statistical analyses were then performed. Bisulfite pyrosequencing was performed as previously described.24Toyota M. Suzuki H. Sasaki Y. Maruyama R. Imai K. Shinomura Y. Tokino T. Epigenetic silencing of microRNA-34b/c and B-cell translocation gene 4 is associated with CpG island methylation in colorectal cancer.Cancer Res. 2008; 68: 4123-4132Crossref PubMed Scopus (577) Google Scholar Briefly, genomic DNA (1 μg) was modified with sodium bisulfite using an EpiTect Bisulfite Kit (Qiagen, Hilden, Germany). Pyrosequencing was then performed using a PSQ96 system with a PyroGold reagent Kit (Qiagen), after which the results were analyzed using Q-CpG software version 1.0.9 (Qiagen). Unsupervised hierarchical clustering, principal component analysis, and correspondence analysis of validation data were performed using JMP version 8 (SAS Institute Inc., Cary, NC). For the statistical analysis, quantitative methylation levels of each gene were converted to z scores, which were defined as follows: (Methylation Level in Each Sample - Mean Methylation Level in All Samples)/(SD of Methylation Levels in All Samples). Primer sequences are listed in Table 1.Table 1Primer Sequences Used in this StudyGenePrimer/TargetForwardReverseProduct size (bp)Methylation Analysis CDKN2APyroseq PCR5′-GGTTGTTTTGGTTGGTGTTTT-3′5′-Bio-ACCCTATCCCTCAAATCCTCTAAAA-3′169Sequence primer5′-TTTTTTTGTTTGGAAAGAT-3′Target5′-ATYGYG-3′ DFNA5Pyroseq PCR5′-GGYGGAGAGAGGGTTYGTT-3′5′-Bio-RAACCCCTCCCRCAACCT-3′91Sequence primer5′-YGGGYGTTTTAGAGT-3′Target5′-YGYGGGATTGGTYGTYG-3′ DKK2Pyroseq PCR5′-GGGTTTTTTGATTAATTAAGAGGAGA-3′5′-Bio-TCTACAATAACTAAAAACAATCAAATAC-3′179Sequence primer5′-TAATTAAGAGGAGAGTTAAA-3′Target5′-TYGTYGAGATTTYGGYG-3′ DLX4Pyroseq PCR5′-GGTTTYGGTTTAGTTTTGGATTTAGTT-3′5′-Bio-CAATTCTACTCCCAAAAAACTCCCA-3′182Sequence primer5′-TGTTTYGTTTTATTTTAAGT-3′Target5′-TGGYGTTATYGTTYG-3′ FZD10Pyroseq PCR5′-GGGATTTATTATAAAAGGAAGAGAAGAT-3′5′-Bio-AATAATCCCCRACACCCCRAAAAC-3′129Sequence primer5′-AAAGGAAGAGAAGATGTATT-3′Target5′-TYGYG-3′ GALNT14Pyroseq PCR5′-GAGYGGGAAAGTTTTTTTAGGTATAG-3′5′-Bio-CCTAAACRCAACTCCCAAACCATC-3′153Sequence primer5′-GAAAGTTTTTTTAGGTATAG-3′Target5′-YGTYGTTTGGYG-3′ IGF2BP1Pyroseq PCR5′-GAAGGGGTTTGTAGAGTTTTAGGGA-3′5′-Bio-CCCACCCACCCTACAAAAAAAAACC-3′157Sequence primer5′-TTGAGTTTTTTATTTTTAGG-3′Target5′-YGGGAGATTATYG-3′ IGFBP7Pyroseq PCR5′-AGGGTTYGGGGTAGGGGATTGGGGAT-3′5′-Bio-AAAACCACACCCCRAAACRATAAAAACAC-3′208Sequence primer5′-YGGGTGTTYGTTTATTTT-3′Target5′-TYGAYGTTAGTAGGAGYGYGYGYG-3′ KCNV1Pyroseq PCR5′-TAAGGAGAGGTAATTTTTTYGGGAGTT-3′5′-Bio-CGCTAAAAAACATCTCTAACCCAATC-3′150Sequence primer5′-GGAGTTYGGGGAATTT-3′Target5′-YGGTYG-3′ LRP1BPyroseq PCR5′-GATGTAAGATTAGAYGTATTTTGTATTG-3′5′-Bio-AACCAATCAACCTTCTCCTACCTAA-3′148Sequence primer5′-TATTGAAAAGTTAAGATATA-3′Target5′-YGGGYGTTTYGTTYGYG-3′ MEOX2Pyroseq PCR5′-TAGAGTTTGGAGGGTAGAGTTGTTGT-3′5′-Bio-ATTCCACTTCCTATCTCCTACTAAAC-3′137Sequence primer5′-GGGTAGAGTTGTTGTTTTT-3′Target5′-TYGGGYG-3′ MINT1Pyroseq PCR5′-GGTTTTTTGTTAGYGTTTGTATTT-3′5′-Bio-ATTAATCCCTCTCCCCTCTAAACTT-3′133Sequence primer5′-TTTAGTAAAAATTTTTTGGG-3′Target5′-GYGTTTGTTGTG-3′ MINT2Pyroseq PCR5′-YGTTATGATTTTTTTGTTTAGTTAAT-3′5′-Bio-TACACCAACTACCCAACTACCTC-3′203Sequence primer5′-TTTTGTTTAGTTAATTGAATTT-3′Target5′-GTYGTYGTTTYGAGTTTTAGG-3′ MINT12Pyroseq PCR5′-YGGGTTATGTTTTATTTTTTGTGTTT-3′5′-Bio-CTCAAAAAAATCAAACAACCAACCAA-3′190Sequence primer5′-TAATTYGGATTTTAAATTAAATA-3′Target5′-AAAYGTTTTTATTTT-3′ MINT31Pyroseq PCR5′-GAYGGYGTAGTAGTTATTTTGTT-3′5′-Bio-CATCACCACCCCTCACTTTAC-3′184Sequence primer5′-TGTAGTTTTAGGAGAGTGAATA-3′Target5′-AYGTTTAGGGGTGATGGTTTTAGTAAA-3′ MIR34BPyroseq PCR5′-GGTYGAGTGATTGTGGYGGGGG-3′5′-Bio-CCTCCATCTTCTAAACRTCTCCCTTA-3′176Sequence primer5′-TAATYGTTTTTGGAATTT-3′Target5′-YGYGGGTYGAGGGGYGGGGYGGGYGYG-3′ MLH1Pyroseq PCR5′-TTGGTATTTAAGTTGTTTAATTAATAGTTG-3′5′-Bio-AAAATACCTTCAACCAATCACCTC-3′119Sequence primer5′-AGTTATAGTTGAAGGAAGAA-3′Target5′-YGTGAGTAYG-3′ RASSF2Pyroseq PCR5′-GGTAGGGGTTGAAAAAGGTTAA-3′5′-Bio-CRCRACTAAAAAACTACTTCAACT-3′177Sequence primer5′-GGYGTTYGGTTTTTA-3′Target5′-GTYGYGYGGTTATYG-3′ RASSF5Pyroseq PCR5′-TYGTTATTAGTYGGGTATGGTTATGG-3′5′-Bio-CRAAACCRCTCAAACTCTATAAATAAC-3′110Sequence primer5′-TATTYGTTATTATTGGATTT-3′Target5′-YGAGTYGTYGYG-3′ SFRP1Pyroseq PCR5′-GTTTTGTTTTTTAAGGGGTGTTGAG-3′5′-Bio-CTCCRAAAACTACAAAACTAAAATAC-3′202Sequence primer5′-GYGTTTGGTTTTAGTAAAT-3′Target5′-TTGYGYGGGGYGGTTTYGAGGGTTYG-3′ SFRP2Pyroseq PCR5′-AATTTYGGATTGGGGTAAAATAAGTT-3′5′-Bio-TTAAACAACAAACAAAAAAACCTAACC-3′182Sequence primer5′-YGTTTTYGTTAGTATTTGG-3′Target5′-TYGYGAGGTYGTTYGYG-3′ SOX5Pyroseq PCR5′-GATTTGGAGGGAGYGGGAGTTTT-3′5′-Bio-CAAAAACAAACAACACAATACRAATACA-3′184Sequence primer5′-GTYGTATTTTTYGGGG-3′Target5′-YGGGYGTYG-3′ WIF1Pyroseq PCR5′-GTTTTYGTAGGTTTTTTGGTATTTAGG-3′5′-Bio-GAACCATACTACTCAAAACCTCCTC-3′174Sequence primer5′-AGGTTTTTTGGTATTTAGG-3′Target5′-TYGGGAGGYGAYGYGTTTAGTYGTTTAAAYG-3′ WNT5APyroseq PCR5′-ATATTTGGGGTTGGAAAGTTTTAATTAT-3′5′-Bio-AACCRACAACAAAAACAAAACCTAATC-3′149Sequence primer5′-GGTTGGAAAGTTTTAATTAT-3′Target5′-YGTYGTYG-3′ ZNF569Pyroseq PCR5′-TAGTYGATTGTAAGAAGGAAGTGTTT-3′5′-Bio-CRCAAAAAAACTCAACCTAAATTTTACA-3′199Sequence primer5′-GGTTTTTGGGAAATGTA-3′Target5′-GTTYGGYG-3′Mutation Analysis AKT1Pyroseq PCR5′-Bio-AGTGTGCGTGGCTCTCACC-3′5′-CATTCTTGAGGAGGAAGTAGCG-3′83Sequence primer5′-GCCAGGTCTTGATGTACT-3′Target5′-YCCCTA-3′Y equals C or T, and R equals A or G.Pyroseq, pyrosequencing. Open table in a new tab Y equals C or T, and R equals A or G. Pyroseq, pyrosequencing. Mutations in codon 600 of BRAF and codons 12 and 13 of KRAS were examined by pyrosequencing using BRAF and KRAS pyrokits (Qiagen), respectively, according to the manufacturer's instructions. Mutation of PIK3CA, AKT2, and PDK1 was analyzed by direct sequencing, as previously described.25Parsons D.W. Wang T.L. Samuels Y. Bardelli A. Cummins J.M. DeLong L. Silliman N. Ptak J. Szabo S. Willson J.K. Markowitz S. Kinzler K.W. Vogelstein B. Lengauer C. Velculescu V.E. Colorectal cancer: mutations in a signalling pathway.Nature. 2005; 436: 792Crossref PubMed Scopus (503) Google Scholar Mutation of TP53 was initially detected by PCR–single-stranded conformational polymorphism analysis, followed by direct sequencing, as previously described.26Sugai T. Habano W. Nakamura S. Uesugi N. Sasou S. Itoh C. A unique method for mutation analysis of tumor suppressor genes in colorectal carcinomas using a crypt isolation technique.Arch Pathol Lab Med. 2000; 124: 382-386Crossref PubMed Google Scholar Mutation of AKT1 was analyzed by pyrosequencing using the primer sequences listed in Table 1. MSI was assessed as previously described,27Sugai T. Habano W. Endoh M. Konishi Y. Akasaka R. Toyota M. Yamano H. Koeda K. Wakabayashi G. Suzuki K. Molecular analysis of gastric differentiated-type intramucosal and submucosal cancers.Int J Cancer. 2010; 127: 2500-2509Crossref PubMed Scopus (25) Google Scholar using primers proposed by the National Cancer Institute Workshop on Microsatellite Instability (BAT25, BAT26, D5S346, D2S123, and D17S250).28Boland C.R. Thibodeau S.N. Hamilton S.R. Sidransky D. Eshleman J.R. Burt R.W. Meltzer S.J. Rodriguez Bigas M.A. Fodde R. Ranzani G.N. Srivastava S. A National Cancer Institute Workshop on Microsatellite Instability for cancer detection and familial predisposition: development of international criteria for the determination of microsatellite instability in colorectal cancer.Cancer Res. 1998; 58: 5248-5257PubMed Google Scholar MSI was defined by the presence, in the tumor sample, of bands that were abnormal in size, compared with a corresponding normal sample. A tumor sample was defined as MSI positive when two or more markers showed instability. Array-based comparative genome hybridization (array CGH) analysis was performed as previously described.29Igarashi S. Suzuki H. Niinuma T. Shimizu H. Nojima M. Iwaki H. Nobuoka T. Nishida T. Miyazaki Y. Takamaru H. Yamamoto E. Yamamoto H. Tokino T. Hasegawa T. Hirata K. Imai K. Toyota M. Shinomura Y. A novel correlation between LINE-1 hypomethylation and the malignancy of gastrointestinal stromal tumors.Clin Cancer Res. 2010; 16: 5114-5123Crossref PubMed Scopus (81) Google Scholar Briefly, 500 ng of genomic DNA from colorectal tumor specimens and sex-matched reference DNA from noncancerous colonic mucosa were labeled with Cy5 and Cy3, respectively, using a Genomic DNA Enzymatic Labeling Kit (Agilent Technologies), and were then hybridized to Human Genome CGH Microarray Kit 180A (G4449A; Agilent Technologies). DNA copy number aberrations (CNAs) were identified using the ADM-2 algorithm included in the Agilent Genomic Workbench software version 5 (Agilent Technologies). A copy number loss was defined as a log 2 ratio of <−0.5, and a copy number gain was defined as a log 2 ratio of >0.5. Unsupervised hierarchical clustering of CNAs was performed using JMP, version 8. The microarray data in this study have been submitted to the Gene Expression Omnibus, and the accession number is GSE35534. To compare differences in continuous variables between groups, t-tests or an analysis of variance with a post hoc Tukey's test was performed. A Fisher's exact test or a χ2 test was used for analysis of categorical data. P < 0.05 (two sided) was considered statistically significant. Statistical analyses were performed using JMP, version 8, and SPSS statistics 18 (IBM Corporation, Somers, NY). To clarify the epigenetic changes occurring early during colorectal tumorigenesis, we first performed global methylation analysis using MCAM in a series of normal colonic tissues (n = 16), precursor lesions (HP, n = 3; SSA, n = 5; TSA, n = 3; tubular adenoma, n = 6; tubulovillous adenoma, n = 10), CISs (n = 14), and CRCs (n = 28). Hierarchical clustering analysis using the MCAM results identified 1010 probe sets that detected frequent hypermethylation in tumor tissues (see Supplemental Figure S2 at http://ajp.amjpathol.org). Subsequent K-means clustering analysis using these probe sets revealed that the samples could be clearly categorized into three subclasses based on the level of their methylation (Figure 1A); subclasses with high and intermediate methylation were presumed to reflect CIMP-H and CIMP-L tumors, respectively. Among the 1010 probe sets, 538 unique genes were in the high-methylation group, whereas 259 genes were in the intermediate-methylation group (see Supplemental Figure S2C and Supplemental Tables S1 and S2 at http://ajp.amjpathol.org). A subset of the precursor lesions could also be categorized into these subclasses, indicating that CIMP-like methylation patterns were already established early during carcinogenesis. To further characterize the genes that acquired methylation progressively during CIMP pathway tumorigenesis, we next performed MCAM analysis with a series of precursor lesions in which CIMP-N flat components were present, along with CIMP-positive protruding components

Ovarian cancer is the most lethal gynecologic malignancy, and clear cell adenocarcinoma of the ovary (OCCA), in particular, has a relatively poor prognosis among the ovarian cancer subtypes because of its high chemoresistance. Chromobox (CBX) 7 is a polycomb repressive complex 1 component that prolongs the lifespan of normal human cells by downregulating the INK4a/ARF expression which promotes cell-cycle progression. However, recent reports studying the relationship between CBX7 expression and patient survival have differed regarding the tumor cell origins, and the precise role of CBX7 in human carcinomas remains obscure. In this study, we analyzed CBX7 expression by immunohistochemistry in 81 OCCA patients and evaluated its association with their clinical outcomes. Both the overall and progression-free survival rates of the CBX7-positive patients were significantly shorter than those of the CBX7-negative patients (p < 0.05). CBX7 knockdown experiments using two OCCA cell lines, TOV21G and KOC-7C, revealed that cell viability was significantly reduced compared to the control cells (p < 0.001). Expression microarray analysis revealed that apoptosis-related genes, particularly tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), were significantly upregulated in CBX7 knockdown cells (p < 0.01). We further confirmed that CBX7 knockdown resulted in TRAIL-induced apoptosis in the OCCA cells. Thus, in this study, we showed for the first time that CBX7 was associated with a decreased OCCA prognosis. We also successfully demonstrated that the TRAIL pathway is a novel target for CBX7 expression modulation in these cells, and therapeutic agents utilizing the TRAIL pathway may be particularly effective for targeted OCCA therapy.

Cyberbullying is a global public health concern with tremendous negative impacts, not only on the physical and mental health of students but also on their well-being and academic performance. However, there are very few studies on cyberbullying among university students, especially in Myanmar. This study aims to determine the percentage of university students who suffered cyberbullying victimization in the last 12 months, and the association between students' socio-demographic characteristics, adverse events following cyberbullying and cyberbullying victimization.A cross-sectional study was conducted among university students aged 18 years and older at one medical university in Magway, Myanmar. A total of 412 students (277 males and 135 females) participated in the study. Data were collected from August to September, 2018 using a self-administered questionnaire. Multiple logistic regression analyses (models I and II) were performed to estimate the unadjusted (UOR) and adjusted odds ratios (AOR), and 95% confidence intervals (CI).In total, 40.8% of males and 51.1% of females in the study had suffered cyberbullying victimization in the past 12 months. In model I, students who had been studying at the university for 3 years or less (AOR = 1.81; 95% CI 1.14-2.85), and who had witnessed psychological, physical or sexual violence, or cyberbullying in their neighborhoods, (AOR = 2.95; 95% CI 1.48-5.91) were more likely to have suffered cyberbullying victimization in the past 12 months. In model II, being a victim of cyberbullying was associated with difficulties in concentrating and understanding lectures (AOR = 3.96; 95% CI 1.72-9.11), and substance abuse (AOR = 2.37; 95% CI 1.02-5.49). Non-resident students were at a higher risk of being cyberbullying victims than their resident peers (AOR = 1.86; 95% CI 1.04-3.34).Two out of five students had suffered cyberbullying victimization in the past 12 months, and only half of the victims discussed their experience(s) with someone else. Students who suffered cyberbullying victimization faced academic difficulties and started or increased smoking, betel chewing or alcohol drinking. Counter measures to prevent and mitigate the adverse events related to cyberbullying victimization are urgently needed among university students in Myanmar. Periodic screening for cyberbullying, counseling services, cyber-safety educational programs, and awareness raising campaigns should be implemented.

Twist, a basic helix--loop-helix transcription factor, has been reported to be associated with the development and progression of human cancer. We examined the distribution and expression of Twist in cervical cancer to examine its clinical significance.We examined the distribution and expression of Twist in 101 cervical cancer specimens and determined the association between their expression and the clinico-pathological features observed, including patient outcome.Of the 101 specimens, 55 cases were negative for Twist immuno-expression, whereas 46 were positive. When categorized into negative versus positive expression, Twist was not associated with any of the clinico-pathological parameters examined. Positive Twist expression significantly predicted poorer overall survival (OS) and progression-free survival (PFS) when compared with negative expression (P < 0.01). In the multivariate analyses, positive Twist expression was an independent prognostic factor for OS (P < 0.05).Our data imply that positive Twist expression seems to be a useful marker in patients with cervical cancer likely to have an unfavorable clinical outcome.

Abstract Background: The molecular mechanism by which Helicobacter pylori infection leads to gastric cancer is not fully understood. Similarly, patients with enlarged-fold (EF+) gastritis, one cause of which is H. pylori infection, have an increased risk for gastric cancer, although again molecular mechanism is unclear. In the present study, we analyzed the methylation status of long interspersed nucleotide elements (LINE-1) and three cancer-related genes in a panel of gastric mucosae, with or without EF+ gastritis. Methods: We used bisulfite pyrosequencing to assess the levels of LINE-1, CDH1, CDH13, and PGP9.5 methylation in 78 gastric mucosa specimens from 48 patients. Results: Levels of LINE-1 methylation were significantly reduced in mucosae from patients with EF+ gastritis. This hypomethylation of LINE-1 was associated with increased methylation of the 5′ CpG islands of the genes, which suggests that, in EF+ gastritis, the methylation of the promoter regions of certain genes is accompanied by global demethylation of repetitive sequences. Conclusions: Our results indicate that genomewide hypomethylation and regional hypermethylation occur in EF+ gastritis and may contribute to the tumorigenesis of diffuse-type gastric cancers. (Cancer Epidemiol Biomarkers Prev 2008;17(10):2555–64)

Indoleamine 2,3-dioxygenase (IDO) is a tryptophan-catabolizing enzyme that induces tolerance to host immune surveillance within the tumor microenvironment. The present study aimed to investigate IDO expression and its prognostic significance in invasive cervical cancer.Immunohistochemical expression of IDO in tumor tissues and its association with clinicopathological factors and survival were analyzed in 112 stage IB-IIB cervical cancer patients treated with radical hysterectomy and pelvic lymphadenectomy.IDO was diffusely expressed in tumor cells in 29 (26%) cases and focally expressed at the invasive front in 29 (26%) cases, while the other 54 (48%) cases were IDO-negative. IDO expression was positively correlated with clinical stage, lymph node metastasis, and lymph-vascular space invasion, but not with histological type. Patients with diffuse IDO expression had significantly reduced overall survival (OS) and disease-free survival (DFS) compared to patients with no IDO expression. The 5-year OS/DFS rates for the IDO-negative, focally positive, and diffusely positive groups were 92.3%/84.9%, 89.5%/75.8%, and 65.5%/51.7%, respectively. When we analyzed patients with stage IB disease alone (n=67), the OS and DFS for the IDO-diffusely positive group were significantly lower than those for the IDO-negative group. In multivariate analysis, diffuse IDO expression was found to be an independent prognostic factor for impaired OS and DFS.Diffuse expression of IDO in the tumor obtained from Stage IB-IIB cervical cancer patients who underwent radical hysterectomy was correlated with an unfavorable clinical outcome. These findings suggest that IDO may be a novel post-operative prognostic indicator for stage IB-IIB cervical cancer.

Cancer cells exhibit two opposing methylation abnormalities: genome-wide hypomethylation and gene promoter hypermethylation. Downregulation of E-cadherin (CDH1) plays a key role in the development of diffuse-type gastric cancer, and DNA methylation is a major cause of the gene's silencing. Hereditary diffuse gastric cancer is caused by germline mutation of CDH1 gene, and DNA methylation frequently serves as the second hit completely inactivating the gene. In sporadic diffuse-type gastric cancer, methylation of CDH1 is more prevalent than mutation of the gene. Epstein-Barr virus (EBV)-associated gastric carcinoma (EBV-associated GC) is characterized by concurrent methylation of multiple genes, and diffuse-type gastric cancer is frequently seen among EBV-associated GCs. Patients with pangastritis or enlarged-fold gastritis, which are both caused by Helicobacter pylori infection, reportedly have an increased risk for diffuse-type gastric cancer. Notably, the gastric mucosa of enlarged-fold gastritis patients exhibits CDH1 hypermethylation and genome-wide hypomethylation. These data suggest that aberrant DNA methylation is an essential promoter of carcinogenesis in individuals at high risk for diffuse-type gastric cancer.

The CpG island methylator phenotype (CIMP) is a distinct form of epigenomic instability. Many CIMP-high colorectal cancers (CRCs) with BRAF mutation are considered to arise from serrated pathway. We recently reported that microRNA-31 (miR-31) is associated with BRAF mutation in colorectal tumors. Emerging new approaches have revealed gradual changes in BRAF mutation and CIMP-high throughout the colorectum in CRCs. Here, we attempted to identify a possible association between miR-31 and epigenetic features in serrated pathway, and hypothesized that miR-31 supports the "colorectal continuum" concept. We evaluated miR-31 expression, BRAF mutation and epigenetic features including CIMP status in 381 serrated lesions and 222 non-serrated adenomas and examined associations between them and tumor location (rectum; sigmoid, descending, transverse and ascending colon and cecum). A significant association was observed between high miR-31 expression and CIMP-high status in serrated lesions with BRAF mutation (p = 0.0001). In contrast, miR-31 was slightly but insignificantly associated with CIMP status in the cases with wild-type BRAF. miR-31 expression in sessile serrated adenomas (SSAs) with cytological dysplasia was higher than that in SSAs, whereas, no significant difference was observed between traditional serrated adenomas (TSAs) and TSAs with high-grade dysplasia. The frequency of miR-31, BRAF mutation CIMP-high and MLH1 methylation increased gradually from the rectum to cecum in serrated lesions. In conclusion, miR-31 expression was associated with CIMP-high status in serrated lesions with BRAF mutation. Our data also suggested that miR-31 plays an important role in SSA evolution and may be a molecule supporting the colorectal continuum.

Inactivation of methylcytosine dioxygenase, ten-eleven translocation (TET) is known to be associated with aberrant DNA methylation in cancers. Tumors with a CpG island methylator phenotype (CIMP), a distinct subgroup with extensive DNA methylation, show characteristic features in the case of colorectal cancer. The relationship between TET inactivation and CIMP in colorectal cancers is not well understood. The expression level of TET family genes was compared between CIMP-positive (CIMP-P) and CIMP-negative (CIMP-N) colorectal cancers. Furthermore, DNA methylation profiling, including assessment of the TET1 gene, was assessed in colorectal cancers, as well as colon polyps. The TET1 was silenced by DNA methylation in a subset of colorectal cancers as well as cell lines, expression of which was reactivated by demethylating agent. TET1 methylation was more frequent in CIMP-P (23/55, 42%) than CIMP-N (2/113, 2%, P < 0.0001) colorectal cancers. This trend was also observed in colon polyps (CIMP-P, 16/40, 40%; CIMP-N, 2/24, 8%; P = 0.002), suggesting that TET1 methylation is an early event in CIMP tumorigenesis. TET1 methylation was significantly associated with BRAF mutation but not with hMLH1 methylation in the CIMP-P colorectal cancers. Colorectal cancers with TET1 methylation have a significantly greater number of DNA methylated genes and less pathological metastasis compared to those without TET1 methylation (P = 0.007 and 0.045, respectively). Our data suggest that TET1 methylation may contribute to the establishment of a unique pathway in respect to CIMP-mediated tumorigenesis, which may be incidental to hMLH1 methylation. In addition, our findings provide evidence that TET1 methylation may be a good biomarker for the prediction of metastasis in colorectal cancer.

Abstract Recent studies have shown that long noncoding RNAs (lncRNAs) have pivotal roles in human malignancies, although their significance in oral squamous cell carcinoma (OSCC) is not fully understood. In the present study, we identified lncRNAs functionally associated with OSCC. By analyzing RNA-seq datasets obtained from primary head and neck squamous cell carcinoma (HNSCC), we identified 15 lncRNAs aberrantly expressed in cancer tissues. We then validated their expression in 18 OSCC cell lines using qRT-PCR and identified 6 lncRNAs frequently overexpressed in OSCC. Among those, we found that knocking down DLEU1 (deleted in lymphocytic leukemia 1) strongly suppressed OSCC cell proliferation. DLEU1 knockdown also suppressed migration, invasion, and xenograft formation by OSCC cells, which is suggestive of its oncogenic functionality. Microarray analysis revealed that DLEU1 knockdown significantly affects expression of a number of cancer-related genes in OSCC cells, including HAS3, CD44, and TP63, suggesting that DLEU1 regulates HA-CD44 signaling. Expression of DLEU1 was elevated in 71% of primary OSCC tissues, and high DLEU1 expression was associated with shorter overall survival of HNSCC patients. These data suggest that elevated DLEU1 expression contributes to OSCC development, and that DLEU1 may be a useful therapeutic target in OSCC.

Interferon regulatory factors (IRFs) are transcription factors known to play key roles in innate and adaptive immune responses, cell growth, apoptosis, and development. Their function in tumorigenesis of gastric cancer remains to be determined, however. In the present study, therefore, we examined epigenetic inactivation of IRF1-9 in a panel of gastric cancer cell lines. We found that expression of IRF4, IRF5, and IRF8 was frequently suppressed in gastric cancer cell lines; that methylation of the three genes correlated with their silencing; and that treating the cells with the demethylating agent 5-aza-2'-deoxycytidine (DAC) restored their expression. Expression of IRF5 in cancer cells was enhanced by the combination of DAC treatment and adenoviral vector-mediated expression of p53, p63, or p73. Interferon-gamma-induced expression of IRF8 was also enhanced by DAC. Moreover, treating gastric cancer cells with DAC enhanced the suppressive effects of interferon-alpha, interferon-beta, and interferon-gamma on cell growth. Among a cohort of 455 gastric cancer and noncancerous gastric tissue samples, methylation of IRF4 was frequently observed in both gastric cancer specimens and noncancerous specimens of gastric mucosa from patients with multiple gastric cancers, which suggests IRF4 methylation could be a useful molecular marker for diagnosing recurrence of gastric cancers. Our findings indicate that epigenetic IRF inactivation plays a key role in tumorigenesis of gastric cancer, and that inhibition of DNA methylation may restore the antitumor activity of interferons through up-regulation of IRFs.

Metachronous gastric cancer (GC) can develop after endoscopic resection of GC and cannot be predicted based on clinical signature. Aberrant DNA methylation in noncancerous gastric mucosa is strongly implicated in gastric carcinogenesis and could be a useful biomarker of GC risk. We evaluated the clinical utility of DNA methylation as a biomarker of metachronous GC risk.We carried out scheduled follow-up endoscopy in 129 patients after curative endoscopic resection of GC. Biopsy specimens were collected from noncancerous mucosa in the gastric antrum and body, after which quantitative methylation analysis of miR-34b/c, SFRP1, SFRP2, SFRP5, DKK2 and DKK3 was carried out using bisulfite pyrosequencing. The utility of the methylation for predicting the risk of metachronous GC development was assessed using Kaplan-Meier and Cox proportional hazards model analyses.During the follow-up period, 17 patients (13%) developed metachronous GCs. The cumulative incidence of metachronous GC was significantly higher among patients with elevated miR-34b/c, SFRP2 and DKK2 methylation in their gastric body. MiR-34b/c showed the strongest association with the risk of metachronous GC, and the cumulative incidence of metachronous GC was much higher in the high-miR-34b/c-methylation group than the low-methylation group. Multivariate analysis adjusted for age, sex, H. pylori status and pathological findings showed miR-34b/c methylation in gastric body to be an independent predictor of metachronous GC risk.Our results suggest that methylation of miR-34b/c in the mucosa of the noncancerous gastric body may be a useful biomarker for predicting the risk of metachronous GC.

Human REV 7 (also known as MAD 2L2 and MAD 2B) is involved in DNA repair, cell cycle regulation, gene transcription, and carcinogenesis. In this study, we evaluated the expression of REV 7 in epithelial ovarian cancer ( EOC ) and analyzed the association between its expression and chemosensitivity in ovarian clear cell carcinoma ( CCC ) cells. Expression of REV 7 in human EOC tissues was assessed by immunohistochemical staining. Expression was detected in the majority of EOC s (92.0%) with especially high levels of expression frequently observed in CCC s (73.5%) compared with that of non‐ CCC s (53.4%). Enhanced immunoreactivity to REV 7 was associated with poor prognosis represented by reduced progression‐free survival in advanced stage (stage II – IV ) EOC as assessed using Kaplan–Meier curves and log–rank tests. The effects of REV 7 knockdown on cell proliferation and chemosensitivity in CCC cells were also analyzed in vitro and in vivo . Knockdown of REV 7 in CCC cells decreased cell proliferation without affecting cell cycle distribution. Additionally, the number of apoptotic cells and DNA damaged cells were increased after cisplatin treatment. In a nude mouse tumor xenograft model, inoculated REV 7‐knockdown tumors showed significantly reduced tumor volumes after cisplatin treatment compared with those of the control group. These findings indicate that depletion of REV 7 enhances sensitivity to cisplatin treatment in CCC , suggesting that REV 7 is a candidate molecular target in CCC management.

Adenylate cyclase 3 (ADCY3) is a widely expressed membrane-associated protein in human tissues, which catalyzes the formation of cyclic adenosine-3',5'-monophosphate (cAMP). However, our transcriptome analysis of gastric cancer tissue samples (NCBI GEO GSE30727) revealed that ADCY3 expression was specifically altered in cancer samples. Here we investigated the tumor-promoting effects of ADCY3 overexpression and confirmed a significant correlation between the upregulation of ADCY3 and Lauren's intestinal-type gastric cancers. ADCY3 overexpression increased cell migration, invasion, proliferation, and clonogenicity in HEK293 cells; conversely, silencing ADCY3 expression in SNU-216 cells reduced these phenotypes. Interestingly, ADCY3 overexpression increased both the mRNA level and activity of matrix metalloproteinase 2 (MMP2) and MMP9 by increasing the levels of cAMP and phosphorylated cAMP-responsive element-binding protein (CREB). Consistent with these findings, treatment with a protein kinase A (PKA) inhibitor decreased MMP2 and MMP9 expression levels in ADCY3-overexpressing cells. Knockdown of ADCY3 expression by stable shRNA in human gastric cancer cells suppressed tumor growth in a tumor xenograft model. Thus, ADCY3 overexpression may exert its tumor-promoting effects via the cAMP/PKA/CREB pathway. Additionally, bisulfite sequencing of the ADCY3 promoter region revealed that gene expression was reduced by hypermethylation of CpG sites, and increased by 5-Aza-2'-deoxycytidine (5-Aza-dC)-induced demethylation. Our study is the first to report an association of ADCY3 with gastric cancer as well as its tumorigenic potentials. In addition, we demonstrate that the expression of ADCY3 is regulated through an epigenetic mechanism. Further study on the mechanism of ADCY3 in tumorigenesis will provide the basis as a new molecular target of gastric cancer.

Abstract Aberrant DNA methylation could potentially serve as a biomarker for colorectal neoplasms. In this study, we assessed the feasibility of using DNA methylation detected in bowel lavage fluid (BLF) for colorectal cancer screening. A total of 508 BLF specimens were collected from patients with colorectal cancer (n = 56), advanced adenoma (n = 53), minor polyp (n = 209), and healthy individuals (n = 190) undergoing colonoscopy. Methylation of 15 genes (miR-1-1, miR-9-1, miR-9-3, miR-34b/c, miR-124-1, miR-124-2, miR-124-3, miR-137, SFRP1, SFRP2, APC, DKK2, WIF1, LOC386758, and ZNF582) was then analyzed in MethyLight assays, after which receiver operating characteristic (ROC) curves were analyzed to assess the diagnostic performance of BLF methylation. Through analyzing BLF specimens in a training set (n = 345), we selected the three genes showing the greatest sensitivity for colorectal cancer detection (miR-124-3, 71.8%; LOC386758, 79.5%; and SFRP1, 74.4%). A scoring system based on the methylation of those three genes (M-score) achieved 82% sensitivity and 79% specificity, and the area under the ROC curve (AUC) was 0.834. The strong performance of this system was then validated in an independent test set (n = 153; AUC = 0.808). No significant correlation was found between M-score and the clinicopathologic features of the colorectal cancers. Our results demonstrate that DNA methylation in BLF specimens may be a useful biomarker for the detection of colorectal cancer. Cancer Prev Res; 7(10); 1002–10. ©2014 AACR.

Objectives We reviewed the clinical outcomes of patients with early-stage epithelial ovarian cancer (EOC) who had undergone fertility-sparing surgery (FSS) to assess recurrence-free survival (RFS). Study design After central pathological review and scanning of the medical records of multiple institutions, a total of 94 patients with stage I EOC (IA: 43 and IC: 51) treated with FSS were analyzed. IC substages were defined as follows: intraoperative spillage (IC1), preoperative capsule rupture or surface invasion (IC2), and positive cytology results (IC3). Results The median age was 30.5 (13–40) years. The median follow-up time was 66.6 months. Fourteen patients (14.9%) showed carcinoma recurrence. Eleven (11.7%) patients died of the disease. The total 5-year RFS rate including all women who received FSS was 84.3%. There was no significant difference in RFS between patients with IC1 and those with stage IA (P = 0.9411). In contrast, the RFS rate of patients with IC2/3 was significantly poorer than in patients with stage IA (IA vs. IC2/3: P = 0.0487, IC1 vs. IC2/3: P = 0.0471). In further analyses according to each histological type and grade, the RFS rate of subjects with the mucinous type was the same as that of those with a clear-cell histology (P = 0.3350). There was a significant difference in RFS of patients with grade 1 (G1) and G2–3 (P = 0.0004). To eliminate selection bias from a number of clinicopathologic factors as thoroughly as possible, the age, FIGO stage, histological type, grade, and postoperative adjuvant chemotherapy were entered into multivariate RFS analyses. Cox multivariable analysis showed that the substage group and grade were independent prognostic factors for RFS. Conclusions Confined to young women with intraoperative rupture, FSS may be proposed, if without tumor-associated dense adhesion. However, those with preoperative rupture, surface invasion, and positive cytology showed a greater risk of recurrence, suggesting that they are not recommended candidates. Although patients with G2–3 tumors showed a poorer prognosis than those with G1, the number of these subjects was so small that the current results should be reconfirmed in the next study.

// Jun Sakata 1 , Fumi Utsumi 1 , Shiro Suzuki 1 , Kaoru Niimi 1 , Eiko Yamamoto 2 , Kiyosumi Shibata 3 , Takeshi Senga 4 , Fumitaka Kikkawa 1 and Hiroaki Kajiyama 1 1 Department of Obstetrics and Gynecology, Graduate School of Medicine, Nagoya University, Nagoya, Japan 2 Department of Healthcare Administration, Graduate School of Medicine, Nagoya University, Nagoya, Japan 3 Department of Obstetrics and Gynecology, Banbuntane Hotokukai, Fujita Health University, Fujita, Japan 4 Division of Tumor Biology, Graduate School of Medicine, Nagoya University, Nagoya, Japan Correspondence to: Hiroaki Kajiyama, email: kajiyama@med.nagoya-u.ac.jp Keywords: epithelial ovarian carcinoma, epithelial-mesenchymal transition, ZEB1, chemoresistance, metastasis Received: June 07, 2017&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; Accepted: July 25, 2017&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; Published: August 10, 2017 ABSTRACT ZEB1, a member of the zinc-finger E-box binding homeobox family, is considered to play a crucial role in cancer progression and metastasis. In the current study, we investigated the role of ZEB1 in metastasis and chronic chemoresistance of epithelial ovarian carcinoma (EOC) cells. Using several EOC and acquired paclitaxel (PTX)-resistant EOC cell lines, we investigated whether silencing ZEB1 led to a reversal of the chemoresistance and metastatic potential in vitro and in vivo . Subsequently, the expression of ZEB1 in EOC tissues and its association with the oncologic outcome were investigated. According to the immunohistochemical staining of EOC tissues, as the positivity of ZEB1 expression was increased, the overall survival of EOC patients became poorer ( P = 0.0022 for trend). Additionally, cell migration and invasion were significantly decreased by ZEB1 silencing in both PTX-sensitive and PTX- resistant cells. Although PTX-sensitivity was not changed by silencing ZEB1 in parental EOC cells, the depletion of ZEB1 made the PTX-resistant EOC cells more sensitive to PTX treatment. In an animal model, mice injected with ZEB1-silencing PTX-resistant cells survived for longer than the control cell-injected mice. Although the intravenous injection of PTX did not affect the tumor weight of shCtrl cells, the tumor weight of shZEB1 cells was significantly reduced by PTX treatment. The current data indicate the possible involvement of ZEB1 in the metastasis and paclitaxel resistance of EOC, and suggest that targeting this molecule may reverse the malignant potential and improve the oncologic outcome for EOC patients.

Two molecular pathways promote the development of colorectal cancer (CRC). One is termed “microsatellite stable” (MSS) whereas the other is characterized by “microsatellite instability” (MSI or MIN). In addition, the CpG island methylation phenotype is known to be an important alteration as a third molecular type. Thus, DNA methylation is thought to provide potential biomarkers for assessment of cancer risk in normal mucosa. In addition, it is also known that colonic location is an important parameter in the development of CRC. We examined the surrounding normal mucosa in three parts of the colon. Next, we quantified DNA methylation levels of SFRP1, SFRP2, SFRP5, DKK2, DKK3, mir34b/c, RASSF1A, IGFBP7, CDKN2A, and MLH1 in isolated cancerous glands and crypts of normal colorectal mucosa adjacent to CRCs using a pyrosequencer. DNA methylation levels of SFRP1, SFRP2, DKK2, and mir34b/c were significantly higher in CRCs with an MSS phenotype than in those with an MSI phenotype. The average level of methylation in normal crypts did not decrease with the distance from the tumor, irrespective of microsatellite status or the tumor location. DNA methylation levels in SFRP1 and SFRP2 genes in normal crypts were significantly higher in left-side than right-side CRC with an MSS phenotype. Finally, the genes were classified into three types based on the methylation frequencies in normal crypts, including type I (SFRP1 and SFRP2I), type II (DKK2 and mir34b/c), and type III (others). Our results showed that DNA methylation of SFRP1 and SFRP2 might be useful to predict cancer risk of surrounding normal mucosa. In addition, a field effect may be present in CRC, affecting both adjacent and non-adjacent normal mucosa.

Long noncoding RNAs (lncRNAs) have emerged as key components in multiple cellular processes, although their physiological and pathological functions are not fully understood. To identify cancer-related lncRNAs, we screened for those that are epigenetically silenced in colorectal cancer (CRC). Through a genome-wide analysis of histone modifications in CRC cells, we found that the transcription start sites (TSSs) of 1,027 lncRNA genes acquired trimethylation of histone H3 lysine 4 (H3K4me3) after DNA demethylation. Integrative analysis of chromatin signatures and the DNA methylome revealed that the promoter CpG islands (CGIs) of 66 lncRNA genes contained cancer-specific methylation. By validating the expression and methylation of lncRNA genes in CRC cells, we ultimately identified 20 lncRNAs, including ZNF582-AS1, as targets of epigenetic silencing in CRC. ZNF582-AS1 is frequently methylated in CRC cell lines (87.5%), primary CRCs (77.2%), colorectal adenomas (44.7%) and advanced adenomas (87.8%), suggesting that this methylation is an early event during colorectal tumorigenesis. Methylation of ZNF582-AS1 is associated with poor survival of CRC patients, and ectopic expression of ZNF582-AS1 suppressed colony formation by CRC cells. Our findings offer insight into the association between epigenetic alterations and lncRNA dysregulation in cancer and suggest that ZNF582-AS1 may be a novel tumor-suppressive lncRNA.

Tumor angiogenesis is an important therapeutic target in colorectal cancer (CRC). We aimed to identify novel genes associated with angiogenesis in CRC. Using RNA sequencing analysis in normal and tumor endothelial cells (TECs) isolated from primary CRC tissues, we detected frequent upregulation of adipocyte enhancer-binding protein 1 (AEBP1) in TECs. Immunohistochemical analysis revealed that AEBP1 is upregulated in TECs and stromal cells in CRC tissues. Quantitative RT-PCR analysis showed that there is little or no AEBP1 expression in CRC cell lines, but that AEBP1 is well expressed in vascular endothelial cells. Levels of AEBP1 expression in Human umbilical vein endothelial cells (HUVECs) were upregulated by tumor conditioned medium derived from CRC cells or by direct coculture with CRC cells. Knockdown of AEBP1 suppressed proliferation, migration, and in vitro tube formation by HUVECs. In xenograft experiments, AEBP1 knockdown suppressed tumorigenesis and microvessel formation. Depletion of AEBP1 in HUVECs downregulated a series of genes associated with angiogenesis or endothelial function, including aquaporin 1 (AQP1) and periostin (POSTN), suggesting that AEBP1 might promote angiogenesis through regulation of those genes. These results suggest that upregulation of AEBP1 contributes to tumor angiogenesis in CRC, which makes AEBP1 a potentially useful therapeutic target.

Indoleamine 2,3-dioxygenase (IDO) is a tryptophan-catabolizing enzyme that induces immune tolerance in mice. Our prior study showed that high tumoral IDO expression in endometrial cancer tissues correlates with disease progression and impaired patient survival. The purpose of the present study was to clarify the functional role of IDO in human endometrial cancer cells and to investigate the therapeutic potential of IDO inhibitors.IDO cDNA was transfected into the human endometrial carcinoma cell line AMEC, resulting in the establishment of stable clones of IDO-overexpressing AMEC cells (AMEC-IDO). AMEC-IDO cells were characterized in vitro as well as in vivo using a mouse xenograft model.There was no significant difference in in vitro cell proliferation, migration, or chemosensitivity to paclitaxel between AMEC-IDO and control vector-transfected cells (AMEC-pcDNA). However, in vivo tumor growth was markedly enhanced in AMEC-IDO-xenografted nude mice when compared with AMEC-pcDNA-xenografted mice. Splenic natural killer (NK) cell counts in AMEC-IDO-xenografted mice were significantly decreased when compared with control mice. Furthermore, conditioned medium obtained from AMEC-IDO cell cultures markedly reduced the NK lysis activity of nude mice. Finally, oral administration of the IDO inhibitor 1-methyl-D-tryptophan in combination with paclitaxel in AMEC-IDO-xenografted mice strongly potentiated the antitumor effect of paclitaxel, resulting in significantly prolonged survival.This is the first evidence showing that IDO overexpression in human cancer cells contributes to tumor progression in vivo with suppression of NK cells. Our data suggest that targeting IDO may be a novel therapeutic strategy for endometrial cancer.

Nongestational pure choriocarcinoma of the ovary is a very rare germ cell tumor. Except in women before menarche, determination of the origin is very difficult without genetic analysis. We present a pure nongestational choriocarcinoma arising in the left ovary of a 19-year-old woman. Following surgery, pathologic findings of the tumor demonstrated pure choriocarcinoma without combination of other germ cell tumor elements. We confirmed its nongestational origin by DNA polymorphism analysis at 15 short tandem repeat loci. Multiple courses of chemotherapy with methotrexate, etoposide, and actinomycin-D were effective for this case. DNA polymorphism analysis is useful to determine genetic origin in pure choriocarcinoma of the ovary

Endoglin is a coreceptor for TGF-β, which is expressed in syncytiotrophoblasts. The soluble form of endoglin (sEng) has been observed to increase in the serum of preeclamptic patients. Several studies have shown that endoglin is involved in cancer invasion. However, the role of endoglin in extravillous trophoblasts (EVT), which have an invasive phenotype, remains unknown. The present study was designed to investigate the expression and role of endoglin in human EVT. We found that endoglin was mainly expressed on cytotrophoblasts within the cell column during the first trimester and its expression decreased in the EVT by immunohistochemistry and immunocytochemistry. The expression of endoglin significantly increased after treatment with TGF-β1 and TGF-β3 in the human EVT cell line, HTR-8/SVneo, as detected by semiquantitative RT-PCR. To investigate the role of endoglin in EVT, the stable knockdown of endoglin was performed by lentiviral short hairpin RNA transfection into the HTR-8/SVneo cells. Although proliferation was not affected, the motility and invasiveness of the HTR-8/SVneo cells significantly increased by the knockdown of endoglin. Both the mRNA expression and secretion of urokinase-type plasminogen activator significantly increased in endoglin knockdown cells. The secretion of sEng was very low in HTR-8/SVneo, and the treatment of endoglin knockdown cells with 10 ng/ml sEng had no effect on their invasiveness. Therefore, the suppression of sEng was not involved in the increased invasiveness of endoglin knockdown cells. These results suggested that EVT increased their invasive function as a result of decreasing expression of transmembrane endoglin.

Abstract Although conventional colonoscopy is considered the gold standard for detecting colorectal tumors, accurate staging is often difficult because advanced histology may be present in small colorectal lesions. We collected DNA present in mucosal wash fluid from patients undergoing colonoscopy and then assessed the methylation levels of four genes frequently methylated in colorectal cancers to detect invasive tumors. We found that methylation levels in wash fluid were significantly higher in patients with invasive than those with noninvasive tumors. Cytologic and K-ras mutation analyses suggested that mucosal wash fluid from invasive tumors contained greater numbers of tumor cells than wash fluid from noninvasive tumors. Among the four genes, levels of mir-34b/c methylation had the greatest correlation with the invasion and showed the largest area under the receiver operating characteristic curve (AUC = 0.796). Using cutoff points of mir-34b/c methylation determined by efficiency considerations, the sensitivity/specificity were 0.861/0.657 for the 13.0% (high sensitivity) and 0.765/0.833 for the 17.8% (well-balanced) cutoffs. In the validation test set, the AUC was also very high (0.915), the sensitivity/specificity were 0.870/0.875 for 13.0% and 0.565/0.958 for 17.8%. Using the diagnostic tree constructed by an objective algorithm, the diagnostic accuracy of the invasiveness of colorectal cancer was 91.3% for the training set and 85.1% for the test set. Our results suggest that analysis of the methylation of DNA in mucosal wash fluid may be a good molecular marker for predicting the invasiveness of colorectal tumors. Cancer Prev Res; 4(5); 674–83. ©2011 AACR.

Abstract Aberrant DNA methylation is implicated in the epigenetic field defect seen in gastric cancer. Our aim in this study was to identify predictive biomarkers by screening for DNA methylation in noncancerous background gastric mucosa from patients with gastric cancer. Using methylated-CpG island amplification coupled with CpG island microarray (MCAM) analysis, we identified 224 genes that were methylated in the noncancerous gastric mucosa of patients with gastric cancer. Among them, RASGRF1 methylation was significantly elevated in gastric mucosa from patients with either intestinal or diffuse type gastric cancer, as compared with mucosa from healthy individuals (8.3% vs. 22.4%, P &amp;lt; 0.001; 8.3% vs. 19.4%, P &amp;lt; 0.001). RASGRF1 methylation was independent of mucosal atrophy and could be used to distinguish both serum pepsinogen test-positive [sensitivity, 70.0%; specificity, 86.7%; area under the receiver operator characteristic (ROC) curve, AUC, 0.763] and -negative patients with gastric cancer (sensitivity, 72.2%; specificity, 87.0%; AUC, 0.844) from healthy individuals. Ectopic expression of RASGRF1 suppressed colony formation and Matrigel invasion by gastric cancer cells, suggesting it may be involved in gastric tumorigenesis. Collectively, our data suggest that RASGRF1 methylation is significantly involved in an epigenetic field defect in the stomach, and that it could be a useful biomarker to identify individuals at high risk for gastric cancer. Cancer Prev Res; 5(10); 1203–12. ©2012 AACR.

<h3>Objectives</h3> To estimate the long-term clinical outcome of patients with recurrent clear cell carcinoma (RCCC) of the ovary in comparison with those with recurrent serous adenocarcinoma (RSAC). <h3>Patients and Methods</h3> In this study, 113 patients with RCCC and 365 patients with RSAC were analyzed. The pathological slides were evaluated under central pathological review. End points were the overall survival (OS), postrecurrence survival (PRS), and timing of death of mortality cases. <h3>Results</h3> The 5-year OS and PRS rates of patients with RCCC were 22.5 and 13.2%, respectively. In both OS and PRS, the prognosis of patients with RCCC was significantly poorer than that of the patients with RSAC (OS: <i>P</i> = 0.0007; PRS: <i>P</i> &lt; 0.0001). Moreover, regardless of the status of the residual tumor (RT) at the initial surgery, the OS and PRS of the patients with RCCC were markedly shorter than those with RSAC (RT [−]: OS, <i>P</i> = 0.0005: PRS, <i>P</i> = 0.0002: RT [+]: OS, <i>P</i> &lt; 0.0001: PRS, <i>P</i> &lt; 0.0001). In multivariable analysis, the histological type was a significantly poorer prognostic indicator for OS and PRS (OS [RCCC vs RSAC]: hazard ratio, 2.302: 95% confidence interval, 1.723–3.076; <i>P</i> &lt; 0.0001: PRS [RCCC vs RSAC]; hazard ratio, 2.353: 95% confidence interval, 1.756–3.155; <i>P</i> &lt; 0.0001). Even in the deceased patients (n = 350), the rate of patients with RCCC dying within 12 months of recurrence was higher than that of RSAC (RCCC, 67.8%; RSAC, 40.7%; [<i>P</i> &lt; 0.0001]). <h3>Conclusions</h3> The long-term clinical outcome of patients with RCCC was extremely poor. We confirmed that RCCC should be investigated as a different malignancy compared with RSAC.

Epigenetic changes, including DNA methylation and histone modification, play key roles in the dysregulation of tumor-related genes, thereby affecting numerous cellular processes, including cell proliferation, cell adhesion, apoptosis, and metastasis. In recent years, numerous studies have shown that noncoding RNAs (ncRNAs) are key players in the initiation and progression of cancer and epigenetic mechanisms are deeply involved in their dysregulation. Indeed, the growing list of microRNA (miRNA) genes aberrantly methylated in cancer suggests that a large number of miRNAs exert tumor-suppressive or oncogenic effects. In addition, it now appears that long ncRNAs may be causally related to epigenetic dysregulation of critical genes in cancer. Dissection of the relationships between ncRNAs and epigenetic alterations may lead to the development of novel approaches to the diagnosis and treatment of cancer.

The relevance of the clinicopathological and molecular features of early gastric cancers (EGCs) having the microsatellite instability (MSI)-high phenotype has not been clearly defined in sporadic gastric carcinogenesis. Here, we examined the clinicopathological and molecular characteristics of EGC according to MSI status in 330 patients with EGC (intestinal-type adenocarcinoma). Tumors were classified as MSI-high (45 cases), MSI-low (9 cases), or microsatellite stable (MSS; 276 cases). The specimens were examined using a combination of polymerase chain reaction (PCR)-microsatellite assays and PCR-pyrosequencing to detect chromosomal allelic imbalances in multiple cancer-related chromosomal loci, MSI, gene mutations (KRAS and BRAF) and methylation status [high methylation epigenome (HME), intermediate methylation epigenome and low methylation epigenome]. In addition, the expression levels of various target proteins were examined using immunohistochemistry. Interestingly, EGC with the MSI phenotype showed distinct papillary features. The expression of gastric mucin was more frequent in EGC with the MSI phenotype, while p53 overexpression was common in EGCs, irrespective of MSI status. The frequency of HME was significantly higher in EGCs with the MSI phenotype than in EGCs with the MSS phenotype. Although there was a low frequency of allelic imbalance in EGCs with the MSI phenotype, some markers of allelic imbalance were more frequently detected in EGCs with the MSI-high phenotype than in EGCs with the MSS phenotype. KRAS and BRAF mutations were rare in EGCs. Thus, the MSI phenotype in EGC is a major precursor lesion in gastric cancer and is characterized by distinct clinicopathological and molecular features.

Our aim was to identify DNA methylation changes associated with the growth pattern and invasiveness of colorectal cancers (CRCs). Comparison of the methylation statuses of large (≥ 20 mm in diameter along the colonic surface) noninvasive tumors (NTs) and small (<20 mm in diameter along the colonic surface) invasive tumors (ITs) using CpG island microarray analysis showed neurotensin receptor 1 (NTSR1) to be hypermethylated in large NTs. Quantitative bisulfite pyrosequencing revealed that NTSR1 is frequently methylated in colorectal tumors, with large NTs exhibiting the highest methylation levels. The higher NTSR1 methylation levels were associated with better prognoses. By contrast, NTSR1 copy number gains were most frequent among small ITs. Methylation of NTSR1 was associated with the gene's silencing in CRC cell lines, whereas ectopic expression of NTSR1 promoted proliferation and invasion by CRC cells. Analysis of primary tumors composed of adenomatous and malignant portions revealed that NTSR1 is frequently methylated in the adenomatous portion, while methylation levels are generally lower in the cancerous portions. These results suggest that NTSR1 methylation is associated with lateral and noninvasive growth of colorectal tumors, while low levels of methylation may contribute to the malignant potential through activation of NTSR1. Our data also indicate that NTSR1 methylation may be a prognostic biomarker in CRC.

Acute malnutrition affects more than 50 million under-five (U5) children, causing 8.0% of global child deaths annually. The prevalence of acute malnutrition (wasting) among U5 children in Afghanistan was 9.5% nationally and 3.7% in Faryab province in 2013. A cross-sectional study was conducted for 600 households in Faryab to find the prevalence and causes of acute malnutrition. Odds ratios (ORs) and 95% confidence intervals (CIs) were estimated using a logistic model. Demographic results of this study showed that 54.0% of the household heads and 92.3% of the mothers had no education. Three-fourths of households had a monthly income ≤ 250 USD. According to the measurement of weight for height Z-score (WHZ), 35.0% (210/600) of the children had acute malnutrition (wasting, WHZ < -2). In more than half of the households, water, sanitation, and hygiene (WASH) conditions were poor. When adjusted, a significant association of acute malnutrition among U5 children was found with the education level of household heads (OR=1.49; 95% CI, 1.02-2.17), age of household heads (OR=2.01; 95% CI, 1.21-3.35), income (OR=1.66; 95% CI, 1.04-2.27), education level of mothers (OR=2.21; 95% CI, 1.00-4.88), age of children (OR=1.99; 95% CI, 1.32-2.93), history of children with diarrhea in the last two weeks of data collection (OR=1.57; 95% CI, 1.10-2.27), feeding frequency (OR=3.01; 95% CI, 1.21-7.46), water sources (OR=1.89; 95% CI, 1.26-2.83), and iodized salt (OR=0.59; 95% CI, 0.39-0.88). The present study indicated that an increase in education level of parents, household income, and quality of WASH would result in a significant decrease in prevalence of wasting among U5 children.

Diacylglycerol kinases (DGKs) are important regulators of cell signaling and have been implicated in human malignancies. Whether epigenetic alterations are involved in the dysregulation of DGKs in cancer is unknown, however. We therefore analyzed methylation of the promoter CpG islands of DGK genes in colorectal cancer (CRC) cell lines. We found that DGKG , which encodes DGKγ, was hypermethylated in all CRC cell lines tested ( n = 9), but was not methylated in normal colonic tissue. Correspondingly, DGKG expression was suppressed in CRC cell lines but not in normal colonic tissue, and was restored in CRC cells by treatment with the DNA methyltransferase inhibitor 5‐aza‐2′‐deoxycytidine (5‐aza‐dC). DGKG methylation was frequently observed in primary CRCs (73/141, 51.8%) and was positively associated with KRAS and BRAF mutations and with the CpG island methylator phenotype (CIMP). DGKG methylation was also frequently detected in colorectal adenomas (89 of 177, 50.3%), which suggests it is an early event during colorectal tumorigenesis. Ectopic expression of wild‐type DGKγ did not suppress CRC cell proliferation, but did suppress cell migration and invasion. Notably, both constitutively active and kinase‐dead DGKγ mutants exerted inhibitory effects on CRC cell proliferation, migration and invasion, and the wild‐type and mutant forms of DGKγ all suppressed Rac1 activity in CRC cells. These data suggest DGKG may play a tumor suppressor role in CRC.

Epigenetic alterations play an important role in the pathogenesis in multiple myeloma, but their biological and clinical relevance is not fully understood. Here, we show that DOT1L, which catalyzes methylation of histone H3 lysine 79, is required for myeloma cell survival. DOT1L expression levels were higher in monoclonal gammopathy of undetermined significance and smoldering multiple myeloma than in normal plasma cells. Treatment with a DOT1L inhibitor induced cell cycle arrest and apoptosis in myeloma cells, and strongly suppressed cell proliferation in vitro The anti-myeloma effect of DOT1L inhibition was confirmed in a mouse xenograft model. Chromatin immunoprecipitation-sequencing and microarray analysis revealed that DOT1L inhibition downregulated histone H3 lysine 79 dimethylation and expression of IRF4-MYC signaling genes in myeloma cells. In addition, DOT1L inhibition upregulated genes associated with immune responses and interferon signaling. Myeloma cells with histone modifier mutations or lower IRF4/MYC expression were less sensitive to DOT1L inhibition, but with prolonged treatment, anti-proliferative effects were achieved in these cells. Our data suggest that DOT1L plays an essential role in the development of multiple myeloma and that DOT1L inhibition may provide new therapies for myeloma treatment.

Myanmar is listed as one of the countries with the highest burden of tuberculosis and HIV infections (TB-HIV) in the world. However, the survival rate and risk factors for mortality among TB-HIV co-infected patients in the country remain unstudied. Therefore, the purpose of this study was to examine these factors.A 12-year retrospective follow-up study was conducted among 3598 TB-HIV co-infected patients (2452 male and 1146 female) aged 15 years and above, enrolled on antiretroviral therapy (ART) from July 1, 2005 to December 31, 2016. Hazard ratios (HR) were estimated using the Cox proportional hazards model. Survival rates at the beginning of ART were calculated using the Kaplan-Meier method.A total of 494 (13.7%) patients died during this period. The survival rate of TB-HIV co-infected patients was 82.0% at 5 years and 58.1% at 10 years. The risk factors for mortality were being bedridden (adjusted hazard ratio (aHR) 2.70, 95% confidence interval (CI) 2.13-3.42), having a low baseline CD4 count (aHR 1.53, 95% CI 1.25-1.87), and being on a second-line ART regimen (aHR 8.12, 95% CI 3.56-18.54).Two out of five TB-HIV patients died within 10 years after ART initiation. Current HIV prevention and treatment programs should focus more on bedridden patients, those on second-line ART, and those with low baseline CD4 counts.

Ubiquitin-like protein containing PHD and RING finger domains 1 (UHRF1) is a major regulator of epigenetic mechanisms and is overexpressed in various human malignancies. In this study, we examined the involvement of UHRF1 in aberrant DNA methylation and gene silencing in colorectal cancer (CRC).CRC cell lines were transiently transfected with siRNAs targeting UHRF1, after which DNA methylation was analyzed using dot blots, bisulfite pyrosequencing, and Infinium HumanMethylation450 BeadChip assays. Gene expression was analyzed using RT-PCR and gene expression microarrays. Depletion of UHRF1 rapidly induced genome-wide DNA demethylation in CRC cells. Infinium BeadChip assays and bisulfite pyrosequencing revealed significant demethylation across entire genomic regions, including CpG islands, gene bodies, intergenic regions, and repetitive elements. Despite the substantial demethylation, however, UHRF1 depletion only minimally reversed CpG island hypermethylation-associated gene silencing. By contrast, the combination of UHRF1 depletion and histone deacetylase (HDAC) inhibition reactivated the silenced genes and strongly suppressed CRC cell proliferation. The combination of UHRF1 depletion and HDAC inhibition also induced marked changes in the gene expression profiles such that cell cycle-related genes were strikingly downregulated.Our results suggest that (i) maintenance of DNA methylation in CRC cells is highly dependent on UHRF1; (ii) UHRF1 depletion rapidly induces DNA demethylation, though it is insufficient to fully reactivate the silenced genes; and (iii) dual targeting of UHRF1 and HDAC may be an effective new therapeutic strategy.

Background Globally, alcohol consumption is a significant public health concern and it is one of the most important risk behaviours among university students. Alcohol consumption can lead to poor academic performance, injuries, fights, use of other substances, and risky sexual behaviours among students. However, the study explored the prevalence of alcohol consumption and the associated risk factors among university students since these have not been fully examined in previous research. Therefore, the aim of this study was to explore the prevalence of alcohol consumption and the associated risk factors among university students in Myanmar. Methods The present cross-sectional study was conducted using a sample of 15-24-year-old university students who were selected from six universities in Mandalay, Myanmar, in August 2018. In total, 3,456 students (males: 1,301 and females: 2,155) were recruited and asked to respond to a self-administered questionnaire. Multiple logistic regression analysis was used to estimate the adjusted odds ratio (AOR) and 95% confidence interval (CI) for alcohol consumption among university students. Results The prevalence of alcohol consumption in the previous 30 days was 20.3% (males: 36.0%, females: 10.8%). The alcohol consumption was significantly higher among males (AOR = 2.3, 95% CI; 1.9–2.9), truant students (AOR = 2.1, 95% CI; 1.3–3.3), smokers (AOR = 7.0, 95% CI; 5.1–9.7), students who reported feeling of hopelessness or sadness (AOR = 1.4, 95% CI; 1.2–1.8), peers' alcohol consumption (AOR = 7.5, 95% CI; 4.8–11.7). Conclusion The present study revealed that males, smokers, peer alcohol consumption, and truant students had higher odds of alcohol consumption among the students. Therefore, effective campus-based counselling, peer education, and national surveillance systems that can monitor risky drinking behaviours among university students should be implemented. Further, government regulations that control the production, sale, promotion, advertising, and restriction of alcohol should be well developed and strengthened, as in the case of other Southeast Asian countries.

Human pregnancy serum and placenta have the ability to degrade uterotonic peptide oxytocin (OT). Placental leucine aminopeptidase (P-LAP), which is also called cystine aminopeptidase, is the only membrane aminopeptidase known to functionally degrade OT as oxytocinase (OTase). P-LAP/OTase hydrolyzes several peptides other than OT including vasopressin and angiotensin III. P-LAP/OTase predicted from cDNA sequence is a type II integral membrane protein, which is converted to a soluble form existing in maternal serum by metalloproteases, possibly ADAM (a disintegrin and metalloproteinase) members. P-LAP/OTase activity increases with normal gestation, while decreases in the patients with preterm delivery and severe preeclampsia. In placenta, P-LAP/OTase is predominantly expressed in differentiated trophoblasts, syncytiotrophoblasts. Activator protein-2 (AP-2) and Ikaros transcription factors play significant roles in exerting high promoter activity of P-LAP/OTase in the trophoblastic cells. Moreover, P-LAP/OTase is transcriptionally regulated in a trophoblast-differentiation-dependent fashion via up-regulation of AP-2, putatively AP-2α. P-LAP/OTase may be involved in maintaining pregnancy homeostasis via metabolizing peptides such as OT and vasopressin.

Gastric cancer cells often show altered Ras signaling, though the underlying molecular mechanism is not fully understood. We examined the expression profile of eight ras-association domain family ( RASSF ) genes plus MST1/2 and found that RASSF2A is the most frequently downregulated in gastric cancer. RASSF2A was completely silenced in 6 of 10 gastric cancer cell lines as a result of promoter methylation, and expression was restored by treating the cells with 5-aza-2′-deoxycytidine. Introduction of RASSF2A into non-expressing cell lines suppressed colony formation and induced apoptosis. These effects were associated with the cytoplasmic localization of RASSF2A and morphological changes to the cells. Complementary DNA microarray analysis revealed that RASSF2A suppresses the expression of inflammatory cytokines, which may in turn suppress angiogenesis and invasion. In primary gastric cancers, aberrant methylation of RASSF2A was detected in 23 of 78 (29.5%) cases, and methylation correlated significantly with an absence of the lymphatic invasion, absence of venous invasion, absence of lymph node metastasis, less advanced stages, Epstein–Barr virus, absence of p53 mutations and the presence of the CpG island methylator phenotype-high. These results suggest that epigenetic inactivation of RASSF2A is required for tumorigenesis in a subset of gastric cancers.

Background In epithelial ovarian cancer (EOC), fertility-sparing surgery (FSS) has mainly been chosen for stage IA disease. The purpose of this study was to clarify the clinical outcome of patients with clear-cell carcinoma of the ovary (CCC) who would usually undergo radical surgery. Cases After a central pathological review and search of the medical records from multiple institutions between 1988 and 2005, a total of 10 CCC patients treated with FSS were retrospectively evaluated in the current study. The mean age was 35.9 years (range: 32–39 years). The median follow-up time was 35.4 months (range: 21.7–153.2 months). The stage was IA in 4 patients, and IC in 6 patients {IC(b) in 5 patients, and IC(2) in one}. Nine patients received adjuvant chemotherapy. Nine patients were alive and one patient with stage IC(2) died of the disease at a follow-up time of 36.8 months. Five pregnancies were observed in 4 patients. Conclusions Although there is no worldwide criterion for FSS in CCC patients at present, it seems that, in selected patients, this surgical approach could be adopted. This should be investigated by additional studies in a larger series.

For successful human placentation, invasion of trophoblast cells into the uterus and its associated vasculature is essential, and the regulation of this process is controlled by many factors at the fetal-maternal interface. N-acetylglucosaminyltransferase V (GnT-V) is a key enzyme that catalyzes beta1, 6-N-acetylglucosamine (beta1-6GlcNAc) branching on asparagine-linked oligosaccharides of cell proteins. GnT-V and its product, beta1-6GlcNAc, are known to regulate cellular transformation and correlate with the metastatic potential of various cancer cells. The aim of the present study was to determine whether extravillous trophoblast (EVT) expressed this molecule and examine the role of GnT-V in the regulation of human trophoblast invasion. Immunohistochemistry showed that GnT-V was strongly expressed within the cytoplasm of EVT in the anchoring villi; this expression was down-regulated in EVTs invading the decidua. Suppression of beta1-6GlcNAc glycosylation by swainsonine enhanced the migratory potential and invasive capability of both primary EVTs and the EVT cell line, HTR-8/SVneo. Down-regulation of GnT-V expression by small interfering RNA in the choriocarcinoma cell line Jar consistently enhanced the migration and invasive capacity of these cells and elevated cellular adhesion to extracellular matrix proteins, such as fibronectin and collagen type I/IV. The extent of beta1-6 branching of alpha5beta1 integrin was significantly reduced in small interfering GnT-V-transfected Jar cells compared with mock transfectants, although the expression of alpha1, alpha5, alpha6, and beta1 integrin on the cell surface was not changed. These results suggest that GnT-V is expressed in human EVT and is involved in regulating trophoblast invasion through modifications of the oligosaccharide chains of alpha5beta1 integrin.

The aim of this study was to clarify the role of global hypomethylation of repetitive elements in determining the genetic and clinical features of multiple myeloma (MM). We assessed global methylation levels using four repetitive elements (long interspersed nuclear element-1 (LINE-1), Alu Ya5, Alu Yb8, and Satellite-α) in clinical samples comprising 74 MM samples and 11 benign control samples (7 cases of monoclonal gammopathy of undetermined significance (MGUS) and 4 samples of normal plasma cells (NPC)). We also evaluated copy-number alterations using array-based comparative genomic hybridization, and performed methyl-CpG binding domain sequencing (MBD-seq). Global levels of the repetitive-element methylation declined with the degree of malignancy of plasma cells (NPC>MGUS>MM), and there was a significant inverse correlation between the degree of genomic loss and the LINE-1 methylation levels. We identified 80 genomic loci as common breakpoints (CBPs) around commonly lost regions, which were significantly associated with increased LINE-1 densities. MBD-seq analysis revealed that average DNA-methylation levels at the CBP loci and relative methylation levels in regions with higher LINE-1 densities also declined during the development of MM. We confirmed that levels of methylation of the 5' untranslated region of respective LINE-1 loci correlated strongly with global LINE-1 methylation levels. Finally, there was a significant association between LINE-1 hypomethylation and poorer overall survival (hazard ratio 2.8, P = 0.015). Global hypomethylation of LINE-1 is associated with the progression of and poorer prognosis for MM, possibly due to frequent copy-number loss.

Background and Aims Sessile serrated adenoma/polyps (SSA/Ps), which are precursor lesions of colorectal cancer (CRC) with BRAF mutation and the CpG island methylator phenotype (CIMP), develop cytologic dysplasia (CD) during the progression of colorectal tumorigenesis. In the present study we aimed to clarify the endoscopic and molecular signatures of SSA/Ps, with and without CD. Methods A series of 208 serrated lesions, including 41 hyperplastic polyps, 90 SSA/Ps, 33 SSA/Ps with CD, and 44 traditional serrated adenomas, were observed and resected using magnifying endoscopy. BRAF and KRAS mutations and methylation of CIMP markers (MINT1, MINT2, MINT12, MINT31, and p16) were analyzed through pyrosequencing. Molecular alterations were then compared with endoscopic and pathologic characteristics. Results Among SSA/Ps without CD, the Type II-Open pit pattern (Type II-O), BRAF mutation, and CIMP were tightly associated with a proximal colon location. SSA/Ps in the distal colon infrequently exhibited Type II-O and CIMP. By contrast, most SSA/Ps with CD showed Type II-O plus adenomatous pit patterns (Type III or IV), BRAF mutation, and CIMP, irrespective of their locations. Conclusions Our results suggest that the Type II-O plus III/IV pit pattern is a common feature of SSA/Ps with CD in both the proximal and distal colon and that this pit pattern is a hallmark of serrated lesions at high risk of developing into CRCs. Sessile serrated adenoma/polyps (SSA/Ps), which are precursor lesions of colorectal cancer (CRC) with BRAF mutation and the CpG island methylator phenotype (CIMP), develop cytologic dysplasia (CD) during the progression of colorectal tumorigenesis. In the present study we aimed to clarify the endoscopic and molecular signatures of SSA/Ps, with and without CD. A series of 208 serrated lesions, including 41 hyperplastic polyps, 90 SSA/Ps, 33 SSA/Ps with CD, and 44 traditional serrated adenomas, were observed and resected using magnifying endoscopy. BRAF and KRAS mutations and methylation of CIMP markers (MINT1, MINT2, MINT12, MINT31, and p16) were analyzed through pyrosequencing. Molecular alterations were then compared with endoscopic and pathologic characteristics. Among SSA/Ps without CD, the Type II-Open pit pattern (Type II-O), BRAF mutation, and CIMP were tightly associated with a proximal colon location. SSA/Ps in the distal colon infrequently exhibited Type II-O and CIMP. By contrast, most SSA/Ps with CD showed Type II-O plus adenomatous pit patterns (Type III or IV), BRAF mutation, and CIMP, irrespective of their locations. Our results suggest that the Type II-O plus III/IV pit pattern is a common feature of SSA/Ps with CD in both the proximal and distal colon and that this pit pattern is a hallmark of serrated lesions at high risk of developing into CRCs.

To better understand progressive changes in gastric cancer (GC), early and advanced GCs (EGC and AGC, respectively) were examined for copy number alterations (CNAs). A crypt isolation method was used to isolate DNA from tumors and normal glands in 20 AGCs, and fresh tumor samples were obtained from 45 EGCs. We assessed CNAs for differentiated-type GCs using an Infinium HumanCytoSNP-12v2.1 BeadChip in EGCs and AGCs. The most frequent aberrations in EGC were gains at 8q23.3 (42.2%) and 8q23.2 (40%), and loss of heterozygosity (LOH) at 3p14.2 (24.2%), suggesting that these CNAs were involved in the development of EGC. On the other hand, the highest frequencies of gains in AGC were found at 8q24.21 (65%) and 8q24.3 (60%). The most frequent LOHs in AGC were at 11q24.3-25, 11q23.2-24.1, 11q14.1, and 12p11.21-13.33, whereas that in EGC was at 3p14.2. In addition, regions of copy-neutral LOHs in AGC were detected at 11q21, 11q13.3-14.3, 11q11, 11p13-15.3, 12q21.1, 12q12-13.3 and 5q33.3-35.1. Comparisons of gains in EGC and AGC showed significant differences at 12q22-q23.2, 12q21.33, 11p12, 11p14.1, 12q21.31-32.32, 3p12.3, 3p14.1, 10p15.1, 1q24.2 and 2q12.1. Copy neutral LOHs were significantly higher in AGC than in EGC at 14q32.11-32.33, 14q21.3, 14q11.2, 5q11.2, 5q 13.3, 14q21.1-23.2, 14q13.2-13.3, 5q12.1-12.3, 5q11.1, and 17p13.3. The total lengths of the CNAs were significantly greater in AGC than in EGC. We found that the pattern of CNAs in AGC was quite different from that in EGC. We suggest that increasing numbers of CNAs are associated with disease progression from EGC to AGC. © 2016 Wiley Periodicals, Inc.

Myanmar transitioned to a civilian government in March, 2011. Although the democratic process has accelerated since then, many problems in the field of healthcare still exist. Since there is a limited overview on the healthcare in Myanmar, this article briefly describes the current states surrounding health services in Myanmar. According to the Census 2014, the population in the Republic of the Union of Myanmar was 51,410,000. The crude birth rate in the previous one year was estimated to be 18.9 per 1,000, giving the annual population growth rate of 0.89% between 2003 and 2014. The Ministry of Health reorganized into six departments. National non-governmental organizations and community-based organizations support healthcare, as well as international non-governmental organizations. Since hospital statistics by the government cover only public facilities, the information on private facilities is limited. Although there were not enough medical doctors (61 per 100,000 population), the number of medical students was reduced from 2,400 to 1,200 in 2012 to ensure the quality of medical education. The information on causes of death in the general population could not be retrieved, but some data was available from hospital statistics. Although the improvement was marked, the figures did not reach the levels set by Millennium Development Goals 4 and 5. A trial prepaid health insurance system started in July 2015, to be followed by evaluation one year later. There are many international donors, including the Japan International Cooperation Agency, supporting health in Myanmar. With these efforts and support, a marked progress is expected in the field of healthcare.

Oxidative stress plays an important role in the pathogenesis of preeclampsia. Recently, molecular hydrogen (H2) has been shown to have therapeutic potential in various oxidative stress-related diseases. The aim of this study is to investigate the effect of H2 on preeclampsia. We used the reduced utero-placental perfusion pressure (RUPP) rat model, which has been widely used as a model of preeclampsia. H2 water (HW) was administered orally ad libitum in RUPP rats from gestational day (GD) 12-19, starting 2 days before RUPP procedure. On GD19, mean arterial pressure (MAP) was measured, and samples were collected. Maternal administration of HW significantly decreased MAP, and increased fetal and placental weight in RUPP rats. The increased levels of soluble fms-like tyrosine kinase-1 (sFlt-1) and diacron reactive oxygen metabolites as a biomarker of reactive oxygen species in maternal blood were decreased by HW administration. However, vascular endothelial growth factor level in maternal blood was increased by HW administration. Proteinuria, and histological findings in kidney were improved by HW administration. In addition, the effects of H2 on placental villi were examined by using a trophoblast cell line (BeWo) and villous explants from the placental tissue of women with or without preeclampsia. H2 significantly attenuated hydrogen peroxide-induced sFlt-1 expression, but could not reduce the expression induced by hypoxia in BeWo cells. H2 significantly attenuated sFlt-1 expression in villous explants from women with preeclampsia, but not affected them from normotensive pregnancy. The prophylactic administration of H2 attenuated placental ischemia-induced hypertension, angiogenic imbalance, and oxidative stress. These results support the theory that H2 has a potential benefit in the prevention of preeclampsia.

Cost information is important for efficient allocation of healthcare expenditure, estimating future budget allocation, and setting user fees to start new financing systems. Myanmar is in political transition, and trying to achieve universal health coverage by 2030. This study assessed the unit cost of healthcare services at two public hospitals in the country from the provider perspective. The study also analyzed the cost structure of the hospitals to allocate and manage the budgets appropriately. A hospital-based cross-sectional study was conducted at 200-bed Magway Teaching Hospital (MTH) and Pyinmanar General Hospital (PMN GH), in Myanmar, for the financial year 2015–2016. The step-down costing method was applied to calculate unit cost per inpatient day and per outpatient visit. The costs were calculated by using Microsoft Excel 2010. The unit costs per inpatient day varied largely from unit to unit in both hospitals. At PMN GH, unit cost per inpatient day was 28,374 Kyats (27.60 USD) for pediatric unit and 1,961,806 Kyats (1908.37 USD) for ear, nose, and throat unit. At MTH, the unit costs per inpatient day were 19,704 Kyats (19.17 USD) for medicine unit and 168,835 Kyats (164.24 USD) for eye unit. The unit cost of outpatient visit was 14,882 Kyats (14.48 USD) at PMN GH, while 23,059 Kyats (22.43 USD) at MTH. Regarding cost structure, medicines and medical supplies was the largest component at MTH, and the equipment was the largest component at PMN GH. The surgery unit of MTH and the eye unit of PMN GH consumed most of the total cost of the hospitals. The unit costs were influenced by the utilization of hospital services by the patients, the efficiency of available resources, type of medical services provided, and medical practice of the physicians. The cost structures variation was also found between MTH and PMN GH. The findings provided the basic information regarding the healthcare cost of public hospitals which can apply the efficient utilization of the available resources.

Vietnam is facing a problem of over-crowding in public hospitals. Long waiting time is a major dissatisfaction for patients. Reducing waiting time benefits not only patients but also hospitals in decreasing overall workload. To identify factors contributing to long waiting time in outpatient clinics, a cross-sectional study was conducted at a national hospital in Vietnam. The time for each process of 7,931 patients who visited the outpatient clinics within Thong Nhat Hospital from 5 to 9 September 2016 was collected from the computerized clinical database. Characteristics of patients (age, sex, address, day of visit, registration time, visited department and test) and waiting times were studied at the outcome measures. Multivariate analyses using the linear regression model was carried out to evaluate the contribution of these factors to the total waiting time. Among 7,931 patients, 52.3% were women, 46.3% were 60-80 years, and 64.8% registered between 5:30 and 9:30. The mean total waiting time was 104.1 minutes. The analysis on total waiting time among 4,564 patients who visited one department without any test showed that department group and registration time were significantly associated with a total waiting time. In 1,259 patients who visited one department with one test, early registration and undergoing blood tests were significantly associated with a longer total waiting time. These results showed that old age, visiting internal medicine departments, early registration time, and undergoing blood tests were factors contributing to a longer total waiting time in the outpatient clinics within Thong Nhat Hospital.

Traditional and complementary medicine (T&CM) has been integrated into the Malaysian public healthcare system since the establishment of the first T&CM unit at a public hospital in 2007. Assessing patient satisfaction is a vital component of health service evaluation. The main objective of this study is to determine the level of patient satisfaction with the utilization of T&CM services at public hospitals in Malaysia and assess the sociodemographic influence on the overall reporting of satisfaction. This study also aims to analyze the response of the patients towards expansion of T&CM services in the public sector in Malaysia. A study was conducted to analyze data on the utilization of T&CM services within public hospitals. Secondary data on 822 patients’ satisfaction with services offered at 15 T&CM units was analyzed to examine the overall levels of satisfaction with T&CM services in public hospitals in Malaysia. Overall, 99.4% of patients were satisfied with T&CM services and most patients (91.8%) felt that T&CM treatment positively impacted their health. Overall satisfaction was not affected by lower levels of satisfaction with subcategories of service, such as the number of treatment sessions received (90.7% satisfied), date to the next appointment (90.7% satisfied), and the absence of adverse effects of treatment received (87.1% satisfied). There were no significant associations between the socioeconomic status of the respondents and the level of satisfaction reported; however, respondents with a monthly salary of Ringgit Malaysia (RM) 1000 to RM 3000 were more than twice as likely to be strongly satisfied with services received (adjusted odds ratios [AOR]: 2.12, 95% CI: 1.19–3.78). This study revealed a high level of satisfaction among patients who had received T&CM treatment at public hospitals in Malaysia. High satisfaction with T&CM treatment validates the integrative management approach adopted in patient care within the public hospitals in Malaysia.

Afghanistan is one of the countries with the poorest maternal mortality ratio in the world. Inadequate utilization of antenatal care (ANC) services increases the risk of maternal mortality. This study aimed to identify the factors associated with ANC visits in Afghanistan. The dataset of the Afghanistan Demographic and Health Survey (AfDHS) 2015 were used for taking the socio-demographic factors, cultural factors, and the number of ANC visits. The subjects of this study were 18,790 women who had at least one live birth in the last five years, and 10,554 women (56.2%) had availed of at least one ANC visit. Most women were 20-29 years old (53.3%), poor (41.7%), had 2-4 children (43.9%), lived in rural areas (76.1%), and had no education (85.0%) or no job (86.7%). Most women answered that husbands made a decision about their healthcare and that getting permission from their husbands was a major challenge. Multivariate analysis showed that age, ethnicity, area of residence, parity, women's education, husband's education, literacy, having a job, wealth, the decision maker for healthcare, and difficulty in getting permission from the husband were significantly correlated with availing of the ANC visits. This study showed that not only the socio-demographic factors but also the cultural factors were associated with ANC visits. The Afghanistan government should improve the education programs at schools and healthcare facilities, for both men and women. To augment women's propensity to take a decision, the programs for women's empowerment need to be supported and extended across the country.

Gestational choriocarcinoma is a gestational trophoblastic neoplasia (GTN) that originates from abnormal trophoblast proliferation. Although chemotherapy is effective for choriocarcinoma, personalized treatment becomes essential when patients develop chemoresistance. Here, we present the clinical course of a case of intractable choriocarcinoma that achieved complete remission with pembrolizumab following cytotoxic chemotherapy.A 38-year-old woman was initially diagnosed with low-risk GTN and treated with single- and multi-agent chemotherapy. She underwent a hysterectomy and was diagnosed with pathological choriocarcinoma with high-risk GTN. She was treated with multiple courses of several chemotherapy regimens. However, she did not achieve remission. Her choriocarcinoma showed high microsatellite instability; therefore, she took ten courses of pembrolizumab, but her hCG value increased. Subsequently, she underwent eight courses of paclitaxel and carboplatin alternating with paclitaxel and etoposide and achieved remission.This case suggests that pembrolizumab may improve the efficacy of subsequent chemotherapy.

Regulation of cytotrophoblast differentiation toward extravillous trophoblasts (EVTs) is critical for establishing successful pregnancy. Previous studies have focused primarily on the factors promoting the differentiation, while inhibitory regulators except hypoxia have been less documented. In this study, to test our hypothesis that angiotensin II (Ang II) would inhibit EVT differentiation, we investigated the effects of Ang II on trophoblast outgrowth and the expression of molecules associated with the proliferation and invasion of trophoblasts using human first trimester villous explant cultures. Ang II increased EVT outgrowth and the number of cells in cell columns. Moreover, Ang II-treated explants exhibited increased Ki67 and integrin alpha5 immunoreactivity in EVTs as well as matrix metalloproteinase-2 activity in the conditioned media, and decreased alpha1 integrin immunoreactivity, which are compatible with the features of the proliferative phenotype EVTs. These effects of Ang II were similar to those of hypoxia (3% O(2)). Ang II stimulated the expression of hypoxia inducible factor-1alpha at both mRNA and protein levels, and also enhanced the expression of plasminogen activator inhibitor-1 (PAI-1). Data presented herein suggest a possible role for Ang II in impairing trophoblast differentiation toward an invasive phenotype, which might be associated with shallow invasion in preeclamptic placentas.

Recent reports have shown that CXCR4 is expressed in various solid tumors and is involved in tumor development and metastasis. We examined the distribution and expression of this molecule in clear cell carcinoma of the ovary to elucidate its clinical significance. Paraffin sections from clear cell carcinoma of the ovary tissues (n = 42) were immunostained with CXCR4 antibody, and the staining intensities were evaluated. The clinicopathologic factors examined were age, FIGO (International Federation of Gynecology and Obstetrics) staging, preoperative value of cancer antigen 125 test, and residual tumor after cytoreductive surgery. Overall survival and progression-free survival were evaluated using the Kaplan-Meier method, and multivariate analysis was completed using Cox proportional hazards analysis. Of the 42 carcinomas, lower level CXCR4 immunoexpression was observed in 21 cases (50.0%) (CXCR4(low) group); and higher level immunoexpression, in 21 cases (50.0%) (CXCR4(high) group). Five-year overall survival was significantly poorer in the CXCR4(high) group than in the CXCR4(low) group (overall survival, CXCR4(low) group [90.2%], CXCR4(high) group [50.3%]; P = .0002). In addition, CXCR4(high) immunoexpression significantly predicted a poorer progression-free survival when compared with lower expression (5-year progression-free survival, CXCR4(low) group [90.5%], CXCR4(high) group [36.2%]; P < .0001). Furthermore, multivariate analyses including the age, preoperative cancer antigen 125 test value, FIGO stage, and CXCR4 expressions revealed that CXCR4(high) expression was an independent prognostic factor for poorer overall survival and progression-free survival of patients with clear cell carcinoma of the ovary (overall survival, P = .0011; progression-free survival, P = .0008, respectively). Our current study suggested that the assessment of CXCR4 immunoreactivity may be a useful prognostic indicator and that CXCR4 may play a critical role in the progression of clear cell carcinoma of the ovary.

Recent studies have shown an activation of the local renin-angiotensin system (RAS) in various tumor tissues, including the abundant generation of angiotensin II (Ang II) by angiotensin-converting enzyme (ACE) and the upregulation of angiotensin II type 1 receptor (AT1R) expression. Thus, considerable attention has been paid not only to the role of the RAS in cancer progression, but also to the blockade of RAS as a new approach to the treatment of human cancer. There is increasing evidence that the Ang II-AT1R pathway is involved in tumor growth, angiogenesis and metastasis in various experimental animal models, suggesting the therapeutic potential of an ACE inhibitor and AT1R blocker. In addition, specific Ang II-degrading enzymes are also expressed in tumors and play a regulatory role in tumor cell proliferation and invasion. This review focuses on the role of the RAS in the progression of gynecologic cancers, such as cervical cancer, endometrial cancer, ovarian cancer, and gestational choriocarcinoma. We present here the clinical potential of blocking the RAS as a novel and promising strategy for the treatment of gynecologic cancers. Keywords: Angiogenesis, Angiotensin II type 1 receptor, Cervical cancer, Endometrial cancer, Ovarian cancer, Reninangiotensin system (RAS), Survival, Vascular endothelial growth factor, Adipocyte-derived leucine aminopeptidase, Aminopeptidase A

Gestational trophoblastic diseases (GTDs) are related to trophoblasts, and human chorionic gonadotropin (hCG) is secreted by GTDs as well as normal placentas. However, the asparagine-linked sugar chains on hCG contain abnormal biantennary structures in invasive mole and choriocarcinoma, but not normal pregnancy or hydatidiform mole. N-acetylglucosaminyltransferase-IV (GnT-IV) catalyses β1,4-N-acetylglucosamine branching on asparagine-linked oligosaccharides, which are consistent with the abnormal sugar chain structures on hCG.We investigated GnT-IVa expression in GTDs and placentas by immunohistochemistry, western blot, and RT-PCR. We assessed the effects of GnT-IVa knockdown in choriocarcinoma cells in vitro and in vivo.The GnT-IVa was highly expressed in trophoblasts of invasive mole and choriocarcinoma, and moderately in extravillous trophoblasts during the first trimester, but not in hydatidiform mole or other normal trophoblasts. The GnT-IVa knockdown in choriocarcinoma cells significantly reduced migration and invasive capacities, and suppressed cellular adhesion to extracellular matrix proteins. The extent of β1,4-N-acetylglucosamine branching on β1 integrin was greatly reduced by GnT-IVa knockdown, although the expression of β1 integrin was not changed. In vivo studies further demonstrated that GnT-IVa knockdown suppressed tumour engraftment and growth.These findings suggest that GnT-IVa is involved in regulating invasion of choriocarcinoma through modifications of the oligosaccharide chains of β1 integrin.

Abstract Aim We aimed to investigate the possibility of an association between a stem‐like hallmark and radiotherapeutic sensitivity in human cervical carcinoma cells. Material and Methods Side‐population ( SP ) cells and non‐ SP ( NSP ) cells in HeLa cells were isolated using flow cytometry and Hoechst 33342 efflux. We performed W estern blot analysis to evaluate the expression of stem cell markers ( CXCR4 , O ct3/4, CD133 , and SOX2 ) and apoptosis markers after irradiation. In addition, SP and NSP cells were injected into nude mice and we assessed subcutaneous tumor formation. To examine tolerance of irradiation, colony formation and apoptosis change were confirmed in the SP and NSP cells. Results SP cells showed a higher expression of CXCR4 , O ct3/4, CD133 , and SOX2 than NSP cells. The colony size of SP cells cultured on non‐coated dishes was larger than that of NSP cells, and NSP cells were easily induced to undergo apoptosis. SP cells tended to form spheroids and showed a higher level of tumorigenicity compared with NSP cells. In addition, nude mice inoculated with SP cells showed greater tumor growth compared with NSP cells. SP cells showed a higher tumorigenicity and lower apoptotic potential, leading to enhanced radiotolerance. Conclusion Tumor SP cells showed higher‐level stem‐cell‐like characters and radioresistance than NSP cells. SP cells may be useful for new therapeutic approaches for radiation‐resistant cervical cancer.

Snakebite is a neglected condition and a common public health problem in Lao People's Democratic Republic (Lao PDR), with a high incidence of up to 1,105 cases per 100,000 persons per year. Snakebite patients with systemic envenoming do not receive effective treatment at local health facilities. Healthcare providers have only limited knowledge in assessing and providing the correct treatment for venomous snakebites. A cross-sectional study was conducted among 119 healthcare providers in Savannakhet Province, Lao PDR, with respect to their socio-demographic characteristics, knowledge of snake identification, and management of snakebite. Data was analyzed using SPSS. Logistic regression was performed to estimate the odds ratios (OR) and 95% confidence intervals (CI) of adequate knowledge for treating snakebites among the healthcare providers. Among 119 participants, 27.7% and 45.4% had an adequate knowledge of snake identification and management of snakebites, respectively. Approximately 59% could correctly identify symptoms of envenomation, and 19.3% expressed confidence in treating snakebites. Study participants who had received training achieved significantly better snake identification results compared to those without training, with an OR of 2.54 (95% CI: 1.02-6.28). In particular, physicians achieved significantly better results compared to nurses in knowing how to manage snakebites, with an OR of 2.31 (95% CI: 1.04-5.12). Given the level of inadequate knowledge of snakebite management among healthcare providers in the province, more training in snakebite management is needed. University and Health Science Colleges should include snakebite management into the curriculum, to ensure that medical graduates have the appropriate knowledge and skills to treat snakebites.

Immunization is one of the most cost-effective interventions to reduce vaccine-preventable diseases morbidity and mortality. Vaccination coverage is very low in Afghanistan; National Risk and Vulnerability Assessment (NRVA) Survey 2008 estimated the coverage of fully immunized children to be 37%. The current study was designed to examine the factors influencing full immunization among children aged 12-23 months. Demographic and vaccination data of 2,561 children of 12-23 months was extracted from the Afghanistan Health Survey (AHS) 2012. The data was analyzed by logistic regression to estimate adjusted odds ratios (AOR) and 95% confidence intervals (CI). The study found that 38.8% of the children were fully immunized. The coverage for specific vaccines was 80.9% for BCG, 72.0% for OPV3, 64.8% for measles, and 50.1% for Penta3. Urban residence (AOR = 0.60, 95% CI 0.40-0.90 relative to rural), children of poorer families (AOR = 1.36, 95% CI 1.10-1.67 relative to poorest), some education (AOR = 1.59, 95% CI 1.20-2.11 relative to no education) and antenatal care (AOR = 1.70, 95% CI 1.44-2.01 relative to not received) were found to be significant predicators of full immunization. This study indicated that the full-immunization rate in Afghanistan was quite low compared to the national target of 90% coverage. Therefore, strategies taking into account the identified factors seem to be vital to improve vaccination coverage.

Colorectal sessile serrated adenoma/polyps (SSA/Ps) are well-known precursors of colorectal cancer (CRC) characterized by BRAF mutation and microsatellite instability.By contrast, the molecular characteristics of traditional serrated adenoma (TSAs) are not fully understood.We analyzed genome-wide DNA methylation in TSAs having both protruding and flat components.We identified 11 genes, including SMOC1, methylation of which progressively increased during the development of TSAs.SMOC1 was prevalently methylated in TSAs, but was rarely methylated in SSA/Ps (p < 0.001).RT-PCR and immunohistochemistry revealed that SMOC1 was expressed in normal colon and SSA/Ps, but its expression was decreased in TSAs.Ectopic expression of SMOC1 suppressed proliferation, colony formation and in vivo tumor formation by CRC cells.Analysis of colorectal lesions (n = 847) revealed that SMOC1 is frequently methylated in TSAs, high-grade adenomas and CRCs.Among these, SMOC1 methylation was strongly associated with KRAS mutation and CpG island methylator phenotype (CIMP)-low.These results demonstrate that epigenetic silencing of SMOC1 is associated with TSA development but is rarely observed in SSA/Ps.SMOC1 expression could thus be a diagnostic marker of serrated lesions, and SMOC1 methylation could play a role in neoplastic pathways in TSAs and conventional adenomas.

Routine vaccination is administered free of charge to all children under one year old in Lao People's Democratic Republic (Lao PDR) and the national goal is to achieve at least 95% coverage with all vaccines included in the national immunization program by 2025. In this study, factors related to the immunization system and characteristics of provinces and districts in Lao PDR were examined to evaluate the association with routine immunization coverage.Coverage rates for Bacillus Calmette-Guerin (BCG), Diphtheria-Tetanus-Pertussis-Hepatitis B (DTP-HepB), DTP-HepB-Hib (Haemophilus influenzae type B), polio (OPV), and measles (MCV1) vaccines from 2002 to 2014 collected through regular reporting system, were used to identify the immunization coverage trends in Lao PDR. Correlation analysis was performed using immunization coverage, characteristics of provinces or districts (population, population density, and proportion of poor villages and high-risk villages), and factors related to immunization service (including the proportions of the following: villages served by health facility levels, vaccine session types, and presence of well-functioning cold chain equipment). To determine factors associated with low coverage, provinces were categorized based on 80% of DTP-HepB-Hib3 coverage (<80% = low group; ≥80% = high group).Coverages of BCG, DTP-HepB3, OPV3 and MCV1 increased gradually from 2007 to 2014 (82.2-88.3% in 2014). However, BCG coverage showed the least improvement from 2002 to 2014. The coverage of each vaccine correlated with the coverage of the other vaccines and DTP-HepB-Hib dropout rate in provinces as well as districts. The provinces with low immunization coverage were correlated with higher proportions of poor villages.Routine immunization coverage has been improving in the last 13 years, but the national goal is not yet reached in Lao PDR. The results of this study suggest that BCG coverage and poor villages should be targeted to improve nationwide coverage.

The government of Mongolia mandates free access to primary healthcare (PHC) for its citizens. However, no evidence is available on the physical presence of PHC services within health facilities. Thus, the present study assessed the capacity of health facilities to provide basic services, at minimum standards, using a World Health Organization (WHO) standardized assessment tool. The service availability and readiness assessment (SARA) tool was used, which comprised a set of indicators for defining whether a health facility meets the required conditions for providing basic or specific services. The study examined all 146 health facilities in Chingeltei and Khan-Uul districts of Ulaanbaatar city, including private and public hospitals, family health centers (FHCs), outpatient clinics, and sanatoriums. The assessment questionnaire was modified to the country context, and data were collected through interviews and direct observations. Data were analyzed using SPSS 21.0, and relevant nonparametric tests were used to compare median parameters. A general service readiness index, or the capacity of health facilities to provide basic services at minimum standards, was 44.1% overall and 36.3, 61.5, and 62.4% for private clinics, FHCs, and hospitals, respectively. Major deficiencies were found in diagnostic capacity, supply of essential medicines, and availability of basic equipment; the mean scores for general service readiness was 13.9, 14.5 and 47.2%, respectively. Availability of selected PHC services was 19.8%. FHCs were evaluated as best capable (69.5%) to provide PHC among all health facilities reviewed (p < 0.001). Contribution of private clinics and sanatoriums to PHC service provisions were minimal (4.1 and 0.5%, respectively). Service-specific readiness among FHCs for family planning services was 44.0%, routine immunization was 83.6%, antenatal care was 56.5%, preventive and curative care for children was 44.5%, adolescent health services was 74.2%, tuberculosis services was 53.4%, HIV and STI services was 52.2%, and non-communicable disease services was 51.7%. Universal access to PHC is stipulated throughout various policies in Mongolia; however, the present results revealed that availability of PHC services within health facilities is very low. FHCs contribute most to providing PHC, but readiness is mostly hampered by a lack of diagnostic capacity and essential medicines.

Abstract The molecular and clinical characteristics of non‐ampullary duodenal adenomas and intramucosal adenocarcinomas are not fully understood because they are rare. To clarify these characteristics, we performed genetic and epigenetic analysis of cancer‐related genes in these lesions. One hundred and seven non‐ampullary duodenal adenomas and intramucosal adenocarcinomas, including 100 small intestinal‐type tumors (90 adenomas and 10 intramucosal adenocarcinomas) and 7 gastric‐type tumors (2 pyloric gland adenomas and 5 intramucosal adenocarcinomas), were investigated. Using bisulfite pyrosequencing, we assessed the methylation status of CpG island methylator phenotype (CIMP) markers and MLH1 . Then using next‐generation sequencing, we performed targeted exome sequence analysis within 75 cancer‐related genes in 102 lesions. There were significant differences in the clinicopathological and molecular variables between small intestinal‐ and gastric‐type tumors, which suggests the presence of at least two separate carcinogenic pathways in non‐ampullary duodenal adenocarcinomas. The prevalence of CIMP‐positive lesions was higher in intramucosal adenocarcinomas than in adenomas. Thus, concurrent hypermethylation of multiple CpG islands is likely associated with development of non‐ampullary duodenal intramucosal adenocarcinomas. Mutation analysis showed that APC was the most frequently mutated gene in these lesions (56/102; 55%), followed by KRAS (13/102; 13%), LRP1B (10/102; 10%), GNAS (8/102; 8%), ERBB3 (7/102; 7%), and RNF43 (6/102; 6%). Additionally, the high prevalence of diffuse or focal nuclear β ‐catenin accumulation (87/102; 85%) as well as mutations of WNT pathway components (60/102; 59%) indicates the importance of WNT signaling to the initiation of duodenal adenomas. The higher than previously reported frequency of APC gene mutations in small bowel adenocarcinomas as well as the difference in the APC mutation distributions between small intestinal‐type adenomas and intramucosal adenocarcinomas may indicate that the adenoma–carcinoma sequence has only limited involvement in duodenal carcinogenesis. © 2020 The Authors. The Journal of Pathology published by John Wiley &amp; Sons, Ltd. on behalf of The Pathological Society of Great Britain and Ireland.

Background In the context of an aging population, quality of life (QOL) is an important consideration for the well-being of the elderly. However, there is limited information on the QOL of the elderly in Myanmar. This study aimed to explore the risk factors for low QOL among the elderly in urban and peri-urban areas of the Yangon Region, Myanmar. Methods A community-based, cross-sectional study was conducted among the elderly aged 60 years or older in two urban and two peri-urban townships in the Yangon Region from July to September 2019. A multi-stage sampling method was used to recruit study participants using a pre-tested questionnaire. A total of 616 (305 males and 311 females) elderly people were interviewed using a face-to-face interview technique. Multiple linear regression analysis was performed on the four domains (physical health, psychological health, social relationship, and environment) of QOL measured with the WHOQOL-BREF. Results Income level and having intimate friends influenced the QOL scores of the elderly in all domains, while education level and marital status influenced psychological health, social relationship, and environment domains. Social interaction with neighbors increased the QOL scores for physical health, social relationship, and environment domains. Living in peri-urban areas was associated with lower QOL scores for physical health, psychological health, and environment, while participation in group activities increased QOL scores in these domains. Having comorbidities affected the QOL for psychological health and environment domains, while the frequency of going out affected physical health, and the frequency of religious performance affected social relationship. Conclusion Residential location, education level, marital status, income, comorbidities, social interactions with neighbors and friends, participation in group activities, and frequencies of going out and religious activities should be considered in planning and implementing programs for the elderly in Myanmar. Peri-urban development, strengthening healthcare and social security systems, and encouraging social interaction and participation in group activities play critical roles in improving the QOL for elderly residing in Myanmar.

Aberrant DNA methylation is prevalent in colorectal serrated lesions. We previously reported that the CpG island of SMOC1 is frequently methylated in traditional serrated adenomas (TSAs) and colorectal cancers (CRCs) but is rarely methylated in sessile serrated lesions (SSLs). In the present study, we aimed to further characterize the expression of SMOC1 in early colorectal lesions.SMOC1 expression was analyzed immunohistochemically in a series of colorectal tumors (n = 199) and adjacent normal colonic tissues (n = 112).SMOC1 was abundantly expressed in normal colon and SSLs while it was significantly downregulated in TSAs, advanced adenomas and cancers. Mean immunohistochemistry scores were as follows: normal colon, 24.2; hyperplastic polyp (HP), 18.9; SSL, 23.8; SSL with dysplasia (SSLD)/SSL with early invasive cancer (EIC), 15.8; TSA, 5.4; TSA with high grade dysplasia (HGD)/EIC, 4.7; non-advanced adenoma, 21.4; advanced adenoma, 11.9; EIC, 10.9. Higher levels SMOC1 expression correlated positively with proximal colon locations and flat tumoral morphology, reflecting its abundant expression in SSLs. Among TSAs that contained both flat and protruding components, levels of SMOC1 expression were significantly lower in the protruding components.Our results suggest that reduced expression of SMOC1 is associated with progression of TSAs and conventional adenomas and that SMOC1 expression may be a biomarker for diagnosis of serrated lesions and risk prediction in colorectal tumors.

<h2>Abstract</h2><h3>Objective</h3> This study aimed to identify the prevalence of postpartum depression (PPD) and the factors associated with PPD in Kampong Chhnang Province, Cambodia. <h3>Study design</h3> A cross-sectional study. <h3>Participants</h3> This study included 440 Cambodian women at 6–8 weeks postpartum who visited health centers between July and September 2021. <h3>Materials and methods</h3> Data were collected through face-to-face interviews by midwives and nurses using a structured questionnaire. The Edinburgh Postnatal Depression Scale (EPDS) in the Khmer language was used to screen for PPD, and suspected PPD was defined as a total EPDS score ≥ 10. Logistic regression analyses were performed to identify the factors associated with suspected PPD. <h3>Findings</h3> The average age of participants was 28.6 years old. The prevalence of suspected-PPD was 30.2 % (<i>n</i> = 133). Factors associated with suspected PPD were income dissatisfaction (adjusted odds ratio (AOR) = 2.66, 95 % confidence interval (CI) 1.27–5.56, <i>P</i> = 0.010), unintended pregnancy (AOR = 1.99, 95 % CI 1.10–3.61, <i>P</i> = 0.023), and a partner employed as a manual laborer (AOR = 3.85, 95 % CI 1.11–13.33, <i>P</i> = 0.034), farmer (AOR = 3.69, 95 % CI 1.11–12.31, P = 0.034), and factory worker (AOR = 5.43, 95 % CI 1.38–21.41, <i>P</i> = 0.016). In addition, poor relationship with partners (AOR = 2.14, 95 % CI 1.17–3.94, <i>P</i> = 0.014), poor relationship with mother-in-law (AOR = 3.51, 95 % CI 1.70–7.21, <i>P</i> < 0.001), and a history of depression before pregnancy (AOR = 6.34, 95 % CI 1.59–25.34, <i>P</i> = 0.009) were significantly associated with suspected-PPD. <h3>Key conclusions and implications for practice</h3> This study highlighted the need for mental health services in primary healthcare settings. Mental health training for healthcare workers, particularly primary-level nurses, should be prioritized and strengthened. Further clinical study on EPDS validation should be carried out to justify the appropriate cut-off EPDS score for Cambodian women. The EPDS should be integrated into routine PNC services to identify women with suspected-PPD. Education on PPD should be provided not only to the nurses and midwives, but also to the women and their families to support the mental health of pregnant and postpartum women.

Since the beginning of the family program in 1998, the proportion of married women who used contraception has fluctuated. An unmet need for contraception among women in Kyrgyzstan drastically increased from 2006 (1.1%) to 2014 (19.1%), and remained unchanged until 2018 (19.0%). This study aims to re-investigate the prevalence of an unmet need for contraception from 2006 to 2018 in a comprehensive manner, and examine the factors associated with an unmet need for contraception among married women over the course of 12 years in the Kyrgyz Republic.This is a cross-sectional study using secondary data that derived from the Multiple Indicator Cluster Survey (MICS). The study employed three datasets from the MICS 2006, 2014, and 2018. The study included a total of 9,229 women aged 15-49 who were married and fecund, and whose status of the met/unmet need for contraception could be identified. Logistic regression was employed to estimate the relationship of an unmet need for contraception with independent factors. A P value < 0.05 was set as statistically significant.The prevalence of an unmet need for contraception was 19.9% in 2006, 20.4% in 2014, and 22.5% in 2018. Across 12 years, all reversible-contraceptive methods for women constantly declined. Although intrauterine devices were the prominent contraceptive method of usage among Kyrgyz women, the trend of usage drastically decreased over time. Factors associated with unmet need for contraception included women's age, area of residence, mother tongue of household head, age of husband, and number of children ever born.The unmet need for contraception among married Kyrgyz women slightly increased, and the trend of modern contraceptive usage declined from 2006 to 2018, particularly the use of pills, injections, and intra-uterine devices. Comprehensive sexual health education for young people and youth-friendly services should be promoted. An effective and reliable supply chain of contraceptive commodities should be prioritized and strengthened. Regular supportive supervision visits are essential to improve the knowledge and skills of healthcare providers to be able to provide intrauterine device service as a contraceptive choice for Kyrgyz women.

Twist is a highly conserved basic helix-loop-helix transcription factor that regulates the expression of E-cadherin and promotes the epithelial-mesenchymal transition, which is critical for tumor infiltration. We examined the distribution and expression of this molecule in clear cell carcinoma of the ovary (CCC) to elucidate their clinical significance.Paraffin sections from CCC tissues (n = 27) were immunostained with Twist antibody and staining intensities were evaluated. Stratified with various clinicopathological factors, overall survival (OS) and progression-free survival (PFS) were evaluated.In the 27 carcinomas, negative Twist immunoexpression was observed in 14 cases (51.9%), and positive immunoexpression in 13 (48.1%). Twist, when categorized into negative versus positive expression, was associated with FIGO stage and peritoneal cytology. In addition, positive Twist expression significantly predicted a poorer OS and PFS compared with negative expression (p < 0.0001). Furthermore, the multivariate analyses revealed that positive Twist expression was an independent prognostic factor for OS and PFS of patients with CCC in this study (p = 0.0077 and 0.0033, respectively).The current findings suggest that the assessment of Twist immunoreactivity may be a useful prognostic indicator and that Twist may play a critical role in the progression of CCC.

The reduced migration/invasion of extravillous trophoblasts (EVTs) is a key feature of the genesis of preeclampsia. We and others previously reported that transcriptional factors activator protein-2 (AP-2) alpha and AP-2gamma act as suppressors of tumor invasion. The present study examined the expressions of AP-2alpha and AP-2gamma in preeclamptic placenta vs. control placenta and investigated their effect on the function of EVTs. The expressions of AP-2alpha and AP-2gamma were elevated in the preeclamptic placentas in comparison with the gestational age-matched control placentas. Their expressions also increased in EVTs of the preeclamptic placentas. Thereafter, we transfected AP-2alpha or AP-2gamma into human EVT cell line, HTR-8/SVneo. The overexpression of AP-2alpha or AP-2gamma decreased the migratory and invasive abilities in HTR-8/SVneo cells. This was followed by the reduction of protease activated receptor-1 and matrix metalloproteinases and a significant induction of plasminogen activator inhibitor-1 and the tissue inhibitor of metalloproteinase-1. AP-2alpha and AP-2gamma were weakly expressed in the cultured EVTs and HTR-8/SVneo cells, whereas they were induced by TNF-alpha, which increases in preeclamptic placenta and impairs trophoblast invasion. In the presence of TNF-alpha, the invasion of the HTR-8/SVneo cells was partially restored by a blocking of AP-2 induction using small interfering RNA of AP-2. The present data suggest that AP-2 may suppress trophoblast migration and invasion, thus leading to a shallow placentation in preeclampsia.

Glypican-3 (GPC3), a membrane-bound heparan sulphate proteoglycan, may play a role in promoting cancer cell growth and differentiation. Recent studies reported that GPC3 is overexpressed in clear cell carcinoma (CCC) of the ovary, and not other ovarian histotypes. However, in CCC patients, the relationship between the overexpression of GPC3 and prognosis has not yet been clarified.To evaluate GPC3 expression by immunohistochemistry in CCC.In 52 CCC patients, GPC3 expression was observed in 40.4%. In cases of CCC, no correlations were identified between GPC3 expression and clinicopathological factors, such as age, FIGO stage, CA125 values, peritoneal cytology, ascitic fluid volume and mortality rate, except for the residual tumour size. GPC3 expression was associated with poor progression-free survival in stage I CCC patients. The numbers of Ki-67-stained cells in GPC3-positive areas were lower than those in GPC3-negative areas. GPC3 expression may be associated with a low proliferation rate in CCC cells. In the early stage of CCC, GPC3-expressing patients tended to be resistant to taxane-based treatment.Results suggest that the overexpression of GPC3 may be related to the low-level proliferation of tumours; it may be associated with resistance to taxane-based chemotherapy and a poor prognosis in CCC of the ovary.

Colorectal cancers (CRCs) exhibit multiple genetic alterations, including allelic imbalances (copy number alterations, CNAs) at various chromosomal loci. In addition to genetic aberrations, DNA methylation also plays important roles in the development of CRC. To better understand the clinical relevance of these genetic and epigenetic abnormalities in CRC, we performed an integrative analysis of copy number changes on a genome-wide scale and assessed mutations of TP53, KRAS, BRAF, and PIK3CA and DNA methylation of six marker genes in single glands isolated from 39 primary tumors. Array-based comparative genomic hybridization (array-CGH) analysis revealed that genomic losses commonly occurred at 3q26.1, 4q13.2, 6q21.32, 7q34, 8p12-23.3, 15qcen and 18, while gains were commonly found at 1q21.3-23.1, 7p22.3-q34, 13q12.11-14.11, and 20. The total numbers and lengths of the CNAs were significantly associated with the aberrant DNA methylation and Dukes' stages. Moreover, hierarchical clustering analysis of the array-CGH data suggested that tumors could be categorized into four subgroups. Tumors with frequent DNA methylation were most strongly enriched in subgroups with infrequent CNAs. Importantly, Dukes' D tumors were enriched in the subgroup showing the greatest genomic losses, whereas Dukes' C tumors were enriched in the subgroup with the greatest genomic gains. Our data suggest an inverse relationship between chromosomal instability and aberrant methylation and a positive association between genomic losses and distant metastasis and between genomic gains and lymph node metastasis in CRC. Therefore, DNA copy number profiles may be predictive of the metastatic behavior of CRCs.

The loss of primordial follicles from gonadal damage caused by chemotherapy results in decreased ovarian reserve. To assess the impact of chemotherapy for patients with gestational trophoblastic neoplasia (GTN) on the ovarian reserve, we evaluated the post-chemotherapy serum anti-Müllerian hormone (AMH) levels.In 22 patients with GTN receiving chemotherapy, serum AMH levels were measured after the administration of chemotherapy and compared with serum AMH levels measured in patients with hydatidiform mole who did not receive chemotherapy, as a control. We also analyzed differences in the serum AMH levels following the administration of different anti-cancer agents.The serum AMH levels measured in the GTN group after chemotherapy was administered (median 1.18, range 0.32-3.94 ng/mL) significantly decreased in comparison to those measured in the control group (median 4.22, range 0.77-6.53 ng/mL, P=0.002). Serum AMH levels were significantly lower in the patients who had received a regimen including etoposide than in the patients who had not received treatment with etoposide (0.71 vs. 1.30 ng/mL, P=0.027).Our results suggest that chemotherapy administered to treat GTN does indeed affect the ovarian reserve, especially in patients who receive a medication regimen that includes etoposide. Measuring their serum AMH levels might therefore be helpful for counseling GTN patients regarding their ovarian reserve.

DNA methylation of CpG islands, together with deacetylation of histone and methylation of histone H3 lysines 9 and 27 (K9/K27), can lead to silencing of tumor-suppressor genes. The mechanisms underlying DNA methylation changes in cancer involve alteration of the activity of DNA methyltransferases (DNMTs), inflammation, and viral infection. DNA methylation affects genes involved in cell-cycle checkpoints, apoptosis, angiogenesis, invasion, immune responses, and cellular signaling. Subsets of cancers show DNA methylation of multiple genes, indicating that these tumors have the CpG island methylator phenotype (CIMP). Cancers with CIMP show distinct genetic changes, including microsatellite instability and mutations in the BRAF Ser/Thr kinase gene. Repetitive sequences such as short and long interspersed repeat elements are often hypomethylated in cancer, and are implicated in chromosomal instability. DNA methylation is a reversible phenomenon, and DNMT inhibitors can induce gene expression due to demethylation.

•Gestational trophoblastic neoplasia can arise during the first trimester originating from trophoblasts of concurrent pregnancy.•Intraplacental choriocarcinoma can be developed from trophoblasts of a previous pregnancy.

Epigenetic alterations, including aberrant DNA methylation and histone modification, play key roles in the dysregulation of tumor-related genes, thereby affecting numerous cellular processes, including cell proliferation, cell adhesion, apoptosis, and metastasis. In recent years, studies have demonstrated that short and long noncoding RNAs (ncRNAs) are key players in the initiation and progression of cancer, and epigenetic mechanisms are deeply involved in their dysregulation. Indeed, the growing list of microRNA (miRNA) genes aberrantly methylated in cancer suggests that a large number of miRNAs act as tumor suppressors or oncogenes. In addition, emerging evidence suggests that dysregulation of long ncRNAs (lncRNAs) plays critical roles in tumorigenesis. And because ncRNAs are involved in regulating gene expression through interaction with epigenetic modifiers, their dysregulation appears causally related to epigenetic alterations in cancer. Dissection of the interrelationships between ncRNAs and epigenetic alterations has the potential to reveal novel approaches to the diagnosis and treatment of cancer.

Objective: Recent studies have shown that cancer-associated fibroblasts (CAFs) and the epithelial-mesenchymal transition (EMT) play important roles in the progression and metastasis of CRC.Although prediction of lymph node metastasis in submucosal invasive colorectal cancer (SiCRC) is important, the relationships of CAF and EMT with lymph node metastasis of SiCRC have not yet been examined.Here, we aimed to analyze the expression patterns of CAF-and EMT-related proteins in SiCRC. Materials and Methods:The expression of CAF-related markers, including α-smooth muscle actin, CD10, podoplanin, fibroblast specific protein 1, and adipocyte enhancer-binding protein 1, and EMT-related proteins [zinc finger protein SNAI2 (ZEB1) and twist-related protein 1 (TWIST1) in SiCRC with (n = 29) or without (n = 80) lymph node metastasis was examined by immunohistochemistry.We examined the expression patterns of biomarkers using hierarchical cluster analysis.Consequently, four subgroups were established based on the expression patterns of CAF-and EMT-related markers, and the associations of these subgroups with clinicopathological variables.Results: In multivariate analysis, subgroup 2, which was characterized by high expression of all markers, was correlated with lymph node metastasis (p < 0.01).Next, we examined the associations of individual biomarkers with lymph node metastasis.Multivariate analysis showed that moderately differentiated adenocarcinoma was significantly associated with lymph node metastasis (p < 0.05).Conclusions: Our findings showed that expression patterns of CAF markers and EMT-related proteins may allow for stratification of patients into risk categories for lymph node metastasis in SiCRC.

Hepatitis B virus (HBV) infection is a major cause of hepatocellular carcinoma (HCC). Nucleos(t)ide analogue (NA) therapy effectively reduces the incidence of HCC, but it does not completely prevent the disease. Here, we show that dysregulation of microRNAs (miRNAs) is involved in post-NA HCC development. We divided chronic hepatitis B (CHB) patients who received NA therapy into two groups: 1) those who did not develop HCC during the follow-up period after NA therapy (no-HCC group) and 2) those who did (HCC group). miRNA expression profiles were significantly altered in CHB tissues as compared to normal liver, and the HCC group showed greater alteration than the no-HCC group. NA treatment restored the miRNA expression profiles to near-normal in the no-HCC group, but it was less effective in the HCC group. A number of miRNAs implicated in HCC, including miR-101, miR-140, miR-152, miR-199a-3p, and let-7g, were downregulated in CHB. Moreover, we identified CDK7 and TACC2 as novel target genes of miR-199a-3p. Our results suggest that altered miRNA expression in CHB contributes to HCC development, and that improvement of miRNA expression after NA treatment is associated with reduced HCC risk.