Yiping Qu

Gene methylation in gastric cancer

Gastric cancer is one of the most common malignancies and remains the second leading cause of cancer-related death worldwide. Over 70% of new cases and deaths occur in developing countries. In the early years of the molecular biology revolution, cancer research mainly focuses on genetic alterations, including gastric cancer. Epigenetic mechanisms are essential for normal development and maintenance of tissue-specific gene expression patterns in mammals. Disruption of epigenetic processes can lead to altered gene function and malignant cellular transformation. Recent advancements in the rapidly evolving field of cancer epigenetics have shown extensive reprogramming of every component of the epigenetic machinery in cancer, including DNA methylation, histone modifications, nucleosome positioning, noncoding RNAs, and microRNAs. Aberrant DNA methylation in the promoter regions of gene, which leads to inactivation of tumor suppressor and other cancer-related genes in cancer cells, is the most well-defined epigenetic hallmark in gastric cancer. The advantages of gene methylation as a target for detection and diagnosis of cancer in biopsy specimens and non-invasive body fluids such as serum and gastric washes have led to many studies of application in gastric cancer. This review focuses on the most common and important phenomenon of epigenetics, DNA methylation, in gastric cancer and illustrates the impact epigenetics has had on this field.

MiR-145 inhibits oral squamous cell carcinoma (OSCC) cell growth by targeting c-Myc and Cdk6

BackgroundMicroRNAs (miRNAs) are a large group of negative gene regulators that potentially play a critical role in tumorigenesis. Increasing evidences indicate that miR-145 acts a tumor suppressor in numerous human cancers. However, its role in oral carcinogenesis remains poorly defined. The aim of this study is to determine expression levels of miR-145 in oral squamous cell carcinomas (OSCCs) and normal mucosa tissues, and explore its biological functions in OSCCs.MethodsReverse transcription quantitative real-time PCR (RT-qPCR) assay was used to evaluate expression levels of miR-145. The biological functions of miR-145 were determined by cell proliferation and colony formation, cell cycle and apoptosis, as well as cell invasion assay.ResultsMiR-145 was frequently down-regulated in OSCCs compared with normal mucosa tissues. Restoring miR-145 expression in OSCC cells dramatically suppressed cell proliferation and colony formation, and induced G1 phase arrest and cell apoptosis. Importantly, our data showed that miR-145 downregulated the expression of c-Myc and Cdk6, which have previously been identified as two direct targets of miR-145.ConclusionsOur data suggest that miR-145 exerts its tumor suppressor function by targeting c-Myc and Cdk6, leading to the inhibition of OSCC cell growth. MiR-145 rescue may thus be a rational for diagnostic and therapeutic applications in OSCC.

Pathogenetic mechanisms in gastric cancer

Gastric cancer (GC) is a major public health issue as the fourth most common cancer and the second leading cause of cancer-related death. Recent advances have improved our understanding of its molecular pathogenesis, as best exemplified by elucidating the fundamental role of several major signaling pathways and related molecular derangements. Central to these mechanisms are the genetic and epigenetic alterations in these signaling pathways, such as gene mutations, copy number variants, aberrant gene methylation and histone modification, nucleosome positioning, and microRNAs. Some of these genetic/epigenetic alterations represent effective diagnostic and prognostic biomarkers and therapeutic targets for GC. This information has now opened unprecedented opportunities for better understanding of the molecular mechanisms of gastric carcinogenesis and the development of novel therapeutic strategies for this cancer. The pathogenetic mechanisms of GC are the focus of this review.

TERT promoter mutations and long telomere length predict poor survival and radiotherapy resistance in gliomas

Increasing evidences have implicated somatic gain-of-function mutations at the telomerase reverse transcriptase (TERT) promoter as one of the major mechanisms that promote transcriptional activation of TERT and subsequently maintain telomere length in human cancers including glioma. To investigate the prognostic value of these mutations and telomere length, individually and their coexistence, in gliomas, we analyzed two somatic mutations C228T and C250T in the TERT promoter, relative telomere length (RTL), IDH1 mutation and MGMT methylation in 389 glioma patients, and explored their associations with patient characteristics and clinical outcomes. Our data showed that C228T and C250T mutations were found in 17.0% (66 of 389) and 11.8% (46 of 389) of gliomas, respectively, and these two mutations were mutually exclusive in this cancer. Moreover, they were significantly associated with WHO grade. We also found that the RTL was significant longer in gliomas than in meningiomas and normal brain tissues (Median, 0.89 vs. 0.44 and 0.50; P < 0.001), and demonstrated that the RTL was strongly correlated with tumor recurrence. Importantly, TERT promoter mutations or long RTL caused a significantly poorer survival than TERT wild-type or short RTL. Coexisting TERT promoter mutations and long RTL were more commonly associated with poor patient survival than they were individually. Notably, the patients with TERT promoter mutations particularly C228T or long RTL were resistant to radiotherapy. Collectively, TERT promoter mutations and long RTL are not only prognostic factors for poor clinical outcomes, but also the predictors of radiotherapy resistance in gliomas.

Low frequency of TERT promoter mutations in a large cohort of gallbladder and gastric cancers

Dear Editors, Telomerase is a ribonucleoprotein that consists of an RNA subunit and a reverse transcriptase catalytic subunit. The latter is encoded by telomerase reverse transcriptase (TERT) gene. Telomerase adds telomeric repeats to chromosome ends, thereby maintaining telomere length. It is frequently upregulated in human cancers, contributing to human tumorigenesis. Mutations in the TERT gene promoter have been reported in melanoma, follicular cellderived thyroid cancer, bladder cancer, glioblastoma and other cancers. Two of them, 1,295,228 C>T and 1,295,250 C>T (termed C228T and C250T hereafter, respectively), are particularly common. These two mutations have been demonstrated to confer the TERT promoter increased transcriptional activity. Importantly, these mutations are found to be absent in benign tumors and normal human subjects, further implicating their potentially critical roles in human tumorigenesis. However, until now, TERT promoter mutations have not been investigated in a large cohort of gallbladder and gastric cancers. Although a previous study has shown no mutations in TERT gene promoter in gallbladder cancer, the limited number (N5 10) yielded an inconclusive results. In this study, a total of 154 gallbladder cancer tissues and 268 gastric cancer tissues were randomly obtained from the First Affiliated Hospital of Xi’an Jiaotong University School of Medicine, with approval by the institutional review board of the Hospital. Clinicopathological data were obtained from the patients’ files or by interview with the patients or their relatives, and were presented in Supporting Information Table 1. Genomic DNA was isolated using standard procedures of protease K digestion, phenol-chloroform extraction and ethanol precipitation. A pyrosequencing assay was performed to examine these two TERT promoter mutations. The following primers were used to amplify a fragment of 162 bp, which contained the sites of C228T and C250T mutations: 50-GCG CTG CCT GAA ACT CGC-30 (sense, biotinylated at the 50-end) and 50-CGT CCT GCC CCT TCA CCT-30 (antisense). PCR was performed with an initial denaturation at 95 C for 3 min, followed by 40 cycles of 95 C denaturation for 30 sec, 59 C annealing for 30 sec and 68 C elongation for 1 min. Quality of PCR products was determined by gel electrophoresis. The primer for pyrosequencing (50-CCC GCC CCC TCC CGA-30) was designed immediately upstream of C250T so that these two mutations are analyzed in the same assay. Pyrosequencing assay was performed on a PyroMark Q24 system using PyroMark Gold Q24 reagent (Qiagen). Sanger sequencing was then performed to confirm the results of pyrosequencing using the same primer pair as for pyrosequencing assay. As shown in Figure 1 and Table 1, similar to the findings in a previous study, the frequency of TERT promoter mutations in esophageal squamous cell carcinoma (ESCC) was very low, we also found low frequency of TERT promoter mutations in primary gallbladder and gastric cancer tissues, and gastric cancer cell lines. C228T and C250T mutations were found in 6 and 8 of 154 (3.9% and 5.2%) gallbladder cancer tissues, respectively, and these two mutations were mutually exclusive in this cancer. Notably, only two C250T mutations were found in 268 gastric cancer tissues, and no C228T or C250T mutations were found in eight gastric cancer cell lines. Given extremely low frequency of TERT promoter mutations in gastric cancer, we only investigated the association of these two mutations with clinicopathological characteristics of gallbladder cancer patients, including gender, age, tumor size, invasion, tumor node metastasis (TNM) stage, lymph node metastasis and survival status. The data showed that TERT promoter mutations did not significantly correlate with any clinicopathological variables in gallbladder cancer (Supporting Information Table 2).

TERT promoter mutations predict worse survival in laryngeal cancer patients.

1 Department of Endocrinology, The First Affiliated Hospital of Xi’an Jiaotong University School of Medicine, Xi’an 710061, People’s Republic of China 2 Department of Otolaryngology, The First Affiliated Hospital of Xi’an Jiaotong University School of Medicine, Xi’an 710061, People’s Republic of China 3 Center for Translational Medicine, The First Affiliated Hospital of Xi’an Jiaotong University School of Medicine, Xi’an 710061, People’s Republic of China

Variable copy number of mitochondrial DNA (mtDNA) predicts worse prognosis in advanced gastric cancer patients.

BackgroundChange of mitochondrial DNA (mtDNA) copy number is widely reported in various human cancers, including gastric cancer, and is considered to be an important hallmark of cancers. However, there is remarkably little consensus on the value of variable mtDNA content in the prognostic evaluation of this cancer.MethodsUsing real-time quantitative PCR approach, we examined mtDNA copy number in a cohort of gastric cancers and normal gastric tissues, and explored the association of variable mtDNA content with clinical outcomes of gastric cancer patients.ResultsOur data showed that the majority of gastric cancer patients had low mtDNA content as compared to control subjects although the relative mean mtDNA content was higher in the former than the latter. Moreover, we found that variable mtDNA content was strongly associated with lymph node metastasis and cancer-related death of the patients with late-stage tumors. Notably, variable mtDNA content did not affect overall survival of gastric cancer patients, however, we found that increased mtDNA content was associated with poor survival in the patients with late-stage tumors.ConclusionIn this study, we demonstrated that variable mtDNA content markedly increased the risk of lymph node metastasis and high mortality of the patients with late-stage tumors. Additionally, we found a strong link between increased mtDNA content and worse survival of the patients with late-stage tumors. Taken together, variable mtDNA content may be a valuable poor prognostic factor for advanced gastric cancer patients.Virtual slidesThe virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/1344721463103353.

Concomitant PIK3CA amplification and RASSF1A or PAX6 hypermethylation predict worse survival in gastric cancer.

OBJECTIVES A large number of genetic and epigenetic alterations have been found in gastric cancer, but there is remarkably little consensus on the value of individual biomarker in diagnosis and prognosis of this cancer. This study was designed to illustrate the value of PIK3CA amplification in combination with promoter methylation of RASSF1A and PAX6 genes in early diagnosis and prognosis of gastric cancer. DESIGN AND METHODS Using real-time quantitative PCR, quantitative methylation-specific PCR (Q-MSP), and methylation-specific PCR (MSP) assays, we examined PIK3CA amplification and promoter methylation of RASSF1A and PAX6 genes in a cohort of gastric cancers, and explored the association of various (epi)genotypes with clinical outcomes of gastric cancer patients. RESULTS We demonstrated PIK3CA gene was specifically amplified in gastric cancers, but not in normal gastric tissues. Moreover, frequent methylation of RASSF1A and PAX6 was also found in gastric cancers. Given the patients harboring diverse (epi)genotypes, we thus investigated the effect of various (epi)genotypes on poor prognosis in gastric cancer. The data showed that concomitant PIK3CA amplification and RASSF1A or PAX6 methylation was closely associated with poor clinical outcomes, particularly survival, as compared to other (epi)genotypes in gastric cancer. CONCLUSIONS We found frequent PIK3CA amplification and promoter methylation of RASSF1A and PAX6 genes in gastric cancers, and demonstrated concomitant PIK3CA amplification and promoter methylation in any one of these two genes were significantly associated with worse survival in gastric cancer. Collectively, such (epi)genotypes may be strong and independent poor prognostic factors for gastric cancer patients.

Low copy number of mitochondrial DNA (mtDNA) predicts worse prognosis in early-stage laryngeal cancer patients.

ObjectivesAlterations in mitochondrial DNA (mtDNA) copy number have been widely reported in various human cancers, and been considered to be an important hallmark of cancers. However, little is known about the value of copy number variations of mtDNA in the prognostic evaluation of laryngeal cancer.Design and methodsUsing real-time quantitative PCR method, we investigated mtDNA copy number in a cohort of laryngeal cancers (n =204) and normal laryngeal tissues (n =40), and explored the association of variable mtDNA copy number with clinical outcomes of laryngeal cancer patients.ResultsOur data showed that the relative mean mtDNA content was higher in the laryngeal cancer patients (11.91 ± 4.35 copies) than the control subjects (4.72 ± 0.70 copies). Moreover, we found that mtDNA content was negatively associated with cigarette smoking (pack-years), tumor invasion, and TNM stage. Notably, variable mtDNA content did not affect overall survival of laryngeal cancer patients. However, when the patients were categorized into early-stage and late-stage tumor groups according to TNM stage, we found that low mtDNA content was strongly associated with poor survival in the former, but not in the latter.ConclusionsThe present study demonstrated that low mtDNA content was strongly correlated with some of clinicopathological characteristics, such as cigarette smoking, tumor invasion and TNM stage. In addition, we found a strong link between low mtDNA content and worse survival of the patients with early-stage tumors. Taken together, low copy number of mtDNA may be a useful poor prognostic factor for early-stage laryngeal cancer patients.Virtual slidesThe virtual slides for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/1841771572115955

Analysis of BRAF V600E mutation and DNA methylation improves the diagnostics of thyroid fine needle aspiration biopsies

BackgroundThyroid nodules with indeterminate cytological features on fine needle aspiration biopsy specimens (FNABs) have a ~20% risk of thyroid cancer. BRAFV600E mutation and DNA methylation are useful markers to distinguish malignant thyroid neoplasm from benign. The aim of this study was to determine whether combined detection of BRAFV600E mutation and methylation markers on FNABs could improve the diagnostic accuracy of thyroid cancer.MethodsUsing pyrosequencing and quantitative methylation-specific PCR (Q-MSP) methods, FNABs from 79 and 38 patients with thyroid nodules in training and test groups, respectively, were analyzed for BRAFV600E mutation and gene methylation.ResultsBRAFV600E mutation was found in 30/42 (71.4%) and 14/20 (70%) FNABs in training and test groups, respectively. All BRAFV600E-positive samples were histologically diagnosed as papillary thyroid cancer (PTC) after thyroidectomy. As expected, BRAF mutation was not found in all benign nodules. Moreover, we demonstrated that the five genes, including CALCA, DAPK1, TIMP3, RAR-beta and RASSF1A, were aberrantly methylated in FNABs. Of them, methylation level of DAPK1 in PTCs was significantly higher than that in benign samples (P <0.0001). Conversely, methylation level of RASSF1A in PTCs was significantly lower than that in benign samples (P =0.003). Notably, compared with BRAF mutation testing alone, combined detection of BRAF mutation and methylation markers increased the diagnostic sensitivity and accuracy of PTC with excellent specificity.ConclusionOur data have demonstrated that combine analysis of BRAF mutation and DNA methylation markers on FNABs may be a useful strategy to facilitate the diagnosis of malignant thyroid neoplasm, particularly PTC.Virtual slidesThe virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/6080878071149177.