Esophageal squamous cell carcinoma (ESCC) is a prevalent and fatal cancer in China and other Asian countries. Epigenetic silencing of key tumor suppressor genes (TSGs) is critical to ESCC initiation and progression. Recently, many novel TSGs silenced by promoter methylation have been identified in ESCC, and these genes further serve as potential tumor markers for high-risk group stratification, early detection, and prognosis prediction. This review summarizes recent discoveries on aberrant promoter methylation of TSGs in ESCC, providing better understanding of the role of disrupted epigenetic regulation in tumorigenesis and insight into diagnostic and prognostic biomarkers for this malignancy.
Tumor suppressor gene; CpG island; promoter methylation; esophageal squamous cell carcinoma; tumor marker
Esophageal squamous cell carcinomas (ESCC) are usually asymptomatic and go undetected until they are incurable. Cytological screening is one strategy to detect ESCC at an early stage and has shown promise in previous studies, although improvement in sensitivity and specificity are needed. Proteases modulate cancer progression by facilitating tumor invasion and metastasis. In the current study, matrix metalloproteinases (MMPs) were studied in a search for new early detection markers for ESCC.
Protein expression levels of MMPs were measured using zymography in 24 cases of paired normal esophagus and ESCC, and in the tumor-associated stroma and tumor epithelium in one sample after laser capture microdissection (LCM). MMP-3 and MMP-10 transcripts in both the epithelium and stroma in five cases were further analyzed by quantitative reverse transcriptase polymerase chain reaction (qRT-PCR).
Gelatin zymography showed bands corresponding in size to MMP-2, MMP-3, MMP-9, and MMP-10 enzymes in each of the 24 cancer cases. MMP levels tended to be higher in tumors than paired normal tissue; however, only the 45 kDa band that corresponds to the activated form of MMP-3 and MMP-10 was strongly expressed in all 24 tumors with little or no expression in the paired normal foci. LCM-based analysis showed the 45 kDA band to be present in both the stromal and epithelial components of the tumor microenvironment, and that MMP-3 and MMP-10 mRNA levels were higher in tumors than paired normal tissues for each compartment.
Increased levels of MMPs occur in ESCC suggesting their up-regulation is important in esophageal tumorigenesis. The up-regulated gene products have the potential to serve as early detection markers in the clinic.
The objective of this study was to characterize the molecular events in the carcinogenesis of esophageal squamous cell carcinoma (ESCC) and to identify biomarkers for early detection of the disease. Matched precancerous and cancerous tissues resected from 34 esophageal cancer patients from Chongqing, southern China, were compared to evaluate the extent of loss of heterozygosity (LOH). Sixteen microsatellite markers on chromosome regions 3p, 4p, 5q, 8p, 9p, 9q, 11p, 13q and 17p were used for PCR-based LOH analysis. The overall frequency of LOH at the 16 microsatellite loci was significantly increased as the pathological status of the resection specimens changed from low-grade dysplasia (LGD) to high-grade dysplasia (HGD) and SCC (P<0.001). A total of 8 markers showed LOH in the LGD samples. In addition, heterozygosity was regained at 4 loci in the SCC samples of 4 patients, respectively, in comparison to the results for these loci in the HGD samples. The overall rate of LOH increased significantly with the deterioration of the lesions, indicating that tumorigenesis of the esophageal squamous epithelia is a progressive process involving accumulative changes in LOH. The 8 loci showing allelic loss in the LGD samples may be involved in the early-stage tumorigenesis of ESCC, and LOH analysis at these loci may help improve the early detection of this disease. Regain of heterozygosity found in certain patients suggests the possibility of genetic heterogeneity in the tumori-genesis of esophageal cancer.
esophageal squamous cell carcinoma; microsatellite; loss of heterozygosity
The aberrant DNA methylation of tumor suppressor genes is well documented in esophageal cancer, including adenocarcinoma (EAC) and squamous cell carcinoma (ESCC) as well as in Barrett's esophagus (BE), a pre-malignant condition that is associated with chronic acid reflux. BE is a well-recognized risk factor for the development of EAC, and consequently the standard of care is for individuals with BE to be placed in endoscopic surveillance programs aimed at detecting early histologic changes that associate with an increased risk of developing EAC. Yet because the absolute risk of EAC in individuals with BE is minimal, a clinical need in the management of BE is the identification of additional risk markers that will indicate individuals who are at a significant absolute risk of EAC so that they may be subjected to more intensive surveillance. The best currently available risk marker is the degree of dysplasia in endoscopic biopsies from the esophagus; however, this marker is suboptimal for a variety of reasons. To date, there are no molecular biomarkers that have been translated to widespread clinical practice. The search for biomarkers, including hypermethylated genes, for either the diagnosis of BE, EAC, or ESCC or for risk stratification for the development of EAC in those with BE is currently an area of active research. In this review, we summarize the status of identified candidate epigenetic biomarkers for BE, EAC, and ESCC. Most of these aberrantly methylated genes have been described in the context of early detection or diagnostic markers; others might prove useful for estimating prognosis or predicting response to treatment. Finally, special attention will be paid to some of the challenges that must be overcome in order to develop clinically useful esophageal cancer biomarkers.
DNA methylation; biomarker; Barrett's esophagus; esophageal adenocarcinoma; esophageal squamous cell carcinoma
In order to characterize the molecular events in the carcinogenesis of esophageal cancer and to identify biomarkers for the early detection of the disease, matched precancerous and cancerous tissues resected from 34 esophageal cancer patients in Chongqing of southern China were compared for the extent of loss of heterozygosity (LOH). Sixteen microsatellite markers on nine chromosome regions were used for the PCR-based LOH analysis. The overall frequency of LOH at the 16 microsatellite loci was significantly increased as the pathological status of the resection specimens changed from low-grade dysplasia (LGD) to high-grade dysplasia (HGD) and squamous cell carcinoma (SCC) (P < 0.001), indicating that tumorigenesis of the esophageal squamous epithelia is a progressive process involving accumulative changes of LOH. A total of eight markers showed LOH in the LGD samples, suggesting that these loci may be involved in the early-stage tumorigenesis of esophageal squamous cell carcinoma (ESCC) and that LOH analysis at these loci may help improve the early detection of this disease. In addition, heterozygosity was regained at four loci in the SCC samples of four patients compared with the HGD samples, suggesting the possibility of genetic heterogeneity in the tumorigenesis of esophageal cancer.
esophageal squamous cell carcinoma; microsatellite; loss of heterozygosity
Objective: Biomarker assay is a noninvasive method for the early detection of esophageal squamous cell carcinoma (ESCC). Searching for new biomarkers with high specificity and sensitivity is very important for the early detection of ESCC. Serum surface-enhanced laser desorption/ionization-time of flight mass spectrometry (SELDI-TOF-MS) is a high throughput technology for identifying cancer biomarkers using drops of sera. Methods: In this study, 185 serum samples were taken from ESCC patients in a high incidence area and screened by SELDI. A support vector machine (SVM) algorithm was adopted to analyze the samples. Results: The SVM patterns successfully distinguished ESCC from pre-cancerous lesions (PCLs). Also, types of PCL, including dysplasia (DYS) and basal cell hyperplasia (BCH), and healthy controls (HC) were distinguished with an accuracy of 95.2% (DYS), 96.6% (BCH), and 93.8% (HC), respectively. A marker of 25.1 kDa was identified in the ESCC patterns whose peak intensity was observed to increase significantly during the development of esophageal carcinogenesis, and to decrease obviously after surgery. Conclusions: We selected five ESCC biomarkers to form a diagnostic pattern which can discriminate among the different stages of esophageal carcinogenesis. This pattern can significantly improve the detection of ESCC.
Biomarker; Esophageal squamous cell carcinoma; Surface-enhanced laser desorption/ionization; Support vector machine
Esophageal squamous cell carcinoma (ESCC) is a common malignancy and one of the more difficult diseases to diagnose in Japan due to its poor prognosis. MicroRNAs are small non-coding RNAs of 21–23 nucleotides that regulate gene expression. MicroRNA-34b (miR-34b) has been reported to be overexpressed in various types of cancer. However, its role in ESCC has yet to be extensively studied. The present study investigated the expression of miR-34b in 88 ESCC patients. The miR-34b expression in ESCC was significantly higher than that in the corresponding normal esophageal mucosa. It was more highly expressed in tumors with more advanced stages. However, its expression did not correlate with the p53 status. Transfection of anti-miR-34b to the ESCC cells suppressed cell growth in vitro. These results suggest an oncogenic role of miR in ESCC.
microRNA-34b; esophageal squamous cell carcinoma; MTT assay
AIM: To study the expression of β-catenin in esophageal squamous cell carcinoma (ESCC) at stage T2-3N0M0 and its relation with the prognosis of ESCC patients.
METHODS: Expression of β-catenin in 227 ESCC specimens was detected by immunohistochemistry (IHC). A reproducible semi-quantitative method which takes both staining percentage and intensity into account was applied in IHC scoring, and receiver operating characteristic curve analysis was used to select the cut-off score for high or low IHC reactivity. Then, correlation of β-catenin expression with clinicopathological features and prognosis of ESCC patients was determined.
RESULTS: No significant correlation was observed between β-catenin expression and clinicopathological parameters in terms of gender, age, tumor size, tumor grade, tumor location, depth of invasion and pathological stage. The Kaplan-Meier survival curve showed that the up-regulated expression of β-catenin indicated a poorer post-operative survival rate of ESCC patients at stage T2-3N0M0 (P = 0.004), especially of those with T3 lesions (P = 0.014) or with stage IIB diseases (P = 0.007). Multivariate analysis also confirmed that β-catenin was an independent prognostic factor for the overall survival rate of ESCC patients at stage T2-3N0M0 (relative risk = 1.642, 95% CI: 1.159-2.327, P = 0.005).
CONCLUSION: Elevated β-catenin expression level may be an adverse indicator for the prognosis of ESCC patients at stage T2-3N0M0, especially for those with T3 lesions or stage IIB diseases.
Esophageal squamous cell carcinoma; β-catenin; Prognosis; Receiver operating characteristic curve; Immunohistochemistry
Hedgehog (Hh) signaling is frequently activated in human cancer, including
esophageal cancer. Most esophageal cancers are diagnosed in the advanced stages,
therefore, identifying the very alterations that drive esophageal carcinogenesis
may help designing novel strategies to diagnose and treat the disease. Analysis
of Hh signaling in precancerous lesions is a critical first step in determining
the significance of this pathway for carcinogenesis. Here we report our data on
Hh target gene expression in 174 human esophageal specimens [28 esophageal
adenocarcinomas (EAC), 19 Barrett’s esophagus, 103 cases of esophageal
squamous cell carcinoma (ESCC), and 24 of squamous dysplastic lesions], and in
two rat models of esophageal cancer. We found that 96% of human EAC express Hh
target genes. We showed that PTCH1 expression is the most reliable biomarker. In
contrast to EAC, only 38% of ESCC express Hh target genes. We found activation
of Hh signaling in precancerous lesions of ESCCs and EACs in different degrees
(21% and 58% respectively). Expression of Hh target genes is frequently detected
in severe squamous dysplasia/ carcinoma in situ (p=0.04) and
Barrett’s esophagus (p=0.01). Unlike EAC, sonic hedgehog (Shh) expression
was rare in ESCCs. Consistent with the human specimen data, we found a high
percentage of Hh signaling activation in precancerous lesions in rat models.
These data indicate that Hh signaling activation is an early molecular event in
the development of esophageal cancer, particularly EAC.
Esophageal adenocarcinoma (EAC); esophageal squamous cell carcinoma (ESCC); hedgehog (Hh); patched-1 (PTCH1 for humans and Ptch1 for animals); Gli2; sFRP-1; human homologue of hedgehog-interaction protein (HHIP); rat model; Barrett’s esophagus (BE)
Esophageal squamous cell carcinoma (ESCC) is one of the most malignant tumors and typically presents at an advanced and rapidly fatal stage. To better understand the role of genetics in the etiology and prevention of ESCC and to identify potential susceptibility genes as well as early detection markers, we previously compared tumor and matched normal tissues from ESCC patients from a high-risk area of China using cDNA expression microarrays and identified 41 differentially-expressed genes (13 over-expressed and 28 under-expressed).
In the current study, we validated and quantitated differential mRNA expression in a sample of nine of these 41 genes, including four that were over-expressed (SPARC, FADD, Fascin, COL7A1), and five that were under-expressed (CK4, TGM3, ECM1, PPL, EVPL), in 75 new ESCC patients using quantitative Real-time RT-PCR and the 2-ΔΔCT method to examine both tumor and matched normal tissue. In addition, we examined expression patterns for these genes by selected demographic and clinical characteristics.
Four previously over-expressed (tumor ≥2-fold normal) genes were all increased in the majority of new ESCC patients: SPARC was increased in 71% of patients, Fascin in 70%, FADD in 63%, and COL7A1 in 57%. Five previously under-expressed (tumor ≤0.5-fold normal) genes similarly showed decreased mRNA expression in two-thirds or more of patients: CK4 was decreased in 83% of patients, TGM3 in 77%, ECM1 in 73%, and PPL and EVPL in 67% each. In subset analyses, associations with age (for COL7A1), family history (for PPL and ECM1), and alcohol use (for SPARC and Fascin) were also noted.
These data indicate that these nine genes have consistent differential mRNA expression, validating results of our previous cDNA array results, and affirming their potential role in the early detection of ESCC.
Radiofrequency ablation (RFA) is safe and effective for eradicating neoplasia in Barrett’s esophagus.
Evaluate RFA for eradicating early esophageal squamous cell neoplasia (ESCN) defined as moderate- and high-grade squamous intraepithelial neoplasia (MGIN, HGIN) and early flat-type esophageal squamous cell carcinoma (ESCC).
Prospective cohort study.
Tertiary referral center.
Esophageal unstained lesions (USLs) were identified using Lugol’s chromoendoscopy. Inclusion: at least 1 flat (type 0-IIb) USL ≥3cm, USL-bearing esophagus ≤12 cm, and a consensus diagnosis of MGIN, HGIN, or ESCC by two expert GI pathologists. Exclusion: prior endoscopic resection or ablation, stricture, or any non-flat mucosa.
Circumferential RFA creating a continuous treatment area (TA) including all USLs. At 3-month intervals thereafter, chromoendoscopy with biopsies, followed by focal RFA of USLs, if present.
Main outcome measures
Complete response (CR) at 12 months, defined as absence of MGIN, HGIN or ESCC in TA; CR after one RFA session; neoplastic progression from baseline; and adverse events.
29 patients (14 male, mean age 60.3 years) with MGIN (18), HGIN (10), or ESCC (1) participated. Mean USL length was 6.2 cm (TA 8.2 cm). At 3-months, after one RFA session, 86% of patients (25/29) were CR. At 12-months, 97% (28/29) of patients were CR. There was no neoplastic progression. There were 4 strictures, all dilated to resolution.
Single center study with limited number of patients.
In patients with early ESCN (MGIN, HGIN, flat-type ESCC), RFA was associated with a high rate of histological complete response (97% of patients), no neoplastic progression, and an acceptable adverse event profile.
Pepsinogens are a class of endopeptidases that are secreted by the gastric epithelium and released into the circulation. Low serum pepsinogen I (PGI) and low serum pepsinogen I / pepsinogen II ratio (PGI/II ratio) are markers of gastric fundic atrophy, and have recently been shown to be associated with increased risk of esophageal squamous cell carcinoma (ESCC). We conducted the current study to test whether these markers are also associated with esophageal squamous dysplasia (ESD), the precursor lesion of ESCC.
We measured serum PGI and PGII, using enzyme-linked immunosorbent (ELISA) assays, in 125 case subjects (patients with moderate or severe ESD) and 250 sex-matched control subjects (no ESD) selected from an endoscopic screening study in Linxian, China. We used conditional logistic regression models adjusted for age, smoking, and place of residence to calculate odds ratios (ORs) and 95% confidence intervals (95% CIs).
Serum PGI showed no statistically significant association with ESD, whether analyzed as a dichotomous, ordinal (quartiles), or continuous variable. Lower serum PGI/II ratio, however, showed a dose-response association with increased risk of ESD, with an adjusted OR (95% CI) of 2.12 (1.08 − 4.18), comparing the lowest versus the highest quartile. The association between lower serum PGI/II ratio and log OR of ESD was nearly linear, and the p-value for the continuous association was 0.03.
Lower serum PGI/II ratio was linearly associated with higher risk of ESD. This result is consistent with recent findings that gastric atrophy may increase the risk of ESCC.
Esophageal cancer; Squamous dysplasia; Pepsinogen; China
The aim of the study was to evaluate the association of immunoglobulin G type of autoantibodies to oxidized low-density lipoprotein (oxLDL-lgG) and oxLDL-lgM with the progression of esophageal squamous cell carcinoma (ESSC).
Residents from Feicheng, China aged 40 to 69 years were screened for esophageal lesions in a screening program conducted during the period of January 2008 to December 2006. There were 33 controls with normal esophageal squamous epithelium cells, 37 patients with basal cell hyperplasia, 47 with esophageal squamous cell dysplasia, and 43 with ESCC. All the participants were diagnosed by biopsy and histopathological examination. Adiponectin, oxidized low-density lipoprotein (oxLDL), autoantibodies against oxLDL (oxLDL-ab), OxLDL-lgG, and OxLDL-lgM were determined by enzyme linked immunosorbent assay (ELISA). Total cholesterol, High-density lipoprotein (HDL), triglyceride, serum albumin, and blood pressure were co-estimated. Analysis of covariance for lipid levels was used to control the influence of covariates.
The level of oxLDL-lgM increased gradually along with esophageal carcinoma progression. The oxLDL-lgM levels in the ESCC group were the highest after possible covariates were controlled. Binary logistic regression showed that oxLDL-lgM had a positive correlation with the development of esophageal carcinoma, while oxLDL and oxLDL-ab had a negative correlation with ESSC. No significant association between the levels of oxLDL-lgG and adiponectin and the different stages of ESSC was observed.
The present study shows that the decreased oxLDL and oxLDL-ab and the elevated oxLDL-lgM serum levels may relate to the development and progression of ESSC.
Esophageal squamous cell carcinoma (ESCC) is one of the most common malignancies. Early diagnosis is critical for guiding the therapeutic management of ESCC. It is imperative to find more effective biomarkers of ESCC.
To identify novel biomarkers for esophageal squamous cell carcinoma (ESCC), specimens from 10 patients with ESCC were subjected to a comparative proteomic analysis. The proteomic patterns of ESCC samples and normal esophageal epithelial tissues (NEETs) were compared using two-dimensional gel electrophoresis. And differentially expressed proteins were identified using MALDI-TOF-MS/MS. For further identification of protein in selected spot, western blotting and immunohistochemistry were employed.
Twelve proteins were up-regulated and fifteen proteins were down-regulated in the ESCC samples compared with the NEET samples. Up-regulation of galectin-7 was further confirmed by western blotting and immunohistochemistry. Furthermore, immunohistochemical staining of galectin-7 was performed on a tissue microarray containing ESCC samples (n = 50) and NEET samples (n = 10). The expression levels of galectin-7 were markedly higher in the ESCC samples than in the NEET samples (P = 0.012). In addition, tissue microarray analysis also showed that the expression level of galectin-7 was related to the differentiation of ESCC.
The present proteomics analysis revealed that galectin-7 was highly expressed in ESCC tissues. The alteration in the expression of galectin-7 was confirmed using a tissue microarray. These findings suggest that galectin-7 could be used as a potential biomarker for ESCC.
Esophageal cancer is a deadly cancer with esophageal squamous cell carcinoma (ESCC) as the major type. Until now there has been a lack of reliable prognostic markers for this malignancy. This study aims to investigate the clinical correlation between Forkhead box M1 (FoxM1) and patients’ parameters in ESCC.
Immunohistochemistry was performed to investigate the expression and localization of FoxM1 in 64 ESCC tissues and 10 nontumor esophageal tissues randomly selected from 64 patients before these data were used for clinical correlations.
Cytoplasmic and nuclear expressions of FoxM1 were found in 63 and 16 of the 64 ESCC tissues, respectively. Low cytoplasmic expression of FoxM1 was correlated with early pathological stage in ESCC (P = 0.018), while patients with nuclear FoxM1 were younger in age than those without nuclear expression (P < 0.001). Upregulation of FoxM1 mRNA was found in five ESCC cell lines (HKESC-1, HKESC-2, HKESC-3, HKESC-4, and SLMT-1) when compared to non-neoplastic esophageal squamous cell line NE-1 using quantitative polymerase chain reaction (qPCR). Except for HKESC-3, all studied ESCC cell lines demonstrated a high expression of FoxM1 protein using immunoblot. A high mRNA level of FoxM1 was observed in all of the ESCC tissues examined when compared to their adjacent nontumor tissues using qPCR.
Cytoplasmic FoxM1 was correlated with pathological stage and might be a biomarker for advanced ESCC.
The etiology of esophageal squamous cell cancer (ESCC) in high prevalence regions of China remains unclear.
Endoscopic biopsies were conducted among 7381 inhabitants aged from 25 to 65 of Anyang, China.
In this study, 2.57, 0.20 and 0.16% of the participants had mild, moderate and severe squamous dysplasia, respectively; 0.19 and 0.08% showed squamous carcinoma in situ and invasive ESCC. Using deep well (depth >100 meters) as water source (odds ratio=0.72, 95% confidence interval: 0.54–0.96) was negatively associated with ESCC and its precursors, whereas tobacco and alcohol use were not significantly associated with ESCC.
Water source and other factors in this region need further evaluation by longitudinal studies.
prevalence; risk factor; esophageal cancer; precursor lesion; China
Esophageal squamous cell carcinoma (ESCC) is the most common histological subtype of cancer in the upper and middle esophagus and is characterized by a high rate of mortality. The incidence of esophageal cancer varies greatly among regions of the world and occurs at a high frequency in Asia and South America.
In our department, a 51-year-old man was diagnosed with ESCC after presenting with extensive disseminated skin nodules. Biopsy of the nodules showed metastatic ESCC. Cutaneous manifestations of esophageal neoplasia are very rare and are mainly described for esophageal adenocarcinoma (EADC). Here we report a very uncommon case of extensive skin metastases of ESCC.
Early biopsies of suspicious skin lesions are important and should be performed in patients with unclear symptoms such as weight loss or dysphagia and especially in patients with a history of cancer, since they can reveal the existence of a distant malignant disease leading to diagnosis and prompt therapy.
Background: Barrett's esophagus (BE) affects up to 12 million Americans and confers an increased risk for development of esophageal adenocarcinoma (EAC). EAC is often fatal unless detected early. Given the high prevalence, high cost of surveillance and relatively low risk of most affected individuals, identification of high-risk patients for additional scrutiny, regular surveillance, or ablative therapy is crucial. The exploration of “field effect” by probing uninvolved esophageal mucosa to predict the risk of EAC has the potential as an improved surveillance and prevention strategy. In this study, we evaluate the ability of nuclear nano-architecture markers from normal squamous esophagus and gastric cardia to detect the “field effect” of esophageal dysplasia and EAC, and their response to endoscopic therapy.
Methods: Patients with normal esophagus, gastroesophageal reflux, BE and EAC were eligible for enrollment. We performed endoscopic cytology brushings of the gastric cardia, ~1-2 cm below the gastroesophageal junction, and of the normal squamous esophageal mucosa at ~20 cm from the incisors and standard cytology slides were made using Thinprep method. Optical analysis was performed on the cell nuclei of cytologically normal-appearing epithelial cells.
Results: The study cohort consisted of 128 patients. The nuclear nano-architecture markers detected the presence of esophageal dysplasia and EAC with statistical significance. The field effect does not exhibit a spatial dependence. These markers reverted toward normal in response to endoscopic therapy.
Conclusions: Optical analysis of gastric cardia and upper squamous esophagus represents a potentially viable method to improve risk stratification and ease of surveillance of patients with Barrett's esophagus and to monitor the efficacy of ablative therapy.
nuclear architecture; nanoscale; esophagus; dysplasia; field effect.
Inactivation of protein tyrosine phosphatase receptor-type O (PTPRO), a new member of the PTP family, by hypermethylation has been described in several forms of cancers. We evaluated PTPRO hypermethylation as a potential epigenetic biomarker in esophageal squamous cell carcinoma (ESCC). PTPRO hypermethylation was observed in 27 (75%) of 36 primary tumors and correlated significantly with depth of invasion (T-stage, P = 0.013). Among matched peripheral blood samples from ESCC patients, 13 (36.1%) of 36 had detectable methylated PTPRO in plasma, and 15 (41.7%) of 36 had it in the buffy coat. No methylated PTPRO was observed in normal peripheral blood samples from 10 healthy individuals. In addition, demethylation by 5-aza-dC treatment led to gene reactivation in PTPRO-methylated and -silenced ESCC cell lines. This is the first report for detection of PTPRO as a non-invasive biomarker for solid tumors in peripheral blood. Our findings suggest that hypermethylated PTPRO occurs frequently in ESCC. Its detection in the peripheral blood of ESCC patients illustrates its potential clinical application as an epigenetic biomarker for noninvasive diagnosis and disease monitoring.
PTPRO; DNA methylation; epigenetics; Cancer surveillance and screening
AIM: To investigate the promoter region methylation status of retinoblastoma protein-interacting zinc finger gene 1 (RIZ1) in the human esophageal squamous cell carcinoma (ESCC) cell lines and tissues and verify the relationship between methylation of RIZ1 and oncogenesis, tumor progression and metastasis etc of ESCC.
METHODS: Methylation-specific polymerase chain reaction (MSP) was used to investigate the promoter region methylation status of RIZ1 in 6 ESCC cell lines. One cell line where RIZ1 promoter region methylation was detected was selected for the next study, where the cell line was treated with 5-aza-CdR. Real-time polymerase chain reaction was used to investigate its influence on the transcription of RIZ1. Experiments using frozen pathological specimens from 47 ESCC patients were performed using the same MSP methodology.
RESULTS: Promoter methylation of RIZ1 gene was detected in TE13, CaEs17 and EC109 cell lines and the cell line TE13 was chosen for further study. The expression of RIZ1 mRNA in TE-13 was up-regulated after treatment with 5-aza-CdR. The rate of methylation in carcinomas tissues was significantly higher than those in matched neighboring normal and distal ending normal tissue, and the deviation of data was statistically significant (χ2 = 24.136, P < 0.01). Analysis of the gender, age familial history, tumour deviation, tumour saturation, lymph gland displacement and clinical staging of 47 samples from ESCC patients showed that the fluctuation of data was not statistically significant.
CONCLUSION: Promoter methylation may play an important role in the epigenetic silencing of RIZ1 gene expression in human ESCC. RIZ1 is considered to be a potential tumor suppressor gene and may be a biological parameter for testing early stage human ESCC.
Retinoblastoma protein-interacting zinc finger gene 1; Tumor suppressor genes; Esophageal squamous cell carcinoma; Promoter methylation; Methylation-specific polymerase chain reaction
The aim of this study was to investigate the correlation between cyclin A expression and efficacy of paclitaxel-based chemotherapy in patients with esophageal squamous cell carcinoma (ESCC). The expression of cyclin A was examined in 48 newly diagnosed ESCC patients prior to treatment using the MaxVision immunohistochemistry method. The patients received four cycles of paclitaxel-based chemotherapy, the short-term treatment efficacy was evaluated and a 3-year follow-up was conducted. The response rate was greater in patients with positive cyclin A expression compared with those with negative expression (54.8 vs. 23.5%; χ2=4.373; P<0.05). Univariate and multivariate Cox analysis revealed that clinicopathological stage, degree of differentiation and expression of cyclin A were independent prognosis factors in patients with ESCC following paclitaxel-based chemotherapy. ESCC patients with positive cyclin A expression demonstrated an increased sensitivity to paclitaxel-based chemotherapy, suggesting that cyclin A may be used as a marker to predict the treatment efficacy of paclitaxel in patients with ESCC.
esophageal squamous cell carcinoma; cyclin A; paclitaxel; immunohistochemistry; chemotherapy
Tumor suppressor maspin is a differentially regulated gene in the progression of many types of cancer. While the biological function of maspin in blocking tumor invasion and metastasis is consistent with the loss of maspin expression at the late stage of tumor progression, the differential expression and the biological significance of maspin in early stage of tumor progression appear to be complex and remain to be elucidated. In the current study, we examined the expression of maspin in 84 esophageal squamous cell carcinoma (ESCC) cases (stages I–III) and 55 non-tumor adjacent esophageal tissue specimens by immunohistochemical (IHC) staining. The correlation of maspin with clinicopathological parameters was analyzed. Compared to normal esophageal squamous tissue where 80% (47/55) of the cases expressed maspin at a low to moderate level, all ESCC specimens (100% (84/84)) were positive for maspin expression at a moderate to high level. ESCC with low or moderate maspin expression had significantly shorter postoperative survival rates compared to those that had high maspin expression (p<0.001). Since the correlation of maspin with ESCC histology and the correlation of maspin with ESCC prognosis seem to be at odds, we further investigated the biological function of maspin in ESCC using the established ESCC cell lines. The expression of maspin in five human esophageal squamous cancer cell lines (T12, E450, KYSE150, EC109, and KYSE510) was examined by the Western blot. ESCC cell line KYSE510 that did not express maspin and was stably transfected by maspin cDNA or an empty vector. The resulting transfected cells were characterized in vitro. Maspin expression significantly inhibited cell proliferation, motility and matrigel invasion. Taken together, our data suggest that the transient up-regulation of maspin in the early development of ESCC may be a defense mechanism against further transition towards more malignant phenotypes, ultimately slowing down ESCC tumor progression.
The purpose of this study was to evaluate the association of multi-genotype polymorphisms with the stepwise progression of esophageal squamous cell cancer (ESCC) and the possibility of predicting those at higher risk.
A total of 1,004 subjects were recruited from Feicheng County, China, between Jan. 2004 and Dec. 2007 and examined by endoscopy for esophageal lesions. These subjects included 270 patients with basal cell hyperplasia (BCH), 262 patients with esophageal squamous cell dysplasia (ESCD), 226 patients with ESCC, and 246 controls with Lugol-voiding area but diagnosed as having normal esophageal squamous epithelial cells by histopathology. The genotypes for CYP2E1 G1259C, hOGG1 C326G, MTHFR C677T, MPO G463A, and ALDH2 allele genes were identified in blood samples collected from all participants.
The alleles ALDH2 and MTHFR C677T were critical for determining individual susceptibility to esophageal cancer. Compared to the ALDH 1*1 genotype, the ALDH 2*2 genotype was significantly associated with increased risks of BCH, ESCD, and ESCC. However, the TT genotype of MTHFR C677T only increased the risk of ESCC. Further analysis revealed that the combination of the high-risk genotypes 2*2/1*2 of ALDH 2 and TT/TC of MTHFR C677T increased the risk of BCH by 4.0 fold, of ESCD by 3.7 fold, and ESSC by 8.72 fold. The generalized odds ratio (ORG) of the two combined genotypes was 1.83 (95%CI: 1.55-2.16), indicating a strong genetic association with the risk of carcinogenic progression in the esophagus.
The study demonstrated that the genotypes ALDH2*2 and MTHFR 677TT conferred elevated risk for developing esophageal carcinoma and that the two susceptibility genotypes combined to synergistically increase the risk.
This study was designed to determine the significance of DNA methyltransferases (DNMTs) in DNA hypermethylation in esophageal squamous cell carcinoma (ESCC) and to identify DNA methylation markers in serum for the early diagnosis of ESCC. A promoter methylation profile of 12 tumor-related genes was assessed using methylation-specific PCR in ESCC and paired non-tumor tissue samples from 47 patients. Expression levels of DNMTs were examined by real-time reverse transcription-PCR and immunohistochemistry. Using MethyLight, the methylation status of five genes was analyzed in serum samples from 45 patients and 15 healthy individuals. A total of 46 (97.9%) of 47 ESCC samples showed methylation in at least one of the examined genes, and methylation was most frequent for RAR-β (46.8%), DAPK (46.8%), p16 (44.7%) and CDH1 (42.6%). Methylation of RASSF1A was significantly correlated with the poorly differentiated tumors and the early pathologic tumor classification (p = 0.035 and p = 0.046, respectively). Tumoral DNMT3b mRNA upregulation was significantly correlated with hypermethylation of multiple tumor-related genes (p = 0.021). In addition, hypermethylation of cell-free serum DNA was common in ESCC patients and diagnostic accuracy was increased when methylation of multiple genes (RAR-β, DAPK, CDH1, p16 and RASSF1A) were analyzed in combination (ROC AUC 0.911, 82.2% sensitivity and 100% specificity). The present study suggests that hypermethylation of multiple tumor-related genes may be involved in the pathogenesis of ESCC and mediated by the increase of DNMT3b expression. A cluster of multiple methylated genes in serum DNA has the potential as a novel biomarker for ESCC diagnosis.
hypermethylation; esophageal squamous cell carcinoma; DNA methyltransferase 3b; serum; biomarker; diagnosis
AIM: To characterize cytogenetic alterations in esophageal squamous cell carcinoma (ESCC) and its metastasis.
METHODS: A total of 37 cases of primary ESCC and 15 pairs of primary ESCC tumors and their matched metastatic lymph nodes cases were enrolled from Linzhou, the high incidence area for ESCC in Henan, northern China. The comparative genomic hybridization (CGH) was applied to determine the chromosomal aberrations on the DNA extracted from the frozen ESCC and metastatic lymph node samples from these patients.
RESULTS: CGH showed chromosomal aberrations in all the cases. In 37 cases of primary ESCC, chromosomal profile of DNA copy number was characterized by frequently detected gains at 8q (29/37, 78%), 3q (24/37, 65%), 5p (19/37, 51%); and frequently detected losses at 3p (21/37, 57%), 8p and 9q (14/37, 38%). In 15 pairs of primary ESCC tumors and their matched metastatic lymph node cases, the majority of the chromosomal aberrations in both primary tumor and metastatic lymph node lesions were consistent with the primary ESCC cases, but new candidate regions of interest were also detected. The most significant finding is the gains of chromosome 6p with a minimum high-level amplification region at 6p12-6q12 in 7 metastatic lymph nodes but only in 2 corresponding primary tumors (P = 0.05) and 20p with a minimum high-level amplification region at 20p12 in 11 metastatic lymph nodes but only in 5 corresponding primary tumors (P < 0.05). Another interesting finding is the loss of chromosome 10p and 10q in 8 and 7 metastatic lymph nodes but only in 2 corresponding primary tumors (P < 0.05).
CONCLUSION: Using the CGH technique to detect chromosomal aberrations in both the primary tumor and its metastatic lymph nodes of ESCC, gains of 8q, 3q and 5p and loss of 3p, 8p, 9q and 13q were specifically implicated in ESCC in Linzhou population. Gains of 6p and 20p and loss of 10pq may contribute to the lymph node metastasis of ESCC. These findings suggest that the gains and losses of chromosomal regions may contain ESCC-related oncogenes and tumor suppressor genes and provide important theoretic information for identifying and cloning novel ESCC-related oncogenes and tumor suppressor genes.
Comparative genomic hybridization; Genetic alterations; Esophageal squamous cell carcinoma; Metastatic lymph nodes