Invasive Esophageal Squamous Cell Carcinoma Research Articles

Down-Regulation of PTEN Expression Modulated by Dysregulated miR-21 Contributes to the Progression of Esophageal Cancer

miR-21, a putative tumor oncomiR, is a frequently overexpressed miRNA in a variety of tumors. Because it targets tumor-suppressor genes it has been linked to tumor progression. In this study we investigated the role of miR-21 in esophageal squamous cell carcinoma (ESCC), and its possible mechanism. Expression of miR-21 was detected by stem-loop RT-PCR in tissue from 76 invasive ESCC at stage I-IV and in their corresponding para-cancerous histological normal tissues (PCHNT). Thirty endoscopic esophageal mucosal biopsy specimens from non-tumor patients were used as controls. Expression of PTEN in 76 paired ESCC and PCHNT was investigated by real-time RT-PCR and an immunohistochemical method, respectively. Paired tumor and PCHNT specimens of 20 ESCC cases were randomly selected for western blot analysis. The effect of miR-21 on PTEN expression was assessed in the ESCC cell line with an miR-21 inhibitor to reduce miR-21 expression. Furthermore, the roles of miR-21 in cell biology were analyzed by use of miR-21 inhibitor-transfected cells. Stem-loop RT-PCR revealed miR-21 was significantly overexpressed in ESCC tissues and cell lines. Overexpression of miR-21 correlated with tumor status, lymph node metastasis, and clinical stage. We demonstrated that knockdown of miR-21 significantly increased expression of PTEN protein. Consequent PTEN expression reduced cell proliferation, invasion, and migration. Our findings suggest that miR-21 could be a potential oncomiR, probably by regulation of PTEN, and a novel prognostic factor for ESCC patients.

Squamous dysplasia--the precursor lesion for esophageal squamous cell carcinoma.

Esophageal squamous cell carcinoma (ESCC) accounts for 80% of all esophageal cancers worldwide, and esophageal squamous dysplasia (ESD) is the only histopathology that predicts the development of ESCC. The prevalence of ESD parallels rates of invasive ESCC and is typically found in 25% or more of adults above the age of 35 years in populations in north central China, where risk for ESCC is among the highest in the world. Results of chemoprevention and early detection studies to prevent progression of ESD suggest that these approaches, coupled with emerging endoscopic therapies, offer promise for the prevention of esophageal cancer mortality in high-risk populations. Future research on ESD and ESCC should focus on finding additional modifiable risk factors and on identifying biomarkers to incorporate into early detection strategies.

Abstract C5: Study of the relationship between RAR-beta2 hypermethylation and invasive esophageal squamous cell carcinoma

Abstract Esophageal squamous cell carcinoma (ESCC) is the sixth leading cause of cancer death among men in Taiwan, and the five-year survival rate is below 10%. The retinoic acid receptor beta2 (RAR-beta2) is frequently deleted or silenced at early stages in tumor progression. Most of RAR-beta2 repressions was associated with promoter hypermethylation in several epithelial carcinomas. The aim of this study is to investigate RAR-beta2 promoter methylation status in different stages of ESCC patients. Through methylation-specific PCR analysis, methylation rate of RAR-beta2 was 21.1% (15/71) in tumor cases, but unmethylated in all normal counterparts. In RAR-beta2 promoter hypermethylation cases, the methylation frequency of lymph nodes invasion and tumor metastasis was twofold increased. Moreover, RAR-beta2 promoter methylation frequency was increased with 14.7% and showed significant decrease survival rate in advanced stage. Esophageal cancer cell lines 81T, 81T 1-1 and 81T 1-4 were treated with 5-aza-dC (DNA methylation inhibitor), the level of RAR-beta2 methylation was decreased in all ESCC cell lines. Moreover, migration ability was inhibited in 81T cell line. The migration ability of 81T 1-1 and 81T 1-4 cell lines were decreased about 50%. Our findings indicate that RAR-beta2 hypermethylation in ESCC could be useful for prognosis marker. RAR-beta2 methylation may become one of the clinical therapy targets and invasion-migration markers. Citation Format: Ruei-Siang Wu, Chun-Chieh Yu, Yue-Yuah Li, Ming-Tsang Wu, Ruei-Nian Li. Study of the relationship between RAR-beta2 hypermethylation and invasive esophageal squamous cell carcinoma. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Invasion and Metastasis; Jan 20-23, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;73(3 Suppl):Abstract nr C5.

Study of the relationship between RARβ2 hypermethylation and invasive esophageal squamous cell carcinoma.

23 Background: Esophageal squamous cell carcinoma (ESCC) is the sixth leading cause of cancer death among men in Taiwan, and the five-year survival rate is below 10%. The retinoic acid receptor beta2 (RARβ2) is frequently deleted or silenced at early stages in tumor progression. Most of RARβ2 repressions are associated with promoter hypermethylation in several epithelial carcinomas. In addition, the degree of whole genome methylation decreases as the lesion progresses from benign to invasive cells. The aim of this study is to investigate RARβ2 promoter methylation status and global methylation level in different stages of ESCC patients. Methods: We examined the methylation frequency of ESCC tissues by the method of methylation-specific PCR and pyrosequencing. The RARβ2 protein expression was determined by western blotting and cell mobility was measured by migration assay. Results: The methylation rate of RARβ2 was 21.1% (15/71) in tumor cases, but the normal control was unmethylated. The frequency of lymph nodes invasion and the frequency of tumor metastasis were 2-fold increased in RARβ2 promoter hypermethylated cases compared with normal counterparts. Furthermore, RARβ2 promoter methylation frequency was increased with 14.7% and showed significant decrease survival rate in advanced stage of ESCC patients. The level of LINE-1 methylation in ESCC tissues (mean 67%) was significantly lower than normal counterparts (mean 80%). But the level of LINE-1 methylation was not associated with tumor stage or survival rate. In vitro, ESCC cell line treated with 5-aza-dC was decreased in RARβ2 methylation level and was increased in RARβ2 protein expression. The migration ability of ESCC cell line was inhibited during demethylation of RARβ2 promoter. Conclusions: Our findings indicate that LINE-1 methylation level and RARβ2 hypermethylation could be useful for prognosis markers and invasion-migration markers in ESCC.

Amplification of the telomerase RNA component gene as a new genetic marker for disease progression and prognosis in esophageal squamous cell carcinoma

Amplification of the human telomerase RNA component (TERC) gene was found in esophageal squamous cell carcinoma (ESCC). However, its roles in the progression and prognosis of ESCC have not been well understood. The amplification of TERC in normal mucosa, low-grade and high-grade intraepithelial neoplasia, and invasive ESCC samples were evaluated using a fluorescence in situ hybridization assay. The amplification of TERC invariably occurred in high-grade intraepithelial neoplasia and invasive ESCC, partially occurred in low-grade intraepithelial neoplasia specimens, and seldom occurred in normal mucosa. The average signal ratio of TERC to chromosome 3 centromere-specific probe (TERC/CSP3) was 1.00 ± 0.01 (average ± standard deviation) in normal mucosas, 1.01 ± 0.08 in low-grade intraepithelial neoplasias, 1.39 ± 0.26 in high-grade intraepithelial neoplasias, and 1.56 ± 0.41 in invasive ESCC. High TERC/CSP3 ratio was positively associated with lymph node metastasis (P = 0.005) and advanced tumor stage (P = 0.045). Patients with high amplification of TERC had poor survival (P = 0.01). The amplification of TERC could be used as a new genomic marker for disease progression and prognosis of ESCC. The amplified TERC gene may be a potential therapeutic target for ESCC.

Treatment Strategy for Superficial Pharyngeal Squamous Cell Carcinoma Synchronously Combined with Esophageal Cancer

Background: Esophageal squamous cell carcinoma (ESCC) is often synchronously accompanied by pharyngeal squamous cell carcinoma (PSCC). However, treatment strategies for these synchronous cancers have not been established. Aim: To evaluate retrospectively the effects of both chemoradiotherapy (CRT) targeted for invasive ESCC on synchronous superficial PSCC and additional endoscopic resection (ER) for PSCC. Patients and Methods: Screening endoscopy in the pharynx was performed in newly diagnosed ESCC patients. CRT combined with 5-fluorouracil (5-FU) and cisplatin (CDDP) was administered to all patients. The effect on superficial PSCC was only evaluated for 5-FU-CDDP chemotherapy that excluded the pharynx from the radiation field. When PSCC was remnant or recurrent in patients evaluated at complete response (CR) of ESCC, ER was performed on the PSCC. Results: Fourteen cases of superficial PSCC (4.0%) were detected in 348 ESCC patients. Three PSCC reached CR in 8 ESCC-CR patients, while all 3 lesions recurred. No treatment response was found in the remaining 11 PSCC. As a second treatment, ER for 8 PSCC was completed in the 8 ESCC-CR patients, with one complication due to pneumonia. Conclusions: Standard 5-FU-CDDP CRT targeted for invasive ESCC did not demonstrate a sufficient efficacy for superficial PSCC, while ER even for PSCC after chemotherapy was curative.

Overexpression of the DEC1 Protein Induces Senescence In Vitro and Is Related to Better Survival in Esophageal Squamous Cell Carcinoma

Esophageal squamous cell carcinoma (ESCC) is a leading cause of cancer-related death in China and has limited effective therapeutic options except for early surgery, since the underlying molecular mechanism driving its precursor lesions towards invasive ESCC is not fully understood. Cellular senescence is the state of the permanent growth arrest of a cell, and is considered as the initial barrier of tumor development. Human differentiated embryo chondrocyte expressed gene 1 (Dec1) is an important transcription factor that related to senescence. In this study, DEC1 immunohistochemical analysis was performed on tissue microarray blocks constructed from ESCC combined with adjacent precursor tissues of 241 patients. Compared with normal epithelia, DEC1 expression was significantly increased in intraepithelial neoplasia and DEC1 expression was significantly decreased in ESCC in comparison with intraepithelial neoplasia. In vitro, DEC1 overexpression induced cellular senescence, and it inhibited cell growth and colony formation in ESCC cell line EC9706. Fresh esophagectomy tissue sections from five ESCC patients were detected by immunohistochemistry of DEC1 and senescence-associated β-galactosidase (SA-β-Gal) activity, and strongly positive expression of DEC1 was correlated to more senescent cells in these fresh tissue sections. Kaplan – Meier method analysis of the 241 patients revealed that DEC1 expression levels were significantly correlated with the survival of ESCC patients after surgery. The expression levels of DEC1 were also correlated with age, tumor embolus, depth of invasion of ESCC, lymph metastasis status and pTNMs. These results suggest that DEC1 overexpression in precursor lesions of ESCC is a protective mechanism by inducing cellular senescence in ESCC initiation, and DEC1 may be a potential prognostic marker of ESCC.

Hypermethylation-modulated down-regulation of CDH1 expression contributes to the progression of esophageal cancer

CDH1, a cell adhesion molecule, which plays a key role in maintaining the epithelial phenotype, is regarded as an invasion-suppressor gene in light of accumulating evidence from in vitro experiments and clinical observations. In an attempt to clarify the mechanism responsible for inactivation of this gene in carcinomas, we investigated the methylation status of the CDH1 gene 5'-CpG islands and its regulatory mechanism in the progression of esophageal squamous cell carcinoma. Real-time methylation-specific polymerase chain reaction (qMSP) and treatment with the demethylating agent 5-aza-2'-deoxycytidine (5-Aza-CdR) were conducted to analyze the methylation status at the CDH1 promoter region in the human esophageal carcinoma cell lines, EC1 and EC9706. A total of 235 invasive esophageal squamous cell carcinomas (ESCC) at stages I-IV and their corresponding normal tissue samples, were included in an immunohistochemistry study and methylation analysis of CDH1. The results demonstrate that in EC1 and EC9706 cells, the CDH1 promoter is methylated and treatment with 5-Aza-CdR restored CDH1 expression. Enhanced CDH1 expression decreased cell migration, invasion ability and increased adhesion ability. Decreased CDH1 expression was detected in 59.6% of ESCC tissues, compared with their adjacent non-neoplastic epithelia, which had a close correlation with the primary tumor status, lymph node status, distant metatasis and clinicopathologic stage. Hypermethylation at the CDH1 promoter was detected in 97.9% of 140 cases of ESCC with low CDH1 expression. The methylation of CDH1 promoters (P=0.929) was closely correlated with the lack of expression of their corresponding proteins. The Cox regression model for survival analysis showed that increases in CDH1 methylation had a greater impact on the prognosis than tumor clinical stage. These findings suggest that CDH1 gene silencing by promoter hypermethylation and the resultant reduction of CDH1 expression may play an important role in the progression of ESCC. CDH1 methylation was a significant predictor of survival in ESCC patients after surgery.

Down-regulation of microRNA 10a expression in esophageal squamous cell carcinoma cells

This study identified significantly down-regulated microRNAs (miRs) specific for esophageal squamous cell carcinoma (ESCC) cells. Total RNA was extracted from ESCC cell lines (OE21 and TE10) and a non-malignant human esophageal squamous cell line (Het1A), and subjected to microarray analysis. Expression levels of miRs that showed significant down-regulation in ESCC cells compared to Het1A cells based on the comprehensive analysis were analyzed by quantitative reverse transcription polymerase chain reaction. Among the significantly down-regulated miRs, miR-10a expression levels in the five ESCC cell lines examined were significantly lower than in Het1A and the esophageal adenocarcinoma cells. Since miR-10a is a specific miR in ESCC, its clinical relevance was examined. Using ESCC tumor samples and non-cancerous tissue obtained endoscopically, the involvement of miR-10a in the clinicopathological findings was examined. MiR-10a expression was comparably down-regulated in the tumors of high-grade intraepithelial neoplasm and non-invasive ESCC, while the expression levels were elevated in the invasive ESCC tumors. Treatment with a demethylating agent, 5-aza-2'-deoxycytidine, restored miR-10a expression in OE21 cells. Only a modest additive or synergistic effect was observed in the presence of a histone deacetylase inhibitor, trichostatin A. These results imply that miR-10a may be differentially expressed in ESCC cells and may be involved in ESCC development and progression. The unique epigenetic regulation of miR-10a expression can be mediated via hypermethylation of the CpG islands proximal to its gene locus, at least in certain ESCC cells.

Abstract 1493: High-resolution comparative genomic hybridization of preneoplastic and invasive esophageal squamous cell carcinoma: Identification of the earliest preneoplastic changes

Abstract Background: Esophageal cancer causes > 400,000 deaths/year in the world. Clinically useful early detection methods must identify these cancers and their precursor lesions at more curable stages. We conducted comparative genomic hybridization analyses of preneoplastic and invasive esophageal squamous cell carcinoma to look for gene copy number patterns that might be useful as early detection markers. The current effort focuses on aberrations associated with mild squamous dysplasia, the earliest histologic form of squamous preneoplasia. Methods: DNA was extracted from either biopsies or surgical resections from 122 esophageal tissues spanning a spectrum including normal mucosa, squamous preneoplasia, and invasive squamous cell carcinoma. High-resolution micro-array based comparative genomic hybridization (Agilent) was used to measure regions of chromosomal gain or loss with an emphasis on identifying changes associated with the transition from normal to mild dysplasia. Results: Chromosomal number aberrations were identified that involve several chromosomes, occur early in the neoplastic process, and progress with disease severity. Specific chromosomal number aberrations associated with mild squamous dysplasia, the earliest form of squamous preneoplasia, were uniquely identified. Conclusions: Comparative genomic hybridization identifies patterns of chromosomal number aberrations that may distinguish patients with and without high-grade squamous dysplasia or cancer. The current effort also identifies specific changes associated with mild squamous dysplasia, the earliest form of preneoplasia, and this may help elucidate mechanisms related to the initiation of carcinogenesis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1493.

Abstract B50: Large scale DNA methylation profiling and high-resolution comparative genomic hybridization of preneoplastic and invasive esophageal squamous cell carcinoma: Discovery of early detection markers

Abstract B50 Background Esophageal cancer causes > 350,000 deaths/year in the world. Clinically useful early detection methods must identify these cancers and their precursor lesions at more curable stages. We conducted genome-wide methylation profiling and comparative genomic hybridization analyses of preneoplastic and invasive esophageal squamous cell carcinoma to look for methylation or gene copy number patterns that might be useful as early detection markers. Methods DNA was extracted from esophageal biopsies and surgical resections from 94 esophageal tissues spanning a spectrum from normal to invasive tumor. Bisulfite genotyping for 800 genes (Illumina GoldenGate methylation BeadArray), was used to measure CpG methylation of 1505 targets. High-resolution microarray based comparative genomic hybridization (Agilent) was used to measure regions of chromosomal gain or loss. Results Analysis of methylation beadarray data identified targets that are differentially methylated between normal and high-grade squamous dysplasia. Chromosome number aberrations were identified that involve several chromosomes, occur early in the neoplastic process, and progress with disease severity. Conclusions Genome-wide methylation profiling and comparative genomic hybridization identify patterns of gene methylation and chromosome number aberrations, respectively, that may distinguish patients with and without high-grade squamous dysplasia or cancer. Citation Information: Cancer Prev Res 2008;1(7 Suppl):B50.

COX-2 ( PTGS2) gene methylation in epithelial, subepithelial lymphocyte and stromal tissue compartments in a spectrum of esophageal squamous neoplasia

Background: Previous studies have shown important effects of stromal elements in carcinogenesis. To explore the tumor–stromal relationship in esophageal neoplasia, we examined methylation of COX-2 ( PTGS2), a gene etiologically associated with the development of gastrointestinal cancers, in adjacent foci of epithelium, subepithelial lymphocytes and non-lymphocytic stromal cells found in sections of normal squamous epithelium, squamous dysplasia and invasive esophageal squamous cell carcinoma. Methods: Adjacent foci of epithelium, subepithelial lymphocytic aggregates and non-lymphocytic stromal tissues were laser microdissected from six fully embedded, ethanol fixed, esophagectomy samples from Shanxi, China, a high-risk region for esophageal cancer. Promoter CpG site-specific hypermethylation status of COX-2 was determined using real-time methylation-specific PCR (qMS-PCR) based on Taqman Chemistry. The methylation status of a subset of samples was confirmed by pyrosequencing. Results: Forty-nine microdissected foci were analyzed. COX-2 gene methylation was significantly more common in subepithelial lymphocytes (12/16 (75% of all foci)) than in epithelial foci (3/16 (19%)) or foci of non-lymphocytic stromal tissues (3/17 (18%)) (Fisher's exact p = 0.05). Two of three epithelial samples and all three stromal samples that showed COX-2 methylation were adjacent to foci of methylated subepithelial lymphocytes. Pyrosequencing confirmed the methylation status in a subset of samples. Conclusions: In these esopohageal cancer patients, COX-2 gene methylation was more common in subepithelial lymphocytes than in adjacent epithelial or stromal cells in both grades of dysplasia and in foci of invasive cancer. These findings raise the possibility that methylation of subepithelial lymphocytes may be important for tumorigenesis. Future studies of gene methylation should consider separate evaluation of epithelial and non-epithelial cell populations.

An expression of squamous cell carcinoma antigen 2 in peripheral blood within the different stages of esophageal carcinogenesis

The malignant transformation of esophageal mucosa is a progressive process, which includes basal cell hyperplasia, dysplasia, carcinoma in situ, and invasive esophageal squamous cell carcinoma (ESCC). The objectives of this study were to prove the relationship of squamous cell carcinoma antigen 2 (SCCA2) mRNA expression in peripheral blood with non-malignant lesion, premalignant lesion, and carcinoma of the esophagus at the same assay, as well as to evaluate whether or not SCCA2 mRNA expression in peripheral blood may be a biomarker for monitoring the premalignant lesion of the disease. The subjects consisted of 50 patients with basal cell hyperplasia, 50 patients with dysplasia, 50 patients with ESCC (12 carcinoma in situ, 38 carcinoma in invasive stage), and 50 controls who were pathologically diagnosed to be normal and whose esophageal mucosa were stained brown by iodine. All the subjects are residents of Feicheng, China, which is considered an area with a high incidence of esophageal cancer. All subjects were diagnosed by two separate histopathologists, and the expression of SCCA2 mRNA in peripheral blood was detected by semiquantitative reverse transcription-polymerase chain reaction (RT-PCR). Furthermore, SCCA2 concentration in the serum was measured using an enzyme-linked immunosorbent assay (ELISA). In the cancer group, SCCA2 mRNA expression was also detected in 20 tissues of esophageal cancer. By using the band intensity ratios of SCCA2 to beta-actin, with a positive cut-off value of > or = 0.4, the positive rates of the SCCA2 mRNA expression in peripheral blood were found to be 82% (41/50), 60% (30/50), 48% (24/50), and 36% (18/50) in the cancer, dysplasia, basal cell hyperplasia, and control groups, respectively. The positive rate of the cancer group was significantly different from the three other groups (P < 0.05), and there was also a significant difference in the SCCA2 mRNA expression between the dysplasia group and the control group (chi(2)=5.769, P= 0.016). In the multinomial logistic regression analysis, the odds ratios (ORs) were 1.71 [95% confidence interval (95% CI), 0.73-3.99] in the basal cell hyperplasia group, 2.77 (95% CI, 1.14-6.71) in the dysplasia group, and 7.87 (95% CI, 2.88-21.55) in the cancer group after being adjusted for age, gender, smoking index, drinking index, and family history of esophageal cancer. The SCCA2 mRNA expression in peripheral blood was then divided into different grades according to the band intensity ratios of SCCA2 to beta-actin. By using a positive cut-off value of > or = 0.4, the testing sensitivities in the basal cell hyperplasia, dysplasia, and cancer groups were found to be 48%, 60%, and 82%, respectively, with the same testing specificity at 64%. On the other hand, SCCA2 mRNA expression in peripheral blood had a 97.5% agreement with that in tissue, and there was a significant correlation between the ELISA SCCA2 levels in the serum and the SCCA2 mRNA expression levels in the peripheral blood (r= 0.80, P= 0.01). The results indicate that SCCA2 mRNA expression in peripheral blood is linked with the different stages of esophageal pathological changes, despite the fact that SCCA2 mRNA was not a biomarker for screening early esophageal cancer. This knowledge may be useful in monitoring the processes of change that occur in esophageal premalignant lesions among subjects who live in a high-incidence area.

No association between HPV infection and the neoplastic progression of esophageal squamous cell carcinoma: Result from a cross‐sectional study in a high‐risk region of China

Esophageal cancer is a leading cause of cancer death, especially in developing countries. In high-risk regions, squamous cell carcinoma is the most common type of esophageal cancer, and its etiology remains poorly understood. The purpose of this study was to evaluate the association between human papillomavirus (HPV) infection and esophageal squamous cell carcinoma (ESCC) and related precursor lesions in a high-risk area of China. We conducted a cross-sectional study among adult inhabitants of Linxian, China. All subjects were interviewed about potential risk factors, had the length of their esophagus sampled by a balloon cytology examination and underwent endoscopy with mucosal iodine staining and biopsy of all unstained lesions. A multivalent HPV hybridization probe, Digene Hybrid Capture II (Gaithersburg, MD), which recognizes high-risk types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59 and 68, was used to determine the HPV infection status of the cytologic specimens, and the endoscopic biopsies were used to classify each subject's esophageal disease. 740 subjects completed the cytologic and endoscopic exams, and 702 had adequate cytologic and biopsy specimens. Using a cutpoint of > or =3.0 pg/ml of HPV DNA to define a positive test, HPV positivity was identified in 13% (61/475) of subjects without squamous dysplasia, 8% (8/102) with mild dysplasia, 7% (6/83) with moderate dysplasia, 16% (6/38) with severe dysplasia and zero (0/4) with invasive ESCC. Changing the cutpoint defining a positive test did not change the association of HPV infection and dysplasia grade. In this high-risk population, infection of esophageal cells with high-risk HPV types occurs in 13% of asymptomatic adults with no evidence of squamous dysplasia and a similar proportion of individuals with mild, moderate or severe dysplasia. This suggests that HPV infection is not a major risk factor for ESCC in this high-risk Chinese population. Further studies are warranted to determine if infection with this agent is associated with neoplastic progression in a subset of cases.

β‐Catenin splice variants and downstream targets as markers for neoplastic progression of esophageal cancer

This study characterizes the frequency of exon 3 CTNNB1 mutations and compares the expression of CTNNB1 transcript variants and downstream targets MYC and WAF1 (p21) across the neoplastic progression of esophageal squamous cell carcinomas (ESCCs). Mutational analysis was performed on 56 tumors and corresponding germline DNA, using primers to exon 3 of CTNNB1 and SSCP DNA sequencing gels. Quantitative Real Time RT-PCR was performed on 45 foci representing the histological spectrum from normal to invasive cancer, using specific primer sets for alternative splice variants that differ by the presence (16A) or absence (16B) of a 159-bp noncoding segment of exon 16 of CTNNB1, in conjunction with downstream targets MYC and WAF1. Two unique mutations were identified, S37F in the SxxxS repeat region, and a germline polymorphism, T59A. Thus, mutation of CTNNB1 exon 3 is a rare event in this population. RT-PCR analysis successfully confirmed the presence of both beta-catenin splice variants in histologically normal and preneoplastic squamous epithelium, and invasive tumors of the esophagus, and identified a significant reduction in the 16A/16B ratio (P = 0.014) and an accompanying significant increase in the MYC/WAF1 expression ratio (P = 0.001) with progression from normal mucosa to dysplasia. This represents the first identification of two CTNNB1 transcripts in histologically "normal" esophageal squamous cells, squamous dysplasia, and invasive ESCC. These results show an increase in the minor mRNA (16B) isoform and changes in the expression of downstream markers consistent with increased transcription during the histological progression from normal to squamous dysplasia.

Differential protein expression between esophageal squamous cell carcinoma and dysplasia, and prognostic significance of protein markers

The aim of the current study was to evaluate the protein expression involved in the progression from dysplasia to invasive esophageal squamous cell carcinomas and to analyze the prognostic value of markers. Immunohistochemistry was performed for cell cycle regulators [p53, p21, p27, p16, cyclin D1, Rb], apoptosis-related proteins [Fas, Fas-L, FADD, TRAIL, DR4, DR5, caspase-8, caspase-3, bcl-2, Bax], tumor suppressor proteins [ β-catenin, E-cadherin, FHIT, Smad 4, VHL, PTEN, KAI-1], and oncoproteins [c-myc, COX-2, EGFR]. Caspase-3, TRAIL, Fas-L, Fas, Smad 4, VHL, E-cadherin, and EGFR revealed significant differences between dysplasia and their corresponding invasive cancer portion in 25 cases. In a total of 118 cases of invasive cancer, proteins with frequent (⩾60% of the cases) alterations were p53 (overexpression in 64% of SCCs), p27 (loss in 91%), p16 (loss in 81%), and FHIT (loss in 75%). Early clinical stage and bcl-2 immunopositivity were related to the survival rate of patients. In conclusion, caspase-3, TRAIL, Fas-L, Fas, Smad 4, VHL, E-cadherin, and EGFR may be involved in the progression from dysplasia to invasive esophageal SCCs. Clinical stage and bcl-2 are independent prognostic factors throughout the multivariate analysis.

Squamous dysplasia is the histologic precursor of invasive esophageal squamous cell carcinoma: results from a 13-year prospective follow-up study in a high-risk population

Squamous dysplasia is the histologic precursor of invasive esophageal squamous cell carcinoma: results from a 13-year prospective follow-up study in a high-risk population

Reduced Fhit Expression Occurs in the Early Stage of Esophageal Tumorigenesis: No Correlation with p53 Expression and Apoptosis

The FHIT gene, encompassing the FRA3B fragile site at chromosome 3p14.2, is a candidate tumor suppressor gene involved in multiple tumors, including esophageal carcinoma. We analyzed Fhit expression using an immunohistochemical method in invasive carcinoma, carcinoma in situ (CIS) and dysplasia, in paraffin sections of 75 esophageal squamous cell carcinomas (ESCs) to further elucidate the role of Fhit protein in esophageal carcinogenesis. In addition, we also examined whether Fhit expression correlated with p53 expression and apoptosis. Compared to adjacent normal mucosa, significant loss or reduction of Fhit expression was noted in 67 of 75 (89.3%) invasive ESCs, in 13 of 19 (68.4%) CIS lesions, and in 10 of 23 (43.5%) dysplastic lesions. There was a progressive loss or reduction of Fhit expression with progressive increases in the severity of histopathological changes (p < 0.001). However, there was no association between Fhit expression and clinicopathological findings, including tumor stage, lymph node metastasis, or overall survival. Moreover, Fhit expression was not significantly associated with p53 expression and apoptosis. These results indicate that abnormal Fhit expression is a common event in the early stage of ESC development and may occur independently of p53 expression and apoptosis mechanisms.