Normal Esophageal Mucosa Samples Research Articles

Paired box 5 increases the chemosensitivity of esophageal squamous cell cancer cells by promoting p53 signaling activity.

Background:Gene promoter methylation is a major epigenetic change in cancers, which plays critical roles in carcinogenesis. As a crucial regulator in the early stages of B-cell differentiation and embryonic neurodevelopment, the paired box 5 (PAX5) gene is downregulated by methylation in several kinds of tumors and the role of this downregulation in esophageal squamous cell carcinoma (ESCC) pathogenesis remains unclear.Methods:To elucidate the role of PAX5 in ESCC, eight ESCC cell lines, 51 primary ESCC tissue samples, and eight normal esophageal mucosa samples were studied and The Cancer Genome Atlas (TCGA) was queried. PAX5 expression was examined by reverse transcription-polymerase chain reaction and western blotting. Cell apoptosis, proliferation, and chemosensitivity were detected by flow cytometry, colony formation assays, and 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide assays in ESCC cell lines with PAX5 overexpression or silencing. Tumor xenograft models were established for in vivo verification.Results:PAX5 methylation was found in 37.3% (19/51) of primary ESCC samples, which was significantly associated with age (P = 0.007) and tumor-node-metastasis stage (P = 0.014). TCGA data analysis indicated that PAX5 expression was inversely correlated with promoter region methylation (r = −0.189, P = 0.011 for cg00464519 and r = −0.228, P = 0.002 for cg02538199). Restoration of PAX5 expression suppressed cell proliferation, promoted apoptosis, and inhibited tumor growth of ESCC cell lines, which was verified in xenografted mice. Ectopic PAX5 expression significantly increased p53 reporter luciferase activity and increased p53 messenger RNA and protein levels. A direct interaction of PAX5 with the p53 promoter region was confirmed by chromatin immunoprecipitation assays. Re-expression of PAX5 sensitized ESCC cell lines KYSE150 and KYSE30 to fluorouracil and docetaxel. Silencing of PAX5 induced resistance of KYSE450 cells to these drugs.Conclusions:As a tumor suppressor gene regulated by promoter region methylation in human ESCC, PAX5 inhibits proliferation, promotes apoptosis, and induces activation of p53 signaling. PAX5 may serve as a chemosensitive marker of ESCC.

Quantitative proteomic profiling of esophageal adenocarcinoma tumors to assess prevalence of approved targets and elucidate novel biomarkers.

343 Background: Esophageal adenocarcinoma (EAC) continues to have extremely poor prognosis despite advances in surgical and oncological regimens. Targeted and immune therapy hold promise to improve outcomes in solid tumors including EAC. At present HER-2 and anti-PD-L1 therapies are approved by the FDA for EAC. Additionally, off-label use of EGFR directed therapy has been reported. The aim of this study is to measure expression of 80+ oncoproteins in EAC using multiplexed mass spectrometry to elucidate expression of currently approved-drug targets and explore promising new targets for drug development. Methods: After Institutional Review Board approval (Mayo Clinic and KUMC), EAC patients whose tumors were resected via esophagectomy before chemotherapy and/or radiation were identified. Normal esophageal mucosa samples with no history of EAC were also retrieved for analysis. Tissue sections were microdissected to exclude stroma from adenocarcinoma cells. Isolated EAC cells were solubilized for mass spectrometry-based quantitation of more than 80 clinically relevant tumor markers. Results: 55 EAC tissues were analyzed. HER2, PD-L1 and EGFR were overexpressed only in 16.3%, 0%, and 0% of patients, respectively. New oncoproteins #2 and #6 were noted to have high (7,634.0 and 27,222.6 attomoles/ug) and consistent (98.1% and 94.5%) overexpression in EAC samples. Expression of HER2, oncoproteins #2 and #6 were significantly higher in EAC compared to normal esophageal mucosa. There was no statistical difference in the expression of PD-L1 and EGFR between EAC and normal samples. Conclusions: Mass spectrometry revealed a very low prevalence of target oncoproteins in EAC for currently used drugs. Our study potentially reveals alternate markers which are near universally present at high levels and should be further explored for targeted treatment.[Table: see text]

STLA101 assay for the detection of cancerous progression in Barrett’s esophagus: A multi-institutional study.

249 Background: Since 1975, Esophageal adenocarcinoma (EAC) incidence has increased more than any other cancer (̃700%). Pre-malignant metaplastic tissue, or Barrett's Esophagus (BE), is the only known precursor for EAC. Periodic histological assessment of endoscopic biopsy along dysplasia-carcinoma sequence is used to assess progression risk. Current surveillance protocols have clinical limitations as 80% of patients still present with stage II-IV EAC. The aim of this study is to compare novel proteomic expression patterns to distinguish stable BE patients from those who developed EAC using the STLA101 assay. Methods: After Institutional Review Board approval patients with BE/GERD were identified. Two cohorts were selected: progressors (progressed to EAC at subsequent interval of 0.5-3 years) and non–progressors (̃10 yrs of f/u surveillance without progression along dysplasia/carcinoma path). 24 normal esophageal mucosa samples with no h/o of BE were also retrieved for baseline expression levels. Tissue sections were microdissected to isolate pure BE cells from surrounding stromal cells. BE cells underwent a multi-step process where tissue was heated and digested to produce a liquid biopsy for mass spec. Isotopically labeled control peptides representing the STLA101 panel were spiked in with digested tissue samples to quantify all of the markers simultaneously using a Triple-quad mass spectrometer. Results: A total of 95 tissues were analyzed. 29 BE biopsies progressed to EAC within a mean of 380 days while the remaining 28 BE and 14 GERD specimens did not progress over a 10-yr period. All 8 biomarkers were expressed at a higher level in BE tissue that progressed to cancer when compared to BE/GERD tissue that remained stable over 10 years. The assay had a 100% technical success rate as no samples yielded “non-detectable” data points. Conclusions: The differential overexpression of the STLA101 panel on progressive and non-progressive BE tissues demonstrates the clinical utility of STLA101 as a predictive biomarker panel for early detection of esophageal carcinogenesis. Non-progressive BE tissues were similar to normal mucosa, therefore, patients whose BE tissue samples resemble this profile may receive a reduced screening cadence. On the other hand, patients with overexpression of the STLA101 panel may be eligible to receive more frequent screening or therapeutic intervention.

S431 Proteomic Assay for Barrett’s Esophagus Progression: A Multi-Institutional Retrospective Study

Introduction: Incidence of Esophageal adenocarcinoma (EAC) in the US has increased at a greater rate than any other cancer. EAC arises from pre-malignant metaplastic tissue - Barrett’s Esophagus (BE). Periodic histological assessment of an endoscopic biopsy is used to assess progression along dysplasia-carcinoma sequence. Current surveillance protocols have clinical limitations as 80% of EAC patients present in advanced cancer stages. The aim of this study is to compare 8 proteomic expression patterns to distinguish stable BE patients from those who developed EAC using the STLA101 assay. Methods: After Institutional Review Board approval (Mayo Clinic and KUMC) patients with BE were identified. 2 cohorts were selected: progressors (progressed to EAC at subsequent interval of 0.5-3 years) and non–progressors (who had 10 yrs of f/u surveillance without progression along dysplasia/carcinoma path). 11 normal esophageal mucosa samples were also retrieved for analysis. Serial tissue sections were microdissected to reduce proteomic noise from surrounding stromal cells. Isolated cells underwent a multi-step process where cross-links were reversed, and proteins were digested and reduced into a mass spec-compatible lysate. Isotopically labeled control peptides of the STLA101 panel were spiked in with digested tissue samples to quantify all of the markers simultaneously using a Q-Exactive HFX mass spectrometer in a CAP/CLIA setting. Results: A total of 42 tissues were analyzed. Cohort tissue descriptions can be found in Table 1. 13 BEs progressed to EAC within a mean of 334 days while the remaining 18 BEs did not progress over a 10-yr period. All 8 biomarkers were expressed at a higher level when comparing dysplastic BE tissue that progressed to cancer to BE tissue that remained stable over 10 yrs and normal esophageal mucosa. There was not a significant difference in age, sex and length of BE between the groups. The assay had a 100% technical success rate as no samples yielded “non-detectable” data points. Conclusion: The overexpression of the STLA101 panel in dysplastic BE tissue that progressed to cancer demonstrates the potential predictability of the assay when assessing molecular hallmarks of carcinogenesis. The expression quantities of the non-progressive BE tissues were closer to normal mucosa and may warrant patients with this profile type to be screened less. Patients with high expression quantities of the STLA101 panel may be candidates for a more frequent screening cadence or therapeutic intervention.Figure 1.: Mass spectrometry-based expression patterns of 2 markers in the STLA101 panel demonstrating clear overexpression of proteins involved in the malignant transformation of Barrett’s precancerous tissue into esophageal adenocarcinoma. The expression mean of non-diseased esophageal squamous mucosa, labeled as “normal”, are represented by blue bars. The green bars represent non-progressive BE tissue that remained stable for over 10-years according to collaborating institutional databases. The red bars represent progressive BE tissue retrieved at a mean of 334 days before adenocarcinoma diagnosis.Table 1.: Specimen types analyzed in this study

Expression of ADAM29 and FAM135B in the pathological evolution from normal esophageal epithelium to esophageal cancer: Their differences and clinical significance.

A Disintegrin And Metalloprotease Domain 29 (ADAM29) and Family with sequence similarity 135 member B (FAM135B) genes have been reported to be associated with a carcinogenic risk of esophageal squamous cell carcinoma (ESCC). However, to the best of our knowledge, the expression of ADAM29 and FAM135B in the pathological evolution from normal esophageal epithelial cells to ESCC has not yet been investigated. The present study aimed to investigate the expression of ADAM29 and FAM135B in normal esophageal mucosal epithelium, low-grade and high-grade esophageal intraepithelial neoplasia, and ESCC. Furthermore, the present study aimed to investigate the role of ADAM29 and FAM135B in the development of esophageal lesions. Immunohistochemistry was performed in order to detect the expression levels of ADAM29 and FAM135B proteins in normal esophageal mucosa samples (40 cases), low-grade intraepithelial neoplasia samples (20 cases), high-grade intraepithelial neoplasia samples (20 cases) and ESCC samples (40 cases). The results of the present study demonstrated that the positive rates of ADAM29 and FAM135B proteins increased gradually from normal esophageal mucosal epithelium and esophageal intraepithelial neoplasia, to ESCC (P<0.05). Furthermore, the expression levels of ADAM29 and FAM135B proteins in ESCC were not associated with age and the tumor size (P>0.05); however, the protein levels were associated with the pathological stage, clinical stage and lymph node metastasis of ESCC (P<0.05). In addition, there was a significant association between the expression levels of ADAM29 protein and FAM135B protein (χ2=60.071; P<0.001). The results of the present study demonstrated that the expression levels of ADAM29 and FAM135B were associated with the tumor behavior characteristics and the progression of esophageal cancer, the expression of which could be used for the diagnosis of early esophageal cancer, and provide the basis for guiding individualized treatment.

Identification of crucial genes associated with esophageal squamous cell carcinoma by gene expression profile analysis.

To uncover the genes associated with the development of esophageal squamous cell carcinoma (ESCC), an ESCC microarray dataset was used to identify genes differentially expressed between ESCC and normal control tissues. The dataset GSE17351 was downloaded from the Gene Expression Omnibus, containing 5 tumor esophageal mucosa samples and 5 adjacent normal esophageal mucosa samples from 5 male patients with ESCC. The differentially expressed genes (DEGs) were identified using the Linear Models for Microarray Data R package. Then, a co-expression network was constructed using the Weighted Correlation Network Analysis (WGCNA) package, and co-expression network modules were obtained with a hierarchical clustering algorithm. Additionally, functional enrichment analyses for DEGs in the top 2 modules with the highest significance were respectively conducted using the WGCNA package and the cluster Profiler package. In total, 487 upregulated and 468 downregulated DEGs were identified. A total of 24 modules were obtained from the co-expression network, and the top 2 modules with the highest significance, designated as ‘blue4’ and ‘magenta’, were further analyzed. In the module blue4, DEGs were significantly enriched in a number of Gene Ontology terms, including ‘spindle organization’ [e.g., ubiquitin conjugating enzyme E2 C (UBE2C) and SAC3 domain containing 1] and ‘cell cycle process’ [e.g., UBE2C, minichromosome maintenance complex component 6 (MCM6) and cell division cycle 20 (CDC20)]. Furthermore, a number of DEGs (e.g., UBE2C, CDC20 and MCM6) were enriched in the ‘cell cycle’ and ‘ubiquitin mediated proteolysis’ pathways. In the module ‘magenta’, a number of DEGs [e.g., transferrin receptor (TFRC) and TEA domain transcription factor 4 (TEAD4)] were enriched in the primary metabolic process and intracellular membrane-bounded organelle. Additionally, 308 upregulated genes and 215 downregulated genes were differentially expressed in the same pattern in another dataset, GSE20347, including UBE2C, CDC20, MCM6, TFRC, TEAD4, protein phosphatase 1 regulatory subunit 3C and MAL, T-cell differentiation protein. These DEGs may function in the development of ESCC.

Inactivation of RUNX3 predicts poor prognosis in esophageal squamous cell carcinoma after Ivor-Lewis esophagectomy.

The inactivation of RUNX3 in various cancers has been reported while the expression of RUNX3 on protein level in esophageal squamous cell carcinoma (ESCC) and its relationship with pathological parameters and prognosis still remained unclear. In this study, we examined the expression of RUNX3 in 158 ESCC samples and 20 normal esophageal mucosa samples by immunohistochemistry and qRT-PCR. The IHC result showed that RUNX3 was detected mainly in the nuclei of basal layer cells in 18 of 20 normal mucosa samples while in 158 ESCC samples, there were 46 with RUNX3 nuclei expression, 37 RUNX3 cytoplasmic expression, and 75 negative expression. The qRT-PCR confirmed the downregulation of RUNX3 mRNA in the RUNX3 protein negative group than in the RUNX3 nuclei and cytoplasmic expression group (P < 0.001), and the methylation-specific PCR showed a low methylation rate in the ESCC tissue samples with RUNX3 protein negative expression (6/40, 15%). The RUNX3 nuclei expression negatively correlated with the lymph node metastasis (P = 0.033) and recurrence status (P = 0.019), and the survival analysis showed that the patients with RUNX3 nuclei expression had a higher 5-year survival rate than the patients with RUNX3 cytoplasmic/negative expression (P = 0.022). The Cox regression analysis showed that the T classification (P = 0.001), lymph node metastasis (P < 0.001), and RUNX3 inactivation (negative/cytoplasmic expression, P = 0.039) were independent risk factor of poor prognosis. In conclusion, we found a frequent inactivation of RUNX3 due to low expression and cytoplasmic dislocalization in ESCC. The inactivation of RUNX3 may be involved in the progression of ESCC, and RUNX3 could be an indicator of prognosis for patients with ESCC after surgery.

Human papillomavirus type 16 infection may be involved in esophageal squamous cell carcinoma carcinogenesis in Chinese Kazakh patients

The aim of this study was to investigate human papillomavirus type 16 (HPV16) prevalence in esophageal squamous cell carcinoma (ESCC) in Xinjiang Kazakh patients and its role in ESCC carcinogenesis. One hundred and fifty cases of ESCC and 150 cases of corresponding normal esophageal mucosa (CNGM) samples were collected from north Xinjiang where the Kazakh ethnic group has lived since ancient times. HPV16 infection in ESCC and CNGM was detected by genotype-specific polymerase chain reaction. HPV16 DNA was detected in 55 of 150 ESCC samples (36.7%) and 24 of 150 corresponding normal esophageal mucosa samples (16%) with significant differences (P < 0.001, odds ratio = 3.039, 95% confidence interval: 1.756-5.260). No statistically significant correlations were found between HPV16 infection and the age or gender of patients, tumor site, tumor cell differentiation, or lymph node metastasis (P > 0.05). HPV16 infection is common in cases of ESCC in the Kazakh ethnic group in Xinjiang and may be involved in ESCC carcinogenesis.

Inhibition of SOX17 by MicroRNA 141 and Methylation Activates the WNT Signaling Pathway in Esophageal Cancer

In this study, we explored the possibility of SOX17 promoter region methylation as an esophageal cancer detection marker, the regulation of SOX17 expression, and the function of SOX17 in the WNT signaling pathway in esophageal cancer. Eight esophageal cancer cell lines, 9 normal esophageal mucosa samples, 60 cases of dysplasia, and 169 cancer tissue samples were included. Methylation-specific PCR, semiquantitative reverse transcription-PCR, immunohistochemistry, luciferase reporter assay, colony formation, and Western blot analysis were used to analyze methylation and function of SOX17 in esophageal cancer. MicroRNA-related detection methods were performed to evaluate microRNA regulation of SOX17. SOX17 methylation was found in progression tendency with 0% of normal mucosa, 39% of grade 1 dysplasia, 48% of grades 2 and 3 dysplasia, and 65% of primary cancer. SOX17 methylation is related to esophageal cancer patients' history of alcohol use and may induce β-catenin expression and redistribution. Loss of SOX17 expression is correlated to promoter region hypermethylation, and re-expression was activated by 5-aza-2'-deoxycytidine treatment in esophageal cancer cell lines. Restoration of SOX17 expression suppresses TCF/β-catenin-dependent transcription and colony formation. MicroRNA 141 was also found to down-regulate SOX17 expression and activate the WNT signal pathway. SOX17 is frequently methylated in esophageal cancer and in a progression tendency during esophageal carcinogenesis. Loss of SOX17 removes the normal inhibition of WNT signaling and promotes esophageal tumorigenesis.

LINE-1 Hypomethylation in Noncancerous Esophageal Mucosae is Associated with Smoking History

Cancer cells exhibit two types of DNA methylation alterations: global DNA hypomethylation and site-specific CpG island promoter hypermethylation. Selected gene promoter hypermethylation in normal esophageal mucosae has attracted attention as a surrogate marker for the epigenetic field defect induced by smoking and/or drinking in esophageal squamous cell carcinoma (ESCC). However, the significance of global DNA hypomethylation for field cancerization remains unclear. By using histologically normal esophageal mucosa samples from 109 ESCC cases and 20 autopsy cases without ESCCs, we measured long interspersed nucleotide element 1 (LINE-1) methylation levels by pyrosequencing, which correlates with global DNA methylation level. LINE-1 methylation levels in normal esophageal mucosae of ESCC patients were significantly lower than those of autopsy individuals (P = 0.017). LINE-1 methylation of noncancerous mucosae ranged 68.3-93.0 on a 0-100 scale (mean 81.7, median 82.2, standard deviation 5.9). LINE-1 hypomethylation was significantly associated with smoking history (P = 0.014 for smoking duration; P = 0.0017 for number of cigarettes per day; P = 0.0002 for tobacco pack-year). LINE-1 methylation was not associated with alcohol drinking or age at diagnosis. A history of tobacco use was significantly associated with LINE-1 hypomethylation in noncancerous esophageal mucosae of ESCC patients. These results support the potential of LINE-1 methylation level as an indicator of epigenetic field for ESCC cancerization, particularly when caused by tobacco smoking.

M1974 Clinicopathological Characteristics of Barrett's Esophageal Cancer in Japanese Patients and Long-Term Results of Therapy

Background: Human papillomavirus (HPV) is increasingly recognized as an important etiological factor of esophageal cancer. However, its prevalence is highly variable between different geographic areas, and is also dependent on the different diagnostic tests such as in situ hybridization, southern blot, and polymerase chain reaction (PCR). Aim: To determine the prevalence of HPV in a selected population of esophageal cancer in Catalonia, Spain. Patients and Methods: Consecutive patients diagnosed with esophageal cancer were selected for the study between February 2007 and September 2008. Paired endoscopic biopsy samples were obtained from normal mucosa and tumour and collected in RNA later. Extracted DNA was amplified using F-HPV typingTM kit PCR (Molgentix, Spain) with a set of 24 fluorescently labelled primers recognizing HPV types 6, 11, 16, 18, 26, 31, 33, 35, 39, 42, 43 , 44, 45, 51, 52, 53, 56, 58, 59, 66, 68, 70, 73 and 82. Results: Twenty patients (mean age: 62 ±11, 16 M/ 4 F) diagnosed with 11 squamous cell carcinoma and 9 with esophageal adenocarcinoma participated in the study. HPV was detected in 6 out of 11 patients (54.5 %) with squamous cell carcinoma and in 1 of 9 patients (11.1%) with adenocarcinoma of esophagus. Medium risk HPV types (53, 70, 73) were detected in 5 out of 7 patients in both tumour and the surrounding normal mucosa. Multi-infection with several medium risk HPV types was found in 4 (57%) of them. Low risk VPH 42 was also present in 5/7 (71%) of cancer samples, three in combination with medium risk VPH, one isolated, and the remaining sample had medium risk HPV 73 in the surrounding normal mucosa. Conclusions: HPV is present in 54% of cases of squamous cell carcinoma in our series. The presence of several medium risk HPV is common in both tumor and normal esophageal mucosa samples from these patients.

M1973 High Prevalence of Human Papillomavirus in a Spanish Population with Esophageal Cancer

Background: Human papillomavirus (HPV) is increasingly recognized as an important etiological factor of esophageal cancer. However, its prevalence is highly variable between different geographic areas, and is also dependent on the different diagnostic tests such as in situ hybridization, southern blot, and polymerase chain reaction (PCR). Aim: To determine the prevalence of HPV in a selected population of esophageal cancer in Catalonia, Spain. Patients and Methods: Consecutive patients diagnosed with esophageal cancer were selected for the study between February 2007 and September 2008. Paired endoscopic biopsy samples were obtained from normal mucosa and tumour and collected in RNA later. Extracted DNA was amplified using F-HPV typingTM kit PCR (Molgentix, Spain) with a set of 24 fluorescently labelled primers recognizing HPV types 6, 11, 16, 18, 26, 31, 33, 35, 39, 42, 43 , 44, 45, 51, 52, 53, 56, 58, 59, 66, 68, 70, 73 and 82. Results: Twenty patients (mean age: 62 ±11, 16 M/ 4 F) diagnosed with 11 squamous cell carcinoma and 9 with esophageal adenocarcinoma participated in the study. HPV was detected in 6 out of 11 patients (54.5 %) with squamous cell carcinoma and in 1 of 9 patients (11.1%) with adenocarcinoma of esophagus. Medium risk HPV types (53, 70, 73) were detected in 5 out of 7 patients in both tumour and the surrounding normal mucosa. Multi-infection with several medium risk HPV types was found in 4 (57%) of them. Low risk VPH 42 was also present in 5/7 (71%) of cancer samples, three in combination with medium risk VPH, one isolated, and the remaining sample had medium risk HPV 73 in the surrounding normal mucosa. Conclusions: HPV is present in 54% of cases of squamous cell carcinoma in our series. The presence of several medium risk HPV is common in both tumor and normal esophageal mucosa samples from these patients.

Silencing of MGMT expression by promoter hypermethylation in the metaplasia–dysplasia–carcinoma sequence of Barrett’s esophagus

To determine the relevance of MGMT in Barrett’s carcinogenesis, we analyzed promotor hypermethylation and expression of MGMT in Barrett’s adenocarcinomas and its paired precursor lesions from 133 patients using a methylation-specific PCR, real-time RT-PCR and immunohistochemistry. Hypermethylation was detected in 78.9% of esophageal adenocarcinomas, in 100% of Barrett’s intraepithelial neoplasia, in 88.9% of Barrett’s metaplasia, but only in 21.4% of normal esophageal mucosa samples ( P < 0.001) and correlated significantly with downregulation of MGMT transcripts ( P = 0.048) and protein expression ( P = 0.02). Decrease of protein expression was significantly correlated with progressed stage of disease, lymph node invasion and tumor size. We conclude, that aberrant promoter methylation of MGMT is a frequent and early event during tumorigenesis of Barrett’s esophagus. High prevalence of MGMT hypermethylation may represent a candidate marker for improved diagnosis and targeted therapy in Barrett’s adenocarcinoma.

Expression of Calcium-Binding Proteins S100A2, S100A4 in Barrett's Adenocarcinomas

In this study, we investigated the mRNA, protein expression of S100A2, S100A4 in adenocarcinomas of the stomach, esophagus. Real-time reverse transcription-polymerase reaction analysis on 72 tumors revealed frequent overexpression of S100A2, S100A4 in Barrett's adenocarcinomas (BAs) (P < .01). Immunohistochemical analysis on tumor tissue microarrays that contained 187 tumors showed absent to weak staining for S100A2 in all normal gastric mucosa samples, whereas normal esophageal mucosa samples demonstrated moderate to strong nuclear staining. Contrary to the nuclear expression of S100A2 in normal esophageal mucosa, two thirds of Barrett's dysplasia, BAs that overexpressed S100A2 demonstrated stronger cytosolic staining than nuclear staining (P < .001). Overexpression of S100A2 protein was more frequently seen in well-differentiated tumors than in others (P = .02). Moderate to strong staining of S100A4 was detected in two thirds of tumors, was frequently observed in the presence of Barrett's esophagus (P = .02). Similar to S100A2, the expression of S100A4 was predominantly cytosolic in two thirds of the tumors (P = .001). There was a significant correlation between S100A4 overexpression, lymph node metastasis (N2-N4) (P = .027). These results demonstrate frequent cytosolic overexpression of S100A2, S100A4 in BAs. Further studies are ongoing to underst, the biological significance of these S100A proteins in Barrett's tumorigenesis.

Safrole–DNA adducts in tissues from esophageal cancer patients: clues to areca-related esophageal carcinogenesis

Epidemiological studies have demonstrated that areca quid chewing can be an independent risk factor for developing esophageal cancer. However, no studies are available to elucidate the mechanisms of how areca induces carcinogenesis in the esophagus. Since the areca nut in Taiwan contains a high concentration of safrole, a well-known carcinogenic agent, we analyzed safrole–DNA adducts by the 32P-postlabelling method in tissue specimens from esophageal cancer patients. In total, we evaluated 47 patients with esophageal cancer (16 areca chewers and 31 non-chewers) who underwent esophagectomy at the National Taiwan University Hospital between 1996 and 2002. Of the individuals with a history of habitual areca chewing (14 cigarette smokers and two non-smokers), one of the tumor tissue samples and five of the normal esophageal mucosa samples were positive for safrole–DNA adducts. All patients positive for safrole–DNA adducts were also cigarette smokers. Such adducts could not be found in patients who did not chew areca, irrespective of their habits of alcohol consumption or cigarette smoking ( p < 0.001, comparing the areca chewers with non-chewers). The genotoxicity of safrole was also tested in vitro in three esophageal cell lines and four cultures of primary esophageal keratinocytes. In two of the esophageal keratinocyte cultures, adduct formation was increased by treatment with safrole after induction of cytochrome P450 by 3-methyl-cholanthrene. This paper provides the first observation of how areca induces esophageal carcinogenesis, i.e., through the genotoxicity of safrole, a component of the areca juice.