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This study examined the in-vivo effect of the NSAID celecoxib on DNA methylation in the promoter region of the tumor-suppressor genes cadherin 13, tissue factor pathway inhibitor 12, and follistatin-like protein 1, and on apoptosis, in esophageal squamous cell carcinoma (ESCC). Forty-five patients who underwent an esophagectomy for ESCC were allocated to either a treatment group (n=22) or a control group (n=23). Patients in the treatment group were administered 800 mg/day of celecoxib for 14 days before surgery. Patients in the control group did not take any type of NSAID. Biopsies of the tumor were collected before surgery and tissue from the resection specimens after surgery. Methylation-specific PCR was used to measure DNA methylation and apoptosis was measured by flow cytometry. There was no difference in the proportion of patients with methylation for each of the genes between the patient groups before treatment. In those patients with pretreatment methylation, there was a significant reduction in the proportion with methylation and a significant increase in the corresponding messenger RNA expression after treatment with celecoxib. In those tissues in which there was a reduction in methylation following celecoxib treatment, there was a significant increase in the percentage of apoptotic cells, but not in the tissues with no change in methylation. In ESCC, in-vivo treatment with celecoxib is associated with a reduction in DNA methylation and increase in messenger RNA expression of tumor-suppressor genes, and increases in apoptosis.

Background Information: Carcinoma of the oesophagus is the sixth leading cause of cancer death in the western world and is associated with a 5-year survival of less than 15%. Recent evidence suggests that stromal-epithelial interactions are fundamental in carcinogenesis. The advent of co-culture techniques permits the investigation of cross-talk between the stroma and epithelium in a physiological setting. We have characterized a histologically representative oesophageal organotypic model and have used it to compare the most commonly used squamous oesophageal cell line, HET-1A, with primary oesophageal squamous cells for use in studies of the oesophageal epithelium in vitro., Results: When grown in an organotypic culture with normal fibroblasts, the oesophageal carcinoma cell lines OE21 (squamous) and OE19 (adenocarcinoma) morphologically resembled the tumour of origin with evidence of stromal invasion and mucus production, respectively. However, HET-1A cells, which were derived from normal squamous oesophageal cells, appeared dysplastic and failed to display evidence of squamous differentiation. By comparison with primary oesophageal epithelial cells, the HET-1A cells were highly proliferative and did not express the epithelial markers E-cadherin or CK5/6 (casein kinase 5/6), or the stratified epithelial marker ΔNp63, but did express the mesenchymal markers vimentin and N-cadherin., Conclusion: Studies of epithelial carcinogenesis will benefit from culture systems which allow manipulation of the stromal and epithelial layers independently. We have developed an organotypic culture using primary oesophageal squamous cells and fibroblasts in which a stratified epithelium with a proliferative basal layer that stains strongly for ΔNp63 develops. This model will be suitable for the study of the molecular events in the development of Barrett's oesophagus. The most commonly used normal oesophageal squamous cell line, HET-1A, does not have the characteristics of normal oesophageal squamous cells and should not be used in models of the normal oesophageal epithelium. Until more representative cell lines are available, future studies in oesophageal cancer will be reliant on the availability and manipulation of primary tissue.

Background: Although most cases of esophageal squamous cell carcinoma (ESCC) in western populations have been attributed to high levels of exposure to tobacco and alcohol, infectious agents have been postulated as possible causes, particularly human papillomavirus (HPV)., Methods: To explore this issue, we analyzed HPV DNA prevalence and HPV types together with lifestyle factors, in relation to tumor stage and survival in a low-incidence population. Archived tumor samples from a nationwide cohort of 222 ESCC patients were tested for the presence of HPV DNA by PCR; positive samples were sequenced to determine HPV type, and p16(INK4a) status was assessed by immunohistochemistry., Results: Of 222 ESCC patients, 8 tested HPV positive (prevalence, 3.6%; 95% confidence interval, 1.1-6.1%), of which 6 were HPV-16 positive and 2 were HPV-35 positive. Four of the eight HPV-positive tumors overexpressed p16(INK4a). None of 55 normal esophageal tissue samples from healthy participants had any detectable HPV. Although the numbers were low, it seemed that patients with HPV-positive ESCC tumors were younger than those with HPV-negative tumors (mean age, 60.8 versus 65.3 years, P = 0.18) and had higher body mass index (BMI) throughout life (mean current BMI of 25.1 for HPV positive, 22.2 for HPV negative, P = 0.08; mean BMI at 20 years of 25.8 for HPV positive, 22.1 for HPV negative, P = 0.003). We found no difference between patients with HPV-positive and HPV-negative tumors with respect to other lifestyle factors., Conclusions: These findings suggest a very low prevalence of HPV DNA in human ESCC., Impact: HPV is very unlikely to be a common cause of ESCC in Australia., ((c)2010 AACR.)

Background: We examined the effect of aspirin on survival following resection for squamous cell carcinoma (SCC) of the esophagus or adenocarcinoma of the gastric cardia., Methods: Patients who underwent esophagectomy for these cancers between May 2000 and December 2002 were allocated to one of three groups and given daily either a low dose of aspirin, placebo, or no tablets., Results: The 5-year survival for all patients on aspirin (445) was 51.2%, placebo (658) 41%, and no tablet (495) 42.3% (P = 0.04 for difference between treatments). The 5-year survival for all SCC patients on aspirin (267) was 49.8%, placebo (433) 42.2%, and no tablet (343) 41.2% (P = 0.26). There was a significant improvement in survival for patients with adenocarcinoma of the cardia on aspirin compared with the two control groups combined (P = 0.029). Survival for T2N0M0 SCC patients was significantly improved with aspirin (71) compared with placebo (167) or no tablet (134) (P = 0.0004). However, there was no significant difference between the survival curves for T2N0M0 adenocarcinoma patients on aspirin (21) and the two control groups combined (65) (P = 0.29)., Conclusions: The results of this preliminary study support further investigation of aspirin as adjuvant therapy to improve survival in subsets of postesophagectomy patients.

In the development and progression of cancer, tumor suppressor genes may be silenced by mechanisms such as methylation. Thus the discovery of new genes silenced by methylation may uncover new tumor suppressor genes, and improve our understanding of cancer biology. In this study we investigated the methylation of 19 genes in esophageal squamous cell carcinoma. Methylation was measured in 10 of these genes in esophageal squamous cell carcinoma cell lines: CDH13, CLDN6, C16orf62, FBN2, FNBP1, ID4, RBP1, RBP4, TFPI2 and TMEFF2. To determine if there was a correlation between DNA methylation and gene silencing, each cell line was cultured with or without the demethylating drug 5-aza-2'-deoxycytidine (aza-dC). For 6 genes (CLDN6, FBN2, RBP1, RBP4, TFPI2 and TMEFF2) there was an association between reduction of methylation and increase in mRNA expression in the demethylated cell lines. The frequency of the methylation of these 6 genes in esophageal squamous cell carcinoma resection specimens was also investigated. All 6 genes showed more frequent methylation in the tumor than the matched proximal resection margin of uninvolved esophagus. There was a significant difference in the frequency of methylation and in the extent of the methylation between the cancer and the margin tissues for CLDN6, FBN2, TFPI2 and TMEFF2 (P=0.0007, P=0.0048 P=0.0002 and P<0.0001, respectively). This is the first report of gene silencing by methylation of CLDN6, FBN2, RBP4, TFPI2 and TMEFF2 in esophageal squamous cell carcinoma.

Although non-steroidal anti-inflammatory drugs (NSAIDs) have been demonstrated to have cancer-preventive effects and induce apoptosis of cancer cells, the mechanism of their effects is not clearly known. We studied the mechanism in human esophageal cancer cell line TE13. The esophageal squamous cell carcinoma cell line TE-13 was cultured with NS-398 at different concentrations or for different times. Proliferation and apoptosis were measured by MTT reduction and flow cytometry. Prostaglandin F(1alpha) was determined with radioimmunoassay. Expression of COX-2 mRNA was measured by RT-PCR and COX-2 protein levels with Western blot analysis. Nuclear NF-kappaB and cytoplasmic IkappaB protein levels were determined by electrophoretic mobility shift assay and Western blot, respectively. NS-398 significantly inhibited cell proliferation and induced apoptosis at concentrations of 0.001, 0.01, 1, and 100 micromol/L. NS-398 dose-dependently decreased the levels of COX-2 mRNA, COX-2 protein, nuclear NF-kappaB protein and production of PGF(1alpha) and increased the cytoplasmic IkappaB protein. In conclusion, NS-398 inhibits the proliferation of, and induced apoptosis in, the cultured TE-13 SCC cell line. These changes correlate with a reduction in COX-2 mRNA and protein expression, prostaglandin synthesis, an inhibition of NF-kappaB nuclear translocation, and an increase in cytoplasmic IkappaB.

Our previous study showed that aspirin induced apoptosis of esophageal cancer cells in vitro by inhibiting the pathway of NF-kappaB downstream regulation of cyclooxygenase-2. The purpose of this study was to determine if similar changes occurred in vivo in the tumors of patients with SCC of the esophagus who were given a preferential COX-2 inhibitor, meloxicam. Fifty-three patients who had an esophagectomy for SCC were allocated randomly to either a Treatment group (n = 25) or a control group (n = 28). Patients in the Treatment group were given 7.5 mg/day of meloxicam, for between 10 and 14 days before surgery. Patients in the control group did not take any type of NSAID during this time interval. Samples of the tumor taken from the resected specimens were collected. Proliferation and apoptosis were measured by flow cytometry. The concentration of 6-keto-prostaglandin F(1)alpha in cancer tissue was determined by radio-immuno-assay. Expression of COX-2 mRNA was measured with RT-PCR and COX-2 protein levels with Western blot analysis. Nuclear NF-kappaB and cytoplasmic I kappaB protein levels were determined by electrophoretic mobility shift assay and Western blot, respectively. There were significantly more apoptotic cells in the tumors of patients who were using meloxicam. It also decreased the levels of COX-2 mRNA, COX-2 protein and nuclear NF-kappaB protein and increased the cytoplasmic I kappaB protein in the cancer. We conclude that meloxicam induces apoptosis in SCC of the esophagus in vivo by inhibiting the pathway of NF-kappaB downstream regulation of COX-2., ((c) 2007 Wiley-Liss, Inc.)

Aim: To measure the frequency of DNA methylation of the tissue inhibitor of metalloproteinase 3 (TIMP3) promoter and relate this to any change of gene expression in esophageal squamous cell carcinoma in patients from a region of high incidence in China., Methods: Cancer cell lines were treated with or without the demethylating reagent 5-aza-2'-deoxycytidine. Methylation of the TIMP3 promoter was assessed in three regions by melt curve analysis and its expression was assessed by real-time RT-PCR. Tumors and proximal resection margins were obtained from 64 patients with esophageal squamous cell carcinoma from a region of high incidence in China. Methylation was assessed by melt curve analysis and expression by immunohistochemistry., Results: Methylation in one of the three promoter regions assessed correlated with gene silencing in esophageal cell lines. A degree of methylation of TIMP3 was found in only four esophageal squamous cell carcinomas, and partial loss of TIMP3 protein expression in just one., Conclusion: Methylation and loss of expression of TIMP3 occurs infrequently in esophageal squamous cell carcinoma in a region of high incidence in China.

Background: Metallothionein 3 (MT3) inhibits growth in a variety of cell types. We measured MT3 gene expression by RT-PCR, and DNA methylation in the MT3 promoter by combined bisulphite restriction analysis, in four oesophageal cancer cell lines and the resected oesophagus from 64 patients with oesophageal squamous cell carcinoma (SCC)., Results: MT3 expression was not detected in one of the four oesophageal cell lines. The MT3 promoter was methylated in all of the oesophageal cell lines, but the degree of methylation was greater in the non-expressing cell line. After treatment with 5-aza-2'-deoxycytidine there was a reduction in the degree of methylation, and an increase in MT3 expression, in each of the cell lines (p < 0.01). Methylation was detected in 52% (33 of 64) of primary SCC and 3% (2 of 62) of histologically normal resection margins. MT3 expression was measured in 29 tumours, 17 of which had methylation of MT3. The expression of MT3 was significantly less in the methylated tumours compared to either the unmethylated tumours (p = 0.03), or the matched margin (p = 0.0005). There was not a significant difference in MT3 expression between the tumour and the margin from patients with unmethylated tumour. No correlations were observed between methylation of MT3 and survival time, patient age, gender, smoking or drinking history, tumour stage, volume, or lymph node involvement., Conclusion: We conclude that MT3 expression is frequently down-regulated in oesophageal SCC, by DNA methylation, but that this is not a prognostic indicator.

Background: Aspirin has potential in the prevention or treatment of oesophageal cancer, the seventh most common cancer in the world, but its mechanism of action is still not certain., Methods: The oesophageal squamous cell carcinoma cell line TE-13 was cultured with aspirin at different concentrations or for different times. Proliferation and apoptosis were measured by MTT reduction and flow cytometry. Expression of COX-2 mRNA was measured by RT-PCR and COX-2 protein levels with Western blot analysis. Nuclear NF-kappaB and cytoplasmic IkappaB protein levels were determined by electrophoretic mobility shift assay and Western blot, respectively., Results: Aspirin significantly inhibited cell proliferation and induced apoptosis at concentrations of 1, 4, 8 mmol/L. Aspirin dose-dependently decreased the levels of COX-2 mRNA, COX-2 protein and nuclear NF-kappaB protein and increased the cytoplasmic IkappaB protein., Conclusion: We conclude that aspirin inhibits the proliferation of, and induced apoptosis in, the cultured TE-13 SCC cell line. These changes correlate with a reduction in COX-2 mRNA and protein expression, prostaglandin synthesis, an inhibition of NF-kappaB nuclear translocation, and an increase in cytoplasmic IkappaB. These results support the further investigation of the cyclooxygenase pathway in investigating the potential of aspirin and similar drugs in cancer prevention and therapy.

Objective: Does papillary squamotransitional cell carcinoma (PSTCC) behave differently from conventional squamous cell carcinoma of the cervix and does PSTCC have true transitional cell differentiation?, Materials and Methods: Twenty cases of PSTCC were identified from archival files. Clinical data were compiled. Immunoperoxidase stains for uroplakin III, p63 and p16 were performed on available tissue blocks., Results: Patients ranged in age from 27 to 85 years. Twelve patients were FIGO Stage I, 4 were Stage II, and 2 were Stage III. Thirteen patients had clinical follow-up ranging from 5 to 132 months. Three patients subsequently had more extensive disease than initial clinical staging indicated. Nine patients had no tumor progression, three had local recurrence and one had metastatic disease. Eight cases were strongly immunoreactive for p63 and p16 and 14 were negative for uroplakin III., Conclusions: PSTCC lacks true transitional cell differentiation and probably shares similar clinicopathologic features with conventional cervical squamous cell carcinoma.

Objective: To evaluate the qualification of a Pap smear classified as atypical squamous cells of undetermined significance (ASCUS) favor reactive or neoplasia as recommended by the Bethesda System., Study Design: The smears from 105 concurrent patients with a cytologic diagnosis of ASCUS not otherwise qualified were reviewed and subclassified as ASCUS favor reactive, low grade squamous intraepithelial lesion (LSIL) or high grade squamous intraepithelial lesion (HSIL) based on the Bethesda System criteria. The cervical biopsy diagnoses were correlated., Results: Of the 105 cases classified as ASCUS, 37 were subclassified as favor reactive, 51 as favor LSIL and 17 as favor HSIL on cytologic review. In the ASCUS favor reactive group, 19 (51%) had reactive changes on biopsy, 17 (46%) had cervical intraepithelial neoplasia (CIN) 1, and 1 (2%) had CIN 3. A total of 48% patients had CIN. In the favor LSIL group, there was CIN 1 in 28 cases (55%), CIN 2 or 3 in 12 (23%) and benign changes in 11 (22%) on biopsy. Seventy-eight percent had CIN. In the 17 cases classified as ASCUS favor HSIL group, all had CIN., Conclusion: Of the total 105 cases of ASCUS, 71% had CIN, 29% had reactive changes on follow-up biopsies, and 48% of patients in the ASCUS favor reactive group had CIN. Qualifiers of ASCUS have questionable utility in patient management.

The clinical staging of carcinoma of the vulva is a predictor of patient survival; however, the significance of other prognostic factors remains somewhat controversial. Length of survival after diagnosis of invasive squamous cell carcinoma was determined for 39 clinically staged and surgically treated patients who were followed at our institution. Clinical stage, tumor type, use of radiotherapy (RT), histopathologic features (invasive pattern, depth of invasion, lymph node status, nuclear grade, adjacent dysplasia, desmoplasia, inflammation) and DNA ploidy (determined by flow cytometry from paraffin-embedded tissue) were evaluated as predictors of survival. Kaplan-Meier survival curves were generated for strata defined by each of the various predictors and compared using the log-rank test. Advanced stage (p = 0.0002), RT use (p = 0.0004), "spray" invasive pattern (p = 0.005), positive lymph node status (p = 0.001), increased positive lymph node number (p = 0.016), and greater depth of invasion (p = 0.039) were associated univariantly with decreased survival time. Spray invasive pattern (p = 0.018), positive lymph node status (p = 0.030), positive lymph node number (p = 0.040), and RT use (p = 0.045) continued to be associated with decreased survival time after controlling for stage. Of the significant factors, invasive pattern stands out as a qualitative feature that may have potential benefit in predicting survival independent of clinical stage in patients with vulvar carcinoma.