ESCC Cases Research Articles

Abstract 1262: Aspirin inhibits the carcinogenesis of esophageal squamous cell carcinoma and enhances its responses to cisplatin

Abstract Esophageal squamous cell carcinoma (ESCC) is one of the most lethal malignancies. Over 70% of ESCC cases occur in China. Unfortunately, the treatment of ESCC has hardly been improved all these years. Several studies suggested that aspirin (ASA) might decrease the risk of ESCC and prolonged the survival of patients with ESCC. However, it is unclear if ASA could prevent ESCC and/or enhance ESCC treatment by chemotherapy. In this study, a N-nitroso-N-methylbenzylamine (NMBzA) - induced ESCC model was employed to prove that aspirin could prevent the growth of esophageal tumor. F344 rats were treated with NMBzA by subcutaneous injection with or without ASA in drinking water (2mg/ml). After 35 week ASA-NMBzA or NMBzA alone treatment, rats were killed and esophageal tumor development were examined. The results showed that F344 rats treated with NMBzA and receiving a daily intake of ASA developed much less tumors than F344 rats treated with NMBA alone both in amount and in size (Tumor count: 2.40 ± 1.57 vs 10.85 ± 3.86 , P < 0.001; Tumor volume: 11.10 ± 13.38 mm3 vs 70.79 ± 41.65 mm3, P < 0.001). Immunohistochemical analysis indicated that a higher rate of apoptosis was observed in the basal layer of esophageal epithelium in ASA-NMBzA treated rats than in NMBzA alone treated rats. These results indicated that ASA prevented development of esophageal tumors in rats induced by NMBzA. Moreover, using in vitro human ESCC cell culture and in vivo xenograft models, we showed that ASA has strong beneficial effects on inhibition of ESCC cell proliferation and colony formation, reduction of ESCC cancer stem cells and enhance of ESCC cell cytotoxicity induced by cisplatin treatment. Biochemical analysis revealed that these effects of ASA on human ESCC cells in vitro and in vivo were due to inhibiting the repairing of DNA damage, decreasing the efflux activity and ALDH1 activity of the tumor cells, and blockades of PI3K/Akt pathway. Thus, our results demonstrated a positive role of aspirin in the prevention and treatment of ESCC. Note: This abstract was not presented at the meeting. Citation Format: Zhigeng Zou, Hongjun Fan, Xiying Yu, Shuming Zhang, Liping Guo, Wei Jiang, Shih-Hsin Lu. Aspirin inhibits the carcinogenesis of esophageal squamous cell carcinoma and enhances its responses to cisplatin [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1262. doi:10.1158/1538-7445.AM2017-1262

ERK expression and its correlation with STAT1 in esophageal squamous cell carcinoma.

BackgroundEsophageal squamous cell carcinoma is one of leading causes of cancer-related deaths in Chaoshan region a high-risk region for esophageal cancer. Extracellular regulated protein kinases (ERK) usually play an important role in cell proliferation and differentiation. However, accumulating evidence has shown that the ERK was aberrantly expressed in cancers and correlated with STAT1 depression.ResultsThe activated ERK downregulates STAT1 expression in ESCC cell lines and U0126 increases expression of STAT1. Our immunohistochemistry result also confirms that the expression of ERK inversely correlated with that of STAT1 in ESCC tumors. In addition, a significantly higher expression of ERK/p-ERK was found in ESCC tissues in comparison with case-matched normal esophageal tissues (p < 0.05). Moreover, the immunohistochemical analysis demonstrated that ERK expression was paralleled with the differentiation and clinical stage. In 74 patients with follow-up data, those with ERKlow tumors survived significantly longer than those with ERKhigh tumors (p = 0.04); patients with ERKlow/STAT1high tumors had the longest survival (p = 0.001).Materials and MethodsTo investigate whether ERK can mediated STAT1 expression in ESCC, we used the MEK plasmid and U0126, a MEK inhibitor, to treat the cell. To further confirm our in-vitro study, we detected the ERK, p-ERK and STAT1 expression in 131 ESCC cases and 22 case-matched normal esophageal tissues adjacent to the tumors specimens.ConclusionsThese findings provide pathological evidence that ERK/p-ERK is negatively correlated with STAT1 in ESCC. Our data suggests that inhibition of ERK and/or restoration of STAT1 expression maybe useful therapeutic strategies for ESCC.

Flap endonuclease-1 rs174538 G>A polymorphisms are associated with the risk of esophageal cancer in a Chinese population.

BackgroundEsophageal cancer has a high mortality rate, particularly in Asia, and there are obvious racial differences in regard to incidence. The purpose of our study was to assess the genetic susceptibility of functional single nucleotide polymorphisms in flap endonuclease‐1 (FEN1) in esophageal squamous cell carcinoma ESCC.MethodsClinical blood samples of 629 ESCC cases and 686 control samples were collected. The ligation detection reaction method was used to determine FEN 1 rs174538 G>A genotypes.ResultsA significantly decreased risk of ESCC was associated with FEN 1 rs174538 GA genotypes among patients under 63 years old.ConclusionsOur results suggest that functional polymorphism FEN 1 rs174538 G>A might affect personal susceptibility to ESCC. This result provides a solid theoretical foundation for further clinical study using larger sample sizes.

Abstract 164: Clonal evolution in noncancerous esophageal mucosa in normal and cancer-bearing individuals

Abstract Background Esophageal squamous cell carcinoma (ESCC) represents the most common form of esophageal cancer worldwide, especially in East Asia, where alcohol drinking and smoking have been implicated in the field carcinogenesis of ESCC. However, the oncogenic process therein has been poorly understood in terms of gene mutations. Patients &amp; Methods A total of 100 samples, including cancer, dysplastic, and non-dysplastic esophageal tissues, were obtained from 24 individual with (N = 14) or without (N = 10) ESCC (a median of 2.5 samples per case: 1−29) either by endoscopy or surgery and were subjected to whole exome sequencing (WES). An additional paired cancer/non-caner samples from 32 patients was analyzed by targeted sequencing (TS). All samples were analyzed for copy number alterations (CNAs) using SNP array- and/or digital sequencing-based karyotyping. Results In WES, clonal evolution in esophageal epithelia, as determined by the presence of somatic mutations, was detected in 21 of 21 cancer, 12 of 12 dysplastic, and 63 of 67 non-dysplastic samples, where the mean number of mutation per sample showed a significant trend to increase in cancer (65) and dysplastic samples (50) compared to non-dysplastic samples (13) (P = 2.1×10-11). CNAs, especially those involving CDKN2A, CCND1, YAP1, and EGFR, were frequently affected in cancer samples, but rarely so in non-dysplastic samples. Non-dysplastic samples tended to have smaller allelic burden and therefore, clone size, compared to dysplastic and cancer samples (P = 2.2×10-16). Mutations had a predominant age-related signature in non-dysplastic samples but increasing APOBEC3A/3B patterns was observed in cancer and dysplastic samples. Shared mutations were found only within cancer tissues but never among dysplastic or non-dysplastic samples, suggesting the latter lesions are clonally independent from each other. In accordance with previous reports, TP53 mutations were found in 21/21 cancer samples and also found in dysplastic (11/12) and non-dysplastic samples at a lower frequency (26/67). Strikingly, non-dysplastic samples harbored a very high frequency of NOTCH1 mutations (51/67), which were also found in cancer (3/21) and non-dysplastic (8/12) samples but at much lower frequencies (P = 6.6×10-7). TS of validation samples confirmed the trend of higher NOTCH1 (84% vs. 25%) and lower TP53 mutation rates (38% vs. 100%) in non-dysplastic samples compared to cancer samples. The number of mutations in non-dysplastic samples was higher in drinkers than non-drinkers. Multiple NOTCH1 mutations were more common in cancer patients and drinkers than non-drinkers. Conclusion Clonal proliferation in non-cancer esophageal epithelia is common even in non-ESCC cases and extensive in ESCC cases. NOTCH1 and TP53 mutations play major roles in clonal evolution in common but may have differential impacts on esophageal carcinogenesis, which is likely to be shaped by APOBEC-induced mutations and CNAs. Citation Format: Akira Yokoyama, Hiromichi Suzuki, Tetsuichi Yoshizato, Kosuke Aoki, Yusuke Shiozawa, Youichi Fujii, Yusuke Sato, Nobuyuki Kakiuchi, Sugi Kin, Keisuke Kataoka, Kenichi Yoshida, Hideki Makishima, Yusuke Amanuma, Shinya Oohashi, Yuichi Shiraishi, Kenichi Chiba, Hiroko Tanaka, Brown J.B., Masashi Sanada, Shigeru Tsunoda, Sachiko Minamiguchi, Yoshiharu Sakai, Hironori Haga, Tsutome Chiba, Satoru Miyano, Manabu Muto, Seishi Ogawa. Clonal evolution in noncancerous esophageal mucosa in normal and cancer-bearing individuals. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 164.

A118G Polymorphism in μ-Opioid Receptor Gene and Interactions with Smoking and Drinking on Risk of Oesophageal Squamous Cell Carcinoma.

To investigate the single nucleotide polymorphism (SNP) of A118G and its interaction with smoking and drinking on oesophageal squamous cell carcinoma (ESCC) risk. A total of 960 subjects (545 males and 415 females) with a mean age of 58.1 ± 13.4 years were selected, including 490 ESCC patients and 470 normal control subjects. A logistic regression model was used to examine the association between A118G and ESCC and its interaction with A118G and current smoking and drinking. The odds ratio (OR) and 95% confident interval (95%CI) were calculated. The frequency for the A allele of A118G was significantly higher in ESCC cases, OR (95%CI) = 1.22 (1.08-1.59). Logistic regression analysis showed a significant association between the A allele in A118G and increased ESCC risk. The ESCC risk was significantly higher in carriers of the A allele of the A118G polymorphism than those with GG (AG + AA vs. GG, adjusted OR (95%CI) = 1.20 (1.05-1.53)). We found that current smokers with AG or AA of the A118G genotype have the highest ESCC risk compared with never smokers with a GG genotype; the OR (95%CI) was 2.57 (1.66-3.33). Current drinkers with AG or AA of the A118G genotype have the highest ESCC risk compared with not currently drinking subjects with the GG genotype, OR (95%CI) = 2.36 (1.47-3.25), after adjusting for covariates. The A allele of A118G and ESCC and additional interaction between the A allele of A118G and smoking or drinking were associated with increased ESCC risk.

Polymorphisms in the AKT1 and AKT2 genes and oesophageal squamous cell carcinoma risk in an Eastern Chinese population.

Ethnic Han Chinese are at high risk of developing oesophageal squamous cell carcinoma (ESCC). Aberrant activation of the AKT signalling pathway is involved in many cancers, including ESCC. Some single nucleotide polymorphisms (SNPs) in genes involved in this pathway may contribute to ESCC susceptibility. We selected five potentially functional SNPs in AKT1 (rs2494750, rs2494752 and rs10138277) and AKT2 (rs7254617 and rs2304186) genes and investigated their associations with ESCC risk in 1117 ESCC cases and 1096 controls in an Eastern Chinese population. None of individual SNPs exhibited an association with ESCC risk. However, the combined analysis of three AKT1 SNPs suggested that individuals carrying one of AKT1 variant genotypes had a decreased ESCC risk [adjusted odds ratio (OR) = 0.60, 95% CI = 0.42–0.87]. Further stratified analysis found that AKT1 rs2294750 SNP was associated with significantly decreased ESCC risk among women (adjusted OR = 0.63, 95% CI = 0.43–0.94) and non‐drinkers (OR = 0.79, 95% CI = 0.64–0.99). Similar protective effects on women (adjusted OR = 0.56, 95% CI = 0.37–0.83) and non‐drinker (adjusted OR = 0.75, 95% CI = 0.60–0.94) were also observed for the combined genotypes of AKT1 SNPs. Consistently, logistic regression analysis indicated significant gene–gene interactions among three AKT1 SNPs (P < 0.015). A three‐AKT1 SNP haplotype (C‐A‐C) showed a significant association with a decreased ESCC risk (adjusted OR = 0.70, 95% CI = 0.52–0.94). Multifactor dimensionality reduction analysis confirmed a high‐order gene–environment interaction in ESCC risk. Overall, we found that three AKT1 SNPs might confer protection against ESCC risk; nevertheless, these effects may be dependent on other risk factors. Our results provided evidence of important gene–environment interplay in ESCC carcinogenesis.

Pathway, in silico and tissue-specific expression quantitative analyses of oesophageal squamous cell carcinoma genome-wide association studies data.

Oesophageal cancer is the fourth leading cause of cancer death in China where essentially all cases are histologically oesophageal squamous cell carcinoma (ESCC). Agnostic pathway-based analyses of genome-wide association study (GWAS) data combined with tissue-specific expression quantitative trait loci (eQTL) analysis and publicly available functional data can identify biological pathways and/or genes enriched with functionally-relevant disease-associated variants. We used the adaptive multilocus joint test to analyse 1827 pathways containing 6060 genes using GWAS data from 1942 ESCC cases and 2111 controls with Chinese ancestry. We examined the function of risk alleles using in silico and eQTL analyses in oesophageal tissues. Associations with ESCC risk were observed for 36 pathways predominantly involved in apoptosis, cell cycle regulation and DNA repair and containing known GWAS-associated genes. After excluding genes with previous GWAS signals, candidate pathways (and genes) for ESCC risk included taste transduction (KEGG_hsa04742; TAS2R13, TAS2R42, TAS2R14, TAS2R46,TAS2R50), long-patch base excision repair (Reactome_pid; POLD2) and the metabolics pathway (KEGG_hsa01100; MTAP, GAPDH, DCTD, POLD2, AMDHD1). We identified and validated CASP8 rs13016963 and IDH2 rs11630814 as eQTLs, and CASP8 rs3769823 and IDH2 rs4561444 as the potential functional variants in high-linkage disequilibrium with these single nucleotide polymorphisms (SNPs), respectively. Further, IDH2 mRNA levels were down-regulated in ESCC (tumour:normal-fold change = 0.69, P = .75E-14). Agnostic pathway-based analyses and integration of multiple types of functional data provide new evidence for the contribution of genes in taste transduction and metabolism to ESCC susceptibility, and for the functionality of both established and new ESCC risk-related SNPs.

Family history of cancer and the risk of squamous cell carcinoma of oesophagus: a case-control study in Kashmir, India.

Background:Only a few studies have examined the association between family history of cancer (FHC) and the risk of oesophageal squamous cell carcinoma (ESCC) in high incidence areas of ESCC. We conducted a case–control study to evaluate the relationship between FHC and ESCC risk in Kashmir, India, with analysis of detailed epidemiological data and information on multiple gene polymorphisms.Methods:We collected detailed information on FHC and a number of socio-demographic and lifestyle factors, and also obtained blood samples for genetic analysis from 703 histopathologically confirmed ESCC cases and 1664 individually matched controls. Conditional logistic regression models were used to calculate odds ratios (ORs) and 95% confidence intervals (95% CIs).Results:Participants who had FHC showed a strong association with ESCC risk, and the risk was stronger when first-degree relatives (FDRs) had FHC (OR=6.8; 95% CI=4.6–9.9). Having a sibling with a cancer showed the strongest association (OR=10.8; 95% CI=6.0–19.3), but having a child with a cancer was not associated with ESCC risk. A history of any cancer in the spouse was also associated with ESCC risk (OR=4.1; 95% CI=1.6–10.2). Those with two or more relatives with FHC were at a higher risk of ESCC. After restricting FHC to familial ESCC only, the above associations were strengthened, except when spouses were affected with ESCC (OR=2.5; 95% CI=0.7–8.9). When we examined the associations between several single-nucleotide polymorphisms and ESCC in those with and without FHC, the associations of variant genotypes in cytochrome P450 (CYP) 2C19 and CYP2D6 and the wild genotype of CYP2E1 with ESCC were much stronger in those with FHC. The FHC had an additive interaction with several risk factors of ESCC in this population.Conclusion:Our results showed that FHC was strongly associated with ESCC risk in Kashmir. It seems both genetic factors and shared environment are involved in this association.

The characteristics of oesophageal squamous cell carcinoma: an analysis of 1317 cases in southeastern China.

Aim of the studyTo research the demographic and histopathological features of ESCC in southeastern China.Material and methodsWe retrospectively reviewed the ESCC cases in the biobank of the National Engineering Centre for Biochip in Shanghai, which cooperates with lots of hospitals and research institutions in southeastern China. The patients were pathologically confirmed as having ESCC. The demographic and histopathological features of these cases were analysed subsequently.ResultsA total of 1317 patients were enrolled. The overall male: female ratio was 2.88: 1. 74.34% of these cases occurred in people aged between 50–70 years. Dysphagia was the most common symptom, which accounted for 93.40% of all the patients. Stage II and III were predominant (79.73%). 72.89% of patients had a tumour length greater than 3 cm. Most of the tumours (65.83%) were located in middle third of the oesophagus. There was a significant difference among the tumour stage, length, and location in different sex groups (P < 0.05), but not between different age groups (P > 0.05). In males, ESCC is usually located in the lower parts, with a longer tumour length and higher tumour stage. 24.15% of patients had lymph nodes ratio (LNR) > 0.2.ConclusionsIn our analysis, dysphagia was more common in ESCC patients, to whom more attention should be paid. Additionally, males had a higher incidence, with longer and more distant disease, which gives a poor prognosis.

Abstract 571: Comparative immunohistochemical study of basaloid and usual squamous cell carcinoma of the esophagus

Abstract Basaloid squamous cell carcinoma (BSCC) is a rare epithelial malignancy variant of the typical SCC, and it has been associated to an aggressive behavior, with a high capacity for developing metastasis and low survival rates. It has been reported that less than 12% of the primary esophageal carcinoma have been diagnosed as BSCC. The study aimed to compare the protein expression of HER-2, EGFR, Ki-67, Cyclins A, B1 and D1 between usual SCC and BSCC. Immunohistochemistry of 94 usual ESCC and 17 EBSCC cases were carried out using Tissue Microarray technique. Immunohistochemical analysis of cyclins and growth factors receptors was based on frequency and intensity of tumor cells in each sample; Ki-67 analysis was based on the frequency of tumor cells showing strong nuclear positive staining. When the clinicopathological data were compared, only histological grade (p=0.000) showed a significant difference between the usual and basaloid SCC groups. No significant difference was found in gender, age, lymphatic, venous, and perineural invasion, pT, pN, pM and TNM staging when the two groups were compared. No significant difference was found between the overall survival rates of both groups. In EBSCC cases, cyclin A expression was positive in 94.1% of the cases, while 5.9% showed no expression. Cyclin B1 expression showed positivity in 23.6% of cases and 76.4% showed no staining. Most of the cases (70.6%) showed no cyclin D1 expression, while 29.4% were considered positive. EGFR expression was positive in 52.9% and negative in 47.1% of the cases. Expression of HER2 was observed in a small proportion of the cases (29.4%), when compared to those (70.6%) with no expression. Considering the KI-67 labeling index, most of the cases (76.4%) were classified as Low Proliferation, while only 11.8% were considered as High Proliferation. Among the ESCC cases, 30.0% were considered negative for cyclin A expression and 70.0% cases were positive. For cyclin B1, 18.4% were positive and 81.6% showed no expression. Cyclin D1 showed no staining in 85.4% of the cases, while 14.6% were positive. EGFR expression was positive in 80.9% and negative in 19.1% of the cases. HER2 expression was negative in 52.8% and 44.7% were considered positive. Analysis of Ki-67 expression showed that most of the cases (70.8%) were classified as Low Proliferation, while 29.2% were considered as High Proliferation. Comparing the markers expression between the two groups, EGFR (p=0.025) and cyclin A (p=0.029) showed significant differences. Positive EGFR expression was more frequent in the usual ESCC than in the EBSCC group. For cyclin A, positivity was more frequent in the EBSCC group, when compared to the usual ESCC cases. Regarding the other markers, there was no significant difference between the two groups. Although some observed differences did not achieve statistical significance, this work has further highlighted possible differences between the molecular pathogenesis of BSCC and SCC. Citation Format: Yukie Sato-Kuwabara, Juliano Jampietro, José Ivanildo Neves, Felipe José F Coimbra, Carolina Parucce, Wilson Luís Costa Junior, Fernando Augusto Soares. Comparative immunohistochemical study of basaloid and usual squamous cell carcinoma of the esophagus. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 571. doi:10.1158/1538-7445.AM2014-571

Abstract 2225: Comprehensive molecular characterization of esophageal squamous cell carcinoma

Abstract Background Esophageal squamous cell carcinoma (ESCC) is a world-wide prevalent cancer, which is particularly common in certain regions of Asia. However, the genetic alterations of this disease have not been fully described. Methods We determined the mutational landscape of ESCC through whole-exome or targeted deep sequencing of 139 tumor/germline pairs. The somatic copy number variations (SCNV) of over 180 ESCC tumors were analyzed by SNP-array or array-CGH. Candidate driver mutations were mathematically calculated by the most updated algorithm MutSigCV, and the protein expression of which were thoroughly evaluated by immunohistochemistry. Finally, biological effects of candidate drivers were examined through functional studies in vitro and in vivo. Results Deep sequencing and computational analysis identified frequent and novel driver mutations affecting genes such as FAT1 (11.5%), ZNF750 (7.2%), EP300 (7.9%) and MLL2 (18.0%). FAT1 and ZNF750 genes were homozygouly deleted in 3.5% and 3.4% ESCCs, respectively, and their RNA transcripts and proteins were expressed at much lower levels in primary tumors compared to adjacent normal esophageal epithelial. Importantly, silencing of wild-type FAT1 or ZNF750 expression with siRNA/shRNAs significantly promoted cell proliferation and decreased cell differentiation, whereas ectopic expression of these genes resulted in opposite biological consequences. These genetic alterations and biological evidence strongly suggest that FAT1 and ZNF750 likely encode tumor suppressors which are frequently disrupted in ESCC. Furthermore, by integrating the landscape of somatic mutation, SCNV and protein dysregulation, we proposed that RTK-PI3K-MAPK pathways, cell cycle and epigenetic regulation are frequently altered by multiple molecular mechanisms in esophageal tumors. Finally, our systematic approaches uncovered many novel druggable candidates in this disease, such as FGFR1, ALK and XPO1. Conclusions By demonstrating the genomic landscape of over 180 ESCC cases, our integrated study unmasks a number of novel genetic lesions in ESCC and provides an important molecular foundation for understanding esophageal tumors and developing therapeutic targets. Citation Format: Dechen Lin, Xuan Meng, Liang Xu, Lingwen Ding, Manoj Garg, Henry Yang, Lizhen Liu, Jiajie Hao, Mingrong Wang, Yasunobu Nagata, Yusuke Sato, Yusuke Okuno, Seishi Ogawa, Phillip Koeffler. Comprehensive molecular characterization of esophageal squamous cell carcinoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2225. doi:10.1158/1538-7445.AM2014-2225

Identification of genomic alterations in oesophageal squamous cell cancer

Oesophageal cancer is one of the most aggressive cancers and is the sixth leading cause of cancer death worldwide. Approximately 70% of global oesophageal cancer cases occur in China, with oesophageal squamous cell carcinoma (ESCC) being the histopathological form in the vast majority of cases (>90%). Currently, there are limited clinical approaches for the early diagnosis and treatment of ESCC, resulting in a 10% five-year survival rate for patients. However, the full repertoire of genomic events leading to the pathogenesis of ESCC remains unclear. Here we describe a comprehensive genomic analysis of 158 ESCC cases, as part of the International Cancer Genome Consortium research project. We conducted whole-genome sequencing in 17 ESCC cases and whole-exome sequencing in 71 cases, of which 53 cases, plus an additional 70 ESCC cases not used in the whole-genome and whole-exome sequencing, were subjected to array comparative genomic hybridization analysis. We identified eight significantly mutated genes, of which six are well known tumour-associated genes (TP53, RB1, CDKN2A, PIK3CA, NOTCH1, NFE2L2), and two have not previously been described in ESCC (ADAM29 and FAM135B). Notably, FAM135B is identified as a novel cancer-implicated gene as assayed for its ability to promote malignancy of ESCC cells. Additionally, MIR548K, a microRNA encoded in the amplified 11q13.3-13.4 region, is characterized as a novel oncogene, and functional assays demonstrate that MIR548K enhances malignant phenotypes of ESCC cells. Moreover, we have found that several important histone regulator genes (MLL2 (also called KMT2D), ASH1L, MLL3 (KMT2C), SETD1B, CREBBP and EP300) are frequently altered in ESCC. Pathway assessment reveals that somatic aberrations are mainly involved in the Wnt, cell cycle and Notch pathways. Genomic analyses suggest that ESCC and head and neck squamous cell carcinoma share some common pathogenic mechanisms, and ESCC development is associated with alcohol drinking. This study has explored novel biological markers and tumorigenic pathways that would greatly improve therapeutic strategies for ESCC.

Contact with animals and risk of oesophageal squamous cell carcinoma: outcome of a case–control study from Kashmir, a high-risk region

BackgroundSeveral studies have reported association between animal contact and some cancer types, including lymphohaematopoietic, colon, pancreatic and neurological malignancies. We aimed to investigate the association between animal contact and risk...

Sex differences in the proportion of esophageal squamous cell carcinoma cases attributable to tobacco smoking and alcohol consumption

Objective: Alcohol and tobacco are the two major established environmental factors associated with squamous cell carcinoma of the esophagus (ESCC). However, the prevalence of these exposures differs substantially between men and women. Moreover, the prevalence of smoking has declined in recent years, whereas per capita consumption of alcohol has remained steady in both sexes. Quantifying the burden of ESCC attributable to these causal factors is necessary to inform potential preventive strategies. Methods: We estimated the population attributable fraction (PAF) of ESCC due to smoking and alcohol, using data from an Australian population based case–control study (305 ESCC cases, 1554 controls). Results: Estimated PAF for ESCC were 49% (95% CI: 38–60) and 32% (95% CI: 25–40) due to smoking and heavy alcohol consumption respectively. More than 75% of the ESCC burden in men could be attributed to smokers with heavy alcohol consumption. The highest burden was among ≥30 pack years smokers who also consumed alcohol heavily (>17 drinks/week); this differed significantly between men (PAF 36%, 95% CI 29–44) and women (PAF 5%, 95% CI 2–10). Among women only, low intakes of fruit and vegetables accounted for about 9% of the ESCC burden. Conclusion: The burden of ESCC attributable to smoking combined with heavy alcohol consumption is remarkably high in men. In women, the burden of ESCC due to these factors is lower, and poor nutrition may also play a role.

Abstract 1487: Evaluation of the relationship between e-cadherin expression and proliferative activity at the invasive front of esophageal squamous cell carcinoma.

Abstract Introduction Esophageal squamous cell carcinoma (ESCC) has a poor prognosis mainly because it is usually in an advanced stage at the time of diagnosis. The epithelium-mesenchyme transition (EMT), when epithelial cells loss their original characteristics and acquire mesenchymal phenotype, is indicated first by the loss of E-cadherin expression. The EMT is considered the main event in tumor progression and metastasis. In vitro studies have demonstrated that e-cadherin downregulation can lead to cell proliferation. This finding has not been evaluated in human tumors. This study aimed to elucidate whether tumor cells that have undergone EMT at the invasive front of the ESCC are in proliferative state. Material and Methods Samples of 58 ESCC cases were double stained by immunohistochemistry (IHQ) for E-cadherin (BD Bioscience) and Ki67 (ROCHE) antibodies using the Automated System Ventana BenchMark XT (ROCHE). IHC analysis was performed using Aperio ScanScope XT (APERIO) with the Algorithm ColorDeconvolution. In each case E-cadherin and Ki67 expression were evaluated in the tumor core and at the invasive front, separately. Positivity was divided into strong, moderate and weak positive according to intensity staining. For each case a score was given based on the formula with the percentages of each positivity group [Score = 1x (% Weak) + 2x (% Moderate) + 3x (% Strong)]. Results and Discussion Analysis of the IHQ double-stain for E-cadherin showed that 51 (87.9%) cases had a high expression at the tumor core when compared with invasive front and in 7 (12.1%) cases were found high expression at the invasion front (p&amp;lt;0,001). The median values for E-cadherin double-stain scores were 184.2 and 163.0 at invasive front and tumor core, respectively (p&amp;lt;0.001). Analyses of the Ki67 double-stain showed that 19 (32.7%) cases showed a high expression at tumor core and 39 (67.3%) cases showed a high expression at the invasive front (p&amp;lt;0,001). Proliferation rates, represented by Ki67 expression, showed median values of 8.1 and 6.8 at the invasive front and tumor core, respectively (p=0.052). Spearman correlation between the IHQ double and single-stain was moderately positive (p &amp;lt;0.001). Conclusion Tumor cells at the invasive front showed an increased proliferation rate as they lose the E-cadherin expression and at the tumor core showed an higher expression of E-cadherin and a lower expression of Ki67. However, some cases showed a different behavior with a higher expression of E-cadherin at the invasive front and a elevated rate proliferation at tumor core. A more complex mechanism in the dynamics of E-cadherin and Ki67 expressions could be acting during EMT. Further studies are necessary to the better understanding of the complexity of this mechanism. Citation Format: Juliano Jampietro, Yukie Sato-Kuwabara, José Humberto H.T.G. Fregnani, Felipe Coimbra, Claudia Malheiros Coutinho Camillo, Fernando Augusto Soares. Evaluation of the relationship between e-cadherin expression and proliferative activity at the invasive front of esophageal squamous cell carcinoma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1487. doi:10.1158/1538-7445.AM2013-1487

Hookah smoking, nass chewing, and oesophageal squamous cell carcinoma in Kashmir, India

Background:Although cigarette smoking is an established risk factor for oesophageal squamous cell carcinoma (ESCC), there is little information about the association between other smoking and smokeless tobacco products, including hookah and nass, and ESCC risk. We conducted a case–control study in Kashmir Valley, India, where hookah smoking, nass chewing, and ESCC are common, to investigate the association of hookah smoking, nass use, and several other habits with ESCC.Methods:We recruited 702 histologically confirmed ESCC cases and 1663 hospital-based controls, individually matched to the cases for age, sex, and district of residence from September 2008 to January 2012. Conditional logistic regression models were used to calculate odds ratios (ORs) and 95% confidence intervals (95% CIs).Results:Ever-hookah smoking (OR=1.85; 95% CI, 1.41–2.44) and nass chewing (OR=2.88; 95% CI, 2.06–4.04) were associated with ESCC risk. These associations were consistent across different measures of use, including intensity, duration, and cumulative amount of use, and after excluding ever users of the other product and cigarette smokers. Our results also suggest an increased risk of ESCC associated with ever-gutka chewing and -bidi smoking. However, the latter associations were based on small number of participants.Conclusion:This study shows that hookah and nass use are associated with ESCC risk. As prevalence of hookah use seems to be increasing among young people worldwide, these results may have relevance not only for the regions in which hookah use has been a traditional habit, but also for other regions, including western countries.

Geographic origin is a significant determinant of human papillomavirus prevalence in oesophageal squamous cell carcinoma: Systematic review and meta-analysis

Since the first reports in 1982 suggesting an aetiological role for human papillomavirus (HPV) in a subset of oesophageal squamous cell carcinomas (ESCC), the literature reporting HPV detection in ESCC has expanded rapidly. However no formal meta-analysis of this literature has been published yet. The objective of this study was to perform a systematic review and formal meta-analysis of the literature reporting HPV detection in ESCC. MEDLINE and Current Contents were searched through March 2012. The effect size was calculated as event rates and their 95% confidence interval (95% CI), with homogeneity testing using Cochran's Q and I² statistics. Meta-regression was used to test the impact of study-level covariates (HPV detection method, geographic origin of study) on effect size, and potential publication bias was estimated using funnel plot symmetry (Begg and Mazumdar rank correlation, Egger's regression, and Duval and Tweedie's trim and fill method). Of the 1177 abstracts found, 152 studies were determined to be eligible for this meta-analysis. These 152 studies covered a total of 10,234 ESCC cases, analysed by different HPV detection methods in different geographic regions. Of these 10,234 cases, 3135 (30.6%) tested HPV-positive, translating to an effect size of 0.372 (95% CI 0.360-0.384; fixed effects model) and 0.290 (95% CI 0.251-0.31; random effects model). When stratified by HPV detection technique, there was a significant heterogeneity between the studies, but importantly, the between-strata summary comparison was not significant (random effects model; p = 0.440). In contrast, there was significant heterogeneity between the studies from the different geographic regions. In the maximum likelihood meta-regression, HPV detection method was not a significant study-level covariate, in contrast to the geographic origin of the study, which had a significant impact (p = 0.00005) on the summary effect size estimates. No evidence for significant publication bias was found in funnel plot symmetry testing. In the sensitivity analysis, all meta-analytic results appeared robust to all (n = 151) one-by-one study removals. These meta-analysis results indicate that the reported wide variability in HPV detection rates in ESCC is not due to the HPV detection techniques, but is explained by the geographic origin of the study. These data substantiate the recently elaborated concept that ESCC might have a different aetiology in low-incidence and high-incidence geographic regions, HPV playing an important role only in the latter.

Abstract LB-91: Transcriptome analysis of esophageal squamous cell carcinoma by SAGE identifies a new biomarker, NRD1

Abstract Esophageal squamous cell carcinoma (ESCC) is one of the most common malignancies worldwide. Because most ESCC is diagnosed at advanced stages, the overall 5-year survival rate of ESCC still remains low in spite of the use of various treatment modalities. Therefore, identification of new diagnostic markers and therapeutic targets for ESCC is important. Serial Analysis of Gene Expression (SAGE) is one of the common comprehensive methods to analyze transcript expression levels, and enables the quantitative and simultaneous analysis of a large number of transcripts. To identify potential diagnostic markers and therapeutic targets for ESCC, the SAGE library was generated from one ESCC sample. A total of 14,430 tags were generated, including 5,765 that were unique. By comparing SAGE tags from the ESCC sample with those from normal human squamous esophagus, several genes that is upregulated in ESCC compared with normal squamous esophagus in ESCC were identified. Among these, we focused on the nardilysin (N-arginine dibasic convertase, NRD1) gene. NRD1 has been reported to be associated with ectodomein shedding of TNF-α through activation of ADAM proteases. There is only one report in the literature mentioning the association of NRD1 with cancer (breast cancer), however, there is no report that links expression of NRD1 and ESCC. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) analysis showed a high level of NRD1 expression in three of five esophageal cancer cell lines and seven of 14 ESCC samples, but not in 14 normal systemic tissues. Western blot analysis also showed upregulation of NRD1 protein in total five esophageal cancer cell lines. Immunohistochemical analysis revealed expression of NRD1 in cytoplasm of 43 out of 109 ESCC cases (39%), as well as heart, skeletal muscle, and testis in agreement with previous reports. In clinicopathological analysis, expression of NRD1 correlated with the advanced T grade, N grade and tumor stage, and was an independent predictor of survival in patients with ESCC. Knockdown of NRD1 in TE5 cells inhibited cell invasion ability. Furthermore, qRT-PCR analysis showed a reduction of matrix metalloproteinase-2 (MMP-2) expression in NRD1 knocked down cells. In conclusion, NRD1 is an independent predictor of survival, and contribute to cell invasion through regulation of MMP-2 in ESCC. NRD1 could be a novel diagnostic and therapeutic target for ESCC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-91. doi:1538-7445.AM2012-LB-91

Overexpression of GPR39 contributes to malignant development of human esophageal squamous cell carcinoma

BackgroundBy using cDNA microarray analysis, we identified a G protein-coupled receptor, GPR39, that is significantly up-regulated in ESCC. The aim of this study is to investigate the role of GPR39 in human esophageal cancer development, and to examine the prevalence and clinical significance of GPR39 overexpression in ESCC.MethodsThe mRNA expression level of GPR39 was analyzed in 9 ESCC cell lines and 50 primary ESCC tumors using semi-quantitative RT-PCR. Immunohistochemistry was used to assess GPR39 protein expression in tissue arrays containing 300 primary ESCC cases. In vitro and in vivo studies were done to elucidate the tumorigenic role of GPR39 in ESCC cells.ResultsWe found that GPR39 was frequently overexpressed in primary ESCCs in both mRNA level (27/50, 54%) and protein level (121/207, 58.5%), which was significantly associated with the lymph node metastasis and advanced TNM stage (P < 0.01). Functional studies showed that GPR39 has a strong tumorigenic ability. Introduction of GPR39 gene into ESCC cell line KYSE30 could promote cell proliferation, increase foci formation, colony formation in soft agar, and tumor formation in nude mice. The mechanism by which amplified GPR39 induces tumorigenesis was associated with its role in promoting G1/S transition via up-regulation of cyclin D1 and CDK6. Further study found GPR39 could enhance cell motility and invasiveness by inducing EMT and remodeling cytoskeleton. Moreover, depletion of endogenous GPR39 by siRNA could effectively decrease the oncogenicity of ESCC cells.ConclusionsThe present study suggests that GPR39 plays an important tumorigenic role in the development and progression of ESCC.

Abstract B27: DNA polymorphisms in sex hormone pathway genes and risk of esophageal squamous cell carcinoma

Abstract Background: Esophageal cancer (EC) is the sixth most common cancer in the world and exhibits a dramatic geographic distribution in incidence and histological subtype. In China, EC is the fourth leading cause of cancer death, the predominant histologic type being squamous cell carcinoma (ESCC), as opposed to adenocarcinoma which is more common in developed counties of the western world. Recently it has been suggested that sex hormones may be associated with the risk of aero-digestive tract cancers including those of the colon, head and neck, and stomach. To evaluate the potential role of sex hormones in ESCC, we examined sex hormone metabolizing gene variants and ESCC risk in a high risk population from north central China. Methods: A combined total of 1027 ESCC cases and 1452 controls from separate studies were genotyped for 752 tagged SNPs in 46 sex hormone-related genes. SNP-, gene-, and pathway-based associations with ESCC risk were evaluated using the adaptive rank-truncated product (ARTP) method and the additive model within unconditional logistic regression adjusted for age and sex. Statistical significance was determined through use of a permutation-based sampling procedure (20,000 permutations). Results: No significant pathway-based association was observed when all 46 sex hormone genes were considered together (P=0.16). Gene-based associations with ESCC were observed for 6 genes: SULT2B1 (ARTP P=0.012), CYP1B1 (P=0.019), CYP3A7 (P=0.03), CYP3A5 (P=0.033), SHBG (P=0.037), and CYP11A1 (P=0.038). Of examined individual SNPs, sixty three SNPs in 21 genes were also statistically significant (P&amp;lt;0.05). Among the strongest effects observed were rs4149455 in SULT2B1 (per allele OR 0.81, 95% CI 0.72-0.91, P=0.0007); rs9341266 in CYP1B1 (OR 1.45, 1.16-1.82, P=0.001); rs17161780 in CYP3A5 (OR 1.19, 1.04-1.35, P=0.009); rs2005548 in CYP3A7 (OR 1.15, 1.02-1.29, P=0.02), and rs727428 in SHBG (OR 0.85, 0.76-0.96, P=0.01). Conclusion: Genetic variation in specific sex hormone-related genes may contribute to ESCC risk among Chinese individuals at high risk for ESCC. Citation Information: Cancer Prev Res 2010;3(12 Suppl):B27.