Abstract B07: Analysis of allele specific expression in esophageal squamous cell carcinoma with combination of exome sequencing and mRNA Sequencing

In the current study, allele specific expression analysis was performed in two subspecies cows (Bos taurus and Bos indicus) at SNP and gene levels. RNA-Seq data of 21,078,477 and 20940063 paired end reads from pooling of whole blood samples (Leukocyte) from 40 US Holstein (Bos Taurus) and 45 Cholistani cows (Bos indicus) obtained from SRA database in NCBI. Quality control and trimming of row RNA-Seq data were processed by FASTQC and Trimmomatic softwares. The transcriptome was assembled by TopHat2 software in two cow's population by aligning and mapping the RNA-Seq reads on bovine reference genome. The SNPs were discovered by Samtools software and ASE analysis was performed by Chi-square test. Results showed that 50183 and 137954 SNPs were discovered on the assembled transcriptome of Holstein and Cholistani cow samples, respectively, and 15308 SNPs were common in both breeds. 10158 SNPs from 50183 (20%) in Holstein and 31523 SNPs from 137954 (23%) in Cholistani cows were identified as ASE-SNPs. Reference allele and alternative allele count in Holstein and Cholistani cows were 3041 and 7155, respectively. Among 131 discovered SNPs in 41 genes with different expression in Holstein and Cholistani cows, 31 ASE-SNPs (5 in Holstein; 26 in Cholistani cows) were discovered.

Differential Allele-Specific Expression Uncovers Breast Cancer Genes Dysregulated by Cis Noncoding Mutations.

Single-cell RNA-seq (scRNA-seq) is emerging as a powerful tool for understanding gene function across diverse cells. Recently, this has included the use of allele-specific expression (ASE) analysis to better understand how variation in the human genome affects RNA expression at the single-cell level. We reasoned that because intronic reads are more prevalent in single-nucleus RNA-Seq (snRNA-Seq), and introns are under lower purifying selection and thus enriched for genetic variants, that snRNA-seq should facilitate single-cell analysis of ASE. Here we demonstrate how experimental and computational choices can improve the results of allelic imbalance analysis. We explore how experimental choices, such as RNA source, read length, sequencing depth, genotyping, etc., impact the power of ASE-based methods. We developed a new suite of computational tools to process and analyze scRNA-seq and snRNA-seq for ASE. As hypothesized, we extracted more ASE information from reads in intronic regions than those in exonic regions and show how read length can be set to increase power. Additionally, hybrid selection improved our power to detect allelic imbalance in genes of interest. We also explored methods to recover allele-specific isoform expression levels from both long- and short-read snRNA-seq. To further investigate ASE in the context of human disease, we applied our methods to a Parkinson’s disease cohort of 94 individuals and show that ASE analysis had more power than eQTL analysis to identify significant SNP/gene pairs in our direct comparison of the two methods. Overall, we provide an end-to-end experimental and computational approach for future studies.

Objective To determine clinical significance of breast eancer anti-estrogen drug resistance protein 1 (BCAR1) expression in sera and tissues of esophageal squamous cell cancer (ESCC).Methods Between Feb.2010 and Jan.2011,serum BCAR1 levels in 46 cases of ESCC and 25 healthy volunteers from our department were detected by using enzyme linked immunosorbent assay (ELISA).A total of 106 ESCC samples and their adjacent normal tissues from our department obtained between Feb.2004 and July 2006 were investigated for BCAR1 protein expression by using tissue microassay (TMA) and immunohistochemistry (IHC).Results The serum BCAR1 levels were significantly higher in ESCC group than in normal control group (P ＜0.01 ).The serum BCAR1 levels in advanced ESCC group were significantly higher than in early ESCC group and normal control group ( P ＜ 0.01 ).The serum BCAR1 levels were decreased after removal of ESCC. BCAR1 protein was strongly expressed in 97％ of ESCC samples ( 103/106),while weekly expressed in 15％ of adjacent normal tissues (16/106) ( P ＜ 0.001 ).There was a significant correlation between BCAR1 positive expression and differentiation (P ＜0.05 ),however,there was no correlation between BCAR1 positive expression and age,gender,lymph node metastasis,depth of invasion and TNM stage ( P ＞ 0.05 ).Survival analysis revealed that BCAR1 low expression was in favor of survival time in comparison with BCAR1 high expression ( P ＜ 0.01 ). BCAR1 expression and lymph node metastasis were independent prognosis indictor by COX regression analysis.Conclusion BCAR1 protein expression is significantly higher in sera and tissues of ESCC than normal control group,and has a correlation with progression,therapeutic efficacy,differentiation and prognosis of ESCC.BCAR1 protein might be used as a new tumor marker for diagnosis and prognosis of ESCC. Key words: Breast cancer anti-estrogen drug resistance protein 1; Esophageal squamous cell carcinoma; Tissue microassay

BackgroundGenetic analysis of gene expression level is a promising approach for characterizing candidate genes that are involved in complex economic traits such as meat quality. In the present study, we conducted expression quantitative trait loci (eQTL) and allele-specific expression (ASE) analyses based on RNA-sequencing (RNAseq) data from the longissimus muscle of 189 Duroc × Luchuan crossed pigs in order to identify some candidate genes for meat quality traits.ResultsUsing a genome-wide association study based on a mixed linear model, we identified 7192 cis-eQTL corresponding to 2098 cis-genes (p ≤ 1.33e-3, FDR ≤ 0.05) and 6400 trans-eQTL corresponding to 863 trans-genes (p ≤ 1.13e-6, FDR ≤ 0.05). ASE analysis using RNAseq SNPs identified 9815 significant ASE-SNPs in 2253 unique genes. Integrative analysis between the cis-eQTL and ASE target genes identified 540 common genes, including 33 genes with expression levels that were correlated with at least one meat quality trait. Among these 540 common genes, 63 have been reported previously as candidate genes for meat quality traits, such as PHKG1 (q-value = 1.67e-6 for the leading SNP in the cis-eQTL analysis), NUDT7 (q-value = 5.67e-13), FADS2 (q-value = 8.44e-5), and DGAT2 (q-value = 1.24e-3).ConclusionsThe present study confirmed several previously published candidate genes and identified some novel candidate genes for meat quality traits via eQTL and ASE analyses, which will be useful to prioritize candidate genes in further studies.

. DNA polymerase iota (Pol iota) is an error-prone DNA polymerase involved in translesion DNA synthesis (TLS) that may play a significant role in the accumulation of DNA mutations. Our previous studies identified the elevated expression of Pol iota in human esophageal squamous cell cancer (ESCC) tissues and revealed that Pol iota contributes to ESCCs’ progression. The present study aimed to investigate the molecular mechanism by which Pol iota enhances the invasiveness and metastasis of ESCC cells. We found that the expression of Pol iota was higher in ESCCs with lymph node metastasis compared to those without lymph node metastasis. Kaplan-Meier analysis revealed a negative correlation between the expression of Pol iota and patients’ prognosis. Furthermore, the expression of matrix metalloproteinase-2(MMP-2) and matrix metalloproteinase-9(MMP-9), both being essential regulators for cells invasion, were associated with that of Pol iota in ESCCs tissue samples. The wound healing and transwells assay revealed that over-expression of Pol iota enhances motility and invasiveness of ESCC cells. In vivo, up-regulation of Pol iota promoted the colonization of ESCC cells in the liver, lung and kidney. Signaling pathway analysis showed that Pol iota induces the expression of MMP-2/9 and enhances the ESCCs progression via JNK-AP-1 cascade. Altogether, our finding demonstrated the underlying mechanism by which Pol iota promotes ESCCs’ development. Citation Format: Shitao Zou, Jinchang Wu, Wei-Qun Ding, Jundong Zhou. High expression of Pol é promotes invasion and metastases in esophageal squamous cell carcinoma. [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 707.

Objective To explore the relationship between serum p53 antibodies and clinicopathological characteristics in patients with esophageal squamous cell carcinoma(ESCC) and to investigate sequential changing regularity of serum p53 antibodies before and after surgical resection. Methods The eligible ESCC cases of this study were consecutively recruited between October 2010 and February 2012 from Luhe Hospital.There were 68 males and 30 females.The serum p53 antibody concentration was detected in ESCC patients and in 30 healthy adults as controls by enzyme linked immunosorbent assay(ELISA).The blood samples were collected on the day before operation and on the 7th, 30th, 90th and 180th days postoperatively. Results (1) The concentration[(338. 96± 104. 14)ng/L vs.(242. 30±39. 79) ng/L]and positive rate(37.8% vs.0)of serum p53 antibodies in patients with ESCC were significantly higher than normal individuals(P< 0. 05).(2) The positive rate of pre-operative serum p53 antibodies in patients with ESCC positively correlated with the pieces of consumed cigarette, cell differentiation and TNM(P< 0. 05).(3) The level of serum p53 antibodies decreased gradually in patients with ESCC underwent radical resection of the cancer, and completely returned to the normal level around the 30th day after operation. Conclusion (1)Serum p53 antibody can be used as a potential marker for early detection of ESCC and predication of prognosis.(2)It has important clinical reference value for detecting the early recurrence or metastasis to monitor serum p53 antibodies level on postoperative patients with ESCC. Key words: Esophageal squamous cell carcinoma; P53 gene; Enzyme linked immunosorbent assay; P53 antibody in serum

Introduction: MicroRNA-210 regulates cancer cell proliferation by targeting fibroblast growth factor receptor-like 1 (FGFRL1) in esophageal squamous cell cancer (ESCC) (JBC 2011). The expression of FGFRL1 has been associated with tumor growth and lymph node metastasis. In the present study, we analyzed and compared the expression of FGFR1, FGFR2, FGFR3, and FGFR4 with that of FGFRL1 using a tissue microarray. We also examined whether antibodies for FGFRL1 inhibited the growth and motility of ESCC cells. Material and Methods: A squamous cell carcinoma tissue microarray established in Toyama University was used to evaluate the expression of the 5 FGFRs. Sixty-nine specimens of ESCC were obtained from 62 male and 7 female patients, with an average age of 64.4 years old, that underwent surgery between 1990 and 2008. The TNM stages of these patients were as follows; stage 1: 7, stage 2a; 10, stage 2b; 10, stage 3; 35, stage 4; 7. The immunohistochemical results of these patients were scored according to intensity and distribution after a careful examination by two independent researchers. The ESCC cell line KYSE520 was used for experimental analyses. Polyclonal and monoclonal antibodies for FGFRL1 were used to evaluate the function of FGFRL1. Results: The percentage of patients who tested positive for the expression of the different FGFRs was as follows: FGFRL-1, 81% (56/69); FGFR1, 65% (37/57); FGFR2, 36% (22/61); FGFR3, 21% (12/57); FGFR4, 39% (22/56). The prognosis of FGFRL1 positive patients was significantly worse than that of FGFRL1 negative patients. (P=0.0311). Both polyclonal and monoclonal antibodies inhibited the growth (50% reduction) and motility (25% reduction) of ESCC cells. However, no association was observed between the expression of the other FGFRs and patient prognosis. Regarding combination analysis of the expression of each FGFR, patients that co-expressed FGFRL1 and FGFR1 had the worst prognosis, while patients who tested negative for the expression of both FGFRL1 and FGFR4 had the best prognosis. Conclusions: Among the 5 FGFRs, the expression of FGFRL1 was the main oncogenic driving factor in ESCC patients. The expression of FGFR1 and FGFR4 may be a sub-driving factor of ESCC. Citation Format: Yutaka Shimada, Tomoyuki Okumura, Takuya Nagata, Yoshinori Takei, Kazuhiro Tsukada, Kazuharu Shimizu. Role of fibroblast growth factor receptors in 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 3447. doi:10.1158/1538-7445.AM2014-3447

Allele-specific expression (ASE) analysis detects the relative abundance of alleles at heterozygous loci as a proxy for cis-regulatory variation, which affects the personal transcriptome and proteome. This study describes the development and application of an ASE analysis pipeline on a unique cohort of 87 well phenotyped and RNA sequenced patients from the Maastricht Cardiomyopathy Registry with dilated cardiomyopathy (DCM), a complex genetic disorder with a remaining gap in explained heritability. Regulatory processes for which ASE is a proxy might explain this gap. We found an overrepresentation of known DCM-associated genes among the significant results across the cohort. In addition, we were able to find genes of interest that have not been associated with DCM through conventional methods such as genome-wide association or differential gene expression studies. The pipeline offers RNA sequencing data processing, individual and population level ASE analyses as well as group comparisons and several intuitive visualizations such as Manhattan plots and protein–protein interaction networks. With this pipeline, we found evidence supporting the case that cis-regulatory variation contributes to the phenotypic heterogeneity of DCM. Additionally, our results highlight that ASE analysis offers an additional layer to conventional genomic and transcriptomic analyses for candidate gene identification and biological insight.

Background: Genome-wide association study (GWAS) have identified over 45 susceptibility loci for lung cancer; many studies including our own group, have focused on low-frequency and rare coding variants using fine mapping and exome sequencing. This strategy, however, has met with limited success as about 90% of GWAS hits are noncoding and act primarily through altering transcriptional regulation in an allele-specific manner. The RNA-Seq based allele-specific expression (ASE) analysis affords an innovative approach to study preferential expression of an allele in direct relationship to its genotype, providing information on cis-regulatory effects for the expression of putative genes. However currently, there are no lung cancer studies that have rigorously evaluated the ASE variation in lung tumor and adjacent tissues. Methods: Leveraging The Cancer Genome Atlas (TCGA) resource, we performed transcriptomic-wide ASE analysis using existing RNA-Seq datasets of paired tumor and adjacent tissues from 54 lung adenocarcinoma patients. We first quantified the RNA read counts of Referent and Alternate alleles of heterozygous variants, then evaluated the allelic imbalance on a per-sample basis using Beta-binomial test, and explored the differential ASE between tumor and adjacent tissues using paired Wilcoxon test. Functional regulatory consequences were generated from Ensembl Variant Effect Predictor. Results: We identified total 208 significant ASEs, including 35 tissue-specific (only in tumor or only in adjacent), 28 sharing, and 145 differential variants. Of the 208 candidates, 41 were from the human leukocyte antigen (HLA) locus (primary DQA2, DQB1, DRB1, H and J), 26 were from the immunoglobulin (IG) superfamily (primary IGH, IGL, IGK and F11R). About 80% candidates were noncoding (mostly in 5’ and 3’ untranslated regions) and with regulatory features (21 promoter, seven enhancer, seven open chromatin region, two induce nonsense-mediated mRNA decay, one CTCF-binding site, and one transcription factor binding site). Other top genes included MDM2, APOL1, and CTSB. Pathway analyses revealed 27 genes involved in immune response pathway, and 12 genes involved in HLA antigen processing and presentation pathway. Conclusion: This study is the first transcriptomics ASE analysis in lung adenocarcinoma. The key somatic cis-regulatory ASE variants identified from this study, especially immunogenic allelic variations from HLA and IG genes, could be used for identifying high-risk individuals for targeted lung cancer checkpoint blockade and related immunotherapies. Citation Format: Yanhong Liu, Spiridon Tsavachidis, Farrah Kheradmand, Margaret R. Spitz, Chris Amos. Transcriptome analysis links immune genes allelic expression imbalances to lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1584.

BackgroundThe simplest definition of cis-eQTLs versus trans, refers to genetic variants that affect expression in an allele specific manner, with implications on underlying mechanism. Yet, due to technical limitations of expression microarrays, the vast majority of eQTL studies performed in the last decade used a genomic distance based definition as a surrogate for cis, therefore exploring local rather than cis-eQTLs.ResultsIn this study we use RNAseq to explore allele specific expression (ASE) in adipose tissue of male and female F1 mice, produced from reciprocal crosses of C57BL/6J and DBA/2J strains. Comparison of the identified cis-eQTLs, to local-eQTLs, that were obtained from adipose tissue expression in two previous population based studies in our laboratory, yields poor overlap between the two mapping approaches, while both local-eQTL studies show highly concordant results. Specifically, local-eQTL studies show ~60% overlap between themselves, while only 15-20% of local-eQTLs are identified as cis by ASE, and less than 50% of ASE genes are recovered in local-eQTL studies. Utilizing recently published ENCODE data, we also find that ASE genes show significant bias for SNPs prevalence in DNase I hypersensitive sites that is ASE direction specific.ConclusionsWe suggest a new approach to analysis of allele specific expression that is more sensitive and accurate than the commonly used fisher or chi-square statistics. Our analysis indicates that technical differences between the cis and local-eQTL approaches, such as differences in genomic background or sex specificity, account for relatively small fraction of the discrepancy. Therefore, we suggest that the differences between two eQTL mapping approaches may facilitate sorting of SNP-eQTL interactions into true cis and trans, and that a considerable portion of local-eQTL may actually represent trans interactions.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2164-15-471) contains supplementary material, which is available to authorized users.

Esophageal squamous cell carcinoma (ESCC) is a highly aggressive malignancy, requiring effective biomarkers for prognosis and therapeutic responsiveness. Histone H3K9 methyltransferases (EHMT1 and EHMT2) are global genome organizers, which are crucial for maintaining the balance state of cells in a tissue-specific manner. It was previously suggested that EHMT1 expression is a predictor of prognosis in several malignant tumors; however, the prognostic significance of EHMT1 expression in ESCC has not been determined. A cohort of 50 ESCC cases and 46 paired normal esophageal tissue samples were evaluated to assess the levels of EHMT1 expression by immunohistochemistry and reverse transcription-polymerase chain reaction. The SPSS software package was used for statistical data analysis. A significantly upregulated EHMT1 expression was observed in squamous preinvasive lesions and ESCC compared to the matched normal esophageal epithelia (52.0 vs. 21.7%, respectively). The expression of EHMT1 was correlated with tumor grade (G), depth of invasion (T) and lymph node metastasis (N) in ESCC. EHMT1 overexpression was found to be associated with poor cancer-specific survival in squamous cell carcinomas (χ2=3.922, P=0.048). The expression of EHMT1 was identified as an independent prognostic factor for overall survival in ESCC patients. In conclusion, EHMT1 expression is upregulated in ESCC and early preinvasive esophageal squamous lesions and the overexpression of EHMT1 is associated with poor prognosis in ESCC. Therefore, the expression of EHMT1 may be an effective prognostic biomarker for ESCC.

Allele-specific expression (ASE) analysis improves the understanding of transcription’s cis-regulation. Herein, we used imputed SNPs along with RNA-Seq data from the Longissiumus thoracis muscle of 190 Nelore steers to identify functional cis-regulatory variants from ASE analysis. Using a Binomial Test, we identified 38,177 SNPs in ASE regions (ASE SNPs; FDR ≤0.05). We then searched for aseQTLs (SNPs potentially regulating the ASE) by comparing their heterozygosity to the measured allelic ratio under a Wilcoxon Rank Sum test. We identified 21,543 aseQTLs potentially regulating a total of 430 ASE SNPs (FDR ≤0.05). Based on a linear model, ASE SNPs and aseQTLs were associated with transcript abundance. We identified 3,333 SNPs acting as cis-eQTLs (FDR≤0.05). Results were integrated with previous ASE, functional regions, and meat quality-related differentially expressed genes data. This study described novel SNPs potentially regulating the transcription of genes that may affect beef traits.

Survivin, a new member of the family of apoptosis inhibitors, is expressed almost exclusively in proliferating cells, above all in cancers. Subcellular localisation and prognostic implications of the survivin protein have not yet been determined in oesophageal squamous cell carcinoma. The survival of 84 patients with oesophageal squamous cell carcinomas was correlated with the extent of immunohistochemical survivin expression in tumour cell nuclei. Tumours were scored positive when >5% cells stained positive. Patients were followed up for at least 5 years or until death. In normal oesophageal squamous cell epithelium, some cytoplasmic survivin expression was detected in the basal cells, whereas proliferating cells showed nuclear staining of survivin. Nuclear expression of survivin was also detected in 67 cancers (80%). The mean survival for patients of this group (28 months, range 20–36) was significantly less than that for patients without survivin expression in the tumour cell nuclei (108 months, range 62–154, P=0.003). Using univariate analysis, nuclear survivin expression (P=0.003), tumour depth (P=0.001), lymph node metastasis (P=0.003) and stage (P<0.001) were the best predictors of survival. In contrast, cytoplasmic survivin staining was noted in 53 (63%) tumours and had no prognostic relevance. In conclusion, the analysis of nuclear survivin expression identifies subgroups in oesophageal squamous cell cancer with favourable (survivin−) or with poor prognosis (survivin+). We suggest that the determination of nuclear survivin expression could be used to individualise therapeutic strategies in oesophageal squamous cell cancer in the future.

Background: c-Met is a receptor tyrosine kinase (RTK) that has been shown to be a potential target of therapy in a variety of human cancers. We investigated if c-Met served as a potential therapeutic target in esophageal squamous cell cancer (ESCC). Methods: Cytotoxicity (MTT) assay, cell migration assay and cell cycle analysis by flow cytometry were conducted in KYSE 510 and 170 ESCC cell lines (kindly provided by Dr. Yutaka Shimada) in response to various concentrations of c-met inhibitors, SU11274 and PHA665752. Apoptosis of ESCC cell lines was detected by TUNEL assay. Cell cycle regulatory proteins, cyclin A and B1, tumor suppressors including Rb, p53 p21 and p27, and intrinsic apoptotic factors were determined by Western blot analysis. Mice inoculated with KYSE 170 ESCC cell line were used to test therapeutic response of c-met inhibitors in vivo. Results: SU11274 and PHA665752 significantly suppressed viability and migration of KYSE 170 and 510 ESCC cells with decreased expression of phosphorylated c-Met (p-c-Met). Expression of p21, p27, p53 and Rb was upregulated in ESCC cell lines treated with c-Met inhibitors while inducing G1/G2 cell cycle arrest and apoptosis. Tumor growth in vivo was significantly suppressed in response to treatment with c-Met inhibitors with decreased expression of p-c-Met within the mice tumors. Conclusion: c-Met inhibitors exerted significant anticancer effects in vitro and in vivo for ESCC cell lines, suggesting that c-Met may serve as a potential 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 2878. doi:1538-7445.AM2012-2878