Jean S. Wang

The MicroRNAs, MiR-31 and MiR-375, as Candidate Markers in Barrett's Esophageal Carcinogenesis

There is a critical need to identify molecular markers that can reliably aid in stratifying esophageal adenocarcinoma (EAC) risk in patients with Barretts esophagus. MicroRNAs (miRNA/miR) are one such class of biomolecules. In the present cross‐sectional study, we characterized miRNA alterations in progressive stages of neoplastic development, i.e., metaplasia–dysplasia–adenocarcinoma, with an aim to identify candidate miRNAs potentially associated with progression. Using next generation sequencing (NGS) as an agnostic discovery platform, followed by quantitative real‐time PCR (qPCR) validation in a total of 20 EACs, we identified 26 miRNAs that are highly and frequently deregulated in EACs (≥4‐fold in >50% of cases) when compared to paired normal esophageal squamous (nSQ) tissue. We then assessed the 26 EAC‐derived miRNAs in laser microdissected biopsy pairs of Barretts metaplasia (BM)/nSQ (n = 15), and high‐grade dysplasia (HGD)/nSQ (n = 14) by qPCR, to map the timing of deregulation during progression from BM to HGD and to EAC. We found that 23 of the 26 candidate miRNAs were deregulated at the earliest step, BM, and therefore noninformative as molecular markers of progression. Two miRNAs, miR‐31 and −31*, however, showed frequent downregulation only in HGD and EAC cases suggesting association with transition from BM to HGD. A third miRNA, miR‐375, showed marked downregulation exclusively in EACs and in none of the BM or HGD lesions, suggesting its association with progression to invasive carcinoma. Taken together, we propose miR‐31 and −375 as novel candidate microRNAs specifically associated with early‐ and late‐stage malignant progression, respectively, in Barretts esophagus.

microRNA 223 Is Upregulated in the Multistep Progression of Barrett's Esophagus and Modulates Sensitivity to Chemotherapy by Targeting PARP1

Purpose: Recent microarray and RNA-sequencing studies have uncovered aberrantly expressed microRNAs (miRNA) in Barretts esophagus–associated esophageal adenocarcinoma. The functional significance of these miRNAs in esophageal adenocarcinoma initiation and progression is largely unknown. Experimental Design: Expression levels of miR-199a/b-3p, -199a-5p, -199b-5p, -200b, -200c, -223, and -375 were determined in microdissected tissues from cardiac mucosa, Barretts esophagus, dysplastic Barretts esophagus, and esophageal adenocarcinoma using quantitative real-time PCR. miR-223 expression was validated in precursors and esophageal adenocarcinomas from 95 patients with esophageal adenocarcinoma by in situ hybridization (ISH). miR-223 was transfected into two esophageal adenocarcinoma cell lines, and in vitro assays were conducted. Target genes were identified using Illumina microarray, and results were validated in cell lines and human specimens. Results: miR-199 family members and miR-223 were significantly overexpressed in esophageal adenocarcinoma, however, only miR-223 showed a stepwise increase during esophageal adenocarcinoma carcinogenesis. A similar trend was observed by ISH, which additionally showed that miR-223 is exclusively expressed by the epithelial compartment. miR-223–overexpressing cells had statistically significantly more migratory and invasive potential than scramble sequence–transfected cells. PARP1 was identified as a direct target gene of miR-223 in esophageal adenocarcinoma cells. Increased sensitivity to chemotherapy was observed in cells with enforced miR-223 expression and reduced PARP1. Conclusions: miR-223 is significantly upregulated during the Barretts esophagus–dysplasia–esophageal adenocarcinoma sequence. Although high miR-223 levels might contribute to an aggressive phenotype, our results also suggest that patients with esophageal adenocarcinoma with high miR-223 levels might benefit from treatment with DNA-damaging agents. Clin Cancer Res; 19(15); 4067–78. ©2013 AACR.

RNA sequencing identifies transcriptionally viable gene fusions in esophageal adenocarcinomas

Esophageal adenocarcinoma is a deadly cancer with increasing incidence in the United States, but mechanisms underlying pathogenesis are still mostly elusive. In addressing this question, we assessed gene fusion landscapes by comprehensive RNA sequencing (RNAseq) of 55 pretreatment esophageal adenocarcinoma and 49 nonmalignant biopsy tissues from patients undergoing endoscopy for Barretts esophagus. In this cohort, we identified 21 novel candidate esophageal adenocarcinoma-associated fusions occurring in 3.33% to 11.67% of esophageal adenocarcinomas. Two candidate fusions were selected for validation by PCR and Sanger sequencing in an independent set of pretreatment esophageal adenocarcinoma (N = 115) and nonmalignant (N = 183) biopsy tissues. In particular, we observed RPS6KB1-VMP1 gene fusion as a recurrent event occurring in approximately 10% of esophageal adenocarcinoma cases. Notably, esophageal adenocarcinoma cases harboring RPS6KB1-VMP1 fusions exhibited significantly poorer overall survival as compared with fusion-negative cases. Mechanistic investigations established that the RPS6KB1-VMP1 transcript coded for a fusion protein, which significantly enhanced the growth rate of nondysplastic Barretts esophagus cells. Compared with the wild-type VMP1 protein, which mediates normal cellular autophagy, RPS6KB1-VMP1 fusion exhibited aberrant subcellular localization and was relatively ineffective in triggering autophagy. Overall, our findings identified RPS6KB1-VMP1 as a genetic fusion that promotes esophageal adenocarcinoma by modulating autophagy-related processes, offering new insights into the molecular pathogenesis of esophageal adenocarcinomas. Cancer Res; 76(19); 5628-33. ©2016 AACR.

Identifying DNA methylation biomarkers for non-endoscopic detection of Barrett’s esophagus

Combining DNA methylation markers with a swallowable device for sampling the distal esophagus effectively detects Barrett’s neoplasias. A test that goes down easy Barrett’s esophagus is a premalignant condition of the distal esophagus that increases the risk of esophageal cancer. Unfortunately, screening for Barrett’s esophagus currently requires endoscopy, an invasive and expensive procedure, and thus, it is not routinely performed. Moinova et al. have now demonstrated a simplified approach to screening by identifying a pair of DNA methylation markers that correlate with the presence of Barrett’s esophagus. The authors also invented a swallowable balloon-based device that can capture DNA samples for methylation analysis and found that it is well tolerated in patients and provides >90% sensitivity and specificity compared to endoscopy, suggesting its potential as a screening method. We report a biomarker-based non-endoscopic method for detecting Barrett’s esophagus (BE) based on detecting methylated DNAs retrieved via a swallowable balloon-based esophageal sampling device. BE is the precursor of, and a major recognized risk factor for, developing esophageal adenocarcinoma. Endoscopy, the current standard for BE detection, is not cost-effective for population screening. We performed genome-wide screening to ascertain regions targeted for recurrent aberrant cytosine methylation in BE, identifying high-frequency methylation within the CCNA1 locus. We tested CCNA1 DNA methylation as a BE biomarker in cytology brushings of the distal esophagus from 173 individuals with or without BE. CCNA1 DNA methylation demonstrated an area under the curve of 0.95 for discriminating BE-related metaplasia and neoplasia cases versus normal individuals, performing identically to methylation of VIM DNA, an established BE biomarker. When combined, the resulting two biomarker panel was 95% sensitive and 91% specific. These results were replicated in an independent validation cohort of 149 individuals who were assayed using the same cutoff values for test positivity established in the training population. To progress toward non-endoscopic esophageal screening, we engineered a well-tolerated, swallowable, encapsulated balloon device able to selectively sample the distal esophagus within 5 min. In balloon samples from 86 individuals, tests of CCNA1 plus VIM DNA methylation detected BE metaplasia with 90.3% sensitivity and 91.7% specificity. Combining the balloon sampling device with molecular assays of CCNA1 plus VIM DNA methylation enables an efficient, well-tolerated, sensitive, and specific method of screening at-risk populations for BE.

Linkage and related analyses of Barrett's esophagus and its associated adenocarcinomas

Familial aggregation and segregation analysis studies have provided evidence of a genetic basis for esophageal adenocarcinoma (EAC) and its premalignant precursor, Barretts esophagus (BE). We aim to demonstrate the utility of linkage analysis to identify the genomic regions that might contain the genetic variants that predispose individuals to this complex trait (BE and EAC).

Genomic regions associated with susceptibility to Barrett’s esophagus and esophageal adenocarcinoma in African Americans: The cross BETRNet admixture study

Background Barrett’s esophagus (BE) and esophageal adenocarcinoma (EAC) are far more prevalent in European Americans than in African Americans. Hypothesizing that this racial disparity in prevalence might represent a genetic susceptibility, we used an admixture mapping approach to interrogate disease association with genomic differences between European and African ancestry. Methods Formalin fixed paraffin embedded samples were identified from 54 African Americans with BE or EAC through review of surgical pathology databases at participating Barrett’s Esophagus Translational Research Network (BETRNet) institutions. DNA was extracted from normal tissue, and genotyped on the Illumina OmniQuad SNP chip. Case-only admixture mapping analysis was performed on the data from both all 54 cases and also on a subset of 28 cases with high genotyping quality. Haplotype phases were inferred with Beagle 3.3.2, and local African and European ancestries were inferred with SABER plus. Disease association was tested by estimating and testing excess European ancestry and contrasting it to excess African ancestry. Results Both datasets, the 54 cases and the 28 cases, identified two admixture regions. An association of excess European ancestry on chromosome 11p reached a 5% genome-wide significance threshold, corresponding to -log10(P) = 4.28. A second peak on chromosome 8q reached -log10(P) = 2.73. The converse analysis examining excess African ancestry found no genetic regions with significant excess African ancestry associated with BE and EAC. On average, the regions on chromosomes 8q and 11p showed excess European ancestry of 15% and 20%, respectively. Conclusions Chromosomal regions on 11p15 and 8q22-24 are associated with excess European ancestry in African Americans with BE and EAC. Because GWAS have not reported any variants in these two regions, low frequency and/or rare disease associated variants that confer susceptibility to developing BE and EAC may be driving the observed European ancestry association evidence.

Conducting a randomized trial in rural and urban safety-net health centers: Added value of community-based participatory research

Background Colorectal cancer (CRC) is the second most common cancer in the US. Despite evidence that screening reduces CRC incidence and mortality, screening rates are sub-optimal with disparities by race/ethnicity, income, and geography. Rural-urban differences in CRC screening are understudied even though approximately one-fifth of the US population lives in rural areas. This focus on urban populations limits the generalizability and dissemination potential of screening interventions. Methods Using community-based participatory research (CBPR) principles, we designed a cluster-randomized trial, adaptable to a range of settings, including rural and urban health centers. We enrolled 483 participants across 11 health centers representing 2 separate networks. Both networks serve medically-underserved communities; however one is primarily rural and one primarily urban. Results Our goal in this analysis is to describe baseline characteristics of participants and examine setting-level differences. CBPR was a critical for recruiting networks to the trial. Patient respondents were predominately female (61.3%), African-American (66.5%), and earned <

Columnar islands in Barrett's esophagus: Do they impact Prague C&M criteria and dysplasia grade?

1200 per month (87.1%). The rural network sample was older; more likely to be female, white, disabled or retired, and have a higher income, but fewer years of education. Conclusions Variation in the samples partly reflects the CBPR process and partly reflects inherent differences in the communities. This confirmed the importance of using CBPR when planning for eventual dissemination, as it enhanced our ability to work within diverse settings. These baseline findings indicate that using a uniform approach to implementing a trial or intervention across diverse settings might not be effective or efficient.

Abstract B104: Medical mistrust and cancer screening in an underserved population

The standard for classifying Barretts metaplasia on endoscopy, the Prague C&M criteria, ignores all islands of metaplastic‐appearing tissue. The aims of the present study were to measure the prevalence of columnar islands, quantify their impact on metaplasia extent, and determine if they harbor advanced dysplasia.

Abstract B17: Challenges tracking underserved participants in a longitudinal study

Racial/ethnic minorities and those of lower socioeconomic status suffer disproportionate rates of cancer incidence and mortality. They are also less likely to engage in preventive screening practices. The potential impact for improved screening rates is great. Screening for breast and colon cancer, two of the top five cancer sites, has been shown to reduce mortality. Results are more controversial for the effect on prostate cancer mortality, another of the top five cancer sites in men. Among reported reasons for lower screening practices are mistrust of the healthcare system, fear of the procedures or results, lack of or inadequate insurance coverage, and fatalistic beliefs. Racial/ethnic minorities are typically thought to exhibit higher levels of medical mistrust than their Caucasian counterparts. The purpose of this analysis was to examine associations between medical mistrust and specific cancer screening behaviors – breast, colon, and prostate cancer screening – in a low-income urban and predominately racial minority adult population. As part of a longitudinal survey study to assess colon cancer screening practices, participants were recruited from federally qualified health centers in an urban Midwest city. Eligibility included age ≥40 and being a patient at a federally qualified health center. Interviewer-administered surveys were used to collect baseline data on self-reported cancer screening practices, health history, insurance status, and demographics. Medical mistrust was assessed using the Group Based Medical Mistrust Scale. The study population included 144 individuals and was 61% male, with an average age of 51 years (±6.73 SD). Participants were predominately African American (87.5%), uninsured (52%), and low income, with 42.4% earning a monthly income Preliminary results indicate that there were no significant differences in medical mistrust by gender, having a healthcare home (defined as one particular doctor9s office individuals visit when sick), or by ever having been screened for colon, breast, or prostate cancer. Bivariate analysis found that having a healthcare home was significantly associated with having had a mammography (p=0.02), CRC screening (p=0.012), and prostate cancer screening (p Medical mistrust may be a less important determinant in nonadherence of low-income, racial/ethnic minority adults to screening guidelines than previously thought. As medical mistrust levels did not differ by gender or healthcare home status, it may be that focusing efforts on connecting low-income individuals with regular sources of care in which they will experience a greater continuity in care has the potential to increase cancer screening and preventive behaviors. This may provide avenues for interventions focused on eliminating disparities in cancer screening rates among minorities and those of lower socioeconomic status. Citation Information: Cancer Epidemiol Biomarkers Prev 2011;20(10 Suppl):B104.