Recurring Translocations in Barrett’s Esophageal Adenocarcinoma

Abstract

Barrett’s esophagus (BE) is a premalignant metaplasia in patients with chronic gastroesophageal reflux disease (GERD). BE can progress to esophageal adenocarcinoma (EA) with less than 15% 5-year survival. Chromosomal aneuploidy, deletions, and duplication are early events in BE progression to EA, but reliable diagnostic assays to detect chromosomal markers in premalignant stages of EA arising from BE are lacking. Previously, we investigated chromosomal changes in an in vitro model of acid and bile exposure-induced Barrett’s epithelial carcinogenesis (BEC). In addition to detecting changes already known to occur in BE and EA, we also reported a novel recurring chromosomal translocation t(10:16) in the BE cells at an earlier time point before they undergo malignant transformation. In this study, we refine the chromosomal event with the help of fluorescence microscopy techniques as a three-way translocation between chromosomes 2, 10, and 16, t(2:10;16) (p22;q22;q22). We also designed an exclusive fluorescent in situ hybridization for esophageal adenocarcinoma (FISH-EA) assay that detects these chromosomal breakpoints and fusions. We validate the feasibility of the FISH-EA assay to objectively detect these chromosome events in primary tissues by confirming the presence of one of the fusions in paraffin-embedded formalin-fixed human EA tumors. Clinical validation in a larger cohort of BE progressors and non-progressors will confirm the specificity and sensitivity of the FISH-EA assay in identifying malignant potential in the early stages of EA.

Gene Expression in Barrett’s Esophagus Cell Lines Resemble Esophageal Squamous Cell Carcinoma Instead of Esophageal Adenocarcinoma

“…The notable observation that (unlike primary BE tissues) BE cell lines are ESCC-like (instead of EAC-like) was subsequently validated in a previously published in vitro BEC model [ 18 , 19 , 20 , 21 , 22 , 23 ], where we treated a BE cell line (BAR-T) with ABS for 5 min/day for 70 weeks ( Figure 5 A). Significant changes in cell morphology were observed at 34 and 46 weeks, and malignant characteristics such as formation of foci and colonies in soft agar and tumors in nude mice were observed beyond 58 weeks, as previously described [ 19 ].…”

“…A previously published BEC model [ 18 , 19 , 20 , 21 , 22 , 23 ] comprised of a non-neoplastic BE cell line (BAR-T) [ 24 ] exposed to acidic (pH4) bile salt mixture (ABS) for 5 min/day for up to 70 weeks. Significant changes in cell morphology were observed at 34 and 46 weeks, and malignant characteristics such as formation of colonies in soft agar and tumors in nude mice were observed after 58 weeks, as previously described [ 19 ].…”

“…We performed RNA-sequencing on a total of 18 samples: 10 samples collected at different time points from the BEC model (4 already transformed and 6 not yet transformed), and 8 untreated samples never exposed to ABS. Sequencing was performed in 3 sets at different sequencing centers: the first set ( Table S1 ) included 4 samples (1 each: untreated BAR-T, BEC20W, BEC40W, and BEC60W) [ 22 , 23 ]; the second set ( Table S2 ) included 10 samples: 3 untreated BAR-T (ctrl_0W_S1-3), 2 untreated BAR-T cells growing for 60 weeks without ABS exposure (ctrl_60W_S6-7), 2 BEC40W, and 3 BEC70W; and the third set ( Table S3 ) included 4 samples: 1 untreated BAR-T (ctrl_0W), 1 untreated BAR-T cells growing for 20 weeks without ABS exposure (ctrl_20W), 1 BEC20W, and 1 BEC50W.…”

“…This was because not only did the genes up in EAC vs. ESCC (EAC hi ) show substantially lower expression in BE cell lines than primary BE tissues, but also the genes up in ESCC vs. EAC (ESCC hi ) had substantially higher expression in BE cell lines than primary BE tissues. We validated this “squamous like” GEP of BE cell lines in a previously published in vitro model of BE carcinogenesis (BEC) [ 18 , 19 , 20 , 21 , 22 , 23 ], where prolonged acid and bile salt (ABS) exposure resulted in increased columnar markers [ 18 ], chromosomal aberrations [ 20 ], and malignant transformation [ 19 ] of a benign BE cell line (BAR-T) [ 24 ] but did not alter the “squamous like” GEP of the BE cell line. GEP of only one EAC case from the Cancer Genome Atlas (TCGA) [ 16 ] resembled the GEP of BE cell lines, and this EAC case was investigated in detail.…”

Barrett’s Epithelium to Esophageal Adenocarcinoma: Is There a “Point of No Return”?

“…biological functions associated with the phenotypes displayed during the same interval. Our previous observations (Bajpai et al, 2012(Bajpai et al, , 2021) also point to chromosomal rearrangements during this critical period in the BEC model that may account for catastrophic changes propelling the commitment of BE cells to malignant progression. There are other pieces of evidence in literature supporting the presence of chromosomal aberrations and stage-specific gene expression signatures associated with disease progression from BE to EAC (Garewal et al, 1989;Barrett et al, 1996;Newell et al, 2019).…”