Next-generation sequencing of endoscopic biopsies identifies ARID1A as a tumor-suppressor gene in Barrett's esophagus

Abstract

The incidence of Barrett's esophagus (BE)-associated esophageal adenocarcinoma (EAC) is increasing. Next-generation sequencing (NGS) provides an unprecedented opportunity to uncover genomic alterations during BE pathogenesis and progression to EAC, but treatment-naive surgical specimens are scarce. The objective of this study was to establish the feasibility of using widely available endoscopic mucosal biopsies for successful NGS, using samples obtained from a BE 'progressor'. Paired-end whole-genome NGS was performed on the Illumina platform using libraries generated from mucosal biopsies of normal squamous epithelium (NSE), BE and EAC obtained from a patient who progressed to adenocarcinoma during endoscopic surveillance. Selective validation studies, including Sanger sequencing, immunohistochemistry and functional assays, were performed to confirm the NGS findings. NGS identified somatic nonsense mutations of AT-rich interactive domain 1A (SWI like) (ARID1A) and PPIE and an additional 37 missense mutations in BE and/or EAC, which were confirmed by Sanger sequencing. ARID1A mutations were detected in 15% (3/20) high-grade dysplasia (HGD)/EAC patients. Immunohistochemistry performed on an independent archival cohort demonstrated ARID1A protein loss in 0% (0/76), 4.9% (2/40), 14.3% (4/28), 16.0% (8/50) and 12.2% (12/98) of NSE, BE, low-grade dysplasia, HGD and EAC tissues, respectively, and was inversely associated with nuclear p53 accumulation (P=0.028). Enhanced cell growth, proliferation and invasion were observed on ARID1A knockdown in EAC cells. In addition, genes downstream of ARID1A that potentially contribute to the ARID1A knockdown phenotype were identified. Our studies establish the feasibility of using mucosal biopsies for NGS, which should enable the comparative analysis of larger 'progressor' versus 'non-progressor' cohorts. Further, we identify ARID1A as a novel tumor-suppressor gene in BE pathogenesis, reiterating the importance of aberrant chromatin in the metaplasia-dysplasia sequence.

Figure 1

A network of mutated tier 1 genes. Genes harboring tier 1 mutations are colored in gray.

Figure 2

Immunohistochemistry for ARID1A (left panels) and p53 (right panels) in representative cores of BE (a, b, 20 × ), EAC (c, d, 20 × ) and lymph node metastasis (LNM; e, f, 20 ×) lesions. In all lesions, nuclear ARID1A expression is completely lost, whereas there is nuclear accumulation of p53. The NSE in image (c, d, black arrows) served as control for staining as ARID1A is present and p53 is absent. The red arrows (c, d) indicate EAC.

Figure 3

(a) Immunohistochemistry results for lesions in which complete ARID1A loss was detected. Nuclear accumulation of p53 was observed in 5/12 EAC ARID1A loss patients. ARID1A loss was observed in 0% (0/76), 4.9% (2/41), 14.3% (4/28), 16% (8/50), 12.2% (12/98) and 6.5% (2/31) of the NSE, BE, LGD, HGD, EAC and lymph node metastasis (LNM), respectively (b). There was a stepwise increase in presence of nuclear accumulation of p53 seen during neoplastic progression of BE (c).

Figure 4

Robust ARID1A expression was detected at the expected size in OE33, JHesoAD1 and HEEpiC cells. ARID1A protein was efficiently knocked down (81% compared with mock OE33) in OE33 cells (a). A significant increase in cell growth (b) and promotion of invasion (c) was observed in the ARID1A KD OE33 cells. The brown staining in photo (d, 20 × ) and (e, 20 × ), which are example photos, depicts incorporation of BrdU in mock and ARID1A KD cells, respectively. Overall, 48.8% and 67.9% of the mock and ARID1A KD cells, respectively, were proliferative 36–48 h after transfection (P <0.001). (f) Shows quantitative reverse transcriptase (qRT)-PCR validation results (four biological replicates) of the microarray experiment. The error bar in the graph indicates the standard error of the mean, whereas the asterisk depicts a P <0.05 between ARID1A KD and mock OE33 cells.

Figure 5

A flowchart depicting the comparison strategy to identify EAC-specific, BE-specific and shared SNVs.