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Study Points to Mutations Present in Pre-cancerous Barrett's Esophagus FFPE Samples

NEW YORK (GenomeWeb) – Sequencing non-cancerous esophageal tissue in individuals with Barrett's esophagus may help to predict which individuals will progress to advanced diseases such as high-grade dysplasia or esophageal cancer, according to a study appearing online today in the Journal of Molecular Diagnostics.

Researchers from the US and Japan used high-throughput sequencing to test formalin-fixed, paraffin-embedded (FFPE), non-neoplastic tissue samples from 28 individuals with a Barrett's esophagus-related condition called Barrett's intestinal metaplasia (BIM), in which cells lining the esophagus start to resemble those found in the small intestine. They also tested samples from four individuals without BIM.

In three-quarters of the successfully sequenced BIM cases that progressed to high-grade dysplasia or esophageal adenocarcinoma, the team uncovered mutations involving cancer-related genes such as TP53, APC, and CDKN2A — alterations that were not picked up in BIM-negative individuals or in cases that didn't progress.

"The ability to detect mutations in non-neoplastic mucosa, quantitatively and with high detection sensitivity, makes it possible to use [next-generation sequencing] mutational testing in the early detection and surveillance of patients who develop [Barrett's esophagus]," corresponding author Antonia Sepulveda, a pathology and cell biology researcher at Columbia University, said in a statement.

Barrett's esophagus — a potential complication of gastroesophageal reflux disease — can significantly raise the risk of developing esophageal adenocarcinoma, the team noted. But beyond sometimes difficult-to-discern pathological examinations of biopsies of Barrett's esophagus mucus collected by endoscopy, there has been no reliable method to track progression to more serious forms of disease.

"Previously evaluated testing approaches for [esophageal adenocarcinoma] risk stratification in [Barrett's esophagus] using esophageal biopsy samples include nuclear DNA content abnormalities, such as aneuploidy and tetraploidy; gene copy number alterations, such as loss of heterozygosity of p16 (CDKN2A) and p53 (TP53); somatic gene mutations; and hyper-methylation of a number of genes," Sepulveda and co-authors noted.

Nevertheless, they noted that little is known about the feasibility of detecting such alterations from the sort of FFPE samples collected during a typical endoscopy procedure on Barrett's esophagus in the clinic.  

To investigate the possibility of using sequencing-based mutation profiling to predict progression with typical clinical specimens, the researchers did targeted cancer gene sequencing on FFPE samples from 32 individuals: 15 individuals with BIM who went on to develop high-grade dysplasia or esophageal adenocarcinoma, 13 individuals with BIM but no progression, and four without BIM.

The team assessed 50 oncogenes or tumor suppressors in 19 of the samples using version 2 of the AmpliSeq Cancer Hotspot panel, followed by sequencing on the Thermo Fisher Scientific Ion Torrent PGM with a 318 chip. It also used the Illumina MiSeq instrument to do targeted sequencing on the 48 genes found in the Illumina TruSeq Cancer Panel for 13 cases, including 10 of those tested with the Ion Torrent instrument.

From these sequences, the researchers found apparently pathogenic mutations in six of the eight BIM-progressor cases for which they had sufficient material for sequencing. Three of these BIM samples involved mutations to TP53 alone, two contained TP53 mutations as well as CDKN2A mutations, and one carried an APC alteration.

For the remaining seven BIM cases that progressed, they profiled mutations in the tumor or dysplastic tissue only, uncovering mutations to TP53, APC, CDKN2A, KRAS, FGFR, and PDGFRA.

In contrast, the team did not detect mutations in the cancer panel genes in non-progressed BIM cases or in individuals who had not developed BIM.

"[O]ur data indicate that DNA from routine endoscopic FFPE samples of non-dysplastic BIM can be efficiently used to simultaneously detect multiple mutations by [next-generation sequencing] with high analytical sensitivity, enabling the application of genomic testing of [Barrett's esophagus] patients for improved [high-grade dysplasia] and [esophageal adenocarcinoma] surveillance in clinical practice," the study's authors concluded.