Application of a multi-gene next-generation sequencing panel to a non-invasive oesophageal cell-sampling device to diagnose dysplastic Barrett’s oesophagus

26 Jul 2017

The early detection and endoscopic treatment of patients with the dysplastic stage of Barrett’s oesophagus is key to preventing progression to oesophageal adenocarcinoma. However, endoscopic surveillance protocols are hampered by the invasiveness of repeat endoscopy, sampling bias and a subjective histopathological diagnosis of dysplasia. In this case-control study we investigated the use of a non-invasive, pan-oesophageal cell-sampling device, the Cytosponge™, coupled with a cancer hotspot panel to identify patients with dysplastic Barrett’s oesophagus. Formalin-fixed, paraffin-embedded (FFPE) Cytosponge samples from 31 patients with non-dysplastic and 28 with dysplastic Barrett’s oesophagus with good available clinical annotation were selected for inclusion. Sample were microdissected and amplicon sequencing performed using a panel covering >2800 COSMIC hotspot mutations in 50 oncogenes and tumour suppressor genes. Strict mutation criteria were determined and duplicates were run to confirm any mutations with an allele frequency <12%. When compared with endoscopy and biopsy as the gold standard the panel achieved a 71.4% sensitivity (95% CI 51.3-86.8) and 90.3% (95% CI 74.3-98.0) specificity for diagnosing dysplasia. TP53 had the highest rate of mutation in 14/28 dysplastic samples (50%). CDKN2A was mutated in 6/28 (21.4%), ERBB2 in 3/28 (10.7%) and 5 other genes at lower frequency. The only gene from this panel found to be mutated in the non-dysplastic cases was CDKN2A in 3/31 cases (9.7%) in keeping with its known loss early in the natural history of the disease. Hence, it is possible to apply a multi-gene cancer hotspot panel and nextgeneration sequencing to microdissected, FFPE samples collected by the Cytosponge, in order to distinguish non-dysplastic from dysplastic Barrett’s oesophagus. Further work is required to maximise the panel sensitivity.