Background and Aim: Widespread applicability of genetic testing for Barrett’s esophagus depends on developing accurate methods for assaying archival formalin-fixed, paraffin-embedded (FFPE) histopathology specimens taken at endoscopy. We sought to develop an accurate mRNA comparative quantification method for FFPE tissues that would detect disease even in patients with Barrett’s intestinal metaplasia, who have small alterations in gene expression compared to patients with dysplasia or cancer.
Methods: mRNA was isolated from formalin-fixed, paraffin-embedded (FFPE) small endoscopic biopsies from 10 patients with IM and 11 control patients with a normal squamous-lined esophagus. IM tissues included only small numbers of glands in a single endoscopic biopsy in some cases. Multiplex tandem PCR (MT-PCR) was used to quantitate genes of interest for Barrett’s esophagus and control genes in duplicate using the Rotor-Gene 6000 system (Corbett Life Sciences).
Results: Significantly increased expression of hTERT, ODC1, MYB, SPARC, RAR-A, and significantly decreased expression of GSTP1 and RAR-G were found in IM compared to normal tissues. The direction and magnitude of the differences were very similar to those found using fresh frozen tissues. Two novel genes identified from a microarray study, CD151 (which encodes a protein that enhances cell motility, invasion and metastasis of cancer cells) and TCIRG1 (T-cell, immune regulator 1, ATPase, H+ transporting, lysosomal V0 subunit A3) were overexpressed in IM.
Conclusions: Accurate mRNA comparative quantification, matching fresh frozen tissue results, can be obtained from even single small formalin-fixed, paraffin-embedded Barrett’s esophagus endoscopic biopsy tissues using a multiplex tandem PCR method.