(A) Overview of the opened resection specimen shows columnar metaplasia across the gastro-oesophageal junction (arrows) and a nodular OAC (arrowhead). (B) H&E-stained cryostat section (left) of the longitudinal strip across the gastro-oesophageal junction reveals columnar metaplasia of the distal esophagus (arrows) and an OAC at the squamocolumnar junction (arrowhead). Submucosal gland complex (asterisk) confirms the oesophageal origin. Cytochrome c oxidase (CCO) staining of this longitudinal strip (right) shows several discontinuous epithelial patches that are CCO-deficient. (C) Overview and high-power photomicrographs of one CCO-deficient epithelial patch and the associated OAC and the mosaic spread of the CCO-deficient clone within the background mucosa. (D) Deep next-generation mitochondrial DNA (mtDNA) sequencing reveals unique mtDNA mutations within the CCO-deficient epithelial patch shown in (C). Barchart shows the variant allele fractions (VAFs) of the mutations in the control (stroma) sample and in material from the clonal expansion, mtDNA mutations are indicated. (E) Sanger resequencing shows that spatially distinct CCO-deficient epithelial patches and the OAC carry the same 16290C> T mtDNA mutation. (F) Consecutive sections of neighbouring glands show cardia-type metaplasia and intestinal metaplasia. Top left: H& E staining shows the absence of goblet cell differentiation in the non-dysplastic cardia-type epithelium (marked by arrowhead), whereas the neighbouring intestinal metaplasia shows abundant goblet cells (marked by an asterisk). Top right: clonal loss of CCO activity in the cardia-type epithelium (marked by arrowhead). Bottom left: CDX2 staining confirms the absence of internalisation in CCO-deficient cardia-type metaplasia. Strong nuclear labelling is seen in neighbouring intestinal metaplasia (marked by arrowhead). Bottom right: low proliferative activity as shown by Ki67 proliferation marker stain, consistent with the morphological absence of dysplasia. Evolution of oesophageal adenocarcinoma from metaplastic columnar epithelium without goblet cells in Barrett's oesophagus: Lavery DL, Martinez P, Gay LJ, Cereser B, Novelli MR, Rodriguez-Justo M, Meijer SL, Graham TA, McDonald SA, Wright NA, Jansen M - Gut (2015). Not altered. CC.