Introduction Intestinal metaplasia of the stomach has been heavily implicated as a premalignant condition that can lead to gastric adenocarcinoma (GA). However, while there have been many studies describing the mutation load within GA, there is little evidence to show its genetic origins from metaplasia. Furthermore previous studies from our laboratory have shown that the development of a field metaplasia depends on the division of metaplastic crypts to form a patch. However there is some controversy about the clonality of such crypts. Here we describe experiments where we define the clonal nature of metaplastic crypts within the human stomach and demonstrate the genetic relationship these have with associated dysplasia.
Methods Blocks of gastric adenocarcinoma with dysplasia-associated metaplasia were used in this study. Sections were stained with a dual enzyme assay for mitochondrial cytochrome c oxidase (CCO) and succinate dehydrogenase (SDH). It has been established that mitochondrial DNA (mtDNA) mutations that result in CCO-deficiency, are a reliable marker of clonality. We laser-captured individual cells from a single CCO-deficient crypt and PCR sequenced the entire mtDNA genome. To determine the genetic relationship between metaplasia and dysplasia we laser captured crypts along a strip of gastric mucosa that contained patches of metaplastic and dysplastic crypts. We then screened these for TP53 and APC mutations by nested PCR sequencing.
Results Every cell within a CCO-deficient metaplastic crypt contained the same mtDNA mutation (G12898A transition in mtCOII, and therefore clona), whereas all cells from neighbouring CCO-positive crypts were wild type. Screening in one patient with GA revealed a truncating APC mutation (c.1450 G insertion). All crypts were laser captured and genotyped for this mutation. We demonstrated that 100% of dysplastic crypts were APC-mutated, interestingly, there were a few metaplastic crypts that also contained the same mutation, indicating the dysplasia did arise from a field of metaplasia. These findings were confirmed in two other patients with APC and TP53 mutations.
Conclusion Contrary to previous reports, we have demonstrated that intestinal metaplastic crypts are clonal. Our work shows that there is a founder mutation within each dysplastic lesion. The same founder mutation can be detected within the surrounding metaplasia proving that dysplasia arises from metaplasia in the human stomach.
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