Spatially defined microsatellite analysis reveals extensive genetic mosaicism and clonal complexity in intestinal metaplastic glands

Abstract

Intestinal metaplasia (IM) has been recognized as the first irreversible precancerous stage of intestinal-type gastric cancer at which genetic instabilities, such as microsatellite (MS) instability and loss of heterozygosity, can already be detected. However, the extent and clonal relationship of these genetic lesions in the precancerous tissues are not fully appreciated. In this work, we have used well established MS markers to analyze the relatedness of spatially separated individual metaplastic glands as well as subsegments within single glands from the same patients. We found that individual IM glands frequently show different marker lengths even for closely apposed IM glands, suggesting that these tissues have already gained the ability to independently evolve their genome regardless of whether or not they share a common origin. Furthermore, within individual IM glands, there is also significant intra-gland diversity in the MS markers. Since most of these cells are not dividing and only have a limited lifespan, this result indicates that in each IM gland, a single dominant clone is rare and new clones are constantly created by either progenitor cells or stem cells. This greatly enhanced ability to create de novo genetic alterations may underlie the importance of this stage in the eventual progression toward cancer. Given the widely observed phenotype switch in the early stages of many solid tumors, whether this associated genetic stability is also an intrinsic property of metaplastic transformation should be extensively characterized to further our understanding of cancer initiation.

Complex clonal mosaicism within microdissected intestinal metaplastic glands without concurrent gastric cancer

“…The fact that each individual IM gland could have multiple coexisting clones suggests that genomic alterations are at a high frequency. These characteristics are consistent with those found with IM glands isolated from resected GA tissues 5. From this perspective, both IM tissues may share similar molecular processes related to genomic instability.…”

“…Among the 375 MS marker measurements in all IM glands, 19.2% showed length changes (see online supplementary table S6). This is comparable to that of IM glands from patients with GA where the overall frequency of MS marker variation was 18.6% 5. However, we note that there is a much higher probability for a length change in the intergenic regions (D2S123, D5S346 and BAT-25: ∼5%–6%) than in the coding regions (BAX: 1.6% and hMSH3: 0%) in these samples.…”

“…Since a single cell only contains ∼6.5 pg of DNA, sufficient amplification is required for the analysis of multiple MS markers 5. Although previous amplification based on degenerate oligonucleotide-primed PCR (DOP-PCR) can be used to this end,10 its coverage is less satisfactory (∼80% success rate) even for the few markers selected here 5. In this study, we found that amplification based on multiple annealing and looping-based cycles (MALBAC) yielded a much improved coverage for the tested markers.…”

“…A recent study of IM tissues from the periphery of resected cancer tissues found that there was considerable genomic instability at this stage 5 6. However, since these tissues coinhabit the same microenvironment as cancer and presumably over a prolonged period time, it is critical to determine whether such pervasive genetic instability is an inherent and early property of IM tissue alone.…”

Evolution of Premalignant Disease

“…The mosaic patterns of genetically and epigenetically diverse clonal populations in premalignant tissues evolve in space as well as time (Tycko 2003;Leedham et al 2008;Guo et al 2015). How do these clones and subclones physically expand in a tissue?…”