Clonal analysis of isolated intestinal metaplastic glands of stomach using X linked polymorphism

Abstract

Background: Monoclonal precancerous cells undergo successive biochemical and genetic changes during the multistep process of carcinogenesis in the gastrointestinal tract. Despite a high association with intestinal-type stomach cancer (differentiated adenocarcinoma of the stomach), the role of intestinal metaplasia is unclear in stomach carcinogenesis.

Aims: To study the clonality of intestinal metaplasia.

Methods: The clonality of 86 single intestinal metaplastic glands isolated by EDTA treatment from gastrectomy specimens from patients with cancer were investigated. The methylation sensitive restriction enzyme HpaII and polymerase chain reaction (PCR) were used to detect a polymorphic human androgen receptor gene locus linked to an inactive X chromosome.

Results: Forty one (48%) intestinal metaplastic glands were heterotypic (mixed cells of different allelic methylation) and 45 (52%) were homotypic (cell population of the same allelic methylation), while almost all the single pyloric glands were homotypic. Eleven of 13 intestinal metaplastic mucosae that were 6 mm in diameter contained glands that had originated from different cells. There were no strong relationships between clonal type and location or histological type of intestinal metaplasia.

Conclusion: Intestinal metaplasia in general is not a lesion that arises or proceeds monoclonally.

Figure 1

Schematic representation of HUMARA gene. There is one polymorphic CAG repeat and two HpaII sites, which are methylation sensitive. These sites are completely methylated on inactive X alleles and unmethylated on active X alleles. The repetition number of the CAG is from 16 to 29 in Asian people.

Figure 2

Single intestinal metaplastic gland. (A) A feature under a stereomicroscope; original magnification × 70. (B)-(D) Sectioned and haematoxylin and eosin stained features of the gland under a microscope; original magnification × 100. These three glands are heterotypic, although there were no interstitial tissues.

Figure 3

Representative clonal analysis. All the samples were analysed in pairs consisting of those not digested and those digested with HpaII. In the ALFred pattern, the PCR product of higher molecular mass is on the right, with the lower molecular mass product on the left. The peaks on the right are products of longer repeats of HUMARA, and those on the left are products of shorter repeats. Gland A, heterotypic. The results after digestion with HpaII are the same as those with no digestion. Gland B, homotypic of longer allelic methylation. Gland C, homotypic of shorter allelic methylation. HpaII(−), not digested with HpaII; HpaII(+), digested with HpaII.

Figure 4

Results for three glands that showed replication errors (RERs). Gland C1-1-1 (case 1, lesion 1, gland 1) is a control gland from the same case, which is heterotypic and does not show RERs. The profiles for the three glands (C1-2-1, C1-2-2, C1-2-3) have one extra small peak on the right when they are not digested with HpaII. The profiles all differ after digestion with HpaII.