Clonal expansion and loss of heterozygosity at chromosomes 9p and 17p in premalignant esophageal (Barrett's) tissue

Abstract

Background: Abnormalities involving the p16 (also known as cyclin-dependent kinase N2 [CDKN2], p16 [INK4a], or MTS1) and p53 (also known as TP53) tumor suppressor genes are highly prevalent in esophageal adenocarcinomas. Loss of heterozygosity (LOH) at 9p21 and 17p13 chromosomes (locations for p16 and p53 genes, respectively) is frequently observed in the premalignant condition, Barrett's esophagus. We studied extensively the distribution and heterogeneity of LOH at 9p and 17p chromosomes throughout the Barrett's segment in patients who have not yet developed esophageal adenocarcinoma.

Methods: We evaluated 404 samples from 61 consecutive patients enrolled in the Seattle Barrett's Esophagus Study from February 1995 through September 1998. All patients had high-grade dysplasia but no diagnosis of cancer. The samples were assayed for LOH at 9p and 17p chromosomes after amplification of genomic DNA by use of polymerase chain reaction and DNA genotyping. The cell fractions were purified by flow cytometry on the basis of DNA content and proliferation-associated antigen labeling. Association between LOH at 9p and LOH at 17p with flow cytometric abnormalities was determined by chi-squared test, and logistic regression models were used to model and test for the extent to which a particular genotype was found in 2-cm intervals.

Results and conclusions: LOH at 9p and 17p chromosomes are highly prevalent somatic genetic lesions in premalignant Barrett's tissue. LOH at 9p is more common than LOH at 17p in diploid samples and can be detected over greater regions of Barrett's epithelium. In most patients with high-grade dysplasia, the Barrett's mucosa contains a mosaic of clones and subclones with different patterns of LOH. Some clones had expanded to involve extensive regions of Barrett's epithelium. LOH at 9p and 17p chromosomes may be useful biomarkers to stratify patients' risk of progression to esophageal cancer.

Fig. 1

Prevalence of loss of heterozygosity (LOH) at chromosomes 9p and 17p in flow cytometrically sorted cell populations from patient biopsy tissues from Barrett’s epithelium. A total of 404 sorted fractions from 60 patients with a final diagnosis of high-grade dysplasia were evaluated, including 53 2N fractions (diploid G₀/G₁), 167 G₁ fractions (Ki67-positive G₁), 108 G₂/M fractions (4N DNA content composed of <6% of the cells in the biopsy), 15 increased 4N fractions (4N DNA content composed of >6% of the cells in the biopsy), and 61 aneuploid populations.

Fig. 2

Loss of heterozygosity (LOH) along the Barrett’s epithelial segment in individual patients. Six examples of patients with different LOH patterns. OS (ora serrata) and LES (lower esophageal sphincter) are endoscopic features used to define the region of Barrett’s within the esophagus. cm = location along the esophagus in centimeters from the incisors; sort = type of sample sorted (either Ki67-positive G₁, Ki67-negative 2N, 4N [normal G₂/M], Inc 4N [increased 4N], or aneuploid); open circles = no LOH; filled circles = LOH; and hatched circles = alternate allele lost. A) Case No. 252, single clone; B) case No. 916, change in ploidy; C) case No. 994, accumulation of loss; D) case No. 368 and E) case No. 790, mutually exclusive clones; and F) case No. 306, alternate allele patterns. Asterisk indicates a different LOH pattern along the same chromosome, i.e., different extent of loss.

Fig. 3

Genotyping by use of fluorescent staining of markers for loss of heterozygosity (LOH) analysis. See Fig. 2 legend for the definitions of OS and LES. Fluorescent traces (graphic) for LOH results for all chromosome 9 loci in case No. 336 are shown. A) The Q^LoH ratio (see “Materials and Methods” section) is given below the peaks. LOH is indicated by an arrow pointing to the lost allele. B) Schematic of all LOH results along the Barrett’s epithelial segment. An extra allele was found in the diploid and aneuploid populations from level 32, but additional alleles were not used as markers of clonality in this study. Asterisk indicates different LOH pattern along the same chromosomes, as indicated by boxed patterns. Open circles = no LOH; filled circles = LOH.

Fig. 4

Expansion of loss of heterozygosity (LOH) at 9p and 17p chromosomes with and without flow-cytometric abnormalities. The levels with LOH (y-axis) in each flow-cytometric category (increased 4N and/or aneuploid [panels A– < C] or diploid [panels D– < F]) were plotted against the number of (2-cm) levels evaluated for each patient (x-axis). The number of 2-cm levels evaluated varies relative to the Barrett’s segment length. Samples with 9p LOH only are plotted on panels A and D, 17p LOH are plotted only on panels B and E, and both 9p LOH and 17p LOH are plotted together on panels C and F. Points on the dashed diagonal line have undergone complete expansion within the Barrett’s segment, points on the horizontal have not undergone expansion, and clones in between have undergone incomplete expansion. The percent of clones with expansion is given in the upper left corner of each graph.