High resolution chromosome 3p, 8p, 9q and 22q allelotyping analysis in the pathogenesis of gallbladder carcinoma

Abstract

Our recent genome-wide allelotyping analysis of gallbladder carcinoma identified 3p, 8p, 9q and 22q as chromosomal regions with frequent loss of heterozygosity. The present study was undertaken to more precisely identify the presence and location of regions of frequent allele loss involving those chromosomes in gallbladder carcinoma. Microdissected tissue from 24 gallbladder carcinoma were analysed for PCR-based loss of heterozygosity using 81 microsatellite markers spanning chromosome 3p (n=26), 8p (n=14), 9q (n=29) and 22q (n=12) regions. We also studied the role of those allele losses in gallbladder carcinoma pathogenesis by examining 45 microdissected normal and dysplastic gallbladder epithelia accompanying gallbladder carcinoma, using 17 microsatellite markers. Overall frequencies of loss of heterozygosity at 3p (100%), 8p (100%), 9q (88%), and 22q (92%) sites were very high in gallbladder carcinoma, and we identified 13 distinct regions undergoing frequent loss of heterozygosity in tumours. Allele losses were frequently detected in normal and dysplastic gallbladder epithelia. There was a progressive increase of the overall loss of heterozygosity frequency with increasing severity of histopathological changes. Allele losses were not random and followed a sequence. This study refines several distinct chromosome 3p, 8p, 9q and 22q regions undergoing frequent allele loss in gallbladder carcinoma that will aid in the positional identification of tumour suppressor genes involved in gallbladder carcinoma pathogenesis.

Figure 1

Representative example of the precise microdissection technique of invasive GBC (A and B) and high-grade dysplastic lesion (C and D) used in this study. Note that only tumour and epithelial cells were microdissected (A, C, before; B, D, after) while stromal tissue is intact.

Figure 2

Patterns of chromosome 3p allele losses in gallbladder carcinoma. The cases have been arranged from left to right in decreasing order of chromosome 3p allele losses. Markers are placed in the predicted order from 3pter-cen. Four regions (R1–R4) having frequent allelic losses are shown. Lower panel: seven autoradiographs showing discrete allele loss at chromosome 3p in a microdissected GBC case (case 8) demonstrating loss and retention of neighbouring alleles. L, lymphocytes or normal stromal cells; T, microdissected invasive GBC. Horizontal bars on the left of the autoradiographs indicate the main allelic bands.

Figure 3

Patterns of chromosome 8p allele losses in gallbladder carcinoma. The cases have been arranged from left to right in decreasing order of chromosome 8p allelic losses. Markers are placed in the predicted order from 8pter-cen. Three regions (R1–R3) having frequent allelic losses are shown. Lower panel: seven autoradiographs showing discrete allele loss at chromosome 8p in a microdissected GBC case (case 20) demonstrating loss and retention of neighbouring alleles. L, lymphocytes or normal stromal cells; T, microdissected invasive GBC. Horizontal bars on the left of the autoradiographs indicate the main allelic bands.

Figure 4

Patterns of chromosome 9q allele losses in gallbladder carcinoma. The cases have been arranged from left to right in decreasing order of chromosome 9q allele losses. Markers are placed in the predicted order from 9qcen-ter. Four regions (R1–R4) having frequent allelic losses are shown. Lower panel: eight autoradiographs showing discrete allele loss at chromosome 9q in a microdissected GBC case (case 17) demonstrating loss and retention of neighbouring alleles. L, lymphocytes or normal stromal cells; T, microdissected invasive GBC. Horizontal bars on the left of the autoradiographs indicate the main allelic bands.

Figure 5

Patterns of chromosome 22q allele losses in gallbladder carcinoma. The cases have been arranged from left to right in decreasing order of chromosome 22q allele losses. Markers are placed in the predicted order from 22qcen-ter. Two regions (R1–R2) having frequent allelic losses are shown. Lower panel: six autoradiographs showing discrete allele loss at chromosome 22q in a microdissected GBC case (case 20) demonstrating loss and retention of neighbouring alleles. L, lymphocytes or normal stromal cells; T, microdissected invasive GBC. Horizontal bars on the left of the autoradiographs indicate the main allelic bands.

Figure 6

Summary of all allelotyping results by chromosome arms in the pathogenesis of GBC. A total of 69 specimens are shown, including all 24 invasive carcinomas and all 45 non-malignant epithelium (17 histologically normal appearing and 28 dysplastic epithelia) accompanying GBCs. The specimens have been sorted from left to right in ascending number chromosome arm allele losses. The sources of the various specimens are coded above the boxes and include normal epithelium (N), dysplasia (D) and tumour (T). Lower panel: eight autoradiographs showing discrete allele loss at chromosomes 3p, 8p, 9q y 22q in two GBCs and their accompanying non-malignant epithelium. L, lymphocytes or normal stromal cells; N, normal epithelium; D, dysplasia; T, microdissected invasive GBC. Horizontal bars on the left of the autoradiographs indicate the main allelic bands. Data analysis shows that allelic losses present in normal and dysplastic epithelia were not random. Analysis of informative samples for all four chromosome arms shows that most normal epithelia demonstrate 8p or 8p+3p losses and the majority of dysplasias have losses on 8p and/or 3p with 9q and/or 22q, sugesting a sequential model of genetic abnormalities that begins with 8p LOH and progresses through 3p, 9q and 22q losses.