Primary Motility  Disorders of the  Esophagus
 The Esophageal
 Esophagogastric  Junction

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Volume: Barrett's Esophagus
Chapter: Markers

What is the in vivo value of chromosomal 9p21 allelic loss and CDKN2 gene mutations in detecting neoplastic progression before aneuploidy and cancer?

D.G. Beer (Ann Arbor)

The p16 cyclin-dependent kinase (CDKN2/MTS1/INK4A) gene is a tumor suppressor gene which is located on chromosome 9p21. The p16/CDKN2 gene product inhibits the phosphorylation of the retinoblastoma protein (pRb) by forming a binary complex with the cyclin-dependent kinases Cdk4 and Cdk6 [1]. By preventing the phosphorylation of Rb, p16/CDKN2 can inhibit the cells entry into the cell cycle, thus loss of p16/CDKN2 function may potentially lead to unregulated cell proliferation. Alterations in p16/CDKN2 gene function, as will be described below, may occur by a number of distinct mechanisms including allelic loss, gene mutation and methylation-induced silencing of the p16 gene promoter.

Detection of p16 allelic loss and mutation

Allelic loss of 9p21 in esophageal adenocarcinomas was first reported by Tarmin et al. [2], and a high frequency loss of heterozygosity of the p16/CDKN2 gene was described by Barrett et al. [3]. Using a polymerase chain reaction (PCR)-based assay, microdissected tissue and two microsatellite markers which flank the p16 gene, Gonzalez et al. [4] observed loss of heterozygosity in (10/11) 89% of Barrett's adenocarcinomas, but not in nondysplastic Barrett's mucosa at these loci. Gonzalez et al., also reported homozygous deletion of p16 in 25% of the adenocarcinomas but mutations were not observed in the second exon of the p16 gene by this group in either the Barrett's metaplasia or adenocarcinomas. In contrast, loss of heterozygosity at 9p21 was reported in 75% (24 of 32) of both premalignant Barrett's esophagus and adenocarcinomas when a the technique of flow-sorted cells from biopsies was utilized [3]. These authors also found 23% (5 of 22) of the remaining p16 allele to contain mutations but no homozygous deletions were detected. The reasons for the different results between these two studies is unclear, but may relate to the methods utilized such as the ability to isolate more homogenous cell populations using flow-sorting. In a later study, Wong et al. [5] found 90% (9 of 10) of patients with Barrett's esophagus to demonstrate 9p21 loss of heterozygosity and 40% (4 of 10) of premalignant Barrett's esophagus and 9% (1 of 11) of Barrett's adenocarcinomas to contain p16 mutations. A recent study by Barrett et al. [6] found 91% of aneuploid cell populations in Barrett's metaplasia to show 9p21 loss of heterozygosity and 26% (11 of 43) of these also contained p16/CDKN2 mutations. An important finding from the studies by Barrett et al. [3, 6] was the observation that 9p21 loss of heterozygosity was an early event in Barrett's metaplasia which preceded the development of aneuploidy and cancer. This suggests these alterations may play a causal role in the neoplastic progression of Barrett's metaplastic cells with p16 changes.

Inactivation of p16 by promoter methylation

Although somatic mutations within the p16 gene appear frequent in some human cancers [7], in esophageal adenocarcinomas they appear less frequent. An alternative mechanism for inactivation of the remaining p16 allele in Barrett's esophagus and esophageal adenocarcinoma following loss of heterozygosity may be promoter hypermethylation [5, 8]. The 5' region of certain genes contain CpG islands, regions often not containing methylated cytosine residues [9]. These CpG islands are often associated with regulatory genes and maintained as unmethylated residues [9]. Hypermethylation of CpG islands may transcriptionally silence the p16 gene promoter. Utilizing flow-sorted cells from aneuploid cell populations of both Barrett's esophagus and esophageal adenocarcinomas and a methylation-specific, PCR-based assay, Wong et al. [5] reported detection of hypermethylation of the p16 promoter in 30% (3 of 10) of patients with premalignant Barrett's esophagus and in 45% (5 of 11) of adenocarcinomas. Eight of 14 (57%) of these patients showing p16 hypermethylation also demonstrated loss of heterozygosity at 9p21. Klump et al. [8] also utilized a methylation-specific, PCR-based assay to examine p16 methylation status in Barrett's esophagus, dysplastic Barrett's and adenocarcinomas. Only 3% (2 of 67) of nondysplastic cases, but 60% (3 of 5) of indefinite for dysplasia, 55.6% of low-grade dysplastic and 75% (3 of 4) high-grade dysplasia (HGD) cases showed p16 promoter hypermethylation and indicated that this is a relatively common event in neoplastic development in Barrett's metaplasia.

Timing of p16 gene alterations in Barrett's metaplasia

The studies by Wong et al. [5] and Klump et al. [8] clearly indicate that hypermethylation of the p16 gene promoter is a frequent event during the neoplastic progression of Barrett's metaplasia. Promoter hypermethylation and allelic deletion appear to be more frequent events than mutation as mechanisms for the loss of function of the p16 tumor suppressor gene. The CDKN2/p16 gene plays a critical role in the regulation of the cell cycle and alterations in cyclin-CDK complexes may contribute to the dysregulation of cell proliferation and may potentially increase genomic instability [10]. The observed 9p21 deletion and p16 promoter hypermethylation in Barrett's metaplasia may directly contribute to increased cell proliferation associated with intestinal-type Barrett's metaplasia [11]. Studies by Galipeau et al. [12] and Barrett et al. [6] using consecutively obtained biopsies from individuals with Barrett's metaplasia have investigated the relationship between alterations in p16 and the evolution of cell lineages in Barrett's metaplasia. These studies indicate that 9p21 loss of heterozygosity, CDKN2/p16 methylation and p16 gene mutations occur in diploid cell populations in Barrett's metaplasia and precede the development of aneuploidy and cancer. Similar to the observed alterations in the p53 tumor suppressor gene (17p loss of heterozygosity) in Barrett's metaplasia, CDKN2/p16 gene alterations are detected prior to the loss of heterozygosity observed at 5q, 18q and 13q. By examining the clonal ordering of these genetic alterations, these studies suggest that there may be no obligate order of 17p and 9p loss of heterozygosity or mutation, and both precede the development of aneuploidy and cancer. The study by Galipeau et al. [12], however, indicates that 9p loss of heterozygosity is more common than 17p loss of heterozygosity and can be detected over a greater region of diploid Barrett's epithelium. When HGD Barrett's epithelium arises in these consecutively biopsied individuals, a mosaic of different cell clones is observed showing different patterns of 9p and 17p loss of heterozygosity.

The potential consequences of p16 gene alterations for neoplastic progression

Although the exact cause or the mechanisms underlying p16 promoter methylation, p16 mutation or 9p21 loss of heterozygosity remain unknown, each event may lead to the subsequent loss of p16 function and potentially allow the expansion of a specific subset of Barrett's metaplastic cells with increased proliferative capabilities. This property of increased cell proliferation or cycling may increase the potential of these cells to sustain additional genetic alterations over time, such as chromosome 17p loss [12] or the subsequent losses observed at 5q, 18q and 13q. In this way, the in vivo detection of p16 alterations in patients with Barrett's esophagus prior to development of aneuploidy, may be a useful genetic biomarker to identify those individuals who may benefit from chemoprevention strategies in addition to standard surveillance protocols. The challenge will be to identify sensitive, and widely usable techniques to detect these p16 alterations from biopsies of Barrett's esophagus.


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Publication date: August 2003 OESO©2015