CpG island methylation profile of pancreatic intraepithelial neoplasia

Abstract

Infiltrating adenocarcinoma of the pancreas is thought to develop through well-defined precursor lesions called pancreatic intraductal neoplasia (PanIN). Despite the exponential growth in our understanding of genetic events that characterize the progression of PanINs to invasive carcinoma, little is known about the role of epigenetic alterations in these precursor lesions. To define the timing and prevalence of methylation abnormalities during early pancreatic carcinogenesis, we investigated the CpG island methylation profile in the various grades of PanINs. Using methylation-specific PCR, we analyzed DNA samples from 65 PanIN lesions for methylation status of eight genes recently identified by microarray approach as aberrantly hypermethylated in invasive pancreatic cancer. Aberrant methylation at any of the eight genes was identified in 68% of all the PanIN lesions examined, and, notably, aberrant methylation was identified in more than 70% of the earliest lesions (PanIN-1A). The average number of methylated loci was 1.1 in PanIN-1A, 0.8 in PanIN-1B, 1.1 in PanIN-2, and 2.9 in PanIN-3 lesions (P=0.01 for PanIN -3 vs earlier PanINs). Among the genes analyzed, NPTX2 demonstrated an increase in methylation prevalence from PanIN-1 to PanIN-2 (P=0.0008), and from PanIN-2 to PanIN-3 for SARP2 (P=0.001), Reprimo (P=0.01), and LHX1 (P=0.03). These results suggest that aberrant CpG island hypermethylation begins in early stages of PanINs, and its prevalence progressively increases during neoplastic progression.

Figure 1

Methylation-specific PCR analysis of SARP2 in various grades of PanINs. The PCR products in lanes U and M indicate the presence of unmethylated and methylated templates, respectively.

Figure 2

Frequency of aberrant CpG island methylation at eight genes in various grades of PanINs. The methylation frequency significantly increased from PanIN-1 to PanIN-2 for NPTX2 (6 vs 46%, P = 0.0008) and from PanIN-2 to PanIN-3 for SARP2 (20 vs 83%, P = 0.001), reprimo (20 vs 67%, P = 0.01), and LHX1 (7 vs 42%, P = 0.03).

Figure 3

Methylation profiles of 10 genes (8 genes analyzed in this study and 2 genes (ppENK and p16) in a previous study) in PanINs determined by MSP. PCa, pancreatic adenocarcinoma; CP, chronic pancreatitis; CBD ca, common bile duct cancer; Amp ca, ampullary cancer; End. T, endocrine tumor; MCN, mucinous cystic tumor. Filled boxes, methylated alleles; open boxes, unmethylated alleles; *, not determined.