preproenkephalin and Carcinoma--Pancreatic-Ductal

CpG islands of the promoter region of some genes are methylated in pancreatic cancer tissue and the detection of this methylation has been suggested to be useful in the diagnosis of various cancers. The aim of this study was to investigate whether the detection of methylated CpG islands in plasma can be used in the diagnosis of pancreatic cancer.. Plasma DNA was collected from patients with pancreatic cancer, chronic pancreatitis, and healthy controls. The methylation status of six genes, UCHL1, NPTX2, SARP2, ppENK, p16, and RASSF1A, was checked by methylation-specific PCR and was subsequently confirmed by direct sequencing after bisulfite treatment.. CpG island methylation was detectable in 13 of 16 patients (81.3%) with pancreatic cancer, 1 of 29 healthy controls (3.5%), and 8 of 13 patients with chronic pancreatitis (61.5%). The mean number of genes with CpG island methylation was 1.6±1.2 in pancreatic cancer, 0.04±0.19 in healthy controls, and 1.2±1.1 in chronic pancreatitis. Among six genes, p16 was more specifically methylated in pancreatic cancer compared with chronic pancreatitis (p=0.016). The methylation status was not correlated with smoking history, tumor size, or cancer stage.. The detection of methylated genes in the plasma may have a role in differentiating between pancreatic cancers and healthy controls but not between pancreatic cancer and chronic pancreatitis.

Topics: Adult; Aged; Biomarkers, Tumor; C-Reactive Protein; Carcinoma, Pancreatic Ductal; Carrier Proteins; Cell Line, Tumor; CpG Islands; DNA Methylation; Enkephalins; Female; Genes, p16; Humans; Intercellular Signaling Peptides and Proteins; Male; Membrane Proteins; Middle Aged; Nerve Tissue Proteins; Pancreatic Neoplasms; Pancreatitis, Chronic; Polymerase Chain Reaction; Protein Precursors; Tumor Suppressor Proteins; Ubiquitin Thiolesterase

Aberrant methylation of CpG islands is a common mechanism for the dysregulation of tumor suppressor genes in a variety of human malignancies. Preproenkephalin ppENK) hypermethylation is recognized in 90% of pancreatic carcinoma (PCa) tissues, but not in normal pancreas. We analyzed ppENK hypermethylation in pure pancreatic juice (PPJ) in patients with PCa, intraductal papillary mucinous neoplasms (IPMN), and chronic pancreatitis (CP), and elucidated its usefulness as a marker in the diagnosis of PCa compared with p53 mutation.. PPJ was collected endoscopically from 28 patients with PCa, 15 patients with IPMN, and 20 patients with CP. DNA was extracted from the supernatant and the sediment of PPJ. Methylation-specific polymerase chain reaction was performed for hypermethylation analysis of ppENK. In addition, single-strand conformation polymorphism and direct sequencing were performed simultaneously to identify p53 mutations.. The incidence of ppENK hypermethylation in the supernatant and/or the sediment of PPJ was 50% (14 of 28) in patients with PCa. In contrast, the incidence of ppENK hypermethylation was 26.7% (4 of 15) in patients with IPMN, and 5% (1 of 20) in patients with CP (P < 0.002). The incidence of p53 mutations in the PPJ was 42.9% (12 of 28) in patients with PCa and 0% (0 of 20) in patients with CP. Furthermore, the incidence of ppENK hypermethylation and/or p53 mutations in the PPJ was enhanced to 67.9% (19 of 28) in patients with PCa in the combination assay.. These results suggest that ppENK hypermethylation in PPJ is specific for cancer, and the combination assay with p53 enhances the genetic diagnosis of PCa.

Topics: Adult; Aged; Aged, 80 and over; Biomarkers, Tumor; Carcinoma, Pancreatic Ductal; DNA Methylation; DNA Mutational Analysis; Enkephalins; Female; Genes, p53; Humans; Male; Middle Aged; Mutation; Pancreatic Juice; Pancreatic Neoplasms; Pancreatitis, Chronic; Polymerase Chain Reaction; Polymorphism, Single-Stranded Conformational; Protein Precursors

Pancreatic intraductal neoplasia (PanIN) is thought to be the precursor to infiltrating pancreatic ductal adenocarcinoma. We have previously shown that the preproenkephalin (ppENK) and p16 genes are aberrantly methylated in pancreatic adenocarcinoma. In this study we define the methylation status of the ppENK and p16 genes in various grades of PanINs. One hundred seventy-four samples (28 nonneoplastic pancreatic epithelia, 7 reactive epithelia, 29 PanIN-1A, 48 PanIN-1B, 27 PanIN-2, 14 PanIN-3, 15 invasive ductal adenocarcinomas, and 6 miscellaneous pancreatic neoplasms) were microdissected from 29 formalin-fixed paraffin-embedded surgically resected pancreata, and were analyzed by methylation-specific polymerase chain reaction. Fourteen of 15 (93.3%) invasive pancreatic ductal adenocarcinomas showed methylation of the ppENK gene and 4 of 15 (26.7%) showed methylation of the p16 gene. Nonneoplastic pancreatic epithelia did not harbor methylation of either gene. The prevalence of methylation of the ppENK gene increased significantly with increasing PanIN grade. A similar nonsignificant trend was noted for p16 methylation. Aberrant methylation of the ppENK gene was found in 7.7% of PanIN-1A, 7.3% of PanIN-1B, 22.7% of PanIN-2, and 46.2% of PanIN-3. Aberrant methylation of the p16 gene was found in 12% of PanIN-1A, 2.6% of PanIN-1B, 4.5% of PanIN-2, and 21.4% of PanIN-3. All but one of the PanINs from the 14 pancreata without pancreatic carcinoma was unmethylated with respect to either the p16 or ppENK gene. Our results suggest that methylation-related inactivation of the ppENK and p16 genes is an intermediate or late event during pancreatic carcinogenesis. Because aberrant methylation of ppENK or p16 was more often detected in similar grade PanINs from patients with pancreatic carcinoma than in those with other pancreatic diseases, it may be a useful indicator of the potential malignancy of epithelial cells of the pancreas.

Topics: Aged; Aged, 80 and over; Carcinoma, Pancreatic Ductal; Chronic Disease; Cyclin-Dependent Kinase Inhibitor p16; DNA Methylation; DNA, Neoplasm; Enkephalins; Epithelium; Female; Humans; Male; Middle Aged; Neoplasm Invasiveness; Pancreas; Pancreatic Neoplasms; Pancreatitis; Protein Precursors

The functional abrogation of several tumor suppressor genes, including p16, DPC4, and p53, is a major mechanism underlying pancreatic ductal carcinogenesis. However, mutational inactivation of these genes is relatively uncommon in intraductal papillary mucinous neoplasms (IPMNs) of the pancreas. We hypothesized that an alternative mechanism for gene inactivation (notably, transcriptional silencing by promoter methylation) could be important in the pathogenesis of IPMNs.. Using methylation-specific polymerase chain reaction, we analyzed the methylation status of 7 CpG islands previously identified as aberrantly methylated in pancreatic adenocarcinoma (including preproenkephalin [ppENK], p16, and thrombospondin 1) in 51 IPMNs of different histologic grades. The relationship between methylation status and expression was evaluated using reverse-transcription polymerase chain reaction for ppENK and immunohistochemistry for p16.. We found that more than 80% of the IPMNs exhibited hypermethylation of at least one of these CpG islands. Hypermethylation of ppENK and p16 was detected at a significant higher frequency in high-grade (in situ carcinoma) IPMNs than in low-grade (adenoma/borderline) IPMNs (ppENK, 82% vs. 28%, P = 0.0002; p16, 21% vs. 0%, P = 0.04). Furthermore, the average number of methylated loci was significantly higher in high-grade IPMNs than in low-grade IPMNs (2.4 vs. 0.9; P = 0.0008). Aberrant methylation of ppENK and p16 was associated with loss of expression.. These results suggest that de novo methylation of multiple CpG islands is one of the critical pathways that contributes to the malignant progression of IPMNs.

Topics: Adenocarcinoma, Mucinous; Adenocarcinoma, Papillary; Adenoma; Adult; Aged; Aged, 80 and over; Carcinoma in Situ; Carcinoma, Pancreatic Ductal; CpG Islands; DNA Methylation; Enkephalins; Female; Genes, p16; Humans; Male; Middle Aged; Pancreatic Neoplasms; Protein Precursors