Diagnosing Pancreatic Cancer Using Methylation Specific PCR Analysis

Fukushima, Noriyoshi; Walter, Kimberly; Ueki, Takashi; Sato, Norihiro; Matsubayashi, Hiroyuki; Cameron, John L.; Hruban, Ralph H.; Canto, Marcia I.; Yeo, Charles J.; Goggins, Michael

doi:10.4161/cbt.183

Cited by 98 publications

(65 citation statements)

References 24 publications

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“…This list includes ppENK, p16, hMLH1, RAR beta (Ueki et al, 2000(Ueki et al, , 2001, cyclin D2 (Matsubayashi et al, 2003) and TSLC1 (Jansen et al, 2002). In addition to understanding the biological significance of aberrant methylation in pancreatic adenocarcinoma, the detection of aberrantly methylated DNA in secondary fluids such as the pancreatic juice of patients with pancreatic disease may aid in the early diagnosis of pancreatic cancer .…”

Section: Discussionmentioning

confidence: 99%

Aberrant methylation of suppressor of cytokine signalling-1 (SOCS-1) gene in pancreatic ductal neoplasms

et al. 2003

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The suppressor of cytokine signalling-1 (SOCS-1) gene is frequently silenced in human hepatocellular carcinoma by aberrant methylation. The aim of this study was to determine if SOCS-1 is inactivated in pancreatic ductal neoplasms, and to investigate if aberrant methylation of this gene affected the Janus kinase/signal transducers and activators of transcription (JAK/STAT) pathway. Aberrant methylation in the CpG island of the SOCS-1 gene was detected in six of 19 (31.6%) human pancreatic cancer cell lines using methylation-specific PCR, and was associated with a loss or reduction of gene expression in five of the six methylated cell lines. Thirteen of 60 pancreatic ductal adenocarcinomas (21.7%) and two of 34 intraductal papillary mucinous neoplasms (IPMNs) (5.9%) had methylated SOCS-1. In contrast, SOCS-1 methylation was not seen in pancreatic normal ductal epithelia (zero out of 15), in pancreatic intraepithelial neoplasia (PanINs) (zero out of 49) or in the IPMNs without infiltrating cancer (zero out of 20). 5-Aza-2 0 -deoxycytidine treatment of the SOCS-1-methylated pancreatic cancer cell lines led to restoration of SOCS-1 gene expression. Interleukin-6, which has been shown to act through the JAK/STAT pathway to increase cell growth, induced modest time and dosedependent cell proliferation in a SOCS-1-methylated cell line (PL10, P ¼ 0.015) but not in two unmethylated cell lines. These results indicate that loss of SOCS-1 gene is associated with transcriptional silencing and may have growth-promoting effects, and that its methylation is a useful marker of pancreatic cancer.

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Section: Discussionmentioning

confidence: 99%

Aberrant methylation of suppressor of cytokine signalling-1 (SOCS-1) gene in pancreatic ductal neoplasms

et al. 2003

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“…29 Three patient populations with high predisposition for pancreatic cancer that might benefit from periodic monitoring of pancreatic duct juice are: patients with chronic pancreatitis, pancreatic cancer families without a known gene defect, or patients from families with documented increased genetic risk (BRCA2, Peutz-Jeghers syndrome, hereditary pancreatitis, and FAMMM). 36 Pancreatic juice can be collected during routine upper GI endoscopy or ERCP using secretin infusion, 37 and in these high risk settings the risk and the expense of the procedure may be justified given the lethality of late detected disease. The volume of pancreatic duct fluid, collected from the operating room in this study, is well within the volume that can be collected during ERCP following secretin stimulation.…”

Section: Discussionmentioning

confidence: 99%

“…The aberrant methylation of specific genes has been reported in pancreatic cancer 46,47 and this methylation can be detected in endoscopically collected pancreatic juice samples. 37 Other DNA point mutations, such as KRAS2 codon 13, KRAS2 codon 61, p53, BRAF, and others 48 may also be useful for early detection, and if warranted by their frequency, will be incorporated into multiplex LigAmp reactions in the future.…”

Section: Methodsmentioning

confidence: 99%

Sensitive and quantitative detection of KRAS2 gene mutations in pancreatic duct juice differentiates patients with pancreatic cancer from chronic pancreatitis, potential for early detection

Shi

Fukushima

Abe

et al. 2008

Cancer Biology & Therapy

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KRAS2 gene mutations are found in 75-90% of infiltrating pancreatic ductal adenocarcinomas but can also be present with other nonneoplastic pancreatic diseases. We recently developed a novel sensitive assay for point mutation detection, called "LigAmp", which can detect one mutant molecule in the presence of 10,000 wild-type molecules and can quantify mutant DNA over a wide dynamic range. We analyzed KRAS2 mutations in surgically-collected pancreatic duct juice samples from patients with pancreatic adenocarcinoma (n = 27) and chronic pancreatitis (n = 9). DNA sequencing demonstrated that 17 of the 27 pancreatic cancers harbored KRAS2 mutations at codon 12, including G12D (GGT → GAT), G12V (GTT), and G12R (CGT). We determined the relative amounts of each KRAS2 mutant by simultaneously quantifying wild-type and mutant KRAS2 DNA. For all pancreatic adenocarcinoma patients, the dominant KRAS2 mutation detected in the pancreatic juice corresponded to that found in the primary cancer. Mutation levels were substantially higher in patients with pancreatic cancer (0.05 to 82% of total KRAS2 molecules) compared to those with chronic pancreatitis (0 to 0.7%). Among patients with mutant KRAS2 positive cancers, all but one (94%) had mutant KRAS2 DNA concentrations of more than 0.5% in their pancreatic juice samples, whereas only 1 of 9 (11%) pancreatic juice samples from patients with chronic pancreatitis had more than 0.5% mutant KRAS2 DNA, corresponding to a sensitivity of 94% and a specificity of 89%. LigAmp quantification of mutant KRAS2 in pancreatic juice differentiates pancreatic adenocarcinoma from chronic pancreatitis, and may be a useful early detection tool for pancreatic cancer.

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“…We and others have previously examined the utility of a panel of DNA methylation markers in pancreatic juice samples for the diagnosis of pancreatic cancer. 4,11,42 The identification of additional aberrantly methylated genes in pancreatic cancers may help efforts to use these markers as diagnostic tools, particularly since using a panel of markers may help ensure high specificity. 11 In conclusion, we find that the MCA array method can detect hundreds of differentially methylated genes in pancreatic adenocarcinoma relative to normal pancreas, is reproducible and can reliably detect DNA methylation profiles using small amounts of input DNA.…”

Section: E S B I O S C I E N C E D O N O T D I S T R I B U T Ementioning

confidence: 99%

Genome-wide profiling at methylated promoters in pancreatic adenocarcinoma

Omura¹,

Li²,

Li³

et al. 2008

Cancer Biology & Therapy

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162

129

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Many genes undergo aberrant methylation in human cancers, and microarray platforms enable more comprehensive profiling of aberrant DNA methylation patterns.Results: 1,010 of 87,922 probes on the 88 K promoter array (606 genes) had a higher signal (log 2 > 2) in the pancreatic cancer line, Panc-1 compared to the non-neoplastic pancreatic duct line, HPDE. Using this cut-off, bisulfite sequencing and/ or MSP confirmed differential methylation of all 27 genes (66 probes) predicted to be methylated by the MCA array. More than half of the genes aberrantly hypermethylated in Panc-1 were not expressed in the pancreatic duct (HPDE) by expression array analysis. Using the 244 K CpG island array, 1,968 CpG islands were differentially methylated in MiaPaca2 compared to normal pancreas. The MCA method was more likely to identify hypermethylation within CpG islands than a cocktail of methylation sensitive restriction enzymes. DNA methylation profiles using 10 ng of DNA were highly correlated with those obtained using 5 mg of DNA (R2 = 0.98). Analysis of 57 pancreatic cancers and 34 normal pancreata using MSP identified MDFI, hsa-miR-9-1, ZNF415, CNTNAP2 and ELOVL4 as methylated in 96%, 89%, 86%, 82% and 68% of the cancers vs. 9%, 15%, 6%, 3% and 9% of normal pancreata, respectively. Methods: We used methylated CpG island amplification (MCA) and Agilent promoter and CpG island microarrays to identify differential DNA methylation patterns in pancreatic cancer vs. normal pancreas. We examined MCA array reproducibility, compared it to methylation profiles obtained using a cocktail of methylation-sensitive restriction enzymes and examined gene expression of methylated genes.Conclusion: Promoter and CpG island array analysis finds aberrant methylation of hundreds of promoters and CpG islands in pancreatic cancer cells.

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Diagnosing Pancreatic Cancer Using Methylation Specific PCR Analysis

Cited by 98 publications

References 24 publications

Aberrant methylation of suppressor of cytokine signalling-1 (SOCS-1) gene in pancreatic ductal neoplasms

Aberrant methylation of suppressor of cytokine signalling-1 (SOCS-1) gene in pancreatic ductal neoplasms

Sensitive and quantitative detection of KRAS2 gene mutations in pancreatic duct juice differentiates patients with pancreatic cancer from chronic pancreatitis, potential for early detection

Genome-wide profiling at methylated promoters in pancreatic adenocarcinoma

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