Introduction: Pancreatic adenocarcinoma is anticipated to become the most fatal gastrointestinal neoplasm by 2017. Because patients who present with symptoms uniformly have advanced disease, 5-year survival is poor. Surveillance programs in the highest risk patients have produced disappointing results and use invasive tests that would not be safe or practical at the population level. Molecular markers, specifically aberrant DNA methylation, show promise in early studies. We aimed to: 1) measure the sensitivity and specificity of aberrant DNA methylation in stools as a minimally invasive way to detect pancreatic cancer; and, 2) identify novel methylation candidate markers highly sensitive and specific for pancreatic cancer.
Methods: To select candidate markers among those reported in the literature, DNA was extracted from unmatched tissue samples of pancreatic cancers and of normal colonic epithelia and assayed for aberrant methylation by methylation specific PCR. The most discriminant candidates and mutant KRAS were then assayed from matched archival stools of patients with pancreatic cancer in comparison to stools from healthy controls. Sensitivity and specificity for pancreatic cancer were determined from multivariate logistic regression models. To identify novel candidate markers, DNA was extracted from matched, archival tissues of pancreas cancer and two control groups (normal pancreas and normal colon) and sequenced using the reduced representation bisulfite technique. Among all mapped regions, those with the highest variance in methylation differences between cases and controls were filtered prior to analysis. Significant regions were then blindly assayed by methylation specific PCR in an independent, matched sample set, where sensitivity and specificity were measured using univariate logistic regression.
Results: In tissues, methylated BMP3, EYA4, UCHL1 and MDFI were highly discriminant for pancreatic cancer in comparison to normal colon samples. However, when assayed in stools, only BMP3 remained significant. In combination with mutant KRAS, the area under the receiver operating characteristics curve for BMP3 in detection of pancreatic cancer, was 0.85, indicating strong association. Results were not significantly influenced by tumor location or stage. Reduced representation bisulfite sequencing identified over 500 differentially methylated regions which met a priori significance thresholds. The top 25 novel candidates were validated in independent samples, showing both strong association with cases, compared to controls and high signal to noise ratio.
Conclusions: We report the first demonstration of feasibility for the detection of pancreatic cancer using assay of aberrantly methylated DNA markers from stool. This is a critical first step in the long-term goal of developing a minimally invasive screening tool to curb the mortality rate of this devastating disease. Because the discriminant candidate markers, methylated BMP3 and mutant KRAS, are not unique to pancreatic cancer, we developed a marker discovery strategy which yielded dozens of highly discriminant, validated, novel candidates, many of which have never before been reported in association with cancer. Further studies are indicated to measure the site-specificity of these markers for pancreatic cancer, compared to other gastrointestinal neoplasms and to study the clinical utility of these novel candidates in distant biologic media, such as blood, stool or pancreatic juice.
|Advisor:||Ahlquist, David A.|
|Commitee:||Limburg, Paul J., Loftus, Edward V., Mahoney, Douglas W.|
|School:||College of Medicine - Mayo Clinic|
|Department:||Clinical and Translational Science|
|School Location:||United States -- Minnesota|
|Source:||MAI 54/03M(E), Masters Abstracts International|
|Subjects:||Molecular biology, Genetics, Oncology|
|Keywords:||Biological markers, DNA methylation, Early detection, Epigenomics, Feces analysis, Pancreatic neoplasms|
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