Sensitive Detection of Colorectal Cancer in Peripheral Blood by Septin 9 DNA Methylation Assay

et al. (2008) Sensitive Detection of Colorectal Cancer in Peripheral Blood by Septin 9 DNA Methylation Assay. PLoS ONE 3(11): e3759. doi:10.1371/journal.pone.0003759 Sensitive Detection of Colorectal Cancer in Peripheral Blood by Septin 9 DNA Methylation Assay Robert Gru tzmann 0 Bela Molnar 0 Christian Pilarsky 0 Jens K. Habermann 0 Peter M. Schlag 0 Hans D. 0 Saeger 0 Stephan Miehlke 0 Thomas Stolz 0 Fabian Model 0 Uwe J. Roblick 0 Hans-Peter Bruch 0 Rainer Koch 0 Volker Liebenberg 0 Theo deVos 0 Xiaoling Song 0 Robert H. Day 0 Andrew Z. Sledziewski 0 Catherine Lofton-Day 0 Joseph Najbauer, City of Hope Medical Center, United States of America 0 1 Department of Visceral-, Thoracic- and Vascular Surgery, University Hospital Carl Gustav Carus Dresden , Dresden, Germany , 2 Semmelweis University , Budapest , Hungary , 3 Department of Surgery, University Hospital Schleswig-Holstein , Campus Lu beck, Lu beck, Germany , 4 Department of Surgery and Surgical Oncology, Charite Campus Berlin Buch , Robert-Ro ssle-Klinik, Berlin, Germany , 5 Department of Gastroenterology, University Hospital Carl Gustav Carus Dresden , Dresden, Germany , 6 Vo lklingen Clinic , Vo lklingen, Germany, 7 Epigenomics AG, Berlin, Germany , 8 Institute of Medical Informatics and Biometrics, Technical University of Dresden , Dresden, Germany , 9 Epigenomics Inc , Seattle, Washington , United States of America Background: Colorectal cancer (CRC) is the second leading cause of cancer deaths despite the fact that detection of this cancer in early stages results in over 90% survival rate. Currently less than 45% of at-risk individuals in the US are screened regularly, exposing a need for better screening tests. We performed two case-control studies to validate a blood-based test that identifies methylated DNA in plasma from all stages of CRC. Methodology/Principal Findings: Using a PCR assay for analysis of Septin 9 (SEPT9) hypermethylation in DNA extracted from plasma, clinical performance was optimized on 354 samples (252 CRC, 102 controls) and validated in a blinded, independent study of 309 samples (126 CRC, 183 controls). 168 polyps and 411 additional disease controls were also evaluated. Based on the training study SEPT9-based classification detected 120/252 CRCs (48%) and 7/102 controls (7%). In the test study 73/126 CRCs (58%) and 18/183 control samples (10%) were positive for SEPT9 validating the training set results. Inclusion of an additional measurement replicate increased the sensitivity of the assay in the testing set to 72% (90/ 125 CRCs detected) while maintaining 90% specificity (19/183 for controls). Positive rates for plasmas from the other cancers (11/96) and non-cancerous conditions (41/315) were low. The rate of polyp detection (.1 cm) was ,20%. Conclusions/Significance: Analysis of SEPT9 DNA methylation in plasma represents a straightforward, minimally invasive method to detect all stages of CRC with potential to satisfy unmet needs for increased compliance in the screening population. Further clinical testing is warranted. - Colorectal cancer (CRC) is one of the most common neoplasms found in men and women in the United States. The American Cancer Society had estimated that 154,000 new cases of CRC would occur in 2007, resulting in more than 52,000 deaths. Screening programs for the identification of early stage CRC and pre-neoplastic conditions can significantly improve disease outcome because of the treatment benefit of early detection [1]. Current non-invasive screening procedures are not very effective, as they require patient compliance to self-collect stool sample analyzed annually for the presence of occult blood (FOBT) [2]. To date, improvements in feces-based tests by making them more sensitive and more user friendly have not increased compliance in CRC screening. Invasive screening tests such as colonoscopy or sigmoidoscopy, although more effective, require extensive bowel preparation, invasion of privacy, and sedation, and do not overcome current compliance issues in CRC screening. There is growing expectation that the new generation of screening tests based on molecular biomarkers present in blood should improve patient compliance in CRC screening as evidenced by the success of other screening programs such as cholesterol/lipids and prostate specific antigen (PSA) [57]. Determination of epigenetic events is a strong candidate for early detection of disease since regulation of gene expression by aberrant DNA methylation is a well-characterized event in tumor biology [8,9], and is extensively described for CRC [1012]. Increased levels of free-circulating methylated DNA in the blood of cancer patients compared to healthy controls have been reported [13,14]. Several laboratories also reported promising DNA methylation-based marker candidates for detection of CRC [1519]. Translating such marker candidates into clinically validated and commercially viable tests has been exceedingly slow and inadequate. To facilitate improvements in biomarker translation medicine Pepe et al. proposed a systematic process for biomarker validation for early detection of cancer with 5 distinct phases, each phase providing increased level of evidence of marker validation [20]. In an initial study we presented the first level of evidence that SEPT9, a DNA methylation-based biomarker, effectively discriminates CRC from normal specimens [21]. The Septin 9 gene belongs to a class of GTPases involved in numerous cellular process [22].The gene has been shown to have multiple alternatively spliced transcripts encoding at least 5 characterized polypeptides designated v1v5 [23], some of which have been associated with cancer. The ratio of v4 and v4* expression, identical proteins encoded by different transcripts, has been shown to be altered in ovarian cancer with v4 being the predominant form expressed in normal cells and v4* expressed in tumors [24]. Recent studies of the v1 isoform suggest that over-expression of this polypeptide may promote tumor progression in mammary tissue [25]. Our previous work describing aberrant methylation in the promoter region of the v2 transcript indicates that methylation in this region is associated with colorectal cancer [21]. Moving forward in the process proposed by Pepe et al, in this report we provide the second level of evidence by presenting results from validation of clinical assay for SEPT9 in two large independent plasma sets demonstrating the potential of this marker for early detection of CRC. Increasing the number of assay replicates tested resulted in high sensitivity for CRC with excellent specificity in healthy controls. Specificity was further evaluated in a number of disease controls. Finally, the SEPT9 methylation of pre-malignant lesions is reported. Patients 700 patient samples collected at 9 sites were measured in the training study. It included patients with all stages of colorectal cancer, individuals without diseases of the colon as ve (...truncated)