Septin9 Publications (please click on title for a short summary)

1. Jin et al. (2014, J. Gastroenterol. Hepatol.): Performance of a second generation methylated SEPT9 test in detecting colorectal neoplasm

Background & Aim: Screening and early detection reduces mortality due to colorectal cancer (CRC). Methylated Septin 9 (SEPT9) is a new blood-based biomarker for CRC. We evaluated the performance of the second generation SEPT9 assay for the detection of colorectal neoplasm, and compared it with fecal immunochemical test (FIT).

Methods: A total of 135 patients with CRC, 169 with adenomatous polyps, 81 with hyperplastic polyps and 91 healthy controls were included. The clinical status of all subjects was verified by colonoscopy. In all patients, peripheral blood samples were taken for SEPT9 testing using Epi proColon 2.0 test. For 177 patients, both SEPT9 and FIT were performed.

The sensitivity and specificity of SEPT9 for CRC were 74.8% (95% CI: 67.0%-81.6%) and 87.4% (vs non-CRC, 95% CI: 83.5%-90.6%), respectively. SEPT9 was positive in 66.7% of stage I, 82.6% of stage II, 84.1% of stage III , and 100% of stage IV CRCs. The sensitivity of SEPT9 for advanced adenomas was 27.4% (95% CI: 18.7%-37.6%). The sensitivity and specificity of FIT for CRC was 58.0% (95% CI: 46.1%-69.2%) and 82.4% (95% CI: 74.4%-88.7%), respectively. SEPT9 showed better performance in CRC detection than FIT, but similar among advanced adenomas.

Conclusion: With improved performance characteristics in detecting CRC, the second generation SEPT9 assay could play an important role in CRC screening and early detection.

Journal of Gastroenterology and Hepataology (JGH), 2014 Dec 4
doi: 10.1111/jgh.12855
2. Molnar et al. (2014, Expert Rev. Mol. Diagn.): Plasma methylated septin 9: a colorectal cancer screening marker
Colorectal cancer (CRC) is a slow-developing cancer (10–15 years) with one of the highest frequencies in the world’s population. Many countries have implemented various CRC screening programs, but have not achieved the desired compliance. Colonoscopy – considered the gold standard for CRC screening – has its limitations as well as the other techniques used, such as irrigoscopy, sigmoidoscopy, fecal blood and hemoglobin tests. The biomarker septin9 has been found to be hypermethylated in nearly 100% of tissue neoplasia specimens and detected in circulating DNA fractions of CRC patients. A commercially available assay for septin 9 has been developed with moderate sensitivity (~70%) and specificity (~90%) and a second generation assay, Epi proColon 2.0 (Epigenomics AG), shows increased sensitivity (~92%). The performance of the assay proved to be independent of tumor site and reaches a high sensitivity of 77%, even in early cancer stages (I and II). Furthermore, septin 9 was recently used in follow-up studies for detection of early recurrence of CRC. This article evaluates the opportunities, known limitations and future perspectives of the recently introduced Epi proColon 2.0 test, which is based on the detection of aberrantly methylated DNA of the v2 region of the septin 9 gene in plasma.

Expert Rev Mol Diagn. 2014 Nov 27:1-14.
doi: 10.1586/14737159.2015.975212
3. Adler et al. (2014, BMC Gastroenterology): Improving compliance to colorectal cancer screening using blood and stool based tests in patients refusing screening colonoscopy in Germany

Background: Despite strong recommendations for colorectal cancer (CRC) screening, participation rates are low. Understanding factors that affect screening choices is essential to developing future screening strategies. Therefore, this study assessed patient willingness to use non-invasive stool or blood based screening tests after refusing colonoscopy.

Methods: Participants were recruited during regular consultations. Demographic, health, psychological and socioeconomic factors were recorded. All subjects were advised to undergo screening by colonoscopy. Subjects who refused colonoscopy were offered a choice of non-invasive tests. Subjects who selected stool testing received a collection kit and instructions; subjects who selected plasma testing had a blood draw during the office visit. Stool samples were tested with the Hb/Hp Complex Elisa test, and blood samples were tested with the Epi proColon® 2.0 test. Patients who were positive for either were advised to have a diagnostic colonoscopy.

Results: 63 of 172 subjects were compliant to screening colonoscopy (37%). 106 of the 109 subjects who refused colonoscopy accepted an alternative non-invasive method (97%). 90 selected the Septin9 blood test (83%), 16
selected a stool test (15%) and 3 refused any test (3%). Reasons for blood test preference included convenience of an office draw, overall convenience and less time consuming procedure.

Conclusions: 97% of subjects refusing colonoscopy accepted a non-invasive screening test of which 83% chose the Septin9 blood test. The observation that participation can be increased by offering non-invasive tests, and that a blood test is the preferred option should be validated in a prospective trial in the screening setting.

BMC Gastroenterology 2014, 14:183
4. Potter et al. (2014, Clin. Chem.): Validation of a Real-Time PCR–Based Qualitative Assay for the Detection of Methylated SEPT9 DNA in Human Plasma

Background: Epi proColon® is a new blood-based colorectal cancer (CRC) screening test designed to determine the methylation status of a promoter region of the SEPT9 (septin 9) gene in cell-free DNA isolated from plasma. We describe the analytical and clinical performance of the test.

Methods: Analytical performance at 4 testing laboratories included determination of limit of detection, precision, and reproducibility of the SEPT9 test. Clinical performance was evaluated in a prospective study by use of samples (n1544) from subjects enrolled in the PRESEPT clinical trial. Results were analyzed by comparison with colonoscopy, the reference standard.

Results: The limit of detection for methylated SEPT9 DNA was 7.8 pg/mL (95% CI 6–11 pg/mL) corresponding to 2 genome copies of methylated SEPT9 per milliliter of plasma. In the prospective clinical trial, sensitivity for all stages of CRC was 68% (95% CI 53%–80%) and for stage I–III CRC, 64% (48%–77%). Adjusted specificity, on the basis of negative colonoscopy findings, was 80.0% (78%–82%).

Significance: The Epi proColon test is a simple, realtime PCR–based assay for the detection of methylated SEPT9 DNA in blood that may provide a noninvasive CRC screening alternative for people noncompliant with current CRC screening guidelines.

Clinical Chemistry, September 2014 vol. 60 no. 9 1183-1191

5. Johnson et al. (2014, PLoS One): Plasma Septin9 versus Fecal Immunochemical Testing for Colorectal Cancer Screening: A Prospective Multicenter Study

Background: Screening improves outcomes related to colorectal cancer (CRC); however, suboptimal participation for available screening tests limits the full benefits of screening. Non-invasive screening using a blood based assay may potentially help reach the unscreened population.

Objective: To compare the performance of a new Septin9 DNA methylation based blood test with a fecal immunochemical test (FIT) for CRC screening.

Design: In this trial, fecal and blood samples were obtained from enrolled patients. To compare test sensitivity for CRC, patients with screening identified colorectal cancer (n = 102) were enrolled and provided samples prior to surgery. To compare test specificity patients were enrolled prospectively (n=199) and provided samples prior to bowel preparation for screening colonoscopy.

Measurements: Plasma and fecal samples were analyzed using the Epi proColon and OC Fit-Check tests respectively.

Results: For all samples, sensitivity for CRC detection was 73.3% (95% CI 63.9–80.9%) and 68.0% (95% CI 58.2–76.5%) for Septin9 and FIT, respectively. Specificity of the Epi proColon test was 81.5% (95% CI 75.5–86.3%) compared with 97.4% (95% CI 94.1–98.9%) for FIT. For paired samples, the sensitivity of the Epi proColon test (72.2% –95% CI 62.5–80.1%) was shown to be statistically non-inferior to FIT (68.0%–95% CI 58.2–76.5%). When test results for Epi proColon and FIT were combined, CRC detection was 88.7% at a specificity of 78.8%.

Conclusions: At a sensitivity of 72%, the Epi proColon test is non- inferior to FIT for CRC detection, although at a lower specificity. With negative predictive values of 99.8%, both methods are identical in confirming the absence of CRC.

PLoS One 2014, 9(6):1-8. e98238
DOI: 10.1371/journal.pone.0098238
6. Heichman K. (2013, Mol. Diagn. Ther.): Blood-Based Testing for Colorectal Cancer Screening

Colorectal cancer (CRC) is the third most common non-skin cancer diagnosed in men and women in the USA and worldwide. While it has been clearly established that screening for CRC, using a variety of methods, is cost effective and has a significant impact on overall survival, screening rates have proven to be sub-optimal.
It has been long conjectured that a simple blood-based test, with a specimen drawn at a routine doctor’s office visit, would encourage those individuals who have refused or ignored screening recommendations to undergo screening.

This article reviews the currently available blood-based screening tests for CRC, including the ColonSentryTM messenger RNA (mRNA) expression panel and the SEPT9 methylated DNA test, and explores newer biomarkers that are near clinical implementation. Also discussed are additional applications for blood-based CRC testing, such as assessing prognosis, disease surveillance, and expansion of screening tests to high-risk populations, such as the estimated 1.4 million individuals in the USA with inflammatory bowel disease.

Molecular Diagnosis and Therapy 2013
DOI 10.1007/s40291-013-0074-z
7. Wasserkort et al. (2013, BMC Cancer): Aberrant septin 9 DNA methylation in colorectal cancer is restricted to a single CpG island

Background: The septin 9 gene (SEPT9) codes for a GTP-binding protein associated with filamentous structures and cytoskeleton formation. SEPT9 plays a role in multiple cancers as either an oncogene or a tumor suppressor gene. Regulation of SEPT9 expression is complex and not well understood; however, hypermethylation of the gene was recently introduced as a biomarker for early detection of colorectal cancer (CRC) and has been linked to cancer of the breast and of the head and neck. Because the DNA methylation landscape of different regions of SEPT9 is poorly understood in cancer, we analyzed the methylation patterns of this gene in distinct cell populations from normal and diseased colon mucosa.

Methods: Laser capture microdissection was performed to obtain homogeneous populations of epithelial and stromal cells from normal, adenomatous, and tumorous colon mucosa. Microdissected samples were analyzed using direct bisulfite sequencing to determine the DNA methylation status of eight regions within and near the SEPT9 gene. Septin-9 protein expression was assessed using immunohistochemistry (IHC).

Results: Regions analyzed in SEPT9 were unmethylated in normal tissue except for a methylation boundary detected downstream of the largest CpG island. In adenoma and tumor tissues, epithelial cells displayed markedly increased DNA methylation levels (>80%, p <0.0001) in only one of the CpG islands investigated. SEPT9 methylation in stromal cells increased in adenomatous and tumor tissues (?50%, p <0.0001); however, methylation did not increase in stromal cells of normal tissue close to the tumor. IHC data indicated a significant decrease (p <0.01) in Septin-9 protein levels in epithelial cells derived from adenoma and tumor tissues; Septin-9 protein levels in stromal cells were low in all tissues.

Conclusions: Hypermethylation of SEPT9 in adenoma and CRC specimens is confined to one of several CpG islands of this gene. Tumor-associated aberrant methylation originates in epithelial cells; stromal cells appear to acquire hypermethylation subsequent to epithelial cells, possibly through field effects. The region in SEPT9 with diseaserelated hypermethylation also contains the CpGs targeted by a novel blood-based screening test (Epi proColon®) providing further support for the clinical relevance of this biomarker.

BMC Cancer 2013, 13:398
8. Ladabaum et al. (2013,  Cancer Epidemiology, Biomarkers & Prevention): Colorectal Cancer Screening with Blood-Based Biomarkers: Cost-Effectiveness of Methylated Septin 9 DNA vs. Current Strategies

Background: Screening reduces colorectal cancer mortality, but many persons remain unscreened. Screening with a blood test could improve screening rates. We estimated the comparative effectiveness and cost effectiveness of colorectal cancer screening with emerging biomarkers, illustrated by a methylated Septin 9 DNA plasma assay (mSEPT9), versus established strategies.

Methods: Weconducted a cost-utility analysis using a validated decision analytic model comparing mSEPT9, fecal occult blood testing (FOBT), fecal immunochemical testing (FIT), sigmoidoscopy, and colonoscopy, projecting lifetime benefits and costs.

Results: In the base case, mSEPT9 decreased colorectal cancer incidence by 35% to 41% and colorectal cancer mortality by 53% to 61% at costs of $8,400 to $11,500/quality-adjusted life year gained versus no screening. All established screening strategies were more effective than mSEPT9. FIT was cost saving, dominated mSEPT9, and was preferred among all the alternatives. Screening uptake and longitudinal adherence rates over time strongly influenced the comparisons between strategies. At the population level, mSEPT9 yielded incremental benefit at acceptable costs when it increased the fraction of the population screened more than it was substituted for other

mSEPT9 seems to be effective and cost-effective compared with no screening. To be cost effective compared with established strategies, mSEPT9 or blood-based biomarkers with similar test performance characteristics would need to achieve substantially higher uptake and adherence rates than the alternatives. It remains to be proven whether colorectal cancer screening with a blood test can improve screening uptake or long-term adherence compared with established strategies.

Impact: Our study offers insights into the potential role of colorectal cancer screening with blood-based biomarkers.

2013 American Association for Cancer Research
Cancer Epidemiol Biomarkers Prev; 22(9); 1567–76.
doi: 10.1158/1055-9965.EPI-13-0204
9. Church et al. (2013, Gut): Prospective evaluation of methylated SEPT9 in plasma for detection of asymptomatic colorectal cancer

Background: As screening methods for colorectal cancer (CRC) are limited by uptake and adherence, further options are sought. A blood test might increase
both, but none has yet been tested in a screening setting.

Objective: We prospectively assessed the accuracy of circulating methylated SEPT9 DNA (mSEPT9) for detecting CRC in a screening population.

Asymptomatic individuals ?50 years old scheduled for screening colonoscopy at 32 US and German clinics voluntarily gave blood plasma samples
before colon preparation. Using a commercially available assay, three independent blinded laboratories assayed plasma DNA of all CRC cases and a stratified random sample of other subjects in duplicate real time PCRs. The primary outcomes measures were standardised for overall sensitivity and specificity estimates.

7941 men (45%) and women (55%), mean age 60 years, enrolled. Results from 53 CRC cases and from 1457 subjects without CRC yielded a standardised sensitivity of 48.2% (95% CI 32.4% to 63.6%; crude
rate 50.9%); for CRC stages I–IV, values were 35.0%, 63.0%, 46.0% and 77.4%, respectively. Specificity was 91.5% (95% CI 89.7% to 93.1%; crude rate 91.4%). Sensitivity for advanced adenomas was low (11.2%).

Conclusions: Our study using the blood based mSEPT9 test showed that CRC signal in blood can be detected in asymptomatic average risk individuals undergoing screening. However, the utility of the test for population screening for CRC will require improved sensitivity for detection of early cancers and advanced adenomas.

Gut 2013, 63:317-325

10. Toth et al. (2012, PLoS One): Detection of Methylated SEPT9 in Plasma Is a Reliable Screening Method for Both Left- and Right-Sided Colon Cancers

Background: Methylated Septin 9 (SEPT9) is a sensitive biomarker for colorectal cancer (CRC) from peripheral blood. However, its relationship to cancer localization, guaiac-based fecal occult blood test (gFOBT) and carcinoembryonic antigen (CEA) have not been described.

Methodology/Principal Findings:
Plasma samples were collected for SEPT9 analysis from patients with no evidence of disease (NED) (n = 92) before colonoscopy and CRC (n = 92) before surgical treatment. DNA was isolated and bisulfiteconverted using Epi proColon kit 2.0. Qualitative determination was performed using Epi proColon 2.0 RT-PCR assay.
Samples for gFOBT and CEA analysis were collected from NED (n = 17 and 27, respectively) and CRC (n = 22 and 27, respectively). SEPT9 test was positive in 15.2% (14/92) of NED and 95.6% (88/92) of CRC, including 100% (67/67) from stage II to stage IV CRC and 84% (21/25) of stage I CRC when a sample was called positive if 1 out of 3 PCR replicates was positive.
In a second analysis (2 out of 3 PCR replicates) specificity improved to 99% (91/92) of NEDs, at a sensitivity of 79.3% (73/92) of SEPT9 positives in CRC. gFOBT was positive in 29.4% (5/17) of NED and 68.2% (15/22) of CRC and elevated CEA levels were detected in 14.8% (4/27) of NED and 51.8% (14/27) of CRC. Both SEPT9 (84.8%) and CEA (85.2%) showed higher specificity than gFOBT (70.6%). SEPT9 was positive in 96.4% (54/56) of left-sided colon cancer (LSCC) cases and 94.4% (34/36) of rightsided colon cancer (RSCC) cases. gFOBT was positive in 83.3% (10/12) of cases with LSCC and 50% (5/10) of cases with RSCC, elevated CEA was detected 60% (9/15) of LSCC and 41.7% (5/12) of RSCC.

Conclusions/Significance: The high degree of sensitivity and specificity of SEPT9 in plasma makes it a better method to detect CRC than gFOBT and CEA, even for the more difficult to detect RSCC.

PLoS One 2013, 7(9): e46000
11. Lofton-Day et al. (2012, Practical Gastroenterology): Opportunities and Limitations of Blood-Based CRC Screening Tests.
Although there are several guideline-recommended options for colorectal cancer (CRC) screening, approximately one-third or 35 million eligible individuals in the United States still do not actively participate in any screening program, falling considerably short of the American Cancer Society’s nationwide goal to reach a CRC screening rate of 75% by 2015. Many experts in the medical community believe an easy to administer, effective blood-based screening test will greatly improve this situation. Recently, blood-based tests have entered the market in both the United States and Europe. This review discusses these newly available tests, their potential and the challenges they must overcome for success.

Practical Gastroenterology, 2012, 36(7):29-34.
12. Warren et al. (2011, BMC Medicine): Septin 9 methylated DNA is a sensitive and specific blood test for colorectal cancer

Background: About half of Americans 50 to 75 years old do not follow recommended colorectal cancer (CRC) screening guidelines, leaving 40 million individuals unscreened. A simple blood test would increase screening
compliance, promoting early detection and better patient outcomes. The objective of this study is to demonstrate the performance of an improved sensitivity blood-based Septin 9 (SEPT9) methylated DNA test for colorectal
cancer. Study variables include clinical stage, tumor location and histologic grade.

Methods: Plasma samples were collected from 50 untreated CRC patients at 3 institutions; 94 control samples were collected at 4 US institutions; samples were collected from 300 colonoscopy patients at 1 US clinic prior to endoscopy. SEPT9 methylated DNA concentration was tested in analytical specimens, plasma of known CRC cases, healthy control subjects, and plasma collected from colonoscopy patients.

Results: The improved SEPT9 methylated DNA test was more sensitive than previously described methods; the test had an overall sensitivity for CRC of 90% (95% CI, 77.4% to 96.3%) and specificity of 88% (95% CI, 79.6% to 93.7%), detecting CRC in patients of all stages. For early stage cancer (I and II) the test was 87% (95% CI, 71.1% to 95.1%) sensitive. The test identified CRC from all regions, including proximal colon (for example, the cecum) and had a
12% false-positive rate. In a small prospective study, the SEPT9 test detected 12% of adenomas with a false-positive rate of 3%.

Conclusions: A sensitive blood-based CRC screening test using the SEPT9 biomarker specifically detects a majority of CRCs of all stages and colorectal locations. The test could be offered to individuals of average risk for CRC who
are unwilling or unable to undergo colonscopy.

BMC Medicine 2011, 9:133
13. Toth et al. (2011, Pathol. Oncol. Res.):  The Influence of Methylated Septin 9 Gene on RNA and Protein Level in Colorectal Cancer

Colorectal cancer is one of the leading death causes in the world. Specificity and sensitivity of the present screening methods are unsuitable and their compliance is too low. Nowadays the most effective method is the colonoscopy, because it gives not only macroscopic diagnosis but therapeutic possibility as well, however the compliance of the patients is very low. Hence development of new diagnostic methods is needed. Altered expression of septin 9 was found in several tumor types including colorectal cancer.

The aim of this study was to detect the methylation related mRNA and protein expression changes of septin 9 in colorectal adenomadysplasia-carcinoma sequence and to analyze its reversibility by demethylation treatment.

Septin 9 protein expression showed significant difference between normal and colorectal cancer (CRC) samples (p<0,001). According to biopsy microarray results, septin 9 mRNA expression decreased in the progression of colon neoplastic disease (p<0,001). In laser microdissected epithelial cells, septin 9 significantly underexpressed in CRC compared to healthy controls (p<0,001). The expression of septin9_v1 region was higher in the healthy samples, while septin9_v2, v4, v4*, v5 overexpression were detected in cancer epithelial cells compared to normal. The septin 9 mRNA and protein levels of HT29 cells increased after demethylation treatment.

The increasing methylation of septin 9 gene during colorectal adenomadysplasia-carcinoma sequence progression is reflected in the decreasing mRNA and protein expression, especially in the epithelium. These changes can be reversed by demethylation agents converting this screening marker gene into therapeutic target.

Pathol. Oncol. Res. (2011) 17:503–509
DOI 10.1007/s12253-010-9338-7
14. Tänzer et al. (2010, PLoS One): Performance of Epigenetic Markers SEPT9 and ALX4 in Plasma for Detection of Colorectal Precancerous Lesions.

Background: Screening for colorectal cancer (CRC) has shown to reduce cancer-related mortality, however, acceptance and compliance to current programmes are poor. Developing new, more acceptable non-invasive tests for the detection of cancerous and precancerous colorectal lesions would not only allow  preselection of individuals for colonoscopy, but may also prevent cancer by removal of precancerous lesions.

Plasma from 128 individuals (cohort I – exploratory study: 73 cases / 55 controls ) was used to test the performance of a single marker, SEPT9, using a real-time quantitative PCR assay. To validate performance of SEPT9, plasma of 76 individuals (cohort II – validation study: 54 cases / 22 controls) was assessed. Additionally, improvement of predictive capability considering SEPT9 and additionally ALX4 methylation was investigated within these patients.

In both cohorts combined, methylation of SEPT9 was observed in 9% of controls (3/33), 29% of patients with colorectal precancerous lesions (27/94) and 73% of colorectal cancer patients (24/33). The presence of both SEPT9 and ALX4 markers was analysed in cohort II and was observed in 5% of controls (1/22) and 37% of patients with polyps (18/49). Interestingly, also 3/5 (60%) patients with colorectal cancer were tested positive by the two marker panel in plasma.

While these data confirm the detection rate of SEPT9 as a biomarker for colorectal cancer, they also show that methylated DNA from advanced precancerous colorectal lesions can be detected using a panel of two DNA methylation markers, ALX4 and SEPT9. If confirmed in larger studies these data indicate that screening for colorectal precancerous lesions with a blood-based test may be as feasible as screening for invasive cancer.

PLoS One. 2010 Feb 4;5(2):e9061.
doi: 10.1371/journal.pone.0009061.
15. Payne (2010, Future Medicine-Epigenomics): From discovery to the clinic: the noval DNA methylation biomarker mSEPT9 for the detectio of colorectal cancer in blood.
Conclusions: Rewiev article describing the whole process from marker discovery to the clinical application.

Detection of colorectal cancer at an early stage has been shown to significantly decrease mortality from the disease, while the advent of effective therapies for late-stage colorectal cancer make the detection of colorectal cancer at any stage a critical step in further reducing colorectal cancer mortality.

Availability of a blood-based test for colorectal cancer is expected to improve screening compliance in the general population. Through DNA methylation-sensitive, restriction enzyme-based biomarker discovery, we identified a region of the Septin 9 gene that is methylated in over 90% of colorectal cancer tissues with little or no methylation seen in normal colon tissue and other controls.

Specific detection of colorectal cancer DNA using the Septin 9 methylation biomarker (mSEPT9) was demonstrated in multiple studies of
plasma from colorectal cancer patients and colonoscopy-verified negative controls. A prospective, population-based trial to determine the clinical performance of mSEPT9 in colorectal cancer screening guideline-eligible individuals has recently been completed, with the results to be published in the near future.
The potential pitfalls and lessons learned in the multiyear process of developing the mSEPT9 biomarker from initial discovery to commercialization are described in this article.

Future Medicine, 2010, Epigenomics (2010) 2(4), 575–585
doi 10.2217/EPI.10.35
16. Weiss and Rösch (2010, European Oncology): Potential of a New Blood Test for Colorectal Cancer Screening - The Septin 9 Gene Biomarker.

Despite clear evidence for a better prognosis when detected early, in most countries colorectal cancer (crc) has a low compliance rate in terms of screening. There are several methods of crc screening ranging from a variety of stool tests, e.g. faecal occult blood test (foBT), to endoscopy (sigmoidoscopy, colonoscopy).
A blood test for crc detection is a new alternative, at least for patients not willing to accept screening colonoscopy or to undergo foBT. The septin 9 biomarker is a potential candidate to fulfil this purpose. It has been validated in several case–control studies, showing a strong association of plasma containing methylated DnA within the septin 9 gene (msePT9) with the presence of crc. If sensitive methylated-DnA-detection technologies are used for msePT9 detection in blood plasma samples, sensitivities of about 50% for stage i, 70–80% for stages ii and iii and 90–100% for stage iV at a specificity of ?90% have been reported in these studies.
Screening experts assume that such a blood-based test will increase compliance to crc screening. further studies are ongoing or have just been completed, including a large prospective screening trial involving 8,000 individuals in the us and germany.
The main objectives of this clinical investigation, called Prospective evaluation of septin 9 Performance for colorectal cancer screening (PresePT), are to determine the performance of the septin 9 test for identification of crc in a screening population and to demonstrate the health economic benefit of septin9 in this setting. results are expected in April 2010. This article presents an update on current analytical and clinical data on msePT9.

European Oncology, 2010;6(1)
17. DeVos et al. (2009, Clin. Chem.): Circulating Methylated SEPT9 DNA in Plasma is a Biomarker for Colorectal Cancer.

Background: The presence of aberrantly methylated SEPT9 DNA in plasma is highly correlated with the occurrence of colorectal cancer. We report the development of a new SEPT9 biomarker assay and its validation in case-control studies. The development of such a minimally invasive blood-based test may help to reduce the current gap in screening coverage.

A new SEPT9 DNA methylation assay was developed for plasma. The assay comprised plasma DNA extraction, bisulfite conversion of DNA, purification of bisulfite-converted DNA, quantification of converted DNA by real-time PCR, and measurement of SEPT9 methylation by real-time PCR. Performance of the SEPT9 assay was established in a study of 97 cases with verified colorectal cancer and 172 healthy controls as verified by colonoscopy. Performance based on predetermined algorithms was validated in an independent blinded study with 90 cases and 155 controls.

The SEPT9 assay workflow yielded 1.9 microg/L (CI 1.3-3.0) circulating plasma DNA following bisulfite conversion, a recovery of 45%-50% of genomic DNA, similar to yields in previous studies. The SEPT9 assay successfully identified 72% of cancers at a specificity of 93% in the training study and 68% of cancers at a specificity of 89% in the testing study.

Conclusion: Circulating
methylated SEPT9 DNA, as measured in the new (m)SEPT9 assay, is a valuable biomarker for minimally invasive detection of colorectal cancer. The new assay is amenable to automation and standardized use in the clinical laboratory.

Clinical Chemistry, July 2009 vol. 55 no. 7 1337-1346
doi: 10.1373/clinchem.2008.115808
18. Lofton-Day et al. (2008, Clin. Chem.): DNA Methylation Biomarkers for Blood-Based Colorectal Cancer Screening.

Sensitive, specific blood-based tests are difficult to develop unless steps are taken to maximize performance characteristics at every stage of marker discovery and development. We describe a sieving strategy for identifying high-performing marker assays that detect colorectal cancer (CRC)-specific methylated DNA in plasma.

We first used restriction enzyme-based discovery methods to identify marker candidates with obviously different methylation patterns in CRC tissue and nonpathologic tissue. We then used a selection process incorporating microarrays and/or real-time PCR analysis of tissue samples to further test marker candidates for maximum methylation in CRC tissue and minimum amplification in tissues from both healthy individuals and patients with other diseases. Real-time assays of 3 selected markers were validated with plasma samples from 133 CRC patients and 179 healthy control individuals in the same age range.

Restriction enzyme-based testing identified 56 candidate markers. This group was reduced to 6 with microarray and real-time PCR testing. Three markers, TMEFF2, NGFR, and SEPT9, were tested with plasma samples. TMEFF2 methylation was detected in 65% [95% confidence interval, 56%-73%] of plasma samples from CRC patients and not detected in 69% (62%-76%) of the controls. The corresponding results for NGFR were 51% (42%-60%) and 84% (77%-89%); for SEPT9, the values were 69% (60%-77%) and 86% (80%-91%).

Conclusion: The stringent criteria applied at all steps of the selection and validation process enabled successful identification and ranking of blood-based marker candidates.

Clinical Chemistry, February 2008 vol. 54 no. 2 414-423
doi: 10.1373/clinchem.2007.095992
19. Grützmann et al. (2008, PLoS One): Sensitive Detection of Colorectal Cancer in Peripheral Blood by Septin9 DNA Methylation Assay.

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%.

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.

PLoS One. 2008;3(11):e3759.
doi: 10.1371/journal.pone.0003759. Epub 2008 Nov 19.