Detection of Colorectal Cancer Using a Simplified SEPT9 Gene Methylation Assay Is a Reliable Method for Opportunistic Screening

SEPT9 gene methylation was validated as a biomarker for colorectal cancer (CRC) for >10 years and available as the Epi proColon test as an aid in CRC detection for >6 years. It was proven to be an accurate, reliable, fast, and convenient molecular test. In this opportunistic screening study, we validated a further simplified SEPT9 gene methylation assay in 1031 subjects in Chinese hospitals. The sensitivity for CRC detection was 76.6% at a specificity of 95.9%, and the results showed a satisfactory detection rate for each CRC stage, including early stages. The new SEPT9 assay, with enhanced technical simplicity, convenience, and lower cost, did not differ in performance compared with Epi proColon 2.0, the commercialized SEPT9 assay. The CRC detection sensitivity was further enhanced when the assay was combined with carcinoembryonic antigen (sensitivity, 86.4%) or fecal immunochemical test (sensitivity, 94.4%), suggesting that the combined tests may be an effective option for future opportunistic screening. In brief, our study has validated a new SEPT9 assay and combined testing as an aid in cancer detection, providing a new approach for opportunistic CRC screening. SEPT9 gene methylation was validated as a biomarker for colorectal cancer (CRC) for >10 years and available as the Epi proColon test as an aid in CRC detection for >6 years. It was proven to be an accurate, reliable, fast, and convenient molecular test. In this opportunistic screening study, we validated a further simplified SEPT9 gene methylation assay in 1031 subjects in Chinese hospitals. The sensitivity for CRC detection was 76.6% at a specificity of 95.9%, and the results showed a satisfactory detection rate for each CRC stage, including early stages. The new SEPT9 assay, with enhanced technical simplicity, convenience, and lower cost, did not differ in performance compared with Epi proColon 2.0, the commercialized SEPT9 assay. The CRC detection sensitivity was further enhanced when the assay was combined with carcinoembryonic antigen (sensitivity, 86.4%) or fecal immunochemical test (sensitivity, 94.4%), suggesting that the combined tests may be an effective option for future opportunistic screening. In brief, our study has validated a new SEPT9 assay and combined testing as an aid in cancer detection, providing a new approach for opportunistic CRC screening. Colorectal cancer (CRC) is the third most common malignancy in men and the second in women.1Schreuders E.H. Ruco A. Rabeneck L. Schoen R.E. Sung J.J. Young G.P. Kuipers E.J. Colorectal cancer screening: a global overview of existing programmes.Gut. 2015; 64: 1637-1649Crossref PubMed Scopus (727) Google Scholar Regular screening, early detection, and early treatment of CRC can achieve effective prevention and even cure. Despite this, 60% to 70% of CRC patients are not diagnosed until late stages, and only 11.8% of cases are detected at early stages.2American Cancer SocietyColorectal Cancer Facts & Figures 2011–2013. American Cancer Society, Atlanta2011Google Scholar It is therefore urgent to reduce the rates of CRC morbidity and mortality by improving the screening rate. In China, fecal occult blood test (FOBT), colonoscopy, and carcinoembryonic antigen (CEA) are currently available for CRC screening. FOBT is widely used because of its low cost and noninvasiveness, but the false-positive rate is relatively high because of interfering factors. 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Recently, the plasma-based SEPT9 gene methylation assay, developed as the Epi proColon test, was reported to be a promising method for the early detection of CRC. This is supported in a number of clinical studies, including case-control and prospective sample collections, to show that the methylated SEPT9 gene is a useful biomarker for early CRC detection. To date, 13 studies that evaluated this assay have exhibited a sensitivity of 56.1% to 79.3% with a specificity of 89.6% to 99.1% (two-thirds algorithm).3Tóth K. Sipos F. Kalmár A. Patai A.V. Wichmann B. Stoehr R. Golcher H. Schellerer V. Tulassay Z. Molnár B. Detection of methylated SEPT9 in plasma is a reliable screening method for both left- and right-sided colon cancers.PLOS ONE. 2012; 7: e46000Crossref PubMed Scopus (152) Google Scholar, 13Lofton-Day C. Model F. Devos T. Tetzner R. Distler J. Schuster M. Song X. Lesche R. Liebenberg V. Ebert M. Molnar B. Grützmann R. Pilarsky C. Sledziewski A. 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Lofton-Day C.E. Tetzner R. Koenig T. Quigley N.B. Weiss G. Validation of a real-time PCR-based qualitative assay for the detection of methylated SEPT9 DNA in human plasma.Clin Chem. 2014; 60: 1183-1191Crossref PubMed Scopus (178) Google Scholar, 21Johnson D.A. Barclay R.L. Mergener K. Weiss G. König T. Beck J. Potter N.T. Plasma Septin9 versus fecal immunochemical testing for colorectal cancer screening: a prospective multicenter study.PLOS ONE. 2014; 9: e98238Crossref PubMed Scopus (126) Google Scholar, 22Tóth K. Wasserkort R. Sipos F. Kalmár A. Wichmann B. Leiszter K. Valcz G. Juhász M. Miheller P. Patai Á.V. Tulassay Z. Molnár B. Detection of methylated septin 9 in tissue and plasma of colorectal patients with neoplasia and the relationship to the amount of circulating cell-free DNA.PLOS ONE. 2014; 9: e115415Crossref PubMed Scopus (82) Google Scholar, 23Jin P. Kang Q. Wang X. Yang L. Yu Y. Li N. He Y.Q. Han X. Hang J. Zhang J. Song L. Han Y. Sheng J.Q. Performance of a second-generation methylated SEPT9 test in detecting colorectal neoplasm.J Gastroenterol Hepatol. 2015; 30: 830-833Crossref PubMed Scopus (107) Google Scholar, 24Lee H.S. Hwang S.M. Kim T.S. Kim D.W. Park do J. Kang S.B. Kim H.H. Park K.U. Circulating methylated septin 9 nucleic acid in the plasma of patients with gastrointestinal cancer in the stomach and colon.Transl Oncol. 2013; 6: 290-296Abstract Full Text PDF PubMed Scopus (70) Google Scholar The clinical performance observed with the Epi proColon 2.0 kit was enhanced compared with the original product (Epigenomics AG, Berlin, Germany), and at present, Epi proColon 2.0 CE is the only commercial kit available for SEPT9 gene methylation analysis globally. Two types of screening programs are available for CRC screening, including population-based mass screening and opportunistic screening. Population-based mass screening programs have definite standards for subject recruitment, and the screening services are checked and monitored by designated individuals. Subjects taking part in population-based mass screening are offered the same services, information, and support. It can be a large-scale screening program that aims at average-risk population or a smaller program that aims at high-risk population. In contrast, opportunistic screening happens when potential subjects come to their doctors for health examination or test because of illness or discomfort. Doctors use this opportunity to encourage these subjects to attend a disease-screening program. Subjects may present various health conditions and symptoms. Unlike a population-based mass screening program, opportunistic screening may not be checked or monitored. Only one large-scale population-based mass screening study (PRESEPT study) was performed so far with SEPT9 assay.19Church T.R. Wandell M. Lofton-Day C. Mongin S.J. Burger M. Payne S.R. Castaños-Vélez E. Blumenstein B.A. Rösch T. Osborn N. Snover D. Day R.W. Ransohoff D.F. PRESEPT Clinical Study Steering Committee, Investigators and Study TeamProspective evaluation of methylated SEPT9 in plasma for detection of asymptomatic colorectal cancer.Gut. 2014; 63: 317-325Crossref PubMed Scopus (512) Google Scholar, 20Potter N.T. Hurban P. White M.N. Whitlock K.D. Lofton-Day C.E. Tetzner R. Koenig T. Quigley N.B. Weiss G. Validation of a real-time PCR-based qualitative assay for the detection of methylated SEPT9 DNA in human plasma.Clin Chem. 2014; 60: 1183-1191Crossref PubMed Scopus (178) Google Scholar This study recruited 7941 subjects, and the clinical performance of the SEPT9 assay was evaluated in 1544 subjects. Thirty of 44 CRC patients were detected by the assay, representing a screening sensitivity of 68%, whereas 1182 of 1500 non-CRC patients [including advanced adenoma, small polyps, and no evidence of diseases (NEDs)] were confirmed to be negative, representing an adjusted specificity of 80%. This prospective study represented the performance of the SEPT9 assay in a real screening background; however, no opportunistic CRC screening was performed so far with the SEPT9 assay. The main disadvantage of opportunistic screening is that it only covers those patients visiting the doctors, leaving patients who do not see a doctor out of the preventive program. However, opportunistic screening is crucial for disease prevention and early detection because patients come to their doctors normally present certain gastrointestinal (GI) symptoms, which may be signs of precancerous or cancerous diseases. Here, we have validated a simplified SEPT9 assay for CRC detection from the techniques developed by Epigenomics AG for Epi proColon 2.0. The improvements included simplified experimental procedures with a single PCR reaction, convenient operational processes with reduced numbers of kit components, an automated plasma DNA extraction procedure, and faster result interpretation, leading to a reduced per test cost. The new assay was validated as an opportunistic CRC screening assay in the hospital setting. We report on a prospective screening study to evaluate the utility of an improved SEPT9 gene methylation assay for opportunistic screening for CRC in the high-risk Chinese population in the hospital setting. This study was registered as RESEPT study at ClinicalTrials.gov (https://www.clinicaltrials.gov, trial registration ID: NCT02540850). Patients and samples with complete information were included, including sample number, sex, age, and clinical diagnosis. Positive samples refer to samples confirmed to be CRC or any other disease with colonoscopy and/or pathologic diagnosis. Normal samples refer to samples confirmed to show NEDs by colonoscopy and/or pathologic diagnosis. Patients and samples with incomplete information, including incomplete history of CRC surgery, were excluded. Patients with other cancer history, or any chemotherapy or radiotherapy, and women who were pregnant were also excluded. The plan for the trial was submitted to the ethics committee of the participating hospitals for review and approval before the start of the clinical trial. All subjects signed the informed consent before blood or stool collection, and they were informed of the usage of plasma and the test results. Clinical status was not determined before blood draw for SEPT9 assay, and blood samples were obtained from all subjects who met the selection criteria. All technicians (D.W., G.Z., P.J., J.Z., S.L., Q.W., G.W., J.S., J.W., L.S., X.H., and J.Q.) were blinded to the clinical information of subjects. To investigate the feasibility of combined screening, samples for CEA and FIT were collected at the will of participating patients and the judgment of physicians. When all planned tests were completed, subjects were divided into the following clinical status groups according to colonoscopy diagnosis and pathology: CRC, adenoma, proliferative polyps, non-CRC GI diseases (including inflammatory bowel diseases, colitis, ulcer, abscess, etc.), non-CRC cancers, and NEDs (Figure 1; Table 1, Table 2).Table 1Number of Enrolled Subjects by Diagnosis Group in the Testing Study (Opportunistic Screening)Diagnosis groupNo.SexAge (years)MaleFemale<5050–5960–69≥70CRC Total29118310853859360 Stage 04401012 Stage I37211698155 Stage II66402610152021 Stage III82493310312714 Stage IV3319149996 Not specified69501914222112Adenoma2131239056736321Polyps11763545134257Non-CRC GI diseases10854546721164Non-CRC cancers7431411NED295118177141954514Total1031545486369312243107CRC, colorectal cancer; GI, gastrointestinal; NED, no evidence of diseases. Open table in a new tab Table 2Positive Detection Rate for Each Diagnosis Group in Opportunistic ScreeningDiagnosis groupPositive detection rate [% (n/N)]95% CICRC Overall76.6 (223/291)0.713–0.814 Stage 025.0 (1/4)0.006–0.806 Stage I64.9 (24/37)0.475–0.798 Stage II72.7 (48/66)0.604–0.830 Stage III79.3 (65/82)0.689–0.874 Stage IV93.9 (31/33)0.798–0.993 Not specified78.3 (54/69)0.667–0.873Adenoma9.8 (21/214)0.062–0.146Proliferative polyps5.2 (6/116)0.019–0.109Non-CRC GI diseases3.7 (4/108)0.010–0.092Non-CRC cancers0.0 (0/7)0.000–0.410NED4.1 (12/295)0.021–0.070Overall25.8 (266/1031)0.232–0.286CRC, colorectal cancer; GI, gastrointestinal; NED, no evidence of diseases. Open table in a new tab CRC, colorectal cancer; GI, gastrointestinal; NED, no evidence of diseases. CRC, colorectal cancer; GI, gastrointestinal; NED, no evidence of diseases. Sample size estimation was based on the following equation for known positive detection rate: N = Z2 × [P (1 − p)]/E2. The parameters were defined as follows: Z is a statistical parameter (Z = 1.96 for 95% CI); E represented the error (5% was chosen in this study), and P represented the probability of a positive (putative positive detection rate). The P value (0.75) was selected from existing literature for SEPT9 sensitivity. From this, an estimated 288 CRC cases were required, so in the study a goal was set to collect complete information for 300 cases, to account for potential incomplete information, tracking, loss of samples, etc. From the estimation that CRC accounts for 30% of high-risk outpatients at least 1000 patients should be included; therefore, the study goal was to recruit 1200 patients, anticipating a 20% loss of follow-up rate (Figure 1). Samples were collected from outpatients or inpatients, and the sample information was recorded in sample collection forms. A 10-mL peripheral blood sample was collected with 10-mL K2EDTA anticoagulant tubes for the SEPT9 assay [BioChain (Beijing) Science and Technology, Inc., Beijing, P.R. China] to ensure the accuracy of the assay. Sample storage and transportation followed the instructions for use of the SEPT9 kit. For subjects who volunteered to provide samples for FIT, stool samples were collected on two consecutive days with the use of the InSure FIT (Enterix Inc., Edison, NJ) collection card, and the cards were returned to hospitals or central laboratories within 14 days from the first collection. Sample tests and data analysis followed the manufacturer's manual. CEA tests were performed in hospitals that routinely run the CEA assay. The new SEPT9 gene methylation assay (PCR fluorescence probe method) is composed of two general steps. First, total cell-free DNA in plasma was extracted from 3.5-mL plasma samples (10-mL whole blood sample) with the use of the plasma processing kit manufactured by BioChain (Beijing) Science and Technology, Inc. The DNA was then incubated with bisulfite, in which unmethylated cytosine is converted to uracil, whereas methylated cytosines are not. In the second step, methylated target sequences in the bisulfite-converted DNA template are amplified by real-time PCR. PCR blocking oligonucleotides and methylation-specific probes work together to distinguish between methylated and unmethylated DNA. The sequence of primers, blockers, and probes for SEPT9 detection used in methylation-specific PCR amplification are as follows: forward primer, 5′-CCCACCAACCATCATAT-3′; reverse primer, 5′-GTAGTAGTTAGTTTAGTATTTATTTT-3′; blocker, 5′-CATCATATCAAACCCCACAATCAACACACAAC-3′; probe1, 5′-GTTCGAAATGATTTTATTTAGTTGC-3′; probe2, 5′-CGTTGATCGCGGGGTTC-3′. Instead of performing three PCR reactions that contained 30 μL each as in the Epi proColon 2.0 assay, a single 60-μL PCR was performed for each sample in the new SEPT9 assay. The thermocycling program is as follows: activation at 94°C for 20 minutes; 45 cycles at 62°C for 5 seconds, 55.5°C for 35 seconds, and 93°C for 30 seconds; and cooling at 40°C for 5 seconds. β-Actin was used as the internal control to evaluate the plasma DNA quality and the validity of PCR amplification. The sequence of primers and probes for β-actin detection used in PCR amplification were as follows: forward primer, 5′-GTGATGGAGGAGGTTTAGTAAGTT-3′; reverse primer, 5′-CCAATAAAACCTACTCCTCCCTTAA-3′; and probe, 5′-ACCACCACCCAACACACAATAACAAACACA-3′. Positive and negative controls were provided in the kit as quality controls and were run in parallel with samples each time. The above-mentioned primers, blockers, probes, and reaction conditions were decided from the optimization experiments, in which three positive control and three negative control samples were used to define the effectiveness of the above-mentioned factors in the new 60-μL PCR reaction system. The kit was designed purposely as close as possible to Epi proColon 2.0 in components, sample preparation, bisulfite conversion, reaction condition, and performance, because it is crucial to know whether double PCR volume (60 μL) with double input of DNA can increase sensitivity. Because positive controls contain a definite quantity of methylated SEPT9 DNA, they were used as the standard in setting up the detection threshold. The selection of threshold should ensure sufficient detection of positive samples and minimal false-positive detection of negative samples. Therefore, negative controls were also used to ensure the specificity of the new assay. The aim of the optimization is that the performance of the new SEPT9 assay should be at least equally effective as Epi proColon 2.0, and the new test achieved the aim. The detection threshold and the above-mentioned reaction factors were further examined and optimized in a case-control study with the use of clinical plasma samples. Training of all staff, including the operation procedure of the assays and the use of kits and instruments, was completed before the start of the trial. This trial was a randomized, single-blind study. Patient recruitment was started in February 2014 and was completed in April 2015. Samples were processed, the SEPT9 assay was performed, and the results for SEPT9 assay were interpreted from the instructions for use. Statistical analysis was performed from the results of the SEPT9 assay and colonoscopy and/or pathologic examination. Samples from patients who volunteered to take the InSure FIT and/or CEA tests were collected, processed, and analyzed according to the instructions for use for each assay. All subjects received colonoscopy and/or pathologic examination to confirm the diagnosis after samples were collected. Colonoscopy and/or pathologic examination are regarded as the gold standard in CRC diagnosis. The data for the SEPT9 assay and the colonoscopy data for all subjects were collected and analyzed. The following parameters were calculated: sensitivity, specificity, PPV, negative predictive value (NPV), and positivity rate. Statistical analysis was performed with SPSS software version 18.0 (IBM Corporation, Armonk, NY). A full explanation of the parameters can be found at ClinicalTrials.gov (https://www.clinicaltrials.gov, trial registration ID: NCT02540850). Data from sensitivity and specificity were used to plot the receiver operating characteristic (ROC) curve. Because most cycle threshold (Ct) values from normal controls were not detected in the PCR reaction, we had to set the Ct values to 45 (the maximal number of PCR cycles we ran in the assay) for those not detected normal controls to plot the curve. This limitation led to the lack of specificity data points for Ct values >45. Therefore, no data were plotted above certain percentage for 1-specificity (the x axis) in the ROC curves. The techniques from Epigenomics AG were used to develop a simplified SEPT9 assay from our understanding of the needs of Chinese doctors and patients. The main improvements were as follows: the number of PCR reactions was reduced from three to one, and the amount of consumables in PCR reaction were also reduced to one-third of the original reaction, whereas the throughput per run increased three times. One PCR reaction per test saves times in preparing the reaction, and it is also easier for result interpretation compared with three reactions. A low-cost automated plasma DNA extraction method was used to facilitate the sample preparation. To achieve the observed test performance, a Ct cutoff value of 41 was established in the training study and validated in the testing study (opportunistic screening). As illustrated in Table 3, which reports the sensitivity and specificity with different threshold values, the Ct cutoff at 41 was optimal. These data suggest that the assay is stable in both studies, and the choice of 41 as the threshold was optimal.Table 3Sensitivity and Specificity in Training and Screening Study at Various Ct ValuesCt valueTraining study [% (n/N)]Opportunistic screening [% (n/N)]SensitivitySpecificitySensitivitySpecificity4075.9 (22/29)100.0 (0/53)70.5 (205/291)97.6 (288/295)4179.3 (23/29)94.3 (3/53)76.6 (223/291)95.9 (283/295)4279.3 (23/29)90.6 (5/53)78.7 (229/291)91.9 (271/295)Ct, cycle threshold. Open table in a new tab Ct, cycle threshold. To ensure the effectiveness of the improved SEPT9 assay in opportunistic screening, a case-control training study was performed to determine performance levels before the start of the trial. These cases are selected from the hospitals where the subsequent opportunistic screening was performed. Blood samples were collected before colonoscopy examination and stored according to the instructions for use. Samples with confirmed colonoscopy or pathologic diagnosis of CRC, adenoma, non-CRC GI diseases, or NEDs were selected to perform the SEPT9 assay. The grouping of samples in the case-control study was based on diagnosis of pathologic examination. Therefore, the samples from the case-control study were selected to examine the performance of the new SEPT9 assay, and this method is fundamentally different to that in the opportunistic screening. Table 4 lists the number of cases selected for each group and the corresponding positive detection rate. The overall sensitivity was 79.3% for CRC detection, and the positive detection rate for each CRC stage was satisfactory. The specificity was 94.3%, because the positive detection rate for NED subjects was only 5.7%, and the detection rate for adenoma and non-CRC GI diseases was also low (9.3% and 0.0%, respectively), indicating that the improved SEPT9 assay is specific for CRC detection. The ROC curve was plotted (Figure 2), and an optimal Ct value of 41 was determined for interpretation of data. The area under the ROC curve was calculated to be 0.873, suggesting a high sensitivity and specificity of the assay in distinguishing CRC from NED subjects. These data suggest that the new SEPT9 assay is not statistically different from Epi proColon 2.0 in case-control studies, and it could be further assessed in the opportunistic screening study.Table 4Number of Enrolled Subjects by Diagnosis Group and Positive Detection Rate for Each Group in the Training StudyDiagnosis groupNo.Positive detection rate [% (n/N)]CRC Total2979.3 (23/29) Stage 00NA Stage I20.0 (0/2) Stage II6100.0 (6/6) Stage III1580.0 (12/15) Stage IV5100.0 (5/5) Not specified10.0 (0/1)Adenoma869.3 (8/86)Polyps0NANon-CRC GI diseases500.0 (0/50)Non-CRC cancers0NANED5394.3 (50/53)Total21815.6 (34/218)CRC, colorectal cancer; GI, gastrointestinal; NA, not applicable; NED, no evidence of diseases. Open table in a new tab CRC, colorectal cancer; GI, gastrointestinal; NA, not applicable; NED, no evidence of diseases. To directly compare the performance of the new SEPT9 assay with Epi proColon 2.0 in detecting CRC and excluding normal subjects, 20 CRC patients and 20 NED subjects were selected from the above case-control training study, and parallel tests were performed in the same cohort. The diagnosis of CRC and NED for these subjects was confirmed by colonoscopy. Table 5 shows the number of positive detection and the concordance rate in each CRC stage for both new SEPT9 assay and Epi proColon 2.0. In all CRC stages, both assays detected the same number of subjects, and the concordance rate was 1