Blood-based SEPT9 Test in Colorectal Cancer Detection

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1 Prof. JIANQIU SHENG, PENG JIN, YING HAN GI UNIT, BEIJING MILITARY GENERAL HOSPITAL Blood-based SEPT9 Test in Colorectal Cancer Detection A Report of Preliminary Study in China Disclosure of Interest: Nothing to Disclose

2 INTRODUCTION Methylated septin 9(SEPT9) can be sensitively and specifically detected in circulating blood. Diagnostic blood-based test for methylated SEPT9 in colon cancer CRC) patients has been documented and case-control studies (in western countries) demonstrated an overall CRC detection rate of around 65%, with a false positive rate of approximately 10%. Recently, the Second generation methylated Septin 9 (SEPT9) test has been available in China. Jorja D Warren, Wei Xiong; Ashley M Bunker. Septin 9 methylated DNA is a sensitive and specific blood test for colorectal cancer. BMC Medicine 2011, 9:133

3 INTRODUCTION Fecal occult blood test (FOBT), especially human hemoglobin-specific fecal immunochemical test (FIT) improved the sensitivity and specificity for detection of CRC,but still limited by the fact that some neoplasms may not bleed. Blood-based SEPT9 test might be more acceptable compared with FOBT: blood sample drawn at a routine checkup, would encourage those who have refused or ignored screening recommendations to undergo screening. Church TR, Wandell M. Prospective evaluation of methylated SEPT9 in plasma for detection of asymptomatic colorectal cancer Gut 2014 Feb;63 (2):

4 AIMS of Study 1. to evaluate the efficacy of blood-based SEPT9 test in detection of CRC/colorectal adenoma (precancer) in Chinese population 2. to compare the effectiveness of SEPT9 with FIT in detection of CRC/colorectal adenoma (precancer) in case control study.

5 ETHICS APPROVAL This study was approved by Beijing Military General Hospital Ethics Committee Informed consent was obtained from each subject.

6 SUBJECTS & METHODS 1. Colonoscopically & pathologically confirmed cases between March 2013 and April 2014 in Beijing Military General Hospital, including: CRC: 135 cases Adenomatous polyps: 169 cases Hyperplastic polyps : 81 case Healthy controls (No Evidence of Disease, NED) : 91 cases 2. IBD, Lynch syndrome, familial adenomatous polyposis (FAP), Peutz-Jeghers syndrome, or other malignant diseases were excluded.

7 None of the patients received chemotherapy, radiotherapy, endoscopic or surgical intervention before colonoscopy. Adenomatous polyps were subclassified into two groups: 1. HGD (high grade dysplasia) n=22, including 5 cases of carcinoma in situ 2. N-HGD ( advanced adenomas size 10 mm without HGD) n=62 N-AA (Non-Advanced Adenomas) n=85 * Of the 135 cases of CRC, 90 underwent surgical treatment after colonoscopy and stages were determined based on the resected specimens.

8 SUBJECTS & METHODS Peripheral blood samples were taken before colonoscopy preparation using 10 ml EDTA tubes. Plasma was isolated from whole blood by repeated centrifugation for 12 min. at 1,350 rcf and stored at -80. Frozen samples were sent to BioChain Medical Laboratory (Beijing, China) for SEPT9 test, according to the manufacturer s instructions described by Tóth et al. Each sample was tested in triplicate (2/3 as positive). The laboratory was blind to the subjects clinical results.

9 SUBJECTS & METHODS Both SEPT9 and FIT were tested simultaneously in 177 cases, including: 69 CRC 65 adenomatous polyps 16 hyperplastic polyps 27 healthy controls

10 SUBJECTS & METHODS Epi procolon 2.0 test (Epigenomics AG, Berlin, Germany) Plasma DNA methylation(sept9) detection kit 1.Epi procolon Plasma Quick Kit 2.Epi procolon Sensitive PCR kit 3.Epi procolon Control kit

11 RESULTS Total SEPT9 + (n, %) SEPT9 (n, %) No Evidence of Disease(NED) 91 3 (3.3%) 88 (96.7%) Hyperplastic polyps 81 5 (6.2%) 76 (93.8%) Adenomatous polyps (20.7%) 134 (79.3%) N-AA (14.1%) 73 (85.9%) N- HGD (22.6%) 48 (77.4%) HGD 22 9 (40.9%) 13 (59.1%) CRC (74.8%) 34 (25.2%)

12 SEPT9 Colonoscopy / Pathology CRC NED(CONTROL) Total Positive 101(A) 3(B) 104 (A+B) Negative 34 (C) 88 (D) 122(C+D) Total 135(A+C) 91 (B+D) Sensitivity=a/(a+c)*100%= 74.8%(95% CI:67.0%~ 81.6%) Specificity=d/(b+d)*100%=96.7%(95% CI: 91.3%~ 99.2 %) 226(A+B+C+D Accuracy=(a+d)/(a+b+c+d)*100%=83.6%(95%CI: 78.4%~ 88.0 %) Positive predictive value=a/(a+b)*100%=97.1% (95%CI : 92.4%~ 99.3 %) Negative predictive value=d/(c+d)*100%=72.1% (95%CI: 63.7%~ 79.5 %) )

13 CRC stage vs. SEPT9 Stage I C R C Stage II Stage III Stage IV SEPT (66.7%) 19 (82.6%) 37 (84.1%) 5 (100%) Total With the progression of CRC stage, the higher positive rate of SEPT9 detected

14 CRC+ Adenoma vs. SEPT9 SEPT9 Sensitivity 95% CI Specificity 95% CI CRC 74.8% 67.0%-81.6% 87.4% 83.5%-90.6% CRC + HGD 70.1% 62.6%-76.8% 89.3% 85.6%-92.4% CRC + N-HGD 56.6% 50.0%-63.1% 92.2% 88.4%-95.1% CRC +Adenoma 44.7% 39.2%-50.4% 95.3% 91.4%-97.8% N-AA 27.4% 18.7%-37.6% Adenomas 20.7% 15.1%-27.3%

15 Total FIT + (n, %) SEPT9 VS.FIT SEPT9 + (n, %) FIT + SEPT9 + FIT + SEPT9 - FIT SEPT9 + FIT SEPT9 - No evidence of disease 27 1 (3.7%) 1 (3.7%) Hyperplastic polyps 16 5 (31.3%) 3(18.8%) Adenomatous polyps 65 13(20.0%) 12(18.5% N-AA 30 3 (10.0%) 3 (10.0%) N- HGD 26 7 (26.9%) 7 (26.9%) HGD 9 3 (33.3%) 2 (22.2%) CRC

16 SEPT9 vs. FIT in CRC detection SEPT9 + SEPT9 - Total FIT + 28 (40.6%) 12(17.4%) 40(58.0%) FIT - 25(36.2%) 4(5.8%) 29(42.0%) P=0.033 Total 53(76.8%) 16(23.2%) 69(100%) Missed detection rate for CRC: FIT 42%(n=25); SEPT9 23.2%(n=12); FIT+SEPT9 5.8%(n=4)

17 SEPT9 vs. FIT in CRC+HGD detection SEPT9 + SEPT9 - Total FIT + 32(30.8%) 18(17.3%) 50(48.1%) FIT - 30(28.8%) 24(23.1%) 54(51.9%) Total 62(59.6%) 42(40.4%) 104(100%)

18 SUMMARY Positive rates of blood-based SEPT9 test increased along with the progression of colorectal neoplasm. Blood based SEPT9 test is a potential candidate for screening of CRC. Combination of blood-based SEPT9 test and FIT increases the detection rate of CRC,in a reciprocal manner. Asymptomatic population screening is needed to evaluate the sensitivity, specificity and accuracy of blood-based SEPT9 test in detection of colorectal neoplasm.

19 THANKS FOR YOUR ATTENTION!

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