Blood based colon cancer screening in Europe. Methylated Septin 9: Biomarker of malignant development in the colon Measured in Tissue and Plasma
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1 Blood based colon cancer screening in Europe Methylated Septin 9: Biomarker of malignant development in the colon Measured in Tissue and Plasma Molnár, Béla M.D., PhD 2nd Dept. of Medicine Semmelweis University Budapest Hungary
2 Continuous colorectal epithelial proliferation is the basis of CRC development Red: PCNA, divided cell nucleus Green: Tunnel reaction, apoptotic cell nucleus Blue: Calm cell nucleus
3 The genetic alterations in the colorectal adenomadysplasia-carcinoma sequence Vogelstein et al, N Engl J Med 1988
4 2nd Department 2nd Department of Internal of Internal Medicine, Medicine, Semmelweis Semmelweis University University Colorectal cancer development: a multistep, multiparameter process. B. Molnar et.al. Experiences in the production of digital slides..
5 Screening of colorectal cancer by imaging Type of screening Method Description Advantages Disadvantages Endoscopic examinations Flexible sigmoidoscopy: Visual examination of the rectum and lower third of the colon by insertion of a flexible tube into the colon. Colonoscopy: Direct visual examination of the entire colon and rectum with removal of polyps Colon capsule endoscopy: Visualization of colon through swallowed pill-sized camera Requires minimum preparation No sedation required Visualizes entire bowel Allows biopsies and polypectomies and therefore may be preventive Complete evaluation of the gastrointestinal tract Invasive Tumors in the upper colon may not be detected Invasive Requires bowel preparation Sedation required Requires bowel preparation Capsule retention After ingesting until excretion powerful electromagnetic fields should be avoided
6 Type of screening Method Description Advantages Disadvantages Computed tomographic colonography (CTC): 2- or 3-dimensional x-ray views of entire colon and rectum Minimally invasive Procedure performed within 10 mins No recovery time No Sedation required Requires bowel preparation Colonoscopy may be performed afterwards to remove any suspicious polyps Radiologic examinations Double contrast barium enema (DCBE): Usually view of entire colon; complete radiological examination of the colon Minimally invasive Detects cancers and majority of polyps Evaluates entire colon May require drinkable contrast solution Radiation exposure Requires bowel preparation Colonoscopy may be performed afterwards to remove any suspicious polyps Radiation exposure
7 Screening of colorectal cancer by stool test Type of screening Method Description Advantages Disadvantages Fecal occult blood tests (FOBT): Guaiac-based (gfobt) Non-invasive Inexpensive Non-bleeding tumors not detected Dietary restrictions for higher accuracy Stool tests Immunochemical (ifobt) Detection of tumor cell DNA shed into large bowel M2-PK: Detection of specific enzyme M2-PK Can detect large bleeding polyps Non-invasive Single stool sample Non-invasive No dietary restrictions Repeat samples needed High costs for stool test High rate of falsepositives
8 Issues of the screening programs in Europe : Capacity of the organisations Costs of the programms Sensitivity/specificity of the methods Patients s compliance and acceptance, attitude Taber JM, et al. : Preferences for blood-based colon cancer screening differ by race/ethnicity. American Journal of Health Behaviour 38, (2014). Blood based cancer screening markers : clinical and routine aspects: Simplicity Patient s acceptance Patient s compliance towards existing methods
9 Tumor (protein) markers in peripheral blood CA19-9 CEA AFP Combination markers of serum tumor markers No Screening application of today Recurrence detection, follow-up examinations only
10 mrna expression based colorectal cancer peripheral blood test Biomarker Method Test Producer Sensitivity [%] Specificity [%] ANXA3, CLEC4D, LMNB1, PRRG4, TNFAIP6, VNN1, IL2RB mrna expression of 7 gene biomarker combination ColonSentry GeneNews (Canada) 72 70
11 Potential plasma biomarkers for future CRC screening: Marker Type of marker Sensitivity Specificity mir-21 microrna in serum BIRC5 mrna in serum SDC2 KIAA1199 ONC107 Methylated biomarker in serum qrt-pcr for measurement of mrna in plasma Mass spectrometry assay of serum proteins
12 plasma DNA concentration (ng/ml) 2nd Department of Internal Medicine, Semmelweis University Circulating free DNA in peripheral blood Healthy IBD Adenoma CRC Mean (ng/ml) 176, , , ,568 SD 82, ,97 111, , Normal IBD Adenoma CRC sample groups Healthy vs. IBD 0,3143 Healthy vs. Adenoma 0,9254 Healthy vs. CRC 0,0391
13 Source of plasma circulating tumor cells and tumor derived DNA
14 Why is methylated Septin 9 a good CRC biomarker? Septin 9 an absolute(100%) colorectal cancer biomarker How is methylated Septin 9 a suitable CRC screening marker? NGS sequencing of Plasma circulating free DNA Methylated Septin9 in plasma EpiproColon is a good CRC screening test? Ver.1 ; Ver. 2.0
15 CpG island 2nd Department of Internal Medicine, Semmelweis University DNA methylation: an alternative mechanism to mutation for gene silencing GENE expression Methylation CpG island Demethylation X GENE repression DNA methylation of CpG islands located in the promoter region can lead to repression of transcription Dysregulation of DNA methylation is proven to contribute to the formation of colorectal cancer DNA methylation is reversible, and can be influenced by demethylation agents (e.g. 5-aza-2'-deoxycytidine)
16 Methylation of Septin 9 in CRC at the tissue level Wasserkort et al. BMC Cancer 2014
17 Septin 9 methylation in CRC tissue Healthy biopsy SEPT9/ACTB ratio Adenoma biopsy SEPT9/ACTB ratio Cancer biopsy SEPT9/ACTB ratio PMR for m SEPT9 in tissue PMR >1% found in NED 7.7% (1/13) Adenoma 100% (10/10) CRC biopsies 100% (16/16) PMR = percent methylation reference Toth et al. Path. Onc. Res. PORE 2012
18 Septin9 RNA levels in CRC laser micro-dissected samples Stroma 1. Cutting of the tissue compartments by focused laser ray 2. Removal of the samples from slide 3. RNA and DNA isolation from the cells 4. Affymetrix whole genomic microarray analysis array (> transcripts) Toth et al. Path. Onc. Res. PORE ,8 2,75 2,7 2,65 2,6 2,55 2,5 2,45 2,4 2,35 2,3 2,25 2,8 2,75 2,7 2,65 2,6 2,55 2,5 2,45 2,4 2,35 2,3 2, Epithelium
19 SEPTIN9 protein expression correlates with methylation and RNA epression in the N-Ad-Dyspl-CRC sequence Toth et al. Path. Onc. Res PORE 2012
20 Septin 9 in colorectal cancer 1. Methylated Septin 9 correlated with decreased protein IHC 2. Methylated Septin 9 correlated with decreased mrna transcription 3. Zero/0 levels of methylated Septin 9 in normal epithelia 4. Laser capture studies confirm results
21 NGS sequencing (Solid) of plasma, free DNA, Septin 9 gene Adenoma CRC Normal IBD pooled specimen, 1000 base pairs resolution
22 NGS sequencing data of circulating free DNA: Septin 9 Cpg island 100 BASE PAIRS ANALYSIS
23 Application of the Epi procolon plasma Methylated Septin9 assay in the clinical workflow
24 Worksteps and components of the Epi procolon kit
25 Semi-Automated Septin 9 PCR workflow Manual sample preparation Prepared PCR plate Automated reaction preparation Quantitative PCR measurements 3x
26 Methods 1. Sample collection 2. Total DNA isolation - plasma: Epi procolon 2.0 plasma DNA preparation kit (Epigenomics AG, Berlin, Germany). 3. Bisulfite conversion - non-methylated cytosines are converted to uracil 4. Quantitative determination - Epi procolon 2.0 Real time-pcr kit
27 Plasma msept9 results in the development phase Publication devos, 2009 devos, 2009 Grützmann, 2008 Test Version Research Kit Research Kit Research Kit Algorith m 1 of 3 2 of 3 2 of 3 Sensitivity, 72% 56% 58% [49-67%] Specificity, 86% 95% 90% [85-93%] CRC I, 53% 26% 36% [17-59%] CRC II, 75% 60% 56% [38-72%] CRC III, 78% 67% 65% [51-77%] CRC IV, 100% 75% 73% [39-94%] Adenoma ND ND 18% [4-43%] Polyp ND ND 9% [2-34%]
28 Plasma msept9 results Epi procolon 1.0 Publicatio n Test Version Algorith m Sensitivity, Specificity, CRC I, CRC II, CRC III, CRC IV, Adenoma Polyp Weiss, 2010 Epi procolon of 2 67% [57-76%] 88% [82-92%] 44/66* 25/37* ND ND Church, 2014 Epi procolon of 2 51% [32-64%] 91% [90-93%] 36% [13-60%] 57% [33-88%] 58% [17-85%] 80% [24-100%] 10% [7-16%] 8% [4-11%] Church, 2014 Epi procolon 1.0 plus additional PCR 1 of 3 64% [48-80%] 88% [87-90%] ND ND ND ND 14% ND
29 Plasma msept9 results: Epi procolon 2.0 Publication Test Version Algorith m Sensitivity, Specificity, CRC I, CRC II, CRC III, CRC IV, Adenoma Polyp Tóth, 2012 Epi procolon of % [ %] 84.8% [ %] 84% 100% 100% 100% ND ND Tóth, 2012 Epi procolon of % [ %] 98.9% [ %] 60% 92.8% 88.6% 77.8% ND ND Potter, 2014 Epi procolon US 1 of 3 68% [53-80%] 80% [78-82%] 64% [48-77%] 100% [57-100%] 22% [18-24%] 20% [16-24%] Johnson, 2014 Epi procolon US 1 of 3 73% [64-81%] 82% [76-86%] 62% [43-78%] 80% [58-92%] 65% [45-81%] 92% [67-100%] ND ND
30 Detection of CRC stages by plasma msept9 assay
31 Laboratory derived tests for Septin 9 Septin 9 LDT RealTime ms9 Colorectal Cancer assay Name Company Sens. Spec. Abbott Molecular Septin 9 LDT (US) ColoVantage Quest Diagnostics Septin 9 LDT (US) SEPT9 LDT ARUP Laboratories Septin 9 LDT (Canada) SEPT9 LDT Gamma Dynacare 70 90
32 BSM_0502 BSM_0503 BSM_0504 BSM_0505 BSM_0506 BSM_0507 BSM_0508 BSM_0509 BSM_0510 BSM_0511 BSM_0512 BSM_0513 BSM_0514 BSM_0515 BSM_0516 BSM_0517 BSM_0518 BSM_0519 BSM_0520 BSM_0521 BSM_0462 BSM_0463 BSM_0464 BSM_0465 BSM_0466 BSM_0467 BSM_0468 BSM_0469 BSM_0470 BSM_0471 BSM_0472 BSM_0473 BSM_0474 BSM_0475 BSM_0476 BSM_0477 BSM_0478 BSM_0479 BSM_0480 BSM_0481 BSM_0482 BSM_0483 BSM_0484 BSM_0485 BSM_0486 BSM_0487 BSM_0488 BSM_0489 BSM_0490 BSM_0491 BSM_0492 BSM_0493 BSM_0494 BSM_0495 BSM_0496 BSM_0497 BSM_0498 BSM_0499 BSM_0500 BSM_0501 2nd Department of Internal Medicine, Semmelweis University Septin 9 methylation in plasma of adenoma/cancer patients Healthy plasma SEPT9/ACTB ratio Adenoma plasma SEPT9/ACTB ratio Cancer plasma SEPT9/ACTB ratio PMR for m SEPT9 in plasma PMR > 0,01% found in NED 10% (2/20) Adenoma 25% (5/20) CRC 85% (17/20) PMR = percent methylation reference
33 Septin 9 colon cancer : test limitations Pregnancy : always positive 100 %! Inflammatory conditions: as normals in the sens. and specs.
34 Technical advances can influence the SEPTIN9 methylated DNA based plasma testing DNA isolation : automation for higher plasma volume, higher DNA yield ( adenoma patients) Bisulfite conversion: decrease in loss of DNA Digital PCR : increased sensitivity and single copy analysis DNA release induction : Proapoptotic induction therapy NSAIDs
35 Methylated Septin9 and the Epi ProColon test: Clear scientific support for a role in colorectal cancer mrna, IHC, DNA studies on Septin9 in tissue Clinically validated biomarker for colorectal cancer screening Real-Time PCR, Next Gen Sequencing - plasma Epi procolon a CE marked IVD for colorectal cancer screening Complete kit for IVD use plasma based screening for colorectal cancer
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