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Journal of Digestive Diseases 2016; 16; 699–712
doi: 10.1111/1751-2980.12299
Systematic Review
Noninvasive DNA methylation biomarkers in colorectal cancer: A systematic review Meng XUE,*,† San Chuan LAI,†,‡ Zhi Peng XU* & Liang Jing WANG*,† *Department of Gastroenterology, The Second Affiliated Hospital, School of Medicine, Zhejiang University, Institute of Gastroenterology, Zhejiang University, and ‡ Department of Gastroenterology, Sir Run Run Shaw Hospital, School of Medicine, Zhejiang University, Hangzhou, Zhejiang Province, China
†
OBJECTIVE: To summarize the current evidence on the biomarkers associated with DNA methylation in the screening and diagnosis of colorectal cancer (CRC). METHODS: A literature search was conducted on the databases of PubMed and Web of Science to identify articles published from 1 January 2000 to 6 June 2015 with language striction. Stuides focusing on the association between noninvasive biomarkers indicating DNA methylation and CRC were included. RESULTS: Altogether 74 studies were finally included in the study. Varied genetic markers in the feces and blood samples were hypermethylated in patients KEY WORDS:
with CRC than in the healthy controls. Some of them could even be detected at the early stage of the tumors. The sensitivity of the genetic markers was superior to that of fecal occult blood test and carcinoembryonic antigen. Multitarget DNA assays using a combination of different methylated genes could improve the diagnostic sensitivity. CONCLUSIONS: Genetic markers might be minimally invasive, economical and accurate for the screening and surveillance of CRC. Large multicenter studies evaluating these biomarkers systematically and prospectively not only in CRC but also in other types of cancers are needed in the future.
colorectal neoplasms, DNA methylation, screening.
INTRODUCTION Colorectal cancer (CRC), a neoplasm that was known to be common only in the developed countries, has been increasingly detected among the populations in the developing countries and regions nowadays, with high incidence and mortality.1 Early screening and Correspondence to: Liang Jing WANG, Department of Gastroenterology, The Second Affiliated Hospital, School of Medicine, Zhejiang University, 88 Jiefang Road, Hangzhou, Zhejiang Province 310009, China. Email: [email protected] Conflict of interest: None. © 2016 Chinese Medical Association Shanghai Branch, Chinese Society of Gastroenterology, Renji Hospital Affiliated to Shanghai Jiaotong University School of Medicine and Wiley Publishing Asia Pty Ltd
diagnosis of CRC brings timely treatment and significantly decreased mortality in these patients. Serum carcinoembryonic antigen (CEA) level,2 fecal occult blood test (FOBT)3 and conventional colonoscopy4 have long been used for the screening and surveillance of CRC. The former two modalities, with a low medical cost, achieve a high compliance among the individuals; however, false positivity and negativity are inevitable.5 On the other hand, colonoscopy has a high sensitivity, but the discomfort and the risks related to bowel preparation and the procedure per se make it less widely accepted.6,7 Both genetic and epigenetic alterations have been found to be involved in the carcinogenesis of CRC.8 The epigenetic modifications include DNA methylation, histone modifications, nucleosomal occupancy and remodeling, chromatin looping and noncoding RNA,9 among which
699
700
M Xue et al.
the CpG island methylator phenotype (CIMP) is known to be the most common molecular feature.10 Chromosomes 1, 5, 6, 8, 11, 13, 18, 19, 21 and 22 are the locations where methylation alterations frequently occur in CRC, as reported by genome-wide DNA methylation analyses.11 A cluster of genes characterized by altered DNA methylation have been identified during the past few decades along these chromosomes, including Zinc finger of the cerebellum 1 (ZIC1) and Klotho (KL), which were first found to be hypermethylated in CRC in our previous studies.12,13 Methylated DNA fragments are detectable in not only primary tumor tissues but also remote media including blood, stool, urine and bowel lavage fluid,14–16 making them eligible to act as a screening tool for CRC in clinical setting. In this review, we aimed to summarize the current evidence on the biomarkers associated with DNA methylation in the screening and diagnosis of CRC. MATERIALS AND METHODS A literature search was conducted on the databases of PubMed and Web of Science for articles published from 1 January 2000 to 6 June 2015. The following terms were used for the search: (“methylation” OR “methylated”) AND (“colorectal cancer” OR “colon cancer” OR “rectal cancer”) AND (“screening” OR “diagnosis”). Stuides focused on the association between noninvasive biomarkers indicating DNA methylation, including those based on blood, stool and urine samples, and CRC were included in the study. Prospective, retrospective or crosssectional studies published in English or Chinese were included. And only those compared CRC with healthy controls, with the sample size of ≥2 were included. The references of the included manuscripts were screened for eligible studies, and abstracts presented at related conferences were also searched. Reviews, comments, consensus, meta-analyses, letters to the editors, case reports and case series, studies on animals or cell lines were excluded from the study. The list of articles generated by the search was manually reviewed by two investigators (Meng XUE and San Chuan LAI). Any disagreement was resolved by the discussion with the other two investigators (Zhi Peng XU and Liang Jing WANG). The quality of the studies was assessed using the Quality Assessment of Diagnostic Accuracy Studies (QUADAS). RESULTS The characteristics of the studies Altogether 2 401 studies were identified during the primary literature search. Among them, 1 093 were
Journal of Digestive Diseases 2016; 16; 699–712 excluded because of the manuscript type (consensus, guidelines, meta-analysis, systematic review, letter to the editor, editorial, case report or case series) (n = 1038) and the language restriction (not published in English or Chinese) (n = 55). Potentially relevant manuscripts were retrieved for further review, of which 1 236 studies were excluded for being irrelevant to the noninvasive DNA methylation biomarkers in CRC. The references of the remaining 72 articles were checked manually, and two articles were further included. Finally, a total of 74 studies15–88 were eligible for this systematic review. The flowchart of study selection is shown in Figure 1. Among all the 74 studies, 40 was conducted in the Western countries15,17,19,24,26,27,30–39,43,48,49,52,56– 59,62,64–67,69–72,75–79,84,85 (including one also performed in West Asia,39 and one also in East Asia52), 30 in East Asia,16,18,20–22,25,28,29,40–42,44–47,50,51,53– 55,60,61,63,73,80–84,87 two in Iran,68,74 one in 23 Singapore, and the remaining one unreported88. In 35 studies, samples were handled and analyzed in a blinded fashion.15,18,31–35,37,38,43,49–51,54–56,58–60,64,65,67– 69,71,74–76,78,80–85 Methylation-specific PCR (MSP), quantitative (qMSP) or nested MSP (nMSP) were applied in most studies,17,20–26,29,31–34,36,40,42,44–48,50,51,53–61,63,64,66– 84,87,88 quantitative allele-specific real-time target and signal amplification (QuARTS) in 3 studies,65,83,84 Methylight or Methyl-BEAMing (beads, emulsion, amplification and magnetics) in 9 studies,16,18,19,27,28,39,43,49,86 commercial methylation assay kit in 3 studies,35,37,38 pyrosequencing in 2 studies,62,87 Melting curve analysis in 2 studies,41,75 and methylation microarray in one study30. The QUADAS was applied to assess the quality of each study. Seven studies18,51,55,58,59,69,74 were marked “yes” to all the 14 questions, suggesting a good quality, while 11–13 questions were marked “yes” for the remaining studies. In 13 studies,18,39,42,51,55,58,59,62,69,70,72,74,88 noninvasive samples were collected either within two weeks before or after colonoscopy for histopathology or from newly diagnosed CRC; therefore, question 4 was marked with “yes”. In those 35 blinded studies,15,18,31–35,37,38,43,49–51,54–56,58–60,64,65,67–69,71,74– 76,78,80–85 DNA methylation status was assessed without histopathological results, and question 10 was labeled as “yes”. The histopathological diagnosis was not avaliable yet at the time of sample collection in two studies,79,88 and whether the pathologists were blinded to the methylation results was unclear; therefore, question 11 was labeled as “unclear” for these two studies.
Journal of Digestive Diseases 2016; 16; 699–712
Figure 1.
Noninvasive methylation markers in CRC
701
Flowchart demonstrating the selection of eligible studies.
Blood-based DNA methylation biomarkers Diagnostic efficiency
Most whole blood DNA is extracted from leukocytes. The association between the low-level methylation of leukocyte DNA and the risk of CRC tends to suggest that genomic-wide DNA is hypomethylated in CRC.89,90 Another pattern of methylation defects in tumor is the hypermethylation of CpG islands in localized promoter regions,91 which allows the researchers to illustrate the role of DNA methylation in the development of cancers. Apoptotic and necrotic tumor cells release DNA into the circulation by macrophages;92 therefore, not only genomic DNA extracted from whole blood but also extracellular free DNA from serum or plasma can be used for blood-based screening of the tumors. Over 20 genes with hypermethylated promoters had been identified in the blood over the last 15 years, and their positive rates in CRC ranged from 6.1% to 95.6% (Table 1).17–48 MutL homolog 1 (MLH1) was the first gene identified to be with a hypermethylated promoter. Grady et al.17 assessed the methylation status of hMLH1 in the serum of 18 consecutive CRC patients by MSP, and found 3 (16.7%) with positive hMLH1 expression accompanied by microsatellite instability. In another study conducted by Leung et al.18 a high-throughput quantitative assay, MethyLight, was applied to detect methylation,93 achieving a sensitivity of 42.9% and 32.7%, respectively, for determining the methylation
status of hMLH1 and helicase-like transcrioption factor (HLTF) in CRC. While the specificity in distinguishing methylation in 41 age-matched controls was 97.6% and 92.7%, respectively. However, combining three methylation markers [adenomatous polyposis coli (APC), MLH1 and HLTF] improved the sensitivity to 57.0%. Serum or plasma cyclin-dependent kinase inhibitor 2A (CDKN2A) methylation was assessed by MSP in four studies.20–23 Altogether 101 (57.4%) of the 176 CRC patients but no healthy volunteers had hypermethylated CDKN2A, yielding a strikingly high specificity of 100%. In addition to CDKN2A, Tan et al.23 also demonstrated a high methylation rate of runt-related transcription factor 3 (RUNX3) in CRC patients, with a sensitivity of 65% and a specificity of 100%, respectively. For methylated vimentin (VIM) in CRC a sensitivity of 59% was reported, as detected by a digital quantification method named BEAMing (beads, emulsion, amplification and magnetics) technology.39 This enabled the identification of a single DNA molecule of methylated VIM mixed with 1 000 copies of unmethylated VIM, thus improving the sensitivity, specificity and standardization among different assays.
Methylated Septin 9 (mSEPT9)
Although it had been discovered for less than 10 years,35 mSEPT9 was emerged as one of the most
702 Table 1.
Genes (panel)
M Xue et al.
Journal of Digestive Diseases 2016; 16; 699–712
Blood-based methylation markers in colorectal cancer (CRC) First author (publication year)
Single gene hMLH1 Grady17 (2001) Leung18 (2005) Wallner19 (2006) CDKN2A (p16) Zou20 (2002) Nakayama21 (2002) Nakayama22 (2003) Tan23 (2007) CDH4 Miotto24 (2004) HLTF Leung18 (2005) Wallner19 (2006) Lee25 (2009) APC Leung18 (2005) ALX4 Ebert26 (2006) Tänzer27 (2010) He28 (2010) HPP1/TPEF Wallner19 (2006) RUNX3 Tan23 (2007) Nishio29 (2010) RASSF1A Tan23 (2007) Cassinotti30 (2012) CDH1 Tan23 (2007) SEPT9 Tänzer27 (2010) He28 (2010) Lofton-Day31 (2008) Grützmann32 (2008) deVos33 (2009) Warren34 (2011) Ahlquist35 (2012) Tóth36 (2012) Church37 (2014) Potter38 (2014) TMEFF2 Lofton-Day31 (2008) He28 (2010) NGFR Lofton-Day31 (2008) VIM Li39 (2009) Shirahata40 (2010) Wif1 Lee25 (2009)
Healthy controls Specimens
Methods
CRC
Total Methylation Total Methylation (N) (n, %) (N) (n, %) AUROC
Serum Serum Serum
MSP MethyLight MethyLight
NA 41 20
NA 1 (2.4) 0 (0)
NA 0.71 NA
NA 0.0001 0.016
Serum Serum Serum Serum
MSP MSP MSP MSP
10 NA NA 10
0 (0) NA NA 0 (0)
20 94 45 17
14 44 31 12
(70.0) (46.8) (68.9) (70.6)
NA NA NA NA
0.001 NA NA NA
Whole blood
MSP
17
0 (0)
46
32 (69.6)
NA
0.001
Serum Serum Plasma
MethyLight MethyLight MSP
41 20 276
49 103 243
16 (32.7) 31 (30.1) 44 (18.1)
0.63 NA NA
0.015 0.005 NA
Serum
MethyLight
41
49
3 (6.1)
0.53
0.21
Serum Plasma Plasma
MethyLight MethyLight MethyLight
30 22 170
30 5 182
25 (83.3) 4 (80.0) 87 (47.8)
0.839 NA NA
0.0001 0.289 0.001
Serum
MethyLight
20
0 (0)
103
13 (12.6)
NA
0.09
Serum Serum
MSP Real-time qMSP
10 54
0 (0) NA
17 344
11 (64.7) 100 (29.1)
NA NA
0.001 0.0001
Serum Plasma
MSP Microarray
10 30
17 30
4 (23.5) 28 (93.3)
NA NA
0.097 0.01
Serum
MSP
10
17
3 (17.6)
NA
0.159
Plasma Plasma Plasma Plasma Plasma Plasma Plasma Plasma Plasma Plasma
MethyLight MethyLight qMSP qMSP qMSP qMSP qMSP Epi proColon Epi proColon Epi proColon
34 170 179 285 327 94 48 92 NA 444
4 6 25 25 45 11 13 14
(11.8) (3.5) (14.0) (8.8) (13.8) (11.7) (27.1) (15.2) NA 97 (21.8)
33 182 133 378 187 50 30 92 53 44
27 (81.8) 136 (74.7) 92 (69.2) 193 (51.1) 138 (73.8) 45 (90.0) 18 (60.0) 88 (95.7) 27 (50.9) 30 (68.2)
NA NA 0.80 NA NA NA NA NA NA NA
Journal of Digestive Diseases 2016; 16; 699–712
doi: 10.1111/1751-2980.12299
Systematic Review
Noninvasive DNA methylation biomarkers in colorectal cancer: A systematic review Meng XUE,*,† San Chuan LAI,†,‡ Zhi Peng XU* & Liang Jing WANG*,† *Department of Gastroenterology, The Second Affiliated Hospital, School of Medicine, Zhejiang University, Institute of Gastroenterology, Zhejiang University, and ‡ Department of Gastroenterology, Sir Run Run Shaw Hospital, School of Medicine, Zhejiang University, Hangzhou, Zhejiang Province, China
†
OBJECTIVE: To summarize the current evidence on the biomarkers associated with DNA methylation in the screening and diagnosis of colorectal cancer (CRC). METHODS: A literature search was conducted on the databases of PubMed and Web of Science to identify articles published from 1 January 2000 to 6 June 2015 with language striction. Stuides focusing on the association between noninvasive biomarkers indicating DNA methylation and CRC were included. RESULTS: Altogether 74 studies were finally included in the study. Varied genetic markers in the feces and blood samples were hypermethylated in patients KEY WORDS:
with CRC than in the healthy controls. Some of them could even be detected at the early stage of the tumors. The sensitivity of the genetic markers was superior to that of fecal occult blood test and carcinoembryonic antigen. Multitarget DNA assays using a combination of different methylated genes could improve the diagnostic sensitivity. CONCLUSIONS: Genetic markers might be minimally invasive, economical and accurate for the screening and surveillance of CRC. Large multicenter studies evaluating these biomarkers systematically and prospectively not only in CRC but also in other types of cancers are needed in the future.
colorectal neoplasms, DNA methylation, screening.
INTRODUCTION Colorectal cancer (CRC), a neoplasm that was known to be common only in the developed countries, has been increasingly detected among the populations in the developing countries and regions nowadays, with high incidence and mortality.1 Early screening and Correspondence to: Liang Jing WANG, Department of Gastroenterology, The Second Affiliated Hospital, School of Medicine, Zhejiang University, 88 Jiefang Road, Hangzhou, Zhejiang Province 310009, China. Email: [email protected] Conflict of interest: None. © 2016 Chinese Medical Association Shanghai Branch, Chinese Society of Gastroenterology, Renji Hospital Affiliated to Shanghai Jiaotong University School of Medicine and Wiley Publishing Asia Pty Ltd
diagnosis of CRC brings timely treatment and significantly decreased mortality in these patients. Serum carcinoembryonic antigen (CEA) level,2 fecal occult blood test (FOBT)3 and conventional colonoscopy4 have long been used for the screening and surveillance of CRC. The former two modalities, with a low medical cost, achieve a high compliance among the individuals; however, false positivity and negativity are inevitable.5 On the other hand, colonoscopy has a high sensitivity, but the discomfort and the risks related to bowel preparation and the procedure per se make it less widely accepted.6,7 Both genetic and epigenetic alterations have been found to be involved in the carcinogenesis of CRC.8 The epigenetic modifications include DNA methylation, histone modifications, nucleosomal occupancy and remodeling, chromatin looping and noncoding RNA,9 among which
699
700
M Xue et al.
the CpG island methylator phenotype (CIMP) is known to be the most common molecular feature.10 Chromosomes 1, 5, 6, 8, 11, 13, 18, 19, 21 and 22 are the locations where methylation alterations frequently occur in CRC, as reported by genome-wide DNA methylation analyses.11 A cluster of genes characterized by altered DNA methylation have been identified during the past few decades along these chromosomes, including Zinc finger of the cerebellum 1 (ZIC1) and Klotho (KL), which were first found to be hypermethylated in CRC in our previous studies.12,13 Methylated DNA fragments are detectable in not only primary tumor tissues but also remote media including blood, stool, urine and bowel lavage fluid,14–16 making them eligible to act as a screening tool for CRC in clinical setting. In this review, we aimed to summarize the current evidence on the biomarkers associated with DNA methylation in the screening and diagnosis of CRC. MATERIALS AND METHODS A literature search was conducted on the databases of PubMed and Web of Science for articles published from 1 January 2000 to 6 June 2015. The following terms were used for the search: (“methylation” OR “methylated”) AND (“colorectal cancer” OR “colon cancer” OR “rectal cancer”) AND (“screening” OR “diagnosis”). Stuides focused on the association between noninvasive biomarkers indicating DNA methylation, including those based on blood, stool and urine samples, and CRC were included in the study. Prospective, retrospective or crosssectional studies published in English or Chinese were included. And only those compared CRC with healthy controls, with the sample size of ≥2 were included. The references of the included manuscripts were screened for eligible studies, and abstracts presented at related conferences were also searched. Reviews, comments, consensus, meta-analyses, letters to the editors, case reports and case series, studies on animals or cell lines were excluded from the study. The list of articles generated by the search was manually reviewed by two investigators (Meng XUE and San Chuan LAI). Any disagreement was resolved by the discussion with the other two investigators (Zhi Peng XU and Liang Jing WANG). The quality of the studies was assessed using the Quality Assessment of Diagnostic Accuracy Studies (QUADAS). RESULTS The characteristics of the studies Altogether 2 401 studies were identified during the primary literature search. Among them, 1 093 were
Journal of Digestive Diseases 2016; 16; 699–712 excluded because of the manuscript type (consensus, guidelines, meta-analysis, systematic review, letter to the editor, editorial, case report or case series) (n = 1038) and the language restriction (not published in English or Chinese) (n = 55). Potentially relevant manuscripts were retrieved for further review, of which 1 236 studies were excluded for being irrelevant to the noninvasive DNA methylation biomarkers in CRC. The references of the remaining 72 articles were checked manually, and two articles were further included. Finally, a total of 74 studies15–88 were eligible for this systematic review. The flowchart of study selection is shown in Figure 1. Among all the 74 studies, 40 was conducted in the Western countries15,17,19,24,26,27,30–39,43,48,49,52,56– 59,62,64–67,69–72,75–79,84,85 (including one also performed in West Asia,39 and one also in East Asia52), 30 in East Asia,16,18,20–22,25,28,29,40–42,44–47,50,51,53– 55,60,61,63,73,80–84,87 two in Iran,68,74 one in 23 Singapore, and the remaining one unreported88. In 35 studies, samples were handled and analyzed in a blinded fashion.15,18,31–35,37,38,43,49–51,54–56,58–60,64,65,67– 69,71,74–76,78,80–85 Methylation-specific PCR (MSP), quantitative (qMSP) or nested MSP (nMSP) were applied in most studies,17,20–26,29,31–34,36,40,42,44–48,50,51,53–61,63,64,66– 84,87,88 quantitative allele-specific real-time target and signal amplification (QuARTS) in 3 studies,65,83,84 Methylight or Methyl-BEAMing (beads, emulsion, amplification and magnetics) in 9 studies,16,18,19,27,28,39,43,49,86 commercial methylation assay kit in 3 studies,35,37,38 pyrosequencing in 2 studies,62,87 Melting curve analysis in 2 studies,41,75 and methylation microarray in one study30. The QUADAS was applied to assess the quality of each study. Seven studies18,51,55,58,59,69,74 were marked “yes” to all the 14 questions, suggesting a good quality, while 11–13 questions were marked “yes” for the remaining studies. In 13 studies,18,39,42,51,55,58,59,62,69,70,72,74,88 noninvasive samples were collected either within two weeks before or after colonoscopy for histopathology or from newly diagnosed CRC; therefore, question 4 was marked with “yes”. In those 35 blinded studies,15,18,31–35,37,38,43,49–51,54–56,58–60,64,65,67–69,71,74– 76,78,80–85 DNA methylation status was assessed without histopathological results, and question 10 was labeled as “yes”. The histopathological diagnosis was not avaliable yet at the time of sample collection in two studies,79,88 and whether the pathologists were blinded to the methylation results was unclear; therefore, question 11 was labeled as “unclear” for these two studies.
Journal of Digestive Diseases 2016; 16; 699–712
Figure 1.
Noninvasive methylation markers in CRC
701
Flowchart demonstrating the selection of eligible studies.
Blood-based DNA methylation biomarkers Diagnostic efficiency
Most whole blood DNA is extracted from leukocytes. The association between the low-level methylation of leukocyte DNA and the risk of CRC tends to suggest that genomic-wide DNA is hypomethylated in CRC.89,90 Another pattern of methylation defects in tumor is the hypermethylation of CpG islands in localized promoter regions,91 which allows the researchers to illustrate the role of DNA methylation in the development of cancers. Apoptotic and necrotic tumor cells release DNA into the circulation by macrophages;92 therefore, not only genomic DNA extracted from whole blood but also extracellular free DNA from serum or plasma can be used for blood-based screening of the tumors. Over 20 genes with hypermethylated promoters had been identified in the blood over the last 15 years, and their positive rates in CRC ranged from 6.1% to 95.6% (Table 1).17–48 MutL homolog 1 (MLH1) was the first gene identified to be with a hypermethylated promoter. Grady et al.17 assessed the methylation status of hMLH1 in the serum of 18 consecutive CRC patients by MSP, and found 3 (16.7%) with positive hMLH1 expression accompanied by microsatellite instability. In another study conducted by Leung et al.18 a high-throughput quantitative assay, MethyLight, was applied to detect methylation,93 achieving a sensitivity of 42.9% and 32.7%, respectively, for determining the methylation
status of hMLH1 and helicase-like transcrioption factor (HLTF) in CRC. While the specificity in distinguishing methylation in 41 age-matched controls was 97.6% and 92.7%, respectively. However, combining three methylation markers [adenomatous polyposis coli (APC), MLH1 and HLTF] improved the sensitivity to 57.0%. Serum or plasma cyclin-dependent kinase inhibitor 2A (CDKN2A) methylation was assessed by MSP in four studies.20–23 Altogether 101 (57.4%) of the 176 CRC patients but no healthy volunteers had hypermethylated CDKN2A, yielding a strikingly high specificity of 100%. In addition to CDKN2A, Tan et al.23 also demonstrated a high methylation rate of runt-related transcription factor 3 (RUNX3) in CRC patients, with a sensitivity of 65% and a specificity of 100%, respectively. For methylated vimentin (VIM) in CRC a sensitivity of 59% was reported, as detected by a digital quantification method named BEAMing (beads, emulsion, amplification and magnetics) technology.39 This enabled the identification of a single DNA molecule of methylated VIM mixed with 1 000 copies of unmethylated VIM, thus improving the sensitivity, specificity and standardization among different assays.
Methylated Septin 9 (mSEPT9)
Although it had been discovered for less than 10 years,35 mSEPT9 was emerged as one of the most
702 Table 1.
Genes (panel)
M Xue et al.
Journal of Digestive Diseases 2016; 16; 699–712
Blood-based methylation markers in colorectal cancer (CRC) First author (publication year)
Single gene hMLH1 Grady17 (2001) Leung18 (2005) Wallner19 (2006) CDKN2A (p16) Zou20 (2002) Nakayama21 (2002) Nakayama22 (2003) Tan23 (2007) CDH4 Miotto24 (2004) HLTF Leung18 (2005) Wallner19 (2006) Lee25 (2009) APC Leung18 (2005) ALX4 Ebert26 (2006) Tänzer27 (2010) He28 (2010) HPP1/TPEF Wallner19 (2006) RUNX3 Tan23 (2007) Nishio29 (2010) RASSF1A Tan23 (2007) Cassinotti30 (2012) CDH1 Tan23 (2007) SEPT9 Tänzer27 (2010) He28 (2010) Lofton-Day31 (2008) Grützmann32 (2008) deVos33 (2009) Warren34 (2011) Ahlquist35 (2012) Tóth36 (2012) Church37 (2014) Potter38 (2014) TMEFF2 Lofton-Day31 (2008) He28 (2010) NGFR Lofton-Day31 (2008) VIM Li39 (2009) Shirahata40 (2010) Wif1 Lee25 (2009)
Healthy controls Specimens
Methods
CRC
Total Methylation Total Methylation (N) (n, %) (N) (n, %) AUROC
Serum Serum Serum
MSP MethyLight MethyLight
NA 41 20
NA 1 (2.4) 0 (0)
NA 0.71 NA
NA 0.0001 0.016
Serum Serum Serum Serum
MSP MSP MSP MSP
10 NA NA 10
0 (0) NA NA 0 (0)
20 94 45 17
14 44 31 12
(70.0) (46.8) (68.9) (70.6)
NA NA NA NA
0.001 NA NA NA
Whole blood
MSP
17
0 (0)
46
32 (69.6)
NA
0.001
Serum Serum Plasma
MethyLight MethyLight MSP
41 20 276
49 103 243
16 (32.7) 31 (30.1) 44 (18.1)
0.63 NA NA
0.015 0.005 NA
Serum
MethyLight
41
49
3 (6.1)
0.53
0.21
Serum Plasma Plasma
MethyLight MethyLight MethyLight
30 22 170
30 5 182
25 (83.3) 4 (80.0) 87 (47.8)
0.839 NA NA
0.0001 0.289 0.001
Serum
MethyLight
20
0 (0)
103
13 (12.6)
NA
0.09
Serum Serum
MSP Real-time qMSP
10 54
0 (0) NA
17 344
11 (64.7) 100 (29.1)
NA NA
0.001 0.0001
Serum Plasma
MSP Microarray
10 30
17 30
4 (23.5) 28 (93.3)
NA NA
0.097 0.01
Serum
MSP
10
17
3 (17.6)
NA
0.159
Plasma Plasma Plasma Plasma Plasma Plasma Plasma Plasma Plasma Plasma
MethyLight MethyLight qMSP qMSP qMSP qMSP qMSP Epi proColon Epi proColon Epi proColon
34 170 179 285 327 94 48 92 NA 444
4 6 25 25 45 11 13 14
(11.8) (3.5) (14.0) (8.8) (13.8) (11.7) (27.1) (15.2) NA 97 (21.8)
33 182 133 378 187 50 30 92 53 44
27 (81.8) 136 (74.7) 92 (69.2) 193 (51.1) 138 (73.8) 45 (90.0) 18 (60.0) 88 (95.7) 27 (50.9) 30 (68.2)
NA NA 0.80 NA NA NA NA NA NA NA
