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1.  Prospective evaluation of methylated SEPT9 in plasma for detection of asymptomatic colorectal cancer 
Gut  2013;63(2):317-325.
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.
Design
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.
Results
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.
Clinical Trial Registration Number:
NCT00855348
doi:10.1136/gutjnl-2012-304149
PMCID: PMC3913123  PMID: 23408352
Colorectal Cancer Screening; Methylation; Colonoscopy; Colorectal Adenomas; Colorectal Neoplasm
2.  Colorectal Cancer Screening for Average-Risk North Americans: An Economic Evaluation 
PLoS Medicine  2010;7(11):e1000370.
An economic analysis of different screening methods for detection of colorectal cancers suggests that in US or Canadian settings, screening with fecal immunochemical testing results in lower health-care costs as compared with other screening approaches.
Background
Colorectal cancer (CRC) fulfills the World Health Organization criteria for mass screening, but screening uptake is low in most countries. CRC screening is resource intensive, and it is unclear if an optimal strategy exists. The objective of this study was to perform an economic evaluation of CRC screening in average risk North American individuals considering all relevant screening modalities and current CRC treatment costs.
Methods and Findings
An incremental cost-utility analysis using a Markov model was performed comparing guaiac-based fecal occult blood test (FOBT) or fecal immunochemical test (FIT) annually, fecal DNA every 3 years, flexible sigmoidoscopy or computed tomographic colonography every 5 years, and colonoscopy every 10 years. All strategies were also compared to a no screening natural history arm. Given that different FIT assays and collection methods have been previously tested, three distinct FIT testing strategies were considered, on the basis of studies that have reported “low,” “mid,” and “high” test performance characteristics for detecting adenomas and CRC. Adenoma and CRC prevalence rates were based on a recent systematic review whereas screening adherence, test performance, and CRC treatment costs were based on publicly available data. The outcome measures included lifetime costs, number of cancers, cancer-related deaths, quality-adjusted life-years gained, and incremental cost-utility ratios. Sensitivity and scenario analyses were performed. Annual FIT, assuming mid-range testing characteristics, was more effective and less costly compared to all strategies (including no screening) except FIT-high. Among the lifetimes of 100,000 average-risk patients, the number of cancers could be reduced from 4,857 to 1,782 and the number of CRC deaths from 1,393 to 457, while saving CAN$68 per person. Although screening patients with FIT became more expensive than a strategy of no screening when the test performance of FIT was reduced, or the cost of managing CRC was lowered (e.g., for jurisdictions that do not fund expensive biologic chemotherapeutic regimens), CRC screening with FIT remained economically attractive.
Conclusions
CRC screening with FIT reduces the risk of CRC and CRC-related deaths, and lowers health care costs in comparison to no screening and to other existing screening strategies. Health policy decision makers should consider prioritizing funding for CRC screening using FIT.
Please see later in the article for the Editors' Summary
Editors' Summary
Background
Colorectal (bowel) cancer is the second leading cause of cancer deaths for both men and women in North America. Colorectal cancer screening is an important means for reducing morbidity and mortality and fulfils the World Health Organization criteria for mass screening. However, a variety of CRC screening approaches are available. Colonoscopy is viewed as the gold standard of colorectal cancer screening as it has a high sensitivity for identifying adenomas and cancer and polyps can be removed during the screening examination. However, colonoscopy is associated with a number of complications and there are also barriers to access. Another type of test, the guaiac fecal occult blood test, has been shown to reduce mortality from colorectal cancer but this test has low sensitivity for identifying colorectal neoplasia, particularly adenomas. Fecal immunochemical tests, which also detect blood in the stool, have improved test performance characteristics (high sensitivity and specificity) and the potential to improve participation rates compared to guaiac fecal occult blood test and flexible sigmoidoscopy. Fecal DNA (a stool test, based on the detection of DNA shed by cancerous tissue) is another screening option, as is computed tomographic colonography (“virtual” colonoscopy), that might rival colonoscopy in detecting advanced adenomas and colorectal cancer but is expensive and requires a full colonic preparation.
Why Was This Study Done?
In the absence of firm comparative evidence to guide the selection of any one screening modality and given the varied test performance characteristics and the significant differences in costs and resources associated with each, a robust cost-effectiveness analysis might help health policy makers in deciding whether or not to offer screening and if so, in selecting the most appropriate and cost effective screening modality. In this study the researchers conducted a full economic evaluation of all relevant colorectal cancer screening modalities in North America.
What Did the Researchers Do and Find?
The researchers used an incremental cost-utility analysis, a sophisticated modeling technique, and two hypothetical patient cohorts (individuals with an “average risk,” i.e., no family history of colorectal cancer, aged 50–64 and 65–75) to compare guaiac-based fecal occult blood test or fecal immunochemical test annually (the researchers considered three distinct fecal immunochemical testing strategies on the basis of assays and collection methods taken from studies that have reported “low,” “mid,” and “high” test performance characteristics), fecal DNA every three years, flexible sigmoidoscopy or computed tomographic colonography every 5 years, and colonoscopy every 10 years. The researchers also included a no screening natural history arm as a comparison to each screening approach. For the baseline data of their model, the researchers used adenoma and colorectal prevalence rates from a recent systematic review and based screening adherence, test performance, and colorectal treatment costs on available data. The researchers found that annual fecal immunochemical testing with mid-range testing characteristics, was more effective and less costly compared to all strategies (including no screening). Using this screening modality, among the lifetimes of 100,000 average-risk patients, the number of cancers could be reduced from 4,857 to 1,393 and the number of deaths from colorectal cancer from 1,782 to 457, while saving CAN$68 per person. Although in the sensitivity and scenario analysis, screening patients using fecal immunochemical testing became more expensive than a strategy of no screening when the test performance of fecal immunochemical testing was reduced, or the cost of managing colorectal cancers was lowered, the researchers found that screening for colorectal cancer with fecal immunochemical testing remained the most economically attractive screening option.
What Do These Findings Mean?
This model-based economic analysis found that fecal immunochemical testing is more effective and less costly than all other colorectal screening strategies, including the most commonly-used stool-based screening test, guaiac-based fecal occult blood testing, and no screening. Furthermore, this study suggests that annual screening with fecal immunochemical testing (assuming mid-range test performance characteristics) reduces the risk of colorectal cancer and colorectal cancer–related deaths, and lowers health care costs in comparison to all other screening strategies and to no screening. Therefore, health policy makers should consider prioritizing funding for fecal immunochemical testing as the screening modality for colorectal cancer.
Additional Information
Please access these Web sites via the online version of this summary at http://dx.doi.org/10.1371/journal.pmed.1000370.
Cancer.org has information for patients on colorectal cancer
The US Centers for Disease Control (CDC) list colorectal screening guidelines
The CDC also provides patient information on colorectal cancer Screening
doi:10.1371/journal.pmed.1000370
PMCID: PMC2990704  PMID: 21124887
3.  Serum M2-pyruvate kinase: A promising non-invasive biomarker for colorectal cancer mass screening 
AIM: To explore the value of serum M2-pyruvate kinase (M2-PK) in colorectal cancer (CRC) mass screening.
METHODS: We conducted a molecular epidemiology study in Hangzhou, China, from year 2006 to year 2008. Serum samples were collected from 93 CRC, 41 advanced adenomas, 137 adenomas, 47 non-adenomatous polyps, and 158 normal participants in a community setting. Serum M2-PK and carcinoembryonic antigen (CEA) were measured using Enzyme-linked immunosorbent assay. SPSS 16.0 software was used to perform data analysis. Area under the receiver operating characteristic curve (AUC), sensitivity, and specificities were estimated for serum M2-PK in diagnosis of colorectal lesions and compared with CEA.
RESULTS: Average serum M2-PK value among 158 normal people was 2.96 U/mL and not affected by gender (P = 0.47) or age (P = 0.59). Average serum M2-PK (U/mL) was 14.75 among stage III and 13.10 among stage I and II CRC patients, about 4 times higher than that among normal people. Average serum M2-PK was 8.58, 6.70, 5.13 and 2.51 U/mL among advanced adenoma, adenomas, non-adenomatous polyps, and inflammatory bowel disease patients, respectively. AUC for serum M2-PK was greater than that for CEA among all colorectal lesions. AUC for serum M2-PK was 0.89 (0.84, 0.94) (95% confidence interval), higher than that for CEA [0.70 (0.62-0.79)] in CRC stage I and II, 0.89 (0.84-0.94) vs 0.73 (0.63-0.83) in CRC stage III, 0.81 (0.74-0.86) vs 0.63 (0.53 - 0.73) in advanced adenomas, 0.69 (0.64-0.76) vs 0.54 (0.47-0.60) in adenomas, and 0.69 (0.62-0.78) vs 0.58 (0.48-0.68) in non-adenomatous polyps. The diagnostic sensitivity for all colorectal lesions increased with decrease in the cut-off value of serum M2-PK. The diagnostic sensitivity (%) of serum M2-PK was 100.00 for CRC, 95.12 advanced adenoma, 82.48 adenoma, and 82.98 non-adenomatous polyp. There were no CRC cases missed and 40.51% of unnecessary colonoscopies were avoided when the cut-off value was 2.00 U/mL.
CONCLUSION: Serum M2-PK can be used as a primary screening test in CRC mass screening. It may be a promising non-invasive biomarker for CRC early detection.
doi:10.4251/wjgo.v4.i6.145
PMCID: PMC3382661  PMID: 22737276
Serum M2-pyruvate kinase; Colorectal cancer screening; Serum biomarker; Carcinoembryonic antigen
4.  Effect of Flexible Sigmoidoscopy-Based Screening on Incidence and Mortality of Colorectal Cancer: A Systematic Review and Meta-Analysis of Randomized Controlled Trials 
PLoS Medicine  2012;9(12):e1001352.
A systematic review and meta-analysis of randomized trials conducted by B. Joseph Elmunzer and colleagues reports that that flexible sigmoidoscopy-based screening reduces the incidence of colorectal cancer in average-risk patients, as compared to usual care or no screening.
Background
Randomized controlled trials (RCTs) have yielded varying estimates of the benefit of flexible sigmoidoscopy (FS) screening for colorectal cancer (CRC). Our objective was to more precisely estimate the effect of FS-based screening on the incidence and mortality of CRC by performing a meta-analysis of published RCTs.
Methods and Findings
Medline and Embase databases were searched for eligible articles published between 1966 and 28 May 2012. After screening 3,319 citations and 29 potentially relevant articles, two reviewers identified five RCTs evaluating the effect of FS screening on the incidence and mortality of CRC. The reviewers independently extracted relevant data; discrepancies were resolved by consensus. The quality of included studies was assessed using criteria set out by the Evidence-Based Gastroenterology Steering Group. Random effects meta-analysis was performed.
The five RCTs meeting eligibility criteria were determined to be of high methodologic quality and enrolled 416,159 total subjects. Four European studies compared FS to no screening and one study from the United States compared FS to usual care. By intention to treat analysis, FS-based screening was associated with an 18% relative risk reduction in the incidence of CRC (0.82, 95% CI 0.73–0.91, p<0.001, number needed to screen [NNS] to prevent one case of CRC = 361), a 33% reduction in the incidence of left-sided CRC (RR 0.67, 95% CI 0.59–0.76, p<0.001, NNS = 332), and a 28% reduction in the mortality of CRC (relative risk [RR] 0.72, 95% CI 0.65–0.80, p<0.001, NNS = 850). The efficacy estimate, the amount of benefit for those who actually adhered to the recommended treatment, suggested that FS screening reduced CRC incidence by 32% (p<0.001), and CRC-related mortality by 50% (p<0.001).
Limitations of this meta-analysis include heterogeneity in the design of the included trials, absence of studies from Africa, Asia, or South America, and lack of studies comparing FS with colonoscopy or stool-based testing.
Conclusions
This meta-analysis of randomized controlled trials demonstrates that FS-based screening significantly reduces the incidence and mortality of colorectal cancer in average-risk patients.
Please see later in the article for the Editors' Summary
Editor's Summary
Background
Colorectal cancer (CRC) is the second leading cause of cancer-related death in the United States. Regular CRC screening has been shown to reduce the risk of dying from CRC by 16%, and CRC screening can identify early stage cancers in otherwise healthy people, which allows for early treatment and management of the disease. Screening for colorectal cancer is frequently performed using a flexible sigmoidoscopy (FS), which is a thin, flexible tube with a tiny camera and light on the end, allowing a doctor to look at the inside wall of the bowel and remove any small growths or polyps. Although screening may detect early cancers, the life-saving and health benefits of screening are uncertain because the polyp may not necessarily progress. This could lead to anxiety and unnecessary interventions and treatments amongst those screened. Randomized controlled trials (RCTs) are needed to determine all of the risks involved in cancer screenings, however the guidelines that recommend FS-based screening do not rely upon RCT data. Recently, the results of four large-scale RCTs evaluating FS screening for CRC have been published. The conflicting results with respect to the incidence and mortality of CRC in these studies have called into question the effectiveness of endoscopic screening.
Why Was This Study Done?
The results of RCTs measuring the risks and outcomes of CRC screening have shown varying estimates of the benefits of using FS screening. If better estimates of the risks and benefits of FS screening are developed, then the current CRC screening guidelines may be updated to reflect this new information. In this study, the authors show the results of a meta-analysis of published RCTs, which more precisely estimates the effects of FS-based screening on the incidence and mortality of colorectal cancer.
What Did the Researchers Do and Find?
The researchers used the Medline and Embase databases to find relevant studies from 1966 to May 28, 2012. After screening 3,319 citations and 29 potentially relevant articles, five RCTs of high methodologic quality and 416,159 total subjects evaluating the effect of FS screening on the incidence and mortality of CRC were identified. The data were extracted and random effects meta-analysis was performed. The meta-analysis revealed that FS-based screening was associated with an 18% relative risk reduction in the incidence of CRC (0.82, 95% CI 0.73–0.91, p<0.001, number needed to screen (NNS) to prevent one case of CRC = 361), a 33% reduction in the incidence of left-sided CRC (RR 0.67, 95% CI 0.59–0.76, p<0.001, NNS = 332), and a 28% reduction in the mortality of CRC (RR 0.72, 95% CI 0.65–0.80, p<0.001, NNS = 850). The amount of benefit for those who adhered to the recommended treatment suggested that FS screening reduced CRC incidence by 32% (p<0.001), and CRC-related mortality by 50% (p<0.001).
What Do These Findings Mean?
This meta-analysis of RCTs evaluating the effect of FS on CRC incidence and mortality demonstrates that a FS-based strategy for screening is very effective in reducing the incidence and mortality of CRC in patients. The current recommendations for endoscopic screening are based on observational studies, which may not accurately reflect the effect of FS-based screening on the incidence and mortality of CRC. Here, the authors performed a systematic review and meta-analysis of five recent RCTs to better estimate the true effect of FS-based screening on the incidence and mortality of CRC. Thus, the results of this meta-analysis may affect health policy, and directly impact patients and clinicians.
Additional Information
Please access these Web sites via the online version of this summary at http://dx.doi.org/10.1371/journal.pmed.1001352.
Cancer research UK provides comprehensive information about screening for colorectal cancers as does the UK National Screening Committee
PubMed Health has general information about colon cancer
The National Cancer Institute also has comprehensive resources on colorectal cancer and treatment
The Mayo Clinic provides an overview of all aspects of colon cancer
doi:10.1371/journal.pmed.1001352
PMCID: PMC3514315  PMID: 23226108
5.  Fecal Occult Blood Test for Colorectal Cancer Screening 
Executive Summary
The colorectal cancer (CRC) screening project was undertaken by the Medical Advisory Secretariat (MAS) in collaboration with the Cancer Care Ontario (CCO).
In November 2007, the Ontario Health Technology Advisory Committee (OHTAC) MAS to conduct an evidence-based analysis of the available data with respect to colorectal cancer diagnosis and prevention. The general purpose of the project was to investigate the effectiveness, cost effectiveness, and safety of the various methods and techniques used for colorectal cancer screening in average risk people, 50 years of age and older.
The options currently offered for colorectal cancer screening were reviewed and five technologies were selected for review:
Computed tomographic (CT) colonography
Magnetic resonance (MR) colonography
Wireless capsule endoscopy (PillCam Colon)
Fecal occult blood test (FOBT)
Flexible sigmoidoscopy
In this review, colonoscopy was considered as the “gold standard” technique by which the effectiveness of all other modalities could be evaluated. An economic analysis was also conducted to determine cost-effectiveness of different screening modalities.
Evidence-based analyses have been prepared for each of these technologies, as well as summary document that includes an economic analysis, all of which are presented at the MAS Web site: http://www.health.gov.on.ca/english/providers/program/mas/tech/techmn.html
Objective
The objective of this evidence review is to examine the effectiveness and cost-effectiveness of fecal occult blood testing (FOBT), including guaiac FOBT (gFOBT) and immunochemical FOBT (iFOBT), for use in colorectal cancer (CRC) screening in asymptomatic, average-risk adults.
Specifically:
Is the use of gFOBT or iFOBT associated with a reduction in CRC and overall mortality?
What are the sensitivity and specificity of gFOBT and iFOBT for the detection of 1) CRC and 2) large polyps (≥ 1 cm)?
Clinical Need
CRC is the most common cause of non-tobacco related cancer death in Canada. It has been estimated that in 2007, 7,800 people were diagnosed with CRC in Ontario and 3,250 died from the disease, making the province’s incidence and mortality rate of CRC amongst the highest in the world.
Description of Technology/Therapy
There are two general types of FOBT that are categorized according to the analyte detected: guaiac FOBT (gFOBT) and immunochemical FOBT (iFOBT). Blood in the stool is a nonspecific finding but may originate from CRC or larger (>1 cm) polyps (small adenomatous polyps do not tend to bleed). Bleeding from cancers and larger polyps may be intermittent and not always detectable in a single sample. The FOBT thus requires regular testing that consists of collecting specimens from consecutive bowel movements. A positive gFOBT or iFOBT involves a diagnostic workup with colonoscopy to examine the entire colon in order to rule out the presence of cancer or advanced neoplasia.
Methods of Evidence-Based Analysis
A literature search was conducted from January 2003 to June 2008 that included OVID MEDLINE, MEDLINE In-Process and Other Non-Indexed Citations, EMBASE, the Cumulative Index to Nursing & Allied Health Literature (CINAHL), The Cochrane Library, and the International Agency for Health Technology Assessment/Centre for Review and Dissemination.
Inclusion Criteria
Patients at average risk for CRC
All patients must be at least 50 years of age
Biennial FOBT as a screening modality and use of colonoscopy as the reference standard
Systematic reviews and randomized controlled trials (RCTs)
Outcomes: CRC mortality, overall mortality, sensitivity, specificity, adverse effects
Exclusion Criteria
Studies involving fewer than 100 patients
Studies that do not report sufficient data for analysis
Comparisons of Interest
Evidence exists for these comparisons of interest:
gFOBT compared with the reference “gold standard” colonoscopy (or double-contrast barium enema where colonoscopy is incomplete or contraindicated)
iFOBT compared with the reference gold standard colonoscopy (or DCBE where colonoscopy is incomplete or contraindicated)
gFOBT compared with iFOBT
The quality of the diagnostic studies was examined according to the ‘GRADE Working Group criteria’ for grading quality of evidence and strength of recommendations for diagnostic tests and strategies.
Summary of Findings
Single-Test Studies
There is limited direct/indirect evidence that iFOBT has sensitivity/specificity superior to that of unrehydrated gFOBT for CRC detection:
sensitivity for gFOBT:
pooled iFOBT sensitivity:
There is evidence that iFOBT and gFOBT have lower sensitivities for adenoma detection than for CRC detection:
sensitivity for rehydrated gFOBT
pooled iFOBT sensitivity
Repeated-Test Studies
No trials have examined CRC mortality outcomes after repeated testing of iFOBT.
Two RCTs from the United Kingdom and Denmark showed significant reduction in CRC mortality using unrehydrated gFOBT biennially
Relative risk reductions of 13% (UK trial) and 16% (Danish trial); absolute difference of 0.1% (UK trial) and 0.2% (Danish trial).
No significant reduction in overall mortality
Interval cancers (CRC that develop in the intervals between routine screening)
United Kingdom trial: 236 CRCs detected by positive test, 236 interval CRCs after negative test
Danish trial: 120 CRCs detected by positive test, 146 interval CRCs after negative test
Unrehydrated gFOBT has low sensitivity for CRC detection (45% in the UK trial and 54% in the Danish trial).
true positive rate
false positive rate
true negative rate
false negative rate
Guaiac FOBT – GRADE Quality of Evidence for Interventions
CRC indicates colorectal cancer; FOBT, fecal occult blood test; GRADE, Grading of Recommendations Assessment, Development and Evaluation; RCT, randomized controlled trial.
Unlikely to be an important uncertainty.
GRADE Quality of Evidence for Diagnostic Tests: Implications of Testing Focusing on Accuracy
Benefit from diagnosis and treatment after confirmatory colonoscopy
Small risk of bowel perforation during colonoscopy
Benefit of reassurance
Anxiety/worry leading up to confirmatory colonoscopy
Small risk of bowel perforation during confirmatory colonoscopy
Detriment from delayed diagnosis
Some uncertainty (until after confirmatory colonoscopy)
No Uncertainty
Uncertainty
Uncertainty
FOBT indicates fecal occult blood test; GRADE, Grading of Recommendations Assessment, Development and Evaluation.
Immunochemical FOBT – GRADE Quality of Evidence for Diagnostic Studies
FN indicates false negative; FOBT, fecal occult blood test; FP, false positive; Development and Evaluation; TN, true negative; TP, true positive.
Uncertainty until after confirmatory colonoscopy
Stress/worry for patient until confirmatory colonoscopy
Detrimental effects due to delayed diagnosis.
For these 3 reasons, downgrade quality from High to Moderate.
For these 3 reasons, downgrade quality from Moderate to Low.
Considerations for the Ontario Health System
Executive Summary Table 4 shows the potential system pressures and benefit/risk analysis for the use of FOBT and colonoscopy to screen for CRC in average-risk adults, ages 50 and over in Ontario.
Summary of Potential System Pressures for FOBT Screening
Prevent and detect
Detect
Every 10 years
Must repeat at regular intervals
Every 2 years
Must repeat at regular intervals
Observational studies
RCTs
Used as gold standard in studies
Intervention GRADE quality: High (gFOBT)
Diagnostic GRADE quality: Low (iFOBT)
No RCTs examining the effectiveness of repeated iFOBT on CRC mortality reduction were identified
Limited direct/indirect evidence that iFOBT has superior sensitivity/specificity to unrehydrated gFOBT for detection of CRC
0.1% risk of serious bleeding and perforation requiring surgery
0.3% risk of serious complications (stroke/bleeding requiring hospitalization/ myocardial infarction)
High interval cancer rate
The small benefit in CRC mortality reduction (absolute difference 0.1% to 0.2%) also coincides with a 0.3% risk of serious complications.
No food 1 day prior to exam
Office/hospital visit
Complete bowel preparation
Sedation
Eliminate citrus fruit and juices and vitamin C from diet for 3 days prior to/during stool collection.
Person applies 2 samples per bowel movement (each occurring on 3 different days) onto test areas of FOBT cards.
Increased demand for colonoscopies and colonoscopists or nurses who perform colonoscopies.
Patient receives kit from family physician, pharmacist
Patients mail completed FOBT kit to participating laboratory
Results sent back to patient
Increased demand for colonoscopies for positive patients
Removal of polyp during colonoscopy or surgery
Referral to colonoscopy
Cost-effective
Cost-effective
2nd of 5 choices in a patient survey study
5th of 5 choices in a patient survey study
FOBT indicates fecal occult blood test;; gFOBT, guaiac FOBT; GRADE, Grading of Recommendations Assessment, Development and Evaluation; iFOBT, immunochemical FOBT; RCT, randomized controlled trial.
PMCID: PMC3377532  PMID: 23074514
6.  Increased plasma levels of the APC-interacting protein MAPRE1, LRG1 and IGFBP2 preceding a diagnosis of colorectal cancer in women 
Longitudinal blood collections from cohort studies provide the means to search for proteins associated with disease prior to clinical diagnosis. We investigated plasma samples from the Women’s Health Initiative (WHI) cohort to determine quantitative differences in plasma proteins between subjects subsequently diagnosed with colorectal cancer (CRC) and matched controls that remained cancer free during the period of follow-up. Proteomic analysis of WHI samples collected prior to diagnosis of CRC resulted in the identification of six proteins with significantly (p <0.05) elevated concentrations in cases compared to controls. Proteomic analysis of two colorectal cancer cell lines showed 5 of the 6 proteins were produced by cancer cells. MAPRE1, IGFBP2, LRG1 and CEA were individually assayed by enzyme linked immunosorbent assay (ELISA) in 58 pairs of newly diagnosed CRC samples and controls and yielded significant elevations (p <0.05) among cases relative to controls. A combination of these four markers resulted in an ROC with an AUC=0.841 and 57% sensitivity at 95% specificity. This combination rule was tested in an independent set of WHI samples collected within 7 months prior to diagnosis from cases and matched controls resulting in 41% sensitivity at 95% specificity. A panel consisting of CEA, MAPRE1, IGFBP2 and LRG1 has predictive value in pre-diagnostic colorectal cancer plasmas.
doi:10.1158/1940-6207.CAPR-11-0412
PMCID: PMC3419141  PMID: 22277732
colorectal cancer; risk markers; Pre-Diagnostic samples
7.  Detection of Colorectal Cancer by Serum and Tissue Protein Profiling: A Prospective Study in a Population at Risk 
Biomarker Insights  2008;3:375-385.
Colorectal cancer (CRC) is the second most common cause of cancer-related death in Europe and its prognosis is largely dependent on stage at diagnosis. Currently, there are no suitable tumour markers for early detection of CRC. In a retrospective study we previously found discriminative CRC serum protein profiles with surface enhanced laser desorption ionisation—time of flight mass spectrometry (SELDI-TOF MS). We now aimed at prospective validation of these profiles. Additionally, we assessed their applicability for follow-up after surgery and investigated tissue protein profiles of patients with CRC and adenomatous polyps (AP). Serum and tissue samples were collected from patients without known malignancy with an indication for colonoscopy and patients with AP and CRC during colonoscopy. Serum samples of controls (CON; n = 359), patients with AP (n = 177) and CRC (n = 73), as well as tissue samples from AP (n = 52) and CRC (n = 47) were analysed as described previously. Peak intensities were compared by non-parametric testing. Discriminative power of differentially expressed proteins was assessed with support vector machines (SVM). We confirmed the decreased serum levels of apolipoprotein C-1 in CRC in the current population. No differences were observed between CON and AP. Apolipoprotein C-I levels did not change significantly within 1 month post-surgery, although a gradual return to normal levels was observed. Several proteins differed between AP and CRC tissue, among which a peak with similar mass as apolipoprotein C-1. This peak was increased in CRC compared to AP. Although we prospectively validated the serum decrease of apolipoprotein C-1 in CRC, serum protein profiles did not yield SVM classifiers with suitable sensitivity and specificity for classification of our patient groups.
PMCID: PMC2688344  PMID: 19578519
biomarkers; colorectal cancer; SELDI-TOF MS; validation
8.  Identification of a Circulating MicroRNA Signature for Colorectal Cancer Detection 
PLoS ONE  2014;9(4):e87451.
Prognosis of patients with colorectal cancer (CRC) is generally poor because of the lack of simple, convenient, and noninvasive tools for CRC detection at the early stage. The discovery of microRNAs (miRNAs) and their different expression profiles among different kinds of diseases has opened a new avenue for tumor diagnosis. We built a serum microRNA expression profile signature and tested its specificity and sensitivity as a biomarker in the diagnosis of CRC. We also studied its possible role in monitoring the progression of CRC. We conducted a two phase case-control test to identify serum miRNAs as biomarkers for CRC diagnosis. Using quantitative reverse transcription polymerase chain reactions, we tested ten candidate miRNAs in a training set (30 CRCs vs 30 controls). Risk score analysis was used to evaluate the diagnostic value of the serum miRNA profiling system. Other independent samples, including 83 CRCs and 59 controls, were used to validate the diagnostic model. In the training set, six serum miRNAs (miR-21, let-7g, miR-31, miR-92a, miR-181b, and miR-203) had significantly different expression levels between the CRCs and healthy controls. Risk score analysis demonstrated that the six-miRNA-based biomarker signature had high sensitivity and specificity for distinguishing the CRC samples from cancer-free controls. The areas under the receiver operating characteristic (ROC) curve of the six-miRNA signature profiles were 0.900 and 0.923 for the two sets of serum samples, respectively. However, for the same serum samples, the areas under the ROC curve used by the tumor markers carcinoembryonic antigen (CEA) and carbohydrate antigen 19-9 (CA19-9) were only 0.649 and 0.598, respectively. The expression levels of the six serum miRNAs were also correlated with CRC progression. Thus, the identified six-miRNA signature can be used as a noninvasive biomarker for the diagnosis of CRC, with relatively high sensitivity and specificity.
doi:10.1371/journal.pone.0087451
PMCID: PMC3977854  PMID: 24709885
9.  Colon Cancer-Specific Antigen-2 May Be Used as a Detecting and Prognostic Marker in Colorectal Cancer: A Preliminary Observation 
PLoS ONE  2014;9(4):e94252.
Background
A specific and sensitive serum marker for colorectal cancer (CRC) detection and surveillance is central to effective treatment. It was preliminarily reported that some nuclear matrix proteins may be served as a specific blood based marker for colon cancer. The objective of this study is to evaluate the value of serum CCSA-2 detection in diagnosis, prognostic estimation and surveillance for CRC.
Method
Serum CCSA-2 protein was measured in 181 various patient populations and 20 healthy donors before surgery. For 106 CRC patients, it was also measured on day 7 after surgery. Among them, 49 CRC patients' CCSA-2 protein were measured during the follow-up period according to NCCN Guideline.
Results
The serum CCSA-2 concentration in CRC patients was significantly higher than which in other patients and healthy individuals. Serum CCSA-2, at the cut-off point of 64.10 ng/mL, had a sensitivity of 98.10% and a specificity of 97.90% in separating CRC populations from all other individuals. The CCSA-2 assay was significantly more sensitive than CEA and CA19-9 assay in CRC detection. After surgery, the serum CCSA-2 level of CRC patients declined significantly, but it rebounded to a high level when recurrences occurred. The pre-operative serum CCSA-2 level in patients who had a relapse within the follow-up period was significantly higher than which in patients without relapse.
Conclusions
Serum CCSA-2 not only may be a potential biomarker using in screening and surveillance of CRC, but also may be an independent prognostic marker for CRC patients. Further clinical trials need to be performed in a larger population of patients to ulteriorly confirm these results.
doi:10.1371/journal.pone.0094252
PMCID: PMC3978011  PMID: 24710115
10.  Fecal immunochemical test accuracy in average-risk colorectal cancer screening 
AIM: To assess the fecal immunochemical test (FIT) accuracy for colorectal cancer (CRC) and advanced neoplasia (AN) detection in CRC screening.
METHODS: We performed a multicentric, prospective, double blind study of diagnostic tests on asymptomatic average-risk individuals submitted to screening colonoscopy. Two stool samples were collected and the fecal hemoglobin concentration was determined in the first sample (FIT1) and the highest level of both samples (FITmax) using the OC-sensor™. Areas under the curve (AUC) for CRC and AN were calculated. The best FIT1 and FITmax cut-off values for CRC were determined. At this threshold, number needed to scope (NNS) to detect a CRC and an AN and the cost per lesion detected were calculated.
RESULTS: About 779 individuals were included. An AN was found in 97 (12.5%) individuals: a CRC in 5 (0.6%) and an advanced adenoma (≥ 10 mm, villous histology or high grade dysplasia) in 92 (11.9%) subjects. For CRC diagnosis, FIT1 AUC was 0.96 (95%CI: 0.95-0.98) and FITmax AUC was 0.95 (95%CI: 0.93-0.97). For AN, FIT1 and FITmax AUC were similar (0.72, 95%CI: 0.66-0.78 vs 0.73, 95%CI: 0.68-0.79, respectively, P = 0.34). Depending on the number of determinations and the positivity threshold cut-off used sensitivity for AN detection ranged between 28% and 42% and specificity between 91% and 97%. At the best cut-off point for CRC detection (115 ng/mL), the NNS to detect a CRC were 10.2 and 15.8; and the cost per CRC was 1814€ and 2985€ on FIT1 and FITmax strategies respectively. At this threshold the sensitivity, NNS and cost per AN detected were 30%, 1.76, and 306€, in FIT1 strategy, and 36%, 2.26€ and 426€, in FITmax strategy, respectively.
CONCLUSION: Performing two tests does not improve diagnostic accuracy, but increases cost and NNS to detect a lesion.
doi:10.3748/wjg.v20.i4.1038
PMCID: PMC3921527  PMID: 24574776
Colorectal neoplasms; Early detection of cancer; Sensitivity and specificity; Adenoma; Occult blood; Cost-benefit analysis
11.  Detection of serological biomarkers by proximity extension assay for detection of colorectal neoplasias in symptomatic individuals 
Background
Although the potential of biomarkers to aid in early detection of colorectal cancer (CRC) is recognized and numerous biomarker candidates have been reported in the literature, to date only few molecular markers have been approved for daily clinical use.
Methods
In order to improve the translation of biomarkers from the bench to clinical practice we initiated a biomarker study focusing on a novel technique, the proximity extension assay, with multiplexing capability and the possible additive effect obtained from biomarker panels. We performed a screening of 74 different biomarkers in plasma derived from a case–control sample set consisting of symptomatic individuals representing CRC patients, patients with adenoma, patients with non-neoplastic large bowel diseases and healthy individuals.
Results
After statistical evaluation we found 12 significant indicators of CRC and the receiver operating characteristic (ROC) curve of Carcinoembryonic antigen (CEA), Transferrin Receptor-1 (TFRC), Macrophage migration inhibitory factor (MIF), Osteopontin (OPN/SPP1) and cancer antigen 242 (CA242) showed additive effect. This biomarker panel identified CRC patients with a sensitivity of 56% at 90% specificity and thus the performance is sufficiently high to further investigate this combination of five proteins as serological biomarkers for detection of CRC. Furthermore, when applying the indicators to identify early-stage CRC a combination of CEA, TFRC and CA242 resulted in a ROC curve with an area under the curve of 0.861.
Conclusions
Five plasma protein biomarkers were found to be potential CRC discriminators and three of these were additionally found to be discriminators of early-stage CRC. These explorative data in symptomatic individuals demonstrates the feasibility of the multiplex proximity extension assay for screening of potential serological protein biomarkers and warrants independent analyses in a larger sample cohort, including asymptomatic individuals, to further validate the performances of our CRC biomarker panel.
doi:10.1186/1479-5876-11-253
PMCID: PMC3827929  PMID: 24107468
Colorectal cancer; Plasma; Biomarkers; Proximity ligation assay; Proximity extension assay
12.  Diagnostic Accuracy of Elevated Serum Carcinoembryonic Antigen for Recurrence in Postoperative Stage II Colorectal Cancer Patients: Comparison With Stage III 
Annals of Coloproctology  2013;29(4):155-159.
Purpose
Elevated levels of serum carcinoembryonic antigen (CEA) following a curative resection of colorectal cancer (CRC) indicate recurrence; however, the levels of CEA may be elevated above the normal limit without recurrence. The aim of this study is to analyze the diagnostic accuracy of elevated serum CEA for predicting recurrence in postoperative stage II and stage III CRC patients.
Methods
A total of 336 stage II and stage III CRC patients who underwent a curative resection between January 2005 and October 2009 were enrolled. Sensitivities, specificities, positive predictive values (PPVs), negative predictive values (NPVs), likelihood ratios and post-test probabilities of recurrence associated with elevated CEA were analyzed and compared.
Results
The median follow-up duration was 45 months (36 to 134 months). Twenty-seven of 189 stage II patients (14.3%) and 52 of 147 stage III patients (35.4%) developed recurrence during the follow-up period. Sensitivities, specificities, PPVs, and NPVs of elevated CEA were 37.0%, 91.4%, 41.7%, and 89.7%, respectively, in stage II patients and 46.2%, 90.5%, 72.7%, and 75.4% in stage III patients. Post-test probabilities of recurrence associated with elevated CEA were 41.8% in stage II patients and 71.9% in stage III patients.
Conclusion
The predictive performance of the probability of recurrence associated with elevated serum CEA after a curative resection in stage II CRC patients is lower than that in stage III CRC patients.
doi:10.3393/ac.2013.29.4.155
PMCID: PMC3767865  PMID: 24032116
Carcinoembryonic antigen; Tumor marker; Colorectal neoplasms; Recurrence; Accuracy
13.  Sensitive Detection of Colorectal Cancer in Peripheral Blood by Septin 9 DNA Methylation Assay 
PLoS ONE  2008;3(11):e3759.
Background
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%.
Conclusions/Significance
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.
doi:10.1371/journal.pone.0003759
PMCID: PMC2582436  PMID: 19018278
14.  Detection of mismatch repair gene germline mutation carrier among Chinese population with colorectal cancer 
BMC Cancer  2008;8:44.
Background
Hereditary nonpolyposis colorectal cancer (HNPCC) is an autosomal dominant syndrome. The National Cancer Institute (NCI) has recommended the Revised Bethesda guidelines for screening HNPCC. There has been a great deal of research on the value of these tests in other countries. However, literature about the Chinese population is scarce. Our objective is to detect and study microsatellite instability (MSI) and mismatch repair (MMR) gene germline mutation carriers among a Chinese population with colorectal cancer.
Methods
In 146 prospectively recruited consecutive patients with clinically proven colorectal cancer, MSI carriers were identified by analysis of tumor tissue using multiplex fluorescence polymerase chain reaction (PCR) using the NCI recommended panel and classified into microsatellite instability-low (MSI-L), microsatellite instability-high (MSI-H) and microsatellite stable (MSS) groups. Immunohistochemical staining for MSH2, MSH6 and MLH1 on tissue microarrays (TMAs) was performed, and methylation of the MLH1 promoter was analyzed by quantitative methylation specific PCR (MSP). Germline mutation analysis of blood samples was performed for MSH2, MSH6 and MLH1 genes.
Results
Thirty-four out of the 146 colorectal cancers (CRCs, 23.2%) were MSI, including 19 MSI-H CRCs and 15 MSI-L CRCS. Negative staining for MSH2 was found in 8 CRCs, negative staining for MSH6 was found in 6 CRCs. One MSI-H CRC was negative for both MSH6 and MSH2. Seventeen CRCs stained negatively for MLH1. MLH1 promoter methylation was determined in 34 MSI CRCs. Hypermethylation of the MLH1 promoter occurred in 14 (73.7%) out of 19 MSI-H CRCs and 5 (33.3%) out of 15 MSI-L CRCs. Among the 34 MSI carriers and one MSS CRC with MLH1 negative staining, 8 had a MMR gene germline mutation, which accounted for 23.5% of all MSI colorectal cancers and 5.5% of all the colorectal cancers. Five patients harbored MSH2 germline mutations, and three patients harbored MSH6 germline mutations. None of the patients had an MLH1 mutation. Mutations were commonly located in exon 7 and 12 of MSH2 and exon 5 of MSH6. Right colonic lesions and mucinous carcinoma were not common in MSI carriers.
Conclusion
Our data may imply that the characteristics of HNPCC in the Chinese population are probably different from those of Western countries. Application of NCI recommended criteria may not be effective enough to identify Chinese HNPCC families. Further studies are necessary to echo or refute our results so as to make the NCI recommendation more universally applicable.
doi:10.1186/1471-2407-8-44
PMCID: PMC2275286  PMID: 18257912
15.  Identification of Kininogen-1 as a Serum Biomarker for the Early Detection of Advanced Colorectal Adenoma and Colorectal Cancer 
PLoS ONE  2013;8(7):e70519.
Background
Serum markers represent potential tools for the detection of colorectal cancer (CRC). The aim of this study was to obtain proteomic expression profiles and identify serum markers for the early detection of CRC.
Methods
Proteomic profiles of serum samples collected from 35 healthy volunteers, 35 patients with advanced colorectal adenoma (ACA), and 40 patients with CRC were compared using Clinprot technology. Using enzyme-linked immunosorbent assays (ELISAs), 366 sera samples were additionally analyzed, and immunohistochemistry studies of 400 tissues were used to verify the expression of kininogen-1 and its value in the early detection of CRC.
Results
Predicting models were established among the three groups, and kininogen-1 was identified as a potential marker for CRC using Clinprot technology. ELISAs also detected significantly higher serum kininogen-1 levels in ACA and CRC patients compared to controls (P<0.05). Furthermore, the area under the receiver operating characteristic curve (AUC) for serum kininogen-1 in the diagnosis of ACA was 0.635 (P = 0.003), and for serum carcinoembryonic antigen (CEA) was 0.453 (P = 0.358). The sensitivity, specificity, and accuracy of serum kininogen-1 for diagnosing Duke’s stage A and B CRC was 70.13%, 65.88%, and 67.90%, respectively, whereas serum CEA was 38.96%, 85.88%, and 63.58%, respectively. Moreover, immunohistochemistry showed that expression of kininogen-1 was significantly higher in CRC and ACA tissues than in normal mucosa (48.39% vs. 15.58% vs. 0%, P<0.05).
Conclusions
These results suggest that Clinprot technology provides a useful tool for the diagnosis of CRC, and kininogen-1 is a potential serum biomarker for the early detection of advanced colorectal adenoma and CRC.
doi:10.1371/journal.pone.0070519
PMCID: PMC3720899  PMID: 23894665
16.  Emerging roles of the ribonucleotide reductase M2 in colorectal cancer and ultraviolet-induced DNA damage repair 
AIM: To investigate the roles of the ribonucleotide reductase M2 (RRM2) subunit in colorectal cancer (CRC) and ultraviolet (UV)-induced DNA damage repair.
METHODS: Immunohistochemical staining of tissue microarray was performed to detect the expression of RRM2. Seven CRC cell lines were cultured and three human colon cancer cell lines, i.e., HCT116, SW480 and SW620, were used. Reverse transcription polymerase chain reaction and Western blotting were performed to determine the mRNA and protein expression levels of RRM2, respectively. Cell proliferation assay, cell cycle analysis were performed. Cell apoptosis was evaluated by double staining with fluorescein isothiocyanate-conjugated Annexin V and propidium iodide (PI) using Annexin V/PI apoptosis kit. The motility and invasion of CRC cells were assessed by the Transwell chamber assay. Cells were irradiated with a 254 nm UV-C lamp to detect the UV sensitivity after RRM2 depletion.
RESULTS: Immunohistochemical staining revealed elevated RRM2 levels in CRC tissues. RRM2 overexpression was positively correlated with invasion depth (P < 0.05), poorly differentiated type (P = 0.0051), and tumor node metastasis stage (P = 0.0015). The expression of RRM2 in HCT116 cells was downregulated after transfection, and HCT116 cell proliferation was obviously suppressed compared to control groups (P < 0.05). In the invasion test, the number of cells that passed through the chambers in the RRM2-siRNA group was 81 ± 3, which was lower than that in the negative control (289 ± 7) and blank control groups (301 ± 7.2). These differences were statistically significant (P < 0.01). Our data suggest that RRM2 overexpression may be associated with CRC progression. RRM2 silencing by siRNA may inhibit the hyperplasia and invasiveness of CRC cells, suggesting that RRM2 may play an important role in the infiltration and metastasis of CRC, which is a potential therapeutic strategy in CRC. In addition, RRM2 depletion increased UV sensitivity.
CONCLUSION: These findings suggest that RRM2 may be a facilitating factor in colorectal tumorigenesis and UV-induced DNA damage repair.
doi:10.3748/wjg.v18.i34.4704
PMCID: PMC3442208  PMID: 23002339
Ribonucleotide reductase M2; Colorectal cancers; Tissue microarray; Ultraviolet irradiation; Cancinogenesis; Metabolic genes
17.  Evaluation of specific fecal protein biochips for the diagnosis of colorectal cancer 
AIM: To develop and initially test a potential fecal protein biochip for the screening of colorectal cancer (CRC).
METHODS: Fecal protein from 20 colorectal cancer patients and 20 healthy controls were extracted from all of the fecal samples and screened for proteomic differences using a Biotin label-based protein array. Candidate proteins were then verified by ELISA. Finally, we will select out the significant protein and a seven-target multiplex fecal protein biochip was generated and tested for 20 fecal samples to determine the effectiveness of the biochip on identifying CRC. And the value of the protein biochip would be discussed.
RESULTS: After tested by protein biochip of the fecal protein from 20 colorectal cancer patients and 20 healthy controls and levels of calprotectin, M2-pyruvatekinase, angiopoietin-2, fibroblast growth factor-23 (FGF-23), proteins of the matrix metalloproteinase, thrombopoietin (TPO) and interleukin-13 (IL-13) were significantly different between CRC and healthy controls. The sensitivity of all the seven proteins combined was 0.7, specificity was 0.4, and area under the receiver operating characteristics was 0.729. The most promising combinations of test proteins were FGF-23, TPO, and IL-13, reaching a sensitivity of 0.7 and a specificity of 0.7. The combination of FGF-23 and TPO scored highest with sensitivity of 0.7 and specificity of 0.8. Its mean that the combination of FGF-23 and TPO has the highest value for the diagnosis of CRC in our study.
CONCLUSION: A protein biochip composed of proteins found to be elevated in the feces of colorectal cancer patients has great potential as a noninvasive diagnostic for colorectal cancer. The addition of new protein biomarkers and technologies, as they are discovered, is an excellent avenue of future research.
doi:10.3748/wjg.v20.i5.1332
PMCID: PMC3921516  PMID: 24574808
Protein biochip; Feces; Colorectal cancer; Fibroblast growth factor-23; Thrombopoietin
18.  A Tissue-Based Comparative Effectiveness Analysis of Biomarkers for Early Detection of Colorectal Tumors 
OBJECTIVES:
We recently identified a six-gene methylation-based biomarker panel suitable for early detection of colorectal cancer (CRC). In this study, we compared the performance of this novel epi-panel with that of previously identified DNA methylation markers in the same clinical tissue sample sets.
METHODS:
Quantitative methylation-specific PCR was used to analyze the promoter region of SEPT9 and VIM in a total of 485 tissue samples, divided into test and validation sets. ITGA4, NTRK2, OSMR, and TUBG2 were also included in the analyses. Receiver operating characteristic (ROC) curves were used to compare the performances of the individual biomarkers with that of the novel epi-panel.
RESULTS:
SEPT9 and VIM were methylated in 82 and 67% of CRCs (n=169) and in 88 and 54% of the adenomas (n=104). Only 3% of the normal mucosa samples (n=107) were methylated for these genes, confirming that the methylation was highly cancer-specific. Areas under the ROC curve (AUC), distinguishing CRCs from normal mucosa, were 0.94 for SEPT9 and 0.81 for VIM. AUC values for separating adenomas from normal mucosa samples were 0.96 and 0.81 for the same genes. In comparison, the novel epi-panel achieved an AUC of 0.98 (CRC) and 0.97 (adenomas).
ITGA4, OSMR, NTRK2, and TUBG2 were methylated in 90, 78, 7, and 1% of the CRCs, and in 76, 77, 3, and 0% of the adenomas. Between 0 and 2% of the normal mucosa samples were methylated for the same genes. ITGA4 and OSMR achieved an AUC of 0.96 and 0.92 (CRC vs. normal mucosa), and 0.93 and 0.92 (adenomas vs. normal mucosa).
CONCLUSIONS:
We have confirmed the high performance of some of the previously identified DNA methylation markers. Furthermore, we showed that a recently reported epi-panel performed better than the individual DNA methylation biomarkers when analyzed in the same tissue samples. This observation was also true for VIM and SEPT9, which are included in commercially available noninvasive tests for CRC. These results further underscore the value of combining a manageable number of individual markers into a panel, which in addition to having a higher sensitivity and specificity might provide a more profound robustness to a noninvasive test compared with single markers.
doi:10.1038/ctg.2012.21
PMCID: PMC3535074  PMID: 23324654
19.  KRAS Mutation Is a Predictor of Oxaliplatin Sensitivity in Colon Cancer Cells 
PLoS ONE  2012;7(11):e50701.
Molecular biomarkers to determine the effectiveness of targeted therapies in cancer treatment have been widely adopted in colorectal cancer (CRC), but those to predict chemotherapy sensitivity remain poorly defined. We tested our hypothesis that KRAS mutation may be a predictor of oxaliplatin sensitivity in CRC. KRAS was knocked-down in KRAS-mutant CRC cells (DLD-1G13D and SW480G12V) by small interfering RNAs (siRNA) and overexpressed in KRAS-wild-type CRC cells (COLO320DM) by KRAS-mutant vectors to generate paired CRC cells. These paired CRC cells were tested by oxaliplatin, irinotecan and 5FU to determine the change in drug sensitivity by MTT assay and flow cytometry. Reasons for sensitivity alteration were further determined by western blot and real-time quantitative reverse transcriptase polymerase chain reaction (qRT -PCR). In KRAS-wild-type CRC cells (COLO320DM), KRAS overexpression by mutant vectors caused excision repair cross-complementation group 1 (ERCC1) downregulation in protein and mRNA levels, and enhanced oxaliplatin sensitivity. In contrast, in KRAS-mutant CRC cells (DLD-1G13D and SW480G12V), KRAS knocked-down by KRAS-siRNA led to ERCC1 upregulation and increased oxaliplatin resistance. The sensitivity of irinotecan and 5FU had not changed in the paired CRC cells. To validate ERCC1 as a predictor of sensitivity for oxaliplatin, ERCC1 was knocked-down by siRNA in KRAS-wild-type CRC cells, which restored oxaliplatin sensitivity. In contrast, ERCC1 was overexpressed by ERCC1-expressing vectors in KRAS-mutant CRC cells, and caused oxaliplatin resistance. Overall, our findings suggest that KRAS mutation is a predictor of oxaliplatin sensitivity in colon cancer cells by the mechanism of ERCC1 downregulation.
doi:10.1371/journal.pone.0050701
PMCID: PMC3508995  PMID: 23209813
20.  MALDI-TOF MS Combined With Magnetic Beads for Detecting Serum Protein Biomarkers and Establishment of Boosting Decision Tree Model for Diagnosis of Colorectal Cancer 
The aim of present study is to study the serum protein fingerprint of patients with colorectal cancer (CRC) and to screen protein molecules that are closely related to colorectal cancer during the onset and progression of the disease with Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). Serum samples from 144 patients with CRC and 120 healthy volunteers were adopted in present study. Weak cation exchange (WCX) magnetic beads and PBSII-C protein chips reader (Ciphergen Biosystems Ins.) were used. The protein fingerprint expression of all the Serum samples and the resulted profiles between cancer and normal groups were analyzed with Biomarker Wizard system. Several proteomic peaks were detected and four potential biomarkers with different expression profiles were identified with their relative molecular weights of 2870.7Da, 3084Da, 9180.5Da, and 13748.8Da, respectively. Among the four proteins, two proteins with m/z 2870.7 and 3084 were down-regulated, and the other two with m/z 9180.5 and 13748.8 were up-regulated in serum samples from CRC patients. The present diagnostic model could distinguish CRC from healthy controls with the sensitivity of 92.85% and the specificity of 91.25%. Blind test data indicated a sensitivity of 86.95% and a specificity of 85%. The result suggested that MALDI technology could be used to screen critical proteins with differential expression in the serum of CRC patients. These differentially regulated proteins were considered as potential biomarkers for the patients with CRC in the serum and of the potential value for further investigation.
PMCID: PMC3020391  PMID: 21234268
MALDI; colorectal cancer; Biomarker; Protein; serum
21.  Next-Generation Stool DNA Test Accurately Detects Colorectal Cancer and Large Adenomas 
Gastroenterology  2011;142(2):248-e26.
BACKGROUND & AIMS
Technical advances have led to stool DNA (sDNA) tests that might accurately detect neoplasms on both sides of the colorectum. We assessed colorectal neoplasm detection by a next-generation sDNA test and effects of covariates on test performance.
METHODS
We performed a blinded, multicenter, case-control study using archived stool samples collected in preservative buffer from 252 patients with colorectal cancer (CRC), 133 with adenomas ≥1 cm, and 293 individuals with normal colonoscopy results (controls); two-thirds were randomly assigned to a training set and one-third to a test set. The sDNA test detects 4 methylated genes, a mutant form of KRAS, and the α-actin gene (as a reference value) using quantitative, allele-specific, real-time target and signal amplification; it also quantifies hemoglobin. We used a logistical model to analyze data.
RESULTS
The sDNA test identified 85% of patients with CRC and 54% of patients with adenomas ≥1 cm with 90% specificity. The test had a high rate of detection for all nonmetastatic stages of CRC (aggregate 87% detection rate for CRC stages I–III). Detection rates increased with adenoma size: 54% ≥1 cm, 63% >1 cm, 77% >2 cm, 86% >3 cm, and 92% >4 cm (P < .0001). Based on receiver operating characteristic analysis, the rate of CRC detection was slightly greater for the training than the test set (P = .04), whereas the rate of adenoma detection was comparable between sets. Sensitivities for detection of CRC and adenoma did not differ with lesion site.
CONCLUSIONS
Early-stage CRC and large adenomas can be detected throughout the colorectum and with high levels of accuracy by the sDNA test. Neoplasm size, but not anatomical site, affected detection rates. Further studies are needed to validate the findings in a larger population and optimize the sDNA test.
doi:10.1053/j.gastro.2011.10.031
PMCID: PMC4017869  PMID: 22062357
QuARTS; Colorectal Cancer Screening; Stool Testing; Precancer Detection
22.  Potentiality of a triple microRNA classifier: miR-193a-3p, miR-23a and miR-338-5p for early detection of colorectal cancer 
BMC Cancer  2013;13:280.
Background
MicroRNAs (miRNAs) are short, non-coding RNA molecules that act as regulators of gene expression. Circulating blood miRNAs offer great potential as cancer biomarkers. The objective of this study was to correlate the differential expression of miRNAs in tissue and blood in the identification of biomarkers for early detection of colorectal cancer (CRC).
Methods
The study was divided into two phases: (I) Marker discovery by miRNA microarray using paired cancer tissues (n?=?30) and blood samples (CRC, n?=?42; control, n?=?18). (II) Marker validation by stem-loop reverse transcription real time PCR using an independent set of paired cancer tissues (n?=?30) and blood samples (CRC, n?=?70; control, n?=?32). Correlation analysis was determined by Pearson’s test. Logistic regression and receiver operating characteristics curve analyses were applied to obtain diagnostic utility of the miRNAs.
Results
Seven miRNAs (miR-150, miR-193a-3p, miR-23a, miR-23b, miR-338-5p, miR-342-3p and miR-483-3p) have been found to be differentially expressed in both tissue and blood samples. Significant positive correlations were observed in the tissue and blood levels of miR-193a-3p, miR-23a and miR-338-5p. Moreover, increased expressions of these miRNAs were detected in the more advanced stages. MiR-193a-3p, miR-23a and miR-338-5p were demonstrated as a classifier for CRC detection, yielding a receiver operating characteristic curve area of 0.887 (80.0% sensitivity, 84.4% specificity and 83.3% accuracy).
Conclusion
Dysregulations in circulating blood miRNAs are reflective of those in colorectal tissues. The triple miRNA classifier of miR-193a-3p, miR-23a and miR-338-5p appears to be a potential blood biomarker for early detection of CRC.
doi:10.1186/1471-2407-13-280
PMCID: PMC3691634  PMID: 23758639
Colorectal cancer; MicroRNA; MiR-193a-3p; MiR-23a; MiR-338-5p
23.  A systems biology approach to the global analysis of transcription factors in colorectal cancer 
BMC Cancer  2012;12:331.
Background
Biological entities do not perform in isolation, and often, it is the nature and degree of interactions among numerous biological entities which ultimately determines any final outcome. Hence, experimental data on any single biological entity can be of limited value when considered only in isolation. To address this, we propose that augmenting individual entity data with the literature will not only better define the entity’s own significance but also uncover relationships with novel biological entities.
To test this notion, we developed a comprehensive text mining and computational methodology that focused on discovering new targets of one class of molecular entities, transcription factors (TF), within one particular disease, colorectal cancer (CRC).
Methods
We used 39 molecular entities known to be associated with CRC along with six colorectal cancer terms as the bait list, or list of search terms, for mining the biomedical literature to identify CRC-specific genes and proteins. Using the literature-mined data, we constructed a global TF interaction network for CRC. We then developed a multi-level, multi-parametric methodology to identify TFs to CRC.
Results
The small bait list, when augmented with literature-mined data, identified a large number of biological entities associated with CRC. The relative importance of these TF and their associated modules was identified using functional and topological features. Additional validation of these highly-ranked TF using the literature strengthened our findings. Some of the novel TF that we identified were: SLUG, RUNX1, IRF1, HIF1A, ATF-2, ABL1, ELK-1 and GATA-1. Some of these TFs are associated with functional modules in known pathways of CRC, including the Beta-catenin/development, immune response, transcription, and DNA damage pathways.
Conclusions
Our methodology of using text mining data and a multi-level, multi-parameter scoring technique was able to identify both known and novel TF that have roles in CRC. Starting with just one TF (SMAD3) in the bait list, the literature mining process identified an additional 116 CRC-associated TFs. Our network-based analysis showed that these TFs all belonged to any of 13 major functional groups that are known to play important roles in CRC. Among these identified TFs, we obtained a novel six-node module consisting of ATF2-P53-JNK1-ELK1-EPHB2-HIF1A, from which the novel JNK1-ELK1 association could potentially be a significant marker for CRC.
doi:10.1186/1471-2407-12-331
PMCID: PMC3539921  PMID: 22852817
24.  Identification of Predictive Markers of Response to the MEK1/2 Inhibitor Selumetinib (AZD6244) in K-ras–Mutated Colorectal Cancer 
Molecular cancer therapeutics  2010;9(12):3351-3362.
Mutant K-ras activity leads to the activation of the RAS/RAF/MEK/ERK pathway in approximately 44% of colorectal cancer (CRC) tumors. Accordingly, several inhibitors of the MEK pathway are under clinical evaluation in several malignancies including CRC. The aim of this study was to develop and characterize predictive biomarkers of response to the MEK1/2 inhibitor AZD6244 in CRC in order to maximize the clinical utility of this agent. Twenty-seven human CRC cell lines were exposed to AZD6244 and classified according to the IC50 value as sensitive (≤0.1 µmol/L) or resistant (>1 µmol/L). All cell lines were subjected to immunoblotting for effector proteins, K-ras/BRAF mutation status, and baseline gene array analysis. Further testing was done in cell line xenografts and K-ras mutant CRC human explants models to develop a predictive genomic classifier for AZD6244. The most sensitive and resistant cell lines were subjected to differential gene array and pathway analyses. Members of the Wnt signaling pathway were highly overexpressed in cell lines resistant to AZD6244 and seem to be functionally involved in mediating resistance by shRNA knockdown studies. Baseline gene array data from CRC cell lines and xenografts were used to develop a k-top scoring pair (k-TSP) classifier, which predicted with 71% accuracy which of a test set of patient-derived K-ras mutant CRC explants would respond to AZD6244, providing the basis for a patient-selective clinical trial. These results also indicate that resistance to AZD6244 may be mediated, in part, by the upregulation of the Wnt pathway, suggesting potential rational combination partners for AZD6244 in CRC.
doi:10.1158/1535-7163.MCT-10-0376
PMCID: PMC3931013  PMID: 20923857
25.  Differential expression of serum miR-126, miR-141 and miR-21 as novel biomarkers for early detection of liver metastasis in colorectal cancer 
Objective
MicroRNAs (miRNAs) have potential as diagnostic biomarkers in cancer. Evaluation of the association between miRNA expression patterns and early detection of liver metastasis in colorectal cancer (CRC) has not been reported.
Methods
We investigated the expression of metastasis-associated miRs-31, 335, 206, 141, 126, 200b, 200c, 21, Let7a, Let7b and Let7c in localized, liver-metastatic and other organ-metastatic CRC (OM-CRC). Expressions of target miRNAs in serum were evaluated in 116 consecutive localized CRC (L-CRC), 72 synchronous liver-metastatic CRC (SLM-CRC) and 36 other OM-CRC by quantitative real-time PCR.
Results
Seven of 11 tested miRNAs could be detected from serum. Four miRNAs, miR-126, Let-7a, miR-141 and miR-21 were identified as metastasis-associated miRNAs. Compared with L-CRC, significant up-regulated expression was observed for miR-141 and miR-21 in SLM-CRC and OM-CRC, down-regulated expression was observed for miR-126 in SLM-CRC and OM-CRC, and up-regulated expression of Let-7a in OM-CRC. The receiver operating characteristic (ROC) curve showed serum miR-126 had a cut-off [log10 relative quantity (log10RQ)=–0.2005] with 77.78% sensitivity and 68.97% specificity with an area under curve (AUC) of 0.7564, miR-141 had a cut-off (log10RQ=–0.2285) with 86.11% sensitivity and 76.11% specificity with an AUC of 0.8279, and miR-21 had a cut-off (log10RQ=–0.1310) with 73.61% sensitivity and 66.38% specificity with an AUC of 0.7479.
Conclusions
We identified liver metastasis-associated miRNAs, suggesting serum miR-126, miR-141 and miR-21 may be novel biomarkers for clinical diagnosis of early stage liver-metastatic CRC.
doi:10.3978/j.issn.1000-9604.2014.02.07
PMCID: PMC3937759  PMID: 24653631
MicroRNA (miRNAs); colorectal cancer (CRC); liver metastasis; diagnosis

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