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Lin JS, Webber EM, Beil TL, et al. Fecal DNA Testing in Screening for Colorectal Cancer in Average-Risk Adults [Internet]. Rockville (MD): Agency for Healthcare Research and Quality (US); 2012 Feb. (Comparative Effectiveness Reviews, No. 52.)

  • This publication is provided for historical reference only and the information may be out of date.

This publication is provided for historical reference only and the information may be out of date.

Executive Summary


Colorectal cancer (CRC) is the third most common cancer in both men and women and is the third leading cause of cancer deaths in the United States.1 Incidence and mortality rates for CRC have declined over the past two decades, corresponding with an increase in self-reported screening rates.1 However, screening rates remain suboptimal. While different U.S. guideline-issuing organizations agree on the majority of recommended CRC screening options, there are differences between some recommended options, such as fecal DNA testing. In 2008, the United States Preventive Services Task Force (USPSTF) found that evidence was insufficient to recommend fecal DNA testing for CRC screening.2,3 However, the American Cancer Society (ACS), the U.S. Multi-Society Task Force (MSTF) on Colorectal Cancer, and the American College of Radiology (ACR) collectively recommended fecal DNA testing as an alternative screening method. The ACS-MSTF-ACR's recommendation was based on a lower threshold of evidence than that of the USPSTF.4,5

Fecal DNA tests are designed to detect molecular abnormalities in cells from cancer or precancerous lesions that are shed into the stool. Fecal DNA testing to screen for CRC has evolved significantly over time, both in improvements in understanding relevant molecular abnormalities associated with CRC and technological advances to allow for improved detection of molecular abnormalities in DNA in the stool.6 Molecular abnormalities that have served as the basis for CRC screening tests have focused on three major genetic mechanisms: chromosomal instability due to abnormalities in mutational hotspots like APC, KRAS, and TP53; microsatellite instability due to loss of function of mismatch repair genes that can result in accumulation of errors within the DNA sequence; and DNA methylation, an epigenetic alteration, in which promoter sites of genes are hypermethylated leading to suppression of gene transcription.7

Thus far a single company, Exact Sciences, has been the major commercial developer of fecal DNA testing in the United States (Table A). Currently, only one fecal DNA test, ColoSure™, is commercially available. This test is a single marker fecal DNA assay for methylated vimentin distributed by LabCorp. Marketing for commercially available fecal DNA testing specifies that the test is intended for individuals who are not eligible (either unable or unwilling) for more invasive CRC screening (i.e., colonoscopy, flexible sigmoidoscopy, or CT colonography).8

Table A. Development of fecal DNA testing for colorectal cancer screening.

Table A

Development of fecal DNA testing for colorectal cancer screening.


This report includes six Key Questions to systematically review the evidence on fecal DNA testing to screen for CRC in average-risk adults (Figure A).

This figure depicts how the Key Questions in this report inter-relate using an Analytic Framework convention. The figure illustrates how screening with fecal DNA testing among asymptomatic adults at average risk for colorectal cancer may result in improved detection of colorectal cancer and/or precancerous lesions, as well as improved health outcomes such as decreased colorectal cancer mortality, decreased all-cause mortality, and decreased colorectal cancer incidence. The figure also illustrates that harms may occur from screening, and management or treatment of cancer or precancerous lesions.

Figure A

Analytic framework of the benefits and harms of fecal DNA testing in screening for colorectal cancer. Note: Numbers and letters correspond to the Key Questions.

Key Question 1. Clinical utility

What is the effectiveness of fecal DNA testing (alone or in combination with other screening tests) to screen for CRC in reducing morbidity (CRC incidence) or mortality (all-cause or CRC-specific)?

Key Question 2. Clinical validity


What are the absolute test-performance characteristics (e.g., sensitivity, specificity) of fecal DNA testing for CRC screening, as compared to colonoscopy?

  1. To detect CRC?
  2. To detect precancerous lesion(s)?

What is the relative test performance of fecal DNA testing as compared to other established screening modalities in current practice?

  1. To detect CRC?
  2. To detect precancerous lesion(s)?

Key Question 3. Interval of Screening

What is the test performance of fecal DNA testing across different screening interval(s)?

Key Question 4. Analytic Validity


What is the analytic validity (analytic sensitivity, specificity, and reproducibility) of currently available fecal DNA assays?


What are the important analytic and pre-analytic factors that can affect fecal DNA assay validity?

Key Question 5. Acceptability of Testing

What is the acceptability and adherence of fecal DNA screening in comparison to other stool-based screening tests, or in comparison to more invasive modalities of screening?

Key Question 6. Harms

What are the potential harms of fecal DNA testing?

Analytic Framework


Input from Stakeholders

This topic was initiated based on a public nomination submitted to the Agency for Healthcare Research Quality Effective Health Care program. Several individuals expressed concern about the optimal timing of this review during public review due to the current development of new fecal DNA screening test. Despite these comments, it was determined that a review would still be helpful to stakeholders in the interim. A Technical Expert Panel (TEP) helped in the refinement of our review protocol and provided details about fecal DNA test development.

Data Sources and Selection

We performed comprehensive literature searches in the following databases from 2000 through August 11, 2011: MEDLINE, Cochrane Database of Systematic Reviews, Cochrane Central Register of Controlled Trials, Database of Abstracts of Reviews of Effects, and the Health Technology Assessments Database. Searches of these databases were supplemented with manual searching of reference lists of relevant review articles and suggestions made by TEP members. We also performed a focused search of the grey literature, including: unpublished data from recent conference abstracts (2009–2011), regulatory documents, and information regarding ongoing and future research via clinical trial registry entries. Additional unpublished literature was sought via a Scientific Information Packet (SIP) request to LabCorp.

Two reviewers independently screened abstracts against a set of a priori inclusion criteria. Included studies were limited to asymptomatic screening populations, published since 2000 in English language. Full-text articles of abstracts meeting inclusion criteria were retrieved and dual-reviewed against the inclusion criteria. Disagreements were resolved with consultation of a third reviewer.

Data Extraction and Quality Assessment

Data from all included studies were abstracted into standardized evidence tables by one reviewer and checked by a second reviewer. Separate abstraction forms were created for key questions. We abstracted important details relating to study design, population characteristics, test and comparators, and all relevant outcomes.

We applied the study design-specific quality criteria of the USPSTF to assess the methodological quality of included studies.9 We supplemented these quality criteria with methods from the Evaluation of Genomic Applications in Practice and Prevention Working Group (specific to genetic testing),10 the Newcastle Ottawa Scale (specific to cohort studies),11 and the QUADAS criteria (specific to diagnostic accuracy studies).12 Two independent reviewers assigned a quality rating of the internal validity for each study. Disagreements were resolved by discussion and consensus or by consulting a third, independent reviewer.

Data Synthesis and Analysis

We conducted qualitative syntheses of study results for each key question. We did not conduct meta-analysis of results due to the limited number of studies for each key question and clinical differences between studies. For qualitative syntheses, we evaluated and summarized clinical and methodological characteristics of included studies, as well as important internal (quality) and external (applicability) study characteristics. The strength of evidence for primary outcomes was graded using the standard process of the Evidence-based Practice Centers, based on four major domains: risk of bias, consistency, directness, and precision of the evidence.13


Our literature search yielded 336 citations from electronic database searches and outside sources (Figure B). Based on the review of title and abstracts, we subsequently reviewed 34 full-text articles for their eligibility. We included 12 articles, three diagnostic accuracy studies (clinical validity) that met inclusion criteria for Key Question 2, three analytic validity studies for Key Question 4, and six studies of acceptability or preference of testing for Key Question 5. For Key Question 2, all three studies reported absolute test performance based on colonoscopy findings (KQ2.1), two of which also reported test performance compared to guaiac-based FOBT (KQ2.2). Two studies for Key Question 2 also reported adherence to testing and are discussed with Key Question 5 results. We found no studies that addressed clinical utility (Key Question 1), intervals of screening (Key Question 3), or specific harms of screening (Key Question 6).

This flow chart summarizes the literature yield and selection of articles. There were 279 citations identified through published literature searches. An additional 57 citations were identified from outside sources such as reference lists. Of these 336, 302 citations were excluded at the title/abstract level. The full-text of the 34 remaining citations were examined for inclusion. Of these, 22 studies were excluded for the following reasons: study design, population, test not currently available, lack of appropriate comparator, analytic validity not vimentin methylation. One article was excluded for different reasons for different key questions. In total, there were no studies included for key questions 1, 3, or 6. There were 3 studies included for key question 2, 3 studies included for key question 4, and 6 studies included for key question 5.

Figure B

Literature flow diagram. * 1 article was excluded for different reasons for different key questions † 2 articles from KQ2 reported adherence to testing (and therefore are also discussed with KQ5)

Key Questions 2 and 6. Diagnostic accuracy and harms of fecal DNA testing

Despite the availability of numerous initial validation studies of fecal DNA testing, we only found three studies that examined the accuracy of fecal DNA testing in screening populations (Table B).14-16 Two fair-quality diagnostic accuracy studies (n=5004) in screening cohorts of average-risk patients undergoing colonoscopy evaluated a fecal DNA test (SDT-1) that was a prototype to a later version that was clinically available as PreGen Plus™ (Table A).14,15 These two studies found different sensitivities for detection of CRC (25 percent [95% CI, 5 to 57] versus 51.6 percent [95% CI, 34.8 to 68.0]) (Table B). Both found similarly low sensitivities for detection of advanced adenomas (Table B). The specificity for detection for CRC or advanced adenomas was approximately 93 to 96 percent (Table B). In one of the diagnostic accuracy studies, the specificity for the prototype to PreGen Plus (SDT-1) and Hemoccult II™ were not statistically significantly different, although the study had limited power to detect a difference (Table C).15 One smaller study (n=441) evaluating the test accuracy of KRAS mutations,16 and a subset analysis (n=217) of the diagnostic accuracy study by Ahlquist and colleagues,14 evaluating a multi-marker test that included methylated vimentin (SDT-2), were both poor quality. None of these studies evaluated fecal DNA tests applicable to the currently available test, ColoSure.

Table B. Diagnostic accuracy of fecal DNA testing in screening populations (KQ2).

Table B

Diagnostic accuracy of fecal DNA testing in screening populations (KQ2).

Table C. Limitations and quality concerns for diagnostic accuracy studies of fecal DNA testing.

Table C

Limitations and quality concerns for diagnostic accuracy studies of fecal DNA testing.

We did not find any studies that specifically evaluated the harms of fecal DNA testing. The major hypothesized harms of fecal DNA testing are the sequelae from diagnostic inaccuracy (false positives and false negatives).

Key Question 4. Analytic validity of fecal DNA testing

We found three poor-quality studies that specifically evaluated the analytic validity of currently available fecal DNA assays, a single-marker test for methylated vimentin.17-19 These studies showed that technological advances (i.e., methyl-BEAMing and methyl-binding domain enrichment) can improve the analytic sensitivity of assays to detect methylated vimentin in stool samples (Table D). None of the studies evaluated the repeatability, reproducibility, or analytic specificity of testing. These three studies were generally of poor quality, and the technological advances evaluated in these studies are not applicable to the previously studied (SDT-2) or currently available test (ColoSure) for methylated vimentin.

Table D. Analytic validity of fecal DNA testing.

Table D

Analytic validity of fecal DNA testing.

Key Question 5. Acceptability and adherence of testing

We found six fair- to poor-quality studies that evaluated the acceptability and two diagnostic accuracy studies that reported the adherence to fecal DNA testing.14,15,20-25 From very limited evidence, it appears that fecal DNA testing is generally acceptable, although an important test attribute for acceptability appears to be the test's accuracy (Table E). In one fair-quality diagnostic accuracy study, fecal DNA adherence was lower than adherence to fecal occult blood test (FOBT).15 No studies have evaluated the relative acceptability or adherence of fecal DNA tests to fecal immunochemical test (FIT) tests. It is likely that future fecal DNA testing will be in test accuracy, and possibly stool collection, such that the currently available evidence on acceptability and adherence to fecal DNA testing will no longer be relevant.

Table E. Patient preferences and acceptability of fecal DNA testing.

Table E

Patient preferences and acceptability of fecal DNA testing.


Strength of Evidence

Despite considerable media attention and expert-based clinical recommendations that include fecal DNA testing for CRC screening, at present, fecal DNA tests have insufficient evidence about their clinical validity (diagnostic accuracy) in patients at average risk for CRC. Due to the differences in tests evaluated and differences in sensitivity between the two studies that evaluated the same test, the evidence for the test accuracy for fecal DNA testing is both inconsistent and imprecise. Fecal DNA test development has evolved significantly over the past decade. There have been advances in the understanding of molecular markers that reflect neoplastic change and advances in technologies to stabilize, extract, and amplify/detect low levels of human target DNA in stool samples. Therefore, the three studies on diagnostic accuracy of fecal DNA tests in screening populations do not reflect the current commercially available fecal DNA test (or soon to be available fecal DNA testing). Likewise, harms and acceptability of and adherence to fecal DNA testing in comparison to other screening modalities also have insufficient evidence and are largely not applicable to currently available fecal DNA tests. Because patients' (and clinicians') preference of test choice is influenced by test performance, acceptability and adherence to testing will need to be reexamined once test accuracy is known. Subtleties in stool collection may also affect acceptability and adherence, and therefore may change if future fecal DNA testing no longer requires a single whole-stool specimen.

Evidence Gaps and Future Research

The most critical evidence gap for fecal DNA testing to screen for CRC is the lack of appropriately designed diagnostic accuracy studies applicable to currently available fecal DNA testing. At a minimum, clinical decision making should be based upon evidence from test validation studies conducted in the intended population (i.e., asymptomatic screening population) for which the test is proposed. Empiric evidence shows that distorted selection of participants (including nonrepresentative patients) and use of case-control study designs overestimate overall test accuracy due to both variation and spectrum bias.26,27 Based on this review, we found discordant results from the three included diagnostic accuracy studies in comparison to the initial validation studies identified but excluded from this review. For example, initial validation studies for the prototype of PreGen Plus had sensitivity for CRC estimates around 90 percent, and subsequent test validation studies in screening populations showed much lower sensitivities (about 25 to 50 percent).28 When better-quality, more-applicable diagnostic accuracy studies in screening populations become available, clinicians and decision makers can use robust models that have been developed by the National Cancer Institute Cancer Intervention and Surveillance Modeling Network for evaluating CRC screening (e.g., MISCAN, SimCRC) to estimate net benefit of testing (of a program of testing, and harms of testing due to diagnostic inaccuracies) and optimal intervals of testing, compared to other currently used or promising screening modalities. Other important evidence gaps include the relative acceptability of and adherence to fecal DNA testing, compared with FIT (which is a stool based test that does not require dietary or medication restrictions), and issues around fecal DNA testing analytic validity, specifically accuracy, and repeatability and reproducibility. In addition, reporting of potentially important details that may affect analytic validity of assays should be routinely reported in clinical evaluation (clinical validity) studies. Especially given the constant changes in test development, test developers and researchers need to be transparent and explicit about differences in the assays evaluated in studies and the actual assays that are clinically available.


The limitations in this review are primarily from the limitations in the primary research (small body of variable, often poor quality studies) and the evolving nature of fecal DNA testing (resulting in a mismatch between primary research and available testing). However, there are few important limitations in the scope and timing of this review. Our review focused on fecal DNA testing to screen for CRC, and therefore did not address other potential roles of fecal DNA testing. Also, our review did not include stool-based testing using RNA or other genetic/genomic based testing in plasma. However, these newer types of genetic/genomic testing to screen for CRC are more developmental than fecal DNA testing. Finally, this review will likely be out of date as new tests and evidence supporting these tests becomes available within the next 2 years.


95% CI

95 percent confidence interval


American College of Radiology


American Cancer Society


Clinical Laboratory Improvement Amendments


Colorectal cancer

CT colonography

Computed tomographic colonography


DNA integrity assay


Deoxyribonucleic acid

EHC Program

Effective Health Care Program


U.S. Food and Drug Administration


Fecal immunochemica test


Fecal occult blood test (usually used to refer to guaiac based tests like Hemoccult II™ or Hemoccult SENSA™ versus immunochemical based tests for hemoglobin)


Key question


Laboratory-developed test


Methyl-binding domain


Not reported


Polymerase chain reaction


Ribonucleic acid


Stool DNA test


Scientific Information Packet


Technical Expert Panel


Absolute test performance

Performance of a test (sensitivity, specificity) when compared to the gold standard.


Ability of assay to measure what it purports to measure determined independently by a reference method.


Benign tumor from epithelial tissue.

Advanced adenomas

Adenomas 1 cm or greater, or with villous components (tubulovillous or villous), or with high-grade or severe dysplasia.


A measured portion of a sample taken for analysis.

Analytic factors

Test methods and performance of procedures, and monitoring and verification of accuracy and reliability of test results.

Analytic sensitivity (lower limit of detection)

Ability of assay to detect all true positive specimens, for quantitative tests this is defined as the smallest quantity of a substance that can be reliably detected or quantified.

Analytic specificity

Ability present in the sample of assay to measure the target substance when potentially interfering or cross-reacting substances are present in the sample.

Analytic validity

An assay's ability to accurately and reliably measure the genotype (or analyte) of interest.


An analysis conducted to verify the presence (and amount) of a substance.

Chromosomal instability

The gain or loss of whole chromosomes or fractions of chromosomes.

Clinical utility

A test's ability to improve clinical outcomes and the test's usefulness and value it adds to patient management decision-making, compared with current management without genetic testing.

Clinical validity

A test's ability to accurately and reliably predict the clinically defined disorder or phenotype of interest.

DNA integrity

Potential biomarker for colorectal cancer because DNA shed from cancer cells have been characterized as having longer DNA fragments as compared to DNA shed from noncancer cells.


Changes in gene expression caused by mechanisms other than changes in the DNA sequence.

Guaiac based fecal occult blood test (FOBT)

An assay to detect the presence of hemoglobin in the feces that is not visibly apparent in which feces is applied to a thick piece of paper attached to a thin film coated with guaiac (a phenolic compound).

Immunochemical based fecal occult blood test (FOBT) or fecal immunochemical test (FIT)

An assay to detect the presence of hemoglobin in feces that is not visibly apparent in which a fecal sample is collected (e.g., with a brush, probe, stick) and transferred to a test card or slide (dry sampling) or deposited into a liquid buffer (wet sampling). Occult blood is then detected using an antibody specific for human hemoglobin.

Initial test validation

study designed to determine ability and diagnostic accuracy of a test in persons with the target condition (as opposed to validation in the test's intended population); for this report in persons with known CRC or colorectal adenomas; these studies are most often case-control studies in which cases are persons with known CRC or colorectal cancer versus healthy controls.


The addition of a methyl group.

Microsatellite instability

DNA damage due to defects in the normal DNA repair process.

Pre-analytic factors

factors that may affect test performance prior to analysis specimen collection, processing, handling, and delivery to testing site.

Relative test performance

Diagnostic accuracy (sensitivity, specificity) when compared to another test that is not the gold standard.


Replication of results when the assay is performed multiple times on a single specimen.


the copying of DNA into mRNA in gene expression.


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Cover of Fecal DNA Testing in Screening for Colorectal Cancer in Average-Risk Adults
Fecal DNA Testing in Screening for Colorectal Cancer in Average-Risk Adults [Internet].
Comparative Effectiveness Reviews, No. 52.
Lin JS, Webber EM, Beil TL, et al.

AHRQ (US Agency for Healthcare Research and Quality)

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