Rationale and current practice

Multiple tests are clinically used to screen for CRC, these include stool based tests (e.g., guaiac based or immunochemical based fecal occult blood testing), endoscopy (e.g., flexible sigmoidoscopy or colonoscopy), and imaging tests (e.g., double contrast barium enema or CT colonography). The decrease in CRC incidence and mortality over the past two decades in the United States corresponds to an increase in self-reported screening rates from less than 25 percent in the 1980s to about 52 percent in 2002 and about 65 percent in 2010.^1,3 Despite increases in CRC screening over time, screening rates remain below optimal. Multiple patient, clinician, and health-care delivery factors have been found to negatively influence CRC screening, including low socioeconomic or educational status, lack of physician recommendation, and lack of insurance or limited access to health care.⁹

Most organizations agree that any CRC screening is better than no screening, and that the age to begin screening in adults at average-risk for CRC is 50 years old. Currently, most U.S. guideline organizations, including the U.S. Preventive Services Task Force (USPSTF), agree that the recommended options in screening for CRC include: colonoscopy every 10 years; annual high-sensitivity guaiac fecal occult blood testing (FOBT) or fecal immunochemical testing (FIT); and flexible sigmoidoscopy every 5 years with or without fecal blood testing (FOBT or FIT).^10,11

Some disagreement occurs between guideline organizations about screening interventions with less evidence to support their use. These tests include: computerized tomography (CT) colonography, double contrast barium enema (DCBE), and fecal or stool-based DNA testing.¹⁰ In 2008, the USPSTF found that evidence was insufficient to recommend fecal DNA testing for CRC screening based on a systematic review of new and established CRC screening modalities.^5,11 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. This discrepancy between recommendations appears to be due to differences in evidence considered. The ACS-MSTF-ACR recommendation was based a lower threshold of evidence than that of the USPSTF.¹² While the American College of Gastroenterology recognized that fecal DNA testing may offer an alternative form of CRC screening, they state that the preferred forms of screening include colonoscopy and FIT, noting the very limited evidence for fecal DNA testing.¹³

Evolution of fecal DNA testing for colorectal cancer screening

Unlike other stool-based screening tests that are designed to detect hemoglobin, fecal DNA tests are designed to detect molecular abnormalities in cancer or precancerous lesions that are shed into the stool. Molecular abnormalities in CRC that have served as the basis for screening tests have focused on three major genetic mechanisms: chromosomal instability due to abnormalities in mutational hotspots like APC, KRAS, and TP53; microsatellite instability (MSI) due to loss of function of mismatch repair genes that can result in accumulation of errors within the DNA sequence called microsatellites; and DNA methylation, an epigenetic alteration, in which promoter sites of genes are hypermethylated leading to suppression of gene transcription.¹⁴ Although the presence of these alterations does not guarantee a progression to cancer, it is thought that these molecular markers can identify the adenomas most likely to develop into cancer, in addition to detecting early stages of CRC.⁷

Fecal DNA testing to screen for CRC has evolved over time. Some of the most common (and well studied) DNA markers in stool include mutations in APC, KRAS, and TP53; methylation of vimentin, SFRP2, MGMT, MLH1; and measurements of long-DNA integrity and microsatellite instability.¹⁵ The feasibility of stool DNA testing was originally demonstrated using a single marker assay for KRAS in the early 1990s. Later work in 2000 led to the development of a panel of markers intended to detect both advanced adenomas and colorectal tumors. Since that time several configurations of multi-marker stool tests have been evaluated. Earlier studies focused on the Wnt signaling pathway and microsatellite instability, but these initial studies proved less successful than anticipated. More recently, studies have incorporated the detection of methylated markers, in recognition that gene hypermethylation is a more common pathway in CRC than previously understood.¹⁴ Based on our audit of initial validation studies, the most commonly evaluated methylated markers appear to be vimentin and SFRP2. Other potentially useful methylated gene targets for fecal DNA testing include TFPI2 and NDRG4, which are both tumor suppressor genes.¹⁶ Due to the molecular heterogeneity of CRC, potential screening tests have generally considered a panel of markers rather than a single marker in an attempt to maximize clinical sensitivity.¹⁷ Industry now believes that they have designed panels of markers that cover 100 percent of the target lesions at the tissue level, as compared to only 67 percent of screen-relevant neoplasms when the tissue samples were examined with the first generation of available testing.¹⁸

In addition to improvements in relevant marker identification, there have also been significant technological advances in the past several years to allow for improved detection of molecular abnormalities in DNA.¹⁶ However, only 0.01 percent of DNA in the feces is of human origin; most fecal DNA is acquired through outside sources including diet and microflora. Therefore, isolating and detecting target human DNA from the stool presents a challenge for fecal DNA test methodologies.¹⁹ Assay development has had to focus on improving the analytic sensitivity (or lower limit of detection) of test methodology and technology. Techniques have been developed to better preserve stool DNA (e.g., buffer to stabilize DNA) and extract DNA from stool. In addition, techniques to enrich target DNA by selective capture from stool followed by digital or emulsion PCR have been developed (e.g., BEAMing and digital melt curve analysis) and seem promising in improving assay sensitivity.^16,18

Thus far a single company, Exact Sciences, has been the major developer and licensor of fecal DNA testing in the United States. To date, only two fecal DNA tests have been commercially available. PreGen Plus™ was based on the first developed prototype (by Exact Sciences) and was available from 2003 to 2008 from LabCorp. Most recently, Exact Science has licensed technology to LabCorp, who manufactures and markets ColoSure™, the only commercially available test in the United States (Table 1a and b). The prototype and the previously available test PreGen Plus included the same 23 molecular markers, whereas the newer versions only include methylation of vimentin plus or minus an assay for DNA integrity. Thus, the currently available test ColoSure shares no markers with the previously available test. In addition to this evolution in the composition of the test there have also been advances in pre-analytic and analytic technologies. Pre-analytic factors include specimen collection, processing, handling, and delivery to testing site. Analytic factors include test methods and performance of procedures, and monitoring and verification of accuracy and reliability of test results. The major pre-analytic advance was the addition of DNA stabilization buffer at the time of stool collection, which prevents the degradation of DNA while the stool is in transport and storage. The major analytic advances included the use of technologies to isolate human DNA targets that improve the analytic sensitivity or lower limit of detection of these assays.

Table 1a

Development of fecal DNA testing for colorectal cancer screening: Market availability and test details.

Table 1b

Development of fecal DNA testing for colorectal cancer screening: Evidence.

Only one fecal DNA test for the detection of adenomas and colorectal tumors is currently commercially available. This test, ColoSure is provided by LabCorp as laboratory-developed test (LDT) regulated by the Centers for Medicare and Medicaid Services under the Clinical Laboratory Improvement Amendments (CLIA) of 1988. 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).³⁵ ColoSure has not obtained clearance or approval from the U.S. Food and Drug Administration (FDA). Currently, there are no fecal DNA tests approved by the FDA for screening or diagnosing of CRC. The FDA has chosen to exercise “enforcement discretion” on LDT testing and historically, the FDA's oversight of genetic testing has been focused on commercial test kits. The FDA, however, is now engaged in dialogue with manufacturers and the public on how it should develop a consistent, reasonable, and fair approach for LDTs so as to ensure safety and promote innovation.³⁶

A new fecal DNA test developed by Exact Sciences is projected to be available in 2012 (Table 1a and b). This test will include a combination of markers (methylation of NDRG4, BMP3; 7 point mutations in KRAS exon 2) and an immunochemical assay for hemoglobin.³⁷ The fecal immunochemical test (FIT), while optimized for this new assay, is similar to other currently available ELISA-based FITs.

Changes in markers and assay technology are extremely important to understand when evaluating the evidence base for fecal DNA testing because over time assays have included different markers and used different technologies, limiting the applicability of the existing evidence base to currently (and soon to be) available testing.