Stool DNA-Based Colorectal Cancer Detection: Finding the Needle in the Haystack
Authors: W. Atkin, J. P. Martin
Journal: JNCI Journal of the National Cancer Institute (2001)

For many centuries, it has been suspected that examination of stool could provide insights into the state of health or disease of an individual. The Egyptians were concerned with the shape and consistency of stool (Eber’s papyrus, 207), and 19 century Americans were concerned with “poisons and gases” (1); more recently, the components of stool (e.g., pathogens, occult blood, fats, and bile acids) have been the subject of scrutiny. Within the past two decades, with the advent of the polymerase chain reaction (PCR) technique, came the ability to amplify tiny numbers of specific DNA sequences. This advance has permitted the identification of not only mutations that accumulate in neoplasms and define their progression to malignancy but also mutations in cells shed from the tumors into stool. Compared with tissue and blood, extraction of DNA from stool presents special problems because human DNA is often degraded and food digestion products and bacterial contaminants inhibit the PCR. As reported in this issue of the Journal, Dong et al. (2) achieved their goal of extracting sufficient high-quality DNA from all of 51 stool samples collected from patients with a diagnosis of colorectal cancer to permit detection of most mutations where they existed in the tumor. This meticulous study focused on mutations in the K-RAS oncogene and the TP53 tumor suppressor gene (each of which is mutated in around 40%–60% of colorectal cancers) as well as the microsatellite instability (MSI) marker BAT26 (which is altered in 15% of sporadic cancers). Dong et al. were able to detect TP53 and BAT26 mutations in stools from all of the patients with these mutations in their tumor; in each case, the mutation in the stool was identical to that in the tumor, confirming its origin. It was possible to identify K-RAS mutations in only eight of 17 stool samples from patients with a K-RAS mutation in their tumor. The three markers together detected 36 (71%) of the 51 cases of colorectal cancer, but TP53 alone detected 30 of these cases. How do these results compare with those from previous studies, and what does this study tell us about the suitability of these markers for colorectal cancer screening? A consistent finding is that the quantity and quality of the DNA extracted from stools are superior in the presence of colorectal neoplasia (3), probably because there is less efficient degradation by apoptosis of cells shed by tumors compared with fully differentiated cells. Indeed, the amount of extracted DNA per gram of stool has been used as a marker of the presence of malignancy (4). Only one study (5) has reported successful DNA extraction from 100% of stool samples from people with endoscopically normal colons. Providing an informative negative result is an essential component of screening and is possible only if DNA can be extracted from all stool samples. When Sidransky et al. (6) first showed that the mutations in cells shed from K-RAS-mutated colorectal cancers are detectable in stool, K-RAS appeared to be an ideal candidate for colorectal cancer screening because most mutations are clustered at codons 12 and 13. However, from the outset, K-RAS has been problematic as a marker. Few studies (6–9) have shown complete sensitivity of K-RAS mutations for colorectal cancer; moreover, K-RAS mutations have been found in up to 14% of stool samples from people with normal colons (3,5,10), a falsepositive rate that would be unacceptable in a screening situation. It is not clear if the finding of K-RAS mutations in stools in the absence of neoplasia has any prognostic significance, although in one study (11) K-RAS mutations were detected in colonic effluent samples from two of five patients with previously resected carcinoma and from one patient 4 years before colorectal cancer was diagnosed. Most of the mutations in the TP53 gene found in colorectal tumors can be detected by analysis of exons 5–8. These exons were analyzed by Dong et al. (2), who found that all patients with mutations in their tumors at these hotspots had identical mutations in their stool samples. BAT26 is a highly sensitive marker of MSI (12), which is a feature of 30%–40% of cancers that develop in the colon proximal to the splenic flexure (13,14). MSI is rarely found in sporadic distal colon cancer. In the only other study to have examined BAT26 in stool (5), the marker was positive in five of 10 proximal cancers and in none of 12 distal cancers. In the study by Dong et al. (2), three (23%) of 13 right-sided tumors and their corresponding stools harbored BAT26 mutations. However two (4%) of the 51 patients in the study had a germline mutation in BAT26, suggesting that this marker may lack specificity. Inherited polymorphisms in BAT26 and BAT25, an alternative marker of MSI, have been found in 8%–12% of African-Americans, although only 3% showed variation in both markers (15,16). These polymorphisms were found in only 0.1% of Caucasians (15,16), but it is important to know the extent to which they affect other populations because those with substantial rates would require additional markers to confirm that the instability is confined to the tumor. Dong et al. (2) have shown that a small panel of genetic markers might potentially detect 70% of colorectal cancers. They advise caution, however, in interpreting this finding as evidence to support the application of molecular screening for the early detection of colorectal cancer because most of the cancers were advanced and the patients were symptomatic. Ideally, colorectal screening should aim to detect the disease in the