Insight into molecular defects in colorectal cancer has not only elucidated the pathogenic role of such lesions but has also allowed the use of new molecular diagnostic tools for early diagnosis, screening, and prevention. Screening effectiveness depends in large part on accessibility and screening tools themselves. Several methods to screen for colorectal cancer in asymptomatic individuals have been available for some time and have proven their efficacy in several controlled trials.62 Screening strategies using stool-based tests have been performed for decades but have had a relatively modest effect in reducing mortality related to colorectal cancer because of low patient compliance and insufficient test sensitivity and specificity.63, 64
DNA-based fecal tests
Recently, focus has been placed on DNA-based stool tests, which promise more accurate alternatives than conventional methods of colorectal cancer screening. It has been shown that DNA shed from tumors is sufficiently stable in stool to be extracted and subjected to amplification by PCR for screening cancer-associated genetic alterations. The first pilot study of this approach attempted to detect fecal k-ras mutations.45 This test is highly feasible and sensitive, because tumor k-ras mutations are shed into the stool and occur at a limited number of mutational “hot spots” in codons 12 and 13. The main limitation is low sensitivity, as k-ras mutations only occur in approximately 50% of colorectal cancers.
Another single gene study looked for APC mutations in fecal DNA.65 Testing for truncating mutations in this gene, the investigators identified APC alterations in 26 of 46 subjects with neoplasia (57%), and in none of the controls.
The same laboratory studied the microsatellite marker BAT-26 in feces of patients with proximal sporadic colorectal cancers, looking for evidence of MSI.66 By using a PCR-based method to detect microsatellite mutations, the researchers selected 18 of 46 cancers that were MSI-H, and identical mutations were observed in stool in 17 of these 18 cases. However, as stated above, genetic alterations in colorectal cancer are highly heterogeneous, and multiple rather than single genes may help achieve a more sensitive assay.
A few studies of tests for multiple genetic mutations in fecal DNA have been published (Table 5). Dong et al. used a panel of three genetic targets—p53, k-ras, and the mononucleotide repeat marker BAT-26—to detect tumor-associated alterations in feces from patients with colorectal cancers.66 The three alterations, which attempt to detect both the suppressor and the mutator pathways, were able to detect 36 of 51 (71%) patients with colorectal cancer in this pilot study.
Table 5. Multitargeted DNA-based Stool Tests for the Detection of Colorectal Cancer
|Ahlquist 200068||22 cancers||k-ras, p53, APC||91% (20/22)||93% (26/28)|
| ||11 adenomas||BAT 26, long-DNA||82% (9/11)|| |
| ||28 controls|| || || |
|Dong 200167||51 cancers||k-ras, p53, BAT26||71% (36/51)||nd|
|Calistri 200369||56 cancers||k-ras, p53, APC,||51% (27/56)||97% (37/38)|
| ||38 controls||5 MSI loci, long DNA||59% (long-DNA, k-ras and p53)|| |
|Imperiale 200470||2507 subjects||k-ras, p53, APC, BAT26||51.6%||nd|
| ||31 cancers||DNA integrity (long DNA)|| || |
|Petko 200572||42 adenomas||methylation of p16||nd||nd|
| ||44 HPs||hMLH1|| || |
| ||25 controls||MGMT|| || |
A study conducted by Ahlquist et al. targeted mutations in k-ras, APC and p53 as well as the MSI marker, BAT-26.67 Hot spot mutations on the k-ras, APC and p53 genes were targeted for detection. In addition, “long DNA,” which is presumably exfoliated by nonapoptotic dysplastic colonocytes into the stool served as a molecular target because of the greater length of these DNA fragments compared with those shed from normal colonocytes. The sensitivity of this study was 91% for colon cancers and 82% for adenomas. Specificity was 93% and increased to 100% when k-ras was excluded.
A study by Calistri et al. sought mutations in p53 exons 5–8, k-ras exons 1–2, four fragments of APC exon 15, and 5 microsatellite loci in feces and tumors of patients with colorectal cancers.68 In addition, long DNA was evaluated by amplifying longer sequences of both p53 and APC. The most frequent alterations in tumors were k-ras (34%), p53 (34%), MSI (13%), and APC mutations (13%). The most frequently detected alterations in stool were long DNA (51%), k-ras mutations (11%), p53 mutations (6%), MSI (6%), and APC mutations (2%). Interestingly, multiple molecular abnormalities in feces of tumor patients were not frequently found. Molecular alterations were not observed in stools of healthy individuals, except in one case with long DNA, demonstrating the specificity of this assay.
DNA-based stool tests have the potential to improve diagnostic yields of conventional colorectal cancer screening methods. Moreover, compared with fecal occult blood tests, DNA-based assays also have the potential to detect asymptomatic adenomas, most of which are not detectable by blood-based testing.67 Imperiale and colleagues compared a high-throughput fecal DNA test to a standard fecal occult blood test.69 The fecal DNA panel consisted of 21 mutations commonly found in colorectal cancer. The fecal DNA test detected 51.6% of 31 invasive cancers, whereas the fecal occult blood test detected only 12.9% of these cases (P = 0.003). The recognition that a large proportion of colorectal cancers progress through the methylator pathway of carcinogenesis suggested yet one more approach to diagnosis. In some proportion of both colon polyps and invasive cancers, promoter methylation of common tumor-suppressor genes can be detected in human DNA extracted from stool.70, 71 However, high specificity not withstanding, the sensitivity of these assays must be improved before they can serve as reliable screening approaches.
In the final analysis, a general application of this approach will require evaluation of the test under clinical conditions, in which stool samples are first collected by patients and sent to the processing laboratories, which may add variables not anticipated in the pilot studies discussed above. A DNA-based stool test is currently commercially available as PreGen-Plus, from Exact Sciences (Marlborough, MA).
Molecular markers and prognosis
Molecular markers have also been used to predict survival and response to chemotherapy. MSI-H cancers have been shown to have a better overall prognosis in terms of disease-free and overall survival when compared with MSS/MSI-L cancers.60 The latter cancers tend to have a worse outcome, particularly when there are alterations on chromosome 18q in Stage III colorectal cancers.43 Similarly, MSI-H cancers have a worse prognosis in the presence of mutations of the target gene TGFβ-RII.
It has been shown in the in vitro setting that MSI-H colorectal cancer cell lines are relatively resistant to 5-FU and other cytotoxic drugs compared with mismatch–repair-proficient cell lines.59, 60 These findings have been confirmed in a retrospective clinical study which showed that 1) untreated MSI-H cancers have a better outcome than MSS cancers, and 2) 5-FU–based chemotherapy does not improve the outcome of MSI-H colorectal cancers, but rather impairs survival, compared with MSS cancers.57, 58 MSI analysis may serve as a predictive factor in the future and may influence selection of treatment strategies. The possibility that a different therapeutic approach may be helpful was suggested in a study investigating the response of MSI-H tumors to irinotecan.72 Patients with MSI-H cancers responded significantly better than patients with MSI-L or MSS tumors, indicating that this treatment appears to be more suitable for colorectal cancers originating from the mutator pathway.
Another approach has been investigated by the Vogelstein laboratory. They studied the role of chromosomal imbalances in 180 colorectal cancer patients with no evidence of lymph-node involvement or distant metastases. DNA from the tumors was tested for imbalances of chromosomes 8p and 18q by digital single-nucleotide polymorphisms (SNP). They found that patients whose tumors retained alleles of both chromosomes had a 100% 5-year survival, compared with 74% of patients whose tumors retained only one allele of 8p or 18q, and 58% of those who had allelic imbalances on both chromosomes, respectively. Most importantly, these findings were independent of tumor stage.73