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Technical Briefs |
1 Division of Clinical Epidemiology and Aging Research, German Cancer Research Center, Heidelberg, Germany; 2 AJ Innuscreen GmbH, Berlin, Germany; 3 Invitek GmbH, Berlin, Germany; 4 Saarland Cancer Registry, Saarbrücken, Germany;
aaddress correspondence to this author at: German Cancer Research Center (DKFZ), Division of Clinical Epidemiology and Aging Research, Bergheimer Strasse 20, 69115 Heidelberg, Germany; fax 49-(0)6221-54-8142, e-mail u.haug{at}dkfz.de
Abstract
Background: Testing for mutant K-ras in stool has been proposed for the detection of pancreatic and colorectal cancer (CRC). Different analytical techniques have been developed, but studies of this biomarker in the general population are lacking. We investigated the prevalence and potential determinants of mutant K-ras in stool in a large sample of unselected older adults and assessed the association with colonoscopic findings.
Methods: In stool samples from 875 older adults (age range 50–75 years) participating in a large-scale population-based cohort study, we used mutant-enriched PCR and allele-specific hybridization reaction to analyze mutations in codons 12 and 13 of the K-ras gene. We assessed the association between mutant K-ras in stool and risk factors for gastrointestinal cancer sites, exocrine pancreatic insufficiency determined by fecal pancreas elastase 1, and colonoscopic findings.
Results: The overall prevalence of mutant K-ras in stool was 8% (95% confidence interval 6%–10%). There was a tentative association between increased fecal pancreas elastase 1 and mutant K-ras in stool (P = 0.09). Patients with advanced colorectal neoplasia diagnosed within 2 years after stool collection (24 with advanced adenomas, 7 with CRC) all tested negative.
Conclusion: The proposed assay identifies mutant K-ras in stool at a higher prevalence than has been reported for other analytical techniques. Our findings do not support the use of this assay for CRC screening, but its potential use for early detection of pancreatic cancer (in combination with other markers) requires further investigation.
Given that somatic mutations of the K-ras gene are observed in 
40% of colorectal cancers (CRCs) (1)(2)(3) and in >80% of pancreatic cancers (4), mutant K-ras in stool specimens has been proposed as a potential component of marker combinations aimed at the early detection of these cancers (5)(6)(7)(8)(9)(10). Conclusions drawn from existing studies must be viewed cautiously, however, because most of the study populations were highly selective and rather small (5)(6)(7)(8)(9)(10), and different analytical techniques were used and may have led to different detection rates (5). For example, assays based on the amplification of DNA by mutant-enriched PCR are supposed to result in higher rates of K-ras-positive samples than other assays (11). Although higher detection rates may optimize sensitivity, another important question is whether specificity may be affected, i.e., whether diminutive amounts of mutant K-ras in stool can also be detected in average-risk people or, preferentially, in people with certain risk factors (e.g., as proposed for smokers) (12). To clarify these questions, which are highly relevant in regard to potential population-wide testing, large-scale investigations in the screening setting are needed.
We investigated prevalence and potential determinants of mutant K-ras in stool (analyzed by mutant-enriched PCR and allele-specific hybridization reaction) in a large sample of unselected older adults and assessed the association with colonoscopic findings.
The study was carried out in the context of the ESTHER study (epidemiological investigations of the chances of preventing, recognizing early, and optimally treating chronic diseases in an elderly population), a large-scale population-based cohort study. Details of ESTHER, which has been approved by local and regional ethics committees, have been reported elsewhere (13). Briefly, 9953 inhabitants of southwest Germany ages 50 to 75 years were recruited by their general practitioner in the context of a general health examination. After informed consent was obtained, all participants were asked to mail a stool sample to the study center and to fill out a standardized questionnaire. In addition, medical data were recorded from the patients’ charts. As part of the 2-year follow-up (response rate 96%), information was collected on colonoscopies performed since baseline recruitment.
Overall, stool samples from 894 ESTHER study participants were analyzed for mutant K-ras. Participants were divided into 2 subgroups. Group A consisted of persons who underwent colonoscopy between baseline and 2-year follow-up and for whom the colonoscopic findings were available. Group B was quasi-randomly selected, with the only selection criterion being a high amount of initially collected stool, which was assumed to be a random variable. This selection criterion was chosen to allow additional measurement of fecal pancreas elastase 1 and subsequent comparison with the K-ras status.
On arrival at the laboratory, stool samples were stored at –70 °C. After baseline recruitment was finished, DNA from 200 mg stool was extracted using the Invisorb® Spin Stool DNA Kit (Invitek). To analyze codons 12 and 13 of the K-ras gene, 5 µL of the DNA extraction product (stool DNA dissolved in elution buffer), derived from 5 mg of initially collected stool, was used for the analyses. Stool DNA was amplified by mutant-enriched PCR, and an allele-specific hybridization reaction assay was carried out according to a published protocol (8)(14). If enough DNA material was left from the stool sample initially designated for K-ras analysis, positive test results were confirmed by gene sequencing.
Fecal pancreas elastase 1 was measured in nonprocessed stool samples with a commercially available ELISA (ScheBo® Biotech AG). The results were categorized according to an established cutoff for exocrine pancreatic insufficiency of <200 µg elastase 1/g stool (13).
All laboratory analyses were done in a blinded fashion.
Overall, 875 participants of the ESTHER study were included in the statistical analyses (in 19 participants stool DNA was not amplifiable). Characteristics of the study population are shown in Table 1A
. Group A (n = 535) and B (n = 340) did not differ from each other or from the whole study population of the ESTHER study, except that more participants of group A reported a 1st-degree relative with CRC.
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Mutations in the K-ras gene were identified in 70 of 875 stool samples, i.e., the overall prevalence was 8% (95% confidence interval, 6%–10%). In group A and B, the prevalence was 7% and 10%, respectively. Type and frequency of identified K-ras gene mutations are detailed in Table 1B
. For 70% of samples that tested positive for mutant K-ras, enough DNA material was left for gene sequencing, which always confirmed the result of the hybridization assay.
Regarding potential determinants, there was a tentative, albeit not statistically significant (P = 0.09), association with exocrine pancreatic insufficiency (Table 1A
).
In group A, 441 participants underwent complete colonoscopy, reaching the cecum. Advanced colorectal neoplasia was present in 31 participants (including 24 patients with advanced adenomas, i.e., adenomas that were at least 1 cm in diameter, adenomas with villous components, or high-grade dysplasia, and 7 patients with invasive CRC). None of these participants tested positive for mutant K-ras in stool. The same applies to the 25 participants bearing hyperplastic polyps. Among participants with nonadvanced adenomas (n = 50) and with unspecified polyps (i.e., without histological examination, n = 35), K-ras mutations were found in 1 and 3 patients, respectively. The highest rate of mutant K-ras (7.5%, 22/293) was detected in participants showing a negative result at colonoscopy.
Testing a large sample of unselected older adults for mutant K-ras in stool with the described assay yielded a prevalence of this marker of 8% in the whole sample as well as among individuals with negative colonoscopy results. Gene sequencing (performed for 70% of positive results) always confirmed the result of the hybridization assay.
With the use of this assay the observed prevalence of mutant K-ras in stool was much higher than that observed with alternative analytical techniques. Covering the same potential mutations but using another assay, the largest study in the field, which was aimed at the evaluation of fecal DNA testing for the detection of colorectal neoplasms, reported a prevalence of only 1.5% (22/1423) among persons with negative colonoscopy results (15). Given that the latter study applied somewhat stricter inclusion criteria compared with the ESTHER study, potential selection bias should also be considered when comparing the findings between both studies. However, additional analyses applying comparably strict inclusion criteria to ESTHER study participants (data not shown) indicated that selection bias is unlikely to explain the observed large differences in prevalence of mutant K-ras in stool. As already suggested by other authors (11), an alternative explanation may be that assays based on mutant-enriched PCR result in a higher rate of K-ras-positive samples. This suggestion is further supported by a study by Kopreski et al., in which 27% of 105 individuals with negative colonoscopy results had positive results for mutant K-ras in plasma samples tested with an assay based on mutant-enriched PCR (16).
To estimate the potential of the proposed assay for screening, an important question is whether it enables identification of individuals who currently bear colorectal or pancreatic neoplasms or who are at increased risk of developing neoplasms at these organs in the future. The lack of associations between important risk factors for the respective cancer sites and mutant K-ras in stool provides more rebutting than supporting evidence regarding the value of the proposed assay to identify high-risk individuals. In regard to colorectal disease, none of the individuals diagnosed with advanced adenomas or CRC within 2 years after stool collection tested positive. In the most important study for comparison, 16% of CRC cases (95% confidence interval, 5%–34%) and 4% of advanced adenoma cases (95% confidence interval, 3%–7%) tested positive for mutant K-ras in stool at the time of diagnosis (15). According to these figures, 2 positive findings would have been expected among the 31 cases with advanced adenomas/CRC in our study. Although the analytical sensitivity of the assay used in our study (i.e., the probability of detecting mutant K-ras in stool if present) could be improved through stool quantity and storage conditions, these measures are expected to go along with a decrease in diagnostic specificity (i.e., the probability that healthy people test negative). Given that for CRC, K-ras has been proposed for parallel testing in combination with further markers, the validity of such a multiple marker panel is highly vulnerable to decreased specificity of any component.
Mutant K-ras in stool may also play a role for the detection of pancreatic cancer. Sequence testing in combination with additional markers has been suggested to distinguish patients with pancreatic cancer from patients with benign pancreatic disease (8)(17). In this context, the high detection rate of the proposed assay may be a minor problem or even be advantageous provided that the right specific combination of markers is found. Whether the combination of fecal pancreas elastase 1 as a marker of exocrine pancreatic insufficiency and mutant K-ras, which are both independent predictive factors for pancreatic cancer development in patients with chronic pancreatitis (17), may be useful remains to be clarified by further studies.
In conclusion, our study results do not support the use of the proposed assay for CRC screening. Its potential for early detection of pancreatic cancer (in combination with other markers) requires further investigation.
Acknowledgments
We greatly appreciate the laboratory work done by Wiebke Hiesener.
References
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