Mucosa Of Colorectal Cancer Patients Research Articles

Colonization of the gut mucosa of colorectal cancer patients by pathogenic mucosa-associated Escherichia coli strains

Some strains of Escherichia coli are known to be involved in the pathogenesis of colorectal cancer (CRC). The aim of current study was to compare the general characteristics of the E. coli from CRC patients and healthy participants. A total of 96 biopsy samples from 48 CRC patients and 48 healthy participants, were studied. The clonality of the E. coli isolates was analyzed by Enterobacterial repetitive intergenic consensus-based PCR (ERIC-PCR) method. The strains were tested by PCR to determine the prevalence of different virulence factors. According to the results of ERIC-PCR analysis, (from the 860 E. coli isolates) 60 strains from CRC patients and 41 strains from healthy controls were identified. Interestingly, the majority of the strains of both groups were in the same cluster. Enteropathogenic E. coli (EPEC) was detected significantly more often in CRC patients (21.6 %) than in healthy participants (2.4 %) (p < 0.05). The Enteroaggregative E. coli (EAEC) was found in 18.33 % of the strains of CRC patients. However, other pathotypes were not found in the E. coli strains of both groups. Furthermore, all the studied genes encoding for virulence factors seemed to be more prevalent in the strains belonging to CRC patients. Among the virulence genes, the statistical difference regarding the frequency of fuyA, chuA, vat, papC, hlyA and cnf1 genes was found significant (p < 0.05). In conclusion, E. coli strains that carry extraintestinal pathogenic E. coli (ExPEC) and diarrheagenic E. coli (DEC) multiple virulence factors colonize the gut mucosa of CRC patients.

Bovine meat and milk factor protein expression in tumor-free mucosa of colorectal cancer patients coincides with macrophages and might interfere with patient survival.

Bovine milk and meat factors (BMMFs) are plasmid-like DNA molecules isolated from bovine milk and serum, as well as the peritumor of colorectal cancer (CRC) patients. BMMFs have been proposed as zoonotic infectious agents and drivers of indirect carcinogenesis of CRC, inducing chronic tissue inflammation, radical formation and increased levels of DNA damage. Data on expression of BMMFs in large clinical cohorts to test an association with co-markers and clinical parameters were not previously available and were therefore assessed in this study. Tissue sections with paired tumor-adjacent mucosa and tumor tissues of CRC patients [individual cohorts and tissue microarrays (TMAs) (n= 246)], low-/high-grade dysplasia (LGD/HGD) and mucosa of healthy donors were used for immunohistochemical quantification of the expression of BMMF replication protein (Rep) and CD68/CD163 (macrophages) by co-immunofluorescence microscopy and immunohistochemical scoring (TMA). Rep was expressed in the tumor-adjacent mucosa of 99% of CRC patients (TMA), was histologically associated with CD68+/CD163+ macrophages and was increased in CRC patients when compared to healthy controls. Tumor tissues showed only low stromal Rep expression. Rep was expressed in LGD and less in HGD but was strongly expressed in LGD/HGD-adjacent tissues. Albeit not reaching statistical significance, incidence curves for CRC-specific death were increased for higher Rep expression (TMA), with high tumor-adjacent Rep expression being linked to the highest incidence of death. BMMF Rep expression might represent a marker and early risk factor for CRC. The correlation between Rep and CD68 expression supports a previous hypothesis that BMMF-specific inflammatory regulations, including macrophages, are involved in the pathogenesis of CRC.

Gilbert's Syndrome and the Gut Microbiota - Insights From the Case-Control BILIHEALTH Study.

The heme catabolite bilirubin has anti-inflammatory, anti-oxidative and anti-mutagenic effects and its relation to colorectal cancer (CRC) risk is currently under evaluation. Although the main metabolic steps of bilirubin metabolism, including the formation of stercobilin and urobilin, take place in the human gastrointestinal tract, potential interactions with the human gut microbiota are unexplored. This study investigated, whether gut microbiota composition is altered in Gilbert’s Syndrome (GS), a mild form of chronically elevated serum unconjugated bilirubin (UCB) compared to matched controls. Potential differences in the incidence of CRC-associated bacterial species in GS were also assessed. To this end, a secondary investigation of the BILIHEALTH study was performed, assessing 45 adults with elevated UCB levels (GS) against 45 age- and sex-matched controls (C). Fecal microbiota analysis was performed using 16S rRNA gene sequencing. No association between mildly increased UCB and the composition of the gut microbiota in this healthy cohort was found. The alpha and beta diversity did not differ between C and GS and both groups showed a typical representation of the known dominant phyla. Furthermore, no difference in abundance of Firmicutes and Proteobacteria, which have been associated with the mucosa of CRC patients were observed between the groups. A sequence related to the Christensenella minuta strain YIT 12065 was identified with a weak association value of 0.521 as an indicator species in the GS group. This strain has been previously associated with a lower body mass index, which is typical for the GS phenotype. Overall, sex was the only driver for an identifiable difference in the study groups, as demonstrated by a greater bacterial diversity in women. After adjusting for confounding factors and multiple testing, we can conclude that the GS phenotype does not affect the composition of the human gut microbiota in this generally healthy study group.

Intracellular Porphyromonas gingivalis Promotes the Proliferation of Colorectal Cancer Cells via the MAPK/ERK Signaling Pathway.

Porphyromonas gingivalis (P. gingivalis) is a keystone pathogen in periodontitis. However, several clinical studies have revealed an enrichment of P. gingivalis in the stool samples and colorectal mucosa of colorectal cancer patients. Thus, the goal of this study was to determine whether P. gingivalis can promote colorectal cancer progression in vitro. We established an acute infection model (24 h, multiplicity of infection =100) of P. gingivalis invasion of colorectal cancer cells to study the alterations induced by P. gingivalis in the proliferation and cell cycle of colorectal cancer cells. We observed that P. gingivalis can adhere and invade host cells a few hours after infection. Once invaded, P. gingivalis significantly promoted colorectal cancer cell proliferation, and the percentage of S phase cells was increased in the cell cycle assay. However, KDP136, a gingipain-deficient mutant of P. gingivalis 33277, showed a decreased ability to promote colorectal cancer cell proliferation, indicating that gingipain is associated with colorectal cancer cell proliferation. Furthermore, we extracted RNA from colorectal cancer cells for high-throughput sequencing analysis and reconfirmed the results by quantitative polymerase chain reaction and western blot analyses. The results suggested that the MAPK/ERK signaling pathway is significantly activated by P. gingivalis, while these changes were not observed for KDP136. In conclusion, P. gingivalis can invade cells and promote the proliferation of colorectal cancer cells by activating the MAPK/ERK signaling pathway. Gingipain is an essential virulence factor in this interaction.

Abstract 1470: Relative telomere length in tumor tissue and adjacent mucosa of colorectal cancer patients

Abstract Colorectal cancer (CRC) represents a major public health problem, being the fourth leading cause of cancer related deaths mainly due to the late diagnosis and a lack of proper prognostic markers. According to the current knowledge, shortening of telomeres is important contributor in cancer onset. Telomeres, repetitive TTAGGG sequences and boud proteins, are structures at the end of all linear eukaryotic chromosomes. Telomeres are double-edged sword regarding to cancer. On one hand, they serve as a potent tumor-supressor mechanism limiting the cells ability of uncontroled growth. However, on the other hand, shortening of telomeres can lead to the formation of anaphase bridges, initiation of breakage-fusion-bridges cycles and eventually to the induction of chromosome instability phenotype. Telomeric regions are composed of microsatellite repeats and DNA mismatch repair (MMR) defects should directly affect telomere length. Studies looking into the relationship between microsatellite instability (MSI) and telomere length have been sparse. We hypothesize that CRC patients with MSI should have better prognosis, longer survival, also due to the MMR defects in telomere regions replication failure. The aim of our present study was to investigate relative telomere length (RTL) in tumor tissue and adjacent mucosa of 650 CRC patients; it is the largest study on RTL in target tissue so far. Moreover, we measured RTL in peripheral blood lymphocytes obtained from 150 cancer patients. Tumor and adjacent non-affected tissue samples were obtained from patients at surgical resection. RTL was measured using monochrome multiplex quantitative PCR method. Despite the analysis is still running, our preliminary results showed significantly shorter RTL in tumor tissues as compared to adjacent mucosa. The shortest RTL was discovered in right, ascendent part of colon (0.68±0.59, 25.5% of cases), followed by left, descendent colon (0.77±0.47, 37.6%) and the longest RTL was in rectal tumors (0.92±0.69, 36.9%). We recorded gradually increasing RTL ratio (RTL in tumor tissue/ RTL in adjacent mucosa) in later stages according to TNM staging system. Interestingly, patients with RTL ratio &amp;gt; 1 had also worse survival. RTL of CRC tissue and adjacent mucosa may provide a valuable prognostic tool in patients treatment management. This study was supported by GACR 15-14789S and by the National Sustainability Program I (NPU I) Nr. LO1503 provided by the Ministry of Education Youth and Sports of the Czech Republic Citation Format: Michal Kroupa, Vaclav Liska, Krishna Rachakonda, Marketa Urbanova, Michaela Schneiderova, Katerina Jiraskova, Ondrej Vycital, Ludmila Vodickova, Rajiv Kumar, Pavel Vodicka. Relative telomere length in tumor tissue and adjacent mucosa of colorectal cancer patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1470.

The Bacteroides fragilis toxin gene is prevalent in the colon mucosa of colorectal cancer patients.

Enterotoxigenic Bacteroides fragilis (ETBF) produces the Bacteroides fragilis toxin, which has been associated with acute diarrheal disease, inflammatory bowel disease, and colorectal cancer (CRC). ETBF induces colon carcinogenesis in experimental models. Previous human studies have demonstrated frequent asymptomatic fecal colonization with ETBF, but no study has investigated mucosal colonization that is expected to impact colon carcinogenesis. We compared the presence of the bft gene in mucosal samples from colorectal neoplasia patients (cases, n = 49) to a control group undergoing outpatient colonoscopy for CRC screening or diagnostic workup (controls, n = 49). Single bacterial colonies isolated anaerobically from mucosal colon tissue were tested for the bft gene with touch-down polymerase chain reaction. The mucosa of cases was significantly more often bft-positive on left (85.7%) and right (91.7%) tumor and/or paired normal tissues compared with left and right control biopsies (53.1%; P = .033 and 55.5%; P = .04, respectively). Detection of bft was concordant in most paired mucosal samples from individual cases or controls (75% cases; 67% controls). There was a trend toward increased bft positivity in mucosa from late- vs early-stage CRC patients (100% vs 72.7%, respectively; P = .093). In contrast to ETBF diarrheal disease where bft-1 detection dominates, bft-2 was the most frequent toxin isotype identified in both cases and controls, whereas multiple bft isotypes were detected more frequently in cases (P ≤ .02). The bft gene is associated with colorectal neoplasia, especially in late-stage CRC. Our results suggest that mucosal bft exposure is common and may be a risk factor for developing CRC.

Folate levels measured by LC-MS/MS in patients with colorectal cancer treated with different leucovorin dosages.

PurposeCalcium folinate (leucovorin), which is converted in vivo into biologically active folate, enhances the potency of 5-fluorouracil (5-FU)-based chemotherapy in colorectal cancer. A common dosage of leucovorin in adjuvant and palliative settings is 60 mg/m2. The aim was to determine the levels of tetrahydrofolate (THF), 5,10-methylenetetrahydrofolate (methyleneTHF), and 5-methyltetrahydrofolate (methylTHF) in tumour and mucosa of colorectal cancer patients who received different dosages of leucovorin intravenously at time of surgery.MethodsEighty patients scheduled for colorectal resection with indication of colorectal cancer were randomised into four groups to receive leucovorin at 0, 60, 200, or 500 mg/m2, respectively. Blood samples were taken 10 and 30 min after leucovorin administration. Biopsy samples from tumour and mucosa were collected and snap-frozen at surgery. The levels of THF, methyleneTHF, and methylTHF in tumour and mucosa were assessed by liquid chromatography electrospray ionisation tandem mass spectrometry (LC–MS/MS) and the results were related to clinical diagnosis and therapeutic regimens.ResultsThe folate levels in tissue revealed extensive inter-individual variability. The mean methyleneTHF value for the four treatment groups were 880, 1,769, 3,024 and 3,723 pmol/gww. Only half of the patients who received 60 mg/m2 leucovorin had higher levels of methyleneTHF in tumour than patients who received 0 mg/m2 leucovorin. Rectal cancer patients had significantly lower levels of methyleneTHF compared with colon cancer patients.ConclusionsThere was a large inter-patient variability of tissue folate levels in colorectal cancer patients after supplementation with leucovorin at standardised dosage. High leucovorin doses were needed to exceed baseline methyleneTHF values, especially in rectal cancer patients. The results indicate that the standardised leucovorin dose may be insufficient to attain the full antitumour effect of 5-FU. Further studies are needed to establish whether higher dosage yields a better treatment response.

Determination of reduced folates in tumor and adjacent mucosa of colorectal cancer patients using LC‐MS/MS

A liquid chromatography electrospray ionization tandem mass spectrometry (LC-MS/MS) method has been developed for the determination of 5,10-methylenetetrahydrofolate (methyleneTHF), tetrahydrofolate (THF) and 5-methyltetrahydrofolate (methylTHF) in colorectal mucosa and tumor tissues. The folate extraction method includes homogenization, heat and folate conjugase treatment to hydrolyze polyglutamyl folate to monoglutamyl folate. Before analysis on LC-MS/MS, simple and fast sample purification with ultrafiltration (molecular weight cut-off membrane, 10 kDa) was performed. Folates were detected and quantified using positive electrospray. The method described in the present paper was successfully applied to determine the level of three folate monoglutamates in mucosa and tumor samples from 77 colorectal cancer patients, starting from a limited amount of tissue. The results showed that the LC-MS/MS method has a great advantage over other previously used methods because of its high sensitivity and selectivity. Significantly higher levels of methyleneTHF and THF were found in tumor compared with matched mucosa tissues. Folate levels in adjacent mucosa were associated with tumor location, age and gender. The correlation between folate levels and tumor site further strengthens the fact that development of right- and left-sided tumors follows different pathways.

Abstract 91: Analysis of global and repeat sequence DNA methylation in the blood and normal colonic mucosa of colorectal cancer patients, healthy individuals and patients with folate deficiency

Abstract Aberrant DNA methylation is an early event in colorectal tumorigenesis and in colorectal cancer (CRC) patients these changes are detected in their normal colonic mucosa. DNA methylation changes may arise from aging or environmental exposures e.g. folate deficiency. We aimed to compare global and repeat-sequence DNA methylation in the colonic mucosa and blood of three groups of individuals – CRC patients, healthy people and patients with folate deficiency. We conducted a multisite cohort study collecting mucosa and blood from 176 patients who had undergone CRC resection and 194 healthy individuals at screening colonoscopy. Blood was also collected from 30 individuals with megaloblastic anaemia secondary to folate deficiency (red cell folate &amp;lt;539nM). Global DNA methylation was measured using liquid chromatography tandem mass spectrometry adapted from Quinlivan &amp; Gregory (2008)1. Alu repeat methylation was determined using a novel, ultra-sensitive real-time PCR assay based upon methylation-sensitive restriction enzyme digestion coupled with a fluorescence-based readout2. Genotyping for a common polymorphism in the folate metabolism enzyme MTHFR (C677T) was performed using pyrosequencing. The mean age of CRC patients (68 ± 14 years) was significantly higher than healthy subjects (55 ± 14 years; p&amp;lt;0.01) but not significantly different from low-folate subjects (72 ± 22 years). The gender distribution was similar in all 3 groups. The prevalence of the variant T allele of MTHFR in CRC subjects (58%) and healthy controls (64%) were comparable to population-based estimates. However, the T allele was over-represented in low folate patients (79%). In the analysis of the first subset of CRC (n=38) and healthy controls (n=38), mean global DNA methylation was significantly lower in the colonic mucosa (CRC n=20: 4.1% ± 0.3; controls n=36: 4.0% ± 0.3) compared to peripheral blood (CRC n=17: 4.4% ± 0.4; controls n=36: 4.4% ± 0.3, p&amp;lt;0.01). No differences in DNA methylation were found in CRC patients compared to healthy subjects. Consistent with global methylation findings, Alu repeats were 2-fold less methylated in colon compared to peripheral blood in healthy (n=38) and CRC cohorts(n=36). Low-folate subjects had a trend towards lower blood global (4.26% ± 0.2) and Alu methylation (1.2-fold hypomethylated) compared to healthy subjects. To date, our results suggest that Alu repeat methylation reflect global DNA methylation and that this holds true across tissue types irrespective of cancer status. Global and Alu repeat demethylation in individuals with folate deficiency and overrepresentation of the T MTHFR allele suggests a possible link between diet and the epigenome that warrants further study. 1. Quinlivan, E.P. &amp; Gregory, J.F., 3rd. (2008) Nucleic acids research 36, e119. 2. Rand, K. &amp; Molloy, P. (2010) BioTechniques 49, xiii-xvii. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 91. doi:10.1158/1538-7445.AM2011-91

Abstract 60: Constitutional MGMT promoter methylation and allelic expression imbalance are associated with the c.-56C&amp;gt;T (rs16906252) variant

Abstract Loss of expression of the DNA repair protein MGMT in cancer is associated with a mutator phenotype and frequently occurs due to promoter methylation. The c.-56C&amp;gt;T SNP (rs16906252), located within an enhancer element in the 5’UTR of MGMT, is strongly associated with promoter methylation in colorectal cancer (CRC) and at lower levels in normal tissues, including normal colorectal mucosa and peripheral blood. We aimed to investigate the mechanistic link between the c.-56C&amp;gt;T SNP and MGMT methylation in CRC development with a focus on the differential methylation and expression of the two alleles in normal tissues from CRC patients and healthy controls. Genotyping revealed the T allele frequency to be similar in CRC cases (7%) and controls (8%). In the peripheral blood DNA of CRC cases, MGMT methylation was detected at a higher frequency in heterozygous cases (66.6%) compared to homozygous C/C cases (17.1%)(p&amp;lt;0.0001), as well as at higher levels (p&amp;lt;0.0001). Similarly in the blood of healthy controls, MGMT methylation was detected at a higher frequency in heterozygous individuals (43.5%) compared to C/C homozygotes (14.9%)(p&amp;lt;0.002), as well as at higher levels (p&amp;lt;0.0001). Furthermore, allelic sequencing of products from methylation-specific PCR demonstrated that MGMT methylation was linked exclusively to the T allele in 6/6 blood and 15/16 normal colorectal mucosa samples from CRC cases. The relative levels of allelic expression derived from the C and T alleles at the c.-56C&amp;gt;T variant were examined in the normal colorectal mucosa from 14 patients (8 C/T, 6 C/C) informative for a second downstream expressible SNP (rs1803965) by allele-quantification pyrosequencing. Allelic expression imbalance was observed in all eight heterozygotes, with allelic expression ratios (C/T) ranging from 1.79-4.51. In contrast, the six C/C cases showed balanced expression levels (range 1.08-1.54), indicating that reduced transcription was significantly associated with the T allele (p&amp;lt;0.001). Sequencing of cloned cDNA products confirmed the allele expressed at lower levels at the downstream SNP was linked exclusively to the T allele at the c.-56C&amp;gt;T SNP. Finally, we found that in six highly methylated (&amp;gt;30%) CRC specimens that retained MGMT immunoexpression, methylation was predominant on the T allele. Our findings indicate the c.-56T allele is associated with constitutively reduced transcriptional activity in the normal colorectal mucosa of CRC patients. This mechanism may predispose to secondary methylation of the T allele, most likely via altered interaction with cis-acting transcription factors. Monoallelic methylation of MGMT may explain the discordance between presence of MGMT methylation and retention of protein expression in tumors. If commonplace, these findings suggest that MGMT methylation status serves as a poor predictor of patient responsiveness to alkylating drugs. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 60. doi:10.1158/1538-7445.AM2011-60

P16INK4a Gene Promoter Hypermethylation in Mucosa as a Prognostic Factor for Patients with Colorectal Cancer

Low gene expression of folylpolyglutamate synthase (FPGS) in colorectal mucosa correlates with low folate levels and poor survival of colorectal cancer (CRC) patients. Because gene-specific hypermethylation is affected by the folate level, the hypermethylation status in mucosa may also be linked to clinical outcome of CRC patients. The tumor suppressor gene p16INK4a (p16) regulates the cell cycle and angiogenic switch. In human neoplastic tissues, the main mechanism of p16 inactivation is promoter methylation. The aim of the study was to determine whether hypermethylation of the p16 promoter could be detected in mucosa of CRC patients (n = 181) and to analyze if hypermethylation was related to survival. The relation between p16 hypermethylation and expression of FPGS and two other folate-associated genes, reduced folate carrier 1 (RFC-1), and thymidylate synthase (TS), was analyzed (n = 63). The results showed that p16 was hypermethylated in 65 (36%) of the mucosa samples and that hypermethylation was age-related (P = 0.029). After adjustment for known risk factors, Cox regression analysis showed that Dukes' A-C patients with p16 hypermethylation in mucosa had an increased risk of cancer-related death (hazard ratio = 2.9, P = 0.007) and shorter disease-free survival (hazard ratio = 2.5, P = 0.015) compared with patients with no p16 hypermethylation. RFC-1 and FPGS gene expression levels were significantly correlated in patients lacking p16 hypermethylation in mucosa (P = 0.0003), but not at all correlated in patients having hypermethylation in mucosa (P = 1.0). In conclusion, p16 hypermethylation in mucosa of CRC patients was identified as an independent prognostic parameter for cancer-specific survival as well as an independent predictor of DFS. The results suggest that there might be a connection between folate-associated gene expression and p16 methylation status.

Sampling technique influences the detection of K-ras mutations in normal appearing mucosa of colorectal cancer patients.

Based on three colorectal cancer cell lines with specified K-ras status, a sensitive PCR-RFLP assay was established detecting one K-ras mutant among 106 wild-type cells. Using this assay for tissues of 124 colorectal cancer patients, 59 tumor (46%) and 11 mucosa samples (9%) were found to harbor a K-ras mutation. When using the same scalpel for collecting tumor and mucosa tissues (group A), 18% of the patients had a matching K-ras mutation in both tissues, but this coincidence was seen in 3% of patients only, when separate scalpels were used (group B). Thus we conclude that the sampling technique used for collecting specimens is a major contributor to the detection of K-ras mutations in normal appearing mucosa when a highly sensitive detection technique is used.

Analysis of mutant K-ras in multiple sites of normal appearing mucosa of colorectal cancer patients

While 50% of colorectal tumors were found to harbor K-ms codon 12 mutation, only 18% of the respective patients contain this mutation in the normal appearing tissue when enriched PCR, a sensitive method that enables detection of one mutant allele in 10(4) normal alleles, is used. To determine whether the lower percentage could be attributed to the low incidence of this mutation or to the method of sampling, we have analyzed multiple normal appearing samples obtained from the same patient. Of 90 non-neoplastic mucosal samples collected from 20 patients with colorectal cancer, K-ras codon 12 mutation was identified in 6 samples taken from 5 patients. These results indicate that only one or two of the multiple samples contain mutant ras alleles. The presence of mutant ras alleles in the normal appearing tissues did not always correlate with that in the tumor site with respect to its presence and/or the type of base pair alterations, indicating independent or late events. While pointing to the importance of proper sampling method, the ability to detect ms mutation in normal mucosal tissues suggests it may serve as a useful biomarker of internal/external exposure which precede colorectal cancer development.

Mutant K-ras in apparently normal mucosa of colorectal cancer patients. Its potential as a biomarker of colorectal tumorigenesis.

The best way to reduce the incidence of colorectal cancer mortality would be to prevent this cancer. However, none of the biomarkers proposed can accurately identify persons at increased risk of colorectal cancer or those at low risk. As a possible genetic biomarker, K-ras mutations, which are frequently found in colorectal cancers, were analyzed in apparently normal colorectal mucosa. Nonneoplastic mucosa and tumor tissues were collected at surgery from 70 patients with colorectal cancer: one sample each from 50 patients (group A) and multiple samples from the other 20 patients (group B). Mutant K-ras codon 12 was analyzed by the enriched polymerase chain reaction (EPCR), by which one mutant can be detected among 10(3) to 10(4) normal alleles. Only with the aid of EPCR was mutant K-ras detected in nonneoplastic mucosa of nine patients (18%) in Group A and five patients (25%) in Group B. This increased incidence could be attributed to the multiple tissue sampling. The presence of mutant K-ras in nonneoplastic mucosae was not consistently correlated with that in the tumors. These findings suggest that the mutant K-ras identified in nonneoplastic mucosa actually represents de novo mutations, which may be initiated by different etiologic factors and at different times. Mutant K-ras detected in apparently normal mucosa should be a useful biomarker for identifying persons at higher risk of colorectal cancer. Our study also emphasizes the need for improving the method for sample collection to achieve true representation of the colorectal mucosa.