Colorectal cancer is the second most common cause of

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1 GASTROENTEROLOGY 2001;121: Ki-ras Proto-oncogene Mutations in Sporadic Colorectal Adenomas: Relationship to Histologic and Clinical Characteristics TERESE MALTZMAN,*, KIRSTEN KNOLL,* MARIA ELENA MARTINEZ,, TIM BYERS, BETH R. STEVENS, # JAMES R. MARSHALL,, MARY E. REID,, JANINE EINSPAHR, NANCY HART, ACHYUT K. BHATTACHARYYA, CHERYL B. KRAMER, RICHARD SAMPLINER,, **, DAVID S. ALBERTS,, and DENNIS J. AHNEN*, *Department of Veterans Affairs Medical Center, Denver; Departments of Medicine and Preventive Medicine and Biometrics, University of Colorado School of Medicine, Denver; Arizona Cancer Center and College of Public Health, University of Arizona, Tucson; # Colorado Foundation for Medical Care, Denver, Colorado; **Department of Veterans Affairs Medical Center, Tucson; and Department of Medicine, University of Arizona, Tucson, Arizona Background & Aims: The goal of this study was to examine the relationship between Ki-ras mutations in colorectal adenomas and characteristics of both the subject (age, gender, and family/personal history of colonic neoplasia) and the adenoma (multiplicity, size, location, and histologic features). Methods: Ki-ras mutations were detected by direct sequencing in 738 adenomatous polyps removed at baseline from 639 participants in a nutritional trial of adenoma recurrence. Results: Ki-ras mutations were detected in 17.2% of the adenomas. Ki-ras mutations were unrelated to gender, family, or personal history of colonic neoplasia, location within the colorectum, or adenoma multiplicity, but were more common in older subjects (P 0.01 for trend), in larger adenomas (P < for trend), in adenomas with villous histology (odds ratio [OR], 3.2; 95% confidence interval [CI ], vs. tubular), and in adenomas with high-grade dysplasia (32.0% vs. 13.6%; OR, 3.0; 95% CI, vs. low-grade dysplasia). Multivariate analysis showed Ki-ras mutations to be independently associated with subject age (P 0.01 for trend), tubulovillous/villous histology (OR, 2.3; 95% CI, ), and high-grade dysplasia (OR, 1.9; 95% CI, ). Adenoma size was not independently related to Ki-ras mutation. Conclusions: Ki-ras mutations are associated with the histologic features of adenoma progression (villous histology and highgrade dysplasia) rather than with adenoma growth. Colorectal cancer is the second most common cause of cancer death in the United States. 1 Nearly all colorectal cancers arise within pre-existent adenomatous polyps. 2 The genetic progression of the adenoma-carcinoma sequence in the colon involves the mutational activation of oncogenes (e.g., Ki-ras) and inactivation of tumor suppressor genes (e.g., adenomatous polyposis coli, p53) 3 and DNA repair genes (e.g., hmlh1 and hmsh2). 4 Mutation of the Ki-ras proto-oncogene is one of the common early genetic events in the progression of colorectal cancer. 5,6 The Ki-ras gene codes for a plasma membrane-bound guanosine triphosphate binding protein that is a key regulatory component of signal transduction pathways that transmit growth stimulatory signals from cell surface receptors to intracellular targets. 7 The majority of Ki-ras mutations in colonic neoplasia are point mutations at codon 12 or 13, which result in decreased intrinsic guanosine triphosphatase activity and constitutive activation of the Ki-ras signaling pathways. 8 The Ki-ras gene is mutated in approximately 50% of colorectal cancers and large ( 1 cm) adenomas. 6,9 Despite this relatively high mutation frequency, little is known about the molecular epidemiology of Ki-ras mutations in colorectal adenomas. If the process of colonic carcinogenesis is driven by sequential mutational events, one would predict that the pathologic characteristics (size, histology, dysplasia) of adenomas would be related to the types of mutations that have accumulated in the adenoma. Similarly, specific patient characteristics (age, gender, family, or personal history of colonic neoplasia) could predispose individuals to acquiring specific types of mutations in adenomas. In this study we investigated the relationship between the presence of Ki-ras mutations in colonic adenomas and selected characteristics of both the patients and the adenomas. Abbreviations used in this paper: CI, confidence interval; OR, odds ratio; PCR, polymerase chain reaction by the American Gastroenterological Association /01/$35.00 doi: /gast

2 August 2001 KI-ras MUTATIONS IN COLORECTAL ADENOMAS 303 Materials and Methods Study Population This study was conducted among participants in the Wheat Bran Fiber trial. 10 Briefly, the clinical trial was a double-blinded, placebo-controlled trial designed to measure the effects of wheat bran fiber supplementation on colonic adenoma recurrence. Men and women aged years, who had a full colonoscopy with removal of 1 or more adenomas within 3 months before study entry, were recruited from 3 clinical sites in the Phoenix metropolitan area. Data on patient characteristics (age, gender, family history of colorectal cancer, and personal history of colonic polyps before the baseline adenoma) were obtained from self-administered questionnaires and data on the characteristics of the baseline adenoma (size, location, and number of adenomas) were obtained from the endoscopy and pathology reports. Sections and paraffin blocks of the initial adenoma(s) were requested for all subjects in the clinical trial. All of the polyp samples were reviewed and classified by the study pathologist (A.K.B.) who classified the adenomas as tubulovillous if any part of the adenoma had villous architecture and villous if 100% of the adenoma had villous features. High-grade dysplasia was defined as architectural crowding of glands along with stratification of nuclei above the mid-level of the adenomatous epithelium. Participants in the clinical trial who had adenomas reported to be 5 mm in size and for which the obtained blocks had sufficient tissue for Ki-ras mutational analysis were included in this portion of the research protocol. The study was approved by the University of Arizona Human Subjects Committee as well as by the human subjects committees of the 3 Phoenix hospitals. DNA Extraction and Amplification As previously described, 11 5-micron sections were cut from formalin-fixed paraffin-embedded blocks of the adenomas. One slide for each adenoma was stained with H&E to guide the microdissection of the adenomatous tissue. The adenomatous tissue was then scraped from the slide and transferred to a microcentrifuge tube. Thirty-five microliters of DNA extraction buffer (0.5 mol/l Tris, 20 mmol/l EDTA, 10 mmol/l NaCl, 2 mg/ml proteinase K, and 5% Tween 20, ph 9) was added to the tissue sample and incubated in a heat block overnight at 56 C. Ten microliters of Low Tris EDTA buffer (3 mmol/l Tris and 0.2 mmol/l EDTA, ph 7.5) containing 5% chelex (Biorad, Hercules, CA) was added and the tubes were heated to 100 C for 10 minutes. After centrifugation at 12,000g for 2 minutes to pellet undigested tissue and chelex, 1 L of the supernatant was used in a polymerase chain reaction (PCR) to amplify the region of exon 1 of Ki-ras containing codons 12 and 13. The PCR reaction was as follows: 94 C, 2 minutes; and 37 cycles of 94 C, 1 minute; 53 C, 1 minute; 72 C, 1 minute; and 72 C, 5 minutes. The PCR reaction buffer (Promega, Madison, WI) contained 1.5 mmol/l MgCl 2, 0.2 mmol/l deoxynucleoside triphosphates, 0.25 mmol/l Ki-ras sense primer (GAGAATTCATGACTGAATA TAA ACTTGT), 0.25 mmol/l Ki-ras antisense primer (ATCGAATTCCTC- TATTGTTGGATCA TATTC), and 1 U TAQ (Promega, Madison, WI). The 118 base pair PCR product was then visualized on a 2% agarose gel. DNA samples that failed to amplify with 1 L were repeated using 5 L of the extracted DNA. For each set of samples extracted, negative control tubes containing no tissue were simultaneously extracted and tested for ability to amplify a product. Only samples for which the corresponding negative control did not amplify a product were used. For quality control purposes, known positive and negative control samples were regularly analyzed alongside the adenoma samples. Ki-ras Sequencing Mutations at codons 12 and 13 of Ki-ras were detected by direct DNA cycle sequencing using the Ki-ras PCR product in the SequiTherm EXCEL II DNA Sequencing Kit (Epicentre Technologies, Madison, WI). One to 4 L of the PCR product was used directly in the sequencing reaction. A primer downstream of codons 12 and 13 (ATTCGTCCACAAAAT- GAT) was end-labeled and used in the sequencing reaction as per the protocol provided by the manufacturer. The sequencing reaction products were run on a 6% denatured polyacrylamide gel. The gel was dried and exposed to film overnight. Mutations in codons 12 and 13 were identified using the Ki-ras sequence visible on the film. A sample of DNA from the SW480 colon cancer cell line that is mutant at the second nucleotide of codon 12 of the Ki-ras gene was run with each set of reactions and was thus present on each gel as a positive control. Known negative controls were also run regularly with the adenoma samples. The results of the ras analyses were read and recorded without knowledge of the patient or adenoma data. An example of the Ki-ras sequencing results has been previously published. 11 Adenomas were classified as being Ki-ras mutant if an abnormal sequence of codon 12 or 13 was detected. Statistical Analysis Only those polyps that were confirmed by the study pathologist (A.K.B.) as adenomatous were used in the analysis. Patients who had multiple adenomas analyzed were classified according to the Ki-ras status of their largest adenoma. The relationship between Ki-ras mutations in adenomas and various characteristics of patients and of the adenomas were assessed by both univariate and multiple logistical analysis using SAS System for Windows, version 8.1 (SAS Institute Inc, Cary, NC). Odds ratios (ORs) were first calculated separately for all of the independent variables (patient and adenoma characteristics) with Ki-ras mutational status (wild-type vs. mutant) as the dependent variable. Multiple logistic models that included all adenoma or all patient characteristics (except number of previous adenomas because of the high rate of missing data for this factor) were then performed for both adenoma-level and person-level analyses. Multiple logistic regression models at the adenoma level were estimated using generalized estimating

3 304 MALTZMAN ET AL. GASTROENTEROLOGY Vol. 121, No. 2 Table 1. Ki-ras Mutations in Codons 12 and 13 in 738 Colorectal Adenomas Codon 12 (AA) Codon 13 (AA) Mutation type n (%) GGT (Gly) GGC(Gly) Wild-type 611 (82.8) Single mutations GTT (Val) Transversion 46 (6.2) GAT (Asp) Transition 30 (4.1) TGT (Cys) Transversion 12 (1.6) AGT (Ser) Transition 11 (1.5) GCT (Ala) Transversion 3 (0.4) CGT (Arg) Transversion 3 (0.4) GAC (Asp) Transition 17 (2.3) GCC (Ala) Transversion 1 (0.1) Double mutations TGT; GTT (Cys; Val) Transversions 2 (0.3) GTT (Val) GCC (Ala) Transversions 1 (0.1) GTT (Val) GAC (Asp) Transversion/ transition 1 (0.1) AA, amino acids. equations to allow for potential correlation among the adenomas within the same individual. Results A comparison of the group of patients analyzed in this study to those in the total population of the participants in the Wheat Bran Fiber Trial has been reported previously. 11 The groups were similar in race and gender distribution, in family and personal history of adenomas, and in all of the dietary factors analyzed. The only statistically significant difference between the subgroup of patients in this study (N 672) compared with the entire group of participants in the clinical trial (N 1429) was that the patients in this study were slightly older than the overall study population (mean age 66.8 vs years; P 0.05). Successful Ki-ras sequence was obtained from 821 of 900 (91.2%) of the tissue samples received from 672 patients. Among these tissue samples, there were 738 adenomas as determined by the study pathologist from 639 patients. The remainder of the samples were hyperplastic polyps or mucosa without adenomatous change. Ki-ras mutations were found in 127 of the 738 adenomas analyzed (17.2%) (Table 1). Of the 127 adenomas with Ki-ras mutations, 105 (83%) had a single mutation at codon 12; 18 (14%) had a single mutation at codon 13; and 4 (3%) had 2 different Ki-ras mutations. Of the 131 Ki-ras mutations identified, 59 (45%) were transitions and 72 (55%) were transversions. The most common single mutation was a GGT to a GTT transversion in codon 12 that resulted in a change from the amino acid glycine to valine. This particular mutation occurred in 6.2% of all adenomas and 36% of the adenomas with Ki-ras mutations. There were no differences in the prevalence of Ki-ras mutations by subject gender, family history of colorectal cancer, or by personal history of previous colorectal adenomas (Table 2). Older participants were more likely to have an adenoma with a Ki-ras mutation in both the univariate and multivariate analysis (P 0.01 for trend). The age-related association occurred for both transition and transversion mutations of Ki-ras. Subjects less than Table 2. Prevalence of Ki-ras Mutations in the Largest Adenomas as From 639 Patients Compared With Patient Characteristics Clinical characteristics n n(%) Ki-ras mutant Univariate OR a (95% CI) Multivariate OR a (95% CI) Total (17.8) Sex Women (18.6) 1.0 (Reference) 1.0 (Reference) Men (17.5) 1.0 ( ) 1.0 ( ) Age 65 yr (13.4) 1.0 (Reference) 1.0 (Reference) yr (13.1) 1.0 ( ) 0.9 ( ) yr (27.0) 2.4 ( ) 2.0 ( ) 75 yr (19.3) 1.6 ( ) b 1.2 ( ) b Family history of colorectal cancer No (18.0) 1.0 (Reference) 1.0 (Reference) Yes (17.0) 0.9 ( ) 1.0 ( ) Personal history of previous adenomas c No (17.3) 1.0 (Reference) NI Yes (15.4) 0.9 ( ) NI NI, not included in the multivariate model. a Each factor in the model alone in univariate model, but present with all other factors except previous adenomas in the table in the multivariate model (n 582). b P value for trend 0.01 (univariate) and 0.01 (multivariate). c Not reported for 61 participants.

4 August 2001 KI-ras MUTATIONS IN COLORECTAL ADENOMAS 305 Table 3. Prevalence of Ki-ras Mutations in 738 Adenomas as Compared With Adenoma Characteristics Adenoma characteristic n n(%) Ki-ras mutant Univariate OR a (95% CI) Multivariate OR a (95% CI) Total (17.2) Histology b Tubular (10.6) 1.0 (Reference) 1.0 (Reference) Tubulovillous/villous (27.7) 3.2 ( ) 2.3 ( ) Dysplasia c Low grade (13.6) 1.0 (Reference) 1.0 (Reference) High grade (32.0) 3.0 ( ) 1.9 ( ) Location d Colon (17.0) 1.0 (Reference) 1.0 (Reference) Rectum (21.9) 1.4 ( ) 1.3 ( ) No. of adenomas (17.7) 1.0 (Reference) 1.0 (Reference) (17.4) 1.0 ( ) 1.0 ( ) (15.6) 0.9 ( ) f 0.8 ( ) f Size e 1.0 cm (11.0) 1.0 (Reference) 1.0 (Reference) cm (19.7) 2.0 ( ) 1.5 ( ) cm (24.4) 2.6 ( ) g 1.6 ( ) 2.0 cm (29.1) 3.3 ( ) g 1.5 ( ) g a Each factor in the model alone in univariate model, but present with all other factors in the table in the multivariate model (n 694). b Not known for 2 adenomas. c Not known for 34 adenomas. d Not known for 6 adenomas. e Not known for 2 adenomas. f P value for trend (univariate) and (multivariate). g P value for trend (univariate) and 0.17 (multivariate). 70 years of age had lower rates of both transitions and transversions (5.7% and 7.2%, respectively) than subjects 70 years old (11.8% for both types). In 80 participants, multiple adenomas were available for analysis; 66 subjects had 2 adenomas, 9 had 3, 4 had 4, and 1 had 5 adenomas analyzed. Of these study subjects, 8 (10%) had Ki-ras mutations in all of the adenomas, 50 (63%) had mutations in none of the adenomas, and 22 (28%) had discordant results with Ki-ras mutations in some, but not all, of their adenomas. If the Ki-ras status of an individual participant was classified on the basis of whether there was any Ki-ras positive adenoma rather than whether their largest adenoma was Ki-ras mutant, 7 subjects would have been reclassified to the Ki-ras mutant group but none of the statistical relationships would be changed. Ki-ras mutations were more frequent in adenomas from the rectum than those from the colon (21.9 % vs. 17.0%), but this difference did not reach statistical significance (OR, 1.4; 95% confidence interval [CI ], ) (Table 3). Within the colon, similar frequencies of Ki-ras mutations were found in the adenomas proximal and distal to the splenic flexure (16% and 17%, respectively). Ki-ras mutations were significantly more common as adenoma size increased (P for trend). A strong relationship was also found between Ki-ras mutations and adenoma histology. Ki-ras mutations were present in 27.7% of the adenomas that were tubulovillous or villous compared with 10.6% of adenomas that were of tubular histology (OR, 3.2; 95% CI, ). Tubulovillous and villous adenomas were combined for this analysis because there were too few purely villous adenomas (n 42) in the study for separate analyses, but the frequency of mutations in the 42 villous adenomas was 26.2%. Ki-ras mutations were present in 32.0% of adenomas that were graded as having high-grade dysplasia, but only in 13.6% of adenomas with low-grade dysplasia (OR, 3.0; 95% CI, ). The associations between adenoma size and histology and Ki-ras mutations were seen for both transition and transversion mutations and for both codon 12 and 13 mutations. Large ( 1 cm) adenomas had higher rates of both Ki-ras transitions and transversions (11.1% and 10.8%, respectively) than small ( 1 cm) adenomas (3.9% transitions, 7.2% transversions). Similarly, tubulovillous and villous adenomas had higher rates of both transitions and transversions (12.9% and 13.9%, respectively) than tubular adenomas (4.4% transitions, 6.2% transversions) and in adenomas with high-grade dysplasia (16.1% transitions, 14.8% transversions) than in those with low-grade dysplasia (5.7% and 7.8%, respectively). Although there were only 18 adenomas with

5 306 MALTZMAN ET AL. GASTROENTEROLOGY Vol. 121, No. 2 codon 13 mutations, the step up in frequency of this mutation in advanced adenomas was similar to that seen for codon 12 mutations. Ki-ras mutations were not more likely to be present in any single adenoma from a participant with multiple adenomas than in subjects with only a single adenoma at the baseline colonoscopy (Table 3). In the multivariate analyses (Table 3), the presence of tubulovillous or villous histology and high-grade dysplasia remained significantly and independently associated with Ki-ras mutations (OR, 2.3; 95% CI, and OR, 1.9; 95% CI , respectively). Size of the adenoma, however, was no longer significantly associated with Ki-ras mutation after adjustment for the histologic characteristics (P 0.17 for trend). Table 4 details the interrelationships between Ki-ras mutations and adenoma size, histologic classification (tubular vs. tubulovillous or villous), and the degree of dysplasia in this series of adenomas. There were highly significant (P 0.001) relationships between size and both villous histology and high-grade dysplasia. Highgrade dysplasia was present in only 7.9% of 317 small ( 1 cm) adenomas but in 52.8% of the 72 large ( 2cm) adenomas. Villous histologic features were present in only 21.9% of small ( 1 cm) adenomas but in 82.3% of large ( 2 cm) adenomas. There was also a significant (P 0.001) association between villous histology and dysplasia. High-grade dysplasia was present in only 10.9% of tubular adenomas, but in 36.8% of tubulovillous or villous adenomas. Figure 1 shows the frequency of Ki-ras mutations as a function of the size and histologic characteristics of the adenomas. The lowest frequency of Ki-ras mutations (7.5%) was found in small ( 1 cm) adenomas without Table 4. Relationship Between Adenoma Size, Villous Histology, and Dysplasia n Dysplasia a High-grade dysplasia (%) n Histology b Tubulovillous/ villous (%) Total Size (cm) c d d Histology Tubular Tubulovillous/villous d a 34 missing. b 2 missing. c 2 missing. d P 0.001, size vs. dysplasia, size vs. histology, and histology vs. dysplasia. Figure 1. Percent of Ki-ras mutations in adenomas as a function of their size and histologic features. All of the adenomas analyzed were classified as large if they were 1 cm, as having high-grade dysplasia if this histology was seen in any area of the adenoma, and as having villous histology if any portion of the adenoma had villous features. The percent of adenomas with Ki-ras mutations in each of the indicated subgroups is plotted. high-grade dysplasia or villous histology. The presence of any 1 of the more advanced characteristics (large size, high-grade dysplasia, or villous histology) alone had a similar effect on Ki-ras mutations rate increasing the frequency to 12% 15%. In adenomas with 2 of the 3 advanced characteristics, the combination of villous histology and high-grade dysplasia was associated with a higher rate of Ki-ras mutation (57%) than the presence of large size with either villous component or high-grade dysplasia (18% 25%). In adenomas with both villous features and high-grade dysplasia, the presence of large size was not associated with a further increase in the rate of Ki-ras mutation. The greater importance of histology was confirmed by the multivariate analysis. After adjustment for both villous histology and degree of dysplasia, size was no longer related to Ki-ras mutation rates (P 0.17 for trend) (Table 3). Discussion Several risk factors for colorectal cancer have been identified by epidemiologic studies, including characteristics of both the host (age, gender, race, and family cancer history) and the environment (diet, smoking, alcohol, and physical activity). Similarly, several characteristics of colorectal adenomas (size, histology, and degree of dysplasia) have been found to be associated with an increased likelihood of an adenoma containing a carcinoma. 9 In parallel, numerous molecular biologic studies have revealed that colonic carcinogenesis results, in part, from the accumulation of genetic alterations, in-

6 August 2001 KI-ras MUTATIONS IN COLORECTAL ADENOMAS 307 cluding activating mutations of proto-oncogene and inactivating mutations of tumor suppressor and DNA repair genes. Relatively little is known about the relationships among host and environmental risk factors, adenoma characteristics, and the genetic events that are thought to drive the process of colonic carcinogenesis. Ki-ras mutations can occur relatively early in the adenoma-carcinoma sequence. 12,13 Whereas numerous large studies have investigated the clinical significance of Ki-ras mutations in colorectal cancers, less is known about the determinants of Ki-ras mutations in adenomas. In this study, we have investigated several aspects of the molecular epidemiology of Ki-ras mutations in colonic adenomas. Our finding that 17.2% of the 738 adenomas had mutations in the Ki-ras proto-oncogene is at the lower end of the 15% 75% prevalence of Ki-ras mutation rate in adenomas reported in previous series. 6,19 31 It is likely that this reflects both differences in adenoma selection and technical issues related to the methods used for assessment of Ki-ras mutations. The adenomas in this study were smaller (46% 1 cm) and less histologically advanced (62% tubular, 79% low-grade dysplasia) than those analyzed in most of the previous series. 22,24,25,27 32 Because our results (Table 3) and those of several previous studies 6,17,18,21 25,28 show a significant correlation between advanced histology and higher rates of Ki-ras mutations, our Ki-ras mutation frequency would be expected to be lower than many of the previous studies of more advanced adenomas. Morris et al. 24 reported that adenomas from patients with colorectal cancer had a higher rate of Ki-ras mutation than those from subjects without colorectal cancer (23% vs. 48%). Patients with previous or concurrent colorectal cancer were also excluded from our adenoma prevention trial, and our Kiras mutation rate may be lower because of our patient selection criteria. The rate of Ki-ras mutation in our series is very similar to those previous studies of less advanced adenomas 6 and the rate reported by Morris et al. 24 for subjects without concurrent colorectal cancer. Another possible reason for the relatively low rate of Ki-ras mutation in adenomas in our series is our decision to use tissue microdissection, PCR amplification, and direct sequencing of Ki-ras. This method can detect Ki-ras mutations in DNA samples containing 10% or more mutated species. 32 Because our goal was to analyze the histologic correlates and potential consequences of Ki-ras mutation in adenomas, we reasoned that if the dominant clone in an adenoma was Ki-ras mutant, this method would be sensitive enough to detect a meaningful mutation without the risk of detecting minor subpopulations of Ki-ras mutant cells. If we had used a more sensitive assay such as mutant allele-specific amplification and oligonucleotide hybridization, 28,29,31 which have been reported to be able to detect 1 Ki-ras mutant cell in the presence of copies of the wild-type allele, 22 a higher mutation rate would likely have been found. These methods were thought to be overly sensitive for our study in that they have been reported to detect Ki-ras mutations in samples of histologically normal-appearing colonic mucosa from patients with colorectal cancer. 22,33 The frequency of Ki-ras mutations in adenomas in this study is similar to that reported in other series using direct sequencing to detect the presence of mutations. 24 The current study is by far the largest series of adenomas examined for Ki-ras mutations (738 adenomas from 639 subjects). The large size of the study allowed us to perform stratified and multivariate analysis of the data to determine which features of the adenoma or of the study subject were independently associated with Ki-ras mutations. This is particularly important for the adenoma characteristics of size, villous histology, and degree of dysplasia, which are highly inter-related (Table 4). In our univariate analysis, large size, villous histology, and high-grade dysplasia were each significantly associated with higher rates of Ki-ras mutation. Previous reports have differed on this point. For example, Andersen et al. 22 also found a significant univariate relationship with adenoma size, villous histology, and degree of dysplasia, but others have reported a relationship with only 2, 18, , 17,27 or none 28,29 of these advanced features. All of the previous studies were relatively small. The largest previous study 29 reported the Ki-ras mutational status of 200 adenomas from 60 patients. The investigators found a progressive increase in the percent of Ki-ras mutations with increasing adenoma size and with villous histology, but the degree of dysplasia was not assessed. Although these relationships were of about the same magnitude as found in the current study, the differences did not reach statistical significance in either the univariate or multivariate analysis. A strong association between Ki-ras mutation and both villous histology and high-grade dysplasia persisted in the multivariate analysis of our data, but size was not found to be associated with Ki-ras mutation after adjustment for the other histologic characteristics. This finding differs from several previous reports of a significant univariate association between adenoma size and Ki-ras mutation. 6,22,23,26 Univariate analysis of our data would have also led to the conclusion that Ki-ras mutations were strongly related to adenoma size. The multivariate anal-

7 308 MALTZMAN ET AL. GASTROENTEROLOGY Vol. 121, No. 2 ysis, however, showed that size was not an independent predictor of the likelihood of Ki-ras mutation in an adenoma, whereas villous histology and high-grade dysplasia were independently related to Ki-ras mutational status. These observations suggest the possibility that Ki-ras mutations may, in part, drive the histologic progression of adenomas toward villous histology and higher grades of dysplasia rather than affecting the adenoma growth rate. We found that individuals over the age of 70 years had about a 2-fold increase in risk of having a Ki-ras mutant adenoma than did younger individuals. This result is in contrast to that of Rashid et al., 29 who found a decreased rate of Ki-ras mutation in adenomas from older subjects. The reason for this difference is not clear, but variance on this point has been reported previously. Morris et al. 24 found a higher rate of Ki-ras mutations in adenomas from older ( 70 years) than from younger ( 70 years) patients with concurrent colorectal cancer (67% vs. 18%). No such difference in adenomas was seen among patients without colorectal cancer (23% in patients 70 years vs. 18% in those 70 years). It has also been previously shown that older individuals on average have more advanced adenomas (larger and more commonly villous), 9 which may serve to confound the association between Ki-ras mutations and age. The absolute frequency of Ki-ras mutations in rectal adenomas was somewhat higher than that in the colon (21.9% vs. 16.5%), but this difference did not reach statistical significance (P 0.10) and there were no detectable differences in the mutation rate of adenomas from different segments within the colon. A difference in the frequency of genetic changes has been previously reported between proximal and distal colorectal cancers, 20 but to our knowledge there are no reports of differential frequencies of Ki-ras mutations as a function of location of the adenomas. Our finding of no association between Ki-ras mutations and the total number of adenomas present is similar to previous studies. 29 Similarly, no differences were found in the frequency of Ki-ras mutations as a function of the study participants family history of colorectal cancer or their personal history of previous adenomas. These results suggest that the genetic and/or environmental factors responsible for the familial or personal clustering of colonic adenomas may act via mechanisms other than through Ki-ras mutation. We had the opportunity to examine the Ki-ras status of multiple adenomas from 80 patients in our study. In 28% of these patients, there were Ki-ras mutations in some but not all of their adenomas. The relatively high discordance rate for Ki-ras mutation in multiple adenomas from the same patient suggests that the presence of this mutation in an individual adenoma would not likely be a good predictor of either synchronous or metachronous adenoma formation. In preliminary analysis of the polyp prevention trial from which these adenomas were obtained, Ki-ras mutation was not found to be an independent predictor of metachronous adenoma formation. The process of colonic carcinogenesis is thought to be driven, in part, by sequential rounds of mutation followed by clonal expansion such that the final histologic and biologic features of the adenoma are the result of the accumulation of a series of mutations or mutation-like events. If this is so, knowledge of the mutational profile of an adenoma might be expected to provide greater insight into its clinical behavior than the histology and/or size of the lesion. Ki-ras mutation is thought to occur as an early step in the adenoma-carcinoma sequence. The substantial step up in the frequency of Ki-ras mutations during the progression from tubular to tubulovillous/villous adenomas and from low-grade to high-grade dysplasia without a relationship to adenoma size suggests that Ki-ras mutation is an important step in the pathway of adenoma progression to more advanced histology rather than in the promotion of adenoma growth. References 1. Greenlee RT, Murray T, Bolden S, Wingo PA. Cancer Statistics, CA Cancer J Clin 2000;50: Winawer SJ, Zauber AG, Ho MN, O Brien MJ, Gottleib SJ, Sternberg SS, Waye JD, Shapiro M, Bond JH, Panish JF. Prevention of colorectal cancer by colonoscopic polypectomy. The National Polyp Workshop. N Engl J Med 1993;329: Fearon ER, Vogelstein B. A genetic model for colorectal tumorigenesis. 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Am J Clin Pathol 1970;53: Gatteschi B, Constantini M, Bruzzi P. Univariate and multivariate analyses of the relationship between adenocarcinoma and solitary and multiple adenomas in colorectal adenoma patients. Int J Cancer 1991;98: Received January 31, Accepted April 18, Address requests for reprints to: Dennis J. Ahnen, M.D., Department of Veterans Affairs Medical Center, 111E, 1055 Clermont Street, Denver, Colorado dennis.ahnen@uchsc.edu; fax: (303) Supported in part by a Department of Veterans Affairs Merit Review grant (to D.J.A.); the Public Health Service grants CA and CA-23074; a Career Development Award (KO7 CA ) from the National Cancer Institute (to T.M.); and a Career Development Award (KO1 CA ) from the National Cancer Institute (to M.E.M.).