REPORT

Associations Between Cigarette Smoking, Lifestyle Factors, and Microsatellite Instability in Colon Tumors

Martha L. Slattery, Karen Curtin, Kristin Anderson, Khe-Ni Ma, Linda Ballard, Sandra Edwards, Donna Schaffer, John Potter, Mark Leppert, Wade S. Samowitz

Affiliations of authors: M. L. Slattery, K. Curtin, K.-N. Ma, S. Edwards (Health Research Center, Department of Family and Preventive Medicine), L. Ballard, M. Leppert (Department of Human Genetics), W. S. Samowitz (Department of Surgical Pathology), University of Utah, Salt Lake City; K. Anderson, University of Minnesota, School of Public Health, Minneapolis; D. Schaffer, Division of Research, Kaiser Permanente Medical Care Program, Oakland, CA; J. Potter, Fred Hutchinson Cancer Research Center, Seattle, WA.

Correspondence to: Martha L. Slattery, Ph.D., M.P.H., Health Research Center, Department of Family and Preventive Medicine, University of Utah, 391 Chipeta Way, Suite G, Salt Lake City, UT 84108 (e-mail: mslatter{at}dfpm.utah.edu).


    ABSTRACT
 Top
 Notes
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
 References
 
Background: Microsatellite instability (MSI) has been reported to occur in approximately 10%–15% of colon tumors. MSI is characterized by the presence of mutations in tandemly repeated DNA sequences known as microsatellites. Some individuals with unstable tumors have inherited mutations in mismatch repair genes, but MSI is also observed in sporadic colon cancer. It is unknown whether lifestyle factors associated with colon cancer, such as physical activity, body size, cigarette smoking, or use of aspirin and/or nonsteroidal anti-inflammatory drugs, contribute to MSI in sporadic tumors. Methods: Data from a population-based, case–control study of colon cancer were used. Case subjects were between 30 and 79 years of age at the time of diagnosis and included both men and women. Questionnaire data were used to obtain information on lifestyle factors. Tumor MSI was determined with the use of a panel of 10 tetranucleotide repeats and two mononucleotide repeats. A total of 1510 case subjects had valid questionnaire data and tumor DNA from which we were able to obtain MSI status. Questionnaire data were compared with lifestyle factors reported by 2410 population-based control subjects. All statistical tests were two-sided. Results: MSI-positive (MSI+) tumors were most common in older people and women and in the proximal colon. Patients with MSI+ tumors were more likely to smoke 20 or more cigarettes a day than case subjects with MSI-negative (MSI-) tumors (odds ratio for being a smoker = 1.6 [95% confidence interval = 1.0–2.5] for men and 2.2 [95% confidence interval = 1.4–3.5] for women). The association between MSI+ tumors and cigarette smoking was strongest among case subjects who started to smoke at a young age, smoked for 35 or more years, and were either current smokers or had stopped fewer than 15 years before diagnosis. A statistically significant linear trend of increased risk of MSI+ tumors was observed with increasing amount smoked (P<.01). Conclusions: This study suggests that smoking cigarettes statistically significantly contributes to MSI in colon tumors. We estimate that approximately 21% of MSI in colon tumors may be attributable to cigarette smoking.



    INTRODUCTION
 Top
 Notes
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
 References
 
Microsatellite instability (MSI) has been reported to occur in 10%–15% of colon tumors, with a predominance of tumors in the proximal colon (13). MSI appears to be more common among individuals diagnosed with colon cancer before age 50 years or after age 70 years (4). Individuals with inherited mutations of the mismatch repair genes exhibit MSI in colon tumors (5), accounting for 5%–10% of colon tumors with MSI (6). Although MSI has been characterized in a variety of populations, few population-based studies have been reported. Examination of lifestyle factors associated with colon cancer in conjunction with MSI in colon tumors has not been done.

In this study, we assess the associations between lifestyle factors and MSI in a large, population-based study of incident cases of adenocarcinoma of the colon. We focus on those lifestyle factors—such as cigarette smoking, physical activity, body size, and use of aspirin and/or nonsteroidal anti-inflammatory drugs (NSAIDs)—that have been reported to be associated with colon cancer in other studies (7). We also assess the relationship between MSI and KRAS gene mutations, since we have shown an inverse relationship between these variables in a previous study (Samowitz WS, Holden JA, Curtin K, Walker AR, Lynch BJ, Edwards SL: unpublished data).


    SUBJECTS AND METHODS
 Top
 Notes
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
 References
 
Study Population

Study participants were black, white, or Hispanic and were from either the Kaiser Permanente Medical Care Program (KPMCP) of Northern California, an eight-county area in Utah (Davis, Salt Lake, Utah, Weber, Wasatch, Tooele, Morgan, and Summit counties), or the Twin Cities Metropolitan Area in Minnesota. Cases were identified with the use of a rapid case ascertainment system that involved study abstractors visiting hospitals on a monthly basis to identified newly diagnosed cases. Eligibility criteria for case subjects included diagnosis with first primary incident colon cancer [International Classification of Diseases for Oncology, 2nd ed. (8), codes 18.0 and 18.2–18.9] during the period from October 1, 1991, through September 30, 1994; age from 30 years through 79 years at the time of diagnosis; and mental competence to complete the interview. Case subjects with adenocarcinoma or carcinoma of the rectosigmoid junction or rectum (defined as the first 15 cm from the anal opening), with known familial adenomatous polyposis, ulcerative colitis, or Crohn's disease were not eligible. Of all case subjects asked to participate (n = 2647), 75.6% cooperated. The Utah portion of this study was previously evaluated in an analysis of MSI and alterations in KRAS and TP53 genes (Samowitz WS, Holden JA, Curtin K, Walker AR, Lynch BJ, Edwards SL, et al.: unpublished data).

In addition to the eligibility criteria for case subjects, control subjects could never have been diagnosed with a colorectal tumor. Control subjects were selected from eligibility lists for KPMCP, driver's license lists for Minnesota, and random-digit-dialing, driver's license lists, or Health Care Finance Administration lists for Utah. These methods have been described in detail previously (9). Of all control subjects, 63.7% participated and had valid data, leaving a final number of 2410.

The study was approved by Institutional Review Boards in all centers. All of the study participants signed an informed consent prior to participation.

Other Data

Anthropometric, lifestyle, and medical history data were collected by trained and certified interviewers (10). The referent period for the study was the calendar year, approximately 2 years before the date of diagnosis. Information was collected on (a) demographic factors, such as age, sex, and study center; (b) anthropometric and lifestyle factors, such as physical activity (9), body size (including usual adult height and weight 2 and 5 years before the diagnosis), use of aspirin and/or NSAIDs, and cigarette-smoking history; and (c) medical history. A measure of long-term (past 20 years) levels of vigorous leisure-time physical activity was used, since it was shown to be a sensitive predictor of cancer risk in this population (9). Body mass index (BMI) of kg/m2 for men and of kg/m1.5 for women was used as an indicator of body size. Current and past cigarette smoking history was determined as well as the usual number of cigarettes smoked in a day during the time when the participant smoked, age first started to smoke, age stopped smoking, and history of smoking pipes and cigars.

Tissue Ascertainment

Methods for ascertaining tumor tissue and extracting DNA have been discussed in detail previously (11). Tumor DNA, obtained from paraffin blocks, was amplified, and the MSI status was determined. Results were obtained for one or more indicators of MSI for 98% of case subjects from whom we were able to isolate tumor DNA. Of the 1836 case subjects with MSI data, 1510 also had valid interview data and are included in these analyses.

Microsatellite Instability

Each tumor was evaluated for MSI with a panel of 10 tetranucleotide repeats used by us in previous studies, the mononucleotide repeat BAT26, and a mononucleotide repeat within the coding region of TGF{beta}RII. The primer sequences and polymerase chain reaction (PCR) conditions for these repeats were as described previously (4,12,13). Both tumoral DNA and normal DNA were PCR amplified with these 12 primer sets. MSI for a given primer set was defined as the appearance of one or more new PCR products either smaller or larger than those produced from normal DNA. For BAT26, we required that a PCR product from the tumor DNA be at least 4 base pairs smaller than that from germline DNA. Results from the tetranucleotide repeat panel were considered to indicate MSI if three or more repeats were unstable. Results were considered to indicate stability if fewer than 30% of the repeats were unstable and at least six of the 10 repeats were typed. Initial analyses of the relationships between MSI determined by either the panel of 10 tetranucleotides, BAT26, or TGF{beta}RII and various clinical and molecular features gave nearly identical results regardless of the way instability was measured (see Table 1Go). To simplify subsequent analyses, we therefore combined these primer sets into a panel of 12 and scored a tumor as unstable if it was unstable with the panel of 10 tetranucleotides (as defined above), BAT26, or TGF{beta}RII. Any associations detected in this way were then further defined by considering instability results with each of these three measures separately.


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Table 1. Description of the colon cancer case subjects by microsatellite instability (MSI) status
 
The evaluation of MSI was performed before the development of the Bethesda Consensus Panel. However, in a recent analysis, we have shown highly statistically significant relationships between the consensus panel and the panel of 10 tetranucleotides, BAT26, and TGF{beta}RII (Samowitz WS, Holden JA, Curtin K, Walker AR, Lynch BJ, Edwards SL, et al.: unpublished data). In addition, instability in BAT26, one of the consensus panel repeats, has been associated with generalized dinucleotide repeat instability (14). Moreover, we have shown that strong relationships exist between tetranucleotide repeat instability and TGF{beta}RII mutations (the most common coding mononucleotide repeat altered in unstable colon cancers) in colon cancer (12) and tetranucleotide repeat instability and the noncoding mononucleotide repeats BAT26 and BAT40 in colon cancer (13).

KRAS Gene

Mutations in codons 12 and 13 of the KRAS gene were detected by PCR amplification and sequencing of exon 1 as described previously (15), except that primers were tailed with the universal primer and the reverse primer for sequencing. PCR products were sequenced with the use of prism Big Dye terminators (Applied Biosystems, Foster City, CA) and cycle sequencing with Taq FS® DNA polymerase. DNA sequence was collected and analyzed on an ABI prism 377 automated DNA sequencer (Applied Biosystems). We considered as mutations only those base-pair changes that were verified by sequencing in both directions.

Statistical Analyses

The distribution of MSI by population characteristics was determined. Logistic regression models were fit with the use of two groups to compare with MSI-positive (MSI+) tumors: population-based control subjects and MSI-negative (MSI-) case subjects. The characteristics of patients with MSI- tumors also were compared with those of population-based control subjects. The "cancer case subject–population-based control subject" comparison was conducted to estimate the relative risk for developing the disease with or without specific genetic mutations. The purpose of the "case subject–case subject" comparison was to evaluate etiologic heterogeneity or differences in risk factors for different tumor genetic characteristics. For these analyses, the logistic regression models were fit with a dichotomous dependent variable of MSI+ or MSI-. Analyses were done with the use of SAS® and BMDP® statistical software packages. All P values are reported to two decimal places.

As shown in Table 1Go, remarkably similar results were obtained with instability determined by either the panel of 10 tetranucleotide repeats, BAT26, or TGF{beta}RII. To simplify subsequent analyses, we therefore combined these primer sets into a panel of 12 and scored a tumor as unstable if it was unstable with the panel of 10 tetranucleotides (as defined above), BAT26, or TGF{beta}RII. Any associations detected in this way were then further defined by considering instability results with each of these three measures separately. All P values are reported for a two-sided test; P values of less than .05 are commonly considered to be statistically significant.


    RESULTS
 Top
 Notes
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
 References
 
Between 12.7% and 17.6% of tumors were unstable by one or more indicator of MSI (Table 1Go). TGF{beta}RII was least often unstable, whereas the panel that included all markers was the most sensitive indicator of MSI. Regardless of the indicator of MSI, subjects younger than 55 years or older than 70 years and who were women were statistically significantly more likely to have unstable tumors. Tumors of the proximal colon were more likely to be unstable than those of the distal colon. There were no differences in MSI by race. Tumors with MSI were statistically significantly less likely to have KRAS gene mutations by all markers of instability (P<.01 for each).

Among men, no vigorous physical activity and not taking NSAIDs or aspirin were associated with both MSI+ and MSI- tumors (Table 2Go). Other variables examined, including having had gallbladder surgery or using vitamin and mineral supplements, also were not associated with MSI in tumors (data not shown in tables). In men, a high BMI was positively associated with having an MSI- tumor but not with having an MSI+ tumor. Cigarette smoking was more strongly associated with MSI+ than with MSI- tumors. Associations with specific markers of MSI (i.e., panel of 10 tetranucleotide repeats, BAT26, or TGF{beta}RII) were almost identical to those reported when instability was defined on the basis of all markers (data not shown in tables); the association with cigarette smoking went from 1.5 for the panel of 12 to 1.6 for each of the other three indicators of instability. Adjustment for other colon cancer risk factors, including use of aspirin, BMI, physical inactivity, and various dietary factors, did not alter the observed associations.


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Table 2. Associations between lifestyle factors and microsatellite instability among men*
 
Patterns of associations among women (Table 3Go) were similar to those among men, in that low levels of physical activity and not using aspirin or NSAIDs were associated with an increased likelihood of both MSI+ and MSI- tumors. Smoking 20 or more cigarettes per day was associated with a twofold increased risk of MSI+ tumors compared with control subjects, although smoking 20 or more cigarettes per day was not associated with MSI- tumors compared with control subjects. Assessment of specific markers in women showed only slight differences in associations. For instance, the odds ratio for cigarette smoking comparing MSI+ with MSI- for the panel of 12 was 2.2 (95% confidence interval [CI] = 1.4–3.5); for the panel of 10, it was 1.7 (95% CI = 1.0–2.9); for BAT26, it was 2.3 (95% CI = 1.4–3.8); and for TGF{beta}RII, it was 2.3 (95% CI = 1.4–23.9). Adjustment for other colon cancer risk factors, including use of aspirin, BMI, physical inactivity, and various dietary factors, did not alter the observed associations.


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Table 3. Associations between lifestyle factors and microsatellite instability among women*
 
Assessment of patterns of cigarette smoking provided additional support that cigarette smoking is associated with MSI+ tumors (Table 4Go). Current cigarette smoking, starting to smoke at a young age, smoking cigarettes for more total years, and smoking cigarettes for more pack-years were associated with twice the likelihood of MSI+ tumors as MSI- tumors. The increased risk associated with cigarette smoking was not observed among those who had quit smoking at least 15 years earlier. Neither pipe nor cigar smoking was associated with MSI+ tumors. Patterns of associations with risk factors were similar for all markers of MSI. Redefining the population into three categories of tumor stability—stable (referent group), low levels of instability, and unstable—did not appreciably alter the associations between cigarette smoking and unstable tumors, and no relationship was observed between cigarette smoking and low levels of instability. Adjustment for other colon cancer risk factors, including use of aspirin, BMI, physical inactivity, and various dietary factors, did not alter the observed associations.


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Table 4. Associations between cigarette smoking and microsatellite instability*
 

    DISCUSSION
 Top
 Notes
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
 References
 
Of the factors examined in this study, we observed that smoking cigarettes was associated with an increased risk of MSI in tumors. This association was observed for both men and women, and the associated risk increased with length of time of smoking, age starting to smoke, and amount smoked. The association was strongest for current smokers, although an increased risk of having an MSI+ tumor only disappeared after having stopped smoking cigarettes for 15 or more years. Other lifestyle factors, such as physical activity patterns, BMI, and use of aspirin or NSAIDs, were less consistently associated with MSI.

Statistically significant associations between smoking cigarettes and colon cancer risk have been reported in only a few studies, with associations being null or weak and risk estimates of around 1.3–1.4 (1619). Studies that have observed a statistically significant association between cigarette smoking and colon cancer incidence indicate that dose, or amount usually smoked per day (17), is an important predictor of risk. Other studies (20,21) have observed stronger associations, with a relative risk of approximately 2.0, for individuals who reported smoking for 45 years or more. The finding that cigarette smoking is associated with MSI in tumors, although not previously reported, is biologically plausible, in that high levels of cigarette smoke could either generate numerous replication errors and overwhelm the mismatch repair machinery or interfere with mismatch repair itself. The observed association between cigarette smoking and MSI also explains the generally weak association between cigarette smoking and colon cancer risk observed in previously reported studies. If cigarette smoking leads to MSI, which is present in approximately 10%–15% of colon tumors, one would expect low levels of risk associated with cigarette smoking at the population level.

MSI was determined in this study by several markers of instability that did not include the Bethesda Consensus Panel markers, since that panel of markers was suggested after the initiation of this study (22). However, we were able to evaluate associations with several markers of instability and observed similar associations regardless of the indicators of MSI used. In addition, we have shown strong relationships between these measures of instability and the Bethesda panel in a previous study (Samowitz WS, Holden JA, Curtin K, Walker AR, Lynch BJ, Edwards SL, et al.: unpublished data).

Our findings provide a possible explanation for differences in the reported prevalence of genetic mutations in colon tumors among different populations. If smoking influenced the genetic pathway taken by a colon cancer, but not the likelihood of the cancer itself, then populations with a lower proportion of cigarette smokers could be expected to have a lower proportion of MSI+ colon tumors and a higher prevalence of KRAS gene mutations. For instance, in this study, where data were collected from three centers, Utah had the lowest rates of MSI (14.7%) and the lowest rates of current smokers or smokers who stopped smoking cigarettes during the 14 years before the referent date (19.7%). For comparison, 16.6% of the tumors from KPMCP patients were MSI+ and 21.4% of the tumors from patients in Minnesota were MSI+. Corresponding percentages of people who were current cigarette smokers or who stopped smoking cigarettes within the last 14 years were 38.8% and 31.6%, respectively, for Kaiser Permanente and Minnesota. In Utah, where a lower percentage of MSI+ tumors was detected, we also observed a statistically significantly higher percentage of KRAS gene mutations in proximal versus distal tumors. Other environmental factors may further define differences in proportion of colon tumors that have MSI observed between populations.

It is possible that results could be attributed in part to selection bias in the study population. MSI data were obtained for 98% of individuals for whom we obtained tumor blocks. We observed no differences in lifestyle characteristics between those for whom we obtained tumor DNA and those for whom we did not have tumor DNA (11). However, it is impossible to determine if differences in lifestyle and other exposure variables exist between those interviewed and those not interviewed. We do know that prevalence of cigarette smoking in the control population is similar to that reported in population-based surveys from the study populations (17).

In summary, results from this study provide support for an observed association between cigarette smoking and colon cancer risk. On the basis of a twofold increased risk associated with cigarette smoking and MSI in tumors—and the fact that 42.9% of individuals with unstable tumors were either current smokers or had stopped smoking for a period of less than 15 years—we estimate that 21% of unstable tumors in this population could be attributed to cigarette smoking. Smoking is perhaps the largest contributor to MSI in tumors identified to date. The observed associations also provide information that can be valuable in describing disease pathways and the role that nongenetic factors play in those pathways.


    NOTES
 
Supported by Public Health Service grants CA48998 and CA61757 from the National Cancer Institute, National Institutes of Health, Department of Health and Human Services (to M. L. Slattery). Also supported by the Utah Cancer Registry, which is funded by Public Health Service contract N01PC67000 from the National Cancer Institute, with additional support from the State of Utah Department of Health and the University of Utah, the Northern California Cancer Registry, and the Sacramento Tumor Registry.

We acknowledge the contributions and support of Dr. Bette Caan, Judy Morse, and Leslie Palmer to the data collection and tissue processing efforts and Melanie Nichols and Kristen Gruenthal for assistance with genetic analyses.


    REFERENCES
 Top
 Notes
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
 References
 

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Manuscript received April 11, 2000; revised August 23, 2000; accepted September 6, 2000.


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