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© 2000 American Society for Clinical Oncology Population-Based Molecular Detection of Hereditary Nonpolyposis Colorectal CancerFrom the Departments of Medical Genetics and Pathology, Haartman Institute, University of Helsinki; the Family Federation of Finland; Second Department of Surgery, Helsinki University Central Hospital; the Folkhälsan Institute of Genetics, Helsinki; Departments of Surgery of the Central Hospitals of Joensuu, Mikkeli, Lappeenranta, Kajaani, Kotka, Savonlinna, and Jyväskylä; Departments of Surgery and Internal Medicine, Kuopio University Hospital, Kuopio, Finland; and the Human Cancer Genetics Program, Comprehensive Cancer Center, Ohio State University, Columbus, OH. Address reprint requests to Albert de la Chapelle, MD, PhD, Division of Human Cancer Genetics, 646 Medical Research Facility, 420 W 12th Ave, Columbus, OH 43210; email delachapelle-1{at}medctr .osu.edu.
PURPOSE: Cancer morbidity and mortality can be dramatically reduced by colonoscopic screening of individuals with the hereditary nonpolyposis colorectal cancer (HNPCC) syndrome, creating a need to identify HNPCC. We studied how HNPCC identification should be carried out on a large scale in a sensitive and efficient manner. PATIENTS AND METHODS: Colorectal cancer specimens from consecutive newly diagnosed patients were studied for microsatellite instability (MSI). Germline mutations in the MLH1 and MSH2 genes were searched for in MSI(+) individuals. RESULTS: Among 535 colorectal cancer patients, 66 (12%) were MSI(+). Among these, 18 (3.4% of the total) had disease-causing germline mutations in MLH1 or MSH2. Among these 18 patients, five were less than 50 years old, seven had a previous or synchronous colorectal or endometrial cancer, and 15 had at least one first-degree relative with colorectal or endometrial cancer. Notably, 17 (94%) of 18 patients had at least one of these three features, which were present in 22% of all 535 patients. Combining these data with a previous study of 509 patients, mutation-positive HNPCC accounts for 28 (2.7%) of 1,044 cases of colorectal cancer, predicting a greater than one in 740 incidence of mutation-positive individuals in this population. CONCLUSION: Large-scale molecular screening for HNPCC can be done by the described two-stage procedure of MSI determination followed by mutation analysis. Efficiency can be greatly improved by using three high-risk features to select 22% of all patients for MSI analysis, whereby only 6% need to have mutation analysis. Sensitivity is only slightly impaired by this procedure.
GERMLINE DEFECTS IN several DNA mismatch repair genes predispose people to colorectal and endometrial cancer as well as to some other cancer types, a syndrome named hereditary nonpolyposis colorectal cancer (HNPCC) or Lynch syndrome.1-3 Tumors associated with HNPCC are often poorly differentiated, with mucinous features and peritumoral lymphocytic infiltration, yet have no distinct or unique features that could be used for diagnosis of the disease. Instead, the definition of HNPCC has been based on its genetic transmission and occurrence at a young age. The widely used Amsterdam criteria4 require that colorectal cancer occurs in at least three closely related individuals in at least two generations. More recently, other sets of criteria have been proposed to accommodate the fact that other cancers, mainly endometrial cancer, are common manifestations of HNPCC and that family size is shrinking.5 However, clearly, many HNPCC cases are not identified by these criteria, prime examples being individuals with small families or limited knowledge about their families and individuals whose parents and/or siblings died at an early age without acquiring cancer. Whether a family fulfills the clinical criteria or not, a definitive diagnosis of HNPCC can only be established by demonstrating a germline mutation.6 Mutation detection using patient DNA or RNA can be done by several different methods, none of which are 100% sensitive.7 We and others have favored direct exon-by-exon genomic sequencing,8 but this may have to be supplemented by Southern hybridization to detect large deletions.9 By far, most HNPCC cases diagnosed to date are caused by mutations in either MLH1 or MSH2, so it is presently reasonable to limit clinical testing to these two genes,10 perhaps with the addition of MSH6.11-13 Screening for cancer is now available and desirable.14 The need to diagnose HNPCC is presently becoming an increasingly important issue because of recent successes in cancer prevention by colonoscopic screening and the prospect of chemoprevention.15 In a recently completed 15-year prophylactic screening project comprising 252 individuals, colonoscopy at 3-year intervals more than halved colorectal cancer risk and decreased overall mortality by approximately 65% in at-risk and mutation-positive members of HNPCC families (Järvinen et al, manuscript submitted for publication).16,17 In mutation-positive families, there is a need to determine who has and who does not have the mutation. Thus, there is a challenge to diagnose as many HNPCC individuals as possible in an efficient and cost-effective manner. Indeed, HNPCC may be one of a handful of genetic conditions in which large-scale mutation screening is meaningful.18 Because mutational analysis of MLH1 and MSH2 is a work-intensive and expensive undertaking, it is highly desirable to prescreen by a simpler method followed by mutational analysis in a high-risk subset. A formalized approach to the problem was recently published.19 A logistic model for patient selection was proposed. The parameters were composed of the following clinical data: young age at diagnosis of colorectal cancer, fulfillment of the Amsterdam criteria, and the presence of endometrial cancer in the kindred. Patients scoring high-risk values would be scrutinized by molecular methods, mainly germline mutation analysis. We previously tested this formula in a series of colorectal cancer probands. If only first-degree relatives and their cancer status was known, the value of the formula was limited.20 If extensive pedigree data were used, the formula19 was able to identify most, but not all, mutation carriers in our series.20 Unfortunately, extensive pedigrees and family histories are not usually available in clinical practice. Microsatellite instability (MSI) is characteristic of HNPCC tumors21 but occurs in a 10% to 15% subset of sporadic colorectal cancers as well.22 Thus, MSI is a relatively sensitive but nonspecific marker for HNPCC. We proposed the following criteria for primary selection of colorectal cancer patients for MSI testing.8 Colorectal tumors should be tested for MSI when the patient is under 50 years of age, or has multiple primary cancers of the colorectum and/or endometrium, or has a first-degree relative with colorectal or endometrial cancer. Mutation analysis should then be performed in individuals whose tumors score MSI(+). These criteria were designed based on the results of a molecular and genealogic analysis of 509 consecutive colorectal cancer probands.8 In the present study, we prospectively tested these criteria in a series of 535 newly diagnosed colorectal cancer patients. Moreover, by combining the results of both series, we were able to assess the frequency of HNPCC based on a total of 1,044 colorectal cancer patients. We propose a model for the population-based molecular screening for HNPCC targeted at all newly diagnosed colorectal cancer patients.
Patients and Tissue Preparation The study was approved by the appropriate ethics review committees. Cancer and normal tissue specimens were derived from 535 consecutive consenting colorectal cancer patients treated at nine large regional hospitals in southeastern Finland (Table 1). The consent rate was greater than 90%. The individuals ranged in age from 29 to 91 years, with a mean age of 67 years. The fresh-frozen samples were collected between March 1996 and June 1998. All lesions were histologically evaluated before DNA extraction to document proportion of tumor tissue. Out of 535 samples, 520 (97%) contained 50% or more carcinoma tissue. The specimens representing normal mucosa were always derived from a separate site, not from tumor margins.
All first-degree relatives (parents, siblings, and children) of each of the 535 patients were identified through official population registries. On average, eight first-degree relatives were identified. Data on patients and relatives were cross-linked with the Finnish HNPCC registry and the Finnish Cancer Registry to reveal possible connections to previously identified HNPCC families and to derive data on occurrence of cancer in the kindreds. The Finnish Cancer Registry has been functioning since 1953 and has almost complete coverage of histologically or cytologically verified cancer.23-25 All individuals whose tumors were MSI(+) were offered a genetic counseling session. Individuals with germline mutations and their families were offered one or two more counseling sessions.
Analysis of MSI
Detection of Germline Mutations All 535 patients were scrutinized for the two most common mismatch repair gene mutations in Finland. Founder mutation 1 is a 3.5-kilobase genomic deletion of MLH1 comprising exon 16, and founder mutation 2 is MLH1 exon 6 splice site mutation IVS5-1G  A at 454 - 1. Together, these two mutations account for more than half (45 out of 82) of mutation-positive HNPCC families so far diagnosed in Finland.33 Mutation 1 was detected by a PCR-based method that has been described previously.34 Mutation 2 was detected by allele-specific oligonucleotide hybridization, as previously described,34 with the following modifications. PCR-products were run in 2% NuScieve agarose gel (FMC Bioproducts, Rockland, ME) to verify the amplification, thus avoiding the need for hybridization with a wild-type probe. PCR-products from three individuals were pooled together onto the filter. Filters were hybridized with a probe containing the mutant sequence (5' CTT CTG TTC AAG TGG AGG AC 3'). If a positive signal was obtained, the respective samples were rehybridized separately on a new filter. If neither of the founder mutations were detected but the patient’s tumor had displayed MSI, mutation analysis of MLH1 and MSH2 was performed by direct genomic sequencing of the coding exons, including the flanking intronic regions and promoter region, as previously described.8
The significance of a previously unreported missense variant, MSH2 exon 12 D603N (1808C
Missense change G322D (G
Sampling On the basis of Finnish Cancer Registry data on regional cancer incidence,25 we estimate that the sample collection covered approximately 60% of all colorectal carcinomas removed between March 1996 and June 1998 in the collaborating hospitals. The patients provide a representative sampling of the entire population in the area because almost no operations for colorectal cancer are performed in other hospitals in the region. We discuss below (see Discussion) why the patients we accrued were unlikely to be biased for or against HNPCC.
MSI
Mutation Analysis
A previously reported change, MSH2 exon 6 G322D (G A),35,36 was present in one of the MSI(+) patients but also in six (6.7%) out of 89 healthy controls. This change seems to be a neutral variant. Thus, altogether 18 mutations were found among the 535 probands (3.4%).
Fulfillment of Risk Criteria Mutation-positive patients. Among the 18 new HNPCC patients, only five were less than 50 years, seven had a previous or synchronous colorectal or endometrial cancer, and 15 fulfilled the criterion that one first-degree relative had colorectal or endometrial cancer (Table 2). Notably, 17 patients (94%) fulfilled at least one of the three criteria. One patient, a 61-year-old woman, did not (patient no. 700, Table 2). None of her six first-degree relatives had colorectal or endometrial cancer, nor had she had cancer previously. However, when her pedigree was extended, typical HNPCC features became apparent because there were distant genealogically related relatives with early-onset colorectal cancer. This patient would not have been diagnosed if the described criteria had been applied. Based on typical family histories in the entire series of patients, only three fulfilled the original Amsterdam criteria,4 and two additional patients fulfilled the more relaxed Amsterdam criteria.5 Among these five patients, one (C576) was MSI(-). Mutations in MLH1 and MSH2 were sought in this patient but not found. The remaining four patients were MSI(+) and mutation-positive. These data illustrate the relatively high specificity but low sensitivity of the Amsterdam criteria. Even when the population registries were used to enlarge the pedigrees of all 18 HNPCC patients to comprise an average of 38 relatives, six did not fulfill the Amsterdam criteria. None of the 469 patients whose tumors were MSI(-) fulfilled the Amsterdam criteria.
Frequency of HNPCC
Our results allow us to calculate the outcome if this study had been conducted by selecting patients for MSI analysis based on age, previous tumor, and positive family history (Fig 1b). Only 117 patients (22%) would have been selected for MSI analysis, and only 34 of them (6% of the total) would have been MSI(+) and subjected to mutation analysis. Among these 34 patients, 17, or half, would have been diagnosed with a germline mutation, ie, HNPCC. One patient (6%) would have been missed. Figures of a similar nature need to be obtained from other populations and circumstances. For instance, it is not clear whether the existence of founder mutations, such as the ones predisposing to HNPCC in Finland, increases the overall HNPCC incidence. It is also possible, at least in principle, that mismatch repair gene mutations show different penetrances in different populations and circumstances. Nevertheless, the results presented here can be tentatively extrapolated into a true population-based diagnostic endeavor, for instance as follows. In the state of Ohio (population, 10 million) colorectal cancer is diagnosed in 6,100 individuals annually.39 The requirement for MSI testing being 22%, only 1,342 such tests would need to be performed and would disclose 6% of the total or 366 MSI(+) results. Thus, only 366 germline mutational analyses would be needed to diagnose the approximately 165 new mutation-positive HNPCC patients (2.7%) that would occur. Our results suggest that the rate of missed mutations would be low. This scenario, although hypothetical at present, illustrates the general feasibility of population-based approaches to the practice of human cancer genetics in a clinical and population setting. In this scenario, account is not taken of several factors that are likely to reduce its feasibility, such as noncompliance of patients,40 inability to provide even first-degree relative family histories and to assess personal risk,41 lack of interest on the part of physicians, other health personnel, and the society,18 fear of discrimination,42 and cost issues.43 The model we propose relies heavily on the MSI test as a primary screen. A high sensitivity is suggested by the fact that 80% to 95% of HNPCC tumors have been shown in the past to be MSI(+).44,45 However, these tumors emanated from members of previously diagnosed typical HNPCC families. It does not automatically follow that the sensitivity of MSI is equally high in tumors from newly diagnosed, apparently sporadic colorectal cancer patients. Our study provided an excellent test of this in that we screened all 1,044 patients for two founder mutations that are so widespread that they account for over half of all HNPCC in the country. Among the total of 128 MSI(+) patients, 19 were found to have one of these mutations, whereas among 916 MSI(-) patients, no such mutation was found. We consider this to indicate that MSI, when adequately determined, shows high sensitivity for mutation-positive HNPCC tumors. These data also confirm that BAT26 alone is a sensitive indicator of MSI as suggested.29-31 However, the specificity of MSI is low mainly because a large proportion of all MSI(+) tumors are caused by epigenetic silencing of the MLH1 gene, a somatic event caused by promoter methylation.46 We show here that the specificity can be enhanced to approximately 50% by selecting patients fulfilling certain clinical criteria. Only two thirds of the mutation-positive individuals fulfilled the Amsterdam criteria for HNPCC4 even when extensive pedigree information was available, ie, when an average of 38 relatives per proband were identified and their cancer status ascertained (data not shown). We do not presently have enough evidence to evaluate whether the cancer penetrance is lower in HNPCC families identified as described in this article compared with data from previously studied large typical HNPCC families.47 Such data will presumably be forthcoming when further studies become available. Is the 3.4% frequency of detectable HNPCC among 535 unselected colorectal cancer patients a reliable estimate? There could have been biases inflating the figure. For instance, even though the protocol called for accrual of every patient, as many as 40% were missed for a variety of reasons, the most common one being vacation of the surgeon or protocol nurse. We considered whether the patients accrued might have been inadvertently enriched for individuals at higher risk of HNPCC. To evaluate this, we compared the proportion of patients under the age of 50 in our series with the proportion of all patients in this age category in Finland25 and found both to be 8%. Thus, a bias in favor of young age, a major predictor of HNPCC48 did not occur in this series, and we are not aware of other biases either. In contrast, there are reasons to suggest that 3.4% is an underestimate. MSI could show false-negative results either for technical reasons or because the specimen did not contain enough cancer cell nuclei. The sensitivity of sequencing to detect heterozygous mutations in these genes is difficult to evaluate, but it certainly is not 100%.36 Moreover, large deletions that are not detectable by most methods except Southern hybridization exist in MSH2 and could account for up to 10% of all mutations.19 Finally, HNPCC (albeit perhaps with somewhat unusual phenotypic features) has recently been shown to be caused by mutations in MSH6,11-13 but how commonly this occurs is unclear. Thus, the 2.7% figure obtained in this study when combined with a previous figure of ours,8 is in all likelihood an underestimate in the population we studied. If HNPCC accounts for 5% to 10% of all colorectal cancer,1 and as the 2.7% proportion found by us is considerably lower, it is increasingly important to recognize the existence of both mutation-positive and mutation-negative HNPCC.49 In this study, only one patient clearly had mutation-negative [and MSI(-)] HNPCC. Such patients may have yet unknown predisposing genes. We do recommend that they and their relevant family members be offered clinical cancer surveillance.
Supported by grants CA67941 and CA16058 from the National Cancer Institute, contract BMH4-CT96–0772 from the European Commission, the Academy of Finland, the Finnish Cancer Society, the Sigrid Juselius foundation, the Federation of the Finnish Insurance Companies, the Ida Montin Foundation, the Jalmari and Rauha Ahokas Foundation, and the Emil Aaltonen Foundation. We gratefully acknowledge contributions by the Finnish Cancer Registry. We thank Siv Lindroos, Kirsi Laukkanen, Sinikka Lindh, Kirsi Pylvänäinen, and Tuula Lehtinen for assistance.
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TOP
ABSTRACT
INTRODUCTION
-P32ATP using T4-polynucleotide kinase (New England Biolabs Inc, Beverly, MA). Filters were hybridized with a probe containing the mutant sequence (5' CTC AGC TAA ATG CTG TTG TC 3'). If a positive signal was obtained, the respective samples were rehybridized separately on a new filter. 
