Recommendations to improve identification of hereditary and familial colorectal cancer in Europe.

Familial colorectal cancer (CRC) accounts for 10-15% of all CRCs. In about 5% of all cases, CRC is associated with a highly penetrant dominant inherited syndrome. The most common inherited form of non-polyposis CRC is the Lynch syndrome which is responsible for about 2-4% of all cases. Surveillance of individuals at high risk for CRC prevents the development of advanced CRC. About 1 million individuals in Western Europe are at risk for Lynch syndrome. We performed a survey to evaluate the strategies currently used to identify individuals at high risk for CRC in 14 Western European countries. Questionnaires were distributed amongst members of a European collaborative group of experts that aims to improve the prognosis of families with hereditary CRC. The survey showed that in all countries obtaining a family history followed by referral to clinical genetics centres of suspected cases was the main strategy to identify familial and hereditary CRC. In five out of seven countries with a (regional or national) CRC population screening program, attention was paid in the program to the detection of familial CRC. In only one country were special campaigns organized to increase the awareness of familial CRC among the general population. In almost all countries, the family history is assessed when a patient visits a general practitioner or hospital. However, the quality of family history taking was felt to be rather poor. Microsatellite instability testing (MSI) or immunohistochemical analysis (IHC) of CRC are usually recommended as tools to select high-risk patients for genetic testing and are performed in most countries in patients suspected of Lynch syndrome. In one country, IHC was recommended in all new cases of CRC. In most countries there are no specific programs on cancer genetics in the teaching curriculum for medical doctors. In conclusion, the outcome of this survey and the discussions within an European expert group may be used to improve the strategies to identify individuals at high risk of CRC. More attention should be given to increasing the awareness of the general population of hereditary CRC. Immunohistochemical analysis or MSI-analysis of all CRCs may be an effective tool for identifying all Lynch syndrome families. The cost-effectiveness of this approach should be further evaluated. All countries with a CRC population screening program should obtain a full family history as part of patient assessment.

Inherited predisposition to colorectal adenomas caused by multiple rare alleles of MUTYH but not OGG1, NUDT1, NTH1 or NEIL 1, 2 or 3.

BACKGROUND: MUTYH-associated polyposis (MAP) is a recessive trait characterised by multiple colorectal adenomas and a high risk of colorectal cancer. MUTYH functions in the DNA base excision repair pathway and has a key role in the repair of oxidative DNA damage. OBJECTIVES: To assess the contribution of inherited variants in genes involved in base excision repair and oxidative DNA damage including MUTYH, OGG1, NEIL1, NEIL2, NEIL3, NUDT1 and NTH1 to the multiple colorectal adenoma phenotype. METHODS: Inherited variants of MUTYH, OGG1, NEIL1, NEIL2, NEIL3, NUDT1 and NTH1 were sought in 167 unrelated patients with multiple colorectal adenomas whose family histories were consistent with recessive inheritance. These variants were also characterised in approximately 300 population controls. RESULTS: Thirty-three patients (20%) and no controls were MUTYH homozygotes or compound heterozygotes (ie, carried two mutations) and therefore had MAP. Eight different pathogenic MUTYH mutations were identified, of which four were novel. MAP cases had significantly more adenomas than non-MAP cases (p = 0.0009; exact test for trends in proportions) and presented earlier (p = 0.013; analysis of variance). Twenty-four protein-altering variants were identified upon screening of OGG1, NEIL1, NEIL2, NEIL3, NUDT1 and NTH1. However, all combinations of two (or more) variants that were identified at an individual locus in patients were also seen in controls, and no variants were significantly over-represented (or under-represented) in cases. CONCLUSION: Multiple rare alleles of MUTYH are associated with autosomal recessive MAP, while OGG1, NEIL1, NEIL2, NEIL3, NUDT1 and NTH1 do not contribute significantly to autosomal recessive polyposis.

Molecular testing for somatic mutations in common cancers: the views of UK oncologists.

BACKGROUND: Somatic mutations are important determinants of cancer behaviour and response to therapy. However, molecular testing in this context has a relatively low profile within the clinical community, despite publicity surrounding targeted therapies such as Herceptin. AIMS: As the testing process affects many stakeholders, especially oncologists, this paper examines current test request patterns and views of such testing. METHODS: A postal questionnaire was mailed to 582 UK oncologists and haematologists, achieving a 20% response rate. RESULTS: The survey revealed that immunohistochemistry and fluorescent in situ hybridisation are the most commonly requested tests (used by 70% and 55% of respondents, respectively), especially for breast cancer. Availability of suitable treatment options is the main factor influencing the decision to test (selected by 62% of respondents). Respondents were generally positive about future demand for immunohistochemistry, fluorescent in situ hybridisation, microarray analysis and DNA-based tests, but uncertain about the prospects for microsatellite instability and ploidy testing. CONCLUSIONS: Overall, respondents thought that somatic mutation testing could have a significant and positive effect on oncology and haematology departments and patient care, especially with better treatment and tumour classification. However, lack of supportive scientific evidence and funding were considered key barriers to widespread testing. Further research is clearly required on both the resource implications of this increase in demand and the best model of service delivery to ensure the most efficient use of health service resources.

Molecular testing for somatic cancer mutations: a survey of current and future testing in UK laboratories.

BACKGROUND: Genetics clinical practice has paid limited attention to non-inherited aspects of cancer, namely mutations occurring during carcinogenesis. These somatic mutations are likely to be the primary determinants of cancer behaviour and treatment response, with a recent example being Her2/Neu gene status and response to Herceptin in breast cancer. AIM: To assess the feasibility of widespread testing of tumours by surveying UK histopathology and genetics laboratories. METHODS: The questionnaire asked: which of the common cancers or other malignancies are routinely assessed; which molecular and cytogenetic methods are used; who orders and funds testing; what is the future demand for somatic testing; and what are the barriers to widespread testing? RESULTS: Of 50 laboratories surveyed, 33 responded, 22 of which are currently using molecular tests. The survey shows that the most common tests are immunohistochemistry, fluorescence in-situ hybridisation and DNA testing of somatic mutations. Most laboratories predict testing will increase over the next 10 years, particularly for DNA testing using microarrays. Respondents perceived the main barriers to expanding molecular testing were a lack of laboratory funding and scientific evidence and testing not considered an NHS priority. CONCLUSION: These results provide important information for healthcare commissioners faced with managing demand for molecular testing of cancers.

Guidelines for the clinical management of familial adenomatous polyposis (FAP).

BACKGROUND: Familial adenomatous polyposis (FAP) is a well-described inherited syndrome, which is responsible for <1% of all colorectal cancer (CRC) cases. The syndrome is characterised by the development of hundreds to thousands of adenomas in the colorectum. Almost all patients will develop CRC if they are not identified and treated at an early stage. The syndrome is inherited as an autosomal dominant trait and caused by mutations in the APC gene. Recently, a second gene has been identified that also gives rise to colonic adenomatous polyposis, although the phenotype is less severe than typical FAP. The gene is the MUTYH gene and the inheritance is autosomal recessive. In April 2006 and February 2007, a workshop was organised in Mallorca by European experts on hereditary gastrointestinal cancer aiming to establish guidelines for the clinical management of FAP and to initiate collaborative studies. Thirty-one experts from nine European countries participated in these workshops. Prior to the meeting, various participants examined the most important management issues according to the latest publications. A systematic literature search using Pubmed and reference lists of retrieved articles, and manual searches of relevant articles, was performed. During the workshop, all recommendations were discussed in detail. Because most of the studies that form the basis for the recommendations were descriptive and/or retrospective in nature, many of them were based on expert opinion. The guidelines described herein may be helpful in the appropriate management of FAP families. In order to improve the care of these families further, prospective controlled studies should be undertaken.

Detection of copy number changes at the NF1 locus with improved high-resolution array CGH.

Neurofibromatosis type 1 (NF1) is a common autosomal dominant disease caused by various types of mutations in the NF1 gene. We have previously developed a locus-specific DNA microarray for detection of copy number changes at the NF1 locus by comparative genomic hybridization (CGH) analysis. The original array contains 183 probes pooled from 444 polymerase chain reaction (PCR) products. In the current work, we have used 493 probes derived from single PCR products (200--998 bp in size) to construct a higher resolution array with a smaller average probe size for molecular diagnosis of NF1. This has improved the average resolution from 12.6 kb in the previous array to 4.5 kb in the current version. The performance of the newly constructed microarray was validated with 14 well-characterized NF1 mutations for CGH analysis. These mutations represent deletions from approximately 7 kb to over 2 Mb in size. Using this array, we examined a total of 55 NF1 patients for copy number changes at the NF1 locus, detecting deletions in four of them. These results demonstrate that a locus-specific microarray constructed from single PCR products can efficiently detect copy number changes at the NF1 locus, providing a simple method for the molecular diagnosis of NF1.

Molecular classification and genetic pathways in hyperplastic polyposis syndrome.

Hyperplastic Polyposis (HPPS) is a poorly characterized syndrome that increases colorectal cancer (CRC) risk. We aimed to provide a molecular classification of HPPS. We obtained 282 tumours from 32 putative HPPS patients with >or= 10 hyperplastic polyps (HPs); some patients also had adenomas and CRCs. We found no good evidence of microsatellite instability (MSI) in our samples. The epithelium of HPs was monoclonal. Somatic BRAF mutations occurred in two-thirds of our patients' HPs, and KRAS2 mutations in 10%; both mutations were more common in younger cases. The respective mutation frequencies in a set of 'sporadic' HPs were 18% and 10%. Importantly, the putative HPPS patients generally fell into two readily defined groups, one set whose polyps had BRAF mutations, and another set whose polyps had KRAS2 mutations. The most plausible explanation for this observation is that there exist different forms of inherited predisposition to HPPS, and that these determine whether polyps follow a BRAF or KRAS2 pathway. Most adenomas and CRCs from our putative HPPS patients had 'classical' morphology and few of these lesions had BRAF or KRAS2 mutations. These findings suggest that tumourigenesis in HPPS does not necessarily follow the 'serrated' pathway. Although current definitions of HPPS are sub-optimal, we suggest that diagnosis could benefit from molecular analysis. Specifically, testing BRAF and KRAS2 mutations, and perhaps MSI, in multiple polyps could help to distinguish HPPS from sporadic HPs. We propose a specific model which would have diagnosed five more of our cases as HPPS compared with the WHO clinical criteria.

Genetic counselling and consent for tumour testing in HNPCC.

Molecular pathological tests are performed on stored tumour material in order to identify individuals with hereditary non-polyposis colorectal cancer. We have previously identified that there is widespread use of this testing and now describe what counselling occurs prior to testing and the approaches in seeking consent. A respondent from every cancer genetic centre in UK offering microsatellite instability and/or immunohistochemistry testing (n= 20, response rate = 100%) was interviewed in order to ascertain pre-test counselling and consent protocols. Individuals providing consent are not always seen in person prior to providing consent but few services had supporting written information. Nine (of 19) consent forms documented consent to perform genetic testing, while the majority (14/19) sought consent to release pathology samples to the genetic service. Less than half of the services routinely seek consent to test samples from a deceased individual. Concerns were raised about spousal consent when the implications of results are for blood relatives. The differences identified between genetic counselling for testing of tumour tissue and for germ-line genetic testing suggest that counselling protocols specific for somatic testing should be developed. The results are discussed in the context of a changing legal environment and anticipated growing demand for testing.

Guidelines for the clinical management of Lynch syndrome (hereditary non-polyposis cancer).

Lynch syndrome (hereditary non-polyposis colorectal cancer) is characterised by the development of colorectal cancer, endometrial cancer and various other cancers, and is caused by a mutation in one of the mismatch repair genes: MLH1, MSH2, MSH6 or PMS2. The discovery of these genes, 15 years ago, has led to the identification of large numbers of affected families. In April 2006, a workshop was organised by a group of European experts in hereditary gastrointestinal cancer (the Mallorca-group), aiming to establish guidelines for the clinical management of Lynch syndrome. 21 experts from nine European countries participated in this workshop. Prior to the meeting, various participants prepared the key management issues of debate according to the latest publications. A systematic literature search using Pubmed and the Cochrane Database of Systematic Reviews reference lists of retrieved articles and manual searches of relevant articles was performed. During the workshop, all recommendations were discussed in detail. Because most of the studies that form the basis for the recommendations were descriptive and/or retrospective in nature, many of them were based on expert opinion. The guidelines described in this manuscript may be helpful for the appropriate management of families with Lynch syndrome. Prospective controlled studies should be undertaken to improve further the care of these families.

Variability and inequity in testing of somatic tissue for hereditary cancer: a survey of UK clinical practice.

A growing body of literature demonstrates the benefits of molecular pathological investigations of tumour material in the identification of individuals with hereditary non-polyposis colorectal cancer and debates the best detection strategies. This testing is novel as it is the first widespread use of somatic tissue testing to inform genetic analysis and requires the co-ordination of both histopathology and molecular genetics laboratories. However, the clinical use and experience of microsatellite instability (MSI) testing and immunohistochemical analysis have not been reported. A respondent from every cancer genetics centre in the UK (n= 24, response rate 100%) and laboratory performing MSI testing (n= 5, response rate 100%) was interviewed by telephone to ascertain test availability, testing methods, eligibility criteria and post-test management. Twenty centres (83%) offer eligible clients at least one form of tumour testing, and all use tumour testing to determine who should have access to germ line genetic testing. However, no two laboratories used the same testing methods, seven different testing strategies were applied and there was considerable variation in eligibility criteria. The implications of these variations are considered, and recommendations for the development of a consistent service for testing of somatic tissue offered.

A prospective study of neurofibromatosis type 1 cancer incidence in the UK.

Neurofibromatosis type 1 (NF1) is an autosomal dominant condition affecting around one in 3000 live births. The manifestations of this condition are extremely variable, even within families, and genetic counselling is consequently difficult with regard to prognosis. Individuals with NF1 are acknowledged to be at increased risk of malignancy. Several studies have previously attempted to quantify this risk, but have involved relatively small study populations. We present prospective data from 448 individuals with NF1 with a total of 5705 years of patient follow-up. These data have been collected via the UK NF1 association for patients. Demographic information on the affected individuals was cross-referenced with UK cancer registry data by the UK Office of National Statistics. The overall risk of cancer was 2.7 times higher in this cohort of NF1 patients than in the general population (95% confidence interval (CI) 1.9-3.7). The cumulative risk of a malignancy by age 50 years was 20% (95% CI 14-29%); beyond this age, the risk of cancer was not significantly elevated (P=0.27). The most frequent types of cancer were connective tissue (14% risk by age 70, 95% CI 7.8-24%) and brain tumours (7.9, 95% CI 3.9-16%). There was no statistically significant excess of cancers at other sites (P=0.22).

Disease severity and genetic pathways in attenuated familial adenomatous polyposis vary greatly but depend on the site of the germline mutation.

BACKGROUND: Attenuated familial adenomatous polyposis (AFAP) is associated with germline mutations in the 5', 3', and exon 9 of the adenomatous polyposis coli (APC) gene. These mutations probably encode a limited amount of functional APC protein. METHODS AND RESULTS: We found that colonic polyp number varied greatly among AFAP patients but members of the same family tended to have more similar disease severity. 5' Mutants generally had more polyps than other patients. We analysed somatic APC mutations/loss of heterozygosity (LOH) in 235 tumours from 35 patients (16 families) with a variety of AFAP associated germline mutations. In common with two previous studies of individual kindreds, we found biallelic changes ("third hits") in some polyps. We found that the "third hit" probably initiated tumorigenesis. Somatic mutation spectra were similar in 5' and 3' mutant patients, often resembling classical FAP. In exon 9 mutants, in contrast, "third hits" were more common. Most "third hits" left three 20 amino acid repeats (20AARs) on the germline mutant APC allele, with LOH (or proximal somatic mutation) of the wild-type allele; but some polyps had loss of the germline mutant with mutation leaving one 20AAR on the wild-type allele. CONCLUSIONS: We propose that mutations, such as nt4661insA, that leave three 20AARs are preferentially selected in cis with some AFAP mutations because the residual protein function is near optimal for tumorigenesis. Not all AFAP polyps appear to need "three hits" however. AFAP is phenotypically and genetically heterogeneous. In addition to effects of different germline mutations, modifier genes may be acting on the AFAP phenotype, perhaps influencing the quantity of functional protein produced by the germline mutant allele.

Systematic review of genetic influences on the prognosis of colorectal cancer.

BACKGROUND: In terms of genetics, colorectal cancer is one of the best understood of all malignant diseases. Genetic influences on prognosis may have far-reaching implications, especially for the design of surgical and chemoradiotherapeutic regimens. However, their significance in determining prognosis remains unclear. This study aimed to review the literature on the specific role of key genes in determining the survival of patients with colorectal cancer. METHODS: A Medline search was carried out to identify all original scientific papers relating colorectal cancer genetics to patient survival, up to December 2002. Cochrane and Embase databases were also searched. Identified articles were retrieved and searched carefully for additional information. This review includes K-ras, p53, DCC, NM23 and DNA mismatch repair genes. RESULTS AND CONCLUSION: Conflicting evidence exists as to the prognostic significance of genes commonly implicated in the pathogenesis of colorectal carcinoma. Possible causes for such discrepancy include differences in study methods and laboratory techniques, variable duration of follow-up, statistical differences in study power, and heterogeneity in study populations. Future studies should adopt standardized protocols to define clinically relevant genetic observations.

The spectrum of p53 mutations in colorectal adenomas differs from that in colorectal carcinomas.

BACKGROUND: p53 mutations are frequently observed in colorectal carcinomas but they have also been found in colorectal adenomas, although considerably less frequently. AIMS: To explore p53 mutations in benign tumours, we have screened 70 colorectal adenomas for allelic loss at, and point mutations in, TP53 by analysis of selected microdissected cell populations. RESULTS: Sixteen (22.8%) adenomas were found to have allelic loss, of which 11 (15.7%) had p53 mutations. In adenomas with mild, moderate, or severe dysplasia, mutation or allelic loss occurred in 4.8%, 16.7%, and 52.6%, respectively (p<0.001). Seven different mutations were found, all missense changes or inframe deletions: one (Thr150Arg) has not been found before while three (Gln144His, Gly245Arg, and Glu285Gln) have not been described previously in colorectal tumours. The other three mutations (Arg175Gly, DeltaPro190, and Gly245Ser) have been found in colorectal carcinomas, the last commonly. Adenomas harboured a spectrum of p53 mutations which was significantly different from cancers as regards the position in the gene and a higher frequency of G-->C/C-->G changes. CONCLUSIONS: Combining our data on adenomas with data already published and in comparison with the spectrum of mutations in colorectal carcinomas, it is suggested that some p53 mutations have a weaker effect than others and are therefore more likely to be found in adenomas which have not progressed to carcinomas.

Methods of molecular analysis: mutation detection in solid tumours.

Most mutation detection techniques are unsuitable for routine use on solid tumours. Important parameters include sensitivity, specificity, efficiency, use of existing resources, and cost. In the UK, < 0.2% of service genetics laboratory activity involves mutation analysis in tumours (usually for family studies), mainly because it is time consuming/labour intensive (thus expensive) and DNA extracted from formalin fixed, paraffin wax embedded tissue is of low quality and yield. The small size of DNA fragments obtained from tissue blocks limits the polymerase chain reaction, the basis of most mutation detection methods. Other, biological, factors include: (1) heterogeneity of mutations within and between tumours, (2) variation in type and site of mutations in any one gene, (3) normal tissue harbouring mutations, (4) few genes are mutated in most of any one tumour type, and (5) few clinically useful correlations with genetic changes have been found. Present research is centred on correlating single gene mutations with various clinicopathological features, but the pattern of mutations in a combination of genes will probably prove more useful. Microsatellite instability, however, appears to be worth testing for in both familial and sporadic tumours, particularly of the colorectum.

Germline APC variants in patients with multiple colorectal adenomas, with evidence for the particular importance of E1317Q.

Mendelian tumour syndromes are caused by rare mutations, which usually lead to protein inactivation. Few studies have determined whether or not the same genes harbour other, more common variants, which might have a lower penetrance and/or cause mild disease, perhaps indistinguishable from sporadic disease and accounting for a considerable proportion of the unexplained inherited risk of tumours in the general population. Germline variants at the APC locus are excellent candidates for explaining why some individuals are predisposed to colorectal adenomas, but do not have the florid phenotype of familial adenomatous polyposis. We have screened 164 unrelated patients with 'multiple' (3-100) colorectal adenomas for germline variants throughout the APC gene, including promoter mutations. In addition to three Ashkenazi patients with I1307K, we found seven patients with the E1317Q variant. E1317Q is significantly associated with multiple colorectal adenomas (OR = 11. 17, 95% CI = 2.30-54.3, p < 0.001), accounting for approximately 4% of all patients with multiple colorectal adenomas. In addition, four patients with truncating APC variants in exon 9 or in the 3' part of the gene were identified. Germline APC variants account for approximately 10% of patients with multiple adenomas. Unidentified predisposition genes almost certainly exist. We argue that it is worthwhile to screen multiple adenoma patients for a restricted number of germline APC variants, namely the missense changes E1317Q and I1307K (if of Ashkenazi descent), and, if there is a family history of colorectal tumours, for truncating mutations 5' to exon 5, in exon 9 and 3' to codon 1580.

Allelic loss at SMAD4 in polyps from juvenile polyposis patients and use of fluorescence in situ hybridization to demonstrate clonal origin of the epithelium.

Juvenile polyposis syndrome (JPS; Online Mendelian Inheritance in Man2 174900) is a rare Mendelian disorder in which individuals have typical hamartomatous polyps within the gastrointestinal tract. The stromal element of the polyps has classically been thought to be the proliferative component, although epithelial malignancies (largely gastrointestinal cancers) occur more frequently than expected in JPS patients. Germ-line mutations in SMAD4 (DPC4) account for about a third of JPS cases. It has been postulated that the apparent paradox of a stromal lesion predisposing to epithelial malignancy can be resolved by the "landscaper" effect: an abnormal stromal environment affects the development of adjacent epithelial cells, and the resulting regeneration of damaged epithelium leads to an increased risk of cancer. We have found allele loss at the SMAD4 locus on 18q in polyps from JPS individuals with a germ-line SMAD4 mutation, showing that SMAD4 is acting as a tumor suppressor gene in JPS polyps, as it does in sporadic cancers of the gastrointestinal tract. Interphase fluorescence in situ hybridization showed deletion of one copy of SMAD4 in the epithelial component of JPS polyps, but not in the inflammatory infiltrate. Fluorescence in situ hybridization also suggested that a single copy of SMAD4 was present in stromal fibroblasts of JPS polyps. Thus, biallelic inactivation of SMAD4 occurs in both the epithelium and some of the stromal cells in these lesions, suggesting a common clonal origin. Epithelial malignancies almost certainly develop in juvenile polyposis through direct malignant progression of the epithelial component of the hamartomas. SMAD4/DPC4 probably acts as a "gatekeeper" tumor suppressor in juvenile polyps, and there is no need to invoke a "landscaper hypothesis."

Desmoids in familial adenomatous polyposis are monoclonal proliferations.

Desmoids are poorly-understood, locally aggressive, non-metastasizing fibromatoses that occur with disproportionate frequency in patients with familial adenomatous polyposis (FAP). Their nature is controversial with arguments for and against a neoplastic origin. Neoplastic proliferations are by definition monoclonal, whereas reactive processes originate from a polyclonal background. We examined clonality of 25 samples of desmoid tissue from 11 female FAP patients by assessing patterns of X-chromosome inactivation to calculate a clonality ratio. Polymerase chain reaction (PCR) amplification of a polymorphic CAG short tandem repeat (STR) sequence adjacent to a methylation-sensitive restriction enzyme site within the human androgen receptor (HUMARA) gene using fluorescent-labelled primers enabled analysis of PCR products by Applied Biosystems Genescan II software. Twenty-one samples from nine patients were informative for the assay. Samples from all informative cases comprised a median of 66% (range 0-75%) clonal cells but from the six patients with a clonality ratio < or =0.5 comprised a median of 71% (65-75%) clonal cells. FAP-associated desmoid tumours are true neoplasms. This may have implications in the development of improved treatment protocols for patients with these aggressive tumours.

Prevalence of the APC E1317Q variant in colorectal cancer patients.

The notion that some common variants of APC might confer an increased colorectal tumour risk is supported by studies of the I1307K polymorphism. Recently it has been proposed that the E1317Q variant is also associated with an increased risk. We have studied the prevalence of E1317Q in 364 colorectal cancer patients and in 290 controls. Two patients were shown to possess E1317Q. Neither had a family history of colorectal cancer or co-existent adenomatous polyps. Two controls also carried E1317Q. This finding suggests that E1317Q is unlikely to be associated with anything more than a moderate increase in risk of colorectal cancer.

The type of somatic mutation at APC in familial adenomatous polyposis is determined by the site of the germline mutation: a new facet to Knudson's 'two-hit' hypothesis.

APC is often cited as a prime example of a tumor suppressor gene. Truncating germline and somatic mutations (or, infrequently, allelic loss) occur in tumors in FAP (familial adenomatous polyposis). Most sporadic colorectal cancers also have two APC mutations. Clues from attenuated polyposis, missense germline variants with mild disease and the somatic mutation cluster region (codons 1,250-1,450) indicate, however, that APC mutations might not result in simple loss of protein function. We have found that FAP patients with germline APC mutations within a small region (codons 1,194-1,392 at most) mainly show allelic loss in their colorectal adenomas, in contrast to other FAP patients, whose 'second hits' tend to occur by truncating mutations in the mutation cluster region. Our results indicate that different APC mutations provide cells with different selective advantages, with mutations close to codon 1,300 providing the greatest advantage. Allelic loss is selected strongly in cells with one mutation near codon 1,300. A different germline-somatic APC mutation association exists in FAP desmoids. APC is not, therefore, a classical tumor suppressor. Our findings also indicate a new mechanism for disease severity: if a broader spectrum of mutations is selected in tumors, the somatic mutation rate is effectively higher and more tumors grow.