APC mutations in FAP-associated desmoid tumours are non-random but not ‘just right’

Human Molecular Genetics, 2007, Vol. 16, No. 1 doi:10.1093/hmg/ddl442 Advance Access published on November 29, 2006 78–82 APC mutations in FAP-associated desmoid tumours are non-random but not ‘just right’ Andrew Latchford1,2, Emmanouil Volikos3, Victoria Johnson2, Pauline Rogers2, Nirosha Suraweera3, Ian Tomlinson3,4, Robin Phillips1 and Andrew Silver3,* 1 Polyposis Registry and 2Colorectal Cancer Genetics, Cancer Research UK, St Mark’s Hospital, Harrow, Middlesex HA1 3UJ, UK, 3Institute of Cell and Molecular Science and Barts and the London Queen Mary’s School of Medicine and Dentistry, London E1 2AT, UK and 4Molecular and Population Genetics Laboratory, London Research Institute, Cancer Research UK, London WC2A 3PX, UK Received September 15, 2006; Revised November 7, 2006; Accepted November 16, 2006 INTRODUCTION Familial adenomatous polyposis (FAP) is an autosomal, dominantly inherited, Mendelian cancer syndrome caused by germline mutations in the adenomatous polyposis coli (APC) gene, which encodes a large multifunctional protein. FAP is characterized by the development of hundreds or thousands of colorectal polyps, which almost inevitably progress to colorectal cancer unless prophylactic colectomy is undertaken. Following surgery, the mortality of patients with FAP remains in excess of the general population, due to extra-colonic manifestations such as upper gastrointestinal polyps and desmoid tumours (1). Desmoid tumours are poorly understood, frequently aggressive tumours of mesenchymal origin, which arise in musculoaponeurotic structures. Intra-abdominal desmoids often appear to be surgically induced—typically by prophylactic colectomy—but can arise in the absence of surgery (2). The incidence of desmoids in FAP has been estimated to be around 850 times that of the general population and 10 –25% of FAP patients will develop at least one desmoid within their lifetime (3 –6). A number of independent predictors of increased desmoid risk have been identified, including female gender, a family history of desmoids and a germline mutation distal to codon 1399 (7,8). APC is a tumour suppressor gene, and consistent with Knudson’s two-hit hypothesis, both alleles are mutated in FAP-associated intestinal tumours: one in the germline and the other in the soma (9 – 12). Somatic APC mutations, *To whom correspondence should be addressed at. Tel: +44 02078822590; Fax: +44 02078822200; Email: # The Author 2006. Published by Oxford University Press. All rights reserved. For Permissions, please email: Analysis of APC mutations in colonic and duodenal tumours from familial adenomatous polyposis (FAP) patients has shown that the site of the first hit, the germline mutation, can predict the type and position of the somatic mutation or ‘second hit’. The two APC mutations are selected on the basis of a ‘just right’ level of beta-catenin signalling in intestinal tumours achieved through retention of some of the seven 20-amino-acid beta-catenin degradation repeats. Desmoids are a life threatening extra-colonic manifestation in FAP patients. These aggressive tumours of mesenchymal origin are, at present, poorly characterized in terms of mutational APC spectra. We have investigated somatic mutations in the largest cohort of FAP-associated desmoids to date, and combined our results with previously published data. Somatic mutations were found to occur non-randomly and the position of the germline mutation shown to be a major determinant of the somatic mutation, a characteristic shared with intestinal tumours from FAP patients. In contrast to colonic polyps, loss of heterozygosity in desmoids involved deletion rather than mitotic recombination. While tumours from the colorectum and upper gastrointestinal tract usually retain one to two and three to four beta-catenin degradation repeats, respectively, most desmoids preferentially retain two repeats (P < 0.001, x 2 test). In addition, most desmoids with two APC hits (87%, 26/30) had one mutated allele with no 20-amino acid repeats (P < 0.001). This feature, unique among FAP tumours, indicates that a mutation deleting all repeats from one allele may be an important component in maintaining appropriate levels of beta-catenin signalling levels in desmoid tumour cells. Human Molecular Genetics, 2007, Vol. 16, No. 1 79 truncating mutations ((0/8) P ¼ 0.002, Fisher’s exact test). The majority of desmoids from the latter group of patients displayed allelic loss as second hit (6/8) compared to none of the desmoids from patients with germline mutations proximal to 1400 (0/9; P ¼ 0.002, Fisher’s exact test). The total number of 20-AARs remaining in desmoid cells after the first and second hit was established for the St Mark’s cohort of 15 samples (Ds1 –15) and for a combined set of 30 tumours which included a further 15 desmoids where the germline and somatic mutations have been reported previously in the literature (summarized in Table 2) (13). Tumours with LOH as the somatic mutation were included only if the mechanism of LOH—deletion or mitotic recombination—was known; allelic loss by deletion was considered to remove all beta-catenin degradation repeats. As with the St Mark’s samples, interdependence between the first hit and second hit was found to be significant (St Mark’s P ¼ 0.01; Combined, P , 0.001; Fisher’s exact test). The majority of desmoids with two APC hits (26/30) were found to have one mutated allele with zero 20-AARs (P , 0.001, Fisher’s exact test) and most null alleles were contributed by the germline (17/26) rather than the soma (9/26), although this was of borderline significance (P ¼ 0.05, Fisher’s exact test). We now examined whether the number of 20-AARs left intact after germline and somatic mutation arose by chance or were determined by factors intrinsic to cell survival and proliferation. For any given germline mutation, the number of 20-AARs left intact by the somatic mutation was not uniformly distributed (P , 0.001, Kolmogorov-Smirnov test). Hence, the position of the somatic mutation was not determined by chance. To investigate this further, the observed and expected number (calculated by assuming a uniform distribution of somatic mutations) of retained repeats were compared using the combined data set and found to be highly significant (x 2 with four degrees of freedom ¼ 58.967; P , 0.0001, chi-squared goodness of fit test; Table 3). Ungrouped data shows that, overall, by far the biggest contribution was for a sum equal to two repeats ((O-E)2/E ¼ 52.071; Table 3); retention of two beta-catenin degradation repeats was observed much more frequently than would be expected by chance. RESULTS DISCUSSION Somatic mutations were identified in 19/23 tumours (83%): truncating somatic mutations in 63% (12/19), allelic loss in 32% (6/19) and exonic deletion in 5% (1/19) of desmoids investigated. LOH was identified in six tumours using microsatellite markers; MLPA analysis was successful in four of these desmoi (...truncated)