[CANCER RESEARCH 63, 7595–7599, November 15, 2003] Advances in Brief Carcinogenesis in MYH-Associated Polyposis Follows a Distinct Genetic Pathway Lara Lipton, 1,2 Sarah E. Halford, 1 Victoria Johnson, 2 Marco R. Novelli, 3 Angela Jones, 1 Carole Cummings, 2 Ella Barclay, 1 Oliver Sieber, 1 Amir Sadat, 2 Marie-Luise Bisgaard, 4 Shirley V. Hodgson, 5 Lauri A. Aaltonen, 6 Huw J. W. Thomas, 2 and Ian P. M. Tomlinson 1,2 1 Molecular and Population Genetics Laboratory, London Research Institute, Cancer Research United Kingdom, London, United Kingdom; 2 Cancer Research United Kingdom Colorectal Cancer Unit, St. Mark’s Hospital, Harrow United Kingdom; 3 Department of Histopathology, University College London Hospitals, London, United Kingdom; 4 Danish Polyposis Registry, Hvidovre Hospital, Copenhagen, Denmark; 5 Department of Clinical Genetics, St. George’s Hospital Medical School, London, United Kingdom; and 6 Department of Medical Genetics, Haartman Institute, University of Helsinki, Finland Abstract Colorectal carcinomas develop according to particular genetic path- ways, including the chromosomal instability (CIN), microsatellite insta- bility (MSI) and MSI CIN routes. We have determined the genetic pathway in patients with MYH-associated polyposis (MAP), a syndrome of colorectal adenomas and cancer that results from defective base excision repair (BER). As in previous studies, MAP tumors showed a high fre- quency of G>T mutations in APC, in accordance with defective BER. We found that K-ras mutations were common in MAP tumors, all of the changes comprising conversion of the first guanine residue of codon 12 to thymidine (G12C, GGT>TGT). We found no BRAF mutations at the codon 599 hotspot or elsewhere in exon 14. Almost all of the MAP cancers were near-diploid (CIN), and none was MSI. A few p53 mutations were found, but these were not predominantly G>T changes. p53 over- expression was, however, frequent. No SMAD4 or TGFBIIR mutations were found. MAP tumors appear to follow a distinct genetic pathway, with some features of both the CIN and MSI pathways. BER deficiency is rarely accompanied by CIN or MSI. The spectrum of somatic mutations in MAP tumors reflects both selection and hypermutation to which certain guanine residues are particularly prone. Introduction Sporadic colorectal carcinomas develop according to particular genetic pathways (1). The most common pathway is characterized by mutations of the APC and p53 genes, by 18q allelic loss, by mutation of K-ras and SMAD4 in some cases, and by an aneuploid/polyploid karyotype. These tumors are often said to have followed the CIN 7 pathway. Other cancers are characterized by MSI, aberrant DNA mismatch repair, a near-diploid karyotype, and lower levels of p53, SMAD4, and K-ras mutation, but higher frequencies of BAX, TGF- BIIR, and BRAF mutation. Yet additional colorectal cancers have neither MSI nor an aneuploid/polyploid karyotype and are termed MSI- CIN- (2). Superimposed on these three pathogenic pathways are additional levels of complexity; some colorectal cancers, for example, have a tendency to high levels of promoter methylation [the so-called CpG island methylator phenotype (CIMP)+ pathway; Ref. 3]. Germline APC mutations generally lead to a phenotype of profuse colonic polyposis (FAP). One or more of these adenomatous polyps usually progresses to cancer (4), probably because of random muta- tions in the same genes involved in the development of sporadic colorectal cancers. It is likely, although not conclusively demon- strated, that FAP polyps can become cancerous because of progres- sion along any of the CIN+, MSI+ or MSI- CIN- pathways (5). In HNPCC, by contrast to FAP, the phenotype is predominantly one of colorectal carcinoma (6). HNPCC is caused by germ-line mutations at the MSH2, MLH1, or MSH6 mismatch repair loci, and cancers in this syndrome generally follow the MSI pathway of tumorigenesis (7). Relatively recently, a FAP-like condition has been found to result not only from germ-line APC mutations, but also from germ-line MYH mutations (8). MYH polyposis (MAP) is usually phenotypically in- distinguishable from a classical or mild form of FAP, but the former is inherited as a recessive trait, with consequent implications for the risk of disease in other family members (9). MYH encodes a glyco- sylase, which is involved in BER and primarily targets oxidative DNA damage. In keeping with the role of MYH, colorectal tumors from MAP patients show an excess of GT transversion mutations in the APC gene because of the failure to repair lesions induced by the variant base 8-oxo-guanine. Whereas changes in MYH expression may have some role in the pathogenesis of sporadic colorectal tumors, there is currently no evidence to show that the gene is mutated or silenced in bowel cancers outside MAP (10). MYH is, in many ways, an unexpected gene for colorectal polyp- osis. Many puzzles regarding its role in tumorigenesis remain. It is not clear, for example, why germ-line MYH mutations lead to tumors of the gastrointestinal tract, or why MAP differs in its phenotype and inheritance from HNPCC. To gain additional clues to explain how MYH mutations lead to multiple colorectal tumors and cancer, we have determined the genetic pathways in 130 colorectal adenomas and 19 carcinomas from 22 MAP patients. Materials and Methods The patients with biallelic germ-line MYH were identified through Family Cancer Clinics and Polyposis Units in the United Kingdom, Finland, Denmark, and Switzerland. MYH mutation testing had been done as part of two previous studies (see Table 1). All of the patients were known to have multiple colorectal adenomas, although precise adenoma counts were not available for some patients. Most of our patient sample had developed one or more colo- rectal cancers, and 19 of 26 of these were available for molecular analysis. Constitutional DNA was extracted from peripheral blood lymphocytes by standard methods. Both fresh-frozen and fixed, paraffin-embedded tumor tissues were used for the analysis, depending on their availability. DNA was extracted from the former using a standard proteinase K and phenol-chloro- form method. For the latter, 5  10 m unstained tumor sections were dewaxed and dissected into an appropriate amount of digestion buffer (1 magnesium-free buffer, 20 g/ml Proteinase K) using a H&E-stained slide as a guide for the area to be microdissected. Received 8/8/03; revised 9/16/03; accepted 9/17/03. Grant support: European Commission (QLG2-CT-2001-01861). L. A. A. was sup- ported by the Academy of Finland (44870, Finnish Center of Excellence Programme 2000 –2005). L. L. and S. H. are Translational Fellows. L. L. is supported by the Bobby Moore Fund. O. S. is funded by Boehringer Ingelheim Fonds. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Requests for reprints: Ian Tomlinson, Molecular and Population Genetics Labora- tory, London Research Institute, Cancer Research United Kingdom, London WC2A 3PX, United Kingdom. 7 The abbreviations used are: CIN, chromosomal instability; MSI, microsatellite in- stability; MAP, MYH-associated polyposis; BER, base excision repair; FAP, familial adenomatous polyposis; HNPCC, hereditary nonpolyposis colon cancer; F-SSCP, fluo- rescent single-stranded conformational polymorphism; dHPLC, denaturing high pressure liquid chromatography; LOH, loss of heterozygosity. 7595 Research. on February 9, 2016. © 2003 American Association for Cancer cancerres.aacrjournals.org Downloaded from