Fundic gland polyps in familial adenomatous polyposis: neoplasms with frequent somatic adenomatous polyposis coli gene alterations

Fundic gland polyps (FGPs) are the most common gastric polyps in patients with familial adenomatous polyposis (FAP). FGPs have traditionally been regarded as nonneoplastic, possibly hamartomatous lesions, but the pathogenesis of FGPs in both FAP and sporadic patients remains unclear. FGPs in FAP can show foveolar dysplasia, and rarely invasive gastric adenocarcinoma has been reported in patients with FAP and fundic gland polyposis. Using direct gene sequencing and allelic loss assays at 5q, we analyzed somatic adenomatous polyposis coli (APC) gene alterations in 41 FAP-associated FGPs (20 with foveolar dysplasia, six indefinite for dysplasia, and 15 nondysplastic) and 13 sporadic FGPs. The foveolar epithelium and dilated fundic glands of the polyps were separately microdissected and analyzed in 25 of 41 FAP-associated FGPs and 13 of 13 sporadic FGPs. Somatic APC gene alterations were identified frequently (21 of 41 cases, 51%) in FAP-associated FGPs. Both the foveolar epithelium and the dilated fundic gland epithelium comprising the FGPs were shown to carry the same somatic APC gene alteration in 24 (96%) of 25 cases. Furthermore, there was no difference in the frequency of somatic APC gene alterations between FGPs with foveolar dysplasia (10 of 20, 50%), indefinite for dysplasia (four of six, 67%), and nondysplastic (seven of 15, 47%) in FAP patients (P: = 0.697). In contrast, FGPs from non-FAP patients showed infrequent (one of 13, 8%) APC gene alterations (P: = 0.008). These results show that FGPs in FAP patients are pathogenetically distinct from sporadic FGPs. Somatic, second-hit APC gene alterations, which precede morphological dysplasia in many FAP-associated FGPs, indicate that FGPs arising in the setting of FAP are neoplastic lesions.     

Sporadic fundic gland polyps with epithelial dysplasia : evidence for preferential targeting for mutations in the adenomatous polyposis coli gene

Gastric fundic gland polyps (FGPs) occur in two distinct clinicopathological scenarios: sporadic and familial adenomatous polyposis (FAP) associated. FAP-associated FGPs arise through somatic second hit alterations of the adenomatous polyposis coli (APC) gene and frequently demonstrate epithelial dysplasia (Am J Pathol 2000, 157:747-754). Sporadic FGPs, in contrast, tend to contain beta-catenin gene mutations and only infrequently show dysplasia (Am J Pathol 2001, 158:1005-1010). However, sporadic FGPs with dysplasia have not been previously investigated. We studied 13 sporadic FGPs with surface/foveolar low-grade dysplasia or changes indefinite for dysplasia for alterations in the APC/beta-catenin pathway, using chromosome 5q allelic loss assays and direct DNA sequencing of the mutation cluster region in exon 15 of APC and the phosphorylation region in exon 3 of beta-catenin. In addition, to evaluate for possible additional genetic alterations in FGPs, all cases were evaluated for microsatellite instability using fluorescent-based amplification of a standard panel of five microsatellite markers. Alterations in APC were present in seven (53.8%) FGPs, including two cases with bi-allelic APC inactivation (truncating intragenic mutation plus 5q allelic loss), two cases with APC mutation only, and three cases with 5q allelic loss only. In contrast, only two (15.4%) FGPs contained stabilizing beta-catenin mutations. All 13 FGPs were microsatellite stable. These results indicate that sporadic FGPs with dysplasia/indefinite for dysplasia are molecularly similar to FAP-associated FGPs, and are dissimilar to the more common sporadic nondysplastic FGPs. Mutations in APC and beta-catenin, despite occurring in the same genetic pathway, show differing biological properties, a phenomenon that has previously been demonstrated in colorectal neoplasms. The lack of microsatellite instability in FGPs in this study and of K-ras mutations in a previous study suggests that secondary genetic alterations are rare in both dysplastic and nondysplastic FGPs.     

β-Catenin mutations in sporadic fundic gland polyps

Fundic gland polyp (FGP) is the most common gastric polyp. It occurs sporadically or in association with familial adenomatous polyposis (FAP). FAP patients carry germline mutations of the adenomatous polyposis coli (APC) gene, and previous studies have revealed frequent somatic mutations of the APC gene in FGPs associated with FAP. Although inactivation of the APC gene contributes to histogenesis of FGPs associated with FAP, this rarely happens in sporadic cases. Loss of the APC gene promotes abnormal accumulation of beta-catenin, and mutation of GSK-3 beta phosphorylation sites in the beta-catenin gene can have a similar effect. To elucidate the contribution of beta-catenin gene mutation to the histogenesis of sporadic FGP, we analyzed beta-catenin gene mutation in exon 3 in 45 FGP lesions obtained from 35 patients. Somatic mutations were found in 29 lesions: 28 were missense mutations and one was an in-frame deletion. All of the missense mutations were confined to the former two serine residues of the GSK-3 beta phosphorylation sites and their flanking residues (codons 32, 33, 34, 37). Analysis in cases with multiple FGPs revealed a different mutation in each lesion, indicating their multicentric origin. Therefore, a significant proportion of sporadic FGPs have genetic alterations involving beta-catenin stabilization, as did FAP-associated FGPs.     

Sporadic fundic gland polyposis: a clinical, histological, and molecular analysis.

Sporadic fundic gland polyposis (SFGP) is defined as multiple fundic gland polyps in patients without familial adenomatous polyposis syndrome (FAP). Although little is known about the genetic changes in SFGP, mutations in the Wnt signaling pathway have been recently linked to fundic gland polyps in other settings: sporadic polyps are linked to activating beta-catenin mutations, whereas FAP-associated fundic gland polyps are caused by second somatic hits in the adenomatous polyposis coli gene. The relationship between SFGP, single sporadic fundic gland polyps, and FAP-associated polyps remains unclear, and SFGP remain poorly characterized at the clinical, histological, and molecular levels. A retrospective study was undertaken of eight patients with SFGP who had >/=10 polyps with at least five endoscopic biopsy specimens available for study. One additional patient with attenuated FAP who underwent partial gastrectomy was included as a control. The medical records and biopsy specimens were reviewed. Mutations of the beta-catenin gene were evaluated in each fundic gland as well as in control nonpolypoid tissue by direct sequencing of a mutational hot spot in exon 3 of the beta-catenin gene, which encodes the GSK-3beta phosphorylation sites, and a HinfI endonuclease digestion assay. The four men and four women in the study were an average of 57 years of age at biopsy. All patients were on acid-suppression therapy, 5/8 with proton-pump inhibitors (PPI) and 3/8 with Zantac. Sixty-two polyps were studied, and all were <10 mm, with most between 2 and 7 mm. The polyps were histologically identical to single sporadic fundic gland polyps. No dysplasia was seen. Forty-seven of 62 polyps (76%) had detectable beta-catenin mutations. Mutations were found in all eight of the patients. All were point mutations in codons 32, 33, 34, and 37 and are either phosphorylation sites or immediately adjacent to phosphorylation sites, findings identical to that seen in single sporadic fundic gland polyps. Each polyp had a single mutation, and each patient had more than one unique mutation (median = 4), indicating a multifocal origin for the polyps. No mutations were found in nonpolypoid control tissue and in polyps from the attenuated FAP patient. The patients with SFGP in this series were all between 40 and 70 years of age and had histories of acid-suppressive therapy. The fundic gland polyps were histologically and genetically identical to single sporadic fundic gland polyps and demonstrated frequent somatic activating mutations in exon 3 of the beta-catenin gene.     

High-grade dysplasia associated with fundic gland polyposis in a familial adenomatous polyposis patient, with special reference to APC mutation profiles.

We report a patient with familial adenomatous polyposis who developed high-grade dysplasia against a background of fundic gland polyposis. Two large high-grade dysplasia lesions were found in the gastric body, where numerous fundic gland polyps were present. In both lesions, the dysplastic epithelium covered non-neoplastic oxyntic glands that occasionally exhibit cystic changes. A genetic analysis for APC (adenomatous polyposis coli) revealed a somatic 50-bp deletion involving codons 1502-1517 and 2-bp deletion at codon 1465 in each lesion of high-grade dysplasia. In contrast, six of the 18 fundic gland polyps were found to harbor an identical mutation: 1-bp insertion at codon 1556. Both lesions of high-grade dysplasia and the fundic gland polyps were similarly located in the fundic gland area and were caused by the inactivation of APC; however, their mutation profiles of APC were different. These results imply that fundic gland polyps and high-grade dysplasia of the stomach have distinct preferences for APC genotypes in their development.     

Frequent CpG island methylation in sporadic and syndromic gastric fundic gland polyps

We studied methylation of 2 tumor suppressor genes (p14, p16) and 4 MINT (methylated in tumor) clones (MINT1, MINT2, MINT25, MINT31) among 51 fundic gland polyps (FGPs) and 27 normal gastric body biopsy samples using bisulfite treatment of genomic DNA followed by methylation-specific polymerase chain reaction. Thirty-two FGPs were syndromic polyps from 14 patients with familial adenomatous polyposis (FAP); 19 were sporadic FGPs from 15 patients without FAP. Significantly higher mean methylation indices were found between FGPs and normal gastric mucosa (P = .012). FGPs arising in a background of proton pump inhibitor (PPI) effect had significantly higher mean methylation indices than those that did not (P = .023). Perhaps because sporadic FGPs were more likely to be associated with PPI effect than were FAP-associated FGPs, they also demonstrated higher mean methylation indices than syndromic polyps (P = .024). Among FAP-associated FGPs, there was no statistical difference in methylation indices between polyps that were dysplastic, indefinite for dysplasia, or nondysplastic (P = .87). Epigenetic alterations involving methylation of CpG islands might have a role in the development of some FGPs, particularly those with a PPI effect. They do not account for the presence or absence of a dysplastic phenotype in FGPs.     

Mutation cluster region,association between germline and somatic mutations and genotype-phenotype correlation in upper gastrointestinal familial adenomatous polyposis

Studies of adenomatous polyposis coli (APC) mutations in familial adenomatous polyposis (FAP) have focused on large bowel disease. It has been found that: 1) germline APC mutations around codon 1300 are associated with severe colorectal polyposis; 2) somatic APC mutations in colorectal tumors tend to cluster approximately between codons 1250 and 1450; and 3) patients with germline mutations close to codon 1300 tend to acquire somatic mutations (second hits) in their colorectal polyps by allelic loss, whereas the tumors of other FAP patients have truncating second hits. Using new and published data, we have investigated how germline and somatic APC mutations influence the pathogenesis of upper gastrointestinal polyps in FAP. We have compared the results with those from colorectal disease. We found that somatic mutations in upper gastrointestinal polyps cluster approximately between codons 1400 and 1580. Patients with germline APC mutations after codon 1400 tend to show allelic loss in their upper gastrointestinal polyps; the tumors of other patients have truncating somatic mutations after codon 1400. Finally, patients with germline mutations after codon 1400 tend to have more severe duodenal polyposis (odds ratio, 5.72; 95% confidence interval, 1.13 to 28.89; P = 0.035). Thus, in both upper gastrointestinal and colorectal tumors, a specific region of the APC gene is associated with severe disease, clustering of somatic mutations, and loss of the wild-type allele. However, the region concerned is different in upper gastrointestinal and colorectal disease. The data suggest that loss of all APC SAMP repeats is probably necessary for duodenal and gastric tumorigenesis in FAP, as it is in colonic tumors. Compared with colonic tumors, however, retention of a greater number of beta-catenin binding/degradation repeats is optimal for tumorigenesis in upper gastrointestinal FAP.     

Somatic mutations in familial adenomatous polyps. Nuclear translocation of beta-catenin requires more than biallelic APC inactivation

Germline mutations of the APC gene cause familial adenomatous polyposis coli (FAP). APC inactivation results in dysregulation of wnt/wingless signaling and contributes to chromosomal instability in vitro. To investigate somatic alterations that follow a known germline mutation and contribute to the transition from normal to neoplastic mucosa, we studied 10 adenomatous polyps from a 27-year-old patient with an APC germline mutation at codon 554. Chromosomal imbalances were analyzed by comparative genomic hybridization; APC and K-ras were screened for somatic mutations. Before DNA analysis, the polyps were bisected to compare the genetic alterations with the corresponding immunohistologic phenotype of beta-catenin, a proto-oncogene product degraded by the APC tumor suppressor. Gains at chromosome 20 were the most frequent chromosomal alterations (6 polyps). Losses were found predominantly at chromosome 4q (3 polyps). A K-ras mutation was seen in 1 polyp, while all polyps displayed somatic intragenic APC mutations. Comparative immunohistologic analysis revealed strong membranous staining for beta-catenin in all adenomatous polyps, but only 1 adenoma showed nuclear accumulation. Our results suggest chromosomal aberrations contribute early to the progression of adenomatous polyps after biallelic APC inactivation. APC inactivation itself is insufficient for immunohistochemically detectable nuclear translocation of beta-catenin.     

Genetic alterations and epithelial dysplasia in juvenile polyposis syndrome and sporadic juvenile polyps.

Juvenile polyps are regarded as hamartomatous polyps and occur in sporadic and familial syndromic settings. There is increased risk of gastrointestinal neoplasia in patients with juvenile polyposis syndrome, but the molecular mechanisms are not known. We therefore studied 78 colorectal juvenile polyposis from 12 patients with juvenile polyps syndrome and 34 sporadic juvenile polyps for epithelial dysplasia and genetic changes associated with colorectal neoplasia. Dysplasia occurred in 31% of syndromic juvenile polyps but not in sporadic juvenile polyps (P < 0.0001). Topographic control of proliferation and expression of the cyclin-dependent kinase inhibitor p21(WAFI/CIP1) seen in native colorectal epithelium was lost in 79% of dysplastic juvenile polyps and in 8% of nondysplastic juvenile polyps (P < 0.000001). Somatic mutations in the adenomatous polyposis coli (APC) gene were demonstrated in 50% of dysplastic juvenile polyps (3 of 6) but not in any of 16 juvenile polyps without dysplasia (P = 0.01). Both sporadic and syndromic juvenile polyps had K-ras mutations (14%) and there was no relationship to dysplasia. p53 gene product overexpression identified by immunohistochemical staining occurred rarely in dysplastic juvenile polyps (2 of 24, 8%). Our results indicate that the multiple genetic alterations involved in usual colorectal neoplasia also play a role in neoplastic transformation of juvenile polyps, predominantly in juvenile polyposis syndrome.     

Analysis of somatic APC mutations in rare extracolonic tumors of patients with familial adenomatous polyposis coli

Patients with familial adenomatous polyposis coli (FAP) carry heterozygous mutations of the APC gene. At a young age, these patients develop multiple colorectal adenomas that consistently display a second somatic mutation in the remaining APC wild-type allele. Inactivation of APC leads to impaired degradation of beta-catenin, thereby promoting continuous cell-cycle progression. The role of APC inactivation in rare extracolonic tumors of FAP patients has not been characterized sufficiently. Among tissue specimen from 174 patients with known APC germ-line mutations, we identified 8 tumors infrequently seen in FAP. To investigate the pathogenic role of APC pathway deregulation in these lesions, they were analyzed for second-hit somatic mutations in the mutational cluster region of the APC gene. Immunohistochemistry was performed to compare the expression pattern of beta-catenin to the mutational status of the APC gene. Exon 3 of the beta-catenin gene (CTNNB1) was analyzed for activating mutations to investigate alternative mechanisms of elevated beta-catenin concentration. Although CTNNB1 mutations were not observed, second somatic APC mutations were found in 4 of the 8 tumors: a uterine adenocarcinoma, a hepatocellular adenoma, an adrenocortical adenoma, and an epidermal cyst. These tumors showed an elevated concentration of beta-catenin. No APC mutations were seen in focal nodular hyperplasia of the liver, angiofibrolipoma, and seborrheic wart. This is the first study reporting second somatic APC mutations in FAP-associated uterine adenocarcinoma and epidermal cysts. Furthermore, our data strengthen a role for impaired APC function in the pathogenesis of adrenal and hepatic neoplasms in FAP patients.     

Fibromatosis of the breast and mutations involving the APC/beta-catenin pathway

Fibromatoses of the breast are nonmetastasizing tumors, but can be infiltrative and locally recurrent. Breast fibromatoses are rare, and their specific genetic alterations have not been elucidated. However, their occasional occurrence in patients with familial adenomatous polyposis (FAP) and their morphologic identification with other deep fibromatoses (desmoid tumors) suggest that alterations of the APC/beta-catenin pathway might be involved in the pathogenesis of sporadic and FAP-associated breast fibromatoses. We analyzed somatic beta-catenin and APC gene mutations in 33 breast fibromatoses (32 sporadic and 1 FAP-associated) using immunohistochemistry for beta-catenin, 5q allelic loss assays, and direct DNA sequencing for exon 3 of the beta-catenin gene and the mutation cluster region of the APC gene. Nuclear accumulation of beta-catenin was present in the stromal tumor cells in most (82%) cases but not in normal stroma or mammary epithelial cells. Somatic alterations of the APC/beta-catenin pathway were detected in 79% of breast fibromatoses, including activating beta-catenin gene mutations in 15 cases and somatic APC alterations (mutation or 5q allelic loss or both) in 11. These findings indicate that alterations of the APC/beta-catenin pathway with resultant nuclear translocation of beta-catenin are important in the pathogenesis of both sporadic and FAP-associated breast fibromatosis. The spectrum of beta-catenin and APC alterations is similar to that described for desmoid tumors of the abdomen, paraspinal region, and extremities.     

Familial infiltrative fibromatosis (desmoid tumours) (MIM135290) caused by a recurrent 3' APC gene mutation

Desmoid tumours are generally very rare but occur about 100 times more frequently in the colorectal cancer predisposition syndrome familial adenomatous polyposis (MIM 175100), being represented in about 10% of patients. In addition to desmoid disease occurring in familial adenomatous polyposis (FAP) there exist familial infiltrative fibromatosis (MIM 135290) kindreds where there is no evidence of FAP. Previously we have described a kindred with familial infiltrative fibromatosis (FIF) in which desmoid tumours were associated with nonpolyposis colorectal cancer. FAP is caused by mutations in the APC gene and various genotype-phenotype relationships have been defined including reports that colorectal polyposis is less severe with mutations 5' to codon 157 and that the risk of desmoid tumours is high in FAP patients with APC gene mutations between codons 1444 and 1598. There is relatively little information on the phenotype of APC gene mutations 3' to codon 1598; however, one large family has been reported with a mutation at codon 1987 which presents with a highly variable phenotype which includes desmoid disease. We screened our original FIF kindred and three further families with a similar phenotype for mutations in the APC gene. A 4 bp frameshift deletion in codon 1962 was identified in the original FIF kindred and two further apparently unrelated families. Haplotype analysis suggests a common origin for the APC mutation in all three families. Affected individuals had no evidence of congenital hypertrophy of the retinal pigment epithelium. Colorectal polyposis was variable, and most affected patients had either none or a few late onset polyps. These findings demonstrate (i) that FAP and FIF are allelic, and (ii) that APC gene mutations which truncate the APC protein distal to the beta-catenin binding domain are associated with desmoid tumours, absent CHRPE and variable but attenuated polyposis expression.      

Frequent beta-catenin mutations in juvenile nasopharyngeal angiofibromas

Juvenile nasopharyngeal angiofibromas (JNAs) are locally aggressive vascular tumors occurring predominantly in adolescent males. The pathogenesis of JNAs is unknown. Recently, JNAs have been reported to occur at increased frequency among patients with familial adenomatous polyposis, suggesting that alterations of the adenomatous polyposis coli (APC)/beta-catenin pathway might also be involved in the pathogenesis of sporadic JNAs. We analyzed somatic beta-catenin and APC gene mutations in 16 sporadic JNAs from nonfamilial adenomatous polyposis patients using immunohistochemistry for beta-catenin, and direct DNA sequencing for exon 3 of the beta-catenin gene and the mutation cluster region of the APC gene. Nuclear accumulation of beta-catenin was diffusely present in the stromal cells but not in the endothelial cells of all 16 JNAs. Activating beta-catenin gene mutations were present in 75% (12 of 16) of JNAs. Six JNA patients also had recurrent tumors after surgery, and in all cases the beta-catenin gene status of the recurrent JNA was identical to the initial tumor. No mutations in the mutation cluster region of the APC gene were detected in the four JNAs without beta-catenin mutations. The high frequency of beta-catenin mutations in sporadic JNAs and the presence of identical beta-catenin gene mutations in recurrent tumors indicates that activating beta-catenin gene mutations are important in the pathogenesis of JNAs. The immunohistochemical localization of beta-catenin only to the nuclei of stromal cells further suggests that the stromal cells, rather than endothelial cells, are the neoplastic cells of JNAs.     

Duodenal gastric heterotopia,sporadic or fundic gland polyp-associated,frequently carries β-catenin mutation

Duodenal gastric heterotopia (DGH) is a benign asymptomatic condition assumed to be of congenital origin. Since DGH is often associated with fundic gland polyps (FGPs) that frequently carry a somatic β-catenin gene mutation, we examined whether DGH, either sporadic or FGP-associated, is attributable to alterations of the Wnt/β-catenin pathway. Genetic analysis revealed frequent somatic β-catenin gene mutations in DGH; some of which showed the same mutation pattern as coexisting FGPs. All missense mutations were confined to codons 32, 33, and 37. No such mutations were observed, however, in any of the specimens from focal gastric foveolar metaplasia (GFM). Therefore, DGH is not a mere congenital lesion due to aberrant migration of normal gastric mucosa or a simple reactive metaplasia after regenerative stimuli of the duodenal mucosa, but a distinct condition based upon molecular genetic changes in the Wnt/β-catenin pathway.     

APC haploinsufficiency, but not CTNNB1 or CDH1 gene mutations, accounts for a fraction of familial adenomatous polyposis patients without APC truncating mutations

Familial adenomatous polyposis (FAP) is an autosomal dominant condition characterized by the development of hundreds to thousands of colorectal adenomatous polyps. In addition to the classic form, there is also attenuated polyposis (attenuated adenomatous polyposis coli; AAPC), which is characterized by a milder phenotype. FAP/AAPC is caused by germline mutations in the adenomatous polyposis coli (APC) gene. Very recently, germline mutations in the base-excision repair gene MYH have been associated with recessive inheritance of multiple colorectal adenomas in a subset of patients. APC pathogenic alterations are mostly (>95%) represented by frameshift or nonsense mutations leading to the synthesis of a truncated protein. We identified 20 APC truncating mutation carriers out of 30 FAP/AAPC patients from different Italian kindreds. In the remaining 10 patients, we searched for alterations other than truncating mutations by enzymatic mutation detection, real-time quantitative RT-PCR, and genotyping of polymorphic markers encompassing the APC locus. Moreover, to assess whether mutations of genes interacting with APC can substitute or act in association with APC alterations, we sequenced both CTNNB1 (beta-catenin) and CDH1 (E-cadherin) genes. No CTNNB1 or CDH1 mutations were found. On the contrary, four patients showed a reduced APC gene expression compared with healthy subjects. In three of the four cases, genotyping results were compatible with a constitutive allelic deletion. In one case this conclusion was confirmed by haplotype segregation analysis. Our results support the notion that FAP/AAPC can result from APC constitutive haploinsufficiency, with gene deletion being a possible cause of reduced gene expression.     

The expression of E-cadherin and catenins in colorectal tumours from familial adenomatous polyposis patients

Familial adenomatous polyposis patients (FAP) harbour a germline mutation of the adenomatous polyposis coli gene (APC), and APC mutations are early events in the development of sporadic colorectal neoplasms. The APC protein interacts with beta-catenin and gamma-catenin and APC mutations are believed to play a role in the altered levels of beta-catenin in colorectal tumours. Immunohistochemical studies have shown changes in the expression and distribution of E-cadherin and catenins in sporadic colorectal neoplasms. This study assessed the expression and distribution of E-cadherin and catenins in colorectal neoplasms and non-neoplastic mucosa from FAP patients. The expression and cellular distribution of E-cadherin and catenins were studied by immunohistochemistry in 61 adenomas, five carcinomas, and non-neoplastic mucosa from 18 FAP patients. mRNA levels in the carcinomas were studied by in situ hybridization. The expression of E-cadherin and catenins was increased in over 80% of the adenomas, with evident cytoplasmic immunoreactivity. There was increased expression of E-cadherin and catenin in the carcinomas, with a notable increase in the levels of mRNA, in comparison with the non-neoplastic mucosa.     

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

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, chi2 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.     

The 'just-right' signaling model: APC somatic mutations are selected based on a specific level of activation of the beta-catenin signaling cascade

According to the classical interpretation of Knudson's 'two-hit' hypothesis for tumorigenesis, the two 'hits' are independent mutation events, the end result of which is loss of a tumor suppressing function. Recently, it has been shown that the APC (adenomatous polyposis coli) gene does not entirely follow this model. Both the position and type of the second hit in familial adenomatous polyposis (FAP) polyps depend on the localization of the germline mutation. This non-random distribution of somatic hits has been interpreted as the result of selection for more advantageous mutations during tumor formation. However, the APC gene encodes for a multifunctional protein, and the exact cellular function upon which this selection is based is yet unknown. In this study, we have analyzed somatic APC point mutations and loss of heterozygosity (LOH) in 133 colorectal adenomas from six FAP patients. We observed that when germline mutations result in truncated proteins without any of the seven beta-catenin downregulating 20-amino-acid repeats distributed in the central domain of APC, the majority of the corresponding somatic point mutations retain one or, less frequently, two of the same 20-amino-acid repeats. Conversely, when the germline mutation results in a truncated protein retaining one 20-amino-acid repeat, most second hits remove all 20-amino-acid repeats. The latter is frequently accomplished by allelic loss. Notably, and in contrast to previous observations, in a patient where the germline APC mutation retains two such repeats, the majority of the somatic hits are point mutations (and not LOH) located upstream and removing all of the 20-amino-acid repeats. These results indicate selection for APC genotypes that are likely to retain some activity in downregulating beta-catenin signaling. We propose that this selection process is aimed at a specific degree of beta-catenin signaling optimal for tumor formation, rather than at its constitutive activation by deletion of all of the beta-catenin downregulating motifs in APC.     

Three submicroscopic deletions at the APC locus and their rapid detection by quantitative-PCR analysis.

We describe three unrelated kindreds, affected by familial adenomatous polyposis (FAP), with 5q submicroscopic deletions that encompass the entire adenomatous polyposis coli (APC) gene and the adjacent DP1 gene. In one family the deletion encompasses also the MCC (mutated in colon cancer) gene. Affected members of these families had dysplastic adenomatous polyps and congenital hypertrophy of the retinal pigment epithelium (CHRPE); no individual was affected by mental retardation or facial dysmorphism. The deletions were detected by linkage analysis with several intragenic and closely flanking polymorphic markers and confirmed by a quantitative PCR analysis. This procedure could have an impact on the detection of the molecular defect in FAP patients in whom mutational analysis fails to identify the specific mutation.