The APC Variant p.Glu1317Gln predisposes to colorectal adenomas by a novel mechanism of relaxing the target for tumorigenic somatic APC mutations

Multiple rare nonsynonymous variants in APC predispose to colorectal adenomas. The mechanisms through which such variants act have been unclear, but it has been proposed that a specific (“just‐right”) level of β‐catenin signaling is required for colorectal tumorigenesis. This appears to be mediated by selection for APC genotypes that retain one, or rarely two, 20 amino acid β‐catenin downregulating repeats (20AARs). We investigated the mechanism through which the variant p.Glu1317Gln (c.3949G>C) contributes to colorectal tumorigenesis. We compared the patterns of somatic APC mutations in tumors from patients with attenuated familial adenomatous polyposis (AFAP) who did, or did not, coinherit p.Glu1317Gln with their AFAP‐causing APC mutations. Only 8.2% (4/49) of tumors carrying p.Glu1317Gln had somatic mutations predicted to result in mutant polypeptides retaining a single 20AAR, compared to 62.1% (36/58) of those which did not carry this variant (P=5.64×10^?9). Furthermore, tumors with p.Glu1317Gln often carried somatic mutations that were unusually early or late (downstream of the second 20AAR) in the APC open reading frame. These data support a novel mechanism in which p.Glu1317Gln in combination with other weak mutant APC alleles (generating polypepetides with zero, two, or three 20AARs) can provide the necessary growth advantage for colorectal tumorigenesis. Hum Mutat 30:1–7, 2009. © 2009 Wiley‐Liss, Inc.     

Chromosomal instability and APC gene mutations in human sporadic colorectal adenomas

The mechanisms by which adenomatous polyposis coli (APC) gene mutations contribute to colorectal tumourigenesis and progression are still not fully understood. Using in vitro mouse embryonic stem cells, APC mutations have been proposed to dysregulate the interactions between kinetochores and microtubules during mitosis, leading to chromosomal instability (CIN) and aneuploidy. A link between APC mutations and aneuploidy in vivo among human sporadic colorectal adenomas has not been reported previously and was therefore investigated in the present series of 61 adenomas. Multi-parameter flow cytometry, based on scattering and fluorescence from the DNA-specific 4,6-diamidino-2-phenylindole-2-hydrochloride (DAPI) dye, which separates epithelial from stromal lymphocyte nuclei, was used to evaluate the DNA index (DI) and to sort epithelial nuclei. Additionally, DNA extracted from these sorted nuclei was used to analyse APC mutations by DNA sequencing. Aneuploidy was present in 20 of 61 adenomas (33%), with 15 of these 20 cases (75%) having a near-diploid DI (DI different from 1 and less than 1.3). APC mutations were detected in 19 adenomas (31%): 12 were within or downstream of the mutation cluster region (MCR), roughly defined by codons 1200-1500, and seven were upstream of the MCR. Overall, the prevalence of aneuploidy in APC wild-type and mutated adenomas was 26% and 47%, respectively, and no statistically significant association was found between APC status and DI (p = 0.142). However, when APC mutations were subdivided into two groups, ie occurring within/downstream of the MCR and upstream of the MCR, the association of APC mutations within and downstream of the MCR with aneuploidy was statistically significant (p = 0.017). In conclusion, the present data suggest that the type of APC mutation may play a role in the origin of CIN in vivo in human sporadic colorectal adenomas and that APC mutations within and downstream of the MCR, and large-scale chromosomal alterations, may co-operate in the progression of a subgroup of adenomas.     

Novel germline mutations in the APC gene of Cypriot patients with familial and sporadic adenomatous polyposis

Familial adenomatous polyposis (FAP) is one of the two commonest familial syndromes that predispose to colorectal cancer. FAP is caused by mutations in the adenomatous polyposis coli (APC) tumour suppressor gene that has a high penetrance. The disease is characterized by the occurrence of hundreds to thousands of colorectal polyps, which if left untreated give rise to colorectal cancer. In Cyprus, there are no molecular data available as yet on families with FAP. This work presents the results of APC analysis in our population for the first time. The APC gene was analyzed in 33 DNA samples from 20 individuals belonging to four FAP families and 13 patients with sporadic polyposis. We identified three truncating mutations, four missense mutations and 11 polymorphisms. It is of interest that two of the three truncating mutations, 2307delA and Q1242X, are novel, which supports the existence of a unique genetic pool in the Cypriot population. This ethnic molecular study in addition to highlighting population heterogeneity also contributes to phenotype-genotype associations that are essential for the clinical management of FAP families in Cyprus.     

No difference in the occurrence of mismatch repair defects and APC and CTNNB1 genes mutation in a multi-racial colorectal carcinoma patient cohort

BACKGROUND AND AIMS: Colorectal cancers of different subtypes involve different pathogenic pathways like the Wnt and the mutator pathways. In this study, we screened 73 colorectal cancer cases from a multi-racial group for genetic and expression profile defects with the aim of correlating these with patients' clinicopathological characteristics. METHODS: Mutation screening of the entire coding region of APC and exon 3 of CTNNB1, loss of heterozygosity (LOH) of APC, and microsatellite instability (MSI) status were assessed for 44 patients with available paired frozen normal and tumour tissues. In addition, 29 cases with available paraffin embedded tumour blocks were screened for mutation in exon 3 of CTNNB1, the APC mutation cluster region (codon 1286-1513), and hMLH1, hMSH2, hMSH6 protein expressions by immunohistochemistry method. RESULTS: In our study, 15/73 cases showed APC mutations (20.5%), 1/73 cases had CTNNB1 mutation (1.4%), 5/32 cases had APC LOH (15.6%), and 16/70 (22.9%) cases revealed at least some form of mismatch repair (MMR) defect. Tumour grade (poor differentiation) was found to correlate significantly with right-sided tumour and mucinous histology (p = 0.01879 and 0.00320, respectively). Patients of younger age (below 45 years) more often had tumours of mucinous histology (p = 0.00014), while patients of older age (above 75 years) more often had tumours on the right side of the colon (p = 0.02448). Tumours of the mucinous histology subtype often had MMR defects (p = 0.02686). There was no difference in the occurrence of APC and CTNNB1 mutations and MMR defects found within our multi-racial colorectal cancer patient cohort. CONCLUSION: Our findings support the notion that racial factor may not be related to the occurrence of MMR defects and APC and CTNNB1 mutations in our multi-racial patient cohort.     

Dependence of colorectal cancer risk on the parent-of-origin of mutations in DNA mismatch repair genes

Genetic diseases associated with dynamic mutations in microsatellite DNA often display parent-of-origin effects (POEs) in which the risk of disease depends on the sex of the parent from whom the disease allele was inherited. Carriers of germline mutations in mismatch repair (MMR) genes have high risks of colorectal carcinoma (CRC). We investigated whether these risks depend on the parent-of-origin of the mutation. We studied 422 subjects, including 89 MMR gene mutation carriers, from 17 population-based families who were each recruited via a CRC case diagnosed before age 45 years and found to carry a MMR gene mutation. The POE hazard ratio (HR(POE)), defined to be the CRC incidence for carriers with maternally derived mutations divided by the corresponding paternal incidence, was estimated using a novel application of modified segregation analysis. HR(POE) (95% confidence interval) was estimated to be 3.2 (1.1-9.8) for males (P = 0.03) and 0.8 (0.2-2.8) for females (P = 0.5) and the corresponding cumulative risks to age 80 years were 88% (54%-100%) for male carriers with maternally derived mutations and 38-48% for all other carriers. If confirmed by larger studies, these results will have important implications for the etiology of CRC and for the clinical management of MMR gene mutation carriers.     

DNA mismatch repair deficiency in sporadic colorectal cancer and Lynch syndrome

Poulogiannis G, Frayling I M & Arends M J
(2010) Histopathology 56, 167–179 DNA mismatch repair deficiency in sporadic colorectal cancer and Lynch syndrome DNA mismatch repair (MMR) deficiency is one of the best understood forms of genetic instability in colorectal cancer (CRC), and is characterized by the loss of function of the MMR pathway. Failure to repair replication‐associated errors due to a defective MMR system allows persistence of mismatch mutations all over the genome, but especially in regions of repetitive DNA known as microsatellites, giving rise to the phenomenon of microsatellite instability (MSI). A high frequency of instability at microsatellites (MSI‐H) is the hallmark of the most common form of hereditary susceptibility to CRC, known as Lynch syndrome (LS) (previously known as hereditary non‐polyposis colorectal cancer syndrome), but is also observed in ～15–20% of sporadic colonic cancers (and rarely in rectal cancers). Tumour analysis by both MMR protein immunohistochemistry and DNA testing for MSI is necessary to provide a comprehensive picture of molecular abnormality, for use in conjunction with family history data and other clinicopathological features, in order to distinguish LS from sporadic MMR‐deficient CRC. Identification of the gene targets that become mutated in MMR‐deficient tumours may explain, at least in part, some of the clinical, pathological and biological features of MSI‐H CRCs and holds promise for developing novel therapeutics.     

Routine testing for mismatch repair deficiency in sporadic colorectal cancer is justified

This study prospectively examines the accuracy of immunohistochemical staining in the identification of mismatch repair defective (MMRD) colorectal cancer in routine clinical practice. The potential impact of this information on decisions regarding adjuvant treatment and germline testing were quantified. A consecutive series of fresh tissue (836 cancers) was obtained from 786 individuals undergoing curative surgery for colorectal cancer at one institution. As part of normal practice, each tumour was screened for the expression of MLH1 and MSH2 by immunohistochemical staining (IHC) and relevant clinicopathological details were documented. Microsatellite instability (MSI) was assessed using standard markers. Overall, 108 (13%) tumours showed loss of staining for either MLH1 (92 tumours) or MSH2 (16 tumours). The positive predictive value of mismatch repair IHC when used alone in the detection of MSI tumours was 88%, and the negative predictive value was 97%. Specificity and positive predictive value were improved by correlation with microsatellite status. Tumour stage (HR 3.5, 95% CI 2.0-6.0), vascular space invasion (HR 1.9, 95% CI 1.2-3.0) and mismatch repair deficiency (HR 0.2, 95% CI 0.05-0.87) were independent prognostic factors in stages II and III disease. Screening by mismatch repair IHC could reasonably have been expected to prevent ineffective treatment in 3.6% of stage II and 7.6% of stage III patients. The frequency of germline mismatch repair mutations was 0.8%, representing six unsuspected hereditary non-polyposis colorectal cancer (HNPCC) cases. Routine screening of colorectal cancers by mismatch repair IHC identifies individuals at low risk of relapse, and can prevent unnecessary adjuvant treatments in a significant number of individuals. Abnormal immunohistochemistry should be confirmed by microsatellite testing to ensure that false-positive results do not adversely impact on treatment decisions.     

Childhood brain tumours due to germline bi-allelic mismatch repair gene mutations

Childhood brain tumours may be due to germline bi-allelic mismatch repair (MMR) gene mutations in MLH1, MSH2, MSH6 or PMS2. These mutations can also lead to colorectal neoplasia and haematological malignancies. Here, we review this syndrome and present siblings with early-onset rectal adenoma and papillary glioneural brain tumour, respectively, due to novel germline bi-allelic PMS2 mutations. Identification of MMR protein defects can lead to early diagnosis of this condition. In addition, assays for these defects may help to classify brain tumours for research protocols aimed at targeted therapies.     

Germline and somatic mutation analyses in the DNA mismatch repair gene MLH3: Evidence for somatic mutation in colorectal cancers

DNA mismatch repair is of considerable scientific and medical importance because of its essential role in maintaining genomic integrity, and its association with hereditary non-polyposis colon cancer (HNPCC). Germline mutations in five mismatch repair genes (MLH1, MSH2, PMS1, PMS2, and MSH6) have been associated with HNPCC susceptibility. Our laboratory recently identified MLH3, a novel DNA mismatch repair gene. We screened the MLH3 coding sequence in 60 probands with increased genetic risk factors for colorectal cancer susceptibility and no mutations in the other candidate genes. No definite MLH3 germline mutations were found. We subsequently screened 36 colon tumors, and discovered an appreciable frequency of somatic MLH3 coding mutations in MSI-H tumors (25%). In four of six tumors, evidence of biallelic inactivation was noted. Furthermore, MLH3 nonsense mutations were identified in two of 12 microsatellite stable (MSS) tumors with 14q24 loss of heterozygosity. While our analyses do not exclude the existence of germline MLH3 mutations in patients with increased genetic risk factors for colorectal cancer susceptibility, they suggest such mutations are uncommon in this patient population. The finding of an appreciable frequency of somatic MLH3 mutations is consistent with a possible role for this gene in the progression of colorectal cancer tumorigenesis. Hum Mutat 17:389-396, 2001. Published 2001 Wiley-Liss, Inc.     

BAT-26 identifies sporadic colorectal cancers with mutator phenotype: a correlative study with clinico-pathological features and mutations in mismatch repair genes

Microsatellite instability (MSI) is present in most colorectal cancers (CRC) associated with hereditary nonpolyposis colorectal cancer (HNPCC). MSI testing in so-called sporadic forms of CRC may become a useful tool in identifying new HNPCC kindred. The aim of this study was to analyse the utility of BAT-26 as a marker to identify CRCs with MSI and to investigate whether sporadic CRCs with MSI have a phenotypic expression similar to HNPCC cases. MSI was detected using two methods, an association of 7 poly(CA) repeats and a poly(A) repeat alone, BAT-26, in a series of 62 patients with apparently sporadic forms of CRC. Germ-line and somatic mutations in the hMSH2, hMLH1, and hMSH6 genes were analysed in patients with MSI+ tumours. Patients with MSI+ at poly(CA) loci and at BAT-26 were younger (p = 0·024 and p = 0·002), had tumours more frequently right sided (p = 0·017 and p = 0·0001) and more often mucinous (p = 0·037 and p = 0·005, respectively) than patients with MSI negative tumours. Mutation analysis allowed the identification of two patients carrying germ-line mutations in the hMLH1 gene (both were BAT-26+) and two other patients who had somatic mutation in the hMSH2 and in hMLH1 genes. In conclusion, the detection of MSI using poly(CA) repeats or BAT-26 alone allowed the identification of a subset of patients with clinico-pathological characteristics similar to those associated to HNPCC. BAT-26 has the advantage of being a simple and less expensive method that might be used as a screening procedure before mutation analysis. Copyright © 1999 John Wiley & Sons, Ltd.     

大肠肿瘤结肠腺瘤病基因突变的特征

目的 研究早期大肠肿瘤结肠腺瘤病（adenomatous polyposis coli,APC)基因突变特征及可能的内在机理。方法 体外蛋白合成试验和序列分析确定115例非家族性腺瘤性息肉病大肠肿瘤的APC突变。结果 76例APC基因突变病例（101个突变点）中,移码突变为多（56％）,91％为1－2bp碱基改变,其中2／3为碱基A,51％点突变在CpG岛。对比遗传性非息肉病性结直肠癌（hereditary nonpolyposis colorectal cancer,HNPCC)和散发性肿瘤的APC基因突变,HNPCC的移码突变明显多（69％对52％,P＜0．05）,点突变在CpG位点分布明显少（31％对50％,P＜0．05）;另突变频率略低（53％对67％,P＞0．05）,在片段3分布也略少（38％对46％,P＞0．05）。15例（20％）病例还检出双突变,间距介于7－1443bp。结论一些APC基因突变特征与DNA错配修复缺陷有关;HNPCC和散发性大肠肿瘤的APC基因突变存在差异;双突变位点间距相差甚远,提示等位与非等位基因突变并存。     

Phenotypes of invasion in sporadic colorectal carcinomas related to aberrations of the adenomatous polyposis coli (APC ) gene

AIMS: To determine whether the dissociation of tumour cells from neoplastic glands in colorectal carcinomas is caused by disruption of the wnt-signalling pathway and whether the adenomatous polyposis coli (APC) protein is implicated in this. METHODS AND RESULTS: In a series of 99 clinically sporadic colorectal carcinomas, APC exon 15 mutations, loss of heterozygosity (LOH) and promoter methylation were found in 49, 20 and 23 cases, respectively. Singly, these APC aberrations were not associated with the degree of tumour cell dissociation, but dissociation was higher for the cases with combined APC mutation and LOH. Immunohistochemical beta-catenin translocation to the nucleus correlated with APC aberrations. Tumour growth pattern (expansive/infiltrative/diffuse) and tumour stroma (desmoplastic common-type versus keloid-like) showed a statistically significant association with tumour cell dissociation and with beta-catenin translocation. Of other molecular alterations tested (p53 mutation; LOH at 17p13, 18q, 9p21; CpG island methylator phenotype), only the highly microsatellite unstable status (n = 11) was negatively associated. CONCLUSIONS: In colorectal carcinomas, wnt dysregulation relates to APC aberrations, but wnt dysregulation and APC aberrations are not strictly required for tumour cell dissociation, and additional and/or alternative factors must play a role. Of these, outside-in signalling by cancer cell-matrix interactions, as partially mirrored in histomorphological features, could be important.     

APC and p53 mutations in de novo colorectal adenocarcinomas

To investigate genetic features in small and flat colorectal carcinomas that arise de novo, we searched for genetic alterations in six sporadic tumors by examining their APC, K-ras, and p53 genes. Two of the six tumors carried detectable mutations within the mutation cluster region (MCR) of the APC gene; both mutations were predicted to cause truncation of the gene product. Four tumors carried mutations of the p53 gene; three were missense mutations in exon 5, and the other was a 3-bp deletion in exon 6. However, neither codon 12 nor codon 13 of K-ras contained detectable mutation in any tumors. Hence, as “adenoma-carcinoma sequence” model of development of colorectal carcinoma, inactivation of the APC and p53 genes appear to be Involved in development of the de novo type of colorectal carcinoma even though the adenoma stage is not observed. © 1994 Wiley-Liss, Inc.     

I1307K APC and hMLH1 mutations in a non-Jewish family with hereditary non-polyposis colorectal cancer

We describe a French Canadian hereditary non-polyposis colorectal cancer (HNPCC) kindred which carries a novel truncating mutation in hMLH1 . Interestingly, the I1307K APC polymorphism, associated with an increased risk of colorectal cancer, is also present in this family. The I1307K polymorphism has previously only been identified in individuals of self-reported Ashkenazi Jewish origins. In addition, in this family, there appears to be no relationship between the I1307K polymorphism and the presence or absence of cancer.     

APC mutations in human colorectal adenomas: analysis of the mutation cluster region with temperature gradient gel electrophoresis and clinicopathological features

To date, the earliest mutations in colorectal adenomas have been found in the adenomatous polyposis coli (APC) gene, considered the ‘gatekeeper’ in tumourigenesis. To study the types of APC gene mutations and their relation to clinicopathological features which are associated with malignant potential, the mutation cluster region of the APC gene was analysed in a series of 32 human sporadic colorectal adenomas from 22 patients. DNA was extracted from frozen sections and two overlapping fragments which cover the mutation cluster region were amplified using the polymerase chain reaction (PCR). Mutations were screened with temperature gradient gel electrophoresis and identified by DNA sequencing. Mutations were found in 14 samples (44 per cent) from 11 patients. The changes could be characterized as point mutations (n=7), deletions of one or more nucleotides (n=6), and insertions (n=1). From five patients, multiple adenoma samples were obtained and the adenomas from two of these patients showed a heterogeneous mutation pattern in the APC gene. All detected mutations are predicted to result in a truncated APC protein. Genetic alterations in the mutation cluster region of the APC gene occurred significantly more frequently in large adenomas and showed a trend towards an increase in villous adenomas and adenomas with moderate and severe dysplasia. It is concluded that an increased proportion of APC gene mutations were found in the adenomas showing clinicopathological features associated with increased proliferation but not with characteristics of malignant potential. The results suggest that in a significant proportion of adenomas, other (genetic) alterations are involved in early tumourigenesis. © 1998 John Wiley & Sons, Ltd.     

APC germline mutations identified in Czech patients with familial adenomatous polyposis

Familial adenomatous polyposis (FAP) is an autosomal dominantly inherited predisposition to colorectal cancer, which is caused by germline mutations in the adenomatous polyposis coli (APC) gene. The APC mutations have been investigated in 46 Czech unrelated FAP families and 9 suspected FAP families using DGGE analysis and direct DNA sequencing. We found 25 germline APC mutations and identified 11 which were not previously reported. Of the identified mutations, 10 were 1 to 5 bp deletions, four were 1 bp insertions and six were nonsense, all leading to the formation of premature stop codon. In addition, we detected two mutations in the splice-donor region of APC intron 11, one missense and two samesense mutations. Phenotypes of patients with known and novel types of mutations are presented and discussed.     

Immunohistochemical detection of truncated APC protein in sporadic human colorectal adenomas and adenocarcinomas

Mutations of the APC gene frequently occur in sporadic forms of colorectal adenomas and adenocarcinomas. Phenotypically, the vast majority of these mutations result in the truncation of the APC protein. To demonstrate the defective APC gene product in human colorectal tumors, rabbit region-specific antisera raised against the APC protein of amino acid sequences between 371 and 390 (SP1) and between 1821 and 1840 (SP3) were used to exhibit the truncated APC protein. In all, 86 lesions from 67 cases of sporadic adenoma and adenocarcinoma were examined; abnormal staining patterns were distinguished in 43 lesions (50%); the incidence of abnormalities was not significantly different between adenomas and carcinomas. The majority, 75% exhibited epitopic change with the SP1-positive and SP3-negative phenotype (type P1), and 25% exhibited neither of these phenotypes (type P2). The staining pattern in all lesions was uniform, and studies of carcinomas arising in adenomas showed the same pattern of staining. These findings supported the view that the APC lesion is a very early event in colorectal carcinogenesis. Furthermore, this simple immunohistochemical approach demonstrated that different adenomas from the same patient showed different staining patterns.     

Mutation analysis of the adenomatous polyposis coli (APC) gene in northwest Spanish patients with familial adenomatous polyposis (FAP) and sporadic colorectal cancer

Germline mutations in the tumor‐suppresor APC gene are associated with hereditary familial adenomatous polyposis (FAP) and somatic mutations are common in sporadic colorectal cancer. In this study, we report the identification of three novel germline mutations: 1682‐1683insA, 3252‐3253insAT, 3544A>T and a new somatic mutation 4130‐4131delTT, all giving rise to truncated APC proteins. The majority of the mutations we found originate a truncated APC protein and cause the FAP phenotype. However, special attention must be given to the missense mutations Asp1822Val and Ser2621Cys since their segregation with the FAP phenotype is questionable. In our FAP families we did not find any genetical alterations at codon 1309, being this mutation the most frequent reported in APC. Differences in the recurrence of pathological mutations in APC could exist among populations. However, epidemiological studies must be performed to confirm this hypothesis. Hum Mutat 18:355, 2001. © 2001 Wiley‐Liss, Inc.     

Prognostic impact of changes in base excision repair machinery in sporadic colorectal cancer

Objective

to evaluate the prognostic value of base excision repair proteins in sporadic colorectal cancer.

Four new mutations in the DNA mismatch repair gene MLH1 in colorectal cancers with microsatellite instability

Hereditary nonpolyposis colorectal cancer (HNPCC) is frequently associated with inherited mutation in one of four DNA mismatch repair genes. Somatic mutations in the same genes are also found in a subset of sporadic colorectal cancers. A defect in DNA mismatch repair results in an RER (replication error) tumor phenotype. We screened 110 archival and 11 prospectively acquired colorectal cancers for the RER phenotype. A total of 22 cancers were RER-positive. RER-positive tumors were investigated for mutations in the DNA mismatch repair gene MLH1 using single-strand-conformation-polymorphism (SSCP) analysis. We identified four previously undescribed mutations in four different samples. Three mutations were exonic: a point mutation at codon 69 (AGG→AAG [arg→lys]); a single base pair deletion at codon 42/43 (GCAAAATCC→GCAAATCC) leading to a new stop codon downstream; and a point mutation at codon 757 (TAA→TAT) [termination→tyr] which extend the MLH1 peptide by 36 amino acids. The fourth mutation was a 1 base pair insertion six base pairs 5′ to the start of exon 14 (tttgtttt→tttggtttt). The mutations were not seen in the patients' constitutional DNA. The somatic MLH1 mutations identified appear to be causally associated with the RER phenotype. Hum Mutat 12:73, 1998. © 1998 Wiley-Liss, Inc.