Functional categories of TP53 mutation in colorectal cancer: results of an International Collaborative Study.

BACKGROUND: Loss of TP53 function through gene mutation is a critical event in the development and progression of many tumour types including colorectal cancer (CRC). In vitro studies have found considerable heterogeneity amongst different TP53 mutants in terms of their transactivating abilities. The aim of this work was to evaluate whether TP53 mutations classified as functionally inactive (< or=20% of wildtype transactivation ability) had different prognostic and predictive values in CRC compared with mutations that retained significant activity. MATERIALS AND METHODS: TP53 mutations within a large, international database of CRC (n = 3583) were classified according to functional status for transactivation. RESULTS: Inactive TP53 mutations were found in 29% of all CRCs and were more frequent in rectal (32%) than proximal colon (22%) tumours (P < 0.001). Higher frequencies of inactive TP53 mutations were also seen in advanced stage tumours (P = 0.0003) and in tumours with the poor prognostic features of vascular (P = 0.006) and lymphatic invasion (P = 0.002). Inactive TP53 mutations were associated with significantly worse outcome only in patients with Dukes' stage D tumours (RR = 1.71, 95%CI 1.25-2.33, P < 0.001). Patients with Dukes' C stage tumours appeared to gain a survival benefit from 5-fluorouracil-based chemotherapy regardless of TP53 functional status for transactivation ability. CONCLUSIONS: Mutations that inactivate the transactivational ability of TP53 are more frequent in advanced CRC and are associated with worse prognosis in this stage of disease.

Cancer risks in LKB1 germline mutation carriers.

BACKGROUND AND AIMS: Germline mutations in the LKB1 gene are known to cause Peutz-Jeghers syndrome, which is an autosomal dominant disorder characterised by hamartomatous polyposis and mucocutaneous pigmentation. This syndrome is associated with an increased risk of malignancies in different organs but there is a lack of data on cancer range and risk in LKB1 germline mutation carriers. PATIENTS AND METHODS: The cumulative incidence of cancer in 149 Peutz-Jeghers syndrome patients with germline mutation(s) in LKB1 was estimated using Kaplan-Meier time to cancer onset analyses and compared between relevant subgroups with log rank tests. RESULTS: Thirty two cancers were found in LKB1 mutation carriers. Overall cancer risks at ages 30, 40, 50, 60, and 70 years were 6%, 18%, 31%, 41%, and 67%, respectively. There were similar overall cancer risks between male and female carriers. However, there were overall cancer risk differences for exon 6 mutation carriers versus non-exon 6 mutation carriers (log rank p=0.022 overall, 0.56 in males, 0.0000084 in females). Most (22/32) of the cancers occurred in the gastrointestinal tract, and the overall gastrointestinal cancer risks at ages 40, 50, 60, and 70 years were 12%, 24%, 34%, and 63%, respectively. In females, the risks for developing gynaecologic cancer at ages 40 and 50 years were 13% and 18%, respectively. CONCLUSIONS: Mutations in exon 6 of LKB1 are associated with a higher cancer risk than mutations within other regions of the gene. Moreover, this study provides age related cumulative risks of developing cancer in LKB1 mutation carriers that should be useful for developing a tailor made cancer surveillance protocol for Peutz-Jeghers syndrome patients.

A novel case with germline p53 gene mutation having concurrent multiple primary colon tumours.

During a search for causative genes in patients with concurrent multiple primary colon tumours, we found a novel case with a germline mutation of the p53 gene, from GCC (Ala) to GTC (Val) at codon 189. Of the six primary colon tumours that this patient had, one large advanced carcinoma exhibited a somatic p53 mutation and a somatic APC mutation, in addition to the germline p53 mutation. Two early carcinomas and three adenomas had somatic APC mutations but no somatic p53 mutation or loss of the p53 allele. K-ras-2 mutations were detected in an advanced carcinoma and an early carcinoma. The present results suggest that a patient with a certain type of germline p53 mutation is predisposed to concurrent multiple colon tumours. It is also suggested that in such a patient, a somatic APC mutation is involved in tumour formation and that an additional somatic p53 mutation contributes to tumour progression.

Alterations of repeated sequences in 5' upstream and coding regions in colorectal tumors from patients with hereditary nonpolyposis colorectal cancer and Turcot syndrome.

One of the characteristics of tumors from patients with germline mutations of DNA mismatch repair genes is instability at microsatellite regions (MSI). We analysed alterations at repeated sequences of coding regions, as well as those of 5' upstream regions, in 29 MSI-High colorectal tumors from patients with hereditary nonpolyposis colorectal cancer (HNPCC) and Turcot syndrome. We found that repeated sequences in 5' upstream regions were altered in these tumors, at considerable frequencies. The (A)10 repeat in the promoter region (position -178 to approximately -169) of the GAPDH gene was altered in 17% of the tumors. The (A)10(TA)9 in the 5' upstream region (position -318 to approximately -291) of the mitochondrial isoleucyl tRNA synthetase gene (IleRS-A), coded in nuclear DNA, was altered in 59% of the tumors, whereas (A)9 in the 5' upstream region (position -859 to approximately -851) of cytoplasmic isoleucyl tRNA synthetase gene (IleRS-B) was not altered. Alteration at repeated sequences in the coding regions were 72% at TGFbetaRII(A)10, 24% at IGFIIR(G)8, 45% at BAX(G)8, 55% at E2F4(CAG)13, 66% at caspase-5 (A)10, 31% at MBD4(A)10, 55% at hMSH3(A)8 and 34% at hMSH6(C)8. The number of altered genes increased with the advancement of carcinoma according to Dukes categories: mean numbers of altered genes within these 10 genes were 2.6 for Dukes A, 4.7 for Dukes B and 7.8 for Dukes C. The mean number for adenomas was 2.0. These results suggest that the MSI phenotype also causes alteration of 5' upstream regions which may affect apoptosis and some mitochondrial functions in HNPCC and Turcot tumors, and that accumulation of altered genes with repeated sequences is associated with the progression of HNPCC and Turcot colorectal tumors.

Frequent activation of the beta-catenin-Tcf signaling pathway in nonfamilial colorectal carcinomas with microsatellite instability.

It has been reported that wild-type APC protein forms a complex with beta-Catenin and GSK3beta, inducing degradation of beta-Catenin in normal cells. Both beta-Catenin and APC gene mutations have recently been shown to activate the same signaling pathway. Frequent mutations of beta-Catenin in hereditary nonpolyposis colorectal carcinomas have also been reported. It was, however, controversial whether the mutation of the beta-Catenin gene was frequent in nonfamilial colorectal carcinomas with high-frequency microsatellite instability (MSI-H). We analyzed the mutations of the APC and beta-Catenin genes in 56 nonfamilial colorectal carcinomas stratified according to the presence or absence of microsatellite instability (MSI). APC mutations were identified in 11 of 22 (50%) cases of MSI-H and 14 of 34 (41%) cases of microsatellite-stable (MSS)/low-frequency microsatellite instability (MSI-L). In contrast, the frequency of beta-Catenin mutations was significantly higher in MSI-H (6/22; 27%) than in MSS/MSI-L (1/34; 3%) (P = 0.01). beta-Catenin mutations were not detected in carcinomas with APC mutation. APC mutation occurred irrespective of MSI status. beta-Catenin mutation, however, occurred frequently in MSI-H carcinomas. Our data suggest that activation of the beta-Catenin-Tcf signaling pathway, through either beta-Catenin or APC mutation, frequently contributes to MSI-H nonfamilial colorectal carcinomas (17/22; 77%).

Molecular evidence for multicentric development of thyroid carcinomas in patients with familial adenomatous polyposis.

Familial adenomatous polyposis is characterized by multiple colorectal adenomas and an increased incidence of colorectal carcinomas. Patients also develop various extracolonic tumors, of which, thyroid carcinoma is common in young females. The occurrence of multiple carcinomas in one thyroid is frequently observed, although some carcinomas are solitary. To clarify whether each carcinoma develops independently or metastatically spreads from the first one formed, we analyzed the adenomatous polyposis coli (APC) gene mutation in each carcinoma. We found that each carcinoma had a different somatic mutation of the APC gene. This is molecular confirmation for the multicentric development of thyroid carcinomas in familial adenomatous polyposis through biallelic inactivation of the APC gene.

Pathogenesis of non-familial colorectal carcinomas with high microsatellite instability.

AIMS: Microsatellite instability (MSI) was first observed in hereditary non-polyposis colorectal carcinoma (HNPCC) and was subsequently seen in non-familial colorectal carcinoma. The relation between MSI and cancer associated genes in non-familial colorectal carcinomas has yet to be evaluated. To clarify this matter, changes in cancer associated genes were examined in non-familial colorectal carcinomas. METHODS: Alterations in the adenomatous polyposis coli (APC), p53, and Ki-ras genes were analysed in 24 MSI high (alterations in four to seven of seven loci), nine MSI low (alterations in one to three of seven loci), and 31 MSI negative non-familial carcinomas. The hMSH2 and hMLH1 genes were also analysed in 24 MSI high carcinomas. RESULTS: Both the frequencies and types of alterations in the APC and p53 genes in MSI high carcinomas were the same as those in MSI low and MSI negative carcinomas; however, they were different from those seen in HNPCC. The frequency of Ki-ras mutation was significantly lower in the MSI high cases (two of 24; 8%) than in the others (15 of 38; 39%). Somatic mutation of hMSH2 or hMLH1 was detected in six of 24 (25%) of the MSI high cases. CONCLUSIONS: These results suggest that APC and p53 alterations occur irrespective of microsatellite instability status in non-familial colorectal carcinomas, and that Ki-ras mutation is not involved in MSI high non-familial colorectal carcinoma. The pathogenesis of these carcinomas may differ from both the usual adenoma-carcinoma sequence and HNPCC carcinogenesis.

Somatic mutations of LKB1 and beta-catenin genes in gastrointestinal polyps from patients with Peutz-Jeghers syndrome.

Peutz-Jeghers syndrome (PJS) is characterized by multiple gastrointestinal hamartomatous polyps, mucocutaneous melanin deposition, and increased risk of cancer, mainly in the gastrointestinal tract. We examined mutations of the LKB1, beta-catenin, APC, K-ras, and p53 genes in 27 gastrointestinal hamartomatous polyps from 10 patients in nine PJS families. Of these hamartomatous polyps, one intestinal polyp had an adenomatous lesion, and one gastric polyp contained adenomatous and carcinomatous lesions. Germ-line mutations of the LKB1 gene were detected in six PJS families. Somatic mutations of the LKB1 gene were found in 5 polyps, whereas loss of heterozygosity (LOH) at the LKB1 locus at 19p was seen in 14 other polyps. In adenomatous lesions microdissected from hamartomatous polyps, both beta-catenin mutation and 19p LOH were detected. Furthermore, a carcinomatous lesion in a gastric hamartomatous polyp was found to contain a mutation of the p53 gene and LOH at the p53 locus in addition to LOH at the LKB1 locus and a beta-catenin mutation. K-ras mutations were detected in a few polyps, whereas no APC mutation or 5q LOH was detected in hamartomatous polyps. These results suggest that gastrointestinal hamartomatous polyps in PJS patients develop through inactivation of the LKB1 gene by germ-line mutation plus somatic mutation or LOH of the unaffected LKB1 allele, and that additional mutations of the beta-catenin gene and p53 gene convert hamartomatous polyps into adenomatous and carcinomatous lesions.

Nuclear localization of immunoreactive beta-catenin is specific to familial adenomatous polyposis in papillary thyroid carcinoma.

Thyroid carcinoma is the first symptom in some patients with familial adenomatous polyposis (FAP). We evaluated the cellular localization of beta-catenin in thyroid carcinomas associated (n = 4) or not associated (n = 173) with FAP, since loss of functional protein of the adenomatous polyposis coli (APC) gene leads to nuclear accumulation of beta-catenin in adenomas and carcinomas of the FAP colon. Immunoreactive beta-catenin was demonstrated at the cell membrane of glandular cells of the non-neoplastic thyroid and non-FAP carcinomas. On the other hand, cytoplasmic and nuclear accumulation of beta-catenin is specific to FAP-associated papillary carcinomas. The abnormality in the APC / beta-catenin pathway is thus also important in FAP-associated thyroid carcinoma, and beta-catenin immunohistochemistry is a feasible screening method to identify occult FAP in young patients with thyroid tumors.

Specific mutation in exon 11 of c-kit proto-oncogene in a malignant gastrointestinal stromal tumor of the rectum.

Gastrointestinal stromal tumor (GIST) in the distal third of the rectum was detected in a 57-year-old man who underwent an abdominoperineal resection of the rectum. Because the tumor expressed CD34 and c-kit gene product, but did not express smooth muscle actin or S-100 protein, it was diagnosed as an uncommitted type of GIST. Moreover, a specific mutation in the sequence coding the juxtamembrane domain in exon 11 of the c-kit proto-oncogene was revealed by a polymerase chain reaction-single-strand conformation polymorphism method. One year after resection, the patient developed multiple liver metastases. It is suggested that a specific mutation in exon 11 of the c-kit proto-oncogene may have played an essential role in the development of the liver metastases.

Recurrent germline mutation in MSH2 arises frequently de novo.

INTRODUCTION: An intronic germline mutation in the MSH2 gene, A-->T at nt942+3, interferes with the exon 5 donor splicing mechanism leading to a mRNA lacking exon 5. This mutation causes typical hereditary non-polyposis colorectal cancer (HNPCC) and has been observed in numerous probands and families world wide. Recurrent mutations either arise repeatedly de novo or emanate from ancestral founding mutational events. The A-->T mutation had previously been shown to be enriched in the population of Newfoundland where most families shared a founder mutation. In contrast, in England, haplotypes failed to suggest a founder effect. If the absence of a founder effect could be proven world wide, the frequent de novo occurrence of the mutation would constitute an unexplored predisposition. METHODS: We studied 10 families from England, Italy, Hong Kong, and Japan with a battery of intragenic and flanking polymorphic single nucleotide and microsatellite markers. RESULTS: Haplotype sharing was not apparent, even within the European and Asian kindreds. Our marker panel was sufficient to detect a major mutation arising within the past several thousand generations. DISCUSSION: As a more ancient founder is implausible, we conclude that the A-->T mutation at nt942+3 of MSH2 occurs de novo with a relatively high frequency. We hypothesise that it arises as a consequence of misalignment at replication or recombination caused by a repeat of 26 adenines, of which the mutated A is the first. It is by far the most common recurrent de novo germline mutation yet to be detected in a human mismatch repair gene, accounting for 11% of all known pathogenic MSH2 mutations.

[Peutz-Jeghers syndrome].

Peutz-Jeghers syndrome(PJS) is an autosomal dominant disease which is characterized by melanin spots on lips and buccal mucosa, and multiple gastrointestinal hamartomatous polyps. Patients are at risk of forming carcinomas in the gastrointestinal tract, uterus, ovary, breast and other organs. The LKB1(STK11) gene at chromosome 19p13.3 has been identified in 1998 as the causative gene for PJS, and germline mutations of this gene have been detected in about 50 PJS families. These mutations include nonsense mutations, frameshift mutations and missense mutations, all of which are assumed to inactivate the LKB1 gene. LKB1 gene appears to be a tumor suppressor gene, and inactivation of this gene through germline mutation and loss of normal allele may develop hamartomatous polyps. However, the function of LKB1 and mechanism of carcinoma formation are still unclear.

[Mechanism of carcinogenesis by germline mutation of protooncogene in hereditary tumors].

Germline mutations in one of two alleles of c-ret, c-met and c-kit protooncogenes have been revealed to be the causes of three autosomal dominant hereditary tumors; multiple endocrine neoplasia type 2(MEN2), hereditary papillary renal cell carcinoma (HPRCC), and familial gastrointestinal stromal tumor(FGIST), respectively. Patients with MEN2A have missense mutations at extracellular cysteine rich domain of c-ret, those with MEN2B have missense mutations at tyrosine kinase domain of c-ret, those with HPRCC have missense mutations at tyrosine kinase domain of c-met, and those with FGIST have in-frame deletion mutations at juxtamembrane domain of c-kit. All of these mutations are assumed to cause constitutive activation of protooncogenes without binding to ligands, resulting in tumor formation.

The relationship between frequencies of extracolonic manifestations and the position of APC germline mutation in patients with familial adenomatous polyposis.

BACKGROUND: Familial adenomatous polyposis (FAP) patients develop various extracolonic lesions; however, the relationship between germline mutation of the APC gene and extracolonic manifestations is mostly unknown. To examine the genotype-phenotype relationship, we compared the APC mutation and clinical data. METHODS: Germline mutations from codon 157 to 1465 of the APC gene were identified in 39 families of FAP and clinical data were collected from 80 patients of these families. Germline mutations were classified into two groups: mutations from exon 4 to 9 (codon 157 to 416, Group 1) and those from exon 10 to 15H (codon 564 to 1465, Group 2). The complication rates of extracolonic manifestations were compared between these two groups. RESULTS: Frequencies of duodenal polyps and gastric adenomas in Group 2 were higher than those in Group 1 (p < 0.0001 and p < 0.0004, respectively) and development of osteoma was more frequent in Group 2 (p = 0.01). The number of colorectal polyps and retinal pigments also correlated with the germline mutation, which was consistent with previous reports. However, such correlations were less obvious with regard to gastric fundic polyps, desmoid tumors, soft tissue tumors and colorectal cancer. CONCLUSION: There are two types with regard to extracolonic manifestations of FAP: one is more severely affected according to the position of germline mutation of the APC gene and the other is not affected.

Malignant transformation and EGFR activation of immortalized mouse liver epithelial cells caused by HBV enhancer-X from a human hepatocellular carcinoma.

We have previously observed that all human hepatocellular carcinomas (HCCs) from HBV carriers examined had the integrated X region. In this study, HBV DNA was isolated from an integration site in one HCC that had a single, very small integrated viral DNA including the X region, but it had no expression of X gene as poly(A)RNA. It was found that HBV DNA was present between alphoid repetitive sequences, and it included Enhancer and X regions, encompassing the adr sequence from 910 to 1811. Nucleotides for 8 amino acids at the 3' end, a stop codon of X gene and a poly(A) signal downstream of X gene were lost by integration, and nucleotides for 7 amino acids and a stop codon were substituted by a connected alphoid sequence. When this cloned HBV DNA was transfected with an expression vector to an immortalized mouse liver epithelial cell line, MLE-10, malignant transformation occurred. Transformants having expressed poly(A)RNA of the X gene showed anchorage-independent growth in soft agar and tumor formation in the subcutis of nude mice. The mRNA level of EGFR was found to be remarkably enhanced in X-transformed cells, in contrast with the absence of this mRNA in parental and ras-transformed MLE-10. Our data provide evidence that the Enhancer-X region alone is the key contributor to the malignant change of pre-malignant liver cells in HBV carriers through activation of some specific genes, such as EGFR.

Frequent mutation of beta-catenin and APC genes in primary colorectal tumors from patients with hereditary nonpolyposis colorectal cancer.

Hereditary nonpolyposis colorectal cancer (HNPCC) is characterized by defective DNA mismatch repair, which results in genetic instability of tumors; however, only a few target genes have been recognized. Our previous study detected a low frequency of APC gene mutation (21%) in colorectal tumors from HNPCC patients, in contrast to a high frequency of APC gene alteration (>70%) in non-HNPCC tumors. Because both beta-catenin and ACP gene mutations have recently been shown to activate the same signaling pathway, we analyzed beta-catenin mutation in HNPCC tumors. A notable frequency of beta-catenin gene mutation (43%, 12 of 28) was found to occur in HNPCC colorectal tumors. Beta-catenin mutations were not detected in tumors with APC mutations. All beta-catenin mutations detected in HNPCC tumors existed within the regulatory domain of beta-catenin. Immunohistochemical staining of tumors with this mutation showed accumulation of beta-catenin protein in nuclei. These and previous data from our laboratory suggest that activation of the beta-catenin-Tcf signaling pathway, through either beta-catenin or APC mutation, contributes to HNPCC colorectal carcinogenesis in approximately 65% of cases.

Rectal cancer in a 13-year-old boy without a detectable germline mutation in FAP and HNPCC genes.

Hereditary non-polyposis colorectal cancer (HNPCC) is characterized by familial clustering and early onset. It is unclear, however, whether the early onset of colorectal cancer necessarily represents HNPCC. A 13-year-old patient had rectal cancer and underwent curative surgery. DNA from this patient was examined for replication errors (RER) and genes related to familial colorectal cancer (APC, hMSH2, and hMLH1). The patient had a negative family history of colorectal cancer, did not show the RER phenotype, and had no germline mutation of the APC, hMSH2, and hMLH1 genes. The present case suggests that an unusually young patient with colorectal cancer is not always an HNPCC proband. Observation over time, however, will be needed, as a first mutator of familial colorectal cancer could be missed.

Suspected hereditary nonpolyposis colorectal cancer: International Collaborative Group on Hereditary Non-Polyposis Colorectal Cancer (ICG-HNPCC) criteria and results of genetic diagnosis.

PURPOSE: The aim of this study was to determine the frequency of mutations in the mismatch repair genes in families suspected of having hereditary nonpolyposis colorectal cancer. METHODS: We devised two criteria for families suspected of having hereditary nonpolyposis colorectal cancer (Criteria I and II). Criteria I consist of at least two first-degree relatives affected with colorectal cancer with at least one of the following: development of multiple colorectal tumors including adenomatous polyp, at least one colorectal cancer case diagnosed before the age of 50, and occurrence of a hereditary nonpolyposis colorectal cancer extracolonic cancer (endometrium, urinary tract, small intestine, stomach, hepatobiliary system, or ovary) in family members. Criteria II consist of one colorectal cancer patient with at least one of the following: early age of onset (<40 years); endometrial, urinary tract, or small intestine cancer in the index patient or a sibling (one aged <50 years); and two siblings with other integral hereditary nonpolyposis colorectal cancer extracolonic cancers (one aged <50 years). A questionnaire was mailed to members of the International Collaborative Group on Hereditary Non-Polyposis Colorectal Cancer to determine the mutation detection rate in mismatch repair genes from the families fulfilling these criteria. For comparison the mutation detection rate for families fulfilling the Amsterdam hereditary nonpolyposis colorectal cancer criteria in each institution was also obtained. RESULTS: Data were obtained from eight different institutions (in 7 different countries). In a total of 123 patients from 123 families (67 families fulfilling Criteria I and 56 families fulfilling Criteria II), genetic testing for germline mismatch repair gene variants was performed. Germline mutations of the hMLH1 or hMSH2 genes were identified in 24 families (20 percent). Of these, the mutation detection rate for families fulfilling Criteria I was 28 percent (19/67). The mutation detection rate for families fulfilling Criteria II was 9 percent (5/56). In these eight institutions, the overall mutation detection rate for families fulfilling the Amsterdam hereditary nonpolyposis colorectal cancer criteria was 50 percent (77/154). CONCLUSION: The Criteria I for suspected hereditary nonpolyposis colorectal cancer have the advantages that they can be applied to nuclear families and they can include extracolonic cancers. The results of this study suggest that families fulfilling Criteria I should be offered genetic testing. The relatively low mutation detection rate in those families fulfilling Criteria II suggests that, using current techniques, genetic testing in these families is not practical.

Somatic mutation of the APC gene in thyroid carcinoma associated with familial adenomatous polyposis.

We report the existence of both germline and somatic mutations of the APC gene in thyroid carcinomas from familial adenomatous polyposis (FAP) patients. One papillary thyroid carcinoma from a 210-year-old woman, with germline mutation of the APC gene (TCA to TGA at codon 1110), showed a somatic mutation of AAAAC deletion between codons 1060 and 1063. Another somatic mutation of CAG to TAG at codon 886 was also found in one of multiple thyroid carcinomas from a 26-year-old woman with attenuated FAP and germline mutation at codon 175 (C deletion). This is the first evidence that total absence of the normal function of the APC gene is involved in development of thyroid carcinomas in FAP.