Angiogenesis and p53 expression in the colorectal adenoma-carcinoma sequence

Angiogenesis, the formation of new vessels, is essential for tumor growth and metastasis. Mutations of p53 tumor suppressor gene are frequent and play an important role in colorectal oncogenesis. A role of p53 as an angiogenesis inhibitor has also been proposed. We evaluated angiogenesis and p53 expression in 16 hyperplastic polyps, 35 solitary tubular and tubulovillous adenomas, and 47 cases of sporadic colorectal carcinomas arising on the basis of preexisting adenomas, with standard immunohistochemical techniques. The mean microvessel density (MVD) in carcinomas was significantly higher compared with the respective adenomatous part of the same tumor (27.9 vs. 7; P=0.0001). Linear regression analysis of MVD between cancerous and adenomatous areas showed a significant correlation (P = 0.0001, r = 0.56), raising the possibility that carcinomas arising from better vascularized adenomas might show increased vascularity. The MVD was significantly higher in stage C compared with stage A cases (P=0.04). p53 positivity was detected in 26 of 47 cancerous (55%) and in 14 of 47 adenomatous areas (30%; P = 0.0002). All carcinomas arising from p53-positive adenomas were also p53 positive. p53 positivity associated with a higher MVD in adenomas (P = 0.02), but not in carcinomas (P = 0.78). We conclude that angiogenesis and p53 play a critical role in colorectal neoplasia, and the process of malignant transformation in tumors arising from highly angiogenic adenomas, particularly those carrying p53 mutations, is accelerated with rapid tumor progression from stage to stage, indicating a more aggressive tumor phenotype.     

p53 gene mutations in human endometrial carcinoma.

Although carcinoma of the uterine endometrium is the most frequently diagnosed malignancy of the female reproductive tract, the molecular genetic features of this tumor have yet to be described in significant detail. Since mutations of the p53 tumor suppressor gene are the single most common genetic alteration found in human malignancies, we examined the hypothesis that p53 mutations occur in human endometrial carcinoma. Sequencing analysis of exons 5-8 revealed point mutations in 3 of 21 (14%) tumors; one mutation was an unusual single-base insertion at codons 176-177, resulting in a premature stop codon, whereas the other two were CGG----TGG transitions at codon 248. Two of these tumors showed reduction to homozygosity at the p53 allele, but one tumor apparently retained heterozygosity. These data indicate that p53 mutations occur in human endometrial carcinoma, although relatively infrequently, and that loss of the normal p53 allele does not necessarily occur with point mutation of the p53 gene in this tumor type.     

p53 Gene mutations in sporadic colorectal carcinoma in Guangxi region

To explore the spectrum of p53 gene mutations of sporadic colorectal carcinoma in Guangxi, a Zhuang autonomous region in southern China, we examined exons 2 approximately 11 and exon-intron junctions of p53 gene from 48 patients with sporadic colorectal carcinoma by using single strand conformation polymorphism (SSCP) and DNA direct sequencing. As a result, p53 gene mutations were found in 31.25 percent (15/48) of our patients. A total of 13 types of p53 gene mutations were found, namely, c.370T > G, c.524G > A, c.528C > G, c.529_546del18, c.536A > G, c.736A > G, c.743G > A, c.770_771del2, c.772G > T, c.814G > A, c.949delC, c.782+1G > A, and c.919+1G > C. Only a double heterozygote for c.370T > G and c.529_546del18 was found. All the other mutations were heterozygous. No homozygote was found. 524G > A was the most common mutation, accounting for 18.75 percent (3/16). c.370T > G could not be found in the whole somatic mutation section of IARC TP 53 mutation database (2004 version). c.528C > G, c.949delC, c.782+1G > A, and c.919+1G > C could not be found in colorectal carcinomas reported in the somatic mutation section of IARC TP 53 mutation database (2004 version). The frequency (45.83 percent) of p53 gene mutations in > or = 60 years old patients was higher than that (16.67 percent) in < 60 year old patients (P < 0.05). The frequency (47.83 percent) of p53 gene mutations in rectal carcinoma was higher than that (16 percent) in colonic carcinoma (P < 0.025).     

p53 tumor suppressor gene mutations predict decreased survival of patients with sporadic colorectal carcinoma

BACKGROUND: Mutations of the p53 tumor suppressor gene play an integral role in sporadic colorectal carcinogenesis but prior studies have failed to show their prognostic significance consistently. METHODS: Fifty-six consecutive sporadic colorectal tumors were analyzed for their p53 status. Polymerase chain reaction amplification with primers for exons 5-9 was conducted and these products were subjected to single strand conformation polymorphism analysis. Suspected mutations were confirmed with DNA sequencing. p53 status was entered into a colorectal clinical database and these patients then were followed prospectively. Patient status with regard to disease recurrence and survival was updated every 6 months. Survival and disease free survival were calculated according to the method of Kaplan and Meier. The association between p53 status and clinical and pathologic factors with survival and recurrence was statistically determined using univariate analysis and the Cox proportional hazards model for multivariate analysis. RESULTS: p53 mutations were detected in 28 of 56 patients (50%). The median follow-up time was 45 months (range, 3-72 months). There were 33 patients (59%) who were alive at last follow-up. Fifteen of the 23 patients who died (65%) had p53 mutations and 8 (35%) had wild-type p53. Thirteen patients developed a disease recurrence, 9 of whom (69%) had tumors with p53 mutations. Overall 4-year survival rates for patients with wild-type p53 and mutant p53 were 71% and 54%, respectively (P = 0.05). The 4-year disease free survival rates for patients with wild-type p53 and mutant p53 were 83% and 62%, respectively (P = 0.09). p53 status and stage were found to be independent significant predictors for survival (p53 negative: P = 0. 02; stage: P = 0.0002.) Stage was found to be the sole significant predictor for disease free survival (P = 0.006). CONCLUSIONS: In this group of colorectal carcinoma patients, p53 mutations were a significant negative prognostic indicator for overall survival. This finding holds prognostic and therapeutic implications for the management of colorectal carcinoma patients.     

p53 gene mutations in colorectal tumors from patients with familial polyposis coli.

The p53 gene has been elucidated as a tumor suppressor gene, and inactivation of this gene caused by deletion or point mutations may play a crucial role in the development of human malignancies. In colorectal carcinomas with an allelic deletion of the p53 gene, the remaining p53 gene was mutated with considerable frequency. It is most difficult to detect point mutations or small deletions of the gene because the mutations occur in diverse regions, although four hot spots have been observed [J.M. Nigro et al., Nature (Lond.), 342: 705-708, 1989]. The polymerase chain reaction and denaturing gradient gel electrophoresis facilitate detection of mutations in the hot spots of the p53 gene. Using these methods, we detected mutations in three adenomatous polyps and one carcinoma from familial polyposis coli patients and three carcinomas of sporadic cases. The DNA sequence analysis confirmed mutations of the p53 gene in 2 adenomas (13 base-pair deletions in one and a point mutation in the other) and 1 carcinoma (point mutation) from familial polyposis coli patients. These results suggest that the p53 gene mutations may be involved in the formation not only of carcinomas but also of adenomas which occur in familial polyposis coli patients.     

VEGF expression in the colorectal adenoma-carcinoma sequence

Angiogenesis is essential for tumor growth and metastasis. It is controlled by multiple factors, one of the most important being vascular endothelial growth factor (VEGF). VEGF and p53 expression were evaluated in 16 hyperplastic polyps, 35 solitary tubular and tubulovillous adenomas, and 47 cases of sporadic colorectal carcinomas arising on the basis of preexisting adenomas, using immunohistochemistry. In parallel, angiogenesis was assessed by the Chalkley score (CS) method. VEGF positivity was detected in 19/47 carcinoma cases (40%). In the respective adenomatous part of the tumor, VEGF positivity was detected in 11/47 cases (23%). Carcinomas arising from VEGF-positive adenomas were mostly VEGF positive (10/11, 91%), whereas in 28/36 (78%) carcinomas arising from VEGF-negative adenomas VEGF expression was not detected. CS was higher in VEGF-positive compared with VEGF-negative carcinomas (9.1 +/- 1.8 and 7.8 +/- 2.3, respectively, p < 0.05), whereas there was no statistically significant difference between the CS in the VEGF-negative and VEGF-positive adenomatous part of the tumor (3.3 +/- 1.8 and 4.3 +/- 2.3, respectively). Nuclear p53 positivity was detected in 26/47 (55%) cases in the cancerous part and in 14/47 (29%) cases in the adenomatous part of the tissue, and no significant correlation with VEGF expression was observed. We conclude that VEGF associates with angiogenesis in colorectal cancer, and its pattern of expression in adenomas is maintained in the arising carcinomas. Further investigation is warranted to clarify whether these findings could be used as indicators of prognosis in screening programs or in patients with limited stage disease where the usefulness of adjuvant therapies with either cytotoxic drugs or inhibitors of angiogenesis is still unclear.     

De novo germline mutations of the p53 gene in young children with sarcomas

Germline p53 mutations are associated with cancer predisposition in Li-Fraumeni families as well as in individuals with component tumors of the syndrome. In the majority of cases these mutations have been shown to be inherited rather than de novo. We screened 59 children with primary bone or soft tissue sarcomas. Germline p53 mutations were identified in 2 patients. Interestingly, analysis revealed that both mutations were de novo. Although the frequency of germline p53 mutations in primary pediatric sarcoma patients is low, there is evidence for the importance of considering pediatric patients for testing for de novo mutations.     

Inherited p53 gene mutations in breast cancer.

Recent evidence has implicated germ-line mutations of the p53 gene as the cause of cancer susceptibility in the Li-Fraumeni syndrome, associated with the development of breast cancer and other neoplasms. Furthermore, somatic mutations of the p53 gene have been detected in a high percentage of non-familial breast cancers. We therefore sought to identify potential carriers of p53 gene mutations in a cohort of patients with early onset breast cancer. We examined 126 consecutive patients who developed breast cancer at or before the age of 40 for mutations of p53 within conserved regions of the gene. One patient with an inherited germ-line mutation of the p53 gene was identified but the functional significance of this mutation was not clear. It thus appears that only a small percentage of patients with breast cancer under the age of 40 carry germ-line mutations of the p53 gene, an observation which has implications for potential screening and risk assessment in such patients.     

The p53 tumor-suppressor gene and ras oncogene mutations in oral squamous-cell carcinoma.

The frequencies of mutations in the p53 tumor-suppressor gene and ras proto-oncogenes were investigated systematically in surgically resected oral squamous-cell carcinomas (SCCs) using single-strand conformation polymorphism (SSCP) and/or dot-blot hybridization analysis of DNA fragments which had been amplified by the polymerase chain reaction (PCR). p53 gene mutations, within the region of exons 5 to 8, were detected in 17 out of 27 (63%) tumor specimens. The role of p53 mutations in cell-line establishment was investigated. p53 gene mutations were detected in 5 out of 6 tissue samples from which cell lines were established and in 4 out of 5 specimens from which cell lines could not be established, suggesting that the presence of p53 gene mutations is not by itself sufficient for cell-line establishment. Tumor samples were also analyzed for point mutational activation of the ras proto-oncogenes. One out of 30 (3%) tumors showed an activating point mutation in codon 12 of H-ras, this being consistent with reports from Europe and USA but not with any from India. Compared to frequencies of the other genetic changes so far reported for oral SCC, the p53 mutations have been observed most often to undergo genetic change. p53 gene mutation is thus intimately involved in the genesis of oral SCC and consequently should be useful as a marker for the diagnosis of this neoplasm.     

Mutation of the p53 gene precedes aneuploid clonal divergence in colorectal carcinoma.

To establish whether p53 mutation precedes or follows clonal divergence in human colorectal carcinomas, 17 tumours were analysed at multiple sites (2-5 each) for single-strand conformation polymorphisms (SSCP) within exons 5-8 of the p53 gene. A previous study had demonstrated subclones of differing DNA ploidy in these tumours, but all showed immunocytochemical evidence for p53 stabilisation, using the monoclonal antibody PAb 1801. Mutations within exons 5-8 of p53 were identified by the presence of an abnormally migrating band in 10 of the 17 carcinomas: five in exon 5, four in exon 7 and one in exon 8. In each of these positive cases, samples from different parts of the carcinoma showed identical gel migration patterns in SSCP analysis. Similarly, the remaining seven tumours were concordant for absence of band shift across all samples of each tumour. Six SSCP-positive cases contained multiple populations differing in DNA ploidy, while four were homogeneously diploid or aneuploid throughout. Very similar proportions were observed in the SSCP-negative cases. In four positive tumours the mutation was confirmed by sequencing or through alteration of nucleotide-specific restriction enzyme cleavage. Identical mutations appeared in every sample from the same tumour. The results provide unequivocal evidence that the same mutant allele of p53 is present throughout each tumour bearing a mutation, regardless of the clonal variation identified by analysis of DNA ploidy. We conclude that in colorectal tumorigenesis mutation of p53 occurs as a single event which precedes and may facilitate the aneuploid clonal divergence of carcinomas.     

Up-regulation of gelatinases in the colorectal adenoma-carcinoma sequence

Gelatinase activity has been associated with colorectal cancer (CRC) invasion and metastasis. However, it remains unresolved whether these proteases participate in early colorectal carcinogenesis. The activity of metalloproteinases (MMP) 2 and 9 were measured by zymography in 122 colorectal adenomas, 22 CRC samples, 12 hyperplasic polyps and in 114 matched normal mucosal samples from 114 patients undergoing colonoscopy. There was a progressive and significant increase of pro-MMP-9 activity from adenoma to CRC tissue, whereas the activity of the latent and active forms of MMP-2 was exclusively up-regulated in CRC samples. Among neoplastic polyps, pro-MMP-9 activity was significantly higher in advanced versus non-advanced adenomas and in those harbouring high grade dysplasia. In addition, a positive correlation was observed between MMP-9 activity and the size of the adenomas. The present study demonstrates that MMP-9 is markedly up-regulated in the adenomatous tissue and suggests that this gelatinase might be a marker for early colorectal carcinogenesis.     

Clinical and pathological associations with p53 tumour-suppressor gene mutations and expression of p21WAF1/Cip1 in colorectal carcinoma.

Inactivation of the p53 tumour-suppressor gene is common in a wide variety of human neoplasms. In the majority of cases, single point mutations in the protein-encoding sequence of p53 lead to positive immunohistochemistry (IHC) for the p53 protein, and are accompanied by loss of the wild-type allele. Recently, the WAF1/Cip1 gene was identified as one of the genes induced by wild-type p53, and increased expression of p21WAF1/Cip1 has been found to reflect the status of the p53 tumour-suppressor pathway. We investigated the inactivation of p53 in a relatively small, but well-characterised, group of 46 colorectal carcinomas that were previously studied for allelic alterations, ras oncogene mutations and DNA aneuploidy. Alterations in p53 were identified by IHC, loss of 17p and DNA sequence analysis of exons 5-8, whereas p21WAF1/Cip1 protein expression was determined by IHC. p53 mutations were identified in 19 of the 46 tumours (41%), whereas positive IHC for p53 was found in 21 of the 46 tumours (46%). Positive IHC for p21WAF1/Cip1 was detected in 16 of 42 cases (38%). We found no relationship between p21WAF1/Cip1 staining and p53 protein expression or p53 mutational status. Inactivating mutations in the p53 gene correlated with LOH at 17p but not with LOH at 5q or 18q, Dukes'' stage, tumour grade or DNA ploidy. There was a higher survival rate independent of Dukes'' stage in the group with no alterations in p53 compared with those with evidence of dysfunction of p53, but the difference was not statistically significant. We conclude that inactivation of p53 and altered expression of p21WAF1/Cip1 are common in colorectal carcinoma but do not correlate with each other or with the clinical or pathological parameters investigated.     

食管癌手术切缘组织p53突变研究

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Infrequent p53 gene mutations in medulloblastomas

Cytogenetic and molecular studies of medulloblastomas have demonstrated frequent loss of sequences from the short arm of chromosome 17, possibly implicating loss or inactivation of the p53 tumor suppressor gene. We amplified exons 5 through 8 of the p53 gene by the polymerase chain reaction technique. These segments, which encompass the regions usually mutated in human tumors, were sequenced to search for p53 mutations in 12 medulloblastoma tumors, 8 xenografts, and 3 permanent cell lines. Mutation of the p53 gene was found in only 1 of 3 cell lines tested and in none of the xenografts or primary tumors studied. Our results suggest that p53 is mutated in an unusual way or that a second tumor suppressor gene on the short arm of chromosome 17 is involved in the pathogenesis of medulloblastoma.     

Serum anti-p53 in relation to mutations across the entire translated p53 gene in colorectal carcinomas

Previous reports have claimed that antibodies to mutated p53 protein indicate poor outcome in malignant disease. The mechanism behind this highly specific process is unclear, although it has been claimed that certain DNA alterations are prone to induction of immune response, since wild-type p53 is almost never immunogenic. The aim of the present analysis was to evaluate whether the presence of anti-p53 was statistically significantly related to any certain DNA alterations in the entirely expressed p53 gene in primary tumors of colorectal cancer. P53 serum antibodies were determined by an enzyme linked immunosorbant assay (ELISA). P53 antibodies were detected in serum of 24 of 88 patients (27%). Twenty-two of 24 (92%) sero-positive patients had mutations in their p53 gene while only 22 of 64 (34%) sero-negative patients had p53 mutations (p<0.01). Mutations were mainly missense with a trend to significantly higher frequency of deletions in sero-negative patients compared to sero-positive subjects (8/25, 32% and 2/22, 9% respectively, p<0.08). Mutations in sero-positive patients were mainly located in exon 5 and 7 and within conserved regions (17 of 22 mutations). In sero-negative patients missense mutations were usually located in exon 5, 7 and 8 being also most frequently located within conserved regions. Most of the p53 deletions in sero-negative patients were however located outside conserved regions (seven of eight deletions). There was no statistical difference between sero-positive and negative patients concerning the spectrum of mutations along the expressed gene. Our study demonstrates that p53 antibodies are usually related to p53 gene mutations but a mutational event is not sufficient to elicit self-immunization. Cellular protein binding to p53 or individual differences of major histocompatibility complex based presentation of p53 protein sequences by immune cells is therefore the most likely explanation between sero-negative and sero-positive patients.     

p53 and RAS gene mutations in multiple myeloma.

We analysed genomic DNA from 30 patients with multiple myeloma (MM), searching for alterations in the p53 and RAS genes by a combination of polymerase chain reaction and single-strand conformation polymorphism techniques. Mutations in the p53 gene were observed in 20% (6 out of 30) of the patients, and were located in conserved sequence blocks within exons 5 and 7. These were single-nucleotide substitutions and consisted predominantly (4/6) of G:C to A:T transitions. Of the six patients with a mutated p53 gene, four were in the terminal phase of the disease. RAS gene mutations were found more frequently since they occurred in 47% (14 out of 30) of the patients. Mutations consisted of single-nucleotide substitutions, located in codons 12, 13 and 61 of either K- or N-RAS, to the exclusion of H-RAS. Moreover, one patient bore two simultaneous mutations, affecting simultaneously the K- and the N-RAS genes. RAS gene mutations were more frequently observed in patients with fulminating disease (10/15, 67%) than in patients with less aggressive forms of the disease (4/15, 26%). We also analysed genomic DNAs from 10 human myeloma cell lines, of which two bore mutations affecting codon 12 of the K-RAS gene, and one codon 12 of the N-RAS gene. The first two cell lines were obtained from freshly explanted tumor cells in which we observed identical mutations. Results presented here show that activating mutations in the RAS genes are, in MM, more frequent than those affecting the p53 gene and suggest that both events are related to terminal phases of the disease.