Mutations and allelic loss of p53 in primary tumor DNA from potentially cured patients with colorectal carcinoma.

PURPOSE: To compare p53 alterations in survivors and nonsurvivors after surgery for colorectal cancer. PATIENTS AND METHODS: Twenty-nine potentially cured patients with colorectal carcinoma, without recurrent disease for more than 6 years after their primary surgery, were selected to match a group of 41 colorectal cancer patients with early metastatic spread to the liver. All patients were screened for mutations in the p53 gene, exons 5 to 9, by denaturing gradient gel electrophoresis and subsequent sequencing. RESULTS: The frequency of p53 mutations was significantly different in cured patients (60%) compared with patients with early relapse (41%, P <.05). A significant difference was found in the distribution of mutations, indicating that potentially cured patients had a different proportion of mutations in conserved regions of p53 (P =.02). This difference was explained by a significantly different frequency of mutations in exon 8 (40% v 15%, P =.03), which is part of the conserved region V. All mutations in region V were codon 273 mutations in cured patients, whereas three of four mutations were located in codon 273 in patients with metastatic disease. Allelic loss of p53 (loss of heterozygosity [LOH]) was demonstrated in 26% of the cured patients and in 39% of patients with metastatic disease (P =.36). The combination of mutation and LOH of p53 was the same (17%) in both groups. CONCLUSION: A large number of p53 mutations in colorectal cancer do not promote disease progression. Some mutations, particularly within conserved regions, may even counteract negative functional effects of other p53 structural alterations. A complete loss of p53 function was not related to survival or progression after curative operation of colorectal carcinoma.     

p53 mutations are associated with 17p allelic loss in grade II and grade III astrocytoma.

Loss of genetic material on the short arm of chromosome 17 is observed in approximately 40% of human astrocytomas (WHO grades II and III) and in approximately 30% of cases of glioblastoma multiforme (WHO grade IV). Previous studies of glioblastoma multiforme have shown that the p53 gene, located on the short arm of chromosome 17, is frequently mutated in these glioblastomas. To explore whether lower-grade astrocytomas are also associated with corresponding mutations of the p53 gene, we have investigated a series of 22 human astrocytomas of WHO grades II and III both for loss of heterozygosity on chromosome 17p and for p53 mutations. Mutations in the conserved regions of the p53 gene were identified by single strand conformation polymorphism analysis of exons 5, 6, 7, and 8 and were verified by direct DNA sequencing of the polymerase chain reaction products. p53 mutations were observed in 3 of 8 grade II astrocytomas and 4 of 14 grade II astrocytomas. In all 22 tumors, allelic loss of the short arm of chromosome 17 was investigated by restriction fragment length polymorphism analysis. One-half of the grade II astrocytomas (4 of 8) and grade III astrocytomas (7 of 14) exhibited allelic loss on chromosome 17p. Mutations in the p53 gene were exclusively observed in tumors with allelic loss on 17p. Our results show that p53 mutations are not restricted to glioblastoma multiforme and may be important in the tumorigenesis of lower-grade astrocytomas and that p53 mutations in lower-grade astrocytomas are associated with loss of chromosome 17p. These findings are consistent with a recessive mechanism of action of p53 in WHO grade II and III astrocytoma tumorigenesis.     

17p allelic deletions and p53 protein overexpression in Barrett's adenocarcinoma.

Barrett's esophagus is a condition in which the stratified squamous epithelium of the esophagus is replaced by metaplastic columnar epithelium that predisposes to the development of esophageal adenocarcinoma. Allelic deletions of 17p and alterations of p53 including elevated p53 protein levels have been observed in many different tumors. To investigate the presence of 17p allelic deletions and p53 protein overexpression in Barrett's adenocarcinomas, we have combined the use of restriction fragment length polymorphism analysis, multiparameter flow cytometry, and DNA content cell sorting. The combined use of these methodologies permits the purification of aneuploid tumor cells for restriction fragment length polymorphism analysis of 17p allelic deletions and the evaluation of p53 protein expression by multiparameter flow cytometry in the same aneuploid tumor cell populations. We analyzed 15 aneuploid populations and one tetraploid populations from 13 Barrett's adenocarcinomas for 17p allelic deletions and p53 protein overexpression to determine whether both of these alterations are involved in carcinogenesis in Barrett's esophagus. Twelve of 13 tumors (92%) had 17p allelic deletions, and 8 of 13 tumors (62%) had p53 protein overexpression. Eight of the 12 tumors (67%) with 17p allelic deletions also had p53 protein overexpression. These data indicate that both 17p allelic deletions and p53 protein overexpression are frequently involved in carcinogenesis in Barrett's esophagus.     

Overexpression of the p53 protein and allele loss at 17p13 in ovarian carcinoma.

Mouse monoclonal antibodies PAb 240 and PAb 1801 which specifically immunoprecipitate p53 protein, were used to examine 27 fresh ovarian tumours (16 serous adenocarcinomas, six endometrioid carcinomas, one mucinous adenocarcinoma, one mucinous borderline tumour and three benign adenomas). Eleven out of 16 (69%) serous adenocarcinomas and one endometrioid tumour showed positive staining with one or both antibodies and none of the mucinous or benign tumours stained with either antibody. DNA from tumour and peripheral blood leukocytes was used to identify allelic deletions on chromosome 17p in tumours. 11/12 positively staining tumours showed less of heterozygosity (LOH) on 17p at the nearest informative locus to the p53 gene. In this series of ovarian tumours, LOH on 17p correlates closely with the aberrant expression of the p53 protein in a high proportion of advanced stage serous adenocarcinomas. This observation suggests that the p53 tumour suppressor gene is involved in the evolution of epithelial ovarian cancer (EOC) and may have prognostic significance.     

p53 gene mutations occur in combination with 17p allelic deletions as late events in colorectal tumorigenesis

Coordinate loss of one copy of the p53 gene and mutation of the remaining copy occur in colorectal carcinomas and in many other human malignancies. However, the prevalence of p53 gene mutations in carcinomas which maintain both parental copies of p53 has not previously been evaluated. Moreover, it is not known whether p53 gene mutations are limited to malignant tumors or whether they can also occur in benign neoplasms. To answer these questions, a total of 58 colorectal tumors have been examined; in each tumor, allelic losses were assessed using restriction fragment length polymorphisms and p53 gene mutations were assessed by sequencing cloned polymerase chain reaction products. The following conclusions emerged: (a) p53 gene mutations occurred but were relatively rare in adenomas, regardless of size and whether the adenomas were derived from patients with familial adenomatous polyposis; (b) In carcinomas as well as in adenomas, p53 gene mutations were infrequently observed in tumors which contain both copies of chromosome 17p (17% of 30 tumors), while tumors which lost one copy of chromosome 17p usually had a mutation in the remaining p53 allele (86% of 28 tumors); (c) p53 gene mutations were found at similar frequencies in primary tumor samples and in cell lines derived from tumors. These and other data suggest that the rate limiting step in p53 inactivation is point mutation and that once a mutation occurs, loss of the remaining wild-type allele rapidly follows. Both mutations and allelic losses generally occur near the transition from benign to malignant growth, and the p53 gene may play a causal role in this progression.     

Hypermethylation of HIC-1 and 17p allelic loss in medulloblastoma

Medulloblastoma is the most common malignant brain tumor in children.Chromosome arm 17p13.3 is reduced to homozygosity in 35-50% of medulloblastomas,making it the most frequent genetic alteration in these tumors. HIC-1 (hypermethylated in cancer) is a putative tumor suppressor gene located in the area of common deletion. HIC-1 resides in a CpG island and is hypermethylated in many different tumor types. Therefore, we studied a series of tumor specimens for hypermethylation and deletion of the region containing the HIC-1 gene to determine whether these two mechanisms of gene inactivation play a complimentary role in medulloblastoma. Southern blotting was performed using the methylation-sensitive restriction endonuclease NotI. Methylation of NotI restriction sites located in HIC-1 was demonstrated in 26 (72%) of 36 tumors and 11 (92%) of 12 specimens of normal brain. Of these 26 tumors, 23 differed significantly from normal brain. A greater proportion of the cells from the tumors showed methylated alleles of the HIC-1 gene. A group of 15 (42%) of 36 tumors exhibited loss of heterozygosity (LOH) for DNA sequences located on chromosome arm 17p. There was no significant correlation between LOH and methylation status (P = 0.19). Methylation in tumors beyond that seen in normal brain predicted poor overall survival independent of clinical risk category (P = 0.014). The results of our study show that methylation of the CpG island that contains the HIC-1 gene is common in medulloblastoma and, together with LOH of 17p, may be a critical event in the formation and aggressiveness of this tumor.     

Mutation of the TP53 gene and allelic imbalance at chromosome 17p13 in ductal carcinoma in situ.

A panel of 36 cases of preinvasive breast lesions, including 35 cases of ductal carcinoma in situ (DCIS), has been examined for mutation of TP53, allelic imbalance (AI) on 17p13, and expression of TP53, in a number of cases, has been studied using immunohistochemistry. Areas of DCIS, with or without adjacent invasive or benign cells, have been separately microdissected from paraffin-embedded sections and analysed by PCR for genetic changes to chromosome 17p13. TP53 mutations and AI on 17p have been identified in cases of ''pure'' DCIS as well as those with associated invasive carcinoma and, furthermore, have been identified in well-differentiated lesions as well as poorly differentiated ones.     

Allelic loss on chromosome 17p and p53 mutations in human endometrial carcinoma of the uterus

To understand the involvement of allelic losses and inactivation of tumor suppressor genes for the development of endometrial carcinoma of the uterus (EC), 24 cases of EC were examined for loss of heterozygosity (LOH) using a total of 57 polymorphic DNA markers covering all 23 pairs of chromosomes. LOH was observed at 27 loci on 10 different chromosomes, i.e., chromosomes 1, 3, 6, 11, 13, 15, 17, 18, 20, and 21, but was not detected at loci on chromosomes 4, 5, 7, 9, 10, 12, 14, 16, and X. It was observed only in seven of 24 cases, and the other 19 cases did not show LOH at any loci examined, including five cases of tumors with a high proportion of adenomatous hyperplasia. Among seven tumors with LOH at one or more loci, five tumors showed LOH at loci on the short arm of chromosome 17. Furthermore, mutations of the p53 gene, which is located on the short arm of chromosome 17, were detected in three of these 24 tumors by a polymerase chain reaction-single strand conformation polymorphism analysis and subsequent DNA sequencing. In two of these three tumors, p53 mutations were accompanied by the loss of wild-type p53 alleles. These results suggest that inactivation of the p53 gene is involved in the development of EC as in the case of several other types of human cancers.     

Prognostic-significance of chromosome 5q and 17p allelic deletion in colorectal adenocarcinomas

Patient survival was analysed for 75 patients after surgery for primary colorectal adenocarcinoma with regards to allelic loss of chromosome 17p and chromosome 5q. Allelic loss of chromosome 17p occurred in 69% of patients and was not significantly associated with a poorer patient prognosis as assessed by log rank analysis of Kaplan-Meier survival plots (p = 0.161). Allelic loss of chromosome 5q occurred in 32% of patients and was significantly associated with a poorer patient prognosis as assessed by log rank analysis of Kaplan-Meier survival plots (p = 0.014). Analysis of the two variables by Cox regression analysis indicated that allelic loss of chromosome 5q was an independent variable for patient prognosis. Entry of Dukes' stage into the model resulted in a final model with Dukes' stage and allelic loss of chromosome 5q as independent significant variables in assessing patient survival. These results show that allelic loss of chromosome 5q, but not chromosome 17p provides additional prognostic information for assessing patient survival, over and above Dukes' stage.     

Involvement of p53 gene in the allelic deletion of chromosome 17p in human ovarian tumors.

Previous reports have shown that one copy of the chromosome 17 was frequently lost in human ovarian cancers (1). The position of the allelic deletion has not been mapped and involvement of p53 gene has not been determined. In this study, we have shown that in human ovarian carcinoma, the commonest region of allelic loss in chromosome 17p is 17p 13.3 (65%) and 17p13.1 (63.7%; 6 out of 9 informative cases). Allelic loss was also observed at region 17p12 - 11.1 but at a lower frequency (38.6% to 37.5%). The pattern of allelic loss of p53 gene was consistent in both primary and secondary metastatic tumors of the same patient. No gross rearrangement of p53 was however observed at the remaining allele using Southern blot analysis. Allelic loss of p53 gene was closely associated with 17p 13.3, the terminal portion of chromosome 17p. The high frequency of allelic loss of p53 gene in ovarian carcinomas conformed with recent findings in cancers of colon, breast, lung and brain suggesting inactivation of p53 gene play a rate limiting step in pathogenesis of human malignancies.     

p53 mutation and allelic loss of chromosome 3p, 9p of preneoplastic lesions in patients with nonsmall cell lung carcinoma

BACKGROUND: An accumulation of mutations can result in carcinogenesis. Comparing genetic alterations in preneoplastic lesions with those seen in cancer in the same patient may be helpful in the early diagnosis of lung carcinoma or preneoplastic lesions. METHODS: To identify genetic alterations that may play a role in the development of nonsmall cell lung carcinoma (NSCLC), the authors examined the p53 gene and microsatellite markers on chromosome 3p (D3S643, D3S1317), 9p (D9S171, IFNA) in 35 bronchial metaplastic lesions and 28 alveolar hyperplastic lesions from 61 patients. RESULTS: A total of 8 metaplastic lesions (1 squamous metaplasia and 7 dysplasias) and 3 alveolar hyperplastic lesions (with atypia) showed genetic alterations, including loss of heterozygosity (LOH) of 3p, 9p and mutations of the p53 gene. In an analysis of microsatellite markers, 5 of 35 cases of squamous cell carcinoma (SCC) and 3 of 26 cases of adenocarcinoma (Ad) showed LOH in both preneoplastic lesions and synchronous cancers. Nine patients (25.7%) with SCC and 6 patients (23.1%) with Ad were shown to have mutations of the p53 gene by single-strand conformation polymorphism. In 2 of these 9 patients with SCC, the same mutation was observed in both dysplasia and SCC. CONCLUSIONS: These findings suggest that several genetic alterations may occur in preneoplastic lesions or the early stage of SCC of the lung, whereas the genetic alterations examined appeared to occur relatively late in the pathogenesis of pulmonary adenocarcinoma.     

Tumor-suppressor genes in colorectal carcinomas - p53 inactivation is highly associated with allelic loss of chromosome 5q

Sixty-four colorectal carcinomas were evaluated for allelic loss of chromosomes 17p and 5q, as well as point mutation of the p53 tumor suppressor gene. Allelic loss of chromosomes 5q and 17p were found to be weakly associated, whereas point mutation of the p53 gene was found to be more strongly associated with chromosome 5q allelic loss. Carcinomas in which both alleles of p53 had been inactivated showed a strong association with allelic loss of chromosome 5q (p=2.3x10(-4)), a relationship confirmed by an association between allelic loss of chromosome 5q and immunostaining with the p53 monoclonal antibody pAb 1801 (p=9.4x10(-3)). In contrast, allelic loss of chromosome 5q was not associated with either the activation of the c-Ki-ras proto-oncogene, or with DNA aneuploidy. The association between allelic loss of chromosome 5q and the inactivation of both alleles of p53 was significantly associated with tumor dissemination (p=7.2x10(-3)). These results suggest that dissemination of colorectal neoplasia may require the coordinate inactivation of at least two suppressor genes.     

Prognostic significance of p53 overexpression in gastric and colorectal carcinoma.

p53 expression was examined in 55 gastric and 107 colorectal carcinomas with an immunoperoxidase technique, using the polyclonal antibody CM1 on routinely fixed, paraffin embedded tissue. p53 protein was detected in 47% gastric and in 46% colorectal carcinomas and found to correlate with stage of disease and unfavourable clinical outcome (P less than 0.001). Thus, the proportion of positively reacting neoplasms increased as the stage progressed, tumours which had invaded regional lymph-nodes overexpressed p53 more frequently than localised carcinomas and an elevated level of p53 was associated with early relapse and death. In colorectal carcinoma p53 positivity was also linked with site and macroscopic configuration of the primary tumour and was most frequently expressed in carcinomas from the rectum and in ulcerative tumours. p53 overexpression was irrespective of tumour grade. Uniform negative reactivity with anti-p53 antibody was seen in normal epithelium adjacent to carcinoma, intestinal metaplasia, atrophic gastritis and in colonic adenomas. There was a good correlation between immunohistochemical staining on paraffin and frozen sections. These studies suggest that in gastric and colorectal carcinoma, immunohistochemical detection of p53 protein in routinely fixed tissue can be used along with other established parameters to assess prognostic outcome, especially to identify patients with poor short-term prognosis.     

p33ING1、p53在结直肠癌中的表达及其相互关系

目的：探讨p33^ING1、p53在结直肠癌中的表达及其相互关系。方法：应用免疫组化SP法检测60例结直肠癌组织及相应正常黏膜组织中p33^ING1、p53的表达。结果：结直肠癌组织、相应正常黏膜组织中p33^ING1蛋白的阳性表达率分别为43．3％（26／60）、100％（60／60）（P〈O．01），p53蛋白分别为51．6％（31／60）、0％（0／60）。p33^ING1在无淋巴结转移组及淋巴结转移组癌组织中的阳性表达率分别为57．6％（19／33）、25．9％（7／27）（P〈0．05）；在DukesA、B期、Dukes C、D期病例中分别为56．7％（17／30）、30．0％（9／30）（P〈0．05）。在p53表达阴性的29例中有12例（41．4％，12／29）p33^ING1表达缺失，而p53阳性的31例病例中有22例（71．0％，22／31）p33^ING1表达阴性（P〈0．05），在p53蛋白表达阳性的病例中p33^ING1蛋白表达明显缺失，两者表达呈负相关。结论：p33^ING1在结直肠癌组织中低表达，与p53互相协同，在结直肠癌的发生、发展中可能起重要作用。     

p53-expression and chromosome 17-copy number in colorectal adenomas

To get better insight into the role of numerical chromosome 17-aberrations and functional loss of the tumor suppressor gene TP53 during the early steps of colorectal carcinogenesis we analyzed paraffin-embedded tumor tissue from 58 colorectal adenomas with different histological features for p53-expression by immunohistology (IH; moAb DO1), #17-copy number by interphase-cytogenetics [nonradioactive in situ-hybridization (NISH) with a centromer-specific DNA-probe (D17Z1)], and DNA-ploidy by flow cytometry (FCM) with special emphasis on histopathological correlation. Seven adenomas (12%) showed nuclear p53-immunoreactivity. p53-expression was correlated with villous/tubulo-villous growth pattern (p=0.005) and grade of atypia (p=0.003) or dysplasia (p=0.0018). #17-aneusomy was present in 31% of the cases (29% deletions, 2% gains). In the FCM-analysis, 33% of the adenomas were DNA-non-diploid. p53-immunoreactivity correlated statistically significantly with FCM-non-diploidy (p=0.004) suggesting a role for the TP53-gene in the aneuploidization process. #17-deletions were associated to p53-immunoreactivity (p=0.046) but no correlation was found between FCM-ploidy and #17-copy number. As for loss of a tumor suppressor gene both alleles have to be affected, our data suggest a multistep process of TP53-inactivation. Whether the subgroup of adenomas with p53-expression might progress faster into invasive carcinoma than p53-negative cases remains to be tested.     

The role of combined allelic imbalance and mutations of p53 in tumor progression and survival following surgery for colorectal carcinoma

The role of p53 mutations in disease progression and survival of colorectal cancer is unclear, since numerous studies have reported different conclusions. However, few reports, if any, have evaluated disease progression and survival in relationship to 'functional' and 'non-functional' p53 status defined by genetic and molecular indications. Malignant colorectal tumors, from 72 unselected patients who underwent primary and potentially curative elective tumor resections, were either classified as p53 functional (p53+/+, p53+/-) or non-functional (p53-/-) based on DNA sequence analysis of all p53 exons, including determination of allelic imbalance of p53 (LOH), according to four DNA markers; 2 within the coding gene and two markers in the immediate flanking regions of p53. Tumor frequency of microsatellite instability was also analyzed according to Dukes' A-D stages. Dukes' staging predicted survival as expected, while the conceptual p53 status, functional p53 vs non-functional p53, did not clear-cut predict disease specific survival. p53 mutations alone or allelic imbalance inside the reading frame of the gene were unpredictive of survival, while allelic imbalance downstream of p53 predicted reduced survival (p < 0.05). The present study demonstrates that base mutations in combination with allelic imbalance within the reading frame of p53 do not predict survival or progression of colorectal cancer, while allelic imbalance upstream coding parts of the gene predicted disease-specific survival in univariate analysis. Thus, structural alterations within the gene seem less important than alterations in regions with potential control elements.     

p53和P-gp蛋白在胃癌中的表达

目的　研究胃癌组织中p5 3和P 糖蛋白表达的意义。方法　应用SP免疫组织化学的方法对 98例胃癌进行检测。结果　在 98例胃癌中 ,5 9.8%的 p5 3表达阳性 ,5 7.1%的P 糖蛋白表达阳性。p5 3和P 糖蛋白与胃癌的分化程度和生存期限有关 (P <0 .0 5 )。p5 3还与胃癌的浸润程度 ,淋巴结转移情况有关。P 糖蛋白表达阳性者 ,在 p5 3表达阳性组 ,明显高于 p5 3表达阴性组。 结论　联合检测 p5 3蛋白和P 糖蛋白对胃癌的诊断、治疗和预后有重要意义     

p53 gene mRNA expression and chromosome 17p allele loss in breast cancer

p53 messenger RNA expression was examined using a cDNA probe in 76 fresh primary breast tumour specimens, 15 of which came from patients treated with toxoxifen prior to surgery. A 2.8 kb mRNA for p53 was expressed in 43 of the 76 specimens. In 19 tumours the levels were similar to those seen in non-malignant (reduction mammoplasty) breast tissue, but in 24 tumours over-expression of mRNA for p53, approaching that seen in three breast cancer cell lines, was found. The cell lines MCF-7, T-47D and MDA-MB-231 expressed three p53 mRNA species of about 2.8 kb and a forth of 1.6 kb. Increased mRNA expression for p53 correlated (P less than 0.05) with loss of genetic material from the short arm of chromosome 17 as demonstrated by allele loss with the VNTR probe YNZ 22.1. There was also statistically significant correlation between increased p53 mRNA expression and low oestrogen receptor protein content in the tumours (P less than 0.05), but not with other clinical parameters. The findings support the view that p53 is involved in breast tumour biology, and suggest that its role may be complex.     

大肠癌组织中p53蛋白和基因的表达及意义

目的:观察以抑癌基因p53编码蛋白在正常肠黏膜、肠上皮化生及结直肠癌组织中的表达,探讨结直肠癌组织中p53和mRNA的表达及意义。方法:用半定量逆转录聚合酶链反应(RT-PCR)检测64例结直肠癌及其癌旁肠上皮化生组织中p53蛋白的表达水平,比较其与结直肠癌组织学分型、临床病理分期的关系。结果:1)结直肠癌组织中p53蛋白的表达明显低于正常组织(66.7%,26/39)。结直肠癌组织p53蛋白mRNA的表达水平与正常黏膜组织相比较,显著偏低(1.12±1.07,2.32±1.05,P<0.05)。2)蛋白和基因表达呈显著正相关(r=0.875,P<0.01)。3)黏液腺癌组为3/4,管状、乳头状腺癌为19/35,p53蛋白和基因表达与结直肠癌临床病理、组织学分型无明显相关性,P>0.05。结论:p53失活或低表达与结直肠癌的发生有密切相关性。提示p53 mRNA表达水平的检测可作为判定结直肠癌发生及侵袭转移能力的一项客观指标。