Genetic aberration in primary hepatocellular carcinoma:correlation between p53 gene mutation and loss-of-heterozygosity on chromosome 16q21-q23 and 9p21-p23

To elucidate the molecular pathology underlying the development of hepatocellular carcinoma (HCC), we used 41 highly polymorphic microsatellite markers to examine 55 HCC and corresponding non-tumor liver tissues on chromosome 9, 16 and 17. Loss-of-heterozygosity (LOH) is observed with high frequency on chromosomal region 17p13 (36/55, 65%), 9p21-p23 (28/55, 51%), 16q21-q23 (27/55, 49%) in tumors. Meanwhile, microsatellite instability is rarely found in these microsatellite loci. Direct sequencing was performed to detect the tentative mutation of tumor suppressor genes in these regions: p53, MTS1/p16, and CDH1/E-cadherin. Within exon 5-9 of p53 gene, 14 out of 55 HCC specimens (24%) have somatic mutations, and nucleotide deletion of this gene is reported in HCC for the first time. Mutation in MTS1/pl6 is found only in one tumor case. We do not find mutations in CDH1/E-cadherin. Furthermore, a statistically significant correlation is present between p53 gene mutation and loss of chromosome region 16q21q23 and 9p21-p23, which indicates that synergism between p53 inactivation and deletion of 16q21-q23 and 9p21-p23 may play a role in the pathogenesis of HCC. Genetic aberration in hepatocellular

Correlation of p53 gene mutation and expression of P53 protein in cholangiocarcinoma

AIM： To characterize the tumor suppressor gene P53 mutations and study the correlation of P53 gene mutation and the expression of P53 protein in cholangiocarcinoma. METHODS： A total of 36 unselected, frozen samples of cholangiocarcinoma were collected, p53 gene status（exon 5-8） and P53 protein were examined by automated sequencing and immunohistochemical staining, combined with the clinical parameters of patients. RESULTS： P53 gene mutations were found in 22 of 36 （61.1%） patients. Nineteen of 36 （52.8%） patients were positive for P53 protein expression. There were significant differences in extent of differentiation and invasion between the positive and negative expression of P53 protein. However, there were no significant differences in pathologic parameters between the mutations and non-mutations. CONCLUSION： The alterations of the P53 gene evaluated by DNA sequence analysis is relatively accurate. Expression of P53 protein could not act as an independent index to estimate the prognosis of cholangiocarcinoma.

p53 gene mutations in primary gastric cancer

AIMS p53 gene is one of the focuses in the study of tu- mour suppressor genes.So far,there is still controversy about the relationship between p53 alterations and clinicolpathological parameters of gastic cancers such as macroscopic classifica- tion,stage,degree of differentiation,depth of tumour invasion and lymphonod metastasis.Tamura has reported that p53 gene mutations mainly occur in the aneuploid tumours.But in China, nothing is reported in this field of study.Our aim is to analyze the relationship between p53 gene mutations and these param- eters including DNA ploidy in Chinese primary gasrtic cancers. METHODS Mutations of the p53 gene in exon5-8 were examined in 20 cases of primary gasric cancer by PCR-SSCP (Polymerase-chain-reaction-single-strand-conforma- tion-polymorphism)analysis. RESULTS Mutations were detected in 8(40%)cases:2 cases in exon5-6,2 cases in exon7,4 cases in exon8.These mutations were detected from stage 0 to stage Ⅲ No significant association was found between p53 gene mutations and the clinicopathological parameters such as macroscopic classifico- tion,degree of histological differentiation,depth of tumour in- vasion and lymphonod metastasis.In addition,66.7%(6 of 9) of aneuploid tumours had p53 mutations and only 18.2%(2 of 11)of diploid tumours had mutations. CONCLUSIONS These results suggest that p53 gene muta- tions are related to DNA ploidy alterations and that p53 gene is one of the important turnout suppressor genes in human gastric cancer.

DETECTION OF p53 GENE MUTATION OF BRONCHOSCOPIC SAMPLIES IN THE PATIENTS SUSPECTED TO LUNG CANCER

Objective: To determine the feasibility of detecting p53 gene mutations for early diagnosis of lung cancer using the samples from bronchoscopic examination. Methods: Point mutations of the exon 5-8 of p53 gene were detected in 85 bronchoscopic samples of 35 patients suspected to be lung cancer using silver staining PCR-SSCP. Results: p53 gene mutations were founded in 10 of 35 patients(28.6%). The incidence of p53 gene mutations (14.9%) was obviously higher than the cytological positive incidence(2.9%) in samples of sputum, bronchoalveolar lavage and brush, especially for the sputum(27.7%). In the bronchoscopic biopsy specimens, the incidence of p53 gene mutations (12.5%) was lower than that of pathologic positive result (50.0%). However, in view of all the bronchoscopic samples, there was no statistically difference between cytopathologic positive results (11.8%) and the incidence of p53 gene mutations (14.1%). Although the p53 mutations were most common in the samples from the patients bronchoscopically manifested as neoplasm compared with other manifestations, there was no statistical difference. It is valuable to notice that 3 patients with p53 gene mutation merely presented as bronchial inflammation in bronchoscope. Conclusion: Results indicated that the value of detecting p53 gene mutation for the diagnosis of lung cancer using the bronchoscopic samples was more superior to cytological examination and detection of p53 gene mutations in post-bronchoscopic sputum was easy and effective, may be used as a valuable method for early diagnosis of lung cancer.

p53 Gene Mutations in Asbestos Associated Cancers

The accumulation of mutant p53 protein in cancer cells was observed by immunohistochemistry analysis. DNA was extracted from paraffin-embedded tissue. Exons 5, 7 and 8 were amplified and studied by PCR-SSCP and sequencing analysis. Ten cases of asbestos associated cancer tissue were studied, of which five cases had adenocarcinoma, and the other five had mesothelioma, squamous carcinoma, small cell lung cancerl adenosquamous carcinoma and malignant lymphoma respectively. Employing monoclonal antibody PAb1801, five cases were found to be mutant p53 protein mpitive. Seven cases were found to have mutations by PCRSSCP. A total of 7 cases (8 mutations) were found to be positive and 4 cases were found to be opitive by both of these analyses. Of the 8 mutations found by SSCP analysis, 4(50%, 4/8)were clustered in exon 8. A high mutation frequency was noticed in adenocarcinoma (80%,4/5). ffequencing analysis on two specimens revealed two hotspot mutations. In codon 234,TAC for tyrooin was mutated to AAC fOr aspar8gine by a T to A transversion of the first letter. In codon 273, CGT for arginine was mutated to AGT for serine by a C to A transversion of the first letter. ln conclusion, the mutation of p53 gene is common in asbestos associated cancers. However, the mutational spectrum of asbestos associated cancers might be different from that of non-asbestos associated cancers.

DETECTION OF p53 GENE MUTATION IN PLASMA OF PATIENTS WITH GASTRIC CANCER

Objective: To investigated p53 gene mutation in plasma of gastric cancer patients. Methods: DNA extracted from plasma and matched tumor and tumor-adjacent non-cancerous tissues of 96 gastric cancer patients, and DNA from 20 healthy volunteers were studied. Exon 5, 6, 7, and 8 of p53 were amplified by Polymerase Chain Reaction (PCR). The mutation status was analyzed by denaturing high-performance liquid chromatography (DHPLC), followed by direct sequencing of cases with aberrant chromatographic patterns. Results: Heterozygous mutations of p53 gene were detected in 19.9% (19/96) of primary tumor tissues and 5.2% (5/96) of corresponding plasma. All p53 gene mutations detected in plasma DNA consisted with mutations in the matched primary tumor samples. Neither the tumor-adjacent gastric mucosa tissues nor control plasma from healthy volunteers showed p53 gene mutation. No correlation was found between p53 mutation status and clinicopathological features of gastric cancer patients. Conclusion: p53 gene mutation in plasma can be detected in tissues and plasma of gastric cancer patients, which could be applied in screening and surveillance of this disease.

Relationship of telomerase activity and p53 gene mutation in cardiac cancer

Objective： To study the relationship of the telomerase activity and the p53 gene mutation in cardiac cancer. Methods： Telomerase activity and the p53 gene mutation were detected in 46 case of cardiac cancer, peri-cancerous and 30 case of normal mucosa by TRAP-ELISA and PCR-SSCP. Results： The rate of expression of telomerase activity in cardiac cancer, peri-cancerous and normal mucosa were 82.61% （38/46）, 43.48% （20/46） and 13.33% （4/30） respectively. The rate of Exon5→,8 of p53 gene mutation were 39,13% （18/46）, 4.35% （2/46） and 0.00% respectively. There was significant difference between group cancer and without cancer （P 〈 0.01）. Mean of A^- value of telomerase is 1.89：1：0.41 in cancer group and were 1.49：1： 0.43, 0.54：1：0.45 respectively in peri-canvcerous and normal mucosa, there were significant differences in cancer group and group of without cancer （P 〈 0.05）. The rate of p53 gene mutations in group of expression of telomerase activity was 44.74% （17/38）, and 12.50% （1/8） in without expression of telomerase activity. There were significant differences between the two groups. Conclusion： The rate of expression of telomerase activity and mean of A^- value of telomerase in cardiac cancer were obviously higher than without cancer, which indicating telomerase activity was closely related with the occurrence of cardiac cancer. P53 gene mutation in cardiac cancer were higher than the tissue of without cancer, and the rate of p53 gene mutation in telomerase activity were obviously higher than the group of without cancer. This shows the p53 gene mutation can loss of function of suppressing cancer and prompt telomerase activity and cause the cardiac cancer.

Infrequent p53 gene mutation and expression of the cardia adenocarcinomas from a high-incidence area of Southwest China

INTRODUCTIONAdenocarcinomas of the cardia are the lesionsarising from the proximal stomach or within 3 cm ofthe gastroesophageal junction.These cancerstended to be advanced at the time of presentation,usually with poor prognosis.In recent decade,the incidence of adenocarcinoma of gastric eardiaand esophagus are increasing steadily,while therehas been a decrease in the proportion of the cancersarising from the distal stomach.The

The point mutation of p53 gene exon7 in hepatocellular carcinoma from Anhui Province,a non HCC prevalent area in China

AIM: In hepatocellular carcinoma (HCC) prevalent areas of China, the point mutation of p53 exon7 is highly correlated with Hepatitis B virus(HBV) infection and aflatoxin B intake. While in non-HCC-prevalent areas of China, these factors are not so important in the etiology of HCC. Therefore, the point mutation of p53 exon7 may also be different than that in HCC-prevalent areas of China. The aim of this study is to investigate the status and carcinogenic role of the point mutation of p53 gene exon7 in hepatocellular carcinoma from Anhui Province, a non-HCC-prevalent area in China. METHODS: PCR PCR-SSCP and PCR-RFLP were applied to analyze the homozygous deletion and point mutation of p53 exon7 in HCC samples from Anhui, which were confirmed by DNA sequencing and Genbank comparison. RESULTS: In the 38 samples of hepatocellular carcinoma, no homozygous deletion of p53 exon7 was detected and point mutations of p53 exon7 were found in 4 cases, which were found to be heterozygous mutation of codon 249 with a mutation rate of 10.53%(4/38). The third base mutation(G-T) of p53 codon 249 was found by DNA sequencing and Genbank comparison. CONCLUSION: The incidence of point mutation of p53 codon 249 is lower in hepatocellular carcinoma and the heterozygous mutation of p53 exon7 found in these patients only indicate that they have genetic susceptibility to HCC. p53 codon 249 is a hotspot of p53 exon7 point mutation, suggesting that the point mutation of p53 exon 7 may not play a major role in the carcinogenesis of HCC in Anhui Province, a non-HCC-prevalent area in China.

THE POTENTIAL DANGER OF HISTOPATHOLOGICALLYNORMAL TISSUES WITH P53 GENE MUTATION INLARYNGEAL CARCINOMA

Objective Possible differences were analyzed between histopathological and molecular biologicalfindings in laryngeal cancer tissues and its adjacent nomal tissues. Methods Laryngeal carcinoma tissues from20 patients were analyzed by PCR-SSCP. Results P53 gene mutations were detected in 25% (5 out of 20) of thecarcinomas. In these patients P53 mutations were detected in histopathologically nomal tissue both within 0.5 cmand more than 0.5cm distant from the nearest cancerous tissue. Conclusion Since the P53 mutation was commonin both cancer tissue the gene mutation may plan an.important role in laryngeal carcinogenesis. Radical excision ofthe tumour is defined by histopathological limits, but"normal" tissue may nevertheless contain the gene mutation,giving rise to the danger of further cancer development. We suggest that the determination of the surgical marginshould be based on a combination of histopathology and molecular biological findings.

Aflatoxin sufferer and p53 gene mutation in hepatocellular carcinoma

IM To study the p53 gene mutation and its relationship to aflatoxin B1 exposure in hepatocellular carcinoma (HCC).METHODS Restriction fragment length polymorphism analysis method was used in 62 HCC samples, and DNA direct sequencing in another 45 HCC samples.RESULTS In HCC and AFB1 high and lowrisk areas, 36/52 (69%) and 2/10 (20%) cases were found losing the HaeⅢ allele respectively, suggesting one of the base G mutation at the p53 gene codon 249. Similar results appeared in DNA direct sequencing, 20/35 (57%) and 1/10 (10%) respectively mutated at the codon 249 third base G to C transversion.CONCLUSION In HCC after AFB1 exposure, mutation of p53 gene is fixed at codon 249 third base and take the form of G to T transversion. This is a definite marker of mutation which is induced by AFB1 mutagen. It is applicable for molecular epidemiologic survey of the sufferers of AFB1 among HCC cases and for discovering more unknown natural AFB1 contaminated areas..

Overexpression and mutations of tumor suppressor gene p53 in hepatocellular carcinoma

AIMS To examine the prevalance of p53 mutations in hepatocellular carcinoma (HCC) from Chongqing area and the relationship between the p53 mutations and clinicopathological features of HCC,as well as the risk factors. METHODS The overexpression and point mutations of tumor suppressor gene p53 in 38 cases of HCC were detected by a sensitive antigen retrieval fluid (ARF) immunohistochemical method and polymerase chain re- action(PCR)-restriction fragment length polymorphism (RFLP),and single strand conformation polymorphism (SSCP)-silver staining analysis. RESULTS The results showed that 16 of 38 HCCs had positive p53 protein (42.1%),7 HCCs had p53 mutation at 249 (18.4 % ) and 2 HCCS had point muta- tion within exon 7 other than 249. Among 9 cases of HCC with mutations,8 cases demonstrated positive p53 protein,its coincidental rate was 88.9%. The overexpression and mutations of p53 were significantly related to the differentiation and metastasis of HCCs. The frequency of p53 mutations was consistent with high prevalence of HBV and a moderate aflatoxin B1 (AFB1) exposure in our area. CONCLUSIONS The results suggest that AFB1 acts synergistically with HBV in the generation of p53 mutations. Furthermore,dietary exposure to AFB1 may mainly contribute to the tumor specific mutation at codon 249,while HBV may account for other scattered mutations in HCC.

THE SIGNIFICANCE OF P53 GENE MUTATIONS AND EXPRESSIONS IN HUMAN COLORECTAL TUMORS

Using a polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) approach we analyzed 18 human colorectal adenocarcinomas for mutations in exons 5,6,7,8 of p53 gene. At the same time,p53 gene product expression was studied immunohistochemically in these 18 case in frozen sections. The expression of p53 protein was also immunohistochemically studied in formalin-fixed paraffin embeded spccimens of 76 colorectal adenocarcinomas and 112colorectal polyps. Eigbt out of 18 cases (44%) showed a variant band indicative of a mutation in exons 5-6 of p53gene 7 out of 8 cascs (88%) with p53 gene mutations were positivelystanined for P53. There was no significant correlation between p53. expression and clinicopathological manifestations and prognosis. but the strongest staining was cncountered in those cases with well differentiated and early stage adenocarcinomas,while weaker staining was encountered in Poorly differentiated and mucoid adenotarcinomas. p53expression was not observed in proliferative polyps and adenomas with low grade dysplasia. The frequency of p53expression reached 88% (p<0.001) when adenoma showed malignant change. Aiuong three types of adenomas, p53 expression was most frequent in villous type (P<0.05). The frequencies of p53 expression in adenoma, adenoma with malignant change and adenocarcinoma were 4%, 88% and 51% respectively.These indicate that genetic changes of p53 gene play an important role in the transformation from benign adeuoma to adenocarcinoma. p53 immunohistochcmistry can be used as a surrogate marker for p53 gene mutation for early discovery of colorectal adenocarcinomas.

P53 Gene Mutations in Non-Hodgkin's Lymphoma

Summary: To understand P53 gene change of non Hodgkin's lymphoma (NHL) and human malignant lymphoma cell lines, the exons 5 7 of 29 patients with NHL and 9 kinds of human malignant lymphoma cell lines were studied by silver staining PCR SSCP technique. Three cases of P53 gene point mutation was found in 29 cases of NHL. Mutation developed in exon 5 in 2 cases, and in exon 6 in 1 case. They were all diffuse lymphoma. In mutation cases, B cell lymphoma accounted for 2 cases and the other one was T cell lymphoma. There was no P53 gene mutation in low grade follicular lymphoma. Seven strains out of 9 kinds of lymphoma cell lines had P53 gene point mutation. One strain had the mutation in exon 5; 5 strains in exon 6 and 1 strain in exons 5, 6, 7. There was a high mutation rate in lymphoma cell lines and low mutation rate in NHL patients. P53 gene plays an important role in lymphoma cell line establishment, cell regeneration and disease evolution.

Codon 249 mutations of p53 gene in non-neoplastic liver tissues

AIM To study the significance of p53 gene in hepatocarcinogenesis through analyzing codon 249 mutations of p53 gene in non neoplastic liver tissues. METHODS Codon 249 mutation was detected using single stranded conformational polymorphism analysis and allele specific PCR in liver tissues from 10 cases of chronic hepatitis, 5 cases of cirrhosis and 20 cases of HCCs. RESULTS The detection rate of codon 249 mutation in chronic hepatitis, cirrhosis and pericancerous tissues was 70% (7/10), 100% (5/5) and 70% (14/20), respectively by AS PCR. These mutations could not be detected by SSCP analysis. The detection rates were 65% (13/20) and 45% (9/20) in cancerous tissues by AS PCR and SSCP analysis. CONCLUSION Codon 249 mutations of p53 gene were very popular in non neoplastic liver tissues though the number of those mutant cells was only in subsection. Those mutations in cancerous tissues might take place in the stage before the formation of tumor.

Codon 249 mutations of p53 gene in development of hepatocellular carcinoma

AIM To investigate the mechanisms of codon 249 mutation of p53 gene in the formation of hepatocellular carcinoma (HCC). METHODS Codon 249 mutation accompanied by loss of heterozygosity (LOH) and its effect on translation and transcription were studied using SSCP, IHC and RT PCR/slot hybridization. RESULTS Codon 249 mutations were detected in 32 9%, LOH detected in 68 4% among the HCC patients. Mutations of condon 249 were accompanied by LOH in 90%. The positive rates of p53 protein and mRNA were 91 3% and 95 7%, in mutational group, both were significantly higher than those in the non mutational group (91 3% vs 19 1% and 95 7% vs 40 4%, respectively, both P <0 01). The translation of p53 gene was strongly related to its transcription by correlation analysis ( r =0 8208). CONCLUSIONS LOH might play an important role in hepatocarcinogenesis of codon 249 mutation, which could increase both transcription and translation of p53 gene. The increased expression of p53 protein mainly depend on the increased transcription of p53 gene.

Analysis of N-ras gene mutation and p53 gene expression in human hepatocellular carcinomas

IM To study the relationship between Nras gene mutation and p53 gene expression in the carcinogenesis and the development of human hepatocellular carcinomas (HCC).METHODS The Nras gene mutation and the p53 gene expression were analyzed in 29 cases of HCC by polymerase chain reactionsingle strand conformation polymorphism (PCRSSCP) and immunohistochemistry.RESULTS Thirteen cases of HCCs were p53 positive (448%), which showed a rather high percentage of p53 gene mutation in Guangxi. The aberrations at Nras codon 2-37 were found in 7931% of HCCs and 8077% of adjacent nontumorous liver tissues. More than 2 point mutations of Nras gene were observed in 22 cases (7586%). Twelve cases (4137%) of HCCs showed both Nras gene mutation and p53 gene expression.CONCLUSIONS Nras gene and p53 gene may be involved in the carcinogenesis and the development of HCC. That 38% of HCCs with Nras gene mutation did not express p53 protein indicates that some other genes or factors may participate in the carcinogenesis and the development of HCC.

Analysis of Genetic Alterations in TP53 Gene in Breast Cancer - A Secondary Publication

Tumor protein p53 (TP53) mediates DNA repair and cell proliferation in growing cells. The TP53 gene is a tumor suppressor that regulates the expression of target genes in response to multiple cellular stress factors. Key target genes are involved in crucial cellular events such as DNA repair, cell cycle regulation, apoptosis, metabolism, and senescence. TP53 genetic variants and the activity of the wild-type p53 protein (WT-p53) have been linked to a wide range of tumorigenesis. Various genetic and epigenetic alterations, including germline and somatic mutations, loss of heterozygosity, and DNA methylation, can alter TP53 activity, potentially resulting in cancer initiation and progression. This study was designed to screen three reported mutations in the DNA-binding domain of the p53 protein in breast cancer, to evaluate the relative susceptibility and risk associated with breast cancer in the local population. Genomic DNA was isolated from 30 breast tumor tissues along with controls. Tetra and Tri ARMS PCR were performed to detect mutations in the TP53 coding region. For SNPs c.637C>T and c.733C>T, all analyzed cases were homozygous for the wild-type allele ‘C,’ while for SNP c.745A>G, all cases were homozygous for the wild-type allele ‘A.’ These results indicate no relevance of these three SNPs to cancer progression in our study cohort. Additionally, the findings from whole exon sequencing will help to predict more precise outcomes and assess the importance of TP53 gene mutations in breast cancer patients.

Study of mutations of p53, APC and K-ras genes in 47 cases of intestinalmetaplasia of gastric mucosa

Objective:To study the role of the mutations of p53, APC and K-ras genes in 47 cases of 3 types of intestinal metaplasia (IM) of gastric mucosa. Methods:In 47 cases of IM, exons 5- 8 of p53 and exons 15 of APC were examined with PCR-SSCP and codon 12 of K-ras with PCR-RFLP to detect the existence of any mutations of these structures. Results:Muta- tions of p53, APC and K-ras were found in 29.8% (14/47),6.4% (3/47) and 6.4% (3/47) respectively in our series of patients who consisted of 33 with types I and II and 14 with type III of IM. The mutation rate of p53 was far higher in patients with type III IM (57.1%,8/14) than in those with types I and II IM(18.2%,6/33)(P <0.05). Though the mutation rate of APC and K-ras was also higher in the patients with type III IM than in those with types I and II IM, it was of no statistical significance (P >0.05). In one case of type III IM, mutation of both p53 and K-ras was found. Conclusion: The molecular changes of 3 types of IM are different. The mutation of p53 may be closely related to carcinogenesis in cases of type III IM and it serve as a sign for the early diagnosis of gastric carcinoma.

Abnormal Change of p53 Gene in Gastric and PrecancerousLesions and APC Gene Deletion in Gastric Carcinoma and Near Tissues

p53 gene mutation (exon4, 5, 6, 7, 8 and intron6) in gastric cancer and precancerous lesions and p53 gene (exon4 and ontron6), APC gene deletion in gastric carcinomas were studied by PCR/SSCP and PCR/RFLP- Results showed mutation rate of p53 in metaplasia, dysplasia and gastric carcinoma was 37. 5 % (3/8), 42. 11 % (8/19), 53. 33 (16/30) respectively- There was significant dif-ference among groups of metaplasia, dysplasia, cancer and normal controls. Noexon8 mutation was found in metaplasia and dysplasia, but 4 cases were found to have exon8 mutation in cancer group. It is suggested that exon8 mutation occurs at the late stage of gastric cancer, but exon 5, 6, 7 mutation occur in the course ofprecancerous lesion to cancer. Loss of heterozygosity (LOH) of exon4, intron6,APC was 47,37 % (9/19), 8. 73% (2/23), 16. 67 % (3/18) respectively. LOH of exon4 had something to do with poor differentiation, lymph node metastasis,depth of invasion- LOH of exon4 may be one of prognostic marker of gastric cancer. We are led to conclude that p53 gene mutation is an early event and perhaps work together with ras oncogene in gastric carcinogenesis