Detect P53 Gene Research Articles

Detection of p53 gene mutation of bronchoscopic samples in the patients suspected to lung cancer

Objective: To determine the feasibility of detecting p53 geue 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.

Staining patterns of p53 immunohistochemistry and their biological significance in colorectal cancer.

Immunohistochemistry (IHC) is a cheap and rapid method to detect p53 inactivation but the results are often discordant with gene mutation analysis. This study aimed to investigate whether there is a difference in the immunohistochemical staining patterns of p53-positive cells on comparing tumours with inactivating gene mutations with those without. Tissues of 142 colorectal cancers were investigated for p53 inactivation simultaneously by IHC and gene analysis using SSCP of exons 4-9 and sequencing. In addition, tumours were investigated immunohistochemically for the expression of mdm-2 protein, known to be transcriptionally transactivated by the wild-type (wt) p53 gene. p53-positive cells of tumours without detectable p53 gene mutations were microdissected using a PALM laser microscope system and subjected to p53 sequence analysis. Among the 142 cases of colorectal cancer (male/female=88/54; mean age 66a+/-11 years, range 24-90 years), 74% (n=105) of tumours were positive by p53 IHC and mutations in the p53 gene were found in 51% (73 patients). In 16% (12 patients) with mutations in the p53 gene, IHC for p53 was negative. In tumours with mutations in the p53 gene and positive p53 IHC, staining of all nuclei of the tumour was more frequently (57/61, 93%) found than in tumours without p53 gene mutations, where staining of scattered single cells was predominantly seen (29/44, 66%; p<0.0001). mdm-2 positivity (n=33) showed only staining of scattered single cells, predominantly (24/33, 82%; p<0.0001) in tumours without gene mutations. Single cell microdissection followed by mutation analysis of scattered p53-positive cells revealed no gene mutations. A scattered positive immunohistochemical reactivity of p53 in colorectal cancer cells might therefore represent a functionally active non-mutated p53 gene and should not be considered as a marker of gene mutation and inactivation.

Myxoid liposarcoma with transition to round-cell lesion-cell cycle regulator genes and telomerase activity characterizing tumor progression: A case report

A mixed myxoid/round cell liposarcoma was macrodissected in its 2 histologic components and investigated for genetic differences between its low-grade myxoid and the high-grade round-cell region. For both, we failed to detect p53 gene mutations, loss of heterozygosity at the p53 or Rb genes, and p53 protein expression. The round-cell component showed a high telomerase activity, and an elevated c-myc mRNA and protein expression. The myxoid component was characterized by a lack of telomerase activity and low c-myc mRNA expression, and immunohistochemistry failed to detect the c-myc protein. There was a higher Mib-1 proliferation index in the round-cell portion. The same specific translocation t(12;16) and the fusion transcript type II in both components confirmed the close relationship between myxoid and round-cell liposarcomas. Telomerase activity and increased c-myc expression seem to be helpful molecular markers for characterizing tumor progression in myxoid liposarcoma.

Detection of gene mutation in sputum of lung cancer patient

Lung cancer is a common malignant tumor, which has a high incidence and mortality rate. Therefore, it is necessary to seek a new method for the diagnosis, especially the early diagnosis of lung cancer. The development of molecular biology makes the gene diagnosis of lung cancer possible. PCR-SSCP was applied to detect p53 gene mutation of lung cancer patients' sputum cells and we have achieved good results.

Detection of p53 gene mutations: analysis by single-strand conformation polymorphism and Cleavase fragment length polymorphism.

We have generated a collection of clones containing single point mutations within the exon 5-9 hot spot regions of the p53 gene by using polymerase chain reaction (PCR) to amplify select regions of the gene from characterized cell lines. These clones were then used to address the sensitivity of mutation detection using slab-gel single-strand conformation polymorphism (SSCP) and Cleavase fragment length polymorphism (CFLP) assay systems. Both methods exhibited high sensitivities for the detection of mutations in cloned p53 mutations in this study: 97% for CFLP and 94% for SSCP. In addition to resulting in higher sensitivity of mutation detection, CFLP has the capability to analyze longer fragments. In this study, CFLP identified five intronic mutations which were not investigated in the exon-specific SSCP assay. These results agree with those found elsewhere and demonstrate that CFLP scanning can have practical advantages when used for the identification of sequence alterations within the p53 gene.

Cleavage of double-stranded copy RNA by RNase 1 and RNase T 1 provides a robust means to detect p53 gene mutations in clinical specimens

Detecting somatic mutations in patient specimens is challenging because of the wide variation in quality and quantity of genomic DNA in clinically derived material. In cancer specimens, the challenge of detecting mutations is usually compounded by the presence of large numbers of nonmutated normal cells that dampen the relative signal that can be obtained from employing any mutation detection strategy. In the case of somatic mutations in the gene encoding the tumor suppressor, p53, a clinically useful mutation detection assay must be able to detect a wide variety of types of mutations scattered over five coding exons and their flanking intron sequences. This study examined the ability of a mutation detection strategy, termed NIRCATM, to identify single-base mutations in the clinically relevant domain of the p53 gene. This strategy relies on RNase digestion-mediated cleavage of double-stranded copy RNA transcribed in vitro from polymerase chain reaction (PCR)-amplified genomic templates to detect mismatched base pairs resulting from hybridization of complimenting mutant and wild-type copy RNA strands. This assay system was found to robustly detect all twelve possible mismatches and the plus one and minus one frame shifts. Furthermore, the assay could detect mutations in clinical specimens when the mutant alleles composed as few as 4% of the total population of alleles isolated in bulk specimen genomic DNA. This mutation detection strategy worked efficiently in bladder, breast, colon and lung tumors as well as sediments from bladder cytology specimens.

Cleavage of double-stranded copy RNA by RNase 1 and RNase T1 provides a robust means to detect p53 gene mutations in clinical specimens.

Detecting somatic mutations in patient specimens is challenging because of the wide variation in quality and quantity of genomic DNA in clinically derived material. In cancer specimens, the challenge of detecting mutations is usually compounded by the presence of large numbers of nonmutated normal cells that dampen the relative signal that can be obtained from employing any mutation detection strategy. In the case of somatic mutations in the gene encoding the tumor suppressor, p53, a clinically useful mutation detection assay must be able to detect a wide variety of types of mutations scattered over five coding exons and their flanking intron sequences. This study examined the ability of a mutation detection strategy, termed NIRCA, to identify single-base mutations in the clinically relevant domain of the p53 gene. This strategy relies on RNase digestion-mediated cleavage of double-stranded copy RNA transcribed in vitro from polymerase chain reaction (PCR)-amplified genomic templates to detect mismatched base pairs resulting from hybridization of complimenting mutant and wild-type copy RNA strands. This assay system was found to robustly detect all twelve possible mismatches and the plus one and minus one frame shifts. Furthermore, the assay could detect mutations in clinical specimens when the mutant alleles composed as few as 4% of the total population of alleles isolated in bulk specimen genomic DNA. This mutation detection strategy worked efficiently in bladder, breast, colon and lung tumors as well as sediments from bladder cytology specimens.

Mutations of p53 gene can be detected in the plasma of patients with large bowel carcinoma.

AIMS: To attempt to detect p53 gene mutations in the plasma of patients with large bowel carcinoma. METHODS: Plasma was collected from 20 control patients with no history of cancer...

Analysis of p53 gene mutations in keloids using polymerase chain reaction-based single-strand conformational polymorphism and DNA sequencing.

Keloids are the result of a dysregulated wound healing process. They are characterized by the formation of excess scar tissue that proliferates beyond the boundaries of the original wound. Somatic mutations of p53 have been implicated as causal events in up to 50% of all human malignancies. In addition, p53 has been shown to play an important role in controlling cell proliferation and apoptosis. We hypothesize that mutations in p53 can lead to a hyperproliferative state that can result in keloid formation. To detect p53 DNA mutations in tissues and cultured fibroblasts from skin lesions of 7 patients with keloids. The polymerase chain reaction followed by single-strand conformational polymorphism analysis and direct DNA sequencing were used to detect p53 gene mutations. The Department of Dermatology, Henry Ford Hospital, Detroit, Mich. Seven patients with keloids seen for routine surgical excision of their lesions. Normal DNA specimens were obtained from buccal smears and healthy skin samples from these patients. Mutations in the p53 were identified in all patients by polymerase chain reaction followed by single-strand conformational polymorphism analysis and subsequently confirmed by DNA sequencing. A mutation in exon 5 resulting in amino acid substitution was found in 1 of the patients in keloid tissue and cultured keloid fibroblasts (codon 156, CGC-->CCC, arginine-->proline). Frameshift mutations in exons 5 and 6 caused by the insertion or deletion of a nucleotide at different positions were found in 6 patients with keloids in both keloid tissues and cultured fibroblasts. Mutations in exon 4 resulting in amino acid substitution were found in all patients in both keloid tissues and cultured fibroblasts (all in codon 72, CGC-->CCC, arginine-->proline). No p53 mutations were detected in buccal smears or cultured fibroblasts from healthy skin samples of any of the patients. Focal mutations in p53 may increase cell proliferation and decrease cell death in the dysregulated growth patterns that have been clinically documented. An understanding of the pattern of all growth dysregulation related to keloids may lead to new therapeutic strategies.

Clinical implication of screening p53 gene mutations in head and neck squamous cell carcinomas.

The role of the tumour-suppressor gene p53 in the tumorigenesis of head and neck cancer has been well established, but the clinical significance of p53 alteration is still unclear. A group of 50 patients with head and neck squamous cell carcinoma (HNSCC) were investigated for p53 alterations. DNA was extracted from fresh tumour samples and polymerase chain reaction/single-strand conformation polymorphism analysis was used to detect p53 gene mutations in the region from exon 5 to exon 9. In addition, p53 protein overexpression was assessed by immunohistochemistry using the monoclonal antibody DO-7 on paraffin-embedded tissue sections. p53 gene mutations were found in 45% and p53 protein expression was detected in 61.2% of tumour samples. While p53 protein expression was not correlated with any clinical factors, p53 gene mutations indicated local regional recurrences of HNSCC. The risk of locoregional recurrence was significantly greater in patients with a p53 gene mutation than in patients with the wild-type p53 gene (P = 0.001). Multivariate analysis confirmed p53 gene mutation to be an independently predictive factor for the tumour recurrence (P = 0.0064). When we analysed p53 gene mutation in 12 patients with primary and recurrent tumours, we found that 4 patients (33.3%) had a different p53 gene mutation in the recurrent tumour from that in the original primary tumour. The results indicate that p53 gene mutations and not protein overexpression are valuable predictors for tumour recurrences and for differential diagnosis of a second primary HNSCC.

Expression of p53, MDM2 protein and Ki-67 antigen in recurrent meningiomas.

Association of p53 gene abnormalities with tumor progression and prognosis of many neoplasms has been demonstrated, but little is known about the clinical significance of p53 abnormalities in meningiomas. The significance of p53 protein expression in recurrent meningiomas and its relationships with MDM2 protein and proliferation activity were investigated by analyzing 39 meningiomas immunohistochemically. p53 protein was expressed in 11 (35%) of 31 non-recurrent and 7 (88%) of 8 recurrent meningiomas. A high frequency of p53 expression was observed in recurrent meningiomas, which tended to have a high p53 positive index (p53 PI), indicating that p53 immunoreactivity may be a marker for predicting tumor recurrence. Four recurrent meningiomas with high p53 PIs were analyzed by the polymerase chain reaction-single strand conformation polymorphism method to detect p53 gene mutations, but none were found in exons 4-8 of this gene. Fifteen (71%) of 21 MDM2-positive and 3 (17%) of 18 MDM2-negative tumors expressed p53 protein, showing that MDM2 expression was more common in meningiomas with p53 expression. p53 immunoreactivity in the absence of mutation may indicate stabilization of the wild type through interaction with the MDM2 protein. The Ki-67/MIB-1 proliferation index (MIB-1 PI) correlated well with recurrence. The p53-positive tumors had a significantly higher mean MIB-1 PI than p53-negative tumors, suggesting that wild-type p53 inactivation by the MDM2 protein may be involved in controlling the proliferative activity in meningiomas. In conclusion, immunohistochemical examination for p53 protein as well as proliferative activity may help predict the malignant potential of tumor recurrence.

Detection ofp53 gene mutation by rapid PCR-SSCP and its association with poor survival in breast cancer

We examined the association between mutation of the p53 gene and survival in a large cohort of breast cancer patients. Using a rapid, non-isotopic single-strand conformation polymorphism (SSCP) method we screened for mutations in exons 4-10 of the p53 gene in 375 primary breast cancers from patients with a median follow-up of 57 months. Mutations were found in 19% of tumours. Statistically significant associations were found between p53 mutation and histological grade, hormone receptor status, ploidy and S-phase fraction. No association was found between p53 mutation and axillary lymph node involvement, histological type, tumour size, vascular invasion or patient age. In univariate survival analysis, p53 mutation was strongly associated with poor prognosis. This was maintained in the lymph node-negative and hormone receptor-positive patient subgroups. In multivariate analysis, p53 mutation was associated with poor survival independent of lymph node status, estrogen receptor status and S-phase fraction. Our results demonstrate the feasibility of using a rapid and simple polymerase chain reaction-SSCP screening procedure to detect p53 gene mutation in breast cancer for the provision of prognostic information.

Detection of p53 gene mutations in cytopathology and biopsy specimens from patients with lung cancer.

In order to ascertain the feasibility of detecting p53 gene mutations in patients with lung cancer in a nonsurgical diagnostic setting before starting treatment, we screened for p53 gene mutations in tumor specimens obtained using diagnostic methods such as fiberoptic bronchoscopy, thoracentesis, and percutaneous needle aspiration. We examined 206 specimens from 66 patients diagnosed with primary lung cancer at Hiroshima University Hospital between October 1991 and July 1993 using the polymerase chain reaction/denaturing gradient gel electrophoresis technique. p53 gene mutations were found in 64 of 159 (40%) cytologically positive specimens, but in none of 47 cytologically negative specimens. The PCR-based assay did not increase the sensitivity of the cytopathologic examination in detecting malignant cells. The type and location of the p53 gene mutation was the same in cytologically positive specimens obtained by different methods, but from the same patient. Of the 66 patients, p53 gene mutations were found in 27 (41%) at the time of the first nonsurgical diagnostic examination: 7 of 12 (58%) with small cell carcinoma, 9 of 20 (45%) with squamous cell carcinoma, and 11 of 34 (32%) with adenocarcinoma of the lung. The incidence of p53 gene mutation for each histologic subtype was comparable to previously published data examining surgically and/or autopsy-obtained specimens. These results indicate that detection of p53 gene mutations in a nonsurgical, diagnostic setting is feasible. This technique will make it possible to assess the significance of p53 gene mutations in relation to survival and response to therapy before starting treatment, in future prospective studies.

Demonstration of human papillomavirus type 16-related DNA and absence of detectable p53 gene mutations in widespread cutaneous squamous cell carcinomas after oral psoralen with UV-A treatment

Demonstration of human papillomavirus type 16-related DNA and absence of detectable p53 gene mutations in widespread cutaneous squamous cell carcinomas after oral psoralen with UV-A treatment

Molecular genetic analysis of clear cell adenocarcinomas of the vagina and cervix associated and unassociated with diethylstilbestrol exposure in utero

Prenatal exposure to the synthetic estrogen diethylstilbestrol (DES) is associated with the subsequent development of clear cell adenocarcinoma of the lower reproductive tract in young women, and data concerning the molecular genetic alterations involved in the etiology of this tumor type have not previously been reported. Such knowledge would be of potential value by providing insight into the molecular mechanisms of hormonal carcinogenesis in general, as well as by suggesting molecular markers for risk assessment in the estrogen-exposed population. A total of 24 samples of clear cell adenocarcinoma of the vagina or cervix, 16 associated with exposure in utero to DES and 8 with no history of DES exposure, were obtained as archival fixed and embedded tissue specimens. DNA was purified from these tissues and used to examine a number of biologically plausible molecular genetic endpoints for tumor specific alterations. No evidence was found for mutations in the K-ras or H-ras protooncogenes, the Wilms' tumor (WT1) tumor suppressor gene, or the estrogen receptor gene. Sporadic overexpression of the p53 tumor suppressor gene was detected in some tumor cell nuclei by immunohistochemistry, but in the absence of detectable p53 gene mutation. Genetic instability as manifested by somatic mutation of microsatellite repeats was widespread in these tumors, with evidence of microsatellite instability in all DES-associated tumors examined, and in 50% of those tumors not associated with DES exposure. These data are consistent with the hypothesis that the induction of genomic instability may be an important mechanism of DES-induced carcinogenesis.

Association of p53 gene mutations with short survival in pancreatic adenocarcinoma.

Mutations of the p53 gene have been found in a variety of human cancers and are implicated in the biologic functions of cancer. To investigate the clinical implications of p53 mutations in pancreatic adenocarcinoma, we examined the association of mutations of the p53 gene with patients’ prognosis. Single‐strand conformational polymorphism analysis and direct DNA sequencing were used to detect p53 gene mutations in 37 pancreatic adenocarcinomas. p53 gene mutations were detected in 16 (43%) of the 37 pancreatic adenocarcinomas. Direct sequencing did not reveal preferential clustering at any specific codon. There was no significant association of the presence of p53 gene mutations with histologic types, extent of tumor invasion, the presence of lymph node metastasis, or tumor stage. Univariate analysis showed that survival of patients with p53‐gene‐mutation tumors was significantly poorer than that of patients with p53‐gene‐nonmutated tumors (p=0.02). Cox's multivariate analysis of ten clinicopathologic features including p53 gene mutations revealed that presence of p53 gene mutations (p= 0.026) and curativity of operation (p=0.014) were independent predictors of survival. Furthermore, the survival of patients with p53‐gene‐mutated tumor was significantly poorer than that of patients with p53‐gene‐nonmutated tumors, both in patients who underwent curative operation (p=0.04) and in patients who underwent non‐curative operation (p=0.01). These results suggested that mutations of the p53 gene might play an important role in cancer aggressiveness and could be a clinically useful predictor of prognosis in patients with pancreatic adenocarcinoma.

Arsenic-related Bowen's disease and paraquat-related skin cancerous lesions show no detectable ras and p53 gene alterations

We have investigated point mutations of condons 12, 13, and 61 in H-, K-, and N- ras oncogenes as well as p53 tumour suppressor gene exon 5 through exon 9 by PCR-SSCP analysis in 26 skin biopsy tissues from 16 arsenic-related Bowen's disease patients and 6 skin samples from 4 paraquat manufacturing workers. No mutation was found. These results are different from findings with UV associated skin cancers. Interestingly, a silent change at condon 27 of H- ras in one allele was detected in all 4 paraquat manufacturing workers and in 2 of 16 arsenic-related Bowen's disease patients. It is likely that the molecular mechanisms involved in arsenic and paraquat induced skin cancers differ from sunlight-related skin maligancies.

A correlative study of p53 protein alteration and p53 gene mutation in glioblastoma multiforme.

The p53 tumor suppressor gene is frequently mutated in glioblastomas. Mutations within the p53 gene often result in aberrant expression of the p53 protein leading to protein accumulation within the nucleus of the cells which can be detected by immunochemistry. Many studies have correlated alterations of p53 protein expression with p53 gene mutations. Positive staining of tumor cells for p53 protein has been widely assumed, perhaps incorrectly, to signify the presence of p53 gene mutations. This study compared the immunostaining patterns for p53 protein in 37 glioblastomas with the molecular genetic data obtained by the single strand conformation polymorphism assay. p53 gene mutations were detected in 46% (17 of 37) of glioblastomas, while 65% (24 of 37) of glioblastomas were positive for protein accumulation by immunohistochemistry. Although 30 of 37 glioblastomas analyzed showed concordance for p53 protein expression and p53 gene mutations, a subset of seven glioblastomas showed discordant accumulation of the p53 protein in the absence of any detectable p53 gene mutations. The mdm-2 gene was assessed in 17 glioblastomas for gene rearrangements or amplification, but none were found. This result suggests that a mechanism other than p53 gene mutation can result in altered p53 protein expression.

Comparative Study of p53 Gene and Protein Alterations in Human Astrocytic Tumors

The p53 gene is a tumor suppressor gene involved in many common malignancies, including astrocytomas. Genetic analysis of the p53 gene and immunohistochemistry of the p53 protein have each been used to screen astrocytomas. To compare these methods, we performed immunohistochemistry with the monoclonal antibody PAb 1801 and single-strand conformational polymorphism (SSCP) with sequence analysis on 34 astrocytic tumors (WHO grades II, III and IV). Seven cases had detectable p53 protein and gene mutations, while twelve cases had neither detectable protein nor gene mutations. Four tumors had frameshift mutations in the p53 gene that were not revealed by immunohistochemistry. One tumor had a genetic polymorphism and no detectable p53 protein. Ten tumors had p53 protein accumulation but no mutations by SSCP; these cases may represent p53 mutations outside of the conserved exons or elevated levels of wild-type p53 protein. Thus, some p53 mutations are missed with PAb 1801 immunohistochemistry alone. p53 immunohistochemistry, however, may reveal p53 accumulation independent of mutations in the conserved portions of the gene. Finally, we suggest that glioblastomas with p53 mutations in the conserved region of the gene may be a subset that are more common in women and in younger patients.

Sensitive detection of p53 gene mutations in esophageal endoscopic biopsy specimens by cell sorting combined with polymerase chain reaction single-strand conformation polymorphism analysis.

For the rapid and sensitive detection of p53 gene mutations in esophageal endoscopic biopsy specimens, we combined cell sorting with the polymerase chain reaction and single‐strand conformation polymorphism (PCR‐SSCP) analysis. Mutations in exons 5–8 of the p53 gene were investigated by FCR‐SSCF analysis using 103 sorted nuclei obtained from each endoscopic biopsy specimen of 16 patients with esophageal cancer. DNAs extracted from their respective surgical specimens were investigated by a conventional method of PCR‐SSCP analysis. Mutations in the biopsy specimens were detected in 6 of the 12 aneuploid tumors but in none of the 4 diploid tumors. After tumor cell enrichment by cell sorting, one mutation in exon 8 became apparent, which could not be detected from the surgical specimen by a conventional method of PCR‐SSCP analysis. This method should improve the sensitivity of detecting p53 gene mutations, and provides additional information concerning the DNA ploidy pattern in the tumors.