Overexpression Of P53 Protein Research Articles

Prognostic Significance of Serine 392 Phosphorylation in Overexpressed p53 Protein in Human Esophageal Squamous Cell Carcinoma

Objective: Posttranscriptional modification involving phosphorylation of wild-type p53 has been considered to play a role in the stabilization of p53. Little is known, however, about the role of phosphorylation of mutant p53 overexpressed in tumors. The aim of this study is to determine the phosphorylation state of p53 in tumors and its contribution to tumor development. Methods: Using immunohistochemical techniques, we examined the phosphorylation of Ser392 and 15 sites in p53 overexpressed in 137 esophageal squamous cell carcinomas (ESCCs). The relationships between the phosphorylation and the expression of cell cycle regulators, Ki-67 labeling index (LI), apoptotic index, clinicopathological factors, and patient prognosis were tested statistically. Results: Of the 137 samples examined, staining for Ser392 phosphospecific p53 antibody was detected in 53 (38.7%) cases, corresponding to 58.9% of 90 p53-positive cases, whereas only 3 (2.2%) were positive for Ser15 phosphospecific p53 antibody. A significant correlation was identified between Ser392 phosphorylation and high levels of Ki-67 LI (p = 0.0271), lymphatic invasion (p = 0.0246), and poorer prognosis for patients with stage II and III advanced tumors (p = 0.0136). Using multivariate analysis, Ser392 phosphorylation was recognized as an independent prognostic factor (p = 0.0136). Conclusions: These findings suggest that p53 protein overexpressed in ESCCs is frequently phosphorylated at Ser392, and that the Ser392 phosphorylation might contribute to tumor progression of ESCCs.

Nasal-type extranodal natural killer/T-cell lymphomas: a clinicopathologic and genotypic study of 42 cases in Singapore

We studied the clinicopathologic features of 42 cases of nasal-type extranodal natural killer (NK)/T-cell lymphoma in Singapore and compared our findings with other series reported in the Asian and Western populations. A panel of immunohistochemical stains, which included CD2, CD3, CD4, CD8, CD56, T-cell intracellular Antigen-1 and granzyme B, and in situ hybridization for Epstein–Barr virus encoded RNA (EBER) were performed. Polymerase chain reaction for T-cell receptor-gamma gene rearrangement using both gel and capillary electrophoresis were evaluated to determine the proportion of tumors which are of true T-cell lineage. We also studied the functional status of the overexpressed p53 protein in these lymphomas by correlating p53 expression with its downstream target protein, p21. In all, 31 out of 42 cases presented in the upper aerodigestive tract. The other sites of involvement included gastrointestinal tract, skin, soft tissue, testis, liver, spleen, bone marrow and brain. The tumors displayed characteristic morphologic features. In situ hybridization for EBER was detected in 41 out of 42 cases (97.6%). The only significant adverse prognostic factor identified was an International Prognostic Index of two or more. A significantly higher proportion of the tumors (27%), compared to previous studies, demonstrated monoclonal T-cell receptor-gamma gene rearrangement. There was, however, no difference in survival or clinicopathologic features between the true NK-cell tumors and their T-cell counterparts. Overexpression of p53 was present in 40% of the cases, but no significant difference in survival rate was detected in patients with p53 overexpression and there was no association between p53 overexpression with large cell morphology, and advanced stage of disease. These findings suggest that molecular aberrations other than those of the p53 pathway may be operative in the pathogenesis of this malignancy.

Biological features of sporadic colorectal carcinoma with high-frequency microsatellite instability: special reference to tumor proliferation and apoptosis.

We evaluated the relationship between biological behavior and microsatellite instability (MSI) status, with or without p53 status, in sporadic colorectal carcinoma. MSI was analyzed with regard to biological features such as cellular proliferation and apoptotic cell death, in addition to clinicopathological features, in 87 patients with sporadic colorectal carcinoma. Fourteen (16.1%) of 87 tumors showed instability at two or more of the five loci examined (high-frequency MSI [MSI-H]). Four demonstrated instability at one locus (low-frequency MSI [MSI-L]), and 69 showed no instability (microsatellite-stable [MSS]). The MSI-H tumors tended to be located in the proximal colon and more often were mucinous carcinoma. The MSI-H tumors also tended to be in patients with multiple colorectal carcinomas and to demonstrate, rarely, an infiltrating growth pattern or venous invasion. The incidence of p53 protein overexpression in the MSI-H tumors was significantly lower than that in the MSI-L/MSS tumors (21% vs 54%). There was no significant difference in the proliferating-cell nuclear antigen (PCNA) labeling index (PI) or apoptotic index (AI) between the MSI-H and MSI-L/MSS tumors. The AI in the MSI-H tumors with p53 overexpression was significantly lower than that in the MSI-H tumors without p53 overexpression, and was also significantly lower than that in the MSI-L/MSS tumors with p53 overexpression. In the MSI-H tumors with p53 overexpression, no expression of BAX protein was found, and there was high expression of bcl-2 protein, resulting in a low BAX/bcl-2 ratio. In sporadic colorectal carcinoma, an MSI-H tumor with p53 protein overexpression may display aggressive biological features.

Large cell neuroendocrine carcinoma of the lung: a comparison with large cell carcinoma with neuroendocrine morphology and small cell carcinoma

Large cell neuroendocrine carcinoma (LCNEC) of the lung is a malignant neuroendocrine tumor clinicopathologically similar to and falling in-between atypical carcinoid tumor and small cell lung carcinoma (SCLC). The diagnosis of LCNEC is based mainly on a characteristic neuroendocrine morphology and biological neuroendocrine differentiation. In order to know the discrepancy between morphological and biological neuroendocrine differentiation, LCNEC was immunohistochemically and molecular biologically compared with large cell carcinoma with neuroendocrine morphology (LCCNM), which lacked only biological neuroendocrine differentiation among the criteria of LCNEC. Immunohistochemically, disruption of the RB pathway, namely a lack of RB expression and simultaneous overexpression of p16 protein, was characteristic of LCNEC, but not LCCNM. In G2/M cell cycle regulation, 14-3-3 sigma expression was markedly reduced in LCNEC. Moreover, the antibody 34 beta E12 recognizing a set of large-sized keratin gave a different staining pattern between LCNEC and LCCNM. The immunohistochemical data suggested that LCNEC has a similar biological marker profile to SCLC and different from LCCNM. However, a loss of heterozygosity (LOH) analysis using microsatellite markers showed a high frequency of LOH at 3p in both LCNEC and LCCNM as well as in SCLC. Morphological neuroendocrine differentiation might not be identical to biological neuroendocrine differentiation in large cell carcinoma of the lung.

Upregulation of p16(INK4A) and Bax in p53 wild/p53-overexpressing crypts in ulcerative colitis-associated tumours.

In ulcerative colitis (UC)-associated tumours, p53 gene mutations and p53 protein overexpression are frequently found in early stages, but the two types of alteration do not always coincide. To clarify this discrepancy, p53 mutations and expression of p53-associated molecules were analysed in UC-associated dysplasias by a combination of microdissection, polymerase chain reaction-direct sequencing and immunohistochemistry at the single crypt level. Mismatch of p53 protein overexpression (+)/mutation (−) or p53 overexpression (−)/gene mutation (+) was found in nine crypts in regenerative mucosa (19 crypts), in 27 in low-grade dysplasia (41), in one in high-grade dysplasia (5) and in 12 in invasive carcinomas (17). Regarding these mismatched crypts of the first type, significant increase in p16INK4A and Bax expression was found. The Ki-67 labelling index was depressed in such p53-diffusely positive lesions with the wild-type p53 gene, compared to their p53-diffusely positive and mutant type counterparts. p16INK4A was upregulated indirectly as part of the negative feedback, and increase in Bax, directly controlled by wild-type p53, indicates upregulation of apoptosis. No significant relation with p53-related gene products was detected with the p53 protein overexpression (−)/p53 mutation (+) mismatch. Therefore, a tumorigenesis pathway independent of p53 dysfunction appears to exist in association with ulcerative colitis.

Coexpression of p53 protein and MDR functional phenotype in leukemias: the predominant association in chronic myeloid leukemia.

One of the best characterized resistance mechanisms of leukemias is multidrug resistance (MDR) mediated by P-glycoprotein (Pgp) and multidrug-resistant related protein (MRP). In addition to Pgp and MRP, p53 mutation or inactivation might play a relevant role in therapeutic failure. Some studies have demonstrated that Pgp and MRP may be activated in association with overexpression of mutant or inactivated p53 protein. The aim of this study was to investigate the association between p53 expression and MDR functional phenotype analyzed by flow cytometry (FCM). Rhodamine-123 assay analyzed by FCM was used to detect the MDR phenotype that was positive in 18 out of 41 (43.9%) cases of chronic myeloid leukemia (CML), 16 out of 28 (57.1%) chronic lymphoid leukemia (CLL) cases, 11 out of 28 (39.3%) acute myeloid leukemia (AML) cases, and four out of 22 (18.2%) acute lymphoid leukemia (ALL) cases. Variable levels of p53 expression were observed in leukemic cells: 12 out of 41 (29.2%) in CML, nine out of 28 (32.1%) in CLL, 15 out of 28 (53.6%) in AML, and eight out of 22 (36.4%) in ALL samples. In our study, no significant association between p53 expression and MDR functional phenotype was observed in ALL, CLL, and AML. On the other hand, a significant association (P = 0.0003) of the coexpression was observed in CML. The p53 overexpression was more frequently seen in the accelerated phase and the blastic phase of this disease. Our results suggest that an MDR functional phenotype could be associated with p53 mutation in the advanced stage of leukemias.

Mutational spectra of p53 in geographically localized esophageal squamous cell carcinoma groups in China.

Esophageal carcinoma is a particularly interesting tumor because of the dramatic difference in its incidence and geographic distribution among populations of similar ethnic origin. Epidemiologic data have suggested that many environmental exposures may be associated with an increased risk of its formation. In this study, 92 samples of esophageal squamous cell carcinoma (ESCC) were collected from patients who resided in 2 geographic areas in China with different incidences of ESCC: Linxian and Zhejiang. Overexpression and mutations of the p53 tumor-suppressor gene were examined by using immunohistochemistry, single-strand conformation polymorphism analysis, and direct sequencing. The rates of point mutation and overexpression of p53 in the ESCC specimens studied were 30.4% (29 of 92 specimens) and 51.1% (47 of 92 specimens), respectively. The overexpression of p53 was associated with tumor metastasis and with 5-year case fatality. Significant differences were found in the rates of overexpression and mutations in patients with clinical T2 tumors between the specimens from Linxian, which is a high-incidence geographic area, and the specimens from Zhejiang, which is a low-incidence area. Furthermore, different mutational spectra were found in the tumor samples from these two geographic areas: In tumor samples from Linxian, the most common substitution mutation was a transversion in exon 5, whereas the most common mutations in tumor samples from Zhejiang were transitions in exon 7. The data suggest that the mutation and overexpression of p53 may play important roles in the development of ESCC. The changes in p53 may reflect environmental exposure to the different combinations of mutagenic factors and genetic instability demonstrated by the populations in Linxian and Zhejiang. The overexpression of p53 protein may have significance as a prognostic factor for patients with esophageal carcinoma.

Application of immunohistochemistry-laser microdissection-PCR technique in detecting p53 gene mutation in paraffin sections of gastric cancer

To evaluate the application of immunohistochemistry-laser microdissection-polymerase chain reaction (IHC-LMD-PCR) technique in detecting p53 gene mutations using paraffin sections of advanced gastric cancers. The expression of p53 protein in 41 paraffin-embedded advanced gastric cancer samples was assessed by immunohistochemistry. The p53-positive or negative carcinoma cells and normal foveolar cells far from the main tumor were then isolated by LMD. The DNA was extracted and p53 gene at exon 5 - 8 amplified by PCR. The products were then analyzed by single-strand conformation polymorphism (SSCP) and automated sequencing. p53 gene was successfully amplified in all the 41 specimens. Overexpression of p53 protein was noted in 11 cases. Mutations of p53 gene were found in 15 cases. Eight of the 11 p53-overexpressed cases had p53 gene mutations. On the other hand, p53 gene mutations were found in only 7 of the 30 p53 protein-negative cases. The presence of p53 gene mutations significantly correlated with p53 protein overexpression (P = 0.004). IHC-LMD-PCR technique can be successfully applied in paraffin sections of gastric cancers for the detection of p53 gene mutations. The results correlate well with overexpression of p53 protein.

P53, Bcl-2, PCNA expression and apoptotic rates during cervical tumorigenesis

5068 Background: In order to estimate the apoptotic rates during the development of cervical cancer, we investigated apoptosis by morphological criteria and by the TUNEL assay, as well as by the expression of proteins related to this process such as p53, Bcl-2 and PCNA in lesions of all phases of tumor progression in the cervical epithelium. Methods: The expression of p53, Bcl-2 and PCNA proteins were immunohistochemically examined using 31 normal cervical epithelium, 10 low grade squamous lesions (CIN I), 28 high grade squamous lesions (CIN II/III) and 36 invasive squamous cell carcinomas (ISCC). The same samples were used for TUNEL assays and HPV detection by polymerase chain reaction (PCR) and sequence. Results:Human papillomavirus (HPV) presence is necessary but not sufficient for cervical cancer development, suggesting that cellular genes participate in tumor progression. PCNA protein expression was significantly increased as the grade of cervical lesion becomes higher from normal epithelium to ISCC. Surprisingly, p53 levels also increased with lesion degree. Apoptosis as determined by TUNEL assay, and PCNA protein expression show a significant positive correlation with increasing histological abnormality. Statistical analysis showed a positive correlation between p53 and PCNA expression in CIN I (r=0.378, P=0.016). A similar correlation was found between p53 and Bcl-2 expression in CIN II/III (r=0.385, P=0.023). A total of 76 of 105 (71.4%) cervical samples were HPV positive, with HPV 16 being the most common high-risk type detected (72.4%). Conclusions:Our results suggest that p53 protein overexpression during cervical tumorigenesis could be playing a pivotal role in cervical tumor progression as a late event. Although a stronger p53 than Bcl-2 expression in CIN II/III was detected, the up-regulation of Bcl-2 in ISCC suggests that protection against apoptosis is important for maintaining cervical neoplasia. PCNA protein expression increase may be a useful marker for diagnosis of various dysplasia grades and cervical cancer. No significant financial relationships to disclose.

P53, Bcl-2, PCNA expression and apoptotic rates during cervical tumorigenesis

5068 Background: In order to estimate the apoptotic rates during the development of cervical cancer, we investigated apoptosis by morphological criteria and by the TUNEL assay, as well as by the expression of proteins related to this process such as p53, Bcl-2 and PCNA in lesions of all phases of tumor progression in the cervical epithelium. Methods: The expression of p53, Bcl-2 and PCNA proteins were immunohistochemically examined using 31 normal cervical epithelium, 10 low grade squamous lesions (CIN I), 28 high grade squamous lesions (CIN II/III) and 36 invasive squamous cell carcinomas (ISCC). The same samples were used for TUNEL assays and HPV detection by polymerase chain reaction (PCR) and sequence. Results:Human papillomavirus (HPV) presence is necessary but not sufficient for cervical cancer development, suggesting that cellular genes participate in tumor progression. PCNA protein expression was significantly increased as the grade of cervical lesion becomes higher from normal epithelium to ISCC. Surprisingly, p53 levels also increased with lesion degree. Apoptosis as determined by TUNEL assay, and PCNA protein expression show a significant positive correlation with increasing histological abnormality. Statistical analysis showed a positive correlation between p53 and PCNA expression in CIN I (r=0.378, P=0.016). A similar correlation was found between p53 and Bcl-2 expression in CIN II/III (r=0.385, P=0.023). A total of 76 of 105 (71.4%) cervical samples were HPV positive, with HPV 16 being the most common high-risk type detected (72.4%). Conclusions:Our results suggest that p53 protein overexpression during cervical tumorigenesis could be playing a pivotal role in cervical tumor progression as a late event. Although a stronger p53 than Bcl-2 expression in CIN II/III was detected, the up-regulation of Bcl-2 in ISCC suggests that protection against apoptosis is important for maintaining cervical neoplasia. PCNA protein expression increase may be a useful marker for diagnosis of various dysplasia grades and cervical cancer. No significant financial relationships to disclose.

Mucin phenotypic expression and background mucosa of esophagogastric junctional adenocarcinoma.

In the West, the incidence of adenocarcinoma of the cardia or esophagogastric junction (EGJ) is increasing. This carcinoma is variously defined, however, and it contains heterogeneous tumors. In Japan, the frequency of this carcinoma is low, and little is known about it. We studied small esophagogastric junctional adenocarcinoma to determine its characteristics in Japanese patients. Fifty-four patients with Siewert type II junctional adenocarcinoma (with tumors <<4 cm in maximum diameter) were studied. The carcinomas were classified into two types: tumors straddling the EGJ (EGJ-type) and tumors occurring entirely below the EGJ (bEGJ-type). Characteristics of the tumors and surrounding mucosae were evaluated. Phenotypic expression of mucin, p53 overexpression, and Helicobacter pylori infection were investigated histologically and immunohistochemically. Twenty-three (43%) patients had EGJ-type and 31 (57%) had bEGJ-type carcinomas. The ratio of advanced cancers was significantly higher in EGJ-type. Some between-group similarities and differences existed in histological type, mucin phenotype, and p53 protein overexpression, but they were not statistically significant. In the mucosa surrounding EGJ-type tumors vs bEGJ-type tumors, oxyntic glands were significantly better preserved, gastric intestinal metaplasia occurred significantly less frequently, and the H. pylori infection rate was lower. Barrett's metaplasia was seen in only 2 patients, with the EGJ-type. EGJ-type and bEGJ-type adenocarcinomas have different background mucosa of the stomach. EGJ-type has less atrophy of the oxyntic glands and less intestinal metaplasia in the stomach. This type appears to be the Western-type junctional adenocarcinoma that is still rare in Japan. Many cases of advanced junctional adenocarcinoma in Japan are of subjunctional origin.

Analysis of mucin, p53 protein and Ki-67 expressions in gastric differentiated-type intramucosal neoplastic lesions obtained from endoscopic mucosal resection samples: a proposal for a new classification of intramucosal neoplastic lesions based on nuclear atypia.

There are differing views between Western and Japanese pathologists on the use of histological criteria to classify gastrointestinal tumors. It is therefore a priority to create a new histological classification of the stomach in order to resolve the confusion. Expression patterns were examined of mucin (MUC2, CD10, MUC5AC, pyloric gland-type mucin), p53 protein, and Ki-67 in tumor cells according to the following new classification system for differentiated-type intramucosal neoplastic lesions of the stomach, based on nuclear atypia: borderline neoplasia (adenoma (including dysplasia), indefinite tumor of adenoma or low-grade cancer, and low-grade cancer) and definite carcinoma (intermediate cancer, and high-grade cancer). The resulting grades were: adenoma, 23; indefinite tumor for adenoma or low-grade cancer, 6; low-grade cancer, 28; intermediate cancer, 48; high-grade cancer, 20. While the frequency of intestinal-type borderline neoplasias was higher than that of definite carcinomas, the mixed-type of definite carcinomas occurred with higher frequency than borderline neoplasias. The p53 protein overexpression and the Ki-67-positive rate increased with an increase in the grade assigned according to the new classification. The correlated expression levels of p53 protein, Ki-67, and various mucins, support the conclusion that this classification of intramucosal neoplastic lesions is useful for obtaining a consensus diagnosis of gastric intramucosal neoplasia between pathologists and gastrointestinal clinicians.

Frequent allelic deletion at the FHIT locus associated with p53 overexpression in squamous cell carcinoma subtype of Taiwanese non-small-cell lung cancers.

The fragile histidine triad (FHIT) gene, encompassing the FRA3B fragile site at chromosome 3p14.2, is a tumour suppressor gene involved in different tumour types including non-small-cell lung cancers (NSCLCs). In the current study, we examined for allelic deletion at the FHIT locus in 58 primary and microdissected NSCLCs, for which a clinicopathologic profile was available. We found a loss of 87.7% in heterozygosity (LOH) frequency at one or more microsatellite markers (D3S1289, D3S2408, D3S1766, D3S1312, D3S1600). Allelic deletion of D3S1766 was related to tumour histology in 10 of 11 squamous cell carcinomas (90.9%) displaying LOH compared with nine of 17 adenocarcinomas (52.9%; P=0.049). Besides, in the subset of adenocarcinomas, a higher rate of LOH at D3S1289 was observed in male (six out of eight, 75%) than in female patients (four out of 17, 23.5%; P=0.028). However, FHIT LOH was not correlated overall with a variety of clinical parameters including sex, smoking status, staging, lymph node metastasis and survival. These results indicated that the high frequency of FHIT gene disruption was important in the development of both squamous cell carcinomas and adenocarcinomas. Furthermore, there was no association between LOH at FHIT and protein expression, suggesting the presence of complex mechanisms of Fhit inactivation. On the other hand, the association between FHIT LOH and p53 protein overexpression assessment reached statistical significance (P=0.026), implying that common alterations affect the two genes in tumour progression.

P53‐Positive Tumor‐Distant Squamous Epithelia of the Head and Neck Reveal Selective Loss of Chromosome 17

The progression of clinically manifest premalignant lesions in the head and neck region to primary or second primary cancer is characterized by numerical and structural chromosomal aberrations. However, many tumors arise from histologically inconspicuous mucosal sites. The objective was to investigate whether chromosomal aberrations can be detected in tumor-distant mucosa and whether they can help predict the risk of second primary malignancy. A retrospective series of 72 clinically healthy, arbitrarily taken mucosal samples from 53 patients with squamous cell carcinoma of the head and neck was studied. A previous analysis of the p53 protein status had revealed both p53-positive and p53-negative samples. The samples were analyzed by fluorescence in situ hybridization (FISH) using centromeric probes specific for the chromosomes 1, 10, 17, and 18. Tumor-distant mucosa generally showed increased numerical chromosomal aberrations, which consisted mainly of monosomies. In the group of patients with p53-positive epithelia, all aberrations including gains of chromosomes (trisomies, tetrasomies) were more frequent. Monosomy for chromosome 17 was most significantly and selectively enhanced in this group (Wilcoxon Scores [rank sum] test, P =.0002). Detectable chromosomal aberrations occur in clinically healthy epithelia in patients at risk for secondary head and neck cancer. Specifically, unbalanced chromosome 17 monosomy in conjunction with p53 protein overexpression may constitute a valuable biomarker for progressive "field cancerization."

Expression of proteins in p53 (p14ARF-mdm2-p53-p21WAF/CIP1) pathway and their significance in exocrine pancreatic carcinoma

To investigate the expression of p14(ARF), p53, mdm2 and p21(WAF/CIP1) proteins and their relationship in exocrine pancreatic carcinoma. Specimens of pancreatic carcinoma, adjacent non-neoplastic pancreatic tissue and pancreatic benign lesions were examined for p14(ARF), p53, mdm2 and p21(WAF/CIP1) protein expression by tissue microarray technique and immunohistochemistry. The expression of p14(ARF), p53, mdm2 and p21(WAF/CIP1) proteins in pancreatic carcinoma were 35.3% (59/167), 57.5% (96/101), 64.1% (107/167) and 39.5% (66/167) respectively. The expression of p53 proteins was increased in pancreatic carcinoma (P < 0.01), while the expression of p14(ARF) and p21(WAF/CIP1) proteins was reduced (P < 0.05), as compared with that in non-neoplastic pancreatic tissue. p21(WAF/CIP1) protein expression in pancreatic carcinoma significantly correlated with the age of patients and perineural invasion (P < 0.05). p53 protein expression correlated significantly with tumor differentiation, lymph node metastasis and perineural invasion (P < 0.05). Mdm2 protein expression correlated significantly with tumor differentiation (P < 0.05), while p14(ARF) protein expression correlated significantly with the age of patients and metastasis (P < 0.05). There was also statistic correlation between the expression of these four genes (P < 0.05). Overexpression of p53 and mdm2 and loss of p14(ARF) and p21(WAF/CIP1) expression may contribute to the pathogenesis of pancreatic carcinoma. These proteins play a critical role in cell cycle arrest and apoptosis after DNA damage through p14(ARF)-mdm2-p53-p21(WAF/CIP1) pathway. Detection of p53 and Mdm2 protein overexpression may be useful in evaluation of the aggressiveness of pancreatic carcinoma.

Molecular biological and immunohistochemical analysis of tp53 in human ameloblastomas

Ameloblastoma is the most frequent epithelial tumor of the jaws. The spread is locally invasive and it tends to recur. Malignant transformation and occurrence of metastases has been described. Immunohistochemical analysis shows an enhanced expression of P53 protein in ameloblastomas. Mutation of the tp53 tumor suppressor gene was verified in several human tumors. In this study histological sections were analyzed for the expression of P53 protein and the tp53 gene was examined for mutations. Tumor DNA from 29 patients with an ameloblastoma was examined for mutations in exons 5 to 8 of the tp53 tumor suppressor gene using PCR, SSCP,- and sequential analysis. Histological sections of the tumors were analyzed immunohistochemically for an overexpression of P53 protein. Two tp53 mutations (6.9%) in ameloblastomas were verified for the first time. In all 58.6% of the tumors showed an immunoreactivity for P53 protein. There was a statistically significant positive correlation (Fisher's exact test p<0.0148) between an increased number of P53-positive tumor cells and the appearance of recurrence. In the face of the uncertain postoperative behavior of ameloblastomas, the immunohistochemical verification of more than 10% P53-positive tumor cells may give a prognostic indication for a tendency to recurrence.

P53 protein overexpression and p53 mutation analysis in patients with intestinal metaplasia of the cardia and Barrett's esophagus

p53 mutation is a common genetic change in human cancers, but the clinical significance is controversial. We studied 68 patients and estimate the prevalence of intestinal metaplasia of cardia (IMC), Barrett's esophagus (BE), and p53 protein overexpression, and described molecular alterations of p53 gene exons 5 to 8. Immunohistochemical analysis showed positive p53 in 56.1–39.1% (IMC) and 60.9% (BE). Molecular analysis showed 36.6% altered cases in exon 5 and 9.8% in exon 7. In conclusion, p53 protein overexpression is common in IMC and BE. The molecular alterations observed may be due to LOH, genomic instability or other unknown alteration.

Standardized approach for microsatellite instability detection in gastric carcinomas

Microsatellite instability (MSI) defines a specific type of genetic instability. Although consensus diagnostic criteria for MSI definition in colorectal cancer have been established, their utility in other tumor types remain to be proven. Previously we developed a mathematical model for MSI definition in colorectal cancer. The aim of this study was to establish diagnostic criteria for MSI evaluation in human gastric cancer. We designed an algorithm for the efficient characterization of MSI and used it to analyze data on 7 microsatellite markers in 35 gastric carcinomas. Theoretical models considering 1, 2, or 3 populations were tested against the data collected. Also, hypermethylation of hMLH1 gene promoter and hMLH1 protein expression were studied. The observed frequencies of MSI in our series of samples best fit a 2-population model: stable and unstable, defined by instability in 2 or more of a minimum of 7 markers analyzed. MSI was observed in 29% of the tumors. Misclassification rate was <4% when any 7 loci were analyzed. MSI(+) tumors inversely associated with p53 protein overexpression. A good correlation between hMLH1 status (either protein or promoter hypermethylation) and MSI classification was observed. We have developed a simple, sensitive, and specific approach to assess the presence of MSI in gastric cancer that may have clinical applications.

Co-mutation of p53,K-rasgenes and accumulation of p53 protein and its correlation to clinicopathological features in rectal cancer

To determine the accuracy of p53 gene mutations predicted by overexpression of p53 protein immunohistochemically, and to investigate the co-mutation of p53 and K-ras genes in rectal cancer and its effect on promoting malignant biologic behaviors of tumors. Ninety-seven specimens of rectal cancer were surgically resected in our hospital from August 1996 to October 1997. The hot mutation areas of p53 gene (in exons 5-8) and K-ras gene (in codon 5/12 and 13) were detected with polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP), and overexpression of p53 protein was detected with immunohistochemistry (IHC) in the 97 specimens of rectal cancer. Correlation between gene mutations and tumor clinicopathologic factors was studied, and survival analysis was penfomed as well. There were 36 cases of p53 gene mutations in 61 p53 protein positive cases, and 21 cases of p53 gene non-mutation in 36 p53 protein negative cases respectively. The coincidence rate of p53 gene mutation by IHC method with PCR-SSCP method was 58.8% (57/97). The mutation rate of p53 gene was 52.6% (51/97), while K-ras gene mutation was observed in codons 12 and 13 in 61 cases with a mutation rate of 62.9% (61/97). Single gene mutation of p53 or K-ras was found in 32 cases. Both p53 and K-ras gene mutation were found in 48 cases. Statistical analysis showed that p53 and K-ras gene mutations were not related to the clinicopathologic factors, including tumor size, gross tumor type, histological classification, differentiation, invasion to intestinal veins, lymphatics and nerves, invasive depth to wall, lymph node metastasis, and Dukes' stages (P>0.05). The survival in patients with no gene mutation, single gene mutation and both gene mutations were similar (P>0.05). IHC has a certain false positive and false negative rate in detecting p53 gene mutations. Malignant biological behaviours of rectal cancer are not enhanced by p53 and K-ras gene mutations. Co-mutation of p53 and K-ras gene has neither synergic carcinogenesis-promoting effect, nor prognostic effect on rectal cancer.

P53 protein expression and cell proliferation in non-neoplastic and neoplastic proliferative skin diseases.

The p53 protein is essential for the regulation of cell proliferation and its aberrant accumulation is usually seen in malignant tumors but also occurs in squamous epithelium of inflammatory skin diseases characterized by hyperproliferation. The aim of this study is to elucidate the role of the p53 tumor suppressor protein in the pathogenesis of different hyperproliferative, non-malignant and malignant skin diseases, and to determine the association between p53 overexpression and cell proliferation. We also investigated the influence of aging on p53 and Ki-67 protein expression. One hundred and fifty skin specimens divided into 30 samples each of normal skin (NS), psoriatic skin (PS), keratoacanthomas (KA), basal cell carcinomas (BCC), and squamous cell carcinomas (SCC) were examined immunohistochemically to assess p53 and Ki-67 protein expression. p53 immunostaining of NS, PS, KA, BCC and SCC was detected in 39.0%, 46.7%, 66.7%, 80% and 86.7% of cases, respectively. Median values and ranges of p53 protein expression were as follows: 0.0% (range, 0.0-1.8%) in NS, 0.0% (range, 0.0-6.5%) in PS, 9.2% (range, 0.0-24.0%) in KA, 19.3% (range, 0.0-48.1%) in BCC and 30.1% (range, 0.0-68.1%) in SCC. p53- and Ki-67-positive cells were present in basal (NS) and suprabasal layers (PS), and not only in cancer nests of KA, BCC and SCC but also in dysplastic and even morphologically normal epidermis adjoining cancers. The positivity of p53 and Ki-67 proteins differed significantly among the groups, with no differences in p53 expression between NS and PS and in Ki-67 expression between PS and KA. Within all groups there was a significant correlation between p53 and Ki-67 expression. Lesion location and patient age, with the exception of location in PS and age in BCC, were significantly related to p53 and Ki-67 expression in all groups. Our findings suggest that p53 overexpression occurs mainly in neoplastic skin lesions, although it may also occur in squamous epithelium of inflammatory skin diseases such as PS, as well as in normal skin epithelium. It is associated with cell proliferation in normal as well as altered epithelium. p53 protein overexpression is an age-related process and significantly associated with sun exposure, especially in NS and PS but also in KA and SCC. Our findings suggest that Ki-67 rate and p53 protein expression reflect the degree of malignancy in the examined cutaneous neoplasms.