Gallbladder carcinoma: the role of p53 protein overexpression and Ki-67 antigen expression as prognostic markers

BackgroundThe overexpression of p53 protein and the expression of Ki-67 antigen may affect the survival of patients with gallbladder carcinoma. This association has been tested in a series of 41 patients with relatively early carcinoma of the gallbladder.MethodsForty-one surgical specimens from patients with a postoperative histological diagnosis of gallbladder carcinoma were studied. All patients were operated by simple cholecystectomy only because the tumours were not advanced and/or their general condition was poor. Patients submitted to radical operations were excluded. p53 expression was calculated from nuclear staining according to the intensity and extent of positive cells, as graded on a scale from 1 to 3; a combined score of >3 was considered as overexpression. Ki-67 expression was calculated by the MIB-I index: the percentage of positively stained tumour cell nuclei out of the total tumour cells counted (n = 1000); >20% of stained cells was considered positive.ResultsTwenty-nine gallbladder carcinomas (71%) overexpressed p53 protein in the cell nuclei. No significant differences were found in relation to cell differentiation on the level of tumour infiltration through the gallbladder wall. Five-year survival of patients with gallbladder carcinoma overexpressing p53 was 17.2%, while survival of patients without p53 overexpression was 30% (not significant). Twenty-four cases (58.5%) were considered positive for the MIB-I index. There were no differences between the grade of cell differentiation and wall infiltration. Five-year survival of the patients with a MIB-I positive index was 9.2% as opposed to 27.7% for those with a negative index (not significant).Conclusionsp53 protein nuclear overexpression and Ki-67 protein expression in gallbladder carcinoma were not related to histological differentiation, level of gallbladder wall invasion or patient survival.     

p53 and Beta-Catenin Expression in Gallbladder Tissues and Correlation with Tumor Progression in Gallbladder Cancer

Background/Aim:

The inactivation of the tumor suppressor gene and activation of the proto-oncogene are key steps in the development of human cancer. p53 and beta-catenin are examples of such genes, respectively. In the present study, our aim was to determine the role of these genes in the carcinogenesis of the gallbladder by immunohistochemistry.

Patients and Methods:

Sections from paraffin-embedded blocks of surgically resected specimens of gallbladder cancer (GBC) (80 cases), chronic cholecystitis (60 cases), and control gallbladders (10 cases) were stained with the monoclonal antibody p53, and polyclonal antibody beta-catenin. Results were scored semiquantitatively and statistical analysis performed. p53 expression was scored as percentage of the nuclei stained. Beta-catenin expression was scored as type of expression–membranous, cytoplasmic, and nuclear staining. Beta-catenin expression was correlated with tumor invasiveness, differentiation, and stage.

Results:

Over-expression of p53 was seen in 56.25% of GBC cases and was not seen in chronic cholecystitis or in control gallbladders. p53 expression in gallbladder cancer was significantly higher than in inflammatory or control gallbladders (P < 0.0001). p53 expression increased with increasing tumor grade (P = 0.039). Beta-catenin nuclear expression was seen in 75% cases of gallbladder cancer and in no case of chronic cholecystitis and control gallbladder. Beta-catenin nuclear expression increased with tumor depth invasiveness, and grade (P = 0.028 and P = 0.0152, respectively).

Conclusion:

p53 and beta-catenin nuclear expression is significantly higher in GBC. p53 expression correlates with increasing tumor grade while beta-catenin nuclear expression correlates with tumor grade and depth of invasion, thus suggesting a role for these genes in tumor progression of GBC.     

p53基因在食管癌组织中的高表达

本文利用免疫组化LSAB法检测了p53基因在35例食管癌组织中的表达情况。35例食管癌标本中，60％（21／35）的标本癌组织都存在有p53基因的高表达，其中18例标本同时存在有癌旁粘膜上皮p53基因在10例癌旁粘膜上皮中也存在高表达。p53的高表达主要位于核内，部分也有胞浆表达。p53基因的高表达与癌组织的分化程度有关，分化越差，阳性率越高（P＜0．0025）。p53基因的表达存在异质性，并与肿瘤细胞的浸润转移及细胞周期的不同有关。我们的结果表明，p53基因的高表达在食管癌的发生中是一个常见的基因改变，它在食管癌的发生发展中起着重要的作用。     

p53 Gene mutations and overexpression of p53 product in cancerous and noncancerous biliary epithelium in patients with pancreaticobiliary maljunction

To investigate the molecular mechanisms of the high incidence of carcinogenesis in the biliary epithelium of patients with pancreaticobiliary maljunction, we examined p53 gene mutations, loss of heterozygosity of p53, and overexpression of p53 gene product in the cancerous and noncancerous biliary epithelium of 27 patients with pancreaticobiliary maljunction. Mutations of the p53 gene were examined by polymerase chain reaction-single strand conformation polymorphism and a direct sequencing method. Loss of heterozygosity of the p53 gene was determined using a double-targeted fluorescence in situ hybridization method. Expression of p53 gene product was examined using immunohistochemical staining. Mutations of the p53 gene were found in 4 of 5 biliary carcinomas (80%) and in 10 of 26 noncancerous biliary lesions (38.5%). Point mutations of the p53 gene were detected at codons 207, 212, and 217 on exons 5 through 8. The incidence of p53 gene mutations on exons 5, 6, 7, and 8 was 12.9%, 36.4%, 0.0%, and 13.8%, respectively. Loss of heterozygosity of p53 was shown in 72% of the cells obtained from the cancerous lesion, and in an average of 14% obtained from the noncancerous lesions. Overexpression of p53 protein was found in 57.1% of carcinoma, and in 31.3% of the noncancerous lesions. These results suggest that p53 gene mutations are involved in the carcinogenesis of biliary epithelium in patients with pancreaticobiliary maljunction.     

肝细胞癌中p53基因突变的检测及新DNA片段的发现

目的：检测原代肝癌组织以及肝癌细胞系中ｐ５３的突变位点和频率,了解ｐ５３突变与肝癌发病和治疗的关系。方法：设计了３对引物,采用ＰＣＲ技术扩增ｐ５３的５－８外显子,然后进行ＤＮＡ测序鉴定,共测定了两个肝癌细胞系（ＢＥＬ－７４０２和２．２．１５）以及两例原代肝癌组织,其中５和６外显子用一对引物串联扩增出来,上游引物序列为５′－ＧＧＡＡＴＴＣＣＴＣＴＴＣＣＴＧＣＡＧ－３′,下游为：５′－ＧＧＡＡＴＴＣＡ     

肝细胞癌患者中p53基因变化的相关因素分析

ｐ５３基因的变化已被认为是肿瘤病因学和分子发病机制的重要线索。为了阐明ＨＣＣ的主要致病因子及其在ＨＣＣ发病机制中的相对意义，本研究对ｐ５３基因变化较常见的中国南部地区的７０例ＨＣＣ患者采用Ｘ＾２检验等，分析一般资料、嗜肝病毒感染和病理资料与ｐ５３基因的ＬＯＨ则与嗜肝病毒感染和癌周组织中的肝硬化程度相关。以上结果提示，ＨＣＣ的主要致病因素是嗜肝病毒感染和高度流行区的某些其它因素，其中嗜肝病毒感染在Ｈ     

Clinical implication of anti-p53 antibodies and p53-protein in pancreatic disease

Summary p53 gene mutations play an important role in the pathogenesis of pancreatic carcinomas. Anti-p53 antibodies and p53 protein have been detected in the sera of patients with pancreatic carcinomas. However, very little is known about the clinical significance of these p53 antibodies. We investigated the relationship between anti-p53 antibodies and the presence of p53 protein in cancer cells and the serum, as well as other clinical factors. Anti-p53 antibodies were detected in 19 (23%) of 82 pancreatic-duct-cell carcinomas, and in one (5%) of 21 cases of chronic pancreatitis. However, no antibodies were detected in mucin-producing tumors or in islet-cell tumors of the pancreas. The anti-p53 antibodies were detected in both early and advanced stages. In those patients undergoing surgical resection for pancreatic duct-cell carcinomas, the prognosis of patients who were negative for the anti-p53 antibodies was better than patients who were positive. Of the 11 cases that were positive for anti-53 antibodies, 8 (73%) were also positive for the immunohistochemical expression of p53 protein in cancer cells. However, there was no significant correlation between the presence of anti-p53 antibodies and the serum p53 protein levels. These results suggest that the benefits of measuring the anti-p53 antibody titier as a screening test to detect pancreatic carcinoma are limited, but the presence of anti-p53 antibodies predicts a poor prognosis for post-operative pancreatic carcinoma patients.     

抑癌基因PTEN及p53在肝细胞肝癌中表达的免疫组化研究

为探讨肝细胞肝癌组织中抑癌基因PTEN及p53蛋白的表达情况及临床病理意义。应用免疫组织化学技术检测了41例肝细胞肝癌及其相应的癌旁组织中PTEN和p53蛋白的表达情况。41例癌旁组织PTEN全部阳性表达,肝细胞肝癌组织中PTEN阳性表达率39％,阳性信号显示于胞浆中。p53阳性表达率51％,PTEN蛋白在肝细胞肝癌组织中的阳性表达与组织分化程度明显相关,高分化癌的阳性率为73％,低分化癌阳性率27％。肝细胞肝癌细胞中存在较高比例的PTEN蛋白阴性表达,说明在肝细胞肝癌的发生发展中PTEN基因失活起着重要作用,它的阳性表达可能有一定的预后意义。     

Expression of p53 in adenocarcinoma of the gallbladder and bile ducts

Abstract: Point mutation of the p53 tumor suppressor gene appears to be an important event in tumor development and progression, and overexpression of the p53 gene product has been widely studied in a variety of neoplasms. Some point mutations of the p53 gene lead to an increase in half-life in the gene product, which accumulates in the nucleus and can be detected by immunohistochemical means. We studied overexpression of p53 protein in specimens from 12 patients with adenocarcinoma of the gallbladder, two gallbladders with epithelial dysplasia without carcinoma, eight carcinomas of the common bile duct, 13 hilar cholangiocarcinomas, and six peripheral cholangiocarcinomas. The monoclonal antibody Ab-2 (Oncogene Science) was used in conjunction with citrate microwave antigen retrieval. Nuclear staining was scored as positive (graded 1 to 3, depending on number of positive nuclei) or negative. Overexpression of p53 protein was present in 7/12 (58%) gallbladder carcinomas, and was seen more often in moderately or poorly differentiated tumors. Intramucosal carcinoma adjacent to invasive carcinoma was positive in three cases, although fewer cells stained than in the carcinoma. Two cases of low-grade dysplasia not associated with carcinoma were negative. Expression of p53 was not an independent prognostic factor when survival was related to grade and stage of tumor. Three of eight (38%) common bile duct carcinomas and 5/13 (38%) hilar cholangiocarcinomas were positive for p53. Slightly fewer (2/6, 33%) peripheral cholangiocarcinomas were positive. No difference in survival relative to p53 expression was demonstrated.     

食管鳞癌中p53与bcl-2的表达及其临床意义

目的 :探讨食管鳞癌中p53和bcl 2的表达情况、相互关系及其临床意义。方法 :应用免疫组化法 (S P)对 1 0 0例食管鳞癌病人的内镜活检标本检测p53和bcl 2的表达。结果 :在癌旁正常鳞状上皮、癌旁不典型增生和癌组织中p53的阳性表达率分别为8.9%、43.6 %和 58.6 % (χ2 =30 .2 ,P <0 .0 1 ) ;bcl 2阳性表达率分别为 6.7%、3 .6 %和 43.0 % (χ2 =57.4,P <0 .0 1 )。p53阳性表达率在不典型增生中已接近癌组织 ,bcl 2在癌旁正常鳞状上皮和不典型增生组织中表达较低 ,在癌组织中表达较高。在分化较好的食管鳞癌中 ,p53的阳性表达率和表达强度等级明显高于bcl 2 ,在低分化癌中 ,p53和bcl 2的阳性表达率和表达强度等级无差异。结论 :p53表达可能是食管鳞癌的早期事件 ,bcl 2表达较晚 ,是癌变的特征。p53和bcl 2表达的相互关系与食管鳞癌的分化程度有关     

Clinical significance of p53 antigen and anti-p53 antibodies in the sera of hepatocellular carcinoma patients

Background. To analyze the clinical significance of serum p53 protein and anti-p53 antibodies as serological markers for hepatocellular carcinoma (HCC). Methods. We studied clinical data, i.e., age, sex, etiology, serum alpha-fetoprotein (AFP) level, TMN staging, and Okuda staging in 141 patients with HCC. The sera of these patients were analyzed for serum p53 protein and serum anti-p53 antibodies by enzyme-linked immunosorbent assay (ELISA). Results. Serum p53 antigen and serum anti-p53 antibodies were detected in the sera of 32 of the 141 (22.7%) patients and 26 of the 141 patients (18.4%), respectively. Of note, the HCC patients who were positive for p53 antigen (32/141) had no circulating anti-p53 antibodies. When both these groups of patients were combined as a serum p53 status-positive group, the total number in this group was 58 (41.1%). Positive status of p53 was not associated with age (P = 0.206), serum alpha-fetoprotein level (P = 0.851), Okuda staging (P = 0.243), or survival (P = 0.078), but was correlated significantly with TMN staging (P = 0.049). Interestingly, a shorter survival time (mean, 3.9 months) was noted in the serum p53 status-positive group, in comparison with the longer survival time (mean, 6.5 months) in the serum p53 status-negative group. Conclusions. Combination of the detection of serum p53 antigen and antibodies by ELISA may represent a suitable noninvasive investigation in assessing the clinical implications and prognoses of patients with HCC. Received: December 8, 2000 / Accepted: June 22, 2001     

Immunohistochemical study of microvessel density, CD44 (standard form), p53 protein and c-erbB2 in gallbladder carcinoma

BACKGROUND: The purpose of the present study was to investigate microvessel density (MVD), and the expression of CD44 adhesion molecule, p53 protein and c-erbB2 in gallbladder carcinoma, and their relation to histological grade and tumor invasiveness. METHODS: Immunohistochemical staining with antibodies against factor VIIIRAg, CD44 standard form (CD44s), p53 protein and c-erbB2 was performed on paraffin sections from 33 cases of gallbladder carcinoma. RESULTS: Significant increase of MVD with increasing depth of invasion (P < 0.02) was observed. No association of MVD with histological differentiation, CD44s, p53 and c-erbB2 protein expression was found. The expression of p53 protein was significantly higher in deeply invasive tumors (P = 0.028) and in moderately and poorly differentiated carcinomas (P < 0.05). The CD44s expression was higher in well-differentiated carcinomas (P < 0.05). c-erbB2 expression was found in 30.4% of tumors studied, but did not relate to any other parameters. CONCLUSION: Microvessel density and p53 protein expression increase progressively with increasing tumor invasiveness in gallbladder carcinomas. Microvessel density, p53 protein, CD44s, and perhaps c-erbB2 expression may be implicated in gallbladder carcinoma evolution.     

Expression of p53 and p21 Are Independent Prognostic Factors in Patients with Serosal Invasion by Gastric Carcinoma

To evaluate whether the expression of p53 andthat of p21 are independent prognostic factors inpatients with advanced gastric cancer, we investigatedclinicopathological factors and the expression of p53 and p21 in 158 patients with gastric cancerthat had invaded the serosa and who had undergonecurative gastrectomy. In multivariate survival analysisof 156 surviving patients, we evaluated the size of the tumor, lymph node metastasis, venousinvasion, lymph node dissection, expression of p53, andexpression of p21 as independent prognostic factors.Moreover, we divided patients into four groups according to the expression of p53 and p21 in theirtumors [group A, p53^-/p21^-, N = 40(one died within 30 days of surgery); group B, p53^-/p21^-, N = 23; group C,p53⁺/p21⁺, N = 58; and group D,p53⁺/p21⁺, N = 37 (one died within 30 days of surgery)]. The 5- and 10-yearsurvival rates of 39 patients in group A were 71.7% and64.3%, those of 23 patients in group B were 81.4% and81.4%, those of 58 patients in group C were 35.6% and 30.2%, and those of 36 patients ingroup D were 67.9% and 60.7%. The prognosis of patientsin group C was poorer than that of patients in the otherthree groups. This result indicates that the evaluation of the expression of both p53 andp21 expression might provide prognostic information thatis more accurate than that provided by evaluation of theexpression of p53 alone.     

p53 gene mutations and expression of p53 and mdm2 proteins in invasive breast carcinoma

The aim of the study was to analyzep53 gene mutations and the expression of p53 and mdm2 proteins in 31 randomly selected invasive breast carcinomas. The results were then correlated with tumor grade, stage, estrogen receptor status, nodal status, and DNA ploidy. The expression of the proteins p53 and mdm2 was determined immunohistochemically using formalin-fixed, paraffin-embedded material. Screening for p53 mutation involved analysis of the highly conserved regions of thep53 gene (exons 5–9) by the polymerase chain reaction/single-strand conformation polymorphism (PCR-SSCP) technique. PCR products with band shifts were directly sequenced. Immunohistochemical staining of p53 was positive in 9 cases (29.0%), only 2 of which showed ap53 gene mutation. These were identified as a CG transversion at the second position of codon 278 in exon 8 and an AG transition at the second position of codon 205 in exon 6. A third case with a mutation was observed (CT transition, position 1 of codon 250 in exon 7) that did not show p53 immunohistochemically. Of the 9 p53-positive tumors, 2 were moderately differentiated (grade II). The remaining tumors were poorly differentiated (7/9). By contrast, p53-negative carcinomas were well differentiated (grade I) in most cases (P=0.02). DNA cytometry in 8 of the 9 p53-positive carcinomas revealed an aneuploid stem line. The majority of the p53-negative tumors were diploid (P=0.01). Mdm2 oncoprotein was detected in 10 tumors (32.2%), 4 of which were p53-positive, including the 3 with mutations. The grading of the mdm2-positive tumors was moderate or poor, G1 carcinomas were always noted to be mdm2-negative (P=0.04). Overexpression of p53 protein is a complex mechanism and does not merely indicate the detection of mutations in thep53 gene. This study has shown that p53 expression correlates with tumor grade and DNA ploidy. Mdm2 expression was also associated with the tumor grade. Immunohistological demonstration of the p53 protein alone is insufficient as a basis for comment on the functional state of thep53 gene and gene product. The interrelation between recognition of the p53 protein and gene mutation needs more careful assessment to define their roles in the control of neoplasia.     

Prognostic influence of p53 nuclear overexpression in colorectal carcinoma

PURPOSE: The aim of this study was to test the prognostic influence of p53 nuclear overexpression in colorectal carcinoma. METHODS: We performed an analysis of the prognostic influence of the nuclear overexpression of p53 with immunohistochemistry in 126 cases of colorectal carcinoma operated on in our hospital between 1987 and 1992, with a minimum follow-up time of 60 months (5 years). RESULTS: Our results show a statistically significant prognostic influence of p53 overexpression on disease-free survival time, but not on the overall survival time, in univariate analysis, but this influence is lost in multivariate analysis. CONCLUSIONS: Our results confirm recent reports by other authors that failed to show the independent prognostic value of p53 in colorectal carcinoma.     

Mutations of p53 Tumor Suppressor Gene, Apoptosis, and Proliferation in Intrahepatic Cholangiocellular Carcinoma of the Liver

This study was performed to examine the correlation between mutations of the p53 tumor suppressor gene, the occurrence of apoptosis, and proliferation in cholangiocellular carcinoma of the liver. The results obtained were compared with pathohistological stage (according to UICC) and grade and with disease related survival rate. In 41 curatively (R0–) resected intrahepatic cholangiocellular carcinomas, the status of the p53 gene was determined by direct sequencing of exons 4–9 and immunohistochemically. Apoptosis was assessed using the in situ end labeling (ISEL) technique in combination with morphological criteria. Proliferation was analyzed by immunohistochemistry of MIB-1 (Ki-67), Proliferating cell nuclear antigen (PCNA), and silver-stained nucleolar organizer regions (AgNOR). The results obtained were compared with pathohistological stage (according to UICC), grade, several other histopathological factors, and survival rate. Mutations of p53 were detected in 15/41 carcinomas examined (37%). The most common change was a GC and CT transition, changing the hot spot amino acid determined by exons 4–8. Of these 15 tumors, 14 were also p53-positive by immunohistochemistry. In each carcinoma examined, we could demonstrate MIB-1, PCNA, and AgNOR dots and also apoptotic cells in variable proportions. The proliferation markers showed a significant correlation among themselves. In univariate survival analysis, the extent of the primary tumor, lymph node status, grade, and p53 were significant factors influencing patient survival. Performing multivariate Cox regression survival analysis, however, only the extent of primary tumor and lymph node status had an independent prognostic impact. Apoptosis was not related to patient prognosis or to other parameters examined. In conclusion, these results indicated that p53 could serve as an additional prognostic parameter that could provide auxiliary information for patient outcome. However, tumor stage and lymph node involvement were the strongest prognostic factors. We failed to establish apoptosis or other pathological parameters as factors predicting the prognosis of patients with cholangiocellular carcinoma.     

Prognostic value of p53 gene mutations and the product expression in de novo acute myeloid leukemia

In acute myeloid leukemia (AML), p53 mutations are reportedly infrequent but associated with a poor prognosis. The majority of mutations are missense mutations, which generally lead to accumulation of nuclear p53 protein. However, the prognostic significance of the accumulation remains unknown in AML. In this study, we compared the prognostic value of p53 mutations versus accumulation of the product. p53 mutations were found in 9 (4.5%) of 200 patients with de novo AML. The p53 mutation detectable (mutation+) group had a worse prognosis (p = 0.0009) than the mutation not detectable (mutation-) group. Multivariate analysis showed that the p53 mutation was an independent factor (p = 0.005) for short overall survival as well as 60 yr or older (p = 0.001) and unfavorable karyotypes (p = 0.001). In 79 of the 200 patients, the expression of p53 was studied by immunocytochemistry (ICC) using anti-p53 monoclonal antibody (DO-7). All samples carrying missense mutations (N = 6) were positive for ICC in over 15% of nuclei of each sample, chosen as the optimized cutoff value of p53 accumulation. Accumulation was thus found in 14 of the 79 patients. However, there was no prognostic difference according to the accumulation, because the mutation-/accumulation+ group (N = 8) tended to have a good prognosis. These findings indicate that molecular detection of p53 mutations yields better prognostic information than ICC. In a subset of AML, p53 protein might be accumulated without mutation presumably due to upstream signals of p53.