Proliferation,DNA ploidy,p53 overexpression and nuclear DNA fragmentation in six equine melanocytic tumours

Melanocytic tumours are a well‐known clinical and pathological entity in horses, but further phenotypic characterization of these tumours is lacking. Six melanocytic tumours from five horses (two metastatic and four benign) were examined by Ki67, PCNA and p53 immunostaining, DNA nick end labelling (Tunel) and Feulgen staining. The stainings were evaluated using quantitative image analysis. The resulting parameters of growth fraction (Ki67), S‐phase index (PCNA), p53 index, apoptotic index, DNA index, nuclear diameter, ploidy balance, proliferation index (Feulgen) and hyperploidy were analysed. The metastatic melanomas showed overexpression of p53 in a large portion of the cells. Apoptosis was also found in the metastatic melanomas. No differences were found in growth fraction, S‐phase index (PCNA) nor in DNA configuration between the metastatic and the benign tumours. No immunohistochemical evidence of mutant p53 could be found in the tumours. In conclusion, melanocytic tumours in horses seem to have different phenotypic characteristics in comparison with melanocytic tumours in dogs, cats and humans, especially with respect to proliferative activity of the benign tumours. Therefore, markers put forward in these other species for predicting the clinical behaviour of the melanomas seem to be of no value in the horse. Moreover, quantitative DNA changes or p53 mutations do not seem to be involved in tumourogenesis in these cases.     

Significance of protein p53 overexpression in the clinical course of high-risk superficial bladder cancer

OBJECTIVES: This is a retrospective study in which the long-term biological behavior of 67 "high-risk" superficial bladder tumors and the prognostic relevance (prediction of disease recurrence and progression) of the determination of the p53 phenotype in these cases were studied. MATERIAL AND METHODS: 67 tumors with a "high-risk" of progression were selected from the 1,103 transurethral resections for bladder cancer carried out in 640 patients in this center between 1987 and 1992. These included 39 T1G3, 14 Tis (isolated or associated with Ta-T1, non-G3 tumors), and 14 Ta-T1, non-G3 tumors with submucosal lymphatic affection (L+). The median follow-up of these cases was 69.7 months. An immunohistochemical technique with monoclonal antibodies (DO-7) was used to detect the p53 phenotype in paraffin-fixed material. RESULTS: Tumor recurrence occurred in 31 patients (46.3%) and local or distant progression in 14 (20.9%). Radical cystectomy was carried out in 16 (23.9%) cases. p53 overexpression of > or =20% ("p53+") was detected in 40 tumors (59.7%). The rate of recurrence and progression, the disease and progression-free intervals, cancer-specific survival, disease-free survival and progression-free survival were similar in the 3 tumor groups (in all cases, p > 0.05). There were no significant differences in the overexpression of protein p53, using the standard cutoff point of 20% stained nuclei, on comparing the same variables in the whole group of 67 patients (in all cases, p > 0.05). CONCLUSION: The detection of protein p53 was not found to be of use in the retrospective prediction of disease progression or survival in "high-risk" superficial bladder cancer.     

The correlation of p53 protein overexpression and p53 antibodies in serum of patients with transitional cell carcinoma of the urinary bladder

BACKGROUND: Mutations of p53 gene were demonstrated in many solid tumors with varying frequency. We analyzed the relationship between p53 protein expression in bladder cancer tissue, p53 autoantibodies in serum and the clinical course of 32 patients with and 10 patients without transitional cell carcinoma of the urinary bladder. MATERIALS AND METHODS: In the 32 patients studied, bladder cancer was diagnosed as pTaG1-2 in 8 cases, pT1G2 in 6, pT1G3 in 7, pT2G2-3 in 7, pT3G2-3 in 3 and pT4 in 1 patient. Anti-p53 antibodies were detected by an enzyme-linked immunosorbent assay. Immunohistochemical staining was performed using a standardized alkaline phosphatase monoclonal anti-alkaline phosphatase method. To prove the statistical significance of tumor grading and staging, the Kruskal-Wallis test was applied (p < 0.01). The mean follow-up was 26 months. RESULTS: We found 12.5% p53 autoantibody-positive sera without a statistically significant correlation with tumor grade (p = 0.0569) and category (p = 0.612). Three of 4 patients who had p53 autoantibody-positive sera died within 9 months. All of these sera-positive patients had p53 protein-positive tumor tissue. Control sera were all negative for p53 autoantibodies. CONCLUSION: This study shows a strong relationship between p53 protein overexpression and the occurrence of p53 autoantibody in bladder cancer. The expression of p53 autoantibodies seems to be an event in cases of bladder cancer with an unfavorable tumor-specific outcome. Because of the small number of cases and the short follow-up time, further quantitative studies will hopefully demonstrate whether this might be of prognostic importance.     

人膀胱癌耐药细胞株p53基因突变与染色体畸变的关系

目的：研究膀胱癌耐药细胞株p53蛋白表达，探讨其与细胞染色体变化特点及耐药性的关系。方法：使用药物深度递增的方法，分别采用阿霉素（ADM）或足叶乙甙（VP－16）建立了人膀胱癌耐药细胞株BIU－87／A和BIU－87／V，采用免疫组织化学的方法对比分析其和亲代细胞株p53蛋白表达情况，并用G显带法研究各株细胞染色体特点。结果：BIU－87／A对ADM、VP－16的耐药倍数分别为57．42、59．40；BIU－87／V对ADM、VP－16的耐药倍数分别为6．45、5．44。BIU－87的p53蛋白表达阴性，而BIU－87／A、BIU－87／V的p53蛋白均阳性，且BIU－87／A的表达量多于BIU－87／V。BIU－87／A和BIU－87／V细胞染色体数目为超三倍体，染色体众数分别为78、73，数目在50－113之间，大多数染色体均有数目异常，变动于1－4条之间，染色体缺失、突变、到位、易位和环状染色体等结构畸形明显多于亲本细胞。结论：膀胱肿瘤耐药细胞株p53的异常表达可导致细胞染色体畸变增加，并与细胞多药耐药性与有一定的关系。     

Numerical aberrations of chromosomes 1 and 7 by fluorescent in situ hybridization and DNA ploidy analysis in breast cancer

The goals of this study were to detect the numerical alterations of chromosomes 1 and 7 in breast cancer and to correlate the findings with DNA ploidy status as well as with parameters of prognostic significance. Fluorescence in situ hybridization (FISH) with centromeric probes for chromosomes 1 and 7 and cellular DNA content measurement by image analysis-based cytophotometry were applied on interface nuclei from fresh tissue imprints of 59 breast ductal carcinomas. Immunohistochemical stainings for estrogen receptor (ER), progesterone receptor (PR), HER-2, p53, and Ki67 were performed on paraffin tumor sections. The correlation between DNA ploidy and chromosomal aberrations revealed a significant association between aneuploidy and aneusomy for both chromosomes 1 (p=0.002) and 7 (p=0.00001), however, a number of diploid tumors were found to be aneusomic, especially for chromosome 1. Chromosome 7 polysomy was significantly associated with a higher incidence of axillary lymph node metastasis (p=0.05), poorly differentiated (grade III) tumors (p=0.03), negative ER and PR status (p=0.02 and 0.001, respectively), as well as p53 protein expression (p=0.05) and a higher Ki67 labeling index (p=0.004). Chromosome 1 aneusomy was only related with HER-2 protein overexpression (p=0.05). No association between chromosome alterations and tumor size was detected. In conclusion, the results of our study indicate that the detection of numerical aberrations of chromosomes 1 and 7 by FISH seems to be more sensitive than DNA ploidy status for the evaluation of abnormal cellular DNA and chromosome 7 aneusomy characterizes tumors with aggressive features and therefore might be a useful predictor of unfavorable biological behavior in breast cancer.     

Expression of p53 protein related to human papillomavirus and DNA ploidy in superficial esophageal carcinoma

We examined the p53 protein and human papilloma virus (HPV) by immunohistochemistry and DNA ploidy by cytofluorometry in paraffin-embedded esophageal carcinoma tissue specimens. Sixty-one patients with superficial esophageal carcinoma were operated on between 1983 and 1991 without any prior treatment. Immunostaining of the anti-p53 protein antibody (CM1) was positive in 32 carcinomas (52%). Patients with p53-positive tumors had a poorer outcome than those with p53-negative tumors (P<0.05). In addition, patients with p53-positive tumors did not have any characteristic site of relapse. Only 5 of the 61 patients (8.2%) had HPV-positive tumors. One of these 5 carcinomas expressed both p53 protein and HPV. Three patients with HPV-positive tumors which had invaded the submucosal layer died of relapse. A determination of DNA ploidy revealed 30 patients with aneuploid tumors, 13 with polyploid tumors and 18 with diploid tumors. The outcome of the patients with aneuploid tumors was worse than that of the patients with diploid tumor (P<0.05). p53 protein expression was not associated with DNA ploidy; however, the 16 patients who had both p53-positive and aneuploid tumors had a worse prognosis than patients with p53-negative and aneuploid tumors (P<0.01). These findings suggest that p53 protein expression in conjunction with DNA ploidy may be a useful indicator in evaluating the prognosis of patients with superficial esophageal carcinoma.     

c-FLIP及突变p53蛋白表达与膀胱癌预后关系的研究

目的研究膀胱癌中细胞型Fas相关死亡域样白介素-1β转换酶抑制蛋白（c-FLIP）、突变p53蛋白的表达与膀胱癌预后的关系。方法采用免疫组织化学方法测定82例膀胱癌组织和10例正常膀胱黏膜中c-FLIP和突变p53蛋白的表达。膀胱癌患者中,男66例,女16例,年龄45～90岁。2002年TNM分期Ta～T129例、T2～T453例。WHO1973年病理分级G1级12例、G2级41例、G3级29例。结合临床资料分析其与膀胱癌分期、分级、复发的相关性。采用SPSS 13.0软件包分析其表达与肿瘤组织学性状的关系以及对患者预后的影响。结果82例膀胱癌组织中c-FLIP的阳性表达率为62.2%（51/82）,其中Ta～T1肿瘤阳性表达率为37.9%,T2～T4肿瘤阳性表达率为75.5%;突变p53蛋白的阳性表达率为45.1%（37/82）,其中Ta～T1肿瘤阳性表达率为24.1%,T2～T4肿瘤阳性表达率为56.6%。10例正常膀胱黏膜中c-FLIP和突变p53蛋白的表达均为阴性。c-FLIP与p53蛋白在不同的分期组间阳性表达差异均有统计学意义（P〈0.05）;生存分析发现c-FLIP与突变p53蛋白表达对生存具有明显影响（P〈0.05）。结论c-FLIP与突变p53蛋白对评价膀胱癌的侵袭性和预后都是一个重要的独立因素。膀胱肿瘤组织中的c-FLIP及突变p53蛋白表达缺失提示患者预后较好。     

膀胱移行细胞癌中Survivin与Ki-67、p53的表达及临床意义

目的 探讨膀胱移行细胞癌中Survivin、p53及Ki-67的表达与膀胱移行细胞癌临床病理特征的关系及其临床意义. 方法 用免疫组织化学方法 检测88例膀胱移行细胞癌与10例正常膀胱黏膜组织中Survivin、p53及Ki-67的表达,并用RT-PCR方法 验证其中30例膀胱移行细胞癌和10例正常膀胱黏膜组织中Survivin的表达. 结果 免疫组织化学方法 显示在膀胱移行细胞癌中Survivin、p53阳性表达率分别为63.6%(56/88)和45.5%(40/88),正常膀胱黏膜组织中均无表达,差异有统计学意义(P<0.01),且Survivin的表达与膀胱癌的临床分期密切相关(P<0.05).膀胱癌组织中Ki-67增殖指数(PI)为20.4士10.7,正常膀胱黏膜组织中为0.RT-PCR方法 显示在30例膀胱移行细胞癌中Survivin阳性表达率为100%,而正常膀胱黏膜组织中均无表达.结论 Survivin与膀胱癌的恶性程度有关,与p53及Ki-67共同参与了膀胱移行细胞癌的发生、发展及进程.     

Synergistic enhancement of resistance to cisplatin in human bladder cancer cells by overexpression of mutant-type p53 and Bcl-2

PURPOSE: The objective of this study was to characterize the effect of mutant-type p53 and Bcl-2 expression on the sensitivity to cisplatin in a human bladder cancer cell line both in vitro and in vivo. MATERIALS AND METHODS: We transfected mutant-type p53 cDNA, Bcl-2 cDNA, or both cDNAs into KoTCC-1, a human bladder cancer cell line that does not express mutant-type p53 or Bcl-2 protein. The effects of the overexpression of mutant-type p53, Bcl-2, or both on the sensitivity to cisplatin and the apoptotic features in vitro were evaluated by the MTT assay, staining with Hoechst 33258 and a DNA fragmentation assay. We then examined the in vivo effects of cisplatin treatment on the transfectants by subcutaneous and intraperitoneal tumor cell injection models in athymic nude mice. RESULTS: The introduction of mutant-type p53 or Bcl-2 conferred resistance to cisplatin on KoTCC-1 cells through the inhibition of apoptosis. This phenotype was more remarkable in the cell line transfected with both mutant-type p53 and Bcl-2 than in the cell lines transfected with either mutant-type p53 or Bcl-2 alone. Furthermore, the KoTCC-1 cells transfected with both mutant-type p53 and Bcl-2 exhibited significantly higher resistance to cisplatin treatment than cells transfected with mutant-type p53 or Bcl-2 alone in experimental models in vivo. CONCLUSIONS: These findings suggest that the overexpression of both mutant-type p53 and Bcl-2 in bladder cancer cells synergistically interferes with the therapeutic effect of cisplatin through the inhibition of the apoptotic pathway.     

Serum anti-p53 antibodies and p53 protein status in the sera and tumors from bladder cancer patients

OBJECTIVES: The purpose of this study was to evaluate the association between the serum anti-p53 antibodies (Abs) status and the p53 protein status in the sera and tumors as well as clinical or pathological parameters in bladder cancer patients retrospectively. METHODS: Serum samples from 100 patients with bladder cancer were assayed for anti-p53 Abs and p53 protein by enzyme-linked immunosorbent assay (ELISA). A monoclonal antibody DO7 was used for immunohistochemical staining of tumor p53 protein. RESULTS: Prevalences of serum anti-p53 Abs, serum p53 protein and tumor p53 protein were 12, 1 and 63%, respectively. There was a significant correlation between serum anti-p53 Abs status and factors including tumor stage, tumor grade, and tumor p53 protein status. In the univariate analysis, tumor stage, tumor grade, serum anti-p53 Abs status, and tumor p53 protein status were significantly associated with an increased risk of death. Multivariate analysis showed that tumor stage was the only independent prognostic factor among the factors examined. CONCLUSIONS: The present study suggests that serum anti-p53 Abs had a limited value as a tumor marker in bladder cancer patients. Further studies to elucidate the mechanism of anti-p53 Abs production will be necessary for a better understanding of the immune status in bladder cancer patients.     

尼古丁对膀胱癌p53基因表达的相关研究

目的：探讨尼古丁与癌基因表达和膀胱癌生物学行为的关系。方法：对40只大鼠以10％BBN为致癌剂膀胱灌注诱发膀胱癌,其中30只分25、15、5mg／kg剂量以咽管灌胃方式给予尼古丁,10只作为对照,应用免疫组化方法对膀胱癌模型中p53蛋白进行检测。结果：不同剂量的尼古丁干预下的p53蛋白表达的阳性率分别为60％、30％、20％。p53蛋白表达阳性率与给药剂量、给药时间呈正相关。结论：尼古丁对癌基因的异常表达及协同作用,在膀胱癌的发生发展中起一定作用。     

流式细胞术分析肝细胞癌DNA倍体异质性的研究

目的 研究肝细胞癌(肝癌)是否存在DNA倍体的异质性。方法 采用流式细胞术检测了29例肝癌标车不同区域的DNA倍体情况。结果 16例为异倍体，13例为二倍体，没有发生二倍体和异倍体并存的肝癌，16例异倍体肝癌中，9例肿瘤内DNA指数相同．其他7例都存在异倍体亚克隆。结论 在肝癌的发生发展过程中二倍体肿瘤和异倍体肿瘤各自按固有的倍体模式增殖，二倍体肝癌一般不转化为异倍体肝癌，但异倍体肝癌可产生新的异倍体亚克隆。     

p53 gene mutation and p53 protein overexpression in a patient with simultaneous double cancer of the gallbladder and bile duct associated with pancreaticobiliary maljunction

Pancreaticobiliary maljunction (PBM) is associated with the occurrence of biliary cancer due to pancreatobiliary reflux. We present a case of simultaneous double cancer of the gallbladder and bile duct. A 77-year-old woman who had jaundice, intra- and extra-hepatic biliary ductal dilatation and a space-occupying lesion in the gallbladder and lower bile duct underwent pancreatoduodenectomy. The gallbladder cancer showed papillary carcinoma without mutation of the K-ras gene and with p53 non-sense mutation of CCA (Pro) to CA (Stop) on codon 301 in exon 8. The bile duct cancer revealed a well-differentiated adenocarcinoma without mutation of the K-ras gene and with p53 miss-sense mutation of GTG (Val) to GAG (Glu) on codon 272 in exon 8. There were no mutations of either the K-ras or p53 gene in non-cancerous epithelia. In contrast, only the mucosa of the common channel had p53 protein accumulation and high cell proliferation activity. Therefore, the genetic pathway might be the same in both the gallbladder and bile duct cancer, and a high potential for carcinogenesis might be present in the epithelium of the common channel in patients with PBM.     

Human papilloma virus DNA and p53 mutation analysis on bladder washes in relation to clinical outcome of bladder cancer

OBJECTIVES: High-risk human papilloma virus (HPV) types stimulate degradation and deactivation of protein associated with the p53 tumour suppressor gene via the ubiquitin-dependent pathway. For a long time, changes of the p53 tumour suppressor gene have been correlated with poor clinical outcome in patients with superficial bladder cancer. We aimed to study the association between presence of (high-risk) HPV DNA, p53 status, and clinical outcome in bladder cancer patients. This study must be seen as a preliminary study to investigate this potentially important problem. MATERIAL AND METHODS: From 107 patients, 166 bladder wash samples were obtained. p53 status was determined by mutation analysis, HPV detection, and genotyping by the SPF(10)-LiPA assay. Clinical data were abstracted from the medical files. RESULTS: The prevalence of all-type and high-risk HPV infection in malignancies of the bladder was 15.2% and 8.1%, respectively. In high-grade tumours this prevalence was 18.2% and 10.6%, respectively. In grade 1, 2 and 3 tumours the infection rate of high-risk HPV types was 0%, 3.3%, and 10.6%, respectively (trend test: p=0.221). In Ta, T1, and T2-T4 tumours the high-risk HPV infection rate was 0%, 12.5% and 18.2%, respectively (trend test: p=0.045). In the p53 wild-type patients who showed progression, 1 of 9 patients had a high-risk type HPV infection. In the group of wild-type patients who showed no progression, 4 of 37 patients had a high-risk type HPV infection (odds ratio: 1.03; 95% confidence interval, 0.1-10.5). CONCLUSIONS: The data of this pilot study show the suggestion of a positive trend in the correlation between tumour grade/stage and high-risk type HPV infection. However, no additional risk for progression is found for p53 wild-type patients with a high-risk HPV infection.