Immunohistochemical detection of CDKN2, retinoblastoma and p53 gene products in primary astrocytic tumors

The expressions of p16(INK4), retinoblastoma (RB) and p53 protein were immunohistochemically examined in 70 primary astrocytic tumors. In 58 patients with high grade astrocytoma (18 anaplastic astrocytomas and 40 glioblastomas), 30 (51.1%) and 15 (25.9%) cases were undetectable for p16(INK4) and pRB, respectively, but their lack occurred infrequently in 12 low grade astrocytomas. The expression of p16(INK4) was inversely correlated with that of PRB, especially in glioblastomas. Accumulation of p53 was detected in 32 (45.7%) of 70 cases without any dependence on the grade. A deregulation of three tumor suppressor gene products most often occurs singly. Only patients with negative staining for pRB were significantly associated with a shorter survival time. Our findings suggest that loss of functional pRB at the G1/S check point may represent an important step in glioblastoma development and have a stronger negative impact on clinical outcome than p16(INK4) or p53 aberrations.     

Analysis of p53 mutation and cyclin d1 expression in breast tumors

P53 and cyclin D1 are interacting regulatory genes and both are frequently altered in breast cancer. We analysed p53 mutation by SSCP and sequencing methods as well as p53 protein accumulation immunohistochemically in 34 consecutively operated breast tumors. None of 4 fibroadenomas revealed p53 mutation or p53 protein accumulation. Mutation of p53 was present in 7 carcinomas. Immunohistochemistry revealed accumulation of p53 protein in 6 carcinomas and there was a significant correlation between p53 mutation and protein accumulation. Overexpression of cyclin D1 protein was observed in 11 carcinomas by immunohistochemistry and no correlation was observed between cyclin D1 overexpression and p53 mutation or accumulation. Our data support the concept that the p53-cyclin D1 signal pathway and the cyclin D1 cascade are disregulated in breast cancer.     

肿瘤DNA含量、p53表达和PCNA表达对预测食管癌预后的意义

目的：探讨预测食管癌预后的生物学指标。方法：选择具有相同ＴＮＭ分期但术后长期生存和短期死亡的两组食管癌及４８例,应用自动化图象分析术测定肿瘤细胞ＤＮＡ含量,用免疫组织化学法测定肿瘤细胞ｐ５３表达和ＰＣＮＡ表达。结果：生存超达１０年的患者,二倍体的检出率是５２．４％（２２／４２）,ｐ５３和ＰＣＮＡ过度表达率各为６０．４％（２９／４８）和３７．５％（１３／４８）。３年内死亡者,二倍体的检出率是３０．９     

Interlaboratory comparison of IDH mutation detection

Isocitrate dehydrogenase (IDH) mutational testing is becoming increasingly important. For this, robust and reliable assays are needed. We tested the variation of results between six laboratories of testing for IDH mutations. Each laboratory received five unstained slides from 31 formalin-fixed paraffin-embedded (FFPE) glioma samples, and followed its own standard IDH diagnostic routine. All laboratories used immunohistochemistry (IHC) with an antibody against the most frequent IDH1 mutation (R132H) as a first step. Three laboratories then sequenced only IHC negative cases while the others sequenced all cases. Based on the overall analysis, 13 samples from 11 tumors had an R132H mutation and one tumor showed an R132G mutation. Results of IHC for IDH1 R132H mutations in all six laboratories were completely in agreement, and identified all R132H mutations. Upon sequencing the results of two laboratories deviated from those of the others. After a review of the entire diagnostic process, on repeat (blinded) testing one laboratory was completely in agreement with the overall result. A change in technique did only partially improve the results in the other laboratory. IHC for the IDH1 R132H mutation is very reliable and consistent across laboratories. IDH sequencing procedures yielded inconsistent results in 2 out of 6 laboratories. Quality assurance is pivotal before IDH testing is made part of clinical management of patients.     

大肠肿瘤cyclin D1、p21WAF1/CIP1、p53和增殖细胞核抗原的免疫组化研究

目的：探讨细胞周期调控因子表达在大肠肿瘤发生发展中的作用及其预后的影响。方法：彩免疫组化染色对１１３例大肠癌、１４例大肠腺瘤、１１例腺瘤癌变组织中ｃｙｃｌｉｎＤ１、ｐ＾２１＾ＷＡＦ１／ＣＩＰＩ、ｐ＾５３和ＰＣＮＡ进行了检测。结果：大肠癌组织ｃｙｃｌｉｎＤ１和ｐ＾５３阳性表达率分别为５４．８７％和５６．６４％,均显著高于正常大肠组织（Ｐ〈０．０５）。高分化腺癌ｐ＾２１蛋白阳性表达率６９．７０％,低分     

Immunohistochemical detection of p53 protein and proliferating cell nuclear antigen (pcna) in salivary-gland tumors

The expression of p53 protein and PCNA was immunohistochemically examined in 161 cases of salivary gland tumors. The p53 protein was positive in 23.8% (24/101) of malignant salivary gland tumors, while only one case of 60 pleomorphic adenomas was positive. p53 protein was frequently detectable in salivary duct carcinoma (60%), basal cell adenocarcinoma (66.7%), acinic cell carcinoma (53.3%) and undifferentiated carcinoma (50%), but was rarely seen in adenoid cystic carcinoma (17.2%) and mucoepidermoid carcinoma (0%). p53 protein was more frequently detectable in PCNA positive cases than in negative cases (p=0.0074).     

The expression and significance of IDH1 and p53 in osteosarcoma

Background

To detect the expression of isocitrate dehydrogenase 1 (IDH1) and transformation-related protein 53 (p53) in osteosarcoma and analyze the correlation between them and the clinico-pathological features.

Methods

The expressions of IDH1 and p53 were detected in human osteosarcoma cell lines (MG-63 and U2OS) by immunocytochemistry, Real-time PCR and Western Blotting. The expressions of IDH1 and p53 in formalin-fixed paraffin-embedded tissue sections from 44 osteosarcoma patients were determined by immunohistochemistry, and the correlation between them and clinicopagthological features were analyzed. None of these patients received chemotherapy prior to surgery.

Results

IDH1 is detected in osteosarcoma cell lines and biopsies. IDH1 expresses higher in U2OS cells with wild type p53 than in MG-63 cells with mutation p53. IDH1 correlates with histological Rosen grade and metastasis negatively. P53 correlates with histological Rosen grade, metastasis and overall survival in clinical osteosarcoma biopsies. Osteosarcoma patients with High IDH1 expression have a very high p53 expression.

Conclusion

IDH1 may correlate with p53 and be a candidate biomarker for osteosarcoma correlate with histological Rosen grade and metastasis.     

p53 and Ki-ras as prognostic factors for Dukes' stage B colorectal cancer

Mutations of the TP53 and Ki-ras genes have been reported to be of prognostic importance in colorectal carcinomas. An increased intracellular concentration of the p53 protein, although not identical to, is sometimes seen in tumours with TP53 mutation and has been correlated with poor prognosis in some tumour types. Previous colorectal cancer studies, addressing the prognostic importance of Ki-ras mutation and TP53 aberrations, yielded contradictory results. The aim of this study was to determine in a clinically and therapeutically homogeneous group of 122 sporadic Dukes' B colorectal carcinomas with a median follow-up of 67 months (3-144 months) whether or not p53 protein expression, TP53 mutation and K-ras mutation correlated with prognosis. p53 staining was performed by immunohistochemistry, using the monoclonal antibody DO7 on paraffin-embedded tissue. Mutations in exons 5-8 of the TP53 gene and in codons 12 and 13 of the K-ras gene were assayed in paraffin-embedded tissue by the single-strand conformation polymorphism (SSCP) assay. Nuclear p53 staining was found in 57 (47%) tumours. Aberrant migration patterns indicating mutation of the TP53 gene were found in 39 (32%) tumours. Forty-six carcinomas (38%) showed a mutation of the Ki-ras codons 12 or 13. In a univariate analysis, patients with wild-type TP53 status showed a trend towards better survival, compared with those with mutated TP53 (log-rank test, P = 0.051). Likewise, tumours immunohistochemically positive for p53 showed a worse prognosis than p53-negative tumours (P = 0.010). The presence or absence of mutations in Ki-ras did not correlate with prognosis (P = 0.703). In multivariate analysis, only p53 immunoreactivity emerged as an independent marker for prognosis hazard ratio (HR) = 2.16, 95% confidence interval (CI) 1.12-4.11, P = 0.02). Assessment of p53 protein expression is more discriminative than TP53 mutation to predict the outcome of Dukes' stage B tumours and could be a useful tool to identify patients who might benefit from adjuvant therapy.     

Immunohistochemical detection of p53 and Bcl-2 in colorectal carcinoma: no evidence for prognostic significance.

To evaluate the prognostic significance of immunohistochemically detected p53 and Bcl-2 proteins in colorectal cancer, tissue sections from 238 paraffin-embedded colorectal carcinomas were immunostained for p53 (MAb DO-7 and CM-1 antiserum) and Bcl-2 (MAb Bcl-2:124). Staining patterns were assessed semiquantitatively and correlated with each other and with sex, age, tumour site, Dukes'' classification, tumour differentiation, mucinous characteristics, lymphocyte and eosinophilic granulocyte infiltration, and patient survival. In our series, 35% of carcinomas showed no nuclear staining and 34% (DO-7) to 40% (CM-1) showed staining in over 30% of tumour cell nuclei. A majority of carcinomas that had been immunostained with CM-1 showed cytoplasmic staining, but this was not observed with DO-7. With respect to Bcl-2, 51% of tumours were completely negative, 32% displayed weak and 15% moderate staining; only 3% showed strong positive staining. No evidence was found for reciprocity between Bcl-2 expression and nuclear p53 accumulation. From 13 cases containing tumour-associated adenoma, four were Bcl-2 negative in premalignant and malignant cells, in another four cases these cells showed similar staining intensities and in the remaining cases only the malignant colorectal cells were Bcl-2 negative. Therefore, our data indicate that Bcl-2 is dispensable in the progression towards carcinoma. Except for an association between nuclear p53 accumulation and mucinous tumours (P = 0.01), no significant correlation was found between the clinicopathological parameters mentioned above and immunostaining pattern of (nuclear or cytoplasmic) p53 or Bcl-2.     

IDH1 mutation detection by droplet digital PCR in glioma

Glioma is the most frequent central nervous system tumor in adults. The overall survival of glioma patients is disappointing, mostly due to the poor prognosis of glioblastoma (Grade IV glioma). Isocitrate dehydrogenase (IDH) is a key factor in metabolism and catalyzes the oxidative decarboxylation of isocitrate. Mutations in IDH genes are observed in over 70% of low-grade gliomas and some cases of glioblastoma. As the most frequent mutation, IDH1(R132H) has been served as a predictive marker of glioma patients. The recently developed droplet digital PCR (ddPCR) technique generates a large amount of nanoliter-sized droplets, each of which carries out a PCR reaction on one template. Therefore, ddPCR provides high precision and absolute quantification of the nucleic acid target, with wide applications for both research and clinical diagnosis. In the current study, we collected 62 glioma tissue samples (Grade II to IV) and detected IDH1 mutations by Sanger direct sequencing, ddPCR, and quantitative real-time PCR (qRT-PCR). With the results from Sanger direct sequencing as the standard, the characteristics of ddPCR were compared with qRT-PCR. The data indicated that ddPCR was much more sensitive and much easier to interpret than qRT-PCR. Thus, we demonstrated that ddPCR is a reliable and sensitive method for screening the IDH mutation. Therefore, ddPCR is able to applied clinically in predicting patient prognosis and selecting effective therapeutic strategies. Our data also supported that the prognosis of Grade II and III glioma was better in patients with an IDH mutation than in those without mutation.     

Functional analysis of p53 gene and the prognostic impact of dominant-negative p53 mutation in endometrial cancer

In addition to the loss of function, mutant p53 can possess a dominant-negative effect on wild-type p53 and may also exert gain-of-function activity. It is not clear whether the functional status of p53 mutation contributes to differences in outcome in endometrial cancer. We collected a total of 92 RNA samples of high quality from endometrial cancer tissues, and the samples were subjected to yeast functional assay and sequencing for p53 mutations. The detected mutant p53 genes were further investigated for their dominant-negative activity using a yeast-based transdominance assay. p53 mutation was found in 24 out of 92 (26.1%) tumors, of which 10 exhibited no dominant-negative activity (recessive mutation) and 14 showed dominant-negative activity. Dominant-negative p53 mutation was related to advanced stages (p = 0.01), nonendometrioid type tumors (p = 0.01) and grade 3 tumors (p = 0.04). The patients with dominant-negative mutation had significantly shorter survival than patients with no mutation (p < 0.0001) and those with a recessive mutation (p = 0.01) in the p53 gene. No difference in survival was found between the patients with tumors harboring a recessive p53 mutation and those with tumors harboring a wild-type p53. Multivariate analysis revealed that dominant-negative p53 mutation (p = 0.019), FIGO stage (p = 0.0037) and histologic subtype (p = 0.014) were independently related to patient survival. Dominant-negative p53 mutation was the most important prognostic factor for stage III/IV endometrial cancer (p = 0.0023). In conclusion, dominant-negative p53 mutation is often found in advanced stages and aggressive histologic subtypes of endometrial cancer and it is a strong predictor of survival of patients with advanced endometrial cancer. To elucidate further the role of p53 mutation in endometrial cancer, it is necessary to investigate gain-of-function activity involving dominant-negative p53 mutant proteins.     

Dysfunction of p53 pathway in human colorectal cancer: analysis of p53 gene mutation and the expression of the p53-associated factors p14ARF, p33ING1, p21WAF1 and MDM2

Mutations of p53 tumor suppressor gene increase with tumor progression in colorectal cancers. In this study, we examined the expressions of p33ING1, p14ARF, MDM2 and p21WAF1 mRNA in 25 advanced colorectal cancers by quantitative RT-PCR method, and compared the expression levels of p33ING1, p14ARF, p21WAF1 and MDM2 in relation to p53 status in the tumors. Fifteen of 25 colorectal cancers (60%) showed abnormal accumulation of p53 protein in the nucleus, and the remaining 10 colorectal cancers (40%) were negative for p53 immunostaining. We found a G --> T transition (nonsense mutation) at the first nucleotide of codon 298 (exon 8) in one p53-negative case, and a frame shift mutation on exon 7 in another p53-negative case. In remaining eight p53-negative cases, there was no mutation in the entire open reading frame of p53 cDNA. Interestingly, in eight cases with p53 wild-type gene, 6 cases (75%) showed a marked down-regulation of p14ARF mRNA, and three cases (37.5%) over-expressed MDM2 mRNA. Only one case with wild-type p53 gene showed normal level expression of p53 regulatory-factors (p33ING1, p14ARF, and MDM2). Thus, p53 tumor suppressor pathway was disrupted in 24 of 25 colorectal cancers (96%).     

p21/WAF1, p53 and PCNA expression and p53 mutation status in hepatocellular carcinoma

The cyclin-dependent kinase inhibitor p21/WAF1 is regulated by p53-dependent and p53-independent pathways. In addition, p21/WAF1 binds with proliferating cell nuclear antigen (PCNA) and inhibits the action of PCNA. To investigate the possible role of p21/WAF1 in human hepatocellular carcinomas (HCCs), we examined the expression of p21/WAF1 and its relation with PCNA and p53 expression in 97 surgically resected HCCs by immunohistochemistry and with the mutation status of p53 in 26 HCCs. p53 mutation status was examined by direct DNA sequencing using 3 sets of primers covering exons 5–9. Six of the 26 tumors showed p53 point mutations and only 33% of these HCCs demonstrated p21/WAF1 expression. In contrast, 75% of HCCs without p53 mutations showed p21/WAF1 expression. Of all 97 HCCs, p21/WAF1 expression was significantly higher in the tumors than in corresponding non-tumorous liver. When the tumors were stratified into 2 groups by the median tumor p21/WAF1 score, those with higher expression were found to have a lower incidence of multiple tumor nodules (p = 0.008) and tumor microsatellite formation (p = 0.050). The tumor p21/WAF1 score was positively associated with tumor PCNA expression (p = 0.036) but not with tumor p53 expression. Thus, in HCC, expression of p21/WAF1 is in part dependent on p53 status, but a p53-independent pathway also plays a significant role in the regulation of p21/WAF1 expression. High p21/WAF1 expression is significantly associated with solitary tumor nodules and, to a lesser extent, tumor microsatellites but may not be enough to suppress tumor progression. Int. J. Cancer (Pred. Oncol.) 79:424–428, 1998. © 1998 Wiley-Liss, Inc.     

Immunohistochemical analysis of p53 in colorectal cancer regarding clinicopathological correlation and prognostic significance

The overexpression of the tumor suppressor gene p53 was investigated immunohistochemically in 144 cases of primary colorectal cancer and in 8 cases with cancer in the corresponding metastatic lymph nodes. Abnormalities in p53 expression were found in 36 cases (25%) of the 144 primary cancer cases. In addition, p53-positive tumors were found to metastasize frequently to the lymph nodes, as compared to p53-negative tumors (61.1% vs. 41.7%, P=0.0428). p53 staining was identical in 7 of 8 (87.5%) cases in primary and metastatic lesions. When the DNA content of the tumor was determined by flow cytometry, the DNA index (mean ± SD) was significantly higher in p53-positive tumors than in p53-negative tumors (1.57 ± 0.38 vs. 1.39 ± 0.37, P=0.012). Therefore, the immunohistochemical data of p53 in colorectal cancer may help in potentially predicting metastatic spread to the lymph nodes. © 1995 Wiley-Liss, Inc.     

Ki-67, p53, ER receptors, ploidy and S phase as long-term prognostic factors in T1 node-negative breast cancer.

BACKGROUND: The aim of the present study was to evaluate a series of biomarkers with regard to long-term prognostic value in patients with T1 (< or =2 cm) node-negative breast cancer. METHOD: The prognostic value of Ki-67, p53, oestrogen receptor (ER) immunohistochemical labelling, flow-cytometric S phase fraction and ploidy was evaluated in 212 patients with pT1N0M0 breast cancer. The median follow-up time was 15.9 years (range 0.2-27.2 years). RESULTS: In an analysis of breast cancer-specific survival up to 5 years, high Ki-67 (> or =10%; p = 0.002), high p53 (> or =20%; p = 0.01), negative ER (<30%; p = 0.01) as well as aneuploidy of the tumour (p = 0.02) were significant prognostic factors. When the follow-up was extended to 10 years, only Ki-67 (p = 0.03) was significantly associated with outcome and beyond 15 years none of the studied markers provided significant prognostic information when analyzed separately. There was a weak but significant difference in long-term survival when patients with a combination of high Ki-67 (> or =10%), high SPF (>3%) and high p53 (> or =20%) were compared to patients with other combinations (p = 0.03). CONCLUSION: According to the results of our series, it seems that several prognostic markers which are associated with short-term survival (< or =5 years) in pT1N0M0 breast cancer may not be significant predictors of long-term (>15 years) breast cancer-specific survival.     

Adenovirus-mediated p53AIP1 gene transfer as a new strategy for treatment of p53-resistant tumors

The p53AIP1 gene, which we recently identified as a novel p53-target, mediates p53-dependent apoptosis. We evaluated the effects of adenovirus-mediated introduction of p53AIP1 (Ad- p53AIP1 ) on 30 human cancer-cell lines in vitro , and two cell lines in vivo , in comparison with the effects of p53 (Ad-p53). In 20 of the 30 cell lines, p53AIP1-induced apoptosis was observed, and in 12 of these p53AIP1-sensitive cancer cell lines, the apoptotic effects of p53AIP1 were greater than those of p53 itself. Cancers with wild-type p53, which were thought to be p53-resistant, were likely to be sensitive to p53AIP1-induced apoptosis. p53-re-sistant cancers such as LS174T (p53+/+) and A549 (p53+/+), in which no increase of p53AIP1 mRNA expression was observed when Ad-p53 was introduced, were killed effectively by Ad- p53AIP1 . Furthermore, co-introduction of p53 and p53AIP1 had a synergistic effect on the induction of apoptosis, regardless of p53 status. Finally, adenovirus-mediated introduction of p53AIP1 suppressed tumor growth in vivo . These results suggested that p53AIP1 gene transfer might become a new strategy for the treatment of p53-resistant cancers. (Cancer Sci 2004; 95: 91–97)     

Proliferating cell nuclear antigen and p53 expression as prognostic factors in T1-2MO prostatic adenocarcinoma

The expression of proliferating cell nuclear antigen and p53 protein was analysed by immunocytochemical methods (PC10, CMI antisera) in 139 patients with T1-2MO prostatic adenocarcinomas followed-up for > 12 years. p53 protein was expressed in 21 (15%) tumours (15%), the fraction of positive nuclei being very low (mean SE, 1% ± 0.7%). Accumulation of p53 protein in epithelial cells was independent of tumour stage and Gleason score, and had no effect on prognosis. In 4 cases, p53 protein was expressed only in stromal cells. The fraction of PCNA-positive nuclei (evaluable in 116 cases) was higher in T2 than in T1 tumours (p < 0.001); furthermore, high Gleason score was positively correlated with PCNA positivity (p < 0.001). A finding of over 5% of PCNA-positive nuclei predicted progression in T and M categories and were a sign of poor outcome. The fraction of PCNA-positive stromal-cell nuclei was related to T-category with a borderline significance (p = 0.06). In a multi-variate analysis of the prognostic factors, independent predictors of survival included Gleason score (p < 0.001), fraction of PCNA-positive nuclei (p = 0.013), observation before therapy (p = 0.05), and T-category (p = 0.07) in that order of significance. The results suggest that overexpression of p53 protein is of marginal prognostic value in local prostatic adenocarcinomas, whereas direct measurement of cell proliferation by PCNA immunolabelling provides important prognostic information in T1-2MO tumours, in addition to the Gleason score.     

p53 expression, DNA ploidy and mitotic index as prognostic factors in patients with epithelial ovarian carcinoma

AIMS AND BACKGROUND: Biological variables linked to genomic instability were examined and related to survival in 52 patients affected by ovarian carcinoma and nine patients with low malignant potential tumors (LMP). METHODS: DNA ploidy was measured by image cytometry in isolated neoplastic cells; the mitotic index was measured in Feulgen-toluidine blue-stained sections and p53 was investigated by immunohistochemistry. RESULTS: Twenty-five tumors (4 LMP) were peridiploid (ploidy < 2.25c), 22 tumors (4 LMP) were hyperdiploid (2.25c > ploidy < 2.9c) and 14 (1 LMP) had high ploidy (> or = 2.9c). MI ranged from 0.3 to 24.2 with a mean of 1.8 for LMP and 6.8 for carcinomas (P<0.001). Widespread p53 overexpression was detected in 49% of carcinomas and in none of the LMP tumors. CONCLUSIONS: Survival analysis performed in patients with carcinomas indicated that, of the examined biological variables, only MI was moderately associated with survival in a subgroup of early-stage patients.