The estimation of proliferative activity by pcna and AgNORs in leukoplakia and squamous-cell carcinoma of the oral cavity

The proliferating activity of leukoplakia and squamous cell carcinoma (SCC) was estimated using proliferating cell nuclear antigen (PCNA) immunostaining and silver-binding argyrophilic nucleolar organizer region (AgNOR) staining. Twenty-eight leukoplakias and 15 SCCs of the oral cavity were used in this study. The mean+/-S.D. of PCNA LI and AgNOR counts were 7.7+/-6.0% and 2.43+/-0.68/nucleus in leukoplakias, and 22.3+/-11.6% and 4.77+/-1.49/nucleus in SCCs. Both of PCNA LI and AgNOR counts were significantly higher in SCCs than in leukoplakias. There was a significant linear correlation between PCNA LI and AgNOR counts (p=0.0022) in leukoplakias, but not in SCCs. In the series of leukoplakias, PCNA LI apparently increased in leukoplakias with dysplasia and malignant transformed cases. Our data suggest that PCNA LI and AgNOR counts are useful markers of proliferating activity, and PCNA LI might be a prognostic factor of leukoplakias.     

肾盂癌p53蛋白、AgNOR和PCNA相关性及临床意义的研究

采用免疫组化和银染色技术对31例肾盂癌组织中p53蛋白，核仁组成区嗜银蛋白（AgNOR）和增殖细胞核抗原（PCNA）进行研究。发现肾盂癌p53表达阳性率，AgNOR颗粒计数和PCNA增殖指数均随肿瘤病理分级、临床分期的上升而增加，且与预后呈负相关。p53蛋白阳性表达组AgNOR颗粒计数和PCNA增殖指数显著高于p53蛋白阴性表达组。肾盂癌AgNOR计数与PCNA增殖指数间存在正相关性。提示p53蛋白表达，AgNOR计数和PCNA免疫染色在肾孟癌生物学行为及预后评估中具有重要意义。     

恶性纤维组织细胞瘤PCNA与AgNOR定量检测及其预后意义

目的为探讨恶性纤维组织细胞瘤的增殖细胞核抗原（PCNA）及核仁组成区嗜银蛋白（AgNOR）定量检测与肿瘤恶性度及预后的关系。方法应用免疫组织化学杂色LSAB法及AgNOR染色法对46例资料完整有随访结果的恶性纤维组织细胞瘤（MFH）手术切除标本进行染色以显示PCNA及AgNOR,彩色病理图像分析系统定量检测。结果肿瘤细胞核PCNA过表达及AgNOR计数与患者生存期呈负相关（rPCNA＝－0．6248、rAgNOR＝－0．5941,P＜0.01）;PCNA及AgNOR定量高值组较之定量低值组患者生存期短（P＜0．01）;肿瘤有转移组PCNA及AgNOR定量高于无转移组（P＜0．01）;肿瘤位置深在组PCNA及AgNOR定量高于肿瘤位置浅表组（P＜0．01）。结论肿瘤细胞核PCNA及AgNOR定量与患者生存期密切相关,与肿瘤转移及位置深浅等反映肿瘤恶性度的指标亦密切相关,提示肿瘤细胞核PCNA及AgNOR定量对于判断恶性纤维组织细胞瘤恶性度及预后的意义值得重视。     

MDM2 and p53 expression in gliomas: a multivariate survival analysis including proliferation markers and epidermal growth factor receptor.

p53 and the murine double minute 2 (MDM2) oncoprotein expression was evaluated in paraffin-embedded tissue from 61 patients with central nervous system gliomas (53 astrocytomas and eight oligodendrogliomas) and related to proliferation-associated markers [i.e. proliferating cell nuclear antigen (PCNA), Ki-67 and nuclear organizer regions (NORs)] and epidermal growth factor receptor (EGFR). We used the monoclonal antibodies PC-10, MIB-1, DO-1, 1B1O and EGFR 113 and the colloid silver nitrate (AgNOR) technique. MDM2 and p53 were co-expressed in 28% of cases. A p53-positive/MDM2-negative phenotype was observed in 15% and a p53-negative/MDM2-positive phenotype in 20% of cases. There was a positive correlation of p53 and MDM2 expression with grade and proliferation indices. Univariate analysis in the group of diffuse astrocytomas showed that older age, high histological grade, high PCNA labelling index (LI) and high AgNOR score were associated with reduced overall survival (P < 0.05). p53 LI, Ki-67 LI, AgNOR score, tumour location and grade influenced disease-free survival (P < 0.05), whereas the only parameters affecting post-relapse survival were histological grade and Ki-67 LI (P < 0.1). Multivariate analysis revealed that age, radiotherapy, PCNA LI and p53 LI were the independent predictors of overall survival. p53 LI, Ki-67 LI, MDM2 LI, EGFR LI, grade and type of therapy were independent predictors of disease-free survival, and grade was the only independent predictor of post-relapse survival. Our results indicate that p53 LI and MDM2 LI, EGFR expression as well as proliferation markers (PCNA and Ki-67) are useful indicators of overall and disease-free survival in diffuse astrocytoma patients.     

Evaluation of argyrophilic nucleolar organizer regions in tongue squamous cell carcinomas

Using a silver staining technique, argyrophilic nucleolar organizer regions (AgNORs) were studied on the biopsy specimens taken from 67 tongue squamous cell carcinomas (SCCs), and the relations of the results of AgNORs to the clinicopathological findings, to the labelling indices of proliferating cell nuclear antigen (PCNA LI), and to the outcomes of the disease were investigated. The mean numbers of AgNORs per cell were 1.67 +/- 0.19 (n = 27) in the control squamous epithelia and 3.58 +/- 1.15 (n = 67) in the tongue SCCs, respectively, and a significant difference was found between the two groups (P < 0.0001). Concerning the clinicopathological findings, the mean numbers of AgNORs in the progressive cases (T3, T4), in the factor N-positive groups (N1, N2), and in the advanced stages (stages III, IV) were statistically higher than those in T1, T2, N0, and earlier stage (stages I, II) tumours, respectively. Similarly, a higher value of the AgNOR count was present in the histological grade III or diffuse invasive tumour. There was also a directly significant correlation between the AgNOR counts and the labelling indices of PCNA (r = 0.53, P < 0.0001). Concerning the outcome of the disease, the mean numbers of AgNORs were higher in the group with local recurrence or with lymph-node metastasis. A lower rate of 5 years' survival was found in the high value group (63.5%) of AgNORs compared with that of the low value group (86.0%), with a significant difference between the two groups (P < 0.05). The results suggest that AgNORs may reflect the degree of malignancy and cellular proliferation in tongue SCCs.     

Comparative Immunohistochemical Studies of p53 and Proliferating Cell Nuclear Antigen Expression and Argyrophilic Nucleolar Organizer Regions in Pancreatic Duct Cell Carcinomas

Thirty-eight cases of pancreatic duct cell carcinoma were examined for p53 expression and proliferating cell nuclear antigen (PCNA) by enhanced immunohistochemistry, as well as for changes in numbers of argyrophilic nucleolar organizer regions (AgNORs). Fifteen cases (39.5%) showed p53 overexpression, which tended to increase in proportion to the histopathological grading of malignancy. However, tumor stage and lymph node status were not correlated to p53 overexpression. PCNA labeling index (LI) increased with both histologically malignant grading and pathological stage, but was not correlated with lymph node status. The expressions of p53 and PCNA thus did not necessarily reflect the degree of malignant development. In contrast, AgNOR number showed statistically significant correlations with these three indicators of malignancy. A comparative analysis of p53, PCNA LI and AgNOR number showed overexpression of p53 to be correlated to PCNA LI and essentially unrelated to AgNOR number. The present results thus indicate a close relation between p53 and PCNA, while AgNORs appear to be regulated separately from either of them.     

Proliferative activity as a prognostic factor in Borrmann type 4 gastric carcinoma.

Proliferative activities in 181 primary Borrmann type 4 gastric carcinomas were investigated using percentage labelling of proliferating cell nuclear antigen (PCNA) and an argyrophilic nucleolar organiser region (AgNOR) count. Tumours with a high proliferative activity often metastasised to lymph nodes (P < 0.01), and these patients had a lower survival rate (P < 0.05). A significant correlation was recognised between the PCNA labelling percentage and AgNOR count (r = 0.452, P < 0.001). Cox''s regression analysis showed that PCNA labelling percentage is an independent prognostic factor. These results indicate that estimating proliferative activity may be useful in predicting lymph node metastasis and patients'' prognosis in cases of Borrmann type 4 gastric carcinoma.     

Nuclear DNA content and nucleolar organizer regions in colorectal cancer

The prognostic value of nuclear DNA content and argyrophilic nucleolar organizer regions (AgNOR) is still controversial in colorectal cancer. Sixty patients with colorectal cancer were studied by flow cytometric DNA analysis and AgNOR measurement, and their prognostic significance was tested. DNA index was closely linked to depth of invasion and lymph node metastasis, while AgNOR count did not correlate with such parameters. The survival curve was stongly influenced by depth of invasion, lymph node metastasis, and Dukes' stage but was not affected by DNA ploidy and AgNOR count. These results indicate that neither DNA ploidy nor AgNOR count correlates with survival of patients, although DNA ploidy is linked to progression of colorectal cancer.     

子宫内膜增生与内膜癌增殖细胞核抗原表达及AgNOR计数关联研究

应用抗增殖细胞核抗原（PCNA）的单克隆抗体，以ABC免疫组织化学方法及银染色方法对112例子宫内膜标本进行测定，并与病理学诊断进行对比分析。结果表明：在各种类型的子宫内膜中，PCNA的表达及AgNOR计数依病变的进展明显递增。子宫内膜癌组PCNA表达及AgNOR计数明显高于正常子宫内膜组及子宫内膜增生组（增殖期例外），各组相互比较有显著差异（P＜0．01）。此种方法是测定子宫内膜增生细胞增殖分数的有效手段。对于判断其生物学行为及预后有重要意义，可做为临床判断良恶性子宫内膜增生的指标之一。     

胃粘膜灶性异型增生的形态研究

３９６例活检胃粘膜炎症性病变中１９．９％找见异型增生灶，其由单个至１０多个细胞构成，可向管腔内或外突起。其检出率为糜烂性胃炎的８．０％，浅表性胃炎的１０．９％，萎缩性胃炎的３８．４％，慢性溃疡的１８．９％。灶性异型增生细胞ＰＣＮＡ阳性表达６１．３％；ＡｇＮＯＲ计数３．８１～８．７２个，提示ＰＣＮＡ和ＡｇＮＯＲ检测可帮助判断胃粘膜灶性异型增生。     

Prognosis for gastric-cancer invading the serosa evaluated by argyrophilic nucleolar organizer region staining

Eighty-five patients with gastric cancer invading the serosa following curative resection were analyzed with respect to clinicopathological features and prognosis by staining the argyrophilic nucleolar organizer region (AgNOR). The AgNOR was counted in 200 cancer cells per tissue and the mean number per cell was identified as the AgNOR score. The AgNOR count varied from 1.79 to 5.71, while the mean value was 2.92 +/- 0.74 and the median score was 2.95. The patients were divided into two groups: the AgNOR high group (AgNOR count greater-than-or-equal-to 2.95, n=44) and the AgNOR low group (AgNOR count < 2.95, n=41). There were no differences between the two groups with respect to sex, age, tumor size, location of the tumor, macroscopic appearance, histological differentiation, growth pattern and vascular involvement. The lymphatic advancement of tumor cells was prominent and the rate of lymph node metastasis was higher (p<0.01) in patients in the AgNOR high group. The AgNOR high group had a higher rate of recurrence and also had a variety of recurrence styles. The prognosis of the AgNOR high group was poorer, with statistical significance (p<0.01), while the 10-year survival rate was 35.3% for the AgNOR high group and 77.2% for the AgNOR low group. Therefore, the AgNOR count was found to be closely related to tumor advancement and thus is considered to have a predictive value for the prognosis of patients with gastric cancer invading the serosa after curative resection.     

Prognostic significance of argyrophilic nucleolar organizer staining in soft-tissue sarcomas.

The utility of argyrophilic stain for nucleolar organizer region (AgNOR) for estimating proliferative activity and prognosis of soft-tissue sarcomas (STS) was examined. Formalin-fixed and paraffin-embedded sections of 38 cases with STS were used; the reaction product of AgNOR stain was observed as dots mainly in the nucleoli. The mean number of AgNOR dots per nucleus of tumor cells was calculated in 200 cells (AgNOR count). The AgNOR count, ranging from 1.4 to 16.1 (mean, 7.5), showed a good correlation with cellularity (r = 0.483, p less than 0.003) and histologic grade (r = 0.626, p less than 0.00005), but less shown with mitotic counts (r = 0.350, p less than 0.04). The prognosis of cases with AgNOR low-count group (5-year survival rate was 74.6%) was much better than those in high count group (33.3%) (p less than 0.0005). The AgNOR count correlated well with reactivity of tumor cells for Ki-67 staining, which was available only in freshly prepared sections. These findings suggested that the AgNOR staining is a simple and useful method for estimating tumor-cell proliferation and prognosis of patients with STS.     

Prognostic value of AgNOR counting

The prognostic significance of the argyrophilic nucleolar organizer regions (AgNORs) in tumour pathology is still a matter of debate. A prospective study was performed in a series of renal cell carcinomas to clarify the prognostic value of AgNOR counting. Sections from 21 renal cell carcinomas were stained in 1990 with the method of Ploton. Black dots within the nucleus from 200 tumour cells were counted: the mean AgNOR count for the whole series was 6.13, the median 5.94 and the SD 1.78. Patients were then followed up for at least 6 years or to death: at the time of the survival analysis (June 1996), 13 patients were alive without evidence of recurrence or metastasis, 6 had died of the disease and 2 of myocardial infarction. All the patients with 5.94 AgNORs per cell or fewer were alive at 6-year follow-up, while only 60% of patients with more than 5.94 AgNORs per cell survived (p=0.01). In the multivariate analysis, only AgNOR count (p=0.015) retained an independent prognostic significance. With the limitation due to the small number of cases, this prospective study clearly indicates that AgNOR count has a significant prognostic role, at least in renal cell carcinoma.     

Prognostic values of proliferating cell nuclear antigen (PCNA) and Ki-67 for radiotherapy of oesophageal squamous cell carcinomas.

The relationship of immunohistochemical indices of proliferating cell nuclear antigen (PCNA) and Ki-67 to local control and survival rates for patients with oesophageal squamous cell carcinomas treated by definitive radiotherapy (RT) was investigated. Biopsy materials before RT were obtained from 65 patients with oesophageal cancer. The median PCNA labelling index (LI) and the median Ki-67 LI were 52% and 45% respectively. The PCNA LI was independent of known prognostic factors on local control for oesophageal cancer, although Ki-67 LI correlated with several prognostic factors. In the univariate analysis, patients with the PCNA LI of < 52% or the Ki-67 LI of < 45% showed significantly higher local recurrence rates than those with higher LIs (both P < 0.05). This difference in local control rate according to LIs was prominent for the patients treated with conventional fractionation. In the multivariate analysis, T-stage (P = 0.0056) and PCNA LI (P = 0.0332) were significant factors for local control in the final model using a stepwise regression procedure. In conclusion, PCNA LI and Ki-67 LI were significantly correlated with local control probabilities in oesophageal squamous cell carcinomas treated by definitive RT.     

乳腺癌雌激素受体表达与p53、PCNA、AgNOR表达关系

目的 研究乳腺癌雌激素受体（ＥＲ）表达与ｐ５３,增殖细胞核抗原（ｐｒｏｌｉｆｅｒａｔｉｏｎ ｃｅｌｌ ｍｕｃｌｅａｒ ａｎｔｉｇｅｎ,ＰＣＮＡ）表达和银染核仁组成区（ＡｇＮＯＲ）计数关系。方法 采用ＡｇＮＯＲ染色技术和免疫组化Ｓ－Ｐ法,用抗ＥＲ,抗ｐ５３（ＤＯ－７）及抗ＰＣＮＡ（ＰＣ１０）单克隆抗体,研究５０例乳腺癌ＡｇＮＯＲ计数及ＥＲ,ｐ５３,ＰＣＮＡ表达。结果 乳腺癌３３例（６６％）ＥＲ表达阳     

Hepatoma-derived growth factor is a novel prognostic factor for gastrointestinal stromal tumors

Proliferating activity as mitotic count is generally accepted as a major prognostic indicator for gastrointestinal stromal tumors (GISTs). Hepatoma-derived growth factor (HDGF) is a novel growth factor and elevated in several types of cancer. Our study was designed to elucidate the expression and prognostic role of HDGF in GISTs. A total 178 surgically resected CD117-positive GISTs specimens were collected for immunohistochemical analysis using antibodies against HDGF. The immunoreactivities were scored as labeling index (LI) and correlated with clinicopathologic parameters of GIST patients. The HDGF immunoreactivities were detected in both nucleus and cytoplasm of GISTs tissues. Besides, the nuclear and cytoplasmic HDGF was parallely upregulated in GISTs (p < 0.001). The nuclear HDGF LI were positively correlated with that of PCNA (p < 0.001) and Ki-67 (p < 0.001), tumor mitosis (p < 0.001), tumor sizes (p = 0.007) and NIH risk categories (p < 0.001). In addition, the cytoplasmic HDGF LI were also positively correlated with that of PCNA (p = 0.031) and Ki-67 (p = 0.038), tumor sizes (p = 0.003) and tumor mitosis (p = 0.015). Patients with higher HDGF levels had earlier tumor recurrence and unfavorable outcome (p < 0.05). In addition to standard prognostic factors (NIH risk categories), the nuclear HDGF LI is an independent prognostic factor for disease free and overall survivals of GIST patients after operation. We conclude that HDGF is a novel prognostic factor for GIST patients.     

Tumour angiogenesis and tumour cell proliferation as prognostic indicators in gastric carcinoma.

Tumour growth depends on neovascularisation and tumour cell proliferation. Factor VIII-related antigen (F-VIII RA) localises to vascular endothelium. Expression of proliferating cell nuclear antigen (PCNA) is correlated with cell proliferation. We investigated the correlation between the expression of these antigens and prognosis in gastric carcinoma. A total of 108 specimens resected from patients with gastric carcinoma were investigated by staining with monoclonal antibodies against F-VIII RA and PCNA. Microvessel count (MVC; the mean number of microvessels in the five areas of highest vascular density at 200 x magnification) and PCNA labelling index (PCNA LI; percentage of positive cells in more than 500 tumour cells) were determined. The results showed that prognosis was significantly worse in patients who had a tumour with a high MVC (16 or greater) or a high PCNA LI (42% or greater) than in those patients who had a tumour with a low MVC (less than 16) or a low PCNA LI (less than 42%). Furthermore, MVC was significantly associated with the risk of hepatic recurrence. In conclusion, both MVC and PCNA LI may be good prognostic indicators in patients with gastric carcinoma.     

Predictive value of Ki-67 and argyrophilic nucleolar organizer region staining for lymph node metastasis in gastric cancer

Proliferative activities in 91 primary gastric carcinomas and 36 corresponding metastatic perigastric lymph nodes were investigated using Ki-67 labeling percentage and an argyrophilic nucleolar organizer region (AgNOR) count. Tumors with a high proliferative activity often metastasized to lymph nodes, and the proliferative activities of the primary lesion and the perigastric lymph node metastases were similar. A significant correlation was recognized between the Ki-67 labeling percentage and the AgNOR count (r = 0.744; P less than 0.001). The Ki-67 labeling percentage and AgNOR count proved to be useful predictors of nodal metastasis regardless of tumor size, depth of invasion, and histological type. Even when tumors are smaller (less than 7 cm) or the stage of the disease is early (pT1, 2), the formation of metastasis increased with an increased Ki-67 labeling percentage or AgNOR count. The combination analysis of depth of invasion with Ki-67 labeling percentage or AgNOR count gives a more precise prediction of nodal metastasis, compared with histological analysis alone.     

Down-regulation of tumor suppressor gene PTEN, overexpression of p53, plus high proliferating cell nuclear antigen index predict poor patient outcome of hepatocellular carcinoma after resection

We aimed to evaluate the interaction of two tumor suppressor genes PTEN and p53 and their relationship with cell cycle protein proliferating cell nuclear antigen (PCNA) in hepatocellular carcinoma (HCC). A total of 124 resected HCC paraffin specimens were collected from 1987 to 1999 for immunohistochemistry. Expression of PTEN, p53 and PCNA in HCC were analyzed for clinicopathologic correlation. The study revealed decreased or absent PTEN immunostaining (PTEN down-regulation) in 42.7% and positive p53 (p53+) immunostaining in 41.9% of HCC. There was a positive correlation between PTEN down-regulation and p53 (+) (P=0.001). PTEN down-regulation or p53 (+) correlated with increased HCC dedifferentiation, advanced pathologic stages and high PCNA labeling index (LI) of tumors (P<0.05). Patients with either PTEN down-regulation, p53 (+), or high PCNA LI had shorter survival and higher recurrence rates than patients with intact PTEN expression, p53 (-), or low PCNA LI respectively (P<0.05). By combining the three genes, patients with all PTEN down-regulation (+)/p53 (+)/high PCNA LI had the shortest overall survival (P<0.001) and the highest recurrence rates (P<0.001), followed by patients with two, one and none of three events accordingly. Combination of PTEN/p53/PCNA represented an independent prognostic factor for tumor recurrence and disease-specific survival (P<0.05). In conclusion, the down-regulated PTEN expression and p53 over-expression are involved in the pathogenesis of HCC. They correlate with high PCNA expression, HCC de-differentiation and advanced HCC stages. A combination of the three genes predicts patient outcome more powerfully than any of the individual genes.