PCNA/AgNOR and Ki-67/ AgNOR double staining in oral squamous cell carcinoma

This study was performed on oral squamous cell carcinomas (OSCC) in order to investigate the relation between the number of interphase silver-stained nucleolar organizer regions (AgNORs) and the immunolabeling of proliferation-associated markers, using antibodies to Ki-67 and proliferating cell nuclear antigen (PCNA). Fifteen consecutive cases of oral squamous cell carcinoma were used and a double staining technique was performed in order to quantify the number of NORs in PCNA-positive and -negative cells as well as in Ki-67-positive and -negative cells. Our results showed a higher mean number of AgNORs in PCNA- and Ki-67-positive cells than in PCNA- and Ki-67-negative cells. We concluded that there is an association between cell proliferation and AgNOR score in OSCC.     

增殖细胞核抗原和Ki-67抗原在涎腺癌中的表达

目的:探讨细胞增殖核抗原和Ki-67抗原在不同类型涎腺癌中的表达及意义。方法:采用SABC和LSAB免疫组化法分别检测PCNA和Ki-67在17例粘液表皮样癌、12例腺样囊性癌、9例恶性多形性腺瘤中的表达和分布。结果:1)PCNA及Ki-67的增殖指数即PI值在粘液表皮样癌和腺样囊性癌的不同分级中表达不同,且有统计学意义(P<0.005)。2)恶性多形性腺瘤PCNA及Ki-67的PI值较高,与腺样囊性癌及粘液表皮样癌相比差异有统计学意义(P<0.001)。3)PCNA及Ki-67的表达水平与涎腺癌的生物学行为(复发和转移)明显相关。结论:涎腺癌中PCNA和Ki-67的表达水平与细胞分化有关,其PI值可作为粘液表皮样癌和腺样囊性癌的组织学分级的重要参数,也是诊断恶性多形性腺瘤的重要指标。PCNA和Ki-67的高表达预示着涎腺癌预后的不良。     

Correlation of proliferative markers (Ki-67 and PCNA) with survival and lymph node metastasis in oral squamous cell carcinoma: a clinical and histopathological analysis of 113 patients

The purposes of this study were to examine the correlations between proliferation markers and survival rate in oral squamous cell carcinoma (OSCC) patients, and to evaluate the efficacy of proliferation markers in predicting lymph node metastasis. The patients' age, gender, T score, clinical stage, PCNA and Ki-67 index were analysed. Univariate analysis showed that T score had a significant influence on survival, and stage 4 group had a significantly lower survival rate. Lymph node metastasis was also a significant predictor of survival. Using a cut-off point of 25%, those patients with lower Ki-67 scores had survival advantage over those with higher Ki-67 scores. PCNA did not show any differences in survival with a cut-off point of 50%. Ki-67 and PCNA were significantly higher in the primary tumours associated with lymph node metastasis (pN+) than in those without lymph node metastasis (pN0). Multivariate analysis showed that clinical stage and Ki-67 were independent prognostic factors for survival in OSCC patients. From this result, it can be postulated that the cancer staging based on the TNM stage was a powerful prognostic variable and Ki-67 had a significant effect on the cumulative survival rate.     

Cell proliferation markers in the odontogenic keratocyst: effect of inflammation

The immunohistochemical expression of PCNA and Ki-67 proteins and the histochemical expression of AgNORs were studied in 20 odontogenic keratocysts in order to assess the relationship between epithelial cell proliferation and inflammation within the capsule. Immunostained cells were quantified by conventional methods, and both quantitative and morphometric analyses of AgNORs were performed by TV image analysis. Non-inflamed odontogenic keratocysts showed a typical epithelial lining and inflamed odontogenic keratocysts were lined also by hyperplastic non-keratinized stratified squamous epithelium. A statistically significant increase of PCNA+ and Ki-67+ cells and of AgNOR numbers was detected in the linings of inflamed odontogenic keratocysts compared to non-inflamed lesions. The results suggest the existence of greater proliferative activity in the epithelial cells of inflamed odontogenic keratocysts, which may be associated with the disruption of the typical structure of odontogenic keratocyst linings.     

Correlation between ploidy status using flow cytometry and nucleolar organizer regions in benign and malignant epithelial odontogenic tumors

ObjectiveDifferentiation between the aggressive benign odontogenic tumors and their malignant counterparts is controversial and difficult. While flow cytometry (FCM) allowed DNA analysis in neoplasia, argyrophilic organizer regions (AgNORs) number and/or size in a nucleus are correlated with the ribosomal gene activity and therefore with cellular proliferation. The aim of this research was to study the diagnostic accuracy of FCM and AgNORs staining in differentiating between benign and malignant epithelial odontogenic tumors and to correlate between these two interventions.DesignSixteen benign cases [8 cases of ameloblastoma (AB) and 8 cases of keratocystic odontogenic tumor (KCOT)] and 13 malignant epithelial odontogenic tumors [8 cases of ameloblastic carcinoma (ABC) and 5 cases of clear cell odontogenic carcinoma(CCOC)] were included in the current study. For FCM analysis, a single cell suspension from Formalin fixed paraffin-embedded (FFPE) tumors was prepared according to a modified method described by Hedley (1989) and AgNORs staining were performed in accordance to the Ploton protocol (1986). Analysis of AgNORs was performed using both quantitative and qualitative methods.ResultsThe work revealed that all the examined tumors were diploid, except for 40% of CCOC cases. The S-phase fraction (SPF) value, AgNORs count and AgNORs area/cell showed statistically significant difference on comparing benign and malignant groups. A weak positive correlation was observed between SPF and AgNORs count.ConclusionThe SPF value was considered to be more sensitive and specific in differentiation between aggressive benign and malignant epithelial odontogenic tumors in comparison to AgNORs counting.     

PCNA and Ki-67 immunoreactivity in multinucleated cells of giant cell fibroma and peripheral giant cell granuloma

Immunohistochemical investigation of PCNA and Ki-67, two diverse nuclear proteins essential to the cell cycle, was undertaken in archival, formalin-fixed and paraffin-embedded specimens of giant cell fibroma (GCF) and peripheral giant cell granuloma (PGCG). GCF multinucleated cell nuclei were mostly PCNA⁺, although there was variability in staining intensity. This indicates heterogeneity in nuclear PCNA metabolism of GCF multinucleated cells, and it is possible that the most intensely stained nuclei have passed through the cell cycle more recently compared to the less immunoreactive nuclei. However, the absence of Ki-67 immunoreactivity in GCF multinucleated cells, and absence of mitoses in GCF multinucleated cells, suggests that cell cycling in the absence of cytokinesis is not involved in GCF multinucleated cell formation. Alternatively, GCF multinucleated cells possibly form by fusion of mononuclear cells previously identified as fibroblasts, although this theory cannot be confirmed by the data presented in this study, and the histogenesis of GCF multinucleated cells remains unclear. In contrast, absence of either PCNA or Ki-67 immunoreactivity in PGCG multinucleated cells is consistent with an osteoclast lineage and formation from differentiated mononuclear cells.     

增殖细胞核抗原和Ki-67抗原在黏液表皮样癌中的表达

目的:检测细胞增殖核抗原(PCNA)和Ki-67抗原在黏液表皮样癌中的表达,探讨其表达量与粘液表皮样癌分级的相关性及意义。方法:对17例黏液表皮样癌的临床病理特点进行回顾性分析,采用SABC和LSAB免疫组化法分别检测PCNA和Ki-67在17例黏液表皮样癌的表达和分布。结果:17例黏液表皮样癌中,高分化型11例(其中1例转移)。低分化型6例(其中4例转移)。PCNA及Ki-67的增殖指数(PI值)在黏液表皮样癌中,低分化型明显高于高分化型(P<0.001)。在发生转移的黏液表皮样癌组中,PCNA及Ki-67的表达水平均明显高于无转移组。结论:黏液表皮样癌中PCNA和Ki-67的表达水平与细胞分化有关,其PI值可作为组织学分级的重要参数。PCNA和Ki-67的高表达预示着黏液表皮样癌预后的不良。     

基于局部可变阈值的无监督定量Ki-67细胞增殖指数模型的初步建立

目的: 开发一种高效率基于Ki-67细胞增殖指数的自动定量模型。方法: 选择口腔鳞癌组织切片进行Ki-67免疫组织化学标记。通过全玻片成像技术获得数字化彩色病理图像,采用基于局部统计的可变阈值方法将图像中的无关浅色背景区域去掉,保留胞核部分。在L^*a^*b^*颜色空间中,采用阈值方法对蓝色（苏木精染色）和棕色（Ki67染色）进行分离,计算增殖指数。采用SPSS 24.0软件包中的配对t检验和Spearman相关系数与人工计数和机器学习中常用的颜色反卷积方法进行比较分析。结果: 建立了一种新的胞核检测和分类模型,可以处理不同大小的病理图像,并有效检测出免疫组织化学阳性细胞和阴性细胞,结果与人工计数结果无显著差异（P>0.05）,但计算速度较快,在处理较大尺寸图像时计算效率优势更加明显,且检测结果优于常用的颜色反卷积方法（P<0.05）。结论: 本研究开发的胞核自动定量分析模型可高效分析口腔鳞癌细胞核Ki-67染色情况,计算相应增殖指数,辅助病理诊断。     

PCNA, Ki-67 and p53 expressions in submandibular salivary gland tumours

Salivary gland tumours are uncommon with a broad heterogeneity. The most common benign tumour is the pleomorphic adenoma, whereas mucoepidermoid carcinoma and adenoid cystic carcinoma predominate among the malignancies. Most salivary gland tumours occur in the parotid, and consequently clinical and biological data are normally derived from this site. This work describes the expressions of PCNA, Ki-67 and p53 in 15 pleomorphic adenomas, 15 mucoepidermoid carcinomas and 15 adenoid cystic carcinomas of the submandibular gland. Our results showed that all pleomorphic adenomas were negative for p53 and Ki-67 with 66.6% being positive for PCNA. Conversely, p53 was positive in 53% of the mucoepidermoid carcinomas and in 20% of the adenoid cystic carcinomas. Ki-67 was expressed in 47.7% of the mucoepidermoid carcinomas and 40% of the adenoid cystic carcinomas. All malignant tumours were positive for PCNA. These results indicate that the proliferative rate analysed with PCNA and Ki-67 and the expression of p53 in pleomorphic adenoma and adenoid cystic carcinoma of the submandibular gland were similar to those described in the parotid and minor salivary glands. However, mucoepidermoid carcinomas showed higher expression of these markers than those of other salivary glands. This work is the first describing the expression of these immunohistochemical markers exclusively in submandibular salivary gland tumours.     

口腔鳞癌组织中Ki-67和p53蛋白的表达

目的 探讨口腔鳞癌组织中Ki-67和p53蛋白的表达及其与临床病理特征的关系。方法采用免疫组织化学S-P法对10例正常口腔黏膜组织、16例口腔白斑（OLK）组织、48例口腔鳞癌（OSCC）组织中的Ki-67和p53蛋白表达进行检测,结合患者临床病理资料进行分析,使用SPSS17.0 软件包对数据进行统计学处理。结果Ki-67蛋白在正常口腔黏膜组织、口腔白斑和口腔鳞癌组织中的阳性表达率分别为30.0%、56.3%和79.2%;p53的阳性表达率分别为0.0%、43.8%和70.8%,Ki-67和p53在正常黏膜组与口腔白斑和口腔鳞癌组差异均具有显著性（P<0.05）;Ki67蛋白在口腔鳞癌组织中的表达与肿瘤的临床分期、分化程度、有无淋巴结转移有关（P<0.05）,p53蛋白的表达与肿瘤的分化程度有关(P<0.05);Ki-67和p53蛋白在口腔鳞癌组织中的表达呈正相关（P<0.05）。结论Ki-67和p53蛋白在口腔鳞癌组织中高表达,可能在口腔鳞癌的发生、发展过程中起着重要作用。     

Analysis of cell proliferation and pattern of invasion in oral squamous cell carcinoma

Cell proliferation markers play an important role in the biological behavior of neoplasms. This study investigated the immunohistochemical expression of PCNA, Ki-67 and Cyclin B1 proteins based on the pattern of cell invasion in oral squamous cell carcinoma (OSCC). A total of 39 OSCC specimens and 13 samples of normal oral mucosa (control) were immunohistochemically analyzed. Protein expression was evaluated according to World Health Organization - Histological Malignancy Grading (WHO-HMG) and a specific grading system for invasion, graded from 1 to 4, varying from a consistently well-defined "pushing" border to diffuse infiltration and cellular dissociation, and was then correlated with clinical features. We found higher expression of Ki-67 and Cyclin B1 in OSCC when compared with the control group. High Ki-67 expression levels were more commonly seen in the floor of the mouth than in the tongue (P = 0.009). Cyclin B1 showed a positive correlation with histological grade, according to WHO-HMG criteria (P = 0.01). Our results suggest that Cyclin B1 is a reliable proliferation marker for indicating degree of tumor proliferation. Correlations between PCNA, Ki-67, Cyclin B1 and invasive tumor front with overall survival were not observed. Further studies are needed in order to elucidate whether cell proliferation activity at the tumor invasion front is related to prognosis.     

成釉细胞瘤肿瘤细胞巢周边及中心区细胞增殖和分化的研究

目的　研究成釉细胞瘤肿瘤细胞巢周边及中心区细胞增殖和分化的特点。方法　选取43例成釉细胞瘤 ,包括滤泡型 15例、丛状型 2 0例和棘皮瘤型 8例。应用免疫组化SP方法检测标本组织中增殖细胞核抗原 (proliferatingcellnuclearantigen ,PCNA)、Ki 6 7、CK10&13和CK19蛋白的表达。结果　PCNA在肿瘤细胞巢周边区的增殖指数 (5 12± 2 76 ) %显著高于中心区 (1 36± 1 0 2 ) % ,P <0 0 0 1;Ki 6 7在肿瘤细胞巢周边区的增殖指数 (3 6 3± 1 80 ) %显著高于中心区 (1 2 6± 0 96 ) % ,P <0 0 0 1;PCNA和Ki 6 7的增殖指数有显著相关关系 (P <0 0 1) ;CK10&13和CK19在成釉细胞瘤中心区细胞表达显著高于周边区 (P <0 0 1)。结论　成釉细胞瘤肿瘤细胞巢周边区为增殖细胞区 ,周边区和中心区细胞的分化程度不同。     

增殖细胞核抗原和Ki67在口腔癌浸润前沿表达及意义

目的:探讨口腔黏膜癌浸润前沿分级、细胞增殖核抗原和Ki67抗原在浸润前沿中的表达及意义.方法:对30例原发口腔黏膜癌进行分级,采用免疫组化SP法分别检测浸润前沿和中心PCNA、Ki67的表达.结果:口腔黏膜癌浸润前沿(ITF)和非前沿WHO(1997)分级差异有显著性(P<0.05);浸润前沿PCNA(P<0.01)、Ki67(P<0.05)的表达均高于非前沿部分,差别有显著性;浸润前沿PCNA标记指数(LI)(P<0.05)与浸润前沿分级(IFG)总分呈正相关关系.结论:口腔黏膜癌浸润前沿肿瘤细胞分化较差,增殖活性明显高于非前沿部分.在浸润前沿中PCNA和Ki67的高表达可以反映肿瘤的高恶性度及高复发转移倾向.     

Reduced expression of nuclear factor kB in oral mucosa undergoing preoperative chemoradiotherapy

Radiotherapy as well as chemotherapy in head and neck cancer induces severe oral mucositis. Even after healing of the mucositis, however, the oral mucosa looking atrophic is known to be susceptible to injury and infection. In order to investigate such vulnerability of mucosa, we immunohistochemically studied the expressions of Ki-67, proliferating cell nuclear antigen (PCNA), cyclin D₁, nuclear factor (NF)-kB, and keratinocyte growth factor (KGF) receptor in the oral mucosal keratinocytes undergoing preoperative concurrent chemoradiotherapy for oral cancer, compared with those of the oral mucosa without such therapy. As a result, the expressions of Ki-67, PCNA, and cyclin D₁ were decreased in the chemoradiotherapy-treated oral keratinocytes. Interestingly, NF-kB expression, which is known to be enhanced in oral mucositis, was reduced after chemoradiotherapy. The chemoradiotherapy had no effect on the expression of KGF receptor in oral keratinocytes. In conclusion, the vulnerability of oral mucosa undergoing chemoradiation may be associated with reduced NF-kB expression and impaired growth activity.     

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目的    探讨P27kipl和Ki-67在口底鳞状细胞癌中的表达水平及其与肿瘤分化程度、淋巴结转移、预后的关系。方法    收集枣庄矿业集团中心医院病理科1998年1月至2004年12月的口底鳞状细胞癌手术根治标本蜡块及其相应的癌旁正常黏膜组织（距肿物1.5 cm）蜡块各45例，应用免疫组织化学染色S-P方法进行P27kipl、Ki-67基因蛋白的检测。结果    癌旁正常黏膜中，P27kipl高表达率为75.56%、Ki-67阳性表达率为8.89%，口底鳞状细胞癌中P27kipl高表达率为22.22%、Ki-67阳性表达率为35.56%，经比较两组间差异均有统计学意义（均P＜0.01）。口底鳞状细胞癌分化较好者，P27kipl高表达率（26.92%）高于分化较差者（15.79%），但差异无统计学意义（P＞0.05）；Ki-67阳性表达率在分化较好者（23.08%）与较差者（52.63%）间差异有统计学意义（P＜0.05）。P27kipl高表达率、Ki-67阳性表达率在有、无淋巴结转移中比较，差异均有统计学意义（均P＜0.05）。P27kipl高表达率、Ki-67阳性表达率在术后存活期中比较，差异亦均有统计学意义（均P＜0.05）。结论    P27kipl、Ki-67在口底鳞状细胞癌中起重要的调控作用，并是判断其预后的重要参考指标。     

Quantitative analysis of argyrophilic nuclear organizer regions in giant cell lesions of jaws

J Oral Pathol Med (2010) 39 : 431–434 Background: Giant cell lesions of the jaws are considerably similar according to histopathologic characteristics yet show different clinical behaviors. These lesions include central giant cell granuloma (CGCG), aneurysmal bone cyst, Cherubism, and Brown tumor associated with hyperparathyroidism. The present study aimed to investigate AgNORs count in these lesions as a proliferative marker and to determine whether it can be used to discriminate between them or not. Methods: Forty‐one cases of giant cell lesions of jaws were retrived from Oral Pathology Department (1987–2007). They included 21 cases of CGCG, eight cases of aneurysmal bone cyst (ABC), six cases of Cherubism, six cases of Brown tumor. The mean AgNORs count was calculated for all cases. To compare mean AgNORs in groups of lesions, ANOVA test was performed. Results: Mean AgNOR counts were: (0/85 ± 0/29) in CGCG, (0/76 ± 0/32) in ABC (0/87 ± 0/10) in Cherubism and (0/82 ± 0/16) in Brown tumor. A significant difference was not observed in AgNOR counts among these groups of lesions. Conclusions: Jaws giant cell containing lesions have no acceptable differences in mean AgNORs.     

Evaluation of p53, PCNA, Ki-67, MDM2 and AgNOR in oral peripheral and central giant cell lesions

OBJECTIVES: Peripheral giant cell lesion (PGCL) and central giant cell lesion (CGCL) of the jaws have a distinct clinical behaviour. Whether such biological differences are supported by a distinct pattern of proliferation markers or cell cycle associated proteins expression is not known. Therefore the purpose of the present study was to compare the immunohistochemical expression of p53, MDM2, Ki-67, PCNA and the histochemical expression of argyrophilic nuclear organiser region (AgNOR) on PGCL and CGCL of the jaws. MATERIALS AND METHODS: Paraffin wax blocks of 14 cases of PGCL and 12 cases of CGCL were retrieved. A biotin-streptavidin amplified system was used for identification of the antigens. The AgNOR number was also evaluated. RESULTS: Ki-67 immunoreactivity was greater in the mononuclear cells of PGCL compared to CGCL. PCNA and AgNOR staining were similar in PGCL and CGCL. Prominent MDM2 immunoreactivity was observed in all tissues investigated. By contrast, there was no p53 immunoreactivity. CONCLUSIONS: Although CGCL present a more aggressive clinical behaviour, it has a decreased proliferative activity compared to PGCL. Finally, p53, MDM2, PCNA, Ki-67 immunohistochemical expression and AgNOR histochemical expression do not reflect their distinct biological behaviour.     

The relationship of the histologic grade at the deep invasive front and the expression of Ki-67 antigen and p53 protein in oral squamous cell carcinoma

BACKGROUND: Although many histopathologic characteristics of oral squamous cell carcinoma (O-SCC) have been identified as prognostic factors, accurate, and unequivocal factors have not been clearly identified. The purpose of this study was to evaluate a potential association between the histologic grade of malignancy at the deep invasive front and the expression of Ki-67 antigen and p53 protein in O-SCC. METHODS: The expression of Ki-67 antigen and p53 at the invasive tumor front area of O-SCC was examined by immunohistochemistry of archived tissue from 62 cases. The mean age of patients was 60.7 years (range: 37-89) and the male-female ratio was 1.6:1 (38 men, 24 women). There were 20, 17, 14, and 11 cases classified as stage I to stage IV, respectively. The correlation between the intensity of immunostaining for Ki-67 antigen and p53 and the histologic grade of malignancy at the deep invasive front (invasive front grade, IFG) was analyzed. The expression of Ki-67 antigen and p53 in normal oral epithelia (10 cases) was also investigated. RESULTS: The mean Ki-67 labeling index (LI) in the O-SCC samples was 32.8 +/- 12.0% (n = 62). The mean total score of IFG (IFG score) was 9.1 +/- 2.7 points (n = 62). There was a significant linear correlation between the IFG score and the Ki-67 antigen (gamma = 0.651, R2 = 0.596, P < 0.0001). Of 50 tumors examined, 27 (54.0%) exhibited p53-positive nuclear immunostaining. The staining patterns for Ki-67 antigen and p53 were similar. Both Ki-67-LI and p53-positive status were significantly correlated with the IFG scores. CONCLUSION: The findings of this study demonstrate that overexpression of Ki-67 antigen and p53 at the deep tumor invasive front of O-SCC is associated with histologic grade of malignancy.     

Aberrant expression of cyclin A and cyclin B1 proteins in oral carcinoma

Cyclins play an important role in regulating the passage of dividing cells through critical checkpoints in cell cycle. Aberrant exprssion of cylcin proteins has been found in a number of human cancers, including carcinomas of the head and neck, where amplification of the cyclin D1 gene is a common finding. The obective of this study was to examine cell cycle kinetics in oral carcinomas by determining the expression the S phase protein cylcin A and the M phase protein cyclin B1. Routinely processed tissue sections of 50 oral squamous cell carcinomas from the floor of the mouth were stained by immunohistochemistry for cyclin A, cyclin B1 and Ki-67 proteins. Ten specimens of normal epithelium from the floor of the mouth were used as controls. Teh number of cells showing nuclear staining for cylin A, cyclin B1 and Ki-67 proteins was determined by computer image analysis. There were 17 well-differentiated, 25 moderately differentiated and 8 poorly differentiated tumours. Mean counts for cylcin A (29.50 ± 4.10, Mean ± 95% CI), cyclin B1 (2.05 ± 0.30) and Ki-67 (49.46 ± 5.91) protiens in the carcinomas were significantly higher than counts for the normal epithelial controls (cyclin A: 930 ± 1.72; cyclin B1: 1.01 ± 0.36; Ki-67: 17.4. ± 4.17). For cyclin A, cyclin B1 and Ki-67, mean staining scores fosr all tumour grades were significantly higher than controsl. There was a strong correlation between ki-67 and cyclin A scores in all tumour groups (r²=0.68); however, the correlations between cyclin B1 and Cyclin A Scores(r²=0.35) and between clylcin B1 and ki-67 scores (r²=0.39) were weak. We conclude that there is overexpression of cyclin A and cyclin B1 proteins in oral carcinoma. Furthermore, the poor correlations for cylcin B1 scores with other cell cycle indices suggest that there may be aberrant cell cycle progression at G2/M checkpoint in oral carcinomas.