Immunohistochemical expression of p27kip1 in metastatic laryngeal squamous cell carcinoma

PurposeLaryngeal squamous cell carcinoma (LSCC) is an interesting diagnostic and therapeutic issue. The diagnostic delay is mainly a consequence of the lack of evident symptoms in the early stage of the disease. The purpose of current studies was the evaluation of the expression of p27^kip1 in primary and metastatic LSCC in correlation with patients’ clinicopathological data.Material/methodsThe indirect immunohistochemical studies were performed on the series of 60 sections (primary tumor: 20 cases of N(0) and 20 cases of N(+), and nodal meta: 20 cases), using primary antibody against p27^kip1 [clone 1B4]. The expression of analyzed protein was performed using automated morphometric methods.ResultsThe p27^kip1 nuclear expression was found in 100% (40/40) cases of primary tumor, and in 85% (17/20) cases of SCC meta at lymph nodes. In primary LSCC N(0) the expression of p27^kip1 was significantly higher compared to N(+) cases (p = 0.036672). However, the p27^kip1 expression in SCC metastases was higher compared to the primary SCC.Moreover, the analyses based on the classification trees revealed the cutoff p27^kip1 expression in primary LSCC (IRS ≤ 76) which was characteristic for N(+) patients. Consequently, our analysis revealed that high expression of p27^kip1 (IRS > 76) was characteristic for N(0) patients.ConclusionsOur results suggest that p27^kip1 might be useful prognostic factor of metastatic potential in laryngeal squamous cell carcinoma.     

肾癌中cyclinD1和p27kip1的表达及其意义

目的探讨cyc lin D1、p27k ip1在普通型肾细胞癌(renal cell carc inom a,RCC)发生、发展中的作用。方法用半定量RT-PCR方法检测25例普通型RCC和10例肿瘤远端的正常肾组织中cyc lin D1的mRNA含量,用免疫组化方法检测76例普通型RCC中cyc lin D1和p27k ip1蛋白的表达,并对cyc lin D1、p27k ip1蛋白表达与临床病理参数的关系进行分析。结果普通型RCC中cyc lin D1的mRNA含量0.488±0.399,高于正常对照组0.089±0.066(P<0.01)。cyc lin D1的表达与肿瘤体积大小有关,体积大者cyc lin D1高表达(P<0.05)。普通型RCC中p27k ip1表达低于正常对照组,随着p27k ip1表达的降低,肿瘤的细胞核Fu-hrm an分级、TNM分期增高。结论cyc lin D1高表达和p27k ip1的低表达与普通型RCC的发生有关;p27k ip1的低表达可能促进肿瘤的演进,p27k ip1的表达可作为评价普通型RCC预后的参考指标。     

Aberrant p27kip1 expression in endocrine and other tumors.

The p27kip1 (p27) gene encodes an inhibitor of cyclin-dependent kinase activity. The expression of p27 protein in normal and neoplastic tissues was investigated by immunoblotting and immunohistochemistry. Immunoblotting studies detected a 27-kd protein band that was decreased in neoplastic pituitary tissues compared with normal pituitary. Immunostaining of 177 tissues showed abundant expression of p27 protein in normal tissues with decreased numbers of immunoreactive cells in adenomas and carcinomas in both endocrine and nonendocrine tissues. p27 expression was inversely related to the proliferation marker Ki-67 antigen detected with monoclonal antibody MIB-1. Parathyroid adenomas and hyperplasias had similar Ki-67 labeling indices; however, hyperplasias had threefold more p27-positive cells than parathyroid adenomas, suggesting that p27 immunostaining may be useful in distinguishing between these two conditions. These results indicate that there is widespread aberrant p27 expression in hyperplastic tissues and in benign and malignant neoplasms compared with normal tissues. Immunohistochemical analysis of p27 along with Ki-67 may be used to assess the biological behavior of various neoplasms, to classify hyperplastic and neoplastic tissues, and to study cell cycle regulation during tumor progression.     

Cyclin D1 and p27 expression as prognostic factor in papillary carcinoma of thyroid: association with clinicopathological parameters

Aim

To determine the prognostic value of cell cycle regulators cyclin D1 and p27 for papillary thyroid carcinomas.

Methods

Analysis included 180 patients with papillary thyroid carcinoma who underwent surgery at Split University Hospital Center between 1999 and 2001. Clinical data were obtained from clinical charts and histopathology reports. Immunohistochemistry was performed on formalin-fixed, paraffin-embedded tissue by antibody p27 and cyclin D1. Quantification was based on the intensity and distribution of nuclear staining.

Results

Univariate analysis showed that sex (P = 0.019) and capsular invasion (P = 0.010) were significant predictors of lymph node metastases, whereas age (P = 0.96), histopathological variant (P = 0.075), size (P = 0.556) and multifocality (P = 0.131) were not. Univariate analysis also showed that overexpression of cyclin D1 (P < 0.001) and underexpression of p27 (P < 0.001) predicted lymph node metastases in papillary thyroid carcinomas. There was a significant correlation between cyclin D1 (P = 0.024) and p27 (P = 0.029) expression in two prognostic groups of low and high risk. Low risk group was cyclin D1 negative and p27 positive, whereas high risk group was cyclin D1 positive and p27 negative. Multivariate analysis confirmed that sex (P = 0.041), capsular invasion (P = 0.027), and p27 (P < 0.001) were strong independent predictors of lymph node metastases in the high-risk group.

Conclusions

Immunohistochemical analysis of p27 expression may be a valuable tool for identifying risk of lymph node metastases and more aggressive behavior of papillary thyroid carcinoma.Papillary thyroid carcinoma is the most common endocrine malignancy (1). It has a low mortality rate – the overall 10 years survival rate being about 95%, but a significant number of patients present with aggressive disease. This is why it is of utmost importance to evaluate the prognostic factors and identify high-risk patients (1). A few studies have described the factors influencing prognosis and long term outcome of thyroid cancer (2,3). Some authors proposed lymph node metastasis as a prognostic factor, but many disagree with it (4). It is still unclear whether lymph node metastasis depends on the same factors that influence survival (5), such as age, sex, tumor size, tumor histopathological variant, tumor focality, and extrathyroidal extension.Cell cycle kinases also play an important role in metastazing of thyroid carcinoma. The cell cycle is controlled by cyclin-dependent kinases (CDK), which are activated by forming complexes with cyclins. Cyclin D1 gene, located on chromosome 11q23, is a positive regulator of the cell cycle. It encodes a nuclear protein that forms complexes with CDKs 4 and 6, which phosphorylate and inactivate the retinoblastoma protein. This allows cell cycle progression from G1 to S phase (6).Tumor suppressor gene p27 is located on the chromosome 12p13. It encodes the CDK-inhibiting nuclear protein and inhibits the formation of cyclinD1/cdk complexes during G0 and early G1 phases of the cell cycle. This inhibits inactivation of the phospho-retinoblastoma protein and prevents G1 to S phase transition (6).Normal thyroid cells do not show nuclear immunoreactivity to cyclin D1, but do show strong immunoreactivity to p27 (7). Cyclin D1 overexpression and p27 underexpression were found in a variety of tumors and were associated with increased tumor aggressiveness, incidence of lymph node metastases, and poorer prognosis (8-10).In the present study, we tested the hypothesis that cyclin D1 overexpression and p27 underexpression would allow us to identify the subgroup of papillary carcinomas with a potential for metastatic behavior.     

stathmin和P27kip1在大肠癌中的表达及意义

目的:探讨stathmin蛋白与p27kip1蛋白在大肠癌组织中的表达及意义.方法:应用免疫组织化学SABC法检测25例正常大肠黏膜组织、25例大肠腺瘤组织、47例大肠癌组织中stathmin蛋白及p27kip1蛋白的表达情况.结果:①stathmin蛋白在正常大肠黏膜组织、大肠腺瘤组织及大肠癌组织中的阳性表达率分别为20％、48％、74.47％;正常大肠黏膜组分别与大肠腺瘤组及大肠癌组比较,差异均有统计学意义(P＜0.05);大肠腺瘤组与大肠癌组比较,差异亦有统计学意义(P＜0.05):stathmin蛋白的表达与肿瘤的分化程度、有无淋巴结转移及TNM分期显著相关(P＜0.05).②p27kap1蛋白在正常大肠黏膜组织、大肠腺瘤组织及大肠癌组织中的阳性表达率分别为92％、80％、31.91％.正常大肠黏膜组与大肠癌组比较,差异有统计学意义(P＜0.05);大肠腺瘤组与大肠癌组比较,差异亦有统计学意义(P＜0.05);正常大肠黏膜组与大肠腺瘤组比较,差异无统计学意义(P＞ 0.05);p27kip1蛋白的表达与肿瘤的分化程度及淋巴结转移有关(P＜0.05).③stathmin蛋白的表达与p27kip1蛋白的表达呈负相关(r=--0.695 3,P＜0.01).结论:stathmin蛋白在大肠癌组织中高表达,其表达程度与肿瘤的分化程度、淋巴结转移及TNM分期显著相关,p27kip1蛋白在大肠癌组织中低表达,其表达程度与肿瘤的分化程度及淋巴结转移显著相关,提示stathmin及p27kip1蛋白共同参与了大肠癌的发生、发展;stathmin蛋白可作为一种判断大肠癌恶性程度及侵袭转移的生物学指标.     

Expression of keratin 19 distinguishes papillary thyroid carcinoma from follicular carcinomas and follicular thyroid adenoma

Keratin expression with the use of chain-specific monoclonal antikeratin antibodies was investigated in normal thyroid tissue (n = 4), colloid nodules (n = 19), follicular thyroid adenomas (n = 18), follicular carcinomas (n = 10), and papillary carcinomas (n = 12). Frozen sections were stained with monoclonal antibodies M20 (keratin 8), M9 (keratin 18), and LP2K (keratin 19) with the use of the indirect immunoperoxidase technique. The immunohistochemical findings showed that the expression of keratins 8 and 18 was equally extensive in all normal, benign, and malignant lesions tested. In contrast, different staining patterns were observed with the use of monoclonal antibody to keratin 19. Follicular carcinomas were only focally stained with this antibody or were not reactive at all. Keratin 19, however, was present in all the tumor cells of papillary tissues and in a moderate amount of cells of nonneoplastic thyroid lesions and follicular adenomas. In papillary carcinoma, an identical homogeneous expression of keratin 19 was observed in both papillary and follicular structures, which suggests a common cellular origin. These results show that immunohistochemical staining with the use of monoclonal antibody against keratin 19 is useful to distinguish papillary thyroid carcinomas from follicular adenomas and follicular thyroid carcinomas.     

Complex regulation of the cyclin-dependent kinase inhibitor p27kip1 in thyroid cancer cells by the PI3K/AKT pathway: regulation of p27kip1 expression and localization

Functional inactivation of the tumor suppressor p27(kip1) in human cancer occurs either through loss of expression or through phosphorylation-dependent cytoplasmic sequestration. Here we demonstrate that dysregulation of the PI3K/AKT pathway is important in thyroid carcinogenesis and that p27(kip1) is a key target of the growth-regulatory activity exerted by this pathway in thyroid cancer cells. Using specific PI3K inhibitors (LY294002, wortmannin, and PTEN) and a dominant active AKT construct (myrAKT), we demonstrated that the PI3K/AKT pathway controlled thyroid cell proliferation by regulating the expression and subcellular localization of p27. Results obtained with phospho-specific antibodies and with transfection of nonphosphorylable p27(kip1) mutant constructs demonstrated that PI3K/AKT-dependent regulation of p27(kip1) mislocalization in thyroid cancer cells occurred via phosphorylation of p27(kip1) at T157 and T198 (but not at S10 or T187). Finally, we evaluated whether these results were applicable to human tumors. Analysis of 100 thyroid carcinomas indicated that p27(kip1) phosphorylation at T157/T198 and cytoplasmic mislocalization were preferentially associated with activation of the PI3K/AKT pathway. Thus the PI3/AKT pathway and its effector p27(kip1) play major roles in thyroid carcinogenesis.     

p53 expression in anaplastic carcinomas arising from thyroid papillary carcinomas.

AIM--To study the expression of p53 tumour suppressor gene in anaplastic carcinomas arising from thyroid papillary carcinomas. METHODS--Formalin fixed, paraffin wax embedded tissues from four cases of anaplastic carcinomas associated with thyroid papillary carcinomas were studied by immunohistochemistry with two different p53 monoclonal antibodies. RESULTS--The anaplastic component showed nuclear immunostaining in two cases, but not in the other two. In all cases the papillary carcinoma component was negative. CONCLUSION--The results support the hypothesis that p53 stimulates tumour progression in thyroid tumours.     

甲状腺乳头状癌中MCM7、CDK2、p27蛋白的表达及意义

目的 探讨MCM7、CDK2及p27蛋白与人甲状腺乳头状癌(papillary thyroid carcinoma,PTC)发生、发展的关系.方法 采用免疫组织化学SP法检测40例PTC、30例甲状腺腺瘤、30例结节性甲状腺肿及20例正常甲状腺组织中MCM7、CDK2、p27蛋白的表达.结果 PTC中MCM7、CDK2蛋白的阳性表达率分别为100.00%(40/40)、80.00%(32/40),两者均明显高于甲状腺腺瘤、结节性甲状腺肿及正常甲状腺组织(P<0.01,P<0.01).PTC中p27蛋白的阳性表达率为22.50%(9/40),明显低于甲状腺腺瘤、结节性甲状腺肿及正常甲状腺组织(P<0.01).PTC中MCM7与CDK2蛋白的表达呈正相关(r=0.550,P<0.01),MCM7、CDK2及p27蛋白的表达呈负相关(r=-0.334,P<0.05;r=-0.413,P<0.01).结论 MCM7、CDK2蛋白的高表达及p27蛋白的低表达变化与PTC可能存在关联,三者联合检测或许可以作为临床早期诊断和评价甲状腺肿瘤细胞增殖活性的潜在的参考指标.     

MAGE-1, GAGE-1/-2 gene expression in FNAB of classic variant of papillary thyroid carcinoma and papillary hyperplasia in nodular goiter.

Differentiation of the papillary variant of papillary thyroid carcinoma (PTC) from papillary hyperplasia in nodular goiter may be difficult in fine-needle aspiration biopsy (FNAB) by means of morphology alone. To improve cytodiagnostic accuracy the occurrence of MAGE-1, GAGE-1/-2 gene expression was analyzed by means of RT-PCR. The genes investigated are recognized by autologous T lymphocytes and are expressed in carcinomas of various sites e.g. lung, ovary, colon but not in non-neoplastic tissue except testis. Routinely obtained smears with cytologic diagnosis of PTC confirmed by histology (n=20) and diagnosis of nodular goiter (n=10) were investigated. The MAGE-1, GAGE-1/-2 PCR products were found in 6/20 of the carcinomas but in none of the benign lesions. To identify PCR products automatic gene-sequencing in all positive cases was performed. The data indicate that MAGE-1, GAGE-1/-2 gene expression may give additional information to delineate PTC from papillary hyperplasia in FNAB.     

p27/kip1 expression in normal epithelium, benign and neoplastic breast lesions.

The development of cancer in the breast and in other sites is a complex process requiring a number of different genetic and epigenetic alterations. The accumulation of the genetic changes is thought to underlie the progression from precancerous lesions to carcinomas. The expression of p27/kip1 protein, a cyclin-dependent kinase inhibitor, was investigated by immunohistochemistry in normal epithelial specimens, benign alterations, and malignant lesions of the breast. The number of p27/kip1-positive cells ranged from none to more than 98% in the overall series. Wide ranges of p27/kip1-positive cells were consistently observed within each histological category, but the median value progressively decreased in typical hyperplasia and fibroadenoma, with an even more marked reduction in malignant lesions, compared with normal epithelium. Moreover, the percentage of cells expressing p27/kip1 in tumours was about three times lower in invasive than in in situ lesions and was inversely related to tumour size, but not to lymph node involvement. In conclusion, the degree to which p27 expression is altered in typical hyperplastic lesions and fibroadenomas indicates that the deregulation of p27 may occur very early on during breast cell transformation, but the usefulness of its determination to categorize subgroups of lesions at different risk of evolution remains somewhat doubtful.     

Expression of p27/kip1 is down-regulated in human prostate carcinoma progression

p27^Kip1 is a cyclin-dependent kinase inhibitor whose down-regulation has been observed in several tumour models, including breast, colorectal, and gastric carcinomas. The purpose of this study was to assess p27^Kip1 protein expression in normal and benign prostatic epithelia as well as the possible existence of abnormalities in prostate carcinoma progression. p27^Kip1 expression was immunohistochemically analysed in 51 normal tissue samples, 11 nodular hyperplasias (NH), 22 high-grade prostatic intraepithelial neoplasias (PIN), 56 localized prostate adenocarcinomas, and 19 metastases. Immunoblotting was performed in ten cases. Normal prostate epithelium and NH showed diffuse and intense p27^Kip1 nuclear expression in most cases. A significant p27^Kip1 down-regulation was observed in many carcinomas when compared with benign epithelium. Forty-seven cases (84 per cent) were low p27^Kip1 expressors (<50 per cent positive cells) and nine cases (16 per cent) were high p27^Kip1 expressors. p27^Kip1 down-regulation was also consistently seen in PIN. Fourteen out of 19 metastases (74 per cent) were low p27^Kip1 expressors. Six metastatic samples had their corresponding primary tumour analysed and three cases showed decreased expression in the metastasis. It is concluded that p27^Kip1 is constitutively expressed in normal and benign prostatic tissue. This expression is clearly down-regulated in neoplastic progression from the preinvasive lesions through invasive carcinoma and metastases and this therefore occurs in early stages of neoplastic transformation. Copyright © 1999 John Wiley & Sons, Ltd.     

Proliferating cell nuclear antigen expression in papillary thyroid carcinoma.

AIMS: To correlate the expression of proliferating cell nuclear antigen (PCNA) with various clinicopathological features of papillary thyroid carcinoma. METHODS: Sections from 131 formalin fixed, paraffin wax embedded papillary thyroid carcinomas were stained with a monoclonal antibody (PC10) directed against PCNA using the avidin-biotin immunoperoxidase (ABC) method. PCNA immunoreactivity was based on the PCNA labelling index (LI) following evaluation of at least 1000 tumour cells, and expressed as follows: grade A (LI < 10%), grade B (10% < or = LI < 25%), and grade C (LI > or = 25%). The relation between PCNA expression in these three groups and other clinicopathological factors, such as sex, age, tumour size, nodal metastases, and histological differentiation, were examined. RESULTS: Based on the labelling index, 57 (43.5%) cases were graded as A, 46 (35.1%) as B, and 28 (21.4%) as C. The female-:male ratios were 6.13:1 for group A, 2.83:1 for group B, and 2.11:1 for group C. The mean (SD) ages of the patients were 39.0 (16.1) in group A, 53.5 (14.4) in group B, and 55.8 (13.3) years in group C. The correlation between age and PCNA grade was strongest in women. CONCLUSIONS: PCNA immunoreactivity is correlated with sex and age in patients with papillary thyroid tumours.     

Apaf-1在甲状腺乳头状癌中的表达及意义

目的:探讨凋亡蛋白酶活化因子1(Apaf-1)在甲状腺乳头状癌(PTC)中的m RNA与蛋白表达及其与细胞增殖的关系。方法:分别采用real-time PCR、Western blot及免疫组化法检测并比较甲状腺乳头状癌和癌旁组织中Apaf-1的m RNA及蛋白表达情况,并分析其表达水平与PTC临床病理学特征的关系;通过下调CGTHW-3细胞中Apaf-1表达量,验证Apaf-1对细胞增殖的影响。结果:在PTC组织中,Apaf-1的m RNA和蛋白表达量均显著低于癌旁组织(P 0. 05);下调Apaf-1表达增强了CGTHW-3细胞的增殖活性(P 0. 05)。结论:Apaf-1在PTC中低表达,抑制其表达可增强CGTHW-3细胞的增殖能力。Apaf-1在甲状腺乳头状癌中可能发挥抑癌作用。     

鼻咽癌中p27kip1表达及其与临床病理特征相关研究

Objective To inrestigate the expression of p27kip1 protein and its association with clinicopathologic features in nasopharyngeal carcinoma. Methods Immunohistochemistry was performed to detect p27kip1 expression in 60 paraffin- embedded nasopharyngeal carcinoma samples and 30 paraffinembedded non-cancer nasopharyngeal tissue samples. Results Nuclear and cytoplasmic p27kip1 staining was detected in nasopharyngeal carcinoma samples. Of 60 nasopharyngeal carcinoma samples, 46.7% (28/60)were positive for p27kip1 in cell nuclei, compared with as found in non-cancer nasopharyngeal tissue [90%(27/30), P＜0.01 ], and 68.3% (41/60) were positive in cytoplasma, compared with 20% (6/30) of noncancer nasopharyngeal tissue (P＜0.01). Both nuclear and cytoplasmic expression of p27kip1 were not associated to tumor size and local invasion, while positive nuclear expression of p27kip1 was significantly associated to nodal and remote metastasis and TNM stage. Cytoplasmic expression of p27kip1 was associated to nodal infiltration and TNM stage. Conclusions Compared to that in non-cancer tissue, p27kip1 is upregulated in cytoplasm and down- regulated in nuclei of nasopharyngeal carcinoma tissue, presenting a mislocalization. Less or no nuclear expression of p27kip1 may be associated to malignant progression of nasopharyngeal carcinoma. Abnormal expression and location of p27kip1 may be associated to nasopharyngeal stage and metastasis.     

鼻咽癌中p27kip1表达及其与临床病理特征相关研究

目的 检测p27kip1蛋白在鼻咽癌中的表达,分析其与临床病理特征的关系.方法 收集60例手术活检切取的鼻咽癌组织蜡块,与30例非肿瘤鼻咽组织石蜡标本作比较.应用免疫组织化学技术检测鼻咽癌组织中p27kip1蛋白的表达,回顾性研究鼻咽癌患者的临床病理特征.结果 p27ksp1蛋白在鼻咽癌细胞核和细胞质中均有表达.鼻咽癌组织中细胞核表达阳性率为46.7%(28/60),低于非肿瘤鼻咽组织细胞核表达的90%(27/30),P＜0.01;而在细胞质中阳性率为68.3%(41/60),显著高于非肿瘤鼻咽组织的细胞质表达的20%(6/30),P＜0.01.未发现p27kip1蛋白在细胞核和细胞质表达与鼻咽癌原发灶的范围和局部侵犯程度有关,但p27kip1蛋白胞核表达与淋巴结转移、远处转移和TNM临床分期有关;细胞质表达仅与淋巴结转移和TNM临床分期有关,可见p27kip1蛋白胞核表达与临床病理的关系更为密切.结论 与非肿瘤鼻咽组织相比,p27kip1蛋白为细胞核低表达、细胞质高表达.鼻咽癌中p27kip1蛋白存在不同于非肿瘤鼻咽组织的错误的核质定位,其在细胞核中表达的减少或丢失可能与鼻咽癌的恶性发展有关,提示p27kip1蛋白的异常表达和定位可能涉及鼻咽癌的分期和转移.     

反转录病毒载体介导的P27kip1促HepG2细胞凋亡简

 目的 研究反转录病毒介导的P27kip1基因过表达对HepG2细胞的影响。 方法 构建携带有人P27kip1基因的反转录病毒载体,经脂质体介导转染PA317包装细胞,G418筛选获得稳定产毒细胞株,病毒感染HepG2细胞,筛选出P27kip1阳性克隆,Western blot检测P27kip1在HepG2中的表达,并通过细胞形态学观察、MTT、FCW等方法,检测P27kip1对HepG2细胞的影响。结果 成功构建了含有人P27kip1基因的pLNCX-P27反转录病毒载体并导入包装细胞获得了稳定产毒细胞株,病毒感染HepG2后P27kip1基因可在细胞内高表达,过表达P27kip1基因的细胞生长速度受阻,较多细胞阻滞于G1期,且凋亡增多。结论 构建的pLNCX-P27载体能稳定高效地将P27kip1基因导入HepG2细胞,过表达P27kip1可抑制细胞生长,加速凋亡。