Immunohistochemical Evaluation of p27 (kip1) in Pleomorphic Adenomas and Adenoid Cystic Carcinomas of the Minor Salivary Glands

Background: p27(kip1), a universal cyclin-dependent kinase inhibitor, is a useful marker for predicting clinical ‍aggressiveness with various human tumors. In this study, p27 expression was investigated in pleomorphic adenomas ‍(PAs) and adenoid cystic carcinomas (ACCs) of minor salivary glands to evaluate its utility for differentiation purposes. ‍At the same time, the correlation between p27 and ACC grading was evaluated. Materials & Methods: ‍Clinicopathological features of 22 patients (11 ACCs, 11 PAs), including age, sex and size of tumor were obtained ‍from medical records. Immunohistochemical staining with p27(kip1) was performed for each specimen and p27 ‍labelling indices were determined with a computer-assisted image-analyzing system (CAS 200). Pearson’s correlation ‍coefficient, Spearman’s correlation coefficient, Students t-test, Kruskal-Wallis test and ANOVA were applied for ‍statistical analyses using SPSS 11.5. Results: p27 LIs for all PAs were above 25% whereas for ACCs they were under ‍25% (except one case). p27 expression (LI and intensity) was significantly lower in ACCs than PAs. The correlation ‍between p27 expression and ACC grading was not significant. Conclusion: Overall, these findings suggest that reduced ‍expression of p27 might be correlated with the development of ACC and could be an indicator of malignant behavior.     

Expression of p53 tumor suppressor gene in adenoid cystic and mucoepidermoid carcinomas of the salivary glands

Seventeen adenoid cystic carcinomas (ACCs) and 27 mucoepidermoid carcinomas (MECs) occurring in the salivary glands were analyzed for p53 tumor suppressor gene alteration (exons 5-8) and protein expression. The cell proliferation activity was also examined by Ki-67 immunohistochemistry. The p53 alterations were detected in three samples (17.6%) of ACC and in four samples (14.8%) of MEC, and were only found in carcinomas arising in the minor salivary glands. The occurrence of the p53 gene alteration is less frequent in ACC and MEC than that in other kinds of tumors, and therefore does not seem to play a critical role in the course of the tumorigenesis in ACC and MEC. All ACC samples arising from the minor salivary glands exhibiting p53 gene alterations showed recurrence/metastasis, thus suggesting a poor outcome of these patients. All ACCs and three out of four MECs samples with p53 gene alterations showed the lowest degree of p53 immunostaining ratio, thus suggesting that no correlation exists between the p53 gene alterations and the p53 immunostaining in these salivary gland carcinomas. No significant relationship was demonstrated between the immunostaining ratio of either p53 or Ki-67 and the morphological growth pattern or patient clinical course in the ACC samples. The p53 immunopositivity in MEC correlated to the histological grade. The Ki-67 immunostaining ratio was also significantly related to the histological grade and the clinical course in MEC.     

Beta-6 Integrin, tenascin-C, and MMP-1 expression in salivary gland neoplasms

Beta-6 Integrin, tenascin-C, and MMP-1 (matrix metalloproteinase-1) are invasion-related proteins that are frequently overexpressed in many human malignancies. The objective of this study was to determine whether there is overexpression of these molecules in three types of salivary neoplasms showing markedly different behavior. A total of 55 formalin-fixed, paraffin-embedded archived specimens comprising 19 adenoid cystic carcinomas (ACC), 18 polymorphous low-grade adenocarcinomas (PLGA) and 18 pleomorphic adenomas (PA) were utilized in this study. A standard immunohistochemical technique was used to determine the expression levels of beta-6 integrin, tenascin-C, and matrix metalloproteinase-1 (MMP-1) proteins. Sections were assessed semiquantitatively, and tumors were divided into two groups, low-expressors (0-1+) and high-expressors (2-3+) for statistical analysis. Staining was graded as 0 (<1% positive tumor cells), 1+ (<25% positive tumor cells), 2+ (25-50% positive tumor cells), and 3+ (>50% positive cells). The results showed that the malignant tumors were higher expressors of beta-6 than the benign tumors. ACCs showed significantly higher expression of beta-6 than PAs (p=0.04). No significant difference was observed between ACCs and PLGAs. beta-6 expression was rarely seen in normal salivary gland epithelium and was occasionally present in mucosa overlying the tumors. PAs were high-expressors of tenascin-C with a significant difference relative to ACCs (p=0.03). A majority of tumors in all three tumor types showed high expression of MMP1 with expression significantly greater in the PAs compared to ACCs (p=0.008). We conclude that ACCs and PLGAs express beta-6, tenascin-C, and MMP-1, but that their expression patterns are not significantly different. beta-6 appears to be more closely associated with the malignant tumors, and MMP-1 more closely associated with the benign tumors. We believe that beta-6, tenascin-C, and MMP-1 proteins are part of the molecular repertoire used by salivary tumors for malignant invasion and benign tumor expansion.     

Expressions of nuclear factor kappaB, inducible nitric oxide synthase, and vascular endothelial growth factor in adenoid cystic carcinoma of salivary glands: correlations with the angiogenesis and clinical outcome.

OBJECTIVE: To evaluate the expressions of nuclear factor kappaB (NF-kappaB p65), inducible nitric oxide synthase enzyme (iNOS), and vascular endothelial growth factor (VEGF) in relation to angiogenesis (microvessel density, MVD) and clinical outcomes in adenoid cystic carcinoma (ACC) of salivary glands. METHODS: Immunohistochemical staining was used to quantify the protein expression levels of NF-kappaB p65, iNOS, and VEGF in 80 surgically resected ACCs and 20 normal salivary tissues. In all cases of ACCs, MVD was evaluated by counting CD34-reactive endothelial cells or endothelial cell clusters. RESULTS: The nuclear localization of NF-kappaB p65 was only detected in ACC cells. Both iNOS and VEGF staining activities in ACCs were more significant than those in normal gland tissues (P < 0.01). MVD had significant correlations with NF-kappaB p65, iNOS, and VEGF expressions (P < 0.01). In three histologic types of ACCs, the NF-kappaB, iNOS, VEGF expressions, and MVD were significantly higher in solid type than in cribriform and tubular types (P < 0.01). The NF-kappaB, iNOS, VEGF expressions, and MVD were significantly correlated with clinical stage, tumor size, vascular invasion, recurrence, and metastasis (P < 0.05). Multivariate analysis showed NF-kappaB, iNOS and VEGF expression, MVD, solid histotype, and perineural invasion had an independent prognostic effect on overall survival. CONCLUSION: The expressions of NF-kappaB p65, iNOS, and VEGF were related with MVD. Clinical outcomes raised the possibility that the overexpression of these cytokines might contribute to tumor angiogenesis and have prognostic value in ACCs.     

Osteopontin expression in salivary gland tumours

Osteopontin (OPN) is expressed in numerous carcinomas and plays a role in tumour development, invasion and metastasis. This study examines by immunohistochemistry the expression of OPN in normal salivary gland tissue and three types of salivary gland tumour: pleomorphic adenoma (PA), adenoid cystic carcinoma (ACC) and polymorphous low grade adenocarcinoma (PLGA). PAs and PLGAs demonstrated higher levels of OPN than normal salivary gland tissue, while ACC, although showing a trend towards increased OPN, was not significantly different. The results of this study indicate that OPN expression is present in normal salivary gland tissue, and is increased in certain salivary gland tumours, but further investigation is necessary to clarify its role.     

胃癌中p27Kip1、p53表达与其浸润、转移和预后的关系

目的研究胃癌组织中p27Kip1和p53的表达与胃癌浸润、转移和预后的关系。方法用免疫组化(二步法)检测100例胃癌组织中的p27Kip1蛋白和p53蛋白的表达。结果100例胃癌组织中p27Kip1和p53蛋白阳性表达率分别为44%和49%。在胃癌深部浸润组、淋巴结转移组和5年内死亡组中p27Kip1呈显著低表达(P<0.05);在胃癌淋巴结转移组和5年内死亡组中p53呈显著高表达(P<0.05)。经单变量分析结果显示p27Kip1高表达组5年生存率为70.59%,显著高于低表达组54.55%,和阴性组26%;p53高表达组5年生存率为19.23%,显著低于低表达组43.75%和阴性组53.19%。多变量Cox回归模型分析显示p27Kip1、p53均是独立的预后指标,但p27Kip1表达对患者预后的相对危险度(RR=3.06)显著大于p53表达的相对危险度(RR=2.33,P<0.01)。结论p27Kip1低表达与p53高表达的癌细胞常更能浸润与转移,使患者生存率明显降低。p27Kip1和p53是胃癌预后的独立指标,其中p27Kip1表达评估胃癌预后的作用优于p53。     

Low level of p27(Kip1) protein expression in gastric adenocarcinoma is associated with disease progression and poor outcome

BACKGROUND AND OBJECTIVES: Low tumor expression of the p27(Kip1) protein, which is involved in cell cycle control and apoptosis, is considered a negative prognostic factor in different types of cancer. The aim of this study was to evaluate the clinical and pathological significance of low p27(Kip1) protein expression in patients who had undergone resection for gastric adenocarcinoma. METHODS: p27(Kip1) protein was studied by immunohistochemistry in formalin-fixed tumor sections from 95 patients who underwent resection for gastric adenocarcinoma between 1991 and 1996. Based on the median value of protein expression, p27(Kip1) protein expression was classified as low or high. RESULTS: Low p27(Kip1) protein expression was significantly associated with tumor de-differentiation, increased penetration through the gastric wall, lymph node metastasis, and advanced tumor stage. In the group of 84 patients who underwent curative surgery, 5-year survival was 74% in cases with high p27(Kip1) protein expression and 38% in those with low p27(Kip1) protein expression (P < 0.001). At multivariate analysis, low p27(Kip1) protein expression was an independent negative prognostic factor for survival (RR = 3.671; P = 0.004). CONCLUSIONS: In gastric adenocarcinoma, low p27(Kip1) protein expression is associated with poorly differentiated and advanced tumors and is a negative prognostic factor of potential clinical value.     

Analysis and significance of c-MET expression in adenoid cystic carcinoma of the salivary gland

Adenoid cystic carcinoma (ACC), a rare salivary gland malignancy, is a histogenetic, morphologic, and clinical heterogeneous disease. Extensive efforts have been made to characterize molecular events associated with these tumors, including the identification of prognostic and predictive biomarkers. Increased copy number gain and amplification of c-Met, the cell surface receptor for hepatocyte growth factor, has been shown to enhance tumor growth and invasiveness and promote metastasis in certain tumor types. In this study, we evaluated the expression of c-Met by immunohistochemistry (IHC) in a large cohort of salivary gland ACCs and examined its clinicopathologic implications. Archival formalin-fixed paraffin-embedded blocks from 200 ACC patients were used in this study. Pathologic patterns and phenotypic expression of c-Met were recorded and compared with clinical factors including gender, age, disease stage at diagnosis, and clinical outcomes. Correlations between c-MET expression and clinical characteristics were assessed by Pearson''s chi-square test or by the 2-tailed Fisher exact test. Curves describing overall survival were generated by Kaplan-Meier product limit method. Strong c-MET expression was seen in inner ductal and outer myoepithelial cells in 53.2% of the cases. There was no correlation between c-Met overexpression and clinicopathologic parameters or patient''s overall survival ( p = .94074). In conclusion, c-MET expression is high in a significant subgroup of ACC patients. While c-MET expression is not a prognostic factor in ACC, its role as a predictive marker of benefit from MET inhibitors deserves further investigation.     

The Relationship between the Expression of p27Kip1, p53 and the Infiltration, Metastasis and Prognosis in Gastric Carcinoma

Objective: To study the relationship between the expression of p27^Kip1 and p53, and the infiltration, metastasis and prognosis in gastric carcinoma. Methods: The expression of p27^Kip1 and p53 at protein level was determined by immunohistochemical assay (two-step method) in 100 cases of gastric carcinoma. Results: Of the 100 cases, the positive rate of p27^Kip1 and p53 expressions were 44% and 49%, respectively. In the group of gastric carcinomas with deep infiltration (infiltration group), lymph nodes metastasis group (metastasis group) and death-within-5-years group (death group), the expression of p27^Kip1 was statistically lower (P<0.05). In the metastasis group and death group, the expression of p53 was significantly higher (P<0.05). The results of the monovariate analysis revealed that the 5-year survival rate of the high p27^Kip1 expression group was 70.59%, which is higher than those of the low p27^Kip1 expression group (54.55%) and the negative experession group (26%). The 5-year survival rate of the high p53 expression group was 19.23%, which was lower than those of the p53 low expression group (43.75%) and the negative group (53.19%). Cox multivariate analysis showed that p27^Kip1, like p53, was an independent prognostic index. But p27^Kip1 protein expression was a stronger independent survival predictor (RR=3.06) than p53 expression (RR=2.33). Conclusion: The low expression of p27^Kip1 and the high expression of p53 reflected the more frequent invasion and metastasis, which resulted in the reduced survival of patients. As an independent markers of the gastric carcinoma, the expression of p27^Kip1 is more useful than that of p53 in the prognosis prediction of gastric carcinoma.     

Adrenocortical carcinoma: clinical, morphologic, and molecular characterization.

PURPOSE: To define multimolecular phenotypes of adrenocortical carcinoma (ACC) and to correlate outcome with morphologic and molecular parameters. PATIENTS AND METHODS: Clinical data were analyzed for 124 patients, histopathologic slides for 67 primary tumors, and tissue specimens for 74 patients (38 primary and 36 metastatic tumors) with ACC and for 38 normal adrenal tissue samples. Molecular expression profiles were investigated by immunohistochemistry. The prognostic significance of 12 gross and histologic parameters in 67 primary ACCs was evaluated. Morphologic and protein expression patterns were correlated with disease-specific survival (DSS). Univariate influence of prognostic factors on DSS was analyzed by log-rank test and multivariate analysis by Cox regression. RESULTS: The median follow-up period was 4.7 years. Significant predictors of DSS included distant metastasis at time of initial presentation; venous, capsular, and adjacent organ invasion; tumor necrosis, mitotic rate, atypical mitosis, and mdm-2 overexpression. Five-year DSS by number (one to six) of adverse histologic parameters was as follows: one to two, 84%; three to four, 37%; more than four, 9% (P =.005).The phenotype Ki-67(-)p53(-)mdm-2(+)cyclinD1(-)Bcl-2(-)p21(-)p27(+) was observed in 83% of normal and 3% of malignant adrenal tissue (P =.01). Molecular phenotypic expression was more heterogeneous in malignant than in normal (10 v five phenotypes) adrenal tissue. CONCLUSION: Meticulous morphologic evaluation, mitotic count, and tumor stage are essential in determining prognosis for patients with ACC. Multimolecular phenotyping demonstrates that the molecular complexity and heterogeneity of these neoplasms are such that targeted therapy needs to be patient specific.     

胃癌中p27~(Kip1)、p53表达与其浸润、转移和预后的关系(英文)

目的研究胃癌组织中p27~(Kipl)和 p53的表达与胃癌浸润、转移和预后的关系。方法用免疫组化(二步法)检测100例胃癌组织中的 p27~(Kipl)蛋白和 p53蛋白的表达。结果 100例胃癌组织中 p27~(Kipl)和 p53蛋白阳性表达率分别为44%和49%。在胃癌深部浸润组、淋巴结转移组和5年内死亡组中p27~(kipl)呈显著低表达(P<0.05);在胃癌淋巴结转移组和5年内死亡组中 p53呈显著高表达(P<0.05)。经单变量分析结果显示p27~(Kipl)高表达组5年生存率为70.59%,显著高于低表达组54.55%和阴性组26%;p53高表达组5年生存率为19.23%,显著低于低表达组43.75%和阴性组53.19%。多变量 Cox 回归模型分析显示p27~(Kipl)、p53均是独立的预后指标,但 p27~(Kipt)表达对患者预后的相对危险度(RR=3.06)显著大于 p53表达的相对危险度(RR=2.33,P<0.01)。结论 p27~(Kipl)低表达与 p53高表达的癌细胞常更能浸润与转移,使患者生存率明显降低。p27~(kipl)和 p53是胃癌预后的独立指标,其中 p27~(Kipl)表达评估胃癌预后的作用优于 p53。     

Effect of p27Kip1 on the ability of invasion and metastasis of an oral cancer cell line

p27Kip1 is a cyclin-dependent kinase inhibitor which regulates progression of cells from G1 into S phase in a cell cycle. p27Kip1 has been reported to be an important prognostic factor in various malignancies. As the ability of invasion and metastasis has been thought to be the most important factor for patient's prognosis, we examined whether up-regulation or down-regulation of p27Kip1 can affect the ability of invasion and metastasis of an oral cancer cell (B88t cell) in vitro and in vivo. We constructed an expression vector containing sense- or antisense-oriented human p27Kip1 cDNA with pcDNA3.1. We transfected B88t cells with the empty vector, the sense- or antisense-oriented expression vector to regulate the expression of p27Kip1 gene. Up-regulation of p27Kip1 protein markedly inhibited the migration of cancer cells in a Boyden chamber. Down-regulation of p27Kip1 protein markedly enhanced the migration of cancer cells in a Boyden chamber, and the tumor invasion in nude mice. Moreover, we detected up-regulation of E-cadherin and down-regulation of Tiam-1 (tumor invasive and metastasis-1) in the sense transfectants, up-regulation of Tiam-1 and down-regulation of E-cadherin in the antisense transfectants by Western blotting. These results suggest that p27Kip1 may play an important role in the invasion and metastasis of an oral cancer cell involved in regulation of E-cadherin and Tiam-1.     

Immunohistochemical evaluation of estrogen and progesterone receptors in adenoid cystic carcinoma of salivary gland origin

Adenoid cystic carcinoma (ACC) is a malignant tumor of salivary gland origin, which is characterized by a high rate of local recurrence and distant metastasis even after aggressive surgical treatment. In several studies using various immunohistochemical techniques, estrogen and progesterone receptor (ER and PgR) proteins in salivary gland ACC have been identified and the possible use of endocrine therapy as a treatment modality suggested. On this basis, 27 samples of formalin-fixed paraffin-embedded salivary gland ACC were immunohistochemically evaluated for the presence of ER and PgR. ER was not detected in any of the tumors and PgR was identified in only two cases. Thus, application of hormone therapy to salivary gland ACC is not supported by the results of the present study.     

胶质瘤p27Kip1、bcl-2和PCNA蛋白的表达

目的　探讨胶质瘤p2 7Kip1,bcl 2和PCNA蛋白表达与肿瘤恶性程度、细胞增殖活性、凋亡程度的关系。方法　采用免疫组化染色S P法检测 66例不同级别的胶质瘤 p2 7Kip1,bcl 2和PCNA蛋白的表达。结果　在 66例胶质瘤中 ,p2 7Kip1表达 18例(2 7% ) ,bcl 2表达 2 0例 (3 0 % ) ,PCNA表达 5 1例 (77% )。p2 7Kip1蛋白表达率随着胶质瘤级别升高而减少 ;bcl 2蛋白表达率随肿瘤级别升高而相应增加 ;PCNA表达随胶质瘤级别升高阳性反应强度增加 ;但Ⅰ、Ⅱ与Ⅲ级和Ⅳ级组间无显著性差异。结论　p2 7Kip1蛋白表达的缺失可能与胶质瘤的发生有关 ;bcl 2基因可能间接抑制细胞凋亡而与胶质瘤的分型、细胞的增殖活性以及潜在的临床行为无直接关系 ;PCNA的表达与星形胶质细胞瘤的恶性行为有关     

DNA copy number gains at loci of growth factors and their receptors in salivary gland adenoid cystic carcinoma.

PURPOSE: Adenoid cystic carcinoma (ACC) is a malignant salivary gland tumor with a high mortality rate due to late, distant metastases. This study aimed at unraveling common genetic abnormalities associated with ACC. Additionally, chromosomal changes were correlated with patient characteristics and survival. EXPERIMENTAL DESIGN: Microarray-based comparative genomic hybridization was done to a series of 18 paraffin-embedded primary ACCs using a genome-wide scanning BAC array. RESULTS: A total of 238 aberrations were detected, representing more gains than losses (205 versus 33, respectively). Most frequent gains (>60%) were observed at 9q33.3-q34.3, 11q13.3, 11q23.3, 19p13.3-p13.11, 19q12-q13.43, 21q22.3, and 22q13.33. These loci harbor numerous growth factor [fibroblast growth factor (FGF) and platelet-derived growth factor (PDGF)] and growth factors receptor (FGFR3 and PDGFRbeta) genes. Gains at the FGF(R) regions occurred significantly more frequently in the recurred/metastasized ACCs compared with indolent ACCs. Furthermore, patients with 17 or more chromosomal aberrations had a significantly less favorable outcome than patients with fewer chromosomal aberrations (log-rank = 5.2; P = 0.02). CONCLUSIONS: Frequent DNA copy number gains at loci of growth factors and their receptors suggest their involvement in ACC initiation and progression. Additionally, the presence of FGFR3 and PDGFRbeta in increased chromosomal regions suggests a possible role for autocrine stimulation in ACC tumorigenesis.     

Frequent downregulation of 14-3-3 sigma protein and hypermethylation of 14-3-3 sigma gene in salivary gland adenoid cystic carcinoma

14-3-3 sigma:, a target gene of the p53 tumour suppressor protein, has been shown to regulate the cell cycle at the G2/M checkpoint. Recent studies have demonstrated that 14-3-3 sigma is downregulated by hypermethylation of the CpG island in several types of cancer. In this study, we investigated the expression and methylation status of 14-3-3 sigma in human salivary gland adenoid cystic carcinoma (ACC) and mucoepidermoid carcinoma (MEC). Immunohistochemical analysis revealed that the positive expression rate of 14-3-3 sigma in ACC (one out of 14) was markedly lower than that in MEC (ten out of 10). Since most of the ACCs carried the wild-type p53 protein, downregulation of 14-3-3 sigma in ACC may not be due to the dysfunction of p53 pathway. Microdissection-methylation-specific PCR revealed that frequent hypermethylation of the 14-3-3 sigma gene was observed in ACC when compared to that in MEC. In cultured-ACC cells, we confirmed the downregulation of 14-3-3 sigma via hemimethylation of the gene by sequencing analysis after sodium bisulphite treatment. Furthermore, re-expression of 14-3-3 sigma in the ACC cells was induced by the treatment with DNA demethylating agent, 5-aza-2'-deoxycytidine. Irradiation apparently induced the enhanced expression of 14-3-3 sigma and G2/M arrest in normal salivary gland cells; however, in the ACC cells, neither induction of 14-3-3 sigma nor G2/M arrest was induced by irradiation. These results suggest that downregulation of 14-3-3 sigma might play critical roles in the neoplastic development and radiosensitivity of ACC.