p53/MDM2 Pathway Aberrations in Parathyroid Tumors: p21(WAF-1) and MDM2 Are Frequently Overexpressed in Parathyroid Adenomas

Parathyroid adenomas (PTAs) are the main cause of primary hyperparathyroidism. Cell cycle regulation in normal parathyroid tissue (NPT) and PTA remains largely unknown. We have systematically explored several components involved in the p53/MDM2/p19^ARF pathway in PTA and compared the results were with NPT. Forty-six PTA and 12 NPT were immunostained with anti-p21^WAF-1, MDM2, p53, and p27^KIP1 antibodies. The slides were processed by cytometry and the results were statistically analyzed using nonparametric methods (Mann-Whitney test). p21^WAF-1 and MDM2 expression were significantly higher in PTA compared with NPT (p<0.05). The opposite results were found for p27^KIP1 (p<0.05). Occasional p53 staining was found in some PTA, albeit no significant difference was found in comparison with NPT. In conclusion, MDM2 and p21^WAF-1 are the proteins more overexpressed in PTA. These findings are surprising taking into account the benign nature of PTA, making them suitable candidates for further molecular analysis.     

Immunohistochemical Analysis of 11-β-Hydroxysteroid Dehydrogenase Type 2 and Glucocorticoid Receptor in Subclinical Cushing’s Disease due to Pituitary Macroadenoma

Subclinical Cushing’s disease (SCD) is characterized by lack of clinically evident Cushingoid features, despite abnormal hypersecretion of ACTH. Nearly half the cases of SCD are due to macroadenomas, and in the majority of them, ACTH secretion is not inhibited even by high-dose dexamethasone. Impaired glucocorticoid (GC) action may be correlated with the proliferation and development of pituitary macroadenomas causing SCD. In this study, immunohistochemical analysis of the resected tumors were performed to evaluate the expression of 11β-hydroxysteroid dehydrogenase type 2 (11βHSD2) and glucocorticoid receptor (GR) in pituitary tissues obtained from two SCD (macroadenomas), eight Cushing’s disease (CD) (microadenomas), nine acromegaly, and nine normal pituitary (NP). Scattered 11βHSD2-immunopositive cells were detected in all NP tissues, but its immunoreactivity was totally absent in any tumorous tissues except two CD. Scattered GR-immunopositive cells were also detected and GR immunostaining was restricted to the cytosol in NP tissue. In contrast, GR-immunopositive cells were abundantly present and GR immunostaining was restricted to the nucleus in all the tumorous tissues. There were marked differences in both expression levels and localization between NP tissues and all the tumors. There may be a mechanism other than that via 11βHSD2 for causes of impaired negative feedback action by GC in SCD and CD, but results of our present study suggest that impaired GC action may be involved, at least in part, in tumorigenesis of SCD and CD.     

Expression of cyclin D1, Ki-67 and PCNA in non-small cell lung cancer: prognostic significance and comparison with p53 and bcl-2

Uncontrolled cell proliferation is the hallmark of malignant tumours. Thus, the proliferative potential of tumour cells is an important prognostic factor. However, evaluation of the prognostic significance of the expression of proteins involved in regulation of cell proliferation remains controversial. In the present study, expression of Ki-67, PCNA and cyclin D1 was estimated in a group of 89 surgically resected non-small cell lung carcinomas using immunohistochemistry. The results were compared with expression of bcl-2 and p53 and with clinicopathological parameters including patients' survival. Ki-67 and PCNA were found to be moderately and highly expressed in 39% and 44% of the tumours, respectively. There was a strong correlation between Ki67 and PCNA expression. Forty five of 88 tumours (51%) showed overexpression of cyclin D1. Surprisingly, cyclin D1 was mainly localized in the cytoplasm and only a small group of tumours (9/88, 10%) showed nuclear staining as well. Bcl-2 and p53 expression was observed in 69% and 30% of the tumours, respectively. All these markers were found to be independent of clinicopathological parameters, except for Ki-67 and bcl-2 expression, which was associated with squamous cell carcinomas. It is concluded that none of the markers that were studied can be used as an independent prognostic factor, whereas the following combinations of markers may have favourable prognostic value: p53 positivity and low Ki-67 expression, p53 positivity and lack of cyclin D1 expression, bcl-2 positivity and low Ki-67 expression, and lack of cyclin D1 expression and low Ki-67 expression.     

Apoptosis and Expression of the Proto-Oncogenes bcl-2 and p53 and the Proliferation Factor Ki-67 in Human Medullary Thyroid Carcinoma

Apoptosis and immunoreactivity forbcl-2, p53, and Ki-67 were studied in 21 patients with medullary thyroid carcinoma (MTC). The DNA nick end labeling method was used to assess apoptosis. The relationships between the different factors were analyzed, as were their relations to clinicopathological data, including survival. More than 80% of the tumors harbored apoptotic cells. Tumors in individuals who had died of the MTC disease had a higher percentage of apoptosis. All cases demonstrated immunoreactivity tobcl-2; disease-free individuals had a higher rate than those with recurrent disease. No obvious pattern could be discerned in the relation of p53 or Ki-67 to the outcome of the MTC disease. An inverse correlation betweenbcl-2 and apoptosis (r=−0.81;p<0.01) was demonstrated.bcl-2 was significantly (p=0.014) associated with apoptosis even after taking both p53 and Ki-67 into consideration, but these two factors were unrelated to apoptosis. None of the factors studied were correlated to crude survival, either in univariate or in multivariate analyses. This study established thatbcl-2 immunoreactivity is closely associated with apoptosis in MTC, suggesting that a downregulation of thebcl-2 protein is related to a more aggressive growth rate and might be a useful marker for the evaluation of MTC.     

Expression of p21<Superscript>cip1</Superscript>, p27<Superscript>kip1</Superscript>, and p16<Superscript>INk4a</Superscript> Cyclin-Dependent Kinase Inhibitors in Papillary Thyroid Carcinoma: Correlation with Clinicopathological Factors

In a variety of human malignancies, aberrant expression of proteins involved in the control of cell-cycle progression has been reported. In this study, p21^cip1, p27^kip1, and p16^INk4a cyclin-dependent kinase inhibitors were analyzed to evaluate their usefulness in clinical management of papillary thyroid carcinoma (PTC). Archived material derived from 46 cases of PTC was analyzed immunohistochemically. Protein expression was ascertained on tissue microarrays, and results were correlated with clinicopathological features of the patients. Positive immunostaining was observed in 14 (30,4%) p21^cip1, 26 (56,5%) p27^kip1, and 14 (30,4%) p16^INk4a cases. No significant correlation between p21^cip1 or p27^kip1 and clinical factors was found. In contrast, p16^INk4a expression showed a significant correlation with initial extension of the disease. Therefore, 45.8% of patients with loco-regional extension were p16^INk4a positive, whereas overexpression was only seen in 15.7% of cases with intrathyroid disease (p < 0.05). Moreover, all patients with simultaneous p16^INk4a positivity and lack of p27^kip1 staining (four patients) presented lymph node metastases. In contrast, only 12 (28.5%) of the remaining patients showed lymph node tumor involvement. In conclusion, p16^INk4a expression suggests extrathyroid neck extension of PTC. This effect is enhanced when p27^kip1 is negative. We think that their analysis by immunohistochemistry could be useful in the management of patients with PTC.     

Distinction Between Lipoma and Liposarcoma by MDM2 Alterations: A Case Report of Simultaneously Occurring Tumors and Review of the Literature

We investigated a lipoma and a well-differentiated/dedifferentiated liposarcoma (WD/DDL), occurring simultaneously in one patient for the possible role of p53 and mdm2 in the molecular oncogenesis of liposarcoma and tumor progression. The hypothesis tested was if there is a continuum in the development from lipoma to liposarcoma. Lipoma was characterized by a lack of p53 mutation, p53 LOH and p53 protein expression, as well as by mdm2 amplification and mdm2 protein expression. p53 mutation and p53 LOH were found neither in the well-differentiated nor in the dedifferentiated parts of the liposarcoma. In contrast, mdm2 amplification and an increase in mdm2 protein expression were found to be associated with malignancy and dedifferentiation, whereas p53 protein expression was only slightly increased. These findings indicate that mdm2 constitutes one of the most common targets for molecular aberration in WD/DDL. We suggest that mdm2 is a marker distinguishing between ordinary lipoma and well-differentiated liposarcoma, and that the genesis of these tumors is different.     

Possible Relation of p53 and mdm-2 Oncoprotein Expression in Thyroid Carcinoma: A Molecular-Pathological and Immunohistochemical Study on Paraffin-Embedded Tissue

Routinely processed tissues from a series of benign and malignant thyroid lesions were immunohistochemically investigated with antibodies against p53 and mdm-2. p53 was immunolocalized in <10% of nuclei in 2/80 nodular goiters, 2/60 follicular adenomas, 26/68 follicular carcinomas, 7/40 papillary carcinomas, 3/10 “insular” carcinomas, and 10/31 anaplastic carcinomas. More than 10% positively stained nuclei were found in 2 widely invasive follicular, 2 insular, and 15 anaplastic carcinomas. All p53-positive cases showed a concomitant immunohistochemical mdm-2 expression; an immunohistochemical colocalization on serial section was demonstrated in 12 anaplastic carcinomas. Screening by polymerase chain reaction single-strand conformation polymorphism (PCR-SSCP) analysis of these 12 cases revealed no relevant mutations in the coding regions of exons 2–11 of the p53 gene. Additionally, 1 follicular adenoma, 6 follicular carcinomas (4 minimally and 2 widely invasive), 1 papillary, and 2 poorly differentiated insular carcinomas were mdm-2 positive without immunohistochemically detectable p53 expression. These results provide evidence that wild-type p53 expression in thyroid carcinomas may be associated with mdm-2 induced formation of stable complexes. However, the role of p53 mutations and p53 protein inactivation owing to other factors (e.g., mdm-2) in the progression of thyroid carcinomas is still poorly understood.     

Expression of Pirh2, a p27(Kip1) ubiquitin ligase, in hepatocellular carcinoma: correlation with p27(Kip1) and cell proliferation

p53-Induced ring-H2 protein (Pirh2), a recently identified ubiquitin-protein ligase, interacts with p27(Kip1) to promote ubiquitination of p27(Kip1) independently of p53. High Pirh2 and low p27(Kip1) immunoreactivity are associated with a poor prognosis in several cancers, including resistant phenotypes. In the present study, we investigated the role of Pirh2 and p27(Kip1) in human hepatocellular carcinoma (HCC) progression. Immunohistochemical analysis was performed on formalin-fixed paraffin sections of 87 specimens. Statistical analysis showed that expression of Pirh2 was negatively related to p27(Kip1) expression (r = 0.787; P < .05), and Pirh2 expression correlated significantly with histologic grade (P < .001), venous invasion (P = .004), tumor size (P = .024), and the presence of multiple tumor-bearing lymph nodes (P = .017), whereas p27(Kip1) expression correlated significantly with histologic grade (P < .001), venous invasion (P = .048), and cirrhosis (P = .028). By Kaplan-Meier analysis, the survival curves of low versus high expressers of Pirh2 and p27(Kip1) showed significant separation (P < .01). Molecular interaction could be demonstrated between Pirh2 and p27(Kip1) in three HCC cell lines. In vitro, following release of two HCC cell lines from serum starvation, the expression of Pirh2 was upregulated, whereas p27(Kip1) was downregulated. Our results suggest that Pirh2 mediates the degradation of p27(Kip1) and participates in cell proliferation in human HCC. These findings provide a rational framework for further development of Pirh2 inhibitors as a novel class of anti-tumor agents.     

High levels of MMP-2, MMP-9, MT1-MMP and TIMP-2 mRNA correlate with poor survival in ovarian carcinoma

The object of this study was to analyze the potential association between the expression of MMP-2, MMP-9, MT1-MMP and TIMP-2, and disease outcome in advanced-stage ovarian carcinomas. Sections from 70 paraffin-embedded blocks (36 primary ovarian carcinomas and 34 metastatic lesions) from 45 patients diagnosed with advanced stage ovarian carcinomas (FIGO stages III–IV) were studied using mRNA in situ hybridization (ISH) technique. Patients were divided retrospectively in two groups based on disease outcome. Long-term survivors (21 patients) and short-term survivors (24 patients) were defined using a double cut-off of 36 months for disease-free survival (DFS) and 60 months for overall survival (OS). Mean follow-up period for patients that were diagnosed with advanced-stage carcinoma was 70 months. The mean values for DFS and OS were 109 and 125 months for long-term survivors, as compared to 3 and 21 months for short-term survivors, respectively. Intense mRNA signals were detected more frequently in tumor cells of short-term survivors with use of all four probes. Comparable findings were observed in peritumoral stromal cells with ISH for MMP-2, MMP-9 and TIMP-2 mRNA. Notably, primary tumors with intense mRNA signal for TIMP-2 (No = 14) were uniformly associated with a fatal outcome. In univariate analysis of primary tumors, mRNA levels of TIMP-2 in stromal cells (P = 0.0002), as well as for MMP-9 (P = 0.012) and TIMP-2 (P = 0.02) in tumor cells, correlated with poor outcome. In univariate analysis of metastatic lesions, mRNA levels of TIMP-2 in stromal cells (P = 0.031), as well as for MMP-2 (P = 0.027) and MT1-MMP (P = 0.008) in tumor cells, correlated with poor outcome. Interestingly, the presence of MT1-MMP in stromal cells correlated with longer survival (P = 0.025). In a multivariate analysis of ISH results for primary tumors, TIMP-2 levels in stromal cells (P = 0.006) and MMP-9 levels in tumor cells (P = 0.011) retained their predictive value. We conclude that MMP-2, MMP-9, MT1-MMP and TIMP-2 are valid markers of poor survival in advanced-stage ovarian carcinoma. This revised version was published online in July 2006 with corrections to the Cover Date.     

P57KIP2 immunostaining and molecular cytogenetics: combined approach aids in diagnosis of morphologically challenging cases with molar phenotype and in detecting androgenetic cell lines in mosaic/chimeric conceptions

Although molar pregnancies are typically defined by morphological, histologic, and genetic criteria, most cases are diagnosed solely on histologic findings. Recently, several studies have demonstrated the usefulness of p57KIP2 immunostaining as an ancillary diagnostic tool for molar pregnancies. The p57KIP2 gene is paternally imprinted and maternally expressed; therefore, the positive staining of its protein indicates the presence of a functional maternal allele. Because complete hydatidiform moles (CHMs) lack a maternal genome, p57KIP2 immunostaining is absent. Previous studies have validated this staining technique by demonstrating differential nuclear expression in CHMs versus non-CHMs; however, these studies have not included cytogenetic analysis. We report on 58 cases of hydropic placentas, correlating cytogenetic and p57KIP2 immunostaining results. In addition, cases with unusual p57KIP2 staining patterns are discussed. Also included are 2 mosaic conceptions (1 diploid/triploid and 1 diploid/tetraploid), 6 chimeric/mosaic conceptions with androgenetic/biparental cell lines, and 2 cases of placental mesenchymal dysplasia.     

Mutation analysis of the <Emphasis Type="Italic">MECP2</Emphasis> gene in patients of Slavic origin with Rett syndrome: novel mutations and polymorphisms

Rett syndrome (RTT), an X-linked dominant neurodevelopmental disorder in females, is caused mainly by de novo mutations in the methyl-CpG-binding protein 2 gene (MECP2). Here we report mutation analysis of the MECP2 gene in 87 patients with RTT from the Czech and Slovak Republics, and Ukraine. The patients, all girls, with classical RTT were investigated for mutations using bi-directional DNA sequencing and conformation sensitive gel electrophoresis analysis of the coding sequence and exon/intron boundaries of the MECP2 gene. Restriction fragment length polymorphism analysis was performed to confirm the mutations that cause the creation or abolition of the restriction site. Mutation-negative cases were subsequently examined by multiple ligation-dependent probe amplification (MLPA) to identify large deletions. Mutation screening revealed 31 different mutations in 68 patients and 12 non-pathogenic polymorphisms. Six mutations have not been previously published: two point mutations (323T>A, 904C>T), three deletions (189_190delGA, 816_832del17, 1069delAGC) and one deletion/inversion (1063_1236del174;1189_1231inv43). MLPA analysis revealed large deletions in two patients. The detection rate was 78.16%. Our results confirm the high frequency of MECP2 mutations in females with RTT and provide data concerning the mutation heterogeneity in the Slavic population.