Expression of cell-cycle regulatory proteins in endometrial carcinomas: correlations with hormone receptor status and clinicopathologic parameters.

PURPOSE: The normal human endometrium is characterized by hormone-dependent variations in the levels of cell-cycle regulatory proteins during the menstrual cycle. As this tightly controlled system is disturbed in endometrial carcinomas, we analyzed which cell-cycle regulators are involved in endometrial carcinogenesis. METHODS: We performed Western blot analysis of five cell-cycle stimulating (cyclins D1, E, B1, cdk2, cdk4) and three cell-cycle inhibiting (p16(INK4a), p21(WAF1), Rb) proteins in 41 endometrial carcinoma specimens. In addition, expression of the estrogen and progesterone receptors (ER, PR), Ki67, and, in selected cases, p16, cyclin E, and cyclin B1 was studied by immunohistochemistry. RESULTS: We found upregulation of all analyzed cell-cycle regulators in most tumors compared to normal endometrial tissue samples. Overexpression of cyclin E, cyclin B1, and p21 was associated with a less differentiated phenotype. In addition, high levels of cyclin E, cdk2, and cdk4 correlated with weak/absent ER expression, and p16 and p21 overexpression was significantly associated with low PR immunoreactivity. Cyclin B1 expression correlated with cyclin E, cdk2, cdk4, p21, Rb, and Ki67, and cyclin E expression with cyclin D1 and Rb. CONCLUSIONS: We conclude that cyclin E and cyclin B1 might be the major cell-cycle regulators involved in proliferation and reduced differentiation of endometrial carcinomas. In addition, p16, p21, and Rb appear to be uncoupled from their normal cell-cycle inhibiting function in many endometrial carcinomas.

Overexpression of the p16 cell cycle inhibitor in breast cancer is associated with a more malignant phenotype.

In order to study the role of the p16INK4A(MTS1/CDKN2a) tumor suppressor in breast cancer, we analyzed p16 protein expression in 60 breast cancer samples which were also analyzed for expression of Rb, Ki67, HER2/neu, and estrogen and progesterone receptors (ER, PR). P16 expression was investigated by two methods: western blotting (WB) followed by densitometry, and immunohistochemistry (IHC). The Rb status was studied by western blotting, and expression of Ki67, HER2/neu, ER, and PR was analyzed immunohistochemically. P16-negative results were found in 18% of the carcinomas by WB, but in only one case by IHC and were not associated with established prognostic parameters. In contrast, p16 overexpression which was detected by WB and IHC in 15% and 25% of the tumors, respectively, was significantly associated with unfavorable prognostic indicators. High p16 expression as detected by both methods correlated significantly with high grading and a negative estrogen receptor status. In addition, a significant association of p16 staining with inverse progesterone receptor status and high Ki67 expression was found with IHC. No correlation of p16 expression with clinical stage, HER2/neu immunostaining, Rb expression or Rb phosphorylation was found. Comparison of western blot results and immunohistochemistry suggests that both nuclear and cytoplasmic immunoreactivity in tumor cells is specific and due to p16 expression. We conclude that high p16 reactivity (both nuclear andcytoplasmic) is indicative of a more undifferentiated, malignant phenotype in mammary carcinomas.

Relaxin-like factor (RLF): a new specific marker for Leydig cells in the ovary.

Relaxin-like factor (RLF), also known as the Leydig cell insulin-like factor (Ley-I-L), is a novel member of the insulin-IGF-relaxin family of hormones and growth factors that has recently been shown to be strongly expressed in testicular Leydig cells. Expression of the RLF peptide in the human ovary and in ovarian tumors has not been studied. In the present study, the expression of the RLF peptide in the human ovary was investigated by immunohistochemistry using a specific antibody raised against human RLF. By this method, RLF was found to be expressed in hilus (Leydig) cells and theca interna cells but absent in granulosa cells, ovarian stromal cells, and surface epithelium. RLF expression was also observed in the corpus luteum, although at a lower level than in theca cells. Thirty-seven sex cord-stromal tumors and five cases of hilar Leydig cell hyperplasia were also investigated for RLF expression. RLF was found to be strongly expressed in hilar Leydig cell hyperplasia and sex cord-stromal tumors with a component of Leydig or luteinized cells. Of the analyzed theca cell tumors, two displayed a diffuse staining pattern. As expected, RLF was not expressed in granulosa cell tumors. In conclusion, RLF appears to be a useful marker for Leydig cells in the human ovary and may be a diagnostic supplement in hyperplasias and tumors derived therefrom.