Karin Milde-Langosch

Clinical significance of p53 alterations in surgically treated prostate cancers

Despite the high number of previous studies, the role of p53 alterations in prostate cancer is not clearly defined. To address the role of p53 alterations in prostate cancer biology, a total of 2514 cancers treated by radical prostatectomy were successfully analyzed by immunohistochemistry in a tissue microarray format. Overall a low rate of p53-positive tumors was found (2.5%). A significant underestimation of p53-positive cases was excluded by subsequent large section analyses and direct sequencing of the p53 gene in subsets of our patients. Large section analysis of 23 cases considered negative on the tissue microarray yielded only one weakly p53-positive tumor. Only 4 out of 64 (6.4%) high-grade tumors, that were considered negative for p53 by immunohistochemistry, presented exon 5–8 mutations. These data suggest a high sensitivity of our immunohistochemistry approach and confirm the overall low frequency of p53 alterations in clinically localized prostate cancer. A positive p53 immunostaining was strongly associated with presence of exon 5–8 mutations (P<0.0001), advanced pT-stage (P<0.0001), high Gleason grade (P<0.0001), positive surgical margins (P=0.03) and early biochemical tumor recurrence (P<0.0001). A higher rate of positive p53 immunostaining was detected in late-stage diseases including metastatic prostate cancer (P=0.0152) and hormone-refractory tumors (P=0.0003). Moreover, p53 expression was identified as an independent predictor of biochemical tumor recurrence in the subgroup of low- and intermediate-grade cancers. In summary, the results of this study show that p53 mutations characterize a small biologically aggressive subgroup of prostate cancers with a high risk of progression after prostatectomy. The rate of p53 alterations increases with prostate cancer progression.

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 and cytoplasmic) is indicative of a more undifferentiated, malignant phenotype in mammary carcinomas.

Expression of cyclin-dependent kinase inhibitors p16MTS1, p21WAF1, and p27KIP1 in HPV-positive and HPV-negative cervical adenocarcinomas

Abstract. Inactivation or down-regulation of the cell-cycle inhibitors p16MTS1, p21WAF1, and p27KIP1 is involved in the carcinogenesis of various human tumors. In cervical squamous cell carcinomas that are associated with human papillomavirus (HPV) infection, the expression or function of these proteins is impaired by the action of viral oncoproteins E6 and E7. Comparably less is known about the role of these cyclin-dependent kinase inhibitors in cervical adenocarcinomas, 15–40% of which are HPV negative. Therefore, we studied the expression of p16MTS1, p21WAF1, and p27KIP1 by immunohistochemistry in 60 cervical adenocarcinomas. HPV infection was determined by PCR, and HPV 16 and 18 E6/E7 oncogene expression was analyzed by RNA-RNA in situ hybridization. We found significant correlations of strong p16 expression with HPV 16/18 infection and HPV 16/18 E6/E7 oncogene expression (P=0.001). Moderate or strong p16 expression was also observed in 41% of HPV-negative carcinomas, indicating that HPV-independent mechanisms might also lead to p16 overexpression. In addition, stronger p21 and p27 expression was significantly associated with the detection of HPV 16 or 18 E6/E7 transcripts (P=0.015 and 0.030, respectively). Obviously, the tumor suppressor action of these proteins can be overcome in HPV-positive lesions. In contrast, absent or low p16, p21, and p27 immunostaining was observed in most HPV-negative cervical adenocarcinomas and might contribute to carcinogenesis in these tumors.

Expression pattern of the AP‐1 family in breast cancer: Association of fosB expression with a well‐differentiated, receptor‐positive tumor phenotype

In the present study, the expression of members of the AP‐1 family of transcription factors in breast tumors (n = 53) was investigated by Western blot with antibodies specific for each of the AP‐1 family members (c‐jun, junB, junD and c‐fos, fosB, fra1 and fra2). The tumors were characterized with regard to grading, staging, histology, steroid‐receptor‐expression status and c‐erbB2/neu expression. For comparison, normal breast‐tissue samples, human breast‐cancer cell lines (T47D and MDA‐MB231) and the transformed human breast epithelial cell line HBL100 were also analyzed. For c‐jun, junB, c‐fos and fra2, a relatively uniform expression pattern without significant differences among tumors was observed. junD‐protein amounts varied strongly in the tumor specimens. fosB‐expression levels also varied strongly in the tumors, weak/absent expression being found in 47%, while 45% exhibited strong/very strong levels of expression. While none of the other AP‐1 family members showed significant correlations with clinico‐pathological tumor parameters or receptor status, expression of fosB was found to correlate significantly with positive steroid‐hormone‐receptor status (in the tumors and the cell lines) and a more differentiated tumor phenotype. Expression of 2 fra‐1‐specific bands of 33 and 36.5 kDa showed significant negative correlation with fosB expression, as well as with estrogen‐receptor status and differentiation. We conclude that strong differences in the expression pattern of AP‐1 family members are present in breast tumors, and that certain members of this family, such as fosB and fra‐1, might be involved in the pathogenesis of these tumors. Int. J. Cancer (Pred. Oncol.) 84:533–538, 1999.

The Role of the AP-1 Transcription Factors c-Fos, FosB, Fra-1 and Fra-2 in the Invasion Process of Mammary Carcinomas

Members of the Fos family of AP-1 transcription factors (c-Fos, FosB, FosB2, Fra-1 and Fra-2) are able to form dimers with Jun proteins which bind to the regulatory sequences of target genes. As many proteases involved in tumor invasion are AP-1-regulated, we assumed that Fos family members might be important for invasion of mammary carcinomas. Therefore, we performed transient transfections with expression vectors for c-Fos, FosB, FosB2, Fra-1 and Fra-2, followed by matrigel invasion assays. Fra-1 transfection resulted in a 2–4-fold increase of invasive cells in both cell lines. In a less degree, the invasive potential of MDA-MB231 cells was stimulated by Fra-2, whereas MCF7 invasion was enhanced by c-Fos and FosB. By double-labelling immunocytochemistry, PAI-1 up-regulation was observed in cells transfected with c-Fos, Fra-1 and Fra-2 expression vectors, whereas MMP1 and MMP9 expression was not affected. Results of cotransfection with a MMP9 promoter construct and AP-1 expression vectors do not indicate a direct up-regulation of MMP9 expression by Fos proteins except a positive effect of c-Fos in MCF7 cells. In parallel, expression of Fos family members as determined by Western Blot analysis in 75 mammary carcinomas was correlated with MMP1, MMP9, PAI-1 and uPAR protein levels in the tumors. Interestingly, high FosB levels were significantly associated with MMP1 overexpression, whereas expression of c-Fos and phosphorylated Fra-1 correlated with MMP9 protein levels. Strong Fra-2 expression correlated with high levels of MMP9, PAI-1, the uPA/PAI-1 complex and early recurrence. These data indicate that Fos proteins, especially Fra-1, c-Fos and Fra-2, might be involved in invasion of breast cancer cells.

Prognostic and Predictive Effects of Immunohistochemical Factors in High-Risk Primary Breast Cancer Patients

Purpose: To analyze prognostic and predictive effects of immunohistochemical factors within a randomized study of high-dose versus standard-dose chemotherapy in high-risk breast cancer with >10 involved lymph nodes. Experimental Design: Histopathologic specimens in 188 of 302 patients were analyzed for Ki-67, p16, maspin, Bcl-2, Her2/neu, and p53. Results: In a univariate analysis after adjustment for therapy, tumor size, and estrogen receptor, Her2/neu positivity (P = 0.001) was a negative and Bcl2 positivity (P = 0.003) was a positive prognostic factor for event-free survival. In a multivariate analysis, Her2/neu positivity (hazard ratio, 3.68; 95% confidence interval, 2.01-6.73; P = 0.0001) had a negative influence on event-free survival, whereas p53 positivity (hazard ratio, 0.57; 95% confidence interval, 0.34-0.95; P = 0.03) and Bcl2 positivity (hazard ratio, 0.35; 95% confidence interval, 0.19-0.64; P = 0.0006) were associated with a better event-free survival. Analyzing the predictive effect of the immunohistochemical factors, an interaction between p53 and treatment could be shown (P = 0.005). The hazard ratio for high-dose chemotherapy versus standard chemotherapy is estimated as 2.3 (95% confidence interval, 0.67-7.92) in p53-negative patients and as 0.46 (95% confidence interval, 0.2-1.07) in p53-positive patients, which indicates a superiority of high-dose chemotherapy in p53-positive patients and an inferiority in p53-negative patients. No interactive effect could be shown for the other factors. Conclusions: Her2/neu and Bcl-2 are prognostic but not predictive factors in patients with high-risk primary breast cancer; p53-positive patients might benefit more from high-dose chemotherapy than from standard chemotherapy, and p53-negative patients might benefit more from standard chemotherapy than from high-dose therapy.

Expression and prognostic relevance of activated extracellular-regulated kinases (ERK1/2) in breast cancer

Extracellular-regulated kinases (ERK1, ERK2) play important roles in the malignant behaviour of breast cancer cells in vitro. In our present study, 148 clinical breast cancer samples (120 cases with follow-up data) were studied for the expression of ERK1, ERK2 and their phosphorylated forms p-ERK1 and p-ERK2 by immunoblotting, and p-ERK1/2 expression in corresponding paraffin sections was analysed by immunohistochemistry. The results were correlated with established clinical and histological prognostic parameters, follow-up data and expression of seven cell-cycle regulatory proteins as well as MMP1, MMP9, PAI-1 and AP-1 transcription factors, which had been analysed before. High p-ERK1 expression as determined by immunoblots correlated significantly with a low frequency of recurrences and infrequent fatal outcome (P=0.007 and 0.008) and was an independent indicator of long relapse-free and overall survival in multivariate analysis. By immunohistochemistry, strong p-ERK staining in tumour cells was associated with early stages (P=0.020), negative nodal status (P=0.003) and long recurrence-free survival (P=0.017). In contrast, expression of the unphosphorylated kinases ERK1 and ERK2 was not associated with clinical and histological prognostic parameters, except a positive correlation with oestrogen receptor status. Comparison with the expression of formerly analysed cell-cycle- and invasion-associated proteins corroborates our conclusion that activation of ERK1 and ERK2 is not associated with enhanced proliferation and invasion of mammary carcinomas.

Multicenter Study Using Paraffin-Embedded Tumor Tissue Testing PITX2 DNA Methylation As a Marker for Outcome Prediction in Tamoxifen-Treated, Node-Negative Breast Cancer Patients

PURPOSE We recently reported DNA methylation of the paired-like homeodomain transcription factor 2 (PITX2) gene to be strongly correlated with increased risk of recurrence in node-negative, hormone receptor-positive, tamoxifen-treated breast cancer patients using fresh frozen specimens. Aims of the present study were to establish determination of PITX2 methylation for routine analysis in formalin-fixed paraffin-embedded (FFPE) breast cancer tissue and to test PITX2 DNA methylation as a biomarker for outcome prediction in an independent patient cohort. PATIENTS AND METHODS Real-time polymerase chain reaction (PCR) technology was validated for FFPE tissue by comparing methylation measurements in FFPE specimens with those in fresh frozen specimens from the same tumor. The impact of PITX2 methylation on time to distant metastasis was then evaluated in FFPE specimens from hormone receptor-positive, node-negative breast cancer patients (n = 399, adjuvant tamoxifen monotherapy). RESULTS Reproducibility of the PCR assay in replicate measurements (r(s) > or = 0.95; n = 150) and concordant measurements between fresh frozen and FFPE tissues (r(s) = 0.81; n = 89) were demonstrated. In a multivariate model, PITX2 methylation added significant information (hazard ratio = 2.35; 95% CI, 1.20 to 4.60) to established prognostic factors (tumor size, grade, and age). CONCLUSION PITX2 methylation can be reliably assessed by real-time PCR technology in FFPE tissue. Together with our earlier studies, we have accumulated substantial evidence that PITX2 methylation analysis holds promise as a practical assay for routine clinical use to predict outcome in node-negative, tamoxifen-treated breast cancer, which might allow, based on future validation studies, the identification of low-risk patients who may be treated by tamoxifen alone.

p16/MTS1 inactivation in ovarian carcinomas: High frequency of reduced protein expression associated with hyper-methylation or mutation in endometrioid and mucinous tumors

Inactivation of the tumor‐suppressor gene p16 (MTS1/CDKN2/INK4a) has been described in various human malignancies. Although p16 deletion has been found in various ovarian tumor cell lines, p16 inactivation by homozygous deletion or mutation has been reported only sporadically in primary ovarian carcinomas. In a comprehensive study, we analyzed p16 protein expression by immuno‐histochemistry (IHC) on paraffin sections of 94 primary ovarian carcinomas of different histological subtype. Loss of expression was detected in 19 primary tumors (20%), mainly mucinous and endometrioid carcinomas. To reveal the cause of suppressed expression, we performed (i) analysis of homozygous deletions by comparative PCR after micro‐dissection, (ii) mutation analysis by single‐strand conformation polymorphism analysis and subsequent direct sequencing and (iii) methylation‐specific PCR to determine the methylation status of 5′‐CpG islands. Loss of or weak p16 expression was caused by hyper‐methylation (12/19 IHC‐negative cases), somatic mutation (10 tumors) or homozygous deletion (1 case). Aberrant p16 results by one of these methods were detected in 71–79% of endometrioid and mucinous, but in only 10% of serous‐papillary, carcinomas. Our data suggest that p16 inactivation is a typical feature of certain subtypes of ovarian carcinoma. Int. J. Cancer (Pred. Oncol.) 75:61–65, 1998.

Expression and Prognostic Value of the Cell-cycle Regulatory Proteins, Rb, p16mts1, p21waf1, p27kip1, Cyclin E, and Cyclin D2, in Ovarian Cancer

To investigate the role of cell-cycle regulatory proteins in ovarian cancer, we performed immunohistochemistry for the cell-cycle promoters cyclin E and cyclin D2 and the cell-cycle inhibitors, Rb, p16MTS1, p21WAF1, and p27 KIP1, in 93 ovarian carcinomas (77 with follow-up data). The results were correlated with clinicopathological parameters and the prognostic value was determined by multivariate analysis. Strong Rb and moderate-high cyclin E immunoreactivity in carcinomas were weakly associated with high expression of the proliferation marker Ki67. By Coxs multivariate analysis, advanced stage (p=0.013), strong Rb expression (p=0.006), and negative-weak p21 staining (p=0.011) were independent prognostic indicators of short overall survival, indicating an apparently paradoxical role of the retinoblastoma protein in these tumors. In addition, trends pointing to an association of higher age (p=0.067) and positive cyclin E immunoreactivity (p=0.093) with an unfavorable prognosis were observed.