Thomas Löning

MED12 mutations in uterine fibroids—their relationship to cytogenetic subgroups

Recurrent chromosomal alterations are found in roughly 20% of all uterine fibroids but in the majority cytogenetic changes are lacking. Recently, mutations of the gene mediator subcomplex 12 (MED12) have been detected in a majority of fibroids but no information is available whether or not they co‐occur with cytogenetic subtypes as, e.g., rearrangements of the genes encoding high mobility group AT‐hook (HMGA) proteins. In a total of 80 cytogenetically characterized fibroids from 50 patients, we were not only able to confirm the frequent occurrence of MED12 mutations but also to stratify two mutually exclusive pathways of leiomyomagenesis with either rearrangements of HMGA2 reflected by clonal chromosome abnormalities affecting 12q14∼15 or by mutations affecting exon 2 of MED12. On average the latter mutations were associated with a significantly smaller tumor size. However, G>A transitions of nucleotides c.130 or c.131 correlate with a significantly larger size of the fibroids compared to other MED12 mutations thus explaining the high prevalence of the former mutations among clinically detectable fibroids. Interestingly, fibroids with MED12 mutations expressed significantly higher levels of the gene encoding wingless‐type MMTV integration site family, member 4 (WNT4). Based on these findings and data from the literature, we hypothesize that estrogen and the mutated MED12 cooperate in activating the Wnt pathway which in turn activates β‐catenin known to cause leiomyoma‐like lesions in a mouse model. The occurrence of a “fibroid‐type mutation” in a rare histologic subtype of endometrial polyps suggests that this mechanism is not confined to uterine leiomyomas.

Keratin polypeptides distribution in normal and diseased human epidermis and oral mucosa

Immune sera against total keratin and keratin polypeptide subunits were induced in guinea pigs, using the different bands of SDS polyacrylamide gel electrophoresis of fibrous proteins of stratum corneum, derived from normal human epidermis. The distribution of the different polypeptides was studied in numerous human biopsies of normal epidermis, normal oral mucosa and epidermal and mucosal inflammatory, premalignant and malignant lesions using the indirect immunoperoxidase method. Antisera against total keratin (TK) and against the keratin polypeptide of M.W. 55,000 dalton (55K) labelled all keratinocytes in normal and pathological conditions. These antisera may be useful for the histodifferentiation in diagnostic pathology. Atisera against the keratin polypeptides of M.W. 67,000 (67K) and 62,000 dalton (62K) identified only keratin antigens in the spinous, granular and keratinized layer of normal epidermis and oral mucosa. No labelling of the basal layer was achieved with these immune sera. However, there were important differences in the distribution of these keratin antigens in altered epithelia which may be of value in the differential diagnosis of inflammatory, premalignant and malignant lesions of the skin and oral mucosa.

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.

Matrix-metalloproteinases 1, 2 and 3 and their tissue inhibitors 1 and 2 in benign and malignant breast lesions: an in situ hybridization study

Abstract Invasive growth requires degradation of extracellular matrix. Altered expression of matrix degrading enzymes may indicate an increased potential for invasive growth. We determined the expression patterns of matrix-metalloproteinases (MMP)-1, -2, and -3 and of the tissue inhibitors of metalloproteinases (TIMP)-1 and -2 by in situ hybridization with isotopically labeled RNA probes in normal breast tissue (n=6), fibrocystic disease (n=20), five cases of which contained radial scars, lobular carcinoma in situ (CLIS; n=5), ductal carcinoma in situ (DCIS; n=9) and invasive carcinomas (n=24). Only a few cells displayed MMP-1- and MMP-2-specific labeling in normal breast tissue and fibrocystic disease. Noninvasive ductal carcinomas showed elevated MMP-2 transcript levels in peritumor stromal cells in the absence of significant MMP-1 specific signals. In general, compared with adjacent normal breast tissue, a gradual increase of MMP-2 was found in noninvasive to invasive cancers. Invasive ductal and lobular carcinomas displayed co-expression of MMP-1 and MMP-2 by stromal cells, mainly of the invasion front, with high signal intensity particularly in high-grade invasive carcinomas. Tumor cells and peritumor stroma showed low MMP-3 transcript levels, especially in medullary carcinomas. TIMP-1 and -2 transcript levels were increased in invasive carcinomas correlating with the histological grade. These RNA expression patterns suggest an increased invasive potential in breast carcinomas even prior to histologically overt invasive growth.

Differential Expression of CD66a (BGP), a Cell Adhesion Molecule of the Carcinoembryonic Antigen Family, in Benign, Premalignant, and Malignant Lesions of the Human Mammary Gland

CD66a, also known as biliary glycoprotein (BGP), is a member of the carcinoembryonic antigen (CEA) family and the human homologue of the rat cell-CAM. There is evidence that aberrant expression or loss of CD66a in tumor tissue is of biological significance. No data about its expression in breast carcinoma cells and only sparse information about the expression of CD66a in normal breast are available thus far. In this study we used monoclonal antibodies to analyze the expression of CD66a and CEA in normal tissue, benign lesions, and in noninvasive and invasive carcinomas of the mammary gland. In normal tissue and benign lesions, CD66a was consistently expressed at the apical sites of epithelial cells and in myoepithelia, whereas CEA was absent or was restricted only to some apical membranes within the ductal tree. The specific staining of myoepithelia was most evident in pseudoinfiltrative radial scars and sclerosing adenosis. However, the apical expression of CD66a disappeared with the development of the malignant phenotype in noninvasive and invasive carcinomas, and changed gradually from low- to high-grade noninvasive carcinomas into a predominant uniform membrane staining all around the atypical cells. CEA expression was irregular in intensity and distribution. The native apical CD66a staining was partially preserved in some highly differentiated invasive carcinomas with a better prognosis, such as tubular and papillary carcinomas. These findings indicate that loss of CD66a expression rather than a change in staining patterns coincides with the development of the malignant phenotype.

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.

One-step nucleic acid amplification-a molecular method for the detection of lymph node metastases in breast cancer patients; results of the German study group.

Sentinel lymph node (SN) biopsy is part of the staging procedure in breast cancer patients. In this study, we compared an intraoperative tool named one-step nucleic acid amplification (OSNA) to our routine histological investigation. OSNA consists of a short homogenization step followed by amplification of cytokeratin (CK) 19 mRNA directly from the lysate. To evaluate the performance of OSNA in comparison to histology, analysis of 343 axillary lymph nodes (ALN) from 93 breast cancer patients was performed with both methods. Discordant samples were subjected to other methods. If these tests supported the OSNA results, these samples were excluded from the study. The concordance rate was 91.8%, sensitivity 98.1%, and specificity 90.8% before and 95.5%, 100%, and 95.6%, respectively, after discordant case investigation. Our results show that OSNA is an excellent method for the detection of metastases in lymph nodes and can be applied as an intraoperative diagnostic approach.

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.