Kerstin Pischinger

Expression of Sex Steroid Receptors and their Co-Factors in Normal and Malignant Breast Tissue: AIB1 is a Carcinoma-Specific Co-Activator

The differential expression pattern of estrogen receptor alpha (ER-α), estrogen receptor beta (ER-β) and their co-activator/co-repressor proteins is thought to modulate estrogenic action and to be present already during the early stages of tumorigenesis. It has therefore been postulated that certain co-activator and co-repressor proteins contribute to the development of breast cancer. There are some reports providing information on gene amplification and mRNA over-expression of certain co-factors in breast cancer, but to date there is only limited knowledge about their respective protein expressions. The aim of this study was to examine the expression of four steroid receptor co-activators (steroid receptor co-activator 1 (SRC-1), transcription intermediary factor 2 (TIF 2), protein 300 kDa/CREB binding protein (p300/CBP), amplified in breast cancer 1 (AIB1)), and of the co-repressor nuclear receptor co-repressor (NCoR), in malignant breast tissues and in matching normal breast biopsies of the same individuals. Protein expression was analyzed by immunohistochemistry and was compared to prognostic parameters such as lymph node involvement, tumor grading and receptor status. All members of the co-regulatory protein family were detected in both, benign and matching malignant tissue samples, except for AIB1, which was found to be expressed exclusively in malignant epithelium. AIB1 was preferentially present in carcinomas with high tumor grade (r = 0.48, p = 0.014), and was co-expressed with p300/CBP (r = 0.54, p = 0.006). TIF 2 correlated significantly to nodal status (r = 0.46, p = 0.025). Furthermore, protein levels of ER-β, p300/CBP and AIB1 were higher in invasive ductal carcinomas than in normal mammary tissue. The tumoral ER-α protein expression was significantly correlated with that of PgR (r = 0.61, p = 0.001) and NCoR (r = 0.4, p = 0.043), whereas ER-β expression was associated with SRC-1 (r = 0.68, p ≤ 0.001), TIF 2 (r = 0.64, p = 0.001) and NCoR (r = 0.48, p = 0.014) protein levels in malignant specimens. In our hands, 20% of ER-β positive tumors did not express ER-α protein, thereby suggesting that a substantial fraction of ER-beta positive tumors is falsely considered to be ‘estrogen receptor negative’ if only ER-α specific antibodies are employed in the histological assessment of the ER status.

Co-expression of ErbB-family members in human breast cancer: Her-2/neu is the preferred dimerization candidate in nodal-positive tumors

Over-expression of members of the ErbB-receptor family has been associated with malignant transformation. The amplification of Her-2/neu in tumor tissue is now an established prognostic factor in breast cancer. In order to initiate signal transduction, ErbB-receptor monomers need to form homo- or heterodimers. The composition of these dimers is thought to influence both quality and quantity of downstream signaling pathways, and to determine the biological response. We have investigated the protein expression pattern of the four ErbB-receptors EGFR, Her-2/neu, Her-3 and Her-4, and correlated it with their putative ligands EGF, TGF-α and HRG in 74 women with invasive breast cancer. Using western blot-analysis on cell membrane isolates, we detected the co-expression of all four ErbB-family members in 79.7% of cases, and of all of the three investigated ligands in 82.4%. We did not observe a correlation between EGFR and Her-2/neu or Her-4 protein expression, EGFR and Her-3 (p = 0.005), and Her-3 and Her-4 (p = 0.05) were clearly co-expressed. The strongest overall correlation, was found between Her-2/neu and Her-3 (p < 0.001) and between Her-2/neu and Her-4 (p = 0.001). This was particularly true in nodal-positive tumors (p <0.001 and p = 0.002) whereas in nodal-negative tumors the co-expression was either less significant (Her-2/neu and Her-3; p = 0.01) or not significant (Her-2/neu and Her-4). The co-expression of EGFR/Her-3 was associated with the expression of all ligands, whereas the Her-2/neu/Her-3 was correlated with HRG (p = 0.002), thereby indicating a functional relation between specific receptor-dimer combinations and putative ligands. Taken together, we have performed the first comprehensive survey of ErbB-system expression in breast cancer, and have demonstrated the presence of a co-regulated receptor/ligand system in vivo. We have further shown that Her-2/neu is the preferred co-expression partner in nodal-positive tumors and thus the most likely dimerization candidate in malignant breast tumors.

Presence of nanobacteria in psammoma bodies of ovarian cancer: evidence for pathogenetic role in intratumoral biomineralization

Aims:  The presence of laminated, calcified extracellular debris known as psammoma bodies is a well‐known histomorphological feature of ovarian adenocarcinomas and other human malignancies. Biomineralization has recently been found to be associated with a group of extremely small Gram‐negative bacteria capable of precipitating calcium salts. The aim of the present study was to evaluate a possible pathogenic link between the development of psammoma bodies and nanobacteria infection.

Detection of hepatitis C virus (HCV) RNA in normal cervical smears of HCV-seropositive patients

The presence of hepatitis C virus (HCV) in normal cervical smears (CS) obtained from 22 HCV-seropositive and 50 HCV-seronegative patients was assessed by reverse-transcriptase-polymerase chain reaction (RT-PCR). The presence of HCV in serum was established by use of enzyme-linked immunosorbent assay, Western blot test, and RT-PCR. HCV was detected in 36.4% (n=8) of CS cells recovered from 22 HCV-seropositive patients, but not in CS samples obtained from 50 HCV-seronegative patients. Furthermore, cells from the CS of 2 seropositive/smear-positive patients and 1 seropositive/smear-negative patient were isolated; HCV RNA was detectable in the cervical lymphocytes of the 2 smear-positive patients, but not in epithelial cells or granulocytes. HCV RNA is detectable in the CS of some HCV-seropositive women. The clinical importance of these data requires further study.

Oncogenic Potential of c-erbB-2 and Its Association with c-K-ras in Premalignant and Malignant Lesions of the Human Uterine Endometrium

The aim of this study was to detect activated c-K-ras by gene point mutation and to find c-erbB-2 gene amplification with p185 expression in association with the c-K-ras gene product p21 in the human endometrium. Specimens obtained from 25 normal, 31 hyperplastic and 72 malignant samples of the human endometrium were examined for point mutation in codons 12, 13 and 61 of the c-K-ras by direct sequencing and c-erbB-2 gene amplification with p185 and p21 expression by differential polymerase chain reaction (DPCR) and immunohistochemistry. Neither the normal endometrium nor endometrial hyperplasias were found to have mutations in the c-K-ras gene, although a double mutation of codons 12 and 13 as a single-point mutation was observed in one case of endometrioid carcinoma (2.8%). In each of two other cases of endometrioid carcinoma (2/72), two single-point mutations of codon 13 (5.6%) were shown. Using DPCR, we found c-erbB-2 to be amplified in 15 premalignant (48%) and 45 malignant (63%) samples. We noticed that nonamplification of the c-erbB-2 gene was associated with the absence of immunoreactivity. Our data indicate that, while c-erbB-2 plays a role in the early development of endometrioid carcinomas, c-K-ras gene activation by point mutation does not.

Detection of human papillomavirus on Papanicolaou-stained cervical smears using indirect in situ polymerase chain reaction hybridization

OBJECTIVE Polymerase chain reaction (PCR) and indirect in situ hybridization were combined to detect human papillomavirus (HPV) DNA on Papanicolaou (PAP)-stained cervical smears. To our knowledge, this is the first report of an experiment using indirect in situ PCR (IS-PCR) on PAP-stained cervical smears. DESIGN We collected native cell specimens from cervicovaginal lavage of 162 patients with squamous intraepithelial lesions. Solution-phase PCR (SP-PCR) was performed as the reference method in the detection of HPV DNA. Indirect IS-PCR was carried out for the same patients to detect the HPV DNA types 6/11 and 16/18 after the PAP-stained smears had been decolorized. Low-risk and high-risk HPV DNA types were also detected by both SP-PCR and indirect IS-PCR. RESULTS In the evaluation by indirect IS-PCR, 48 of 81 PAP-stained cell smears of low-grade squamous intraepithelial lesions were positive for HPV DNA, as compared to 40 positive cell smears determined by indirect SP-PCR (sensitivity of indirect IS-PCR compared to SP-PCR, 98.1%). Forty-two of 42 high-grade squamous intraepithelial lesion samples were positive for HPV DNA, as determined by both methods (sensitivity of IS-PCR, 100%). Cell lines investigated in this study as positive or negative controls for HPV DNA were confirmed by indirect IS-PCR and SP-PCR. CONCLUSIONS Our data show that in comparison to SP-PCR, indirect IS-PCR is a highly sensitive method to detect HPV DNA in cell smears from the uterine cervix. The advantages of indirect IS-PCR are (a) low numbers of cells needed, (b) the possibility of using PAP-stained specimens, and (c) cytologic details of smears can be preserved.