Double- and competitive-differential PCR for gene dosage quantitation.

An association of a loss of DNA replication control and the activation of erbB oncogenes can be deduced from studies with different cancers (1-5). In the first study on ovarian cancer 26% of the tumors had c-erbB-2 amplifications (6). The correlation between c-erbB-2 amplification and expression on mRNA and protein level was perfect. The median survival time of patients with ovarian cancers was negatively correlated to the degree of amplification. The association of egfr (c-erbB-1) amplification and overexpression with ovarian cancer prognosis has not been investigated as extensively as for c-erbB-2. EGF-R is expressed in normal ovarian epithelium and patients whose ovarian cancer continues to express EGF-R have a worse prognosis (7). Rearrangements of the egfr gene in ovarian cancer were also described, as identified by Southern blot analysis (8). Bauknecht submitted that a failure of chemotherapy was seen for ovarian cancers with low EGF-R expression (8).

c-erbB-2/EGFR as dominant heterodimerization partners determine a motogenic phenotype in human breast cancer cells.

Separate mechanisms for oncogenesis and metastasis have been postulated. We show here that prolonged and invasive cell migration, a key mechanism in cancer metastasis, is linked to c-erbB-2 signaling. Cell lines with c-erbB-2 and EGFR expression and transphosphorylation activity display a high transendothelial invasiveness in an endothelial-extracellular matrix model mimicking a capillary vessel wall in vitro. Tyrosine-phosphorylated c-erbB-2 receptors and EGFR are localized predominantly in areas of the cell with high membrane extension activity. On the molecular level, there is a subtle cross talk between the transmembrane signaling molecule c-erbB-2 and the actin cytoskeleton at multiple levels, including the generation of the second messenger PIP2 and the mobilization of the actin-regulatory protein gelsolin. Our data strongly suggest that c-erbB-2, especially in a heterodimer with EGFR, is closely involved in signaling pathways, inducing alterations in cell morphology that are required for a human breast cancer cell to become motile and conceivably metastatic.

Selection of potentially metastatic subpopulations expressing c-erbB-2 from breast cancer tissue by use of an extravasation model.

Overexpression of the crbB-2 gene-encoded p185(c-erbB-2) is correlated with early onset of metastasis in breast cancer patients. Furthermore, the detection of blood-borne epithelium-derived clustered cells expressing p185(c-erbB-2) was related to advanced stages in breast cancer. To further elucidate the receptor's function in the metastatic process of human breast cancers, we analyzed disaggregated cells and cell clusters from freshly dissected breast cancer tissues. We studied whether their capability of extravasation is correlated with their expression of c-erbB-2. A model for the venular wall was constructed by growing human umbilical vein endothelial cells (HUVECs) on porous membranes coated with basement membrane extracellular matrix. In four control breast cancer cell lines (SK-BR-3, MCF-7, MDA-MB-468, and MDA-MB-468, the latter transfected with a full-length c-erbB-2 cDNA vector) producing different levels of the c-erbB-2 receptor, the expression level correlated positively with the invasiveness of the cells. The invasive, predominantly clustered cells from 14 of 23 tumors were positively stained for p185(c-erbB-2) by immunocytochemistry. Furthermore, we show that the invasive cell populations express the metastasis-associated proteins matrix metalloproteinase MMP-2, CD44, and integrins alpha(v)beta3 and alpha6. In this first study on the behavior of cells and cell clusters from disaggregated operated cancers in an extravasation model, we could demonstrate the presence of c-erbB-2-expressing cell subpopulations within the individual breast cancers that are presumably of high metastatic potential.

Isolation of blood-borne epithelium-derived c-erbB-2 oncoprotein-positive clustered cells from the peripheral blood of breast cancer patients.

Clinical studies including thousands of breast cancer patients have shown that c-erbB-2 is amplified and overexpressed in 20-30% of invasive human breast cancers and that it is associated with distant metastasis in specified patient subgroups. To isolate and characterize hematogeneously spreading c-erbB-2-positive epithelium-derived cells from the peripheral blood of breast cancer patients, a combined buoyant density gradient and immuno-magnetic separation method has been used. The method utilizes a biotinylated anti-cytokeratin monoclonal antibody (MAb) for capturing the epithelium-derived cells. The expression of c-erbB-2 by the captured cells was detected using an anti-c-erbB-2 rabbit antibody (21N) coupled to an anti-rabbit gold-labeled anti-body, whereby immunoenzymatic cytokeratin staining was performed using a silver-enhanced immunogold double staining protocol. In total, 29 of the 46 patients tested had either cytokeratin (24/29) or cytokeratin/c-erbB-2 (19/29) positive clustered cells in their peripheral blood. We thus report here the presence and the frequency of clone-specifically stained clustered cells in the peripheral blood of breast cancer patients. The frequency of cytokeratin/c-erbB2 double-positive clustered cells in the peripheral blood was on average 10 times higher than that of double-positive single cells. The numbers of cytokeratin/c-erbB-2 double-positive clustered cells were positively correlated with the stage of tumors. Results of in vitro motility experiments using single and clustered cells from primary breast cancer tissue strongly support the assumption that cytokeratin/c-erbB-2 double-positive clustered cells have a high potential for locomotion. We suggest that blood-borne epithelium-derived c-erbB-2-positive clustered cells are the possible precursor cells responsible for the formation of distant metastases and bone marrow micrometastases.

Competitive-differential polymerase chain reaction for gene dosage estimation of erbB-1 (egfr), erbB-2, and erbB-3 oncogenes.

Increases in the average gene copy number (AGCN) of the erbB oncogenes, especially the erbB-2 gene, have been found in a variety of human cancers. Such information is useful with respect to prognosis of the disease. Poor reproducibility and DNA damage complicate quantitative polymerase chain reaction (PCR)-based methods. Therefore, we describe a quantitative PCR method for the estimation of AGCN in the oncogenes erbB-1 (egfr), erbB-2, and erbB-3. The method comprises a competitive and differential PCR in a one-tube reaction (competitive-differential PCR, or cdPCR). Genomic sequences of the oncogene and the human beta-globin (HBB) reference gene and two competitors for the oncogene and reference gene were amplified with two primer pairs simultaneously. The competitors were chosen to be amplified with the same efficiency as the genomic sequences. Using this method, we confirmed egfr and erbB-2 amplification in cancer cell lines and tumor tissue, and we also detected erbB-3 amplifications. Furthermore, gene dosage decreases were detectable, e.g., an erbB-2 hemizygosity in MCF-7 cells. Thus, cdPCR facilitates the detection of both increases and decreases in AGCN with high reproducibility, sensitivity, and accuracy. This method is therefore suitable for clinical studies on erbB oncogene dosage deviations.