Detection of HER-2/neu oncogene amplification in flow cytometry-sorted breast ductal cells by competitive polymerase chain reaction

Abstract

Background. The amplification and/or overexpression of the HER‐2/neu oncogene has been proposed as an important prognostic marker in breast cancer. However, contradictory results from various groups regarding whether there is statistical significance in HER‐2 amplification or overexpression in predicting overall and disease free survival in node positive versus node negative patients exist in the literature. Current assays on quantifying the HER‐2 oncogene rely on DNA extracted from homogenized breast tissue. Not only is a large amount of tissue required, but also, the DNA extract is contaminated with DNA from stromal cells and leukocytes, leading to decreased specificity and sensitivity of the HER‐2 assay. Improving the specificity (DNA from breast ductal cells) and the sensitivity (competitive polymerase chain reaction [PCR]) of the HER‐2 amplification detection assay will help resolve some of these controversies. Methods. Using multiparameter flow cytometry (FCM), ductal cells from breast biopsies and fine needle aspirations (FNAs) are identified and selectively sorted using anti‐cytokeratin, anti‐HER‐2 antibody labeling and DNA staining. HER‐2 amplification in these sorted cells is then quantified by competitive DNA PCR using a competitive reference standard mutant template that is susceptible to the restriction enzyme Sma‐1. Results. Applying this strategy, SK‐BR‐3, an HER‐2 amplified breast cancer cell line, was found to have approximately 9X baseline HER‐2 oncogene copies. In addition, MCF‐7, a known HER‐2 nonamplified breast cancer cell line, was found to have baseline HER‐2 oncogene copies. In the 10 clinical breast samples tested, 4 of the 10 breast cancers were HER‐2 amplified using as few as 1000 cells. The cytokeratin positive cells of these cancers, in contrast to the cytokeratin negative cells, have detectably higher HER‐2 amplification (7.2 ± 2.8X versus 3.2 ± 1.1X, respectively). Hence, HER‐2 gene amplification would have been underestimated if unsorted cells were used because of stromal dilution. In the cytokeratin positive cells that were HER‐2 oncogene amplified, corresponding HER‐2 oncoprotein overexpression was detected by FCM. Conclusions. Using FCM, the ductal cell subpopulation of a breast specimen can be successfully sorted from breast biopsy and FNA specimens. Moreover, by applying the technique of competitive PCR, improved specificity and sensitivity in HER‐2 oncogene amplification detection is achieved. The entire procedure can be accomplished in 1 day, allowing for a cost‐effective assay and rapid turnaround time. Cancer 1994; 73:2771–8.

Fusion to Listeriolysin O and Delivery by Listeria monocytogenes Enhances the Immunogenicity of HER-2/neu and Reveals Subdominant Epitopes in the FVB/N Mouse

“…ϩ cell depletion CD8 ϩ T cells were depleted in NT-2 tumor-bearing mice with 0.5 mg of 2.43 (21) on days 6,7,8,11,14,17,20, and 23 posttumor injection. The 2.43 (anti-CD8 ϩ T cell) Ab was affinity purified from ascites on a protein G-Sepharose column (Amersham Biosciences).…”

“…It consists of an extracellular domain, a transmembrane domain, and an intracellular domain, which is known to be involved in cellular signaling (1)(2)(3)(4). It is overexpressed in 25-40% of all breast cancers and is also overexpressed in many cancers of the ovaries, lung, pancreas, and gastrointestinal tract (5)(6)(7). The overexpression of HER-2/neu is associated with uncontrolled cell growth and signaling, both of which contribute to the development of tumors (2,8).…”

Developments in Quantitative PCR

“…measurement of c-erbB-2 oncogene amplification is reported in Figure 4A. This technique is highly sensitive and allows the measurement in isolated cells obtained by flow sorting (91), or even in samples with degraded DNA, as in the case of archived samples (47,85,86).…”

Evaluation of Listeria Monocytogenes Based Vaccines for HER-2/neu in Mouse Transgenic Models of Breast Cancer

“…In addition to this, over 1500 men are diagnosed with breast cancer each year in the United States alone. 20-40% of all breast cancers overexpress the tumor associated antigen, HER-2/neu, and it is also overexpressed in cancers of the ovaries, lung, pancreas, and gastrointestinal tract (Disis et al, 1997;Knutson et al, 1999;Li et al, 1994). The overexpression of HER-2/neu in breast adenocarcinomas has been associated with the progression of focal tumors to metastatic cancer and the subsequent poor prognosis of patients (Knutson et al, 1999;Treurniet et al, 1992).…”