Borderline HER‐2 breast cancer cases: Histochemical versus real‐time PCR analysis and impact of different cut‐off values

Background. HER-2 is a key molecule serving as the therapeutic target, prognostic biomarker, and classification marker in breast cancer. Accurate microRNA profilings had not been conducted in purified tumor cells of HER-2-negative and HER-2-positive tissue specimens obtained from breast cancer patients. Methods. (i) Differential expression microRNA discovery using laser capture microdissection- (LCM-) assisted specimen preparation and microRNA array chips on HER-2 overexpressing and triple-negative breast carcinoma (TNBC) subtype tissues, (ii) differential expression microRNA validation by quantitative real-time PCR, and (iii) independent validation on tissue microarray. Results. Five microRNAs (miR-20a-5p, miR-221-3p, miR-362-5p, miR-502-3p, and miR-222-3p) were screened and validated as upregulated microRNAs in TNBC cells comparing to HER-2 overexpressing cells using a microRNA array (5 cases in each group) and quantitative real-time PCR (20 cases in each group). The expression difference of miR-362-5p had the most significant statistical significance (p=0.0016) among the five microRNAs. The expression of miR-362-5p and its target gene Sema3A was further analyzed using in situ hybridization (ISH) and immunohistochemistry on standard tissue sections (n=150). 70.8% of HER-2-negative cells showed moderate expression of miR-362-5p whereas 20.4% HER-2-negative cells correlated with strong expression of miR-362-5p (p<0.0001). The proportion of patients with moderate/strong miR-362-5p expression in luminal, HER-2 overexpressing, and TNBC subtypes were 53.2%, 22.2%, and 74.3%, respectively (p=0.0002). High miR-362-5p expressers had shorter overall survival in the univariate analysis (p=0.046). There was a significant negative correlation between miR-362-5p and Sema3A expression (p<0.0001). The patients with negative/weak Sema3A protein expression had poorer prognosis than those with moderate (HR: 3.723, p=0.021) or strong (HR: 3.966, p=0.013) Sema3A protein expression in the multivariate analysis. Conclusions. miR-362-5p/Sema3A might provide a promising therapeutic pathway and represents a candidate therapeutic target of the TNBC subtype.

Download Full-text

HER-2/neu gene amplification assessment in breast cancer patients in Isfahan province by real time PCR, differential PCR and immunohistochemistry

Gene ◽

10.1016/j.gene.2012.01.025 ◽

2012 ◽

Vol 497 (2) ◽

pp. 237-242 ◽

Cited By ~ 6

Author(s):

Zohreh Hojati ◽

Elham Orangi

Keyword(s):

Breast Cancer ◽

Real Time ◽

Cancer Patients ◽

Gene Amplification ◽

Real Time Pcr ◽

Breast Cancer Patients ◽

Isfahan Province ◽

Her 2

Download Full-text

HER-2/neu Gene Copy Number Quantified by Real-Time PCR: Comparison of Gene Amplification, Heterozygosity, and Immunohistochemical Status in Breast Cancer Tissue

Clinical Chemistry ◽

10.1373/49.2.219 ◽

2003 ◽

Vol 49 (2) ◽

pp. 219-229 ◽

Cited By ~ 41

Author(s):

Melanie Königshoff ◽

Jochen Wilhelm ◽

Rainer M Bohle ◽

Alfred Pingoud ◽

Meinhard Hahn

Keyword(s):

Breast Cancer ◽

Real Time ◽

Cell Lines ◽

Real Time Pcr ◽

Gene Copy ◽

Protein Overexpression ◽

Tissue Samples ◽

Cancer Pathogenesis ◽

Her 2 ◽

Gene Status

Abstract Background: Amplification of the oncogene HER-2/neu influences breast cancer pathogenesis, and therapy and prognosis may be affected by the degree of amplification. The extent of amplification or protein overexpression typically is analyzed by fluorescence in situ hybridization or immunohistochemistry (IHC), but quantitative PCR techniques have been described that may provide alternatives to these methods. Methods: We developed a rapid-cycle, real-time PCR assay for quantification of HER-2/neu gene status. We compared results obtained with this assay with short tandem repeat findings by capillary electrophoresis (CE) and with protein overexpression assessments by IHC. Accuracy and linearity were tested on cell lines and with simulation experiments. We analyzed the amplification of HER-2/neu in 51 clinical tissue samples from patients with suspected breast cancer. Results: The intra- and interrun CVs for HER-2/neu quantification by real-time PCR were 12% and 18%, and the CV for different simulated amplification and deletion experiments was <7%. The results for HER-2/neu gene status in cell lines matched the values reported in literature. We detected HER-2/neu amplification by real-time PCR in 11 samples, all from patients with invasive ductal carcinoma. Allelic imbalances were found by CE analyses in three samples and by protein overexpression in six samples; five of these were also detected by real-time PCR. Comparison of the quantification results with known prognostic indices yielded results similar to those reported in several other published studies. Conclusions: The assay is suitable for accurate and precise quantification of HER-2/neu copy numbers in tumor tissue samples obtained in routine clinical practice.

Download Full-text