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Real-time reverse transcription-PCR assay for future management of ERBB2-based clinical applications
I Bièche1, P Onody, I Laurendeau
1Laboratoire de Génétique Moléculaire, Faculté des Sciences Pharmaceutiques et Biologiques de Paris, 4 Avenue de l'Observatoire, F-75006 Paris, France.
Background:
Gene amplification/overexpression of ERBB2 (HER2, neu) is a major event in human breast tumorigenesis. ERBB2-based therapeutic agents and ERBB2-specific gene therapy are under development. These new perspectives call for a sensitive and accurate method to screen breast cancer patients for ERBB2 alterations.
Methods:
We have developed and validated a real-time quantitative reverse transcription (RT)-PCR assay, based on fluorescent TaqMan methodology, to quantify ERBB2 gene expression at the mRNA level in breast tumors. This recently developed method of nucleic acid quantification in homogeneous solutions has the potential for a wide dynamic range, interlaboratory agreement, and high-throughput capacity without tedious post-PCR processing. The ERBB2 mRNA signal was normalized to the signal for TATA box-binding protein mRNA.
Results:
The dynamic range was >1000-fold. The relationship between C(t) and log starting concentration was linear (r(2) >/=0.99). The mean (SD) normalized expression of ERBB2 in healthy breast tissue was 0.95 (0.37). Overexpression (>5 SD above mean for healthy breast) of the ERBB2 gene was observed (at 3.2- to 135-fold) in 23 (17%) of 134 breast tumor RNA samples. As expected, ERBB2 overexpression was present in all tumors with ERBB2 gene amplification but was uncommon and at a low ratio (<5) in breast cancers without gene amplification.
Conclusions:
This new simple, rapid, semi-automated assay is a major alternative to fluorescence in situ hybridization and immunochemistry for gene alteration analysis in human tumors and may be a powerful tool for large randomized, prospective cooperative group trials and to support future ERBB2-based biological and gene therapy approaches.
Insights
A new real-time quantitative reverse transcription PCR assay accurately measures ERBB2 (HER2) gene expression in breast tumors. This method aids in screening patients for ERBB2 alterations, crucial for developing targeted therapies.
Area of Science:
- Oncology
- Molecular Biology
- Genetics
Background:
- ERBB2 (HER2, neu) gene amplification/overexpression is a key factor in breast cancer development.
- Emerging ERBB2-targeted therapies necessitate precise methods for detecting ERBB2 alterations in patients.
- Accurate screening for ERBB2 alterations is vital for advancing breast cancer treatment strategies.
Purpose of the Study:
- To develop and validate a sensitive and accurate real-time quantitative reverse transcription PCR (RT-PCR) assay for quantifying ERBB2 gene expression at the mRNA level in breast tumors.
- To establish a reliable method for screening breast cancer patients for ERBB2 alterations, supporting the development of ERBB2-based therapies.
Main Methods:
- A real-time quantitative RT-PCR assay utilizing fluorescent TaqMan methodology was developed and validated.
- ERBB2 mRNA levels were quantified and normalized against TATA box-binding protein mRNA for accurate comparison.
- The assay demonstrated a wide dynamic range (>1000-fold) and linear relationship (r(2) >/=0.99) between C(t) and log starting concentration.
Main Results:
- ERBB2 overexpression, defined as >5 standard deviations above the mean in healthy breast tissue, was detected in 17% (23/134) of breast tumor samples, with fold changes ranging from 3.2 to 135.
- The assay showed high precision, with a mean normalized expression of 0.95 (0.37) in healthy breast tissue.
- ERBB2 overexpression correlated with gene amplification, being present in all tumors with amplification and uncommon in those without.
Conclusions:
- The developed RT-PCR assay offers a simple, rapid, and semi-automated alternative to traditional methods like fluorescence in situ hybridization (FISH) and immunohistochemistry (IHC) for analyzing gene alterations in tumors.
- This assay is a powerful tool for large-scale clinical trials and supports the advancement of ERBB2-targeted biological and gene therapies.
- The method provides a sensitive and accurate means for screening breast cancer patients, facilitating personalized treatment approaches.