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Updated: Oct 15, 2026
Optimization of a Multiplex RNA-based Expression Assay Using Breast Cancer Archival Material
Published on: August 1, 2018
Detection of gene amplification in archival breast cancer specimens by laser-assisted microdissection and
U Lehmann1, S Glöckner, W Kleeberger
1Institute of Pathology, Medizinische Hochschule Hannover, Germany. ulrich@mh-hannover.de
Abstract:
Gene amplification is one of the most important mechanisms leading to deregulated gene expression in cancer. The exact quantitative detection of this frequent genomic alteration in solid tumors is often hampered by an admixture of nonneoplastic bystander and stroma cells. To overcome this obstacle and to develop an objective quantitative method we have combined laser-assisted microdissection of tumor cells with the novel 5'-exonuclease-based real-time polymerase chain reaction (PCR) assay. The latter method enables the highly reproducible exact quantification of minute amounts of nucleic acids. As a model system amplification of c-erbB2/Her-2/neu gene and the adjacent topoisomerase IIalpha gene was determined in paraffin-embedded breast cancer specimens (n = 23) after immunohistochemical labeling and laser-based microdissection of tumor cells. The high sensitivity of real-time PCR enabled the reliable and objective detection of low-level amplifications in as few as 50 cells from archival tissue sections. Low-level amplifications were shown to escape from detection unless tumor cells were isolated by microdissection. In selected cases intratumor heterogeneity was demonstrated using areas of approximately 50 to 100 cells. This novel approach combining immunohistochemistry, laser microdissection, and quantitative kinetic PCR allows morphology-guided studies in archival tissue specimens and will enable the exact quantification of gene copy numbers in even small and precancerous lesions.
Insights
Gene amplification drives cancer by deregulating gene expression. A new method combining laser microdissection and real-time PCR accurately quantifies gene amplification in cancer cells from tissue samples.
Area of Science:
- Oncology
- Molecular Biology
- Genetics
Background:
- Gene amplification is a key mechanism in cancer development and progression.
- Detecting gene amplification in solid tumors is challenging due to non-tumor cell contamination.
- Accurate quantification of gene copy number alterations is crucial for cancer diagnosis and treatment.
Purpose of the Study:
- To develop an objective and quantitative method for detecting gene amplification in solid tumors.
- To overcome limitations of traditional methods in quantifying genomic alterations in mixed cell populations.
- To enable precise gene copy number analysis in archival tissue specimens.
Main Methods:
- Combined laser-assisted microdissection of tumor cells with 5'-exonuclease-based real-time polymerase chain reaction (PCR).
- Used immunohistochemical labeling to identify tumor cells for microdissection.
- Quantified amplification of c-erbB2/Her-2/neu and topoisomerase IIalpha genes in paraffin-embedded breast cancer specimens.
Main Results:
- The novel method accurately quantified gene amplification in as few as 50 cells from archival tissue.
- Low-level gene amplifications were detected only after microdissection, highlighting the importance of cell isolation.
- Demonstrated intratumor heterogeneity of gene amplification in selected cases.
- Achieved highly reproducible quantification of minute nucleic acid amounts.
Conclusions:
- The integrated approach of immunohistochemistry, laser microdissection, and quantitative kinetic PCR enables morphology-guided studies.
- This method allows for the exact quantification of gene copy numbers in small lesions and precancerous tissues.
- The technique provides a reliable tool for studying genomic alterations in archival cancer specimens.
