Detection of drug resistance in human tumors by in vitro enzymatic amplification
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M Kashani-Sabet1, J J Rossi, Y Lu
1Department of Medical Oncology, City of Hope National Medical Center, Duarte, California 91010.
Abstract:
Both acquired and natural resistance to chemotherapy agents have proved problematic in the treatment of neoplasia. Thymidylate synthase, which catalyzes the synthesis of thymidine precursors, has been shown to be amplified in response to a variety of chemotherapeutic agents. The detection of such amplification could prove beneficial in the development of alternative clinical protocols. In this study we report the use of existing enzymatic amplification methods in order to detect incipient amplification of the thymidylate synthase gene upon resistance to cisplatin. The assay utilizes a modification of the polymerase chain reaction in which a sequence of the thymidylate synthase gene is amplified including two flanking oligonucleotides acting as primers for DNA synthesis. This method exhibits greater sensitivity than conventional nucleic acid detection methods and requires less than 100 ng of total RNA from patient tumors and no in vitro culturing of patient cells.
Insights
Detecting thymidylate synthase gene amplification can help predict chemotherapy resistance in cancer. This study developed a sensitive polymerase chain reaction assay for early detection, aiding alternative treatment strategies.
Area of Science:
- Oncology
- Molecular Biology
- Genetics
Background:
- Chemotherapy resistance in neoplasia is a significant clinical challenge.
- Thymidylate synthase gene amplification is a known mechanism of resistance to certain chemotherapeutic agents.
- Early detection of gene amplification can inform clinical treatment protocols.
Purpose of the Study:
- To develop a sensitive method for detecting thymidylate synthase gene amplification.
- To assess this method's utility in identifying incipient resistance to cisplatin.
- To provide a tool for developing alternative clinical strategies in cancer treatment.
Main Methods:
- Utilized a modified polymerase chain reaction (PCR) assay.
- The assay amplifies a specific sequence of the thymidylate synthase gene using flanking oligonucleotides.
- Employed enzymatic amplification for enhanced sensitivity.
Main Results:
- The developed assay demonstrated high sensitivity for detecting thymidylate synthase gene amplification.
- The method successfully detected incipient gene amplification associated with cisplatin resistance.
- The assay requires minimal sample input (less than 100 ng total RNA) and no in vitro cell culturing.
Conclusions:
- The modified PCR assay is a sensitive and efficient tool for detecting thymidylate synthase gene amplification.
- This detection method can aid in the early identification of chemotherapy resistance.
- The findings support the development of alternative clinical protocols for neoplasia treatment.