Related Experiment Video
Updated: May 15, 2026
Simple and Fast Rolling Circle Amplification-Based Detection of Topoisomerase 1 Activity in Crude Biological Samples
Published on: December 2, 2022
Development and validation of an immunoassay for quantification of topoisomerase I in solid tumor tissues
Thomas D Pfister1, Melinda Hollingshead, Robert J Kinders
1Laboratory of Human Toxicology and Pharmacology, Applied/Developmental Research Support Directorate, SAIC-Frederick, Inc., Frederick National Laboratory for Cancer Research, Frederick, Maryland, United States of America. pfistert@mail.nih.gov
Background:
Topoisomerase I (Top1) is a proven target for cancer therapeutics. Recent data from the Fluorouracil, Oxaliplatin, CPT-11: Use and Sequencing (FOCUS) trial demonstrated that nuclear staining of Top1 correlates with chemotherapeutic efficacy. Such a correlation may help identify patients likely to respond to Top1 inhibitors and illuminate their mechanism of action. Cellular response to Top1 inhibitors is complex, but Top1 target engagement is a necessary first step in this process. This paper reports the development and validation of a quantitative immunoassay for Top1 in tumors.
Methodology/Principal Findings:
We have developed and validated a two-site enzyme chemiluminescent immunoassay for quantifying Top1 levels in tumor biopsies. Analytical validation of the assay established the inter-day coefficient of variation at 9.3%±3.4% and a 96.5%±7.3% assay accuracy. Preclinical fit-for-purpose modeling of topotecan time- and dose-effects was performed using topotecan-responsive and -nonresponsive xenografts in athymic nude mice. Higher baseline levels of Top1 were observed in topotecan-responsive than -nonresponsive tumors. Top1 levels reached a maximal decrease 4 to 7 hours following treatment of engrafted mice with topotecan and the indenoisoquinoline NSC 724998.
Conclusions/Significance:
Our analysis of Top1 levels in control and treated tumors supports the previously proposed mechanism of action for Top1 inhibitor efficacy, wherein higher baseline Top1 levels lead to formation of more covalent-complex-dependent double-strand break damage and, ultimately, cell death. In contrast, xenografts with lower baseline Top1 levels accumulate fewer double-stand breaks, and may be more resistant to Top1 inhibitors. Our results support further investigation into the use of Top1 levels in tumors as a potential predictive biomarker. The Top1 immunoassay described in this paper has been incorporated into a Phase I clinical trial at the National Cancer Institute to assess pharmacodynamic response in tumor biopsies and determine whether baseline Top1 levels are predictive of response to indenoisoquinoline Top1 inhibitors.
Insights
A new immunoassay accurately measures Topoisomerase I (Top1) in tumors. Higher baseline Top1 levels predict better response to Top1 inhibitors, supporting its use as a predictive biomarker in cancer therapy.
Area of Science:
- Oncology
- Molecular Biology
- Biochemistry
Background:
- Topoisomerase I (Top1) is a validated target for cancer therapeutics.
- Nuclear Top1 levels correlate with chemotherapeutic efficacy, suggesting potential as a predictive biomarker.
- Understanding Top1 target engagement is crucial for cellular response to Top1 inhibitors.
Purpose of the Study:
- To develop and validate a quantitative immunoassay for measuring Topoisomerase I (Top1) levels in tumor biopsies.
- To investigate the correlation between baseline Top1 levels and response to Top1 inhibitors in preclinical models.
- To support the use of Top1 levels as a predictive biomarker for patient selection in clinical trials.
Main Methods:
- Development and validation of a two-site enzyme chemiluminescent immunoassay for Top1 quantification.
- Analytical validation including inter-day coefficient of variation and assay accuracy.
- Preclinical modeling using topotecan-responsive and -nonresponsive xenografts in athymic nude mice to assess Top1 levels and drug effects.
Main Results:
- The developed immunoassay demonstrated good analytical performance (CV 9.3%±3.4%, accuracy 96.5%±7.3%).
- Higher baseline Top1 levels were observed in topotecan-responsive xenografts compared to non-responsive ones.
- Top1 levels decreased significantly 4-7 hours post-treatment with topotecan or NSC 724998.
Conclusions:
- Higher baseline Top1 levels are associated with increased double-strand break damage and cell death, supporting Top1 inhibitors' mechanism of action.
- Lower baseline Top1 levels may indicate resistance to Top1 inhibitors.
- The Top1 immunoassay is being utilized in a Phase I clinical trial to assess its predictive value for patient response to indenoisoquinoline Top1 inhibitors.
