420P - An exosome-based ESR1 monitoring RT-qPCR kit that rapidly and accurately detects acquired resistance variants at ≤ 0.1% frequency in liquid biopsy samples

Background

Breast cancer is a complex disease requiring routine monitoring and management to ensure the best possible patient outcomes. The updated NCCN guidelines for breast cancer, which call for ESR1 mutation testing and the use of elacestrant upon detection underscore the need for sensitive, accurate, and accessible assays to detect ESR1 resistance mutations. We developed the QuantideX® qPCR ESR1 exoMutation Kit, which interrogates 11 key ESR1 mutations associated with endocrine therapy resistance in HR+/HER2- mBC. The kit analyzes mutations in plasma utilizing both circulating exosomal RNA (exoRNA) and cell-free DNA (cfDNA) to enable sensitive detection on RT-qPCR platforms. Here we characterize the analytical performance of the kit on a cohort of metastatic breast cancer (mBC) subjects and contrived samples.

Methods

Plasma was collected from a cohort of 50 subjects with stage IV, HR+/HER2-, mBC. The cohort was supplemented with contrived samples comprising blends of synthetic nucleic acids with known variants. Plasma samples were subjected to an in-house isolation method optimized to co-enrich exoRNA and cfDNA (included within the kit). Multiplex RT-qPCR target enrichment was performed using the QuantideX qPCR ESR1 exoMutation Kit and evaluated on widely used thermal cycler and qPCR instruments.

Results

We report on the results of studies into the kit’s performance characteristics. Analytical sensitivity was seen at the equivalent of 5 copies of mutation per mL plasma input. High precision of ≥90% was shown across replicates at 0.1% allele fraction. Analytical specificity of ≥90% negative results in mutation-negative replicates was maintained. All mutant positive samples were verified via an orthogonal method.

Conclusions

Our solution to detect ESR1 resistance mutations enables single-day sample-to-answer resulting for liquid biopsies with multiplexed RT-qPCR. Enhancing mutation detection sensitivity by complementing cfDNA with exoRNA will facilitate future research into breast cancer treatment options, further supported by the updated breast cancer NCCN guidelines showcasing the immediate need for timely and sensitive ESR1 mutation detection.

Clinical trial identification

Editorial acknowledgement

Legal entity responsible for the study

Asuragen, Inc. a bio-techne brand.

Funding

Asuragen, Inc. a bio-techne brand.

Disclosure

S. Statt: Financial Interests, Institutional, Stocks/Shares: Bio-Techne. J.R. Thibert: Financial Interests, Institutional, Stocks/Shares: Bio-Techne. M. Church: Financial Interests, Institutional, Stocks/Shares: Bio-Techne. J. Myers: Financial Interests, Institutional, Stocks/Shares: Bio-Techne. H. Dale: Financial Interests, Institutional, Stocks/Shares: Bio-Techne. B. Caughron: Financial Interests, Institutional, Stocks/Shares: Bio-Techne. K. Kelnar: Financial Interests, Institutional, Stocks/Shares: Bio-Techne. B. Haynes: Financial Interests, Institutional, Stocks/Shares: Bio-Techne.