Abstract PD2-08: Detecting subtype switching in metastatic breast cancer with circulating tumor DNA methylation profiling

Abstract Background: Subtype switching in metastatic breast cancer (BC), for example switching between ER positive and triple negative subtypes, is a well-recognized change in tumor phenotype which can be a major source of treatment failure if not identified. Guidelines1,2 recommend repeat tissue biopsy of metastasis to retest ER+/PR+/HER2+ status at recurrence. Yet, such biopsies may not always be clinically feasible, single-site tumour biopsies sample only a single metastasis, and subtype may change later during metastatic therapy. We developed a non-invasive blood test to monitor the evolution of metastatic BC subtype based on circulating tumor DNA (ctDNA) methylation profiling. Methods: A capture based enzymatic ctDNA based methylation profile previously described3 was used to develop three independent binary Machine learning classifiers to discriminate between ER positive v ER negative, and HER-2 positive v HER-2 negative and TNBC vs non-TNBC from ctDNA methylation profiles. A 10-fold stratified nested cross-validation was used to assess classifier characteristics. Liquid biopsy subtype was compared with metastatic tissue biopsy subtype, in independent test folds, split by whether the subtype was the same or different in the original primary cancer. Subtype was defined by immunohistochemistry (IHC). Results: There was a total of 191 metastatic liquid biopsy samples taken from 86 patients where multiple biopsies had been taken, with a median 63 months between first and repeat tissue biopsy (range 20-203 months). Of these 37/191 (19%) samples were from 17 patients that had switched subtype based on tissue. The liquid biopsy correctly identified the subtype switch in 89.2% (33/37) of samples, with plasma samples taken a median of 7 months from the tissue biopsy. Focussing on the samples that were initially ER+HER2-. Of the cancers that were ER+HER2- on both tissue biopsies, 96% (129/134) were liquid ER+HER2- and 0% (0/134) were liquid TNBC. Conversely of the cancers that were ER+HER2- on initial biopsy and TNBC on the later biopsy, only 9% (1/11) were liquid ER+HER2-, and 91% (10/11) were liquid TNBC including one sample that was mixed ER+/TNBC liquid subtype (p0.0001). Focussing on the samples that were initially TNBC. Of the cancers that were TNBC on both tissue biopsies, 88% (8/9) were liquid TNBC and 11% (1/9) were liquid ER+HER2-. Conversely of the cancers that were TNBC on initial biopsy and ER+HER2- on the later biopsy, 0% were liquid TNBC and 100% (10/10) were liquid ER+HER2- (p=0.0002). Similar results were obtained for other subtype switching, including robust identification of cancers that acquired HER2 positivity. Furthermore in 8% (3/37) of samples both the original and the new subtype were co-detected in liquid biopsy, suggesting both subtypes may co-exist. Conclusion: We demonstrate identification of tumors that switched subtype using a liquid biopsy and reveal that multiple subtypes may co-exist in the same tumor/patient. Subject to further validation, clinical trials are warranted to assess whether liquid biopsy subtyping has the potential to direct metastatic BC treatment. Citation Format: N. Pasha, R. Cutts, A. Amenssag, S. Hrebien, N. Cunningham, C. Swift, P. Proszek, A. Gulati, G. Qiong, K. Dunne, R. Roylance, A. tutt, R. Baird, I. MacPherson, A. Ring, S. Johnston, A. Okines, M. hubank, S. Haider, I. Garcia-Murillas, S. Filippi, N. Turner. Detecting subtype switching in metastatic breast cancer with circulating tumor DNA methylation profiling abstract. In: Proceedings of the San Antonio Breast Cancer Symposium 2025; 2025 Dec 9-12; San Antonio, TX. Philadelphia (PA): AACR; Clin Cancer Res 2026;32(4 Suppl):Abstract nr PD2-08.