Abstract PS2-07-22: Serial, multi-omic, multi-analyte, liquid biopsy monitoring of metastatic lobular breast cancer to detect clinically-relevant heterogeneity and evolution

Abstract Precision and immuno-oncology therapies commonly rely on molecular biomarker information obtained from single tissue biopsies, largely missing tumor heterogeneity and evolution. CtDNA offers a minimally invasive, yet limited approach to mitigate tissue shortcomings in monitoring the evolving tumor clonal architecture since it is characterized by low tumor fraction and provides only an aggregate picture of the genomic landscape. We hypothesized that serial, single-cell genomic profiling of circulating tumor cells (CTCs) complements the molecular information obtained from tissue and ctDNA. Invasive lobular carcinoma (ILC) of the breast, a high CTC producing malignancy offers an ideal setting to study the contribution of CTCs. We analyzed 119 individual CTCs and 15 leukocyte controls, 26 ctDNAs, and 24 tissue samples over time from 15 CTC-positive patients with metastatic ILC undergoing various therapies. CTCs were enriched/isolated with the tandem CellSearch®/DEPArray™ system and genomically profiled to detect mutations, copy number alterations, and in a novel application, tumor mutation burden (TMB) and microsatellite instability (MSI) at the single cell level. Molecular findings were combined with clinical information. We confirmed the presence of high CTC levels in metastatic ILC, where almost 80% of patients had 5 CTC/7.5mL blood. Single-cell profiling had a 2.7% rate of artifactual mutation detection and a 16.5% combined rate of miscalling the zygosity of a heterozygous mutation compared to bulk sequencing as truth. CTC copy number alterations were concordant with tissue (R = 0.77) and even allowed detection of single-cell intragenic copy number differences at the single amplicon level. Only 20/36 (56%) targetable genomic alterations were shared in all three specimen types (tissue, ctDNA and CTC). The rest were shared in either 2 or 1 specimen types, except ctDNA, which had no exclusive alterations and had particularly low detection rate of copy number alterations. Adding CTC analysis to tissue, ctDNA or both, contributed 0.49 - 0.69 additional targetable alterations per patient. Interestingly, 1 patient with plentiful CTCs had no detectable ctDNA. Our novel CTC TMB analysis revealed that in only 2/5 patients that were TMB-high by any specimen type, there was agreement between all three specimen types. Notably, one of the other 3 patients was TMB-high in CTCs only, while another in CTC and ctDNA only, and the last in tissue and ctDNA, but not CTCs. Remarkably, TMB heterogeneity among individual CTCs was common, with 3/5 TMB-high patients having CTCs with greater than, and fewer than 10 mut/Mb. Data from one patient’s 6 tissue, 5 ctDNA, and 28 individual CTC samples over 7 timepoints from primary cancer to hospice revealed in unprecedented detail the heterogeneity and evolution in response to endocrine, chemo and immunotherapy pressures not appreciable in ctDNA and tissue. Another patient harbored CTCs of two different clones matching tissue from two different lesions, only one of whom was TMB-high and expressed higher cytokeratin levels. Another patient, displaying ERBB2 copy amplification and HER2 overexpression by IHC, who was treated with anti-HER2 therapy, later showed disappearance of the ERBB2 amplification in CTCs but not in ctDNA. Taken together, these data support the non-invasive monitoring of advanced lobular breast cancer patients by liquid biopsy that incorporates CTCs to complement tissue and ctDNA in informing resistance mechanisms and subsequent treatment approaches. Citation Format: A. K. Cani, E. M. Dolce, E. P. Darga, K. Hu, C. Liu, D. Robinson, Y. Wu, C. Paoletti, G. D. Luker, S. A. Tomlins, A. M. Udager, N. C. Henderson, D. G. Thomas, J. M. Rae, C. M. Arul, E. F. Cobain, D. F. Hayes. Serial, multi-omic, multi-analyte, liquid biopsy monitoring of metastatic lobular breast cancer to detect clinically-relevant heterogeneity and evolution 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 PS2-07-22.