Serial monitoring of circulating tumor cells and circulating tumor DNA in metastatic lobular breast cancer identifies intra-tumor heterogeneity and precision and immuno-oncology biomarkers of therapeutic importance

Abstract Clinical decisions on precision and immuno-oncology therapies are based on predictive biomarkers commonly obtained from a single metastatic biopsy or archived primary tumor tissue. Circulating genomic biomarkers offer a minimally invasive approach to monitor intra-patient tumor heterogeneity and detect in real-time the clinically-relevant evolving clonal architecture. Although currently underutilized, we hypothesize that single-cell DNA next generation sequencing (scNGS) of circulating tumor cells (CTC) is a particularly well-suited method to complement biomarker information obtained from tissue and cell-free circulating tumor DNA (ctDNA). In this study we analyzed 113 individual CTC, 21 ctDNA, and 15 white blood cells (WBC) samples, from 15 CTC-positive lobular breast cancer patients, four of whom had CTC available at both metastatic baseline and after progression on a variety of therapies chosen at their physician’s discretion. Clinical NGS data from 15 tumor tissue biopsies obtained using a ~1700-gene DNA panel and whole transcriptome sequencing were available for comparison. CTC were enriched with the CellSearch® system and isolated as single cells with the DEPArray™ system. Whole genome amplified CTC and WBC, as well as ctDNA underwent scNGS with the Oncomine Comprehensive Assay covering ~500 genes and 1.1Mb of genomic space to detect mutations, copy number alterations, tumor mutation burden (TMB) and microsatellite instability (MSI). 99.1% of single cells and 95.2% of ctDNA samples were informative, with a mean sequencing depth of 664x. Using our previously developed, CTC-based precision medicine reporting platform, MI-CTCSeq, CTC in 9 of 15 patients (60%) had mutations that were actionable by FDA-approved targeted therapies including in the oncogenes PIK3CA and FGFR2 and HER2. 3 of these 9 patients (33%) harbored actionable alterations not shared between all 3 analyte types (tissue, CTC and ctDNA). These included 3 actionable mutations found in CTC and ctDNA only, 1 in tissue and ctDNA only, and 1 in ctDNA only. However, 2 of those ctDNA mutations were identified near the limit of detection and with a priori knowledge of their presence from tissue or CTC. Further, 1 patient with plentiful CTC had no detectable ctDNA and one patient’s tissue biopsy was inadequate for sequencing while both liquid biopsy analytes were abundant. 13 patients (87%) displayed intra-patient, inter-CTC genomic heterogeneity of putative driver mutations. 1 of 4 (25%) patients with CTC available in >1 timepoint displayed fluctuations in their CTC subclonal makeup between timepoints. Data from this patient’s 2 tissue biopsies, 3 ctDNA samples, and 27 individual CTC over 4 timepoints combined to reveal in unprecedented detail inter-metastatic lesion and inter-CTC heterogeneity and tumor evolution in response to endocrine and immunotherapy selective pressures. ScNGS of CTC helped provide an additional level of detail not appreciated by sequencing of the other two analyte types. In another patient, CTC were composed of 2 subclones which were indistinguishable by ctDNA, 1 of which appears to have not been sampled by the tissue biopsy. Using a novel method, we enabled detection of single-cell CTC TMB and MSI. CTC TMB scores (dichotomized as above/below 10 mutations/Mb) were 100% concordant with those measured in the corresponding tissue biopsies. Further, in a novel observation, we detected intra patient, inter-CTC heterogeneity of TMB and MSI, which has potential implications for immunotherapy response and development of resistance. Taken together, these data support the non-invasive biomarker interrogation and monitoring by liquid biopsy that incorporates CTC scNGS and complements tissue in informing precision and immuno-oncology approaches. This may have important implications for appropriate treatment selection and identification of therapeutic resistance mechanisms. Citation Format: Andi Cani, Emily Dolce, Alissa Turnbull, Kevin Hu, Chia-Jen Liu, Elizabeth Darga, Dan Robinson, Yi-Mi Wu, Dafydd G. Thomas, Costanza Paoletti, Scott Tomlins, James Rae, Aaron Udager, Arul Chinnaiyan, Erin F. Cobain, Daniel F. Hayes. Serial monitoring of circulating tumor cells and circulating tumor DNA in metastatic lobular breast cancer identifies intra-tumor heterogeneity and precision and immuno-oncology biomarkers of therapeutic importance [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P4-02-04.