Characterization of disease evolution in sequential sampled metastatic breast cancer using liquid biopsy

Background: It is established that tumors evolve over time due to treatment pressure or physiological constraints. Tissue biopsies give a one-time window into the characteristics of a primary tumor or metastatic nodule. Their main pitfall is procedure risk, pain, and cost, which is why they are rarely repeated. Liquid biopsies, however, are performed with minimal discomfort and risk, providing an easier way to monitor disease. Circulating tumor cells (CTCs) and cell-free DNA (cfDNA) have proven to be of high prognostic and diagnostic value. Here we evaluate 17 longitudinal blood draws collected from a metastatic breast cancer patient over 4 years and assess the power of single-cell analysis relative to analysis of tissue biopsies and cfDNA. Methods: CTCs were enumerated using the high-definition single-cell assay (HD-SCA) and single CTCs were isolated by micromanipulation. DNA of single cells underwent whole genome amplification and Illumina library construction for copy number variation (CNV) analysis. Genomic DNA from microdissected FFPE tissue and cfDNA extracted from plasma were converted into Illumina sequencing libraries for CNV analysis. CA27.29 levels were reported by the clinic. Results: Levels of CTCs and circulating tumor DNA (ctDNA) fractions were positively correlated with CA27.29. CNV analysis of single tumor cells at time of enrollment revealed 3 morphometrically indistinguishable clones that persisted during the 4-year follow-up. Genomic analysis of primary breast tissue and bone metastasis detected at time of diagnosis matched CNV data of the evolutionary first clone, while a later liver metastasis was comprised solely of clone 2. Remarkably, CTCs in clone 2 were identified years before discovery of the liver metastasis. To date, we have not detected a metastatic site harboring clone 3. We found that CNV profiles from cfDNA represented the aggregate of CTC clones. Conclusion: Longitudinal blood sampling enabled tracing of tumor evolution robustly and less invasively than solid tumor biopsies. Metastasis associated clones were identified years before measureable disease, providing an opportunity for earlier interventions. While plasma-based assays detected the presence of tumor-derived DNA, the single-cell high-content resolution of the HD-SCA workflow was required to deconvolute 3 genomically distinct clones present in this patient. We propose that enumeration of CTCs and analysis of ctDNA could be used as additional clinical markers to monitor disease recurrence, adding valuable morphogenomic information about the primary tumor and metastatic sites. Additionally, we are currently analyzing the results of single nucleotide variation analysis of single CTCs, cfDNA and FFPE tissue from this patient. Citation Format: Lisa Welter, Liya Xu, Dillon McKinley, Sara Restrepo-Vassalli, Angel Dago, Mariam Rodriguez Lee, Anand Kolatkar, James Hicks, Jorge Nieva, Peter Kuhn. Characterization of disease evolution in sequential sampled metastatic breast cancer using liquid biopsy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2963.