Abstract 2405: Harnessing liquid biopsy and genetic barcoding in evaluating diversity within triple-negative breast cancer
Authors: Ceresa Davide , Cardinali Barbara , Daga Antonio , Tasso Roberta , Sciutto Andrea , Appolloni Irene , Marubbi Daniela , Carli Franca , Fregatti Piero , Malatesta Paolo , Mastro Lucia Del 
Published: March 2024

Abstract
Introduction: While liquid biopsy shows promise as a non-invasive precision medicine tool, its ability to comprehensively represent the complexity of a tumor or only a subset of its clones remains uncertain. This issue is particularly crucial in the context of triple-negative breast cancer (TNBC), a type of cancer known for its significant heterogeneity. The goal of this study is to examine the clonal diversity within Circulating Biomaterials (CBMs): namely circulating tumor cells (CTCs), extracellular vesicles RNA (evRNA), and circulating tumor DNA (ctDNA), employing genetic barcoding and patient-derived TNBC xenografts.
Methodology: Cells from TNBC patients' resected tumors were tagged with a unique barcode sequence through vector transduction. These barcoded cells were subsequently introduced into immunodeficient mice to form xenografts. When these xenografts grew to a certain size (15mm in diameter), the mice were humanely euthanized, and the tumor tissues as well as CBMs from their blood and plasma were collected. CTCs were isolated through negative enrichment and DEPArray technology; evRNA was extracted using ultracentrifugation and TRIzol™ Plus RNA Purification method; ctDNA was retrieved using the QIAamp DNA Micro Kit. Tumor tissues were processed for RNA and DNA using RNeasy micro kit and Qiamp DNA micro kit, respectively. RNA sequencing of the tumor tissues was carried out using the Lexogen - CORALL Total RNA-Seq Kit and Illumina NextSeq high output reagents.
Results: Analysis of xenografts from four patients showed that ctDNA was the predominant genetic material detected through liquid biopsy. Crucially, clonal analysis using Next Generation Sequencing (NGS) demonstrated that ctDNA provides an accurate reflection of the primary tumor's clonal heterogeneity. The method efficiently identified both main tumor clones and subclones, even those constituting as little as 1% of the tumor, with a 50% detection probability. Transcriptomic analysis of the tumor tissues uncovered a distinct mesenchymal and pro-metastatic signature, more pronounced in tumors with higher ctDNA release. Clonal analysis of CTCs and evRNA is still ongoing.
Conclusions: This investigation affirms the effectiveness of ctDNA analysis in liquid biopsy for identifying subclonal diversity in TNBC. Our results suggest that liquid biopsy via ctDNA is capable of managing TNBC's extensive intra-tumoral heterogeneity, including the identification of smaller subclones. Furthermore, a link was identified between the release of ctDNA and a mesenchymal, prometastatic gene expression pattern in the tumors.
Citation Format: Davide Ceresa, Barbara Cardinali, Antonio Daga, Roberta Tasso, Andrea Sciutto, Irene Appolloni, Daniela Marubbi, Franca Carli, Piero Fregatti, Paolo Malatesta, Lucia Del Mastro. Harnessing liquid biopsy and genetic barcoding in evaluating diversity within triple-negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2405.