Summary Our knowledge of copy number evolution during the expansion of primary breast tumors is limited 1 , 2 . To investigate this process, we developed a single cell, single-molecule DNA sequencing method and performed copy number analysis of 16,178 single cells from 8 triple-negative breast cancers (TNBCs) and 4 cell lines. Our data shows that breast tumors and cell lines are comprised of a large milieu of subclones (7–22) that are organized into a few (3–5) major superclones. Evolutionary analysis suggests that after clonal TP53 mutations, multiple LOH events and genome doubling, there was a period of transient genomic instability followed by ongoing copy number evolution during the primary tumor expansion. By subcloning single daughter cells in culture, we show that tumor cells re-diversify their genomes and do not retain isogenic properties. These data show that TNBCs continue to evolve chromosome aberrations and maintain a reservoir of subclonal diversity during primary tumor growth.

Our knowledge of copy number evolution during the expansion of primary breast tumors is limited. To investigate this process, we developed a single-cell, single-molecule DNA sequencing method and performed copy number analysis of 13,808 single cells from six triple-negative breast cancers (TNBCs) and two breast cancer cell lines. Our data shows that breast tumors and cell lines are comprised of a large milieu (17-26) of subclones that are organized into a few (4-8) major superclones. Phylogenetic analysis and mathematical modeling show that copy number evolution was ongoing during the expansion of the primary tumor, after the initial punctuated burst of genomic instability. By expanding single daughter cells in culture, we show that cancer cells re-diversify their genomes and do not retain isogenic properties. These data elucidate modes of evolution and show that TNBCs maintain a vast reservoir of subclonal copy number diversity during the expansion of the primary tumor mass. Citation Format: Darlan Conterno Minussi, Michael Nicholson, Hanghui Ye, Alexander Davis, Kaile Wang, Emi Sei, Haowei Du, Mashiat Rabbani, Cheng Peng, Min Hu, Shanshan Bai, Thomas McDonald, Aislyn Schalck, Anna Casasent, Angelica Barrera, Hui Chen, Bora Lim, Banu Arun, Funda Meric-Bernstam, Franziska Michor, Nicholas Navin. Breast tumors maintain a reservoir of subclonal diversity during primary expansion [abstract]. In: Proceedings of the AACR Virtual Special Conference on Tumor Heterogeneity: From Single Cells to Clinical Impact; 2020 Sep 17-18. Philadelphia (PA): AACR; Cancer Res 2020;80(21 Suppl):Abstract nr PO-133.