Adaptive and Acquired Evolution in Response to Chemotherapy in Triple-Negative Breast Cancer

Triple-negative breast cancer (TNBC) is an aggressive subtype that displays extensive intratumor heterogeneity and frequently develops resistance to neoadjuvant chemotherapy (NAC). An unresolved question is whether resistance is caused by the adaptive selection of rare mutations in pre-existing subclones (adaptive resistance), or alternatively through the acquisition of new mutations induced by the therapeutic agents. To investigate this question, we applied single cell DNA and RNA sequencing, in addition to bulk deep-exome sequencing, to study matched longitudinal samples from 20 TNBC patients during NAC. Deep-exome sequencing identified 11 patients in which NAC led to clonal extinction, and 9 patients in which clones persisted during NAC and established the resistant tumor mass. Single cell DNA sequencing of 1000 cells from 8 patients showed that clones with chemoresistant copy number profiles were pre-existing and selected in response to NAC, following an adaptive resistance model. In contrast, single cell RNA sequencing of 8,500 cells in 8 patients, showed that NAC induced transcriptional reprogramming in response to NAC, including the upregulation of MTORC, interferon response, MYC, EMT and Angiogenesis signatures. Our data suggests chemoresistance evolution is a two-step process that involves both the adaptive selection of genotypes, and acquired resistance of chemoresistant phenotypes.