Localized breast cancer (LBC) embraces multiple genetic mutagenic events during the tumour development. Each mutation, at both gene copy number variant (CNV) and single-nucleotide variant (SNV) levels, may generate distinct cellular subclones within the same tumour, resulting in intra-tumoral heterogeneity (ITH). Although ITH is a risk factor for therapy-resistance and relapse, its intrinsic nature at the single-cell level remains obscure. Here, we investigate the genetic mechanism underlying ITH in a cohort of hormone receptor-positive/HER2-negative (HR+/HER2–) LBC.
We employed single-cell DNA sequencing (scDNA-seq) to dissect ITH in patients with HR+/HER2–, node positive LBC treated with adjuvant chemo-hormone therapy. ScDNA-seq of individual samples was performed on intact nuclei isolated from the frozen tissues using TAPESTRI platform. ITH was assessed as the number of clusters detected based on CNV and SNV variations. Potential associations between these clusters and clinical data, including molecular subtypes, relapse and survival rate were additionally examined.
Overall 138 HR+/HER2– LBC patients, of whom 36.7% (n = 51) were luminal A and 62.3% (n = 86) luminal B. Overall, 7.3% (n = 10) and 27.5% (n = 38) of patients developed local and distant relapses with a survive rate of 63.8% (n = 88). ScDNA-seq using approximately 3500 nuclei/ sample was highly robust with an average of 83% sequencing reads mapped to the genome, indicating a good mapping quality. We observed several intra-tumour CNV clusters, varying from 1 to 5, with 76.3% patients (n = 103) harbouring at least 2 clusters. In addition, we captured several genes driving the CNV-based clusterisation: NCOR1, FGFR1 and KRAS in 3; BRCA2 in 4; and RB1 in 8 patients each. CNV clusters showed no statistical association with disease subtype, relapse or survival. Further analysis of SNV-based clusterisation is still underway.
CNV-clustering based ITH was highly prevalent in HR+/HER2– LBC patients. Particularly, CNV of a few driver genes might differentiate clusterisation. Furthermore, our finding showed that scDNA-seq may be effectively applied to unveil ITH mechanism, potentially guiding to investigate a new predictive and prognostic tool.
Gustave Roussy.
Gustave Roussy.
F. André: Financial Interests, Institutional, Research Grant: AstraZeneca, Lilly, Novartis, Pfizer, Roche, Daiichi Sankyo; Other, Personal, Other, Founder: Pegacsy. All other authors have declared no conflicts of interest.