2O - Dissecting intratumoral heterogeneity in localized HR+/HER2- breast cancer using single-cell DNA sequencing (scDNA-seq)

Background

Localized breast cancer (LBC) embraces multiple genetic mutagenic events during tumor development. Copy number variant (CNV) and single-nucleotide variant (SNV) generated distinct cellular subclones within the same tumor, leading to intra-tumoral heterogeneity (ITH). Although ITH is a risk factor for therapy-resistance and relapse, its mechanism at the single-cell resolution remains unknown. Here, we study the genetic bases underlying ITH in a cohort of HR⁺/HER2^–, node-positive LBC.

Methods

ScDNA-seq was performed on intact nuclei isolated from frozen tumors using TAPESTRI platform. ITH was assessed using CNV- and SNV-based clusterization (CBC and SBC, respectively). Correlations between ITH and clinical data, including molecular subtypes, relapse and survivorship were examined.

Results

The cohort contains 138 patients, in which 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 survival rate of 63.8% (n = 90). Intra-tumor clusters varied from 1 to 4 in CBC and from 1 to 5 in SBC, with 63.8 % patients (n = 88) in CBC and 73.9% patients (n = 102) in SBC having at least 2 clusters. In CBC, specific gene amplifications and deletions occurred mostly in tumors having 2 and 3 clusters, in which deletion events were more prevalent in the 3-cluster tumors and in luminal B than luminal A. We captured CBC-specific genes including AKT1, ATM, KRAS, and PIK3CA, which associated with 2-cluster tumors; and BRCA1, ERBB2, GATA3, IDH1 and NRAS with 3-cluster tumors. In SBC, tumors having more than 2 clusters occurred more frequently in luminal B than luminal A (Wilcoxon test, p = 0.015). Notably, mutational phylogenies in luminal A and B tumors were different, implying SNV-derived ITH may emerge and evolve independently via distinct mutations and clonal patterns.

Conclusions

CNV- and SNV-clustering based ITH highly prevailed in HR⁺/HER2^– LBC patients at the single-cell resolution. Luminal B tumors carried more deletions and more SNV-clusters than luminal A. By uncovering distinct genetic bases leading to CBC and SBC between the 2 subtypes, our findings could potentially contribute to precision medicine as an early diagnosis and prognosis tool.

Editorial acknowledgement

Clinical trial identification

Legal entity responsible for the study

Institut Gustave Roussy.

Funding

Institut Gustave Roussy.

Disclosure

All authors have declared no conflicts of interest.