Single Cell Expression Analysis of Ductal Carcinoma in Situ Identifies Complex Genotypic-Phenotypic Relationships Altering Epithelial Composition

To identify mechanisms underlying the growth of ductal carcinoma in situ (DCIS) and properties that lead to progression to invasive cancer, we performed single-cell RNA-sequencing (scRNA-seq) on DCIS lesions and matched synchronous normal breast tissue.

Using inferred copy number variations (CNV), we identified neoplastic epithelial cells from the clinical specimens which contained a mixture of DCIS and normal ducts.

Phylogenetic analysis based on the CNVs demonstrated intratumoral clonal heterogeneity was associated with significant gene expression differences.

We also classified epithelial cells into mammary cell states and found that individual genetic clones contained a mixture of cell states suggesting an ongoing pattern of differentiation after neoplastic transformation.

Cell state proportions were significantly different based on estrogen receptor (ER) expression with ER-DCIS more closely resembling the distribution in the normal breast, particularly with respect to cells with basal characteristics.

Using deconvolution from bulk RNA-seq in archival DCIS specimens, we show that specific alterations in cell state proportions are associated with progression to invasive cancer.

Loss of an intact basement membrane (BM) is the functional definition of invasive breast cancer (IBC) and scRNA-seq data demonstrated that ongoing transcription of key BM genes occurs in specific subsets of epithelial cell states.

Examining BM in archival microinvasive breast cancers and an in vitro model of invasion, we found that passive loss of BM gene expression due to cell state proportion alterations is associated with loss of the structural integrity of the duct leading to an invasive phenotype.

Our analyses provide detailed insight into DCIS biology.

SIGNIFICANCE: Single cell analysis reveals that preinvasive breast cancer is comprised of multiple genetic clones and there is substantial phenotypic diversity both within and between these clones.

Ductal carcinoma in situ (DCIS) of the breast is a non-invasive condition commonly identified through mammographic screening.

A primary diagnosis of DCIS carries little mortality risk on its own, but its presence is a risk factor for subsequent clonally related invasive breast cancer (IBC) (1–5).