Unveiling Inter- and Intra-Tumor Heterogeneity in Ductal Carcinoma in Situ (DCIS) and Invasive Ductal Carcinoma (IDC) through Integration of Unified Single-Cell Whole Genome Copy Number and RNA Expression Data

Tia A. Tate1, Isai Salas-González1, Katie Kennedy1, Jamie Remington1, Durga Arvapalli1, Swetha Velivela1, Jeffrey R. Marks2, E. Shelley Hwang2, Victor J. Weigman1, Jon S. Zawistowski1

1 BioSkryb Genomics, Durham, NC, USA 2 Department of Surgery, Duke University Medical Center, Durham, NC

Introduction: Ductal Carcinoma in situ (DCIS) is a precursor to invasive ductal carcinoma (IDC) with complex heterogeneity influenced by genomic alterations and the tumor microenvironment. This study aims to uncover gene dysregulation mechanisms in breast cancer patients with a history of DCIS, using a unified single-cell copy number and RNA expression workflow. Insights gained will illuminate the molecular landscape of DCIS to IDC progression, revealing tumor evolution and heterogeneity mechanisms.

Methods: DCIS and IDC samples were collected from 11 patients post-mastectomy. Cells were isolated and either enriched for EpCAM using FACS or analyzed without enrichment. Unified whole genome and transcriptome amplification were performed on approximately 100 cells per sample (1225 cells in total) using ResolveOMETM. DNA libraries were enriched for exomes and sequenced on an Illumina NovaSeq6000. Data was analyzed using the BaseJumperTM platform.

Results: Our analysis of 11 patient biopsies provide evidence of potential characteristics underlying tumoral evolution in IDC. We observed vast heterogeneity among the tumor profiles, with some displaying a more quiescent nature. Across the 11 profiles, a range of 1.5-17% copy number events were identified, carrying potential genomic consequences. Importantly, these findings confirmed known DCIS copy number profiles, including 1q gains in 7 out of 11 cases and 16q loss in all cases.

We identified additional copy signatures occurring in low proportions of patients. Notable alterations included 17p loss in 5 of 11 patients, chromosome 7 gain in 4 of 11 patients and 11q loss in 6 of 11 patients. These characteristic copy number alterations are associated with key genes, involved in cell cycle regulation and progression (CCND1, CKS1B), regulation of cell growth and proliferation (BRAF, NF1), migration (BRAF, PTK2), immune response (JAK1), and tumor invasion and metastasis (MMP7).

Conclusions: Our findings emphasize the assorted copy number profiles associated with invasive breast cancer and highlight the potential involvement of key genes in tumoral evolution. Furthermore, single-cell amplification of genome and transcriptome underscore the limitations of bulk data analysis, as these profiles would often be missed. By elucidating these mechanisms at the single-cell level, we gain valuable insights into the underlying processes driving tumoral evolution from DCIS to IDC, providing implications for personalized treatment strategies by identifying aggressive clonal cell populations and expanding treatment options.