Abstract P2-05-01: Single cell sequencing analysis of formalin-fixed paraffin-embedded ductal carcinomas in situ and invasive breast cancers reveals clonal selection in the progression from in situ to invasive disease
Authors: Martelotto LG , Baslan T , Kendall J , Rodgers L , Cox H , King TA , Weigelt B , Hicks J , Reis-Filho JS 
Published: May 2016

Background: Ductal carcinoma in situ (DCIS) is considered to be a precursor of invasive breast cancer (IBC), and is found synchronously in over 45% of patients with invasive disease. Whether the progression from DCIS to IBC results in clonal shifts and the genomic imbalances that may drive this process remain to be elucidated. Single-cell sequencing constitutes a powerful approach to address these questions as it enables the phylogenetic reconstruction of subpopulations of cancer cells at single cell resolution. To date, single-cell sequencing approaches remain limited to fresh/frozen samples, precluding the use of archival formalin-fixed and paraffin-embedded (FFPE) samples, the largest source of tumor material in pathology departments. Here, we describe the development of a methodology for single-cell copy number (CN) profiling of single nuclei derived from FFPE tumor samples and subsequently employed this approach to define whether DCIS displays intra-lesion genetic heterogeneity and if clonal shifts are observed in the progression from DCIS to IBC.

Methods: DCIS and IBC areas were independently microdissected from archival FFPE samples. Microdissected tissue fragments were subsequently reverse-crosslinked, and their extracellular matrix was digested. Intact individual diploid nuclei from cells in G1 were FACS-sorted into individual wells of 96-well plates. DNA was extracted from each cell and subsequently repaired and amplified. Sequencing libraries were prepared using standard protocols followed by whole genome sequencing on a HiSeq 2000. Single-cell sequencing data were analyzed to define the CN profiles for each cell, to infer the clonal composition of each DCIS and IBC, and to trace the genomic events that occurred during the progression from in situ to invasive disease.

Results: We performed single-cell sequencing of 192 cells derived from two synchronous FFPE DCIS and IBCs. One pair of DCIS and IBC was diploid and the other was tetraploid. Principal component analysis discriminated neoplastic from normal cells and resulted in the identification of clonal cancer cell populations. Genome-wide CN profiling of single cells from DCIS and IBC components demonstrated the extent of intra-tumor genetic heterogeneity present in these samples. In addition, founder CN events present in these lesions, including TP53 and RB1 losses and FGFR1/BRF2 and CCND1 amplifications, were already detected in the DCIS samples. Phylogenetic analysis based on CN profiles from single cells revealed that the invasive cancer likely stemmed from minor subclones from the DCIS and provided evidence of branched evolution in the progression from DCIS to IBC.

Conclusions: We developed a robust procedure to perform single-cell massively parallel sequencing of individual nuclei isolated from FFPE samples. This approach revealed that the progression from DCIS to IBC results in clonal shifts, suggesting that this biological phenomenon may constitute an evolutionary bottleneck.

Citation Format: Martelotto LG, Baslan T, Kendall J, Rodgers L, Cox H, King TA, Weigelt B, Hicks J, Reis-Filho JS. Single cell sequencing analysis of formalin-fixed paraffin-embedded ductal carcinomas in situ and invasive breast cancers reveals clonal selection in the progression from in situ to invasive disease. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P2-05-01.