Abstract 2989: Intra-tumor heterogeneity and clonal changes in the progression of DCIS to invasiveness: Combined tumor bulk and single cell analysis
Authors: Sakr Rita A. , Martelotto Luciano G. , Baslan Timour , Ng Charlotte KY , Kendall Jude , Rodgers Linda , Cox Hilary , Riggs Mike , D'Itali Sean , Stepansky Asya , Olvera Narciso , King Tari A. , Weigelt Britta , Reis-Filho Jorge S. , Hicks James 
Published: August 2015

Abstract
INTRODUCTION: Ductal carcinoma in situ (DCIS) is a clonal intraductal proliferation of epithelial cells which acts as a non-obligate precursor of invasive breast cancer (IBC), yet the genetic events leading to the acquisition of invasive behavior remain unclear. We hypothesize that DCIS is composed of mosaics of genetically diverse tumor cell clones, and that the process of invasion is an evolutionary bottleneck. To test this hypothesis we performed a detailed characterization of the repertoire of genetic alterations and intra-tumor genetic heterogeneity in synchronously diagnosed DCIS and IBC using NextGen sequencing of bulk tumor and single cells.
METHODS: DNA extracted from fresh frozen, microdissected DCIS, IBC and adjacent normal tissue was subjected to whole exome sequencing on an Illumina HiSeq2000. Reads were aligned to the reference human genome hg19. Single nucleotide variants (SNVs) were called by MuTect, and gene copy number alterations were determined using VarScan2. Single nuclei were isolated from 100μm serial sections microdissected to separate DCIS and IBC. Individual nuclei were FACS-sorted into a 96-well plate, lysed and whole genome amplified. Amplified DNA samples were barcoded, pooled and sequenced on a HiSeq2000. Single cell sequencing data were mapped to the reference genome and uniquely mapped reads were allocated into bins, normalized, segmented and CN values generated.
RESULTS: In 6 cases of synchronous DCIS and IBC, a median of 41 and 47 non-synonymous mutations were found in each component, respectively. The somatic mutations identified in both DCIS and adjacent IBC components affected known driver breast cancer genes, including AKT1, PIK3CA, GATA3, MAP2K4 and TP53. Interestingly, we also found mutations restricted to either DCIS or IBC: ATRX (IBC-3); ALK and PKD2 (IBC-5); ESR1 (DCIS-5). The gene copy number profiles of matched DCIS and IBC were similar in all 6 pairs, however we also identified gene copy number alterations restricted either to DCIS or IBC: 1q gain (IBC-5); 3p and 3q losses (DCIS-6); 12p homozygous deletion (DCIS-4). Single cell sequencing of two cases (3 and 4) revealed that the majority of cells from both DCIS and IBC were derived from a common precursor lineage with shared copy number losses and gains. Both sets of DCIS-IBC pairs in these cases displayed elements of a subclonal structure with dominant clones alongside genetically diverse derivatives as well as genetic heterogeneity reflected in variable copy number alterations and non-modal clones.
CONCLUSION: Synchronous DCIS and IBC share founder genetic events, but also harbor somatic genetic alterations restricted to either the DCIS or IBC components, demonstrating that although DCIS is a precursor of IBC, intra-tumor genetic heterogeneity is present at the DCIS stage. Changes in clonal composition likely take place in the progression from DCIS to IBC.
Citation Format: Rita A. Sakr, Luciano G. Martelotto, Timour Baslan, Charlotte KY Ng, Jude Kendall, Linda Rodgers, Hilary Cox, Mike Riggs, Sean D'Itali, Asya Stepansky, Narciso Olvera, Tari A. King, Britta Weigelt, Jorge S. Reis-Filho, James Hicks. Intra-tumor heterogeneity and clonal changes in the progression of DCIS to invasiveness: Combined tumor bulk and single cell analysis. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2989. doi:10.1158/1538-7445.AM2015-2989