Abstract 4735: Single-cell sequencing from formalin-fixed paraffin-embedded breast cancers: a powerful tool to address intratumor genetic heterogeneity
Authors: Martelotto Luciano G. , Sakr Rita A. , Baslan Timour , Rodgers Linda , Cox Hilary , Kendall Jude , King Tari A. , Weigelt Britta , Hicks James , Reis-Filho Jorge S. 
Published: August 2015

Abstract
Background: At diagnosis, tumors often consist of multiple, genotypically distinct cell populations. Genome sequencing of single cells has opened new avenues of investigation, yet the application of this technology remains limited to fresh/frozen tissue samples. Here, we describe and validate a method to perform single-cell massively parallel sequencing using DNA extracted from individual nuclei from FFPE tumor samples.
Methods: Tissue sections are deparaffinized and the area of interest microdissected, DNA is reverse-crosslinked and the extracellular matrix is digested. Extracted single nuclei are then FACS-sorted, lysed and the whole genome repaired using a broad-spectrum DNA-repair enzyme cocktail. Repaired DNA is whole genome amplified (WGA) using GenomePlex WGA4 (Sigma-Aldrich) with modifications for heavily damaged genomic templates. Illumina sequencing libraries are generated using standard approaches followed by multiplex sequencing on an Illumina HiSeq2000. To obtain copy number (CN) profiles, single-cell sequencing data are mapped to the reference genome with PCR duplicates removed. Uniquely mapped reads are allocated and counted in genomic intervals of variable length (bins) with CN states roughly proportional to the number of allocated sequencing reads. Bin counts are then normalized on the basis of GC-content, segmented and transformed to CN values to identify long contiguous regions of equivalent CN.
Results: As a proof-of-principle we performed single-cell CN profiling on two aneuploid synchronous ductal carcinomas in situ (DCIS)/invasive ductal carcinomas (IDCs), where both FFPE and frozen material was available. Using our novel methodology, 24 single nuclei were multiplexed/lesion. An average of 3 million reads/cell (average coverage of 0.1X/cell) was obtained, providing sufficient data to infer CN profiles of single cells accurately. These data were successfully employed to identify non-neoplastic cells and distinct clonal lineages of neoplastic cells within each lesion. The data obtained from the analysis of FFPE samples were concordant with those obtained from the analysis of matched frozen samples.
Conclusions: We developed a robust procedure to perform single-cell massively parallel sequencing of individual nuclei isolated from FFPE samples, providing the opportunity to unlock pathology archives for studies aiming to catalog and dissect the biological and clinical relevance of intra-tumor genetic heterogeneity.
Citation Format: Luciano G. Martelotto, Rita A. Sakr, Timour Baslan, Linda Rodgers, Hilary Cox, Jude Kendall, Tari A. King, Britta Weigelt, James Hicks, Jorge S. Reis-Filho. Single-cell sequencing from formalin-fixed paraffin-embedded breast cancers: a powerful tool to address intratumor genetic heterogeneity. [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 4735. doi:10.1158/1538-7445.AM2015-4735