Abstract 3617: Digital sorting rescues low-cellularity tumor FFPE samples for genome-wide copy-number profiling
Authors: Forcato Claudio , Lalibertè Julie , Bolognesi Chiara , Cataldo Ivana , Buson Genny , Mangano Chiara , Cantù Cinzia , Fontana Francesca , Tononi Paola , Medoro Gianni , Harkins Tim , Lawlor Rita T. , Scarpa Aldo , Manaresi Nicolò 
Published: July 2016

Introduction: The analysis of formalin-fixed paraffin embedded (FFPE) cancer specimens is particularly challenging due to minute amount of tissue, low-tumor cellularity and heterogeneity, associated with low quality DNA. This results in an imprecise characterization of somatic variants and copy-number alterations (CNA). Here we show how digital sorting combined with low-pass whole genome sequencing (WGS) can resolve genome-wide copy-number profiling even for the more challenging FFPE samples.

Methods: An archival FFPE sample from one pancreatic cancer patient, rejected by the International Cancer Genome Consortium (ICGC) criteria due to low tumor content (&lt;25%), was dissociated into a single cell suspension. Using the DEPArray™ digital sorter, tumor and normal stromal cell subpopulations (range = 70-77) were recovered using Keratin/Vimentin immunofluorescence and DNA ploidy, from which 0.4-0.5ng DNA was obtained. After lysis, Illumina®-compatible libraries were prepared from one pool of pseudo-diploid sorted tumor cells, one pool of stromal cells and one unsorted sample (1500 cells, i.e. about 10ng gDNA) using the Accel-NGS® 2S DNA Library Kit from Swift Biosciences. Libraries were used for low-pass whole genome sequencing on Illumina® MiSeq. Paired-end (2×100) reads were aligned to hg19 human reference using BWA software and copy-number profiles were generated with Control-FREEC.

Results: A total of 11M paired-end reads and a mean coverage of 0.12x were obtained from low-pass WGS, enabling the detection of CNAs at good resolution. The resulting copy-number profiles clearly show the difference between the stromal and tumor populations, with the first characterized by a flat profile (0 gains, 0 losses) and the second presenting several somatic copy-number alterations (6 gains, 22 losses). As expected due to dilution by normal cells, CNA profiles of unsorted samples missed most of the gains and losses (only 5/28 = 18% of aberrant genomic regions were detected). At a single base level, the difference between unsorted and sorted samples was more significant as the unsorted fraction missed &gt;99% of CNA bases found in the sorted tumor population.

Conclusions: DEPArray™ sorting combined with Accel-NGS® 2S kits and Illumina® low-pass whole genome sequencing enables high quality genome-wide profiling of pure tumor and stromal populations. The capacity of this method to deal with low DNA input (0.5ng) from archival FFPE samples characterized by low cancer cell content (&lt;25%) makes it a valuable and easily accessible tool for studying tumor CNA profiles.

Citation Format: Claudio Forcato, Julie Lalibertè, Chiara Bolognesi, Ivana Cataldo, Genny Buson, Chiara Mangano, Cinzia Cantù, Francesca Fontana, Paola Tononi, Gianni Medoro, Tim Harkins, Rita T. Lawlor, Aldo Scarpa, Nicolò Manaresi. Digital sorting rescues low-cellularity tumor FFPE samples for genome-wide copy-number profiling. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3617.