Abstract 5345: Complementary NGS approaches on digitally sorted pure tumor cells reveal hidden molecular characteristics in low tumor content FFPE specimens

Introduction: Precise characterization of tumor cell populations is an essential requirement for guiding the cancer care, allowing patients to receive personalized therapies. Poor biopsies with low-tumor content represent a significant barrier for sample enrollment in clinical trials. Here we describe a multi-level approach for precisely characterizing the genetic mutation landscape of pure tumor cell populations sorted by the DEPArray™ technology from low-cellularity FFPE samples. Methods: 50μm sections of FFPE from breast infiltrating ductal adenocarcinoma with 10% tumor content were processed by DEPArray sorting protocol. Illumina-compatible libraries were prepared from sorted stromal (n=497) and tumor (n=419) cell pools, and from the unsorted sample. An aliquot of these libraries was processed using SeqCap EZ MedExome enrichment kit (Roche) for whole-exome sequencing (WES) with a HiSeq 2500, reaching a mean coverage of 27x for tumor and 25x for stromal libraries. A second aliquot was used for low-pass (≈1M fragments per sample) whole-genome sequencing (WGS) on a MiSeq to analyze copy-number alterations (CNA). Other cell lysates from stromal (n=104, n=112) and pure tumor (n=75) populations were used in DEPArray OncoSeek amplicon-based assay for focused analysis of clinically relevant somatic variants and copy-number alterations. Results: In sorted pure tumor populations, WES analysis of B-allele frequencies of germline heterozygous SNPs clearly outlined an aberrant profile, precisely revealing several Loss-of-Heterozygosity (LOH) and copy-altered regions. Conversely, unsorted gDNA showed a flat profile non-distinguishable from sorted stromal cells, as expected for a low-cellularity tumor sample. Similar results were obtained by low-pass WGS, where the huge number of copy number aberrations (≈1.2 Gbp) in tumor is contrasted by lack of gains and losses in stromal cells and unsorted gDNA. Noteworthy, WES, low-pass and targeted sequencing by OncoSeek panel highlighted a focal amplification of ERBB2 gene (>13 copies), which was just barely detectable as a 1-copy gain in bulk gDNA. Genetic analyses showed a high concordance between WES and targeted panel data, with two non-synonymous homozygous somatic mutations found in TP53 (p.L111R) and ERBB2 (p.D769Y). In the unsorted sample, the TP53 mutation was missed because allelic frequency was below the limit of detection due to normal-cell dilution, while the ERBB2 mutation was still detectable only because of the