Single-cell clarity and heterogeneity in copy number profiles in primary synovial and Ewing sarcoma with ResolveDNA® genomic amplification

Katherine A. Kennedy1, Jon S. Zawistowski1, Viren R. Amin1, Durga M. Arvapalli1, Isai Salas-González1, Natalie Jäger2, Hanno Glimm3, Gary L. Harton1, Stefan Fröhling2, Jason A.A. West1, Stefan Pfister2.

  1. BioSkryb Genomics, Inc., Durham, NC.
  2. Hopp Children’s Cancer Center Heidelberg (KiTZ) and German Cancer Consortium (DKTK), Heidelberg, Germany
  3. NCT Dresden and German Cancer Consortium (DKFZ), Dresden, Germany

Soft tissue sarcomas are a diverse set of mesenchymal malignancies frequently driven by translocation events, resulting in expression of aberrant fusion gene products that fuel cell proliferation and tumor growth. Examples of these, including Ewing sarcoma and synovial sarcoma, have limited data surrounding the broader genomic alterations, and intra-tumoral heterogeneity, that synergizes with and contributes to disease aggression and prognosis.

In a collaboration with the clinical, translational and basic research sarcoma expertise at the Hopp Children’s Cancer Center Heidelberg (KiTZ), we utilized ResolveDNA kit to amplify the genomes of individual sarcoma cells with an aim to resolve copy number aberration (CNA) at the single cell level in primary sarcoma samples. The ResolveDNA chemistry attenuates amplicon size, redirecting the amplification primer to the primary DNA template of interest and avoiding exponential copying of amplicons. This results in unprecedented genomic coverage and uniformity, with high SNV precision and sensitivity, allelic balance, and the enhanced ability to accurately call CNA.

Our initial study comprised cells from two patient samples: a putative synovial sarcoma and a Ewing sarcoma that were FACS sorted as single cells in each well. We defined CNA using the Ginkgo algorithm and identified discrete profiles both between and within sarcoma samples. The vast majority of putative synovial sarcoma single cells displayed a highly disrupted genome, with stark focal, sub-chromosomal gains and losses suggestive of chromothripsis. While rare, isolated cells demonstrated a lack of any apparent CNA. Intriguingly, single-cell heterogeneity was notable in multiple sub-chromosomal regions including 4p and 16q. An SS18:SSX translocation was not identified in this patient, suggesting that these cells may have originated from a secondary sarcoma as opposed to a synovial sarcoma. In contrast, the Ewing sarcoma single cells harbored discrete, full-chromosomal gains and losses. Concurrently, we have extended these findings to catalog alterations at the single nucleotide level using whole exome sequencing (WES) from the same single cells that were profiled by CNV. We also analyzed primary samples from additional sarcoma subtypes to define genomic evolution and clonal lineage contributing to these pathologies.

The heterogeneity observed in this study demonstrates the power of single cell vs bulk analysis to reveal the nature of genomic instability and aid in the characterization of sarcoma subtypes at unprecedented levels of resolution. These abilities are particularly crucial to understand disease development and progression in orphan sarcomas, especially those with complex genome rearrangements and/or poor clinical outcome.