Abstract P051

Genetic tumor cell heterogeneity and genomic instability in classic Hodgkin lymphoma

Classic Hodgkin lymphoma (cHL) is one of the most prevalent types of lymphomas in the Western world and one of the most frequent cancers in young adults. Hodgkin and Reed-Sternberg (HRS) lymphoma cells are highly heterogeneous and typically show multiple chromosomal abnormalities. The rarity of HRS cells has hampered their analysis over the last decades, but understanding the tumor cell heterogeneity is essential to reveal mechanisms of therapy resistance. Therefore, we developed an advanced workflow to perform whole-exome sequencing (WES) of single HRS cells to reveal signs of intraclonal tumor evolution and the underlying mutagenic processes.

Frozen cHL lymph node suspensions were processed using an antibody-based staining method, and single HRS cells were transferred to a 96-well plate using a fluorescence-activated cell sorter. We used a single-cell whole-genome amplification kit to barcode the DNA prior to the whole-exome capturing. Bulk non-tumor cells were simultaneously collected to later on distinguish germline from somatic mutations. The single-cell WES workflow was tested with a mixture of cells from two HL cell lines. By comparing cell line-specific mutations, the index-sorted cells could be explicitly assigned to its particular cell line, showing the robustness of this pipeline in identifying distinct mutations in subsets of cells. We further validated the method using a primary cHL case, which had previously been characterized in a bulk WGS study. Most of the identified exonic mutations from the bulk analysis were also identified in at least a subset of single cells, and subsequent genealogical analysis revealed first insights into the mutational development of this HL case. Six additional HL cases are currently being processed.

A detailed characterization of mutation patterns at the single-cell level may provide profound insights into the genetic complexity and heterogeneity of the unique HRS cancer cells. This characterization is expected to reveal novel insights into lymphoma progression and the underlying mutagenic processes and is a pivotal step towards our long-term objective of elucidating the mechanisms of genetic instability in HRS cells, to identify markers of treatment resistance and develop new diagnostic and therapeutic approaches.