Flow-sorting and Exome Sequencing of the Reed-Sternberg Cells of Classical Hodgkin Lymphoma.

TitleFlow-sorting and Exome Sequencing of the Reed-Sternberg Cells of Classical Hodgkin Lymphoma.
Publication TypeJournal Article
Year of Publication2017
AuthorsReichel JB, McCormick J, Fromm JR, Elemento O, Cesarman E, Roshal M
JournalJ Vis Exp
Date Published2017 06 10
KeywordsB-Lymphocytes, DNA Copy Number Variations, Exome, Flow Cytometry, Hodgkin Disease, Humans, Polymerase Chain Reaction, Reed-Sternberg Cells, T-Lymphocytes

The Hodgkin Reed-Sternberg cells of classical Hodgkin lymphoma are sparsely distributed within a background of inflammatory lymphocytes and typically comprise less than 1% of the tumor mass. Material derived from bulk tumor contains tumor content at a concentration insufficient for characterization. Therefore, fluorescence activated cell sorting using eight antibodies, as well as side- and forward-scatter, is described here as a method of rapidly separating and concentrating with high purity thousands of HRS cells from the tumor for subsequent study. At the same time, because standard protocols for exome sequencing typically require 100-1,000 ng of input DNA, which is often too high, even with flow sorting, we also provide an optimized, low-input library construction protocol capable of producing high-quality data from as little as 10 ng of input DNA. This combination is capable of producing next-generation libraries suitable for hybridization capture of whole-exome baits or more focused targeted panels, as desired. Exome sequencing of the HRS cells, when compared against healthy intratumor T or B cells, can identify somatic alterations, including mutations, insertions and deletions, and copy number alterations. These findings elucidate the molecular biology of HRS cells and may reveal avenues for targeted drug treatments.

Alternate JournalJ Vis Exp
PubMed ID28654052
PubMed Central IDPMC5608337
Grant ListR01 CA194547 / CA / NCI NIH HHS / United States