Flow-sorting and exome sequencing of the reed-sternberg cells of classical Hodgkin lymphoma Academic Article uri icon

Overview

MeSH Major

  • Immunophenotyping
  • Ki-67 Antigen
  • Lymphoma, AIDS-Related
  • Lymphoma, Large B-Cell, Diffuse

abstract

  • 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.

publication date

  • June 10, 2017

Research

keywords

  • Academic Article

Identity

Language

  • eng

Digital Object Identifier (DOI)

  • 10.3791/54399

PubMed ID

  • 28654052

Additional Document Info

volume

  • 2017

number

  • 124