Characterization and Purification of Neoplastic Cells of Nodular Lymphocyte Predominant Hodgkin Lymphoma from Lymph Nodes by Flow Cytometry and Flow Cytometric Cell Sorting

We report the flow cytometric (FC) identification and characterization of lymphocyte predominant (LP) cells from tissues involved by nodular lymphocyte predominant Hodgkin lymphoma (NLPHL). First, we immunophenotyped the NLPHL cell line (DEV) confirming a germinal center immunophenotype, lack of expression of CD32 and CD58, and expression of CD54. Nineteen of 26 lymph nodes involved by NLPHL demonstrated a population with an LP immunophenotype (73%), which included expression of germinal center markers (CD75/Bcl-6-positive, CD32-weak/negative without CD10), a B-cell immunophenotype (CD19/CD20/CD40+), IgD and/or IgM expression (67%), and lack of programmed death-ligand 1/ligand 2. The LP cells demonstrated an adhesion macromolecule expression pattern distinct from Hodgkin and Reed-Sternberg cells of classical Hodgkin lymphoma (CHL) (uniform CD50 and variable CD58 for NLPHL; minimal CD50, bright CD58 expression for CHL). A two-tube consensus assay identified LP cells in all seven NLPHL cases examined and only one non-NLPHL case (94 cases evaluated). Finally, FC cell sorting studies confirm that FC-defined populations have an LP cytomorphology. Taken together, these findings demonstrate a two-tube consensus assay can be used to immunophenotype NLHPL with high specificity and sensitivity and rapidly purify LP cells for genetic studies. This study also confirms aneuploidy in LP cells, provides antigens that may be helpful in distinguishing NLPHL from CHL, and suggests that T cells interact less avidly with LP cells than with Hodgkin and Reed-Sternberg cells.