Gene Expression Profiling of Microdissected Hodgkin Reed Sternberg Cells: Molecular Subtypes and Treatment Outcome Correlations.

Classical Hodgkin lymphoma (cHL) is unique among lymphomas due to the scarcity of the malignant Hodgkin Reed Sternberg (HRS) cells, which are derived from clonal germinal center B cells. Investigations using laser capture microdissection permit more detailed analysis of these cells. However, most recent studies were limited by low case numbers and lack of available clinical data.

We studied 29 cases of cHL and the 5 HL lines KMH2, HDLM2, L428, L540, and L1236 by gene expression profiling. All patients were treated at the BC Cancer Agency between 1984 and 2006 and received at least 4 cycles of polychemotherapy and stage-dependent radiotherapy. The cohort also included 5 biopsies taken at relapse. Treatment failure was defined as disease progression or relapse at any time (n=14) after initiation of treatment; treatment success as absence of progression (n=15). We used laser microdissection (Molecular Machines & Industries Cellcut with Nikon Eclipse TE2000-S microscope) to study the enriched HRS cell compartment separately from the microenvironment. RNA extraction was performed on pools of 1000 microdissected HRS cells in each case. Gene expression profiles were generated using Affymetrix HG UA133 2.0 Plus arrays using two-cycle labeling reactions. HRS cell profiles were compared to microdissected germinal centers (GC), and HL cell line profiles compared to enriched tonsillar CD77+ centroblasts (MACS cell separation, Miltenyi). Furthermore, we compared gene expression profiles of treatment failures to those of treatment successes.

We identified 1342 differentially expressed probesets (fold change >5, False Discovery Rate (FDR) adjusted p value <0.001) between HRS and GC cells. Using overrepresentation analysis we found genes involved in NFκB, JAK/Stat, IL-6, IL-9 signaling, cytotoxic T lymphocyte-mediated apoptosis and IL-15 production to be significantly over-expressed in HRS cells and genes involved in B cell, T cell receptor signaling and many transcriptional regulators such as FOXO1, E2F5, IRF8, NFATC1, NFYB, POU2AF1 to be significantly under-expressed in HRS cell. Comparison of these data to differentially expressed genes in the HL cell lines (1004 genes, fold change >5, FDR-adjusted p value <0.001) showed significant overlap of genes involved in proliferation, apoptosis, IL15 signaling and B cell receptor signaling, while overrepresentation of metabolism genes was unique to the cell lines. Hierarchical clustering of all 29 primary HL cases identified 3 separate clusters characterized by 1) a cytotoxic signature, 2) TNF/TGFB receptor signaling or 3) a residual B cell signature. Dichotomizing the profiles into the two treatment outcome groups demonstrated that NFγB signaling, complement system genes and genes involved in the developmental process of hematopoietic progenitor cells, macrophages and blood vessels were overexpressed in treatment failure samples.

Using microdissection of HRS cells in a large number of cases we were able to further characterize the unique expression program of HL and refine the data inventory about dysregulated cellular functions and pathways in this disease. Overexpression of genes associated with NFκB, complement and hematopoietic progenitor cells proliferation correlate strongly with treatment failure. Further study using immunohistochemistry is currently ongoing to validate these findings and to develop clinically useful biomarkers.

Gascoyne:Roche Canada: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding.