Serial Analysis of Gene Expression Revealed Consistent Downregulation of More Than 100 Genes in Hodgkin Lymphoma.

Hodgkin Lymphoma (HL) is unique in its cellular composition, consisting of a minority of tumor cells in an inflammatory background. The tumor cells (Reed-Sternberg cells, RS cells) are thought to be derived from germinal center B cells (centroblasts, CB) that have escaped apoptosis during antigen selection. Global gene expression studies in HL have been hampered by the fact that RS cells only represent less than 1% of the cells in the tumor tissue. Only a few large-scale gene expression studies are available, mostly using cell lines derived from patients with classical HL. To get more insight into the gene expression profile of RS cells we applied the Serial Analysis of Gene Expression Technique (SAGE) on several HL cell lines. SAGE was performed on the cell lines L428 and L1236 (classical cell lines, cHL), DEV (Nodular Lymphocyte Predominant cell line, NLP) and on CB as normal counterparts.

In total, about 100.000 tags were sequenced (Table 1). After normalization and exclusion of tags present only once, 3.635 genes were retained and used for further analyses. A five-fold difference in tag frequency between the HL cell lines and CB was used to select for up- or downregulated genes. Although many genes were upregulated in every individual HL cell line compared to CB, only few genes were consistently upregulated. In contrast, comparison of the downregulated genes revealed that 125 were downregulated in all three HL cell lines compared to CB (Table 2). Tags corresponding to Fascin, TARC/CCL17 and BIC, known to be highly expressed in HL indeed were observed in the SAGE libraries. Among the genes downregulated in L428 a loss of B lineage-specific genes was observed confirming the previous findings in L1236. Interestingly our SAGE results suggest that the NLP cell line DEV has an intermediate loss of B-lineage specific genes compared to CB. To gain more insight in the biology, differentially expressed genes were grouped according to gene ontology’s (based on biological process or molecular function) and pathways. We have now selected more than 70 genes that will be subjected to quantitative RT-PCR analysis to confirm our SAGE results and to study their expression in tumor cell enriched HL samples.

In conclusion: Downregulation of gene expression seems to be important in the pathogenesis of HL since we observed a consistent downregulation of more than 100 genes in HL compared to CB.

Table 1. Overview of Hodgkin lymphoma SAGE libraries

Table 2. Number of up- and downregulated genes in Hodgkin lymphoma compared to centroblasts