Abstract
The origin of the Reed–Sternberg cell of Hodgkin's disease remained clouded in mystery for almost a century after its discovery in 1898. The major obstacle to its understanding is that, unlike other cancers, the malignant cell of Hodgkin's disease is vastly outnumbered by surrounding non-neoplastic cells at approximately 1000:1. We have devised several strategies to isolate Reed–Sternberg T-cells to determine their origin, global gene expression and, ultimately, their pathogenesis. This has increased the number of genes known to be expressed in Reed–Sternberg cells by >100-fold to over 12,000. Approaches such as density gradients, microdissection, and cell sorting help to enrich Reed–Sternberg cells for genomic DNA analysis. However, single-cell micromanipulation of living Reed–Sternberg cells was required to determine the genome-wide gene expression profile of these cells. Combined analysis of single cells and cell lines revealed the expression of 2666 named genes. Further analysis with high-density gene expression microarrays has demonstrated the expression of approximately 12,000 genes by Reed–Sternberg cells. The gene expression profile is that of an aberrant germinal center B-lymphocyte that resists apoptosis through CD40 signaling and NFkB activation. Gene expression analysis of Hodgkin's disease is an extreme test case demonstrating the application of high-throughput gene expression studies even to individual cells from clinical samples.
Hodgkin's disease is the most common cancer among adolescents and young adults in the United States, yet its basic biology has remained poorly understood. The majority of cells in tissues involved by Hodgkin's disease are not neoplastic but rather are composed primarily of an inflammatory infiltrate. Therefore, bulk extraction of RNA or DNA from Hodgkin's disease tissues reveals little, if anything, about the genetics or gene expression of the rare neoplastic Reed–Sternberg cell, which represents 0.01% of the cell population (Cossman et al. 1998). To overcome this hurdle, we applied several approaches to isolate Reed–Sternberg cells, beginning with density gradient centrifugation of cell suspensions derived from minced Hodgkin's tissues. This provided an enriched cell population (approximately 5%) sufficient for Southern blot analysis, which showed rearrangement of immunoglobulin genes in the Reed–Sternberg cell-enriched fractions (Sundeen et al. 1987). This finding strongly suggested that the Reed-Sternberg cell is clonal and argued in favor of a B-cell origin. However, these studies did not reveal information regarding the gene expression of Reed–Sternberg cells. Gene expression information was limited to that which could be obtained by in situ techniques such as immunohistochemistry. IHC studies collectively had reported the expression of approximately 50 different genes, at the protein level, in Hodgkin's disease.
To accelerate progress in understanding the gene expression profile of Reed–Sternberg cells, we applied a technique to create cDNA libraries from single cells. To collect viable single Reed–Sternberg cells with intact mRNA, we identified and collected living cells under inverted-phase microscopy using micromanipulation (Trumper et al. 1993). From six cases of Hodgkin's disease, 78 single-cell cDNA libraries were successfully prepared. These included 48 from individual Reed–Sternberg cells and the remainder from single B-or T-lymphocytes. Using PCR, the expression of approximately 50 genes was determined from individual Reed–Sternberg cells. For example, we identified expression (and mutation) of p53, as well as many genes involved in cell signaling and apoptosis control (Trumper et al. 1993; Messineo et al. 1998)
Despite the advances in understanding signaling pathways and other genes in Reed–Sternberg cells using PCR on the single-cell cDNA libraries, the pace of determining the gene expression profile was slow. To increase the rate of discovery, we turned to high-throughput gene sequencing of cDNAs from the libraries. cDNA libraries from four individual Reed–Sternberg cells taken from two patients were subjected to high-throughput sequencing and genomic bioinformatic analysis. In all, more than 27,000 cDNAs were sequenced and the expression of 2666 named genes was shown in single Reed–Sternberg cells and cell lines in a study conducted with Human Genome Sciences (Cossman et al. 1999). This expression profile was compared to that determined for both germinal center B-cells and dendritic cells. The Reed–Sternberg cells most closely aligned with germinal center B-cells, although they were clearly different.
High-throughput sequencing of cDNAs significantly extended the number of genes known to be expressed by Reed–Sternberg cells. However, this approach lacks the breadth of coverage of genes available through high-density gene expression microarrays. Using a human gene expression array containing 42,000 sequences (Affymetrix), the gene expression profile of two Hodgkin's disease Reed–Sternberg cell lines was determined and revealed the expression of nearly 12,000 genes in these cells. It confirmed the expression profile obtained by high-throughput sequencing and again corroborated the understanding that the Reed–Sternberg cell is of B-cell lineage. This microarray study, conducted in collaboration with Gene Logic (Gaithersburg, MD) applied novel bioinformatics tools enabling the comparison of the gene expression profiles of Reed–Sternberg cells to other cell types and showed the close association to germinal center B-cells. Through the application of visualization and analytical tools, the regulation of specific metabolic and signaling pathways within the Reed–Sternberg cell was determined. This information provides a basis for identifying therapeutic and diagnostic targets in Hodgkin's disease (Annunziata et al. 2000).