BRIEF REPORT |
Correspondence to: Jeffrey Cossman, Oscar Benwood Hunter Professor of Pathology, Georgetown U. Medical Center, NW 103 MedicalDental Building, 3900 Reservoir Rd. NW, Washington, DC 20007.
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Summary |
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The origin of the ReedSternberg 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 ReedSternberg 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 ReedSternberg cells by >100-fold to over 12,000. Approaches such as density gradients, microdissection, and cell sorting help to enrich ReedSternberg cells for genomic DNA analysis. However, single-cell micromanipulation of living ReedSternberg 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 ReedSternberg cells. The gene expression profile is that of an aberrant germinal center B-lymphocyte that resists apoptosis through CD40 signaling and NFB 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. (J Histochem Cytochem 49:799800, 2001)
Key Words: Hodgkin's disease, ReedSternberg cells, gene expression, B-lymphocyte
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Introduction |
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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 ReedSternberg cell, which represents 0.01% of the cell population (
Despite the advances in understanding signaling pathways and other genes in ReedSternberg 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 ReedSternberg 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 ReedSternberg cells and cell lines in a study conducted with Human Genome Sciences (
High-throughput sequencing of cDNAs significantly extended the number of genes known to be expressed by ReedSternberg 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 ReedSternberg 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 ReedSternberg 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 ReedSternberg 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 ReedSternberg cell was determined. This information provides a basis for identifying therapeutic and diagnostic targets in Hodgkin's disease (
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Footnotes |
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Presented in part at the Joint Meeting of the Histochemical Society and the International Society for Analytical and Molecular Morphology, Santa Fe, NM, February 27, 2001.
Received for publication January 12, 2001; accepted February 16, 2001.
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Literature Cited |
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