Transcriptome based analysis of Hodgkin lymphoma : insights into the microenvironment

This thesis is focused on the analysis of Hodgkin lymphoma (HL), in particular the complex interaction between the malignant cells and the abnormally behaving bystander cells, and this complexity of the inflammatory infiltrate in HL constantly challenge scientists. The common thread in this thesis is the usage of gene expression profiling of whole tumour tissues, laser-captured Hodgkin Reed Sternberg (H-RS) cells and of cell-lines.

EBV+ and EBV- tumour were compared to germinal centre cells in order to identify EBV dependent genes. By overlapping the results with the gene expression profiles from the comparision between a normal HL derived cell-line and the same with introduced EBNA1+, and a set of EBV dependent genes could be identified. Among them CCL20. CCL20 is a chemokine that binds to CCR6, a receptor present on regulatory T-cells. Cell-based experiments showed that CCL20 attracted regulatory Tcells from peripheral blood. This finding could contribute to the understanding how EBV infection affects the way the H-RS cell orchestrates the infiltrating tumour stroma towards an anergic state.

HL is divided into subtypes and the most common ones are nodular sclerosis (NS) and mixed cellularity (MC). Gene expression profiling identified a set of genes that distinguished each the two subtypes. In NS macrophages and fibroblasts were positive for the NS subtype specific genes and in MC the macrophages and lymphocytes stained positive for the MC subtype specific genes . Only two of the studied genes were expressed by the H-RS cells, Cathepsin K at rare occasions and Connective tissue growth factor.

Connective tissue growth factor (CTGF) had a higher expression in the NS subtype. Immunohistochemistry showed a subtype specific expression of the gene in fibroblasts and macrophages but also a subtype independent expression in the malignant cells. Due to CTGFs association with apoptosis and prognosis in other cancer types we evaluated the expression in the H-RS cells and identified three different expression patterns. A larger cohort is needed if one is to draw any conclusions about prognostic from the study.

HL derived cell-lines have been used as a model for HL for a long time. However it is well known that tissue culture only approximate part of the malignancy. For example, the microenvironment is lacking, as well as tissue polarity. In order to improve the cellline model of HL, the cell-line L1236 was cultured in a three dimensional (3D) matrix consisting of self-organizing peptides. Gene expression profiles of the 3D cultured L1236 were compared to conventionally grown L1236, gene expression profiles from tumours, and laser captured H-RS cells.

List of scientific papers

I. Baumforth KR, Birgersdotter A, Reynolds GM, Wei W, Kapatai G, Flavell JR, Kalk E, Piper K, Lee S, Machado L, Hadley K, Sundblad A, Sjoberg J, Bjorkholm M, Porwit AA, Yap LF, Teo S, Grundy RG, Young LS, Ernberg I, Woodman CB, Murray PG (2008). "Expression of the Epstein-Barr virus-encoded Epstein-Barr virus nuclear antigen 1 in Hodgkins lymphoma cells mediates Up-regulation of CCL20 and the migration of regulatory T cells." Am J Pathol 173(1): 195-204. Epub 2008 May 23
https://pubmed.ncbi.nlm.nih.gov/18502823

II. Birgersdotter A, Baumforth KRN, Porwit A, Sjöberg J, Wei W, Björkholm M, Murray PG, Ernberg I (2008). "Inflammation and tissue repair markers distinguish the nodular sclerosis subtypes and mixed cellularity subtypes of classical Hodgkin lymphoma." British Journal of Cancer (Submitted)

III. Birgersdotter A, Baumforth KRN, Sjöberg J, Wei W, Murray PG, Björkholm M, Porwit A, Ernberg I (2008). "Connective tissue growth factor (CTGF) is expressed in the malignant cells of Hodgkin lymphoma, but not in those of other mature B-cell lymphomas." American Journal of Clinical Pathology (Submitted)

IV. Birgersdotter A, Baumforth KR, Porwit A, Sundblad A, Falk KI, Wei W, Sjöberg J, Murray PG, Björkholm M, Ernberg I (2007). "Three-dimensional culturing of the Hodgkin lymphoma cell-line L1236 induces a HL tissue-like gene expression pattern." Leuk Lymphoma 48(10): 2042-53
https://pubmed.ncbi.nlm.nih.gov/17917972