Transcriptional Analysis of Cell-Sorted B-cell Lymphomas From High-throughput to Focused Identification of Molecular Targets

Sammanfattning: B cell lymphomas (BCLs) are a group of severe cancers, afflicting both men and women at different ages. Gene expression profiling (GEP), have during the latest decades fundamentally contributed to a better classification and biological understanding of the different BCLs. Most studies using GEP have analyzed mRNA from tumor tissue constituted of both tumor cells and non-malignant bystander cells. This thesis is based on four original papers where pure, cell-sorted BCL cells are analyzed by GEP. This procedure allows identification of differences in tumor cells rather than variations due to cellular composition. These studies focus on identifying new prognostic, diagnostic and/or functional markers for BCLs, using four different approaches. In the first study, GEP of pure mantle cell lymphoma (MCL) cells were used to identify MCL-associated targets genes. Subsequent immunohistochemistry (IHC) using antibodies raised against a unique protein epitope signature (PrEST) was performed. Using this high-throughput strategy we were able to identify proteins either uniquely or highly expressed in MCL compared to normal lymphoid tissue. This study confirmed the usage of transcriptional screening to identify molecular tumor-associated targets, as well as suggest the PrEST-approach as a novel efficient technique to identify molecular targets with both a known and unknown identity. The indolent growing follicular lymphoma (FL) cells are dependent on interplay with cells in the microenvironment for survival. We analyzed, in a second study, pure FL cells for highly expressed genes encoding surface bound proteins. An aberrant expression of CX3CR1 on the surface, not only on FL cells but also on tumor cells in several other BCLs was identified. CX3CR1 is suggested to be involved in site-specific dissemination, the possibility to use CX3CR1 as a target for antibody-mediated intervention needs however further investigations. The indolent FL often transforms to diffuse large B-cell lymphoma (DLBCL-tr). Previous studies analyzing tumor tissue for markers involved in this high-grade transformation have revealed results with low concordance. In a, we analyzed purified FL and DLBCL-tr tumor cells and found that large inter-tumor heterogeneity still was observed. Despite the heterogenous gene expression, this third study identified distinct differences between tumor entities rather than differences due to various composition of bystander cell and two proteins, galectin-3 and NEK2 pinpointing a subgroup of DLBCL-tr over FL were identified. In the last study, we used a unique material of eight different B cell lymphomas and identified for the first time unique transcription factors for each of these highly purified entities. The identified TFs are not only potential new molecular targets but are also partly responsible for the differences in global gene expression in the different BCLs as demonstrated by unsupervised clustering. In conclusion, analyzing pure lymphoma cells, dramatically enhance the resolution of analyses and allowed for identification of tumor-cell related genes using different approaches. Downstream analyzes of these genes allow for identification of proteins with a major biological importance for BCLs and potentially of new biomarkers.

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