The aim of this thesis was to set up a pilot study in regard to screening mutations in Diffuse Large B-cell Lymphoma with the goal to elucidate the prognostic and predictive impact as well as pathogenetic understanding. This was conducted with focus on a particular gene EZH2, as a novel somatic mutation was identified in the GCB-subtype of Diffuse Large B-cell Lymphoma, by Morin et al. in 2010. The somatic mutation, referred to as EZH2(Y641) was the only one identified across 19 exons and arose only in a codon, coding for Tyrosine 641, placed in the catalytic site of EZH2. To obtain the aims of examination, a PCR-based method, known as High Resolution Melting was applied for mutation screening, followed by sequencing. Samples screened were a broad cohort of lymphomas, but mainly Diffuse Large B-cell Lymphoma, from the retrospective collaborative translational trial termed CHEPRETRO. Samples found mutated was then further analysed in regard to genomic alterations, by examining genomic tumour DNA with Cyto2.7M array from Affymetrix and gene profiling tumor RNA by Affymetrix U_133_plus2 micro array. Lastly a clinical outcome analysis was conducted, comparing mutated EZH2 samples to those with EZH2 wild type, within the first five years following diagnosis.
The detection of the EZH2(Y641) mutation through high resolution melting analysis, was considered successful as all the samples displaying a heteroduplex melting curve, similar to that of the EZH2(Y641) mutated cell line DB, had their mutational status verified through sequencing. This was achieved using three different templates, gDNA from snap-frozen OCT tissue, gDNA from formalin fixed paraffin embedded tissue and cDNA, synthesized from RNA, proving the method applicable for mutation screening across various templates.
It was not possible to detect a genomic profile distinct to EZH2(Y641) mutants using the data utilized from the Cyto2.7M array.
Gene expression analysis by the U_133_plus2 microarray platform, revealed 25 differentially expressed genes for patients with the identified EZH2(Y641) mutation when compared to EZH2(wild type) of the GCB-subtype of Diffuse Large B-cell Lymphoma.
The clinical outcome analyses performed in the available cohort of patients showed no significant difference, between patients mutated in EZH2(Y641) compared to EZH2(wt), in regard to the GCB-subtype, treatment with R-CHOP and in general to patients with Diffuse Large B-cell Lymphoma, disregarding subtype.