Cytogenetic Profiling of B-Cell Lymphomas

CYTOGENETIC PROFILING OF B-CELL LYMPHOMAS

Bech Rasmussen, Sophie

4. term

Medical Biotechnology, Master

2011

2011-12-06

79

Formålet med dette pilotstudie var at etablere en metode til at screene for genmutationer i diffust storcellet B-celle-lymfom (DLBCL) og undersøge deres prognostiske og prædiktive betydning samt sygdomsmekanismer. Fokus var på genet EZH2, hvor en somatisk mutation, EZH2(Y641), er beskrevet i GCB-subtypen af DLBCL. Mutationen sidder i kodonet for tyrosin 641 i enzymets katalytiske område. Vi anvendte en PCR-baseret metode, højopløsningssmeltning (HRM), til at finde mutationer og bekræftede fundene med sekventering. Prøverne kom fra en bred lymfomkohort, primært DLBCL, fra det retrospektive samarbejdsstudie CHEPRETRO. For prøver med mutation blev der undersøgt genomiske ændringer i tumor-DNA med Affymetrix Cyto2.7M-array og genekspression i tumor-RNA med Affymetrix U_133_plus2 microarray. Derudover blev kliniske udfald sammenlignet mellem patienter med og uden EZH2-mutation i de første fem år efter diagnose, herunder efter subtype (GCB) og behandling (R-CHOP). HRM-metoden identificerede EZH2(Y641)-mutationen pålideligt; alle prøver med den karakteristiske smeltekurve fik mutationen bekræftet ved sekventering. Metoden virkede på tværs af forskellige prøvetyper: gDNA fra snap-frosset OCT-væv, gDNA fra formalin-fikseret paraffinindlejret (FFPE) væv og cDNA syntetiseret fra RNA. Der blev ikke identificeret en særskilt genomprofil for EZH2(Y641)-mutanter med Cyto2.7M-data. Genekspressionsanalysen fandt 25 gener, der var forskelligt udtrykt hos patienter med EZH2(Y641) sammenlignet med EZH2(wild type) inden for GCB-subtypen. De kliniske analyser viste ingen signifikant forskel i udfald inden for fem år mellem patienter med og uden mutationen, hverken i GCB-subtypen, ved R-CHOP-behandling eller generelt for DLBCL.

This pilot study aimed to establish a method to screen for gene mutations in Diffuse Large B-cell Lymphoma (DLBCL) and to explore their prognostic and predictive value as well as disease mechanisms. We focused on the EZH2 gene, where a somatic mutation, EZH2(Y641), has been reported in the GCB subtype of DLBCL. The mutation affects the codon for tyrosine 641 within the enzyme’s catalytic site. We used a PCR-based technique, high-resolution melting (HRM), to detect mutations and confirmed findings by sequencing. Samples were drawn from a broad lymphoma cohort, mainly DLBCL, within the retrospective collaborative translational study CHEPRETRO. For mutated samples, we assessed genomic alterations in tumor DNA using the Affymetrix Cyto2.7M array and gene expression in tumor RNA using the Affymetrix U_133_plus2 microarray. We also compared clinical outcomes within the first five years after diagnosis between patients with and without the EZH2 mutation, including by subtype (GCB) and treatment (R-CHOP). HRM reliably identified the EZH2(Y641) mutation; all samples with the characteristic melting curve had the mutation confirmed by sequencing. The method worked across different sample templates: gDNA from snap-frozen OCT tissue, gDNA from formalin-fixed paraffin-embedded (FFPE) tissue, and cDNA synthesized from RNA. No distinct DNA alteration profile specific to EZH2(Y641) cases was found using Cyto2.7M data. Gene expression analysis identified 25 genes differentially expressed in patients with EZH2(Y641) compared to EZH2(wild type) within the GCB subtype. Clinical analyses showed no significant differences in five-year outcomes between patients with and without the mutation, whether in the GCB subtype, with R-CHOP treatment, or across DLBCL overall.

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