Characterisation of molecular alterations in diffuse large b-cell lymphoma with respect to transformation, progression and prognostic markers

Abstract: Diffuse large B-cell lymphoma (DLBCL) is the most common lymphoma type, and comprises nearly 500 newly diagnosed cases in Sweden every year. DLBCL is a group of aggressive lymphomas that are extremely heterogeneous and differ in morphology, immunophenotype, molecular and clinical features as well as in response to therapy. The transformation of low malignant lymphoma, most commonly follicular lymphoma (FL), to DLBCL is associated with a progressive disease and poor prognosis. The overall aim of this thesis was to identify the genetic events underlying the progression and transformation of indolent FL to aggressive DLBCL and to outline molecular alterations distinguishing DLBCL with germinal center (GC) and non-GC phenotype. In search for novel markers in lymphoma, we have investigated an anti-apoptotic protein, HS-associated protein X-1 (HAX-1), at the protein and transcript level in malignant lymphomas (paper I). We found high levels of HAX-1 mRNA and protein expression in Bcell lymphomas. We also identified a positive association between proliferation (Ki67) and HAX-1 expression, as well as an inverse correlation between Bcl-2 and HAX-1 demonstrated on a transcript and protein level in FL, which indicates the potential role of HAX-1 as a Bcl-2 homolog. In papers II and III we studied paired tumours of FL and its transformed DLBCL (tDLBCL) counterparts and compared with de novo DLBCL (dnDLBCL) using arraycomparative genome hybridisation (array-CGH) in order to outline genetic alteration of importance for the transformation process. We identified a gain of 2p15-16.1 as a potential prognostic marker, and that this alteration may appear already in the FL prior to transformation. This chromosomal region encompasses, among others, genes involved in NF- κB pathway, such as REL, USP34, COMMD1, OTX1 and of known importance for B-cell development, such as BCL11A. We have also performed whole exome sequencing (WES) results in a smaller group of patients with consecutive lymphoma samples. A comparison with array-CGH data showed that these two methods complement and strengthen each other in pinpointing crucial genetic events. Furthermore, our studies revealed that clonal evolution of transformed tumours occurs according to the branching model. A number of mutations identified in the peri-transformation phases – defined as FL appearing prior and tDLBCL directly after transformation - affected genes involved in histone modifications, cell cycle, apoptosis, PI-3 kinase pathway, and Ras signalling pathway. In Paper IV we performed proteomic profiling of DLBCL subtypes i.e. non-GC and GC, and identified 91 proteins present exclusively in the non-GC type of DLBCL. We focused on two proteins of importance for the NF-κB pathway (BiP and Hsp90) as well as on Cyclin B2. We observed increased expression of these three proteins in DLBCL and also in non-GC vs GC type of DLBCL. These findings suggest a potential relevance of these proteins as prognostic markers and may lead to new treatment options.