Identification of novel prognostic biomarkers in diffuse large B cell lymphoma in the rituximab era

Diffuse large B cell lymphoma (DLBCL) is the most common form of lymphoma and is characterized by marked clinical and genetic heterogeneity. Approximately 60% of patients with DLBCL are cured with multi-agent chemotherapy consisting of rituximab, cyclophosphamide, hydroxyldaunomycin, oncovin and prednisone (R-CHOP). R-CHOP represents the current standard of care throughout the world for the treatment of DLBCL. The international prognostic index (IPI) is a clinical tool that can help risk-stratify patients at the time of diagnosis but it fails to identify 50% of patients who will relapse and provides no insights into the biology of the disease. The aim of this work was to identify prognostic markers that would complement the IPI, would reflect the underlying tumour biology and could be easily translated into the clinical setting. We used flow cytometry (FCM) to study the protein expression of the CD20 antigen, the target of rituximab on lymphoma cells and performed fluorescence in situ hybridization on DLBCL biopsies to identify the presence of genomic rearrangements in the BCL2 and MYC oncogenes. We also used DNA sequencing to determine if somatic mutations involving the rituximab binding epitope of CD20 had any impact upon patients failing therapy. We determined that recurrent mutations of exon 5 of CD20 do not explain rituximab resistance in clinical cases of DLBCL. In contrast, we could demonstrate that both a reduced expression of CD20 by FCM and the presence of concurrent de-regulation of MYC and BCL2 expression are independently associated with an inferior survival in DLBCL patients treated with R-CHOP. Importantly, both maintain their prognostic significance in multivariate analysis, independent of the IPI. Furthermore, these biomarkers reveal important novel insights into DLBCL biology and provide rational targets for therapy. As such they should be investigated and validated prospectively for future use in the clinical setting.Medicine, Faculty ofPathology and Laboratory Medicine, Department ofGraduat