The molecular landscape of rare aggressive lymphomas

Posttransplant lymphoproliferative disorder (PTLD) is a rare but life threatening disorder following solid organ and hematopoietic stem cell transplantation. The disorder is characterized by an uncontrolled proliferation of lymphocytes and caused by medication induced diminished immune surveillance. Although not required for diagnosis of PTLD, Epstein Barr virus (EBV) plays a major role in the pathogenesis of about 2/3 of PTLDs.  To gain a more accurate understanding of complex genomic aberrations and to identify key gain or loss key regions, we are currently performing next generation sequencing studies on samples of patients diagnosed with PTLD. During the last 25-30 years more than 200 cases of biopsy proven PTLD were diagnosed and samples collected in our center, providing a challenging opportunity for further genomic studies. Clinical characteristics and gene expression profiling studies on a subset of these cases have been performed and published before. Since we currently have no clear view on the role of chromosomal aberrations versus mutations or small insertions/deletions in PTLD, we sequenced the full genomes of 17 monomorphic (both EBV+ and EBV-) diffuse large B cell lymphoma PTLDs at 50x coverage. Currently mapping has been completed and we will now start detailed analysis of these data. This will allow us to get insight in the spectrum of mutations that are present in these lymphoma samples, and will also make it possible to detect large chromosomal aberrations such as translocations, duplications and deletions. (a) If point mutations are common: we will analyze more samples using exome sequencing, as this technology is well suited to detect point mutations. (b) If structural variations occur frequently: we will include a low coverage genome sequence analysis, which will allow us to detect chromosomal rearrangements.  (c) RNA-sequencing: for those cases where also RNA is available, we will consider to perform RNA sequencing to detect additional relevant genetic alterations. This validated and innovative technique allows for the simultaneous detection of mutations, splicing variants, fusion transcripts and extended regions of hemizygosity.

In a second step, we will try to validate aberrations found in the first set of samples in a larger cohort by performing targeted sequencing. Dependent on the aberrations found in these sequencing studies, further attempts will be made to determine their significance and role in diagnosis, classification, treatment and prognosis. These further studies will include immunohistochemical analysis of expressed proteins, incorporation of antibodies directed against components of key signaling pathways and determining importance of specific cellular localization of involved proteins.

Finally, after validation, the exact role of genomic aberrations can be further unraveled by studying their significance on frozen material, and by knock-down or overexpression experiments in cell-based models and xenograft models. An exact description of these experiments will depend on the initial findings.

In conclusion, the results of these analyses should lead to (1) improved insights in molecular and genetic pathogenesis of these disorders, (2) development of targeted therapies and (3) detection of early diagnostic and/or prognostic biomarkers.