Exploring next-generation sequencing in chronic lymphocytic leukemia

Sammanfattning: Next-generation sequencing (NGS) techniques have led to major breakthroughs in the characterization of the chronic lymphocytic leukemia (CLL) genome with discovery of recurrent mutations of potential prognostic and/or predictive relevance. However, before NGS can be introduced into clinical practice, the precision of the techniques needs to be studied in better detail. Furthermore, much remains unknown about the genetic mechanisms leading to aggressive disease and resistance to treatment. Hence, in Paper I, the technical performance of a targeted deep sequencing panel including 9 genes was evaluated in 188 CLL patients. We were able to validate 143/155 (92%) selected mutations through Sanger sequencing and 77/82 mutations were concordant in a second targeted sequencing run, indicating that the technique can be introduced in clinical practice. In Paper II we screened 18 NF-κB pathway genes in 315 CLL patients through targeted deep sequencing which revealed a recurrent 4 base-pair deletion in the NFKBIE gene. Screening of NFKBIE in 377 additional cases identified the mutation in ~6% of all CLL patients. We demonstrate that the lesion lead to aberrant NF-κB signaling through impaired interaction with p65 and is associated with unfavorable clinical outcome. In Paper III we sought to delineate the genetic lesions that leads to relapse after fludarabine, cyclophosphamide, and rituximab treatment. Through whole-exome sequencing of pre-treatment and relapse samples from 41 cases we found evidence of frequent selection of subclones harboring driver mutations and subsequent clonal evolution following treatment. We also detected mutations in the ribosomal protein RPS15 in 8 cases (19.5%) and characterization of the mutations through functional assays point to impaired p53 regulation in cells with mutated RPS15. Paper IV aimed at characterizing 70 patients assigned to three major subsets (#1, #2, and #4) through whole-genome sequencing. Besides recurrent exonic driver mutations, we report non-coding regions significantly enriched for mutations in subset #1 and #2 that may facilitate future molecular studies. Collectively, this thesis supports the potential of targeted sequencing for mutational screening of CLL in clinical practice, provides novel insight into the pathobiology of aggressive CLL, and demonstrates the clinical outcome and cellular effects of NFKBIE and RPS15 mutations.