PF376 DIFFERENTIAL GENOMIC AND TRANSCRIPTOMIC EVENTS ASSOCIATED WITH HIGH-GRADE TRANSFORMATION OF CHRONIC LYMPHOCYTIC LEUKEMIA

Abstract

Background: The high-grade transformation of chronic lymphocytic leukemia (CLL), also called Richter's Syndrome (RS), carries a dismal prognosis. RS shares the histological characteristics with diffuse large B-cell lymphoma (DLBCL), but it has a distinct molecular profile compared to de novo DLBCL. Heterogeneous mutational profiles characterize RS, but studies report 90% of the tumours to involve molecular lesions in at least one of the TP53, CDKN2A, MYC or NOTCH1 genes. Being chemotherapy resistant, RS remains an area of high unmet clinical need. Aims: Our aim was to perform an integrative analysis of paired CLL and RS phases using a combination of coding and non-coding single nucleotide variants (SNVs), small insertions and deletions (InDels) and copy number variants (CNVs) detected by whole genome sequencing (WGS). Genes and pathways implicated in transformation were interrogated by RNA expression profiling of 800 cancer-related genes. Methods: We performed WGS on paired CLL and RS phase samples from 17 patients enrolled in our phase II trial of ofatumumab, cyclophosphamide, doxorubin, vincristine and prednisolone (O-CHOP). Through integrated analysis of SNVs, InDels and CNVs, we searched for differences in the somatic mutation spectrum between the two disease phases. We also employed targeted DNA and RNA expression profiling (NanoString) on an extended patient cohort (n = 38) to confirm our findings. Results: A significantly larger mutation burden (SNVs and InDels) was detected in RS (31.2 (±22.5) variants/subject) compared to CLL phase (20.8 (±12.7) variants/subject) (p = 0.031) and significantly more genes were mutated in RS (n = 457 genes; 29.6 (±21.3) mutated genes/subject) compared to CLL phase (n = 306 genes; 19.8 (±12.4) mutated genes/subject) (p = 0.022). Most mutated genes and variants detected were missense mutations and non-recurrent (i.e. detected in a single subject/phase). As in previous studies, lesions in the TP53, NOTCH1 and CDKN2A genes were common. We also detected mutations in XPO1 (35.3%) and TRAF3 (35.3%) genes more frequently than previously reported in RS. We next investigated the combined impact of mutated genes on cellular pathways and found MAPK pathway gene lesions to be enriched in RS vs CLL phase. Lesions in at least one MAPK pathway gene were detected in 9 (52.9%) CLL and 15 (88.2%) RS samples. In addition, at least one PI3K pathway gene lesion was detected in 10 (58.8%) CLL and 14 (82.4%) RS samples. Analysis of clonal evolution across different pathways demonstrated a high expansion probability for clones containing MAPK mutations. Next, we performed RNA expression profiling and pathway enrichment analysis, showing the JAK-STAT, PI3K and P53 pathways were the most likely to harbor differentially expressed genes. Five pathways (PI3K, cell cycle and apoptosis, RAS, PANC Drivers and MAPK) had 10 or more differentially expressed genes. Finally, we explored the evolution in the mutation landscape of non-coding regions during transformation finding 5 patients that carried mutation clusters in a region previously characterized as a functionally active PAX5 enhancer in CLL. Three of these occurred in the RS phase only. Expanded analysis on all filtered mutations in the entire PAX5 enhancer region showed 10 samples each with 1–8 mutations, of which 6 had mutations only in the RS phase. Summary/Conclusion: In conclusion, we show that integrated analysis of WGS combined with RNA expression profiling identifies a number of directly targetable pathways that merit clinical evaluation in RS.