Molecular analysis of different clinical presentations of chronic lymphocytic leukemia reveals novel molecular predictors and molecular heterogenety of different anatomical compartments

Chronic lymphocytic leukemia (CLL) and Small lymphocytic lymphoma (SLL) are different manifestation of the same lymphoproliferative disease. Our study aims at evaluating molecular markers that predispose to chemorefractoriness in CLL and to identify molecular landscape of the different anatomical compartments of SLL. Fludarabine, cyclophosphamide, and rituximab (FCR) is the most effective chemoimmunotherapy regimen for young and fit CLL patients devoid of TP53 disruption. A cohort of 287 patients receiving first-line FCR was analyzed by next generation sequencing (NGS). By univariate analysis, BIRC3 mutations identify a poor prognostic subgroup of patients failing FCR (p<0.001) as cases harboring TP53 mutations (p<0.001). BIRC3 mutations maintained an independent association with an increased risk of progression (p=0.004) in multivariate analysis. By immunoblotting analysis, we showed that the non-canonical NF-KB pathway is active in BIRC3 mutated cell lines and in primary CLL samples. In vitro results indicate that BIRC3 mutated primary CLL cells are less sensitive to fludarabine. BIRC3 mutations may be used as a new molecular predictor to select high-risk patients for novel drugs. Regarding SLL, we investigated 12 SLL patients, provided with: cell free DNA (CfDNA) from plasma, genomic DNA (gDNA) from LNF biopsies, gDNA from CD19+ cells and from CD3+ cells, by using NGS. By comparing mutations identified, SLL genotyping on the cfDNA does not recapitulate SLL genetics and lacks 65.2% identified in the PB CD19+ cells and in the LNF. By considering the 44 mutations identified in the LNF and in the PB CD19+ cells, 20.4% of mutations were unique to the LNF biopsy, 36.4% were unique to the PB CD19+ cells and only 43.2% were shared. The analysis of the LNF only or of the PB CD19+ cell only may miss mutations with potential clinical relevance, suggesting that both these two compartments should be tested to have a comprehensive view of the SLL genetics relevance.