Extensive next-generation sequencing analysis in chronic lymphocytic leukemia at diagnosis: clinical and biological correlations

Gian Matteo Rigolin, Elena Saccenti, Cristian Bassi, Laura Lupini, Francesca Maria Quaglia, Maurizio Cavallari, Sara Martinelli, Luca Formigaro, Enrico Lista, Maria Antonella Bardi, Eleonora Volta, Elisa Tammiso, Aurora Melandri, Antonio Urso, Francesco Cavazzini, Massimo Negrini, Antonio Cuneo

In chronic lymphocytic leukemia (CLL), next-generation sequencing (NGS) analysis represents a sensitive, reproducible, and resource-efficient technique for routine screening of gene mutations. We performed an extensive biologic characterization of newly diagnosed CLL, including NGS analysis of 20 genes frequently mutated in CLL and karyotype analysis to assess whether NGS and karyotype results could be of clinical relevance in the refinement of prognosis and assessment of risk of progression. The genomic DNA from peripheral blood samples of 200 consecutive CLL patients was analyzed using Ion Torrent Personal Genome Machine, a NGS platform that uses semiconductor sequencing technology. Karyotype analysis was performed using efficient mitogens. Mutations were detected in 42.0 % of cases with 42.8 % of mutated patients presenting 2 or more mutations. The presence of mutations by NGS was associated with unmutated IGHV gene (p = 0.009), CD38 positivity (p = 0.010), risk stratification by fluorescence in situ hybridization (FISH) (p < 0.001), and the complex karyotype (p = 0.003). A high risk as assessed by FISH analysis was associated with mutations affecting TP53 (p = 0.012), BIRC3 (p = 0.003), and FBXW7 (p = 0.003) while the complex karyotype was significantly associated with TP53, ATM, and MYD88 mutations (p = 0.003, 0.018, and 0.001, respectively). By multivariate analysis, the multi-hit profile (≥2 mutations by NGS) was independently associated with a shorter time to first treatment (p = 0.004) along with TP53 disruption (p = 0.040), IGHV unmutated status (p < 0.001), and advanced stage (p < 0.001). Advanced stage (p = 0.010), TP53 disruption (p < 0.001), IGHV unmutated status (p = 0.020), and the complex karyotype (p = 0.007) were independently associated with a shorter overall survival. At diagnosis, an extensive biologic characterization including NGS and karyotype analyses using novel mitogens may offer new perspectives for a better refinement of risk stratification that could be of help in the clinical management of CLL patients.