1215 - Unveiling the Spectrum of Genetic Alterations in Relapsed/Refractory CLL Patients on Targeted Inhibitors: A Prospective Unicentric Study

Introduction: Next-generation sequencing (NGS) has been instrumental in characterizing the genomic landscape of Chronic Lymphocytic Leukemia (CLL). While a wealth of predictive data has been derived predominantly from large phase 3 trials, comprehending the practical implications of CLL mutational landscape continues to present challenges due to the presence of variants of uncertain significance (VUS) and minor clone presence. Real-world studies that trace clonal evolution can serve as a bridge to this knowledge gap, offering insights into the pathology of CLL. Therefore, our goals were to map genetic mutations, track clonal evolution, specify associated parameters, and uncover their potential associations.

Patients and

Methods: This prospective unicentric study included patients with relapsed or refractory CLL who were initiating treatment with oral kinase inhibitors between 2019 and 2022 at our hematologic center. CLL cells were isolated using the RosetteSep method.

Following cell separation, DNA samples were analyzed using a custom capture-based panel (SureSelect XT HS by Agilent) for CLL-associated genes. This NGS analysis was performed using the Illumina MiSeq platform. Further sample collection after 12 months of therapy and at disease progression was planned.

Results: We studied a cohort of 45 CLL patients with a median age of 72 years, 71% male. A significant proportion 76% of patients had unmutated IGHV, 40% displayed TP53 mutation and/or del17p; del11q was identified in 33% of patients. Complex karyotype was detected in 11% of cases. The median follow–up was 22 months. With regard to treatment, 56% received Brutone tyrosine kinase (BTK) inhibitors, others received Bcl-2 inhibitor venetoclax (31%) or PI3K inhibitor idelalisib (13%). Basic characteristics and prognostic factors are summarized in Table 1.

Molecular profiling of the 63 samples from 45 patients revealed a broad spectrum of gene mutations and variants. The most frequent oncogenic mutations were observed in the TP53 gene (40% of cases, n=25) and the SF3B1 gene (37% of cases, n=23). NOTCH1 and ATM mutations were also notable, each appearing in 19% of instances (n=12 each). Oncogenic variants were identified also in the following genes: ASXL1, ATM, BCL2, BCOR, BIRC3, BRAF, BTK, CARD11, DDX41, ETV6, FBXW7, IKZF3, KIT, KRAS, MED12, NFKBIE, NOTCH1, NRAS, PLCG2, POT1, RPS15, SAMHD1, SF3B1, STAG2, TET2, TP53, TRAF2, WT1, XPO1, ZRSR2. Upon further analysis, VUS were found in several genes, including ATM, GATA2, KIT, NOTCH1, RPS15, STAG2, TP53, and ZRSR2.

Clonal evolution was assessed in 16 patients, with genomic changes in 15 (94%). New mutations emerged in 11 patients (69%), with observed genes including XPO1, KRAS, BIRC3, TRAF2, NRAS, BRAF, TP53, RPS15, ATM, MED12, SF3B1, TET2, BTK, and NOTCH1. Meanwhile, 5 patients (31%) experienced the disappearance of certain mutations, specifically those in NOTCH1, STAG2, WT1, KIT, KRAS, TP53, IKZF3 and SAMHD1. In one patient, a benign BCL2 mutation was initially detected and followed an oncogenic variant in the same gene in a subsequent sample. Additionally, one patient demonstrated disease progression associated with the emergence of a BTK mutation.

Conclusion: This interim analysis points to the dynamic and complex nature of genomic alterations in CLL, with implications for disease progression and therapeutic responses. Our findings highlight the importance of longitudinal genomic monitoring to uncover evolving mutational profiles and their clinical implications.