Through the European Research Initiative on chronic lymphocytic leukemia (CLL) (ERIC), we screened 3490 patients with CLL for mutations within the NOTCH1 (n=3334), SF3B1 (n=2322), TP53 (n=2309), MYD88 (n=1080) and BIRC3 (n=919) genes, mainly at diagnosis (75%) and before treatment (>90%). BIRC3 mutations (2.5%) were associated with unmutated IGHV genes (U-CLL), del(11q) and trisomy 12, whereas MYD88 mutations (2.2%) were exclusively found among M-CLL. NOTCH1, SF3B1 and TP53 exhibited variable frequencies and were mostly enriched within clinically aggressive cases. Interestingly, as the timespan between diagnosis and mutational screening increased, so too did the incidence of SF3B1 mutations; no such increase was observed for NOTCH1 mutations. Regarding the clinical impact, NOTCH1 mutations, SF3B1 mutations and TP53 aberrations (deletion/mutation, TP53ab) correlated with shorter time-to-first-treatment (P<0.0001) in 889 treatment-naive Binet stage A cases. In multivariate analysis (n=774), SF3B1 mutations and TP53ab along with del(11q) and U-CLL, but not NOTCH1 mutations, retained independent significance. Importantly, TP53ab and SF3B1 mutations had an adverse impact even in U-CLL. In conclusion, we support the clinical relevance of novel recurrent mutations in CLL, highlighting the adverse impact of SF3B1 and TP53 mutations, even independent of IGHV mutational status, thus underscoring the need for urgent standardization/harmonization of the detection methods.

Key Points• Whole-exome sequencing of CLL patients who relapsed after FCR treatment revealed frequent mutations in RPS15.• RPS15 mutations are likely to be early clonal events and confer poor prognosis.Fludarabine, cyclophosphamide, and rituximab (FCR) is first-line treatment of medically fit chronic lymphocytic leukemia (CLL) patients; however, despite good response rates, many patients eventually relapse. Although recent high-throughput studies have identified novel recurrent genetic lesions in adverse prognostic CLL, the mechanisms leading to relapse after FCR therapy are not completely understood. To gain insight into this issue, we performed whole-exome sequencing of sequential samples from 41 CLL patients who were uniformly treated with FCR but relapsed after a median of 2 years. In addition to mutations with known adverse-prognostic impact (TP53, NOTCH1, ATM, SF3B1, NFKBIE, and BIRC3), a large proportion of cases (19.5%) harbored mutations in RPS15, a gene encoding a component of the 40S ribosomal subunit. Extended screening, totaling 1119 patients, supported a role for RPS15 mutations in aggressive CLL, with one-third of RPS15-mutant cases also carrying TP53 aberrations. In most cases, selection of dominant, relapse-specific subclones was observed over time. However, RPS15 mutations were clonal before treatment and remained stable at relapse. Notably, all RPS15 mutations represented somatic missense variants and resided within a 7 amino-acid, evolutionarily conserved region. We confirmed the recently postulated direct interaction between RPS15 and MDM2/MDMX and transient expression of mutant RPS15 revealed defective regulation of endogenous p53 compared with wild-type RPS15. In summary, we provide novel insights into the heterogeneous genetic landscape of CLL relapsing after FCR treatment and highlight a novel mechanism underlying clinical aggressiveness involving a mutated ribosomal protein, potentially representing an early

Mansouri et al. applied targeted deep sequencing to identify mutations within NF-κB core complex genes in CLL. NFKBIE, the gene encoding the inhibitory IκBε molecule, was most frequently mutated, especially in poor-prognostic subgroups of CLL. The authors show that NFKBIE mutations were associated with significantly reduced IkBε expression and p65 inhibition, ultimately leading to NF-κB activation and a more aggressive disease.

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Recent studies have revealed recurrent mutations of the NOTCH1, SF3B1 and BIRC3 genes in chronic lymphocytic leukemia (CLL), especially among aggressive, chemorefractory cases. Nevertheless, it is currently unknown whether their presence may differ in subsets of patients carrying stereotyped B-cell receptors and also exhibiting distinct prognoses. Here, we analyzed the mutation status of NOTCH1, SF3B1 and BIRC3 in three subsets with particularly poor prognosis, that is, subset #1, #2 and #8, aiming to explore links between genetic aberrations and immune signaling. A remarkably higher frequency of SF3B1 mutations was revealed in subset #2 (44%) versus subset #1 and #8 (4.6% and 0%, respectively; P<0.001). In contrast, the frequency of NOTCH1 mutations in subset #2 was only 8%, lower than the frequency observed in either subset #1 or #8 (19% and 14%, respectively; P=0.04 for subset #1 versus #2). No associations were found for BIRC3 mutations that overall were rare. The apparent non-random association of certain mutations with stereotyped CLL subsets alludes to subset-biased acquisition of genomic aberrations, perhaps consistent with particular antigen/antibody interactions. These novel findings assist in unraveling specific mechanisms underlying clinical aggressiveness in poor-prognostic stereotyped subsets, with far-reaching implications for understanding their clonal evolution and implementing biologically oriented therapy.

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Patients with chronic lymphocytic leukemia (CLL) progressing on ibrutinib constitute an unmet clinical need. Though BTK and PLCG2 mutations are associated with ibrutinib resistance, their frequency and relevance to progression are not fully understood. In this multicenter retrospective observational study, we analyzed 98 patients with CLL treated with ibrutinib (49 relapsing after an initial response and 49 still responding after ≥1 year of continuous treatment) using a next-generation sequencing (NGS) panel (1% sensitivity) comprising 13 CLL-relevant genes including BTK and PLCG2. BTK hotspot mutations were validated by droplet digital PCR (ddPCR) (0.1% sensitivity). By integrating NGS and ddPCR results, 32/49 relapsing cases (65%) carried at least 1 hotspot BTK mutation and/or PLCG2 mutation(s); in 6/32, BTK mutations were only detected by ddPCR [variant allele frequency (VAF) 0.1-1.2%]. BTK/PLCG2 mutations were also identified in 6/49 responding patients (12%; 5/6 VAF &lt;10%), of whom 2 progressed at later time points. Among the relapsing patients, the BTK-mutated (BTKmut) group was enriched for EGR2 mutations, while BTK-wildtype (BTKwt) cases more frequently displayed BIRC3 and NFKBIE mutations. Using an extended capture-based panel, only BRAF and IKZF3 mutations showed a predominance in relapsing cases, who were enriched for del(8p) (n=11; 3 BTKwt). Finally, no difference in TP53 mutation burden was observed between BTKmut versus BTKwt relapsing cases, and ibrutinib treatment did not appear to favor selection of TP53-aberrant clones. In conclusion, we show that BTK/PLCG2 mutations were absent in a substantial fraction (35%) of a real-world cohort failing ibrutinib, and propose additional mechanisms contributing to resistance.