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by targeted next-generation sequencing of 24 recurrently mutated genes in CLL. By univariate analysis adjusted for multiple comparisons BIRC3 mutations identify a poor prognostic subgroup of patients in whom FCR treatment fails (median progression-free survival: 2.2 years, P<0.001) similar to cases harboring TP53 mutations (median progression-free survival: 2.6 years, P<0.0001). BIRC3 mutations maintained an inde- pendent association with an increased risk of progression with a hazard ratio of 2.8 (95% confidence interval 1.4-5.6, P=0.004) in multivariate analysis adjusted for TP53 mutation, 17p deletion and IGHV mutation sta- tus. If validated, BIRC3 mutations may be used as a new molecular predictor to select high-risk patients for novel frontline therapeutic approaches.
BIRC3 mutations in CLL
Introduction
Nuclear factor-κB (NF-κB) signaling is a key component of the development and evolution of chronic lymphocytic leukemia (CLL).1 Two NF-κB pathways exist, namely the canonical and non-canonical pathways.2 The former is triggered by B-cell receptor signaling via Bruton tyrosine kinase (BTK), while the latter is activated by members of the tumor necrosis factor (TNF) cytokine family.3 Upon receptor binding, the TRAF3/MAP3K14-TRAF2/BIRC3 negative regulatory complex of non-canonical NF-κB sig- naling is disrupted, MAP3K14 (also known as NIK), the central activating kinase of the pathway, is released and activated to induce the phosphorylation and proteasomal processing of p100, thereby leading to the formation of p52-containing NF-κB dimers. The p52 protein dimerizes with RelB to translocate into the nucleus, where it regu- lates gene transcription. BIRC3 (Baculoviral IAP Repeat Containing 3) is a negative regulator of non-canonical NF- κB. Physiologically, BIRC3 (also known as cIAP2) cat- alyzes MAP3K14 protein ubiquitination in a manner that is dependent on the E3 ubiquitinine ligase activity of its C- terminal RING domain. MAP3K14 ubiquitination results in its proteasomal degradation.4
B-cell neoplasia often pirates signaling pathways by molecular lesions to promote survival and proliferation. Although according to bioinformatics criteria BIRC3 is one of the candidate driver genes of CLL, the functional impli- cations of BIRC3 mutations are partially unexplored.5-7 Furthermore, little is known about the prognostic impact of BIRC3 mutations in CLL cohorts homogeneously treat- ed first-line with fludarabine, cyclophosphamide, and rit- uximab (FCR).7
FCR is the most effective chemoimmunotherapy regi- men for the management of CLL in young and fit patients devoid of TP53 disruption.8 Survival after FCR is, howev- er, variable, and is affected by the molecular characteris- tics of the CLL clone.9 Deletion of 17p and TP53 mutations are present in most, but not all patients who are refractory to chemo-immunotherapy, which prompts the identifica- tion of additional biomarkers associated with early failure of FCR.10-12
(qRT-PCR) was utilized to analyze the non-canonical NF-κB signa- ture. Primary CLL were exposed to fludarabine and venetoclax for 24-48 h and apoptosis was measured using the eBioscience Annexin V Apoptosis Detection Kit APC (ThermoFisher). Details are supplied in the Online Supplementary Methods.
Cancer personalized profiling by deep sequencing
A retrospective multicenter cohort of 287 untreated CLL patients receiving first-line therapy with FCR was analyzed for mutations, including 173 patients from a previously published multicenter clinical series and 114 new patients not included in our previous report.10 The study was approved by the Ethical Committee of the Ospedale Maggiore della Carità di Novara asso- ciated with the Amedeo Avogadro University of Eastern Piedmont (study number CE 67/14). Further information is provided in the Online Supplementary Methods. A targeted resequencing gene panel was designed to include: (i) coding exons plus splice site of 24 genes known to be implicated in CLL pathogenesis and/or prog- nosis; (ii) 3’UTR of NOTCH1; and (iii) enhancer and promoter region of PAX5 (size of the target region: 66627bp) (Table S1).6,7 The next-generation sequencing libraries for genomic DNA (gDNA) were constructed using the KAPA Library Preparation Kit (Kapa Biosystems) and those for RNA were constructed using the RNA Hyper Kit (Roche). Multiplexed libraries (n=10 per run) were sequenced using 300-bp paired-end runs on a MiSeq sequencer (Illumina) to obtain a coverage of at least 2000x in >90% of the tar- get region (66627 bp) in 80% of cases (Online Supplementary Table S2). A robust and previously validated bioinformatics pipeline was used for variant calling (Online Supplementary Appendix).
Statistical analysis
Progression-free survival (PFS) was the primary endpoint. Survival analysis was performed with the Kaplan-Meier method and compared between strata using the log-rank test. To account for multiple testing, adjusted P values were calculated using the Bonferroni correction. The adjusted association between exposure variables and PFS was estimated by Cox regression. Internal vali- dation of the multivariate analysis was performed using a boot- strap approach. Statistical significance was defined as a P value <0.05 (Online Supplementary Appendix).
Results
BIRC3 mutations associate with activation of non-canonical nuclear factor-κB signaling
In order to map unique BIRC3 mutations in CLL com- prehensively, we compiled somatically confirmed variants identified in the current CLL study cohort with those identified in previous studies13 or listed in public CLL mutation catalogues (Figure 1A). Virtually all BIRC3 muta- tions were frameshift mutations or stop codons clustering in two hotspot regions comprised between amino acids
Methods
Functional studies
The human CLL cell line MEC1, the splenic marginal zone lym- phoma cell lines SSK41 and VL51, the mantle cell lymphoma cell lines MAVER-1, Z-138 and JEKO-1, the human HEK-293T cell line, as well as primary CLL cells were used in functional experiments. The entire non-canonical NF-κB pathway was assessed by western blot analysis. Quantitative real-time polymerase chain reaction
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