P622: double-hit traf3 deletion and mutation identified by high-throughput sequencing define a new independent prognostic factor in chronic lymphocytic leukemia

Background: Chromosome 14q abnormalities involving the IGH gene are present in 5-15% of chronic lymphocytic leukemia (CLL) patients. Besides IGH translocations, interstitial 14q32.33/IGH deletions have been reported at lower frequencies (2%), and their clinical implications and underlying molecular mechanisms remain unknown. In other B-cell malignancies such as multiple myeloma, TRAF3 gene, proximal to the IGH locus, may be encompassed in the deletion, with mutations in the remaining allele. However, the molecular status of TRAF3 in CLL and their impact in prognosis have not been studied in depth. Aims: To analyze the genetic landscape of CLL patients with IGH deletion and evaluate the molecular status of TRAF3 to elucidate their impact in CLL pathogenesis and prognosis. Methods: A total of 897 CLLs were analyzed by FISH with the IGH break-apart probe and the 4-probe CLL panel (ATM, CEP12, D13S319 and TP53) (Vysis). 324 untreated CLLs (54 with IGH deletion (del-3’IGH) and 270 as the control group) were sequenced by targeted-NGS, with a custom panel including 54 CLL-related genes (Nextseq platform, Illumina). Copy number variations (CNVs) of TRAF3 were assessed by a NGS approach and validated by SNP Array 6.0. Results: FISH analyses identified a total of 54 out of 897 CLLs (6%) with a 300 kb deletion of the centromeric side of IGH (del-3’IGH CLLs). Moreover, our results showed that 76% of del-3’IGH CLLs had additional FISH abnormalities, being the most frequent the loss of 13q (42%), +12 (22%), del(11q) (11%) and 17p- (9%). NGS analyses showed that the most recurrently mutated genes in del-3’IGH CLL were NOTCH1 (30%), ATM (20%) and TRAF3 (13%). Notably, the mutational frequency of TRAF3 mutations was significantly higher in del-3’IGH CLLs than in the control group (13% vs. 0.5%, p<0.001). Given the surprisingly incidence of TRAF3 mutations in this subgroup, we further assessed its CNV status, noticing that TRAF3 loss was enriched in del-3’IGH CLLs when compared to the control group (11/54, 20% vs. 4/270, 1.5%, p<0.001). By integrating mutational and CNV information, we observed that 7 out of 11 patients with a TRAF3 loss harbored mutations in this gene. The deletion was present in a higher percentage of cells than the mutation (mean: 70.5% vs. 15.6%), which may suggest that TRAF3 mutation appears as a subclonal event, leading to a biallelic inactivation of this gene. Our results showed a novel biallelic alteration in CLL, reminding the double null event that occurs in other gene drivers such as TP53 and ATM. Of note, this double-hit on TRAF3 contributed to a shorter time to first treatment (TFT) in del-3’IGH CLL patients (5 vs. 54 months, p<0.001). Furthermore, TRAF3 biallelic inactivation had a similar TFT than TP53 or ATM-altered patients with an adverse prognosis (median TRAF3: 5 vs. 59 months, p<0.001, ATM: 2 vs 59, p<0.001 and TP53: 12 vs 59, p=0.002), being also an independent risk factor in the multivariate analyses in the CLL cohort (n=324) (HR=0.21, 95% CI=0.05-0.85, p=0.029) (Table 1). Image:Summary/Conclusion: Our work demonstrates that TRAF3 mutations and deletions are highly enriched in CLL patients with del-3’IGH, resulting in the biallelic inactivation of this gene. The presence of this double-hit on TRAF3 worsen the prognosis of CLL patients, being and independent prognostic factor with similar impact on TFT than the biallelic loss of TP53. Funding: PI21/00983; FI19/00191 (CPC); CD19/00222 (MHS); FEHH (MQÁ); USAL (RBS)