CO13 | Dissection of biallelic NOTCH1 disruption in chronic lymphocytic leukemia reveals chromosome 9 copy neutral-loss of heterozygosity

Abstract

Introduction. NOTCH1 is frequently mutated in CLL, with an incidence of 12–25%, and shorter TTFT and OS compared to wild-type cases, with subclonal mutations also exerting a residual prognostic impact. However, the prognostic significance of NOTCH1 disruption with biallelic involvement (VAF > 50%) has not yet been specifically evaluated.

Methods. A total of 4222 peripheral blood samples from 3564 CLL patients referred to the Clinical and Experimental Onco-Hematology Unit, CRO Aviano, were screened for NOTCH1 mutations by NGS (exon 34 and part of 3′UTR). Copy number analysis of NOTCH1 was performed using TaqMan probes for exon 34 (Hs00124234_cn) and exon 31 (Hs00088335_cn) by ddPCR/qPCR, or FISH on fixed nuclei. Whole-exome sequencing (WES) was conducted on sorted CD19+ (tumor) and CD3+ (germline) fractions from frozen samples. The PureCN R package was used to estimate copy number from exome sequencing (WES) data.

Results. NOTCH1 mutations were identified in 753 samples (17%), including 46 (6%) with VAF > 50% (high-burden). Copy number analysis was performed to assess biallelic involvement and to exclude Chr9 deletions. ddPCR demonstrated that most high-burden samples were diploid, with only 3/46 (6.5%) showing deletions. FISH confirmed these findings, revealing 19/20 (95%) diploid samples without Chr9 deletions and 1 sample with a 56% loss. However, NGS of NOTCH1 exon 34 revealed a peculiar polymorphic unbalancing of D2185D rs2229974 SNP (e.g. VAF 0.1-0.4 or 0.6-0.9) in heterozygous samples. Most of such samples with NOTCH1 mutations above 50% were unbalanced (13/20). To investigate the hypothesis of tumor-restricted allelic imbalance, separate sequencing on FACS-sorted CD19+ and CD3+ fractions from 7 high-burden unbalanced samples (plus 1 deleted control) showed that both NOTCH1 mutation and D2185D unbalancing were confined to CD19+ cells. Complete allelic loss of 9q34 was observed in 3 samples and partial loss in the other 3 samples. However confirmed a diploid (2n) status in both tumor and germline fractions (1n in the deleted control). WES revealed strong allelic imbalance across multiple loci on Chr9 compared to germinal controls, suggesting chromosomal deletions, whereas PureCN predicted diploid status in 5 samples and confirmed deletion only in the control. FISH on 9q22 confirmed diploid status in these samples. WES also detected alterations on other chromosomes (Chr8q gain/loss, del11q, del13q, del22), all confirmed by FISH with specific probes. Overall, NGS data suggest presence of a D2185D imbalance, and thus likely Chr9 alterations, in 150 (3.5%) samples, independently of NOTCH1 mutations.

Conclusion. This study describes, for the first time in CLL, a non-rare copy-neutral loss of heterozygosity (cn-LOH) mechanism on chromosome 9, where high-burden NOTCH1 disruption coexists with a normal (2n) copy number. Ongoing analyses aim to clarify the clinical implications of cn-LOH in both NOTCH1-mutated and wild-type CLL cases.