The pathogenesis of chronic lymphocytic leukemia (CLL) remains incompletely understood.1 This includes the level of understanding of the role of single gene mutations in CLL biology and clinical behavior, and knowledge in this area is evolving.2,3 Recently, using massively parallel sequencing, stabilizing mutations in NOTCH1 were identified in CLL,4 and preliminary associations of NOTCH1 mutations and CLL disease characteristics were reported. The latter included detection of an enrichment of NOTCH1 mutations in IgVH unmutated CLL, results indicating worse overall survival (OS) and an association of NOTCH1 exon 34 mutations with disease transformation (Richter’s transformation).5
To complement existing knowledge of the frequency, characteristics and clinical impact of NOTCH1 exon 34 mutations in CLL, and to provide guidance for future research, we resequenced NOTCH1 exon 34 in a well-characterized, prospectively enrolled cohort of 257 CLL patients, of which 200 were untreated and 57 relapsed at study enrollment.6 Sequence analysis using Sanger sequencing was performed on DNA isolated from FACS-sorted CD19 + cells, cryopreserved at the time of study enrollment. The somatic nature of mutations was confirmed using paired DNA isolated from FACS-sorted CD3 + cells.
A total of 21 NOTCH1 exon 34-mutated cases were identified, resulting in a mutation frequency estimate of 8% in this cohort. Data are summarized in Table 1. We identified 18 cases with NOTCH1 exon 34 frameshift mutations, the majority (13/21 = 62%) of which displayed the reported hotspot dinucleotide deletion (c.7541_7542het_delCT), and 3 cases with nonsense mutations. Interestingly, NOTCH1 exon 34 mutations were substantially enriched in CLL samples that were relapsed at enrollment (10/57 = 18%), whereas the frequency in untreated CLL was substantially lower (11/200 = 6%).
Table 1.
Characteristics of CLL cases with NOTCH1 exon 34 mutations
| CLL number | NOTCH1 nucleotide (genomic, build GRCh37/hg19) | NOTCH1 nucleotide (cDNA) | Mutation effect on NOTCH1 protein | FISH25a results | TP53 exon 2–10 | SNP 6.0 array-based aCNA | IgVH status | ZAP70 status (% positive) | CD38 status (% positive) |
|---|---|---|---|---|---|---|---|---|---|
| CLL-002 | g.chr9:139390781het_delC | c.7410het_delC | 5 incorrect aa after Ser2470, premature stop codon 2476 | Trisomy 12 | Wt | 0 | UM | 86 | 24 |
| CLL-033 | g.chr9:139390649_139390650het_delCT | c.7541_7542het_delCT | 3 incorrect aa after Ser2513, premature stop codon 2517. | 11q | M | 4 | UM | 65 | unknown |
| CLL-055 | g.chr9:139390649_139390650het_delCT | c.7541_7542het_delCT | 3 incorrect aa after Ser2513, premature stop codon 2517. | Normal | Wt | 0 | UM | 91 | 59 |
| CLL-077 | g.chr9:139390649_139390650het_delCT | c.7541_7542het_delCT | 3 incorrect aa after Ser2513, premature stop codon 2517. | 13q | Wt | 0 | UM | 39 | 22 |
| CLL-078 | g.chr9:139390816C>C/T | c.7375C>C/T | p.Gln2459>Gln/STOP | 13q | Wt | 1 | UM | 82 | 72 |
| CLL-083 | g.chr9:139390649_139390650het_delCT | c.7541_7542het_delCT | 3 incorrect aa after Ser2513, premature stop codon 2517. | Trisomy 12 | Wt | 0 | UM | 41 | 95 |
| CLL-085 | g.chr9:139390649_139390650het_delCT | c.7541_7542het_delCT | 3 incorrect aa after Ser2513, premature stop codon 2517. | Trisomy 12 | Wt | 1 | UM | 77 | 31 |
| CLL-094 | g.chr9:139390649_139390650het_delCT | c.7541_7542het_delCT | 3 incorrect aa after Ser2513, premature stop codon 2517. | Normal | Wt | 2 | M | 16 | 61 |
| CLL-096 | g.chr9:139390649_139390650het_delCT | c.7541_7542het_delCT | 3 incorrect aa after Ser2513, premature stop codon 2517. | 13q | Wt | 1 | M | 22 | 1 |
| CLL-117 | g.chr9:139390649_139390650het_delCT | c.7541_7542het_delCT | 3 incorrect aa after Ser2513, premature stop codon 2517. | 13q | Wt | 2 | UM | 80 | 60 |
| CLL-134 | g.chr9:139390649_139390650het_delCT | c.7541_7542het_delCT | 3 incorrect aa after Ser2513, premature stop codon 2517. | Trisomy 12 | Wt | 0 | UM | 77 | 28 |
| CLL-148 | g.chr9:139390649_139390650het_delCT | c.7541_7542het_delCT | 3 incorrect aa after Ser2513, premature stop codon 2517. | 13q | Wt | 1 | UM | 42 | 24 |
| CLL-149 | g.chr9:139390649_139390650het_delCT | c.7541_7542het_delCT | 3 incorrect aa after Ser2513, premature stop codon 2517. | Trisomy 12 | Wt | 0 | UM | 58 | 95 |
| CLL-150 | g.chr9:139390861C>C/T | c.7330C>C/T | p.Gln2444>Gln/STOP | Trisomy 12 | Wt | 2 | UM | 88 | 81 |
| CLL-172 | g.chr9:139390649_139390650het_delCT | c.7541_7542het_delCT | 3 incorrect aa after Ser2513, premature stop codon 2517. | Trisomy 12 | Wt | 0 | M | 85 | 99 |
| CLL-179 | g.chr9:139390747het_delC | c.7444het_delC | p.Leu2482>Leu/STOP | Trisomy 12 | Wt | 0 | UM | 76 | 98 |
| CLL-189 | g.chr9:139390649_139390650het_delCT | c.7541_7542het_delCT | 3 incorrect aa after Ser2513, premature stop codon 2517. | Trisomy 12 | Wt | 1 | UM | 6 | 29 |
| CLL-201 | g.chr9:13939068>C4C/T | c.7507C>C/T | p.Gln2503>Gln/STOP | 11q, Trisomy 12 | Wt | 1 | UM | 53 | 24 |
| CLL-206 | g.chr9:139391366het_insA | c.6825het_insA | 78 incorrect aa after Ser2274, premature stop codon 2353 | 11q, Trisomy 12 | M | 1 | UM | 87 | 24 |
| CLL-209 | g.chr9:139390820het_delG | c.7371het_delG | 17 incorrect aa after Pro2458, premature stop codon 2476 | 11q | Wt | 1 | UM | 61 | 86 |
| CLL-257 | g.chr9:139391748_139391760het_delGCTACCTGGGCAG | c.6431_3443het_delGCTACCTGGGCAG | 99 incorrect aa after Asn2143, premature stop codon 2243. | 11q, 17p, 13q | M | 20 | UM | 61 | 91 |
Abbreviations: aCNA, acquired subchromosomal copy number aberrations; CLL, chronic lymphocytic leukemia; FISH, fluorescence in situ hybridization; M, mutated; UM, unmutated.
FISH findings present in ≥25% of nuclei; characteristics of CLL cases with NOTCH1 exon 34 mutations. Transcript accession ID: NM_017617.3.
Next, we reviewed in detail the characteristics of the 21 CLL cases with NOTCH1 exon 34 mutations. CLL with NOTCH1 exon 34 mutations were highly enriched for IgVH unmutated status (18/21 = 86%), ZAP70 positivity >20% (19/21 = 90%) and CD38 positivity >30% (12/20 = 60%), indicating strong co-occurrence with biological traits associated with progressive and proliferative CLL (see Table 1).
In contrast to these very strong associations, NOTCH1 exon 34-mutated CLL was rarely associated with del17p (1/21 = 5%), and was associated with TP53 mutations (3/21 = 14%) or del11q (5/21 = 24%) at frequencies expected for random associations. However, trisomy 12 was detected at frequencies that were substantially higher than expected (11/21 = 52%), possibly indicating cooperation between these two genomic aberrations.
We also analyzed SNP 6.0 array-based acquired subchromosomal copy number aberrations (aCNA) in CLL with NOTCH1 exon 34 mutations (SNP 6.0 arrays, Affymetrix, Santa Clara, CA, USA). Data, summarized in Table 1, indicate that most such cases had relatively stable genomes (≥2 aCNA was found in 5/21 = 24%: a frequency similar to those identified in the entire cohort).6
The aforementioned data suggested to us that NOTCH1 exon 34 mutations as detected through direct sequencing occurred late in CLL disease evolution and usually in the setting of known factors associated with proliferative and progressive CLL, but at frequencies suggesting random association with genomically high-risk CLL (which is aggressive CLL with short OS; here it is defined as the presence of either del17p, del11q, elevated single-nucleotide polymorphism array-based aCNA count or TP53 mutations).
Next, we determined effects of NOTCH1 exon 34 mutations on outcome in CLL, restricting the analysis to the CLL cohort that was untreated at the date of trial enrollment and using the trial enrollment date (equal to the sample procurement date) as the reference date for analysis. We performed multivariate analysis based on proportional hazard modeling, using OS as the primary endpoint and incorporating variables known to affect the clinical course of CLL patients. Data are summarized in Table 2 (data for the modeling using the CLL diagnosis date as a reference is included in Table 2, right columns). From this data, known strong predictors of short OS in CLL, including TP53 mutations, del17p, or elevated single-nucleotide polymorphism array-based genomic complexity, emerged with substantially elevated hazard ratios, whereas NOTCH1 exon 34 mutations did not emerge as either strong or significant predictors (all hazard ratios close to 1, P-values not significant).
Table 2.
Results of multivariate analysis of patients untreated at trial enrollment
| Variable | N | HR | CI | P-value |
|---|---|---|---|---|
| (a) Untreated patient group OS from the enrollment date | ||||
| Del11q present vs not | 14/176 | 2.3 | 0.7–7.6 | 0.15 |
| Del17p present vs not | 15/175 | 4.9 | 1.7–14.7 | <0.01 |
| Rai stage 1–4 vs 0 | 106/84 | 1.6 | 0.7–3.4 | 0.27 |
| ZAP70 ≥20% vs <20% | 82/108 | 0.8 | 0.3–1.9 | 0.56 |
| IgVH UM vs M | 82/108 | 1.6 | 0.7–4 | 0.3 |
| NOTCH1 mutated vs wt | 11/179 | 1 | 0.2–4.9 | 0.99 |
| TP53 exon 2–10 mutated vs not | 24/166 | 4 | 1.7–9 | <0.01 |
| Rai stage 1–4 vs 0 | 106/84 | 1.5 | 0.7–3.3 | 0.29 |
| ZAP70 ≥20% vs <20% | 82/108 | 0.8 | 0.4–2 | 0.69 |
| IgVH UM vs M | 82/108 | 2 | 0.9–4.9 | 0.1 |
| NOTCH1 mutated vs wt | 11/179 | 0.7 | 0.2–3.4 | 0.67 |
| Number of SNP 6.0 array genomic lesions ≥3 vs <3 | 30/158 | 3.9 | 1.7–8.5 | <0.01 |
| Rai stage 1–4 vs 0 | 106/82 | 1.4 | 0.6–3 | 0.41 |
| ZAP70 ≥20% vs <20% | 82/106 | 0.9 | 0.4–2 | 0.77 |
| IgVH UM vs M | 82/106 | 1.8 | 0.8–4.1 | 0.18 |
| NOTCH1 mutated vs wt | 11/177 | 0.9 | 0.2–4.5 | 0.94 |
| (b) Untreated patient group OS from the diagnosis date | ||||
| Del11q present vs not | 14/176 | 1.9 | 0.6–6.3 | 0.28 |
| Del17p present vs not | 15/175 | 3.4 | 1.1–10.6 | 0.03 |
| Rai stage 1–4 vs 0 | 106/84 | 1.8 | 0.8–4 | 0.16 |
| ZAP70 ≥20% vs <20% | 82/108 | 1 | 0.4–2.4 | 0.94 |
| IgVH UM vs M | 82/108 | 2.3 | 0.9–5.9 | 0.08 |
| NOTCH1 mutated vs wt | 11/179 | 1 | 0.2–5 | 1 |
| TP53 exon 2–10 mutated vs not | 24/166 | 3.6 | 1.5–8.8 | <0.01 |
| Rai stage 1–4 vs 0 | 106/84 | 1.8 | 0.8–4 | 0.13 |
| ZAP70 ≥20% vs <20% | 82/108 | 1 | 0.4–2.4 | 0.94 |
| IgVH UM vs M | 82/108 | 2.6 | 1.1–6.4 | 0.03 |
| NOTCH1 mutated vs wt | 11/179 | 0.8 | 0.2–3.8 | 0.78 |
| Number of SNP 6.0 array genomic lesions ≥3 vs <3 | 30/158 | 3.8 | 1.6–8.9 | <0.01 |
| Rai stage 1–4 vs 0 | 106/82 | 1.7 | 0.8–3.9 | 0.16 |
| ZAP70 ≥20% vs <20% | 82/106 | 0.9 | 0.4–2.2 | 0.89 |
| IgVH UM vs M | 82/106 | 2.5 | 1–5.9 | 0.04 |
| NOTCH1 mutated vs wt | 11/177 | 1 | 0.2–4.8 | 1 |
Abbreviations: CI, confidence intervals; HR, hazard ratio; UM vs M, unmutated vs mutated; wt, wild type.
Finally, given the reported association of NOTCH1 exon 34 mutations with Richter’s transformation, we assessed the 21 cases with NOTCH1 exon 34 mutations in our cohort for such an event. Overall, we identified three cases with disease transformation (one patient with secondary Hodgkin’s lymphoma following prior therapies for CLL and two cases of large B-cell lymphoma) that resulted in the deaths of these patients. The frequency of disease transformation in this cohort of 21 NOTCH1 exon 34-mutated CLL was 3/21 = 14%.
In summary, our data provide important confirmatory data on the frequency of NOTCH1 exon 34 mutations in CLL. We provide novel detailed insights into the association of NOTCH1 exon 34-mutated CLL with biological markers and describe strong associations with proliferative CLL, but fail to confirm associations with survival. However, given the relatively low frequency of NOTCH1 exon 34 mutations in untreated CLL, much larger studies are needed to confirm these findings. Future work on CLL with NOTCH1 exon 34 mutations likely will focus on the role of mutated NOTCH1 in CLL cell proliferation or survival and testing NOTCH1 exon 34 mutations as a novel target for NOTCH–NOTCH ligand axis-directed therapeutics.
Acknowledgments
Supported by a National Institutes of Health Grants CA136537 (SM) and the Translational Research Program of the Leukemia and Lymphoma Society of America (SM). We are grateful for services provided by the microarray core of the University of Michigan Comprehensive Cancer Center.
Footnotes
CONFLICT OF INTEREST
The authors declare no conflict of interest.
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