DNMT3A and NPM1 Double Mutated AML Is a Phenomenon of the Younger Patient and Could Represent a New Entity

Background. The occurrence of clonal hematopoiesis increases with age and is associated with increased risk of hematologic malignancies (Jaiswal et al, NEJM 2014). Recent work identified pre-leukemic mutations in DNMT3A, ASXL1 and TET2 in AML that persist in remission and are not associated with survival (Jongen-Lavrencic et al. NEJM 2018). NPM1 mutation (NPM1^mut) identifies a WHO AML entity which accounts for ~30% of all AML and is associated with younger age. Mutant DNMT3A was shown to be the most frequent co-mutation in NPM1^mut AML, which is counterintuitive given its known increase with age. We aimed to dissect the age association of known co-mutations in NPM1^mut AML vs wild type NPM1 (NPM1^wt) AML in a large cohort.

Methods. Between 2005 and 2018, 9010 patients (pts) were diagnosed with AML by cytomorphology at our institution. For 6990/9010 pts (78%) NPM1 was studied at diagnosis by gene scan analysis, melting curve analysis or next-generation sequencing (NGS, NPM1 testing performed: NPM1^p). Samples were sequenced for AML-related genes, including: ASXL1, DNMT3A, TET2, IDH1/2, WT1, FLT3, NRAS, PTPN11, SF3B1, SRSF2, TP53, CEBPA, RUNX1. At least two genes other than NPM1 were sequenced in 6872/6990 (98%) cases. FLT3-ITD was analyzed by gene scan in 6851/6990 (98%) pts. For 6488/6990 NPM1^p pts (93%), chromosome banding analysis (CBA) was performed. The NPM1^p cohort did not differ from the whole AML cohort nor from the general AML population in terms of age distribution (z-score comparison). Differences across age groups were evaluated by chi-square testing and. ANOVA tests were applied on continuous variables.

Results. In the NPM1^p cohort, 2097/6990 pts (30%) were NPM1^mut. As expected, NPM1^mut was more common in younger pts (<60 yrs, 37%) compared to older pts (27%, odds ratio, OR=1.5, p<0.0001). NPM1 mutational frequency peaked in the age group from 30 to 59 years (yrs, 38%), it was significantly lower in younger (20-29 yrs, 18%, p<0.0001) and older pts (60-79 yrs, 28% and >80 yrs 21%, p<0.0001). The most commonly co-mutated genes were: DNMT3A (47%), FLT3-ITD (41%), TET2 (23%) and IDH2 (only R140, 18%), in line with previous reports (Ivey et al. NEJM 2016).

As expected, the common CHIP-related genes ASXL1 and TET2 were more frequently mutated in pts ≥60 yrs vs <60 yrs (ASXL1: 4% vs 0.5%, OR= 8.3, p=0.001; TET2: 29% vs 16%, OR=2.1, p<0.0001). Interestingly, DNMT3A mutations were significantly more frequent in younger pts (52% vs 43%, OR=1.5, p<0.0001). Regarding mutation subtypes, DNMT3A-R882 was more frequent than non-R882 in younger pts compared to the older ones (68% of all DNMT3A+ cases in <60 yrs vs 50% in ≥60 yrs, OR=2, p<0.0001). The mean age at diagnosis was 58 yrs (range: 22-86 yrs) for R882 vs 63.3 yrs (26-88 yrs) for non-R882 (p<0.0001). Other gene mutations associated with younger age included: WT1 (9% vs 2%, OR=4, p<0.0001), NRAS (16% vs 11%, OR=1.5, p=0.04) and PTPN11 (11 vs 1%, OR= 8.5, p=0.024).

To determine the clonal structure, we compared the variant allele frequency (VAF) of NPM1 and DNMT3A±5%, 131/164 (80%) had a DNMT3A VAF higher than NPM1 and only 3 (2%) a DNMT3A VAF lower than NPM1. This suggests that DNMT3A is the first hit and NPM1^mut is acquired during clonal evolution in the majority of cases. VAF clonal relationships were constant across age groups.

For 1904/2097 NPM1^mut pts with available CBA analysis (91%), 250/1904 (13%) had an aberrant karyotype which was significantly associated with older age in NPM1^mut AML (8% in 20-29 yrs old pts vs 20% in ≥80 yrs old pts, p<0.0001; age <60yrs: 10% vs age ≥60 yrs: 15%, p=0.0043).

As a control, we analyzed the age dependency of mutations in NPM1^wt AML pts (4893/6990): ASXL1, TET2, DNMT3A (both R882 and non-R882), IDH1 and IDH2R140 mutations increased continuously with age, while FLT3-ITD was more frequent in young pts (16% vs 9%, p<0.0001).

Conclusion. In a large AML cohort, prototypic CHIP mutations such as ASXL1 and TET2 increase with age regardless of NPM1 status, whereas in NPM1^mut AML mutated DNMT3A was a phenomenon of the younger patient (essentially due to the R882 subtype). VAF comparison indicates that DNMT3A is a pre-leukemic mutation in NPM1^mut AML and thus represents the first hit in NPM1^mut AML pathogenesis. We conclude that NPM1^mut/DNMT3A^mut double mutated AML could represent a distinct biologic entity which needs further investigation.