Similarities and Differences of DNMT3A, TET2 and ASXL1 Mutations across Ccus, MDS and AML
Authors: Huber Sandra , Hutter Stephan , Baer Constance Regina , Meggendorfer Manja , Hoermann Gregor , Kern Wolfgang , Haferlach Torsten , Haferlach Claudia 
Published: December 2024

Background:
The most frequently mutated genes in clonal hematopoiesis (CH) occurring during aging are DNMT3A, TET2, and ASXL1 (DTA). DTA mutations (mut) also recurrently occur in overt myeloid malignancies.
Aim:
Analyze similarities and differences regarding incidence, co-aberrations and clonal hierarchy across CCUS, MDS and AML in patients with DTAmut.
Methods:
We analyzed 1,604 samples (received 2005-2021) of patients (median age: 71 years [2-93]; female: 42%) diagnosed with CCUS (n=222) or a non-therapy related myeloid malignancy (MDS: n=637; AML: n=745). All samples were analyzed by WGS (median coverage: 100x) in addition to standard routine diagnostics. To evaluate clonal hierarchy variant allele frequency (VAF) estimates were considered using a VAF difference cutoff of ≥5%.
Results:
In CCUS DTAmut were detected in 67% (149/222) (DNMT3A: 33%, TET2: 28%, ASXL1: 15%), in MDS in 48% (304/637) (DNMT3A: 12%, TET2: 26%, ASXL1: 19%) and in AML in 46% (340/745) (DNMT3A: 20%, TET2: 20%, ASXL1: 15%). The median VAF of all DTAmut increased from CCUS to MDS to AML (DNMT3A: 6%, 31%, 44%; TET2: 25%, 39%, 45%; ASXL1: 21%, 39%, 46%).
In DTAmut CCUS, 48% (71/149) only had either DNMT3A (n=45) or TET2 (n=20) or ASXL1 (n=6) as the sole aberration, while sole DTAmut in MDS and AML were rare (MDS: 18/304, 6%; AML: 1/340, 0.3%). In detail, DNMT3Amut in CCUS was the sole abnormality in 61%, while in MDS co-aberrations were present in 88% (top 3: SF3B1: 37%, del(5q): 35%, TET2: 20%) and in AML in 100% (top 3: NPM1: 59%, FLT3: 44%, TET2: 23%). In MDS DNMT3Amut were significantly more frequent in low blast MDS (MDS-5q: 23%; MDS-SF3B1: 18%; MDS-LB: 11%) than in MDS with increased blasts (17% vs. 5%; p&lt;0.001). In AML DNMT3Amut were mostly found within AML-NPM1 (52%) and AML defined by differentiation (42%) and less frequently in all other subtypes (&lt;17%).
TET2mut were the sole abnormality in CCUS in 32% and recurrently showed co-mut in SRSF2 (18%) and ASXL1 (18%). In MDS TET2mut was mainly accompanied by mutations in SF3B1 (32%), SRSF2 (25%) or ASXL1 (23%). Like DNMT3Amut, TET2mut also occurred in 18% of low blast MDS (MDS-SF3B1: 27%; MDS-5q: 14%; MDS-LB: 14%), however in contrast, significantly more frequently in MDS-IB (43%, p&lt;0.001). In AML the most frequent co-mut of TET2 were NPM1 (31%), DNMT3A (25%) and SRSF2 (24%). The highest frequencies of TET2mut were detected in AML-CEBPA (34%) and AML-NPM1 (27%). Interestingly, 54% (207/382) of all TET2mut were multi-hit (CCUS: 45%; MDS: 59%; AML: 53%).
ASXL1mut in CCUS were sole in 18%, most frequently accompanied by TET2 (32%) or SRSF2 (21%) mutations. In MDS ASXL1mut were significantly more often (36% vs. 11%, p&lt;0.001) detected in MDS-IB than in low blast MDS (MDS-SF3B1: 6%; MDS-5q: 15%; MDS-LB: 14%). The most frequent co-mut were TET2 (32%), SRSF2 (29%) and STAG2 (27%). In AML ASXL1mut were mostly detected in AML-MR (35%) or AML-MECOM (22%), the most frequent co-mut were SRSF2 (41%), RUNX1 (35%) and TET2 (27%).
Notably, DTAmut co-occurred with each other in 19% (150/793) of DTAmut patients (CCUS: 14%; MDS: 19%; AML: 21%; 2/150: D+T+A). Of those with two DTAmut the most frequent (53%) combination was TA (DT: 38%; DA: 9%).
The clonal hierarchy was analyzed for the most frequent co-mut (NPM1, SF3B1, SRSF2). In AML-NPM1 DNMT3A or TET2 mutations had a higher VAF than NPM1mut in 46%, while only in 12% DNMT3Amut or TET2mut were secondary events (42%: &lt;5% VAF difference). Regarding SF3B1 and DTA, DNMT3Amut (29%), TET2mut (28%) and SF3B1mut (39%) occurred as the clear primary event with similar frequencies, in contrast to ASXL1mut showing a higher VAF than SF3B1mut in only 5%. In combination with SRSF2, DTAmut were the clear primary event in 34-50%, SRSF2mut only in 7-24%.
Conclusions:
DTAmut show significant differences in frequencies and co-mutation patterns across CCUS, MDS and AML. Our data suggest that DNMT3Amut as a sole abnormality with low VAF is sufficient for causing CCUS, whereas TET2mut and ASXL1mut may not be sufficient alone, respectively, for causing CCUS as these are more frequently accompanied by additional abnormalities. In MDS, all DTAmut frequently show co-aberrations. While DNMT3Amut is more common in low blast MDS, TET2mut and ASXL1mut are more common in increased blast MDS. In AML, DNMT3Amut and TET2mut share NPM1 as the most common co-mutation, while ASXL1mut has a completely different co-mutation pattern. Common to all DTAmut is their rare co-occurrence with TP53mut and AML-defining fusion genes.