DNMT3A mutational analysis in primary myelofibrosis, chronic myelomonocytic leukemia and advanced phases of myeloproliferative neoplasms

Recent reports have identified somatic mutations in DNMT3A in 4–22% of patients with de novo acute myeloid leukemia.^{1, 2} These mutations, thought to result in a loss-of-function, were found to be predominantly heterozygous mutations in the methyltransferase domain, with a recurrent mutation at the R882 codon. In addition to DNMT3A mutations in acute myeloid leukemia, Walter et al.³ have found DNMT3A mutations in 8% (12/150) of myelodysplastic syndromes patients as well.³ Importantly, both Ley et al. and Walter et al. found that mutations in DNMT3A were associated with a worsened overall survival in both de novo acute myeloid leukemia and myelodysplastic syndromes, respectively.

Given these finding, we sequenced DNMT3A in a clinically and genetically well-annotated cohort of patients with primary myelofibrosis (PMF), the myeloproliferative neoplasm (MPN)/myelodysplastic syndromes overlap syndrome chronic myelomonocytic leukemia (CMML) and advanced phases of MPNs in order to determine the genetic and clinical implications of DNMT3A mutations in this class of myeloid maligancies.

Study samples from 94 patients were recruited from the Mayo Clinic (n=94; 46 PMF, 22 post-polycythemia vera/essential thrombocythemia MF, 11 blast-phase MPN and 15 CMML). High throughput DNA resequencing was used to seqeunce bone marrow-derived DNA for all coding regions of DNMT3A (NM_175629). In addition, these samples were all previously sequenced for all coding regions of EZH2 (NM_015338), ASXL1 (NM_004456), and TET2 (NM_017628) and the regions of known mutations in IDH1, IDH2, JAK2 and MPL as previously reported.⁴ CMML samples were also screened for known FLT3, K Ras and N Ras mutations. Non-synonymous alterations not in dbSNP were censored from analysis.

We identified three somatic mutations in DNMT3A in this cohort of patients (Table 1). All three mutations were heterozygous and occurred in patients with PMF resulting in a DNMT3A mutational frequency of 7% (3/46) in PMF. In addition to the R882H methyltransferase domain mutation found in one PMF patient, the other patients had heterozygous nucleotide deletions outside of the known domains of DNMT3A resulting in the occurrence of premature stop codons. All three PMF patients found to have a mutation in DNMT3A were also found to have a co-occuring mutation in another gene known to be mutated in MPNs, including co-occurences with mutations in JAK2, TET2 and ASXL1 (Table 1). In addition to the three somatic DNMT3A mutations, we found five single nucleotide variants, which we were unable to delineate as somatic missense mutations versus unannotated SNPs. These variants, some of which were also noted by Ley et al.,² may be delineated in future studies containing more samples with paired normal tissue and are as follows: DNMT3A E30A, P99S, P569A, R659H and R899C.

Table 1.

Mutations in DNMT3A and co-occurring genetic abnormalities in a cohort of PMF patients^a

Sample	DNMT3a mutation (nucleotide)	DNMT3a mutation (protein)	Karyotype	Other genetic analysis
				JAK2	TET2	ASXL1	MPL	EZH2	IDH1/2
PMF #1	700_hetDelC	G120fsX40	20q- (all metaphases)	Mutant	WT	WT	WT	WT	WT
PMF #2	1603_hetDelT	P419fsX230	Normal	Mutant	Mutant	WT	WT	WT	WT
PMF #3	2646 G>GA	R882H	Normal	WT	WT	Mutant	WT	WT	WT

Abbreviation: PMF, primary myelofibrosis.

^aWT: wild type.

None of the three PMF patients found to have DNMT3A mutations underwent leukemic transformation during the time of follow-up, and the patients had variable Dynamic International Prognostic Scoring System (DIPPS) prognostic scores at presentation.⁵ PMF patient 1 was a 56-year-old man at time of diagnosis with a low DIPSS prognostic score at presentation and marked splenomegaly. He has been alive at 64 months without leukemic progression. In contrast, PMF patients 2 and 3 died at 13 and 27 months after presentation (neither developed leukemic transformation). PMF patient 2 was a 79-year-old man at time of diagnosis with a high DIPSS prognostic score at presentation and marked splenomegaly. PMF patient 3 was a 57-year-old woman at time of diagnosis with an intermediate-2 DIPSS prognostic score at presentation, and marked splenomegaly.

We found no mutations in DNMT3A in CMML, blast-phase MPN’s or post-PV/ET MF suggesting that DNMT3A mutations may not be common events in blast-phase transformation of chronic MPNs or in the pathogenesis of CMML. However, larger patient cohorts will be needed to resolve this question more thoroughly and to determine if DNMT3A mutations are important in the pathogenesis of PV, ET and/or post-PV/ET MF. The recent discovery of DNMT3A mutations in acute myeloid leukemia, myelodysplastic syndromes and now MPNs, underscores the profound heterogeneity and promiscuity of mutations amongst the myeloid malignancies. Larger studies of patients with uniform diagnoses and comprehensive genetic analysis will hopefully allow for greater understanding of the potential clinical impact of these new mutations to MPN prognosis.