Table 1.

DNMT3A mutation status segregates patients into differentiation and cytotoxic responses

Subjec	FLT3 Genotype	Best marrow blast (%)	Baseline karyotype response	Karyotype at response	Cooperating mutations
Differentiation response
1009-01	FLT3-ITD	10	46,XY	46,XY	DNMT3A*, NPM1, ASXL1, IDH1
1009-02	FLT3-ITD	15	46,XX	46,XX	DNMT3A, NPM1, TET2
1009-14	FLT3-ITD	<5	46,XY	46,XY	DNMT3A, NPM1
1009-04	FLT3-ITD	<5	46,XX	46,XX	DNMT3A, NPM1, WT1, ATM*
1009-07	FLT3-ITD	<5	46,XX	46,XX	DNMT3A, NPM1
1009-09	FLT3-ITD	<5	46,XY	46,XY	DNMT3A, NPM1, TET2
1009-11	FLT3-ITD	<5	46,XX	46,XX	DNMT3A, NPM1, TET2
1009-10	FLT3-WT	<5	46,XY	46,XY	TET2
1009-21	FLT3-ITD	10	46,XY,+11	46,XY,+11	DNMT3A, ASXL1
Cytotoxic response
1009-06	FLT3-ITD	<5	Complex	46,XY	Not evaluable
1009-03	FLT3-ITD	<5	Hyperdiploid/complex	46,XX	DNMT3A, RUNX1
1009-13	FLT3-WT	<5	Complex	46,XY	TP53, NOTCH1
1009-12	FLT3-WT	<5	Complex	46,XY	Not evaluable
1009-17	FLT3-WT	<5	Complex	Complex	TP53, JAK2
1009-19	FLT3-ITD	<5	Complex	No growth	No mutations
1009-08	FLT3-ITD	<10	Complex	Complex	RUNX1
1009-18	FLT3-ITD	<5	46,XY, t(8;21),(q22;q22) with multiple additional abnormalities	46,XY, t(8;21),(q22;q22) with multiple additional abnormalities	TET2*
1009-16	FLT3-ITD	15	46,XY,del(5)(q23q33)	46,XY,del(5)(q23q33)	ATM*
1009-15	FLT3-ITD	70	47,XX,+8,del(16)(q13)	47,XX,+8,del(16)(q13)	DNMT3A, NPM1

Abbreviations: FLT3, Fms-like tyrosine kinase 3; ITD, internal tandem duplication. Genetic and cytogenetic studies at baseline and best response are shown. Responding patients with normal karyotype uniformly showed differentiation response. Patients with differentiation response had a statistically significant increase in the incidence of NPM1 mutations (odds ratio (OR) = 24.5, P<0.01) and/or DNMT3A (OR = 10.5, P<0.05, Fisher’s exact test). One subject with rapidly progressive leukemia and one with unclassifiable response due to low baseline blast content are not shown. FLT3-ITD and D835 mutations were detected using fluorescent primers and multiplexed PCR amplification followed by capillary electrophoresis and/or EcoRV digestion resistance assay by published methods. Allelic ratios of FLT3-ITD to the WT sequence were calculated using peak area (GeneMapper Software, Applied Biosystems, Grand Island, NY, USA). DNA was additionally extracted from cryopreserved blast samples and analyzed by next-generation sequencing for 23 genes commonly mutated in AML.¹⁵ Briefly, sequencing was done using an amplicon-based capture protocol (Luminex, Grand Island, NY, USA) with sequencing using MiSeq. Average depth of coverage was over 2000X and minimum depth was 250X. Sensitivity for mutations was 4% for all mutations other than FLT3-ITD, which was sensitive to a variant allele frequency of 1%. With the exception of variants of unclear significance (VOUS)—as indicated by asterisk—all gene mutations listed are recurrent pathogeneic sequence alterations as referenced by public databases, including dbSNP (http://www.ncbi.nlm.nih.gov/SNP/), the Catalog of Somatic Mutations in Cancer (http://cancer.sanger.ac.uk/cosmic) and the 1000 Genomes Project (http://www.1000genomes.org). Statistical analyses did not include VOUS.