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. Author manuscript; available in PMC: 2016 Dec 1.
Published in final edited form as: Leukemia. 2015 Nov 20;30(6):1422–1425. doi: 10.1038/leu.2015.320

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.