Table 2.

Validation of sequence variants in 13 selected AML genes as detected by HAMLET.

Gene	Region	HAMLET component	HAMLET:	mut	wt	mut	wt	Sensitivity (%)	Specificity (%)
			Validation:	mut	mut	wt	wt
NPM1	Hotspot	VARSCAN		35	0	0	65	100	100
DNMT3A	Hotspot	VARSCAN		15	0	0	85	100	100
	Outside hotspot	VARSCAN		17	0	0	17	100	100
FLT3	Hotspot ex20	VARSCAN		8	0	0	17	100	100
	Outside hotspot	VARSCAN		12	0	0	13	100	100
ASXL1	Hotspot	VARSCAN		13	1	0	18	92.9	100
CEBPA	Entire gene	VARSCAN		18	1	0	18	94.7	100
IDH1	Hotspot	VARSCAN		7	0	0	14	100	100
	Outside hotspot	VARSCAN		7	0	0	16	100	100
IDH2	Hotspot	VARSCAN		12	0	0	12	100	100
	Outside hotspot	VARSCAN		4	0	0	22	100	100
KIT	Hotspot	VARSCAN		7	0	0	13	100	100
	Outside hotspot	VARSCAN		3	0	0	8	100	100
NRAS	Hotspot	VARSCAN		14	0	0	15	100	100
RUNX1	Entire gene	VARSCAN		21	0	0	28	100	100
TET2	Entire gene	VARSCAN		34	0	0	34	100	100
TP53	Entire gene	VARSCAN		3	0	0	12	100	100
WT1	Entire gene	VARSCAN		9	0	0	11	100	100
TOTAL		VARSCAN		239	2	0	418	99.2	100
FLT3	Hotspot ex14-15	ReSCU		34	0	2	64	100	97.0
KMT2A	Hotspot ex2-13	ReSCU		8	0	0	4	100	100
TOTAL		ReSCU		42	0	2	68	100	97.1
TOTAL	VARSCAN and ReSCU			281	2	2	486	99.3	99.6

Validation of NPM1 mutations and FLT3-ITD was performed by PCR on genomic DNA and electrophoretic fragment size analysis. Validation of other variants was performed by RT-PCR on cDNA followed by Sanger sequencing or NGS using MiSeq. Gene hotspots are defined in Table SVI.

mut variant detected, wt wild type.