A novel non-invasive monitoring assay of 5-azacitidine efficacy using global DNA methylation of peripheral blood in myelodysplastic syndrome

Figures & data

Table 1 Methylation index of patients with myelodysplastic syndrome and post-MDS AML

UPN	Sex	Age (y)	FAB	IPSS	IPSS-R	PB blasts (%)	BM blasts (%)	Karyotype	New cytogenetic scoring	Methylation index (%)*
										Whole blood		N		MNC
I. Bone marrow blasts <5% (n=15) (mean ± SD)										64.8±9.0	(63.0: 49.3~82.7)	61.6±14.8	(63.7: 28.9~78.3)	59.3±13.7	(60.2: 32.8~79.1)
10^a	M	60	RA	Int-1	Int	0	3.6	del(20q)	Good	60.6		78.3		73
101	F	83	RA	Int-1	L	0	0	NC	Good	63.0		28.9		57
103	F	77	RA	Int-1	Int	0	4	NC	Good	66.0		55.1		77.6
104	M	81	RA	Int-1	Int	0	0	NC	Good	57.2		40.6		32.8
107	M	68	RARS	Int-1	Int	0	0.8	−9,+mar	Int	59.8		75.4		66.4
18^a	M	75	RCMD	Int-2	H	0	0	complex:-Y, del(5q), del(17p)	Very poor	67.9		62.8		60.6
20^a	M	62	RCMD	Int-1	Int	1	1.2	NC	Good	61.1		66.1		52.1
25^a	M	72	RCMD	Int-2	Int	0.5	2	del 7, +marker	Int	64.6		ND		ND
31^a	M	66	RCMD	Int-2	Int	0	4.4	add(21q)	Good	82.0		ND		ND
106	M	41	RCMD	Int-1	Int	0	0	NC	Good	57.7		75.2		60.2
108	M	44	RCMD	Int-1	Int	0	0	del(9q)/N	Int	72.0		47.7		55.5
119	F	74	RCMD	L	L	0	0	NC	Good	58.9		63.5		38.5
120	M	77	RCMD	Int-1	Int	0	0	der(1;7)	Int	49.3		72.4		79.1
105	M	82	MDS-U	Int-1	Int	0	0	del(20q),I (17q)	Int	68.8		63.7		52.4
130	M	72	MDS-U	Int-1	H	0	0	del(3p)	Int	82.7		70.7		65.9
II. Bone marrow blasts ≥5%, <20% (n=13) (mean ± SD)										64.4±10.1	(60.9: 53.9~83.0)	59.6±12.5	(57.7: 42.8~76.7)	65.4±9.2	(61.5: 57.8~80.0)
5^a	M	68	RAEB1	Int-2	VH	0	7	der(1;7)	Int	71.9		76.7		77.7
7^a	M	70	RAEB1	Int-2	VH	8	6	complex	Very poor	75.7		75.7		66.9
1	F	70	RAEB2	H	VH	8.5	11	complex: del(7q),del(5q), −13	Very poor	83.3		52.9		71.3
4	M	55	RAEB2	H	VH	0	16	complex	Very poor	80.2		71.8		80
8	F	61	RAEB2	Int-2	VH	2.5	10.4	complex: del(5q),del(7q), del(12p)	Very poor	54.2		59.2		59.9
9	M	23	RAEB2	Int-2	VH	3	14	NC	Good	54.6		46.3		59.3
12	M	50	RAEB2	Int-2	VH	0	14.6	NC	Good	57.1		ND		ND
15^a	F	80	RAEB2	H	Int	0	10.5	NC	Good	53.9		53.4		59.4
21^a	M	71	RAEB2	Int-2	VH	0	19	del(6q), −Y	Int	59.7		42.8		52.8
26^a	M	55	RAEB2	H	VH	14	17	complex	Very poor	63.6		ND		ND
30^a	M	63	RAEB2	H	VH	0	15	del(20q), +2, +8	Int	61.5		ND		ND
118	M	76	RAEB2	Int-2	VH	3.5	8	der(1;7)	Int	60.1		57.7		61.5
125	M	78	RAEB2	Int-2	VH	0	10	NC	Good	60.9		ND		ND
III. Bone Marrow Blasts >20% (n=6) (mean ± SD)										66.6±8.0	(64.8: 57.3~80.1)	62.0±10.1	(61.5: 47.3~73.7)	59.3±7.5	(59.0: 48.2~68.4)
3	F	42	Post-MDS AML			12	29	+9		66.2		58.6		57.5
6	M	58	Post-MDS AML			25	32.5	der(1;7)		63.3		61.5		59
13^a	M	70	Post-MDS AML			0	20.5	NC		61.9		47.3		48.2
22	M	77	Post-MDS AML			5	43.2	complex: del(5q), del(20q), −Y		57.3		68.7		63.2
27^a	F	62	Post-MDS AML			1	28.8	complex		80.1		ND		ND
131	M	67	Post-MDS AML			0.5	21	+8, −15		70.5		73.7		68.4
Controls (healthy volunteers; n=13)										82.8±5.9	(84.1: 71.8~90.1)	79.7±9.3	(71.8: 48.2~88.0)	70.0 ±8.3	68.8(59.8~86.4)

Notes: *Methylation index (%) is shown as mean ± standard deviation(S.D.). Median, minimum and maximam are shown in parentheses.

F, female; M, male; IPSS, International Prognostic Scoring System; IPSS-R, revised IPSS. ^aThirteen patients treated with AZA whose methylation index was subsequently measured.

Abbreviations: L, low; H, high; VH, very high; Int, intermediate; N, neutrophils; MNC, mononuclear cells; MDS, myelodysplastic syndrome; AML, acute myeloid leukemia; PB, peripheral blood; BM, bone marrow; NC, normal karyotypes; Methylation index,percent of methylated DNA in the sample measured by a single molecule methylation assay.

Figure 1 Methylation index (MI) measured using a single-molecule methylation assay (SMMA). The percent of methylation DNA in the sample is shown. (A). MI of whole blood. (B). MI of the neutrophil fraction. (C). MI of mononuclear cells. (D). MI is significantly increased in MDS patients with a very poor cytogenetic score according to the Revised International Prognostic Scoring System.

Abbreviations: MDS, myelodysplastic syndrome; post MDS-AML, post myelodysplastic acute myeloid leukemia.

Table 2 Clinical response to AZA in 13 patients whose methylation index was subsequently measured

UPN	Sex	Age	Dx	IPSS	IPSS-R	AZA cycles	Clinical response	Outcome	Mutations*
5	M	68	RAEB1	Int-2	H	10	HI-P, HI-E → relapse	Dead	DNMT3A, TET2, EZH2
7	M	70	RAEB1	Int-2	H	4	HI-E → relapse	Dead	TP53
10	M	60	RA	Int-1	L	9	failure	Alive	not detected
13	M	70	post-MDS AML			15	HI-P, HI-N→relapse→HI-P,HI-E,HI-N	Alive	not detected
15	F	80	RAEB2	H	Int	12	HI-P→relapse	Alive	RAD21, RUNX1
18	M	75	RCMD	Int-2	H	6	HI-P, HI-E→relapse	Alive	DNMT3A, KMT2A
20	M	62	RCMD	Int-1	Int	5	Failure	Alive	TET2, EZH2
21	M	71	RAEB2	Int-2	H	4	Failure	Alive	EED, TP53
25	M	72	RCMD	Int-2	Int	9	Failure	Alive	DNMT3A, TET2, SETBP1, KRAS
26	M	55	RAEB2	H	vH	5	failure	Dead	TP53
27	F	62	post-MDS AML			4	failure	Dead	SH2B3, TP53, STAG2
30	M	63	RAEB2	H	vH	6	HI-E, HI-N	Alive	TET2, RUNX1
31	M	66	RCMD	Int-2	Int	6	failure	Alive	ASXL1

Note: *Detailed results of mutation analysis using a the GeneRead DNAseq Targeted Panel V2 (Human Myeloid Neoplasms. Panel, QIAGEN) are shown in Supplementary file 1.

Abbreviations: UPN, unique patient number; IPSS, International Prognostic Scoring System; IPSS-R, IPSS revised; AZA, 5-azacitidine; Dx, diagnosis; HI-P, hematological improvement in platelets; HI-E, HI in erythrocytes; HI-N, HI in neutrophils

Figure 2 The restoration rate of methylation for AZA-treated patients. (A). ∆MI at day 28 (MIday²⁸ – MI^day0) for AZA responders was significantly higher than that of non-responders (P=0.0352). (B). ∆MI at day 120 was significantly higher than that of non-responders (P=0.0251). (C). For AZA responders, ∆MI at day 120 was significantly higher than at day 28 (P=0.0216). (D). For AZA non-responders, there was no significant difference between ∆MI at day 28 and at day 120 (P=0.2961).

Note: *Represents a significant difference.
Abbreviation: MI, methylation index.

Table S1 Mutation analysis using the GeneRead DNAseq Targeted Panel V2 (Human Myeloid Neoplasms Panel; Qiagen)

Transcription/Other							RAS	Splicing	Chromatin Modification						DNA Methylation							Group
STAG2	RUNX1	TP53			SH2B3	RAD21	KRAS	SETBP1	ASXL1	EED	KMT2A	EZH2			TET2				DNMT3A			Gene
X	21	17	17	17	12	8	12	18	20	11	11	7	7	7	4	4	4	4	2	2	2	Chr
123,189,997	36,252,940	7,577,120	7,578,190	7,577,081	111,884,785	117,866,542	25,380,285	42,531,913	31,023,483	85,988,145	118,375,276	148,523,579	148,507,436	148,508,731	106,193,748	106,180,819	106,155,755	106,196,829	25,469,981	25,467,449	25,463,285	Position
mut	mut	mut	mut	mut	mut	mut	mut	mut	mut	mut	mut	mut	mut	mut	mut	inframe_del	del	mut	mut	mut	del	Variant
T406A	S141L	R273H	Y220C	E286V	E292K	G368V	T58I	G870S	E990*	W364R	R2890P	Y292N	N673I	E645K	R1404*	S1284del	H222W fs	L1721W	F354S	G543C	R736P fs	AA Influence
														31.7				42.1			29.6	UPN5
				37.3																		UPN7
																						UPN1 0
																						UPN1 3
	8.5					5.6																UPN1 5
											39.4									37.7		UPN1 8
												42.3	39.6				41.4					UPN2 0
			15							29.2												UPN2 1
							9.5	39.7										45.5	38.2			UPN2 5
		24.6																				UPN2 6
39.8		59.1			47.8																	UPN2 7
	44.1														10	41.1						UPN3 0
									11.2													UPN3 1
																						UPN3 4

Prebet T, Sun Z, Figueroa ME, et al. Prolonged administration of azacitidine with or without entinostat for myelodysplastic syndrome and acute myeloid leukemia with myelodysplasia-related changes: results of the US Leukemia Intergroup trial E1905. J Clin Oncol. 2014;32(12):1242–1248. doi:10.1200/JCO.2013.50.310224663049

 PubMed Web of Science ®Google Scholar