Hypermethylation of secreted frizzled-related proteins predicts poor prognosis in non-M3 acute myeloid leukemia

Figures & data

Table 1 Correlations of hypermethylation of SFRPs gene promoter with clinical features in non-M3 AML patients

Patient’s parameters	SFRP1 methylation			SFRP2 methylation			SFRPs methylation			Total (n=139)
	High (n=42)	Low (n=97)	P-value	High (n=38)	Low (n=101)	P-value	High (n=72)	Low (n=67)	P-value
Sex, male/female	27/15	59/38	0.710	22/16	64/37	0.563	44/28	42/25	0.863	86/53
Median age (range) (years)	56 (18–86)	49.5 (18–85)	0.316	56.5 (18–86)	47 (18–85)	0.095	55.5 (18–86)	45.5 (18–85)	0.106	52 (18–86)
Median WBCs (range) (×10^9/L)	27.7 (0.7–528.0)	18.45 (0.8–300.0)	0.671	17.7 (0.7–528.0)	21.5 (0.8–528.0)	0.439	27.7 (0.7–528.0)	16.85 (0.8–300)	0.740	18.7 (0.7–528.0)
Median hemoglobin (range) (g/L)	83 (32–114)	72 (34–147)	0.411	71 (32–119)	74.5 (33–147)	0.401	75 (33–123)	74 (32–147)	0.303	74 (32–147)
Median platelets (range) (×10^9/L)	31 (6–190)	41.5 (3–399)	0.319	36 (7–264)	39 (3–399)	0.817	37 (6–264)	39 (3–399)	0.881	38 (3–399)
BM blasts (range) (%)	46 (15.5–99)	52.5 (6–94.5)	0.299	68 (10.5–97.5)	47.0 (6–99)	0.062	52.0 (10.5–99)	51.25 (6–94.5)	0.476	51.5 (6–99)
Age (years)			0.526			0.011			0.041
<50	16 (38%)	47 (48%)		11 (29%)	52 (51%)		26 (36%)	37 (55%)		63 (45%)
≥50	24 (57%)	47 (48%)		27 (71%)	44 (44%)		44 (61%)	27 (40%)		71 (51%)
No data	2 (5%)	3 (4%)		0 (0%)	5 (5%)		2 (3%)	3 (5%)		5 (4%)
FAB			0.356			0.078			0.482
M0	0 (0%)	1 (1%)		1 (3%)	0 (0%)		1 (1%)	0 (0%)		1 (1%)
M1	3 (7%)	12 (12%)		8 (21%)	7 (7%)		8 (11%)	7 (10%)		15 (11%)
M2	17 (41%)	50 (52%)		18 (47%)	49 (49%)		30 (42%)	37 (56%)		67 (48%)
M4	11 (26%)	20 (21%)		7 (18%)	24 (24%)		20 (28%)	11 (16%)		31 (22%)
M5	8 (19%)	12 (12%)		4 (11%)	16 (16%)		10 (14%)	10 (15%)		20 (14%)
M6	3 (7%)	2 (2%)		0 (0%)	5 (5%)		3 (4%)	2 (3%)		5 (4%)
Karyotype classification			0.588			0.615			0.899
Favorable	8 (19%)	11 (11%)		3 (8%)	16 (16%)		9 (13%)	10 (15%)		19 (14%)
Intermediate	27 (64%)	70 (73%)		28 (73%)	69 (69%)		50 (68%)	47 (70%)		97 (69%)
Poor	4 (10%)	11 (11%)		4 (11%)	11 (11%)		9 (13%)	6 (9%)		15 (11%)
No data	3 (7%)	5 (5%)		3 (8%)	5 (5%)		4 (6%)	4 (6%)		8 (6%)
Karyotype			0.714			0.780			0.989
Normal	24 (57%)	54 (56%)		22 (57%)	56 (56%)		41 (57%)	37 (56%)		78 (56%)
t(8;21)	7 (17%)	11 (11%)		3 (8%)	15 (15%)		8 (11%)	10 (15%)		18 (13%)
11q23	1 (2%)	1 (1%)		1 (3%)	1 (1%)		1 (1%)	1 (1%)		2 (1%)
Complex	2 (5%)	9 (9%)		3 (8%)	8 (8%)		6 (8%)	5 (7%)		11 (8%)
Others	5 (12%)	17 (18%)		6 (16%)	16 (16%)		12 (17%)	10 (15%)		22 (16%)
No data	3 (7%)	5 (5%)		3 (8%)	5 (5%)		4 (6%)	4 (6%)		8 (6%)
Gene mutation*
C/EBPA (±)	6/34 (15%)	19/76 (20%)	0.630	6/32 (16%)	19/78 (20%)	0.637	10/60 (14%)	15/50 (23%)	0.267	25/110 (19%)
NPM1 (±)	4/36 (10%)	14/81 (15%)	0.585	7/31 (18%)	11/86 (11%)	0.398	10/60 (14%)	8/57 (12%)	0.804	18/117 (13%)
FLT3-ITD (±)	2/38 (5%)	9/86 (9%)	0.506	4/34 (11%)	7/90 (7%)	0.728	5/65 (7%)	6/59 (9%)	0.758	11/124 (8%)
DNMT3A (±)	1/39 (3%)	7/88 (7%)	0.435	2/36 (5%)	6/91 (6%)	1.000	3/67 (4%)	5/60 (8%)	0.481	8/127 (6%)
IDH1/2 (±)	3/37 (8%)	8/87 (8%)	1.000	4/34 (11%)	7/90 (7%)	0.728	6/64 (9%)	5/60 (8%)	1.000	11/124 (8%)
C-KIT (±)	2/38 (5%)	10/85 (11%)	0.352	2/36 (5%)	10/87 (10%)	0.509	3/67 (4%)	9/56 (14%)	0.070	12/123 (9%)
N/K-RAS (±)	9/31 (23%)	8/87 (8%)	0.043	5/33 (13%)	12/85 (12%)	1.000	11/59 (16%)	6/59 (9%)	0.306	17/118 (13%)
CR (±)	11/19 (37%)	25/38 (40%)	0.823	9/18 (33%)	27/39 (41%)	0.640	18/35 (34%)	18/22 (45%)	0.292	36/57 (39%)

Note:

* Percentage was equal to the number of mutated patients divided by total cases in each group. Bold values indicate positive results.

Abbreviations: SFRPs, secreted frizzled-related proteins; AML, acute myeloid leukemia; WBCs, white blood cells; BM, bone marrow; FAB, French-American-British; CR, complete remission.

Table 2 Primer sequences of SFRPs for RQ-PCR and RQ-MSP

Primer	Sequence	Size (bp)	T^1 (°C)	T^2 (°C)
Expression
SFRP1-F	5′-AGATGCTTAAGTGTGACAAGTTCC-3′	130	64	85
SFRP1-R	5′-TCAGATTTCAACTCGTTGTCACAG-3′
SFRP2-F	5′-TGAGTGCGACCGTTTCCC-3′	298	62	81
SFRP2-R	5′-GAGCCACAGCACCGATTT-3′
SFRP4-F	5′-GTACAGGAAAGGCCTCTTGATG-3′	154	68	80
SFRP4-R	5′-TGCAGCCACTCCTCTGCAC-3′
SFRP5-F	5′-GCGCCCAGTGTGAGATGG-3′	115	65	82
SFRP5-R	5′-TTCCGGTCCCCATTCTCTATC-3′
Methylation
SFRP1-M-F	5′-TGTAGTTTTCGGAGTTAGTGTCGCGC-3′	126	65	82
SFRP1-M-R	5′-CCTACGATCGAAAACGACGCGAACG-3′
SFRP1-U-F	5′-GTTTTGTAGTTTTTGGAGTTAGTGTTGTGT-3′	135	60
SFRP1-U-R	5′-CTCAACCTACAATCAAAAACAACACAAACA-3′
SFRP2-M-F	5′-GGGTCGGAGTTTTTCGGAGTTGCGC-3′	138	68	83
SFRP2-M-R	5′-CCGCTCTCTTCGCTAAATACGACTCG-3′
SFRP2-U-F	5′-TTTTGGGTTGGAGTTTTTTGGAGTTGTGT-3′	144	65
SFRP2-U-R	5′-AACCCACTCTCTTCACTAAATACAACTCA-3′
SFRP4-M-F	5′-GGGTGATGTTATCGTTTTTGTATCGAC-3′	112	67	81
SFRP4-M-R	5′-CCTCCCCTAACGTAAACTCGAAACG-3′
SFRP4-U-F	5′-GGGGGTGATGTTATTGTTTTTGTATTGAT-3′	116	67
SFRP4-U-R	5′-CACCTCCCCTAACATAAACTCAAAACA-3′
SFRP5-M-F	5′-AAGATTTGGCGTTGGGCGGGACGTTC-3′	136	68	85
SFRP5-M-R	5′-ACTCCAACCCGAACCTCGCCGTACG-3′
SFRP5-U-F	5′-GTAAGATTTGGTGTTGGGTGGGATGTTT-3′	141	69
SFRP5-U-R	5′-AAAACTCCAACCCAAACCTCACCATACA-3′

Notes: T¹: annealing temperature; T²: temperature of fluorescence collection.

Abbreviations: SFRPs, secreted frizzled-related proteins; RQ-PCR, real-time quantitative polymerase chain reaction; RQ-MSP, real-time quantitative methylation-specific polymerase chain reaction.

Figure 1 Electrophoresis results of RQ-MSP products in leukemic cell lines.

Abbreviations: RQ-MSP, real-time quantitative methylation-specific polymerase chain reaction; M, methylation; U, unmethylation.

Figure 2 Electrophoresis results of RQ-MSP products of SFRPs in AML patients.

Notes: 1: GeneRuler™ 100-bp DNA ladder; 2 and 3: two controls; 4–11: samples of non-M3 AML patients (four patients with high SFRP1/SFRP2 expression and the other four with low SFRP1/SFRP2 expression); 12: positive control; 13: negative control.
Abbreviations: RQ-MSP, real-time quantitative methylation-specific polymerase chain reaction; SFRPs, secreted frizzled-related proteins; AML, acute myeloid leukemia; M, methylation; U, unmethylation.

Figure 3 Overall survival of non-M3 AML patients: (A) all patients; (B) cytogenetically normal patients.

Abbreviations: AML, acute myeloid leukemia; SFRPs, secreted frizzled-related proteins.

Figure 4 HL60 with 5-aza-dC treatment.

Notes: Relative changes in (A) SFRP1 and (B) SFRP2 expression levels and (C) SFRP1 and (D) SFRP2 methylation levels before and after treatment of HL60 cell line with the demethylating agent 5-aza-dC for 72 h at 0 μM, 0.1 μM, 1 μM, 10 μM, and 50 μM concentrations. Expression levels of SFRP1 and SFRP2 were determined by RQ-PCR and normalized to the housekeeping gene ABL. Methylation levels of SFRP1 and SFRP2 were determined by RQ-MSP and normalized to the housekeeping gene ALU. Error bars indicate standard deviation.
Abbreviations: 5-aza-dC, 5-aza-2′-deoxycytidine; RQ-PCR, real-time quantitative polymerase chain reaction; RQ-MSP, real-time quantitative methylation-specific polymerase chain reaction.

Figure 5 Correlation between SFRP1 hypermethylation and expression level in non-M3 AML patients and HL60 cell line (since the value was lg function conversion, methylation and expression level at zero was excluded).

Abbreviation: AML, acute myeloid leukemia.