RASSF1A hypermethylation is associated with ASXL1 mutation and indicates an adverse outcome in non-M3 acute myeloid leukemia

Figures & data

Table 1 Analysis of clinical characteristics and outcome in two groups

Variables	Total	Hypermethylation (n)	Non-methylation (n)	P-value
Patients (N)	226	52	174	NS
Age	43 (12–94)	38 (14–87)	43 (12–94)	0.229
Sex	137 (M)/89 (F)	35 (M)/17 (F)	102 (M)/72 (F)	0.261
M2	54	11	43	0.597
M4	43	15	32	0.103
M5	60	14	46	0.944
Unclassified AML	41	8	33	0.557
WBC (×10^9/L)	21.74 (1.9–76.3)	19.95 (1.98–76.3)	22.32 (1.9–74)	0.443
Hemoglobin (g/L)	72 (26–131)	73 (34–131)	71 (26–126)	0.529
Platelets (×10^9/L)	41 (1–146)	43 (1–141)	40 (4–146)	0.414
Marrow blasts (%)	70 (26–98)	68 (39–95)	71 (26–98)	0.179
Induction therapy
IA	70	15	55	0.705
DA	84	24	60	0.126
MA	72	13	59	0.226
Allo-HSCT	29	8	21	0.524
Auto-HSCT	26	5	21	0.627

Abbreviations: NS, nonsignificant; AML, acute myeloid leukemia; WBC, white blood cell; IA, idarubicin and cytarabine; DA, daunorubicin and cytarabine; MA, mitoxantrone and cytarabine; allo-HSCT, allogeneic hematopoietic stem cell transplantation; auto-HSCT, autologous hematopoietic stem cell transplantation.

Figure 1 (A) Relative expression of the RASSF1A gene was detected in the patients with RASSF1A hypermethylation and the cases with non-methylation, and significant difference was found between the two groups. ^*Singular value. (B) There was a negative correlation between RASSF1A methylation levels and RASSF1A transcript levels (R=−0.464, P=0.028).

Table 2 Comparison of genetic alterations between patients with acute myeloid leukemia with or without hypermethylation of the RASSF1A promoter

Variant	Total (n)	Hypermethylation (n)	Non-methylation (n)	P-value
Cytogenetic riska
Favorable	20	4	16	0.955
Intermediate	151	30	121	0.111
Unfavorable	55	18	37	0.049
Cytogenetic characteristicsb
t(8;21)	12	1	11	0.374
inv(16)/t(16;16)	8	3	5	0.573
11q23 abnormalities	15	6	9	0.193
Complex karyotype	33	13	20	0.016
Normal karyotype	122	23	99	0.108
Gene mutationsc
IDH1	23	7	16	0.372
ASXL1	22	9	13	0.036
FLT3	25	4	21	0.377
KIT	8	1	7	0.771
TET2	16	4	12	0.846
SF3B1	9	1	8	0.645
CEBPA	11	1	10	0.449
DNMT3A	19	7	12	0.225
NPM1	49	10	39	0.625
NRAS	13	2	11	0.739
SRSF2	13	3	10	0.995
U2AF1	18	5	13	0.834
RUNX1	16	5	11	0.162

Notes:

a Cytogenetic abnormalities were grouped according to published criteria adopted by the Southwest Oncology Group as favorable, intermediate, and unfavorable. Favorable: inv(16)/t(16;16)/del(16q), t(15;17) with/without secondary aberrations, t(8;21) lacking del(9q), or complex karyotypes; unfavorable: del(5q)/−5, del(7q)/−7, abnormalities of 3q, 9q, 11q, 20q, and 17p, t(6;9), t(9;22), and complex karyotypes; intermediate: normal karyotype and other abnormalities.

b Patients may be counted more than once because of coexistence of more than one cytogenetic abnormality in the leukemic clone.

c Patients may be counted more than once because of coexistence of more than one mutation in the leukemic clone.

Figure 2 Spectrum of gene mutations in 226 non-M3 AML patients with hypermethylation and non-methylation of the RASSF1A gene.

Figure 3 (A and B) Among non-M3 AML patients, those with RASSF1A hypermethylation (n=52) had inferior relapse-free survival and overall survival compared to those with no hypermethylation (n=174) (P=0.012 and P=0.014, respectively). (C and D) Patients with higher RASSF1A methylation levels (n=13) did not show different relapse-free survival and overall survival compared to individuals with lower methylation levels (n=39) (P=0.968 and P=0.798, respectively).

Table 3 Univariate and multivariate analysis of clinical and molecular variables for RFS and OS in non-M3 AML patients

Variables	Univariate analysis				Multivariate analysis
	RFS		OS		RFS		OS
	P-value	OR (95% CI)	P-value	OR (95% CI)	P-value	OR (95% CI)	P-value	OR (95% CI)
Agea	0.006	2.730 (1.739–4.286)	0.009	2.720 (1.734–4.267)	0.002	2.540 (1.522–4.238)	0.005	2.536 (1.516–4.242)
Unfavorable karyotypeb	0.041	1.617 (1.021–2.562)	0.058	1.60 (0.985–2.473)	0.033	1.701 (1.045–2.768)	0.041	1.663 (1.022–2.706)
RASSF1A hypermethylation	0.012	1.782 (1.125–2.822)	0.014	1.758 (1.110–2.785)	0.040	1.622 (0.979–2.687)	0.060	1.593 (0.962–2.637)
ASXL1 mutation	0.008	2.238 (1.237–4.052)	0.007	2.277 (1.257–4.123)	0.058	1.863 (0.978–3.547)	0.083	1.780 (0.927–3.417)
FLT3-ITD	0.004	3.009 (1.735–5.533)	0.004	3.078 (1.725–5.493)	0.021	3.518 (1.921–6.442)	0.025	3.510 (1.907–6.460)
RUNX1 mutation	0.002	3.278 (1.563–6.877)	0.001	3.341 (1.592–7.010)	0.005	3.078 (1.407–6.733)	0.004	3.179 (1.461–6.995)
DNMT3A mutation	0.006	2.306 (1.275–4.172)	0.007	2.244 (1.241–4.058)	0.033	2.577 (1.380–4.812)	0.034	2.505 (1.340–4.684)
U2AF1 mutation	0.011	2.295 (1.214–4.338)	0.011	2.273 (1.203–4.295)	0.036	2.122 (1.051–4.286)	0.052	2.018 (0.994–4.097)

Notes:

a Patients aged >60 years vs others.

b Unfavorable cytogenetics vs others.

Abbreviations: RFS, relapse-free survival; OS, overall survival; OR, odds ratio; CI, confidence interval.

Table S1 Primers and probes for detection of MYOD1 and RASSF1A methylation levels

Gene	Designation	Sequence (5′–3′) and labeling
MYOD1	Forward primer	GTGATAAAATATTAAATGTGTTTGGTAAGTTTA
MYOD1	Reverse primer	ATTTTTCTAAAAACTTCCTCAAAACTATCATC
MYOD1	FAM-MGB probe	ATTGTAAAGGTAATTTGATGATAG
RASSF1A	Forward primer	TGGTTTTTAGAAATACGGGTATTTTCGCGTG
RASSF1A	Reverse primer	AAAACCCGAAAACGAAACTAAACGCGCT
RASSF1A	FAM-MGB probe	CGTGGTGTTTTGCGGTCGTCGTCGT

Table S2 Primers and probes for detection of ABL1 and RASSF1A mRNA levels

Gene	Designation	Sequence (5′–3′) and labeling
ABL1	Forward primer	AGGCTGCCCAGAGAAGGTCTA
ABL1	Reverse primer	TGTTTCAAAGGCTTGGTGGAT
ABL1	FAM-MGB probe	TGGAATCCCTCTGACCGG
RASSF1A	Forward primer	CCTGCATGTGCTGTCACGCACAAGG
RASSF1A	Reverse primer	CTCATCATCCAACAGCTTCCGCAAGTACAC
RASSF1A	FAM-MGB probe	CACGTGAAGTCATTGAGGCCCTGCTG

Table S3 Primers of gene mutations for sequencing

Gene	Primer F (5′–3′)	Primer R (5′–3′)
ASXL1	TGATGCTGCCTCGAGTTGTC	TTGGTCAAACCCAGCTTCTGT
	AGAAGCTGGGTTTGACCAAAGA	GTGGCTTTTCGGTGTGAACA
	CCCCGGCTTGAAGATCGT	GTGAGTCCAACTGTAGCCCTCTGT
	GGCACCACTTCCTGGGAAA	TGCTTCAGAGTCTCCGTTGATTT
	GAGGCCACTAACCCACTTGTG	CCCTTGGCCTGTAACATTGC
DNTM3A	GAAGACCCCTGGAACTGCTACA	GAAGTAGCGGGCCCTGTGT
	TCTACCGCCTCCTGCATGAT	TGGGTGCTGATACTTCTCTCCAT
FLT3	TCCTGTTTCTCGGATGGATACC	TGGGTCATCATCTTGAGTTCTGA
NRAS	GCCGCATGACTCGTGGTT	GGCGTATTTCTCTTACCAGTGTGTAA
SF3B1	TCAACACTTAGTCCAGAAGAGCAAA	ACTTTCTGCTGCTCATCCACAA
	GCCATCTTGCCACATCTTAGAAGT	CCATCAATCAGTTGTTCTTCAAGTTT
	TCTTCCTCCCTTTTTTAAACACTTCT	GTGGAGTCATCTTATGCATACCTATGA
SRSF2	CGACGCTGAGGACGCTATG	AAGACCTACCCCAAATCCCATT
TET2	ATCACTCACCCATCGCATACC	TCATTGTCCCTGCAGTCTGTATGT
	ATGACATACAGACTGCAGGGACAA	TGTTGCAAAAGGTGTGAGTTTGA
	CCAGTGTTGAAACAGCACTTGAA	GGCACAGGAAAAACATTTGCA
KIT	TACATGGACATGAAACCTGGAGTT	AATGGTCTACCACGGGCTTCT
NPM1	AGGAGGAGGATGTGAAACTCTTAAGTAT	AACACGGTAGGGAAAGTTCTCACT
CEBPA	AAGAAGTCGGTGGACAAGAACAG	GCAGGCGGTCATTGTCACT
RUNX1	GTCATTTCCTTCGTACCCACAGT	GTGTGGGCTGACCCTCATG
U2AF1	TGTGGAGATGCAGGAACACT	AATGGCATGGCTCAGAATCG
IDH1	CCAAGTCACCAAGGATGCTG	CCATGTCGTCGATGAGCCTA