Deregulation of CircANXA2, Circ0075001, and CircFBXW7 Gene Expressions and Their Predictive Value in Egyptian Acute Myeloid Leukemia Patients

Figures & data

Figure 1 (A) Relative quantification of gene expression of CircANXA2 (B) relative quantification of gene expression of Circ0075001. (C) Relative quantification of gene expression of CircFBXW7. (D) Melting curve analysis for CircANXA2 expression. (E) Melting curve analysis for Circ0075001 expression. (F) Melting curve analysis for CircFBXW7 expression.

Table 1 Comparison Between the Two Studied Groups According to Different Parameters

	AML (n = 66)		Control (n = 54)		Test of Sig.	p
	No.	%	No.	%
Sex
Male	37	56.1	29	53.7	χ^Citation2=0.067	0.796
Female	29	43.9	25	46.3
Age (years)
Min. – Max.	19.0–75.0		17.0–69.0		t=1.242	0.217
Mean ± SD.	51.05 ± 15.30		47.80 ± 12.88
Median (IQR)	51.50 (40.0–65.0)		49.0(38.0–60.0)
TLC x 10^3
Min. – Max.	0.80–23.0		4.20–11.0		U=1710.50	0.706
Mean ± SD.	8.92 ± 6.57		7.06 ± 2.18
Median (IQR)	7.25(2.70–15.0)		6.25 (5.30–9.50)
Hb g/dl
Min. – Max.	3.0–11.80		11.0–15.10		t=18.782	<0.001*
Mean ± SD.	8.35 ± 1.85		13.38 ± 1.04
Median (IQR)	8.55 (7.0–9.90)		13.50 (12.50–14.0)
PLT x 10^3
Min. – Max.	10.0–150.0		154.0–354.0		U=0.0	<0.001*
Mean ± SD.	55.17 ± 29.32		253.22 ± 50.19
Median (IQR)	55.0 (30.0–72.0)		266.0 (205.0–300.0)
CircANXA2
Min. – Max.	0.02–596.66		0.11–4.37		U=628.0	<0.001*
Mean ± SD.	87.17 ± 148.47		1.37 ± 1.13
Median (IQR)	12.97 (1.80–89.88)		1.0 (0.39–1.71)
Circ0075001
Min. – Max.	0.02–825.85		0.08–2.50		U=517.50	<0.001*
Mean ± SD.	72.20 ± 174.93		0.84 ± 0.53
Median (IQR)	3.01 (1.70–12.54)		0.99 (0.57–1.0)
CircFBXW7
Min. – Max.	0.01–2.50		0.17–4.90		U= 619.50	<0.001*
Mean ± SD.	0.81 ± 0.76		2.41 ± 1.51
Median (IQR)	0.60 (0.08–1.20)		1.80 (1.0–4.20)

Notes: p, p-value for comparing between the studied groups; *statistically significant at p ≤ 0.05.

Abbreviations: IQR, interquartile range; SD, standard deviation; χ², Chi-square test; t, Student’s t-test; U, Mann Whitney test.

Table 2 Distribution of the Studied Cases According to Clinical Data in AML Group (n = 66)

	No.	%
Splenomegaly	26	39.4
Hepatomegaly	25	37.9
LN	4	6.1
CNS involvement	2	3.0
B.M BLASTS
Min. – Max.	20.0–90.0
Mean ± SD.	42.71 ± 23.40
Median (IQR)	30.0 (30.0–60.0)
FAB
M1	8	12.1
M2	13	19.7
M3	11	16.7
M4	11	16.7
M5	11	16.7
M6	5	7.6
M7	7	10.6
Overall survival (months)
Min. – Max.	1.0–12.0
Mean ± SD.	7.95 ± 4.32
Median (IQR)	9.50 (3.0–12.0)
Outcome
Died	29	43.9
Alive	31	47.0
Lost during follow up	6	9.1
Cytogenetic criteria
Favorable	33	50.0
Intermediate	16	24.2
Adverse	17	25.8
CR achievement	31	47.0
ELN CLASS
Favorable cytogenetic
t(8:21)+ve	7	10.6
inv(16)+ve	6	9.1
PML-RARA+VE	11	16.7
Mutated NPM1 FLT3-ITD-/low	9	13.6
Intermediate cytogenetic
t (9:11)	6	9.1
Mutated NPM1 FLT3-ITD high	10	15.2
Adverse cytogenetic
Inv (3) +ve	3	4.5
Wild NPM1 FLT3-ITD high	14	21.2

Abbreviations: IQR, interquartile range; SD, standard deviation.

Table 3 Validity (AUC, Sensitivity, Specificity) for CircANXA2, Circ0075001 and CircFBXW7to Discriminate AML (n = 66) from Control (n = 54)

	AUC	p	95% C. I	Cut off	Sensitivity	Specificity	PPV	NPV
CircANXA2	0.824	<0.001*	0.746–0.901	>1.87	74.24	88.89	89.1	73.8
Circ0075001	0.855	<0.001*	0.778–0.932	>1.16	83.33	79.63	83.3	79.6
CircFBXW7	0.826	<0.001*	0.755–0.898	≤1.2	75.76	74.07	78.1	71.4

Abbreviations: AUC, area under a curve; p value, probability value; CI, confidence intervals; NPV, negative predictive value; PPV, positive predictive value; *, statistically significant at p ≤ 0.05.

Figure 2 (A) ROC curve for CircANXA2, Circ0075001, and CircFBXW7 to discriminate AML from control. (B) Relation between high and low CircANAX2 expression with FAB classification in AML patients. (C) Relation between high and low CircANAX2 expression with Cytogenetic criteria in AML patients. (D) Relation between high and low CircANAX2 expression with Overall survival in AML patients. (E) Relation between high and low Circ0075001expression with FAB classification in AML. (F) Relation between high and low Circ0075001 expression with Cytogenetic criteria in AML. *Statistically significant at p ≤ 0.05.

Table 4 Relation Between High and Low Expression of CircANAX2 Expression with Clinical Data in AML Patients (N = 66)

	CircANAX2 Expression				Test of Sig.	p
	Low Median (<12.97) (n=33)		High Median (>12.97) (n=33)
	No.	%	No.	%
Sex
Male	18	54.5	19	57.6	χ^Citation2=0.062	0.804
Female	15	45.5	14	42.4
Age (years)
Min. – Max.	19.0–75.0		22.0–75.0		t=1.039	0.303
Mean ± SD.	49.09 ± 15.16		53.0 ± 15.42
Median (IQR)	50.0		56.0
Splenomegaly	8	24.2	18	54.5	χ^Citation2=6.346	0.012*
Hepatomegaly	8	24.2	17	51.5	χ^Citation2=5.216	0.022*
Lymphadenopathy	2	6.1	2	6.1	χ^Citation2=0.0	^FEp=1.000
CNS involvement	0	0.0	2	6.1	χ^Citation2=2.063	^FEp=0.492
B.M BLASTS
Min. – Max.	20.0–90.0		20.0–90.0		U=461.0	0.278
Mean ± SD.	40.52 ± 23.24		44.91 ± 23.70
Median	30.0		33.0
FAB
M1	3	9.1	5	15.2	χ^Citation2=23.474	^MCp=0.001*
M2	8	24.2	5	15.2
M3	11	33.3	0	0.0
M4	6	18.2	5	15.2
M5	3	9.1	8	24.2
M6	2	6.1	3	9.1
M7	0	0.0	7	21.2
Overall survival (months)
Min. – Max.	1.0–12.0		1.0–12.0		U=261.50	<0.001*
Mean ± SD.	10.03 ± 3.45		5.88 ± 4.13
Median	12.0		4.0
Outcome
Died	7	21.2	22	66.7	χ^Citation2=15.265	^MCp<0.001*
Alive	23	69.7	8	24.2
Lost FU	3	9.1	3	9.1
Cytogenetic criteria
Favorable	24	72.7	9	27.3	χ^Citation2=17.0	<0.001*
Intermediate	7	21.2	9	27.3
Adverse	2	6.1	15	45.4
CR achievement	23	69.7	8	24.2	χ^Citation2=13.687	<0.001*
TLC x 10^3
Min. – Max.	0.80–23.0		1.10–21.0		U=538.50	0.939
Mean ± SD.	8.88 ± 6.60		8.95 ± 6.65
Median	7.50		7.0
Hb gm/dl
Min. – Max.	3.0–11.80		4.90–11.30		t=0.411	0.682
Mean ± SD.	8.45 ± 1.86		8.26 ± 1.86
Median	8.30		8.80
PLT x 10^3
Min. – Max.	17.0–120.0		10.0–150.0		U=521.0	0.763
Mean ± SD.	55.61 ± 25.84		54.73 ± 32.83
Median	55.0		56.0

Notes: p, p-value for comparing between different parameters *statistically significant at p ≤ 0.05.

Abbreviations: χ², chi-square test; MC, Monte Carlo; FE, Fisher Exact; t, Student’s t-test, U; Mann Whitney test.

Table 5 Relation Between Low and High Expression of Circ0075001expression with Clinical Data in AML Patients (N = 66)

	Circ0075001 Expression				Test of Sig.	p
	Low Median (<3.01) (n=33)		High Median (>3.01) (n=33)
	No.	%	No.	%
Sex
Male	21	63.3	16	48.5	χ^Citation2=1.538	0.215
Female	12	36.4	17	51.5
Age (years)
Min. – Max.	24.0–75.0		19.0–75.0		t=1.236	0.221
Mean ± SD.	48.73 ± 13.17		53.36 ± 17.06
Median	49.0		58.0
Splenomegaly	5	15.2	21	63.6	χ^Citation2=16.246	<0.001*
Hepatomegaly	5	15.2	20	60.6	χ^Citation2=14.488	<0.001*
Lymphadenopathy	1	3.0	3	9.1	χ^Citation2=1.065	^FEp=0.613
CNS involvement	0	0.0	2	6.1	χ^Citation2=2.063	^FEp=0.492
B.M BLASTS
Min. – Max.	20.0–90.0		20.0–90.0		U=522.50	0.775
Mean ± SD.	44.88 ± 24.77		40.55 ± 22.11
Median (IQR)	32.0		30.0
FAB
M1	3	9.1	5	15.2	χ^Citation2=22.144	^MCp=0.001*
M2	12	36.4	1	3.0
M3	8	24.2	3	9.1
M4	6	18.2	5	15.2
M5	2	6.1	9	27.3
M6	1	3.0	4	12.1
M7	1	3.0	6	18.2
Overall survival (months)
Min. – Max.	1.0–12.0		1.0–12.0		U=273.0	<0.001*
Mean ± SD.	10.0 ± 3.44		5.91 ± 4.17
Median	12.0		4.0
Outcome
Died	10	30.3	19	57.6	χ^Citation2=10.693	^MCp=0.004*
Alive	22	66.7	9	27.3
Lost FU	1	3.0	5	15.2
Cytogenetic criteria
Favorable	23	69.6	10	30.4	χ^Citation2=10.25	P=0.005*
Intermediate	5	15.2	11	33.3
Adverse	5	15.2	12	33.3
CR achievement	23	69.7	8	24.2	χ^Citation2=13.687	<0.001*
TLC x 10^3
Min. – Max.	0.80–23.0		1.10–21.0		U=519.50	0.748
Mean ± SD.	9.32 ± 6.93		8.51 ± 6.28
Median	7.50		6.20
Hb gm/dl
Min. – Max.	5.0–11.80		3.0–11.30		t=2.197	0.032*
Mean ± SD.	8.84 ± 1.54		7.87 ± 2.02
Median	9.0		7.50
PLT x 10^3
Min. – Max.	17.0–150.0		10.0–100.0		U=472.0	0.352
Mean ± SD.	58.97 ± 30.77		51.36 ± 27.74
Median	55.0		50.0

Notes: p, p-value for comparing between different parameters *statistically significant at p ≤ 0.05.

Abbreviations: χ², chi-square test; MC, Monte Carlo; FE, Fisher Exact t: Student’s t-test; U, Mann Whitney test.

Table 6 Relation Between Low and High Expression of circFBXW7 Expression with Clinical Data in AML Patients (N = 66)

	circFBXW7 Expression				Test of Sig.	p
	Low Median (<0.60) (n=33)		High Median (>0.60) (n=33)
	No.	%	No.	%
Sex
Male	21	63.3	16	48.5	χ^Citation2=1.538	0.215
Female	12	36.4	17	51.5
Age (years)
Min. – Max.	19.0–75.0		22.0–72.0		t=0.973	0.334
Mean ± SD.	52.88 ± 15.60		49.21 ± 15.0
Median (IQR)	54.0		50.0
Splenomegaly	14	42.4	12	36.4	χ^Citation2=0.254	0.614
Hepatomegaly	14	42.4	11	33.3	χ^Citation2=0.580	0.447
Lymphadenopathy	4	12.1	0	0.0	χ^Citation2=4.258	^FEp=0.114
CNS involvement	2	6.1	0	0.0	χ^Citation2=2.063	^FEp=0.492
B.M BLASTS
Min. – Max.	20.0–90.0		20.0–90.0		U=475.0	0.366
Mean ± SD.	39.09 ± 20.74		46.33 ± 25.59
Median	30.0		30.0
FAB
M1	3	9.1	5	15.2	χ^Citation2=2.844	^MCp=0.856
M2	6	18.2	7	21.2
M3	5	15.2	6	18.2
M4	6	18.2	5	15.2
M5	6	18.2	5	15.2
M6	4	12.1	1	3.0
M7	3	9.1	4	12.1
Overall survival (months)
Min. – Max.	1.0–12.0		1.0–12.0		U=485.0	0.419
Mean ± SD.	8.45 ± 4.28		7.45 ± 4.36
Median (IQR)	12.0		7.0
Outcome
Died	14	42.4	15	45.5	χ^Citation2=0.999	^MCp=0.672
Alive	17	51.5	14	42.4
Lost FU	2	6.1	4	12.1
Cytogenetic criteria
Favorable	17	51.5	16	48.5	χ^Citation2=1.55	0.458
Intermediate	6	18.2	10	30.3
Adverse	10	30.3	7	21.2
CR achievement	15	45.5	16	48.5	χ^Citation2=0.061	0.805
TLC x 10^3
Min. – Max.	0.80–20.10		1.20–23.0		U=539.0	0.944
Mean ± SD.	9.18 ± 6.83		8.65 ± 6.40
Median	10.0		6.70
Hb gm/dl
Min. – Max.	4.90–11.80		3.0–11.30		t=0.624	0.535
Mean ± SD.	8.49 ± 1.63		8.21 ± 2.05
Median	8.30		8.80
PLT x 10^3
Min. – Max.	10.0–120.0		10.0–150.0		U=446.0	0.206
Mean ± SD.	50.24 ± 25.42		60.09 ± 32.40
Median	50.0		66.0

Abbreviations: χ², chi-square test; MC, Monte Carlo; FE, Fisher Exact; t, Student’s t-test; U, Mann Whitney test p, p-value for comparing between different parameters.

Table 7 Univariate and Multivariate Logistic Regression Analysis for the Parameters Affecting CR Achievement (31 Vs 35)

	Univariate		^#Multivariate
	p	OR (95%C.I)	p	OR (95%C.I)
Female	0.851	1.098(0.415–2.908)
Age (years)	0.006*	0.950(0.916–0.986)	0.297	0.973 (0.924–1.024)
TLC	0.590	0.980(0.909–1.055)
Hb	0.011*	1.490(1.095–2.028)	0.095	1.469 (0.936–2.306)
PLT	0.064	1.017(0.999–1.036)
CircANXA2 (Low <12.97)	<0.001*	7.187 (2.420–21.347)	0.024*	5.470 (1.250–23.947)
Circ0075001 (Low <3.01)	<0.001*	7.187 (2.420–21.347)	0.537	1.629 (0.346–7.660)
CircFBXW7 (High >0.60)	0.805	1.129 (0.429–2.971)

Notes: ^#All variables with p<0.05 was included in the multivariate. *Statistically significant at p ≤ 0.05.

Abbreviations: OR, odds ratio; C.I, confidence interval; LL, lower limit; UL, upper limit.

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