Role of matrix metalloproteinase MMP-2, MMP-9 and tissue inhibitor of metalloproteinase (TIMP-1) in the clinical progression of pediatric acute lymphoblastic leukemia

Figures & data

Figure 1. Detection of Metrix metalloproteinase (MMP) in pediatric acute lymphoblastic leukemia case. (A): Forwards/side gating on blat population. (B): Fluorescein isothiocyanate (FITC) Isotype control, (C): phycoerythrin (PE) isotype control, (D): MMP-9, (E): Tissue inhibitor of metalloproteinases 1 (TIMP-1) and (F): MMP-2. (G): represent the stem cells gating on the blast population. (H): select the CD34+CD38− population gated on the whole blast population R1.

Table 1. Clinical characteristics of ALL patients.

Patients’ characteristics		Frequency (%)
TLC (median & range) × 10^9/l		22.5 (0.8–325)
BM blast at diagnosis (median & range)		87 (48–98)%
BMA- day 15 (median & range)		0 (0–77%)%
Age	mean ± SD	8.68 ± 5.4 years
	median (range)	6.5 (1–18) years
Gender	male
	female	29 (38.2%)
Imunophenotype	T ALL	6 (7.9%)
	preB ALL	49 (64.5%)
	Common B	21 (27.6%)
Risk stratification	LR	26 (34.2%)
	SR	19 (25.0%)
	HR	31 (40.8%)
MRD-day 15	<0.01	22 (30.1%)
	≥0.01	51 (69.9%)
MRD-day 42	<0.01	31 (57.4%)
	≥0.01	23 (42.6%)
CR	no	9 (11.8%)
	yes	67 (88.2%)
DNA index	standard	66 (86.8%)
	favorable	10 (13.2%)
CD34	negative	40 (52.6)
	positive	36 (47.4%)
lymphadenopathy	negative	39 (51.3%)
	positive	37 (48.7%)
Testicular	negative	68 (89.5%)
	positive	8 (10.5%)
CNS	negative	72 (94.7%)
	positive	4 (5.3%)
Hepatomegaly	negative	23 (30.3%)
	positive	53 (69.7%)
Splenomegaly	negative	15 (19.7%)
	positive	61 (80.3%)
Relapse	negative	59 (77.6%)
	positive	17 (22.4%)
Death	negative	56 (73.7%)
	positive	20 (26.3%)

Figure 2. Expression levels of (A) TIMP-1, (B) MMP-2, (C) MMP-9 and (D) CSCs CD38+CD34− in ALL patients compared to control subjects.

Figure 3. ROC analysis for diagnosis of ALL patients using (A) TIMP-1, MMP-2, MMP-9 and (B) CSCs CD34⁺CD38⁻. (C) association between MMP-9/TIMP-1 ratio and complete remission of pediatric ALL patients. (D) Correlation among MMP-2, MMP-9, TIMP-1 and CD34+CD38− cells expressions in the control group. (E) Correlation among MMP-2, MMP-9, TIMP-1 and CD34+CD38− cells expressions in pediatric ALL patients.

Table 2. ROC curve analysis of TIMP1, MMP2, MMP9 and CD34+CD38− stem cells for identification of ALL patients.

Variable(s)	Area	Cutoff	Sensitivity	Specificity	P-value	95% Confidence Interval
						Lower	Upper
MMP9	0.660	1.07	53.9%	40%	0.053	0.511	0.808
MMP2	0.568	0.841	80.3%	53.3%	0.404	0.414	0.723
TIMP1	0.892	1.133	78.9%	100%	P < 0.001	0.827	0.957
CD34+ CD38− CSCs	0.855	0.55	78.9%	73.3%	P < 0.001	0.759	0.951

Table 3. Association between MMP9, MMP2 and clinical features of the patients.

	MMP9		P-value	MMP2		P-value
	Low-expression	Overexpression		Low-expression	Overexpression
Age (years)	6.5 (1–18)	6 (1–17)	0.584	8 (1–14)	6 (1–18)	0.911
TLC (×10^9/l)	15.5 (2.8–325)	27 (0.8–283)	0.125	18 (3–114)	25 (0.8–325)	0.928
BM blast at diagnosis	85 (63-93)%	87 (60–98)%	0.156	82.5 (63–98)%	87 (60–96)%	0.047
BM blast-day-15	0 (0–40)%	0 (0–77)%	0.739	0 (0–4)%	1 (0–77)%	0.001
Gender
male	21 (61.8%)	34 (59.6%)	0.842	13 (52.0%)	42 (63.6%)	0.344
female	13 (38.2%)	23 (40.4%)		12 (48.0%)	24 (36.4%)
Phenotype
T-ALL	4 (15.4%)	2 (4.0%)	0.091	3 (15.8%)	3 (5.3%)	0.188
Pre B-ALL	13 (50.0%)	36 (72.0%)		13 (68.4%)	36 (63.2%)
Common B	9 (34.6%)	12 (24.0%)		3 (15.8%)	18 (31.6%)
Risk stratfication
LR	11 (42.3%)	15 (30.0%)	0.326	7 (36.8%)	19 (33.3%)	0.921
SR	4 (15.4%)	15 (30.0%)		5 (26.3%)	14 (24.6%)
HR	11 (42.3%)	20 (40.0%)		7 (36.8%)	24 (42.1%)
MRD-day 15
<0.01	8 (30.8%)	14 (29.8%)	0.930	10 (52.6%)	12 (22.2%)	0.020
≥0.01	18 (69.2%)	33 (70.2%)		9 (47.4%)	42 (77.8%)
MRD-day 42
<0.01	12 (66.7%)	19 (52.8%)	0.392	9 (50.0%)	22 (61.1%)	0.561
≥0.01	6 (33.3%)	17 (47.2%)		9 (50.0%)	14 (38.9%)
CR
no	3 (11.5%)	6 (12.0%)	0.953	0 (0.0%)	9 (15.8%)
yes	23 (88.5%)	44 (88.0%)		19 (100.0%)	48 (84.2%)	0.102
CD34
negative	12 (46.2%)	28 (56.0%)	0.473	11 (57.9%)	29 (50.9%)	0.791
positive	14 (53.8%)	22 (44.0%)		8 (42.1%)	28 (49.1%)
Testicular
negative	24 (92.3%)	44 (88.0%)	0.708	19 (100.0%)	49 (86.0%)	0.189
positive	2 (7.7%)	6 (12.0%)		0 (0.0%)	8 (14.0%)
CNS
negative	23 (88.5%)	49 (98.0%)	0.113	18 (94.7%)	54 (94.7%)	1.000
positive	3 (11.5%)	1 (2.0%)		1 (5.3%)	3 (5.3%)
Hepatomegaly
negative	9 (34.6%)	14 (28.0%)	0.604	8 (42.1%)	15 (26.3%)	0.251
positive	17 (65.4%)	36 (72.0%)		11 (57.9%)	42 (73.7%)
Splenomegaly
negative	4 (15.4%)	11 (22.0%)	0.559	2 (10.5%)	13 (22.8%)	0.330
positive	22 (84.6%)	39 (78.0%)		17 (89.5%)	44 (77.2%)
Lymphadenopathy
negative	11 (42.3%)	28 (56.0%)	0.335	10 (52.6%)	29 (50.9%)	0.895
positive	15 (57.7%)	22 (44.0%)		9 (47.4%)	28 (49.1%)
DNA index
standard	23 (88.5%)	43 (86.0%)	1.000	18 (94.7%)	48 (84.2%)	0.436
favorable	3 (11.5%)	7 (14.0%)		1 (5.3%)	9 (15.8%)

Table 4. Association between TIMP1, CD34+ CD38− and clinical features of the patients.

	TIMP-1		P-value	CD34^+CD38^- CSCs		P-value
	Low-expression	Overexpression		Low-expression	Overexpression
Age (years)	4 (1–18)	7(1–17)	0.217	6 (1–18)	6 (1–17)	0.243
TLC (×10^9/l)	13.1 (4–93.7)	26.4 (0.8–325)	0.076	16.7 (3–93.7)	25.7 (0.8–325)	0.579
BM blast at diagnosis	81 (72–98)%	87 (60–96)%	0.033	78 (63–98)%	87 (60–96)%	0.005
BM blast-day15	0 (0–2)%	1 (0–77)%	0.001	0 (0–9)%	1 (0–77)%	0.003
Gender
male	10 (62.5%)	37 (61.7%)	0.951	11 (40.7%)	44 (68.8%)	0.01
female	6 (37.5%)	23 (38.3%)		16 (59.3%)	20 (31.3%)
Phenotype
T_ALL	1 (6.3%)	5 (8.3%)	0.915	1 (6.3%)	5 (8.3%)	0.268
preB_ALL	11 (68.8%)	38 (63.3%)		13 (81.3%)	36 (60.0%)
commonB	4 (25.0%)	17 (28.3%)		2 (12.5%)	19 (31.7%)
Risk stratification
LR	13 (81.3%)	13 (21.7%)	<0.001	9 (56.3%)	17 (28.3%)	0.109
SR	2 (12.5%)	17 (28.3%)		3 (18.8%)	16 (26.7%)
HR	1 (6.3%)	30 (50.0%)		4 (25.0%)	27 (45.0%)
MRD day 15
<0.01	12 (75.0%)	10 (17.5%)	<0.001	9 (56.3%)	13 (22.8%)	0.015
≥0.01	4 (25.0%)	47 (82.5%)		7 (43.8%)	44 (77.2%)
MRD day 42
<0.01	13 (100%)	18 (43.9%)	<0.001	7 (58.3%)	24 (57.1%)	0.941
≥0.01	0 (0.0%)	23 (56.1%)		5 (41.7%)	18 (42.9%)
CR
no	0 (0.0%)	9 (15.0%)	0.191	1 (6.3%)	8 (13.3%)	0.675
yes	16 (100%)	51 (85.0%)		15 (93.8%)	52 (86.7%)
Testicular
negative	16 (100%)	52 (86.7%)	0.191	16 (100%)	52 (86.7%)	0.191
positive	0 (0.0%)	8 (13.3%)		0 (0.0%)	8 (13.3%)
CNS
negative	15 (93.8%)	57 (95.0%)	0.842	15 (93.8%)	57 (95.0%)	0.842
positive	1 (6.3%)	3 (5.0%)		1 (6.3%)	3 (5.0%)
Hepatomegaly
negative	7 (43.8%)	16 (26.7%)	0.226	5 (31.3%)	18 (30.0%)	0.923
positive	9 (56.3%)	44 (73.3%)		11 (68.8%)	42 (70.0%)
Splenomegaly
negative	5 (31.3%)	10 (16.7%)	0.286	3 (18.8%)	12 (20.0%)	0.911
positive	11 (68.8%)	50 (83.3%)		13 (81.3%)	48 (80.0%)
Lymphadenopathy
negative	5 (31.3%)	34 (56.7%)	0.094	6 (37.5%)	33 (55.0%)	0.266
positive	11 (68.8%)	26 (43.3%)		10 (62.5%)	27 (45.0%)
DNA index
standard	14 (87.5%)	52 (86.7%)	0.930	15 (93.8%)	51 (85.0%)	0.678
favorable	2 (12.5%)	8 (13.3%)		1 (6.3%)	9 (15.0%)

Figure 4. Risk stratification of the patients according to TIMP-1 expression.

Figure 5. Analysis of disease-free survival and overall survival rates of the ALL patients.

Table 5. Multivariate logistic regression analysis for the incidence of ALL.

	Univariate logistic regression			Multivariate logistic regression
	OR	95% CI	Sig.	OR	95% CI	Sig.
MMP2	2.000	.629	6.355	0.240
MMP9	2.198	.717	6.733	0.168
TIMP-1	8.625	2.403	30.961	0.001	4.224	1.024	17.422	0.046
CD34^+CD38^− CSCs	10.312	2.895	36.733	<0.001	6.873	1.783	26.500	0.005