Clinical Features of the Patients
The current study included 76 patients, of them 47 (61.8%) were males and 29 (38.2%) were females, with a median age of 6.5 (range: 1-18) years. The median BM blast at diagnosis was 87%, with a range of 48% to 98%, while it was 0 (range: 0-77%) at day 15 of treatment. The IPT analysis showed that 49 (64.5%) patients had (Pre-B) phenotype, 21 (27.6%) had common B, and 6 (7.9%) patients had T-ALL phenotype. Patients were classified according to risk stratification of ALL  into low risk (LR) patients in 26 (34.2%), standard risk (SR) in 19 (25.0%) patients, and high risk (HR) in 31 (40.8%) patients. Regarding MRD at day 15, there were 22 (30.1%) patients had MRD<0.01, and 51 (69.9%) patients had MRD ≥0.01. While for MRD at day 42, there were 31 (57.4%) patients had MRD<0.01, and 23 (42.6%) patients had MRD ≥0.01. Complete remission was achieved in 67 (88.2%) patients, and DNA index was favorable (>1) in 10/76 (13.2%) patients. There were 53 (69.7%) patients presented with hepatomegaly, 61 (80.3%) had splenomegaly, 37 (48.7%) had lymphadenopathy, 8 (10.5%) patients had testicular affection, and 4 (5.3%) patients had central nervous system (CNS) involvement. (Table 1).
Expression levels of TIMP-1, MMP-2, MMP-9 and CSCs CD38+CD34- in ALL patients
There was a significant increase in TIMP-1 expression in ALL patients compared to control subjects [1.52 (0.41-10) versus 0.91(0.6-1.12); respectively, P<0.001], while there were no significant differences between ALL patients and control group regarding the expression levels of MMP-2 [1.12 (0.21-3.5) versus 1.08 (0.42-2.04); respectively, P= 0.404], and MMP-9 [1.12 (0.38-4.07) versus 0.84 (0.57-1.48); respectively, P=0.053]. on the other hand, there was a significant increase in CSCs CD84+CD38- in ALL patients [1 (0.03-18.6)] compared to control group [0.3 (0.01-1.1), P<0.001, Figure 2].
Identification of ALL patients using TIMP-1, MMP-2, MMP-9 and CSCs CD34+CD38-
ROC curve analysis was performed to evaluate the diagnostic power of the studied markers to detect ALL patients. The sensitivity, specificity, AUC of MMP-2 were (80.3%, 53.3% and 0.568; respectively) at a cutoff value of 0.841 (P=0.404), and that of MMP-9 were (53.9%, 40% and 0.660) at a cutoff value of 1.07 (P=0.053). While the sensitivity, specificity, AUC of TIMP-1 were (78.9%, 100% and 0.892; respectively) at a cutoff value of 1.133 (P<0.001), and that of CSCs CD34+ CD38- were (78.9%, 73.3% and 0.855; respectively) at a cutoff value of 0.55 (P<0.001, Figure 3A, B). Patients were classified into low-expression and overexpression of the assessed markers according to the appropriate cutoff values obtained by the ROC curve (Table 2).
Association between MMP-2, MMP-9 expression and the clinical features of the patients
Patients with MMP-2 overexpression showed a significant increase in the BM blast cell count at diagnosis and at day-15 of treatment, as well as a significant increase in MRD at day-15, compared to those with MMP-2 low expression (P=0.047, P=0.001 and P=0.020; respectively). There was no significant association between MMP-9 expression levels and the assessed relevant clinic-pathological features of the patients (Table 3).
On the other hand, increased TIMP-1 expression associated significantly with increased BM blast cell count at diagnosis and at day-15 of treatment (P=0.033 and P=0.001; respectively). Also, TIMP-1 associated significantly with the high-risk stratification of the patients, as out of 60 patients who had TIMP-1 overexpression, 30 (50.0%) patients had high-risk disease, 17 (28.3%) had standard risk, and 13 (21.7%) patients had low-risk disease (P<0.001). There was a significant association between increased TIMP-1 expression and increased MRD at day 15 and day 42 (P<0.001 for both). Regarding the MMP-9/TIMP-1 ratio, it was observed that all assessed ALL patients who did not achieve complete remission (CR), showed MMP-9/TIMP-1 lower than 0.96, however this association is of a borderline significance (P=0.05, Figure 3C).
Regarding CD34+CD38- CSCs, it associated significantly with increased BM blast cell count at diagnosis and at day-15 of treatment (P=0.005 and P=0.003; respectively). Also, it associated significantly with increased MRD at day-15 (P=0.015, Table 4).
Correlation among MMP-2, MMP-9, TIMP-1 and CD34+CD38- cells expressions
The present results showed that there was no significant correlation among MMP-2, MMP-9, TIMP-1and CD34+CD38- cells expressions under normal condition in control subjects (Figure 3D). However, in ALL pediatric patients, there was a significant correlation between TIMP-1 expression and MMP-2 (r=0.325, P=0.004), as well as MMP-9 (r=0.326, P=0.004). Similarly, CD34+CD38- cells correlated significantly with MMP-2 (r=0.736, P<0.001), MMP-9 (r=0.379, P=0.001), and TIMP-1 (r=0.567, P<0.001, Figure 3E).
Disease-free survival and overall survival rates of ALL patients
There was a significant association between TIMP-1 overexpression and shorter DFS and OS rates of ALL patients (P=0.009 and P=0.048; respectively). however, no significant association detected between the expression of MMP-2, MMP-9, CD34+CD38- cells and survival (DFS, OS) rates of the patients (P>0.05, Figure 4).
Univariate and Multivariate logistic regression analysis for diagnosis of ALL
Univariate analysis revealed that the incidence of pediatric ALL associated significantly with increased expression of TIMP-1 [OR: 8.625, P=0.001], and CD34+CD38- CSCs [OR: 10.312, P<0.001], while there was no significant association with MMP-2 and MMP-9 expression levels. Multivariate logistic regression analysis showed that both TIMP-1 [OR: 4.224, P=0.046], and CD34+CD38- CSCs [OR: 6.873, P=0.005] are independent diagnostic factors for pediatric ALL (Table 5).