Effects of Surgery Combined with Different Chemotherapy on Matrix Metalloproteinase-9 and Tissue Inhibitors of Metalloproteinase-1 in Children with Neuroblastoma

Background Neuroblastoma (NB) is a common extracranial malignancy in children and accounts for 15% of all cancer-related deaths in children, with the 5-year survival of patients in an advanced stage being lower than 40%. Preoperative adjuvant chemotherapy has been reported to facilitate surgical resection and improve the 2-year survival of patients. Objective To analyze the efficacy of surgery plus different chemotherapy on children with NB and to investigate the correlation of matrix metalloproteinase-9 (MMP-9) and tissue inhibitors of metalloproteinase-1 (TIMP-1) with chemotherapy efficacy. Methods From April 2005 to May 2017, a total of 92 cases of NB treated in our hospital were assessed for eligibility and recruited. They were assigned at a ratio of 1: 1 to receive either CAV (cyclophosphamide + vincristine + adriamycin) (group A) and EP (etoposide + cisplatin) alternately or TOPO (topotecan) + CTX (cytoxan) + CiE (etoposide + cisplatin) + CPV (cyclophosphamide + pirarubicin + vincristine) (group B). The outcome measures include chemotherapy efficacy, surgical resection rates, complications, 2-year recurrence, and 2-year survival. The levels of NK cells, CD4+/CD8+ cells, MMP-9, TIMP-1, and urine catecholamine (VMA) in peripheral blood of patients before and after initial chemotherapy were determined to analyze the correlation of MMP-9, TIMP-1, and VMA with the efficacy of chemotherapy. Results The two groups had similar efficacy (84.00% vs. 95.24%) and surgical resection rates (60.00% vs. 61.90%) after the initial chemotherapy (P > 0.05). Surgery for all eligible patients was successful after second chemotherapy. All eligible patients showed myelosuppression after chemotherapy, including 48 cases with stages I-II (52.17%) and 44 cases with stages III-IV (47.83%). The ratio of CD4+/CD8+ cells, MMP-9, TIMP-1, and VMA expression levels in peripheral blood of patients decreased (P < 0.05) after chemotherapy, and the ratio of CD4+/CD8+ cells was further reduced after surgery (P < 0.05), while natural killer (NK) cells levels increased (P < 0.05). However, intergroup differences were absent in the incidence of myelosuppression, CD4+/CD8+ cell ratio, NK cells, MMP-9, TIMP-1, and VMA expression levels (P > 0.05). MMP-9 and TIMP-1 were positively correlated with VMA (P < 0.05), and the expression levels of MMP-9 and TIMP-1 and VMA after chemotherapy were negatively correlated with chemotherapy efficiency (P < 0.05). Patients with high expressions of MMP-9, TIMP-1, and VMA were associated with lower 2-year survival versus those with low expressions (P < 0.05). Conclusion Surgery plus chemotherapy for children with NB yields a promising clinical efficacy and a favorable surgical resection outcome. MMP-9 and TIMP-1 may be the potential biological indicators for chemotherapy efficiency and have a reference value for following surgical treatment of patients.


Introduction
Neuroblastoma (NB), also known as neurocytoma, is one of the most common extracranial malignant solid tumors in children, accounting for 6%-10% of all pediatric tumors [1,2]. It accounts for 15% of all childhood cancer-related deaths and is the embryonal tumor with the lowest 5-year survival [3]. Although treatment approaches such as chemotherapy, surgery, and stem cell transplantation achieved improved prognosis for patients, patients in the advanced stage are associated with a 5-year survival lower than 40% [4,5].
Surgery is the mainstay for the treatment of NB, and the resection of the primary lesion is of great significance for improving the therapeutic efficiency and prognosis and prolonging survival [6]. However, NB is associated with a high degree of malignancy and a big tumor size; nearly 2/3 of the patients are predisposed to multiple early metastases, with unfavorable complete surgical resection outcomes [7]. At present, controversy exists in the timing and scope of surgery for high-risk patients, which necessitates the amelioration of the treatment modalities. Previous research studies have reported that preoperative chemotherapy can reduce the primary lesion to facilitate surgical resection. Zhang et al. [8] used preoperative OPAC chemotherapy to improve surgical resection of NB with stages III and IV to 89% and 79%, respectively. Johnston et al. [9] also reported better surgical resection (60%) of NB after preoperative chemotherapy. e combination of surgery and chemotherapy in children with NB has been marginally explored, and the evaluation of preoperative chemotherapy efficiency mostly relies on the iconography observation of tumor size, which is impractical for routine analysis. Matrix metalloproteinase-9 (MMP-9) and tissue inhibitors of metalloproteinase-1 (TIMP-1) play important roles in the proliferation and metastasis of NB tumor cells [10,11].
Accordingly, this study was conducted to analyze the efficacy of surgery plus different chemotherapy on children with NB and to investigate the correlation of MMP-9 and TIMP-1 with chemotherapy efficacy.

Research Subjects.
From April 2005 to May 2017, a total of 92 NB patients aged 1-15 years admitted to our institution which includes 61 males and 31 females were assessed for eligibility and recruited. Inclusion criteria were as follows: all patients were diagnosed with NB in the International Neuroblastoma Staging System (INSS) stages III-IV by pathology or by bone marrow hemocytology with unremovable tumors at first diagnosis and all patients' conditions were evaluated by clinicians and treated with preoperative chemotherapy combined with surgical resection, with complete medical records and follow-up data. Exclusion criteria were as follows: patients with other severe diseases or tumors, with coagulation disorders, with autoimmune diseases or long-term use of immunosuppressants, and with communication disorders or cognition impairment. is study has been approved by the ethical committee of our hospital, and all guardians of the patients have signed the informed consent and cooperated with medical staff to complete relevant diagnosis and treatment.

erapies.
Of the 92 eligible patients included in this study, 50 patients receiving alternative CAV or EP regimens were in group A and 42 eligible patients receiving TOPO + CTX + CiE + CPV regimen were assigned to group B. e efficacy of chemotherapy was evaluated. In the case of effective chemotherapy results with resectable tumors after assessments, the patients were given surgery and postoperative chemotherapy was intensified. Otherwise, CV chemotherapy was continued for 2-4 courses, followed by one course of CE treatment with the dose of DDP adjusted to 6.6 mg/kg i.v. gtt d1-d3 and the dose of VP16 adjusted to 5 mg/kg i.v. gtt d1-d3 if the CV regimen was ineffective. e specific chemotherapy scheme is given in Table 1.

Analysis of Chemotherapy Efficacy.
e efficacy of chemotherapy in the two groups was observed and recorded. e efficacy of chemotherapy was divided into complete response (CR), partial response (PR), stable disease (SD), and progressive disease (PD). CR refers to all found lesions that were disappeared and the condition lasts for more than 4 weeks. PR refers to the sum of the largest single diameter of the tumor that was reduced by more than 30% and the condition lasts for more than 4 weeks. SD refers to the sum of the largest single diameter of the tumor that did not decrease by more than 30% or the increase did not exceed 20%, and the condition lasts 4 weeks. PD refers to the sum of the largest single diameter of the tumor increased by more than 20% or new lesions appeared.

Outcome Measures.
e range of surgery, complications of initial chemotherapy, 2-year recurrence, and 2-year survival of the patients were recorded. e levels of NK cells, CD4+/CD8+ cells, MMP-9, TIMP-1, and urine vanillylmandelic acid (VMA) in peripheral blood of patients before and after initial chemotherapy were determined to analyze the correlation of MMP-9, TIMP-1, and VMA with the efficacy of chemotherapy.

Detection
Methods. Enzyme-linked immunosorbent assay (ELISA) was used to determine the expression levels of MMP-9 and TIMP-1 in peripheral blood of patients, and the specific steps were following the kit instruction. MMP-9 and TIMP-1 assay kits were purchased from Abcam in the United States, with article no. of Ab100610 and ab187394, respectively. Urine VMA levels in both groups were determined by a high-performance liquid chromatography-mass spectrometer (LC-MS, ermoFisher). Backman CytoFLEX flow cytometry (Beckman Coulter) was used to determine the levels of NK cells and CD4+/CD8+ cells in peripheral blood. Urine samples were random urine in 24 hours, and peripheral blood samples were morning fasting blood.

Statistical
Analysis. SPSS 19.0 (Chicago, IL, the United States) was applied for statistical analysis. e distribution of outcomes was evaluated and presented as mean (SD) for continuous outcomes and frequency (%) for categorical outcomes. Normality was evaluated by the Kolmogorov-Smirnov test. If data were not normally distributed, nonparametric corollary for continuous outcomes and the χ 2 test for categorical outcomes were used. Group comparisons of baseline participant characteristics were made using a ttest or nonparametric corollary for continuous outcomes and a χ 2 test for categorical outcomes. Spearman and Pearson test was used for correlation analysis. e K-M curve and log-rank test were used to analyze the survival of patients. GraphPad Prism 8.0 (La Jolla, CA) was used for image rendering. An analysis of covariance (ANCOVA) model adjusting for the baseline value of the outcome was performed with the treatment group and time (baseline/ post-intervention) as factors. For within-group difference, the changes in MMP from baseline to week 9 were examined by a paired t-test, with a level of significance set at P < 0.05. Repeated measures of ANCOVA on the trial outcomes were a 2 × 2 (time: baseline, postintervention) analysis.

General Data.
In this study, 92 patients were included as per the inclusion and exclusion criteria, including 61 males and 31 females, aged (6.6 ± 1.9) years. Among them, there were 38 cases in the INSS stage III and 54 cases in stage IV. Other specific data are given in Table 2. ere was no significant statistical difference in the general data of patients in group A and group B (P > 0.05).

Chemotherapy Efficacy.
e total effective rate was 84.00% in group A and 95.24% in group B, and there was no significant difference between the two groups (P > 0.05), as given in Table 3.

Surgical Resection.
e lesion resection status of the patients was obtained from the surgical records. e resection rate of the patients in group A after initial chemotherapy was 60.00% and that in group B was 61.90%.
ere were no significant difference between the two groups (P > 0.05). After the second chemotherapy, all patients successfully received surgery, as given in Table 4.

Analysis of Complications.
e incidence of myelosuppression after chemotherapy was analyzed. All patients showed myelosuppression after chemotherapy, including 48 patients with stages I-II (52.17%) and 44 patients with stages III-IV (47.83%). e results of peripheral blood immune cell classification also showed that the CD4+/CD8+ cell ratio in peripheral blood of patients after chemotherapy decreased (P < 0.05), and the ratio of CD4+/CD8+ cells was further reduced after surgery (P < 0.05), while NK cells levels were increased (P < 0.05). However, there was no significant difference in the incidence of myelosuppression, CD4+/ CD8+ cell ratio, and NK cell level between the two groups (P > 0.05) ( Table 5).

3.5.
Changes of MMP-9, TIMP-1, and VMA Levels before and after Chemotherapy. After chemotherapy, MMP-9, TIMP-1, and VMA levels in peripheral blood of patients in the two groups were significantly decreased (P < 0.05), but intergroup differences in MMP-9, TIMP-1 and VMA levels between group A and group B before and after chemotherapy were absent (P > 0.05). Pearson correlation analysis showed that both MMP-9 and TIMP-1 were positively correlated with VMA (P < 0.05) (Figure 1).

e Correlation of MMP-9, TIMP-1, and VMA with the
Efficacy of Chemotherapy after Chemotherapy. In this study, all patients who achieved CR in chemotherapy successfully underwent complete resection. erefore, the patients were divided into a CR group and a PR + SD + PD group. Spearman correlation analysis showed that the expression levels of MMP-9, TIMP-1, and VMA after chemotherapy were negatively correlated with the efficacy of chemotherapy (P < 0.05) (Figure 2).

Discussion
INSS is one of the most commonly used NB staging systems in clinical practice. Patients with NB in the INSS stages III-IV usually suffer from tumor infiltration or lymph node and   distant metastases, and simple surgical treatment is considered insufficient for complete removal of the lesion, resulting in poor prognosis [12,13]. Prior research has found that preoperative adjuvant chemotherapy can effectively facilitate surgical resection and prolong the 5-year survival of the patients [14]. However, the efficacy of the combination of surgery and chemotherapy for NB has been marginally explored, and there has been no unified view on preoperative chemotherapy for advanced NB patients. With the increasingly serious multidrug resistance of tumors and the continuous development of chemotherapy drugs, there exists an urgent need to analyze the differences in the efficacy of current preoperative chemotherapy in NB.
is study compared the efficacy of two preoperative chemotherapy regimens (CAV and EP alternative therapy or TOPO + CTX + CiE + CPV therapy) in NB. In the second   Journal of Oncology scheme, the dosage of CTX, VCR, and VP16 was adjusted, and TOPO and pirarubicin were added to replace ADM. e latter three drugs all acted on DNA or RNA to exert antitumor effects [15][16][17]. e results of this study showed that there was no difference in the efficacy of chemotherapy and the incidence of myelosuppression between the two schemes. e regimens had significant inhibitory effects on the cellular immune function of patients, as evidenced by decreased CD4+/CD8+ cell ratio. In this study, the level of NK cells increased significantly after chemotherapy, and the cellular immune function of patients was further weakened by the further stressful stimulus of surgery. Similar results have been found in many studies, in which the cellular immune function of cancer patients is reduced after chemotherapy and surgical treatment [18,19]. In the study of Xie and Wang [20], chemotherapy plus surgery mildly inhibits the cellular immune function of NB patients versus chemotherapy alone, and the recurrence of NB is closely related to the decreased immune function of patients. is also reflects the advantages of the combination approach for the treatment of NB. In addition, the results of the present study showed no statistical differences in the surgical resection range between the two groups. In the study of Valteau-Couanet and Kushner et al. [21,22], CAV and EP regimens are suitable for preoperative treatment of NB patients, and about 80% of patients can get a complete or partial response after receiving the chemotherapy combined with surgical treatment. However, the treatment of this regimen only lasts for 5 courses, as further treatment is associated with unimproved efficacy and increased toxicity. In this study, CAV and EP were alternated for 5 courses of treatment, while TOPO + CTX + CiE + CDV were alternated for 6 courses of treatment. is may suggest another option for patients with a strong constitution. Notwithstanding a longer chemotherapy cycle, TOPO + CTX + CiE + CDV adopts a combination of more drugs, which can effectively reduce the incidence of drug resistance [23].
Similar to the research results by Xie and Wang [20], the results of this study also showed decreased expression of MMP-9 and TIMP-1 after chemotherapy. MMPs usually form complexes with TIMPs in noncovalent bonds, which inhibits the tumor metastasis and the transformation of malignant phenotypes [24,25]. Moreover, reduced levels of VMA in peripheral blood after chemotherapy were observed. VMA is one of the most commonly used indicators in the diagnosis of NB, which is related to high-risk characteristics of NB and can monitor chemotherapy response [26,27]. e results of this study showed that VMA was negatively correlated with the efficiency of chemotherapy in patients. In addition, MMP-9 and TIMP-1 also present a similar trend of changes, indicating the great potential of MMP-9 and TIMP-1 as two potential biological indicators for clinical determination of the chemotherapy efficacy. In addition, MMP-9, TIMP-1, and VMA were related to the 2-year survival of patients, which further provided evidence for the monitoring of therapeutic effect on NB patients by using MMP-9 and TIMP-1 [8,9,14].
ere are still some deficiencies in this study. e number of patients included in this study is small, and the results obtained still require more evidence. In addition, the follow-up time of this study is relatively short, which will be extended to 5-year survival observation in future studies.
In conclusion, surgery plus chemotherapy for children with NB yields a promising clinical efficacy and a favorable surgical resection outcome. MMP-9 and TIMP-1 may be the potential biological indicators for chemotherapy efficiency and have a reference value for following surgical treatment of patients.

Data Availability
e datasets used during the present study are available from the corresponding author upon request.

Conflicts of Interest
e authors declare that they have no conflicts of interest.