Matrix metalloproteinase-9 (MMP-9) is a class of enzymes related to cancer pathogenesis because of its involvement in the degradation of extracellular matrix (Peisker et al., 2017). MMP-9 participates in tumorigenesis, and its high expression indicates poor prognosis in ESCC (Li et al., 2013; Gu et al., 2005). Serum MMP-9 is an effective biomarker in the diagnosis and prognosis of ESCC (?ukaszewicz-Zajac et al., 2009; Mroczko et al., 2008). Esophagectomy is the most common standard treatment for localized ESCC. However, recurrence rate remains high (Alidina et al., 2004). Although surgical resection is a potential mainstay for curable ESCC, locoregional recurrence is up to 23.8 to 58.0% of cases (Miyata et al., 2011; Lu et al., 2013; Guo et al., 2014). Therefore, the correlation of serum MMP-9 with recurrence in patients with locally advanced resectable ESCC should be determined.

This study investigates the prognosis of serum MMP-9 in recurrent patients with advanced ESCC after curative esophagectomy followed by chemotherapy or concurrent radiotherapy and evaluates the potential of serum MMP-9 as a recurrence predictor in patients with locally advanced resectable ESCC.

Patients

From January 2012 to June 2016, 173 patients (stages III and IV) with advanced ESCC confirmed by histopathology and who underwent R0 resection were enrolled at Jiangsu Cancer Hospital (Nanjing, China). This study was performed in accordance with the Declaration of Helsinki and approved by the Biomedical Research Ethics Committee of Jiangsu Cancer Hospital. All participants provided informed consent.

Treatment modality

At the beginning, patients did not receive any treatment (before treatment). Then, all patients received chemotherapy at four different intervals, with about 1 to 2 months interval. A total of 57 patients received concurrent radiotherapy at the first course of chemotherapy with radiotherapy schemes, such as GTV60-65Gy/28-33f, CTV50-55Gy/28-33f and PTV50-66Gy/28-33f (after therapies). Chemotherapy was consistent with the previous description and included taxane combined with platinum, 5 fluorouracil and its derivatives combined with platinum, and gemcitabine combined with platinum (Ye et al., 2020). Five months after the end of chemotherapy, 57 patients had recurrence at the original lesion or metastasis, including lymph node and distal metastases, and classified as recurrent patients (recurrence patient group). Others were classified as nonrecurrent patients (nonrecurrence patient group). The recurrence patient group received further treatments, including chemotherapy or concurrent radiotherapy, again.

Serum samples and serum MMP-9 detection

Blood samples before and after chemotherapies (that is, the 1st, 2nd, 3rd and 4th cycles of treatment) were collected. The samples of recurrence patients were still collected at recurrence (re-0 cycle) and at course of further treatment (re-1 and re-2 cycle). Samples were stored at -80°C after centrifugation.

Human cytokine/chemokine panel (that is, MMP-9) was purchased from Millipore (CAT no. HMMP2MAG-55K-01; Millipore, USA) and performed in accordance with the manufacturer’s instructions. Serum MMP-9 level was determined using the Luminex FLEXMAP 3D instruments and software supplied by Luminex Corporation (Austin, USA). The preparation of blood samples, setting of detection parameters, and calculation of serum MMP-9 concentrations were conducted in accordance with a previous study (Ma et al., 2017).

Statistical analysis

The concentrations of MMP-9 in serum were presented as mean ± SD. P < 0.05 indicated significance. Differences between two groups were analyzed using unpaired t-test. Comparisons among three or more groups were performed by ANOVA followed by pairwise comparisons by using the Bonferroni post hoc test. The follow-up ended on April 25, 2020, and the overall survival (OS) was calculated. Recurrence-free survival rates were calculated from the date of operation to the date of recurrence via the Kaplan-Meier method, and the significance of comparisons between groups was measured through the log-rank test. Cutoff values for OS and recurrence-free survival of serum MMP-9 were assessed by the receiver operating characteristic (ROC). The area under curve (AUC), sensitivity, and specificity were calculated. Statistical analyses were performed using a commercially available statistical software (that is, GraphPad Prism 5).

Serum MMP-9 decreased in recurrent and nonrecurrent patients with locally advanced resectable ESCC after therapies

The clinical characteristics of 173 patients with locally advanced resectable ESCC are detailed in Table 1. Serum MMP-9 levels were approximately equal between recurrent and nonrecurrent patients before treatment (P > 0.05, Figure 1A). However, serum MMP-9 levels significantly decreased in 116 nonrecurrent and 57 recurrent patients with locally advanced resectable ESCC after therapies compared with those before treatment (P < 0.001, Figure 1B). Furthermore, 57 recurrent patients maintained lower serum MMP-9 levels at recurrence than those before treatment (P < 0.001, Figure 1C). Serum MMP-9 levels did not increase until the third cycle of treatment in recurrent and nonrecurrent patients with locally advanced resectable ESCC after therapies (Figure 2A).  Compared  with  that  at  recurrence,  serum MMP-9 levels no longer decreased in recurrent patients during further treatments (P > 0.001, Figure 2B).  No difference in serum MMP-9 level after therapies was observed between recurrent patients treated with chemotherapy and concurrent radiotherapy. However, nonrecurrent patients treated with concurrent radiotherapy had low serum MMP-9 than those treated with chemotherapy (P < 0.05, Figure 2C). Nonrecurrent and recurrent patients treated with concurrent radiotherapy at the 2nd, 3rd, and 4th cycles of treatments had significantly  decreased serum MMP-9 levels compared with those before treatment (P < 0.001, Figures 2D and 2E). Nonrecurrent patients treated with chemotherapy had significantly lower serum MMP-9 levels at the 2nd, 3rd and 4th cycles of treatments than those before treatment (P < 0.001), whereas the opposite was observed in recurrent patients (P > 0.05, Figures 2D and E).

Only non-recurrent patients with low serum MMP-9 had long survival time

The OS values of recurrent and non-recurrent patients were approximately equal (P > 0.05, Figure 3A). However, for recurrent patients, serum MMP-9 ≥ 980 ng/mL at recurrence, MMP-9 ≥ 521 ng/mL after treatment, and MMP-9 ≥ 521 ng/mL after further treatment were not independent factors for OS (P > 0.05; Figure 3B to D). Furthermore, in recurrent patients with locally advanced resectable ESCC, concurrent radiotherapy did not prolong OS compared with chemotherapy (P > 0.05, Figure 4A). Recurrent patients with serum MMP-9 < 635 ng/mL before treatment and MMP-9 < 521 ng/mL after therapies had long OS. However, serum MMP-9 ≥ 635 ng/mL before treatment and ≥ 521 ng/mL after therapies were still  not  determined  as independent factors for OS(P > 0.05, Figures 4B and 4C). However, nonrecurrent patients treated with concurrent radiotherapy had significantly longer OS compared with patients treated with chemotherapy (P < 0.05, Figure 4D). Survival times in nonrecurrent patients with MMP-9 ≥ 475 ng/mL and MMP-9 < 475 ng/mL before treatment were not different (P > 0.05, Figure 4E). However, serum MMP-9 ≥ 700.25 ng/mL after therapies was an independent factor for OS in nonrecurrent patients with locally advanced resectable ESCC. Nonrecurrent patients with low serum MMP-9 (< 700.25 ng/mL) had significantly longer OS than those with high serum MMP-9 (≥ 700.25 ng/mL) after therapies (P < 0.001, Figure 4F).

Serum MMP-9 was associated with recurrence-free survival rate in patients with locally advanced resectable ESCC

Among 173 patients with locally advanced resectable ESCC, patients with serum MMP-9 < 577 ng/mL had significantly higher recurrence-free survival rates than those with MMP-9 ≥ 577 ng/mL before treatment (P = 0.0411, Figure 5A). Furthermore, compared with those with MMP-9 ≥ 700.25 ng/mL after therapies, 173 patients with MMP-9 < 700.25 ng/mL had higher recurrence-free survival rates (P < 0.001, Figure 5B). For recurrent patients, the difference in recurrence-free survival rate was significant between MMP-9 ≥ 635 ng/mL and MMP-9 < 635 ng/mL before treatment (P = 0.0483, Figure 5C). However, no significant difference in recurrence-free survival rate was observed between 57 nonrecurrent patients with MMP-9 ≥ 521 ng/mL and MMP-9 < 521 ng/mL after therapies (P = 0.2613, Figure 5D).

Esophagectomy is a common standard for the treatment of localized disease. However, local recurrence rate is high in patients with EC after operation (Alidina et al., 2004). Neoadjuvant radiochemotherapy improves the 5-year survival rate in patients with EC (van Hagen et al., 2012; Shapiro et al., 2015). Neoadjuvant chemoradiotherapy followed by surgery prolongs survival time over surgery alone in patients with locally advanced ESCC (Yang et al., 2018). In the present study, 173 patients with advanced ESCC have undergone esophagectomy followed by chemotherapy or concurrent radiotherapy. A total of 57 recurrent patients after cycles of chemotherapy or concurrent radiotherapy are observed. Postoperative nonrecurrent patients who received concurrent radiotherapy have longer survival time than those treated with chemotherapy alone indicating that locally advanced resectable ESCC patients without recurrence obtains benefits from concurrent radiotherapy. Nonrecurrent patients treated with concurrent radiotherapy have maintained lower serum MMP-9 levels than those treated with chemotherapy alone during the progress of treatments. However, serum MMP-9 levels are not significantly low in recurrent patients treated with concurrent radiotherapy compared with those treated with chemotherapy. This finding means that serum MMP-9 is related to survival time and can be used as a potential prognostic factor. Without regard for recurrence, concurrent radiotherapy is a suitable therapeutic regimen for patients with locally advanced resectable ESCC.

This study has limitations. This study is a retrospective analysis based on existing data rather than selecting specific recurrent patients into the group. Therefore, few other risk factors are analyzed when predicting recurrence through serum MMP-9 in patients with locally advanced resectable ESCC. In addition, patients with recurrence at the original lesion or metastasis are classified as recurrence patient group in this study rather than patients with ESCC and local recurrence merely after curative esophagectomy followed by chemotherapy or concurrent radiotherapy.

Although serum MMP-9 after therapies is not a direct marker of recurrence, serum MMP-9 level before treatment is negatively associated with recurrence-free survival in locally advanced resectable ESCC patients, suggesting that serum MMP-9 before treatment is a potential risk factor for recurrence in ESCC patients after curative esophagectomy followed by chemotherapy or concurrent radiotherapy.

The authors have not declared any conflict of interests.

The present study was supported by projects of Xuzhou Science and Technology Bureau (KC19183).