Med Oncol (2014) 31:91 DOI 10.1007/s12032-014-0091-8
ORIGINAL PAPER
Increased MMP-21 expression in esophageal squamous cell carcinoma is associated with progression and prognosis Zhengwei Zhao • Lingling Yan • Shisen Li Hao Sun • Yongan Zhou • Xiaofei Li
•
Received: 17 April 2014 / Accepted: 17 June 2014 / Published online: 12 July 2014 Ó Springer Science+Business Media New York 2014
Abstract Esophageal squamous cell carcinoma (ESCC) is a highly aggressive malignancy, requiring effective biomarkers for prognosis and therapeutic responsiveness. In this retrospective study of banked pathology material, we investigated the protein expression of MMP-21 in ESCC and its association with clinical significance. MMP-21 protein expression was investigated in 311 cases of ESCC by immunohistochemistry assay. Statistical analysis was utilized to evaluate the association of MMP-21 expression with clinicopathological characteristics and overall survival of patients with ESCC. Results showed that MMP-21 expression was significantly increased in ESCC (P \ 0.001). It was also found that MMP-21 expression in ESCC was associated with tumor invasion (P \ 0.001), lymph node metastasis (P \ 0.001), distant metastasis (P \ 0.001) and TNM stage (P \ 0.001). Kaplan–Meier analysis showed MMP-21 expression was associated with overall survival of patients with ESCC for patients with tumors of positive MMP-21
Zhengwei Zhao and Lingling Yan have contributed equally to this work. Z. Zhao Y. Zhou (&) X. Li (&) Department of Thoracic Surgery, Tangdu Hospital, Fourth Military Medical University, Xi’an, Shaanxi, China e-mail: [email protected] X. Li e-mail: [email protected] L. Yan Sanatorium for Retired Cadres, Bayi Street, Shaanxi Military Region, Xi’an, Shaanxi, China S. Li H. Sun Department of Digestive Surgery, Xijing Hospital of Digestive Diseases, Fourth Military Medical University, Xi’an, Shaanxi, China
staining tend to have worse overall survival (P \ 0.001). Multivariate analysis proved that MMP-21 was an independent prognostic factor for overall survival for patients with ESCC (P \ 0.001). These results suggested the potential role of MMP-21 in tumor progression and prognosis predication of human ESCC. It might also be a novel molecular target for therapeutic intervention. Keywords Matrix metalloproteinase-21 Esophageal squamous cell carcinoma Immunohistochemistry Overall survival
Introduction Esophageal cancer is one of the most common human malignancies worldwide, which causes an estimated 400,000 deaths per year all over the world [1]. Although there are several subtypes of esophageal cancer, primary esophageal squamous cell cancer (ESCC) accounts for approximately 90–95 % of all esophageal cancer [2]. ESCC is more common in Asian countries, such as China and Japan, compared with that in Western countries [3, 4]. In China alone, newly diagnosed ESCC cases each year account for nearly 50 % of that in the world [4, 5]. ESCC is a highly aggressive malignancy, with early metastasis to the lymph nodes and to distant organs [6–8]. Due to this aggressive nature, despite the advances made in diagnosis, surgical techniques and treatments, the prognosis of ESCC remains poor, with the median survival time for patients with ESCC ranging from 1 to 2 years [9–11]. The development and progression of ESCC is a complex process with multiple molecular factors participating [12–14]. Currently, TNM staging system is the best way to predict the prognosis of patients with ESCC [15, 16]. However,
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understanding of the molecular basis of this disease and identifying molecular risk markers may lead to improved diagnostic accuracy, prognosis predication and consequent treatment strategies [17, 18]. Matrix metalloproteinases (MMPs) are a group of zincdependent proteins which constitute a family of highly homologous enzymes, engaged in the degradation and remodeling of extracellular matrix (ECM) [19]. Till now, at least 28 human MMPs have been distinguished [20]. These enzymes can be divided into six groups, such as collagenases, gelatinases, stromelysins, matrilysins, membrane-type and a sixth group encompassing several other MMPs not classified in the previous categories, differing in structure, cellular localization and substrate specificity [21]. Due to their ECM-degrading capacity, MMPs are implicated in cancer invasion and metastasis with different classes of MMPs being associated more frequently with cancers of varying origin. Indeed, high expression levels of MMPs have been correlated with tumor aggressiveness of various human malignancies [22]. Among all MMPs identified, MMP-21 is a recently identified member of the MMPs family, which is a secreted 57-kDa pro-protein convertase-activated MMP and implicated in development, stromal remodeling, inflammation and cancer [23–27]. The MMP-21 gene has a unique structure that it has only seven exons and most of the splice sites are not conserved, whereas other MMP genes usually have ten exons. MMP-21 is also different from other MMPs in regulation since MMP-21 is not increased in keratinocytes (KCs) in response to tissue injury while only transforming growth factor (TGF)-b1 can induce MMP-21 expression in KCs [25]. It has also been reported that MMP-21 is expressed in cancer cells that are in the invasive front of tumor rather than dysplastic cells [26]. In addition, MMP-21 is also found to be expressed by macrophages and fibroblasts both in vivo and in vitro [28]. These results suggested a different expression pattern of MMP-21 from other MMPs. However, to our knowledge, the association of MMP-21 expression with clinicopathological characteristics and prognosis of ESCC has not been addressed yet. Therefore, in the present study, we investigated the protein expression of MMP-21 in clinical ESCC specimens and evaluated its association with clinicopathological characteristics and overall survival of patients.
Materials and methods Patients and specimens The protocol of the present study was approved by the ethics committee of the Fourth Military Medical
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University according to the ethical guidelines of the 1975 Declaration of Helsinki. Surgical specimens of ESCC were retrospectively collected from 311 patients undergone surgery at Xijing Hospital and Tangdu Hospital, Fourth Military Medical University between January 2002 and December 2005. Only patients who did not receive neoadjuvant chemotherapy or radiotherapy prior to surgery were recruited. Histomorphology of all the primary tumors specimens and regional lymph nodes was confirmed with hematoxylin-eosin staining by Department of Pathology, Fourth Military Medical University. Clinical parameters such as gender, age, differentiation status, lymph node metastasis and TNM stage were collected and entered in a database. Complete follow-up was made available for at least 5 years. All patients who died of other diseases than ESCC or unexpected events were excluded from the study. Informed consent was obtained from all patients, and specimens were handled anonymously according to the ethical and legal standards. Follow-up information of all participants was updated every 3 months by telephone visit and questionnaire letters. Death of participants was ascertained by reporting from the family and verified by review of public records. All specimens were fixed in 10 % formalin, embedded in paraffin.
Immunohistochemistry assay Four-micrometer-thick sections cut from the paraffin embedded tissue samples were deparaffinized and rehydrated. For antigen retrieval, sections were placed in citrate buffer and heated at 90 °C in a microwave oven for 15 min. The sections then were treated with 3 % hydrogen peroxide for 15 min at room temperature. After washing in phosphate-buffered saline (PBS), endogenous biotin was blocked with 30 % egg white for 20 min at room temperature. After that, non-specific binding was blocked with normal serum. These sections were then incubated with rabbit polyclonal MMP-21 antibody (1:200) in PBS at 4 °C overnight. After rinsing with PBS, secondary goat antirabbit IgG (1:400) were added, and the sections were incubated for 1 h at room temperature. After rinsing with PBS, the ready-to-use streptavidin–horseradish peroxidase complexes were added and incubated for 20 min at 37 °C. The color was developed by 0.05 % 30 ,30 -diaminobenzidine tetrahydrochloride plus 0.01 % hydrogen peroxide for 5 min at room temperature. The nucleus was counterstained with hematoxylin for 2 min at room temperature. Negative controls were performed by replacing the primary antibody with pre-immune serum. Appropriate positive and negative controls were performed in each run of immunohistochemistry assay.
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Evaluation of staining The tissue specimens were viewed separately by two pathologists without prior knowledge of the clinical or clinicopathological status of the specimens. The staining was evaluated by scanning the entire tissue specimen under low magnification (409), and then confirmed under high magnification (2009 and 4009). An immunoreactivity score (IRS) system was applied. The extensional standard: A, number of positive stained cell B5 % scored 0; 6–25 % scored 1; 26–50 % scored 2; 51–75 % scored 3; [75 % scored 4. B, intensity of stain: colorless scored 0; pallideflavens scored 1; yellow scored 2; brown scored 3. Multiply A and B. The staining score was stratified as - (0 score, absent), ?(1–4 score, weak), ??(5–8 score, moderate) and ???(9–12 score, strong) according to the proportion and intensity of positively stained cancer cells. Specimens will be rescored if difference of scores from two pathologists was more than 3 [29]. As the sample size of strong positive group was relatively small, specimens with weak, moderate and strong positive staining were combined as positive group (?).
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staining (-) of MMP-21. Therefore, statistical analysis revealed that the difference of MMP-21 staining between normal and ESCC tissues was statistically significant (P \ 0.001), indicating that the protein expression of MMP-21 in ESCC was significantly increased compared with that in normal tissues. The relationship of MMP-21 to clinicopathological characteristics of patients with ESCC Based on staining evaluation, we further investigated the correlation between the MMP-21 staining and clinicopathological characteristics, as shown in Table 1. In ESCC with different invasion status, positive staining of MMP-21 was more frequently detected in T3 and T4 tumors compared with T1 and T2 tumors(P \ 0.001), indicating that MMP-21 expression was increased in more invasive tumors. Then, we further analyzed the relation between
Table 1 Relation between MMP-21 staining and clinicopathological characteristics n
Statistical analysis Associations between MMP-21 protein immunohistochemical staining and clinicopathological characteristics were analyzed by Mann–Whitney test and Kruskal–Wallis test, as appropriate. Survival curves were estimated using the Kaplan–Meier method and differences in survival distributions were evaluated by the log-rank test. Cox’s proportional hazards modeling of factors potentially related to survival was performed in order to identify which factors might have a significant influence on survival, and controlling for age, gender and differentiation status. Differences with a P value of 0.05 or less were considered to be statistically significant.
Immunohistochemical staining of MMP-21 in ESCC specimens In the present study, 311 cases of ESCC tissues were investigated. MMP-21 staining was found to be predominantly located at cytoplasm of tumor cells, which is in consistence with previous investigation. Based on the IRS system on MMP-21 staining, 185 cases were defined as positive staining (?) while 126 cases were defined as negative staining (-) of MMP-21. By contrast, among matched normal tissues, 90 cases were defined as positive staining (?), while 221 cases were defined as negative
P
Negative (-)
Positive (?)
311
126
185
Male
195
75
120
Female
116
51
65
\60
170
68
102
C60
141
58
83
Total Gender
0.34*
Age
0.839*
0.192
Differentiation Poor Moderate Well
69
34
35
151
60
91
91
32
59 \0.001*
Invasion T1 ? T2
Results
MMP-21 staining
146
87
59
165
39
126
Negative
189
96
93
Positive
122
30
92
Negative
279
124
155
Positive
32
2
30
T3 ? T4 Node metastasis
\0.001*
\0.001*
Distant metastasis
\0.001
TNM stage I
69
44
25
II
98
54
44
III
112
26
86
IV
32
2
30
* P value was estimated by Mann–Whitney test
P value was estimated by Kruskal–Wallis test
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Fig. 1 Kaplan–Meier survival curve for patterns of patients with ESCC and MMP-21 staining
MMP-21 staining and node status with results showing that positive MMP-21 staining was more frequently detected in ESCC with lymph node metastasis compared with those without (P \ 0.001). In addition, positive MMP-21 staining was also more frequently detected in ESCC with distant metastasis (P \ 0.001). As far as TNM stage was concerned, MMP-21 expression was found to be closely associated with TNM stage for positive ratio of MMP-21 which tends to increase from stage I to stage IV tumors (P \ 0.001). However, MMP-21 staining was not correlated to patient’s gender, age or differentiation status (Table 1). The association of MMP-21 with overall survival of patients with ESCC During the follow-up period, the median overall survival time of all recruited ESCC patients was 37 months (95 % CI 32.2–43.4). Kaplan–Meier postoperative analysis was used to analyze the survival rate of patients with ESCC of hierarchical MMP-21 staining. Results showed that patients with ESCC of positive MMP-21 staining tend to have worse overall survival with median overall survival time to be 27 months (95 % CI 19.3–33.9), while those with ESCC of negative MMP-21 staining tend to have better overall survival with median overall survival time to be 48 months (95 % CI 39.3–57.5). Kaplan–Meier postoperative survival curve is shown in Fig. 1 (log-rank test: P \ 0.001). When unadjusted hazard ratio (HR) was considered with negative MMP-21 staining as reference (1.00), the unadjusted HR of patients with positive MMP-21 staining was 2.05 (95 % CI 1.53–2.74; P \ 0.001), indicating that patients with ESCC of positive MMP-21 staining had a 2.05-fold higher risk of death
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compared with those with ESCC of negative MMP-21 staining. Moreover, differentiation status and TNM stage were also found to be associated with overall survival of patients with ESCC. However, sex or age had no prognostic value on overall survival (Table 2). In multivariate analysis, Cox’s proportional hazards model adjusted for age, gender, differentiation status and TNM stage was utilized to identify independent prognostic factor. Results showed that the adjusted HR of patients with ESCC of positive MMP-21 staining was 1.73 (95 % CI 1.28–2.34; P \ 0.001), indicating that patients with ESCC of positive MMP-21 staining had a 1.73-fold higher risk of death compared with those with ESCC of negative MMP-21 staining, without regard to other clinicopathological characteristics. This finding suggested that MMP21 expression could serve as an independent prognostic factor of overall survival for patients with ESCC without consideration of other prognostic factors. In addition, TNM stage was also found to be an independent prognostic factor for overall survival of patients. However, sex, age or differentiation status was not found to be independently associated with prognosis of patients in multivariate analysis (Table 2).
Discussion In China, ESCC accounts for most esophageal malignant tumors, where it bears more than half of the global burden. It is widely accepted that ESCC is prone to invade adjacent regions and to metastasize to lymph nodes and distant organs, which accounts for its poor prognosis. The mechanism of ESCC progression has not been fully elucidated and, despite the advances in diagnosis, surgical techniques and other treatments, its prognosis remains poor. To develop novel treatments and cures, it is imperative to address the factors underlying prognosis for biomarkers predictive prognosis, which may help to design more effective and targeted therapies for ESCC. Since chemotherapy or chemoradiation is effective only in around 50 % of patients, it is better for patients with good prognosis to avoid potential overtreatment so as to escape treatment toxicities. Currently, utilized TNM staging system is not able to provide sufficient prognostic characterization of patients with ESCC. Thus, assessment of prognosis on the basis of molecular characteristics would help inform decisions and tailor therapy to ESCC individuals so as to achieve the best possible outcome. Although a variety of molecular markers have been identified to determine prognosis of ESCC, sensitive and specific biomarkers for accurate indicators for ESCC prognosis are still unavailable. Therefore, to further improve the survival rate in patients with ESCC, novel prognostic and molecular
Med Oncol (2014) 31:91 Table 2 Association of MMP21 and clinical factors with overall survival
Page 5 of 7 91 Unadjusted HRa (95 % CI)
Adjusted HRb (95 % CI)
P
P
MMP-21 Negative
–
Weak positive
2.05 (1.53–2.74)
– \0.001
\0.001
1.73 (1.28–2.34)
Sex Female
–
–
Male
1.06 (0.74–1.58)
0.674
1.18 (0.89–1.87)
0.425
Age B 60
–
[ 60
1.09 (0.78–1.59)
– 0.537
1.19 (0.91–1.92)
0.251
Differentiation status
a
Hazard ratios in univariate models
Well
–
Moderately
1.48 (1.03–2.29)
0.036
– 1.25 (0.93–1.97)
0.259
Poor
2.21 (1.42–4.15)
0.005
1.47 (0.96–2.53)
0.238
– 2.21(1.36–4.51)
0.002
– 2.18(1.35–4.84)
0.001
TNM stage
b
I II III
3.52 (1.99–7.14)
\0.001
3.46 (1.96–6.63)
\0.001
HR hazard ratio, 95 % CI 95 % confidence interval
IV
5.46 (2.65–11.85)
\0.001
5.33 (2.41–11.12)
\0.001
Hazard ratios in multivariable models
targets for therapeutic intervention are critically needed for patients with ESCC. For this purpose, we investigated the expression pattern and prognostic role of MMP-21 in ESCC in this study. Based on immunohistochemistry assay and staining score system, we found that the protein expression of MMP-21 was significantly increased in ESCC compared with that in normal tissues, indicating that MMP-21 might play an oncogenic role in ESCC carcinogenesis. Moreover, our study demonstrated a strong association between MMP-21 expression and ESCC invasion status, metastasis and TNM stage. It was found that positive MMP-21 staining was more frequently to be detected in ESCC of deep invasion, lymph node metastasis, distant metastasis and advanced TNM stage, which suggested a promotive role of MMP-21 in ESCC invasion and metastasis. However, MMP-21 staining was not found to be correlated with patient’s gender, age or differentiation status. These results are in well consistence with previous publications on other human malignancies [30–34]. As tumor invasion and metastasis is closely related to prognosis of ESCC, we further analyzed the prognostic role of MMP-21 in patients with ESCC. Kaplan–Meier analysis of the survival curves showed a significant association between MMP-21 expression and overall survival of patients that patients with positive MMP-21 staining had a worse overall survival. Cox’s proportional hazards model adjusted for age, gender, differentiation status and TNM stage showed the
same trend as Kaplan–Meier postoperative survival analysis, indicating that MMP-21 expression could serve as an independent prognostic factor for patient with ESCC without regard to other clinicopathological characteristics. The potential mechanism of the oncogenic role of MMP-21 on ESCC might be due to its ECM-degrading capacity. Previous investigations found that MMP-21 can degrade denatured collagen and type IV, V, VII, IX and X collagens, and therefore facilitate malignant tumor cells invasion and metastasis, which finally cause death of patients. These results suggested that MMP-21 could constitute a useful prognostic marker to identify patients who have higher risk of death and are, thus, good candidates to receive a more aggressive treatment. However, future prospective studies are still needed to determine the accuracy and efficiency of MMP-21 on predicting the prognosis of patients with ESCC. In conclusion, in this retrospective study of banked pathology material, we found for the first time that MMP-21 expression in ESCC was related to tumor invasion, metastasis and TNM stage. In addition, it also proved for the first time that MMP-21 could be an independent prognostic marker for patients with ESCC. These findings suggested that MMP-21 may be a diagnostic marker and even a potential therapeutic target for patients with ESCC. Conflict of interests
We declared no conflict of interests.
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ORIGINAL PAPER
Increased MMP-21 expression in esophageal squamous cell carcinoma is associated with progression and prognosis Zhengwei Zhao • Lingling Yan • Shisen Li Hao Sun • Yongan Zhou • Xiaofei Li
•
Received: 17 April 2014 / Accepted: 17 June 2014 / Published online: 12 July 2014 Ó Springer Science+Business Media New York 2014
Abstract Esophageal squamous cell carcinoma (ESCC) is a highly aggressive malignancy, requiring effective biomarkers for prognosis and therapeutic responsiveness. In this retrospective study of banked pathology material, we investigated the protein expression of MMP-21 in ESCC and its association with clinical significance. MMP-21 protein expression was investigated in 311 cases of ESCC by immunohistochemistry assay. Statistical analysis was utilized to evaluate the association of MMP-21 expression with clinicopathological characteristics and overall survival of patients with ESCC. Results showed that MMP-21 expression was significantly increased in ESCC (P \ 0.001). It was also found that MMP-21 expression in ESCC was associated with tumor invasion (P \ 0.001), lymph node metastasis (P \ 0.001), distant metastasis (P \ 0.001) and TNM stage (P \ 0.001). Kaplan–Meier analysis showed MMP-21 expression was associated with overall survival of patients with ESCC for patients with tumors of positive MMP-21
Zhengwei Zhao and Lingling Yan have contributed equally to this work. Z. Zhao Y. Zhou (&) X. Li (&) Department of Thoracic Surgery, Tangdu Hospital, Fourth Military Medical University, Xi’an, Shaanxi, China e-mail: [email protected] X. Li e-mail: [email protected] L. Yan Sanatorium for Retired Cadres, Bayi Street, Shaanxi Military Region, Xi’an, Shaanxi, China S. Li H. Sun Department of Digestive Surgery, Xijing Hospital of Digestive Diseases, Fourth Military Medical University, Xi’an, Shaanxi, China
staining tend to have worse overall survival (P \ 0.001). Multivariate analysis proved that MMP-21 was an independent prognostic factor for overall survival for patients with ESCC (P \ 0.001). These results suggested the potential role of MMP-21 in tumor progression and prognosis predication of human ESCC. It might also be a novel molecular target for therapeutic intervention. Keywords Matrix metalloproteinase-21 Esophageal squamous cell carcinoma Immunohistochemistry Overall survival
Introduction Esophageal cancer is one of the most common human malignancies worldwide, which causes an estimated 400,000 deaths per year all over the world [1]. Although there are several subtypes of esophageal cancer, primary esophageal squamous cell cancer (ESCC) accounts for approximately 90–95 % of all esophageal cancer [2]. ESCC is more common in Asian countries, such as China and Japan, compared with that in Western countries [3, 4]. In China alone, newly diagnosed ESCC cases each year account for nearly 50 % of that in the world [4, 5]. ESCC is a highly aggressive malignancy, with early metastasis to the lymph nodes and to distant organs [6–8]. Due to this aggressive nature, despite the advances made in diagnosis, surgical techniques and treatments, the prognosis of ESCC remains poor, with the median survival time for patients with ESCC ranging from 1 to 2 years [9–11]. The development and progression of ESCC is a complex process with multiple molecular factors participating [12–14]. Currently, TNM staging system is the best way to predict the prognosis of patients with ESCC [15, 16]. However,
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understanding of the molecular basis of this disease and identifying molecular risk markers may lead to improved diagnostic accuracy, prognosis predication and consequent treatment strategies [17, 18]. Matrix metalloproteinases (MMPs) are a group of zincdependent proteins which constitute a family of highly homologous enzymes, engaged in the degradation and remodeling of extracellular matrix (ECM) [19]. Till now, at least 28 human MMPs have been distinguished [20]. These enzymes can be divided into six groups, such as collagenases, gelatinases, stromelysins, matrilysins, membrane-type and a sixth group encompassing several other MMPs not classified in the previous categories, differing in structure, cellular localization and substrate specificity [21]. Due to their ECM-degrading capacity, MMPs are implicated in cancer invasion and metastasis with different classes of MMPs being associated more frequently with cancers of varying origin. Indeed, high expression levels of MMPs have been correlated with tumor aggressiveness of various human malignancies [22]. Among all MMPs identified, MMP-21 is a recently identified member of the MMPs family, which is a secreted 57-kDa pro-protein convertase-activated MMP and implicated in development, stromal remodeling, inflammation and cancer [23–27]. The MMP-21 gene has a unique structure that it has only seven exons and most of the splice sites are not conserved, whereas other MMP genes usually have ten exons. MMP-21 is also different from other MMPs in regulation since MMP-21 is not increased in keratinocytes (KCs) in response to tissue injury while only transforming growth factor (TGF)-b1 can induce MMP-21 expression in KCs [25]. It has also been reported that MMP-21 is expressed in cancer cells that are in the invasive front of tumor rather than dysplastic cells [26]. In addition, MMP-21 is also found to be expressed by macrophages and fibroblasts both in vivo and in vitro [28]. These results suggested a different expression pattern of MMP-21 from other MMPs. However, to our knowledge, the association of MMP-21 expression with clinicopathological characteristics and prognosis of ESCC has not been addressed yet. Therefore, in the present study, we investigated the protein expression of MMP-21 in clinical ESCC specimens and evaluated its association with clinicopathological characteristics and overall survival of patients.
Materials and methods Patients and specimens The protocol of the present study was approved by the ethics committee of the Fourth Military Medical
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University according to the ethical guidelines of the 1975 Declaration of Helsinki. Surgical specimens of ESCC were retrospectively collected from 311 patients undergone surgery at Xijing Hospital and Tangdu Hospital, Fourth Military Medical University between January 2002 and December 2005. Only patients who did not receive neoadjuvant chemotherapy or radiotherapy prior to surgery were recruited. Histomorphology of all the primary tumors specimens and regional lymph nodes was confirmed with hematoxylin-eosin staining by Department of Pathology, Fourth Military Medical University. Clinical parameters such as gender, age, differentiation status, lymph node metastasis and TNM stage were collected and entered in a database. Complete follow-up was made available for at least 5 years. All patients who died of other diseases than ESCC or unexpected events were excluded from the study. Informed consent was obtained from all patients, and specimens were handled anonymously according to the ethical and legal standards. Follow-up information of all participants was updated every 3 months by telephone visit and questionnaire letters. Death of participants was ascertained by reporting from the family and verified by review of public records. All specimens were fixed in 10 % formalin, embedded in paraffin.
Immunohistochemistry assay Four-micrometer-thick sections cut from the paraffin embedded tissue samples were deparaffinized and rehydrated. For antigen retrieval, sections were placed in citrate buffer and heated at 90 °C in a microwave oven for 15 min. The sections then were treated with 3 % hydrogen peroxide for 15 min at room temperature. After washing in phosphate-buffered saline (PBS), endogenous biotin was blocked with 30 % egg white for 20 min at room temperature. After that, non-specific binding was blocked with normal serum. These sections were then incubated with rabbit polyclonal MMP-21 antibody (1:200) in PBS at 4 °C overnight. After rinsing with PBS, secondary goat antirabbit IgG (1:400) were added, and the sections were incubated for 1 h at room temperature. After rinsing with PBS, the ready-to-use streptavidin–horseradish peroxidase complexes were added and incubated for 20 min at 37 °C. The color was developed by 0.05 % 30 ,30 -diaminobenzidine tetrahydrochloride plus 0.01 % hydrogen peroxide for 5 min at room temperature. The nucleus was counterstained with hematoxylin for 2 min at room temperature. Negative controls were performed by replacing the primary antibody with pre-immune serum. Appropriate positive and negative controls were performed in each run of immunohistochemistry assay.
Med Oncol (2014) 31:91
Evaluation of staining The tissue specimens were viewed separately by two pathologists without prior knowledge of the clinical or clinicopathological status of the specimens. The staining was evaluated by scanning the entire tissue specimen under low magnification (409), and then confirmed under high magnification (2009 and 4009). An immunoreactivity score (IRS) system was applied. The extensional standard: A, number of positive stained cell B5 % scored 0; 6–25 % scored 1; 26–50 % scored 2; 51–75 % scored 3; [75 % scored 4. B, intensity of stain: colorless scored 0; pallideflavens scored 1; yellow scored 2; brown scored 3. Multiply A and B. The staining score was stratified as - (0 score, absent), ?(1–4 score, weak), ??(5–8 score, moderate) and ???(9–12 score, strong) according to the proportion and intensity of positively stained cancer cells. Specimens will be rescored if difference of scores from two pathologists was more than 3 [29]. As the sample size of strong positive group was relatively small, specimens with weak, moderate and strong positive staining were combined as positive group (?).
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staining (-) of MMP-21. Therefore, statistical analysis revealed that the difference of MMP-21 staining between normal and ESCC tissues was statistically significant (P \ 0.001), indicating that the protein expression of MMP-21 in ESCC was significantly increased compared with that in normal tissues. The relationship of MMP-21 to clinicopathological characteristics of patients with ESCC Based on staining evaluation, we further investigated the correlation between the MMP-21 staining and clinicopathological characteristics, as shown in Table 1. In ESCC with different invasion status, positive staining of MMP-21 was more frequently detected in T3 and T4 tumors compared with T1 and T2 tumors(P \ 0.001), indicating that MMP-21 expression was increased in more invasive tumors. Then, we further analyzed the relation between
Table 1 Relation between MMP-21 staining and clinicopathological characteristics n
Statistical analysis Associations between MMP-21 protein immunohistochemical staining and clinicopathological characteristics were analyzed by Mann–Whitney test and Kruskal–Wallis test, as appropriate. Survival curves were estimated using the Kaplan–Meier method and differences in survival distributions were evaluated by the log-rank test. Cox’s proportional hazards modeling of factors potentially related to survival was performed in order to identify which factors might have a significant influence on survival, and controlling for age, gender and differentiation status. Differences with a P value of 0.05 or less were considered to be statistically significant.
Immunohistochemical staining of MMP-21 in ESCC specimens In the present study, 311 cases of ESCC tissues were investigated. MMP-21 staining was found to be predominantly located at cytoplasm of tumor cells, which is in consistence with previous investigation. Based on the IRS system on MMP-21 staining, 185 cases were defined as positive staining (?) while 126 cases were defined as negative staining (-) of MMP-21. By contrast, among matched normal tissues, 90 cases were defined as positive staining (?), while 221 cases were defined as negative
P
Negative (-)
Positive (?)
311
126
185
Male
195
75
120
Female
116
51
65
\60
170
68
102
C60
141
58
83
Total Gender
0.34*
Age
0.839*
0.192
Differentiation Poor Moderate Well
69
34
35
151
60
91
91
32
59 \0.001*
Invasion T1 ? T2
Results
MMP-21 staining
146
87
59
165
39
126
Negative
189
96
93
Positive
122
30
92
Negative
279
124
155
Positive
32
2
30
T3 ? T4 Node metastasis
\0.001*
\0.001*
Distant metastasis
\0.001
TNM stage I
69
44
25
II
98
54
44
III
112
26
86
IV
32
2
30
* P value was estimated by Mann–Whitney test
P value was estimated by Kruskal–Wallis test
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Fig. 1 Kaplan–Meier survival curve for patterns of patients with ESCC and MMP-21 staining
MMP-21 staining and node status with results showing that positive MMP-21 staining was more frequently detected in ESCC with lymph node metastasis compared with those without (P \ 0.001). In addition, positive MMP-21 staining was also more frequently detected in ESCC with distant metastasis (P \ 0.001). As far as TNM stage was concerned, MMP-21 expression was found to be closely associated with TNM stage for positive ratio of MMP-21 which tends to increase from stage I to stage IV tumors (P \ 0.001). However, MMP-21 staining was not correlated to patient’s gender, age or differentiation status (Table 1). The association of MMP-21 with overall survival of patients with ESCC During the follow-up period, the median overall survival time of all recruited ESCC patients was 37 months (95 % CI 32.2–43.4). Kaplan–Meier postoperative analysis was used to analyze the survival rate of patients with ESCC of hierarchical MMP-21 staining. Results showed that patients with ESCC of positive MMP-21 staining tend to have worse overall survival with median overall survival time to be 27 months (95 % CI 19.3–33.9), while those with ESCC of negative MMP-21 staining tend to have better overall survival with median overall survival time to be 48 months (95 % CI 39.3–57.5). Kaplan–Meier postoperative survival curve is shown in Fig. 1 (log-rank test: P \ 0.001). When unadjusted hazard ratio (HR) was considered with negative MMP-21 staining as reference (1.00), the unadjusted HR of patients with positive MMP-21 staining was 2.05 (95 % CI 1.53–2.74; P \ 0.001), indicating that patients with ESCC of positive MMP-21 staining had a 2.05-fold higher risk of death
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compared with those with ESCC of negative MMP-21 staining. Moreover, differentiation status and TNM stage were also found to be associated with overall survival of patients with ESCC. However, sex or age had no prognostic value on overall survival (Table 2). In multivariate analysis, Cox’s proportional hazards model adjusted for age, gender, differentiation status and TNM stage was utilized to identify independent prognostic factor. Results showed that the adjusted HR of patients with ESCC of positive MMP-21 staining was 1.73 (95 % CI 1.28–2.34; P \ 0.001), indicating that patients with ESCC of positive MMP-21 staining had a 1.73-fold higher risk of death compared with those with ESCC of negative MMP-21 staining, without regard to other clinicopathological characteristics. This finding suggested that MMP21 expression could serve as an independent prognostic factor of overall survival for patients with ESCC without consideration of other prognostic factors. In addition, TNM stage was also found to be an independent prognostic factor for overall survival of patients. However, sex, age or differentiation status was not found to be independently associated with prognosis of patients in multivariate analysis (Table 2).
Discussion In China, ESCC accounts for most esophageal malignant tumors, where it bears more than half of the global burden. It is widely accepted that ESCC is prone to invade adjacent regions and to metastasize to lymph nodes and distant organs, which accounts for its poor prognosis. The mechanism of ESCC progression has not been fully elucidated and, despite the advances in diagnosis, surgical techniques and other treatments, its prognosis remains poor. To develop novel treatments and cures, it is imperative to address the factors underlying prognosis for biomarkers predictive prognosis, which may help to design more effective and targeted therapies for ESCC. Since chemotherapy or chemoradiation is effective only in around 50 % of patients, it is better for patients with good prognosis to avoid potential overtreatment so as to escape treatment toxicities. Currently, utilized TNM staging system is not able to provide sufficient prognostic characterization of patients with ESCC. Thus, assessment of prognosis on the basis of molecular characteristics would help inform decisions and tailor therapy to ESCC individuals so as to achieve the best possible outcome. Although a variety of molecular markers have been identified to determine prognosis of ESCC, sensitive and specific biomarkers for accurate indicators for ESCC prognosis are still unavailable. Therefore, to further improve the survival rate in patients with ESCC, novel prognostic and molecular
Med Oncol (2014) 31:91 Table 2 Association of MMP21 and clinical factors with overall survival
Page 5 of 7 91 Unadjusted HRa (95 % CI)
Adjusted HRb (95 % CI)
P
P
MMP-21 Negative
–
Weak positive
2.05 (1.53–2.74)
– \0.001
\0.001
1.73 (1.28–2.34)
Sex Female
–
–
Male
1.06 (0.74–1.58)
0.674
1.18 (0.89–1.87)
0.425
Age B 60
–
[ 60
1.09 (0.78–1.59)
– 0.537
1.19 (0.91–1.92)
0.251
Differentiation status
a
Hazard ratios in univariate models
Well
–
Moderately
1.48 (1.03–2.29)
0.036
– 1.25 (0.93–1.97)
0.259
Poor
2.21 (1.42–4.15)
0.005
1.47 (0.96–2.53)
0.238
– 2.21(1.36–4.51)
0.002
– 2.18(1.35–4.84)
0.001
TNM stage
b
I II III
3.52 (1.99–7.14)
\0.001
3.46 (1.96–6.63)
\0.001
HR hazard ratio, 95 % CI 95 % confidence interval
IV
5.46 (2.65–11.85)
\0.001
5.33 (2.41–11.12)
\0.001
Hazard ratios in multivariable models
targets for therapeutic intervention are critically needed for patients with ESCC. For this purpose, we investigated the expression pattern and prognostic role of MMP-21 in ESCC in this study. Based on immunohistochemistry assay and staining score system, we found that the protein expression of MMP-21 was significantly increased in ESCC compared with that in normal tissues, indicating that MMP-21 might play an oncogenic role in ESCC carcinogenesis. Moreover, our study demonstrated a strong association between MMP-21 expression and ESCC invasion status, metastasis and TNM stage. It was found that positive MMP-21 staining was more frequently to be detected in ESCC of deep invasion, lymph node metastasis, distant metastasis and advanced TNM stage, which suggested a promotive role of MMP-21 in ESCC invasion and metastasis. However, MMP-21 staining was not found to be correlated with patient’s gender, age or differentiation status. These results are in well consistence with previous publications on other human malignancies [30–34]. As tumor invasion and metastasis is closely related to prognosis of ESCC, we further analyzed the prognostic role of MMP-21 in patients with ESCC. Kaplan–Meier analysis of the survival curves showed a significant association between MMP-21 expression and overall survival of patients that patients with positive MMP-21 staining had a worse overall survival. Cox’s proportional hazards model adjusted for age, gender, differentiation status and TNM stage showed the
same trend as Kaplan–Meier postoperative survival analysis, indicating that MMP-21 expression could serve as an independent prognostic factor for patient with ESCC without regard to other clinicopathological characteristics. The potential mechanism of the oncogenic role of MMP-21 on ESCC might be due to its ECM-degrading capacity. Previous investigations found that MMP-21 can degrade denatured collagen and type IV, V, VII, IX and X collagens, and therefore facilitate malignant tumor cells invasion and metastasis, which finally cause death of patients. These results suggested that MMP-21 could constitute a useful prognostic marker to identify patients who have higher risk of death and are, thus, good candidates to receive a more aggressive treatment. However, future prospective studies are still needed to determine the accuracy and efficiency of MMP-21 on predicting the prognosis of patients with ESCC. In conclusion, in this retrospective study of banked pathology material, we found for the first time that MMP-21 expression in ESCC was related to tumor invasion, metastasis and TNM stage. In addition, it also proved for the first time that MMP-21 could be an independent prognostic marker for patients with ESCC. These findings suggested that MMP-21 may be a diagnostic marker and even a potential therapeutic target for patients with ESCC. Conflict of interests
We declared no conflict of interests.
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