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Prognostic value of matrix metalloproteinase 9 expression in patients with non-small cell lung cancer
Clinica Chimica Acta 413 (2012) 1121–1126
Contents lists available at SciVerse ScienceDirect
Clinica Chimica Acta journal homepage: www.elsevier.com/locate/clinchim
Prognostic value of matrix metalloproteinase 9 expression in patients with non-small cell lung cancer Wen-Jia Peng a, 1, Jun-Qing Zhang b, 1, Bing-Xiang Wang a, Hai-Feng Pan a, Man-Man Lu a, Jing Wang a,⁎ a b
Department of Epidemiology and Biostatistics, School of Public Health, Anhui Medical University, Hefei, China Center for Disease Control and Prevention of Hefei City, Hefei, China
a r t i c l e
i n f o
Article history: Received 12 February 2012 Received in revised form 12 March 2012 Accepted 12 March 2012 Available online 20 March 2012 Keywords: MMP-9 Prognosis Non-small cell lung cancer Meta-analysis Hazard ratio
a b s t r a c t Background: The role of matrix metalloproteinase 9 (MMP-9) expression in non-small cell lung cancer (NSCLC) remains controversial. We performed a systematic review of the literature with meta-analysis. Methods: Electronic databases were used to identify published studies before December 1, 2011. Pooled hazard ratio (HR) with 95% confidence interval (95% CI) was used to estimate the strength of the association between MMP-9 expression survival of NSCLC patients. Heterogeneity and publication bias were also assessed. Results: The final analysis of 2029 NSCLC cases from 17 studies is presented. The combined HR of 1.84 (95% CI: 1.62–2.09) suggested that MMP-9 over-expression had a poor prognosis in patients with NSCLC. Subgroup analyses also detected significant association. Heterogeneity and publication bias was absent in current meta-analysis. Sensitivity analyses suggested that the summary statistics obtained should approximate the actual average. Conclusion: High MMP-9 expression is associated with a poor prognosis in patients with NSCLC. © 2012 Elsevier B.V. All rights reserved.
1. Introduction Lung cancer (LC) remains the most frequent and the most lethal human malignancy, accounting for 1.1 million deaths annually worldwide [1]. Despite diagnostic and therapeutic improvements, the prognosis for lung cancer patients is generally poor, with an overall five-year survival rate of approximately 15% [2]. Non-small cell lung cancer (NSCLC) accounts for approximately 85% of all LC cases, comprising adenocarcinoma (AD), squamous cell carcinoma (SQ) and carcinoid [3]. Prognostic factors for survival are useful in the management of NSCLC patients. Several independent prognostic factors for the survival of NSCLC have been identified such as performance status, TNM stage, age, sex, and weight loss [4]. These factors are useful when choosing the treatment on an individual basis, principally disease stage and performance status. The biological factors involved in carcinogenesis should also be considered as potential survival prognostic factors. In recent years, substantial efforts have been made to identify prognostic factors in order to individualize treatment and improve prognosis. Many molecular markers have been implicated both in the pathogenesis and prognosis of NSCLC, such as MMP-2 [5], Bcl-2 [6], and P27 [7]. Matrix metalloproteinases (MMPs) are able to degrade extracellular matrix (ECM) components and promote the formation of
⁎ Corresponding author at: Department of Epidemiology and Biostatistics, School of Public Health, Anhui Medical University, 81 Meishan Road, Hefei, Anhui 230032, China. Tel.: + 86 551 5161175; fax: + 86 551 5161126. E-mail address: [email protected] (J. Wang). 1 Contributed equally to this work. 0009-8981/$ – see front matter © 2012 Elsevier B.V. All rights reserved. doi:10.1016/j.cca.2012.03.012
metastasis. MMPs have an important role in tumor invasion and angiogenesis [8]. Among all identified MMPs, MMP-9 is a collagenase with a molecular mass of 92 kDa which catalyzes type IV collagen, a major structural protein of basement membranes, including the blood vessel basement membrane, and is chemically and it is genetically distinct from stroma collagen types I and III and cartilage collagen type II. In addition, its degradation plays an important role in tumor cell invasion of the vasculature [9,10]. Therefore, MMP-9 is considered to play a critical role in metastasis. The synthesis and secretion of MMP-9 can be stimulated by a variety of stimuli, including cytokines, during various pathological processes such as tumor invasion, atherosclerosis, inflammation, and rheumatoid arthritis [11]. Increased MMP-9 levels have been associated with poor prognosis in stage IV gastrointestinal cancer [12]. In colorectal cancer the tissue levels of MMP-9 predicted relapse and survival [13]. Therefore, MMP-9 should be a promising prognostic marker. Enhanced expression of MMP-9 in cancer cells has been noticed to be a significant factor to predict poor survival in NSCLC [14,15]. However, the results are inconsistent. 2. Materials and methods 2.1. Identification of studies The electronic database of PubMed, Embase and Web of science were searched for studies that investigated the prognostic significance of MMP-9 in NSCLC to be included in the present metaanalysis. References therein were also manually reviewed for articles potentially missed in the initial search. Studies were examined
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Table 1 Main characteristics and results of the eligible studies. First author
Year
Histology
Stage
No. of pts
Method
Cut off
High expression
HR estimation
Cox G Swinson DEB Chen XL Kim SJ Swinson DEB Shou Y Takemoto N (1) Takemoto N (2) Ishikawa S Cox G Pinto CA Sienel W Shao WL Martins SJ Grossi F Shimanuki Y Swinson DEB
2001 2004 2008 2005 2004 2001 2007 2007 2004 2000 2003 2003 2011 2009 2010 2005 2002
NSCLC (SQ: 103, AD: 50, LCC: 14) NSCLC (AD: 47, SQ: 101, LCC: 14, Other:4) NSCLC (SQ: 21, AD: 20) NSCLC (SQ: 38, AD: 26, LCC: 6, Other: 4) NSCLC (SQ: 107, AD: 49, LCC: 12, Other: 4) NSCLC (AD: 80, SQ: 37, ADC: 1, LCC: 1) AD SQ NSCLC (SQ: 77, AD: 122, LCC: 11, Other: 8) NSCLC (SQ: 105, AD: 50, LCC: 14) AD NSCLC (AD: 60, SQ: 57, Other: 26) NSCLC (SQ: 21, Other: 125) AD NSCLC (AD: 33, SQ: 50, LCC: 4) NSCLC (AD: 43, SQ: 15, LCC: 1, Other: 4) NSCLC (AD: 49, LCC: 14, SQ: 111, Other: 4)
I–IIIA I–IIIA I–III I–II I–IIIA I–III I–IIIA I–IIIA I–IIIA I–IIIA I–III NA IA IIIB–IV IIIA I–IIIA I–IIIA
167 166 41 74 172 119 46 37 218 169 152 143 146 51 87 63 178
IHC IHC IHC IHC IHC IHC RT-PCR RT-PCR IHC IHC IHC IHC IHC IHC IHC IHC IHC
20% 20% 50% 30% 20% NA NA NA 25% 20% 20% 95% 25% 80% 20% NA 20%
87 (52.10%) 87 (52.41%) 27 (65.85%) 51 (68.92%) 84 (48.84%) 75 (67.57%) 28 (60.87%) 14 (37.84%) NA 92 (54.44%) 76 (50.00%) 26 (18.18%) 89 (60.96%) 15 (29.41%) 43 (49.43%) 30 (49.18%) 88 (49.44%)
HR + 95% CI HR + 95% CI Available data Available data HR + 95% CI Survival curve HR + 95% CI HR + 95% CI HR + 95% CI HR + 95% CI HR + 95% CI HR + 95% CI HR + 95% CI HR + 95% CI Survival curve Survival curve HR + 95% CI
IHC: Immunohistochemistry; RT-PCR: reverse transcriptase-polymerase chain reaction. SQ: Squamous cell cancer; AD: Adenocarcinoma; LCC: Large cell carcinoma; ADC: Adenosquamous carcinoma. Pts: patients.
without language limits imposed with an updating search on December 1, 2011. Search terms were “non-small cell lung cancer” or “NSCLC”, “MMP-9” or “matrix metalloproteinase-9”, or “type IV collagenase” or “Gelatinase-B”, “prognosis”. To be eligible for inclusion in this systematic review, a study must meet the following criteria: (1) The histologic type of the tumors was NSCLC, (2) it assesses the relationship between MMP-9 and survival, and has been published as a full paper, (3) it has a follow-up time exceeding 3 years, (4) hazard ratio (HR) and its 95% confidence interval (95% CI) comparing patients with MMP-9 expression with patients without expression were described or statistically extractable from data in the article. When duplicate articles were published, only the newest or most informative single article was selected. 2.2. Data extraction To minimize the bias and to improve the reliability, 2 reviewers checked all potentially relevant studies independently. Potential disagreements were resolved by consensus. The following characteristics were extracted from eligible studies: name of first author, year of publication, sample size, test method, cut-off value, tumor stage, histologic type and HR estimation. 2.3. Statistical analysis HR with 95% CI was used to combine the data. When described in original articles, these values were obtained directly. When these statistical variables were not provided, they were calculated from available numerical data or Kaplan–Meier survival curve. In these cases, after dividing the time axis into non-overlapping intervals, log HR and its variance for each interval were calculated. These estimated values were combined in a stratified manner to obtain the overall HR and 95% CI [16]. The statistical heterogeneity within studies was tested with the chi-squared based Q-test, and absence of heterogeneity across studies was identified, then the fixed-effects model was used. Otherwise, the random-effects model was used. We also quantified the effect of heterogeneity using I2 statistic which measured the degree of heterogeneity. I2 value ranges from 0% to 100% (I2 = 0–25%, no heterogeneity; I 2 = 25–50%, moderate heterogeneity; I2 = 50–75%, large heterogeneity; I 2 = 75–100%, extreme heterogeneity) [17]. Evidence of publication bias was sought by the methods of Egger et al. [18] and Begg et al. [19]. The significance of the intercept was determined by the t-test suggested by Egger (pb 0.05 was considered representative of statistically
significant publication bias). All of the calculations were performed using STATA version 9.0 (Stata Corp., College Station, TX). 3. Results 3.1. Study characteristics A total of 24 studies were identified that met the inclusion criteria. When extracting data, three studies were excluded because of investigating MMP-9 expression with LC survival. Five studies were excluded because of insufficient information to calculate the HR. One study [20] provided information about AD and SQ, which we treated independently. Therefore, 17 studies [14,15,20–33] containing 2029 NSCLC patients were subjected to final analysis. Of these studies, the sample sizes ranged from 31 to 178 patients. The positive rate of MMP-9 expression varied from 18.18% to 68.92%. Expression of MMP-9 was evaluated by Immunohistochemistry (IHC) in fifteen studies [14,15,21–33], by reverse transcriptase polymerase chain reaction (RTPCR) in 2 studies [20]. In 12 of the 17 studies [14,15,20–22,25,27– 30,33], the useful data for calculation were obtained directly from the original articles, and data were calculated from available information in 2 original articles [23,24], in other 3 studies [26,31,32], HRs had to be extrapolated from Kaplan–Meier survival curve. HRs obtained from multivariate analyses were more accurate than HRs obtained from univariate analyses (if both were available for the studies) because intermixed factors were included in the multivariate analyses. The main characteristics of the 17 eligible publications are reported in Table 1. 3.2. Main results of meta-analysis The main results of the meta-analysis are summarized in Table 2 and Figs. 1–5. Overall, the combined HR for all 17 studies was 1.84 (95% CI: 1.62–2.09), and the test for heterogeneity was nonsignificant (Q = 24.33, I 2 = 34.2%, P = 0.082), suggesting that MMPTable 2 Main results of meta-analysis.
Overall MMP-9 MMP-9 MMP-9 MMP-9
in in in in
NSCLC by IHC adenocarcinoma stages I–IIIA cut off: 20%
No. of studies
Combined HR (95% CI)
Heterogeneity test (Q, I2, P)
17 15 3 9 7
1.84 1.82 2.67 1.66 1.86
24.33, 23.08, 0.19, 13.40, 7.30,
(1.62–2.09) (1.60–2.08) (1.78–4.01) (1.42–1.95) (1.57–2.19)
34.2%, 0.082 39.3%, 0.059 0.0%, 0.909 40.3%, 0.099 17.80%, 0.294
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Fig. 1. Forest plot of meta-analysis of the effect of MMP-9 expression on survival in NSCLC patients.
9 over-expression had a poor prognosis factor for survival in NSCLC. The subgroup analysis was performed according to histology, extent of the disease, test method, and threshold. When we limited the method used to detect MMP-9 to immunohistochemistry (IHC), the result was significant association (HR: 1.82, 95% CI: 1.60–2.08). Three trials for AD were assessable. The aggregation with fixed-
effect model produced an HR of 2.67 (95% CI: 1.78–4.01). To treat separately nine studies reporting on stages I–IIIA patients, we still observed a statistically detrimental effect (HR: 1.66, 95% CI: 1.42–1.95). Finally, according to the threshold of 20%, HR for the seven studies assessing MMP-9 expression with fixed-effect model was 1.86 (95% CI: 1.57– 2.19). Heterogeneity was absent in each subgroup.
Fig. 2. Forest plot of the subgroup analysis by test method (IHC) of MMP-9 expression.
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Fig. 3. Forest plot of the subgroup analysis by histology (AD).
3.3. Publication bias
4. Discussion
Both Begg's funnel plot and Egger's test were performed to assess the publication bias. Begg's funnel plot did not reveal any evidence of obvious asymmetry in the overall meta-analysis (Fig. 6). There was no indication of publication bias from either Egger's (P = NS) or Begg's (P = NS) tests.
Meta-analysis is a quantitative approach in which individual study findings on the same topic are statistically integrated and analyzed. Recently, it has been used for the evaluation of cancer prognostic markers [34]. In the current meta-analysis, we have combined 17 eligible studies including 2029 cases comparing the survival of NSCLC according to MMP-9 expression level of the primary tumor, indicating that MMP-9 over-expression has a significant correlation with poor survival in patients with NSCLC. Our data help to clarify the results of individual studies arguing with the hypothesis that high MMP-9 expression is a prognostic factor for non-small cell lung cancer, in addition to improve understanding of lung cancer biology and identification of highrisk subgroup patients for whom specific therapy might be necessary.
3.4. Sensitivity analysis A sensitivity analysis, in which one study was removed at a time, was performed to evaluate result stability. The corresponding pooled HRs were not significantly altered, suggesting stability of our results (Table 3).
Fig. 4. Forest plot of the subgroup analysis by disease stage (I–IIIA).
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Fig. 5. Forest plot of the subgroup analysis by threshold (20%) of MMP-9 positive.
In fact, penetration of basement membranes and degradation of ECM are essential steps in the dissemination of tumor cells from primary tumors to distant organs. MMP-9 has been associated with invasion and metastasis because of its potency to degrade gelatin and type IV collagen as major components of basement membranes [15]. Many experiments reported that it was a poor prognostic factor for survival in NSCLC. Pinto et al. [28] suggested that tumor staining for MMP-9 in resected adenocarcinoma of the lung is strongly related to survival. Shao et al. [29] found that MMP-9 expression maintained independent prognostic influence on overall survival. Our meta-analysis also demonstrated a statistical significance of MMP-9 expression as a poor prognostic factor for overall survival in patients with NSCLC. Tumor stage is an important prognostic indicator for NSCLC [35]. Survival probability continues to be influenced by the stage of NSCLC at diagnosis. Subgroup analysis of I–IIIA stages showed a significant association was found for the prognosis of NSCLC, which revealed that high MMP-9 expression might be associated with poor prognosis in early stage. Though heterogeneity across studies in our study was nonsignificant, we could not fully neglect potential heterogeneity. The
Begg's funnel plot with pseudo 95% confidence limits
Table 3 Results of the sensitivity analyses.
log[var1]
2
1
0
-1 0
.2
.4
.6
methods used for the assessment of the level of MMP-9 in NSCLC patients differed among these studies. The most common applied method was IHC. IHC results depend on the primary antibody used. In this review, a range of antibodies was used to detect the protein. The dilution of the antibody also differed, leading to a potential problem, because the sensitivity of the method can depend on the antibody concentration. Due to the small groups of studies evaluating the same antibody, subgroup analysis could not be performed to explore this technical problem. Besides, there were some differences among these studies in cut-off of defining the specimens as MMP-9 positivity. We performed the subgroup analysis by threshold of 20%, heterogeneity was reduced. There was not a common threshold value in defining positive expression in assessment of MMP-9 in NSCLC patients, which might produce heterogeneity. Other factors could be dilution of antibody, length of follow-up, and inconsistency of clinical and pathological features. Publication bias is a major concern for all forms of meta-analysis. Positive results tend to be accepted by journals, while negative results are often rejected or not even submitted. The present analysis did not support publication bias, so the obtained summary statistics may approximate the actual average. However, it should be noted that our
.8
s.e. of: log[var1] Fig. 6. Begg's funnel plot of the 17 eligible studies assessing MMP-9 in NSCLC.
Excluded studies
HR (95% CI)
Cox G Swinson DEB Chen XL Kim SJ Swinson DEB Shou Y Takemoto N (1) Takemoto N (2) Ishikawa S Cox G Pinto CA Sienel W Shao WL Martins SJ Grossi F Shimanuki Y Swinson DEB
1.79 1.85 1.81 1.86 1.88 1.85 1.83 1.83 1.95 1.86 1.80 1.84 1.79 1.82 1.82 1.83 1.89
(1.56–2.05) (1.61–2.12) (1.59–2.07) (1.63–2.12) (1.64–2.15) (1.62–2.11) (1.61–2.09) (1.60–2.08) (1.70–2.23) (1.62–2.13) (1.57–2.05) (1.61–2.10) (1.56–2.06) (1.59–2.07) (1.60–2.08) (1.60–2.09) (1.64–2.17)
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meta-analysis could not completely exclude biases. For example, the study was restricted to articles published in English and Chinese, which probably led to bias. Besides, some studies, due to lack of the data necessary for aggregation, were excluded from the meta-analysis which also brought out bias. We had to state the method for extrapolating the HR. If not reported in original articles, HRs were calculated from the data available in the published article and, if this was not possible, extrapolated from the survival curves according to the method of Parmar et al. [16]. Though reading the survival rates on the graphical representation of the survival curves was performed independently by two authors, this strategy did not completely eliminate inaccuracy in the extracted survival rates. Consequently, the estimated HR might be less reliable than when obtained from published statistics. However, we compared our estimated HRs and their statistical significance with published results and did not identify any major contradiction. In conclusion, our study suggests that MMP-9 over-expression is a poor prognostic factor for survival in patients with NSCLC. Our results were based on the aggregation of data obtained in univariate survival analyses performed in retrospective studies. Considering the heterogeneities of included studies and bias of meta-analysis, our conclusions are to be tempered. In order to become a useful prognostic factor in the clinical practice, a standardization of the IHC technique is needed, particularly concerning the positivity threshold. In addition, the present results need to be confirmed by an adequately designed prospective study with an appropriate multivariate analysis taking into account the classical well-defined prognostic factors for NSCLC. Acknowledgment This work was partly supported by grants from the Academic Leader Foundation of Anhui Medical University and the Key Project of the Education Department of Anhui Province Natural Science Research (Code: KJ2012A165). References [1] Jemal A, Siegel R, Ward E, et al. Cancer statistics, 2009. CA Cancer J Clin 2009;59: 225–49. [2] Alberg AJ, Ford JG, Samet JM, American College of Chest Physicians. Epidemiology of lung cancer: ACCP evidence-based clinical practice guidelines (2nd edition). Chest 2007;132(3 Suppl.):29S–55S. [3] Esposito L, Conti D, Ailavajhala R, Khalil N, Giordano A. Lung cancer: are we up to the challenge? Curr Genomics Nov 2010;11(7):513–8. [4] Paesmans M, Sculier JP, Libert P, et al. Prognostic factors for survival in advanced non-small-cell lung cancer: univariate and multivariate analyses including recursive partitioning and amalgamation algorithms in 1,052 patients. The European Lung Cancer Working Party. J Clin Oncol 1995;13:1221–30. [5] Qian Q, Wang Q, Zhan P, et al. The role of matrix metalloproteinase 2 on the survival of patients with non-small cell lung cancer: a systematic review with metaanalysis. Cancer Invest 2010;28:661–9. [6] Martin B, Paesmans M, Berghmans T, et al. Role of Bcl-2 as a prognostic factor for survival in lung cancer: a systematic review of the literature with meta-analysis. Br J Cancer 2003;89(1):55–64. [7] Zhuang Y, Yin HT, Yin XL, Wang J, Zhang DP. High p27 expression is associated with a better prognosis in East Asian non-small cell lung cancer patients. Clin Chim Acta 2011;412(23–24):2228–31. [8] Basset P, Bellocq JP, Wolf C, et al. A novel metalloproteinases gene specifically expressed in stromal cells of breast carcinomas. Nature 1990;348:699–704. [9] Fujise N, Nanashima A, Taniguchi Y, et al. Prognostic impact of Cathepsin B and matrix metalloproteinase-9 in pulmonary adenocarcinomas by immunohistochemical study. Lung Cancer 2000:19–26.
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Contents lists available at SciVerse ScienceDirect
Clinica Chimica Acta journal homepage: www.elsevier.com/locate/clinchim
Prognostic value of matrix metalloproteinase 9 expression in patients with non-small cell lung cancer Wen-Jia Peng a, 1, Jun-Qing Zhang b, 1, Bing-Xiang Wang a, Hai-Feng Pan a, Man-Man Lu a, Jing Wang a,⁎ a b
Department of Epidemiology and Biostatistics, School of Public Health, Anhui Medical University, Hefei, China Center for Disease Control and Prevention of Hefei City, Hefei, China
a r t i c l e
i n f o
Article history: Received 12 February 2012 Received in revised form 12 March 2012 Accepted 12 March 2012 Available online 20 March 2012 Keywords: MMP-9 Prognosis Non-small cell lung cancer Meta-analysis Hazard ratio
a b s t r a c t Background: The role of matrix metalloproteinase 9 (MMP-9) expression in non-small cell lung cancer (NSCLC) remains controversial. We performed a systematic review of the literature with meta-analysis. Methods: Electronic databases were used to identify published studies before December 1, 2011. Pooled hazard ratio (HR) with 95% confidence interval (95% CI) was used to estimate the strength of the association between MMP-9 expression survival of NSCLC patients. Heterogeneity and publication bias were also assessed. Results: The final analysis of 2029 NSCLC cases from 17 studies is presented. The combined HR of 1.84 (95% CI: 1.62–2.09) suggested that MMP-9 over-expression had a poor prognosis in patients with NSCLC. Subgroup analyses also detected significant association. Heterogeneity and publication bias was absent in current meta-analysis. Sensitivity analyses suggested that the summary statistics obtained should approximate the actual average. Conclusion: High MMP-9 expression is associated with a poor prognosis in patients with NSCLC. © 2012 Elsevier B.V. All rights reserved.
1. Introduction Lung cancer (LC) remains the most frequent and the most lethal human malignancy, accounting for 1.1 million deaths annually worldwide [1]. Despite diagnostic and therapeutic improvements, the prognosis for lung cancer patients is generally poor, with an overall five-year survival rate of approximately 15% [2]. Non-small cell lung cancer (NSCLC) accounts for approximately 85% of all LC cases, comprising adenocarcinoma (AD), squamous cell carcinoma (SQ) and carcinoid [3]. Prognostic factors for survival are useful in the management of NSCLC patients. Several independent prognostic factors for the survival of NSCLC have been identified such as performance status, TNM stage, age, sex, and weight loss [4]. These factors are useful when choosing the treatment on an individual basis, principally disease stage and performance status. The biological factors involved in carcinogenesis should also be considered as potential survival prognostic factors. In recent years, substantial efforts have been made to identify prognostic factors in order to individualize treatment and improve prognosis. Many molecular markers have been implicated both in the pathogenesis and prognosis of NSCLC, such as MMP-2 [5], Bcl-2 [6], and P27 [7]. Matrix metalloproteinases (MMPs) are able to degrade extracellular matrix (ECM) components and promote the formation of
⁎ Corresponding author at: Department of Epidemiology and Biostatistics, School of Public Health, Anhui Medical University, 81 Meishan Road, Hefei, Anhui 230032, China. Tel.: + 86 551 5161175; fax: + 86 551 5161126. E-mail address: [email protected] (J. Wang). 1 Contributed equally to this work. 0009-8981/$ – see front matter © 2012 Elsevier B.V. All rights reserved. doi:10.1016/j.cca.2012.03.012
metastasis. MMPs have an important role in tumor invasion and angiogenesis [8]. Among all identified MMPs, MMP-9 is a collagenase with a molecular mass of 92 kDa which catalyzes type IV collagen, a major structural protein of basement membranes, including the blood vessel basement membrane, and is chemically and it is genetically distinct from stroma collagen types I and III and cartilage collagen type II. In addition, its degradation plays an important role in tumor cell invasion of the vasculature [9,10]. Therefore, MMP-9 is considered to play a critical role in metastasis. The synthesis and secretion of MMP-9 can be stimulated by a variety of stimuli, including cytokines, during various pathological processes such as tumor invasion, atherosclerosis, inflammation, and rheumatoid arthritis [11]. Increased MMP-9 levels have been associated with poor prognosis in stage IV gastrointestinal cancer [12]. In colorectal cancer the tissue levels of MMP-9 predicted relapse and survival [13]. Therefore, MMP-9 should be a promising prognostic marker. Enhanced expression of MMP-9 in cancer cells has been noticed to be a significant factor to predict poor survival in NSCLC [14,15]. However, the results are inconsistent. 2. Materials and methods 2.1. Identification of studies The electronic database of PubMed, Embase and Web of science were searched for studies that investigated the prognostic significance of MMP-9 in NSCLC to be included in the present metaanalysis. References therein were also manually reviewed for articles potentially missed in the initial search. Studies were examined
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Table 1 Main characteristics and results of the eligible studies. First author
Year
Histology
Stage
No. of pts
Method
Cut off
High expression
HR estimation
Cox G Swinson DEB Chen XL Kim SJ Swinson DEB Shou Y Takemoto N (1) Takemoto N (2) Ishikawa S Cox G Pinto CA Sienel W Shao WL Martins SJ Grossi F Shimanuki Y Swinson DEB
2001 2004 2008 2005 2004 2001 2007 2007 2004 2000 2003 2003 2011 2009 2010 2005 2002
NSCLC (SQ: 103, AD: 50, LCC: 14) NSCLC (AD: 47, SQ: 101, LCC: 14, Other:4) NSCLC (SQ: 21, AD: 20) NSCLC (SQ: 38, AD: 26, LCC: 6, Other: 4) NSCLC (SQ: 107, AD: 49, LCC: 12, Other: 4) NSCLC (AD: 80, SQ: 37, ADC: 1, LCC: 1) AD SQ NSCLC (SQ: 77, AD: 122, LCC: 11, Other: 8) NSCLC (SQ: 105, AD: 50, LCC: 14) AD NSCLC (AD: 60, SQ: 57, Other: 26) NSCLC (SQ: 21, Other: 125) AD NSCLC (AD: 33, SQ: 50, LCC: 4) NSCLC (AD: 43, SQ: 15, LCC: 1, Other: 4) NSCLC (AD: 49, LCC: 14, SQ: 111, Other: 4)
I–IIIA I–IIIA I–III I–II I–IIIA I–III I–IIIA I–IIIA I–IIIA I–IIIA I–III NA IA IIIB–IV IIIA I–IIIA I–IIIA
167 166 41 74 172 119 46 37 218 169 152 143 146 51 87 63 178
IHC IHC IHC IHC IHC IHC RT-PCR RT-PCR IHC IHC IHC IHC IHC IHC IHC IHC IHC
20% 20% 50% 30% 20% NA NA NA 25% 20% 20% 95% 25% 80% 20% NA 20%
87 (52.10%) 87 (52.41%) 27 (65.85%) 51 (68.92%) 84 (48.84%) 75 (67.57%) 28 (60.87%) 14 (37.84%) NA 92 (54.44%) 76 (50.00%) 26 (18.18%) 89 (60.96%) 15 (29.41%) 43 (49.43%) 30 (49.18%) 88 (49.44%)
HR + 95% CI HR + 95% CI Available data Available data HR + 95% CI Survival curve HR + 95% CI HR + 95% CI HR + 95% CI HR + 95% CI HR + 95% CI HR + 95% CI HR + 95% CI HR + 95% CI Survival curve Survival curve HR + 95% CI
IHC: Immunohistochemistry; RT-PCR: reverse transcriptase-polymerase chain reaction. SQ: Squamous cell cancer; AD: Adenocarcinoma; LCC: Large cell carcinoma; ADC: Adenosquamous carcinoma. Pts: patients.
without language limits imposed with an updating search on December 1, 2011. Search terms were “non-small cell lung cancer” or “NSCLC”, “MMP-9” or “matrix metalloproteinase-9”, or “type IV collagenase” or “Gelatinase-B”, “prognosis”. To be eligible for inclusion in this systematic review, a study must meet the following criteria: (1) The histologic type of the tumors was NSCLC, (2) it assesses the relationship between MMP-9 and survival, and has been published as a full paper, (3) it has a follow-up time exceeding 3 years, (4) hazard ratio (HR) and its 95% confidence interval (95% CI) comparing patients with MMP-9 expression with patients without expression were described or statistically extractable from data in the article. When duplicate articles were published, only the newest or most informative single article was selected. 2.2. Data extraction To minimize the bias and to improve the reliability, 2 reviewers checked all potentially relevant studies independently. Potential disagreements were resolved by consensus. The following characteristics were extracted from eligible studies: name of first author, year of publication, sample size, test method, cut-off value, tumor stage, histologic type and HR estimation. 2.3. Statistical analysis HR with 95% CI was used to combine the data. When described in original articles, these values were obtained directly. When these statistical variables were not provided, they were calculated from available numerical data or Kaplan–Meier survival curve. In these cases, after dividing the time axis into non-overlapping intervals, log HR and its variance for each interval were calculated. These estimated values were combined in a stratified manner to obtain the overall HR and 95% CI [16]. The statistical heterogeneity within studies was tested with the chi-squared based Q-test, and absence of heterogeneity across studies was identified, then the fixed-effects model was used. Otherwise, the random-effects model was used. We also quantified the effect of heterogeneity using I2 statistic which measured the degree of heterogeneity. I2 value ranges from 0% to 100% (I2 = 0–25%, no heterogeneity; I 2 = 25–50%, moderate heterogeneity; I2 = 50–75%, large heterogeneity; I 2 = 75–100%, extreme heterogeneity) [17]. Evidence of publication bias was sought by the methods of Egger et al. [18] and Begg et al. [19]. The significance of the intercept was determined by the t-test suggested by Egger (pb 0.05 was considered representative of statistically
significant publication bias). All of the calculations were performed using STATA version 9.0 (Stata Corp., College Station, TX). 3. Results 3.1. Study characteristics A total of 24 studies were identified that met the inclusion criteria. When extracting data, three studies were excluded because of investigating MMP-9 expression with LC survival. Five studies were excluded because of insufficient information to calculate the HR. One study [20] provided information about AD and SQ, which we treated independently. Therefore, 17 studies [14,15,20–33] containing 2029 NSCLC patients were subjected to final analysis. Of these studies, the sample sizes ranged from 31 to 178 patients. The positive rate of MMP-9 expression varied from 18.18% to 68.92%. Expression of MMP-9 was evaluated by Immunohistochemistry (IHC) in fifteen studies [14,15,21–33], by reverse transcriptase polymerase chain reaction (RTPCR) in 2 studies [20]. In 12 of the 17 studies [14,15,20–22,25,27– 30,33], the useful data for calculation were obtained directly from the original articles, and data were calculated from available information in 2 original articles [23,24], in other 3 studies [26,31,32], HRs had to be extrapolated from Kaplan–Meier survival curve. HRs obtained from multivariate analyses were more accurate than HRs obtained from univariate analyses (if both were available for the studies) because intermixed factors were included in the multivariate analyses. The main characteristics of the 17 eligible publications are reported in Table 1. 3.2. Main results of meta-analysis The main results of the meta-analysis are summarized in Table 2 and Figs. 1–5. Overall, the combined HR for all 17 studies was 1.84 (95% CI: 1.62–2.09), and the test for heterogeneity was nonsignificant (Q = 24.33, I 2 = 34.2%, P = 0.082), suggesting that MMPTable 2 Main results of meta-analysis.
Overall MMP-9 MMP-9 MMP-9 MMP-9
in in in in
NSCLC by IHC adenocarcinoma stages I–IIIA cut off: 20%
No. of studies
Combined HR (95% CI)
Heterogeneity test (Q, I2, P)
17 15 3 9 7
1.84 1.82 2.67 1.66 1.86
24.33, 23.08, 0.19, 13.40, 7.30,
(1.62–2.09) (1.60–2.08) (1.78–4.01) (1.42–1.95) (1.57–2.19)
34.2%, 0.082 39.3%, 0.059 0.0%, 0.909 40.3%, 0.099 17.80%, 0.294
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Fig. 1. Forest plot of meta-analysis of the effect of MMP-9 expression on survival in NSCLC patients.
9 over-expression had a poor prognosis factor for survival in NSCLC. The subgroup analysis was performed according to histology, extent of the disease, test method, and threshold. When we limited the method used to detect MMP-9 to immunohistochemistry (IHC), the result was significant association (HR: 1.82, 95% CI: 1.60–2.08). Three trials for AD were assessable. The aggregation with fixed-
effect model produced an HR of 2.67 (95% CI: 1.78–4.01). To treat separately nine studies reporting on stages I–IIIA patients, we still observed a statistically detrimental effect (HR: 1.66, 95% CI: 1.42–1.95). Finally, according to the threshold of 20%, HR for the seven studies assessing MMP-9 expression with fixed-effect model was 1.86 (95% CI: 1.57– 2.19). Heterogeneity was absent in each subgroup.
Fig. 2. Forest plot of the subgroup analysis by test method (IHC) of MMP-9 expression.
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Fig. 3. Forest plot of the subgroup analysis by histology (AD).
3.3. Publication bias
4. Discussion
Both Begg's funnel plot and Egger's test were performed to assess the publication bias. Begg's funnel plot did not reveal any evidence of obvious asymmetry in the overall meta-analysis (Fig. 6). There was no indication of publication bias from either Egger's (P = NS) or Begg's (P = NS) tests.
Meta-analysis is a quantitative approach in which individual study findings on the same topic are statistically integrated and analyzed. Recently, it has been used for the evaluation of cancer prognostic markers [34]. In the current meta-analysis, we have combined 17 eligible studies including 2029 cases comparing the survival of NSCLC according to MMP-9 expression level of the primary tumor, indicating that MMP-9 over-expression has a significant correlation with poor survival in patients with NSCLC. Our data help to clarify the results of individual studies arguing with the hypothesis that high MMP-9 expression is a prognostic factor for non-small cell lung cancer, in addition to improve understanding of lung cancer biology and identification of highrisk subgroup patients for whom specific therapy might be necessary.
3.4. Sensitivity analysis A sensitivity analysis, in which one study was removed at a time, was performed to evaluate result stability. The corresponding pooled HRs were not significantly altered, suggesting stability of our results (Table 3).
Fig. 4. Forest plot of the subgroup analysis by disease stage (I–IIIA).
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Fig. 5. Forest plot of the subgroup analysis by threshold (20%) of MMP-9 positive.
In fact, penetration of basement membranes and degradation of ECM are essential steps in the dissemination of tumor cells from primary tumors to distant organs. MMP-9 has been associated with invasion and metastasis because of its potency to degrade gelatin and type IV collagen as major components of basement membranes [15]. Many experiments reported that it was a poor prognostic factor for survival in NSCLC. Pinto et al. [28] suggested that tumor staining for MMP-9 in resected adenocarcinoma of the lung is strongly related to survival. Shao et al. [29] found that MMP-9 expression maintained independent prognostic influence on overall survival. Our meta-analysis also demonstrated a statistical significance of MMP-9 expression as a poor prognostic factor for overall survival in patients with NSCLC. Tumor stage is an important prognostic indicator for NSCLC [35]. Survival probability continues to be influenced by the stage of NSCLC at diagnosis. Subgroup analysis of I–IIIA stages showed a significant association was found for the prognosis of NSCLC, which revealed that high MMP-9 expression might be associated with poor prognosis in early stage. Though heterogeneity across studies in our study was nonsignificant, we could not fully neglect potential heterogeneity. The
Begg's funnel plot with pseudo 95% confidence limits
Table 3 Results of the sensitivity analyses.
log[var1]
2
1
0
-1 0
.2
.4
.6
methods used for the assessment of the level of MMP-9 in NSCLC patients differed among these studies. The most common applied method was IHC. IHC results depend on the primary antibody used. In this review, a range of antibodies was used to detect the protein. The dilution of the antibody also differed, leading to a potential problem, because the sensitivity of the method can depend on the antibody concentration. Due to the small groups of studies evaluating the same antibody, subgroup analysis could not be performed to explore this technical problem. Besides, there were some differences among these studies in cut-off of defining the specimens as MMP-9 positivity. We performed the subgroup analysis by threshold of 20%, heterogeneity was reduced. There was not a common threshold value in defining positive expression in assessment of MMP-9 in NSCLC patients, which might produce heterogeneity. Other factors could be dilution of antibody, length of follow-up, and inconsistency of clinical and pathological features. Publication bias is a major concern for all forms of meta-analysis. Positive results tend to be accepted by journals, while negative results are often rejected or not even submitted. The present analysis did not support publication bias, so the obtained summary statistics may approximate the actual average. However, it should be noted that our
.8
s.e. of: log[var1] Fig. 6. Begg's funnel plot of the 17 eligible studies assessing MMP-9 in NSCLC.
Excluded studies
HR (95% CI)
Cox G Swinson DEB Chen XL Kim SJ Swinson DEB Shou Y Takemoto N (1) Takemoto N (2) Ishikawa S Cox G Pinto CA Sienel W Shao WL Martins SJ Grossi F Shimanuki Y Swinson DEB
1.79 1.85 1.81 1.86 1.88 1.85 1.83 1.83 1.95 1.86 1.80 1.84 1.79 1.82 1.82 1.83 1.89
(1.56–2.05) (1.61–2.12) (1.59–2.07) (1.63–2.12) (1.64–2.15) (1.62–2.11) (1.61–2.09) (1.60–2.08) (1.70–2.23) (1.62–2.13) (1.57–2.05) (1.61–2.10) (1.56–2.06) (1.59–2.07) (1.60–2.08) (1.60–2.09) (1.64–2.17)
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meta-analysis could not completely exclude biases. For example, the study was restricted to articles published in English and Chinese, which probably led to bias. Besides, some studies, due to lack of the data necessary for aggregation, were excluded from the meta-analysis which also brought out bias. We had to state the method for extrapolating the HR. If not reported in original articles, HRs were calculated from the data available in the published article and, if this was not possible, extrapolated from the survival curves according to the method of Parmar et al. [16]. Though reading the survival rates on the graphical representation of the survival curves was performed independently by two authors, this strategy did not completely eliminate inaccuracy in the extracted survival rates. Consequently, the estimated HR might be less reliable than when obtained from published statistics. However, we compared our estimated HRs and their statistical significance with published results and did not identify any major contradiction. In conclusion, our study suggests that MMP-9 over-expression is a poor prognostic factor for survival in patients with NSCLC. Our results were based on the aggregation of data obtained in univariate survival analyses performed in retrospective studies. Considering the heterogeneities of included studies and bias of meta-analysis, our conclusions are to be tempered. In order to become a useful prognostic factor in the clinical practice, a standardization of the IHC technique is needed, particularly concerning the positivity threshold. In addition, the present results need to be confirmed by an adequately designed prospective study with an appropriate multivariate analysis taking into account the classical well-defined prognostic factors for NSCLC. Acknowledgment This work was partly supported by grants from the Academic Leader Foundation of Anhui Medical University and the Key Project of the Education Department of Anhui Province Natural Science Research (Code: KJ2012A165). References [1] Jemal A, Siegel R, Ward E, et al. Cancer statistics, 2009. CA Cancer J Clin 2009;59: 225–49. [2] Alberg AJ, Ford JG, Samet JM, American College of Chest Physicians. Epidemiology of lung cancer: ACCP evidence-based clinical practice guidelines (2nd edition). Chest 2007;132(3 Suppl.):29S–55S. [3] Esposito L, Conti D, Ailavajhala R, Khalil N, Giordano A. Lung cancer: are we up to the challenge? Curr Genomics Nov 2010;11(7):513–8. [4] Paesmans M, Sculier JP, Libert P, et al. Prognostic factors for survival in advanced non-small-cell lung cancer: univariate and multivariate analyses including recursive partitioning and amalgamation algorithms in 1,052 patients. The European Lung Cancer Working Party. J Clin Oncol 1995;13:1221–30. [5] Qian Q, Wang Q, Zhan P, et al. The role of matrix metalloproteinase 2 on the survival of patients with non-small cell lung cancer: a systematic review with metaanalysis. Cancer Invest 2010;28:661–9. [6] Martin B, Paesmans M, Berghmans T, et al. Role of Bcl-2 as a prognostic factor for survival in lung cancer: a systematic review of the literature with meta-analysis. Br J Cancer 2003;89(1):55–64. [7] Zhuang Y, Yin HT, Yin XL, Wang J, Zhang DP. High p27 expression is associated with a better prognosis in East Asian non-small cell lung cancer patients. Clin Chim Acta 2011;412(23–24):2228–31. [8] Basset P, Bellocq JP, Wolf C, et al. A novel metalloproteinases gene specifically expressed in stromal cells of breast carcinomas. Nature 1990;348:699–704. [9] Fujise N, Nanashima A, Taniguchi Y, et al. Prognostic impact of Cathepsin B and matrix metalloproteinase-9 in pulmonary adenocarcinomas by immunohistochemical study. Lung Cancer 2000:19–26.
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