Clinicopathological significance of CXCR4 in non-small cell lung cancer

Introduction

Lung cancer is the second most common cancer and is the leading cause of cancer-related death in both men and women in the United States and throughout the world.¹ Non-small cell lung cancer (NSCLC) – which includes the following histologic types: adenocarcinoma (AD), squamous cell carcinoma (SCC), large cell carcinoma, and mixed histologies – accounts for approximately 85% of all lung cancer and is the leading cause of cancer-related deaths worldwide.^2,3 Despite the advances in early detection, radical cure operations, and multimodal therapeutic modalities, at diagnosis, there are about 80% of NSCLC cases in advanced stage, for which systemic chemotherapy remains the standard care but provides marginal improvement in survival.⁴ There is a major unmet medical need for effective and well-tolerated treatment options for patients with advanced NSCLC. Therefore, investigation into the mechanism of initiation and progression, as well as identification of prognostic markers, is still needed, and will help to identify patients with a high chance of lung cancer recurrence and provide better prognosis and individualization of treatment.

Chemokines are a group of small proteins that play a role in the immune system and in the progression of tumors; they are associated with cytoskeletal rearrangements, cell migration during development and physiology, and host immune response via interactions with G protein-coupled receptors.^5,6 The expression of chemokines leading to aberrant chemokine receptor signaling is altered in many malignancies. C-X-C chemokine receptor type 4 (CXCR4), also known as fusin or CD184, is an alpha-chemokine receptor specific for stromal-derived-factor-1 (SDF-1, also called CXCL12).^7,8 CXCR4 belongs to the superfamily of seven transmembrane domain heterotrimeric G protein-coupled receptors, is functionally expressed on the cell surface of various types of cancer cells, and plays a role in the cell proliferation and migration of these cells.^9–11 Recently, CXCR4 has been reported to play an important role in cell survival, proliferation, and migration, as well as in metastasis of several cancers including breast,^12–14 cervical,^15,16 colon or rectal,^17,18 esophageal,^18–20 gall bladder,²¹ kidney,^22–24 liver,^25,26 ovarian,^27,28 pancreatic,²⁹ prostate,^30–32 stomach,^33,34 uterine cancer,³⁵ multiple myeloma,^36,37 non-Hodgkin lymphoma,³⁸ etc.

A number of studies also showed that CXCR4 is also overexpressed in NSCLC and may account for its progression, metastasis, and prognosis.^39–43 However, there still exists controversy. Wagner et al found that positive cytomembranous staining for CXCR4 is an independent indicator of poor prognosis, while nuclear staining confers a survival benefit to patients with AD of the lung.⁴⁴ In addition, the roles of CXCR4 in NSCLC and clinical significance have not been thoroughly investigated. Meta-analysis has great advantages in confirming prognostic and pathological factors in cancer patients by avoiding the disadvantages of small sample size. Therefore, it is necessary to perform a pooled analysis using the results of published articles. In this study, we have analyzed and updated the published clinical investigations regarding the effect of CXCR4 on patients with NSCLC.

Materials and methods

Search strategy and selection criteria

We searched PubMed, Embase, and ISI Web of Knowledge to identify studies from January 1, 2000 to June 1, 2014 using the search terms: “lung” and “cancer” or “tumor” or “neoplasm” or “carcinoma”; “expression” and “CXCR4” or “C-X-C chemokine receptor type 4”; and “prognosis” or “prognostic” or “outcome”. We also hand-searched the reference lists of the retrieved articles and reviews for additional articles. Conference abstracts were not selected for our analysis due to the insufficient data reported in them. After exclusion of nonrelevant and/or redundant publications from the different databases, the remaining papers were evaluated in the full text version for inclusion and exclusion criteria and for relevant articles in the reference lists. All searched data were retrieved. Authors’ bibliographies and references of selected studies were also searched for other relevant studies. The most complete study was chosen to avoid duplication if the same patient populations were reported in several publications.

The criteria that an eligible study had to meet were as follows: 1) CXCR4 expression evaluated in the primary NSCLC tissues; 2) researches revealed the relationship between CXCR4 expression and NSCLC clinicopathological parameters and prognosis; and 3) studies provided sufficient information to estimate hazard ratio (HR) about overall survival (OS) and 95% confidence interval (CI). The exclusion criteria included the following: 1) publications that were letters, reviews, case reports, conference abstracts, editorials, or expert opinion; and 2) all publications regarding in vitro/ex vivo studies, cell lines, and human xenografts.

Data extraction and methodological assessment

Two authors (XMZ and LH) independently reviewed and extracted data from eligible studies. Disagreements were resolved by discussion and consensus. Two authors (XZ and LH) reviewed all of the articles that fit the inclusion and exclusion criteria. The following information was recorded for each study: the first author name, year of publication, sample source, number of cases, clinicopathological parameters, cancer tumor node metastasis stage, immunohistochemical staining method, antibody source, percentage rate of expression, and follow-up. Data for study characteristics and clinical responses were summarized and the data turned into table format. Heterogeneity of investigation was evaluated to determine whether the data of the various studies could be analyzed for a meta-analysis.

For the methodological evaluation of the studies, three investigators (XZ, LH, and GH) read through each publication independently, and assessed and scored them according to REMARK guidelines and the ELCWP quality scale.^45,46 The three readers provided the quality scores and compared them, and then reached a consensus value for each item.

Statistical analysis

Analysis was conducted using the STATA 12.0 (StataCorp LP, College Station, TX, USA) and Review Manager 5.2 (Cochrane Collaboration, Oxford, UK). The pooled frequency of CXCR4 expression and 95% CI were estimated. The frequency of CXCR4 expression was compared in different tumor characteristics. Heterogeneity among studies was evaluated with Cochran’s Q test⁴⁷ and the I² statistic.^48,49 When heterogeneity was not an issue (I² values <50%), a fixed effect model was used to calculate parameters. If there was substantial heterogeneity (I² values ≥50%), a random-effects model was used to pool data and attempt to identify potential sources of heterogeneity based on subgroup analyses. The pooled odds ratio (OR) was estimated for the association between CXCR4 expression and clinicopathological features. P-values tailed less than 0.05 were considered statistically significant.

Publication bias was assessed by using a method reported by Egger et al.⁵⁰ We also explored reasons for statistical heterogeneity using metaregression, subgroup analysis, and sensitivity analysis. The analysis of metaregression and publication bias was performed by using STATA version 10.0.

Results

Identification of relevant studies

Sixty-five publications were identified by the search method as described above. Fifty-two of those were excluded due to being laboratory studies, non-original articles (review), or studies irrelevant to the current analysis. Eventually, there were 13 studies included in final meta-analysis, as shown in Figure 1.

Figure 1 Flow chart of study selection.

Study characteristics

Thirteen studies published from 2004 to 2014 were eligible for meta-analysis. A total of 1,446 NSCLC patients from People’s Republic of China, Japan, France, Germany, Canada, and USA were enrolled. Their basic characteristics are summarized in Table 1. Positive CXCR4 expression was defined by immunohistochemistry. Most of studies defined positive CXCR4 expression as when any part of nucleus or cytoplasm was stained. There was difference in defining the cutoff value of positive CXCR4 expression. The majority of studies defined the cutoff value by combining intensity and percentage of CXCR4 expression, while the rest merely used the percentage of stained cells.

Table 1 Basic characteristics of the included studies Notes: Abcam Ltd.: Abcam Limited Biotechnology Europe, UK. Beijing Boao Sen: Beijing Boao Sen Biotechnology Co., Ltd. PRC. Biotrend: Biotrend Chemicals, LLC, Destin FL, USA. Boster: Boster, Pleasanton, CA, USA. Leinco Technologies: Leinco Technologies, Inc., St. Louis, MO, USA. R&D: R&D Systems, Inc., Minneapolis, MN, USA. Abbreviations: IHC, immunohistochemistry; RT-PCR, reverse transcription polymerase chain reaction; PRC, People’s Republic of China; CXCR4, chemokine receptor type 4;+, positive.

The correlation of CXCR4 expression with clinicopathological features

Increased CXCR4 expression in NSCLC

We first determined whether CXCR4 expression is significantly higher in NSCLC than in normal lung tissue. The pooled OR from five studies including 380 NSCLC and 118 normal lung tissue, as shown in Figure 2 (OR=12.86, 95% CI=3.63–45.59, P<0.0001), indicated that CXCR4 expression is significantly higher in NSCLC than normal lung tissue.

Figure 2 The pooled OR from five studies including 380 NSCLC and 118 normal lung tissue. Notes: OR=12.86, 95% CI=3.63–45.59, P<0.0001. Abbreviations: CI, confidence interval; M-H, Mantel-Haenszel; NSCLC, non-small cell lung cancer; OR, odds ratio.

Relationship between the frequency of CXCR4 expression and smoking status

We then determined whether CXCR4 expression rate in NSCLC patients without a smoking history is significantly higher than that in patients with a smoking history. The pooled OR from four studies including 323 and 173 NSCLC with or without smoking history, respectively, as shown in Figure 3 (OR=1.22, 95% CI=0.79–1.89, P=0.37), indicated that CXCR4 expression was not strongly associated with the smoking status in NSCLC patients.

Figure 3 Four hundred and ninety-six NSCLC patients with the smoking status pooled in four studies. Notes: Aberrant CXCR4 expression was not strongly associated with the smoking status in NSCLC patients (OR=1.22, 95% CI=0.79–1.89, P=0.37). Abbreviations: CI, confidence interval; M-H, Mantel-Haenszel; NSCLC, non-small cell lung cancer; OR, odds ratio; CXCR4, chemokine receptor type 4.

Relationship between the frequency of CXCR4 expression and type of pathology

As previously reported, there was an obvious difference between CXCR4 expression in AD and SCC, indicating that CXCR4 expression in SCC may be associated with the development of these tumors.^51,52 The pooled OR from eleven studies including 296 SCC and 486 AD is shown in Figure 4 (OR=1.46, 95% CI=0.75–2.83, P=0.26), and indicated that CXCR4 expression does not play a big role in the pathogenesis of SCC and AD.

Figure 4 The pooled OR from eleven studies including 296 squamous cell carcinomas and 486 ADs. Notes: OR=1.46, 95% CI=0.75–2.83, P=0.26, which indicated that CXCR4 expression does not play a big difference in the pathogenesis of SCCs and AD. Abbreviations: AD, adenocarcinomas; CI, confidence interval; M-H, Mantel-Haenszel; NSCLC, non-small cell lung cancer; OR, odds ratio; SCCs, squamous cell carcinomas; CXCR4, chemokine receptor type 4.

The role of CXCR4 expression in NSCLC progression

We analyzed 736 NSCLC patients pooled from eight studies to assess whether CXCR4 expression in NSCLC was associated with advanced stage. As shown in Figure 5A, aberrant CXCR4 expression was significantly higher in advanced NSCLC (stages III and IV) than in early-stage NSCLC (stages I and II) (OR=2.33, 95% CI=1.13–4.82, P=0.02). In addition, as shown in Figure 5B, aberrant CXCR4 expression was not significantly higher in poorly differentiated NSCLC than in moderately and highly differentiated NSCLC (OR=1.33, 95% CI=0.89–2.00, P=0.17). These results suggest that CXCR4 expression may not associate with tumor’s differentiated status, but may play an important role in NSCLC progression and development.

Figure 5 Seven hundred and thirty-six NSCLC patients pooled from eight studies to assess whether CXCR4 expression in NSCLC was associated with advanced stage. Notes: (A) CXCR4 expression was significantly higher in advanced NSCLC (stages III and IV) compared to in early-stage NSCLC (stages I and II) (OR=2.33, 95% CI=1.13–4.82, P=0.02). (B) CXCR4 expression was not significantly high in poorly differentiated NSCLC compared to moderately and highly differentiated NSCLC (OR=1.33, CI=0.89–2.00, P=0.17). Abbreviations: CI, confidence interval; M-H, Mantel-Haenszel; NSCLC, non-small cell lung cancer; OR, odds ratio; CXCR4, chemokine receptor type 4.

The role of CXCR4 expression in metastatic NSCLC

We then analyzed 1,049 NSCLC patients pooled from eleven studies to assess whether CXCR4 expression in NSCLC was associated with metastatic status. As shown in Figure 6, aberrant CXCR4 expression was significantly higher in metastatic NSCLC than in nonmetastatic NSCLC (OR=3.74, 95% CI=1.71–8.19, P=0.0009). These results suggest that CXCR4 expression is strongly correlated with metastatic status in NSCLC patients.

Figure 6 One thousand and forty-nine NSCLC patients pooled from eleven studies to assess whether CXCR4 expression in NSCLC was associated with metastatic status. Notes: CXCR4 expression was significantly higher in metastatic NSCLC compared to nonmetastatic NSCLC (OR=3.74, 95% CI=1.71–8.19, P=0.0009). Abbreviations: CI, confidence interval; M-H, Mantel-Haenszel; NSCLC, non-small cell lung cancer; OR, odds ratio; CXCR4, chemokine receptor type 4.

CXCR4 expression as a prognostic factor for NSCLC

Only four studies estimated the relationship between CXCR4 expression and OS in NSCLC. The pooled HR for OS showed that CXCR4 expression was significantly associated with worse survival in NSCLC patients as shown in Figure 7 (HR=3.26, 95% CI=2.22–4.79, P<0.00001).

Figure 7 Four of the included studies investigated the relationship between overall survival (OS) and CXCR4 expression. Notes: The pooled HR for OS showed that CXCR4 expression was significantly associated with worse survival in NSCLC (HR=3.26, 95% CI=2.22–4.79, P<0.00001). Abbreviations: CI, confidence interval; HR, hazard ratio; IV, inverse variance; NSCLC, non-small cell lung cancer; OS, overall survival; SE, standard error; CXCR4, chemokine receptor type 4.

Sensitivity analyses and publication bias

A sensitivity analysis, in which one study was removed at a time, was conducted to assess the stability of the results. The pooled ORs and HRs were not significantly changed, indicating the stability of our analyses. The funnel plots were largely symmetric (Figure 8) suggesting there were no publication biases in the meta-analysis of CXCR4 expression and clinicopathological features.

Figure 8 Funnel plots. Notes: The funnel plots were largely symmetric suggesting there were no publication biases in the meta-analysis of CXCR4 expression and clinicopathological features. (A) The funnel plot from four studies comparing NSCLC and normal lung tissue. (B) The funnel plot from four studies in determining CXCR4 expression and smoking status in NSCLC patients. (C) The funnel plot from eight studies comparing CXCR4 expression between squamous cell carcinoma and adenocarcinoma. (D) The funnel plot from eight studies in determining CXCR4 expression for different stages of NSCLC. (E) The funnel plot from six studies in determining CXCR4 expression in different differentiated NSCLC. (F) The funnel plot from eleven studies in determining the relationship between CXCR4 expression and metastatic status in NSCLC. (G) The funnel plot from four studies in determining the relationship between CXCR4 expression and overall survival in NSCLC. Abbreviations: NSCLC, non-small cell lung cancer; OR, odds ratio; CXCR4, chemokine receptor type 4.

Discussion

Recently, many studies have shown that the presence of CXCR4 on lymphocytes and/or cancer cells can activate chemotaxis and signify proliferation, invasion, metastasis, and angiogenesis in several cancers, including NSCLC.^{9,13,41,53,54} To date, there have been some studies describing the precise expression and prognostic impact of CXCR4 in NSCLC; however, the roles and clinical significance of CXCR4 expression in NSCLC have not been thoroughly investigated. We conducted the meta-analysis to determine the correlation between CXCR4 expression and clinicopathological characteristics in NSCLC. Analyses of the pooled data showed that NSCLC had a higher CXCR4 expression than normal lung tissue. Aberrant CXCR4 expression was significantly higher in advanced NSCLC (stages III and IV) than in early-stage NSCLC (stages I and II). Aberrant CXCR4 expression was significantly higher in metastatic NSCLC than in nonmetastatic NSCLC, and NSCLC patients with CXCR4 expression had a lower survival rate than those without CXCR4 expression. However, CXCR4 expression is not strongly associated with smoking status in NSCLC patients. CXCR4 expression does not make a big difference in the pathogenesis of SCC and AD. In addition, aberrant CXCR4 expression was not significantly higher in poorly differentiated NSCLC than in moderately and highly differentiated NSCLC. The results from the current study demonstrate that the expression rate of CXCR4 in NSCLC was significantly higher than that in the normal lung tissues, indicating that CXCR4 expression was common in NSCLC. CXCR4 expression may not be associated with tumor’s differentiated status, but may play an important role in NSCLC progression and development. In addition, CXCR4 expression is strongly correlated with metastatic status and prognostic outcome in NSCLC patients. Information about the prognostic and predictive value of CXCR4 in NSCLC is limited. To our knowledge, this present meta-analysis is the first study to systematically evaluate the association between CXCR4 expression, clinicopathological features, and prognostic factors in NSCLC.

CXCR4 is pleiotropic during tumor suppression. CXCR4 mediates actin polymerization and pseudopodia formation, as well as induces chemotactic and invasive responses.¹¹ Hence, CXCR4 plays an important role between tumor cells and the tumor microenvironment, with the interaction influencing the adhesion, migration, and invasion of tumor cells, reflecting the strong association of CXCR4 with cancer metastasis. CXCR4 expression on tumor cells is upregulated by hypoxia and angiogenic factors, such as vascular endothelial growth factor (VEGF) and hypoxia-inducible factor 1 (HIF-1); thus, CXCR4 could be a novel target of VEGF-, HIF-1-, and hypoxia-mediated angiogenesis.^55–57 In addition, SDF-1α is the ligand for CXCR4, and SDF-1α, when highly expressed in NSCLC, has a significant role in tumor-relevant stem cell recruitment.^41,58 Therefore, CXCR4 can be considered as an oncogene, and its activation could contribute to tumor progression and poor prognosis. Although only four studies evaluated the relationship between OS and CXCR4 expression in NSCLC, they showed very similar results.^44,51,59,60 Based on this meta-analysis, we may consider that CXCR4 expression in NSCLC tends to indicate a poor prognosis.

Consistent results were shown in sensitivity analyses, and no evidence of publication bias was found. This study has several potential limitations. First, the possibility of information and selection biases as well as unidentified confounders could not be completely excluded because all of the included studies were observational. Second, the searching strategy was restricted to articles published in English and Chinese. Articles with potentially high-quality data that were published in other languages were not included because of anticipated difficulties in obtaining accurate medical translation. Third, the samples and studies were limited by a presence of heterogeneity between the studies. Statistical heterogeneity among the studies may be due to the differences in the baseline characteristics of patients, source of samples, normalization controls, technical platforms, source of antibodies, cut-off values, duration of follow-up, and other technical issues. In addition, the percentage of Asian population in the enrolled patients in some studies (Figures 2 and 5B) reached 90%, raising a question of whether these results apply to other populations. Hence, cautions should be taken when our findings are interpreted among the general populations and in various ethnic populations.

Conclusion

Our meta-analysis showed that high CXCR4 expression was significantly higher in NSCLC than in normal lung tissue. In addition, CXCR4 expression was significantly associated with clinical stages, metastatic status, and OS in NSCLC patients. The results indicate that the aberrant CXCR4 expression plays an important role in the carcinogenesis and metastasis of NSCLC. It is thus safe to say that the remarkable potential of CXCR4 is as a prognostic biomarker for patients with NSCLC. Further large-scale studies, especially multicenter and well-matched cohort research, will provide more insight into the role of CXCR4 in the prognosis and clinical implementation of NSCLC patients.

Disclosure

The authors report no conflicts of interest in this work.

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