1 SOMPS, Pitié Salpetrière Hospital, Paris; 2 Départment de Médecine, Institut Gustave Roussy, Villejuif; 3 CEA-DSV/DRR/LRO, Fontenay aux Roses Cedex; 4 Laboratoire dImmunologie Cellulaire, Faculté de Médecine Pitié-Salpétrière; 5 Avicenne Hospital, Bobigny Cedex, France
Received 23 October 2003; revised 19 November 2003; accepted 22 December 2003
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ABSTRACT |
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The expression of CXCR4 has been implicated in metastatic dissemination in different models of breast cancer and melanoma. In the present study, we evaluated CXCR4 expression in non-small-cell lung cancer (NSCLC) and the relationship between CXCR4 expression and the prognosis of stage I disease.
Patients and methods:
Using immunohistochemical analysis, we retrospectively analyzed CXCR4 expression in specimens from 61 patients with completely resected pathologically confirmed stage I NSCLC for whom clinical follow-up data were available.
Results:
In the present study, we have shown that: CXCR4 is expressed by tumor cells in stage I NSCLC; CXCR4 is located in the nucleus and/or in the cytoplasm of tumor cells; strong nuclear staining was observed in 17 cases (29.8%); patients whose tumors had CXCR4-positive nuclear staining had a significantly longer duration of survival than patients whose tumors had no nuclear expression (P = 0.039, log-rank test). Interestingly, the 5-year metastasis rates were 23.5% and 34.1% in patients with CXCR4-positive and CXCR4-negative nuclear expression, respectively (P = 0.2).
Conclusion:
Strong CXCR4-positive nuclear staining was associated with a significantly better outcome in early-stage NSCLC. The mechanisms underlying this clinically and biologically important finding need to be further explored.
Key words: chemokines, CXCR4, immunohistochemistry, lung cancer, prognostic factor
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Introduction |
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Chemokines are low chemotactic factors that regulate the development and migration of various cell types [4]. Over 40 chemokines have been identified. Chemokines can be classified into four groups (CXC, CX3C, CC and C) based on the position of the first two highly conserved cysteines of the amino acid sequence. Chemokine receptors are seven transmembrane G protein-coupled receptors. To date, 19 chemokine receptors have been identified. CXCR4 is a chemokine receptor initially described to be involved in the homing of lymphocytes in inflammatory tissues [5, 6]. The CXCR4 ligand, i.e. CXCL12/SDF1, is expressed in lung, bone and liver, and is known to chemoattract lymphocytes in these organs [7]. Based on the lymphocyte homing model, some investigators hypothesized that CXCR4 could be involved in the occurrence of metastasis. CXCR4 has also been shown to be expressed by breast, thyroid, renal and small-cell lung cancer tumor cells, and it appears to be most probably a ubiquitous receptor [811]. CXCR4 expression has been implicated in metastatic spread in animal models of breast cancer and melanoma [8, 12]. Indeed, CXCR4 expression on breast or melanoma tumor cell lines is associated with the development of lung metastases, and binding the receptor with an exogeneous antibody decreases the formation of lung metastases [8]. It has recently been shown that CXCR4 mediates the capacity of NSCLC cell lines to metastasize to the lung in mouse models [13]. Based on these data, we hypothesized that CXCR4 expression could be associated with the prognosis in stage I NSCLC.
In the present study, we evaluated CXCR4 expression in NSCLC and the relationship between CXCR4 expression and the prognosis of stage I disease.
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Patients and methods |
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Immunohistochemical staining for CXCR4
Paraffin-embedded, 5-µm-thick tissue sections from all primary tumors were stained for the CXCR4 protein using a primary rabbit polyclonal antibody (Abcam, clone 2074; Cambridge, UK). Slides were deparaffinized through a series of xylene baths. Rehydration was performed through graded alcohols. The sections were then immersed in methanol containing 0.3% hydrogen peroxidase for 20 min to block endogenous peroxidase activity and incubated in 2.5% blocking serum to reduce nonspecific binding. Sections were incubated overnight at 4°C with primary anti-CXCR4 antibody at dilutions of 1:1000. The sections were then processed using standard avidinbiotin immunohistochemistry according to the manufacturers recommendations (Vector Laboratories, Burlingame, CA, USA). Diaminobenzidine was used as a chromogen, and commercial hematoxylin was used for counterstaining. CXCR4 expression was evaluated at the cytoplasmic and at the nuclear level. The intensity of staining (13) and percentage of positive cells (1 = <10%, 2 = 1050%, 3 = >50%) were taken into account to define a composite score. Positive nuclear staining was defined as a nuclear score of 6 or 9 [i.e. any slide with >50% of the cells expressing nuclear staining (3) with intermediate or strong intensity (2 or 3)]. Cells were counted in at least three fields (at x400) in the tumor areas.
Antibody specificity was assessed using a specific blocking peptide (Abcam, ab8126; Cambridge, UK). Incubating the peptide with an equal volume of antibody for 30 min at 37°C usually completely blocks the signal obtained with anti-CXCR4 (ab2074) by immunohistochemistry. Furthermore, specificity was also assessed by performing a western blot analysis with the protein extract of the A-549 NSCLC cell line.
Statistical analysis
Survival was calculated by the KaplanMeier method, and the resulting curves were compared using the log-rank test. Fishers exact test and the 2 test were used to analyze the association between two categorical variables. P <0.05 was considered to be statistically significant. Immunohistochemical analysis was performed in a blinded manner with respect to the clinical data concerning the subjects.
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Results |
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CXCR4 expression: correlation with outcome
Positive nuclear staining was associated with better survival compared with negative nuclear staining (5-year OS = 93% versus 52.8%, P = 0.036) (Figure 3). The 5-year metastasis rates were 23.5% and 34.1% in patients with positive and negative nuclear expression, respectively (P = 0.2). There was no correlation between nuclear staining and the metastatic site, although a trend was seen for lung metastases alone (three out of four versus three out of 15, P = 0.06). Interestingly, among the four patients with CXCR4-positive nuclear staining (three with a score of 6 and one with a score of 9) and a metastatic relapse, three are still alive (lung relapse alone). Two of these relapses occurred within 2 years and two after 5 years (thus potentially being second primary tumors). In contrast, 14 out of the 15 patients with a metastatic relapse and CXCR4-negative nuclear staining have died.
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Discussion |
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CXCR4 has been found to be expressed in breast cancer [8], thyroid cancer [9], rhabdomyosarcoma [14], pancreatic cancer [15], hepatocellular carcinoma [16] and leukemia [17]. The present study is the first to report CXCR4 expression in NSCLC, like that found in small-cell lung cancer [11]. It is noteworthy that CXCR4 is not found to be expressed in normal lung tissues. Our data therefore suggest that the expression of CXCR4 by tumor cells is associated with malignant transformation in the lung. That CXCR4 may be upregulated during the malignant process is also suggested in other tumor models. Indeed, in breast cancer, CXCR4 expression was found in malignant areas, but not in normal tissue [8]. Similar findings have been reported in thyroid cancers [9], glioma [18] and pancreatic cancers [15]. Another argument implicating CXCR4 expression in carcinogenesis is the finding that the interaction of CXCR4 with SDF1 mediates the activation of PI3 kinase and Akt and that of cell proliferation [19, 20]. Our data, in line with those previously reported, suggest that CXCR4 expression by tumor cells is an event that may participate in malignant transformation.
A striking finding was the presence of CXCR4 in the nucleus in specimens from 27% of the patients included in our series. CXCR4 is a serpentine transmembrane protein that mediates transduction signaling and has been described to be present either on the cell membrane [20] or in the cytoplasm. To date, only one study has reported nuclear localization of CXCR4, and this was in hepatocellular carcinoma [16]. The mechanism mediating the nuclear expression of CXCR4 has yet to be elucidated.
CXCR4-positive nuclear staining was associated with better survival in our study. Indeed, the 5-year OS rates were 93% and 52% for patients with strong and weak nuclear staining, respectively. This is the first demonstration that the location of the expression of a chemokine receptor is associated with outcome. Other authors have previously reported on the prognostic value of specific chemokines such as CCR2 (MCP-1) in breast cancer or CCR7 in gastric cancer [21, 22]. Our finding raises several questions: Are these clinical data supported by experimental findings (in vitro or in animal models)? What are the clinical implications of these data? We have stated previously that CXCR4 is a transmembrane protein whose activity is mediated via interaction with a soluble chemokine (SDF1). It could be speculated that the nuclear location of CXCR4 inhibits its ability to mediate the signal provided by SDF1, thereby decreasing cell proliferation and metastasis. Zeelenberg et al. [23] reported that the retention of CXCR4 in intracellular compartments (endoplasmic reticulum) of T-cell hybridoma reduced metastasis and increased the survival of mice. In another study, Kollet et al. [24] reported that hematopoietic stem cells exhibiting intracytoplasmic but not transmembrane CXCR4 were unable to migrate in bone marrow. These studies highlight the fact that the intracellular retention of CXCR4 is associated with decreased homing of malignant and normal cells.
It has recently been shown that cisplatin-based chemotherapy improves the 5-year OS rates of patients presenting with stage IIII NSCLC [3]. However, the absolute benefit provided by chemotherapy is modest since 5-year OS rates were, respectively, 45% and 40% for patients treated with chemotherapy and for those who were not [3]. As no clinical characteristic was correlated with the benefit afforded by chemotherapy, there is a need to find biomarkers that could be used to select patients for chemotherapy. In our study, strong CXCR4-positive nuclear staining was associated with a 93% 5-year OS rate in patients with stage I NSCLC, compared with a 52% OS rate in patients with weak nuclear staining. This suggests that patients with strong nuclear staining for CXCR4 are not candidates for adjuvant chemotherapy. This finding needs to be confirmed in a larger series since only 61 patients were included in the present study.
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Acknowledgements |
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FOOTNOTES |
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