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Tumor-associated angiogenesis is important for tumor growth and metastasis. Interleukin (IL)-8 was recently reported to be an important angiogenic factor both in vitro and in vivo. In this study we evaluated, for the first time, IL-8 messenger RNA (mRNA) expression in non-small-cell lung cancer (NSCLC), using real-time quantitative reverse-transcription-polymerase chain reaction, and correlated IL-8 mRNA expression in tumor and nontumor lung samples from 58 patients with NSCLC (29 with squamous cell carcinoma and 29 with adenocarcinoma, of whom 20 had Stage I, 10 had Stage II, and 28 had Stage III disease) with these patients' clinicopathologic characteristics, angiogenesis, and outcome. IL-8 protein expression and tumor microvessel count (MC) were assessed immunohistochemically. IL-8 mRNA expression was significantly greater in tumor tissue; high expression was highly associated with tumor in advanced stages (p = 0.03), distant lymph node metastasis (p = 0.02), high tumor MC (> 123) (p = 0.00003), short survival (< 26 mo) (p < 0.00001), and early relapse (< 16 mo) (p < 0.00001). Tumor MC correlated strongly with IL-8 mRNA expression (r = 0.56, p < 0.001). Multivariate analysis showed IL-8 mRNA expression and intratumor MC to be the most important predictors of patient survival and relapse. Thus, in NSCLC, IL-8 mRNA expression is strongly associated with tumor progression, tumor angiogenesis, survival, and time to relapse, suggesting its use as a prognostic indicator.
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ABSTRACT
INTRODUCTION
METHODS
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Angiogenesis is required for the growth and progression of solid tumors, and is important for their metastasis (1, 2). A good correlation between the degree of angiogenesis induced by a tumor with advancement of the tumor and systemic metastasis of disease has been reported in breast (3), prostate (4), and lung (5, 6) cancer, as well as in a variety of malignant tumors (7, 8).
Angiogenesis is a complicated process, involving degradation of the basement membrane and invasion of the stroma by endothelial cells, and their proliferation, migration, and organization into a capillary structure (9). This process is regulated by the local activity of a variety of angiogenic factors, including basic fibroblast growth factor and vascular endothelial growth factor (VEGF) (10, 11).
Interleukin- (IL)-8, a cytokine of the C-X-C chemokine family that consists of small (8 to ~ 10 kD), basic, heparin-binding proteins, was originally classified as a neutrophil chemoattractant with inflammatory activity (12, 13). However, it was recently shown to be a potential angiogenic factor that can induce endothelial cell chemotaxis and proliferation in vitro and in vivo (14). Recent studies have shown that IL-8 is expressed in several human cancer cell lines derived from astrocytoma (15), hepatoma (16), transitional cell carcinoma (17), and melanoma (18), and that it is associated with angiogenesis and the metastatic potential of human melanoma cell lines in nude mice (18, 19). IL-8 overexpression has been demonstrated in specimens of several human solid cancers, such as squamous cell carcinoma of the head and neck (HNSCC) (20), colorectal cancer (21), glioblastoma (22), and melanoma (23). High expression of IL-8 receptors (IL-8 RA and IL-8 RB) in microvessel endothelial cells and in tumor cells has also been reported in HNSCC (24), as well as in breast cancer (25). In primary lung cancer, however, studies of IL-8 expression are limited (26), and the role of IL-8 overexpression in tumor-associated angiogenesis, tumor progression, and patient prognosis in NSCLC has never been studied.
The complete sequence of IL-8 mRNA has been determined from human peripheral blood monocyte complementary DNA (cDNA); it consists of a 101-base, 5'-untranslated region, a 297-base coding region, and a long, 1.2-kb 3'-untranslated region (12). This information makes it possible to use quantitative reverse transcription-polymerase chain reaction (RT-PCR) to assess IL-8 mRNA expression.
Recently, real-time quantitative RT-PCR has been established as a rapid and sensitive technique for accurate quantification of mRNA in tissues and cells (27). Using the 5'-nuclease activity of Taq polymerase and a dual-labeled fluorogenic hybridization probe, real-time RT-PCR can quantify the target sequence while PCR is still in the log phase of amplification. This method is more reliable than endpoint PCR product measurement for quantifying starting copy number (29).
In this study we used this approach to evaluate IL-8 mRNA expression in NSCLC, with the goals of evaluating correlations between IL-8 mRNA expression and: (1) the clinicopathologic characteristics of NSCLC patients; and (2) tumor-associated angiogenesis, and also assessed the significance of IL-8 mRNA expression as a prognostic indicator in NSCLC.
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Fifty-eight patients who underwent resection for clinical stage I, II, or IIIA NSCLC (30) at our institution between May 1994 and September 1996 were included in the study. Three others were excluded because their tissue specimens were preserved inappropriately or most of the specimen was necrotic and unsuitable for immunohistochemical staining.
Paraffin-embedded, formalin-fixed surgical specimens were collected for histologic examination and immunohistochemical studies of IL-8 protein expression and intratumor microvessel density. Specimens of lung cancer tissue and the nontumorous part of the lung, obtained at surgery and immediately frozen, were used for analysis of IL-8 mRNA expression.
Histologic classification of the tumors was based on the World Health Organization criteria (31). Tumor size, adjacent tissue invasion, and nodal status were determined by pathologic examination. The final staging of the disease was determined from a combination of surgical and pathologic findings, according to the tumor-nodes-metastasis (TNM) system (30) and the guidelines of the American Joint Committee on Cancer Staging (32).
Follow-up data were obtained from the patients' medical charts. The timing of postoperative relapse was calculated from the date of operation to the date of detection of local recurrence or distant metastasis. The patients' survival time was calculated from the date of operation to the date of death. Early relapse was defined as relapse occurring before the median postoperative relapse time, and short survival was defined as survival for less than the median value for patient survival.
Use of Real-Time Quantitative RT-PCR to Assess IL-8 mRNA Expression
Total mRNA was extracted from resected lung tissue with an RNA extraction kit (Rneasy Mini Kit; Qiagen, Valencia, CA). The primers and the probe used to amplify and quantify IL-8 mRNA were designed by using sequences within exon 1 to exon 2. The forward primer used for real-time RT-PCR of IL-8 mRNA was: 5'-CTC TTG GCA GCC TTC CTG ATT-3' (exon 1); and the reverse primer was: 5'-TAT GCA CTG ACA TCT AAG TTC TTT AGCA-3' (exon 2), both of which were chosen according to IL-8 complementary DNA (cDNA) sequence data (12). The sequence of the probe used to detect and quantify the RT-PCR product was 5'-CTT GGC AAA ACT GCA CCT TCA CAC AGA-3'. This sequence, which is specific to IL-8 cDNA and does not hybridize with other chemokine family members (33), was chosen to span the exon 1-exon 2 junction so as to prevent quantification of the PCR product from contaminating IL-8 genomic DNA.
Each amplification mixture (50 µl) contained 50 ng of sample RNA; 5× TaqMan EZ buffer (10 µl) (Perkin-Elmer, Foster City, CA); 25 mM manganese acetate (6 µl); 300 µM adenosine triphosphate, deoxycytosine triphosphate, and deoxyguanosine triphosphate, and 600 µM deoxyuridine triphosphate; 5 U of rTth DNA polymerase; 0.5 U of AmpErase uracil N-glycosylase; 200 nM IL-8 forward and reverse primer; and 100 nM dual-labeled fluorogenic IL-8 probe (Perkin-Elmer). The rTth DNA polymerase has both reverse transciptase and Taq polymerase activity. For RT, the mixtures were incubated at 50° C for 2 min, 60° C for 30 min, and 95° C for 5 min for deactivation. The subsequent thermal cycling profile consisted of 40 cycles of denaturation at 94° C for 20 s and primer annealing and extension at 62° C for 1 min. Each assay included a standard curve, a no-template control, and triplicate total RNA samples. Any samples with a coefficient of variance (CV) greater than 10% were retested.
-Actin mRNA (internal control) was quantified in the same way
as IL-8 mRNA, using the forward and reverse primers 5'-CGC CCA
GCA CGA TGA AA-3' and 5'-CCG CCG ATC CAC ACA GA-3',
respectively, and the probe 5'-AAG ATC ATT GCT CCT CCT
GAG CGC AAG T-3' (34).
The fluorescence emitted by the reporter dye (FAM-6-carboxy-fluorescein; peak fluorescence emission at 518 nm) was detected on-line in real-time with the ABI prism 7700 sequence detection system
(Perkin-Elmer Applied Biosystems). The threshold cycle (CT) was
defined as the fractional cycle number at which the fluorescence generated by cleavage of the probe exceeded a fixed threshold above
baseline (Figures 1A and 1B); at a given threshold, a higher CT value
indicates a lower starting copy number. The relative amount of tissue
IL-8 mRNA, standardized against the amount of -actin mRNA, was
expressed as 
CT = [CTIL-8 CT
-actin]. The ratio of IL-8 mRNA
copies to -actin mRNA copies was then calculated as 2
CT × K (K = constant). We analyzed CT as both a continuous and as a dichotomous variable, and assigned a value of 9.86 (mean value) as the cutoff
between low- and high-level IL-8 mRNA expression.
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Immunohistochemical Staining for IL-8 Protein
IL-8 protein was detected immunohistochemically with a modified avidin-biotin-peroxidase complex method (35). Briefly, paraffin-embedded tumor samples were cut into 5-µm-thick slices, which were then mounted on poly-L-lysine-coated slides, deparaffinized with xylene, and rehydrated with 100%, 90%, 80%, 70%, and 50% ethyl alcohol. The slides were then immersed in 0.1 M sodium citrate buffer (pH = 6.0) and heated in a microwave oven for 10 min (600 W, 110 V). Endogenous peroxidase was blocked by incubation for 20 min with 0.3% hydrogen peroxide in methanol. With intervening washes in phosphate-buffered saline (PBS), the slides were sequentially incubated at room temperature for 60 minutes with monoclonal anti-IL-8 antibody (1:200 dilution; Endogen, Woburn, MA), for 30 min with a rabbit antimouse Ig IgG antibody (1:10), and for 30 min with avidin- biotin-peroxidase complex, the color being developed with diaminobenzidine tetrahydrochloride (Zymed, San Francisco, CA). The tumor specimen was then counterstained with Mayer's hematoxylin solution, with a brown color indicating areas of IL-8 protein expression. Bronchial epithelium and alveolar macrophages adjacent to the tumors were used as positive controls (26). Negative controls consisted of tumor specimens treated identically except for the absence of the primary antibody.
Microvessel Staining and Counting
Microvessels were stained by using mouse polyclonal anti-CD34 antibody (1:20 dilution) (Novocastra, Newcastle, UK) as the primary antibody, with binding visualized through the avidin-biotin-peroxidase-complex method. Any brown-immunostained endothelial cell separated from adjacent microvessels was considered as representing a single microvessel. The tumor sample was first examined at a low power (×100) to identify the area with the highest density of microvessels, after which all microvessels in three ×200-power fields (×20 objective and ×10 ocular magnification, 0.785 mm2 per field) in this area were counted, and the average of these three readings was taken as the microvessel count (MC).
Counting was done in a blind manner by two independent observers, with the interobserver agreement being good (r = 0.91, p < 0.001). MC was analyzed as both a continuous and as a dichotomous variable, using a cutoff value of 123 vessels per ×200-power field (mean value), which fell between the low and high MC values.
Statistical Analyses
All statistical analyses were performed with SPSS for Windows software, Version 8 (SPSS Inc., Chicago, IL). The chi-square and Mann- Whitney tests were used to compare the clinicopathologic characteristics of tumors (and patients) with high and low IL-8 mRNA expression (36). The relationship between IL-8 mRNA expression and MC was analyzed through linear regression. Survival curves were obtained by the Kaplan-Meier method (37), and the difference in survival between low- and high-IL-8 expression groups was analyzed with the log-rank test, as was the difference in relapse time. Univariate analysis was used to identify clinicopathologic variables associated with a poor prognosis. Multivariate analysis with a stepwise Cox regression model (36) was then used to select and identify the most important independent predictors of survival and relapse. Values of p < 0.05 were considered statistically significant. Where appropriate, data are presented as the mean ± SD.
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Patients
The patients consisted of 40 men and 18 women (age 60 ± 10 years [mean ± SD]), 29 of whom had squamous cell carcinoma and 29 of whom had adenocarcinoma. The surgical-pathologic stage of disease was I in 20 patients, II in 10 patients, and IIIA or IIIB in 28 patients. Tumor status was T1 in 10 patients, T2 in 28 patients, T3 in 19 patients, and T4 in one patient. Twenty-eight patients had no lymph node metastasis (N0), and 30 had regional or mediastinal lymph node metastasis (N1 in 14 patients, N2 in 14 patients, and N3 in two patients). Thirty-eight patients were treated only with surgery, whereas 20 received radiation and/or chemotherapy during the follow-up periods.
Follow-up, ranging from 32 to 60 mo, lasted until May 1999. The postoperative relapse time ranged from 1 to 49 mo (median: 16.0 mo), and the survival time ranged from 2 to 49 mo (median: 26.0 mo). Twenty-eight patients had an early relapse and 28 had a short survival.
IL-8 mRNA Expression
The CT values for IL-8 mRNA in tumor samples are shown in
Figures 1A and 1B. The RT-PCR products for IL-8 mRNA
and -actin mRNA were, respectively, cDNAs of 87 bp and
67 bp. IL-8 mRNA expression in the 58 tumor samples, standardized with -actin mRNA (CT), ranged from 5.4 to
15.7, the mean value (9.86 ± 2.20) being significantly higher
than that in the patients' normal lung tissue (8.7 ± 2.7; p = 0.012, paired t test). With the mean value used as the cutoff point, 32 tumors showed low IL-8 mRNA expression (CT < 9.86) and 26 showed high expression.
Immunohistochemical Staining of IL-8 Protein and Microvessel Endothelium
IL-8 protein was seen predominantly in the cytoplasm of lung cancer cells in the tumor specimens (Figures 2A and 2B). A little staining was seen in infiltrating inflammatory cells, whereas other stromal cells showed negative staining.
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The immunostained microvessels appeared as brown linear fragments, with or without an internal lumen (Figure 2C). The MC in tumors ranged from 22 to 238 per ×200 field (mean: 123 ± 60.4), with a median value of 115 per ×200 field.
Correlation of Clinicopathologic Characteristics with IL-8 mRNA Expression
Table 1 shows a comparison of the clinicopathologic characteristics in the high-IL-8 mRNA expression (32 patients) and
low-IL-8 mRNA expression (26 patients) groups. Members of
the high-expression group were more likely than those of the
low-expression group to have advanced (Stage IIIA or IIIB)
disease (p = 0.03), distant lymph node metastasis (N2-N3) (p = 0.02), a high (
123) tumor MC (p = 0.00003), short survival
(< 26 mo) (p < 0.00001), and early relapse (< 16 mo) (p < 0.00001). There was no statistical difference between adenocarcinoma and squamous cell carcinoma in IL-8 mRNA expression.
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Relationship Between Microvessel Counts and IL-8 mRNA Expression
The MC in tumors correlated strongly with tumor IL-8 mRNA expression (linear regression: r = 0.56, p < 0.0001) (Figure 3).
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IL-8 mRNA Expression and Prognosis
The high-expression group had a significantly shorter median survival (16 mo; 95% confidence interval [CI]: 11.8 to 20.1 mo) than did the low-expression group (45 mo; 95% CI: 34.4 to 55.6 mo) (log-rank test, p < 0.0001) (Figure 4A). The median duration to postoperative recurrence was also shorter in the high-expression group (5 mo; 95% CI: 4.2 to 5.8 mo) than in the low-expression group (28 mo; 95% CI: 17.8 to 38.2 mo) (log-rank test, p < 0.0001) (Figure 4B).
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Univariate analysis (log-rank test) showed that stage of disease, tumor status, nodal status, IL-8 mRNA expression, and MC were the greatest prognostic factors for survival, whereas age, nodal status, IL-8 mRNA expression, and MC were prognostic factors for early relapse (Table 2). After multivariate analysis with a Cox regression model, IL-8 mRNA expression (p < 0.0001) and MC (p = 0.0153) remained the most significant and independent prognostic factors for survival, whereas IL-8 mRNA expression (p = 0.0001) and MC (p = 0.0175) were the most significant factors for prediction of recurrence (Table 3).
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IL-8, an 8-kD protein produced by a variety of cells, is a potent leukocyte chemotactic factor. Recently, it was also shown to be a potent angiogenic factor (14, 38). Recombinant IL-8 mediates endothelial cell chemotaxis and proliferation both in vitro and in vivo (38, 39). IL-8 is reported to play a role in tumor progression in several types of cancer. Eisma and colleagues (20) showed that the level of IL-8 protein in squamous cell carcinoma of the head and neck is significantly greater than in normal cells, and Uede and coworkers (21) found that serum levels of IL-8 in patients with colorectal cancer correlated positively with liver and lung metastasis.
To the best of our knowledge, our study represented the first use of real-time quantitative RT-PCR to measure IL-8 mRNA expression in NSCLC. IL-8 mRNA expression in tumor tissue was shown to be significantly higher than in corresponding nontumorous lung tissue. Using immunohistochemical staining, we found that IL-8 protein was predominantly expressed in cancer cells. IL-8 mRNA expression was highly correlated with intratumor MC, a marker for angiogeneic activity. High IL-8 mRNA expression was associated with advanced stage (IIIA or IIIB) disease, distant lymph node metastasis, shorter patient survival, and early postoperative relapse. Multivariate analysis showed that IL-8 mRNA expression and intratumor MC were the most significant prognostic factors in NSCLC. These results imply that in NSCLC, IL-8 mRNA expression plays an important role in angiogenesis and tumor progression, and that it is also associated with an adverse prognosis. This correlation of IL-8 mRNA expression with clinicopathologic characteristics and patient outcome in NSCLC has not been previously reported.
Our findings are supported by several studies done by other investigators. Smith and colleagues (41) showed that a bronchogenic carcinoma homogenate could induce endothelial cell chemotaxis and corneal neovascularization, and that these effects were attenuated by a neutralizing anti-IL-8 antibody, Yatsunami and associates (26) showed that IL-8 immunostaining was positive in 36 of 56 (64%) NSCLC samples and negative in most small-cell carcinoma samples, and Arenberg and coworkers (42) showed that anti-IL-8 antibody reduced tumor growth and intratumor MCs in xenografts of the A549 cell line (adenocarcinoma) in mice with severe combined immunodeficiency. These results indicate that IL-8 is one of the major angiogenic factors secreted by NSCLC cells.
Another possible mechanism for the association of IL-8 with tumor progression is through an autocrine pathway. Several studies have shown that IL-8 receptors (IL-8 RA and IL-8 RB) are expressed in both tumor cells and microvessel endothelial cells in HNSCC (24), breast cancer (25), and melanoma (43). IL-8 is secreted by a variety of cancer cells (20). Schadendorf and colleagues (43) showed that the IL-8 produced by human malignant melanoma stimulates the tumor cells themselves to proliferate in vitro. Singh and coworkers (18) found that IL-8 mRNA expression correlated with the metastatic potential of a melanoma cell line in nude mice. However, in NSCLC, the addition of a neutralizing anti-IL-8 antibody to the culture medium did not reduce tumor cell proliferation when tested on several lung cancer cell lines in vitro (26, 42), and IL-8 receptors have not been reported to be expressed in lung cancer cells. The role of IL-8 as an autocrine growth factor in NSCLC requires further study. Richards and colleagues (24) showed that IL-8 RA and RB are also strongly expressed on endothelial cells, with IL-8 RB expression being high in microvessels and IL-8 RA expression high in large vessels (24, 25). Our study and those of other groups have provided in vivo and in vitro evidence that IL-8 expression is linked to tumor-associated angiogenesis, and that the increased angiogenesis might contribute to tumor progression and poor patient prognosis in NSCLC.
IL-8 is a well-known chemoattractant factor for neutrophils, and neutrophil infiltration is sometimes seen in NSCLC.
These recruited inflammatory cells could enhance angiogenesis by secreting several cytokines, such as tumor necrosis
factor (TNF)-
. Thus, in addition to its direct angiogenic
effect, the IL-8 secreted by tumor cells might also indirectly
induce angiogenesis by recruiting inflammatory cells to the
tumor tissues.
Desbaillets and coworkers (44) have shown that exposure of a human glioblastoma cell line to anoxic stress causes upregulation of mRNA expression for both IL-8 and VEGF, but with different time courses. Using immunohistochemistry, Ferrer and associates (45) showed coexpression of IL-8 and VEGF in prostate cancer cells, whereas benign prostatic hyperplasia and normal prostate cells displayed little staining for either of these angiogenic factors; in addition, they showed that IL-8 and VEGF could be induced, respectively, by IL-1 and TNF in a prostate cancer cell line grown in culture. Using immunohistochemistry and enzyme-linked immunoassay, Eisma and colleagues (20) also demonstrated coexpression of IL-8 and VEGF protein in 35 HNSCC samples; as in an unpublished study of VEGF mRNA expression in NSCLC done by ourselves, there was good correlation between the expression of IL-8 mRNA and VEGF mRNA (r = 0.63, p < 0.0001). These results imply that although both IL-8 mRNA and VEGF mRNA are probably upregulated by different pathways, these two angiogenic factors could be simultaneously upregulated in cancer cells by a common upstream inducer or by crosstalk of these two signal-transduction pathways.
In the present study we used real-time quantitative RT-PCR to assess IL-8 mRNA expression in lung cancer. This approach has several advantages over conventional quantitative RT- PCR. First, it provides a real-time method for detecting specific amplification products. In addition, the CT value used for quantification of mRNA is measured when PCR amplification is in the log phase of PCR product accumulation, thus providing a more reliable measure of starting copy number than the endpoint measurement used with conventional quantitative RT- PCR (46, 47). Real-time quantitative RT-PCR also has a wider dynamic range, reducing the need for serial dilution. In addition, there is no need for sample handling after PCR, or even for transfer and blotting. Additionally, real-time PCR provides more accurate and reproducible quantification of nucleic acids than do current quantitative RT-PCR methods (27), and the amount of starting total RNA used (50 ng) is far less than that used in conventional quantitative RT-PCR (1 µg).
We therefore conclude that in NSCLC patients, high IL-8 mRNA expression in cancer tissue correlates with intense angiogenesis, advanced disease, distant lymph node metastasis, short survival, and early relapse. These findings suggest that quantification of IL-8 mRNA expression in tumor tissue could be used as a prognostic indicator for patients with NSCLC. IL-8 could also be used as a target for antiangiogenic therapy in NSCLC. Further studies, including a larger number of cases of NSCLC, are planned to verify this association between IL-8 expression and poor prognosis.
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Supported by grant NSC-88-2314-B-002-207 from the National Science Council of the Republic of China, and grant NTUH88-M110 from the National Taiwan University Hospital.
Correspondence and requests for reprints should be addressed to Pan-Chyr Yang, M.D., Ph.D., National Taiwan University Hospital, No 7, Chung-Shan South Road, Taipei 100, Taiwan. E-mail: pcyang{at}ha.mc.ntu.edu.tw
(Received in original form February 23, 2000 and in revised form May 1, 2000).
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References |
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