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Introduction

Primary lung cancer is one of the most common types of cancer worldwide, and non-small cell lung cancers (NSCLCs) account for approximately 85% of all primary lung cancers. Surgical resection is the only potentially curative treatment for patients with early disease. However, the 5-year survival rate after surgical resection remains unsatisfactory. Improved survival of patients with NSCLC requires better clinical predictors of outcomes and of response to specific therapeutic interventions.

Insulin-like growth factor-1 receptor (IGF-1R) is a trans-membrane heterotetrametric protein encoded by the IGF-1R gene located on chromosome 15q25-q26. IGF-1R promotes oncogenic transformation, growth and survival of cancer cells (1–4). The binding of insulin-like growth factor (IGF)-1 and IGF-2 to the extracellular subunit domain of IGF-1R activates the tyrosine kinase activity of IGF-1R and triggers a cascade of reactions involving signal transduction pathways, including components such as Ras, Raf, mitogen-activated protein kinase and phosphoinositol-3-kinase (PI3K)/AKT/BAD (Bcl-xL/Bcl2-associated death promoter) (5). IGFs are synthesized together with six molecular species of specific binding proteins [IGF binding protein (IGFBP)-1 to -6]. IGFBPs modulate IGF-1 and IGF-2 bioavailability in both circulation and the cellular microenvironment. In several malignancies, IGF-1R overexpression promotes tumor growth, progression, invasion and metastasis (6). Increased metastatic activity was reported in mice after intrasplenic injection of lung cancer cell lines transfected with IGF-1R (7).

Matrix metalloproteinases (MMPs) are a family of highly conserved enzymes that are capable of degrading the extra-cellular matrix (ECM). Over 25 well-characterized members of this proteinase family have been identified. MMPs play key roles not only in normal processes, but also in tissue remodeling associated with inflammatory disease, cancer invasion and metastasis (8,9). Substantial evidence indicates that overexpression of MMPs correlates with a more aggressive tumor-cell phenotype, as well as with poor outcomes in patients with cancer.

MMP-7 is the smallest (28 kDa) member of the MMP family. It has broad substrate specificity against ECM components and is produced by tumor cells. The functions of MMP-7 include destruction of basement membrane components, which is a crucial event in tumor cell invasion and metastasis. Increased expression of MMP-7 in cancer cells is associated with tumor progression and metastasis in various types of cancer (10,11). To date, few studies have examined the expression of MMP-7 in lung cancer (12–14).

Miyamoto et al (15) reported that MMP-7 possesses IGFBP-3 protease activity. MMP-7-induced proteolysis of IGFBP-3 plays a crucial role in regulating IGF-I bioavailability and promotes cell survival. These findings suggest that MMP-7 may augment carcinogenesis and the progression of tumors that express IGF-1R. Adachi et al (16) found that IGF/IGF-1R upregulated MMP-7 expression in a gastrointestinal cancer cell line, suggesting that a positive feedback loop involving IGF-1R and MMP-7 may have a part in tumor progression.

The aim of this study was to evaluate the expression levels of IGF-1R and MMP-7 in resected NSCLC, and to examine the relations of such levels to clinical characteristics and survival.

Patients and methods

Patients

This study was performed in 78 consecutive patients with pathological (p)-stage I to III NSCLC who underwent complete tumor resection and nodal dissection without any pre-operative therapy at the Respiratory Center, Yokohama City University Medical Center, between January 1, 2000 and November 30, 2003.

The subjects were 54 men and 24 women with a mean age of 64.7 years (range 19–82; median 65) (Table I). The most common histological type of tumor was adenocarcinoma (57.7%; 45 cases), followed by squamous cell carcinoma (33.3%; 26 cases), large-cell carcinoma (6.4%; 5 cases), typical carcinoid (1 case) and pulmonary blastoma (1 case). The stage of the primary tumor was T1 in 35 patients (44.9%), T2 in 29 (37.1%), T3 in 9 (11.5%) and T4 in 5 (6.4%). Thirty-eight (48.7%) patients had no metastasis to regional lymph nodes (N0), whereas 11 (14.1%) had metastatic involvement of the hilar lymph nodes (N1), and 29 (37.1%) had metastases to the mediastinal nodes (N2, N3). Thirty-two tumors (41.0%) were classified as stage I, 15 (19.2%) were stage II and 31 (39.7%) were stage III. At the end of follow-up, 36 patients (46.1%) were alive and 42 (53.8%) had died.

Table I. Patient characteristics.

Table I.

Patient characteristics.

Characteristics	No. of patients (%)
Total	78 (100)
Age (mean ± SD), years	64.7±10.8
Gender
Male	54 (69.2)
Female	24 (30.7)
Histological type
Adenocarcinoma	45 (57.7)
Squamous cell carcinoma	26 (33.3)
Large-cell carcinoma	5 (6.4)
Typical carcinoid	1 (1.3)
Pulmonary blastoma	1 (1.3)
Pathological stage
I	32 (41.0)
II	15 (19.2)
III	31 (39.7)
Smoking status
Smoker	56 (71.8)
Non-smoker	22 (28.2)
T-factor
T1	35 (44.9)
T2	29 (37.2)
T3	9 (11.5)
T4	5 (6.4)
N-factor
N0	38 (48.7)
N1	11 (14.1)
N2	28 (35.9)
N3	1 (1.3)
Recurrence
(+)	36 (46.2)
(−)	42 (53.8)

Histological subgroups were determined according to the World Health Organization classification. Pathological tumor-node-metastasis classification and staging were assigned in accordance with the International Staging System. The mean follow-up was 1,466 days (range 106–3,328). Informed consent was obtained from each patient and the Yokohama City Medical Committee approved this study.

Immunohistochemistry

Formalin-fixed, paraffin-embedded tissue specimens were cut into 4-μm thick sections and mounted on slides. The sections were deparaffinized and rehydrated.

For IGF-1R, the slides were heated in a microwave for 10 min in a 10-μmol/l citrate buffer solution at pH 6.0 and cooled to room temperature for 20 min. After quenching the endogenous peroxidase activity with 3% H2O2 for 5 min, the sections were incubated for 60 min at room temperature with the primary antibody diluted at 1:100 for IGF-1R (a rabbit polyclonal antibody, clone 1161; Signalway Antibody, Pearland, TX, USA). Peroxidase-Labeled Polymer EnVision+ kit (Dako, Glostrup, Denmark) was used for specific staining.

For MMP-7, after the endogenous peroxidase activity was blocked, the sections were incubated for 90 min at room temperature with the primary antibody for MMP-7 (a mouse monoclonal antibody, clone 141-7B2; Daiichi Fine Chemicals, Toyama, Japan), diluted at 20 μg/ml. Endogenous biotin was blocked by Dako’s Biotin Blocking system (Dako), according to the manufacturer’s specifications. After rinsing, specific staining was visualized with the use of an LSAB+ system-HRP system (Dako).

Color was produced by the application of 3,3’-diaminobenzidine for 10 min. The sections were counterstained with Meyer’s hematoxylin (Muto Pure Chemicals, Tokyo, Japan).

All sections were scored semi-quantitatively and qualitatively, without knowledge of the clinical data. Expression levels were measured by immunohistochemical analysis on the basis of staining intensity, scored from 0 to 4 as follows: 0, negative; 1, trace; 2, weak; 3, moderate; and 4, strong. The percentage of stained cells (0–100%) was multiplied by the staining intensity (0–4). The final score ranged from 0 to 400. Staining of the sample was considered high when the score was equal to the median value or higher, or was otherwise considered as low.

Statistical analysis

Univariate analysis was performed by the χ2 test and Mann-Whitney U test. Continuous data were compared using the Student’s t-test. The postoperative survival rate was analyzed by the Kaplan-Meier method, and differences in survival rates were assessed with the log-rank test. A Cox proportional hazard regression model was used for multivariate analyses. Death from any cause was included in the calculation of postoperative survival. Differences were considered significant at P<0.05. All statistical manipulations were performed using the SPSS version 17.0 for Windows (SPSS Inc., Chicago, IL, USA).

Results

Expression of IGF-1R and MMP-7 according to immunohistochemical analysis

Immunohistochemical expression of IGF-1R was detected in the tumor cell membrane. Staining for MMP-7 was characterized by a heterogeneous cytoplasmic pattern (Fig. 1). Among the 78 carcinomas studied, median expression scores were 210 for IGF-1R and 140 for MMP-7. A total of 41 (52.6%) carcinomas were classified as IGF-1R-high, and 42 (53.8%) were classified as MMP-7-high.

Figure 1. Immunostaining of NSCLC in serial sections. (A) Strong staining (intensity 4) of IGF-1R, and (B) weak staining (intensity 2) of IGF-1R. (C) Strong staining (intensity 4) of MMP-7, and (D) weak staining (intensity 2) of MMP-7.

Relation between expression of IGF-1R and MMP-7 and clinicopathological factors

High expression of IGF-1R was related to lymph node metastasis (P=0.034) and recurrence (P=0.006). MMP-7 expression status did not significantly correlate with any clinicopathological factor (Table II). There was no significant correlation between the expression of IGF-1R and that of MMP-7 (P=0.184).

Table II. Characteristics of the NSCLC patients and IGF-1R and MMP-7 expression.

Table II.

Characteristics of the NSCLC patients and IGF-1R and MMP-7 expression.

	IGF-1R expression			MMP-7 expression
	High	Low	P-value	High	Low	P-value
No. of patients	41	37		42	36
Age (mean ± SD)	64.8±10.0	64.7±11.7	0.960	65.3±10.0	64.3±11.5	0.687
Gender
Male	27	27	0.496	27	27	0.307
Female	14	10		15	9
Histological type
Adenocarcinoma	21	24	0.466	23	22	0.945
Squamous cell carcinoma	17	9		15	11
Large-cell carcinoma	2	3		3	2
Other	1	1		1	1
Smoking status
Smoker	29	27	0.826	28	28	0.277
Non-smoker	12	10		14	8
T-factor
T1	17	18	0.321	19	16	0.986
T2	17	12		15	14
T3	3	6		5	4
T4	4	1		3	2
N-factor
N0	14	24	0.034	21	17	0.788
N1	6	5		6	5
N2	20	8		14	14
N3	1	0		1	0
Recurrence
(+)	25	11	0.006	19	17	0.861
(−)	16	26		23	19

Relation between expression of IGF-1R and MMP-7 and overall survival

Overall survival was significantly worse in patients with IGF-1R-high tumors than in those with IGF-1R-low tumors (P=0.011) (Table III, Fig. 2). The 5-year survival rate was 34.1% in patients with IGF-1R-high tumors, as compared to 63.0% in those with IGF-1R-low tumors. Overall survival was slightly, but not significantly, worse in patients with MMP-7-high tumors than in those with MMP-7-low tumors. The 5-year survival rate was 37.5% in patients with MMP-7-high tumors, as compared to 58.3% in those with MMP-7-low tumors (P=0.220) (Fig. 2). Subsequently, we conducted subset analyses to investigate the prognostic significance of IGF-1R and MMP-7. IGF-1R-high was associated with worse overall survival than IGF-1R-low in patients who were male and in those with adenocarcinoma (P=0.022 and 0.016, respectively) (Table IV).

Figure 2. Overall survival of 78 patients with NSCLC. (A) IGF-1R-high vs. IGF-1R-low NSCLC; (B) MMP-7-high vs. MMP-7-low NSCLC.

Table III. Univariate analysis of overall survival in NSCLC.

Table III.

Univariate analysis of overall survival in NSCLC.

Variables	5-year survival rate (%)	P-value
Gender		0.015
Male	40.1
Female	64.8
Histological type		0.176
Adenocarcinoma	54.2
Squamous cell carcinoma	37.5
T-factor		0.002
T1	67.2
T2,3,4	32.5
N-factor		0.280
N0	57.2
N1,2,3	38.9
Smoking status		0.109
Smoker	43.6
Non-smoker	57.7
IGF-1R expression		0.011
High	34.1
Low	63.0
MMP-7 expression		0.220
High	37.5
Low	58.3

Table IV. Subset analysis of overall survival in NSCLC.

Table IV.

Subset analysis of overall survival in NSCLC.

Variables	5-year survival rate (%)
	IGF-1R-high	IGF-1R-low	P-value	MMP-7-high	MMP-7-low	P-value
Gender
Male	25.9	54.5	0.022	24.0	55.5	0.051
Female	50.0	87.5	0.064	62.2	66.6	0.955
Histological type
Adenocarcinoma	38.1	68.4	0.016	48.9	59.0	0.608
Squamous cell carcinoma	29.4	55.5	0.586	23.3	54.5	0.478
Pathological stage
I	53.8	72.8	0.182	61.1	71.4	0.601
II, III	25.0	53.5	0.072	22.2	50.0	0.246
Smoking status
Smoker	31.0	57.6	0.050	28.8	57.1	0.104
Non-smoker	41.6	77.7	0.087	54.5	62.5	0.913

Multivariate analysis of overall survival in patients with NSCLC included the following factors: IGF-1R expression, T-factor, N-factor, gender and MMP-7 expression. Male gender (HR=2.598; 95% CI 1.198–5.638, P=0.016), T2–4 disease (HR=2.540; 95% CI 1.291–4.997, P=0.007) and high expression of IGF-1R (HR=2.322; 95% CI 1.215–4.436, P=0.011) were significantly associated with worse overall survival (Table V).

Table V. Multivariate analysis of overall survival in NSCLC.

Table V.

Multivariate analysis of overall survival in NSCLC.

Variables	P-value	Hazard ratio	95% confidence interval
Gender	0.016	2.598	1.198–5.638
T-factor	0.007	2.540	1.291–4.997
IGF-1R	0.011	2.322	1.215–4.436

Discussion

Overexpression of IGF-1R has been reported to promote tumor growth, invasion and metastasis in several types of malignancies (6). Despite previous studies, however, the clinical significance of IGF-1R expression in NSCLC remains unclear. MMP-7 has been reported to have multiple biologic functions related to tumor behavior, such as growth, invasion, proliferation and apoptosis. In addition, a relation between MMP-7 expression and postoperative outcomes has been reported (12–14), but definitive evidence is lacking. Therefore, we studied immunohistochemically the expression of IGF-1R and MMP-7 in post-surgical patients with NSCLC.

We assessed the association of IGF-1R and MMP-7 expression with clinicopathological features. Dziadziuszko et al (17) found that IGF-1R expression was higher in squamous cell carcinomas than in other histological types and was associated with disease stage. Cappuzzo et al (18) reported that a positive IGF-1R expression was significantly associated with squamous cell histology and grade III differentiation. Ludovini et al (19) reported that IGF-1R protein overexpression was associated with larger tumor size. Merrick et al (20) showed that higher IGF-1R scores were associated with adenocarcinoma and never-smokers. Our results showed that high IGF-1R expression was significantly related to higher N-factor (P= 0.034) and recurrence (P=0.006). As for histological type, IGF-1R expression was slightly, but not significantly, higher in squamous cell carcinomas.

Concerning MMP-7 expression, Liu et al (12) showed that MMP-7 expression was significantly higher in squamous cell carcinomas than in adenocarcinomas. Leinonen et al (13) reported that high MMP-7 expression was related to lower T-factor and well-differentiated tumors; moreover, MMP-7 expression was higher in adenocarcinomas than in other histological subtypes. Sasaki et al (14) demonstrated a trend toward higher MMP-7 mRNA expression levels in NSCLCs with lymph node metastasis. By contrast, our results showed no correlation between MMP-7 and any clinicopathological factor.

We also analyzed the relationship of IGF-1R and MMP-7 expression to post-surgical outcomes. Previously, Merrick et al (20) analyzed IGF-1R expression in 184 surgically treated patients with stage I to IV NSCLC. In stage I disease, high IGF-1R expression was associated with significantly shorter survival than low IGF-1R expression. Dziadziuszko et al (17) evaluated 189 NSCLCs and showed that the IGF-1R gene copy number is of prognostic value; nonetheless, IGF-1R protein expression upon immunohistochemical analysis was not related to survival. Ludovini et al (19) reported that IGF-1R protein expression alone was not significantly associated with survival, although high co-expression of both IGF-1R and epidermal growth factor receptor was associated with shorter disease-free survival in resected NSCLC. Cappuzzo et al (18) concluded that IGF-1R expression does not represent a prognostic factor in resected NSCLC patients.

In the present study, overall survival was significantly poorer in patients with IGF-1R-high tumors than in those with IGF-1R-low tumors, and multivariate analysis showed IGF-1R expression as an independent indicator of poor outcomes. As for MMP-7, Liu et al (12) reported that the overall survival rate was significantly lower in patients with MMP-7-positive NSCLC than in those with MMP-7-negative NSCLC. On the other hand, Leinonen et al (13) reported that MMP-7 had no prognostic value in NSCLC. Our results showed a trend toward poorer survival in patients with MMP-7-high tumors than in those with MMP-7-low, but the difference fell short of reaching statistical significance.

In conclusion, our results suggest that overexpression of IGF-1R is a useful predictor of lymph node metastasis and recurrence in patients with NSCLC. Overexpression of IGF-1R may thus be an important prognostic factor along with gender and T-factor in patients with NSCLCs.

References