Introduction

Lung cancer is the leading cause of cancer-related death; every year, there are 1.35 million new lung cancer cases in the world [1]. Non-small cell lung cancer (NSCLC) accounts for approximately 75–80 % of cases of lung cancer [2]. Despite the advances in early detection, radical cure operation, and multimodal therapeutic modalities in the past decades, the median survival of patients with untreated metastatic NSCLC is only 4–5 months with a 1-year survival rate of only 10 % [3]. About 30–40 % of stage I patients will relapse after surgical resection [4]. Moreover, prospective randomized data showed that adjuvant chemotherapy in stage IB has not achieved a significant survival benefit and even a detrimental effect was observed in stage IA [5]. Therefore, there is an urgent need to identify novel biomarkers that will help select the patients with high chance of lung cancer recurrence and provide better prognosis and individualization treatment.

Chitinase 3-like 1 (CHI3L1) is a 40-kDa mammalian glycoprotein which is related in amino acid sequence to the chitinase protein family but contains no enzymatic property. CHI3L1 was found to be a major secreted protein of human articular cartilage chondrocytes and synovial cells [6, 7]. In addition, CHI3L1 has been detected in sera of patients with various diseases including inflammatory bowel disease, pulmonary sarcoidosis, systemic sclerosis, and liver fibrosis [7–10]. Several studies in human solid tumors such as cervical cancer and breast cancer have shown the association of high serum CHI3L1 level with poor patient prognosis and short survival time [11, 12].

The relationship between tumor vascularity and prognosis of NSCLC has been investigated at present [13]. Brock reported that patient survival was significantly correlated with microvessel density (MVD), while some other studies reported that MVD did not correlate with metastasis [14]. Chaturvedi concluded that triple-negative breast cancer outgrow their vascular supply and develop chronic hypoxia, including a decrease of proliferation [15]. Finally, a report showed an inverse correlation of MVD with metastasis and prognosis in NSCLC [16].

Thus, in our present study, we used immunohistochemical method to investigate CHI3L1 expression and MVD in NSCLC samples and analyzed the relationships of CHI3L1 expression and MVD with variable clinicopathologic features and prognosis in patients with NSCLC.

Materials and methods

Patients and tumor sample

Tissue specimens were obtained from 95 patients who had undergone surgical resection for NSCLC in the Department of Cardiothoracic Surgery, Changhai Hospital, from October 2005 to November 2008. All patients had no preoperative radiotherapy or chemotherapy and had no distant metastases. The data on their clinicopathologic features and follow-up were complete. Forty-six cases of adjacent tissue samples were taken from about 0.5 cm away from the outer edge of the lung tumor tissues, and the other 38 cases were taken more than 5 cm away from the tumor margin of normal lung tissues as negative controls. For quantitative real-time PCR (qRT-PCR) analysis, nine matched pairs of tumor tissues and adjacent non-tumor tissue samples were obtained from pulmonary lobectomy specimens of patients diagnosed with NSCLC immediately after surgery between August 2013 and October 2013 in our department, and stored at −80 °C. For all patients, histological type and grade of cancer cell differentiation were reevaluated and determined by the classification system of the World Health Organization modified in 2004, and postsurgical pathological staging was determined based on the international staging system. This study was approved by the Ethical Committee of The Second Military Medical University prior to its initiation.

Immunohistochemical (IHC) analysis

The tissues were 10 % formalin-fixed, paraffin-embedded, and cut into 4-μm thickness. All slides were then deparaffinized in xylene and dehydrated with descending-grade alcohol. Endogenous peroxidase activity was quenched by brooding in methanol containing 3 % hydrogen peroxide for 30 min at room temperature. After blocking with recommended serum for 20 min at room temperature, the slides were incubated with a primary antibody CHI3LI or CD34 (diluted 1:400, Abcam) overnight at 4 °C and a homologous secondary antibody for 1 h at room temperature in a humidified box. Then the sections were stained with freshly dispensed diaminobenzidine solution (DAB) for observation under a microscope. In the process, the slides were all rinsed three times with phosphate-buffered saline (PBS, pH 7.2) before each step, except for the procedure of serum blocking to incubation with the primary antibody.

Evaluation of IHC staining

For immunostaining of CHI3L1, the area within the diagnostic area was scored by three independent observers and a reproducible semiquantitative method that considered both staining intensity (0, negative; 1, weak; 2, moderate; and 3, strong) and the percentage of positively stained cells (0, 0–5 %; 1, 6–25 %; 2, 26–50 %; 3, 51–75 %; 4, >76 %) was adopted [17]. For the evaluation of the positive staining of CHI3L1, at least three sections or areas from each sample should be scored. Conflicting scores were resolved by choosing the value consistent between two observers or the average of the scores.

MVD was determined with CD34-stained slides using the procedure of Weidner et al. [18]. Individual microvessels were counted in the area of highest vascularity at ×200 in three selected microscopic fields. Any brown staining endothelial cell or cluster that was separated from other nearby microvessels was counted. The presence of a vascular lumen was not necessary to identify a microvessel. Large anastomosing sinusoidal vessels were counted as a single vessel. Large vessels with thick muscular walls were excluded from the count. The microvessel count was expressed as the mean number of vessels in the selected area.

The cutoff value for high and low expression was determined based on a heterogeneity value measured through log-rank statistical analysis with respect to overall survival [19]. The staining index score 4 was chosen as a cutoff point for discrimination between CHI3L1 low and high expression. And the staining index score ≥4 defined tumors with high CHI3L1 expression, and the staining index score <4 indicated low CHI3L1 expression. Tumors with microvessels ≥35 were classified as high MVD, while tumors with microvessels <35 were classified as low MVD.

Quantitative real-time PCR (qRT-PCR)

Total RNA samples from surgical specimens were extracted using TRIzol Reagent (Invitrogen, Carlsbad, USA) according to the manufacturer’s instructions. The extracted RNA was pretreated with RNase-free DNase, and 2 μg RNA from each sample was used for complementary DNA (cDNA) synthesis primed with random hexamers. For the PCR amplification of CHI3L1 cDNA, an initial amplification step using CHI3L1-specific primers was performed with denaturation at 95 °C for 10 min, followed by 28 denaturation cycles at 95 °C for 60 s, primer annealing at 58 °C for 30 s, and a primer extension phase at 72 °C for 30 s. Upon the completion of the cycling steps, a final extension step at 72 °C for 5 min was performed before the reaction mixture was stored at 4 °C. Real-time PCR was then employed to determine the fold increase of CHI3L1 messenger RNA (mRNA) in each of the NSCLC relative to the paired normal lung tissue taken from the same patient. Expression data were normalized to the GAPDH to control the variability in expression levels, and all experiments were performed in triplicate.

Statistical analysis

All statistical analyses were performed using SPSS version 18.0 software (SPSS Inc., Chicago, USA). Chi-square test was used to test the correlation between CHI3L1 expression and MVD and the associations between CHI3L1 expression or MVD and clinicopathological factors. Kaplan-Meier method was used to calculate the survival curves, and log-rank test was used to compare the difference between the survivals of patient subgroups. Multivariate Cox regression analysis was used to identify significant independent prognostic factors. Values of P < 0.05 were considered statistically significant.

Results

CHI3L1 is overexpressed in NSCLC tissues

We detected the expression of CHI3L1 protein in tumor tissues and adjacent non-tumor tissues and normal lung tissues by IHC. As shown in Fig. 1a, diffuse cytoplasm staining of CHI3L1 protein at various intensities was observed in cancer cells, but CHI3L1 was barely detected in adjacent non-tumor tissues. However, we observed no statistically significant correlation between CHI3L1 protein expression and any clinicopathological features of NSCLC tissues (P > 0.05, Table 1). The mean value of CHI3L1 expression in 95 NSCLC tissues was 53.68 %, significantly higher than those in adjacent non-tumor tissues and normal lung tissues (34.78 and 31.58 %, respectively, P < 0.05, Table 2).

Fig. 1
figure 1

The expression pattern of CHI3L1 in NSCLC tissues. a ICH analysis of CHI3L1 in NSCLC and normal tissues (×200). b qRT-PCR analyses of CHI3L1 mRNA in nine pairs of NSCLC tissues and matched non-tumor tissues with GAPDH as a loading control in both panels. c Intratumoral microvessels were stained as brown by the anti-CD34 monoclonal antibody in NSCLC tissues

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Table 1 Relationship between CHI3L1 expression or MVD and clinicopathological variables in NSCLC patients
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Table 2 The expression of CHI3L1 in NSCLC tissues, adjacent non-tumor tissues, and normal tissues
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To investigate the status of CHI3L1 gene expression in NSCLC, we used qRT-PCR to measure the mRNA expression in nine pairs of NSCLC tissues and adjacent non-tumor tissues. Compared with their adjacent non-tumor specimens, six of nine NSCLC had up-regulated expression of CHI3L1 (Fig. 1).

MVD is associated with NSCLC clinical features

Intratumoral MVD was quantified by counting CD34-positive endothelial cells in cancer tissues (Fig. 1c), and the staining intensity of MVD ranged broadly from 8 to 96 microvessels/200 × magnification field. We found that MVD was significantly correlated with invasion depth (P < 0.05) but not with other clinicopathologic factors of NSCLC (P > 0.05, Table 1).

High CHI3L1 expression predicts poor prognosis in NSCLC patients

Of the 95 patients, 49 (51.6 %) cases died within 5 years after operation, and tumor relapse developed during follow-up in 56 (58.9 %) patients. Kaplan-Meier analyses compared by the log-rank test were used to calculate the effect of the clinicopathologic factors on overall survival and disease-free survival. Univariate analysis demonstrated that high MVD and CHI3L1 protein overexpression significantly predicted decreased overall 5-year survival (P < 0.05). In addition, high MVD and CHI3L1 protein overexpression indicated a higher risk of recurrence (P < 0.05, Fig. 2); furthermore, multivariate analysis identified CHI3L1 overexpression (P = 0.012) and MVD (P = 0.028) as independent prognostic factors for progression-free survival. However, only CHI3L1 overexpression retained its significance as an independent prognostic factor for overall as well as progression-free survival (P = 0.009, Table 3).

Fig. 2
figure 2

Kaplan-Meier curves of disease-free and overall survival stratified according to the status of CHI3L1 protein expression and MVD

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Table 3 Univariate and multivariate analyses of prognostic variables
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Correlation between CHI3L1 overexpression and MVD

We examined the survival differences of patients stratified for low MVD and high MVD according to CHI3L1 protein expression status. For patients without CHI3L1 overexpression, we found a highly significant inferior overall survival (OS) and disease-free survival (PFS), respectively, in patients with high MVD compared with patients with low MVD (P = 0.005 for OS and P = 0.002 for PFS, Table 4). However, there were no significant differences in survival between low-MVD group and high-MVD group for patients with CHI3L1 overexpression (P > 0.05, Table 4). And statistical analysis demonstrated that there was significantly more MVD in tumors with CHI3L1 protein overexpression than in those with CHI3L1 protein non-overexpression (P < 0.05, Mann-Whitney U test, Fig. 3).

Table 4 Survival differences stratified by low MVD and high MVD in patients with or without CHI3L1 overexpression
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Fig. 3
figure 3

Intratumoral MVD in relation to CHI3L1 protein immunoreactivity. Mann-Whitney U test demonstrated that tumors with CHI3L1 protein high expression showed higher intratumoral MVD than tumors with CHI3L1 protein low expression (P < 0.05)

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Discussion

The ability to proliferate uncontrollably is the dominant characteristic of many types of cancer cells. Evaluation of molecular prognostic factors is an important area of cancer research. Several studies have shown that CHI3L1 is overexpressed in several types of cancer and suggest the oncogenic role of CHI3L1 [20, 21]. However, few studies have investigated the expression and significance of CHI3L1 in NSCLC, especially for the prognosis of NSCLC.

In the present study, our results indicated that there was no significant correlation between CHI3L1 expression and the clinicopathological features of NSCLC in the statistical analysis. However, high MVD was significantly associated with T status in NSCLC but not associated with age, gender, smoking, histology, and histological grade. To a certain extent, T stage is a critical process of tumor development, and the difference in the correlation between MVD and clinicopathological features may reflect that the microvessel density is an important aspect of tumor development. However, we found no significant difference in CHI3L1 expression between lung adenocarcinoma and lung squamous cell carcinoma by immunohistochemical staining.

Noteworthy, our data indicated that CHI3L1 is overexpressed, and it was common in early NSCLC tissues and significantly associated with increased angiogenic activity measured as intratumoral MVD, suggesting that CHI3L1 plays a key role in NSCLC tumorigenesis by the induction and/or promotion of tumor angiogenesis. Previous reports have demonstrated that CHI3L1 was up-regulated in many types of tumor. Harving reported that CHI3L1 was significantly higher in tumors from patients with soft tissue sarcomas and the staining intensity was higher in highly malignant tumors than in low-malignancy tumors [20]. Libreros detected CHI3L1 expression in breast cancer patients and found that the enhanced expression of CHI3L1 was closely correlated with pro-angiogenic molecules such as monocyte chemoattractant protein-1 (MCP-1) and matrix metalloproteinases-9 (MMP-9) expression [21]. Özdemir showed the relationship between serum level of CHI3L1 and Gleason score, grade and stage of the disease, in patients with prostate cancer and demonstrated that elevated serum level of CHI3L1 was strongly associated only with metastatic stage of the prostate cancer [22]. Isabella reported that CHI3L1 expression predicts shorter overall and disease-free survival in anal carcinoma patients [23]. Although multiple growth factors have been demonstrated to regulate angiogenesis and vascular development, little is known about the complex regulation mechanism of gene expression and translation [24]. Our results highlight the potential role of CHI3L1 in tumor angiogenesis.

Our survival analysis indicated that high MVD and CHI3L1 protein overexpression significantly predicted poor overall 5-year survival and a higher recurrence rate. Moreover, further analysis using the Cox regression model confirmed that CHI3L1 expression and MVD were independent factors in predicting progression-free survival for NSCLC patients, suggesting that CHI3L1 protein and MVD may be potential prognostic factors for the relapse of early stage NSCLC patients. However, in multivariate analysis, only CHI3L1 expression could independently and significantly predict overall 5-year survival, despite the finding that high MVD was significantly associated with tumor recurrence. Our data showed that CHI3L1 overexpression had an overwhelming influence on the OS and PFS, while MVD showed a significant effect on the OS and PFS only in the patients without CHI3L1 overexpression.

In conclusion, this study demonstrated that CHI3L1 protein is overexpressed in early stage NSCLC and correlate with tumor angiogenesis and relapse. Moreover, CHI3L1 overexpression is an independent prognostic factor for NSCLC patients. As a result, these data suggested that CHI3L1 may be a candidate biomarker for NSCLC prognosis and a target for new therapies.