Abstract

Background:

Cyclin B1 has an important role in the G2–M phase transition of the cell cycle. Wee1 delays mitosis by suppressing the activity of the Cyclin B1/cdc2 complex. The objective of the present study was to elucidate the clinicopathological and prognostic significance of Cyclin B1 and Wee1 expression in non-small-cell lung cancer (NSCLC).

Patients and methods:

An immunohistochemical assessment of Cyclin B1 and Wee1 expression was performed in 79 patients with NSCLC.

Results:

The expression of Cyclin B1 was correlated with differentiation (P = 0.0423) and vascular invasion (P = 0.001). Patients with overexpression of Cyclin B1 had higher mean values for both the Ki-67 proliferative index (Ki-Index) (P <0.0001) and proliferating cell nuclear antigen labeling index (PCNA-LI) (P <0.0001), and a poorer prognosis (P = 0.0068). Patients lacking expression of Wee1 had a higher recurrence rate (P = 0.0084) and a poorer prognosis (P = 0.0457), and tended to have higher Ki-Index and PCNA-LI values. Multivariate analysis suggested that both Cyclin B1 (P = 0.0244) and Wee1 (P = 0.0444) expression were significant prognostic factors.

Conclusions:

These findings suggest that Cyclin B1 expression could be a significant prognostic parameter in NSCLC. The loss of Wee1 expression may have a potential role in promoting tumor progression and may be a significant prognostic indicator in NSCLC.

Received 28 March 2003; revised 22 October 2003; accepted 6 November 2003

Introduction

Cyclin and cyclin-dependent kinase complexes play an important role in control of the cell cycle [1]. Cyclin B1/cdc2 complex has a role as a maturation/mitosis-promoting factor in the G2–M phase transition during the cell cycle [1]. Thus, dysregulation of the expression of Cyclin B1 may be involved in uncontrolled cell growth and malignant transformation. Overexpression of Cyclin B1 has been reported in various malignant tumors [26] and has been shown to be a poor prognostic factor in some of them [46]. The activity of Cyclin B1/cdc2 complex is inhibited by phosphorylation of the tyrosine 15 residue (Tyr15) on cdc2 [7]. The phosphorylation of Tyr15 is achieved by Wee1 kinase, a nuclear protein that delays mitosis until the completion of DNA replication in cells with DNA damage [8]. In colon carcinoma cells, Wee1 expression is reduced, suggesting that it acts as a tumor suppressor [9].

We carried out immunohistochemical analysis to assess the clinicopathological and prognostic significance, and the interrelationship, of Cyclin B1 and Wee1 expression. Furthermore, we examined by differential PCR analysis whether patients with non-small-cell lung cancer (NSCLC) have amplification or deletion of the Cyclin B1 and Wee1 genes.

Patients and methods

Patients

The study focused on 79 patients with NSCLC who had undergone lobectomy with lymph node dissection between 1989 and 2001 at the Department of Surgery I, Gunma University Faculty of Medicine. The lung cancers were classified according to the TNM Classification of the International Union Against Cancer, 5th edition [10]. Patients who had received irradiation or chemotherapy prior to surgery, or for whom follow-up had been considered inadequate, were not included in the study. The pathological staging of this group was: stage I in 53, stage II in nine and stage III in 17 cases. Among the 79 patients, 16 had disease recurrence after surgery.

Immunohistochemistry

Immunohistochemical staining was performed according to a previously described method [11]. The tissue sections were placed in 10 mM citrate buffer (pH 6.0 for Cyclin B1 and Ki-67, pH 7.0 for Wee1) and heated in an autoclave oven. The sections were incubated with either normal rabbit serum for the Cyclin B1, Ki-67 and proliferating cell nuclear antigen (PCNA) samples, or 4% Block Ace Solution (Dainippon Pharmaceutical Corp., Osaka, Japan) for the Wee1 samples. The sections were incubated with anti-Cyclin B1 monoclonal antibody (Novocastra Laboratories, Newcastle-upon-Tyne, UK) at a dilution of 1:200, and with anti-Wee1 monoclonal antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-Ki-67 monoclonal antibody (DAKO, Glostrup, Denmark) and anti-PCNA monoclonal antibody (DAKO) all at a dilution of 1:100.

When >15% of tumor cells were immunoreactive [6], the sample was regarded as Cyclin B1-positive. The Wee1 labeling index defined the percentage of nuclear-stained tumor cells, and was classified as follows: 0 = <10%, 1+ = 10–30%, 2+ = 30–50% and 3+ = >50% of cell staining. Because this division was considered the most reliable prognostic indicator, we regarded a specimen with a score 0 or 1+ as negative. The Ki-67 proliferative index (Ki-Index) and PCNA labeling index (PCNA-LI) were defined as the percentages of nuclear-stained tumor cells.

Differential PCR analysis for the expression of Cyclin B1 and Wee1 genes

We studied 18 of the 79 patients whose specimens were subjected to immunohistochemical analysis. Total DNA was extracted from each fresh-frozen human lung tumor tissue and normal tissue using a DNeasy Tissue Kit (Qiagen, Valencia, CA, USA). For differential PCR analysis, a 150-bp fragment of the Cyclin B1 gene was co-amplified with an 82-bp fragment of the interferon-γ (IFNG) gene as an internal control [12]. Moreover, the same analysis was performed for a 440-bp fragment of the Wee1 gene and the IFNG gene. Specific primers used were as follows: 5′-CCTGCAAATGCCTGGTTTAT-3′ and 5′-CATGCTTCGATGTGGCATAC-3′ for Cyclin B1; 5′-ACTGATAGAATCCAGTTTGC-3′ and 5′-ATGCAATGCCTACAAAGTGC-3′ for Wee1 [13]; and 5′-GCAGAGCCAAATTGTCTCCT-3′ and 5′-GGTCTCCACACTCTTTTGGA-3′ for IFNG [12]. Differential PCR was performed by 25 cycles of denaturation at 94°C for 1 min, annealing at 55°C for 1 min and extension at 72°C for 1 min.

The PCR products were electrophoresed in 3% agarose gel with ethidium bromide and autoradiographed by Fluor-S MAX2 MultiImager (Bio-Rad, Hercules, CA, USA). Analysis of gene density was performed using Quantity One v. 4.2.1 software (Bio-Rad). The ratio of the total target gene density relative to the total control gene density was calculated for each sample. Furthermore, using this result we analyzed the ratio of lung tumor tissue relative to normal tissue (T/N ratio) in each patient.

Statistical analysis

Statistical analysis was performed according to a previously described method [11], and Cox’s proportional hazards model for risk ratio was used to assess the results of multivariate survival analysis. Differences were considered to be statistically significant at P <0.05.

Results

Cyclin B1 expression was predominantly located with in the cytoplasm of the tumor cells (Figure 1A). Positive expression of Cyclin B1 was observed in 13 out of 79 patients (16.5%). Positive staining for Wee1 was observed in the nuclei of the tumor cells (Figure 1B). Absence of Wee1 expression was seen in 52 patients (65.8%). The relationship between the expression of Cyclin B1 and Wee1 and the clinicopathological features of NSCLC, including Ki-Index and PCNA-LI values, is shown in Table 1. Poorly differentiated tumors showed significantly higher Cyclin B1 expression than well and moderately differentiated tumors (P = 0.0423). Furthermore, there was a significant correlation between Cyclin B1 expression and vascular invasion (P = 0.001). Both the Ki-Index and PCNA-LI values were significantly higher in Cyclin B1-positive tumors than negative tumors (P <0.0001 and P <0.0001, respectively). There was no significant correlation between Wee1 expression and the various clinicopathological factors. The Ki-Index and PCNA-LI values tended to be higher in Wee1-negative tumors than positive tumors. No significant correlation was recognized between the expression of Cyclin B1 and Wee1 (Table 2).

The patients with Wee1-negative tumors had a significantly higher recurrence rate than those with Wee1-positive tumors (Table 3; P = 0.0084). The postoperative survival rate of patients with Cyclin B1-positive tumors was significantly lower than that of patients with Cyclin B1-negative tumors (Figure 2A; P = 0.0068). The postoperative survival rate of patients with Wee1-negative tumors was also significantly lower than that of patients with Wee1-positive tumors (Figure 2B; P = 0.0457). The result of the multivariate analysis is shown in Table 4. This result suggests that the expression of Cyclin B1 (P = 0.0244) and Wee1 (P = 0.0444) are both significant prognostic factors.

The mean value of the T/N ratio in the Cyclin B1 gene and the Wee1 gene was 0.97 ± 0.17 and 0.95 ± 0.18, respectively. Therefore, there appeared to be no difference in the expression of Cyclin B1 and Wee1 genes between lung tumor tissue and normal tissue.

Discussion

In this study, overexpression of Cyclin B1 was associated with grade of differentiation and vascular invasion, and also with significantly higher Ki-Index and PCNA-LI values. The expression of Ki-67 and PCNA are generally well established as markers of cell proliferation and malignant potential in NSCLC [14, 15]. Thus, our data suggest that overexpression of Cyclin B1 may indicate an increased malignant potential, and may be a significant prognostic indicator in patients with NSCLC. Soria et al. [6] reported that there was a clinical relationship between overexpression of Cyclin B1 and early-stage NSCLC, particularly the squamous subtype. Some investigators have suggested that overexpression of Cyclin B1 is associated with aggressive tumor behavior, and that this is a useful prognostic marker in patients with squamous cell carcinoma [46]. However, other authors have reported that overexpression of Cyclin B1 is associated with less aggressive tumor behavior, and is not a useful prognostic parameter in patients with adenocarcinoma [2, 3]. Although >70% of the patients in our study had adenocarcinoma, our data suggest that expression of Cyclin B1 is a significant prognostic indicator. Although the data supporting this argument are not shown, overexpression of Cyclin B1 was associated with a poor prognosis in patients with adenocarcinoma. The reason that other reports have differed from ours may be due to organ specificity, disease stage or heterogeneity. About one-third of the patients in this study were at an advanced stage, and overexpression of Cyclin B1 was observed more frequently in stage III tumors.

Similar to a previous study [16], our data also indicate that patients with NSCLC have no amplification or deletion of the Cyclin B1 gene. This result suggests that Cyclin B1 expression at the protein level may be regulated by other mechanisms, for example transcription, DNA methylation or translation, and not by gene expression at the DNA level. The activity of the Cyclin B1/cdc2 complex is negatively controlled by Wee1 kinase activity [8]. Thus, loss of Wee1 confers an advantage to neoplastic cells by allowing faster progression through the cell cycle. In this study, absence of Wee1 expression was observed immunohistochemically in about two-thirds of the NSCLC patients. The reduction in Wee1 expression was associated with a poorer prognosis and a higher recurrence rate. These tumors also tended to have higher Ki-Index and PCNA-LI values. This suggests that Wee1 may be only a weak suppressor of tumor progression in NSCLC. As for Cyclin B1, Wee1 expression at the protein level may be regulated by other mechanisms, and not by gene expression at the DNA level.

In conclusion, we suggest that assessment of Cyclin B1 expression could be useful for evaluating tumor progression and prognosis in NSCLC patients. Loss of Wee1 expression may play a role in promoting tumor aggressiveness, and may be a useful prognostic indicator.

Acknowledgements

The authors thank Dr Takaaki Sano for useful comments on histopathological analysis.

*

Correspondence to: Dr T. Yoshida, Department of Surgery I, Gunma University Faculty of Medicine, 3-39-22, Showa-machi, Maebashi, Gunma, Japan. Tel: +81-27-220-8224; Fax: +81-27-220-8230; E-mail: ytakeshi@med.gunma-u.ac.jp

Figure 1. (A) Photograph of tissue section immunostained for Cyclin B1 (×200). Cyclin B1 was detected in the cytoplasm or nuclei of the tumor cells, or in both. (B) Photograph of tissue section immunostained for Wee1 (×200). Expression of Wee1 was detected in the nuclei of the tumor cells.

Figure 1. (A) Photograph of tissue section immunostained for Cyclin B1 (×200). Cyclin B1 was detected in the cytoplasm or nuclei of the tumor cells, or in both. (B) Photograph of tissue section immunostained for Wee1 (×200). Expression of Wee1 was detected in the nuclei of the tumor cells.

Figure 2. (A) Postoperative survival curves for patients with non-small-cell lung cancer (NSCLC) compared with the Cyclin B1 expression status. The survival curve for patients with positive expression of Cyclin B1 was significantly lower than those for patients with negative expression. (B) Postoperative survival curves for patients with NSCLC compared with the Wee1 expression status. The survival curve for patients who had Wee1-negative tumors was significantly lower than those for patients who had Wee1-positive tumors.

Figure 2. (A) Postoperative survival curves for patients with non-small-cell lung cancer (NSCLC) compared with the Cyclin B1 expression status. The survival curve for patients with positive expression of Cyclin B1 was significantly lower than those for patients with negative expression. (B) Postoperative survival curves for patients with NSCLC compared with the Wee1 expression status. The survival curve for patients who had Wee1-negative tumors was significantly lower than those for patients who had Wee1-positive tumors.

Table 1.

The correlation between clinicopathalogical factors and Cyclin B1 and Wee1 expression in patients with NSCLC

Parameter n Cyclin B1  Wee1 
  – P  – P 
Age (years) (mean ± SD)  65.7 ± 9.9 64.5 ± 11.0  0.698  66.3 ± 10.6 63.7 ± 8.6 0.2755 
Sex         
 Male 54 44 10  0.4674  36 18 0.8162 
 Female 25 22  3   16  9  
Histology         
 Adenocarcinoma 56 50  6  0.0986  36 20 0.8981 
 Squamous cell carcinoma 20 14  6   14  6  
 Large cell carcinoma  3  2  1    2  1  
Differentiation         
 Well + moderately 56 50  6  0.0423  34 22 0.1186 
 Poorly 20 14  6   16  4  
Tumor status         
 pT1 42 37  5  0.1387  28 14 0.3551 
 pT2 24 21  3   14 10  
 pT3  5  3  2    5  0  
 pT4  8  5  3    5  3  
Nodal status         
 pN– 63 55  8  0.0739  43 20 0.366 
 pN+ 16 11  5    9  7  
Lymphatic invasion         
 ly– 37 31  6  0.957  28  9 0.0831 
 ly+ 42 35  7   24 18  
Vascular invasion         
 v– 50 47  3  0.001  34 16 0.5921 
 v+ 29 19 10   18 11  
Stage         
 I 53 47  6  0.0788  35 18 0.9541 
 >II 26 19 10   18 11  
Ki-Index (mean ± SD)  27.1 ± 18.9 58.8 ± 26.7 <0.0001  35.6 ± 23.8 25.9 ± 21.6 0.0789 
PCNA-LI (mean ± SD)  36.0 ± 25.5 75.4 ± 31.2 <0.0001  46.9 ± 29.7 34.0 ± 29.4 0.0694 
Parameter n Cyclin B1  Wee1 
  – P  – P 
Age (years) (mean ± SD)  65.7 ± 9.9 64.5 ± 11.0  0.698  66.3 ± 10.6 63.7 ± 8.6 0.2755 
Sex         
 Male 54 44 10  0.4674  36 18 0.8162 
 Female 25 22  3   16  9  
Histology         
 Adenocarcinoma 56 50  6  0.0986  36 20 0.8981 
 Squamous cell carcinoma 20 14  6   14  6  
 Large cell carcinoma  3  2  1    2  1  
Differentiation         
 Well + moderately 56 50  6  0.0423  34 22 0.1186 
 Poorly 20 14  6   16  4  
Tumor status         
 pT1 42 37  5  0.1387  28 14 0.3551 
 pT2 24 21  3   14 10  
 pT3  5  3  2    5  0  
 pT4  8  5  3    5  3  
Nodal status         
 pN– 63 55  8  0.0739  43 20 0.366 
 pN+ 16 11  5    9  7  
Lymphatic invasion         
 ly– 37 31  6  0.957  28  9 0.0831 
 ly+ 42 35  7   24 18  
Vascular invasion         
 v– 50 47  3  0.001  34 16 0.5921 
 v+ 29 19 10   18 11  
Stage         
 I 53 47  6  0.0788  35 18 0.9541 
 >II 26 19 10   18 11  
Ki-Index (mean ± SD)  27.1 ± 18.9 58.8 ± 26.7 <0.0001  35.6 ± 23.8 25.9 ± 21.6 0.0789 
PCNA-LI (mean ± SD)  36.0 ± 25.5 75.4 ± 31.2 <0.0001  46.9 ± 29.7 34.0 ± 29.4 0.0694 

NSCLC, non-small-cell lung cancer; Ki-Index, Ki-67 proliferative index; PCNA-LI, proliferating cell nuclear antigen labeling index.

Table 2.

The correlation between Cyclin B1 and Wee1 expression in patients with NSCLC

Wee1 n  Cyclin B1   
   – P 
– 52  43 0.7768 
27  23  
Wee1 n  Cyclin B1   
   – P 
– 52  43 0.7768 
27  23  

NSCLC, non-small-cell lung cancer.

Table 3.

The correlation between the recurrence rate and Cyclin B1 and Wee1 expression in patients with NSCLC

Recurrence n Cyclin B1  Wee1 
  – P  – P 
– 63 55 0.0739  37 26 0.0084 
16 11   15  1  
Recurrence n Cyclin B1  Wee1 
  – P  – P 
– 63 55 0.0739  37 26 0.0084 
16 11   15  1  

NSCLC, non-small-cell lung cancer.

Table 4.

Multivariate analysis on prognosis of patients with NSCLC

Parameter Regression coefficient OR (95%CI) P 
pT (1 versus 2–4) 0.938  8.366 (1.332–52.557) 0.0235 
pN (– versus +) 1.005  0.653 (0.091–4.682) 0.6718 
Lymphatic invasion  (– versus +) 0.809  2.441 (0.5–11.915) 0.2699 
Vascular invasion  (– versus +) 0.824  0.801 (0.159–4.029) 0.7873 
Stage (I versus II–III) 0.947  0.915 (0.143–5.862) 0.9257 
Cyclin B1 expression  (– versus +) 1.052 10.669 (1.357–83.886) 0.0244 
Wee1 expression  (– versus +) 1.102  9.169 (1.057–79.526) 0.0444 
Parameter Regression coefficient OR (95%CI) P 
pT (1 versus 2–4) 0.938  8.366 (1.332–52.557) 0.0235 
pN (– versus +) 1.005  0.653 (0.091–4.682) 0.6718 
Lymphatic invasion  (– versus +) 0.809  2.441 (0.5–11.915) 0.2699 
Vascular invasion  (– versus +) 0.824  0.801 (0.159–4.029) 0.7873 
Stage (I versus II–III) 0.947  0.915 (0.143–5.862) 0.9257 
Cyclin B1 expression  (– versus +) 1.052 10.669 (1.357–83.886) 0.0244 
Wee1 expression  (– versus +) 1.102  9.169 (1.057–79.526) 0.0444 

NSCLC, non-small-cell lung cancer; OR, odds ratio; CI, confidence interval.

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