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Prognostic significance of cyclin D1 expression in resected stage I, II non-small cell lung cancer in Arabs
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     a Department of Surgery, Faculty of Medicine, Kuwait University, Chest Diseases Hospital, P.O. Box 24923, Safat 13110, Kuwait

    b Department of Pathology, Kuwait Cancer Center, Ministry of Health, Kuwait

    Abstract

    The aim of this study was to evaluate the prognostic significance of cyclin D1 expression in primary, resected stage I and II non-small cell lung cancer (NSCLC) in Arabs. A longitudinal cohort study of 98 consecutive patients with resected stage I and II NSCLC were evaluated immunohistochemically for the expression of cyclin D1 and determined its prognostic significance by comparison with follow-up data from January 1994 to December 1999. This study included 76 male and 22 female patients. They represented 31 stage I NSCLC and 67 stage II tumors. Expression of cyclin D1 was detected immunohistochemically in 48 (49%) of the 98 NSCLC. Cyclin D1 expression had significantly higher positive results in patients with chronic obstructive pulmonary disease (P=0.001), poorly differentiated carcinoma (P=0.001), presence of vascular invasion (P=0.003), and visceral pleural invasion (P=0.005). Patients with cyclin D1-positive tumors had shorter survival than those with cyclin D1-negative tumors (5-year survival rates, 48% and 74%, respectively; P=0.006 by the log-rank test). In conclusion, a higher percentage of NSCLC with visceral pleural invasion, vascular invasion, poor differentiation of the tumor, patients with chronic obstructive pulmonary disease have positive cyclin D1 expression than other lung tumors.

    Key Words: Cyclin D1 expression; Non-small cell lung cancer; Prognostic factor

    1. Introduction

    Lung cancer is one of the leading causes of cancer death throughout the world. Whereas the treatment of non-small cell lung cancer (NSCLC) has improved, there is no evidence to suggest that therapeutic advances have resulted in a marked increase in survival rates, and the overall 5-year survival rate remains <15% [1]. Therefore, there is an urgent need to improve our understanding of properties of cancer cells and to define prognostic factors. This would help to choose the best therapeutic procedures. Many studies have highlighted the aberrant expression, due to genetic alterations and occasionally with prognostic significance in primary lung cancer [2–4]. Cyclin D1 overexpression has been discovered in many types of human cancers including NSCLC as a result of gene amplification or translocations targeting the D1 locus on human chromosome 11q13 [5]. However, evaluation of the relevance of cyclin D1 in NSCLC is not yet conclusive.

    In Kuwait, lung cancer is the leading cause of cancer in males, accounting for 28.2% of all cancers in males. Therefore, in this study, we investigate the expression of cyclin D1 by immunohistochemistry in 98 surgically resected stages I, II NSCLC. Various clinical parameters of the patients as well as the pathological characteristics of the tumors were collected for correlation with the extent of expression of cyclin D1.

    2. Materials and methods

    2.1. Lung cancer patients and specimens

    Ninety-eight lung cancer specimens were obtained from patients who underwent complete surgical resection for NSCLC at the Chest Diseases Hospital, which is the only center doing thoracic surgery in the country, during the period from January 1994 to December 1999. New histological sections of 3–4 μm were cut and stained with hematoxylin and eosin and with Mucicarmine stains to classify the tumors into different histological types. According to the World Health Organization (WHO 1999) [6], they were histopathologically diagnosed as squamous cell carcinoma (n=49) and adenocarcinoma (n=49). All of the specimens were formalin fixed and sectioned for microscopic examination after staining with hematoxylin-eosin. Histological diagnosis and pathological features were obtained. Surgical staging was done according to Mountain (1997) based on tumor size and involvement of lymph node metastasis [7].

    The clinical data of these patients including, sex, age, smoking status, and presence or absence of chronic obstructive pulmonary disease (COPD) were recorded and analyzed with the result of cyclin D1 expression in each tumor. COPD is defined when a compatible clinical picture exists, when there is a chronic obstruction to the airflow and when other conditions with similar symptoms are ruled out. Obstruction to the airflow was confirmed when the post-bronchodilator as forced expiratory volume in 1 s (FEV1) <70% predicted or FEV1/FVC (forced vital capacity) <70% predicted [8]. Complete clinical and follow-up information was available for this cohort.

    2.2. Immunohistochemistry

    For immunohistochemical demonstration of cyclin D1 in the tumor tissue, 3–4 μm thick sections mounted on poly-L-lysine coated slides were deparaffinized in xylene and rehydrated through graded alcohol series followed by washing with distilled water 3 times for 2 min each. Endogenous peroxidase in each section was blocked by treating the slides in boiling 0.01 M citrate buffer and allowed to cool down for at least 15–20 min at room temperature; the slides were, however, rinsed with phosphate-buffered saline (PBS). Primary antibodies (mouse anti-cyclin D1) were added and incubated for about 30–60 min at room temperature and subsequently rinsed with PBS. Secondary antibodies and enzyme conjugate were added and incubated for 10 min each. Chromogen, 3,3'-diaminobenzidine (DAB) was added and incubated for 5–10 min at room temperature; this is followed by counter staining with Meyer's hematoxylin. One slide that was known to be positive for the antigen detected was used as positive control, and one slide incubated without primary antibody was used as negative control for each staining batch. Cyclin D1 positive cancer cells were those that expressed either nuclear positivity or strong cytoplasmic positivity (Fig. 1).

    2.3. Statistical analysis

    Data were expressed as mean ± S.D.; data analyses were made using SPSS software windows version 8 packages (SPSS, Chicago, IL). The cut-off level for statistical significance was P<0.05. The unpaired Student's t-test was used to assess the significance between means of variables in the groups. The Pearson 2-test was used to ascertain the significance of association between two categorical variables. The 2-test was replaced by Fisher's exact test if the cell frequencies of any of the 2x2 contigency tables went below 5. Survival curves were estimated using the Kaplan–Meier method, and differences were evaluated by the log-rank test. The stepwise multivariate analysis was used according to Cox's model.

    3. Results

    This study included 76 male and 22 female patients, and the mean age was 52.2 years (range, 29–77 years). They represented 31 stage I NSCLC and 67 stage II tumors. Thirty-eight patients (39%) had COPD. The FEV1 values were: mean 74.5 (S.D.16.9) % Predicted, (range, 40–104% Pred.). The FEV1/FVC values were: mean 80.49 (S.D.17.2)% Predicted (range, 47–109% Pred.).

    In the current study, cyclin D1 expression was detected in 48 of the 98 lung tumors (49%). Simultaneous nuclear and cytoplasmic staining was observed in 4/98 (4%) of the cases. The relationship of clinical parameters of the patients with the presence of cyclin D1 expression is shown in Table 1. There was no significant difference of cyclin D1 expression in relation to gender and smoking status. Compared with various pathological characteristics of the tumor, cyclin D1 had significantly higher positive results in patients with vascular invasion (P=0.003), poor differentiation (P=0.001), and visceral pleural invasion (P=0.005), (Table 2).

    Patients with cyclin D1 positive tumors survived shorter than patients with cyclin D1 negative tumors (5-year survival rates, 48% and 74% respectively, P=0.006) (Fig. 2). By single variate analysis of survival function, cyclin D1 (Fig. 2), (P=0.006), in addition to COPD (P=0.01), vascular invasion (P=0.01), poor differentiation (P<0.0001), and visceral pleural invasion (P<0.0001), were significant prognostic factors affecting survival. By multiple variate analysis, only visceral pleural invasion (P<0.0001) and poor differentiation (P=0.04) remained as independent, significant prognostic factors. Squamous cell carcinoma patients with cyclin D1-positive tumors were found to have significantly shorter survival than those with cyclin D1-negative tumors (mean survival 46.1 vs. 56 months; P=0.007 by the log-rank test). In contrast, we observed no difference for adenocarcinoma between cyclin D1-positive and cyclin D1-negative tumors (mean survival 49 vs. 51 months; P=0.2 by the log-rank test).

    Further analysis investigated the influence of cyclin D1 on patient's survival in different pathological stages of lung cancer. The results showed cyclin D1 expression was an independent, significant prognostic factor only in stage I patients (Fig. 3), but not in stage II patients (Fig. 4).

    4. Discussion

    Several studies have proposed that cyclins could act as oncogens by forcing cells to evade the normal growth control [2,9]. Over-expression or amplification of cyclin D1 has been demonstrated in lung cancer patients [3,5,10–13]. We found cyclin D1 to be expressed in 49% of our specimens. This was similar to that reported by Betticher et al. [10] who found cyclin D1 expression in 25 of 53 (47%) NSCLC. These authors describe cyclin D1 to be predominantly present in the cytoplasm of lung carcinoma cells (22/25 cases). This finding is also in agreement with those of Nguyen et al. [5].

    In this study, a higher rate of cyclin D1 expression was noted in lung cancer patients with age under 45 years. It is suggested that alteration of cyclin D1 expression might play a more significant role in the pathogenesis of primary NSCLC in the younger age group. Although the relationship between smoking and cyclin D1 expression in lung cancer has been reported, the difference in cyclin D1 positivity in lung cancer patients with smoking habits was only slightly higher than non-smokers (51% vs. 44%) in the current study. Ratschiller et al. [14] found that the carcinomas of smokers expressed cyclin D1 in 77% of cases while carcinomas of non-smokers expressed this protein only 57% of the time (P<0.01). The correlation between smoking and cyclin D1 expression in this study and other studies was maintained when the analysis was limited to squamous cell lung carcinoma [14,15].

    Regarding the relationship of cyclin D1 expression with pathological characteristics of the tumor, the results reported in the literature have been controversial [10–13,15]. In this study, we found that NSCLC with vascular invasion and visceral pleural invasion has a significantly higher rate of cyclin D1 expression. There was also a significant correlation between cyclin D1 expression and tumor differentiation which was similar to previous reports in the literature [10,11]. The relationship of cyclin D1 expression with the prognosis of lung cancer has been studied but the results of research have been conflicting [4,10–13]. The reasons for such a controversial difference are not yet clear, but several possible causes might be imposed, including varying method of detection, antibodies used for staining, heterogeneity of the patients and cut-off values for positive expression. In this study which is done in Arabs with a homogenous group of NSCLC and stage I, II, and longer periods of observation, we found cyclin D1 expression to be an independent prognostic factor after surgical resection only in the stage I NSCLC patients. Caputi and associates [3] reported cyclin D1 expression to be a negative prognostic marker whose expression correlates with a shorter patient survival time. Betticher et al. [10] reported that cyclin D1 expression correlates with the grade of differentiation of the tumor, with amount of lymphocytic infiltration, and with a reduction in local relapse data, but not with patient outcomes. Other studies on lung cancer patients have reported cyclin D1 expression with a shorter survival time or stating the usefulness of cyclin D1 as a prognostic marker [13,15].

    Cyclin D1 expression was frequently found in tumors with altered p53 protein expression; cyclin D1 positive tumors showed higher proliferation activity determined by the ki-67 index than cyclin D1 negative tumors. Furthermore, the effect of a p53 mutation or overexpression is significant when associated with an abnormality of cyclin D1 of itself [4,12,13]. Mishina et al. [12] reported that a combination of cyclin D1 overexpression and p53 mutation or p53 protein overexpression was associated with a poor survival because of its association with higher-grade tumors. Similarly, Burke and associates [4] showed that both abnormalities of cyclin D1 and the p53 pathway play substantial roles in the development of NSCLC and have a significant effect on patient survival.

    In conclusion, the results of the present study show an inverse relationship between cyclin D1 expression and the prognosis of NSCLC. A higher percentage of NSCLC with visceral pleural invasion, vascular invasion, poor differentiation of the tumor, patients with COPD or younger age group have positive cyclin D1 expression than other lung tumors. Additional study of a larger number of patients is required to confirm the role of cyclin D1 in lung cancer.

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