Cyclin E plays a pivotal role in the regulation of G1-S transition and relates to malignant transformation of cells. However, the clinical significance of cyclin E in patients with laryngeal squamous cell carcinoma (LSCC) remains unknown. We examined the expression of cyclin E in 102 patients with LSCC and analyzed its relation to clinicopathological parameters, cell proliferation, and clinical outcome. Cyclin E overexpression was observed in 54 cases (52.94%) of LSCC and was significantly correlated with the tumor site(P = 0.012), tumor size (P =0.006), poor differentiation (P = 0.026), lymph node metastasis (P = 0.012), and advanced stage(P = 0.002). A positive correlation between the cyclin E expression and proliferative activity of tumor cells was found(r = 0.896; P < 0.0001). Kaplan-Meier analysis showed that shorter disease-free and overall survival was significantly associated with proliferating cell nuclear antigen (PCNA) overexpression and cyclin E overexpression. When PCNA and cyclin E are combined, the patients with both PCNA overexpression and cyclin E overexpression had the poorest prognoses when compared with the other cases. Additionally, in early stage (I–II) cases,cyclin E was also revealed to possess a significant prognostic role. By multivariate analysis, lymph node metastasis and cyclin E overexpression were independent prognostic factors for disease-free survival, and tumor size, lymph node metastasis, advanced stage, as well as cyclin E overexpression were independent prognostic factors for overall survival. These findings indicate that cyclin E overexpression is associated with unfavorable clinicopathological parameters and represents an independent marker for cell proliferation and prognosis of LSCC.

LSCC3 is one of the most common malignant neoplasms of the head and neck. Its incidence varies widely in different countries, and increasing trends have been observed in many previous studies (1). Currently, prognostic evaluation is mainly based on the clinical stage,tumor site, and histopathological grade. Recent studies have suggested that other factors, such as the molecular and cellular characteristics of the primary tumor, may improve our ability to prognosticate (2).

Cyclins are prime cell cycle regulators and play a central role in the control of cell proliferation by forming complexes with different CDKs (3). These complexes enzymatically phosphorylate cell cycle regulatory elements, such as retinoblastoma protein. Cyclin D1 and E are responsible for activation of CDKs during the G1 phase and rate-limiting factors for the G1-S transition (4). In these interactions, CDKs and cyclins act as catalytic subunits and regulatory molecules, respectively, peaking in specific phases of the cell cycle (5). Cyclin E, the focus of our study, is a highly conserved protein that was first identified by virtue of its ability to rescue G1 cyclin-defective budding yeast (6), which, along with its catalytic subunit CDK2, is involved in phosphorylation of the retinoblastoma protein. Increased expression of cyclin E has been shown to reduce the length of G1 and accelerate the transition into S-phase (7). Abnormalities in cell cycle regulators and subsequent deregulation of the G1-S transition may be one of the most important biological events in the malignant transformation of cells. Cyclin E overexpression has been found in several cancers including breast (8, 9, 10), colorectal (11), gastric (12), and lung (13, 14) cancers.

Proliferation activity is a potent biological marker that estimates the growth of neoplasms quantitatively and can aid in determining the prognosis of patients with carcinomas (15, 16). PCNA is a nonhistone nuclear protein with a molecular weight of Mr 36,000 and functions as an auxiliary factor of DNA polymerase δ, an enzyme playing a key role in DNA replication (17). This protein is expressed specifically in the cell nucleus from late G1 to S phase (18). Several authors have reported that PCNA expression is a significant prognostic factor in LSCCs (15, 16). On the other hand, Dutta et al.(10) have proposed that cyclin E can be used as a proliferative marker in breast tumor because of its cell cycle-specific expression. However, to the best of our knowledge, the status of cyclin E expression in LSCC, including its possible clinical significance and the correlation with cell proliferation, has not been examined. Therefore, in view of these facts and to gain better insight of the clinical relevance of cyclin E, we investigated the expression of cyclin E immunohistochemically in 102 LSCCs and analyzed its relation to clinicopathological factors, including PCNA expression and the prognostic value of cyclin E and PCNA.

Patients.

For this retrospective study, a total of 102 patients were randomly chosen from 458 patients who underwent surgery for primary LSCC at the Department of Otolaryngology, First Affiliated Hospital of China Medical University, between 1988 and 1993. The main clinical and pathological parameters of the patients are shown in Table 1. Eighty-six patients were male and 16 were female; their ages ranged from 38 to 89 years (mean, 63.49 years;SD, 10.90). According to the Union International Contre Cancer Tumor-Node-Metastasis staging system (1987), 58 cases were supraglottic and 44 were glottic carcinomas. Fourteen cases were in stage I, 19 in stage II, 47 in stage III, and 22 in stage IV. There were 35 well-differentiated (G1), 30 moderately differentiated (G2), and 37 poorly differentiated (G3, including 2 with undifferentiation) squamous cell carcinomas. The patients with T3-T4 tumors underwent postsurgical adjuvant loco-regional radiotherapy (at 50 Gy). The follow-up time was 5 years for each patient ranging from 3 to 60 months (mean, 44.85 months; SD, 22.14). Thirty-three patients died from LSCC (12 patients as a result of local failure, 19 patients as a result of failure in regional lymph nodes, and 2 patients as a result of distant metastasis). Thirty-four patients exhibited recurrence. All tumor samples were obtained by biopsy or surgery before any particular therapy and were fixed in 10% buffered formalin and embedded in paraffin for immunohistochemical analysis.

Immunohistochemistry.

Paraffin sections (4 μm) of all 102 specimens were deparaffinized in xylene and then rehydrated through graded alcohol. Endogenous peroxidase activity was blocked with 0.3% hydrogen peroxide for 30 min. To reduce nonspecific binding, the sections were incubated with 10% goat (for polyclonal antibody) or horse (for monoclonal antibody)serum for 30 min at room temperature. Anti-cyclin E (M-20) rabbit polyclonal antibody (Santa Cruz Biotechnology, Santa Cruz, CA; 1:500)and anti-PCNA (PC10) mouse monoclonal antibody (Santa Cruz Biotechnology; 1:1000) were incubated at 4°C overnight. For each case, a corresponding section was incubated with nonimmune rabbit or mouse serum as a negative control. Immunostaining was performed by the avidin-biotin peroxidase complex (ABC) method using a Vectastain ABC kit (Vector Laboratories). Staining was visualized with 3,3-diaminobenzidine tetrachloride. The nuclei was counterstained lightly with Mayor’s hematoxylin. As positive control samples for cyclin E and PCNA, we used human epithelial ovarian tumors and hepatocellular carcinoma, respectively (19, 20).

The immunoreactive cells were independently evaluated by two of our members (Y. D. and L. S.), who were unaware of the clinicopathological factors and the patients’ clinical outcome. At least 20 high-power fields were chosen randomly, and 2000 cells were counted for each case. Only cells with brown-colored nuclear staining were considered as positive. The cyclin E and PCNA index was counted as a ratio of immunoreactive positive cells to the total number of cells counted. The cyclin E scores are defined as follows: −, no positive cells or positively stained cells <5%; +, 6–25% of tumor cells stained positively; 2+, 26–50% of tumor cells stained positively; 3+, >50% of tumor cells stained positively. Normal laryngeal epithelium adjacent to the tumor provided a negative internal control for immunoreaction. The positive cutoff value of PCNA index was defined as >25%, which was in accordance with the cutoff value of Liu et al.(16).

Statistical Analysis.

We performed a statistical analysis to investigate the relationship between cyclin E expression and clinicopathological parameters by means of the χ2 test. Regression analysis was used to investigate the correlation between cyclin E expression and proliferative activity measured by PCNA index. Survival analysis was undertaken using the Kaplan-Meier method, and curves were compared using log-rank analysis. Multivariate analysis, using the Cox proportional hazards model, was to identify which factors were independent predictors of patients’ prognoses. Statistical significance was defined as P < 5%. All statistical analyses were performed using JMP software version 3.2.5 (SAS Institute, Inc.)

Expression of Cyclin E and Its Correlation with Clinicopathological Parameters.

By using the anti-cyclin E antibody, no or only few weakly positive cells were observed occasionally in non-neoplastic epithelium adjacent to the tumor, which showed a low rate of nuclear staining in <25% of the epithelial cells. Hence, we defined overexpression of cyclin E in LSCC as being >2+ (>25%). Expression of cyclin E was heterogeneously distributed in a large number of LSCCs. Immunostaining to cyclin E was exclusively localized in the nuclei of carcinoma cells (Fig. 1, A and B). Overall, 88.24% (90 of 102) of the tumors were positive for cyclin E,and 52.94% (54/102) showed an overexpression pattern. We analyzed the correlation between cyclin E overexpression and clinicopathological parameters. A significant correlation between cyclin E overexpression and tumor site (P = 0.012), tumor size(P = 0.006), poor differentiation (P =0.026), lymph node metastases (P = 0.012), and advanced stage (P = 0.002) was revealed in LSCC.

Correlation between Cyclin E Expression and Proliferative Activity.

Using the anti-PCNA antibody (PC10), nuclear staining of tumor cells was detected, and 62.75% (64 of 102) cases was revealed an overexpression pattern (Fig. 1,C). An increase of cyclin E expression rate was accompanied by a proportional increase in PCNA index. A positive correlation was obtained between the cyclin E index and the PCNA index. The regression line was Y = 0.851X+ 10.413, r = 0.896 (P < 0.0001),which indicated a tight correlation between cyclin E expression and proliferation activity of tumor cells (Fig. 2).

Prognostic Significance of Cyclin E and PCNA Expression.

By using the Kaplan-Meier’s analysis, patients with PCNA overexpression and cyclin E overexpression was associated with worse disease-free survival and overall survival than those without(P = 0.036, P = 0.0006, P = 0.0023, and P = 0.002,respectively; Fig. 3, A and B; Fig. 4, A and B). When cyclin E and PCNA were combined, patients with both PCNA overexpression and cyclin E overexpression revealed the poorest disease-free survival and overall survival (P = 0.0001 and P < 0.0001, respectively; Figs. 3 C and 4C).

To further investigate the prognostic role of cyclin E expression in early-stage patients, the Kaplan-Meier’s analysis was performed on 33 cases with stage (I–II), and the results showed that cyclin E overexpression was still associated with poorer prognosis, both in disease-free survival and overall survival (P = 0.022 and P = 0.034, respectively; Fig. 5).

Multivariate Analysis Using the Cox’s Proportional Hazards Models.

Among age, sex, tumor site, tumor grade, tumor size, lymph node metastasis, clinical stage, and PCNA overexpression, as well as cyclin E overexpression, lymph node metastasis, and cyclin E overexpression,were independent prognostic factors for disease-free survival. Tumor size, lymph node metastasis, clinical stage, and cyclin E overexpression were independent prognostic factors for patients’overall survival (Table 2).

The current study demonstrated large variations of cyclin E expression in LSCC. This is, to our knowledge, the first study to show the correlation between cyclin E expression and clinical relevance. A total of 88.24% (90 of 102) of the tumor samples were positive for cyclin E, and 52.94% (54 of 102) showed an overexpression pattern. It has been reported that cyclin E overexpression was observed in several malignancies, including breast (8, 9, 10), colorectal (11), gastric (12), and lung (13, 14) cancer. In particular, Mishina et al.(13) and Lonardo et al.(21) have reported recently that cyclin E is often overexpressed in squamous cell carcinomas of lung and bronchus of the respiratory system and is associated with increasing grade, lymph node metastasis, and advanced stage (8, 9, 12, 14, 22). In this study also, a close correlation was found between cyclin E overexpression and tumor site,tumor size, poor differentiation, and lymph node metastasis, as well as advanced stage. Thus, these findings suggested that cyclin E protein frequently exhibits dysregulated overexpression and may contribute to the malignant transformation of the laryngeal squamous epithelia and the possible association with the stronger aggressive behavior in LSCC.

On the other hand, the proliferative activity is a known risk factor for cancer, and proliferative activity of tumor cells is correlated with progression and prognosis of the tumors (15). PCNA expression has been considered to reflect the proliferation rate of tumor cells and has been suggested as a prognostic indicator in LSCC (15, 16). The previous studies by Keyomarsi et al.(9) and Fukuse et al.(14) have found no correlation between PCNA overexpression and cyclin E overexpression. However, studies by Dutta et al.(10) and Porter et al.(22) have found a correlation between cyclin E overexpression and Ki-67 overexpression, which is one of the proliferation markers in breast cancer. In this study, we found that a high expression of cyclin E may also be associated with a high PCNA index. A positive correlation was obtained between cyclin E expression and PCNA index (r = 0.896; P <0.0001), which can suggest that cyclin E expression is associated with tumor cell proliferation.

Recently, abnormal expression of cyclins has been considered as one of the most important factors in the tumorigenesis of various human malignancies. The cause can be explained by the fact that cyclin E is a late G1 cyclin to form a complex with CDK2 (3, 23) and is a candidate for controlling the G1-S transition. Its overexpression with an inducible system accelerates entry into the S-phase (24). Abnormalities in cell cycle regulators and subsequent deregulation of the G1-S transition may be one of the most important biological events in malignant transformation of cells (9, 10). In vitrostudies using carcinogen-induced transformation of immortalized human bronchial epithelial cells suggest that cyclin E plays a key role in the transformation of bronchial epithelial cells (24). Recently, Spruck et al.(25) have proposed that cyclin E overexpression can induce chromosome instability, which is involved in the development and progression of tumors. Furthermore, cyclin E is also altered in other types of solid tumors as well as leukemias and lymphomas (26, 27, 28, 29). The data obtained from this study suggest that cyclin E overexpression could be associated with cancer cell proliferation, promoted cancer cells toward more advanced stages, and caused stronger invasion into deeper tissues and/or the lymphatic system, as well as resulted in poorer clinical courses.

With regard to prognosis, cyclin E is a potential prognostic factor because its overexpression is associated with a poor prognosis in a number of different malignancies, including breast (8, 9, 10, 20), colorectal (11), gastric (12),and lung (13, 14) carcinomas. In this study, the survival analysis by the Kaplan-Meier method revealed that PCNA overexpression and cyclin E overexpression were significantly associated with shorter disease-free survival and overall survival. When cyclin E and PCNA were combined, the patients with both cyclin E and PCNA overexpression revealed the poorest disease-free survival and overall survival. In addition, to further investigate the prognostic role of cyclin E expression in early-stage patients, we analyzed cyclin E overexpression in 33 patients with stage I–II, and the results showed that cyclin E overexpression was still associated with poorer prognosis, both in disease-free survival and overall survival, but the PCNA overexpression was not (data not shown). Moreover, the multivariate analysis revealed that PCNA overexpression was not an independent prognostic indicator. Cyclin E overexpression and lymph node metastasis were independent prognostic indicators for disease-free survival, and cyclin E overexpression, tumor size, and lymph node metastasis, as well as advanced stage were proven to be independent prognostic indicators for overall survival. These results suggested that cyclin E is more sensitive as a prognostic factor than PCNA, which is consistent with the results of lung cancer (14). On the other hand, the multivariate analysis confirmed that accurate Tumor-Node-Metastasis staging system still remains the most useful predictive indicator of prognosis of LSCC. Therefore, the immunohistochemical evaluation of cyclin E expression, alone or combined with PCNA index, may further add new information for patients’ prognoses. Because cyclin E and PCNA protein level may be examined in preoperative biopsied specimens, such information could provide better planning of appropriate treatment strategies, especially for the functional neck dissection. Accordingly, these data also suggested that the patients with cyclin E overexpression and/or higher PCNA index should have suitable management after surgery.

In conclusion, we demonstrate large variations of cyclin E expression,which showed a close correlation with clinical relevance and identified that cyclin E as a new marker for tumor cell proliferation. If our results are confirmed in a larger study, cyclin E expression may play a pivotal role for the biological behavior of LSCC, and that cyclin E overexpression may be a potential new prognostic marker for LSCC.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

        
1

This work was supported by Grants-in-Aid for Scientific Research from the Ministry of Education, Science, Sports and Culture of Japan.

                
3

The abbreviations used are: LSCC, laryngeal squamous cell carcinoma; CDK, cyclin-dependent kinase; PCNA,proliferating cell nuclear antigen.

Fig. 1.

Immunohistochemical staining patterns for cyclin E and PCNA in LSCC. A, positive immunostaining was found exclusively in the nuclei with a cyclin E labeling index of 88%. B, weak immunostaining for cyclin E labeling index of 4%. C, positive immunostaining patterns for PCNA was found exclusively in the nuclei with a labeling index of 85%. Bar, 20 μm.

Fig. 1.

Immunohistochemical staining patterns for cyclin E and PCNA in LSCC. A, positive immunostaining was found exclusively in the nuclei with a cyclin E labeling index of 88%. B, weak immunostaining for cyclin E labeling index of 4%. C, positive immunostaining patterns for PCNA was found exclusively in the nuclei with a labeling index of 85%. Bar, 20 μm.

Close modal
Fig. 2.

Correlation between cyclin E expression and PCNA index in LSCC. A linear correlation was detected between the expression of both proteins in the same neoplastic foci. Regression line:Y = 0.851X + 11.413, r = 0.896, P < 0.0001.

Fig. 2.

Correlation between cyclin E expression and PCNA index in LSCC. A linear correlation was detected between the expression of both proteins in the same neoplastic foci. Regression line:Y = 0.851X + 11.413, r = 0.896, P < 0.0001.

Close modal
Fig. 3.

A, disease-free survival curves of cases with LSCC according to PCNA expression. B,disease-free survival curves of cases with LSCC according to cyclin E expression. C, disease-free survival curves of cases with LSCC according to cyclin E and PCNA expression status.

Fig. 3.

A, disease-free survival curves of cases with LSCC according to PCNA expression. B,disease-free survival curves of cases with LSCC according to cyclin E expression. C, disease-free survival curves of cases with LSCC according to cyclin E and PCNA expression status.

Close modal
Fig. 4.

A, overall survival curves of cases with LSCC according to PCNA expression. B, overall survival curves of cases with LSCC according to cyclin E expression. C, overall survival curves of cases with LSCC according to cyclin E and PCNA expression status.

Fig. 4.

A, overall survival curves of cases with LSCC according to PCNA expression. B, overall survival curves of cases with LSCC according to cyclin E expression. C, overall survival curves of cases with LSCC according to cyclin E and PCNA expression status.

Close modal
Fig. 5.

A, disease-free survival curves of cases with stage I–II LSCCs according to cyclin E expression. B, overall survival curves of cases with stage I–II LSCCs according to cyclin E expression.

Fig. 5.

A, disease-free survival curves of cases with stage I–II LSCCs according to cyclin E expression. B, overall survival curves of cases with stage I–II LSCCs according to cyclin E expression.

Close modal
Table 1

%Cyclin E expression and clinicopathological parameters

ParametersTotalCyclin E scoreP
>25%(%)≤25%(%)
Age       
≤60 45 28 62.22 17 37.78 0.095 
>60 57 26 45.61 31 54.39  
Sex       
Female 16 56.25 43.75 0.773 
Male 86 45 52.33 41 47.67  
Tumor site       
Glottic 44 17 38.79 27 61.36 0.012 
Supraglottic 58 37 63.79 21 36.21  
Tumor grade       
G1–2 65 29 44.62 36 55.38 0.026 
G3 37 25 67.57 12 32.43  
Tumor size       
T1–2 45 17 37.78 28 62.22 0.006 
T3–4 57 37 64.91 20 35.09  
Lymph node       
N0 59 25 42.37 34 57.63 0.012 
N+ 43 29 67.44 24 32.56  
Clinical stage       
I–II 33 10 30.30 23 69.70 0.002 
III–IV 69 44 63.77 25 36.23  
ParametersTotalCyclin E scoreP
>25%(%)≤25%(%)
Age       
≤60 45 28 62.22 17 37.78 0.095 
>60 57 26 45.61 31 54.39  
Sex       
Female 16 56.25 43.75 0.773 
Male 86 45 52.33 41 47.67  
Tumor site       
Glottic 44 17 38.79 27 61.36 0.012 
Supraglottic 58 37 63.79 21 36.21  
Tumor grade       
G1–2 65 29 44.62 36 55.38 0.026 
G3 37 25 67.57 12 32.43  
Tumor size       
T1–2 45 17 37.78 28 62.22 0.006 
T3–4 57 37 64.91 20 35.09  
Lymph node       
N0 59 25 42.37 34 57.63 0.012 
N+ 43 29 67.44 24 32.56  
Clinical stage       
I–II 33 10 30.30 23 69.70 0.002 
III–IV 69 44 63.77 25 36.23  
Table 2

%Multivariate Cox model analysis of disease-free survival and overall survival

Disease-free survivalOverall survival
Hazards ratio95% CIaPHazards ratio95% CIaP
Age (>60/≤60) 0.93 0.65–1.32 0.692 1.01 0.71–1.46 0.937 
Sex (male/female) 1.17 0.71–1.79 0.505 1.11 0.64–1.75 0.684 
Tumor site (SG/GL)b 1.00 0.67–1.54 0.984 0.96 0.65–1.48 0.860 
Tumor grade (G3/G1,2) 1.31 0.91–1.89 0.147 1.44 0.99–2.12 0.056 
Tumor size (T3,4/T1,21.31 0.89–1.99 0.179 1.65 1.07–2.74 0.022 
Lymph node (+/−) 1.81 1.21–2.78 0.003 1.96 1.32–3.01 0.001 
Stage (III–IV/I–II) 1.46 0.91–2.53 0.122 1.71 1.02–3.27 0.040 
PCNA (>25%/≤25%) 1.05 0.63–1.70 0.849 1.21 0.71–2.21 0.504 
Cyclin E (>25%/≤25%) 1.64 1.06–2.68 0.026 1.62 1.03–2.71 0.036 
Disease-free survivalOverall survival
Hazards ratio95% CIaPHazards ratio95% CIaP
Age (>60/≤60) 0.93 0.65–1.32 0.692 1.01 0.71–1.46 0.937 
Sex (male/female) 1.17 0.71–1.79 0.505 1.11 0.64–1.75 0.684 
Tumor site (SG/GL)b 1.00 0.67–1.54 0.984 0.96 0.65–1.48 0.860 
Tumor grade (G3/G1,2) 1.31 0.91–1.89 0.147 1.44 0.99–2.12 0.056 
Tumor size (T3,4/T1,21.31 0.89–1.99 0.179 1.65 1.07–2.74 0.022 
Lymph node (+/−) 1.81 1.21–2.78 0.003 1.96 1.32–3.01 0.001 
Stage (III–IV/I–II) 1.46 0.91–2.53 0.122 1.71 1.02–3.27 0.040 
PCNA (>25%/≤25%) 1.05 0.63–1.70 0.849 1.21 0.71–2.21 0.504 
Cyclin E (>25%/≤25%) 1.64 1.06–2.68 0.026 1.62 1.03–2.71 0.036 
a

CI, confidence interval.

b

SG, supraglottic; GL, glottic.

We thank Drs. Yu Gang and Guan Chao, Department of Otolaryngology, First Affiliated Hospital of China Medical University, Shenyang, China, for collection and storage of laryngeal tumor specimens and cooperation, Jennifer Ann Tai for proofreading the manuscript, and the staff members of the Department of Physiology, Kagawa Medical University, for technical assistance.

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