Med Oncol (2012) 29:742–749 DOI 10.1007/s12032-011-9971-3
ORIGINAL PAPER
Prognostic significance of galectin-3 and cyclin D1 expression in undifferentiated nasopharyngeal carcinoma Mustafa Fuat Acikalin • Durmus Etiz • Melek Kezban Gurbuz • Erkan Ozudogru Funda Canaz • Ertugrul Colak
•
Received: 21 April 2011 / Accepted: 25 April 2011 / Published online: 7 May 2011 Ó Springer Science+Business Media, LLC 2011
Abstract Galectin-3 was shown to be involved in various biological events, including cell growth, adhesion, differentiation, angiogenesis, apoptosis, tumorigenesis, and metastasis. The prognostic significance of galectin-3 expression has already been evaluated in several cancers. However, its prognostic role has not been investigated in nasopharyngeal carcinoma. The loss of cell cycle control is one of the critical steps in the development of nasopharyngeal carcinoma. Cyclin D1 is one of the key proteins involved in cell cycle control and is essential for G1/S phase transition. Overexpression of cyclin D1 has been observed in several human cancers. In the present study, the expression of galectin-3 and cyclin D1 was evaluated with immunohistochemical analysis in 45 patients diagnosed as undifferentiated nasopharyngeal carcinoma and expression of these proteins was correlated with clinicopathological parameters and prognosis. Multivariate analysis showed that
M. F. Acikalin F. Canaz Department of Pathology, Eskisehir Osmangazi University Medical Faculty, 46480 Eskisehir, Turkey D. Etiz Department of Radiation Oncology, Eskisehir Osmangazi University Medical Faculty, 46480 Eskisehir, Turkey M. K. Gurbuz E. Ozudogru Department of Otorhinolaryngology, Eskisehir Osmangazi University Medical Faculty, 46480 Eskisehir, Turkey E. Colak Department of Biostatistics, Eskisehir Osmangazi University Medical Faculty, 46480 Eskisehir, Turkey M. F. Acikalin (&) ¨ niversite Evleri, C5 Blok Daire 4, Gu¨ltepe Mah., U Eskisehir, Turkey e-mail:
[email protected]
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older age ([50 vs. B50) (P = 0.028), distant metastasis at presentation (M1 vs. M0) (P = 0.001), and increased galectin-3 expression ([5% vs. B5%) (P = 0.025) were independently correlated with poor overall survival. We found no statistically significant correlation between cyclin D1 immunoexpression and disease outcome. The Spearman’s correlation coefficient revealed a significant correlation between galectin-3 and cyclin D1 expression (r = 0.425; P = 0.004). Our findings suggested that the immunohistochemical analysis of galectin-3 might be useful in predicting prognosis in nasopharyngeal carcinoma. Keywords Nasopharyngeal carcinoma Galectin-3 Cyclin D1 Prognosis
Introduction Nasopharyngeal carcinoma (NPC) is a distinctive type of head and neck cancer characterized by a geographically well-defined distribution worldwide. While rare in most parts of the world, its incidence is considerably higher in China, Southeast Asia, Northern Africa, and Arctic region [1]. When the disease is diagnosed and treated at an early stage, most NPC patients can be cured. However, because the disease is usually asymptomatic at early stages, the NPC patients tend to present at a more advanced stage and the long-term survival rate of these patients is poor. In a recent large series consisting of 1,706 NPC cases, the 5-year overall survival rates were found to be 100, 75.9, 66.5, and 49.3% for stage I, II, III, and IV respectively [2]. Although the standard treatment for NPC is radiation therapy, a high rate of treatment failure is observed in patients with advanced disease. Concurrent adjuvant chemotherapy improves survival rates for patients with advanced NPC.
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The accurate prediction of prognosis is crucial for optimizing therapy in patients with NPC. While many factors have been evaluated as potential prognostic indicators, such as stage of disease [3, 4], nodal involvement [3], distant metastasis [4], histopathologic type [4, 5], tumor volume [6], age [5], and parapharyngeal extension [6], they have limited value in determining the treatment modality. Owing to variability in the patient outcome, identification of novel prognostic factors that more reliably predict the biological behavior of the tumors would help identify patients for whom adjuvant systemic therapy is useful. Galectins are a growing family of proteins defined by their affinity for b-galactosides and by conserved sequence elements [7]. Galectin-3 is one of the most extensively investigated members of this family concerning with cancer. Galectin-3 was shown to be involved in various biological events, including cell growth [8], adhesion [8], differentiation [8], angiogenesis [8, 9], apoptosis [8], tumorigenesis [10], and metastasis [11]. Unlike many galectins, such as galectin-1, -7, and -9, galectin-3 was reported to be an antiapoptotic molecule [12]. Overexpression of galectin-3 has been shown to correlate with tumor progression and metastasis in renal [13], tongue [14], thyroid [15], and prostatic cancers [16]. In contrast, galectin-3 down-regulation has been reported during the progression of cancers of the breast [17] and uterus [18]. There are several studies investigating the expression of galectin-3 in head and neck squamous cell carcinomas (HNSCCs) and most of these studies demonstrated the relationship between the galectin-3 expression and the biological behavior in tumors from the various head and neck sites [14, 19, 20]. To the best of our knowledge, there are only one study in the literature analyzing the expression of galectin-3 in NPC. Wu et al. [21] observed that galectin-3, fibronectin, and plasminogen activator inhibitor 1 were highly expressed in NPC biopsies, but weakly or not expressed in normal nasopharyngeal tissues. But our study is the first to evaluate the prognostic significance of galectin-3 expression in NPC. The molecular mechanisms underlying the development of NPC are complex, involving the aberrations of various pathways and the alterations in expression of numerous proteins [22]. Like other cancers, the loss of cell cycle control is one of the critical steps in the development of NPC. Cyclin D1 is one of the key proteins involved in cell cycle control and is essential for G1/S phase transition. Cyclin D1 interacts with cyclin-dependent kinase 4/6 and forms a complex that inactivates pRb through phosphorylation, allowing passage through the restriction point and progression through the G1 phase [23]. Cells overexpressing cyclin D1 have a shortened G1 phase and are able to transverse the G1/S transition under conditions that limit the growth of normal cells. Overexpression of cyclin D1
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has been observed in several human cancers [24–28]. There are conflicting data about the prognostic relevance of cyclin D1 expression in NPC [29–31]. In order to clarify the role of galectin-3 and cyclin D1 in the prognostic evaluation of NPC, we investigated the correlation of galectin-3 and cyclin D1 expression with clinicopathologic parameters and disease outcome.
Materials and methods Patients and tissue specimens The study included 45 patients diagnosed as undifferentiated NPC at the Department of Pathology of Osmangazi University Medical Faculty Hospital between 2003 and 2010. Follow-up information was obtained from the files of Departments of Otorhinolaryngology and Radiation Oncology. Of 45 patients, 28 were men and 17 were women with age range from 13 to 85 years (mean 49.4, median 53 years). Regional metastases were found in 37 (82.2%) patients at the time of initial treatment. Eight patients (17.8%) had distant metastases at the time of diagnosis. The tumors were staged according to American Joint Committee on Cancer criteria. Two (4.4%) patients had stage I, seven (15.6%) patients stage II, twelve (26.7%) patients stage III, and twenty-four (53.3%) patients stage IV disease. All the patients were treated with standard therapy based on the clinical stage. Briefly, patients with stage I–II disease were treated with radiotherapy alone, and those with stage III–IVB disease with concurrent cisplatin (80 mg/m2/day, 1,22,43 days) chemotherapy. Dose prescription for curative aimed patients was 70 Gray (Gy) during 7 weeks to the gross tumor, and 50–60 Gy for elective treatment of potential risk sites. Palliative patients (Stage IVC) received 30 Gy (in 2 weeks, 10 fractions) radiation dose. The follow-up interval ranged from 4 to 88 months (mean 32, median 29 months). Table 1 lists clinical characteristics of patients. All materials were biopsy specimens taken at the time of diagnosis before treatment. Histopathologic classification was based on World Health Organization (WHO) classification system. All hematoxylin and eosin (H&E)—stained slides were reviewed, and the diagnoses were confirmed. Only patients diagnosed as undifferentiated NPC were included. Immunohistochemistry For the immunohistochemical study, 4-micrometer-thick sections were obtained from formalin-fixed and paraffinembedded tissue samples. Tissue sections were deparaffinized in xylene, rehydrated in descending ethanol series, and washed with phosphate-buffered saline (PBS) (pH 7.4).
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Table 1 Clinical characteristics of patients Characteristics
No. of patients (%)
Gender Male
28 (62.2)
Female
17 (37.8)
Age Mean
49.4
Median
53
Range
13–85
Smoking No
26 (57.8)
Yes
19 (42.2)
T classification 1
8 (17.8)
2
22 (48.9)
3
7 (15.6)
4
8 (17.8)
N classification 0
8 (17.8)
1
6 (13.3)
2
13 (28.9)
3
18 (40.0)
M classification M0
37 (82.2)
M1
8 (17.8)
AJCC stage I
2 (4.4)
II
7 (15.6)
III IV
12 (26.7) 24 (53.3)
Locoregional recurrence No
37 (82.2)
Yes
8 (17.8)
Tumor-related death No
30 (66.7)
Yes
15 (33.3)
Endogenous peroxidases were then blocked with 3% hydrogen peroxide for 10 min. Heat-induced antigen retrieval was performed in 10 mM citrate buffer for 2 min at 100°C. To reduce nonspecific binding, block solution (ScyTek, Utah, USA) was applied for 5 min. The sections were incubated with either mouse monoclonal antibody to galectin-3 (clone 9C4, dilution 1:80, Thermo Fisher, Fremont, USA) or a rabbit monoclonal antibody to cyclin D1 (clone SP4, dilution 1:30, Thermo Fisher, Fremont, USA) for 1 h at room temperature in a moist chamber. The sections were incubated with Biotinylated Link Antibody (ScyTek, Utah, USA) for 20 min, and then, Streptavidin/ HRP label was applied for 20 min at room temperature. The immunostaining was visualized with AEC chromogen.
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Fig. 1 High expression of galectin-3 in tumor cells of nasopharyngeal carcinoma (9200)
Finally, sections were counterstained with hematoxylin. All counts were assessed without knowledge of the patient’s outcome and any other pertinent data and were done by two independent investigators (M.F.A, F.C). For galectin-3, both cytoplasmic and nuclear staining, for cyclin D1 only nuclear staining of tumor cells was considered. The expression of galectin-3 was evaluated on a 9400 field, and the average percentage of five randomly selected and defined fields was determined. Any welldefined, brown-staining tumor cell that was clearly separate from neighboring dendritic cells was counted. A cutoff point of 5% stained tumor cells within the tumor cell population was chosen, as proposed by several investigators [19, 20]. Galectin-3 expression was considered to be positive when samples demonstrated [5% reactivity and negative when samples demonstrated B5% reactivity. Cyclin D1 expression was calculated by estimating the proportion of the tumor cells with stained nuclei within 500 representative tumor cells. Cyclin D1 expression was considered positive if [10% of tumor cells exhibited nuclear staining and negative if B10% exhibited nuclear staining, as previously reported [32]. In the immunohistochemical analysis of galectin-3 and cyclin D1, disagreement between the investigators was observed in four (8.8%) and three (6.6%) cases, respectively. In these cases, consensus was reached by simultaneous re-evaluation using a double-headed light microscope. Examples of the immunohistochemical staining of galectin-3 and cyclin D1 are shown in Figs. 1 and 2. Statistical analysis All statistical analyses were performed using SPSS 15.0 for Windows. Chi-square tests (Yates’ chi-square, Pearson chisquare and Fisher’s exact test) were used to examine the
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utilizing backward stepwise (wald) method. A P value of less than 0.05 was considered to indicate statistical significance. The study was performed in accordance with local laws, and approval was obtained from the Ethics Committee of the Medical Faculty, University of Eskisehir Osmangazi, Turkey.
Results Among the 45 cases, 32 (71.1%) demonstrated galectin-3 expression in more than 5% of tumor cells and were considered as galectin-3-positive. The mean value of galectin3 expression was 16.4%, and the median value was 10.0%. There was no significant difference between the galectin3-negative and the galectin-3-positive group with respect to gender, age, smoking, tumor extent, nodal status, distant metastasis, and stage (Table 2). Positive expression ([10%) of cyclin D1 was observed in 39 of 45 cases (86.6%). The mean value of cyclin D1 expression was 43.3%, and the median value was 42.0%. The relationship between cyclin D1 expression and various clinicopathological parameters is summarized in Table 2. No significant differences in cyclin D1 expression were found for any clinicopathological parameters.
Fig. 2 Example of nasopharyngeal carcinoma showing high cyclin D1 expression in tumor cells (9200)
distribution of galectin-3 and cyclin D1 status according to different clinicopathological parameters. Direct correlations between galectin-3 and cyclin D1 expressions were evaluated by determining the Spearman’s rank correlation coefficient. Survival curves were calculated using the Kaplan–Meier method, and the differences were analyzed by using the log-rank test. Multivariate analysis was performed by the Cox proportional hazard regression model
Table 2 Correlation between expression of galectin-3 and cyclin D1 and clinicopathological features
Clinicopathological features
Galectin-3 staining B5%
Gender Male
P
[5%
Cyclin D1 staining B10%
[10%
0.511
0.385
7
21
5
23
6
11
1
16
B50
5
15
2
18
[50
8
17
4
21
Female Age
0.854
Smoking
0.678
0.185
0. 686
No
10
16
3
23
Yes
3
16
3
16
T1-T2
10
20
4
26
T3-T42
3
12
2
13
0
8
6
31
4
33
2
6
T classification
0.492
N classification 4
4
N1-N2-N3
9
28
M classification M0 M1
1.000
0.202
N0
0. 572
0. 672 10
27
3
5
AJCC stage
P
0.286
0.094
0.323
1–2
5
4
0
9
3–4
8
28
6
30
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Fig. 3 Kaplan–Meier analysis of overall survival according to distant metastasis. Distant metastasis at the time of presentation (M1) was significantly correlated with shorter overall survival (P = 0.007)
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76 months for galectin-negative group (B5%). Using the log-rank test, no statistical significance in recurrence-free survival was found to correlate with clinicopathological parameters such as gender, age, smoking, tumor extent, nodal status, distant metastasis at presentation, stage, expression of galectin-3, and cyclin D1. All clinicopathological parameters were included to Cox proportional hazards regression model utilizing the stepwise method to assess their independent predictive value for recurrence-free and overall survival. Multivariate analysis ruled out any statistically significant association between locoregional recurrence and gender, age, smoking, tumor extent, nodal status, distant metastasis at presentation, stage, expression of galectin-3 or cyclin D1 (Table 3). Multivariate analysis showed that older age ([50 vs. B50) (P = 0.028), distant metastasis at presentation (M1 vs. M0) (P = 0.001), and increased galectin-3 expression ([5% vs. B5%) (P = 0.025) were independently correlated with poor overall survival (Table 4). The most important independent predictor of poor overall survival was the distant metastasis; patients with distant metastasis (M1) were 8.24 times more likely to die than patients without distant metastasis (M0). The risk ratio of poor overall survival was 6.18 among patients with positive galectin-3 expression
Table 3 Multivariate analysis of prognostic variables influencing disease-free survival
Fig. 4 Kaplan–Meier analysis of overall survival according to galectin-3 expression. There was a trend toward a less favorable survival rate in the galectin-positive group ([5%), which was not significant (P = 0.100)
Only distant metastasis at the time of presentation was significantly correlated with shorter overall survival in Kaplan–Meier analysis and log-rank test (P = 0.007) (Fig. 3). Mean time to tumor-related death for patients with distant metastasis was 32 months, whereas it was 56 months for patients without distant metastasis. There was a trend toward a less favorable survival rate in the galectin-positive group ([5%), which was not significant (P = 0.100) (Fig. 4). Mean time to tumor-related death in galectin-positive group was 45 months, whereas it was
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Variables
Hazards ratio
95% CI
P
Age ([50 vs. B50)
2.14
0.46–10.02
0.336
Gender (male vs. female)
2.55
0.39–16.45
0.327
Smoking (yes vs. no) T classification (T3-T4 vs. T1-T2)
0.67 0.91
0.13–3.42 0.19–4.31
0.631 0.904
N classification (N1-N2-N3 vs. N0)
1.41
0.05–36.73
0.836
M classification (M1 vs. M0)
0.97
0.09–10.71
0.977
AJCC stage (3–4 vs. 1–2)
1.07
0.03–36.50
0.970
Galectin-3 staining ([5% vs. B5%)
4.57
0.41–51.70
0.219
Cyclin D1 staining ([10% vs. B10%)
0.42
0.08–2.29
0.313
CI confidence interval
Table 4 Multivariate analysis of prognostic variables influencing overall survival Variables
Hazards ratio
95% CI
P
Age ([50 vs. B50)
3.89
1.15–13.14
0.028
M classification (M1 vs. M0)
8.24
2.40–28.24
0.001
Galectin-3 staining ([5% vs. B5%)
6.18
1.26–30.34
0.025
CI confidence interval
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Fig. 5 The Spearman’s correlation coefficient showing a significant correlation between galectin-3 and cyclin D1 (r = 0.425; P = 0.004)
versus those with negative galectin-3 expression. We found no statistically significant correlation between cyclin D1 immunoexpression and disease outcome in terms of recurrence-free and overall survival. The Spearman’s correlation coefficient revealed a significant correlation between galectin-3 and cyclin D1 expressions (r = 0.425; P = 0.004) (Fig. 5).
Discussion In patients with NPC, many factors including stage of disease, nodal involvement, distant metastasis, histopathologic type, tumor volume, age, and parapharyngeal extension have been evaluated as potential prognostic indicators. However, these factors have limited value in determining low- and high-risk groups and the treatment modality. In recent years, the search for novel prognostic factors that more reliably predict the biological behavior of the tumors has focused on the role of the various molecular biomarkers. Hence, we investigated the prognostic relevance of galectin-3 and cyclin D1 expression in patients with NPC. Among the 45 patients diagnosed as undifferentiated NPC, multivariate analysis showed that patient age (P = 0.028), distant metastasis at the time of presentation (P = 0.001), and galectin-3 expression (P = 0.025) allow discrimination between high-risk and low-risk patients in terms of overall survival. The risk ratio of tumor-related death was found to be 8.24, 6.18, and 3.89 for patients with distant metastasis, positive galectin-3 expression, and increased age, respectively. Multivariate analysis showed that any parameters were correlated with locoregional recurrence. Although patients with positive galectin-3 expression were 4.57 times more likely to recur than
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patients with negative galectin-3 expression, it did not reach significance. Our findings suggested that the immunohistochemical analysis of galectin-3 might be useful in predicting prognosis in NPC. Recent researches showed that galectin-3 can be a useful and reliable marker for predicting the aggressiveness of different tumors due to its involvement in cell growth, adhesion, differentiation, angiogenesis, apoptosis, tumorigenesis, and metastasis. However, it seems that the involvement of galectin-3 in tumorigenesis heavily depends on the histological origin of the tissue. Several studies investigating the expression of galectin-3 in HNSCCs demonstrated the close relationship between the galectin-3 expression and the biological behavior in various types of HNSCCs [1, 19, 20]. Honjo et al. [14] observed that enhanced expression of galectin-3 in the cytoplasm was associated with a reduced level of disease-free survival in patients with tongue cancers. Piantelli et al. [19] found a significant correlation between galectin-3 tumor positivity and longer relapse-free periods and overall survival in patients with node-negative laryngeal squamous cell carcinomas. Plzak et al. [20] showed that decreased levels of galectin-3 expression are associated with shortened relapse-free and overall survival in oropharyngeal and laryngeal cancers. To our knowledge, there are only one study in the literature analyzing the expression of galectin-3 in NPC. In this study, Wu et al. [21] investigated secreted proteomes of two NPC cell lines to identify biomarkers for early NPC diagnosis and they observed that three proteins (fibronectin, galectin-3 and plasminogen activator inhibitor 1) were highly expressed in NPC biopsies, but weakly or not expressed in normal nasopharyngeal tissues. But our study is the first to evaluate the prognostic role of galectin-3 expression in NPC. Further studies involving a larger patient population with longer follow-up period are required to provide more information regarding galectin-3 expression in NPC. Several studies showed association between overexpression of cyclin D1 with worse prognosis in various cancers [24, 27, 28]. Overexpression of cyclin D1 is one of the most consistent alterations identified in HNSSC and is correlated with the presence of lymph node metastases, advanced clinical stage, disease recurrence, and poor prognosis [24, 28]. However, potential significance of cyclin D1 expression in NPC has not yet been adequately evaluated and there are conflicting data about its prognostic role. In the present study, we found no association between overexpression of cyclin D1 and poor prognosis in terms of shorter disease free and overall survival. There was also no statistically significant relationship between cyclin D1 expression and clinicopathological parameters. Similar results were reported by Lin et al. [31] who examined the expression level of cyclin D1 in recurrent NPC that have failed previous treatment with radiation ± chemotherapy.
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The results of this study failed to support the association between overexpression of cyclin D1 with increased risk of recurrence. However, other investigators reported different results about the significance of cyclin D1 expression in NPC. In a series of 65 patients, Hwang et al. [29] observed that the patients with low levels of cyclin D1 exhibited a significantly higher rate of local recurrence than patients with high levels of cyclin D1 after radiotherapy for NPC. On the contrary, in another study, patients with high levels of cyclin D1 was found to show significantly higher rates of early local recurrence and poorer prognosis concerning 10-year survival [30]. It is well-known that galectin-3 plays a pivotal role in the regulation of cancer-related gene expression. In the previous studies, it was shown that galectin-3 stimulates cell proliferation through cyclin D1 activation as cyclin D1 is a final target of Wnt pathway [33, 34]. In the present study, we found a significant correlation between galectin3 and cyclin D1 expressions in NPC (r = 0.425; P = 0.004), suggesting that galectin-3 mediates cyclin D1 activation in NPC. In accordance with our findings, it has been shown that nuclear and cytoplasmic expression of galectin-3 is strongly associated with an increase in cyclin D1 and b-catenin expressions in thyroid carcinomas [34]. Similarly, it was shown that galectin-3 upregulates cyclin D1 promoter activity in human breast epithelial cells independent on cell adhesion [35]. Ferrazzo et al. [36] found that the superexpression of nuclear cyclin D1 is related to an intense expression of galectin-3 in adenoid cystic carcinoma of salivary gland. In summary, our data suggest that the immunohistochemical analysis of galectin-3 might be a meaningful tool to identify patients with high-risk disease and those patients who should benefit from aggressive therapy. In view of the small group of patients with relatively short follow-up in our series, the results need to be supported by wider series with longer follow-up before more definitive conclusions can be made. Conflict of interest
None.
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