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IOS Press. Prognostic significance of beta-2 adrenergic receptor in oral squamous cell carcinoma. Diego Mauricio Bravo-Calderóna, Denise Tostes Oliveiraa,∗, ...
Cancer Biomarkers 10 (2011/2012) 51–59 DOI 10.3233/CBM-2012-0228 IOS Press

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Prognostic significance of beta-2 adrenergic receptor in oral squamous cell carcinoma Diego Mauricio Bravo-Calder´ona, Denise Tostes Oliveiraa,∗ , Aparecido Nilceu Maranab , Suely Nonogakic, Andr´e Lopes Carvalhod and Luiz Paulo Kowalskie Department of Stomatology, Area of Pathology, Bauru School of Dentistry, University of S˜ao Paulo, Bauru, Brazil Department of Computing, School of Sciences, S˜ao Paulo State University (UNESP), Bauru, Brazil c Adolfo Lutz Institute, Pathology Division, S˜ao Paulo, Brazil d Department of Head and Neck Surgery, Fundac¸a˜ o Pio XII – Cancer Hospital of Barretos, Barretos, S˜ao Paulo, Brazil e Department of Head and Neck Surgery and Otorhinolaryngology, Cancer Hospital A. C. Camargo, S˜ao Paulo, Brazil a

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Abstract. The aim of this study was to evaluate the expression of β2-adrenergic receptor (β2-AR) in oral squamous cell carcinoma (OSCC) and to investigate the correlations between expression level and clinical characteristics, outcome, and patient prognosis. A total of 106 OSCC patients underwent surgical treatment at the A.C. Camargo Cancer Hospital, S˜ao Paulo, Brazil, were analyzed for clinicopathological data, treatment, tumor outcome, prognosis and immunohistochemical expression of β2-AR. The β2-AR expression was statistically analyzed relative to clinicopathological variables and survival using the Chi-square test, Kaplan-Meier curves and Cox regression model. Most OSCC (72.6%) exhibited malignant cells with strong cytoplasmatic and membranous β2-AR expression. β2-AR expression was significantly associated with alcohol (p = 0.021), simultaneous consumption of alcohol and tobacco (p = 0.014) and T stage (p = 0.07). In addition, OSCC patients who exhibited strong β2-AR expression demonstrated a higher rate of overall survival (p = 0.001) and cancer specific survival (p = 0.004) compared to patients with weak/negative β2-AR expression. The Cox regression model demonstrated that strong β2-AR expression was an independent favorable prognostic factor for OSCC patients. These results suggest that the strong malignant cell β2-AR expression is a favorable prognostic factor for OSCC patients and could be used as a target for new anti-neoplastic pharmacological strategies. Keywords: Oral cancer, adrenergic receptor, stress

1. Introduction Recent evidence has demonstrated that chronic stress, via activation of adrenergic receptors by catecholamines, mediates increased tumor growth and influences cancer development and progression [1,4,11, 15,17,18]. Previously reported studies have proved that the activation of β2-adrenergic receptor (β2-AR), which is expressed on human tumor cells, regulates ∗ Address for correspondence: Denise Tostes Oliveira, Faculdade ´ de Odontologia de Bauru, Area de Patologia, Alameda Oct´avio Pinheiro Brisolla, 9-75, CEP 17012-901, Bauru, S˜ao Paulo, Brazil. Tel.: +55 21 14 32358251; Fax: +55 21 14 32234679; E-mail: [email protected].

diverse cell mechanisms including proliferation, angiogenesis, migration and metastases [1,3,4,8–11,15, 17–20]. The effects of stress related hormones, particularly during tumor cell proliferation, can be either stimulatory or inhibitory depending on the type of hormone and type of tumor [16]. For example, in esophageal and pancreatic cancer cells, β2-AR stimulation by epinephrine increases cell proliferation [8, 20]. By contrast, pirbuterol, a pharmacological agonist of β2-AR, causes inhibition of growth and induction of apoptosis, leading to tumor regression in mouse xenograft models of breast cancer [3]. To date, few studies have investigated the relationship between stress and/or β2-AR expression and oral squamous cell carcinoma (OSCC) behavior [1,11].

ISSN 1574-0153/11/12/$27.50  2011/2012 – IOS Press and the authors. All rights reserved

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D.M. Bravo-Calder´on et al. / Significance of β2-AR expression in oral squamous cell carcinoma

More specifically, some authors have presented interesting data indicating that β2-AR stimulation by norepinephrine in oral squamous cell carcinoma cell lines increases cell proliferation and the production of IL6 [1,11]. Additionally, it has been previously reported that malignant cell expression of β2-AR significantly correlates with age, lymph node metastases, tumor size, and the clinical stage of oral squamous cell carcinoma, and that β2-AR may play an important role in the formation and metastasis of oral cancer [11]. The present study was designed to evaluate the association between malignant cell β2-AR expression and patient clinicopathological characteristics in order to determine whether this protein is a potentially useful marker to predict the presence of nodal metastasis, tumor invasion, clinical outcome and prognosis for patients with oral cancer. 2. Patients and methods 2.1. Patients and tumor samples This retrospective study was based on the analysis of 106 patients who received surgical treatment for primary oral squamous cell carcinoma at the Head and Neck Surgery and Otorhinolaryngology Department of the A.C. Camargo Cancer Hospital, S˜ao Paulo, Brazil from 1970 to 2000. Patients with other simultaneous primary tumors, distant metastases at the time of admission, or those undergoing preoperative chemotherapy and/or radiotherapy were excluded from this study. Patient clinical data were collected from hospital records, and included age, gender, ethnic group, tobacco and alcohol consumption, tumor location, disease stage according to the TNM system of the International Union Against Cancer (UICC) [13], treatment (surgery, postoperative adjuvant radiotherapy) and clinical follow-up (recurrence, occurrence of second primary tumor, or death). The Research Ethics Committee of the A.C. Camargo Cancer Hospital, S˜ao Paulo, Brazil, approved this study (# 1385/10). Surgically resected specimens of OSCC were obtained from the Department of Pathology archives, and the formalin-fixed, paraffin-embedded tissue blocks were cut into 3 μm sections for hematoxylin and eosin (H&E) staining and immunohistochemistry analysis of β2-AR. The tumor sections were examined by two pathologists (DMBC, DTO) without knowledge of clinical data, and the histopathological malignancy grade was determined according to the Bryne system [2]. Tumor infiltration of adjacent structures, vascular embolization, and lymph node metastases (pN+) were also reported.

2.2. β2-adrenergic receptor expression in oral squamous cell carcinoma Sections were deparaffinized in xylene and hydrated using graded alcohol/water baths. Antigen retrieval was performed using 10 mM citrate buffer (pH 6.0) in a domestic pressure cooker (Nigro, model Eterna 41/2L, Araraquara, SP, Brazil) for 4 min, and then endogenous peroxidase activity was blocked by incubation in 3% H2 O2 for 30 min. Tissue sections were incubated for 18 hours at 4◦ C in a humidified chamber with the anti-β2-adrenergic receptor primary antibody (Santa Cruz Biotechnology, sc-9042, Santa Cruz, CA, USA), diluted 1:50 in phosphate buffered saline (PBS) with bovine serum albumin solution (Sigma, A9647, St Louis, MO, USA) to block non-specific reactions. Next, the tumor sections were sequentially incubated with Post Primary Block (Novocastra, NovoLink Max Polymer, RE7260-K, Newcastle Upon Tyne, UK) for 30 min, followed by incubation with Polymer from the same kit. The antigen-antibody reactions were revealed using 3,3’ diaminobenzidine tetrahydrochloride (DAB/Sigma, D-5637, St. Louis, MO, USA) for 5 min in the dark. Sections were counterstained with Harris hematoxylin before being dehydrated and prepared with a cover slip. The vascular smooth muscle within the sections served as the positive internal control. For a negative control, the primary antibody was omitted during immunohistochemical staining. Microscopic images of ten fields of the invasive tumor front using x400 magnification were captured to analyze the immunohistochemical expression of β2AR in OSCC. The images were digitally obtained with a camera (Axiocam MRc, ZEISS, Jena, Germany) integrated to light microscope (Axioskop 2 Plus, ZEISS, Jena, Germany) and saved by a computer program system (Axiovision 4.6, ZEISS, Jena, Germany). Prior to the evaluation of the immunohistochemical malignant cell expression of β2-AR, the software ImageJ (Java-based image processing and analysis program of public-domain developed by Wayne Rasband NIH, Bethesda, MD, USA) was used to segment representative tumor images. Variations in the brown intensity of malignant cells positive for β2-AR expression were categorized according to the following RGB channel value ranges: 1) 2) 3) 4) 5)

R (red) channel – from 90 to 194; G (green) channel – from 50 to 140; B (blue) channel – from 45 to 147; The R value should be greater than the B value; The G value should be greater than the B value.

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Fig. 1. Analysis of the immunohistochemical expression of β2-adrenergic receptor in oral squamous cell carcinoma. (A): OSCC with weak/ negative expression of β2-AR, (C): OSCC with strong expression of β2-AR, (B and D): Red areas represent the automatic segmented cells immunostained by β2-AR using the software developed in MATLAB computer language. (A and C: immunohistochemistry β2-AR, Original magnification X400).

The ten images captured of fields of the invasive tumor front were automatically segmented using MATLAB computing language-based software according to the criteria listed above. This software also calculated statistical measurements based on the number of pixels within each of the segmented areas (determining the β2-AR immunopositive regions) (Fig. 1). After performing this computer-assisted immunohistochemistry analysis, the average of the β2-AR expression levels of the ten tumor images was calculated. The averages of the 106 specimens of OSCC were placed in ascending order and the median was established as the cutoff point serving to classify the specimens as exhibiting weak/negative (averages, 0.35 to 29.94) or strong (averages, 30.39 to 80.39) β2-AR expression. This measurement of malignant cell β2-AR expression was then subjectively confirmed by two investigators (DMBC, DTO) without knowledge of the tumor histopathologic features and patient clinical status. There were differences between the subjective analysis and the automatic computer-assisted immunohistochemical analysis in 13% of the tumors. The criterion of the subjective analysis was retained because this assessment involved the entire invasive tumor front.

2.3. Statistical analysis All statistical analysis was performed using SPSS 13.0 for windows software (SPSS Inc., Chicago, IL, USA). The association between β2-AR expression and clinicopathological variables was analyzed by the Chi-square test or Fisher’s exact test. Overall and cancer-specific survival rates were calculated using the Kaplan-Meier method and the comparison of the survival curves was performed using the log-rank test. Prognostic factors were evaluated by multivariate analysis using the Cox proportional hazard regression model. The follow-up period was the time between surgery and death or last patient information date. For all tests, p  0.05 was considered to represent a statistically significant result.

3. Results Our findings demonstrated that β2-AR expression was detected in the oral epithelium adjacent to the tumor, with the exception of the basal and corneum lay-

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Table 1 Associations between malignant cell expression of β2-adrenergic receptor and features of the 106 patients with oral squamous cell carcinoma

Variable Gender Ethnic group Age Tumor site

Tobacco* Alcohol* Tobacco + alcohol* T stage N stage Clinical stage Recurrence** Metastases Radiotherapy Second primary tumor

Male Female White Not white  58 years > 58 years Tongue Floor of mouth Retromolar area Inferior gingiva Yes No Yes No Yes No T1/T2 T3/T4 N+ N0 II III/IV Yes No Yes No Yes No Yes No

β2-adrenergic receptor Weak/negative (N = 29) Strong (N = 77) N % N % 25 86.2 65 84.4 4 13.8 12 15.6 26 89.7 67 87.0 3 10.3 10 13.0 19 65.5 36 46.8 10 34.5 41 53.2 12 41.4 45 58.4 10 34.5 18 23.4 5 17.2 8 7.8 2 6.9 6 10.4 23 95.8 66 95.7 1 4.2 3 4.3 23 92.0 46 68.7 2 8.0 21 31.3 22 91.7 44 65.7 2 8.3 23 34.3 10 34.5 49 63.6 19 65.5 28 36.4 16 55.2 40 51.9 13 44.8 37 48.1 6 20.7 24 31.2 23 79.3 53 68.8 14 48.3 30 39.0 15 51.7 47 61.0 0 0.0 3 3.9 29 100.0 74 96.1 17 58.6 36 46.8 12 41.4 41 53.2 4 13.8 13 16.9 25 86.2 64 83.1

p 0.999 0.999 0.085 NA

0.999 0.021 0.014 0.007 0.767 0.206 0.386 0.560 0.276 0.999

N = number of cases; p = p value obtained by chi-square test or Fisher’s exact test; NA = not available, *Excluding patients with lost records; **Local and/or regional recurrence.

ers (Fig. 2). In addition, immunohistochemical analysis results demonstrated that the salivary gland ducts, inflammatory cells, and smooth muscle fibers of the arteries exhibited strong β2-AR expression. Seventy seven (72.6%) of the 106 oral squamous cell carcinoma cases exhibited strong positivity for β2-AR. Expression of β2-AR was identified in the cytoplasm and/or membrane of cancer cells in the invasive tumor front, but keratin pearls were completely negative (Fig. 3). Patients were divided into strong or weak/negative β2-AR expression levels. Tables 1 and 2 display the associations between β2-AR expression and clinicopathological factors of the 106 patients with OSCC. The OSCC patient malignant cell β2-AR expression levels were significantly associated with alcohol consumption (p = 0.021), simultaneous consumption of alcohol and tobacco (p = 0.014), and with clinical T stage (p = 0.07), as described in Table 1. Strong β2-

AR expression were more frequently detected in early clinical stage T1/T2 and in patients who exhibited alcohol consumption or alcohol and tobacco consumption. In contrast, β2-AR expression levels did not correlate with the other histopathological characteristics, including the grade of malignancy, lymph node involvement (pN+), vascular embolization and perineural, muscular or bone infiltration (Table 2). 3.1. Survival analysis Clinical follow-up for the 106 patients with OSCC ranged from 0.03 to 288.03 months (mean 57.97 months ± 45.5 standard deviation). Survival analyses indicated statically significant differences in the 5- and 10-year overall and cancer-specific survival rates among the strong and weak/negative β2AR expression groups. As shown in Figs 4 and 5, the weak/negative β2-AR expression group demonstrated

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Table 2 Associations between malignant cell expression of β2-adrenergic receptor and histopathological parameters of the 106 patients with oral squamous cell carcinoma

Variable Malignancy grading Vascular embolization Perineural infiltration Muscular infiltration Bone infiltration* Lymph node involvement

More differentiated Less differentiated Yes No Yes No Yes No Yes No pN+ pN0

β2-adrenergic receptor Weak/negative Strong (N = 29) (N = 77) N % N % 23 79.3 67 87.0 6 20.7 10 13.0 17 58.6 43 55.8 12 41.4 34 44.2 18 62.1 55 71.4 11 37.9 22 28.6 24 82.8 64 83.1 5 17.2 13 16.9 5 17.9 10 13.3 23 82.1 65 86.7 16 55.2 38 49.4 13 44.8 39 50.6

p 0.366 0.797 0.354 0.999 0.545 0.593

N = number of cases; p = p value obtained by chi-square test or Fisher’s exact test; *Excluding patients with lost records.

Fig. 2. Expression of β2-adrenergic receptor in oral epithelium adjacent to tumor. Note the negativity of corneum and basal layers limited by red line. (Immunohistochemistry β2-AR, Original magnification X100).

Fig. 3. Strong malignant cell immunoexpression of β2-adrenergic receptor in oral squamous cell carcinoma. Note the negativity of keratin pearls (Immunohistochemistry β2-AR, Original magnification X100).

a significantly higher risk of death for OSCC patients according to overall (p = 0.001) and cancer specific (p = 0.004) survival rates. Moreover, the Kaplan-Meier test revealed that age (p = 0.027), N stage (p = 0.048), and T stage (p = 0.053) also significantly influenced the overall and cancer-specific survival rates of the 106 patients with OSCC. Multivariate analysis (or the Cox regression model) revealed that the β2-AR expression level was an independent prognostic factor when matched by age and N stage (overall survival rate) or T stage (cancer-specific survival rate), as shown in Table 3. Regarding overall survival, OSCC patients with strong β2-AR expression

exhibited a hazard ratio of 0.343 (95% CI: 0.206–0.569, p = 0.000) compared to patients with weak/negative β2-AR expression. Concerning cancer specific survival, tumors with strong β2-AR expression exhibited a hazard ratio of 0.454 (95% CI: 0.240–0.858, p = 0.015) compared to the weak/negative tumors for this protein. 4. Discussion In the present study, a group of patients with oral squamous cell carcinoma treated with surgical resection was selected to assess the predictive value of β2-AR

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D.M. Bravo-Calder´on et al. / Significance of β2-AR expression in oral squamous cell carcinoma

Fig. 4. Cumulative overall survival probability curves by groups of patients with oral squamous cell carcinoma presenting strong and weak/negative malignant cell expression of β2-adrenergic receptor (β2-AR).

Fig. 5. Cumulative cancer-specific survival probability curves by groups of patients with oral squamous cell carcinoma presenting strong and weak/negative malignant cell expression of β2-adrenergic receptor (β2-AR).

expression for nodal metastasis, clinical outcome, and prognosis using immunohistochemical staining. We demonstrated that strong β2-AR immunoexpression by malignant cells occurred in most oral squamous cell carcinomas. This result reinforces previous findings of β2-AR expression levels in oral cancer demonstrated by RT-PCR assay, Western blot analysis, and immunohistochemistry [1,11]. The immunoexpression of β2-AR in oral cancer cells was negative in keratin pearls (Fig. 3), which is in con-

cordance with a pattern of expression in the oral epithelium in which the corneum layer exhibits negativity for this protein (Fig. 2). In addition, we found that normal cells and tissues in proximity to the tumor also demonstrated positive β2-AR expression, including inflammatory cells, salivary gland ducts, smooth muscle fibers, and oral epithelium, as has also been previously described by others [1,5–7,11,12,14]. Our immunohistochemistry analysis of β2-AR expression in OSCC was performed using comparative

D.M. Bravo-Calder´on et al. / Significance of β2-AR expression in oral squamous cell carcinoma Table 3 Multivariate analysis of overall and cancer-specific survival rates of the 106 patients with oral squamous cell carcinoma Factor Overall survival Age ( 58 years vs > 58 years) N stage (N0 vs N+) β2-AR (weak/negative vs strong) Cancer-specific survival T stage (T1/T2 vs T3/T4) β2-AR (weak/negative vs strong)

HR

95% CI

p

2.138 (1.335–3.423) 0.002 1.891 (1.187–3.013) 0.007 0.343 (0.206–0.569) 0.000 1.565 (0.854–2.870) 0.147 0.454 (0.240–0.858) 0.015

HR: Hazard ratio; CI: Confidence interval; p = p value obtained by Cox proportional hazard regression model; β2-AR = beta-2 adrenergic receptor.

automatic computer-assisted analysis based in a MATLAB computer language software, as well as by subjective analysis carried out by investigators. Although the software has optimized this analysis, there were differences between the automated and subjectively assessed β2-AR immunoexpression in 13% of the tumors. Probalbly, this diference resulted of the interference caused by the immunopositivity of other cells in the tumor microenvironment that also express β2-AR, thus increasing the pixels detected by the software in each microscopic field. For this reason, we emphasize that the results of automatic analysis of immunohistochemistry biomarkers in cancer should be confirmed by subjective analysis of the tumor. The survival analyses provided evidence that weak/ negative malignant cell expression of β2-AR indicates a poor prognosis in OSCC patients. Patients with strong β2-AR tumor cell expression demonstrated a higher rate of overall (p = 0.001) and cancer specific (p = 0.004) survival compared to those with β2-AR weak/negative tumors. Additionally, strong β2-AR expression was an independent favorable prognosis factor when other variables such as age, N stage (overall survival rate) or T stage (cancer-specific survival rate) were also taken into consideration. These findings, which indicate that strong β2-AR expression by cancer cells is a favorable prognostic factor for OSCC patients, are in concordance with previous findings obtained by Yu et al. [19] in prostate cancer patients in which the disease free survival rate for patients with low β2-AR expression levels was significantly worse compared to patients exhibiting a high expression level for this protein. Furthermore, the present study is the first to have demonstrated the influence of β2-AR immunoexpression in oral cancer patient prognosis; the comparison of these results with others is impracticable because most investigations of β2-AR in cancer were carried out in

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vitro using different malignant cell lines [1,3,8–10,15, 17,18,20]. Taken together, our clinical results initially seem paradoxical considering the effect, in vitro, of stress related hormones on cancer. For example, several studies previously performed in different human cancer cell lines have demonstrated that stress via activation of β2-AR by catecholamines upregulates cell proliferation, migration, metastases, and formation of angiogenesis related molecules [1,3,8–11,15,17,18,20]. Particularly in oral cancer, the stimulation of β2-AR by norepinephrine has been shown to activate the tumor cell cAMP-protein Kinase A signaling pathway, in turn leading to increased cell proliferation and migratory activity of oral squamous cell carcinoma cell lines [1,11] In contrast, our clinical results confirmed that strong β2-AR expression by malignant cells can help identify patients at a lower risk for oral cancer. Further studies are warranted to confirm our clinical findings and to elucidate whether, after long periods of exposure to stress hormones, the receptors for these molecules, such as β2-AR, which is expressed on malignant cells, exhibit a causal effect on growth and progression of oral cancer in OSCC lines and animal models. According to the Chi-square test and Fisher’s exact test, there was no statistically significant association between β2-AR expression level and the histopathological malignancy grade (Byrne’s grading system) [2], lymph nodes involvement (pN+), and tumor invasion, including vascular, perineural, muscular or bone infiltration (Table 2). Interestingly, we found a significant association between malignant cell β2-AR expression level of OSCC patients and alcohol consumption (p = 0.021) or simultaneous alcohol/tobacco consumption (p = 0.014), as shown in Table 1. In addition, expression of β2-AR also significantly correlated with clinical T stage (p = 0.007); patients with tumors less than 4 cm in greatest dimension, clinically classified as T1 or T2, exhibited a higher frequency of strong β2-AR expression. Despite the associations that we demonstrated in the present investigation among the risks factors for OSCC, clinical T stage, and β2-AR expression levels, we did not find the significant correlations with clinical T3-T4 stage and with the occurrence of lymph node metastases in oral cancer patients reported by Shang, Liu and Liang [11]. The significant association that we demonstrated between positive β2-AR expression and alcohol consumption or tobacco use is certainly a relevant issue. However, immunohistochemistry does not indi-

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cate whether the neurotransmitter receptor expressed by the malignant cells in these OSCC patients is functionally active. Therefore, the activation of the β2AR pathway by alcohol or tobacco needs to be further investigated in OSCC cell lines. In addition, to our knowledge there are not previous studies investigating the effect of β2-AR activation on the levels of cytokines and/or growth factors in serum and/or tumors of OSCC patients. Our research group is considering the implementation of a future prospective clinical trial to measure the blood, saliva, and tumor levels of catecholamines and cytokines, and to compare these values with the tumor expression levels of β2-AR and with the clinical outcomes for OSCC patients. In conclusion, although our results demonstrated that malignant cell β2-AR expression potentially represents a favorable prognosis for OSCC patients, further clinical studies are warranted to confirm the role of β2-AR activation in oral squamous cell carcinoma progression and to elucidate its influence on clinical outcome.

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Acknowledgements The authors would like to thank F´atima Aparecida Silveira Camargo for technical support and Cleyton Zanardo de Oliveira for statistical collaboration. This study was supported by Conselho Nacional de Desenvolvimento Cient´ıfico e Tecnol´ogico (CNPq – grants #476139/2010-4, #500991/2010-3 and #142790/20117) and by Fundac¸a˜ o de Amparo a` Pesquisa do Estado de S˜ao Paulo (FAPESP – grant #2010/06333-3).

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