Tumor Biol. DOI 10.1007/s13277-016-4971-8

ORIGINAL ARTICLE

Toll-like receptor 5 and 7 expression in adenoid cystic carcinoma of major salivary glands K. Hirvonen 1,5 & L. Bäck 1 & C. Haglund 2 & I. Leivo 3 & L. Jouhi 1 & A. A. Mäkitie 1 & J. Hagström 4

Received: 2 December 2015 / Accepted: 2 February 2016 # International Society of Oncology and BioMarkers (ISOBM) 2016

Abstract Adenoid cystic carcinoma (ACC) of the salivary glands has a poor long-term prognosis and high metastatic rate. Toll-like receptors (TLRs) have been related to tumour progression but have also tumour growth-inhibiting responses. To the best of our knowledge, they have not been s t u d i e d p r e v i o u s l y i n A C C . We s t u d i e d t h e immunoexpression of TLR 5 and 7 in ACC of the major salivary glands. From a cohort of 54 patients with ACC of the major salivary glands treated at the Department of Otorhinolaryngology-Head and Neck Surgery, Helsinki University Hospital, Helsinki, Finland in 1974–2009, there were 34 primary tumours and six metastases available for immunohistochemical analysis. Immunohistochemical expression of TLR 5 and 7 were correlated to clinicopathological A. A. Mäkitie and J. Hagström contributed equally to this work. Electronic supplementary material The online version of this article (doi:10.1007/s13277-016-4971-8) contains supplementary material, which is available to authorized users. * K. Hirvonen [email protected]

1

Department of Otorhinolaryngology-Head and Neck Surgery, University of Helsinki and Helsinki University Hospital, Helsinki, Finland

2

Department of Surgery, Helsinki University Hospital, Helsinki, Finland and Research Programs Unit, Translational Cancer Biology, University of Helsinki, Helsinki, Finland

3

Department of Pathology, University of Turku, Turku, Finland

4

Department of Pathology, University of Helsinki and Helsinki University Hospital, Helsinki, Finland

5

Department of Otorhinolaryngology-Head and Neck Surgery, Helsinki University Hospital, P.O. Box 220, FI-00029 HUS Helsinki, Finland

findings and patient survival. Both TLR 5 and 7 were expressed in ACCs and their metastases, mostly on the cell membranes. The expression was heterogeneous in individual tumours. TLR 5 was expressed less in male samples, and TLR 7 had lower expression in ACCs with solid growth pattern. No correlation with survival was found. In the normal salivary gland, the TLR 5 and 7 expression was mainly negative. Both TLR 5 and 7 are expressed in salivary adenoid cystic carcinoma on the cell membranes as well as in cytoplasm. Keywords Head and neck cancer . Salivary gland cancer . Immunohistochemistry . Metastasis

Abbreviations ACC Adenoid cystic carcinoma TLR Toll-like receptor OS Overall survival DSS Disease-specific survival DFS Disease-free survival DAMP Danger-associated molecular pattern PAMP Pathogen-associated molecular pattern LPS Lipopolysaccharide PRR Pattern-recognition receptor

Introduction Salivary gland cancers comprise approximately 4 % of all head and neck cancers [1]. In a Finnish population-based patient material from 1991 to 1996, adenoid cystic carcinoma (ACC) was the most common type of salivary gland cancer (27 %), followed by mucoepidermoid carcinoma (19 %) and acinic cell carcinoma (17 %) [2]. The treatment protocol for ACC includes surgery followed by radiotherapy if necessary.

Tumor Biol.

ACC is notorious for late metastases and poor prognosis. In our earlier study of 54 patients with ACC of the major salivary glands over a 35-year period, disease recurrence or appearance of metastatic disease following treatment with curative intent occurred in 61.5 % of patients, and 10-year disease-specific survival was 61.5 % [3]. The published survival rates have not improved significantly over the years, and therefore, it would be important to learn about the molecular pathogenesis and markers of this malignancy to gain understanding of its behaviour and to find new alternative treatment modalities. Toll-like receptors (TLRs) are pattern-recognition receptors (PRRs) that participate in immunological first-line host defence against pathogens by recognizing pathogen-associated molecular patterns (PAMPs). They can also detect endogenous alert signals from the dying or damaged cells through danger-associated molecular patterns (DAMPs). In humans, there are ten known TLRs. TLRs 1, 2, 4, 5, 6, and 10, which are mainly located on the cell surface, recognize bacterial lipopolysaccharides (LPS) and lipoproteins. TLRs 3, 7, 8, and 9 detect bacterial DNA and viral RNA mainly in intracellular endosome-lysosomes, where these components undergo processing [4, 5]. In the skin and in the mucosa of the respiratory, gastrointestinal, and genitourinary tract, TLRs regulate innate and adaptive immunoresponses [5–7]. The association between chronic inflammation and development of cancer is well established [8–11]. The role of TLRs is important in defence mechanisms against inflammatory processes. The role of TLRs in cancer is however, complex and currently a subject of intensive research. TLR activation may result in a chronically inflamed microenvironment, and may therefore promote cancer. Activation of TLRs in tumour cells may promote cancer-cell survival, chemoresistance, and by this process, tumour progression. On the other hand, the activation of TLRs may also mediate responses against cancer, probably via different cellular activation pathways than in tumorigenesis. To study TLR-mediated signaling for possible use in tumour immunotherapy, it is important to document the expression of each TLR in different tumour types [4, 5, 7]. In the salivary gland, expression of TLRs has been studied in Sjögren’s syndrome [12, 13]. So far, little is known about the function of TLRs in salivary gland carcinomas. In mucoepidermoid carcinoma, high TLR 9 expression has been associated with improved survival [14]. In another study, TLR 5 promoted cell migration and invasive growth in salivary gland adenocarcinoma in vitro [15]. TLR 5 and 7 expression has been studied earlier in oropharyngeal squamous cell carcinoma and other head and neck cancers [15–19]. We studied the expression of these TLRs to clarify their role in ACC of the major salivary glands. We also compared the immunoexpression of TLR 5 and 7 with clinical and clinicopathological features and patient survival.

Materials and methods Patients The study population included 54 patients with ACC of the major salivary glands treated at the Helsinki University Hospital between 1974 and 2009. Staging of tumours was performed according to the International Union Against Cancer tumour-node-metastases (TNM) classification (UICC 2009, 7th edition). The study design was approved by the institutional Research Ethics Board (Dnro 31/13/03/02/2010, 01 February 2010). Fifty-two patients had been treated with curative intent, and all except one had surgery as primary treatment. More detailed information on these patients has been reported in our earlier study [3]. Demographic patient data are presented in Table 1. Thirty-four primary tumour samples and six metastases were available for immunohistochemical evaluation.

Immunohistochemistry Tissue samples were cut into 4 μm sections. Deparaffinization was carried out in xylene and rehydration in a graded series of alcohols. The tissue slides were heated in a PreTreatment module (Lab Vision Corp., UK Ltd, UK) in Tris-HC1 buffer for TLR 7 (pH 8.5) and Tris-EDTA buffer for TLR 5 (pH 9.0), and endogenous peroxidase was blocked with 0.3 % Dako REAL Peroxidase-Blocking Solution. For immunostaining, Table 1

Characteristics of 32 major salivary gland ACC patients N (%)

Gender

Tumour site

T class

N class

Stage

Male Female

18 (56) 14 (44)

Median age, years (range) Parotid Submandibular

58 (25–80) 18 (56) 12 (38)

Sublingual T1 T2 T3 T4a T4b N0 N1 N2 N3 I II III IVA IVB

2 (6) 9 (28) 6 (19) 9 (28) 7 (22) 1 (3) 28 (88) 4 (13) 0 0 9 (28) 6 (19) 9 (28) 7 (22) 1 (3)

Tumor Biol.

monoclonal mouse anti-human antibody for TLR 5 (1:200, IMG-664A Imgenex, Cruz Biotechnology, USA) and polyclonal rabbit anti-human antibody for TLR 7 (1:300, IMG581A Imgenex, USA) were used. Further incubation was carried out with the Dako REAL Antibody Diluent S2022 and staining was visualized with Dako REAL DAB+ Chromogen. Washing between each step was done with PBS-0.04 %Tw e e n 2 0 . M e y e r ’s h a e m a t o x y l i n w a s u s e d f o r counterstaining, and slides were mounted with PERTEX (Histolab). As a positive control, we used tissue of tonsillar squamous cell carcinoma, and as a negative control, we used slides where primary antibody was changed to PBS.

Table 2 Immunoexpression of TLR 5 and TLR 7 in primary major salivary gland ACC tumours and metastases Number of valid samples Immunoscore

TLR 5 Primary

TLR 5 Metastases

TLR 7 Primary

TLR 7 Metastases

0

8 (26 %)

0 (0 %)

1 (3 %)

1 (17 %)

1 2

11 (35 %) 9 (29 %)

4 (67 %) 1 (17 %)

7 (22 %) 16 (50 %)

2 (33 %) 2 (33 %)

3

3 (10 %)

1 (17 %)

8 (25 %)

1 (17 %)

Total

31

6

32

6

Immunoscoring

TLR 5

Two independent researchers scored the slides without information on the clinical data. We first assessed whether TLR 5 and 7 were expressed in the cytoplasm, nucleus, or cell membrane, and the scoring was then performed by estimating the percentage of positively stained cells. We grouped the immunoscores as 0 for 0–10 % (negative or very mild), 1 for 11–40 % (mild), 2 for 41–70 % (moderate), and 3 for 71–100 % positivity (strong). To validate the expression in normal tissues, we used normal salivary gland tissue found on tumour slides.

In normal salivary gland tissue, TLR 5 expression was mostly negative, except for areas immediately adjacent to the tumour area, where some positivity on nuclear membrane was seen in ductal structures and acini. In ACCs, TLR 5 was expressed mainly on cell membranes (n = 28; 90 %) but in addition, nuclear (n = 11; 36 %) and cytoplasmic positivity (n = 17; 55 %) was seen in some cases (Fig. 3). The cellular location of TLR 5 positivity varied in the three different growth patterns: the cribriform areas revealed more cell membrane staining (Fig. 1). In solid and tubular growth patterns, the staining was more cytoplasmic. In metastases, the expression of TLR 5 was seen on cell membranes in all the samples and in the cytoplasm in 83 % of cases, expression was mostly mild (Table 2). Nuclear expression was detectable in two out of 6 cases (33 %).

Statistical analysis We used SPSS version 21.0 software (SPSS Inc., Chicago, IL, USA) to explore the association of TLR expression on clinicopathological variables such as tumour size, T class, stage, growth pattern, perineural invasion and presence of neck or distant metastastases. We used the chi-square test with exact p values to evaluate the differences in TLR staining according to clinicopathological variables. We calculated survival curves using the Kaplan-Meier method with the log-rank test and observed the relationship of TLR scores to overall survival (OS), disease-specific survival (DSS), and disease-free survival (DFS). Follow-up time was defined as the period between the last day of treatment and the last day of follow-up or death of any cause in OS, death to disease in DSS or of any form of disease recurrence in DFS. For comparison, the tumours were grouped in low (0 and 1) and high (2 and 3), as well as negative (0) to positive (1–3) categories. A two-sided p value less than 0.05 was considered statistically significant.

Results The expression percentage of TLR 5 and 7 in primary tumours and metastases are presented in Table 2.

TLR 7 Normal salivary gland tissue was usually negative for TLR 7. In some specimens ductal and acinar structures of the normal salivary gland showed 10 to 50 % positivity in cell membranes. In certain tumours, TRL 7 was expressed in lymphocytes of the salivary gland adjacent to the tumour. Nerves showed TLR 7 positivity in both tumour area and normal tissues (Fig. 2). In tumours, TLR 7 expression was extremely heterogeneous (Fig. 3). In all tumours, the areas of strong expression and totally negative areas were detected. TLR 7 was expressed on cell membranes in all tumours (n = 32; 100 %). In addition, in part of the tumours, nuclear (n = 10; 31 %) and cytoplasmic (n = 2; 6 %) expression was detectable. In metastases, TLR 7 expression appeared in cell membranes in all six samples. One sample had cytoplasmic expression.

Tumor Biol. Fig. 1 TLR 5 expression in ACC; cribriform area of the tumour (a, b) and normal salivary gland tissue (c). Magnification ×200

The association of immunostaining and clinicopathological data TLR 5 In males, TLR 5 expression was lower (mean expression 0.89) compared with females (mean expression 1.69) (p = 0.022). TLR 7 In ACCs with solid growth pattern, TLR 7 expression was lower (mean expression 1.25) compared with ACCs with cribriform or tubular growth patterns (mean expression 2.21) (p = 0.003). No other correlations emerged with either TLR 5 or TLR 7. Expression of neither TLR correlated with disease recurrence or patient survival.

Discussion We investigated the expression of TLR 5 and 7 in a series of the major salivary gland ACC. These TLRs Fig. 2 TLR 7 expression in ACC; cribriform area (a), solid area (b) and normal salivary gland tissue (c). Arrows point to salivary gland tissue and double arrow to nerves (c). Magnification ×200

were expressed in both primary tumours and metastases. Both the expression intensity (negative to strong) as well as the expression pattern (nuclear, membranous, and cytoplasmic) did range even within the same tumour. Low TLR 5 expression correlated with male gender, and low TLR 7 expression correlated with solid growth pattern. Neither of the TLRs explored correlated with disease recurrence nor patient survival. ACC has three different histologic growth patterns often coexisting in different proportions in individual tumours. Those with predominantly cribriform pattern comprise approximately 45 % of specimens. The predominantly tubular pattern (35 %) is associated with the best prognosis. A predominantly solid pattern is the rarest (approximately 20 % of ACC) and has the worst prognosis [1]. The solid pattern has been pointed out as an independent prognostic factor for metastases [20]. Also in the present limited series, the solid pattern correlated with neck lymph node metastases, but not with survival. One tumour often includes areas with different growth patterns. Metastases may have either one or more of the growth patterns, dependent on the tumour clones giving rise to the metastasis. The solid growth pattern of ACC correlated with low TLR 7 expression.

Tumor Biol.

In summary, the present study shows that both TLR 5 and TLR 7 are expressed in ACC. No correlation with survival was found. Further studies are needed to clarify the role of TLRs in salivary gland cancers. Compliance with ethical standards Conflicts of interest None.

References Fig. 3 ACC tissue showing heterogeneity of immunoexpression of TLR 7 (a) and TLR 5 (b). Magnification ×100

1.

2.

Not many studies exist on TLR 7 in head and neck tumours. In juvenile angiofibroma TLR 7 expression was seen in cell membranes without clinical correlations [19]. In oropharyngeal squamous cell carcinoma high TLR 7 expression and low TLR 5 expression have been shown in HPV positive cancers, which are known to have better response to radiotherapy [16]. To our knowledge, TLR 7 has not been previously studied in salivary gland cancers. In lung cancer, high TLR 7 correlates with resistance to neoadjuvant chemotherapy and poor clinical outcome [21]. TLR 7 is usually expressed intracellularly in endosomes, lysosomes, and endoplasmic reticulum. It recognizes viral single-stranded RNA [4, 7, 22]. In oropharyngeal squamous cell carcinoma, TLR 7 is expressed on the nuclear membrane and in nuclei [16]. In our study, TLR 7 was expressed in the plasma membrane in all samples. The role of TLR 7 expressed on the plasma membrane is still unclear. TLR 5 has earlier been described in both normal salivary gland and in adenocarcinomas. It has been suggested that bacterial flagellin promotes invasion and migration by TLR 5-induced pathway in salivary gland adenocarcinomas [15]. In squamous cell carcinoma of the tongue, high TLR 5 is associated with old age, female gender and DSS, and it is an independent predictor of recurrence and mortality [17]. Similar to our study, lower TLR 5 expression correlated with male gender. TLR 5 has been found to express not only on the plasma membrane, but also in the cytoplasm [16, 17, 22]. These findings are in line with our findings of TLR 5 being mainly expressed not only on plasma membranes but also in subcellular locations. To clarify the role of differently expressed TLR 5, future studies are needed. The limitation of our study is the small sample size. The enlargement of the study series will be, however, challenging with the given rarity of this disease. We will thus promote multi-institutional studies in our further research on ACC.

3.

4. 5.

6. 7.

8. 9.

10.

11. 12.

13.

14.

15.

16.

Concus PA, Tran NT, DeLacure DM. Malignant diseases of the salivary glands. In: Lalwani KA, editor. Current diagnosis & treatment, otolaryngology head and neck surgery. New York: Mc Graw Hill Lange; 2008. p. 311–5. Luukkaa H, Klemi P, Leivo I, Koivunen P, Laranne J, Makitie A, et al. Salivary gland cancer in Finland 1991–96: an evaluation of 237 cases. Acta Otolaryngol. 2005;125:207–14. Hirvonen K, Back L, Saarilahti K, Leivo I, Hagstrom J, Makitie AA. Pattern of recurrent disease in major salivary gland adenocystic carcinoma. Virchows Arch. 2015;467(1):19–25. Ioannou S, Voulgarelis M. Toll-like receptors, tissue injury, and tumourigenesis. Mediat Inflamm. 2010. doi:10.1155/2010/581837. Yu L, Wang L, Chen S. Dual character of toll-like receptor signaling: pro-tumorigenic effects and anti-tumor functions. Biochim Biophys Acta. 1835;2013:144–54. Akira S, Uematsu S, Takeuchi O. Pathogen recognition and innate immunity. Cell. 2006;124:783–801. Basith S, Manavalan B, Yoo TH, Kim SG, Choi S. Roles of toll-like receptors in cancer: a double-edged sword for defense and offense. Arch Pharm Res. 2012;35:1297–316. Bargen JA. Chronic ulcerative colitis associated with malignant disease. 1928. Dis Colon Rectum. 1994;37:727–30. Beasley RP, Hwang LY, Lin CC, Chien CS. Hepatocellular carcinoma and hepatitis B virus. A prospective study of 22 707 men in Taiwan. Lancet. 1981;2:1129–33. Parsonnet J, Friedman GD, Vandersteen DP, Chang Y, Vogelman JH, Orentreich N, et al. Helicobacter pylori infection and the risk of gastric carcinoma. N Engl J Med. 1991;325:1127–31. zur Hausen H. Condylomata acuminata and human genital cancer. Cancer Res. 1976;36:794. Spachidou MP, Bourazopoulou E, Maratheftis CI, Kapsogeorgou EK, Moutsopoulos HM, Tzioufas AG, et al. Expression of functional toll-like receptors by salivary gland epithelial cells: increased mRNA expression in cells derived from patients with primary Sjogren’s syndrome. Clin Exp Immunol. 2007;147:497–503. Kawakami A, Nakashima K, Tamai M, Nakamura H, Iwanaga N, Fujikawa K, et al. Toll-like receptor in salivary glands from patients with Sjogren’s syndrome: functional analysis by human salivary gland cell line. J Rheumatol. 2007;34:1019–26. Korvala J, Harjula T, Siirila K, Almangush A, Aro K, Makitie AA, et al. Toll-like receptor 9 expression in mucoepidermoid salivary gland carcinoma may associate with good prognosis. J Oral Pathol Med. 2014;43:530–7. Park JH, Yoon HE, Kim DJ, Kim SA, Ahn SG, Yoon JH. Toll-like receptor 5 activation promotes migration and invasion of salivary gland adenocarcinoma. J Oral Pathol Med. 2011;40:187–93. Jouhi L, Datta N, Renkonen S, Atula T, Makitie A, Haglund C, et al. Expression of toll-like receptors in HPV-positive and HPV-negative oropharyngeal squamous cell carcinoma-an in vivo and in vitro study. Tumour Biol. 2015;36(10):7755–64.

Tumor Biol. 17.

Kauppila JH, Mattila AE, Karttunen TJ, Salo T. Toll-like receptor 5 (TLR5) expression is a novel predictive marker for recurrence and survival in squamous cell carcinoma of the tongue. Br J Cancer. 2013;108:638–43. 18. Omar AA, Korvala J, Haglund C, Virolainen S, Hayry V, Atula T, et al. Toll-like receptors -4 and -5 in oral and cutaneous squamous cell carcinomas. J Oral Pathol Med. 2015;44:258–65. 19. Renkonen S, Cardell LO, Mattila P, Lundberg M, Haglund C, Hagstrom J, et al. Toll-like receptors 3, 7, and 9 in juvenile nasopharyngeal angiofibroma. APMIS. 2015;123:439–44. 20. Bhayani MK, Yener M, El-Naggar A, Garden A, Hanna EY, Weber RS, et al. Prognosis and risk factors for early-stage adenoid cystic carcinoma of the major salivary glands. Cancer. 2011;118(11):2872–8. 21. Chatterjee S, Crozet L, Damotte D, Iribarren K, Schramm C, Alifano M, et al. TLR7 promotes tumor progression, chemotherapy

22.

resistance, and poor clinical outcomes in non-small cell lung cancer. Cancer Res. 2014;74:5008–18. Kumar H, Kawai T, Akira S. Toll-like receptors and innate immunity. Biochem Biophys Res Commun. 2009;388:621–5.

Highlights 1. TLR 5 and 7 are shown to be expressed in salivary gland adenoid cystic carcinoma for the first time. 2. TLR 5 and 7 are expressed on cell membranes and in cytoplasm in ACC. 3. TLR 5 correlated with male gender and TLR 7 with solid growth pattern in ACC.