Oral Oncology 51 (2015) 96–102
Contents lists available at ScienceDirect
Oral Oncology journal homepage: www.elsevier.com/locate/oraloncology
Predictive role of toll-like receptors 2, 4, and 9 in oral tongue squamous cell carcinoma Laura K. Mäkinen a,⇑, Timo Atula a, Valtteri Häyry a, Lauri Jouhi a, Neeta Datta b, Sanna Lehtonen b, Abdirisak Ahmed c, Antti A. Mäkitie a, Caj Haglund d,e,1, Jaana Hagström b,f,1 a Department of Otorhinolaryngology – Head and Neck Surgery, Helsinki University Central Hospital and University of Helsinki, P.O. Box 220, Haartmaninkatu 4E, FI-00029 HUS, Helsinki, Finland b Department of Pathology, Haartman Institute, University of Helsinki, P.O. Box 21, Haartmaninkatu 3, FI-00014 University of Helsinki, Helsinki, Finland c Institute of Dentistry, Biomedicum 1, University of Helsinki, P.O. Box 63, Haartmaninkatu 8, FI-00014 University of Helsinki, Helsinki, Finland d Department of Surgery, Helsinki University Central Hospital, Haartmaninkatu 4, P.O. Box 440, FI-00029 HUS, Helsinki, Finland e Research Programs Unit, Translational Cancer Biology, University of Helsinki, P.O. Box 440, FI-00014 University of Helsinki, Helsinki, Finland f Department of Pathology, HUSLAB, Helsinki University Central Hospital, P.O. Box 400, Haartmaninkatu 3, FI-00029 HUS, Helsinki, Finland
a r t i c l e
i n f o
Article history: Received 13 June 2014 Received in revised form 12 August 2014 Accepted 24 August 2014 Available online 26 September 2014 Keywords: Tongue carcinoma Oral cancer Immunohistochemistry Prognosis Recurrence Survival Invasion Metastasis p16 protein
s u m m a r y Objectives: The clinical behavior of oral tongue squamous cell carcinoma (OTSCC) can be unpredictable, and even small tumors may behave aggressively. Toll-like receptors (TLRs) are pattern-recognition molecules involved in innate immunity, and they are also expressed in many types of cancer. TLRs play an apparently pivotal role in some cancers related to tumor progression and, conversely, cancer inhibition, however their role in oral cancer is unclear. We therefore studied the expression of TLR-2, -4, -5, -7, and -9 in early-stage OTSCC. Materials and methods: Tissue microarray technique and immunohistochemistry was employed to define the expression of TLRs from the tumors of 73 consecutive patients with Stage I-II OTSCC. Immunoexpression scores were compared with patient and tumor related characteristics and survival. Results: All TLRs were expressed in OTSCC tissue. High/strong TLR-2, -4, and -9 expression correlated with deeper tumor invasion. Cytoplasmic TLR-2 and -4 also correlated significantly with higher tumor grade, whereas high TLR-5 expression associated with lower tumor grade. High expression of TLR-9 correlated with advanced tumor size. Negative or mild TLR-5 expression predicted poor disease-specific survival. Conclusion: All the studied TLRs showed high expression in early-stage OTSCC. More importantly, TLR-2, -4, and -9 seemed to predict invasive tumor growth. Ó 2014 Elsevier Ltd. All rights reserved.
Introduction The behavior of early-stage oral tongue squamous cell carcinoma (OTSCC) can be unpredictable despite improvements in
Abbreviations: DFS, disease-free survival; DSS, disease-specific survival; HNSCC, head and neck squamous cell carcinoma; HPV, human papilloma virus; LPS, lipopolysaccharide; OS, overall survival; OSCC, oral squamous cell carcinoma; OTSCC, oral tongue squamous cell carcinoma; TLR, toll-like receptor. ⇑ Corresponding author. Tel.: +358 40 5678687; fax: +358 9 471 75010. E-mail addresses: laura.k.makinen@helsinki.fi (L.K. Mäkinen), timo.atula@hus.fi (T. Atula), valtteri.hayry@fimnet.fi (V. Häyry), lauri.jouhi@helsinki.fi (L. Jouhi), neeta.datta@helsinki.fi (N. Datta), sanna.h.lehtonen@helsinki.fi (S. Lehtonen), abdirisak.ahmed@helsinki.fi (A. Ahmed), antti.makitie@helsinki.fi (A.A. Mäkitie), caj.haglund@hus.fi (C. Haglund), jaana.hagstrom@hus.fi (J. Hagström). 1 Equal contribution. http://dx.doi.org/10.1016/j.oraloncology.2014.08.017 1368-8375/Ó 2014 Elsevier Ltd. All rights reserved.
diagnostic methods and treatment modalities [1,2]. Even small tumors may behave aggressively. The 5-year survival rate of early-stage (I–II) OTSCC is still modest [3]. To adjust the treatment more accurately, improved knowledge of the underlying mechanisms behind aggressiveness of the disease as well as new predictive markers are needed. Intense research studies are currently addressing the relevance of innate immunity response in cancer initiation and progression. Cancer immunotherapy is expected to be the fourth weapon against cancer along with surgery, radiation, and chemotherapy [4]. It is well known that environmental carcinogens (tobacco, alcohol) are major contributors to the development of head and neck squamous cell carcinomas whereas tumor progression could be partly due to a failure of the innate immune response against cancer [5].
97
L.K. Mäkinen et al. / Oral Oncology 51 (2015) 96–102
Toll like receptors (TLRs) are a major class of pattern-recognition receptors that are expressed by cells of the humane immune system and epithelial cells located near host-environment boundary. In addition, they are also present in many types of cancer. Ten human TLRs have been identified, each recognizing a specific microbial component. There are two kinds of ligands for TLRs: exogenous pathogen associated molecular patterns (PAMPs), which are components of microbes, and endogenous damageassociated molecular patterns (DAMPs) released from injured or inflamed tissues [6]. TLR-2 and TLR-4 recognize lipopolysaccharide (LPS, a membrane component of Gram-negative bacteria), TLR-5 recognizes bacterial flagellin, TLR-7 recognizes double or single stranded RNA, and TLR-9 recognizes bacterial DNA [7,8]. TLRs detecting bacterial LPS and lipoproteins are located on the cell surface (TLR-1, TLR-2, TLR-4, TLR-5, and TLR-6), whereas TLR-3, -7, -8, and -9 that mainly recognize viral RNA and bacterial DNA are located in endosomes and lysosomes, where these materials are processed [6]. Thus, TLRs trigger signals activate innate and adaptive immune responses [6]. The actual role of TLRs in tumorigenesis remains unknown, and it is related to cancer progression as well as inhibition [8–10]. TLR-mediated tumor progression is considered to result from the activation of TLRs in tumor cells, whereas tumor regression is thought to derive from the activation of host immune responses [8]. The function of each TLR is complex and variable and is affected by multiple factors. Little is known about TLRs in head and neck squamous cell carcinoma (HNSCC). Various HNSCCs express TLR-2, -3, -4, -5, -7, and -9 both in vitro [11–18] and in vivo [11,13–15,19–22]. To further clarify the role of TLRs in early-stage oral cancer we studied TLR-2, -4, -5, -7, and -9 in small OTSCCs. The surrogate marker for human papilloma virus (HPV), p16, is widely utilized in the evaluation of oropharyngeal carcinomas [23]. Thus p16INK4a staining of tongue carcinoma tumors was performed here, and findings compared with TLR expression.
Material and methods Patients The present study assesses a series of early-stage OTSCC patients with tumors clinically defined as T1N0M0 or T2N0M0 which were treated at the Helsinki University Central Hospital between 1992 and 2002. The patient material has been described in more detail previously [24,25]. Demographic data of the material is described in Table 1. Tumor samples from 73 patients were available for immunohistochemistry (36 males and 37 females, median age 59 years, range 23–95). Thirty-five tumors (48%) were clinically classified as T1 and 38 (52%) as T2. All patients had been treated with curative intent including surgical resection of the primary tumor. An experienced head and neck pathologist re-evaluated all the original histological tumor specimens. According to histopathological classification, 52 tumors (71%) were classified as pT1 and 21 (29%) as pT2. Tumor invasion depth was measured from the level of the proximate normal mucosal surface. All patients in this study were treated according to the Finnish national guidelines for the treatment of Head and Neck cancer. Postoperative radiation therapy was given only to patients with deeply invasive tumors, and to those with neck metastases in neck dissection specimens. Consequently, 31 patients received no further primary treatment for the neck, 41 patients underwent elective neck dissection, and one patient received radiotherapy without surgery. Thus, radiotherapy had no effect on evaluation
Table 1 Demographic and clinicopathological features of 73 patients with oral tongue squamous cell carcinoma.
*
Clinicopathological variable
No. of patients (%)
Age, years 660 >60 Range Median
40 (55) 33 (45) 23–95 59
Sex Male Female
36 (49) 37 (51)
Grade I II III
24 (33) 35 (48) 14 (19)
Clinical T stage (mm) cT1 (620) cT2 (21–40)
35 (48) 38 (52)
Pathological T stage (mm) pT1 (620) pT2 (21–40)
52 (71) 21 (29)
Pathological node positivitya pN0 pN+
26 (36) 15 (21)
Pathological stage I II III IV
46 (63) 12 (16) 12 (16) 3 (4)
Invasion depth (mm) 64 >4
29 (40) 44 (60)
41/73 (56%) of the patients had elective neck dissection.
of tumor characteristics, nor on the evaluation of occult metastases, except possibly in one patient. Of all patients, 34 received post-operative radiotherapy, including the neck in 33 patients. Twenty-four patients (33%) had occult neck metastases, i.e. lymph node metastases in the elective neck dissection specimen (n = 15), or neck metastasis during follow up without failure at the primary site (n = 9). During follow-up, ten patients developed local recurrence. Only two patients were diagnosed with distant metastases, both after locoregional recurrence. All patients, but one, had a minimum follow-up time of five years or until death. Median follow-up time was 7.6 years (range, 0.3–17.2), during which 19 patients died of tongue cancer and 22 of other causes. Dates and causes of death were provided by the national agency of population statistics, Statistics Finland. The study design was approved by the institutional Research Ethics Board. Tissue array blocks Tissue microarray (TMA) technique was used as previously described [26]. Representative tumor tissue was lacking in seven patients for TLR-2, in six patients for TLR-4 and -9, in eight patients for TLR-7, and in ten patients for TLR-5 and p16INK4a evaluation. Immunohistochemistry Tissue slides as well as TMA slides were cut into 4–5 lm thick sections. Deparaffinization was done in xylene and rehydration with graded alcohol series. Slides were treated in a PreTreatment-module (Lab Vision Corp., UK Ltd, UK) in Tris–HCl buffer (pH 8.5) at 98 °C for 20 min and with 0.3% Dako REAL Peroxidase-Blocking Solution for 5 min to block endogenous
98
L.K. Mäkinen et al. / Oral Oncology 51 (2015) 96–102
peroxidase. Autostainer 480 (Lab Vision Corp.) was used for the immunostaining process by adding primary antibodies (Table 2) for one hour, followed by a 30 min incubation with Dako REAL EnVision/HRP detection system, including Rabbit/Mouse (ENV) reagent. Staining was visualized by Dako REAL DAB + Chromogen for 10 min. Washing between each step was performed with PBS-0.04%–Tween20. Slides were counterstained with Meyer’s hematoxylin and mounted in mounting medium (Aquamount, BDH, Poole, UK). Tissue known to be highly positive for studied proteins was used as a positive control, and for a negative control the specimens were processed without primary antibody. In addition, TLR immunostainings of non-neoplastic tongue tissue was performed from ten patients. Evaluation of immunostainings TLR immunopositivity in tumor cells was scored by two independent investigators (L.K.M. and J.H.) who were blinded for the clinical data (staining patterns, see Table 2 and Fig. 1). Cytoplasmic staining intensity of TLR-2, -5 and -9 was scored as 0 for negative, 1 for mild, 2 for moderate, and 3 for strongly positive. Nuclear TLR-2 staining, cytoplasmic TLR-4 staining, and nuclear membranous TLR-7 staining was scored according to the percentage of positive tumor cells as follows: 0 = no positive cells, 1 = positive cells up to 10% (low), 2 = 11–50% (moderate), 3 = 51–80% (high), and 4 = over 80% (very high). p16INK4a was classified positive when over 70% of cells were stained. Each patient was expected to have six tumor spots; for each patient the highest immunoscore was selected for further statistical analysis. Cell culture and preparation of total cell lysates Human oral tongue cancer cell line (HSC-3), received from the Institute of Dentistry, University of Helsinki, was propagated in equivalent amount of DMEM medium with 4.5 g/l glucose and Ham’s F12 Nutrient mixture supplemented with 10% fetal calf serum (FCS), penicillin and streptomycin, sodium pyruvate and hydrocortisone (Sigma–Aldrich, St. Louis, MO). Cells were lysed in Nonidet P-40 (NP-40) lysis buffer (1% NP-40, 20 mM HEPES, pH 7.5, 150 mM NaCl) supplemented with 50 mM NaF, 1 mM Na3VO4 and 1 Complete Proteinase Inhibitor Cocktail (Roche, Basel, Switzerland) at 4 °C for 30 min. Detergent-insoluble material was removed by centrifugation (16,000g at 4 °C for 15 min). Protein concentrations were measured by using the Bradford assay (Bio-Rad Laboratories, Hercules, CA, USA). Western blotting Fifty lg of total cell lysates were separated by 10% SDS–PAGE, transferred to PVDF-FL membranes (Millipore, Billerica, MA) and blocked with Odyssey blocking buffer (LI-COR, Lincoln, NE) diluted 1:1 with PBS. The membranes were incubated with rabbit antiTLR-2 (Santa Cruz Biotechnology, Inc.), rabbit anti-TLR-4 (Santa Cruz Biotechnology, Inc.) and mouse anti-actin (Sigma) IgGs, followed by Alexa Fluor 680 (Invitrogen) and IRDye 800 (LI-COR Biosciences) anti-mouse or anti-rabbit IgGs. The signal was
detected using an Odyssey Infrared Imager (LI-COR) and subsequently quantified using Odyssey software. Statistical analysis SPSS statistics 22.0 software (SPSS Inc., Chicago, IL, USA) was used. Immunoexpression scores of TLRs were compared with clinicopathological variables: size (diameter and pT-stage), invasion depth, grade, presence of occult neck metastases, and patients’ age and sex. Similarly, the scores of TLR stainings were compared with p16INK4a staining scores. TLR immunoexpression scores were evaluated in regard to disease-specific survival (DSS), overall survival (OS), and disease-free survival (DFS) time by the Kaplan–Meier estimates and the logrank test. DSS was defined as the interval between primary surgical treatment and death from OTSCC or end of follow-up, whereas for OS the end-point was death from any cause or end of follow-up. Correspondingly, the end-point for DFS time was detection of recurrent disease or end of follow-up. For survival analysis, TLR expression scores were dichotomized due to limited number of patients. Concerning scoring by cytoplasmic staining intensity the groups were: 0 = score 0 and 1, 1 = score 2 and 3, and respectively, scoring by the percentage of positive tumor cells: 0 = score 0, 1, and 2, 1 = score 3 and 4. Correlations between categorical variables and differences between staining patterns of TLRs were analyzed by Fisher’s exact test and Spearman correlation. Kaplan–Meier estimates and logrank test were used for survival analyses. A two-sided p-value of less than 0.05 was considered statistically significant. The following variables were included in a multivariate analysis (Cox regression): pT-stage, grade, presence of occult neck metastases, invasion, and TLR-5 immunoscores. Variables were selected in a backward stepwise manner, and a p-value of 0.05 was the limit for inclusion of a covariate. Also covariates that earlier have been shown to have prognostic value in OTSCC were selected. Results All TLRs examined were expressed in OTSCC tissue. Score distribution of different TLRs is shown in detail in Figs. 2 and 3. Nuclear and cytoplasmic TLR-2 and cytoplasmic TLR-4 immunoexpression were present in most of the tumors (95%, 94%, and 97%, respectively). The majority of the cases showed moderate or strong/high or very high expression. TLRs were also expressed in non-neoplastic tongue tissue, but expression was partly different compared with cancer tissue. TLR-2 positivity was observed in nucleus and in cytoplasm in both non-neoplastic and carcinoma tissue, but expression was stronger in cancer. Nuclear expression of TLR-4 was stronger in non-neoplastic tissue samples compared with OTSCC, which had so scarce positivity of TLR-4 in nucleus that it was not scored. Cytoplasmic TLR-4 positivity was seen more often in carcinoma than in benign tissue. Western blot analysis confirmed that TLR-2 and -4 were expressed in the HSC-3 tongue carcinoma cell line, and that their molecular weights corresponded with full-length proteins (Fig. 4).
Table 2 Antibodies used. Antigen
Primary antibody
Dilution
Staining pattern
TLR-2 TLR-4 TLR-5 TLR-7 TLR-9 p16INK4a
H-175, Santa Cruz Biotechnology, Inc., CA, USA, polyclonal H-80, Santa Cruz Biotechnology, Inc., CA, USA, polyclonal IMG-664A, Imgenex, San Diego, CA, USA, monoclonal IMG-581A, Imgenex, San Diego, CA, USA, polyclonal H-100, Santa Cruz Biotechnology, Inc., CA, USA, polyclonal Clone E6H4, 9511, CINtechÒ Histology Kit, Roche, Germany, monoclonal
1:50 1:50 1:200 1:300 1:100 Ready-to-use
Cytoplasmic and/or nuclear Cytoplasmic Cytoplasmic Nuclear membranous Cytoplasmic Cytoplasmic
99
L.K. Mäkinen et al. / Oral Oncology 51 (2015) 96–102 Table 3 Association of TLRs with clinicopathological variables in OTSCC patients (p-value, Spearman’s rho).
Age (years, continuous variable) Sex Grade (I–III) Tumor size (mm, continuous variable) Tumor size (pT1 6 20 mm, pT2 21–40 mm) Invasion depth (mm, continuous variable) Invasion depth (categorical, 0–4, >4 mm) Occult neck metastasis (N0/N+)a
TLR-2 (cytoplasmic)
TLR-2 (nuclear)
TLR-4
TLR-5
TLR-7
TLR-9
NS NS 0.021 NS NS 0.026 NS NS
NS NS NS NS NS NS NS NS
NS NS 0.005 NS NS 0.008 0.018 NS
NS NS 0.039 NS NS NS NS NS
NS NS NS NS NS NS NS NS
NS NS NS 0.003 NS 0.019 0.012 NS
NS, not significant. A p-value of less than 0.05 was considered statistically significant. a Lymph node positivity in pathological examination or neck metastasis during follow up without a local recurrence.
Fig. 1. Immunohistochemical staining for TLRs in oral tongue squamous cell carcinoma tissue. Mild (a), moderate (b), and strong (c) cytoplasmic TLR-2 staining showing also nuclear TLR-2 positivity. Mild, moderate, and strong cytoplasmic TLR-5 (d–f) and TLR-9 (g–i) staining. Cytoplasmic TLR-4 (j) and membranous TLR-7 staining (k). Magnification 400.
TLR-5 immunoexpression was present in 98% of the tumor samples, most tumors showing moderate or strong expression as was also seen in benign tissue. TLR-7 was expressed in 92% of the tumors and mostly at low or moderate level and positivity was nuclear membranous both in non-neoplastic and malignant tissue. TLR-9 expression was found in 96% of the tumors, mainly mild or moderate. Only six tumors showed strong TLR-9 expression and expression was cytoplasmic both in benign and malignant tissue. p16INK4a was positive in 9% (n = 6/65) of the tumors.
Both high cytoplasmic TLR-2 and TLR-4 expression correlated with deeper tumor invasion (p = 0.026 and p = 0.008, respectively, Spearman’s rho, Table 3) and higher tumor grade (p = 0.021 and p = 0.005, Spearman’s rho), whereas high TLR-5 expression correlated with lower tumor grade (p = 0.039, Spearman’s rho). High TLR-9 expression correlated with deeper tumor invasion (p = 0.019, Spearman’s rho) and larger tumor diameter (p = 0.003, Spearman’s rho). None of the TLRs studied correlated with disease recurrence. p16INK4a did not correlate with any of the
100
L.K. Mäkinen et al. / Oral Oncology 51 (2015) 96–102
Fig. 2. Score distribution (i.e. percentage of positive tumor cells) of TLRs 2, 5, and 9 (No. of patients) in oral tongue squamous cell carcinoma tissue.
Fig. 3. Score distribution (i.e. Intensity of cytoplasmic staining) of TLRs 2, 5, and 9 (No. of patients) in oral tongue squamous cell carcinoma tissue.
Fig. 4. Expression of TLR-2 and TLR-4 in HSC-3 tongue carcinoma cell line. Western blotting of total cell lysates prepared of HSC-3 cells with TLR-2 and TLR-4 antibodies, reveals that both proteins were expressed, and that their molecular weights corresponded with full-length proteins. Actin is included as a loading control. The lanes represent two biological replicate samples. mw, molecular weight; WB, Western blotting.
clinicopathological factors nor with TLR expressions. Nuclear expression of TLR-2 or membranous expression of TLR-7 did not correlate with any variables. In survival analyses, negative or mild (group 0, n = 4) TLR-5 expression predicted poor DSS (p = 0.014, log-rank test, Fig. 5) and poor OS (p = 0.005, log-rank test, data not shown), yet, multivariate analysis failed to show TLR-5 expression as an independent prognostic factor in OTSCC. Survival analyses revealed no other significant correlations. Discussion The role of TLRs in OSCC remains elusive. The present study shows that TLR-2, -4, and -9 are associates associated with the invasive potential of early-stage OTSCC tumors, and that TLR-9
correlates with advanced tumor size. Tumors with high TLR-2 or -4 expression were more often poorly differentiated, whereas strong TLR-5 expression was associated with well differentiated tongue cancer. In survival analyses negative or mild TLR-5 expression was related to worse survival. We found TLRs to be extensively expressed in tongue carcinoma tissue. This is in accordance with previous studies of TLRs in HNSCC [11,13–15,19–22]. All the studied TLRs were also expressed in nonneoplastic tongue tissue, but staining patterns were somewhat different compared with carcinoma tissue. Cytoplasmic TLR-2 expression was lower in non-neoplastic tongue tissue compared with tumors, but both benign and malignant samples showed vast nuclear positivity. A study by Ng et al. [27] demonstrated clearly higher nuclear and cytoplasmic TLR-2 expression in chronic inflammatory cells, in endothelial cells in the microenvironment of OSCC, and in dysplasia as compared with hyperplastic epithelium, suggesting that immune surveillance is activated against altered cells. In our series, TLR-4 expression was widely present in both malignant and benign tissues, but expression was mainly nuclear in benign and cytoplasmic in cancer tissue. Szczepanski et al. [15] also found cytoplasmic TLR-4 expression in normal tissue, but it was weaker than in laryngeal or in oral squamous cell carcinoma tissue. Sun et al. [16] also showed TLR-4 expression, although weak, in normal mucosa adjacent to OSCC. Both nonneoplastic and malignant tissues in the present series had strong cytoplasmic TLR-5 expression. On the other hand, Kauppila et al. [21] found increased cytoplasmic TLR-5 expression in OTSCC compared with normal epithelium. TLR-7 staining was nuclear and/or nuclear membranous both in non-neoplastic tissue and in tongue cancer. To our knowledge, there are no previous studies on TLR-7 expression in OSCC tissue samples or normal oral mucosa. In our series, TLR-9 was mildly or moderately positive in cytoplasm of both benign and malignant tissues, whereas Min et al. [22] showed, that TLR-9 expression was higher in OSCC tissue than in paired adjacent normal tissue. p16INK4a was positive only in a minor part (9%) of the tumors, which is in accordance with previous studies of p16INK4a in OSCC [28–30], and no correlation with clinicopathological variables nor with TLR expressions was found. It is worth noting that even though p16 is used as a surrogate marker for HPV in oropharyngeal carcinoma, it does not strictly correlate with HPV positivity [23]. We showed that TLR-2 and -4 are expressed in an OTSCC cell line at their expected molecular weights of 90 kDa and 91 kDa, respectively. This demonstrates that these TLRs, that often are reported to be expressed on the cell surface [6], are present in this tongue cancer cell-line in their full-length forms even though they are cytoplasmic. Szczepanski et al. [15] also studied TLR-4 in laryngeal and oral SCC tumors as well as in HNSCC cell lines and showed, that TLR-4 was expressed in all tumors and cell lines. On the other hand, Pries et al. [13] measured TLRs 1–10 in eight different HNSCC cell lines, but TLR-3 was the only TLR expressed. We showed that, high cytoplasmic expression of TLR-2, -4, and 9 correlates with deeper tumor invasion. A recent study of Yang et al. [31] of TLR-2 in gastric carcinoma showed that increased TLR-2 expression notably promoted the transcription of genes related to angiogenesis and invasion, and that the invasive capacity of the studied gastric cancer cells was strikingly advanced by TLR-2 stimulation in Transwell invasion assay. Immunohistochemical TLR-2 expression also correlated with the occurrence of metastases in gastric cancer. However, Park et al. [12] could not find TLR-2 or TLR-5 activation affecting the invasion of OSCC cells. Szczepanski et al. [15] demonstrated that exposure of HNSCC cell lines to TLR-4 ligand LPS enhanced proliferation of tumor cells and that TLR-4 activation protected tumor cells from lysis mediated by NK-92 cells. They concluded that TLR-4 ligation on tumor cells supports HNSCC progression. Therefore, our finding of TLR-4
L.K. Mäkinen et al. / Oral Oncology 51 (2015) 96–102
101
Fig. 5. Low TLR-5 expression correlates with poor survival in early-stage oral tongue squamous cell carcinoma. For survival analysis TLR-5 expression scores were dichotomized into two groups as follows: none and mild = 0, moderate and strong = 1. Patients in group 0 (n = 4) had worse disease specific survival than patients in group 1 (n = 59) (p = 0.014, log-rank test).
correlating with tumor invasion is in line with this previous study. Supporting our observation of TLR-9 expression correlating with tumor invasion, a recent study by Ruan et al. [18] showed activation of TLR-9 to induce migration and invasion of oral cancer cells. It would be interesting to investigate whether the expression of TLR-2, -4, and -9 is strongest especially in the invasive front of the tumor, but this should be evaluated from whole tissue sections and not from tissue microarray blocks, which were used in the present study. We found that strong cytoplasmic TLR-2 and high TLR-4 expression correlated with poor differentiation grade tumors, whereas strong TLR-5 expression was seen in well-differentiated tumors. Conversely, Szczepanski et al. [15] reported TLR-4 expression intensity in laryngeal squamous cell carcinoma to correlate inversely with tumor grade; Sun et al. [16] made a similar finding in OSCC. Chuang et al. [14] found that high TLR-3 expression correlated with poorly differentiated OSCC. It is important to note that grade has not been shown to reliably predict prognosis in OSCC, mainly due to the subjective nature of its assessment [32]. High TLR-9 expression correlated with advanced tumor size, which is in accordance with a previous study of TLR-9 in OSCC by Min et al. [22]. They also demonstrated in vitro, that stimulation of OSCC cells with TLR-9 agonist CpG-ODN significantly increased tumor proliferation. Various treatment modalities may pose a confounding factor for retrospective outcome analyses. All patients in this series, however, were treated according to the Finnish national treatment guidelines for Head and Neck cancer. For example, all patients with occult neck metastases also received postoperative radiotherapy. The present survival analyses showed that patients with none or mild TLR-5 expression will have worse DSS than patients with moderate or strong TLR-5 expression. Our finding differs from the report of Kauppila et al. [21], who showed strong TLR-5 expression to be an independent predictor of OTSCC mortality. One explanation could be that their study on whole tissue sections included tongue carcinomas of different stages, whereas our study using tissue microarray technique focused on clinically early-stage
disease. More importantly, the present statistical analysis showed that there were no differences between pathological Stages I and II (pN0) and pathological Stage III-IVA (pN+) patients when compared with TLR expression scores. Therefore, the role of TLRs in predicting survival of HNSCC remains inconclusive. Conclusions All TLRs examined showed high expression in tissue samples from early-stage tongue carcinoma. TLR-2, -4, and -9 associated with invasive growth of the tumor. Conflict of interest statement None declared. Acknowledgements We thank Päivi Peltokangas and Elina Aspiala for excellent technical assistance and M.Sc. Timo Pessi for statistical advice. This study had financial support from the National Graduate School of Clinical Investigation, the University of Helsinki, Ida Montin Foundation (LM), and The Sigrid Juselius Foundation (CH). References [1] Bello IO, Soini Y, Salo T. Prognostic evaluation of oral tongue cancer: means, markers and perspectives (I). Oral Oncol 2010;46:630–5. [2] Bello IO, Soini Y, Salo T. Prognostic evaluation of oral tongue cancer: means, markers and perspectives (II). Oral Oncol 2010;46:636–43. [3] Makitie AA, Koivunen P, Keski-Santti H, Tornwall J, Pukkila M, Laranne J, et al. Oral tongue carcinoma and its treatment in Finland. Eur Arch Otorhinolaryngol 2007;264:263–7. [4] Couzin-Frankel J. Breakthrough of the year 2013. Cancer immunotherapy. Science 2013;2013(342):1432–3. [5] Duray A, Demoulin S, Hubert P, Delvenne P, Saussez S. Immune suppression in head and neck cancers: a review. Clin Dev Immunol 2010;2010:701657. [6] Ioannou S, Voulgarelis M. Toll-like receptors, tissue injury, and tumourigenesis. Mediators Inflamm 2010;2010. http://dx.doi.org/10.1155/ 2010/581837. Epub 2010 Sep 14.
102
L.K. Mäkinen et al. / Oral Oncology 51 (2015) 96–102
[7] Miller LS, Modlin RL. Toll-like receptors in the skin. Semin Immunopathol 2007;29:15–26. [8] Yu L, Wang L, Chen S. Dual character of toll-like receptor signaling: pro-tumorigenic effects and anti-tumor functions. Biochim Biophys Acta 2013;1835:144–54. [9] 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. [10] Pradere JP, Dapito DH, Schwabe RF. The yin and yang of toll-like receptors in cancer. Oncogene 2013. [11] Rydberg C, Mansson A, Uddman R, Riesbeck K, Cardell LO. Toll-like receptor agonists induce inflammation and cell death in a model of head and neck squamous cell carcinomas. Immunology 2009;128:e600–11. [12] Park JH, Yoon HE, Jeon DI, Ahn SG, Yoon JH. Activation of TLR2 and TLR5 did not affect tumor progression of an oral squamous cell carcinoma, YD-10B cells. J Oral Pathol Med 2010;39:781–5. [13] Pries R, Hogrefe L, Xie L, Frenzel H, Brocks C, Ditz C, et al. Induction of c-mycdependent cell proliferation through toll-like receptor 3 in head and neck cancer. Int J Mol Med 2008;21:209–15. [14] Chuang HC, Huang CC, Chien CY, Chuang JH. Toll-like receptor 3-mediated tumor invasion in head and neck cancer. Oral Oncol 2012;48:226–32. [15] Szczepanski MJ, Czystowska M, Szajnik M, Harasymczuk M, Boyiadzis M, KrukZagajewska A, et al. Triggering of toll-like receptor 4 expressed on human head and neck squamous cell carcinoma promotes tumor development and protects the tumor from immune attack. Cancer Res 2009;69:3105–13. [16] Sun Z, Luo Q, Ye D, Chen W, Chen F. Role of toll-like receptor 4 on the immune escape of human oral squamous cell carcinoma and resistance of cisplatininduced apoptosis. Mol Cancer 2012;11:33,4598–11-33. [17] Ahn MY, Kwon SM, Cheong HH, Park JH, Lee J, Min SK, et al. Toll-like receptor 7 agonist, imiquimod, inhibits oral squamous carcinoma cells through apoptosis and necrosis. J Oral Pathol Med 2012;41:540–6. [18] Ruan M, Zhang Z, Li S, Yan M, Liu S, Yang W, et al. Activation of toll-like receptor-9 promotes cellular migration via up-regulating MMP-2 expression in oral squamous cell carcinoma. PLoS One 2014;9:e92748. [19] Szczepanski M, Stelmachowska M, Stryczynski L, Golusinski W, Samara H, Mozer-Lisewska I, et al. Assessment of expression of toll-like receptors 2, 3 and 4 in laryngeal carcinoma. Eur Arch Otorhinolaryngol 2007;264:525–30. [20] Luo Q, Hu S, Yan M, Sun Z, Chen W, Chen F. Activation of toll-like receptor 3 induces apoptosis of oral squamous carcinoma cells in vitro and in vivo. Int J Biochem Cell Biol 2012;44:1266–75.
[21] 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. [22] Min R, Zun Z, Siyi L, Wenjun Y, Lizheng W, Chenping Z. Increased expression of toll-like receptor-9 has close relation with tumour cell proliferation in oral squamous cell carcinoma. Arch Oral Biol 2011;56:877–84. [23] Mirghani H, Amen F, Moreau F, Guigay J, Ferchiou M, Melkane AE, et al. Human papilloma virus testing in oropharyngeal squamous cell carcinoma: what the clinician should know. Oral Oncol 2014;50:1–9. [24] Keski-Santti H, Atula T, Tornwall J, Koivunen P, Makitie A. Elective neck treatment versus observation in patients with T1/T2 N0 squamous cell carcinoma of oral tongue. Oral Oncol 2006;42:96–101. [25] Makinen LK, Hayry V, Hagstrom J, Sorsa T, Passador-Santos F, Keski-Santti H, et al. Matrix metalloproteinase-7 and matrix metalloproteinase-25 in oral tongue squamous cell carcinoma. Head Neck 2013. [26] Makinen LK, Hayry V, Atula T, Haglund C, Keski-Santti H, Leivo I, et al. Prognostic significance of matrix metalloproteinase-2, -8, -9, and -13 in oral tongue cancer. J Oral Pathol Med 2012;41:394–9. [27] Ng LK, Rich AM, Hussaini HM, Thomson WM, Fisher AL, Horne LS, et al. Tolllike receptor 2 is present in the microenvironment of oral squamous cell carcinoma. Br J Cancer 2011;104:460–3. [28] Garcia-de Marcos JA, Perez-Zafrilla B, Arriaga A, Arroyo-Rodriguez S, Poblet E. Human papillomavirus in carcinomas of the tongue: clinical and prognostic implications. Int J Oral Maxillofac Surg 2013. [29] Dunkel J, Vaittinen S, Grenman R, Kinnunen I, Irjala H. Prognostic markers in stage I oral cavity squamous cell carcinoma. Laryngoscope 2013;123: 2435–41. [30] Deng Z, Hasegawa M, Aoki K, Matayoshi S, Kiyuna A, Yamashita Y, et al. A comprehensive evaluation of human papillomavirus positive status and p16INK4a overexpression as a prognostic biomarker in head and neck squamous cell carcinoma. Int J Oncol 2014;45:67–76. [31] Yang H, Wang B, Yan J, Wang T, Zhou XN, Wen HY, et al. Toll-like receptor 2 promotes invasion by SGC-7901 human gastric carcinoma cells and is associated with gastric carcinoma metastasis. Ann Clin Lab Sci 2014;44: 158–66. [32] Woolgar JA. Histopathological prognosticators in oral and oropharyngeal squamous cell carcinoma. Oral Oncol 2006;42:229–39.
Contents lists available at ScienceDirect
Oral Oncology journal homepage: www.elsevier.com/locate/oraloncology
Predictive role of toll-like receptors 2, 4, and 9 in oral tongue squamous cell carcinoma Laura K. Mäkinen a,⇑, Timo Atula a, Valtteri Häyry a, Lauri Jouhi a, Neeta Datta b, Sanna Lehtonen b, Abdirisak Ahmed c, Antti A. Mäkitie a, Caj Haglund d,e,1, Jaana Hagström b,f,1 a Department of Otorhinolaryngology – Head and Neck Surgery, Helsinki University Central Hospital and University of Helsinki, P.O. Box 220, Haartmaninkatu 4E, FI-00029 HUS, Helsinki, Finland b Department of Pathology, Haartman Institute, University of Helsinki, P.O. Box 21, Haartmaninkatu 3, FI-00014 University of Helsinki, Helsinki, Finland c Institute of Dentistry, Biomedicum 1, University of Helsinki, P.O. Box 63, Haartmaninkatu 8, FI-00014 University of Helsinki, Helsinki, Finland d Department of Surgery, Helsinki University Central Hospital, Haartmaninkatu 4, P.O. Box 440, FI-00029 HUS, Helsinki, Finland e Research Programs Unit, Translational Cancer Biology, University of Helsinki, P.O. Box 440, FI-00014 University of Helsinki, Helsinki, Finland f Department of Pathology, HUSLAB, Helsinki University Central Hospital, P.O. Box 400, Haartmaninkatu 3, FI-00029 HUS, Helsinki, Finland
a r t i c l e
i n f o
Article history: Received 13 June 2014 Received in revised form 12 August 2014 Accepted 24 August 2014 Available online 26 September 2014 Keywords: Tongue carcinoma Oral cancer Immunohistochemistry Prognosis Recurrence Survival Invasion Metastasis p16 protein
s u m m a r y Objectives: The clinical behavior of oral tongue squamous cell carcinoma (OTSCC) can be unpredictable, and even small tumors may behave aggressively. Toll-like receptors (TLRs) are pattern-recognition molecules involved in innate immunity, and they are also expressed in many types of cancer. TLRs play an apparently pivotal role in some cancers related to tumor progression and, conversely, cancer inhibition, however their role in oral cancer is unclear. We therefore studied the expression of TLR-2, -4, -5, -7, and -9 in early-stage OTSCC. Materials and methods: Tissue microarray technique and immunohistochemistry was employed to define the expression of TLRs from the tumors of 73 consecutive patients with Stage I-II OTSCC. Immunoexpression scores were compared with patient and tumor related characteristics and survival. Results: All TLRs were expressed in OTSCC tissue. High/strong TLR-2, -4, and -9 expression correlated with deeper tumor invasion. Cytoplasmic TLR-2 and -4 also correlated significantly with higher tumor grade, whereas high TLR-5 expression associated with lower tumor grade. High expression of TLR-9 correlated with advanced tumor size. Negative or mild TLR-5 expression predicted poor disease-specific survival. Conclusion: All the studied TLRs showed high expression in early-stage OTSCC. More importantly, TLR-2, -4, and -9 seemed to predict invasive tumor growth. Ó 2014 Elsevier Ltd. All rights reserved.
Introduction The behavior of early-stage oral tongue squamous cell carcinoma (OTSCC) can be unpredictable despite improvements in
Abbreviations: DFS, disease-free survival; DSS, disease-specific survival; HNSCC, head and neck squamous cell carcinoma; HPV, human papilloma virus; LPS, lipopolysaccharide; OS, overall survival; OSCC, oral squamous cell carcinoma; OTSCC, oral tongue squamous cell carcinoma; TLR, toll-like receptor. ⇑ Corresponding author. Tel.: +358 40 5678687; fax: +358 9 471 75010. E-mail addresses: laura.k.makinen@helsinki.fi (L.K. Mäkinen), timo.atula@hus.fi (T. Atula), valtteri.hayry@fimnet.fi (V. Häyry), lauri.jouhi@helsinki.fi (L. Jouhi), neeta.datta@helsinki.fi (N. Datta), sanna.h.lehtonen@helsinki.fi (S. Lehtonen), abdirisak.ahmed@helsinki.fi (A. Ahmed), antti.makitie@helsinki.fi (A.A. Mäkitie), caj.haglund@hus.fi (C. Haglund), jaana.hagstrom@hus.fi (J. Hagström). 1 Equal contribution. http://dx.doi.org/10.1016/j.oraloncology.2014.08.017 1368-8375/Ó 2014 Elsevier Ltd. All rights reserved.
diagnostic methods and treatment modalities [1,2]. Even small tumors may behave aggressively. The 5-year survival rate of early-stage (I–II) OTSCC is still modest [3]. To adjust the treatment more accurately, improved knowledge of the underlying mechanisms behind aggressiveness of the disease as well as new predictive markers are needed. Intense research studies are currently addressing the relevance of innate immunity response in cancer initiation and progression. Cancer immunotherapy is expected to be the fourth weapon against cancer along with surgery, radiation, and chemotherapy [4]. It is well known that environmental carcinogens (tobacco, alcohol) are major contributors to the development of head and neck squamous cell carcinomas whereas tumor progression could be partly due to a failure of the innate immune response against cancer [5].
97
L.K. Mäkinen et al. / Oral Oncology 51 (2015) 96–102
Toll like receptors (TLRs) are a major class of pattern-recognition receptors that are expressed by cells of the humane immune system and epithelial cells located near host-environment boundary. In addition, they are also present in many types of cancer. Ten human TLRs have been identified, each recognizing a specific microbial component. There are two kinds of ligands for TLRs: exogenous pathogen associated molecular patterns (PAMPs), which are components of microbes, and endogenous damageassociated molecular patterns (DAMPs) released from injured or inflamed tissues [6]. TLR-2 and TLR-4 recognize lipopolysaccharide (LPS, a membrane component of Gram-negative bacteria), TLR-5 recognizes bacterial flagellin, TLR-7 recognizes double or single stranded RNA, and TLR-9 recognizes bacterial DNA [7,8]. TLRs detecting bacterial LPS and lipoproteins are located on the cell surface (TLR-1, TLR-2, TLR-4, TLR-5, and TLR-6), whereas TLR-3, -7, -8, and -9 that mainly recognize viral RNA and bacterial DNA are located in endosomes and lysosomes, where these materials are processed [6]. Thus, TLRs trigger signals activate innate and adaptive immune responses [6]. The actual role of TLRs in tumorigenesis remains unknown, and it is related to cancer progression as well as inhibition [8–10]. TLR-mediated tumor progression is considered to result from the activation of TLRs in tumor cells, whereas tumor regression is thought to derive from the activation of host immune responses [8]. The function of each TLR is complex and variable and is affected by multiple factors. Little is known about TLRs in head and neck squamous cell carcinoma (HNSCC). Various HNSCCs express TLR-2, -3, -4, -5, -7, and -9 both in vitro [11–18] and in vivo [11,13–15,19–22]. To further clarify the role of TLRs in early-stage oral cancer we studied TLR-2, -4, -5, -7, and -9 in small OTSCCs. The surrogate marker for human papilloma virus (HPV), p16, is widely utilized in the evaluation of oropharyngeal carcinomas [23]. Thus p16INK4a staining of tongue carcinoma tumors was performed here, and findings compared with TLR expression.
Material and methods Patients The present study assesses a series of early-stage OTSCC patients with tumors clinically defined as T1N0M0 or T2N0M0 which were treated at the Helsinki University Central Hospital between 1992 and 2002. The patient material has been described in more detail previously [24,25]. Demographic data of the material is described in Table 1. Tumor samples from 73 patients were available for immunohistochemistry (36 males and 37 females, median age 59 years, range 23–95). Thirty-five tumors (48%) were clinically classified as T1 and 38 (52%) as T2. All patients had been treated with curative intent including surgical resection of the primary tumor. An experienced head and neck pathologist re-evaluated all the original histological tumor specimens. According to histopathological classification, 52 tumors (71%) were classified as pT1 and 21 (29%) as pT2. Tumor invasion depth was measured from the level of the proximate normal mucosal surface. All patients in this study were treated according to the Finnish national guidelines for the treatment of Head and Neck cancer. Postoperative radiation therapy was given only to patients with deeply invasive tumors, and to those with neck metastases in neck dissection specimens. Consequently, 31 patients received no further primary treatment for the neck, 41 patients underwent elective neck dissection, and one patient received radiotherapy without surgery. Thus, radiotherapy had no effect on evaluation
Table 1 Demographic and clinicopathological features of 73 patients with oral tongue squamous cell carcinoma.
*
Clinicopathological variable
No. of patients (%)
Age, years 660 >60 Range Median
40 (55) 33 (45) 23–95 59
Sex Male Female
36 (49) 37 (51)
Grade I II III
24 (33) 35 (48) 14 (19)
Clinical T stage (mm) cT1 (620) cT2 (21–40)
35 (48) 38 (52)
Pathological T stage (mm) pT1 (620) pT2 (21–40)
52 (71) 21 (29)
Pathological node positivitya pN0 pN+
26 (36) 15 (21)
Pathological stage I II III IV
46 (63) 12 (16) 12 (16) 3 (4)
Invasion depth (mm) 64 >4
29 (40) 44 (60)
41/73 (56%) of the patients had elective neck dissection.
of tumor characteristics, nor on the evaluation of occult metastases, except possibly in one patient. Of all patients, 34 received post-operative radiotherapy, including the neck in 33 patients. Twenty-four patients (33%) had occult neck metastases, i.e. lymph node metastases in the elective neck dissection specimen (n = 15), or neck metastasis during follow up without failure at the primary site (n = 9). During follow-up, ten patients developed local recurrence. Only two patients were diagnosed with distant metastases, both after locoregional recurrence. All patients, but one, had a minimum follow-up time of five years or until death. Median follow-up time was 7.6 years (range, 0.3–17.2), during which 19 patients died of tongue cancer and 22 of other causes. Dates and causes of death were provided by the national agency of population statistics, Statistics Finland. The study design was approved by the institutional Research Ethics Board. Tissue array blocks Tissue microarray (TMA) technique was used as previously described [26]. Representative tumor tissue was lacking in seven patients for TLR-2, in six patients for TLR-4 and -9, in eight patients for TLR-7, and in ten patients for TLR-5 and p16INK4a evaluation. Immunohistochemistry Tissue slides as well as TMA slides were cut into 4–5 lm thick sections. Deparaffinization was done in xylene and rehydration with graded alcohol series. Slides were treated in a PreTreatment-module (Lab Vision Corp., UK Ltd, UK) in Tris–HCl buffer (pH 8.5) at 98 °C for 20 min and with 0.3% Dako REAL Peroxidase-Blocking Solution for 5 min to block endogenous
98
L.K. Mäkinen et al. / Oral Oncology 51 (2015) 96–102
peroxidase. Autostainer 480 (Lab Vision Corp.) was used for the immunostaining process by adding primary antibodies (Table 2) for one hour, followed by a 30 min incubation with Dako REAL EnVision/HRP detection system, including Rabbit/Mouse (ENV) reagent. Staining was visualized by Dako REAL DAB + Chromogen for 10 min. Washing between each step was performed with PBS-0.04%–Tween20. Slides were counterstained with Meyer’s hematoxylin and mounted in mounting medium (Aquamount, BDH, Poole, UK). Tissue known to be highly positive for studied proteins was used as a positive control, and for a negative control the specimens were processed without primary antibody. In addition, TLR immunostainings of non-neoplastic tongue tissue was performed from ten patients. Evaluation of immunostainings TLR immunopositivity in tumor cells was scored by two independent investigators (L.K.M. and J.H.) who were blinded for the clinical data (staining patterns, see Table 2 and Fig. 1). Cytoplasmic staining intensity of TLR-2, -5 and -9 was scored as 0 for negative, 1 for mild, 2 for moderate, and 3 for strongly positive. Nuclear TLR-2 staining, cytoplasmic TLR-4 staining, and nuclear membranous TLR-7 staining was scored according to the percentage of positive tumor cells as follows: 0 = no positive cells, 1 = positive cells up to 10% (low), 2 = 11–50% (moderate), 3 = 51–80% (high), and 4 = over 80% (very high). p16INK4a was classified positive when over 70% of cells were stained. Each patient was expected to have six tumor spots; for each patient the highest immunoscore was selected for further statistical analysis. Cell culture and preparation of total cell lysates Human oral tongue cancer cell line (HSC-3), received from the Institute of Dentistry, University of Helsinki, was propagated in equivalent amount of DMEM medium with 4.5 g/l glucose and Ham’s F12 Nutrient mixture supplemented with 10% fetal calf serum (FCS), penicillin and streptomycin, sodium pyruvate and hydrocortisone (Sigma–Aldrich, St. Louis, MO). Cells were lysed in Nonidet P-40 (NP-40) lysis buffer (1% NP-40, 20 mM HEPES, pH 7.5, 150 mM NaCl) supplemented with 50 mM NaF, 1 mM Na3VO4 and 1 Complete Proteinase Inhibitor Cocktail (Roche, Basel, Switzerland) at 4 °C for 30 min. Detergent-insoluble material was removed by centrifugation (16,000g at 4 °C for 15 min). Protein concentrations were measured by using the Bradford assay (Bio-Rad Laboratories, Hercules, CA, USA). Western blotting Fifty lg of total cell lysates were separated by 10% SDS–PAGE, transferred to PVDF-FL membranes (Millipore, Billerica, MA) and blocked with Odyssey blocking buffer (LI-COR, Lincoln, NE) diluted 1:1 with PBS. The membranes were incubated with rabbit antiTLR-2 (Santa Cruz Biotechnology, Inc.), rabbit anti-TLR-4 (Santa Cruz Biotechnology, Inc.) and mouse anti-actin (Sigma) IgGs, followed by Alexa Fluor 680 (Invitrogen) and IRDye 800 (LI-COR Biosciences) anti-mouse or anti-rabbit IgGs. The signal was
detected using an Odyssey Infrared Imager (LI-COR) and subsequently quantified using Odyssey software. Statistical analysis SPSS statistics 22.0 software (SPSS Inc., Chicago, IL, USA) was used. Immunoexpression scores of TLRs were compared with clinicopathological variables: size (diameter and pT-stage), invasion depth, grade, presence of occult neck metastases, and patients’ age and sex. Similarly, the scores of TLR stainings were compared with p16INK4a staining scores. TLR immunoexpression scores were evaluated in regard to disease-specific survival (DSS), overall survival (OS), and disease-free survival (DFS) time by the Kaplan–Meier estimates and the logrank test. DSS was defined as the interval between primary surgical treatment and death from OTSCC or end of follow-up, whereas for OS the end-point was death from any cause or end of follow-up. Correspondingly, the end-point for DFS time was detection of recurrent disease or end of follow-up. For survival analysis, TLR expression scores were dichotomized due to limited number of patients. Concerning scoring by cytoplasmic staining intensity the groups were: 0 = score 0 and 1, 1 = score 2 and 3, and respectively, scoring by the percentage of positive tumor cells: 0 = score 0, 1, and 2, 1 = score 3 and 4. Correlations between categorical variables and differences between staining patterns of TLRs were analyzed by Fisher’s exact test and Spearman correlation. Kaplan–Meier estimates and logrank test were used for survival analyses. A two-sided p-value of less than 0.05 was considered statistically significant. The following variables were included in a multivariate analysis (Cox regression): pT-stage, grade, presence of occult neck metastases, invasion, and TLR-5 immunoscores. Variables were selected in a backward stepwise manner, and a p-value of 0.05 was the limit for inclusion of a covariate. Also covariates that earlier have been shown to have prognostic value in OTSCC were selected. Results All TLRs examined were expressed in OTSCC tissue. Score distribution of different TLRs is shown in detail in Figs. 2 and 3. Nuclear and cytoplasmic TLR-2 and cytoplasmic TLR-4 immunoexpression were present in most of the tumors (95%, 94%, and 97%, respectively). The majority of the cases showed moderate or strong/high or very high expression. TLRs were also expressed in non-neoplastic tongue tissue, but expression was partly different compared with cancer tissue. TLR-2 positivity was observed in nucleus and in cytoplasm in both non-neoplastic and carcinoma tissue, but expression was stronger in cancer. Nuclear expression of TLR-4 was stronger in non-neoplastic tissue samples compared with OTSCC, which had so scarce positivity of TLR-4 in nucleus that it was not scored. Cytoplasmic TLR-4 positivity was seen more often in carcinoma than in benign tissue. Western blot analysis confirmed that TLR-2 and -4 were expressed in the HSC-3 tongue carcinoma cell line, and that their molecular weights corresponded with full-length proteins (Fig. 4).
Table 2 Antibodies used. Antigen
Primary antibody
Dilution
Staining pattern
TLR-2 TLR-4 TLR-5 TLR-7 TLR-9 p16INK4a
H-175, Santa Cruz Biotechnology, Inc., CA, USA, polyclonal H-80, Santa Cruz Biotechnology, Inc., CA, USA, polyclonal IMG-664A, Imgenex, San Diego, CA, USA, monoclonal IMG-581A, Imgenex, San Diego, CA, USA, polyclonal H-100, Santa Cruz Biotechnology, Inc., CA, USA, polyclonal Clone E6H4, 9511, CINtechÒ Histology Kit, Roche, Germany, monoclonal
1:50 1:50 1:200 1:300 1:100 Ready-to-use
Cytoplasmic and/or nuclear Cytoplasmic Cytoplasmic Nuclear membranous Cytoplasmic Cytoplasmic
99
L.K. Mäkinen et al. / Oral Oncology 51 (2015) 96–102 Table 3 Association of TLRs with clinicopathological variables in OTSCC patients (p-value, Spearman’s rho).
Age (years, continuous variable) Sex Grade (I–III) Tumor size (mm, continuous variable) Tumor size (pT1 6 20 mm, pT2 21–40 mm) Invasion depth (mm, continuous variable) Invasion depth (categorical, 0–4, >4 mm) Occult neck metastasis (N0/N+)a
TLR-2 (cytoplasmic)
TLR-2 (nuclear)
TLR-4
TLR-5
TLR-7
TLR-9
NS NS 0.021 NS NS 0.026 NS NS
NS NS NS NS NS NS NS NS
NS NS 0.005 NS NS 0.008 0.018 NS
NS NS 0.039 NS NS NS NS NS
NS NS NS NS NS NS NS NS
NS NS NS 0.003 NS 0.019 0.012 NS
NS, not significant. A p-value of less than 0.05 was considered statistically significant. a Lymph node positivity in pathological examination or neck metastasis during follow up without a local recurrence.
Fig. 1. Immunohistochemical staining for TLRs in oral tongue squamous cell carcinoma tissue. Mild (a), moderate (b), and strong (c) cytoplasmic TLR-2 staining showing also nuclear TLR-2 positivity. Mild, moderate, and strong cytoplasmic TLR-5 (d–f) and TLR-9 (g–i) staining. Cytoplasmic TLR-4 (j) and membranous TLR-7 staining (k). Magnification 400.
TLR-5 immunoexpression was present in 98% of the tumor samples, most tumors showing moderate or strong expression as was also seen in benign tissue. TLR-7 was expressed in 92% of the tumors and mostly at low or moderate level and positivity was nuclear membranous both in non-neoplastic and malignant tissue. TLR-9 expression was found in 96% of the tumors, mainly mild or moderate. Only six tumors showed strong TLR-9 expression and expression was cytoplasmic both in benign and malignant tissue. p16INK4a was positive in 9% (n = 6/65) of the tumors.
Both high cytoplasmic TLR-2 and TLR-4 expression correlated with deeper tumor invasion (p = 0.026 and p = 0.008, respectively, Spearman’s rho, Table 3) and higher tumor grade (p = 0.021 and p = 0.005, Spearman’s rho), whereas high TLR-5 expression correlated with lower tumor grade (p = 0.039, Spearman’s rho). High TLR-9 expression correlated with deeper tumor invasion (p = 0.019, Spearman’s rho) and larger tumor diameter (p = 0.003, Spearman’s rho). None of the TLRs studied correlated with disease recurrence. p16INK4a did not correlate with any of the
100
L.K. Mäkinen et al. / Oral Oncology 51 (2015) 96–102
Fig. 2. Score distribution (i.e. percentage of positive tumor cells) of TLRs 2, 5, and 9 (No. of patients) in oral tongue squamous cell carcinoma tissue.
Fig. 3. Score distribution (i.e. Intensity of cytoplasmic staining) of TLRs 2, 5, and 9 (No. of patients) in oral tongue squamous cell carcinoma tissue.
Fig. 4. Expression of TLR-2 and TLR-4 in HSC-3 tongue carcinoma cell line. Western blotting of total cell lysates prepared of HSC-3 cells with TLR-2 and TLR-4 antibodies, reveals that both proteins were expressed, and that their molecular weights corresponded with full-length proteins. Actin is included as a loading control. The lanes represent two biological replicate samples. mw, molecular weight; WB, Western blotting.
clinicopathological factors nor with TLR expressions. Nuclear expression of TLR-2 or membranous expression of TLR-7 did not correlate with any variables. In survival analyses, negative or mild (group 0, n = 4) TLR-5 expression predicted poor DSS (p = 0.014, log-rank test, Fig. 5) and poor OS (p = 0.005, log-rank test, data not shown), yet, multivariate analysis failed to show TLR-5 expression as an independent prognostic factor in OTSCC. Survival analyses revealed no other significant correlations. Discussion The role of TLRs in OSCC remains elusive. The present study shows that TLR-2, -4, and -9 are associates associated with the invasive potential of early-stage OTSCC tumors, and that TLR-9
correlates with advanced tumor size. Tumors with high TLR-2 or -4 expression were more often poorly differentiated, whereas strong TLR-5 expression was associated with well differentiated tongue cancer. In survival analyses negative or mild TLR-5 expression was related to worse survival. We found TLRs to be extensively expressed in tongue carcinoma tissue. This is in accordance with previous studies of TLRs in HNSCC [11,13–15,19–22]. All the studied TLRs were also expressed in nonneoplastic tongue tissue, but staining patterns were somewhat different compared with carcinoma tissue. Cytoplasmic TLR-2 expression was lower in non-neoplastic tongue tissue compared with tumors, but both benign and malignant samples showed vast nuclear positivity. A study by Ng et al. [27] demonstrated clearly higher nuclear and cytoplasmic TLR-2 expression in chronic inflammatory cells, in endothelial cells in the microenvironment of OSCC, and in dysplasia as compared with hyperplastic epithelium, suggesting that immune surveillance is activated against altered cells. In our series, TLR-4 expression was widely present in both malignant and benign tissues, but expression was mainly nuclear in benign and cytoplasmic in cancer tissue. Szczepanski et al. [15] also found cytoplasmic TLR-4 expression in normal tissue, but it was weaker than in laryngeal or in oral squamous cell carcinoma tissue. Sun et al. [16] also showed TLR-4 expression, although weak, in normal mucosa adjacent to OSCC. Both nonneoplastic and malignant tissues in the present series had strong cytoplasmic TLR-5 expression. On the other hand, Kauppila et al. [21] found increased cytoplasmic TLR-5 expression in OTSCC compared with normal epithelium. TLR-7 staining was nuclear and/or nuclear membranous both in non-neoplastic tissue and in tongue cancer. To our knowledge, there are no previous studies on TLR-7 expression in OSCC tissue samples or normal oral mucosa. In our series, TLR-9 was mildly or moderately positive in cytoplasm of both benign and malignant tissues, whereas Min et al. [22] showed, that TLR-9 expression was higher in OSCC tissue than in paired adjacent normal tissue. p16INK4a was positive only in a minor part (9%) of the tumors, which is in accordance with previous studies of p16INK4a in OSCC [28–30], and no correlation with clinicopathological variables nor with TLR expressions was found. It is worth noting that even though p16 is used as a surrogate marker for HPV in oropharyngeal carcinoma, it does not strictly correlate with HPV positivity [23]. We showed that TLR-2 and -4 are expressed in an OTSCC cell line at their expected molecular weights of 90 kDa and 91 kDa, respectively. This demonstrates that these TLRs, that often are reported to be expressed on the cell surface [6], are present in this tongue cancer cell-line in their full-length forms even though they are cytoplasmic. Szczepanski et al. [15] also studied TLR-4 in laryngeal and oral SCC tumors as well as in HNSCC cell lines and showed, that TLR-4 was expressed in all tumors and cell lines. On the other hand, Pries et al. [13] measured TLRs 1–10 in eight different HNSCC cell lines, but TLR-3 was the only TLR expressed. We showed that, high cytoplasmic expression of TLR-2, -4, and 9 correlates with deeper tumor invasion. A recent study of Yang et al. [31] of TLR-2 in gastric carcinoma showed that increased TLR-2 expression notably promoted the transcription of genes related to angiogenesis and invasion, and that the invasive capacity of the studied gastric cancer cells was strikingly advanced by TLR-2 stimulation in Transwell invasion assay. Immunohistochemical TLR-2 expression also correlated with the occurrence of metastases in gastric cancer. However, Park et al. [12] could not find TLR-2 or TLR-5 activation affecting the invasion of OSCC cells. Szczepanski et al. [15] demonstrated that exposure of HNSCC cell lines to TLR-4 ligand LPS enhanced proliferation of tumor cells and that TLR-4 activation protected tumor cells from lysis mediated by NK-92 cells. They concluded that TLR-4 ligation on tumor cells supports HNSCC progression. Therefore, our finding of TLR-4
L.K. Mäkinen et al. / Oral Oncology 51 (2015) 96–102
101
Fig. 5. Low TLR-5 expression correlates with poor survival in early-stage oral tongue squamous cell carcinoma. For survival analysis TLR-5 expression scores were dichotomized into two groups as follows: none and mild = 0, moderate and strong = 1. Patients in group 0 (n = 4) had worse disease specific survival than patients in group 1 (n = 59) (p = 0.014, log-rank test).
correlating with tumor invasion is in line with this previous study. Supporting our observation of TLR-9 expression correlating with tumor invasion, a recent study by Ruan et al. [18] showed activation of TLR-9 to induce migration and invasion of oral cancer cells. It would be interesting to investigate whether the expression of TLR-2, -4, and -9 is strongest especially in the invasive front of the tumor, but this should be evaluated from whole tissue sections and not from tissue microarray blocks, which were used in the present study. We found that strong cytoplasmic TLR-2 and high TLR-4 expression correlated with poor differentiation grade tumors, whereas strong TLR-5 expression was seen in well-differentiated tumors. Conversely, Szczepanski et al. [15] reported TLR-4 expression intensity in laryngeal squamous cell carcinoma to correlate inversely with tumor grade; Sun et al. [16] made a similar finding in OSCC. Chuang et al. [14] found that high TLR-3 expression correlated with poorly differentiated OSCC. It is important to note that grade has not been shown to reliably predict prognosis in OSCC, mainly due to the subjective nature of its assessment [32]. High TLR-9 expression correlated with advanced tumor size, which is in accordance with a previous study of TLR-9 in OSCC by Min et al. [22]. They also demonstrated in vitro, that stimulation of OSCC cells with TLR-9 agonist CpG-ODN significantly increased tumor proliferation. Various treatment modalities may pose a confounding factor for retrospective outcome analyses. All patients in this series, however, were treated according to the Finnish national treatment guidelines for Head and Neck cancer. For example, all patients with occult neck metastases also received postoperative radiotherapy. The present survival analyses showed that patients with none or mild TLR-5 expression will have worse DSS than patients with moderate or strong TLR-5 expression. Our finding differs from the report of Kauppila et al. [21], who showed strong TLR-5 expression to be an independent predictor of OTSCC mortality. One explanation could be that their study on whole tissue sections included tongue carcinomas of different stages, whereas our study using tissue microarray technique focused on clinically early-stage
disease. More importantly, the present statistical analysis showed that there were no differences between pathological Stages I and II (pN0) and pathological Stage III-IVA (pN+) patients when compared with TLR expression scores. Therefore, the role of TLRs in predicting survival of HNSCC remains inconclusive. Conclusions All TLRs examined showed high expression in tissue samples from early-stage tongue carcinoma. TLR-2, -4, and -9 associated with invasive growth of the tumor. Conflict of interest statement None declared. Acknowledgements We thank Päivi Peltokangas and Elina Aspiala for excellent technical assistance and M.Sc. Timo Pessi for statistical advice. This study had financial support from the National Graduate School of Clinical Investigation, the University of Helsinki, Ida Montin Foundation (LM), and The Sigrid Juselius Foundation (CH). References [1] Bello IO, Soini Y, Salo T. Prognostic evaluation of oral tongue cancer: means, markers and perspectives (I). Oral Oncol 2010;46:630–5. [2] Bello IO, Soini Y, Salo T. Prognostic evaluation of oral tongue cancer: means, markers and perspectives (II). Oral Oncol 2010;46:636–43. [3] Makitie AA, Koivunen P, Keski-Santti H, Tornwall J, Pukkila M, Laranne J, et al. Oral tongue carcinoma and its treatment in Finland. Eur Arch Otorhinolaryngol 2007;264:263–7. [4] Couzin-Frankel J. Breakthrough of the year 2013. Cancer immunotherapy. Science 2013;2013(342):1432–3. [5] Duray A, Demoulin S, Hubert P, Delvenne P, Saussez S. Immune suppression in head and neck cancers: a review. Clin Dev Immunol 2010;2010:701657. [6] Ioannou S, Voulgarelis M. Toll-like receptors, tissue injury, and tumourigenesis. Mediators Inflamm 2010;2010. http://dx.doi.org/10.1155/ 2010/581837. Epub 2010 Sep 14.
102
L.K. Mäkinen et al. / Oral Oncology 51 (2015) 96–102
[7] Miller LS, Modlin RL. Toll-like receptors in the skin. Semin Immunopathol 2007;29:15–26. [8] Yu L, Wang L, Chen S. Dual character of toll-like receptor signaling: pro-tumorigenic effects and anti-tumor functions. Biochim Biophys Acta 2013;1835:144–54. [9] 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. [10] Pradere JP, Dapito DH, Schwabe RF. The yin and yang of toll-like receptors in cancer. Oncogene 2013. [11] Rydberg C, Mansson A, Uddman R, Riesbeck K, Cardell LO. Toll-like receptor agonists induce inflammation and cell death in a model of head and neck squamous cell carcinomas. Immunology 2009;128:e600–11. [12] Park JH, Yoon HE, Jeon DI, Ahn SG, Yoon JH. Activation of TLR2 and TLR5 did not affect tumor progression of an oral squamous cell carcinoma, YD-10B cells. J Oral Pathol Med 2010;39:781–5. [13] Pries R, Hogrefe L, Xie L, Frenzel H, Brocks C, Ditz C, et al. Induction of c-mycdependent cell proliferation through toll-like receptor 3 in head and neck cancer. Int J Mol Med 2008;21:209–15. [14] Chuang HC, Huang CC, Chien CY, Chuang JH. Toll-like receptor 3-mediated tumor invasion in head and neck cancer. Oral Oncol 2012;48:226–32. [15] Szczepanski MJ, Czystowska M, Szajnik M, Harasymczuk M, Boyiadzis M, KrukZagajewska A, et al. Triggering of toll-like receptor 4 expressed on human head and neck squamous cell carcinoma promotes tumor development and protects the tumor from immune attack. Cancer Res 2009;69:3105–13. [16] Sun Z, Luo Q, Ye D, Chen W, Chen F. Role of toll-like receptor 4 on the immune escape of human oral squamous cell carcinoma and resistance of cisplatininduced apoptosis. Mol Cancer 2012;11:33,4598–11-33. [17] Ahn MY, Kwon SM, Cheong HH, Park JH, Lee J, Min SK, et al. Toll-like receptor 7 agonist, imiquimod, inhibits oral squamous carcinoma cells through apoptosis and necrosis. J Oral Pathol Med 2012;41:540–6. [18] Ruan M, Zhang Z, Li S, Yan M, Liu S, Yang W, et al. Activation of toll-like receptor-9 promotes cellular migration via up-regulating MMP-2 expression in oral squamous cell carcinoma. PLoS One 2014;9:e92748. [19] Szczepanski M, Stelmachowska M, Stryczynski L, Golusinski W, Samara H, Mozer-Lisewska I, et al. Assessment of expression of toll-like receptors 2, 3 and 4 in laryngeal carcinoma. Eur Arch Otorhinolaryngol 2007;264:525–30. [20] Luo Q, Hu S, Yan M, Sun Z, Chen W, Chen F. Activation of toll-like receptor 3 induces apoptosis of oral squamous carcinoma cells in vitro and in vivo. Int J Biochem Cell Biol 2012;44:1266–75.
[21] 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. [22] Min R, Zun Z, Siyi L, Wenjun Y, Lizheng W, Chenping Z. Increased expression of toll-like receptor-9 has close relation with tumour cell proliferation in oral squamous cell carcinoma. Arch Oral Biol 2011;56:877–84. [23] Mirghani H, Amen F, Moreau F, Guigay J, Ferchiou M, Melkane AE, et al. Human papilloma virus testing in oropharyngeal squamous cell carcinoma: what the clinician should know. Oral Oncol 2014;50:1–9. [24] Keski-Santti H, Atula T, Tornwall J, Koivunen P, Makitie A. Elective neck treatment versus observation in patients with T1/T2 N0 squamous cell carcinoma of oral tongue. Oral Oncol 2006;42:96–101. [25] Makinen LK, Hayry V, Hagstrom J, Sorsa T, Passador-Santos F, Keski-Santti H, et al. Matrix metalloproteinase-7 and matrix metalloproteinase-25 in oral tongue squamous cell carcinoma. Head Neck 2013. [26] Makinen LK, Hayry V, Atula T, Haglund C, Keski-Santti H, Leivo I, et al. Prognostic significance of matrix metalloproteinase-2, -8, -9, and -13 in oral tongue cancer. J Oral Pathol Med 2012;41:394–9. [27] Ng LK, Rich AM, Hussaini HM, Thomson WM, Fisher AL, Horne LS, et al. Tolllike receptor 2 is present in the microenvironment of oral squamous cell carcinoma. Br J Cancer 2011;104:460–3. [28] Garcia-de Marcos JA, Perez-Zafrilla B, Arriaga A, Arroyo-Rodriguez S, Poblet E. Human papillomavirus in carcinomas of the tongue: clinical and prognostic implications. Int J Oral Maxillofac Surg 2013. [29] Dunkel J, Vaittinen S, Grenman R, Kinnunen I, Irjala H. Prognostic markers in stage I oral cavity squamous cell carcinoma. Laryngoscope 2013;123: 2435–41. [30] Deng Z, Hasegawa M, Aoki K, Matayoshi S, Kiyuna A, Yamashita Y, et al. A comprehensive evaluation of human papillomavirus positive status and p16INK4a overexpression as a prognostic biomarker in head and neck squamous cell carcinoma. Int J Oncol 2014;45:67–76. [31] Yang H, Wang B, Yan J, Wang T, Zhou XN, Wen HY, et al. Toll-like receptor 2 promotes invasion by SGC-7901 human gastric carcinoma cells and is associated with gastric carcinoma metastasis. Ann Clin Lab Sci 2014;44: 158–66. [32] Woolgar JA. Histopathological prognosticators in oral and oropharyngeal squamous cell carcinoma. Oral Oncol 2006;42:229–39.
