APMIS 123: 439–444
© 2015 APMIS. Published by John Wiley & Sons Ltd. DOI 10.1111/apm.12366
Toll-like receptors 3, 7, and 9 in Juvenile nasopharyngeal angiofibroma SUVI RENKONEN,1 LARS-OLAF CARDELL,2 PETRI MATTILA,1 MARIE LUNDBERG,1 1,2, € € 3,* and ANTTI A. MAKITIE CAJ HAGLUND,3,4,5 JAANA HAGSTROM * 1
Department of Otorhinolaryngology - Head and Neck Surgery, Helsinki University Central Hospital and University of Helsinki, Helsinki, Finland; 2Division of Ear, Nose and Throat Diseases, Department of Clinical Sciences, Intervention and Technology, Karolinska Institutet, Stockholm, Sweden; 3Department of Pathology, Haartman Institute and HusLab, University of Helsinki and Helsinki University Central Hospital, Helsinki; 4Department of Surgery, Helsinki University Central Hospital, Helsinki; and 5Research Programs Unit, Translational Cancer Biology, University of Helsinki, Helsinki, Finland
Renkonen S, Cardell L-O, Mattila P, Lundberg M, Haglund C, Hagstr€ om J, M€ akitie AA. Toll-like receptors 3, 7, and 9 in Juvenile nasopharyngeal angiofibroma. APMIS 2015; 123: 439–444. Juvenile nasopharyngeal angiofibroma (JNA) is a rare, benign tumor affecting adolescent males. The etiology of JNA as well as the causes determining the variable growth patterns of individual tumors remains unknown. Toll-like receptors (TLRs) are part of the innate immune response to microbes; by recognition of distinct features, they link to induction of pro-inflammatory signaling pathways. We immunostained TLR 3, 7, and 9 in 27 JNA specimens of patients treated at the Helsinki University Central Hospital, Helsinki, Finland, during the years 1970–2009. Results: TLR 3, 7, and 9 expressions were found in stromal and endothelial cells of JNA, and their expression levels varied from negative to very strong positive. TLR 3 expression was found to have a significant correlation with the clinical stage of JNA. Conclusions: The present results propose a putative role of TLRs in the growth process of JNA. Key words: Neoplasm; head and neck; immunohistochemistry; tumor; immunology; juvenile nasopharyngeal angiofibroma; toll-like receptors. Suvi Renkonen, Haartman Institute, PO Box 21, FIN-00014 University of Helsinki, Helsinki, Finland. e-mail: suvi.renkonen@helsinki.fi
*Equal contribution
Juvenile nasopharyngeal angiofibroma (JNA) is a rare, highly vascular tumor, located in the nasopharynx (1). Histologically, it is composed of stromal and endothelial compartments, the neoplastic cell population being uncertain. The etiology of this tumor is unknown, as are also the specific factors determining the variable growth patterns and outcomes of individual tumors (2). Several studies have aimed to elucidate the factors and processes underlining JNAs initiation and growth, and various growth factors, hormonal receptors and certain tumor suppressors and oncogenes have been discussed to possibly play an important role (3–8). Previously, we managed to demonstrate that genes regulating the immune system were overexpressed in the high-stage JNA compared with low-stage Received 11 September 2014. Accepted 23 December 2014
JNA (9). In our samples, both mast cells and lymphocytes were discovered referring to putative chronic inflammatory process. Toll-like receptors (TLRs) are a vital part of the initiation and signaling mechanisms leading to inflammation (10). These proteins are a group of specific pattern recognition molecules that bind to microbial components (pathogen-associated molecular patterns, PAMPs) and to damage-associated molecular patterns (DAMPs) to trigger innate immunity and direct adaptive immunity in the face of immunological danger (11). TLRs are expressed by cells of the immune system as well as by epithelial and endothelial cells that provide the first line defense against microbes (11). By their location, TLRs are divided into surface proteins (TLR 1, 2, 4, 5, 6, and 10) and intracellular proteins (TLR 3, 7, 8, 9), activated by self- and microbial-derived 439
RENKONEN et al.
molecules. Under normal conditions, TLRs also participate in tissue repair and regeneration. Under abnormal conditions, however, TLRs are reported to participate in the pathogenesis of a large number of inflammatory and/or metabolic disorders, such as asthma and allergy (12), inflammatory bowel disease (13), and atherosclerosis (14). Chronic inflammation is also known to be a preferable microenvironment for tumor development (15). Based on our earlier finding of the putative role of immune responses in determining the different outcomes of JNA, we wanted to explore the possible presence of inflammation linked TLR proteins in JNA tissue. We chose to investigate the presence of TLR 3, 7, and 9, known to be intracellular and activated by nucleic acids and thus participating in the process of viral detection (16). Viral infections have been reported to have a role in the initiation of certain head and neck, such as HPV-related oropharyngeal tumor and EBV-related nasopharyngeal cancer (17, 18). MATERIALS AND METHODS Patients Clinicopathological data from 27 patients treated for histologically verified JNA between 1970 and 2009 at the Helsinki University Central Hospital (HUCH), Finland were retrospectively reviewed. The referral area of this tertiary care academic institution currently covers an area of approximately 1.6 million people. Surgical and discharge registries of the hospital as well as the database of the Department of Pathology were used to identify the patients. All patients had undergone surgical resection of the primary tumor. Tumors were classified according to Andrews staging system, where Stage I tumors are limited to nose, nasopharynx, and sinuses and Stages II–IV represent tumors invading bony structures. Detailed data of the clinical parameters of these 27 JNAs are presented in our earlier work, as are also the vessel densities of each tumor sample. For vessel density, vessels marked by CD31 were counted and the mean counts per mm2 were calculated (19).
corporation), or rabbit polyclonal TLR 9 (sc-25468) antibody (Santa Cruz Biotechnology, Inc) for one hour, followed by a 30 min incubation with Dako REAL EnVision/HRP detection system, Rabbit (ENV) reagent. Slides were finally visualized by Dako REAL DAB+ Chromogen (Dako, Glostrup, Denmark) for 10 min. PBS0.04%–Tween20 washing was accomplished between each step. Slides were counterstained with Meyer’s hematoxylin and mounted in mounting medium (Aquamount, BDH, Poole, UK).
Evaluation of immunostaining All stained samples (n = 27) were evaluated, but insufficient samples were excluded from the analyses. Two investigators (JH and SR) evaluated the immunostainings independently and without knowledge of clinical data, by counting the percentage of positive tumor cells. Each whole tissue slide was evaluated, and an average staining percentage was estimated. No positivity was scored as 0, up to 30% positive cells as 1 (low), 30–50% as 2 (moderate), and 50–80% as 3 (high). The scoring system has been described earlier by H€ ayry et al. (20). The expression was scored separately for endothelial cells and stromal cells. Due to the long period of sample collecting (1970–2009), not all specimens were representative for evaluation and were thus excluded (6/8 for TLR 3, 4/2 for TLR 7, and 2/3 for TLR 9, as for stromal and endothelial scoring). As positive controls, tonsillar carcinoma was used for TLR 3, normal tongue for TLR 9 and squamous cell carcinoma of the skin, as well as normal skin for TLR 7. As negative control, a specimen processed without primary antibody was used.
Statistical analyses Categorical variables were cross-tabulated and analyzed using the Fisher’s exact test or Spearman correlation. We used SPSS version 15.0 software (SPSS, IL, USA) for all statistical analyses.
RESULTS The expressions of TLR 3, TLR 7, and TLR 9 in JNA specimens were as follows (Table 1).
Immunohistochemistry For immunohistochemistry, formalin-fixed and paraffinembedded samples were cut into 4-micrometer thick sections, deparaffinized in xylene, and rehydrated through a graded alcohol series. For antigen retrieval, slides were treated in a PT-module for 20 min at 98 °C. Immunohistochemical stainings were performed in Autostainer 480 (LabVision) using Dako REAL EnVision Detection System, Peroxidase/DAB+, Rabbit/Mouse (Dako, Glostrup, Denmark). Slides were treated with 0.3% Dako REAL Peroxidase-Blocking Solution to block endogenous peroxidase activity followed by primary antibody incubation with rabbit polyclonal IgG TLR 3 (sc-10740) antibody (Santa Cruz Biotechnology, Inc, Heidelberg, Germany), rabbit polyclonal TLR 7 (IMG-581A) antibody (Imgenex
440
Tlr 3
TLR 3 showed granular staining in the cytoplasm. Expression was detected in stromal cells in 67% of the samples and in endothelial cells in 89% of the JNA samples (Fig. 1). Tlr 7
Juvenile nasopharyngeal angiofibroma samples showed less TLR 7 than TLR 3 expression. TLR 7 expression was detected in stromal cells in 52% of the samples and only in 17% in endothelial cells. © 2015 APMIS. Published by John Wiley & Sons Ltd
JUVENILE NASOPHARYNGEAL ANGIOFIBROMA
Table 1. Immunoexpression of TLR-3, TLR-7, and TLR-9 in JNA samples Number of cases TLR3 en TLR3 st TLR7 en Negative 7 (33%) 2 (11%) 19 (83%) Low 9 (43%) 12 (63%) 3 (13%) Moderate 3 (14%) 3 (16%) 1 (4%) High 2 (10%) 2 (11%) 0 (0%) Representative cases 21 19 23 en, endothelial staining; st, stromal staining.
Fig. 1. JNA tissue showing TLR 3 immunostaining in both stromal cells and endothelial cells of slit like vessels (x400).
TLR7 st 12 (48%) 9 (36%) 2 (8%) 2 (8%) 25
TLR9 en 11 (44%) 8 (32%) 4 (16%) 2 (8%) 25
TLR9 st 8 (33%) 9 (42%) 4 (17%) 3 (13%) 24
Fig. 3. Endothelial and stromal staining of TLR 9 in JNA tissue (x400).
Correlations between histology, immunohistochemistry, and clinical parameters
We compared the protein expression levels of stromal and endothelial TLR 3, TLR 7, and TLR 9 with vessel density, tumor stage, duration of symptoms before diagnosis, and with each other. Negativity of endothelial TLR 3 expression had a statistically significant correlation with higher tumor stage (Fisher’s exact p =
© 2015 APMIS. Published by John Wiley & Sons Ltd. DOI 10.1111/apm.12366
Toll-like receptors 3, 7, and 9 in Juvenile nasopharyngeal angiofibroma SUVI RENKONEN,1 LARS-OLAF CARDELL,2 PETRI MATTILA,1 MARIE LUNDBERG,1 1,2, € € 3,* and ANTTI A. MAKITIE CAJ HAGLUND,3,4,5 JAANA HAGSTROM * 1
Department of Otorhinolaryngology - Head and Neck Surgery, Helsinki University Central Hospital and University of Helsinki, Helsinki, Finland; 2Division of Ear, Nose and Throat Diseases, Department of Clinical Sciences, Intervention and Technology, Karolinska Institutet, Stockholm, Sweden; 3Department of Pathology, Haartman Institute and HusLab, University of Helsinki and Helsinki University Central Hospital, Helsinki; 4Department of Surgery, Helsinki University Central Hospital, Helsinki; and 5Research Programs Unit, Translational Cancer Biology, University of Helsinki, Helsinki, Finland
Renkonen S, Cardell L-O, Mattila P, Lundberg M, Haglund C, Hagstr€ om J, M€ akitie AA. Toll-like receptors 3, 7, and 9 in Juvenile nasopharyngeal angiofibroma. APMIS 2015; 123: 439–444. Juvenile nasopharyngeal angiofibroma (JNA) is a rare, benign tumor affecting adolescent males. The etiology of JNA as well as the causes determining the variable growth patterns of individual tumors remains unknown. Toll-like receptors (TLRs) are part of the innate immune response to microbes; by recognition of distinct features, they link to induction of pro-inflammatory signaling pathways. We immunostained TLR 3, 7, and 9 in 27 JNA specimens of patients treated at the Helsinki University Central Hospital, Helsinki, Finland, during the years 1970–2009. Results: TLR 3, 7, and 9 expressions were found in stromal and endothelial cells of JNA, and their expression levels varied from negative to very strong positive. TLR 3 expression was found to have a significant correlation with the clinical stage of JNA. Conclusions: The present results propose a putative role of TLRs in the growth process of JNA. Key words: Neoplasm; head and neck; immunohistochemistry; tumor; immunology; juvenile nasopharyngeal angiofibroma; toll-like receptors. Suvi Renkonen, Haartman Institute, PO Box 21, FIN-00014 University of Helsinki, Helsinki, Finland. e-mail: suvi.renkonen@helsinki.fi
*Equal contribution
Juvenile nasopharyngeal angiofibroma (JNA) is a rare, highly vascular tumor, located in the nasopharynx (1). Histologically, it is composed of stromal and endothelial compartments, the neoplastic cell population being uncertain. The etiology of this tumor is unknown, as are also the specific factors determining the variable growth patterns and outcomes of individual tumors (2). Several studies have aimed to elucidate the factors and processes underlining JNAs initiation and growth, and various growth factors, hormonal receptors and certain tumor suppressors and oncogenes have been discussed to possibly play an important role (3–8). Previously, we managed to demonstrate that genes regulating the immune system were overexpressed in the high-stage JNA compared with low-stage Received 11 September 2014. Accepted 23 December 2014
JNA (9). In our samples, both mast cells and lymphocytes were discovered referring to putative chronic inflammatory process. Toll-like receptors (TLRs) are a vital part of the initiation and signaling mechanisms leading to inflammation (10). These proteins are a group of specific pattern recognition molecules that bind to microbial components (pathogen-associated molecular patterns, PAMPs) and to damage-associated molecular patterns (DAMPs) to trigger innate immunity and direct adaptive immunity in the face of immunological danger (11). TLRs are expressed by cells of the immune system as well as by epithelial and endothelial cells that provide the first line defense against microbes (11). By their location, TLRs are divided into surface proteins (TLR 1, 2, 4, 5, 6, and 10) and intracellular proteins (TLR 3, 7, 8, 9), activated by self- and microbial-derived 439
RENKONEN et al.
molecules. Under normal conditions, TLRs also participate in tissue repair and regeneration. Under abnormal conditions, however, TLRs are reported to participate in the pathogenesis of a large number of inflammatory and/or metabolic disorders, such as asthma and allergy (12), inflammatory bowel disease (13), and atherosclerosis (14). Chronic inflammation is also known to be a preferable microenvironment for tumor development (15). Based on our earlier finding of the putative role of immune responses in determining the different outcomes of JNA, we wanted to explore the possible presence of inflammation linked TLR proteins in JNA tissue. We chose to investigate the presence of TLR 3, 7, and 9, known to be intracellular and activated by nucleic acids and thus participating in the process of viral detection (16). Viral infections have been reported to have a role in the initiation of certain head and neck, such as HPV-related oropharyngeal tumor and EBV-related nasopharyngeal cancer (17, 18). MATERIALS AND METHODS Patients Clinicopathological data from 27 patients treated for histologically verified JNA between 1970 and 2009 at the Helsinki University Central Hospital (HUCH), Finland were retrospectively reviewed. The referral area of this tertiary care academic institution currently covers an area of approximately 1.6 million people. Surgical and discharge registries of the hospital as well as the database of the Department of Pathology were used to identify the patients. All patients had undergone surgical resection of the primary tumor. Tumors were classified according to Andrews staging system, where Stage I tumors are limited to nose, nasopharynx, and sinuses and Stages II–IV represent tumors invading bony structures. Detailed data of the clinical parameters of these 27 JNAs are presented in our earlier work, as are also the vessel densities of each tumor sample. For vessel density, vessels marked by CD31 were counted and the mean counts per mm2 were calculated (19).
corporation), or rabbit polyclonal TLR 9 (sc-25468) antibody (Santa Cruz Biotechnology, Inc) for one hour, followed by a 30 min incubation with Dako REAL EnVision/HRP detection system, Rabbit (ENV) reagent. Slides were finally visualized by Dako REAL DAB+ Chromogen (Dako, Glostrup, Denmark) for 10 min. PBS0.04%–Tween20 washing was accomplished between each step. Slides were counterstained with Meyer’s hematoxylin and mounted in mounting medium (Aquamount, BDH, Poole, UK).
Evaluation of immunostaining All stained samples (n = 27) were evaluated, but insufficient samples were excluded from the analyses. Two investigators (JH and SR) evaluated the immunostainings independently and without knowledge of clinical data, by counting the percentage of positive tumor cells. Each whole tissue slide was evaluated, and an average staining percentage was estimated. No positivity was scored as 0, up to 30% positive cells as 1 (low), 30–50% as 2 (moderate), and 50–80% as 3 (high). The scoring system has been described earlier by H€ ayry et al. (20). The expression was scored separately for endothelial cells and stromal cells. Due to the long period of sample collecting (1970–2009), not all specimens were representative for evaluation and were thus excluded (6/8 for TLR 3, 4/2 for TLR 7, and 2/3 for TLR 9, as for stromal and endothelial scoring). As positive controls, tonsillar carcinoma was used for TLR 3, normal tongue for TLR 9 and squamous cell carcinoma of the skin, as well as normal skin for TLR 7. As negative control, a specimen processed without primary antibody was used.
Statistical analyses Categorical variables were cross-tabulated and analyzed using the Fisher’s exact test or Spearman correlation. We used SPSS version 15.0 software (SPSS, IL, USA) for all statistical analyses.
RESULTS The expressions of TLR 3, TLR 7, and TLR 9 in JNA specimens were as follows (Table 1).
Immunohistochemistry For immunohistochemistry, formalin-fixed and paraffinembedded samples were cut into 4-micrometer thick sections, deparaffinized in xylene, and rehydrated through a graded alcohol series. For antigen retrieval, slides were treated in a PT-module for 20 min at 98 °C. Immunohistochemical stainings were performed in Autostainer 480 (LabVision) using Dako REAL EnVision Detection System, Peroxidase/DAB+, Rabbit/Mouse (Dako, Glostrup, Denmark). Slides were treated with 0.3% Dako REAL Peroxidase-Blocking Solution to block endogenous peroxidase activity followed by primary antibody incubation with rabbit polyclonal IgG TLR 3 (sc-10740) antibody (Santa Cruz Biotechnology, Inc, Heidelberg, Germany), rabbit polyclonal TLR 7 (IMG-581A) antibody (Imgenex
440
Tlr 3
TLR 3 showed granular staining in the cytoplasm. Expression was detected in stromal cells in 67% of the samples and in endothelial cells in 89% of the JNA samples (Fig. 1). Tlr 7
Juvenile nasopharyngeal angiofibroma samples showed less TLR 7 than TLR 3 expression. TLR 7 expression was detected in stromal cells in 52% of the samples and only in 17% in endothelial cells. © 2015 APMIS. Published by John Wiley & Sons Ltd
JUVENILE NASOPHARYNGEAL ANGIOFIBROMA
Table 1. Immunoexpression of TLR-3, TLR-7, and TLR-9 in JNA samples Number of cases TLR3 en TLR3 st TLR7 en Negative 7 (33%) 2 (11%) 19 (83%) Low 9 (43%) 12 (63%) 3 (13%) Moderate 3 (14%) 3 (16%) 1 (4%) High 2 (10%) 2 (11%) 0 (0%) Representative cases 21 19 23 en, endothelial staining; st, stromal staining.
Fig. 1. JNA tissue showing TLR 3 immunostaining in both stromal cells and endothelial cells of slit like vessels (x400).
TLR7 st 12 (48%) 9 (36%) 2 (8%) 2 (8%) 25
TLR9 en 11 (44%) 8 (32%) 4 (16%) 2 (8%) 25
TLR9 st 8 (33%) 9 (42%) 4 (17%) 3 (13%) 24
Fig. 3. Endothelial and stromal staining of TLR 9 in JNA tissue (x400).
Correlations between histology, immunohistochemistry, and clinical parameters
We compared the protein expression levels of stromal and endothelial TLR 3, TLR 7, and TLR 9 with vessel density, tumor stage, duration of symptoms before diagnosis, and with each other. Negativity of endothelial TLR 3 expression had a statistically significant correlation with higher tumor stage (Fisher’s exact p =
