AM ER IC AN JOUR NA L OF OTOLARY NG OLOG Y –H EA D A N D N E CK ME D I CI NE AN D SUR G E RY 3 5 ( 2 0 14 ) 58 2–5 8 8
Available online at www.sciencedirect.com
ScienceDirect www.elsevier.com/locate/amjoto
The presence of tumor-infiltrating IL-17-producing cells in juvenile nasopharyngeal angiofibroma tumor microenvironment is a poor prognostic factor☆ Xicai Sun, MD 1 , Limin Guo, MD 1 , Huan Wang, MD 1 , Huapeng Yu, MD 1 , Jingjing Wang, MD, Xinjun Meng, MD, Zhuofu Liu, MD, Juan Liu, MD, Li Hu, MD, Han Li, MD, Dehui Wang, MD⁎ Department of Otolaryngology, Eye, Ear, Nose and Throat Hospital, Shanghai Medical College, Fudan University, Shanghai, China
ARTI CLE I NFO
A BS TRACT
Article history:
Background: Although juvenile nasopharyngeal angiofibroma (JNA) is a benign tumor
Received 12 March 2014
histologically, it demonstrates aggressive propensity of locally destructive growth causing bone erosion. The patients with JNA remain high recurrence rate after surgical excision. Th17 cells secrete the proinflammatory cytokine interleukin-17 (IL-17), and play an important role in carcinogenesis and tumor progression. So far, no studies have focused on the significance of IL17-producing cells in the JNA tumor microenvironment. The current study was designed to investigate the localization and level of tumor-infiltrating IL-17-producing cells in JNA microenvironment. The presence and number of IL-17-producing cells were further analyzed for a possible association with clinicopathological features and disease outcome. Materials and methods: Immunohistochemistry was used to analyze the expression of IL-17 in a tissue microarray from 70 patients with JNA and 10 control subjects. Correlations between the levels of IL-17 expression and clinicopathologic variables, as well as tumor recurrence, were assessed. Results: In vessels, the IL-17-producing cells were identified in pericytes and irregular smooth muscle cells, but the matured vascular endothelial cells showed no IL-17 reactivity. The expression of IL-17 in stromal cells was concentrated in the less differentiated and plump cells that contained a central hypochromatic nucleus and single small nucleolus. Chi-square test showed that tumor stage (p = 0.09), operation history (p = 0.828), operation approach (p = 0.159), and volume of intraoperative hemorrhage (p = 0.352) were not associated with the expression of IL-17 in JNA patients. However, intratumoral IL-17producing cells were negatively associated with patient's age (p = 0.004). Furthermore, we found that patients with extensive infiltration of IL-17-producing cells had significantly higher recurrence rates than those with less infiltration of IL-17-producing cells (p = 0.028). Log rank analysis showed that JNA patients with high levels of IL-17 had significantly shorter disease free survival (DFS) than those with low levels of IL-17 (p = 0.004). Univariate Cox regression analysis suggested that IL-17 and patient's age were significantly associated
☆
Declaration of interest: The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper. ⁎ Corresponding author at: Department of Otolaryngology, Eye, Ear, Nose and Throat Hospital, Shanghai Medical College, Fudan University, 83 Fenyang Road, Shanghai, 200031, China. Tel.: + 86 21 64 377 134 788; fax: + 86 21 64 377 151. E-mail address: [email protected] (D. Wang). 1 Drs. Xicai Sun, Limin Guo, Huan Wang, and Huapeng Yu contributed equally to this work. http://dx.doi.org/10.1016/j.amjoto.2014.06.001 0196-0709/© 2014 Elsevier Inc. All rights reserved.
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with DFS. Multivariate analysis indicated that high infiltration with IL-17-producing cells was associated with poor DFS. Of all clinicopathological features, IL-17 level was an independent factor predicting the patient's prognosis. Conclusion: In JNA patients, a high level of IL-17-producing cells was negatively associated with patient's age. Patients with extensive infiltration of IL-17-producing cells had significantly higher tumor recurrence rates. High infiltration of IL-17-producing cells in JNA microenvironment is an independent poor prognostic factor for shorter disease-free survival. Future studies further focusing on the role of IL-17 may provide more promising therapeutic methods for extensive JNA tumors. © 2014 Elsevier Inc. All rights reserved.
1.
Introduction
Juvenile nasopharyngeal angiofibroma (JNA) is a rare, highly vascularized benign tumor originating from the superior margin of the sphenopalatine foramen or pterygoid canal [1]. It accounts for approximately 0.05% of all head and neck tumors, and occurs almost exclusively in adolescent males. Although JNA is a benign tumor histologically, it has a propensity to grow destructively and erode bone. Surgery is the mainstay of treatment, but the recurrence rate after surgical excision of JNA is high and fairly heterogeneous, varying from 20% to over 50% [2–7]. Histopathology of JNA shows that it is composed of stromal cells and proliferating irregular vascular components. The typical stromal cells in JNA are fibroblasts in a dense collagen matrix [8,9]. However, it has been found that JNA is also infiltrated by numerous inflammatory cells. Substantial evidence indicates that mast cells and T-lymphocytes are identified as major cellular populations in JNA as they represented 30% of the cells in studied tumor specimens [10]. It has been shown that the inflammatory microenvironment plays a critical role in tumor progression [11,12]. Tumor cells and host immune cells interact with each other to create the tumor microenvironment. CD4+ T cells are the major component of immune cells in the tumor microenvironment, and play an important role in tumor regression and evasion [13,14]. CD4+ T helper 17 (Th17) cells have been identified as a novel subset of CD4+ T-lymphocytes, and are characterized by its signature cytokine interleukin-17 (IL-17). IL-17 is a proinflammatory cytokine, and has been implicated in inflammatory and autoimmune disease. Furthermore, several studies on skin cancer, hepatocellular carcinoma, gallbladder carcinoma, laryngeal, gastric, breast, colorectal, pancreatic and lung cancer have demonstrated that IL-17 exhibits tumorpromoting functions [15–23]. However, in acute leukemia, advanced epithelial ovarian cancer and melanoma patients, the expression of IL-17 was reported to have an anti-tumor effect by promoting cytotoxic T-lymphocytes' (CTL) activities, reducing angiogenesis and the recruitment of effector Th1 cells to the tumor [24–26]. This contradictory result may be attributed to variant tumor immunogenicity and a different role of IL-17 in different tumor stages [27]. Recently, Wendler et al. showed that infiltration of JNA tumors by CD4+ and CD8+ T-lymphocytes was evident in immunofluorescent stainings [10]. So far, no studies have focused on the significance of IL-17-producing cells in the JNA tumor microenvironment and the role of IL-17 in JNA remains unclear. Aim of the
present study was to investigate the level of tumor-infiltrating IL17-producing cells in JNA microenvironment, and analyze its association with clinicopathological features and disease outcome.
2.
Materials and methods
2.1.
Patients and tissue specimens
Paraffin-embedded specimens from 70 patients with JNA who underwent surgical resection in the Eye, Ear, Nose and Throat Hospital of Fudan University between 2003 and 2007 were enrolled in the study. All patients suffered from a histologically proven JNA tumor. Clinical data were obtained from patients' medical records. All JNA patients were male and ranged in age from 9 to 41 years old (median age, 17 years) at the time of surgery. The normal middle turbinates of 10 male patients with a median age of 21.4 years (range, 13–26 years) were used as normal controls. The clinicopathological characteristics of patients with JNA are summarized in Table 1. Tumor staging of JNA was based on the classification of Radkowski and colleagues [28]. The study protocol was approved by the institutional review board of Fudan University.
3.
Patients' follow-up
Until August 31, 2013, complete follow-up data were available for 55 patients. The follow-up period ranged from 69 to 127 months, with a mean of 95 months. Completeness of the resection was defined as no macroscopic residual tumor and was further confirmed with histology of surgical margins, as well as postoperative CT scan or MRI scan. Recurrence was defined as clinically or radiologically demonstrating tumor presence in the nasopharynx, nasal cavity, or neighboring structures, with confirmed major symptoms including epistaxis or nasal obstruction after the initial surgical treatment. There are distinct therapeutic principles and surgical techniques for JNA in different hospitals, which may interfere with the analysis of relapse of JNA. So, the history of previous operations at other hospitals was not considered for the recurrence analysis in this study.
3.1.
Tissue microarray and immunohistochemical assay
A tissue microarray for JNA was prepared in our previous report [3]. The tissue samples included 70 JNA tumor specimens and 10 normal middle turbinates. Briefly,
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Table 1 – Clinicopathological characteristics and their correlation with IL-17 immunoreactivity in JNA patients assessed with chi-square test. Variables
Total number Sex Male Female Median age, years ≤ 17 >17 Tumor stage Stage Ia Stage Ib Stage IIa Stage IIb Stage IIc Stage IIIa Stage IIIb Operation history No Yes Operation approach With endoscope Without endoscope Volume of intraoperative hemorrhage (ml) ≤ 800 >800
No. of patients
Levels of IL-17 expressions (scores)
p Value
0
1
2
3
70 0 17 39 31
0 1
4 13
15 11
20 6
0.004
4 7 6 7 34 5 7
0 0 1 0 0 0 0
0 3 1 0 8 1 4
1 1 2 2 16 3 1
3 2 2 5 10 1 2
0.09
40 30
1 0
10 7
14 12
15 11
0.828
17 53
1 0
2 15
6 20
8 18
0.159
26 44
1 0
7 10
7 19
11 15
0.352
70
Abbreviation: JNA, juvenile nasopharyngeal angiofibroma.
formalin-fixed and paraffin-embedded surgical tissue samples were obtained from the Department of Otolaryngology– Head and Neck Surgery, Eye, Ear, Nose, and Throat Hospital, Fudan University, China. Two histopathologically representative regions of the tumor specimen were defined on the respective hematoxylin-eosin stained sections and marked on the surface of the standard paraffin block. Two cores from each tumor block were punched using a biopsy needle (1.5 mm in diameter). All tissue cores were put into a tissue microarray (TMA). Samples were processed in collaboration with Shanghai Biochip Company, Shanghai, China. Serial 4 μm sections were cut and processed for immunohistochemistry. Tissue sections were processed for immunohistochemical staining as previously described [3]. Briefly, sections were deparaffinized, hydrated, and treated with 3% hydrogen peroxide for 15 minutes at room temperature to block the endogenous peroxidase activity. The sections were then rinsed twice with phosphate-buffered saline (PBS) buffer for 5 minutes and incubated with 10% nonimmune serum (goat) for 30 minutes. Monoclonal antibodies against IL-17 (1:100; Abcam, UK) were used for incubating sections at 4 °C overnight. Slides were washed twice with PBS, and then incubated with biotinylated secondary antibody for 30 min. After secondary antibody staining, 0.05% diaminobenzidine (DAB) as chromagen (DAB; DAKO, Denmark) was used, and then the nuclei were counterstained with hematoxylin. The distribution of IL-17-stained cells and quantitation of staining results were performed using light microscopy by two independent investigators without knowledge of the clinical data. The investigators had different scores on three samples. In case of
different scores, the consensus score was determined and the mean value of the 2 cores was considered representative of 1 patient. A 4-tier scale system was used to evaluate the intensity of cytoplasmic staining. Immunoreactivity was scored as follows: 0 = negative result; 1 = weak immunoreactivity; 2 = moderate immunoreactivity; and 3 = strong immunoreactivity. For further analysis, the patients were divided into two groups, low IL-17 expression (0–1) versus high IL-17 expression (2–3).
3.2.
Statistical analysis
Associations between clinicopathologic variables and immunostaining results were analyzed by chi-square test. Overall survival was calculated by the Kaplan–Meier method. The impact of the expression of IL-17 on the time to recurrence of JNA patients was investigated by Kaplan–Meier survival curves. The significance of the difference between groups was compared using the log-rank test. The Cox proportional hazards model served for multivariate survival analysis. A p value stage IIa) Volume of intraoperative hemorrhage (ml) (≤800 ml vs. >800 ml) JNA operation history (yes vs. no) Operation approach (with endoscope vs. without endoscope)
Univariate
Multivariate
HR
95% CI
p
HR
95% CI
p
6.379 0.859 7.197 2.469 1.888 0.758
1.480–27.494 0.755–0.977 0.965–53.682 0.904–6.745 0.794–4.489 0.253–2.270
0.013 0.021 0.054 0.078 0.150 0.620
4.488 0.896
1.011–19.924 0.781–1.028
0.048 0.117
HR = hazard ratio; CI = confidence interval at 95% level; DFS, disease free survival; JNA, juvenile nasopharyngeal angiofibroma.
in pericytes and irregular smooth muscle cells, but the matured vascular endothelial cells showed no IL-17 reactivity. The majority of stromal cells have an undifferentiated phenotype which can show smooth muscle or endothelial differentiation [9]. The immunoreaction of IL-17 in stromal components was concentrated in the less differentiated and plump cells. It has been known that TGF-β is essential for the initial differentiation and expansion of Th17 cells [54]. In JNA specimens, activated TGF-β was identified both in stromal and vascular cells, which indicates that TGF-β may play an important role in the differentiation and expansion of Th17 cells associated with JNA [43]. Moreover, we evaluated the correlation between the levels of IL-17 expressions and clinicopathologic features in JNA. Previous studies suggested that patient's age was one of the factors that had an effect on tumor recurrence of JNA [2–4]. Our study showed that IL-17-producing cells in JNA were negatively associated with patient's age, and patients with more infiltration of IL-17-producing cells had significantly higher recurrence rates than those with less infiltration of IL-17producing cells, which suggests that IL-17-producing cells may carry direct or indirect pro-tumor effect in JNA. Studies on gastric cancer demonstrated that the increased level of Th17 cells was associated with clinical stage [55]. In this study, the level of IL-17 was not associated with tumor stage in patients with JNA. This result may be attributed to a small number of patients collected in our study. We further assessed the prognostic value of IL-17 expression in JNA. In univariate Cox regression analysis, the level of IL-17 expression and patient's age were significantly associated with DFS. Multivariate analysis indicated that patients with high infiltration of IL-17-producing cells had significantly poorer DFS than those with low infiltration of IL-17-producing cells. Of all clinicopathological features, IL-17 level showed to be an independent factor predicting prognosis. In conclusion, our data demonstrated that a high level of IL-17-producing cells was negatively associated with patient's age in JNA. Patients with high infiltration of IL-17-producing cells had significantly higher tumor recurrence rates. Extensive infiltration of IL-17-producing cells in JNA microenvironment is an independent poor prognostic factor for shorter disease-free survival. IL-17-producing cells have been identified in pericytes, irregular smooth muscle cells, and undifferentiated stromal cells in JNA, which suggests a possible role for IL-17 in the pathogenesis of JNA. Future study of the role of IL-17 may provide more promising therapeutic methods for curing extensive JNA.
Acknowledgments The authors would like to thank Dr. Lichun Zhang and Peter van Maanen for their valuable contributions to the revision of the manuscript. This work was supported by the National Science Foundation of China grant no. 81371077.
REFERENCES
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[35] Schuon R, Brieger J, Heinrich UR, et al. Immunohistochemical analysis of growth mechanisms in juvenile nasopharyngeal angiofibroma. Eur Arch Otorhinolaryngol 2007;264:389–94. [36] Ngan BY, Forte V, Campisi P. Molecular angiogenic signaling in angiofibromas after embolization: implications for therapy. Arch Otolaryngol Head Neck Surg 2008;134:1170–6. [37] Renkonen S, Heikkila P, Haglund C, et al. Tenascin-C, GLUT-1, and syndecan-2 expression in juvenile nasopharyngeal angiofibroma: correlations to vessel density and tumor stage. Head Neck 2013;35:1036–42. [38] Silveira SM, Custodio DMA, Butugan O, et al. Tumor microenvironmental genomic alterations in juvenile nasopharyngeal angiofibroma. Head Neck 2012;34:485–92. [39] Gramann M, Wendler O, Haeberle L, et al. Expression of collagen types I, II and III in juvenile angiofibromas. Cells Tissues Organs 2009;189:403–9. [40] Gramann M, Wendler O, Haeberle L, et al. Prominent collagen type VI expression in juvenile angiofibromas. Histochem Cell Biol 2009;131:155–64. [41] Sternberg SS. Pathology of juvenile nasopharyngeal angiofibroma; a lesion of adolescent males. Cancer 1954;7:15–28. [42] Zhang PJ, Weber R, Liang HH, et al. Growth factors and receptors in juvenile nasopharyngeal angiofibroma and nasal polyps: an immunohistochemical study. Arch Pathol Lab Med 2003;127:1480–4. [43] Dillard DG, Cohen C, Muller S, et al. Immunolocalization of activated transforming growth factor beta1 in juvenile nasopharyngeal angiofibroma. Arch Otolaryngol Head Neck Surg 2000;126:723–5. [44] Nagai MA, Butugan O, Logullo A, et al. Expression of growth factors, proto-oncogenes, and p53 in nasopharyngeal angiofibromas. Laryngoscope 1996;106:190–5. [45] Ponti G, Losi L, Pellacani G, et al. Wnt pathway, angiogenetic and hormonal markers in sporadic and familial adenomatous polyposis-associated juvenile nasopharyngeal angiofibromas (JNA). Appl Immunohistochem Mol Morphol 2008;16:173–8. [46] Renkonen S, Hayry V, Heikkila P, et al. Stem cell-related proteins C-KIT, C-MYC and BMI-1 in juvenile nasopharyngeal angiofibroma — do they have a role. Virchows Arch 2011;458:189–95. [47] Duerr S, Wendler O, Aigner T, et al. Metalloproteinases in juvenile angiofibroma — a collagen rich tumor. Hum Pathol 2008;39:259–68. [48] Nonogaki S, Campos HG, Butugan O, et al. Markers of vascular differentiation, proliferation and tissue remodeling in juvenile nasopharyngeal angiofibromas. Exp Ther Med 2010;1:921–6. [49] Schlauder SM, Knapp C, Steffensen TS, et al. Aromatase may play a critical role in the pathogenesis of juvenile nasopharyngeal angiofibroma. Fetal Pediatr Pathol 2009;28:232–8. [50] Bettelli E, Korn T, Oukka M, et al. Induction and effector functions of T(H)17 cells. Nature 2008;453:1051–7. [51] Korn T, Oukka M, Kuchroo V, et al. Th17 cells: effector T cells with inflammatory properties. Semin Immunol 2007;19:362–71. [52] Stockinger B, Veldhoen M. Differentiation and function of Th17 T cells. Curr Opin Immunol 2007;19:281–6. [53] Tong Z, Yang XO, Yan H, et al. A protective role by interleukin-17 F in colon tumorigenesis. PLoS One 2012;7:e34959. [54] Qi W, Huang X, Wang J. Correlation between Th17 cells and tumor microenvironment. Cell Immunol 2013;285:18–22. [55] Iida T, Iwahashi M, Katsuda M, et al. Tumor-infiltrating CD4+ Th17 cells produce IL-17 in tumor microenvironment and promote tumor progression in human gastric cancer. Oncol Rep 2011;25:1271–7. http://dx.doi.org/10.3892/or.2011.1201 [Epub 2011 Mar 1].
Available online at www.sciencedirect.com
ScienceDirect www.elsevier.com/locate/amjoto
The presence of tumor-infiltrating IL-17-producing cells in juvenile nasopharyngeal angiofibroma tumor microenvironment is a poor prognostic factor☆ Xicai Sun, MD 1 , Limin Guo, MD 1 , Huan Wang, MD 1 , Huapeng Yu, MD 1 , Jingjing Wang, MD, Xinjun Meng, MD, Zhuofu Liu, MD, Juan Liu, MD, Li Hu, MD, Han Li, MD, Dehui Wang, MD⁎ Department of Otolaryngology, Eye, Ear, Nose and Throat Hospital, Shanghai Medical College, Fudan University, Shanghai, China
ARTI CLE I NFO
A BS TRACT
Article history:
Background: Although juvenile nasopharyngeal angiofibroma (JNA) is a benign tumor
Received 12 March 2014
histologically, it demonstrates aggressive propensity of locally destructive growth causing bone erosion. The patients with JNA remain high recurrence rate after surgical excision. Th17 cells secrete the proinflammatory cytokine interleukin-17 (IL-17), and play an important role in carcinogenesis and tumor progression. So far, no studies have focused on the significance of IL17-producing cells in the JNA tumor microenvironment. The current study was designed to investigate the localization and level of tumor-infiltrating IL-17-producing cells in JNA microenvironment. The presence and number of IL-17-producing cells were further analyzed for a possible association with clinicopathological features and disease outcome. Materials and methods: Immunohistochemistry was used to analyze the expression of IL-17 in a tissue microarray from 70 patients with JNA and 10 control subjects. Correlations between the levels of IL-17 expression and clinicopathologic variables, as well as tumor recurrence, were assessed. Results: In vessels, the IL-17-producing cells were identified in pericytes and irregular smooth muscle cells, but the matured vascular endothelial cells showed no IL-17 reactivity. The expression of IL-17 in stromal cells was concentrated in the less differentiated and plump cells that contained a central hypochromatic nucleus and single small nucleolus. Chi-square test showed that tumor stage (p = 0.09), operation history (p = 0.828), operation approach (p = 0.159), and volume of intraoperative hemorrhage (p = 0.352) were not associated with the expression of IL-17 in JNA patients. However, intratumoral IL-17producing cells were negatively associated with patient's age (p = 0.004). Furthermore, we found that patients with extensive infiltration of IL-17-producing cells had significantly higher recurrence rates than those with less infiltration of IL-17-producing cells (p = 0.028). Log rank analysis showed that JNA patients with high levels of IL-17 had significantly shorter disease free survival (DFS) than those with low levels of IL-17 (p = 0.004). Univariate Cox regression analysis suggested that IL-17 and patient's age were significantly associated
☆
Declaration of interest: The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper. ⁎ Corresponding author at: Department of Otolaryngology, Eye, Ear, Nose and Throat Hospital, Shanghai Medical College, Fudan University, 83 Fenyang Road, Shanghai, 200031, China. Tel.: + 86 21 64 377 134 788; fax: + 86 21 64 377 151. E-mail address: [email protected] (D. Wang). 1 Drs. Xicai Sun, Limin Guo, Huan Wang, and Huapeng Yu contributed equally to this work. http://dx.doi.org/10.1016/j.amjoto.2014.06.001 0196-0709/© 2014 Elsevier Inc. All rights reserved.
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with DFS. Multivariate analysis indicated that high infiltration with IL-17-producing cells was associated with poor DFS. Of all clinicopathological features, IL-17 level was an independent factor predicting the patient's prognosis. Conclusion: In JNA patients, a high level of IL-17-producing cells was negatively associated with patient's age. Patients with extensive infiltration of IL-17-producing cells had significantly higher tumor recurrence rates. High infiltration of IL-17-producing cells in JNA microenvironment is an independent poor prognostic factor for shorter disease-free survival. Future studies further focusing on the role of IL-17 may provide more promising therapeutic methods for extensive JNA tumors. © 2014 Elsevier Inc. All rights reserved.
1.
Introduction
Juvenile nasopharyngeal angiofibroma (JNA) is a rare, highly vascularized benign tumor originating from the superior margin of the sphenopalatine foramen or pterygoid canal [1]. It accounts for approximately 0.05% of all head and neck tumors, and occurs almost exclusively in adolescent males. Although JNA is a benign tumor histologically, it has a propensity to grow destructively and erode bone. Surgery is the mainstay of treatment, but the recurrence rate after surgical excision of JNA is high and fairly heterogeneous, varying from 20% to over 50% [2–7]. Histopathology of JNA shows that it is composed of stromal cells and proliferating irregular vascular components. The typical stromal cells in JNA are fibroblasts in a dense collagen matrix [8,9]. However, it has been found that JNA is also infiltrated by numerous inflammatory cells. Substantial evidence indicates that mast cells and T-lymphocytes are identified as major cellular populations in JNA as they represented 30% of the cells in studied tumor specimens [10]. It has been shown that the inflammatory microenvironment plays a critical role in tumor progression [11,12]. Tumor cells and host immune cells interact with each other to create the tumor microenvironment. CD4+ T cells are the major component of immune cells in the tumor microenvironment, and play an important role in tumor regression and evasion [13,14]. CD4+ T helper 17 (Th17) cells have been identified as a novel subset of CD4+ T-lymphocytes, and are characterized by its signature cytokine interleukin-17 (IL-17). IL-17 is a proinflammatory cytokine, and has been implicated in inflammatory and autoimmune disease. Furthermore, several studies on skin cancer, hepatocellular carcinoma, gallbladder carcinoma, laryngeal, gastric, breast, colorectal, pancreatic and lung cancer have demonstrated that IL-17 exhibits tumorpromoting functions [15–23]. However, in acute leukemia, advanced epithelial ovarian cancer and melanoma patients, the expression of IL-17 was reported to have an anti-tumor effect by promoting cytotoxic T-lymphocytes' (CTL) activities, reducing angiogenesis and the recruitment of effector Th1 cells to the tumor [24–26]. This contradictory result may be attributed to variant tumor immunogenicity and a different role of IL-17 in different tumor stages [27]. Recently, Wendler et al. showed that infiltration of JNA tumors by CD4+ and CD8+ T-lymphocytes was evident in immunofluorescent stainings [10]. So far, no studies have focused on the significance of IL-17-producing cells in the JNA tumor microenvironment and the role of IL-17 in JNA remains unclear. Aim of the
present study was to investigate the level of tumor-infiltrating IL17-producing cells in JNA microenvironment, and analyze its association with clinicopathological features and disease outcome.
2.
Materials and methods
2.1.
Patients and tissue specimens
Paraffin-embedded specimens from 70 patients with JNA who underwent surgical resection in the Eye, Ear, Nose and Throat Hospital of Fudan University between 2003 and 2007 were enrolled in the study. All patients suffered from a histologically proven JNA tumor. Clinical data were obtained from patients' medical records. All JNA patients were male and ranged in age from 9 to 41 years old (median age, 17 years) at the time of surgery. The normal middle turbinates of 10 male patients with a median age of 21.4 years (range, 13–26 years) were used as normal controls. The clinicopathological characteristics of patients with JNA are summarized in Table 1. Tumor staging of JNA was based on the classification of Radkowski and colleagues [28]. The study protocol was approved by the institutional review board of Fudan University.
3.
Patients' follow-up
Until August 31, 2013, complete follow-up data were available for 55 patients. The follow-up period ranged from 69 to 127 months, with a mean of 95 months. Completeness of the resection was defined as no macroscopic residual tumor and was further confirmed with histology of surgical margins, as well as postoperative CT scan or MRI scan. Recurrence was defined as clinically or radiologically demonstrating tumor presence in the nasopharynx, nasal cavity, or neighboring structures, with confirmed major symptoms including epistaxis or nasal obstruction after the initial surgical treatment. There are distinct therapeutic principles and surgical techniques for JNA in different hospitals, which may interfere with the analysis of relapse of JNA. So, the history of previous operations at other hospitals was not considered for the recurrence analysis in this study.
3.1.
Tissue microarray and immunohistochemical assay
A tissue microarray for JNA was prepared in our previous report [3]. The tissue samples included 70 JNA tumor specimens and 10 normal middle turbinates. Briefly,
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Table 1 – Clinicopathological characteristics and their correlation with IL-17 immunoreactivity in JNA patients assessed with chi-square test. Variables
Total number Sex Male Female Median age, years ≤ 17 >17 Tumor stage Stage Ia Stage Ib Stage IIa Stage IIb Stage IIc Stage IIIa Stage IIIb Operation history No Yes Operation approach With endoscope Without endoscope Volume of intraoperative hemorrhage (ml) ≤ 800 >800
No. of patients
Levels of IL-17 expressions (scores)
p Value
0
1
2
3
70 0 17 39 31
0 1
4 13
15 11
20 6
0.004
4 7 6 7 34 5 7
0 0 1 0 0 0 0
0 3 1 0 8 1 4
1 1 2 2 16 3 1
3 2 2 5 10 1 2
0.09
40 30
1 0
10 7
14 12
15 11
0.828
17 53
1 0
2 15
6 20
8 18
0.159
26 44
1 0
7 10
7 19
11 15
0.352
70
Abbreviation: JNA, juvenile nasopharyngeal angiofibroma.
formalin-fixed and paraffin-embedded surgical tissue samples were obtained from the Department of Otolaryngology– Head and Neck Surgery, Eye, Ear, Nose, and Throat Hospital, Fudan University, China. Two histopathologically representative regions of the tumor specimen were defined on the respective hematoxylin-eosin stained sections and marked on the surface of the standard paraffin block. Two cores from each tumor block were punched using a biopsy needle (1.5 mm in diameter). All tissue cores were put into a tissue microarray (TMA). Samples were processed in collaboration with Shanghai Biochip Company, Shanghai, China. Serial 4 μm sections were cut and processed for immunohistochemistry. Tissue sections were processed for immunohistochemical staining as previously described [3]. Briefly, sections were deparaffinized, hydrated, and treated with 3% hydrogen peroxide for 15 minutes at room temperature to block the endogenous peroxidase activity. The sections were then rinsed twice with phosphate-buffered saline (PBS) buffer for 5 minutes and incubated with 10% nonimmune serum (goat) for 30 minutes. Monoclonal antibodies against IL-17 (1:100; Abcam, UK) were used for incubating sections at 4 °C overnight. Slides were washed twice with PBS, and then incubated with biotinylated secondary antibody for 30 min. After secondary antibody staining, 0.05% diaminobenzidine (DAB) as chromagen (DAB; DAKO, Denmark) was used, and then the nuclei were counterstained with hematoxylin. The distribution of IL-17-stained cells and quantitation of staining results were performed using light microscopy by two independent investigators without knowledge of the clinical data. The investigators had different scores on three samples. In case of
different scores, the consensus score was determined and the mean value of the 2 cores was considered representative of 1 patient. A 4-tier scale system was used to evaluate the intensity of cytoplasmic staining. Immunoreactivity was scored as follows: 0 = negative result; 1 = weak immunoreactivity; 2 = moderate immunoreactivity; and 3 = strong immunoreactivity. For further analysis, the patients were divided into two groups, low IL-17 expression (0–1) versus high IL-17 expression (2–3).
3.2.
Statistical analysis
Associations between clinicopathologic variables and immunostaining results were analyzed by chi-square test. Overall survival was calculated by the Kaplan–Meier method. The impact of the expression of IL-17 on the time to recurrence of JNA patients was investigated by Kaplan–Meier survival curves. The significance of the difference between groups was compared using the log-rank test. The Cox proportional hazards model served for multivariate survival analysis. A p value stage IIa) Volume of intraoperative hemorrhage (ml) (≤800 ml vs. >800 ml) JNA operation history (yes vs. no) Operation approach (with endoscope vs. without endoscope)
Univariate
Multivariate
HR
95% CI
p
HR
95% CI
p
6.379 0.859 7.197 2.469 1.888 0.758
1.480–27.494 0.755–0.977 0.965–53.682 0.904–6.745 0.794–4.489 0.253–2.270
0.013 0.021 0.054 0.078 0.150 0.620
4.488 0.896
1.011–19.924 0.781–1.028
0.048 0.117
HR = hazard ratio; CI = confidence interval at 95% level; DFS, disease free survival; JNA, juvenile nasopharyngeal angiofibroma.
in pericytes and irregular smooth muscle cells, but the matured vascular endothelial cells showed no IL-17 reactivity. The majority of stromal cells have an undifferentiated phenotype which can show smooth muscle or endothelial differentiation [9]. The immunoreaction of IL-17 in stromal components was concentrated in the less differentiated and plump cells. It has been known that TGF-β is essential for the initial differentiation and expansion of Th17 cells [54]. In JNA specimens, activated TGF-β was identified both in stromal and vascular cells, which indicates that TGF-β may play an important role in the differentiation and expansion of Th17 cells associated with JNA [43]. Moreover, we evaluated the correlation between the levels of IL-17 expressions and clinicopathologic features in JNA. Previous studies suggested that patient's age was one of the factors that had an effect on tumor recurrence of JNA [2–4]. Our study showed that IL-17-producing cells in JNA were negatively associated with patient's age, and patients with more infiltration of IL-17-producing cells had significantly higher recurrence rates than those with less infiltration of IL-17producing cells, which suggests that IL-17-producing cells may carry direct or indirect pro-tumor effect in JNA. Studies on gastric cancer demonstrated that the increased level of Th17 cells was associated with clinical stage [55]. In this study, the level of IL-17 was not associated with tumor stage in patients with JNA. This result may be attributed to a small number of patients collected in our study. We further assessed the prognostic value of IL-17 expression in JNA. In univariate Cox regression analysis, the level of IL-17 expression and patient's age were significantly associated with DFS. Multivariate analysis indicated that patients with high infiltration of IL-17-producing cells had significantly poorer DFS than those with low infiltration of IL-17-producing cells. Of all clinicopathological features, IL-17 level showed to be an independent factor predicting prognosis. In conclusion, our data demonstrated that a high level of IL-17-producing cells was negatively associated with patient's age in JNA. Patients with high infiltration of IL-17-producing cells had significantly higher tumor recurrence rates. Extensive infiltration of IL-17-producing cells in JNA microenvironment is an independent poor prognostic factor for shorter disease-free survival. IL-17-producing cells have been identified in pericytes, irregular smooth muscle cells, and undifferentiated stromal cells in JNA, which suggests a possible role for IL-17 in the pathogenesis of JNA. Future study of the role of IL-17 may provide more promising therapeutic methods for curing extensive JNA.
Acknowledgments The authors would like to thank Dr. Lichun Zhang and Peter van Maanen for their valuable contributions to the revision of the manuscript. This work was supported by the National Science Foundation of China grant no. 81371077.
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