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Relationship between inflammation and the severity of Recurrent Respiratory Papillomatosis
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Relationship between inflammation and the severity of Recurrent Respiratory Papillomatosis Vivian Narana Ribeiro El Achkar (DDS)a, Andressa Duarteb, Román Carlosc, Jorge Esquiche Leónd, Alfredo Ribeiro-Silvae, Shirley Shizue Nagata Pignatarif, ⁎ Estela Kaminagakuraa, a
Department of Bioscience and Oral Diagnosis, Institute of Science and Technology, São Paulo State University - UNESP, São José dos Campos, Brazil Department of Pathology, Ribeirão Preto School of Medicine, University of São Paulo, Ribeirão Preto, Brazil c Pathology Department, Hospital Herrera-Llerandi, Guatemala City, Guatemala d Oral Pathology, Department of Stomatology, Public Oral Health and Forensic Dentistry, School of Dentistry of Ribeirão Preto, University of São Paulo, Ribeirão Preto, Brazil e Department of Pathology and Legal Medicine, Ribeirão Preto School of Medicine, University of São Paulo, Ribeirão Preto, Brazil f Department of Otorhinolaryngology and Head and Neck Surgery, São Paulo Federal University, Brazil b
A R T I C LE I N FO
A B S T R A C T
Keywords: Laryngeal papillomatosis Human papillomavirus Inflammatory cells Squamous cell papillomatosis
Objective: To characterize inflammatory cells in Recurrent Respiratory Papillomatosis (RRP) and to correlate it with severity using the Derkay laryngoscopic scale. Materials and methods: The data and biopsies from 36 patients with Juvenile (JRRP) and 56 patients with Adult (ARRP) were collected and analyzed under light microscopy. The patients were separated into groups according to the Derkay index: ≥20 for the most severe and < 20 for the less severe cases. Immunohistochemical analysis using CD3, CD4, CD8, CD15, CD20, CD68, FoxP3 and MUM-1 antibodies was performed, and the inflammatory cells were quantified. All the clinicopathological characteristics and the results of the immunohistochemical analysis were compared among the groups proposed using the Chi-Square test and correlated through the Spearman correlation test. Results: The ARRP showed significantly higher quantities of CD3+, CD8+ and MUM1+ cells (p < .05) than the JRRP samples. The presence of CD15+ cells showed positive correlation with the Derkay index (p < .05), while the MUM-1+ cells showed an inverse correlation (p = .01). Conclusion: There are differences between the inflammatory cells population in the juvenile and adult groups and it can be related to disease severity.
1. Introduction Recurrent Respiratory Papillomatosis (RRP) is a serious disease that occurs in two clinical forms: juvenile (JRRP) and adult (ARRP); the former occurs predominantly before 5 years of age and affects both females and males equally; the latter is prevalent in male patients between 20 and 40 years of age [1]. Human papillomavirus (HPV) infection is responsible for the development of the disease, primarily the low-risk types HPV 6 and 11 [1,2]. HPV type 6 is the most common [3,4] and type 11 is predominant in the most aggressive cases [1,5]. There are scales that assist in therapeutic decision-making and in
the identification of aggressive cases, including: Voice Handicap Index10 (VHI-10) [6], Roughness, Breathiness, Asthenia, Strain (GRBAS) [7] and the laryngoscopic scale of Derkay et al. (1998) [8]. The immune system is important in preventing lesion progression and encouraging HPV-infection clearance [9]. There are few studies correlating the aggressiveness of RRP to inflammation and the results are not uniform [10,11]. The severity of RRP has been correlated with greater numbers of CD83+ mature dendritic cells (DC) in the epithelium and there is evidence of the involvement of migration of these cells and their retention in the epithelial layer [11]. In RRP, the imbalance between Th1 and Th2 immune responses may contribute to the persistence of infection and recurrence of this disease [10].
⁎
Corresponding author at: Department of Bioscience and Oral Diagnosis, Science and Technology Institute, São Paulo State University - UNESP, Engenheiro Francisco José Longo, 777 Jardim São Dimas, São José dos Campos, SP 12245-000, Brazil. E-mail addresses: [email protected] (V.N.R. El Achkar), [email protected] (A. Duarte), [email protected] (R. Carlos), [email protected] (J.E. León), [email protected] (A. Ribeiro-Silva), [email protected] (S.S.N. Pignatari), [email protected] (E. Kaminagakura). https://doi.org/10.1016/j.amjoto.2019.102321 Received 24 September 2019 0196-0709/ © 2019 Elsevier Inc. All rights reserved.
Please cite this article as: Vivian Narana Ribeiro El Achkar, et al., Am J Otolaryngol, https://doi.org/10.1016/j.amjoto.2019.102321
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The membranous and/or cytoplasmic labeling was quantified by the investigator (VNREA, AD), in a blind count, without knowledge of any distinction of the groups and samples. The discrepancy count was analyzed separately by another investigator (EK). The TMA slides were scanned, using a Pannoramic DESK device (3DHistech®, Budapest, Hungary). For counting, the Pannoramic Viewer program (3DHistech®, Budapest, Hungary) was used. Ten areas representative of the lesion were used for the count magnified by 40×. For the results, the numbers obtained from the mean of this count were used [16,17].
Noting the important role of inflammation, or its absence, related to the above-mentioned disease, the present study aims to identify possible differences in the population of inflammatory cells secreting proinflammatory cytokines and chemokines between patients with low and high Derkay indexes. The results may detect prognostic factors that lead to the early recognition of aggressive cases, and also determine possible causes that lead to the varied behavior of this disease that may occur in isolated cases, multiple relapses or spontaneous remission. 2. Materials and methods
2.4. Statistical analysis 2.1. Samples Statistical analyses were performed using GraphPad Prism version 7.00 software (La Jolla, California-USA). The final results were presented as mean, standard deviation (SD) and percentage (%). Differences were tested by the t-Student, q-square test (x2) or exact Fisher test and correlated by the Spearman test. p < .05 results were considered statistically significant.
Retrospective samples of JRRP and ARRP were collected at different centers: 3 samples from the Pediatric Otorhinolaryngology Outpatient Clinic of the Paulista Medical School/UNIFESP; 70 from the Ribeirão Preto Medical School/USP and 19 from the Head and Neck Clinical Center in Guatemala. A total of 36 juvenile samples and 56 adults' samples were used, of which 46 were from Brazil and 10 from Guatemala, and the sampling period was from 1997 to 2016. The Research Ethical Committee of the São Paulo State University approved this study (CAAE: 50419615.1.0000.0077, approval number: 1.419.232). Archived formalin-fixed paraffin-embedded tissue blocks from biopsies or surgical specimens of all cases were available, and when required, data was recorded in clinical records and reports. Clinical data such as age at the time of diagnosis, sex, location of lesions, relapses, adjuvant treatment (HPV vaccine and interferon-alfa) signs and symptoms were collected from the patients' medical records. Patients aged ≤16 years were included in the JRRP group and > 16 years in the ARRP group [12]. The H&E slides, obtained from biopsies or surgical specimens, were reviewed and described. In addition, the laryngoscopic scale of Derkay et al. (1998) [8] was applied to obtain the score for each case at the time when the first biopsy has taken. The samples were separated into groups to distinguish the cases with greater (score ≥ 20) and lesser (score < 20) severity [12,13].
3. Results 3.1. Clinical findings From the clinical data, for the juvenile group, the mean age was 4.94 years, ranging from 1 to 15 years, with 18 (50.00%) cases in males and 18 (50.00%) in females (ratio of 1:1). Additionally, 6 (16.67%) patients received some adjuvant treatment and 1 (2.78%) patient was receiving treatment for asthma, and 6 (18.18%) required tracheostomy. The mean age of the adult group was 41.62 years, ranging from 19 to 74 years of age; 24 (50%) patients were over 40 years old, with 41 (73.21%) cases in males and 15 (26.78%) in females (ratio of 1: 2.73), 2 (4.34%) cases there was a need to perform tracheostomy. The clinical data is summarized in Table 2. Among the adult cases there were 2 (4.34%) patients who had human immunodeficiency virus (HIV). In 1 (2.17%) patient, malignant transformation was observed. These cases were excluded from the statistical analysis, because this variation may imply a change in cell count. Regarding the Derkay index, the mean for the ARRP group was 8.26 (min. 3 and max. 27); for the JRRP group it was 10.97 (min. 4 and max. 31), being statistically significant for the t-test with p = .02 (Table 3) and for the Spearman correlation test (p < .05). The latter demonstrated that the lower the age, then the higher the Derkay score, that is, the greater the severity. Among the cases selected, the larynx and oropharynx were most commonly affected, with additional lesions in the soft palate (1.08%), pharynx (1.08%) and base of tongue (1.08%). The most commonly affected sites were the true vocal cords in JRRP (50.24%), followed by false vocal cords (12.68%); in adult patients the true vocal cords were most commonly affected (p < .05).
2.2. Construction of the Tissue Micro-Array (TMA) After a morphological review of all the cases, tumor regions were demarcated from at least one original block for each case and 4 areas for each case were selected, in which there was a representative area of inflammation, as follows: predominantly epithelial or predominantly underlying connective tissue. The tissue microarray (TMA) blocks were constructed as previously described [14]. 2.3. Immunohistochemistry (IHC) The IHC method is similar to that reported by Kaminagakura et al (2007) [15]. All information about the primary antibodies is reported in Table 1.
Table 1 List of primary antibodies including: dilution, clone, positive control, and manufacturer. Antibody
Dilution
Clone
Control +
Manufacturer
CD3 CD4 CD8 CD20 CD68 CD15 MUM-1 FoxP3
1:100 1:500 1:100 1:400 1:3000 1:40 1:100 1:100
Policlonal 4B12 C8/144B L26 KP1 BY87 EAU32 SP97
Tonsil Tonsil Tonsil Lymph node Tonsil Tonsil Tonsil Tonsil
DakoCytomation, Glostrup, Denmark Leica Biosystems, Wetzlar, Germany DakoCytomation, Glostrup, Denmark DakoCytomation, Glostrup, Denmark DakoCytomation, Glostrup, Denmark Leica Biosystems, Wetzlar, Germany Leica Biosystems, Wetzlar, Germany Spring Bioscience, Pleasanton, California
2
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Table 2 Clinical data of all JRRP and ARRP samples. Clinicopathological features #
Age ± SD Female n/t (%) Male n/t (%) Tracheostomy n/t (%) Adj treat§ n/t (%) Death n/t (%)
Table 5 Spearman correlation test between the Derkay index and the number of cells. Spearman r negative values represent an inverse proportional relationship, while a positive value denotes a proportional relationship.
JRRP
ARRP
4.94 ± 3.30 18/36 (50.00) 18/36 (50.00) 6/33 (18.18) 6/36 (16.67) 1/36 (2.78)
41.62 ± 14.01 15/56 (26.78) 41/56 (73.21) 2/46 (4.34) 2/46 (4.34) 1/46 (2.17)
Legend: #standard deviation; §adjuvant treatment; n/t: number of samples/total of samples. Table 3 Mean and standard deviation of the total number of quantifications of inflammatory cells and comparison between JRRP and ARRP. Cells
JRRP (Mean ± SD)
ARRP (Mean ± SD)
P
CD3 CD4 CD8 CD15 CD20 CD68 FoxP3 MUM-1
10.55 ± 6.97 4.07 ± 3.35 3.09 ± 3.90 5.33 ± 5.72 1.34 ± 2.12 5.21 ± 4.21 3.45 ± 4.16 2.62 ± 5.54
23.35 ± 18.59 7.08 ± 7.27 9.94 ± 11.39 4.89 ± 4.87 4.18 ± 10.26 4.20 ± 2.99 5.59 ± 7.91 12.24 ± 15.30
< 0.05 0.23 < 0.05 0.87 0.33 0.28 0.42 < 0.05
Table 4 Spearman correlation test between the age and number of cells. Spearman r negative values represent an inverse proportion relationship, while a positive value denotes a proportional relationship. P
CD3 CD4 CD8 CD15 CD20 CD68 FoxP3 MUM-1
77 78 76 79 77 77 77 76
0.38 0.20 0.39 −0.05 0.28 −0.03 0.14 0.57
< 0.05 0.07 < 0.05 0.63 0.01 0.77 0.19 < 0.05
P
CD3 CD4 CD8 CD15 CD20 CD68 FoxP3 MUM-1
77 78 76 79 77 77 77 76
0.02 −0.07 −0.16 0.32 −0.13 −0.00 −0.08 −0.28
0.80 0.53 0.15 < 0.05 0.22 0.97 0.46 0.01
RRP is an uncommon disease that can develop a severe clinical course leading to death [1,18]. Such aggressiveness is related to age, time of evolution and type of HPV [19,20]. Additionally, the immune system may also play an important role in the clinical course of the disease [11]. CD3+ T cells represents the total number of T lymphocytes [17]. In the present study, these cells were present in greater numbers in adult patients; the group that presented less severe RRP. CD3+ T cells are important to prevent malignant transformation of leukoplakia, since patients who developed head and neck squamous cell carcinoma (HNSCC) had significantly fewer CD3+ cells than those in both the dysplastic epithelium and subepithelial connective tissue [21]. In our study, the least quantity of CD8+ cells was observed in juvenile patients, which was the group that showed a more severe clinical course similar to previous results [22,23]. The critical period for the development of the immune system occurs between 4 and 6 years of age [11]; this period covers the mean age of JRRP patients in our study. The immaturity of the juvenile immune system can lead to a decrease in the processing and/or presentation of antigens or a decrease in the secretion of pro-inflammatory cytokines, which could contribute to the severity of RRP. However, some authors have reported an inversion in the proportion of CD4+/CD8+ cells and high production of cytokines mRNA by the CD56+/NK cells in RRP when compared to healthy controls. These findings suggest that a dysregulated antiviral response can be a reason for persistent infection in RRP patients [24,25]. Additionally, we have demonstrated a tendency for a higher number of CD8+ cells in low-grade dysplasia, while the number of CD4+ cells was similar in the different degrees of dysplasia. On the other hand, in lesions caused by high risk HPV, the results are controversial; for example, in cervical intraepithelial neoplasia (CIN), a high viral load leads to the predominance of CD8+ T lymphocytes [25]. Meanwhile, a high expression of CD8 cells, B cells and IL2 was positively correlated with an improved prognosis of HPV+ HNSCC [26]. Regulatory T cells (Tregs) are a subpopulation of T cells responsible for immune self-tolerance, effectively suppressing T cell activation [27]. There are several subtypes of CD4+ Tregs, such as T helper, Natural Killer (NK)-T and CD4 + CD25 + FoxP3+ [28], and the FoxP3 function stabilizes Tregs function [29]. In the present study, a tendency was observed for a smaller number of these cells (FoxP3+) in the connective tissue of the cases that have presented relapses. Effective host responses to HPV in genital warts are characterized by an active cell-mediated immune response, in which CD4+ lymphocytes and macrophages predominate, which is consistent with a delayed-type hypersensitivity reaction to foreign antigens [30]. T cell activity can be inhibited by a high frequency of neutrophils, because neutrophils and tumor cells promote an IL6 and IL8 rich environment in cervical cancer [9]. A high infiltration of neutrophils was negatively correlated with HPV+ HNSCC survival [26] and was
The mean of the cell quantification was used to compare the JRRP and ARRP groups (Table 3), to correlate age (Table 4) and the Derkay index (Table 5) with the number of inflammatory cells and also to correlate the inflammatory cells with each characteristic. The Derkay subgroups < 20 and ≥ 20, irrespective of age, were also correlated with the presence of inflammatory cells. Regarding the dysplasia grade, CD3+ and CD8+ cells were correlated with low-grade, while CD68+ cells were related to high-grade of dysplasia. The immunohistochemical findings obtained concerning the inflammatory cells of the JRRP and ARRP groups are shown in Fig. 1. HPV 6 was detected in 53 (66.3%) samples and was significantly more prevalent in the 34 (77.3%) ARRP individuals compared with 19 (52.8%) JRRP patients (p = .03). HPV 11 was detected in 30 (37.5%) samples and was significantly more prevalent in the JRRP group of individuals (52.8%) compared with ARRP cases (25.0%) (p = .02). Twelve cases were excluded from DNA extraction and HPV genotyping due to the quality of the material from the paraffin blocks (unpublished data). No statistically significant association was found between the HPV genotyping and inflammatory cells type.
Spearman r
Spearman r
4. Discussion
3.2. Correlation between inflammatory cells and clinical and histological characteristics and HPV genotyping
n
n
P values in bold are statistically significant.
P values in bold are statistically significant.
Cells
Cells
P values in bold are statistically significant. 3
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Fig. 1. a. Microphotography of JRRP showing few CD3+ cells in the epithelium and in connective tissue. b. CD3+ cells dispersed in the epithelium and in large amounts in connective tissue of ARRP. c. negative expression of CD8+ in juvenile sample. d. membranous positivity of CD 8+ in epithelium of ARRP group. e. absence of MUM1+ cells in JRRP while these cells were observed in connective tissue of ARRP (f). g. exocytosis area demonstrated by CD15+ cells in JRRP. 1 h. CD15+ were negative in ARRP. (DAB stain, Mayer's counterstain).
In more severe cases of JRRP, a smaller quantity of CD3+, CD8+ and MUM1+ cells was observed. The immune system imbalance can play an important role in the severity of RRP.
correlated with an increased size of colorectal carcinoma [31]. In our study, these CD15+ cells were present in the epithelium and in the connective tissue of the most RRP severe cases, especially in ulceration areas causing edema and pain. Cytokines are also important mediators regulating the tumor microenvironment. IL12 and IFNγ delivered by T cells are possibly involved in myeloid cell accumulation and macrophage polarization [26]. The presence of the M2-macrophage phenotype performs functions that are different from the M1 phenotype, acting as an immunity regulator and as an aid in tissue repair [32], in addition to stimulating tumor growth, since it is an angiogenesis promoter [33]. The presence of CD68+ cells in RRP was associated with high-degree dysplasia, and in JRRP there was an inverse proportional correlation between CD68+ and CD3+ cells. This suppressive role of the adaptive immune response of M2-macrophages is already known [33] and in this study, by means of the CD68 expression, this role was observed in juvenile patients, in whom the disease has shown a higher Derkay index. B cells with terminal differentiation and plasma cells (MUM1+) can produce antibodies [34]. In RRP, MUM1+ cells were correlated with the lowest Derkay index mainly in adult patients; the immune role of this cell was related to lower severity and may help to combat the disease. The quality of B cell responses generated by natural infection varies from person to person [35]. However, vaccination increases natural HPV immunity by boosting the antibodies level and B memory cell numbers and efficiency. Persons previously infected with HPV vaccines types, such as HPV 6, 11, 16 and 18, could also benefit from vaccination [35].
Funding For the financial support received from FAPESP (São Paulo Research Foundation, Grant: 16/24019-0 to EK) and CAPES (Coordination for the Improvement of Higher Education Personnel to VNREA). Declaration of competing interest All the authors declare that there is no conflict of interest. References [1] Richardson M, Gale N, Hille J, Zidar N. Squamous cell papilloma and squamous cell papilomatosis. In: El-Naggar AK, Chan JK, Grandis JR, Takata T, Slotweg PJ, editors. WHO classification head and neck tumours. Lyon: IARC Press; 2017. p. 93–5. [2] Davids T, Muller S, Wise JC, Johns MM, Klein A. Laryngeal Papillomatosis associated dysplasia in the adult population: an update on prevalence and HPV subtyping. Ann Otol Rhinol Laryngol 2014;123:402–8. https://doi.org/10.1177/ 0003489414526848. [3] Auborn KJ, Little RD, Platt THK, Vaccariello MA, Schildkraut CL. Replicative intermediates of human papillomavirus type 11 in laryngeal papillomas: site of replication initiation and direction of replication. Proc Natl Acad Sci U S A 1994;91:7340–4. https://doi.org/10.1073/pnas.91.15.7340. [4] Vancurova I, Wu R, Miskolci V, Sun S. Increased p50/p50 NF-KB activation in human papillomavirus type 6- or type 11-induced laryngeal papilloma tissue. J Virol 2002;76:1533–6. https://doi.org/10.1128/jvi.76.3.1533-1536.2002. [5] Mounts P, Kashima H. Association of human papillomavirus subtype and clinical course in respiratory papillomatosis. Laryngoscope 1984;94:28–33. https://doi.org/ 10.1002/lary.5540940106. [6] Rosen CA, Lee AS, Osborne J, Zullo T, Murry T. Development and validation of the voice handicap index-10. Laryngoscope 2004;114:1549–56. https://doi.org/10. 1097/00005537-200409000-00009. [7] Kuet ML, Pitman MJ. Photoangiolytic laser treatment of recurrent respiratory
5. Conclusion In summary, our results found a correlation of CD3+ and CD8+ cells with low-grade dysplasia and CD68+ with high-grade dysplasia. 4
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Am J Otolaryngol journal homepage: www.elsevier.com/locate/amjoto
Relationship between inflammation and the severity of Recurrent Respiratory Papillomatosis Vivian Narana Ribeiro El Achkar (DDS)a, Andressa Duarteb, Román Carlosc, Jorge Esquiche Leónd, Alfredo Ribeiro-Silvae, Shirley Shizue Nagata Pignatarif, ⁎ Estela Kaminagakuraa, a
Department of Bioscience and Oral Diagnosis, Institute of Science and Technology, São Paulo State University - UNESP, São José dos Campos, Brazil Department of Pathology, Ribeirão Preto School of Medicine, University of São Paulo, Ribeirão Preto, Brazil c Pathology Department, Hospital Herrera-Llerandi, Guatemala City, Guatemala d Oral Pathology, Department of Stomatology, Public Oral Health and Forensic Dentistry, School of Dentistry of Ribeirão Preto, University of São Paulo, Ribeirão Preto, Brazil e Department of Pathology and Legal Medicine, Ribeirão Preto School of Medicine, University of São Paulo, Ribeirão Preto, Brazil f Department of Otorhinolaryngology and Head and Neck Surgery, São Paulo Federal University, Brazil b
A R T I C LE I N FO
A B S T R A C T
Keywords: Laryngeal papillomatosis Human papillomavirus Inflammatory cells Squamous cell papillomatosis
Objective: To characterize inflammatory cells in Recurrent Respiratory Papillomatosis (RRP) and to correlate it with severity using the Derkay laryngoscopic scale. Materials and methods: The data and biopsies from 36 patients with Juvenile (JRRP) and 56 patients with Adult (ARRP) were collected and analyzed under light microscopy. The patients were separated into groups according to the Derkay index: ≥20 for the most severe and < 20 for the less severe cases. Immunohistochemical analysis using CD3, CD4, CD8, CD15, CD20, CD68, FoxP3 and MUM-1 antibodies was performed, and the inflammatory cells were quantified. All the clinicopathological characteristics and the results of the immunohistochemical analysis were compared among the groups proposed using the Chi-Square test and correlated through the Spearman correlation test. Results: The ARRP showed significantly higher quantities of CD3+, CD8+ and MUM1+ cells (p < .05) than the JRRP samples. The presence of CD15+ cells showed positive correlation with the Derkay index (p < .05), while the MUM-1+ cells showed an inverse correlation (p = .01). Conclusion: There are differences between the inflammatory cells population in the juvenile and adult groups and it can be related to disease severity.
1. Introduction Recurrent Respiratory Papillomatosis (RRP) is a serious disease that occurs in two clinical forms: juvenile (JRRP) and adult (ARRP); the former occurs predominantly before 5 years of age and affects both females and males equally; the latter is prevalent in male patients between 20 and 40 years of age [1]. Human papillomavirus (HPV) infection is responsible for the development of the disease, primarily the low-risk types HPV 6 and 11 [1,2]. HPV type 6 is the most common [3,4] and type 11 is predominant in the most aggressive cases [1,5]. There are scales that assist in therapeutic decision-making and in
the identification of aggressive cases, including: Voice Handicap Index10 (VHI-10) [6], Roughness, Breathiness, Asthenia, Strain (GRBAS) [7] and the laryngoscopic scale of Derkay et al. (1998) [8]. The immune system is important in preventing lesion progression and encouraging HPV-infection clearance [9]. There are few studies correlating the aggressiveness of RRP to inflammation and the results are not uniform [10,11]. The severity of RRP has been correlated with greater numbers of CD83+ mature dendritic cells (DC) in the epithelium and there is evidence of the involvement of migration of these cells and their retention in the epithelial layer [11]. In RRP, the imbalance between Th1 and Th2 immune responses may contribute to the persistence of infection and recurrence of this disease [10].
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Corresponding author at: Department of Bioscience and Oral Diagnosis, Science and Technology Institute, São Paulo State University - UNESP, Engenheiro Francisco José Longo, 777 Jardim São Dimas, São José dos Campos, SP 12245-000, Brazil. E-mail addresses: [email protected] (V.N.R. El Achkar), [email protected] (A. Duarte), [email protected] (R. Carlos), [email protected] (J.E. León), [email protected] (A. Ribeiro-Silva), [email protected] (S.S.N. Pignatari), [email protected] (E. Kaminagakura). https://doi.org/10.1016/j.amjoto.2019.102321 Received 24 September 2019 0196-0709/ © 2019 Elsevier Inc. All rights reserved.
Please cite this article as: Vivian Narana Ribeiro El Achkar, et al., Am J Otolaryngol, https://doi.org/10.1016/j.amjoto.2019.102321
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The membranous and/or cytoplasmic labeling was quantified by the investigator (VNREA, AD), in a blind count, without knowledge of any distinction of the groups and samples. The discrepancy count was analyzed separately by another investigator (EK). The TMA slides were scanned, using a Pannoramic DESK device (3DHistech®, Budapest, Hungary). For counting, the Pannoramic Viewer program (3DHistech®, Budapest, Hungary) was used. Ten areas representative of the lesion were used for the count magnified by 40×. For the results, the numbers obtained from the mean of this count were used [16,17].
Noting the important role of inflammation, or its absence, related to the above-mentioned disease, the present study aims to identify possible differences in the population of inflammatory cells secreting proinflammatory cytokines and chemokines between patients with low and high Derkay indexes. The results may detect prognostic factors that lead to the early recognition of aggressive cases, and also determine possible causes that lead to the varied behavior of this disease that may occur in isolated cases, multiple relapses or spontaneous remission. 2. Materials and methods
2.4. Statistical analysis 2.1. Samples Statistical analyses were performed using GraphPad Prism version 7.00 software (La Jolla, California-USA). The final results were presented as mean, standard deviation (SD) and percentage (%). Differences were tested by the t-Student, q-square test (x2) or exact Fisher test and correlated by the Spearman test. p < .05 results were considered statistically significant.
Retrospective samples of JRRP and ARRP were collected at different centers: 3 samples from the Pediatric Otorhinolaryngology Outpatient Clinic of the Paulista Medical School/UNIFESP; 70 from the Ribeirão Preto Medical School/USP and 19 from the Head and Neck Clinical Center in Guatemala. A total of 36 juvenile samples and 56 adults' samples were used, of which 46 were from Brazil and 10 from Guatemala, and the sampling period was from 1997 to 2016. The Research Ethical Committee of the São Paulo State University approved this study (CAAE: 50419615.1.0000.0077, approval number: 1.419.232). Archived formalin-fixed paraffin-embedded tissue blocks from biopsies or surgical specimens of all cases were available, and when required, data was recorded in clinical records and reports. Clinical data such as age at the time of diagnosis, sex, location of lesions, relapses, adjuvant treatment (HPV vaccine and interferon-alfa) signs and symptoms were collected from the patients' medical records. Patients aged ≤16 years were included in the JRRP group and > 16 years in the ARRP group [12]. The H&E slides, obtained from biopsies or surgical specimens, were reviewed and described. In addition, the laryngoscopic scale of Derkay et al. (1998) [8] was applied to obtain the score for each case at the time when the first biopsy has taken. The samples were separated into groups to distinguish the cases with greater (score ≥ 20) and lesser (score < 20) severity [12,13].
3. Results 3.1. Clinical findings From the clinical data, for the juvenile group, the mean age was 4.94 years, ranging from 1 to 15 years, with 18 (50.00%) cases in males and 18 (50.00%) in females (ratio of 1:1). Additionally, 6 (16.67%) patients received some adjuvant treatment and 1 (2.78%) patient was receiving treatment for asthma, and 6 (18.18%) required tracheostomy. The mean age of the adult group was 41.62 years, ranging from 19 to 74 years of age; 24 (50%) patients were over 40 years old, with 41 (73.21%) cases in males and 15 (26.78%) in females (ratio of 1: 2.73), 2 (4.34%) cases there was a need to perform tracheostomy. The clinical data is summarized in Table 2. Among the adult cases there were 2 (4.34%) patients who had human immunodeficiency virus (HIV). In 1 (2.17%) patient, malignant transformation was observed. These cases were excluded from the statistical analysis, because this variation may imply a change in cell count. Regarding the Derkay index, the mean for the ARRP group was 8.26 (min. 3 and max. 27); for the JRRP group it was 10.97 (min. 4 and max. 31), being statistically significant for the t-test with p = .02 (Table 3) and for the Spearman correlation test (p < .05). The latter demonstrated that the lower the age, then the higher the Derkay score, that is, the greater the severity. Among the cases selected, the larynx and oropharynx were most commonly affected, with additional lesions in the soft palate (1.08%), pharynx (1.08%) and base of tongue (1.08%). The most commonly affected sites were the true vocal cords in JRRP (50.24%), followed by false vocal cords (12.68%); in adult patients the true vocal cords were most commonly affected (p < .05).
2.2. Construction of the Tissue Micro-Array (TMA) After a morphological review of all the cases, tumor regions were demarcated from at least one original block for each case and 4 areas for each case were selected, in which there was a representative area of inflammation, as follows: predominantly epithelial or predominantly underlying connective tissue. The tissue microarray (TMA) blocks were constructed as previously described [14]. 2.3. Immunohistochemistry (IHC) The IHC method is similar to that reported by Kaminagakura et al (2007) [15]. All information about the primary antibodies is reported in Table 1.
Table 1 List of primary antibodies including: dilution, clone, positive control, and manufacturer. Antibody
Dilution
Clone
Control +
Manufacturer
CD3 CD4 CD8 CD20 CD68 CD15 MUM-1 FoxP3
1:100 1:500 1:100 1:400 1:3000 1:40 1:100 1:100
Policlonal 4B12 C8/144B L26 KP1 BY87 EAU32 SP97
Tonsil Tonsil Tonsil Lymph node Tonsil Tonsil Tonsil Tonsil
DakoCytomation, Glostrup, Denmark Leica Biosystems, Wetzlar, Germany DakoCytomation, Glostrup, Denmark DakoCytomation, Glostrup, Denmark DakoCytomation, Glostrup, Denmark Leica Biosystems, Wetzlar, Germany Leica Biosystems, Wetzlar, Germany Spring Bioscience, Pleasanton, California
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Table 2 Clinical data of all JRRP and ARRP samples. Clinicopathological features #
Age ± SD Female n/t (%) Male n/t (%) Tracheostomy n/t (%) Adj treat§ n/t (%) Death n/t (%)
Table 5 Spearman correlation test between the Derkay index and the number of cells. Spearman r negative values represent an inverse proportional relationship, while a positive value denotes a proportional relationship.
JRRP
ARRP
4.94 ± 3.30 18/36 (50.00) 18/36 (50.00) 6/33 (18.18) 6/36 (16.67) 1/36 (2.78)
41.62 ± 14.01 15/56 (26.78) 41/56 (73.21) 2/46 (4.34) 2/46 (4.34) 1/46 (2.17)
Legend: #standard deviation; §adjuvant treatment; n/t: number of samples/total of samples. Table 3 Mean and standard deviation of the total number of quantifications of inflammatory cells and comparison between JRRP and ARRP. Cells
JRRP (Mean ± SD)
ARRP (Mean ± SD)
P
CD3 CD4 CD8 CD15 CD20 CD68 FoxP3 MUM-1
10.55 ± 6.97 4.07 ± 3.35 3.09 ± 3.90 5.33 ± 5.72 1.34 ± 2.12 5.21 ± 4.21 3.45 ± 4.16 2.62 ± 5.54
23.35 ± 18.59 7.08 ± 7.27 9.94 ± 11.39 4.89 ± 4.87 4.18 ± 10.26 4.20 ± 2.99 5.59 ± 7.91 12.24 ± 15.30
< 0.05 0.23 < 0.05 0.87 0.33 0.28 0.42 < 0.05
Table 4 Spearman correlation test between the age and number of cells. Spearman r negative values represent an inverse proportion relationship, while a positive value denotes a proportional relationship. P
CD3 CD4 CD8 CD15 CD20 CD68 FoxP3 MUM-1
77 78 76 79 77 77 77 76
0.38 0.20 0.39 −0.05 0.28 −0.03 0.14 0.57
< 0.05 0.07 < 0.05 0.63 0.01 0.77 0.19 < 0.05
P
CD3 CD4 CD8 CD15 CD20 CD68 FoxP3 MUM-1
77 78 76 79 77 77 77 76
0.02 −0.07 −0.16 0.32 −0.13 −0.00 −0.08 −0.28
0.80 0.53 0.15 < 0.05 0.22 0.97 0.46 0.01
RRP is an uncommon disease that can develop a severe clinical course leading to death [1,18]. Such aggressiveness is related to age, time of evolution and type of HPV [19,20]. Additionally, the immune system may also play an important role in the clinical course of the disease [11]. CD3+ T cells represents the total number of T lymphocytes [17]. In the present study, these cells were present in greater numbers in adult patients; the group that presented less severe RRP. CD3+ T cells are important to prevent malignant transformation of leukoplakia, since patients who developed head and neck squamous cell carcinoma (HNSCC) had significantly fewer CD3+ cells than those in both the dysplastic epithelium and subepithelial connective tissue [21]. In our study, the least quantity of CD8+ cells was observed in juvenile patients, which was the group that showed a more severe clinical course similar to previous results [22,23]. The critical period for the development of the immune system occurs between 4 and 6 years of age [11]; this period covers the mean age of JRRP patients in our study. The immaturity of the juvenile immune system can lead to a decrease in the processing and/or presentation of antigens or a decrease in the secretion of pro-inflammatory cytokines, which could contribute to the severity of RRP. However, some authors have reported an inversion in the proportion of CD4+/CD8+ cells and high production of cytokines mRNA by the CD56+/NK cells in RRP when compared to healthy controls. These findings suggest that a dysregulated antiviral response can be a reason for persistent infection in RRP patients [24,25]. Additionally, we have demonstrated a tendency for a higher number of CD8+ cells in low-grade dysplasia, while the number of CD4+ cells was similar in the different degrees of dysplasia. On the other hand, in lesions caused by high risk HPV, the results are controversial; for example, in cervical intraepithelial neoplasia (CIN), a high viral load leads to the predominance of CD8+ T lymphocytes [25]. Meanwhile, a high expression of CD8 cells, B cells and IL2 was positively correlated with an improved prognosis of HPV+ HNSCC [26]. Regulatory T cells (Tregs) are a subpopulation of T cells responsible for immune self-tolerance, effectively suppressing T cell activation [27]. There are several subtypes of CD4+ Tregs, such as T helper, Natural Killer (NK)-T and CD4 + CD25 + FoxP3+ [28], and the FoxP3 function stabilizes Tregs function [29]. In the present study, a tendency was observed for a smaller number of these cells (FoxP3+) in the connective tissue of the cases that have presented relapses. Effective host responses to HPV in genital warts are characterized by an active cell-mediated immune response, in which CD4+ lymphocytes and macrophages predominate, which is consistent with a delayed-type hypersensitivity reaction to foreign antigens [30]. T cell activity can be inhibited by a high frequency of neutrophils, because neutrophils and tumor cells promote an IL6 and IL8 rich environment in cervical cancer [9]. A high infiltration of neutrophils was negatively correlated with HPV+ HNSCC survival [26] and was
The mean of the cell quantification was used to compare the JRRP and ARRP groups (Table 3), to correlate age (Table 4) and the Derkay index (Table 5) with the number of inflammatory cells and also to correlate the inflammatory cells with each characteristic. The Derkay subgroups < 20 and ≥ 20, irrespective of age, were also correlated with the presence of inflammatory cells. Regarding the dysplasia grade, CD3+ and CD8+ cells were correlated with low-grade, while CD68+ cells were related to high-grade of dysplasia. The immunohistochemical findings obtained concerning the inflammatory cells of the JRRP and ARRP groups are shown in Fig. 1. HPV 6 was detected in 53 (66.3%) samples and was significantly more prevalent in the 34 (77.3%) ARRP individuals compared with 19 (52.8%) JRRP patients (p = .03). HPV 11 was detected in 30 (37.5%) samples and was significantly more prevalent in the JRRP group of individuals (52.8%) compared with ARRP cases (25.0%) (p = .02). Twelve cases were excluded from DNA extraction and HPV genotyping due to the quality of the material from the paraffin blocks (unpublished data). No statistically significant association was found between the HPV genotyping and inflammatory cells type.
Spearman r
Spearman r
4. Discussion
3.2. Correlation between inflammatory cells and clinical and histological characteristics and HPV genotyping
n
n
P values in bold are statistically significant.
P values in bold are statistically significant.
Cells
Cells
P values in bold are statistically significant. 3
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Fig. 1. a. Microphotography of JRRP showing few CD3+ cells in the epithelium and in connective tissue. b. CD3+ cells dispersed in the epithelium and in large amounts in connective tissue of ARRP. c. negative expression of CD8+ in juvenile sample. d. membranous positivity of CD 8+ in epithelium of ARRP group. e. absence of MUM1+ cells in JRRP while these cells were observed in connective tissue of ARRP (f). g. exocytosis area demonstrated by CD15+ cells in JRRP. 1 h. CD15+ were negative in ARRP. (DAB stain, Mayer's counterstain).
In more severe cases of JRRP, a smaller quantity of CD3+, CD8+ and MUM1+ cells was observed. The immune system imbalance can play an important role in the severity of RRP.
correlated with an increased size of colorectal carcinoma [31]. In our study, these CD15+ cells were present in the epithelium and in the connective tissue of the most RRP severe cases, especially in ulceration areas causing edema and pain. Cytokines are also important mediators regulating the tumor microenvironment. IL12 and IFNγ delivered by T cells are possibly involved in myeloid cell accumulation and macrophage polarization [26]. The presence of the M2-macrophage phenotype performs functions that are different from the M1 phenotype, acting as an immunity regulator and as an aid in tissue repair [32], in addition to stimulating tumor growth, since it is an angiogenesis promoter [33]. The presence of CD68+ cells in RRP was associated with high-degree dysplasia, and in JRRP there was an inverse proportional correlation between CD68+ and CD3+ cells. This suppressive role of the adaptive immune response of M2-macrophages is already known [33] and in this study, by means of the CD68 expression, this role was observed in juvenile patients, in whom the disease has shown a higher Derkay index. B cells with terminal differentiation and plasma cells (MUM1+) can produce antibodies [34]. In RRP, MUM1+ cells were correlated with the lowest Derkay index mainly in adult patients; the immune role of this cell was related to lower severity and may help to combat the disease. The quality of B cell responses generated by natural infection varies from person to person [35]. However, vaccination increases natural HPV immunity by boosting the antibodies level and B memory cell numbers and efficiency. Persons previously infected with HPV vaccines types, such as HPV 6, 11, 16 and 18, could also benefit from vaccination [35].
Funding For the financial support received from FAPESP (São Paulo Research Foundation, Grant: 16/24019-0 to EK) and CAPES (Coordination for the Improvement of Higher Education Personnel to VNREA). Declaration of competing interest All the authors declare that there is no conflict of interest. References [1] Richardson M, Gale N, Hille J, Zidar N. Squamous cell papilloma and squamous cell papilomatosis. In: El-Naggar AK, Chan JK, Grandis JR, Takata T, Slotweg PJ, editors. WHO classification head and neck tumours. Lyon: IARC Press; 2017. p. 93–5. [2] Davids T, Muller S, Wise JC, Johns MM, Klein A. Laryngeal Papillomatosis associated dysplasia in the adult population: an update on prevalence and HPV subtyping. Ann Otol Rhinol Laryngol 2014;123:402–8. https://doi.org/10.1177/ 0003489414526848. [3] Auborn KJ, Little RD, Platt THK, Vaccariello MA, Schildkraut CL. Replicative intermediates of human papillomavirus type 11 in laryngeal papillomas: site of replication initiation and direction of replication. Proc Natl Acad Sci U S A 1994;91:7340–4. https://doi.org/10.1073/pnas.91.15.7340. [4] Vancurova I, Wu R, Miskolci V, Sun S. Increased p50/p50 NF-KB activation in human papillomavirus type 6- or type 11-induced laryngeal papilloma tissue. J Virol 2002;76:1533–6. https://doi.org/10.1128/jvi.76.3.1533-1536.2002. [5] Mounts P, Kashima H. Association of human papillomavirus subtype and clinical course in respiratory papillomatosis. Laryngoscope 1984;94:28–33. https://doi.org/ 10.1002/lary.5540940106. [6] Rosen CA, Lee AS, Osborne J, Zullo T, Murry T. Development and validation of the voice handicap index-10. Laryngoscope 2004;114:1549–56. https://doi.org/10. 1097/00005537-200409000-00009. [7] Kuet ML, Pitman MJ. Photoangiolytic laser treatment of recurrent respiratory
5. Conclusion In summary, our results found a correlation of CD3+ and CD8+ cells with low-grade dysplasia and CD68+ with high-grade dysplasia. 4
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