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Surgical outcomes and unique histological features of tonsils after tonsillectomy in adults with periodic fever, aphthous stomatitis, pharyngitis, and cervical adenitis syndrome
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Surgical outcomes and unique histological features of tonsils after tonsillectomy in adults with periodic fever, aphthous stomatitis, pharyngitis, and cervical adenitis syndrome Kohei Yamahara a,*, Kana Lee b, Yuki Egawa c, Noriyuki Nakashima d, Satoshi Ikegami a a
Department of Otolaryngology, Head and Neck Surgery, Shizuoka City Shizuoka Hospital, Shizuoka, 420-8630, Japan Department of Otolaryngology, Shin-Suma General Hospital, Kobe, Hyogo 654-0048, Japan c Department of Pathology, Shizuoka City Shizuoka Hospital, Shizuoka, 420-8630, Japan d Department of Physiology, Kurume University School of Medicine, 67 Asahi-Machi, Kurume, 830-0011, Japan b
A R T I C L E I N F O
A B S T R A C T
Article history: Received 10 May 2019 Accepted 21 August 2019 Available online xxx
Objectives: Data on the adult-onset periodic fever, aphthous stomatitis, pharyngitis, and cervical adenitis (PFAPA) syndrome are scarce. European studies reported that unlike pediatric-onset PFAPA, tonsillectomy is ineffective for adult-onset PFAPA. The aims of this study were (1) to assess the response to tonsillectomy in a cohort of Japanese adult-onset PFAPA patients and (2) to evaluate the histologic appearance of tonsils in adult-onset PFAPA patients and to compare them with those of tonsils from age- and sex-matched controls with chronic tonsillitis. Methods: In this retrospective cohort study, 5 adults with PFAPA and 15 controls who had undergone tonsillectomy were recruited. The size of the tonsil germinal centers was measured by hematoxylin and eosin staining, and the number and density of B and T lymphocytes in germinal centers were measured by immunohistochemistry, using CD3, CD4 and CD8 as T cell markers and CD20 as B cell marker. Results: All patients had complete remission of the symptoms after surgery. PFAPA patients had significantly smaller germinal center areas than controls. The number and density of CD8+ cells in germinal centers were significantly lower in tonsils from PFAPA compared with controls. No differences were found between the two groups in CD3+, CD4+, and CD20+ cells. These results are compatible with the tonsillar features of pediatric-onset PFAPA. Conclusion: Our report demonstrates that tonsillectomy might be effective for adult-onset PFAPA and that tonsils of adult- and pediatric-onset PFAPA share the same histological features. These results suggest that the pathogenic mechanisms of adult- and pediatric-onset PFAPA are identical. © 2019 Elsevier B.V. All rights reserved.
Keywords: PFAPA Adult-onset PFAPA Germinal center area CD8
1. Introduction
* Corresponding author at: Department of Otolaryngology, Head and Neck Surgery, Shizuoka City Shizuoka Hospital 10-93 Oute-cho, Aoi-ku, Shizuoka, Shizuoka 420-8630, Japan. E-mail address: [email protected] (K. Yamahara).
PFAPA (periodic fever, aphthous stomatitis, pharyngitis, and cervical adenitis) syndrome, first described in 1987, is the most common periodic fever syndrome of childhood [1]. The syndrome is diagnosed according to clinical criteria, the hallmarks being recurrent, regular, stereotypical episodes of fever with pharyngitis, cervical adenopathy, and aphthous
https://doi.org/10.1016/j.anl.2019.08.009 0385-8146/© 2019 Elsevier B.V. All rights reserved.
Please cite this article in press as: Yamahara K, et al. Surgical outcomes and unique histological features of tonsils after tonsillectomy in adults with periodic fever, aphthous stomatitis, pharyngitis, and cervical adenitis syndrome. Auris Nasus Larynx (2019), https://doi.org/10.1016/j. anl.2019.08.009
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stomatitis [2]. Although the pathogenesis of this syndrome has not yet been well elucidated, it is considered an autoinflammatory disease [3,4]. Patients may be managed medically with episodic doses of corticosteroids or with prophylactic cimetidine or colchicine, but each of these treatments has limitations [5,6]. Although tonsillectomy is not always helpful, complete resolution of PFAPA after tonsillectomy has been reported in several case series and in two randomized trials [7–9]. Histological analysis of the tonsils from PFAPA patients could provide clues to the pathogenesis of PFAPA, and some studies revealed unique histological features of tonsils from children with PFAPA (pediatric-onset PFAPA), suggesting that tonsils are the primary site of immune dysregulation in children with PFAPA [10,11]. These histological features included the following: (1) smaller germinal center areas ;[10] (2) a lower number of CD8+ cells in germinal centers [11]. PFAPA usually arises in young children before the age of 5 [12], while reports of cases of adult-onset PFAPA have recently increased in Europe [12–15]. Few data are available on adult-onset PFAPA, but its clinical characteristics seem to be similar to those of pediatric-onset PFAPA, while the response to tonsillectomy is different: Some studies reported that tonsillectomy was ineffective in adult-onset patients [12–14]. This raises the question whether the pathogenic mechanisms of adult- and pediatric-onset PFAPA differ. Determining these differences in pathogenesis could contribute to the development of the treatment of adult-onset PFAPA. To date, there have been no reports on the histological features of the tonsils of adult-onset PFAPA patients. The aims of this study were (1) to assess the response to tonsillectomy in a cohort of Japanese adult-onset PFAPA patients and (2) to evaluate the histologic and immunohistological appearance of tonsils in such patients and to compare them with those of tonsils from a control group of age-and sex-matched adults operated for chronic tonsillitis. 2. Materials and methods Approval was obtained from the Shizuoka City Shizuoka Hospital and Shin-Suma Hospital Review Boards. The patients provided written informed consent to participate in the study. 2.1. Recruitment of adult-onset PFAPA cases This is a retrospective study of adults with PFAPA syndrome who underwent tonsillectomy at Shizuoka City Shizuoka Hospital and Shin-Suma Hospital from 2014 to 2018. PFAPA was diagnosed according to Padeh’s diagnostic criteria :[12] monthly cyclic fever in any age group, possible aphthous stomatitis, cervical lymphadenitis, exudative tonsillitis with negative throat culture, completely asymptomatic during intervals, and rapid response to glucocorticoid. The inclusion criteria for adult-onset PFAPA patients were the following: (1) age of onset of PFAPA episodes: 18 years or older, (2) Japanese race, (3) meeting Padeh’s diagnostic criteria, (4) exclusion of cyclic neutropenia, (5) exclusion of hereditary periodic fevers by genetic testing, (6) tonsillectomy performed to treat PFAPA. Cyclic neutropenia was excluded by serial measurements of neutrophils between two subsequent febrile episodes. All
patients who were suspected of having PFAPA underwent genetic testing, which found no mutations in the genes responsible for the most common types of hereditary periodic fevers (HPF), namely familial Mediterranean fever (FMF) and TNF receptor-associated periodic syndrome (TRAPS). Tonsillectomy was offered to patients with PFAPA if the febrile episodes lasted for several months and the febrile pattern showed no signs of remission in spite of medical treatment (cimetidine 800 mg daily and oral predonisone 60 mg at the time of the febrile attacks). To evaluate the outcome after surgery, a follow-up interview was conducted at least 12 months after surgery. 2.2. Recruitment of chronic tonsillitis patients Subjects with chronic tonsillitis were identified from patients undergoing tonsillectomy between 2014 and 2018. We chose three patients with chronic tonsillitis as controls for each PFAPA case. Inclusion criteria for chronic tonsillitis patients were the following: (1) same sex as the matched PFAPA patient, (2) Japanese race, (3) age-matched by 1 year to the corresponding PFAPA patient, (4) not meeting Padeh’s diagnostic criteria. 2.3. Surgical procedure All adults with PFAPA and controls were operated during an afebrile period. Tonsillectomy was performed with the classical cold knife method, with emphasis on blunt dissection. Hemostasis was obtained by clipping the hemorrhage site and using silk sutures. 2.4. Histologic measurements Archived paraffin-embedded, formalin-fixed tonsil blocks from the recruited subjects were obtained. Five-micrometer sections were prepared from each patient’s tissue block and stained with hematoxylin and eosin (H&E). To avoid introducing bias in the selection of regions for histologic measurement, sections were selected from the middle region between the upper and the lower pole for all tonsils. Crosssectional areas of both whole tonsils and all germinal centers were measured by ImageJ software (NIH). The areas of all germinal centers were averaged for each tonsil. The ratio of germinal center areas to tonsil area was expressed as percentage (%). For immunohistochemistry, the immunohistochemical Bond Refine Detection, a biotin-free polymeric horseradish peroxidase-linker antibody conjugate system (Bond Polymer Refine kit: Vision Biosystems Ltd., Newcastle Upon Tyne, UK), was used on sections for the detection of B (CD20/L26; Novocastra Ltd., Newcastle Upon Tyne, UK) and T (CD3, CD4 and CD8/ PS1; Novocastra Ltd., Newcastle Upon Tyne, UK) lymphocytes on the Bond-automated system. To count the number of positive cells in germinal centers by immunohistochemistry, five germinal centers were randomly selected in each subject’s tonsil and the number of positive cells was averaged among them. The density of CD3+, CD4+ or CD8+ cells was calculated by dividing the number of positive cells by each
Please cite this article in press as: Yamahara K, et al. Surgical outcomes and unique histological features of tonsils after tonsillectomy in adults with periodic fever, aphthous stomatitis, pharyngitis, and cervical adenitis syndrome. Auris Nasus Larynx (2019), https://doi.org/10.1016/j. anl.2019.08.009
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germinal center area, expressed as number of positive cells per mm2, then averaged over the germinal centers. 2.5. Statistical analysis Student’s t-test was used for comparisons between two groups. The data were expressed as mean standard error (SE); p values below 0.05 were considered statistically significant. 3. Results 3.1. Clinical characteristics and outcome A total of 10 adults who met the Padeh’s diagnostic criteria were identified. Out of 10 patients, 6 underwent tonsillectomy. Of these, one patient was excluded because of lack of genetic testing. Finally, five adult-onset PFAPA patients were considered, all of Japanese race. Patients in the PFAPA group were 28, 31, 33, 36, and 37 years old, and their clinical characteristics are listed in Table 1. Fifteen patients with chronic tonsillitis were recruited (mean age, 32.9 years), all of Japanese ethnicity. All patients of chronic tonsillitis had tested positive for throat culture before surgery, indicating that they were not PFAPA patients. There were four males and one female in the PFAPA group, and twelve males and three females in the control group. The mean follow-up after surgery was 22 months (specifically 13, 20, 22, 25, and 30 months) in the PFAPA group, and 26 months in the control group. No postoperative complications were reported. Unlike in other reports [14,15], in all adult-onset PFAPA patients the febrile episodes of PFAPA ceased after surgery during the follow-up period. 3.2. Histological examination of the tonsils of adult-onset PFAPA patients PFAPA patients had significantly smaller germinal center areas compared with chronic tonsillitis patients (0.12 mm2 vs. 0.26 mm2, p < 0.05, Fig. 1A). In addition, PFAPA patients had significantly lower germinal center to tonsil area ratio compared with patients with chronic tonsillitis (5.0% vs. 12.6%, p < 0.05, Fig. 1B). These histological features are the same as those of the tonsils of pediatric-onset PFAPA, as reported by Manthriram et al. [10]. Other histological findings, such as Mantle width, interfollicular distance, or crypt width, were not significantly different between the two groups (data not shown). Representative sections are shown in Fig. 1C and D.
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3.3. Immunohistochemical examination The number of CD8+ cells in germinal centers was significantly lower in tonsils from PFAPA patients compared with controls (2.04 vs. 19.0, p < 0.05, Fig. 2A). Next, the density of CD8+ cells in germinal centers was compared and was also found to be significantly lower in the PFAPA group compared to controls (13.6/mm2 vs. 78.3/mm2, p < 0.05, Fig. 2B). Representative sections are shown in Fig. 2C and D. No significant differences were found between the groups when comparing the number of CD3+ or CD4+ cells in tonsillar germinal centers (Fig. 3A–D, and G–J). The germinal centers were deeply populated with CD20+ cells in both PFAPA and controls, too many to be counted accurately, and no apparent difference in the density of CD20+ cells between the two groups could be detected (Fig. 3E and F). These are also the same histological features of tonsils of pediatric-onset PFAPA as reported by Manthiram et al. [10] and Forsvoll et al. [11]. 4. Discussion In the current study, we demonstrated that tonsillectomy completely resolved the symptoms in all adult-onset patients, just as in pediatric-onset PFAPA, and that the histological features are quite similar to those of pediatric-onset PFAPA. As the comparison group, we evaluated tonsils from patients with chronic tonsillitis, rather than obstructive sleep apnea (OSA), although previous studies of pediatric-onset PFAPA chose OSA tonsils as controls [10,11]. We compared the histological features of tonsils from chronic tonsillitis with those from OSA and confirmed that there were no apparent differences between them (data not shown). Both PFAPA and chronic tonsillitis have recurrent tonsil inflammation, but inflammation in PFAPA is induced by some non-infectious mechanism. Thus, examining the histological differences between tonsils from patients with PFAPA and those with chronic tonsillitis could provide clues to the pathogenic mechanisms of PFAPA. In addition, a current problem in clinical practice is that almost all PFAPA patients are misdiagnosed as having chronic tonsillitis. Key pathological differences could be exploited to reduce these diagnostic errors. The diagnostic criteria for PFAPA syndrome are based only on clinical history and physical findings, so that different diseases could possibly meet these criteria and be misdiagnosed as PFAPA. Hereditary periodic fevers (HPF) are the most difficult diseases to differentiate from PFAPA [15]. It is now
Table 1 Clinical characteristics of 5 adults who underwent tonsillectomy due to periodic fever, aphthous stomatitis, pharyngitis and cervical adenitis syndrome. Patient
Gender
Age at surgery
Average duration of each episode (days)
Average interval of each episode (days)
Days from last steroid dose to tonsillectomy
Days from last episode to tonsillectomy
Months of follow-up
Response to tonsillectomy
1 2 3 4 5
Male Male Male Female Male
28 36 33 31 37
4.9 4.3 4.2 4.4 4.6
31.8 31 30.8 32.1 32.3
46 50 38 40 39
14 22 10 13 12
20 25 22 30 13
Complete Complete Complete Complete Complete
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Fig. 1. Comparison of tonsils from patients with PFAPA and chronic tonsillitis by hematoxylin and eosin staining. A. Germinal center areas in tonsils of adult patients with PFAPA were significantly smaller than those of chronic tonsillitis. B. The ratio of germinal center area to the tonsil area was significantly lower in the PFAPA group than that in the control group. C, D. Representative hematoxylin and eosin-stained histologic tonsil section from patients with PFAPA (C) and chronic tonsillitis (D). Scale bars: 1 mm (C–D). Data are expressed as mean SE; *p < 0.05 by Student’s t-test.
well-established that HPF patients could have adult onset of the symptoms, and such late onset is probably secondary to heterozygosity or hypomorphic mutations. In these cases, patients may present with a milder clinical picture, lacking the most typical clinical characteristics of HPF, and further complicating the differential diagnosis between PFAPA and HPF. Among HPFs, FMF and TRAPS are reported to occur in adults. Thus, in this study, all adult patients who fulfilled PFAPA diagnostic criteria underwent genotype evaluation of the MEFV and TNFRSF1A genes, respectively causing FMF and TRAPS. No patient had mutations in these genes, so we could confirm that they were indeed PFAPA patients. Just as in our study, Cantarini et al. conducted genetic tests for HPF in all patients with a presumed PFAPA clinical phenotype, and they included patients whose genetic tests were negative [14,16]. In their study, tonsillectomy was performed in 9 adult-onset PFAPA patients, leading to a response in just 2, while it was ineffective in the other cases [14]. This difference in the effectiveness of tonsillectomy is not well understood. A
possible reason for this is the ethnic difference: our patients were Japanese, while those of Cantarini et al. were Caucasian. While a larger sample size would be needed to conclusively determine whether tonsillectomy is effective for adult-onset PFAPA, our study suggests that this might be indeed the case. We compared the histologic features and immune cell composition of the tonsils of adult patients with PFAPA and age-matched patients with chronic tonsillitis. Our study demonstrated that (1) Adult-onset PFAPA patients had significantly smaller germinal center areas compared with chronic tonsillitis patients and that (2) the number of CD8+ cells in germinal centers was significantly lower in tonsils from PFAPA compared with those from chronic tonsillitis. These results are in agreement with those concerning pediatric-onset PFAPA described by some reports [10,11], suggesting that adult- and pediatric-onset PFAPA share the same pathogenesis. We also examined the histology of the tonsils of pediatric-onset PFAPA with the qualitative assessments described in the Materials and Methods, and confirmed that the histologic features are
Please cite this article in press as: Yamahara K, et al. Surgical outcomes and unique histological features of tonsils after tonsillectomy in adults with periodic fever, aphthous stomatitis, pharyngitis, and cervical adenitis syndrome. Auris Nasus Larynx (2019), https://doi.org/10.1016/j. anl.2019.08.009
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Fig. 2. Comparison of tonsils from patients with PFAPA and chronic tonsillitis by CD8 immunohistochemical staining. A. The number of CD8+ cells in germinal centers was significantly lower in tonsils from PFAPA compared to controls. B. The density of CD8+ cells in germinal centers was also significantly lower in the PFAPA group compared with controls. C, D. Immunohistochemical staining of CD8+ cells in representative tonsils from patients with PFAPA (C) and chronic tonsillitis (D). Scale bars: 200 mm (C–D). Data are expressed as mean SE; *p < 0.05 by Student’s t-test.
compatible with those reported in prior studies [10,11] (Supplementary Fig. 1A–D). However, a consensus regarding the histological features of pediatric-onset PFAPA is still lacking: Other groups have assessed the histology of tonsils from pediatric-onset PFAPA with results differing from ours. Berlucchi et al. reported lymphoid hyperplasia in PFAPA tonsils in the absence of other unusual pathological findings [17], and Peridis et al. reported that they could not find any difference in tonsillar histology between PFAPA and chronic tonsillitis [7]. However, the study by Berlucchi et al. was performed with no control group for comparison [17]. In addition, the assessment method used by Peridis et al. is not described, suggesting the possibility that they did not perform qualitative assessment. Considering these facts, it is possible that their studies would have obtained the same results as ours if they analyzed the tonsils of pediatric-onset PFAPA with our methods. We examined the percentage of B lymphocytes (CD20+) and T lymphocytes (CD3+, CD4+, and CD8+) by histological analysis. In contrast, Dytrych et al. assessed lymphocyte population by flow cytometry and showed that tonsils from
children with PFAPA had a lower percentage of B lymphocytes, switched B cells, and lower levels of CD4+ cells with high PD-1 expression (an inhibitory molecule) compared with those of control patients [18]. Our result showed no significant differences between the groups when comparing the number of CD4+ cells in tonsillar germinal centers. In the current study, we counted the whole number of CD4+ cells, not only CD4+ cells with PD-1 expression. CD4+ cells are not a unitary set of cells but represent a series of distinct cell populations with different functions [19]. It is possible that we would have obtained the same result as Dytrych et al. had we analyzed CD4+ cells with high PD-1 expression. Manthiram et al. speculated that low levels of CD4+ cells with high PD-1 expression suggest that PFAPA tonsils have fewer T follicular helper cells (specialized CD4+ cells that help germinal center B cells develop and proliferate), which correlated with the smaller germinal centers seen in PFAPA [10,20]. In our study, there were no apparent differences in the density of CD20+ cells between groups. However, considering that the areas of the germinal centers in PFAPA tonsils were significantly smaller
Please cite this article in press as: Yamahara K, et al. Surgical outcomes and unique histological features of tonsils after tonsillectomy in adults with periodic fever, aphthous stomatitis, pharyngitis, and cervical adenitis syndrome. Auris Nasus Larynx (2019), https://doi.org/10.1016/j. anl.2019.08.009
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Fig. 3. Comparison of tonsils from patients with PFAPA and chronic tonsillitis by CD3, CD4, and CD20 immunohistochemical staining. A, B. Immunohistochemical staining of CD3+ cells in representative tonsils from patients with PFAPA (A) and chronic tonsillitis (B). C, D. Immunohistochemical staining of CD4+ cells in representative tonsils from patients with PFAPA (C) and chronic tonsillitis (D). E, F. Immunohistochemical staining of CD20+ cells in representative tonsils from patients with PFAPA (E) and chronic tonsillitis (F). G, H. The number (G) and density (H) of CD3+ cells in a germinal center were compared between the PFAPA and control groups, and no significant differences were found. I, J. The number (I) and density (J) of CD4+ cells in a germinal center were compared between the PFAPA and control groups, and no significant differences were found. Scale bars: 200 mm (A–F).
than in controls, it is possible that the number of CD20+ cells in PFAPA tonsils were also significantly lower than controls, which might result in the smaller germinal centers seen in the PFAPA group, as Manthiram et al. speculated. The role of CD8 + cells in tonsillar germinal centers is not well understood, but a subset of CD8+ cells are reported to work as antigenexperienced T cells supporting B cell survival and immunoglobulin G production [21], and the presence of regulatory CD8 + cells in germinal centers has been described [22]. Some studies suggested that regulatory CD8+ cells trigger important
downregulatory actions, and these cells have been implied in the regulation of autoimmunity [23,24]. Forsvoll described how the absence or reduction of certain regulatory or supporting stimuli provided by these cells may cause altered function of the B cell follicules, and then contribute to the bursts of inflammation seen during febrile episodes of PFAPA [11]. However, further studies are required to elucidate the pathogenesis of PFAPA. This study has several limitations, and our results must be interpreted with caution. A major limitation of our study is the
Please cite this article in press as: Yamahara K, et al. Surgical outcomes and unique histological features of tonsils after tonsillectomy in adults with periodic fever, aphthous stomatitis, pharyngitis, and cervical adenitis syndrome. Auris Nasus Larynx (2019), https://doi.org/10.1016/j. anl.2019.08.009
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small PFAPA sample size (n = 5). Adult-onset PFAPA is still a rare entity and in Japan, only 3 patients with adult-onset with PFAPA have been reported other than our patients [25–27]. Due to the small sample size, we might have failed to detect minor differences between adults with PFAPA and controls. Another limitation of this study is that no data were available about the histology of the PFAPA tonsils for which tonsillectomy was ineffective, because tonsillectomy was effective in all our adult-onset patients. If these tonsils showed different histological features from those of our cohort, histological examination of tonsils could help clinicians understand the prognosis of adult-onset PFAPA patients who had tonsillectomy. In spite of the small PFAPA sample size in our study, our data showed significant statistical differences, allowing us to reach some conclusions regarding the course of the disease in adult patients. Further studies of tonsil tissues are needed to understand the mechanism behind the histologic differences we observed. Increased awareness of PFAPA will result in an increased number of adult-onset PFAPA patients and clarify the pathogenic mechanisms of this rare disease.
5. Conclusion To the best of our knowledge, this study was the first to analyze the histological features of the tonsils of adult-onset PFAPA patients. Our report demonstrates that tonsillectomy might be effective for adult-onset PFAPA patients and that tonsils of adult- and pediatric-onset PFAPA share unique histological features. Adult-onset PFAPA is still a rare entity and we described only five patients. Although our cohort is small and further studies are needed, our results suggest that the pathogenic mechanisms of adult- and pediatric-onset PFAPA are identical. In the future, the histological differences between the PFAPA tonsils for which tonsillectomy was effective vs. non-effective should be examined.
Disclosure statement The authors declare that they have no competing interests. Ethical statement This manuscript has not been published or presented elsewhere in part or in entirety and is not under consideration by another journal. All study participants provided written informed consent. We have read and understood your journal’s policies, and we believe that neither the manuscript nor the study violates any of these. There are no conflicts of interest to declare. Acknowledgments We would like to thank and Editage (www.editage.jp) for English language editing.
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Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.anl.2019.08.009.
References [1] Marshall GS, Edwards KM, Butler J, Lawton AR. Syndrome of periodic fever, pharyngitis, and aphthous stomatitis. J Pediatr 1987;110:43–6. [2] Thomas KT, Feder Jr HM, Lawton AR, Edwards KM. Periodic fever syndrome in children. J Pediatr 1999;135:15–21. [3] Forsvoll J, Kristoffersen EK, Oymar K. Incidence, clinical characteristics and outcome in Norwegian children with periodic fever, aphthous stomatitis, pharyngitis and cervical adenitis syndrome; a populationbased study. Acta paediatrica (Oslo Norway: 1992) 2013;102:187–92. [4] Hofer M, Pillet P, Cochard MM, Berg S, Krol P, Kone-Paut I, et al. International periodic fever, aphthous stomatitis, pharyngitis, cervical adenitis syndrome cohort: description of distinct phenotypes in 301 patients. Rheumatology (Oxford England) 2014;53:1125–9. [5] Wurster VM, Carlucci JG, Feder Jr HM, Edwards KM. Long-term follow-up of children with periodic fever, aphthous stomatitis, pharyngitis, and cervical adenitis syndrome. J Pediatr 2011;159:958–64. [6] Feder HM, Salazar JC. A clinical review of 105 patients with PFAPA (a periodic fever syndrome). Acta paediatrica (Oslo Norway: 1992) 2010;99:178–84. [7] Peridis S, Koudoumnakis E, Theodoridis A, Stefanaki K, Helmis G, Houlakis M. Surgical outcomes and histology findings after tonsillectomy in children with periodic fever, aphthous stomatitis, pharyngitis, and cervical adenitis syndrome. Am J Otolaryngol 2010;31:472–5. [8] Garavello W, Romagnoli M, Gaini RM. Effectiveness of adenotonsillectomy in PFAPA syndrome: a randomized study. J Pediatr 2009;155:250–3. [9] Renko M, Salo E, Putto-Laurila A, Saxen H, Mattila PS, Luotonen J, et al. A randomized, controlled trial of tonsillectomy in periodic fever, aphthous stomatitis, pharyngitis, and adenitis syndrome. J Pediatr 2007;151:289–92. [10] Manthiram K, Correa H, Boyd K, Roland J, Edwards K. Unique histologic features of tonsils from patients with periodic fever, aphthous stomatitis, pharyngitis, and cervical adenitis (PFAPA) syndrome. Clin Rheumatol 2018;37:1309–17. [11] Forsvoll J, Janssen EA, Moller I, Wathne N, Skaland I, Klos J, et al. Reduced number of CD8+ cells in tonsillar germinal centres in children with the periodic fever, aphthous stomatitis, pharyngitis and cervical adenitis syndrome. Scand J Immunol 2015;82:76–83. [12] Padeh S, Stoffman N, Berkun Y. Periodic fever accompanied by aphthous stomatitis, pharyngitis and cervical adenitis syndrome (PFAPA syndrome) in adults. Isr Med Assoc J: IMAJ 2008;10:358–60. [13] Cantarini L, Vitale A, Galeazzi M, Frediani B. A case of resistant adultonset periodic fever, aphthous stomatitis, pharyngitis and cervical adenitis (PFAPA) syndrome responsive to anakinra. Clin Exp Rheumatol 2012;30:593. [14] Cantarini L, Vitale A, Bartolomei B, Galeazzi M, Rigante D. Diagnosis of PFAPA syndrome applied to a cohort of 17 adults with unexplained recurrent fevers. Clin Exp Rheumatol 2012;30:269–71. [15] Cattalini M, Soliani M, Rigante D, Lopalco G, Iannone F, Galeazzi M, et al. Basic characteristics of adults with periodic fever, aphthous stomatitis, pharyngitis, and adenopathy syndrome in comparison with the typical pediatric expression of disease. Mediat Inflammation 2015;2015:570418. [16] Rigante D, Vitale A, Natale MF, Lopalco G, Andreozzi L, Frediani B, et al. A comprehensive comparison between pediatric and adult patients with periodic fever, aphthous stomatitis, pharyngitis, and cervical adenopathy (PFAPA) syndrome. Clin Rheumatol 2017;36:463–8. [17] Berlucchi M, Meini A, Plebani A, Bonvini MG, Lombardi D, Nicolai P. Update on treatment of Marshall’s syndrome (PFAPA syndrome): report of five cases with review of the literature. Ann Otol Rhinol Laryngol 2003;112:365–9.
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[18] Dytrych P, Krol P, Kotrova M, Kuzilkova D, Hubacek P, Krol L, et al. Polyclonal, newly derived T cells with low expression of inhibitory molecule PD-1 in tonsils define the phenotype of lymphocytes in children with Periodic Fever, Aphtous Stomatitis, Pharyngitis and Adenitis (PFAPA) syndrome. Mol Immunol 2015;65:139–47. [19] Zhu J, Paul WE. CD4 T cells: fates, functions, and faults. Blood 2008;112:1557–69. [20] Crotty S. T follicular helper cell differentiation, function, and roles in disease. Immunity 2014;41:529–42. [21] Quigley MF, Gonzalez VD, Granath A, Andersson J, Sandberg JK. CXCR5+ CCR7- CD8 T cells are early effector memory cells that infiltrate tonsil B cell follicles. Eur J Immunol 2007;37:3352–62. [22] Kim HJ, Verbinnen B, Tang X, Lu L, Cantor H. Inhibition of follicular T-helper cells by CD8(+) regulatory T cells is essential for self tolerance. Nature 2010;467:328–32.
[23] Ramiscal RR, Vinuesa CG. T-cell subsets in the germinal center. Immunol Rev 2013;252:146–55. [24] Chang JH, Chung Y. Regulatory T cells in B cell follicles. Immune Netw 2014;14:227–36. [25] Kutsuna S, Ohmagari N, Tanizaki R, Hagino N, Nishikomori R, Ujiie M, et al. The first case of adult-onset PFAPA syndrome in Japan. Modern Rheumatol 2016;26:286–7. [26] Yasuura S, Harada Y. A case of adult-onset periodic fever, aphthous stomatitis, pharyngitis, and adenitis syndrome in Japan. J Gen Family Med 2017;18:261–4. [27] Ota K, Kwak-Kim J, Takahashi T, Mizunuma H. Pregnancy complicated with PFAPA (periodic fever, aphthous stomatitis, pharyngitis and cervical adenitis) syndrome: a case report. BMC Pregnancy Childbirth 2018;18:207.
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Surgical outcomes and unique histological features of tonsils after tonsillectomy in adults with periodic fever, aphthous stomatitis, pharyngitis, and cervical adenitis syndrome Kohei Yamahara a,*, Kana Lee b, Yuki Egawa c, Noriyuki Nakashima d, Satoshi Ikegami a a
Department of Otolaryngology, Head and Neck Surgery, Shizuoka City Shizuoka Hospital, Shizuoka, 420-8630, Japan Department of Otolaryngology, Shin-Suma General Hospital, Kobe, Hyogo 654-0048, Japan c Department of Pathology, Shizuoka City Shizuoka Hospital, Shizuoka, 420-8630, Japan d Department of Physiology, Kurume University School of Medicine, 67 Asahi-Machi, Kurume, 830-0011, Japan b
A R T I C L E I N F O
A B S T R A C T
Article history: Received 10 May 2019 Accepted 21 August 2019 Available online xxx
Objectives: Data on the adult-onset periodic fever, aphthous stomatitis, pharyngitis, and cervical adenitis (PFAPA) syndrome are scarce. European studies reported that unlike pediatric-onset PFAPA, tonsillectomy is ineffective for adult-onset PFAPA. The aims of this study were (1) to assess the response to tonsillectomy in a cohort of Japanese adult-onset PFAPA patients and (2) to evaluate the histologic appearance of tonsils in adult-onset PFAPA patients and to compare them with those of tonsils from age- and sex-matched controls with chronic tonsillitis. Methods: In this retrospective cohort study, 5 adults with PFAPA and 15 controls who had undergone tonsillectomy were recruited. The size of the tonsil germinal centers was measured by hematoxylin and eosin staining, and the number and density of B and T lymphocytes in germinal centers were measured by immunohistochemistry, using CD3, CD4 and CD8 as T cell markers and CD20 as B cell marker. Results: All patients had complete remission of the symptoms after surgery. PFAPA patients had significantly smaller germinal center areas than controls. The number and density of CD8+ cells in germinal centers were significantly lower in tonsils from PFAPA compared with controls. No differences were found between the two groups in CD3+, CD4+, and CD20+ cells. These results are compatible with the tonsillar features of pediatric-onset PFAPA. Conclusion: Our report demonstrates that tonsillectomy might be effective for adult-onset PFAPA and that tonsils of adult- and pediatric-onset PFAPA share the same histological features. These results suggest that the pathogenic mechanisms of adult- and pediatric-onset PFAPA are identical. © 2019 Elsevier B.V. All rights reserved.
Keywords: PFAPA Adult-onset PFAPA Germinal center area CD8
1. Introduction
* Corresponding author at: Department of Otolaryngology, Head and Neck Surgery, Shizuoka City Shizuoka Hospital 10-93 Oute-cho, Aoi-ku, Shizuoka, Shizuoka 420-8630, Japan. E-mail address: [email protected] (K. Yamahara).
PFAPA (periodic fever, aphthous stomatitis, pharyngitis, and cervical adenitis) syndrome, first described in 1987, is the most common periodic fever syndrome of childhood [1]. The syndrome is diagnosed according to clinical criteria, the hallmarks being recurrent, regular, stereotypical episodes of fever with pharyngitis, cervical adenopathy, and aphthous
https://doi.org/10.1016/j.anl.2019.08.009 0385-8146/© 2019 Elsevier B.V. All rights reserved.
Please cite this article in press as: Yamahara K, et al. Surgical outcomes and unique histological features of tonsils after tonsillectomy in adults with periodic fever, aphthous stomatitis, pharyngitis, and cervical adenitis syndrome. Auris Nasus Larynx (2019), https://doi.org/10.1016/j. anl.2019.08.009
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stomatitis [2]. Although the pathogenesis of this syndrome has not yet been well elucidated, it is considered an autoinflammatory disease [3,4]. Patients may be managed medically with episodic doses of corticosteroids or with prophylactic cimetidine or colchicine, but each of these treatments has limitations [5,6]. Although tonsillectomy is not always helpful, complete resolution of PFAPA after tonsillectomy has been reported in several case series and in two randomized trials [7–9]. Histological analysis of the tonsils from PFAPA patients could provide clues to the pathogenesis of PFAPA, and some studies revealed unique histological features of tonsils from children with PFAPA (pediatric-onset PFAPA), suggesting that tonsils are the primary site of immune dysregulation in children with PFAPA [10,11]. These histological features included the following: (1) smaller germinal center areas ;[10] (2) a lower number of CD8+ cells in germinal centers [11]. PFAPA usually arises in young children before the age of 5 [12], while reports of cases of adult-onset PFAPA have recently increased in Europe [12–15]. Few data are available on adult-onset PFAPA, but its clinical characteristics seem to be similar to those of pediatric-onset PFAPA, while the response to tonsillectomy is different: Some studies reported that tonsillectomy was ineffective in adult-onset patients [12–14]. This raises the question whether the pathogenic mechanisms of adult- and pediatric-onset PFAPA differ. Determining these differences in pathogenesis could contribute to the development of the treatment of adult-onset PFAPA. To date, there have been no reports on the histological features of the tonsils of adult-onset PFAPA patients. The aims of this study were (1) to assess the response to tonsillectomy in a cohort of Japanese adult-onset PFAPA patients and (2) to evaluate the histologic and immunohistological appearance of tonsils in such patients and to compare them with those of tonsils from a control group of age-and sex-matched adults operated for chronic tonsillitis. 2. Materials and methods Approval was obtained from the Shizuoka City Shizuoka Hospital and Shin-Suma Hospital Review Boards. The patients provided written informed consent to participate in the study. 2.1. Recruitment of adult-onset PFAPA cases This is a retrospective study of adults with PFAPA syndrome who underwent tonsillectomy at Shizuoka City Shizuoka Hospital and Shin-Suma Hospital from 2014 to 2018. PFAPA was diagnosed according to Padeh’s diagnostic criteria :[12] monthly cyclic fever in any age group, possible aphthous stomatitis, cervical lymphadenitis, exudative tonsillitis with negative throat culture, completely asymptomatic during intervals, and rapid response to glucocorticoid. The inclusion criteria for adult-onset PFAPA patients were the following: (1) age of onset of PFAPA episodes: 18 years or older, (2) Japanese race, (3) meeting Padeh’s diagnostic criteria, (4) exclusion of cyclic neutropenia, (5) exclusion of hereditary periodic fevers by genetic testing, (6) tonsillectomy performed to treat PFAPA. Cyclic neutropenia was excluded by serial measurements of neutrophils between two subsequent febrile episodes. All
patients who were suspected of having PFAPA underwent genetic testing, which found no mutations in the genes responsible for the most common types of hereditary periodic fevers (HPF), namely familial Mediterranean fever (FMF) and TNF receptor-associated periodic syndrome (TRAPS). Tonsillectomy was offered to patients with PFAPA if the febrile episodes lasted for several months and the febrile pattern showed no signs of remission in spite of medical treatment (cimetidine 800 mg daily and oral predonisone 60 mg at the time of the febrile attacks). To evaluate the outcome after surgery, a follow-up interview was conducted at least 12 months after surgery. 2.2. Recruitment of chronic tonsillitis patients Subjects with chronic tonsillitis were identified from patients undergoing tonsillectomy between 2014 and 2018. We chose three patients with chronic tonsillitis as controls for each PFAPA case. Inclusion criteria for chronic tonsillitis patients were the following: (1) same sex as the matched PFAPA patient, (2) Japanese race, (3) age-matched by 1 year to the corresponding PFAPA patient, (4) not meeting Padeh’s diagnostic criteria. 2.3. Surgical procedure All adults with PFAPA and controls were operated during an afebrile period. Tonsillectomy was performed with the classical cold knife method, with emphasis on blunt dissection. Hemostasis was obtained by clipping the hemorrhage site and using silk sutures. 2.4. Histologic measurements Archived paraffin-embedded, formalin-fixed tonsil blocks from the recruited subjects were obtained. Five-micrometer sections were prepared from each patient’s tissue block and stained with hematoxylin and eosin (H&E). To avoid introducing bias in the selection of regions for histologic measurement, sections were selected from the middle region between the upper and the lower pole for all tonsils. Crosssectional areas of both whole tonsils and all germinal centers were measured by ImageJ software (NIH). The areas of all germinal centers were averaged for each tonsil. The ratio of germinal center areas to tonsil area was expressed as percentage (%). For immunohistochemistry, the immunohistochemical Bond Refine Detection, a biotin-free polymeric horseradish peroxidase-linker antibody conjugate system (Bond Polymer Refine kit: Vision Biosystems Ltd., Newcastle Upon Tyne, UK), was used on sections for the detection of B (CD20/L26; Novocastra Ltd., Newcastle Upon Tyne, UK) and T (CD3, CD4 and CD8/ PS1; Novocastra Ltd., Newcastle Upon Tyne, UK) lymphocytes on the Bond-automated system. To count the number of positive cells in germinal centers by immunohistochemistry, five germinal centers were randomly selected in each subject’s tonsil and the number of positive cells was averaged among them. The density of CD3+, CD4+ or CD8+ cells was calculated by dividing the number of positive cells by each
Please cite this article in press as: Yamahara K, et al. Surgical outcomes and unique histological features of tonsils after tonsillectomy in adults with periodic fever, aphthous stomatitis, pharyngitis, and cervical adenitis syndrome. Auris Nasus Larynx (2019), https://doi.org/10.1016/j. anl.2019.08.009
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germinal center area, expressed as number of positive cells per mm2, then averaged over the germinal centers. 2.5. Statistical analysis Student’s t-test was used for comparisons between two groups. The data were expressed as mean standard error (SE); p values below 0.05 were considered statistically significant. 3. Results 3.1. Clinical characteristics and outcome A total of 10 adults who met the Padeh’s diagnostic criteria were identified. Out of 10 patients, 6 underwent tonsillectomy. Of these, one patient was excluded because of lack of genetic testing. Finally, five adult-onset PFAPA patients were considered, all of Japanese race. Patients in the PFAPA group were 28, 31, 33, 36, and 37 years old, and their clinical characteristics are listed in Table 1. Fifteen patients with chronic tonsillitis were recruited (mean age, 32.9 years), all of Japanese ethnicity. All patients of chronic tonsillitis had tested positive for throat culture before surgery, indicating that they were not PFAPA patients. There were four males and one female in the PFAPA group, and twelve males and three females in the control group. The mean follow-up after surgery was 22 months (specifically 13, 20, 22, 25, and 30 months) in the PFAPA group, and 26 months in the control group. No postoperative complications were reported. Unlike in other reports [14,15], in all adult-onset PFAPA patients the febrile episodes of PFAPA ceased after surgery during the follow-up period. 3.2. Histological examination of the tonsils of adult-onset PFAPA patients PFAPA patients had significantly smaller germinal center areas compared with chronic tonsillitis patients (0.12 mm2 vs. 0.26 mm2, p < 0.05, Fig. 1A). In addition, PFAPA patients had significantly lower germinal center to tonsil area ratio compared with patients with chronic tonsillitis (5.0% vs. 12.6%, p < 0.05, Fig. 1B). These histological features are the same as those of the tonsils of pediatric-onset PFAPA, as reported by Manthriram et al. [10]. Other histological findings, such as Mantle width, interfollicular distance, or crypt width, were not significantly different between the two groups (data not shown). Representative sections are shown in Fig. 1C and D.
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3.3. Immunohistochemical examination The number of CD8+ cells in germinal centers was significantly lower in tonsils from PFAPA patients compared with controls (2.04 vs. 19.0, p < 0.05, Fig. 2A). Next, the density of CD8+ cells in germinal centers was compared and was also found to be significantly lower in the PFAPA group compared to controls (13.6/mm2 vs. 78.3/mm2, p < 0.05, Fig. 2B). Representative sections are shown in Fig. 2C and D. No significant differences were found between the groups when comparing the number of CD3+ or CD4+ cells in tonsillar germinal centers (Fig. 3A–D, and G–J). The germinal centers were deeply populated with CD20+ cells in both PFAPA and controls, too many to be counted accurately, and no apparent difference in the density of CD20+ cells between the two groups could be detected (Fig. 3E and F). These are also the same histological features of tonsils of pediatric-onset PFAPA as reported by Manthiram et al. [10] and Forsvoll et al. [11]. 4. Discussion In the current study, we demonstrated that tonsillectomy completely resolved the symptoms in all adult-onset patients, just as in pediatric-onset PFAPA, and that the histological features are quite similar to those of pediatric-onset PFAPA. As the comparison group, we evaluated tonsils from patients with chronic tonsillitis, rather than obstructive sleep apnea (OSA), although previous studies of pediatric-onset PFAPA chose OSA tonsils as controls [10,11]. We compared the histological features of tonsils from chronic tonsillitis with those from OSA and confirmed that there were no apparent differences between them (data not shown). Both PFAPA and chronic tonsillitis have recurrent tonsil inflammation, but inflammation in PFAPA is induced by some non-infectious mechanism. Thus, examining the histological differences between tonsils from patients with PFAPA and those with chronic tonsillitis could provide clues to the pathogenic mechanisms of PFAPA. In addition, a current problem in clinical practice is that almost all PFAPA patients are misdiagnosed as having chronic tonsillitis. Key pathological differences could be exploited to reduce these diagnostic errors. The diagnostic criteria for PFAPA syndrome are based only on clinical history and physical findings, so that different diseases could possibly meet these criteria and be misdiagnosed as PFAPA. Hereditary periodic fevers (HPF) are the most difficult diseases to differentiate from PFAPA [15]. It is now
Table 1 Clinical characteristics of 5 adults who underwent tonsillectomy due to periodic fever, aphthous stomatitis, pharyngitis and cervical adenitis syndrome. Patient
Gender
Age at surgery
Average duration of each episode (days)
Average interval of each episode (days)
Days from last steroid dose to tonsillectomy
Days from last episode to tonsillectomy
Months of follow-up
Response to tonsillectomy
1 2 3 4 5
Male Male Male Female Male
28 36 33 31 37
4.9 4.3 4.2 4.4 4.6
31.8 31 30.8 32.1 32.3
46 50 38 40 39
14 22 10 13 12
20 25 22 30 13
Complete Complete Complete Complete Complete
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Fig. 1. Comparison of tonsils from patients with PFAPA and chronic tonsillitis by hematoxylin and eosin staining. A. Germinal center areas in tonsils of adult patients with PFAPA were significantly smaller than those of chronic tonsillitis. B. The ratio of germinal center area to the tonsil area was significantly lower in the PFAPA group than that in the control group. C, D. Representative hematoxylin and eosin-stained histologic tonsil section from patients with PFAPA (C) and chronic tonsillitis (D). Scale bars: 1 mm (C–D). Data are expressed as mean SE; *p < 0.05 by Student’s t-test.
well-established that HPF patients could have adult onset of the symptoms, and such late onset is probably secondary to heterozygosity or hypomorphic mutations. In these cases, patients may present with a milder clinical picture, lacking the most typical clinical characteristics of HPF, and further complicating the differential diagnosis between PFAPA and HPF. Among HPFs, FMF and TRAPS are reported to occur in adults. Thus, in this study, all adult patients who fulfilled PFAPA diagnostic criteria underwent genotype evaluation of the MEFV and TNFRSF1A genes, respectively causing FMF and TRAPS. No patient had mutations in these genes, so we could confirm that they were indeed PFAPA patients. Just as in our study, Cantarini et al. conducted genetic tests for HPF in all patients with a presumed PFAPA clinical phenotype, and they included patients whose genetic tests were negative [14,16]. In their study, tonsillectomy was performed in 9 adult-onset PFAPA patients, leading to a response in just 2, while it was ineffective in the other cases [14]. This difference in the effectiveness of tonsillectomy is not well understood. A
possible reason for this is the ethnic difference: our patients were Japanese, while those of Cantarini et al. were Caucasian. While a larger sample size would be needed to conclusively determine whether tonsillectomy is effective for adult-onset PFAPA, our study suggests that this might be indeed the case. We compared the histologic features and immune cell composition of the tonsils of adult patients with PFAPA and age-matched patients with chronic tonsillitis. Our study demonstrated that (1) Adult-onset PFAPA patients had significantly smaller germinal center areas compared with chronic tonsillitis patients and that (2) the number of CD8+ cells in germinal centers was significantly lower in tonsils from PFAPA compared with those from chronic tonsillitis. These results are in agreement with those concerning pediatric-onset PFAPA described by some reports [10,11], suggesting that adult- and pediatric-onset PFAPA share the same pathogenesis. We also examined the histology of the tonsils of pediatric-onset PFAPA with the qualitative assessments described in the Materials and Methods, and confirmed that the histologic features are
Please cite this article in press as: Yamahara K, et al. Surgical outcomes and unique histological features of tonsils after tonsillectomy in adults with periodic fever, aphthous stomatitis, pharyngitis, and cervical adenitis syndrome. Auris Nasus Larynx (2019), https://doi.org/10.1016/j. anl.2019.08.009
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Fig. 2. Comparison of tonsils from patients with PFAPA and chronic tonsillitis by CD8 immunohistochemical staining. A. The number of CD8+ cells in germinal centers was significantly lower in tonsils from PFAPA compared to controls. B. The density of CD8+ cells in germinal centers was also significantly lower in the PFAPA group compared with controls. C, D. Immunohistochemical staining of CD8+ cells in representative tonsils from patients with PFAPA (C) and chronic tonsillitis (D). Scale bars: 200 mm (C–D). Data are expressed as mean SE; *p < 0.05 by Student’s t-test.
compatible with those reported in prior studies [10,11] (Supplementary Fig. 1A–D). However, a consensus regarding the histological features of pediatric-onset PFAPA is still lacking: Other groups have assessed the histology of tonsils from pediatric-onset PFAPA with results differing from ours. Berlucchi et al. reported lymphoid hyperplasia in PFAPA tonsils in the absence of other unusual pathological findings [17], and Peridis et al. reported that they could not find any difference in tonsillar histology between PFAPA and chronic tonsillitis [7]. However, the study by Berlucchi et al. was performed with no control group for comparison [17]. In addition, the assessment method used by Peridis et al. is not described, suggesting the possibility that they did not perform qualitative assessment. Considering these facts, it is possible that their studies would have obtained the same results as ours if they analyzed the tonsils of pediatric-onset PFAPA with our methods. We examined the percentage of B lymphocytes (CD20+) and T lymphocytes (CD3+, CD4+, and CD8+) by histological analysis. In contrast, Dytrych et al. assessed lymphocyte population by flow cytometry and showed that tonsils from
children with PFAPA had a lower percentage of B lymphocytes, switched B cells, and lower levels of CD4+ cells with high PD-1 expression (an inhibitory molecule) compared with those of control patients [18]. Our result showed no significant differences between the groups when comparing the number of CD4+ cells in tonsillar germinal centers. In the current study, we counted the whole number of CD4+ cells, not only CD4+ cells with PD-1 expression. CD4+ cells are not a unitary set of cells but represent a series of distinct cell populations with different functions [19]. It is possible that we would have obtained the same result as Dytrych et al. had we analyzed CD4+ cells with high PD-1 expression. Manthiram et al. speculated that low levels of CD4+ cells with high PD-1 expression suggest that PFAPA tonsils have fewer T follicular helper cells (specialized CD4+ cells that help germinal center B cells develop and proliferate), which correlated with the smaller germinal centers seen in PFAPA [10,20]. In our study, there were no apparent differences in the density of CD20+ cells between groups. However, considering that the areas of the germinal centers in PFAPA tonsils were significantly smaller
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Fig. 3. Comparison of tonsils from patients with PFAPA and chronic tonsillitis by CD3, CD4, and CD20 immunohistochemical staining. A, B. Immunohistochemical staining of CD3+ cells in representative tonsils from patients with PFAPA (A) and chronic tonsillitis (B). C, D. Immunohistochemical staining of CD4+ cells in representative tonsils from patients with PFAPA (C) and chronic tonsillitis (D). E, F. Immunohistochemical staining of CD20+ cells in representative tonsils from patients with PFAPA (E) and chronic tonsillitis (F). G, H. The number (G) and density (H) of CD3+ cells in a germinal center were compared between the PFAPA and control groups, and no significant differences were found. I, J. The number (I) and density (J) of CD4+ cells in a germinal center were compared between the PFAPA and control groups, and no significant differences were found. Scale bars: 200 mm (A–F).
than in controls, it is possible that the number of CD20+ cells in PFAPA tonsils were also significantly lower than controls, which might result in the smaller germinal centers seen in the PFAPA group, as Manthiram et al. speculated. The role of CD8 + cells in tonsillar germinal centers is not well understood, but a subset of CD8+ cells are reported to work as antigenexperienced T cells supporting B cell survival and immunoglobulin G production [21], and the presence of regulatory CD8 + cells in germinal centers has been described [22]. Some studies suggested that regulatory CD8+ cells trigger important
downregulatory actions, and these cells have been implied in the regulation of autoimmunity [23,24]. Forsvoll described how the absence or reduction of certain regulatory or supporting stimuli provided by these cells may cause altered function of the B cell follicules, and then contribute to the bursts of inflammation seen during febrile episodes of PFAPA [11]. However, further studies are required to elucidate the pathogenesis of PFAPA. This study has several limitations, and our results must be interpreted with caution. A major limitation of our study is the
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small PFAPA sample size (n = 5). Adult-onset PFAPA is still a rare entity and in Japan, only 3 patients with adult-onset with PFAPA have been reported other than our patients [25–27]. Due to the small sample size, we might have failed to detect minor differences between adults with PFAPA and controls. Another limitation of this study is that no data were available about the histology of the PFAPA tonsils for which tonsillectomy was ineffective, because tonsillectomy was effective in all our adult-onset patients. If these tonsils showed different histological features from those of our cohort, histological examination of tonsils could help clinicians understand the prognosis of adult-onset PFAPA patients who had tonsillectomy. In spite of the small PFAPA sample size in our study, our data showed significant statistical differences, allowing us to reach some conclusions regarding the course of the disease in adult patients. Further studies of tonsil tissues are needed to understand the mechanism behind the histologic differences we observed. Increased awareness of PFAPA will result in an increased number of adult-onset PFAPA patients and clarify the pathogenic mechanisms of this rare disease.
5. Conclusion To the best of our knowledge, this study was the first to analyze the histological features of the tonsils of adult-onset PFAPA patients. Our report demonstrates that tonsillectomy might be effective for adult-onset PFAPA patients and that tonsils of adult- and pediatric-onset PFAPA share unique histological features. Adult-onset PFAPA is still a rare entity and we described only five patients. Although our cohort is small and further studies are needed, our results suggest that the pathogenic mechanisms of adult- and pediatric-onset PFAPA are identical. In the future, the histological differences between the PFAPA tonsils for which tonsillectomy was effective vs. non-effective should be examined.
Disclosure statement The authors declare that they have no competing interests. Ethical statement This manuscript has not been published or presented elsewhere in part or in entirety and is not under consideration by another journal. All study participants provided written informed consent. We have read and understood your journal’s policies, and we believe that neither the manuscript nor the study violates any of these. There are no conflicts of interest to declare. Acknowledgments We would like to thank and Editage (www.editage.jp) for English language editing.
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Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.anl.2019.08.009.
References [1] Marshall GS, Edwards KM, Butler J, Lawton AR. Syndrome of periodic fever, pharyngitis, and aphthous stomatitis. J Pediatr 1987;110:43–6. [2] Thomas KT, Feder Jr HM, Lawton AR, Edwards KM. Periodic fever syndrome in children. J Pediatr 1999;135:15–21. [3] Forsvoll J, Kristoffersen EK, Oymar K. Incidence, clinical characteristics and outcome in Norwegian children with periodic fever, aphthous stomatitis, pharyngitis and cervical adenitis syndrome; a populationbased study. Acta paediatrica (Oslo Norway: 1992) 2013;102:187–92. [4] Hofer M, Pillet P, Cochard MM, Berg S, Krol P, Kone-Paut I, et al. International periodic fever, aphthous stomatitis, pharyngitis, cervical adenitis syndrome cohort: description of distinct phenotypes in 301 patients. Rheumatology (Oxford England) 2014;53:1125–9. [5] Wurster VM, Carlucci JG, Feder Jr HM, Edwards KM. Long-term follow-up of children with periodic fever, aphthous stomatitis, pharyngitis, and cervical adenitis syndrome. J Pediatr 2011;159:958–64. [6] Feder HM, Salazar JC. A clinical review of 105 patients with PFAPA (a periodic fever syndrome). Acta paediatrica (Oslo Norway: 1992) 2010;99:178–84. [7] Peridis S, Koudoumnakis E, Theodoridis A, Stefanaki K, Helmis G, Houlakis M. Surgical outcomes and histology findings after tonsillectomy in children with periodic fever, aphthous stomatitis, pharyngitis, and cervical adenitis syndrome. Am J Otolaryngol 2010;31:472–5. [8] Garavello W, Romagnoli M, Gaini RM. Effectiveness of adenotonsillectomy in PFAPA syndrome: a randomized study. J Pediatr 2009;155:250–3. [9] Renko M, Salo E, Putto-Laurila A, Saxen H, Mattila PS, Luotonen J, et al. A randomized, controlled trial of tonsillectomy in periodic fever, aphthous stomatitis, pharyngitis, and adenitis syndrome. J Pediatr 2007;151:289–92. [10] Manthiram K, Correa H, Boyd K, Roland J, Edwards K. Unique histologic features of tonsils from patients with periodic fever, aphthous stomatitis, pharyngitis, and cervical adenitis (PFAPA) syndrome. Clin Rheumatol 2018;37:1309–17. [11] Forsvoll J, Janssen EA, Moller I, Wathne N, Skaland I, Klos J, et al. Reduced number of CD8+ cells in tonsillar germinal centres in children with the periodic fever, aphthous stomatitis, pharyngitis and cervical adenitis syndrome. Scand J Immunol 2015;82:76–83. [12] Padeh S, Stoffman N, Berkun Y. Periodic fever accompanied by aphthous stomatitis, pharyngitis and cervical adenitis syndrome (PFAPA syndrome) in adults. Isr Med Assoc J: IMAJ 2008;10:358–60. [13] Cantarini L, Vitale A, Galeazzi M, Frediani B. A case of resistant adultonset periodic fever, aphthous stomatitis, pharyngitis and cervical adenitis (PFAPA) syndrome responsive to anakinra. Clin Exp Rheumatol 2012;30:593. [14] Cantarini L, Vitale A, Bartolomei B, Galeazzi M, Rigante D. Diagnosis of PFAPA syndrome applied to a cohort of 17 adults with unexplained recurrent fevers. Clin Exp Rheumatol 2012;30:269–71. [15] Cattalini M, Soliani M, Rigante D, Lopalco G, Iannone F, Galeazzi M, et al. Basic characteristics of adults with periodic fever, aphthous stomatitis, pharyngitis, and adenopathy syndrome in comparison with the typical pediatric expression of disease. Mediat Inflammation 2015;2015:570418. [16] Rigante D, Vitale A, Natale MF, Lopalco G, Andreozzi L, Frediani B, et al. A comprehensive comparison between pediatric and adult patients with periodic fever, aphthous stomatitis, pharyngitis, and cervical adenopathy (PFAPA) syndrome. Clin Rheumatol 2017;36:463–8. [17] Berlucchi M, Meini A, Plebani A, Bonvini MG, Lombardi D, Nicolai P. Update on treatment of Marshall’s syndrome (PFAPA syndrome): report of five cases with review of the literature. Ann Otol Rhinol Laryngol 2003;112:365–9.
Please cite this article in press as: Yamahara K, et al. Surgical outcomes and unique histological features of tonsils after tonsillectomy in adults with periodic fever, aphthous stomatitis, pharyngitis, and cervical adenitis syndrome. Auris Nasus Larynx (2019), https://doi.org/10.1016/j. anl.2019.08.009
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[18] Dytrych P, Krol P, Kotrova M, Kuzilkova D, Hubacek P, Krol L, et al. Polyclonal, newly derived T cells with low expression of inhibitory molecule PD-1 in tonsils define the phenotype of lymphocytes in children with Periodic Fever, Aphtous Stomatitis, Pharyngitis and Adenitis (PFAPA) syndrome. Mol Immunol 2015;65:139–47. [19] Zhu J, Paul WE. CD4 T cells: fates, functions, and faults. Blood 2008;112:1557–69. [20] Crotty S. T follicular helper cell differentiation, function, and roles in disease. Immunity 2014;41:529–42. [21] Quigley MF, Gonzalez VD, Granath A, Andersson J, Sandberg JK. CXCR5+ CCR7- CD8 T cells are early effector memory cells that infiltrate tonsil B cell follicles. Eur J Immunol 2007;37:3352–62. [22] Kim HJ, Verbinnen B, Tang X, Lu L, Cantor H. Inhibition of follicular T-helper cells by CD8(+) regulatory T cells is essential for self tolerance. Nature 2010;467:328–32.
[23] Ramiscal RR, Vinuesa CG. T-cell subsets in the germinal center. Immunol Rev 2013;252:146–55. [24] Chang JH, Chung Y. Regulatory T cells in B cell follicles. Immune Netw 2014;14:227–36. [25] Kutsuna S, Ohmagari N, Tanizaki R, Hagino N, Nishikomori R, Ujiie M, et al. The first case of adult-onset PFAPA syndrome in Japan. Modern Rheumatol 2016;26:286–7. [26] Yasuura S, Harada Y. A case of adult-onset periodic fever, aphthous stomatitis, pharyngitis, and adenitis syndrome in Japan. J Gen Family Med 2017;18:261–4. [27] Ota K, Kwak-Kim J, Takahashi T, Mizunuma H. Pregnancy complicated with PFAPA (periodic fever, aphthous stomatitis, pharyngitis and cervical adenitis) syndrome: a case report. BMC Pregnancy Childbirth 2018;18:207.
Please cite this article in press as: Yamahara K, et al. Surgical outcomes and unique histological features of tonsils after tonsillectomy in adults with periodic fever, aphthous stomatitis, pharyngitis, and cervical adenitis syndrome. Auris Nasus Larynx (2019), https://doi.org/10.1016/j. anl.2019.08.009