Histiocytic necrotizing lymphadenitis in children: A clinical and immunohistochemical comparative study with adult patients

International Journal of Pediatric Otorhinolaryngology 77 (2013) 429–433

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Histiocytic necrotizing lymphadenitis in children: A clinical and immunohistochemical comparative study with adult patients Jae-Hyun Seo a, Jun-Myung Kang a, HeeJeong Lee b, WeonSun Lee c, Se-Hwan Hwang a, Young-Hoon Joo a,* a

Department of Otolaryngology, Head and Neck Surgery, College of Medicine, The Catholic University of Korea, Seoul, Republic of Korea Department of Hospital Pathology, College of Medicine, The Catholic University of Korea, Seoul, Republic of Korea c Institute of Clinical Medicine Research, Bucheon St. Mary’s Hospital, The Catholic University of Korea, Seoul, Republic of Korea b

A R T I C L E I N F O

A B S T R A C T

Article history: Received 27 August 2012 Received in revised form 30 November 2012 Accepted 4 December 2012 Available online 29 December 2012

Objectives: Limited information is available regarding the characteristics of histiocytic necrotizing lymphadenitis (HNL) in children. This study compares the clinical and laboratory features as well as the immunohistochemical findings of HNL in children with those of adults. Study design: Retrospective analysis. Methods: Thirty patients who underwent a biopsy of a cervical lymph node and were histologically proven to have HNL were enrolled in this study. There were 13 children and 17 adults. CD68, CD163 and myeloperoxidase expression were analyzed by immunohistochemical staining. Results: Children had more bilateral lymphadenopathy (P = 0.045) and a higher expression of CD68 (P = 0.043) than did the adult patients. However, there was no significant difference between the groups in the following variables: patient gender, presence of fever, size and necrosis of enlarged lymph node, multiplicity of lymphadenopathy, WBC count, ESR, CRP, recurrence, and expression of myeloperoxidase and CD163. Conclusions: The clinical and immunohistological characteristics of HNL in pediatric patients are similar to those of adults. Bilateral involvement of lymph nodes and a high expression of CD68 were the only features significantly associated with children with HNL. ß 2012 Elsevier Ireland Ltd. All rights reserved.

Keywords: Histiocytic necrotizing lymphadenitis Child Sign and symptoms Immunohistochemistry Antigen CD

1. Introduction Histiocytic necrotizing lymphadenitis (HNL), also known as Kikuchi–Fujimoto disease, was first described independently by both Kikuchi and Fujimoto et al. in 1972 [1,2]. This rare and unusual form of lymphadenitis preferentially affects young females, with a majority of patients under the age of 30 years old, in a 3–4:1 ratio [3,4]. A variable percentage of patients (30– 50%) may develop a low grade fever associated with upper respiratory symptoms in addition to the lymphadenopathy [5]. Extranodal involvement is uncommon, but skin rash, hepatitis, arthritis, oral ulcers, and eye involvement have been reported [5,6]. Laboratory tests may reveal high C-reactive protein level (CRP) or erythrocyte sedimentation rate (ESR), leukopenia, and atypical lymphocytes. Definitive diagnosis depends on lymph node biopsy. Three histopathological variants have been reported: proliferative,

* Corresponding author at: Department of Otolaryngology, Head and Neck Surgery, Bucheon St. Mary’s Hospital, College of Medicine, The Catholic University of Korea, 2 Sosa-dong, Wonmi-gu, Bucheon, Kyounggi-do 420-717, Republic of Korea. Tel.: +82 32 340 7207; fax: +82 32 340 2674. E-mail address: [email protected] (Y.-H. Joo). 0165-5876/$ – see front matter ß 2012 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.ijporl.2012.12.003

necrotizing, and xanthomatous. The etiopathogenesis of HNL is still unknown. Infectious agents (Epstein–Barr virus, herpes virus 6 and 8, toxoplasma, yersinia, brucella, human immunodeficiency virus, and human T-cell lymphotropic virus type 1) and genetic associations (human leukocyte antigen class-2) have been implicated [7]. HNL affects individuals of all ages, particularly young women, but there are few descriptions of this disease in the pediatric literature [8–12]. Therefore, the aims of the present study include evaluation of the clinical, laboratory, and immunohistochemical features of HNL in Korean children and comparison of these findings with those of Korean adults diagnosed with HNL. 2. Methods 2.1. Patients and tumor samples The clinical and pathological data of 13 children who underwent excisional biopsy and were diagnosed with HNL at the Department of Otolaryngology, HNS, The Catholic University of Korea, Bucheon, Korea, from April 2008 to December 2011, were reviewed. The criteria for enrolment included HNL may vary but often meet the following criteria. Microscopically, the

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affected nodes showed focal, well-circumscribed, paracortical necrotizing lesions. There were abundant karyorrhectic debris, scattered fibrin deposits, and collections of mononuclear cells. Plasma cells and meutrophils were very scanty. The exclusion criteria were the following: (1) history of excisional or incisional biopsy of HNL for diagnosis; (2) history of prior HNL; (3) coexistent cervical tuberculosis or other granulomatous lesions; (4) other coexistent systemic diseases such as systemic lupus erythematosus. The mean age of the children was 12.3 years (range 5–17 years). All of the patients received antibiotic treatment for sustained fever or enlarged lymph nodes. We also studied, as a comparative group, 17 adult patients, between the ages of 18 and 63 (mean 28.8 years). Biopsy of a lymph node was usually performed in the outpatient department after several weeks of follow-up. The interval between onset of symptoms and cervical lymph node excision biopsy ranged from 6 days to 8 weeks. The Institutional Review Board of Bucheon St. Mary’s Hospital approved the retrospective review of medical records and use of archived tumor specimens.

60 min. To block the endogenous hydroperoxidase activity, UV INHIBITOR was performed at 37 8C for 4 min before the detect primary antibody. The primary antibodies for CD168, CD163, and myeloperoxidase were diluted in Dako Antibody Diluent (Dako Cytomation, Glostrup, Denmark) with background-reducing components to the following dilutions: CD68 – 1:100 dilution (Dako), CD163 – 1:100 dilution (Abcam Ltd., Cambridge, UK), and myeloperoxidase – 1:100 dilution (Abcam Ltd.). Then, the primary antibodies were incubated for 32 min at 37 8C, while HRPlabeled secondary antibody was incubated for 8 min at 37 8C. To visualize the signal for protein, the HRP-labeled secondary antibody was exposed to UV DAB with UV DAB H2O2 for 8 min and UV COPPER for 4 min (UV COPPER changes the DAB color to a reddish brown). Lastly, the slides were counterstained with Hematoxylin II (Ventana) for 4 min and Bluing Reagent (Ventana) for 4 min.

2.2. Immunohistochemistry

The immunohistochemical staining was interpreted by two independent pathologists who were blind to the lymph node status of the patients corresponding to the sections. The expression of each antibody was quantified based on the extent of staining. Inflammatory cells which showed distinct cytoplasmic staining were considered positive. The percentage of positive cells was graded on the following scale: grade 0 (negative), grade 1 (1–30% positive cells), grade 2 (31–70% positive cells), or grade 3 (71–100% positive cells).

All archival tissue samples were routinely fixed in formalin and embedded in paraffin. Immunohistochemistry was performed on 3-mm paraffin sections using an automated immunohistochemical stainer (Ventana Medical Systems, Inc., Tucson, AZ, USA). Sections were deparaffinized using EZ PrepTM (Ventana) solution. Deparaffinized tissue sections were pretreated with cell conditioning solution (Ventana) at 95 8C for

2.3. Semiquantitative analyses of immunohistochemical staining

Table 1 Demographic profiles of histiocytic necrotizing lymphadenitis patients. Case

Age (years)

Gender

Fever (>37.5 8C)

WBC (/mm3)

ESR (mm/h)

CRP (mg/dL)

Bilate-rality

Multi-plicity

CD68

CD163

Myeloperoxidase

Children 1 2 3 4 5 6 7 8 9 10 11 12 13

5 6 9 11 13 13 13 13 14 14 15 17 17

M F M M F F M M F M F M F

+ + + +

3800 3810 3130 3730 7210 4230 5600 3460 4520 4730 2210 4300 5850

20 14 10 35 23 28 24 3 17 22 33 18 6

7.23 16.90 1.40 7.30 2.11 10.29 40.00 0.10 0.52 0.46 27.60 10.20 3.74

+ +

+ + + + + + + + + + + + +

3+ 2+ 3+ 2+ 2+ 2+ 3+ 2+ 2+ 3+ 3+ 3+ 3+

0 0 0 0 0 0 0 1+ 0 0 0 0 0

1+ 1+ 1+ 2+ 0 1+ 1+ 1+ 1+ 0 1+ 1+ 1+

Adults 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30

18 18 19 22 22 23 23 25 26 29 31 32 32 32 26 29 63

F F F F M F F F M M F F F M F F F

4460 3200 3080 6430 4050 5910 4200 2880 4490 2830 6200 6230 6120 7340 3340 5450 7030

31 21 23 17 17 8 37 20 2 7 34 28 2 46 29 37 33

0.80 16.41 9.80 8.70 37.72 0.34 71.00 10.10 2.06 1.25 1.62 13.60 0.45 21.70 0.50 15.20 2.61

+

+

+

+ + +

0 2+ 2+ 2+ 3+ 3+ 1+ 2+ 1+ 2+ 2+ 3+ 1+ 1+ 2+ 3+ 3+

0 0 0 0 0 1+ 0 0 1+ 0 0 0 0 0 0 0 0

0 2+ 0 1+ 1+ 1+ 1+ 0 1+ 0 1+ 2+ 1+ 1+ 1+ 1+ 1+

+

+ +

+ + +

+

+

+

+

+

+ + +

+ + +

+ + + + + + + +

WBC, white blood cell count; ESR, erythrocyte segmentation rate; CRP, C-reactive protein; level 0, no expression; 1+, weak expression; 2+, moderate expression; 3+, marked expression.

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Fig. 1. Immunohistochemical analysis in histiocytic necrotizing lymphadenitis: weak expression for CD68 (A), negative immunostaining for CD163 (C), and myeloperoxidase (E) in the cytoplasm; marked expression for CD68 (B), positive immunostaining for CD163 (D), and myeloperoxidase (F) in the cytoplasm (original magnification 400).

2.4. Statistical analysis

3. Results

Continuous variables (size of lymphadenopathy, WBC count, etc.) were recorded as means  standard deviations and analyzed using the t-test. To analyze statistically significant relationships among the distribution of categorical values (gender, presence of CD68 and myeloperoxidase, etc.), the chi-square test or Fisher’s exact test were used. P < 0.05 was considered statistically significant. All calculations were performed using SPSS software version 13.0 (SPSS, Chicago, IL, USA).

3.1. Clinical findings There were seven male child patients and six female child patients with a male–female ratio of 1.2:1. Their ages ranged from five to 17 years with a mean age of 12.3  3.7 years. Clinical data are summarized in Table 1. Lymphadenopathy with prolonged fever was observed in seven patients (53.8%). The mean size of affected lymph nodes on biopsy was 16.5  4.1 mm. Necrosis was defined by the

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visualization of low attenuation on the inside of lymph nodes on enhanced scans, and in this series, five patients had necrotic lymph nodes. Unilateral and bilateral cervical lymphadenopathy was identified in 46.2% and 53.8% of patients, respectively. All patients had involvement of multiple cervical lymph nodes. The most frequent sites of cervical lymphadenopathy were: level II (92.3%), level III (92.3%), level IV (61.5%), level V (61.5%), and level I (30.8%). Leukopenia with a white blood cell (WBC) count of <3900/mm3 was noted in six patients (46.2%). Six patients had elevated ESR (>20 mm/h) and seven had elevated CRP (>5 mg/dL). During a mean follow-up period of 6.8 months, one patient experience recurrence. 3.2. Immunohistochemical findings CD68, CD163, and myeloperoxidase immunoreactivity was detected mainly in the cytoplasm and membranes of lymphocytes (Fig. 1). All 13 children demonstrated moderate to strong cytoplasmic staining for CD68 (2+ to 3+). Of 13 child patients, myeloperoxidase reactivity was detected in 11 (84.6%), and CD163 reactivity was detected in one (7.7%). Table 2 depicts complete immunohistochemistry results. 3.3. Differences between children and adults with histiocytic necrotizing lymphadenitis Bilateral lymphadenopathy was significantly different between child and adult patient groups, with seven children demonstrating bilateral node involvement (53.8%) compared to only three adults (17.6%) (P = 0.045) (Table 3). There was a significant difference between groups in CD68 expression (P = 0.043), however, there was no significant difference in myeloperoxidase and CD163 expression between these groups. A higher percentage of the child patient group was male, however, this result did not reach statistical significance (P = 0.093). Variables which were not significantly different between the groups included fever, lymph node size and necrosis, node multiplicity, WBC count, ESR, and CRP. Treatment results also did not differ significantly between the adult and child patient groups. 4. Discussion The most common presentation of HNL is cervical lymphadenitis, although generalized lymphadenitis also occurs. Other symptoms include low-grade fever, malaise, weight loss, nausea, vomiting, fatigue, and diarrhea [13]. The most common sites of cervical lymphadenitis include the posterior cervical triangle and jugular carotid triangle [14]. Bilateral cervical lymph node enlargement occurred in 11% [13]. In this study, we analyzed clinical and radiologic features of cervical lymphadenitis. The majority of our patients presented with multiple node involvement, and one third presented with bilateral lymphadenopathy. There was a significant difference in bilateral node involvement between the pediatric and adult populations; bilateral involvement of the neck was more common in children. In this study, Table 2 Expression of immunohistochemical markers in histiocytic necrotizing lymphadenitis. Marker (intensitya)

CD 68 (2+) CD 163 (1+) Myeloperoxidase (1+)

Fraction of expression (%) Children (N = 13)

Adults (N = 17)

P value

100.0 7.7 84.6

70.6 11.8 76.5

0.043 0.603 0.469

a The immunostaining results were evaluated semiquantitatively using grades 0 to 3+.

Table 3 Comparative clinical and laboratory findings of histiocytic necrotizing lymphadenitis. Parameter Age (years) Gender Male Female Fever (>37.5 8C) Yes No Size of lymphadenopathy (mm) Necrosis of lymphadenopathy Yes No Multiplicity of lymphadenopathy Yes No Laterality Bilateral neck Unilateral neck WBC (mm3) ESR (mm/h) CRP (mg/dL) Recurrence Yes No

Children (N = 13) 12.3  3.7

Adults (N = 17)

P value

28.8  10.8 0.093

7 (53.8%) 6 (46.2%)

4 (23.5%) 13 (76.5%)

7 (53.8%) 6 (46.2%) 16.5  4.1

6 (35.3%) 11 (64.7%) 17.0  6.1

0.260

0.500 0.642 5 (38.5%) 8 (61.5%)

8 (47.1%) 9 (52.9%)

13 (100%) 0 (0%)

14 (82.4%) 3 (17.6%)

0.045

0.135 7 (53.8%) 3 (17.6%) 6 (46.2%) 14 (82.4%) 4352.3  1291.7 4896.5  1534.5 0.167 19.5  9.7 23.06  13.0 0.197 9.8  12.0 12.6  18.7 0.452 0.261 1 (7.7%) 4 (23.5%) 12 (92.3%) 13 (76.5%)

more than half of the pediatric patients with HNL demonstrated bilateral lymphadenitis, thus a diagnosis of HNL should not be excluded when lymphadenitis affects both sides of the neck in children. HNL, which typically affects young women between 20 and 30 years of age, is a rare disease in the pediatric population and is known to affect genders differently in different age groups. The disease is more prevalent in males under the age of 12 and females over the age of 13 [11,15–17]. The cause of this pattern is unknown but may involve hormonal changes after puberty [11]. In our study, M:F sex ratios were 7:6 in children (up to age 18) and 6:11 in adults. In the pediatric population, the ratio was 3:1 under the age of 12 and 4:5 between the ages of 13 and 17. These ratios are similar to previous reports. Although of no diagnostic value for HNL, laboratory findings demonstrate leukopenia with WBC < 4000/mm3 in 25–50% of patients and slightly elevated CRP [11,13,15]. In our study, leukopenia with WBC < 4000/mm3 was discovered in 46.2% of children and 29.4% of adults. ESR and CRP were mildly elevated in both groups, but this was not statistically significant. It has been suggested that the clinical pathogenesis of HNL is an infectious disease, particularly a viral infection, though its immediate cause remains unknown [18]. Histologically, the lymph nodes affected by HNL exhibit focal proliferations of transformed lymphocytes and histiocytes (tissue macrophages) along with apoptotic cells and nuclear debris [19]. Immunohistochemically, the histiocytic component is characterized by the expression of the CD68 antigen, whereas the lymphoid component carries a T-cell phenotype with a prevalence of CD8+ cytotoxic cells [20,21]. CD68 is a glycoprotein used as a monocyte-macrophage marker. It is used to distinguish diseases of otherwise similar appearance, such as the monocyte-macrophage and lymphoid forms of leukemia (the latter being CD68 negative). In this study, 100% of 13 children with HNL expressed CD68, whereas 70.6% of 17 adults expressed the antigen. We cannot explain why the expression of CD68 is significantly increased in children. Viral infections such as Epstein– Barr virus, cytomegalovirus, type 1 human T cell lymphotropic virus, human herpesvirus 6, and parvovirus B-19 have been suspected to be linked to HNL [7,18]. HNL may be a hyperimmune

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reaction of the monocyte-macrophage lineage to viral infections in children. The present study has some limitations, including the retrospective analysis, small sample size, unequal distribution of age groups, and short median follow-up period. Accordingly, we suggest that an additional prospective investigation be conducted in a larger number of patients. 5. Conclusion We found that the bilateral involvement of lymphadenopathy in children and adults differed, but that other clinical features were equivalent in both patient groups. HNL in children was also characterized by a high expression of CD68. Long-term follow-up studies will lead to a better understanding of the natural history and pathogenesis of HNL in childhood. Conflict of interest None. Acknowledgment This work was supported, in part, by grants from the alumni associated of the Department of Otolaryngology Head and Neck Surgery, The Catholic University of Korea References [1] M. Kikuchi, Lymphadenitis showing focal reticulum cell hyperplasia with nuclear debris and phagocytes: a clinicopathological study, Nippon Ketsueki Gakkai Zassho 35 (1972) 379–380. [2] Y. Fujimoto, Y. Kojima, K. Yamaguchi, Cervical subacute necrotizing lymphadenitis, Naika 30 (1972) 920–927. [3] C.E. Garcı´a, H.V. Girdhar-Gopal, D.M. Dorfman, Kikuchi–Fujimoto disease of the neck. Update, Ann. Otol. Rhinol. Laryngol. 102 (1993) 11–15.

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