924 Brief Reports
Development of Langerhans Cell Histiocytosis Associated With Chronic
Active Epstein–Barr Virus Infection
Naoki Sakata, MD,1* Naomi Toguchi, MD,1 Masatomo Kimura, MD,2 Masahiro Nakayama,
Keisei Kawa, MD,4 and Tsukasa Takemura, MD1
Chronic active Epstein–Barr virus (CAEBV) infection is character-
ized by a status of lymphoproliferative disease of EBV-infected cells,
resulting in chronic or recurrent infectious mononucleosis-like
symptoms. CAEBV is always accompanied by life-threatening
complications. We report the case of a 2-year-old female patient
with CAEBV who subsequently developed Langerhans cell histiocy-
Key words: chronic active EB virus (CAEBV) infection; Epstein–Barr virus (EBV); Langerhans cell histiocytosis (LCH)
INTRODUCTION
Primary Epstein–Barr virus (EBV) infection is usually asymp-
tomatic, but sometimes it results in infectious mononucleosis, which
resolves spontaneously after the emergence of EBV-specific
immunity [1]. However, EBV causes chronic infections in
apparently immunocompetent hosts. Chronic active EBV (CAEBV)
infection is characterized by chronic or recurrent infectious
mononucleosis-like symptoms persisting over a long time, and by
an unusual pattern of anti-EBV antibodies [2]. Some studies have
reported that patients with CAEBV infection have high viral loads,
as assessed by real-time quantitative polymerase chain reaction
(RQ-PCR) [3]. Moreover, accumulating evidence suggests that the
harboring of EBV by T or natural killer (NK) cells results in clonal
expansion of these cells, which causes a status of hypercytokinemia,
hemophagocytic syndrome, and lymphoproliferative disease [4–6].
Langerhans cell histiocytosis (LCH), a rare disease of children
involving the bone, skin, soft tissue, and other organs, has highly
variable biological behavior and clinical severity ranging from
solitary lytic bone lesions with a good prognosis to a disseminated
disorder with diffuse organ involvement [7]. Phenotypic characters
of the cell proliferating at the lesion are similar to those of
Langerhans cells of the epidermis, including expression of CD1a
and langerin (CD207) and positivity for S-100 and Birbeck granules
[7,8]. Using X-chromosome-linked DNA markers, it has been
demonstrated that the CD1aþ cells in LCH lesions are clonal [9,10],
suggesting a neoplastic origin of these cells. In contrast, it has been
argued that the proliferation of these cells in LCH is a
physiologically appropriate response to viral infection and malig-
nancy [11–13]. We report the clinical course of a patient with
CAEBV subsequently developing bilateral orbital masses, which
were diagnosed as LCH.
CASE REPORT
A 1-year-old female patient was referred to our hospital
presenting with prolonged fever, anemia (hemoglobin, 5.9 g/dl),
and remarkable splenomegaly. Serial investigations, such as tumor
markers, bone marrow examination, bone and gallium scintigraphy,
and systemic computed tomography (CT), failed to find hematolo-
gical and malignant disorders. However, we found an increase of
EBV-DNA copies measured by RQ-PCR (1
104 copies/106 WBC)
in her peripheral blood and the serum level of soluble interleukin-2
ß 2007 Wiley-Liss, Inc.
DOI 10.1002/pbc.21249
MD,
3
tosis (LCH) presenting with bilateral exophthalmos, bone, and skin
involvement. In situ hybridization for EBER revealed EBV-infected B-
cells present in lesional tissue implying that interactions between
EBV-infected B-cells and lesional Langerhans cells may be associated
with the development of LCH. Pediatr Blood Cancer 2008;50:924–
927. ß 2007 Wiley-Liss, Inc.
receptor (10,600 U/ml), suggesting EBV-induced hypercytokine-
mia. The levels of LDH and fibrinogen were 585 IU/L and 130 mg/
dl, respectively; however we could not diagnose EBV-associated
hemophagocytic lymphohistiocytosis because repeated bone mar-
row examinations demonstrated normal cellularity and no phago-
cytosis, and the serum levels of ferritin or triglyceride were normal.
Acyclovir and intravenous gammaglobulin were started, but no
effect on her clinical signs. Therefore, she was started on
prednisolone followed by resolution of her symptoms and signs.
On weaning of prednisolone, however, the splenomegaly and fever
recurred and cyclosporine A (CsA) was added to her therapy.
Three months after the patient’s admission, clinical signs were
controlled with the use of prednisolone and CsA; however, the EBV-
DNA copy number was still high (1.2
104) and serum titers against
VCA (IgG) and EBNAwere 2,560 and <10, respectively, raising the
possibility of CAEBV. Next, we investigated which lymphocyte
population was harboring EBV. We purified each lymphocyte
population (CD4þ, CD8þ, CD56þ, CD19þ) from the patient’s
peripheral blood by FACS (FACSVantage; Beckton-Dickinson),
and then looked for EBV-DNA by qualitative PCR [14] (Fig. 1A).
An experiment showed that EBV was harbored by T-cells in addition
to B-cells, and that, among T-cells, the EBV load was somewhat
greater in CD8þ than in CD4þ cells. Next, we examined the T-cell
receptor (TCR) Vb repertoire using monoclonal antibodies (IO Test
Beta Mark, TCR Vb repertoire kit; Coulter) and found a skewed
pattern in CD8þ cells (Fig. 1B). Together, these findings indicated
that EBV had infected the patient’s CD8þ cells and that this was
followed by clonal expansion of these cells, which is consistent with
the criteria for the identification of CAEBV [15]. Patient’s parents
did not accept to give her more intensive therapy for CAEBV, such
as chemotherapy or stem cell transplantation; she, therefore,
received prednisolone and CsA for a year.
—
1
—————
Department of Pediatrics, Kinki University School of Medicine,
Osaka; 2Department of Pathology, Kinki University School of
Medicine, Osaka; 3Division of Pathology, Osaka Medical Center and
Research Institute for Maternal and Child Health, Izumi, Osaka;
4
Division of Hematology/Oncology, Osaka Medical Center and
Research Institute for Maternal and Child Health, Osaka
*Correspondence to: Naoki Sakata, 377-2 Onohigashi Osakasayama,
Osaka 589-8511, Japan. E-mail: nsakata@med.kindai.ac.jp
Received 22 January 2007; Accepted 26 March 2007
 Brief Reports 925

Fig. 1. Results of qualitative PCR assay and the pattern of the TCR Vb repertoire. A: Different lymphocyte populations (CD4þ, CD8þ, CD56þ,
CD19þ) were purified from the patient’s peripheral blood using a fluorescence-activated cell sorter (FACSVantage; Beckton–Dickinson). EBV
DNA (162 bp) was detected by PCR. As positive control (P.C), DNA extracted from B95.8 cell line was used. B: Peripheral blood lymphocytes were
stained with various anti-TCR Vb monoclonal antibodies (IO Test Beta Mark, TCR Vb repertoire kit; Coulter) in addition to anti-CD4 and CD8
monoclonal antibodies. Three-color flow analysis was performed using a flow cytometer (FACScan; Beckton–Dickinson). Oligoclonal
proliferation was detected only in CD8þ population. [Color figure can be viewed in the online issue, which is available at www.interscience.
wiley.com.]

At 2 years of age, she presented with bilateral exophthalmos. CT
scan revealed bilateral orbital tumors with osteolysis of the right
temporal bone. A biopsy of the right orbital tumor showed small
round cells, histiocytes, and neutrophils (Fig. 2A). Some of the small
round cells had renal-shaped nuclei and were positive for S-100,
CD1a, and langerin. The findings were consistent with those of
LCH. In situ hybridization using an EBER probe revealed that
CD1aþ cells were negative for EBER (Fig. 2B and C). However,
immunostaining demonstrated a population of EBERþ B-cells
(CD20þ, CD79aþ). On examination, she had a typical skin eruption
in the post-auricular area and investigation revealed additional
punched-out lesions in cranial and ischial bones. After 5 months, she
achieved complete remission with chemotherapy as reported by
Koyama et al. [16] and her viral load of EBV decreased below the
detection limit of RQ-PCR (Fig. 2D).
DISCUSSION
The pathogenesis of LCH is still enigmatic and it certainly
remains unclear whether EBV infection causes LCH. McClain et al.
failed to find evidence of genomes for EBV in 56 cases of LCH [17].
On the contrary, Shimakage et al. showed positive hybridization
signals for EBER1 RNA using in situ hybridization in paraffin
sections from 17 cases of LCH [18]. These differences may be
dependent on the sensitivity of the examination or the endemic
distribution of EBV-associated malignant disease. In our case,
EBER in situ hybridization revealed no positivity of CD1aþ cells for
EBER, suggesting that EBV genomes are not integrated into these
cells. However, we could not rule out an association of EBV
infection with the development of LCH because a number of EBV-
infected B-cells infiltrated the tumor tissue, suggesting that
Pediatr Blood Cancer DOI 10.1002/pbc
interactions between EBV-infected B-cells and lesional cells,
including CD1aþ cells in the LCH lesion, might contribute to the
pathological development of LCH. Egeler et al. suggested that the
expression of CD40 and CD40 ligand (CD40L) in LCH lesions
along with CD40–CD40L interactions might play an important role
in activating both the lesional cells of LCH (CD40þ) and T-cells
(CD40Lþ). This resulted in increased expression of costimulatory
and adhesion molecules, proliferation, and the production of
proinflammatory cytokines and proteolytic enzymes, all features
of LCH [19]. In contrast, Imadone et al. showed immunostaining
results indicating that the distribution of CD3þ T-cells in the
patient’s tumor tissue was sparse (data not shown), which implies
that the main source of CD40L may have been EBV-infected B-cells
that had infiltrated the tumor tissue, because EBV infection induced
expression of CD40L on B-cells [20].
In regard to our patient’s treatment, we employed more intensive
chemotherapy for patients with EBV-associated T/NK-LPD [16]
than that for patients with LCH. EBV-DNA load decreased with the
chemotherapy, eventually becoming non-detectable by RQ-PCR;
the bilateral orbital masses resolved.
In conclusion, the association between chronic EBV infection
and the subsequent occurrence of LCH remains obscure; however,
we suggest that EBERþ B-cells detected in the tumor tissue may
trigger the activation of Langerhans-like lesional cells and
eventually the development of LCH in some cases.
ACKNOWLEDGMENT
The authors thank Akihisa Sawada and Shunichi Takeshima
(Osaka Medical Center for Maternal and Child Health) for
performing flow cytometry or immunostaining.
926 Brief Reports

Fig. 2. Pathological findings of right orbital tumor and EBV-DNA copy number in the clinical course. A: HE (hematoxylin and eosin) staining.
B: Immunohistological findings of CD1a staining; cells staining brown were positive for CD1a. C: EBER in situ hybridization; cells staining black
were positive for EBER. D: PRD, prednisolone; CsA, cyclosporine A; *, onset of LCH; VP, etoposide 100 mg/m2 per week; CHOP,
cyclophosphamide, 750 mg/m2 (day 1); adriamycin, 25 mg/m2 (days 1, 2); vincristine, 2 mg/m2 (day1); prednisolone, 50 mg/m2 (days 1-5); CA,
cytosine arabinoside, 3 g/m2 every 12 h (days 1, 2); l-asparaginase, 10,000 U/m2 (day 2); prednisolone, 30 mg/m2 (days 1, 2); HDCA, cytosine
arabinoside, 1.5 g/m2 every 12 hr (days 1–6); prednisolone, 30 mg/m2 (days 1–6). [Color figure can be viewed in the online issue, which is available
at www.interscience.wiley.com.]

REFERENCES 4. Jones JF, Shurin S, Abramowsky C, et al. T-cell lymphomas

1. Cohen JI. Epstein–Barr virus infection. N Engl J Med 2000;343:
481–492.
2. Rickinson AB. Chronic, symptomatic Epstein–Barr virus infec-
tion. Immunol Today 1986;7:13–14.
3. Kimura H, Morita M, Yabuta Y, et al. Quantitative analysis of
Epstein–Barr virus load by using a real-time PCR assay. J Clin
Microbiol 1999;37:132–136.
containing Epstein–Barr viral DNA in patients with chronic
Epstein–Barr virus infections. N Engl J Med 1988;318:733–741.
5. Kikuta H, Taguchi Y, Tomizawa K, et al. Epstein–Barr virus
genome-positive T lymphocytes in a boy with chronic active EBV
infection associated with Kawasaki-like disease. Nature 1988;
333:455–457.
6. Kanegane H, Bhatia K, Gutierrez M, et al. A syndrome of
peripheral blood T-cell infection with Epstein–Barr virus (EBV)

Pediatr Blood Cancer DOI 10.1002/pbc


followed by EBV-positive T-cell lymphoma. Blood 1998; 91:
2085–2091.
7. Favara BE. Langerhans’ cell histiocytosis pathobiology and
pathogenesis. Semin Oncol 1991;18:3–7.
8. Mizumoto N, Takashima A. CD1a and langerin: Acting as more
than Langerhans cell markers. J Clin Invest 2004;113:658–660.
9. Willman CL, Busque L, Griffith BB, et al. Langerhans’-cell
histiocytosis (histiocytosis X)—a clonal proliferative disease. N
Engl J Med 1994;331:154–160.
10. Yu RC, Chu C, Buluwela L, et al. Clonal proliferation of
Langerhans cells in Langerhans cell histiocytosis. Lancet 1994;
343:767–768.
11. Chen CJ, Ho TY, Lu JJ, et al. Identical twin brothers concordant for
Langerhans’ cell histiocytosis and discordant for Epstein–Barr
virus-associated haemophagocytic syndrome. Eur J Pediatr 2004;
163:536–539.
12. Glotzbecker MP, Dormans JP, Pawel BR, et al. Langerhans cell
histiocytosis and human herpes virus 6 (HHV-6), an analysis by
real-time polymerase chain reaction. J Orthop Res 2006;24:313–
320.
13. Raj A, Bendon R, Moriarty T, et al. Langerhans cell histiocytosis
following childhood acute lymphoblastic leukemia. Am J Hematol
2001;68:284–286.
Central Nervous System Juvenile Xanthogranuloma
With Malignant Transformation
1 2
Andrea Orsey, MD, Michele Paessler, MD,
Juvenile xanthogranuloma (JXG) is a rare histiocytic disorder that
typically manifests in the skin. Here, we describe a patient with JXG
diffusely involving the central nervous system (CNS), whose disease
responded to therapy but subsequently underwent dissemination to
the peritoneum and bone marrow. Repeat biopsy at dissemination
revealed pleomorphic histiocytes with tetraploidy, suggesting
Key words: central nervous system; histiocytic sarcoma; juvenile xanthogranuloma
INTRODUCTION
The histiocytoses are proliferative disorders of dendritic cells
(DC) that occur predominately in children. These disorders include
Langerhans cell histiocytosis (LCH), which is characterized by the
accumulation of abnormal DC containing Birbeck granules within
affected tissues, and non-Langerhans cell histiocytoses, which are
associated with the proliferation and/or activation interstial/dermal
DC and macrophages. Juvenile xanthogranuloma (JXG) is a non-
Langerhans cell histiocytosis that usually occurs as a spontaneously
regressing cutaneous nodule on the trunk, scalp, face or extremities.
In rare patients, JXG involves extracutaneous sites, such as the liver,
lungs, spleen, kidney, gastrointestinal tract, pancreas, bone or CNS
[1]. Unlike cutaneous JXG, which is typically self-limited, JXG of
the CNS is often difficult to control. Here, we report a patient with
JXG involving the leptomeningeal surfaces and deep white matter.
Despite initial improvement after chemo- and radiation therapy, this
patient’s disease spread to the peritoneal cavity and bone marrow.
Repeat biopsy at dissemination demonstrated an unusual histiocytic
proliferation characterized by marked cytologic atypia and tetra-
ploidy.
ß 2007 Wiley-Liss, Inc.
DOI 10.1002/pbc.21252
Brief Reports 927
14. Uhara H, Sato Y, Mukai K, et al. Detection of Epstein–Barr virus
DNA in Reed-Sternberg cells of Hodgkin’s disease using the
polymerase chain reaction and in situ hybridization. Jpn J Cancer
Res 1990;81:272–278.
15. Kawa K. Diagnosis and treatment of Epstein–Barr virus-associated
natural killer cell lymphoproliferative disease. Int J Hematol
2003;78:24–31.
16. Koyama M, Takeshita Y, Sakata A, et al. Cytotoxic chemotherapy
successfully induces durable complete remission in 2 patients with
mosquito allergy resulting from Epstein–Barr virus-associated T-/
natural killer cell lymphoproliferative disease. Int J Hematol 2005;
82:437–440.
17. McClain K, Jin H, Gresik V, et al. Langerhans cell histiocytosis:
Lack of a viral etiology. Am J Hematol 1994;47:16–20.
18. Shimakage M, Sasagawa T, Kimura M, et al. Expression of
Epstein–Barr virus in Langerhans’ cell histiocytosis. Hum Pathol
2004;35:862–868.
19. Egeler RM, Favara BE, Laman JD, et al. Abundant expression of
CD40 and CD40-ligand (CD154) in paediatric Langerhans cell
histiocytosis lesions. Eur J Cancer 2000;36:2105–2110.
20. Imadome K, Shirakata M, Shimizu N, et al. CD40 ligand is a critical
effector of Epstein–Barr virus in host cell survival and transforma-
tion. Proc Natl Acad Sci USA 2003;100:7836–7840.
1
Beverly J. Lange, MD, and Kim E. Nichols, MD1*
evolution to a clonal histiocytic neoplasm. Despite further chemo-
therapy, the patient died of disease progression. This case highlights
the clinical and pathological heterogeneity of JXG and the difficulty
of treating multi-focal CNS disease. Pediatr Blood Cancer 2008;50:
927–930. ß 2007 Wiley-Liss, Inc.
CASE REPORT
An 11-year-old Caucasian male presented to an outside hospital
with headache, nausea, emesis and blurred vision. Examination
revealed papilledema and bradycardia. Laboratory values and an
unenhanced MRI of the brain were normal. The patient underwent
serial lumbar punctures, which revealed elevated opening pressures,
ranging from 20 to 55 cm of water. Cerebrospinal fluid (CSF)
specimens demonstrated elevated protein levels and a leukocytosis
consisting of monocytes and macrophages. The patient was
——————
This article contains Supplementary Material available at http://www.
interscience.wiley.com/jpages/1545-5009/suppmat.
1
Division of Pediatric Oncology, Children’s Hospital of Philadelphia,
Philadelphia, Pennsylvania; 2Department of Pathology, Children’s
Hospital of Philadelphia, Philadelphia, Pennsylvania
*Correspondence to: Kim E. Nichols, Children’s Hospital of
Philadelphia, Division of Pediatric Oncology, Wood Building, 4th
Floor Mail Box, 34th Street and Civic Center Boulevard, Philadelphia,
PA 19104. E-mail: nicholsk@email.chop.edu
Received 26 February 2007; Accepted 3 April 2007