Children were eligible to participate if they met the diagnostic criteria for HLH with a positive EBV infection and had not received treatment for this disease. The diagnostic criteria were: fever persisting beyond seven days with peak temperature ≥38.5℃, splenomegaly, cytopenia affecting at least two lineages in the peripheral blood not caused by a hypocellular or dysplastic bone marrow, and hypertriglyceridemia (fasting triglycerides >3 SD or 177 mg/dL) or hypofibrinogenemia (fibrinogen <3 SD or 150 mg/dL) that raised strong suspicion of hemophagocytic syndrome (2, 3). Bone marrow aspiration was performed in all patients and a liver biopsy was performed in one patient (Patient 3) for cytomorphological analysis.

EBV infection was demonstrated by serology, polymerase chain reaction (PCR), and in situ hybridization.

Serological tests for EBV infection were based on viral capsid antigen (VCA)-IgG/IgM, early antigen (EA)-DR-IgG/IgM and Epstein-Barr virus nuclear antigen (EBNA) antibody titers.

Bone marrow cells of patients were examined for the presence of EBV DNA by PCR. DNA was extracted from bone marrow cells with 100 µL of 5% Chelax resin (Bio-Rad Laboratories, Hercules, CA, U.S.A.) and two cycles of heating (up to 100℃) and cooling. Five microliters of the DNA eluate was used for amplification of EBV DNA, with 250 nM concentration of sense and antisense primers (5'-TGATAACCATGGACGAGGAC-3' and 5'-GCAGCCAATGCAACTTGGAC-3', respectively) that correspond to the 139 base-pair fragment of the Epstein Barr Nuclear Antigen-1 (EBNA-1), and Accupower® PCR PreMix (Bioneer, Daejoen, Korea). PCR was performed using 5 min of denaturation at 95℃, 35 cycles of 1 min at 94℃, 1.5 min at 57℃, 2 min at 72℃, and finally 10 min at 72℃. The PCR products were analyzed by electrophoresis using 2% agarose gels. DNA from EBV-transformed human B-cell lines (IM-9) and human T-leukemic cell lines (Molt-4) were used as positive and negative controls, respectively.

In situ hybridization of EBV-encoded small RNA (EBER) was performed according to a modification of a previously described cDNA-mRNA in situ hybridization method, using the manual Microbe System (Fisher Scientific, Pittsburgh, PA, U.S.A.) (13). Briefly, 5 µm thick bone marrow sections were placed on the distal one-third of Probe-On positively charged slides. The specimens were deparaffinized with Hemode (xylene substitute) at a ratio of 3:1 for 5 min, placed in 100% alcohol for 45 sec, and rehydrated with 1 Automation Buffer (Biomeda, Foster City, CA, U.S.A.). The specimens were digested with pepsin (pH 2.0) for 6 min at 110℃ and immediately washed in distilled water for 30 sec. The biotinylated cDNA oligonucleotide probe (Research Genetics, Huntsville, AL, U.S.A.) to EBER-mRNA was reconstituted to 1 µg/µL solution in 10 mM Tris (pH 7.5), diluted to a concentration of 200 ng/mL in the probe cocktail containing 12.5% (v/v) formamide, 0.5% (w/v) chondroitin sulfate, 675 mM sodium chloride, 138 mM sodium citratem, 12 mM sodium phosphate monobasic, 63 mM sodium dibasic, 0.1% (v/v) Tween 20 (JT Baker, Phillipsburg, NJ, U.S.A.) and 1:100,000 diluted random 9-mers (Research Genetics). This probe mixture was applied for 10 min at 92℃ and 30 min at 45℃ to hybridize with target nucleic acids in the specimens. After hybridization, the specimens were washed in 5×SSC for 3 min and 2×SSC for 3 min at room temperature. Detection of hybridized biotin probes was performed with avidin-alkaline phosphatase (Dako, Carpenteria, CA, U.S.A.), diluted 1:200 in Tris buffer (pH 8.0), and signal localization was obtained with the naphthol AS-MX phosphate/Fast Red TR chromogen solution. The slides were washed with distilled water, counterstained with hematoxylin for 5 sec, and coverslips applied with Universal Mount (Research Genetics).

The core elements of induction chemotherapy were steroids and etoposide (VP-16). The starting doses were 10 mg/m² per day oral dexamethasone and 150 mg/m² per dose intravenous etoposide twice a week. Remission induction therapy, either closely or entirely, followed the HLH-94 protocol in all patients (1-3).

The aim of continuation treatment is to sustain resolution of the disease (2, 3). After eight weeks of induction chemotherapy, a complete response (CR) was defined as an unequivocal resolution of clinical signs and symptoms and the normalization of laboratory findings, including data obtained from imaging studies and resolution of hemophagocytosis on bone marrow study. Patients with persistent fever and other symptoms of HLH, abnormally high serum ferritin concentration or remaining hemophagocytosis on bone marrow study were considered to have a partial response (PR) and were placed on intensified or maintenance regimens until CR was induced. The continuation therapy from week 9 comprised 10 mg/m² dexamethasone pulses for three days every second week and 150 mg/m² etoposide infusions on alternating weeks in combination with daily oral cyclosporin (CSA) (1-3).

The mean age at diagnosis was 5.2 yr (range 2.3-13.0); two patients were boys and two were girls. The patients had prolonged fever, splenomegaly, hepatomegaly, lymphadenopathy and/or neurological symptoms with cytopenia. None of the patients had a family history of HLH (Table 1).

At the time of diagnosis, the serum ferritin concentrations were 364 ng/mL-24,295 ng/mL (normal range 15-332 ng/mL) and the serum lactate dehydrogenase levels were 2,357 IU/L-5,540 IU/L (normal range 180-460 IU/L) (Table 2). Serological testing for EBV showed an elevated VCA-IgG (normal range 0.9-1.09 U/mL), with increased EA-IgG titers (normal range 0.9-1.09 U/mL) indicating reactivated EBV infections in all patients (Table 3). EBV DNA was detected by PCR in Patient 1 and 4 (Fig. 1) and EBER was detected by in situ hybridization in bone marrow specimens of the three patients (Fig. 2). Hemophagocytosis was shown in the bone marrow aspirates of all patients (Fig. 3) and liver biopsy specimen of Patient 3 (data not shown). There were no chromosomal anomalies of peripheral blood mononuclear cells or bone marrow cells (data not shown).

During the first eight weeks of therapy, the four patients exhibited PR and were placed on continuation treatment. Patients were provided therapy for a median of seven months (range 4-10 months) (Table 4).

On hospital day 2, Patient 1 suffered a pulmonary hemorrhage requiring ventilator care; immunochemotherapy by HLH-94 and continuous CSA infusion were started. Severe neutropenia (absolute neutrophil count 48/µL) without evidence of bacterial infection during remission induction led to temporary withdrawal of all cytoreductive therapy and continuous infusions of CSA for 11 days. On hospital day 27, the patient achieved hematopoietic recovery and was able to restart his scheduled chemotherapy. However, this patient was considered to have a PR after induction treatment, because serum ferritin concentration was 374 ng/mL, which was above the normal range. After continuation treatment, the patient achieved CR and his serum ferritin concentration normalized.

Patient 2 had a seizure and loss of consciousness at admission. She presented with neurological deterioration and a brain MRI showed increased signal in the head of the caudate nucleus, medial portion of the thalamus, right lentiform nucleus, cortex and subcortical white matter of the right temporal lobe, and periaqueductal gray matter. Tests for serum antinuclear antibody and anti-double-stranded DNA were negative. She received medication including antibiotics, antiviral agents, and antifungal agents. On hospital day 2, pancytopenia developed and her bone marrow aspirate was characterized by infiltration of hemophagocytosis. Immunochemotherapeutic agents including dexamethasone, etoposide, CSA, and intrathecal methotrexate were administered immediately and her neurological symptoms improved. However, all immunochemotherapy except dexamethasone was stopped because of severe bone marrow suppression. Six days after the cessation of immunochemotherapy, cranial nerve VI palsy occurred suddenly, but it was treated successfully with etoposide and high dose dexamethasone. There was no hemophagocytosis in the bone marrow after one month of chemotherapy. Abnormal brain imaging remained after induction therapy, and she was treated by continuation therapy with HLH-94 protocol. The brain MRI abnormalities resolved after continuation treatment for 10 months. Dexamethasone was discontinued because of progressive abdominal striae and she showed poor compliance to oral CSA. Although CR was attained, she dropped out of the follow-up 20 months after the onset of disease.

Patient 3 received prednisolone and cyclophosphamide and switched to induction treatment by the HLH-94 regimen from hospital day 2. His liver biopsy demonstrated some histiocytic infiltration in the liver sinusoid. Although the patient had shown persistent hepatomegaly over five months, hemophagocytosis subsided after induction treatment.

Patient 4 was admitted with prolonged fever, hepatosplenomegaly, leukopenia, and thrombocytopenia. Immunochemotherapy by HLH-94 from hospital day 5 appeared to successfully induce CR. However, at the end of induction treatment, she presented with recurrent fever, hepatosplenomegaly, and pancytopenia; hemophagocytic histiocytes were noted again in bone marrow, indicating reactivation. The induction treatment was restarted and clinical symptoms and hemophagocytosis of bone marrow disappeared after one month of treatment, after which she was maintained on continuation treatment with no evidence of reactivation thereafter.