Key words : Acute graft-versus-host disease, Allogeneic hematopoietic cell transplant, Haploidentical transplanta-tion, Natural killer cell, Pregnancy

Introduction

Chronic active Epstein-Barr virus infection (CAEBV) is an intractable EBV-infected T cell or natural killer (NK) cell lymphoproliferative disorder characterized by recurrent infectious mononucleosis-like symptoms, including persistent fever, lymphadenopathy, and hepatosplenomegaly. Allogeneic hematopoietic cell transplant (allo-HCT) is considered the only curative treatment for CAEBV; however, a high incidence of graft failure (GF) (13%-50%) poses a formidable barrier.^1-3 Limited data are available regarding salvage allo-HCT for GF in CAEBV patients.^1-3

During the past few decades, human leukocyte antigen (HLA)-haploidentical donors have emerged as a promising alternative stem cell source for patients without HLA-matched donors. Several retrospective studies have demonstrated encouraging results of salvage HLA-haploidentical allo-HCT with post-transplant cyclophosphamide (PTCy-haplo) for GF in malignant and nonmalignant hematological disorders.^4-6 However, to our knowledge, there have been no reports regarding PTCy-haplo as a salvage treatment for GF in CAEBV patients. Herein, we present the case of an adult patient with CAEBV who received salvage PTCy-haplo for GF after allo-HCT from an unrelated HLA-matched donor.

Case Report

Written informed consent was obtained from the patient for publication of this case report and any accompanying images.

A 32-year-old woman who was pregnant with twins (gestational age, 22 weeks) was admitted to our hospital due to pancytopenia and hepatosp­lenomegaly. She had a history of more than 10 years of intermittent symptoms of fever and cold, including the births of her first 2 children when she was 28 and 30 years of age, respectively. However, the cause of these symptoms remained unclear due to absence of any blood EBV-DNA polymerase chain reaction assay. Neither she nor her family had a history of hypersensitivity to mosquito bites.

On admission, the patient was awake and alert, with a body temperature of 37.2 °C, a heart rate of 96 beats/min, blood pressure of 107/71 mm Hg, and oxygen saturation of 97% at ambient air. A blood test revealed pancytopenia (white blood cell count, 2300 cells/μL; hemoglobin level, 8.4 g/dL; and platelet count, 9.5 × 104 cells/μL) and elevated aspartate aminotransferase (99 U/L; reference, 13-30 U/L), C-reactive protein (2.85 mg/dL; reference, 0-0.4 mg/dL), and soluble interleukin-2 receptor (885 U/mL; reference, 204-587 U/mL).

The EBV antibody tests demonstrated a past infection pattern: EBV-viral capsid antigen antibody (VCA)-immunoglobulin M, <10-fold; VCA-immunoglobulin G, ×1280; EBV nuclear antigen, ×40; and EBV-early antigen immunoglobulin G, ×40. The EBV-DNA level in her peripheral blood mononuclear cells (PBMCs) was 5.6 × 10⁴ copies/μg DNA. Bone marrow aspiration displayed no abnormality, with a G-banding analysis that showed a normal karyotype. Computed tomography showed hepatosplenomegaly but no lymphadenopathy. Flow cytometry in situ hybridization with EBV-encoded small RNA (EBER) revealed that EBV-infected lymphocyte cells in PBMCs were negative for CD3, CD4, CD5, CD8, CD20, and T-cell receptor ?β chains (Figure 1, a-e), with slightly higher expression levels of CD16 and CD56 in comparison with EBER-negative lymphocytes (Figure 1f). Clonal T-cell receptor β-chain gene rearrangement and immunoglobulin heavy chain gene rearrangement were negative, whereas Southern blots of EBV-terminal repeats in a whole blood revealed a monoclonal band (Figure 1g). Therefore, the patient was diagnosed with NK-cell type CAEBV.

Since no growth disturbance was observed in twin fetuses via transabdominal ultrasonography and electronic fetal heart rate monitoring and maternal CAEBV showed a stable clinical course, the obstetricians judged that the patient could continue pregnancy. However, she underwent caesarean section at 31 weeks of gestation due to the appearance of persistent fever and gradual liver transaminase elevation.

At the time of birth, no abnormal findings were detected in the neonatal twin siblings, and a cord blood test showed no EBV viremia. After delivery, treatment for CAEBV was initiated with immunotherapy, including prednisolone and cyclophosphamide, followed by chemotherapy with CHOP (vincristine, cyclophosphamide, doxorubicin, prednisolone) and ESCAP (etoposide, cytosine arabinoside, L-asparaginase, methylprednisolone, prednisolone).⁷ Although the EBV-DNA level in PBMCs did not decrease during the chemoimmunotherapy (Figure 2), the symptoms and laboratory findings (including body temperature, liver function, and C-reactive protein levels) were stable.

Subsequently, the patient received reduced intensity conditioning that consisted of etoposide, cytosine arabinoside, fludarabine, anti-thymocyte globulin (ATG), and methylprednisolone,⁷ followed by bone marrow allo-HCT from an unrelated HLA-matched female donor with total nucleated cells of 2.04 × 108 cells/kg. On day 27 after allo-HCT, she experienced grade III acute graft-versus-host disease (aGVHD) with skin (stage 2) and gastrointestinal (stage 3) involvement, which responded to systemic corticosteroid treatment. The absolute neutrophil count increased from the week 4 but decreased afterward. An assessment of T-cell chimerism in the peripheral blood on day 31 revealed 96% recipient cells, and a bone marrow biopsy showed severe hypocellularity without hemophagocytosis, which suggested autologous hematopoietic recovery with primary GF.

Three months after the first allo-HCT, she underwent salvage PTCy-haplo with reduced intensity conditioning using peripheral blood stem cells from her 59-year-old father, which contained CD34+ cells of 4.09 × 10⁶ cells/kg. She received no treatment for EBV prior to the salvage PTCy-haplo. The conditioning regimen included fludarabine, cyclophosphamide, busulfan, and total body irradiation. The GVHD prophylaxis consisted of cyclophosphamide (50 mg/kg, day 3 and day 4), tacrolimus, and mycophenolate mofetil.⁸ The patient had no detectable donor-specific anti-HLA antibodies against the HLA-haploidentical donor. Neutrophils and platelets successfully engrafted on day 19 and day 113 with full donor chimerism in peripheral T cells on day 26 and day 110 after salvage PTCy-haplo. The patient experienced grade I aGVHD with skin involvement (stage 2) and mild chronic GVHD (cGVHD) of the skin (quiescent form) but did not show any severe transplant-related complications. The EBV-DNA in PBMCs decreased to an undetectable or very low level, and flow cytometry in situ hybridization revealed no evident EBV-infected NK cells or T cells at 10 months after salvage PTCy-haplo (Figure 2).

Discussion

We encountered a successful case of PTCy-haplo as rescue therapy for GF in a CAEBV patient. Insufficient preconditioning (only 2 cycles of chemotherapy), a high EBV-DNA load (3.2 × 10⁴ copies/μg DNA), and splenomegaly may have caused GF after the first allo-HCT.^1-3,9,10 However, subsequent PTCy-haplo resulted in favorable hematopoietic engraftment without disease recurrence or severe transplant-related complications. The development of aGVHD following the first allo-HCT without engraftment may have been caused by residual donor-derived T cells for the following reasons. A prior study reported that only a T-cell dose of 1.0 × 10⁵ cells/kg could be sufficient to induce aGVHD,¹¹ and our patient had a volume of donor CD3+ cells greater than 1.0 × 10⁵ cells/kg at the onset of aGVHD. Furthermore, we observed autologous hematopoietic recovery after the first allo-HCT, although the persistent high EBV load could have led to subsequent suppression of the recipient’s fragile hematopoietic recovery.¹²

Several retrospective studies have investigated the feasibility of salvage PTCy-haplo in patients with hematological disorders (other than CAEBV). Prata and colleagues reported that 79% and 59% of patients achieved neutrophil and platelet engraftment at day 30 and day 50 after salvage PTCy-haplo with a median duration to engraftment of 18 and 29 days, respectively.⁴ The cumulative incidence of grade II-IV aGVHD (at day 100) and cGVHD (at 1 year) was 14% and 31%, respectively.⁴ Harada and colleagues reported comparable results, with neutrophil and platelet engraftment observed in 81.8% (at day 30) and 30.3% (at day 60), respectively, and grade II-IV and grade III-IV aGVHD (at day 100) developing in 15.2% and 6.1% of the evaluable patients, respectively.⁶ Albert and colleagues investigated 12 pediatric patients with nonmalignant disorders undergoing salvage PTCy-haplo and demonstrated that all patients obtained neutrophil and platelet engraftment with a median interval of 18 and 39 days from PTCy-haplo, respectively.⁵ In this study, only 1 patient (8%) experienced grade I aGVHD and mild cGVHD with skin involvement.⁵ Although our case showed delayed platelet engraftment (113 days), fast neutrophil engraftment and effective prevention of severe GVHD by PTCy were in line with the previous results.

When a donor is needed for salvage allo-HCT in CAEBV, umbilical cord blood (UCB) may be a viable alternative source, given its prompt availability. However, the data regarding cord blood transplant (CBT) in adult-onset CAEBV are limited, and a major concern is the higher incidence of GF.^1-3 Surprisingly, a recent study on adult-onset EBV?associated T?cell and NK?cell lymphoproliferative disorders revealed that 4 of 8 CAEBV patients (50%) experienced GF after CBT, suggesting a potential risk of recurrent GF after salvage CBT.³ A registry-based study from Japan compared the outcomes of salvage allo-HCT using UCB and an HLA-haploidentical donor in various hematological disorders and demonstrated that the haploidentical donor group showed earlier neutrophil engraftment and lower nonrelapse mortality than the UCB group.¹³ In that study, only 10 of 129 patients (7.8%) received PTCy and the other patients predominantly received ATG as GVHD prophylaxis in the haploidentical donor group; however, the results nevertheless suggested the superiority of PTCy-haplo over CBT in the setting of salvage allo-HCT, as PTCy showed a better rate of survival than ATG following haploidentical allo-HCT.¹⁴

Furthermore, HLA-mismatched donor and ATG use have been reported as substantial risk factors for EBV viremia and EBV-associated posttransplant lymphoproliferative disorder.¹⁵ Thus, in terms of reducing the risk of EBV reactivation, PTCy-based GVHD prophylaxis may have a further advantage over ATG in CAEBV patients undergoing salvage allo-HCT from an HLA-haploidentical donor.

Conclusions

The present case suggests that PTCy-haplo could be an alternative option for patients with CAEBV who develop GF after allo-HCT. Recipients with CAEBV following allo-HCT are at higher risk of GF, although PTCy-haplo has a potential advantage in the time-sensitive situation from the viewpoint of easy donor availability and the efficacy and the safety profile of PTCy.

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