We report a case of Epstein–Barr virus infection with the subsequent development of haemophagocytic lymphohistiocytosis in a teenage Crohn's disease patient treated with azathioprine. We found that the early introduction of the anti-B cell monoclonal antibody rituximab precipitated a rapid fall in circulating B-cells and EBV viral load, resulting in a prompt and sustained recovery from what is a potentially fatal complication of azathioprine therapy in Crohn's disease patients.
Haemophagocytic lymphohistiocytosis (HLH) is an uncommon and potentially fatal clinical syndrome of immune dysfunction and dysregulation. It is associated with reduced cytotoxic and apoptotic activity in natural killer and cytotoxic T-cells in tandem with sustained T-lymphocyte and macrophage activation and expansion.1 There is a resultant pro-inflammatory hypercytokinaemia (interferon gamma,2 tumour necrosis factor alpha3 and interleukin 64) which underlies the pathogenesis of the disease. Lymphocytes and macrophages infiltrate bone marrow, lymph nodes, splenic and CNS tissue where the latter, phagocytose erythrocytes, leukocytes, platelets and their precursor cells.5 Although pathognomonic this aberrant behaviour is not always demonstrable at diagnosis.6
HLH exists in two subsets with common clinical and pathobiological characteristics, namely familial haemophagocytic lymphohistiocytosis (FHLH) and secondary haemophagocytic lymphohistiocytosis (SHLH). FHLH is an autosomal recessive immune disorder which presents in infancy, and may also occur in association with immune disorders such as X-linked lymphoproliferative disease,7 Griscelli syndrome8 and Chédiak–Higashi syndrome.9 FHLH is invariably fatal if untreated;10 however bone marrow transplantation may be curative.11 SHLH most commonly occurs in immunocompromised patients, e.g. those with T-cell lymphomas,12 and those in receipt of immunosuppressant drugs such as azathioprine.13 SHLH may occur in any age group and may present as an acute fulminant illness or take a more insidious course.
HLH diagnosis is based on fulfilling 5 of the following 8 criteria: fever, splenomegaly, bicytopoenia, hypertriglyceridaemia (≥ 265 mg/dL) and/or hypofibrinogenaemia (≤ 1.5 g/L), haemophagocytosis, low/absent NK-cell-activity, hyperferritinaemia (≥ 500 ng/mL) and high-soluble interleukin-2-receptor levels. A positive family history of HLH or molecular diagnosis consistent with HLH may supplant the above criteria.6
HLH-2004 is the established treatment guideline for HLH, and it advocates treatment with etoposide, dexamethasone and cyclosporine A, with the addition of intrathecal methotrexate and corticosteroids in patients with CNS symptoms.6 HLH in both populations may be triggered by acute infection, which is usually viral (frequently EBV), although cases involving bacterial, fungal and parasitic infections have also been reported.14
EBV is a well documented cause of lymphoproliferative disorders including SHLH in both paediatric and adult inflammatory bowel disease (IBD) patients treated with azathioprine and/or infliximab.15–17 A recent study has shown that paediatric IBD patients are at a 100-fold greater risk for the development of HLH compared to the general population.18 We describe a case where rituximab, a monoclonal antibody directed against B cell CD20 antigen, was used to successfully treat EBV associated HLH in a teenage Crohn's disease patient being treated with azathioprine.
Our patient was diagnosed with Crohn's disease at age 10. Azathioprine had been commenced shortly after her diagnosis and had gradually been increased from 1.5 mg/kg/day to 2.5 mg/kg/day to maintain clinical remission. She presented aged 14 with pyrexia (40.5 °C), headache, fever, myalgia, abdominal pain and vomiting. She had attended her GP the previous day, who had stopped her azathioprine due to low white cells and platelets. Examination demonstrated a raised erythematous rash on her face and arms, hepato-splenomegaly, bilateral cervical lymphadenopathy pharyngitis, glossitis and tonsillitis with exudate.
Her bloods on admission were as follows: Hb 10.4 g/L, WCC 1.6 × 109/L (neutrophils 1.3 × 109/L, lymphocytes 0.2 × 109/L), Plt 41 × 109/L, Bili 72 μmol/L, ferritin 5325 ng/mL, fibrinogen 0.85 g/L, Alk Phos 217 iμ/L, ALT 149 iμ/L, AST 332 iμ/L, albumin 24 g/L, CRP 68 mg/L and triglycerides 233 mmol/L. Monospot test was positive. An initial diagnosis of progressive EBV infection was made. Lymphocyte subsets showed an absence of natural killer cells and a high proportion of activated T-lymphocytes. EBV PCR from peripheral blood had a viral load of 210,385 unit load/ml. Her 6 thioguanine levels were within therapeutic levels at 264 pmol/8 × 10eRBC. An abdominal ultrasound revealed splenomegaly with multiple focal hypoechoic nodules.
A bone marrow aspirate performed shortly after admission revealed increased numbers of macrophages and evidence of haemophagocytosis consistent with a diagnosis of haemophagocytic lymphohistiocytosis. She was commenced on rituximab (10 mg/kg) on the day of diagnosis and received two further doses of rituximab at 6 days, and then a further 10 days after the first dose. The introduction of rituximab precipitated a rapid fall in the patients EBV viral load, her blood results slowly began to normalise, and she made a full clinical recovery. Within 72 hours of administration her temperature had settled and she remained apyrexial thereafter. EBV viral load was just detectable after the second dose. By the time the patient received her final dose of rituximab lymphocyte cytometry had confirmed the absence of B-cells in her peripheral circulation (anti-CD19 antibodies were used in the flow cytometry analysis). A repeat abdominal ultrasound confirmed resolution of the lesion in her spleen and her blood count and liver function tests returned to normal.
The patient was initially found to have reduced perforin expression, and genetic studies revealed that she was heterozygous for a nucleotide substitution in PRF1 gene. Repeat testing demonstrated normal perforin expression. A subsequent granule release assay was normal. The pathogenicity of this mutation has not been determined and in the absence of a second mutation a diagnosis of SHLH rather than FHLH was confirmed.
Three months after her admission the patient contracted H1N1. She received a 5 day course of oseltamivir and made a full and speedy recovery. Her Crohn's disease was subsequently treated with a low dose of maintenance of prednisolone until CD relapse 18 months after her episode of HLH. She was subsequently successfully maintained on methotrexate with no further episodes of relapse or EBV/HLH while on methotrexate. Her last B-cell count was within normal parameters 486 (22%) but with no evidence of EBV recurrence as assessed by serial EBV PCR measurements.
Azathioprine is effective in prolonging remission of paediatric Crohn's disease and is consequently often introduced early in a patient's disease course.19,20 However as well as these clear therapeutic benefits, there is a clearly documented link between azathioprine therapy in IBD patients and the development of EBV-associated lymphoproliferative disorders such as HLH and lymphomas.13,15,21–23 In affected patients azathioprine induces an immune deficiency favouring expansion of EBV infected B-cells, which in turn facilitate the secondary expansion of T cells and macrophages which underlies the development of HLH and lymphomas. Azathioprine's pivotal role in the development of these disorders in supported by cases in which lymphoma resolution only occurred after the drug was withdrawn.24
EBV associated HLH recently has had a reported 5-year survival rate in excess of 80%.25 This is a considerable improvement on 5-year historical survival rates from the early 1990's which were reported to be 55%.11 Such an improvement is best explained by the widespread adoption of the HLH-1994 treatment protocol and its successor HLH-2004, along with earlier recognition and diagnosis of the condition.6,11 Despite the success of HLH-1994/2004 in improving survival rates, both treatment protocols lack specific targeted therapy against the principal molecular effectors of the disorder, namely EBV infected B-lymphocytes.
We report a case in which a female paediatric Crohn's disease patient on azathioprine developed EBV associated HLH and was successfully treated with rituximab. We believe the early recognition of the diagnosis and the institution of rituximab were important factors in the successful outcome in this case. By specifically targeting and eliminating EBV infected B-cells, rituximab suppresses the immune dysfunction driving the disease process. Rituximab is capable of eliminating in excess of 80% of circulating B-cells within 24–72 hours of administration, and it may take up to 6 months or longer to return to previous levels.26 Rituximab has previously been used to successfully treat azathioprine induced EBV associated SHLH in a 53 year old woman with Crohn's disease27 and is also the treatment of choice in other EBV associated lymphoproliferative disorders such as B-cell lymphomas, post transplantation lymphoproliferative disorders and HLH in X-linked lymphoproliferative disease.28–30
In the 72 hours following administration of rituximab our patient made marked clinical improvements, with resolution of pyrexia, reduction of lymphadenopathy, improvement of transaminitis, normalisation of synthetic liver function and reduction in EBV viral load. Rituximab terminated the progression of EBV associated HLH and consequently our patient did not require treatment as per the HLH 2004 protocol thus avoiding further immunosuppression. Our experience is in contrast with Ross et al. who report the unsuccessful use of rituximab in EBV associated lymphoproliferative disease in a paediatric Crohn's disease patient who had been receiving azathioprine.23
The patient was found to have reduced perforin expression and genetic studies revealed she was heterozygous for a common PRF1 polymorphism Ala91Val. Repeat perforin expression studies were normal. The Ala91Val allele is however found in 4–17% of the general population.31,32 The exact role of Ala91Val in HLH is controversial; however several cases of late-presentation FHLH have been linked to Ala91Val homozygosity. Wild-type/Ala91Val or heterozygous individual's such as this patient have been shown to have only mildly reduced perforin activity compared with wild-type perforin homozygotes.33 Further genetic tests were conducted, a granule release assay returned as normal which excludes defects in munc 13-4, syntacin 11 munc 18-2 as well as Chédiak–Higashi, Griscelli and HP2 syndromes and makes the possibility of FHLH therefore highly unlikely.
In light of increasing recognition of the risk posed by primary EBV infection to paediatric patients on azathioprine, further studies are required to establish the benefits of establishing patient's EBV status before commencing azathioprine therapy, as at present this practise is not recommended by current guidelines.34 In the interim patients and their families should be educated about the symptoms of EBV infection and the danger it poses to paediatric patients on azathioprine, as early recognition and treatment with rituximab can potentially terminate disease progression and avoid the need for further treatment.
Currently our patient is under surveillance with regular EBV DNA viral load quantitative PCR and lymphocyte cytometry. Normalisation of B-cells post rituximab brings with it the theoretical risk of relapse of HLH, and persistent detectable EBV DNA is associated with increased risk of developing lymphomas; however, this has not been apparent in this case.35
Progressive EBV infection with associated HLH is a rare and potentially fatal complication of azathioprine therapy in paediatric Crohn's disease patients. From our experiences we advocate a high degree of clinical suspicion for disease development in susceptible patients, as early recognition, discontinuation of all immunosuppressive medications and treatment an anti-CD20 monoclonal antibody such as rituximab may terminate the disease progression thus heralding clinical recovery and avoiding the need for further treatment.
Conflict of interest
No authors have a relevant conflict of interest to declare.
RKR has received support from a Medical Research Council (MRC) patient research cohorts initiative grant (G0800675) for PICTS and is supported by an NHS Research Scotland career fellowship award. The work of the IBD team at Yorkhill is supported by the Catherine McEwan Foundation and the Yorkhill IBD fund.