Abstract

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.

Introduction

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.1517 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.

Case report

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.

Discussion

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.13,15,2123 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.2830

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

Summary

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.

Acknowledgements

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.

References

1
Egeler
R.M.
Shapiro
R.
Loechelt
B.
Filipovich
A.
Characteristic immune abnormalities in hemophagocytic lymphohistiocytosis
J Pediatr Hematol Oncol
 
18
4
1996
340
345
2
Ohga
S.
Matsuzaki
A.
Nishizaki
M.
Nagashima
T.
Kai
T.
Suda
M.
et al
Inflammatory cytokines in virus-associated hemophagocytic syndrome. Interferon-gamma as a sensitive indicator of disease activity
Am J Pediatr Hematol
 
15
3
1993
291
298
3
Watanabe
M.
Shimamoto
Y.
Yamaguchi
M.
Inada
S.
Miyazaki
S.
Sato
H.
Viral-associated haemophagocytosis and elevated serum TNF-alpha with parvovirus-B19-related pancytopenia in patients with hereditary spherocytosis
Clin Lab Haematol
 
16
2
1994
179
182
4
Henter
J.I.
Elinder
G.
Sder
O.
Hansson
M.
Andersson
B.
Andersson
U.
Hypercytokinemia in familial hemophagocytic lymphohistiocytosis
Blood
 
78
11
1991
2918
2922
5
Janka
G.E.
Familial hemophagocytic lymphohistiocytosis
Eur J Pediatr
 
140
3
1983
221
230
6
Henter
J.
Horne
A.
Aric
M.
Egeler
R.M.
Filipovich
A.
Imashuku
S.
et al
HLH-2004: diagnostic and therapeutic guidelines for hemophagocytic lymphohistiocytosis
Pediatr Blood Cancer
 
48
2
2007
124
131
7
Arico
M.
Imashuku
S.
Clementi
R.
Hibi
S.
Teramura
T.
Danesino
C.
et al
Hemophagocytic lymphohistiocytosis due to germline mutations in SH2D1A, the X-linked lymphoproliferative disease gene
Blood
 
97
4
2001
1131
1133
8
Meeths
Clinical presentation of Griscelli syndrome type 2 and spectrum of RAB27A mutations
Pediatr Blood Cancer
 
54
4
2010
563
572
9
Jayaranee
S.
Menaka
N.
Chediak–Higashi syndrome: a case report
Malays J Pathol
 
26
1
2004
53
57
10
Gencik
A.
Signer
E.
Mller
H.
Genetic analysis of familial erythrophagocytic lymphohistiocytosis
Eur J Pediatr
 
142
4
1984
248
252
11
Henter
J.
Samuelsson-Horne
A.
Aric
M.
Egeler
R.M.
Elinder
G.
Filipovich
A.
et al
Treatment of hemophagocytic lymphohistiocytosis with HLH-94 immunochemotherapy and bone marrow transplantation
Blood
 
100
7
2002
2367
2373
12
Chang
C.S.
Wang
C.H.
Su
I.J.
Chen
Y.C.
Shen
M.C.
Hematophagic histiocytosis: a clinicopathologic analysis of 23 cases with special reference to the association with peripheral T-cell lymphoma
J Formos Med Assoc
 
93
5
1994
421
428
13
N'guyen
Y.
Andreoletti
L.
Patey
M.
Lecoq-Lafon
C.
Cornillet
P.
Lon
A.
et al
Fatal Epstein–Barr virus primo infection in a 25-year-old man treated with azathioprine for Crohn's disease
J Clin Microbiol
 
47
4
2009
1252
1254
14
Rouphael
N.
Talati
N.
Vaughan
C.
Cunningham
K.
Moreira
R.
Gould
C.
Infections associated with haemophagocytic syndrome
Lancet Infect Dis
 
7
12
2007
814
822
15
Serrano
A.G.
Martn
F.P.
Guerrero Igea
F.J.
Muoz
J.G.
Gil
S.P.
Fatal infectious mononucleosis during azathioprine treatment in Crohn's disease
Gastroenterol Hepatol
 
23
1
2000
7
8
16
Francolla
K.
Altman
A.
Sylvester
F.
Hemophagocytic syndrome in an adolescent with Crohn disease receiving azathioprine and infliximab
J Pediatr Gastroenterol Nutr
 
47
2
2008
193
195
17
Deneau
M.
Wallentine
J.
Guthery
S.
O'Gorman
M.
Bohnsack
J.
Fluchel
M.
et al
Natural killer cell lymphoma in a pediatric patient with inflammatory bowel disease
Pediatrics
 
126
4
October 1 2010
e977
e981
18
Biank
V.F.
Sheth
M.K.
Talano
J.
Margolis
D.
Simpson
P.
Kugathasan
S.
et al
Association of Crohn's disease, thiopurines, and primary Epstein–Barr virus infection with hemophagocytic lymphohistiocytosis
J Pediatr
 
159
5
2011
808
812
[11]
19
Jaspers
G.
Verkade
H.
Escher
J.
de Ridder
L.
Taminiau
J.A.J.M.
Rings
E.H.H.M.
Azathioprine maintains first remission in newly diagnosed pediatric Crohn's disease
Inflamm Bowel Dis
 
12
9
2006
831
836
20
Markowitz
J.
Grancher
K.
Kohn
N.
Lesser
M.
Daum
F.
A multicenter trial of 6-mercaptopurine and prednisone in children with newly diagnosed Crohn's disease
Gastroenterology
 
119
4
2000
895
902
[10]
21
Posthuma
E.F.
Westendorp
R.G.
van der Sluys Veer
A.
Kluin-Nelemans
J.C.
Kluin
P.M.
Lamers
C.B.
Fatal infectious mononucleosis: a severe complication in the treatment of Crohn's disease with azathioprine
Gut
 
36
2
1995
311
313
22
Beaugerie
L.
Brousse
N.
Bouvier
A.M.
Colombel
J.F.
Lémann
M.
Cosnes
J.
et al
Lymphoproliferative disorders in patients receiving thiopurines for inflammatory bowel disease: a prospective observational cohort study
Lancet
 
374
9701
2009
1617
1625
[11/7–13]
23
Ross
Epstein–Barr virus-associated lymphoproliferative disorder in Crohn disease treated with azathioprine
J Pediatr Gastroenterol Nutr
 
51
2
2010
229
231
24
Larvol
L.
Soule
J.C.
Le Tourneau
A.
Reversible lymphoma in the setting of azathioprine therapy for Crohn's disease
N Engl J Med
 
331
13
1994
883
884
25
Ishii
E.
Ohga
S.
Imashuku
S.
Yasukawa
M.
Tsuda
H.
Miura
I.
et al
Nationwide survey of hemophagocytic lymphohistiocytosis in Japan
Int J Hematol
 
86
1
2007
58
65
26
Maloney
D.G.
Liles
T.M.
Czerwinski
D.K.
Waldichuk
C.
Rosenberg
J.
Grillo-Lopez
A.
et al
Phase I clinical trial using escalating single-dose infusion of chimeric anti-CD20 monoclonal antibody (IDEC-C2B8) in patients with recurrent B-cell lymphoma
Blood
 
84
8
1994
2457
2466
27
Serrate
C.
Silva-Moreno
M.
Dartigues
P.
Poujol-Robert
A.
Sokol
H.
Gorin
N.C.
et al
Epstein–Barr virus-associated lymphoproliferation awareness in hemophagocytic syndrome complicating thiopurine treatment for Crohn's disease
Inflamm Bowel Dis
 
15
10
2009
1449
1451
28
Dayharsh
G.A.
Loftus
J.
Edward
V.
Sandborn
W.J.
Tremaine
W.J.
Zinsmeister
A.R.
et al
Epstein–Barr virus-positive lymphoma in patients with inflammatory bowel disease treated with azathioprine or 6-mercaptopurine
Gastroenterology
 
122
1
2002
72
77
[1]
29
Milone
M.
Tsai
D.
Hodinka
R.
Silverman
L.
Malbran
A.
Wasik
M.
et al
Treatment of primary Epstein–Barr virus infection in patients with X-linked lymphoproliferative disease using B-cell-directed therapy
Blood
 
105
3
2005
994
996
30
Milpied
N.
Vasseur
B.
Parquet
N.
Garnier
J.L.
Antoine
C.
Quartier
P.
et al
Humanized anti-CD20 monoclonal antibody (rituximab) in post transplant B-lymphoproliferative disorder: a retrospective analysis on 32 patients
Ann Oncol
 
11
Suppl. 1
2000
113
116
[1]
31
zur Stadt
U.
Beutel
K.
Weber
B.
Kabisch
H.
Schneppenheim
R.
Janka
G.
et al
A91V is a polymorphism in the perforin gene not causative of an FHLH phenotype
Blood
 
104
6
September 15 2004
1909
1910
32
Lee
S.M.
Villanueva
J.
Sumegi
J.
Zhang
K.
Kogawa
K.
Davis
J.
et al
Characterisation of diverse PRF1 mutations leading to decreased natural killer cell activity in North American families with haemophagocytic lymphohistiocytosis
J Med Genet
 
41
2
2004
137
144
33
Voskoboinik
I.
Trapani
J.
Addressing the mysteries of perforin function
Immunol Cell Biol
 
84
1
2006
66
71
34
Rahier
J.F.
Ben-Horin
S.
Chowers
Y.
Conlon
C.
De Munter
P.
D'Haens
G.
et al
European evidence-based consensus on the prevention, diagnosis and management of opportunistic infection in inflammatory bowel disease
J Crohns Colitis
 
3
2009
47
91
35
Savoie
A.
Perpte
C.
Carpentier
L.
Joncas
J.
Alfieri
C.
Direct correlation between the load of Epstein–Barr virus-infected lymphocytes in the peripheral blood of pediatric transplant patients and risk of lymphoproliferative disease
Blood
 
83
9
1994
2715
2722