Abstract

A 31-year-old lady of Chinese ethnicity was admitted with a six-day history of fever, night sweats and abdominal discomfort. She had no respiratory, urinary or gastrointestinal symptoms to suggest a source of infection. There was no weight loss or constitutional symptoms prior to her presentation. She had no history of travel or infective contacts and reported no joint pains, alopecia, rashes, headaches or photophobia. There was no significant past medical history, and she was not on any regular medications. She had no known family history of malignancy or autoimmune disease. Physical examination revealed mild hepatosplenomegaly: both liver and spleen palpable at 2 and 3 cm from the costal margin respectively, but no palpable lymphadenopathy, rashes or skin lesions. Examination of the cardiac and respiratory systems and oropharynx was unremarkable. Based on the history of fever and night sweats with hepatosplenomegaly, possible differential diagnoses included infective and neoplastic causes, with inflammatory and autoimmune disorders also being considerations. Given the relatively short duration of symptoms and age of the patient, infective causes were considered more likely. Viral infections which can present in this manner include Epstein–Barr Virus (EBV) cytomegalovirus (CMV) and human immunodeficiency virus (HIV).1 Other less likely infective causes include bacterial, parasitic and fungal infections. Leptospirosis, brucellosis and tuberculosis1 are potential bacterial causes although the short duration of symptoms makes the latter less likely. Given the absence of a travel history and the patient's geographic location, parasitic infections such as malaria and leishmaniasis are less likely.1 Fungal infections such as histoplasmosis are also a consideration however this would be unusual in the absence of a history of immune suppression.1 Although a lymphoid malignancy was a consideration in view of the hepatosplenomegaly, the short duration of symptoms would be unusual. Similarly, the absence of joint or skin symptoms made an autoimmune disorder less likely. Initial investigations revealed pancytopenia: hemoglobin 10.3 g/dL, total white cell count 1.49 × 109/L (absolute neutrophil count 1.20 × 109/L), platelet count 40 × 109/L. There was also significant derangement of her liver function: alkaline phosphatase 518 U/L, alanine transaminase (ALT) 239 U/L, aspartate transaminase (AST) 277 U/L. There was also a marked elevation of the lactate dehydrogenase (LDH) at 2008 U/L. A chest radiograph showed no significant abnormality and urinalysis showed no pyuria or haematuria. Two sets of blood cultures were negative for bacterial growth. In summary, this patient had a short history of pyrexia and night sweats, with hepatosplenomegaly, along with pancytopenia and deranged liver function. Possible differential diagnoses have been summarized in Table 1. Acute infection remained the most likely possibility with EBV, CMV, severe dengue and chikungunya being considerations. So, HIV, which may present in the acute setting with fever, can also cause hepatosplenomegaly, pancytopenia and deranged liver function. Another possible infective cause would be acute viral hepatitis, which can cause aplastic anemia, resulting in pancytopenia, however this usually affects young men, and occurs 2–3 months after an episode of acute hepatitis.2 No features in the patient's history support this possibility, which makes it less likely. Other differentials to consider include haematologic neoplasms, such as lymphoproliferative disorders and acute leukemias which can both present with pancytopenia, hepatosplenomegaly, and deranged liver function with a raised LDH. Given the persistent fever, pancytopenia and organomegaly, haemophagocytic lymphohistiocytosis (HLH) was also considered as a plausible differential.3 Notably, polymerase chain reaction (PCR) detected EBV DNA at 1.6 × 104 copies/mL in the peripheral blood, while PCR for Parvovirus, CMV, and Chikungunya were negative. The patient tested negative for EBV Early Antigen (Ea) IgM antibodies, although serological tests for EBV Viral Capsid Antigen (VCA) IgM and IgG antibodies were not done. Similarly, serology for dengue, HIV, hepatitis A, B and C viruses also returned negative. No malarial parasites were seen on a thick peripheral blood film. Importantly, the peripheral blood film showed no evidence of microangiopathy, circulating atypical lymphoid cells or blasts. Interestingly the patient's ferritin was markedly elevated at > 7500µg/L and her triglycerides were also increased at 3.75 mmol/L (ULN 2.2 mmol/L). Autoimmune screen including anti-nuclear antibody, anti-double stranded DNA, and anti-ENA were negative. The suspicion of HLH was raised in view of the patient's fever, hepatosplenomegaly, pancytopenia, hyperferritinaemia and hypertriglyceridaemia. Given the EBV viral load, EBV-associated HLH and related lymphoproliferative disorders were high on the list of differential diagnoses.1, 4 A decision was therefore made to proceed with bone marrow aspirate (BMA) and trephine biopsy. Bone marrow (BM) specimens were also drawn for flow cytometry and karyotyping studies. The BMA showed a reduction in normal hematopoiesis along with atypical mature lymphoid cells comprising 8% of nucleated cells (Figure 1A). There was also evidence of haemophagocytic activity although this was not overt (Figure 1B). The BM trephine showed atypical T cells which were positive for EBV-encoded RNA (EBER). These T cells were CD8+ and CD56- by immunohistochemistry. Double staining for EBER and CD8 confirmed that the EBER positive cells were CD8+ T cells (Figure 2). Flow cytometry revealed a population of CD8+ T cells showing an abnormally high side scatter along with an aberrant CD5 heterogeneous expression (Figure 3A, B). There was no other aberrant antigen expression or loss of normal T-cell antigens in this population which comprised 1.7% of nucleated cells. Karyotyping of the bone marrow showed no cytogenetic abnormality. In summary, the BM studies showed an atypical EBER positive, CD8+ T-cell population associated with haemophagocytosis. Differential diagnoses include acute primary EBV infection, or a T-cell lymphoproliferative disorder presenting with HLH, such as EBV associated HLH, systemic EBV+ T cell lymphoma of childhood (SE-TLC), chronic active EBV (CAEBV), or EBV positive peripheral T-cell lymphoma (PTCL-NOS EBV+).5 As the aberrant cells were of T lineage (CD3+, CD56-), natural killer T cell lymphoma (NKTL) and aggressive natural killer (NK) cell leukemia were less likely although not impossible as a subset of NKTL can arise from cytotoxic T cells.5 A computed tomography (CT) scan was done which showed no lymphadenopathy or extranodal masses while her fever and abdominal discomfort resolved spontaneously 2 days from admission. Her cytopaenias began to improve and ferritin also began to downtrend during this time. As her blood counts were returning close to the normal range and her fever had resolved, the patient was discharged with outpatient follow up after 5 days of inpatient monitoring. During outpatient follow up, she remained clinically well with normalization of blood counts 3 weeks from discharge. Bone marrow studies were repeated 3 months post-discharge and showed no morphologic or flow cytometric evidence of a clonal T cell population. Note, EBV-PCR in the blood was negative at this point, and the patient was clinically well, with normal blood counts 1 year post discharge. A T-cell receptor gene rearrangement analysis was not performed given the spontaneous resolution of her symptoms and absence of a clonal T-cell population on follow up BM assessment. Based on the clinical and laboratory findings, a diagnosis of reactive HLH as a result of primary EBV infection was made. Epstein–Barr virus is a double stranded DNA virus of the herpes virus family.6 The incidence of EBV infection in the community is high with over 90% of the world's population being EBV carriers.6 It is transmitted by infected saliva and establishes latency in peripheral blood B cells without causing disease in healthy individuals.6 In some cases, primary EBV infection can present as infectious mononucleosis, with transient symptoms including fever, hepatosplenomegaly, pharyngitis, and lymphadenopathy. The majority of EBV infections result in a self-limiting illness, however a subset of patients develop EBV-related malignancies.7, 8 The most common EBV related lymphoid malignancies are B-cell lymphomas such as EBV-associated diffuse large B-cell lymphoma (DLBCL), Burkitt lymphoma and post-transplant lymphoproliferative disorders.7, 8 Less commonly, EBV infects T and NK cells leading to a spectrum of rare disorders with variable clinicopathological phenotypes.7, 8 The WHO 2016 classification recognizes six entities under this category7, 8 (Table 7, 8). These include aggressive malignancies as well as more indolent cutaneous disorders and provisional entities which are being further characterized. Haemophagocytic lymphohistiocytosis is a severe systemic inflammatory syndrome characterized by abnormal activation of macrophages.10 Primary HLH occurs when there is a known causative gene mutation, specifically PRF1, STXBP2, RAP27, STXII, UNC13D genes, and X-linked lymphoproliferative disorder.10 Thus, HLH more commonly occurs secondary to infection, malignancy, or inflammatory disorders.10 The most common infectious cause of secondary HLH is EBV.10 The diagnosis of HLH is based on the presence of at least five of eight criteria, described in the HLH-2004 study, which were updated in the HLH 2009 protocol3, 11 (Table 3). This patient met five out of eight criteria for HLH, however, it is noteworthy that many of these criteria are non-specific and can occur in other acute inflammatory states. The diagnosis can therefore be challenging and a high index of clinical suspicion is required. The EBV-associated HLH (EBV-HLH) is a heterogenous condition that can develop as a result of primary EBV infection, but also in association with EBV-associated lymphoproliferative diseases.10, 12 In our patient, the challenge was to make the distinction between EBV-HLH arising from primary EBV infection and SE-TLC, disorders with significant clinicopathologic overlap but markedly different prognoses. Although EBV typically infects B-cells, EBV-HLH arising from EBV infection can present as a fulminant infection of CD8 + T-cells,12 and has been reported to be associated with aberrant and even clonal T-cell populations.12 Spontaneous resolution has been reported in a proportion of patients11 suggesting this is a transient activation of T cells rather than a true lymphoproliferative disorder.11, 12 Systemic EBV-positive T-cell lymphoma of childhood is an extremely rare, aggressive disease reported mainly in east Asians.13 Most cases are characterized by rapid deterioration following acute EBV infection. Despite being more recognized in the pediatric population, SE-TLC has also been reported in young adults.12 Thus, SE TLC is typically associated with cytopenias and splenomegaly, features which are also found in EBV-HLH. Histological features include haemophagocytosis and atypical T cells in the bone marrow.14 The presence of EBV positive clonal T cells,12 is a key feature of SE-TLC which has a grim prognosis with reports of 100% mortality in some series.12 Since SE-TLC and EBV-HLH from primary EBV infection have overlapping clinical features and can both be associated with EBV positive clonal CD8+ T cells, they can be difficult to distinguish. As EBV typically infects B-cells, TCR analysis and karyotyping should be considered in patients whom primary EBV infection of T cells is suspected. Smith et al. demonstrated that karyotypic abnormalities were associated with 100% mortality in patients presenting with clinical features of EBV-HLH/SE-TLC.12 Monoclonality by T-cell receptor (TCR) gene rearrangements was found in 62% of patients with a fatal outcome. These data suggest that the presence of karyotypic abnormalities and TCR gene clonality may be helpful in making the distinction between these entities but is clearly not definitive. Table S1 summarizes the karyotypic abnormalities of HLH and SE-TLC patients reported by Smith et al.12 In keeping with these reports, our patient had no karyotypic abnormalities and had a benign outcome. In view of this, our patient was given a final diagnosis of reactive HLH arising from primary EBV infection. Regardless of the diagnosis, the first step in management of both conditions is to focus on the treatment of HLH. The distinction between EBV-HLH from primary EBV infection and SE-TLC becomes important as the former may be managed conservatively, while SE-TLC requires more aggressive treatment. Patients with SE-TLC have a high mortality rate and often succumb to HLH.15 These patients should be treated according to the HLH 2009 protocol,11 with the goal of proceeding to hematopoietic stem cell transplant.15 In addition to EBV-HLH and SE-TLC, systemic disorders associated with EBV-related T and NK cell lymphoproliferative disease include Extranodal NK/T cell lymphoma (ENKTL), Aggressive Natural Killer Leukemia (ANKL), nodal EBV positive PTCL and systemic CAEBV.5 It is probable that there is some overlap between the WHO categories of EBV related T/NK LPD (Figure 4). Distinguishing between these disorders can be difficult given their rarity as well as overlap in clinicopathological features. Both ENKTL and ANKL are EBV driven lymphomas arising from NK cells or rarely cytotoxic T-cells. They are relatively more common in East Asia and Latin America, but rare in the West.4, 14 Nodal EBV-positive PTCL is a subtype of PTCL NOS and is a relatively recently described entity.10 While EBV is known to be positive in by stander B-cells in some subtypes of PTCL such as angioimmunoblastic T-cell lymphoma, it typically does not infect the malignant T-cells.16 Nodal EBV positive PTCL is unique in that the malignant T-cells are EBV positive. This neoplasm has a transcriptomic profile distinct to ENKTL and is characterized by loss of chromosome 14q11.2.17 Chronic Active EBV of the T or NK cell type is defined by persistent infectious mononucleosis-like symptoms lasting more than 3 months with increased EBV DNA (> 102.5 copies/mg) detected in the peripheral blood.5 Histologic evidence of EBV protein or RNA in affected organs is also a requirement for diagnosis.5 Cutaneous entities include Hydroa vacciniforme-like LPD (HVLPD), and severe mosquito bite allergy (SMBA), both of which are most commonly seen in the pediatric population. Although some patients have an indolent course, both these disorders can evolve into a T or NK cell lymphoma.16 Patterns of EBV-lymphoproliferative disorders differ between east and west in terms of prevalence, the type of cell that EBV infects, and the incidence of HLH.10 The EBV-associated lymphoproliferative disorders in western countries more commonly involve B-cells, whereas EBV-associated NK and T cell lymphoproliferative diseases are more prevalent in Asia, Central and South America.10 In CAEBV patients from Japan, EBV infects T and NK cells, whereas in the United States, EBV typically infects B cells.10 Notably, the incidence of EBV-associated HLH appears to be higher in Asia (approximately three-quarters of HLH cases) as compared to the United States (approximately one-third of HLH cases).10 Given the strong ethnic predisposition to these entities, genetic susceptibility traits are an important area to be addressed in future studies. Sheng et al. recently reported a case of EBV associated HLH in a Chinese female with concomitant STXBP2, LYST as well as other mutations associated with susceptibility to HLH.24 This suggests that susceptibility to EBV associated T and NK cell LPD may be polygenic. Studies in larger cohorts are required to definitively answer this question. The significant overlap in clinicopathological features of EBV-associated T and NK cell LPD necessitates great attention to detail in establishing an accurate diagnosis. The poor prognosis associated with many of these disorders underscores the need for a better understanding of their biology. There is also an urgent need for novel therapeutics for the more aggressive subtypes of EBV associated T/NK LPD. While the activity of the PD-1 inhibitor nivolumab in refractory EBV-HLH,25 has been encouraging, larger studies are required. Given the rarity of these entities, international collaboration is called for to systematically study these diseases and enable more informed patient management. Dr. Ng Siok Bian is supported by the National Medical Research Council (NMRC), Clinician Scientist Award, (CSAINV17nov016, WBS R-179-000-063-213), NMRC Open Fund Large Collaborative Grant, Singapore IYMPHoma translatiONal study (SYMPHONY) (NMRC OF-LCG18May-0028). The authors declare no relevant conflicts of interest. Informed consent has been obtained from the patient for the purposes of publishing this case report. Data sharing is not applicable to this article as no new data were created or analyzed in this study. Table S1 Karyotypic Abnormalities from cases reported by Smith et al (2014)1 Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.

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