PB2052 PHENOTYPIC IDENTIFICATION OF EBV‐INFECTED LYMPHOCYTES FOR THE DIFFERENTIAL DIAGNOSIS OF HLH, CAEBV AND PTLD; A CLINICALLY APPLICABLE, FLOW‐CYTOMETRY‐ BASED PERIPHERAL BLOOD ASSAY
Authors: Martinez‐Calle N. , Shannon‐Lowe C. , Collins P. , Lewis D. , Fox C.P. 
Published: June 2019

Background:

Epstein–Barr virus (EBV) is a ubiquitous lymphotropic virus which preferentially infects B‐cells1,2, but can also infect T‐cells and NK‐cells. EBV can cause B‐cell or T/NK lymphoproliferative (LPD) disorders and EBV‐related haemophagocytic lymphohistiocytosis (HLH)2. These rare but distinct diseases share clinical features of lymphadenopathy, fever, malaise, organomegaly and night sweats1. High levels of EBV DNA, as assessed by qPCR on whole blood or plasma are typical but, per se, have limited clinical value. EBV copy number in individual lymphocyte subsets offers much greater clinical value but is technically challenging and hence rarely investigated.

Aims:

We have applied a dual in‐situ hybridisation‐flow cytometry technique (EBER‐FlowRNA) allowing the detection and phenotypic characterisation of specific lymphocyte subsets infected by EBV, impacting the clinical management of patients with EBV‐associated lymphoproliferative disease.

Methods:

Following informed consent, blood samples from patients with suspected EBV‐related LPD were obtained at diagnosis. All patients were part of the observational ‘Studies of EBV‐associated NK/T‐cell diseases and formation of a registry’ (07/H1208/62). All analyses were performed at the Institute for Immunology and Immunotherapy (Birmingham University, UK). Clinical information for the cases was retrospectively obtained from clinical records.

Peripheral blood mononuclear cells (PBMC) were isolated from fresh patient blood and subjected to multicolour cell surface staining using up to 10 individual fluorophores. The cells were then fixed/permeabilised and subjected to EBER in‐situ hybridisation (PrimeFlowRNA) using pairs of 20‐mer specific probes, previously amplified and loaded with up to 400 fluorescent molecules; Beta‐2‐microglobulin probe pairs were used as positive control. Samples were processed by multicolour flow cytometry (BD Fortessa) and analysed with FlowJo software.

Results:

We present 4 representative clinical cases. Clinical characteristics described in Table 1. Flow cytometry of the samples was performed within 48 h of sample collection. Case 1 reflected a CD3+/CD4+/EBER+ population compatible with relapsed CAEBV, excluding the alternative diagnosis of EBV‐related B‐cell PTLD. Case 2 was an unusual CAEBV with gastric involvement and a CD3lo/CD4+/EBER+ population in peripheral blood, consistent with EBER positive malignant clone in the gastric biopsy. Case 3 presented with florid HLH and a CD19+/EBER+ clone consistent with primary EBV infection in the context of immunosuppression, treated successfully with Rituximab. Case 4 presented with a systemic illness and hepatic impairment, with identification of a CD56+/CD8+/EBER+ clone representing aggressive NK cell leukaemia. In cases 1, 2 and 4, EBER‐flowRNA prevented inappropriate use of empirical rituximab for raising EBV DNA‐aemia, whereas in case 3, EBER‐flowRNA excluded a T/NK LPD and supported rituximab use.

Summary/Conclusion:

In line with previously reported methodology4,5, our customised flow‐cytometry approach successfully identified the EBV‐infected lymphocyte subsets with a short turnaround time (48 hours sample‐to‐result). Additionally, successful EBER fluorescent labelling allowed for cell sorting and subsequent genomic and transcriptomic studies of EBV infected cells. Importantly, flow cytometry findings directed appropriate therapeutic approaches. Application of this technique in a larger cohort is warranted. We propose EBER‐FlowRNA as a relevant, rapid and feasible differential diagnostic tool for EBV‐related LPD.

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