A differential diagnosis method for systemic CAEBV and the prospect of EBV-related immune cell markers via flow cytometry

Figures & data

Table 1. Diagnostic criteria for EBV-positive T/NK cell proliferation.

Disease	Criteria	Criteria/Supplement	Reference
Sys CAEBV	Include all the following conditions from (1) to (4): (1) IM-like symptoms persisting for > 3 months (2) Increased EBV-DNA (>10^2.5 copies/µg) in PB (3) Histological evidence of organ disease (4) Demonstration of EBV RNA or viral protein in affected tissues in patients without known immunodeficiency, malignancy, or autoimmune disorders	MHLW research group of Japan: (1) Sustained or recurrent IM-like symptoms persist for more than 3 months (2) Elevated EBV genome load in PB or the tissue lesion (3) EBV infection of T or NK cells in the affected tissues or the PB (4) Exclusion of other possible diagnoses: primary infection of EBV (infectious mononucleosis), autoimmune diseases, congenital immunodeficiencies, HIV, and other immunodeficiencies requiring immunosuppressive therapies or underlying diseases with potential immunosuppression. Patients who fulfilled criteria (1)–(4) were diagnosed with CAEBV	[15,21]
EBV-HLH	(1) Clinical criteria (fever and splenomegaly) (2) Laboratory criteria (cytopenia affecting 2 of 3 lineages in the peripheral blood, hypertriglyceridemia, and/or hypofibrinogenemia) (3) Histological criteria (hemophagocytosis in the BM, spleen, or lymph nodes)	Exclusion of Congenital immunodeficiency including familial HLH, neoplasia-associated HLH	[6,25]
sEBV(+)TCL	(1) Illness or symptoms including fever, persistent hepatitis, lymphadenopathy, hepatosplenomegaly, hemophagocytosis, and interstitial pneumonia (2) Can occur shortly after primary EBV infection or in the setting of CAEBV (3) Monoclonal expansion of EBV-infected T cells with an activated cytotoxic phenotype in tissues or peripheral blood	(1) Progressive clinical course with features of HLH (2) Overt infiltrates of CD8+ and CD56 − cytotoxic T-lymphocytes with small or occasionally medium to large-sized or large nuclei (3) Overt lymphoma or rearrangements of T-cell receptor (TCR) genes	[15,26]
ANKL	(1) Cells with a slightly immature-looking morphology with broad, pale cytoplasm and azurophilic granules, a slightly fine nuclear chromatin, and the occasional presence of nucleoli (2) sCD3–, CD56+, CD16, and CD57– immunophenotype (3) Genetically, germline configuration TCR- and IgH genes (4) Functionally, a high or sometimes absent non-major histocompatibility complex–restricted cytotoxicity.		[27–29]
ENKTL	(1) Vascular damage and destruction, prominent necrosis (2) Cytotoxic phenotype (3) Association with EBV	Histopathological diagnosis: CD56, EBER, and cytotoxic molecules are positive	[30,31]
EBV + PTCL, NOS	(1) A T follicular helper (TFH) cell phenotype, as defined by the expression of at least two (ideally three) of the following markers: CD10, BCL6, PD1, CXCL13, CXCR5, ICOS, and SAP (2) Exclusion from any of the specifically defined entities of mature T-cell lymphoma in the current classification	EBV+, included within the category of peripheral T-cell lymphoma, NOS.	[32]

Figure 1. Proposed diagram illustrating the various approaches for diagnosing and managing EBV infection. Sys-CAEBV: systemic CAEBV, LPD: lymphoproliferative disorders, HLH: haemophagocytic lymphohistiocytosis, IM: Infectious monocytosis. FCM: flow cytometry Dotted arrow(): possible, red arrow () : reconfirmed.

Table 2. Immunophenotype of cells concerned.

A. T-series universal markers (CD2, sCD3, cCD3, CD4, CD5, CD7, and CD8)
Entity	Year	n	Country	Abnormal NK cells percentages	Immunophenotype
					CD2	sCD3	CD4	CD5	CD7	CD8
ANKL	2013	43 [114]	China	0.06–76 %	95% +++	/	/	98% -	(47 ± 44)% -	98% -
	2014	29 [124]	China	0.8%-87.7%	97% +++	93% -	97% -	–	48% loss	71% loss
	2022	14 [93]	China	/	+	–	–	–	-/+ het	-/dim
ENKTL	2013	11 [114]	China	0.06–76 %	+++	/	/	/	(45 ± 39)% -	0%
	2022	23 [93]	China	/	+	–	-/rarely	–	-/+het	–
ANKL(n = 2) ENKTL(n = 10)	2015	12 [121]	Portugal	/	83%	0%	0%	0%	42%	22%

Table

B. NK lineage-specific markers (CD16, CD56), differentiation markers (CD94, CD57, CD161),
Entity	Year	n	Country	Immunophenotype
				CD16	CD56	CD94	CD57	CD161
ANKL	2013	43 [114]	China	(27 ± 34)% −	88% +++	/	0%	/
	2014	29 [124]	China	67% loss	+++	79% +++	–	71% loss
	2022	14 [93]	China	+dim/−	bri	bri	–	/
ENKTL	2013	11 [114]	China	(14 ± 13)% −	+++	/	0%	/
	2018	12 [123]	Singapore	41% +/ dim	75% bright	/	8% +/ dim	/
	2022	23 [93]	China	–	bri	bri	-/+het	/
ANKL(n = 2) ENKTL(n = 10)	2015	12 [121]	Portugal	10%	>90%	>85%	0%	31%

Table

C. Activation markers (CD30, CD38, HLA-DR), functional markers of cytotoxicity (Granzyme B and Perforin), proliferation markers (Ki-67),
Entity	Year	n	Country	Immunophenotype
				CD30	CD38	HLA-DR	Granzyme B	Perforin	Ki67
ANKL	2013	43 [114]	China	98% −	/	/	/	(21 ± 11)%	/
	2014	29 [124]	China	/	/	/	/	/	typically > 40%
	2022	14 [93]	China	–	+	+/−	+het/−	+het/−	+/−
ENKTL	2013	11 [114]	China	/	/	/	/	(19 ± 12)%	/
	2022	23 [93]	China	–	+/bri	+/−	+het/−	+het/−	/
ANKL(n = 2) ENKTL(n = 10)	2015	12 [121]	Portugal	/	80%	>50%	/	/	/

Table

D. Clonality markers (KIRs: CD158a, CD158b, CD158e1, CD158i); Other markers to determine NK cell abnormalities: leukocyte common antigen CD45, CD45RA, CD45RO, TCRs
Entity	Year	n	Country	Immunophenotype
				CD158a	CD45	CD45RA	CD45RO	TCR
ANKL	2014	29 [124]	China	7% restricted KIR isoform	52% stronger than normal	/	/	–
	2022	14 [93]	China	/	/	+/-	-/+	/
ENTKL	2022	23^9 3	China	-/+het	/	+/-	-/+	/
ANKL(n = 2) ENKTL(n = 10)	2015	12 [121]	Portugal	0%	100%	>70%	>85%	0%

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Data availability statement

Data sharing is not applicable to this article as no new data were created or analysed in this study.