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An exploratory study on the differential diagnostic indicators between adult systemic EBV-positive T-cell lymphoproliferative disorders and angioimmunoblastic T-cell lymphoma with multiple EBV infections

Infectious Agents and Cancer volume 20, Article number: 3 (2025) Cite this article

Abstract

Background

The differential diagnosis between adult systemic EBV-positive T-cell lymphoproliferative disorders (EBV+ T-LPD) and angioimmunoblastic T-cell lymphoma (AITL) with multiple EBV infections is difficult, and distinguishing between the two has become a diagnostic challenge for pathologists. Given that the clinical treatment plans are different, an accurate diagnosis is a prerequisite to ensure effective treatment, therefore, it is extremely necessary and meaningful to find effective pathological indicators for distinguishing between two diseases.

Methods

We present a retrospective study comparing 7 cases of adult EBV+ T-LPD and 16 cases of AITL with multiple EBV infections diagnosed at our institution from 2017 to 2022. Differences in immunophenotype, type of EBV-infected cells, clonality and gene mutations between the two groups of cases were compared by immunohistochemical staining, double-label staining, TCR gene rearrangement and next-generation sequencing analysis.

Results

7 cases of adult EBV+ T-LPD: all cases had no more than 1 T follicular helper (THF) marker was expressed, and there were significantly more EBER+/CD3 + cells than EBER+/CD20 + cells; 5 cases had mutation detection results, in which only 1 had the characteristic KMT2D mutation, 2 had TET2 mutations, and no common mutations such as DDX3X were detected.16 cases of AITL with multiple EBV infections: all cases were found to express at least 2 TFH markers, with 87% of them expressing at least 3 TFH markers., and had significantly more EBER+/CD20 + cells than EBER+/CD3 + cells; 4 cases had mutation test results, with mutated high-frequency genes being TET2 (100%, and all of them had 2 or more TET2 mutations) and RHOA G17V (100%), DNMT3A mutation occurred in 2 cases (50%), and IDH2 R172 mutation occurred in 1 case (25%).

Conclusions

We found that the expression pattern of TFH markers, the types of cells predominantly infected by EBV and the different mutations can all be used as effective pathological indicators for distinguishing between two diseases.

Introduction

Epstein-Barr virus (EBV), a member of the herpesvirus gamma subfamily, is a double-stranded DNA virus originally discovered by Epstein and Barr in a Burkitt’s lymphoma cell line in 1964 [1]. EBV is primarily transmitted by saliva, over 90% of people are infected with EBV and the infection persists throughout life. Most people infected with the virus have no symptoms, some experience only mild symptoms similar to the cold, while others may present with obvious systemic symptoms and follow a self-limiting clinical course, such as infectious mononucleosis (IM) [2]; some are directly associated with tumours, such as Burkitt’s lymphoma, NK/T-cell lymphoma and Hodgkin’s lymphoma; others can infect epithelial cells and cause nasopharyngeal and gastric cancer; in addition, EBV is also associated with a group of lineage-specific lymphoproliferative disorders characterized by a long-term, recurrent clinical course that can progress to lymphoma, such as chronic active EBV infection (CAEBV).

The name EBV-positive lymphoproliferative disorders (EBV+ -LPD) was formally proposed at the International Conference on the Classification of EBV Lymphoproliferative Disorders in 2008 [3]. EBV+ -LPD refers to diseases with proliferative, borderline, and neoplastic lineages, including polyclonal, oligoclonal and monoclonal growth of similar cells. It is further subdivided into EBV+ B-LPD and EBV+ T/NK-LPD according to the main cells infected by EBV in the lesion. EBV+ B-LPD is less common and occurs mainly in western countries [4], whereas EBV+ T/NK-LPD occurs mainly in Asia, such as Japan, Korea, and China, Latin America, such as Mexico, and among indigenous peoples of Central and South America [3, 5]. The 2016 revision of the World Health Organization (WHO) Classification of Tumours of Haematopoietic and Lymphoid Tissues [6] pointed out that EBV+ T/NK-LPD, including systemic EBV+ T-cell lymphoma of childhood and CAEBV infection (systemic and cutaneous lesions, hydroa vacciniforme-like lymphoproliferative disorder (HV-like-LPD) and severe mosquito bite allergy (SMBA)). In the “5th edition of the WHO classification of haematolymphoid tumours” revised in 2022 [7] and the international consensus classification [8], there was a small change in EBV+ T/NK-LPD and lymphomas of childhood. HV-LPD replaced the previous HV-like-LPD, and the disease was divided into classical and systemic; CAEBV disease(CAEBVD)replaced CAEBV infection and was restricted to cases with T and NK cell phenotypes, excluding B-cell cases. Systemic EBV+ T-cell lymphoma of childhood has a sudden and aggressive clinical course, with most cases being associated with hemophagocytic syndrome (HPS). It is characterized by monoclonal proliferation of EBV-infected cytotoxic T cells, often leading to severe complications and even death within a few days to weeks; Both HV-LPD and SMBA have characteristic clinical manifestations and pathological features, whereas systemic EBV+ T/NK-LPD (sCAEBVD) is often a diagnostic difficulty for pathologists.

sCAEBVD is characterized by persistent, recurrent flu-like symptoms, including fever, lymphadenopathy, and hepatosplenomegaly etc., lasting for more than 3 months. It occurs mainly in children and adolescents [6]. However, in recent years, an increasing number of adult cases have been reported with a more aggressive clinical course and poorer prognosis [9,10,11], and the diagnosis is more challenging, because the pathological morphology and immunophenotype of the disease are similar to those of other T/NK-cell lymphomas (e.g. extranodal NK/T-cell lymphoma, angioimmunoblastic T-cell lymphoma (AITL), and other T/ NK-cell lymphomas, and EBV+ nodal T/NK-cell lymphoma listed separately in the 5th edition of the WHO), and clinical history must be considered for accurate diagnosis. For example, the distinction between sCAEBVD and NK/T-cell lymphoma cannot be made on the basis of morphology, immunophenotype or even molecular genetics, whereas clinical information is crucial. While sCAEBVD typically presents with systemic symptoms from the onset and progresses over months to years, NK/T-cell lymphoma often begins with localized lesions before spreading throughout the body. Recent genetic studies have suggested a potential precursor relationship between sCAEBVD and NK/T-cell lymphoma. New genetic studies [12, 13] have shown that sCAEBVD is similar to somatic mutations (e.g., DDX3X, KMT2D) in NK/T-cell lymphoma, suggesting that it may be a precursor lesion of NK/T-cell lymphoma. However, some T-cell lymphomas with EBV infection also present with systemic systemic symptoms at the onset of disease and are very similar in morphology, we do have difficulties in differentiating them in our diagnostic workup, for example, with AITL.

Adult systemic EBV+ T-LPD and AITL have considerable overlap in clinical manifestations, histology and immunophenotype. The differential diagnosis can be based on the quantity and type of EBV-infected cells. In general or typical cases, the number of EBV-infected cells in AITL is variable, often lower and predominantly infecting B cells. In systemic EBV+ T-LPD, EBV infects T cells, and a large number of cells are infected. However, cases of AITL with multiple EBV infections have been observed, so it is necessary to investigate the cell types of EBV infection. If the infected cells are predominantly B cells, this may help to differentiate between two diseases, whereas if they are predominantly T cells, the distinction becomes more difficult. Therefore, we need to actively search for other meaningful indicators that can help distinguish between the two. Tumour cells in AITL express T follicular helper (TFH) cell markers [6, 14]. However, there are no reports in the national and international literature on whether T cells in systemic EBV+ T-LPD express TFH cell markers. If T cells in this lesion do not express TFH cell markers, this can be used as an effective indicator to distinguish between the two. In addition, the molecular genetic changes of the two are also different [15,16,17,18,19]. We reviewed 7 cases of adult systemic EBV+ T-LPD and 16 cases of AITL with multiple EBV infection as controls. We studied the expression of TFH cell markers, the types of EBV-infected cells, and the molecular genetic differences between the two lesions in order to better differentiate them in the future.

Materials and methods

Cases selection

A total of 390 archival cases diagnosed with EBV+ T/NK-LPD and 493 archival cases diagnosed with AITL from the database of the Department of Pathology, Beijing Friendship Hospital, Capital Medical University/ Lymphoma Diagnostic Research Center of Beijing Institute of Clinical Medicine from 2017 to 2022 were collected as the primary research objects.

Criteria for case inclusion: Adult systemic EBV+ T-LPD (research group): ①Age of onset ≥ 30 years old; ②The site is lymph node (complete lymph node sample); ③Fulfil the diagnostic criteria of CAEBV; ④Systemic lesions, excluding two localized skin lesions; ⑤EBV mainly infects T cells; ⑥Complete case data. AITL with multiple EBV infections (control group):①The site is lymph node (complete lymph node sample); ②Multiple EBV infections(>50/HPF);③The diagnosis of AITL has been confirmed; ④Complete case data. Diagnostic criteria for CAEBV: Previously, the diagnostic criteria recommended by the Japanese Association for Research on EBV and Related Diseases [20, 21]: ①Persistent or recurrent IM-like symptoms for more than 3 months; ②Detection of an increased number of EBV genomes or elevated antibody titres in peripheral blood (PB) and/or affected tissues, at least 1 of the following 4 items must be fulfilled: Detection of EBV DNA by Southern hybridisation; Detection of EBER-positive cells; Elevated EBV DNA load in PB (DNA > 102.5copies/mg); High EBV-related antibody titres measured by fluorescent antibody tests (VCA-IgG ≥ 640 and EA-IgG ≥ 160); ③Currently known autoimmune, neoplastic and immunodeficiency diseases excluded. In 2022, the Ministry of Health Labor and Welfare research team in Japan revised the diagnostic criteria for CAEBV [22]: ①Persistent or recurrent IM-like symptoms for more than 3 months; ②Detection of an increased number of EBV genomes in PB and/or affected tissues; ③Detection of EBV-infected T or NK cells in PB and/or affected tissues; ④Chronic disease that cannot be explained by other known disease processes at the time of diagnosis. Diagnosis of CAEBV now requires confirmation of a high copy number of the EBV genome and EBV-infected T or NK cells. An EBV DNA load ≥ 10,000 IU/mL in whole blood is proposed as the diagnostic cut-off for CAEBV in this updated guideline. High EBV-related antibody titres are not necessary for the diagnosis of CAEBV, as some patients do not have high antibody titres.

Immunohistochemical staining

Immunohistochemistry (IHC) staining was performed manually on formalin-fixed, paraffin- embedded (FFPE) tissue for immunophenotypic analysis. The EliVision two-step method was used. Sections were stained with monoclonal antibodies against CD21、CD20、cytoplasmic CD3、CD5、CD4、CD8、CD56、Granzyme B、TIA1、Ki-67、CD10、BCL6、PD-1、ICOS、CXCL13、CD30、EBNA2 and others, cytoplasmic CD3 was the product of Dako, the rest of the antibodies used were from Maxim-Bio (Cat. No. KIT-5910/5931), Fuzhou, China. Positive and negative controls were analysed according to the manufacturer’s instructions.

EBER‑in situ hybridization

The EBV probe in situ hybridisation (ISH) kit (Triplex International Biosciences (China) Co. Ltd., Fuzhou, China) was used to detect EBERs. Details of this procedure were described in our previous report [23]. The positive signal was located in the nucleus and was brown in colour. EBV-positive extranodal NK/ T-cell lymphoma was used as a positive control, and EBV-negative tonsillar lymphoid tissue hyperplasia was used as a negative control. EBER-positive cells were counted: the area with the densest positive cells was first determined at low magnification, then the positive cells were counted at high magnification.

Double-labelled ISH and IHC staining

An IHC plus EBER-ISH dual-staining technique was performed using the Leica Bond MAX autostainer (Leica, Melbourne, Australia). Section (2-µm thick) were first stained for EBER with DAB staining (brown), then for CD3 (LN10, RTU; Lecia), CD20 (L26, RTU; Lecia) followed by visualization of the red staining after application of amino-ethylcarbazole (Bond Polymer Refine Red Detection kit, Leica) as chromogen.

T cell receptor gene clonality analysis

T cell receptor (TCR) gene rearrangement analysis was performed using polymerase chain reaction (PCR) based on the “Biomed-2” primers (InVivo Scribe Technologies, San Diego, CA, USA) [24]. DNA was extracted from FFPE tissue samples using the TIANamp FFPE DNA Kit (DP331) (TIANGEN, Beijing, China). For the gene rearrangement assay, PCR was carried out in a 25-µl volume containing 22.5-µl of master mix, 0.13-µl of Ampli Taq Gold DNA polymerase, and 100ng of genomic DNA. The cycling profile used for all reactions was as follows: 95℃ for 7 min; 35 cycles of 95℃ for 45s, 60℃ 45s, 72℃ 90s; and a 10-min final extension at 72℃. After amplification, PCR products were denatured at 94℃ for 5 min followed by a quick chill to reanneal the PCR products at 4℃ for at least 60 min. The PCR products were electrophoresed in 6% polyacrylamide gels (BioRad) in 1 × TBE buffer at 120 V for approximately 65 min. The gel was then soaked for 20 min in 100 ml of 0.1 M NaCl solution containing 10 µl of 10 mg/ml Gel Red (Biotium, USA) and photographed using ultraviolet illumination.

DNA sequencing and mutation analysis

Sample purity, quantity and degradation were assessed using a NanoDrop spectrophotometer (260/280: 1.6–2.3, 260/230 > 1.6), Qubit fluorometric quantification (≥ 100 ng/µL) and electrophoresis (size of DNA fragments ≥ 300 bp). We performed capture-based sequencing using the IDT xGen Exome Research Panel v1.0 on Illumina NovaSeq 6000 sequencing instruments (Illumina, San Diego, CA, United States) according to the manufacturer’s protocol. The coding regions of 300 genes known to be frequently mutated in haematological malignancies were analysed. The average sequencing depth was 300x. A minimum coverage of 100x and a variant allele frequency (VAF) > 5% were used as thresholds for calling single nucleotide variants and short insertions or deletions (InDels). Alignment of the reads to the human reference (hg 19 assembly) and variant calling and annotation were performed using GATK and ANNOVAR. All variants with a population minor allele frequency ≥ 1% in the 1000 Genomes Project and genomAD East Asian databases were filtered.

Results

Clinical features

The 7 patients with adult systemic EBV+ T-LPD were 4 males and 3 females, with an average age of 38 years and a median age of 41 years (range 31–43 years). The mean and median interval from disease onset to biopsy was 13 and 6 months, respectively (range 3–48 months). All 7 patients (100%) presented with fever、generalised lymphadenopathy, 6 patients (85.7%) with hepatosplenomegaly、upper respiratory tract symptoms (sore throat, swollen tonsils) or pneumonia, 2 patients (28.6%) with rash; laboratory data: 5 patients (71.4%) had liver dysfunction and pancytopenia, 4 patients (57.1%) had bone marrow hemophagocytosis, and the proportion of eosinophils in peripheral blood was increased in 2 cases (28.6%). All 7 patients were consultation cases without EBV serum immunological test results, and 6 patients had serum EBV DNA load test results ranging from 2.09 × 104 to 7.25 × 107.

The 16 cases of AITL with multiple EBV infections were 12 males and 4 females, with an average age of 69years and a median age of 68 years (range 46–80 years), and a mean and median interval from onset to biopsy of 5 and 4 months, respectively (range 2–12 months). All 16 patients had multiple lymphadenopathy (100%), 8 patients had fever (50%), 6 patients had hepatosplenomegaly and rash (37.5%); laboratory data were available in 10 cases: 5 cases had pancytopenia (50%) and 2 cases had elevated peripheral eosinophils (20%). All 16 patients were consultation cases and the results of serum EBV immunology and EBV DNA load were unknown.

Morphological features

7 cases of adult systemic EBV+ T-LPD: The lymph node structure was destroyed in 6 cases, preserved in 1 case with enlargement of the paracortical region, extracapsular infiltration was observed in 3 cases, and multifocal necrosis occurred in 3 cases. In all cases there was a mixed cellular composition characterised by predominantly small to medium-sized tumour cells with slightly irregular nuclei and inconspicuous nucleoli, together with an increased presence of histocytes and vessels (Fig. 1A, B).

Fig. 1
figure 1

Morphology and immunohistochemical/in-situ hybridisation markers for adult systemic EBV+ T-LPD (A, B, H&E; C, CD4; D, CD8; E, PD1; F, ICOS; G, CXCL13; H, CD10; I, Bcl-6; J, EBER; K, EBER/CD3; L, EBER/CD20). A, B The lymph node structure is destroyed, mixed cellular composition characterised by predominantly small to medium-sized tumour cells with slightly irregular nuclei, with an increased presence of histocytes and vessels. C, D Tumour cells are predominantly CD4-positive T cells. E-I The expression of TFH cell markers, except that some cells in the residual germinal center are positive, tumour cells barely express TFH markers, a few cells can weakly-moderately express ICOS. J EBER-ISH positive, with a predominance of small and medium cells. K Double in-situ hybridisation and immunohistochemistry for EBER and CD3 showed EBV-infected mainly T cells (EBER, brown; CD3, red). L, Double in-situ hybridisation and immunohistochemistry for EBER and CD20 showed EBV-infected fewer B cells (EBER, brown; CD20, red)

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16 cases of AITL with multiple EBV infections: In 15 cases, the lymph node structure was destroyed, while the subcapsular lymphoid follicular structure was preserved in 1 case, extracapsular infiltration was observed in 13 cases, all cases had obvious vascular proliferation and abundant histocytes, but no necrosis was found; in 7 cases, the tumour cells were of medium size, with irregular nuclei, empty cytoplasm and mitoses were easily visible, in 9 cases, the tumour cells were composed of large, medium and small cells, with slightly irregular nuclei and small nucleoli (Fig. 2A, B).

Fig. 2
figure 2

Morphology and immunohistochemical/in-situ hybridisation markers for AITL (A, B, H&E; C, CD4; D, CD8; E, PD1; F, ICOS; G, CXCL13; H, CD10; I, Bcl-6; J, EBER; K, EBER/CD3; L, EBER/CD20). A, B The lymph node structure is destroyed and has obvious vascular proliferation and abundant histocytes, the tumour cells are medium-large size, with irregular nuclei, small nucleoli, empty cytoplasm and mitoses are easily visible. C, D Tumour cells are predominantly CD4-positive T cells. E-I Tumour cells strongly and diffusely express TFH cell markers. J EBER-ISH positive, multiple EBER-positive cells with large, medium and small cells, K, L Double in-situ hybridisation and immunohistochemistry for EBER and CD3/CD20 showed EBV-infected mainly B cells and fewer T cells (EBER, brown; CD3/CD20, red)

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Immunohistochemical and in situ hybridization findings

7 cases of adult systemic EBV+ T-LPD: T-cell markers CD3, CD5, CD4 and CD8 were expressed in most tumour cells of all cases, CD5 antigen expression was lost in 6 cases (85.7%); 6 cases (85. 7%) were predominantly CD4-positive T cells and 1 case was predominantly CD8-positive T cells (Fig. 1C, D); all cases (100%) expressed the cytotoxic molecules TIA-1 and Granzyme B, with TIA-1 expression being more significant; CD56 expression was scattered in 5 cases; the Ki67 index ranged from 10 to 80%; CD21 showed a small amount of residual disordered and slightly irregular follicular dendritic cell (FDC) networks in 4 cases and a small amount of regular FDC networks in 3 cases.

16 cases of AITL with multiple EBV infection: The tumour cells expressed the T-cell markers CD3, CD5, CD4 and CD8 in all cases and were dominated by CD4-positive T-cells (100%) (Fig. 2C, D). CD5 antigen expression was lost in 12 cases (75%); cytotoxic molecular markers were tested in 3 cases, and TIA-1 expression was higher than Granzyme B expression. The Ki-67 index ranged from 50 to 90%; in 10 cases, CD21 showed a significant increase in disordered and irregular FDC networks, 5 cases had only mild proliferation of FDC networks, and 1 case had only a small amount of residual FDC networks.

The expression of TFH cell markers (PD1, ICOS, CXCL13, CD10, BCL-6) in the research group (Fig. 1E-I) and in the control group (Fig. 2E-I) is shown in Tables 1 and 2.

Table 1 TFH marker coexpression in the studied entities
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Table 2 Percentage (no.) Of individual TFH marker expression in the studied entities
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All cases were positive for EBER, with the proportion of positive nuclei ranging from 50 to 200 cells per HPF (Fig. 1J) in the research group and 50 to 300 cells per HPF (Fig. 2J) in the control group. Positive cells in both groups included large, medium and small cells, with a predominance of small and medium cells.

Double-labelled ISH and IHC staining for EBER and CD3 showed that EBER-positive cells were also CD3-positive (Fig. 1K) and mixed with fewer CD20-positive B cells (Fig. 1L) in all cases of the research group; conversely, EBER-positive cells were mainly CD20-positive cells in all cases of the control group (Fig. 2K, L).

PCR for TCR gene rearrangements

T cells could be oligoclonal, polyclonal or monoclonal, according to sCAEBVD clonality analysis [21, 25, 26], this ancillary test was not helpful in differentiating sCAEBVD from other EBV+ T/NK- cell lymphomas. In our study of 7 cases of adult systemic EBV+ T-LPD, all showed monoclonal TCR rearrangements (both single and multiple-tube rearrangements), mainly manifested as monoclonal rearrangements of TCRG (Fig. 3); of the 16 cases of AITL with multiple EBV infections, 8 underwent TCR gene rearrangement testing and all showed monoclonal TCR rearrangements (both single and multiple-tube rearrangements).

Fig. 3
figure 3

TCR gene rearrangements of adult systemic EBV+ T-LPD reveals monoclonal rearrangements of TCR GA (Green fluorescent signal within the effective detection range of 145–255 bp shows a distinct amplification peak). The red florescent signals are sizes at 100 bp, 200 bp and 300 bp, respectively

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Results of whole exome sequencing

7 cases of adult systemic EBV+ T-LPD: All cases were submitted for 300 types of blood tumour gene mutation screening tests, of which 2 cases could not undergo Whole Exome Sequencing (WES) due to severe DNA fragmentation, and the test results of the remaining 5 cases are shown in Table 3.

Table 3 Mutation status of the 7 cases of adult systemic EBV+ T-LPD
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16 cases of AITL with multiple EBV infections: 7 cases were submitted for 300 types of blood tumour gene mutation screening tests, of which 3 cases could not undergo WES due to severe DNA fragmentation and insufficient DNA concentration, and the test results of the remaining 4 cases are shown in Table 4.

Table 4 Mutation status of the 7 cases of AITL
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Follow-up

Of the 7 cases of adult systemic EBV+ T-LPD patients who were followed up, 6 were followed up regularly and 1 was lost to follow-up. 5 patients died between 2 and 20 months after diagnosis. The mean survival time was 10.8 months and the median survival time was 10.5 months. Of note, 1 patient who is still alive underwent allogeneic haematopoietic stem cell transplantation (allo-HSCT).

Of the 16 cases of AITL with multiple EBV infections observed, 13 cases were followed up and 3 cases were lost to follow-up. 11 patients died between 2 and 15 months after diagnosis, The mean survival time was 7 months and the median survival time was 5.5 months. Two survivors have received chemotherapy and are currently in good health.

Discussion

EBV+ T/NK-LPD is most common in East Asian countries and was initially identified as a group of childhood diseases. As physicians’ understanding of the disease has deepened, more and more adult cases have been recorded, with the highest number of cases reported from Japan [9,10,11, 27], including some notable large-scale reports: Kawomoto K et al. [11] summarised a total of 54 adult cases in Japan from 2005 to 2015 (all patients were aged ≥ 15 years, of which 22 were aged ≥ 50 years). Yonese I et al. [27] summarised 100 EBV+ T/NK-LPD cases diagnosed between 2003 and 2016, with more adult cases (≥ 20 years) than children in this study (< 9 years: 23 cases, 9–45 years: 64 cases, ≥ 45 years: 13 cases). Actually, it was not rare in China, Luo Ling et al. [28] retrospectively reviewed and analysed all adult-onset EBV+ T/NK-LPD cases admitted to Peking Union Medical College Hospital between 2012 and 2016. There were 28 patients with adult-onset EBV+ T/NK-LPD. The median age was 45 years (range, 20–81 years). A total of 162 adult EBV+ T/NK-LPD cases (≥ 20 years) were diagnosed in the database of our institution (Department of Pathology, Beijing Friendship Hospital Affiliated to Capital Medical University/Lymphoma Diagnosis Research Center of Beijing Institute of Clinical Medicine) during the 5 years from 2017 to 2022.

In adult EBV+ T/NK-LPD, our diagnostic challenge is to diagnose systemic EBV+ T/NK-LPD (sCAEBVD). In addition to fulfilling the diagnostic criteria for CAEBV, EBV-associated T/NK-cell lymphomas must be excluded, especially to differentiate from NK/T-cell lymphoma. A growing body of literature [12, 13] has proved that CAEBVD originates from EBV-infected lymphoprogenitor cells that acquire DDX3X and other mutations, leading to clonal evolution involving multiple cell lineages. Notably, CAEBVD patients had frequent intragenic deletions in the EBV genome that were also common in extranodal NK/T-cell lymphoma, but were not detected in IM or post-transplant lymphoproliferative disorders, suggesting that these mutations could play a unique role in the neoplastic proliferation of EBV-infected cells. CAEBVD can be a progression to NK/T -cell lymphoma; In addition, we have also found that in a few cases of AITL, a large number of EBER-positive cells may be present (in most cases with a few or scattered EBER-positive cells). At this time, we need to distinguish it from EBV+ T-LPD.

It is usually difficult to distinguish the two on the basis of clinical history or histological morphology. According to literature reports [9, 27, 29], sCAEBVD in children and adults has different characteristics, with cases under 20 years of age being more common in males, and cases over 45 years of age being more common in females, while there is no gender difference in cases between 20 and 45 years of age. AITL occurs mainly in older men, with an average age of 60 years. Our two groups of cases included 7 adult systemic EBV+ T-LPD (> 20 years old) with a male to female ratio of 4:3 and a slight predominance of males. The average age was 38 years and all cases were < 45 years; 16 cases of AITL, with a male to female ratio of 3:1 and an average age of 69 years. All were consistent with literature reports. Most cases of sCAEBVD presented with fever, lymphadenopathy and hepatosplenomegaly, hemophagocytic lymphohistiocytosis (HLH) and pancytopenia were present in approximately 1/3 of cases [27]; AITL is often accompanied by B symptoms, systemic lymphadenopathy, hepatosplenomegaly, rash, hypergammaglobulinemia, anemia, eosinophilia, etc. [30, 31], the clinical manifestations of our two groups of cases were consistent with those reported in the literature. In addition, the histomorphology of the two diseases is relatively similar (see above for details). Therefore, there was no significant difference in the clinical manifestations and morphological characteristics of the two diseases, which could not be used as indicators for differential diagnosis.

We tried to distinguish the two diseases by immunophenotype, the type of EBV-infected cells and molecular genetic changes.

Most cases of tumour cells in AITL are positive for CD4 and negative for CD8, and the T cells in sCAEBVD are often positive for CD4, while a minority express CD8 and cytotoxic markers such as TIA1 and granzyme B [21, 32]. Therefore, the proportion of CD4-and CD8-positive cells (the type of T cells) in the lesion cannot be used to distinguish the two. AITL is a T-cell lymphoma of TFH cell origin, the tumour cells frequently express various markers of TFH cell, among them CD10, BCL6, CXCL13, PD1 and ICOS were the most common, all AITL cases had ≥ 2 TFH cell markers expressed [33]. The expression rates of each TFH cell marker vary in AITL according to literature reports from different countries [30,31,32,33,34]: CD10(30-89%)、BCL6(29-91%)、CXCL13 (44-96%)、PD1(62-100%), and ICOS(94-98%). However, the differential expression of individual TFH-associated markers and their co-expression in a broad group of benign and malignant lymphoid proliferations that may mimic AITL has not been well studied. For systemic EBV+ T-LPD, there have been no literature reports seen on whether it expresses TFH cell markers, only single study [35] has discussed the expression patterns of TFH immunohistochemical markers in AITL and other morphologically similar benign and neoplastic lesions. The results of this study showed that cells strongly express the TFH cell markers in the germinal center of normal follicles, and in rare cases, scattered positive cells were found outside the follicles; Atypical hyperplasia of cells in the paracortex, ICOS showed the highest level of expression with immunoreactivity in 29% of cases, BCL-6, PD1, CXCL13, and CD10 were expressed in 10%, 7%, 3% and 3% of cases respectively, in addition 13% of cases showed co-expression of more than 1 marker, however more than 85% of cases were negative or expressed only 1 of the markers; PD-1、ICOS、CXCL13、BCL6 and CD10 were strongly and diffusely expressed in 74%、89%、89%、42% and 47% of AITL, respectively; 74% of AITL co-expressed 3 or more markers.

In our study, the positive rates of tumour cells (≥ 5%) of PD-1, ICOS, CXCL13, BCL6 and CD10 were 100%, 88%, 81%, 69% and 50%, respectively, in the control group of 16 AITL cases, all cases expressed ≥ 2 TFH cell markers and 87% of them expressed ≥ 3 TFH cell markers, which is consistent with the literature; 7 cases of EBV+ T-LPD in the research group, except that some cells in the residual germinal center expressed TFH cell markers, only 1 case expressed PD-1 (diffuse cell distribution, about 6%, weakly positive), 2 cases expressed ICOS (diffuse cell distribution, less than 10%, weakly positive), 1 case expressed BCL6 (the cells were dispersed, about 6%, moderately positive). The results of previous studies and our study showed that TFH cell markers are occasionally expressed in benign and neoplastic T-cell proliferative lesions. Therefore, we should not make a diagnosis of T-cell lymphoma of TFH cell origin (TCL-TFH) on the basis of TFH cell markers expression alone without careful consideration of other features. Overall, 100% of the AITL in our cohort expressed ≥ 2 markers, whereas none of the EBV+ T-LPD cases expressed more than 1 marker. Therefore, the expression of TFH cell markers, especially the combined expression of TFH cell markers, can be used as an effective indicator to differentiate one from the other. In addition, AITL cases usually present with proliferative and disregulated FDC networks, whereas EBV+ T-LPD cases often have a small amount of residual FDC networks, which can sometimes be used for differential diagnosis.

EBV-infected B cells are commonly observed in AITL; EBV-infected B cells were found in 66% of cases in the Japanese retrospective study [34], 91% in the French-Swiss study [31], and 74% in the COMPLETE study [36]. Nevertheless, EBV-infected T cells have been very rarely reported in the literature, with only two cases of nodal T follicular helper (TFH) cell lymphoma (nTFHL), with dominant EBV-infected CD4 + T cells containing neoplastic TFH cells and non-neoplastic background cells reported by Wang Zhe et al. [37] in 2023. The results of double-labelled staining in 16 cases of AITL with multiple EBV infections showed that most of the infected cells were non-neoplastic B cells, and a few T cells were infected. In the “5th edition of the WHO classification of haematolymphoid tumours” revised in 2022, CAEBVD was restricted to T cell and NK cell phenotypes, the double-labelled staining results of 7 cases of systemic EBV+ T-LPD in this group showed that most of the EBV-infected cells were T cells and only a few B cells were infected. According to the literature and our study, EBV-infected T cells in AITL are very rare, with only two cases reported to date. Therefore, we can distinguish them by identifying the type of EBV-infected cells by double-labelled staining.

The molecular alterations of AITL have been extensively reported in the literature. The common mutated genes of AITL include TET2, DNMT3A, IDH2R172, RHOAG17V et al. [30, 38,39,40,41], the most common mutations are epigenetic (TET2, DNMT3A, IDH2) and genes associated with T cell signaling (RHOA, PLCG1, CD28, FYN, VAV1), the incidence of TET2 mutation is the highest, up to 80%, and most of them (66%) are accompanied by more than 2 TET2 mutations, the frequency of DNMT3A mutation is about 10-40% [31, 42], IDH2 mutation rate is 20-45% [42, 43], and RHOA mutation rate is 50-70% [44, 45], the mutation of RHOA p.G17V and/or IDH2 p.R172 are specific for AITL, which are very helpful for diagnosis. It is currently hypothesised that deleterious mutations in the TET-2 and DNMT3A genes, which occur in the early stages of haematopoietic development, are the initial events to promote tumourigenesis (the first hit), and subsequently the appearance of specific mutations such as RHOA p.G17V and IDH2 p.R172 (the second hit), which regulate the differentiation and proliferation of clonal TFH cells, leading to the development of AITL [46]; Systemic EBV+ T-LPD has almost no germline mutations, and common somatic mutations include DDX3X, KMT2D, BCOR/BCORL1, TET2 and KDM6A. According to the report [12], DDX3X and KMT2D have been reported to confer survival or growth advantages to cells, and are thought to be driver mutations. The study also found that these mutations in CAEBVD were associated with a poor prognosis. The samples in this study were analyzed by blood tumour mutation group (300 genes, including the common mutated genes of AITL and sCAEBVD mentioned above). In the 4 cases of AITL in this group, the high-frequency mutated genes were TET2 (100%, and all had 2 or more TET2 mutations) and RHOA (100%), the locus of RHOA mutation was G17V in all 4 samples, with DNMT3A mutation in 2 cases (50%) and IDH2 p.R172 mutation in 1 case (25%), which was consistent with literature reports, it is noteworthy that one of them had KMT2B mutation (25%), which is not a common high-frequency mutated gene in AITL, whether it is a feature of multiple EBV infections cases needs to be studied in more cases. Unfortunately, in this group of 5 cases of EBV+ T-LPD, only 1 case had a relatively characteristic KMT2D mutation, 2 cases had TET2 mutation, and no common gene mutations such as DDX3X were detected. Of course, the main reason is that our case size is too small and the representation is inadequate. However, these 5 cases of EBV+ T-LPD did not have the common gene mutations associated with AITL, which can also be used to exclude the diagnosis of AITL.

AITL is associated with a poor overall prognosis. Data from the International T-cell Lymphoma Project (ITCLP) and other large collaborative studies showed that 32–41% and 18–38% of AITL patients survive and remain event-free at 5 years from diagnosis, respectively [34, 47, 48]. Newly published data from a subset of 282 AITL cases enrolled in the ITCLP from 2006 to 2018 showed overall survival (OS) and progression-free survival (PFS) at 5 years of 44% and 32%, respectively [49], this is consistent with the previous findings. In parallel, the same study identified disease progression within 24 months of diagnosis (POD-24) as a strong predictor of prognosis in patients with AITL, with an estimated 5-year OS of 63% versus only 6% for patients with and without POD-24. In addition, national research has shown that a large number of EBV-positive cells (> 50/HPF) is associated with a significantly worse prognosis and is an independent prognostic indicator for OS and PFS [50]. In this group, all 16 cases of AITL were accompanied by a large number of infected cells (> 50/HPF), and the mortality rate was as high as 77% (10/13), all of them died within 24 months (8 cases died within 12 months), 3 patients are still alive, with a survival period of 25–60 months, and no disease progression was observed within 24 months. Systemic EBV+ T-LPD is also a progressive disease, and the prognosis of adult-onset (> 20 years) CAEBVD has been reported to be worse than that of childhood-onset CAEBVD [9, 11, 27], however, the reason for this has not yet been determined. In this group of 7 adult patients with systemic EBV+ T/NK-LPD, the mortality rate was 83% (5/6). The prognosis of both disease groups is poor, and correct diagnosis is a prerequisite to ensure that patients receive appropriate treatment, thus providing an opportunity to improve the prognosis. Currently, allo-HSCT is considered the only curative treatment for CAEBVD. Chemotherapy is used to reduce viral load and control disease activity in CAEBVD prior to HSCT, as the OS rate in patients with inactive disease is significantly higher than in patients with active disease at the time of HSCT [21]. In addition, chemotherapy may reduce the risk of HSCT-related complications. A combination of immunochemotherapy to reduce EBV-infected T/NK cells prior to allo-HSCT has been proposed [51, 52]. Currently, HSCT is the most common treatment for CAEBVD, while chemotherapy is the main treatment for AITL in China.

The mechanism by which EBV induces T/NK-cell proliferation remains unknown. Previous studies [53,54,55,56] have shown that several pathways, such as NF-κB, mTOR and JAK/STAT3, are activated in EBV-positive T/N-cell lines from patients with CAEBVD. EBV may contribute to the development of lymphoma in T/NK cells through the activation of these signaling pathways, which could be considered as novel targets for therapeutic intervention. In particular, STAT3 mediates the molecular signaling of the receptors of a large number of inflammatory cytokines. This study has showed that JAK inhibitors, such as ruxolitinib, inhibit both STAT3 activity and cytokine production. STAT3-mediated pathways may be attractive therapeutic targets, not only for resolving inflammation but also for eradicating EBV-infected cells.

Conclusion

Differential diagnosis between adult systemic EBV+ T-LPD and AITL with multiple EBV infections is challenging. With the increasing incidence of systemic EBV+ T/NK-LPD in adults, the distinction between the two has become a diagnostic challenge for pathologists. The clinical management of the two diseases is different, and accurate diagnosis is essential for effective treatment. Therefore, it is very necessary and meaningful to find helpful pathological indicators for their differentiation. In our study, we found that the expression pattern of TFH cell markers (AITL: ≥3 markers widely positive; systemic EBV+ T-LPD: <2 markers focally positive), the type of EBV-infected cells (AITL: mainly infected B cells; systemic EBV+ T-LPD: mainly infected T cells), as well as the different genetic mutations (AITL: TET2, DNMT3A, IDH2R172, RHOAG17V et al.; systemic EBV+ T-LPD: KMT2D, TET2 et al.) could be effective pathological indicators for differentiation.

Data availability

No datasets were generated or analysed during the current study.

Abbreviations

Allo-HSCT:

Allogeneic haematopoietic stem cell transplantation

AITL:

Angioimmunoblastic T-cell lymphoma

CAEBV:

Chronic active EBV infection

EBV:

Epstein-Barr virus

EBV+-LPD:

EBV-positive lymphoproliferative disorders

FDC:

Follicular dendritic cell

HLH:

Hemophagocytic lymphohistiocytosis

HPS:

Hemophagocytic syndrome

HV-like-LPD:

Hydroa vacciniforme-like lymphoproliferative disorder

IHC:

Immunohistochemistry

IM:

Infectious mononucleosis

InDels:

Insertions or deletions

ITCLP:

International T-cell Lymphoma Project

nTFHL:

Nodal T follicular helper cell lymphoma

OS:

Overall survival

PB:

Peripheral blood

PFS:

Rogression-free survival

POD-24:

Progression within 24 months of diagnosis

SMBA:

Severe mosquito bite allergy

TFH:

T follicular helper

VAF:

Variant allele frequency

WHO:

World Health Organization

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Acknowledgements

The authors would like to thank the following institutions and pathologists for providing slides or wax blocks of consultation cases: Dr Chen Zhihong, Department of Pathology, Xiangtan Central Hospital. Dr Liu Yifei, Department of Pathology, Affiliated Hospital of Nantong University. Dr Wang Hongkun, Department of Pathology, The First Hospital of Shanxi Medical University. Dr Liu Yong and Dr Zou Wanwan, Department of Pathology, Jiangxi Cancer Hospital. Dr Sun Jirui, Department of Pathology, Baoding No.1 Central Hospital. Dr Gu Yonghui, Department of Pathology, The First Affiliated Hospital of Guangxi Medical University. Dr Long Weiguo, Department of Pathology, Affiliated Hospital of Jiangsu University. Dr Song Xudong, Department of Pathology, Affiliated Hospital of North China University of Science and Technology. Dr Han Hongxiu and Dr Xie Ping, Department of Pathology, Shanghai Tongji Hospital.

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  1. Mulan Jin and Xiaoge Zhou contributed equally to this work.

Authors and Affiliations

  1. Department of Pathology, Beijing Friendship Hospital, Capital Medical University, Beijing, China

    Xiaodan Zheng, Yuanyuan Zheng, Yanlin Zhang, Jianlan Xie, Xiaojing Teng, Kuo Bi, Lan Sun, Xiaowen Huang & Xiaoge Zhou

  2. Department of Pathology, Beijing Chaoyang Hospital, Capital Medical University, Beijing, China

    Xiaodan Zheng & Mulan Jin

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Contributions

Xiaodan Zheng contributed to the manuscript drafting. Jianlan Xie and Yuanyuan Zheng contributed to the collection of documents. Yanlin Zhang contributed to quality assessment of tissue samples for histology, IHC and ISH. Xiaojing Teng, Kuo Bi and Lan Sun contributed to molecular detection. Xiaowen Huang contributed to image collation. Xiaoge Zhou and Mulan Jin revised the manuscript. All authors read and approved the final manuscript. Xiaoge Zhou and Mulan Jin were the co-corresponding authors.

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13027_2024_627_MOESM1_ESM.png

Supplementary Figure S3: Summary of TCR gene rearrangement 6-tube results from adult systemic EBV+ T-LPD. TCR GA shows monoclonal rearrangements, TCRD (Green and blue fluorescent signals within the effective detection range of 120-280bp) shows oligoclonal rearrangements, TCRGB (Green and black fluorescent signals within the effective detection range of 80-140bp, 160-220bp), TCRBA (black and blue fluorescent signals within the effective detection range of 240-285bp), TCRBB (blue fluorescent signals within the effective detection range of 240-285bp) and TCRBC (black and blue fluorescent signals within the effective detection range of 170-210bp, 285-325bp) show polyclonal rearrangements

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Zheng, X., Zheng, Y., Zhang, Y. et al. An exploratory study on the differential diagnostic indicators between adult systemic EBV-positive T-cell lymphoproliferative disorders and angioimmunoblastic T-cell lymphoma with multiple EBV infections. Infect Agents Cancer 20, 3 (2025). https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s13027-024-00627-x

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  • DOI: https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s13027-024-00627-x

Keywords

Infectious Agents and Cancer

ISSN: 1750-9378

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