Epstein–Barr virus latency in kidney specimens from transplant recipients

Luis Fernando Arias1, Susana Hernández3, Dolores Prats2, Ana Sanchez-Fructoso2, María Márques2, Teresa Alvarez1, Alberto Barrientos2 and Julia Blanco1

1Department of Pathology and 2Department of Nephrology, Hospital Clínico San Carlos, Madrid and 3National Center for Oncologic Research (CNIO), Madrid, Spain

Correspondence and offprint requests to: Julia Blanco, Department of Pathology, Hospital Clínico San Carlos, C/ de Martín Lagos s/n 28040, Madrid, Spain. Email: mblanco.hcsc{at}salud.madrid.org



   Abstract
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
Background. Epstein–Barr virus (EBV) infection is common in immunosuppressed patients and can lead to life threatening lymphoproliferative diseases. Small numbers of cells infected by EBV have been detected in human tissues, transplanted or non-transplanted. Little is known about EBV latency in the allograft kidneys of patients without post-transplant lymphoproliferative disease (PTLD). The aims of this study were to look for the presence of EBV-encoded small RNAs (EBER) in allograft kidneys and to quantify their expression.

Methods. We analysed 62 allograft nephrectomies and 20 native kidneys to determine the presence of EBV; we also quantified its expression and calculated its ratios to CD45 and CD20 cells. The techniques used were: tissue microarray, EBER-1- and 2-specific in situ hybridization and immunohistochemistry.

Results. EBER expression was detected in 30.6% of transplanted kidneys and 5% of non-transplanted kidneys. In the positive specimens, a mean of 8.2 cells/1.57 mm2 expressed the EBERs (range 1–38 cells). The ratios of EBER-positive (+) cells to CD45 or CD20 cells were 1.7 ± 2.4% (range 0.1–8.1%) and 8.4 ± 10.9% (range 0.5–34.4%), respectively. No relationship was found between anti-T-cell treatment and EBER expression in the failed allografts.

Conclusions. In failed kidney allografts, a small number of lymphocytes can express EBV latency. The number of EBER+ cells is smaller than in PTLD. Studies of functioning grafts are necessary to better understand the clinical relevance of this expression.

Keywords: EBER; Epstein–Barr virus; in situ hybridization; kidney transplantation; tissue microarray



   Introduction
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
Epstein–Barr virus (EBV) infection is associated with lymphoproliferative diseases in both non-immunocompromised and immunocompromised individuals, including recipients of organ transplant. The process entails the infection of B-lymphocytes by EBV, which leads to proliferation and transformation of infected cells. As T-cells provide immunosurveillance over EBV-infected cells [1], immunosuppressive agents used in organ transplantation tip the balance in favour of viral proliferation [2,3].

EBV-associated post-transplant lymphoproliferative disease (PTLD) develops in ~1% of renal transplant recipients, and evidence of EBV-encoded small RNA (EBER) expression is found in the majority of the constituent affected cells [4,5]. In situ hybridization (ISH) for EBERs probably is the most sensitive method for detecting EBV infection in tissues, and remains the gold standard with which to confirm that a histopathologic lesion is EBV-related [4,6]. EBER 1 and EBER 2 are small nuclear RNAs that are expressed in large numbers (up to 107 copies/cell) in cells with latent infections, thus providing an abundant target for ISH [7]. Several authors have suggested that EBERs may act to promote host cell survival and virus growth and that they may participate in some aspect of replication, transcription, or RNA processing in EBV-transformed cells [7]. Small numbers of EBV-infected cells have been detected in a variety of tissues from individuals with different inflammatory and non-inflammatory disorders [8,9]. Of the biopsies from transplanted solid organs without PTLD, 10–34% show EBER expression, while this expression is detected in <10% of lymphoid cells [4,6,10,11,12].

The aims of this study were: to look for the presence of EBER 1 and EBER 2 in transplanted kidneys without PTLD; to quantify them for a better understanding of the biology of EBV infection; and to facilitate the interpretation of EBER ISH in the diagnosis of PTLD.



   Subjects and methods
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 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
We reviewed H&E stained sections from all allograft nephrectomies at our institution, and selected the ones with non-necrotic renal tissue and lymphocytic infiltrates in order to construct a tissue microarray (TMA) according to a previous description [13]. We found 62 specimens with such characteristics, among 113 cases received between 1984 and 2001. The H&E sections were used to identify areas with relatively heavier lymphocytic infiltrates; and from the corresponding selected regions of the paraffin-embedded kidney specimens we took two cores per case, each 1 mm in diameter. The samples were precisely arrayed into a new recipient paraffin block (40 x 23 mm) with a manual tissue arrayer (Beecher Instruments, Silver Spring, MD) using an edge-to-edge spacing of 0.5 mm. With a microtome, 5 µm sections of the resulting TMA block were cut, and were subjected to histochemical, immunohistochemical and ISH procedures. Background clinical data were collected for each case. Samples from 20 nephrectomies, from non-transplanted patients which had inflammatory mononuclear infiltration also were included in the TMA. We used a specimen from a grafted kidney that had confirmed PTLD and was positive for EBER 1 and EBER 2 ISH as a positive control.

The laboratory case number for each core and its position in the TMA block were entered into a computer-generated grid for exact identification. A total of 166 cores were taken from the 83 cases included in the study (62 grafted kidneys, 20 non-transplanted kidneys, and one PTLD for ISH control).

TMA sections were stained with H&E, Masson’s trichromic, PAS and methenamine silver for histologic characterization. ISH was carried out using a commercially available probe (EBER PNA Probe/FITC. Dako, Denmark) to detect EBER 1 and EBER 2. TMA sections were deparaffinized and then were digested with proteinase K for 20 min at 37°C. The hybridization was performed at 55°C for 1.5 h. Subsequent visualization was carried out using the Dako PNA ISH Detection Kit. The slides were counterstained with Mayer’s haematoxylin. Brown granular staining was considered positive only if it was in the nucleus.

To identify CD45 and CD20 cells, immunohistochemistry was performed using monoclonal antibodies (Dako), diluted 1:200 and 1:500, respectively. The EnVisionä system (Dako) was used to locate the positive cells for colour visualization with diaminobenzidine. Quantification of the EBER-positive (EBER+), CD45 and CD20 cells in the two core cylinders (total cross sectional area: 1.57 mm2) were performed in parallel sections. The ratios of EBER+ cells to CD45 or CD20 cells were calculated using percentages.

Statistical analysis
Data are expressed as mean ± SD and ranges. Fisher’s exact test was used to compare percentages. The Student’s t-test was applied to assess the mean differences of CD45 and CD20 cells, post-transplant time, and interval between the last anti-T-cell or antiviral treatment and nephrectomy; each variable having been tested previously for normality using the Kolomogorov-Smirnov test. Differences were considered statistically significant if P was <0.05 in two-tailed distributions. Calculations were performed using SPSS® software, version 11.5 (SPSS, Chicago, IL).



   Results
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 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
The main histopathologic diagnoses found in the 62 allograft nephrectomy specimens were: acute rejection (37 cases), chronic/sclerosing allograft nephropathy (CAN) (19 cases), acute tubular necrosis (two cases) and one case each of perirenal abscess, cytomegalovirus infection, obstructive nephropathy and papillary carcinoma. The specimens were from 45 males and 17 females. Patient age when the grafted kidney was removed ranged from 19 to 71 years (mean 43 ± 14). Time between transplantation and nephrectomy ranged from 2 to 4380 days (mean 458 ± 987). The immunosuppressive treatments used were: prednisone, mycophenolate mofetil or azathioprine, and cyclosporine or tacrolimus. Patients with acute rejection received high doses of immunosuppressive therapy with pulse doses of steroids, ATG or OKT3, and optimization of cyclosporine or tacrolimus.

The main histopathologic diagnoses found in the non-transplanted nephrectomies were: renal carcinoma (seven cases), obstructive chronic nephropathy (six cases), segmental dysplasia (one case) and autosomal dominant polycystic disease (one case). The five remaining kidneys were from cadaveric donors, and had not been grafted due to severe angiosclerosis with a high percentage of glomerulosclerosis (three cases) or technical reasons [2]. Neoplastic or infectious diseases were not detected in any of them. The sources of the kidneys ranged in age from 2.5 to 85 years (mean 54 ± 38), and 14 of the 20 were men.

EBERs were demonstrated by ISH in 19 out of the 62 transplanted renal tissues (30.6%) and in one out of the 20 non-transplanted kidneys (5%) (P = 0.033). In the transplanted kidneys, the number of labelled cells ranged from 1 to 38 in the two cores of the tissue array (mean 8.2 ± 9.4), and 14 out of 19 had less than 10 labelled cells (Table 1). Based on morphologic features, nuclear labeling was found only in mononuclear leukocytes (Figure 1).


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Table 1. Kidney allografts with EBER expression

 


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Fig. 1. (a) EBER+ control: PTLD, nuclear staining in >80% of the neoplastic cells (ISH for EBERs, x200). (b) Case 9: there are numerous EBER+ cells in the interstitium (x400). (c) Case 15: dispersed EBER+ cells among EBER- lymphocytes (x400). (d) Case 16: only one EBER+ cell was identified in this case. There is prominent mononuclear infiltrate (x400).

 
The quantification of CD45 and CD20 was considered to be exact when less than 200 labelled cells were counted per core. When more than 200, evaluation was made difficult by the superposition of cellular membranes, and exact quantification was impossible. The total number of CD45 or CD20 cells in the different groups and the comparison of means are shown in Table 2. In transplanted patients with EBER expression, the ratio of EBER+ cells to CD45 cells ranged from 0.1 to 8.1% (mean 1.7 ± 2.4). In 11 cases this ratio was <=1%. The ratio of EBER+ cells to CD20 cells ranged from 0.5 to 34.4% (mean 8.4 ± 10.9) (Table 1). The post-transplant intervals are shown in Table 2. In three positive cases, the post-transplant period was shorter than 2 weeks.


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Table 2. Comparative characteristics in transplanted and non-transplanted cases

 
The EBER+ control with PTLD contained abundant EBER labelled cells (>80% of the total lymphoid cell population). The non-transplanted EBER+ case had five labelled cells (2.3% of the total of CD45 cells counted and 7% of the CD20 cells).

Of the 19 patients with EBER+ cells in their grafts 12 had received anti-T-cell therapy (five with ATG, seven with OKT3) in the 6 months preceding nephrectomy, whereas 21 out of the 43 patients without EBER expression had received that therapy (12 with ATG, nine with OKT3) (P = 0.4). The interval between the last anti-T-cell treatment and the nephrectomy ranged from zero to 40 days (mean 13.6 ± 12.8) in EBER+ cases, and from 1 to 115 days (mean 29.8 ± 30.4) in EBER-negative (EBER-) cases (P = 0.042). Of the 19 patients with EBER expression five and eight of the 43 without EBER expression had received antiviral treatments (P = 0.513). The interval between the last antiviral treatment and nephrectomy ranged from 0 to 30 days in EBER+ cases (mean 8.2 ± 12.9) and from 0 to 120 days in EBER- cases (mean 34.5 ± 41.0) (P = 0.13).

The number of cases with histopathologic diagnoses of acute rejection or CAN in the allograft nephrectomy specimens was not significantly different between EBER+ and EBER- cases (P = 1.0). There were no differences between patients with high EBER expression (EBER+ cell/CD20 cell ratio >10%) and low expression with regard to clinical, histopathologic and immunohistochemical characteristics. Follow-up after nephrectomy ranged from 15 days to 14 years. Of the patients studied, five EBER+ and 16 EBER- ones received subsequent transplants in our centre, and none has developed PTLD.



   Discussion
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 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
After primary infection, EBV establishes a lifelong, persistent infection and elicits a reactive proliferation of EBV-specific T-cells. The immune response to EBV-infected lymphocytes is complex, involving both humoral and cell-mediated immune mechanisms. Despite clinical recovery and specific immune responses to EBV the virus is not eliminated from the host. EBV shares the capacity for latency with other members of the herpes virus group [1], and in its latent state it is not easily detected in normal individuals. In EBV-seropositive immunocompetent persons, an estimated one in 105–106 of B-cells contains the latent EBV genome [14]. Taking advantage of the abundance of EBER transcripts in latently infected cells, we analysed the expression of EBERs by using ISH on kidney samples from 62 transplanted and 20 non-transplanted patients. The available literature points to a variable expression of EBV in tissue samples from non-PTLD specimens. Niedobitek et al. [15] reported 62% positivity in 29 cases of reactive lymphadenitis, and the relative number of labelled cells was low—although in some cases it could be as high as 200 cells/0.5 cm2. The same authors reported EBER expression in each of nine cases with HIV-associated lymphadenitis, and the number of positive cells was >1000/0.5 cm2. Chang et al. [16] did not detect EBV in any of the 201 non-lymphoid tissue sections from their non-transplanted patients. In transplanted livers, Barkholt et al. [12] detected EBER expression in 10 of 18 (56%) samples from patients without PTLD. Hubscher et al. [11] found EBER positivity in 19.4% of 67 liver allografts and in 42.6% of 47 non-transplanted livers with inflammatory diseases. The proportion of labelled lymphocytes was <1% in all of the cases studied. Finn et al. [10] failed to show lymphocytes positive for EBER in biopsy samples of non-lymphoid tissues from seropositive recipients of kidneys, livers, lungs and hearts.

At high infective loads, up to 25% of EBV-exposed B-lymphocytes express EBV infection [17], and between one in 105 and one in 107 circulating lymphoid cells are permanently infected [18]. In our study and others [10,11], the identification of EBV expression shows that the proportion of infected cells in transplanted organs exceeds that observed in peripheral blood lymphocytes. A likely explanation for this finding could be the proliferation of EBV-infected cells as a consequence of immunosuppression. The finding could also reflect a migration of infected circulating cells to the graft.

Our observations agree with previous studies [6,10,11,12], and indicate that low-grade EBER expression is common in transplanted patients. We have shown that EBER expression in transplanted kidneys occurs at a higher rate than in non-transplanted kidneys (30.6 vs 5%). In agreement with Randhawa et al. [4], we also found that EBER+ cells made up <10% of leukocytes. In contrast, in cases with PTLD EBER is expressed in 50% or more of the cells studied [4].

We determined the ratios of EBER+ cells to CD45 or CD20 cells, because lymphocytes, specifically B cells, are the main cells that express EBV latency. The EBV receptor has been identified as the receptor for the ‘d’ region of the third component of complement C3d (also known as CR2 or CD21) in the B-cell surface [19]. A small proportion of immature T-cells and non-B, non-T lymphocytes can express this receptor and be infected by the virus [1,19]. Rarely, EBV-associated T-lymphomas have been reported [20]. It is not possible for us to specify the exact type of infected lymphoid cells, because we did not perform double immunolabelling. The mean number of CD45 cells in our samples of kidney allografts was significantly higher in the EBER+ cases than in the EBER- ones (P = 0.006), which can be a factor influencing the results. EBER detection is more likely in tissues with larger numbers of lymphoid cells, specifically B-cells. Individuals with latent EBV infections have small numbers of circulating infected cells, and the number increases along with the total number of lymphocytes [15]. However, in our study there were no statistical differences between the mean numbers of CD20 cells in the different groups.

None of the cases developed PTLD; still, it is not possible to determine whether or not these patients were at a higher risk of developing PTLD, due to the fact that immunosuppressive therapy was stopped after nephrectomy. In the present study, the histopathologic diagnoses in kidney tissues from EBER+ and EBER- cases do not suggest an association between EBV infection and acute rejection or CAN. Furthermore, in our study with failed grafts, it is not possible to determine if EBV infection is associated with graft dysfunction. In a previous study of liver transplanted patients, Hubscher et al. [11] showed that small numbers of EBV-infected cells are present in a wide range of liver diseases, and that the occasional identification of infected cells in post-transplant liver biopsies does not predict progression to PTLD. They were unable to show an obvious association between EBV infection and graft dysfunction. In contrast, in transplantations of the small intestine, Finn et al. [10] believe that it is rare to detect EBER+ cells in intestinal biopsies; but also that when found, they reflect decreased immunodestruction and could portend the development of PTLD. Randhawa et al. [6] demonstrated the expression of EBER 1 in 71% of liver samples from their patients who subsequently developed PTLD, suggesting that such expressions may permit early identification of patients at risk for PTLD.

We had three EBV-infected specimens obtained as early as the first 2 weeks after transplantation. The significance of this fact is unclear; however, it could reflect the intensity of cellular immunosuppression [12]. In our study, the number of patients with anti-T-cell treatment and patients with antiviral therapy among EBER+ and EBER- cases were not statistically different. Barkholt et al. [12] reported, however, that liver transplanted patients with anti-CD3-cell antibody (OKT3) treatment tended to develop an EBV infection more often than patients without anti-CD3 therapy. In our cases the interval between the last anti-T-cell treatment and nephrectomy was significantly shorter in EBER+ cases. Perhaps more recent intense immunosuppression facilitates EBV detection, but we do not know the exact relevance of this finding.

In this study, we do not have adequate serologic data to determine if the allograft recipients had a primary or a secondary EBV infection. EBV-specific antibodies are not routinely measured at our institution.

Finally, all the samples in our study are grafts that failed; therefore, the data only indicate the prevalence of EBER+ cells in failed kidney allografts, not in allografts in general. More studies of functioning grafted kidneys are necessary to better understand EBV graft infection.

In summary, EBER expression in small numbers can be common in transplanted kidneys without PTLD, and that may be due to the proliferation of EBV-infected cells resulting from immunosuppression. Although EBER+ cells were more common in grafted kidneys than in native kidneys, PTLD was not found to have occurred after stopping immunosuppressive therapy. The increased EBER expression in grafted tissue does not presently have diagnostic or therapeutic implications, but it is an important finding which raises questions about EBV latency and replication in transplant recipients, and it can help us differentiate PTLD from other inflammatory conditions.



   Acknowledgments
 
We thank Miss Lydia Sanchez, National Center for Oncologic Research (CNIO), Madrid, Spain, for the technical support in the preparation of tissue arrays and ISH elaboration; Mrs Angeles Bermúdez for her histochemical work; Miguel A. Zuluaga for assistance with statistical analysis; and Miss Heather Rogers for assistance with manuscript preparation. Luis F. Arias is a PhD candidate supported by the University of Antioquia, Medellín, Colombia.

Conflict of interest statement. None declared.



   References
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 

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Received for publication: 20.12.02
Accepted in revised form: 13. 6.03





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