Quantification of Epstein-Barr Virus DNA Is Helpful for Evaluation of Chronic Active Epstein-Barr Virus Infection

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1 Tohoku J. Exp. Med., 2012, 227, Quantification of EBV DNA in Chronic Active EBV Infection 307 Quantification of Epstein-Barr Virus DNA Is Helpful for Evaluation of Chronic Active Epstein-Barr Virus Infection Yuichi Sakamoto, 1 Yasushi Mariya 1 and Kohmei Kubo 2 1 Department of Laboratory Medicine, Aomori Prefectural Central Hospital, Aomori, Japan 2 Department of Hematology, Aomori Prefectural Central Hospital, Aomori, Japan Chronic active Epstein-Barr virus infection (CAEBV) presents with chronic or recurrent infectious mononucleosis-like symptoms, such as low-grade fever, liver dysfunction, lymphadenopathy, and hepatosplenomegaly. Immunological methods are useful for the diagnosis of viral infections. However, CAEBV patients do not necessarily have high titers of Epstein-Barr virus (EBV)-specific antibodies. Hosts that are immunocompromised after hematopoietic stem cell transplantations sometimes suffer from systemic EBV-associated hemophagocytic lymphohistiocytosis (EBV-HLH) and EBV-positive lymphoma. Patients with EBV-associated diseases are often diagnosed by analyses of bone marrow. Cytomegalovirus (CMV) can cause serious pneumonia or retinitis in immunocompromised hosts. In order to noninvasively understand the clinical status of patients with EBV-associated diseases, we conducted real-time polymerase chain reaction (PCR) methods in their peripheral blood in order to quantify EBV and CMV DNA levels, which reflect viral activity. Here, we describe a 30-year-old Japanese female patient with CAEBV. The patient had repeated fever, fatigue, and liver dysfunction. The histopathological results of liver biopsies were positive for EBV-encoded RNA-1. Acute hepatitis was associated with the EBV infection. The whole-blood EBV DNA levels were high and above copies/ml. After immunosuppressive and antiviral therapies, EBV DNA levels lowered. However, she had to receive bone marrow transplantation because of her EBV-HLH. As the number of lymphocytes increased in the post-transplantation period, EBV DNA levels gradually increased again. The simultaneous detection of CMV DNA was more sensitive than the CMV antigenemia test that is often used to diagnose CMV infections. Unfortunately, the patient died due to a fungal infection. Observing EBV DNA levels closely with real-time quantitative PCR methods is helpful for evaluating the changes in the clinical course. Keywords: chronic active Epstein-Barr virus infection; Cytomegalovirus; Epstein-Barr virus; Japan Molecular Center of Excellence; real-time quantitative polymerase chain reaction Tohoku J. Exp. Med., 2012, 227 (4), Tohoku University Medical Press Epstein-Barr virus (EBV) infections sometimes lead to the clinical syndrome known as infectious mononucleosis (IM) in young adults. IM is a benign disease and is associated with proliferation of B cells. About 90% of all adults acquire EBV and are seropositive to EBV (Cohen 2000; de Boer et al. 2003). Chronic active EBV infection (CAEBV) presents with chronic or recurrent IM-like symptoms, such as low-grade fever, liver dysfunction, lymphadenopathy, and hepatosplenomegaly. Other symptoms include pancytopenia, disseminated intravascular coagulation (DIC), and chronic illnesses that cannot be explained. Patients with CAEBV exhibit abnormal patterns of anti-ebv antibodies with high titers to the EBV-capsid antigen (VCA) and the early antigen (EA) and low titers to the EBV nuclear antigen (EBNA). Increased levels of the EBV genome are detected in affected tissues and peripheral blood (Straus 1988; Kimura et al. 2001; Wakiguchi 2002; Okano et al. 2005). EBV infects T and natural killer cells to cause systemic lymphoproliferative diseases, such as CAEBV, EBVassociated hemophagocytic lymphohistiocytosis (EBV- HLH), and EBV-positive lymphoma (Kasahara et al. 2001; Imashuku 2007). These diseases have a poor prognosis. We previously measured the mrna levels of several genes in patients with hematological malignancies using quantitative real-time polymerase chain reaction (RQ-PCR) methods in our hospital (Sakamoto et al. 2009, 2011). Here, we describe a 30-year-old Japanese female patient who was diagnosed with CAEBV after presenting with recurrent IM-like symptoms. Because of hematopoietic stem cell transplantation, the clinical course of this patient was divided into the following 2 phases: CAEBV in the pretransplantation period and EBV-HLH in the post-trans- Received February 6, 2012; revision accepted for publication June 29, doi: /tjem Correspondence: Yuichi Sakamoto, Department of Laboratory Medicine, Aomori Prefectural Central Hospital, Higashitsukurimichi, Aomori-shi, Aomori , Japan ys@jichi.ac.jp 307

2 308 Y. Sakamoto et al. plantation period. RQ-PCR methods for determining EBV and CMV DNA levels were used to understand the patient s condition and to determine a treatment strategy. Materials and Methods DNA extraction Samples of whole blood were collected with EDTA from the patient. DNA was extracted from 200 μl of whole blood using QIAmp DNA Blood Mini Kits (QIAGEN GmbH, Hilden, Germany). The quality of DNA was checked by a spectrophotometer-optical density instrument, the Gene Quant pro (GE Healthcare Biosciences, Tokyo, Japan). Quantitative real-time PCR and data analysis RQ-PCR and fluorescence measurements were performed with a COBAS TaqMan48 instrument (Roche Diagnostics, Basel, Switzerland). The Japan Molecular Center of Excellence (JMCoE; Tokyo, Japan) EBV primer probe set, the JMCoE DNA master mix, and 50 μl of extracted DNA were used. The fluorescence probe was labeled with reporter dye at the 5 -end and with quencher dye at the 3 -end. RQ-PCR was performed on the COBAS TaqMan48 instrument (Roche Diagnostics) as follows: denaturation at 95 C for 2 min; 16 cycles of denaturation at 95 C for 15 sec; annealing at 62 C for 44 sec; extension at 40 C for 2 min, and final extension at 40 C for 2 min. Corrective calculations with JMCoE standards included 10 2, 10 3, and 10 4 copies/ml of standards. The resulting data were analyzed with UCDAS software (Roche). Immunohistochemical analysis Formalin-fixed and paraffin-embedded sections of tissue were deparaffinized and rehydrated. Sections were stained with primary antibodies against CD3 (clone 2GV6, prediluted, Roche), CD4 (clone 1F6, 1:10, Novocastra, Newcastle, UK), CD8 (clone SP57, prediluted, Roche), CD79a (clone SP18, prediluted, Roche), and Granzyme B (clone GrB-7, 1:50, Dako UK Ltd., Cambridgeshire, UK). Those incubated with anti-phospho-specific probes were incubated at optimal temperatures according to the manufacturer s protocols. Sections were then stained with biotinylated secondary antibodies, and the antibody complexes were detected with the avidin-biotin complex method using 3 -diaminobenzidine as the chromogen and hematoxylin as the counterstain. Positive and negative controls were run in parallel with the samples. Detection of EBV-encoded small RNA 1 by in situ hybridization In situ hybridization (ISH) with EBV-encoded RNA-1 (EBER- 1) oligonucleotides was performed in order to detect EBV small RNA that were expressed in latently infected cells in formalin-fixed and paraffin-embedded sections using Ribo Map Kits and Blue Map Kits (Roche) in accordance with the manufacturer s instructions. Sequences of 2 oligo RNA probes that were labeled with digoxigenin for EBER-1 detection were as follows: sense, 5 -GUCUCCUCC CUAGCAAAACC-3 ; antisense, 5 -AACCACAGACAC CGUCCUCA-3. Sequences of sense probes are complementary sequences of antisense probes. The slides were incubated at 37 C for 6 h before being washed with probe wash and developed with an ISH detection system (Ventana Discovery, Roche). Ethical considerations This protocol was approved by the ethical committee of Aomori Prefectural Central Hospital. Clinical Findings In February 2009, a 30-year-old Japanese woman complained of liver dysfunction. Her past history included 2 incidents of liver dysfunction of unknown origin. Doctors performed evaluations of the liver dysfunction. A biopsy of the liver that was performed in the hospital showed positive cytotoxic T lymphocytes and highly positive EBV-DNA ( copies/μg RNA). She was diagnosed with CAEBV and was sent to our hospital to receive therapy. Upon admission, the patient had liver dysfunction and hypergammaglobulinemia with the following blood chemistry results: alkaline phosphatase, 3,361 IU/L; aspartate aminotransferase, 773 IU/L; alanine aminotransferase, 426 IU/L; lactate dehydrogenase, 707 IU/L; IgA, mg/dl; IgG, 1,925.3 mg/dl; and IgM, mg/dl. She was suspected of having some chronic inflammatory or viral liver diseases. She was negative for Hepatitis B virus, Hepatitis C virus, and Human immunodeficiency virus infection. Enzyme-linked immunosorbent assays revealed anti-vca IgG, anti-ea IgG, and anti-ebna IgG against EBV were 1:80, 1:160, and 1:10, respectively. These anti-ebv antibodies indicated a reactivation of EBV. Anti-Cyto megalovirus (CMV) IgG and IgM were positive. The whole-blood EBV DNA levels were very high and above copies/ml (cutoff value, copies/ ml). The histopathological findings of the liver biopsy performed upon admission are shown in Fig. 1. There was cellular infiltration with lymphocytes and neutrophils in the Glissonian pedicles. Severe liver damage with infiltration of inflammatory cells indicated acute hepatitis. Most of the lymphocytes in the Glissonian pedicles and hepatic lobules were CD3-positive T cells. There were few CD79a-positive cells that were highly specific for B cells. There were more CD4-positive T cells than CD8-positive T cells. Granzyme B, the cytotoxic serine protease from CD8-positive T cells, was positive in about 30% of the lymphocytes. ISH with an EBER-1 probe showed the infiltration of small lymphocytes that were positive for EBER-1 in the portal area. This hepatitis was caused by EBV-infected lymphocytes. These results showed that the EBV infection caused severe hepatitis with many lymphocytic infiltrates. The patient was treated with immunosuppressive therapy, including cyclosporine and prednisolone. Optimal doses of acyclovir, ganciclovir, and foscarnet were used during her clinical course. The effectiveness of these drugs could be observed on the viral loads. During the clinical course, a subarachnoid hemorrhage occurred, and antiviral treatment was continued. The levels of EBV DNA decreased. However, she developed fever again. Bone marrow aspiration biopsy data showed EBV-HLH. Therefore, an allogenic bone marrow transfusion (BMT) was performed. Graft failure was followed by peripheral blood stem cell transfusion (PBSCT). EBV and CMV DNA levels were low during the early phase in the post-trans-

3 Quantification of EBV DNA in Chronic Active EBV Infection 309 Fig. 1. Histological findings of liver biopsy. (A1-3) (Hematoxylin & Eosin, Azan and Ag stain, 400 ): There was cellular infiltration with lymphocytes, eosinophils, and neutrophils in the Glissonian pedicles and hepatic lobules. Eosinophils were scattered among the hepatocytes (A1, arrows). Steatosis was accompanied by vascular congestion (A2-3, arrows). The infiltrated lymphocytes and neutrophils and apoptosis of the hepatocytes indicated acute interface hepatitis around the Glissonian pedicles (A1-3). (B) CD3 + stain, 400 : Most of the lymphocytes were CD3-positive T cells in the Glissonian pedicles and hepatic lobules. (C) CD79a + stain, 400 : There were few CD79a-positive B cells. (D) CD4 +, (E) CD8 + stain, 400 : There were more CD4-positive T cells than CD8-positive T cells. (F) Granzyme B, 400 : Granzyme B was positive in about 30% of the lymphocytes. (G) EBV-encoded RNA-1 (EBER-1) in situ hybridization, 400 : EBER-1 positive signals were observed in the lymphocytes (arrow) in the Glissonian pedicles and hepatic lobules, suggesting Epstein-Barr virus (EBV) infection. The EBER-1-positive cells appeared to be T cells. These results showed that EBV infection caused severe hepatitis with infiltration of lymphocytes. plantation period. However, EBV DNA levels gradually increased as lymphocytes increased. A CMV antigenemia test, which is often used to diagnose CMV infection, was negative. QR-PCR methods were too sensitive to detect CMV DNA levels above the cutoff. Nevertheless, even though her EBV and CMV DNA levels were observed closely, she finally died due to a fungal infection and multiple organ failure (Fig. 2). An autopsy showed the existence of pericarditis, bacterial and fungal infection in both lungs, liver failure, acute renal failure, and DIC. CD3-positive T cells infiltrated the lymph nodes, liver, and spleen. The bone marrow was hypoplastic. The EBER-1-positive cells corresponded to CD3-positive T cells. CD3-positive T cells infiltrated the liver, spleen, and lymph nodes. There were a few EBER-1- positive T cells in the spleen. There were more CD8- positive T cells, most of which were cytotoxic T cells, than CD4-positive T cells. The accumulation of fibrin in the pericardium sac suggested pericarditis. There were pulmonary congestion and fungal colonies in both lungs. Necrosis in the hepatic lobules caused liver failure. Necrosis of the tubular epithelium in the kidney suggested acute renal failure. Discussion CAEBV infection, which is a disorder with a rare pathogenesis, is more prevalent in East Asian countries where patients have a high morbidity and mortality (Kimura

4 310 Y. Sakamoto et al. Fig. 2. Clinical course and viral load. After immunotherapy and anti-virus treatment were begun, EBV DNA levels decreased. On the 87 th day, EBV DNA was not detected after allo-bmt. After allo-pbsct against graft failure, EBV DNA and lymphocytes increased. CMV DNA was detected since the early stage after hematopoietic stem cell transplantation. Multiple organ failure with fungal infection caused her to die on 178 th day. ACV, Acyclovir; Allo-BMT, allogenic bone marrow transplantation; Allo- PBSCT, allogenic peripheral blood stem cell transplantation; CYA, cyclosporin; CMV IG, Cytomegalovirus immunoglobulin; FOS, foscarnet; PSL, prednisolone; WBC, white blood cell. et al. 2001; Shibuya et al. 2003). If a patient presents with a series of unexplained symptoms that are suspicious of CAEBV, EBV DNA and EBER-1 levels should be tested. CAEBV patients have high titers to EBV-VCA-IgG and EBV-EA-IgG and low titers to EBNA (Straus 1988; Wakiguchi 2002; Okano et al. 2005). RQ-PCR analysis of EBV DNA in whole blood is a valuable diagnostic method for CAEBV. Viral loads of more than 10 3 to 10 4 copies/μg DNA have been demonstrated in the peripheral blood mononuclear cells of patients with CAEBV. The patients with more than 10 1 copies/μg of peripheral blood mononuclear cell DNA showed a CAEBV state (Kimura et al. 1999). Previous studies have reported that not all patients had high VCA-IgG titers (Kimura et al. 2001; de Boer et al. 2003; Shibuya et al. 2003). Okano et al. (2005) reported that anti-ebv antibodies with raised anti-vca and anti-ea levels ordinarily consist of VCA-IgG 1:640 and EA-IgG 1:160; positive IgA antibodies to VCA and/or EA are often demonstrated in the proposed guidelines and are recommended specific tests. Such immunological abnormalities have also been detected in some of their parents, suggesting that CAEBV might be associated with certain genetic background-related immunological disorders (Fujieda et al. 1993). However, the EBV-antibody titers of this patient did not meet these criteria (Okano et al. 2005). The clinical course of this patient was divided into 2 phases. The former phase was the pretransplantation period. The patient in this case was an immunocompetent host who had no specific history. EBV-infected T cells and natural killer cells have been found to play an important role in CAEBV patients (Kawa et al. 2002). A disturbance in the host-virus balance and helper T cell imbalance has been suggested to be related to CAEBV. Most patients are treated with antiviral agents (acyclovir and ganciclovir), cytokines (Interferon-α and Interleukin-2), and chemotherapy (corticosteroids, etoposide, and cyclosporine). Recently, promising results have been obtained with autologous EBV-specific cytotoxic T cell therapy and hematopoietic cell transplantation (Savoldo et al. 2002; Gotoh et al. 2008; Sato et al. 2008). The chemotherapy reduced the EBV DNA levels of this patient. However, the patient became immunocompromised, and the CMV was reactivated. The RQ-PCR method was highly positive for CMV DNA. The latter phase was the post-transplantation period. This period indicated that the patient was immunocompromised. Allogeneic peripheral blood or bone marrow stem cell transplantation can be the treatment of choice because successful results were reported in CAEBV patients (Okamura et al. 2000; Kawa et al. 2002; Taketani et al. 2002). The lymphocytes of this patient after BMT and

5 Quantification of EBV DNA in Chronic Active EBV Infection 311 PBSCT gradually increased. Whereas RQ-PCR could detect CMV infection, the CMV antigenemia test was negative in a few blood cells within the post-transplantation period. Clinical physicians can start antiviral therapy by the early detection of CMV infection. Conclusion The quantification of EBV and CMV DNA was helpful for understanding the clinical course and for determining a treatment strategy in a case of CAEBV. Conflict of Interest All authors declare no conflict of interest. References Cohen, J.I. (2000) Epstein-Barr virus infection. N. Engl. J. Med., 343, de Boer, M., Mol, M.J., Bogman, M.J., Galama, J.M. & Raymakers, R.A. (2003) Chronic active Epstein-Barr virus infection in an adult with no detectable immune deficiency. Neth. J. Med., 61, Fujieda, M., Wakiguchi, H., Hisakawa, H., Kubota, H. & Kurashige, T. (1993) Defective activity of Epstein-Barr virus (EBV) specific cytotoxic T lymphocytes in children with chronic active EBV infection and in their parents. Acta Paediatr. Jpn., 35, Gotoh, K., Ito, Y., Shibata-Watanabe, Y., Kawada, J., Takahashi, Y., Yagasaki, H., Kojima, S., Nishiyama, Y. & Kimura, H. (2008) Clinical and virological characteristics of 15 patients with chronic active Epstein-Barr virus infection treated with hematopoietic stem cell transplantation. Clin. Infect. Dis., 46, Imashuku, S. (2007) Systemic type Epstein-Barr virus-related lymphoproliferative diseases in children and young adults: challenges for pediatric hemato-oncologists and infectious disease specialists. Pediatr. Hematol. Oncol., 24, Kasahara, Y., Yachie, A., Takei, K., Kanegane, C., Okada, K., Ohta, K., Seki, H., Igarashi, N., Maruhashi, K., Katayama, K., Katoh, E., Terao, G., Sakiyama, Y. & Koizumi, S. (2001) Differential cellular targets of Epstein-Barr virus (EBV) infection between acute EBV-associated hemophagocytic lymphohistiocytosis and chronic active EBV infection. Blood, 98, Kawa, K., Okamura, T., Yasui, M., Sato, E. & Inoue, M. (2002) Allogeneic hematopoietic stem cell transplantation for Epstein-Barr virus-associated T/NK-cell lymphoproliferative disease. Crit. Rev. Oncol. Hematol., 44, Kimura, H., Morita, M., Yabuta, Y., Kuzushima, K., Kato, K., Kojima, S., Matsuyama, T. & Morishima, T. (1999) Quantitative analysis of Epstein-Barr virus load by using a real-time PCR assay. J. Clin. Microbiol., 37, Kimura, H., Hoshino, Y., Kanegane, H., Tsuge, I., Okamura, T., Kawa, K. & Morishima, T. (2001) Clinical and virologic characteristics of chronic active Epstein-Barr virus infection. Blood, 98, Okamura, T., Hatsukawa, Y., Arai, H., Inoue, M. & Kawa, K. (2000) Blood stem-cell transplantation for chronic active Epstein-Barr virus with lymphoproliferation. Lancet, 356, Okano, M., Kawa, K., Kimura, H., Yachie, A., Wakiguchi, H., Maeda, A., Imai, S., Ohga, S., Kanegane, H., Tsuchiya, S., Morio, T., Mori, M., Yokota, S. & Imashuku, S. (2005) Proposed guidelines for diagnosing chronic active Epstein- Barr virus infection. Am. J. Hematol., 80, Sakamoto, Y., Mariya, Y., Sasaki, S., Teshiromori, R., Oshikiri, T., Segawa, M., Ogura, K., Akagi, T., Kubo, K., Kaimori, M. & Funato, T. (2009) WT1 mrna level in periopheral blood is a sensitive biomarker for monitoring minimal residual disease in acute myeloid leukemia. Tohoku J. Exp. Med., 219, Sakamoto, Y., Mariya, Y., Oshikiri, T., Sasaki, S., Segawa, M., Teshiromori, R., Ogura, K., Akagi, T., Kaimori, M. & Kubo, K. (2011) Monitoring twenty-six chronic myeloid leukemia patients by BCR-ABL mrna level in bone marrow: a single hospital experience. Acta Med. Okayama, 65, Sato, E., Ohga, S., Kuroda, H., Yoshiba, F., Nishimura, M., Nagasawa, M., Inoue, M. & Kawa, K. (2008) Allogeneic hematopoietic stem cell transplantation for Epstein-Barr virusassociated T/natural killer-cell lymphoproliferative disease in Japan. Am. J. Hematol., 83, Savoldo, B., Huls, M.H., Liu, Z., Okamura, T., Volk, H.D., Reinke, P., Sabat, R., Babel, N., Jones, J.F., Webster-Cyriaque, J., Gee, A.P., Brenner, M.K., Heslop, H.E. & Rooney, C.M. (2002) Autologous Epstein-Barr virus (EBV)-specific cytotoxic T cells for the treatment of persistent active EBV infection. Blood, 100, Shibuya, A., Tsuchihashi, T., Watanabe, M., Nakazawa, T., Takeuchi, A., Sakurai, K., Mitomi, H. & Saigenji, K. (2003) Severe chronic active Epstein-Barr virus infection associated with multiple necrotic lesions in the liver. Hepatol. Res., 25, Straus, S.E. (1988) The chronic mononucleosis syndrome. J. Infect. Dis., 157, Taketani, T., Kikuchi, A., Inatomi, J., Hanada, R., Kawaguchi, H., Ida, K., Oh-Ishi, T., Arai, T., Kishimoto, H. & Yamamoto, K. (2002) Chronic active Epstein-Barr virus infection (CAEBV) successfully treated with allogeneic peripheral blood stem cell transplantation. Bone Marrow Transplant., 29, Wakiguchi, H. (2002) Overview of Epstein-Barr virus-associated diseases in Japan. Crit. Rev. Oncol. Hematol., 44,