Epstein-Barr Virus Infection Associated With Bone Marrow Fibrin-Ring Granuloma

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1 Hematopathology / EBV-Associated BM Fibrin-Ring Granuloma Epstein-Barr Virus Infection Associated With Bone Marrow Fibrin-Ring Granuloma Hee-Jung Chung, MD, Hyun-Sook Chi, MD, Seongsoo Jang, MD, and Chan-Jeoung Park, MD Key Words: Epstein-Barr virus; EBV; Bone marrow; Fibrin-ring granuloma; Clinical course; Hemophagocytic lymphohistiocytosis DOI: /AJCPB7SX7QXASPSK Abstract We previously reported 24 cases of marrow fibrin-ring granuloma (FRG) encountered in 1 institution and concluded that, contrary to previous studies showing marrow FRG as a diagnostic marker for Q fever, Epstein-Barr virus (EBV) was the most common proven cause of such FRG. The present study characterized patients with EBV-associated marrow FRG. We retrospectively reviewed 17 cases of EBV-associated FRG (43% of 40 cases with marrow FRG) diagnosed by bone marrow biopsy. Patients with EBV-associated hemophagocytic lymphohistiocytosis (5/17 patients) and chronic active EBV infection (4/17) constituted 53% of patients with EBV-associated FRG. Three patients had lymphoma without marrow involvement. All patients except 1 presented first with fever; splenomegaly was the next most common clinical finding (12/17). Cytopenia was observed in the 17 cases: anemia in 16, thrombocytopenia in 9, and leukopenia in 8. Patients with EBV-associated FRG showed lower survival outcomes than did patients without EBV (median, 3.0 vs 11.8 months; P =.009). Patients with bone marrow FRG accompanied by fever require careful evaluation to discern whether active EBV infection is involved because the prognosis is poor. Fibrin-ring granuloma (FRG) has been considered to be confined to patients with Q fever because of the unique FRG morphologic features. 1-3 An epithelioid histiocyte, neutrophil, and lymphocyte infiltrate is found in the shape of a doughnut encasing a vacuole or with an empty central space, and the outer rim is surrounded by eosinophilic fibrinoid material Image However, additional studies revealed that FRG could be observed in patients with diseases other than Q fever, such as viral infections, 7,8 allopurinol hypersensitivity hepatitis, 9 Hodgkin lymphoma, and peripheral T-cell lymphoma. 10 Previously, we reviewed 24 cases of marrow FRG treated in a tertiary care hospital and concluded that Epstein-Barr virus (EBV) was the major proven cause of marrow FRG, 6 unlike what was seen in many earlier reports. The present study aimed to characterize patients with EBV-infected bone marrow (BM) and FRG and is an extension of our previous research. Materials and Methods Subjects Among patients on whom BM biopsies were performed in the Asan Medical Center, Seoul, South Korea, from 1995 to 2008, patients diagnosed with BM FRG were selected by electronic chart review. Among 26,648 patients on whom BM biopsies were performed, 40 (0.15%) showed BM FRG. Among these, 17 (43%) had EBV-associated BM FRG. This work received institutional review board approval. Determination of EBV-Associated BM FRG We retrospectively examined electronic medical records to determine likely causes of FRG. We checked patient histories for administration of allopurinol; infection by Coxiella burnetii, 300 Am J Clin Pathol 2010;133: DOI: /AJCPB7SX7QXASPSK

2 Hematopathology / Original Article A B C Image 1 Marrow fibrin-ring granuloma. A (Case 6), Multiple fibrin-ring granulomas in bone marrow clot section (H&E, 400). Epstein-Barr virus (EBV) polymerase chain reactions on marrow aspirate samples were positive. Reproduced with permission. 6 B (Case 9), EBV-encoded small RNA (EBER) seen by in situ hybridization of a bone marrow trephine biopsy specimen revealing a strongly positive reaction ( 200). C and D (Case 2), Bone marrow clot section specimen showing a fibrin-ring granuloma. Prominent eosinophilic fibrinoid material around the doughnut-shaped infiltrate of epithelioid histiocytes, neutrophils, and lymphocytes is noticeable on Masson trichrome stain (C, H&E, 400; D, Masson trichrome, 400). D cytomegalovirus, or hepatitis A virus; lymphoma; and experience of other less common infections (including those caused by bacteria in general, viruses, acid-fast bacilli, and fungi) as determined by classic culture methods or molecular techniques such as polymerase chain reaction (PCR). EBV-associated BM FRG was determined by one of the following criteria within 3 days of BM biopsy. The criteria were positivity for both Epstein-Barr nuclear antigen (EBNA) by serology and EBV by PCR from BM aspirates, or positive serology for EBNA and EBV-encoded small RNA (EBER) by in situ hybridization of BM samples spread on trephine biopsy slides. 8 Qualitative PCR followed the method of Niesters and colleagues. 11 Prognosis of Patients With EBV-Associated BM FRG We analyzed the survival probability of patients with EBV-associated BM FRG by the Kaplan-Meier approach, using SPSS (version 13.5, SPSS, Chicago, IL). Between-group comparisons were made using the log-rank test with pairwise analysis over strata, and statistical significance was defined when the P value was less than.05. Am J Clin Pathol 2010;133: DOI: /AJCPB7SX7QXASPSK 301

3 Chung et al / EBV-Associated BM Fibrin-Ring Granuloma Results Clinical and Laboratory Characterization of EBV-Associated BM FRG The median age of patients with EBV-associated BM FRG was 42.4 years (range, 3-70 years). Of all patients, 16 (94%) were male and 1 (6%) was female. The most common symptoms were fever (16/17 [94%]), followed by myalgia (5/17 [29%]), chills (4/17 [24%]), and gastrointestinal symptoms (2/17 [12%]). Based on radiography and physical examination, splenomegaly was observed in 12 (71%) of 17 cases, hepatomegaly in 11 (65%), and lymph node enlargement in 7 (41%). Among laboratory findings, cytopenia was the most common, and increases in alanine aminotransferase and aspartate aminotransferase levels were observed in 15 and 14 cases, respectively Table 1. Among 17 cytopenic cases, 5 (29%) showed pancytopenia and 6 (35%), bicytopenia. When cytopenias were analyzed, anemia was observed in 16 cases (94%), thrombocytopenia in 9 (53%), and leukopenia in 8 (47%). Leukocytosis was observed in 3 cases (18%). Pattern of EBV Association The 17 cases of active EBV BM infection showed variable phenotypes; 5 cases (29%) were EBV-associated hemophagocytic histiocytosis (EBV-HLH), 4 (24%) were chronic active EBV (CAEBV), and 3 (18%) were lymphoma. The remaining 5 cases were 1 each of acute EBV infection, EBV and HIV coinfection, Langerhans cell histiocytosis, EBV-associated posttransplantation lymphoproliferative disorder, and adult-onset Still disease (Table 1). In 1 case, the patient with an acute EBV infection was simultaneously positive by serology to viral capsid antigen IgM, IgG, and EBNA and to EBER by in situ hybridization tests on marrow trephine biopsy sections. Among the other 16 cases with EBV infection, 8 tested positive for EBV by PCR, for EBER using in situ hybridization, and by serology for EBNA; 4 were positive for EBV by PCR and serology for EBNA; and the remaining 4 were positive for EBV by PCR and for EBER by in situ hybridization. Prognoses of Patients With EBV-Associated BM FRG When survival probability was compared among groups, a significant difference was observed between the EBVassociated group and the non EBV-associated group (P =.009). The follow-up period was 120 months. In the EBVassociated group, 11 (65%) of 17 patients died during followup, and median patient survival was 3.0 months (95% confidence interval, months). In the non EBV-associated group, 6 (26%) of 23 patients died during follow-up, and median patient survival was 11.8 months (95% confidence interval, months). Table 1 Clinical and Laboratory Data for 17 Patients With EBV-Associated Bone Marrow Fibrin-Ring Granuloma * Clinical Feature Laboratory Data Case No./ WBC Count Hemoglobin Platelet Count Sex/Age (y) Diagnosis Fever Hepatomegaly Splenomegaly LAP (/μl) (g/dl) ( 10 3 /μl) 1/M/34 EBV-HLH , /M/60 EBV-HLH , /M/3 EBV-HLH , /M/61 EBV-HLH , /F/5 EBV-HLH + + 3, /M/37 CAEBV + + 3, /M/41 CAEBV , /M/46 CAEBV , /M/62 CAEBV , /M/50 Acute EBV infection + + 5, /M/60 AIDS + 1, /M/70 Extranodal NK/T-cell lymphoma , /M/38 Extranodal NK/T-cell lymphoma + + 2, /M/35 T-cell lymphoma on skin, positive , for EBV ISH 15/F/8 LCH , /M/36 EBV-associated PTLD , /M/62 Adult onset Still disease , ALT, alanine aminotransferase; AST, aspartate aminotransferase; CAEBV, chronic active EBV infection; EBV, Epstein-Barr virus; EBV-HLH, EBV-associated hemophagocytic lymphohistiocytosis; ISH, in situ hybridization; LAP, lymphadenopathy; LCH, Langerhans cell histiocytosis; LDH, lactate dehydrogenase; ND, not done; PTLD, posttransplantation lymphoproliferative disorder; +, present;, absent. * These data permitted diagnosis of bone marrow fibrin-ring granuloma. Laboratory values are given in conventional units; conversions to Système International units are as follows: WBC count ( 10 9 /L), multiply by 0.001; hemoglobin (g/l), multiply by 10.0; platelet count ( 10 9 /L), multiply by 1.0; AST (μkat/l), multiply by ; ALT (μkat/l), multiply by ; LDH, (μkat/l), multiply by Am J Clin Pathol 2010;133: DOI: /AJCPB7SX7QXASPSK

4 Hematopathology / Original Article Discussion A substantial number of our FRG cases (36%) also showed EBV infections. This proportion is similar to that seen in our previous review of 40 cases of BM FRG. 6 In the present study, among patients with BM FRG, only 1 patient had Q fever. The initial serum IgM titer in this patient was 1:1,024, decreasing to less than 1:16 after 12 weeks of treatment. However, as we neither measured anti C burnetii serum IgG titer in all patients nor performed PCR tests for Q fever in all patients, we cannot exclude the possibility that other patients may also have had Q fever. Clinically, Q fever manifests as a cold accompanied by pneumonia or hepatitis, and only 1 patient (with hepatic failure; detailed data not shown) showed symptoms of pneumonia or hepatitis. As C burnetii invades the liver to an extent greater than BM, the probability that we failed to detect Q fever is low, and Q fever frequency is not expected to be as high as that of liver FRG. We confirmed that active EBV infection was the most frequent cause of granuloma formation in our FRG cohort. We excluded other infectious agents such as cytomegalovirus, herpes simplex virus, Salmonella typhi, and Mycobacterium tuberculosis through serologic testing and PCR. EBV is a human herpesvirus of class 4, with a double-stranded genome of 172 kilobases. 12 EBV infection occurs worldwide, and, in South Korea, all citizens aged 10 to 15 years are serologically Laboratory Data AST (U/L) ALT (U/L) LDH (U/L) Outcome ,136 Died Died ND Died 5,094 1, Died Alive ND Died ND Died ,170 Died Died ,659 Alive Alive Alive ,011 Alive Died ND Alive ,563 Died Alive positive for EBV IgG. 13 A granulomatous lesion attributed to EBV infection was formerly described by Fiala and colleagues in These authors suggested that infectious mononucleosis detected by clinical and serologic testing could be the cause of multiple granuloma of the BM. 14 In our study, however, only 1 of 17 EBV infections was in the acute phase, whereas 11 were in chronic phases, including CAEBV. This suggests that chronic infection may be more frequent than acute infection. Four cases satisfied the diagnostic criteria for CAEBV established by Straus. 15 All 4 patients were immunocompetent and had no underlying disease. They had high fever continuously for more than 3 months and were refractory to etoposide/dexamethasone treatment. Their infections progressed rapidly, and the patients died 6, 7, 18, and 26 days after diagnosis of infection. Five patients with EBV-HLH were treated with HLH-94 or HLH-2004 chemotherapy but died at an average of 47 days after diagnosis. In summary, only 6 of 17 patients with FRG accompanied by EBV survived, corresponding to a mortality rate of 65%. The average survival duration of the 11 patients who died was very short (1.6 ± 1.8 months). In cases 8 and 14, EBV was detected by quantitative PCR from BM aspirate samples obtained at the time of diagnosis, and the virus was present at 102 copies/ml and 15,720 copies/ ml, respectively. In case 14, subsequent virus quantification in BM aspirates showed a decrease to 2,490 copies/ml after 10 days. Because patient 8 died 8 days after diagnosis, quantitative follow-up was not possible. Case 10 was suspected to have acute EBV infection despite his age (50 years). The patient was positive for viral capsid antigen IgM and IgG on serology, suggesting a primary infection, but was also positive for EBNA, which is usually observed only 2 months after EBV infection. Atypical lymphocytes were found in a peripheral blood smear. This patient complained of fever, sore throat, and lymphadenopathy. Laboratory findings and symptoms were consistent with acute or subacute EBV infection. In Korea, the first report of FRG in the BM and multiple granulomatous liver lesions in a patient with an active EBV infection was published in 1995 by Kang and colleagues 16 of our group. Although the FRG morphologic features were atypical, infiltration of multiple granuloma and fibrin strands surrounding a small vacuole was observed, and BM examination revealed an increase in mature histiocytes phagocytosing various types of hematopoietic cells. 16 In addition, lymphocyte infiltrations into portal veins, hepatocellular necrosis, and hepatocellular mitosis, all common findings of EBV hepatitis, were seen in the patient s liver biopsy specimen. 16 That patient was also enrolled in this study as case 7, showing CAEBV combined with chronic fever for 7 months, constituting further evidence of EBV infection and splenomegaly. Worldwide, there have been about 10 reports of 14 cases Am J Clin Pathol 2010;133: DOI: /AJCPB7SX7QXASPSK 303

5 Chung et al / EBV-Associated BM Fibrin-Ring Granuloma of BM FRG. 2,4,5,7,8,17-19 Kvasnicka and Thiele 5 described a patient developing BM FRG after treatment for Lyme disease, and a similar finding was described in our previous work. 6 EBV-associated BM FRG is associated with a worse prognosis than FRG alone. Thus, in patients with FRG, steps should be taken to exclude EBV infection, as well as other infectious agents, using routine screening tests. Clinical pathologists should take an active interest in ruling out EBV infection by requesting molecular and serologic tests. From the Department of Laboratory Medicine, University of Ulsan College of Medicine and Asan Medical Center, Seoul, Republic of Korea (South Korea). Address reprint requests to Dr Chi: Dept of Laboratory Medicine, University of Ulsan College of Medicine and Asan Medical Center, Pungnap-2dong, Songpa-gu, Seoul, Republic of Korea (South Korea) References 1. Okun DB, Sun NC, Tanaka KR. Bone marrow granulomas in Q fever. Am J Clin Pathol. 1979;71: Voigt JJ, Delsol G, Fabre J. Liver and bone marrow granulomas in Q fever. Gastroenterology. 1983;84: Bonilla MF, Kaul DR, Saint S, et al. Ring around the diagnosis. N Engl J Med. 2006;354: Blanco P, Viallard JF, Parrens M, et al. Bone-marrow fibrinring granuloma. Lancet. 2003;362: Kvasnicka HM, Thiele J. Differentiation of granulomatous lesions in the bone marrow. Pathologe. 2002;23: Chung HJ, Chi HS, Cho YU, et al. Bone marrow fibrinring granuloma: review of 24 cases. Korean J Lab Med. 2007;27: Young JF, Goulian M. Bone marrow fibrin ring granulomas and cytomegalovirus infection. Am J Clin Pathol. 1993;99: Glazer E, Ejaz A, Coley CJN, et al. Fibrin ring granuloma in chronic hepatitis C: virus-related vasculitis and/or immune complex disease? Semin Liver Dis. 2007;27: Vanderstigel M, Zafrani ES, Lejonc JL, et al. Allopurinol hypersensitivity syndrome as a cause of hepatic fibrin-ring granulomas. Gastroenterology. 1986;90: Raya Sánchez JM, Argüelles HA, Brito Barroso ML, et al. Bone marrow fibrin-ring (doughnut) granulomas and peripheral T-cell lymphoma: an exceptional association. Haematologica. 2001;86: Niesters HG, van Esser J, Fries E, et al. Development of a real-time quantitative assay for detection of Epstein-Barr virus. J Clin Microbiol. 2000;38: Cohen JI. Epstein-Barr virus infection. N Engl J Med. 2000;343: Oh SH, Lee YA, Moon WY, et al. Prevalence of Epstein-Barr virus (EBV) antibody in Korean children. J Korean Pediatr Soc. 1994;37: Fiala M, Colodro I, Talbert W, et al. Bone marrow granulomas in mononucleosis. Postgrad Med J. 1987;63: Straus SE. The chronic mononucleosis syndrome. J Infect Dis. 1988;157: Kang ES, Gong GY, Chi HS. A case of fibrin-ring granuloma in bone marrow and liver associated with Epstein-Barr virus infection. Korean J Clin Pathol. 1995;15: Delsol G, Pellegrin M, Voigt JJ, et al. Diagnostic value of granuloma with fibrinoid ring [letter]. Am J Clin Pathol. 1980;73: Rexroth G, Rösch W, Altmannsberger M. Bone marrow granulomatosis in Q-fever [in German]. Med Klin (Munich). 2000;95: Srigley JR, Vellend H, Palmer N, et al. Q-fever. The liver and bone marrow pathology. Am J Surg Pathol. 1985;9: Am J Clin Pathol 2010;133: DOI: /AJCPB7SX7QXASPSK