American Journal ofPathology, Vol. 136, No. 3, March 1990 Copyright © American Association ofPathologists

The Detection of Epstein-Barr Virus in Hairy Cell Leukemia Cells by In Situ Hybridization

Barbara C. Wolf,t Alvin W. Martin, Richard S. Neiman,t Anthony J. Janckila, Lung T. Yam, Annmarie Caracansi,* Brett A. Leav,* Richard Winpenny,* Daniel S. Schultz,* and Hubert J. Wolfe* From the Department of Pathology,* Tufts University School ofMedicine, Boston, Massachusetts; Hematopathology Section,t Mallory Institute ofPathology and Department ofPathology, Boston University School of Medicine, Boston, Massachusetts; and the Departments of Pathology, Medicine and Hematology,* University of Louisville and VeteransAdministration Medical Center,

Louisville, Kentucky

Epstein-Barr virus (EBV) has been implicated in the pathogenesis of several B-cell lymphoidproliferations. Becuse patients with hairy cell leukemia (HCL) have a high incidence of seropositivity for EBV antigens, we studied the cells of HCL for evidence of EBV infection using in situ hybridization techniques. EBV mRNA was detected in the tumor cells in four of six cases using a radiolabeled RNA probe. Confirmatory serologic data were available in three cases in which the viral DNA was detected and in one negative case. Our results suggest that EBV infection may have a pathogenetic role in this disorder. (Am JPathol 1990, 136: 717- 723)

Epstein-Barr virus (EBV) infection has been suggested as an etiologic factor in a variety of benign and malignant disorders. Epidemiologic evidence has indicated that EBV infection is associated with infectious mononucleosis and nasopharyngeal carcinoma, as well as African Burkitt's lymphoma, from which the virus was isolated.1 2 EBV has been implicated in the pathogenesis of B-cell lymphoid malignancies arising in patients with immunosuppressed conditions,3-12 and Southern blotting and in situ hybridization studies have detected EBV DNA in Hodgkin's disease, as well as other lymphoid malignancies and angioimmunoblastic lymphadenopathy.13'15 Because patients with hairy cell leukemia (HCL), a disorder of presumed B-cell origin, have a high incidence of sero-

positivity for EBV,16-20 we used in situ hybridization techniques to study hairy cells for evidence of EBV infection.

Materials and Methods Patients Hairy cells were obtained from six patients with HCL according to the procedure described below. The patients were all male, with a mean age at presentation of 45.3 years (range, 33 to 59 years). At the time cells were taken all patients had been splenectomized and three had subsequently undergone leukapheresis, one of whom had failed to respond to interferon therapy.

Cell Separation Mononuclear cells from spleen cell suspensions (2 cases) or from postsplenectomy peripheral blood (1 case) or leukophereses product (3 cases) were isolated by FicollPaque flotation and subsequently separated into two fractions over a discontinuous Percoll gradient (Pharmacia Fine Chemicals, Uppsala, Sweden) according to a method previously described.21 Cells at the gradient interface of 1.050 to 1.070 (fraction A) contained from 80% to 90% hairy cells, as identified morphologically and by tartrate resistant acid phosphatase (TRAP) activity. Using indirect immunocytochemical methods, these cells were shown to express surface antigens characteristic of hairy cells, including HLA-DR, CR2 (receptor for C3d component of complement), interleukin-2 receptor, and CD20 and CD22 (pan-B cell markers), as well as monoclonal surface immunoglobulin. Cells at the gradient interface of 1.070 to 1.085 (fraction B) contained 80% to 90% lymphocytes, with the remainder being mostly hairy cells and a few polymorphonuclear leukocytes. Only 0.02% of the total number of hairy cells recovered in this process are contained in fraction B.21 The cells were cryopreserved at -70 C in a medium of 40% RPMI-1 640, 50% calf serum, and 10% dimethyl sulfoxide. Accepted for publication November 6, 1989. Address reprint requests to Barbara C. Wolf, MD, Mallory Institute of Pathology, 784 Massachusetts Avenue, Boston, MA 02118.

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In Situ Hybridization In situ hybridization studies were performed on cytocentrifuge smears of thawed, washed cells of fractions A and B using a radioactive 35S-labeled antisense RNA probe to detect EBV mRNA.22 The probe (a gift from Drs. D. Staunton and D. A. Thorley-Lawson) was derived from a 2.8-kb cDNA of the BNLF1 open reading frame, which encodes for the most abundant mRNA in EBV-infected lymphoblastoid cell lines. The clone was isolated from a gtl 1 library by screening with monoclonal antibodies to the BNLF1 gene product. The details of the methodology have been described previously.23 The cDNA was subcloned, initially into Bluescript (Stratagene, La Jolla, CA), and then as an EcoR1 fragment into a pBSM13+ plasmid, and linearized by the restriction enzyme Smal. This modified the insert, allowing a 0.7-kb fragment to be transcribed as the antisense probe using a T7 RNA polymerase and 35S-labeled uridine triphosphate (UTP). Because Smal cut the insert at multiple sites during linearization, a sense probe for use as a negative control could not be produced. The transcribed fragment encodes for a protein termed p63, which is detected in the plasma membranes of infected cells (latent membrane protein).24-26 Cytocentrifuge smears were prepared on gelatinized glass slides, fixed in 4% paraformaldehyde in phosphatebuffered saline (PBS), and incubated at 42 C overnight. Before hybridization the slides were incubated at room temperature with PBS (0.1 M pH 7.2; 3 minutes), 0.1 M glycine in PBS (twice, 3 minutes each), and PBS (3 minutes). They were then treated with Proteinase K (Sigma Chemical, St. Louis, MO) (1 ,ug/ml in 0.1 M Tris buffer [pH 8.0]) containing 50mM EDTA at 37 C for 30 minutes, followed by incubation at room temperature in 4% paraformaldehyde (5 minutes), PBS (twice, 3 minutes each), acetic anhydride (0.5 ml/200 ml 0.1 M triethanolamine, 10 minutes), and at 37 C 50% deionized formamide/2X saline sodium citrate (SSC) (10 minutes). Hybridization was performed using 106 counts per minute of radiolabeled probe in hybridization buffer composed of 50% formamide, 2X SSC, 10% dextran sulfate, 0.25% bovine serum albumin, 0.25% Ficoll 400, 0.25% polyvinyl pyrrolidone 360, 250 mM Tris (pH 7.5), 0.5% sodium pyrophosphate, 0.5% SDS, and 250,ug/ml denatured salmon sperm DNA. The slides were incubated at 42 C overnight, washed in 4X SSC at 37 C for 1 hour, and digested with RNAse (20 ,g RNAse/ml in 0.5M NaCI/1 OmM Tris-HCI, 1 mM EDTA [pH 8.0]) at 37 C for 30 minutes. They were washed sequentially in 2X SSC and 0.1 X SSC for 30 minutes each at 37 C and in graduated solutions of ethanol/0.3 M ammonium acetate (70%, 95%, 100%, and 100%) at room temperature for 5 minutes each. After air drying, the slides were dipped in liquid emulsion (Kodak NTB2, Kodak, Rochester, NY), air dried, and kept at 4 C for 8 days, then developed with D-19 developer (Kodak) and counterstained

with hematoxylin and eosin. The positive control was an EBV-infected lymphoblastoid cell line and the negative control was an EBV-negative erythroleukemia line.

Serologic Studies Antibody titers to the EBV viral capsid antigen, early antigen, and nuclear antigen were determined on sera from four patients using standard complement-fixation techniques.

Electron Microscopy Electron microscopic studies were performed on formalin- or glutaraldehyde-fixed spleen sections in all cases. Sections were dehydrated in acetone and embedded in Epon. Thick sections were stained with methylene blue to assess adequacy of tissue and to select appropriate areas for examination. Thin sections were stained with lead nitrate and uranium acetate and examined under a Siemmens 101 transmission electron microscope on copper grids.

Results The cells of fraction A (hairy cell enriched) obtained from four of the six patients showed hybridization with the radiolabeled RNA probe (Figures 1 and 2). The silver grains were concentrated over the nuclei of the cells and were markedly increased over the low background levels. The hairy cells of the remaining two patients showed no hybridization (Figure 3). In contrast, none of the lymphoid cells of the B fractions (hairy cell depleted) showed hybridization. The serologic tests for EBV antigens suggested a chronic or reactivation infection in three of the four patients for whom the data were available (Table 1). This was evidenced by elevated titers of the immunoglobulin G antibody to the viral capsid antigen (> 1 :1280) as well as antibodies to the early (.1:20) and nuclear (21:8) antigens. The hairy cells of these three patients showed hybridization with the RNA probe. The serologic studies were negative in the fourth case and the cells of this patient did not show hybridization. Ultrastructural examination of the spleens revealed typical pseudosinuses lined by hairy cells. The hairy cells showed abundant surface projections and ribosome lamella complexes were identified in all cases. Careful examination failed to reveal viral particles in any case. Follow-up data indicate that the four patients with EBV mRNA detected in their tumor cells are alive, one having responded to splenectomy only, while the other three

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Figure 1. In situ hybridization for EBV mRNA in one case. There is strong labeling of tumor cell nuclei (A) and no labeling of the cells of the hairy cell-depletedfraction (B) (Magnification, X 1000).

have required splenectomy, leukapheresis, and interferon therapy to control disease progression. One of the patients without detectable EBV mRNA is alive after splenectomy alone; the other died of a myocardial infarction while on interferon therapy.

Discussion Epstein-Barr virus, a member af the herpes virus group, infects a small number of peripheral blood B lymphocytes.27'28 In vitro the virus also infects B cells, causing polyclonal proliferation and activation and giving rise to immortal cell lines.2932 The receptor for the virus has been shown to be the receptor for the C3d component of complement (CD21 ).33 Since the advent of molecular probe techniques, EBV has been implicated in the pathogenesis of an ever-increasing number of lymphoid proliferations. Staal and colleagues,13 using Southern blotting techniques, detected

the EBV genome in tissues of 8 of 28 patients with Hodgkin's disease, 3 of 37 with diffuse large-cell lymphoma, and 1 with angioimmunoblastic lymphadenopathy. Weiss and coworkers14 localized the viral genome to the ReedSternberg cell in five cases of Hodgkin's disease using a radiolabeled DNA probe. EBV DNA has also been identified in tissues of B-cell lymphomas arising in patients with the acquired immunodeficiency syndrome and in posttransplant lymphoproliferative disorders, as well as central nervous system lymphomas and thymomas.3 12,3639 Once a subject of controversy, HCL is now considered a malignancy of B-cell origin, as evidenced by clonal immunoglobulin gene rearrangements in the neoplastic cells.40 Patients with this disorder have a high incidence of seropositivity for EBV, with elevated titers of antibodies to the viral capsid and early antigens."'6 Titers of antibodies to the early antigen subside and may become undetectable in otherwise healthy patients with infectious mononucleosis, and thus the persistent elevation of antibody titers in HCL patients suggests a chronic or recur-

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Figure 2. A second case showing labeling of the hairy cells (A) and unlabeled lymphoid cells(B) (Magnification, X 1000).

rent infection.26 Some HCL cell lines bind antibodies to the EBV nuclear antigen and others have been shown to contain EBV DNA by in situ hybridization.41'42 Sarienji and colleagues43 demonstrated the EBV receptor on 3% to 45% of leukemic cells in patients with HCL but could not detect the viral genome in hairy cells using Southern blotting techniques. EBV has been reported to infect and transform a subset of hairy leukemic cells.20 We used a radiolabeled antisense RNA probe to detect EBV mRNA in the tumor cells of patients with HCL. In situ hybridization techniques allow visualization of the cells containing the nucleic acid of interest, which is not possible with filter hybridization techniques. We chose the radiolabeled RNA probe because of its greater sensitivity compared with a commercially available biotinylated double-stranded DNA probe.22 We detected the presence of EBV mRNA in the hairy cells of four of six patients, but we could not detect the EBV nucleic acid in other lymphoid cells of the same patients. In three of the four positive cases in which the data

were available, serologic studies indicated a chronic infection. EBV mRNA was not found in either the hairy cells or lymphoid fractions of two patients, one of whom showed no serologic evidence of infection. Although one previous electron microscopic study reported the finding of mature viral particles in cultured hairy cells,44 we did not identify viral particles in the spleens of any of our patients. Thus our studies suggest that in most cases of HCL, EBV infection is latent and viral replication is not evident. It is possible that hairy cells may, in some way, permit transcription of the viral DNA but be nonpermissive for viral

replication. Our findings indicate that HCL leukemia may be another B-cell lymphoid malignancy associated with latent EBV infection. The presence of the viral genome in tumor cells of patients with this disorder, in addition to seropositivity for viral antigens and the presence of the EBV receptor on hairy cells, suggest a direct link between the viral infection and neoplastic transformation. However, because serologic evidence of EBV infection is common in

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Figure 3. In situ hybridization for EBV mRNA in a third case showing nopositivlity in either the hairy cell fraction (A) or the lymphoid fraction (B) above the low background levels (Magnification, X 1000).

the adult population45 and because the viral genome is not detectable in some cases, it is possible that EBV acts as an etiologic factor in only a subset of patients with HCL. On the other hand, as suggested by Staal and colleagues,13 the virus may act as a promoter or cofactor rather than as the proximate cause of neoplastic transformation. It may be postulated that if the viral DNA is integrated in the genome of a B cell, this may leave the lymTable 1. Serologic Tests with EBV-associatedAntigens Hybridization Patient with RNA probe VCA EA EBNA 1 Positive 1:2560 .1:20 1:20 2 Positive 1:1280 1:20 .1:8 3 Positive 1:2560 .1:20 .1:8 4 1:2 Negative 1:280 .1:20 5 Positive NA 6 NA Negative VCA = lgG anti-viral capsid antigen (starting dilution, 1:80). EA = antiearly antigen (starting dilution, 1:20). EBNA (starting dilution, 1:2). NA = not available.

=

anti-nuclear antigen

phocyte at risk for a second transforming event. The cells of Burkitt's lymphoma are characterized by a translocation between chromosome 8 and, usually, chromosome 14 that confers immortality on the cells and activates the c-myc oncogene.4647 Although no consistent karyotypic abnormalities have been identified in HCL, it is possible that EBV integration in the genome of B cells of patients who develop this disorder may cause similar activation of an as yet-unidentified oncogene. Immunodeficiency appears to be a common variable among the EBV-associated lymphoproliferative disorders. Individuals with chronic EBV infections have circulating cytotoxic T cells that can lyse EBV-transformed B cells.48 Recent studies have indicated that p63 may induce this specific cytotoxic T-cell response.49 African Burkitt's lymphoma occurs in regions endemic for malaria, which is associated with depressed T-cell function. 50 Similarly, Hodgkin's disease is associated with defective cell-mediated immunity.51'52 Patients with HCL may have a monocytopenia and defective macrophage function, in addition

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to a profound neutropenia.5354 They lack virus-neutralizing antibodies, suggesting a relative immunodeficiency.19 It is uncertain, however, whether the immunodeficiency in HCL patients is secondary to EBV infection or whether a pre-existing relative immunodeficiency results in the host's inability to limit the effects of a viral infection. The significance of the presence of EBV mRNA in the cells of HCL has yet to be elucidated. Although our series of patients is too small to draw firm conclusions, a direct relationship seems to exist between the expression of EBV mRNA in tumor cells and anti-EBV antibodies in the patients' sera. However, although we could not detect EBV transcripts in the hairy cells in two cases, it is possible that these patients could still harbor the EBV genome integrated at low copy number, which might be identified using a different probe or by more sensitive techniques such as the polymerase chain reaction, or in an episomal form. Three of our four patients in whom EBV mRNA was demonstrable have had difficult clinical courses requiring multiple treatment strategies. It may be that viral transcription, even in the absence of replication, heralds unstable disease. Further studies are warranted to determine whether correlations exist between clinical indicators of disease activity in HCL and the presence of the EBV genome in the hairy cells.

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