291
Detection of Human Herpesvirus 6 in Tissues Involved by Sinus Histiocytosis with Massive Lymphadenopathy (Rosai-Dorfman Disease) Paul H. Levine, Nazma Jahan, Pamela Murari,* Mark Manak, and Elaine S. Jaffe
National Cancer Institute. National Institutes of Health. Bethesda. and Biotech Research Laboratories. Rockville. Maryland; Art/led Forces Institute of Pathology. Washington. DC
After preliminary serologic data demonstrated elevated antibody titers to human herpesvirus (HHV) 6 in patients with sinus histiocytosis with massive lymphadenopathy (SHML) or RosaiDorfman disease, tissues were examined from 9 patients with classical SHML to search for evidence of HHV-6 infection. Involved tissues from 7 of the 9 patients had detectable HHV-6 by in situ hybridization: Tissue from 1 had detectable Epstein-Barr virus genome but no HHV-6 and tissue from another had no detectable HHV-6 or Epstein-Barr virus. These studies suggest that HHV-6 and, to a lesser extent, Epstein-Barr virus may be involved in the etiology of SHML.
Materials and Methods Identification of patients. Paraffin blocks of formalin-fixed biopsies from 7 patients with classical clinical and pathologic
Received 24 January 1992; revised 30 March 1992. Grant support: National Institutes of Health (AI-82502). Reprints or correspondence: Dr. Paul H. Levine, National Cancer Institute, National Institutes of Health, Bldg. EPN, Rm. 434, Bethesda. MD 20892. * Present affiliation: Department of Pathology. Reynolds Army Community Hospital. Fort Sill, Oklahoma.
The Journal of Infectious Diseases
1992;166:291-5
1992 by The University of Chicago. All rights reserved. 0022-1899/92/6602-00 I0$0 1.00 @
features of SHML were selected from the files of the Armed Forces Institute of Pathology, and 2 were obtained from the National Institutes of Health (NIH) (table I). Control samples. For each experiment, several controls were used for specificity. Three preparations of the HSB-2 cell line (one uninfected, one heavily infected, and one lightly infected with HHV-6) were used in each experiment as negative and. positive controls. The controls for the EBV reactivity included the Raji cell line, which contains EBV in a latent form but has no replicating virus, and a clone of the P3HR-I cell line (designated P3H3) , which contains replicating EBV that produces both early antigen and viral capsid antigen. The cytomegalovirus (CMV) controls were slides prepared with infected human fibroblasts HFW 1-38 (ATCC: CCL 75, Pandata). Human tissue controls included clinically uninvolved lymph node, parotid gland, and tissues from 45 non-Hodgkin's lymphoma patients evaluated in a concurrent study [24]. Sample preparation. Cultures of infected or uninfected cells were gently harvested, centrifuged at 100 g for 5 min, and washed with PBS (Quality Biological, Gaithersburg, MD). A total of 5 X 104 cells were spotted per 6-mm well on heavy Tefloncoated microscope slides. The cells were air-dried for 30-60 min, fixed with 4%paraformaldehyde for 1-2 min, and stored at - 20°C in 70% ethanol. Slides of paraffin-embedded cultured cells were also prepared from cell pellets. From 750 to 1000 mL of cells (l X 106 cells/ mL) were pelleted by centrifugation and washed with PBS. Pellets were gently dislodged from the bottom of the centrifuge and fixed overnight in 2 mL of 10% formalin in PBSat room temperature. The fixed pellets were embedded in paraffin, and thin sections were mounted on slides (National Cancer Institute-Frederick [MD] Research and Development Center; FCRDC) for use as controls for in situ hybridization. Tissue sections (3- to 6-JLm thick) were cut from paraffin-embedded tissues that had been fixed in 10% neutral buffered formalin. Sections were mounted on glass slides precoated with 2% 3-aminopropyltriethoxysilane (Sigma, St. Louis) in acetone. Hapten labeling ofprobes. The plasmid pZVH 14 containing an 8.7-kb portion of theHHV-6 genome, the BamHI W fragment of EBV, and a cloned CMV fragment (Digene, College Park, MD) were used for the HHV-6, EBV, and CMV probes.
Downloaded from http://jid.oxfordjournals.org/ at Monash University on March 7, 2015
Sinus histiocytosis with massive lymphadenopathy (SHML) or Rosai-Dorfman disease is an illness of unknown etiology, primarily manifested by painless cervicallymphadenopathy. It most commonly affects children and young adults [ 1-3]. The clinical course is usually benign, characterized by a chronic course marked by exacerbations and remissions. Fatalities have been reported [3, 4], however, particularly in patients with immunologic abnormalities [5], and therapy can be lifesaving [6]. Although pathologic similarities to malignant lymphoma have been noted [7], it is now apparent that SHML is usually not premalignant (5 of 423 patients in a registry ofSHML patients had associated hematologic malignancies) [3] and is likely to represent an exaggerated immunologic response to an infectious agent. Because herpesviruses, particularly Epstein-Barr virus (EBV) and the more recently described human herpesvirus (HHV) 6 [8], cause lymphoproliferative disease [9-12] and have been associated with human lymphoma [12-23], we used in situ hybridization to detect HHVs in paraffin-embedded tissues to see if any specific candidate agents could be detected in SHML biopsies.
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Table 1. Case information and biopsy sites for 9 patients with classicalclinical and pathologic features of sinus histiocytosis with massive lymphadenopathy. Case no.• biopsy date
I. 1985 2. 1986 3. 1986 4. 1986 5. 1986 6.1989 7. 1989 8. 1982 9.1983
Age. sex. race
Probe results Site studied
19. M. B 73. F.? 39. M.? 64.M. W 39. M. B II. M. A 67.M. W 25. M. W 6. M.?
Left cervical lymph node Soft tissue mass. right hip Left axillary lymph node Left inguinal lymph node Left cervical lymph node Large neck mass Left inguinal lymph node Maxillary sinus Cervical lymph node
HHV-6
+ + +
EBV
+ +
+ + + +
respectively. The Bluescript KS- plasmid vector (Stratagene, La Jolla, CA) without insert was used as a negative control. The inserts containing the virus sequences were cut from the plasmids by restriction endonuclease digestion and isolated on agarose gels. They were then labeled with digoxigenin by random priming using the "Genius" kit from Boehringer-Mannheim (Indianapolis) for use as probes. In situ hybridization. Slides of formalin-fixed, paraffin-embedded tissue sections were dewaxed by gentle heating and immersion in xylene and rehydrated by immersion in graded alcohols (95%,80%, and 70%ethanol). The slides were incubated for 15-30 min in 25 JLg/mL proteinase Kin TE (10 mM TRIS, pH 7.6, I mM EDTA) and 0.1%SDS at 37°C for 15 min and postfixed for 5 min in freshly prepared 4% paraformaldehyde in PBS at room temperature. The slides were prehybridized in 50%formamide, 4X standard saline citrate (SSC), 3.75% dextran sulfate, 250 JLg/mL sheared and denatured salmon sperm DNA, 250 JLg/mL tRNA, and 5X Denhardt's solution (I X = 0.02% ficoll, 0.02% polyvinylpyrrolidone, 0.02% bovine serum albumin) for 1-3 hat 48-50°C. The hybridization solution was similar to the prehybridization solution but contained 1-4 JLg/mL digoxigenin-Iabeled probe. Slides were then heated to 100°C for 4 min, chilled on ice, and incubated for 3 h at 47°C in a humidity chamber. After hybridization, slides were washed twice each with 2x SSC plus 0.1%SDS and IX SSC and 0.1%SDS at room temperature. The final two washes were at 54°C in 0.2x SSC and 0.1 % SDS. All washes were for 15-30 min with agitation. For detection of digoxigenin-Iabeled probes, the tissues were blocked for I h in blocking agent supplied by the manufacturer. exposed to antidigoxigenin for I h, and developed with 5-bromo-4-chloro-3-indolyl phosphate/4-nitroblue tetrazolium according to manufacturer's directions (Boehringer-Mannheim). Immunophenotyping. Immunophenotyping was done on fixed, paraffin-embedded tissue sections, using the avidin-biotin complex technique as previously described [25, 26]. The antibody panel included UCHL-I (CD45RO), L-26 (CD20), KP-I
(CD68) lysozyme, SIOO (all from DAKO, Santa Barbara, CA) and Leu-MI (CDI5; Becton-Dickinson, Mountain View, CA). Serology. Antibodies to HHV -6 and EBV were detected by immunofluorescence [8, 27].
Results Control sections prepared from packed HHV-6-infected culture cells and subjected to in situ hybridization showed intense purple staining of the nuclei, indicative of HHV-6 infection. Uninfected HSB-2 cells did not stain. The HHV-6 probes were specific for HHV-6-infected cells only and did not hybridize with EBV- or CMV-infected control cells. Similarly, the EBV probe was specific for EBV-infected cells only and did not cross-hybridize with HHV-6- or CMV-infected cells. Tissues from 9 cases ofSHML were examined for the presence ofEBV and HHV-6 by in situ hybridization. Seven had significant reactivity with the HHV-6 probe (table I). One biopsy specimen (case 5) reacted with EBV but not with HHV-6, another (case 3) reacted with both the HHV-6 and EBV probes, and a third sample (case I) did not react with either. The HHV-6 sequences were not uniformly distributed throughout the tissue, tending to be localized in discrete regions. Cells that stained positively were observed especially in sinuses containing abnormal histiocytes (figure I). In some individuals, multiple tissues from the same patient were available for study. All three tissue samples for case 2 and the two samples for case 4 were positive for HHV6 sequences only. Of the six samples of case 7 examined, however, four were positive for HHV-6 and two were negative. Immunoperoxidase studies were confirmatory for the diagnosis of SHML. All cases showed diffuse cytoplasmic reactivity with the S 100 protein marker. The principal cells often, but not always, had the phenotype of'histiocytes, demonstrating positive reactivity for lysozyme and KP-I. The B cell marker, L-26, was negative as was the granulocyte-associated antigen CD 15. Focal positivity for UCHL-I was seen on some histiocytic cells. In the 1 patient studied for HHV-6 and EBV antibodies, the viral capsid antibody titers to both viruses were 1:160.
Discussion SHML has been considered to be a disease characterized by abnormal immunologic response to a precipitating agent, possibly a virus. EBV has been thought to be an etiologic agent, but not all patients with this illness have antibodies to this virus [3, 5]. In a small series of patients we studied (see Acknowledgments), all had antibodies to both EBV and to HHV-6, several in high titers. In this biopsy series, a serum sample was available for only I NIH case, and antibody titers to both viruses were within the normal range.
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NOTE. Cases 1-7 were from the Armed Forces Institute of Pathology; 8 and 9 from the National Cancer Institute. HHV-6. human herpesvirus 6; EBV. Epstein-Barr virus; B. black; W. white; A. Asian.
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Human Herpesvirus 6 and Rosai-Dorfman Disease
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Figure 1. Histopathology of sinus histiocytosis with massive lymphadenopathy (SHML). Large histiocytes with abundant cytoplasm fill distended sinus. Histiocytes contain intact lymphocytes (emperipoleis), characteristic feature of SHML (arrow). (Stained with hematoxylin-eosin; original magnification X400. Bar = 10 ~m.)
Figure 2. In situ hybridization for human herpesvirus 6 in sinus histiocytosis with massive lymphadenopathy (case 2). Granular positivity in nuclei of histiocytes within distended sinus. (Original magnification X400: no counterstain. Bar = 10 ~m.)
sequelae ofHHV-6 have been identified: exanthem subitum (roseola infantum) [31, 32], heterophile negative infectious mononucleosis [l l], and hepatitis [33]; preliminary data also suggest a role in pneumonitis occurring in bone marrow transplant recipients [34]. Elevated antibody titers may be found in Kawasaki disease [35], sarcoidosis [36], malignant lymphoproliferative diseases [37], and chronic fatigue syndrome [38], but these elevations are not consistent and may be due to immunologic perturbations that allow increased viral replication rather than reflecting an etiologic relationship (for a recent review, see [I 2]). We used an in situ hybridization technique to examine> 100 other tissues, including those from patients with Burkitt's lymphoma, chronic fatigue syndrome, and a variety of Hodgkin's and non-Hodgkin's lymphomas. Although HHV-6-staining cells were found in some patients from each category, most tissues were negative
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Identification ofvirus in pathologic specimens is often considered one of the most important parameters in determining viral etiology ofdisease. The consistent identification ofEBV genome in nasopharyngeal carcinoma [28] provides important evidence towards this etiologic relationship. However, even in diseases where the virus is frequently, but not always, identified (e.g., EBV in Burkitt's lymphoma [ 13-15] and human T cell leukemia virus type I in adult T cell leukemia/ lymphoma [29]), the associated virus is generally considered to be the predominant etiologic agent but more than one precipitating cause can result in a similar pathologic lesion [l4, 29, 30]. In this study, the detection of HHV -6 in 13 of 17 tissues involved by Rosai-Dorfman disease, particularly in the abnormal cells within sinuses (figure 2), argues for a pathogenetic role for this new HHV. Thus far, at least three clinical
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Acknowledgments
We thank the following contributors for allowing us to study their cases: St. Croix Community Hospital (St. Croix Falls, WI; case I); Hye-Kung Kim, Merrithew Memorial Medical Center (Martinez, CA; case 2); F. Azizi, Kaiser Foundation Hospitals (Fontana, CA; case 3); Sheng-CW, Sharon General Hospital (Sharon, PA; case 4); A. R. Summerlin, Columbus Medical Center (Columbus, GA; case 5); Chansuda Wongsrichanalai, Department ofImmunologyjAfrins (San Francisco; case 6); and S. Jurco, South Lake Memorial Hospital (Clermont, FL; case 7). We also thank Ernest Baden, Ronald Dorfman, Elliott Foucar, Bertil E. Gader, Andrew Raubitschek, Juan Rosai, and V. Shanta for sera from patients with Rosai-Dorfman disease, which led to this current study; Gary Pearson (Georgetown University, Washington, DC) for testing these sera; and the FCRDC Document Processing Department for typing support.
JID 1992;166 (August)
References I. Rosai J, Dorfman RF. Sinus histiocytosis with massive lymphadenopathy. A newly recognized benign clinicopathological entity. Arch Pathol 1969;87:63-70. 2. Rosai J, Dorfman RF. Sinus histiocytosis with massive lymphadenopathy: a pseudolymphomatous benign disorder. Analysis of 34 cases. Cancer 1972;30: I 174-88. 3. Foucar E, Rosai J, Dorfman RF. Sinus histiocytosis with massive lymphadenopathy (Rosai-Dorfman disease): review of the entity. Semin Diagn PathoI1990;7:19-73. 4. Foucar E, Rosai J, Dorfman RF. Sinus histiocytosis with massive lymphadenopathy. An analysis of 14 deaths occurring in a patient registry. Cancer 1984;54: 1834-40. 5. Foucar E, Rosai J, Dorfman RF, Eyman JM. Immunologic abnormalities and their significance in sinus histiocytosis with massive lymphadenopathy. Am J Clin Pathol 1984;82:515-25. 6. Komp OM. The treatment ofsinus histiocytosis with massive lymphadenopathy (Rosai-Dorfman disease). Semin Diagn Pathol 1990;7:836. 7. Dorfman RF, Warnke R. Lymphadenopathy simulating the malignant lymphomas. Hum Pathol 1974;5:519-50. 8. Salahuddin SZ, Ablashi DV, Markham PO, et at. Isolation of a new virus, HBLV, in patients with Iymphoproliferative disorders. Science 1986;234: 596-60 I. 9. Henle G, Henle W, Diehl V. Relation of Burkitt's tumor-associated herpes type virus to infectious mononucleosis. Proc Natl Acad Sci USA 1968;59:94-10 I. 10. Purtilo DT, Okano M, Grierson HL. Immune deficiency as a risk factor in Epstein-Barr virus-induced malignant diseases. Environ Health Perspect 1990;88:225-30. II. Steeper T A, Horwitz CA, Ablashi DV, et at. The spectrum of clinical and laboratory findings resulting from human herpesvirus-6 (HHV6) in patients with mononucleosis-like illnesses not resulting from Epstein-Barr virus or cytomegalovirus. Am J Clin Pathol 1990; 93:776-83. 12. Levine PH, Jarrett R, Clark D. The epidemiology of human herpesvirus-6. In: Ablashi DV, Krueger GRF, Salahuddin SZ, eds. Human herpesvirus-6: epidemiology, molecular biology and clinical pathology. Amsterdam: Elsevier Science, 1992:9-23. 13. Zur Hausen H, Schulte-Holthausen H, Klein E, et at. EBV DNA in biopsies of Burkitt tumours and anaplastic carcinomas of the nasopharynx. Nature 1970;228: 1056-8. 14. Shiramizu B, Barriga F. Neequaye J. et at. Patterns of chromosomal breakpoint locations in Burkitt's lymphoma: relevance to geography and Epstein-Barr virus association. Blood 1991 ;77: 1516-26. 15. Henle W, Henle G, Gunven P, et at. Patterns of antibodies to EpsteinBarr virus-induced early antigens in Burkitt's lymphoma. Comparison of dying patients with long-term survivors. J Natl Cancer Inst 1973;50: 1163-73. 16. Levine PH. Ablashi DV, Berard CWo et at. Elevated antibody titers to Epstein-Barr virus in Hodgkin's disease. Cancer 1971 :27:416-21. 17. Weiss LM, Movahed LA, Warnke RA, Sklar J. Detection of EpsteinBarr viral genomes in Reed-Sternberg cells of Hodgkin's disease. N Engl J Med 1989;320:502-6. 18. Bignon YJ, Bernard D. Cure H, et at. Detection of Epstein-Barr viral genomes in lymph nodes of Hodgkin's disease patients. Mol Carcinog 1990;3:9-11. 19. Pallesen G, Hamilton-Dutoit SJ, Rowe M, Young LS. Expression of Epstein-Barr virus latent gene products in tumour cells of Hodgkin's disease. Lancet 1991;337:320-2. 20. Josephs SF. Buchbinder A. Streicher H, et at. Detection of human B Iymphotropic vii-us (human herpes virus 6) sequence in B cell lymphoma tissue of three patients. Leukemia 1988;2: 132-5. 21. Jarrett RF. Gledhill S. Qureshi F. et at. Identification of human herpes-
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and we did not observe any clear association between HHV6 and other diseases (unpublished data, [39]). Additional control tissues from nonspecific lymphatic hyperplasia were negative for HHV-6 sequences. Since the region of HHV -6 infection is not uniform throughout tissue, it could be argued that some tissue sections simply miss the infected region. Thus, it may be possible that in case 7, where only four of six tissue samples were positive for HHV-6, we missed the areas of infection in the two negative tissues. We did not have access to additional sections from cases I or 5 to rule out the possibility that other areas of tissue may have contained viral sequences. The detection ofEBV in the I patient negative for HHV-6 is also of interest; whether EBV was present as a passenger virus or was a trigger for this striking immunologic response (SHML) is yet to be determined. EBV has been identified in some biopsies from Hodgkin's disease patients [17-19, 23], and there is evidence that other agents, including HHV -6, may be associated with other cases [22]. The immunotypic findings in this study are similar to those previously reported and support a histiocytic origin for the cells of SHML [40]. Although the S I00 protein is found characteristically in interdigitating reticulum cells, previous studies of reactive lymph nodes have shown positivity in sinus histiocytes when the lymph nodes showed active sinus histiocytosis [26]. Therefore, the presence of S I00 may reflect a state of activation in lesional cells. In summary, these findings suggest but do not prove that HHV-6 and, on occasion, EBV can trigger SHML. There continues to be the possibility that the viruses detected in the cells result from secondary infection, particularly since infection of a cell by one virus can make the cell susceptible to others that ordinarily cannot infect these cells [41]. An expansion of these studies to larger case series should help to resolve these issues.
at.
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22.
23.
24.
25. 26. 27.
29. 30.
31.
32.
virus 6-specific DNA sequences in two patients with non-Hodgkin's lymphoma. Leukemia 1988;2:496-502. Torelli G, Marasca R, Luppi M, et al. Human herpesvirus-6 in human lymphomas: identification of specific sequences in Hodgkin's lymphomas by polymerase chain reaction. Blood 1991;77:2251-8. Wu TC, Mann RB, Charache P, et al. Detection of EBV gene expression in Reed-Sternberg cells of Hodgkin's disease. Int J Cancer 1990;46:801-4. Shen Y, Ming HA, Jahan N, Manak M, Jaffe ES, Levine PH. In situ hybridization detection of human herpesvirus-6 in biopsies from Chinese patients with non-Hodgkin's lymphoma. Arch Pathol Lab Med (in press). Hsu SM, Jaffe ES. Leu M I and peanut agglutinin stain the neoplastic cells of Hodgkin's disease. Am J Clin PathoI1984;82:29-32. Jaffe ES. Histiocytoses oflymph nodes: biology and differential diagnosis. Semin Diagn Pathol 1988;5:376-90. Henle G, Henle W. Immunofluorescence in cells derived from Burkitt's lymphoma. J Bacteriol 1966;91: 1248-56. Raab-Traub N, Flynn K, Pearson G, et al. The differentiated form of nasopharyngeal carcinoma contains Epstein-Barr virus DNA. Int J Cancer 1987;39:25-9. Tajima K, T- and B-cell Malignancy Study Group. Japanese National Survey. Int J Cancer 1990;45:237-43. Jaffe ES, Blattner WA, Blayney DW, et al. The pathologic spectrum of HTLV-associated leukemia/lymphoma in the United States. Am J Surg Pathol 1984;8:263-75. Yamanishi K, Okuno T, Shiraki K, et al. Identification of human herpes virus-6 as a causal agent for exanthem subitum. Lancet 1988; I: 1065-7. Suga S, Yoshikawa T, Asano Y, Yazaki T, Hirata S. Human herpes-
33. 34.
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virus-6 infection (exanthem subitum) without rash. Pediatrics 1989;83: 1003-6. Irving WL, Cunningham AL. Serological diagnosis of infection with human herpesvirus-6. BMJ 1990;300: 156-9. Carrigan DR. Human herpesvirus-6 and bone marrow transplantation. In: Ablashi DV, Krueger GRF, Salahuddin SZ, eds. Human herpesvirus-6: epidemiology, molecular biology and clinical pathology. Amsterdam: Elsevier Science, 1992:281-30 I. Okano M, Luka J, Thiele GM, Sakiyama Y, Matsumoto S, Purtilo DT. Human herpesvirus 6 infection and Kawasaki disease. J Clin MicrobioI 1989;27:2379-80. Biberfeld P, Petren AL, Eklund A, et al. Human herpesvirus-6 (HHV-6, HBLV) in sarcoidosis and Iymphoproliferative disorders. J Virol Methods 1988;21:49-59. Clark DA, Alexander FE, McKinney PA, et al. The seroepidemiology of human herpesvirus-6 (HHV-6) from a case-control study of leukaemia and lymphoma. Int J Cancer 1990;45:829-33. Levine PH, Krueger GRF, Kaplan M, et al. The post-infectious chronic fatigue syndrome. In: Ablashi DV, Huang AT, Pagano JS, Pearson GR, Yang CS, eds. Epstein-Barr virus and human disease. Clifton, NJ: Humana Press, 1989:405-38. Krueger GRF, Manak M, Bourgeois N, et al. Persistent active herpes virus infection associated with atypical polyclonal Iymphoproliferation (APL) and malignant lymphoma. Anticancer Res 1990;9: 145776. Eisen RN, Buckley PJ, Rosai J. Immunophenotypic characterization of sinus histiocytosis with massive lymphadenopathy (Rosai-Dorfman disease). Semin Diagn Pathol 1990;7:74-82. Schonnebeck M, Krueger GRF, Braun M, et al. Human herpesvirus-6 infection may predispose cells to superinfection by other viruses. In Vivo 1991;5:255-64.
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28.
Human Herpesvirus 6 and Rosai-Dorfman Disease
Detection of Human Herpesvirus 6 in Tissues Involved by Sinus Histiocytosis with Massive Lymphadenopathy (Rosai-Dorfman Disease) Paul H. Levine, Nazma Jahan, Pamela Murari,* Mark Manak, and Elaine S. Jaffe
National Cancer Institute. National Institutes of Health. Bethesda. and Biotech Research Laboratories. Rockville. Maryland; Art/led Forces Institute of Pathology. Washington. DC
After preliminary serologic data demonstrated elevated antibody titers to human herpesvirus (HHV) 6 in patients with sinus histiocytosis with massive lymphadenopathy (SHML) or RosaiDorfman disease, tissues were examined from 9 patients with classical SHML to search for evidence of HHV-6 infection. Involved tissues from 7 of the 9 patients had detectable HHV-6 by in situ hybridization: Tissue from 1 had detectable Epstein-Barr virus genome but no HHV-6 and tissue from another had no detectable HHV-6 or Epstein-Barr virus. These studies suggest that HHV-6 and, to a lesser extent, Epstein-Barr virus may be involved in the etiology of SHML.
Materials and Methods Identification of patients. Paraffin blocks of formalin-fixed biopsies from 7 patients with classical clinical and pathologic
Received 24 January 1992; revised 30 March 1992. Grant support: National Institutes of Health (AI-82502). Reprints or correspondence: Dr. Paul H. Levine, National Cancer Institute, National Institutes of Health, Bldg. EPN, Rm. 434, Bethesda. MD 20892. * Present affiliation: Department of Pathology. Reynolds Army Community Hospital. Fort Sill, Oklahoma.
The Journal of Infectious Diseases
1992;166:291-5
1992 by The University of Chicago. All rights reserved. 0022-1899/92/6602-00 I0$0 1.00 @
features of SHML were selected from the files of the Armed Forces Institute of Pathology, and 2 were obtained from the National Institutes of Health (NIH) (table I). Control samples. For each experiment, several controls were used for specificity. Three preparations of the HSB-2 cell line (one uninfected, one heavily infected, and one lightly infected with HHV-6) were used in each experiment as negative and. positive controls. The controls for the EBV reactivity included the Raji cell line, which contains EBV in a latent form but has no replicating virus, and a clone of the P3HR-I cell line (designated P3H3) , which contains replicating EBV that produces both early antigen and viral capsid antigen. The cytomegalovirus (CMV) controls were slides prepared with infected human fibroblasts HFW 1-38 (ATCC: CCL 75, Pandata). Human tissue controls included clinically uninvolved lymph node, parotid gland, and tissues from 45 non-Hodgkin's lymphoma patients evaluated in a concurrent study [24]. Sample preparation. Cultures of infected or uninfected cells were gently harvested, centrifuged at 100 g for 5 min, and washed with PBS (Quality Biological, Gaithersburg, MD). A total of 5 X 104 cells were spotted per 6-mm well on heavy Tefloncoated microscope slides. The cells were air-dried for 30-60 min, fixed with 4%paraformaldehyde for 1-2 min, and stored at - 20°C in 70% ethanol. Slides of paraffin-embedded cultured cells were also prepared from cell pellets. From 750 to 1000 mL of cells (l X 106 cells/ mL) were pelleted by centrifugation and washed with PBS. Pellets were gently dislodged from the bottom of the centrifuge and fixed overnight in 2 mL of 10% formalin in PBSat room temperature. The fixed pellets were embedded in paraffin, and thin sections were mounted on slides (National Cancer Institute-Frederick [MD] Research and Development Center; FCRDC) for use as controls for in situ hybridization. Tissue sections (3- to 6-JLm thick) were cut from paraffin-embedded tissues that had been fixed in 10% neutral buffered formalin. Sections were mounted on glass slides precoated with 2% 3-aminopropyltriethoxysilane (Sigma, St. Louis) in acetone. Hapten labeling ofprobes. The plasmid pZVH 14 containing an 8.7-kb portion of theHHV-6 genome, the BamHI W fragment of EBV, and a cloned CMV fragment (Digene, College Park, MD) were used for the HHV-6, EBV, and CMV probes.
Downloaded from http://jid.oxfordjournals.org/ at Monash University on March 7, 2015
Sinus histiocytosis with massive lymphadenopathy (SHML) or Rosai-Dorfman disease is an illness of unknown etiology, primarily manifested by painless cervicallymphadenopathy. It most commonly affects children and young adults [ 1-3]. The clinical course is usually benign, characterized by a chronic course marked by exacerbations and remissions. Fatalities have been reported [3, 4], however, particularly in patients with immunologic abnormalities [5], and therapy can be lifesaving [6]. Although pathologic similarities to malignant lymphoma have been noted [7], it is now apparent that SHML is usually not premalignant (5 of 423 patients in a registry ofSHML patients had associated hematologic malignancies) [3] and is likely to represent an exaggerated immunologic response to an infectious agent. Because herpesviruses, particularly Epstein-Barr virus (EBV) and the more recently described human herpesvirus (HHV) 6 [8], cause lymphoproliferative disease [9-12] and have been associated with human lymphoma [12-23], we used in situ hybridization to detect HHVs in paraffin-embedded tissues to see if any specific candidate agents could be detected in SHML biopsies.
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Table 1. Case information and biopsy sites for 9 patients with classicalclinical and pathologic features of sinus histiocytosis with massive lymphadenopathy. Case no.• biopsy date
I. 1985 2. 1986 3. 1986 4. 1986 5. 1986 6.1989 7. 1989 8. 1982 9.1983
Age. sex. race
Probe results Site studied
19. M. B 73. F.? 39. M.? 64.M. W 39. M. B II. M. A 67.M. W 25. M. W 6. M.?
Left cervical lymph node Soft tissue mass. right hip Left axillary lymph node Left inguinal lymph node Left cervical lymph node Large neck mass Left inguinal lymph node Maxillary sinus Cervical lymph node
HHV-6
+ + +
EBV
+ +
+ + + +
respectively. The Bluescript KS- plasmid vector (Stratagene, La Jolla, CA) without insert was used as a negative control. The inserts containing the virus sequences were cut from the plasmids by restriction endonuclease digestion and isolated on agarose gels. They were then labeled with digoxigenin by random priming using the "Genius" kit from Boehringer-Mannheim (Indianapolis) for use as probes. In situ hybridization. Slides of formalin-fixed, paraffin-embedded tissue sections were dewaxed by gentle heating and immersion in xylene and rehydrated by immersion in graded alcohols (95%,80%, and 70%ethanol). The slides were incubated for 15-30 min in 25 JLg/mL proteinase Kin TE (10 mM TRIS, pH 7.6, I mM EDTA) and 0.1%SDS at 37°C for 15 min and postfixed for 5 min in freshly prepared 4% paraformaldehyde in PBS at room temperature. The slides were prehybridized in 50%formamide, 4X standard saline citrate (SSC), 3.75% dextran sulfate, 250 JLg/mL sheared and denatured salmon sperm DNA, 250 JLg/mL tRNA, and 5X Denhardt's solution (I X = 0.02% ficoll, 0.02% polyvinylpyrrolidone, 0.02% bovine serum albumin) for 1-3 hat 48-50°C. The hybridization solution was similar to the prehybridization solution but contained 1-4 JLg/mL digoxigenin-Iabeled probe. Slides were then heated to 100°C for 4 min, chilled on ice, and incubated for 3 h at 47°C in a humidity chamber. After hybridization, slides were washed twice each with 2x SSC plus 0.1%SDS and IX SSC and 0.1%SDS at room temperature. The final two washes were at 54°C in 0.2x SSC and 0.1 % SDS. All washes were for 15-30 min with agitation. For detection of digoxigenin-Iabeled probes, the tissues were blocked for I h in blocking agent supplied by the manufacturer. exposed to antidigoxigenin for I h, and developed with 5-bromo-4-chloro-3-indolyl phosphate/4-nitroblue tetrazolium according to manufacturer's directions (Boehringer-Mannheim). Immunophenotyping. Immunophenotyping was done on fixed, paraffin-embedded tissue sections, using the avidin-biotin complex technique as previously described [25, 26]. The antibody panel included UCHL-I (CD45RO), L-26 (CD20), KP-I
(CD68) lysozyme, SIOO (all from DAKO, Santa Barbara, CA) and Leu-MI (CDI5; Becton-Dickinson, Mountain View, CA). Serology. Antibodies to HHV -6 and EBV were detected by immunofluorescence [8, 27].
Results Control sections prepared from packed HHV-6-infected culture cells and subjected to in situ hybridization showed intense purple staining of the nuclei, indicative of HHV-6 infection. Uninfected HSB-2 cells did not stain. The HHV-6 probes were specific for HHV-6-infected cells only and did not hybridize with EBV- or CMV-infected control cells. Similarly, the EBV probe was specific for EBV-infected cells only and did not cross-hybridize with HHV-6- or CMV-infected cells. Tissues from 9 cases ofSHML were examined for the presence ofEBV and HHV-6 by in situ hybridization. Seven had significant reactivity with the HHV-6 probe (table I). One biopsy specimen (case 5) reacted with EBV but not with HHV-6, another (case 3) reacted with both the HHV-6 and EBV probes, and a third sample (case I) did not react with either. The HHV-6 sequences were not uniformly distributed throughout the tissue, tending to be localized in discrete regions. Cells that stained positively were observed especially in sinuses containing abnormal histiocytes (figure I). In some individuals, multiple tissues from the same patient were available for study. All three tissue samples for case 2 and the two samples for case 4 were positive for HHV6 sequences only. Of the six samples of case 7 examined, however, four were positive for HHV-6 and two were negative. Immunoperoxidase studies were confirmatory for the diagnosis of SHML. All cases showed diffuse cytoplasmic reactivity with the S 100 protein marker. The principal cells often, but not always, had the phenotype of'histiocytes, demonstrating positive reactivity for lysozyme and KP-I. The B cell marker, L-26, was negative as was the granulocyte-associated antigen CD 15. Focal positivity for UCHL-I was seen on some histiocytic cells. In the 1 patient studied for HHV-6 and EBV antibodies, the viral capsid antibody titers to both viruses were 1:160.
Discussion SHML has been considered to be a disease characterized by abnormal immunologic response to a precipitating agent, possibly a virus. EBV has been thought to be an etiologic agent, but not all patients with this illness have antibodies to this virus [3, 5]. In a small series of patients we studied (see Acknowledgments), all had antibodies to both EBV and to HHV-6, several in high titers. In this biopsy series, a serum sample was available for only I NIH case, and antibody titers to both viruses were within the normal range.
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NOTE. Cases 1-7 were from the Armed Forces Institute of Pathology; 8 and 9 from the National Cancer Institute. HHV-6. human herpesvirus 6; EBV. Epstein-Barr virus; B. black; W. white; A. Asian.
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Figure 1. Histopathology of sinus histiocytosis with massive lymphadenopathy (SHML). Large histiocytes with abundant cytoplasm fill distended sinus. Histiocytes contain intact lymphocytes (emperipoleis), characteristic feature of SHML (arrow). (Stained with hematoxylin-eosin; original magnification X400. Bar = 10 ~m.)
Figure 2. In situ hybridization for human herpesvirus 6 in sinus histiocytosis with massive lymphadenopathy (case 2). Granular positivity in nuclei of histiocytes within distended sinus. (Original magnification X400: no counterstain. Bar = 10 ~m.)
sequelae ofHHV-6 have been identified: exanthem subitum (roseola infantum) [31, 32], heterophile negative infectious mononucleosis [l l], and hepatitis [33]; preliminary data also suggest a role in pneumonitis occurring in bone marrow transplant recipients [34]. Elevated antibody titers may be found in Kawasaki disease [35], sarcoidosis [36], malignant lymphoproliferative diseases [37], and chronic fatigue syndrome [38], but these elevations are not consistent and may be due to immunologic perturbations that allow increased viral replication rather than reflecting an etiologic relationship (for a recent review, see [I 2]). We used an in situ hybridization technique to examine> 100 other tissues, including those from patients with Burkitt's lymphoma, chronic fatigue syndrome, and a variety of Hodgkin's and non-Hodgkin's lymphomas. Although HHV-6-staining cells were found in some patients from each category, most tissues were negative
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Identification ofvirus in pathologic specimens is often considered one of the most important parameters in determining viral etiology ofdisease. The consistent identification ofEBV genome in nasopharyngeal carcinoma [28] provides important evidence towards this etiologic relationship. However, even in diseases where the virus is frequently, but not always, identified (e.g., EBV in Burkitt's lymphoma [ 13-15] and human T cell leukemia virus type I in adult T cell leukemia/ lymphoma [29]), the associated virus is generally considered to be the predominant etiologic agent but more than one precipitating cause can result in a similar pathologic lesion [l4, 29, 30]. In this study, the detection of HHV -6 in 13 of 17 tissues involved by Rosai-Dorfman disease, particularly in the abnormal cells within sinuses (figure 2), argues for a pathogenetic role for this new HHV. Thus far, at least three clinical
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Acknowledgments
We thank the following contributors for allowing us to study their cases: St. Croix Community Hospital (St. Croix Falls, WI; case I); Hye-Kung Kim, Merrithew Memorial Medical Center (Martinez, CA; case 2); F. Azizi, Kaiser Foundation Hospitals (Fontana, CA; case 3); Sheng-CW, Sharon General Hospital (Sharon, PA; case 4); A. R. Summerlin, Columbus Medical Center (Columbus, GA; case 5); Chansuda Wongsrichanalai, Department ofImmunologyjAfrins (San Francisco; case 6); and S. Jurco, South Lake Memorial Hospital (Clermont, FL; case 7). We also thank Ernest Baden, Ronald Dorfman, Elliott Foucar, Bertil E. Gader, Andrew Raubitschek, Juan Rosai, and V. Shanta for sera from patients with Rosai-Dorfman disease, which led to this current study; Gary Pearson (Georgetown University, Washington, DC) for testing these sera; and the FCRDC Document Processing Department for typing support.
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References I. Rosai J, Dorfman RF. Sinus histiocytosis with massive lymphadenopathy. A newly recognized benign clinicopathological entity. Arch Pathol 1969;87:63-70. 2. Rosai J, Dorfman RF. Sinus histiocytosis with massive lymphadenopathy: a pseudolymphomatous benign disorder. Analysis of 34 cases. Cancer 1972;30: I 174-88. 3. Foucar E, Rosai J, Dorfman RF. Sinus histiocytosis with massive lymphadenopathy (Rosai-Dorfman disease): review of the entity. Semin Diagn PathoI1990;7:19-73. 4. Foucar E, Rosai J, Dorfman RF. Sinus histiocytosis with massive lymphadenopathy. An analysis of 14 deaths occurring in a patient registry. Cancer 1984;54: 1834-40. 5. Foucar E, Rosai J, Dorfman RF, Eyman JM. Immunologic abnormalities and their significance in sinus histiocytosis with massive lymphadenopathy. Am J Clin Pathol 1984;82:515-25. 6. Komp OM. The treatment ofsinus histiocytosis with massive lymphadenopathy (Rosai-Dorfman disease). Semin Diagn Pathol 1990;7:836. 7. Dorfman RF, Warnke R. Lymphadenopathy simulating the malignant lymphomas. Hum Pathol 1974;5:519-50. 8. Salahuddin SZ, Ablashi DV, Markham PO, et at. Isolation of a new virus, HBLV, in patients with Iymphoproliferative disorders. Science 1986;234: 596-60 I. 9. Henle G, Henle W, Diehl V. Relation of Burkitt's tumor-associated herpes type virus to infectious mononucleosis. Proc Natl Acad Sci USA 1968;59:94-10 I. 10. Purtilo DT, Okano M, Grierson HL. Immune deficiency as a risk factor in Epstein-Barr virus-induced malignant diseases. Environ Health Perspect 1990;88:225-30. II. Steeper T A, Horwitz CA, Ablashi DV, et at. The spectrum of clinical and laboratory findings resulting from human herpesvirus-6 (HHV6) in patients with mononucleosis-like illnesses not resulting from Epstein-Barr virus or cytomegalovirus. Am J Clin Pathol 1990; 93:776-83. 12. Levine PH, Jarrett R, Clark D. The epidemiology of human herpesvirus-6. In: Ablashi DV, Krueger GRF, Salahuddin SZ, eds. Human herpesvirus-6: epidemiology, molecular biology and clinical pathology. Amsterdam: Elsevier Science, 1992:9-23. 13. Zur Hausen H, Schulte-Holthausen H, Klein E, et at. EBV DNA in biopsies of Burkitt tumours and anaplastic carcinomas of the nasopharynx. Nature 1970;228: 1056-8. 14. Shiramizu B, Barriga F. Neequaye J. et at. Patterns of chromosomal breakpoint locations in Burkitt's lymphoma: relevance to geography and Epstein-Barr virus association. Blood 1991 ;77: 1516-26. 15. Henle W, Henle G, Gunven P, et at. Patterns of antibodies to EpsteinBarr virus-induced early antigens in Burkitt's lymphoma. Comparison of dying patients with long-term survivors. J Natl Cancer Inst 1973;50: 1163-73. 16. Levine PH. Ablashi DV, Berard CWo et at. Elevated antibody titers to Epstein-Barr virus in Hodgkin's disease. Cancer 1971 :27:416-21. 17. Weiss LM, Movahed LA, Warnke RA, Sklar J. Detection of EpsteinBarr viral genomes in Reed-Sternberg cells of Hodgkin's disease. N Engl J Med 1989;320:502-6. 18. Bignon YJ, Bernard D. Cure H, et at. Detection of Epstein-Barr viral genomes in lymph nodes of Hodgkin's disease patients. Mol Carcinog 1990;3:9-11. 19. Pallesen G, Hamilton-Dutoit SJ, Rowe M, Young LS. Expression of Epstein-Barr virus latent gene products in tumour cells of Hodgkin's disease. Lancet 1991;337:320-2. 20. Josephs SF. Buchbinder A. Streicher H, et at. Detection of human B Iymphotropic vii-us (human herpes virus 6) sequence in B cell lymphoma tissue of three patients. Leukemia 1988;2: 132-5. 21. Jarrett RF. Gledhill S. Qureshi F. et at. Identification of human herpes-
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and we did not observe any clear association between HHV6 and other diseases (unpublished data, [39]). Additional control tissues from nonspecific lymphatic hyperplasia were negative for HHV-6 sequences. Since the region of HHV -6 infection is not uniform throughout tissue, it could be argued that some tissue sections simply miss the infected region. Thus, it may be possible that in case 7, where only four of six tissue samples were positive for HHV-6, we missed the areas of infection in the two negative tissues. We did not have access to additional sections from cases I or 5 to rule out the possibility that other areas of tissue may have contained viral sequences. The detection ofEBV in the I patient negative for HHV-6 is also of interest; whether EBV was present as a passenger virus or was a trigger for this striking immunologic response (SHML) is yet to be determined. EBV has been identified in some biopsies from Hodgkin's disease patients [17-19, 23], and there is evidence that other agents, including HHV -6, may be associated with other cases [22]. The immunotypic findings in this study are similar to those previously reported and support a histiocytic origin for the cells of SHML [40]. Although the S I00 protein is found characteristically in interdigitating reticulum cells, previous studies of reactive lymph nodes have shown positivity in sinus histiocytes when the lymph nodes showed active sinus histiocytosis [26]. Therefore, the presence of S I00 may reflect a state of activation in lesional cells. In summary, these findings suggest but do not prove that HHV-6 and, on occasion, EBV can trigger SHML. There continues to be the possibility that the viruses detected in the cells result from secondary infection, particularly since infection of a cell by one virus can make the cell susceptible to others that ordinarily cannot infect these cells [41]. An expansion of these studies to larger case series should help to resolve these issues.
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