Title: Expression of Human Herpesvirus-6 Antigens in Benign and Malignant Lymphoproliferative Diseases
Abstract: Immunohistochemistry was used to look for the expression of human herpesvirus-6 (HHV-6) antigens in a well characterized series of benign, atypical, and malignant lymphoid lesions, which tested positive for the presence of HHV-6 DNA. A panel of specific antibodies against HHV-6 antigens, characteristic either of the early (p41) or late (p101K, gp106, and gp116) phases of the viral cycle, was applied to the lymphoid tissues from 15 non-Hodgkin's lymphomas, 14 Hodgkin's disease cases, 5 angioimmunoblastic lymphadenopathies with dysproteinemia, 14 reactive lymphadenopathies, and 2 cases of sinus histiocytosis with massive lymphadenopathy (Rosai-Dorfman disease). In lymphomatous tissues, the expression of late antigens was documented only in reactive cells, and mainly in plasma cells. Of interest, the expression of the early p41 antigen was detected in the so-called “mummified” Reed-Sternberg cells, in two Hodgkin's disease cases. In reactive lymphadenopathies, the HHV-6 late antigen-expressing cells were plasma cells, histiocytes, and rare granulocytes distributed in interfollicular areas. In both cases of Rosai-Dorfman disease, the p101K showed an intense staining in follicular dendritic cells of germinal centers, whereas the gp106 exhibited an intense cytoplasmic reaction in the abnormal histiocytes, which represent the histological hallmark of the disease. The expression of HHV-6 antigens is tightly controlled in lymphoid tissues. The lack of HHV-6 antigen expression in neoplastic cells and the limited expression in degenerating Reed-Sternberg cells argue against a major pathogenetic role of the virus in human lymphomagenesis. The detection of a rather unique pattern of viral late antigen expression in Rosai-Dorfman disease suggests a possible pathogenetic involvement of HHV-6 in some cases of this rare lymphoproliferative disorder. Immunohistochemistry was used to look for the expression of human herpesvirus-6 (HHV-6) antigens in a well characterized series of benign, atypical, and malignant lymphoid lesions, which tested positive for the presence of HHV-6 DNA. A panel of specific antibodies against HHV-6 antigens, characteristic either of the early (p41) or late (p101K, gp106, and gp116) phases of the viral cycle, was applied to the lymphoid tissues from 15 non-Hodgkin's lymphomas, 14 Hodgkin's disease cases, 5 angioimmunoblastic lymphadenopathies with dysproteinemia, 14 reactive lymphadenopathies, and 2 cases of sinus histiocytosis with massive lymphadenopathy (Rosai-Dorfman disease). In lymphomatous tissues, the expression of late antigens was documented only in reactive cells, and mainly in plasma cells. Of interest, the expression of the early p41 antigen was detected in the so-called “mummified” Reed-Sternberg cells, in two Hodgkin's disease cases. In reactive lymphadenopathies, the HHV-6 late antigen-expressing cells were plasma cells, histiocytes, and rare granulocytes distributed in interfollicular areas. In both cases of Rosai-Dorfman disease, the p101K showed an intense staining in follicular dendritic cells of germinal centers, whereas the gp106 exhibited an intense cytoplasmic reaction in the abnormal histiocytes, which represent the histological hallmark of the disease. The expression of HHV-6 antigens is tightly controlled in lymphoid tissues. The lack of HHV-6 antigen expression in neoplastic cells and the limited expression in degenerating Reed-Sternberg cells argue against a major pathogenetic role of the virus in human lymphomagenesis. The detection of a rather unique pattern of viral late antigen expression in Rosai-Dorfman disease suggests a possible pathogenetic involvement of HHV-6 in some cases of this rare lymphoproliferative disorder. Human herpesvirus-6 (HHV-6) is ubiquitous in the human adult population throughout the world, with seroconversion occurring early in life.1Okuno T Takahashi K Balachandran K Shiraki K Yamanishi K Takahashi M Baba K Seroepidemiology of human herpesvirus 6 infection in normal children and adults.J Clin Microbiol. 1989; 27: 651-653PubMed Google Scholar Primary infection with HHV-6 in young children may cause exanthem subitum2Yamanishi K Okuno T Shiraki T Takahashi M Kondo T Asano Y Kurata T Identification of human herpesvirus-6 as a causal agent for exanthem subitum.Lancet. 1988; i: 1065-1067Abstract Scopus (1288) Google Scholar and acute febrile illness,3Pruksananonda P Breese-Hall C Insel RA McIntyre K Pellett PE Long CE Schnabel KC Pincus PH Stamey FR Dambaugh TR Stewart JA Primary human herpesvirus 6 infection in young children.N Engl J Med. 1992; 326: 1445-1450Crossref PubMed Scopus (248) Google Scholar whereas in adults it may cause, although very rarely, hepatitis,4Irving WL Cunningham AL Serological diagnosis of infection with human herpesvirus 6.Br Med J. 1990; 300: 156-159Crossref PubMed Scopus (61) Google Scholar encephalitis,5Merelli E Sola P Barozzi P Torelli G An encephalitic episode in a multiple sclerosis patient with human herpesvirus 6 latent infection.J Neurol Sci. 1996; 137: 42-46Abstract Full Text PDF PubMed Scopus (22) Google Scholar and a mononucleosis-like syndrome.6Steeper TA Horwitz CA Ablashi DV Salahuddin SZ Saxinger C Saltzman R Schwartz B The spectrum of clinical and laboratory findings resulting from human herpesvirus 6 (HHV-6) in patients with mononucleosis-like illness not resulting from Epstein-Barr virus or cytomegalovirus.Am J Clin Pathol. 1990; 93: 776-783PubMed Google Scholar Like all other human herpesviruses, HHV-6 is capable of remaining latent in host cells subsequent to primary infection and then reactivating in an immunocompromised state. 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Indeed, HHV-6 efficiently replicatesin vitro and induces a cytopathic effect in CD4+ T lymphocytes, but it has been also shown to be tropic for other hematopoietic cell types, namely CD8+ T lymphocytes, B cells, natural killer cells, monocytes/macrophages, and megakaryocytes.9Ablashi DV Lusso P Lung CL Salahuddin SZ Josephs SF Llana T Kramarsky B Biberfeld P Markham PD Gallo RC Utilization of human hematopoietic cell lines for the propagation and characterization of HBLV (human herpesvirus 6).Int J Cancer. 1988; 42: 787-791Crossref PubMed Scopus (130) Google Scholar, 10Kondo K Kondo T Okuno T Takahashi M Yamanishi K Latent human herpesvirus 6 infection of human monocytes: macrophages.J Gen Virol. 1991; 72: 1401-1408Crossref PubMed Scopus (318) Google Scholar, 11Levy JA Ferro F Lennette ET Oshiro L Poulin L Characterization of a new strain of HHV-6 (HHV-6 SF) recovered from the saliva of an HIV infected individual.Virology. 1990; 178: 113-121Crossref PubMed Scopus (67) Google Scholar, 12Lusso P Markham PD Tschachler ET di Marzo Veronese F Salahuddin SZ Ablashi DV Pahwa S Korhn K Gallo RC In vitro cellular tropism of human B-lymphotropic virus (human herpesvirus 6).J Exp Med. 1988; 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85: 1263-1271Crossref PubMed Google Scholar HHV-6 may be well considered an oncogenic virus, given the ability of specific viral genomic fragments to transform animal and human cell lines in vitro, which in turn may induce tumors in animal models.17Razzaque A Oncogenic potential of human herpesvirus-6 DNA.Oncogene. 1990; 5: 1365-1370PubMed Google Scholar, 18Razzaque A Williams O Wang J Rhim JS Neoplastic transformation of immortalized human epidermal keratinocytes by two HHV-6 DNA clones.Virology. 1993; 195: 113-120Crossref PubMed Scopus (50) Google Scholar, 19Thompson J Choudhury S Kashanchi F Doniger J Bernemann Z Frenkel N Rosenthal LJ A transforming fragment within the direct repeat region of human herpesvirus type 6 that transactivates HIV-1.Oncogene. 1994; 9: 1167-1175PubMed Google Scholar The first specific oncoprotein of HHV-6, called ORF-1, has also been recently identified.20Kashanchi F Araujo J Doniger J Muralidhar S Hoch R Khleif S Mendelson E Thompson J Azumi N Brady J Luppi M Torelli G Rosenthal LJ Human herpesvirus (HHV-6) ORF-1 transactivating gene exhibits malignant transforming activity and its protein binds to p53.Oncogene. 1997; 14: 359-367Crossref PubMed Scopus (79) Google Scholar Furthermore, since its first isolation from the peripheral blood of patients with lymphomas,21Salahuddin SZ Ablashi DV Markham PD Josephs SF Sturzenegger S Kaplan M Halligan M Biberfeld P Wong-Staal F Kramarsky B Gallo RC Isolation of a new virus, HBLV, in patients with lymphoproliferative disorders.Science. 1986; 234: 596-600Crossref PubMed Scopus (1225) Google Scholar serological22Torelli G Marasca R Luppi M Selleri L Ferrari S Narni F Mariano MT Federico M Ceccherini-Nelli L Bendinelli M Montagnani G Montorsi M Artusi A Human herpesvirus-6 in human lymphomas: identification of specific sequences in Hodgkin's lymphomas by polymerase chain reaction.Blood. 1991; 77: 2251-2258PubMed Google Scholar, 23Levine PH Ebbesen P Ablashi DV Saxinger WC Nordentoft A Connelly RR Antibodies to human herpesvirus-6 and clinical course in patients with Hodgkin's disease.Int J Cancer. 1992; 51: 53-57Crossref PubMed Scopus (29) Google Scholar and molecular22Torelli G Marasca R Luppi M Selleri L Ferrari S Narni F Mariano MT Federico M Ceccherini-Nelli L Bendinelli M Montagnani G Montorsi M Artusi A Human herpesvirus-6 in human lymphomas: identification of specific sequences in Hodgkin's lymphomas by polymerase chain reaction.Blood. 1991; 77: 2251-2258PubMed Google Scholar, 24Jarrett RF Gledhill S Qureshi F Crae SH Madhock R Brown I Evans I Krajewski A O'Brien CJ Cartwright RA Venables P Onions DE Identification of human herpesvirus-6 specific DNA sequences in two patients with non-Hodgkin's lymphoma.Leukemia. 1988; 2: 496-502PubMed Google Scholar, 25Josephs SF Buchbinder A Streicher HZ Ablashi DV Salahuddin SZ Guo H-G Wong-Staal F Cossman J Raffeld M Sundeen J Levine P Biggar R Krueger GRF Fox RI Gallo RC Detection of human B lymphotropic virus (human herpesvirus 6) sequences in B cell lymphoma tissues of three patients.Leukemia. 1988; 2: 132-135PubMed Google Scholar, 26Luppi M Marasca R Barozzi P Artusi T Torelli G Frequent detection of human herpesvirus-6 sequences by polymerase chain reaction in paraffin-embedded lymph nodes from patients with angioimmunoblastic lymphadenopathy and angioimmunoblastic lymphadenopathy-like lymphoma.Leuk Res. 1993; 17: 1003-1011Abstract Full Text PDF PubMed Scopus (49) Google Scholar, 27Di Luca D Dolcetti R Mirandola P de Re V Secchiero P Carbone A Boiocchi M Cassai E Human herpesvirus 6: a survey of presence and variant distribution in normal peripheral lymphocytes and lymphoproliferative disorders.J Infect Dis. 1994; 99: 533-535Google Scholar studies have suggested an association between HHV-6 infection and lymphoproliferative diseases. At present, it is still largely unknown which cell types harbor HHV-6 in lymphoid tissues in vivo. The limited number of viral genomes in infected cells also hampers the application of DNA in situ hybridization techniques to identify the target cells of HHV-6 infection, either reactive or neoplastic, in pathological lymphoid tissues positive for the presence of HHV-6 DNA, as determined by polymerase chain reaction (PCR). The detection of specific HHV-6 antigens by immunohistochemistry has provided a unique and sensitive tool for the identification of infected cells both in fresh cellular populations and in archival tissue sections. Recently, this technical approach has been revealed to be successful in localizing HHV-6-infected cells in brain tissues from patients with multiple sclerosis, providing the first strong evidence toward an etiological relationship.28Challoner PB Smith KT Parker JD MacLeod DL Coulter SN Rose TM Schultz E Bennett JL Garber RL Chang M Schad PA Stewart PM Nowinski Brown JP Burmer GC Plaque-associated expression of human herpesvirus 6 in multiple sclerosis.Proc Natl Acad Sci USA. 1995; 92: 7440-7444Crossref PubMed Scopus (563) Google Scholar Thus, we judged it appropriate to use immunohistochemistry with a panel of antibodies for different viral antigens to look for the presence and distribution of HHV-6 antigen-expressing cells in the lymphoid tissues positive for the presence of HHV-6 DNA from a well characterized series of patients with benign, atypical, and malignant lymphoproliferative diseases. Tissues from 15 non-Hodgkin's lymphomas (NHL), 14 Hodgkin's disease (HD) cases, 5 angioimmunoblastic lymphadenopathies with dysproteinemia (AILD), 14 reactive lymphadenopathies, and 2 cases of sinus histiocytosis with massive lymphadenopathy (Rosai-Dorfman disease) were included in the study. In all cases of NHL and HD, the diagnosis was determined on the basis of the histological analysis of a lymph node biopsy, and revised according to the REAL classification.29Harris NL Jaffe ES Stein H Banks PM Chan JKC Cleary ML Delsol G De Wolf-Peeters C Falini B Gatter KC Grogan TM Isaacson PG Knowles DM Mason DY Muller-Hermelink H-K Pileri S Piris MA Ralfkiaer E Warnke RA A revised European-American classification of lymphoid neoplasms: a proposal from the International Lymphoma Study Group.Blood. 1994; 84: 1361-1392PubMed Google Scholar All of the NHL cases were subjected to standard immunophenotyping procedures as well as to Southern blot analysis of immunoglobulin (Ig) heavy and light chain genes and of T-cell receptor β-chain gene, to confirm either the B- or T-cell lineage.22Torelli G Marasca R Luppi M Selleri L Ferrari S Narni F Mariano MT Federico M Ceccherini-Nelli L Bendinelli M Montagnani G Montorsi M Artusi A Human herpesvirus-6 in human lymphomas: identification of specific sequences in Hodgkin's lymphomas by polymerase chain reaction.Blood. 1991; 77: 2251-2258PubMed Google Scholar The distribution of NHL cases according to lymphoma subtype was as follows: 4 follicular center, 2 mantle cell, 2 marginal zone B cell, 3 diffuse large B cell, 1 Burkitt's, 2 peripheral T cell, and 1 anaplastic large cell. The distribution of HD cases according to subtype was as follows: 6 mixed cellularity, 5 nodular sclerosis, 2 lymphocyte predominance, and 1 lymphocyte depletion. The series of 14 reactive lymphadenopathies consisted of 5 cases of florid follicular hyperplasia, 4 cases with a predominantly paracortical lesion, 4 cases with sinus histiocytosis, and 1 histiocytic necrotizing lymphadenitis. All of these tissue samples harbored HHV-6 DNA sequences, as detected by PCR, which was performed as previously reported.22Torelli G Marasca R Luppi M Selleri L Ferrari S Narni F Mariano MT Federico M Ceccherini-Nelli L Bendinelli M Montagnani G Montorsi M Artusi A Human herpesvirus-6 in human lymphomas: identification of specific sequences in Hodgkin's lymphomas by polymerase chain reaction.Blood. 1991; 77: 2251-2258PubMed Google Scholar One NHL,30Torelli G Barozzi P Marasca R Cocconcelli P Merelli E Ceccherini-Nelli L Ferrari S Luppi M Targeted integration of human herpesvirus 6 in the p arm of chromosome 17 of human peripheral blood mononuclear cells in vivo.J Med Virol. 1995; 46: 178-188Crossref PubMed Scopus (102) Google Scholar two HD,22Torelli G Marasca R Luppi M Selleri L Ferrari S Narni F Mariano MT Federico M Ceccherini-Nelli L Bendinelli M Montagnani G Montorsi M Artusi A Human herpesvirus-6 in human lymphomas: identification of specific sequences in Hodgkin's lymphomas by polymerase chain reaction.Blood. 1991; 77: 2251-2258PubMed Google Scholar and the five AILD26Luppi M Marasca R Barozzi P Artusi T Torelli G Frequent detection of human herpesvirus-6 sequences by polymerase chain reaction in paraffin-embedded lymph nodes from patients with angioimmunoblastic lymphadenopathy and angioimmunoblastic lymphadenopathy-like lymphoma.Leuk Res. 1993; 17: 1003-1011Abstract Full Text PDF PubMed Scopus (49) Google Scholar cases have been previously described. In one NHL case and in two HD cases, the viral copy number was so high that it was detectable also by Southern blot analysis.22Torelli G Marasca R Luppi M Selleri L Ferrari S Narni F Mariano MT Federico M Ceccherini-Nelli L Bendinelli M Montagnani G Montorsi M Artusi A Human herpesvirus-6 in human lymphomas: identification of specific sequences in Hodgkin's lymphomas by polymerase chain reaction.Blood. 1991; 77: 2251-2258PubMed Google Scholar, 30Torelli G Barozzi P Marasca R Cocconcelli P Merelli E Ceccherini-Nelli L Ferrari S Luppi M Targeted integration of human herpesvirus 6 in the p arm of chromosome 17 of human peripheral blood mononuclear cells in vivo.J Med Virol. 1995; 46: 178-188Crossref PubMed Scopus (102) Google Scholar Genotype characterization, performed by a PCR assay,26Luppi M Marasca R Barozzi P Artusi T Torelli G Frequent detection of human herpesvirus-6 sequences by polymerase chain reaction in paraffin-embedded lymph nodes from patients with angioimmunoblastic lymphadenopathy and angioimmunoblastic lymphadenopathy-like lymphoma.Leuk Res. 1993; 17: 1003-1011Abstract Full Text PDF PubMed Scopus (49) Google Scholar showed the HHV-6 variant B genome in all cases, with the exception of three AILD cases, showing HHV-6 variant A genome in two cases and a mixture of HHV-6 variant A and B genomes in one case, as previously reported.26Luppi M Marasca R Barozzi P Artusi T Torelli G Frequent detection of human herpesvirus-6 sequences by polymerase chain reaction in paraffin-embedded lymph nodes from patients with angioimmunoblastic lymphadenopathy and angioimmunoblastic lymphadenopathy-like lymphoma.Leuk Res. 1993; 17: 1003-1011Abstract Full Text PDF PubMed Scopus (49) Google Scholar Furthermore, tissues from five NHL, four HD, and five reactive lymphadenopathy cases, which tested negative for HHV-6 sequences by PCR, were also included in the study to serve as negative controls in immunohistochemical experiments. Sections from formalin-fixed, paraffin-embedded tissues were stained with an avidin-biotin complex immunodetection system in a TechMate instrument (BioTek Solutions, Santa Barbara, CA). Sections (4 μm) mounted on positively charged slides were deparaffinized with xylene and rehydrated, immersed for 10 minutes in 3% hydrogen peroxide/methanol to quench endogenous peroxidase, and microwaved for 10 minutes in citrate buffer.28Challoner PB Smith KT Parker JD MacLeod DL Coulter SN Rose TM Schultz E Bennett JL Garber RL Chang M Schad PA Stewart PM Nowinski Brown JP Burmer GC Plaque-associated expression of human herpesvirus 6 in multiple sclerosis.Proc Natl Acad Sci USA. 1995; 92: 7440-7444Crossref PubMed Scopus (563) Google Scholar After slow cooling, the sections were incubated successively with primary antibody, biotin-labeled goat anti-mouse or anti-rabbit Ig, streptavidin-biotin peroxidase complex, and 3,3′-diaminobenzidine tetrahydrochloride chromogen. Tissues were counterstained with hematoxylin. Mouse IgG1 monoclonal antibody (mAb) to HHV-6B virion protein p101K (late antigen) was obtained from P. Pellett (Centers for Disease Control and Prevention, Atlanta, GA) and from Chemicon International (Temecula, CA) and used at a dilution of 1:200.28Challoner PB Smith KT Parker JD MacLeod DL Coulter SN Rose TM Schultz E Bennett JL Garber RL Chang M Schad PA Stewart PM Nowinski Brown JP Burmer GC Plaque-associated expression of human herpesvirus 6 in multiple sclerosis.Proc Natl Acad Sci USA. 1995; 92: 7440-7444Crossref PubMed Scopus (563) Google Scholar, 31Pellett PE Sánchez-Martínez D Dominguez G Black JB Anton E Greenamoyer C Dambaugh TR A strongly immunoreactive virion protein of human herpesvirus 6 variant B strain Z29: identification and characterization of the gene and mapping of a variant-specific monoclonal antibody reactive epitope.Virology. 1993; 196: 521-531Crossref Scopus (73) Google Scholar Mouse IgG2a mAb C5 to DNA binding protein p41 (early antigen) was used at a dilution of 1:50 (Biodesign International, Kennebunkport, ME).28Challoner PB Smith KT Parker JD MacLeod DL Coulter SN Rose TM Schultz E Bennett JL Garber RL Chang M Schad PA Stewart PM Nowinski Brown JP Burmer GC Plaque-associated expression of human herpesvirus 6 in multiple sclerosis.Proc Natl Acad Sci USA. 1995; 92: 7440-7444Crossref PubMed Scopus (563) Google Scholar, 32Agulnick AD Thompson JR Iyengar S Pearson G Ablashi DV Ricciardi RP Identification of a DNA-binding protein of human herpesvirus 6, a putative DNA polymerase stimulatory factor.J Gen Virol. 1993; 74: 1003-1009Crossref PubMed Scopus (42) Google Scholar Mouse IgG2b mAb to gp106 (late antigen)33Okuno T Shao H Asada H Shiraki K Takahashi M Yamanishi K Analysis of human herpesvirus 6 glycoproteins recognized by monoclonal antibody OHV1.J Gen Virol. 1992; 73: 443-447Crossref PubMed Scopus (47) Google Scholar and IgG2b mAb to gp116 (late antigen) (both from Advanced Biotechnologies, Inc., Columbia, MD) were used at a dilution of 1:50. The specificity of these antibodies was confirmed by testing isotype-matched control mouse mAbs against human IgG (IgG1, X931; IgG2a, X943; and IgG2b, X944; Dakopatts, Glostrup, Denmark) in selected HHV-6-positive cases. Control and test antibodies were used at the same IgG concentrations. Antibodies to other herpesviruses included the anti-human cytomegalovirus mAbs DDG9 and CCH2 (Dakopatts) to early and immediate-early antigens, rabbit polyclonal anti-herpes simplex virus 1 antibody B114 (Dakopatts), and anti-Epstein-Barr virus (EBV) mAb to latent membrane protein-1 (LMP-1) (Dakopatts). The antibody panel also included CD3, UCHL-1 (CD45RO), L-26 (CD20), KP1 (CD68), S-100, CD21, Ber-H2 (CD30), and EMA/E29 (all from Dakopatts), and Leu-M1 (CD15; Becton-Dickinson, San Jose, CA). All antibodies were used as recommended by the manufacturers. The expression of HHV-6 antigens was investigated by immunohistochemistry in 15 NHL cases, which tested positive for HHV-6 DNA by PCR. One of these cases was also positive by Southern blot analysis, indicating high copy number latent integration of the HHV-6 genome, as previously reported.30Torelli G Barozzi P Marasca R Cocconcelli P Merelli E Ceccherini-Nelli L Ferrari S Luppi M Targeted integration of human herpesvirus 6 in the p arm of chromosome 17 of human peripheral blood mononuclear cells in vivo.J Med Virol. 1995; 46: 178-188Crossref PubMed Scopus (102) Google Scholar Neoplastic cells were consistently negative for HHV-6 antigens in all NHL tissues examined, even in the B-NHL case (follicular center type) with so a high copy number that it tested positive for HHV-6 DNA by Southern blot analysis. Cytoplasmic staining was observed with the antibody p101K in rare plasma cells interspersed among neoplastic cells and in some isolated spindle-shaped stromal cells located in the lymph node capsule and in the surrounding fibroadipose tissue. No reactivity was observed with antibodies gp106, gp116, and p41. In five cases of AILD, previously shown to be positive for HHV-6 DNA by PCR,26Luppi M Marasca R Barozzi P Artusi T Torelli G Frequent detection of human herpesvirus-6 sequences by polymerase chain reaction in paraffin-embedded lymph nodes from patients with angioimmunoblastic lymphadenopathy and angioimmunoblastic lymphadenopathy-like lymphoma.Leuk Res. 1993; 17: 1003-1011Abstract Full Text PDF PubMed Scopus (49) Google Scholar the immunostaining was observed with antibody p101K, gp106, and gp116 in scattered plasma cells. In two cases, focal collections of positively staining plasma cells were present (Figure 1A). No reactivity was observed with antibody p41. The expression of HHV-6 antigens was investigated by immunohistochemistry in 14 lymph nodes affected by HD that were positive for HHV-6 DNA by PCR. Two of these cases were positive also by Southern blot analysis, as previously reported.22Torelli G Marasca R Luppi M Selleri L Ferrari S Narni F Mariano MT Federico M Ceccherini-Nelli L Bendinelli M Montagnani G Montorsi M Artusi A Human herpesvirus-6 in human lymphomas: identification of specific sequences in Hodgkin's lymphomas by polymerase chain reaction.Blood. 1991; 77: 2251-2258PubMed Google Scholar In all cases, complete effacement of the lymph node architecture was observed, and no remnants of previous normal structure could be detected. Only rare cells were found to exhibit cytoplasmic reaction for HHV-6 antigens, as evidenced by the p101K and gp116 antibodies. No staining was observed with the gp106 antibody. The morphology of the positive cells was that of reactive histiocytes and plasma cells. Isolated granulocytes also showed a positive reaction. Reed-Sternberg (R-S) and Hodgkin cells were consistently negative for HHV-6 antigens. The p41 antibody stained rare plasma cells and isolated granulocytes in all cases. Of interest, in the two cases with a high enough copy number of HHV-6 sequences to be detectable by Southern blot analysis, this antibody also showed positive immunohistochemical staining in R-S cells with shrunken cytoplasm and pyknotic nuclei. These were considered to represent degenerating “mummified” cells34Henry K Neoplastic disorders of lymphoreticular tissue: thymus, lymph nodes, spleen and lymphatics.in: Symmers WSC Systemic Pathology. vol 7. Churchill Livingstone, London1992: 868-879Google Scholar (Figure 1, B and C). No staining was observed in viable R-S and Hodgkin cells with antibody p41 (Figure 1D). In the same cases, there was clear evidence of staining of rare plasma cells and granulocytes in the reactive component, in addition to the positive mummified cells. The HD cases were also examined with anti-EBV mAb to LMP-1. Positive reactivity was observed in seven cases and restricted only to R-S and Hodgkin cells, whereas reactive lymphoid cells were consistently negative (not shown). Of note, in the two HD cases with mummified cells positive for HHV-6 p41, immunostaining for EBV LMP-1 was negative both in viable R-S and Hodgkin cells and in mummified cells. Moreover, using immunohistochemistry for EBV LMP-1 and for four HHV-6 antigens, we could not demonstrate the co-infection of the two viruses in lymphoid cells, as the only lymphoid cells expressing HHV-6 antigens were rare plasma cells, which invariably stained negative for LMP-1. Fourteen nonneoplastic lymph nodes, including five cases of florid follicular hyperplasia, four cases with a predominantly paracortical lesion, four cases with sinus histiocytosis, and one histiocytic necrotizing lymphadenitis as well as two cases of Rosai-Dorfman disease, were studied for the expression of HHV-6 antigens. All of the examined cases were positive for HHV-6 DNA sequences by PCR. In most cases, only isolated cells stained positive with the HHV-6-specific antibodies p101K, gp106 and gp116. Antibody p41 showed no reaction. The number of immunopositive cells was always less than 1% of the total lymph node cell burden, being usually lower than 0.5%. No staining was observed in the single case of histiocytic necrotizing lymphadenitis. Positive cells were found to correspond to plasma cells and histiocytes, whereas endothelial cells, follicular dendritic cells, and different types of lymphoid cells (lymphocytes, centrocytes, centroblasts, and immunoblasts) were consistently immunonegative. Isolated granulocytes, present in dilated sinuses in two cases of reactive lymphadenopathy with sinus histiocytoses, showed a positive reaction. Positive cells were not homogeneously distributed in the nodal tissue but appeared to be scattered in interfollicular regions (Figure 2A). Hyperplastic germinal centers were usually negative, showing a bland, doubtful staining only in one case. An interesting reactivity with p101K and gp106 antibodies was observed in both cases of Rosai-Dorfman disease. The p101K antibody exhibited an intense staining in follicular dendritic cells of germinal centers (Figure 2B), both hyperplastic and burned out, present in areas in which the normal nodal architecture was still retained. In the two Rosai-Dorfman disease cases, adjacent tissue sections were stained either with anti-HHV-6 p101K or with CD21 antibodies, respectively. The same distribution of positive cells in the follicles was observed with both antibodies, providing further evidence that the HHV-6-positive cells in the follicles corresponded to follicular dendritic cells. With antibody gp106, an intense granular positive reaction was observed in the cytoplasm of abnormal histiocytes located within distended sinuses (Figure 2, C and D). Emperipolesis was present inside so