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lymphoid neoplasms, especialy Hodgkin's dis- ease. To investigate this point, we evaluated the presence and distribution of HHV-6 DNA by. Southern blot ...
American Journal of Pathology, Vol. 149, No. 5, November 1996 Copyright © American Society for Investigative Pathology

Human Herpesvirus 6 and Epstein-Barr Virus in Hodgkin's Disease A Controlled Study by Polymerase Chain Reaction and in Situ Hybridization

Guido Valente,* Paola Secchiero,t Paolo Lusso,* Maria Cristina Abele,* Cristina Jemma,* Gigliola Reato,* Simonetta Kerim,* Robert C. Gallo,t and Giorgio Palestro* From the Department of Biomedical Sciences and Oncology,* School of Medicine and San Giovanni Hospital, University of Torino, Torino, Italy; the Laboratory of Tumor Cell Biology,t National Cancer Institute, Bethesda, Maryland; and the Unit of Human Virology,t Department of Biological and Technological Research, San Raffaele Scientific Institute, Milano, Italy

Human herpesvirus-6 (HHV-6), a T-lymphotropic double-stranded DNA virus highly endemic in human populations, has been suggested to play a possible role in the development of

lymphoid neoplasms, especialy Hodgkin's disease. To investigate this point, we evaluated the presence and distribution of HHV-6 DNA by Southern blot, nested polymerase chain reaction, and in situ hybridization in a series of lympho-

proliferative disorders including 73 Hodgkin's disease cases, 15 non-Hodgkin lymphomas, and 19 reactive lymph nodes. A high prevalence of HHV-6 infection was observed within the Hodgkin's disease category by polymerase chain reaction (38 of52, 73%) and in situ hybridization (47 of 57, 82.4%); however, a similar prevalence was found in non-Hodgkin's lymphomas (10 of 15, 66.6%) and reactive lymph nodes (13 of 19, 68.4%). In no case did Southern blot detect viral DNA, suggesting that the neoplastic tissue contained a low number of HHV-6 copies. In situ hybridization showed that the HHV-6 positivity was restricted to lymphocytes, whereas Hodgkin and Reed-Sternberg ceUs were consistently negative. Immunohistochemical staining with specific

monoclonal antibodies against viral structural proteins was also negative, indicating the absence of a productive infection. No relationship was observed between HHV-6positivity and histological type, clinical parameters, and outcome ofthe disease. In the same series, a high proportion of cases (39 of52, 75%) showed the presence of the Epstein-Barr virus (EBV) genome by polymerase chain reaction, in situ hybridization for Epstein-Barr-virus-encoded smaU RNA and immunohistochemical detection of latent membrane protein-1 gave similar results (73.6% ofpositive cases with both methods). In 54.9% of the cases, both sequences of HHV-6 and Epstein-Barr virus DNA were found, suggesting that a synergism of the two viruses may occur. However, the lack of detectable HHV-6 DNA in Reed-Sternberg and Hodgkin's ceUs seems to argue against such an interpretation. Based on these results, HHV-6 does not appear to play a specific role in the pathogenesis of Hodgkin's disease. (Am J Pathol 1996, 149:1501-1510)

Human herpesvirus 6 (HHV-6) is an enveloped virus approximately 200 nm in diameter. Its icosadeltahedral nucleocapsid surrounds an electron-dense core containing a large double-stranded DNA genome.) HHV-6 has a primary tropism for CD4+ T cells,2-4 but it can also replicate in CD8+ T lymphocytes,5 Epstein-Barr virus (EBV)-infected B cells,6 macrophages,7 and fibroblasts.' Seroepidemiological surSupported by Associazione Italiana Ricerca sul Cancro (Milano, Italy) and CNR of Italy (Progetto Finalizzato ACRO). M. C. Abele is a fellow of Comitato Gigi Ghirotti. Accepted for publication June 18, 1996. Address reprint requests to Dr. Guido Valente, Dipartimento di Scienze Biomediche e Oncologia Umana, Via Santena 7, 1-10126 Torino, Italy.

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veys have shown that HHV-6 is highly prevalent in human populations in different geographic areas.9 Isolates have been classified into two major subgroups (A and B) on the basis of specific restriction endonuclease polymorphism patterns, reactivity with specific antisera, and cellular tropism.10-12 Epidemiology of the A variant is still largely undefined, but the B variant apparently is common in human populations and associated with the only disorder conclusively linked to HHV-6, exanthema subitum.13 Recently, some benign and malignant lymphoproliferative disorders have been supposed to be pathogenetically associated with HHV-6 infection. HHV-6 has been identified by virus isolation and/or molecular techniques in cases of mononucleosis-like syndrome,14 non-Hodgkin's lymphomas (NHLs) associated or not with acquired immunodeficie~ncy

syndrome (AIDS),15-18 angioimmunoblastic lymphadenopathy and angioimmunoblastic-lymphadenopathy-like lymphomas,19 Kikuchi's lymphadenitis,20 and Rosai-Dorfman disease.21 Moreover, high levels of anti-HHV-6 antibodies have been observed in patients with Hodgkin's disease (HD),22 although the same authors showed the presence of HHV-6 DNA sequences with polymerase chain reaction (PCR) in only 3 of 28 (12%) of the neoplastic tissues. These 3 cases were also positive for EBV and showed the same histological subtype (nodular sclerosis with lymphocyte depletion) as well as an unfavorable clinical course. To further investigate the role of HHV-6 in HD, we studied a series of 73 HD cases using Southern blot analysis and PCR on frozen tissue, and in situ hybridization (ISH) on paraffin-embedded material. The main aims of the study were to elucidate whether HHV-6 frequently infects HD lymph nodes and whether the neoplastic cells of HD are infected by the virus, to confirm the previously suggested relationship between HHV-6 infection and clinical course, and to determine the entity of the EBV/HHV-6 co-infection in HD.

Materials and Methods

Samples A total of 73 cases of HD were studied (Table 1). The histological diagnoses of all cases were reviewed and classified according to British National Lymphoma Investigation.23'24 The main clinical parameters (stage, presence of bulky disease, response to therapy, and overall and disease-free survival) were obtained from the clinical files. In 52 cases, frozen material was stored at -80°C and available for DNA extraction; PCR analysis for HHV-6 and EBV DNA

Table 1.

Cases of Hodgkin's Disease Included in the Study

Number of cases Lymphocyte predominance NS NS II NS interfollicular Mixed cellularity Lymphocyte depletion Total

2 32 18 4 14 3 73

was carried out in these cases, and Southern blots for HHV-6 DNA in 30 cases. Frozen tissues from 15 cases of B and T NHL and from 19 reactive lymph nodes were used as controls. Immunohistochemical staining with a pool of monoclonal antibodies against HHV-6 structural proteins was performed on fresh tissue in 25 cases. A total of 57 cases, including 20 with no available frozen tissue for DNA extraction, were tested by ISH for HHV-6 DNA and Epstein-Barr-encoded small RNA (EBER) on paraffin sections. Immunohistochemical detection of EBV latent membrane protein-1 (LMP1) was also carried out in the same cases.

Southern Blot Analysis High molecular weight DNA, extracted from lymph node samples by standard methods, was digested with HindlIl and EcoRI restriction endonucleases (Boehringer Mannheim, Mannheim, Germany), sizefractionated by electrophoresis through 0.8% agarose gel, and transferred to nitrocellulose membranes (Hybond C-extra, Amersham, Little Chalfont, UK). Membranes were hybridized with randomly primed 32P-labeled probe pZVH14.25

PCR Analysis Detection and variant characterization of the HHV-6 genome was performed by nested PCR on 1 ,Ag of purified genomic DNA, as previously reported.26 Variant B subclasses B1 and B2 were distinguished from their restriction pattern after BgIll endonuclease digestion of the PCR product.26 The sensitivity of our test was between 5 and 10 HHV-6 genome equivalents/,tg of DNA. PCR amplification of EBV DNA was performed using a reaction mixture containing 1 j,g of genomic DNA, 1 ,umol/L of each primer (TC70 and TC72; Research Genetics, Huntsville, AL), 400 ,tmol/L of each dNTP, 1X PCR buffer (10 mmol/L Tris/HCI, pH 8.3, 50 mmol/L KCI), 1.7 mmol/L MgCI2, and 2.5 U of Taq polymerase (Perkin-Elmer Cetus,

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Norwalk, CT) in 100 p.l total volume. DNA extracted from the Raji cell line was used as a positive control for each reaction. PCR was performed using a Perkin-Elmer Cetus thermal cycler with the following setting: denaturation at 920C for 1 minute, annealing at 650C for 1 minute, and extension at 720C for 1 minute for 45 cycles. Primers TC70 and TC72 amplify a 265-bp fragment of BMLF1, a single-copy gene of the EBV genome.27 Experiments to assess the sensitivity of the PCR showed that the specific amplified product of the 265-bp BMLF1 gene region was detected in as little as 3 pg of Raji cell DNA, corresponding to 0.3 Raji cells or 15 copies of the EBV genome.28,29 A 10-,ul aliquot of the amplification product was run on 2% agarose gel and stained with ethidium bromide. The strictest precautions were taken to avoid false positive results due to PCR contamination. The cases/DNAs that proved negative were shown to be amplifiable when tested by ,B-globin PCR using primers PCO4 and GH20 (Perkin Elmer Cetus) as previously described.30

(washing buffer) for 10 minutes. Localization of the hybridized digoxigenin-labeled probe was detected as follows. Slides were incubated with a polyclonal sheep anti-digoxigenin antibody conjugated with alkaline phosphatase diluted 1:500 (Boehringer Mannheim) for 2 hours in a humid box. They were then washed twice with washing buffer and flooded with freshly prepared alkaline phosphatase development reagent (X-phosphate, 5-bromo-4-chloro-3-indolyl phosphate toluidine salt, plus nitroblue tetrazolium). After development of the blue end-point in the dark for 16 hours, the slides were placed in stop buffer, rinsed in PBS, and mounted in aqueous mounting medium. The specificity of the reaction was checked on cytospins of a cell line infected by HHV-6. Negative controls were performed by omitting probe from the hybridization mixture and/or by using an unrelated probe from the murine virus X-MuLV.

In Situ Hybridization for HHV-6 DNA

Oligonucleotides (a mixture of two 30-mers) obtained from sequences of EBV mRNA (EBER 1-2) were used as probe (Dako, Glostrup, Denmark). The method was carried out as previously reported31 with some modifications. Briefly, paraffin-embedded tissue sections, once dewaxed and rehydrated, were digested with 0.003 mg/ml proteinase K at 370C for 30 minutes and then hybridized with EBER probes (30 p.1/slide) for 2 hours at 370. After a rapid wash in Tris-buffered saline, slides were incubated first with an alkaline-phosphatase-conjugated rabbit F(ab) antibody for 30 minutes at room temperature and then with the alkaline phosphatase substrate 5-chromo-4-chloro-3-indolyl phosphate/nitroblue tetrazolium for 60 minutes in the dark. The reaction was stopped in running tap water and slides were counterstained and mounted as for DNA ISH.

Paraffin sections were dewaxed and rehydrated through a series of clean xylene and ethanol washings, rinsed in PBS, immersed in 0.01% Triton X-100 in phosphate-buffered saline (PBS) for 90 seconds, and rinsed again in PBS. The slides were incubated in 0.25 mg/ml proteinase K (Sigma Chemical Co., St. Louis, MO) at 370C for 12 minutes, and the digestion was stopped by rinsing the slides in cold PBS. After protein digestion, the slides were covered with 50 ,ul of prehybridization mixture (1X Denhardt's solution, 50% formamide, 4X standard saline citrate (SSC), 500 p.g/ml salmon sperm DNA, 1.5% dextran sulfate, 250 p.g/ml tRNA; Boehringer Mannheim) and incubated for 2 hours at room temperature in a humid box, and 10 p.l of the same mixture, containing 10 ng of digoxigenin-labeled probe (hybridization mixture) was placed on a coverslip and transferred to the slide. The probe used was a 9.0-kb insert obtained from the probe pZVH14 as previously described25 and digoxigenin labeled with the random prime method. Tissue DNA was denaturated at this stage by heating the slides at 950C for 10 minutes. Hybridization was then allowed to proceed overnight at 370C. The coverslips were then removed and the slides washed with the following post-hybridization schedule: 2X SSC for 1 hour at room temperature, 1 X SSC for 1 hour at room temperature, 0.5X SSC for 30 minutes at 370C, and 0.1X SSC for 30 minutes at room temperature. Slides were then transferred to 100 mmol/L Tris/HCI, pH 7.5, 150 mmol/L NaCI buffer

In Situ Hybridization for EBV mRNAs

Immunohistochemistry EBV LMP1 was immunohistochemically detected on paraffin sections incubated with the specific antibody (Dako), using an ABC method.32 Cryostat sections were fixed in cold acetone and incubated with a pool of monoclonal antibodies against HHV-6 proteins. They included 7A2 (anti-gplO2) and C5 (antigp41/38) (ABI, Rockville, MD), and P11G1-G9C7 (Biotech, Rockville, MD) (all of which react against both HHV-6 A and B). As above, an ABC method was used for detection, with diaminobenzidine tetrachloride as chromogen.

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Table 2. Results of PCR, in Situ Hybridization, and Immunobistocbemistry in Hodgkin's Disease Cases HHV-6-DNA-positive cases

Histology NDLP NS NS II NSIF MC LD Total

Number of cases

PCR (%)

ISH (%)

2 32 18 4 14 3 73

0/1 (0) 20/23 (86.9) 8/13 (61.5) 2/4 (50) 7/10 (70) 1/1 (100) 38/52 (73)

0/1 (0) 18/21 (85.7) 12/16 (75)

3/4 (75)

11/12 (91.6) 3/3 (100)

47/57 (82.4)

EBER-positive cases Both HRSC/L

HHV-6-positive EBV-DNA-positive Only HRSC cases, MAb (%) cases, PCR (%) (%) ND

1/1 (100) 17/23 (73.9) 10/13 (76.9) 2/4 (50) 8/10 (80) 1/1 (100) 39/52 (75)

0/10 (0) 0/8 (0) 2/2 (100)* 0/5 (0) ND

2/25 (8)

0/1 (0/1)

4/21 (19.1) 4/16 (25) 1/4 (25) 0/12 (0) 3/3 (100) 12/57 (21)

LMP-positive

(%)

cases, MAb (%)

1/1 (100) 11/21 (52.3)

1/1 (100) 15/21 (71.4) 12/16 (75) 2/4 (50) 9/12 (75) 3/3 (100) 42/57 (73.7)

8/16 (50) 1/4 (25)

9/12 (75) 3/3 (100) 33/57 (57.9)

MAb, monoclonal antibody; ND, not done; NDLP, nodular and diffuse lymphocyte predominance; NSIF, NS with interfollicular pattern; MC, mixed cellularity; LD, lymphocyte depletion; HRSC/L, Hodgkin and Reed-Sternberg cells and surrounding lymphocytes. *Positivity in residual germinal centers.

Statistical Analysis

subtype, in 70% of the mixed cellularity type, and in the single case of lymphocyte depletion. Expression of B1 and B2 variants showed a different distribution within NS subtypes; B2 was observed in 70% of the NS cases whereas Bl was found in 62.5% of the NS 11 cases (Table 3). Of 15 NHL cases, 10 (66.6%), including 8 B-cell and 2 T-cell lymphomas, were positive for HHV-6 DNA by PCR. Of 19 reactive lymph nodes used as controls, 13 (68.4%) were positive for HHV-6 DNA sequences.

A univariate analysis was carried out considering for each case the positivity for HHV-6 and EBV and the main clinical parameters, including overall and disease-free survival, clinical stage, and presence of bulky disease. The results were analyzed with the BMPD Statistical Software (Los Angeles, CA).

Results HHV-6 Studies

In Situ Hybridization

PCR Analysis

Of 57 cases investigated by ISH, 47 (82.4%) were positive for HHV-6 DNA (Table 2). Good agreement was seen between ISH and PCR results; 84.7% of the cases tested with both methods were found to be positive. The main discordance concerned 5 NS cases positive by PCR and negative by ISH. The distribution of positive cases in the histological subtypes of HD largely overlapped that observed by PCR; HHV-6 sequences were observed in 80.4% of the NS cases, 75% of the NSIF cases, 91.6% of the mixed cellularity cases, and 100% of the lymphocyte depletion cases. The positive nuclei of the lymphoid cells showed no specific localization in the neoplas-

The presence of HHV-6 DNA sequences was investigated by PCR in 52 cases of HD (Table 2). In 38 of 52 cases (73%), HHV-6 DNA was detected (Figure 1, A and B), with high predominance of the variant B (36 cases, 94%); in only 2 cases was the presence of both A and B observed. Variant B subclasses Bi and B2 were detected in 15 cases (39.5%) and in 23 cases (60.5%), respectively, including the 2 cases with co-expression of A and B (Table 3). Concerning the histological subtypes of HD, HHV-6 DNA sequences were demonstrated in 73.1% of the nodular sclerosis (NS) cases, in 50% of the interfollicular

M 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 1819 M C+

A Figure 1. PCR detection and variant characterization of HHV-6 genomes in lymph node biopsies. A: Etbidium-bromide-stained gel of amplification products obtained by nested PCR. Lane M, DNA marker (123-bp ladder); lanes 1, 5, 11, 15, and 19, negative controls (samples without DNA); lanes 2 to 4, 6 to 10, 12 to 14, and 16 to 18, different HD patients. B: The nested PCR products were analyzed by gel electrophoresis after cleavage with the restriction endonucleases HindIII (H), BglII (B), and TaqI( TI. Profiles are shown representative of HHV-6 variant B1 (sample 1), variant B2 (sample 2), co-presence of variant A and B2 (samples 3 and 4) obtained from the lymph node samples analysis, and variant A (HHV-6 GS strain).

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3

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H B T M

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HHV-6 in Hodgkin's Disease 1505 AJP November 1996, Vol. 149, No. 5

Figure 2. Hodgkin's disease mixed-cellularity type (A) and NSI-type (B). randomly distributed lymphoid cells positiveforHHV-6DNA. In the NS I type, the positive cells occur predominantly arotund bundles offibrous tissue. ISH with digoxigenin-tailed probe; counterstaining, methyl green; original

magziffication, X 250.

tic tissue (Figure 2A); however, in the NS cases, an increased number of positive cells was observed close to the bundles of sclerosis (Figure 2B). No Hodgkin and Reed-Sternberg cells (HRSCs) were positive in any case (Figure 3).

Southern Blot Analysis No positive samples were detected by hybridizing lymph node DNA with the pZVH14 probe, suggesting that the number of HHV-6 genomes was below the detection limit of Southern blot (approximately 10,000 copies). Table 3. PCR Analysis of HHV-6 Variant B1/B2 Positivity in Hodgkin's Disease Cases

Histological Number Variant B Variant B2 of cases positivity (%) positivity (%) type NS 20 6/20 (30) 14/20 (70) NS II 8 5/8 (62.5) 3/8 (37.5) NSIF 2 1/2 (50) 1/2 (50) MC 7 3/7 (42.8) 4/7 (57.2) LD 1/1 (100) 0/1 (0) Total 38 15/38 (39.4) 23/38 (60.5) NSIF, NS with interfollicular pattern; MC, mixed cellularity; LD, lymphocyte depletion.

Figure 3. Hodgkin's disease mixed-cellularity-type: lymphoid cells positive for HHV-6 DNA surrounding some negative HRSCs. ISH with digoxigenin-tailed probe; counterstaining, methyl green; original magnification, X 250.

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Figure 4. Detectioni oJ EBV DNA with BMLF1 priiners in Raji cells, for assessment ofPC'K senisitivity, anid ini HD samples. A, lanes 1 throuigh 7: Serial 10-fold dilutions of Rali cell DNA. Lane 1, 300 nzg (:3 x 10" cells); lane 2, 30ng (-.3 X 103); lane 3, 7g (- 3 X 102 cells); lane 4, 300 pg ( 30 cells); lane 5, 30pg (-3 cells); lane 6, 3 pg (- 3 x 10 1 cells); lane 7, .300JA (-3 x 10-2 cells); lane 8, negative cotitrol (sample without DNA). B: Lane M, DNA marker ( 123-hp ladder); lane C+, positive conitrol (Raji cell DNA); lanes 6, 12, 13, 18, and 23, negative conitrols (samples uwithout DNA); lanes 1 to 5, 7 to 11, 14 to 17, and 19 to 22, DNA from lymph node biopsies of HD patients.-

A

1

265 bp

1

2

3 4

5

6 7

8

11

2 3 4 5 6 7 8 9 10 11 12 M C+ 13 14 1516 17 18 1920 2122 23 M C+

B

Immunohistochemistry A pool of monoclonal antibodies against different structural proteins of HHV-6 was tested in 25 HD cases positive for HHV-6 DNA by PCR. None of them were positive, thus indicating a nonproductive infection.

EBV Studies PCR Analysis EBV DNA sequences were detected in 39 of 52 (75%) cases of HD (Figure 2, A and B). The distribution of positive cases in the histological subtypes was the following: NS cases, 72.5%; NS interfollicular cases, 50%; mixed cellularity, 80%; and lymphocyte depletion (one case), 100%. The reactive samples and T-cell NHLs were consistently negative, whereas a single B-cell NHL case was positive. In Situ Hybridization EBER was detected in 42 of 57 (73.6%) cases. In all of the positives, a variable number of HRSCs were strongly positive, and in 33 of these cases, surrounding lymphocytes were also stained (Figure 5). Only 1 of 10 NHLs (also positive by PCR for EBV DNA) were EBER positive. In the cases studied by both methods, no discrepancies were observed between PCR and ISH.

ISH, 32 of 57 cases (59.6%) were positive for HHV-6 DNA and EBER. This technique showed the presence of EBER in the lymphocyte compartment in addition to HRSCs in 22 of these cases (38.5%).

Relationship with Clinical Parameters No statistical relationships were observed between HHV-6 or EBV positivity and overall or dis-

ease-free survival, staging and bulkiness of disease, or response to therapy.

Discussion One of the main aims of this study was to ascertain the incidence of HHV-6 infection in a large series of HD cases. We used different molecular and immunological techniques for the detection of HHV-6, including PCR, Southern blot, ISH, and immunohistochemistry. Using a highly sensitive PCR analysis on 1 ,ug of lymph node DNA, the presence of HHV-6

Immunohistochemistry All cases found positive for EBER (42 of 57) were also positive for LMP1. In contrast to ISH results, a strong reaction was localized in HRSCs (Figure 6), whereas surrounding lymphocytes were consistently negative, apart from some sporadic cells.

Co-Infection by HHV-6 and EBV Sequences of both HHV-6 and EBV DNA were found by PCR in 28 of 51 cases (54.9%), whereas by

Figure 5. Hodgkins disease NS-II-tlpe: inumertous HRSCs alnd sur rounding ymnphocytes are positiv'e for EBER. ISH witb digoxigenintailed probe; iio counterstaining. methyl green; origiiicl maglificatio/i, X300

.f~to

Figure 6. Hodgkin's disease NS-II-Hvpe: numerous HRSCs shou cvtoplasm positive for LMP1. ABC immunoperoxidase; counterstaining, hemalum; original magnffication, X300.

detected in 73% of our HD cases. This result is different from those reported by other authors using PCR. Torelli et a122 found the presence of HHV-6 DNA sequences in only approximately 12% of their HD cases, whereas Di Luca et al33 detected HHV-6 in 29% of the cases. Furthermore, all of the HHV-6positive cases described by Torelli et a122 had a similar histological pattern (NS with lymphocyte depletion) and an aggressive outcome. This observation has led them to speculate that HHV-6 infection, possibly in cooperation with EBV, could contribute to the development of unfavorable immunological and/or clinical events. Our results, obtained on a larger number of cases, are not consistent with a direct pathogenetic role of HHV-6 in HD for several reasons: 1) the percentage of HHV-6-positive cases in the group of reactive lymph nodes was not significantly different from that of HD (68% versus 73%); 2) HHV-6 sequences were demonstrated by PCR analysis and ISH, but none of the cases was positive in Southern blots, suggesting the presence of a relatively low number of copies; 3) by ISH, we observed HHV-6 in randomly scattered small lymphocytes but, different from EBV, not in the HRSCs, which are regarded as the neoplastic cells in HD; 4) in no case was HHV-6 infection productive, as the neoplastic tissue was not positive for antibodies against viral structural proteins; and 5) in no case was

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was HHV-6 positivity correlated with clinical outcome, clinicopathological characteristics, or aggressive histological subtypes. HHV-6 is highly prevalent in human populations. More than 80% of blood donors and other healthy adults have been shown to be seropositive either by enzyme-linked immunosorbent assay or indirect fluorescent antibody tests.3435 Our results are consistent with a widespread presence of HHV-6 infection in human lymphoid tissue. In agreement with our data, Sumiyoshi et al36 recently detected HHV-6 by PCR in a high percentage of HD cases. The discrepancies between our results and others in the literature, however, deserve some comment. The possibility of cross-contamination between the samples under study and positive controls is excluded by the strict precautions adopted in carrying out nested PCR procedures37-39; moreover, ISH for HHV-6 DNA, a technique less affected by crosscontamination, showed a percentage of positive cases similar to that found by PCR. Although some differences have been demonstrated in the geographical distribution of HHV-6,4° the contrasting results are more likely explained by the wide spectrum of sensitivity of the techniques used in different laboratories. The threshold of sensitivity of our PCR method is less than 10 copies of viral DNA.26 The amount of DNA analyzed could also account for the discrepancies between different laboratories. A high prevalence of HHV-6 DNA has been detected by nested PCR in blood cells of healthy individuals by amplifying 5 [kg of DNA, whereas a lower prevalence was found when 1 ,ug of the same DNA was processed.3 With regard to the prevalence of the HHV-6 B subclass in our series (94% of the cases), this result does not appear to be linked to any clinical or pathological parameter. The different distribution of the two variants B1 and B2 within NS cases, ie, prevalence of B1 in NS 11 and of B2 in NS 1, is interesting because of the different prognostic values of these two histological subtypes, but it remains, at present, unexplained. Another aim of our study was to determine the extent of co-infection by EBV and HHV-6 and to see whether any synergism between the two viruses occurs in the pathogenesis of HD. EBV DNA was detected by PCR in the majority of our HD cases (75%), whereas all of the reactive lymph nodes and NHLs (except one) were negative. This result is intermediate between those reported in other western countries and in Japan (19 to 60%),41 46 and those reported in developing countries (90 to 100%).47.48 Although again we cannot exclude differences in

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PCR sensitivity, this result may indicate that the involvement of EBV in HD is higher in Italy than in other western countries. Strong agreement was observed between the detection of EBV DNA and positivity for EBER and LMP1. As the latter are indices of latent infection, these data show that EBV positivity in our series may reflect more than simple seropositivity of the patients. Moreover, the positivity for EBER in the lymphoid cells, as well as in HRSCs, in 33 of 57 cases tested by ISH, accords with the observation that the reactive cellular fraction of the Hodgkin's tissue is consistently associated with EBV.49 In a substantial proportion of our cases (54.9%), EBV and HHV-6 DNA were simultaneously present, although we did not demonstrate the presence of both viruses in the same individual cells. Considering that HRSCs were always negative for HHV-6, a comparison between the two viruses should concern only the lymphocyte compartment; on this basis, the lymphocyte population of only 38.5% of the cases was positive for both HHV-6 and EBV, which is a percentage more or less expected considering the prevalence of the two viruses separately. Although HHV-6 may be involved in the neoplastic transformation of established cell lines50 and high titers of antibodies against HHV-6 have been detected in the serum of patients with HD,51 the high positivity in our control cases suggests that HHV-6 does not play a specific role in HD. This view is supported by the lack of detectable HHV-6 sequences in HRSCs by ISH. Although this technique is less sensitive than PCR, and negative results may be due to the incapacity to detect single copies of HHV-6, the probe we used was obtained from that used for PCR, and our findings were uniform in the whole series of 57 cases. This suggests that, as a rule, the virus infects the surrounding lymphocytes but not HRSCs. Despite these considerations, there is evidence that EBV immortalization induces susceptibility to HHV-6 infection in lymphoblastoid B cells, and this characteristic could depend on the induction of a cellular receptor for HHV-6.6 Recently, it has been shown that HHV-6 activates the replicative cycle of EBV upon superinfection of EBV-immortalized Blymphoblastoid cell lines in vitro.52 However, the difference in the cellular localization of the two viruses, and the lack of a demonstrable HHV-6 latency, are consistent with a different role in HD. Recently, it has been shown that HHV-6 can induce the production of inflammatory cytokines, such as tumor necrosis factor-a and interleukin1 53; a similar mechanism may occur in HD, in

which cytokines may play a role in processes of immunological dysregulation.5455

Acknowledgments We thank Dr. Francesco Negro for his help in preparing the HHV-6 probe, Mrs. Marilena Abbadini, and Mr. Nino Ferraro for skillful technical assistance. We are also grateful to Dr. Bob Milne for revising the manuscript.

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