Human herpesvirus type 8 and Epstein±Barr virus-associatedcutaneous lymphoma taking anaplastic large cell morphologyin a man with HIV infection

K.NAKAMURA, H.KATANO,* Y.HOSHINO,² T.NAKAMURA,³ O.HOSONO,§A.MASUNAGA,¶ S.MORI,* A.IWAMOTO² AND K.TAMAKI

Department of Dermatology, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan

Departments of *Pathology, ²Infectious Diseases, ³Infectious Diseases and Applied Immunology,

§Clinical Immunology and AIDS Research Centre, and ¶Laboratory of Medicine, Institute of Medical Science,University of Tokyo, Tokyo, Japan

Accepted for publication 11 February 1999

Summary Human herpesvirus type 8 (HHV-8, Kaposi's sarcoma-associated herpesvirus)-positive lymphoma

taking anaplastic large cell morphology in the skin is described in a 46-year-old man with AIDS.Multiple erythematous nodules appeared on the trunk and extremities during the treatment of AIDS.

Histological examination of cutaneous nodules showed dense in®ltration of CD30� atypical

lymphoid cells in the deep dermis. Immunoglobulin JH gene rearrangement was detected in theselymphoma cells. Both Epstein±Barr virus-encoded small RNA and HHV-8 mRNA (T1.1/nut-1) were

detected in these lymphoma cells by in situ hybridization. Remarkable retention of the pericardial

¯uid was observed at the same time that cutaneous lesions grew, and lymphoma cells in thepericardial ¯uid showed the same phenotype as the cutaneous lymphoma. Chemotherapy with

cyclophosphamide, doxorubicin, vincristine and prednisone effectively reduced both the cutaneous

nodules and pericardial ¯uid. However, the patient died 4 months after diagnosis because ofcytomegalovirus infection. As far as we know, this is the ®rst report of an HHV-8-positive cutaneous

lymphoma taking anaplastic large cell morphology. This case suggests the association of AIDS-related anaplastic large cell lymphoma with HHV-8.

Key words: anaplastic large cell lymphoma, Epstein±Barr virus, human herpesvirus type 8, human

immunode®ciency virus, Kaposi's sarcoma-associated herpesvirus, primary effusion lymphoma

Non-Hodgkin's lymphoma (NHL) is the most frequent

neoplasm in Japanese AIDS patients.1 The incidence isreported to be 4±10% among Western AIDS patients,

but exceeds 20% among Japanese.1±3 Most AIDS-

related NHLs are diffuse, aggressive B-cell lymphomas,namely, small non-cleaved cell lymphoma (Burkitt's

lymphoma), large cell and large cell immunoblastic

plasmacytoid lymphoma.4±6 Of AIDS-related NHLs,10±15% have been reported using the term AIDS-

related anaplastic large cell lymphoma (ALCL).7,8

ALCL is a clinically heterogeneous population ofNHL.8±11 ALCL is classi®ed as a T/null-cell type lym-

phoma in the revised European±American classi®cation

of lymphoid neoplasms (REAL);10 however, there aresome B-cell lymphomas exhibiting anaplastic large cell

morphology. p80NPM/ALK protein, a fusion protein of

translocation (2;5), is identi®ed in many ALCLs, andcases expressing p80 have been reported to have a good

prognosis.12

Primary effusion lymphoma (PEL) is a distinct clin-icopathological subtype of NHL, which mainly occurs in

HIV-positive homosexual men.13±15 PEL mainly occurs

in the pleural, pericardial and abdominal cavities, with-out in®ltration into solid tissues. Histologically, lym-

phoma cells of PEL show marked pleomorphism,

expressing CD30, epithelial membrane antigen (EMA)and HLA-DR. PEL shows clonal immunoglobulin gene

rearrangements, and is generally involved in B-cell

lymphomas.13,14 In addition to HIV, the presence ofEpstein±Barr virus (EBV) and human herpesvirus type

8 (HHV-8, Kaposi's sarcoma-associated herpesvirus)

has been identi®ed in lymphoma cells of PEL.14,15

British Journal of Dermatology 1999; 141: 141±145.

141q 1999 British Association of Dermatologists

Correspondence: K.Nakamura. E-mail: makamura-der@h.u-tokyo.ac.jp

HHV-8 encodes various genes such as v-cyclinD, vbcl-2,

MIP1, T0.7, T1.1/nut-1 and vIL-6. Among these, v-cyclinD,GPCR, T0.7 and K12 have been reported to have the

ability to transform and affect the growth of cells.

We describe a case of AIDS-related, p80-negative,CD30� cutaneous lymphoma taking anaplastic large

cell morphology. The latter proved to be a case of skin

involvement of PEL, which originated from pericardialeffusion. Both EBV-encoded small RNA (EBER) and

HHV-8 mRNA (T1.1/nut-1) were detected.

Case report

A 46-year-old homosexual man was admitted in October

1996 with recurrent pneumonia, and diagnosed as

having HIV infection and a respiratory infection withPneumocystis carinii. In August 1996, his CD4� cell

count had been 8/mm3 (normal 700±1000). InNovember 1996, the patient noticed a slight fever and

increased general malaise. Echocardiogram revealed

abnormal uptake in the pericardial cavity. Hepato-splenomegaly and pancytopenia were detected. No

obvious lymphadenopathy was observed. In December

1996, a large amount of pericardial ¯uid was noted.Aspiration of the pericardial cavity was performed and

this ¯uid showed in®ltration of atypical lymphoid cells

(Fig. 1). At the same time, multiple erythematousnodules appeared on the chest, abdomen and upper

and lower limbs, and these tumours developed progres-

sively. The most prominent nodule of the abdomen wasa 3´2 ´ 4 cm, relatively well demarcated, elastic-soft,

slightly elevated tumour (Fig. 2a). His haemoglobin

was 3´6 g/dL (normal 14´0±18´0) and white bloodcell count 1´43 ´ 109/L (normal 4´5±11´0 ´ 109),

with 73% polymorphonuclear leucocytes, 4% lympho-

cytes and 21% eosinophils. Anti-EBV antibodies were asfollows: EBV antiviral capsid antigen (VCA) IgG 160,

VCA IgM <10, EBV-encoded nuclear antigen (EBNA)

10.Biopsy of the cutaneous tumour of the abdomen

showed diffuse in®ltration of atypical lymphoid cells

142 K.NAKAMURA et al.

q 1999 British Association of Dermatologists, British Journal of Dermatology, 141, 141±145

Figure 1. Giemsa staining of effusion lymphoma in the pericardial

¯uid. Lymphoma cells consist of large immunoblastic cells with round

or oval-shaped nuclei (original magni®cation ´ 800).

Figure 2. (a) Erythematous nodules are

evident on the chest and abdomen

(arrows). (b) Photomicrograph of a biopsyof a nodule of the trunk. Diffuse in®ltration

of atypical lymphoid cells is observed from

the reticular dermis through the fat tissue

(haematoxylin and eosin (H&E); originalmagni®cation ´ 40). (c) High power view of

the lymphoma. Atypical large lymphoid

cells consisted of large blastic cells with

round, and sometimes cleaved nuclei andabundant cytoplasm. Some mitotic ®gures

were observed (H&E; original magni®cation

´ 400). (d) Immunohistochemical stainingof CD30 (Ki-1) in atypical lymphoid cells of

the cutaneous nodule. Almost all atypical

cells are positive for CD30 (original

magni®cation ´ 400).

extending from the reticular dermis through fat tissue(Fig. 2b). These atypical lymphoid cells consisted of

large blastic cells with abundant cytoplasm, large

round nuclei and prominent nucleoli (Fig. 2c). Themitotic count was 5 per high power ®eld. There were

no atypical lymphoid cells in the epidermis. Immuno-

histochemical staining of large atypical lymphoid cellsin the cutaneous nodules revealed positivity for CD30

(Fig. 2d) and CD45RO, but did not show positivity for

CD3, CD20, EMA or p80.In situ hybridization (ISH) was performed to detect

EBER and HHV-8 mRNA in the cutaneous tumour. As a

probe for the EBER gene a digoxigenin (DIG)-labelled30-base oligonucleotide, 50-AGA CAC CGT CCT CAC

CAC CCG GGA CTT GTA-30, was used, and for HHV-8

(T1.1/nut-1) a DIG-labelled 50-base oligonucleotide, 50-GAG CGC TCC CAG CTG CCG CAC ACC ACT TTA GTC

CAA TGT TCT TAC ACG AC-30, was used.16 The DIG

oligonucleotide 30-tailing kit (Boehringer Mannheim,Mannheim, Germany) was used for labelling the

probe. Brie¯y, the paraf®nized slides were digested

with proteinase K (10 mg/mL) and treated with0´2 mol/L HCl. Hybridization was performed in a

hybridization mixture consisting of Denhardt's solution,

dextran sulphate and DIG-labelled oligonucleotideprobe. After hybridization, the slides were rinsed and

incubated with anti-DIG antibody. Detection was per-formed with nitroblue tetrazolium salt. Twenty to 30

per cent of lymphoma cells were positive for EBER (data

not shown), whereas only 1±2% were positive for HHV-8 T1.1/nut-1 mRNA (Fig. 3a). Both EBER and HHV-8/

T1.1 were identi®ed in the nuclei of the atypical lym-

phoid cells of the cutaneous nodule. DNA fragmentsobtained from the cutaneous nodule showed the re-

arranged band of the joining region (JH) of the immuno-

globulin gene (Fig. 3b), but not of T-cell receptor-b gene(data not shown).

Based on histological ®ndings, clinical and laboratory

data, the cutaneous tumour was diagnosed as a B-celllymphoma taking anaplastic large cell morphology,

complicated with PEL in the pericardial ¯uid. Translo-

cation (2;5) associated with p80 was absent in thepatient. The patient was immediately treated with ®ve

courses of cyclophosphamide, doxorubicin, vincristine

and prednisone (CHOP) for both cutaneous lymphomaand PEL in the pericardial ¯uid, and both lesions

regressed during two courses of the CHOP therapy.

The patient died 4 months after the diagnosis becauseof cytomegalovirus infection. According to retrospective

studies, PEL cells in the pericardial ¯uid were positive for

CD30,CD45 and CD45RO, but negative for CD3, CD4,

CD5, CD8, CD10, CD14, CD19, CD20, CD21, CD56 and

CD68 and immunoglobulin g, m, a, d, l and k (data not

shown). In addition, Southern blot analysis of PEL cellsusing JH immunoglobulin gene probes showed a re-

arranged pattern similar to that in the atypical lym-

phoid cells in the cutaneous tumours (Fig. 3b). EBERand HHV-8 mRNA were detected in PEL cells.

Discussion

We have described a homosexual man with AIDS whohad an HHV-8-positive cutaneous lymphoma taking

anaplastic large cell morphology, complicated with

PEL in the pericardial ¯uid. We diagnosed this cuta-neous lymphoma as AIDS-related ALCL based on the

HHV-8/EBV LYMPHOMA AND HIV 143

q 1999 British Association of Dermatologists, British Journal of Dermatology, 141, 141±145

Figure 3. (a) Detection of human herpesvirus type 8 (T1.1/nut-1)

mRNA in nuclei of atypical lymphoid cells of the cutaneous nodule by

in situ hybridization. (b) Southern blot using biotin-labelled JH probe.

DNA of skin tumours (S), tumours in pericardial effusion (P) andnegative control (N) were digested with BamHI and HindIII. Germline

bands (G) in are 5´6 kb in size. Rearrangement is indicated by re-

arranged band (R).

histopathological features of the lymphoma cells, posi-tivity of CD30 and response to chemotherapy. The

cutaneous lymphoma was a null-cell lymphoma

immunohistologically, but a B-cell lymphoma geneti-cally. The diagnostic criteria for ALCL are still under

debate. While B-cell lymphomas taking anaplastic large

cell morphology were excluded from ALCL under theREAL classi®cation,10 they have been included in ALCL

in other classi®cations such as the updated Kiel classi-

®cation.17 Strictly, our case cannot be included in ALCLunder the REAL classi®cation, but according to the

updated Kiel classi®cation, the diagnosis was AIDS-

related ALCL.ALCL consists of pathogenetically heterogeneous

entities. In about half of ALCLs, the chimeric protein

p80 is expressed. ALCLs with p80 generally occur inyoung patients and tend to be associated with a benign

prognosis, compared with p80-negative ALCLs.12

Beylot-Barry et al. detected p80 in primary cutaneousALCLs, using nested polymerase chain reaction and

ISH.18 Our case did not exhibit p80 in either cutaneous

tumours or the pericardial ¯uid, and was considered tobe an aggressive type of ALCL.

We ®nally diagnosed this cutaneous lymphoma as a

skin involvement of PEL for the following reasons: ®rst,both the cutaneous lymphoma and the pericardial PEL

showed similar immunophenotypes; secondly, EBV andHHV-8 were detected in both lymphomas; thirdly, the

immunoglobulin gene rearrangement pattern of the

cutaneous lymphoma was similar to that of the PEL;fourthly, CHOP chemotherapy reduced both lymphomas

simultaneously and effectively; and lastly, the cutaneous

lymphoma occurred as multiple lesions near the chest.Almost 30 cases of PEL have been reported; however,

the involvement of solid tissues is quite rare. Only a few

cases involving tissues such as lung, submandibulargland and large intestine have been reported.19,20 Our

case was characterized by the simultaneous appearance

of tumours of the skin and pericardial ¯uid. Our case wasdifferent from subcutaneous T-cell lymphoma21 because

the lymphoma cells mainly in®ltrated in the dermis and

did not show panniculitis histologically. In addition, thesecells did not exhibit a T-cell immunophenotype.

HHV-8 is a newly described human herpesvirus that

is a causal factor of Kaposi's sarcoma.22 Recently, DNAsequences of HHV-8 have been discovered in PEL from

HIV-positive patients.23 HHV-8 (T1.1/nut-1) mRNA is

generally transcribed in lytic cells when PEL cells arecultured with phorbol ester in vitro.24 Thus, HHV-8

(T1.1/nut-1) might play an important part in nuclear

processing during lytic growth. Our case had HHV-8

(T1.1/nut-1) mRNA in lymphoma cells of both cuta-neous nodules and pericardial effusion. As far as we

know, this is the ®rst report of the detection of HHV-8

(T1.1/nut-1) mRNA in cutaneous AIDS-related ALCL.Strong expression of HHV-8 (T1.1/nut-1) suggests a

rapid growth of HHV-8 in the lymphoma cells of both

regions.EBERs are transcribed in high copy numbers (107 per

cell) in infected cells, and are used as markers for latent

EBV infection.25 EBERs are detected in a higher propor-tion of EBV-infected cells than are latent membrane

protein-1 and EBNA-2. We detected EBERs in both

skin and pericardial ¯uid by ISH. Most cases of AIDS-related ALCL are associated with EBV infection.26,27 In

addition, PEL often correlates with EBV infection.14,15

There were fewer HHV-8 (T1.1/nut-1) mRNA-positivecells than EBER-positive cells in our case of cutaneous

ALCL, because EBER is expressed in the latent phase,

while HHV-8 T1.1/nut-1 is speci®c for the lytic phase.We did not detect lytic EBV in our case. These data

suggest that the latent EBV, as well as HHV-8, may be

associated with the pathogenesis of AIDS-related ALCL.We suggest that HHV-8 may act in conjunction with

EBV to generate full transformation.

In summary, we report a case of HHV-8-positive,CD30 � lymphoma taking anaplastic large cell mor-

phology, which developed in the skin. Our case differsfrom previous reports in which PEL did not involve solid

tissues. Multiple cutaneous nodules developed along

with the appearance of PEL in our case. Further inves-tigations will elucidate the pathogenetic role of HHV-8

and EBV in AIDS-related ALCL and PEL.

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