Simultaneous Epstein Barr Virus and Cytomegalovirus Infection Accompanied by Leiomyomatous Change in a Well-Differentiated Liposarcoma in a Patient With Long-Term Corticosteroid Treatment

Patient. A 59-year-old woman presented with a large tumour of the abdominal wall. She had been taking corticosteroids for severe chronic obstructive pulmonary disease for 15 years. On CT scan the tumour had the characteristics of lipomatous tissue with a dense core. Results. Histology showed a well-differentiated liposarcoma with a core of benign fibroleiomyomatous differentiation. Within the core, a third component was observed, characterized by more pleomorphism and the presence of an inflammatory infiltrate. In this component, immunoperoxidase stains and in situ hybridization demonstrated cytomegalovirus (CMV) and Epstein Barr virus (EBV) infection in large and small cells, respectively. Discussion. Long-term corticosteroid use for pulmonary disease may extend the list of immunosuppressed states associated with the development of leiomyomatous tumours with EBV infection, previously described in AIDS patients and liver transplant recipients. The role of CMV is uncertain.


Introduction
Liposarcom as constitute 16± 18% of the soft tissue sarcom as with a peak incidence between the ages of 40 and 60 years. T here are four m ajor types of liposarcom a: m yxoid liposarcom a, round cell liposarcom a, well-differentiated liposarcom a and pleom orphic liposarcom a. 1 T he well-differentiated subtype of liposarcom a m ay show dedifferentiation resulting in a high-grade sarcom a often showing a m orphology resem bling m alignant ® brous histiocytom a. 2,3 Heterologous differentiation w ithin the dedifferentiated component towards skeletal m uscle, sm ooth m uscle, bone and osteoid or vessels has previously been described. These dedifferentiated com ponents generally show m alignant features, but m ay also appear benign, especially in the case of sm ooth m uscle differentiation. 2± 8 The aetiology of soft tissue sarcom as is still unknown. Sarcom as m ay develop in scar tissue, especially after burns, and after intensive radiotherapy. 1,9 Exposure to chem ical products, such as dioxin and herbicides, m ight play a role as w ell. 10,11 Recently, there have been reports suggesting a relation between Epstein Barr virus (EBV ) infection and the developm ent of smooth m uscle tum ours in patients with AID S and liver transplant recipients. 12± 16 W e report a case of a patient with a welldifferentiated liposarcom a which showed central leiom yom atous change concurrent with both EBV and cytomegalovirus (C M V) infection.

Case history
A 59-year-old C aucasian fem ale was adm itted to another hospital because of an aggravation of urem ia and asthma not responding to oral steroids and antibiotics. She had suffered from chronic obstructive pulm onary disease for over 40 years for which she had used steroids orally for 15 years and an inhaler for m ore than a decade; the daily steroid dosage depended on the patient' s pulm onary status. Previous history further revealed hypertension, gastric ulcer and thyroidectomy because of a m ultinodular goitre. Upon routine physical exam ination, there was wheezing over both lungs and a large m ass was found in the right lower abdom en. She was treated for her pulm onary com plaints w ith high 1357-714 X/97/010055± 04 Ó 1997 Journals Oxford Ltd 70% ), then digested with 20 m g m l 2 1 of proteinase-K for 60 m in at 37°C. After blocking, rinsing and dehydration the slides were covered by a m ixture of either CM V (British T echnology) or EBV (EBER; D AKO ) speci® c oligonucleotides. CM V oligonucleotides were labelled with digoxigenin (DIG) and EBV oligonucleotides were labelled with F IT C. T he slides were heated for 5 m in at 92°C and incubated overnight at 37°C . D etection was carried out using anti-FITC ± alkaline phosphatase for EBV and anti-D IG± alkaline phosphatase for CM V . Final colour was developed by the BCIP m ethod.

Results
T he m ost signi® cant ® ndings at postm ortem were bronchopneum onia in the upper lobes of both lungs and a tum our originating from the low er right abdom inal wall without relation to the intra-abdom inal organs. T he tum our, which had a m axim um diam eter of 18 cm, had a sm ooth and lobulated surface and was com pletely surrounded by a capsule. O n cross-se ction the periphery had the asp ect of adipose tissue with a central core of ® rm w hite tissue with a diameter of 7 cm. There were no signs of m etastases.
M icroscopic exam ination revealed foci of Aspergillus infection in both lungs. T he alveolar epithelium show ed eosinophilic intranuclear inclusions typical of a viral infection (Fig. 2). T hese cells were imm unoreactive for CM V protein and show ed a positive signal for the CM V genome with in situ hyb ridization. In addition, im munoreactivity for EBV protein was dem onstrated and there was a positive signal for the EBV genome with in situ hyb ridization. The peripheral part of the tumour of the abdominal wall showed adipose tissue w ith foci of ® brosis (Fig. 3). The fat vacuoles were of different size. Convincing m ulti-vacuolated tum our cells were not observed and there was no mitotic activity. H owever, som e fat cells show ed ® nely vacuolated cytoplasm and/or hyp erchrom atic nuclei. T hese doses of intravenous steroids, ipratropium, fenoterol, theophylline and antibiotics. After her pulm onary condition improved, a CT scan was perform ed to evaluate the abdom inal m ass. T his showed a tum our of the abdom inal w all with a diam eter of 18 cm with the density of lipom atous tissue in the periphery and a denser core (Fig. 1).
The patient was referred to the departm ent of surgical oncology of our hospital for further evaluation of the tum our. A tru-cut biopsy showed a m esenchym al tum our, which was dif® cult to classify. Shortly after this procedure, her condition deteriorated and she developed severe dyspnoea and acute heart failure w ith anuria. She died one day after transfer to our hospital. A postm ortem examination was perform ed.

M ethods
T he tru-cut biopsy and sam ples from the tum our taken at postm ortem w ere exam ined on H&Estained paraf® n sections. Standard im m unperoxidase stains were perform ed using antibodies to vimentin, sm ooth m uscle actin and desm in on paraf® n and frozen sections. Im m unohistological stains for the detection of EBV (LM P, clone CS 1± 4; D AK O) and C M V (IEA, clone E13; Argene Biosoft) proteins were perform ed on paraf® n sections and frozen sections after high tem perature antigen retrieval, using the m ultilink-label technology of Biogenex. F inal colour was developed by the BCIP/NBT (b -chloro-indolyl-phosphate/nitro blue tetrazolium ) m ethod.
In situ hybridization studies were perform ed on standard paraf® n-em bedded tissue. Tissue sections      of the lesion resem bled a ® broleiomyom atous tum our without evidence of m alignancy. Im m unoperoxidase stains for vim entin, actin and desm in were positive. A third com ponent w as present in the central part of the tumour and form ed the only constituent of the tru-cut biopsy. T he m ost rem arkable feature was the presence of large cells with prominent eosinophilic intranuclear inclusions embedded in a spindle cell background (Fig. 5). A prominent in¯am m atory in® ltrate was present, largely consisting of polym orphonuclear leucocytes, as well as foci of necrosis. Im m unoperoxidase stains for CM V proteins were strongly positive in the large cells (Fig. 6); the imm unoperoxidase reaction for LM P-1 EBV was slightly positive in sm aller tum our cells. In situ hybridization show ed a positive signal for the CM V genom e in the large cells, which w ere also CM V protein im m unoreactive. EBER-1 mRN A was demonstrated in sm aller cells (Fig. 7).

D iscussion
T he current report describes a tum our consisting of well-diffe rentiated liposarcom a with a central core m orphological features, together with the deep location and the size of the tum our, led to a diagnosis of w ell-differentiated liposarcom a rather than lipom a. T he central part of the tum our w as com posed of bundles of spindle-sh aped cells, som e of which displayed hyperchrom atic irregular nuclei (Fig. 4); no m itotic activity or necrosis was observed. This part of leiom yom a. T he latter part appeared to be affected by both a CM V and an EBV infection. D edifferentiation of well-differentiated liposarcom as is a w ell-established phenom enon and several reports have described the occurrence of heterologous components such as skeletal m uscle, sm ooth m uscle, bone or osteoid, or vessels. 2± 8 In m ost cases, these heterologous com ponents are clearly m alignant. In our case the central leiomyom atous com ponent of the tum our showed no signs of malignancy.
Recently, several reports 12± 15 have described the occurrence of leiomyom as and leiomyosarcom as in H IV -positive patients and M cClain et al. 12 have suggested a relation betw een EBV infection and the developm ent of leiomyom as in these patients. Sim ilar observations have been reported in liver transplant recipients. 16 T hese ® ndings lead to the suggestion that the leiom yom atous differentiation and the EBV infection in our case of welldifferentiated liposarcom a are also related. A parallel between AID S patients, transplant recipients and our patient is a m arked decrease in imm unologic surveillance, w hich in our patient was brought about by the use of steroids for chronic obstructive pulm onary disease for 15 years. This also explains her A spergillus and C M V bronchopneum onia. T o the best of our knowledge, a relation between sm ooth m uscle tum ours and CM V infection has not yet been described. However, the presence of a CM V infection restricted to the leiom yomatous part of her tum our suggests the possibility of a relation between C M V and the form ation of sm ooth m uscle tumours as well.