# EBV-Associated Smooth Muscle Neoplasms:  Solid Tumors Arising in the Presence of Immunosuppression and Autoimmune Diseases

Authors: Kimberly Moore Dalal; Cristina R. Antonescu; Ronald P. DeMatteo; Robert G. Maki
Journal: Sarcoma
 (2008)

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## Abstract

Background. Epstein-Barr virus (EBV)-related smooth muscle neoplasms (SMNs) have been associated with immune dysregulation, most notably in patients who have undergone solid organ transplantation or in patients with HIV/AIDS. Objective. to report our experience with EBV-related neoplasms as well as describing the first EBV-related SMN in the setting of administration of glucocorticoids and the tumor necrosis factor inhibitor etanercept. Design.  We have case reports, of minimum 3-year follow-up,  2002–2005. Setting.  It was held in an academic and tertiary referral cancer center. Patients. Patients are with dysregulated immunity after solid organ transplantation, HIV/AIDS, or with psoriasis after treatment with etanercept. Interventions.  There were discontinuation of etanercept, right hepatic trisegmentectomy, and  chemotherapy. Measurements.  We use survival as a measurement here. Results. Patients who were able to withstand reduction in immunosuppression survived. Surgical resection or chemotherapy was successful in delaying progression of disease. Limitations.  There was a  relatively short follow-up for these slow-growing neoplasms. Conclusion. EBV-related SMNs have variable aggressiveness. While chemotherapy may slow disease progression, resection and improving the host immune status provide the best opportunity for primary tumor control.


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## Body

## 1. Introduction

Immunocompromised patients have an
increased incidence of  immunosuppression-associated malignancies. While
unusual, smooth muscle neoplasms (SMNs), such as leiomyomas and
leiomyosarcomas, occur at a greater frequency in immune-dysregulated patients
compared to the general population [1, 2].
SMNs have been reported in immunodeficient patients after solid organ
transplants [2–5], with acquired immune deficiency syndrome (AIDS) [6], or with
congenital conditions [7–10]. SMNs are strongly associated with Epstein-Barr virus
(EBV) [4, 6, 9].We report three cases of EBV-associated
SMN, one in a patient with psoriasis who had received the tumor necrosis factor
(TNF) antagonist etanercept, and describe our institution’s recent experience
with this entity.Patient 1. A 55-year-old Asian man presented in 2002 with hepatitis B, hypertension, penile
carcinoma status postresection and lymphadenectomy in 1988, and chronic renal
insufficiency. He had undergone cadaveric renal transplant in 1994 and remained
rejection free on cyclosporine, azathioprine, mycophenolate mofetil, and
prednisone.In March 2002, he
developed abdominal pain and a 15-pound weight loss.  Computed tomography (CT) scans (7/02)
revealed an unresectable 14 cm caudate-based mass; multiple pulmonary nodules
were visualized. Alpha-fetoprotein level was normal. Core needle biopsy
demonstrated a well-differentiated SMN. His immunosuppressive therapy was not
reduced because of the likelihood of transplant rejection. The patient refused
systemic chemotherapy.  By November 2002,
he developed jaundice and increasing debilitation. CT scan revealed rapid
disease progression with pulmonary metastases and ascites. He died later that
month.Patient 2. An 18-year-old woman diagnosed with congenital
AIDS began to experience abdominal pain at age 11, in 1995. Colonoscopy was initially
negative, but laparoscopy in 1997 revealed a well-differentiated SMN arising
from the rectosigmoid, unchanged in 1998 and 1999 on subsequent colonoscopies. In
October 2002, during an evaluation for persistent abdominal pain and areflexia
in the left ankle and knee, magnetic resonance imaging (MRI) revealed multiple
abdominal and paraspinal masses. She underwent resection of the lumbar
paraspinal tumor and rod placement. Pathology demonstrated an EBV-related SMN.By 2003, CD4
count was 8, and viral load was 15 000. Medications included stavudine and
lamivudine. She then presented, wheel-chair bound, with fatigue and refractory
abdominal pain, weighing 19 kg. Eastern Cooperative Oncology Group (ECOG)
performance status was 4. CT scans (3/03) demonstrated low-attenuation lesions
in the liver and a 9 cm mass arising between the stomach and the liver. She demonstrated
marked clinical improvement after starting doxorubicin in October 2003, and 5–10% decrease in
masses on reimaging.  She interrupted
therapy in mid 2004, starting dacarbazine in November 2004 for persistent disease
affecting the chest, abdomen, and paraspinal areas. Despite her noncompliance
and neutropenia, the tumors decreased in size further, her weight doubled, she
began to walk, and has an ECOG performance status of 1 as of January 2006. She
is still alive with disease (AWD) but without progression as of last follow-up in
October 2008.Patient 3. The patient is a 25-year-old woman with left kidney agenesis, history of
psoriasis since childhood, and seven episodes of shingles. In August 2004, she
began intravenous etanercept. One month later, she developed left cervical
lymphadenopathy and right lower extremity numbness and tingling. A positive
monospot test led to a diagnosis of mononucleosis. Etanercept was discontinued;
her symptoms resolved. In October, she began prednisone (40 mg daily) treatment
for increasing dyspnea attributed to asthma; this was tapered off in January
2005 when she saw her primary care provider and chest radiograph revealed
bilateral infiltrates. Chest CT
demonstrated infiltrates and
three liver lesions. Lung biopsy revealed Pneumocystis
carinii pneumonia. CT scan (1/05) confirmed three liver lesions, the
largest measuring 7 cm with central necrosis.
Fine needle aspiration of a liver mass revealed a well-differentiated SMN.
CT scan (4/05) noted that the largest liver lesion was 7.5 cm in size. Subsequent CT
scan (8/05)
revealed no change. Positron emission tomography (PET) scan (9/05) showed
tracer uptake in the liver lesions (Figure 1). In October 2005, she underwent
an extended right hepatectomy and cholecystectomy. Pathology revealed multicentric
EBV-associated SMNs (Figure 2). She remains without disease as of October 2008.CT and PET scans of EBV-related SMN of liver. CT scan of the abdomen and pelvis (8/05) from Patient 3 revealed multiple masses, mainly in the right lobe and involving segment 4. The largest mass measured6.8×5.8 cm and was located in the right inferior lobe (a). FDG-PET (9/05) demonstrated areas of hypermetabolism in the inferior aspect of the right hepatic lobe (SUV max 5.7), and two other neighboring foci (SUV max 5) (b).
(a)(b)Gross
and microscopic findings from EBV-related SMN of liver. Liver specimen from
Patient 3 measured17×13.2×7.6 cm. Four nodules were found, ranging in size
from 0.4 cm to 6.7 cm (a). Histologic features on H&E staining included
moderate increased cellularity (with areas containing rounder, less fusiform
cells), lack of nuclear pleomorphism, and a low mitotic rate (1 mitotic
figure/50 high-powered field)  (b). Nearly all cells were positive for
EBV-EBER by ISH, although intensity of the staining varied from cell to cell (c). Electron microscopy revealed spindle cells with abundant cytoplasmic
aggregates of actin microfilaments, including fusiform dense bodies and
attachment plaques, confirming the diagnosis of a well-differentiated smooth
muscle proliferation (d).
(a)(b)(c)(d)


## 2. Methods

### 2.1. Light Microscopy and Immunohistochemistry (ihc)

Formalin-fixed,
paraffin-embedded tissue blocks were sectioned (4μm) and stained with
hematoxylin and eosin for conventional histology. IHC analysis was performed
using standard techniques with the following  antibodies (prediluted from
Ventana medical Systems Inc, Tucson, AZ, USA, except where noted): HHF-35, smooth muscle actin (SMA),
desmin (DAKO, 1:50), CD117 (DAKO, 1:500), S100 protein (1:500,  Dako, Glostrup, Denmark), and HMB-45.


### 2.2. in Situ Hybridization for Eber

Detection of EBV-encoded
early RNA transcripts (EBER 1 and 2) was performed using biotin-conjugated
oligonucleotides (PNA probe, Dako), using standard protocols according to the manufacturer’s
instructions. The positive control used was a posttransplant EBV-associated
diffuse large cell lymphoma. All steps were performed under RNAse-free
conditions.


### 2.3. Electron Microscopy (em)

Tissue for EM was available in Case 3.
Representative fresh tumor was fixed in 3% formaldehyde—3% glutaraldehyde, postfixed in 1% osmium
tetroxide, embedded in epoxy resin, and stained with uranyl acetate-lead
citrate using standard procedures. The thin sections were examined using a
Philips-410 electron microscope.


## 2.1. Light Microscopy and Immunohistochemistry (ihc)

Formalin-fixed,
paraffin-embedded tissue blocks were sectioned (4μm) and stained with
hematoxylin and eosin for conventional histology. IHC analysis was performed
using standard techniques with the following  antibodies (prediluted from
Ventana medical Systems Inc, Tucson, AZ, USA, except where noted): HHF-35, smooth muscle actin (SMA),
desmin (DAKO, 1:50), CD117 (DAKO, 1:500), S100 protein (1:500,  Dako, Glostrup, Denmark), and HMB-45.


## 2.2. in Situ Hybridization for Eber

Detection of EBV-encoded
early RNA transcripts (EBER 1 and 2) was performed using biotin-conjugated
oligonucleotides (PNA probe, Dako), using standard protocols according to the manufacturer’s
instructions. The positive control used was a posttransplant EBV-associated
diffuse large cell lymphoma. All steps were performed under RNAse-free
conditions.


## 2.3. Electron Microscopy (em)

Tissue for EM was available in Case 3.
Representative fresh tumor was fixed in 3% formaldehyde—3% glutaraldehyde, postfixed in 1% osmium
tetroxide, embedded in epoxy resin, and stained with uranyl acetate-lead
citrate using standard procedures. The thin sections were examined using a
Philips-410 electron microscope.


## 3. Results

### 3.1. Microscopic and Electron Microscopic Findings

All patient
samples were positive for EBV-EBER by ISH (Figure2), and the vast majority of
cells were positive for EBER by ISH. IHC from biopsies of the liver tumor from
Patient 1 was positive for SMA and desmin, but negative for CD117 (ckit),
HMB-45, and S-100. Histologic features from the colonic SMN from Patient 2
included mild to moderate increase in cellularity, low mitotic count (<1/10
HPF), and no areas of necrosis. IHC was positive for desmin and SMA, but
negative for CD117 and S-100.The liver
specimen from Patient 3 revealed multicentric EBV-associated SMNs consisting of
four nodules ranging in size from 0.4 cm to 6.7 cm (Figure2).  Moderate increased cellularity (with areas of
somewhat rounder, less fusiform cells), focal areas of necrosis, lack of
nuclear pleomorphism, and low mitotic rate (1 mitotic figure/50 high-powered
field) were noted. The tumors were positive for SMA, HHF35, desmin and negative
for CD117.  EM from Patient 3 revealed spindle
cells with abundant cytoplasmic aggregates of actin microfilaments, including
fusiform dense bodies and attachment plaques (Figure 2). Linear arrangements of
pinocytotic vesicles were noted in many cells. Cytoplasmic organelles were
mainly represented by mitochondria, while rough endoplasmic reticulum cisternae
were sparse. These ultrastructural features confirmed the diagnosis of a
well-differentiated SMN. No nuclear viral particles were detected.


## 3.1. Microscopic and Electron Microscopic Findings

All patient
samples were positive for EBV-EBER by ISH (Figure2), and the vast majority of
cells were positive for EBER by ISH. IHC from biopsies of the liver tumor from
Patient 1 was positive for SMA and desmin, but negative for CD117 (ckit),
HMB-45, and S-100. Histologic features from the colonic SMN from Patient 2
included mild to moderate increase in cellularity, low mitotic count (<1/10
HPF), and no areas of necrosis. IHC was positive for desmin and SMA, but
negative for CD117 and S-100.The liver
specimen from Patient 3 revealed multicentric EBV-associated SMNs consisting of
four nodules ranging in size from 0.4 cm to 6.7 cm (Figure2).  Moderate increased cellularity (with areas of
somewhat rounder, less fusiform cells), focal areas of necrosis, lack of
nuclear pleomorphism, and low mitotic rate (1 mitotic figure/50 high-powered
field) were noted. The tumors were positive for SMA, HHF35, desmin and negative
for CD117.  EM from Patient 3 revealed spindle
cells with abundant cytoplasmic aggregates of actin microfilaments, including
fusiform dense bodies and attachment plaques (Figure 2). Linear arrangements of
pinocytotic vesicles were noted in many cells. Cytoplasmic organelles were
mainly represented by mitochondria, while rough endoplasmic reticulum cisternae
were sparse. These ultrastructural features confirmed the diagnosis of a
well-differentiated SMN. No nuclear viral particles were detected.


## 4. Discussion

Multiple lines of evidence implicate EBV
in the formation of SMNs [4, 8].   EBV
DNA and RNA have been demonstrated in SMNs in immunocompromised patients [4, 5]
and in our patients. Conversely, EBV is absent in sporadic smooth muscle tumors
in immunocompetent patients [7, 11].
Pathogenesis appears to be related to infection and transformation of
smooth muscle cells by EBV; fusion of EBV-superinfected lymphoblastoid cells
and human embryonic fibroblasts may be the vehicle by which smooth muscle cells
are infected [12]. CD21 expression may play a role, in pathogenesis, as CD21
has been described in 16 of 20 EBV-associated SMNs in patients with AIDS [4, 6, 9].  Moreover, five to ten percent of
smooth muscle cells display strong immunofluorescent staining of CD21 [13]. Based on viral genomic analyses of tumors,
multifocal lesions appear to result from multiple independent infection events
rather than metastasis [14]. It appears that the virus is not overtly virulent,
since EBV seems to effect change in smooth muscle cells of immunocompromised
patients rather than immunocompetent patients.
Perhaps this is why these tumors tend to be low grade.Histologically, EBV-associated SMNs have
a mild to moderate increase in cellularity, low mitotic count (<1/10 HPF),
and little to no areas of necrosis. Others have reported foci of primitive-rounded
cells and intratumoral T lymphocytes [14, 15], but these were not seen in our
three cases.  Immunohistochemically, SMNs
stain positively for desmin and SMA. On EM, SMNs demonstrate spindle cells with
abundant cytoplasmic actin microfilaments with linearly arranged pinocytic
vesicles. SMNs appear to be relatively well differentiated with only a modest
degree of atypia [14].In a literature review of 19 reports of
EBV-associated SMNs following organ transplantation [16], SMNs occurred after a
latency of one to six years as a single tumor, or more often, as multifocal or
multicentric lesions in multiple organs [16]. The most common locations were
liver, lung, heart, and colon [16, 16–18].
As the behavior of SMNs appears related to immune status rather than to
specific histologic features, the mainstay of treatment was the reduction of
immunosuppression; however, five of the seven patients who survived with
follow-up of 10 months to 12 years underwent surgical resection [16].Immunosuppression predisposes patients to
the development of SMNs, first described 40 years ago [19]. Initially reported,
following renal transplantation and immunosuppression in the pediatric
population, SMNs are observed in patients after solid organ and stem cell
transplants [2–5], with AIDS [6], or with congenital immunodeficiency [7–10].
Reversal of immunosuppression, effective in treatment of posttransplant
lymphoproliferative disorder, appears critical to improved outcomes of
EBV-associated SMNs [18] (e.g., reduction or change of immunosuppressive agent
in transplant patients or use of famciclovir for high-risk patients [17]).In our case
series, the three patients with SMNs were immunodeficient for different reasons
(Table1). We
describe the first EBV-associated SMN in a human patient receiving etanercept,
although immune dysregulation associated with her psoriasis was more likely an
important predisposing condition.
Etanercept competitively binds TNF to reduce TNF activity from excessive
inflammatory levels to normal physiologic levels, effectively treating
rheumatoid arthritis and psoriasis within months [20]. Known side effects of
etanercept include an increased risk of lymphoma in these patients [20–22],
although it has been reported that long-term treatment with TNF-alpha
inhibitors does not increase EBV load in patients with rheumatoid arthritis
[23]. Psoriasis patients treated with etanercept have also been diagnosed with
noncutaneous solid tumors, nonmelanoma skin cancer, and melanoma as well as an
increased risk of liver cancer [24, 25]. Anti-TNF-alpha treatment has been
associated with vascular smooth muscle cell proliferation in a rabbit model
[26].Table 1
Three different clinical scenarios in which EBV-related SMNs developed: patient summary. Dx = diagnosis. SMN = smooth muscle neoplasm. DOD = dead of disease. AWD = alive with disease. NED = no evidence of disease.
#Age at Dx (year)Cause of immune dysregulationDate of start of immune dysregulationTime to SMNSiteTreatmenStatus follow-up155MCadaveric renal transplant prednisone, cyclosporine, azathioprine, mycophenolate mofetil19948 yearsLiver (caudate 14 cm); lungObservation (declined systemic therapy) (7/02)DOD 5 months218FAIDS CD4 count = 8Birth11 yearsColon, chest, paraspinal/ vertebral, liver, gluteal, perigastric areasAntiretrovirals doxorubicin, then dacarbazine (10/03)AWD without progression, 5 years325FPsoriasis, etanercept therapy20+ years, 8/0420+ years, 5 months after etanercept startedLiverStopped immunosuppressives; hepatic resection (10/05)NED, 3 yearsIn summary, these cases highlight the
variable aggressiveness and clinical outcomes of SMNs in patients with
immunosuppression on the basis of autoimmunity, iatrogenic immunosuppression,
and that caused by HIV. Chemotherapy and surgery both play a role in patients
with these usually indolent diseases. It remains to be seen whether effects on
EBV itself with antiviral agents or with EBV-specific T cell therapy [27] might
have salutary effects against this rare soft tissue neoplasm.



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