Natural killer T cell immunotherapy combined with oncolytic vesicular stomatitis virus or reovirus treatments differentially increases survival in mouse models of ovarian and breast cancer metastasis.

BACKGROUND: Oncolytic viruses reduce tumor burden in animal models and have generated promising results in clinical trials. However, it is likely that oncolytic viruses will be more effective when used in combination with other therapies. Current therapeutic approaches, including chemotherapeutics, come with dose-limiting toxicities. Another option is to combine oncolytic viruses with immunotherapeutic approaches. METHODS: Using experimental models of metastatic 4T1 breast cancer and ID8 ovarian peritoneal carcinomatosis, we examined natural killer T (NKT) cell-based immunotherapy in combination with recombinant oncolytic vesicular stomatitis virus (VSV) or reovirus. 4T1 mammary carcinoma cells or ID8 ovarian cancer cells were injected into syngeneic mice. Tumor-bearing mice were treated with VSV or reovirus followed by activation of NKT cells via the intravenous administration of autologous dendritic cells loaded with the glycolipid antigen α-galactosylceramide. The effects of VSV and reovirus on immunogenic cell death (ICD), cell viability and immunogenicity were tested in vitro. RESULTS: VSV or reovirus treatments followed by NKT cell activation mediated greater survival in the ID8 model than individual therapies. The regimen was less effective when the treatment order was reversed, delivering virus treatments after NKT cell activation. In the 4T1 model, VSV combined with NKT cell activation increased overall survival and decreased metastatic burden better than individual treatments. In contrast, reovirus was not effective on its own or in combination with NKT cell activation. In vitro, VSV killed a panel of tumor lines better than reovirus. VSV infection also elicited greater increases in mRNA transcripts for proinflammatory cytokines, chemokines, and antigen presentation machinery compared with reovirus. Oncolytic VSV also induced the key hallmarks of ICD (calreticulin mobilization, plus release of ATP and HMGB1), while reovirus only mobilized calreticulin. CONCLUSION: Taken together, these results demonstrate that oncolytic VSV and NKT cell immunotherapy can be effectively combined to decrease tumor burden in models of metastatic breast and ovarian cancers. Oncolytic VSV and reovirus induced differential responses in our models which may relate to differences in virus activity or tumor susceptibility.

Phase I Study of Oncolytic Vaccinia Virus GL-ONC1 in Patients with Peritoneal Carcinomatosis.

Purpose: Peritoneal carcinomatosis is common in advanced tumor stages or disease recurrence arising from gastrointestinal cancers, gynecologic malignancies, or primary peritoneal carcinoma. Because current therapies are mostly ineffective, new therapeutic approaches are needed. Here, we report on a phase I study designed to assess safety, MTD, and antitumor activity of intraperitoneal administration of oncolytic vaccinia virus GL-ONC1 in advanced stage peritoneal carcinomatosis patients.Patients and Methods: GL-ONC1 was administered intraperitoneally every 4 weeks for up to four cycles at three different dose levels (107-109 pfu) following a standard 3+3 dose escalation design. GL-ONC1 was infused via an indwelling catheter that enabled repetitive analyses of peritoneal fluid biopsies. The primary study objective was safety of GL-ONC1 according to Common Terminology Criteria for Adverse Events, version 4.0 (CTCAEv4.0).Results: Patients with advanced-stage peritoneal carcinomatosis (n = 7) or advanced peritoneal mesothelioma (n = 2) received 24 doses of GL-ONC1. Adverse events were limited to grades 1-3, including transient flu-like symptoms and increased abdominal pain, resulting from treatment-induced peritonitis. No DLT was reported, and the MTD was not reached. Furthermore, no signs of viral shedding were observed. Importantly, in 8 of 9 study patients, effective intraperitoneal infections, in-patient replication of GL-ONC1, and subsequent oncolysis were demonstrated in cycle 1. All patients developed neutralizing activities against GL-ONC1.Conclusions: GL-ONC1 was well tolerated when administered into the peritoneal cavity of patients with advanced stage peritoneal carcinomatosis. Efficient tumor cell infection, in-patient virus replication, and oncolysis were limited to treatment cycle 1 (ClinicalTrials.gov number, NCT01443260). Clin Cancer Res; 24(18); 4388-98. ©2018 AACR.

Telomerase-specific oncolytic adenovirus expressing TRAIL suppresses peritoneal dissemination of gastric cancer.

Peritoneal dissemination is the most common condition of metastasis in gastric cancer. The survival duration of a patient with advanced stage gastric cancer, may be improved by gene therapy. In this study, we used an oncolytic adenovirus vector (Ad/TRAIL-E1) that expresses both the TRAIL and E1A genes under the control of a tumor-specific promoter. We evaluated the anti-tumor effect of Ad/TRAIL-E1 on gastric cancer cells in vitro, as well as in vivo in a xenograft peritoneal carcinomatosis mouse model. Our data showed that Ad/TRAIL-E1 induced TRAIL-mediated apoptosis in gastric cancer cell lines, but not in the normal cell lines. In addition, Ad/TRAIL-E1 significantly inhibited peritoneal metastasis and prolonged the survival of mice without treatment-related toxicity. Therefore, tumor-specific TRAIL expression from an oncolytic adenovirus vector may provide a novel therapeutic approach for the treatment of advance stage gastric cancer with peritoneal dissemination.

Vesicular stomatitis virus is a potent agent for the treatment of malignant ascites.

Cancer cells in ascites are usually exposed to a hypoxia tumor microenvironment and utilize enhanced glycolysis which produces energy and metabolizes nutrients to support proliferation. Vesicular stomatitis virus (VSV) is an oncolytic virus that relies on the host cellular metabolism for replication. We tested the efficacy of VSV on peritoneal carcinomatosis and assessed VSV replication in cancer cells from ascites. BALB/c female mice bearing peritoneal H22 or MethA cells received an i.p. administration of 1x108 PFU VSV or 1x108 PFU equivalent of UV-inactivated VSV on day 10, 12 and 14 after incubation. Administration of VSV resulted in a significant inhibition of ascites formation and prolonged survival of the treated mice. The replication of VSV was obviously enhanced in the cancer cells from the ascites. Considering the central carbon metabolic pathways, cancer cells in the malignant ascites provided more exogenous glucose, glutamine and pyruvate after VSV infection due to its unregulated glycolytic activity and glutamine metabolism. Pharmacologically, inhibition of the glycolytic pathway and glutamine metabolism reduced VSV replication, and this inhibited replication was rescued by the addition of multiple tricarboxylic acid (TCA) cycle intermediates. Our results demonstrated that metabolic adaptive processes in peritoneal carcinoma, such as high glycolytic activity and glutamine metabolism, favor VSV replication. These results suggest the clinical potency of VSV in the treatment of malignant ascites and provide new insights into the further exploration of the potential application of VSV in the treatment of hypoxia ascites cancer cells.

Imaging and Antitumoral Effect of a Cyclo-oxygenase 2-specific Replicative Adenovirus for Small Metastatic Gastric Cancer Lesions.

BACKGROUND: Long-term outcomes of patients with peritoneal dissemination of gastric cancer remain unsatisfactory despite advances in treatment modalities. Internal luminescence conditionally replicative adenovirus (CRAd) presents a novel approach for cancer treatment and imaging. MATERIALS AND METHODS: 3CL is a modified cyclooxygenase-2 (COX2) promoter-driven CRAd which contains the luciferase expression gene for bioluminescence imaging. The visualizing and therapeutic effect of 3CL was evaluated in a mouse model of peritoneal dissemination. RESULTS: Intraperitoneal injection of 3CL achieved the shrinkage and reduction of lesions of peritoneal dissemination. Six model mice treated with 3CL had a significantly longer mean survival time than 6 mock-treated mice (85.7 versus 34.3 days, p=0.0005). By whole-body bioluminescent imaging, the sensitivity and specificity of peritoneal dissemination detection through macroscopic inspection were 58.1% and 83.2%, respectively, whereas 3CL viral imaging modality yielded corresponding values of 78.8% and 99.3%. Peritoneal lesions detected by imaging histologically contained cancer cells and necrotic tissue, which originated from viral oncolytic effects. CONCLUSION: Cox2 CRAds with 5/3 chimeric-fiber modification, therefore, appear to be a promising imaging and therapeutic tools for peritoneal dissemination of gastric cancer.

Assessment of the Na/I symporter as a reporter gene to visualize oncolytic adenovirus propagation in peritoneal tumours.

PURPOSE: In vivo imaging of the spread of oncolytic viruses using the Na/I symporter (NIS) has been proposed. Here, we assessed whether the presence of NIS in the viral genome affects the therapeutic efficacy of the oncolytic adenovirus dl922-947 following intraperitoneal administration, in a mouse model of peritoneal ovarian carcinoma. METHODS: We generated AdAM7, a dl922-947 oncolytic adenovirus encoding the NIS coding sequence. Iodide uptake, NIS expression, infectivity and cell-killing activity of AdAM7, as well as that of relevant controls, were determined in vitro. In vivo, the propagation of this virus in the peritoneal cavity of tumour-bearing mice was determined using SPECT/CT imaging and its therapeutic efficacy was evaluated. RESULTS: In vitro infection of ovarian carcinoma IGROV-1 cells with ADAM7 led to functional expression of NIS. However, the insertion of NIS into the viral genome resulted in a loss of efficacy of the virus in terms of replication and cytotoxicity. In vivo, on SPECT/CT imaging AdAM7 was only detectable in the peritoneal cavity of animals bearing peritoneal ovarian tumours for up to 5 days after intraperitoneal administration. Therapeutic experiments in vivo demonstrated that AdAM7 is as potent as its NIS-negative counterpart. CONCLUSION: This study demonstrated that despite the detrimental effect observed in vitro, insertion of the reporter gene NIS in an oncolytic adenovirus did not affect its therapeutic efficacy in vivo. We conclude that NIS is a highly relevant reporter gene to monitor the fate of oncolytic adenovectors in live subjects.

Synchronous hepatic, mesenteric and pulmonary Epstein-Barr virus-associated smooth muscle tumors in a renal transplant recipient.

Epstein-Barr virus-associated smooth muscle tumors (EBV-SMT) are distinct lesions that occur in immunocompromised patients. EBV-SMT following solid organ transplantation are rare and generally have an indolent biological behavior. Post-transplant EBV-SMT have been reported in various anatomical locations. This report describes a synchronous and multicentric development of EBV-SMT in liver, mesentery, and lung of a 33-yr-old male patient, 10 yr after a deceased allograft renal transplantation. The hepatic and mesenteric tumors were available for study. These tumors were composed of bland looking, desmin-positive, spindle-shaped cells which showed a strong nuclear staining for EBV with in situ hybridization technique. A literature review of post solid organ transplant EBV-SMT in the liver and lung, particularly regarding their pathogenesis, synchronicity and biological behavior would be provided.

Intraperitoneal administration of telomerase-specific oncolytic adenovirus sensitizes ovarian cancer cells to cisplatin and affects survival in a xenograft model with peritoneal dissemination.

Despite tremendous development in chemotherapy for ovarian cancer over the past few decades, the prognosis of advanced cases with massive peritoneal dissemination is still unsatisfactory, and novel treatment modalities that can combine with chemotherapy are urgently needed. We recently developed virotherapy for solid tumors using telomerase-specific replication-selective adenoviruses (Telomelysin: OBP-301), in which the human telomerase reverse transcriptase (hTERT) gene promoter has been inserted to direct tumor-specific E1 gene expression. In this study, we investigated the anti-tumor effects of OBP-301, combined with cisplatin (CDDP), on ovarian cancer cells. In vitro treatment of SKOV3 cells with OBP-301 at a multiplicity of infection (MOI) of 0.01-100 induced significant cell death in a dose-dependent manner, with moderate cytotoxicity at an MOI of 1-10 and maximal cytotoxicity at an MOI of 100. In contrast, OBP-301 treatment of normal human cells showed no significant cell death at an MOI of 1-10 and exhibited modest cytotoxicity at an MOI of 100. The effects of low-dose CDDP at 0.5-1 microM, which induced only 20% cell death, were significantly augmented by combination with OBP-301 at an MOI of 1-10, finally achieving 40% cell death. Such enhancement of CDDP sensitivity was also observed in CDDP-resistant ovarian cancer cells. The combinatorial effects were further tested using a xenograft mouse model of SKOV3 with peritoneal dissemination. After intraperitoneal administration of OBP-301, we confirmed that injected OBP-301 fused with the green fluorescent protein (GFP) gene (OBP-401) was preferentially localized to peritoneal disseminations, as determined by fluorescence imaging. Treatment of mice with CDDP at low dose (0.5 mg kg(-1)) had modest effects, showing a 10% decrease in disseminations, whereas combination with intraperitoneal administration of OBP-301 at an MOI of 10 led to enhanced effects, achieving an approximately 80% decrease in disseminations. Kaplan-Meier analysis showed improved overall survival of mice treated with CDDP plus OBP-301 compared with CDDP alone. These findings support the therapeutic potential of intraperitoneal administration of OBP-301 to sensitize ovarian cancer cells to CDDP.

Reovirus inhibits the peritoneal dissemination of pancreatic cancer cells in an immunocompetent animal model.

The prognosis of pancreatic cancer with peritoneal dissemination has not improved. The aim of this study was to clarify whether oncolytic reovirus is effective against the peritoneal dissemination of pancreatic cancer in an immunocompetent animal model. The hamster pancreatic cancer cells HaP-T1 were inoculated into the peritoneal cavity of the hamster and reovirus (1x10(8) plaque-forming units) was administered into the peritoneal cavity on days 1, 3, 5 and 7 after HaP-T1 inoculations. The number and weight of the disseminated nodules in each group were recorded. Reovirus protein in the disseminated nodules was examined by immunohistochemical staining. The tumor volumes of peritoneal dissemination in the treatment group were significantly less than those in the control group (p<0.05). In addition, the amount of ascites was decreased in the treatment group in comparison to the control group. Immunohistochemical examination revealed that reovirus replication was seen only in the disseminated nodules but not in surrounding normal tissues. There were no serious side effects observed in this study. These data suggested that intraperitoneal administration of reovirus might be an effective form of oncolytic viral therapy for peritoneal dissemination of pancreatic cancer.

Regional administration of oncolytic Echovirus 1 as a novel therapy for the peritoneal dissemination of gastric cancer.

The dissemination of malignant gastric cells to the peritoneum occurs frequently, usually as an early event in disease, and results in poor patient prognosis. Surgery and chemotherapy offer limited therapeutic success. The low-pathogenic human enterovirus, Echovirus 1 (EV1), is an oncolytic virus that selectively targets and destroys malignant prostate and ovarian cancer xenografts in vivo. Lytic EV1 infection requires the cell surface expression of alpha(2)beta(1), an integrin involved in the dissemination of gastric cancer cells to the peritoneum. Herein, we evaluated the capacity of EV1 for anti-neoplastic cell action in gastric peritoneal carcinomatosis. Flow cytometric analysis demonstrated that alpha(2)beta(1) was abundantly surface expressed on a panel of gastric cancer cell lines, rendering the majority of lines highly susceptible to in vitro lytic EV1 infection and supportive of efficient viral progeny production. A bioluminescent MKN-45-Luc SCID mouse model of peritoneal dissemination was developed to allow real-time non-invasive monitoring of peritoneal tumor burden. Employing this mouse model, we demonstrated a therapeutic dose-response for escalating oncolytic EV1 doses. Taken together, these results emphasize the exciting potential for EV1 as a single or adjunct therapy for the control of the peritoneal dissemination of gastric cancer.

Low grade peritoneal mucinous carcinomatosis associated with human papilloma virus infection: case report.

Pseudomyxoma peritonei is a clinical syndrome characterized by peritoneal dissemination of a mucinous tumor with mucinous ascites. The vast majority of the pseudomyxoma peritonei are associated with mucinous neoplasms of the appendix. We describe a case of pseudomyxoma peritonei associated with mucinous adenocarcinoma of the cervix in a 60-year-old woman. The patient developed low grade mucinous peritoneal carcinomatosis 8 years after hysterectomy for cervical adenocarcinoma. No other primary mucinous tumor was identified and peritoneal carcinomatosis tested positive for high-risk human papilloma virus (HPV), showing both integrated and episomal pattern. HPV has been previously associated with development of cervical carcinomas (both squamous and mucinous) but neither has cervical adenocarcinoma nor HPV been implicated in development of pseudomyxoma peritonei. To the best of our knowledge, this is the first description of HPV-associated malignancy presenting as pseudomyxoma peritonei.

Cellular and molecular parameters of mesothelioma.

Malignant mesotheliomas (MM) are neoplasms arising from mesothelial cells that line the body cavities, most commonly the pleural and peritoneal cavities. Although traditionally recognized as associated with occupational asbestos exposures, MMs can appear in individuals with no documented exposures to asbestos fibers, and emerging data suggest that genetic susceptibility and simian virus 40 (SV40) infections also facilitate the development of MMs. Both asbestos exposure and transfection of human mesothelial cells with SV40 large and small antigens (Tag, tag) cause genetic modifications and cell signaling events, most notably the induction of cell survival pathways and activation of receptors, and other proteins that favor the growth and establishment of MMs as well as their resistance to chemotherapy. Recent advances in high-throughput technologies documenting gene and protein expression in patients and animal models of MMs can now be validated in human MM tissue arrays. These have revealed expression profiles that allow more accurate diagnosis and prognosis of MMs. More importantly, serum proteomics has revealed two new candidates (osteopontin and serum mesothelin-related protein or SMRP) potentially useful in screening individuals for MMs. These mechanistic approaches offer new hope for early detection and treatment of these devastating tumors.

Using sindbis viral vectors for specific detection and suppression of advanced ovarian cancer in animal models.

We studied the therapeutic value of Sindbis vectors for advanced metastatic ovarian cancer by using two highly reproducible and clinically accurate mouse models: a SCID xenograft model, established by i.p. inoculation of human ES-2 ovarian cancer cells, and a syngenic C57BL/6 model, established by i.p. inoculation of mouse MOSEC ovarian cancer cells. We demonstrate through imaging, histologic, and molecular data that Sindbis vectors systemically and specifically infect/detect and kill metastasized tumors in the peritoneal cavity, leading to significant suppression of the carcinomatosis in both animal models. Use of two different bioluminescent genetic markers for the IVIS Imaging System permitted demonstration, for the first time, of an excellent correlation between vector delivery and metastatic locations in vivo. Sindbis vector infection and growth suppression of murine MOSEC tumor cells indicate that Sindbis tumor specificity is not attributable to a species difference between human tumor and mouse normal cells. Sindbis virus is known to infect mammalian cells using the Mr 67,000 laminin receptor. Immunohistochemical staining of tumor cells indicates that laminin receptor is elevated in tumor versus normal cells. Down-regulated expression of laminin receptor with small interfering RNA significantly reduces the infectivity of Sindbis vectors. Tumor overexpression of the laminin receptor may explain the specificity and efficacy that Sindbis vectors demonstrate for tumor cells in vivo. We show that incorporation of antitumor cytokine genes such as interleukin-12 and interleukin-15 genes enhances the efficacy of the vector. These results suggest that Sindbis viral vectors may be promising agents for both specific detection and growth suppression of metastatic ovarian cancer.

Human herpes virus 8-negative primary effusion lymphoma in a patient with a ventriculoperitoneal shunt tube.

A 60-year-old woman was referred to our hospital in 1996 due to an abdominal distension in the right lower quadrant. She had undergone a partial resection of a cholesteatoma at the right temporal lobe of the cerebrum 30 years previously, and a ventriculoperitoneal shunt (VPS) tube had been placed with drainage into the right lower peritoneal cavity. The patient developed paralytic ileus in December 1966, and ultrasound and computed tomography of the abdomen revealed a cystic mass in the right lower quadrant without lymphadenopathies or masses. Cytologic examinations of the fluid in the cystic mass revealed signs of malignant lymphoma. After the resection of the cystic mass, lymphoma cells were detected in the fluid, but the wall of the cyst consisted of only fibrous tissues. Results of immunophenotypic analysis of the lymphoma cells by immunocytochemistry or flow cytometry were positive for CD19, CD20, CD22, CD45, and HLA-DR but negative for CD45RO, CD3, CD4, and CD8. The genome of human herpes virus (HHV)-8 was not detected in the lymphoma cells, but Epstein-Barr (EB) nuclear antigen 1 and EB virus (EBV)-encoded small nuclear RNAs were detected. Chromosome analysis by the G-banding method showed complicated abnormalities including der(8)t(2;8)(q31;q24), but Southern blotting analysis suggested that the c-myc oncogene did not participate in the lymphomagenesis. The patient's disease was diagnosed as HHV-8-negative primary effusion lymphoma (PEL). The long-standing inflammatory stimulation by a VPS tube might have contributed to the clonal evolution of EBV-infected lymphocytes. resulting in the development of PEL.

HHV8-negative primary effusion lymphoma of the peritoneal cavity presenting with a distinct immunohistochemical phenotype.

Primary effusion lymphoma (PEL) has been recognized as a body-cavity-based lymphoma that was originally reported to be associated with human herpes virus 8 (HHV8) infection, and was frequently found in human immunodeficiency virus-positive (HIV) patients. Here we describe an autopsy case of PEL of the peritoneal cavity in an immunocompetent patient. Cytological analysis of tumor cells within ascites revealed immunocytochemical features of keratin positivity and CD45 negativity. At autopsy, the presence of a massive volume of ascites as well as diffuse tumor cell infiltrates within the serosa of the intestine and mesenterium were observed. Tumor cells were morphologically similar to anaplastic large-cell lymphoma, but were immunohistochemically positive for keratin and epithelial membrane antigen (EMA). They also showed no reactivity to representative lymphocyte surface markers including CD45, in addition to being negative for CD30 and p80NPM/ALK. Molecular analysis of the tumor cells revealed monoclonality of the immunoglobulin heavy-chain gene rearrangement which demonstrated a lymphoma of the B-cell lineage. Furthermore, HHV8 was not detected by immunohistochemical analysis, PCR or nested PCR technique. Based on these results, we consider the present case to be an HHV8-negative PEL with keratin and EMA positivity.

Effect of preexisting anti-herpes immunity on the efficacy of herpes simplex viral therapy in a murine intraperitoneal tumor model.

HSV-1716, a replicating nonneurovirulent herpes simplex virus type 1, has shown efficacy in treating multiple types of human tumors in immunodeficient mice. Since the majority of the human population has been previously exposed to herpes simplex virus, the efficacy of HSV-based oncolytic therapy was investigated in an immunocompetent animal tumor model. EJ-6-2-Bam-6a, a tumor cell line derived from h-ras-transformed murine fibroblast, exhibit a diffuse growth pattern in the peritoneal cavity of BALB/c mice and replicate HSV-1716 to titers observed in human tumors. An established intraperitoneal (ip) tumor model of EJ-6-2-Bam-6a in naive and HSV-immunized mice was used to evaluate the efficacy of single or multiple ip administrations of HSV-1716 (4 x 10(6) pfu/treatment) or of carrier cells, which are irradiated, ex vivo virally infected EJ-6-2-Bam-6a cells that can amplify the viral load in situ. All treated groups significantly prolonged survival versus media control with an approximately 40% long-term survival rate (cure) in the multiply treated, HSV-naive animals. Prior immunization of the mice with HSV did not significantly decrease the median survival of the single or multiply treated HSV-1716 or the carrier cell-treated groups. These studies support the development of replication-selective herpes virus mutants for use in localized intraperitoneal malignancies.

Simian virus 40: the link with human malignant mesothelioma is well established.

Mesotheliomas are malignancies of the pleural, pericardial, and peritoneal surfaces with a mean survival of less than 1 year from the time of diagnosis (1). While mesotheliomas were extremely rare in the first half of this century, the incidence of these tumors has increased enormously in the last several decades. Presently, 2-3 thousand people in the US develop and die of mesothelioma each year (1). It is estimated that approximately 80% of mesotheliomas develop in people with a history of occupational asbestos exposure or in individuals with family member(s) professionally exposed to asbestos that brought home fibers on their clothing (1). Although conventional wisdom dictates that asbestos is the most commonly associated "environmental" factor with mesothelioma, asbestos does not transform human mesothelioma cells in tissue culture (2). This suggests that additional carcinogens act in concert with asbestos to cause mesothelioma. Recent evidence indicated that Simian Virus 40 (SV40) preferentially causes mesotheliomas in hamsters, and that SV40 is present in up to 80% of human mesotheliomas in the US and in Europe (reviewed in ref. 3 and 4).

[Smooth muscle mesentery associated with Epstein-Barr virus (EBV). Report of a case in a child with a liver transplant under FK506 regimen]. / Tumeur musculaire lisse mésentérique associée au virus d'Epstein-Barr (EBV). A propos d'un cas chez un enfant transplanté hépatique sous FK 506.

Smooth-muscle tumors, benign and malignant, are increasingly recognized in children who are immunocompromised because of HIV infection and organ transplantation. We report a case of an EBV-associated smooth-muscle tumor, of unusual location arising in a seven-year-old post-transplant patient who was previously treated for a lymphoproliferative disease. Five years after liver transplantation, a mesenteric tumor was diagnosed. The tumor was composed of spindle cells with smooth-muscle features. Immunohistochemical analysis was positive for muscle-specific actin and desmin, negative for EBV latent membrane protein (LMP-1). In situ hybridization revealed nuclear EBV sequences. This case underlines the role of EBV infection in the development of unusual smooth-muscle tumors after organ transplantation. The evolution of these rare tumors is uncertain.