BET-Inhibitors Disrupt Rad21-Dependent Conformational Control of KSHV Latency.

Kaposi's Sarcoma-associated Herpesvirus (KSHV) establishes stable latent infection in B-lymphocytes and pleural effusion lymphomas (PELs). During latency, the viral genome persists as an epigenetically constrained episome with restricted gene expression programs. To identify epigenetic regulators of KSHV latency, we screened a focused small molecule library containing known inhibitors of epigenetic factors. We identified JQ1, a Bromodomain and Extended Terminal (BET) protein inhibitor, as a potent activator of KSHV lytic reactivation from B-cells carrying episomal KSHV. We validated that JQ1 and other BET inhibitors efficiently stimulated reactivation of KSHV from latently infected PEL cells. We found that BET proteins BRD2 and BRD4 localize to several regions of the viral genome, including the LANA binding sites within the terminal repeats (TR), as well as at CTCF-cohesin sites in the latent and lytic control regions. JQ1 did not disrupt the interaction of BRD4 or BRD2 with LANA, but did reduce the binding of LANA with KSHV TR. We have previously demonstrated a cohesin-dependent DNA-loop interaction between the latent and lytic control regions that restrict expression of ORF50/RTA and ORF45 immediate early gene transcripts. JQ1 reduced binding of cohesin subunit Rad21 with the CTCF binding sites in the latency and lytic control regions. JQ1 also reduced DNA-loop interaction between latent and lytic control regions. These findings implicate BET proteins BRD2 and BRD4 in the maintenance of KSHV chromatin architecture during latency and reveal BET inhibitors as potent activators of KSHV reactivation from latency.

EBV-associated but HHV8-unrelated double-hit effusion-based lymphoma.

Effusion-based lymphoma is a rare and unique type of large B-cell lymphoma presenting in effusion without a mass lesion. It shares many clinicopathological features with primary effusion lymphoma (PEL), but is distinct from PEL by the absence of HHV8 association. Double hit lymphoma (DHL) is an aggressive B-cell lymphoma, defined by concurrent rearrangement of MYC and BCL2 or BCL6. DHL often presents as lymphadenopathy or an extranodal mass, but rarely occurs in effusion. Here we report a 61-year-old male with alcoholic cirrhosis presenting as massive ascites and left pleural effusion. He has no HIV, HBV or HCV infection and no mass lesion by CT scans. Cytology of both pleural effusion and ascites show large lymphoma cells with plasmablastic morphology characterized by pleomorphic and eccentric nuclei, prominent nucleoli and frequent mitoses. Immunohistochemical study with cell block shows that the lymphoma cells express plasma cell-related markers (CD138, MUM-1 and EMA), but not CD3, CD30, CD45, B-cell markers (CD19, CD20, CD79a, and PAX5), HHV8, ALK or cytokeratin. EBER is positive in most lymphoma cells. Fluorescence in situ hybridization reveals rearrangement at the IGH, BCL2, and MYC loci, but not at BCL6. It is diagnosed as an EBV-associated but HHV8-unrelated double hit effusion-based lymphoma with plasmablastic features. The patient passed away soon after diagnosis without chemotherapy. This is the first reported case of double-hit effusion-based lymphoma with MYC and BCL2 rearrangement. This case illustrates the importance of integrating clinical, cytological, immunophenotypical, and molecular findings to reach a correct diagnosis. Diagn. Cytopathol. 2017;45:257-261. © 2016 Wiley Periodicals, Inc.

Susceptibility of KSHV-Infected PEL Cell Lines to the Human Complement System.

Pleural effusion lymphoma (PEL) is a rare B-cell lymphoma that has a very poor prognosis with a median survival time of around 6 months. PEL is caused by Kaposi's sarcoma-associated herpesvirus, and is often co-infected with the Epstein Barr virus. The complement system is fundamental in the innate immune system against pathogen invasion and tumor development. In the present study, we investigated the activation of the complement system in PEL cells using human serum complements. Interestingly, two widely used PEL cell lines, BCP-1 and BCBL-1, showed different susceptibility to the complement system, which may be due to CD46 expression on their cell membranes. Complement activation did not induce apoptosis but supported cell survival considerably. Our results demonstrated the susceptibility of PEL to the complement system and its underlying mechanisms, which would provide insight into understanding the pathogenesis of PEL.

Primary effusion lymphoma presenting as a cutaneous intravascular lymphoma.

Primary effusion lymphoma (PEL) is a rare and aggressive lymphoma that arises in the context of immunosuppression and is characterized by co-infection with Epstein-Barr virus (EBV) and human herpesvirus-8/Kaposi sarcoma-associated herpesvirus (HHV-8/KSHV). It was originally described as arising in body cavity effusions, but presentation as a mass lesion (extracavitary PEL) is now recognized. Here, we describe a case of PEL with an initial presentation as an intravascular lymphoma with associated skin lesions. The patient was a 53-year-old man with human immunodeficiency virus (HIV)/acquired immune deficiency syndrome (AIDS) who presented with fevers, weight loss and skin lesions concerning for Kaposi sarcoma (KS). A skin biopsy revealed no evidence of KS; however, dermal vessels contained large atypical cells that expressed CD31 and plasma cell markers but lacked most B- and T-cell antigens. The atypical cells expressed EBV and HHV-8. The patient subsequently developed a malignant pleural effusion containing the same neoplastic cell population. The findings in this case highlight the potential for unusual intravascular presentations of PEL in the skin as well as the importance of pursuing microscopic diagnosis of skin lesions in immunosuppressed patients.

Self-limited effusion large B-cell lymphoma: two cases of effusion lymphoma maintaining remission after drainage alone.

We report two cases of human herpesvirus-8 (HHV-8)-negative large B-cell lymphoma involving pericardial and/or pleural effusion that regressed after drainage alone. Case 1 is a 70-year-old man showing massive pericardial effusion. Cytology of the drained effusion showed monotonous infiltration of CD3-, CD20+, CD79a+, and CD138- large B-cells. Monoclonality was shown by Southern blot analysis. Case 2 is a 70-year-old man with massive pericardial and bilateral pleural effusion. Cytology of pericardial effusion showed infiltration of CD20+, CD45RO-, CD138-, immunoglobulin lambda chain+, and kappa chain- large B cells. In both cases, effusion resolved after drainage and no relapse has been observed. HHV-8 was not demonstrated in either case. Clinical presentation of our two cases resembled primary effusion lymphoma (PEL), but cytomorphology, immunophenotype, and prognosis were clearly distinct from those of PEL. HHV-8-negative effusion lymphomas might include prognostically favorable self-limited tumors that could regress without any cytotoxic therapy.

Prognosis in primary effusion lymphoma is associated with the number of body cavities involved.

Primary effusion lymphoma (PEL) is a rare lymphoma associated with Kaposi sarcoma-associated herpesvirus (KSHV), also known as human herpesvirus 8 (HHV8), and characterized by a malignant body cavity effusion without solid organ or nodal involvement. Prognostic factors in patients with PEL have not been systematically studied. We conducted a literature search for patients with HHV8-positive PEL to identify potential prognostic factors for survival. Our search identified 147 patients, among which 104 patients were HHV8-positive. The median overall survival was 9 months. The median age was 57 years with a male predominance (6:1). Pathologically, 33% of the patients expressed CD20 and 69% expressed CD30. Patients with PEL with > 1 body cavity involved had a median overall survival (OS) of 4 months compared with 18 months in patients with only one cavity involved (p = 0.003). Additionally, in patients with one involved body cavity, pericardial involvement was associated with a longer median OS than pleural followed by peritoneal involvement (40, 27 and 5 months, respectively; p = 0.04). In conclusion, our study suggests that the number and location of body cavities involved are prognostic in patients with PEL.

A new cancer cell detection method using an infectivity-enhanced adenoviral vector.

Cytological examination is widely used as a diagnostic tool because of the ease of collecting cells from the involved area. However, the diagnostic yield of cytological examination is unsatisfactory; the reasons include sampling error, poorly prepared samples, small numbers of malignant cells, and low grades of cellular atypia. In this study, we focused on the high infectivity of adenovirus towards epithelial cells and applied the luciferase- expressing adenoviral vector to a new cancer cell detection tool. In addition, adenoviral infectivity was enhanced by modifying viral fiber proteins. The sensitivity of the diagnostic tool was tested using the NCI-H1299 lung cancer cell line, and validated in body fluid samples from cancer patients with a variety of etiology. Results showed that the adenovirus efficiently transfected NCI-H1299 with high sensitivity. Only 10 cancer cells were sufficient for detection of luciferase signals. In body fluid samples, the adenovirus confirmed the diagnosis for malignant and benign cancer, but not in non-epithelial cell derived samples. This study provides proof-of-concept for a more reliable and sensitive diagnostic tool for epithelium-derived cancer.

[Primary effusion lymphoma in two kidney transplant recipients]. / Lymphome des séreuses chez le transplanté rénal: deux cas.

BACKGROUND: Primary effusion lymphoma (PEL) is a highly malignant non-Hodgkin lymphoma associated with Kaposi's sarcoma-associated herpesvirus/human herpesvirus-8 infection (KSHV/HHV-8). It is chiefly seen in HIV patients and is rare in transplant recipients, possibly going unrecognized. OBSERVATION: We describe two male kidney transplant recipients, aged 47 and 51 years, followed for Kaposi's sarcoma in skin, lymph nodes, gastrointestinal (GI) tract and lung whose disease was poorly controlled by sirolimus and chemotherapy. Recurrent pleural effusion contrasted with reduction of cutaneous Kaposi lesions. KHSV viral loads were negative or very low in plasma, were negative or very low, whereas those in pleural effusion were high. Lymphoma cells were discovered only seven to nine months after the initial effusion despite repeated needle biopsies. In one patient, tumour cells were co-infected with Epstein-Barr virus. CONCLUSION: The contrast between a very low KHSV viral load in plasma and a very high viral load pleural effusion should alert physicians and prompt suspicion of PEL in Kaposi's sarcoma patients with recurrent serous effusion. The potential inhibitory role of sirolimus on PEL progression is discussed.

Cytology of metastatic cervical squamous cell carcinoma in pleural fluid: report of a case confirmed by human papillomavirus typing.

Cervical squamous cell carcinomas are rarely the cause of malignant effusions. Their identification can be relatively easy when keratinizing atypical squamous cells are present, but may be very difficult when only nonkeratinizing malignant cells are present. We present the case of a 47-year-old woman who presented with a large left pleural effusion after having recently completed chemoradiation therapy for stage IIB cervical squamous cell carcinoma. Cytologic examination of the fluid showed a uniform population of single atypical cells with finely vacuolated cytoplasm, ectoendoplasmic demarcation, cell-in-cell arrangements, and short rows of cells with intervening "windows," all features reminiscent of mesothelial cells. No keratinization or three-dimensional cell clusters were identified. A panel of immunohistochemical stains was performed on the cell block material, and the atypical cells were positive for cytokeratin 5/6, p63, and p16 but not for cytokeratin 7, calretinin, WT1, or Ber-EP4 or TTF1. These findings were consistent with metastatic squamous cell carcinoma. HPV DNA determination and typing by PCR confirmed the presence of HPV16 in an aliquot of pleural fluid. This is to our knowledge the first reported case of pleural fluid involved by metastatic squamous cell carcinoma where HPV DNA testing was used to confirm the origin of the metastasis. Despite its rarity, metastatic nonkeratinizing squamous cell carcinoma should be considered when a single cell population of large atypical cells is found in effusions. Immunoperoxidase stains and HPV testing can be performed to establish the diagnosis and confirm the origin from a cervical primary.

Human herpes virus 8-unrelated primary effusion lymphoma-like lymphoma: report of a rare case and review of the literature.

A rare case of human herpes virus 8-unrelated primary effusion lymphoma-like lymphoma: a report and review of the literature. APMIS 2009; 117:222-29. Primary effusion lymphoma (PEL) is a very rare type of lymphoma usually confined to the body cavities predominantly in immunosuppressed patients infected with human herpes virus 8 (HHV-8). The new term for HHV-8 independent PEL is HHV8-unrelated PEL-like lymphoma. We describe an 89-year-old human immunodeficiency virus (HIV)-negative male patient with HHV8-unrelated PEL-like lymphoma in the pleura. No hepatosplenomegaly or lymphadenopathy was detected. Chest radiography and computed tomography revealed right pleural effusion, but no evidence of tumor mass or lymph node enlargement. Cytological analysis of the pleural effusion revealed a high-grade lymphoma with round nuclei, prominent nucleoli and abundant cytoplasm with immunophenotypes positive for CD45, CD30, CD38, CD7 and CD71. Because of the advanced age, no chemotherapy was given. Effusion resolved spontaneously. One year after the diagnosis, a new pleural effusion developed at the left side. Following thoracentesis and pleurodesis, the patient remained in complete remission for 40 months. To date, 30 cases of HHV8-unrelated PEL-like lymphoma/HIV negative have been reported in the literature. The outcome of the HHV8-unrelated PEL-like lymphoma patients who were HIV negative seems to be better than HIV- and HHV-8-positive PEL.

Role of viral induced vascular endothelial growth factor (VEGF) production in pleural effusion and malignant mesothelioma.

Vascular endothelial growth factor (VEGF) plays a key role in formation of pleural effusions and in tumorigenesis and progression of malignant mesothelioma. Mesothelial cells (MC) express the viral receptors Toll-like receptor 3 (TLR3), RIG-I and MDA5. Activation of these receptors by viral RNA exemplified by poly(I:C) RNA leads to a time- and dose-dependent increase of mesothelial VEGF synthesis. To show the specific effect of viral receptors knockdown experiments with siRNA for TLR3, RIG-I and MDA5 were performed. This finding of viral induced mesothelial VEGF synthesis may indicate a novel link between viral infections and formation of pleural effusions and progression of malignant mesothelioma.

Measles virus induces oncolysis of mesothelioma cells and allows dendritic cells to cross-prime tumor-specific CD8 response.

Despite conventional medical and surgical treatments, malignant pleural mesothelioma (MPM) remains incurable. Oncovirotherapy (i.e., the use of replication-competent virus for cancer treatment) is currently explored in clinical trials. In this study, we investigated the antineoplastic potential of a new oncolytic viral agent, a live-attenuated measles virus (MV) strain derived from the Edmonston vaccine lineage (Schwarz strain). We evaluated both oncolytic activity and immunoadjuvant properties of the MV vaccine strain on mesothelioma tumor cells. Infectivity, syncytium formation, and cytolytic activity of MV were studied on a panel of mesothelioma cells derived from pleural effusions of MPM patients. We observed that MV infected preferentially MPM cell lines in comparison with nontransformed mesothelial cells, leading to an efficient killing of a significant fraction of tumor cells. A cytoreductive activity was also evidenced through formation of multinuclear cellular aggregates (syncytia). The susceptibility of MPM cell lines to measles infection was assessed by the analysis of cell surface expression of the MV vaccine receptor (CD46). We also evaluated whether MV infection of mesothelioma cells could elicit an autologous antitumor immune response. We showed that MV Schwarz strain induced apoptotic cell death of infected mesothelioma cells, which were efficiently phagocytosed by dendritic cells (DC). Loading of DCs with MV-infected MPM cells induced DC spontaneous maturation, as evidenced by the increased expression of MHC and costimulatory molecules along with the production of proinflammatory cytokines. Priming of autologous T cells by DCs loaded with MV-infected MPM cells led to a significant proliferation of tumor-specific CD8 T cells. Altogether, these data strongly support the potential of oncolytic MV as an efficient therapeutic agent for mesothelioma cancer.

Extracavitary tumor after primary effusion lymphoma: relapse or second distinct lymphoma?

HHV-8-associated solid lymphomas which develop in extracavitary sites during the course of primary effusion lymphoma (PEL) could represent the relapse of original PEL tumors in different anatomical sites, or newly occurring distinct HHV-8-associated lymphomas, such as multicentric Castleman disease-related microlymphomas. HHV-8 episome clonality might help identify which event takes place.

Epstein-Barr virus inhibits Kaposi's sarcoma-associated herpesvirus lytic replication in primary effusion lymphomas.

The majority of AIDS-associated primary effusion lymphomas (PEL) are latently infected with both Kaposi's sarcoma-associated herpesvirus (KSHV) and Epstein-Barr virus (EBV). PELs harboring two viruses have higher oncogenic potential, suggesting functional interactions between EBV and KSHV. The KSHV replication and transcription activator (K-RTA) is necessary and sufficient for induction of KSHV lytic replication. EBV latent membrane protein 1 (LMP-1) is essential for EBV transformation and establishment of latency in vitro. We show EBV inhibits chemically induced KSHV lytic replication, in part because of a regulatory loop in which K-RTA induces EBV LMP-1 and LMP-1 in turn inhibits K-RTA expression and furthermore the lytic gene expression of KSHV. Suppression of LMP-1 expression in dually infected PEL cells enhances the expression of K-RTA and lytic replication of KSHV upon chemical induction. Because LMP-1 is known to inhibit EBV lytic replication, KSHV-mediated induction of LMP-1 would potentiate EBV latency. Moreover, KSHV infection of EBV latency cells induces LMP-1, and K-RTA is involved in the induction. Both LMP-1 and K-RTA are expressed during primary infection by EBV of KSHV latency cells. Our findings provide evidence that an interaction between EBV and KSHV at molecular levels promotes the maintenance and possibly establishment of viral latency, which may contribute to pathogenesis of PELs.

Potential role of natural killer cells in controlling growth and infiltration of AIDS-associated primary effusion lymphoma cells.

Natural killer (NK) cells are an important component of the innate immune response against microbial infections and tumors. Direct involvement of NK cells in tumor growth and infiltration has not yet been demonstrated clearly. Primary effusion lymphoma (PEL) cells were able to produce tumors and ascites very efficiently with infiltration of cells in various organs of T-, B- and NK-cell knock-out NOD/SCID/gammac(null) (NOG) mice within 3 weeks. In contrast, PEL cells formed small tumors at inoculated sites in T- and B-cell knock-out NOD/SCID mice with NK-cells while completely failing to infiltrate into various organs. Immunosupression of NOD/SCID by treatment with an antimurine TM-beta1 antibody, which transiently abrogates NK cell activity in vivo, resulted in enhanced tumorigenicity and organ infiltration in comparison with non-treated NOD/SCID mice. Activated human NK cells inhibited tumor growth and infiltration in NOG mice. Our results suggest that NK cells play an important role in growth and infiltration of PEL cells, and activated NK cells could be a promising immunotherapeutic tool against tumor or virus-infected cells either alone or in combination with conventional therapy. The rapid and efficient engraftment of PEL cells in NOG mice also suggests that this new animal model could provide a unique opportunity to understand and investigate the mechanism of pathogenesis and malignant cell growth.

Effusion and solid lymphomas have distinctive gene and protein expression profiles in an animal model of primary effusion lymphoma.

Lymphoma usually forms solid tumours in patients, and high expression levels of adhesion molecules are observed in these tumours. However, Kaposi's sarcoma-associated herpesvirus (KSHV)-related primary effusion lymphoma (PEL) does not form solid tumours and adhesion molecule expression is suppressed in the cells. Inoculation of a KSHV-associated PEL cell line into the peritoneal cavity of severe combined immunodeficiency mice resulted in the formation of effusion and solid lymphomas in the peritoneal cavity. Proteomics using two-dimensional difference gel electrophoresis and DNA microarray analyses identified 14 proteins and 105 genes, respectively, whose expression differed significantly between effusion and solid lymphomas. Five genes were identified as having similar expression profiles to that of lymphocyte function-associated antigen 1, an important adhesion molecule in leukocytes. Among these, coronin 1A, an actin-binding protein, was identified as a molecule showing high expression in solid lymphoma by both DNA microarray and proteomics analyses. Western and northern blotting showed that coronin 1A was predominantly expressed in solid lymphomas. Moreover, KSHV-encoded lytic proteins, including viral interleukin-6, were highly expressed in effusion lymphoma compared with solid lymphoma. These data demonstrate that effusion and solid lymphomas possess distinctive gene and protein expression profiles in our mouse model, and suggest that differences in gene and protein expression between effusion and solid lymphomas may be associated with the formation of effusion lymphoma or invasive features of solid lymphoma. Furthermore, the results obtained using this combination of proteomics and DNA microarray analyses indicate that protein synthesis partly reflects, but does not correlate strictly with, mRNA production.

KSHV G protein-coupled receptor inhibits lytic gene transcription in primary-effusion lymphoma cells via p21-mediated inhibition of Cdk2.

Kaposi sarcoma (KS) remains the most common AIDS-associated malignancy worldwide. In sub-Saharan Africa especially, this aggressive endothelial-cell tumor is a cause of widespread morbidity and mortality. Infection with Kaposi sarcoma-associated herpesvirus (KSHV) is now known to be an etiologic force behind KS and primary-effusion lymphoma (PEL). Over time, KSHV has pirated many human genes whose products regulate angiogenesis, inflammation, and the cell cycle. One of these, the KSHV vGPCR, is a lytic product that is a constitutively active homolog of the IL-8 receptor. Although it is considered a viral oncogene and causes KS-like lesions in mice, vGPCR expression results in cell-cycle arrest of KSHV-infected PEL cells. In the present study, we show that this arrest is mediated by p21 in a p53-independent manner; the resulting Cdk2 inhibition decreases the efficiency of chemical induction of KSHV lytic transcripts ORF 50 and 26. Importantly, Cdk2 activity is also essential for replication in other human herpesviruses. The ability of vGPCR to delay or abort KSHV replication may explain how despite being a lytic product, this potent signaling molecule has a vital role in tumor formation via its induction of various KS-associated cytokines.