KSHV infection of B cells primes protective T cell responses in humanized mice.

Kaposi sarcoma associated herpesvirus (KSHV) is associated with around 1% of all human tumors, including the B cell malignancy primary effusion lymphoma (PEL), in which co-infection with the Epstein Barr virus (EBV) can almost always be found in malignant cells. Here, we demonstrate that KSHV/EBV co-infection of mice with reconstituted human immune systems (humanized mice) leads to IgM responses against both latent and lytic KSHV antigens, and expansion of central and effector memory CD4+ and CD8+ T cells. Among these, KSHV/EBV dual-infection allows for the priming of CD8+ T cells that are specific for the lytic KSHV antigen K6 and able to kill KSHV/EBV infected B cells. This suggests that K6 may represent a vaccine antigen for the control of KSHV and its associated pathologies in high seroprevalence regions, such as Sub-Saharan Africa.

Daratumumab induces cell-mediated cytotoxicity of primary effusion lymphoma and is active against refractory disease.

Primary effusion lymphoma (PEL), an aggressive non-Hodgkin lymphoma caused by Kaposi sarcoma-associated herpesvirus (KSHV), lacks standard therapy and has a median survival of 10-22 months with combination chemotherapy. PEL is a tumor of plasmablast-like B cells generally expressing CD38, the target of daratumumab (Dara). Initially, we assessed PEL cells from eight patients and established that each expressed high levels of CD38 by flow cytometry. PEL cell lines were also evaluated and most had high CD38 expression. We then assessed Dara's effects on complement-dependent cytotoxicity (CDC) and antibody-dependent cell-mediated cytotoxicity (ADCC) of PEL cell lines as well as its clinical benefits on two patients with PEL. Despite high CD38 expression, Dara did not induce CDC of PEL cell lines, due in part to high levels of the complement-inhibitory proteins, CD55 and CD59. However, Dara induced significant and dose-dependent increases in ADCC, particularly in those lines with high CD38 levels. Two FDA-approved drugs, all trans-retinoic acid (ATRA) and pomalidomide (Pom), significantly increased surface CD38 levels in low-CD38 expressing PEL cell lines, resulting in increased Dara-induced ADCC. Two patients with refractory PEL were treated with Dara alone or in combination with Pom. One patient with leptomeningeal PEL had a complete response to Dara and Pom combination treatment. Others had improvement in performance status and resolution of malignant ascites with Dara alone. Together, these data support the use of Dara monotherapy or in combination with ATRA or Pom as a potential therapeutic option for PEL.

Pomalidomide restores immune recognition of primary effusion lymphoma through upregulation of ICAM-1 and B7-2.

Pomalidomide (Pom) is an immunomodulatory drug that has efficacy against Kaposi's sarcoma, a tumor caused by Kaposi's sarcoma-associated herpesvirus (KSHV). Pom also induces direct cytotoxicity in primary effusion lymphoma (PEL), a B-cell malignancy caused by KSHV, in part through downregulation of IRF4, cMyc, and CK1α as a result of its interaction with cereblon, a cellular E3 ubiquitin ligase. Additionally, Pom can reverse KSHV-induced downregulation of MHCI and co-stimulatory immune surface molecules ICAM-1 and B7-2 on PELs. Here, we show for the first time that Pom-induced increases in ICAM-1 and B7-2 on PEL cells lead to an increase in both T-cell activation and NK-mediated cytotoxicity against PEL. The increase in T-cell activation can be prevented by blocking ICAM-1 and/or B7-2 on the PEL cell surface, suggesting that both ICAM-1 and B7-2 are important for T-cell co-stimulation by PELs. To gain mechanistic insights into Pom's effects on surface markers, we generated Pom-resistant (PomR) PEL cells, which showed about 90% reduction in cereblon protein level and only minimal changes in IRF4 and cMyc upon Pom treatment. Pom no longer upregulated ICAM-1 and B7-2 on the surface of PomR cells, nor did it increase T-cell and NK-cell activation. Cereblon-knockout cells behaved similarly to the pomR cells upon Pom-treatment, suggesting that Pom's interaction with cereblon is necessary for these effects. Further mechanistic studies revealed PI3K signaling pathway as being important for Pom-induced increases in these molecules. These observations provide a rationale for the study of Pom as therapy in treating PEL and other KSHV-associated tumors.

Kaposi Sarcoma With Intravascular Primary Effusion Lymphoma in the Skin: A Potential Pitfall in HHV8 Immunohistochemistry Interpretation.

Primary effusion lymphoma is a rare, clinically aggressive large B-cell neoplasm universally associated with human herpesvirus 8 that occurs in the setting of immune compromise. It is classically described as a lymphomatous effusion occurring within body cavities. Recently, however, solid tumor masses, and rarely an intravascular form, have been described. We report a case of a cutaneous intravascular primary effusion lymphoma occurring within ectatic vascular spaces of a Kaposi sarcoma skin lesion in a human immunodeficiency virus-positive adult. Human herpesvirus 8 immunohistochemistry was positive in the nuclei of the Kaposi sarcoma spindled cells as well as within large intravascular plasmacytoid cells. This unusual case highlights the importance of careful assessment of the nature of human herpesvirus 8-positive staining cells in an otherwise typical Kaposi sarcoma. A careful search for dual pathology in immune-compromised patients as well as the importance of histologic assessment of skin lesions in human immunodeficiency virus-positive patients is also highlighted.

Reconsideration of the first recognition of breast implant-associated anaplastic large cell lymphoma: A critical review of the literature.

The current literature credits Keech and Creech with the first report of breast implant-associated anaplastic large cell lymphoma (BIA-ALCL) in 1997. Here we discuss what we consider is the first ever description of BIA-ALCL, a recently recognized entity by the WHO. We unearthed the description of a patient that was diagnosed with primary effusion lymphoma (PEL), surrounding a breast implant in 1996. In light of the current state of knowledge, we evaluated the evidence presented in 1996 and consider that BIA-ALCL is a more appropriate diagnosis rather than PEL. We base our proposal on the following features: 1). clinical presentation of effusion around a breast implant, 2). occurring in an HIV negative patient, 3). absence of EBV co-infection, and 4). a historically questionable demonstration of HHV8. In effect we further support that HHV8 is not related with BIA-ALCL based on the following facts: 1). An extensive review of the literature did not disclose a similar case in the next 24 years, 2). Use of state of the art HHV8 by immunohistochemistry did not disclose any positive case among 30 randomly tested cases. We believe this matter is of importance because in the current WHO, the assertion that PEL is a possible complication of breast implants may lead to a diagnosis with poor prognosis and susceptible of morbidity related with aggressive therapy, in contrast with BIA-ALCL that can be cured with surgery alone.

Viral, immunologic, and clinical features of primary effusion lymphoma.

Primary effusion lymphoma (PEL) is an aggressive HIV-associated lymphoma with a relatively poor prognosis in the era of effective HIV therapy. Kaposi sarcoma herpesvirus (KSHV) is the etiologic agent, and ∼80% of tumors are coinfected with Epstein-Barr virus (EBV). A better understanding of how KSHV-related immune dysregulation contributes to the natural history of PEL will improve outcomes. Twenty patients with PEL diagnosed between 2000 and 2013, including 19 treated with modified infusional etoposide, vincristine, and doxorubicin with cyclophosphamide and prednisone (EPOCH), were identified. We compared their clinical, virologic, and immunologic features vs 20 patients with HIV-associated diffuse large B-cell lymphoma and 19 patients with symptomatic interleukin (IL)-6 related KSHV-associated multicentric Castleman disease. Survival analyses of treated patients with PEL were then performed to identify prognostic factors and cancer-specific mortality. Compared with HIV-associated diffuse large B-cell lymphoma, PEL was associated with significant hypoalbuminemia (P < .0027), thrombocytopenia (P = .0045), and elevated IL-10 levels (P < .0001). There were no significant differences in these parameters between PEL and KSHV-associated multicentric Castleman disease. Median overall survival in treated patients with PEL was 22 months, with a plateau in survival noted after 2 years. Three-year cancer-specific survival was 47%. EBV-positive tumor status was associated with improved survival (hazard ratio, 0.27; P = .038), and elevated IL-6 level was associated with inferior survival (hazard ratio, 6.1; P = .024). Our analysis shows that IL-6 and IL-10 levels contribute to the natural history of PEL. Inflammatory cytokines and tumor EBV status are the strongest prognostic factors. Pathogenesis-directed first-line regimens are needed to improve overall survival in PEL.

Primary effusion lymphoma of the pleural space: Report of a rare complication of cardiac transplant with review of the literature.

Primary effusion lymphoma (PEL) is a rare mature B-cell non-Hodgkin's lymphoma arising in body cavities and presenting with effusions. It has been described predominantly in patients with impaired immunity from the acquired immunodeficiency syndrome and is associated with the Human Herpesvirus-8 (HHV-8). Seldom has PEL been diagnosed in persons negative for the human immunodeficiency virus (HIV), and in such cases it has occurred primarily in the setting of posttransplant immunosuppression. We report an instructive case of a Caribbean-American HIV-negative orthotopic heart transplant recipient with a history of HHV-8-associated Kaposi's sarcoma who developed HHV-8 viremia and PEL of the pleural space early in the posttransplant course. This case highlights the importance of considering PEL in the differential diagnosis of a new pleural effusion in a transplant recipient at risk for HHV-8-associated disease.

Age and CD20 Expression Are Significant Prognostic Factors in Human Herpes Virus-8-negative Effusion-based Lymphoma.

Human herpes virus-8 (HHV-8)-negative effusion-based lymphoma (HHV-8-negative EBL) can be distinguished from primary effusion lymphoma based on clinical and pathologic findings. Although the morphology between the 2 is similar and they both originate from body cavities with serous effusions and are characterized by lack of tumor masses, HHV-8-negative EBL generally occurs in older patients, and has favorable response to therapy and better prognosis than primary effusion lymphoma. However, no systematic studies have investigated prognostic factors in patients with HHV-8-negative EBL. In this report, clinical and pathologic characteristics of 67 cases of HHV-8-negative EBL, including 2 of our own cases, were analyzed. Univariate analyses revealed older age (70 y and above), Japanese ethnicity, pericardial effusion, CD20 expression, and chemotherapy with rituximab were significantly favorable prognostic factors. Peritoneal effusion was identified as an unfavorable prognostic factor. In the multivariate analysis, age and CD20 expression were independent prognostic factors (P=0.013 and 0.003, respectively). A past history of induced fluid overload, hepatitis C viral infection, and peritoneal effusion were significantly correlated with patients aged below 70 years, while pericardial and pleural effusions were significantly correlated with patients aged 70 years and above. A comparison of cases with and without CD20 expression revealed that Japanese ethnicity and pericardial effusion were significantly correlated with CD20 expression, whereas a past history of induced fluid overload and peritoneal effusion were significantly correlated with the absence of CD20. We concluded that older age and CD20 expression are significant and favorable independent prognostic factors of HHV-8-negative EBL.

Biology and management of primary effusion lymphoma.

Primary effusion lymphoma (PEL) is a rare B-cell malignancy that most often occurs in immunocompromised patients, such as HIV-infected individuals and patients receiving organ transplantation. The main characteristic of PEL is neoplastic effusions in body cavities without detectable tumor masses. The onset of the disease is associated with latent infection of human herpes virus 8/Kaposi sarcoma-associated herpes virus, and the normal counterpart of tumor cells is B cells with plasmablastic differentiation. A condition of immunodeficiency and a usual absence of CD20 expression lead to the expectation of the lack of efficacy of anti-CD20 monoclonal antibody; clinical outcomes of the disease remain extremely poor, with an overall survival at 1 year of ∼30%. Although recent progress in antiretroviral therapy has improved outcomes of HIV-infected patients, its benefit is still limited in patients with PEL. Furthermore, the usual high expression of programmed death ligand 1 in tumor cells, one of the most important immune-checkpoint molecules, results in the immune escape of tumor cells from the host immune defense, which could be the underlying mechanism of poor treatment efficacy. Molecular-targeted therapies for the activating pathways in PEL, including NF-κB, JAK/STAT, and phosphatidylinositol 3-kinase/AKT, have emerged to treat this intractable disease. A combination of immunological recovery from immune deficiency, overcoming the immune escape, and the development of more effective drugs will be vital for improving the outcomes of PEL patients in the future.

Rapidly progressive intravascular primary effusion lymphoma in an HIV-positive renal transplant recipient.

We describe the clinical and post-mortem findings of a case of rapidly progressive, ultimately fatal primary effusion lymphoma (PEL) arising in an HIV-positive man 2 years after renal transplantation. Disseminated multi-organ involvement associated with a peculiar intravascular pattern of growth, as seen in this case, has only been reported once previously. This is also, to our knowledge, the first detailed description of a lymphoma arising post-transplant in an HIV-positive patient.

Nuclear Localization and Cleavage of STAT6 Is Induced by Kaposi's Sarcoma-Associated Herpesvirus for Viral Latency.

Emerging evidence implies that STAT6 plays an important role in both the adaptive and innate immune responses to virus infection. Kaposi's sarcoma-associated herpesvirus (KSHV) is an oncogenic γ-herpesvirus agent associated with several human malignancies, including Kaposi's sarcoma (KS) and primary effusion lymphomas (PELs). Previously, we demonstrated that KSHV blocks IL-4-induced STAT6 phosphorylation and retains a basal IL-13/STAT6 constitutive activation for cell survival and proliferation. However, the mechanism by which KSHV regulates STAT6 remains largely unknown. Here, we found that KSHV-encoded LANA interacts with STAT6 and promotes nuclear localization of STAT6 independent of the tyrosine 641-phosphorylation state. Moreover, nuclear localization of STAT6 is also dramatically increased in KS tissue. The latent antigen LANA induces serine protease-mediated cleavage of STAT6 in the nucleus, where the cleaved STAT6 lacking transactivation domain functions as a dominant-negative regulator to repress transcription of Replication and Transcription Activator (RTA) and in turn shut off viral lytic replication. Blockade of STAT6 by small interference RNA dramatically enhances expression of RTA, and in turn reduces KSHV-infected endothelial cell growth and colony formation. Taken together, these results suggest that nuclear localization and cleavage of STAT6 is important for modulating the viral latency and pathogenesis of KSHV.

Quercetin induces apoptosis and autophagy in primary effusion lymphoma cells by inhibiting PI3K/AKT/mTOR and STAT3 signaling pathways.

Quercetin, a bioflavonoid contained in several vegetables daily consumed, has been studied for long time for its antiinflammatory and anticancer properties. Quercetin interacts with multiple cancer-related pathways such as PI3K/AKT, Wnt/ß-catenin and STAT3. These pathways are hyperactivated in primary effusion lymphoma (PEL), an aggressive B cell lymphoma whose pathogenesis is strictly linked to the oncogenic virus Kaposis' Sarcoma-associated Herpesvirus (KSHV). In this study, we found that quercetin inhibited PI3K/AKT/mTOR and STAT3 pathways in PEL cells, and as a consequence, it down-regulated the expression of the prosurvival cellular proteins such as c-FLIP, cyclin D1 and cMyc. It also reduced the release of IL-6 and IL-10 cytokines, leading to PEL cell death. Moreover, quercetin induced a prosurvival autophagy in these cells and increased the cytotoxic effect of bortezomib, a proteasomal inhibitor, against them. Interestingly, quercetin decreased also the expression of latent and lytic KSHV proteins involved in PEL tumorigenesis and up-regulated the surface expression of HLA-DR and calreticulin, rendering the dying cells more likely detectable by the immune system. The results obtained in this study indicate that quercetin, which does not exert any cytotoxicity against normal B cells, may represent a good candidate for the treatment of this aggressive B cell lymphoma, especially in combination with autophagy inhibitors or with bortezomib.

Azidothymidine Sensitizes Primary Effusion Lymphoma Cells to Kaposi Sarcoma-Associated Herpesvirus-Specific CD4+ T Cell Control and Inhibits vIRF3 Function.

Kaposi sarcoma-associated herpesvirus (KSHV) is linked with the development of Kaposi sarcoma and the B lymphocyte disorders primary effusion lymphoma (PEL) and multi-centric Castleman disease. T cell immunity limits KSHV infection and disease, however the virus employs multiple mechanisms to inhibit efficient control by these effectors. Thus KSHV-specific CD4+ T cells poorly recognize most PEL cells and even where they can, they are unable to kill them. To make KSHV-infected cells more sensitive to T cell control we treated PEL cells with the thymidine analogue azidothymidine (AZT), which sensitizes PEL lines to Fas-ligand and TRAIL challenge; effector mechanisms which T cells use. PELs co-cultured with KSHV-specific CD4+ T cells in the absence of AZT showed no control of PEL outgrowth. However in the presence of AZT PEL outgrowth was controlled in an MHC-restricted manner. To investigate how AZT sensitizes PELs to immune control we first examined BJAB cells transduced with individual KSHV-latent genes for their ability to resist apoptosis mediated by stimuli delivered through Fas and TRAIL receptors. This showed that in addition to the previously described vFLIP protein, expression of vIRF3 also inhibited apoptosis delivered by these stimuli. Importantly vIRF3 mediated protection from these apoptotic stimuli was inhibited in the presence of AZT as was a second vIRF3 associated phenotype, the downregulation of surface MHC class II. Although both vFLIP and vIRF3 are expressed in PELs, we propose that inhibiting vIRF3 function with AZT may be sufficient to restore T cell control of these tumor cells.

Altering the Anti-inflammatory Lipoxin Microenvironment: a New Insight into Kaposi's Sarcoma-Associated Herpesvirus Pathogenesis.

Lipoxins are host anti-inflammatory molecules that play a vital role in restoring tissue homeostasis. The efficacy of lipoxins and their analog epilipoxins in treating inflammation and its associated diseases has been well documented. Kaposi's sarcoma (KS) and primary effusion lymphoma (PEL) are two well-known inflammation related diseases caused by Kaposi's sarcoma-associated herpesvirus (KSHV). Controlling inflammation is one of the strategies adopted to treat KS and PEL, a primary motivation for exploring and evaluating the therapeutic potential of using lipoxins. This study documents how KSHV manipulates and downregulates the secretion of the anti-inflammatory lipoxin A4 in host cells and the viral factors involved in this process using in vitro KS and PEL cells as models. The presence of the lipoxin A4 receptor/formyl peptidyl receptor (ALX/FPR) in KS patient tissue sections and in vitro KS and PEL cell models offers a novel possibility for treating KS and PEL with lipoxins. Treating de novo KSHV-infected endothelial cells with lipoxin and epilipoxin creates an anti-inflammatory environment by decreasing the levels of NF-κB, AKT, ERK1/2, COX-2, and 5-lipoxygenase. Lipoxin treatment on CRISPR/CAS9 technology-mediated ALX/FPR gene deletion revealed the importance of the lipoxin receptor ALX for effective lipoxin signaling. A viral microRNA (miRNA) cluster was identified as the primary factor contributing to the downregulation of lipoxin A4 secretion in host cells. The KSHV miRNA cluster probably targets enzyme 15-lipoxygenase, which is involved in lipoxin A4 synthesis. This study provides a new insight into the potential treatment of KS and PEL using nature's own anti-inflammatory molecule, lipoxin. IMPORTANCE: KSHV infection has been shown to upregulate several host proinflammatory factors, which aid in its survival and pathogenesis. The influence of KSHV infection on anti-inflammatory molecules is not well studied. Since current treatment methods for KS and PEL are fraught with unwanted side effects and low efficiency, the search for new therapeutics is therefore imperative. The use of nature's own molecule lipoxin as a drug is promising. This study opens up new domains in KSHV research focusing on how the virus modulates lipoxin secretion and warrants further investigation of the therapeutic potential of lipoxin using in vitro cell models for KS and PEL.

Primary Effusion Lymphoma: Cytological Diagnosis of a Rare Entity--Report of Two Cases in HIV-Uninfected Patients from a Single Institution.

BACKGROUND: Primary effusion lymphoma (PEL) is a Kaposi's sarcoma-associated herpesvirus (KSHV)-related large B-cell lymphoma often affecting immunocompromised adults. However, several cases in nonimmunocompromised elderly patients have been reported. It usually occurs as a massive, serosal effusion without tumoral masses and lymphadenopathies. The most frequent diagnostic materials are cytological smears, displaying large lymphocytes with variable morphological aspects but with a specific 'null-cell' phenotype associated with the expression of KSHV-related latency-associated nuclear antigen. CASES: We report 2 cases of PEL that occurred in elderly, HIV-uninfected patients. In the first case, an 83-year-old man presented with severe dyspnea of 1 week duration. Radiological images documented pericardial effusion causing cardiac tamponade, without lymphadenopathies and solid masses. In the second case, an HIV-uninfected 94-year-old man was affected by anorexia, fatigue, shortness of breath and nonproductive cough. Radiological studies revealed bilateral pleural effusion, without evidence of abnormal lymphadenopathies and lung and/or pleural masses. Thoracentesis was immediately performed. Cytological evaluations of pericardial (first case) and pleural (second case) fluid effusion showed anaplastic and immunoblastic lymphocytes, respectively, expressing LCA/CD45, CD30 and KSHV. CONCLUSION: Albeit rare, PEL should be kept in mind in the diagnostic algorithm of serosal cytological evaluation in elderly HIV-uninfected patients.

Targeting HGF/c-MET induces cell cycle arrest, DNA damage, and apoptosis for primary effusion lymphoma.

Kaposi sarcoma-associated herpesvirus (KSHV) is a principal causative agent of primary effusion lymphoma (PEL) with a poor prognosis in immunocompromised patients. However, it still lacks effective treatment which urgently requires the identification of novel therapeutic targets for PEL. Here, we report that the hepatocyte growth factor (HGF)/c-MET pathway is highly activated by KSHV in vitro and in vivo. The selective c-MET inhibitor, PF-2341066, can induce PEL apoptosis through cell cycle arrest and DNA damage, and suppress tumor progression in a xenograft murine model. By using microarray analysis, we identify many novel genes that are potentially controlled by HGF/c-MET within PEL cells. One of the downstream candidates, ribonucleoside-diphosphate reductase subunit M2 (RRM2), also displays the promising therapeutic value for PEL treatment. Our findings provide the framework for development of HGF/c-MET-focused therapy and implementation of clinical trials for PEL patients.

Capsaicin triggers immunogenic PEL cell death, stimulates DCs and reverts PEL-induced immune suppression.

Capsaicin, the pungent alkaloid of red pepper has been extensively studied for its many properties, especially the anti-inflammatory and anti-oxidant ones. It binds to vanilloid receptor 1, although it has been reported to be able to mediate some effects independently of its receptor. Another important property of Capsaicin is the anticancer activity against highly malignant tumors, alone or in combination with other chemotherapeutic agents. In this study, we found that Capsaicin induced an apoptotic cell death in PEL cells correlated with the inhibition of STAT3. STAT3 pathway, constitutively activated in PEL cells, is essential for their survival. By STAT3 de-phosphorylation, Capsaicin reduced the Mcl-1 expression level and this could represent one of the underlying mechanisms leading to the Capsaicin-mediated cell death and autophagy induction. Next, by pharmacological or genetic inhibition, we found that autophagy played a pro-survival role, suggesting that its inhibition could be exploited to increase the Capsaicin cytotoxic effect against PEL cells. Finally, we show that Capsaicin induced DAMP exposure, as for an immunogenic cell death, directly promoted DC activation and, more importantly, that it counteracted the immune-suppression, in terms of DC differentiation, mediated by the PEL released factors.