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Background/Aim: Prostate cancer (PCa) is one of the most common types of cancer in men. Prostate-specific antigen (PSA) is currently the only biomarker used to screen for the risk of developing PCa. Because PSA tests may show false positives, identifying novel PCa-specific biomarkers would improve prediction and diagnosis at an early stage. Previously, we identified a number of genes/microRNAs (miRNAs) in prostate tissue as potential biomarkers of chronic prostatitis in a rat model of chemical-induced prostatitis. The current study aimed to evaluate their potential for use as translational, diagnostic markers in humans. Materials and Methods: We performed quantitative polymerase chain reaction analysis using pathologically clear (normal) or confirmed PCa tissue samples from the same patients (N=18 per group). Results: Levels (relative fold changes) of bone morphogenetic protein 7 (BMP7) transcripts were significantly lower in PCa tissues, compared with clear tissues, in a paired t-test (p=0.0075). Although neural cell adhesion molecule 1 (NCAM1) transcripts tended to be altered in PCa tissues, statistically insignificant differences were observed (p=0.0521). No statistically significant differences were observed for the other genes/miRNAs analyzed in PCa tissues due to a high degree of individual variance in expression. Conclusion: Similar to the results previously observed in rats, changes in the levels of BMP7 and NCAM1 transcripts were evident in human PCa tissues, suggesting that these genes may serve as potential diagnostic biomarkers during the early stages of PCa. Further studies are needed to determine the potential use of these molecules as biomarkers.

Morreton virus (MORV) is an oncolytic Vesiculovirus, genetically distinct from vesicular stomatitis virus (VSV).To report that MORV induced potent cytopathic effects (CPEs) in cholangiocarcinoma (CCA) and hepatocellular carcinoma (HCC) in vitro models.In preliminary safety analyses, high intranasal doses (up to 10Our findings indicate that wild-type MORV is safe and can induce potent tumor regression via immune-mediated and immune-independent mechanisms in HCC and CCA animal models without dose limiting adverse events. These data warrant further development and clinical translation of MORV as an oncolytic virotherapy platform.

The outbreak of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which is the causative pathogen for the coronavirus disease 2019 (COVID-19) pandemic, has greatly stressed our healthcare system. In addition to severe respiratory and systematic symptoms, several comorbidities increase the risk of fatal disease outcomes, including chronic viral infections. Increasing cases of lytic reactivation of human herpesviruses in COVID-19 patients and vaccinated people have been reported recently. SARS-CoV2 coinfection, COVID-19 treatments, and vaccination may aggravate those herpesvirus-associated diseases by reactivating the viruses in latently infected host cells. In this review, we summarize recent clinical findings and limited mechanistic studies regarding the relationship between SARS-CoV-2 and different human herpesviruses that suggest an ongoing potential threat to human health in the postpandemic era.

Oncolytic viruses can effectively unwrap a multimodal anti-tumor activity, encompassing a selective tumor cell killing and promoting a systemic anti-tumor immunity, making them a formidable foe against cancer. Among these, several members of the Rhabdoviridae family are particularly attractive as oncolytic agents due to their natural tumor selectivity and non-pathogenicity in humans. In this study, we demonstrated that intratumorally (IT) administration of Jurona virus (JURV), a novel oncolytic Rhabdovirus, induces dynamic tumor regression in human HCC xenograft and syngeneic models. Our data shows that IT injections of JURV trigger the recruitment and activation of cytotoxic T (CTLs) and decrease the tumor-associated macrophages (TAM) infiltration leading to tumor growth delay in both local and distant murine HCC tumors in a syngeneic model. Moreover, when administered concomitantly, JURV and anti-PD-1 therapy profoundly modulate the tumor microenvironment (TME) via enhanced infiltration of CTLs, suggesting that immune checkpoint blockade therapy could potentiate the immunomodulatory effect of JURV and potentially provide durable anti-tumor immunity. Our analysis of the molecular and cellular mechanism of JURV-medicated anti-cancer activity unveiled that JURV and anti-PD-1 antibodies activate different effectors of the immune system but have complementary anti-tumor activities. Furthermore, our results indicate that the abscopal effect induced by JURV is likely mediated by the mechanism regulating the T helper cell responses. Our work supports the further development of JURV as a novel immunovirotherapy platform for hepatocellular carcinoma.

An outbreak of the novel coronavirus SARS-CoV-2, the causative agent of Coronavirus Disease-2019 (COVID-19), a respiratory disease, has infected almost one hundred million people since the end of 2019, killed over two million, and caused worldwide social and economic disruption. Because the mechanisms of SARS-CoV-2 infection of host cells and its pathogenesis remain largely unclear, there are currently no antiviral drugs with proven efficacy. Besides severe respiratory and systematic symptoms, several comorbidities increase risk of fatal disease outcome. Therefore, it is required to investigate the impacts of COVID-19 on pre-existing diseases of patients, such as cancer and other infectious diseases. In the current study, we report that SARS-CoV-2 encoded proteins and some currently used anti-COVID-19 drugs are able to induce lytic reactivation of Kaposi’s sarcoma-associated herpesvirus (KSHV), one of major human oncogenic viruses, through manipulation of intracellular signaling pathways. Our data indicate that those KSHV + patients especially in endemic areas exposure to COVID-19 or undergoing the treatment may have increased risks to develop virus-associated cancers, even after they have fully recovered from COVID-19., Chen et al. find that SARS-CoV-2 encoded proteins and some anti-COVID-19 drugs can induce lytic reactivation of Kaposi’s sarcoma-associated herpesvirus (KSHV), one of the major human oncogenic viruses. This study suggests that KSHV-positive patients exposed to COVID-19 or undergoing its treatment may have increased risks to develop virus-associated cancers, even after they have fully recovered from COVID-19.

Recently, remdesivir and molnupiravir were approved for treating COVID-19 caused by SARS-CoV-2 infection. However, little is known about the impact of these drugs on other viruses preexisted in COVID-19 patients. Here we report that remdesivir but not molnupiravir induced lytic reactivation of Kaposi's sarcoma-associated herpesvirus (KSHV) and Epstein-Barr virus (EBV), two major oncogenic herpesviruses. Remdesivir induced mature virion production from latently infected cells. Mechanistic studies showed that remdesivir induced KSHV and EBV reactivation by regulating several intracellular signaling pathways.

Morreton virus (MORV) is a novel oncolyticVesiculovirus, genetically distinct from vesicular stomatitis virus (VSV). we report that MORV induced potent cytopathic effects in a panel of cholangiocarcinoma (CCA) and hepatocellular carcinoma (HCC) cell lines. In addition, high intranasal doses of MORV were not associated with significant adverse effects and were well tolerated in mice bearing liver tumor xenografts and syngeneic liver cancers. Furthermore, single intratumoral treatments with MORV (1 x 107TCID50) triggered a robust antitumor immune response leading to substantial tumor regression and disease control in a syngeneic CCA model, using 10-fold lower dose compared to VSV (1 x 108TCID50). In addition, MORV and VSV both induced prominent tumor growth delay in immunodeficient mice bearing Hep3B hepatocellular carcinoma (HCC) but not in mice bearing HuCCT-1 CCA xenografts. Our findings indicate that wild-type MORV is safe and can induce potent tumor regression in HCC and CCA animal models without adverse events via immune-mediated and immune-independent mechanisms. Further development and clinical translation of MORV as virotherapy for liver cancers are warranted.

Primary effusion lymphoma (PEL) is an aggressive malignancy with poor prognosis even under chemotherapy. Kaposi sarcoma–associated herpesvirus (KSHV), one of the human oncogenic viruses, is the principal causative agent. Currently, there is no specific treatment for PEL; therefore, developing new therapies is of great importance. Sphingolipid metabolism plays an important role in determining the fate of tumor cells. Our previous studies have demonstrated that there is a correlation between sphingolipid metabolism and KSHV+ tumor cell survival. To further develop sphingolipid metabolism-targeted therapy, after screening a series of newly synthesized ceramide analogs, here, we have identified compounds with effective anti-PEL activity. These compounds induce significant PEL apoptosis, cell-cycle arrest, and intracellular ceramide production through regulation of ceramide synthesizing or ceramide metabolizing enzymes and dramatically suppress tumor progression without visible toxicity in vivo. These new compounds also increase viral lytic gene expression in PEL cells. Our comparative transcriptomic analysis revealed their mechanisms of action for inducing PEL cell death and identified a subset of novel cellular genes, including AURKA and CDCA3, controlled by sphingolipid metabolism, and required for PEL survival with functional validation. These data provide the framework for the development of promising sphingolipid-based therapies against this virus-associated malignancy.

Kaposi's sarcoma-associated herpesvirus (KSHV) causes several human cancers, including Kaposi's sarcoma (KS) and primary effusion lymphoma (PEL), which are mostly seen in immunocompromised patients such as HIV+ individuals. Tuberculosis (TB), caused by the bacterial pathogen Mycobacterium tuberculosis (Mtb), remains one of the deadliest infectious diseases in the world. The risk of developing TB is dramatically higher in people living with HIV than among those without HIV infection. Case reports link cutaneous or pulmonary KS in HIV+ patients with mycobacterial co-infections, however, impacts of Mtb infection or its products on KSHV-infected cells are not known. We report here that ESAT-6, a secreted Mtb virulence factor, induces viral reactivation from KSHV-infected cells. KSHV-infected pulmonary endothelial cells were resistant to ESAT-6 induced inhibition of cell growth. Our data demonstrate that Mtb virulence factors influence the biology of KSHV-infected cells, highlighting the need to study the interactions between these two pathogens commonly found in people living with HIV. This article is protected by copyright. All rights reserved.

The anti-CSPG4 monoclonal antibodies (mAbs) have shown anti-tumor activity and therapeutic potential for treating breast cancer. In addition, CSPG4 is a dominant tumor-associated antigen that is also involved in normal-tissue development in humans. Therefore, the potential for off-tumor activity remains a serious concern when targeting CSPG4 therapeutically. Previous work suggested that glycans contribute to the binding of specific anti-CSPG4 antibodies to tumor cells, but the specificity and importance of this contribution are unknown. In this study, the reactivity of anti-CSPG4 mAbs was characterized with a peptide mimetic of carbohydrate antigens expressed in breast cancer. ELISA, flow cytometry, and microarray assays were used to screen mAbs for their ability to bind to carbohydrate-mimicking peptides (CMPs), cancer cells, and glycans. The mAb VT68.2 displayed a distinctly strong binding to a CMP (P10s) and bound to triple-negative breast cancer cells. In addition, VT68.2 showed a higher affinity for N-linked glycans that contain terminal fucose and fucosylated lactosamines. The functional assays demonstrated that VT68.2 inhibited cancer cell migration. These results define the glycoform reactivity of an anti-CSPG4 antibody and may lead to the development of less toxic therapeutic approaches that target tumor-specific glyco-peptides.

In humans, the adaptive immune system uses the exchange of information between cells to detect and eliminate foreign or damaged cells; however, the removal of unwanted cells does not always require an adaptive immune system1,2. For example, cell selection in Drosophila uses a cell selection mechanism based on 'fitness fingerprints', which allow it to delay ageing3, prevent developmental malformations3,4 and replace old tissues during regeneration5. At the molecular level, these fitness fingerprints consist of combinations of Flower membrane proteins3,4,6. Proteins that indicate reduced fitness are called Flower-Lose, because they are expressed in cells marked to be eliminated6. However, the presence of Flower-Lose isoforms at a cell's membrane does not always lead to elimination, because if neighbouring cells have similar levels of Lose proteins, the cell will not be killed4,6,7. Humans could benefit from the capability to recognize unfit cells, because accumulation of damaged but viable cells during development and ageing causes organ dysfunction and disease8-17. However, in Drosophila this mechanism is hijacked by premalignant cells to gain a competitive growth advantage18. This would be undesirable for humans because it might make tumours more aggressive19-21. It is unknown whether a similar mechanism of cell-fitness comparison is present in humans. Here we show that two human Flower isoforms (hFWE1 and hFWE3) behave as Flower-Lose proteins, whereas the other two isoforms (hFWE2 and hFWE4) behave as Flower-Win proteins. The latter give cells a competitive advantage over cells expressing Lose isoforms, but Lose-expressing cells are not eliminated if their neighbours express similar levels of Lose isoforms; these proteins therefore act as fitness fingerprints. Moreover, human cancer cells show increased Win isoform expression and proliferate in the presence of Lose-expressing stroma, which confers a competitive growth advantage on the cancer cells. Inhibition of the expression of Flower proteins reduces tumour growth and metastasis, and induces sensitivity to chemotherapy. Our results show that ancient mechanisms of cell recognition and selection are active in humans and affect oncogenic growth.

Although the Eukaryotic Translation Initiation Factor 4 Gamma 1 (EIF4G1) has been found overexpressed in a variety of cancers, its role in non–small cell lung cancers (NSCLC) pathogenesis especially in immunoregulatory functions, its clinical relevance and therapeutic potential remain largely unknown. By using cancer patients tissue assays, the results indicate that EIF4G1 expressional levels are much higher in NSCLC tissues than in adjacent or normal lung tissues, which are also associated with NSCLC patient survival. By using an RNA‐Sequencing based pipeline, the data show that EIF4G1 has a significant association with immune checkpoint molecules such as PD‐1/PD‐L1 in NSCLC. EIF4G1 small‐molecule inhibitors effectively repress NSCLC growth in cell culture and xenograft animal models. Protein array results identify the signature of proteins controlled by EIF4G1 in NSCLC cells, in which new candidates such as MUC1 and NRG1 are required for NSCLC survival and tumorigenesis with clinical relevance. Taken together, these results have for the first time demonstrated the immunoregulatory functions, clinical relevance and therapeutic potential of the EIF4G1 network in NSCLC, which may represent a promising and novel target to improve lung cancer treatment.

Kaposi's sarcoma-associated herpesvirus (KSHV) infection causes several human cancers, including Kaposi's sarcoma (KS), one of the most common AIDS-associated tumors. The involvement of the oral cavity represents one common clinical manifestation of AIDS-KS individuals with periodontal diseases and an oral carriage of a variety of pathogenic bacteria, including Porphyromonas gingivalis. In the current study, we report the clinical relevance of P. gingivalis and KSHV coinfection in the oral cavity of a cohort of HIV+ patients. Furthermore, we found that P. gingivalis conditioned medium or derived lipopolysaccharide effectively induced KSHV lytic reactivation from infected oral cells. This reactivation requires TLR4 as well as the activities of p38 and Jun N-terminal kinase- mitogen-activated protein kinase signaling pathways. Our findings reveal the mechanisms through which coinfected periodontal pathogens potentially promote oncogenic virus pathogenesis in the unique niche of immunocompromised patients.

Kaposi’s sarcoma-associated herpesvirus (KSHV) causes several human cancers, such as Kaposi’s sarcoma (KS) and primary effusion lymphoma (PEL). Current treatment options for KSHV infection and virus associated diseases are sometimes ineffective, therefore, more effectively antiviral agents are urgently needed. As a herpesvirus, lytic replication is critical for KSHV pathogenesis and oncogenesis. In this study, we have established a high-throughput screening assay by using an inducible KSHV+ cell-line, iSLK.219. After screening a compound library that consisted of 1280 Food and Drug Administration (FDA)-approved drugs, 15 hit compounds that effectively inhibited KSHV virion production were identified, most of which have never been reported with anti-KSHV activities. Interestingly, 3 of these drugs target histamine receptors or signaling. Our data further confirmed that antagonists targeting different histamine receptors (HxRs) displayed excellent inhibitory effects on KSHV lytic replication from induced iSLK.219 or BCBL-1 cells. In contrast, histamine and specific agonists of HxRs promoted viral lytic replication from induced iSLK.219 or KSHV-infected primary cells. Mechanistic studies indicated that downstream MAPK and PI3K/Akt signaling pathways were required for histamine/receptors mediated promotion of KSHV lytic replication. Direct knockdown of HxRs in iSLK.219 cells effectively blocked viral lytic gene expression during induction. Using samples from a cohort of HIV+ patients, we found that the KSHV+ group has much higher levels of histamine in their plasma and saliva than the KSHV- group. Taken together, our data have identified new anti-KSHV agents and provided novel insights into the molecular bases of host factors that contribute to lytic replication and reactivation of this oncogenic herpesvirus., Author summary As a major oncogenic human herpesviruses, KSHV infection causes several cancers mostly seen in immunocompromised patients. Currently, effective antiviral treatments are still lacking. The “old drugs, new tricks” approach may serve as a feasible strategy for high-throughput screening of novel agents against lytic replication of KSHV. Here we screened an FDA-approved drug library and identified 15 new anti-KSHV agents. Interestingly, several of these candidates target histamine receptors, implying the involvement of histamine-related signaling in lytic replication and reactivation of KSHV. This involvement was directly demonstrated using antagonists and agonists specific for individual histamine receptors as well as RNAi. The downstream signaling pathways required for histamine-mediated promotion of KSHV lytic replication was also identified. Clinical data from a cohort of HIV+ patients confirm the relevance and elevation of histamine in the microenvironment of HIV+/KSHV+ patients. Thus, our findings provide new clues for developing anti-KSHV treatments, and identify novel mechanisms through which histamine and related signaling pathways function as important host factors facilitating KSHV lytic reactivation and pathogenesis.

Background Despite increased usage of multiparameter flow cytometry (MFC) to assess diagnosis, prognosis, and therapeutic efficacy (minimal residual disease, MRD) in plasma cell neoplasms (PCNs), standardization of methodology and data analysis is suboptimal. We investigated the utility of using the mean and median fluorescence intensities (FI) obtained from MFC to objectively describe parameters that distinguish plasma cell (PC) phenotypes. Methods In this retrospective study, flow cytometry results from bone marrow aspirate specimens from 570 patients referred to the Myeloma Institute at UAMS were evaluated. Mean and median FI data were obtained from 8-color MFC of non-neoplastic, malignant, and mixed PC populations using antibodies to CD38, CD138, CD19, CD20, CD27, CD45, CD56, and CD81. Results Of 570 cases, 252 cases showed only non-neoplastic PCs, 168 showed only malignant PCs, and 150 showed mixed PC populations. Statistical analysis of median FI data for each CD marker showed no difference in expression intensity on non-neoplastic and malignant PCs, between pure and mixed PC populations. ROC analysis of the median FI of CD expression in non-neoplastic and malignant PCs was used to develop an algorithm to convert quantitative FI values to qualitative assessments including "negative," "positive," "dim," and "heterogeneous" expression. Conclusions FI data derived from 8-color MFC can be used to define marker expression on PCs. Translation of FI data from Infinicyt software to an Excel worksheet streamlines workflow and eliminates transcriptional errors when generating flow reports. © 2018 International Clinical Cytometry Society.

ABO blood group antigens are expressed on von Willebrand factor (VWF) and glycosylation patterns influence circulating VWF levels. The aim of this study was to examine the effect of ABO blood type on tissue-associated VWF protein levels. We selected 35 formalin-fixed paraffin-embedded pulmonary tissue blocks obtained at autopsy from decedents who died from pulmonary embolism with known ABO blood groups (O, A, B and AB phenotypes), prepared tissue microarrays (TMAs) and stained TMAs with antibodies to VWF and platelet/endothelial cell adhesion marker-1 (PECAM-1) as a marker of endothelial cells. A pixel count scoring algorithm was used to quantify VWF and PECAM-1 staining intensity in pulmonary arterioles in digitised images. Compared with type O, non-O individuals have a significantly higher amount of endothelial cell-associated VWF protein expression. VWF protein levels associated with pulmonary vascular endothelial cells is influenced by ABO antigenic determinants.

An outbreak of the novel coronavirus SARS-CoV-2, the causative agent of Coronavirus Disease-2019 (COVID-19), a respiratory disease, has infected over 75,000,000 people since the end of 2019, killed over 1,600,000, and caused worldwide social and economic disruption. Because the mechanisms of SARS-CoV-2 infection of host cells and its pathogenesis remain largely unclear, there are currently no antiviral drugs with proven efficacy. Besides severe respiratory and systematic symptoms, several comorbidities increase risk of fatal disease outcome. Therefore, it is required to investigate the impacts of COVID-19 on pre-existing diseases of patients, such as cancer and other infectious diseases. Kaposi's sarcoma-associated herpesvirus (KSHV) is the etiologic agent of several human cancers, such as Kaposi's sarcoma (KS) and Primary effusion lymphoma (PEL), mostly seen in immunosuppressed patients especially people with HIV infection. In the current study, we for the first time report that SARS-CoV-2 encoded spike (S) and nucleocapsid (N) proteins and some currently used anti-COVID-19 drugs (especially Azithromycin and Nafamostat mesylate) are able to induce lytic reactivation of KSHV from virus infected normal and tumor cells, through manipulation of intracellular signaling pathways. In addition, we found the obvious upregulation of ACE2 expression, the major receptor of SARS-CoV-2, in AIDS-KS tissues, indicating that KSHV may potentially increase SARS-CoV-2 co-infection. Together, our data indicate that those KSHV+ patients especially in endemic areas exposure to COVID-19 or undergoing the treatment may have increased risks to develop these virus-associated cancers, even after they have fully recovered from COVID-19. Citation Format: Zhiqiang Qin, Jungang Chen, Lu Dai, Lindsey Barrett, Steven Post. SARS-CoV-2 proteins and anti-COVID-19 drugs induce lytic reactivation of an oncogenic virus [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr LB072.

Classic Hodgkin lymphoma (CHL) characteristically shows few malignant cells in a microenvironment comprised of mixed inflammatory cells. Although CHL is associated with a high cure rate, recent studies have associated poor prognosis with absolute monocyte count in peripheral blood and increased monocyte/macrophages in involved lymph nodes. Thus, the role of monocytic infiltration and macrophage differentiation in the tumor microenvironment of CHL may be more relevant than absolute macrophage numbers to defining prognosis in CHL patients and potentially have therapeutic implications. Most studies identify tumor-associated macrophages (TAMs) using markers (e.g., CD68) expressed by macrophages and other mononuclear phagocytes, such as monocytes. In contrast, Class A Scavenger Receptor (SR-A/CD204) is expressed by tissue macrophages but not monocytic precursors. In this study, we examined SR-A expression in CHL (n = 43), and compared its expression with that of other macrophage markers. We confirmed a high prevalence of mononuclear cells that stained with CD68, CD163, and CD14 in CHL lymph nodes. However, SR-A protein expression determined by immunohistochemistry was limited to macrophages localized in sclerotic bands characteristic of nodular sclerosis CHL. In contrast, SR-A protein was readily detectable in lymph nodes with metastatic tumor, extra-nodal CHL, T cell/histiocyte-rich large B cell lymphoma, and resident macrophages in non-malignant tissues, including spleen, lymph node, liver and lung. The results of SR-A protein expression paralleled the expression of SR-A mRNA determined by quantitative RT-PCR. These data provide evidence that tumor-infiltrating monocyte/macrophages in CHL have a unique phenotype that likely depends on the microenvironment of nodal CHL.

Collectively, viruses are the principal cause of cancers arising in patients with immune dysfunction, including human immunodeficiency virus (HIV)-positive patients. Kaposi sarcoma (KS) etiologically linked to Kaposi sarcoma-associated herpesvirus (KSHV) continues to be the most common AIDS-associated tumor. The involvement of the oral cavity represents one of the most common clinical manifestations of this tumor. HIV infection incurs an increased risk among individuals with periodontal diseases and oral carriage of a variety of pathogenic bacteria. However, whether interactions involving periodontal bacteria and oncogenic viruses in the local environment facilitate replication or maintenance of these viruses in the oral cavity of HIV-positive patients remain largely unknown. We previously showed that pathogen-associated molecular patterns (PAMPs) from specific periodontal bacteria promoted KSHV entry into oral cells and subsequent establishment of latency. In the current study, we demonstrate that Staphylococcus aureus, one of common pathogens causing infection in HIV-positive patients, and its PAMPs can effectively induce KSHV lytic reactivation from infected oral cells, through the Toll-like receptor reactive oxygen species and cyclin D1-Dicer-viral microRNA axis. This investigation provides further clinical evidence about the relevance of coinfection due to these 2 pathogens in the oral cavities of a cohort HIV-positive patients and reveals novel mechanisms through which these coinfecting pathogens potentially promote virus-associated cancer development in the unique niche of immunocompromised patients.

Kaposi's sarcoma-associated herpesvirus (KSHV) causes several cancers such as Kaposi's sarcoma (KS) and primary effusion lymphoma (PEL). PD-1/PD-Ls immune checkpoint molecules play important roles in cancer cell immune escape. The expression of PD-1/PD-Ls and their regulation by oncogenic viruses, in particular KSHV, remain largely undefined. Here we demonstrate strong PD-1/PD-L1/PD-L2 expression in KS tissues from a cohort of HIV + patients. We found that induction of KSHV lytic reactivation significantly upregulates PD-L1 expression on infected tumor cells, potentially through several major cellular signaling pathways and IL-1β, which may represent a novel mechanism for virus-associated tumor cell immune escape.

Factors contributing to aggressive behavior in vulvar squamous cell carcinoma (vSCC) are poorly defined; however, a recent study has shown that vSCCs with an infiltrative pattern of invasion and fibromyxoid stroma are associated with worse outcomes than tumors with a pushing or nested pattern of invasion and lymphoplasmacytic stroma. Epithelial-mesenchymal transition (EMT) has been associated with tumor progression in a number of malignancies, and this study proposes that EMT contributes to tumor aggressiveness in this subset of vSCC. Immunohistochemistry was used to detect nuclear localization of β-catenin, loss of E-cadherin, and presence of vimentin in 58 cases of vSCC. The association of these phenotypic changes with pathologic features and clinical outcomes was tested using Fisher’s exact and χ2 analyses (significance at p ≤0.05). EMT-associated features were identified in 45 of 58 cases (78%) with 28 cases exhibiting more than one feature. Nuclear β-catenin and presence of vimentin were significantly more likely to occur in tumors with an infiltrative pattern of invasion or a fibromyxoid stromal response. Loss of E-cadherin was significantly associated with an infiltrative pattern, but not a fibromyxoid stroma. Risk for tumor recurrence was significantly increased in tumors with nuclear localization of β-catenin alone or in tumors displaying multiple EMT-associated features. These results suggest that the development of an EMT may be a mechanism by which infiltrative vulvar tumors with a fibromyxoid stromal response behave more aggressively and convey worse outcomes than tumors that do not exhibit these pathologic features.

We previously showed that the prothrombin time (PT), but not activated partial thromboplastin time (APTT), is correlated with serum immunoglobulin level in patients with plasma cell neoplasms.To determine if the observed effect of serum immunoglobulins on PT was reagent and/or method dependent, PT and APTT were measured in plasma samples obtained from patients referred to the Myeloma Institute using mechanical (STAR Evolution; Diagnostica Stago) and optical clot detection (ACL TOP 500 analyzer; Instrumentation Laboratory) and manufacturer provided reagents.A total of 97 patients were included in this study. Twelve patients had abnormal coagulation test results. An isolated prolonged PT was found in 8 patients and an isolated prolonged PTT was detected in 4 patients.The PT, but not APTT, was positively correlated with serum paraprotein level and this correlation was observed regardless of the reagents and instrumentation used to assess clot time.

Background Vulvar squamous cell carcinoma (vSCC) is a rare but debilitating disease. One vSCC variant comprises tumor cells that grow and expand as a cohesive sheet of cells that “pushes” and compresses the associated lymphoplasmacytic (LPC) stroma. Another vSCC variant features tumor cells that grow in loose association with one another and infiltrate the associated fibromyxoid (FMX) stroma consisting mainly of extracellular matrix. Clinically, infiltrative vSCC with FMX stroma (Inf/FMX) is significantly associated with lymph node metastases and recurrence. Methods An unbiased proteomic approach was used to identify pathways involved in the development of the different vSCC variants. Proteins extracted from formalin-fixed and paraffin-embedded tissues of 10 cases of pushing vSCC with LPC stroma (Push/LPC) and eight cases of Inf/FMX were subjected to liquid chromatography-tandem mass spectrometry (LC–MS/MS). Results Analysis identified 2265 different proteins in the 18 samples of vSCC. Of these, 282 proteins were differentially expressed between vSCC variants. Of these, 45 were higher and 237 lower in Inf/FMX compared to Push/LPC tumors. Consistent with the desmoplastic morphology and increased picrosirius red staining, expression of subunits of several collagens (Col 1, 3, 6, 14) was higher in the more aggressive Inf/FMX tumors. In contrast, signal transducer and activator of transcription 1 (STAT1), an important regulator of several inflammatory pathways, was expressed at lower levels in the Inf/FMX tumors. This finding was confirmed by immunohistochemistry using an antibody to STAT1. Informatics analysis of the differing profiles identified differences in pathways associated with integrin signaling and inflammation mediated by chemokines and cytokines. Conclusions Comparing the proteomic profiles of vSCC morphologic variants indicates that increased expression of collagen subunits and decreased expression of STAT1 are associated with a more aggressive tumor variant, defined by increased incidence of nodal metastases and tumor recurrence. Informatic analyses further identify that both alterations in cell interaction with matrix and immune function differ with tumor aggressiveness. Identification of these pathways provides a molecular basis for understanding aggressiveness of vSCC. Electronic supplementary material The online version of this article (10.1186/s12014-017-9175-8) contains supplementary material, which is available to authorized users.

Patterns of invasion and stromal response are understudied in vulvar squamous cell carcinoma. The aim of this study was to explore whether histologic features such as an infiltrative pattern of invasion and fibromyxoid stromal response (FMX-SR) are meaningful prognostic factors. We reviewed 143 vulvar squamous cell carcinoma resections and correlated patterns of invasion and stromal response with patient age, ethnicity, depth of invasion, tumor size, perineural invasion (S100/AE1/3 stain), lymph node involvement (LNI), extranodal extension, margin status, pathologic stage, and recurrence. Univariate analyses of continuous variables were performed using t tests, whereas Pearson χ tests were used for categorical variables. Logistic regression analyses examined the relationship between histopathologic characteristics and clinical outcomes. There was a statistically significant association between infiltrative tumors and an FMX-SR in comparison with noninfiltrative tumors (P

Objectives Vulvar squamous cell carcinoma (vSCC) is a gynecologic malignancy diagnosed in nearly 4500 women in the United States each year. Current criteria for treatment planning provide inadequate assessment of aggressive vSCC cases, resulting in insufficient use of adjuvant treatments and high rates of vSCC recurrence. Perineural invasion (PNI) is a pathologic feature inconsistently included in the assessment of vSCC, because its relevance to clinical outcomes in these women is not well defined. The purpose of this study was to determine the association between PNI and relevant clinical parameters such as recurrence. Methods A total of 103 cases of vSCC were evaluated for PNI using pathology report review and immunohistochemistry dual-chromogen staining for S100 and AE1/3. Medical records were reviewed for clinical and follow-up data. Data were analyzed using univariate and multivariate logistic regression statistical methods. Results Patients with vSCC containing PNI had a greater risk for cancer recurrence than those whose tumors did not contain PNI (odds ratio=2.8, P=0.0290). There was no significant correlation between the presence of PNI and nodal involvement, stage, or lymphovascular invasion. Tumors with PNI had greater depth of invasion (DOI) (P=0.0047); however, DOI was not associated with recurrence (P=0.2220). When analyzed using a multivariable logistic regression model, PNI was an independent predictor of recurrence in vSCC (adjusted odds ratio=2.613, P=0.045). Conclusions PNI is an independent indicator of risk for recurrence in vSCC. The association of PNI with increased risk for recurrence, independent of DOI, nodal involvement, lymphovascular invasion, or stage, should encourage practicing pathologists to thoroughly search for and report the presence of PNI in vSCC.

BACKGROUND There is no widely accepted rapid on-site evaluation (ROSE) reporting system for endobronchial ultrasound-guided transbronchial needle aspiration. At the University of Arkansas for Medical Sciences, ROSE reporting was unstructured. The goal was to evaluate, compare, and improve upon 2 structured approaches proposed in the literature. METHODS One hundred eighteen consecutive nodal aspirates were retrospectively reviewed by a pathology resident and a staff cytopathologist, both of whom were blinded to the original unstructured readings. Each reviewer interpreted every specimen with 2 different structured criteria proposed in the literature: criteria from the University of Minnesota (the Minnesota [MN] criteria) and criteria from the North Shore Long Island Jewish Health System (the New York [NY] criteria). The data allowed a comparison of the original unstructured ROSE system with the MN and NY scoring schemes and the final diagnosis. RESULTS Original on-site adequacy (OSA) had been assessed at 96%. Three cases were false-adequate according to the original unstructured approach; these had been called adequate on site, but a subsequent slide review including cell blocks did not show definite nodal tissue. OSA dropped to 86% with the MN criteria and to 85% with the NY criteria. No false-adequate on-site diagnoses would have been rendered with the application of either structured criteria. There were no significant differences between the MN and NY criteria with respect to the determination of OSA. An assessment of ease of application favored the NY criteria. With respect to diagnostic categories, each of the systems (MN and NY) was felt to have a category of value not used by the other system. CONCLUSIONS A standardized intra- and inter-institutional system for ROSE reporting is needed. On the basis of comparative analyses and consensus, modifications to prior criteria have been proposed in the hope of approaching this goal. Cancer (Cancer Cytopathol) 2015;123:362–72. © 2015 American Cancer Society

Inflammation is associated with modification of the extracellular environment, changes in cytokine expression, and the accumulation of immune cells. Such modifications create ligands that support SR-A-mediated macrophage adhesion and retention. This may be particularly important in settings, such as atherosclerosis and diabetes, as modified lipoproteins and gluc-collagen are ligands for SR-A. SR-A-mediated adhesion requires the PLA2-dependent generation of AA and its metabolism by 12/15 LOX. In contrast, the inhibition of the COX-dependent conversion of AA to PG had no effect on SR-A-mediated adhesion. In this study, macrophages were isolated from SR-A+/+ and SR-A−/− mice and plated on gluc-collagen to test the hypothesis that COX-derived PGs are produced during SR-A-mediated adhesion and regulate macrophage function. SR-A-mediated binding to gluc-collagen induced a rapid but transient increase in PG production, which required the activation of PLA2 and Src kinase but not PI3K. SR-A+/+ macrophages cultured on gluc-collagen for 24 h secreted a similar amount of TNF-α and 2.5-fold more IL-10 than SR-A−/− macrophages. The inhibition of COX substantially increased TNF-α production but reduced IL-10 levels in SR-A+/+ macrophages. These effects of COX inhibition were reversed by exogenous PGE2 and mimicked by specific antagonism of the EP4 receptor. Thus, in addition to the enhancement of macrophage adhesion, SR-A binding to gluc-collagen stimulates PG production, which in turn, differentially regulates the expression of inflammatory cytokines.

Objectives: Modified LDL uptake by macrophages (MΦ) leading to foam cell formation is an early event in atherosclerosis. Recent studies suggest that MΦ polarization contributes to the progression of atherosclerosis. Therefore, we investigated the hypothesis that macrophage plasticity affects modified LDL uptake and subsequent foam cell formation. Approach & Results: Bone marrow-derived MΦ were treated with LPS and IFNγ or IL-4 to induce MΦ polarization to M(LPS/IFNγ or M1) and M (IL4 or M2) phenotypes. Foam cell formation was determined after incubating M(LPS/IFNγ) and M(IL4) MΦ with oxidized-LDL. Our results show that formation of foam cells was marginally increased in M(IL4) MΦ compared to un-polarized M(-) MΦ. However, accumulation of neutral lipids was higher in M(LPS/IFNγ) MΦ compared to M(IL4) MΦ indicating that foam cell formation is enhanced by polarization to pro-inflammatory M(LPS/IFNγ) MΦ. Interestingly, mRNA and protein expression of CD36, one of the major scavenger receptors, were lower in M(LPS/IFNγ) MΦ compared to un-polarized MΦ. To assess the specific contribution of CD36, foam cell formation was assessed in MΦ isolated from CD36-deficient mice. Notably, oxidized-LDL uptake and foam cell formation in CD36-deficient M(LPS/IFNγ) MΦ was similar to M(LPS/IFNγ) MΦ from wild type mice. This indicates that CD36 does not mediate the increased foam cell formation in M(LPS/IFNγ) MΦ. We therefore examined expression of LOX-1, another scavenger receptor implicated in foam cell formation. We found that both mRNA and protein expression for LOX-1 increased several fold in M(LPS/IFNγ) MΦ. We also showed that TLR4-mediated NF-κB activation is the primary mechanism for the increased LOX-1 expression and foam cell formation in M(LPS/IFNγ) polarized MΦ. Conclusion: Macrophage plasticity, specifically polarization of MΦ to pro-inflammatory M(LPS/IFNγ) phenotype promotes foam cell formation, which has implications for the progression of atherosclerosis.

Scavenger receptors constitute a large family of proteins that are structurally diverse and participate in a wide range of biological functions. These receptors are expressed predominantly by myeloid cells and recognize a diverse variety of ligands including endogenous and modified host-derived molecules and microbial pathogens. There are currently eight classes of scavenger receptors, many of which have multiple names, leading to inconsistencies and confusion in the literature. To address this problem, a workshop was organized by the United States National Institute of Allergy and Infectious Diseases, National Institutes of Health, to help develop a clear definition of scavenger receptors and a standardized nomenclature based on that definition. Fifteen experts in the scavenger receptor field attended the workshop and, after extensive discussion, reached a consensus regarding the definition of scavenger receptors and a proposed scavenger receptor nomenclature. Scavenger receptors were defined as cell surface receptors that typically bind multiple ligands and promote the removal of nonself or altered-self targets. They often function by mechanisms that include endocytosis, phagocytosis, adhesion, and signaling that ultimately lead to the elimination of degraded or harmful substances. Based on this definition, nomenclature and classification of these receptors into 10 classes were proposed. This classification was discussed at three national meetings and input from participants at these meetings was requested. The following manuscript is a consensus statement that combines the recommendations of the initial workshop and incorporates the input received from the participants at the three national meetings.

The circulating blood platelet as a participant in inflammatory pathways has been the focus of intense investigation and the topic of several reviews and other chapters in this book (Franco et al. Blood. 126:582–588, 2015). Inflammation underlies the pathophysiologic response in many diseases, and therefore defining the role of the platelet in inflammatory responses is paramount to understanding the molecular basis of the disease process (Leslie. Science 328(5978):562–564, 2010). In this chapter, we focus on the historical background describing the role of platelets in connective tissue inflammation, with emphasis on studies linking platelets and rheumatoid arthritis. A link between platelets and the inflammatory response starts with an appreciation for platelets being a specific cellular entity restricted to higher mammals. Lower vertebrates, such as fish and birds, do not have platelets, but instead have a dual-functioning cell, the thrombocyte, that has both immune and hemostatic properties (see Momi and Wiwanitkit 2017). Thus, the divergence of thrombocyte function to multiple cell types, such as neutrophils, monocytes, and platelets, might be expected to result in functional overlap with some similar properties retained by each individual cell type in higher species. The participation of platelets in inflammation needs careful consideration given the widespread use of antithrombotic agents for prevention of cardiovascular events. Understanding the molecular basis of the platelet/inflammatory axis will also ensure the best practice decisions will be made for the treatment of cardiovascular disease and additional inflammatory conditions.

In this issue of Blood, de Stoppelaar et al further unravel the relevance of platelets in a mouse model of pneumonia-derived sepsis by illustrating how platelets dynamically modulate infection and the inflammatory response.

SummaryIntroduction Abnormal screening coagulation tests are frequently observed in asymptomatic patients with multiple myeloma and other plasma cell neoplasms. Methods Prothrombin time (PT), activated partial thromboplastin time (APTT) and fibrinogen activity were correlated with clinical history and disease parameters in patients referred to the Myeloma Institute for Research and Therapy. Results An isolated prolonged PT was the most common abnormal coagulation test (25%). Prolonged PT was more frequently observed in patients with multiple myeloma (n = 157) compared to MGUS patients (n = 34) or other diagnostic categories of plasma cell dyscrasia. There were no differences in age, gender, previous chemotherapy, or immunoglobulin isotype in patients with isolated prolonged PT (n = 62) compared to those with normal screening coagulation tests (n = 173). Fibrinogen activity was significantly lower in patients with prolonged PT; however, there was no correlation between fibrinogen activity and PT. Serum M protein concentrations were significantly greater in patients with prolonged PT and were positively correlated with PT. Conclusion An association between disease severity and prolonged PT is suggested by our finding that patients with multiple myeloma were more likely to have prolonged PT than patients with other plasma cell neoplasms. Of the factors examined, the monoclonal protein level was significantly higher in patients with isolated prolonged PT and correlated with PT.

The objective of the study was to evaluate the expression pattern of interleukin-6 (IL-6) to determine its utility in differentiating Castleman Disease subtypes and reactive lymphadenopathies. Paraffin-embedded tissue blocks from 20 cases referred for assessment of Castleman Disease (CD) and 4 cases of reactive hyperplasia were selected for immunohistochemical staining with an IL-6 antibody. Six pathologists evaluated the hematoxylin and eosin stained tissue sections and IL-6 expression pattern. Of 20 CD referral cases, the pathologic diagnosis was CD in 14 cases and included 6 hyaline-vascular (HV-CD), 6 plasma cell (PC-CD) and 2 "mixed type"-CD cases. The remaining 6 referral cases showed morphologic features consistent with reactive lymphadenopathy. Patients with non-CD, reactive lymphadenopathies had clinical and/or laboratory features of systemic lupus erythematosus, Hashimoto's disease, viral infection or chronic cellulitis. The pattern of IL-6 expression differed between CD subtypes and non-CD cases. In PC-CD, IL-6 expression was detected in plasma cells and vascular endothelial cells; whereas IL-6 immunoreactivity was detected primarily in vascular endothelial cells in HV-CD. Interfollicular plasma cells were prominent in PC-CD and reactive lymphadenopathies; however, IL-6 expression was significantly increased in PC-CD compared to reactive lymph nodes. Together with morphologic features, the expression pattern of IL-6 detected by immunohistochemistry is helpful to distinguish CD subtypes and reactive mimics.

A novel mechanism for enhancing cell adhesion through recruitment of SR-A to the cell surface. The importance of cytoplasmic motifs in differentially regulating SR-A function was demonstrated by deleting the first 49 cytoplasmic aa (SR-AΔ1–49), which abolished SR-A-mediated ligand internalization without reducing cell adhesion. To identify additional cytoplasmic motifs within the first 49 aa that regulate SR-A function, the acidic residues in a conserved motif (EDAD) were changed to their amide derivatives (SR-AQNAN). The function and regulation of SR-AQNAN were compared with that of SR-AΔ1–49 and SR-A in transfected HEK-293 cells. Blocking PI3K activation inhibited SR-A, but not SR-AΔ1–49- or SR-AQNAN-mediated cell adhesion. Although deleting (SR-AΔ1–49) or mutating (SR-AQNAN) the EDAD motif abolished the PI3K sensitivity of SR-A-mediated cell adhesion, these mutations did not affect ligand internalization or PI3K activation during cell adhesion. To define the mechanism by which PI3K regulates SR-A-mediated cell adhesion, the cellular localization of wild-type and mutant SR-A was examined. PI3K inhibition reduced surface localization of SR-A but not of SR-AΔ1–49 or SR-AQNAN. The regulation of SR-A surface localization by PI3K was confirmed in peritoneal macrophages, which endogenously express SR-A. Together, these results suggest a pathway in which SR-A binding to an immobilized ligand activates PI3K to recruit more receptor to the plasma membrane and enhances cell adhesion.

In the current study, we examined whether diabetes affected the ability of HDL to stimulate nitric oxide (NO) production. Using HDL isolated from both diabetic humans and diabetic mouse models, we found that female HDL no longer induced NO synthesis, despite containing equivalent amounts of estrogen as nondiabetic controls. Furthermore, HDL isolated from diabetic females and males prevented acetylcholine-induced stimulation of NO generation. Analyses of both the human and mouse diabetic HDL particles showed that the HDLs contained increased levels of myristic acid. To determine whether myristic acid associated with HDL particles was responsible for the decrease in NO generation, myristic acid was added to HDL isolated from nondiabetic humans and mice. Myristic acid-associated HDL inhibited the generation of NO in a dose-dependent manner. Importantly, diabetic HDL did not alter the levels of endothelial NO synthase or acetylcholine receptors associated with the cells. Surprisingly, diabetic HDL inhibited ionomycin-induced stimulation of NO production without affecting ionomycin-induced increases in intracellular calcium. Further analysis indicated that diabetic HDL prevented calmodulin from interacting with endothelial NO synthase (eNOS) but did not affect the activation of calmodulin kinase or calcium-independent mechanisms for stimulating eNOS. These studies are the first to show that a specific fatty acid associated with HDL inhibits the stimulation of NO generation. These findings have important implications regarding cardiovascular disease in diabetic patients.

Class A scavenger receptors (SR-A) participate in multiple macrophage functions including macrophage adhesion to modified proteins. SR-A-mediated adhesion may therefore contribute to chronic inflammation by promoting macrophage accumulation at sites of protein modification. The mechanisms that couple SR-A binding to modified proteins with increased cell adhesion have not been defined. In this study, SR-A expressing HEK cells and SR-A+/+ or SR-A−/− macrophages were used to delineate the signaling pathways required for SR-A-mediated adhesion to modified protein. Inhibiting Gi/oactivation, which decreases initial SR-A-mediated cell attachment, did not prevent the subsequent spreading of attached cells. In contrast, inhibition of Src kinases or PI3-kinase abolished SR-A-dependent cell spreading without affecting SR-A-mediated cell attachment. Consistent with these results, the Src kinase Lyn and PI3-kinase were sequentially activated during SR-A-mediated cell spreading. Furthermore, activation of both Lyn and PI3-kinase was required for enhancing paxillin phosphorylation. Activation of a Src kinase-PI3-kinase-Akt pathway was also observed in cells expressing a truncated SR-A protein that does not internalize indicating that SR-A-mediated activation of intracellular signaling cascades following adhesion to MDA-BSA is independent of receptor internalization. Thus SR-A binding to modified protein activates signaling cascades that have distinct roles in regulating initial cell attachment and subsequent cell spreading.