Colleen M. Cebulla

Germline BAP1 mutation predisposes to uveal melanoma, lung adenocarcinoma, meningioma, and other cancers.

Objective To investigate the potential contribution of germline sequence alterations in the BAP1 gene in uveal melanoma (UM) patients with possible predisposition to hereditary cancer. Design A total of 53 unrelated UM patients with high risk for hereditary cancer and five additional family members of one proband were studied. Mutational screening was carried out by direct sequencing. Results Of the 53 UM patients studied, a single patient was identified with a germline BAP1 truncating mutation, c. 799 C→T (p.Q267X), which segregated in several family members and was associated with UM and other cancers. Biallelic inactivation of BAP1 and decreased BAP1 expression were identified in the UM, lung adenocarcinoma and meningioma tumours from three family members with this germline BAP1 mutation. Germline BAP1 variants of uncertain significance, likely non-pathogenic, were also identified in two additional UM patients. Conclusion This study reports a novel hereditary cancer syndrome caused by a germline BAP1 mutation that predisposes patients to UM, lung carcinoma, meningioma, and possibly other cancers. The results indicate that BAP1 is the candidate gene in only a small subset of hereditary UM, suggesting the contribution of other candidate genes.

Targeting Hypoxia, a Novel Treatment for Advanced Retinoblastoma

PURPOSE The purpose of this study was to evaluate the presence and extent of hypoxia in murine retinoblastoma tumors and the feasibility of targeting hypoxic cells as a novel therapeutic strategy. METHODS Hypoxic and vascular areas in LH(BETA)T(AG) mouse retinal tumors were measured using immunohistochemistry. The glycolytic inhibitor 2-deoxy-d-glucose (2-DG) was used to test the efficacy of targeting hypoxic cells in retinoblastoma. Sixteen-week-old LH(BETA)T(AG) mice received injections of saline, carboplatin (31.25 microg/20 microL), 2-DG (500 mg/kg), and carboplatin (31.25 microg/20 microL) + 2-DG (500 mg/kg). Carboplatin was administered through biweekly subconjunctival injections to right eyes only for 3 weeks. 2-DG was administered through intraperitoneal injection three times a week for 5 weeks. Saline was administered using both methods. Eyes were enucleated at 21 weeks of age and examined for residual tumor. RESULTS Hypoxic regions were observed in tumors larger than 3.28 mm(2). When 2-DG was combined with carboplatin, a marked decrease in tumor burden was observed that was significantly more pronounced than when either agent was given alone. The hypoxic tumor cell population as measured by pimonidazole was markedly reduced by carboplatin + 2-DG (P < 0.01) and by 2-DG alone (P < 0.01), but not by carboplatin alone, indicating that 2-DG effectively killed hypoxic retinoblastoma cells in vivo. CONCLUSIONS Treatment with glycolytic inhibitors as adjuvants to chemotherapy has the potential to increase the efficacy of chemotherapy in advanced retinoblastoma. This approach may have benefits for children with this disease and should be further investigated.

Expanding the clinical phenotype of hereditary BAP1 cancer predisposition syndrome, reporting three new cases

The clinical phenotype of BAP1 hereditary cancer predisposition syndrome (MIM 614327) includes uveal melanoma (UM), cutaneous melanoma (CM), renal cell carcinoma (RCC), and mesothelioma. However, the frequency of the syndrome in patients with UM and the association with other cancers are still not clear. In this study, we screened 46 previously untested, unrelated UM patients with high risk for hereditary cancer for germline mutation in BAP1. We also studied four additional patients with a personal or family history suggestive of BAP1 hereditary cancer syndrome. We identified three patients with germline pathogenic mutations (c.2050 C>T, pGln684*; c.1182C>G, p.Tyr394*, and c.1882_1885delTCAC, p. Ser628Profs*8) in BAP1. Two of these three patients presented with UM and the third with a metastatic adenocarcinoma likely from a hepatic cholangiocarcinoma. Reported family histories included UM, mesothelioma, RCC, CM, and several other internal malignancies. The results of this study confirm the association between germline BAP1 mutation and predisposition to UM, mesothelioma, CM and RCC. However, other cancers, such as cholangiocarcinoma and breast carcinoma may be part of the phenotype of this hereditary cancer predisposition syndrome. In addition, the results support the existence of other candidate genes in addition to BAP1 contributing to hereditary predisposition to UM.

Viral Inhibition of Interferon Signal Transduction

The type I and II interferons (IFNs) are potent stimulators of antigen processing and presentation and are essential in antiviral immunity. IFNs upregulate the transcription of major histocompatibility complex (MHC) class I and II molecules, associated antigen-processing proteins, and induce the production of direct antiviral effector molecules such as 2′,5′-oligoadenylate synthetase, double-stranded-RNA-dependent protein kinase and Mx proteins. It is increasingly evident that viruses have evolved mechanisms to globally inhibit the actions of IFNs through disruption of their signal transduction pathways. Herein, we review the ability and novel mechanisms of several diverse viruses to inhibit IFN-induced JAK/STAT signal transduction.

Comprehensive review of BAP1 tumor predisposition syndrome with report of two new cases

The BRCA1‐associated protein‐1 (BAP1) tumor predisposition syndrome (BAP1‐TPDS) is a recently identified hereditary cancer syndrome. Germline mutations in this tumor suppressor gene predispose families to the development of various malignancies. The molecular functions of the gene as well as the clinical phenotype of the syndrome are still being clarified. We sought to conduct a comprehensive review of published research into BAP1‐TPDS to more thoroughly delineate the clinical implications of germline BAP1 mutations. We also report two additional families with germline BAP1 mutations. Current evidence demonstrates that germline BAP1 mutations predispose families to uveal melanoma, renal cell carcinoma, malignant mesothelioma, cutaneous melanoma, and possibly to a range of other cancers as well. Some of these cancers tend to be more aggressive, have a propensity to metastasize, and onset earlier in life in patients with BAP1 mutations as compared to non‐predisposed patients with equivalent cancers. Although further research is necessary, this information can aid in the management, diagnosis, and therapy of these patients and their families, and highlights the importance of genetic counseling.

Human Cytomegalovirus Protein pp71 Disrupts Major Histocompatibility Complex Class I Cell Surface Expression

ABSTRACT The human cytomegalovirus tegument protein pp71 is the product of the UL82 gene. Roles for pp71 in stimulating gene transcription, increasing infectivity of viral DNA, and the degradation of retinoblastoma family proteins have been described. Here we report a novel function for pp71 in limiting accumulation of cell surface major histocompatibility complex (MHC) class I complexes. MHC molecules were analyzed in glioblastoma cells exposed to a replication-defective adenovirus expressing UL82 (Adpp71) or after transient transfection of the UL82 gene. Accumulation of cell surface MHC class I levels diminished in a specific and dose-dependent manner after exposure to Adpp71 but not after exposure to an adenovirus expressing β-galactosidase (Adβgal). UL82 expression did not interfere with accumulation of either MHC class I heavy-chain transcript or protein, nor did UL82 expression correlate with markers of apoptosis. Rather, UL82 expression correlated with an increased proportion of MHC class I molecules exhibiting sensitivity to endoglycosidase H treatment. Finally, we show that, in cells infected with recombinant virus strain missing all of the unique short region MHC class I evasion genes, disruption of UL82 expression by short, interfering RNAs led to increased accumulation of cell surface MHC class I complexes. These findings support a novel role for HCMV pp71 in disruption of the MHC class I antigen presentation pathway.

Human Cytomegalovirus Disrupts Constitutive MHC Class II Expression

CD8+ and CD4+ T lymphocytes are important in controlling human CMV (HCMV) infection, but the virus has evolved protean mechanisms to inhibit MHC-based Ag presentation and escape T lymphocyte immunosurveillance. Herein, the interaction of HCMV with the MHC class II Ag presentation pathway was investigated in cells stably transfected with class II transactivator. Flow cytometry experiments demonstrate that HCMV infection decreases cell-surface MHC class II expression. HCMV down-regulates MHC class II surface expression without a significant effect on class II RNA or steady-state protein levels. SDS-stability and confocal microscopy experiments demonstrate normal levels of steady-state peptide-loaded class II molecules in infected cells and that class II molecules reach late endosomal and HLA-DM positive peptide-loading compartments. However, MHC class II positive vesicles are retained in an abnormal perinuclear distribution. Finally, experiments with a mutant HCMV strain demonstrate that this novel mechanism of decreased MHC class II expression is not mediated by one of the known HCMV immunomodulatory genes. These defects in MHC class II expression combined with previously identified CMV strategies for decreasing MHC class I expression enables infected cells to evade T lymphocyte immunosurveillance.

Retinal tumor imaging and volume quantification in mouse model using spectral-domain optical coherence tomography

We have successfully imaged the retinal tumor in a mouse model using an ultra-high resolution spectral-domain optical coherence tomography (SD-OCT) designed for small animal retinal imaging. For segmentation of the tumor boundaries and calculation of the tumor volume, we developed a novel segmentation algorithm. The algorithm is based on parametric deformable models (active contours) and is driven by machine learning-based region classification, namely a Conditional Random Field. With this algorithm we are able to obtain the tumor boundaries automatically, while the user can specify additional constraints (points on the boundary) to correct the segmentation result, if needed. The system and algorithm were successfully applied to studies on retinal tumor progression and monitoring treatment effects quantitatively in a mouse model of retinoblastoma.

Cytomegalovirus induces sialyl Lewis(x) and Lewis(x) on human endothelial cells.

BACKGROUND Cytomegalovirus (CMV) is the primary viral cause of complications in transplant recipients. We sought to understand the mechanisms of its dissemination and induction of vascular disease, which may lead to transplant complications. Sialyl Lewis(x) (sLe(x)) and Lewis(x) (Le(x)) are known for their roles in mediating cell adhesion and as tumor-associated carbohydrate antigens. Herein we explore whether CMV induces surface expression of these important molecules in endothelial cells (EC). METHODS Flow cytometry was used to detect surface expression of sLe(x) and Le(x) on CMV-infected human umbilical vein endothelial cells (HUVEC), with or without ultraviolet inactivation of the virus. To elucidate mechanisms of CMV-mediated induction, mRNA coding for predominant HUVEC sialyltransferases (ST) and fucosyltransferases (FT), key enzymes in sLe(x) and Le(x) synthesis, was analyzed by Northern blot. Dual immunohistochemical staining for sLe(x) and Le(x) expression of human colon and placental tissue was performed to investigate in vivo relevance. RESULTS sLe(x) expression on CMV-infected HUVEC was strongly up-regulated by 8 days after inoculation. Le(x) expression was detectable earlier and increased steadily over time. In contrast, ultraviolet-inactivated CMV did not induce expression of these molecules. Northern blot assays demonstrated higher levels of important EC glycosyltransferases ST-IV, FT-III, and FT-IV in CMV-infected EC. Finally, high levels of sLe(x) and Le(x) were expressed in CMV-infected EC in vivo. CONCLUSIONS Given the known biologic functions of sLe(x) and Le(x), we suggest that CMV induction of these molecules may have widespread consequences ranging from CMV dissemination to induction of CMV-associated vascular disease, including thrombosis.

Human Cytomegalovirus Inhibition of Major Histocompatibility Complex Transcription and Interferon Signal Transduction

Pathogens have evolved diverse mechanisms for escaping host innate and adaptive immunity. Viruses that maintain a persistent infection are particularly effective at disabling key arms of the host immune response. For example, the herpesviruses establish a persistent infection in human and animal hosts, in part through critical immunoevasive strategies. Cytomegalovirus, a beta-herpesvirus, impairs major histocompatibility complex (MHC) class I and class II antigen presentation by decreasing MHC expression on the surface of the infected cell, thus enabling infected cells to escape CD8+ and CD4+ T lymphocyte immunosurveillance. Moreover, cytomegalovirus blocks the interferon signal transduction pathway, thereby limiting the direct and indirect antiviral effects of the interferons. In this review, we focus on an emerging paradigm in which the effectiveness of viruses, particularly human cytomegalovirus, to escape antiviral immune responses is significantly enhanced by their ability to inhibit MHC transcription and interferon (IFN)-stimulated (JAK/STAT) signal transduction.