Viral oncoprotein antibodies as a marker for recurrence of Merkel cell carcinoma: A prospective validation study.

BACKGROUND: Merkel cell carcinoma (MCC) is an aggressive skin cancer with a recurrence rate of >40%. Of the 2000 MCC cases per year in the United States, most are caused by the Merkel cell polyomavirus (MCPyV). Antibodies to MCPyV oncoprotein (T-antigens) have been correlated with MCC tumor burden. The present study assesses the clinical utility of MCPyV-oncoprotein antibody titers for MCC prognostication and surveillance. METHODS: MCPyV-oncoprotein antibody detection was optimized in a clinical laboratory. A cohort of 219 patients with newly diagnosed MCC were followed prospectively (median follow-up, 1.9 years). Among the seropositive patients, antibody titer and disease status were serially tracked. RESULTS: Antibodies to MCPyV oncoproteins were rare among healthy individuals (1%) but were present in most patients with MCC (114 of 219 patients [52%]; P < .01). Seropositivity at diagnosis independently predicted decreased recurrence risk (hazard ratio, 0.58; P = .04) in multivariate analyses adjusted for age, sex, stage, and immunosuppression. After initial treatment, seropositive patients whose disease did not recur had rapidly falling titers that became negative by a median of 8.4 months. Among seropositive patients who underwent serial evaluation (71 patients; 282 time points), an increasing oncoprotein titer had a positive predictive value of 66% for clinically evident recurrence, whereas a decreasing titer had a negative predictive value of 97%. CONCLUSIONS: Determination of oncoprotein antibody titer assists in the clinical management of patients with newly diagnosed MCC by stratifying them into a higher risk seronegative cohort, in which radiologic imaging may play a more prominent role, and into a lower risk seropositive cohort, in which disease status can be tracked in part by oncoprotein antibody titer. Cancer 2017;123:1464-1474. © 2016 American Cancer Society.

CD8+ lymphocyte intratumoral infiltration as a stage-independent predictor of Merkel cell carcinoma survival: a population-based study.

OBJECTIVES: Intratumoral CD8+ lymphocytes (IT-CD8s) have shown promise as a prognostic indicator for Merkel cell carcinoma (MCC). We tested whether IT-CD8s predict survival among a population-based MCC cohort. METHODS: One hundred thirty-seven MCC cases that had not previously been analyzed for IT-CD8s were studied. RESULTS: Three-year MCC-specific survival rates were 56%, 72%, and 100% for patients with absent (n = 46), low (n = 85), and moderate or strong (n = 6) IT-CD8s, respectively. Increased IT-CD8s were associated with improved MCC-specific survival in a multivariate competing risk-regression analysis including stage, age, and sex (hazard ratio [HR] = 0.5; 95% confidence interval [CI] = 0.3-0.9). Although a similar trend was observed for overall survival, statistical significance was not reached (HR = 0.8; 95% CI = 0.6-1.0), likely because of the high rate of non-MCC deaths among older patients. CONCLUSIONS: This study of prospectively captured MCC cases supports the concept that cellular immunity is important in MCC outcome and that CD8+ lymphocyte infiltration adds prognostic information to conventional staging.

Downregulation of MHC-I expression is prevalent but reversible in Merkel cell carcinoma.

Merkel cell carcinoma (MCC) is an aggressive, polyomavirus-associated skin cancer. Robust cellular immune responses are associated with excellent outcomes in patients with MCC, but these responses are typically absent. We determined the prevalence and reversibility of major histocompatibility complex class I (MHC-I) downregulation in MCC, a potentially reversible immune-evasion mechanism. Cell-surface MHC-I expression was assessed on five MCC cell lines using flow cytometry as well as immunohistochemistry on tissue microarrays representing 114 patients. Three additional patients were included who had received intralesional IFN treatment and had evaluable specimens before and after treatment. mRNA expression analysis of antigen presentation pathway genes from 35 MCC tumors was used to examine the mechanisms of downregulation. Of note, 84% of MCCs (total n = 114) showed reduced MHC-I expression as compared with surrounding tissues, and 51% had poor or undetectable MHC-I expression. Expression of MHC-I was lower in polyomavirus-positive MCCs than in polyomavirus-negative MCCs (P < 0.01). The MHC-I downregulation mechanism was multifactorial and did not depend solely on HLA gene expression. Treatment of MCC cell lines with ionizing radiation, etoposide, or IFN resulted in MHC-I upregulation, with IFNs strongly upregulating MHC-I expression in vitro, and in 3 of 3 patients treated with intralesional IFNs. MCC tumors may be amenable to immunotherapy, but downregulation of MHC-I is frequently present in these tumors, particularly those that are positive for polyomavirus. This downregulation is reversible with any of several clinically available treatments that may thus promote the effectiveness of immune-stimulating therapies for MCC.

Clear-cell meningioma presenting as an infiltrative external auditory canal mass.

We present a case of clear-cell meningioma occurring in a 72-year-old female presenting with an infiltrative external auditory canal mass (with both intracranial and extracranial extension and extensive leptomeningeal involvement) and facial nerve paralysis.

Transcriptome-wide studies of merkel cell carcinoma and validation of intratumoral CD8+ lymphocyte invasion as an independent predictor of survival.

PURPOSE: Merkel cell carcinoma (MCC) is a polyomavirus-associated skin cancer that is frequently lethal and lacks established prognostic biomarkers. This study sought to identify biomarkers that improve prognostic accuracy and provide insight into MCC biology. PATIENTS AND METHODS: Gene expression profiles of 35 MCC tumors were clustered based on prognosis. The cluster of genes overexpressed in good-prognosis tumors was tested for biologic process enrichment. Relevant mRNA expression differences were confirmed by quantitative polymerase chain reaction and immunohistochemistry. An independent set of 146 nonoverlapping MCC tumors (median follow-up, 25 months among 116 living patients) was employed for biomarker validation. Univariate and multivariate Cox regression analyses were performed. RESULTS: Immune response gene signatures were prominent in patients with good prognoses. In particular, genes associated with cytotoxic CD8+ lymphocytes were overexpressed in tumors from patients with favorable prognoses. In the independent validation set, cases with robust intratumoral CD8+ lymphocyte infiltration had improved outcomes (100% MCC-specific survival, n = 26) compared with instances characterized by sparse infiltration (60% survival, n = 120). Only stage and intratumoral CD8 infiltration (but not age, sex, or CD8+ lymphocytes localized to the tumor-stroma interface) were significant in both univariate and multivariate Cox regression analyses. Notably, traditional histologic identification of tumor-infiltrating lymphocytes was not a significant independent predictor of survival. CONCLUSION: Intratumoral CD8+ lymphocyte infiltration can be readily assessed on paraffin-embedded tissue, is independently associated with improved MCC-specific survival, and therefore, may provide prognostic information that enhances established MCC staging protocols.

PIPKI gamma 90 negatively regulates LFA-1-mediated adhesion and activation in antigen-induced CD4+ T cells.

T cell activation requires the formation and maintenance of stable interactions between T cells and APCs. The formation of stable T cell-APC contacts depends on the activation of the integrin LFA-1 (CD11aCD18). Several positive regulators of LFA-1 activation downstream of proximal TCR signaling have been identified, including talin; however, negative regulators of LFA-1 activity remain largely unexplored. Extended isoform of phosphatidylinositol phosphate kinase type I γ (PIPKIγ90) is a member of the type I phosphatidylinositol phosphate kinase family that has been shown previously to modulate talin activation of integrins through production of phosphatidylinositol 4,5-bisphosphate and direct binding to talin. In this study, we show that PIPKIγ90 negatively regulates LFA-1-mediated adhesion and activation of T cells. Using CD4(+) T cells from PIPKIγ90-deficient mice, we show that CD4(+) T cells exhibit increased LFA-1-dependent adhesion to ICAM-1 and increased rates of T cell-APC conjugate formation with enhanced LFA-1 polarization at the synapse. In addition to increased adhesiveness, PIPKIγ90-deficient T cells exhibit increased proliferation both in vitro and in vivo and increased production of IFN-γ and IL-2. Together, these results demonstrate that PIPKIγ90 is a negative regulator of Ag-induced T cell adhesion and activation.

Calpain 4 is not necessary for LFA-1-mediated function in CD4+ T cells.

BACKGROUND: T cell activation and immune synapse formation require the appropriate activation and clustering of the integrin, LFA-1. Previous work has reported that the calpain family of calcium-dependent proteases are important regulators of integrin activation and modulate T cell adhesion and migration. However, these studies have been limited by the use of calpain inhibitors, which have known off-target effects. METHODOLOGY/PRINCIPAL FINDINGS: Here, we used a LoxP/CRE system to specifically deplete calpain 4, a small regulatory calpain subunit required for expression and activity of ubiquitously expressed calpains 1 and 2, in CD4+ T cells. CD4+ and CD8+ T cells developed normally in Capn4(F/F):CD4-CRE mice and had severely diminished expression of Calpain 1 and 2, diminished talin proteolysis and impaired casein degradation. Calpain 4-deficient T cells showed no difference in adhesion or migration on the LFA-1 ligand ICAM-1 compared to control T cells. Moreover, there was no impairment in conjugation between Capn4(F/F):CD4-CRE T cells and antigen presenting cells, and the conjugates were still capable of polarizing LFA-1, PKC-theta and actin to the immune synapse. Furthermore, T cells from Capn4(F/F):CD4-CRE mice showed normal proliferation in response to either anti-CD3/CD28 coated beads or cognate antigen-loaded splenocytes. Finally, there were no differences in the rates of apoptosis following extrinsic and intrinsic apoptotic stimuli. CONCLUSION/SIGNIFICANCE: Our findings demonstrate that calpain 4 is not necessary for LFA-1-mediated adhesion, conjugation or migration. These results challenge previous reports that implicate a central role for calpains in the regulation of T cell LFA-1 function.

Gene related to anergy in lymphocytes (GRAIL) expression in CD4+ T cells impairs actin cytoskeletal organization during T cell/antigen-presenting cell interactions.

GRAIL (gene related to anergy in lymphocytes), is an E3 ubiquitin ligase with increased expression in anergic CD4+ T cells. The expression of GRAIL has been shown to be both necessary and sufficient for the induction of T cell (T) anergy. To date, several subsets of anergic T cells have demonstrated altered interactions with antigen-presenting cells (APC) and perturbed TCR-mediated signaling. The role of GRAIL in mediating these aspects of T cell anergy remains unclear. We used flow cytometry and confocal microscopy to examine T/APC interactions in GRAIL-expressing T cells. Increased GRAIL expression resulted in reduced T/APC conjugation efficiency as assessed by flow cytometry. Examination of single T/APC conjugates by confocal microscopy revealed altered polarization of polymerized actin and LFA-1 to the T/APC interface. When GRAIL expression was knocked down, actin polarization to the T/APC interface was restored, demonstrating that GRAIL is necessary for alteration of actin cytoskeletal rearrangement under anergizing conditions. Interestingly, proximal TCR signaling including calcium flux and phosphorylation of Vav were not disrupted by expression of GRAIL in CD4+ T cells. In contrast, interrogation of distal signaling events demonstrated significantly decreased JNK phosphorylation in GRAIL-expressing T cells. In sum, GRAIL expression in CD4+ T cells mediates alterations in the actin cytoskeleton during T/APC interactions. Moreover, in this model, our data dissociates proximal T cell signaling events from functional unresponsiveness. These data demonstrate a novel role for GRAIL in modulating T/APC interactions and provide further insight into the cell biology of anergic T cells.

Adhesions ring: a structural comparison between podosomes and the immune synapse.

Podosomes and the immune synapse are integrin-mediated adhesive structures that share a common ring-like morphology. Both podosomes and immune synapses have a central core surrounded by a peripheral ring containing talin, vinculin and paxillin. Recent progress suggests significant parallels between the regulatory mechanisms that contribute to the formation of these adhesive structures. In this review, we compare the structures, functions and regulation of podosomes and the immune synapse.

Talin1 regulates TCR-mediated LFA-1 function.

The leukocyte integrin LFA-1 plays a critical role in T cell trafficking and T cell adhesion to APCs. It is known that integrin-mediated adhesion is regulated by changes in integrin ligand-binding affinity and valency through inside-out signaling. However, the molecular mechanisms involved in TCR-mediated LFA-1 regulation are not well understood. In this study, we show that the cytoskeletal protein talin1 is required for TCR-mediated activation of LFA-1 through regulation of LFA-1 affinity and clustering. Depletion of talin1 from human T cells by small interfering RNAs impairs TCR-induced adhesion to ICAM-1 and T cell-APC conjugation. TCR-induced LFA-1 polarization, but not actin polarization, is defective in talin1-deficient T cells. Although LFA-1 affinity is also reduced in talin1-deficient T cells, rescue of LFA-1 affinity alone is not sufficient to restore LFA-1 adhesive function. Together, our findings indicate that TCR-induced up-regulation of LFA-1-dependent adhesiveness and resulting T cell-APC conjugation require talin1.

The conserved C-terminal I/LWEQ module targets Talin1 to focal adhesions.

The cytoskeletal protein Talin1 is a critical link between integrins and the actin cytoskeleton, where it is required for the structural and signaling functions of integrin-containing adhesion complexes. However, the elements in Talin1 that are responsible for localizing it to adhesion complexes are not known. In this report we have used a series of constructs based on the modular structure of Talin1 to determine the structural elements that specify the subcellular localization of Talin1. We show that the conserved actin-binding I/LWEQ module at the C-terminus of Talin1 is necessary and sufficient for targeting to focal adhesion complexes. We also used truncation and site-directed mutagenesis to demonstrate that this novel targeting function correlates with, but is separable from, the actin-binding properties of the Talin1 I/LWEQ module. In addition, we have shown that focal adhesion targeting, unlike actin binding, is not conserved among I/LWEQ module proteins. Finally, we have demonstrated that the subcellular localization of the Talin1 I/LWEQ module is regulated by an intrasteric interaction with an upstream alpha-helix, suggesting that both the actin binding and adhesion-targeting elements are masked in full-length Talin1. Our results define a novel role for the I/LWEQ module as the primary adhesion-complex targeting determinant of Talin1 and suggest that pathways that can relieve inhibition of I/LWEQ module function will be important for regulating the structural and signaling properties of adhesion complexes.

TRPM7 regulates cell adhesion by controlling the calcium-dependent protease calpain.

m-Calpain is a protease implicated in the control of cell adhesion through focal adhesion disassembly. The mechanism by which the enzyme is spatially and temporally controlled is not well understood, particularly because the dependence of calpain on calcium exceeds the submicromolar concentrations normally observed in cells. Here we show that the channel kinase TRPM7 localizes to peripheral adhesion complexes with m-calpain, where it regulates cell adhesion by controlling the activity of the protease. Our research revealed that overexpression of TRPM7 in cells caused cell rounding with a concomitant loss of cell adhesion that is dependent upon the channel of the protein but not its kinase activities. Knockdown of m-calpain blocked TRPM7-induced cell rounding and cell detachment. Silencing of TRPM7 by RNA interference, however, strengthened cell adhesion and increased the number of peripheral adhesion complexes in the cells. Together, our results suggest that the ion channel TRPM7 regulates cell adhesion through m-calpain by mediating the local influx of calcium into peripheral adhesion complexes.

Development of type 1 diabetes in wild bank voles associated with islet autoantibodies and the novel ljungan virus.

Wild bank voles (Clethrionomys glareolus) may develop diabetes in laboratory captivity. The aim of this study was to test whether bank voles develop type 1 diabetes in association with Ljungan virus. Two groups of bank voles were analyzed for diabetes, pancreas histology, autoantibodies to glutamic acid decarboxylase (GAD65), IA-2, and insulin by standardized radioligand-binding assays as well as antibodies to in vitro transcribed and translated Ljungan virus antigens. Group A represented 101 trapped bank voles, which were screened for diabetes when euthanized within 24 hours of capture. Group B represented 67 bank voles, which were trapped and kept in the laboratory for 1 month before being euthanized. Group A bank voles did not have diabetes. Bank voles in group B (22/67; 33%) developed diabetes due to specific lysis of pancreatic islet beta cells. Compared to nondiabetic group B bank voles, diabetic animals had increased levels of GAD65 (P < .0001), IA-2 (P < .0001), and insulin (P = .03) autoantibodies. Affected islets stained positive for Ljungan virus, a novel picorna virus isolated from bank voles. Ljungan virus inoculation of nondiabetic wild bank voles induced beta-cell lysis. Compared to group A bank voles, Ljungan virus antibodies were increased in both nondiabetic (P < .0001) and diabetic (P = .0015) group B bank voles. Levels of Ljungan virus antibodies were also increased in young age at onset of newly diagnosed type 1 diabetes in children (P < .01). These findings support the hypothesis that the development of type 1 diabetes in captured wild bank voles is associated with Ljungan virus. It is speculated that bank voles may have a possible zoonotic role as a reservoir and vector for virus that may contribute to the incidence of type 1 diabetes in humans.

Expression of the vesicular inhibitory amino acid transporter in pancreatic islet cells: distribution of the transporter within rat islets.

gamma-Aminobutyric acid (GABA) is stored in microvesicles in pancreatic islet cells. Because GAD65 and GAD67, which catalyze the formation of GABA, are cytoplasmic, the existence of an islet vesicular GABA transporter has been postulated. Here, we test the hypothesis that the putative transporter is the vesicular inhibitory amino acid transporter (VIAAT), a neuronal transmembrane transporter of GABA and glycine. We sequenced the human VIAAT gene and determined that the human and rat proteins share over 98% sequence identity. In vitro expression of VIAAT and immunoblotting of brain and islet lysates revealed two forms of the protein: an approximately 52-kDa and an approximately 57-kDa form. By immunoblotting and immunohistochemistry, we detected VIAAT in rat but not human islets. Immunohistochemical staining showed that in rat islets, the distribution of VIAAT expression parallels that of GAD67, with increased expression in the mantle. GABA, too, was found to be present in islet non-beta-cells. We conclude that VIAAT is expressed in rat islets and is more abundant in the mantle and that expression in human islets is very low or nil. The rat islet mantle differs from rat and human beta-cells in that it contains only GAD67 and relatively increased levels of VIAAT. Cells that express only GAD67 may require higher levels of VIAAT expression.

Immune reactivity to GAD25 in type 1 diabetes mellitus.

While both isoforms of glutamic acid decarboxylase (GAD) function as important autoantigens in autoimmune diabetes mellitus-GAD65 in humans and GAD67 in the NOD mouse-GAD67 is not synthesized in human pancreatic islets and is thought not to be an autoantigen in human diabetes. We have recently shown, however, that human islets contain a GAD67 splice variant: GAD25. Given the evidence that GAD67 could be a key diabetogenic autoantigen in the NOD mouse and the high prevalence of GAD65 autoantibodies in human type 1 diabetes, it became important to ask whether there is also immune reactivity to GAD25 in type 1 diabetes-possibly implicating it in the pathogenesis of the disease-and whether GAD25 reactivity could, like GAD65 reactivity, function as a clinically useful marker for the disease. We also hypothesized that the presence of autoantibodies to the smaller splice variant could be a cause of the up to 30% prevalence of GAD67 autoreactivity associated with type 1 diabetes. We therefore analyzed GAD25 reactivity in 105 newly-diagnosed children with type 1 diabetes and 74 control subjects. While 14 (13%) of the diabetic subjects were positive for GAD67 autoantibodies, only 3 (3%) were positive for GAD25 reactivity, none of which were GAD67 antibody-positive. Analysis of reactivity to a GAD67 chimera was consistent with GAD67 binding activity being due to cross-reactive GAD65 antibodies. Immunostaining confirmed the presence of GAD25 in human islets, revealing GAD25-positive cells to be sparse. Our results indicate that autoreactivity to GAD25 is rare in newly diagnosed type 1 diabetes and does not underlie GAD67 reactivity.