Olga K. Afanasiev

Endogenous Wnt/β-Catenin Signaling Is Required for Cardiac Differentiation in Human Embryonic Stem Cells

Background Wnt/β-catenin signaling is an important regulator of differentiation and morphogenesis that can also control stem cell fates. Our group has developed an efficient protocol to generate cardiomyocytes from human embryonic stem (ES) cells via induction with activin A and BMP4. Methodology/Principal Findings We tested the hypothesis that Wnt/β-catenin signals control both early mesoderm induction and later cardiac differentiation in this system. Addition of exogenous Wnt3a at the time of induction enhanced cardiac differentiation, while early inhibition of endogenous Wnt/β-catenin signaling with Dkk1 inhibited cardiac differentiation, as indicated by quantitative RT-PCR analysis for β-myosin heavy chain (β-MHC), cardiac troponin T (cTnT), Nkx2.5, and flow cytometry analysis for sarcomeric myosin heavy chain (sMHC). Conversely, late antagonism of endogenously produced Wnts enhanced cardiogenesis, indicating a biphasic role for the pathway in human cardiac differentiation. Using quantitative RT-PCR, we show that canonical Wnt ligand expression is induced by activin A/BMP4 treatment, and the extent of early Wnt ligand expression can predict the subsequent efficiency of cardiogenesis. Measurement of Brachyury expression showed that addition of Wnt3a enhances mesoderm induction, whereas blockade of endogenously produced Wnts markedly inhibits mesoderm formation. Finally, we show that Wnt/β-catenin signaling is required for Smad1 activation by BMP4. Conclusions/Significance Our data indicate that induction of mesoderm and subsequent cardiac differentiation from human ES cells requires fine-tuned cross talk between activin A/BMP4 and Wnt/β-catenin pathways. Controlling these pathways permits efficient generation of cardiomyocytes for basic studies or cardiac repair applications.

Merkel polyomavirus-specific T cells fluctuate with merkel cell carcinoma burden and express therapeutically targetable PD-1 and Tim-3 exhaustion markers.

Purpose: The persistent expression of Merkel cell polyomavirus (MCPyV) oncoproteins in Merkel cell carcinoma (MCC) provides a unique opportunity to characterize immune evasion mechanisms in human cancer. We isolated MCPyV-specific T cells and determined their frequency and functional status. Experimental Design: Multiparameter flow cytometry panels and HLA/peptide tetramers were used to identify and characterize T cells from tumors (n = 7) and blood (n = 18) of patients with MCC and control subjects (n = 10). PD-1 ligand (PD-L1) and CD8 expression within tumors were determined using mRNA profiling (n = 35) and immunohistochemistry (n = 13). Results: MCPyV-specific CD8 T cells were detected directly ex vivo from the blood samples of 7 out of 11 (64%) patients with MCPyV-positive tumors. In contrast, 0 of 10 control subjects had detectable levels of these cells in their blood (P < 0.01). MCPyV-specific T cells in serial blood specimens increased with MCC disease progression and decreased with effective therapy. MCPyV-specific CD8 T cells and MCC-infiltrating lymphocytes expressed higher levels of therapeutically targetable PD-1 and Tim-3 inhibitory receptors compared with T cells specific to other human viruses (P < 0.01). PD-L1 was present in 9 of 13 (69%) MCCs and its expression was correlated with CD8-lymphocyte infiltration. Conclusions: MCC-targeting T cells expand with tumor burden and express high levels of immune checkpoint receptors PD-1 and Tim-3. Reversal of these inhibitory pathways is therefore a promising therapeutic approach for this virus-driven cancer. Clin Cancer Res; 19(19); 5351–60. ©2013 AACR.

Merkel Cell Polyomavirus-Specific CD8+ and CD4+ T-cell Responses Identified in Merkel Cell Carcinomas and Blood

Purpose: Merkel cell polyomavirus (MCPyV) is prevalent in the general population, integrates into most Merkel cell carcinomas (MCC), and encodes oncoproteins required for MCC tumor growth. We sought to characterize T-cell responses directed against viral proteins that drive this cancer as a step toward immunotherapy. Experimental Design: Intracellular cytokine cytometry, IFN-γ enzyme-linked immunospot (ELISPOT) assay, and a novel HLA-A*2402–restricted MCPyV tetramer were used to identify and characterize T-cell responses against MCPyV oncoproteins in tumors and blood of MCC patients and control subjects. Results: We isolated virus-reactive CD8 or CD4 T cells from MCPyV-positive MCC tumors (2 of 6) but not from virus-negative tumors (0 of 4). MCPyV-specific T-cell responses were also detected in the blood of MCC patients (14 of 27) and control subjects (5 of 13). These T cells recognized a broad range of peptides derived from capsid proteins (2 epitopes) and oncoproteins (24 epitopes). HLA-A*2402–restricted MCPyV oncoprotein processing and presentation by mammalian cells led to CD8-mediated cytotoxicity. Virus-specific CD8 T cells were markedly enriched among tumor infiltrating lymphocytes as compared with blood, implying intact T-cell trafficking into the tumor. Although tetramer-positive CD8 T cells were detected in the blood of 2 of 5 HLA-matched MCC patients, these cells failed to produce IFN-γ when challenged ex vivo with peptide. Conclusions: Our findings suggest that MCC tumors often develop despite the presence of T cells specific for MCPyV T-Ag oncoproteins. The identified epitopes may be candidates for peptide-specific vaccines and tumor- or virus-specific adoptive immunotherapies to overcome immune evasion mechanisms in MCC patients. Clin Cancer Res; 17(21); 6671–80. ©2011 AACR.

Immunobiology of Merkel Cell Carcinoma: Implications for Immunotherapy of a Polyomavirus-Associated Cancer

Merkel cell carcinoma (MCC) is an aggressive skin malignancy with a high mortality rate and an increasing incidence. The recent discovery of Merkel cell polyomavirus has revolutionized our understanding of MCC pathogenesis. Viral oncoproteins appear to play a critical role in tumor progression and are expressed in the majority of MCC tumors. Virus-specific humoral and cellular immune responses are detectable in MCC patients and are linked to the natural history of the disease. Despite persistent expression of immunogenic viral proteins, however, MCC tumors are able to evade the immune system. Understanding of the mechanisms of immune evasion employed by MCC tumors is rapidly increasing and offers opportunities for development of rational immune therapies to improve patient outcomes. Here we review recent discoveries in MCC with a special focus on the pathogenic role of Merkel cell polyomavirus and the immunobiology of this virus-associated disease.

Regression of metastatic Merkel cell carcinoma following transfer of polyomavirus-specific T cells and therapies capable of re-inducing HLA class-I

Chapuis, Afanasiev, and colleagues show that the combined regimen of local tumor-targeted preconditioning and systemic immune therapies elicited a durable complete response in two of three lesions with a prolonged period without development of additional distant metastasis. Merkel cell carcinoma (MCC) is an aggressive skin cancer that typically requires the persistent expression of Merkel cell polyomavirus (MCPyV) oncoproteins that can serve as ideal immunotherapeutic targets. Several immune evasion mechanisms are active in MCC, including downregulation of HLA class-I expression on tumor cells and dysfunctional endogenous MCPyV-specific CD8 T-cell responses. To overcome these obstacles, we combined local and systemic immune therapies in a 67-year-old man, who developed metastatic MCPyV-expressing MCC. Intralesional IFN-β-1b or targeted single-dose radiation was administered as a preconditioning strategy to reverse the downregulation of HLA-I expression noted in his tumors and to facilitate the subsequent recognition of tumor cells by T cells. This was followed by the adoptive transfer of ex vivo expanded polyclonal, polyomavirus-specific T cells as a source of reactive antitumor immunity. The combined regimen was well tolerated and led to persistent upregulation of HLA-I expression in the tumor and a durable complete response in two of three metastatic lesions. Relative to historical controls, the patient experienced a prolonged period without development of additional distant metastases (535 days compared with historic median of 200 days; 95% confidence interval, 154–260 days). The transferred CD8+ T cells preferentially accumulated in the tumor tissue, remained detectable and functional for more than 200 days, persisted with an effector phenotype, and exhibited evidence of recent in vivo activation and proliferation. The combination of local and systemic immune stimulatory therapies was well tolerated and may be a promising approach to overcome immune evasion in virus-driven cancers. Cancer Immunol Res; 2(1); 27–36. ©2013 AACR.

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

Paulson and colleagues report that 84% of Merkel cell carcinoma (MCC) tumors downregulated MHC-I expression, and MCC patients treated with intralesional IFNs had increased MHC-I expression on their tumor cells, thus promoting the use of immune-stimulating therapies for MCC. 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. Cancer Immunol Res; 2(11); 1071–9. ©2014 AACR.

Vascular E-Selectin Expression Correlates with CD8 Lymphocyte Infiltration and Improved Outcome in Merkel Cell Carcinoma

Merkel cell carcinoma (MCC) is an aggressive, polyomavirus-linked skin cancer. While CD8 lymphocyte infiltration into the tumor is strongly correlated with improved survival, these cells are absent or sparse in most MCCs. We investigated whether specific mechanisms of T-cell migration may be commonly disrupted in MCC tumors with poor CD8 lymphocyte infiltration. Intratumoral vascular E-selectin, critical for T-cell entry into skin, was downregulated in the majority (52%) of MCCs (n=56), and its loss was associated with poor intratumoral CD8 lymphocyte infiltration (p<0.05; n=45). Importantly, survival was improved in MCC patients whose tumors had higher vascular E-selectin expression (p<0.05). Local nitric oxide (NO) production is one mechanism of E-selectin downregulation and it can be tracked by quantifying nitrotyrosine, a stable biomarker of NO-induced reactive nitrogen species (RNS). Indeed, increasing levels of nitrotyrosine within MCC tumors were associated with low E-selectin expression (p<0.05; n=45) and decreased CD8 lymphocyte infiltration (p<0.05, n=45). These data suggest that one mechanism of immune evasion in MCC may be restriction of T cell entry into the tumor. Existing therapeutic agents that modulate E-selectin expression and/or RNS generation may restore T cell entry and could potentially synergize with other immune-stimulating therapies.

Abstract LB-430: Anti-polyomavirus CTL targeting Merkel cell carcinoma combined with MHC class I upregulation induces tumor regression

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Merkel cell carcinoma (MCC) represents a highly aggressive neuroendocrine skin malignancy with 46% 5 year disease-associated mortality making it 3x as lethal as melanoma. Merkel cell polyomavirus (MCPyV), a recently discovered viral agent related to SV40, is clonally integrated into ∼80% of MCC tumors. MCPyV produces T Antigen (T-Ag) oncoproteins that are persistently expressed, necessary for survival and proliferation of most MCCs, representing an optimal target for immunotherapy. An HLA A*2402-restricted T-Ag epitope recognized by autologous CD8+ T-cells was identified in a patient with MCC. The presence of a small endogenous population (0.1% of CD8+ T-cells) of MCC-specific CD8+ T-cells failed to prevent new metastases, and responses may have been impaired by MHC class I down-regulation observed in the patients tumor. As adoptively transferred antigen-specific T cells (CTL) have demonstrated clinical benefit in other tumor settings, we set out to examine the safety, in vivo persistence and anti-tumor effect of CTL targeting MCC. Autologous polyclonal antigen-specific CD8+ T cells targeting the HLA A*2402-restricted epitope of the MCPyV T-Ag oncoprotein were generated for this patient. Ex-vivo cultures were supplemented with the γc-chain cytokine IL-21, which has been demonstrated to promote less terminal differentiation of cultured cells as a means to increase CTL in vivo survival. To induce tumor MHC class I up-regulation with minimal confounding therapeutic effect, the patient received a single intra-lesional injection of 3.3 x 106 IU IFNβ 1B to 1 of 3 measurable metastatic lesions 24h prior to a first infusion of 1010/m2 of polyclonal multimer sorted MCPyV-specific CTL. Four weeks later, a single 8Gy dose of radiation to detectable disease was followed 24h later by a second T cell infusion. Both infusions were followed by low-dose IL-2 (500,000 IU/m2 s.c. x 14 days). The patient experienced no treatment-associated side effects. Four weeks after the first infusion, the metastatic lesion infused with IFNβ 1B regressed by 40% whereas the non-infused lesions increased by an average of 32%. Eight weeks after the second infusion, all lesions had regressed by an average of 62% with complete disappearance of the metastatic lesion previously infused with IFNβ 1B, and no development of new systemic lesions. Infused CTL peaked at 8% of total CD8+ T cells 7 days after infusions and persisted at ≥1% for at least 6 weeks. Infused CTL remained polyfunctional (secretion of IFNγ, TNFα and IL-2 in response to cognate antigen) over the observed period. Overall, this first-in-human trial suggests infusion of IL-21 derived polyclonal CTL targeting MCPyV is safe, and combined with up-regulation of tumor MHC class I, promotes in vivo tumor regression. Plans are underway to extend this investigational treatment to MCC patients with a broader array of MHC-restricted viral epitopes. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-430. doi:1538-7445.AM2012-LB-430

Track and Attack: Emerging Prognostic and Therapeutic Approaches

Merkel cell carcinoma (MCC) is an aggressive skin malignancy with an increasing incidence and high mortality rate. In the majority of MCC tumors, Merkel cell polyomavirus (MCPyV) oncoproteins appear to drive tumor progression and their ongoing expression is likely required. Thus, MCPyV is a promising tumor-associated target for disease tracking and immunotherapy. Indeed, virus-specific humoral and cellular immune responses are detectable in MCC patients, are linked to the natural history of the disease, and can be used for prognosis and early detection of disease recurrence. Learning how to therapeutically exploit the immune system to generate long-lasting and effective antitumor responses will be an ongoing challenge, but as our knowledge of the synergistic effects of therapies increases, the translational use of these strategies will be become more feasible for MCC patients.

Abstract IA17: Merkel cell carcinoma therapy with viral oncoprotein-specific T cells in combination with immunostimulatory adjuvants.

Persistent expression of Merkel cell polyomavirus (MCPyV) T antigen oncoproteins is required for the growth of most Merkel cell carcinomas (MCCs). MCPyV oncoproteins are thus attractive tumor antigens for evaluating the feasibility of antigen-specific adoptive T-cell therapy in MCC. Importantly, unlike mammalian tumor associated antigens that have some degree of expression within normal tissues, MCPyV oncoprotein expression is restricted to MCCs. We have recently identified several dozen MCC-associated viral peptides recognized by T cells. However, a major challenge for T cell therapy is that MCCs often downregulate HLA-I expression (51% of 114 tumors). We report that a single dose of either radiotherapy (2-8 Gy) or intralesional interferon reverses HLA-I downregulation in MCCs and can be used as an adjuvant in combination with virus-specific T cells for a multi-modality approach to target MCC. The feasibility and efficacy of this combined treatment has been explored in a 68 year-old man. He initially presented with a primary MCC on the hip that expressed low levels of HLA-I, high levels of MCPyV oncoprotein, and contained MCPyV-specific CD8 T cells. Based on rising antibody titers to MCPyV T-antigen, he was later found to have peri-pancreatic metastases. Blood-derived virus-specific T cells failed to respond to peptide challenge and expressed high levels of T cell exhaustion markers PD-1 and Tim-3. We used a novel approach to generate functional, polyclonal virus-specific therapeutic T cells. Lymphocytes from blood were cultured with peptide-loaded autologous dendritic cells, IL-2, IL-7, and IL-21, then sorted using an HLA-I/peptide tetramer and further expanded. 86% of these polyclonal CD8+ T cells were virus-specific and killed appropriate target cells. 24h after injecting interferon-beta into a pancreatic metastasis, the patient received 10^10 T cells/m2. Four weeks later, a single 8 Gy dose of radiation was given to the tumor area and was followed by a second T cell infusion 24h later. Treatment was well tolerated. After 2 treatments, all 3 metastatic lesions had markedly responded (>60% shrinkage by RECIST). No new distant disease has been detected at 456 days after the first metastasis (far beyond a median of 200 days for historical stage-matched controls). At >140 days after initiation of therapy, each of the following parameters remained markedly increased (relative to pre-treatment baseline) among virus-specific T cells in the blood: frequency, proliferation (Ki-67) and antigen-induced interferon-gamma production. TCR sequence analysis was used to track the prevalence and tumor infiltration of infused T cell clonotypes. After treatment, an increased fraction of virus-specific T cells was observed in the metastasis compared to a pre-treatment biopsy of the primary tumor. The use of polyclonal T cells may have been important because we observed that only a subset of the infused clones effectively infiltrated the sampled metastasis. This study represents a first-in-man treatment of a carcinoma using T cells targeting an oncogenic viral antigen and benefitted from the ability to accurately track adoptively transferred T cells specific for a non-self tumor antigen. Citation Format: Olga Afanasiev, Aude Chapuis, Jayasri Iyer, Kotaro Nagase, Kelly Paulson, Aaron Seo, Ivy Lai, Ilana Roberts, Erik Farrar, Joo Ha Hwang, Upendra Parvathaneni, David Koelle, Cassian Yee, Paul Nghiem. Merkel cell carcinoma therapy with viral oncoprotein-specific T cells in combination with immunostimulatory adjuvants. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology: Multidisciplinary Science Driving Basic and Clinical Advances; Dec 2-5, 2012; Miami, FL. Philadelphia (PA): AACR; Cancer Res 2013;73(1 Suppl):Abstract nr IA17.