A microRNA Prognostic Signature in Patients with Diffuse Intrinsic Pontine Gliomas through Non-Invasive Liquid Biopsy.

Diffuse midline gliomas (DMGs) originate in the thalamus, brainstem, cerebellum and spine. This entity includes tumors that infiltrate the pons, called diffuse intrinsic pontine gliomas (DIPGs), with a rapid onset and devastating neurological symptoms. Since surgical removal in DIPGs is not feasible, the purpose of this study was to profile circulating miRNA expression in DIPG patients in an effort to identify a non-invasive prognostic signature with clinical impact. Using a high-throughput platform, miRNA expression was profiled in serum samples collected at the time of MRI diagnosis and prior to radiation and/or systemic therapy from 47 patients enrolled in clinical studies, combining nimotuzumab and vinorelbine with concomitant radiation. With progression-free survival as the primary endpoint, a semi-supervised learning approach was used to identify a signature that was also tested taking overall survival as the clinical endpoint. A signature comprising 13 circulating miRNAs was identified in the training set (n = 23) as being able to stratify patients by risk of disease progression (log-rank p = 0.00014; HR = 7.99, 95% CI 2.38-26.87). When challenged in a separate validation set (n = 24), it confirmed its ability to predict progression (log-rank p = 0.00026; HR = 5.51, 95% CI 2.03-14.9). The value of our signature was also confirmed when overall survival was considered (log-rank p = 0.0021, HR = 4.12, 95% CI 1.57-10.8). We have identified and validated a prognostic marker based on the expression of 13 circulating miRNAs that can shed light on a patient's risk of progression. This is the first demonstration of the usefulness of nucleic acids circulating in the blood as powerful, easy-to-assay molecular markers of disease status in DIPG. This study provides Class II evidence that a signature based on 13 circulating miRNAs is associated with the risk of disease progression.

Early Effector T Lymphocytes Coexpress Multiple Inhibitory Receptors in Primary Non-Small Cell Lung Cancer.

Clinical efficacy of PD-1/PD-L1 targeting relies upon the reactivation of tumor-specific but functionally impaired PD-1+ T cells present before therapy. Thus, analyzing early-stage primary tumors may reveal the presence of T cells that are not yet functionally impaired. In this study, we report that activated (HLA-DR+) T cells with an effector memory (TEM) profile are enriched in such lesions. Tumor-infiltrating lymphocytes coexpressed PD-1 with the inhibitory receptors TIM-3, CTLA-4, LAG-3, and TIGIT, but also displayed a recently activated, nonexhausted phenotype. We also identified a subset of CD8+PD-1+FOXP3+ T lymphocytes at the earliest phase of functional differentiation after priming, termed "early effector cells" (EEC), which also exhibited an activated nonexhausted phenotype, but was less differentiated and associated with coexpression of multiple inhibitory receptors. In response to autologous tumor, EECs upregulated CD107a, produced IL2 and IFNγ, and were competent for differentiation. The identification of EECs marked by inhibitory receptor expression at tumor sites will enable investigations of early stages of adaptive antitumor immunity, as well as support the rationale for administering immunotherapy in early-stage non-small cell lung cancer. Cancer Res; 77(4); 851-61. ©2016 AACR.

NFATc2 is a potential therapeutic target in human melanoma.

The identification of intracellular signaling pathways that promote cell proliferation and resistance to cell death may lead to the development of improved treatment for advanced melanoma. Here we show that the calcineurin/nuclear factor of activated T cells c2 (NFATc2) pathway has an antiapoptotic role in melanoma cells. Expression of NFATc2 was constitutive in vitro and in vivo in human melanoma, and cyclosporin A (CsA) treatment of melanoma cells led to downmodulation of NFATc2. Inhibition of the calcineurin/NFAT pathway by CsA, or by NFATc2 silencing, led to modulation of cell cycle inhibitors and apoptosis-related proteins such as Apollon, and promoted caspase-dependent apoptosis of neoplastic cells. Calcineurin/NFATc2 targeting significantly enhanced melanoma cell death induced by antitumor agents, such as MEK- or BRAF(V600E)-specific inhibitors, and tumor necrosis factor-related apoptosis-inducing ligand, which trigger the intrinsic or extrinsic pathway of apoptosis, respectively. These findings identify NFATc2 as a potential therapeutic target in melanoma.

AMPK activators inhibit the proliferation of human melanomas bearing the activated MAPK pathway.

Raf/MEK/ERK signaling can inhibit the liver kinase B1-AMP-activated protein kinase (LKB1-AMPK) pathway, thus rendering melanoma cells resistant to energy stress conditions. We evaluated whether pharmacological reactivation of the AMPK function could exert antitumor effects on melanoma cells bearing this pathway constitutively active because of a mutation in NRAS or BRAF genes. Nine melanoma cell lines were treated with the AMPK activators 5-aminoimidazole-4-carboxamide-ribonucleoside (AICAR) and phenformin. The activation of AMPK enzymatic activity, phosphorylation of AMPK and acetyl-CoA carboxylase kinase, in-vitro proliferation, cell cycle, and in-vivo growth of xenografts in nude mice were evaluated. AICAR and phenformin promoted phosphorylation and enzymatic activity of AMPK, as well as phosphorylation of the AMPK downstream target acetyl-CoA carboxylase. Drug treatment of either BRAF-mutant or NRAS-mutant melanomas, at doses not inducing cell death, was accompanied by a dose-dependent decrease in melanoma cell proliferation because of cell cycle arrest in either the G0/G1 or the S phase, associated with an increased expression of the p21 cell cycle inhibitor. Melanomas isolated from subcutaneously implanted mice, 25 days from treatment with AICAR, showed increased staining of the senescence-associated marker ß-galactosidase, high p21 expression, and evidence of necrosis. Altogether, these results indicate that pharmacological activators of AMPK-dependent pathways inhibit the cell growth of melanoma cells with active Raf/MEK/ERK signaling and provide a rationale for further investigation on their use in combination therapies.

Role of Apollon in human melanoma resistance to antitumor agents that activate the intrinsic or the extrinsic apoptosis pathways.

PURPOSE: To assess the role of Apollon in melanoma resistance to intrinsic and extrinsic pathways of apoptosis and to identify strategies to reduce its expression. EXPERIMENTAL DESIGN: Apollon expression was assessed in melanoma cells in vitro and in vivo. Apollon modulation and melanoma apoptosis were evaluated by Western blot and/or flow cytometry in response to cytotoxic drugs, mitogen-activated protein/extracellular signal-regulated kinase (MEK)-, BRAF(V600E)-, and mTOR-specific inhibitors, TRAIL and anti-HLA class II monoclonal antibodies (mAb). Mitochondrial depolarization, caspase activation, apoptosis assays, and gene expression profiling were used to test effects of Apollon silencing, by siRNA, on melanoma response to antitumor agents. RESULTS: Apollon was constitutively expressed by melanoma cells, in vitro and in vivo, and at higher levels than in benign melanocytic lesions. Melanoma apoptosis correlated significantly with Apollon protein downmodulation in response to cytotoxic drugs, MEK, or BRAF(V600E)-specific inhibitors. Combinatorial treatment with MEK and mTOR inhibitors and HLA class II ligation, by a specific mAb, promoted Apollon downmodulation and enhanced melanoma apoptosis. Apollon downmodulation induced by antitumor agents was caspase independent, but proteasome dependent. Knockdown of Apollon, by siRNA, triggered apoptosis and/or significantly enhanced melanoma cell death in response to cytotoxic drugs, MEK- and BRAF(V600E)-specific inhibitors, and soluble or membrane-bound TRAIL. Apollon silencing promoted mitochondrial depolarization and caspase-2, caspase-8, caspase-9, and caspase-3 activation in response to different antitumor agents and altered the profile of genes modulated by MEK or BRAF(V600E)-specific inhibitors. CONCLUSIONS: Targeting of Apollon may significantly improve melanoma cell death in response to antitumor agents that trigger the intrinsic or the extrinsic apoptosis pathways.

Tumor-reactive CD8+ early effector T cells identified at tumor site in primary and metastatic melanoma.

CD8(+) T cells at the earliest stage of effector generation have not been identified at tumor site of melanoma patients. Such early effectors, if present, should be characterized by a specific phenotype, distinct from that expressed at later stages of the antigen-induced differentiation program, by short-lived effector cells, memory precursors, and terminal effectors. Here, we show that neoplastic tissues from primary and metastatic lesions of melanoma patients contain a subset of CD8(+) T cells expressing FOXP3. CD8(+) FOXP3(+) CD25(+) T lymphocytes were found in tumor-invaded lymph nodes (TILN), s.c. metastases, and advanced primary lesions. Their frequency was significantly higher in TILN compared with tumor-free lymph nodes or with peripheral blood and in primary tumors compared with TILN. CD8(+) FOXP3(+) T cells did not express markers of regulatory [CTLA-4, CCL4, interleukin-10 (IL-10), transforming growth factor-ß1], exhausted (PD-1), or senescent (CD57) CD8(+) T lymphocytes. Instead, this subset showed an antigen-experienced "EM1" phenotype (CCR7(-) CD45RA(-) CD28(+) CD27(+)) and exhibited a CD127(-), KLRG1(-), HLA-DR(+), CD38(+), T-bet(+), perforin(+) "early effector" profile predicted by current models. CD8(+) FOXP3(+) T cells produced IFN-γ on short in vitro activation, recognized autologous tumor by CD107a mobilization, and expressed Ki-67 on ex vivo analysis. In response to autologous tumor plus IL-2/IL-15, the CD8(+) FOXP3(+) T cells proliferated promptly and showed competence for differentiation (downregulation of CD27 and upregulation of T-bet). These results suggest development of early phases of antitumor immunity even in advanced melanoma. Moreover, the CD8(+) FOXP3(+) "early effector" subset may be an invaluable tool for monitoring immunity at tumor site.

Impaired STAT phosphorylation in T cells from melanoma patients in response to IL-2: association with clinical stage.

PURPOSE: To assess the extent of signal transducer and activator of transcription (STAT) activation in response to interleukin 2 (IL-2) in melanoma patients' T cells, along with clinical stage of tumor progression. EXPERIMENTAL DESIGN: T lymphocytes from peripheral blood of healthy donors and of American Joint Committee on Cancer stage I to IV melanoma patients, as well as from metastatic lymph nodes of patients, were evaluated for responsiveness to IL-2. CFSE assays and single-cell phospho-STAT-specific flow cytometry screening were used. Results. T cells from advanced melanoma patients, in comparison with healthy donors, showed reduced proliferation to IL-2 and IL-15, but not to anti-CD3 monoclonal antibody. Impaired response occurred in CCR7(+) and CCR7(-) T-cell subsets, but not in CD3(-) CD8(+) natural killer (NK) cells, and was not explained by induction of apoptosis, increased cytokine consumption, or altered IL-2R subunit expression in patients' T lymphocytes. By phospho-specific flow cytometry, defective STAT1 and STAT5 activation in response to IL-2 was found mainly in T lymphocytes from peripheral blood and/or tumor site of American Joint Committee on Cancer stage III and IV patients, compared with stage I and II patients and to donors, and in melanoma antigen-specific T cells isolated from metastatic lymph nodes. At tumor site, impaired STAT activation in T cells did not correlate with frequency of CD4(+) CD25(+) Foxp3(+) T cells. Serum from advanced melanoma patients inhibited IL-2-dependent STAT activation in donors' T cells and a neutralizing monoclonal antibody to transforming growth factor beta1 counteracted such inhibition. CONCLUSIONS: These results provide evidence for development of impaired STAT signaling in response to IL-2, along with clinical evolution of the disease, in melanoma patients' T cells.

Peptides with dual binding specificity for HLA-A2 and HLA-E are encoded by alternatively spliced isoforms of the antioxidant enzyme peroxiredoxin 5.

Peptides with dual binding specificity for classical HLA class I and non-classical HLA-E molecules have been identified in virus-encoded proteins, but not in cellular proteins from normal or neoplastic cells. Expression screening of a melanoma cDNA library with a CTL clone recognizing an HLA-A2-restricted tumor-specific epitope encoded by mutant peroxiredoxin 5 (Prdx5), a stress-inducible peroxidase, led to the identification of two alternatively spliced isoforms of the same gene. These isoforms, which lack the catalytic cysteine fundamental for enzymatic activity, showed widespread expression in neoplastic and normal tissues but were unstable at the protein level, being detectable, following transient transfection, only after lactacystin treatment to inhibit proteasomal degradation. Isoform-specific sequences which formed, respectively, as result of exon 1 splicing to either exon 3 or 4, encoded two distinct nonapeptides (AMAPIKTHL and AMAPIKVRL, not present in the full-length protein) with anchor residues for HLA-A2 and HLA-E molecules and able to stabilize HLA-A2 and HLA-E cell surface expression. HLA-E+ targets, loaded with these peptides, were not recognized by NK cells expressing CD94/NKG2A inhibitory or CD94/NKG2C activatory receptors. However, both peptides were recognized, although with low avidity, by HLA-E-restricted CD8+ CTL. The nonapeptide AMAPIKVRL was used to elicit HLA-A2-restricted CTL clones that killed peptide-pulsed lymphoblastoid cell lines and melanoma cells expressing the corresponding Prdx5 isoform. Our results suggest that alternatively spliced isoforms of Prdx5, through the generation of HLA-E- and HLA-A2-restricted peptides may be part of immune-mediated stress response contributing to the detection and elimination of damaged normal or neoplastic cells.

The effect of artificial antigen-presenting cells with preclustered anti-CD28/-CD3/-LFA-1 monoclonal antibodies on the induction of ex vivo expansion of functional human antitumor T cells.

BACKGROUND: Adoptive cell therapy with ex vivo expanded autologous antitumor cytotoxic T lymphocytes represents an important therapeutic option as an anticancer strategy. In order to identify a reliable method for producing adequate amounts of functional antitumor cytotoxic T lymphocytes with a potentially long in vivo lifespan, we tested the T-cell expansion efficiency of a new artificial antigen-presenting cell-based system. DESIGN AND METHODS: Our artificial antigen-presenting cells were generated with activating (anti-CD3), co-stimulating (anti-CD28) and adhesion (anti-LFA-1) biotinylated monoclonal antibodies preclustered in microdomains held on a liposome scaffold by neutravidin rafts. The co-localization of T-cell ligands in microdomains and the targeting of an adhesion protein, increasing the efficiency of immunological synapse formation, represent the novelties of our system. The activity of our artificial antigen-presenting cells was compared with that of anti-CD3/-CD28 coated immunomagnetic microbeads and immobilized anti-CD3 monoclonal antibody (OKT3 clone), the only two commercially available artificial systems. RESULTS: Our artificial antigen-presenting cells expanded both polyclonal T cells and MART-1-specific CD8(+) T cells in a more efficient manner than the other systems. Stimulation with artificial antigen-presenting cells allows for the generation of viable T cells displaying an immunophenotype consistent with in vivo potential for persistence, without increasing the frequency of regulatory T cells. The starting specificity of anti MART-1 CD8(+) T cells was preserved after stimulation with artificial antigen-presenting cells and it was statistically greater when compared to the activity of the same cells expanded with the other systems. Finally, our artificial antigen-presenting cells proved to be suitable for large-scale application, minimizing the volume and the costs of T-cell expansion. CONCLUSIONS: Our artificial antigen-presenting cells might represent an efficient tool to rapidly obtain a sufficient number of functional T cells for adoptive immunotherapy in patients with cancer.

Coexpression of NRASQ61R and BRAFV600E in human melanoma cells activates senescence and increases susceptibility to cell-mediated cytotoxicity.

Activating mutations in BRAF and NRAS oncogenes in human melanomas are mutually exclusive. This finding has suggested an epistatic relationship but is consistent even with synthetic lethality. To evaluate the latter possibility, a mutated NRAS(Q61R) oncogene was expressed, under a constitutive or a doxycycline-regulated promoter, in a metastatic melanoma clone (clone 21) harboring an activated BRAF(V600E) oncogene. After the first 10 to 12 in vitro passages, the constitutive NRAS(Q61R) transfectant displayed progressive accumulation in G(0)-G(1) phase of the cell cycle and stained for the senescence-associated beta-galactosidase activity (SA-beta-Gal). Inducible expression of NRAS(Q61R), by the Tet-Off system, in clone 21 cells (21NRAS(61ON)) led to overactivation of the RAS/RAF/mitogen-activated protein kinase signaling pathway and, after the 10th in vitro passage, led to promotion of senescence. This was documented by reduced proliferation, flattened cell morphology, reduced growth in Matrigel, positive staining for SA-beta-Gal, and expression of AMP-activated protein kinase and of the cell cycle inhibitor p21(waf1/Cip1). These effects were detected neither in 21 cells with silenced NRAS(Q61R) (21NRAS(61OFF)) nor in cells transfected with an inducible wild-type NRAS gene (21NRAS(WTON)). In addition, when compared with parental 21 cells, or with 21NRAS(61OFF), 21NRAS(61ON) and constitutive NRAS(Q61R) transfectants cells showed increased susceptibility to cytotoxicity by both HLA class I antigen-restricted and nonspecific T cells and up-regulation of several MHC class I antigen processing machinery components. These results suggest a relationship of synthetic lethality between NRAS and BRAF oncogenes, leading to selection against "double-mutant" cells.

Association of antigen-processing machinery and HLA antigen phenotype of melanoma cells with survival in American Joint Committee on Cancer stage III and IV melanoma patients.

Because changes in the expression level of antigen-processing machinery (APM) components and HLA class I and II antigens in melanoma cells are expected to affect their interactions with the immune system of the host, we assessed the clinical relevance of quantitative variations in the expression of these molecules in melanoma lesions. Short-term (<10 in vitro passages) melanoma cell lines isolated from 85 American Joint Committee on Cancer (AJCC) stage III and IV patients were stained with APM component and HLA class I antigen-specific and HLA class II antigen-specific monoclonal antibodies and analyzed by flow cytometry. The phenotype of all tumors was characterized by intertumor and intratumor heterogeneity in the expression of all the markers and by significant correlations in the level of expression of markers belonging to the HLA class I antigen-processing and presentation pathway. Hierarchical clustering of the mean fluorescence intensity data defined two main clusters of tumors. The corresponding groups of patients differed significantly in the overall survival but not in other relevant clinical variables, including AJCC stage and therapy received after surgery. Cox regression analysis showed that beta2-microglobulin and HLA class II antigen expression were significantly associated with patients' survival. This evidence was corroborated by the immunohistochemical analysis for HLA class II antigen expression of melanoma lesions from an unrelated group of 52 AJCC stage III and IV patients. These results suggest that quantitative variations in APM component and HLA expression in melanoma lesions from AJCC stage III and IV patients may have an effect on the clinical course of the disease.

Apoptosis protease activator protein-1 expression is dispensable for response of human melanoma cells to distinct proapoptotic agents.

Loss of expression of the apoptosis protease activator protein-1 (APAF-1) in human melanoma is thought to promote resistance to programmed cell death by preventing caspase-9 activation. However, the role of the APAF-1-dependent pathway in apoptosis activated by cellular stress and/or DNA damage has been recently questioned. We investigated APAF-1 expression in a large panel of human melanomas and assessed cellular response to several proapoptotic agents in tumors expressing or lacking APAF-1 protein. In two melanomas with wild-type p53 but with differential expression of APAF-1, treatment with camptothecin, celecoxib, or an nitric oxide synthase inhibitor (1400W) significantly modulated expression of 36 of 96 genes in an apoptosis-specific cDNA macroarray, but APAF-1 mRNA levels were not induced (in APAF-1(-) cells) nor up-regulated (in APAF-1(+) cells), a finding confirmed at the protein level. Treatment with cisplatin, camptothecin, etoposide, betulinic acid, celecoxib, 1400W, and staurosporine promoted enzymatic activity not only of caspases -2, -8, and -3 but also of caspase-9 in both APAF-1(+) and APAF-1(-) tumor cells. Moreover, drug-induced caspase-9 enzymatic activity could be not only partially but significantly reduced by caspase-2, -3, and -8 -specific inhibitors in both APAF-1(+) and APAF-1(-) tumor cells. In response to 1 to 100 micromol/L of cisplatin, camptothecin, or celecoxib, APAF-1(+) melanomas (n = 12) did not show significantly increased levels of apoptosis compared with APAF-1(-) tumors (n = 7), with the exception of enhanced apoptosis in response to a very high dose (100 micromol/L) of etoposide. These results suggest that the response of human melanoma cells to different proapoptotic agents may be independent of their APAF-1 phenotype.

The paradox of T-cell-mediated antitumor immunity in spite of poor clinical outcome in human melanoma.

Human melanoma is hardly ever curable at an advanced stage, but overwhelming evidence from untreated or vaccinated patients indicates that this tumor is highly antigenic and frequently immunogenic. Here, we review recent results indicating that CD8(+) T cell-mediated antitumor immunity is activated at the systemic and tumor level in the early clinical stages (AJCC stages I and II) and continues to be promoted, in a fraction of patients, even in metastatic disease (stages III and IV). This evidence was obtained by looking at frequency, differentiation phenotype, and function of antitumor T cells in periphery and tumor site of melanoma patients. On the other hand, the paradox of immunity in spite of poor clinical evolution of the disease, points toward a model of concurrent evolution of immunity and tumor escape. As melanoma progresses to metastatic disease, powerful mechanisms of tumor evasion from immune recognition, and of immunosuppression, are activated, thus tilting the balance between immunity and escape in favor of tumor resistance to host defense. Nevertheless, recent developments in our understanding of regulation of T cell-mediated immunity can provide clues to the prospects for improved immunotherapy approaches. By integrating the information from basic research in immunology, from murine tumor models, and from trials of immunotherapy, we discuss how the most relevant steps of the antitumor response should be manipulated with greater efficacy by future clinical trials.