Wen-Rong Lie

IL-12p70–producing patient DC vaccine elicits Tc1-polarized immunity

BACKGROUND Systemic administration of IL-12p70 has demonstrated clinical activity in cancer patients, but dose-limiting toxicities have hindered its incorporation in vaccine formulations. Here, we report on the immunological and clinical outcomes upon vaccination with CD40L/IFN-γ-matured, IL-12p70-producing DCs. METHODS 7 HLA-A*0201+ newly diagnosed stage IV melanoma patients were immunized against the gp100 melanoma antigen using autologous peptide-pulsed, CD40L/IFN-γ-matured DCs. PBMCs were taken weekly for immune monitoring by tetramer analysis and functional assays. CT imaging was performed at baseline, week 9, and week 18 for clinical assessment using RECIST. RESULTS 6 of 7 treated patients developed sustained T cell immunity to all 3 melanoma gp100 antigen-derived peptides. 3 of the 6 immunological responders developed confirmed clinical responses (1 complete remission >4 years, 2 partial response). Importantly, DC vaccine-derived IL-12p70 levels positively correlated with time to progression (P = 0.019, log-rank), as did T-cytotoxic 1 (Tc1) immunity, as assessed by IFN-γ/IL-13 and IFN-γ/IL-5 ratios (P = 0.035 and P = 0.030, respectively, log-rank). In contrast, a pathway-specific defect in IL-12p35 transcription was identified upon CD40L/IFN-γ activation in clinical nonresponder patient DCs, and gp100-specific T cells from these patients displayed a Tc2 phenotype. Incorporation of TLR3 and TLR8 agonists into the CD40L/IFN-γ activation protocol corrected the IL-12p70 production defect in DCs derived from clinical nonresponder patients. CONCLUSION These findings underscore the essential role of IL-12p70 in the development of therapeutic type 1 antigen-specific CD8+ T cell immunity in humans with cancer.

Amino-Terminal Extended Peptide Single-Chain Trimers Are Potent Synthetic Agonists for Memory Human CD8+ T Cells

Upon Ag exposure, most memory T cells undergo restimulation-induced cell death. In this article, we describe a novel synthetic agonist, an N-terminal extended decamer peptide expressed as a single-chain trimer, the amino-terminal extended peptide MHC class I single-chain trimer (AT-SCT), which preferentially promotes the growth of memory human CD8+ T cells with minimal restimulation-induced cell death. Using CMV pp65 and melanoma gp100 Ags, we observe the in vitro numerical expansion of a clonally diverse polyfunctional population of Ag-specific CD8+ T cells from healthy individuals and vaccinated melanoma patients, respectively. Memory CD8+ T cells stimulated with AT-SCT presented on MHC class I/II-null cells show reduced cytokine production, slower kinetics of TCR downregulation, and decreased cell death compared with native nonamer MHC class I single-chain trimer (SCT)-activated T cells. However, both ERK phosphorylation and cell cycle kinetics are identical in AT-SCT– and SCT-activated T cells. Probing of SCT and AT-SCT peptide–MHC complexes using fluorochrome-conjugated TCR multimers suggests that nonamer- and decamer-linked peptides may be anchored differently to the HLA-A2 peptide-binding groove. Our findings demonstrate that modified peptide–MHC structures, such as AT-SCT, can be engineered as T cell agonists to promote the growth and expansion of memory human CD8+ T cells.

Abstract 1730: Expression of serum biomarkers related to epithelial-to-mesenchymal transition in non-small cell lung cancer recurrence

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Objective: Recurrent disease in stage I non-small cell lung cancer (NSCLC) is primarily attributed to metastatic dissemination at the time of surgery undetected by current staging practices. We hypothesized that metastatic progression is driven by epithelial-to-mesenchymal transition (EMT) resulting in differences in tumor-shed protein biomarkers. The objective of this study was to evaluate the difference in expression of these biomarkers in patients with recurrent stage 1 NSCLC. Methods: We used the Luminex immunobead platform, including the MILLIPLEX map human angiogenesis/growth factor magnetic bead panel, to evaluate 80 biomarkers related to EMT against a total of 75 patients who underwent a complete anatomic resection. Patients were divided into the following cohorts: a) stage I NSCLC without recurrence in 2 years (n=50), and b) stage I NSCLC with recurrence within 2 years of follow up (n=25). Peripheral blood was collected and processed using standard phlebotomy techniques. Specimens were obtained in compliance with institutional IRB standards and consent. The Mann-Whitney test and receiver operator characteristics (ROC) curves were used to assess differences in biomarker concentrations between cohorts. Results: Univariate analysis revealed 19 biomarkers with significant (ROC >0.6) differences in expression between the patient cohorts including: angiopoietin 2, MCP-1, MIP-IB, TNF-R1, IGFBP-5, VEGF-D, IGF-1, IGFBP-3, follistatin, sICAM-1, sE-SELECTIN, CYFRA 21.1, RANTES, IL-Ira, M-CSF, IGFBP-1, IGFBP-6, HB-EGF, and PGF. The Mann-Whitney test revealed five biomarkers highly significant (p<0.05) for recurrence in stage 1 NSCLC, including MCP-1, VEGF-D, follistatin, sICAM-1, and placental growth factor (PGF). Evaluation of these biomarkers with the Ingenuity Pathway Analysis (IPA) suite identified several highly significant (p<1x10−5) biological themes, including ten IPA-defined processes associated with development (e.g. embryonic development and cardiovascular system development), seven processes associated with pathological processes (e.g. cancer, cell death, and respiratory disease), and seven processes associated with metastasis (e.g. cellular movement, immune cell trafficking, and cell-to-cell signaling and interaction). Random Forest analysis generated a 6-analyte panel consisting of MCP-1, IP-10, sICAM-1, IGFBP2, RANTES, and IGFBP3 that provided 71.1% classification accuracy with 66.1% sensitivity and 73.3% specificity. Conclusions: Here we report observations concerning the expression of EMT pathway members that may provide key insights into the role of circulating biomarkers related to recurrence in stage 1 NSCLC. Upon further validation, these biomarkers may serve as convenient surrogates to help guide molecular diagnostics and treatment strategies. 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 1730. doi:1538-7445.AM2012-1730

Abstract 2218: Quantitative multiplex analysis of immune checkpoint protein expression in circulation and in the tumor microenvironment

Immune checkpoint inhibitors have been proven to be an effective method in improving antitumor immune response. Many immune checkpoint proteins are expressed as soluble forms in circulation and in the tumor and tumor microenvironment. Here we report the development of bead-based Luminex multiplex assays for the quantitative profiling of co-inhibitory and co-stimulating immune checkpoint proteins CTLA-4, PD-1, TIM-3, LAG-3, HVEM. GITRL, BTLA, CD27, CD28, CD40, GITR, PD-L1, B7-1/CD80, B7-2/CD86, and ICOS. In order to explore the use of soluble immune checkpoint proteins as putative cancer biomarkers, we used these multiplex assays to measure checkpoint protein levels in serum samples from breast cancer patients, colon cancer patients, and a corresponding set of normal serum samples. Analysis of the soluble checkpoint protein signatures generated from this multiplex approach revealed a significantly elevated level of soluble TIM-3 protein in the breast cancer serum samples and in the colon cancer serum samples compared to the healthy serum controls (p Citation Format: Wen-Rong Lie, Jehangir Mistry. Quantitative multiplex analysis of immune checkpoint protein expression in circulation and in the tumor microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2218. doi:10.1158/1538-7445.AM2017-2218

Abstract 58: Metformin impacts IGF-1R/ Akt signaling and energy metabolism in lung adenocarcinoma

Introduction: Clinical outcomes for type II diabetes patients diagnosed with NSCLC are superior for those receiving metformin relative to other diabetic medications, though the mechanism for this effect remains incompletely elucidated. We hypothesized that metformin induces changes in Akt-based signaling via AMPK that increases glycolytic flux, and adversely affects survival in cell-line models of lung adenocarcinoma. The objective for these studies is to investigate therapeutic strategies involving metformin that have the potential to synergize with strategies targeting IGF-1R/Akt signaling. Methods: The lung adenocarcinoma cell lines, A549 and H358, were treated with 0, 1, 5, 10 mM metformin for 24, 48, and 72 hours and evaluated for changes in expression and activity of IGF-1R/Akt signaling, as well as the expression of 17 enzymes involved in glycolytic and oxidative energy metabolism, all using Luminex® immunobead assays. In parallel, MTT assays were performed for up to 5 days to document changes in cellular proliferation/ death rates. Extracellular acidification was also monitored as a measure of glycolytic flux. All findings were collated and statistically evaluated using SPSS v19 (Chicago, IL) using independent t-tests or one-way ANOVA. Results: We observed that metformin decreased IGF-1R/Akt signaling while increasing the expression of the protein levels of the signaling intermediates. Treatment with metformin increased the expression of Akt, IRS-1, PTEN, GSK3alpha and TSC2 (p Conclusion: This study links changes with Akt signaling with changes in glycolytic flux and oxidative metabolism. This study suggests metformin may make an attractive adjunct for advanced NSCLC patients receiving therapy that mechanistically involves targeting Akt signaling and metabolic control, such as IGF1R inhibition. Citation Format: Gabriela C. Lobato, Steve Li, Imad Tarhoni, Wen-Rong Lie, Jeffrey A. Borgia. Metformin impacts IGF-1R/ Akt signaling and energy metabolism in lung adenocarcinoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 58.

Abstract 205: Analysis of resistance to RTK inhibitors using a novel RTK multiplex assay

A novel multiplex immunoassay was developed for examining acquired resistance to Receptor Tyrosine Kinase (RTK) inhibitors. RTKs are transmembrane proteins which act as receptors for growth factors, neurotrophic factors and other extracellular signaling molecules. These cell-surface kinases are activated by extracellular ligands leading to receptor dimerization and tyrosine phosphorylation at specific residues in the cytoplasmic tails to initiate RTK-mediated signal transduction. Of the >500 known protein kinases in the human genome there are approximately 60 RTKs. They are central components of cell signaling networks and play crucial roles in normal physiological processes and disease processes ranging from diabetes to cancer. Many RTKs, such as EGFR, HER2, c-Kit, PDGFR and VEGFR, have been used as targets for drug development and RTK-targeted therapies have illustrated the utility of these treatments for selected cancers. However, in many cases, compensatory RTK signaling enables cancer cells to acquire resistance to RTK inhibitors that selectively target a single RTK. For example, some EGFR and HER2 inhibitors have led to resistance and are associated with increased expression of IGF1R. In order to understand the mechanisms of resistance to RTK inhibitors, we have developed a bead-based multiplex immunoassay capable of simultaneously detecting phosphorylation of 18 different RTKs. First we demonstrate detection of RTK phosphorylation in response to growth factor stimulation using various cell lines. Next we examined the specificity of two inhibitors targeting EGFR and HER2. These inhibitors specifically reduced growth factor-stimulated phosphorylation of EGFR and HER2, without inhibiting the activation of other RTKs. Although we did not observe compensatory activation of other RTKs in response to the two EGFR and HER2 inhibitors we tested, this study demonstrates the feasibility of using this novel 18-plex RTK panel for examining the mechanism of resistance to single RTK inhibitors. In summary, the RTK multiplex panel allowed for simultaneous detection of multiple tyrosine phosphorylated RTKs in a specific, sensitive, and reproducible manner. Citation Format: Lu Chen, Anthony J. Saporita, Wen-Rong Lie, Reeti Maheshwari, Melissa Schluter, Jehangir Mistry, Joseph Hwang. Analysis of resistance to RTK inhibitors using a novel RTK multiplex assay. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 205.

Abstract B01: Profiling of serum and plasma angiogenic biomarkers in breast cancer and colon cancer using Luminex xMAP technology-based human angiogenesis multiplex immunoassay panels

Tumors require neovascularization for growth. Consequently, the angiogenesis process is key to understanding tumor development and progression, as well as diagnosis and treatment. Measuring the levels of pro- and anti-angiogenic factors in plasma or serum in cancer patients may provide prognostic information concerning disease processes and mechanisms and the elucidation of potential biomarkers for epidemiological studies or clinical trials. The Luminex xMAP technology, a bead-based multiplex immunoassay for the simultaneous measurement of multiple analytes in a small sample size, has been widely used in cancer biomarker discoveries. We report here the use of two newly developed multiplex human angiogenesis panels to profile the circulating biomarkers commonly used in the angiogenesis pathway analysis. The two panels are 1) MILLIPLEX MAP Human Angiogenesis Panel 1, a 17-plex multiplex immunoassay for the simultaneous quantification of the following 17 angiogenesis biomarkers: Angiopoietin-2, BMP-9, EGF, Endoglin, Endothelin-1, FGF-1, FGF-2, Follistatin, G-CSF, HB-EGF, HGF, IL-8, Leptin, PLGF, VEGF-A, VEGF-C, and VEGF-D, and 2) MILLIPLX MAP Human Angiogenesis Panel 2, a 20-plex multiplex immunoassay for simultaneous quantitation of the following 20 human angiogenesis biomarkers: Angiostatin, soluble E-Selectin, Osteopontin, PDGF-AB/BB, PECAM-1/sCD31, Tenascin C, Thrombospondin-2 (TSP-2), soluble AXL, soluble c-Kit, soluble EGFR, soluble Her2/ErbB2, soluble Her3/ErbB3, soluble HGFR/c-Met, soluble IL-6Rα, soluble Neuropilin-1, soluble Tie-2, soluble urokinase receptor (suPAR), soluble VEGFR1, soluble VEGFR2, and soluble VEGFR3. In this study, we report the use of these 37 angiogenic, growth factor and soluble receptor biomarkers to profile serum samples and plasma samples from a panel of breast cancer and colon cancer patients, with a similar number of normal serum and plasma samples, as a corresponding set, to generate a tumor profile of angiogenic factors using the Luminex xMAP platform. The results of this study demonstrated the utility of these human angiogenesis panels in studying cancer development and cancer biomarker discovery, as well as its potential application in translational research. Citation Format: Wen-Rong Lie, Deborah Droll, Jehangir Mistry. Profiling of serum and plasma angiogenic biomarkers in breast cancer and colon cancer using Luminex xMAP technology-based human angiogenesis multiplex immunoassay panels. [abstract]. In: Proceedings of the AACR Special Conference: Tumor Angiogenesis and Vascular Normalization: Bench to Bedside to Biomarkers; Mar 5-8, 2015; Orlando, FL. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl):Abstract nr B01.

Abstract 3995: Quantitative protein profiling of tumor angiogenesis and metastasis biomarkers in mouse and human models

Tumor and stromal cells secrete a variety of proteins acting as extracellular signals and creating a supportive microenvironment for tumor development, angiogenesis, and metastasis. We used the MILLIPLEX MAP cytokine/chemokine, cancer, angiogenesis, metastasis, MMP, bone metabolism, adipocyte, and cell signaling multi-pathway panels, based on the Luminex xMAP technology, to quantitatively evaluate the protein biomarker expression profiles on cultured mouse and human tumor cells and stromal cells as well as in vivo mouse and human tumor tissues. In the mouse system, biomarker profiling was performed to characterize secreted protein levels in Lewis Lung carcinoma (LLC) cells, B16.10 melanoma cells, b.End3 endothelial cells, and LLC tumors. The in vivo characterization, using primary tumors from mice bearing metastatic LLC, has identified differential expression of a panel of biomarkers, including amphiregulin (AREG), EGF, PECAM-1, KC, MCP-1, MIP-2, MMP-12, and resistin, between the high-fat (45% fat) and AIN-93G (15% fat) fed mice (p Citation Format: Wen-Rong Lie, Jonathan Lipsey, Tim Warmke, Lin Yan, Jehangir Mistry. Quantitative protein profiling of tumor angiogenesis and metastasis biomarkers in mouse and human models. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3995. doi:10.1158/1538-7445.AM2014-3995

Abstract LB-157: IL-12p70 producing dendritic cell vaccine elicits Tc1 polarized T cells and extends time to progression in metastatic melanoma.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Systemic administration of IL-12p70 has demonstrated clinical activity in cancer patients but dose-limiting toxicities have hindered its incorporation in vaccine formulations. In this proof-of-concept phase I clinical trial ([NCT00683670][1]), we report on the immunological and clinical outcomes upon vaccination of newly diagnosed stage IV melanoma patients with CD40L/IFN-γ matured IL-12p70 producing dendritic cells (mDC). HLA-A*0201+ individuals with treatment naive metastatic melanoma were vaccinated with mDC pulsed with melanoma gp100-derived peptides, every 3 weeks by intravenous infusion for six doses, after a single dose of cyclophosphamide (300 mg/m2 iv). CT imaging was performed at baseline, week 9 and 18 for clinical assessment using RECIST. PBMC were taken weekly for immune monitoring by tetramer analysis and functional assays. Six of seven treated patients develop sustained T cell immunity to all three melanoma gp100 antigen-derived (G154, G209-2M and G280-9V) peptides, while one patient had transient immunity only to the G209-2M peptide. Three of the six immunological responders developed a radiographic response by RECIST criteria and all three individuals had a time to progression (TTP) >11.5 mo. A Cox regression analysis followed by likelihood-ratio test revealed a positive correlation (p=0.0198) between DC derived IL-12p70 and TTP. Moreover, among clinical responders, vaccine-induced gp100-specific T cells displayed a Tc1 phenotype. In contrast, a selective defect in IL-12p35 transcription was identified in clinical non-responder patient DC and gp100-specific T cells from these patients displayed the Tc2 phenotype. Incorporation of TLR3 and TLR8 agonists into the CD40L/IFN-γ maturation protocol corrected the IL-12p70 production defect in DC derived from clinical non-responder patients. These findings underscore the essential role of IL-12p70 in the development of type-1 immunity in humans with cancer and provide evidence-based rationale for incorporating IL-12p70 into the next generation of cancer vaccine formulations. Citation Format: Beatriz M. Carreno, Michelle Becker-Hapak, Alexander Huang, Megan Chan, Amer Alyasiry, Wen-Rong Lie, Rebecca L. Aft, Lynn A. Cornelius, Katherine M. Trinkaus, Gerald P. Linette. IL-12p70 producing dendritic cell vaccine elicits Tc1 polarized T cells and extends time to progression in metastatic melanoma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr LB-157. doi:10.1158/1538-7445.AM2013-LB-157 [1]: /lookup/external-ref?link_type=CLINTRIALGOV&access_num=NCT00683670&atom=%2Fcanres%2F73%2F8_Supplement%2FLB-157.atom

Abstract 409: In vitro and in vivo characterization of protein biomarkers of metastasis in melanoma.

Metastasis is a major cause of death in cancer patients. We report here the use of a multiplex proteomic analysis approach to in vitro and in vivo identify biomarkers involved in melanoma invasion/metastasis. An in vitro quantitative profile of the secretome (>200 proteins) was done on non-metastatic melanoma cell lines (A375, LH, M14, DM.6, Lox, and WUmel19) and their metastatic RhoC-expressing counterparts (A375-RhoC, LH-RhoC, M14-RhoC, DM.6-RhoC, Lox-RhoC, and WUmel19-RhoC). A heat map cluster analysis was performed to characterize protein expression levels on metastatic vs. the non-metastatic melanoma cells and identify secreted markers with significantly altered expression that may be involved in tumor invasion, angiogenesis, and metastasis. An in vivo characterization, using an animal model of human melanoma pulmonary metastasis, has identified differential expression of tissue and circulating protein biomarkers in tumor and sera, respectively. Moreover, use of species-specific assays has allowed us to dissect the contribution of stroma (mouse) and tumor (human). Increased expression of a panel of markers, including IL-8, PLGF, FGF-2, follistatin, MMP-1, MMP-9, TIMP-1, TIMP-2, GDF15, NSE, OPN, and MIF was detected in vivo in both tumor and sera, indicating their roles in inducing angiogenesis, tumor invasion and metastasis. Cell signaling pathway multiplex analyses revealed the activation of PI3K/mTOR pathway in the tumor. Elevated levels of phosphorylated targets of PI3K/mTOR pathway, p70S6K (pT412), GSK3α (pS21), GSK3β (pS9), RPS6 (pS235/236), TSC2 (pS939) and mTOR (pS2448) were detected in the pulmonary tumor metastasis. Hence this human melanoma pulmonary metastasis model may prove to be a useful animal model for studying the actions of PI3K/mTOR inhibitors in melanoma metastasis. Altogether, these in vitro and in vivo studies using multiplex proteomic analysis may assist in the discovery of biomarkers and therapeutic targets for human melanoma interventions. Citation Format: Wen-Rong Lie, Michelle Becker-Hapak, Megan Chan, James Graham, Jehangir Mistry, Gerald Linette, Beatriz Carreno. In vitro and in vivo characterization of protein biomarkers of metastasis in melanoma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 409. doi:10.1158/1538-7445.AM2013-409