Ena Wang

Mitochondrial Membrane Potential Identifies Cells with Enhanced Stemness for Cellular Therapy.

Long-term survival and antitumor immunity of adoptively transferred CD8(+) T cells is dependent on their metabolic fitness, but approaches to isolate therapeutic T cells based on metabolic features are not well established. Here we utilized a lipophilic cationic dye tetramethylrhodamine methyl ester (TMRM) to identify and isolate metabolically robust T cells based on their mitochondrial membrane potential (ΔΨm). Comprehensive metabolomic and gene expression profiling demonstrated global features of improved metabolic fitness in low-ΔΨm-sorted CD8(+) T cells. Transfer of these low-ΔΨm T cells was associated with superior long-term in vivo persistence and an enhanced capacity to eradicate established tumors compared with high-ΔΨm cells. Use of ΔΨm-based sorting to enrich for cells with superior metabolic features was observed in CD8(+), CD4(+) T cell subsets, and long-term hematopoietic stem cells. This metabolism-based approach to cell selection may be broadly applicable to therapies involving the transfer of HSC or lymphocytes for the treatment of viral-associated illnesses and cancer.

Ultra-low Dose Interleukin-2 Promotes Immune-modulating Function of Regulatory T Cells and Natural Killer Cells in Healthy Volunteers

Low-dose interleukin-2 (IL-2) expands regulatory T cells (Tregs) and natural killer (NK) cells after stem cell transplantation (SCT) and may reduce graft-versus-host disease (GVHD). We hypothesized that ultra-low dose (ULD) IL-2 could serve as an immune-modulating agent for stem cell donors to prevent GVHD following SCT. However, the safety, dose level, and immune signatures of ULD IL-2 in immune-competent healthy subjects remain unknown. Here, we have characterized the phenotype and function of Tregs and NK cells as well as the gene expression and cytokine profiles of 21 healthy volunteers receiving 50,000 to 200,000 units/m(2)/day IL-2 for 5 days. ULD IL-2 was well tolerated and induced a significant increase in the frequency of Tregs with increased suppressive function. There was a marked expansion of CD56(bright) NK cells with enhanced interferon-γ (IFN-γ) production. Serum cytokine profiling demonstrated increase of IFN-γ induced protein 10 (IP-10). Gene expression analysis revealed significant changes in a highly restricted set of genes, including FOXP3, IL-2RA, and CISH. This is the first study to evaluate global immune-modulating function of ULD IL-2 in healthy subjects and to support the future studies administrating ULD IL-2 to stem cell donors.

Cish actively silences TCR signaling in CD8+ T cells to maintain tumor tolerance

Palmer et al. find that Cish, a member of the SOCS family, is induced by TCR stimulation in CD8+ T cells and inhibits their functional avidity against tumor. The authors uncover a novel mechanism of suppression for a SOCS member.

The immune‐related role of BRAF in melanoma

The existence of a dichotomy between immunologically active and quiescent tumor phenotypes has been recently recognized in several types of cancer. The activation of a Th1 type of immune signature has been shown to confer better prognosis and likelihood to respond to immunotherapy. However, whether such dichotomy depends on the genetic make‐up of individual cancers is not known yet. BRAF and NRAS mutations are commonly acquired during melanoma progression. Here we explored the role of BRAF and NRAS mutations in influencing the immune phenotype based on a classification previously identified by our group.

Type I cytokines synergize with oncogene inhibition to induce tumor growth arrest

Acquavella, Clever, and colleagues show that IFNγ and TNFα synergize with vemurafenib to induce tumor growth arrest, supporting further study of the intersection between immunologic and oncogenic signaling in cancer cells and of treatment strategies combining vemurafenib and T-cell–based immunotherapy. Both targeted inhibition of oncogenic driver mutations and immune-based therapies show efficacy in treatment of patients with metastatic cancer, but responses can be either short lived or incompletely effective. Oncogene inhibition can augment the efficacy of immune-based therapy, but mechanisms by which these two interventions might cooperate are incompletely resolved. Using a novel transplantable BRAFV600E-mutant murine melanoma model (SB-3123), we explored potential mechanisms of synergy between the selective BRAFV600E inhibitor vemurafenib and adoptive cell transfer (ACT)–based immunotherapy. We found that vemurafenib cooperated with ACT to delay melanoma progression without significantly affecting tumor infiltration or effector function of endogenous or adoptively transferred CD8+ T cells, as previously observed. Instead, we found that the T-cell cytokines IFNγ and TNFα synergized with vemurafenib to induce cell-cycle arrest of tumor cells in vitro. This combinatorial effect was recapitulated in human melanoma–derived cell lines and was restricted to cancers bearing a BRAFV600E mutation. Molecular profiling of treated SB-3123 indicated that the provision of vemurafenib promoted the sensitization of SB-3123 to the antiproliferative effects of T-cell effector cytokines. The unexpected finding that immune cytokines synergize with oncogene inhibitors to induce growth arrest has major implications for understanding cancer biology at the intersection of oncogenic and immune signaling and provides a basis for design of combinatorial therapeutic approaches for patients with metastatic cancer. Cancer Immunol Res; 3(1); 37–47. ©2014 AACR. See related commentary by Riddell, p. 23

Molecular magnetic resonance imaging in cancer

The ability to identify key biomolecules and molecular changes associated with cancer malignancy and the capacity to monitor the therapeutic outcome against these targets is critically important for cancer treatment. Recent developments in molecular imaging based on magnetic resonance (MR) techniques have provided researchers and clinicians with new tools to improve most facets of cancer care. Molecular imaging is broadly described as imaging techniques used to detect molecular signature at the cellular and gene expression levels. This article reviews both established and emerging molecular MR techniques in oncology and discusses the potential of these techniques in improving the clinical cancer care. It also discusses how molecular MR, in conjunction with other structural and functional MR imaging techniques, paves the way for developing tailored treatment strategies to enhance cancer care.

The Ovarian Cancer Chemokine Landscape Is Conducive to Homing of Vaccine-Primed and CD3/CD28–Costimulated T Cells Prepared for Adoptive Therapy

Purpose: Chemokines are implicated in T-cell trafficking. We mapped the chemokine landscape in advanced stage ovarian cancer and characterized the expression of cognate receptors in autologous dendritic cell (DC)–vaccine primed T cells in the context of cell-based immunotherapy. Experimental Design: The expression of all known human chemokines in patients with primary ovarian cancer was analyzed on two independent microarray datasets and validated on tissue microarray. Peripheral blood T cells from five HLA-A2 patients with recurrent ovarian cancer, who previously received autologous tumor DC vaccine, underwent CD3/CD28 costimulation and expansion ex vivo. Tumor-specific T cells were identified by HER2/neu pentamer staining and were evaluated for the expression and functionality of chemokine receptors important for homing to ovarian cancer. Results: The chemokine landscape of ovarian cancer is heterogeneous with high expression of known lymphocyte-recruiting chemokines (CCL2, CCL4, and CCL5) in tumors with intraepithelial T cells, whereas CXCL10, CXCL12, and CXCL16 are expressed quasi-universally, including in tumors lacking tumor-infiltrating T cells. DC-vaccine primed T cells were found to express the cognate receptors for the above chemokines. Ex vivo CD3/CD28 costimulation and expansion of vaccine-primed Tcells upregulated CXCR3 and CXCR4, and enhanced their migration toward universally expressed chemokines in ovarian cancer. Conclusions: DC-primed tumor-specific T cells are armed with the appropriate receptors to migrate toward universal ovarian cancer chemokines, and these receptors are further upregulated by ex vivo CD3/CD28 costimulation, which render T cells more fit for migrating toward these chemokines. Clin Cancer Res; 21(12); 2840–50. ©2015 AACR.

T1rho MRI and CSF biomarkers in diagnosis of Alzheimer's disease

In the current study, we have evaluated the performance of magnetic resonance (MR) T1rho (T1ρ) imaging and CSF biomarkers (T-tau, P-tau and Aβ-42) in characterization of Alzheimers disease (AD) patients from mild cognitive impairment (MCI) and control subjects. With informed consent, AD (n = 27), MCI (n = 17) and control (n = 17) subjects underwent a standardized clinical assessment and brain MRI on a 1.5-T clinical-scanner. T1ρ images were obtained at four different spin-lock pulse duration (10, 20, 30 and 40 ms). T1ρ maps were generated by pixel-wise fitting of signal intensity as a function of the spin-lock pulse duration. T1ρ values from gray matter (GM) and white matter (WM) of medial temporal lobe were calculated. The binary logistic regression using T1ρ and CSF biomarkers as variables was performed to classify each group. T1ρ was able to predict 77.3% controls and 40.0% MCI while CSF biomarkers predicted 81.8% controls and 46.7% MCI. T1ρ and CSF biomarkers in combination predicted 86.4% controls and 66.7% MCI. When comparing controls with AD, T1ρ predicted 68.2% controls and 73.9% AD, while CSF biomarkers predicted 77.3% controls and 78.3% for AD. Combination of T1ρ and CSF biomarkers improved the prediction rate to 81.8% for controls and 82.6% for AD. Similarly, on comparing MCI with AD, T1ρ predicted 35.3% MCI and 81.9% AD, whereas CSF biomarkers predicted 53.3% MCI and 83.0% AD. Collectively CSF biomarkers and T1ρ were able to predict 59.3% MCI and 84.6% AD. On receiver operating characteristic analysis T1ρ showed higher sensitivity while CSF biomarkers showed greater specificity in delineating MCI and AD from controls. No significant correlation between T1ρ and CSF biomarkers, between T1ρ and age, and between CSF biomarkers and age was observed. The combined use of T1ρ and CSF biomarkers have promise to improve the early and specific diagnosis of AD. Furthermore, disease progression form MCI to AD might be easily tracked using these two parameters in combination.

Human melanomas and ovarian cancers overexpressing mechanical barrier molecule genes lack immune signatures and have increased patient mortality risk

ABSTRACT We have identified eight genes whose expression in human melanoma metastases and ovarian cancers is associated with a lack of Th1 immune signatures. They encode molecules with mechanical barrier function in the skin and other normal tissues and include filaggrin (FLG), tumor-associated calcium signal transducer 2 (TACSTD2), and six desmosomal proteins (DST, DSC3, DSP, PPL, PKP3, and JUP). This association has been validated in an independent series of 114 melanoma metastases. In these, DST expression alone is sufficient to identify melanomas without immune signatures, while FLG and the other six putative barrier molecules are overexpressed in a different subset of melanomas lacking immune signatures. Similar associations have been identified in a set of 186 ovarian cancers. RNA-seq data from 471 melanomas and 307 ovarian cancers in the TCGA database further support these findings and also reveal that overexpression of barrier molecules is strongly associated with early patient mortality for melanoma (p = 0.0002) and for ovarian cancer (p < 0.01). Interestingly, this association persists for FLG for melanoma (p = 0.012) and ovarian cancer (p = 0.006), whereas DST overexpression is negatively associated with CD8+ gene expression, but not with patient survival. Thus, overexpression of FLG or DST identifies two distinct patient populations with low immune cell infiltration in these cancers, but with different prognostic implications for each. These data raise the possibility that molecules with mechanical barrier function in skin and other tissues may be used by cancer cells to protect them from immune cell infiltration and immune-mediated destruction.

Future perspectives in melanoma research

The sixth “Melanoma Bridge Meeting” took place in Naples, Italy, December 1st–4th, 2015. The four sessions at this meeting were focused on: (1) molecular and immune advances; (2) combination therapies; (3) news in immunotherapy; and 4) tumor microenvironment and biomarkers. Recent advances in tumor biology and immunology has led to the development of new targeted and immunotherapeutic agents that prolong progression-free survival (PFS) and overall survival (OS) of cancer patients. Immunotherapies in particular have emerged as highly successful approaches to treat patients with cancer including melanoma, non-small cell lung cancer (NSCLC), renal cell carcinoma (RCC), bladder cancer, and Hodgkin’s disease. Specifically, many clinical successes have been using checkpoint receptor blockade, including T cell inhibitory receptors such as cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4) and the programmed cell death-1 (PD-1) and its ligand PD-L1. Despite demonstrated successes, responses to immunotherapy interventions occur only in a minority of patients. Attempts are being made to improve responses to immunotherapy by developing biomarkers. Optimizing biomarkers for immunotherapy could help properly select patients for treatment and help to monitor response, progression and resistance that are critical challenges for the immuno-oncology (IO) field. Importantly, biomarkers could help to design rational combination therapies. In addition, biomarkers may help to define mechanism of action of different agents, dose selection and to sequence drug combinations. However, biomarkers and assays development to guide cancer immunotherapy is highly challenging for several reasons: (i) multiplicity of immunotherapy agents with different mechanisms of action including immunotherapies that target activating and inhibitory T cell receptors (e.g., CTLA-4, PD-1, etc.); adoptive T cell therapies that include tissue infiltrating lymphocytes (TILs), chimeric antigen receptors (CARs), and T cell receptor (TCR) modified T cells; (ii) tumor heterogeneity including changes in antigenic profiles over time and location in individual patient; and (iii) a variety of immune-suppressive mechanisms in the tumor microenvironment (TME) including T regulatory cells (Treg), myeloid derived suppressor cells (MDSC) and immunosuppressive cytokines. In addition, complex interaction of tumor-immune system further increases the level of difficulties in the process of biomarkers development and their validation for clinical use. Recent clinical trial results have highlighted the potential for combination therapies that include immunomodulating agents such as anti-PD-1 and anti-CTLA-4. Agents targeting other immune inhibitory (e.g., Tim-3) or immune stimulating (e.g., CD137) receptors on T cells and other approaches such as adoptive cell transfer are tested for clinical efficacy in melanoma as well. These agents are also being tested in combination with targeted therapies to improve upon shorter-term responses thus far seen with targeted therapy. Various locoregional interventions that demonstrate promising results in treatment of advanced melanoma are also integrated with immunotherapy agents and the combinations with cytotoxic chemotherapy and inhibitors of angiogenesis are changing the evolving landscape of therapeutic options and are being evaluated to prevent or delay resistance and to further improve survival rates for melanoma patients’ population. This meeting’s specific focus was on advances in immunotherapy and combination therapy for melanoma. The importance of understanding of melanoma genomic background for development of novel therapies and biomarkers for clinical application to predict the treatment response was an integral part of the meeting. The overall emphasis on biomarkers supports novel concepts toward integrating biomarkers into personalized-medicine approach for treatment of patients with melanoma across the entire spectrum of disease stage. Translation of the knowledge gained from the biology of tumor microenvironment across different tumors represents a bridge to impact on prognosis and response to therapy in melanoma. We also discussed the requirements for pre-analytical and analytical as well as clinical validation process as applied to biomarkers for cancer immunotherapy. The concept of the fit-for-purpose marker validation has been introduced to address the challenges and strategies for analytical and clinical validation design for specific assays.