Zipei Feng

OX40, PD-1 and CTLA-4 are selectively expressed on tumor-infiltrating T cells in head and neck cancer

The tumor microenvironment of squamous cell carcinoma of the head and neck (SCCHN) has been shown to be immune suppressive. Therefore, strategies aimed at overcoming this issue could have a positive therapeutic impact. Hence, we investigated the expression of the known immune‐modulatory proteins OX40, programmed cell death protein 1 (PD‐1) and cytotoxic T‐lymphocyte‐associated protein 4 (CTLA‐4) in SCCHN on different T‐cell subsets of tumor‐infiltrating lymphocytes (TIL) to ascertain whether these proteins could potentially be targeted alone or in combination for future clinical trials. T cells from peripheral blood (PBL) and tumor were analyzed for the expression of OX40, PD‐1 and CTLA‐4 in 29 patients undergoing surgery. These proteins were all expressed significantly higher in T‐cell subsets isolated from tumors compared with PBL of the same patient. OX40 expression was significantly greater in the TIL regulatory T‐cell (Treg) population relative to conventional CD4 and CD8 TIL or the Treg isolated from PBL. PD‐1 expression was increased in all T‐cell subsets relative to PBL. CTLA‐4 was also increased in all TIL subsets relative to blood, and similar to OX40, its highest level of expression was observed in the Treg TIL. The highest frequency of PD‐1, CTLA‐4 and OX40 triple‐positive cells were found in the Treg population isolated from the tumor. We analyzed both human papilloma virus‐positive and ‐negative patients and found similar levels and expression patterns of these two patient populations for all three proteins. These data suggest that there may be therapeutic advantages of targeting these pathways independently or in combination for patients with this disease.

Timing of PD-1 Blockade Is Critical to Effective Combination Immunotherapy with Anti-OX40.

Purpose: Antibodies specific for inhibitory checkpoints PD-1 and CTLA-4 have shown impressive results against solid tumors. This has fueled interest in novel immunotherapy combinations to affect patients who remain refractory to checkpoint blockade monotherapy. However, how to optimally combine checkpoint blockade with agents targeting T-cell costimulatory receptors, such as OX40, remains a critical question. Experimental Design: We utilized an anti-PD-1–refractory, orthotopically transplanted MMTV-PyMT mammary cancer model to investigate the antitumor effect of an agonist anti-OX40 antibody combined with anti-PD-1. As PD-1 naturally aids in immune contraction after T-cell activation, we treated mice with concurrent combination treatment versus sequentially administering anti-OX40 followed by anti-PD-1. Results: The concurrent addition of anti-PD-1 significantly attenuated the therapeutic effect of anti-OX40 alone. Combination-treated mice had considerable increases in type I and type II serum cytokines and significantly augmented expression of inhibitory receptors or exhaustion markers CTLA-4 and TIM-3 on T cells. Combination treatment increased intratumoral CD4+ T-cell proliferation at day 13, but at day 19, both CD4+ and CD8+ T-cell proliferation was significantly reduced compared with untreated mice. In two tumor models, sequential combination of anti-OX40 followed by anti-PD-1 (but not the reverse order) resulted in significant increases in therapeutic efficacy. Against MMTV-PyMT tumors, sequential combination was dependent on both CD4+ and CD8+ T cells and completely regressed tumors in approximately 30% of treated animals. Conclusions: These results highlight the importance of timing for optimized therapeutic effect with combination immunotherapies and suggest the testing of sequencing in combination immunotherapy clinical trials. Clin Cancer Res; 23(20); 6165–77. ©2017 AACR. See related commentary by Colombo, p. 5999

Multiparametric immune profiling in HPV– oral squamous cell cancer

Evaluation of T lymphocyte frequency provides prognostic information for patients with oral squamous cell cancer (OSCC). However, the effect of simultaneously evaluating T cell frequency and assessing suppressive elements and defects in antigen-processing machinery (APM) has not been clarified. Simultaneous characterization of CD3+, CD8+, FoxP3+, CD163+, and PD-L1+ cells using multispectral imaging was performed on sections from 119 patients with HPV- OSCC. Expression of β2-microglobulin, MHC class I heavy chain, and large multifunctional peptidase 10 was quantified, and all data were correlated with patient outcome. We found that, consistent with previous reports, high numbers of CD8+ T cells at the invasive margin correlated significantly with prolonged overall survival (OS), while the number of FoxP3+ or PD-L1+ cells did not. Compiling the number of FoxP3+ or PD-L1+ cells within 30 μm of CD8+ T cells identified a significant association with a high number of suppressive elements close to CD8+ T cells and reduced OS. Integrating this information into a cumulative suppression index (CSI) increased correlation with OS. Incorporating tumor expression levels of APM components with CSI further improved prognostic power. This multiparametric immune profiling may be useful for stratifying patients with OSCC for clinical trials.

OX40 signaling in head and neck squamous cell carcinoma: Overcoming immunosuppression in the tumor microenvironment

OX40 is a member of the tumor necrosis factor (TNF) receptor family and a potent co-stimulatory pathway that when triggered can enhance T-cell memory, proliferation and anti-tumor activity in patients with metastatic cancer. Ongoing investigations at our institution have demonstrated that OX40 expressing T cells are found in abundance in the tumors of patients with advanced stage head and neck squamous cell carcinoma (HNSCC). This has led to the initiation of human clinical trials investigating OX40-directed therapy for patients with HNSCC in both the metastatic and curative setting. The purpose of this review is to explore what is known about OX40 signaling and discuss how this pathway potentially can be modulated to improve outcome for patients with HNSCC.

Multispectral Imaging of T and B Cells in Murine Spleen and Tumor.

Recent advances in multiplex immunohistochemistry techniques allow for quantitative, spatial identification of multiple immune parameters for enhanced diagnostic and prognostic insight. However, applying such techniques to murine fixed tissues, particularly sensitive epitopes, such as CD4, CD8α, and CD19, has been difficult. We compared different fixation protocols and Ag-retrieval techniques and validated the use of multiplex immunohistochemistry for detection of CD3+CD4+ and CD3+CD8+ T cell subsets in murine spleen and tumor. This allows for enumeration of these T cell subsets within immune environments, as well as the study of their spatial distribution.

Immunoprofiling as a predictor of patient’s response to cancer therapy—promises and challenges

Immune cell infiltration is common to many tumors and has been recognized by pathologists for more than 100 years. The application of digital imaging and objective assessment software allowed a concise determination of the type and quantity of immune cells and their location relative to the tumor and, in the case of colon cancer, characterized overall survival better than AJCC TNM staging. Subsequently, expression of PD-L1, by 50% or more tumor cells, identified NSCLC patients with double the response rate to anti-PD-1. Soon, automated staining methods will improve reproducibility of multiplex staining and allow for CLIA standards so that multiplex staining can be used to make clinical decisions. Ultimately, machine-learning algorithms will help interpret data from tissue images and lead to improved delivery of precision medicine.

Phase II calm extension study: Coxsackievirus A21 delivered intratumorally to patients with advanced melanoma induces immune-cell infiltration in the tumor microenvironment

Meeting abstracts CAVATAK, an oncolytic immunotherapy, is a bio-selected oncolytic strain of Coxsackievirus A21 (CVA21). Following intratumoral (IT) injection, CVA21 preferentially infects ICAM-1 expressing tumor cells, resulting in viral replication, cell lysis, and a systemic anti-tumor immune

Utilizing quantitative immunohistochemistry for relationship analysis of tumor microenvironment of head and neck cancer patients

Meeting abstracts Analysis of tumor-infiltrating immune cells using quantitative immunohistochemistry (IHC) has proved to be a powerful prognostic biomarker in colon cancer [[1][1], [2][2]]. Similar observations have been made in patients with oral, head and neck squamous cell carcinoma (OHNSCC),

Preliminary analysis of immune responses in patients enrolled in a Phase II trial of Cyclophosphamide with Allogenic DRibble Vaccine Alone (DPV-001) or with GM-CSF or Imiquimod for adjuvant treatment of Stage IIIA or IIIB NSCLC

Preliminary analysis of immune responses in patients enrolled in a Phase II trial of cyclophosphamide with allogenic dribble vaccine alone (DPV-001) or with GM-CSF or imiquimod for adjuvant treatment of stage IIIa or IIIb NSCLC Rachel Sanborn, Brian Boulmay, Rui Li, Bradley Spieler, Kyle Happel, Christopher Paustian, Tarsem Mougdil, Zipei Feng, Christopher Dubay, Brenda Fisher, Yoshinobu Koguchi, Sandra Aung, Eileen Mederos, Carlo Bifulco, Michael McNamara, Keith S Bahjat, William Redmond, Augusto C Ochoa, Hong Ming Hu, Bernard Fox, Walter Urba, Traci Hilton

Developing an immunotherapy strategy for the effective treatment of patients with squamous cell carcinoma of the head and neck

Squamous cell carcinoma of the head and neck (HNSCC) arises in the oral cavity, oropharynx, larynx or hypopharynx, and is the 6th leading cause of cancer by incidence worldwide. Approximately 600,000 cases arise each year, and only 40-50% of these patients will survive for 5 years.7 HNSCC can express PD-L1 and suppressive factors that interfere with the differentiation and activation of dendritic cells and effector function of T cells. Similar to observations in other malignancies, CD8 effector T cell infiltration or transcriptional signature consistent with an ongoing T cell response is associated with prolonged survival. This suggests that HNSCC lacking a T cell infiltrate may benefit from immune interventions that induce and/or augment a tumor-specific immune response. To develop a strategy to address this problem we have created a HNSCC tumor bank that is cryopreserving enzymatically isolated viable cells from resected tumors (39 specimens). We are also attempting to develop primary cell lines (4 lines established) and are isolating tumor-infiltrating lymphocytes from these specimens. We are looking to compare immunohistochemical, flowcytometric and functional analyses of these specimens. Consistent with previous studies, results document that isolated TIL secrete IFN-γ when stimulated with their autologous tumor cells (n=2). Since a central aim of these studies is to develop a strategy to create therapeutic immunity in patients lacking T cell infiltrates, we plan to investigate which antigens are recognized and whether, antigens present in a novel DRibble vaccine, that expresses antigens common to HNSCC, are recognized by these TIL. This may provide a strategy to monitor anti-tumor immunity in these patients and possibly provide an antigen source for combination immunotherapy of patients with HNSCC.