"Immunoscore" as a Biomarker for Oral, Head and Neck Squamous Cell Carcinoma

R. Bryan Bell DDS, MD, FACS, Providence Oral, Head and Neck Cancer Program and Clinic, Providence Cancer Center, Portland, OR
Zipei Feng BS, Earle A. Chiles Research Institute, Portland, OR
Tarsem Moudgil PhD, Earle A. Chiles Research Institute, Portland, OR
Allen Cheng DDS, MD, Providence Oral, Head and Neck Cancer Program and Clinic, Providence Cancer Center, Portland, OR
Christopher Paustian PhD, Earle A. Chiles Research Institute, Portland, OR
Christopher Dubay PhD, Earle A. Chiles Research Institute, Portland, OR
Hong-Ming Hu PhD, Earle A. Chiles Research Institute, Portland, OR
Tuan G. Bui DMD, MD, FACS, Providence Oral, Head and Neck Cancer Program and Clinic, Providence Cancer Center, Portland, OR
Tyler Hulett BS, Earle A. Chiles Research Institute, Portland, OR
Rom Leidner MD, Providence Oral, Head and Neck Cancer Program and Clinic, Providence Cancer Center, Portland, OR
Traci Hilton PhD, Earle A. Chiles Research Institute, Portland, OR
Carlo B Bifulco MD, PhD, Department of Pathology, Providence Cancer Center, Portland, OR
Bernard A Fox PhD, Earle A. Chiles Research Institute, Portland, OR
Statement of the Problem: Recently, the delineation of tumor-infiltrating immune cells using standard immunohistochemistry (IHC) coupled with digital imaging and computer algorithms, termed “Immunoscore”, has proved to be a powerful prognostic biomarker, providing significantly (p<0.001) more prognostic power than TNM staging in patients with colon cancer.1,2 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 in patients with oral, head and neck squamous cell carcinoma (OHNSCC). This suggests that OHNSCC 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 an OHNSCC tumor bank that is cryopreserving enzymatically isolated viable cells from resected tumors. Furthermore, we are attempting to develop primary tumor cell lines and are isolating tumor-infiltrating lymphocytes from theses specimens.The purpose of this preliminary investigation was to apply digital imaging and objective assessment techniques to characterize infiltrating immune cells and to coordinate these data with a flow cytometric analysis of the cells isolated from the tumor and evaluate for possible correlation with tumor-specific function.

Materials and methods: 102 subjects with biopsy proven OHNSCC from a variety of subsites underwent surgery with curative intent and were enrolled into this prospective, IRB approved trial. Samples of tumor were obtained from either the primary tumor or from a metastatic lymph node and processed utilizing mechanical and enzymatic digestion to dissociate cell types contained in the tumor specimen. These cells are being analyzed for CD3, CD4, CD8, CD20 and FoxP3 using flow cytometry. In parallel, formalin fixed parafin embedded (FFPE) tissue from the same specimens are being stained for the specified markers, scanned and digital images are being assessed using the Definiens software platform.

Methods of Data Analysis: Descriptive statistics

Results:  Preliminary results have documented consistent staining and potential of the IHC and FACS-based systems to identify infiltrating cells. Definiens software provides a consistent method to assess numbers of IHC stained cells in sections of tumor specimens. Ten confirmed tumor cell lines are established thus far and T cells from 9 of 11 TIL cultures tested to-date secreted IFN-g when stimulated with their autologous tumor cells.

Conclusion: While still early, both methodologies can provide insights into the type, number, function, specificity or relative location of immune cells present in the tumor microenvironment. Planned studies will assess these markers using IHC in larger cohorts of patients with long-term follow-up. While we anticipate that a strong infiltration of immune cells (immunoscore positive) will be associated with better clinical outcomes, the value of the current study will be the availability of isolated and cryopreserved tumor and tumor-infiltrating immune cells for functional studies. Assessment of the tumor cells from immunoscore negative tumors may provide insights into the mechanisms responsible for the absence of infiltrating immune cells. These insights may be exploited to develop novel therapeutic strategies that will reverse the negative immunoscore and improve outcomes of patients with OHNSCC. 

1. Pages F, Berger A, Camus M, Sanchez-Cabo F, Costes A, et al. Effector memory Tcells, early metastasis, and survival in colorectal cancer. N Engl J Med. Dec 22;353(25):2654-66, 2005

2. Galon J, Costes A, Sanchez-Cabo F, Kirilovsky A, et al. Type, density, and location of immune cells within human colorectal tumors predict clinical outcome. Science. Sept 29;313, 2006

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