Towards characterizing the “Immunoscore” in the tumor microenvironment in oral, head and neck cancer

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.^1,2Given these promising findings in colon cancer and a number of other cancers, we have started an objective assessment of immune cell numbers in biopsy specimens from patients with OHNSCC. We are comparing these results with a flow cytometric analysis of cells isolated by mechanical and enzymatic digestion of tumor 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.

Materials and methods: 31 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 specimen 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. Additional studies are evaluating dendritic cell numbers and function. 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.

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.  

Conclusion: While still early, both methodologies can provide insights into the type, number, function 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 T cells, 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