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Introduction

Melanoma is an aggressive cancer with 76,250 new cases of invasive melanoma and 55,560 cases of melanoma in situ in 2012 . The incidence continues to steadily rise at the fastest rate of any malignancy in the U.S and abroad . Head and neck melanoma, which comprises 18% of primary lesions, has a worse prognosis compared to other locations . Ten-year survivability is dependent on various factors including primary location , vertical growth phase, thickness, level of invasion, ulceration, and density of mitotic figures . According to the American Joint Committee on Cancer (AJCC) the most important prognostic factor is the number of nodal metastases . Overall 35%–5% of patients with regional metastases develop distant disease . Furthermore, the status of the sentinel lymph nodes (SLNs) in patients with clinically negative lymph nodes (LNs) is the most important prognostic factor for recurrence . In patients with nodal disease, but no distant metastases (AJCC stage III), the outcomes are remarkably varied in terms of ultimate distant metastases and mortality . This demonstrates the challenge in prognosticating and managing melanoma.

The SLN is in the regional LN basin that receives direct lymphatic flow from the primary lesion and therefore the first metastases. It can be identified by dye, gamma probe, lymphoscintigraphy or a combination at a rate of 94% . SLNs may contain macrometastases, which are evident on exam or imaging, or sub-clinical micrometasteses. Micrometastases occur in 15%–20% of head and neck melanomas . These can be identified on standard hematoxylin and eosin staining or may require specific immunohistochemical (IHC) staining or reverse transcriptase polymerase chain reactions (PCR).

SLN biopsy has become the standard of care to determine subclinical LN metastases and has replaced elective lymph node dissection (ELND) in melanoma. This is supported by a lack of survival benefit from up-front ELND versus SLN biopsy . This has also been confirmed in the head and neck population . A positive SLN leads to a completion neck dissection (CND) that still results in a 13.1% nodal recurrence and a 39.7% total recurrence probability . This suggests that a positive SLN biopsy is an indicator that the melanoma has already spread systemically. Despite the use of SLN biopsy, the death rate from melanoma continued to rise from 1990 to 2006 . Therefore, the presence of positive SLN suggests not only the need for CND, but also the need to consider adjuvant therapies and closer surveillance to detect recurrence.

There are likely other critical factors in SLNs that correlate with clinical outcomes. One factor may be regulatory T cells (Tregs). Tregs exhibit a CD4 + CD25 + FoxP3 + phenotype and are immunosuppressive. Forkhead box P3 (FoxP3) is a transcription repressor expressed on activated Tregs . Tregs can become part of the tumor microenvironment and suppress proliferation and activity of effector T cells and thereby allow tumor immune escape. Increased infiltration of Tregs has been reported in various types of tumors and SLNs . There are no studies comparing Treg expression in SLN with outcome specifically in the head and neck melanoma population. Increased Treg in SLN may identify a subset of patients that have worse clinical outcomes after CND.

Measuring Treg prevalence in SLN may identify patients that would benefit from treatments targeting Treg. One such agent is Ipilimumab, a human monoclonal antibody that blocks cytotoxic T lymphocyte antigen 4 (CTLA-4). CTLA-4 down-regulates T cell activation and is found on cytotoxic T cells and Tregs. Ipilumumab has recently shown an improvement in overall survival in previously treated metastatic melanoma . Several other agents active against Tregs are being studied in the basic science, translational and clinical settings .

Ipilumumab has a substantial benefit in some metastatic melanoma patients, but many derive no therapeutic benefit from it . Other immunologic factors can affect Treg function. Both tumors and native immune cells can express indoleamine 2,3-dioxygenase (IDO), an enzyme that activates Tregs to promote tumor immune escape. IDO acts as an intracellular enzyme that catabolizes tryptophan degradation. IDO knockout mammalian models and those given IDO inhibitors have slower melanoma growth and longer survival when treated with Ipilumumab . New agents against IDO are actively being studied, some in conjunction with Treg inhibitors .

Our objectives were to 1) assess FoxP3/IDO immunoreactivity in head and neck melanoma SLNs and 2) correlate FoxP3/IDO with SLN metastasis positivity and clinical outcome. This could identify a subset of patients that may benefit from future trials and treatment for melanoma through Treg and IDO suppression.