Our study reveals a highly significant concordance of membranous expression of B7-H1 by nevus and melanoma cells with the presence of immune infiltrates and provides direct evidence of geographic colocalization. Furthermore, expression of the inflammatory cytokine IFN-γ, known to be a primary cytokine driving B7-H1 expression, was detected specifically at the interface of B7-H1+
tumor cells and infiltrating immune cells, but not in B7-H1−
tumors. These findings suggest that infiltrating immune cells can produce factors driving B7-H1 expression as a negative feedback mechanism, resulting in what may be considered adaptive immune resistance exerted by the tumor. This mechanism is distinct from the conventional view of immune resistance by either immune escape via mutation of tumor-specific epitopes (31
) or constitutive oncogene-driven up-regulation of immune-inhibitory ligands (33
). IFN-γ is secreted by human melanoma–specific T lymphocytes, and this cytokine rapidly induces B7-H1 in cultured melanomas. Although our studies associate IFN-γ production with B7-H1+
melanomas in vivo, other secreted factors in the tumor microenvironment such as IL-10, IL-6, and common γ-chain cytokines may also be involved (34
). These findings help to explain why immunogenic melanoma fails to be controlled by the immune system, even when boosted by antigen-based cancer vaccines, and implicate a new immune escape mechanism in the cancer microenvironment.
The up-regulation of B7-H1 in the cancer microenvironment should not be interpreted simply as the dominance of immune suppression because this up-regulation is also associated with endogenous inflammatory immune responses. The balance of the host’s immune response and negative feedback inhibition will therefore determine outcome. We did not detect a significant correlation between overall survival and B7-H1 expression in patients with primary invasive melanomas. In contrast, we documented a significantly improved overall survival in patients with B7-H1+
metastatic lesions. With the exception of one published report in cervical cancer (36
), this finding is different from a number of reports from other tumor types that have claimed B7-H1 expression as a negative overall prognostic factor (24
This seemingly paradoxical observation, in which expression of an immunosuppressive molecule correlates with improved outcomes, may be resolved if B7-H1 expression is viewed as reflecting the presence of endogenous antitumor immunity (infiltrating lymphocytes producing cytokines in response to tumor-specific or tumor-selective antigens) among melanoma patients who are candidates for immunotherapy. Indeed, 43% of the patients with metastatic disease in our study received some form of immunotherapy after diagnostic tumor biopsy, including IFN-α, high-dose IL-2, vaccine, PD-1 blockade, or combinations thereof. A growing literature suggests the positive correlation of an inflamed tumor microenvironment with response to immunotherapies such as cancer vaccines or ipilimumab (37
), and our findings further suggest that these immunotherapies should be interpreted as protection or potentiation of ongoing immunity rather than the generation of de novo immunity against cancer. Note that medical center–specific treatment practices may be critical to these observations. Indeed, about one-third of patients with metastatic melanoma show evidence of objective tumor regressions in response to anti–PD-1 therapy (39
). Ongoing studies in our laboratories are investigating whether tumor regressions in this series of patients correlate with tumor cell B7-H1 expression and the presence and type of TILs.
Although intratumoral lymphocytes are a common feature in many different types of cancer, the prognostic significance of this finding varies by tumor type. For example, TILs are associated with a better prognosis in patients with colorectal, ovarian, pancreatic, esophageal, and small-cell lung carcinoma (40
), but are an adverse prognostic feature in patients with renal cell carcinoma (45
). Notably, in colon cancer, the type, density, and location of immune infiltrates have been shown to be a better predictor of survival than TNM classification (46
). In melanoma, TILs may be seen in all stages of disease, but are not prognostically significant in early stages, that is, in in situ or radial growth phase melanomas; they do reach significance in vertical growth phase melanomas and those that have metastasized (47
). Our findings provide an interpretation for these seemingly contradictory observations. Although the presence of TILs may indicate immunogenicity of tumors and ongoing immune responses, clinical outcomes may depend on B7-H1 expression in addition to other factors such as composition of infiltration, persistence of inflammation, and the treatment received.
We observed only rare cases showing diffuse tumor cell B7-H1 expression, unrelated or out of proportion to TILs. These may represent melanomas that have developed intrinsic pathways driving B7-H1 gene expression. PTEN (phosphatase and tensin homolog deleted from chromosome 10) deficiency has been shown to support posttranscriptional B7-H1 expression in gliomas (34
), but factors driving constitutive expression in melanoma have not been defined. Distinct histologic subtypes of melanoma are associated with characteristic tumorigenic signaling pathways: for example, superficial spreading and nodular melanomas are more likely to have activating BRAF mutations than acral lentiginous melanomas, which are often associated with c-Kit mutations. In our study, 45% of superficial spreading and nodular melanomas expressed B7-H1, whereas other subtypes including acral lentiginous melanoma, lentigo maligna melanoma, and desmoplastic melanoma did not, suggesting the presence of distinct signaling pathways. Studies focused on the transcriptional regulation of B7-H1 expression in nodular and superficial spreading melanomas and metastatic lesions, as well as studies of cytokine expression profiles in microdissected boundary areas between tumor cells and TILs, are currently under way in our laboratories to explore these issues. Notably, the correlation between IFN-γ and B7-H1 expression was 100% among the microdissected samples tested. However, roughly one-third of lesions with observable TIL (on IHC and/or H&E staining) did not express B7-H1, suggesting that a T helper 1 (TH
1) pattern of cytokine expression characterized by IFN-γ may be needed to induce B7-H1 expression.
The clinical significance of B7-H1 expression in melanoma has not yet been firmly established. Two reports have been published to date, with conflicting results (49
), perhaps because of different histologic subtypes studied or methodologies used. Gadiot et al.
used the polyclonal antibody 4059 for IHC detection, scored a 1% threshold of expression by melanocytes as a positive result, and did not distinguish membranous and cytoplasmic patterns of cellular expression. In that study, most patients had superficial spreading or nodular-type melanomas, and B7-H1 expression by melanocytes was associated with a trend toward better survival. In contrast, Hino et al.
used mAb clone 27A2 for IHC and compared “high” to “low” cytoplasmic expression based on automated color density measurements. Most patients had the acral lentiginous subtype of melanoma, which is associated with c-Kit mutations, rather than with BRAF mutations, which predominate in cohorts of European descent (51
). Hino et al.
reported high-intensity B7-H1 expression as an independent risk factor for decreased survival. Both studies had about a 4- to 5-year median follow-up time. Neither reported the geographic association of B7-H1 expression with TILs or B7-H1 expression by other cells in the tumor microenvironment.
Both cytoplasmic and membranous B7-H1 staining have been reported in multiple tumor types; however, B7-H1 is a type I transmembrane molecule (6
). Cytoplasmic staining may represent intracellular stores of B7-H1, which may be deployed to the cell surface depending on appropriate stimulation. Our previous study describing B7-H1 expression in various human cancers included a small sample size of melanomas (n
= 22), among which all were deemed at least focally positive in frozen sections (5
); however, the previous study assessed both cytoplasmic and membranous positivity, whereas the current study assesses only membranous expression. We hypothesize that cell surface expression of B7-H1 is the most immediately biologically relevant as a potential biomarker predicting clinical response to PD-1 blockade (29
These findings highlight a therapeutic opportunity in administering agents blocking the PD-1/B7-H1 pathway to patients with metastatic melanoma. B7-H1 is a critical immunomodulating component within the melanoma microenvironment. Expression of B7-H1 on tumor cells at the interface with immune infiltrates, in comparison to constitutive tumor expression in areas devoid of infiltrates, may represent distinct underlying immune resistance mechanisms and warrant further study on the molecular level. A deeper understanding of mechanisms driving the observed expression patterns is essential for developing rational treatment combinations with mAbs blocking PD-1 or B7-H1, which are already in clinical trials, and other potentially synergistic therapeutic agents.