Immune cells in the melanoma microenvironment hold information for prediction of the risk of recurrence and response to treatment

Abstract

Melanoma is an immunogenic tumor and immunotherapy treatment has established an increase in disease-free and overall survival in melanoma patients. However, a complex network of immunosuppressive mechanisms has been demonstrated to occur at the tumor site and in locoregional immune districts, such as sentinel lymph nodes (SLNs). The interplay between tumor cells and the local microenvironment leads to a tumor-driven shaping of the immune response that results in a heterogeneous cellular and molecular composition of tumor infiltrating lymphocytes (TILs). Several studies have reported the potential prognostic value of TILs infiltrating primary tumors and the association of ‘immune signature’ in SLNs and in melanoma metastases with prognosis and responsiveness to immunotherapeutic approaches. However, a systematic and deeper characterization of the local immunological status of TILs and SLNs is still required to refine melanoma stage classification.

Keywords::

• immunotherapy
• melanoma
• prognosis
• sentinel node
• tumor-infiltrating lymphocytes

Melanoma is known as an immunogenic tumor, and signs of immunosurveillance, such as spontaneous regression with the simultaneous onset of vitiligo, have been reported to occasionally occur in this tumor type [1]. The peripheral blood of melanoma patients possesses a relatively large amount of T cells or antibodies that recognize melanoma antigens. An active anti-tumor response is also present at the tumor site [2], and tumor-infiltrating lymphocytes (TILs) enriched in anti-tumor T cells have been used with a certain degree of success for adoptive immunotherapy [3]. Thus, immunotherapy aimed at boosting or inducing ex novo melanoma-directed immunity represents a promising approach for melanoma treatment, and important results in terms of disease-free and overall survival have been recently achieved [4]. However, a complex network of immune-suppressive mechanisms has been demonstrated to occur at the tumor site and in locoregional immune districts, such as sentinel lymph nodes (SLNs). Impaired antigen-presenting cell maturation, T-cell anergy, the induction/recruitment of regulatory T lymphocytes (Tregs) and immunosuppressive cells of myeloid origin [5,6] are some of the crucial mechanisms that strongly limit an active anti-tumor response. Moreover, the cellular and molecular composition of TILs is not homogeneous because they comprise immune cell subsets that exert a variety of pro- or anti-tumor activities. Additionally, the quality of the host immune response differs between patients with early microscopic and more advanced disease [5]. The continuous interplay between tumor cells and the local microenvironment leads to the tumor-driven shaping of the immune response during tumor development and progression. Various approaches, including TIL characterization and lesion gene expression profile analysis, have been performed to identify markers that are able to predict responses to treatment and clinical outcomes. The role of TILs and their relationship with prognosis have been extensively studied, and emerging evidence suggests that a careful analysis of the local immunological status of TILs and SLNs may improve melanoma stage classification.

The prognostic value of TILs in primary melanomas

The grading for TILs as absent, brisk or non-brisk is defined by their distribution and quantity [7]. Majority of the earlier studies on primary melanomas demonstrated that the presence of brisk TILs in the vertical growth phase of the tumor, but not peritumoral, was strongly and independently associated with disease-free and overall survival [8]. These findings have been recently confirmed in a large series of primary melanomas, in which the grade of lymphocyte infiltration predicted survival independent of age, sex, tumor site and, importantly, tumor stage [9]. Although these data strongly indicate that TILs provide additional information that could potentially improve melanoma classification, the presence of TILs is not currently included in the American Joint Committee on Cancer/Tumor-Node-Metastasis (TNM) cancer staging system. Several items prevent the inclusion of this parameter. First, there is no complete agreement on the favorable prognostic role of TILs in melanoma, and examples have been reported showing that TILs may even promote tumor outgrowth and metastasis. These discordant results may be, in part, due to differences in the cohort of patients examined, bearing tumors with different thicknesses and in different growth phases. Second, there is a failure in differentiating the precise localization of the immune cells as either infiltrating or not infiltrating the tumor nests. Moreover, in the majority of studies, no precise multi-color immune phenotype of TILs has been taken into consideration. More recent research has focused on infiltrating CD8⁺ cells as potential indicators of the cytotoxic destruction of transformed cells; however, CD8⁺ cells can be anergic or inactivated and, thus, are not functionally active against tumors [10]. Additionally, CD4⁺ T cells are heterogeneous, as they comprise different subsets of lymphocytes with opposite roles, such as Th1, Th2 or Tregs. In addition to CD4 and CD8, other subsets of infiltrating immune cells with either tolerogenic or immunogenic properties, such as B and NK cells, are emerging as important regulators of the tumor microenvironment. Thus, to include TILs as prognostic markers in melanoma, it is mandatory to have insight into their immunophenotypic features. Phenotypic and functional information should then be matched with TIL localization in respect to tumor cells using precise immunohistochemical analysis. After being processed with algorithms, these data could be finalized in a standardized consensus method. This work has been recently initiated and organized in a computer-assisted approach known as ‘Immunoscore’, a quantifiable scoring system that is based on an automated cell counting method of in situ markers of immune infiltrates. Immunoscore can potentially be used as a new tool for cancer classification. It showed strong prognostic significance in a retrospective study in colorectal cancer patients and may also help in predicting response to therapy [11]. Based on these results, an international consortium has been established to validate and promote the use of Immunoscore in routine clinical settings for different tumors, including melanoma [11].

The gene signature of metastatic melanoma is predictive of the response to immunotherapy

Over the last decade, several gene expression studies were published that aimed to investigate the molecular patterns associated with responsiveness to immunotherapeutic approaches in melanoma metastases [12]. Pre- and post-treatment biopsies obtained from stage III and IV patients enrolled in different immunotherapy trials were tested. Analysis of the pre-treatment tumors indicated that a pre-existing status of immune activation could favor a clinical response. In fact, elevated baseline expression levels of immune-related genes have been observed in metastatic lesions from patients showing clinical responses to immunotherapy. The gene signature reported to predict responsiveness to immunotherapy included interferon-stimulated genes, CXCR3/CCR5 ligands, chemokine genes and genes associated with immune effector functions. The transcriptional profiles of the post-treatment samples from the responding patients were qualitatively similar to those of the pre-treatment samples, but they showed quantitative differences, as the expression levels of the immune signature genes were considerably increased. Taken together, these results clearly indicate that in metastatic melanoma, immune responsiveness is a predetermined status that can be enhanced via immunotherapeutic intervention to promote tumor rejection. Moreover, melanoma tumors defined via gene expression profiling analysis as ‘highly immune responsive’ are derived from patients with a good prognosis [13,14], thus suggesting that the immune signature is endowed with prognostic value and may be useful for selecting patients who are responsive to immunotherapy regimens. Presently, the definition of a gene signature classifier able to identify a subpopulation of melanoma patients that may benefit from immunotherapy is ongoing in a Phase III trial (DERMA). However, it should be mentioned that in a Phase III non-small cell lung cancer trial (MAGRIT), the investigation of this endpoint failed due to the lack of clinical benefit of the immunotherapeutic treatment [15].

The immune signature of the SLN has a prognostic value

The SLN is the first lymph node (LN) to which cancer cells are most likely to spread because it receives lymphatic drainage from the primary tumor. Histological evaluation of the presence or absence of tumor cells in the SLN in melanoma and breast cancer is routinely performed for the staging and management of these tumor types. Conversely, few data are available on the prognostic value of the immunological status of the SLN, although the tumor immune microenvironment in the SLN may reflect the status of systemic immunity. In fact, a negative association between elevated Tregs in the SLN and prognosis has been reported in melanoma [16] and breast cancer patients [17]. Furthermore, in breast cancer patients, the presence of both CD4⁺ T cells and CD1a+ dendritic cells, potential tumor antigen-presenting cells, was predictive of disease-free survival [18], and tumor-promoting immune gene patterns in tumor-positive SLNs are tightly correlated with those found in the blood immune cells [19]. Thus, the immunological features of tumor-draining LNs may anticipate the conditions of systemic immunity in cancer patients, and the fine characterization of immune cells in the SLN may provide information about clinical outcomes. This result was recently shown in a pilot study reporting that gene expression profiles distinguish tumor-positive SLNs obtained from patients with progressing melanoma from those obtained from patients with good prognosis. Of note, the profiles identified in tumor-positive SLNs are not determined by a different tumor burden in the samples, and they do not discriminate between tumor-negative and tumor-positive SLNs that were derived from patients with good outcomes. It was found that most of the genes that are differentially expressed between tumor-positive SLNs with a good or a poor prognosis are immune-related genes [20]. In this context, CD30⁺ lymphocytes were shown to be positively associated with melanoma progression, and higher numbers of CD30⁺ cells were found in tumor-positive SLNs from patients with further metastatic nodes and disease recurrence compared with SLNs from patients showing no further metastatic nodes or disease relapse. CD30⁺ cells in the paracortical area of the SLN included lymphocytes staining for Foxp3 or PD1 markers of tolerogenic or exhausted T cells, and in vitro studies demonstrated a poor effector function of CD30⁺ lymphocytes from melanoma-invaded LNs. Moreover, the frequencies of CD30⁺ lymphocyte subpopulations were found to be increased in the peripheral blood of melanoma patients at advanced disease stages, further supporting the association of CD30⁺ cells with disease progression.

Overall, these results indicate that the SLN contains information regarding the immune response processes that are associated with metastatic disease progression and that immunological prognostic signatures for the molecular stratification of melanoma patients can be developed from the transcriptional profiles of the SLN.

Conclusion

The currently available data point to the immune cells of the tumor microenvironment, including the tumor-draining LNs, as players in determining disease dissemination and, ultimately, clinical outcome. The type of immune infiltrate in melanoma lesions has been associated with prognosis. However, systematic studies to identify algorithms that consider the localization, phenotype and functional status of TILs are still required. Special attention should be paid to the precise dissection of the immunological status of the SLN as the elective immunological site where anti-tumor immunity is settled and, thus, where markers endowed with prognostic potential can likely be found.

Acknowledgement

The authors acknowledge the financial support by Associazione Italiana Ricerca sul Cancro (MR IG-13335; CC IG-10615).

Financial & competing interests disclosure

The authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.

No writing assistance was utilized in the production of this manuscript.