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PIGMENT CELL & MELANOMA Research Prognostic and predictive significance of immune cells infiltrating cutaneous melanoma Andrea Ladányi

DOI: 10.1111/pcmr.12371 Volume 28, Issue 5, Pages 490–500 If you wish to order reprints of this article, please see the guidelines here

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REVIEW

Pigment Cell Melanoma Res. 28; 490–500

Prognostic and predictive significance of immune cells infiltrating cutaneous melanoma
nyi Andrea Lada Department of Surgical and Molecular Pathology, National Institute of Oncology, Budapest, Hungary CORRESPONDENCE Andrea Lad
anyi, e-mail: [email protected]

KEYWORDS melanoma/tumor-infiltrating immune cells/ lymphocytes/dendritic cells/prognostic marker/predictive marker PUBLICATION DATA Received 2 January 2015, revised and accepted for publication 16 March 2015, published online 26 March 2015 doi: 10.1111/pcmr.12371

Summary The tumor microenvironment is shaped by interactions between malignant cells and host cells representing an integral component of solid tumors. Host cells, including elements of the innate and adaptive immune system, can exert both positive and negative effects on the outcome of the disease. In melanoma, studies on the prognostic impact of the lymphoid infiltrate in general, and that of T cells, yielded controversial results. According to our studies and data in the literature, a high peritumoral density of activated T cells, increased amount of B lymphocytes and mature dendritic cells (DCs) predicted longer survival, while intense infiltration by plasmacytoid DCs or neutrophil granulocytes could be associated with poor prognosis. Besides its prognostic value, evaluation of the components of immune infiltrate could provide biomarkers for predicting the efficacy of the treatment and disease outcome in patients treated with immunotherapy or other, non-immune-based modalities as chemo-, radio-, or targeted therapy.

Introduction Malignant melanoma of the skin is considered as one of the most immunogenic tumor types. Similar to other solid tumors, it frequently contains considerable amount of immune cells, which may reflect a reaction of the host against the tumor. Tumor progression, however, can occur in the presence of significant lymphoid infiltrate or even a documented antitumor immune response, indicating that in most cases, the immune system is not capable of efficiently controlling tumor growth. Several mechanisms may be responsible for this inefficiency. Suboptimal activation of tumor antigen-specific T lymphocytes due to lack or low level of expression of these antigens or molecules involved in their presentation can hamper the development of antitumor immune response (Anichini et al., 2004; Marincola et al., 2000). Even in the case of optimal T-cell activation, lack of the proper chemoattractants in the tumor area or their receptors on T lymphocytes, as well as abnormal expression of endothelial adhesion molecules within the tumors, may inhibit the homing of T cells to the tumor. Finally, immune effector function can be hampered locally by suppressor

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factors secreted by tumor cells or neighboring host cells, or by suppressor cells in the tumor microenvironment (Anichini et al., 2004; Gajewski, 2007). In the past 1–2 decades, it became clear that, besides their antitumor activity, cells of the immune system may also exert tumor promoting effects, partly via their role in the development and maintenance of inflammatory processes frequently associated with tumors (Coussens and Werb, 2002; Solinas et al., 2010). Macrophages, which are generally the most abundant tumor-infiltrating immune cells besides T lymphocytes, are key mediators of these processes. Tumor-associated macrophages (TAM) can promote tumor growth and progression via secretion of growth factors, cytokines, proangiogenic, as well as immune suppressor factors (Allavena et al., 2008; Coffelt et al., 2009; Ribatti and Crivellato, 2009; Sica et al., 2006). Similar effects have been described in the case of neutrophil granulocytes and mast cells (Houghton, 2010; Khazaie et al., 2011; Piccard et al., 2012; Ribatti and Crivellato, 2009, 2012). The presence of lymphoid infiltrate in tumors (without assessing the different lymphocyte subsets by immunohistochemistry), as well as intense T-cell infiltration, was

ª 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd

Prognostic significance of immune cells in melanoma

found to correlate with favorable disease outcome in the case of numerous tumor types (Gooden et al., 2011; Senovilla et al., 2012). Infiltration of dendritic cells (DCs), the most efficient antigen-presenting cells, has also shown correlation with survival in most solid tumors tested. On the other hand, high amount of TAM or neutrophils often predicts unfavorable prognosis. Evidence accumulating from both experimental and clinical studies shows that, besides controlling the development and progression of malignant tumors, the antitumor immune response can influence the efficacy of various cancer treatment modalities including different immunotherapeutic and also non-immunological approaches as conventional cytotoxic chemotherapy, targeted therapy, and radiotherapy (Denkert et al., 2011; Zitvogel et al., 2008, 2011). These therapeutic modalities can affect the immune system in many ways, which in turn may contribute to the success of the therapeutic agents. As growing evidence demonstrates the importance of the local immune status in shaping the antitumor immune response (Malyguine et al., 2012), several recent studies aimed at evaluating the density of immune cell types at tumor sites as potential predictors of response to anticancer therapies. In various cancers, the amount of certain T-cell subsets in pre- or post-treatment tumor samples proved predictive of response to chemo-, chemoradiotherapy or targeted therapy (Denkert et al., 2011; Zitvogel et al., 2011). The prognostic value of immune cell infiltration has been the subject of an excellent, very comprehensive recent review by Senovilla et al. (2012), involving data on solid malignancies. The present work focuses on malignant melanoma of the skin, summarizing results of investigations on the prognostic correlations of tumorinfiltrating immune cell types.

Prognostic importance of immune cell types infiltrating primary melanomas Lymphocytes The presence of tumor-infiltrating lymphocytes (TIL) in melanoma has mostly been determined based on hematoxylin–eosin staining (Cipponi et al., 2011). While some studies reported that a prominent lymphocytic infiltrate was an independent parameter associated with good prognosis (Azimi et al., 2012; Sondergaard and Schou, 1985), especially when lymphocytes infiltrating the tumor in vertical growth phase (VGP) were taken into consideration (Clark et al., 1989; Clemente et al., 1996), in others, partly based on similar methodology, no significant correlation was found (Barnhill et al., 1996; Taylor et al., 2007), or lymphocyte infiltration was not an independent predictor of survival (Burton et al., 2011; Larsen and €rn et al., 1994). A Grude, 1978; Mandala et al., 2009; Tho possible explanation for the discordant results is that the influence of TIL on disease outcome may be stronger in the case of advanced melanomas, because studies demonstrating survival advantage of cases with abundant ª 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd

TIL generally involved a high percentage of tumors thicker than 1.7 or 2.0 mm, while in studies failing to show such difference thinner tumors dominated (Taylor et al., 2007). In some of the investigations that did not find correlation with survival, the absence or low amount of TIL proved to be an independent predictor of sentinel lymph node positivity (Burton et al., 2011; Mandal a et al., 2009; Taylor et al., 2007). On the other hand, there was no difference in the frequency of distant metastases, which may be an alternative explanation for the lack of survival benefit associated with the presence of TIL (Taylor et al., 2007). T lymphocytes T lymphocytes are key players in cell-mediated immunity considered of primary importance in antitumor immune response. CD4+ as well as CD8+ T cells recognizing tumor-associated antigens could be isolated from most solid tumors. Infiltration of T lymphocytes or their subsets (e.g., CD8+, CD45RO+) proved to be positive prognostic factors in several cancer types (Gooden et al., 2011; Senovilla et al., 2012). In the case of melanoma, however, relatively few studies have been published, most of them involving rather small case numbers and yielding conflicting results (Table 1). In two smaller studies (34 and 47 patients, respectively), patient survival showed positive correlation with the intratumoral amount of CD8+ or CD4+ T cells (van Houdt et al., 2008; Piras et al., 2005), while a third one (58 patients) found inverse correlation with intratumoral CD3+ T-cell infiltration and no association with CD8+ lymphocyte number (Hillen et al., 2008). Peritumoral T-cell densities were not studied or were found without prognostic significance. Possible explanations for the conflicting data could be the relatively small number of patients involved in these studies, as well as methodological differences, for example, evaluating infiltration in the whole tumor (Hillen et al., 2008) versus that of the tumor base at the VGP (Piras et al., 2005). According to a recent investigation involving 186 cases, there was no association between survival and the amount of CD8+ T lymphocytes infiltrating the VGP (Jensen et al., 2012). Similarly, in a smaller study, we could not find significant correlation between the number of CD3+ or CD8+ cells and survival, although there was a trend toward lower cell densities in melanomas which developed visceral metastases within 5 yrs compared to non-metastatic cases (Kiss et al., 2007). Activated T cells T cells in the tumor microenvironment are often functionally defective, with depressed proliferation capacity, cytokine production, or cytotoxicity (Anichini et al., 2004; Gajewski, 2007). Therefore, when assessing their prognostic role, besides quantitative determination of tumorinfiltrating T lymphocytes, it is important to characterize their activation state. In our earlier studies on 76 cutaneous melanoma samples, we chose two markers for assessing the activation state of T lymphocytes, CD25 (IL-2 receptor 491


nyi Lada Table 1. Prognostic associations of infiltrating immune cells in melanoma

Study Piras et al. (2005) van Houdt et al. (2008) Hillen et al. (2008) Jensen et al. (2012) Kiss et al. (2007) Lad
anyi et al. (2004) Miracco et al. (2007) Lad
anyi et al. (2010) Lad
anyi et al. (2011) Lad
anyi et al. (2007) Emri et al. (2013) Jensen et al. (2009) Storr et al. (2012) Schadendorf et al. (1995)
th et al. To (2000)

Patient number 47 34 58 186 52 76

T cells Good (CD4, CD8) Good (CD4, CD8) Poor (CD3) none (CD8) None (CD8)

Activated T cells

Treg cells

Plasmacytoid DCs

Macrophages

Neutrophils

Mast cells

Good (GrB) None (CD69)

None

None

None (CD68) Good (CD208)

None (CD3, CD8)

Poor

Poor None (CD68)

Good (CD25, CD134) Poor

97

None Good

82 46 190 202

Good (CD1a, CD208) Good (CD1a)

Poor (CD68, CD163) Poor (CD68, CD163) None (CD68)

44

None

35

Poor

a) and CD134 (OX40). Both of these molecules have functional significance; the former plays a crucial role in stimulating T-cell proliferation, while the latter is a costimulatory receptor expressed primarily on recently activated CD4+ T lymphocytes. Lymphocytes carrying the activation markers were more numerous in the peritumoral infiltrate, but they were also found inside melanoma cell nests. High peritumoral density of these cells was associated with favorable prognosis (Lad
anyi et al., 2004). Another, smaller scale study (34 patients) found a correlation between a favorable disease outcome and intratumoral appearance of granzyme B expressing CD8+ T lymphocytes (van Houdt et al., 2008). The amount of T cells carrying the CD69 marker, on the other hand, did not show significant association with survival (Hillen et al., 2008) (Table 1). Moreover, programmed death 1 (PD-1) expression on CD8+ TIL, tested in 37 melanoma samples, €nig et al., 2012). did not correlate with patient survival (Kro The expression of this coinhibitory molecule on both CD4+ and CD8+ TIL was found higher in melanoma metastases compared to primary tumors (Chapon et al., €nig et al., 2012) and, similarly to that of other 2011; Kro coinhibitory molecules, has been considered as a sign of T-cell exhaustion. However, a recent report described PD-1 as a marker identifying the clonally expanded tumor492

Dendritic cells (DCs)

None (CD68)

66

106

B cells

reactive CD8+ T-cell population (Gros et al., 2014), and it is possible that cells expressing the ‘exhaustion-specific’ markers are in fact highly activated but incompletely differentiated T lymphocytes (Haymaker et al., 2012). Regulatory T cells Of suppressor cell types potentially contributing to the decrease in antitumor immune functions, the best characterized ones are CD4+CD25+FOXP3+ regulatory T (Treg) cells. Treg cells are crucial in preventing autoimmune responses and are also implicated in inhibition of antitumor immune response. Accumulation of these cells in the tumor tissue has been demonstrated in the case of many cancer types (reviewed in Knutson et al., 2007). The prognostic role of tumor-infiltrating Tregs is, however, controversial. While in some cancer types, their increased level has been correlated with poor outcome, in others, on the contrary, a marked infiltration by Tregs showed correlation with improved survival, and in some tumor types, no impact on disease outcome could be found (Gooden et al., 2011; deLeeuw et al., 2012; Senovilla et al., 2012). Few investigations have addressed the prognostic relevance of Treg infiltration in melanoma (Table 1). A study performed on 66 melanoma cases with VGP found ª 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd

Prognostic significance of immune cells in melanoma

that high intra- and peritumoral percentage of Tregs predicted local recurrence, but association with survival was not reported (Miracco et al., 2007). In another study involving 58 patients, no correlation between the number of FOXP3+ lymphocytes and Breslow index or patient survival was found (Hillen et al., 2008). Similarly, in our study on samples from 97 patients with melanoma, we did not find association of FOXP3+ cell density with disease outcome or with any other clinicopathological parameter (Lad
anyi et al., 2010). Natural killer and Natural killer T cells Natural killer (NK) cells, effector cells of the innate immune system, play an important role in the destruction of virus-infected and tumor cells (Smyth et al., 2002). Moreover, they participate in regulation of adaptive immune reactions through interactions with DCs (DegliEsposti and Smyth, 2005). The activation state of NK cells is determined by a balance of signaling pathways triggered by activating (NKG2D, DNAM-1, NKp30, NKp44, NKp46, etc.) and inhibitory receptors (members of the killer cell immunoglobulin-like receptor family, CD94/NKG2A, etc.). Their amount in solid tumors is generally low, and NK cells supposedly play a role mainly in systemic antitumor defense in hampering the development of hematogenous metastases. Furthermore, tumor-infiltrating NK cells are often characterized by low expression of activating receptors and decreased functional activity (Fregni et al., 2012; Platonova et al., 2011). This could be caused by the effect of immune suppressive factors produced by tumor cells or neighboring stromal cells, which has been described in several tumor types including melanoma (Balsamo et al., 2009; Pietra et al., 2012). On the contrary, elevated number of NK cells was associated with favorable outcome in some tumor types (Fregni et al., 2012; Senovilla et al., 2012). It has to be noted, however, that most studies applied antibodies against the CD56 or CD57 markers, which are expressed by other cell types besides NK cells, while the use of the NKp46 marker, considered more NK-specific, has been gaining ground only recently (Platonova et al., 2011; Remark et al., 2013). Although their presence was documented in close proximity to melanocytic lesions (Balsamo et al., 2012; Fregni et al., 2013; McKay et al., 2011), the prognostic impact of NK cell infiltration in melanoma has not been investigated. Natural killer T cells express, besides NK cell markers (such as CD56), markers characteristic of T cells, for example, CD3 and the classic (ab) T-cell receptor. Similar to NK lymphocytes, these cells are generally rare in solid tumors, and few reports are available on their prognostic role (Senovilla et al., 2012). B lymphocytes With a few exceptions, B lymphocytes are generally present in solid tumors in a relatively low amount. The role of antibody-dependent immunity in antitumor ª 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd

immune response is not clear, and it is still a matter of debate how systemic B-cell response or in situ B-cell accumulation influences the biological behavior of tumors. In some experimental tumor models, B lymphocytes were shown to enhance tumor growth or progression (Qin et al., 1998; de Visser et al., 2005); other studies, however, led to opposite results (DiLillo et al., 2010). The discrepancy regarding the systemic pro- or antitumor effects of B lymphocytes may be related to the diversity of their functional activities (Nelson, 2010). They can promote tumor growth via antibodies or immune complexes and secrete other immune inhibitory factors; on the other hand, B lymphocytes may also function as effective antigen-presenting cells promoting antitumor T-cell response. Similar to other tumor types, T lymphocytes predominate in melanoma infiltrates, with B-cell frequencies reported between ~15 and 20% of all infiltrating lymphocytes (Hillen et al., 2008; Hussein et al., 2006). In our studies on 106 primary melanomas, the overwhelming majority of B lymphocytes expressing the CD20 marker showed peritumoral location, predominantly dispersed in the stroma surrounding tumor deposits, while B cells organized in dense, follicle-like aggregates were also observed in approximately one-quarter of the samples (Lad
anyi et al., 2011, 2014). These B-cell clusters were associated with T lymphocytes, forming ectopic lymphoid structures, a phenomenon described in several cancer types and in melanoma metastases (Cipponi et al., 2012; Coronella-Wood and Hersh, 2003; Dieu-Nosjean et al., 2008; Messina et al., 2012). A network of CD21+ follicular DCs was demonstrated in some of the follicle-like structures, and MECA-79+ HEV-like venules were also observed neighboring these in the majority of cases (Lad
anyi et al., 2014). We found that the density of B lymphocytes in the melanoma samples correlated with that of activated (CD25+ or OX40+) T cells. Furthermore, intense B-cell infiltration provided significant survival advantage, and combined analysis of peritumoral densities of B cells and activated T lymphocytes revealed very poor prognosis in the case of low amount of both cell types (Lad
anyi et al., 2011). The appearance of B-cell aggregates did not show association with the outcome of the disease, although a trend for their higher prevalence was observed in thicker tumors (Lad
anyi et al., 2014). An earlier study involving smaller number of cases did not find the amount of infiltrating B lymphocytes of prognostic impact (Hillen et al., 2008) (Table 1). Dendritic cells As the most potent antigen-presenting cells, DCs play key roles in the immune response against tumors via processing of tumor-associated antigens and their presentation to CD4+ and CD8+ T lymphocytes. DCs constitute a complex system of cells displaying considerable heterogeneity in phenotype, anatomical location, and functional abilities 493


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(Yanofsky et al., 2013). In the skin, DC types of myeloid origin include Langerhans cells and dermal DCs. The origin (myeloid versus lymphoid) of plasmacytoid DCs (pDCs), the third subset of DCs, is debated (Facchetti et al., 2003; Yanofsky et al., 2013). After acquiring and processing the antigens and after exposure to microbial products, cytokines or CD40L, DCs undergo maturation, resulting in enhanced expression of MHC and costimulatory molecules, cytokine secretion, and increased ability to efficiently prime T cells. Immature or incompletely matured DCs, however, may mediate tolerance instead of immune activation, a phenomenon that has been described in patients with melanoma among others (Enk et al., 1997). In accordance with their major role in the development of antitumor immune response, the presence of a dense DC infiltration has shown positive correlation with survival in the case of most human tumor types studied (Senovilla et al., 2012). In melanoma, our study on primary tumor samples from 82 patients (Lad
anyi et al., 2007) demonstrated that CD1a+ DCs can be found both infiltrating melanoma cell nests and in the surrounding stroma, while mature DCs expressing the DC-LAMP marker are generally confined to the peritumoral areas, associated with lymphocytic infiltrates. For both DC markers, the amount of labeled cells showed strong inverse correlation with the thickness of melanomas, however, the association with patients’ survival was more pronounced in the case of mature DCs (Lad
anyi et al., 2007). The connection between DC-LAMP+ DC infiltration and survival was supported by a recent study involving 186 patients with melanoma (Jensen et al., 2012) (Table 1). Recently, peritumoral presence of CD1a+ DCs was found to correlate with the absence of hematogenous spread (Emri et al., 2013). Similar to that observed in the case of B lymphocytes, the density of DCs showed correlation with that of activated T cells. Combined evaluation of the amount of CD1a+ or DC-LAMP+ DCs and CD25+ or OX40+ lymphocytes demonstrated favorable outcome of high DC/high activated T-lymphocyte density cases compared to other subgroups (Lad
anyi et al., 2007). Solid tumors also contain plasmacytoid DCs which, besides their antigen presentation capacity, may also promote antitumor immune response via secretion of type I interferons (Salio et al., 2003). However, pDCs in the tumor microenvironment are supposed to be predominantly immune suppressive (Demoulin et al., 2013). Similar to results obtained with other tumor types, a study involving 186 primary melanoma cases found high density of CD123+ pDCs in the tumor nests and tumor stroma a predictor of dismal prognosis (Jensen et al., 2012) (Table 1).

processes frequently associated to tumors. Macrophages form a heterogeneous group of cells and, depending on the nature of stimuli they receive, possess diverse functional characteristics. Classical (M1) macrophages possess high antigen-presenting capacity; they can stimulate type I polarized T-cell response and efficiently contribute to immune defense against viral and microbial infections and tumors. They also exert direct antitumor cytotoxic activity by releasing toxic substances (Allavena et al., 2008; Sica et al., 2006). M2 macrophages, on the other hand, possess weak antigen-presenting capacity and have a predominant role in scavenging debris, tissue remodeling, and angiogenesis (Allavena et al., 2008; Sica et al., 2006). They promote Th2 and Treg type polarization, stimulating antibody production but inhibiting CD8+ T-cell and NK cell activity. The simplified M1/M2 classification represents the two extremes in a wide spectrum of functional activity of macrophages. According to the prevailing view, TAM resemble M2 macrophages due to factors expressed in the tumor microenvironment, favoring M2 polarization (Allavena et al., 2008; Sica et al., 2006). They can promote tumor growth directly, or indirectly, by stimulating angiogenesis (Allavena et al., 2008; Dirkx et al., 2006), increase tumor cell migration, invasion, and metastasis formation via releasing matrix degrading enzymes, cytokines, and chemokines. Furthermore, they may hamper development of antitumor immune response by secreting immune suppressive substances (Allavena et al., 2008; Coffelt et al., 2009; Sica et al., 2006). In line with their above-mentioned activity favoring tumor growth and progression, strong infiltration of TAM was found to be a sign of unfavorable disease outcome in most tumor types, although the opposite effect was also observed in some cases (Senovilla et al., 2012; Zhang et al., 2012), presumably due to the diverse functional activity of macrophages. In the case of cutaneous melanoma, earlier smaller scale investigations did not show significant association with patients’ survival (Hillen et al., 2008; Kiss et al., 2007; Piras et al., 2005) (Table 1). In the study of Jensen et al. (2009) on a larger cohort of patients (190 cases), high amount of CD68+ macrophages at the invasive front and high stromal density of those expressing the CD163 marker proved to be poor prognostic factors. These results were corroborated by a recent report (Emri et al., 2013). Another large scale investigation (202 patients), however, did not find significant correlation between TAM number and survival, although it did reveal association with certain clinicopathologic parameters known as adverse prognostic factors such as tumor thickness and ulceration (Storr et al., 2012). Similar to some other tumor types, according to the latter study, macrophage density correlated with microvascular density (Storr et al., 2012).

Macrophages Most solid tumors are abundantly infiltrated by macrophages, playing a major role in chronic inflammatory

Granulocytes and mast cells Besides macrophages, neutrophil granulocytes are also key mediators of tumor-associated inflammatory

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Prognostic significance of immune cells in melanoma

processes. Although in an activated state they are able to destroy malignant cells via production of cytotoxic mediators, the effect of tumor-associated neutrophils is skewed in the direction of promoting tumor progression. They produce, for example, cytokines and growth factors increasing tumor growth directly or via stimulating angiogenesis, and matrix degrading proteases, thus fostering invasion and metastasis formation (Houghton, 2010; Piccard et al., 2012; Ribatti and Crivellato, 2009). Accordingly, high amount of infiltrating neutrophil granulocytes was found to predict poor prognosis in most tumor types studied (Senovilla et al., 2012). Similar results were obtained by a study of Jensen et al. (2012), based on detection of CD66b in primary melanoma samples from 186 patients (Table 1). The presence of eosinophil granulocytes, on the other hand, is generally associated with favorable prognosis (Senovilla et al., 2012); therefore, they are supposed to predominantly exert antitumor effects mediated by secretion of cytotoxic substances or via activation of other effector cells; the exact mechanisms, however, are not known yet. Their prognostic effect in melanoma has not been studied, and their prevalence in this tumor type is low. Infiltration by basophil granulocytes is uncommon in tumors. On the other hand, mast cells that resemble basophils in several aspects and are considered their tissue-resident counterparts are frequently found in the tumor microenvironment and play an important role in the regulation of inflammatory and immune processes. Being multifunctional cells, mastocytes exert diverse effects on tumor development. They can contribute to elimination of tumor cells via secretion of cytotoxic molecules or indirectly, through stimulating other immune cells. However, they can also exert protumor functions directly by producing growth factors and cytokines or via promoting the development of chronic inflammation. Moreover, they secrete numerous proangiogenic factors and matrix degrading enzymes, stimulating vascularization, invasion, and metastasis (Khazaie et al., 2011; Ribatti and Crivellato, 2009, 2012). Data on the prognostic impact of mast cell infiltration in tumors are controversial (Khazaie et al., 2011; Ribatti and Crivellato, 2012; Senovilla et al., 2012). Several studies demonstrated correlation of their amount with tumor progression and also with microvascular density, while others showed association with favorable outcome of the disease. Few studies, performed on relatively small cohorts of patients, have been reported on cutaneous melanoma (Table 1). Schadendorf et al. (1995) did not find connection between peritumoral mast cells and tumor thickness or disease outcome. On the contrary,
th et al. (2000) demonstrated higher number of these To cells in aggressive melanomas and found a correlation with microvascular density. ª 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd

Prognostic importance of immune cells infiltrating melanoma metastases Few studies, performed mostly on lymph node, cutaneous, and subcutaneous metastatic samples, have dealt with the prognostic role of immune cells infiltrating melanoma metastases. The amount of total TIL (Bogunovic et al., 2009; Mihm et al., 1996; Taube et al., 2012) or CD3+ T cells (Bogunovic et al., 2009) showed correlation with patients’ survival. In another study based on a larger cohort of patients (147 cases), increased number of CD3+ and CD8+ T lymphocytes as well as that of CD20+ B cells predicted favorable outcome, while no association was found in the case of cells expressing other markers (CD4, CD56, CD163, DC-LAMP) (Erdag et al., 2012).

Role of tumor-infiltrating immune cells in predicting the efficacy of melanoma immunotherapy Besides its prognostic role, characterization of immune cells in the tumoral microenvironment may also be useful in predicting efficacy of various types of cancer immunotherapy. Response to interleukin-2-based immunotherapy of patients with metastatic melanoma was associated with increased T-cell and macrophage infiltration in posttreatment biopsies (Rubin et al., 1989). The occurrence of CD4+, but not of CD8+ or CD11c+ cells in pretreatment biopsies correlated with therapeutic benefit of IFN-a treatment of systemic or regional metastatic melanoma (H akansson et al., 1996). Higher numbers of CD4+ and CD8+ T cells, as well as granzyme B+ lymphocytes, were found in metastases of non-progressing patients compared to progressors in a recent study with a melanoma cell vaccine (Tjin et al., 2014). Interestingly, activation of immune-related genes and increased T-cell infiltration was observed in regressing lesions compared to progressing ones in patients with melanoma treated with interferon or a melanoma vaccine (Carretero et al., 2012). Recently, with the introduction of immune checkpoint inhibitors in the treatment of melanoma and other cancers (Pardoll, 2012), considerable effort has been made to find biomarkers that could help identify patients likely to react to the treatment. Most experience has been gained with ipilimumab, a monoclonal antibody blocking cytotoxic T-lymphocyte-associated antigen-4 (CTLA-4). Several potential biomarkers have been investigated, mainly with regard to peripheral blood immune cells or serum factors (Ascierto et al., 2013). The predictive role of immune cell infiltration at tumor sites was less intensely studied. In melanoma, ipilimumab treatment enhanced the expression of immune-related genes in metastatic tumor samples, and high baseline expression level of these genes predicted clinical response (Ji et al., 2012). In the same trial, treatment-induced increase in TIL, as well as strong baseline staining for FOXP3 and IDO, was found to correlate with clinical activity, while no 495


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such association was observed in the case of pretreatment TIL, CD4, CD8, CD45RO, granzyme B, or perforin (Hamid et al., 2011). Tendency for correlation of baseline or post-treatment density of CD20+ B lymphocytes with clinical response and survival was found in patients receiving neoadjuvant ipilimumab, with no associations observed for other markers studied (CD4, CD8, CD11c, CD45RO, CD86, FOXP3) (Tarhini et al., 2014). Tremelimumab, another anti-CTLA-4 antibody, also enhanced the infiltration of CD4+, CD8+, HLA-DR+/CD45RO+, and FOXP3+ cells in metastatic melanoma biopsy specimens, irrespective of response to treatment (Huang et al., 2011). In the case of antibodies blocking the PD-1 pathway, the other most frequently used class of immune checkpoint inhibitors, most studies focused on examination of tumor cell expression of PD-L1, the major PD-1 ligand, which was generally found predictive of treatment response in patients with melanoma or various types of cancer (Taube et al., 2014; Topalian et al., 2012; Weber et al., 2013). Recently, Taube et al. (2014) demonstrated in patients treated with nivolumab that tumor cell PD-L1 expression correlated with the intensity of immune cell infiltration, the presence of CD20+ B cells and of lymphoid aggregates, and also with PD-1 expression by infiltrating immune cells. None of the studied markers of immune activity, however, showed significant association with response to treatment (Taube et al., 2014). Evidence has also been accumulating on the contribution of immune mechanisms to the effect of non-immune based anticancer therapies, in part from preclinical studies using agents known to induce immunogenic cell death, showing better therapeutic responses in immunocompetent compared with immune deficient mice (Zitvogel et al., 2008). In the clinical setting, the efficacy of several currently used anticancer agents was demonstrated to depend on the contribution of immune mechanisms (Denkert et al., 2011; Zitvogel et al., 2008, 2011). In the case of melanoma, dacarbazine chemotherapy was shown to increase T-cell infiltration, which was associated with longer survival (Nardin et al., 2011). BRAF inhibitor treatment also induced T-cell infiltration and enhanced melanoma antigen expression in metastatic melanoma (Frederick et al., 2013; Wilmott et al., 2012).

Conclusions Tumor microenvironment is shaped by interactions between malignant cells and host cells representing an integral component of solid tumors. Immune cells can exert both positive and negative effects on the outcome of the disease. Tumor-associated inflammation, characterized by accumulation of leukocytes, the presence of cytokines and chemokines secreted by these (and other) cells, active tissue remodeling and angiogenesis, creates conditions favoring tumor growth, invasion, and metastasis. In the case of TAM, mast cells and neutrophil granulocytes, playing important roles in these processes, 496

effects promoting tumor progression predominate, and their intense infiltration in tumors generally indicates poor prognosis. Development of an efficient antitumor immune response, on the other hand, decreases the probability of tumor recurrence and metastasis formation, thus positively influencing the outcome of the disease. Accordingly, high density of antigen-presenting DCs and T lymphocyte subsets, playing key roles in this process, is mostly accompanied by favorable prognosis. For several immune cell types, the effects on disease outcome may depend on the type and stage of the tumor, and on the location of the infiltrate within and around the lesions. Moreover, the impact of the presence of immune cells is influenced by their functional heterogeneity, and by interactions with each other and with other constituents of the tumor microenvironment. Investigation of tumorinfiltrating immune cell types at any time point could provide only a snapshot on a tumor microenvironment, which constantly changes in the course of the disease and its treatment. In melanoma, most studies on the prognostic impact of immune cells were based on determination of the amount of lymphoid infiltrate in general (without distinguishing lymphocyte subsets), yielding rather controversial results. Similarly, definite conclusions cannot be drawn from the few, mostly small-scale studies aiming at determining the prognostic correlates of the density of T lymphocytes (Table 1). In our studies, high peritumoral density of T cells expressing the activation markers CD25 or CD134 correlated with favorable prognosis. Similar to other tumor types, T lymphocytes represent the dominating cell type in the lymphoid infiltrate of melanoma, but significant amount of B cells could also be observed in the majority of cases, and their increased infiltration predicted favorable prognosis in our study. Density of B lymphocytes correlated with that of activated T cells, and subgroups obtained by combined analysis of the two cell types had different survival probability, raising the possibility that B lymphocytes in the tumor microenvironment may function as antigenpresenting cells promoting the antitumor activity of T cells. Infiltration of DCs, considered the most efficient antigen-presenting cells, and especially that of mature DCs, was also found associated with disease outcome. Similar to B cells, their density correlated with that of activated T lymphocytes, and concordant high number of the two cell types defined a patient subgroup with significant survival advantage. According to data from the literature, intense infiltration by plasmacytoid DCs or neutrophil granulocytes could be associated with shorter survival, while no unambiguous conclusions can be drawn from studies on the prognostic value of other immune cell types (Table 1). Taken together, these results indicate that a prominent infiltration of mature DCs, B cells, and activated T lymphocytes can be considered as favorable, while that of plasmacytoid DCs or neutrophil granulocytes as ª 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd

Prognostic significance of immune cells in melanoma

unfavorable prognostic factors in malignant melanoma. Peritumoral B-cell or DC density in combination with the number of activated T lymphocytes could identify patient subgroups with different disease outcome, suggesting a possible role of these immune cell types in a functional immune response against melanoma, reflected in better outcome of the disease. Besides its potential prognostic relevance, understanding the mechanisms and key players in the development of an efficient antitumor immune response could also be useful in identifying new targets for immunotherapeutic approaches as well as biomarkers for predicting response to therapy. Although no validated predictive biomarkers are available yet for use in the various immunotherapy protocols, several candidates have been suggested, with much effort being put lately on immune checkpoint inhibitor treatment. Data collected so far indicate the presence of an immunologically more active tumor microenvironment in immunotherapy responders, with a simultaneous presence of immune suppressive factors, probably representing natural negative feedback mechanisms of the immune system. Future studies on larger patient cohorts are needed to corroborate results obtained on immune biomarkers as well as to search for potential new markers that can predict disease outcome in the case of the various immunotherapeutic modalities. Recent studies demonstrated the immune stimulatory capacity of conventional anticancer therapies, which, until recently, were believed to cause immune suppression, and a role of immune activation in influencing their therapeutic effects. Understanding the mechanisms of interplay between the immune system and cytotoxic or targeted tumor therapies could provide immunotherapy targets for application in combination with these treatment modalities. Besides their importance in giving new insights in the mechanism of action of antineoplastic therapies, studies evaluating tumor-infiltrating immune cells in patients treated with chemo-, radio-, immune or targeted therapy could reveal new biomarkers that can be used in predicting the efficacy of the treatment and the outcome of the disease.

Acknowledgements The author would like to thank Dr. Klara Balint for critical review of the manuscript. The work was supported by the National Scientific Research Fund OTKA grant 105132.

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