To the best of our knowledge, this study assessed, for the first time, the abundance and distribution of innate and adaptive cellular elements according to CD8 T cells, FoxP3 Treg and CD68 macrophages in a series of 258 patients with HNSCC in order to define a more global immune contexture. Then, we investigated their potential prognostic value separately and in combination to stratify patients using a low immunoscore corresponding to a longer RFS and OS, whereas a high immunoscore was related to a poorer prognosis. Our results confirm that the establishment of an immunoscore has a higher prognostic value than that of TNM staging system and histological grade.
For many years, clinical research around head and neck cancers has been constantly asking for new prognostic biomarkers to better guide patient management. Given the complexity of the interactions between immune infiltrates and the TME, the tumour must no longer be considered as a single entity but must be studied in relation to its microenvironment and the host immune response in order to bring clinical relevance and value in determining the tumour progression and the patient prognosis. As such, many clinicians and researchers have been interested in the infiltration of immune cells in head and neck cancers. In most cases, massive infiltration of CD8 + T lymphocytes in HPV- as well as in HPV + oropharyngeal carcinomas correlates positively with patient prognosis (Nordfors et al. 2013; Oguejiofor et al. 2015; Ward et al. 2014; Sivars et al. 2017; Poropatich et al. 2017). CD8 + TILs have also been established as an independent prognostic marker in patients diagnosed with oropharyngeal squamous cell carcinoma (De Meulenaere et al. 2017). Recently, Echarti et al. confirmed these findings by quantifying CD8 + and FoxP3 + T lymphocytes in epithelial and ST compartments (Echarti et al. 2019). The relationship between tumour-infiltrating Treg and patient prognosis has been evaluated in many malignancies and associated with different prognostic response according to the sites of primary cancer. The prognostic significance of FoxP3 + Tregs has been extensively studied (Shang et al. 2015) and high Treg cell recruitment is reported to correlated with poor prognosis in breast, liver, pancreatic, ovarian, cervical and renal cancer (Merlo et al. 2009; Fu et al. 2007; Hiraoka et al. 2006; Curiel et al. 2004; Shah et al. 2011; Siddiqui et al. 2007) whereas it may also correlate with longer survival as demonstrated in colorectal, bladder cancer, HNSCC and lymphoma (Seminerio et al. 2019; Salama et al. 2009; Winerdal et al. 2011; Badoual et al. 2006; Tzankov et al. 2008). The meta-analysis of Shang et al. compares 76 datasets which highlight the prognostic role of FoxP3 + in 17 cancer types (Shang et al. 2015). For HNSCC, Treg infiltration is highly controversial with conflicting results. While several studies underline the deleterious impact of massive infiltration by Tregs on prognosis, we and others have already showed the opposite (Badoual et al. 2006; Bron et al. 2013). Indeed, we previously reported that FoxP3 + infiltration was associated with longer RFS and OS of patients suffering from HNSSC (Seminerio et al. 2019; Kindt et al. 2017; Shang et al. 2015). The concomitance which is often observed between Treg and cytotoxic T infiltrates could tip the balance towards a favourable immune orientation and consequently participate in the good paradoxical prognostic value of this population. In addition, HNSCC are heterogeneous tumours located in certain anatomical sites rich in lymphoid tissue such as oropharynx, which may explain a greater recruitment of Treg in this site. Importantly, a crucial point of the debate was elucidated in 2016 when Saito et al. demonstrated the existence of two populations of FoxP3 + Tregs in colorectal cancers (Saito et al. 2016). The first one was the immunosuppressive FoxP3high-expressing cells classically associated with a poor prognosis and the second was characterized by non-suppressive capacities and the absence of expression of CD45 (FoxP3low CD45RA-), the naïve T cell marker. These two populations are significantly correlated with opposing prognoses in colorectal cancer. While the first is associated with a poor prognosis, the infiltration of FoxP3low, which secrete pro-inflammatory cytokines such as IL-12 and TGFβ, is characteristic of better patient survival (Saito et al. 2016). In addition, we suggest another hypothesis that could explain the infiltration of FoxP3 + Tregs in head and neck cancers. Indeed, they grow in a septic environment in contact with a resident microbiota, just like colon cancers which are also associated with a better prognosis when the number of Treg is high. This microbiota interacts and may modulate the host oral immune cells and such alterations in Treg functions have already been observed in oral infections (Ladoire, Martin, and Ghiringhelli 2011; Pandiyan et al. 2019). Moreover, studies demonstrated the protective effects of oral FoxP3 + Treg in some local infections (Yamazaki et al. 2012). Given the crosstalk between these immunosuppressive regulatory cells and the cytotoxic lymphocytes, which are the anticancer mediators of the immune system, it seems crucial to consider these two entities in combination in order to better understand the reasons for the contradictory results reported in the literature.
Macrophages also constitute an important partner in innate and adaptive inflammatory responses. Beyond the binary classification of pro-inflammatory M1 and anti-inflammatory M2 macrophages, it is now accepted that these two phenotypes are only the extremes of a continuum of polarization, in which there is a spectrum of differentiated macrophages (Palma et al. 2018). Among these differentiated macrophages, TAMs are involved in immune tolerance, inflammatory disease and cancer (Aras and Zaidi 2017) and are considered as pro-tumorigenic immune cells. They stimulate Treg differentiation and secrete several factors (e.g. TGFβ, TNFα and IL-10) to create a favourable environment for tumour progression and to inhibit the anti-tumour effects of immune cells (Jérôme R. Lechien et al. 2020). Regarding prognosis, tumour infiltration by TAMs is reported to be an unfavourable parameter for patient survival (Esch et al. 2015; Davis, Van Waes, and Allen 2016; Costa et al. 2013). Our recent study has shown that a high recruitment of CD68 + macrophages in a population of 110 HNSCC was correlated with shorter patient RFS and OS. Moreover, the analysis of the M1/M2 ratio in the TME, with a double staining using anti-CD68/anti-CD163 antibodies, revealed that 80% of the macrophage population had an M2 phenotype (Seminerio et al. 2018). Interestingly, it appears that TAMs can secrete IL-10 in order to induce the differentiation of T lymphocytes into Treg (Murai et al. 2009) and thus participate in immune cell evasion.
The existence of complex regulatory loops between these three major mediators of the immune system has led us to quantify their recruitment in ST and IT localizations in a large cohort of head and neck tumours. Indeed, we hypothesized that analysing each tumour compartment (ST versus IT) may provide distinct and complementary prognostic information. This was also supported in the context of rectal cancer where the location of CD8 + T cells and FoxP3 + Treg cells in distinct compartments (epithelium versus stroma) result in different prognostic responses (Posselt et al. 2016). Combining the three markers in an immunoscore signature, we found that a low immunoscore was significantly associated with a longer RFS and a prolonged OS. Based on the calculated optimal cut-offs, the IT immune infiltrations associated with a better prognosis correspond to a high number of CD8 and FoxP3 and a low number of CD68 macrophages. Conversely, in the ST, a better prognosis was observed in patients with a low CD8 infiltration but always a high number of FoxP3. Because of the tumour lysis capacity of CD8 cells, these anticancer actors are strong allies for cancer patients. On the other hand, despite their anti-tumour response suppressor characteristics, Tregs infiltrates have been found to be associated with a favourable outcome, which may be partially attributed to a downregulation of the inflammatory process (Shang et al. 2015; Badoual et al. 2006). A correlation had also been found between a higher number of Treg in the stroma and an absence of metastatic lymph nodes which means that Treg could generate pro-inflammatory processes in the tumour microenvironment favouring a delay in the tumour evolution and consequently would generate a better prognosis of the patients (Bron et al. 2013). Furthermore, Khoury et al. recently proposed that IT TILs were distinct from ST TILs in their biological behaviour (Khoury et al. 2018). Indeed, ST is constituted of many components which can impair host immune responses, such as fibroblasts, macrophages, or endothelial cells, underlining the difference in CD8 TILs located in the ST from CD8 + TILs within tumour cells.
Finally, our multivariate analysis, evaluating the tumour stage, the histological grade and the immunoscore regarding to OS, revealed an impressive HR for immunoscore which can significantly predict the OS of patients. By contrast, in head and neck cancer, only a few studies have identified an immunoscore positively correlating with RFS and OS. In two studies, immunoscore only included TILs, and more precisely CD3 + and CD8 + cells (Lechner et al. 2017; Zhang et al. 2018). A recent study confirmed the positive prognostic impact of TILs in oral squamous cell carcinomas by increasing the immune signature to seven markers that can predict patient survival (Zhou et al. 2020).