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Original article
Prognostic role of tumour-associated macrophages and regulatory T cells in EBV-positive and EBV-negative nasopharyngeal carcinoma
  1. Marc L Ooft1,
  2. Jolique A van Ipenburg2,
  3. Maxime E Sanders1,
  4. Mariette Kranendonk1,
  5. Ingrid Hofland3,
  6. Remco de Bree4,
  7. Senada Koljenović2,
  8. Stefan M Willems1
  1. 1 Department of Pathology, University Medical Center Utrecht, Utrecht, The Netherlands
  2. 2 Department of Pathology, Erasmus MC, University Medical Center Rotterdam, Rotterdam, The Netherlands
  3. 3 Department of Pathology, Core facility Molecular pathology and Biobanking, Netherlands Cancer Institute Antoni van Leeuwenhoek, Amsterdam, The Netherlands
  4. 4 Department of Head and Neck Surgical Oncology, UMC Utrecht Cancer Center, University Medical Center Utrecht, Utrecht, The Netherlands
  1. Correspondence to Stefan M Willems, Department of Pathology, University Medical Center Utrecht, Heidelberglaan 100, 3584 CX Utrecht, The Netherlands; S.M.Willems-4{at}umcutrecht.nl

Abstract

Aims Tumour-associated macrophages (TAMs) and regulatory T cells (Tregs) form a special niche supporting tumour progression, and both correlate with worse survival in head and neck cancers. However, the prognostic role of TAM and Tregs in nasopharyngeal carcinoma (NPC) is still unknown. Therefore, we determined differences in TAMs and Tregs in different NPC subtypes, and their prognostic significance.

Methods Tissue of 91 NPCs was assessed for TAMs and Tregs by determination of CD68, CD163, CD206 and FOXP3 expression in the tumour microenvironment. Clinicopathological correlations were assessed using Pearson X2 test, Fisher’s exact test, analysis of variance and Mann-Whitney U test. Survival was analysed using Kaplan-Meier curves and Cox regression.

Results CD68 and FOXP3 counts were higher in Epstein-Barr virus (EBV)-positive NPC, while CD68−/FOXP3−, CD163+/FOXP3− and CD206+/FOXP3− infiltrates were more common in EBV-negative NPC. In the whole NPC group, CD68−/FOXP3− correlated with worse overall survival (OS), and after multivariate analysis high FOXP3 count showed better OS (HR 0.352, 95% CI 0.128 to 0.968). No difference in M2 counts existed between EBV-positive and negative NPC.

Conclusions FOXP3, a Treg marker, seems to be an independent prognostic factor for better OS in the whole NPC group. Therefore, immune-based therapies targeting Tregs should be carefully evaluated. M2 spectrum macrophages are probably more prominent in EBV-negative NPC with also functional differences compared with EBV-positive NPC.

  • oncology
  • immunohistochemistry
  • immunopathology
  • head and neck cancer

https://doi.org/10.1136/jclinpath-2017-204664

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    Introduction

    Nasopharyngeal carcinoma (NPC) is a histologically distinct type of head and neck squamous carcinoma (HNSCC) with a peculiar racial and geographical distribution.1 NPC can be divided into keratinising, non-keratinising and basaloid histological subtypes according to the WHO, but it can also be divided into Epstein-Barr virus (EBV)-positive and negative subgroups.2 3 NPC, especially the non-keratinising subtype, is characterised by dense lymphoid infiltrates, though not much is known about the tumour infiltrating lymphocytes nor about infiltrating cells from the monocyte lineage. It is postulated that the more intense infiltration of non-keratinising NPC is due to EBV which triggers lytic infection in macrophages and creates a feedback loop for T lymphocyte recruitment.4 5 Although tumour-associated macrophages (TAMs) are associated with better survival in certain solid tumours (ie, colon cancer, lung cancer6–9), a worse survival in others is observed (ie, gastric cancer,10 clear cell renal cell carcinoma11 12 and breast cancer13). In HNSCC, TAMs are predominantly related to worse survival.14–20 This is probably due to M2 (alternatively activated) macrophages, which promote tumour growth. TAMs are known to induce regulatory T cells (Tregs) in the tumour microenvironment,21 22 and together they form a special niche which promotes tumour progression.23 Also, targeting and re-polarising of TAMs is seen as a therapeutic strategy.24 This study characterises TAMs and Tregs in the tumour microenvironment of NPC and its different subtypes, and their association with prognosis.

    Materials and methods

    This study complies with the STROBE statement (https://strobe-statement.org). Sequentially diagnosed formalin-fixed paraffin-embedded NPC specimens were collected at the University Medical Center Utrecht (UMCU) and the Erasmus Medical Center (EMC) in the Netherlands. The clinicopathological data were retrieved from electronic medical records. The Dutch national guidelines state that no separate and additional ethical approval is required for the use of anonymous leftover tissue (ww.federa.org), and this is also part of a standard treatment agreement with patients at the UMCU and EMC.25 All H&E histological slides were reviewed by head and neck pathologists (SW and SK) and a pathology resident researcher (MO) experienced in evaluating NPCs. The date at which tissue for diagnosis was obtained was defined as the date of diagnosis. Disease-free survival (DFS) was defined as the period at which recurrence was determined by a physician. The end date for overall survival (OS) was defined as the date of death.

    Tissue microarray construction

    Tissue microarrays (TMAs) from 96 specimens were constructed with a TMA Grand Master instrument (3D HISTECH, Budapest, Hungary). Tumour areas were marked by a pathologist (SW) and pathology resident (MO). Three cores (0.6 mm) (central tumour areas) were punched from the marked tumour areas and arrayed into a recipient TMA receptor block.

    EBV status

    EBV status was determined by applying EBV-encoded RNA (EBER) in situ hybridization (ISH) to the TMA. A BenchMark ULTRA automated staining instrument (Ventana Medical systems, Tuscon, Arizona, USA) was used for ISH of the TMA using a EBV-specific probe (INFORM EBER (EBV Early RNA) PROBE, Ventana Medical systems) and ISH iVIEW Blue detection kit (Ventana Medical Systems) for staining according to the manufacturer’s instructions.

    Immunohistochemistry

    The BenchMark ULTRA automated staining instrument (Ventana Medical systems) was used for immunohistochemical staining of the four antibodies. CD68, an antigen expressed by cytoplasmic lysosomes in M1 and M2 macrophages26 (Novacastra, CC1 24’, mouse polyclonal, 1:1600); CD163, a transmembrane protein highly expressed by M2 macrophages27 (Novacastra, CCL 24’, mouse polyclonal, 1:400); CD206, a mannose receptor expressed on also M2 macrophages28 (Sigma Aldrich, CC1, rabbit monoclonal, MRC1, 1:500); and FOXP3, a protein expressed by Tregs functioning to suppress the immune system (Abcam, mouse monoclonal, 1:2000) were stained. We additionally investigated densities of macrophage markers and FOXP3, as FOXP3 recruitment by macrophages has been reported,21 22 and because TAMs and Treg co-expression is a possible component of an effective treatment strategy against cancer.29

    For CD68, CD163 and FOXP3, TMA sections of 4 µm thickness were cut, then heated to 75ᵒC for 8 min and deparaffinised by an EZ prep solution. The samples were pretreated with Cell Conditioner at 100ᵒC for 16 min and then a peroxidase inhibitor for 4 min. The subsequent step was applying the primary antibodies on the TMA and incubating it for 32 min. Following application and incubation of the primary antibody, the slides were incubated with Optiview HQ Universal Linker and Optiview HRP multimer (Ventana Medical Systems) for 8 min. The final steps were application of hydrogen peroxide and 3,3'-diaminobenzidine (DAB), and counterstaining with haematoxylin. During each consecutive step of the staining process, the slides were rinsed with reaction buffer. A positive control of normal (anonymised) tonsil tissue was used. For CD206, the TMA paraffin sections were heated at 75°C for 28 min and deparaffinised with EZ prep solution (Ventana Medical Systems). Heat-induced antigen retrieval was carried out using Cell Conditioning 1 (CC1, Ventana Medical Systems) for 64 min at 95ᵒC. Bound antibody was detected using the OptiView DAB Detection Kit (Ventana Medical Systems), and the slides were subsequently counterstained with Hematoxylin II and Bluing Reagent (Ventana Medical Systems). The images of the stains are depicted in figure 1.

    Figure 1
    Figure 1

    Positive Epstein-Barr virus-encoded RNA stain (A) in NPC (×40). Nuclear FOXP3 stain (B), cytoplasmic CD68 stain (C), membranous CD163 stain (D) and membranous CD206 stain (E) (×40). H&E of a non-keratinising NPC ×40 (F). The number of positive staining cells was manually counted in fixed tissue area (ie, tissue microarray cores with a diameter of 0.6 mm each). NPC, nasopharyngeal carcinoma.

    Quantification of tumour infiltrating lymphocytes

    A pathologist (SW) and researcher (MS) blinded to the clinical characteristics evaluated the stained slides. The number of positive cells were quantitatively counted at ×200 magnification in fixed areas (ie, cores with 0.6 mm diameters). If at least half of a core was available, the data were extrapolated to represent a full core. If less than half a core was available, data for that core were excluded from further analysis. To be included in the data analysis, at least one core per sample was needed. The mean count per sample was calculated. A receive operator characteristic (ROC) curve analysis, optimised for OS, was used to determine a cut-off value per marker as has been done before.30–32 The cut-off was validated by determining optimal significance of the split in the Kaplan-Meier plot.

    Statistical analysis

    IBM SPSS Statistics software V. 22 (SPSS, Chicago, Illinois, USA) was used to analyse the data. Distributions were tested for normality using the Shapiro-Wilks test (if n<50) or the Kolmogorov-Smirnov test (if n>50), and parametric or non-parametric tests were used accordingly. The likelihood of univariable independence between EBV-positive and negative groups was performed using the Pearson X2 test (or the Fisher’s exact test) and analysis of variance. To assess differences in counts per marker, we used the Mann-Whitney U test. Survival was assessed using the Kaplan-Meier method, log-rank test and Cox proportional hazard model. Cases with M1 stage were excluded from survival analysis. Two tailed p Values<0.05 were considered statistically significant. The following categories were further dichotomised for survival analysis: age (≤53 to >53 years), T1/2-T3/4, N0-N1/2/3, smoking, alcohol usage, NPC histology and EBV status. To correct for multiple testing, the Holm-Bonferroni correction was also applied.

    Results

    Clinicopathological characteristics

    Five samples had quantitatively inadequate material for evaluation. Thus, 91 biopsy specimens were included for further analysis (see table 1). The follow-up time for the EBV-negative NPCs was 43 months for OS and 36 months for DFS, for the EBV-positive NPCs 57 months for OS and 50 months for DFS, and for the whole NPC group 52 months for OS and 45 months for DFS. There was no significant difference in follow-up time between EBV-positive and negative NPCs.

    View this table:
    Table 1

    Clinicopathological features

    Microenvironment markers

    The optimal cell count cut-offs calculated with ROC curves were 114 for CD68, 61 for CD163, 29 for CD206 and 42 for FOXP3.

    Whole NPC group. CD68 correlated with CD163 (p<0.001) and FOXP3 (p=0.004). CD163 correlated with FOXP3 (p=0.004). CD206 correlated with CD68 (p=0.041)and CD163 (p=0.008). See annex 1.

    EBV-positive NPC. CD68 correlated with CD163 (p=0.012). CD163 correlated with FOXP3 (p=0.015). CD206 correlated with CD68 (p=0.025), CD163 (p=0.006) and FOXP3 (p=0.017). See annex 1.

    EBV-negative NPC. CD68 correlated with CD163 (p=0.012) and FOXP3 (p=0.009). See annex 1.

    Tumour and microenvironment markers

    EBV-positive NPC compared with EBV-negative NPC. EBV-positive NPCs contained significantly higher CD68 and FOXP3 counts (see figure 2), and after the data were dichotomised, CD68 count (p=0.001, 71% vs 33%) and FOXP3 count (p=0.004, 77% vs 44%) remained higher. CD68+/FOXP3+ (p=0.009, 53% vs 24%), CD163+/FOXP3+ (p=0.004, 65% vs 32%) and CD206+/FOXP3+ (p=0.004, 55% vs 35%) were also more common in EBV-positive NPC. CD68−/FOXP3− (p<0.001, 45% vs 7%), CD163+/FOXP3− (p=0.016, 32% vs 11%) and CD206+/FOXP3− (p=0.003, 35% vs 7%) were more common in EBV-negative NPC. No significant differences were found for the mean values of CD206 and CD163 between the EBV-positive and negative NPC.

    Figure 2
    Figure 2

    To assess differences in CD68 and FOXP3 counts between the EBV-positive and EBV-negative NPCs, we used the Mann-Whitney U test. EBV, Epstein-Barr virus; NPC, nasopharyngeal carcinoma.

    Clinicopathological features and microenvironment markers

    The mean values of CD68 and FOXP3 were significantly higher in non-keratinising compared with keratinising NPC (see figure 3). CD68−/FOXP3− (p=0.006, 50% vs 14%) and CD163+/FOXP3− (p=0.009, 46% vs 15%) were more common in keratinising compared with non-keratinising NPC.

    Figure 3
    Figure 3

    To assess differences in CD68 and FOXP3 counts between keratinising and non-keratinising NPC, the Mann-Whitney U test was used. NPC, nasopharyngeal carcinoma.

    Whole NPC group. Higher FOXP3 (p=0.005, 80% vs 50%) and CD163+/FOXP3+ (p=0.020, 67% vs 41%) were associated with T1/2 stage, while CD68−/FOXP3− (p=0.002, 34% vs 7%), CD163−/FOXP3− (p=0.010, 24% vs 4%) and CD206+/FOXP3− (p=0.021, 31% vs 10%) were more common in T3/4 stage. CD163−/FOXP3− was associated with no alcohol usage (p=0.020, 21% vs 0%). When there was a positive smoking history, CD163+/CD206+ infiltrates were more common (p=0.027, 85% vs 55%).

    EBV-positive NPC. CD68−/FOXP3− was more common at older age (p=0.016, 18% vs 0%) and in higher T stage (p=0.014, 20% vs 0%).

    EBV-negative NPC. Higher CD68 (p=0.019, 56% vs 0%) and FOXP3 (p=0.007, 62% vs 0%) counts correlated with lymph node metastasis. CD68−/FOXP3− (p=0.001, 100% vs 22%) correlated with lymph node metastasis (N1/2/3), while CD163+/FOXP3− (p=0.021, 71% vs 18%) was more common when there was no lymph node metastasis.

    Survival and microenvironment markers

    NPC group as a whole. A higher FOXP3 count was associated with a better OS (see figure 4a). T3/4 stage was associated with a worse OS (see figure 4b). When FOXP3 and T stage were entered into a multivariate analysis for OS, only FOXP3 remained significant (see table 2). CD68−/FOXP3− infiltrates showed worse OS (see figure 4c) and DFS (see figure 4d). CD163−/FOXP3− (see figure 4e) and CD206−/FOXP3− (see figure 4f) infiltrates had also worse OS.

    Figure 4
    Figure 4

    Rates of survival were calculated with the Kaplan-Meier method and comparison of survival by log-rank test. Two tailed p values below 0.05 were considered statistically significant. EBV, Epstein-Barr virus; NPC, nasopharyngeal carcinoma.

    View this table:
    Table 2

    Cox regression analysis

    EBV-positive NPC. CD206−/FOXP3− infiltrate correlated with worse OS (see figure 4g).

    EBV-negative NPC. CD163−/FOXP3− infiltrates correlated with worse DFS (see figure 4h). Positive smoking history correlated with better DFS (see figure 4i). All HRs are depicted in table 2.

    The individual macrophage markers (ie, CD68, CD163 and CD206) did not correlate with survival.

    Results after the Holm-Bonferroni correction

    Microenvironment markers. In the whole NPC group, CD68 correlated with CD163.

    Tumour and microenvironment markers. EBV-positive NPC contained higher CD68 and FOXP3 counts than EBV-negative NPC. In EBV-positive NPC, CD68 count (71% vs 33%) was higher than in EBV-negative NPC. CD68−/FOXP3− (45% vs 7%) and CD206+/FOXP3− (35% vs 7%) were more common in EBV-negative NPC compared with EBV-positive NPC.

    Clinicopathological features and microenvironment markers. In the whole NPC group, CD68−/FOXP3− was more common in higher T stage (T3 + 4) (34% vs 7%). In EBV-negative NPC, CD68−/FOXP3− (100% vs 22%) infiltrates were more common in cases with no lymph node metastasis.

    Survival and microenvironment markers. In the NPC group as a whole, patients with CD206−/FOXP3− infiltrates had worse OS. In EBV-negative NPC, a positive smoking history correlated with better DFS.

    Discussion

    TAMs are important in cancer development.14 33 34 However, there is conflicting literature regarding the prognostic role of TAMs in cancer. In HNSCC, TAMs are associated with a poor prognosis.14–20 However, in NPC, a special type of HNSCC, the role of TAM is not known. TAMs have predominantly been studied through detection of CD68, which is an antibody recognising macrophages at the M1 (classically activated) through the M2 (alternatively activated) spectrum.35 36 However, through detection of CD163 and CD206, M2 phenotypic spectrum markers,37 38 a distinction can be made from the M1 spectrum. This is important because M1 and M2 TAMs have different roles in the tumour microenvironment with M1, through production of cytokines such as interleukin (IL)-12, IL-1β, tumor necrosis factor (TNF)-α, IL-6 and IL-23, protecting against tumorigenesis. The M1 macrophages protect against tumorigenesis by suppressing activities which dampen the tumour-specific immune response, suppress tumour growth/invasion/metastasis and angiogenesis through various mechanisms,39 40 and induce phagocytosis. On the contrary, macrophages with the M2 line of differentiation, which have been isolated in solid tumours and are probably induced by IL-4 and IL-13 production, promote tumour growth through their association with IL-1, IL-1β, vascular endothelial growth factor and matrix metalloproteinasis.39 The M2 macrophages can subsequently be divided further into M2a, M2b and M2c macrophages depending on their chemokine and chemokine receptor profiles.41

    There is possible co-stimulation between macrophages and FOXP3 as all of the macrophage markers (ie, CD68, CD163 and CD206) correlated with FOXP3 expression. Therefore, induction of FOXP3, indicating promotion of Tregs by TAMs, as has been previously described,21 22 is also the case in NPCs. Furthermore, it has been reported that Tregs together with TAMs form a special niche which promotes tumour progression and metastasis,23 but high co-densities of macrophages and Tregs with metastasis was not found in this study. Co-stimulation of CD68 and FOXP3 might also be a possible component of an effective treatment strategy against cancer.29 Therefore, we assessed the simultaneous high densities of the macrophage markers with FOXP3 staining in relation to prognosis. Positive simultaneous high densities and thus possible co-stimulation, however, did not show a correlation with survival, but we did find that FOXP3 was an independent prognosticator for better OS. However, FOXP3-positive Tregs42 43 are able to suppress immune attack against tumour cells by inhibiting CD4, CD8, natural killer (NK) cell, B cell and antigen-presenting cell function.44 45 FOXP3 counts have additionally been shown to work synergistically with programmed death-ligand 1 (PDL1),46 which is induced in EBV-positive NPC by latent membrane protein 1 (LMP1) through oncogenic pathways and immune modulation.47 Thus, FOXP3-positive Tregs are hypothetically associated with poor prognosis, and thus contradicting the results of this study. An explanation for the favourable correlation between FOXP3 and survival would be the same phenomenon as seen in colorectal cancer48 with the Tregs attenuating the effect of Th17 cells in promoting tumour growth through the IL-6–Stat3 pathway.49 The latter is additionally supported by the fact that in the whole NPC group there was worse survival if macrophage markers and FOXP3 were low. Blockade of Tregs as a therapeutic option in NPC50 should therefore be cautiously studied as FOXP3 has also been shown to be a tumour suppressor.51

    A positive correlation existed in EBV-positive NPCs between the panmacrophage marker CD68 and the M2 phenotypic markers (ie, CD163 and CD206), but no difference in the M2 phenotypic markers was observed between the EBV-positive and negative NPCs. Thus, the higher CD68 count in EBV-positive NPCs with no increase in M2 phenotypic markers leads us to believe that EBV-negative NPC contains higher numbers of anti-inflammatory macrophages, or that M1 phenotypic macrophages may be more prominent in EBV-positive NPCs. The lack of M2 differentiation in EBV-positive NPC is possibly due to the production of BamHI-A rightward frame 1 (BARF1), a viral protein, which has been shown to inhibit macrophage colony stimulating factor, and thus M2 differentiation of macrophages.52 But, it has also been shown that TAMs correlate with EBV infection titres in NPC,53 and in a recent study CD163 was found to correlate with EBV positivity and worse survival in a patient population in China,54 and these findings might indicate patient population differences regarding (epi)genetics, viral (epi)genotypes or environmental factors which could influence the tumour microenvironment. There was a positive correlation between the macrophage markers and FOXP3 as well, which also suggests co-stimulation in this NPC subgroup. Lack of simultaneous CD206 and FOXP3 high densities correlated with worse OS, further indicating a possible role in co-stimulation of FOXP3 and macrophages when it comes to a better prognosis. We furthermore saw a decrease of the immune infiltrate with lower CD68 and FOXP3 counts at older age and higher T stage indicating possible problems with the immune response in these conditions.

    EBV-negative NPC correlated with higher expression of M2 phenotypic markers when FOXP3 expression was low (CD163+/FOXP3− and CD206+/FOXP3−), indicating M2 phenotypic macrophages to play a more prominent role in EBV-negative NPCs. There are also possible functional differences within the M2 subset between EBV-positive and negative NPCs, with EBV negative NPCs being unable to recruit Tregs, as there was no difference in the M2 markers between EBV-positive and negative NPCs, but there was a difference in the number of Tregs, with EBV-positive NPC having higher Treg counts. Thus, different M2 subtypes with other cytokine profiles probably exist between the EBV-negative and positive NPCs.41 Mantovani et al 41 described three different M2 macrophages (ie, M2a, M2b and M2c), with M2b being induced by immune complex exposure, and the M2c phenotype not being associated with Treg recruitment. As has been reported, EBV-positive NPC has a characteristic serological profile with antibodies against the EBV viral capsid antigen and early antigen IgA,55 which could induce M2b polarisation. This argument is supported by the high Treg count in EBV-positive NPC, as M2b macrophages recruit Tregs.41 And as there is a possible recruitment problem of Tregs in EBV-negative NPC, the most likely TAMs in this subgroup would then be the M2c macrophages.

    The results of this study bring us closer to understanding the role of TAMs in NPC, but a few limitations do need to be discussed. Possible tumour heterogeneity could be present due to evaluation of only TMA cores, but because the TMAs were made from biopsy specimens taken during diagnostic workup, almost all available tumour tissues were evaluated, making the results representative of what is normally available during diagnostic workup. The limited number of EBV-negative NPCs evaluated could have led to false-negative results. Independence of variables could not be evaluated adequately due to the low number of events in the survival analysis. Another limitation of this study is that the macrophages were categorised only through protein expression. It is also important to recognise that categorisation of macrophages in M1 and M2 subsets is an oversimplification, and that there is considerable range between the two subsets. TAMs in central tumour areas were studied as these have shown to be of prognostic significance,56 but the role of peritumoural TAMs needs further investigation.

    Conclusion

    FOXP3, a Treg marker, seems to be an independent prognostic factor for better OS in the whole NPC group. Therefore, immune-based therapies targeting Tregs should be carefully evaluated. M2 spectrum macrophages are probably more prominent in EBV-negative NPC with also functional differences compared with EBV-positive NPC.

    Take home messages

    • FOXP3, a Treg marker, seems to be an independent prognostic factor in nasopharyngeal carcinoma (NPC) for better survival.

    • M2 macrophages in Epstein-Barr virus (EBV)-positive and negative NPCs could have functional differences.

    • M2 phenotypic macrophages play a prominent role in EBV-negative NPCs.

    Acknowledgments

    The authors would like to thank Domenico Castigliego for the help with the tissue microarrays and immunohistochemical staining.

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    Footnotes

    • Handling editor Runjan Chetty.

    • Competing interests None declared.

    • Provenance and peer review Not commissioned; externally peer reviewed.