Immune cell infiltration patterns and survival in head and neck squamous cell carcinoma

Abstract

Purpose: This study examines the tumour-host immune interactions in head and neck squamous cell carcinoma (HNSCC) and their relationship to human papillomavirus (HPV) infectivity and patient survival.

Methods: The adaptive and innate immune profile of surgical tumour specimens obtained from HNSCC patients was determined using qRT-PCR and immunohistochemistry. Intratumoural and invading margin leukocyte populations (CD3, CD8, CD16, CD20, CD68, FoxP3 and HLA-DR) were quantified and compared with patient disease-specific survival. Additionally, the expression of 41 immune activation- and suppression-related genes was evaluated in the tumour microenvironment. Tumour cells were also assessed for expression of HLA-A, HLA-G and HLA-DR. HPV infectivity of tumour biopsies was determined using HPV consensus primers (MY09/MY11 and GP5+/GP6+) and confirmed with p16 immunohistochemistry.

Results: HPV⁺ patient samples showed a significantly increased infiltration by intratumoural CD20⁺ B cells, as well as by invasive margin FoxP3⁺Treg, compared with HPV^- patient samples. There was also a trend towards increased intratumoural CD8⁺ T cells and HLA-G expression on tumour cells in HPV⁺ samples. qRT-PCR data demonstrated a general pattern of increased immune activation and suppression mechanisms in HPV⁺ samples. Additionally, a combined score of intratumoural and invasive margin FoxP3 infiltration was significantly associated with disease-specific survival (P < 0.05).

Conclusions: These data demonstrate significant differences in the immune cell profile of HPV⁺ and HPV^- HNSCC. This study identifies several possible targets for immunotherapy and possible prognostic markers (FoxP3 and HLA-G) that may be specific to HNSCC.

Figure 1

IHC staining of tumour specimens for (a) immune cell populations: CD3⁺ T cells, CD8⁺ T cells, FoxP3⁺Treg (nuclear stain), CD20⁺ B cells, CD16⁺ NK cells, CD68⁺ macrophages and HLA-DR⁺ APCs on patient tumour specimens (200× original magnification) or (b) MHC class I markers: HLA-A, HLA-G and HLA-DR (top row shows positively-stained tumours, bottom row shows negatively-stained tumours; 200× original magnification).

Figure 2

HPV screening of tumour specimens. Samples were screened for HPV infection using PCR, along with confirmation by p16 staining. (a) Sample PCR results demonstrating amplification of a 150-bp product (GP5+/GP6+) in lane 2 and a 450-bp product (MY09/MY11) in lane 3; HeLa cell line was used as a positive control. The negative control (H₂O) for each primer set can be seen in lanes 4 and 5. An HPV⁺ patient sample loaded in lanes 6 and 7 demonstrates amplification using GP5+/GP6+ primers (Lane 6), but the sample does not show amplification using MY09/MY11 primers (Lane 7). (b) p16 staining on an HPV⁺ and (c) HPV⁻ patient sample (200× original magnification).

Figure 3

Pattern of immune gene expression in HPV⁺ and HPV⁻ HNSCC tumours. A heatmap displaying gene expression data for HuRNA, adjacent normal tissue specimens, HPV⁻ patient tumour samples and HPV⁺ patient tumour samples. Gene-specific amplification values were normalized to GAPDH within each sample, and the mean of the log₁₀% GAPDH for each group is shown (green shading indicates GAPDH = 0.001, red shading indicates % GAPDH = 10).

Figure 4

Effect of HPV infection on tumour infiltration by immune cells. (a) Intratumoural immune cell infiltration in HPV⁺ and HPV⁻ patient tumour samples. The mean number of positive cells per HPF is shown +SEM. CD20 was coded as a categorical variable, and the proportion of samples with high levels of infiltration (greater than median) is shown. (b) Invasive margin immune cell infiltration in HPV⁺ and HPV⁻ patient tumour samples. The mean percentage of positive immune cells is displayed, as well as the SEM. FoxP3 was coded on a categorical scale of 0–3 (by quartile). (c) The ratio of CD8⁺ T cells to FoxP3⁺Treg intratumourally and at the invasive margin. (d) The proportion of patient samples positive (>10% of nucleated cells) for HLA-A, HLA-G and HLA-DR. For a–d, * indicates P < 0.05.

Figure 5

Relation of immune infiltrates with HNSCC-specific mortality. (a) Kaplan–Meier curve of survival in HPV⁺ versus HPV⁻ patients. (b) HRs comparing samples with higher levels of immune cell infiltration (top 50%) to those with lower levels of immune cell infiltration (bottom 50%). HRs are reported for intratumoural, invasive margin (peritumoural) and combined populations. HR < 1 indicates decreased and HR > 1 indicates increased HNSCC-specific mortality. (c) Kaplan–Meier curve of survival in subjects with high, moderate or low levels of FoxP3 expression (high = high invasive margin and tumour cell nest infiltration; moderate = high infiltration in one location, low in the other and low = low invasive margin and tumour cell infiltration) (P < 0.05). (d) Kaplan–Meier curve of survival in subjects relative to the extent of intratumoural CD8⁺ infiltrate.