Characterization of the tumor immune microenvironment in human papillomavirus-positive and -negative head and neck squamous cell carcinomas

Abstract

Approximately 15% of advanced head and neck squamous cell carcinomas (HNSCC) respond to anti-PD-(L)1 monotherapies. Tumor PD-L1 expression and human papillomavirus (HPV) status have been proposed as biomarkers to identify patients likely to benefit from these treatments. We aimed to understand the potential immune effects of HPV in HNSCC and to characterize additional potentially targetable immune-regulatory pathways in primary, treatment-naïve tumors. CD3, CD4, CD8, CD20, CD68, FoxP3, PD-1, PD-L2, LAG-3, IDO-1, and GITR cell densities were determined in 27 HNSCC specimens. IHC for PD-L1 assessed percentage of positive tumor cells and immune cells separately or as a combined positive score (CPS), and whether PD-L1 was expressed in an adaptive or constitutive pattern (i.e., PD-L1+ tumor cells juxtaposed to TILs or in the absence of TILs, respectively). HPV testing with p16 IHC was confirmed by HPV genotyping. When compared to HPV(-) tumors (n = 14), HPV+ tumors (n = 13) contained significantly higher densities of CD3+, CD4+, CD8+, CD20+, and PD-1+ cells (P < 0.02), and there was a trend towards increased density of FoxP3 + cells. PD-L1 expression patterns did not vary by tumor viral status, suggesting possible heterogeneous mechanisms driving constitutive vs adaptive PD-L1 expression patterns in HNSCC. IDO-1 expression was abundant (> 500 IDO-1+ cells/mm² in 17/27 specimens) and was found on tumor cells as well as immune cells in 12/27 (44%) cases (range 5-80% tumor cells+). Notably, the studied markers varied on a per-patient basis and were not always related to the degree of T cell infiltration. These findings may inform therapeutic co-targeting strategies and raise consideration for a personalized treatment approach.

Keywords: GITR; HPV; Head and neck squamous cell carcinoma (HNSCC); IDO; PD-1; PD-L1.

Fig. 1

Patterns of PD-L1 expression in HNSCC. Representative examples of a constitutive adaptive, b adaptive, and c combined constitutive and adaptive (mixed) patterns of tumor cell PD-L1 expression are shown. PD-L1 staining is marked by brown chromogen. Purple arrows mark areas of adaptive tumor cell PD-L1 expression at the interface with infiltrating lymphocytes. d There was no significant correlation between tumor cell PD-L1 expression and infiltrating CD3+ T cell densities (r = − 0.0358, P = 0.86) or PD-L1 CPS and CD3+ cell densities (r = 0.21, P = 0.29, see Supplementary Fig. 1 for CPS figure), consistent with the finding of constitutive tumor cell PD-L1 expression in many cases. e Distinct patterns of tumor cell PD-L1 expression in HNSCC specimens did not correlate with tumor viral status. 400×, 100×, and 200×, original magnifications for panels a, b, and c, respectively

Fig. 2

Patterns and prevalence of immune cell infiltrates and coregulatory molecule expression vary by HNSCC specimen. Individual tumor specimens showed varied expression of different markers. For example, both specimens #4 and #8 (top and bottom rows, respectively) are CD3+ T cell inflamed tumors with prominent PD-1 expression. However, specimen #8 also shows LAG-3+ and GITR+ immune cell populations proportionate to PD-1 expression, while specimen #4 has notably less LAG-3 expression. Low-level GITR expression by tumor cells is also present in specimen #4 (black arrow). Additionally, IDO-1 staining patterns differed across specimens. For example, specimen #8 shows constitutive tumor cell IDO-1 expression, whereas specimen #4 shows prominent IDO-1 display at the tumor–host interface in an adaptive pattern

Fig. 3

Patterns of IDO expression in the TME of HNSCC. Distinct patterns of IDO cell expression were observed including a complete interface pattern (tumor islands ringed by IDO + tumor cells and macrophages), b focal interface pattern (predominantly immune cell expression with rare tumor cell expression), and c constitutive tumor cell expression. There was no statistical significance of d tumor cell expression or e expression pattern between HPV+ and HPV(−) HNSCC specimens

Fig. 4

Correlation of degree of intratumoral CD3+ T cell infiltration with immune cell subset densities and coregulatory marker expression. Colors indicate density of marker-positive immune cells. All 27 HNSCC specimens are ranked by CD3+ T cell densities, and specimen IDs are provided to the left of the figure. HPV status is provided to the right

Fig. 5

Immune cell subsets and coregulatory molecule expression on immune cells in HPV+ vs. HPV(−) HNSCC. Protein marker expression was detected with immunohistochemistry and analyzed digitally, as detailed in “Materials and methods”. When compared to HPV(−) tumors, HPV+ tumors contained significantly higher densities of CD3+, CD4+, CD8+, CD20+, and PD-1+ cells; there was also a trend towards an increased density of FOXP3+ cells in HPV+ tumors. Expression of IDO-1 and GITR by immune cells was robust, but did not differ by tumor viral status. P-values, 2-sided Wilcoxon rank-sum test with exact distribution. Error bars display mean ± SEM