Characterization of the immune profile of oral tongue squamous cell carcinomas with advancing disease

Abstract

We investigated whether a unique immune response was instigated with the development of oral tongue squamous cell carcinomas (OTSCC), with/without nodal involvement, with/without recurrent metastatic disease, or within tumor involved nodes. One hundred and ten formalin-fixed paraffin-embedded samples were collected from a retrospective cohort of 67 OTSCC patients and 10 non-cancerous tongue samples. Targets including CD4, CD8, FOXP3, PD-L1, and PD-1 were analyzed by immunohistochemistry. The Nanostring PanCancer Immune Profiling Panel was used for gene expression profiling. Data were externally validated in the The Cancer Genome Atlas (TCGA) head and neck (HNSCC), melanoma and lung squamous cell carcinoma (LSCC) cohorts. A 24-immune gene signature was identified that discriminated more aggressive OTSCC cases, and although not prognostic in HNSCC was associated with survival in other TCGA cohorts (improved survival for melanoma, P < .001 and worse survival for LSCC, P = .038). OTSCC exhibited concordant gene and immunohistochemical (IHC) features characterized by a TH-2 biased, proinflammatory profile with upregulated B cell and neutrophil gene activity and increased CD4, FOXP3, and PD-L1 expression (P < .001 for all by IHC). Compared to less advanced disease, nodal involvement and recurrent OTSCC did not induce a different immune response although recurrent disease was characterized by significantly higher PD-L1 expression (P = .004 by SP263, P = .013 by 22C3, P = .004 for gene expression). Identification of a gene signature associated with different prognostic effects in other cancers highlights common pathways of immune dysregulation that are impacted by the tumor origin. The significant immunosuppressive signaling in OTSCC indicates primary failure of immune system to control carcinogenesis emphasizing the need for early, combination therapeutic approaches.

Keywords: FOXP3; Immune signature; PD-L1; oral tongue squamous cell carcinoma.

FIGURE 1

Summary of oral tongue squamous cell carcinomas (OTSCC) vs non‐cancer analysis. A, Unsupervised hierarchical cluster analysis of OTSCC vs non‐cancer cases according to the log2 fold change of gene expression. B, Differentially expressed genes in OTSCC relative to non‐cancer cases. Genes that were identified as differentially expressed >1.5 log fold change by 2 or more data analyses packages were included. C, Supervised hierarchical cluster analysis of OTSCC vs non‐cancer cases according to the log2 fold change of gene expression

FIGURE 2

A, Patient survival according to the presence of the cancer signature in the The Cancer Genome Atlas (TCGA) melanoma and lung squamous cell carcinoma (LSCC) cohorts. Kaplan‐Meier curves for the B, TCGA melanoma cohort (n = 472), and C, TCGA LSCC cohort (n = 551) according to the presence of the full cancer signature vs part of the cancer signature and no cancer signature

FIGURE 3

Representative images of immune expression markers in oral tongue squamous cell carcinomas (OTSCC) and non‐cancer cases. Histology for OTSCC (Panel A) and non‐cancer cases (Panel B) assessed by H&E A, with similar expression between OTSCC and non‐cancer cases for CD8 B, CD56 C, and PD‐1 D, and increased expression in OTSCC for CD3 E, CD4 F, FOXP3 G and PD‐L1 assessed by SP263 Ventana antibody H

FIGURE 4

Summary of tumor (T) vs matched lymph nodes (LN). A, Unsupervised hierarchical cluster analysis of tumor and matched, involved lymph nodes from oral tongue squamous cell carcinomas (OTSCC) cases according to the log2 fold change of gene expression. B, Differentially expressed genes in primary tumor compared to matched nodes. C, Supervised hierarchical cluster analysis of tumor (T) and matched, involved LN from OTSCC cases according to the log2 fold change of gene expression (*with the exception of one lymph node sample)