Tumour inflammation signature and expression of S100A12 and HLA class I improve survival in HPV-negative hypopharyngeal cancer

Abstract

Hypopharyngeal squamous cell carcinoma (HPSCC) has a very poor prognosis. Local surgery may increase survival, but is often avoided due to significant post-op co-morbidities. Since prognostic markers are lacking, the aim was to find predictive biomarkers that identify patients whose response to oncological treatment is poor and who may benefit from primary surgery to increase survival. Pretreatment biopsies from 23 HPSCC patients, 3 human papillomavirus (HPV) positive and 20 HPV-negative, were analyzed for expression of 750 mRNAs using the Nanostring nCounter IO360 panel in relation to 3-year survival. Validation was performed through immunohistochemistry (IHC) for HLA class I and S100A12 in 74 HPV-negative HPSCC samples. Clustering identified a subset of HPV-negative HPSCC with favorable prognosis and a gene expression signature overexpressing calgranulins and immune genes, distinct from that of HPV-positive HPSCC. Enrichment analysis showed immune signaling, including the tumor inflammation signature, to be enriched in surviving patients. IHC validation confirmed high S100A12 and HLA class I expression to correlate with survival in HPV-negative HPSCC. This shows that immune activity is strongly related to survival in HPV-negative HPSCC. Enrichment of the tumor inflammation signature indicates a potential benefit of immunotherapy. Low expression of both HLA class I and S100A12 could be used to select patients for local surgery.

Figure 1

(A) Heatmap of all samples. Genes are rows and samples columns. (NED = No evidence of disease, DOD = dead of disease). (B) Volcano plot of transcripts differing between HPV-positive and HPV-negative samples. Transcripts higher in HPV-positive samples are marked red. The horizontal line indicates adjusted p value of 0.05. Blue dots represent genes in the tumor inflammation signature. Analyses of mRNA expression were performed in R 3.6.2. Heatmaps were plotted with the R package ComplexHeatmap. (https://www.bioconductor.org/packages/release/bioc/html/ComplexHeatmap.html).

Figure 2

(A) Heatmap of all HPV-negative samples. Genes are rows and samples columns. (B) Volcano plot of transcripts differing between surviving and non-surviving patients with HPV-negative HPSCC. Transcripts higher in survivors are marked red. Unadjusted p values are presented and the horizontal line indicates an unadjusted p value of 0.05. Blue dots represent genes in the tumor inflammation signature. Analyses of mRNA expression were performed in R 3.6.2. Heatmaps were plotted with the R package ComplexHeatmap. (https://www.bioconductor.org/packages/release/bioc/html/ComplexHeatmap.html).

Figure 3

(A) Heatmap of all HPV-negative samples for the 18 genes in the tumor inflammation signature. The black box marks samples from cluster 1 in (B) Gene set enrichment plot for the 18-gene signature in relation to survival. Genes in the signature are marked on the x-axis and ranked from left to right based on their log2-fold change between tumors responding to treatment ant non-responding. Analyses of mRNA expression were performed in R 3.6.2. Heatmaps were plotted with the R package ComplexHeatmap. (https://www.bioconductor.org/packages/release/bioc/html/ComplexHeatmap.html).

Figure 4

Kaplan–Meier curves presenting progression free (A,D,G), disease free (B,E,H) and overall survival (C,E,I) for patients with HPV-negative HPSCC in relation to S100A12 or HLA class I expression. (A–C) S100A12 expression dichotomized between fraction of positive cells more (red) or less (blue) than 33%. (D-F) HLA class I expression dichotomized between absent/low (blue) and medium/high (red). (G–I) Combined expression of S100A12 and HLA class I. Both high (green), one high, one low (red) or both low (blue). Notches denotes censored.