Comparative analysis of immune infiltrates in head and neck cancers across anatomical sites

Abstract

BackgroundThe response rate to immune checkpoint inhibitors targeting programmed cell death 1 (PD-1) receptor is 13%–18% for patients with recurrent or metastatic head and neck squamous cell carcinoma (HNSCC). Detailed understanding of the tumor immune microenvironment (TIME) is crucial in order to explain and improve this response rate. HNSCCs arise at various anatomical locations including the oral cavity, hypopharynx, larynx and oropharynx. Studies directly comparing immune infiltration between anatomical sites are scarce. Since the distinct locations could drive deviating microenvironments, we questioned whether the immune composition varies across these HNSCC sites.MethodsHere, we characterized the TIME of 76 fresh tumor specimens using flow cytometry and performed single-cell RNA-sequencing on nine head and neck tumor samples.ResultsWe found major differences in the composition of the TIME between patients. When comparing anatomical sites: tumors originating from the oral cavity had higher T cell infiltrates than tumors from other anatomical sites. The percentage of tumor-infiltrating T-lymphocytes positive for the immune checkpoint PD-1 varied considerably between patients, with the highest fraction of PD-1+ T cells found in larynx squamous cell carcinomas (SCCs). While we had hypothesized that the anatomical sites of tumor origin would drive sample clustering, our data showed that the type of TIME was more dominant and was particularly driven by the fraction of T cells positive for PD-1. Moreover, a high proportion of PD-1+ CD8+ T cells associated with an improved overall survival. Using single-cell RNA-sequencing, we observed that PD-1 expression was highest in the CD8-ENTPD1 tissue resident memory T cell/exhausted T cell and CD4-CXCL13 type 1 T helper cell clusters.ConclusionsWe found that oral cavity SCCs had the highest frequencies of T cells. We also observed considerable interpatient heterogeneity for PD-1 on T cells, with noticeably higher frequencies of PD-1+ CD4+ T helper cells in larynx SCCs. Within the entire cohort, a higher fraction of CD8+ T cells positive for PD-1 was linked to improved overall survival. Whether the fraction of PD-1+ T cells within the TIME enables immune checkpoint inhibitor response prediction for patients with head and neck cancer remains to be determined.

Immune cell topography of head and neck cancer

“…It seemed that the immune composition of HSCC was more comparable with LSCC, which might be explained by the anatomically closer proximity. Interestingly, in concordance with our work using multiparameteric flow cytometry on fresh HNSCC single-cell suspensions, 21 we found higher CD4+T cell densities in tumors originating from the oral cavity compared with HSCC and LSCC. Of note, patients from that study 21 and the current (partly) overlap ( online supplemental table 2 ), but a different tumor sample was examined.…”

“…Interestingly, in concordance with our work using multiparameteric flow cytometry on fresh HNSCC single-cell suspensions, 21 we found higher CD4+T cell densities in tumors originating from the oral cavity compared with HSCC and LSCC. Of note, patients from that study 21 and the current (partly) overlap ( online supplemental table 2 ), but a different tumor sample was examined. Additionally, a different technique was employed to investigate the TIME, each with its own advantages and limitations.…”

“…We previously reported differences in the immune composition between anatomical sites using multiparametric flow cytometry. 21 Therefore we here compared the spatial TIME of 29 surgical resection specimens originating from the oral cavity (n=12), hypopharynx (n=9), and larynx (n=8, figure 7 ). While interpatient heterogeneity was observed in the immune composition of tumors, OCSCCs demonstrated higher levels of T-cell infiltrates, which could be attributed to higher densities of Tregs, in comparison to HSCC and LSCC (p=0.046, p=0.019, figure 7A–D ).…”

“…When fresh tumor material was available, matched treatment-naive tumor specimens were digested as previously described. 21 In total 1×10 5 single cells were cultured in 100 µL Roswell Park Memorial Institute 1640 medium (Lonza) supplemented with 10% fetal bovine serum (heat-inactivated FBS, Biological Industries), penicillin, streptomycin and L-glutamine (Lonza) at 37°C and 5% CO 2 . After 24 hours, 90 µL conditioned medium was collected, centrifuged for 5 min at 300 g and 80 µL was stored at −20°C until further use.…”

Type I conventional dendritic cells and CD8+ T cells predict favorable clinical outcome of head and neck squamous cell carcinoma patients

“…Additionally, whole tumor infiltration of PD-1 + CD8 + T cells was associated with prolonged OS in the Hypox15 low subcohort and displaying a similar trend in the whole cohort, too. According to the literature, a high frequency of PD-1 + CD8 + T cells was linked to favorable OS in patients with HPV- HNSCC, whereby the 25 th lowest percentile of these immune cell infiltrate pattern was used for grouping the patients ( 84 ).…”