Identifying Predictive Biomarkers for Head and Neck Squamous Cell Carcinoma Response

Abstract

The 5-year survival rate for head and neck squamous cell carcinoma (HNSCC) is approximately 65%. In addition to radio-chemotherapy, immunotherapy is an approach in the treatment of advanced HNSCC. A better understanding of the immune context would allow personalized treatment by identifying patients who are best suited for different treatment options. In our discovery cohort, we evaluated the expression profiles of CMTM6, PD-L1, CTLA-4, and FOXP3 in 177 HNSCCs from Caucasian patients of all tumor stages and different treatment regimens, correlating marker expression in tumor and immune cells with outcomes. Patients with CMTM6^high-expressing tumors had a longer overall survival regardless of treatment. This prognostic benefit of CMTM6 in HNSCC was validated in an independent cohort. Focusing on the in the discovery cohort (n = 177), a good predictive effect of CMTM6^high expression was seen in patients receiving radiotherapy (p = 0.07; log rank), but not in others. CMTM6 correlated with PD-L1, CTLA-4 and FOXP3 positivity, with patients possessing CMTM6^high/FOXP3^high tumors showing the longest survival regardless of treatment. In chemotherapy-treated patients, PD-L1 positivity was associated with longer progression-free survival (p < 0.05). In the 27 patients who received immunotherapy, gene expression analysis revealed lower levels of CTLA-4 and FOXP3 with either partial or complete response to this treatment, while no effect was observed for CMTM6 or PD-L1. The combination of these immunomodulatory markers seems to be an interesting prognostic and predictive signature for HNSCC patients with the ability to optimize individualized treatments.

Keywords: CMTM6; HNSCC; PD-L1; cisplatin; immune-checkpoint inhibitor; radiotherapy; tumor microenvironment.

Figure 1

Kaplan–Meier survival curves of the HNSCC patients. The discovery (n = 177) and validation cohort (n = 286) included HNSCC patients with comparable clinicopathological parameters receiving first- or second-line treatment or best supportive care. (A) Progression-free survival (PFS) and overall survival (OS) for the patients from both cohorts. Treatment regimens included radiotherapy (RT), radio-chemotherapy (RCT), chemotherapy, immune-oncology (IO), or ICI (immune checkpoint inhibition). (B) Treatment-related OS in the discovery cohort (n = 177) stratified according to the specific regimen. (left) Cisplatin-based chemotherapy (n = 64), RT (n = 29) or RCT (n = 13). (right) ICI mono (n = 14 and 4, respectively) or ICI-based combination regimens (n = 8). (C) Treatment-related OS in the validation cohort (n = 286). Log-rank analysis was performed to study differences between the individual regimens. n.s.—not significant.

Figure 2

Kaplan–Meier survival curves of HNSCC patients. For the biomarker-driven and treatment-related prognosis, analysis was performed on n = 177 patients (discovery cohort) receiving first- or second-line treatment. Treatment regimens included chemotherapy, radiotherapy (RT), radio-chemotherapy (RCT), or ICI (immune checkpoint inhibition). (A,B) Overall survival (OS) according to PD-L1 or CMTM6 in all patients (left) and stratified according to HPV status (middle and right). (C) Treatment-related OS stratified according to the specific regimen and PD-L1 status. (D) Treatment-related OS stratified according to the specific regimen and CMTM6 status. Numbers of patients for each treatment group, i.e., cisplatin, RT, RCT, or ICI are given in their respective graphs. Log-rank analysis was performed to study any differences between the individual regimens. n.s.—not significant.

Figure 3

Kaplan–Meier survival curves of HNSCC patients from the discovery cohort. For biomarker-driven and treatment-related prognosis, analysis was performed on n = 177 patients receiving first- or second-line treatment. Treatment regimens included chemotherapy, radiotherapy (RT), radio-chemotherapy (RCT), or ICI (immune checkpoint inhibition). (A) Overall survival (OS) of patients according to FOXP3 and combined FOXP3/CMTM6 status. (B) Treatment-related OS stratified according to the specific regimen and FOXP3/CMTM6 status. Cisplatin-based chemotherapy, RT, RCT, or ICI. Log-rank analysis was performed to study any differences between the individual regimens. n.s.—not significant.

Figure 4

Gene signatures in HNSCC patients from the discovery group with their response to immune checkpoint inhibitors. (A) Heatmap representing the down- and upregulated genes involved in antitumor immune activity by unsupervised hierarchical clustering. Scores are scaled to have a mean of zero and a standard deviation of one. The standardized signature scores are truncated at ±3 standard deviations (99% of the data should fall within ±3 standard deviations of the mean). (B) Box plot indicating a significantly lower CTLA-4 signature in patients with a response to ICI as assessed by univariate analysis (p = 0.02). (C) Volcano plot of all gene signatures displays no significant difference between the ICI responders and non-responders in a multivariate analysis. Up indicates upregulated and down indicates downregulated genes. (D) Survival volcano plot of all gene signatures displays no significant difference between the signature’s hazard ratios in a multivariate analysis. Signatures further to the right are associated with a decreased risk of an event relative to the baseline. (E) Box plot indicating a significantly higher IFN downstream signature in patients whose tumors showed a CPS ≥ 1 for PD-L1 immunohistochemistry as assessed by univariate analysis (p = 0.04). (F) Spaghetti plot displaying a decrease in antitumor immune activity from initial diagnosis to r/m disease with start of ICI until the time point of progression under ICI in non-responders, and (G) an increase in CTLA-4 in recurrent tumor material from a patient who initially had a partial response to ICI. * p < 0.05. CPS—combined positive score; Pre-ICI—sample taken just before the start of ICI; Post-ICI—sample taken just after progression under ICI.