DNA methylation profiles differ in responders versus non-responders to anti-PD-1 immune checkpoint inhibitors in patients with advanced and metastatic head and neck squamous cell carcinoma

Background Biomarkers for response prediction to anti-programmed cell death 1 (PD-1) immune checkpoint inhibitors (ICI) in patients with head and neck squamous cell carcinoma (HNSCC) are urgently needed for a personalized therapy approach. We investigated the predictive potential of inflammatory parameters and DNA methylation profiling in patients with HNSCC treated with anti-PD-1 ICI.Methods We identified patients with HNSCC that were treated with anti-PD-1 ICI therapy in the recurrent or metastatic setting after progression to platinum-based chemotherapy in two independent centers. We analyzed DNA methylation profiles of >850.000 CpG sites in tumor specimens of these patients by Infinium MethylationEPIC microarrays, immune cell density in the tumor microenvironment (CD8, CD3, CD45RO, forkhead box P3 (FOXP3), CD68), PD-1 and programmed cell death ligand 1 (PD-L1) expression by immunohistochemistry, and blood inflammation markers (platelet-to-lymphocyte ratio, leucocyte-to-lymphocyte ratio, monocyte-to-lymphocyte ratio, neutrophil-to-lymphocyte ratio). DNA methylation profiles and immunological markers were bioinformatically and statistically correlated with radiological response to anti-PD-1 ICI.Results 37 patients with HNSCC (median age of 62 years; range 49–83; 8 (21.6%) women, 29 (78.4%) men) were included (Center 1 N=26, 70.3%; Center 2 N=11, 29.7%). Median number of prior systemic therapies was 1 (range 1–4). Five out of 37 (13.5%) patients achieved an objective response to ICI. Median progression-free survival and median overall survival times were 3.7 months (range 0–22.9) and 9.0 months (range 0–38.8), respectively. Microarray analyses revealed a methylation signature including both hypomethylation and hypermethylation which was predictive for response to ICI and included several genes involved in cancer-related molecular pathways. Over-represented differentially methylated genes between responders and non-responders were associated with ‘Axon guidance’, ‘Hippo signaling’, ‘Pathways in cancer’ and ‘MAPK signaling’. A statistically significant correlation of PD-L1 expression and response was present (p=0.0498).Conclusions Our findings suggest that tumor DNA methylation profiling may be useful to predict response to ICI in patients with HNSCC

Supplementary Table S1 from Frequent Overexpression of HER3 in Brain Metastases from Breast and Lung Cancer

Protocols used for IHC stainings on the Ventana Benchmark ULTRA device</p

Supplementary Table S3 from Frequent Overexpression of HER3 in Brain Metastases from Breast and Lung Cancer

Differentially methylated CpG sites in HER3+ vs HER3- breast cancer samples (HER2+ cohort)</p

Supplementary Table S2 from Frequent Overexpression of HER3 in Brain Metastases from Breast and Lung Cancer

Baseline characteristics of patients used for genome-wide DNA-methlyation analysis, stratified into HER3-positive and HER3-negative</p

Supplementary Table S5 from Frequent Overexpression of HER3 in Brain Metastases from Breast and Lung Cancer

Differentially methylated CpG sites in HER3+ vs HER3- breast cancer samples (TNBC cohort)</p

Supplementary Table S4 from Frequent Overexpression of HER3 in Brain Metastases from Breast and Lung Cancer

Differentially methylated CpG sites in HER3+ vs HER3- breast cancer samples (Luminal cohort)</p

Supplementary Table S6 from Frequent Overexpression of HER3 in Brain Metastases from Breast and Lung Cancer

Pathway enrichment analysis of genes associated with differentially methylated CpG sites in HER3+ vs. HER3-</p

Supplementary Figure S1 from Frequent Overexpression of HER3 in Brain Metastases from Breast and Lung Cancer

Further evaluation of HER3 expression in BM of breast and lung cancer.</p

Supplementary Figure S4 from Frequent Overexpression of HER3 in Brain Metastases from Breast and Lung Cancer

Selected CpG sites covering the genomic locus of HER3 (ERBB3) were evaluated for their methylation state (A).</p

Supplementary Figure S2 from Frequent Overexpression of HER3 in Brain Metastases from Breast and Lung Cancer

Kaplan-Meier curves of patients with BM of A) Luminal-BCa B) HER2+ BCa C) TN-BCa, and D) NSCLC patients stratified according to the intensity of HER3 expression determined by IHC.</p