BackgroundSome sarcomas respond to immune checkpoint inhibition, but predictive biomarkers are unknown. We analyzed tumor DNA methylation profiles in relation to immunological parameters and response to anti-programmed cell death 1 (anti-PD-1) immune checkpoint inhibitor (ICI) therapy in patients with sarcoma.Patients and methodsWe retrospectively identified adult patients who had received anti-PD-1 ICI therapy for recurrent sarcoma in two independent centers. We performed (1) blinded radiological response evaluation according to immune response evaluation criteria in solid tumors (iRECIST) ; (2) tumor DNA methylation profiling of >850,000 probes using Infinium MethylationEPIC microarrays; (3) analysis of tumor-infiltrating immune cell subsets (CD3, CD8, CD45RO, FOXP3) and intratumoral expression of immune checkpoint molecules (PD-L1, PD-1, LAG-3) using immunohistochemistry; and (4) evaluation of blood-based systemic inflammation scores (neutrophil-to-lymphocyte ratio, leucocyte-to-lymphocyte ratio, monocyte-to-lymphocyte ratio, platelet-to-lymphocyte ratio). Response to anti-PD-1 ICI therapy was bioinformatically and statistically correlated with DNA methylation profiles and immunological data.Results35 patients (median age of 50 (23–81) years; 18 females, 17 males; 27 soft tissue sarcomas; 8 osteosarcomas) were included in this study. The objective response rate to anti-PD-1 ICI therapy was 22.9% with complete responses in 3 out of 35 and partial responses in 5 out of 35 patients. Adjustment of DNA methylation data for tumor-infiltrating immune cells resulted in identification of methylation differences between responders and non-responders to anti-PD-1 ICI. 2453 differentially methylated CpG sites (DMPs; 2043 with decreased and 410 with increased methylation) were identified. Clustering of sarcoma samples based on these DMPs revealed two main clusters: methylation cluster 1 (MC1) consisted of 73% responders and methylation cluster 2 (MC2) contained only non-responders to anti-PD-1 ICI. Median progression-free survival from anti-PD-1 therapy start of MC1 and MC2 patients was 16.5 and 1.9 months, respectively (p=0.001). Median overall survival of these patients was 34.4 and 8.0 months, respectively (p=0.029). The most prominent DNA methylation differences were found in pathways implicated in Rap1 signaling, focal adhesion, adherens junction Phosphoinositide 3-kinase (PI3K)-Akt signaling and extracellular matrix (ECM)–receptor interaction.ConclusionsOur data demonstrate that tumor DNA methylation profiles may serve as a predictive marker for response to anti-PD-1 ICI therapy in sarcoma.

BackgroundBiomarkers 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.MethodsWe 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.Results37 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).ConclusionsOur findings suggest that tumor DNA methylation profiling may be useful to predict response to ICI in patients with HNSCC.

Purpose: The treatment of oligodendroglioma consists of tumor resection and radio-chemotherapy. The timing of radio-chemotherapy remains unclear and predictive biomarkers are limited. Methods: Adult patients diagnosed with isocitrate dehydrogenase (IDH)-mutated, 1p/19q-codeleted CNS WHO grade 2 and 3 oligodendroglioma at the Medical University of Vienna and the Kepler University Hospital Linz (Austria) in 1992-2019 were included. Progression-free (PFS) and overall survival (OS) between early postoperative treatment and initial observation were compared using propensity score-weighted Cox regression models. DNA methylation analysis of tumor tissue was performed using Illumina MethylationEPIC 850k microarrays. Results: 131/201 (65.2%) patients with CNS WHO grade 2 and 70/201 (34.8%) with grade 3 oligodendroglioma were identified. 83/201 (41.3%) patients underwent early postoperative treatment, of whom 56/83 (67.5%) received radio-chemotherapy, 15/84 (18.1%) radiotherapy (RT) only and 12/83 (14.5%) chemotherapy only. Temozolomide-based treatment was administered to 64/68 (94.1%) patients, while RT + procarbazine, lomustine (CCNU) and vincristine (PCV) was applied in 2/69 (3.5%) patients. Early treatment was not associated with PFS (adjusted hazard ratio (HR) 0.74; 95%CI: 0.33-1.65, p=0.459) or OS (adjusted HR: 2.07; 95%CI: 0.52-8.21, p=0.302) improvement. Unsupervised clustering analysis of DNA methylation profiles from patients receiving early treatment revealed two methylation clusters correlating with PFS, whereas no association of clustering with O6-methylguanine methyltransferase (MGMT) promoter methylation, CNS WHO grade, extent of resection, and treating center could be observed. Conclusions: In this retrospective study, early postoperative treatment was not associated with improved PFS/OS in oligodendroglioma. The potentially predictive value of whole-genome methylation profiling should be validated in prospective trials.

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

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