Abstract Background/Introduction Immune‐related adverse events (irAEs) remain unpredictable complications of immune checkpoint inhibitor (ICI) therapy. Current biomarkers, such as inflammatory blood parameter lack specificity and clinical robustness. Because DNA methylation reflects stable immune programming and predisposition to autoimmune diseases, blood methylation profiling may offer a tumour‐agnostic approach for irAE prediction. Material & methods We analysed baseline leukocyte DNA methylation in a prospectively enrolled real‐world cohort of advanced pan‐cancer patients treated with ICI in a palliative setting. Genome‐wide methylation was profiled using Infinium MethylationEPIC v1.0 BeadChips. Differentially methylated positions (DMPs) were identified by rank‐based comparison and tested for Gene Ontology enrichment. Blood‐cell deconvolution using EpiDISH was applied to exclude compositional bias. Results Among 195 patients (median age 65 years; 34.9% female), 32.8% experienced at least one irAE, most frequently involving thyroid, skin, or lung. Despite globally concordant DNA methylation between groups ( ρ = 0.9996), DMP analysis revealed subtle but distinct site‐specific differences. A methylation‐based classifier discriminated patients who developed irAEs with good accuracy (area under the curve [AUC] 0.831, 95% confidence interval [CI] 0.735–0.927), which remained robust in an internal validation subset (AUC 0.874, 95% CI 0.757–0.992). EpiDISH confirmed no significant variation in immune cell fractions between groups. Functional enrichment of top‐ranked DMPs showed significant overrepresentation of neuronal development, cell‐cell signalling, and epithelial morphogenesis pathways, networks linked to immune‐synapse formation, cytokine signalling, and tissue repair responses. Discussion Baseline blood DNA methylation captures systemic immune‐developmental states predisposing to irAEs. The enrichment of cell‐cell signalling and epithelial morphogenesis pathways suggests an epigenetically encoded predisposition for dysregulated immune‐tissue communication. Blood‐based methylation profiling thus represents a promising, tumour‐agnostic biomarker approach for individualised irAE risk prediction in cancer immunotherapy.

PURPOSE:Given the early recurrence of brain metastasis (BM), identifying factors that drive BM progression is of clinical interest. This study investigates genetic, epigenetic, and inflammatory signatures in progressive BM following different therapeutic approaches. METHODS:A total of 153 patients who underwent surgical resection for progressive BM were grouped according to the therapeutic strategies prior to the first BM resection: prior radiation (n = 43), systemic therapy (n = 37), combined radiation and systemic treatment (n = 10), and treatment-naive patients (n = 63). Among the treatment-naive patients, 35/63 (55.5%) experienced another intracranial relapse and underwent a second resection (=relapse group), enabling paired analyses. Of these, 23/35 (65.7%) received no therapy between resections; 12/35 (34.3%) received CNS-directed radiotherapy. Tissue samples were analysed using whole-exome sequencing, DNA methylation profiling, and immunohistochemistry. RESULTS:BM resected after progression following prior cranial radiotherapy (43/153, 28.1%) showed significantly lower densities of CD3 + , CD8 + , and CD45RO + cells together with increased FOXP3 + cell density compared with treatment-naïve BM (63/153, 41.2%; median CD3 +: 71 vs. 494 cells/mm²; CD8 +: 44 vs. 187 cells/mm²; CD45RO+: 104 vs. 302 cells/mm²; FOXP3 +: 215 vs. 41 cells/mm²). In the paired analyses, progressive specimen after prior radiation were likewise associated with significantly reduced CD3 + , CD8 + , and CD45RO + and increased FOXP3 + cell densities compared with the matched baseline specimen. In contrast, no genetic alterations or differences in DNA methylation patterns between irradiated and non-irradiated matched samples were identified. CONCLUSION:Progressive BM following cranial radiotherapy demonstrated a distinct immune marker profile consistent with a more immunoregulatory, rather immunosuppressive tumour microenvironment. No therapy-associated genetic or epigenetic alterations were identified. Further prospective studies are warranted to determine whether these immune alterations reflect treatment-related effects or biological features associated with resistance following radiotherapy.

Background Subtype is a key prognostic factor for breast cancer. In HER2-positive disease, HER2-directed therapies have improved outcomes, though they can cause cardiac side effects, potentially leading to emergency department (ED) visits. Objectives To assesses reasons for ED presentations in breast cancer patients, highlighting subtype-specific differences in reason for presentation, 3-month mortality (3MM) and the possible association of cardiologic visits with HER2-directed therapy. Methods In this retrospective study, visits by breast cancer patients at an Austrian tertiary care ED were analysed. Subtype frequency rates and subtype-specific 3MM rates were calculated using Chi-Square tests (separately for early and advanced disease). A possible association between HER2-directed therapies and cardiologic ED visits was investigated using a Fisher’s exact test and a multinomial logistic regression controlling for age. Results There was a total of 463 ED visits among 322 patients between August 2016 and December 2019. Subtype distribution was as follows: 42% (n=135) luminal B-like/HER2- negative, 23% (n=74) triple negative, 16% (n=50) luminal B-like/HER2-positive, 10% (n=33) luminal A-like and 9% (n=30) HER2-positive (non-luminal). In patients with advanced BC, subtype was significantly associated with 3MM (p=0.006), with the highest mortality rate observed in TNBC (54%). Active HER2-directed therapy (n=70) was associated with increased cardiologic ED visits (OR = 4.536 [95%CI, 1.850- 11.125]). Conclusions BC subtype influenced the frequency of ED visits and patient survival. HER2-directed therapy was associated with an increased risk for cardiologic ED visits among real-world cancer patients, emphasizing the need for tailored cardio-oncologic care strategies to optimize tolerability and reduce healthcare burdens.

Multinucleated giant cells (MGCs) are a hallmark pathological feature of a wide range of diseases, yet the mechanisms underlying their formation and function remain poorly understood. Although it is recognized that MGCs arise from a heterogeneous pool of myeloid precursors, how a committed MGC fate develops therein remains unknown. Here, combining temporal in vitro and in vivo differentiation of bone marrow-derived myeloid precursors with single cell and bulk RNA sequencing as well as CRISPR/Cas9-mediated gene editing, we shed insight into how MGCs emerge. Our findings reveal that coordinated upregulation of cell fusion genes and cellular metabolism, particularly oxidative phosphorylation, regulate the transition from macrophage progenitors to fusion competent cells. In vivo fate-mapping unveils Ms4a3 -, and Cd11c -but not Cx3cr1 -traced cells as the predominant precursor populations for MGCs in a lung granuloma formation model. Notably, transcription factor profiling in the progression from early myeloid precursors to pre-MGCs identifies IRF4 as a key molecular switch driving MGC generation. IRF4+ MGCs are present in different pathologies, including Schistosoma mansoni egg induced granulomas, Aspergillus fumigatus conidia mediated allergic airway inflammation and human head and neck squamous cell carcinomas. Mechanistically, IRF4 controls critical fusion related genes such as Dcstamp and Ocstamp . Consequently, Irf4 deficient cells are unable to develop into MGCs. Collectively, our work delineates the trajectory of MGC differentiation, establishing IRF4 as a defining transcription factor required for the generation of fusion-competent progenitors that ultimately give rise to MGCs. ### Competing Interest Statement The authors have declared no competing interest. Christian Doppler Research Association, https://ror.org/00mv8h305, CDL Arginine Metabolism FWF Austrian Science Fund, https://ror.org/013tf3c58, SFB F83, 10.55776/PAT5446023 Deutsche Forschungsgemeinschaft, VO944/11-1, CRC1181 project A2 Austrian Academy of Sciences, DOC fellowship

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

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

Neuroendocrine tumors (NET) of the lung constitute a rare entity of primary lung malignancies that often exhibit an indolent clinical course. Epigenetics-related differences have been described previously for lung NET, but the clinical significance remains unclear. In this study, we performed genome-wide methylation analysis using the Infinium MethylationEPIC BeadChip technology on FFPE tissues from lung NET treated at two academic centers. We aimed to investigate the methylation profiles of known prognostic subgroups. In total, 54 tissue samples from primary lung NET were analyzed, of which 37 were typical carcinoids (TC) and 17 atypical carcinoids (AC). Overall, 25/53 patients (47.2%) developed metastases throughout the disease course, 14/26 (53.8%) had a positive somatostatin receptor (SSTR) scan, and 7/28 patients (25.0%) had documented endocrine activity. Analysis of the DNA methylation data showed substantial differences between TC and AC samples and revealed three distinct clusters (C1–C3): C3 ( n = 29) with 100% TC and 89.7% non-metastasized, C2 ( n = 22) with 63.6% AC and 95.5% metastasized, and C1 with three AC samples (2/3 metastasized). In subgroup analyses, distinct methylation patterns were observed based on histology, metastases, SSTR status, and endocrine activity. In the functional gene classification, the genes affected by differential methylation were mainly involved in cell signaling. DNA methylation could potentially aid in the diagnostic process of lung NET. The differences in methylation observed with respect to clinical features like SSTR expression and endocrine activity could translate into improved management of lung NET.

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.

The prognosis of diffuse gliomas previously classified as “lower-grade” is heterogeneous and complicates clinical decisions. We aimed to investigate the molecular profile of clinical outliers to gain insight into biological drivers of long and short-term survivors. Here, patients aged ≥ 18 years and diagnosed with diffuse glioma, WHO grade II/2 or III/3 were included. Short-term survivors (STS) were defined as overall survival (OS) < 1 years, and long-term survivors (LTS) as OS > 10 years. DNA methylation profiling was performed using the Illumina EPIC 850k platform. In total, 385 patients (294 LTS, 91 STS) were included. Median overall survival was 234 months (95

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

Immune cell infiltration in BM of the three subtypes of BCa: Luminal (A), HER2-positive (B) and Triple-negative breast cancer (TNBC) (C).

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

Protocols used for IHC stainings on the Ventana Benchmark ULTRA device

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

Metabolic reprogramming and cell cycle deregulation are hallmarks of cancer cells. The cell cycle kinase CDK6 has recently been implicated in a wide range of hematopoietic malignancies. We here investigate the role of CDK6 in the regulation of cellular metabolism in BCR::ABL1+ leukemic cells. Our study, using gene expression data and ChIP-Seq analysis, highlights the contribution of CDK6 kinase activity in the regulation of oxidative phosphorylation. Our findings imply a competition for promoter interaction of CDK6 with the master regulator of mitochondrial respiration, NRF-1. In line, cells lacking kinase active CDK6 display altered mitochondria morphology with a defective electron transport chain. The enhanced cytoplasm/mitochondria ATP ratio paralleled by high pyruvate and lactate levels indicate a metabolic switch to glycolysis. Accordingly, combinatorial treatment of leukemic cells including imatinib resistant cells with the CDK4/6 inhibitor palbociclib and the glycolysis inhibitor 2-deoxyglucose (2-DG) enhanced apoptosis, while blocking cell proliferation in leukemic cells. These data may open a new therapeutic avenue for hematologic malignancies with high CDK6 expression by exploiting metabolic vulnerabilities unmasked by blocking CDK6 kinase activity that might even be able to overcome imatinib resistance.

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

Tumor-infiltrating lymphocytes (TILs) and tumor-associated macrophages (TAMs) have increasingly been reported to impact the brain metastatic process of solid tumors. However, data on intra-individual differences between primary tumor and brain metastasis (BM), as well as their correlation with clinical outcome parameters, is scarce. We retrospectively identified patients who received resection of the primary tumor and BM between 01/1990 and 10/2022. Density quantification of TAMs (CD68+, CD163+) and TILs (CD3+, CD8+, CD45RO+, FOXP3+) was performed by immunohistochemical staining of matched tumor tissue samples. Images were processed with QuPath software and heterogeneity of generated heatmaps was measured by Shannon Entropy. Time-to-BM (TTBM) was defined as the time from diagnosis of the primary tumor until the first diagnosis of BM. In total, 104 patients (46.2

Objective: We analyzed the impact of amino acid (AA) availability on the inflammatory response in arthritis.Methods: We stimulated rheumatoid arthritis (RA) fibroblast-like synoviocytes (FLSs) with tumor necrosis factor (TNF) in the presence or absence of proteinogenic AAs and measured their response by QuantSeq 3' messenger RNA sequencing, quantitative polymerase chain reaction, and enzyme-linked immunosorbent assay. Signal transduction events were determined by Western blot. We performed K/BxN serum transfer arthritis in mice receiving a normal and a low-protein diet and analyzed arthritis clinically and histologically.Results: Deprivation of AAs decreased the expression of a specific subset of genes, including the chemokines CXCL10, CCL2, and CCL5 in TNF-stimulated FLSs. Mechanistically, the presence of AAs was required for the TNF-induced activation of an interferon regulatory factor 1 (IRF1)-STAT1 signaling circuit that drives the expression of chemotactic factors. The expression of IRF1 and the IRF1-dependent gene set in FLSs was highly correlated with the presence of inflammatory cells in human RA, emphasizing the important role of this AA-dependent pathway in inflammatory cell recruitment to the synovial tissue. Finally, we show that mice receiving a low-protein diet expressed less IRF1 in the inflamed synovium and consequently developed reduced clinical and histologic signs of arthritis.Conclusion: AA deprivation reduces the severity of arthritis by suppressing the expression of IRF1-STAT1-driven chemokines, which are crucial for leukocyte recruitment to the arthritic joint. Overall, our study provides novel insights into critical determinants of inflammatory arthritis and may pave the way for dietary intervention trials in RA.