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BackgroundHead and neck squamous cell carcinoma (HNSCC) remain a substantial burden to global health. Cell-free circulating tumour DNA (ctDNA) is an emerging biomarker but has not been studied sufficiently in HNSCC.MethodsWe conducted a single-centre prospective cohort study to investigate ctDNA in patients with p16-negative HNSCC who received curative-intent primary surgical treatment. Whole-exome sequencing was performed on formalin-fixed paraffin-embedded (FFPE) tumour tissue. We utilised RaDaRTM, a highly sensitive personalised assay using deep sequencing for tumour-specific variants, to analyse serial pre- and post-operative plasma samples for evidence of minimal residual disease and recurrence.ResultsIn 17 patients analysed, personalised panels were designed to detect 34 to 52 somatic variants. Data show ctDNA detection in baseline samples taken prior to surgery in 17 of 17 patients. In post-surgery samples, ctDNA could be detected at levels as low as 0.0006% variant allele frequency. In all cases with clinical recurrence to date, ctDNA was detected prior to progression, with lead times ranging from 108 to 253 days.ConclusionsThis study illustrates the potential of ctDNA as a biomarker for detecting minimal residual disease and recurrence in HNSCC and demonstrates the feasibility of personalised ctDNA assays for the detection of disease prior to clinical recurrence.

Background Epidermal growth factor receptor (EGFR) is both a driver oncogene and a therapeutic target in advanced head and neck squamous cell carcinoma (HNSCC). However, response to EGFR treatment is inconsistent and lacks markers for treatment prediction. This study investigated EGFR-induced epithelial-to-mesenchymal transition (EMT) as a central parameter in tumor progression and identified novel prognostic and therapeutic targets, and a candidate predictive marker for EGFR therapy response. Methods Transcriptomic profiles were analyzed by RNA sequencing (RNA-seq) following EGFR-mediated EMT in responsive human HNSCC cell lines. Exclusive genes were extracted via differentially expressed genes (DEGs) and a risk score was determined through forward feature selection and Cox regression models in HNSCC cohorts. Functional characterization of selected prognostic genes was conducted in 2D and 3D cellular models, and findings were validated by immunohistochemistry in primary HNSCC. Results An EGFR-mediated EMT gene signature composed of n  = 171 genes was identified in responsive cell lines and transferred to the TCGA-HNSCC cohort. A 5-gene risk score comprising DDIT4, FADD, ITGB4, NCEH1, and TIMP1 prognosticated overall survival (OS) in TCGA and was confirmed in independent HNSCC cohorts. The EGFR-mediated EMT signature was distinct from EMT hallmark and partial EMT (pEMT) meta-programs with a differing enrichment pattern in single malignant cells. Molecular characterization showed that ITGB4 was upregulated in primary tumors and metastases compared to normal mucosa and correlated with EGFR/MAPK activity in tumor bulk and single malignant cells. Preferential localization of ITGB4 together with its ligand laminin 5 at tumor-stroma interfaces correlated with increased tumor budding in primary HNSCC tissue sections. In vitro, ITGB4 knock-down reduced EGFR-mediated migration and invasion and ITGB4-antagonizing antibody ASC8 impaired 2D and 3D invasion. Furthermore, a logistic regression model defined ITGB4 as a predictive marker of progression-free survival in response to Cetuximab in recurrent metastatic HNSCC patients. Conclusions EGFR-mediated EMT conveyed through MAPK activation contributes to HNSCC progression upon induction of migration and invasion. A 5-gene risk score based on a novel EGFR-mediated EMT signature prognosticated survival of HNSCC patients and determined ITGB4 as potential therapeutic and predictive target in patients with strong EGFR-mediated EMT.

Receptor-interacting serine/threonine-protein kinase 1 (RIPK1) is a critical regulator of cell death and inflammation, but its relevance for human disease pathogenesis remains elusive. Studies of monogenic disorders might provide critical insights into disease mechanisms and therapeutic targeting of RIPK1 for common diseases. Here, we report on eight patients from six unrelated pedigrees with biallelic loss-of-function mutations in RIPK1 presenting with primary immunodeficiency and/or intestinal inflammation. Mutations in RIPK1 were associated with reduced NF-κB activity, defective differentiation of T and B cells, increased inflammasome activity, and impaired response to TNFR1-mediated cell death in intestinal epithelial cells. The characterization of RIPK1-deficient patients highlights the essential role of RIPK1 in controlling human immune and intestinal homeostasis, and might have critical implications for therapies targeting RIPK1.

Epstein-Barr virus positive (EBV ) smooth muscle tumors (SMTs) constitute a very rare oncological entity. They usually develop in the context of secondary immunodeficiency caused by human immunodeficiency virus infection or immunosuppressive treatment after solid organ transplantation. However, in a small fraction of predominantly pediatric patients, EBV SMTs may occur in patients with primary immunodeficiency disorders (PIDs), such as GATA2 and CARMIL2 deficiency. In secondary immunodeficiencies and when the underlying condition can not be cured, the treatment of EBV SMTs is based on surgery in combination with antiretroviral and reduced or altered immunosuppressive pharmacotherapy, respectively. Importantly, without definitive reconstitution of cellular immunity, long-term survival is poor. This is particularly relevant for patients with EBV SMTs on the basis of PIDs. Recently, allogeneic hematopoietic stem cell transplantation resulted in cure of immunodeficiency and EBV SMTs in a GATA2-deficient patient. We propose that in the absence of secondary immunodeficiency disorders patients presenting with EBV SMTs should be thoroughly evaluated for PIDs. Allogeneic hematopoietic stem cell transplantation should be taken into consideration, ideally in the setting of a prospective clinical trial.

Rubella virus (RuV) has recently been found in association with granulomatous inflammation of the skin and several internal organs in patients with inborn errors of immunity (IEI). The cellular tropism and molecular mechanisms of RuV persistence and pathogenesis in select immunocompromised hosts are not clear. We provide clinical, immunological, virological, and histological data on a cohort of 28 patients with a broad spectrum of IEI and RuV-associated granulomas in skin and nine extracutaneous tissues to further delineate this relationship. Combined immunodeficiency was the most frequent diagnosis (67.8%) among patients. Patients with previously undocumented conditions, i.e., humoral immunodeficiencies, a secondary immunodeficiency, and a defect of innate immunity were identified as being susceptible to RuV-associated granulomas. Hematopoietic cell transplantation was the most successful treatment in this case series resulting in granuloma resolution; steroids, and TNF-α and IL-1R inhibitors were moderately effective. In addition to M2 macrophages, neutrophils were identified by immunohistochemical analysis as a novel cell type infected with RuV. Four patterns of RuV-associated granulomatous inflammation were classified based on the structural organization of granulomas and identity and location of cell types harboring RuV antigen. Identification of conditions that increase susceptibility to RuV-associated granulomas combined with structural characterization of the granulomas may lead to a better understanding of the pathogenesis of RuV-associated granulomas and discover new targets for therapeutic interventions.

BackgroundThe NLRP3 inflammasome integrates several danger signals into the activation of innate immunity and inflammation by secreting IL-1β and IL-18. Most published data relate to the NLRP3 inflammasome in immune cells, but some reports claim similar roles in parenchymal, namely epithelial, cells. For example, podocytes, epithelial cells critical for the maintenance of kidney filtration, have been reported to express NLRP3 and to release IL-β in diabetic kidney disease, contributing to filtration barrier dysfunction and kidney injury. We questioned this and hence performed independent verification experiments.MethodsWe studied the expression of inflammasome components in human and mouse kidneys and human podocytes using single-cell transcriptome analysis. Human podocytes were exposed to NLRP3 inflammasome agonists in vitro and we induced diabetes in mice with a podocyte-specific expression of the Muckle-Wells variant of NLRP3, leading to overactivation of the Nlrp3 inflammasome (Nphs2Cre;Nlrp3A350V) versus wildtype controls. Phenotype analysis included deep learning-based glomerular and podocyte morphometry, tissue clearing, and STED microscopy of the glomerular filtration barrier. The Nlrp3 inflammasome was blocked by feeding ß-hydroxy-butyrate.ResultsSingle-cell transcriptome analysis did not support relevant NLRP3 expression in parenchymal cells of the kidney. The same applied to primary human podocytes in which NLRP3 agonists did not induce IL-1β or IL-18 secretion. Diabetes induced identical glomerulomegaly in wildtype and Nphs2Cre;Nlrp3A350V mice but hyperfiltration-induced podocyte loss was attenuated and podocytes were larger in Nphs2Cre;Nlrp3A350V mice, an effect reversible with feeding the NLRP3 inflammasome antagonist ß-hydroxy-butyrate. Ultrastructural analysis of the slit diaphragm was genotype-independent hence albuminuria was identical.ConclusionPodocytes express low amounts of the NLRP3 inflammasome, if at all, and do not produce IL-1β and IL-18, not even upon introduction of the A350V Muckle-Wells NLRP3 variant and upon induction of podocyte stress. NLRP3-mediated glomerular inflammation is limited to immune cells.

Patients with head‐and‐neck cancer can develop both lung metastasis and primary lung cancer during the course of their disease. Despite the clinical importance of discrimination, reliable diagnostic biomarkers are still lacking. Here, we have characterised a cohort of squamous cell lung (SQCLC) and head‐and‐neck (HNSCC) carcinomas by quantitative proteomics. In a training cohort, we quantified 4,957 proteins in 44 SQCLC and 30 HNSCC tumours. A total of 518 proteins were found to be differentially expressed between SQCLC and HNSCC, and some of these were identified as genetic dependencies in either of the two tumour types. Using supervised machine learning, we inferred a proteomic signature for the classification of squamous cell carcinomas as either SQCLC or HNSCC, with diagnostic accuracies of 90.5% and 86.8% in cross‐ and independent validations, respectively. Furthermore, application of this signature to a cohort of pulmonary squamous cell carcinomas of unknown origin leads to a significant prognostic separation. This study not only provides a diagnostic proteomic signature for classification of secondary lung tumours in HNSCC patients, but also represents a proteomic resource for HNSCC and SQCLC. Synopsis Differentiation between head‐and‐neck cancer metastasis and primary lung cancer is clinically important for therapy selection. A novel diagnostic proteomic signature allows differentiation between these tumour types, and a proteomic resource for squamous cell tumours is here provided. The protein expression profiles of 63 squamous cell lung and 49 head‐and‐neck tumours were analysed by quantitative mass spectrometry yielding a proteomic resource covering 6,214 quantified proteins. 518 proteins were found to be differentially expressed between squamous cell lung and head‐and‐neck cancers which are known to share genomic and morphological features. A diagnostic proteomic signature for differentiation between primary squamous cell lung cancers and head‐and‐neck cancer metastases was identified by quantitative mass‐spectrometry‐based proteomics and validated in independent patient cohorts. Graphical Abstract Differentiation between head‐and‐neck cancer metastasis and primary lung cancer is clinically important for therapy selection. A novel diagnostic proteomic signature allows differentiation between these tumour types, and a proteomic resource for squamous cell tumours is here provided.

Despite advances in multimodal therapy, survival in advanced head and neck squamous cell carcinoma (HNSCC) has improved only modestly. Tumor-informed circulating tumor DNA (ctDNA) assays allow early detection of molecular residual disease (MRD) and recurrence after curative treatment. We analyzed ctDNA in plasma from 76 and saliva from 54 HNSCC patients before and after curative-intent surgery, testing 656 plasma and 128 saliva samples longitudinally. High preoperative ctDNA shedding correlated with advanced pathological stage, lymph node involvement, adverse histologic features, and molecular markers including PD-1 expression and tumor mutational burden. Transcriptomic profiling showed associations between high shedding and increased proliferation, EGFR/MAPK pathway activity, and upregulation of EGFR-related invasion and metastasis genes. Plasma ctDNA detected ≥14 days post-surgery identified 91.3% of recurrences, with lead times up to 500 days before clinical confirmation. These results highlight the value of serial ctDNA monitoring for early relapse detection and potentially improved treatment guidance in HNSCC.

Objectives Cold ischemia during kidney transplantation induces ischemia-reperfusion injury with endothelial dysfunction, capillary leak, and impaired perfusion. Its duration critically determines graft outcome. Dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) enables noninvasive assessment of renal microcirculation and may indicate ischemic injury. We evaluated the impact of ischemia duration on DCE-MRI-derived perfusion parameters in renal transplants in mice. Materials and methods Procedures were approved by the local institutional animal care and use committee. A total of 15 C57BL/6 mice underwent kidney transplantation and were assigned to a short or prolonged cold ischemia group. DCE-MRI was performed to assess renal perfusion. Imaging was conducted at a mean of 268 ± 30 days (mean ± standard deviation) after transplantation. Perfusion parameters were calculated using the Patlak model, which provides the plasma volume fraction (v p ), reflecting renal blood volume and perfusion, and the volume transfer constant (K trans ), characterizing the rate of contrast agent extravasation from capillaries into the extravascular extracellular space. Results Significant differences were observed in the K trans parameter of transplanted kidneys between groups. The median K trans (mL/100 mL/min) was significantly higher in the 16-h group (2.87, interquartile range 2.45–3.03) versus the 30-min group (0.91, 0.90–1.42; p  = 0.008). Median v p (mL/100 mL/min) was non-significantly lower in the 16-h group (21.89, 17.28–23.22) versus the 30-min group (29.02, 24.99–37.15; p  = 0.151). Conclusion Cold ischemia with 16-h duration was associated with significantly higher K trans values in kidney transplants, reflecting significantly increased vascular permeability. DCE-MRI provides a sensitive tool for detecting ischemia-induced microvascular dysfunction. Relevance statement Quantitative DCE-MRI detects microvascular injury after 16-h cold ischemia in kidney transplants in mice, supporting its potential as a noninvasive tool to assess graft integrity and guide interventions aimed at improving long-term transplant outcomes. Key Points The duration of ischemia critically affects endothelial integrity and perfusion characteristics in a mouse kidney transplant model. Prolonged 16-h ischemia leads to increased vascular permeability, indicating more severe endothelial and microcirculatory injury in transplanted kidneys. DCE-MRI enables sensitive detection of subtle ischemia-related microvascular alterations, supporting its value for noninvasive graft assessment. Graphical Abstract