Martin Werner

Background/Objectives: HER2-directed antibody–drug conjugates have expanded the clinical relevance of low-level HER2 expression. However, the prevalence and clinical significance of HER2-ultralow expression in gynecologic malignancies remain insufficiently defined. This study characterized the full spectrum of HER2 expression in endometrial carcinoma (EC) and high-grade serous ovarian carcinoma (HGSOC), including matched metastatic lesions. Methods: HER2 expression was assessed by immunohistochemistry in tissue microarrays from 117 EC and 52 HGSOC patients. Available matched metastatic lesions were analyzed in 23 EC and 18 HGSOC cases. HER2 expression was categorized as zero, ultralow, 1+, 2+, or 3+. Associations with clinicopathological parameters and progression-free survival were evaluated. Results: HER2-ultralow expression was common in primary tumors, occurring in 49/117 EC cases (41.9%) and 11/52 HGSOC cases (21.2%), whereas HER2 3+ expression was rare (EC: 2/117, 1.7%; HGSOC: 1/52, 1.9%). HER2 expression in primary tumors was associated with progression-free survival in both entities, with HER2-zero tumors showing the longest estimated progression-free survival. HER2 score discordance between matched primary tumors and metastases occurred in 16/23 EC cases (69.6%) and 8/18 HGSOC cases (44.4%). Conclusions: HER2-ultralow expression represents a frequent and previously underrecognized category in EC and HGSOC. While its prognostic impact remains limited, the high prevalence of HER2-ultralow tumors suggests potential relevance for future HER2-targeted therapeutic strategies. Prospective studies are needed to determine whether this subgroup may benefit from a HER2-targeted therapy.

Background/Objectives: Purinergic signaling, which involves extracellular ATP (eATP), its metabolites, purinergic receptors and ectonucleotidases, plays a pivotal role in the tumor microenvironment (TME), impacting tumor progression and the antineoplastic immune response. In this study, the CD39, CD73, P2X4, and P2X7 expression in NSCLC tumor cells and the surrounding stroma of 139 resected patients was examined. Methods: The study included tissue samples from 139 NSCLC patients. Tissue microarrays (TMAs) were constructed using 1.0 mm cores from annotated tumor regions. Immunohistochemical staining for CD39, CD73, P2X4, and P2X4 was performed on 2 µm sections. TMA slides were digitized and analyzed with QuPath, where staining intensity was evaluated using a semi-quantitative H-score. Statistical analysis, including survival analysis, was performed using R, to assess the impact of biomarker expression on patient outcomes. Results: High CD39 expression in both tumor and stromal cells was significantly associated with prolonged PFS (respectively: p = 0.0058 and p = 0.0067), particularly in adenocarcinoma (ADC) patients (respectively: p = 0.01 and p = 0.023). In the multivariable Cox model, low CD73 expression in tumor cells correlated with longer PFS (HR: 0.47; 95% CI: [0.28, 0.8], p = 0.005), while low CD73 expression in stromal cells was linked to increased progression risk (HR: 4.81; 95% CI: [1.61, 14.4], p = 0.001). Neither P2X7 nor P2X4 demonstrated a consistent effect on PFS in univariable analyses; however, multivariable analyses suggested that P2X4 might play a prognostic role in NSCLCs (HR: 0.37; 95% CI: [0.19, 0.73], p = 0.003). Conclusions: These findings underscore the importance of purinergic signaling in NSCLC prognosis and highlight the role of the ectonucleotidases CD39 and CD73 as potential therapeutic targets to enhance antineoplastic immune responses.

Immunohistochemical (IHC) studies of formalin-fixed paraffin-embedded (FFPE) samples are a gold standard in oncology for tumor characterization, and the identification of prognostic and predictive markers. However, despite the abundance of archived FFPE samples, their research use is limited due to the labor-intensive nature of IHC on large cohorts. This study aimed to create a high-throughput workflow using modern technologies to facilitate IHC biomarker studies on large patient groups. Semiautomatic constructed tissue microarrays (TMAs) were created for two tumor patient cohorts and IHC stained for seven antibodies (ABs). AB expression in the tumor and surrounding stroma was quantified using the AI-supported image analysis software QuPath. The data were correlated with clinicopathological information using an R-script, all results were automatically compiled into formatted reports. By minimizing labor time to 7.7%—compared to whole-slide studies—the established workflow significantly reduced human and material resource consumption. It successfully correlated AB expression with overall patient survival and additional clinicopathological data, providing publication-ready figures and tables. The AI-assisted high-throughput TMA workflow, validated on two patient cohorts, streamlines modern histopathological research by offering cost and time efficiency compared to traditional whole-slide studies. It maintains research quality and preserves patient tissue while significantly reducing material and human resources, making it ideal for high-throughput research centers and collaborations.

Stimulated Raman Histology (SRH) employs the stimulated Raman scattering (SRS) of photons at biomolecules in tissue samples to generate histological images. Subsequent pathological analysis allows for an intraoperative evaluation without the need for sectioning and staining. The objective of this study was to investigate a deep learning-based classification of oral squamous cell carcinoma (OSCC) and the sub-classification of non-malignant tissue types, as well as to compare the performances of the classifier between SRS and SRH images. Raman shifts were measured at wavenumbers k₁ = 2845 cm⁻¹ and k₂ = 2930 cm⁻¹. SRS images were transformed into SRH images resembling traditional H&E-stained frozen sections. The annotation of 6 tissue types was performed on images obtained from 80 tissue samples from eight OSCC patients. A VGG19-based convolutional neural network was then trained on 64 SRS images (and corresponding SRH images) and tested on 16. A balanced accuracy of 0.90 (0.87 for SRH images) and F1-scores of 0.91 (0.91 for SRH) for stroma, 0.98 (0.96 for SRH) for adipose tissue, 0.90 (0.87 for SRH) for squamous epithelium, 0.92 (0.76 for SRH) for muscle, 0.87 (0.90 for SRH) for glandular tissue, and 0.88 (0.87 for SRH) for tumor were achieved. The results of this study demonstrate the suitability of deep learning for the intraoperative identification of tissue types directly on SRS and SRH images.

Glomerular disease due to podocyte malfunction is a major factor in the pathogenesis of chronic kidney disease. Identification of podocyte-specific signaling pathways is therefore a prerequisite to characterizing relevant disease pathways and developing novel treatment approaches. Here, we employed loss of function studies for EPB41L5 (Yurt) as a central podocyte gene to generate a cell type-specific disease model. Loss of Yurt in fly nephrocytes caused protein uptake and slit diaphragm defects. Transcriptomic and proteomic analysis of human EPB41L5 knockout podocytes demonstrated impaired mechanotransduction via the YAP/TAZ signaling pathway. Further analysis of specific inhibition of the YAP/TAZ-TEAD transcription factor complex by TEADi led to the identification of ARGHAP29 as an EPB41L5 and YAP/TAZ-dependently expressed podocyte RhoGAP. Knockdown of ARHGAP29 caused increased RhoA activation, defective lamellipodia formation, and increased maturation of integrin adhesion complexes, explaining similar phenotypes caused by loss of EPB41L5 and TEADi expression in podocytes. Detection of increased levels of ARHGAP29 in early disease stages of human glomerular disease implies a novel negative feedback loop for mechanotransductive RhoA—YAP/TAZ signaling in podocyte physiology and disease.

About 50% of colorectal cancer patients develop liver metastases. Patients with metastatic colorectal cancer have 5-year survival rates below 20% despite new therapeutic regimens. Tumor heterogeneity has been linked with poor treatment response and clinical outcome, but was so far mainly studied via bulk genomic analyses. In this study we performed spatial proteomics via MALDI mass spectrometry imaging on six patient-matched CRC primary tumor and liver metastases to characterize interpatient, intertumor and intratumor hetereogeneity. We found several peptide features that were enriched in vital tumor areas of primary tumors and liver metastasis and tentatively derived from tumor cell specific proteins such as annexin A4 and prelamin A/C. Liver metastases of colorectal cancer showed higher heterogeneity between patients than primary tumors while within patients both entities show similar intratumor heterogeneity sometimes organized in zonal pattern. Together our findings give new insights into the spatial proteomic heterogeneity of primary CRC and patient-matched liver metastases.

Human papillomavirus (HPV)-driven head and neck squamous cell carcinomas (HNSCC) generally have a more favourable prognosis. We hypothesized that HPV-associated HNSCC may be identified by an miRNA-signature according to their specific molecular pathogenesis, and be characterized by a unique transcriptome compared to HPV-negative HNSCC. We performed miRNA expression profiling of two p16/HPV DNA characterized HNSCC cohorts of patients treated by adjuvant radio(chemo)therapy (multicentre DKTK-ROG n = 128, single-centre LMU-KKG n = 101). A linear model predicting HPV status built in DKTK-ROG using lasso-regression was tested in LMU-KKG. LMU-KKG tumours (n = 30) were transcriptome profiled for differential gene expression and miRNA-integration. A 24-miRNA signature predicted HPV-status with 94.53% accuracy (AUC: 0.99) in DKTK-ROG, and 86.14% (AUC: 0.86) in LMU-KKG. The prognostic values of 24-miRNA- and p16/HPV DNA status were comparable. Combining p16/HPV DNA and 24-miRNA status allowed patient sub-stratification and identification of an HPV-associated patient subgroup with impaired overall survival. HPV-positive tumours showed downregulated MAPK, Estrogen, EGFR, TGFbeta, WNT signaling activity. miRNA-mRNA integration revealed HPV-specific signaling pathway regulation, including PD−L1 expression/PD−1 checkpoint pathway in cancer in HPV-associated HNSCC. Integration of clinically established p16/HPV DNA with 24-miRNA signature status improved clinically relevant risk stratification, which might be considered for future clinical decision-making with respect to treatment de-escalation in HPV-associated HNSCC.

Steroid-resistant nephrotic syndrome (SRNS) frequently leads to end-stage renal disease, ultimately requiring kidney replacement therapies. SRNS is often caused by hereditary monogenic mutations, specifically affecting specialized epithelial cells (podocytes) of the glomerular filtration barrier. Mutations in several components of the nuclear pore complex, including NUP133 and NUP107, have been recently identified to cause hereditary SRNS. However, underlying pathomechanisms, eliciting podocyte-specific manifestations of these nucleoporopathies, remained largely elusive. Here, we generated an in vitro model of NUP133-linked nucleoporopathies using CRISPR/Cas9-mediated genome editing in human podocytes. Transcriptome, nuclear pore assembly, and cytoskeleton regulation of NUP133 loss-of-function, mutant, and wild-type podocytes were analyzed. Loss of NUP133 translated into a disruption of the nuclear pore, alterations of the podocyte-specific transcriptome, and impaired cellular protrusion generation. Surprisingly, comparative analysis of the described SRNS-related NUP133 mutations revealed only mild defects. Am impaired protein interaction in the Y-complex and decrease of NUP133 protein levels might be the primary and unifying consequence of mutant variants, leading to a partial loss-of-function phenotype and disease manifestation in susceptible cell types, such as podocytes.

We describe a sequential multistaining protocol for immunohistochemistry, immunofluorescence and CyTOF imaging for formalin-fixed, paraffin-embedded specimens (FFPE) in the formalin gas-phase (FOLGAS), enabling sequential multistaining, independent from the primary and secondary antibodies and retrieval. Histomorphologic details are preserved, and crossreactivity and loss of signal intensity are not detectable. Combined with a DAB-based hydrophobic masking of metal-labeled primary antibodies, FOLGAS allows the extended use of CyTOF imaging in FFPE sections.

Background: Ectopic thyroid tissue in the iris, also known as a thyroid glandular epithelial choristoma of the iris, has only been described twice in the literature. In both cases it remained asymptomatic. Case presentation: A 67-year-old female patient presented for the first time in mid-2017 with corneal endothelial decompensation, with a history of complicated cataract surgery and IStent® implantation. Slit lamp microscopy showed endothelial decompensation, pseudophakia, anterior synechiae and a whitish iris tumour adhering to the endothelium. The latter had existed since childhood. Given these findings, reduced visual acuity of hand movement perception and an intraocular pressure of 23 mmHg, we performed a keratoplasty combined with an en bloc resection of the iris tumour at 9 o’clock and sector iridectomy at the end of 2019. Histological and immunohistological examination of the iris tumour unexpectedly revealed thyroid tissue. After the procedure described above, the patient had an increase in visual acuity while the graft stayed clear and the eye showed no evidence of tumour recurrence or other complications. Conclusions: We report a third case of ectopic thyroid tissue in the iris. Both previous cases remained asymptomatic, whereas in our case, size and location of the ectopic thyroid tissue contributed to a more complex cataract surgery resulting in endothelial decompensation. Therefore, in such cases appropriate patient information should be provided prior to cataract surgery. Furthermore, careful histological examination and examination of the thyroid is important to exclude malignant diagnoses such as a metastasis of a follicular thyroid carcinoma.