The immune composition of the tumor microenvironment has a major impact on the therapy response in patients with colorectal cancer. Here, we built an atlas with 4.27 million single cells from 1,670 patient samples and complemented it with single-cell profiles from 266 patients, including cells with low mRNA content, spatial transcriptomics from 3.7 million cells, and protein profiles from 0.7 million cells. The analysis of the atlas allows tumor classification into immune desert, B cell enriched, T cell enriched, and myeloid cell enriched immune phenotypes. Within the myeloid compartment, we identify consensus myeloid gene expression programs with four immunomodulatory programs, and uncover a subpopulation of neutrophils with antigen-presenting properties. Moreover, functional experiments using patient-derived organoids show KRAS-dependent pro-tumorigenic polarization of neutrophils. Further, spatial multimodal single-cell profiling reveals niches with IL-1 signaling-based neutrophil-fibroblast interaction. Finally, using an orthotopic mouse model, we show that cancer-derived signals modify neutrophil production in the bone marrow.

Background: Advances in molecular pathology have transformed NSCLC (Non-Small Cell Lung Cancer) diagnosis, prognosis, and treatment by enabling precise tumor characterization and targeted therapeutic strategies. We review key genomic alterations in NSCLC, including EGFR (epidermal growth factor receptor) mutations, ALK (anaplastic lymphoma kinase) and ROS1 (ROS proto-oncogene 1) rearrangements, BRAF (B-Raf proto-oncogene serine/threonine kinase) mutations, MET (mesenchymal-epithelial transition factor) alterations, KRAS (Kirsten rat sarcoma) mutations, HER2 (human epidermal growth factor receptor 2) alterations and emerging NTRK (neurotrophic receptor tyrosine kinase) fusions and AXL-related pathways. Methods: A total of 48 patients with NSCLC was analyzed, including 22 women and 26 men (mean age 70 years, range 44-86). Tumor specimens were classified histologically as adenocarcinomas (n = 81%) or squamous cell carcinomas (n = 19%). Smoking history, PD-L1 (programmed death-ligand 1) expression, and genetic alterations were assessed. NGS (Next-generation sequencing) identified genomic variants, which were classified according to ACMG (American College of Medical Genetics and Genomics) guidelines. Results: The cohort consisted of 29 former smokers, 13 current smokers, and 5 non-smokers (12%), with a mean smoking burden of 33 pack years. PD-L1 TPS (tumor proportion score) was ≥50% in 10 patients, ≥1-<50% in 22, and <1% in 15 patients. In total, 120 genomic variants were detected (allele frequency ≥ 5%). Of these, 52 (43%) were classified as likely pathogenic or pathogenic, 48 (40%) as variants of unknown significance, and 20 (17%) as benign or likely benign. The most frequently altered genes were TP53 (tumor protein p53) (31%), KRAS and EGFR (15% each), and STK11 (serine/threonine kinase 11) (12%). Adenocarcinomas accounted for 89% of all alterations, with TP53 (21%) and KRAS (15%) being most common, while squamous cell carcinomas predominantly harbored TP53 (38%) and MET (15%) mutations. In patients with PD-L1 TPS ≥ 50%, KRAS mutations were enriched (50%), particularly KRAS G12C and G12D, with frequent co-occurrence of TP53 mutations (20%). No pathogenic EGFR mutations were detected in this subgroup. Conclusions: Comprehensive genomic profiling in NSCLC revealed a high prevalence of clinically relevant mutations, with TP53, KRAS and EGFR as the dominant drivers. The strong association of KRAS mutations with high PD-L1 expression, irrespective of smoking history, highlights the interplay between genetic and immunological pathways in NSCLC. These findings support the routine implementation of broad molecular testing to guide precision oncology approaches in both adenocarcinoma and squamous cell carcinoma patients.

Recently, we suggested the combination of chemotherapy and P2RX4 inhibition as a promising novel therapeutic approach for P2RX4-expressing epithelial tumors to prevent paracrine resistance. Here, we aimed to assess whether determining P2RX4 expression status in colorectal and pancreatic cancer patients would allow stratification of potentially responsive patients. Therefore, P2RX4 expression levels were determined by RNA sequencing and immunohistochemistry. Subcellular localization of P2RX4 isoforms was analyzed in HeLa cells and patient-derived tumor organoids. In contrast to its RNA expression profile, P2RX4 protein levels exhibited differential regulation in human colorectal and pancreatic cancer epithelia due to alternative splicing. Interpatient heterogeneity was greater in colorectal cancer than in pancreatic cancer. Notably, these variations in expression did not correlate with overall patient survival. Alternative P2RX4 transcripts gave rise to functionally distinct protein isoforms that differed in subcellular localization and total protein abundance. Only the correctly spliced, canonical P2RX4 isoform was localized to the plasma membrane and was capable of mediating downstream signaling. Accordingly, P2RX4 inhibition in combination with chemotherapy was effective exclusively in patient-derived tumor organoids expressing the canonical P2RX4 transcript. In summary, immunohistochemical, but not transcriptomic, assessment of P2RX4 expression enabled the prediction of sensitivity to combinatorial treatment and facilitated the identification of patients who may benefit from P2RX4 inhibition during chemotherapy. Given the lower degree of heterogeneity observed in pancreatic cancer, this tumor entity may represent a promising candidate for early-phase clinical evaluation of chemotherapy combined with P2RX4 inhibition. © 2026 The Author(s). The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

BACKGROUND:Tumor spread through air spaces (STAS) has been proposed as a histopathological marker of aggressive tumor biology in adenocarcinoma of the lung (ADCL). Its independent prognostic significance and clinical implications regarding surgical strategy remain controversial. This study evaluated clinicopathological correlates and the prognostic impact of STAS in a homogeneous cohort of resected ADCL. METHODS:We retrospectively analyzed 100 patients with primary ADCL resected between 2009 and 2018. STAS was classified as absent, low (1-4 clusters), or high (≥5) by an experienced pathologist. Associations between STAS and clinical, surgical, and pathological variables were tested with univariate analyses and multivariable logistic regression. Overall survival (OS) was evaluated using Kaplan-Meier and Cox regression. RESULTS:STAS was present in 46% of tumors and was significantly associated with a higher pathological N category (pN0-pN3; p = 0.005), more advanced UICC stage (p = 0.049), lymphovascular invasion (LVI; p = 0.008), and perineural invasion (PnI; p = 0.012). In univariate survival analysis, patients with STAS had shorter OS than patients without STAS (p = 0.047). After limited resection, OS did not differ (p = 0.864), whereas after radical anatomical resection, patients with STAS showed reduced OS (p = 0.034). In multivariable Cox regression analysis, STAS did not retain independent prognostic significance. CONCLUSIONS:STAS is frequent in resected ADCL and correlates with adverse pathological features and reduced OS in univariate models. In multivariate analysis, STAS did not emerge as an independent prognostic factor. These findings support the interpretation of STAS as a marker of aggressive tumor biology rather than an independent determinant of prognosis or surgical decision-making.

INTRODUCTION:IDH1 mutations occur in approximately 10%-20% of intrahepatic cholangiocarcinoma (iCCA) and constitute an established target for molecularly guided therapy. As routine molecular diagnostics are commonly based on a single tumour sample, intratumoral heterogeneity could affect the reliable detection of actionable mutations. This study assessed the spatial heterogeneity of IDH1 mutations in iCCA. METHODS:Patients with histologically confirmed iCCA who underwent routine next-generation sequencing (NGS) and had multiple available formalin-fixed paraffin-embedded (FFPE) tumour samples were retrospectively analysed. Baseline IDH1 status was determined by NGS. All available tumour regions were subsequently tested using the Idylla™ IDH1-2 Mutation Assay. Blocks where the Idylla™ IDH1 mutational status was discordant with baseline status were validated by digital polymerase chain reaction (dPCR). RESULTS:Thirty-five patients with iCCA were included, yielding 117 FFPE samples from spatially distinct tumour regions. Baseline NGS identified IDH1 mutations in 22.8% (8/35) of patients. Idylla™ testing revealed discordant results in 5 of 117 samples (4.2%). Validation by dPCR demonstrated that these discordances were attributable to technical limitations or assay-related errors rather than true biological heterogeneity. Following validation, all IDH1-mutated tumours showed concordant mutation status across all analysed tumour regions. CONCLUSIONS:IDH1 mutations appear to be spatially homogeneous in iCCA, supporting their role as an early clonal event. These findings indicate that single-sample molecular testing is sufficient for reliable determination of IDH1 status and patient selection for IDH1-targeted therapies.

INTRODUCTION: Extrapulmonary tuberculosis (TB) and non-tuberculous mycobacterial (NTM) disease remain a diagnostic challenge and conventional methods such as mycobacterial culture or molecular methods (e.g. GeneXpert) performed on formalin-fixed paraffin embedded (FFPE) specimens are often unsuccessful. In this study, we assessed the diagnostic accuracy of chip hybridization from FFPE specimens in a cohort with predominantly extrapulmonary mycobacterial disease. METHODS: FFPE specimens from patients with presumed mycobacterial disease underwent chip hybridization with two different assays (MYCO Direct 1.7 and MYCO Chip Vision Array 2.0) in addition to conventional diagnostic methods for TB and NTM disease including mycobacterial culture, molecular methods and conventional histology. The different techniques were assessed with regards to sensitivity, specificity and species identification. RESULTS: Overall, 184 samples were assessed by one of the two chip hybridization techniques (MYCO Direct 1.7 n=65 and MYCO Chip Vision Array n=119). Samples were mainly extrapulmonary (160/184, 87.0%). Of all samples, 32 (17.4%) turned positive in chip hybridization and 74/184 patients (40.2%) were diagnosed with clinically relevant mycobacterial disease (59 with TB and 15 with NTM disease). For the detection of Mycobacterium tuberculosis complex (MTBC), sensitivity was 38.9% when compared to mycobacterial culture from the same specimen. For the detection of NTM, sensitivity was 50% against NTM culture. Species identification was overall consistent with conventional methods but did not allow for species differentiation of members of the MTBC or Mycobacterium avium complex, respectively. CONCLUSION: As extrapulmonary TB and NTM disease remain a diagnostic challenge, the investigated chip hybridization techniques might present a complementation of the diagnostic portfolio. However, with a low sensitivity to detect MTB from extrapulmonary FFPE specimens, they should not supplant conventional diagnostic methods but could be used to rule in TB when native specimens are not available.

Background: Cholangiocarcinoma, a malignancy originating from the bile ducts, poses significant treatment challenges due to its typically late diagnosis and limited therapeutic options. However, recent advances in molecular genetics enable more personalized treatment approaches. A notable breakthrough in this context is the identification of isocitrate dehydrogenase (IDH) mutations, particularly IDH1 and IDH2, which occur in a subset of cholangiocarcinoma patients. Those with IDH1/2 mutations may benefit from targeted therapies. For instance, Ivosidenib, an IDH1 inhibitor, has shown efficacy in clinical trials, offering a new therapeutic option for patients with IDH1-mutant cholangiocarcinoma. Developing and implementing standardized protocols for testing and reporting mutation status are crucial for consistency and accuracy in clinical practice. Both the Idylla™ IDH1-2 Mutation Assay Kit as a FastTrack method and Next-Generation Sequencing (NGS) panels play critical roles in molecular characterization of cholangiocarcinoma. Methods: Under this aspect, a set of cholangiocarcinomas was tested using the Idylla™ platform regarding the respective recommended guidelines and standards of DIN EN ISO:17020 and DIN EN ISO:15198. Results: Overall, 25 clinically diagnosed intrahepatic cholangiocarcinomas or Adeno-CUPs were analyzed. IDH1/2 mutations were identified in 68% (17/25) of cases using both methods, with high concordance between NGS and Idylla™ results. Discrepancies were observed in two samples, where Idylla™ detected no mutations, but NGS reported IDH1 and IDH2 mutations, respectively. Conclusions: IdyllaTM offers a rapid, user-friendly, and specific method for detecting IDH1/2 mutations, ideal for immediate clinical needs. NGS, while more time-consuming and costly, provides comprehensive genetic profiles valuable for personalized medicine and research. The choice between these methods should be guided by the clinical context, resource availability, and individual patient needs. For routine diagnostics, we recommend an algorithmic approach starting with the FastTrack method followed by NGS for wildtype cases.

Head and neck cancers (HNC) represent an extremely heterogeneous group of diseases with a poorly predictable therapy outcome. Patient-derived tumor organoids (PDTO) offer enormous potential for individualized therapy testing and a better mechanistic understanding of the main HNC drivers. Here, we have established a comprehensive molecularly and functionally characterized head and neck organoid biobank (HNOB) recapitulating the clinically relevant subtypes of TP53 mutant and human papillomavirus type 16 (HPV 16) infection-driven HNC. Organoids were exposed to radiotherapy, and responses were correlated with clinical data. Genetically engineered normal and tumor organoids were used for testing the direct functional consequences of TP53-loss and HPV infection. The HNOB consisting of 18 organoid models, including 15 tumor models, was generated. We identified subtype-associated transcriptomic signatures and pathological features, including sensitivity to TP53 stabilization by the MDM2 inhibitor Nutlin-3. Furthermore, we describe an in vitro radio response assay revealing phenotypic heterogeneity linked to the individual patient’s treatment outcome, including relapse probability. Using genetically engineered organoids, the possibility of co-existence of both cancer drivers was confirmed. TP53 loss, as well as HPV, increased growth in normal and tumor organoids. TP53 loss-of-function alone was insufficient to promote radiation resistance, whereas HPV 16 oncogenes E6/E7 mediated radiosensitivity via induction of cell cycle arrest. Our results highlight the translational value of the head and neck organoid models not only for patient stratification but also for mechanistic validation of therapy responsiveness of specific cancer drivers.

Supplementary Figure S1 shows light microscopic images of CRC organoids and CAFs. Supplementary Figure S2 shows immunostaining and RNA sequencing analysis of cultured CAFs. Supplementary Figure S3 shows chromosomal copy number changes in tumors and matched organoids. Supplementary Figure S4 shows the transcriptional variation among tumors and organoids. Supplementary Figure S5 shows the classification of cancer intrinsic subtypes (CRIS) in tumors, matched organoids and xenotransplants. Supplementary Figure S6 shows the tissue microarray analysis of the of tumor samples from the colorectal cancer organoid-stroma biobank cohort. Supplementary Figure S7 shows the association of growth characteristics with molecular features of colorectal cancer organoid-stroma biobank. Supplementary Figure S8 shows the shows CMS and CRIS classifications of tumors and of matched organoids in different contexts. Supplementary Figure S9 describes the establishment of a drug screening workflow in 3D organoid-stroma co-cultures. Supplementary Figure S10 describes the development of a dual luciferase assay to study cell viability simultaneously in organoids and CAFs. Supplementary Figure S11 demonstrates that the MET inhibitor BAY-474 sensitizes Gefitinib resistant co-cultures. Supplementary Figure S12 shows the association of identified sensitivity and resistance signatures with gene expression and prognosis in different CMS.

Background/Objectives: The non-small-cell lung cancer (NSCLC) therapeutic landscape has undergone a profound transformation with the introduction of multiple personalized treatment options. Mutations in ERBB2 (HER2) have recently emerged as promising novel targets for the treatment of non-squamous NSCLC (nsNSCLC). Accurate, rapid, and efficient molecular profiling is crucial for identifying patients who may benefit from targeted therapies, including HER2-directed agents. Materials and Methods: Here, we aimed to retrospectively assess the performance of the Oncomine™ Precision Assay* (OPA) in combination with the Ion Torrent Genexus™ Integrated Sequencer* (Thermo Fisher Scientific. Waltham, MA, USA) for detecting ERBB2 mutations in nsNSCLC. A total of 108 archived nsNSCLC samples, consisting of biopsies, resections, and cytological specimens, were used to assess concordance with in-house-validated orthogonal tests. Results: The OPA showed high sensitivity and specificity with an overall accuracy of 100% for single-nucleotide variants (SNVs) and insertions and deletions (Indels). SNVs and Indels with allele frequencies as low as 5% were correctly identified across samples with a tumor cell content ranging from 5% to 95%. Additionally, the assay demonstrated high reproducibility across the six participating laboratories. The turnaround time of the OPA was notably shorter compared to traditional orthogonal methods, facilitating rapid molecular report generation. Conclusions: The OPA in combination with the Ion Torrent Genexus™ System allows for highly sensitive and specific detection of relevant ERBB2 mutations. The assay’s streamlined workflow, coupled with its automated data analysis pipeline, enables a fast turnaround time for testing across a range of sample types. This includes samples with reduced tumor cell content and limited available input. This study demonstrates the future potential of using this assay in a clinical setting.

The newly discovered histone methyltransferase KMT9 serves as an epigenetic regulator of carcinogenesis in various cancer entities. For the first time, we investigated the presence of KMT9α in cholangiocarcinoma, the association with histologic subtypes, and its impact on survival. A tissue microarray cohort of all CCA patients who underwent surgical resection with curative intent between 08/2005 and 12/2021 at the University Hospital Frankfurt was immunohistochemically analyzed with the KMT9α antibody. For overall survival, Kaplan–Meier curves and Cox-regression analyses were performed. In total, 174 patients were suitable for IHC analysis. Of the patients, 35.1

Individualisierung und Standardisierung sind scheinbar widersprüchliche Anforderungen in der Medizin. In der Tumorbehandlung von Kopf-Hals-Karzinomen haben beide Begriffe direkten Einfluss auf die Diagnostik, welche üblicherweise in pathologischen Instituten durchgeführt wird. Im vorliegenden Referat wird das Spannungsfeld zwischen technischen Untersuchungen, regulatorischen Anforderungen, strukturellen Änderungen durch Digitalisierung und dem Einzug der personalisierten Medizin beleuchtet. Ziel ist zum einen, durch ein Verständnis der Herausforderungen den interdisziplinären Austausch zu fördern, zum anderen sollen dem HNO-Arzt ganz praktisch die gängigen pathologischen Untersuchungen nähergebracht werden. Am Beispiel der Pathologie lässt sich so schlussendlich zeigen, dass die Standardisierung von Verfahren einer Verbesserung der individuellen Behandlung dient. Zugleich lassen sich aber auch die Herausforderungen ablesen: Trotz umfassender Regularien sowie einer Laborumgebung mit digitaler Unterstützung ist die Standardisierung sehr zeit- und kostenaufwendig. Sollen ähnliche Standardisierungsansätze beispielsweise in einem operativen Umfeld wie der HNO-Chirurgie umgesetzt werden, darf der Aufwand aufgrund der „menschlichen Komponente“ als gleichwertig oder höher eingeschätzt werden.

Pleural mesothelioma (PM) is a rare yet aggressive and heterogeneous cancer type with very poor survival rates. Due to its long latency period and nonspecific symptoms, the disease is usually detected at advanced stages, limiting available treatment options and leading to poor survival rates. So far, the disease can only be confirmed through invasive thoracoscopic biopsy, and proposed circulating protein biomarkers lack sensitivity and specificity, highlighting the urgent need for novel approaches. In our previous work, we characterized the transcriptomic profile of extracellular vesicles secreted by PM cells and demonstrated their feasibility as a source of circulating biomarkers. To further investigate the role of circulating RNA in tumor progression and its potential use in non-invasive testing for PM, we present a detailed characterization of RNA cargo carried by PM exosomes - a distinct subset of extracellular vesicles known to serve as key mediators of intercellular communication. By utilizing primary cell cultures established from tissue samples of 11 PM and 6 non-PM patients, followed by exosome isolation, and total RNA sequencing of RNA from isolated exosomes, matching cells, and tissues, we provide new evidence on exosomal RNA secretion, regulation of PM-exosome cargo genes, and their potential functions in recipient cells. We show that PM-exosomal cargo is enriched in genes associated with proliferation, whose key transcriptional factors are SETDB1, FOXM1 and GATA2 . We identified Pcbp2, Srsf1 and Srsf9, RNA-binding proteins, which may be involved in selective cargo sorting into PM-exosomes. Our analysis identified six secreted genes, including GAS5, AL031666 . 3, RSLD1D1, AC103740 . 2, ADAM10 , and AC020892 . 1 , whose exosomal expression was associated with patient survival, as promising biomarkers for patient diagnosis, stratification, and prognosis assessment. Finally, we identify potential target genes of candidate LncRNAs biomarkers and processes in which they are involved. ### Competing Interest Statement The authors have declared no competing interest.

Background The mitochondrial metabolism in prostate cancer (PCa) is of great importance due the unique metabolic shift from glycolysis to oxidative phosphorylation. In this study, we aimed to analyze the expression level of mitochondrial markers TOM20, DRP1 and OPA1 in benign and malignant tissue, to assess if these markers are associated with different grade and stage of PCa. Materials and Methods This study assessed TOM20, DRP1, and OPA1 expression in formalin-fixed, paraffin-embedded prostate tissue samples, including benign and malignant tissue specimen. Immunohistochemistry on tissue microarrays was conducted, with staining intensities scored semi-quantitatively. Statistical analyses evaluated associations with PCa grade and stage. A survival analysis for biochemical recurrence (RFS), overall survival (OS) and disease specific survival (DSS) was performed using multivariate Cox regression analysis to assess prognostic properties of the markers. Results In total, 527 patients were included in our analysis, which composed of 45 (8.5%) benign prostate hyperplasia (BPH) and 482 (91.5%) PCa samples (436 localized (90.5%) and 46 (9.5%) metastatic). Immunoreactivity for TOM20, DRP1 and OPA1 was strong in 2 of 43 (4.7%), 1 of 43 (2.3%) and 0 of 43 (0%) of BPH control tissue. Strong marker expression was significantly increased in radical prostatectomy specimen (TOM20: 111/371 (29.9%), DRP1: 89/373 (23.9%), OPA1: 60/371 (16.2%), p<0.001) and in metastatic tissue (TOM20: 22/42 (52.4%), DRP1: 14/42 (33.3%), OPA1: 21/41 (51.2%), p<0.001). None of the markers demonstrated prognostic properties for RFS, OS, and DSS. Conclusion A strong association between the expression of the mitochondrial markers TOM20, DRP1 and OPA1 and PCa aggressiveness was demonstrated. However, these markers were not found to be prognostic regarding RFS, OS and DSS. Future studies are needed focusing on the underlying mechanisms of the upregulation of mitochondrial metabolism in aggressive PCa and evaluate potential therapeutic implications.

The occurrence of cervical cancer is often linked to a previous infection with a human papillomavirus (HPV). In order to detect HPV infections in cervical smears, a broad range of tests can be used. This study compares the two hybridisation-based DNA-microarray systems “HPV 3.5 LCD-Array” (Chipron GmbH) and “PapilloCheck®” (Greiner Bio-One GmbH), based on their ability to detect and differentiate HPV infections in 42 different cervical smears. PapilloCheck® can detect and individually identify 24 HPV types, whereas the 3.5 LCD-Array can differentiate among 32 HPV genotypes. However, both systems include all 13 high-risk (HR)-classified types. With Chipron having already stopped the production of the 3.5 LCD-Array test, quite a few laboratories are confronted with the need to establish a new HPV testing method. The two methods were found to have a high agreement regarding the clinical significance of the detected HR HPV types. Discrepant cases were additionally validated with the help of a third test (VisionArray® HPV, ZytoVision GmbH). The results of the VisionArray® test corresponded rather well with the results of the 3.5 LCD-Array.

Supplementary Table S1 shows the clinical data of the CRC organoid-stroma cohort. Supplementary Table S2 shows the inventory of available materials and molecular analyses. Supplementary Table S3 shows the selected variant types for whole exome analysis. Supplementary Table S4 summarizes the genetic characterization of CRC models. Supplementary Table S5 shows the classification according to the consensus molecular subtypes (CMS). Supplementary Table S6 shows classification according to the CRC intrinsic subtypes (CRIS). Supplementary Table S7 summarizes the functional data in mono- and co-cultures. Supplementary Table S8 shows drug sensitivity of CRC organoids in co-culture with autologous and heterologous fibroblasts. Supplementary Table S9 show the drug sensitivity in all biobank models in mono- and co-culture. Supplementary Table S10 shows the cell viability data of the chemogenomic library screens in resistant co-culture models. Supplementary Table S11 shows the identified drug sensitivity and resistance signatures. Supplementary Table S12 lists all antibodies and primer sequences used in this study.