Classification of tumors in neuro-oncology today relies on molecular patterns (mostly DNA methylation) and their machine learning-supported interpretation. Understanding the process of algorithmic interpretation is essential for safe application in clinical routine. This is paradigmatically true for the most common primary intracranial tumor in adults, meningioma. Here, by applying multiomic profiling and multiple lines of orthogonal computational evaluation in multiple independent datasets, we found that not only tumor cell characteristics but also incremental changes in the tumor microenvironment (TME) have impact on epigenetic meningioma classification and clinical outcome. Besides revealing the decisive role of non-neoplastic cells in the CNS methylation classifier, this challenges the model of distinct meningioma subgroups toward a TME-determined risk continuum. This refines current controversies in molecular meningioma subtyping. In addition, we apply these learnings to devise and validate a simple diagnostic approach for increased clinical prediction accuracy based on immunohistochemistry, which is also applicable in resource-limited settings.

Breast cancer brain metastases (BCBM) are a severe condition with high demand for improved personalized treatment, but a comprehensive understanding of BCBM immune-microenvironment heterogeneity and susceptibility to immunotherapy is lacking. Here, we multimodally profile the immune niche in a clinically well-annotated cohort of 156 BCBM applying tissue cytometry, bulk and single nuclei RNA-sequencing, flow cytometry, and spatial transcriptomics, complemented by functional studies in patient-derived models. Integrative analyses reveal two immune landscapes predicting prolonged patient survival and that are not deducible from paired primary tumors: 1) BCBM with a high proportion of CD8+ tissue-resident-like memory T cells as major players of tumor immune control. 2) BCBM containing tertiary lymphoid structures. Surrogate signatures of these landscapes are prognostic in independent BCBM and primary breast cancer cohorts, are associated with fewer metastases, and predict immunotherapy response. Our work provides critical insights into anti-tumor immunity in BCBM and identifies novel biomarkers with translational relevance.

Genomics-guided precision oncology has improved survival in cancer entities with actionable mutations but cannot capture oncogenic signaling that manifests at the protein level. Here, we report a prospective, real-world pan-cancer study profiling proteomes and phosphoproteomes of 1,998 tumor samples from adults and children with rare or advanced cancers enrolled in the German precision oncology programs DKFZ/NCT/DKTK MASTER, CATCH and INFORM and their molecular tumor boards (MTBs). We developed tumor proteome activity status (TOPAS) scores for 46 clinically relevant kinases, an immune activity score capturing antigen presentation and T-cell activation and identified therapeutically targetable cell-surface proteins for 94% of patients. These readouts enhance MTB recommendations by exposing actionable non-genomic kinase activity, refining interpretation of oncogenic genome alterations, and highlighting cell-surface treatment options. Three proof-of-concept analyses indicate clinical utility including kinase activity-stratified pazopanib response in sarcoma, immune activity score-tracked checkpoint-inhibitor outcomes pan-cancer, and a phosphoproteomic biomarker distinguishing EGFR-inhibitor response in chordoma.

Abstract Extrachromosomal DNA (ecDNA) is increasingly recognized as a driver of cellular plasticity, yet its role in therapy adaptation in pancreatic ductal adenocarcinoma (PDAC) remains poorly defined. In patient-derived PDAC models, we identify extrachromosomal ABCB1 amplification as a mechanism of resistance to paclitaxel and KRAS inhibitors and show that ABCB1 copy number dynamically adjusts to selective pressure. Notably, paclitaxel-resistant cells remain primed for rapid de novo ecDNA generation: after eliminating pre-existing ABCB1 ecDNA through single-cell cloning, new and structurally distinct ABCB1 ecDNA rapidly emerges, indicating treatment-induced molecular changes that prime these cells for ecDNA formation. In metastatic breast cancer, taxane-associated ABCB1 amplification is likewise observed and can be tracked in cell-free DNA (cfDNA), underscoring clinical relevance beyond PDAC. Finally, gemcitabine co-treatment suppresses ABCB1 ecDNA generation, suggesting potential strategies to counteract ecDNA-mediated resistance. Together, these findings demonstrate that dynamic ecDNA modulation and inducible ecDNA biogenesis enable rapid, reversible drug resistance, providing a rationale for ecDNA-targeted combination therapies and longitudinal monitoring. Citation Format: Tim Vorberg, Manuel Reitberger, Bernardo Rodriguez Martin, Maja Starostecka, Dominique Schulz, Arlou K. Angeles, Kate I. Glennon, Tasneem Cheytan, Roberto Würth, Vera Thiel, Paul Schwerd-Kleine, Verena Thewes, Laura Michel, Ewgenija Gutjahr, Simon J. Ogrodnik, Heike Conrad, Steffi O. Mehlhorn, Vanessa Vogel, Corinna Klein, Albrecht Stenzinger, Peter Lichter, Andreas Schneeweiss, Martin Granzow, Marc Zapatka, Anna Jauch, Holger Sültmann, Jan Korbel, Andreas Trumpp, Martin R. Sprick. Dynamic copy number changes and de novo generation of extrachromosomal DNA modulate therapy resistance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 1803.

T cells that recognize tumor-specific mutations are crucial for cancer immunosurveillance and in adoptive transfer of TILs or transgenic-TCR T cell products. However, their challenging identification and isolation limits their use in clinical practice. Therefore, novel approaches to isolate tumor-specific T cells are needed. Here, we report the isolation of neoantigen-specific CD8+ T cells from a vaccination site of a metastatic breast cancer patient who received a personalized vaccine. Based on the somatic mutations, potential MHC binding epitopes were predicted, of which 17 were selected to generate a peptide vaccine. Cutaneous biopsies were processed after the fifth vaccination cycle to obtain infiltrating lymphocytes from the vaccination site (VILs). IFNγ ELISpot revealed reactivity to four peptides used in the vaccine. Reactive T cells from VILs were non-overlapping with those detected in the blood and the tumor-microenvironment. ScTCR Seq analysis revealed the presence of a clonotype in VILs that further expanded after a round of in vitro stimulation and validated to be specific against a private mutation, namely NCOR1L1475R, presented in the context of HLA-B * 07:02, with no reactivity to the wild-type peptide. Our study shows, for the first time, that tumor mutation – specific T cells are generated at high frequencies in the vaccination site and can be isolated with standard methods for TCR screening. The easy and safe accessibility of skin biopsies overcomes the major hurdles of current TCR screening approaches and present exciting opportunities for the development of innovative immunotherapeutic strategies.

Metastatic breast cancer remains largely incurable, partly due to our incomplete understanding of its intricate underlying mechanisms. Notably, intercellular communication mediated by small extracellular vesicles and particles (sEVPs) has emerged as a key feature of metastasis. While tumor-derived sEVPs have been extensively studied and are known to be pro-metastatic, the role of sEVPs from metastasis-prone normal tissue sites remains primarily undefined. Here, we characterized and studied the function of sEVPs secreted from tumor-free pre-metastatic organs (TuFMO-sEVPs) such as the brain and lungs in both immunocompetent and patient-derived xenograft models. TuFMO-sEVPs from the brain of mammary tumor-bearing mice were found to have a distinct protein content as compared to brain-sEVPs from tumor-free mice, suggesting that the primary tumor can systemically influence the cargo of TuFMO-sEVPs. Importantly, mice orthotopically injected with breast cancer cells which had been educated with either brain or lung TuFMO-sEVPs prior to transplantation showed significantly increased metastasis to the respective organ. We further demonstrated that TuFMO-sEVPs induced the expression of the enzyme dihydrofolate reductase (DHFR) upon uptake by breast cancer cells, leading to their enhanced metastatic capacity. Organ-specific signatures generated from TuFMO-sEVP educated tumor cells were found to be increased in metastatic samples from breast cancer patients as compared to the primary tumor or normal tissue samples and these signatures also significantly correlated with poorer patient outcome. Collectively, our data reveals a novel facet of the metastatic cascade, implicating a role for TuFMO-sEVPs in directing metastasis and providing a potential therapeutic strategy for targeting this process.

The ability to establish organoids composed exclusively of tumour rather than healthy cells is essential for their implementation into clinical practice. Organoids have recently emerged as a powerful tool to expand patient material in culture and generate modifiable 3D models derived from humans or animal models. For translational research, they enable the creation of model systems for an ever-increasing number of cell types and diseases. And in personalised medicine, they potentially allow for functional drug testing with high predictive power in certain settings. We found that using biopsy material from untreated, early-stage primary breast cancer patients poses significant challenges for consistently culturing tumour cells as organoids. Specifically, we observed frequent outgrowth of genetically normal, non-cancerous epithelial cells. We analysed >100 biopsy samples from early-stage breast cancer and present our large collection of >70 organoid lines. We also show methods of assessing successful tumour cell culture in a time, and cost-efficient manner, proving a high rate (>85%) of normal cell overgrowth in early-stage breast cancer organoids. Finally, we show a number of successful attempts to culture cancer organoids from mastectomy-derived tissue of advanced, metastatic breast cancer. We conclude that the usefulness of organoids from early breast cancer for translational research and personalised medicine, especially guidance of adjuvant or post-surgical maintenance therapy, is strongly limited by the low success rate of culturing cancerous cells under organoid conditions.

The International Cancer Genome Consortium Accelerating Research in Genomic Oncology (ICGC ARGO) project is an international initiative to sequence germline and tumour genomes from 100,000 cancer patients across 13 countries and 22 tumour types. By integrating genomic data with comprehensive clinical information including treatment outcomes, lifestyle, environmental exposures and family history, ICGC ARGO aims to accelerate the application of genomic insights in cancer diagnosis, treatment and prevention. However, a major challenge is harmonizing clinical data from diverse tumour types worldwide. To address this, the ICGC ARGO Data Dictionary was developed to ensure consistent high-quality clinical data collection by defining a minimal set of clinical fields within an event-based data model to capture clinical relationships and support longitudinal data collection. Grounded in international standardized terminology, it is interoperable with other data standards such as Minimal Common Oncology Data Elements (mCODE). Its adoption by global initiatives such as the European-Canadian Cancer Network (EUCANCan) and the Marathon of Hope Cancer Centres Network (MOHCCN) underscores its broad impact on advancing precision oncology research.

DNA methylation-based classification is now central to contemporary neuro-oncology, as highlighted by the World Health Organization (WHO) classification of central nervous system (CNS) tumors. We present the Heidelberg CNS Tumor Methylation Classifier version 12.8 (v12.8), trained on 7,495 methylation profiles, which expands recognized entities from 91 classes in version 11 (v11) to 184 subclasses. This expansion is a result of newly identified tumor types discovered through our large online repository and global collaborations, underscoring CNS tumor heterogeneity. The random forest-based classifier achieves 95% subclass-level accuracy, with its well-calibrated probabilistic scores providing a reliable measure of confidence for each classification. Its hierarchical output structure enables interpretation across subclass, class, family, and superfamily levels, thereby supporting clinical decisions at multiple granularities. Comparative analyses demonstrate that v12.8 surpasses previous versions and conventional WHO-based approaches. These advances highlight the improved precision and practical utility of the updated classifier in personalized neuro-oncology.

CATCH is a prospective precision oncology registry trial that exploits whole-genome/exome- and RNA-sequencing to enable actionable biomarker detection in metastatic breast cancer (mBC) patients of any subtype. We herein report long-term follow-up of the first 558 patients consecutively recruited into CATCH in a monocentric setting between June 2017 and October 2021. Main outcome measures were the rate of implementation of molecular tumor board (MTB) recommended treatments and treatment response as assessed by disease control rate, objective response rate and PFS ratio. Out of the recruited patients, 412 (54.9% HR+/HER2-, 31.3% TNBC, 6.8% HR-/HER2+ and 7.0% HR+/HER2+) were reviewed in the MTB. An appropriate molecularly guided anti-cancer treatment as recommended by MTB was implemented in 183 (44.4%) patients. Gene expression and computationally derived composite biomarkers further expanded treatment options in up to every second patient as compared to genomic sequencing data alone. The outcome was assessed in 152 patients and showed a Disease Control Rate (DCR) of 58.6% and an Objective Response Rate (ORR) of 27.0%. One in three patients (32.8%) showed at least a 50% longer PFS with molecularly guided therapy compared to the previous standard therapy. Notably, 86.4% of the MTB-driven implementations were off-label. CATCH highlights the impact of whole-genome/exome in combination with RNA sequencing to detect clinically relevant biomarkers in mBC. Omics-guided targeted therapy in a real-world setting allows high treatment implementation rates yielding outcome benefit for one-third of the patients.

The development of breast cancer brain metastases (BCBM) is among the most critical predictive factors for patient survival. However, understanding the drug sensitivity of BCBM cells to various drugs, particularly in terms of how effectively they eliminate specific tumor cell clones responsible for the disease, remains limited. Acquiring this knowledge is crucial for improving drug selection and personalizing treatment strategies for individual patients. For this purpose, we generated novel patient-derived BCBM cell lines from three individuals with triple-negative breast cancer and three with HER2-enriched breast cancer. Whole genome and transcriptome analyses verified their resemblance to the original tumor tissues. We performed high-throughput drug screening to evaluate responses to over 250 drugs, testing more than 10 dosage points within our framework HeiDePEx (Heidelberg Drug Screening Platform for Experimental Neurosurgery). The findings were further explored in relation to patient-matched multi-omics data generated as part of the CATCH study, a metastatic breast cancer precision oncology program, which integrates drug screening results, clinical data, and molecular information. Overall, the newly established BCBM cell lines exhibited drug responses that closely reflected the individual patient and breast cancer subtype. Furthermore, they enhanced the clinically assessed multi-omics data by providing extensive insights into functional cellular vulnerabilities across a wide range of targeted anticancer drugs. Taken together, these findings emphasize the critical importance of personalized therapeutic strategies for BCBM patients and demonstrate the value of integrating functional drug screening with multi-omics data. Ultimately, the newly developed and characterized BCBM cell lines serve as a valuable resource for future research.

Chronic lymphocytic leukemia is a malignant lymphoproliferative disorder for which primary or acquired drug resistance represents a major challenge. To investigate the underlying molecular mechanisms, we generate a mouse model of ibrutinib resistance, in which, after initial treatment response, relapse under therapy occurrs with an aggressive outgrowth of malignant cells, resembling observations in patients. A comparative analysis of exome, transcriptome and proteome of sorted leukemic murine cells during treatment and after relapse suggests alterations in the proteasome activity as a driver of ibrutinib resistance. Preclinical treatment with the irreversible proteasome inhibitor carfilzomib administered upon ibrutinib resistance prolongs survival of mice. Longitudinal proteomic analysis of ibrutinib-resistant patients identifies deregulation in protein post-translational modifications. Additionally, cells from ibrutinib-resistant patients effectively respond to several proteasome inhibitors in co-culture assays. Altogether, our results from orthogonal omics approaches identify proteasome inhibition as potentially attractive treatment for chronic lymphocytic leukemia patients resistant or refractory to ibrutinib. The molecular mechanisms underlying resistance to therapy in Chronic lymphocytic leukemia (CLL) remain to be explored. Here, the authors perform multi-omics analysis in a mouse model of ibrutinib resistance and suggest proteasome inhibition for overcoming it.

As part of the COGNITION diagnostic registry program, residual tumor material after neoadjuvant therapy (NAT) of patients with early breast cancer (eBC), who are still at high-risk for relapse after NAT, is analyzed by next generation sequencing to identify biomarkers and actionable alterations. This strategy aims to stratify patients for subsequent genomics-guided therapies to reduce the significant risk of metastatic dissemination and hence to improve disease-free survival. COGNITION-GUIDE is a multicenter umbrella phase-II-trial to translate molecular biomarker profiles generated in the COGNITION platform into six molecular-guided post-neoadjuvant therapeutic options in addition to standard-of-care treatment. Patients can be allocated to The primary endpoint is invasive disease-free survival (IDFS) four years after surgery. Secondary endpoints include IDFS in each study arm separately, distant disease-free survival, overall survival and safety. 240 patients will be enrolled within four years. The COGNITION-GUIDE trial, which was activated in June 2023 and will recruit in different centers in Germany, empowers a risk-adapted, biomarker-guided therapy escalation algorithm in eBC patients who are still at high risk of metastasis.

Circulating tumor cells (CTCs) drive metastasis, the leading cause of death in individuals with breast cancer. Due to their low abundance in the circulation, robust CTC expansion protocols are urgently needed to effectively study disease progression and therapy responses. Here we present the establishment of long-term CTC-derived organoids from female individuals with metastatic breast cancer. Multiomics analysis of CTC-derived organoids along with preclinical modeling with xenografts identified neuregulin 1 (NRG1)–ERBB2 receptor tyrosine kinase 3 (ERBB3/HER3) signaling as a key pathway required for CTC survival, growth and dissemination. Genome-wide CRISPR activation screens revealed that fibroblast growth factor receptor 1 (FGFR1) signaling serves a compensatory function to the NRG1–HER3 axis and rescues NRG1 deficiency in CTCs. Conversely, NRG1–HER3 activation induced resistance to FGFR1 inhibition, whereas combinatorial blockade impaired CTC growth. The dynamic interplay between NRG1–HER3 and FGFR1 signaling reveals the molecular basis of cancer cell plasticity and clinically relevant strategies to target it. Our CTC organoid platform enables the identification and validation of patient-specific vulnerabilities and represents an innovative tool for precision medicine. Trumpp and colleagues develop a method to obtain long-term circulating tumor cell-derived organoids from individuals with metastatic breast cancer and identify the neuregulin 1–HER3 axis as important for organoid growth and a promising therapeutic target.

Despite major advances in immunotherapy, respective treatments often show only limited benefit in patients with chronic lymphocytic leukemia (CLL). Immune checkpoint blockade, which has shown durable responses in multiple malignancies, has yielded inferior efficacy in CLL. Similarly, adoptive CAR-T cell therapy, which has achieved remarkable success in other B cell malignancies, resulted in lower response rates in CLL. The lack of success of these immunotherapies is likely due to the profound T cell dysfunction and immune suppressive microenvironment, typical characteristics of this disease. The so-called “exhaustion” of T cells is marked by impaired effector capacity, sustained inhibitory receptor expression, and loss of progenitor exhausted (TPEX) populations, cell subsets which are critical for responses to PD-1 blockade. This suggests that reversing T cell exhaustion could be key to improving outcomes. Emerging evidence implicates the immunoregulatory cytokine interleukin-10 (IL-10) as a context-dependent modulator of T cell exhaustion. While traditionally viewed as an immunosuppressive mediator that limits tissue damage during chronic inflammation and suppresses antiviral immunity, IL-10 may, in cancer, enhance tumor-infiltrating CD8⁺ T cell function, prolong their survival, and delay T cell exhaustion. Using the Eµ-TCL1 adoptive transfer (TCL1-AT) mouse model of CLL, we previously identified a role for IL-10 receptor (IL-10R)–STAT3 signaling in sustaining CD8⁺ TPEX cells, preventing terminal exhaustion, with loss of signaling accelerating CLL progression (Hanna et al., Immunity 2021). As IL-10 expression is linked to better outcome in CLL patients and IL-10R loss is associated with DLBCL development early in life, we hypothesize that IL-10 improves the fitness of cytotoxic T cells thereby preventing immune escape in CLL and likely other B cell malignancies. To test the potential of IL-10 in modulating T cell responses against CLL, we generated a stabilized IL-10 fusion protein (IL-10-Fc) and demonstrated that treatment of TCL1-AT mice with IL-10-Fc slowed CLL progression, reduced exhausted effector T cells, and preserved TPEX populations. To dissect T cell-intrinsic effects, we employed an in vitro exhaustion assay by repetitive antigen-specific stimulation of T cell receptor (TCR)-transgenic murine CD8⁺ T cells with peptide-loaded target cells. IL-10 treatment maintained effector T cells in these cocultures, reduced terminal exhaustion markers TIM-3 and CD39, and promoted a polyfunctional phenotype with robust cytokine production and CD107a expression, demonstrating a direct effect of IL-10 on T cell fitness. To gain a broader view of IL-10-mediated immune modulation in the TCL1-AT model, we performed spectral flow cytometry and single-cell RNA-sequencing following short-term IL-10-Fc treatment. Both T cell and myeloid compartments displayed therapy-induced alterations, including shifts in regulatory T cells, CD8⁺ T cells, neutrophils, and monocytes. Notably, IL-10-Fc reduced PD-L1⁺ patrolling monocytes, which were linked to immune suppression in CLL, and increased inflammatory monocytes, which suggests a reversal of pro-tumoral myeloid activity. Finally, a pilot combination treatment study revealed that IL-10-Fc synergized with the BTK inhibitor ibrutinib, achieving profound disease control in the TCL1-AT mouse model beyond either agent alone. These findings position IL-10 as a promising immunomodulatory therapy in CLL, capable of restoring T cell function and remodeling the suppressive tumor microenvironment.

Burkitt lymphoma (BL) is an aggressive germinal center B-cell-derived malignancy. Historically, sporadic, endemic, and immunodeficiency-associated variants were distinguished, which differ in the frequency of Epstein-Barr virus (EBV) positivity. Aiming to identify subgroups based on DNA methylation patterns, we here profiled 96 BL cases, 17 BL cell lines, and six EBV-transformed lymphoblastoid cell lines using Illumina BeadChip arrays. DNA methylation analyses clustered the cases into four subgroups: two containing mostly EBV-positive cases (BL-mC1, BL-mC2) and two containing mostly EBV-negative cases (BL-mC3, BL-mC4). The subgroups BL-mC1/2, enriched for EBV-positive cases, showed increased DNA methylation, epigenetic age, and, in part, proliferation history compared to BL-mC3/4. CpGs hypermethylated in EBV-positive BLs were enriched for polycomb repressive complex 2 marks, while the CpGs hypomethylated in EBV-negative BLs were linked to, for example, B-cell receptor signaling. EBV-associated hypermethylation affected regulatory regions of genes frequently mutated in BL (e.g., CCND3, TP53) and impacted superenhancers. This finding suggests that hypermethylation may compensate for the lower mutational burden of pathogenic drivers in EBV-positive BLs. Though minor, significant differences were also observed between EBV-positive endemic and sporadic cases (e.g., at the SOX11 and RUNX1 loci). Our findings suggest that EBV status, rather than epidemiological variants, drives the DNA methylation-based subgrouping of BL.

The T-box transcription factor T-bet is known as a master regulator of T-cell response but its role in malignant B cells is not sufficiently explored. Here, we conducted single-cell resolved multi-omics analyses of malignant B cells from patients with chronic lymphocytic leukemia (CLL) and studied a CLL mouse model with genetic knockout of TBX21. We found that T-bet acts as a tumor suppressor in malignant B cells by decreasing their proliferation rate. NF-κB activity induced by inflammatory signals provided by the microenvironment, triggered T-bet expression which impacted on promoter proximal and distal chromatin co-accessibility and controlled a specific gene signature by mainly suppressing transcription. Gene set enrichment analysis identified a positive regulation of interferon signaling, and a negative control of proliferation by T-bet. In line, we showed that T-bet represses cell cycling and is associated with longer overall survival of CLL patients. Our study uncovers a novel tumor suppressive role of T-bet in malignant B cells via its regulation of inflammatory processes and cell cycling which has implications for stratification and therapy of CLL patients. Linking T-bet activity to inflammation explains the good prognostic role of genetic alterations in inflammatory signaling pathways in CLL.