A subset of patients evaluated for immune dysregulation presents with increased proportions of TCR γδ cells despite normal total lymphocyte counts. Corresponding to this phenotype, the authors report a case of a somatic TCR γδ–specific STAT5B mutation that is molecularly amenable to tailored immune modulation. This raises awareness of druggable TCR γδ–intrinsic, genetically determined immune dysregulation.

Within a prospective cohort of patients with immune dysregulation, we identified several individuals with chronically increased proportions of TCR γδ cells but normal peripheral lymphocyte counts. Among those, we identified one individual with a TCR γδ cell-specific heterozygous p.Y665F STAT5B gain-of-function mutation. Recurrent oral aphthous lesions, susceptibility to infection, arthralgia, and fatigue, were linked to relatively elevated numbers of γδ T cells expressing a Vγ9Vδ2 TCR, displaying hyperphosphorylation of STAT5 upon in vitro IL-2 stimulation. The TCR Vγ9Vδ2 cells exhibited enhanced proliferative response to (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate and dysregulated cytokine production. The TCR γδ cell transcriptome revealed the suppression of the default Th17 program, along with inhibition of RORC and MAF expression. The JAK inhibitor baricitinib improved clinical features of the observed immune dysregulation and reduced the frequency of peripheral TCR Vγ9Vδ2 cells. Thus, functionally altered TCR γδ cells may underlie chronic immune dysregulation of unknown molecular cause, demonstrated here to be amenable to tailored immune modulation.

Abstract In women with high-grade serous ovarian cancer, chemotherapy remains the primary standard treatment, despite growing recognition of the disease as highly heterogeneous. Here, we examine the feasibility and clinical utility of comprehensive multimodal molecular profiling to inform treatment decisions. We analyze blood, single-cell and bulk tumor tissue, and malignant ascites using up to eleven technologies (DNA, RNA, protein, and functional assays) within a four-week turnaround time. Hypothetical treatment recommendations are altered for 76% of patients, and multi-omics-guided maintenance therapy is associated with prolonged overall survival in a subset of patients. Subsequent cohort analysis reveals distinct cellular and molecular profiles in ascites-derived single-cells compared to solid tumor tissue, unique per-patient ex vivo drug responses, and a marked increase in cancer cell heterogeneity following chemotherapy exposure. This coincides with genomic signature alterations in whole-genome-amplified patients. Our data suggest that molecularly guided treatments should be tested as adjuvant therapies prior to chemotherapy in the future.

Effective management of advanced epithelial ovarian cancer (EOC) is challenging, with most patients relapsing despite standard therapies, highlighting the need for personalized treatment. Functional ex vivo drug testing enables matching patients to effective therapies based on drug sensitivity. We present DRUGSENS, a scalable, clinically adaptable ex vivo screening platform that quantitatively assesses single-cell, on-target drug effects in cell lines and patient-derived samples. DRUGSENS offers high-resolution analysis of responses to single agents and relevant drug combinations, guiding personalized treatment across tumor types. The semi-automated assay integrates immunofluorescence-based confocal imaging with computational analysis using QuPath, Fiji, and a dedicated R package. We profiled 21 samples from 17 patients in chemotherapy-naive and relapsed states, achieving 100% culture success and delivering results within 10 days. Most samples showed limited sensitivity to multiple agents, indicating multidrug resistance. Notably, ex vivo drug responses correlated significantly with progression-free and overall survival. DRUGSENS supports rapid, clinically relevant functional profiling in EOC, providing a valuable tool to inform treatment recommendations and prioritize drugs for clinical application.

BackgroundHistiocytic sarcoma (HS) is a rare and aggressive hematopoietic malignancy characterized by the proliferation of cells resembling mature histiocytes. It typically presents in extranodal sites such as the skin, the gastrointestinal tract, and soft tissues and is often accompanied by systemic symptoms including fever and weight loss. HS occurs de novo or results from transformation/transdifferentiation from other hematological neoplasms, such as low-grade B-cell lymphomas. To date, only four cases of HS arising from marginal zone lymphomas (MZL) have been documented.Case presentationWe describe a 66-year-old female patient who presented primarily with abdominal pain and constitutional symptoms. The clinical evaluation showed significant hepatosplenomegaly and lymphadenopathy. A liver biopsy demonstrated a sinus-associated spread of HS. The patient died of suspected hemorrhagic shock before the diagnostic results were finalized and before rescue treatment could be initiated. The autopsy findings confirmed a widespread metastatic HS and concurrent MZL. The molecular analysis showed that both neoplasmas were clonally related, supporting the hypothesis of transformation/transdifferentiation of MZL into HS.ConclusionWe have reported the fifth unusual transformation of a MZL into a HS. Transformed/transdifferentiated HS is a rare and aggressive neoplasm. Evidence from the published case reports suggests that its clinical course may be more severe than de novo HS. This underscores the importance of investigating rare presentations and considering the possibility of an underlying pre-existing hematological malignancy.

Introduction There is an urgent need to better understand how information from circulating tumour DNA (ctDNA) can be integrated into routine care for patients with advanced solid cancer.Methods and analysis The implementation of liquid biopsies in routine care of patients with advanced solid cancer trial (LIQPLAT) is a single-centre, single-arm trial investigating the implementation of ctDNA in the routine care of patients with advanced solid cancer. We present a mixed-methods process evaluation embedded in the LIQPLAT trial, following Medical Research Council guidance and the Reach, Effectiveness, Adoption, Implementation, Maintenance framework. We show a logic model, which details the causal chain and related assumptions from recruiting patients into the trial to the goal of improving quality of life and survival. Data collection is longitudinal and includes: semistructured interviews with healthcare professionals (pathologists, biologists, oncologists; planned n=20) and patients (planned n=15) to identify implementation barriers and facilitators; recordings of molecular tumour board meetings to analyse clinical decision-making; the 23-item Normalisation MeAsure Development survey for healthcare professionals (planned n=20) at four time points. Quantitative data from hospital records will be used to assess implementation outcomes like patient acceptance rates and ctDNA workflow success. Qualitative data will undergo thematic and content analysis, and quantitative data will be analysed using a Bayesian framework.Ethics and dissemination The LIQPLAT trial was approved by the regional ethics committee of Northwestern and Central Switzerland (BASEC 2024-00358). The qualitative aspects of the process evaluation were exempted from ethics review according to the Swiss Human Research Act. We follow guidelines for data security, confidentiality and information governance. Results will be submitted for publication in peer-reviewed journals and discussed at conferences.Trial registration number NCT06367751, SNCTP000005844.

Neoadjuvant chemoimmunotherapy offers promise to improve outcomes for patients with resectable non-small cell lung cancer (NSCLC). Yet not all patients derive treatment benefits, and reliable biomarkers of response are still lacking. We here assess the long-term clinical outcome of neoadjuvant chemotherapy and perioperative anti-PD-L1 inhibition in resectable stage IIIA NSCLC in the SAKK 16/14 trial and provide a comprehensive characterization of anti-tumor immune responses for biomarker-based treatment personalization. We report secondary outcomes of median event-free survival (EFS) of 4.0 years and median overall survival not being reached after a median follow-up of 5.4 years. Computer-aided spatial image analysis emphasizes the importance of CD8+ T cell positioning in tumors, and larger tertiary lymphoid structures in pre-treatment biopsies correlate with improved EFS. Genomic techniques reveal the association of intratumoral TCR diversity with response. Finally, circulating proliferating CD39+ PD-1+ CD8+ T cells and elevated levels of CCL15 post-treatment are seen in patients with sustained therapeutic benefit. NCT02572843.

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.

There is a wide range of tissue changes that may arise after exposition to ionizing radiation. Most of these changes fall in the non-neoplastic category. Nevertheless, due to the damaging effect radiation has on the DNA, there is a risk of developing secondary tumours, usually high-grade sarcomas. Here, we present a case of an elderly man who developed recurrent fibroepithelial polyps of the pharyngeal mucosa. He had a history of ipsilateral squamous cell carcinoma of the tonsil 17 years before treated by complete surgical resection followed by adjuvant radiotherapy. Histologically, these polyps consisted of relatively well-defined, partially eroded mucosal projections with a fibro-myxoid and inflamed stroma lacking evident signs of malignancy. The 4th recurrence was submitted to DNA panel sequencing, which identified three pathogenic variants in the PTEN, DNMT3A, and TERT genes that were not present in the local normal tissue before radiotherapy. On further analysis, well-known and established radiation induced changes such as copy number (CN) alterations or a radiation signature were not detected. The clinical presentation and the mutations detected argue against a purely reactive, but rather a likely benign neoplastic process that is thought to have developed post-actinically, without clear evidence of malignancy (absence of atypia, no invasive growth, flat CN profile). This case report raises awareness of a possible association between radiotherapy and the subsequent development of a benign-appearing low-grade mucosal soft tissue neoplasm.

Mast cell (MC) disorders result from inappropriate release of mediators and/or excessive accumulation of MCs, leading to symptoms of various organs and systems. Clonal MC disorders are defined by the presence of phenotypically aberrant and/or KIT-mutated MCs, and if aggregates of MCs are detectable, are designated as mastocytosis. Systemic mastocytosis (SM) affects mainly the bone marrow, with or without skin involvement. It is associated with the activating mutation D816V in the KIT gene. Other activating KIT gene variants are also observable in SM; activating KIT mutations are recognized as a minor diagnostic SM-criterion. We report a novel KIT variant in a patient with indolent SM, an in-frame deletion-insertion affecting amino acids D816 to N819 (D816_N819delinsll), creating an aliphatic pouch similar to that resulting from the D816V mutation, and leading to MC activation as suggested by the symptoms of the patient and the positivity for phosphorylated STAT5 in the clonal MCs.

INTRODUCTION:Loss of MTAP serves as a potential predictive marker of response to cooperative PRMT5 inhibitors and as a negative predictor of response to immune checkpoint inhibitors. We investigated the prevalence of MTAP deficiency by immunohistochemistry (IHC) in NSCLC as a surrogate for MTAP loss. METHODS:MTAP IHC was performed on 698 NSCLC samples. Data from routine next-generation sequencing, analyzed with the Oncomine Precision Assay (OPA-NGS, Thermo Fisher), were available in 426 cases, including CDKN2A copy number variation (CNV) data in 411 cases. Our findings were compared with data from The Cancer Genome Atlas (TCGA). RESULTS:MTAP deficiency by IHC was found in 18.2% of NSCLC. CDKN2A loss by OPA-NGS, used as a surrogate for MTAP loss, was significantly associated with MTAP deficiency by IHC, but it was found in only 28.4% of the MTAP-deficient NSCLC analyzed for CDKN2A CNV. In the TCGA cohort, only 72.9% of NSCLC with CDKN2A loss had a concurrent MTAP loss defined by CNV. CONCLUSION:MTAP IHC seems to be better suited than OPA-NGS to assess the MTAP status in NSCLC, especially as the MTAP gene is not specifically covered within this panel. CDKN2A loss is not a reliable MTAP loss surrogate, as it overestimates MTAP loss in more than 25% of the cases in the TCGA cohort.

BACKGROUND:Aggressive Variant Prostate Cancers (AVPCs) are incurable malignancies. Platinum-based chemotherapies are used for the palliative treatment of AVPC. The Polycomb Repressive Complex 2 (PRC2) promotes prostate cancer progression via histone H3 Lysine 27 tri-methylation (H3K27me3). EZH2 encodes the catalytic subunit of PRC2. A recently developed nucleosome capture technology (Nu.QⓇ).measures H3K27me3 levels in biological fluids. EZH2 inhibitors (EZH2i) are being tested in clinical trials. We hypothesize that epigenetic reprogramming via EZH2i improves the efficacy of Carboplatin in AVPC and that EZH2i activity can be measured via both cellular- and cell-free nucleosomal H3K27me3 (cf-H3K27me3) levels. METHODS:We studied the expression of PRC2 genes in clinical prostate cancer cohorts (bioinformatics). We determined the effect of EZH2i on cellular- and cf-H3K27me3 levels. We measured dose-dependent effects of Carboplatin with/without EZH2i on AVPC cell viability (IC50). We used RNA-Seq to study how EZH2i modulates gene expression in AVPC cells. RESULTS:PRC2 genes were significantly up-regulated in AVPC vs other prostate cancer types. EZH2i reduced both cellular and cf-H3K27me3 levels. EZH2i significantly reduced Carboplatin IC50. EZH2i reduced the expression of DNA repair genes and increased the expression of p53-dependent pro-apoptotic factors. CONCLUSIONS:EZH2i plus Carboplatin is a promising combination treatment for AVPC.

BACKGROUND/AIM:Extracellular vesicle DNA (EV-DNA) has emerged as a novel biomarker for tumor mutation detection using liquid biopsies, exhibiting biological advantages compared to cell-free DNA (cfDNA). This study assessed the feasibility of EV-DNA and cfDNA extraction and sequencing in old serum samples of patients with breast cancer (BC). PATIENTS AND METHODS:A total of 28 serum samples of 27 patients with corresponding clinical information were collected between 1983 and 1991. EV-DNA was extracted using Exo-GAG kit (Nasabiotech) and cfDNA using QIAsymphony DSP Virus/Pathogen Midi Kit (Qiagen), respectively. Subsequently, 10 matched samples (EV-DNA n=5, cfDNA n=5) of five patients were subjected to sequencing using the Oncomine™ Breast cfDNA Research Assay v2 (Thermo Fisher Scientific). RESULTS:Samples were collected on median 1.9 years after primary diagnosis [interquartile range (IQR)=0.2-7.2]. Median follow-up was 9.5 years (IQR=5.2-14.2). Median age of serum samples was 36.1 years (IQR=34.5-37.3). EV-DNA and cfDNA were extracted from 100% (28/28) of the included samples. Both, DNA quantity and concentration were comparable between EV-DNA and cfDNA. Sequencing was successfully performed in 100% (10/10) of the included samples. Two matched analyses yielded equivalent results in EV-DNA and cfDNA (no mutations, n=1; PIK3CA mutation, n=1), whilst in two analyses, PIK3CA mutation was only found in cfDNA, and in one analysis, a TP53 mutation was only found in EV-DNA. CONCLUSION:EV-DNA extraction and sequencing in old serum samples of patients with BC is feasible and has the potential to address clinically relevant questions in longitudinal studies.

BackgroundRichter transformation refers to the progression of an initially slow-growing small lymphocytic lymphoma/chronic lymphocytic leukemia (SLL/CLL) into an aggressive lymphoma, typically diffuse large B-cell lymphoma (DLBCL) or Hodgkin lymphoma.Case presentationThe patient presented with a rapid onset of localized cervical swelling, accompanied by monoclonal B-cell lymphocytosis displaying a CLL immunophenotype. The histopathological analysis identified a Burkitt lymphoma (BL) located in the submandibular gland and adjacent lymph node. The patient’s bone marrow displayed a minor infiltration of monoclonal B-cells with a CLL immunophenotype (< 10%). Molecular analysis demonstrated the presence of the same monoclonal rearrangement in the framework region (FR3 region) of the immunoglobulin heavy chain (IGH) locus. High-throughput sequencing of the immunoglobulin heavy and light chains also confirmed the presence of the same rearrangement in SLL/CLL and in the Burkitt lymphoma sample, but also highlighted the presence of a second rearrangement in the Burkitt lymphoma cells, not shared with the SLL/CLL cells in the bone marrow. The patient was treated with DA-EPOCH-R, which lead to a complete metabolic response.ConclusionThis report provides an exceptionally rare description of a CLL-type monoclonal B-cell lymphocytosis transforming into a very aggressive Burkitt lymphoma in a treatment naïve patient.