Glioblastoma isocitrate dehydrogenase-wild-type (GBM) is the most lethal and, concurrently, the most common malignant primary brain tumor. Its aggressive behavior is associated with the presence of a subpopulation of glioblastoma stem cells (GSCs) that drive resistance to therapy and early relapse. P-element-induced wimpy testis-interacting RNAs (piRNAs), which are characteristically expressed in undifferentiated cells, are thought to play an important role in GSC biology. To identify piRNAs associated with GSCs, we selected 24 paired GSC and non-GSC cultures from patients with GBM based on the expression of stem cell markers CD133 and Sox2, ability of GSCs to form spheres in culture, and to differentiate and replicate tumors in immunodeficient mice. Global piRNA profiling using next-generation sequencing revealed 98 significantly differentially expressed piRNAs in GSCs compared with non-GSCs, including piR-9491, whose dysregulation in GBM has been previously described. Downregulation of piR-9491 in GSCs led to decreased viability, growth, invasion, and increased apoptosis. Significance of piRNA pathway for GBM pathology was further highlighted by identification of 34 piRNAs connected to patients' survival. These molecules were further used for the establishment of a piRNA signature predicting survival of patients with GBM. Our results not only confirm the important role of piR-9491 in GSCs but also indicate a potential significant role of piRNAs in the biology of GBM.

Abstract Background: G protein-coupled receptor class C group 5 member D (GPRC5D) has emerged as a promising immune target in multiple myeloma (MM). Talquetamab, a GPRC5D-directed bispecific antibody, has shown an efficacy of 70% in triple class refractory MM patients however, genomic loss mediated relapse is often observed. To better understand the interplay of talquetamab, GPRC5D loss and T cells within the immune microenvironment, we performed single cell sequencing on a coculture of GPRC5D loss models and healthy T cells in presence and absence of talquetamab. Methods: GPRC5D mono- and biallelic knockout models were generated using CRISPR-Cas9 technology. Cocultures were established using healthy T cells with or without talquetamab for 24 hours. CITE-seq was performed. Cell hashing with TotalSeq-C antibodies enabled multiplexing. Pooled cells underwent 10x Genomics 5’ GEM-X workflow and were sequenced on Illumina NovaSeq X Plus. Differential gene expression was assessed using the Wilcoxon rank-sum test, and interferon response scores were calculated using AddModuleScore. Results: Pronounced transcriptomic changes in MM cells were observed with both mono- and bi-alleic loss of GPRC5D via CITE-seq. Based on the expression of canonical markers (IL2RA, IRF4, TNFRSF4-18, TNF, IFNG), CD4 and CD8 activated T cells were only observed in presence of talquetamab and GPRC5D+ cells and completely disappeared when stimulated with GPRC5DDel/Del models. A significant induction of interferon stimulated genes (GBP2-4-5, IFI44, IFIT2, ISG15) in presence of talquetamab with GPRC5D on MM cells (p-value < 0.001, absolute log2FC > 2) was observed across T cell subpopulations. Interferon signaling genes (STAT1, IRF1) were found to be upregulated in T cells by talquetamab alone even in absence of GPRC5D+ cells in coculture. Additionally, we also identified genes only upregulated in presence of GPRC5D and talquetamab, including SOCS3, CD69, JUNB, BATF, CISH, and PRDM1, indicating a unique activation status of these T cells. Gene set enrichment analysis performed on T cell sub types indicated an enrichment of IFN-γ and IFNA-signaling (NES>2.0, FDR 0,0) in CD4, CD8, Tcm and Treg populations. Additionally, IL2-STAT5 signaling and inflammatory response (NES>2.0, FDR 0.0) pathways were found to be enriched only in presence of talquetamab and GPRC5D+ cells. Of note, in the absence of talquetamab, T cells transcriptomic profile did not change in response to GPRC5D presence or absence on MM cells. Conclusions: Our findings demonstrate that talquetamab upregulates the expression of interferon signaling genes in T cells even in the absence of GPRC5D and this response is enhanced when GPRC5D is present on MM cells. Moreover, talquetmab alters the activation profile across T cells subsets and both abundance and distribution of different T cells subpopulations is changed by GPRC5D status of the MM cells in coculture. Citation Format: Umair Munawar, Alexander Leipold, Seungbin Han, Silvia Nerreter, Shilpa Kurian, Emma Besant, Nina Rein, Johanna Lehmann, Max Koeppel, Xiang Zhou, Ondrej Slaby, Hermann Einsele, Leo Rasche, Emmanual Saliba, Johannes Waldschmidt, Martin Kortuem. T cell transcriptome is altered by Talquetamab in presence of GPRC5D on MM cells [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 5550.

ABSTRACT Background Programmed death‐ligand 1 (PD‐L1) has emerged as a potential biomarker for prognosis and treatment response in various solid tumors. However, its clinical relevance in ovarian carcinoma (OC) remains unclear, with published studies reporting inconsistent associations with platinum sensitivity and survival. These discrepancies are likely related to the biological heterogeneity of OC and methodological variability in PD‐L1 evaluation. In the absence of a standardized, OC‐specific assessment methodology, the Combined Positive Score (CPS) with cutoffs of ≥ 1 and ≥ 10, applied in other tumor types, represents a pragmatic approach. Methods We retrospectively analyzed a real‐world cohort of 146 patients with ovarian carcinoma treated with primary platinum‐based chemotherapy at University Hospital Brno between 2010 and 2020. PD‐L1 expression was assessed using the 22C3 antibody and CPS methodology on archival, treatment‐naive tumor samples obtained from primary tumors or metastatic lesions. Associations between PD‐L1 status, platinum treatment‐free interval (TFIp), and disease‐specific survival (DSS) were evaluated, including subgroup analyses by histological subtypes. Results Among the 144 evaluable cases, 65 patients (45%) demonstrated positive PD‐L1 expression (CPS ≥ 1), while 35 patients (24%) exhibited strong positive expression (CPS ≥ 10). In the high‐grade serous carcinoma (HGSC) subgroup, CPS ≥ 1 and CPS ≥ 10 were detected in 58/121 (48%) and 30/121 (25%) cases, respectively. PD‐L1 positivity did not differ significantly between HGSC and non‐HGSC subtypes or between primary and metastatic lesions. PD‐L1 positivity defined as CPS ≥ 10 was significantly associated with platinum sensitivity (TFIp ≥ 6 months; p = 0.035 overall; p = 0.028 in HGSC) and longer DSS (p = 0.029). No associations were observed when PD‐L1 positivity was defined using a CPS ≥ 1 cutoff. Conclusions PD‐L1 expression assessed by CPS, particularly at a cutoff of ≥ 10, may have prognostic relevance in ovarian cancer and could help identify patients more likely to benefit from platinum‐based chemotherapy. Prospective validation is warranted.

Gastroesophageal reflux disease (GERD) and its complications – reflux esophagitis (RE), Barrett’s esophagus (BE), and esophageal adenocarcinoma (EAC) – have been associated with bacterial dysbiosis. However, comprehensive characterization of the gastrointestinal tract (GIT) bacteriome across these GERD complications is lacking. This multicenter observational study aimed to fill this gap. Biopsies from patient-matched pathological esophageal tissues, gastric body and antrum, duodenum, and oral and rectal swabs were collected from 151 patients with histologically confirmed RE, BE, or EAC, respecting their pharmacotherapy with proton pump inhibitors (PPIs) status. DNA isolates were analyzed by qPCR and 16S rRNA amplicon sequencing. Basic GIT bacteriome profiles were similar between PPI users and non-users with EAC. In PPI users, the genus Porphyromonas in oral swabs was associated with EAC (P < 0.05). Of esophageal biopsies, EAC samples showed the highest bacterial load and alpha diversity among PPI users (P < 0.05). In PPI users, the relative abundance of genus Streptococcus in esophageal samples was significantly higher for RE than for BE and EAC (P < 0.05), while the opposite was observed for genus Lachnoanaerobaculum in both esophageal and duodenal samples (P < 0.05). Interestingly, BE samples showed distinct bacteriome features deviating from the expected trend reflecting the severity of the pathology in individual groups. This study revealed significant associations of some bacteriome characteristics with GERD complications, especially in the oral swabs and esophageal biopsies.

Idiopathic scoliosis (IS) is the most common pediatric spinal deformity, yet no biomarker currently enables early diagnosis or reliable prediction of progression to guide individualized treatment. Circulating microRNAs (miRNAs) are promising non‑invasive biomarkers reflecting multifactorial disease mechanisms. In our prospective monocentric study, a Czech cohort comprising 114 pediatric IS patients at the time of diagnosis and 89 age‑matched healthy controls was studied. Risk groups were defined based on the final Cobb angle at the end of follow-up at skeletal maturity. Plasma miRNA profiles were obtained by small RNA sequencing and analyzed for differential expression. Logistic regression models were used to construct miRNA diagnostic and prognostic signatures, validated by leave‑one‑out cross‑validation (LOOCV). Differential expression analysis identified 48 miRNAs with significantly different expression in the blood plasma of IS patients and controls (adj. p < 0.05), and plasma miR-4451 to have decreased levels in high-risk compared to low- and medium-risk IS patients (adj. p < 0.01). A 28‑miRNA diagnostic signature distinguished IS patients from controls with AUC = 0.95 (sensitivity 88

ABSTRACT Aicardi–Goutières syndrome type 6 (AGS6) is a genetically determined autoinflammatory disorder, classically inherited in an autosomal recessive manner. We report a Czech child with a heterozygous ADAR1 NM_001111.5:c.3019G>A variant causing AGS6. In silico analysis supports a dominant‐negative effect, underscoring the need to consider dominant inheritance in AGS6 diagnosis and management.

BACKGROUND:Identifying novel diagnostic and prognostic biomarkers for pediatric inflammatory bowel diseases (PIBD), including Crohn's disease (pCD) and ulcerative colitis (pUC), is essential for enhancing treatment outcomes. MicroRNAs (miRNAs) have been recognized for their broader relevance in PIBD pathogenesis. This study investigates their diagnostic potential and clinical utility in PIBD. METHODS:This prospective, monocentric study, with retrospective validation, involved 119 PIBD patients (58 pCD, 61 pUC) and 39 non-IBD controls. Small RNA next-generation sequencing was performed on fresh-frozen gut biopsies, targeting histopathologically confirmed inflamed areas. Twenty-five dysregulated miRNA candidates were validated via RT-qPCR in formalin-fixed, paraffin-embedded gut biopsies. Logistic regression was used to establish diagnostic and prognostic miRNA expression signatures. RESULTS:A diagnostic signature of 5 miRNAs (miR-223-3p, miR-34a-5p, miR-194-5p, miR-215-5p, miR-338-3p) distinguished pCD from non-IBD with 96.49% accuracy. Two miRNAs (miR-223-3p, miR-194-5p) differentiated pUC from non-IBD with 100% accuracy, and miR-215-5p distinguished pCD from pUC specimens with 83.54% accuracy. For treatment-naïve pCD patients, 7 miRNAs predicted residual disease activity at 3 months with 100% accuracy. Additionally, a distinct signature predicted the risk of relapse within 12 months with an accuracy of 84.21%. CONCLUSIONS:In this study, we have established tissue miRNA expression signatures with significant diagnostic and prognostic potential for use in PIBD. These findings aid in stratifying disease severity and risk, paving the way for more precise and personalized management of pediatric IBD.

Radiotherapy (RT) plays a central role in the management of glioblastoma, often in combination with other treatment modalities. While RT can enhance both local and systemic tumor control, especially when used alongside immunotherapy, it is also associated with lymphopenia - a reduction in lymphocyte count - which has been linked to poorer treatment outcomes and reduced survival. This retrospective study aimed to examine the relationship between radiation dose delivered to brain vessels and the severity of lymphopenia in patients with newly diagnosed glioblastoma treated at a tertiary cancer center in 2021. Brain vessels were manually contoured using MRI data, and dose-volume analysis was conducted. Lymphopenia severity was graded according to CTCAE v5.0, and statistical analyses were performed to identify any correlations. Among the 28 patients analyzed, 32% developed grade 1-3 lymphopenia. No significant correlation was found between the radiation dose to brain vessels and the degree of lymphopenia. The median volume of irradiated vessels did not differ significantly between patients with and without lymphopenia. In glioblastoma patients, multiple factors contribute to decreased lymphocyte count - e.g., chemotherapy and corticosteroid use. Although no definitive link was identified, the study underscores the importance of preserving lymphocyte counts during glioblastoma treatment and supports the need for further prospective research to explore strategies like lymphocyte-sparing RT and to better understand the mechanisms behind treatment-related lymphopenia.

Background: We present a case of a 12-month-old female patient with metastatic neuroblastoma who was transferred to our institution after disease progression following two cycles of standard treatment in Ukraine. Upon admission, the child was critically ill, presenting with a large abdominal mass, severe cachexia, deep vein thrombosis with limb oedema, and oxygen dependency due to limited chest excursions. Case report: After initial stabilization, comprehensive genomic profiling revealed an SRGAP3:NTRK2 gene fusion in the tumor sample. An individualized combination of standard chemotherapy, targeted treatment with larotrectinib, and metronomic chemotherapy was administered. This regimen resulted in a radiologically confirmed partial response and a notable clinical improvement. Conclusion: This case documents a rare instance of NTRK2 fusion-driven neuroblastoma successfully treated with a regimen containing a pan-TRK inhibitor that proved to be well-tolerated and effective.

BACKGROUND:Emerging evidence suggests that tumour morphological heterogeneity may influence mutational profiles relevant to therapy response. In this pilot study, we aimed to assess whether mutations identified within specific morphological patterns or at the invasion front correlate with shorter time to progression after anti-EGFR therapy, as compared to whole-tissue analysis. METHODS:We investigated genetic mutations in 142 samples from primary tumours of 39 KRAS wild-type metastatic colorectal cancer (CRC) patients receiving anti-EGFR therapy. Deep next-generation sequencing was performed on whole-tumour sections and six morphology-defined tumour regions. RESULTS:Mutations in genes linked to anti-EGFR therapy response (KRAS, BRAF, NRAS, PTEN and PI3KCA) were found uniquely in the non-responder group, with substantial variability across morphological sub-regions. BRAF mutations were aligned with serrated and mucinous morphologies, while KRAS mutations (p.Lys147Glu and p.Ala146Thr) were associated with mucinous and desmoplastic morphologies. In all cases, the cumulative mutational profile from sub-regions provided more details than that of the whole-tumour profile. CONCLUSION:Our findings highlight that comprehensive analysis, considering morphological heterogeneity, is crucial for personalised CRC treatment strategies.

High-grade serous ovarian carcinoma (HGSOC) remains one of the most lethal gynecologic malignancies due to its aggressive nature, frequent late-stage diagnosis, and the development of treatment resistance. Although platinum-based chemotherapy remains the cornerstone of therapy, the underlying genomic heterogeneity complicates the prediction of treatment response and the development of effective therapies. We performed comprehensive genomic profiling of 523 cancer-associated genes using the TruSight Oncology 500 HT panel in a retrospective cohort of 42 HGSOC patients, including 22 platinum-sensitive (Pt–S) and 20 primary platinum-resistant (Pt-R) cases. In 14 cases, paired tumor samples collected before and after recurrence or neoadjuvant chemotherapy were analyzed to assess the changes in clinically relevant and actionable alterations. Genomic profiling revealed significant heterogeneity in molecular alterations between Pt–S and Pt-R tumors, with CCNE1 amplification confirmed as more frequent in Pt-R cases. Actionable findings ranked in ESCAT tiers I-III were identified in 54.5

This retrospective study aims to show a real-life single-center experience with clinical management of relapsed pediatric ependymomas using results from comprehensive molecular profiling. Eight relapsed ependymomas were tested by whole exome sequencing, RNA sequencing, phosphoproteomic arrays, array comparative genome hybridization, and immunohistochemistry staining for PD-L1 expression and treated with an individualized approach implementing targeted inhibitors, immunotherapy, antiangiogenic metronomic treatment, or other agents. Treatment efficacy was evaluated using progression-free survival (PFS), overall survival (OS), survival after relapse (SAR), and PFS ratios. Genomic analyses did not reveal any therapeutically actionable alterations. Surgery remained the cornerstone of patient treatment, supplemented by adjuvant radiotherapy. Empiric agents were chosen quite frequently, often involving drug repurposing. In six patients, prolonged PFS after relapse was seen because of immunotherapy, MEMMAT, or empiric agents and is reflected in the PFS ratio ≥ 1. The 5-year OS was 88

Brain metastases (BMs) are frequent and devastating complications of systemic malignancies, necessitating accurate diagnosis and origin identification for effective treatment strategies. Invasive biopsies are currently required for definitive diagnosis, highlighting the need for less invasive diagnostic approaches and robust biomarkers. Circulating microRNAs (miRNAs) have demonstrated potential as sensitive and specific diagnostic biomarkers in various cancers. Thus, our objective was to identify and compare miRNA profiles in BM tissue, cerebrospinal fluid (CSF), and plasma, with a specific focus on liquid biopsies for diagnostic purposes. Total RNA enriched for miRNAs was isolated from histopathologically confirmed BM tissues (n = 30), corresponding plasma samples (n = 30), and CSF samples (n = 27) obtained from patients with diverse BM types. Small RNA sequencing was employed for miRNA expression profiling. Significantly differentially expressed miRNAs were observed in BM tissues, enabling the differentiation of primary origins, particularly breast, colorectal, renal cell carcinoma, and melanoma metastases. The heterogeneity observed in lung carcinomas also manifested in the corresponding BMs, posing challenges in accurate discrimination from other BMs. While tissue-specific miRNA signatures exhibited the highest precision, our findings suggest low diagnostic potential of circulating miRNAs in CSF and blood plasma for BM patients. Our study represents the first analysis of miRNA expression/levels in a unique set of three biological materials (tissue, blood plasma, CSF) obtained from the same BM patients using small RNA sequencing. The presented results underscore the importance of investigating aberrant miRNA expression/levels in BMs and highlight the low diagnostic utility of circulating miRNAs in patients with BMs.

DNA methylation-based classification of (brain) tumors has emerged as a powerful and indispensable diagnostic technique. Initial implementations used methylation microarrays for data generation, while most current classifiers rely on a fixed methylation feature space. This makes them incompatible with other platforms, especially different flavors of DNA sequencing. Here, we describe crossNN, a neural network-based machine learning framework that can accurately classify tumors using sparse methylomes obtained on different platforms and with different epigenome coverage and sequencing depth. It outperforms other deep and conventional machine learning models regarding accuracy and computational requirements while still being explainable. We use crossNN to train a pan-cancer classifier that can discriminate more than 170 tumor types across all organ sites. Validation in more than 5,000 tumors profiled on different platforms, including nanopore and targeted bisulfite sequencing, demonstrates its robustness and scalability with 99.1% and 97.8% precision for the brain tumor and pan-cancer models, respectively.

ABSTRACT Background Molecular tumor boards (MTBs) support the development of personalized treatment strategies for patients with various cancer types based on comprehensive genomic profiling (CGP) of tumor tissue. Despite the unprecedented results demonstrated in many molecularly driven clinical trials, access to matched therapy remains a significant challenge in routine clinical practice worldwide. Methods In this study, we analyzed the MTB cohort from University Hospital Brno in the Czech Republic. Between February 2021 and April 2025, a total of 553 cancer patients with limited therapeutic options underwent CGP of tumor tissue and were subsequently presented at the MTB. Results The median age of the patients was 61.1 years, and 62.2% were female. The most frequently tested diagnoses were colorectal cancer (n = 88; 15.9%), cholangiocarcinoma (n = 66; 11.9%), and pancreatic cancer (n = 65; 11.8%). The median number of prior lines of standard systemic therapy before CGP testing was two. MTB‐recommended matched therapy for 326 (59.0%) out of 553 tested patients, based on 545 unique molecular alterations. The most frequently recommended drugs included immunotherapy (162/545; 29.7%), tyrosine kinase inhibitors (140/545; 25.7%), and poly (ADP‐ribose) polymerase inhibitors (63/545; 11.6%). Reimbursement was requested from healthcare insurance providers in 115 cases, with 87 (75.7%) approvals. Together with other reimbursement forms, a total of 96 (17.4%) out of 553 patients initiated matched therapy. A progression‐free survival ratio (PFS2/PFS1) of ≥ 1.3 was observed in 29 (41.4%) of the 70 evaluable pretreated patients. Conclusion This is the first study to report on a real‐world MTB cohort from the Czech Republic, demonstrating a diagnostic yield comparable to previously published studies, good availability of recommended drugs, and clinical benefit in evaluable patients.