Supplementary Table S7, Table showing source data and statistics for non-high-throughput experiments such as flow cytometry, quantitative PCR, protein experiments and other molecular and cellular assays.

Lymph node (LN) function requires the organization of cells into higher-order spatial units. However, the principles governing LN architecture in health and disease remain poorly understood. Here, we used single-cell and spatial mapping to investigate the mechanisms directing immune cell organization in human LNs and its disruption in architecturally distinct lymphoma entities: indolent follicular lymphoma (FL) and aggressive diffuse large B cell lymphoma (DLBCL). Our data substantiate the central role of LN-resident stromal cells in chemokine-driven lymphocyte zonation and reveal an inflammatory feedback loop fueled by tumor-reactive T cells that triggers stromal remodeling, progressive loss of homeostatic chemokine gradients, and tissue disorganization from a non-malignant state to FL and DLBCL. Loss of homeostatic chemokines was associated with adverse patient survival, identifying the underlying architectural rearrangement as a key event during lymphomagenesis. Collectively, our results highlight the principles of LN organization and suggest how lymphoma-induced microenvironmental reprogramming drives the loss of tissue organization.

Supplementary Table S3, Quantitative whole proteome analysis of pooled sorted GCB-cells from Fbxo45 homozygous knockout mice compared with identically isolated cells from wild-type littermates.

Supplementary Table 1 from Epigenetic Inactivation of the Groucho Homologue Gene TLE1 in Hematologic Malignancies

Supplementary Table S2, Mass spectrometric (MS) identification of GEF-H1-derived peptides in FBXO45 immunoprecipitation. FLAG-tagged FBXO45 expressing B-cell lymphoma cell lines were subjected to immunoprecipitation (IP) with anti-FLAG resin. MS analysis of co-purified endogenous proteins revealed the presence of numerous GEF-H1-derived peptides.

Abstract Background Severe, early-onset obesity is a monogenic disorder in a subset of patients. Recently, rare protein-truncating variants (PTVs) in BSN and APBA1 have been implicated as monogenic causes of obesity. Truncating variants in these two genes have been associated with adult-onset monogenic obesity. Here, we aimed to investigate the occurrence of rare PTVs in APBA1 and BSN in minors with severe obesity manifesting in early childhood or adolescence. Methods Previously obtained exome data of 209 children and adolescents (0–18 years) with severe obesity manifesting in early childhood or adolescence genetically tested at the Ulm University Medical Center between April 2022 and May 2024 were mined for rare PTVs in APBA1 (NM_ 001163.4; NP_ NP_001154.2) and BSN (NM_003458.4; NP_003449.2). In all patients, monoallelic, and in the case of autosomal-recessive inheritance, biallelic likely pathogenic and pathogenic variants in well-known obesity-associated genes were excluded. Clinical re-evaluation was performed in two identified cases with rare potentially protein-truncating variants in APBA1. Results The first patient (male, 15 years) presented with a BMI of 33.7 kg/m2 (BMI z-score: 2.7) after nine years of excessive weight gain despite life-style interventions. He had a body fat percentage of 49.5% and showed hyperphagia. The patient displayed impaired expressive language development, dyslexia and an IQ of 84. His mother also had obesity and dyslexia while his father and younger sister were unaffected. We identified the heterozygous, likely pathogenic variant APBA1:c.814C > T;p.(Gln272*) in the index patient and his mother. The second patient (male, 6 years) presented with a BMI of 30.0 kg/m2 (BMI z-score: 3.8), a body fat percentage of 43.6% and hyperphagia. The child had upslanting palpebral fissures and showed impaired expressive language development. Both parents of the boy were affected by severe obesity and underwent bariatric surgery. We identified the heterozygous variant of unknown significance APBA1:c.2442 + 3A > C predicted to impair proper splicing in the index patient and his mother. Conclusion We conclude that rare potentially protein-truncating variants in APBA1 are also found in some minors with early-onset and severe obesity. Therefore, those variants might not be restricted to adult-onset disease. We therefore propose to include APBA1 in diagnostic genetic testing for monogenic obesity in minors and adults.

Extranodal NK/T-cell lymphoma (ENKTL) and primary Epstein-Barr virus (EBV)-positive nodal T/NK-cell lymphoma (nodal-TNKL) are aggressive lymphomas with overlapping clinicopathologic features but distinct underlying biology. While their genomic landscapes have been increasingly defined, comparative epigenetic characterization remains limited. We performed methylated DNA immunoprecipitation sequencing (MeDIP-seq) on formalin-fixed paraffin-embedded samples from ENKTL, nodal-TNKL, ENKTL cell lines, and control tissues. ENKTL displayed extensive promoter hypermethylation associated with repression of tumor suppressor genes, lineage regulators, and lymphoid signaling genes, including LEF1 and BANK1, together with focal hypomethylation of immune- and interferon-responsive genes such as IFITM1. In contrast, nodal-TNKL showed global hypomethylation, particularly affecting cytotoxicity, immune-response, and antigen-presentation pathways; TET2-mutated nodal-TNKL cases exhibited locus-specific methylation changes. Across all samples, global DNA methylation levels correlated with genomic instability. Unsupervised clustering identified two epigenetically distinct ENKTL subgroups, one characterized by higher global methylation, TP53 loss, increased copy number alterations and loss of heterozygosity and significantly poorer overall survival. Together, this study defines fundamental epigenetic differences between ENKTL and nodal-TNKL and links DNA methylation dynamics to genomic instability and clinical outcome, highlighting the value of methylation profiling for refined classification, risk stratification and therapeutic guidance.

Melanocortin 4 receptor (MC4R) deficiency is the most common monogenic cause of obesity, yet remains underdiagnosed. Patients with monogenic obesity often undergo a frustrating diagnostic and therapeutic odyssey of years of ineffective lifestyle interventions before a causal diagnosis is made. We report a four-generation family where genetic testing in a child identified a likely pathogenic MC4R variant also carried by three ancestors. The studied family included a 7-year-old index patient, her mother, grandmother, and great-grandmother with a history of early-onset obesity. Panel sequencing of monogenic obesity genes was performed in the index patient whereas in the relatives targeted analysis of the familial MC4R variant was performed by Sanger sequencing. The index patient developed severe obesity by age 2 years, with hyperphagia, tall stature, and dyslipidemia. Despite lifestyle interventions, her body mass index (BMI) continued to increase. At the age of 7 years, genetic panel testing identified a rare monoallelic variant in the MC4R gene c.913C > T; p.Arg305Trp, previously shown to impair receptor function. Treatment with liraglutide (3.0 mg/day) was initiated at age 8 years, resulting in marked reduction in BMI during the first year of treatment. Subsequent genetic testing of family members identified the same variant in her mother, grandmother, and great-grandmother, all of whom had a history of early-onset obesity and related comorbidities, consistent with segregation of the variant within the family. This case underscores the importance of early genetic testing in severe childhood obesity to avoid ineffective treatments and enable targeted therapies (e.g., GLP-1 analogues). Diagnosing (likely) pathogenic MC4R variants can also identify at-risk relatives, providing psychological and clinical benefits across generations.

Background/Objectives: B-cell receptor (BCR) signalling is implicated in Burkitt lymphoma (BL) lymphomagenesis, although its activation may differ across biological and epidemiological settings. While surface IgM is the predominant immunoglobulin isotype of BCR in BL, cases expressing IgA transcripts or lacking detectable surface immunoglobulin (sIg) expression have been reported, suggesting a more complex pattern of sIg expression than previously recognized. This study aimed to evaluate sIg heavy-chain expression by immunohistochemistry in a relatively large series of BL cases from endemic and sporadic settings and to investigate the mutational landscape of surface immunoglobulin-undetectable (sIg-UND) cases. Methods: sIg heavy-chain expression was assessed by immunohistochemistry in 55 formalin-fixed paraffin-embedded BL samples. Targeted next-generation sequencing was performed on four sIg-UND cases using an Illumina capture-based custom panel covering 74 lymphoma-related genes. KRAS and NRAS hotspot mutations were additionally assessed by real-time PCR. Results: Overall, 41/55 cases showed IgM expression, including 31 IgM-positive and 10 IgM+/UND cases, whereas 8/55 cases showed IgA expression (IgA+/UND). IgA-expressing cases were significantly enriched at mucosal sites, particularly the oral cavity and gastrointestinal tract, consistent with the relevance of these anatomical sites to mucosal IgA production. Six cases lacked detectable sIg expression, showing negativity for all tested immunoglobulin heavy chains in more than 90% of neoplastic cells. Four sIg-UND cases were available for sequencing. These cases harbored mutations affecting genes commonly altered in BL, together with alterations in genes less commonly represented in recurrent BL series, such as MEF2B, CREBBP, PRDM1, PIM1, ARID3A, HIST1H1B, and HIST1H1C. Conclusions: Our study provides a systematic immunohistochemical characterization of surface immunoglobulin heavy-chain expression in a relatively large series of endemic and sporadic BL. We identified a subset of IgA-expressing BL cases enriched at mucosal sites, whereas rarer cases lacked detectable surface immunoglobulin expression. The mutational profile of sIg-UND cases highlights alterations affecting genes involved in epigenetic regulation, chromatin organization, and B-cell differentiation, which may contribute to the biological heterogeneity of these cases. No KRAS or NRAS hotspot mutations were detected in the four sequenced sIg-UND cases, as assessed by targeted NGS and real-time PCR. Further functional studies are needed to clarify the biological and clinical significance of sIg-UND BL cases and to determine the functional relevance of the identified alterations.

Abstract An undifferentiated status and the epigenetic inactivation of tumor-suppressor genes are hallmarks of transformed cells. Promoter CpG island hypermethylation of differentiating genes, however, has rarely been reported. The Groucho homologue Transducin-like Enhancer of Split 1 (TLE1) is a multitasked transcriptional corepressor that acts through the acute myelogenous leukemia 1, Wnt, and Notch signaling pathways. We have found that TLE1 undergoes promoter CpG island hypermethylation–associated inactivation in hematologic malignancies, such as diffuse large B-cell lymphoma and AML. We also observed a mutual exclusivity of the epigenetic alteration of TLE1 and the cytogenetic alteration of AML1. TLE1 reintroduction in hypermethylated leukemia/lymphoma cells causes growth inhibition in colony assays and nude mice, whereas TLE1-short hairpin RNA depletion in unmethylated cells enhances tumor growth. We also show that these effects are mediated by TLE1 transcriptional repressor activity on its target genes, such as Cyclin D1, Colony-Stimulating Factor 1 receptor, and Hairy/Enhancer of Split 1. These data suggest that TLE1 epigenetic inactivation contributes to the development of hematologic malignancies by disrupting critical differentiation and growth-suppressing pathways. [Cancer Res 2008;68(11):4116–22]

Supplementary Data file shows all the supplementary figures, figure legends and supplementary table legends.

Supplementary Table S6, Details of the FISH probes used to identify copy number variations (CNV) affecting FBXO45 and ARHGEF2 in clinical samples.

Supplementary Table S1 shows FBXO45 interactions by immunoprecipitation–tandem mass spectrometry. Normalized Spectral Abundance Factor (NSAF) was calculated as previously published (63). Average NSAFs (n = 3) for the indicated proteins are shown, and the data were obtained from at least three independent experiments.

Abstract Background Genetic screening for maturity-onset diabetes of the young (MODY) involves sequencing of the coding regions of known disease-associated genes. We describe the complex and challenging diagnostic journey of a patient with early-onset diabetes with a novel, intronic HNF1A variant likely affecting a branching site. Case presentation The patient was diagnosed with diabetes at the age of 10 years after incidental hyperglycemia (HbA1c 8.1%, C-peptide 3.0 μg/dl), without polyuria, polydipsia, or weight loss. Type 1 diabetes associated autoantibodies were negative, but the patient had a strong family history of early-onset diabetes (classified as type 1 or type 2 diabetes). Initial genetic testing for HNF4A, GCK, HNF1A, and HNF1B coding regions (including exon/intron boundaries ± 20 bp) and MLPA were negative. Sulfonylureas provided good glycemic control until age 16, when insulin was added. At age 18, an expanded targeted next-generation sequencing (NGS) panel for MODY was also negative. At age 24, whole-exome-sequencing via NGS and additional analysis was conducted, focusing on synonymous and intronic variants, and revealed a heterozygous HNF1A variant (c.327-28A > G;p.?) in the patient and four affected relatives. The variant co-segregated with diabetes, and was predicted to affect splicing via branching site disruption, suggesting pathogenicity. Conclusion In summary, this case highlights the importance of a comprehensive diagnostic approach that combines clinical, biochemical, and extended genetic evaluation. When MODY is strongly suspected despite negative targeted testing, broader sequencing—including intronic and regulatory regions—should be pursued. Accurate variant interpretation remains essential to prevent misclassification and to optimize diagnosis, treatment, and understanding of the genetic complexity of monogenic diabetes.

Supplementary Table S4, Whole genome sequencing of FL, DLBCL and transformed DLBCL reveal loss of FBXO45 as well as copy number gain encompassing ARHGEF2, the gene that encodes GEF H1 protein. Table showing frequency of genomic loss of FBXO45 and gain of ARHGEF2 in DLBCL respectively.

Primary intestinal T-cell lymphomas (ITCLs), comprising enteropathy-associated T-cell lymphoma (EATL) and monomorphic epitheliotropic intestinal T-cell lymphoma (MEITL), are rare aggressive tumors. The role of DNA mismatch repair (MMR) deficiency (dMMR) and microsatellite instability (MSI) in the development of ITCLs remains largely unexplored. Here, we investigated the incidence, molecular mechanisms and clinical relevance of dMMR/MSI in 86 ITCLs (30 EATLs, 56 MEITLs) using whole-exome sequencing, PCR-based MSI testing, DNA methylation profiling, and immunohistochemistry for MLH1, MSH2, MSH6 and PMS2. MMR deficiency was detected in 3 of 53 MEITLs (6%) but in none of the EATLs. dMMR MEITLs showed the highest tumor mutational burden (8.3-17.1 mutations/Mb), compared to median TMBs of 1.9 in MEITL and 2.4 in EATL. The complete loss of MLH1/PMS2 expression in two MSI-high tumors and isolated PMS2 loss in the third case were all associated with biallelic deletions of the affected loci. Notably, MLH1 deletions significantly co‑occurred with SETD2 deletions (p = 0.001), the latter representing a major driver of MEITL tumorigenesis. dMMR MEITLs lacked distinctive clinicopathologic features. These findings indicate that dMMR/MSI occurs in a subset of MEITLs probably during tumor progression, rather than being an initiating driver event, and provide a biological rationale to explore the efficacy of immune checkpoint inhibitors in some of this unfavorable subtype of ITCL.

Supplementary Table S5, FISH analysis to identify copy number variations (CNV) affecting genes of interest within the corresponding genomic loci.

BACKGROUND:Malignant rhabdoid tumors occasionally develop along cranial nerves, but clinical, histopathological, and molecular features have not been examined in larger series. PROCEDURE:We retrospectively interrogated data from the European Rhabdoid Registry, EU-RHAB, to identify malignant rhabdoid tumors affecting cranial nerves. We retrieved clinical information and reviewed magnetic resonance imaging (MRI) data. Furthermore, histopathological review and molecular profiling were performed. RESULTS:Among 425 patients, we identified a total of 14 harboring malignant rhabdoid tumors with cranial nerve involvement. Median age at diagnosis was 28 months (range: 0-13 years). Various cranial nerves were affected, the trigeminal nerve (n = 4) and the facial and/or vestibulocochlear nerve (n = 5) being most frequently involved. In most cases, the initial clinical and neuroradiological suspicion was schwannoma. Neuroradiology review of magnetic resonance imaging studies confirmed a tumor along the cranial nerve, but signal characteristics with restricted diffusion were rather suggestive of a malignant tumor of high cellularity. Histopathological examination, using among others neurofilament staining confirmed the diagnosis and infiltration of nerve fascicles. DNA methylation profiles demonstrated high similarity with ATRT-MYC as well as extracranial malignant rhabdoid tumors (median calibrated scores: 1.00). CONCLUSIONS:Malignant rhabdoid tumors of the cranial nerves represent a small but clinically distinct group, which initially is often not included in the differential diagnoses of pediatric cranial nerve tumors. Restricted diffusion on MRI may provide an early diagnostic clue. Histopathology and molecular signature are characteristic, but the developmental origin of malignant rhabdoid tumors of the cranial nerves remains to be determined.