While most individuals with familial medullary thyroid carcinoma (fMTC) carry RET mutations, in some instances the causative mutations remain unknown. We studied two related families with RET -negative fMTC in 21 affected individuals through linkage analysis, exome/genome sequencing, and high-density array comparative genomic hybridization. We identified a novel heterozygous 40kb intragenic SLC30A9 deletion which segregated with the disease in all affected individuals. The mutant transcript escaped nonsense-mediated decay and resulted in the production of N-terminally truncated proteins via translation reinitiation from in-frame AUG codons located downstream of the deletion. These proteins showed increased stability and their expression in an MTC cell line increased cell proliferation and clonogenic capacity, supporting an oncogenic role. These findings expand the genetic background of fMTC beyond RET mutations and implicate translation reinitiation in the etiology of cancer susceptibility syndromes secondary to structural genomic variants.

Reliable detection of structural variants (SVs) and copy number variations (CNVs) is crucial in the contemporary diagnostics of pediatric B-cell acute lymphoblastic leukemia (B-ALL). However, limitations of commonly used conventional and molecular cytogenetic methods may hinder the accurate genetic characterization of patients. Optical genome mapping (OGM) offers a reliable alternative by enabling high-resolution, genome-wide detection of CNVs and SVs. Chromosomal aberrations were screened using OGM in 51 children with B-ALL. The results were compared with those of karyotyping, fluorescence in situ hybridization (FISH), digital multiplex ligation-dependent probe amplification (digitalMLPA), and targeted RNA sequencing (RNA-seq). OGM data showed high congruency with karyotyping and FISH findings, detecting clinically relevant variants beyond G-banding results and unraveling a complex KMT2A fusion undetected by FISH. Gene fusions involved in complex ETV6::RUNX1 translocations, but not detected by RNA-seq, were confirmed using FISH. Normalization of OGM copy number values with DNA-index-improved concordance with FISH-derived copy numbers in near-tri/tetraploid cases. In the peripheral regions of OGM variants (fringe-zones), a novel evaluation strategy called 'FriZone' was applied, which significantly improved the concordance between OGM and digitalMLPA. In addition, a co-segregation analysis revealed strong associations between ETV6::RUNX1 fusion and deletions of ETV6, RAG2, and NR3C2. OGM uncovered complex rearrangements undetected by widely used methods in 15% of cases, improving genetic classification and risk stratification in 10% of the patients. The FriZone analysis and normalization by DNA-index provide a refined, more accurate approach to OGM variant interpretation, facilitating the efficient application of OGM in clinical diagnostics. © 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.

Risk-adapted treatment protocols conferred remarkable improvement in the survival rates of pediatric acute lymphoblastic leukemia/lymphoma (ALL/LBL). Nevertheless, clinical management is still challenging in certain molecular subgroups and in the presence of alterations associated with an increased rate of relapse. In this study, disease-relevant genomic and transcriptomic profiles were established in a prospective, multicenter, real-world cohort involving 192 children diagnosed with ALL/LBL. Gene fusions were detected in 34.9% of B-ALL and 46.4% of T-ALL patients, with novel chimeric genes involving JAK2, KMT2A, PAX5, RUNX1, and NOTCH1, and with KMT2A-rearranged patients displaying the worst 3-year event-free survival (P 1/4 .019). Nonsynonymous mutations were uncovered in 74.9% of the analyzed patients, and pairwise scrutiny of genetic lesions revealed recurrent clonal selection mechanisms commonly converging on the same pathway (eg, Ras, JAK/ STAT, and Notch) in individual patients. Investigation of matched diagnostic and relapse samples unraveled complex subclonal variegation, and mutations affecting the NT5C2, TP53, CDKN2A, and PIK3R1 genes, emerging at the time of relapse. TP53 and CREBBP mutations, even as subclonal aberrations, were associated with shorter 3-year event-free survival among all patients with B-ALL (TP53 mutant vs wild-type: P = .008, CREBBP mutant vs wild-type: P = .010), and notably, B-ALL patients showing no measurable residual disease on day 33 could be further stratified based on TP53 mutational status (P < .001). Our in-depth molecular characterization performed across all risk groups identified novel opportunities for molecularly targeted therapy in 55.9% of high-risk and 31.6% of standard/intermediate-risk patients. (c) 2025 THE AUTHORS. Published by Elsevier Inc. on behalf of the United States & Canadian Academy of Pathology. This is an open access article under the CC BY-NC-ND license (http://creativecommons. org/licenses/by-nc-nd/4.0/).

We report that ~1.8% of all mesothelioma patients and 4.9% of those younger than 55, carry rare germline variants of the BRCA1 associated RING domain 1 ( BARD1) gene that were predicted to be damaging by computational analyses. We conducted functional assays, essential for accurate interpretation of missense variants, in primary fibroblasts that we established in tissue culture from a patient carrying the heterozygous BARD1 V523A mutation. We found that these cells had genomic instability, reduced DNA repair, and impaired apoptosis. Investigating the underlying signaling pathways, we found that BARD1 forms a trimeric protein complex with p53 and SERCA2 that regulates calcium signaling and apoptosis. We validated these findings in BARD1 -silenced primary human mesothelial cells exposed to asbestos. Our study elucidated mechanisms of BARD1 activity and revealed that heterozygous germline BARD1 mutations favor the development of mesothelioma and increase the susceptibility to asbestos carcinogenesis. These mesotheliomas are significantly less aggressive compared to mesotheliomas in asbestos workers.

Cancer is caused by an accumulation of somatic mutations and copy number alterations (CNAs). Besides mutations, these copy number changes are key characteristics of cancer development. Nonetheless, some tumors show hardly any CNAs, a remarkable phenomenon in oncogenesis. Head and neck squamous cell carcinomas (HNSCCs) arise by either exposure to carcinogens, or infection with the human papillomavirus (HPV). HPV-negative HNSCCs are generally characterized by many CNAs and frequent mutations in CDKN2A, TP53, FAT1, and NOTCH1. Here, we present the hallmarks of the distinct subgroup of HPV-negative HNSCC with no or few CNAs (CNA-quiet) by genetic profiling of 802 oral cavity squamous cell carcinomas (OCSCCs). In total, 73 OCSCC (9.1%) are classified as CNA-quiet and 729 as CNA-other. The CNA-quiet group is characterized by wild-type TP53, frequent CASP8 and HRAS mutations, and a less immunosuppressed tumor immune microenvironment with lower density of regulatory T cells. Patients with CNA-quiet OCSCC are older, more often women, less frequently current smokers, and have a better 5-year overall survival compared to CNA-other OCSCC. This study demonstrates that CNA-quiet OCSCC should be considered as a distinct, clinically relevant subclass. Given the clinical characteristics, the patient group with these tumors will rapidly increase in the aging population. HPV-negative head and neck squamous cell carcinomas (HNSCCs) are generally characterized by many copy number alterations (CNAs) and mutations. Here, the authors characterize a subgroup of HPV-negative HNSCC with no or few CNAs (CNA-quiet) by genomic profiling of 802 oral cavity squamous cell carcinomas.

S222to CFr is a nitrogen-recycling bacteria, we detected the concentration of NH4+ in MM patient's feces and serum, and found that NH4+ increased significantly in RM, and the relative abundance of CFr was positively correlated with NH4+ in feces and serum of RM.Subsequently, we confirmed that NH4+ can promote the drug resistance of MM cells to bortezomib (BTZ) through in vitro and in vivo experiments.Meanwhile, we constructed a CFr deaminase gene deletion (CFr-KO) strain, and found that CFr produces a large fraction of NH4+ by expressing deaminases, and NH4+ molecules produced in the intestinal tract subsequently enter the circulation and eventually travel to the bone marrow, causing MM cells to become resistant to BTZ.Finally, we treated MM cells with NH4+ and detected the expression of resistance related protein.It was found that NH4+ can upregulate the expression of NEK2, and NH4+ can increase the acetylation of NEK2 and reduce its ubiquitin degradation, thus maintaining its protein stability.SLC12A2 as the key transmembrane transporter that mediates the uptake of NH4+ by MM cells, we discovered that furosemide sodium (Fus) can downregulate the expression of SLC12A2 in MM cells.Fus inhibits NH4+ uptake in MM cells and reduces the BTZ resistancepromoting effects of NH4+ supplementation in vitro and in vivo.Next, we analyzed the effect of Fus treatment in MM patients, finding that the MM patients treated with Fus achieved longer progression-free survival and higher curative effect scores.Finally, to explore additional strategies for tackling drug resistance in MM, we performed single Clostridium butyricum (CBu) and triple probiotic (TPro) transplantation by gavage, and we found that in keeping with the effect of CBu transplantation, TPro, an FDA-approved clinical drug, exhibited consistent efficacy in alleviating BTZ resistance.Conclusions: In summary, we identify, for the first time, CFr and NH4+ as key modulators of MM relapse, unveil novel molecular mechanisms for drug resistance in MM patients, and provide new therapeutic strategies for the intervention of MM progression and drug resistance.

Supplementary Table 1. List of antibodies and primer sets used in the study.

Supplementary Materials and Methods

Supplementary Figure 4. Effect of KAT6B shRNA-mediated depletion in the growth rate according to the XTT assay

PDF file 236K, Table S1. List of the 121 lung cancer cell lines screened for MAX alterations. Information about the histopathology of each cell line, and the presence of alterations at MYC and BRG1 is also included. Grey boxes indicate that no information is available. Table S2. List of genes that are up-regulated or down-regulated upon MAX reconstitution. The values represent the n-fold change in the level of gene expression of each of the lung cancer cell lines infected with the wild type MAX relative to the controls (?). Table S3. List of genes that are up-regulated or down-regulated upon depletion of BRG1. The values represent the n-fold change in the level of gene expression of each of the lung cancer cell lines infected with the shBRG1 relative to the controls ?. Table S4. List of the cell lines included in Figure 5. The information about alterations at the indicated genes was obtained from different sources, as indicated. For data extracted from databases we applied the following criteria to define a mutation: i) mutations at tumor suppressor genes (BRG1, SMARCB1, MAX, ARID1A, PRBM1, and MGA) should be homozygous and predictive of truncated proteins, ii) for amplification at the MYC family of oncogenes, only very high levels of gene amplification have been considered to be positive. Other genes, related to MYC/MAX or to the SWI/SNF complex, have also been searched for alterations (i.e., ARID1B, ARID2, MXI, MXDs) but either no alterations were reported in the databases or the changes did not fulfill our selection criteria. CCLE, Cancer Cell Line Encyclopedia (Broad-Novartis Cancer Cell Line Encyclopedia; website, http://www.broadinstitute.org/ccle/). COSMIC, Catalogue of Somatic Mutations in Cancer (Trust Sanger Institute's Cancer Cell Line Project; website, http://cancer.sanger.ac.uk/)

PDF file 422K, Fig. S1. Radio charts of the multiplex ligation-dependent probe amplification (MLPA), depicting intragenic deletions at the indicated lung cancer cell lines and lung primary tumor. The five exons of the MAX gene and the names and the relative location of the different probes used in the MLPA are also shown. The ratio charts depicting a the analysis of a normal DNA, an intragenic deletion of exons from 3 to 5 for the Lu134 cell line and deletion of the entire coding region for the Lu165 cell line are included. The radio chart for the tumorgraft from the same individual as the Lu134 cells is also depicted, ruling out the possibility of it being a cellculture artifact. Fig. S2. A, Cell proliferation, measured using MTT assays, was significantly greater in the cells carrying the empty vector (? control cells) than in those expressing ectopic MAX, from the parental H1417 cells. Lines represent the number of viable cells relative to the total number of cells at 0 h. Error bars, standard deviation *p<0.001. B, Western blot of total lysates of MAX in H1299tr-BRG1wt and H1299tr-BRG1mut cells, treated with (1 ng/μl) to allow BRG1 expression, depicts the ectopic expression of MAX in a hormone-free (HF) environment, or in the presence of glucocorticoids (GC) at the indicated concentrations. In the left panel, cells transfected with the 5'UTR-MAX constructs exhibit decreased levels of ectopic MAX in the BRG1mut cells compared with BRG1wt cells. On the right, cells transfected with the MAX construct lacking 5'UTR show similar levels of ectopic MAX regardless of the status of BRG1. C, Western blots illustrate the reduction of MAX upon depletion of BRG1 in the indicated lung cancer cells carrying amplification of MYC in an HF environment and at the indicated hours after treatment with GC (5 μM). TUBULIN is included as a loading control. Fig. S3. Enriched gene ontology (GO) classifications (p<0.05 for all categories shown) among genes up-regulated in the MAX gene-expression profile (from Table S2). Enrichment (represented by several GO categories) is significant for genes annotated as being involved in neural development and in glucose/carbohydrate metabolism

Background Recurrent genetic lesions provide basis for risk assessment in pediatric acute lymphoblastic leukemia (ALL). However, current prognostic classifiers rely on a limited number of predefined sets of alterations. Methods Disease-relevant copy number aberrations (CNAs) were screened genome-wide in 260 children with B-cell precursor ALL. Results were integrated with cytogenetic data to improve risk assessment. Results CNAs were detected in 93.8% ( n = 244) of the patients. First, cytogenetic profiles were combined with IKZF1 status ( IKZF1 normal , IKZF1 del and IKZF1 plus ) and three prognostic subgroups were distinguished with significantly different 5-year event-free survival (EFS) rates, IKAROS-low ( n = 215): 86.3%, IKAROS-medium ( n = 27): 57.4% and IKAROS-high ( n = 18): 37.5%. Second, contribution of genetic aberrations to the clinical outcome was assessed and an aberration-specific score was assigned to each prognostically relevant alteration. By aggregating the scores of aberrations emerging in individual patients, personalized cumulative values were calculated and used for defining four prognostic subgroups with distinct clinical outcomes. Two favorable subgroups included 60% of patients ( n = 157) with a 5-year EFS of 96.3% (excellent risk, n = 105) and 87.2% (good risk, n = 52), respectively; while 40% of patients ( n = 103) showed high ( n = 74) or ultra-poor ( n = 29) risk profile (5-year EFS: 67.4% and 39.0%, respectively). Conclusions PersonALL, our conceptually novel prognostic classifier considers all combinations of co-segregating genetic alterations, providing a highly personalized patient stratification.

Recent advances in immunotherapy have highlighted that in multiple myeloma (MM), besides risk-determining copy number alterations (CNAs) there is a need for investigation of CNAs in CAR-T cell therapy-related targets (e.g. BCMA, GPRC5D, FcRH5). DigitalMLPA technology has been successfully used for characterization of recurrent CNAs on chromosome arms 1p, 1q, 13q and 17p in newly diagnosed and relapsed MM cases, as well as in cancer cell lines of MM origin. Adding probes aimed at CAR-T cell therapy-related target genes complements the already included predictive target probes ( IKZF1/3, IRF4, MYC, RPL5, SLAMF7 and BRAF V600E point mutation) in SALSA® digitalMLPA™ Probemix D006 Multiple Myeloma. In addition, due to a large overlap of affected genomic regions between hematologic neoplasms, we aimed to demonstrate usefulness of this multiplex assay not only on cell lines of MM origin, but also of myeloid and lymphoid lineages. Methods. D006 Multiple Myeloma digitalMLPA probemix contains (i) probes targeting chromosomal arms with recurrent CNAs, including 1p (33 probes), 1q (29 probes), 13q (23 probes), 17p (20 probes); (ii) probes for promising predictive targets such as BCMA, CRBN, GPRC5D, FcRH5, IKZF1/3, IRF4, MYC, RPL5, SLAMF7 - 2-4 probes per gene, and BRAF V600E mutation-specific probe; (iii) 278 probes for subtelomeric, pericentromeric and middle regions of chromosomal arms for all chromosomes; and (iv) control probes for quality control and troubleshooting. DigitalMLPA reactions were performed with 20-40 ng DNA from 29 cancer cell lines of lymphoid and myeloid lineage, 16 of which were of MM origin. Commercial blood-derived genomic DNA samples from healthy individuals were used as references for data normalization using data analysis software Coffalyser digitalMLPA™. Results. Losses of 1p were among the most frequent CNAs in MM cell lines (n=13) and were mainly local (1p21.1-p22.1), including homozygous deletion of CDKN2C in MM cell lines co-occurring with TENT5C subclonal loss in 2 of those. 13q loss (encompassing predominantly the whole arm) was detected in 13 MM cell lines. 1q and 17p losses were present in 12 MM cell lines. One MM cell line showed loss of BCMA (LOPRA-1) and one of CRBN (EJM), 6 had loss of GPRC5D, 9 had MYC gain. None of the MM cell lines carried the BRAF V600E mutation. In the EOL-1 myeloid lineage cell line, trisomy of chromosomes 4, 6, 8 and 19 were correctly identified by increased ratios for all probes covering these chromosomes: 24, 27, 20 and 11 probes respectively; and in the HG-3 lymphoid lineage cell line 11q25 gain and 13q loss was detected. Examples of CNAs detected by D006 Multiple Myeloma probemix are described in Table 1. Copy number ratios of predictive target genes in MM cell lines are shown in Table 2. Ratios between 0.7-1.3 (green cells) indicate normal copy number, ratios below 0.7-1.3 range indicate copy number loss (red cells) and higher ratios indicate gains (blue cells). CNAs detected by digitalMLPA were highly concordant with those reported in public databases. Conclusions. digitalMLPA technology is well suited for multiplex CNA detection of routinely analyzed genomic regions in MM and provides a unique opportunity to research molecular genetic markers of emerging significance in MM in the same reaction. In addition, this MM assay also gives the possibility to analyze gross CNAs in other hematopoietic neoplasms with overlapping affected regions. Input DNA requirements that are a fraction of current WGS, simple protocol and possibility of combining of digitalMLPA libraries with other NGS libraries make D006 Multiple Myeloma digitalMLPA probemix a reliable, cost-effective and robust method to detect well-established and emerging CNAs.

PDF file 48K, Description of the methodology for: -Lung tumor specimens and cancer cell lines. -Screening for MAX gene alterations: Sanger direct sequencing and Multiplex ligation dependent probe amplification (MLPA). -Expression vectors and lentiviral production. -Chromatin immunoprecipitation (ChIP) assays. -Antibodies and western blots. -Treatments and MTT assays. -Microarray global gene expression analysis. -Statistical and bioinformatic analysis

Supplementary Figure Legends

Supplementary Table 3. The twenty-eight HDAC inhibitors used in the study and their specificity

Supplementary Data from Genomic Profiling of Chondrosarcoma: Chromosomal Patterns in Central and Peripheral Tumors