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PubMed2026

Sequencing of Fibroblast Growth Factor Receptor Inhibitors in Cholangiocarcinoma: A Review of Published Cases.

Cholangiocarcinomas (CCAs) are aggressive biliary tumors that can develop within the intrahepatic (iCCA) or perihilar and distal bile ducts. The prognosis of patients with iCCA is poor due to its relative resistance to chemotherapy. Comprehensive genomic profiling of CCA biopsies by next-generation sequencing has revealed a rich landscape of genomic alterations, including fibroblast growth factor receptor (FGFR) gene fusions and rearrangements that are constitutively active and oncogenic. Several FGFR inhibitors (FGFRis) targeting these FGFR genomic alterations have been developed as potential treatments for iCCA, each of which are highly potent but differ in structure, mechanism of inhibition (ie, adenosine triphosphate-competitive reversible v covalent/irreversible v allosteric), pharmacologic/pharmacodynamic profiles, and selectivity for the four FGFR isoforms. Because of these differences, and due to resistance mutations acquired during FGFRi treatment, determining the optimal sequencing of FGFRis for the treatment of CCA remains contentious and is the subject of ongoing debate. To address this question, this review conducted an analysis of the literature on the FGFRi currently approved or in development, focusing on their distinct mechanisms of action and FGFR selectivity. Publicly available data from case reports on FGFRi sequencing in second and later lines of treatment were compiled from a PubMed search of published congress abstracts and articles. The results support a hypothesis that strategic sequencing of reversible followed by irreversible FGFRi may potentially prolong the duration of treatment benefit from FGFR inhibition compared with nonsequenced treatments. A hypothetical treatment-sequencing algorithm for reversible and irreversible FGFRi is discussed.

Humans↗
PubMed2026

Characterisation of Bordetella pertussis virulence and macrolide resistance in Australia by targeted culture-independent sequencing: a genomic epidemiology study.

BACKGROUND: Bordetella pertussis continues to circulate globally despite widespread vaccination, with a notable epidemic in 2024. Its resurgence is confounded by the emergence of pertactin-deficient, macrolide-resistant B pertussis strains in Asia and Europe, which are under-recognised by conventional diagnostics. We aimed to apply targeted culture-independent next-generation sequencing (tNGS) of respiratory specimens to improve global B pertussis diagnostic capability and genomic surveillance. METHODS: We did a nationwide genomic epidemiology study of B pertussis RT-PCR-positive respiratory specimens that were retrospectively and prospectively collected by diagnostic and public health laboratories in six of seven states and territories of Australia. Specimens underwent tNGS and macrolide-resistant B pertussis-specific PCR, and an opportunistic subset from New South Wales and Queensland were cultured for confirmatory susceptibility testing and whole-genome sequencing. Sequencing data were analysed for genome recovery, virulence profiles, and macrolide resistance mutations, and were compared with international macrolide-resistant B pertussis genomes and ancestral Australian genomes. The performance of the tNGS approach was assessed with logistic regression relative to RT-PCR cycle threshold values, and sensitivity and specificity values were calculated. FINDINGS: 255 respiratory specimens positive for B pertussis were included in the study. 64 (25%) were retrospectively collected between Jan 12, 2012, and Dec 31, 2023, and 191 (75%) were prospectively collected between Jan 1 and Oct 28, 2024. Of these 255 specimens, 148 (58%) yielded near-complete B pertussis genomes through tNGS. Seven co-circulating lineages of B pertussis were documented, including two associated with macrolide-resistance. Eight epidemiologically unrelated and geographically dispersed cases of macrolide-resistant B pertussis with a 23S rRNA 2037A→G mutation were identified by tNGS and confirmed by whole-genome sequencing. Three of these were further validated by phenotypic testing. The estimated prevalence of macrolide resistance among Australian cases positive for B pertussis was 4% (eight of 188). INTERPRETATION: tNGS can recover near-complete B pertussis genomes directly from clinical specimens, enabling identification of macrolide resistance mutations and high-resolution phylogenetic analysis. These findings show that tNGS complements PCR-based surveillance by providing genome-wide assessment of resistance, virulence, and genomic diversity in a single workflow. FUNDING: NSW Health Prevention Research Support Program.

Macrolides↗
PubMed2026

Expanding the TBL1XR1 Disease Spectrum: Generalized Dystonia Associated with a New Genetic Variant.

BACKGROUND: A growing number of identified genes increasingly reveal genetic overlaps between neurodevelopmental disorders and combined dystonia syndromes. CASE REPORT: We report a 61-year-old man with a neurodevelopmental disorder, mild ataxic signs and generalized dystonia who had been misdiagnosed with cerebral palsy for 40 years. Whole-exome sequencing identified a novel heterozygous pathogenic frameshift variant in TBL1XR1. DISCUSSION: TBL1XR1 variants are classically associated with Pierpont syndrome and autism spectrum disorder. Although movement disorders have been reported, this case suggests generalized dystonia as a possible additional manifestation. It highlights the value of retrospective genetic phenotyping and next-generation sequencing in adults with long-standing neurodevelopmental diagnoses.

Humans↗
PubMed2026

DirectASRM: uncovering allele-specific post-transcriptional RNA modifications through direct RNA sequencing.

SUMMARY: We developed DirectASRM, a comprehensive database for the systematic identification, integration, and annotation of allele-specific RNA modifications (ASRMs) from direct RNA sequencing data. DirectASRM enables single-base, transcript-level detection of ASRMs across multiple RNA modification types, diverse organisms and condition-specific contexts. The database further evaluates the confidence of each ASRM-SNP pair association within isoform context by jointly considering statistical evidence of allelic modification imbalance and independent support from external next-generation sequencing (NGS) - based RNA modification resources. DirectASRM also provides extensive functional annotations for ASRMs and their associated variants, including intra-sample transcript-level allele-specific expression (ASE) and allele-specific splicing, as well as additional post-transcriptional regulatory features such as miRNA binding, circRNA, RNA-protein interactions, and disease relevance. Overall, DirectASRM serves as a comprehensive resource that supports systematic investigation of the potential functional impact of genetic variants in epitranscriptomic regulation. AVAILABILITY AND IMPLEMENTATION: DirectASRM database is freely accessible at http://modinfor.com/DirectASRM/. DirectASRM pipeline is available at GitHub (https://github.com/jiayin1101/DirectASRM_pipeline) and Zenodo (DOI: https://doi.org/10.5281/zenodo.19876077).

Alleles↗
PubMed2026

pSTRminer: integrated bioinformatic software for genome-wide identification and population-scale evaluation of polymorphic short tandem repeats.

Animal forensic genetics plays a critical role in criminal investigations by providing crucial evidence through domestic animal individualization and wildlife species identification. While human forensic genetics benefits from standardized short tandem repeats (STR) genotyping systems, animal forensic applications encounter significant challenges, including the limited availability of validated STR markers, the prevalence of error-prone dinucleotide STRs (di-STRs), and insufficient integration of population data. To address these challenges, we developed pSTRminer, an integrated bioinformatic tool that automates genome-wide STR mining and polymorphism evaluation. By applying pSTRminer to domestic cattle (Bos taurus), we identified 775,444 STRs de novo from the reference genome and genotyped them using whole-genome sequencing data from 60 Chinese and 111 African cattle to evaluate polymorphism across diverse genetic backgrounds. This led to the development of the cattle STR database (CSDB), comprising loci with a genotyping success rate&#x2009;&#x2265;&#x2009;40% and polymorphism information content (PIC)&#x2009;&#x2265;&#x2009;0.5. Experimental validation of 30 randomly selected tetranucleotide STRs (tetra-STRs) and 33 di-STRs via next-generation sequencing in a local Chinese cattle population (n&#x2009;=&#x2009;145) confirmed marker reliability. Although tetra-STRs had lower average polymorphism levels, they exhibited significantly lower stutter ratios (p&#x2009;<&#x2009;0.05), providing a viable path for identifying discriminative markers with fewer artifacts. Systematic screening revealed that certain tetra-STRs could surpass di-STRs in polymorphism. In conclusion, pSTRminer provides a scalable framework for developing standardized STR panels, facilitating the identification of robust and informative markers in forensic applications.

Bioinformatic software↗
PubMed2024

Phage Immunoprecipitation and Sequencing-a Versatile Technique for Mapping the Antibody Reactome.

Characterizing the antibody reactome for circulating antibodies provide insight into pathogen exposure, allergies, and autoimmune diseases. This is important for biomarker discovery, clinical diagnosis, and prognosis of disease progression, as well as population-level insights into the immune system. The emerging technology phage display immunoprecipitation and sequencing (PhIP-seq) is a high-throughput method for identifying antigens/epitopes of the antibody reactome. In PhIP-seq, libraries with sequences of defined lengths and overlapping segments are bioinformatically designed using naturally occurring proteins and cloned into phage genomes to be displayed on the surface. These libraries are used in immunoprecipitation experiments of circulating antibodies. This can be done with parallel samples from multiple sources, and the DNA inserts from the bound phages are barcoded and subjected to next-generation sequencing for hit determination. PhIP-seq is a powerful technique for characterizing the antibody reactome that has undergone rapid advances in recent years. In this review, we comprehensively describe the history of PhIP-seq and discuss recent advances in library design and applications.

Humans↗
PubMed2026

VisPan: real-time visualisation of multiplex amplicon-based sequencing panels for rapid syndromic surveillance and pathogen detection.

MOTIVATION: Infectious diseases persist as a major global public health challenge. Diverse factors, including climate change, globalization, deforestation, human-animal interactions, lifestyle choices, and various biological factors, can contribute to their emergence and reemergence. Rapid detection and characterization of (re)emerging pathogens are therefore critical for effective outbreak management and for enhancing our understanding of epidemics by monitoring the transmission, spread, evolution, and genomics of pathogens. In this context, next-generation sequencing technologies (NGS), particularly long-read platforms such as Oxford Nanopore Technologies (ONT), have opened new avenues for real-time pathogen monitoring. However, the bioinformatics bottleneck remains a challenge, emphasizing the need for efficient, accessible, and user-friendly analysis tools. RESULTS: Here, we present a tool adapted from the RAMPART software that enables real-time data visualisation of multiplex PCR syndromic panels combined with Oxford Nanopore sequencing. This real-time analysis enables rapid pathogen detection, from raw data acquisition to taxonomic assignment, within minutes. The interface offers dynamic visual tracking of the sequencing run and amplicon coverage, facilitating immediate insights during diagnostic workflows. Validation experiments confirmed the system's reliability, accurately identifying all pathogens present in complex clinical or environmental samples. This tool provides an integrated, user-friendly solution for genomic pathogen surveillance in field or clinical settings.

Software↗
PubMed2025

Doblin: inferring dominant clonal lineages from high-resolution DNA barcoding time series.

MOTIVATION: The lineage dynamics and history of cells in a population reflect the interplay of evolutionary forces they experience, including mutation, drift, and selection. When the population is polyclonal, lineage dynamics also manifest the extent of clonal competition among co-existing mutational variants. If the population exists in a community of other species, the lineage dynamics could also reflect the population's ecological interaction with the rest of the community. Recent advances in high-resolution lineage tracking via DNA barcoding, coupled with next-generation sequencing of bacteria, yeast, and mammalian cells, allow for precise quantification of clonal dynamics in these organisms. RESULTS: In this work, we introduce Doblin, an R suite for identifying dominant barcode lineages based on high-resolution lineage tracking data. We first benchmarked Doblin's accuracy using lineage data from evolutionary simulations, showing that it recovers the clones' identity and relative fitness in the simulation. Next, we applied Doblin to analyze clonal dynamics in laboratory evolutions of Escherichia coli populations undergoing antibiotic treatment and in colonization experiments of the gut microbial community. Doblin's versatility allows it to be applied to lineage time-series data across different experimental setups. AVAILABILITY AND IMPLEMENTATION: Doblin is available on CRAN (https://CRAN.R-project.org/package=doblin) and Github (https://github.com/dagagf/doblin).

DNA Barcoding, Taxonomic↗
PubMed2026

Population-level genomic surveillance of human norovirus using wastewater-based whole-genome sequencing.

Wastewater-based surveillance has garnered increasing attention as a valuable approach for capturing community-level infection dynamics that are often difficult to detect through clinical reporting systems alone. In this study, we analyzed human norovirus genotype distributions and whole-genome-level variations in wastewater samples collected in Gwangju, Korea. These results were interpreted in conjunction with a documented foodborne outbreak to evaluate the epidemiological relevance of wastewater-based monitoring. Human norovirus concentrations were quantified using TaqMan Array Card-based RT-qPCR, and whole-genome next-generation sequencing (NGS) was performed to obtain viral read counts and reads per kilobase per million filtered reads values. Overall, strong correlations were observed between RT-qPCR-based concentrations and NGS-derived metrics. Genotype dynamics varied among wastewater treatment plants, reflecting differences in catchment size and local population characteristics. In particular, the relative abundance of GII.17[P17] increased during epidemiological week 50, temporally coinciding with a documented local foodborne outbreak. Variant analysis revealed that wastewater samples exhibited mixed nucleotide patterns, with multiple alleles coexisting at varying relative frequencies rather than fixed substitutions. Notably, some nonsynonymous variants detected in clinical samples were also observed in wastewater samples collected surrounding the outbreak period. Together, these findings demonstrate that wastewater-based whole-genome surveillance can capture both genotype-level shifts and nucleotide-level dynamics at the population scale, highlighting its potential as a complementary tool for monitoring community-level norovirus circulation and outbreak-associated genotype dynamics.IMPORTANCEWastewater-based surveillance is increasingly recognized as a promising approach for capturing community-level infection dynamics that are often missed by clinical surveillance. In this study, we applied whole-genome sequencing to wastewater samples collected in Gwangju, South Korea, to comprehensively characterize human norovirus genotype distributions and genetic variation. Distinct genotype patterns were observed across wastewater treatment plants, reflecting differences in catchment population size and local characteristics. Notably, an increase in the GII.17[P17] genotype detected in wastewater coincided with a foodborne outbreak investigated in Gwangju, demonstrating the potential of wastewater surveillance to reflect ongoing community transmission and emerging outbreak-associated genotypes. In addition, wastewater samples contained diverse and coexisting genetic variants, capturing population-level viral diversity and evolutionary dynamics that are not readily detected through clinical surveillance alone. These findings highlight the value of wastewater-based whole-genome surveillance for monitoring community-level viral circulation and support its integration as a complementary strategy to existing clinical surveillance systems.

genotype dynamics↗
PubMed2025

Methylation-based droplet digital polymerase chain reaction shows high concordance with chronic lymphocytic leukemia IGHV somatic mutation status.

OBJECTIVE: Somatic hypermutation at immunoglobulin heavy chain variable (IGHV) genes, an established prognostic and predictive biomarker for chronic lymphocytic leukemia (CLL), is assessed by gene sequencing. We developed a single methylation-specific droplet digital polymerase chain reaction (methyl-ddPCR) to predict IGHV status in patients with CLL. METHODS: The CLL methylation array and IGHV data from the International Cancer Genome Consortium (ICGC) were used for biomarker discovery. Top-ranked candidate regions were manually screened for PCR primer and probe binding sites. A single methyl-ddPCR was evaluated on an internal cohort of CLLs with mutated (M), unmutated (U), and inconclusive IGHV results originally determined by next-generation sequencing (NGS). RESULTS: Analysis of ICGC data identified array probe cg23844018 as a candidate for the PCR. The corresponding CpG site showed high methylation levels in U-CLL and lower levels in M-CLL. On the internal cohort, a single optimal cutoff correctly classified 104 of 115 U- and M-CLLs (90.4%; area under the curve&#x2005;=&#x2005;0.96). The PCR data correlated with some prognostic fluorescence in situ hybridization and CLL subset groupings. Limited analysis suggests that the PCR may be able to stratify some patients with CLL who have inconclusive results on IGHV NGS testing. CONCLUSIONS: The methyl-ddPCR showed high concordance with CLL IGHV status in an internal cohort.

Humans↗
PubMed2025

Allele Level Sequencing of Killer Cell Immunoglobulin-Like Receptor Genes Using Oxford Nanopore Long Read Sequencing.

The human Killer cell Immunoglobulin-like Receptor (KIR) genes, found on chromosome 19, encode for cell surface protein receptors that, through interaction with their ligand, modulate the action of Natural Killer (NK) cells and some subsets of T lymphocytes. KIR genes exhibit extensive variation through variable gene content, copy number, and allele polymorphism. The combination of KIR genes and their ligands is implicated in various clinical settings including haematopoietic stem cell and solid organ transplant, and infectious disease progression. KIR gene content has been used in the selection of optimal stem cell donors with haplotype variations in recipient and donor giving differential clinical outcomes. With the introduction of massively parallel clonal next generation sequencing and single molecule long read third generation sequencing, allele level determination of KIR genotypes has become feasible. We describe a method for amplicon-based long read sequencing on the Oxford Nanopore Technologies platform that provides largely unambiguous allele level typing of KIR genes. The method was validated using DNA extracted from 48 10th International Histocompatibility Workshop (IHWS) cell lines with previously published allele level KIR genotypes and 176 Western Australian samples previously tested for the presence or absence of KIR genes. Our long-read sequencing method was able to accurately determine KIR alleles with an overall concordance of 97%-99% with the published data. Importantly, phasing ambiguity caused by the inability to phase heterozygous base positions over long stretches of gene sequence was resolved in several samples. Thus, our long read PCR sequencing strategy can be used to determine KIR genotypes at allele resolution level.

Humans↗
PubMed2026

Protocol for Duplex Sequencing of Mitochondrial DNA in Single Human Oocytes.

Oocytes are densely packed with mitochondria, the energy-producing organelles that contain their own genome, mitochondrial DNA (mtDNA). Each cell contains multiple copies of mtDNA, with copy number varying among tissue types. Oocytes possess the highest mtDNA copy number, containing hundreds of thousands of mtDNA molecules per cell. Because mitochondria are inherited exclusively through the maternal lineage, accurate detection of mtDNA variants is essential for studies of inheritance, aging, and disease. The presence of multiple mtDNA copies allows wild-type and mutant molecules to coexist within the same cell, a condition known as heteroplasmy, in which low-frequency and de novo variants may occur at frequencies below 1%. Conventional next-generation sequencing (NGS) lacks sufficient accuracy to reliably distinguish these rare variants from errors introduced during library preparation and sequencing. Here, we present a protocol for enriching mtDNA from single human oocytes using Exonuclease V to remove linear DNA, followed by duplex sequencing library preparation for highly accurate mtDNA analysis. This workflow enables error-corrected sequencing of individual oocytes, facilitating reliable detection of low-frequency mtDNA variants and analysis of heteroplasmy and de novo mutagenesis. The protocol provides a reproducible approach for investigating mitochondrial genome variation in single oocytes using Illumina-compatible sequencing platforms.

Humans↗
PubMed2025

TRIM63 Overexpression in FISH-Negative MiTF Family Altered Renal Cell Carcinoma (MiTF RCC).

TFE3 and TFEB break-apart fluorescent in situ hybridization (FISH) assays are the "gold standard" for diagnostic confirmation of microphthalmia-associated transcription factor (MiTF) family-altered renal cell carcinoma (MiTF RCC), which includes TFE3-rearranged RCC and TFEB-altered RCC. However, FISH assays, for multiple reasons, may lead to equivocal or false-negative results, especially in cryptic fusions resulting from intrachromosomal inversions involving 5' partner genes, such as non-POU domain-containing octamer-binding protein (NONO); GRIPI-associated protein 1 (GRIPAP1); RNA-binding motif protein, X chromosome (RBMX); and RNA-binding motif protein 10 (RBM10). When FISH results are negative in cases with strong morphological suspicion of the listed tumor entities, pathologists may recommend targeted RT-PCR or panel-based RNA fusion sequencing for diagnostic confirmation. Our recent RNA in situ hybridization (RNA ISH)-based study demonstrated RNA expression of the tripartite motif containing 63 (TRIM63) to be highly enriched in TFE3-rearranged RCC and TFEB-altered RCC, including 2 FISH false-negative RCC cases harboring RBM10::TFE3 fusion. Based on these observations, we hypothesized that TRIM63 positivity could aid in diagnosing cases that are negative by conventional FISH assay but remain morphologically suspicious, representing an unmet clinical need in this area. We collected 20 RCC cases with morphological suspicion (with equivocal/indeterminate immunohistochemistry panel) of MiTF RCC, which were TRIM63 positive, negative/equivocal for TFE3/TFEB gene rearrangement by FISH, and underwent next-generation sequencing (NGS). On NGS correlation, 14 of 20 (70%) FISH-negative TRIM63-positive tumors harbored an MiTF gene rearrangement. In the remaining 6 cases, we were unable to fully ascertain the MiTF rearrangement status due to the inherent limitation of the NGS panel utilized. The cases with MiTF gene rearrangement include TFE3 rearrangement in 60% (12/20) and TFEB low-level copy gains (with an additional missense mutation in 1 case) in 10% (2/20) of samples. RBM10:TFE3 fusion was seen in 67% (8/12) of TFE3-rearranged RCC in this cohort. TRIM63 RNA ISH assay could aid in identifying cases that harbor TFE3 or TFEB rearrangement associated with false-negative or equivocal TFE3/TFEB FISH results, especially those involving gene fusions with a paracentric Xp11 inversion. Overall, employment of TRIM63 RNA ISH coupled with TFE3/TFEB FISH assays and follow-up genomic interrogation enhanced diagnostic accuracy for patients with MiTF RCC.

Carcinoma, Renal Cell↗
PubMed2025

Complexity of schistosome vector bulinine snails in Kenya: Insights from nuclear genome size variation, complete mitochondrial genome sequence, and morphometric analysis.

Investigations of nuclear genome size, complete mitochondrial genome (mitogenome) sequence, and morphometrics were conducted on specimens of Bulinus snails (Gastropoda: Planorbidae) collected from 14 locations across the east coast, central Kenya, and western Kenya around the Lake Victoria region (November 2013 and January 2024). Flow cytometry measurements of DNA content (C-value) revealed unexpected variation in nuclear genome size, with diploid Bulinus africanus and B. forskalii species groups showing C-values ranging from 0.76 to 1.98 pg, while tetraploid B. truncatus had a C-value of 1.82 pg. Additionally, C-values for six B. globosus specimens from different localities ranged from 1.43 to 1.98 pg. These findings suggest that bulinine snails, particularly the B. africanus species group, have undergone genome expansion, whole genome duplication (polyploidization), or both, which have not been previously recognized. Next-generation sequencing was performed to determine and annotate 14 complete mitogenome sequences. Despite the well-conserved arrangement of protein-coding genes, two versions of mtDNA genome structure, distinguished by the tRNA-D (Asp) location, were found, designated as DCF (Asp-Cys-Phe) type (in the B. forskalii group and the B. truncatus/tropicus complex) and CF (Cys-Phe) type (in the B. africanus group). Phylogenetic analyses based on complete mtDNA sequences of bulinines from Kenya, along with cytochrome c oxidase subunit I (COX1) sequences from various localities across Africa, contributed to resolving species identities and provided further support for the presence of multiple or cryptic species in the taxon B. globosus. A landmark-based morphometric analysis was ineffective in distinguishing these species. This study reveals unexpected nuclear genome size variation, provides new mitogenome sequences, and highlights the limitations of morphological analysis. It offers valuable insights into the cytogenetics, polyploidy, genomics, taxonomy, and evolution of bulinines, which serve as intermediate hosts for schistosomes responsible for human urogenital schistosomiasis and intestinal schistosomiasis in domestic and wild mammals.

Animals↗
PubMed2026

Yes-Associated Protein (YAP)1 and &#x3b2;-Catenin Immunohistochemistry as a Surrogate Marker for GTF2I-Mutant Type A/AB Thymomas.

Thymomas are rare thymic epithelial tumors classified by the World Health Organization into type A/AB thymomas, which commonly harbor GTF2I mutations and behave indolently, and type B thymomas and thymic carcinomas, in which these mutations are less common. Type A and AB thymomas are uniquely enriched for a recurrent somatic hotspot mutation in GTF2I p. L424H; yet, this gene is rarely included in clinical sequencing panels, limiting its diagnostic utility. Yes-associated protein (YAP)1, the principal effector of the Hippo signaling pathway, and &#x3b2;-catenin, the central transcriptional effector of the Wnt pathway, have emerging roles in thymoma biology; however, their relationship to GTF2I mutation status and histologic subtype has not been systematically characterized. We analyzed The Cancer Genome Atlas thymoma data set and an institutional cohort of 38 thymic epithelial tumors to evaluate YAP1 and &#x3b2;-catenin immunohistochemistry (IHC) as surrogate markers for GTF2I mutation status and histologic classification. In The Cancer Genome Atlas data set, YAP1 and CTNNB1 mRNA expression were markedly elevated in type A/AB thymomas relative to type B and carcinoma subtypes, and GTF2I-mutant tumors exhibited significantly higher YAP1 mRNA expression than GTF2I-wildtype tumors. Targeted next-generation sequencing of our institutional cohort confirmed enrichment of the canonical GTF2I p. L424H hotspot in indolent subtypes. By IHC, both nuclear YAP1 positivity and cytoplasmic &#x3b2;-catenin localization were significantly more frequent in indolent thymomas. Cytoplasmic &#x3b2;-catenin demonstrated high specificity (94%) for indolent histology, supporting its use in diagnostically challenging cases such as type A versus type B3 distinction on small biopsies. YAP1 IHC showed a high negative predictive value for GTF2I mutations, such that a YAP1-negative result reliably excludes a GTF2I-mutant tumor. These findings implicate crosstalk between Hippo and Wnt signaling in GTF2I-mutant thymomas and position YAP1 and &#x3b2;-catenin IHC as accessible, cost-effective surrogates for molecular subtyping in a tumor where standard sequencing panels have limited coverage.

GTF2I↗
PubMed2025

Genetic Newborn Screening for Retinoblastoma: A Belgian Initiative Baby Detect.

Baby Detect Project, started in September 2022, aimed to create a newborn screening test using targeted next-generation sequencing for all early-onset, treatable, and serious conditions. The elaborated gene panel covers 405 genes, associated with 165 genetic conditions, and includes RB1, linked to retinoblastoma, the only oncological disease tested for. Germline RB1 mutations concern around 50% of all retinoblastoma cases and 100% of the most severe, bilateral cases. Ninety percent of them occur de novo, which delays the diagnosis by about a year with subsequent loss of vision and sometimes the eye itself. Detecting children with germline RB1 mutation at birth would greatly improve functional and anatomic outcomes, limiting invasive treatments and general anesthesias through early childhood. We discuss herein the novel approach of population screening, the rationale for newborn testing for RB1 mutations, the incidence of expected cases, the reliability of the test and its costs. The next step is to move to a nation-scale population; this initiative marks a landmark in retinoblastoma patients' care.

Humans↗
PubMed2025

Biallelic antigen escape is a mechanism of resistance to anti-CD38 antibodies in multiple myeloma.

Monoclonal antibodies targeting CD38 are a therapeutic mainstay in multiple myeloma (MM). Although they have contributed to improved outcomes, most patients still experience disease relapse, and little is known about tumor-intrinsic mechanisms of resistance to these drugs. Antigen escape has been implicated as a mechanism of tumor-cell evasion in immunotherapy. Yet, it is unknown whether MM cells can develop permanent resistance to anti-CD38 antibodies by acquiring genomic events leading to biallelic disruption of the CD38 gene locus. Here, we analyzed whole-genome and whole-exome sequencing data from patients 701 newly diagnosed MM, 67 patients at relapse with naivety to anti-CD38 antibodies, and 50 patients collected at relapse after anti-CD38 antibodies. We report a loss of CD38 in 10 of 50 patients (20%) after CD38 therapy, 3 of whom exhibited a loss of both copies. Two of these cases showed convergent evolution in which distinct subclones independently acquired similar advantageous variants. Functional studies on missense mutations involved in biallelic CD38 events revealed that 2 variants, L153H and C275Y, decreased binding affinity and antibody-dependent cellular cytotoxicity of the commercial antibodies daratumumab and isatuximab. However, a third mutation, R140G, conferred selective resistance to daratumumab, while retaining sensitivity to isatuximab. Clinically, patients with MM are often rechallenged with CD38 antibodies after disease progression and these data suggest that next-generation sequencing may play a role in subsequent treatment selection for a subset of patients.

Humans↗