Fusion Sequence Research Articles

Measurement of spherical tokamak plasma compression in the PCS-16 magnetized target fusion experiment

Abstract A sequence of magnetized target fusion (MTF) devices built by General Fusion have compressed magnetically confined deuterium plasmas inside imploding aluminum liners. Here we describe the best-performing compression experiment, PCS-16, which was the fifth of the most recent experiments that compressed a spherical tokamak plasma configuration. In PCS-16 the plasma remained axisymmetric with δBpol/Bpol < 20% to a high radial compression factor (CR > 8) with significant poloidal flux conservation (77% up to CR = 1.7, and ≈30% up to CR = 8.65) and a total compression time of 167 μs. Magnetic energy of the plasma increased from 0.96 kJ poloidal and 17 kJ toroidal to a peak of 1.14 kJ poloidal and 29.9 kJ toroidal during the compression, while thermal energy was in the range of 350 ± 25 J. Plasma equilibrium was a low-β state with βtor ≈ 4% and βpol ≈ 15%. Ingress of impurities from the lithium coated aluminum wall was not a dominant effect. Neutron yield from D-D fusion increased significantly during compression. Thermodynamics during the early phase of compression (CR < 1.7) were consistent with increasing Ohmic heating of the electrons due to a geometric rise in the current density at near-constant resistivity, and with increasing ion cooling that approximately matched ion compressional heating power. Ion cooling by electrons was significant because the electrons were cooler than the ions (Te = 200 eV, Ti = 600 eV). Magnetohydrodynamic simulation was used to model the emergence of instabilities that increased electron thermal transport in the final phase of compression. Conditions for ideal stability were actively maintained during compression through a current ramp applied to the central shaft, and after this current ramp reached its peak two-thirds of the way through compression we measured a transition in plasma behavior across multiple diagnostics.

UUD-Fusion: An unsupervised universal image fusion approach via generative diffusion model

Image fusion is a classical problem in the field of image processing whose solutions are usually not unique. The common image fusion methods can only generate a fixed fusion result based on the source image pairs. They tend to be applicable only to a specific task and have high computational costs. Hence, in this paper, a two-stage unsupervised universal image fusion with generative diffusion model is proposed, termed as UUD-Fusion. For the first stage, a strategy based on the initial fusion results is devised to offload the computational effort. For the second stage, two novel sampling algorithms based on generative diffusion model are designed. The fusion sequence generation algorithm (FSGA) searches for a series of solutions in the solution space by iterative sampling. The fusion image enhancement algorithm (FIEA) greatly improves the quality of the fused images. Qualitative and quantitative evaluations of multiple datasets with different modalities demonstrate the great versatility and effectiveness of UUD-Fusion. It is capable of solving different fusion problems, including multi-focus image fusion task, multi-exposure image fusion task, infrared and visible fusion task, and medical image fusion task. The proposed approach is superior to current state-of-the-art methods. Our code is publicly available at https://github.com/xiangxiang-wang/UUD-Fusion.

Convergent evolution links molybdenum insertase domains with organism-specific sequences

In all domains of life, the biosynthesis of the pterin-based Molybdenum cofactor (Moco) is crucial. Molybdenum (Mo) becomes biologically active by integrating into a unique pyranopterin scaffold, forming Moco. The final two steps of Moco biosynthesis are catalyzed by the two-domain enzyme Mo insertase, linked by gene fusion in higher organisms. Despite well-understood Moco biosynthesis, the evolutionary significance of Mo insertase fusion remains unclear. Here, we present findings from Neurospora crassa that shed light on the critical role of Mo insertase fusion in eukaryotes. Substituting the linkage region with sequences from other species resulted in Moco deficiency, and separate expression of domains, as seen in lower organisms, failed to rescue deficient strains. Stepwise truncation and structural modeling revealed a crucial 20-amino acid sequence within the linkage region essential for fungal growth. Our findings highlight the evolutionary importance of gene fusion and specific sequence composition in eukaryotic Mo insertases.

Angiomyolipomatous Lesions of the Nasal Cavity (Sinonasal Angioleiomyoma with Adipocytic Differentiation): A Multi-Institutional Immunohistochemical and Molecular Study.

Mesenchymal neoplasms composed of vascular, smooth muscle, and adipocytic components are uncommon in the nasal cavity. While angioleiomyoma (AL) is a smooth muscle tumor in the Head & Neck WHO classification, it is considered of pericytic origin in the Skin as well as Soft Tissue and Bone classifications. For nasal AL with an adipocytic component, the terms AL with adipocytic differentiation and angiomyolipoma (AML) have been applied, among others. AML is a type of perivascular epithelioid cell tumor (PEComa), most often arising in the kidney, sometimes associated with the tuberous sclerosis complex (TSC). It is uncertain whether nasal cavity AML and AL are best considered hamartomas or neoplasms, as their genetics are largely unexplored. We performed a multi-institutional retrospective study of nasal cavity mesenchymal lesions. Patient demographics, clinical histories, and histologic and immunohistochemical findings were collected. DNA and RNA were extracted from formalin-fixed, paraffin-embedded tissue and analyzed by SNP-based chromosomal microarray, targeted RNA fusion sequencing, and whole-exome sequencing. Fifteen lesions (3-42mm) were identified, predominantly in male (87%) patients with a median age of 60. Patients typically presented with obstructive symptoms, and none had a history of TSC. One AL was a recurrence from six years prior; 11 cases showed no recurrence (median 4.7 years, range: 0.88-12.4). Morphologically, 11 AML contained 30-80% smooth muscle, 10-25% vasculature, and 2-60% adipose tissue, while four AL contained 70-80% smooth muscle and 20-30% vasculature. Other histologic observations included ulceration, thrombosis, inflammation, myxoid change, senescent nuclei, and extramedullary hematopoiesis; no well-developed epithelioid cell morphology was identified. Immunohistochemically, all cases were positive for smooth muscle markers (actin, desmin, and/or caldesmon) and negative for melanocytic markers. Molecular analysis revealed loss of 3p and 11q in a single AML. No other known pathogenic copy number or molecular alterations were seen, including in TSC1/2, TFE3, or NOTCH2. Nasal cavity AML lacks morphologic, immunophenotypic, and genetic features of PEComa family AML. The significant histologic overlap between nasal AML and AL without distinguishing molecular features in either entity suggests "sinonasal angioleiomyoma with adipocytic differentiation" may be the most appropriate terminology for hybrid vascular and smooth muscle lesions containing adipocytic components.

Angiomyolipomatous Lesions of the Nasal Cavity (Sinonasal Angioleiomyoma with Adipocytic Differentiation): A Multi-Institutional Immunohistochemical and Molecular Study.

Mesenchymal neoplasms composed of vascular, smooth muscle, and adipocytic components are uncommon in the nasal cavity. While angioleiomyoma (AL) is a smooth muscle tumor in the Head & Neck WHO classification, it is considered of pericytic origin in the Skin as well as Soft Tissue and Bone classifications. For nasal AL with an adipocytic component, the terms AL with adipocytic differentiation and angiomyolipoma (AML) have been applied, among others. AML is a type of perivascular epithelioid cell tumor (PEComa), most often arising in the kidney, sometimes associated with the tuberous sclerosis complex (TSC). It is uncertain whether nasal cavity AML and AL are best considered hamartomas or neoplasms, as their genetics are largely unexplored. We performed a multi-institutional retrospective study of nasal cavity mesenchymal lesions. Patient demographics, clinical histories, and histologic and immunohistochemical findings were collected. DNA and RNA were extracted from formalin-fixed, paraffin-embedded tissue and analyzed by SNP-based chromosomal microarray, targeted RNA fusion sequencing, and whole-exome sequencing. Fifteen lesions (3 to 42 mm) were identified predominantly in male (87%) patients with a median age of 60. Patients typically presented with obstructive symptoms, and none had a history of TSC. One AL was a recurrence from six years prior; 11 cases showed no recurrence (median 4.7 years, range: 0.88-12.4). Morphologically, 11 AMLs contained 30-80% smooth muscle, 10-25% vasculature, and 2-60% adipose tissue, while four ALs contained 70-80% smooth muscle and 20-30% vasculature. Other histologic observations included surface ulceration, vascular thrombosis, chronic inflammation, and myxoid change; no well-developed epithelioid cell morphology was identified. Immunohistochemically, all cases were positive for smooth muscle markers (actin and/or desmin) and negative for melanocytic markers. Molecular analysis revealed loss of 3p and 11q in a single AML. No other known pathogenic copy number or molecular alterations were seen, including in TSC1/2, TFE3, or NOTCH2. Nasal cavity AML lacks morphologic, immunophenotypic, and genetic features of PEComa family AMLs. The significant histologic overlap between nasal AML and AL without distinguishing molecular features in either entity suggests "sinonasal angioleiomyoma with adipocytic differentiation" may be the most appropriate terminology for hybrid vascular and smooth muscle lesions containing adipocytic components.

Postnatal Skull Development Reveals a Conservative Pattern in Living and Fossil Vizcachas Genus Lagostomus (Rodentia, Chinchillidae).

The plains vizcacha, Lagostomus maximus, is the only living species in the genus, being notably larger than fossil congeneric species, such as Lagostomus incisus, from the Pliocene of Argentina and Uruguay. Here, we compare the skull growth allometric pattern and sexual dimorphism of L. maximus and L. incisus, relating shape and size changes with skull function. We also test whether the ontogenetic trajectories and allometric trends between both sexes of L. maximus follow the same pattern. A common allometric pattern between both species was the elongation of the skull, a product of the lengthening of rostrum, and chondrogenesis on the spheno-occipitalis synchondrosis and coronalis suture. We also detected a low proportion of skull suture fusion. In some variables, older male specimens did not represent a simple linear extension of female trajectory, and all dimorphic traits were related to the development of the masticatory muscles. Sexual dimorphism previously attributed to L. incisus would indicate that this phenomenon was present in the genus since the early Pliocene and suggests social behaviors such as polygyny and male-male competition. Ontogenetic changes in L. incisus were similar to L. maximus, showing a conservative condition of the genus. Only two changes were different in the ontogeny of both species, which appeared earlier in L. incisus compared to L. maximus: the development of the frontal process of the nasals in a square shape, and the straight shape of the occipital bone in lateral view. Juveniles of L. maximus were close to adult L. incisus in the morphospace, suggesting a peramorphic process. The sequence of suture and synchondroses fusion showed minor differences in temporozygomatica and frontonasalis sutures, indicating major mechanical stress in L. maximus related to size. We suggest a generalized growth path in Chinchillidae, but further analyses are necessary at an evolutionary level, including Lagidium and Chinchilla.

Utilizing Immunoinformatics for mRNA Vaccine Design against Influenza D Virus

Background: Influenza D Virus (IDV) presents a possible threat to animal and human health, necessitating the development of effective vaccines. Although no human illness linked to IDV has been reported, the possibility of human susceptibility to infection remains uncertain. Hence, there is a need for an animal vaccine to be designed. Such a vaccine will contribute to preventing and controlling IDV outbreaks and developing effective countermeasures against this emerging pathogen. This study, therefore, aimed to design an mRNA vaccine construct against IDV using immunoinformatic methods and evaluate its potential efficacy. Methods: A comprehensive methodology involving epitope prediction, vaccine construction, and structural analysis was employed. Viral sequences from six continents were collected and analyzed. A total of 88 Hemagglutinin Esterase Fusion (HEF) sequences from IDV isolates were obtained, of which 76 were identified as antigenic. Different bioinformatics tools were used to identify preferred CTL, HTL, and B-cell epitopes. The epitopes underwent thorough analysis, and those that can induce a lasting immunological response were selected for the construction. Results: The vaccine prototype comprised nine epitopes, an adjuvant, MHC I-targeting domain (MITD), Kozaq, 3′ UTR, 5′ UTR, and specific linkers. The mRNA vaccine construct exhibited antigenicity, non-toxicity, and non-allergenicity, with favourable physicochemical properties. The secondary and tertiary structure analyses revealed a stable and accurate vaccine construct. Molecular docking simulations also demonstrated strong binding affinity with toll-like receptors. Conclusions: The study provides a promising framework for developing an effective mRNA vaccine against IDV, highlighting its potential for mitigating the global impact of this viral infection. Further experimental studies are needed to confirm the vaccine’s efficacy and safety.

Visual tracking based on spatiotemporal transformer and fusion sequences

Currently, Transformer-based visual tracking methods have exhibited impressive performance. However, despite their widespread adoption, they still have certain limitations. For example, the design of the Transformer framework is somewhat original and redundant, resulting in lower efficiency. In addition, their application methods lack time consideration, and there is a lack of spatiotemporal correlation between tracking video sequences and predicting coordinate sequences, making it difficult to effectively integrate, and the robustness of corresponding tracking templates is insufficient. To address these issues, we propose a new visual tracking method (STFS). Firstly, it introduces a novel Flatten Transformer architecture, which, in comparison to previous modules, offers enhanced efficiency and expressiveness. Secondly, it takes multi frame feature maps and bounding box coordinates as inputs, integrates spatiotemporal information through the spatiotemporal sequence attention module, and provides relevant sequences for historical trend prediction. Finally, it uses diffusion methods to construct tracking templates and improve stability. To verify the performance of the tracker, we conducted experiments on benchmark datasets including GOT-10 K, LaSOT, TrackingNet, VOT2020, OTB100, and UAV123. The results demonstrate that STFS has achieved competitive experimental results.

Preoperative prediction of perineural invasion of rectal cancer based on a magnetic resonance imaging radiomics model: A dual-center study.

Perineural invasion (PNI) has been used as an important pathological indicator and independent prognostic factor for patients with rectal cancer (RC). Preoperative prediction of PNI status is helpful for individualized treatment of RC. Recently, several radiomics studies have been used to predict the PNI status in RC, demonstrating a good predictive effect, but the results lacked generalizability. The preoperative prediction of PNI status is still challenging and needs further study. To establish and validate an optimal radiomics model for predicting PNI status preoperatively in RC patients. This retrospective study enrolled 244 postoperative patients with pathologically confirmed RC from two independent centers. The patients underwent pre-operative high-resolution magnetic resonance imaging (MRI) between May 2019 and August 2022. Quantitative radiomics features were extracted and selected from oblique axial T2-weighted imaging (T2WI) and contrast-enhanced T1WI (T1CE) sequences. The radiomics signatures were constructed using logistic regression analysis and the predictive potential of various sequences was compared (T2WI, T1CE and T2WI + T1CE fusion sequences). A clinical-radiomics (CR) model was established by combining the radiomics features and clinical risk factors. The internal and external validation groups were used to validate the proposed models. The area under the receiver operating characteristic curve (AUC), DeLong test, net reclassification improvement (NRI), integrated discrimination improvement (IDI), calibration curve, and decision curve analysis (DCA) were used to evaluate the model performance. Among the radiomics models, the T2WI + T1CE fusion sequences model showed the best predictive performance, in the training and internal validation groups, the AUCs of the fusion sequence model were 0.839 [95% confidence interval (CI): 0.757-0.921] and 0.787 (95%CI: 0.650-0.923), which were higher than those of the T2WI and T1CE sequence models. The CR model constructed by combining clinical risk factors had the best predictive performance. In the training and internal and external validation groups, the AUCs of the CR model were 0.889 (95%CI: 0.824-0.954), 0.889 (95%CI: 0.803-0.976) and 0.894 (95%CI: 0.814-0.974). Delong test, NRI, and IDI showed that the CR model had significant differences from other models (P < 0.05). Calibration curves demonstrated good agreement, and DCA revealed significant benefits of the CR model. The CR model based on preoperative MRI radiomics features and clinical risk factors can preoperatively predict the PNI status of RC noninvasively, which facilitates individualized treatment of RC patients.

Two recurrent types of IGH::5′ BCL2 breakpoints representing cytogenetic ins(14;18)(q32;q21q21) and t(14;18)(q32;q21), mediated by the VDJ and class switch recombination processes, respectively

We describe two types of IGH::BCL2 breakpoints involving the 5′ region of BCL2 (5′ BCL2). One was ins(14;18)(q32;q21q21) observed in 2 follicular lymphoma (FL) cases, in which IGH was cleaved at 3’ of IGHD and 5’ of IGHJ and BCL2 was cleaved at 5’ BCL2 and downstream regions, and a 281- or 201-kilobase pair fragment containing the BCL2 protein-coding sequences was invertedly inserted into IGH. In another type observed in 2 FL and 2 chronic lymphocytic leukemia (CLL) cases, breakage and reunion occurred within the switch region associated with IGHM (Sµ) and 5′ BCL2, creating IGH Sµ::5′ BCL2 fusion sequences on der(18)t(14;18)(q32;q21). The former is considered to be mediated by VDJ-recombination, while the latter by the class switch recombination process. There were no particular features in FL or CLL cases with IGH::5′ BCL2 breakpoints compared with those with t(14;18)(q32;q21)/IGH::BCL2 involving the 3′ breakpoint cluster regions.

Abstract 2853: Comparison of methods for detection of hemispheric gliomas of infancy and their characteristic oncogenic tyrosine kinase gene fusions

Abstract Introduction High grade hemispheric gliomas in infancy (IHG) are characterized by a more favorable outcome compared to high grade gliomas in older children. We have shown in previous studies that these tumors are characterized by a stable genome. The occurrence of tyrosine kinase gene fusions in high-grade hemispheric gliomas of infancy may represent therapeutic targets. Methods: 33 high-grade hemispheric gliomas occurring in children younger than three years were retrieved from the archives of the Brain Tumor Reference Center, Institute of Neuropathology, University of Bonn. DNA and RNA were extracted from FFPE tumor samples. Methylation-based classification was done by hybridization to Illumina methylation arrays and subsequent use of the Heidelberg brain tumor classifier versions v11b4, v12.5 and v12.8. In addition UMAP plots were used to visualize similarities of methylation profiles. Gene fusions were identified on the DNA level by FISH using break-apart probes for ALK, NTRK1, -2, -3, ROS1 and MET and Molecular Inversion Probe (MIP) methodology. On the RNA level, fusion transcripts were detected by targeted RNA sequencing as well as Nanostring assay with fusion-specific probes. Results: 32 IHG occurred in the first year of life, one at the age of two years. 23/33 IHG were classified as such by methylation arrays; 6 further cases could be annotated to the IHG group by UMAP plots. 18/33 cases (54.5 %) showed fusions of ALK to different partners; all occurred in the first year of life. Fusions of ROS1 were found in 4, MET in 2, NTRK1 and -3 in 7 cases. 2 cases showed no fusions. The different methods for fusion detection led to comparable results. Only recurrent fusions with known fusion partners were detectable with Nanostring probes designed for the fusion sequence, and library construction for targeted RNA sequencing was not successful in a fraction of cases. Break-apart FISH led to reliable results on the next day, and MIP technology represented the most sensitive method for analysis of FFPE samples. Conclusions: Gene fusions involving the tyrosine kinase genes ALK, MET, ROS1 and NTRK1 and -3 occurred in 93 % of high grade hemispheric gliomas of infancy; the most frequent were ALK fusions occurring in children younger than 1 year. DNA-based MIP technology represented the most robust and sensitive assay. A combination of RNA- and DNA-based methods to detect these fusions with high reliability is recommended. Citation Format: Torsten Pietsch, Gerrit Gielen, Andreas Waha, Evelyn Dörner, Verena Dreschmann, Christof Kramm, Christian Vokuhl. Comparison of methods for detection of hemispheric gliomas of infancy and their characteristic oncogenic tyrosine kinase gene fusions [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2853.

Panel-based RNA fusion sequencing improves diagnostics of pediatric acute myeloid leukemia

New methods like panel-based RNA fusion sequencing (RNA-FS) promise improved diagnostics in various malignancies. We here analyzed the impact of RNA-FS on the initial diagnostics of 241 cases with pediatric acute myeloid leukemia (AML). We show that, compared to classical cytogenetics (CCG), RNA-FS reliably detected risk-relevant fusion genes in pediatric AML. In addition, RNA-FS strongly improved the detection of cryptic fusion genes like NUP98::NSD1, KMT2A::MLLT10 and CBFA2T3::GLIS2 and thereby resulted in an improved risk stratification in 25 patients (10.4%). Validation of additionally detected non-risk-relevant high confidence fusion calls identified PIM3::BRD1, C22orf34::BRD1, PSPC1::ZMYM2 and ARHGAP26::NR3C1 as common genetic variants and MYB::GATA1 as recurrent aberration, which we here describe in AML subtypes M0 and M7 for the first time. However, it failed to detect rare cytogenetically confirmed fusion events like MNX1::ETV6 and other chromosome 12p-abnormalities. As add-on benefit, the proportion of patients for whom measurable residual disease (MRD) monitoring became possible was increased by RNA-FS from 44.4 to 75.5% as the information on the fusion transcripts’ sequence allowed the design of new MRD assays.

Long-Term Strategies for Poorly Water-Soluble Peptides: Combining Fatty Acid Modification with PAS Fusion.

Bulevirtide, an entry inhibitor for the hepatitis B virus (HBV) and hepatitis D virus (HDV), is currently available on the European market. However, its clinical application is constrained by its short half-life and poor water solubility, rendering it unsuitable for fatty acid modification, aimed at achieving long-term effects. To address this limitation, we integrated a polypeptide chain consisting of Pro, Ala, and Ser at the C-terminus, which increased its hydrophilicity. To obtain the fusion sequence of A1 and A2, encompassing amino acids 1-47 of Bulevirtide and PAS, we used Escherichia coli fermentation expression. Subsequently, the N-terminal myristoyl groups of A1 and A2 were modified to yield Myr-A1 and Myr-A2, respectively. Five fatty acid moieties with the same hydrophilic spacers and different fatty acids were conjugated to analogs, generating 10 bioconjugations. The bioconjugates were then evaluated for their anti-HBV activity. Among them, HB-10 was selected for pharmacokinetic analysis and demonstrated a significantly prolonged half-life, with 5.88- and 13.18-fold increases in beagle dogs and rats, respectively. Additionally, higher drug doses resulted in substantially elevated liver concentrations. In conclusion, via fatty acid incorporation and PASylation, we successfully developed a novel Bulevirtide bioconjugate, HB-10, that exhibits an extended action duration. This compound holds substantial promise as a prospective long-acting entry inhibitor, warranting further investigation.

Independent Prognostic Value of DNA-Based Minimal Residual Disease Assessment in Unselected Population-Based Cohort of Pediatric Acute Myeloid Leukemia

Monitoring of minimal residual disease (MRD) in childhood acute myeloid leukemia (AML) can assess response to treatment and predict relapse. Current MRD monitoring methods in childhood AML - flow cytometry and quantitation of fusion transcripts - have significant shortcomings and do not cover the entire spectrum of patients. We demonstrated (Lukes et al, Hemasphere 2020) the feasibility of MRD monitoring using DNA-based primary targets in selected subgroups of AML. Now we extended this approach to the unselected consecutive population of pediatric AML. We included 133 children (0-18) out of 135 consecutively diagnosed with AML in Czechia (2012-2022), all treated on non-MRD-based protocols. For genetic characterization, we used algorithm consisting of screening for recurrent gene fusions and mutations by PCR and targeted-NGS followed by transcriptome sequencing (WTS). Targeted-NGS (or PCR) was performed to identify genomic fusion sequences. 130 patients were eligible for MRD monitoring (3 patients died early). MRD was preferentially monitored by DNA-based qPCR (alternatively by amplicon NGS or qRT-PCR). MRD levels were expressed relative to diagnosis. Primary genetic aberration was found by targeted screening in 102 children; remaining 31 were further investigated using WTS. In 27 children, WTS identified rare, novel or atypical primary genetic aberrations not covered by the targeted screening. Primary genetic aberration was found in 97% of cases (129/133). Majority of AMLs (81%) were classified into common subtypes: AML with KMT2Ar, PML::RARA, RUNX1::RUNX1T1, CBFB::MYH11, mutation (m) of GATA1 , CEBPA or NPM1. Recurrent but rare aberrations in pediatric AML were found in 16 patients: UBTFm, RUNX1m, HOXA10 translocation, KAT6A::CREBBP, KAT6A::LEUTX, DEK::NUP214, BCR::ABL1, NUP98::NSD1 and CBFA2T3::GLIS2. In 5 patients, fusion genes were found, previously described sporadically (SPFQ::ZFP36L216, XPO1::TNRC1817) or not at all in AML (ETV6::CTNNB1, FUS::FEV, ZEB2::RUNX1). Only secondary aberrations were identified in remaining 4 patients. In 82 patients (of 84 examined), genomic fusion sequence was found and qPCR designed and used. Of 41 children carrying genetic aberrations other than fusions, in 29 quantification system was successfully implemented. Altogether, quantification system for MRD monitoring was established in 122 patients, with primary aberrations as DNA targets in 116 patients, reaching sensitivity of 10 -4 in 112 and 5x10 -4 in 4 patients. Four and two children were monitored with sensitivity of 10 -4 using secondary aberrations as DNA targets or by qRT-PCR-based quantification of fusion transcripts, respectively. MRD clearance significantly differed among genetic subtypes: while GATA1m AML had the fastest response, in contrast to patients with prognostically favorable genetic subtypes - CBFB::MYH11, RUNX1::RUNX1T1, CEBPAm and NPM1m - who were treated predominantly on the standard risk (SR) arm of the AML-BFM 2012 Registry Protocol and none achieved molecular remission (mREM) at D28 (70% vs 0%, p &amp;lt; 0.0001). Significant proportion of patients (25-89% within individual subtypes) did not achieve mREM even after the last block of chemotherapy. The response of patients with KMT2Ar AML and AML classified into remaining subtypes (predominantly intermediate- and high-risk) was overall faster compared to prognostically favorable subtypes on SR arm (KMT2Ar D28 mREM 33% vs 0% at D28, p &amp;lt; 0.0001). KMT2A::MLLT10/3 had faster clearance then other KMT2Ar (mREM D28 44% vs. 0% in other KMT2Ar, p = 0.02). In a multivariate analysis including (cyto)genetic risk and treatment, D28 MRD was the only significant predictor of outcome using both the 10 -3 (p = 0.006 for EFS and 0.012 for OS) and 10 -2 levels (p = 0.004 for EFS and 0.01 for OS) for stratification. Similarly, in an alternative multivariate analysis model, D56 MRD (positive at any level versus negative) was also the only significant predictor of outcome independent of risk and treatment (p = 0.005 for EFS and 0.01 for OS) In summary, we present a strategy for MRD monitoring in pediatric AML that is technologically feasible, real-life applicable to vast majority of all patients, and has clear prognostic significance. Supported by National Institute of Cancer Research No. LX22NPO5102 funded by the European Union-Next Generation EU, and AZV grant NU20-07-00322.

Partner Gene Associated Trinary Fusion and Orchestration of Multiple Molecular Events in Acute Promyelocytic Leukemia with Variant Rara Fusion Genes

Background All-trans retinoic acid (ATRA) has been used with great success in acute promyelocytic leukemia (APL) cases with PML::RARA fusion gene (FG). There are still PML::RARA negative cases manifest as APL. So far, 19 RARA fusion partner genes have been reported, with all reported as 5′ partners ( X::RARA). Intriguingly, a significant proportion of X::RARA cases were resistant to ATRA, and mechanisms are yet to be elucidated. Here we report the finding of a novel type of trinary fusion and the orchestrating of multiple molecular events that confer leukemogenesis and ATRA resistance in X::RARA-APL. Methods A total of 23 cases (Fig. 1A) with RNA-seq and WGS data from 10 centers were enrolled. We used Arriba for routine FG calling and manually investigated the nonconventional fusion sequence. RT-PCR, Sanger sequencing, and optical genome mapping (OGM) were used to validate the FGs and to determine whether the RARA 5′ and 3′ fusion events were located on the same cistron. The responsiveness of the fusion proteins to ATRA was evaluated using an optimized UAS/GAL4 reporter system. Results Among the cases enrolled, there were 17 males and 6 females, aged 3-66 (median 32) years. Morphological and immunophenotype analyses all showed features of APL. We first identified the STAT3::RARA fusion event in an ATRA-resistant case (the index case, #A1). Moreover, we noticed another fusion event of truncated RARA exon 9 and a 3′ partner sequence that did not map to any annotated protein-coding genes. Further analysis determined it was the RC sequence of the transposable element (TE) LTR40a with the annotated loci at 17p11.2. Both OGM (at the genome level) and RT-PCR (Fig. 1A) confirmed that the two RARA fusion events were in the same cistron. Thus, the fusion transcript being STAT3::RARA::LTR40a-RC spliced in tandem, but not separate transcripts of STAT3::RARA and RARA::LTR40a-RC. We named this novel form of tandem splicingtrinary fusion. We, therefore, recruit X::RARA-APL cases to investigate the recurrence of RARA 3′ fusion. We identified 9 RARA 5′ partner genes in 23 cases (Fig. 1B), with TTMV -ORF2, STAT5B, STAT3, IRF2BP1, THRAP3, and TNRC18 in multiple cases. Ten (43.5%) cases also harbor RARA 3′ fusion partners. The partner of case #A1_8 were TEs, and the partner of case #A9_10 was the same gene as their 5′ one. Thirteen cases did not harbor 3′ fusions. Notably, both TNRC18::RARA cases (#15_16) carry mutations on their RARA ligand binding domain (LBD) at the primary onset. Gene expression analysis confirmed the aberrant activation of the involved TEs. All TEs involved in the RARA 3' fusions confer a poly_A signal sequence, which was essential for a mRNA. In-depth analysis indicated that TEs participate in forming RARA-FGs through a transposition mechanism rather than a translocation mechanism. The molecular events addition to RARA 5′ splicing showed a 5′ partner associated pattern: all of the 4 STAT3::RARA and 4 STAT5B::RARA cases harbor TEs as their 3′ partner, both TNRC18::RARA cases carry LBD mutation, while all 7 TTMV -ORF2::RARA cases did not harbor 3′ splicing or LBD mutation. Remarkably, although the RARA 3' splicing sites varied, they all resulted in partial or complete exon 9 truncation. RARA exon 9 encodes helix 11_12 (H11_12) of its LBD, which is pivotal in allosteric response to ATRA. The RARA trinary fusion will result in LBD-H12 or H11_12 castration due to the 3' splicing, then lead to losing responsiveness to ATRA via an allosteric disability mechanism (Fig. 1C, 1D). Experiments confirmed that cells transfected with STAT3::RARA fusion with intact LBD responded well to ATRA, while both the fusion in case #A1 and artificial fusion with LBD-H12 castration showed no response to physiological and pharmacological concentrations of ATRA (Fig. 1E). Conclusions This is the first report of pathological trinary fusion, the TEs and transposition mechanism participating in composing pathological fusion genes, and the RARA LBD castration confers ATRA resistance in X::RARA-APL. Also, there might be another synergistic mechanism of RARA 5' fusion and LBD mutation. The formation of trinary fusion and gain of LBD mutation, in addition to X::RARA fusion, requires more genome splicing or transposition or mutation events, which explains the rarity of X::RARA-APL. This study highlights mechanisms of partner gene-associated trinary fusion and the orchestrating of multiple molecular events in the leukemogenesis in X::RARA-APL cases.

Trinary Fusion with Ligand Binding Domain Castration Is Crucial in Rarg-Driven APL and Render Unresponsive to ATRA Via Allosteric Disability Mechanism

Introduction All-trans retinoic acid (ATRA) has been used with great success in acute promyelocytic leukemia (APL) cases with PML::RARA fusion gene (FG). There are still PML::RARA negative cases that manifest as APL, and they often carry FGs involved in RARA, RARB, or RARG. X::RARG positive APL ( RARG-APL) cases have been increasingly reported in recent years, thanks to the utilization of transcriptome sequencing (RNA-seq) and whole genome sequencing (WGS). Almost all evaluable RARG-APL cases were resistant to ATRA, and no mechanism has been elucidated. Intriguingly, cells transformed by artificial X::RARG were extremely sensitive to ATRA (PMID: 25510432), indicating intricate mechanisms unrevealed. This study first and systematically unveils the distinctive features and pivotal molecular mechanism of RARG-driven APL. Methods A total of 22 RARG-APL cases with RNA-seq and WGS data from 11 centers were enrolled (Fig. 1A). We used Arriba for routine FG calling and manually investigated the non-coding fusion sequence. Reverse transcription PCR (RT-PCR) and Sanger sequencing were used to validate the FGs and to determine whether the RARG 5′ and 3′ fusion events were located on the same cistron. The responsiveness of the fusion proteins to ATRA was evaluated using an optimized UAS/GAL4 reporter system. Results Among the cases enrolled, there were 15 males and 7 females, aged 0.9-69 (median 38) years. Morphological and immunophenotype analyses all showed features of APL. Routine FGs calling reported RARG fusion to a 5′ partner in each case, with variable RARG splicing sites (Fig. 1A). There were 7 RARG 5' partner genes, with CPSF6, HNRNPC, and NUP98 observed in multiple cases. In the 3 index cases, we identified that they all had RARG 3‘ fusion events besides the 5‘ one (Fig. 1A). RT-PCR confirmed that the 5′ and 3′ RARG fusion events were in the same cistron in each case. Such as, the fusion transcript is the insertion of RARG-e4_9 (e refers to exon) between NPM1-e4 and e11 in case #G1, rather than two separate transcripts of NPM1-e4:: RARG-e4 and RARG-9:: NPM1-e11. We named this novel form of tandem splicingtrinary fusion. Analysis of the 19 validation cases further confirmed that all RARG-APL cases had RARG 3′ fusion events (Fig. 1A). We also validated that the 5′ and 3′ RARG fusion events were in the same cistron in 3 more cases, of which archived cDNA were available. Remarkably, the 3' splicing sits of all cases were consistently at the end of RARG-e9, leading to RARG-e10 truncation. The RARG 3' partner gene was the same as its 5' partner in 11 cases. But in the other 11 cases, the RARG 3' partner was a transposon element (TE) sequence, the most common being a LINE-L2a (8 cases). Sequences analysis indicated that the committed locus of the involved LINE-L2a was at 4.2 Kbp upstream of the RARG gene. Gene expression analysis confirmed the aberrant activation of the involved TEs. All TEs involved in the RARG 3' fusions confer a poly_A signal sequence to the fusion transcript, which was essential for a mRNA. In-depth analysis indicated that TEs participate in the formation of RARG-FGs through a transposition mechanism rather than a translocation mechanism. RARG-e10 encodes helix 11_12 (H11_12) of its ligand binding domain (LBD), which plays a pivotal role in allosteric response to ATRA (PMID: 31178221). The trinary fusion protein with RARG LBD-H11_12 castration will lose responsiveness to ATRA via an allosteric disability mechanism (Fig. 1B, 1C). Experiments confirmed that cells transfected with X::RARG fusion with intact LBD responded well to ATRA, while fusion with LBD-H11_12 castration showed no response to physiological and pharmacological concentrations of ATRA (Fig. 1D, 1E). Conclusions This study clearly revealed that trinary fusion and LBD castration are crucial molecular etiologies in RARG-APL. The trinary fusion orchestrates the aberrant activation of RARG (5‘ splicing) and the complete allosteric disability in responding to ATRA (3‘ splicing). Thus, this study elegantly explains the mechanism of leukemogenesis and the extensive ATRA resistance of RARG-APL. The formation of trinary fusion requires two steps of genome splicing or transposition events, which explains the rarity of RARG-APL. This also clarifies that RARG-APL has a significant disparity in molecular mechanism from PML::RARA-positive APL, and we suggest that it should be considered as a separate entity of acute myeloid leukemia.

Monitoring and control framework for intelligent real-time optimization of printing sequence of powder bed fusion

Monitoring and control framework for intelligent real-time optimization of printing sequence of powder bed fusion

MODL-31. USING GENETICALLY-ENGINEERED MOUSE MODELS OF TREATMENT-NAÏVE AND -EXPERIENCED NTRK FUSION-DRIVEN GLIOMAS FOR PRE-CLINICAL TRIALS OF TYROSINE KINASE INHIBITORS

Abstract Gene fusions that contain the kinase domain of the three neurotrophic tyrosine kinase receptor (NTRK) genes are frequently observed in infantile high and low-grade gliomas. While these tumors initially respond to treatment with NTRK tyrosine kinase inhibitors (TKIs), treatment resistance and recurrence eventually occur. Treatment-resistant tumors frequently harbor either on-target (in the fusion's kinase domain) or off-target mutations (in downstream effectors of mitogen-activated signaling pathways). Therefore, overcoming this resistance requires a three-pronged strategy: 1) identifying new kinase inhibitors capable of targeting NTRK fusions with on-target kinase domain mutations, 2) identifying druggable targets in the downstream pathways activated by these fusions, and 3) targeting persister cells that contribute to disease recurrence. We utilized the RCAS/tv-a system to establish several mouse models of NTRK1/2/3 fusion-driven gliomas. We found that all analyzed fusions were strong oncogenic drivers on their own, while the loss of additional tumor suppressors (Cdkn2a or Pten) further increased the aggressiveness of these tumors. These mouse models exhibit similar treatment responses as human tumors, initially responding to various TKIs but eventually recurring due to the presence of treatment-resistant persister cells. To mimic the emergence of resistance-associated mutations, we directly introduced different kinase domain mutations into the NTRK fusion sequence or co-expressed secondary mutations (BRAF-V600E or KRAS-G12D). These modifications altered the response to TKI treatment both in vitro and in vivo. Overall, these models serve as valuable tools for studying the biology of NTRK fusion tumors, their treatment response in a treatment-naïve or -experienced state, and the mechanisms underlying treatment resistance.

Clinical Validation of FusionPlex RNA Sequencing and Its Utility in the Diagnosis and Classification of Hematologic Neoplasms

Recurrent gene rearrangements result in gene fusions that encode chimeric proteins, driving the pathogenesis of many hematologic neoplasms. The fifth edition World Health Organization classification and International Consensus Classification 2022 include an expanding list of entities defined by such gene rearrangements. Therefore, sensitive and rapid methods are needed to identify a broad range of gene fusions for precise diagnosis and prognostication. In this study, we validated the FusionPlex Pan-Heme panel analysis using anchored multiplex PCR/targeted RNA next-generation sequencing for routine clinical testing. Furthermore, we assessed its utility in detecting gene fusions in myeloid and lymphoid neoplasms. The validation cohort of 61 cases demonstrated good concordance between the FusionPlex Pan-Heme panel and other methods, including chromosome analysis, fluorescence in situ hybridization, RT-PCR, and Sanger sequencing, with an analytic sensitivity and specificity of 95% and 100%, respectively. In an independent cohort of 28 patients indicated for FusionPlex testing, gene fusions were detected in 21 patients. The FusionPlex Pan-Heme panel analysis reliably detected fusion partners and patient-specific fusion sequences, allowing accurate classification of hematologic neoplasms and the discovery of new fusion partners, contributing to a better understanding of the pathogenesis of the diseases.

Representation of operators using fusion frames

To solve operator equations numerically, matrix representations are employing bases or more recently frames. For finding the numerical solution of operator equations a decomposition in subspaces is needed in many applications. To combine those two approaches, it is necessary to extend the known methods of matrix representation to the utilization of fusion frames. In this paper, we investigate this representation of operators on a Hilbert space H with Bessel fusion sequences, fusion frames and fusion Riesz bases. Fusion frames can be considered as a frame-like family of subspaces. Taking the particular property of the duality of fusion frames into account, this allows us to define a matrix representation in a canonical as well as an alternate way, the later being more efficient and well behaved in respect to inversion. We will give the basic definitions and show some structural results, like that the functions assigning the alternate representation to an operator is an algebra homomorphism. We give formulas for pseudo-inverses and the inverses (if existing) of such matrix representations. We apply this idea to Schatten p-class operators. Consequently, we show that tensor products of fusion frames are fusion frames in the space of Hilbert-Schmidt operators. We will show how this can be used for the solution of operator equations and link our approach to the additive Schwarz algorithm. Consequently, we propose some methods for solving an operator equation by iterative methods on the subspaces. Moreover, we implement the alternate Schwarz algorithms employing our perspective and provide small proof-of-concept numerical experiments. Finally, we show the application of this concept to overlapped convolution and the non-standard wavelet representation of operators.