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Background: Novel protein-coding genes were considered to be born by re-organization of pre-existing genes, such as gene duplication and gene fusion. However, recent progress of genome research revealed that more protein-coding genes than expected were born de novo, that is, gene origination by accumulating mutations in non-genic DNA sequences. Nonetheless, the in-depth process (scenario) for de novo origination is not well understood. Results: We have conceived bioinformatic analysis for sketching a scenario for de novo origination of protein-coding genes. For each de novo protein-coding gene, we firstly identified an edge of a given phylogenetic tree where the gene was born based on parsimony. Then, from a multiple sequence alignment of the de novo gene and its orthologous regions, we constructed ancestral DNA sequences of the gene corresponding to both end nodes of the edge. We finally revealed statistical features observed in evolution between the two ancestral sequences. In the analysis of the Saccharomyces cerevisiae lineage, we have successfully sketched a putative scenario for de novo origination of protein-coding genes. (1) In the beginning was GC-rich genome regions. (2) Neutral mutations were accumulated in the regions. (3) ORFs were extended/combined, and then (4) translation signature (Kozak consensus sequence) was recruited. Interestingly, as the scenario progresses from (2) to (4), the specificity of mutations increases. Conclusion: To the best of our knowledge, this is the first report outlining a scenario of de novo origination of protein-coding genes. Our bioinformatic analysis can capture events that occur during a short evolutionary time by directly observing the evolution of the ancestral sequences from non-genic to genic. This property is suitable for the analysis of fast evolving de novo genes. [ABSTRACT FROM AUTHOR]

Chronic myeloid leukemia (CML), characterized by the presence of the BCR::ABL1 fusion gene, has undergone a transformative shift with the introduction of tyrosine kinase inhibitors (TKIs). The current availability of six different TKIs (imatinib, dasatinib, nilotinib, bosutinib, ponatinib, and asciminib) in clinical practicemakes it important to know their efficacy and toxicity profile for treatment optimization. This review examines the latest insights regarding the use of bosutinib in CML treatment. Clinical trials have demonstrated the effectiveness of bosutinib, positioning it as a first-line treatment that can induce sustained molecular responses. Importantly, it can also be effective in patients who have experienced treatment failure or intolerance with prior TKIs, revealing the potential of bosutinib also in secondand later-line settings. Even in the advanced phase of CML, bosutinib has demonstrated its capacity to achieve molecular responses, expanding its usefulness. Real-world evidence studies echo these findings, emphasizing bosutinib's effectiveness in achieving deep molecular responses, maintaining remissions, and serving as an alternative for patients intolerant or resistant to other TKIs as a second-line therapy. Notably, one of the greatest strengths of bosutinib is its favorable safety profile, in particular the low incidence of vascular complications with its use, which is undoubtedly a comparative advantage over other TKIs. In summary, the latest research highlights the versatility of bosutinib in CML treatment and underscores its pivotal role in optimizing patient management in challenging cases. Continuing research and investigation will further establish bosutinib's place in the evolving landscape of CML therapy, offering an alternative for CML patients across different treatment stages. [ABSTRACT FROM AUTHOR]

Background: Pralsetinib, a targeted inhibitor of the RET enzyme, plays a critical role in the treatment of adult patients with locally advanced or metastatic non-small cell lung cancer (NSCLC) characterized by RET gene fusion mutations following platinum-based chemotherapy. Nevertheless, impurities resulting from the manufacturing and degradation of pralsetinib have the potential to impact its therapeutic effectiveness and safety profile. Methods: To address this issue, a liquid chromatography method was developed and validated for the specific identification of pralsetinib and its related impurities. The separation of pralsetinib and its related impurities was achieved via a Waters X Bridge Ci8 column with dimensions of 4.6 mm x 250 mm and a particle size of 5 ^m. Mobile phase A was composed of 20 mmol/L potassium dihydrogen phosphate (KH2PO4) and acetonitrile (ACN) at a volume ratio of 19:1, while mobile phase B consisted solely of ACN, utilizing a gradient elution technique. Detection was performed at a wavelength of 260 nm, with an injection volume of 10 ^L and a flow rate of 1.0 mL/min. Results: The chromatographic method established in this study was validated according to the ICH Q2 (R1) guidelines. The method demonstrated excellent linearity over a specific concentration range (imp-A: 0.035-10.21 ^ig/mL; imp-B: 0.09-10.16 ^g/mL; imp-C: 0.15-10.19 μg/mL; pralsetinib: 0.04-10.32 ^g/mL). Additionally, the method possesses high sensitivity, with detection limits for impurities A, B, C, and pralsetinib of 0.01, 0.03, 0.015, and 0.013 ^g/mL, respectively, and quantification limits of 0.035, 0.09, 0.05, and 0.04 ^ig/mL, respectively. In terms of specificity, stability, repeatability, accuracy, and robustness, the method met the validation acceptance criteria. Overall, the chromatographic technique established in this study can effectively separate pralsetinib and its impurities, providing reliable assurance for the accurate detection and quantification of impurities. Conclusion: The chromatographic method developed in this study can be utilized for the detection of pralsetinib and its impurities, offering a crucial reference for research on the quality of pralsetinib. [ABSTRACT FROM AUTHOR]

The EML4-ALK oncogene is a fusion of the EML4 and ALK genes and is found in approximately 5– 6% of the cases of non-small cell lung cancer (NSCLC). Herein, we present a unique case of lung adenocarcinoma with metastases to the bilateral ovaries harboring a rare EML4-ALK fusion gene variant in a 52-year-old patient. The patient had initially received a diagnosis of ovarian cancer, then had undergone neo-adjuvant chemotherapy followed by a surgical resection. Despite two cycles of adjuvant chemotherapy consisting of carboplatin and gemcitabine, CT revealed that the pleural effusion had increased from it before chemotherapy, and the shortness of breath worsened. Molecular profiling revealed an EML4-ALK rearrangement containing ALK -EML4 and ALK -NPR2 fusion genes. The diagnosis was changed to primary lung adenocarcinoma with metastases to the bilateral ovaries based on a pathological reevaluation. Treatment with alectinib, a second-generation ALK-tyrosine kinase inhibitor, led to a partial response of 18 months' duration, and the shortness of breath improved. No adverse events related to the alectinib therapy occurred. To assess the unique structure of the fusion genes, RNA sequencing was performed. An intronic sequence from both ALK and EML4 was found between ALK and EML4 exon, possibly because of an unusual insertion of a gene fragment derived from NRP2, indicated by the panel sequencing results. Variations in the drug response among EML4-ALK fusion variants highlight the importance of understanding their molecular structure. Further investigation is warranted to refine fusion gene detection methods and assess the therapeutic implications of rare fusion variants. [ABSTRACT FROM AUTHOR]

Many bacteria co-exist and produce antibiotics, yet we know little about how they cope and occupy the same niche. The purpose of the present study was to determine if and how two potent antibiotic-producing marine bacteria influence the secondary metabolome of each other. We established an agar- and broth-based system allowing co-existence of a Phaeobacter species and Pseudoalteromonas piscicida that, respectively, produce tropodithietic acid (TDA) and bromoalterochromides (BACs). Co-culturing of Phaeobacter sp. strain A36a-5a on Marine Agar with P. piscicida strain B39bio caused a reduction of TDA production in the Phaeobacter colony. We constructed a transcriptional gene reporter fusion in the tdaC gene in the TDA biosynthetic pathway in Phaeobacter and demonstrated that the reduction of TDA by P. piscicida was due to the suppression of the TDA biosynthesis. A stable liquid co-cultivation system was developed, and the expression of tdaC in Phaeobacter was reduced eightfold lower (per cell) in the co-culture compared to the monoculture. Mass spectrometry imaging of co-cultured colonies revealed a reduction of TDA and indicated that BACs diffused into the Phaeobacter colony. BACs were purified from Pseudoalteromonas; however, when added as pure compounds or a mixture they did not influence TDA production. In co-culture, the metabolome was dominated by Pseudoalteromonas features indicating that production of other Phaeobacter compounds besides TDA was reduced. In conclusion, co-existence of two antibiotic-producing bacteria may be allowed by one causing reduction in the antagonistic potential of the other. The reduction (here of TDA) was not caused by degradation but by a yet uncharacterized mechanism allowing Pseudoalteromonas to reduce expression of the TDA biosynthetic pathway. [ABSTRACT FROM AUTHOR]

Non‐small‐cell lung cancer (NSCLC) accounts for the majority of lung cancer cases worldwide, with a significant proportion of patients harbouring actionable oncogenic alterations. Among these alterations, the ROS1 rearrangement represents a distinct subset with therapeutic implications. Here, we present the case of a 52‐year‐old man diagnosed with advanced NSCLC harbouring the ROS1 fusion gene. Despite the initial poor response to conventional chemotherapy, the patient exhibited an exceptional and sustained response to crizotinib, with a progression‐free survival of 94 months and complete metabolic response on PET scan. This case underscores the importance of molecular profiling in guiding treatment decisions and highlights the efficacy of targeted therapies for ROS1‐positive NSCLC. [ABSTRACT FROM AUTHOR]

Backgroud: Supernatants from various cytological samples, including body cavity effusion, sputum, bronchoalveolar lavage fluid (BALF), and needle aspiration, have been validated for detecting genetic alterations using cell‐free DNA (cfDNA) in patients with non‐small cell lung cancer (NSCLC). However, the sensitivity of fusion variations detection remains challenging. The protection of cell‐free RNA (cfRNA) is critical for resolving the issue. Methods: A protective solution (PS) was applied for preserving cfRNA in cytological supernatant (CS), and the quality of protected cfRNA was assessed by cycle threshold (CT) values from reverse transcription quantitative polymerase chain reaction (RT‐qPCR). Furthermore, we collected an additional set of malignant cytological and matched tumor samples from 84 NSCLC patients, cfDNA & cfRNA extraction and double detection for driver gene mutations was validated using the multi‐gene mutations detection by RT‐qPCR. Results: Under the optimal protection system, 91.0% (101/111) of cfRNA were protected effectively. Among the 84 NSCLC patient samples, seven cytological samples failed the tests. In comparison with tumor samples, the overall sensitivity and specificity of detecting driver genes of supernatant cfDNA and cfRNA were 93.8% (74/77) and 100% (77/77), respectively. Notably, when focusing exclusively on patients with fusion gene changes, both sensitivity and specificity reached 100% (11/11) for EML4‐ALK, ROS1, RET fusions, and MET ex14 skipping. Conclusion: These findings suggest that cfDNA & cfRNA extraction and double detection strategy recommended in this study improve the accuracy of driver genes mutations test, especially for RNA‐based assay. [ABSTRACT FROM AUTHOR]

Background: Diagnostic classification of thyroid malignancy is primarily accomplished through examination of histomorphological features and may be substantiated and clarified by molecular data. Individual molecular drivers show relatively robust and specific associations with histological subtypes of thyroid malignancy, including BRAF sequence variants and kinase gene fusions in papillary thyroid carcinoma, predominantly RAS variants in follicular-patterned neoplasia, and additional "late" mutations affecting TERT promoter, TP53, and the PI3K/AKT/PTEN pathway in high-grade malignancies. Given the oncogenic role of FGFR, particularly FGFR1-3, the goal of this study was to explore the role of FGFR in thyroid carcinoma biology. Methods: We completed a multicenter retrospective observational study for thyroid carcinomas with pathogenic alterations in the FGFR gene family. We performed this study by querying the molecular data accumulated for thyroid carcinomas from each center. Results: Overall, 5030 sequenced thyroid malignancies were reviewed, yielding 17 tumors with FGFR alterations, including 11 where FGFR was the primary molecular driver and 6 where FGFR was a secondary pathogenic alteration, with a subset for which there was available clinical follow-up data. Of the 11 carcinomas with an FGFR driver, 9 were gene fusions involving FGFR2:VCL (4 tumors), TG::FGFR1 (3 tumors), FGFR2::CIT, and FGFR2::SHTN1, and the remaining 2 were driven by FGFR1 amplification. In the 6 tumors where a canonical driver of thyroid neoplasia was present (5 cases) or no clear primary driver was detected (1 case), sequencing detected secondary FGFR2 p.W290C, p.Y375C, and p.N549K, as well as FGFR1 p.N546K in the respective tyrosine kinase domains, some at subclonal variant allele frequencies. Conclusions: This study presents the first description of a collection of thyroid carcinomas grouped by primary driver alterations in FGFR, as well as a cohort of thyroid tumors with secondary alterations that potentially lead to tumor progression or resistance to targeted therapy. Given the availability of small molecular inhibitors targeting oncogenic FGFR, this study emphasizes the significant implications for patients from identification of FGFR alterations as they are currently under-recognized in the literature and, most importantly, have potential novel treatment options. [ABSTRACT FROM AUTHOR]

Background: The important challenge to the biotechnology is to find new effective fusion partners that enable to improve solubility, expression, and optimize the subsequent fine purification of the target protein. Results: The most invariant part of the most immunogenic region of the surface virulence factor A of Streptococcus pneumoniae was selected as a model target protein, while the thermostable chemotaxis polypeptide of W-type from Thermotoga petrophila was used as a fusion partner. The genes encoding fusion variants of these proteins were constructed and cloned into a plasmid vector under the control of the strong bacteriophage T7 transcription regulatory system. Effective Escherichia coli producer strains were obtained, and optimal conditions were chosen for the production of resulting constructs. The optimal pH and temperature ranges of recombinant proteins were determined, and three-dimensional shapes of their molecules were also predicted. Methods of low-stage protein purification were improved. Some of the isolated proteins demonstrated a high level of expression, solubility and purity. Conclusions: Novel chimeric proteins were obtained which had not previously been observed in nature in such domain combinations. It was shown that the biotechnologically valuable characteristics of the hybrid proteins were more marked when the thermal-resistant partner was combined with the Nterminus of pneumococcal protein. The principles of their low-stage purification were performed which does not require any special equipment. That is a basis for significant reduction of prices for diagnostic test-systems components and subunit vaccine production. How to cite: Grishin DV, Sidorov NG, Parfenova OK, et al. Construction, heterological expression and a simple purification of the BP region of the pneumococcal surface protein a fused in different orientations to the chemotaxis adaptor protein CheW from Thermotoga petrophila. Electron J Biotechnol 2024;71. [ABSTRACT FROM AUTHOR]

Selective RET inhibitors, such as selpercatinib and pralsetinib, have revolutionized the treatment of cancers with RET gene alterations. These inhibitors have shown remarkable clinical efficacy, particularly in RET-driven lung cancer, medullary thyroid cancer, and other solid tumors driven by RET gene fusions. The assessment of treatment response in oncology has been greatly enhanced by Fluorodeoxyglucose Positron Emission Tomography (FDG-PET), a valuable tool that measures tumor metabolism and provides early indicators of treatment effectiveness. This work explores the effectiveness of selective RET inhibitors in targeting RET-positive cancers and investigates the utility of FDG-PET in assessing treatment response. The paper includes insightful case studies that highlight the successful application of RET inhibitors in the treatment of RET-positive cancers. The findings suggest that FDG-PET has the potential to serve as a non-invasive biomarker for monitoring treatment response in patients with RET-positive cancers. However, further research is required to establish standardized criteria for interpreting FDG-PET scans in the context of selective RET inhibitors and to uncover the broader applications of FDG-PET in precision oncology. [ABSTRACT FROM AUTHOR]

Thermomyces dupontii harbors two P450 paralogs (P450S and P450L) in the gene cluster for the biosynthesis of prenylated indole alkaloids (PIAs) and correponding iron chelators with P450L assigned as one protein containing a CYP like domain fused with a FAD-binding domain-containing oxidoreductase. Genetic manipulation and metabolic profile analysis indicated both P450S and P450L were involved in transforming simple PIAs to their corresponding iron chelators. Moreover, P450S is responsible for bolstering simple PIAs to complex PIAs, and P450L for reinforcing conjugating unsaturated systems in complex PIAs. Chemical investigation led to isolation and characterization of novel complex PIA metabolites with more oxidations. P450L also contributed to forming the third iron-chelating core in iron chelators. A series of iron bioassays and infrastructure analysis revealed that lack of these P450 genes caused strongly elevated Fe3+ levels but attenuated Fe2+ levels, together with abnormal mitochondria in mycelia and lipid droplets and vacuoles in conidia. Phenotype analysis revealed that P450S and P450L facilitated fungal colony pigments, conidial formation and germination via bolstering conidiophores and cell walls in response to temperature reduction. [ABSTRACT FROM AUTHOR]

The molecular characterization of soft tissue and bone tumors is a rapidly evolving field that has changed the perspective of how these tumors are diagnosed today. Morphology and clinico-radiological context still represent the cornerstone of diagnostic considerations but are increasingly complemented by molecular data that aid in objectifying and confirming the classification. The spectrum of analyses comprises mutation or gene fusion specific immunohistochemical antibodies, fluorescence in situ hybridization, DNA and RNA sequencing as well as CpG methylation profiling. This article provides an overview of which tools are presently available to characterize bone and soft tissue neoplasms molecularly, what limitations should be considered, and what conclusions can be drawn from the individual findings. [ABSTRACT FROM AUTHOR]

NUT carcinoma is a rare, aggressive malignancy defined as a carcinoma with a chromosomal rearrangement affecting the nuclear protein in testis (NUTM1) gene. This small round blue cell tumor classically exhibits focal abrupt keratinization and immunohistochemical positivity for keratin and squamous markers. However, keratinization is not always present and reports of positivity for other markers that may obscure the diagnosis are increasing. It is also noteworthy that gene fusions involving NUTM1 are not restricted to NUT carcinoma. Herein, we report a NUT carcinoma arising in the mediastinum of a male patient in his 40 s with morphological and immunohistochemical overlap with Ewing family sarcoma and poorly differentiated synovial sarcoma given a round cell morphology, diffuse strong immunoreactivity for CD99, and patchy strong immunoreactivity for TLE1. Squamous differentiation by morphology and p40 expression were notably absent in this case. Classification as NUT carcinoma was ultimately possible when the morphological and immunohistochemical findings were considered in the context of a BRD4::NUTM1 gene fusion identified by next-generation sequencing. While the patient initially responded to palliative radiotherapy, he died approximately one month later. To our knowledge, this is the first report of TLE1 immunoreactivity in NUT carcinoma. This case highlights a potential diagnostic pitfall and emphasizes the need for molecular confirmation in equivocal situations. [ABSTRACT FROM AUTHOR]

ETV6::ABL1 fusion gene is a rare but recurrent genomic rearrangement in hematological malignancies with poor prognosis. Here, we report 1 case of Ph negative myeloid/lymphoid neoplasms with eosinophilia and tyrosine kinase gene fusions (MLN-TK) who carry ETV6::ABL1 fusion gene. The patient achieved clinical remission after treatment with imatinib. However, disease progression of blast crisis was observed around 2 years later. The patient was treated with second-generation tyrosine kinase inhibitor of flumatinib, yielded a short term second therapeutic response. ETV6::ABL1 positive myeloid/lymphoid neoplasms with eosinophilia and tyrosine kinase gene fusions (MLN-TK) is rare and may be misdiagnosed by conventional cytogenetical analysis. Early treatment with TKIs, particularly second-generation TKIs, may be beneficial to improve treatment results. [ABSTRACT FROM AUTHOR]

Acute myeloid leukemia (AML) with t(8;21) (q22;q22), which forms RUNX1::RUNX1T1 fusion gene, is classified as a favorable-risk group. However, the presence of mutations in KIT exon 17 results in an adverse prognosis in this group. Avapritinib, a novel tyrosine kinase inhibitor, was designed to target KIT mutation. We report a retrospective study of four pediatric patients with AML with t(8:21) and KIT exon 17 mutation who were treated with avapritinib, three of them failed to demethylate drugs and donor lymphocyte infusion targeting RUNX1::RUNX1T1-positivity after allogeneic hematopoietic stem cell transplantation (allo-HSCT). So far, all patients with RUNX1::RUNX1T1 positivity had turned negative after 1, 9, 7, 2 months of avapritinib treatment. The common adverse effect of avapritinib is neutropenia, which is well-tolerated. This case series indicates that avapritinib may be effective and safe for preemptive treatment of children with AML with t(8;21) and KIT mutation after allo-HSCT, providing a treatment option for preventing relapse after allo-HSCT. [ABSTRACT FROM AUTHOR]

The E2A-PBX1 gene fusion is a common translocation in B-cell acute lymphoblastic leukaemia. Patients harbouring the E2A-PBX1 fusion gene typically exhibit an intermediate prognosis. Furthermore, minimal residual disease has unsatisfactory prognostic value in E2A-PBX1 B-cell acute lymphoblastic leukaemia. However, the mechanism of E2A-PBX1 in the occurrence and progression of B-cell acute lymphoblastic leukaemia is not well understood. Here, we mainly review the roles of E2A and PBX1 in the differentiation and development of B lymphocytes, the mechanism of E2A-PBX1 gene fusion in B-cell acute lymphoblastic leukaemia, and the potential therapeutic approaches. [ABSTRACT FROM AUTHOR]

Minimal residual disease (MRD) based risk stratification criteria for specific genetic subtypes remained unclear in childhood acute lymphoblastic leukemia (ALL). Among 723 children with newly diagnosed ALL treated with the Chinese Children Leukemia Group CCLG-2008 protocol, MRD was assessed at time point 1 (TP1, at the end of induction) and TP2 (before consolidation treatment) and the MRD levels significantly differed in patients with different fusion genes or immunophenotypes (P all < 0.001). Moreover, the prognostic impact of MRD varied by distinct molecular subtypes. We stratified patients in each molecular subtype into two MRD groups based on the results. For patients carrying BCR::ABL1 or KMT2A rearrangements, we classified patients with MRD < 10–2 at both TP1 and TP2 as the low MRD group and the others as the high MRD group. ETV6::RUNX1+ patients with TP1 MRD < 10–3 and TP2 MRD-negative were classified as the low MRD group and the others as the high MRD group. For T-ALL, We defined children with TP1 MRD ≥ 10–3 as the high MRD group and the others as the low MRD group. The 10-year relapse-free survival of low MRD group was significantly better than that of high MRD group. We verified the prognostic impact of the subtype-specific MRD-based stratification in patients treated with the BCH-ALL2003 protocol. In conclusion, the subtype-specific MRD risk stratification may contribute to the precise treatment of childhood ALL. [ABSTRACT FROM AUTHOR]

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Abstract Background Anaplastic lymphoma kinase (ALK) test in advanced non‐small cell lung cancer (NSCLC) can help physicians provide target therapies for patients harboring ALK gene rearrangement. This study aimed to investigate the real‐world test patterns and positive rates of ALK gene rearrangements in advanced NSCLC. Methods In this real‐world study (ChiCTR2000030266), patients with advanced NSCLC who underwent an ALK rearrangement test in 30 medical centers in China between October 1, 2018 and December 31, 2019 were retrospectively analyzed. Interpretation training was conducted before the study was initiated. Quality controls were performed at participating centers using immunohistochemistry (IHC)‐VENTANA‐D5F3. The positive ALK gene rearrangement rate and consistency rate were calculated. The associated clinicopathological characteristics of ALK gene rearrangement were investigated as well. Results The overall ALK gene rearrangement rate was 6.7% in 23,689 patients with advanced NSCLC and 8.2% in 17,436 patients with advanced lung adenocarcinoma. The quality control analysis of IHC‐VENTANA‐D5F3 revealed an intra‐hospital consistency rate of 98.2% (879/895) and an inter‐hospital consistency rate of 99.2% (646/651). IHC‐VENTANA‐D5F3 was used in 53.6%, real‐time polymerase chain reaction (RT‐PCR) in 25.4%, next‐generation sequencing (NGS) in 18.3%, and fluorescence in‐situ hybridization (FISH) in 15.9% in the adenocarcinoma subgroup. For specimens tested with multiple methods, the consistency rates confirmed by IHC‐VENTANA‐D5F3 were 98.0% (822/839) for FISH, 98.7% (1,222/1,238) for NGS, and 91.3% (146/160) for RT‐PCR. The overall ALK gene rearrangement rates were higher in females, patients of ≤ 35 years old, never smokers, tumor cellularity of > 50, and metastatic specimens used for testing in the total NSCLC population and adenocarcinoma subgroup (all P < 0.05). Conclusions This study highlights the real‐world variability and challenges of ALK test in advanced NSCLC, demonstrating a predominant use of IHC‐VENTANA‐D5F3 with high consistency and distinct clinicopathological features in ALK‐positive patients. These findings underscore the need for a consensus on optimal test practices and support the development of refined ALK test strategies to enhance diagnostic accuracy and therapeutic decision‐making in NSCLC.

ETV6::ABL1 gene fusion is a rare recurrent genomic rearrangement associated with hematologic malignancies, and frequently occurs with additional anomalies. Due to the opposite chromosome orientations of the ETV6 and ABL1 genes, an oncogenic in-frame ETV6::ABL1 gene fusion cannot be formed by a simple translocation. The molecular mechanism of the ETV6::ABL1 fusion and the significance of co-occurring anomalies are not fully understood. We characterized genomic alterations in an individual with ETV6::ABL1 gene-fusion-positive myeloid neoplasm using various genomic technologies. Our findings uncovered a molecular mechanism of the ETV6::ABL1 fusion, in which a paracentric inversion within the short arm of chromosome 12 (12p) and a translocation between the long arm of a chromosome 9 and the 12p with the inversion were involved. In addition, we detected multiple additional anomalies in the individual, and our findings suggested that the ETV6::ABL1 fusion occurred as a secondary event in a subset of cells with the additional anomalies. We speculate that the additional anomalies may predispose to further pathogenic changes, including ETV6::ABL1 fusion, leading to neoplastic transformation.

Oxford Nanopore Technologies sequencing, also referred to as Nanopore sequencing, stands at the forefront of a revolution in clinical genetics, offering the potential for rapid, long read, and real‐time DNA and RNA sequencing. This technology is currently making sequencing more accessible and affordable. In this comprehensive review, we explore its potential regarding precision cancer diagnostics and treatment. We encompass a critical analysis of clinical cases where Nanopore sequencing was successfully applied to identify point mutations, splice variants, gene fusions, epigenetic modifications, non‐coding RNAs, and other pivotal biomarkers that defined subsequent treatment strategies. Additionally, we address the challenges of clinical applications of Nanopore sequencing and discuss the current efforts to overcome them. [ABSTRACT FROM AUTHOR]

Hairy cell leukemia (HCL) is an uncommon mature B-cell malignancy characterized by a typical morphology, immunophenotype, and clinical profile. The vast majority of HCL patients harbor the canonical BRAF V600E mutation which has become a rationalized target of the subsequently deregulated RAS-RAF-MEK-MAPK signaling pathway in HCL patients who have relapsed or who are refractory to front-line therapy. However, several HCL patients with a classical phenotype display non-canonical BRAF mutations or rearrangements. These include sequence variants within alternative exons and an oncogenic fusion with the IGH gene. Care must be taken in the molecular diagnostic work-up of patients with typical HCL but without the BRAF V600E to include investigation of these uncommon mechanisms. Identification, functional characterization, and reporting of further such patients is likely to provide insights into the pathogenesis of HCL and enable rational selection of targeted inhibitors in such patients if required. [ABSTRACT FROM AUTHOR]

Mesenchymal tumors may display morphologic and immunohistochemical overlap with melanocytic tumors, presenting a pitfall for misdiagnosis. We report a 62‐year‐old woman who presented with a recurrent dermal and subcutaneous tumor over the Achilles tendon 15 years following complete excision. Both the primary and the recurrent tumors were characterized by nests and sheets of epithelioid and spindle cells with eosinophilic cytoplasm and uniform ovoid nuclei. The tumor was positive for S100, SOX10, HMB45, cathepsin K, and p63 (weak), while negative for Melan‐A, MiTF, smooth muscle actin, and desmin. Gene fusion analysis of the recurrent tumor revealed a NONO::TFE3 fusion which has been recently reported in two similar cutaneous cases. Our case highlights the potential of a NONO::TFE3 cutaneous epithelioid and spindle cell tumor to recur after a prolonged disease‐free interval without evidence of high‐grade transformation or distant metastasis. Our findings support its classification as a cutaneous mesenchymal neoplasm of intermediate malignancy. [ABSTRACT FROM AUTHOR]

NRAS Q61 mutations are driver genetic alterations associated with common melanocytic nevi. Herein, we describe a case of NRAS‐mutant melanocytic tumor with a blue nevus‐like morphology. A 71‐year‐old Japanese man presented with a 4.6‐mm nodule on his back. Histopathological examination revealed a dense distribution of spindle‐shaped melanocytes in the upper dermis and a sparse distribution of dendritic melanocytes in the mid‐dermis. The vertical periadnexal extension reached the deep dermis at the center of the tumor. A small junctional component, hyperpigmentation, sclerotic stroma, mild nuclear atypia, and a few mitotic figures were observed. Immunohistochemical examination revealed no PRAME expression and preserved p16 expression. Diffuse RASQ61R immunoreactivity was observed in these tumor cells. Nuclear β‐catenin expression was not observed. Targeted RNA sequencing revealed two mutations, NRAS c.182A>G (Q61R) and FGFR2 c.‐157A>G, but no other pathogenic alterations such as BRAF, GNAQ, GNA11, CTNNB1, PRKAR1A, or IDH1 mutations or kinase gene fusions. The histopathology fits that of compound‐type blue nevus, which is called "Kamino nevus"; however, this tumor was genetically considered to be on the spectrum of conventional acquired melanocytic nevi but not on that of blue nevi. Morphologically, NRAS‐driven melanocytic nevi resemble blue nevi without IDH1R132C coexistence. [ABSTRACT FROM AUTHOR]

A 50-year-old male presented with neck and shoulder pain. Chest CT and 18F-FDG PET/CT revealed osteolytic bone destruction in the left first rib and thoracic vertebrae with increased FDG uptake. Rib biopsy pathology indicated desmoplastic small round cell tumor (DSRCT).18F-FDG PET/CT can accurately locate the distribution of DSRCT and further guide the location of needle biopsy to assist the DSRCT. [ABSTRACT FROM AUTHOR]

Canalicular-like pleomorphic adenomas are a relatively recently described entity, that possess features of both canalicular adenomas and pleomorphic adenomas. The presence of unusual HMGA2 -fusion partners (most commonly HMGA2::WIF1 gene fusions) has established canalicular-like pleomorphic adenoma as a distinct entity. The use of intraoperative frozen section analysis and surrogate HMGA2 IHC are 2 tools that can provide the surgical team with valuable insight into intraoperative decision making and final classification of rare tumors of the parotid gland, respectively. We present a case of canalicular-like pleomorphic adenoma and characterize its appearance on frozen section analysis. HMGA2 IHC staining was retroactively performed, assisting in the confirmation of the tumor subtype. [ABSTRACT FROM AUTHOR]

The diagnosis of low-grade fibroblastic/myofibroblastic tumors of acral sites can be challenging. These tumors encompass a diverse group of neoplasms with a spectrum of biologic potential ranges from benign to overtly malignant. They often demonstrate significant clinical, radiologic, and immunophenotypic overlap, in which the molecular phenotype may play an important diagnostic role to arrive at the final diagnosis. Herein, we report a case of soft tissue mass lesion presented on the palm of an adult patient for four months. Histologically, the tumor consisted of primarily low-grade spindle cells expressing smooth muscle actin. Molecular testing revealed a novel SREBF1::USP6 fusion gene, confirming the final diagnosis of nodular fasciitis and ultimately expanding its molecular profile. This case highlights the diagnostic value of single, cost-effective, targeted molecular panel to arrive at an accurate diagnosis and provide helpful therapeutic information. [ABSTRACT FROM AUTHOR]

Abstract The incidence of esophageal adenocarcinoma (EAC) has surged by 600% in recent decades, with a dismal 5-year survival rate of just 15%. Barrett’s esophagus (BE), affecting about 2% of the population, raises the risk of EAC by 40-fold. Despite this, the transcriptomic changes during the BE to EAC progression remain unclear. Our study addresses this gap through comprehensive transcriptomic profiling to identify key mRNA signatures and genomic alterations, such as gene fusions. We performed RNA-sequencing on BE and EAC tissues from 8 individuals, followed by differential gene expression, pathway and network analysis, and gene fusion prediction. We identified mRNA changes during the BE-to-EAC transition and validated our results with single-cell RNA-seq datasets. We observed upregulation of keratin family members in EAC and confirmed increased levels of keratin 14 (KRT14) using immunofluorescence. More differentiated BE marker genes are downregulated during progression to EAC, suggesting undifferentiated BE subpopulations contribute to EAC. We also identified several gene fusions absent in paired BE and normal esophagus but present in EAC. Our findings are critical for the BE-to-EAC transition and have the potential to promote early diagnosis, prevention, and improved treatment strategies for EAC.

Thermomyces dupontii harbors two P450 paralogs (P450S and P450L) in the gene cluster for the biosynthesis of prenylated indole alkaloids (PIAs) and correponding iron chelators with P450L assigned as one protein containing a CYP like domain fused with a FAD-binding domain-containing oxidoreductase. Genetic manipulation and metabolic profile analysis indicated both P450S and P450L were involved in transforming simple PIAs to their corresponding iron chelators. Moreover, P450S is responsible for bolstering simple PIAs to complex PIAs, and P450L for reinforcing conjugating unsaturated systems in complex PIAs. Chemical investigation led to isolation and characterization of novel complex PIA metabolites with more oxidations. P450L also contributed to forming the third iron-chelating core in iron chelators. A series of iron bioassays and infrastructure analysis revealed that lack of these P450 genes caused strongly elevated Fe3+ levels but attenuated Fe2+ levels, together with abnormal mitochondria in mycelia and lipid droplets and vacuoles in conidia. Phenotype analysis revealed that P450S and P450L facilitated fungal colony pigments, conidial formation and germination via bolstering conidiophores and cell walls in response to temperature reduction.

Lung cancer is the malignant tumor with the highest incidence and mortality rate worldwide. For lung adenocarcinoma, identifying specific gene mutations, fusions, and giving corresponding targeted drugs can greatly improve the survival time of the patients. Among them, anaplastic lymphoma kinase (ALK) fusion occurs in 3%-7% of non-small cell lung cancer (NSCLC). In clinical practice, a variety of detection methods can be used to determine the ALK fusion status, but false negative test results are possible. This paper retrospectively analyzed the diagnosis and treatment of a patient with lung adenocarcinoma, judged the ALK fusion status by various detection methods. Among them, immunohistochemistry (IHC)(Ventana D5F3), RNA based next-generation sequencing (RNA-based NGS) confirmed positive echinoderm microtubule associated protein like 4 (EML4)-ALK fusion, while DNA-based NGS was negative. This paper analyzed the detection methods of ALK fusion, in order to clarify which detection method is the most accurate and simple to choose in different clinical cases and guide the subsequent treatment.

Background: Pulmonary sarcomatoid carcinoma is a rare tumor that is resistant to cytotoxic agents. This observational study aimed to evaluate the detection rate of driver gene alteration and the efficacy of targeted therapy for pulmonary sarcomatoid carcinoma. Methods: We established a database of patients with pulmonary sarcomatoid carcinoma and their clinical information, including EGFR mutation, ALK fusion gene, ROS1 fusion gene, BRAF mutation, and MET exon 14 skipping mutation. The present study retrieved and analyzed the data of patients with pulmonary sarcomatoid carcinoma in whom driver gene alterations were evaluated, and the survival duration after the initiation of treatment with targeted therapy was examined. Results: A total of 44 patients were included in the present study. The EGFR mutation, ALK fusion gene, and MET exon 14 skipping mutation were detected in 2/43 patients (4.7%), 2/34 patients (5.9%), and 2/16 patients (12.5%), respectively. The ROS1 fusion gene (0/18 patients) and BRAF mutation (0/15 patients) were not detected. Female patients (P = 0.063, Fisher's exact test) and patients without smoking history (P = 0.025, Fisher's exact test) were the dominant groups in which any driver mutation was detected. Five patients with driver gene alterations were treated with targeted therapy. Progression-free survival (PFS) was 1.3 months and 1.6 months in 2 of the patients treated with gefitinib. Two patients with the ALK fusion gene showed 2.1 and 14.0 months of PFS from the initiation of treatment with crizotinib, and a patient with the MET exon 14 skipping mutation showed 9.7 months of PFS from the initiation of treatment with tepotinib. Conclusion: The EGFR mutation, ALK fusion gene, and MET exon 14 skipping mutation were detected in patients with pulmonary sarcomatoid carcinoma in clinical practice, and some patients achieved long survival times after receiving targeted therapy. Further investigation is necessary to evaluate the efficacy of targeted therapy for pulmonary sarcomatoid carcinoma. [ABSTRACT FROM AUTHOR]

The human insulin gene modified with a C-peptide was synthesized according to the plant-preferred codon, and a fusion gene expression vector of insulin combined with green fluorescent protein (GFP) was constructed. The optimization of the flax callus culturing was undertaken, and a more efficient Agrobacterium-mediated genetic transformation of the flax hypocotyls was achieved. The critical concentration values of hygromycin on the flax hypocotyl development, as well as on its differentiated callus, were explored by the method of antibiotic gradient addition, and the application of antibiotic screening for the verification of positive calluses was assessed. The fusion gene of insulin and GFP was successfully inserted into the flax genome and expressed, as confirmed through polymerase chain reaction and Western blotting. In conclusion, we have established a flax callus culture system suitable for insulin expression. By optimizing the conditions of the flax callus induction, transformation, screening, and verification of a transgenic callus, we have provided an effective way to obtain insulin. Moreover, the herein-employed flax callus culture system could provide a feasible, cheap, and environmentally friendly platform for producing bioactive proteins. [ABSTRACT FROM AUTHOR]

Objective: Patients who carry NUP98::NSD1 or FLT3/ITD mutations are reported to have poor prognosis. Previous studies have confidently reported that the poor outcome in younger AML patients is owning to dual NUP98::NSD1 and FLT3/ITD positivity, with a high overlap for those two genetic lesions. In this study, we assessed the prognostic value of the presence of both NUP98::NSD1 and FLT3/ITD in pediatric AML patients. Methods: We screened a large cohort of 885 pediatric cases from the COG-National Cancer Institute (NCI) TARGET AML cohort and found 57 AML patients with NUP98 rearrangements. Results: The frequency of NUP98 gene fusion was 10.8% in 529 patients. NUP98::NSD1 fusion was the most common NUP98 rearrangement, with a frequency of 59.6%(34 of 57). NUP98::NSD1 -positive patients who carried FLT3/ITD mutations had a decreased CR1 or CR2 rate than those patients carried FLT3/ITD mutation alone (P = 0.0001). Moreover, patients harboring both NUP98::NSD1 fusion and FLT3/ITD mutation exhibited inferior event-free survival (EFS, P < 0.001) and overall survival (OS, P = 0.004) than patients who were dual negative for these two genetic lesions. The presence of only NUP98::NSD1 fusion had no significant impact on EFS or OS. We also found that cases with high FLT3/ITD AR levels (> = 0.5) with or without NUP98::NSD1 had inferior prognosis. Multivariate analysis demonstrated that the presence of both NUP98::NSD1 and FLT3/ITD was an independent prognostic factors for EFS (hazard ratio: 3.2, P = 0.001) in patients with pediatric AML. However, there was no obvious correlation with OS (hazard ratio: 1.3, P = 0.618). Stem cell transplantation did not improve the survival rate of cases with NUP98 fusion or NUP98::NSD1 AML in terms of EFS or OS. Conclusion: Presence of both NUP98::NSD1 and FLT3/ITD was found to be an independent factor for dismal prognosis in pediatric AML patients. Notably, lack of FLT3/ITD mutations in NUP98::NSD1 -positive patients did not retain its prognostic value. [ABSTRACT FROM AUTHOR]

Chronic myeloid leukemia (CML) is characterized by the Philadelphia chromosome, leading to the BCR::ABL1 fusion gene and hyper-proliferation of granulocytes. Tyrosine kinase inhibitors (TKIs) are effective, and minimal residual disease (MRD) monitoring is crucial. Digital PCR platforms offer increased precision compared to quantitative PCR but lack comparative studies.Eighty CML patient samples were analyzed in parallel using digital droplet PCR (ddPCR) (QXDx™ BCR-ABL %IS Kit) and chip digital PCR (cdPCR) (Dr. PCR™ BCR-ABL1 Major IS Detection Kit).Overall, qualitative and quantitative agreement was good. Sensitivity analysis showed positive percentage agreement and negative percentage agreement were both ≥90 %, and the quadratic weighted kappa index for molecular response (MR) level categorization was 0.94 (95 %CI 0.89, 0.98). MR levels subgroup analysis showed perfect categorical agreement on MR level at MR3 or above, while 35.4 % (17/48) of patient samples with MR4 or below showed discordant categorizations. Overall, Lin’s concordance correlation coefficient (CCC) for the ratio of %BCR::ABL1/ABL1 converted to the International Scale (BCR::ABL1IS) was almost perfect quantitative agreement (Lin’s CCC=0.99). By subgroups of MR levels, Lin’s CCC showed a quantitative agreement of BCR::ABL1IS decreased as MR deepened.Both cdPCR and ddPCR demonstrated comparable performance in detecting BCR::ABL1 transcripts with high concordance in MR3 level or above. Choosing between platforms may depend on cost, workflow, and sensitivity requirements. [ABSTRACT FROM AUTHOR]

Background: The current standard of care treatments for medulloblastoma are insufficient as these do not take tumor heterogeneity into account. Newer, safer, patient-specific treatment approaches are required to treat high-risk medulloblastoma patients who are not cured by the standard therapies. Immunotherapy is a promising treatment modality that could be key to improving survival and avoiding morbidity. For an effective immune response, appropriate tumor antigens must be targeted. While medulloblastoma patients with subgroup-specific genetic substitutions have been previously reported, the immunogenicity of these genetic alterations remains unknown. The aim of this study is to identify potential tumor rejection antigens for the development of antigen-directed cellular therapies for medulloblastoma. Methods: We developed a cancer immunogenomics pipeline and performed a comprehensive analysis of medulloblastoma subgroup-specific transcription profiles (n = 170, 18 WNT, 46 SHH, 41 Group 3, and 65 Group 4 patient tumors) available through International Cancer Genome Consortium (ICGC) and European Genome-Phenome Archive (EGA). We performed in silico antigen prediction across a broad array of antigen classes including neoantigens, tumor-associated antigens (TAAs), and fusion proteins. Furthermore, we evaluated the antigen processing and presentation pathway in tumor cells and the immune infiltrating cell landscape using the latest computational deconvolution methods. Results: Medulloblastoma patients were found to express multiple private and shared immunogenic antigens. The proportion of predicted TAAs was higher than neoantigens and gene fusions for all molecular subgroups, except for sonic hedgehog (SHH), which had a higher neoantigen burden. Importantly, cancer-testis antigens, as well as previously unappreciated neurodevelopmental antigens, were found to be expressed by most patients across all medulloblastoma subgroups. Despite being immunologically cold, medulloblastoma subgroups were found to have distinct immune cell gene signatures. Conclusions: Using a custom antigen prediction pipeline, we identified potential tumor rejection antigens with important implications for the development of immunotherapy for medulloblastoma. [ABSTRACT FROM AUTHOR]

Rhabdomyosarcoma (RMS) is the most common childhood soft tissue sarcoma. For the alveolar subtype (ARMS), the presence of the PAX3::FOXO1 fusion gene and/or metastases are strong predictors of poor outcome. Metastatic PAX3::FOXO1+ ARMS often responds to chemotherapies initially, only to subsequently relapse and become resistant with most patients failing to survive beyond 8 years post-diagnosis. No curative intent phase II or phase III clinical trial has been available for patients in the past 10 years (ARST0921). Thus, metastatic ARMS represents a significantly unmet clinical need. Chemotherapy resistance in ARMS has previously been attributed to PAX3::FOXO1-mediated cell cycle checkpoint adaptation, which is mediated by an HDAC3-SMARCA4-miR-27a-PAX3::FOXO1 circuit that can be disrupted by HDAC3 inhibition. In this study, we investigated the therapeutic efficacy of combining the epigenetic regulator entinostat, a Class I Histone Deacetylase (HDAC1-3) inhibitor, with RMS-specific chemotherapies in patient derived xenograft (PDX) models of RMS. We identified single agent, additive or synergistic relationships between relapse-specific chemotherapies and clinically relevant drug exposures of entinostat in three PAX3::FOXO1+ ARMS mouse models. This preclinical data provides further rationale for clinical investigation of entinostat, already known to be well tolerated in a pediatric phase I clinical trial (ADVL1513). [ABSTRACT FROM AUTHOR]

Aims: Compared to primary breast sarcoma (BSs), radiotherapy-induced sarcoma (RIS) is a less frequent type of secondary breast sarcoma. Undifferentiated pleomorphic sarcoma (UPS) is an even rarer occurrence within the RIS category. This study aimed to present the clinicopathologic and molecular features of breast radiotherapy-induced UPS. Methods: A retrospective study was conducted at the Third Affiliated Hospital of Soochow University to analyze three patients with radiation-induced undifferentiated pleomorphic sarcoma (UPS) following breast cancer, spanning from 2006 to 2023. The clinical and pathological variables were extracted from the medical records, while immunohistochemistry was employed to analyze the immunophenotypes of these tumors. Genomic characteristics were assessed through DNA and RNA sequencing techniques. Another 15 cases from the literature were also reviewed to better characterize the tumor. Results: The affected areas encompass the chest wall and breasts, with an incubation period ranging from 6 to 17 years. The tumor cells exhibit pleomorphism and demonstrate a high degree of pathological mitosis. Notably, two cases displayed an accelerated disease progression, characterized by recurrent tumors and metastases occurring within short intervals of 48 and 7 months respectively subsequent to the initial diagnosis. The two prevailing identified genes were TP53 (2/3, 66.7%) and RB1 (1/3, 33.3%). Through analysis of somatic copy number variation (CNV), it was discovered that two oncogenes, MCL1 (1/3, 33.3%) and MYC (1/3, 33.3%), had experienced gains in CNV. The Tumor Mutational Burden (TMB) values for case 1, case 2, and case 3 were 5.9 mut/Mb, 1.0 mut/Mb, and 3.0 mut/Mb, respectively. Moreover, the analysis of RNA-NGS (next-generation sequencing) revealed the presence of a novel gene fusion, named COL3A1-GULP1, in case 2. Conclusions: Based on our thorough analysis of research findings and previous reports, it is evident that radiotherapy-induced UPS exhibits a highly diverse and frequently severe clinical and biological behavior. Identifying tumor formation using genome sequencing can help understand its biological behavior and determine personalized treatments. [ABSTRACT FROM AUTHOR]

Adenoid cystic carcinoma (AdCC) is a salivary gland neoplasm that infrequently appears in the sinonasal region. The aim of this study was to evaluate the outcome and clinicopathological parameters of sinonasal AdCC. A retrospective analysis was conducted on all cases of AdCC affecting the nasal cavity or paranasal sinuses between 2000 and 2018 at the University Hospital Zurich. Tumor material was examined for morphological features and analyzed for molecular alterations. A total of 14 patients were included. Mean age at presentation was 57.7 years. Sequencing revealed MYB::NFIB gene fusion in 11/12 analyzable cases. Poor prognostic factors were solid variant (p < 0.001), histopathological high-grade transformation (p < 0.001), and tumor involvement of the sphenoid sinus (p = 0.02). The median recurrence-free survival (RFS) and OS were 5.2 years and 11.3 years. The RFS rates at 1-, 5-, and 10-year were 100%, 53.8%, and 23.1%. The OS rates at 1-, 5-, and 10- years were 100%, 91.7%, and 62.9%, respectively. In Conclusion, the solid variant (solid portion > 30%), high-grade transformation, and sphenoid sinus involvement are negative prognostic factors for sinonasal AdCC. A high prevalence of MYB::NFIB gene fusion may help to correctly classify diagnostically challenging (e.g. metatypical) cases. [ABSTRACT FROM AUTHOR]

Background: Clear cell odontogenic carcinoma (CCOC) is an odontogenic carcinoma characterized by sheets and islands of vacuolated and clear cells. The diagnosis of atypical CCOC can pose a challenge when tumor cells deviate from their characteristic clear morphology, even with the aid of genetic profiling for CCOC identification. Case presentation: In this manuscript, we detailed the inaugural instance of a recurrently recurring clear cell odontogenic carcinoma (CCOC) with pronounced squamous differentiation in a 64-year-old male. The primary tumor in this individual initially displayed a biphasic clear cell phenotype. However, subsequent to the third recurrence, the clear tumor cells were entirely supplanted by epidermoid cells characterized by eosinophilic cytoplasm, vesicular chromatin, and prominent nucleoli. Notable aggressive attributes such as necrosis, conspicuous cytological malignancy, perineural dissemination, and vascular invasion were noted. Additionally, the tumor progressed to manifest lung metastases. The tumor cells exhibited positive immunoreactivity for AE1/AE3, KRT19, Pan-CK, EMA, P40, P63, CK34βE12, and P53, while they tested negative for CK35βH11, KRT7, S-100, and neuroendocrine markers. The Ki-67 proliferation index was calculated at an average of 15%. Furthermore, FISH analysis unveiled the presence of the EWSR1::ATF1 gene fusion. Conclusions: This case illustrated a rare and aggressive case of CCOC characterized by significant squamous differentiation upon recurrence of the tumor. [ABSTRACT FROM AUTHOR]

Background: Collagen triple helix repeat containing-1 (CTHRC1), an extracellular matrix protein, is highly expressed in hepatocellular carcinoma (HCC) and linked to poor prognosis. Nevertheless, the precise mechanism of CTHRC1 in HCC is unclear. Methods: Agena MassARRAY® Methylation Analysis assessed the methylation level of CTHRC1 in the promoter region. Functional assays were conducted to investigate the effects of CTHRC1 knockdown in Hep3B2.1 cells. RNA sequencing identified differentially expressed genes and lncRNAs associated with angiogenesis after CTHRC1 knockdown. Furthermore, differential alternative splicing (AS) and gene fusion events were analyzed using rMATS and Arriba. Results: In HCC cell lines, CTHRC1 was highly expressed and associated with hypomethylation. Downregulation of CTHRC1 inhibited Hep3B2.1 cell proliferation, migration, and invasion, blocked cells in the G1/S phase, and promoted apoptosis. We obtained 34 mRNAs and 7 lncRNAs differentially expressed between the NC and CTHRC1 inhibitor groups. Additionally, we found 4 angiogenesis-related mRNAs and lncRNAs significantly correlated with CTHRC1. RT-qPCR results showed that knockdown of CTHRC1 in Hep3B2.1 cells resulted in significantly aberrant expression of CXCL6, LINC02127, and AC020978.8. Moreover, the role of CTHRC1 in HCC development may be associated with events, like 12 AS events and 5 pairs of fusion genes. Conclusions: High expressed CTHRC1 is associated with hypomethylation and may promote HCC development, involving events like angiogenesis, alternative splicing, and gene fusion. [ABSTRACT FROM AUTHOR]

The incidence of esophageal adenocarcinoma (EAC) has surged by 600% in recent decades, with a dismal 5-year survival rate of just 15%. Barrett's esophagus (BE), affecting about 2% of the population, raises the risk of EAC by 40-fold. Despite this, the transcriptomic changes during the BE to EAC progression remain unclear. Our study addresses this gap through comprehensive transcriptomic profiling to identify key mRNA signatures and genomic alterations, such as gene fusions. We performed RNA-sequencing on BE and EAC tissues from 8 individuals, followed by differential gene expression, pathway and network analysis, and gene fusion prediction. We identified mRNA changes during the BE-to-EAC transition and validated our results with single-cell RNA-seq datasets. We observed upregulation of keratin family members in EAC and confirmed increased levels of keratin 14 (KRT14) using immunofluorescence. More differentiated BE marker genes are downregulated during progression to EAC, suggesting undifferentiated BE subpopulations contribute to EAC. We also identified several gene fusions absent in paired BE and normal esophagus but present in EAC. Our findings are critical for the BE-to-EAC transition and have the potential to promote early diagnosis, prevention, and improved treatment strategies for EAC. [ABSTRACT FROM AUTHOR]

Summary Rearrangement of NUTM1 gene (NUTM1r) is one of the most frequent aberrations occurring in infants (younger than 1 year at diagnosis) with B‐cell precursor Acute Lymphoblastic Leukaemia (BCP‐ALL). In this study we had the unique opportunity to analyze the umbilical cord blood (UCB) sample from one infant patient with NUTM1r BCP‐ALL. Herein we reported for the first time that NUTM1r infant ALL arise prenatally, as both the patient‐specific CUX1::NUTM1 fusion gene, as well as two IG/TR leukaemic markers were already present and detectable in the patient's UCB at birth. Our results clearly demonstrate the prenatal origin of NUTM1r infant BCP‐ALL. [ABSTRACT FROM AUTHOR]

Background: The concept of gene fusion describes the process of fusing two genes into one, which is closely linked to tumour occurrence and development and may even be the direct cause of some tumours. Due to their tumour‐specific expression and ability to drive tumour occurrence and development, there is great potential for fusion genes to be used as diagnostic markers and targets for specific types of tumours. Main body: Although many fusion genes have been detected so far, they mainly focus on a small number of highly recurrent fusion genes detected in patients' tumours. There are few studies on the functional mechanism and clinical relevance of rare gene fusions. Our review discusses the generation mechanisms, detection methods, biological functions, and mechanisms of action of fusion genes. Additionally, we discuss the clinical significance of fusion gene detection in some tumour types. Conclusion: The function mechanism research of rare gene fusion is very necessary, and more functions of fusion genes independent of unfused/normal genes can be explored in future studies. There is still a long way to go in implementing precision tumour therapy targeting gene fusion. [ABSTRACT FROM AUTHOR]

Myositis ossificans, a benign tumor composed of spindle cells and osteoblasts, can clinically and radiologically mimic osteosarcoma. While recognition and accurate diagnosis of myositis ossificans can be a challenge, this is critical as it may allow a conservative surgical approach to maximize functional outcomes. Herein, we present a patient with surface myositis ossificans confirmed genetically by the presence of COL1A1::USP6 gene fusion, along with a literature review. Due to the enhanced visualization of the bone matrix, computed tomography (CT) imaging may be a superior imaging modality to magnetic resonance (MR) imaging. Staged biopsies with samples obtained from the periphery and center of the lesions may allow pathologists to discern the zonal distribution histologically. Furthermore, immunohistochemistry fluorescence in situ hybridization and molecular testing can aid in the distinction of myositis ossificans from mimics. Because of their resemblance to other bone tumors, these cases of myositis ossificans highlight the importance of a multidisciplinary approach integrating clinical, radiologic, and pathologic analysis and involving serial imaging, sampling, and judicious use of ancillary immunohistochemical and molecular testing. [ABSTRACT FROM AUTHOR]

The plethora of genome sequences produced in the postgenomic age has not resolved many of our most pressing biological questions. Correlating gene expression with an interrogatable and easily observable characteristic such as the surrogate phenotype conferred by a reporter gene is a valuable approach to gaining insight into gene function. Many reporters including lacZ, amdS, and the fluorescent proteins mRuby3 and mNeonGreen have been used across all manners of organisms. Described here is an investigation into the creation of a robust, synthetic, fusion reporter system for Cryptococcus neoformans that combines some of the most useful fluorophores available in this system with the versatility of the counter-selectable nature of amdS. The reporters generated include multiple composition and orientation variants, all of which were investigated for differences in expression. Evaluation of known promoters from the TEF1 and GAL7 genes was undertaken, elucidating novel expression tendencies of these biologically relevant C. neoformans regulators of transcription. Smaller than lacZ but providing multiple useful surrogate phenotypes for interrogation, the fusion ORF serves as a superior whole-cell assay compared to traditional systems. Ultimately, the work described here bolsters the array of relevant genetic tools that may be employed in furthering manipulation and understanding of the WHO fungal priority group pathogen C. neoformans. [ABSTRACT FROM AUTHOR]

RNA-based next-generation sequencing (RNA-seq) represents the gold standard for detecting gene fusion in non-small cell lung cancer (NSCLC). Despite this, RNA instability makes the management of tissue samples extremely complex, resulting in a significant number of test failures with missing data or the need to switch to other techniques. In the present study, we analyzed pre-analytical variables in 140 tumor tissue samples from patients affected by NSCLC to detect features that increase the chances of successful RNA-seq. We found that the success rate of the analysis positively correlates with the RNA concentration and fragmentation index. Interestingly, small biopsies were more suitable samples than surgical specimens and cell blocks. Among surgical specimens, wedge resections demonstrated better results than lobectomy. Moreover, samples stored for less than 30 days (1 month) had a better chance of success than older samples. Defining the role of pre-analytical variables in RNA-seq allows the detection of more suitable samples for analysis and more effective planning of molecular-based diagnostic approaches in NSCLC. [ABSTRACT FROM AUTHOR]

OBJECTIVE: Porcine interferon-γ (poIFN-γ) and porcine granulocyte-macrophage colony-stimulating factor (poGM-CSF) are multifunctional cytokines that exhibit robust antiviral activity against porcine reproductive and respiratory syndrome virus (PRRSV). In this study, the immunoadjuvant effects of recombinant poIFN-γ-poGM-CSF fusion protein in inactivated PRRSV vaccine administered to piglets were assessed. ANIMALS: Twenty-eight 4-week-old specific pathogen-free piglets. METHODS: The experimental piglets were divided into control, highly pathologic PRRSV, PRRSV killed virus vaccine (KV), poIFN-γ-poGM-CSF, KV + 1.0 mg poIFN-γ-poGM-CSF, KV + 2.0 mg poIFN-γ-poGM-CSF, and KV + 4.0 mg poIFN-γ-poGM-CSF groups. A recombinant poIFN-γ-linker-poGM-CSF fusion gene was constructed via splicing by overlap extension PCR and prepared using an Escherichia coli expression system, after which its adjuvant activity in the context of PRRSV KV administration was assessed. RESULTS: This analysis revealed the successful construction of the poIFN-γ-linker-poGM-CSF fusion gene via splicing by overlap extension PCR, with recombinant poIFN-γ-linker-poGM-CSF successfully being prepared in E coli with a plasmid vector for expressing thioredoxin fusion proteins with an enterokinase site. Importantly, the coadministration of poIFN-γ-linker-poGM-CSF and PRRSV KV significantly increased neutralizing antibody titers, accelerated viral clearance, reduced clinical symptoms, and prevented highly pathogenic PRRSV infection. [ABSTRACT FROM AUTHOR]

While Acanthella acuta Schmidt 1862, a common demosponge found in the Mediterranean Sea and Atlantic Ocean, is morphologically similar to other sponges, its mitochondrial DNA (mtDNA) is unique within the class. In contrast to all other studied demosponges, the mtDNA of A. acuta is inferred to be linear and displays several unusual features such as inverted terminal repeats, group II introns in three mitochondrial genes, and two unique open reading frames (ORFs): one of which (ORF1535) combines a DNA polymerase domain with a DNA-directed RNA polymerase domain, while the second bears no discernible similarity to any reported sequences. The group II intron within the cox2 gene is the first such intron reported in an animal. Our phylogenetic analyses indicate that the cox1 intron is related to similar introns found in other demosponges, while the cox2 intron is likely not of animal origin. The two domains found within ORF1535 do not share a common origin and, along with the cox2 intron, were likely acquired by horizontal gene transfer. The findings of this paper open new avenues of exploration in the understanding of mtDNA linearization within Metazoa. [ABSTRACT FROM AUTHOR]

Numerous studies have been devoted to individual cases of horizontally acquired genes in fungi. It has been shown that such genes expand the hosts' metabolic capabilities and contribute to their adaptations as parasites or symbionts. Some studies have provided an extensive characterization of the horizontal gene transfer (HGT) in Dikarya. However, in the early diverging fungi (EDF), a similar characterization is still missing. In order to fill this gap, we have designed a computational pipeline to obtain a statistical sample of reliable HGT events with a low false discovery rate. We have analyzed 44 EDF proteomes and identified 829 xenologs in fungi ranging from Chytridiomycota to Mucoromycota. We have identified several patterns and statistical properties of EDF HGT. We show that HGT is driven by bursts of gene exchange and duplication, resulting in highly divergent numbers and molecular properties of xenologs between fungal lineages. Ancestrally aquatic fungi are generally more likely to acquire foreign genetic material than terrestrial ones. Endosymbiotic bacteria can be a source of useful xenologs, as exemplified by NOD-like receptors transferred to Mortierellomycota. Closely related fungi have similar rates of intronization of xenologs. Posttransfer gene fusions and losses of protein domains are common and may influence the encoded proteins' functions. We argue that there is no universal approach for HGT identification and inter- and intra-kingdom transfers require tailored identification methods. Our results help to better understand how and to what extent HGT has shaped the metabolic, adaptive, and immune capabilities of fungi. [ABSTRACT FROM AUTHOR]

Dermatofibrosarcoma protuberans (DFSP) is a rare cutaneous sarcoma characterized by the COL1A1‐PDGFB fusion gene. This study utilized single‐cell RNA sequencing to dissect the cellular and molecular landscape of primary DFSP. Distinct DFSP cell clusters, exhibiting fibroblast‐like traits, revealed variations in pathways associated with proliferation, inflammation and metabolism. Differential gene expression analysis during the differentiation from tumour stem cells to DFSP cells unveiled SMOC2, DCN and TGFBR3 as potential regulators of tumour invasion and immune infiltration through VEGF/TGF‐β signalling modulation. Cellular communication analysis highlighted interactions within DFSP cell clusters and with endothelial cells, implicating molecules such as NAMPT, ANGPT2 and PTN in pathogenesis and treatment resistance. These findings offer insights into DFSP intratumour heterogeneity, elucidate molecular mechanisms underlying tumour behaviour, and suggest potential therapeutic targets. [ABSTRACT FROM AUTHOR]