Comprehensive Molecular Analysis Research Articles

SeptAsTERS- SeptiCyte® RAPID as assessment tool for early recognition of sepsis - a prospective observational study.

Early recognition of sepsis is critical to patient outcome, with mortality increasing with every hour of delay in treatment. The aim of this study was to investigate the use of a point-of-care molecular host response assay to differentiate sepsis from inflammation after surgery. Three molecular host response assays (SeptiCyte® RAPID) were performed in 61 patients after major abdominal surgery with admission to the intensive care unit and drawn blood cultures. The first (T0) was taken ± 3h around the time of obtaining blood cultures, the second 24h later (T24) and the third at discharge from the intensive care unit (Tex). The primary endpoint was the agreement of SeptiCyte® RAPID results with the diagnosis of sepsis. SeptiScore® indicates sepsis probability (low risk 0 - high risk 15). Patients were retrospectively classified into sepsis and inflammation by three blinded experts. 25 (42.4%) patients were categorized as "inflammation" and 34 (57.6%) patients as "sepsis". At T0 and T24 septic patients showed significantly higher mean SeptiScores® of 8.0 (± 2.2 SD) vs. 6.3 (± 2.1 SD) and 8.5 (± 2.1 SD) vs. 6.2 (± 1.8 SD), respectively. The Receiver Operating Curves (ROC) for the ability to discriminate between sepsis and inflammation had an Area Under the Curve (AUC) of 0.71 (T0) and 0.80 (T24). Embedded in a comprehensive diagnostic algorithm molecular host response analysis could broaden the possibilities for infection diagnostics to differentiate between sepsis and inflammatory response after surgery. But validation in larger cohorts is needed.

On some new and poorly-known Chrysillini from arid western South Africa (Araneae, Salticidae)

Following a rapid biodiversity assessment of spiders in the arid western interior of South Africa, we report on the occurrence of some poorly known and new species of chrysilline jumping spiders. Helafricanus patellaris (Simon, 1901), Heliocapensis capensis (Wesołowska, 1986), H. mirabilis (Wesołowska, 1986) and Menemerus lesserti Lawrence, 1927 are recorded from the Northern Cape Province for the first time, and Heliocapensis maluti (Wesołowska & Haddad, 2014) (Lesotho) and Heliophanus deformis Wesołowska, 1986 (Angola) are recorded from South Africa for the first time, both also from the Northern Cape. The hitherto unknown females of Heliocapensis mirabilis (Wesołowska, 1986) and Icius pulchellus Haddad & Wesołowska, 2011 and the male of M. lesserti are described for the first time. Three new species are described: Icius jacksonisp. nov. (♂), Menemerus foordisp. nov. (♂) and Natta triguttatasp. nov. (♂♀). One new combination, Afraflacilla matabelensis (Wesołowska, 2011), comb. nov. (ex Pseudicius Simon, 1885), is proposed. We present the first comprehensive molecular analysis of South Africa Chrysillini jumping spiders, based on the cytochrome oxidase I (COI) gene, which supports the monophyly of all but two genera (Helafricanus Wesołowska, 1986 and Heliophanus C.L. Koch, 1833), which we briefly discuss.

Predictive Analysis of Yi-Gai-San's Multifaceted Mechanisms for Tremor-dominant Parkinson's Disease via Network Pharmacology and Molecular Docking Validation.

Based on comprehensive network-pharmacology and molecular docking analysis, this study was intended to unveil the multiple mechanisms of Yi- Gai-San (YGS) in treating the tremor-dominant subtype of Parkinson's disease (PD-DT). The compounds of YGS were meticulously analyzed, selected, and standardized with references to their pharmacological attributes. Its components included Gouteng (Uncaria rhynchophylla), Chaihu (Radix Bupleuri), Chuanxiong (Chuanxiong Rhizoma), Danggui (Angelicae sinensis radix), Fuling (Wolfiporia extensa), Baizhu (Atractylodis macrocephalae rhizoma), and Gancao (Licorice, Glycyrrhizae radix). We identified 75 active compounds within YGS. From these, we predicted 110 gene targets, which exhibited a direct association with PD-DT. PPI network results highlighted core target proteins, including TP53, SLC6A3, GAPDH, MAOB, AKT, BAX, IL6, BCL2, PKA, and CASP3. These proteins potentially alleviate PD-DT by targeting inflammation, modulating neuronal cell apoptosis, and regulating the dopamine system. Furthermore, GO and KEGG enrichment analyses emphasized that YGS might influence various mechanisms, such as the apoptotic process, mitochondrial autophagy, Age-Rage signaling, and dopaminergic and serotonergic synapses. The core proteins from the PPI analysis were selected for the docking experiment. The docking results demonstrated that the most stable ligand-receptor conformations were kaempferol with CASP3 (-9.5 kcal/mol), stigmasterol with SLC6A3 (-10.5 kcal/mol), shinpterocarpin with BCL2L1 (-9.6 kcal/mol), hirsutine with MAOB (-9.7 kcal/mol), hederagenin with PRKACA (-9.8 kcal/mol), and yatein with GAPDH (-9.8 kcal/mol). These results provide us with research objectives for future endeavors in extracting single compounds for drug manufacturing or in-depth studies on drug mechanisms. From these computational findings, we suggested that YGS might mitigate PD-DT via "multi-compounds, multi-targets, and multi-pathways."

PATH-66. MOLECULAR FEATURES RELATED TO LONG-TERM SURVIVAL IN GLIOBLASTOMA

Abstract Glioblastoma Multiforme (GBM) remains the most common malignant primary brain tumor in adults, with a dismal prognosis that rarely exceeds beyond 2 years of survival despite multimodal treatment. While small fractions of GBM patients seem to display extended survival, the underlying mechanisms for this peculiarity remain largely unknown. Previously published investigations of long-term survivors (LTS) usually incorporate small numbers of patients, non-uniform definition of LTS and rarely provide a comprehensive molecular analysis. With advances in multi-omics technologies and their integration with disease diagnostics, numerous prognostic molecular markers have been proposed. So far, only O6-methylguanine-DNA-methyltransferase (MGMT) gene promotor hypermethylation was linked to outcome and therapy response. We aim to investigate omics characteristics associated with LTS. Molecularly confirmed Isocitrate Dehydrogenase (IDH) wildtype GBM patients, >18 years old, operated and treated at our institution between 2013 and 2020 and living ≥5 years post-diagnosis (LTS = 62) or 1-2 years (STS = 62) were identified. Clinical and demographic characteristics were matched. Tumor tissues was analyzed with targeted next generation sequencing (NGS) and compared between LTS and STS. In addition, methylation analysis was done for all LTS. Results will be presented. We hope to shed a broader light on the molecular drivers behind the LTS in GBM patients, that will provide prognostic markers and potential targets for novel treatments.

Deciphering STAT3’s negative regulation of LHPP in ESCC progression through single-cell transcriptomics analysis

BackgroundEsophageal Squamous Cell Carcinoma (ESCC) remains a predominant health concern in the world, characterized by high prevalence and mortality rates. Advances in single-cell transcriptomics have revolutionized cancer research by enabling a precise dissection of cellular and molecular diversity within tumors.ObjectiveThis study aims to elucidate the cellular dynamics and molecular mechanisms in ESCC, focusing on the transcriptional influence of STAT3 (Signal Transducer and Activator of Transcription 3) and its interaction with LHPP, thereby uncovering potential therapeutic targets.MethodsSingle-cell RNA sequencing was employed to analyze 44,206 cells from tumor and adjacent normal tissues of ESCC patients, identifying distinct cell types and their transcriptional shifts. We conducted differential gene expression analysis to assess changes within the tumor microenvironment (TME). Validation of key regulatory interactions was performed using qPCR in a cohort of 21 ESCC patients and further substantiated through experimental assays in ESCC cell lines.ResultsThe study revealed critical alterations in cell composition and gene expression across identified cell populations, with a notable shift towards pro-tumorigenic states. A significant regulatory influence of STAT3 on LHPP was discovered, establishing a novel aspect of ESCC pathogenesis. Elevated levels of STAT3 and suppressed LHPP expression were validated in clinical samples. Functional assays confirmed that STAT3 directly represses LHPP at the promoter level, and disruption of this interaction by promoter mutations diminished STAT3's repressive effect.ConclusionThis investigation underscores the central role of STAT3 as a regulator in ESCC, directly impacting LHPP expression and suggesting a regulatory loop crucial for tumor behavior. The insights gained from our comprehensive cellular and molecular analysis offer a deeper understanding of the dynamics within the ESCC microenvironment. These findings pave the way for targeted therapeutic interventions focusing on the STAT3-LHPP axis, providing a strategic approach to improve ESCC management and prognosis.

Targeted microscopic rare cell retrieval yields sufficient deoxyribonucleic acid for molecular testing in fixed tissue with low tumor fraction

Abstract Introduction/Objective DNA extracted from formalin-fixed paraffin-embedded tissue (FFPE) blocks is often utilized for molecular oncologic testing. Cases with low tumor fraction often fail to obtain adequate tumor for molecular testing. We hypothesized that targeted retrieval of visualized tumor cells from FFPE sections, including those with low tumor fraction, could yield sufficient DNA for molecular testing. Methods/Case Report Unstained sections of various thicknesses were cut from low tumor fraction blocks and visualized by light microscopy and the RareCyte CyteFinder II (Seattle, WA). Microscopic cell retrieval was performed on the RareCyte using 40 µm diameter needles from identified regions of interest. Samples were deparaffinized using Zymo Research (Tustin, CA) solution, and digested and purified using Zymo-Spin Column technology. Sample DNA was quantified with NanoDrop Spectrophotometer (Chino, CA). Results (if a Case Study enter NA) Regions of interest were morphologically identifiable on 4-10 µm thick sections and harder to visualize on 12 µm thick sections. Targeted microscopic cell retrieval was performed with 40 µm diameter needles. Retrieval was performed from 26 to 34 regions of interest per sample and pooled for molecular testing. Samples (n=4) yielded a mean of 81.0 ng of DNA ± 18.4 ng (1.8 ng/µL ± 0.4 ng/µL) and a median of 87.75 ng (1.95 ng/µL). Conclusion Targeted microscopic cell retrieval from low tumor fraction FFPE slides using the RareCyte CyteFinder II can provide sufficient DNA material for molecular testing. Application of this technology has the possibility of recovering sufficient tumor DNA for molecular testing from specimens that may be deemed unsuitable for molecular testing according to current standards. We aim to focus on increasing sample size and completing comprehensive molecular analysis as we continue to expand this work.

A review of genetic resources and trends of omics applications in donkey research: focus on China.

Omics methodologies, such as genomics, transcriptomics, proteomics, metabolomics, lipidomics and microbiomics, have revolutionized biological research by allowing comprehensive molecular analysis in livestock animals. However, despite being widely used in various animal species, research on donkeys has been notably scarce. China, renowned for its rich history in donkey husbandry, plays a pivotal role in their conservation and utilization. China boasts 24 distinct donkey breeds, necessitating conservation efforts, especially for smaller breeds facing extinction threats. So far, omics approaches have been employed in studies of donkey milk and meat, shedding light on their composition and quality. Similarly, omics methods have been utilized to explore the molecular basis associated with donkey growth, meat production, and quality traits. Omics analysis has also unraveled the critical role of donkey microbiota in health and nutrition, with gut microbiome studies revealing associations with factors such as pregnancy, age, transportation stress, and altitude. Furthermore, omics applications have addressed donkey health issues, including infectious diseases and reproductive problems. In addition, these applications have also provided insights into the improvement of donkey reproductive efficiency research. In conclusion, omics methodologies are essential for advancing knowledge about donkeys, their genetic diversity, and their applications across various domains. However, omics research in donkeys is still in its infancy, and there is a need for continued research to enhance donkey breeding, production, and welfare in China and beyond.

Multi-Target Mechanisms of Si-Ni-San on Anxious Insomnia: An Example of Network-pharmacology and Molecular Docking Analysis.

Based on comprehensive network-pharmacology and molecular docking analysis, this study was intended to unveil the multiple mechanisms of Si-Ni-San (SNS) in treating anxious insomnia. The compounds of SNS were meticulously analyzed, selected and standardized with references to their pharmacological attributes. The components included chaihu (Bupleurum chinense DC.), baishao (Paeonia lactiflora Pall.), zhishi (Citrus aurantium L.) and gancao (Glycyrrhiza uralensis Fisch. ex DC.). We used the Traditional Chinese Medicine System Pharmacology (TCMSP) Database, Traditional Chinese Medicines Integrated Database (TCMID), GeneCards database, therapeutic target database (TTD) and comparative toxicogenomic database (CTD) to construct the components-compounds-targets networks and used Cytoscape 3.9.1 software to visualize the outcome. Afterwards, the STRING database and Cytoscape 3.9.1 software were utilized to construct and visualize the protein-protein interaction (PPI) network analysis. In addition, the Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis were also conducted through the Database for Annotation, Visualization, and Integrated Discovery (DAVID). The molecular docking program was carried out using AutoDock 4.2 software to understand interactions between target receptors and compound ligands selected for study. We thoroughly sorted and filtered 31 pharmacologically active compounds from SNS. Subsequently, several potential target genes were predicted, of which there were 59 target genes distinctly associated with anxious insomnia. The PPI analysis indicated that the core target proteins included AKT1, IL6, TNF, SLC6A4, MAOA and GABRA2. The results of our study indicated that SNS potentially remediates anxious insomnia by reducing inflammation, neurodegeneration, and cell apoptosis of neurons. In addition, GO and KEGG enrichment analysis results indicated that SNS could modulate multiple aspects of anxious insomnia through mechanisms related to pathways of neuroactive ligand-receptor interaction. These pathways include various kinds of synaptic transmission pathways, and anti-inflammatory activity associated with response pathways. When we compared the components-compounds-targets networks and the compounds-targets-synaptic pathways networks, the five active compounds, including beta-Sitosterol, Kaempferol, Tetramethoxyluteolin, Isorhamnetin and Shinpterocarpin, were selected to conduct molecular docking experiments. Eleven target proteins, (AKT1, SLC6A4, ADRB2, MAOA, ACHE, ESR1, CYP3A4, CHRNA7, GABRA2, HTR2A and NOS3), which also play significant roles in regulating serotonergic, cholinergic, dopaminergic and GABAergic systems in the PPI network, were selected to act as receptors in molecular docking trials. The results showed that docking pairs isorhamnetin-AKT1, isorhamnetin-SLC6A4, β-sitosterol-MAOA, β- sitosterol-ACHE, isorhamnetin-CHRNA7 and shinpterocarpin-GABRA2 provided the most stable conformations of ligand-receptor binding between key compounds and core target proteins in the SNS. In the study, we offer a computational result, revealing that SNS may alleviate sleep disorders associated with anxiety through a "multi-compounds, multi-targets, and multi-pathways" mechanism. The network-pharmacology and molecular docking outcomes could theoretically confirm the anti-anxiety and anti-insomnia effects of SNS. Although this research is purely statistical and systematic without empirical validation, it serves as a stepping stone and cornerstone for subsequent experimental investigations.

Primary Melanoma of the Urinary Bladder: Clinical, Histopathologic, and Comprehensive Molecular Analysis of a Rare Tumor.

Primary melanoma of the urinary bladder is extremely rare and generally has a poor prognosis. The histopathological diagnosis can be challenging as tumors can be unpigmented and of varying morphology. Here we report a rare example of primary urinary bladder melanoma with clinical, imaging, gross anatomical, histopathologic, immunohistochemical, and molecular findings to illustrate the utility of an integrated approach in establishing the diagnosis and guide therapy. A comprehensive, integrated approach, including molecular studies, may be helpful in further establishing an accurate diagnosis and informing therapies of this rare but poorly behaved entity.

Lissonema sicki, an emerging air sac nematode of European owls: introduction, host switching and rapid establishment on a Mediterranean island.

In recent years, air sac parasitic helminths have been reported to cause severe disease in birds. In addition, various species appear to be expanding and infecting new avian hosts in various regions worldwide. In this context, an air sac nematode was initially detected in 2014 infecting the Eurasian scops owl, hospitalized in the local wildlife hospital in Mallorca (Balearic Islands, Spain). Years later, the parasite was detected in 2 other owl species. Air sac nematodes had never been reported in the Mallorcan Strigiformes before. A comprehensive molecular and morphological characterization analysis, including scanning electron microscopy, was required for species confirmation. The species was identified as Lissonema sicki, a parasite infrequently reported in South American owls. Since its first introduction to Mallorca, it has dramatically increased in prevalence in hospitalized birds, being highly prevalent in the Eurasian scops owl (41%), in the long-eared owl (11%) and in the barn owl (4%). The introduction pathway of this parasite to Europe remains unknown. This discovery underscores the expanding range and impact of L. sicki, emphasizing the importance of ongoing surveillance and research to comprehend and manage the implications of its emergence in new territories.

A comprehensive molecular analysis of cannabidiol: From solid state to antioxidant potential

Cannabis sativa contains approximately 540 natural compounds, including terpenes and flavonoids, and approximately 100 of these compounds are phytocannabinoids, of which the most studied are Δ9-tetrahydrocannabinol and cannabidiol. Due to potential therapeutic properties and psychoactive effects dissociated from cannabis consumption, cannabidiol has received significant attention. This study focuses on molecular modeling and the electronic properties of cannabidiol and cannabidiol-type compounds using chemical reactivity descriptors, aiming to understand the potential antioxidant properties. The radicals formed during free radical scavenging processes were evaluated in thermodynamic and electronic terms through the mechanisms of hydrogen atom transfer and one-electron transfer. The topological parameters showed that the interactions are of the closed-shell type and have a van der Waals character, except where the OH group rotation occurs, resulting in an H bond. Calculations of OH bond dissociation enthalpy and ionization potential showed that CBD-C5 has the best antioxidant potential among cannabidiol-type compounds.

Genetic stability evaluation of caladium somaclonal variants by morphological, cytological, and SSR analysis in three successive generations

Somaclonal variants with valuable agronomic traits play a crucial role in crop breeding, provided that they are stably passed down in subsequent generations. Although numerous somaclonal variants have been identified and studied among in vitro regenerated plants in the foliage plant caladium (Caladium × hortulanum Birdsey), no research has been carried out to evaluate their genetic stability in subsequent generations. This study aimed to evaluate the genetic stability of three types of diploid caladium somaclonal variants, previously derived from in vitro callus cultures, over three successive tuber-propagated generations. The analysis of quantitative morphological characteristics revealed a greater degree of variation among the established plants in the first generation (G1) compared to the second (G2) and third generations (G3). Seven plants exhibiting distinct leaf coloration changes were identified in the G1 generation and were found to be stably passed down to the subsequent G2 and G3 generations. These findings indicate a wide range of morphological variation in the G1 generation, followed by relative stability in subsequent generations. A comprehensive cytological and molecular analysis revealed that five of the seven newly observed variants displayed notable differences in relative nuclear DNA content, chromosome number, or SSR (simple sequence repeat) banding patterns compared to their corresponding normal counterparts. The findings of this study will be instrumental in developing new cultivars with distinctive plant morphology through the exploitation of somaclonal variation in caladium.

Age-dependent variation of aedeagal morphology in Agabusuliginosus and the status of A.lotti (Coleoptera, Dytiscidae).

A doubt has arisen about the taxonomic status of Agabuslotti within the Agabusuliginosus species group due to morphological similarities and lack of molecular data. In this study, a comprehensive morphological and molecular analysis of specimens from Central Europe was conducted, focusing on the Hungarian population. Morphological comparisons of genital structures revealed age-dependent variations, suggesting a gradual transition from A.lotti to A.uliginosus. Molecular analysis of COI sequences further supported this hypothesis, showing minimal genetic differences among most specimens, with only one individual exhibiting distinctiveness. Therefore, A.lotti syn. nov. must be regarded as a junior synonym of A.uliginosus. Our findings also highlight the need for additional multi-marker studies covering a broader geographic range and including both molecular and morphological approaches to elucidate the taxonomic and phylogenetic relationships within this species group. The inclusion of Hungarian samples notably enriched the diversity of haplotypes, emphasizing the importance of expanding sampling efforts in future research.

Mapping Transmission Dynamics and Drug Resistance Surveillance in the Cyprus HIV-1 Epidemic (2017-2021).

The human immunodeficiency virus type 1 (HIV-1) epidemic has been a major public health threat on a global scale since the early 1980s. Despite the introduction of combination antiretroviral therapy (cART), the incidence of new HIV-1 infections continues to rise in some regions around the world. Thus, with the continuous transmission of HIV-1 and the lack of a cure, it is imperative for molecular epidemiological studies to be performed, to monitor the infection and ultimately be able to control the spread of this virus. This work provides a comprehensive molecular epidemiological analysis of the HIV-1 infection in Cyprus, through examining 305 HIV-1 sequences collected between 9 March 2017 and 14 October 2021. Employing advanced statistical and bioinformatic techniques, the research delved deeply into understanding the transmission dynamics of the HIV-1 epidemic in Cyprus, as well as the monitoring of HIV-1's genetic diversity and the surveillance of transmitted drug resistance. The characterization of Cyprus's HIV-1 epidemic revealed a diverse landscape, comprising 21 HIV-1 group M pure subtypes and circulating recombinant forms (CRFs), alongside numerous uncharacterized recombinant strains. Subtypes A1 and B emerged as the most prevalent strains, followed by CRF02_AG. The findings of this study also revealed high levels of transmitted drug resistance (TDR) patterns, raising concerns for the efficacy of cART. The demographic profiles of individuals involved in HIV-1 transmission underscored the disproportionate burden borne by young to middle-aged Cypriot males, particularly those in the MSM community, who reported contracting the virus in Cyprus. An assessment of the spatiotemporal evolutionary dynamics illustrated the global interconnectedness of HIV-1 transmission networks, implicating five continents in the dissemination of strains within Cyprus: Europe, Africa, Asia, North America, and Oceania. Overall, this study advances the comprehension of the HIV-1 epidemic in Cyprus and highlights the importance of understanding HIV-1's transmission dynamics through continuous surveillance efforts. Furthermore, this work emphasizes the critical role of state-of-the-art bioinformatics analyses in addressing the challenges posed by HIV-1 transmission globally, laying the groundwork for public health interventions aimed at curbing its spread and improving patient outcomes.

Unraveling the Molecular Landscape of Uterine Tumor Resembling Ovarian Sex Cord Tumor: Insights From A Clinicopathological, Morphologic, Immunohistochemical, and Molecular Analysis of 35 Cases

Uterine tumor resembling ovarian sex cord tumor (UTROSCT) is a rare tumor of uncertain lineage and low malignant potential. Most tumors behave in a benign manner, but a subset of UTROSCT exhibit an aggressive clinical course with recurrences and metastases. The recurrent molecular alterations in UTROSCT mostly represent gene fusions involving NCOA1-3. We performed a comprehensive clinicopathological, morphologic, immunohistochemical, and molecular analysis on a cohort of 35 UTROSCT. The tumors exhibited various architectural patterns (diffuse, corded/trabecular, tubular, sertoliform, fascicular, whorled, nested, microfollicular, and pseudoglandular), often in combination. The immunohistochemical analysis confirmed the polyphenotypic immunoprofile, often with coexpression of sex cord–stromal, smooth muscle, and epithelial markers, as well as hormone receptors. Next-generation sequencing RNA analysis revealed recurrent NCOA1-3 gene fusions in 22/32 analyzed cases (69%), including ESR1::NCOA3 (11/22), GREB1::NCOA2 (7/22), ESR1::NCOA2 (3/22), and GREB1::NCOA1 (1/22). Tumor mutation burden was low in all cases. The fusion-positive cases exhibited statistically significant association with whorled architecture, conversely necrosis was associated with fusion-negative status. We did not find a significant relationship between any architectural pattern and GREB1 alterations, but the NCOA2-altered tumors were associated with pseudoglandular architecture. The GREB1-altered cases occurred in older patients and tended to be more often intramural masses compared with ESR1-altered cases. On the contrary, the ESR1-altered cases presented more often like submucosal or polypoid tumors. Two tumors exhibited aggressive behavior with recurrent disease. Both of these cases harbored a GREB1::NCOA2 fusion. Unsupervised hierarchical cluster analysis of our cohort revealed 2 main clusters. The tumors with GREB1 or NCOA2 fusion cluster together, suggesting that there are underlying molecular differences between these cases and cases with ESR1::NCOA3 fusion or without fusion. Our findings contribute to the growing knowledge about a rare neoplasm with currently uncertain biological behavior.

Rapid molecular profiling utilising minimal quantities of endobronchial ultrasound-guided aspirates for the detection of Epidermal Growth Factor Receptor, KRAS, ALK, ROS1, RET, NTRK and MET gene alterations from patients with non-small-cell lung carcinomas on the Biocartis Idylla™ platform.

Comprehensive molecular analysis for patients with non-small-cell lung carcinoma (NSCLC) is essential for managing modern targeted therapies. This study sought to establish the feasibility of utilising real-time PCR to perform rapid and comprehensive profiling on minimal amounts of endobronchial ultrasound-guided (EBUS) aspirates as a fast, tissue-sparing route of predictive profiling. A volume of 500 μL of EBUS aspirate and fixative from patients with NSCLC was decanted, and 80 μL (<1% of total specimen received) was utilised for analysis. Biocartis Idylla™ cartridges for epidermal growth factor receptor (EGFR) mutations, KRAS mutations and a GeneFusion cartridge (ALK, ROS1, RET, NTRK1/2/3 rearrangements & MET 14 exon skipping) were analysed for each case to provide molecular data on the main clinically relevant targets as per UK guidelines. A total of 62 cases were included; all of which had successful DNA analysis (EGFR and KRAS cartridges). RNA analysis (GeneFusion cartridge) was successful for 42 of 51 (82%) with initial approach, with 11 of 11 (100%) achieving a successful result with modified protocol. In all, 23 KRAS mutations (37%), 5 EGFR mutations (8%) and 1 ROS fusion (2%) were identified. Average time from specimen receipt to molecular read-out was 5 h. Real-time PCR utilising the Idylla™ platform is rapid, utilises minimal amounts of tissue and provides accurate results. We propose this is a useful ancillary method to utilise alongside next-generation sequencing (NGS) in cases of urgent clinical requirement or EBUS aspirates with inadequate quantities of tissue for NGS.

Deciphering the role of lipid metabolism and acetylation in osteosarcoma: A comprehensive molecular analysis.

Osteosarcoma, known for its rapid progression and high metastatic potential, poses significant challenges in adolescent oncology. This study delves into the roles of lipid metabolism and acetylation genes in the disease's pathogenesis. Utilizing gene set variation analysis, we examined 14 lipid metabolism-related pathways in osteosarcoma patients, identifying significant variances in three pathways between metastatic and primary cases. Additionally, differences in four acetylation genes between these groups were observed. A comprehensive analysis pinpointed 62 lipid metabolism-related genes, with 39 exhibiting significant correlations with acetylation genes, termed lipid metabolism acetylation (LMA) genes. Employing machine learning techniques like Lasso+RSF and GBM, we developed a predictive model for overall survival based on LMA genes. This model, with an average c-index of 0.771, focuses on three key genes: CYP2C8, PAFAH2, and ACOX3, whose prognostic value was confirmed through survival and receiver operating characteristic curve analyses. Quantitative RT-PCR results indicated higher expression levels of ACOX3 and PAFAH2 in OS cells (143B, HOS, MG63) than in osteoblasts (hFOB1.19), while CYP2C8 was lower in OS cells. Furthermore, drug sensitivity analysis through the pRRophetic algorithm suggested potential targeted therapies, revealing drugs with differential sensitivity based on LMA scores and varied treatment responses related to the expression of core genes. This study not only highlights the crucial role of lipid metabolism and acetylation in osteosarcoma but also offers a foundation for personalized treatment strategies, marking a notable advancement in combating this severe form of adolescent cancer.

The Impact of Li-Fraumeni and Germline Retinoblastoma Mutations on Leiomyosarcoma Initiation, Outcomes, and Genetic Testing Recommendations.

Leiomyosarcomas (LMS) are clinically and molecularly heterogeneous, occurring mostly in sporadic but also syndromic settings. The role of pathogenic germline variants (PGV) as LMS drivers and their impact on outcomes remains uncertain. We performed a comprehensive clinicopathologic and molecular analysis using a tumor-normal DNA next-generation sequencing assay (Memorial Sloan Kettering-Integrated Mutational Profiling of Actionable Cancer Targets) of germline-associated LMS compared with sporadic LMS. Among 285 LMS [120 soft-tissue LMS (STLMS) and 165 uterine LMS (ULMS)] with germline testing, 78 (27%, 43 STLMS and 35 ULMS) cases harbored PGV, with 35/78 (45%) of PGV carriers showing biallelic inactivation of the corresponding gene in the tumor (26 STLMS and nine ULMS). The most frequent germline predispositions were TP53 (Li-Fraumeni syndrome; 17 patients, 16 in STLMS) and RB1 (retinoblastoma; 13 patients, 11 in STLMS). Germline TP53 and somatic RB1 alterations often co-occurred in the tumor andvice versa. Other biallelically inactivated PGV were enriched in DNA damage repair-related genes: CHEK2, MSH2, MSH6, RAD51D, BRCA2, and FANCA. Monoallelic PGV were mostly in ULMS and associated with co-occurring TP53 and RB1 somatic alterations. Patients with STLMS with biallelic but not monoallelic PGV were significantly younger than patients with sporadic STLMS (median ages 38 vs. 52 vs. 58 years). No differences in disease-specific or progression-free survival were observed in germline-associated versus sporadic LMS regardless of biallelic status. Although patients with ULMS had a relatively low proportion of PGV, a high percentage of patients with STLMS with PGV had tumor biallelic status, indicating that PGV drive tumorigenesis in these individuals. These findings have significant implications for genetic testing recommendations.

Extracranial metastatic oligodendroglioma with molecular progression, case presentation

BackgroundExtraneural metastasis of central nervous system tumors is generally rare and most often reported in glioblastomas and medulloblastomas, whereas oligodendrogliomas seem to have the lowest risk of extracranial metastasis. Given its infrequent occurrence, both the diagnosis and therapy of metastatic oligodendroglioma is often challenging.Case presentationThis case study presents an oligodendroglioma, the isocitrate dehydrogenase 1 (IDH1) mutant, 1p/19q-codeleted tumor with bone marrow metastasis. The significance of this case lies in the comprehensive molecular analysis conducted for both the primary tumor and the metastasis. Chromosome 7 trisomy and chromosome 10 monosomy (+ 7/-10) were detected in the metastasis indicating molecular progression, which, to the best of our knowledge, has not been previously documented in metastatic oligodendroglioma.ConclusionsThis case study serves additional information for better understanding of the metastatic capabilities of CNS tumors.

Real-World Clinical Utility of Targeted RNA Sequencing in Leukemia Diagnosis and Management.

Gene fusions are key drivers in acute leukemia, impacting diagnosis and treatment decisions. We analyzed 264 leukemia patients using targeted RNA sequencing with conventional karyotyping and reverse transcription polymerase chain reaction (RT-PCR). Leukemic fusions were detected in 127 patients (48.1%). The new guidelines introduced additional diagnostic criteria, expanding the spectrum of gene fusions. We discovered three novel fusions (RUNX1::DOPEY2, RUNX1::MACROD2, and ZCCHC7::LRP1B). We analyzed recurrent breakpoints for the KMT2A and NUP98 rearrangements. Targeted RNA sequencing showed consistent results with RT-PCR in all tested samples. However, when compared to conventional karyotyping, we observed an 83.3% concordance rate, with 29 cases found only in targeted RNA sequencing, 7 cases with discordant results, and 5 cases found only in conventional karyotyping. For the five cases where known leukemic gene rearrangements were suspected only in conventional karyotyping, we conducted additional messenger RNA sequencing in four cases and proved no pathogenic gene rearrangements. Targeted RNA sequencing proved advantageous for the rapid and accurate interpretation of gene rearrangements. The concurrent use of multiple methods was essential for a comprehensive evaluation. Comprehensive molecular analysis enhances our understanding of leukemia's genetic basis, aiding diagnosis and classification. Advanced molecular techniques improve clinical decision-making, offering potential benefits.