Base Modifications Research Articles

Single-Molecule DNA Methylation Reveals Unique Epigenetic Identity Profiles of T Helper Cells.

Both identity and plasticity of CD4 T helper (Th) cells are regulated in part by epigenetic mechanisms. However, a method that reliably and readily profiles DNA base modifications is still needed to finely study Th cell differentiation. Cytosine methylation in CpG context (5mCpG) and cytosine hydroxymethylation (5hmCpG) are DNA modifications that identify stable cell phenotypes, but their potential to characterize intermediate cell transitions has not yet been evaluated. To assess transition states in Th cells, we developed a method to profile Th cell identity using Cas9-targeted single-molecule nanopore sequencing. Targeting as few as 10 selected genomic loci, we were able to distinguish major invitro polarized murine T cell subtypes, as well as intermediate phenotypes, by their native DNA 5mCpG patterns. Moreover, by using off-target sequences, we were able to infer transcription factor activities relevant to each cell subtype. Detection of 5mCpG and 5hmCpG was validated on intestinal Th17 cells escaping transforming growth factor β control, using single-molecule adaptive sampling. A total of 21 differentially methylated regions mapping to the 10-gene panel were identified in pathogenic Th17 cells relative to their nonpathogenic counterpart. Hence, our data highlight the potential to exploit native DNA methylation profiling to study physiological and pathological transition states of Th cells.

Nanopore Direct RNA Sequencing for Modified Uridine Nucleotides Yields Signals Dependent on the Physical Properties of the Modified Base

AbstractSequencing for RNA modifications with the nanopore direct RNA sequencing platform provides ionic current levels, helicase dwell times, and base call data that differentiate the modifications from the canonical form. Herein, model RNAs were synthesized with site‐specific uridine (U) base modifications that enable the study of increasing an alkyl group size, halogen identity, or a change in base acidity to impact the nanopore data. The analysis concluded that increases in alkyl size trend with greater current blockage but a similar change in base‐call error was not found. The addition of a halogen series to C5 of U revealed that the current levels recorded a trend with the water‐octanol partition coefficient of the base, as well as the base call error. Studies with U modifications that are deprotonated (i. e., anionic) under the sequencing conditions gave broad current levels that influenced the base call error. Some modifications led to helicase dwell time changes. These insights provide design parameters for modification‐specific chemical reagents that can shift nanopore signatures to minimize false positive reads, a known issue with this sequencing approach.

Identification of dMMR in metastatic colorectal and non-colorectal cancers in a private Mexican institution.

177 Background: DNA mismatch repair is a process in which normal cells maintain their original genomic information avoiding errors in mismatched nucleotides or base modifications from recombination processes. The failure of this process could drive carcinogenesis due to increased mutation rate as well as high microsatellite instability. It is estimated that the global average prevalence of dMMR/MSI-H across tumor types and stages is approximately 16%, with higher rates reported for early stages. However, the prevalence for colorectal (CRC) and non-colorectal cancers for this biomarker in Mexico has not been reported. Objective:To explore the prevalence of dMMR in metastatic CRC (mCRC) and non-CRC (gastric, esophageal, and endometrial cancer) from a private Mexican institution. Methods: In this retrospective study, the dMMR status of 215 samples were analyzed from a primary database of stored biopsies from patients diagnosed with metastatic CRC, gastric, esophageal, or endometrial cancers between January 2017 and December 2020. 65 records had information about MMR status. Samples were analyzed using a commercial kit to detect dMMR (BioSb). Prevalence of dMMR was stratified by age and gender and expressed as frequencies. Statistical analysis was conducted in SPSS. Results: dMMR was found in 18.5% (N=27) endometrial cancer cases, 12.7% (N=150) of CRC cases, and 8.3% (N=24) of gastric cancer cases. However, it was not detected in any cases of esophageal cancer (N=14). In CRC, we found a higher percentage of incidence in men than in women, 15.6% (n = 14/90) vs. 8.3% (n = 5/60) respectively. By age groups, the highest total percentage (20%) was observed among those 41-50 years old. The age group with the lowest dMMR rate was 31- 40 yo, with 7.7%. All cases of CRC patients over 91 yo (n=3), were pMMR. Conclusions: Even though this data is not representative for the entire Mexican population, it provides preliminary prevalence dMMR estimates for some key cancers. Additional research is needed to give the limited sample sizes available for non-CRC.

Catalytic activity of OGG1 is impaired by Zinc deficiency

Oxidative stress-induced DNA base modifications, if unrepaired, can increase mutagenesis and genomic instability, ultimately leading to cell death. Cells predominantly use the base excision repair (BER) pathway to repair oxidatively-induced non-helix distorting lesions. BER is initiated by DNA glycosylases, such as 8-oxoguanine DNA glycosylase (OGG1), which repairs oxidatively modified guanine bases, including 7,8-dihydro-8-oxoguanine (8-oxoG) and ring-opened formamidopyrimidine lesions, 2,6-diamino-4-hydroxy-5-formamidopyrimidine (FapyG). The OGG1 protein contains a C2H2 zinc (Zn) finger DNA binding domain. However, the impact of dietary Zn deficiency on OGG1 catalytic activity has not been extensively studied. Zn is a common nutrient of concern with increasing age, and the prevalence of oxidative DNA damage is also concurrently increased during aging. Thus, understanding the potential regulation of OGG1 activity by Zn is clinically relevant. The present study investigates the impact of a range of Zn statuses, varying from severe Zn deficiency to exogenous Zn-supplementation, in the context of young and aged animals to determine the impact of dietary Zn-status on OGG1 activity and oxidative DNA damage in mice. Our findings suggest that nutritional Zn deficiency impairs OGG1 activity and function, without altering gene expression, and that aging further exacerbates these effects. These results have important implications for nutritional management of Zn during aging to mitigate age-associated DNA damage.

C2CDB: an advanced platform integrating comprehensive information and analysis tools of cancer-related circRNAs.

Circular RNAs (circRNAs) play important roles in gene expression and their involvement in tumorigenesis is emerging. circRNA-related database is a powerful tool for researchers to investigate circRNAs. However, existing databases lack advanced platform integrating comprehensive information and analysis tools of cancer-related circRNAs. We developed a comprehensive platform called CircRNA to Cancer Database (C2CDB), encompassing 318 158 cancer-related circRNAs expressed in tumors and adjacent tissues across 30 types of cancers. C2CDB provides basic details such as sequence and expression levels of circRNAs, as well as crucial insights into biological mechanisms, including miRNA binding, RNA-binding protein interaction, coding potential, base modification, mutation, and secondary structure. Moreover, C2CDB collects an extensive compilation of published literature on cancer circRNAs, extracting and presenting pivotal content encompassing biological functions, underlying mechanisms, and molecular tools in these studies. Additionally, C2CDB offers integrated tools to analyse three potential mechanisms: circRNA-miRNA ceRNA interaction, circRNA encoding, and circRNA biogenesis, facilitating investigators with convenient access to highly reliable information. To enhance clarity and organization, C2CDB has meticulously curated and integrated the previously chaotic nomenclature of circRNAs, addressing the prevailing confusion and ambiguity surrounding their designations. C2CDB is freely available at http://pengyonglab.com/c2cdb.

Base-modified factor VIII mRNA delivery with galactosylated lipid nanoparticles as a protein replacement therapy for haemophilia A.

The bleeding disorder hemophilia A (HemA) requires systemic functional factor VIII protein infusions on prophylactic schedules. Recently, chemically modified mRNAs have emerged as promising protein replacement therapies to reduce repeated infusions and improve safety profiles. However, the influence of base modifications on mRNA translation kinetics to specific cell types remains unexplored. In this study, towards developing mRNA therapeutics for haemophilia A, we synthesized chemically modified mRNAs with commercially available base modifications of adenine, guanine, uridine, and cytidine, and evaluated in vitro transcription yield and translation kinetics in hepatic cell lines using reporter eGFP mRNA. Our findings demonstrated that mRNA with N1-methyl pseudouridine (m1Ψ) showed a 5-12-fold increase in translation efficiency in both hepatic and endothelial cell lines. As a proof of concept for developing mRNA therapy for HemA, where FVIII is deficient, we developed a m1Ψ modified functional FVIII mRNA with our liver-targeting lipid nanoparticle (Gal-LNP) system. We evaluated its delivery efficiencies in both hepatic cell lines and the HemA mouse model. The m1Ψ-FVIII mRNA showed high therapeutic efficacy up to 15 days in vivo in the HemA mouse model. Gal-LNPs were found to be safe for systemic administration. Our study reveals that incorporating m1Ψ base modifications on mRNAs could improve therapeutic efficacy in liver- and endothelial-based therapeutics. Optimized mRNA synthesis for superior expression kinetics in hepatic cells and its delivery with liver-targeted nanoparticles may emerge as protein replacement therapies for monogenic liver disorders.

Approaches for Determining DNA Persistence Length Using Atomic Force Microscopy.

Atomic force microscopy (AFM) is widely used to image and study biological molecules. As an example, we have utilized AFM to investigate how the mechanical properties of DNA polymers depend on electrostatics and the strength of DNA base stacking by studying double-stranded DNA molecules incorporating several different neutral and charged base modifications. Here we describe ten complementary approaches for determining DNA persistence length by AFM imaging. The combination of different approaches provides increased confidence and statistical reliability over existing methods utilizing only a single approach.

ON SOME NONTYPICAL DERIVATIONAL PROCESSES IN SERBIAN

This paper dicsusses nonconcatenative morphological processes in the modern Serbian language. More precisely, it focuses on derivational capacity of the processes of reduplication, circumfixation, infixation, base modification, and conversion. After representing the difference between concatenative and nonconcatenative morphological processes, each of the mentioned derivational processes is analysed from a crosslinguistic perspective, with special attention paid to the situation in Serbian. Besides the brief overview of the derivational capacity of the nonconcatenative processes, the goal of the paper is to enrich the terminology of the domestic word formation science (and generally of the Serbian grammar tradition).

BG4 antibody can recognize telomeric G-quadruplexes harboring destabilizing base modifications and lesions.

BG4 is a single-chain variable fragment antibody shown to bind various G-quadruplex (GQ) topologies with high affinity and specificity, and to detect GQ in cells, including GQ structures formed within telomeric TTAGGG repeats. Here, we used ELISA and single-molecule pull-down (SiMPull) detection to test how various lengths and GQ destabilizing base modifications in telomeric DNA constructs alter BG4 binding. We observed high-affinity BG4 binding to telomeric GQ independent of telomere length, although three telomeric repeat constructs that cannot form stable intramolecular GQ showed reduced affinity. A single guanine substitution with 8-aza-7-deaza-G, T, A, or C reduced affinity to varying degrees depending on the location and base type, whereas two G substitutions in the telomeric construct dramatically reduced or abolished binding. Substitution with damaged bases 8-oxoguanine and O6-methylguanine failed to prevent BG4 binding although affinity was reduced depending on lesion location. SiMPull combined with FRET revealed that BG4 binding promotes folding of telomeric GQ harboring a G to T substitution or 8-oxoguanine. Atomic force microscopy revealed that BG4 binds telomeric GQ with a 1:1 stoichiometry. Collectively, our data suggest that BG4 can recognize partially folded telomeric GQ structures and promote telomeric GQ stability.

Differential microRNA editing may drive target pathway switching in human temporal lobe epilepsy.

MicroRNAs have emerged as important regulators of the gene expression landscape in temporal lobe epilepsy. The mechanisms that control microRNA levels and influence target choice remain, however, poorly understood. RNA editing is a post-transcriptional mechanism mediated by the adenosine acting on RNA (ADAR) family of proteins that introduces base modification that diversifies the gene expression landscape. RNA editing has been studied for the mRNA landscape but the extent to which microRNA editing occurs in human temporal lobe epilepsy is unknown. Here, we used small RNA-sequencing data to characterize the identity and extent of microRNA editing in human temporal lobe epilepsy brain samples. This detected low-to-high editing in over 40 of the identified microRNAs. Among microRNA exhibiting the highest editing was miR-376a-3p, which was edited in the seed region and this was predicted to significantly change the target pool. The edited form was expressed at lower levels in human temporal lobe epilepsy samples. We modelled the shift in editing levels of miR-376a-3p in human-induced pluripotent stem cell-derived neurons. Reducing levels of the edited form of miR-376a-3p using antisense oligonucleotides resulted in extensive gene expression changes, including upregulation of mitochondrial and metabolism-associated pathways. Together, these results show that differential editing of microRNAs may re-direct targeting and result in altered functions relevant to the pathophysiology of temporal lobe epilepsy and perhaps other disorders of neuronal hyperexcitability.

Alkylation of Toluene to Mono‐benzylated Toluene over Micro‐mesoporous Zeolite: Effect of Post‐synthetic Treatment and Catalytic Application

AbstractPost‐synthetic modification using acid (HNO3) and base (NaOH) are utilised to synthesize hierarchical micro‐mesoporous zeolites with different pore structure. To investigate structural change in materials, X‐ray diffraction (XRD), Scanning electron microscope (SEM), Inductive coupled plasma‐optical emission spectrometry (ICP‐OES), 29Si and 27Al MAS NMR, and N2 adsorption‐desorption isotherms techniques were employed. Acid base titration, and ammonia‐temperature programmed desorption (NH3‐TPD) was performed to evaluate acidic characteristics of materials. As revealed by characterisation, the structure was well preserved after the modification. Hierarchical materials along with parent materials have been evaluated in the toluene alkylation with benzyl alcohol producing mono‐benzylated toluene. All zeolite samples were proved to be catalytically active by their result in the alkylation reaction. Acid treated samples were compared with base treated and parent samples. The results indicate that, base modification significantly increases the mesoporous pore volume in all zeolite as compared to acid modification. Moreover, the highest benzyl alcohol conversion was found to be 98 % in the base modified Beta, with the highest mono‐benzylated toluene selectivity (69 %). Reaction optimization was also studied by altering reaction duration, temperature, and catalyst amount. The reaction products were analysed using Gas Chromatography (GC) and confirmed using Gas chromatography mass spectrometry (GC‐MS).

The pivotal role of uridine modifications in the development of mRNA technology

In 2023, Katalin Karikó and Drew Weissman were awarded the Nobel Prize in Physiology or Medicine for their nucleoside base modifications research that later enabled mRNA vaccine development against COVID-19. This paper briefly reviews these achievements in the context of the development of mRNA technology and its enormous potential for medicine in the prevention of various infectious diseases and cancer treatment, including personalised therapies. Without a doubt, Karikó and Weissman's discoveries were vital in getting around one of the biggest problems with using mRNA molecules in real life: they had to be recognised by endosomal Toll-like receptors and the innate immune response that came after, causing exogenous mRNA molecules to have less translational activity and break down.. Although the Nobel Prize for Karikó and Weissman is fully justified, it must be stressed that mRNA technology would never unfold its potential for public health without a collective scientific effort encompassing over 40 years of research.

Influence of N1-Methylpseudouridine in Guide RNAs on CRISPR/Cas9 Activity

At present, there are many strategies to improve the activity of CRISPR/Cas9. A well-known and effective approach is guide RNA modification. Many chemical guide RNA modifications have been studied, whereas naturally occurring RNA modifications are largely unexplored. N1-methylpseudouridine (m1Ψ) is an RNA base modification widely used in mRNA therapy, and it holds great promise for application in genome editing systems. The present study focuses on investigating the effect of N1-methylpseudouridine on the functioning of CRISPR/Cas9. In vitro cleavage assays helped determine the level of m1Ψ guide RNA modification that is sufficient to cleave the target substrate. By analyzing FAM-labeled dsDNA substrate cleavage, we calculated the kinetic parameters and the specificity scores of modified guide RNAs. Neon transfection and digital PCR enabled us to assess the activity of modified guide RNAs in mammalian cells. Our study shows that the presence of m1Ψ in guide RNAs can help preserve on-target genome editing while significantly reducing the off-target effects of CRISPR/Cas9 in vitro. We also demonstrate that Cas9 complexes with guide RNAs containing m1Ψ allow for genome editing in human cells. Thus, the incorporation of m1Ψ into guide RNAs supports CRISPR/Cas9 activity both in vitro and in cells.

The role of epigenetic and epitranscriptomic modifications in plants exposed to non-essential metals.

Contamination of the soil with non-essential metals and metalloids is a serious problem in many regions of the world. These non-essential metals and metalloids are toxic to all organisms impacting crop yields and human health. Crop plants exposed to high concentrations of these metals leads to perturbed mineral homeostasis, decreased photosynthesis efficiency, inhibited cell division, oxidative stress, genotoxic effects and subsequently hampered growth. Plants can activate epigenetic and epitranscriptomic mechanisms to maintain cellular and organism homeostasis. Epigenetic modifications include changes in the patterns of cytosine and adenine DNA base modifications, changes in cellular non-coding RNAs, and remodeling histone variants and covalent histone tail modifications. Some of these epigenetic changes have been shown to be long-lasting and may therefore contribute to stress memory and modulated stress tolerance in the progeny. In the emerging field of epitranscriptomics, defined as chemical, covalent modifications of ribonucleotides in cellular transcripts, epitranscriptomic modifications are postulated as more rapid modulators of gene expression. Although significant progress has been made in understanding the plant's epigenetic changes in response to biotic and abiotic stresses, a comprehensive review of the plant's epigenetic responses to metals is lacking. While the role of epitranscriptomics during plant developmental processes and stress responses are emerging, epitranscriptomic modifications in response to metals has not been reviewed. This article describes the impact of non-essential metals and metalloids (Cd, Pb, Hg, Al and As) on global and site-specific DNA methylation, histone tail modifications and epitranscriptomic modifications in plants.

Performance of acid- and base-modified biochars for the removal of antibiotics from water under dynamic conditions

The increased recycling of treated municipal wastewater (TMW) to agricultural land is potentially hampered by the presence of antibiotic compounds, which may lead to the spread of antibiotic resistance. Biochar-based adsorption has been widely assessed as a means of polishing TMW to remove antibiotics prior to use; however, relatively little is known about how acid and base modification of biochar impacts its antibiotic retention capabilities, especially under the dynamic (flow-through) conditions inherent in proposed polishing systems. Using small columns of sand mixed with dairy manure or rice husk biochars (initial pH = 8.48 and 8.91, respectively) modified with acid or base, the retention of trimethoprim (TMP), sulfamethoxazole (SMZ), and sulfapyridine (SPD) was assessed. For SMZ and SPD, retention by the columns was consistently markedly lower than that measured in batch studies, indicating that equilibrium data may overestimate the retention of more realistic (dynamic) systems. TMP was completely retained by all biochars under both conditions. In columns, SMZ retention was strongly controlled by its pH-dependent speciation (greater retention at low pH, i.e., in acid modified biochars). SPD retention was strongly controlled by biochar surface area, which was markedly increased in the acid-modified dairy manure biochar and base-modified rice husk biochar. The findings indicate that acid or base modification of certain biochars may improve retention of sulfonamide antibiotics (which are known to be particularly difficult to remove from TMW) even under dynamic conditions. Biochar-based polishing systems comprising such biochars may offer potential in mitigating the spread of antibiotics, and hence antibiotic resistance, in agricultural settings.

A Novel Approach: Nanopore Sequencing of Native Cell-Free DNA in Diffuse-Large B-Cell Lymphoma Patients

Introduction: Nanopore sequencing (NS), a third generation long-read sequencing technology, is increasingly being used to study cancer genomes. NS has many advantages over next-generation sequencing, including the capability to simultaneously extract base sequence as well as base modifications in the same run without any further library modification. Notably, its portability and ability to provide real-time data make it a potentially powerful tool for clinical applications. This study explores the use of NS to directly sequence native cell free DNA (cfDNA) from patients with diffuse-large B-cell lymphoma (DLBCL), the most common type of non-Hodgkin lymphoma. Methods: For this exploratory study, 20 patients with either a new diagnosis or relapsed DLBCL were enrolled. We adapted NS methods for cfDNA and sequenced cfDNA before each chemotherapy cycle using both an Illumina platform (as a control) and the Nanopore platform. For differential methylation, we also sequenced plasma cfDNA from five healthy donors on the Nanopore. Results: Our findings demonstrate that we can directly sequence cfDNA on the Nanopore platform, obtaining a whole genome coverage (average 1.2x) with deeper coverage in some regions and also observe ~60% global DNA methylation level in CG context. From this data we are now analyzing regions that are differentially methylated (DMRs) between normal and DLBCL, determining SNV/Indel profiles, and comparing copy number variants (CNVs). These results will be available and presented at the time of the meeting. Conclusions: NS is capable of detecting cfDNA and measuring methylation levels, offering a promising and potentially clinically applicable new approach for the study of cfDNA. Future directions: As we continue this research, our vision is to leverage the portability and real-time data capabilities of NS for clinical applications. By simultaneously combining multiple genomic features with machine learning, we aim to develop a method for measuring lymphoma cfDNA-based MRD in DLBCL patients to be used in clinical settings.

Screening The Anticancer Activity for New Schiff Bases of Natural Steroids

The significance of cholesterol and ergosterol (provitamin d2) in biological systems has prompted interest in these compounds as pharmaceuticals. It has been reported on the effective straightforward synthesis of Schiff base modifications of cholesterol aldehyde and ergosterol aldehyde and their assessment as possible agents against cancer (esophagus natural cell lines and human cancer cells). Through a process called Steglish esterification, the initial cholesterol and ergosterol are converted into their respective aldehyde derivatives, by reacting with amines such as 2-amino 6-fluoro benzothiazole, carbohydrazide, and thiosemicarbazide, these aldehyde derivatives are transformed into the appropriate Schiff base derivatives, and then their anticancer activity is checked using the MTT (Microculture Tetrazolium Assay) assay method. For the purpose of analyzing their cytotoxic effects, these compounds were tested on cancerous (SK-GT-4, human esophageal adenocarcinoma) and normal (normal cell line, Rat Embryonic fibroblast (REF)) cell lines. The MTT assay results revealed that the compounds (a1, a2, c1, c2, compound 1) were more harmful to human esophageal cancer cell line than to healthy cell line other than compounds (t1, t2, compound 2, ergosterol and cholesterol alone). Overall, our research indicated that provitamin D2 (ergosterol), a1, a2, c2, t2, compound 1, had a growth-inhibiting effect on both cell lines compared to c1, t1, compound 2, and cholesterol alone.

Hairpin amplicon cleavage PCR (HAC-PCR): A novel, highly sensitive and cost-effective approach for detection of low-frequency DNA mutations

Highly sensitive detection of gene variants holds immense significance for cancer diagnosis and treatment. However, the presence of highly redundant wild DNA poses a substantial challenge when identifying low-frequency mutant circulating tumor DNA (ctDNA) in cancer cases. Here, a novel technique called hairpin amplicon cleavage PCR (HAC-PCR) for detecting low-level DNA mutations with exceptional sensitivity was presented. What sets HAC-PCR apart is its ability to achieve high sensitivity independently of strand-displacement, base modification, or the use of additional reagents. The discrimination between wild-type targets (WT) and mutant targets (MT) is significantly improved by introducing uniquely designed hairpin structures. These hairpins hinder the amplification of WT and are cleaved during the elongation of MT. For the first time, the unique mechanism of hairpin amplicon cleavage by Taq polymerase was exploited for mutation identification. The optimized HAC-PCR consistently demonstrates the capability to discriminate mutant DNA at levels as low as 0.01% variant allele fraction (VAF) for five mutation types. Furthermore, this impressive sensitivity of 0.01% VAF has also been achieved in a multiplex assay. When applied to the testing of tissue DNA from 24 patients with thyroid cancers and plasma ctDNA from 12 patients with non-small cell lung cancers (NSCLCs), HAC-PCR exhibited a 100% concordance rate with the existing commercial kit and droplet digital PCR (ddPCR). Therefore, HAC-PCR stands as a cost-effective and highly effective tool for variant identification in clinical testing.

DDDR-19. FIRST-IN-CLASS CANCER THERAPEUTICS TARGETING THE DNA DAMAGE RESPONSE

Abstract Hemispherian is advancing first-in-class therapeutics for cancers that are historically difficult to treat. Glioblastoma multiforme (GBM) is the primary indication for our lead compound GLIX1, which is currently in late-stage preclinical development. GLIX1 is developed together with a biomarker-based companion diagnostic assay to pair diagnostics and optimal treatment. It is Hemispherian’s mission to provide superior treatment options for brain cancer patients. Hemispherian’s proprietary molecules selectively target the DNA damage response in cancer cells. These molecules take advantage of a pre-existing condition of all cancer cells – nearly all cancer cells have a reduction in levels of the DNA base modification, 5-hydroxymethylcytosine. Hemispherian scientists have found that restoration of this base modification to normal cells activates the DNA damage response resulting in cancer cell death. Targeting of the enzyme responsible for maintaining genomic 5-hydroxymethylcytosine, TET2, led to the discovery of the GLIX family of molecules. Hemispherian’s lead GLIX molecules, GLIX1 and GLIX5, show excellent efficacy in vivo in xenograft models, have a superior safety profile, are orally bioavailable and are blood brain barrier penetrant. GLIX efficacy against a panel of patient-derived cell lines representing a broad spectrum of pediatric and adult brain tumor diagnoses will be discussed. Taken together, these molecules have an exceptional profile for treating a broad range of cancers including cancers of the brain.

PATH-64. COMPREHENSIVE CNS TUMOUR MOLECULAR DIAGNOSTICS USING THIRD GENERATION SEQUENCING

Abstract The WHO classification of CNS tumours 2021 recommends reporting a wide range of molecular alterations for WHO-compatible diagnoses. Conventional workflows warrant considerable investment and long turnaround times limited by batching. Nanopore sequencing enables long read sequencing, real-time targeting and simultaneous base modification detection with compact devices. Rapid-CNS2- a rapid, comprehensive adaptive sampling based sequencing pipeline with a turnaround time of 5 days was previously described. The ad-hoc methylation classification model employed by Rapid-CNS2 is limited to MNP v11 classes. This study presents an optimised Rapid-CNS2 and validation cohort that successfully reports hard-to-detect variants. We also present a whole genome sequencing workflow that affords direct upload to the MNP website for MNP v12 classification. We sequenced and analysed 156 samples from the Department of Neuropathology, University Hospital Heidelberg using Rapid-CNS2. RESULTS: were compared to NGS panel sequencing and methylation array analyses. Rapid-CNS2 showed improved resolution over NGS panel sequencing for CNAs. High accuracy was obtained for detection of SNV/Indels. Long reads spanning the breakpoint identified clinically relevant fusions in 10/10 cases. Subclonal SVs like EGFR vIII missed by NGS panel sequencing were detected with high confidence. Complete concordance was achieved for MGMT promoter methylation status and methylation classification for MNP v11 classes. The optimized Rapid-CNS2 workflow shortened the turnaround time to 30 hours from sample receipt and minimum input DNA required was reduced to 500ng. For WGS, samples were sequenced for 72h on a Promethion device. WGS enabled accurate reporting of SNVs, CNAs, SVs, MGMT promoter status in addition to MNP v12 classification directly from the website eliminating the need for ad-hoc training of methylation classification models. The presented nanopore sequencing based workflows provide feasible single assay alternatives to conventional molecular diagnostic approaches with the aim to make such analyses more accessible.