Mutational Analysis Research Articles

Optimized Second-Generation IL-4Rα Inhibition: Structural and Molecular Dynamics Properties of Rademikibart Fab-IL-4Rα Complex

Introduction: Global Burden of Disease studies showed atopic dermatitis (AD) and asthma are the top two immune-mediated inflammatory diseases at younger age [1]. Dupilumab was the initial first-generation interleukin-4 receptor alpha (IL-4Rα) inhibitor for treating both type I and type II IL-4Rα-dependent inflammatory disorders. Following recent clinical trials, rademikibart (previously, CBP-201) emerges as an optimized next-generation human monoclonal antibody with higher binding affinity to IL-4Rα compared to dupilumab [2]. It demonstrated better effect in inhibiting STAT6 intracellular signaling in vitro and provided similar potency inhibiting both IL-4 induced TARC release and IL-4 induced B cell activation [2]. Materials & Methods: X-ray crystallography was used to determine the atomic resolution 3D structure of rademikibart fragment antigen binding (Fab) bound to IL-4Rα. This structure was analyzed and compared computationally with the 2.82 Å resolution crystal structure of dupilumab Fab bound to IL-4Rα (Protein Data Bank Code 6WGL). Molecular dynamics studies on rademikibart and dupilumab bound to IL-4Rα examined the stability of the complexes and effects of amino acid mutations on complex formation. Results: The x-ray crystal structure of rademikibart Fab bound to IL-4Rα was determined at 2.71Å and compared to the complex of dupilumab Fab and IL-4Rα. The rotation angle between dupilumab and rademikibart bound to IL- 4Rα is 59.17°. This rotation enables the epitope of rademikibart, but not dupilumab, on IL-4Rα to overlap more closely with the conserved binding interface utilized by IL-4 and IL-13 cytokines. Molecular dynamics simulations of rademikibart Fab and dupilumab Fab complexed with IL-4Rα showed the third interface loop (residues 148 to 152 in domain 2) of IL-4Rα interacts directly with rademikibart, which is absent in dupilumab/IL- 4Rα complex. This finding is confirmed by analysis of the hydrogen bond interactions at the interface between the antibodies and IL-4Rα, demonstrating superior binding energy for rademikibart. Through single amino acid mutation analysis on rademikibart, we identified residue Y50 on rademikibart as the key residue interacting with IL- 4Rα’s third interface loop. Conclusion: Our data provide a molecular and structural rationale for the enhanced IL-4Rα inhibition by rademikibart over dupilumab, confirming rademikibart as an optimized second-generation IL-4Rα inhibitor.

Abstract A042: Desmosome mutations in the microenvironment regulate melanoma proliferation

Abstract Desmosomes are transmembrane protein complexes critical for cell-cell adhesion in epithelial tissues and other cell types. In skin cancers like melanoma, epithelial cells called keratinocytes are the predominant cells in the primary melanoma microenvironment, yet it remains largely unknown whether this cell type is subject to genetic alterations during melanoma development and its role in early melanoma progression. In this study, using an integrative analysis of tumor mutations and protein biophysical interactions, we identify a high frequency of genetic alterations in desmosome-related genes in human cancers, with the highest occurrences in cutaneous melanoma. Notably, over 70% of cutaneous melanoma cases exhibit mutations in desmosome genes and desmosome gene mutations are associated with lowered expression of the desmosome complex in melanoma. Using spatial transcriptomics and immunohistochemistry analyses, we find that the decrease in desmosome gene expression predominantly occurs in keratinocytes within the tumor microenvironment rather than in the melanoma cells themselves. To study the functional significance of this alteration in melanoma development, we used a human melanoma/keratinocyte co-culture system using primary melanoma cell lines. Our studies show that knockdown of desmosome genes in keratinocytes markedly increased proliferation of adjacent melanoma cells in melanoma/keratinocyte co-cultures. Additionally, melanoma cell proliferation is enhanced when exposed to media preconditioned by desmosome-deficient keratinocytes. These observations suggest that the gradual accumulation of mutations in desmosome genes within neighboring keratinocytes may create a conducive environment that primes melanoma cells for neoplastic transformation. This study underscores the critical role of genetic alterations in the tumor microenvironment in melanoma progression and highlights the potential for targeting desmosome-related pathways in therapeutic strategies against melanoma. Citation Format: Mohita Tagore. Desmosome mutations in the microenvironment regulate melanoma proliferation [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor-body Interactions: The Roles of Micro- and Macroenvironment in Cancer; 2024 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(22_Suppl):Abstract nr A042.

Detection of BRCA-1 gene mutations in epithelial ovarian tumors at tertiary care hospital Karachi

ABSTRACT Background: Ovarian cancer is the most common and lethal gynecological malignancy. Ovarian malignancy is the 3rd most common cancer in Pakistan. More than 90% ovarian cancers are of epithelial cell origin, about 24-40% such cancers have hereditary or sporadic mutation in the BRCA-1 or BRCA-2 genes. The present study was designed to detect BRCA-1 gene mutations in different histological sub-types of epithelial origin of benign, borderline and malignant ovarian tumors. Methods: This cross-sectional study was based on the analysis of BRCA-1 gene mutations in the epithelial origin of benign, borderline and malignant ovarian tumors received at the department of Pathology, Basic Medical Sciences Institute, Jinnah Post graduate Medical Centre, from 01-01-2011 to 31-12-2015. A total of 80 diagnosed cases were selected and analyzed for PCR. Results: BRCA-1 gene mutations were detected in 22 (27.5%) cases out of the 80 randomly selected epithelial ovarian tumors. Serous cyst adenocarcinoma was the commonest including 63% cases. BRCA-1 gene mutations were also positive in other epithelial ovarian tumors, including Mucinous cyst adenocarcinoma (13.6%), Endometroid adenocarcinoma and Mixed Mullerian tumor (4.5% each). In addition mutations were also detected in borderline tumors including Mucinous borderline tumor (9.09%) and Seromucinous borderline tumors (4.5%).The observations and results of the study were elaborated with the assistance of tables, figures and photomicrographs. Conclusion: BRCA-1 gene mutations manifestations were identified in a large number of serous malignant ovarian tumor cases. Small percentages of borderline and malignant mucinous tumors, endometroid adenocarcinoma and mixed mullerian tumor were also positive for BRCA-1 gene mutations.

Precise Genotyping Via Surface‐Enhanced Raman Spectroscopy‐Based Optical Sensing Chip for Guiding Targeted Therapy in Lung Cancer

AbstractThe emergence of “precision medicine” marks a notable shift in cancer treatment, moving from a tumor type–oriented approach to a more targeted, gene‐oriented approach. Detecting low‐abundance mutant genes in blood is challenging but crucial for personalized treatment plans. Herein, a novel platform combining catalytic hairpin self‐assembly (CHA)‐mediated self‐calibrating surface‐enhanced Raman spectroscopy (SERS) with a high‐throughput Raman system (CCSPS) was designed. This platform enables ultrasensitive and rapid genotype analysis of gene mutations. The development of CCSPS specifically targets EGFR mutations, which serve as crucial therapeutic targets for precision therapy in lung cancer. This system shows excellent sensitivity and selectivity, capable of detecting multiple EGFR mutations (Del‐19, L858R, and T790M) with a detection limit as low as attomolar levels. Additionally, precise genotyping analysis was successfully conducted on 42 clinical samples using the CCSPS, yielding results consistent with those obtained through next‐generation sequencing. These results underscore the efficacy of the CCSPS in noninvasively identifying circulating tumor DNA (ctDNA) mutations, facilitating immediate therapeutic decision making at the bedside. In summary, the CCSPS is a fast, accurate, versatile, and compact testing system capable of precisely screening individuals who stand to benefit from targeted therapy, thus promoting personalized and precise healthcare.

A systematic literature review on clonal evolution events preceding relapse in multiple myeloma.

Despite considerable treatment advances, multiple myeloma (MM) remains an incurable hematological cancer due to treatment resistance. A systematic literature search was conducted to identify determinants for clonal evolution driving relapse and drug resistance in MM. A total of 631 non-duplicate publications were screened of which 28 articles were included for data extraction. Genetic alterations, mutational signatures, evolutionary trajectories, and non-genetic determinants were identified as key topics to characterize clonal evolution in relapsed MM. A variety of factors led to clonal diversification and increased tumor mutation burden, such as MAPK-Ras mutations and incremental changes related to chromosomal bands 1 and 17, while mutational signature analyses revealed that APOBEC activity and melphalan treatment leave a distinct impact on the clonal composition in MM genomes. To capture and dissect tumor heterogeneity, our review suggests combining methods or using technical approaches with high resolution to assess the impact of clonal evolution.

Base-excision repair pathway shapes 5-methylcytosine deamination signatures in pan-cancer genomes.

Transition of cytosine to thymine in CpG dinucleotides is the most frequent type of mutation in cancer. This increased mutability is commonly attributed to the spontaneous deamination of 5-methylcytosine (5mC), which is normally repaired by the base-excision repair (BER) pathway. However, the contribution of 5mC deamination in the increasing diversity of cancer mutational signatures remains poorly explored. We integrate mutational signatures analysis in a large series of tumor whole genomes with lineage-specific epigenomic data to draw a detailed view of 5mC deamination in cancer. We uncover tumor type-specific patterns of 5mC deamination signatures in CpG and non-CpG contexts. We demonstrate that the BER glycosylase MBD4 preferentially binds to active chromatin and early replicating DNA, which correlates with lower mutational burden in these domains. We validate our findings by modeling BER deficiencies in isogenic cell models. Here, we establish MBD4 as the main actor responsible for 5mC deamination repair in humans.

Expression levels of some genes in the MAPK pathway (DUSP1, DUSP2, DUSP4, DUSP6 and DUSP10) in eyelid tumor tissue

Abstract Objectives To control mitogen-activated protein kinases (MAPK) signaling pathways involved in the onset and progression of cancer, dual specificity phosphatases (DUSPs/MKP) are essential. This study seeks to detect the correlation between eyelid tumors and the genes DUSPs, known for their influence on MAPK signaling pathways. Additionally, we aim to juxtapose our findings with analyses from various bioinformatics databases. Methods Expression levels of relevant genes in cDNA samples were determined by quantitative PCR method. Open-access databases were used for mutation analysis of relevant genes, mRNA expression changes, and survival analyses, and the STRING database was used for protein-protein interactions. Results It was found that the expression of DUSP1 and DUSP2 showed a significant decrease in the tumor tissue, while a significant increase was detected in the DUSP4 and DUSP6 genes. Additionally, when we compared the study genes with the Cancer Genome Atlas program cancer cohorts, it was found that the DUSP1 and DUSP10 gene expression profiles were downregulated in uveal melanoma compared to other cancer cohorts. Conclusions Significant and obvious changes were observed in the DUSP genes we studied in eyelid tumors. However, the relationship between these genes and cancer must be studied more. Considering that these enzymes are effective in cell proliferation, differentiation, and apoptosis, it would be appropriate to plan comprehensive studies on their interactions with other proteins they interact with in the MAPK pathway.

Molecular alterations influencing the prognostic outcome in small pancreatic cancer (≤ 2 cm)

Abstract Purpose Pancreatic cancer (PC) is the most lethal cancer. The prognosis of small PC (tumor smaller than 20 mm) is better than that of larger PC tumors, indicating the importance of detecting early-stage PC for patient outcome. The aim of this study was to elucidate the molecular features in small PC (≤ 20 mm). Methods This study included 79 PC tumors (≤ 20 mm in pathological examination) resected between 2004 and 2022. c-Myc, Caveolin-1, Smad4, and Thrombospondin-1 were examined by immunostaining. These molecular alterations were compared in PC patients with tumor size ≤10 mm (n = 11) (14%) and 10 mm < tumor size ≤20 mm (n = 68) (86%). Mutation analyses of KRAS, PIK3CA, and BRAF were performed by pyrosequencing in 22 PCs. Results PC with 10 mm < tumor size ≤20 mm showed significantly worse overall survival and disease-free survival than PC with tumor size <10 mm (P = 0.024 and P = 0.028). Tumor c-Myc and stromal Caveolin-1 expressions were significantly increased in tumors larger than 10 mm (P = 0.02 and P = 0.04). c-Myc and Caveolin-1 expressions were associated with poor disease-free survival and overall survival. KRAS, PIK3CA, and BRAF mutation status did not differ between the two groups. Conclusions Tumor c-Myc and stromal Caveolin-1 overexpressions were detected in tumors larger than 10 mm. Their overexpressions were associated with worse prognosis even in small PC. These molecular alterations in small PC may be a clue for the detection of early-stage PC.

Influenza A virus shedding and reinfection during the post-weaning period in swine: longitudinal study of two nurseries

IntroductionInfluenza A virus in swine (IAV-S) is common in the United States commercial swine population and has the potential for zoonotic transmission.ObjectiveTo elucidate influenza shedding the domestic pig population, we evaluated two commercial swine farms in Illinois, United States, for 7 weeks. Farm 1 had a recent IAV-S outbreak. Farm 2 has had IAV-S circulating for several years.MethodsForty post-weaning pigs on Farm 1 and 51 pigs from Farm 2 were individually monitored and sampled by nasal swabs for 7 weeks.ResultsRT-PCR results over time showed most piglets shed in the first 2 weeks post weaning, with 91.2% shedding in week one, and 36.3% in week two. No difference in the number of pigs shedding was found between the two nurseries. Reinfection events did differ between the farms, with 30% of piglets on Farm 1 becoming reinfected, compared to 7.8% on Farm 2. In addition, whole genome sequencing of nasal swab samples from each farm showed identical viruses circulating between the initial infection and the reinfection periods. Sequencing also allowed for nucleic and amino acid mutation analysis in the circulating viruses, as well the identification of a potential reverse zoonosis event. We saw antigenic site mutations arising in some pigs and MxA resistance genes in almost all samples.ConclusionThis study provided information on IAV-S circulation in nurseries to aid producers and veterinarians to screen appropriately for IAV-S, determine the duration of IAV-S shedding, and predict the occurrence of reinfection in the nursery period.

SARS-CoV-2 main protease (M-pro) mutational profiling: An insight into mutation coldspots

SARS-CoV-2 and the COVID-19 pandemic have marked a milestone in the history of scientific research worldwide. To ensure that treatments are successful in the mid-long term, it is crucial to characterize SARS-CoV-2 mutations, as they might lead to viral resistance. Data from >5,700,000 SARS-CoV-2 genomes available at GISAID was used to report SARS-CoV-2 mutations. Given the pivotal role of its main protease (M-pro) in virus replication, a detailed analysis of SARS-CoV-2 M-pro mutations was conducted, with particular attention to mutation-resistant residues or mutation coldspots, defined as those residues that have mutated in five or fewer genomes. 32 mutation coldspots were identified, most of which mediate interprotomer interactions or funneling interaction networks from the substrate-binding site towards the dimerization surface and vice versa. Besides, mutation coldspots were virtually conserved in all main proteases from other CoVs. Our results provide valuable information about key residues to M-pro structure that could be useful in rational target-directed drug design and establish a solid groundwork based on mutation analyses for the inhibition of M-pro dimerization, with a potential applicability to future coronavirus outbreaks.

Targeted Forward Genetics: Saturating Mutational Analyses of Specific Target Loci Within the Genome.

Precise allele replacement by homologous recombination (also known as "gene targeting" or "genome editing") allows scientists to engineer altered DNA sequences, insertions, or deletions at specific locations in the genome. Such reverse genetics provides powerful tools to elucidate the structure and function of regulatory DNA elements, genes, RNAs, and proteins within their natural, endogenous context. Here, we describe in detail the methodology for Targeted Forward Genetics (TFG), which supports population-scale, saturating screens of allele replacements spanning thousands of base pairs at a specific target locus in the genome. The overall approach and detailed protocols, developed for the fission yeast Schizosaccharomyces pombe, are extensible to other organisms in which gene targeting is feasible.

An epidemiologic surveillance study based on wastewater and respiratory specimens reveals influenza a virus prevalence and mutations in Taiyuan, China during 2023-2024.

Influenza is a major cause of morbidity and mortality. Influenza A virus (IAV) is one of the most important pathogens causing influenza and often causes global pandemics due to its tendency to mutate. We aim to use epidemiology based on wastewater and respiratory specimens to understand the occurrence of influenza A virus infections in Taiyuan City. A retrospective epidemiology surveillance was carried out at the First Hospital of Shanxi Medical University (FHSMU) and five wastewater treatment plants (WTPs) in Taiyuan city from 2023 to 2024. Reverse transcription real-time fluorescence quantitative polymerase chain reaction (RT-qPCR) was used to detect influenza A viruses in wastewater and respiratory specimens. High-throughput whole genome sequencing was performed on 17 strains obtained in this study, and subsequent analyses included characterization, phylogenetic construction, amino acid mutation analysis, and antigenic structural variability assessment. 520 wastewater samples and 1,203 throat swab samples were collected. We detected RNA concentration from pH1N1 and H3N2 viruses in wastewater and got 17 genome sequences (5 of pH1N1 and 12 of H3N2) in respiratory specimens. Whole-genome sequencing showed co-prevalence of pH1N1 viruses in the branches of 6B.1A.5a.2a.1 and H3N2 viruses in the branches of 3C.2a1b.2a.2a.3a.a in Taiyuan from 2023 to 2024. Moreover, a HA mutation (N138D), predicted to be of high phenotypic consequence, was found in 8 Taiyuan H3N2 sequences. This study highlights the predominant presence of pH1N1 and H3N2 strains in Taiyuan. The analysis also identified amino acid site variations in the HA antigenic epitopes in H3N2 strains, which may contribute to immune escape.

Characterization and organization of telomeric-linked helicase (tlh) gene families in Fusarium oxysporum.

Telomere-linked RecQ helicase (tlh) genes have been reported in several fungi and a choanoflagellate in the regions adjacent to the terminal telomere repeats. In this study, we explored the Telomere-linked RecQ helicase (tlh) genes in four strains of Fusarium oxysporum, offering new insights into their genomic structure, functional motifs, and impact on chromosomal ends. We conducted a comprehensive analysis, comparing the tlh genes of F. oxysporum with those previously identified in other organisms and uncovering significant similarities. Through comparative genomics, we identified conserved protein motifs across these genes, including a TLH domain, C2H2, and RecQ helicase motifs. Our phylogenetic analysis positions the F. oxysporum tlh genes in a cluster with other known tlhs, suggesting a shared evolutionary origin. Mutation analysis revealed a relatively low level of deleterious mutations in tlh gene paralogs, with a notable proportion of full-size structures maintained across strains. Analysis of subtelomeric sequences indicates that a region with almost identical sequences flanks the majority of chromosome ends, termed tlh-containing region (TLHcr), across these strains. The presence of TLHcrs at chromosome ends, either as single entities or in arrays, underscores their potential role in telomere function and genome stability. Our findings provide a detailed examination of tlh genes in four strains of F. oxysporum, laying the groundwork for future studies on their biological significance and evolutionary history in fungal genomes.

BIOM-31. MOLECULAR ANALYSIS OF RESECTED BREAST CANCER BRAIN METASTASES REVEALS CEREBELLAR LOCATION AND TRIPLE NEGATIVE SUBTYPE ARE NEGATIVE PROGNOSTIC FACTORS

Abstract BACKGROUND Brain metastases (BMs) are a significant cause of morbidity and mortality in metastatic breast cancer (MBC), yet prognostic and predictive biomarkers remain poorly understood. STUDY DESIGN 90 consecutive patients with breast cancer BMs who underwent surgical resection from 2006-2022 were retrospectively identified. Review of the electronic medical record and institutional cancer registry were completed to identify baseline clinical parameters and outcomes. BM mutation analysis was performed using a CLIA-certified targeted DNA mutation sequencing assay as part of routine clinical care. RESULTS All patients were female, median age at first BM resection was 57.9 years (range 27.0-81.3 years), and receptor subtypes included HR+/HER2- (n=27, 30.0%), HER2+ (n=33, 36.7%), and triple negative BC (TNBC; n=30, 33.3%). At time of first BM resection, most patients had a solitary BM (n=55, 61.1%), with anatomic locations including cerebellar (n=29, 32.2%), frontal (n=26, 28.9%), and parietal (n=17, 18.9%) sites. BM DNA mutation analysis (n= 20) revealed pathogenic alterations in TP53 (n=17, 85.0%), ERBB2 amplification (n=7, 35.0%), PIK3CA (n=4, 20.0%), BRCA1 (n=3, 15.0%), and BRCA2 (n=2, 10.0%). Most patients received postoperative radiation (n=82, 90.8%) and/or systemic therapy (n=67, 82.7%). Cerebellar location was significantly associated with the risk of developing leptomeningeal disease compared to other BM resection sites (HR = 2.75, p=0.02). Among all patients, median overall survival (OS) from first BM resection was 27.8 months (range 0.1-196.6 months). HR+/HER2- (median 37 months) or HER2+ (median 36 months) MBC was associated with significantly increased OS compared to TNBC (14.4 months) (p<0.01, log-rank test). CONCLUSIONS Patients with breast cancer BMs exhibited a median OS of >2 years following BM resection, and patients with TNBC had a significantly worse prognosis. Targetable mutations were observed in 40% of resected BM specimens, supporting the utility of molecular analysis of resected BMs to help guide clinical management.

DNAR-16. TARGETING APOBEC CYTIDINE DEAMINASES TO ENHANCE RADIOSENSITIVITY IN GLIOMA

Abstract Glioma is the most common and aggressive primary adult brain tumor. Radiation therapy (RT) is a critical part of glioma treatment, but radioresistance severely limits its therapeutic efficacy. To better understand how radioresistance develops, we analyzed 212 paired primary and recurrent gliomas from the Glioma Longitudinal Analysis Consortium. We found a significant increase in mutational signatures associated with the activity of the Apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like (APOBEC) family of cytidine deaminases in RT-treated recurrent gliomas (p<0.001, paired Wilcoxon signed-rank test). Further analysis of post-treatment mutations showed that RT increased APOBEC mutational signatures independent of other factors (p<0.001, multivariable log-linear regression), suggesting that APOBEC proteins become activated by radiation. However, their role in promoting tumor evolution and treatment resistance is currently unknown. Further analyses of extended signature contexts and expression data nominated APOBEC3B (A3B) and APOBEC3G (A3G) as candidates for creating these mutational signatures. To investigate their roles in RT response, we knocked out (KO) A3B and A3G individually in patient-derived glioma cell lines before RT. Compared to Control cells, irradiated A3B-KO and A3G-KO cells acquired significantly fewer APOBEC mutational signatures by whole genome sequencing (p<0.05, Kruskal–Wallis test) and exhibited enhanced radiosensitivity in vitro. Interestingly, the KO cells displayed a significant decrease in mutational signatures associated with Non-Homologous End-Joining (NHEJ), one of the main pathways for repairing the RT-induced DNA damage (p<0.05, Kruskal–Wallis test), suggesting that APOBEC deletion impaired DNA repair. This was further supported by persistent gH2AX DNA damage foci 48h post-RT and reduced autophosphorylation of DNA-dependent protein kinase (DNA-PK), a vital component of the NHEJ pathway, particularly in A3G KO cells. Overall, our results identify APOBEC proteins as potential targets for radiosensitizing gliomas and highlight the clinical utility of using mutational signatures to predict and monitor response to treatment.

CNSC-06. NAVIGATING BRAIN CANCER: PATIENT STORIES AND MEDICAL BREAKTHROUGH

Abstract The complex interplay of genetic mutation, dys regulated signalling pathways, Epigenetic modifications and Interaction with tumor microenvironment are the major aggravating factors for Brain Tumors. Neurological complications involve Increased Intracranial pressure, Seizures, Motor and Sensory deficits, Aphasia, Hydrocephaly, Behavioral and personality changes, Cerebellar Symptoms. It is diagnosed in a sequential steps. Collecting good history from patient in primary then examining the patient all through and making some investigations to come to a final diagnosis. On asking history to a patient he complains of recurrent convulsions, Personality changes and some motor activity deficit and loss of few senses. On examination we can reveal defect in cranial nerve functions, Motor or sensory deficits, loss of reflexes and coordination. Then Investigations being the next step for diagnosis, We ask the patient to undergo for a test for Blood Biomarkers and CSF analysis in case of suspection of metastatic Brain tumors. Biopsy to obtain for Histopathological Examination. Histopathological examination is done for analysis of genetic mutations and molecular markers. The other imaging techniques like MRI, MRS, CT, PET, EEG are done. This gives a clear picture of what actually caused tumor and shows a way for treatment. Now the treatment involves both Medical and Surgical treatment. Medical treatment includes Chemotherapy along with radiation therapy where the drugs given are Temozolomide and targeted therapy included EGFR BRAF inhibitors for glioblastoma and low grade gliomas respectively. Tumor treating fields- Optune is given. Surgical treatment involves Craniotomy for removal of primary Brain tumors, Neuro Endoscopy if tumor is present in ventricles or base of Brain, Laser interstitial Thermal therapy for deap seated and recurrent brain tumors. This is all about the treatment and now the patient management is very important for further clinical help. Physical, occupational and speech therapy helps patients recover. Symptomatic approach for inoperable tumors Provide comprehensive support for patients nearing end of life, Emphasise confidence and Quality of life.

PATH-20. CLINICAL FINDINGS AND PROGNOSIS OF ADULT GLIOBLASTOMA HARBORING ABRAF V600E MUTATION

Abstract BACKGROUND AND AIM Although the BRAF V600E mutation is commonly associated with pediatric low-grade gliomas, glioblastomas (GB) with this mutation (BRAF-GB) have also been reported. The recent emergence of RAF and MEK inhibitors for BRAF V600E-positive tumors highlights the potential for targeted therapy in a subset of GB patients. This study aimed to characterize the clinical features and prognosis of BRAF-mutant glioblastomas. MATERIALS AND METHODS A retrospective analysis was conducted on a cohort of patients diagnosed with GB (confirmed by 2021 WHO criteria) above 16 years of age. Targeted genetic analysis for mutations in BRAF V600E, TERT promoter, and CDKN2A/B was performed on the GB samples. Clinical features of BRAF-GB were analyzed, and patients were further categorized into three groups: BRAF-GB, typical GB, and molecular GB. Subsequent analysis compared the prognosis among these groups. RESULTS Our study identified 11 cases (5.1%) harboring the BRAF V600E mutation among 215 adult GB patients. Patients with BRAF-GB exhibited a significantly younger median age of onset (38-year-old) compared to the typical GB (N = 167, 64-year-old) and molecular GB (N = 37, 61-year-old) groups. Further analysis of the BRAF-GB cohort (n=11) revealed a high prevalence (82%) of homozygous CDKN2A/B deletions, and only one case (9%) harbored a TERT promoter mutation. Interestingly, three BRAF-GB patients presented with hemorrhagic onset. Four cases showed evidence of prior lesions, with one case diagnosed histologically as a low-grade glioma 14 years earlier. The BRAF-GB group exhibited significantly longer median progression-free survival (32.8 months) and overall survival (84.2 months) compared to both the typical GB group (9.6 months and 18.9 months, respectively) and the molecular GB group (14.1 months and 32.1 months, respectively). CONCLUSIONS Early-age onset, with hemorrhagic onset or presence of prior lesions are factors that could lead to recommendation of BRAF analysis for adult GB patients.

PATH-49. INTRA-OPERATIVE GENETIC ANALYSES FOR DECISION-MAKING DURING TUMOR REMOVAL OF ADULT-TYPE DIFFUSE GLIOMA USING RAPID QUANTITATIVE PCR DEVICE

Abstract BACKGROUND Recent comprehensive molecular analyses of central nervous system (CNS) tumors identified several diagnostic or prognostic markers. Especially, for adult-type diffuse glioma, IDH mutation, TERT promoter (pTERT) mutation and CDKN2A homozygous deletion are quite important markers for diagnosis and prognosis of this type of glioma. Intra-operative identification of these molecular alterations could be effective for decision-making of tumor removal strategy. METHODS In this study, we established intra-operative gene analysis method using rapid quantitative PCR device, GeneSoC, by which we could obtain genotype of these important genes for 20 minutes in an operating room. Using GeneSoC, we could perform 50 cycles of quantitative PCR for approximately 12 minutes. Results; We established rapid gene analyses of mutations of IDH1 R132H, pTERT C225T, pTERT C250T and homozygous deletion of CDKN2A for adult-type diffuse gliomas. For elution of DNA, we incubated at least 1mg of tumor tissue at 95°for 5 minutes. Combined this boiling method with GeneSoC, we could obtain those genetic alterations for 20 minutes after collection of tumor tissue. In analyses of 111 adult-type diffuse glioma cases, sensitivity and specificity for detection of IDH1 p.R132H are 98% and 98%, respectively, and those of pTERT (C225T or C250T) are 100% and 100%, respectively. In analyses of 50 cases, sensitivity and specificity for detection of CDKN2A homozygous deletion are 100% and 83%, respectively. Conclusion; In this study, we could obtain the genotype of important molecular markers intra-operatively, not only during tumor removal but even during tumor biopsy, using GeneSoC device. This rapid genetic analysis might be effective not only for intra-operative diagnosis but also for detection of boundary between tumor and normal tissue.

STK19 facilitates the clearance of lesion-stalled RNAPII during transcription-coupled DNA repair.

Transcription-coupled DNA repair (TCR) removes bulky DNA lesions impeding RNA polymerase II (RNAPII) transcription. Recent studies have outlined the stepwise assembly of TCR factors CSB, CSA, UVSSA, and transcription factor IIH (TFIIH) around lesion-stalled RNAPII. However, the mechanism and factors required for the transition to downstream repair steps, including RNAPII removal to provide repair proteins access to the DNA lesion, remain unclear. Here, we identify STK19 as a TCR factor facilitating this transition. Loss of STK19 does not impact initial TCR complex assembly or RNAPII ubiquitylation but delays lesion-stalled RNAPII clearance, thereby interfering with the downstream repair reaction. Cryoelectron microscopy (cryo-EM) and mutational analysis reveal that STK19 associates with the TCR complex, positioning itself between RNAPII, UVSSA, and CSA. The structural insights and molecular modeling suggest that STK19 positions the ATPase subunits of TFIIH onto DNA in front of RNAPII. Together, these findings provide new insights into the factors and mechanisms required for TCR.

A Systematic Exploration of Mutation‐Based Fault Localization Formulae

ABSTRACTFault localization (FL) aims to automatically find the location of bugs in a software program. In the family of FL approaches, spectrum‐based fault localization (SBFL) is the most widely used and has been extensively studied, which computes the suspicious scores of a code element to be buggy via test coverage information. Mutation‐based fault localization (MBFL) further analyses the relationship between mutants and test results. Although MBFL involves more information, it also faces the following limitations. (1) To precisely evaluate suspicious sources, SBFL approaches proposed dozens of formulae based on coverage, while only a few of them have been adopted by MBFL. (2) The current MBFL approaches are based on the assumption that the capability in localizing bugs of each mutant is the same, so they assign the same weight to the mutants that change the test outputs and consider the mutant from the same code element equivalent. In this paper, we intend to enrich the MBFL family by the following two approaches. First, we collect 25 typical SBFL formulae and transform them into MBFL versions. Second, we propose new assumptions that the mutants should have different weights in computing suspicious sources and propose BLMu, a novel MBFL approach that treats mutants differently. We also propose novel metrics for MBFL by considering the high cost of mutation analysis. We perform large‐scale experiments by evaluating all the MBFL approaches against 395 real‐world Java bugs of the Defects4J benchmark. Our evaluation results reveal that BLMu demonstrates a substantial improvement over both MUSE and Metallaxis, the most popular MBFL approaches, at both the statement level and the method level. Specifically, in terms of Top‐1, BLMu improves by 106% at the statement level and 69% at the method level. When compared with other types of FL approaches, MBFL outperforms typical SBFL approaches while still far behind the state‐of‐the‐art learning‐based FL approaches.