Next-generation Sequencing Analysis Research Articles

Epitope binning for multiple antibodies simultaneously using mammalian cell display and DNA sequencing

Epitope binning, an approach for grouping antibodies based on epitope similarities, is a critical step in antibody drug discovery. However, conventional methods are complex, involving individual antibody production. Here, we established Epitope Binning-seq, an epitope binning platform for simultaneously analyzing multiple antibodies. In this system, epitope similarity between the query antibodies (qAbs) displayed on antigen-expressing cells and a fluorescently labeled reference antibody (rAb) targeting a desired epitope is analyzed by flow cytometry. The qAbs with epitope similar to the rAb can be identified by next-generation sequencing analysis of fluorescence-negative cells. Sensitivity and reliability of this system are confirmed using rAbs, pertuzumab and trastuzumab, which target human epidermal growth factor receptor 2. Epitope Binning-seq enables simultaneous epitope evaluation of 14 qAbs at various abundances in libraries, grouping them into respective epitope bins. This versatile platform is applicable to diverse antibodies and antigens, potentially expediting the identification of clinically useful antibodies.

Reference-free inferring of transcriptomic events in cancer cells on single-cell data

BackgroundCancerous cells’ identity is determined via a mixture of multiple factors such as genomic variations, epigenetics, and the regulatory variations that are involved in transcription. The differences in transcriptome expression as well as abnormal structures in peptides determine phenotypical differences. Thus, bulk RNA-seq and more recent single-cell RNA-seq data (scRNA-seq) are important to identify pathogenic differences. In this case, we rely on k-mer decomposition of sequences to identify pathogenic variations in detail which does not need a reference, so it outperforms more traditional Next-Generation Sequencing (NGS) analysis techniques depending on the alignment of the sequences to a reference.ResultsVia our alignment-free analysis, over esophageal and glioblastoma cancer patients, high-frequency variations over multiple different locations (repeats, intergenic regions, exons, introns) as well as multiple different forms (fusion, polyadenylation, splicing, etc.) could be discovered. Additionally, we have analyzed the importance of less-focused events systematically in a classic transcriptome analysis pipeline where these events are considered as indicators for tumor prognosis, tumor prediction, tumor neoantigen inference, as well as their connection with respect to the immune microenvironment.ConclusionsOur results suggest that esophageal cancer (ESCA) and glioblastoma processes can be explained via pathogenic microbial RNA, repeated sequences, novel splicing variants, and long intergenic non-coding RNAs (lincRNAs). We expect our application of reference-free process and analysis to be helpful in tumor and normal samples differential scRNA-seq analysis, which in turn offers a more comprehensive scheme for major cancer-associated events.

Beyond histology: A tissue algorithm predictive of post-surgical recurrence in hepatocellular carcinomas, including TERT promoter mutation.

Surgical resection for hepatocellular carcinoma (HCC) is burdened with a high recurrence rate and a lack of reliable prognostic factors. The aim of this study was to integrate the HCC pathological features with gene mutations to improve the prognostic role of pathological analysis.This is a monocentric prospective study, including 67 patients resected for HCC. All clinical data and histological features were collected, including tumor grade, architecture, margins, microvascular invasion, and microscopic portal vascular invasion (MPVI). Next-generation sequencing (NGS) was performed using a laboratory-developed multi-gene panel, allowing to amplify 330 amplicons (21.77kb), covering the relevant targets for solid tumor analysis. The most represented mutations were TERT promoter (n = 41, 61.2%), TP53 (n = 18, 26.9%) and CTNNB1 (n = 17, 25.4%). At follow-up, 13 (19.4%) patients experienced HCC recurrence: at multivariate analysis, tumor dimensions (p = 0.040), MPVI (p = 0.010), and TERT mutation (p = 0.034) correlated with recurrence. Dimensions ≥ 4.5cm (very close to AJCC stage pT3; 9 recurrences, p = 0.041, odd-ratio = 3.7), MPVI (9 recurrences, p = 0.062, OR = 3.3), and TERT (11 recurrences, p = 0.049, OR = 4.4) correlated with disease-free survival also at univariate analysis. The concomitant occurrence of these three variables was present in 7 cases, among which 5 recurred (p = 0.002, OR = 15.94).In conclusion, NGS analysis in resected HCC could not only be used for future therapies but should be integrated with histopathology to predict the risk of tumor recurrence after surgical resection: TERT mutation is among the strongest predictors of tumor recurrence, together with tumor stage (dimensions) and the occurrence of MPVI, which should always be reported separately from the classic MVI.

P09 Next-generation sequencing: on-and off-target analysis of gene editing in fibroblasts from patients with recessive dystrophic epidermolysis bullosa

Abstract Introduction and aims Base editors (BEs) and prime editors (PEs) have proved to have advantages over traditional clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 in correcting pathogenic mutations. We previously demonstrated 94.6% efficiency using adenine base editor (ABE)8e in correcting nonsense mutation (c.5047 C>T) in fibroblasts, which causes recessive dystrophic epidermolysis bullosa (RDEB). However, safety profile and cell-type-specific efficiencies of gene editing techniques have not been characterized. The aim of our research is to establish corrections of c.5047 C>T pathogenic variant in fibroblasts and compare off-target effects and efficiency of CRISPR-Cas9, BE and PE systems. Methods Primary fibroblasts harbouring heterozygous C>T pathogenic variant on exon 54 will be transfected with CRISPR-Cas9, ABE8e and PE using electroporation or lipofectamine. Both Sanger sequencing and next-generation sequencing (NGS) will be conducted to confirm pathogenic variant corrections. Galaxy platform which incorporates different NGS analysis tools will be used to analyse the NGS data. Subsequently, whole-exome sequencing and targeted amplicon sequencing will be used for off-target analysis. Results On-target efficiency for ABE8e to correct c.5047 C>T pathogenic variant in patient primary fibroblasts was determined first by Sanger sequencing showing 100% editing in contrast to 84% determined by NGS. Moving forward, we plan to correct the same pathogenic variant with PEs. Initially experiments are under way with PEmax mRNA and engineered PE guide RNAs (epegRNA) to compare different PEs. Furthermore, we will investigate the off-target effects of CRISPR-Cas9, ABE8e and PEs to edit RDEB primary fibroblasts using NGS Methods and conduct functional studies. Conclusions Here, we showed that the c.5047 C>T pathogenic variant in RDEB fibroblasts could be successfully edited using ABE8e. We demonstrated that NGS is required to precisely determine the editing efficacy. Taken together, we have established a pipeline for base editing in RDEB and we aim to achieve the same for prime editing in the future.

Paracrinal regulation of neutrophil functions by coronaviral infection in iPSC-derived alveolar type II epithelial cells

Coronaviruses (CoVs) are enveloped single-stranded RNA viruses that predominantly attack the human respiratory system. In recent decades, several deadly human CoVs, including SARS-CoV, SARS-CoV-2, and MERS-CoV, have brought great impact on public health and economics. However, their high infectivity and the demand for high biosafety level facilities restrict the pathogenesis research of CoV infection. Exacerbated inflammatory cell infiltration is associated with poor prognosis in CoV-associated diseases. In this study, we used human CoV 229E (HCoV-229E), a CoV associated with relatively fewer biohazards, to investigate the pathogenesis of CoV infection and the regulation of neutrophil functions by CoV-infected lung cells. Induced pluripotent stem cell (iPSC)-derived alveolar epithelial type II cells (iAECIIs) exhibiting specific biomarkers and phenotypes were employed as an experimental model for CoV infection. After infection, the detection of dsRNA, S, and N proteins validated the infection of iAECIIs with HCoV-229E. The culture medium conditioned by the infected iAECIIs promoted the migration of neutrophils as well as their adhesion to the infected iAECIIs. Cytokine array revealed the elevated secretion of cytokines associated with chemotaxis and adhesion into the conditioned media from the infected iAECIIs. The importance of IL-8 secretion and ICAM-1 expression for neutrophil migration and adhesion, respectively, was demonstrated by using neutralizing antibodies. Moreover, next-generation sequencing analysis of the transcriptome revealed the upregulation of genes associated with cytokine signaling. To summarize, we established an in vitro model of CoV infection that can be applied for the study of the immune system perturbations during severe coronaviral disease.

Chromosomal and genomic analysis suggests single origin and high molecular differentiation of the B chromosome of Abracris flavolineata

Supernumerary chromosomes (B chromosomes) have been an intriguing subject of study. Our understanding of the molecular differentiation of B chromosomes from an interpopulation perspective remains limited, with most analyses involving chromosome banding and mapping of a few sequences. To gain insights into the molecular composition, origin, and evolution of B chromosomes, we conducted cytogenetic and next-generation sequencing analysis of the repeatome in the grasshopper Abracris flavolineata across various populations. Our results unveiled the presence of B chromosomes in two newly investigated populations and described new satellite DNA sequences. While we observed some degree of genetic connection among A. flavolineata populations, our comparative analysis of genomes with and without B chromosomes provided evidence of two new B chromosome variants. These variants exhibited distinct compositions of various repeat classes, including transposable elements and satellite DNAs. Based on shared repeats, their chromosomal location, and the C-positive heterochromatin content on the B chromosome, these variants likely share a common origin but have undergone distinct molecular differentiation processes, resulting in varying degrees of heterochromatinization. Our data serves as a detailed example of the dynamic and differentiated nature of B chromosome molecular content at the interpopulation level, even when they share a common origin.

BRAF Mutations in Patients with Myeloid Neoplasms: A Cancer Center Multigene Next-Generation Sequencing Analysis Experience.

BRAF mutations are rare in myeloid neoplasms and are reported to be associated with poor treatment outcomes. The purpose of our study is to characterize BRAF mutations in myeloid neoplasms using a next-generation sequencing (NGS) panel based on the experiences of a single cancer center. We conducted a retrospective review of patients with myeloid neoplasms who underwent the HopeSeq studies between January 2018 and September 2023. A total of 14 patients with myeloid neoplasms carrying BRAF mutations were included in our cohort. The clinical, pathological, and molecular features of these patients were investigated. Our study indicates that BRAF mutations are rare in myeloid neoplasms, constituting only 0.53% (14/2632) of all myeloid neoplasm cases, with the most common BRAF mutation being BRAF V600E (4/14; 28.6%). Interestingly, we observed that six out of seven patients with acute myeloid leukemia (AML) exhibited AML with monocytic differentiation, and all the patients with AML exhibited an extremely poor prognosis compared to those without BRAF mutations. TET2 (5/14; 35.7%), ASXL1 (4/14; 28.6%), and JAK2 (4/14; 28.6%) were the three most frequently co-mutated genes in these patients. Moreover, we noted concurrent KMT2A gene rearrangement with BRAF mutations in three patients with AML (3/7; 42.9%). Our study suggests that although BRAF mutations are rare in myeloid neoplasms, they play a crucial role in the pathogenesis of specific AML subtypes. Furthermore, RAS pathway alterations, including BRAF mutations, are associated with KMT2A gene rearrangement in AML. However, these findings warrant further validation in larger studies.

The stress-responsive cytotoxic effect of diesel exhaust particles on lymphatic endothelial cells

Diesel exhaust particles (DEPs) are very small (typically < 0.2 μm) fragments that have become major air pollutants. DEPs are comprised of a carbonaceous core surrounded by organic compounds such as polycyclic aromatic hydrocarbons (PAHs) and nitro-PAHs. Inhaled DEPs reach the deepest sites in the respiratory system where they could induce respiratory/cardiovascular dysfunction. Additionally, a previous study has revealed that a portion of inhaled DEPs often activate immune cells and subsequently induce somatic inflammation. Moreover, DEPs are known to localize in lymph nodes. Therefore, in this study we explored the effect of DEPs on the lymphatic endothelial cells (LECs) that are a constituent of the walls of lymph nodes. DEP exposure induced cell death in a reactive oxygen species (ROS)-dependent manner. Following exposure to DEPs, next-generation sequence (NGS) analysis identified an upregulation of the integrated stress response (ISR) pathway and cell death cascades. Both the soluble and insoluble components of DEPs generated intracellular ROS. Three-dimensional Raman imaging revealed that DEPs are taken up by LECs, which suggests internalized DEP cores produce ROS, as well as soluble DEP components. However, significant cell death pathways such as apoptosis, necroptosis, ferroptosis, pyroptosis, and parthanatos seem unlikely to be involved in DEP-induced cell death in LECs. This study clarifies how DEPs invading the body might affect the lymphatic system through the induction of cell death in LECs.

Abstract PO2-08-05: ERBB2 mutated breast cancer detected by next generation sequencing: integrated analysis based on real-world data and The Cancer Genome Atlas

Abstract Background ERBB2 mutation has been recognized as an actionable target for pan-HER tyrosine kinase inhibitors in the era of precision medicine for solid tumors. In our real-world cohort utilizing next-generation sequencing (NGS), we successfully identified cases of ERBB2-mutated breast cancer. Furthermore, we investigated the clinical and pathological characteristics of these cases by integrating our data with The Cancer Genome Atlas (TCGA). Methods Our real-world cohort with NGS included 423 breast cancer patients treated at Gangnam Severance Hospital in Seoul, Republic of Korea, between April 2017 and January 2023. Among these patients, 13 were confirmed to have ERBB2 mutation through NGS analysis. We also incorporated data from TCGA database, which included 818 cases with ERBB2 sequencing. We compared the clinical characteristics (age, sex, stage, and ER, PR, and HER2 status) between the two groups. Furthermore, we examined the frequency of two of the most common mutations, PIK3CA and TP53, in both groups. Additionally, we investigated the distribution of PAM50 subtypes in the TCGA cohort. Results This study analyzed a total of 1,244 cases with NGS data. Among these cases, 31 (2.5%) exhibited ERBB2 mutation, with 13 patients identified in our dataset and 18 in the TCGA data. Among the 31 cases with ERBB2 mutation, 21 (67.7%) had HER2-negative disease. No significant differences were observed in other clinical characteristics based on ERBB2 status. Within the group of 31 ERBB2-mutated cases, TP53 mutations were found in 12 cases (38.7%), while PIK3CA mutations were found in 12 cases (38.7%). In the TCGA dataset, 4 cases (22.2%) with ERBB2 mutation were classified as HER2-E subtype, and the rate of HER2-E subtype was significantly higher in the ERBB2-mutated group compared to the ERBB2-wild group (22.2% vs. 7.6%, p=0.024). Conversely, 14 cases (77.8%) with ERBB2 mutation exhibited a non-HER2 enriched subtype. Conclusions Consistent with previous knowledge, ERBB2 mutations can be detected in HER2-negative breast cancer, despite their rarity. Furthermore, a considerable proportion of ERBB2-mutated breast cancer may depend on various oncogenic signaling pathways other than the HER2 pathway. Further studies are warranted to optimize therapeutic strategies for ERBB2-mutated breast cancer. Citation Format: Junghyun Kim, Yoonwon Kook, Seung Ho Baek, Min Ji Kim, Sohyun Moon, Seung Eun Lee, Jee Hung Kim, Soong June Bae, Joon Jeong, Sung Gwe Ahn. ERBB2 mutated breast cancer detected by next generation sequencing: integrated analysis based on real-world data and The Cancer Genome Atlas [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO2-08-05.

Influences of Ipomoea batatas Anti-Cancer Peptide on Tomato Defense Genes.

This study investigates the impact of IbACP (Ipomoea batatas anti-cancer peptide) on defense-related gene expression in tomato leaves, focusing on its role in plant defense mechanisms. Previously, IbACP was isolated from sweet potato leaves, and it was identified as a peptide capable of inducing an alkalinization response in tomato suspension culture media. Additionally, IbACP was found to regulate the proliferation of human pancreatic adenocarcinoma cells. Elucidate IbACP's molecular influence on defense-related gene expression in tomato leaves using next-generation sequencing analysis. To assess the impact of IbACP on defense-related gene expression, transcriptome data were analyzed, encompassing various functional categories such as photosynthesis, metabolic processes, and plant defense. Semi-quantitative reverse-transcription polymerase chain reaction analysis was employed to verify transcription levels of defense-related genes in tomato leaves treated with IbACP for durations ranging from 0 h (control) to 24 h. IbACP induced jasmonic acid-related genes (LoxD and AOS) at 2 h, with a significant up-regulation of salicylic acid-dependent gene NPR1 at 24 h. This suggested a temporal antagonistic effect between jasmonic acid and salicylic acid during the early hours of IbACP treatment. Downstream ethylene-responsive regulator genes (ACO1, ETR4, and ERF1) were consistently down-regulated by IbACP at all times. Additionally, IbACP significantly up-regulated the gene expressions of suberization-associated anionic peroxidases (TMP1 and TAP2) at all time points, indicating enhanced suberization of the plant cell wall to prevent pathogen invasion. IbACP enhances the synthesis of defense hormones and up-regulates downstream defense genes, improving the plant's resistance to biotic stresses.

Abstract PS11-03: Comparison of next-generation sequencing (NGS) results from the cerebrospinal fluid, peripheral blood, and systemic metastatic tumor tissue of patients with metastatic breast cancer (MBC) and leptomeningeal disease (LMD)

Abstract Introduction. Leptomeningeal disease (LMD) is a devastating complication of advanced malignancies and occurs in 5-15% of patients (pts) with metastatic breast cancer (MBC). Molecular characterization of the primary tumor to identify actionable mutations is standard of care, and targeted therapies have significantly improved survival. In contrast, metastatic tumors to the central nervous system (CNS) are not routinely assessed for the presence of actionable mutations, so the frequency and concordance of actionable mutations centrally vs. peripherally is not well characterized. In this single-center prospective study, we collected cerebrospinal fluid (CSF) in pts with known or suspected LMD and performed next generation sequencing (NGS) to evaluate the status of actionable mutations in the CSF and compared these results with NGS from matched peripheral blood and systemic metastatic tumor samples. Methods. We enrolled sixteen (16) pts with MBC and suspected or confirmed LMD at a single academic center from 2021-2023 in this non-therapeutic prospective study and collected CSF, peripheral blood, and archival systemic tumor samples from each pt. We used NGS to evaluate the status of actionable biomarkers in the CSF, blood, and tissue using the following NGS platforms: CNSide (Biocept) for CSF; Guardant 360, Foundation One CDx and CNSide for blood; and Caris and the UCSF CLIA-validated UCSF500 panel for the systemic metastatic tumor samples. We also evaluated CSF tumor cell enumeration and HER2 status in a subset of pts (CNSide). Results. Of the sixteen pts with MBC and suspected or confirmed LMD, 81% (13/16) were ultimately determined to be LMD-positive based on positive CSF cytology and/or convincing MRI imaging, including 6 pts with HR+/HER2- MBC, 6 pts with metastatic triple negative breast cancer (mTNBC), and 1 pt with HR+/HER2+ MBC; 3 pts were determined to be LMD-negative based on similar criteria. Of the 6 pts with HR+/HER2- MBC and LMD, NGS detected actionable mutations in the CSF in 67% (4/6), including two pts with PIK3CA E545E, one pt with both PIK3CA E542K and ERBB2 amplification, and 1 pt with ESR1 Y537S. Matched NGS analysis of the peripheral blood and/or systemic metastatic tissue showed concordance with PIK3CA E542K detected across tissue types in two pts, but discordance across tissue types in the other pts, including ESR1 detected systemically in two pts but not in the CSF. Of the 6 pts with mTNBC and LMD, CSF NGS detected actionable mutations in the CSF in 17% (1/6) of pts, including one pt with PIK3CA E542K; analysis of blood NGS for this pt is pending. 3 pts (50%) had mutations in TP53 in the CSF (1 pt with R273H and two pts with K132R), which were concordant across tissue types in all 3 pts. NGS did not detect any variants in the CSF of the one pt with HR+/HER2+ MBC and LMD. CSF tumor cell enumeration and central HER2 status will also be reported. Conclusion. This study demonstrates that NGS can detect actionable mutations in the CSF of pts with MBC and LMD. We observed concordance and heterogeneity in the status of actionable mutations between the CSF, peripheral blood, and systemic metastatic tumor tissue. Larger studies are needed to assess the clinical utility of these observations, particularly with the development of several novel targeted agents that are CNS-penetrant. Citation Format: Laura Huppert, Lindy Her, Christine Hodgdon, Susie Brain, Carol Simmons, Jo Chien, Melanie Majure, Hope Rugo, Mark Jesus Magbanua, Ron Balassanian, Michelle Melisko. Comparison of next-generation sequencing (NGS) results from the cerebrospinal fluid, peripheral blood, and systemic metastatic tumor tissue of patients with metastatic breast cancer (MBC) and leptomeningeal disease (LMD) [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PS11-03.

First parechovirus reported case in Saudi Arabia in hospitalized immunocompromised adult patient

Human parechovirus, a member of the Picornaviridae family (PeVs), can lead to severe infections, including severe meningitis, meningoencephalitis, and sepsis-like syndrome. We report a case of human parechovirus-related encephalitis in a 52-year-old woman diagnosed with glioblastoma multiforme. She underwent surgical resection in June 2022. Unfortunately, her disease recurred, and she underwent a second resection in August 2022, followed by radiation therapy and Temozolomide therapy. She presented to the hospital with acute confusion followed by seizures, necessitating intubation for airway support. A cerebrospinal fluid (CSF) sample was obtained and processed using the Biofire FilmArray, which reported the detection of HSV-1. Despite being on Acyclovir, the patient did not show signs of improvement. Consequently, a second CSF sample was obtained and sent for next-generation sequencing (NGS), which returned a positive result for Parechovirus. In this presented case, the patient exhibited symptoms of an unknown infectious cause. The utilization of NGS and metagenomic analysis helped identify Parechovirus as the primary pathogen present, in addition to previously identified HSV. This comprehensive approach facilitated a thorough assessment of the underlying infection and guided targeted treatment. In conclusion, the application of NGS techniques and metagenomic analysis proved instrumental in identifying the root cause of the infection.

Abstract PO4-05-03: Cell-free tumor DNA analysis in advanced or metastatic breast cancer patients enrolled in the German registry study PRAEGNANT

Abstract Background Accurate molecular profiling is crucial for optimal treatment decisions in advanced or metastatic breast cancer. Liquid biopsies, such as circulating cell-free tumor DNA (cfDNA, cftDNA or ctDNA), provide a non-invasive approach to monitor molecular alterations in the tumor. The German PRAEGNANT registry study aims to investigate such molecular biomarkers for precision medicine and their practical integration into clinical practice. In this context, cftDNA testing was performed as part of a PRAEGNANT research subproject, with the objectives of understanding the physician's rationale for initiating cftDNA testing, identifying detected alterations, and assessing the clinical impact of the associated findings Methods Patients with advanced or metastatic breast cancer were prospectively enrolled in the PRAEGNANT registry study (NCT02338167). The decision to undergo cftDNA testing was based on the physician's discretion. Blood samples were collected using Streck cfDNA BCT tubes and sent to the GUARDANT Health central laboratory for cftDNA extraction and subsequent next-generation sequencing analysis, employing the FDA-approved GUARDANT360 CDx test, a 74 gene panel including all guideline-recommended biomarkers, including single nucleotide variants (SNVs), indels, fusions and amplifications. Recruitment of patients occurred at four PRAEGNANT study centers between April 2022 and July 2023. The GUARDANT360 CDx report, along with a questionnaire regarding the testing intent and clinical impact of the results, was provided to the treating physician. Results 46 patients were eligible for analysis as per clinical characteristics. CftDNA analysis was successfully performed for all 46 cases. 38 (83%) harbored at least one somatic alteration in the analyzed genes. Fifteen patients (33%) harbored alterations in TP53, thirteen (28%) in PIK3CA. Five patients (11%) harbored mutations in ESR1. Somatic mutations in BRCA1 or BRCA2 were detected for five (11%) and six (13%) patients, respectively. One BRCA2 mutation was categorized as synonymous and one BRCA2 and three BRCA1 alterations as variants of uncertain significance. Eight patients (17%) harbored mutations in ATM. Most patients included were HR+HER2- (N=27, 58%). In this subgroup, eight (30%) patients had a PIK3CA alteration, five (19%) with an indication for treatment with alpelisib, eight (30%) a mutation in ATM, BRCA1 or BRCA2 and four (15%) in ESR1. Questionnaires regarding intention of testing and clinical impact were completed by 43 treating physicians (93%). Among them, 33% of patients had current indications for PARP inhibitor treatment, and 61% would consider it at the next change of therapy if a BRCA1 or BRCA2 mutation was detected. Additionally, 21% of patients had a current indication for alpelisib treatment if a relevant PIK3CA mutation was found, and 58% would consider it in the next line of treatment. Overall, cfDNA testing influenced the current or future treatment decisions in 35% of patients. Discussion The high prevalence of somatic alterations in TP53, PIK3CA, ESR1, and BRCA1/2 genes, identified through cftDNA testing with GUARDANT360 CDx, highlights their potential as biomarkers for targeted therapies in advanced/metastatic breast cancer. Detection of specific mutations influenced treatment decisions, such as eligibility for alpelisib or PARP inhibitors and might further facilitate treatment with elacestrant in future treatment lines after its approval in Germany. These findings demonstrate the clinical impact of cftDNA testing in guiding personalized treatment selection. Additionally, the identification of such somatic alterations within a registry study like PRAEGNANT presents a unique opportunity to consider enrolling these patients into biomarker-guided clinical trials. Citation Format: Hanna Hübner, Pauline Wimberger, Elena Laakmann, Eugen Ruckhäberle, Matthias Ruebner, Theresa Link, Erik Belleville, Iris Faull, Marcus Hausch, Diethelm Wallwiener, Andreas Schneeweiss, Hans Tesch, Sara Brucker, Peter A. Fasching, Volkmar Müller, Tanja Fehm. Cell-free tumor DNA analysis in advanced or metastatic breast cancer patients enrolled in the German registry study PRAEGNANT [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO4-05-03.

Identification of Long Noncoding RNAs Expression Profiles Between Gallstone and Gallbladder Cancer Using Next-Generation Sequencing Analysis

Identification of Long Noncoding RNAs Expression Profiles Between Gallstone and Gallbladder Cancer Using Next-Generation Sequencing Analysis

MITO END-3: efficacy of avelumab immunotherapy according to molecular profiling in first-line endometrial cancer therapy

Immunotherapy combined with chemotherapy significantly improves progression-free survival compared to first-line chemotherapy alone in advanced endometrial cancer, with a much larger effect size in microsatellite-instability high (MSI-H) cases. New biomarkers might help to select patients that may have benefit among those with a microsatellite-stable (MSS) tumor. In a pre-planned translational analysis of the MITO END-3 trial, we assessed the significance of genomic abnormalities in patients randomized to standard carboplatin/paclitaxel without or with avelumab. Out of 125 randomized patients, 109 had samples eligible for next-generation sequencing (NGS) analysis, and 102 had MSI tested. According to The Cancer Genome Atlas (TCGA), there were 29 cases MSI-H, 26 MSS TP53 wild-type (wt), 47 MSS TP53 mutated (mut), and one case with POLE mutation. Four mutated genes were present in more than 30% of cases: TP53, PIK3CA, ARID1A, and PTEN. Eleven patients (10%) had a BRCA1/2 mutation (five in MSI-H and six in MSS). High TMB (≥10 Muts/Mb) was observed in all MSI-H patients, in four out of 47 MSS/TP53 mut, and no case in the MSS/TP53 wt category. The effect of avelumab on progression-free survival significantly varied according to TCGA categories, being favorable in MSI-H and worst in MSS/TP53 mut (P interaction=0.003); a similar non-significant trend was seen in survival analysis. ARID1A and PTEN also showed a statistically significant interaction with treatment effect, which was better in the presence of the mutation (ARID1A P interaction=0.01; PTEN P interaction=0.002). The MITO END-3 trial results suggest that TP53 mutation is associated with a poor effect of avelumab, while mutations of PTEN and ARID1A are related to a positive effect of the drug in patients with advanced endometrial cancer.

Investigation of the marine bacterial community along the coastline of the Gulf of Thailand

The Gulf of Thailand provides many services to the Thai population, and human activities may influence the diversity of microorganisms in the seawater. Information of the microorganisms’ profile which inhabit the coastline can be used to monitor the water quality. This study aimed to investigate the bacterial community in the waters along the coastline provinces, including Rayong, Chonburi, Prachuap Kiri Khan, and Nakhon Sri Thammarat. Seawater samples were collected at each site, and the conductivity, pH, salinity, temperature, and turbidity were measured. The samples were subjected to whole DNA extraction, 16S rRNA amplification, next-generation sequencing, and statistical analysis to identify the bacterial diversity and analyse the effects of water parameters on the bacterial community. The dominant bacterial phyla found were Proteobacteria, Bacteroidota, and Cyanobacteria. Spearman rank correlation analysis revealed a high correlation of Pseudoalteromonas, the NS5 marine group, Lachnospiraceae, Marinobacterium, Mariviven, and Vibrio with the seawater parameters. The predatory bacteria Peredibacter and Halobacteriovorax were reported among these bacterial communities for the first time in the Gulf of Thailand. Interestingly, Akkermansia, a novel candidate for next-generation probiotics to improve human health, was also found in the sample from Nakhon Sri Thammarat Province. Although the rich-ness and diversity of the bacterial communities differed among sampling sites, it is a possible source of many valuable bacteria for future use.

Combined hurdle effects of pulsed electric field and ultraviolet-C irradiation on microbial load reduction and composition of hemeproteins from Asian seabass gills

The combined effects of two non-thermal technologies, namely pulsed electric field (PEF) and ultraviolet-C (UV-C) irradiation, on microbial load reduction and composition of crude hemeprotein extract (CHPE) from Asian seabass gills were investigated. PEF at two intensities (15 kV and 17.5 kV) for different times (0, 1, 2.5, 5 and 10 min) was employed to treat CHPE solution. Subsequently freeze-dried PEF treated powders were exposed to UV-C for varying durations (0, 15 and 30 min). Microbial community in gill and selected CHPE powders was also determined using next generation sequencing (NGS). PEF treatment effectively reduced the initial microbial load (8.0 log10 CFU/g) when high PEF intensity (17.5 kV) for longer treatment time (5 min) was employed, leading to microbial load reduction to 5.5 log10 CFU/g. However, prolonged PEF treatment adversely affected CHPE properties, as witnessed by the decreased heme iron content and alterations in protein patterns. Subsequent UV-C irradiation of freeze-dried PEF-treated CHPE powder for 15 min demonstrated a synergistic effect on further reduction of microbial counts to 3.0 log10 CFU/g. NGS analysis showed the dominance of Flavobacteriaceae families in the microbial community composition in gill, untreated CHPE powder and PEF-UV-C treated CHPE powder. Nevertheless, no Vibrio parahaemolyticus and other Vibrio spp. were detected in all samples. The study revealed the potential of combined non-thermal hurdle technologies under appropriate conditions in ensuring microbiological safety and maintaining quality of CHPE. CHPE powder could therefore be used for fortification in food products as an iron supplement to alleviate iron-deficient anaemia.

Osteoblastoma of the thumb with a novel PRSS44::ALK fusion and literature review of osteoblastoma of hands and feet bones.

Osteoblastomas (OBs) are benign neoplasms constituting approximately 1% of primary bone tumors with a predilection for the spine and sacrum. We describe an OB of the proximal phalanx of the left thumb in a 38-year-old female. MRI of left hand demonstrated a 29-mm mildly expansile enhancing lesion involving the entire proximal phalanx of the first digit. Histology displayed a bone-forming tumor consisting of trabeculae of remodeled woven bone framed by plump osteoblasts in a vascularized background. Next-generation sequencing analysis identified a PRSS44::ALK fusion gene.

Antimicrobial susceptibility and resistome of Actinobacillus pleuropneumoniae in Taiwan: a next-generation sequencing analysis

Actinobacillus pleuropneumoniae infection causes a high mortality rate in porcine animals. Antimicrobial resistance poses global threats to public health. The current study aimed to determine the antimicrobial susceptibilities and probe the resistome of A. pleuropneumoniae in Taiwan. Herein, 133 isolates were retrospectively collected; upon initial screening, 38 samples were subjected to next-generation sequencing (NGS). Over the period 2017–2022, the lowest frequencies of resistant isolates were found for ceftiofur, cephalexin, cephalothin, and enrofloxacin, while the highest frequencies of resistant isolates were found for oxytetracycline, streptomycin, doxycycline, ampicillin, amoxicillin, kanamycin, and florfenicol. Furthermore, most isolates (71.4%) showed multiple drug resistance. NGS-based resistome analysis revealed aminoglycoside- and tetracycline-related genes at the highest prevalence, followed by genes related to beta-lactam, sulfamethoxazole, florphenicol, and macrolide. A plasmid replicon (repUS47) and insertion sequences (IS10R and ISVAp11) were identified in resistant isolates. Notably, the multiple resistance roles of the insertion sequence IS10R were widely proposed in human medicine; however, this is the first time IS10R has been reported in veterinary medicine. Concordance analysis revealed a high consistency of phenotypic and genotypic susceptibility to florphenicol, tilmicosin, doxycycline, and oxytetracycline. The current study reports the antimicrobial characterization of A. pleuropneumoniae for the first time in Taiwan using NGS.

Exploring genetic variants in congenital monosaccharide-disaccharide metabolism: Carrier ratiosandphenotypic insights.

Adverse food reactions, often underestimated, encompass congenital monosaccharide-disaccharide metabolism disorders, yielding diverse outcomes such as abdominal pain, diarrhea, bleeding disorders, and even death. This study retrospectively scrutinized genetic variants linked to these disorders in a cohort subjected to whole-exome sequence analysis (WES), determining carrier frequencies and genotype-phenotype correlations. Data from 484 patients, were retrospectively analyzed using a gene panel (ALDOB, FBP1, GALE, GALK1, GALM, GALT, LCT, SLC2A2, SLC5A1, SI) for congenital monosaccharide-disaccharide metabolism disorders. WES was performed on patients using the xGen Exome Research Panel v2 kit, utilizing Next Generation Sequence Analysis (NGS). The study encompassed pathogenic, likely pathogenic, and variant of uncertain significance (VUS) variants. Among 484 patients (244 female, 240 male), 17.35% carried 99 variants (67 distinct) in the analyzed genes. Pathogenic/likely pathogenic allele frequency stood at 0.013, while VUS allele frequency was 0.088. Notably, 44% (37/84) of patients harboring mutations manifested at least one relevant phenotype. Carriage frequencies ranged from 1:25 (SI gene) to 1:968 (GALE gene), with the estimated disease frequency spanning from 1:2500 to 1:3748000. Our study underscores clinical manifestations in heterozygous carriers of recessive genetic disorders, addressing gaps in carrier frequencies and phenotypic effects for congenital monosaccharide-disaccharide metabolism disorders. This knowledge can improve these conditions'diagnosis and management, potentially preventing adverse food reactions and their associated complications.