NGS-based Analysis Research Articles

Performance Characteristics of Next-Generation Sequencing–Based Engraftment Monitoring and Microchimerism Detection in Allogeneic Hematopoietic Cell Transplantation: A Practical Approach for Clinical Assay Validation

Chimerism analysis by next-generation sequencing (NGS) is an emerging method for engraftment monitoring after allogeneic hematopoietic cell transplantation. A high-sensitivity method is required for the detection of microchimerism (<1% chimerism), which may have clinical utility in early relapse detection, allograft monitoring in organ transplantation, and other allogeneic cellular therapies (such as microtransplantations). As more clinical laboratories adopt this method, a thorough assessment of performance is needed. This study evaluated one such NGS-based assay that uses both single-nucleotide polymorphisms and insertions/deletions as genetic markers. An assessment of accuracy, linearity, sensitivity, and reproducibility was performed. Analytical sensitivity was 0.2% donor for single donor and 0.5% donors for double donors. The assay showed a high degree of reproducibility over a full range of chimerism. Comparison to short-tandem-repeat (STR) PCR showed high concordance; yet <5% chimerism was consistently detected by NGS, but not by STR-PCR. Comparison to real-time quantitative PCR showedhigh concordance, but with lower correlation in the midrange (40% to 60% chimerism). Overall, the assay showed consistent performance with high sensitivity and accuracy compared with STR-PCR and real-time quantitative PCR across a full range of chimerism in the setting of single-donor and multidonor transplantations. In addition, criteria for quality metrics were established for sequencing performance and data analysis and considerations made for clinical laboratory validation of NGS-based chimerism assay andanalysis software.

P-142 The developmental time to reach the blastocyst stage in the human is an indicator of diploid mono-pronuclear embryos

Abstract Study question Is it possible to determine diploid mono-pronuclear (1PN) human embryos by the speed of development to blastocyst formation? Summary answer The developmental time to reach the blastocyst stage is a discrimination indicator for diploid 1PN embryos. What is known already We have previously reported that 80.7% of 1PN zygotes had a biparental chromosome using a Live-Cell imaging technique, and some of these developed to the blastocyst stage (Tokoro et al. ASRM 2013). Furthermore, we have reported that these blastocysts can result in a healthy live birth (Tsuji et al. ASRM 2020), and there was no effect on growth up to 5 years of age (Tsuji et al. ASRM 2023). On the other hand, it was also shown that a few of the 1PN embryos that developed into blastocysts were haploid embryos. Study design, size, duration This was a retrospective study that included 28 blastocysts derived from a 1PN zygote. The time period was 72 months (January 2017 to December 2022). These embryos are used for research following obtaining informed consent. Participants/materials, setting, methods The frequency rate of diploid embryos was compared from time of PN fading to blastocyst formation (Gardner’s classification: ≥ expansion grade 3). Two development time range groups were examined and designated: fast-developing embryos (65.7-78.7h) and slow-developing embryos (116.8-139.7h). Ploidy status was determined using whole blastocyst cells and NGS-based SNP analysis. Statistical significance was determined using Fisher’s exact test (level of P&amp;lt;0.05). Main results and the role of chance There was no significant difference in the average age (years) of the two development time groups (fast-developing embryos group; 35.5 +/- 5.5, slow-developing embryos group; 34.1 +/- 2.8). The frequency of diploid status in 1PN embryos with fast developing embryos was 100% (14/14), and in 1PN embryos designated as having slow development, the frequency of diploid embryos was only 14.3% (2/14), haploid embryos 71.4% (10/14), and other was 14.3% (2/14). As a result, the diploid embryo rate was significantly higher in fast-developing embryos than in slow-developing embryos (P&amp;lt;0.001). Limitations, reasons for caution This study was limited to extremely fast or slow-developing embryos to see trends. It is important to study the threshold time between diploidy and haploidy in 1PN embryos. Wider implications of the findings These results confirmed that fast developing 1PN embryos have a high diploid rate and can be used as transferable embryos in addition to 2PN embryos. Conversely, slow developing 1PN embryos should be considered to have a lower priority for transfer because of the high rate of haploid embryos. Trial registration number not applicable

P-528 Establishing sequencing coverage depth for preimplantation genetic testing for monogenic disorders (PGT-M) using a specific targeted next generation sequencing (tNGS)

Abstract Study question What is the sufficient sequencing coverage read depth (RD) for direct variant analysis for PGT-M using a specific tNGS assay? Summary answer Downsampling modelling experiments identified a minimum sequencing depth of 50X that is recommended for PGT-M with this specific tNGS assay. What is known already Properly developed and validated multiplex PCR amplification and NGS-based analysis reliably produces PGT-M diagnoses with low allele and amplification failure at RD of hundreds of reads when analyzing a 3-10 cell trophectoderm (TE) biopsy. However, the RD can be affected by amplification efficiency in a specific genomic region and therefore affect the reproducibility of variant detection: the higher the number of aligned reads, the higher the confidence of base calling at a particular position. Downsampling is a powerful bioinformatics tool for modelling comparisons between samples or between replicates, identifying analytical sequencing depth limits for accurate clinical diagnosis. Study design, size, duration This is a retrospective comparative diagnostic test accuracy (DTA) and verification study. Twenty-three embryos from 8 patients that had undergone PGT-M with clinically established Taqman qPCR genotyping were used as positive controls. The primary outcome was to establish the lowest RD point of down sampling where allelic frequency is reliably detected at heterozygosity between 20-80% in known positive controls. A further 30 PGT-M clinical cases were used to verify analytical performances under clinical conditions. Participants/materials, setting, methods Embryo biopsies were pre-amplified with a multiplex primer pool for PGT-M and PGT-A, using a two-step PCR strategy. Sequencing was performed on Illumina NextSeq550. Reads were aligned to a human reference genome (GRCh37/hg19) and processed with an in house bioinformatic pipeline. The RD and alternative allele frequencies (AAF) for PGT-M was calculated. The reads for the heterozygous variants were downsampled to 10X, 20X, 30X, 50X, 100X, 200X, 300X, 500X, and 1000X to establish the cutoff. Main results and the role of chance One or two TE biopsies (N = 44) of 23 embryos that were 100% concordant with previous PGT-M results were tested for 1 to 3 of the 12 variants (MLH1 c.199G&amp;gt;A, RB1 c.1589A&amp;gt;G, SLC16A2 c.604G&amp;gt;A, GNPTAB c.1123C&amp;gt;T, GNPTAB c.3314A&amp;gt;G, WFS1 c.1391C&amp;gt;G, USH2A c.10073G&amp;gt;A, USH2A del exons 5-11, F5 c.1601G&amp;gt;A, GAA c.693-1G&amp;gt;C, GAA c.875A&amp;gt;G, and HEXA c.1274_1277dupTATC). PGT-A analysis of multiple biopsies from the same embryo demonstrated consistent karyotypes (N = 44). The 19 heterozygous variants from 17 biopsies exhibited average RD of 763X (238X -1980X) with average AAF of 50% (44%- 56%). The reads for the heterozygous variants were downsampled to different levels (100,000 replicates). The distribution of the AAF were compared among the different depth levels. At a RD of 50X, the AAF displayed an interquartile range (IQR) of 8% (46%-54%) between the maximum and minimum values from 20% to 78%. Compared to 30X (IQR: 43%-57%, with minimum 13% and maximum 90%), and 100X (IQR: 47%-54%, with minimum 30% and maximum 71%), the RD at 50X generated reliable heterozygous calls with minimum depth requirement. 245 biopsies from 30 clinic cases were analyzed by tNGS-based PGT-M. 90 samples with heterozygous variant calls showed average RD of 979X (57X-5803X) with average AAF of 47% (29%-585). Limitations, reasons for caution The 50X cutoff is based on the heterozygous variants with theoretical 50% AFF and is specific for this PGT-A assay. This evaluation should be corroborated for complicated variants with homologous genes, which might require different thresholds. The aneuploidy at specific chromosome can cause the deviation of AFF from 50%. Wider implications of the findings This specific tNGS-based PGT-M can provide direct variant analysis at a 50X read depth with a high level of consistency compared to established methodologies while allowing for simultaneous aneuploidy screening using the same biopsy and procedural optimization. Trial registration number not applicable

Diagnostic potential of vitreoretinal lymphoma by detection of gene mutations with NGS in 25 Chinese patients

BackgroundVitreoretinal lymphoma (VRL) is a rare malignant lymphoproliferative tumor. Our study aimed to investigate the mutational profile of VRL distinguishing from uveitis using next-generation sequencing (NGS) analysis on small amounts of vitreous fluid. MethodsVitreous samples from twenty-six eyes of twenty VRL patients and six eyes of five uveitis patients were enrolled. All vitreous samples underwent cytology, immunocytochemistry for B-cell markers, cytokines analysis of IL-10 and IL-6, and flow cytometry. NGS was performed in vitreous specimens from the 25 patients using 82 DLBCL-targeted mutation panels. Vitreous fluids from 8 cases were performed paired NGS-based mutation analysis on both cell-free DNA (cfDNA) and genomic DNA. ResultsThe sensitivity and accuracy rates for vitreous cytology were 70 % and 76 %, and for cytokine analysis (IL-10/IL-6 > 1) were 65 % and 72 %, respectively. Overall, the common mutations in VRL were PIM1 (88.5 %), IGLL5 (88.5 %), KMT2C (73 %), MYD88 (77 %), CD79B (50 %) and TBL1XR1 (46.2 %). In addition, the genetic mutation in cfDNA was consistent with that in genomic DNA in eight VRL cases. ConclusionsThe mutation analysis of 82 DLBCL-targeted spectrum mutation panels by NGS on the vitreous samples is a sensitive and specific tool for distinguishing VRL from uveitis. Utilizing cfDNA for NGS analysis may serve as a liquid biopsy to aid in the diagnosis of VRL, particularly when using small-volume aspirate.

An unsupervised H&amp;E-based machine-learning approach for precise prediction of tumor microenvironment subtypes.

e13593 Background: Precision oncology increasingly relies on accurate classification of the tumor microenvironment (TME), which influences therapeutic responses and prognostication in cancer. While histological observation via H&amp;E staining remains the gold standard due to the detailed spatial context and morphological information provided, its lack of reproducibility limits its use in clinics. Next-generation sequencing (NGS) reliably provides valuable insights into the molecular landscape of tumors. Here we debut an unsupervised machine-learning approach that leverages both the rich detail in H&amp;E slides and the reproducibility of NGS to enhance TME classification. Methods: An ML-based image analysis algorithm was developed using a convolutional neural network (CNN) deep learning model. Using over 600 images from the TCGA COAD and READ datasets and from 139 colorectal cancer (CRC) samples in an internal cohort, we trained the CNN model to curate features from small patches of whole slide images (WSIs). Then, clusters were created from these H&amp;E WSIs without prior labeling. The clusters were validated by experienced pathologists to confirm cluster homogeneity and filter artifacts. We trained a classifier to categorize molecular features from the WSIs into TME subtypes. Performance was measured using the area under the receiver operating characteristic curve (AUC-ROC), based on the model's ability to correctly classify the Fibrotic (F) and Immune Desert (D) TME subtypes (1), as well as MSI status identified by whole exome sequencing. The F subtype corresponds with high stromal content, low immune infiltration, and the worst prognosis. The D subtype corresponds with a cold immune TME and an intermediate prognosis. Progression-free survival (PFS) data from TCGA were used to examine prognostic significance. Results: We obtained 0.86, 0.79, 0.72, and 0.71 AUC scores for predicting the ID and F TMEs, MSI status, and KRAS mutation status, respectively. PFS data analysis showed our H&amp;E-based approach to yield similar or better prognostic significance compared to NGS-based TME classification (ID vs others, p=0.05 (H&amp;E) and 0.2 (NGS); F vs others, p=0.07 (H&amp;E) and 0.09 (NGS)). Our findings showed a notable correlation with RNA- and DNA-based predictions of ID and F TMEs and MSI, with enhanced precision and more robust subtype identification while achieving high embedding consistency. Conclusions: By harnessing the inherently rich detail in histological images, our H&amp;E-based approach complements NGS-based TME analysis. Our findings advocate for the integration of advanced image analysis into the standard TME characterization workflow to refine prognostic and therapeutic strategies for CRC. We will expand this algorithm for use with other cancer types to realize its potential as a promising adjunct to NGS for precise TME classification. 1. Bagaev et al., 2021.

Abstract PO3-19-06: MRD Assay evaluates Recurrence and response via a tumor Informed Assessment: MARIA-Breast Observational Trial

Abstract Background: Detectable ctDNA in patients with solid tumors has been associated with disease prognosis pre-treatment, assessing response to therapy in the form of minimal residual disease (MRD), and monitoring for recurrence after curative intent treatment. Utilizing patient-specific genomic mutation profiling of an individual’s cancer from a tissue sample, in conjunction with the patient’s germline DNA, to create a personalized sequencing panel to analyze for a subset of these genetic mutations from ctDNA in blood is a strategy that has high sensitivity for detecting MRD. Studies have shown that pretreatment levels of ctDNA using this approach are a potential early indicator of disease recurrence after surgery, that ctDNA clearance may be an early predictor of favorable outcomes and has been shown to correlate with pathologic complete response (Forde et al. N Engl J Med. 2022, PMID:35403841), and that this approach has high sensitivity for detecting recurrence for patients in advance of the current standard of care (Abbosh et al. Cancer Res (2020) 80 (16_Supplement): CT023). Methods: This is a multi-site, prospective, observational trial in the United States of 200 patients with early stage breast cancer using a patient-specific tumor-informed MRD assay for ctDNA analysis. Participants are asked to provide study specimens prior to initial treatment intervention, after curative intent surgical resection, during adjuvant therapy (as applicable) and pre-recurrence follow-up. ctDNA will be analyzed with an NGS-based, tumor-informed MRD assay that identifies somatic mutations from DNA obtained from the patient’s tumor tissue, subtracts germline variants via NGS-based analysis of the patient’s germline DNA, and detects a selected set of between 18-50 tumor-specific ctDNA in their blood. All primary tumor specimens will undergo full exome sequencing using the Personalized Cancer Monitoring (PCM) assay. Impact of results of this CLIA-approved MRD assay on clinical decision making will be captured. The primary objective is to assess the ability of MRD to predict post-treatment recurrence. Further objectives are to correlate MRD status with pathologic complete response, determine the lead time to detection of recurrence compared to standard of care, and the association of MRD status with overall survival. Active enrollment started in March, 2022. Support: Invitae. Clinical trial information: NCT05219734. Citation Format: Edward Esplin, Kelli Swan, Lee Ifhar, Brandie Heald, Sarah Nielsen, Daniel Pineda-Alvarez, William O'Callaghan, Robert Daber, Darrel Ross, Carlos Vieira, Aadel Chaudhuri, W. Korn. MRD Assay evaluates Recurrence and response via a tumor Informed Assessment: MARIA-Breast Observational Trial [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 PO3-19-06.

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.

Abstract 7507: Capturing live single circulating tumor cells platform for comprehensive genomic profiling with paired ctDNA in breast cancer patients

Abstract Background: Single circulating tumor cells (sCTC) capture without cell fixation with no blood contaminants like leukocytes are highly challenging. sCTC genome transcriptomics anticipates new avenues in new discovery, targeted therapies, and inclusion of patients in clinical trials where the tissue is inaccessible. We report, an assay to selectively capture live sCTCs at singularity with NGS-based comprehensive mutational profiling in breast cancer (BC) patients. Further, paired ctDNA outcomes using blood with mutational heterogeneity scores of sCTCs adds comprehensive genomics for establishing an accelerated treatment outcomes and resistance signatures. Methods: Retrospectively, sCTC were isolated from 70 breast cancer (BC) patients using OncoRadar platform consisting affinity-based glass beads mediated by anti EpCAM antibody. CTCs were selectively captured and released without fixing and contamination of any leukocytes. Platform possess cleavable disulfide spacer at pH 8.0, between glass beads (diam. 2 mm, area 12.57 mm2) and a ligand. Sensitivity, specificity, positive predictive value (PPV), negative prediction value (NPV), and accuracy were evaluated using MCF-7 cells and CTCs. Assay to isolate CTC has been developed in 96 well plate, for downstream NGS-based comprehensive gene panel analysis using hybridization-based target enrichment. Interestingly, no mutations detected in blood samples (~20%) for CtDNA versus CTC DNA offered non-chip implicated mutations. Results:. Analytical validation using OncoRadar showed higher cell capture efficiency (99%) with spiked MCF-7 cells/ml of blood. Sensitivity and specificity values were 97% and 98% respect, while PPV was 99% and NPV was 7.5% with the accuracy parameter of 99%. Sensitivity, specificity, PPV, and NPV parameters when assessed for CTC isolation from 70 BC patients. NGS-based mutational analysis, sCTC showed the presence of PIK3CA, ESR1, and NF1 mutations, which were absent from paired ctDNA samples. Conclusion: Detection of ctDNA obscure mutations in paired sCTC samples suggests high sensitivity of sCTC-based genomic assays compared to current standard of practice. Heterogeneity scores amongst inter and intra CTCs with MATH scores offer new dimensions in genomics. Combination of ctDNA and sCTC DNA assay offers a comprehensive realistic genomic outcome for more significant therapy decisions, especially when guideline based options may exhausts. Citation Format: Gowhar Shafi, Atul Bharde, Ganesh Khutale, Saloni Andhari, Richa Deshpande, Bhagwat Jadhav, Sangeeta Prajapati, Moubeen Fauzul, Kanchan Hariramani, Madhura Basavalingegowda, Jayant Khandare. Capturing live single circulating tumor cells platform for comprehensive genomic profiling with paired ctDNA in breast cancer patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7507.

Abstract 5035: Use of Streck nucleic acid BCT with plasma nucleic acid next-generation sequencing workflows

Abstract Introduction: Liquid biopsies, which interrogate body fluids in search of analytes released by cancer cells, are becoming increasingly common in clinical oncology. Foremost among the technologies used are those that employ nucleic acids, such as cell-free DNA (cfDNA) and cell-free RNA (cfRNA). Currently, many NGS-based tests perform near the limit of assay detection, making the identification of critical mutations, fusions, or expression patterns like finding a needle in a haystack. Further hindering successful assay development is the variability in analyte availability and detectability that can occur prior to analysis due to blood collection, transport, and storage. Here, we demonstrate that Nucleic Acid BCT stabilizes draw-time levels of plasma nucleic acids for downstream use with NGS-based assays. Methods: Blood from self-proclaimed healthy donors was drawn into EDTA and Nucleic Acid BCT and stored at ambient temperature for up to 7 days before plasma isolation with a general double-spin protocol and storage at -80 °C. Plasma nucleic acids were isolated using the MAGicBead cfDNA Kit (Zymo, cfDNA) or the QIAamp Circulating Nucleic Acid Kit (QIAGEN, cfRNA). Library prep for cfDNA was carried out with added synthetic mutant spike-in oligonucleotides using the Illumina TruSight Tumor 15 kit (Illumina). cfRNA library prep was done with the NEBNext Ultra II RNA Library Prep Kit for Illumina with upfront rRNA depletion (New England Biolabs). Sequencing was performed using an Illumina NextSeq 500 system, and all data were saved in and analyzed with Illumina BaseSpace applications. Results: When blood samples were stored in EDTA tubes for up to 7 days, spiked mutant alleles (EGFRL858R, EGFRdel19, KRASG12D, and PIK3CAE545K) were no longer detected, likely due to a dilution of the spiked DNA concentration following the release of genomic DNA containing wildtype alleles during white blood cell degradation. In contrast, equivalent blood samples stored in Nucleic Acid BCTs displayed mutant allele frequencies reflective of draw-time levels. Interrogation of plasma RNA expression patterns in samples stored in Nucleic Acid BCT versus EDTA revealed similar results, where differentially expressed plasma transcripts in blood samples stored in EDTA were found at high levels (&amp;gt;1,000 transcripts) versus donor-matched blood samples stored in Nucleic Acid BCT (&amp;lt;50 transcripts). Conclusions: Our data demonstrate that Nucleic Acid BCT maintains mutant allele frequencies and the plasma transcriptome profile of blood samples during ambient temperature storage. This provides precious sample integrity during whole blood storage and transport, offering laboratories and assay developers reduced preanalytical variability for NGS-based analysis of gene mutations, fusions, indels, and the plasma transcriptome. Nucleic Acid BCT is for Research Use Only. Not for use in diagnostic procedures. Citation Format: Nicholas M. George, Lisa Bartron. Use of Streck nucleic acid BCT with plasma nucleic acid next-generation sequencing workflows [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5035.

Evaluation of the Impact of Near-Infrared Multiwavelength Locked System Laser Therapy on Skin Microbiome in Atopic Dogs.

Photobiomodulation (PBM) is a newly adopted consensus term to replace the therapeutic application of low-level laser therapy. It has been suggested that PMB influences the microbiome which, in turn, has increasingly been shown to be linked with health and disease. Even though the use of PBM has also grown dramatically in veterinary medicine, there is still a lack of evidence supporting its effect in vivo. Our objective was to investigate the impact of a dual-wavelength near-infrared laser source (Multiwavelength Locked Laser System, MLS®) on the skin microbiome in atopic dogs. Twenty adult-client-owned atopic dogs were enrolled in the study. The dogs were treated with MLS® laser therapy on one half of the abdominal region, whereas the contralateral side was left untreated and served as a control. Skin microbiome samples were collected before and after MLS® treatments, and then subjected to NGS-based ITS and 16S rRNA analysis. The results showed that while microbiome composition and diversity were not significantly affected, PBM could play a role in modulating the abundance of specific bacterial species, in particular Staphylococcus, that represent a major skin pathogenic strain. To the best of the authors' knowledge, this is the first study to investigate the potential impact of MLS® laser therapy on the skin microbiome in atopic dogs.

Assessment of Bone Marrow Involvement in B-Cell non-Hodgkin Lymphoma Using Immunoglobulin Gene Rearrangement Analysis with Next-Generation Sequencing.

Assessment of bone marrow involvement (BMI) in non-Hodgkin lymphoma (NHL) is crucial for determining patient prognosis and treatment strategy. We assessed the prognostic value of next-generation sequencing (NGS)-based immunoglobulin (Ig) gene clonality analysis as an ancillary test for BMI evaluation in NHL. A retrospective cohort of 124 patients newly diagnosed with B-cell NHL between 2019 and 2022 was included. NGS-based Ig clonality analysis was conducted using LymphoTrak IGH FR1 Assay and IGK Assay (Invivoscribe Technologies, San Diego, CA, USA) on BM aspirate samples, and the results were compared with those of histopathological BMI (hBMI). Among the 124 patients, hBMI was detected in 16.9% (n = 21). The overall agreement of BMI between Ig clonality analyses and histopathological analysis for IGH, IGK, and either IGH or IGK was 86.3%, 92.7%, and 90.3%. The highest positive percent agreement was observed with clonal rearrangements of either IGH or IGK gene (90.5%), while the highest negative percent agreement was observed with clonal rearrangement of IGK gene (96.1%). For the prediction of hBMI, positive prediction value ranged between 59.1% and 80.0% and the negative prediction value ranged between 91.3% and 97.9%. NGS-based clonality analysis is an analytic platform with a substantial overall agreement with histopathological analysis. Assessment of both IGH and IGK genes for the clonal rearrangement analysis could be considered for the optimal diagnostic performance of BMI detection in B-cell NHL.

Next-Generation Sequencing-Based Copy Number Variation Analysis in Chinese Patients with Primary Ciliary Dyskinesia Revealed Novel DNAH5 Copy Number Variations.

The online version contains supplementary material available at 10.1007/s43657-023-00130-0.

OcBSA: An NGS-based bulk segregant analysis tool for outcross populations

Constructing inbred lines for self-incompatible species and species with long generation times is challenging, making the use of F1 outcross/segregating populations the main strategy for genetic studies of such species. However, there is a lack of dedicated algorithms/tools for rapid quantitative trait locus (QTL) mapping using the F1 populations. To this end, we have designed and developed an algorithm/tool called OcBSA specifically for QTL mapping of F1 populations. OcBSA transforms the four-haplotype inheritance problem from the two heterozygous diploid parents of the F1 population into the two-haplotype inheritance problem common in current genetic studies by removing the two haplotypes from the heterozygous parent that do not contribute to phenotype segregation in the F1 population. Testing of OcBSA on 1800 simulated F1 populations demonstrated its advantages over other currently available tools in terms of sensitivity and accuracy. In addition, the broad applicability of OcBSA was validated by QTL mapping using seven reported F1 populations of apple, pear, peach, citrus, grape, tea, and rice. We also used OcBSA to map the QTL for flower color in a newly constructed F1 population of potato generated in this study. The OcBSA mapping result was verified by the insertion or deletion markers to be consistent with a previously reported locus harboring the ANTHOCYANIN 2 gene, which regulates potato flower color. Taken together, these results highlight the power and broad utility of OcBSA for QTL mapping using F1 populations and thus a great potential for functional gene mining in outcrossing species. For ease of use, we have developed both Windows and Linux versions of OcBSA, which are freely available at: https://gitee.com/Bioinformaticslab/OcBSA.

Expanding the screening of newborns for detecting inborn errors in metabolism using next generation sequencing following mass spectrometry/immunoassay

Inborn errors of metabolism are rare inherited disorders which leads to significant morbidity and mortality in patients. Very few studies have been conducted in India to assess prevalence of Inborn Errors of Metabolism (IEM) in newborns. We proposed testing by TMS/TR-FIA followed by NGS. This pilot study would be one of the first expanded NBS studies in India.The aim of this study was to determine the prevalence of IEM in newborns based on the samples received at Metropolis Global Reference Lab, India. Next-generation sequencing (NGS) was done as a confirmational analysis for patients tested presumptive positive on Newborn screening using Tandem Mass spectrometry (TMS) and Time-resolved fluoroimmunoassay (TR-FIA). Two years retrospective study was conducted based on incidences of IEM using TMS and TR-FIA. NGS testing was performed on presumptive positive newborns for cystic fibrosis (CF), galactosemia and urea cycle disorder/ organic academia (UCD /OA) who had undergone NBS by TMS and TR-FIA. Highestprevalence of 1.98% &amp; 1.58% was detected for G6PD and TSH respectively by TR-FIA. Prevalence of AA disorders (3.20%), OA (1.60%) and UCD (1.43%) was observed to be the highest amongst the diseases detected by TMS. Presumptive positive case of Argininemia and Cystic Fibrosis were found to be concordant with NGS. Out of three presumptive positive cases, one presumptive positive case of CF and two of galactose were found discordant. Our prevalence study showed similarities to the prevalence reports published by other Asian countries. Expanded NBS program can be improved by including NGS as a first follow-up test after detection of abnormal metabolites in DBS. This approach will help in reducing the encumbrance of false-positive as well as false-negative cases. Our study will be influential in conducting more prospective studies and routine implementation of NGS-based analysis in NBS in India.

Cell-Free DNA and Next-Generation Sequencing for Prenatal Diagnosis.

Deep sequencing by NGS of targeted amplicons can identify rare genetic variants in a pool of DNA where the vast majority of genomic DNA does not contain the variant. This approach can be used to detect a previously described paternally inherited, fetal variant in cell-free DNA (cfDNA) in maternal plasma. This is useful in cases where risk for the fetus is contingent upon inheritance of a paternal variant that the woman does not have. Both pathogenic and non-pathogenic variants that the woman does not have can be detected. In cases of compound heterozygosity, presence of the paternal pathogenic variant also requires detection of the maternal variant for risk assessment, which requires a chorion villus biopsy.We have used this approach to focus on detection of fetal blood groups in cases of presence of maternal alloantibodies against blood group antigens in pregnancy, to predict whether the fetus has inherited a blood group antigen that is targeted by the alloantibodies. In cases of maternal alloantibodies against blood group antigens, the fetus is at risk of hemolytic disease of the fetus and newborn (HDFN). With a known specificity of the maternal antibodies and if the fetal blood group can be determined in the pregnancy, then it can be ascertained if the fetus is at risk of HDFN and rational pregnancy care can be instituted. A noninvasive procedure avoids risks for the fetus. We have reported a procedure based on NGS analysis of PCR amplified cfDNA from maternal plasma. Some fetuses may die as early as week 18. We use this approach to predict fetal K, k, RhC, Rhc, RhE, and ABO blood groups in cases with a risk of HDFN due to the corresponding maternally produced antibodies.The NGS-based analysis can predict the presence or absence of incompatible antigens on the fetal RBCs.In this chapter, a noninvasive method for predicting some fetal blood groups early in pregnancy is described. There is a clinical need for such assays, and they may be a useful tool for management of pregnancies complicated by these alloantibodies within the field of precision medicine.

Comprehensive Molecular Genetic Analysis in Glioma Patients by Next Generation Sequencing.

Glioma is caused by multiple genomic alterations. The evolving classification of gliomas emphasizes the significance of molecular testing. Next generation sequencing (NGS) offers the assessment of parallel combinations of multiple genetic alterations and identifying actionable mutations that guide treatment. This study comprehensively analyzed glioma patients using multi-gene NGS panels, providing powerful insights to inform diagnostic classification and targeted therapies. We conducted a targeted panel-based NGS analysis on formalin-fixed and paraffin-embedded nucleic acids extracted from a total of 147 glioma patients. These samples underwent amplicon capture-based library preparation and sequenced using the Oncomine Comprehensive Assay platform. The resulting sequencing data were then analyzed using the bioinformatics tools. A total of 301 mutations, were found in 132 out of 147 tumors (89.8%). These mutations were in 68 different genes. In 62 tumor samples (42.2%), copy number variations (CNVs) with gene amplifications occurred in 25 genes. Moreover, 25 tumor samples (17.0%) showed gene fusions in 6 genes and intragenic deletion in a gene. Our analysis identified actionable targets in several genes, including 11 with mutations, 8 with CNVs, and 3 with gene fusions and intragenic deletion. These findings could impact FDA-approved therapies, NCCN guideline-based treatments, and clinical trials. We analyzed precisely diagnosing the classification of gliomas, detailing the frequency and co-occurrence of genetic alterations and identifying genetic alterations with potential therapeutic targets by NGS-based molecular analysis. The high-throughput NGS analysis is an efficient and powerful tool to comprehensively support molecular testing in neurooncology.

Practical and Flexible Genome Profiling Study Using the Halo-Shape Annealing and Defer-Ligation Enrichment (HANDLE) System: HM-Screen-JAPAN02

Background Acute myeloid leukemia (AML) is known to have a relatively small number of genomic mutations compared to solid tumors. With the development and clinical application of drugs targeting genetic mutations involved in the pathogenesis of AML, the Japanese Society of Hematology has defined “fast-track mutations” as those that should be reported promptly due to clinical necessity. A multicenter genomic profiling study (HM-SCREEN-JAPAN01: HM01 study) was conducted to comprehensively sequence 406 DNA mutations and 265 RNA aberrations in 177 AML patients, suggesting its usefulness in clinical practice, but the turnaround time (TAT) until results are reported remains an issue (Hosono, et al. Cancer Sci, 2023). Aims We have launched the HM-SCREEN-JAPAN02 (HM02) study, a practice-oriented genome profiling study that improves on the previous HM01 study and uses a newly developed targeted sequencing method. The HM02 study adopted the novel Amoy Myeloid Panel® method, which uses the HANDLE (Halo-shape ANnealing and Defer-Ligation Enrichment) system to significantly shorten TAT and facilitate specimen submission, and was designed to be completed in 7 days from sequencing to reporting. The HM02 study allows mutation analysis at any treatment timing, such as at initial presentation, during treatment, or at relapse, as well as the capability to track clonal changes through repeat analysis; the HM02 study was designed to determine whether such flexible analysis is useful in determining treatment strategy. Results The study was conducted at 23 centers in Japan, enrolling a total of 154 cases from 2020 to 2022, with 191 samples analyzed to assess the usefulness of the study. Repetitive mutation analysis was performed in 21 cases, and clonal changes could be tracked. Among the mutations detected, we selected those that were determined to be pathogenic or likely pathogenic by ClinVar. Of the 154 cases analyzed, none of the pathogenic mutations were detected in 9 cases, and 6 were of the normal karyotype. The most frequently observed mutations were TET2 (51%), FLT3 (30%), TP53 (18%), and NPM1 (17%) (Figure 1). In multivariate analysis adjusted for age, gender, and stem cell transplantation status, TP53 and NRAS mutations were associated with higher risk for death (HR=6.82 and 14.98, respectively). Of the 19 cases in which pathological mutations were repeatedly investigated, 8 were useful in determining minimal/measurable residual disease (MRD) (2 cases of MRD loss and 6 cases of MRD retained), 6 cases showed the development of new clones and 5 cases showed clonal evolution, indicating that repeated mutation analysis can be used to evaluate detailed disease status. In terms of clinical utility, HM02 mutation analysis was useful in 62% of cases (98 of 158), particularly for risk assessment and determining the indication for stem cell transplantation (Figure 2). Conclusion Flexible analysis using the novel Amoy Myeloid Panel® method suggests that it can be used to assess the risk of relapse, evaluate the need for transplantation, and select optimal therapeutic agents. NGS-based mutation analysis provides useful information at any point in AML treatment and is expected to improve outcomes.

Reverse Engineering of Antigenic Peptides in LGL to Decipher Disease Triggers

One of the hypotheses as to the pathogenesis of T-cell large granular lymphocytic leukemia (LGL) posits its origin from the initially polyclonal stimulation by unknown antigens, e.g., viral, autoimmune or tumor antigens. LGL evolves then via oligoclonal and ultimately monoclonal cytotoxic T-cell expansion. This process may be accompanied by acquisition of STAT3 (or rarely STAT5) mutations in 40% of cases. Identification of driver antigens is a key research question in LGL as it could both clarify its triggers as well as the mechanisms of cytopenias. We investigated a cohort of patients with this disease (n=195) by combining HLA allotypes and NGS-based TCR analysis to identify marker TCR Vβ clonotypes with the goal to reverse engineer or deduce peptides and therefore antigenic targets. For this purpose we used in silico and machine-learning modelling to capture structural features predictive of CDR3 Vβ epitope interactions in specific HLA allotype context. To focus on specific HLA/TCR interactions we have analyzed HLA for the presence of common haplotypes. First, we generated HLA haplotype calls using whole-exome sequencing data. We have found that HLA-A*02 was the most common class I allele, but not overrepresented in LGL vs controls. We have also identified HLA-B*44 as a risk allele present in 36% LGL with STAT3 mutation which was further enriched when considering HLA-A*02/HLA-B*44 together (Fig.1A). After TCR NGS we selected a threshold of 5.0% (based on study of 900 controls) to define an expanded (co)dominant clonotypes: 145 expanded clones were identified, of which 143 were unique/private. To identify possible consensus sequence across the CDR3 profile, we clustered the CDR3 AA sequences based on global alignment scores (Needleman-Wunsch) using BLOSUM62 substitution matrix and identified 9 clusters. However, multiple-sequence alignment within each CDR3 cluster did not show any distinct consensus sequence patterns overall and within cases sharing HLA-A*02. Cross-referencing with CDR3 databases of known conditions yielded 87 CDR3β (co)dominant LGL clonotypes. About 50% of those LGL clonotypes were shared in 2 common disease: type I diabetes, Celiac disease We also mined the VDJdb[2] for known CDR3-pHLA associations. Filtering to include high quality annotations, matches and HLA-A*02 restriction; LGL-derived TCR were found to recognize multiple epitopes. Prominent examples include CMV and EBV derived peptidic antigens from pp65, BRLF1, and BMLF1 (NLVPMVATV, RPPIFIRRL) present in 44% LGL expanded clonotypes (Fig.1B). In order to reverse engineer the identity of antigenic peptides based on the CDR3β sequence and HLA type (high affinity peptides restricted to HLA A2, a machine-learning method was developed as predictive modelling tool that quantifies CDR3β/peptide binding. The previously developed LLM trained on AA sequences was used for that purpose[3] whereby LLM embeddings serve as input to a deep CNN-based model, which is trained on IEDB pos/neg datasets[4]. This method was augmented with control CDR3 sequences as negative data points. As a result, our model showed an AUC of 0.94 in cross-validation. As a proof of concept of the proposed model in epitope discovery, CMV pp65-derived peptides were fitted into specific LGL CDR3 and CMV specific CDR3 clonotypes were found to match e.g., pp65 epitope NLVPMVATV among several others overlapping with the above-described findings. In conclusion, our analysis shows that a significant number of expanded clonotypes may be derived from known epitopes e.g., in autoimmune disease and viral infection pointing towards molecular mimicry. However, the specificity of a large fraction of the LGL spectrum remains unknown. For the discovery of corresponding peptides we generated ML-based antigen simulation method allowing to identify the best fitting peptides into the corresponding HLA allele and CDRVβ clonotype. Fig. Global map of co-occurrence of HLA allotypes showing the delta frequency compared to healthy controls and Sankey plot of identified CDR3β - Epitope associations (a) Using the whole-exome sequencing based HLA genotype calls, we calculated the frequency of co-occurrence of different HLA loci and investigated the differences in regards to controls (b) Using the VDJdb, we conducted sequence similarity based search and extracted the CDR3-pHLA annotations.

Low-burden TP53 mutations represent frequent genetic events in CLL with an increased risk for treatment initiation.

TP53 aberrations predict chemoresistance and represent a contraindication for the use of standard chemoimmunotherapy in chronic lymphocytic leukaemia (CLL). Recent next-generation sequencing (NGS)-based studies have identified frequent low-burden TP53 mutations with variant allele frequencies below 10%, but the clinical impact of these low-burden TP53 mutations is still a matter of debate. In this study, we aimed to scrutinise the subclonal architecture and clinical impact of TP53 mutations using a sensitive, NGS-based mutation analysis in a 'real-world' cohort of 901 patients with CLL. In total, 225 TP53 mutations were identified in 17.5% (158/901) of the patients; 48% of these alterations represented high-burden mutations, while 52% were low-burden TP53 mutations. Low-burden mutations as sole alterations were identified in 39% (62/158) of all mutated cases with 82% (51/62) of these being represented by a single low-burden TP53 mutation. Patients harbouring low-burden TP53 mutations had significantly lower time to first treatment compared to patients with wild-type TP53. Our study has expanded the knowledge on the frequency, clonal architecture, and clinical impact of low-burden TP53 mutations. By demonstrating that patients with sole low-burden TP53 variants represent more than one-third of patients with TP53 mutations and have an increased risk for treatment initiation, our findings strengthen the need to redefine the threshold of TP53 variant reporting to below 10% in the routine diagnostic setting.

First-Line Venetoclax Combinations in Fit Patients with CLL: 4-Year Follow-up and NGS-Based MRD Analysis from the Phase 3 GAIA/CLL13 Trial

Background The primary endpoint analysis of the GAIA trial showed superior progression-free survival (PFS) and undetectable MRD (uMRD) rates for venetoclax-obinutuzumab (GV) and GV + ibrutinib (GIV) compared to chemoimmunotherapy (CIT) (Eichhorst et al., NEJM 2023). With additional follow-up, outcomes of the venetoclax (ven)-containing arms were compared and NGS-based MRD results were analyzed. Methods The phase 3 GAIA trial compared 3 different time-limited ven-based combinations against CIT in fit, treatment-naïve patients (pts) with CLL without TP53 aberrations. Pts were randomized to CIT (FCR ≤65 years; BR &amp;gt;65 years), GV, GIV or ven-rituximab (RV). In addition to MRD by flow cytometry (FCM), exploratory MRD analyses were performed using the amplicon-based EuroClonality NGS assay. Reported p values have a descriptive character. Results In total 926 pts were randomized (CIT: 229, RV: 237, GV: 229, GIV: 231). After a median observation time of 50.7 months (interquartile range 44.6-57.9), all pts are now off study treatment. PFS continued to be superior for GV and GIV compared to CIT (GV: median not reached [NR] vs 59.4 months; hazard ratio [HR] 0.47 [97.5% CI 0.32-0.69], p&amp;lt;0.001; GIV: NR vs 59.4 months, HR 0.30 [97.5% CI 0.19-0.47], p&amp;lt;0.001, Figure 1A). PFS with GV and GIV was also superior compared to RV (GV: NR vs 63.2 months; HR 0.57 [97.5% CI 0.38-0.84], p=0.001; GIV: NR vs 63.2 months, HR 0.38 [97.5% CI 0.24-0.59], p&amp;lt;0.001). PFS between GIV and GV was not significantly different (both NR, HR 0.63 [97.5% CI 0.39-1.02], p&amp;gt;0.025), however, GIV was associated with longer PFS compared to GV in pts with unmutated IGHV (HR 0.58 [95% CI 0.36-0.94]) but not in pts with mutated IGHV (HR 0.87 [95% CI 0.33-2.31]). Estimated 4-year PFS rates were 62.0% (CIT), 70.1% (RV), 81.8% (GV) and 85.5% (GIV). The estimated 4-year rates for time to next treatment were 77.2% (CIT), 86.2% (RV), 90.4% (GV) and 96.0% (GIV). Of the 111 pts with subsequent therapies for CLL-type progression (excluding 12 pts with treatment for Richter's transformation as second line), 60 (54.1%) received BTKi-based therapies, 30 (27.0%) ven-based treatments, 12 (10.8%) ven + BTKi and 5 (5.4%) CIT as second-line treatments. No differences in overall survival were observed between the treatment arms (4-year OS rates, CIT 93.5%; RV 96.2%; GV 95.1%; GIV 95.0%). In a multivariate analysis, unmutated IGHV (HR 2.86 [95% CI 1.64-5.01], p&amp;lt;0.001) and bulky disease (any lymph node ≥ 5 cm, HR 1.73 [95% CI 1.11-2.69], p=0.016) were independently associated with shorter PFS in the pooled GV/GIV arms. NGS-based MRD data in PB was available for 816 pts at month 15. Of these, 22.7% (52 pts, CIT), 23.6% (56 pts, RV), 60.3% (138 pts, GV) and 66.2% (153 pts, GIV) achieved uMRD &amp;lt;10 -6 (uMRD6, Figure 1B). In all treatment arms, PFS was shorter in pts with MRD ≥10 -6 compared to those with uMRD6 (CIT: HR 9.98 [95% CI 3.64-27.38], RV: HR 6.57 [95% CI 2.72-16.77], GV: HR 3.93 [95% CI 2.18-7.09], GIV: HR 2.10 [95% CI 1.03-4.28]). Pts who achieved uMRD below the conventional cut-off of 10 -4 by FCM but still had low levels of detectable MRD (≥10 -6 &amp; &amp;lt;10 -4) by NGS had shorter PFS than pts achieving uMRD6 in the pooled GV/GIV arms (HR 2.18 [95% CI 1.32-3.61], Figure 1C). A similar correlation was seen with CIT (HR 4.49 [95% CI 1.53-13.14]) and RV (HR 3.40 [95% CI 1.29-8.98]). In pts with uMRD6 at MO15, clinical response (partial/complete response) did not influence PFS. Grade ≥3 infections were highest in GIV and CIT (CIT: 45 pts [20.8%], RV: 27 [11.4%], GV: 34 [14.9%], GIV: 51 [22.1%]) and cardiac disorders most frequent with GIV (CIT: 14 pts [6.5%], RV: 19 [8.0%], GV: 18 [7.9%], GIV: 41 [17.7%]). Fatal adverse events occurred in 16 (7.4%, CIT), 8 (3.4%, RV), 9 (3.9%, GV) and 11 (4.8%, GIV) pts. The rate of second primary malignancies was higher with CIT (4.19/1000 patient-months) compared to RV (2.34), GV (2.39) and GIV (2.88). When excluding non-melanoma skin cancer, the incidence rates were 2.21 (CIT) 1.21 (RV), 1.16 (GV) and 2.36 (GIV). Conclusions With more than 4 years of follow-up, GV and GIV show superior PFS compared to CIT and RV. Pts with unmutated IGHV have longer PFS with GIV compared to GV. A majority of pts treated with time-limited GV or GIV (60.3% and 66.2%) achieves uMRD6 at MO15. NGS-based MRD assessment identifies pts with very long PFS and appears to improve prognostication in pts with uMRD &amp;lt;10 -4 by conventional FCM. Unmutated IGHV and bulky disease were independently associated with shorter PFS in pooled GV/GIV.