Biology subjects

Nachmanson, D.

Publications and source records attributed to Nachmanson, D..

9 recordsLinked to original sources

bioRxiv2025

Alignment between Duplex Sequencing and transgenic rodent mutation assay data in the assessment of in vivo NDMA-induced mutagenesis

The nitrosamine N-nitrosodimethylamine (NDMA) is a mutagen and rodent carcinogen that has been identified as a process impurity in some commercially available medicines, leading to market withdrawals and new impurity control measures. Error-corrected DNA sequencing techniques, such as Duplex Sequencing (DS), have error rates low enough to revolutionise genetic toxicology testing by directly measuring in vivo mutagenesis within days of exposure. Here, DS was performed on liver samples from an OECD-compliant, Transgenic Rodent Gene Mutation Assay (TGR) conducted under GLP standards. MutaMouse specimens were orally dosed with NDMA using either a repeat-dose 28-day regimen (0.02-4 mg/kg(bw)/day) or single bolus doses of either 5 or 10 mg/kg(bw) administered on day one. Dose-dependent increases in mutation frequency were detected by DS in liver, enabling a No-Observed Genotoxic Effect Level (NOGEL) of 0.07 mg/kg(bw)/day to be determined, supported by mechanistic analyses of trinucleotide mutation spectra. Benchmark dose (BMD) modelling determined similar BMD50 values from either DS or TGR, demonstrating concordance across the two techniques albeit with greater precision from DS due to smaller inter-animal variation. DS offers a fundamental change in mutagenicity assessments enabling more precise point-of-departure determinations with mechanistic clarity and 3Rs advantages compared to the standard TGR approach.

pharmacology and toxicology↗

bioRxiv2024

Protein structure, a genetic encoding for glycosylation

Unlike DNA, RNA, and protein biosynthesis, dogma describes glycosylation as primarily determined by intrinsic cellular limitations, such as glycosyltransferase expression and precursor availability. However, this cannot explain the commonly-observed differences between glycans on the same protein. By examining site-specific glycosylation on diverse human proteins, we detected associations between protein structure and glycan structure, broadly generalizable to human-expressed glycoproteins. Through structural analysis of site-specific glycosylation data, we found protein-sequence and structural features consistently correlated with specific glycan features. To quantify these relationships, we present a new amino acid substitution matrix describing "glycoimpact", i.e., the association of primary protein structure and glycosylation. High-glycoimpact amino acids co-evolve with glycosites, and glycoimpact is high when estimates of amino acid conservation and variant pathogenicity diverge. We report thousands of disease variants near glycosites with high-glycoimpact, including several with known links to aberrant glycosylation (e.g., Oculocutaneous Albinism, Jakob-Creutzfeldt disease, Gerstmann-Straussler-Scheinker, and Gauchers Disease). Finally, glycoimpact quantification is validated by studying oligomannose-complex glycan ratios on HIV ENV, differential sialylation on IgG3 Fc, differential glycosylation on SARS-CoV-2 Spike, and fucose-modulated function of a tuberculosis monoclonal antibody. Finally, to test the causality of protein-glycan associations, we created 5 glycoimpact-designed novel Rituximab variants, 4 of which substantially changed glycoprofiles as predicted. In all, we report that site-specific glycan biosynthesis is influenced by underlying protein structure, enabling glycan structure prediction and genetic sequence-guided glycoengineering.

genetics↗

bioRxiv2023

Tumor microenvironmental determinants of high-risk DCIS progression

ABSTRACT/SUMMARYDuctal carcinoma in situ (DCIS) constitutes an array of morphologically recognized intraductal neoplasms in the mammary ductal tree defined by an increased risk for subsequent invasive carcinomas at or near the site of biopsy detection. However, only 15-45% of untreated DCIS cases progress to invasive cancer, so understanding mechanisms that prevent progression is key to avoid overtreatment and provides a basis for alternative therapies and prevention. This study was designed to characterize the tumor microenvironment and molecular profile of high-risk DCIS that grew to a large size but remained as DCIS. All patients had DCIS lesions >5cm in size with at least one additional high-risk feature: young age (<45 years), high nuclear grade, hormone receptor negativity, HER2 positivity, the presence of comedonecrosis, or a palpable mass. The tumor immune microenvironment was characterized using multiplex immunofluorescence to identify immune cells and their spatial relationships within the ducts and stroma. Gene copy number analysis and whole exome DNA sequencing identified the mutational burden and driver mutations, and quantitative whole-transcriptome/gene expression analyses were performed. There was no association between the percent of the DCIS genome characterized by copy number variants (CNAs) and recurrence events (DCIS or invasive). Mutations, especially missense mutations, in the breast cancer driver genes PIK3CA and TP53 were common in this high-risk DCIS cohort (47% of evaluated lesions). Tumor infiltrating lymphocyte (TIL) density was higher in DCIS lesions with TP53 mutations (p=0.0079) compared to wildtype lesions, but not in lesions with PIK3CA mutations (p=0.44). Immune infiltrates were negatively associated with hormone receptor status and positively associated with HER2 expression. High levels of CD3+CD8-T cells were associated with good outcomes with respect to any subsequent recurrence (DCIS or invasive cancer), whereas high levels of CD3+Foxp3+ Treg cells were associated with poor outcomes. Spatial proximity analyses of immune cells and tumor cells demonstrated that close proximity of T cells with tumor cells was associated with good outcomes with respect to any recurrence as well as invasive recurrences. Interestingly, we found that myoepithelial continuity (distance between myoepithelial cells surrounding the involved ducts) was significantly lower in DCIS lesions compared to normal tissue (p=0.0002) or to atypical ductal hyperplasia (p=0.011). Gene set enrichment analysis identified several immune pathways associated with low myoepithelial continuity and a low myoepithelial continuity score was associated with better outcomes, suggesting that gaps in the myoepithelial layer may allow access/interactions between immune infiltrates and tumor cells. Our study demonstrates the immune microenvironment of DCIS, in particular the spatial proximity of tumor cells and T cells, and myoepithelial continuity are important determinants for progression of disease.

cancer biology↗

bioRxiv2023

Adopting Duplex Sequencing Technology for Genetic Toxicity Testing: A Proof-of-Concept Mutagenesis Experiment with N-Ethyl-N-Nitrosourea (ENU)-Exposed Rats

Duplex sequencing (DuplexSeq) is an error-corrected next-generation sequencing (ecNGS) method in which molecular barcodes informatically link PCR-copies back to their source DNA strands, enabling computational removal of errors by comparing grouped strand sequencing reads. The resulting background of less than one artifactual mutation per 107 nucleotides allows for direct detection of somatic mutations. TwinStrand Biosciences, Inc. has developed a DuplexSeq-based mutagenesis assay to sample the rat genome, which can be applied to genetic toxicity testing. To evaluate this assay for early detection of mutagenesis, a time-course study was conducted using male Hsd:Sprague Dawley SD rats (3 per group) administered a single dose of 40 mg/kg N-ethyl-N-nitrosourea (ENU) via gavage, with mutation frequency (MF) and spectrum analyzed in stomach, bone marrow, blood, and liver tissues at 3 h, 24 h, 7 d, and 28 d post-exposure. Significant increases in MF were observed in ENU-exposed rats as early as 24 h for stomach (site of contact) and bone marrow (a highly proliferative tissue) and at 7 d for liver and blood. The canonical, mutational signature of ENU was established by 7 d post-exposure in all four tissues. Interlaboratory analysis of a subset of samples from different tissues and time points demonstrated remarkable reproducibility for both MF and spectrum. These results demonstrate that MF and spectrum can be evaluated successfully by directly sequencing targeted regions of DNA obtained from various tissues, a considerable advancement compared to currently used in vivo gene mutation assays. HIGHLIGHTSO_LIDuplexSeq is an ultra-accurate NGS technology that directly quantifies mutations C_LIO_LIENU-dependent mutagenesis was detected 24 h post-exposure in proliferative tissues C_LIO_LIMultiple tissues exhibited the canonical ENU mutation spectrum 7 d after exposure C_LIO_LIResults obtained with DuplexSeq were highly concordant between laboratories C_LIO_LIThe Rat-50 Mutagenesis Assay is promising for applications in genetic toxicology C_LI

genomics↗

bioRxiv2022

Integrated Molecular Characterization of Intraductal Papillary Mucinous Neoplasms: An NCI Cancer Moonshot Precancer Atlas Pilot Project

Intraductal papillary mucinous neoplasms (IPMNs) are cystic precursor lesions to pancreatic ductal adenocarcinoma (PDAC). IPMNs undergo multistep progression from low grade (LG) to high grade (HG) dysplasia, culminating in invasive neoplasia. While patterns of IPMN progression have been analyzed using multi-region sequencing for somatic mutations, there is no integrated assessment of molecular events, including copy number alterations (CNAs) and transcriptomics changes, that accompany IPMN progression. We performed laser capture microdissection on surgically resected IPMNs of varying grades of histological dysplasia obtained from 24 patients (total of 74 independent histological lesions), followed by whole exome and whole transcriptome sequencing. Overall, HG IPMNs displayed a significantly greater aneuploidy score than LG lesions, with chromosome 1q amplification, in particular, being associated with HG progression and with cases that harbored cooccurring PDAC. Furthermore, the combined assessment of single nucleotide variants (SNVs) and CNAs identified both linear and branched evolutionary trajectories, underscoring the heterogeneity in the progression of LG lesions to HG and PDAC. At the transcriptome level, upregulation of MYC-regulated targets and downregulation of transcripts associated with the MHC class I antigen presentation machinery was a common feature of progression to HG. Taken together, this work emphasizes the role of 1q copy number amplification as a putative biomarker of high-risk IPMNs, underscores the importance of immune evasion even in non-invasive precursor lesions, and supports a previously underappreciated role of CNA-driven branching evolution as an avenue for IPMN progression. Our study provides important molecular context for risk stratification and cancer interception opportunities in IPMNs. SignificanceIntegrated molecular analysis of genomic and transcriptomic alterations in the multistep progression of intraductal papillary mucinous neoplasms (IPMNs), which are bona fide precursors of pancreatic cancer, identifies features associated with progression of low-risk lesions to high-risk lesions and cancer, which might enable patient stratification and cancer interception strategies.

cancer biology↗

bioRxiv2021

The breast pre-cancer atlas illustrates the molecular and micro-environmental diversity of ductal carcinoma in situ

Micro-environmental and molecular factors mediating the progression of Breast Ductal Carcinoma In Situ (DCIS) are not well understood, impeding the development of prevention strategies and the safe testing of treatment de-escalation. We addressed methodological barriers and characterized the mutational, transcriptional, histological and microenvironmental landscape across 85 multiple micro-dissected regions from 39 cases. Most somatic alterations, including whole genome duplications, were clonal, but genetic divergence increased with physical distance. Phenotypic and subtype heterogeneity frequently associated with underlying genetic heterogeneity and regions with low-risk features preceded those with high-risk features according to the inferred phylogeny. B- and T-lymphocytes spatial analysis identified 3 immune states, including an epithelial excluded state located preferentially at DCIS regions, and characterized by histological and molecular features of immune escape, independently from molecular subtypes. Such breast pre-cancer atlas with uniquely integrated observations will help scope future expansion studies and build finer models of outcomes and progression risk.

genomics↗

bioRxiv2020

Mutational profiling of micro-dissected pre-malignant lesions from archived specimens

BackgroundSystematic cancer screening has led to the increased detection of pre-malignant lesions (PMLs). The absence of reliable prognostic markers has led mostly to over treatment resulting in potentially unnecessary stress, or potentially insufficient treatment and avoidable progression. Importantly, most mutational profiling studies have relied on PML synchronous to invasive cancer, or performed in patients without outcome information, hence limiting their utility for biomarker discovery. The limitations in comprehensive mutational profiling of PMLs are in large part due to the significant technical and methodological challenges: most PML specimens are small, fixed in formalin and paraffin embedded (FFPE) and lack matching normal DNA. MethodsUsing test DNA from a highly degraded FFPE specimen, multiple targeted sequencing approaches were evaluated, varying DNA input amount (3-200 ng), library preparation strategy (BE: Blunt-End, SS: Single-Strand, AT: A-Tailing) and target size (whole exome vs cancer gene panel). Variants in high-input DNA from FFPE and mirrored frozen specimens were used for PML-specific variant calling training and testing, respectively. The resulting approach was applied to profile and compare multiple regions micro-dissected (mean area 5 mm2) from 3 breast ductal carcinoma in situ (DCIS). ResultsUsing low-input FFPE DNA, BE and SS libraries resulted in 4.9 and 3.7 increase over AT libraries in the fraction of whole exome covered at 20x (BE:87%, SS:63%, AT:17%). Compared to high-confidence somatic mutations from frozen specimens, PML-specific variant filtering increased recall (BE:85%, SS:80%, AT:75%) and precision (BE:93%, SS:91%, AT:84%) to levels expected from sampling variation. Copy number alterations were consistent across all tested approaches and only impacted by the design of the capture probe-set. Applied to DNA extracted from 9 micro-dissected regions (8 PML, 1 normal epithelium), the approach achieved comparable performance, illustrated the data adequacy to identify candidate driver events (GATA3 mutations, ERBB2 or FGFR1 gains, TP53 loss) and measure intra-lesion genetic heterogeneity. ConclusionAlternate experimental and analytical strategies increased the accuracy of DNA sequencing from archived micro-dissected PML regions, supporting the deeper molecular characterization of early cancer lesions and achieving a critical milestone in the development of biology-informed prognostic markers and precision chemo-prevention strategies.

genomics↗

bioRxiv2018

Ultra-sensitive sequencing for cancer detection reveals progressive clonal selection in normal tissue over a century of human lifespan

High accuracy next-generation DNA sequencing promises a paradigm shift in early cancer detection by enabling the identification of mutant cancer molecules in minimally-invasive body fluid samples. We demonstrate 80% sensitivity for ovarian cancer detection using ultra-accurate Duplex Sequencing to identify TP53 mutations in uterine lavage. However, in addition to tumor DNA, we also detect low frequency TP53 mutations in nearly all lavages from women with and without cancer. These mutations increase with age and share the selection traits of clonal TP53 mutations commonly found in human tumors. We show that low frequency TP53 mutations exist in multiple healthy tissues, from newborn to centenarian, and progressively increase in abundance and pathogenicity with older age across tissue types. Our results illustrate that subclonal cancer evolutionary processes are a ubiquitous part of normal human aging and great care must be taken to distinguish tumor-derived, from age-associated mutations in high sensitivity clinical cancer diagnostics.

cancer biology↗

bioRxiv2017

CRISPR-DS: An efficient, low DNA input method for ultra-accurate sequencing

Current next-generation sequencing techniques suffer from inefficient target enrichment and frequent errors. To address these issues, we have developed a targeted genome fragmentation approach based on CRISPR/Cas9 digestion. By designing all fragments to similar lengths, regions of interest can be size-selected prior to library preparation, increasing hybridization capture efficiency. Additionally, homogenous length fragments reduce PCR bias and maximize read usability. We combine this novel target enrichment approach with ultra-accurate Duplex Sequencing. The result, termed CRISPR-DS, is a robust targeted sequencing technique that overcomes the inherent challenges of small target enrichment and enables the detection of ultra-low frequency mutations with small DNA inputs.

genomics↗