RESUMO
The presence of measurable residual disease (MRD) is strongly associated with treatment outcomes in acute myeloid leukemia (AML). Despite the correlation with clinical outcomes, MRD assessment has yet to be standardized or routinely incorporated into clinical trials and discrepancies have been observed between different techniques for MRD assessment. In 62 patients with AML, aged 18-60 years, in first complete remission after intensive induction therapy on the randomized phase III SWOG-S0106 clinical trial (clinicaltrials gov. Identifier: NCT00085709), MRD detection by centralized, high-quality multiparametric flow cytometry was compared with a 29-gene panel utilizing duplex sequencing (DS), an ultrasensitive next-generation sequencing method that generates double-stranded consensus sequences to reduce false positive errors. MRD as defined by DS was observed in 22 (35%) patients and was strongly associated with higher rates of relapse (68% vs. 13%; hazard ratio [HR] =8.8; 95% confidence interval [CI]: 3.2-24.5; P<0.001) and decreased survival (32% vs. 82%; HR=5.6; 95% CI: 2.3-13.8; P<0.001) at 5 years. DS MRD strongly outperformed multiparametric flow cytometry MRD, which was observed in ten (16%) patients and marginally associated with higher rates of relapse (50% vs. 30%; HR=2.4; 95% CI: 0.9-6.7; P=0.087) and decreased survival (40% vs. 68%; HR=2.5; 95% CI: 1.0-6.3; P=0.059) at 5 years. Furthermore, the prognostic significance of DS MRD status at the time of remission for subsequent relapse was similar on both randomized arms of the trial. These findings suggest that next-generation sequencing-based AML MRD testing is a powerful tool that could be developed for use in patient management and for early anti-leukemic treatment assessment in clinical trials.
Assuntos
Transplante de Células-Tronco Hematopoéticas , Leucemia Mieloide Aguda , Adulto , Humanos , Leucemia Mieloide Aguda/diagnóstico , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/terapia , Resultado do Tratamento , Prognóstico , Recidiva , Neoplasia Residual/diagnóstico , Citometria de Fluxo/métodosRESUMO
Mutations, the fuel of evolution, are first manifested as rare DNA changes within a population of cells. Although next-generation sequencing (NGS) technologies have revolutionized the study of genomic variation between species and individual organisms, most have limited ability to accurately detect and quantify rare variants among the different genome copies in heterogeneous mixtures of cells or molecules. We describe the technical challenges in characterizing subclonal variants using conventional NGS protocols and the recent development of error correction strategies, both computational and experimental, including consensus sequencing of single DNA molecules. We also highlight major applications for low-frequency mutation detection in science and medicine, describe emerging methodologies and provide our vision for the future of DNA sequencing.
Assuntos
Genômica/métodos , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Mutação , Análise Mutacional de DNA/métodos , HumanosRESUMO
Polyguanine tracts (PolyGs) are short guanine homopolymer repeats that are prone to accumulating mutations when cells divide. This feature makes them especially suitable for cell lineage tracing, which has been exploited to detect and characterize precancerous and cancerous somatic evolution. PolyG genotyping, however, is challenging because of the inherent biochemical difficulties in amplifying and sequencing repetitive regions. To overcome this limitation, we developed PolyG-DS, a next-generation sequencing (NGS) method that combines the error-correction capabilities of duplex sequencing (DS) with enrichment of PolyG loci using CRISPR-Cas9-targeted genomic fragmentation. PolyG-DS markedly reduces technical artifacts by comparing the sequences derived from the complementary strands of each original DNA molecule. We demonstrate that PolyG-DS genotyping is accurate, reproducible, and highly sensitive, enabling the detection of low-frequency alleles (<0.01) in spike-in samples using a panel of only 19 PolyG markers. PolyG-DS replicated prior results based on PolyG fragment length analysis by capillary electrophoresis, and exhibited higher sensitivity for identifying clonal expansions in the nondysplastic colon of patients with ulcerative colitis. We illustrate the utility of this method for resolving the phylogenetic relationship among precancerous lesions in ulcerative colitis and for tracing the metastatic dissemination of ovarian cancer. PolyG-DS enables the study of tumor evolution without prior knowledge of tumor driver mutations and provides a tool to perform cost-effective and easily scalable ultra-accurate NGS-based PolyG genotyping for multiple applications in biology, genetics, and cancer research.
Assuntos
Linhagem da Célula , DNA/genética , Guanina/química , Neoplasias/genética , Poli G/genética , Diferenciação Celular , Evolução Clonal , DNA/química , Genótipo , HumanosRESUMO
The use of error-corrected Next Generation Sequencing (ecNG) to determine mutagenicity has been a subject of growing interest and potentially a disruptive technology that could supplement, and in time, replace current testing paradigms in preclinical safety assessment. Considering this, a Next Generation Sequencing Workshop was held at the Royal Society of Medicine in London in May 2022, supported by the United Kingdom Environmental Mutagen Society (UKEMS) and TwinStrand Biosciences (WA, USA), to discuss progress and future applications of this technology. In this meeting report, the invited speakers provide an overview of the Workshop topics covered and identify future directions for research. In the area of somatic mutagenesis, several speakers reviewed recent progress made with correlating ecNGS to classic in vivo transgenic rodent mutation assays as well as exploring the use of this technology directly in humans and animals, and in complex organoid models. Additionally, ecNGS has been used for detecting off-target effects of gene editing tools and emerging data suggest ecNGS potential to measure clonal expansion of cells carrying mutations in cancer driver genes as an early marker of carcinogenic potential and for direct human biomonitoring. As such, the workshop demonstrated the importance of raising awareness and support for advancing the science of ecNGS for mutagenesis, gene editing, and carcinogenesis research. Furthermore, the potential of this new technology to contribute to advances in drug and product development and improve safety assessment was extensively explored.
Assuntos
Sequenciamento de Nucleotídeos em Larga Escala , Mutagênicos , Animais , Humanos , Londres , Mutagênese , Mutação , Carcinogênese , GenômicaRESUMO
Mutagenicity testing is an essential component of health safety assessment. Duplex Sequencing (DS), an emerging high-accuracy DNA sequencing technology, may provide substantial advantages over conventional mutagenicity assays. DS could be used to eliminate reliance on standalone reporter assays and provide mechanistic information alongside mutation frequency (MF) data. However, the performance of DS must be thoroughly assessed before it can be routinely implemented for standard testing. We used DS to study spontaneous and procarbazine (PRC)-induced mutations in the bone marrow (BM) of MutaMouse males across a panel of 20 diverse genomic targets. Mice were exposed to 0, 6.25, 12.5, or 25 mg/kg-bw/day for 28 days by oral gavage and BM sampled 42 days post-exposure. Results were compared with those obtained using the conventional lacZ viral plaque assay on the same samples. DS detected significant increases in mutation frequencies and changes to mutation spectra at all PRC doses. Low intra-group variability within DS samples allowed for detection of increases at lower doses than the lacZ assay. While the lacZ assay initially yielded a higher fold-change in mutant frequency than DS, inclusion of clonal mutations in DS mutation frequencies reduced this discrepancy. Power analyses suggested that three animals per dose group and 500 million duplex base pairs per sample is sufficient to detect a 1.5-fold increase in mutations with > 80% power. Overall, we demonstrate several advantages of DS over classical mutagenicity assays and provide data to support efforts to identify optimal study designs for the application of DS as a regulatory test.
Assuntos
Medula Óssea , Taxa de Mutação , Masculino , Camundongos , Animais , Procarbazina/toxicidade , Mutagênicos/toxicidade , Mutação , Testes de Mutagenicidade/métodos , Camundongos Transgênicos , Óperon LacRESUMO
The ability to accurately measure mutations is critical for basic research and identifying potential drug and chemical carcinogens. Current methods for in vivo quantification of mutagenesis are limited because they rely on transgenic rodent systems that are low-throughput, expensive, prolonged, and do not fully represent other species such as humans. Next-generation sequencing (NGS) is a conceptually attractive alternative for detecting mutations in the DNA of any organism; however, the limit of resolution for standard NGS is poor. Technical error rates (â¼1 × 10-3) of NGS obscure the true abundance of somatic mutations, which can exist at per-nucleotide frequencies ≤1 × 10-7 Using duplex sequencing, an extremely accurate error-corrected NGS (ecNGS) technology, we were able to detect mutations induced by three carcinogens in five tissues of two strains of mice within 31 d following exposure. We observed a strong correlation between mutation induction measured by duplex sequencing and the gold-standard transgenic rodent mutation assay. We identified exposure-specific mutation spectra of each compound through trinucleotide patterns of base substitution. We observed variation in mutation susceptibility by genomic region, as well as by DNA strand. We also identified a primordial marker of carcinogenesis in a cancer-predisposed strain of mice, as evidenced by clonal expansions of cells carrying an activated oncogene, less than a month after carcinogen exposure. These findings demonstrate that ecNGS is a powerful method for sensitively detecting and characterizing mutagenesis and the early clonal evolutionary hallmarks of carcinogenesis. Duplex sequencing can be broadly applied to basic mutational research, regulatory safety testing, and emerging clinical applications.
Assuntos
Carcinogênese/genética , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Mutagênese/genética , Animais , Carcinógenos/toxicidade , Análise por Conglomerados , DNA/genética , Genes ras , Loci Gênicos , Genoma , Humanos , Camundongos Transgênicos , Mutação/genética , Neoplasias/genética , Oncogenes , Fenótipo , Transcrição GênicaRESUMO
Exposure to environmental mutagens increases the risk of cancer and genetic disorders. We used Duplex Sequencing (DS), a high-accuracy error-corrected sequencing technology, to analyze mutation induction across twenty 2.4 kb intergenic and genic targets in the bone marrow of MutaMouse males exposed to benzo(a)pyrene (BaP), a widespread environmental pollutant. DS revealed a linear dose-related induction of mutations across all targets with low intra-group variability. Heterochromatic and intergenic regions exhibited the highest mutation frequencies (MF). C:G > A:T transversions at CCA, CCC and GCC trinucleotides were enriched in BaP-exposed mice consistent with the known etiology of BaP mutagenesis. However, GC-content had no effect on mutation susceptibility. A positive correlation was observed between DS and the "gold-standard" transgenic rodent gene mutation assay. Overall, we demonstrate that DS is a promising approach to study in vivo mutagenesis and yields critical insight into the genomic features governing mutation susceptibility, spectrum, and variability across the genome.
Assuntos
Benzo(a)pireno , Mutagênicos , Animais , Benzo(a)pireno/toxicidade , Genômica , Masculino , Camundongos , MutaçãoRESUMO
Human colorectal cancers (CRCs) contain both clonal and subclonal mutations. Clonal driver mutations are positively selected, present in most cells, and drive malignant progression. Subclonal mutations are randomly dispersed throughout the genome, providing a vast reservoir of mutant cells that can expand, repopulate the tumor, and result in the rapid emergence of resistance, as well as being a major contributor to tumor heterogeneity. Here, we apply duplex sequencing (DS) methodology to quantify subclonal mutations in CRC tumor with unprecedented depth (104) and accuracy (<10-7). We measured mutation frequencies in genes encoding replicative DNA polymerases and in genes frequently mutated in CRC, and found an unexpectedly high effective mutation rate, 7.1 × 10-7. The curve of subclonal mutation accumulation as a function of sequencing depth, using DNA obtained from 5 different tumors, is in accord with a neutral model of tumor evolution. We present a theoretical approach to model neutral evolution independent of the infinite-sites assumption (which states that a particular mutation arises only in one tumor cell at any given time). Our analysis indicates that the infinite-sites assumption is not applicable once the number of tumor cells exceeds the reciprocal of the mutation rate, a circumstance relevant to even the smallest clinically diagnosable tumor. Our methods allow accurate estimation of the total mutation burden in clinical cancers. Our results indicate that no DNA locus is wild type in every malignant cell within a tumor at the time of diagnosis (probability of all cells being wild type, 10-308).
RESUMO
Next-generation sequencing methods suffer from low recovery, uneven coverage, and false mutations. DNA fragmentation by sonication is a major contributor to these problems because it produces randomly sized fragments, PCR amplification bias, and end artifacts. In addition, oligonucleotide-based hybridization capture, a common target enrichment method, has limited efficiency for small genomic regions, contributing to low recovery. This becomes a critical problem in clinical applications, which value cost-effective approaches focused on the sequencing of small gene panels. To address these issues, we developed a targeted genome fragmentation approach based on CRISPR/Cas9 digestion that produces DNA fragments of similar length. These fragments can be enriched by a simple size selection, resulting in targeted enrichment of up to approximately 49,000-fold. Additionally, homogenous length fragments significantly reduce PCR amplification bias and maximize read usability. We combined this novel target enrichment approach with Duplex Sequencing, which uses double-strand molecular tagging to correct for sequencing errors. The approach, termed CRISPR-DS, enables efficient target enrichment of small genomic regions, even coverage, ultra-accurate sequencing, and reduced DNA input. As proof of principle, we applied CRISPR-DS to the sequencing of the exonic regions of TP53 and performed side-by-side comparisons with standard Duplex Sequencing. CRISPR-DS detected previously reported pathogenic TP53 mutations present as low as 0.1% in peritoneal fluid of women with ovarian cancer, while using 10- to 100-fold less DNA than standard Duplex Sequencing. Whether used as standalone enrichment or coupled with high-accuracy sequencing methods, CRISPR-based fragmentation offers a simple solution for fast and efficient small target enrichment.
Assuntos
Sistemas CRISPR-Cas , Neoplasias Ovarianas/genética , Análise de Sequência de DNA/métodos , Proteína Supressora de Tumor p53/genética , DNA/genética , Fragmentação do DNA , Feminino , Sequenciamento de Nucleotídeos em Larga Escala , HumanosRESUMO
Cumulative evidence indicates that a significant proportion of cancer evolution may occur before the development of histological abnormalities. While recent improvements in DNA sequencing technology have begun to reveal the presence of these early preneoplastic clones, the concept of 'premalignant field' was already introduced by Slaughter more than half a century ago. Also referred to as 'field effect', 'field defect' or 'field cancerization', these terms describe the phenomenon by which molecular alterations develop in normal-appearing tissue and expand to form premalignant patches with the potential to progress to dysplasia and cancer. Field effects have been well-characterized in ulcerative colitis, an inflammatory bowel disease that increases the risk of colorectal cancer. The study of the molecular alterations that define these fields is informative of mechanisms of tumor initiation and progression and has provided potential targets for early cancer detection. Herein, we summarize the current knowledge about the molecular alterations that comprise the field effect in ulcerative colitis and the clinical utility of these fields for cancer screening and prevention.
Assuntos
Colite Ulcerativa/complicações , Neoplasias Colorretais , Lesões Pré-Cancerosas , Animais , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/metabolismo , Transformação Celular Neoplásica/patologia , Neoplasias Colorretais/etiologia , Neoplasias Colorretais/genética , Neoplasias Colorretais/patologia , Humanos , Lesões Pré-Cancerosas/etiologia , Lesões Pré-Cancerosas/genética , Lesões Pré-Cancerosas/patologiaRESUMO
Intratumor genetic heterogeneity reflects the evolutionary history of a cancer and is thought to influence treatment outcomes. Here we report that a simple PCR-based assay interrogating somatic variation in hypermutable polyguanine (poly-G) repeats can provide a rapid and reliable assessment of mitotic history and clonal architecture in human cancer. We use poly-G repeat genotyping to study the evolution of colon carcinoma. In a cohort of 22 patients, we detect poly-G variants in 91% of tumors. Patient age is positively correlated with somatic mutation frequency, suggesting that some poly-G variants accumulate before the onset of carcinogenesis during normal division in colonic stem cells. Poorly differentiated tumors have fewer mutations than well-differentiated tumors, possibly indicating a shorter mitotic history of the founder cell in these cancers. We generate poly-G mutation profiles of spatially separated samples from primary carcinomas and matched metastases to build well-supported phylogenetic trees that illuminate individual patients' path of metastatic progression. Our results show varying degrees of intratumor heterogeneity among patients. Finally, we show that poly-G mutations can be found in other cancers than colon carcinoma. Our approach can generate reliable maps of intratumor heterogeneity in large numbers of patients with minimal time and cost expenditure.
Assuntos
Neoplasias do Colo/genética , DNA de Neoplasias/genética , Mutação , Adenocarcinoma/etiologia , Adenocarcinoma/genética , Adenocarcinoma/secundário , Adulto , Idoso , Idoso de 80 Anos ou mais , Carcinogênese/genética , Diferenciação Celular/genética , Estudos de Coortes , Neoplasias do Colo/etiologia , Neoplasias do Colo/patologia , Heterogeneidade Genética , Humanos , Repetições de Microssatélites , Pessoa de Meia-Idade , Mitose/genética , Filogenia , Poli G/genéticaRESUMO
Mitochondrial DNA (mtDNA) is believed to be highly vulnerable to age-associated damage and mutagenesis by reactive oxygen species (ROS). However, somatic mtDNA mutations have historically been difficult to study because of technical limitations in accurately quantifying rare mtDNA mutations. We have applied the highly sensitive Duplex Sequencing methodology, which can detect a single mutation among >10(7) wild type molecules, to sequence mtDNA purified from human brain tissue from both young and old individuals with unprecedented accuracy. We find that the frequency of point mutations increases ~5-fold over the course of 80 years of life. Overall, the mutation spectra of both groups are comprised predominantly of transition mutations, consistent with misincorporation by DNA polymerase γ or deamination of cytidine and adenosine as the primary mutagenic events in mtDNA. Surprisingly, G â T mutations, considered the hallmark of oxidative damage to DNA, do not significantly increase with age. We observe a non-uniform, age-independent distribution of mutations in mtDNA, with the D-loop exhibiting a significantly higher mutation frequency than the rest of the genome. The coding regions, but not the D-loop, exhibit a pronounced asymmetric accumulation of mutations between the two strands, with G â A and T â C mutations occurring more often on the light strand than the heavy strand. The patterns and biases we observe in our data closely mirror the mutational spectrum which has been reported in studies of human populations and closely related species. Overall our results argue against oxidative damage being a major driver of aging and suggest that replication errors by DNA polymerase γ and/or spontaneous base hydrolysis are responsible for the bulk of accumulating point mutations in mtDNA.
Assuntos
Envelhecimento/genética , DNA Mitocondrial/genética , Mutação , Estresse Oxidativo/genética , Idoso , Idoso de 80 Anos ou mais , Envelhecimento/fisiologia , Autopsia , Encéfalo/metabolismo , Dano ao DNA , DNA Mitocondrial/fisiologia , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos , Lactente , Recém-Nascido , Mitocôndrias/genética , Mitocôndrias/patologia , Mutagênese , Espécies Reativas de Oxigênio/metabolismoRESUMO
Genome instability is regarded as a hallmark of cancer. Human tumors frequently carry clonally expanded mutations in their mitochondrial DNA (mtDNA), some of which may drive cancer progression and metastasis. The high prevalence of clonal mutations in tumor mtDNA has commonly led to the assumption that the mitochondrial genome in cancer is genetically unstable, yet this hypothesis has not been experimentally tested. In this study, we directly measured the frequency of non-clonal (random) de novo single base substitutions in the mtDNA of human colorectal cancers. Remarkably, tumor tissue exhibited a decreased prevalence of these mutations relative to adjacent non-tumor tissue. The difference in mutation burden was attributable to a reduction in C:G to T:A transitions, which are associated with oxidative damage. We demonstrate that the lower random mutation frequency in tumor tissue was also coupled with a shift in glucose metabolism from oxidative phosphorylation to anaerobic glycolysis, as compared to non-neoplastic colon. Together these findings raise the intriguing possibility that fidelity of mitochondrial genome is, in fact, increased in cancer as a result of a decrease in reactive oxygen species-mediated mtDNA damage.
Assuntos
Neoplasias Colorretais/genética , DNA Mitocondrial/genética , Mutagênese , Mutação Puntual/genética , Idoso , Idoso de 80 Anos ou mais , Dano ao DNA/genética , Feminino , Instabilidade Genômica , Glucose/metabolismo , Glicólise/genética , Humanos , Masculino , Pessoa de Meia-Idade , Estresse Oxidativo , Espécies Reativas de Oxigênio/metabolismoRESUMO
Next-generation DNA sequencing promises to revolutionize clinical medicine and basic research. However, while this technology has the capacity to generate hundreds of billions of nucleotides of DNA sequence in a single experiment, the error rate of ~1% results in hundreds of millions of sequencing mistakes. These scattered errors can be tolerated in some applications but become extremely problematic when "deep sequencing" genetically heterogeneous mixtures, such as tumors or mixed microbial populations. To overcome limitations in sequencing accuracy, we have developed a method termed Duplex Sequencing. This approach greatly reduces errors by independently tagging and sequencing each of the two strands of a DNA duplex. As the two strands are complementary, true mutations are found at the same position in both strands. In contrast, PCR or sequencing errors result in mutations in only one strand and can thus be discounted as technical error. We determine that Duplex Sequencing has a theoretical background error rate of less than one artifactual mutation per billion nucleotides sequenced. In addition, we establish that detection of mutations present in only one of the two strands of duplex DNA can be used to identify sites of DNA damage. We apply the method to directly assess the frequency and pattern of random mutations in mitochondrial DNA from human cells.
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Sequenciamento de Nucleotídeos em Larga Escala/métodos , Metagenoma/genética , Mutação/genética , Neoplasias/genética , Projetos de Pesquisa/estatística & dados numéricos , Dano ao DNA/genética , Humanos , Oligonucleotídeos/genéticaRESUMO
Importance: Persistence of FLT3 internal tandem duplication (ITD) in adults with acute myeloid leukemia (AML) in first complete remission (CR) prior to allogeneic hematopoietic cell transplant (HCT) is associated with increased relapse and death after transplant, but the association between the level of measurable residual disease (MRD) detected and clinical outcome is unknown. Objective: To examine the association between pre-allogeneic HCT MRD level with relapse and death posttransplant in adults with AML in first CR. Design, Setting, and Participants: In this cohort study, DNA sequencing was performed on first CR blood from patients with FLT3-ITD AML transplanted from March 2013 to February 2019. Clinical follow-up was through May 2022. Data were analyzed from October 2022 to December 2023. Exposure: Centralized DNA sequencing for FLT3-ITD in pre-allogeneic HCT first CR blood using a commercially available kit. Main Outcomes and Measures: The primary outcomes were overall survival and cumulative incidence of relapse, with non-relapse-associated mortality as a competing risk post-allogeneic HCT. Kaplan-Meier estimations (log-rank tests), Cox proportional hazards models, and Fine-Gray models were used to estimate the end points. Results: Of 537 included patients with FLT3-ITD AML from the Pre-MEASURE study, 296 (55.1%) were female, and the median (IQR) age was 55.6 (42.9-64.1) years. Using the variant allele fraction (VAF) threshold of 0.01% or greater for MRD positivity, the results closely aligned with those previously reported. With no VAF threshold applied (VAF greater than 0%), 263 FLT3-ITD variants (median [range] VAF, 0.005% [0.0002%-44%]), and 177 patients (33.0%) with positive findings were identified. Multivariable analyses showed that residual FLT3-ITD was the variable most associated with relapse and overall survival, with a dose-dependent correlation. Patients receiving reduced-intensity conditioning without melphalan or nonmyeloablative conditioning had increased risk of relapse and death at any given level of MRD compared with those receiving reduced-intensity conditioning with melphalan or myeloablative conditioning. Conclusions and Relevance: This study provides generalizable and clinically applicable evidence that the detection of residual FLT3-ITD in the blood of adults in first CR from AML prior to allogeneic HCT is associated with an increased risk of relapse and death, particularly for those with a VAF of 0.01% or greater. While transplant conditioning intensification, an intervention not available to all, may help mitigate some of this risk, alternative approaches will be necessary for this high-risk population of patients who are underserved by the current standard of care.
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Transplante de Células-Tronco Hematopoéticas , Leucemia Mieloide Aguda , Neoplasia Residual , Transplante Homólogo , Tirosina Quinase 3 Semelhante a fms , Humanos , Tirosina Quinase 3 Semelhante a fms/genética , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/terapia , Leucemia Mieloide Aguda/mortalidade , Leucemia Mieloide Aguda/sangue , Feminino , Masculino , Pessoa de Meia-Idade , Transplante de Células-Tronco Hematopoéticas/métodos , Adulto , Sequências de Repetição em Tandem , Idoso , Duplicação Gênica , Estudos de CoortesRESUMO
Regulators and industry are actively seeking improvements and alternatives to current models and approaches to evaluate potential carcinogenicity of gene therapies (GTs). A meeting of invited experts was organized by NC3Rs/UKEMS (London, March 2023) to discuss this topic. This article describes the consensus reached among delegates on the definition of vector genotoxicity, sources of uncertainty, suitable toxicological endpoints for genotoxic assessment of GTs, and future research needs. The collected recommendations should inform the further development of regulatory guidelines for the nonclinical toxicological assessment of GT products.
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Terapia Genética , Terapia Genética/efeitos adversos , Terapia Genética/métodos , Humanos , Fatores de Risco , Animais , Vetores Genéticos/efeitos adversos , Consenso , Neoplasias/terapia , Neoplasias/genética , Medição de RiscoRESUMO
Mutation rate is an important determinant of evolvability. The optimal mutation rate for different organisms during evolution has been modeled in silico and tested in vivo, predominantly through pairwise comparisons. To characterize the fitness landscape across a broad range of mutation rates, we generated a panel of 66 DNA polymerase I mutants in Escherichia coli with comparable growth properties, yet with differing DNA replication fidelities, spanning 10(3)-fold higher and lower than that of wild type. These strains were competed for 350 generations in six replicate cultures in two different environments. A narrow range of mutation rates, 10- to 47-fold greater than that of wild type, predominated after serial passage. Mutants exhibiting higher mutation rates were not detected, nor were wild-type or antimutator strains. Winning clones exhibited shorter doubling times, greater maximum culture densities, and a growth advantages in pairwise competition relative to their precompetition ancestors, indicating the acquisition of adaptive phenotypes. To investigate the basis for mutator selection, we undertook a large series of pairwise competitions between mutator and wild-type strains under conditions where, in most cases, one strain completely overtook the culture within 18 days. Mutators were the most frequent winners but wild-type strains were also observed winning, suggesting that the competitive advantage of mutators is due to a greater probability of developing selectably advantageous mutations rather than from an initial growth advantage conferred by the polymerase variant itself. Our results indicate that under conditions where organism fitness is not yet maximized for a particular environment, competitive adaptation may be facilitated by enhanced mutagenesis.
Assuntos
Evolução Biológica , DNA Polimerase I/genética , Escherichia coli/enzimologia , Mutação , Escherichia coli/genéticaRESUMO
Error-corrected sequencing of genomic targets enriched by probe-based capture has become a standard approach for detecting single-nucleotide variants (SNVs) and small insertion/deletions (indels) present at very low variant allele frequencies. Less attention has been given to comparable strategies for rare structural variant (SV) junctions, where different error mechanisms must be addressed. Working from samples with known SV properties, we demonstrate that duplex sequencing (DuplexSeq), which demands confirmation of variants on both strands of a source DNA molecule, eliminates false SV junctions arising from chimeric PCR. DuplexSeq could not address frequent intermolecular ligation artifacts that arise during Y-adapter addition prior to strand denaturation without requiring multiple source molecules. In contrast, tagmentation libraries coupled with data filtering based on strand family size greatly reduced both artifact classes and enabled efficient and specific detection of single-molecule SV junctions. The throughput of SV capture sequencing (svCapture) and base-level accuracy of DuplexSeq provided detailed views of the microhomology profile and limited occurrence of de novo SNVs near the junctions of hundreds of newly created SVs, suggesting end joining as a possible formation mechanism. The open source svCapture pipeline enables rare SV detection as a routine addition to SNVs/indels in properly prepared capture sequencing libraries.
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The presence of measurable residual disease (MRD) is strongly associated with treatment outcomes in acute myeloid leukemia (AML). Despite the correlation with clinical outcomes, MRD assessment has yet to be standardized or routinely incorporated into clinical trials. Discrepancies have been observed between different techniques for MRD assessment and there remains a need to compare centralized, high-quality multiparametric flow cytometry (MFC) and ultrasensitive next-generation sequencing (NGS) in AML patients with diverse mutational profiles. In 62 patients with AML, aged 18-60, in first complete remission after intensive induction therapy on the randomized phase 3 SWOG-S0106 clinical trial, MRD detection by MFC was compared with a 29 gene panel utilizing duplex sequencing (DS), an NGS method that generates double-stranded consensus sequences to reduce false positive errors. Using DS, detection of a persistent mutation utilizing defined criteria was seen in 22 (35%) patients and was strongly associated with higher rates of relapse (68% vs 13% at year 5; HR, 8.8; 95% CI, 3.2-24.5; P<0.001) and decreased survival (32% vs 82% at year 5; HR, 5.6; 95% CI, 2.3-13.8; P<0.001). MRD as defined by DS strongly outperformed MFC, which was observed in 10 (16%) patients and marginally associated with higher rates of relapse (50% vs 30% at year 5; HR, 2.4; 95% CI, 0.9-6.7; P=0.087) and decreased survival (40% vs 68% at year 5; HR, 2.5; 95% CI, 1.0-6.3; P=0.059). Furthermore, the prognostic significance of DS MRD status at the time of remission was similar on both randomized arms of the trial, predicting S0106 clinical trial outcomes. These findings suggest that DS is a powerful tool that could be used in patient management and for early treatment assessment in clinical trials.
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Duplex sequencing (DS) is an error-corrected next-generation sequencing method in which molecular barcodes informatically link PCR-copies back to their source DNA strands, enabling computational removal of errors in consensus sequences. 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 DS-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 tissuesâ , a considerable advancement compared to currently used in vivo gene mutation assays.