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While radioactive iodine therapy (RAIT) has been an effective treatment for thyroid cancer, its link to clonal hematopoiesis (CH) has been yet underexplored. In this study, error‐corrected sequencing (median depth: 1926×) of 93 CH‐related genes was performed from the blood samples of 358 thyroid cancer patients, including 110 controls (no RAIT) and 248 RAIT recipients. RAIT recipients were stratified into low‐ and high‐dose groups using a 7.4 GBq cutoff. Multivariable logistic regression revealed that the high‐dose group had a higher CH prevalence with variant allele frequency (VAF) higher than 2% compared to controls, especially in patients aged ≥50 (OR = 2.44, CI = 1.04–6.00, P = 0.04). Thirteen genes had mutations with VAF >2%, with DNMT3A, TET2, and PPM1D being the most common. Notably, only the PPM1D mutations were significantly linked to RAIT, occurring more frequently in the high‐dose group (13%) compared to the low‐dose group (5%) or controls (2%) at a VAF cutoff of 0.5%. In silico analyses indicated that truncating PPM1D mutations confer a selective advantage under high‐dose RAIT and with older age. Although the prognostic implications of PPM1D‐mutated CH remain to be further elucidated, these findings offer valuable insights for optimizing RAIT dosing in thyroid cancer patients. In thyroid cancer patients, high‐dose (≥7.4 GBq) radioactive iodine therapy (RAIT) was associated with a higher prevalence of clonal hematopoiesis (variant allele frequency >2%) in individuals aged ≥50 years (OR = 2.44). In silico analyses showed that truncating PPM1D mutations conferred a selective advantage under these conditions. These findings underscore the need to optimize RAIT dosing.

Human liver-derived metabolically competent HepaRG cells have been successfully employed in both two-dimensional (2D) and 3D spheroid formats for performing the comet assay and micronucleus (MN) assay. In the present study, we have investigated expanding the genotoxicity endpoints evaluated in HepaRG cells by detecting mutagenesis using two error-corrected next generation sequencing (ecNGS) technologies, Duplex Sequencing (DS) and High-Fidelity (HiFi) Sequencing. Both HepaRG 2D cells and 3D spheroids were exposed for 72 h to N-nitrosodimethylamine (NDMA), followed by an additional incubation for the fixation of induced mutations. NDMA-induced DNA damage, chromosomal damage, and mutagenesis were determined using the comet assay, MN assay, and ecNGS, respectively. The 72-h treatment with NDMA resulted in concentration-dependent increases in cytotoxicity, DNA damage, MN formation, and mutation frequency in both 2D and 3D cultures, with greater responses observed in the 3D spheroids compared to 2D cells. The mutational spectrum analysis showed that NDMA induced predominantly A:T → G:C transitions, along with a lower frequency of G:C → A:T transitions, and exhibited a different trinucleotide signature relative to the negative control. These results demonstrate that the HepaRG 2D cells and 3D spheroid models can be used for mutagenesis assessment using both DS and HiFi Sequencing, with the caveat that severe cytotoxic concentrations should be avoided when conducting DS. With further validation, the HepaRG 2D/3D system may become a powerful human-based metabolically competent platform for genotoxicity testing.

Background Risk stratification in multiple myeloma (MM) patients is crucial, and molecular genetic studies play a significant role in achieving this objective. Enrichment of plasma cells for next-generation sequencing (NGS) analysis has been employed to enhance detection sensitivity. However, these methods often come with limitations, such as high costs and low throughput. In this study, we explore the use of an error-corrected ultrasensitive NGS assay called positional indexing sequencing (PiSeq-MM). This assay can detect somatic mutations in MM patients without relying on plasma cell enrichment. Method Diagnostic bone marrow aspirates (BMAs) and blood samples from 14 MM patients were used for exploratory and validation sets. Results PiSeq-MM successfully detected somatic mutations in all BMAs, outperforming conventional NGS using plasma cells. It also identified 38 low-frequency mutations that were missed by conventional NGS, enhancing detection sensitivity below the 5% analytical threshold. When tested in an actual clinical environment, plasma cell enrichment failed in most BMAs (14/16), but the PiSeq-MM enabled mutation detection in all BMAs. There was concordance between PiSeq-MM using BMAs and ctDNA analysis in paired blood samples. Conclusion This research provides valuable insights into the genetic landscape of MM and highlights the advantages of error-corrected NGS for detecting low-frequency mutations. Although the current standard method for mutation analysis is plasma cell-enriched BMAs, total BMA or ctDNA testing with error correction is a viable alternative when plasma cell enrichment is not feasible.

Background Error-corrected next-generation sequencing (ecNGS) enables the sensitive detection of chemically induced mutations. Matsumura et al. reported Hawk-Seq™, an ecNGS method, demonstrating its utility in clarifying mutagenicity both qualitatively and quantitatively. To further promote the adoption of ecNGS-based assays, it is important to evaluate their inter-laboratory transferability and reproducibility. Therefore, we evaluated the inter-laboratory reproducibility of Hawk-Seq™ and its concordance with the transgenic rodent mutation (TGR) assay. Results The Hawk-Seq™ protocol was successfully transferred from the developer’s laboratory (lab A) to two additional laboratories (labs B, C). Whole genomic mutations were analyzed independently using the same genomic DNA samples from the livers of gpt delta mice exposed to benzo[ a ]pyrene (BP), N -ethyl- N -nitrosourea (ENU), and N -methyl- N -nitrosourea (MNU). In all laboratories, clear dose-dependent increases in base substitution (BS) frequencies were observed, specific to each mutagen (e.g. G:C to T:A for BP). Statistically significant increases in overall mutation frequencies (OMFs) were identified at the same doses across all laboratories, suggesting high reproducibility in mutagenicity assessment. The correlation coefficient (r 2 ) of the six types of BS frequencies exceeded 0.97 among the three laboratories for BP- or ENU-exposed samples. Thus, Hawk-Seq™ provides qualitatively and quantitatively reproducible results across laboratories. The OMFs in the Hawk-Seq™ analysis positively correlated (r 2  = 0.64) with gpt mutant frequencies (MFs). The fold induction of OMFs in the Hawk-Seq™ analysis of ENU- and MNU-exposed samples was at least 14.2 and 4.5, respectively, compared to 6.1 and 2.5 for gpt MFs. Meanwhile, the fold induction of OMFs in BP-exposed samples was ≤ 4.6, compared to 8.2 for gpt MFs. These observations suggest that Hawk-Seq™ demonstrates good concordance with the transgenic rodent (TGR) gene mutation assay, whereas the induction of mutation frequency by each mutagen might not directly correspond. Conclusions Hawk-Seq™-based whole-genome mutagenicity evaluation demonstrated high inter-laboratory reproducibility and concordance with gpt assay results. Our results contribute to the growing evidence that ecNGS assays provide a suitable, or improved, alternative to the TGR assay.

Error-corrected sequences (ECSs) that utilize double-stranded DNA sequences are useful in detecting mutagen-induced mutations. However, relatively higher frequencies of G:C > T:A (1 × 10 −7  bp) and G:C > C:G (2 × 10 −7  bp) errors decrease the accuracy of detection of rare G:C mutations (approximately 10 −7  bp). Oxidized guanines in single-strand (SS) overhangs generated after shearing could serve as the source of these errors. To remove these errors, we first computationally discarded up to 20 read bases corresponding to the ends of the DNA fragments. Error frequencies decreased proportionately with trimming length; however, the results indicated that they were not sufficiently removed. To efficiently remove SS overhangs, we evaluated three mechanistically distinct SS-specific nucleases (S1 Nuclease, mung bean nuclease, and RecJf exonuclease) and found that they were more efficient than computational trimming. Consequently, we established Jade-Seq™, an ECS protocol with S1 Nuclease treatment, which reduced G:C > T:A and G:C > C:G errors to 0.50 × 10 −7  bp and 0.12 × 10 −7  bp, respectively. This was probably because S1 Nuclease removed SS regions, such as gaps and nicks, depending on its wide substrate specificity. Subsequently, we evaluated the mutation-detection sensitivity of Jade-Seq™ using DNA samples from TA100 cells exposed to 3-methylcholanthrene and 7,12-dimethylbenz[a]anthracene, which contained the rare G:C > T:A mutation (i.e., 2 × 10 −7  bp). Fold changes of G:C > T:A compared to the vehicle control were 1.2- and 1.3-times higher than those of samples without S1 Nuclease treatment, respectively. These findings indicate the potential of Jade-Seq™ for detecting rare mutations and determining the mutagenicity of environmental mutagens.

Next generation sequencing (NGS) methods have allowed for unprecedented genomic characterization of acute myeloid leukemia (AML) over the last several years. Further advances in NGS-based methods including error correction using unique molecular identifiers (UMIs) have more recently enabled the use of NGS-based measurable residual disease (MRD) detection. This review focuses on the use of NGS-based MRD detection in AML, including basic methodologies and clinical applications.

Background Error-corrected next-generation sequencing (ecNGS) technologies have enabled the direct evaluation of genome-wide mutations after exposure to mutagens. Previously, we reported an ecNGS methodology, Hawk-Seq™, and demonstrated its utility in evaluating mutagenicity. The evaluation of technical transferability is essential to further evaluate the reliability of ecNGS-based assays. However, cutting-edge sequencing platforms are continually evolving, which can affect the sensitivity of ecNGS. Therefore, the effect of differences in sequencing instruments on mutation data quality should be evaluated. Results We assessed the performance of four sequencing platforms (HiSeq2500, NovaSeq6000, NextSeq2000, and DNBSEQ-G400) with the Hawk-Seq™ protocol for mutagenicity evaluation using DNA samples from mouse bone marrow exposed to benzo[ a ]pyrene (BP). The overall mutation (OM) frequencies per 10 6 bp in vehicle-treated samples were 0.22, 0.36, 0.46, and 0.26 for HiSeq2500, NovaSeq6000, NextSeq2000, and DNBSEQ-G400, respectively. The OM frequency of NextSeq2000 was significantly higher than that of HiSeq2500, suggesting the difference to be based on the platform. The relatively higher value in NextSeq2000 was a consequence of the G:C to C:G mutations in NextSeq2000 data (0.67 per 10 6 G:C bp), which was higher than the mean of the four platforms by a ca. of 0.25 per 10 6 G:C bp. A clear dose-dependent increase in G:C to T:A mutation frequencies was observed in all four sequencing platforms after BP exposure. The cosine similarity values of the 96-dimensional trinucleotide mutation patterns between HiSeq and the three other platforms were 0.93, 0.95, and 0.92 for NovaSeq, NextSeq, and DNBSeq, respectively. These results suggest that all platforms can provide equivalent data that reflect the characteristics of the mutagens. Conclusions All platforms sensitively detected mutagen-induced mutations using the Hawk-Seq™ analysis. The substitution types and frequencies of the background errors differed depending on the platform. The effects of sequencing platforms on mutagenicity evaluation should be assessed before experimentation.

Background Pediatric leukemias have a diverse genomic landscape associated with complex structural variants, including gene fusions, insertions and deletions, and single nucleotide variants. Routine karyotype and fluorescence in situ hybridization (FISH) techniques lack sensitivity for smaller genomic alternations. Next-generation sequencing (NGS) assays are being increasingly utilized for assessment of these various lesions. However, standard NGS lacks quantitative sensitivity for minimal residual disease (MRD) surveillance due to an inherently high error rate. Methods Primary bone marrow samples from pediatric leukemia ( n  = 32) and adult leukemia subjects ( n  = 5), cell line MV4–11, and an umbilical cord sample were utilized for this study. Samples were sequenced using molecular barcoding with targeted DNA and RNA library enrichment techniques based on anchored multiplexed PCR (AMP®) technology, amplicon based error-corrected sequencing (ECS) or a human cancer transcriptome assay. Computational analyses were performed to quantitatively assess limit of detection (LOD) for various DNA and RNA lesions, which could be systematically used for MRD assays. Results Matched leukemia patient samples were analyzed at three time points; diagnosis, end of induction (EOI), and relapse. Similar to flow cytometry for ALL MRD, the LOD for point mutations by these sequencing strategies was ≥0.001. For DNA structural variants, FLT3 internal tandem duplication (ITD) positive cell line and patient samples showed a LOD of ≥0.001 in addition to previously unknown copy number losses in leukemia genes. ECS in RNA identified multiple novel gene fusions, including a SPANT-ABL gene fusion in an ALL patient, which could have been used to alter therapy. Collectively, ECS for RNA demonstrated a quantitative and complex landscape of RNA molecules with 12% of the molecules representing gene fusions, 12% exon duplications, 8% exon deletions, and 68% with retained introns. Droplet digital PCR validation of ECS-RNA confirmed results to single mRNA molecule quantities. Conclusions Collectively, these assays enable a highly sensitive, comprehensive, and simultaneous analysis of various clonal leukemic mutations, which can be tracked across disease states (diagnosis, EOI, and relapse) with a high degree of sensitivity. The approaches and results presented here highlight the ability to use NGS for MRD tracking.

Background Mutagenicity, the ability of chemical agents to cause mutations and potentially lead to cancer, is a critical aspect of substance safety assessment for protecting human health and the environment. Metabolic enzymes activate multiple mutagens in living organisms, thus in vivo animal models provide highly important information for evaluating mutagenicity in human. Rats are considered suitable models as they share a similar metabolic pathway with humans for processing toxic chemical and exhibit higher responsiveness to chemical carcinogens than mice. To assess mutagenicity in rats, transgenic rodents (TGRs) are widely used for in vivo gene mutation assays. However, such assays are labor-intensive and could only detect transgene mutations inserted into the genome. Therefore, introducing a technology to directly detect in vivo mutagenicity in rats would be necessary. The next-generation sequencing (NGS) based error-corrected sequencing technique is a promising approach for such purposes. Results We investigated the applicability of paired-end and complementary consensus sequencing (PECC-Seq), an error-corrected sequencing technique, for detecting in vivo mutagenicity in the rat liver samples. PECC-Seq allows for the direct detection of ultra-rare somatic mutations in the genomic DNA without being constrained by the genomic locus, tissue, or organism. We tested PECC-Seq feasibility in rats treated with diethylnitrosamine (DEN), a mutagenic compound. Interestingly, the mutation and mutant frequencies between PECC-Seq and the TGR assay displayed a promising correlation. Our results also demonstrated that PECC-Seq could successfully detect the A:T > T:A mutation in rat liver samples, consistent with the TGR assay. Furthermore, we calculated the trinucleotide mutation frequency and proved that PECC-Seq accurately identified the DEN treatment-induced mutational signatures. Conclusions Our study provides the first evidence of using PECC-Seq for in vivo mutagenicity detection in rat liver samples. This approach could provide a valuable alternative to conventional TGR assays as it is labor- and time-efficient and eliminates the need for transgenic rodents. Error-corrected sequencing techniques, such as PECC-Seq, represent promising approaches for enhancing mutagenicity assessment and advancing regulatory science.