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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. [ABSTRACT FROM AUTHOR]

Background: Assessment of measurable residual disease (MRD) is an essential prognostic tool for B-lymphoblastic leukaemia (B-ALL). In this study, we evaluated the utility of next-generation sequencing (NGS)–based MRD assessment in real-world clinical practice. Method: The study included 93 paediatric patients with B-ALL treated at our institution between January 2017 and June 2022. Clonality for IGH or IGK rearrangements was identified in most bone marrow samples (91/93, 97.8%) obtained at diagnosis. Results: In 421 monitoring samples, concordance was 74.8% between NGS and multiparameter flow cytometry and 70.7% between NGS and reverse transcription-PCR. Elevated quantities of clones of IGH alone (P < 0.001; hazard ratio [HR], 22.2; 95% confidence interval [CI], 7.1–69.1), IGK alone (P = 0.011; HR, 5.8; 95% CI, 1.5–22.5), and IGH or IGK (P < 0.001; HR, 7.2; 95% CI, 2.6–20.0) were associated with an increased risk of relapse. Detection of new clone(s) in NGS was also associated with inferior relapse-free survival (P < 0.001; HR, 18.1; 95% CI, 3.0–108.6). Multivariable analysis confirmed age at diagnosis, BCR::ABL1-like mutation, TCF3::PBX1 mutation, and increased quantity of IGH or IGK clones during monitoring as unfavourable factors. Conclusion: In conclusion, this study highlights the usefulness of NGS-based MRD as a routine assessment tool for prognostication of paediatric patients with B-ALL. [ABSTRACT FROM AUTHOR]

Introduction: Acute myeloid leukemia (AML) is a complex hematologic malignancy characterized by uncontrolled proliferation of myeloid precursor cells within bone marrow. Despite advances in understanding of its molecular underpinnings, AML remains a therapeutic challenge due to its high relapse rate and clonal evolution. Methods: In this retrospective study, we analyzed data from 24 AML patients diagnosed at a single institution between January 2017 and August 2023. Comprehensive genetic analyses, including chromosomal karyotyping, next-generation sequencing, and gene fusion assays, were performed on bone marrow samples obtained at initial diagnosis and relapse. Clinical data, treatment regimens, and patient outcomes were also documented. Results: Mutations in core genes of FLT3, NPM1, DNMT3A, and IDH2 were frequently discovered in diagnostic sample and remained in relapse sample. FLT3-ITD, TP53, KIT, RUNX1, and WT1 mutation were acquired at relapse in one patient each. Gene fusion assays revealed stable patterns, while chromosomal karyotype analyses indicated a greater diversity of mutations in relapsed patients. Clonal evolution patterns varied, with some cases showing linear or branching evolution and others exhibiting no substantial change in core mutations between diagnosis and relapse. Conclusions: Our study integrates karyotype, gene rearrangements, and gene mutation results to provide a further understanding of AML heterogeneity and evolution. We demonstrate the clinical relevance of specific mutations and clonal evolution patterns, emphasizing the need for personalized therapies and measurable residual disease monitoring in AML management. By bridging the gap between genetics and clinical outcome, we move closer to tailored AML therapies and improved patient prognoses. [ABSTRACT FROM AUTHOR]

Background: BCR::ABL1 fusion has significant prognostic value and is screened for chronic myeloid leukemia (CML) disease monitoring as a part of routine molecular testing. To overcome the limitations of the current standard real-time quantitative polymerase chain reaction (RQ-PCR), we designed and validated a next-generation sequencing (NGS)-based assay to quantify BCR::ABL1 and ABL1 transcript copy numbers. Methods: After PCR amplification of the target sequence, deep sequencing was performed using an Illumina Nextseq 550Dx sequencer and in-house–designed bioinformatics pipeline. The Next-generation Quantitative sequencing (NQ-seq) assay was validated for its analytical performance, including precision, linearity, and limit of detection, using serially diluted control materials. A comparison with conventional RQ-PCR was performed with 145 clinical samples from 77 patients. Results: The limit of detection of the NQ-seq was the molecular response (MR) 5.6 [BCR::ABL1 0.00028% international scale (IS)]. The NQ-seq exhibited excellent precision and linear range from MR 2.0 to 5.0. The IS value from the NQ-seq was highly correlated with conventional RQ-PCR. Conclusions: We conclude that the NQ-seq is an effective tool for monitoring BCR::ABL1 transcripts in CML patients with high sensitivity and reliability. Prospective assessment of the unselected large series is required to validate the clinical impact of this NGS-based monitoring strategy. [ABSTRACT FROM AUTHOR]

Background: We intended to evaluate diagnostic utility of a targeted gene sequencing by using next generation sequencing (NGS) panel in patients with intractable early-onset epilepsy (EOE) and find the efficient analytical step for increasing the diagnosis rate. Methods: We assessed 74 patients with EOE whose seizures started before 3 years of age using a customized NGS panel that included 172 genes. Single nucleotide variants (SNVs) and exonic and chromosomal copy number variations (CNVs) were intensively examined with our customized pipeline and crosschecked with commercial or pre-built software. Variants were filtered and prioritized by in-depth clinical review, and finally classified according to the American College of Medical Genetics and Genomics guidelines. Each case was further discussed in a monthly consensus meeting that included the participation of all laboratory personnel, bioinformaticians, geneticists, and clinicians. Results: The NGS panel identified 28 patients (37.8%) with genetic abnormalities; 25 patients had pathogenic or likely pathogenic SNVs in 17 genes including SXTBP1 (n = 3), CDKL5 (n = 2), KCNQ2 (n = 2), SCN1A (n = 2), SYNGAP1 (n = 2), GNAO1 (n = 2), KCNT1 (n = 2), BRAT1, WWOX, ZEB2, CHD2, PRICKLE2, COL4A1, DNM1, SCN8A, MECP2, SLC9A6 (n = 1). The other 3 patients had pathogenic CNVs (2 duplications and 1 deletion) with varying sizes (from 2.5 Mb to 12 Mb). The overall diagnostic yield was 37.8% after following our step-by-step approach for clinical consensus. Conclusions: NGS is a useful diagnostic tool with great utility for patients with EOE. Diagnostic yields can be maximized with a standardized and team-based approach. [ABSTRACT FROM AUTHOR]

Background: We evaluated the incidence and spectrum of pathogenic and likely pathogenic variants of cancer susceptibility genes in BRCA1/2 mutation-negative Korean patients with a high risk for hereditary breast cancer using a comprehensive multigene panel that included 35 cancer susceptibility genes.Methods: Samples from 120 patients who were negative for BRCA1/2 mutations, but had been diagnosed with breast cancer that was likely hereditary, were prospectively evaluated for the prevalence of high-penetrance and moderate-penetrance germline mutations.Results: Nine patients (7.5%) had at least one pathogenic or likely pathogenic variant. Ten variants were identified in these patients: TP53 in two patients, PALB2 in three patients, BARD1 in two patients, BRIP1 in two patients, and MRE11A in one patient. We also identified 30 types of 139 variants of unknown significance (VUS). High-penetrance germline mutations, including TP53 and PALB2, tended to occur with high frequency in young (< 35 years) breast cancer patients (4/19, 21.1%) than in those diagnosed with breast cancer at ≥35 years of age (1/101, 1.0%; p = 0.003).Conclusions: These combined results demonstrate that multigene panels offer an alternative strategy for identifying veiled pathogenic and likely pathogenic mutations in breast cancer susceptibility genes. [ABSTRACT FROM AUTHOR]