Genomic insights into inherited bone marrow failure syndromes in a Korean population.

Inherited bone marrow failure syndromes (IBMFS) pose significant diagnostic challenges due to overlapping symptoms and variable expressivity, despite evolving genomic insights. The study aimed to elucidate the genomic landscape among 130 Korean patients with IBMFS. We conducted targeted next-generation sequencing (NGS) and clinical exome sequencing (CES) across the cohort, complemented by whole genome sequencing (WGS) and chromosomal microarray (CMA) in 12 and 47 cases, respectively, with negative initial results. Notably, 50% (n = 65) of our cohort achieved a genomic diagnosis. Among these, 35 patients exhibited mutations associated with classic IBMFSs (n = 33) and the recently defined IBMFS, aplastic anaemia, mental retardation and dwarfism syndrome (AmeDS, n = 2). Classic IBMFSs were predominantly detected via targeted NGS (85%, n = 28) and CES (88%, n = 29), whereas AMeDS was exclusively identified through CES. Both CMA and WGS aided in identifying copy number variations (n = 2) and mutations in previously unexplored regions (n = 2). Additionally, 30 patients were diagnosed with other congenital diseases, encompassing 13 distinct entities including inherited thrombocytopenia (n = 12), myeloid neoplasms with germline predisposition (n = 8), congenital immune disorders (n = 7) and miscellaneous genomic conditions (n = 3). CES was particularly effective in revealing these diverse diagnoses. Our findings underscore the significance of comprehensive genomic analysis in IBMFS, highlighting the need for ongoing exploration in this complex field.

Analytical Performance Evaluation of a Digital Real-Time PCR for Quantifying Major BCR::ABL1 Transcripts.

BACKGROUND: Accurate quantification of the BCR::ABL1 transcripts is essential for measurable residual disease (MRD) monitoring in chronic myeloid leukemia (CML) after tyrosine kinase inhibitor (TKI) treatment. This study evaluated the newly developed digital real-time PCR method, Dr. PCR, as an alternative reverse transcription-PCR (qRT-PCR) for MRD detection. METHODS: The performance of Dr. PCR was assessed using reference and clinical materials. Precision, linearity, and correlation with qRT-PCR were evaluated. MRD levels detected by Dr. PCR were compared with qRT-PCR, and practical advantages were investigated. RESULTS: Dr. PCR detected MRD up to 0.0032%IS (MR4.5) with excellent precision and linearity and showed a strong correlation with qRT-PCR results. Notably, Dr. PCR identified higher levels of MRD in 12.7% (29/229) of patients than qRT-PCR, including six cases of MR4, which is a critical level for TKI discontinuation. Dr. PCR also allowed for sufficient ABL1 copies in all cases, while qRT-PCR necessitated multiple repeat tests in 3.5% (8/229) of cases. CONCLUSION: Our study provides a body of evidence supporting the clinical application of Dr. PCR as a rapid and efficient method for assessing MRD in patients with CML under the current treatment regimen.

Impaired binding affinity of YTHDC1 with METTL3/METTL14 results in R-loop accumulation in myelodysplastic neoplasms with DDX41 mutation.

DEAD box helicase 41 (DDX41) mutations are the most prevalent predisposition to familial myelodysplastic syndrome (MDS). However, the precise roles of these variants in the pathogenesis of MDS have yet to be elucidated. Here, we discovered a novel mechanism by which DDX41 contributes to R-loop-induced DNA damage responses (DDR) in cooperation with the m6A-METTL complex (MAC) and YTHDC1 using DDX41 knockout (KO) and DDX41 knock-in (KI, R525H, Y259C) cell lines as well as primary samples from MDS patients. Compared to wild type (WT), DDX41 KO and KI led to increased levels of m6A RNA methylated R-loop. Interestingly, we found that DDX41 regulates m6A/R-loop levels by interacting with MAC components. Further, DDX41 promoted the recruitment of YTHDC1 to R-loops by promoting the binding between METTL3 and YTHDC1, which was dysregulated in DDX41-deficient cells, contributing to genomic instability. Collectively, we demonstrated that DDX41 plays a key role in the physiological control of R-loops in cooperation with MAC and YTHDC1. These findings provide novel insights into how defects in DDX41 influence MDS pathogenesis and suggest potential therapeutic targets for the treatment of MDS.

Prognostic value of European LeukemiaNet 2022 criteria and genomic clusters using machine learning in older adults with acute myeloid leukemia.

This study aimed to validate the new European Leukemia Net (ELN) 2022 criteria for genetic risk stratification in older adults with acute myeloid leukemia (AML) and to determine the most likely set of clusters of similar cytogenetic and mutation properties correlated with survival outcomes in three treatment groups: intensive chemotherapy (IC), hypomethylating agents (HMA) alone, and HMA plus venetoclax (HMA/VEN). The study included 279 patients (aged ≥60 years) who received IC (N=131), HMA (N=76), and HMA/VEN (N=72) between July 2017 and October 2021. No significant differences were observed in survival among the groups according to ELN 2022 risk stratification. Unsupervised hierarchical clustering analysis identified nine genomic clusters (C1-9) with varying survival outcomes depending on treatment type. For example, C4 (predominant for core binding factor-AML) displayed a favorable prognosis in the IC group, but not in the HMA or HMA/VEN groups. The HMA/VEN group had better outcomes than the HMA group in many clusters (C1, 2, 3, and 5); however, the addition of VEN to HMA or IC did not improve the survival outcomes compared with those of HMA alone in C7 and C9 (predominant for -5, del(5q), -7, -17/abn(17p), complex karyotypes, and mutated TP53). The study highlights the limitations of ELN genetic risk stratification in older adults with AML. It emphasizes the need for a more comprehensive approach that considers co-occurring somatic mutations to guide treatment selection in older adults with AML.

Shared decision-making in multiple sclerosis physical symptomatic care: a systematic review.

Background: Multiple sclerosis (MS) is a chronic autoimmune inflammatory, demyelinating, and neurodegenerative disease affecting young adults. People with MS are highly interested in engaging in physical symptom management and decision-making but are often not actively engaged in symptom management discussions. Research examining the benefit of shared decision-making in the management of physical MS symptoms is sparse. Objectives: This study aimed to identify and synthesize the evidence on the use of shared decision-making in physical MS symptom management. Design: This study is a systematic review of published evidence on the use of shared decision-making in physical MS symptom management. Data sources and methods: MEDLINE, CINAHL, EMBASE, and CENTRAL databases were searched in April 2021, June 2022, and April 2, 2023, for primary, peer-reviewed studies of shared decision-making in the management of MS physical symptoms. Citations were screened, data extracted, and study quality assessed according to Cochrane guidelines for systematic reviews, including risk of bias assessment. Statistical synthesis of the included study results was not appropriate; results were summarized in a nonstatistical manner using the vote-counting method to estimate beneficial versus harmful effects. Results: Of 679 citations, 15 studies met the inclusion criteria. Six studies addressed shared decision-making in the management of pain, spasms, neurogenic bladder, fatigue, gait disorder, and/or balance issues, and nine studies addressed physical symptoms in general. One study was a randomized controlled trial; most studies were observational studies. All study results and study author conclusions indicated that shared decision-making is important to the effective management of physical MS symptoms. No study results suggested that shared decision-making was harmful or delayed the management of physical MS symptoms. Conclusion: Reported results consistently indicate that shared decision-making is important in effective MS symptomatic care. Further rigorous randomized controlled trials are warranted to investigate the effectiveness of shared decision-making associated with MS physical symptomatic care. Registration: PROSPERO: CRD42023396270.

Shared decision-making among people with Multiple Sclerosis (MS) and their healthcare providers in the management of the physical symptoms of MS. Shared decision-making is suggested to be a key mechanism in promoting optimal symptomatic care related to Multiple Sclerosis (MS). Shared decision-making is mostly done and studied in relation to choosing therapies that may slow disease progression but not usually for symptomatic care. There are a few studies highlighting the effect of utilizing shared decision-making in symptomatic care of MS. We performed this study to identify all the published data about using shared decision-making in symptomatic care in MS to answer the research question: What is the evidence on shared decision-making in managing physical MS symptoms? We performed a systematic search for all the related published study results in four large literature databases. We found 15 studies on the use of shared decision-making in the management of MS-related symptoms. We synthesized the study results relating to the use of shared decision-making in symptomatic care of MS. The studies used several different designs and included a wide range of study rigor and quality. The results of our systematic review are: All the studies were consistent in their conclusions that shared decision-making is important for effective MS-related symptom management.Several studies found that symptomatic care is of the highest priority to people with MS, but not often a priority to their health care providers.The use of a shared decision-making model can promote discussion of symptoms in clinical consultations and align the goals of people with MS and their health care providers.Education of people with MS regarding their symptoms and available treatments will promote effective shared decision-making discussions. The available evidence supports that the use of shared decision-making is beneficial to the management of physical symptoms of MS. Further studies using a randomized controlled study design are required to establish the degree of benefit of utilizing shared decision-making associated with MS symptomatic care.

Genetic and Clinical Characteristics of Korean Chronic Lymphocytic Leukemia Patients with High Frequencies of MYD88 Mutations.

Chronic lymphocytic leukemia (CLL) is the most common adult leukemia in Western countries. However, CLL is relatively rare in Asia; its genetic features are rarely studied. Here, we aimed to genetically characterize Korean CLL patients and to elucidate the genetic and clinical associations based on data obtained from 113 patients at a single Korean institute. We used next-generation sequencing to explore the multi-gene mutational data and immunoglobulin heavy chain variable gene clonality with somatic hypermutation (SHM). MYD88 (28.3%), including L265P (11.5%) and V217F (13.3%), was the most frequently mutated gene, followed by KMT2D (6.2%), NOTCH1 (5.3%), SF3B1 (5.3%), and TP53 (4.4%). MYD88-mutated CLL was characterized by SHM and atypical immunophenotype with fewer cytogenetic abnormalities. The 5-year time to treatment (TTT) of the overall cohort was 49.8% ± 8.2% (mean ± standard deviation) and the 5-year overall survival was 86.2% ± 5.8%. Patients with SHM, isolated del(13q), TP53-wild type, and NOTCH1-wild type showed better results than those without these conditions. In the subgroup analyses, patients with SHM and L265P presented shorter TTT than patients with SHM but not L265P. In contrast, V217F was associated with a higher SHM percentage and showed a favorable prognosis. Our study revealed the distinct characteristics of Korean CLL patients with high frequencies of MYD88 mutations and their clinical relevance.

FLT3-ITD Measurable Residual Disease Monitoring in Acute Myeloid Leukemia Using Next-Generation Sequencing.

The in-frame internal tandem duplication (ITD) of the FMS-like tyrosine kinase 3 (FLT3) gene is an important negative prognostic marker in acute myeloid leukemia (AML). FLT3-ITD monitoring is essential for patients at relapse or those receiving FLT3-targeted therapies. Fragment analysis (FA) is commonly used to detect and quantify FLT3-ITDs; however, detecting low-burden FLT3-ITDs after a treatment is challenging. We, therefore, developed a customized, next-generation sequencing (NGS)-based FLT3-ITD assay that includes a new ITD-tracing algorithm, "SEED", optimized for measurable residual disease (MRD) monitoring. NGS-SEED showed an enhanced sensitivity (0.001%) and has a superior performance over conventional fragment analysis. We further investigated the prognostic impact of MRD analyzed by NGS-SEED in AML patients who underwent allogeneic hematopoietic stem cell transplantation (HSCT). Our assay showed that the MRD assessed before and after HSCT were significantly associated with a risk of relapse and a poor overall survival, respectively, in a time-dependent analysis. Thus, this report highlighted the prognostic value of serial MRD monitoring using a sensitive method in a clinical setting of AML patients with FLT3-ITD.

Spectrum of Genetic Mutations in Korean Pediatric Acute Lymphoblastic Leukemia.

The wide application of next-generation sequencing (NGS) technologies has led to the discovery of multiple genetic alterations in pediatric acute lymphoblastic leukemia (ALL). In this work, we aimed to investigate the mutational spectrum in pediatric ALL. We employed a St. Mary's customized NGS panel comprising 67 leukemia-related genes. Samples were collected from 139 pediatric ALL patients. Eighty-five patients (61.2%) harbored at least one mutation. In B-cell ALL, the RAS pathway is the most involved pathway, and the three most frequently mutated genes were NRAS (22.4%), KRAS (19.6%), and PTPN11 (8.4%). NRAS and PTPN11 were significantly associated with a high hyperdiploidy karyotype (p = 0.018 and p < 0.001, respectively). In T-cell ALL, the three most frequently mutated genes were NOTCH1 (37.5%), FBXW7 (16.6%), and PTEN (6.2%). Several pairs of co-occurring mutations were found: NRAS with SETD, NRAS with PTPN11 in B-cell ALL (p = 0.024 and p = 0.020, respectively), and NOTCH1 with FBXW7 in T-cell ALL (p < 0.001). The most frequent newly emerged mutation in relapsed ALL was NT5C2. We procured comprehensive genetic information regarding Korean pediatric ALL using NGS technology. Our findings strengthen the current knowledge of recurrent somatic mutations in pediatric ALL.

Clinical implication of minimal residual disease assessment by next-generation sequencing-based immunoglobulin clonality assay in pediatric B-acute lymphoblastic leukemia.

Measuring minimal residual disease (MRD) during treatment is valuable to identify acute lymphoblastic leukemia (ALL) patients who require intensified treatment to avert relapse. We performed the next-generation sequencing (NGS)-based immunoglobulin gene (Ig) clonality assay and evaluated its clinical implication in pediatric B-ALL patients to assess MRD. Fifty-five patients who were diagnosed and treated with de novo (n = 44) or relapsed/refractory B-ALL (n = 11) were enrolled. MRD assessment was performed using the LymphoTrack® Dx IGH and IGK assay panels. The percentage of the clonal sequences per total read count was calculated as MRD (% of B cells). The data were normalized as the proportion of total nucleated cells (TNC) by LymphoQuant™ Internal control or the B-cell proportion in each sample estimated by flow cytometry or immunohistochemistry. Clonal Ig rearrangement was identified in all patients. The normalized MRD value was significantly lower than the unnormalized MRD value (p < 0.001). When categorizing patients, 27 of 50 patients (54%) achieved normalized MRD <0.01%, while 6 of them did not achieve MRD <0.01% when applying the unnormalized value. The normalized post-induction MRD value of 0.01% proved to be a significant threshold value for both 3-year event-free survival (100% for MRD <0.01% vs. 60.9% ± 10.2% for MRD ≥0.01%, p = 0.007) and 3-year overall survival (100% for MRD <0.01% vs. 78.3% ± 8.6% for MRD ≥0.01%, p = 0.011). However, unnormalized MRD was not a significant factor for outcome in this cohort. Our study demonstrated that MRD assessment by NGS-based Ig clonality assay could be applied in most pediatric B-ALL patients. Normalized post-induction MRD <0.01% was a significant prognostic indicator.

Clinical Features, Gene Alterations, and Outcomes in Prefibrotic and Overt Primary and Secondary Myelofibrotic Patients.

The Philadelphia-negative myeloproliferative neoplasms (MPNs) are divided in three major groups: polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF). The 2016 WHO classification incorporates also prefibrotic PMF (pre-PMF) and overt PMF. This study aimed to discriminate the clinical features, genetic alterations, and outcomes in patients with prefibrotic, overt PMF, and secondary MF (SMF). This study included 229 patients with diagnosed myelofibrosis (MF). Among 229 patients, 67 (29%), 122 (53%), and 40 (18%) were confirmed as SMF, overt PMF, and pre-PMF, respectively. The JAK2 V617F mutation was differentially distributed in SMF and PMF, contradictory to CALR and MPL mutations. Regarding nondriver mutations, the occurrence of ASXL1 mutations differed between PMF and SMF or pre-PMF. The three-year overall survival was 91.5%, 85.3%, and 94.8% in SMF, overt PMF, and pre-PMF groups. Various scoring systems could discriminate the overall survival in PMF but not in SMF and pre-PMF. Still, clinical features including anemia and thrombocytopenia were poor prognostic factors throughout the myelofibrosis, whereas mutations contributed differently. Molecular grouping by wild-type SF3B1 and SRSF2/RUNX1/U2AF1/ASXL1/TP53 mutations showed inferior progression-free survival (PFS) in PMF, SMF, and pre-PMF. We determined the clinical and genetic features related to poor prognosis in myelofibrosis.

Discontinuation of tyrosine kinase inhibitors based on BCR-ABL1 monitoring by digital droplet PCR in pediatric chronic myeloid leukemia.

Lifelong treatment of pediatric chronic myeloid leukemia (CML) patients with tyrosine kinase inhibitors (TKIs) can affect their growth and development. For these reasons, clinical trials have explored the feasibility of TKI discontinuation in children with a sufficient TKI response. We evaluated the analytical performance of digital droplet PCR (ddPCR) to quantify BCR-ABL1 and compared the results with reverse transcription quantitative polymerase chain reaction (RT-qPCR). We further investigated whether ddPCR could be used to determine TKI discontinuation in a clinical setting. Performance of ddPCR was evaluated using standard materials for BCR-ABL1, and a total of 197 clinical samples from 45 pediatric CML patients was included for comparison with RT-qPCR. ddPCR showed excellent analytical sensitivity with 0.001% international scale (IS) and linearity with R 2 > 0.99 in log scale. BCR-ABL1 % IS results correlated well with those of RT-qPCR (R 2 = 0.9435), however, they showed a moderate strength for agreement with a Cohen's kappa of 0.41 due to higher sensitivity of ddPCR. Among 45 pediatric CML patients, 42 were treated with first-line TKIs including imatinib (n = 27, 64%) and dasatinib (n = 12, 29%), and three patients that were started with imatinib were switched to dasatinib. When we evaluated whether follow-up samples fulfilled ABL1 copies ≥ 10,000 required for deep molecular response (DMR), all samples were acceptable by ddPCR, whereas 18% by RT-qPCR did not reached acceptable ABL1 copies. Moreover, 52 and 13% reached ABL1 copies ≥ 32,000 required for MR4.5 by ddPCR and RT-qPCR, respectively. Seven patients discontinued TKI and the median TKI treatment duration was 73 months prior to discontinuation. Prior to discontinuation, the median duration of sustained undetected BCR-ABL1 was 60 months. Two patients experienced loss of major MR (MMR) during follow-up and restarted dasatinib 5 months after discontinuation. They achieved MMR again and maintained better than DMR afterward. Results from those patients demonstrated that RT-qPCR did not match the need for adequate ABL1 copies for MR4.5 while majority of ddPCR could. Therefore, ddPCR was technically more acceptable to decide and monitor pediatric CML patients before and after TKI discontinuation.

Genetic Characteristics According to Subgroup of Acute Myeloid Leukemia with Myelodysplasia-Related Changes.

Acute myeloid leukemia with myelodysplasia-related changes (AML-MRC) includes heterogeneous conditions such as previous history and specific cytogenetic and morphological properties. In this study, we analyze genetic aberrations using an RNA-based next-generation sequencing (NGS) panel assay in 45 patients with AML-MRC and detect 4 gene fusions of KMT2A-SEPT9, KMT2A-ELL, NUP98-NSD1, and RUNX1-USP42 and 81 somatic mutations. Overall, all patients had genetic aberrations comprising of not only cytogenetic changes, but also gene fusions and mutations. We also demonstrated several characteristic genetic mutations according to the AML-MRC subgroup. TP53 was the most commonly mutated gene (n = 11, 24%) and all were found in the AML-MRC subgroup with myelodysplastic syndrome-defining cytogenetic abnormalities (AML-MRC-C) (p = 0.002). These patients showed extremely poor overall survival not only in AML-MRC, but also within the AML-MRC-C subgroup. The ASXL1 (n = 9, 20%) and SRSF2 (n = 7, 16%) mutations were associated with the AML-MRC subgroup with >50% dysplasia in at least two lineages (AML-MRC-M) and were frequently co-mutated (55%, 6/11, p < 0.001). Both mutations could be used as surrogate markers to diagnose AML-MRC, especially when the assessment of multilineage dysplasia was difficult. IDH1/IDH2 (n = 13, 29%) were most commonly mutated in AML-MRC, followed by CEBPA (n = 5, 11%), PTPN11 (n = 5, 11%), FLT3 (n = 4, 9%), IDH1 (n = 4, 9%), and RUNX1 (n = 4, 9%). These mutations were not limited in any AML-MRC subgroup and could have more significance as a risk factor or susceptibility marker for target therapy in not only AML-MRC, but also other AML categories.

Characteristics of RAS pathway mutations in juvenile myelomonocytic leukaemia: a single-institution study from Korea.

Juvenile myelomonocytic leukaemia (JMML), a rare clonal haematopoietic disorder of childhood, is characterised as a myelodysplastic/myeloproliferative neoplasm. Despite ground-breaking genetic discoveries, JMML remains difficult to diagnose given its diverse clinical features and disease course. A total of 24 patients with JMML were diagnosed and treated at a single institution, and their genetic profiles and association with clinical and laboratory characteristics were analysed. In all, 22 of the patients received allogeneic haematopoietic stem cell transplantation after myeloablative conditioning, mostly from a haploidentical family donor. RAS pathway mutations were identified in 88% of patients: PTPN11 [nine (38%)], NRAS [nine (38%)], KRAS [two (8%)], NF1 [five (21%)] and CBL [one (4%)]. Secondary mutations were found in 25% of patients: SETBP1, JAK3, ASXL1, GATA2, KIT, KDM6A, and BCOR. Six patients showed cytogenetic abnormalities, including three with monosomy 7. The estimated 5-year event-free survival (EFS) and overall survival (± standard error) of the entire cohort were 58·9 (10·9)% and 73·5 (10·8)% respectively. NRAS (+) patients had a higher 5-year EFS than NRAS (-) patients [72·9 (16·5)% vs. 52·5 (13·1)%, P = 0·127]. NRAS (+) patients had a better 5-year EFS than PTPN11 (+) patients [41·7 (17·3)%, P = 0·071]. Our study revealed the genetic characteristics of Korean JMML patients with RAS pathway and secondary mutations.