BACKGROUND:Measurable residual disease (MRD) is a key prognostic marker in adult B-cell precursor acute lymphoblastic leukemia (B-ALL), yet inter-assay discordance may complicate MRD-guided decisions, particularly around allogeneic hematopoietic cell transplantation (allo-HCT). METHODS:We retrospectively assessed MRD by multiparametric flow cytometry (MFC), next-generation sequencing (NGS), and BCR::ABL1 quantitative reverse transcription PCR (RT-qPCR) in 182 adults with B-ALL, generating 646 paired MRD samples for inter-method comparison. Clinical outcome analyses were performed in 100 patients with serial MRD assessments after remission induction (TP1), after consolidation (TP2), and at pre-HCT. RESULTS:MRD levels correlated strongly across methods, with the highest concordance between MFC and NGS. Discordance was frequent at deep response levels, most prominently in BCR::ABL1-positive disease, where RT-qPCR often remained positive despite negative MFC/NGS, consistent with multilineage or CML-like features. MRD measured after TP2 was predictive of overall survival outcomes. Pre-HCT poor molecular response (>0.1%) was associated with the worst survival despite allo-HCT. Major molecular response (detectable MRD < 0.1%) showed outcomes comparable to complete molecular response (CMR; undetectable MRD at assay-specific sensitivity limits) by MFC or NGS. In contrast, pre-HCT MRD assessment by RT-qPCR did not significantly discriminate survival outcomes under contemporary preemptive tyrosine kinase inhibitor strategies. Importantly, across both BCR::ABL1-positive and -negative ALL, discordant CMR characterized by NGS positivity despite negative RT-qPCR or MFC consistently showed inferior survival, indicating greater clinical relevance, whereas discordant negativity by other assays may not reflect durable remission. CONCLUSIONS:Integrated MRD assessment, with focused interpretation of discordant results, may improve risk stratification and optimization of MRD-guided strategies in adult B-ALL.

Background:In the 5th edition of the WHO Classification of Hematopoietic Neoplasms (WHO-HAEM5) and the International Consensus Classification (ICC), acute myeloid leukemia (AML) diagnostic criteria were revised to incorporate genetic abnormalities, such as variants in myelodysplasia-related (MR) genes. As the implications of these changes on AML reclassification in Korean patients had not yet been fully evaluated, we comprehensively investigated them in this large-scale, multicenter study. Methods:We retrospectively analyzed data from 2,668 patients with AML aged ≥ 18 yrs from seven institutions who were originally diagnosed according to WHO-HAEM4R criteria. Clinical, cytogenetic, and targeted next-generation sequencing data (including MR genes and TP53 variants) were collected and analyzed. All cases were reassessed according to both WHO-HAEM5 and ICC criteria. Results:Overall, 28.5% of patients harbored at least one MR gene variant. Among the MR genes, variants in RUNX1 and ASXL1 were most frequently detected. Compared with the original diagnoses, 20.3% and 29.9% of patients were reclassified to different categories according to the WHO-HAEM5 and ICC criteria, respectively. Discordance between WHO-HAEM5 and ICC classifications was 13.2%, which is largely attributed to the inclusion of "AMLs with TP53" variants in the ICC. AML-MR was associated with older age, male predominance, and poorer survival compared with AML, not otherwise specified. Patients with recurrent fusion genes largely retained their original classification. Conclusions:Incorporating MR gene variant data and TP53-variant status in diagnosis influenced AML classification and risk stratification in our cohort, highlighting the potential importance of comprehensive molecular profiling in AML characterization.

B-lymphoblastic leukemia with BCR::ABL1-like features (BCR::ABL1-like ALL) is a high-risk subtype of B-ALL driven by diverse kinase-activating genetic alterations. Accurate detection of these lesions is essential for molecular classification and guiding targeted therapies. We evaluated multiple RNA-based platforms to establish an optimized diagnostic strategy in a cohort enriched for suspected BCR::ABL1-like ALL.Among 340 newly diagnosed B-ALL patients, 33 harbored fluorescence in situ hybridization (FISH)-detected kinase gene abnormalities. Eighteen patients with sufficient RNA were included for multiplex RT-PCR, a targeted RNA panel, and whole-transcriptome RNA sequencing (WTS). WTS data were analyzed using two fusion workflows—DRAGEN and Arriba. Transcriptome profiles were classified using ALLCatchR based on TPM expression matrices.Across all platforms, 20 distinct genetic lesions were identified. Multiplex RT-PCR detected 4 events (20%), reflecting its restriction to primer-covered targets. The targeted RNA panel identified 9 lesions (45%); however, it showed panel-dependent limitations, including misclassification in 5 cases (28%) when partner genes were not included in the assay design. WTS captured 17 genetic lesions (85%) when DRAGEN and Arriba results were combined enabled recognition of both known and novel rearrangements. ALLCatchR assigned 14 of the 18 analyzable cases (78%) to the BCR::ABL1-like subtype, providing subtype resolution beyond structural variant identification.WTS, combined with robust fusion pipelines and transcriptomic classification, delivers the most comprehensive assessment of kinase-activating lesions in BCR::ABL1-like ALL. These results support optimized strategy in which WTS is prioritized for cases not fully resolved by FISH or targeted RNA panels, enabling more accurate molecular classification and guiding targeted therapy.

BACKGROUND:Polycythemia vera (PV) is characterized by the abnormal proliferation of red blood cells (RBCs). Thrombosis and associated cardiovascular diseases are leading causes of mortality in patients with PV. This study aimed to investigate the association of Lutheran/BCAM (CD239) and other RBC antigens with thrombosis in PV. MATERIALS AND METHODS:This single-center, prospective study consecutively enrolled 50 PV patients, 39 with secondary polycythemia, and 20 healthy controls (HC) who visited the apheresis unit for phlebotomy between May 2022 and September 2023. The normalized expression levels of Lutheran/BCAM (CD239), Indian (CD44), LW/ ICAM (CD242), and Rh-related integrin-associated protein (IAP, CD47) antigens were assessed by flow cytometry. JAK2V617F expression was quantified and coagulation parameters were analyzed. Laboratory and clinical data were retrieved from the medical records. RESULTS:PV patients exhibited significantly higher mean fluorescence intensity (MFI) for Lutheran/BCAM: 45.2 ± 32.8 vs. 33.0 ± 14.4, p = 0.047, Indian (CD44): 13.5 ± 18.4 vs. 8.6 ± 1.1, p = 0.195, and IAP (CD47): 604.8 ± 193.2 vs. 514.9 ± 63.2, p = 0.036 compared to HC. The Indian (CD44) antigen was identified as a risk factor for thrombosis with an odds ratio (OR) of 1.359 (95% confidence interval [CI]: 1.003 - 1.842) in a multivariable model. Positive JAK2 measurable residual disease (JAK2-MRD) (expression was detected in 100% (25/25) of PV patients assessed, with a median variant allele frequency of 51.8% (95% CI: 45.4 - 65.0%). CONCLUSIONS:Higher expression of Indian (CD44) MFI levels in RBCs were associated with thrombotic events in patients with PV. Assessing RBC Indian (CD44) expression may serve as a potential biomarker for thrombotic risk stratification and prevention in PV.

Pediatric bone marrow failure (BMF) comprises heterogeneous inherited and immune-mediated disorders, yet many cases remain genetically unclassified despite comprehensive genomic evaluation. To identify shared transcriptional features beyond protein-coding variants, we performed total RNA sequencing on bone marrow samples from pediatric patients with BMF (n=12) and controls (n=2). Based on whole-genome sequencing, patients were stratified into genetically defined BMF (g-BMF, n=4) and genetically undefined BMF (u-BMF, n=8). Comparative transcriptomic analyses revealed minimal differences in protein-coding genes and long non-coding RNAs (lncRNAs) between g-BMF and u-BMF, indicating strong transcriptional concordance. Compared with controls, 55.2% of differentially expressed protein-coding genes were shared between g-BMF and u-BMF and were predominantly upregulated. Pathway enrichment analyses consistently identified immune activation and cellular stress-response pathways, suggesting a shared inflammatory transcriptional state across pediatric BMF irrespective of genetic classification. lncRNA profiling showed that 69.6% of differentially expressed lncRNAs were common to both BMF groups. ATP1A1-AS1, USP3-AS1, and SNHG32 were reproducibly overexpressed and validated by real-time quantitative polymerase chain reaction. Correlation-based pathway analyses associated ATP1A1-AS1 and USP3-AS1 with immune-related and biosynthetic programs, whereas SNHG32 showed a distinct co-expression pattern. Collectively, these findings suggest shared coding and non-coding transcriptomic patterns in pediatric BMF that appear largely independent of genetic classification within this cohort.

Here we systematically investigated genomic alterations from the initiation of induced pluripotent stem (iPS) cell generation to induced mesenchymal stromal/stem cell differentiation. We observed a total of ten copy number alterations (CNAs) and five single-nucleotide variations (SNVs) during the phases of reprogramming, differentiation and passaging. We identified a higher frequency of CNAs and SNVs in iPS cells generated using the Sendai virus (SV) method compared with those generated with episomal vectors (Epi). Specifically, all SV-iPS cell lines exhibited CNAs during the reprogramming phase, while only 40% of Epi-iPS cells showed such alterations. Additionally, SNVs were observed exclusively in SV-derived cells during passaging and differentiation, with no SNVs detected in Epi-derived lines. Gene expression analysis revealed upregulation of chromosomal instability-related genes in late-passage SV-iPSCs, further indicating increased genomic instability. Notably, TP53 mutations were identified, underscoring the vulnerability of the gene and the critical need for careful genomic scrutiny when preparing iPS cells and derived cell lines.

In this study, we present the development of the Mutation tagging by CRISPR-based Ultra-precise Targeted Elimination in Sequencing (MUTE-Seq) method. We engineered a highly precise advanced-fidelity FnCas9 variant, named FnCas9-AF2, to effectively discriminate single-base mismatches at all positions of the single guide RNA (sgRNA) target sequences. FnCas9-AF2 exhibited significantly lower off-target effects compared to existing high-fidelity CRISPR-Cas9 variants. MUTE-Seq leverages FnCas9-AF2 for the enrichment of mutant DNA through the exclusive cleavage of perfectly matched wild-type DNA, allowing for sensitive detection of low-frequency cancer-associated mutant alleles. MUTE-Seq enabled sensitive monitoring of minimal residual disease (MRD) from the bone marrow of patients with Acute Myeloid Leukemia (AML). Furthermore, MUTE-Seq was applied in a multiplexed manner on cell-free DNA (cfDNA) from patients diagnosed with non-small cell lung cancer (NSCLC) and pancreatic cancer. This approach demonstrated a significant improvement in the sensitivity of simultaneous mutant detection and highlighted its clinical utility for early-stage cancer patients with extremely low levels of circulating tumor DNA (ctDNA). We anticipate that the FnCas9-AF2-based MUTE-Seq could offer a valuable clinical tool to facilitate improved molecular diagnosis, prognosis evaluation, and treatment planning for cancers in various stages.

Microbial contamination of platelet concentrates (PC) remains a persistent challenge in transfusion medicine, necessitating robust preventive measures prior to product release. Differentially expressed gene (DEG) analysis of microbe inoculated PC offers a promising approach to identifying potential biomarkers for contamination detection. Within PC, each S. aureus (ATCC 29213) and S. epidermidis (ATCC 12228) was inoculated in a 103 CFU/mL concentration. Total RNA was extracted from the samples at predetermined time points (0-, 1-, 3-, and 6-hours post-inoculation), followed by high-throughput RNA sequencing. DEG, gene enrichment, and pathway analysis were conducted. Diagnostic potential was evaluated through the calculation of area under the curve (AUC) values and the assessment of additional performance metrics. DEG identified 5884 and 974 DEGs in S. aureus and S. epidermidis samples, respectively. Pathway analysis revealed distinct biological responses: S. aureus-inoculated samples showed prominent enrichment in ribosomal and spliceosome pathways, while S. epidermidis-inoculated samples demonstrated significant activation of mitogen-activated protein kinase (MAPK) signaling pathways and natural killer (NK) cell-mediated cytotoxicity pathways. ROC analysis of the commonly differentially expressed genes in both S. aureus and S. epidermidis-inoculated samples demonstrated significant diagnostic potential. The genes H19, CAVIN1, A2M, and EPAS1 exhibited statistically significant adjusted p-values and AUC values exceeding 0.8, with the exception of the H19 gene in S. epidermidis, suggesting their utility as potential biomarkers for staphylococcal contamination detection. Interaction between PC and microbial contaminants resulted in DEG and genes could be analyzed for microbial contamination of PC. However, to establish the robustness and broader applicability of these findings, further studies encompassing a more diverse range of microbial species are necessary.

Background: Data on human leukocyte antigen (HLA) typing using modern high-resolution methods are increasingly needed. Previous HLA studies in Korea were limited by small sample sizes or low-resolution data. We analyzed high-resolution (eight-digit) HLA data obtained through next-generation sequencing from healthy volunteers from Korea registered for hematopoietic stem cell transplantation, providing a large-scale dataset for population-based genetic analysis. Methods: We comprehensively analyzed HLA data from 2,726 volunteers from Korea. HLA-A,-B,-C, and-DRB1 alleles were identified using a GenDX NGSgo kit. Allele frequency (AF), haplotype frequency (HF), and equilibrium status were analyzed using the PyPop software. Principal component, Euclidean distance, and Nei's standard genetic distance analyses were performed using R software to compare data from Korea with that from other East Asian populations. Results: AF analysis identified 222 alleles (52 HLA-A, 70 HLA-B, 59 HLA-C, and 41 HLA-DRB1); HLA-A *24:02:01:01, HLA-B*15:01:01:01, HLA-C*01:02:01:01, and HLA-DRB1*09:01:02:01 were the most common. HF analysis revealed 632 haplotypes in HLA class I, and 2,848 in combined classes I and II; HLA-A*33:03:01:01,B*44:03:01:0 1,C*14:03:01:01 and HLA-A*33:03:01:01,B*44:03:01:01,C*14:03:01:01, DRB1*13:02:01:02 were the most common. Principal component and Euclidean distance analyses revealed genetic similarities among the individuals from Korea, Japan, and China. Conclusions: This study revealed key population genetic trends and relationships with East Asian neighbors, reflecting both shared and distinctive HLA characteristics. These data can provide valuable insights for anthropological and clinicopathological studies, as well as for understanding genetic diversity within the East Asian region.

Background. TP53 mutations are associated with poor prognosis in myelodysplastic neoplasm (MDS) and AML. The updated 5th WHO classification and International Consensus Classification (ICC) categorize TP53-mutated MDS and AML as unique entities. We conducted a multicenter study in Korea to investigate the characteristics of TP53-mutated MDS and AML, focusing on diagnostic aspects based on updated classifications. Methods. This study included patients aged >= 18 yrs who were diagnosed as having MDS (N=1,244) or AML (N=2,115) at six institutions. The results of bone marrow examination, cytogenetic studies, and targeted next-generation sequencing, including TP53, were collected and analyzed. Results. TP53 mutations were detected in 9.3% and 9.2% of patients with MDS and AML, respectively. Missense mutation was the most common, with hotspot codons R248/R273/G245/Y220/R175/C238 accounting for 25.4% of TP53 mutations. Ten percent of patients had multiple TP53 mutations, and 78.4% had a complex karyotype. The median variant allele frequency (VAF) of TP53 mutations was 41.5%, with a notable difference according to the presence of a complex karyotype. According to the 5th WHO classification and ICC, the multi-hit TP53 mutation criteria were met in 58.6% and 75% of MDS patients, respectively, and the primary determinants were a TP53 VAF >50% for the 5th WHO classification and the presence of a complex karyotype for the ICC. Conclusions. Collectively, we elucidated the molecular genetic characteristics of patients with TP53-mutated MDS and AML, highlighting key factors in applying TP53 mutation-related criteria in updated classifications, which will aid in establishing diagnostic strategies.

Multiple myeloma (MM), a hematological malignancy, is characterized by malignant plasma cell proliferation in the bone marrow. Recent treatment advances have significantly improved patient outcomes associated with MM. In this study, we aimed to develop comprehensive, evidence-based guidelines for the diagnosis, prognosis, and treatment of MM. We identified 12 key clinical questions essential for MM management, guiding the extensive literature review and meta-analysis of the study. Our guidelines provide evidence-based recommendations by integrating patient preferences with survey data. These recommendations include current and emerging diagnostic tools, therapeutic agents, and treatment strategies. By prioritizing a patient-centered approach and rigorous data analysis, these guidelines were developed to enhance MM management, both in Korea and globally.

BACKGROUND:Tisagenlecleucel (tisa-cel), a CD19-targeted chimeric antigen receptor T-cell (CAR-T) therapy, is an approved treatment for relapsed or refractory (R/R) diffuse large B-cell lymphoma (DLBCL) and B-cell precursor acute lymphoblastic leukemia (B-ALL). While pivotal trials demonstrated its efficacy, real-world evidence from Asian populations remains scarce. In particular, data on manufacturing feasibility, clinical outcomes, and predictive biomarkers in Korean patients are limited. OBJECTIVE:This study aimed to evaluate the clinical outcomes and prognostic factors of tisa-cel therapy in Korean patients with R/R DLBCL and B-ALL, with a specific focus on apheresis product quality and baseline immune markers. STUDY DESIGN:A single-center retrospective study was conducted including 91 patients (DLBCL = 72; B-ALL = 19) intended to receive tisa-cel between April 2022 and April 2024. Apheresis data, treatment responses, survival outcomes, and toxicity profiles were analyzed. The intention-to-treat (ITT) cohort was defined from the time of apheresis. Cox regression was used to identify predictors of survival and relapse, and separate analyses were performed for DLBCL and B-ALL. RESULTS:Out of 91 patients, 72 received tisa-cel infusion. One-year overall survival (OS) and disease-free survival (DFS) for the entire ITT cohort were 45.9% and 37.5%, respectively. In 19 B-ALL after tisa-cel infusion, 1-year OS and DFS were 68.4% and 52.6%, with no non-relapse mortality (NRM). In 53 DLBCL after tisa-cel infusion, 1-year OS and DFS were 46.8% and 41.6%, respectively, with 29.6% cumulative incidence of relapse among responders. NRM was 9.5% mostly observed in elderly patients. In the DLBCL subgroup, poor OS was independently associated with third-or-later-line salvage therapy (hazard ratio [HR] 2.39), high peripheral blood monocyte proportion at apheresis (>10%, HR 2.57), low CD3+ T-cell concentration in apheresis product (<1.0×10⁸/mL, HR 4.31), and occurrence of immune effector cell-associated neurotoxicity syndrome (HR 2.47). B-ALL patients demonstrated favorable survival and lower toxicity, but no significant prognostic markers were identified. CONCLUSIONS:Tisa-cel therapy was feasible and effective in Korean patients with R/R B-cell malignancies. Pre-treatment immune parameters such as monocyte burden and CD3+ T-cell quality in apheresis products significantly influenced outcomes in DLBCL. These findings suggest that optimizing patient selection and refining manufacturing strategies may enhance CAR-T efficacy, particularly in resource-constrained or heterogeneous real-world settings.

Background: Aplastic anemia (AA) is a rare bone marrow failure syndrome characterized by notably short telomere length, which is associated with treatment responses. In this study, we measured telomere lengths in Korean pediatric AA patients using flow-fluorescence in situ hybridization (Flow-FISH) and explored their shortening in relation to disease characteristics, genetic conditions and patient outcomes. Methods: We analyzed peripheral blood samples from 75 AA patients and 101 healthy controls. Telomere lengths were measured using Flow-FISH, and relative telomere length (RTL) and delta RTL assessments were conducted. Genetic evaluations included karyotyping, chromosome breakage tests and clinical exome sequencing (CES) to identify inherited bone marrow failure syndrome (IBMFS)-associated genetic variants. Results: Telomere lengths in AA patients were significantly lower than those of age-adjusted healthy controls. Patients receiving immunosuppressive therapy tended to have long telomeres, as indicated by high delta RTL values. Patients with genetic abnormalities, including karyotype abnormalities (n = 2) and genetic variants (n = 11) such as carrier genes of IBMFS or variants of unclear significance, showed significantly short telomere lengths. Conclusions: This study reinforces the importance of telomere length as a biomarker in acquired AA. Utilizing Flow-FISH, we were able to accurately measure telomere lengths and establish confidence in this method as an appropriate laboratory test. We found significant reduction in telomere lengths in AA patients, and importantly, longer telomeres were correlated with better outcomes in immunosuppressive therapy. Additionally, our genetic analysis underscored the relevance of variants in IBMFS-associated genes to the pathophysiology of short telomeres.

ABSTRACT 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.

Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is a potential cure for myelodysplastic neoplasms (MDSs) and other hematologic malignancies. This study investigates post-transplantation genetic evolution and telomere dynamics in hematopoietic cells, with a focus on clonal hematopoiesis (CH). We conducted a longitudinal analysis of 21 MDS patients who underwent allo-HSCT between September 2009 and February 2015. Genetic profiles of hematopoietic cells from both recipients and donors were compared at equivalent pre- and post-transplantation time points. Targeted sequencing identified CH-associated mutations, and real-time quantitative PCR measured telomere length. Furthermore, we compared CH incidence between recipients and age-matched controls from the GENIE cohort from routine health checkups. Post-allo-HSCT, 38% of recipients developed somatic mutations not detected before transplantation, indicating de novo CH originating from donor cells. Compared to age-matched healthy controls, recipients showed a significantly higher incidence of CH, suggesting increased susceptibility to genetic changes post-transplant. Telomere length analysis also revealed accelerated shortening in transplanted cells, highlighting the heightened stress and proliferation demands in the new microenvironment. Our findings reveal a notable incidence of donor-derived CH in allo-HSCT recipients, alongside significant telomere attrition. This suggests the potential influence of the marrow microenvironment on genetic and molecular changes in hematopoietic cells.

We evaluated the analytical performance of ID NOW™ COVID-19 2.0 assay versus conventional real-time reverse transcription-polymerase chain reaction (RT-PCR) using a total of 792 clinical samples from nasopharyngeal and oropharyngeal swabs, stored in frozen universal transport medium samples. Positive percent agreement (PPA) and negative percent agreement of ID NOW were 97.6 % and 100 %, respectively. The overall percent agreement between ID NOW and RT-PCR was 99.5 %. The PPA of ID NOW in detecting SARS-CoV-2 in 164 RT-PCR positive patients, all of whom had symptoms related COVID-19, was 97.1 % within 8 days since symptom onset, 97.9 % from 8 to 14 days since symptom onset, and 97.6 % after 14 days since symptom onset, with no significant difference between the days since symptom onset. The ID NOW assay demonstrated good performance, providing a rapid and randomly accessible alternative to conventional RT-PCR for timely SARS-CoV-2 detection, particularly in situations requiring rapid results for patient care.

Introduction: Measurable residual disease (MRD) is the strongest independent prognostic factor in B lymphoblastic leukemia (B-ALL). The methods used for MRD monitoring include multiparametric flow cytometry (MFC), next-generation sequencing (NGS) of immunoglobulin gene rearrangements, and quantitative PCR (qPCR). In this study, we aim to evaluate the concordance between these three well-defined MRD assessment methods to better characterize the biology of MRD in adult B-ALL patients. Method: This study involved adult patients aged 19 or older with B-ALL, treated with modified hyper-CVAD (hyperfractionated cyclophosphamide, vincristine, doxorubicin, and dexamethasone) and allogeneic hematopoietic stem cell transplant (allogeneic-HSCT) at Catholic Hematology Hospital from May 2022 to June 2024. A total of 646 bone marrow aspirates were collected from 182 patients (96 Ph-negative ALL, 86 Ph-positive ALL) after remission induction [Time-point 1 (TP1), 4-6 weeks], after first [Time-point 2 (TP2), 10-12 weeks] and second consolidation [Time-point 3 (TP3), 16-20 weeks], and at 3-month interval after transplantation. Poor MRD response was defined as > 0.1%, and complete MRD response as < 0.001%, and poor MRD responders were treated with MRD-directed therapy using blinatumomab or next-generation tyrosine kinase inhibitors. MFC-MRD utilized DuraClone RE ALB tubes (Beckman Coulter, Marseille, France), complemented with additional antibodies (CD22, CD66c, CD73, CD123, and CD304). NGS-MRD was performed using the LymphoTrack® IGH and IGK assay panels (InVivoScribe, San Diego, CA). The qPCR-MRD employed the Real-Q BCR-ABL Quantification Kit (BioSewoom, Seoul, Republic of Korea). All three methods demonstrated a virtual sensitivity of 10-6. Results: The study showed strong correlation between the MRD assessment methods: MFC-MRD and NGS-MRD (r = 0.8459, P <0.0001), MFC-MRD and qPCR (r = 0.8470, P <0.0001), and NGS-MRD and qPCR (r = 0.8319, P <0.0001). At a 0.1% MRD threshold, the concordance rates were 94.3%, 85.3%, and 87.0%, respectively, while at a 0.01%, they were 89.6%, 78.6%, and 79.6%. A significant discordance was noted with 49 samples from 25 patients showing qPCR-MRD positivity but MFC and NGS negativity. Among these discordant cases, seven patients had more than three consecutive discordant results, two of which involved p210 (b2a2 and b3a2) and the others p190 (e1a2), suggesting the possibility of lymphoblastic phase of chronic myeloid leukemia (CML) or CML-like B-ALL. We also identified qPCR method could more detect poor MRD responders compared to MFC or NGS in Ph-positive ALL, but the detection power was similar between MFC and NGS in Ph-negative ALL. Our MRD-directed therapy suggested prognostically significant MRD time point was TP3 at 16-20 weeks after treatment, and poor MRD responders (> 0.1%) at TP3 by any detection methods showed significantly poor survival outcome even after allogeneic-HSCT in both Ph-negative and positive ALL. Major MRD responders (0.001%-0.1%) showed similar survival outcome compared to complete MRD responders (< 0.001%) at any time points. Conclusion: Our data suggested all MRD detection methods showed acceptable power and good concordance rates, but the detection power was different between Ph-positive and Ph-negative ALL. We also suggested MRD-directed therapeutic strategies might predict the significant time point of MRD for the prediction of survival outcomes.