We explored the utility of cerebrospinal fluid (CSF) circulating tumor DNA (ctDNA) sequencing as a noninvasive diagnostic tool for detecting central nervous system (CNS) involvement in patients with diffuse large B-cell lymphoma (DLBCL). Secondary CNS involvement in DLBCL, although rare (~5% of cases), presents diagnostic and prognostic challenges during systemic disease progression or relapse. Effective treatment is impeded by the blood–brain barrier. This was a prospective cohort study (Samsung Lymphoma Cohort Study III) involving 17 patients with confirmed CNS involvement. High-throughput sequencing was conducted using targeted gene panels designed to detect low-frequency variants and copy number alterations pertinent to lymphomas in ctDNA extracted from archived CSF samples. Despite challenges such as low DNA concentrations affecting library construction, the overall variant detection rate was 76%. Detected variants included those in genes commonly implicated in CNS lymphoma, such as MYD88. The study highlights the potential of CSF ctDNA sequencing to identify CNS involvement in DLBCL, providing a promising alternative to more invasive diagnostic methods such as brain biopsy, which are not always feasible. Further validation is necessary to establish the clinical utility of this method, which could significantly enhance the management and outcomes of DLBCL patients with suspected CNS involvement.

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.

Background: Circulating tumor DNA (ctDNA) is a potential biomarker in pancreatic ductal adenocarcinoma (PDAC). However, studies on residual ctDNA in patients post-chemotherapy are limited. We assessed the prognostic value of residual ctDNA in metastatic PDAC relative to that of carbohydrate antigen 19-9 (CA19-9). Methods: ctDNA analysis using a targeted next-generation sequencing panel was performed at baseline and during chemotherapy response evaluation in 53 patients. Progression-free survival (PFS) and overall survival (OS) were first evaluated based on ctDNA positivity at baseline. For further comparison, patients testing ctDNA-positive at baseline were subdivided based on residual ctDNA into ctDNA responders (no residual ctDNA post-chemotherapy) and ctDNA non-responders (residual ctDNA post-chemotherapy). Additional survival analysis was performed based on CA19-9 levels. Results: The baseline ctDNA detection rate was 56.6%. Although clinical outcomes tended to be poorer in patients with baseline ctDNA positivity than in those without, the differences were not significant. Residual ctDNA post-chemotherapy was associated with reduced PFS and OS. The prognosis of ctDNA responders was better than that of non-responders but did not significantly differ from that of ctDNA-negative individuals (no ctDNA both at baseline and during post-chemotherapy). Compared with ctDNA responses to che-motherapy, a ≥ 50% decrease in the CA19-9 level had less effect on both PFS and OSbased on hazard ratios and significance levels. ctDNA could be monitored in half of the patients whose baseline CA19-9 levels were within the reference range. Conclusions Residual ctDNA analysis post-chemotherapy is a promising approach for predicting the clinical outcomes of patients with metastatic PDAC.

Background: FISH is the standard method for detecting cytogenetic abnormalities (CAs) in patients with multiple myeloma, and pre-enrichment of plasma cells is recommended to increase detection rates. However, optimal strategies to ensure sufficient plasma cell retrieval when standard enrichment techniques fail remain underexplored. We investigated factors influencing the success of fluorescence-activated cell sorting (FACS) and assessed the use of direct FISH in cases in which FACS failed. Methods: A retrospective analysis was conducted on 457 bone marrow samples submitted for FISH between November 2016 and May 2022. FACS was considered successful when plasma cells (CD38+ and CD138+ cells) constituted > 1% of the total number of cells. Direct FISH was performed for samples with FACS failure. Results: FACS was successful in 70.9% of cases and had a high positivity rate (94.8%).Shorter sample transfer times significantly improved FACS success, with a 77.1% success rate for transfer times < 2 hrs, compared with 67.8% for longer times (P = 0.0388). Plasma cell percentage was a strong determinant of FACS success, with a median of 31.2% in successful cases versus 8.5% in failures (P < 0.0001). Even when FACS failed, direct FISH detected CAs in 43.6% of cases. Conclusions Plasma cell percentage and sample transfer time are critical factors influencing FACS success. While FACS-FISH demonstrates superior sensitivity in detecting CAs, direct FISH serves as a valuable alternative when FACS fails. These findings highlight the importance of optimizing sample handling and FISH protocols for accurate cytogenetic analysis of multiple myeloma.

Background: Facioscapulohumeral muscular dystrophy (FSHD) is a common form of muscular dystrophy that mainly affects skeletal muscle. FSHD1 accounts for 95% of all FSHD cases and can be diagnosed based on the pathogenic contraction of the D4Z4-repeat array on chromosome 4q35. Genetic diagnosis of FSHD1 is challenging because of the large size and repetitive nature of the D4Z4 region. We evaluated the clinical applicability of optical genome mapping (OGM) for the genetic diagnosis of FSHD1. Methods: We included 25 individuals with clinically confirmed or suspected/probable FSHD and their families. Ultra-high-molecular-weight DNA from peripheral blood was labeled, stained, and imaged using a single-molecule OGM platform (Bionano Genomics Saphyr system). D4Z4 repeat size and haplotype information were analyzed using the manufacturer’s dedicated pipeline. We also compared the workflow and test time between Southern blot analysis and OGM. Results: We obtained concordant OGM and Southern blot results with 10 samples from patients with clinically confirmed FSHD. The D4Z4 repeat size differed within 1 unit between the Southern blot analysis and OGM. Among nine patients with clinically suspected or probable FSHD, six patients were confirmed to have pathogenic contractions by OGM.In our cohort, one de novo mosaic FSHD1 patient was successfully diagnosed with OGM.Moreover, OGM has a more straightforward and less time-consuming workflow than Southern blot analysis. Conclusions OGM enables accurate and reliable detection of pathogenic contraction of the D4Z4-repeat array and is a valuable tool for the genetic diagnosis of FSHD1.

Background: Facioscapulohumeral muscular dystrophy (FSHD) is a common form of muscular dystrophy that mainly affects skeletal muscle. FSHD1 accounts for 95% of all FSHD cases and can be diagnosed based on the pathogenic contraction of the D4Z4-repeat array on chromosome 4q35. Genetic diagnosis of FSHD1 is challenging because of the large size and repetitive nature of the D4Z4 region. We evaluated the clinical applicability of optical genome mapping (OGM) for the genetic diagnosis of FSHD1. Methods: We included 25 individuals with clinically confirmed or suspected/probable FSHD and their families. Ultra-high-molecular-weight DNA from peripheral blood was labeled, stained, and imaged using a single-molecule OGM platform (Bionano Genomics Saphyr system). D4Z4 repeat size and haplotype information were analyzed using the manufacturer’s dedicated pipeline. We also compared the workflow and test time between Southern blot analysis and OGM. Results: We obtained concordant OGM and Southern blot results with 10 samples from patients with clinically confirmed FSHD. The D4Z4 repeat size differed within 1 unit between the Southern blot analysis and OGM. Among nine patients with clinically suspected or probable FSHD, six patients were confirmed to have pathogenic contractions by OGM.In our cohort, one de novo mosaic FSHD1 patient was successfully diagnosed with OGM.Moreover, OGM has a more straightforward and less time-consuming workflow than Southern blot analysis. Conclusions OGM enables accurate and reliable detection of pathogenic contraction of the D4Z4-repeat array and is a valuable tool for the genetic diagnosis of FSHD1.

Background: Facioscapulohumeral muscular dystrophy (FSHD) is a common form of muscular dystrophy that mainly affects skeletal muscle. FSHD1 accounts for 95% of all FSHD cases and can be diagnosed based on the pathogenic contraction of the D4Z4-repeat array on chromosome 4q35. Genetic diagnosis of FSHD1 is challenging because of the large size and repetitive nature of the D4Z4 region. We evaluated the clinical applicability of optical genome mapping (OGM) for the genetic diagnosis of FSHD1. Methods: We included 25 individuals with clinically confirmed or suspected/probable FSHD and their families. Ultra-high-molecular-weight DNA from peripheral blood was labeled, stained, and imaged using a single-molecule OGM platform (Bionano Genomics Saphyr system). D4Z4 repeat size and haplotype information were analyzed using the manufacturer’s dedicated pipeline. We also compared the workflow and test time between Southern blot analysis and OGM. Results: We obtained concordant OGM and Southern blot results with 10 samples from patients with clinically confirmed FSHD. The D4Z4 repeat size differed within 1 unit between the Southern blot analysis and OGM. Among nine patients with clinically suspected or probable FSHD, six patients were confirmed to have pathogenic contractions by OGM.In our cohort, one de novo mosaic FSHD1 patient was successfully diagnosed with OGM.Moreover, OGM has a more straightforward and less time-consuming workflow than Southern blot analysis. Conclusions OGM enables accurate and reliable detection of pathogenic contraction of the D4Z4-repeat array and is a valuable tool for the genetic diagnosis of FSHD1.

Background: Facioscapulohumeral muscular dystrophy (FSHD) is a common form of muscular dystrophy that mainly affects skeletal muscle. FSHD1 accounts for 95% of all FSHD cases and can be diagnosed based on the pathogenic contraction of the D4Z4-repeat array on chromosome 4q35. Genetic diagnosis of FSHD1 is challenging because of the large size and repetitive nature of the D4Z4 region. We evaluated the clinical applicability of optical genome mapping (OGM) for the genetic diagnosis of FSHD1. Methods: We included 25 individuals with clinically confirmed or suspected/probable FSHD and their families. Ultra-high-molecular-weight DNA from peripheral blood was labeled, stained, and imaged using a single-molecule OGM platform (Bionano Genomics Saphyr system). D4Z4 repeat size and haplotype information were analyzed using the manufacturer’s dedicated pipeline. We also compared the workflow and test time between Southern blot analysis and OGM. Results: We obtained concordant OGM and Southern blot results with 10 samples from patients with clinically confirmed FSHD. The D4Z4 repeat size differed within 1 unit between the Southern blot analysis and OGM. Among nine patients with clinically suspected or probable FSHD, six patients were confirmed to have pathogenic contractions by OGM.In our cohort, one de novo mosaic FSHD1 patient was successfully diagnosed with OGM.Moreover, OGM has a more straightforward and less time-consuming workflow than Southern blot analysis. Conclusions OGM enables accurate and reliable detection of pathogenic contraction of the D4Z4-repeat array and is a valuable tool for the genetic diagnosis of FSHD1.

Background: The three best-known NUP214 rearrangements found in leukemia (SET::NUP214, NUP214::ABL1, and DEK::NUP214) are associated with treatment resistance and poor prognosis. Mouse experiments have shown that NUP214 rearrangements alone are insufficient for leukemogenesis; therefore, the identification of concurrent mutations is important for accurate assessment and tailored patient management. Here, we characterized the demographic characteristics and concurrent mutations in patients harboring NUP214 rearrangements. Methods: To identify patients with NUP214 rearrangements, RNA-sequencing results of diagnostic bone marrow aspirates were retrospectively studied. Concurrent targeted nextgeneration sequencing results, patient demographics, karyotypes, and flow cytometry information were also reviewed. Results: In total, 11 patients harboring NUP214 rearrangements were identified, among whom four had SET::NUP214, three had DEK::NUP214, and four had NUP214::ABL1. All DEK::NUP214-positive patients were diagnosed as having AML. In patients carrying SET::NUP214 and NUP214::ABL1, T-lymphoblastic leukemia was the most common diagnosis (50%, 4/8). Concurrent gene mutations were found in all cases. PFH6 mutations were the most common (45.5%, 5/11), followed by WT1 (27.3%, 3/11), NOTCH1 (27.3%, 3/11), FLT3-internal tandem duplication (27.3%, 3/11), NRAS (18.2%, 2/11), and EZH2 (18.2%, 2/11) mutations. Two patients represented the second and third reported cases of NUP214::ABL1-positive AML. Conclusions We examined the characteristics and concurrent test results, including gene mutations, of 11 leukemia patients with NUP214 rearrangement. We hope that the elucidation of the context in which they occurred will aid future research on tailored monitoring and treatment.