Utility of Next-Generation Sequencing-Based Chimerism Analysis for Early Relapse Prediction following Allogenic Hematopoietic Cell Transplantation.

Chimerism monitoring following allogeneic hematopoietic cell transplantation (HCT) plays a pivotal role in evaluating engraftment status and identifying early indicators of relapse. Recent advancements in next-generation sequencing (NGS) technology have introduced AlloSeq HCT as a more sensitive alternative to short tandem repeat (STR) analysis. This study aimed to compare AlloSeq HCT with STR, focusing on the prediction of early relapse post-allogeneic HCT. Chimerism levels in 29 HCT recipients were assessed using both STR and NGS, employing a total of 125 whole blood or bone marrow aspirate samples (68 post-HCT and 57 pre-HCT samples from recipients or donors). AlloSeq HCT exhibited high concordance with STR and demonstrated the potential for early detection of chimeric changes, particularly at extremely low levels. The combined advantages of high sensitivity and automated data analysis offered by AlloSeq HCT substantiate its clinical adoption for effective chimerism monitoring.

Comparison of Measurable Residual Disease in Pediatric B-Lymphoblastic Leukemia Using Multiparametric Flow Cytometry and Next-Generation Sequencing.

Measurable residual disease (MRD) testing, a standard procedure in B-lymphoblastic leukemia (B-ALL) diagnostics, is assessed using multiparametric flow cytometry (MFC) and next-generation sequencing (NGS) analysis of immunoglobulin gene rearrangements. We evaluated the concordance between eight-color, two-tube MFC-MRD the LymphoTrack NGS-MRD assays using 139 follow-up samples from 54 pediatric patients with B-ALL. We also assessed the effect of hemodilution in MFC-MRD assays. The MRD-concordance rate was 79.9% (N=111), with 25 (18.0%) and 3 (2.2%) samples testing positive only by NGS-MRD (MFC-NGS+MRD) and MFC-MRD (MFC+NGS-MRD), respectively. We found a significant correlation in MRD values from total nucleated cells between the two methods (r=0.736 [0.647-0.806], P<0.001). The median MRD value of MFC-NGS+MRD samples was estimated to be 0.0012% (0.0001%-0.0263%) using the NGS-MRD assays. Notably, 14.3% of MFC-NGS+MRD samples showed NGS-MRD values below the limit of detection in the MFC-MRD assays. The percentages of hematogones detected in MFC-MRD assays significantly differed between the discordant and concordant cases (P<0.001). MFC and NGS-MRD assays showed relatively high concordance and correlation in MRD assessment, whereas the NGS-MRD assay detected MRD more frequently than the MFC-MRD assay in pediatric B-ALL. Evaluating the hematogone percentages can aid in assessing the impact of sample hemodilution.

Clinical Practice Guideline for Blood-based Circulating Tumor DNA Assays.

Circulating tumor DNA (ctDNA) has emerged as a promising tool for various clinical applications, including early diagnosis, therapeutic target identification, treatment response monitoring, prognosis evaluation, and minimal residual disease detection. Consequently, ctDNA assays have been incorporated into clinical practice. In this review, we offer an in-depth exploration of the clinical implementation of ctDNA assays. Notably, we examined existing evidence related to pre-analytical procedures, analytical components in current technologies, and result interpretation and reporting processes. The primary objective of this guidelines is to provide recommendations for the clinical utilization of ctDNA assays.

Analysis of HTT CAG repeat expansion among healthy individuals and patients with chorea in Korea.

BACKGROUND: Although the epidemiology of Huntington's disease (HD) in Korea differs notably from that in Western countries, the genetic disparities between these regions remain unclear. OBJECTIVE: To investigate the characteristics and clinical significance of cytosine-adenine-guanine (CAG) repeat size associated with HD in the Korean population. METHODS: We analyzed the CAG repeat lengths of the HTT gene in 941 healthy individuals (1,882 alleles) and 954 patients with chorea (1,908 alleles) from two referral hospitals in Korea. We presented normative CAG repeat length data for the Korean population and computed the reduced penetrance (36-39 CAG) and intermediate allele (27-35 CAG) frequencies in the two groups. Furthermore, we investigated the relationship between intermediate alleles and chorea development using logistic regression models in individuals aged ≥55 years. RESULTS: The mean (±standard deviation) CAG repeat length in healthy individuals was 17.5 ± 2.0, with a reduced penetrance allele frequency of 0.05 % (1/1882) and intermediate allele frequency of 0.69 % (13/1882). We identified 213 patients with genetically confirmed HD whose CAG repeat length ranged from 39 to 140, with a mean of 45.2 ± 7.9 in the longer allele. Compared with normal CAG repeat alleles, intermediate CAG repeat alleles were significantly related to a higher risk of developing chorea (age of onset range, 63-84 years) in individuals aged ≥55 years. CONCLUSIONS: This study provides insights into the specific characteristics of CAG repeat lengths in the HTT gene in the Korean population. The reduced penetrance and intermediate allele frequencies in the Korean general population seem to be lower than those reported in Western populations. The presence of intermediate alleles may increase the risk of chorea in the Korean elderly population, which requires further large-scale investigations.

Acral Pigmentation in Peutz-Jeghers Syndrome: Dermoscopic Findings and Treatment with the Q-Switched Nd:YAG Laser.

Peutz-Jeghers syndrome (PJS; MIM 175200) is an autosomal dominant multiple-organ cancer syndrome. It is characterized by brown macules distributed in the perioral skin, oral mucosa, hands and feet, and hamartomatous gastrointestinal polyps that can eventually lead to intestinal obstruction, abdominal pain, bleeding, and anemia. Patients with PJS are at a higher risk of ovarian, testicular, breast, lung, and pancreatic cancers. This predisposition is due to the pathogenic variant in serine/threonine kinase 11 (STK11) gene located on chromosome 19p13.3. Here, we present the dermoscopic findings, histopathologic features of acral pigmentation, and DNA sequencing results of the patient with PJS. We also report a successful removal of acral pigmentation using the Q-switched Nd:YAG laser (QSNYL) treatment. Our results suggest that QSNYL therapy could be a treatment option for acral pigmentation in patients with PJS.

The Korean Genetic Diagnosis Program for Rare Disease Phase II: outcomes of a 6-year national project.

The Korean Genetic Diagnosis Program for Rare Disease (KGDP) enrolled 1890 patients with rare diseases between March 2017 and October 2022. Children and adolescents accounted for the majority of the patients, and systemic disease was the most common presenting symptom. The exome-based virtual disease-specific multigene panel was the most frequently used analytical method, with an overall diagnostic yield of 33.3%. A total of 629 positive cases were diagnosed, involving 297 genes. All 297 genes identified in these cases were confirmed to be known genes listed in the OMIM database. The nationwide KGDP network and its cooperation with the Korean Undiagnosed Diseases Program (KUDP) provide a more comprehensive genetic analysis of undiagnosed cases. The partnership between the KGDP and KUDP has the potential to improve the diagnosis and treatment options for patients. In conclusion, KGDP serves as the primary access point or gateway to KUDP.

Distinct mutational pattern of T-cell large granular lymphocyte leukemia combined with pure red cell aplasia: low mutational burden of STAT3.

T-cell large granular lymphocyte leukemia (T-LGL) is often accompanied by pure red cell aplasia (PRCA). A high depth of next generation sequencing (NGS) was used for detection of the mutational profiles in T-LGL alone (n = 25) and T-LGL combined with PRCA (n = 16). Beside STAT3 mutation (41.5%), the frequently mutated genes included KMT2D (17.1%), TERT (12.2%), SUZ12 (9.8%), BCOR (7.3%), DNMT3A (7.3%), and RUNX1 (7.3%). Mutations of the TERT promoter showed a good response to treatment. 3 of 41 (7.3%) T-LGL patients with diverse gene mutations were revealed as T-LGL combined with myelodysplastic syndrome (MDS) after review of bone marrow slide. T-LGL combined with PRCA showed unique features (low VAF level of STAT3 mutation, low lymphocyte count, old age). Low ANC was detected in a STAT3 mutant with a low level of VAF, suggesting that even the low mutational burden of STAT3 is sufficient for reduction of ANC. In retrospective analysis of 591 patients without T-LGL, one MDS patient with STAT3 mutation was revealed to have subclinical T-LGL. T-LGL combined with PRCA may be classified as unique subtype of T-LGL. High depth NGS can enable sensitive detection of concomitant MDS in T-LGL. Mutation of the TERT promoter may indicate good response to treatment of T-LGL, thus, its addition to an NGS panel may be recommended.

Prevalence and founder effect of DRC1 exon 1-4 deletion in Korean patients with primary ciliary dyskinesia.

Primary ciliary dyskinesia (PCD) is a genetically heterogeneous disorder affecting ciliary structure and function. PCD exhibiting dynein regulatory complex subunit 1 (DRC1) exon 1-4 deletion has been reported in several Japanese PCD patients; however, no large scale studies have been performed. Here, we aimed to determine the prevalence and founder effect of this variant in the Korean population. Using an in-house copy number variation tool, we screened for DRC1 exon 1-4 deletion in 20 patients with PCD and exome data of 1435 patients in the Seoul National University Hospital repository. In cases of suspected DRC1 deletion, confirmatory gap-PCR was performed. In a PCD cohort, three of 20 (15%) patients were positive for DRC1 exon 1-4 deletion (NM_145038.5(DRC1): c.1-3952_540 + 1331del27748-bp) while pathogenic variants were found in CCDC39 (N = 1), DNAAF6 (N = 1), DNAH9 (N = 1). In the 1,435-sample exome data, seven patients (0.49%) were confirmed to have DRC1 exon 1-4 deletion. A chimeric sequence including the junction was searched from the 1000 Genomes Project data repository. One Japanese patient (0.96%) was found to have the same DRC1 exon 1-4 deletion, which was absent in other populations. This study demonstrated that the DRC1 exon 1-4 deletion is a founder mutation based on haplotype analysis. In summary, the prevalence of PCD based on DRC1 exon 1-4 deletion is particularly high in Korean and Japanese populations, which is attributed to the founder effect. Genetic testing for DRC1 exon 1-4 deletion should be considered as an initial screening tool for Korean and Japanese patients with PCD.

Clinical application of the Panbio™ COVID-19 Ag rapid test device and SSf-COVID19 kit for the detection of SARS-CoV-2 infection.

OBJECTIVE: We evaluated the sensitivity and specificity of the Panbio™ COVID-19 Ag rapid test device using nasal swabs and those of the SSf-COVID19 kit, one of RT-PCR tests, using saliva specimens. These tests were compared with RT-PCR tests using nasopharyngeal swabs for the diagnosis of SARS-CoV-2 infection. The three diagnostic tests were simultaneously conducted for patients aged ≥ 18 years, who were about to be hospitalized or had been admitted for COVID-19 confirmed by RT-PCR in two research hospitals from August 20 to October 29, 2021. Nasal swabs were tested using the Panbio™ COVID-19 Ag rapid test device. More than 1 mL of saliva was self-collected and tested using the SSf-COVID19 kit. RESULTS: In total, 157 patients were investigated; 124 patients who were about to be hospitalized and 33 patients already admitted for COVID-19. The overall sensitivity and specificity of the Panbio™ COVID-19 Ag rapid test device with nasal swabs were 64.7% (95% confidence interval [CI] 47.9-78.5%) and 100.0% (95% CI 97.0-100.0%), respectively. The median time to confirm a positive result was 180 s (interquartile range 60-255 s). The overall sensitivity and specificity of the SSf-COVID19 kit with saliva specimens were 94.1% (95% CI 80.9-98.4%) and 100.0% (95% CI 97.0-100.0%), respectively.

Parallel Analysis of Pre- and Postoperative Circulating Tumor DNA and Matched Tumor Tissues in Resectable Pancreatic Ductal Adenocarcinoma: A Prospective Cohort Study.

BACKGROUND: Circulating tumor DNA (ctDNA) is a promising biomarker for early tumor detection and minimal residual disease (MRD) assessment in early-stage cancer, but quantifying minute amounts of ctDNA is challenging and well-designed studies on ctDNA in early-stage cancer are still lacking. Here, we adapted a sensitive next-generation sequencing (NGS) technology and performed parallel analysis of pre- and postoperative ctDNA and matched tumor tissues in a prospective cohort of patients with resectable pancreatic ductal adenocarcinoma (PDAC). METHODS: In total, 70 consecutive patients undergoing curative resection for resectable PDAC were enrolled. We performed integrated digital error suppression-enhanced cancer personalized profiling by deep sequencing NGS of triple-matched samples (pre/postoperative plasma cell-free DNA [cfDNA], tumor tissue, and genomic DNA) targeting 77 genes. RESULTS: Preoperative ctDNA was detected in 37.7% of the evaluable patients, with a median variant allele frequency of 0.09%. Twelve additional oncogenic mutations were detected exclusively in preoperative ctDNA but not in tissue. When quantitative concentrations of ctDNA were estimated in haploid genome equivalents per milliliter (hGE/mL), the risk of early recurrence was high in patients with postoperative ctDNA >1 hGE/mL. cfDNA variants from 24.5% of patients had features compatible with clonal hematopoiesis. CONCLUSIONS: An optimized NGS approach might add value beyond tissue analysis through the highly sensitive detection of minute amounts of ctDNA in resectable PDAC. Postoperative ctDNA concentration could be a tool for MRD assessment. Moreover, parallel analyses of matched tissues and leukocytes might be required to accurately detect clinically relevant ctDNA.

Clinical staging and genetic profiling of Korean patients with primary lymphedema using targeted gene sequencing.

Lymphedema is a progressive disease caused by lymphatic flow blockage in the lymphatic pathway. Primary (hereditary) lymphedema is caused by genetic mutations without secondary causes. We performed clinical profiling on Korean primary lymphedema patients based on their phenotypes using lymphoscintigraphy and made genetic diagnoses using a next-generation sequencing panel consisting of 60 genes known to be related to primary lymphedema and vascular anomalies. Of 27 patients included in this study, 14.8% of the patients had lymphedema of the upper extremities, 77.8% had lymphedema of the lower extremities and 7.4% had 4-limbs lymphedema. Based on the International Society of Lymphology staging, 14, 10, and 3 patients had stage 3, 2, and 1 lymphedema, respectively. Only one family was genetically confirmed to harbor likely pathogenic variants in CELSR1. The proband was carrying two likely pathogenic variants in CELSR1, while her symptomatic mother was confirmed to carry only one of the variants. Furthermore, two other variants of uncertain significance in CELSR1 were detected in other patients, making CELSR1 the most commonly altered gene in our study. The clinical and genetic profile of hereditary lymphedema reported here is the first such data series reported for South Korea.

SARS-CoV-2 shedding dynamics and transmission in immunosuppressed patients.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern have been emerging. However, knowledge of temporal and spatial dynamics of SARS-CoV-2 is limited. This study characterized SARS-CoV-2 evolution in immunosuppressed patients with long-term SARS-CoV-2 shedding for 73-250 days, without specific treatment. We conducted whole-genome sequencing of 27 serial samples, including 26 serial samples collected from various anatomic sites of two patients and the first positive sample from patient 2's mother. We analysed the intrahost temporal dynamics and genomic diversity of the viral population within different sample types. Intrahost variants emerging during infection showed diversity between individual hosts. Remarkably, N501Y, P681R, and E484K, key substitutions within spike protein, emerged in vivo during infection and became the dominant population. P681R, which had not yet been detected in the publicly available genome in Korea, appeared within patient 1 during infection. Mutually exclusive substitutions at residues R346 (R346S and R346I) and E484 (E484K and E484A) of spike protein and continuous turnover of these substitutions occurred. Unique genetic changes were observed in urine samples. A household transmission from patient 2 to his mother, at least 38 days after the diagnosis, was characterized. Viruses may differently mutate and adjust to the host selective pressure, which could enable the virus to replicate efficiently for fitness in each host. Intrahost variants could be candidate variants likely to spread to the population eventually. Our findings may provide new insights into the dynamics of SARS-CoV-2 in response to interactions between the virus and host.

Immunohistochemical Staining to Identify Concomitant Systemic Mastocytosis in Acute Myeloid Leukemia with RUNX1::RUNX1T1.

Systemic mastocytosis with associated hematological neoplasm (SM-AHN) poses diagnostic challenges because of the coexistence of atypical mast cell proliferation and hematological neoplasms. We assessed the presence of SM-AHN in patients with acute myeloid leukemia (AML) with RUNX1::RUNX1T1 from 2014 to 2020. Bone marrow (BM) samples were evaluated for mast cell aggregates using CD117 and CD25 immunohistochemical (IHC) staining. The KIT D816V variant burden at diagnosis and post induction was assessed using droplet digital PCR. Among 23 patients diagnosed as having AML with RUNX1::RUNX1T1, four (17.4%) were also diagnosed as having SM-AHN. No significant differences in clinical characteristics or overall survival (P=0.565) were observed between patients with or without SM-AHN, except for the presence of KIT variants (P=0.040). After induction therapy, IHC staining revealed the presence of mast cell aggregates in the BM, and the KIT D816V variant burden decreased with decreasing blast count and was similar in BM aspirates, smear slides, and sections. Concomitant SM-AHN was not infrequent in AML patients with RUNX1::RUNX1T1. This study showed the importance of CD117 and CD25 IHC staining after induction chemotherapy for SM-AHN screening, especially in patients with KIT variants.

Epidemiologic Trends of Thalassemia, 2006-2018: A Nationwide Population-Based Study.

Thalassemia is the most common form of hereditary anemia. Here, we aimed to investigate the 13-year trend of the epidemiologic profiles and risk of comorbidities in thalassemia using a nationwide population-based registry in Korea. Diagnosis of thalassemia, the comorbidities and transfusion events in patients with thalassemia were identified in the Korean National Health Insurance database, which includes the entire population. The prevalence of thalassemia increased from 0.74/100,000 in 2006 to 2.76/100,000 in 2018. Notably, the incidence rate has nearly doubled in the last 2 years from 0.22/100,000 in 2016 to 0.41/100,000 in 2018. The annual transfusion rate gradually decreased from 34.7% in 2006 to 20.6% in 2018. Transfusion events in patients with thalassemia were significantly associated with the risk of comorbidities (diabetes: odds ratio [OR] = 3.68, 95% confidence interval [CI] = 2.59-5.22; hypertension: OR = 3.06, 95% CI = 2.35-4.00; dyslipidemia: OR = 1.72, 95% CI = 1.22-2.43; atrial fibrillation: OR = 3.52, 95% CI = 1.69-7.32; myocardial infarction: OR = 3.02, 95% CI = 1.09-8.38; stroke: OR = 3.32, 95% CI = 2.05-5.36; congestive heart failure: OR = 2.83, 95% CI = 1.62-4.97; end-stage renal disease: OR = 3.25, 95% CI = 1.96-5.37). Early detection of comorbidities and timely intervention are required for the management of thalassemia.