Monitoring measurable residual disease in paediatric acute lymphoblastic leukaemia using immunoglobulin gene clonality based on next-generation sequencing.

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.

Comprehensive insights into AML relapse: genetic mutations, clonal evolution, and clinical outcomes.

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.

Common genes and recurrent causative variants in 957 Asian patients with pediatric epilepsy.

OBJECTIVE: We aimed to identify common genes and recurrent causative variants in a large group of Asian patients with different epilepsy syndromes and subgroups. METHODS: Patients with unexplained pediatric-onset epilepsy were identified from the in-house Severance Neurodevelopmental Disorders and Epilepsy Database. All patients underwent either exome sequencing or multigene panels from January 2017 to December 2019, at Severance Children's Hospital in Korea. Clinical data were extracted from the medical records. RESULTS: Of the 957 patients studied, 947 (99.0%) were Korean and 570 were male (59.6%). The median age at testing was 4.91 years (interquartile range, 1.53-9.39). The overall diagnostic yield was 32.4% (310/957). Clinical exome sequencing yielded a diagnostic rate of 36.9% (134/363), whereas the epilepsy panel yielded a diagnostic rate of 29.9% (170/569). Diagnostic yield differed across epilepsy syndromes. It was high in Dravet syndrome (87.2%, 41/47) and early infantile developmental epileptic encephalopathy (60.7%, 17/28), but low in West syndrome (21.8%, 34/156) and myoclonic-atonic epilepsy (4.8%, 1/21). The most frequently implicated genes were SCN1A (n = 49), STXBP1 (n = 15), SCN2A (n = 14), KCNQ2 (n = 13), CDKL5 (n = 11), CHD2 (n = 9), SLC2A1 (n = 9), PCDH19 (n = 8), MECP2 (n = 6), SCN8A (n = 6), and PRRT2 (n = 5). The recurrent genetic abnormalities included 15q11.2 deletion/duplication (n = 9), Xq28 duplication (n = 5), PRRT2 deletion (n = 4), MECP2 duplication (n = 3), SCN1A, c.2556+3A>T (n = 3), and 2q24.3 deletion (n = 3). SIGNIFICANCE: Here we present the results of a large-scale study conducted in East Asia, where we identified several common genes and recurrent variants that varied depending on specific epilepsy syndromes. The overall genetic landscape of the Asian population aligns with findings from other populations of varying ethnicities.

Utility of Plasma Microbial Cell-Free DNA Whole-Genome Sequencing for Diagnosis of Invasive Aspergillosis in Patients With Hematologic Malignancy or COVID-19.

BACKGROUND: We evaluated the clinical accuracy and utility of whole-genome sequencing (WGS) of plasma microbial cell-free DNA (cfDNA) as a novel noninvasive method in diagnosing invasive aspergillosis (IA) in patients with hematologic malignancy (HM) or coronavirus disease 2019 (COVID-19). METHODS: Adults with HM or COVID-19 and suspected IA were recruited. IA cases were retrospectively diagnosed according to EORTC/MSG definitions and ECMM/ISHAM criteria for HM and COVID-19 patients, respectively. The results of cfDNA WGS were compared with the conventional diagnosis. RESULTS: Microbial cfDNA WGS was performed 53 times from 41 participants (19 from HM, 16 from COVID-19, and 7 from the control group). In participants with HM, Aspergillus cfDNA was detected in 100% of proven IA and 91.7% of probable IA cases. In participants with COVID-19, 50.0% of probable IA were positive for Aspergillus in cfDNA WGS. Concordance between Aspergillus cfDNA detection and proven/probable IA conventional diagnosis was significantly higher in participants with HM than in those with COVID-19. IA diagnosed using EORTC/MGS definitions showed significantly high concordance between Aspergillus cfDNA detection and proven/probable IA. CONCLUSIONS: Aspergillus cfDNA detection strongly correlated with proven/probable IA diagnosed using EORTC/MSG definitions and could be used as an additional diagnostic tool for IA.

Copy-number analysis by base-level normalization: An intuitive visualization tool for evaluating copy number variations.

Next-generation sequencing (NGS) facilitates comprehensive molecular analyses that help with diagnosing unsolved disorders. In addition to detecting single-nucleotide variations and small insertions/deletions, bioinformatics tools can identify copy number variations (CNVs) in NGS data, which improves the diagnostic yield. However, due to the possibility of false positives, subsequent confirmation tests are generally performed. Here, we introduce Copy-number Analysis by BAse-level NormAlization (CABANA), a visualization tool that allows users to intuitively identify candidate CNVs using the normalized single-base-level read depth calculated from NGS data. To demonstrate how CABANA works, NGS data were obtained from 474 patients with neuromuscular disorders. CNVs were screened using a conventional bioinformatics tool, ExomeDepth, and then we normalized and visualized those data at the single-base level using CABANA, followed by manual inspection by geneticists to filter out false positives and determine candidate CNVs. In doing so, we identified 31 candidate CNVs (7%) in 474 patients and subsequently confirmed all of them to be true using multiplex ligation-dependent probe amplification. The performance of CABANA was deemed acceptable by comparing its diagnostic yield with previous data about neuromuscular disorders. Despite some limitations, we expect CABANA to help researchers accurately identify CNVs and reduce the need for subsequent confirmation testing.

Development and validation of sensitive BCR::ABL1 fusion gene quantitation using next-generation sequencing.

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.

The Genotype-Phenotype Correlation in Human 5α-Reductase Type 2 Deficiency: Classified and Analyzed from a SRD5A2 Structural Perspective.

The phenotype of the 5α-reductase type 2 deficiency (5αRD2) by the SRD5A2 gene mutation varies, and although there have been many attempts, the genotype-phenotype correlation still has not yet been adequately evaluated. Recently, the crystal structure of the 5α-reductase type 2 isozyme (SRD5A2) has been determined. Therefore, the present study retrospectively evaluated the genotype-phenotype correlation from a structural perspective in 19 Korean patients with 5αRD2. Additionally, variants were classified according to structural categories, and phenotypic severity was compared with previously published data. The p.R227Q variant, which belongs to the NADPH-binding residue mutation category, exhibited a more masculine phenotype (higher external masculinization score) than other variants. Furthermore, compound heterozygous mutations with p.R227Q mitigated phenotypic severity. Similarly, other mutations in this category showed mild to moderate phenotypes. Conversely, the variants categorized as structure-destabilizing and small to bulky residue mutations showed moderate to severe phenotypes, and those categorized as catalytic site and helix-breaking mutations exhibited severe phenotypes. Therefore, the SRD5A2 structural approach suggested that a genotype-phenotype correlation does exist in 5αRD2. Furthermore, the categorization of SRD5A2 gene variants according to the SRD5A2 structure facilitates the prediction of the severity of 5αRD2 and the management and genetic counseling of patients affected by it.

Real-world data on prognostic value of measurable residual disease assessment by fragment analysis or next-generation sequencing in multiple myeloma.

Measurable residual disease (MRD) negativity is a strong prognostic indicator in multiple myeloma (MM). However, the optimal use of MRD in daily clinical practice has been hampered by the limited feasibility of MRD testing. Therefore, we examined the clinical relevance of commercially available MRD modalities based on clonality assays by fragment analysis with IdentiClone® (n = 73 patients) and next-generation sequencing (NGS) with LymphoTrack® (n = 116 patients) in newly diagnosed patients with MM who received autologous stem cell transplantation (ASCT). MRD was assessed at the end of induction (pre-ASCT) and/or at 100 days after ASCT (post-ASCT). MRD could not predict survival when assessed by fragment analysis. However, NGS-based MRD negativity at pre- or post-ASCT was beneficial in terms of progression-free and overall survival. Moreover, NGS-based MRD negativity was independently associated with improved progression-free and overall survival, and MRD-positive patients both pre- and post-ASCT had worst outcome. Indeed, initial adverse prognostic features by high-risk cytogenetics could be mitigated upon achieving MRD negativity by NGS. We demonstrate the feasibility and clinical benefit of achieving MRD negativity by commercially available clonality-based MRD assays in MM and support incorporating NGS, but not fragment analysis, to tailor therapeutic strategies in real-world practice.

Analytical and Clinical Validation of Cell-Free Circulating Tumor DNA Assay for the Estimation of Tumor Mutational Burden.

BACKGROUND: Ultra-deep sequencing to detect low-frequency mutations in circulating tumor-derived DNA (ctDNA) increases the diagnostic value of liquid biopsy. The demand for large ctDNA panels for comprehensive genomic profiling and tumor mutational burden (TMB) estimation is increasing; however, few ctDNA panels for TMB have been validated. Here, we designed a ctDNA panel with 531 genes, named TMB500, along with a technical and clinical validation. METHODS: Synthetic reference cell-free DNA materials with predefined allele frequencies were sequenced in a total of 92 tests in 6 batches to evaluate the precision, linearity, and limit of detection of the assay. We used clinical samples from 50 patients with various cancers, 11 healthy individuals, and paired tissue samples. Molecular barcoding and data analysis were performed using customized pipelines. RESULTS: The assay showed high precision and linearity (coefficient of determination, r2 =0.87) for all single nucleotide variants, with a limit of detection of 0.24%. In clinical samples, the TMB500 ctDNA assay detected most variants present and absent in tissues, showing that ctDNA could assess tumor heterogeneity in different tissues and metastasis sites. The estimated TMBs correlated well between tissue and blood, except in 4 cases with extreme heterogeneity that showed very high blood TMBs compared to tissue TMBs. A pilot evaluation showed that the TMB500 assay could be used for disease monitoring. CONCLUSIONS: The TMB500 assay is an accurate and reliable ctDNA assay for many clinical purposes. It may be useful for guiding the treatment of cancers with diverse genomic profiles, estimating TMB in immune therapy, and disease monitoring.

Caspase-10 affects the pathogenesis of primary biliary cholangitis by regulating inflammatory cell death.

Primary biliary cholangitis (PBC) is an autoimmune disease that involves chronic inflammation and injury to biliary epithelial cells. To identify critical genetic factor(s) in PBC patients, we performed whole-exome sequencing of five female siblings, including one unaffected and four affected sisters, in a multi-PBC family, and identified 61 rare heterozygote variants that segregated only within the affected sisters. Among them, we were particularly interested in caspase-10, for although several caspases are involved in cell death, inflammation and autoimmunity, caspase-10 is little known from this perspective. We generated caspase-10 knockout macrophages, and then investigated the obtained phenotypes in comparison to those of its structurally similar protein, caspase-8. Unlike caspase-8, caspase-10 does not play a role during differentiation into macrophages, but after differentiation, it regulates the process of inflammatory cell deaths such as necroptosis and pyroptosis more strongly. Interestingly, caspase-10 displays better protease activity than caspase-8 in the process of RIPK1 cleavage, and an enhanced ability to form a complex with RIPK1 and FADD in human macrophages. Higher inflammatory cell death affected the fibrotic response of hepatic stellate cells; this effect could be recovered by treatment with UDCA and OCA, which are currently approved for PBC patients. Our findings strongly indicate that the defective roles of caspase-10 in macrophages contribute to the pathogenesis of PBC, thereby suggesting a new therapeutic strategy for PBC treatment.

Application of CRISPR/Cas9-based mutant enrichment technique to improve the clinical sensitivity of plasma EGFR testing in patients with non-small cell lung cancer.

BACKGROUND: Approximately 50%-60% of secondary resistance to primary EGFR- tyrosine kinase inhibitors (TKI) therapy is caused by acquired p.Thr790Met (T790M) mutation; however, highly fragmented, low-quantity circulating tumor DNA is an obstacle for detecting mutations. Therefore, more sensitive mutation detection techniques are required. Here, we report a new mutant enrichment technology, the CRISPR system combined with post-polymerase chain reaction (PCR) cell-free DNA (cfDNA) (CRISPR-CPPC) to detect the T790M mutation using droplet digital PCR (ddPCR) from cfDNA. METHODS: The CRISPR-CPPC process comprises the following three steps: (1) cfDNA PCR, (2) assembly of post-PCR cfDNA and CRISPR/CRISPR associated protein 9 complex, and (3) enrichment of the target DNA template. After CRISPR-CPPC, the target DNA was detected using ddPCR. We optimized and validated CRISPR-CPPC using reference cfDNA standards and cfDNA from patients with non-small cell lung cancer who underwent TKI therapy. We then compared the detection sensitivity of CRISPR-CPPC assay with the results of real-time PCR and those of ddPCR. RESULTS: CRISPR-CPPC aided detection of T790M with 93.9% sensitivity and 100% specificity. T790M mutant copies were sensitively detected achieving an approximately 13-fold increase in the detected allele frequency. Furthermore, positive rate of detecting a low T790M copy number (< 10 copies/mL) were 93.8% (15/16) and 43.8% (7/16) for CRISPR-CPPC assay and ddPCR, respectively. CONCLUSIONS: CRISPR-CPPC is a useful mutant enrichment tool for the sensitive detection of target mutation. When tested in patients with progressive disease, the diagnostic performance of CRISPR-CPPC assay is exceptionally better than that of any other currently available methods.

Optical genome mapping identifies clinically relevant genomic rearrangements in prostate cancer biopsy sample.

BACKGROUND: Prostate cancer (PCa) is characterized by complex genomic rearrangements such as the ETS oncogene family fusions, yet the clinical relevance is not well established. While paneled genetic tests of DNA repair genes are recommended in advanced PCa, conventional genomic or cytogenetic tools are not ideal for genome-wide screening of structural variations (SVs) such as balanced translocation due to cost and/or resolution issues. METHODS: In this study, we tested the feasibility of whole-genome optical genomic mapping (OGM), a newly developed platform for genome-wide SV analysis to detect complex genomic rearrangements in consecutive unselected PCa samples from MRI/US-fusion targeted biopsy. RESULTS: We tested ten samples, and nine (90%) passed quality check. Average mapping rate and coverage depth were 58.1 ± 23.7% and 157.3 ± 97.7×, respectively (mean ± SD). OGM detected copy number alterations such as chr6q13 loss and chr8q12-24 gain. Two adjacent tumor samples were distinguished by inter/intra-chromosomal translocations, revealing that they're from the same ancestor. Furthermore, OGM detected large deletion of chr13q13.1 accompanied by inter-chromosomal translocation t(13;20)(q13.1;p13) occurring within BRCA2 gene, suggesting complete loss of function. CONCLUSION: In conclusion, clinically relevant genomic SVs were successfully detected in PCa samples by OGM. We suggest that OGM can complement panel sequencing of DNA repair genes BRCA1/2 or ATM in high-risk PCa.

Application of Multigene Panel Testing in Patients With High Risk for Hereditary Colorectal Cancer: A Descriptive Report Focused on Genotype-Phenotype Correlation.

BACKGROUND: The genetic test solely based on the clinical features of hereditary colorectal cancer has limitations in clinical practice. OBJECTIVE: This study aimed to analyze the results of comprehensive multigene panel tests based on clinical findings. DESIGN: This was a cross-sectional study based on a prospectively compiled database. SETTING: The study was conducted at a tertiary hospital. PATIENTS: A total of 381 patients with high risk for hereditary colorectal cancer syndromes were enrolled between March 2014 and December 2019. MAIN OUTCOME MEASURES: The primary outcome was to describe the mutational spectrum based on genotype-phenotype concordance and discordance. RESULTS: Germline mutations were identified in 89 patients for polyposis hereditary colorectal cancer genes (76 in APC; 4 in PTEN; 4 in STK11; 3 in BMPR1A; 1 in POLE; 1 in POLD1), 89 patients for nonpolyposis hereditary colorectal cancer genes (41 in MLH1; 40 in MSH2; 6 in MSH6; and 2 in PMS2), and 12 patients for other cancer predisposition genes (1 in ATM; 2 in BRCA1; 1 in BRCA2; 1 in BRIP1; 1 in MLH3; 1 in NBN; 1 in PMS1; 1 in PTCH1; 1 in TP53; and 2 in monoallelic MUTYH). If we had used direct sequencing tests of 1 or 2 major genes based on phenotype, 48 (25.3%) of 190 mutations would not have been detected due to technical differences (12.1%), less frequent genotype (4.2%), unclear phenotype (3.7%), and genotype-phenotype discordance (4.7%). The genotype-phenotype discordance is probably linked to compound heterozygote, less distinctive phenotype, and insufficient information for colorectal cancer risk. LIMITATIONS: This study included a small number of patients with insufficient follow-up duration. CONCLUSIONS: A comprehensive multigene panel is expected to identify more genetic mutations than phenotype-based direct sequencing, with special utility for unclear phenotype or genotype-phenotype discordance. See Video Abstract at http://links.lww.com/DCR/B844. APLICACIN DE PRUEBAS DE PANEL MULTIGNICO EN PACIENTES CON ALTO RIESGO DE CNCER COLORRECTAL HEREDITARIO INFORME DESCRIPTIVO ENFOCADO EN LA CORRELACIN GENOTIPOFENOTIPO: ANTECEDENTES:La prueba genética basada únicamente en la característica clínica del cáncer colorrectal hereditario tiene limitaciones en la práctica clínica.OBJETIVO:Este estudio tuvo como objetivo analizar el resultado de pruebas integrales de panel multigénico basadas en hallazgos clínicos.DISEÑO:Este fue un estudio transversal basado en una base de datos recopilada prospectivamente.AJUSTE:El estudio se realizó en un hospital terciario.PACIENTES:Se inscribió un total de 381 pacientes con alto riesgo de síndromes de cáncer colorrectal hereditario entre marzo del 2014 y diciembre del 2019.PRINCIPALES MEDIDAS DE RESULTADO:El resultado principal fue describir el espectro mutacional basado en la concordancia y discordancia genotipo-fenotipo.RESULTADOS:Se identificaron mutaciones de la línea germinal en 89 pacientes para genes de cáncer colorrectal hereditario con poliposis (76 en APC; 4 en PTEN; 4 en STK11; 3 en BMPR1A; 1 en POLE; 1 en POLD1), 89 pacientes para genes de CCR hereditario sin poliposis (41 en MLH1; 40 en MSH2; 6 en MSH6; y 2 ​​en PMS2) y 12 pacientes por otro gen de predisposición al cáncer (1 en ATM; 2 en BRCA1; 1 en BRCA2; 1 en BRIP1; 1 en MLH3; 1 en NBN; 1 en PMS1; 1 en PTCH1; 1 en TP53; y 2 ​​en MUTYH monoalélico). Si hubiéramos utilizado pruebas de secuenciación directa de uno o dos genes principales basados ​​en el fenotipo, 48 (25,3%) de 190 mutaciones no se habrían detectado debido a diferencias técnicas (12,1%), genotipo menos frecuente (4,2%), fenotipo poco claro (3,7%) y discordancia genotipo-fenotipo (4,7%). La discordancia genotipo-fenotipo probablemente esté relacionada con el heterocigoto compuesto, el fenotipo menos distintivo y la información insuficiente para el riesgo de cáncer colorrectal.LIMITACIONES:Este estudio incluyó una pequeña cantidad de pacientes con una duración de seguimiento insuficiente.CONCLUSIONES:Se espera que un panel multigénico completo identifique más mutaciones genéticas que la secuenciación directa basada en el fenotipo, con especial utilidad para la discordancia de fenotipo o genotipo-fenotipo poco clara. Consulte Video Resumen en http://links.lww.com/DCR/B844. Traducción- Dr. Francisco M. Abarca-Rendon).

Chimerism Assay Using Single Nucleotide Polymorphisms Adjacent and in Linkage-Disequilibrium Enables Sensitive Disease Relapse Monitoring after Hematopoietic Stem-Cell Transplantation.

BACKGROUND: Short tandem repeat (STR)-based chimerism analysis has been widely used for chimerism monitoring after hematopoietic stem-cell transplantation (HSCT), but technical artifacts can be problematic. We designed a chimerism assay using single nucleotide polymorphisms (SNPs) adjacent and in linkage-disequilibrium (CASAL), which doubly checked for SNP pairs, and thus could reduce background errors and increase analytical sensitivity. METHODS: CASAL targeted 84 SNP pairs within 10 bp distance and in perfect linkage-disequilibrium. Using undiluted and serially diluted samples, baseline error rates, and linearity was calculated. Clinical performance of CASAL was evaluated in comparison with a conventional STR assay, using 191 posttransplant samples from 42 patients with HSCT. RESULTS: CASAL had ∼10 times lower baseline error rates compared to that of ordinary next-generation sequencing. Limit of detection and quantification of CASAL were estimated to be 0.09 and 0.39%, respectively, with a linear range of 0.1-100%. CASAL correlated well with STR assay (r2 = 0.99) and the higher sensitivity enabled detection of low-level recipient chimerism and earlier prediction of relapse. CONCLUSIONS: CASAL is a simple, analytically sensitive and accurate assay that can be used in clinical samples after HSCT with a higher performance compared to that of traditional assays. It should also be useful in other forensic and archeological testing.

Exosome-based detection of EGFR T790M in plasma and pleural fluid of prospectively enrolled non-small cell lung cancer patients after first-line tyrosine kinase inhibitor therapy.

BACKGROUND: The exosomal nucleic acid (exoNA) from the plasma and pleural fluid can potentially provide means to identify genomic changes in non-small cell lung cancer (NSCLC) patients who develop resistance to targeted epidermal growth factor receptor (EGFR) inhibitor therapy. METHODS: We compared the performance of the following tools to detect EGFR mutations in 54 plasma samples and 13 pleural fluid using cfDNA, combined TNA (exoTNA + cfTNA), or total cellular DNA: droplet digital PCR (ddPCR), the Cobas® EGFR Mutation Test v2 (Cobas) and NGS with Oncomine Pan-Cancer Cell-Free Assay. RESULTS: All three of these platforms demonstrated 100% specificity in the detection of EGFR mutations in the plasma. In the detection of an activating mutation (exon 19 deletion and L858R), Cobas using cfDNA, ddPCR using combined TNA, and NGS using combined TNA showed a sensitivity of 93, 95.3, and 93.8%, respectively. For T790M mutation detection, the Cobas, ddPCR, and NGS showed a sensitivity of 64.7, 88.2, and 93.3%, respectively. Pleural fluid analysis revealed enrichment of the T790M mutant copies in the exosomes. ddPCR using exoTNA showed higher sensitivity than did total cellular DNA from the pleural fluid. CONCLUSION: These results demonstrated that combined TNA in the plasma and exoTNA in the pleural fluid can be used to evaluate low-abundant EGFR mutant copies in NSCLC.

Evaluation of Combined Cancer Markers With Lactate Dehydrogenase and Application of Machine Learning Algorithms for Differentiating Benign Disease From Malignant Ovarian Cancer.

BACKGROUND: The differential diagnosis of ovarian cancer is important, and there has been ongoing research to identify biomarkers with higher performance. This study aimed to evaluate the diagnostic utility of combinations of cancer markers classified by machine learning algorithms in patients with early stage ovarian cancer, which has rarely been reported. METHODS: In total, 730 serum samples were assayed for lactate dehydrogenase (LD), neutrophil-to-lymphocyte ratio (NLR), human epididymis protein 4 (HE4), cancer antigen 125 (CA125), and risk of ovarian malignancy algorithm (ROMA). Among them, 53 were diagnosed with early stage ovarian cancer, and the remaining 677 were diagnosed with benign disease. RESULTS: The areas under the receiver operating characteristic curves (ROC-AUCs) of the ROMA, HE4, CA125, LD, and NLR for discriminating ovarian cancer from non-cancerous disease were .707, .680, .643, .657, and .624, respectively. ROC-AUC of the combination of ROMA and LD (.709) was similar to that of single ROMA in the total population. In the postmenopausal group, ROC-AUCs of HE4 and CA125 combined with LD presented the highest value (.718). When machine learning algorithms were applied to ROMA combined with LD, the ROC-AUC of random forest was higher than that of other applied algorithms in the total population (.757), showing acceptable performance. CONCLUSION: Our data suggest that the combinations of ovarian cancer-specific markers with LD classified by random forest may be a useful tool for predicting ovarian cancer, particularly in clinical settings, due to easy accessibility and cost-effectiveness. Application of an optimal combination of cancer markers and algorithms would facilitate appropriate management of ovarian cancer patients.

Copy number variations and multiallelic variants in Korean patients with Leber congenital amaurosis.

Purpose: We comprehensively evaluated the mutational spectrum of Leber congenital amaurosis (LCA) and investigated the molecular diagnostic rate and genotype-phenotype correlation in a Korean cohort. Methods: This single-center retrospective case series included 50 Korean patients with LCA between June 2015 and March 2019. Molecular analysis was conducted using targeted panel-based next-generation sequencing, including deep intronic and regulatory variants or whole exome sequencing. The molecular diagnosis was made based on the inheritance pattern, zygosity, and pathogenicity. Results: Among the 50 patients, 27 patients (54%) were male, and 11 (22%) showed systemic features. Genetic variants highly likely to be causative were identified in 78% (39/50) of cases and segregated into families. We detected two pathogenic or likely pathogenic variants in a gene linked to a recessive trait without segregation analysis in three cases (6.0%). GUCY2D (20%), NMNAT1 (18%), and CEP290 (16%) were the most frequently mutated genes in Korean LCA. Copy number variations were found in three patients, which accounted for 6% of LCA cases. A possible dual molecular diagnosis (Senior-Løken syndrome along with Leigh syndrome, and Joubert syndrome with transposition of the great arteries) was made in two patients (4%). Three of 50 patients were medically or surgically actionable: one patient for RPE65 gene therapy and two patients with WDR19 Senior-Løken syndrome for early preparation for kidney and liver transplantations. Conclusions: This study demonstrated that approximately 4% of patients may have dual molecular diagnoses, and 6% were surgically or medically actionable in LCA. Therefore, accurate molecular diagnosis and careful interpretation of next-generation sequencing results can be of great help in patients with LCA.

Selecting short length nucleic acids localized in exosomes improves plasma EGFR mutation detection in NSCLC patients.

BACKGROUND: Exosomal nucleic acid (exoNA) is a feasible target to improve the sensitivity of EGFR mutation testing in non-small cell lung cancer patients with limited cell-free DNA (cfDNA) mutant copies. However, the type and size of target exoNA related to the sensitivity of EGFR mutation testing has not been explored extensively. METHODS: The type and size of target exoNA related to the sensitivity of EGFR mutation testing was evaluated using ddPCR. A total of 47 plasma samples was tested using short-length exoTNA (exosomal DNA and RNA) and cfDNA. RESULTS: The sensitivity of short-length exoTNA (76.5%) was higher than that of cfDNA (64.7%) for detecting EGFR mutations in NSCLC patients. In EGFR-mutant NSCLC patients with intrathoracic disease (M0/M1a) or cases with low-copy T790M, the positive rate was 63.6% (N = 7/11) and 45.5% (N = 5/11) for short-length exoTNA and cfDNA, respectively. On average, the number absolute mutant copies of short-length exoTNA were 1.5 times higher than that of cfDNA. The mutant allele copies (Ex19del and T790M) in short-length exoTNA were relatively well preserved at 4 weeks after storage. The difference (%) in absolute mutant allele copies (Ex19del) between 0 days and 4 weeks after storage was - 61.0% for cfDNA. CONCLUSION: Target nucleic acids and their size distribution may be critical considerations for selecting an extraction method and a detection assay. A short-length exoTNA (200 bp) contained more detectable tumor-derived nucleic acids than exoDNA (~ 200 bp length or a full-length) or cfDNA. Therefore, a short-length exoTNA as a sensitive biomarker might be useful to detect EGFR mutants for NSCLC patients with low copy number of the mutation target.

Emergence of multidrug-resistant Providencia rettgeri isolates co-producing NDM-1 carbapenemase and PER-1 extended-spectrum ß-lactamase causing a first outbreak in Korea.

BACKGROUND: Nosocomial outbreak due to carbapenem-resistant Enterobacteriaceae has become serious challenge to patient treatment and infection control. We describe an outbreak due to a multidrug-resistant Providencia rettgeri from January 2016 to January 2017 at a University Hospital in Seoul, Korea. METHODS: A total of eight non-duplicate P. rettgeri isolates were discovered from urine samples from eight patients having a urinary catheter and admitted in a surgical intensive care unit. The ß-lactamase genes were identified using polymerase chain reaction and direct sequencing, and strain typing was done with pulsed-field gel electrophoresis (PFGE). RESULTS: All isolates showed high-level resistance to extended-spectrum cephalosporins, aztreonam, meropenem, ertapenem, ciprofloxacin, and amikacin. They harbored the blaNDM-1 carbapenemase and the blaPER-1 type extended-spectrum ß-lactamases genes. PFGE revealed that all isolates from eight patients were closely related strains. CONCLUSIONS: The 13-month outbreak ended following reinforcement of infection control measures, including contact isolation precautions and environmental disinfection. This is the first report of an outbreak of a P. rettgeri clinical isolates co-producing NDM-1 and PER-1 ß-lactamase.

Diagnostic application of clinical exome sequencing in Leber congenital amaurosis.

PURPOSE: Leber congenital amaurosis (LCA) is a hereditary retinal dystrophy with wide genetic heterogeneity. Next-generation sequencing (NGS) targeting multiple genes can be a good option for the diagnosis of LCA, and we tested a clinical exome panel in patients with LCA. METHODS: A total of nine unrelated Korean patients with LCA were sequenced using the Illumina TruSight One panel, which targets 4,813 clinically associated genes, followed by confirmation using Sanger sequencing. Patients' clinical information and familial study results were obtained and used for comprehensive interpretation. RESULTS: In all nine patients, we identified pathogenic variations in LCA-associated genes: NMNAT1 (n=3), GUCY2D (n=2), RPGRIP1 (n=2), CRX (n=1), and CEP290 or SPATA7. Six patients had one or two mutations in accordance with inheritance patterns, all consistent with clinical phenotypes. Two patients had only one pathogenic mutation in recessive genes (NMNAT1 and RPGRIP1), and the clinical features were specific to disorders associated with those genes. Six patients were solved for genetic causes, and it remains unclear for three patients with the clinical exome panel. With subsequent targeted panel sequencing with 113 genes associated with infantile nystagmus syndrome, a likely pathogenic allele in CEP290 was detected in one patient. Interestingly, one pathogenic variant (p.Arg237Cys) in NMNAT1 was present in three patients, and it had a high allele frequency (0.24%) in the general Korean population, suggesting that NMNAT1 could be a major gene responsible for LCA in Koreans. CONCLUSIONS: We confirmed that a commercial clinical exome panel can be effectively used in the diagnosis of LCA. Careful interpretation and clinical correlation could promote the successful implementation of clinical exome panels in routine diagnoses of retinal dystrophies, including LCA.