Search

Background Hepatocellular carcinoma (HCC) is the second-most-common cause of cancer-related deaths worldwide, and an accurate and non-invasive biomarker for the early detection and monitoring of HCC is required. We assessed pathogenic variants of HCC driver genes in cell-free DNA (cfDNA) from HCC patients who had not undergone systemic therapy. Methods Plasma cfDNA was collected from 20 HCC patients, and deep sequencing was performed using a customized cfDNA next-generation sequencing panel, targeting the major HCC driver genes (TP53, CTNNB1, TERT) that incorporates molecular barcoding. Results In 13/20 (65%) patients, we identified at least one pathogenic variant of two major HCC driver genes (TP53 and CTNNB1), including 16 variants of TP53 and nine variants of CTNNB1. The TP53 and CTNNB1 variants showed low allele frequencies, with median values of 0.17% (range: 0.06%-6.99%) and 0.07% (range: 0.05%-0.96%), respectively. However, the molecular coverage of variants was sufficient, with median values of 5,543 (range: 2,317-9,088) and 7,568 (range: 2,400-9,633) for TP53 and CTNNB1 variants, respectively. Conclusions Our targeted DNA sequencing successfully identified low-frequency pathogenic variants in the cfDNA from HCC patients by achieving high coverage of unique molecular families. Our results support the utility of cfDNA analysis to identify somatic gene variants in HCC patients.

Background Minimal residual disease (MRD) monitoring is a powerful tool to predict the risk of relapse. Herein, we present an MRD monitoring strategy for B-cell lymphoblastic leukemia (B-ALL) using high-throughput sequencing (HTS) of immunoglobulin (Ig) clonality before implementation into routine practice. Methods We selected 74 bone marrow (BM) specimens from 47 patients who were diagnosed with B-ALL. Ig clonality was analyzed using both fragment analysis and HTS. The performance of Ig clonality was evaluated through comparison of the results from real-time quantitative polymerase chain reaction (qPCR) of leukemia-specific fusion transcripts and flow cytometry. Results IGH clonality was observed in all patients, and the sum of clonal burden varied (9.47%–96.77%). IGK clonality was identified in 70% of patients and availed in cases with low IGH clonal burden. The total IGH clonal burden was significantly correlated with the proportion of leukemic blasts, leukemia-specific fusion transcripts, and flow cytometry. We recognized the different responses of each clone and emerging clones originating from the trace of Ig rearrangement presented in the initial specimen. IGH clonal burden after chemotherapy represented patient outcomes well. IGH assay also provided information of repertoire diversity of IGH rearrangement. Conclusion The Ig clonality assay via HTS will be a promising tool for MRD monitoring of B-ALL through an adequate strategy to identify and monitor individual clones and determine repertoire diversity.

Background & Aim Multiple myeloma (MM) is a hematologic malignancy characterized by neoplastic proliferation of clonal plasma cells in the bone marrow and its pathogenesis is not fully understood. Recent genomic studies have shown that the RAS/MAPK pathway (NRAS, KRAS, BRAF, FGFR3) is the most frequently mutated pathway in patients with MM, 40% to 55% of newly diagnosed patients with MM have driver mutations in NRAS or KRAS. About of patients with MM harbored the BRAF mutation at diagnosis, mostly on amino acid V600, increasing to 18% at relapse. To understand the prevalance the somatic mutation of the RAS/MAPK pathway (NRAS, KRAS, and BRAF) in MM, we performed the mutation analysis from 50 patients with MM. Methods, Results & Conclusion All bone marrow samples were from initial diagnostic specimens, evaluated by a hematologist at St Mary`s hospital, Seoul, between 2009 and 2012. The clinical and demographic characteristics of the 50 MM patients are summarized in Table 1. BRAF V600E was screened by allele-specific real-time PCR (AS-PCR). Using pyrosequencing, bone marrow were retrospectively analysed for RAS status that is, for mutations within KRAS and NRAS mutation. RAS mutations were detcted in 8(16.0%) of 50 patients, including 4 patients with KRAS mutation (2 patients; G12V, 2 patients; G13D), 4 patients with NRAS mutation (Q61R). Among 4 patients with KRAS mutaion, 1 patient had t(8;14) genetic aberrattion. Among 4 patients with NRAS Q61R mutaion, 3 patients had hyperdiploidy. The point mutations of NRAS and KRAS genes occurs at a rate of 20% of cases of MM. BRAF V600E mutation was detected in 8 (16.0%) of 50 MM patients. In these patients, 4 patients had IgA monoclonal protein and 3 patients had light chain (kappa) monoclonal protein, and 1 patient had IgG(kappa) monoclonal protein. In these patients 3 patients (37.5%, 3/8) had 1q gain, 3 patietns (37.5%, 3/8) had t(11;14), 1 patient (12.5%, 1/8) had t(8;14), and 1 patient (12.5%, 1/8) had add (14)(q32). Compared with BRAF wild type, those carrying the BRAF V600E mutation had higher frequency of IgA, and patients with mutated the BRAF V600E genotype had higher level of calcium (P = 0.0154) and beta-2 microglobulin (P=0.0491) (Table 2). BRAF and RAS mutation screening is important in the molecular diagnostics of MM and it provide a rationale for clinical development of targeted treatment in MM.

Corneal dystrophy typically refers to a group of rare hereditary disorders with a heterogeneous genetic background. A comprehensive molecular genetic analysis was performed to characterize the genetic spectrum of corneal dystrophies in Korean patients. Patients with various corneal dystrophies underwent thorough ophthalmic examination, histopathologic examination, and Sanger sequencing. A total of 120 probands were included, with a mean age of 50 years (SD = 18 years) and 70% were female. A total of 26 mutations in five genes (14 clearly pathogenic and 12 likely pathogenic) were identified in 49 probands (41%). Epithelial-stromal TGFBI dystrophies, macular corneal dystrophy and Schnyder corneal dystrophy (SCD) showed 100% mutation detection rates, while endothelial corneal dystrophies showed lower detection rates of 3%. Twenty six non-duplicate mutations including eight novel mutations were identified and mutations associated with SCD were identified genetically for the first time in this population. This study provides a comprehensive characterization of the genetic aberrations in Korean patients and also highlights the diagnostic value of molecular genetic analysis in corneal dystrophies.

We evaluated the characteristics of bone marrow stromal cells (BMSCs) and hematopoietic cells (HCs) from patients of myelodysplastic syndrome (MDS, n = 21) and acute myeloid leukemia (AML, n = 58), and compared the results with control BMSCs derived from healthy donors (n = 8). The patient BMSCs had lower proliferative activity than that of the controls due to increased senescence. This retarded proliferation induced failure to obtain enough metaphase cells for karyotyping in patient BMSCs (10%). Patient BMSCs were genetically altered which was demonstrated by chromosome abnormalities in 5% of the patients (one MDS and three AML), whereas no clonal abnormalities were detected in the controls. The most common abnormality of the BMSCs was an extra chromosome 5, followed by an extra chromosome 7 and balanced translocations. The proportion of the abnormal metaphase cells was low (17.8%). We also analyzed the epigenetic changes of long interspersed nucleotide element 1 (LINE-1) repetitive element and CDKN2B using pyrosequencing. The quantitative measurement of global LINE-1 methylation demonstrated that patient BMSCs revealed global hypomethylation (68.2 ± 3.8) compared with controls (72.9 ± 3.4, P