Analytical Performance Evaluation of a Digital Real-Time PCR for Quantifying Major BCR::ABL1 Transcripts.

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.

Analytical performance of the Abbott ID NOW 2.0 assay for SARS-CoV-2 detection in clinical samples from symptomatic patients.

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.

Letter to the Editor: Performance Analysis of the Aptima HBV Quant Assay for Hepatitis B virus DNA Quantification in Plasma.

The quantification of hepatitis B virus (HBV) DNA is critical for the diagnosis and management of HBV infections. We evaluated the performance of the Aptima HBV Quant assay for quantitative HBV DNA analysis. The intra-assay coefficient of variation for this assay was 2.08% (mean 3.45 log IU/mL) and 1.10% (mean 5.23 log IU/mL). Linearity ranged from 1.03 to 8.20 log IU/mL. The limit of detection was estimated at 4.31 IU/mL, which corresponded to the 4.29 IU/mL claimed by the manufacturer. All 25 other viral infections were determined to be negative. Passing-Bablok regression analysis showed no significant deviations between Aptima HBV Quant assay and Abbott RealTime HBV assay. The Aptima HBV Quant assay demonstrated comparable performance to the Abbott assay.

Long-term outcome of minimally invasive thymectomy versus open thymectomy for locally advanced cases.

OBJECTIVES: Our goal was to compare the oncological outcomes and efficacy between minimally invasive thymectomy (MIT) and open thymectomy (OT) in patients with early or locally advanced thymoma using a multicentre study database. METHODS: We retrospectively collected data from 1,239 patients who underwent thymectomy between January 2000 and December 2013, as recorded in the database of the Korean Association for Research on Thymus. We compared the postoperative outcomes of the MIT and OT groups using unmatched and propensity score (PS) matched data. RESULTS: We excised the thymoma using MIT and OT in 455 and 784 patients, respectively. We matched 378 patients with Masaoka-Koga stage I or II thymoma by their PS. The operative time, duration of hospital stay and complications were significantly shorter in the MIT group than in the OT group (all P < 0.005). In the PS matched data, the groups did not show significant differences in the 10-year survival rate (87.7% in OT vs 85.5% in MIT, stage II, mean follow-up duration: 12.9 years in OT vs 11.1 years in MIT), recurrence-free survival (94.0% in OT vs 86.4% in MIT) and R0 resection (97.35% in OT and MIT, P = 0.59). CONCLUSIONS: Compared with OT, MIT was associated with shorter operative times, shorter durations of hospital stay and fewer complications. Long-term survival, recurrence-free survival and complete resection were not significantly different between the OT and MIT groups. Our findings may help physicians track the progress of patients with early or locally advanced thymomas and design treatment plans for them.

Analytical and Clinical Performance of Droplet Digital PCR in the Detection and Quantification of SARS-CoV-2.

BACKGROUND AND OBJECTIVE: Since the initial coronavirus disease outbreak in late 2019 (COVID-19), reverse-transcription real-time polymerase chain reaction (RT-qPCR) has become the gold standard test to detect severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2). However, a more sensitive and accurate diagnostic tool was required. Therefore, droplet digital polymerase chain reaction (ddPCR) was suggested as an alternative method. Here, we evaluated the performance of ddPCR to detect SARS-CoV-2 and compared it to the performance of RT-qPCR. METHODS: The analytical performances, including limit of blank and limit of detection, were established using positive and negative SARS-CoV-2 reference materials. A total of 366 RNA extracts (173 positive and 193 negative by RT-qPCR) were collected from four institutions and tested with a Bio-Rad SARS-CoV-2 ddPCR kit that detects the SARS-CoV-2 genome using primers for N1 and N2. RESULTS: Limit of blank was set at 0, and the limits of detection of N1 and N2 were 1.99 copies/µL and 5.18 copies/µL, respectively. Linearity was evaluated using serial dilution samples, which demonstrated good results (R2: 0.999, linear range: 5.88-6825.25 copies/µL for N1 and R2: 0.999, 5.53-5855.47 copies/µL for N2). The results of ddPCR and RT-qPCR revealed substantial agreement (Cohen's kappa: 0.639, p < 0.01). The 63 samples with positive ddPCR but negative RT-qPCR showed low copy numbers, and 55% of them had COVID-19-related symptoms. CONCLUSIONS: Droplet digital polymerase chain reaction demonstrated excellent sensitivity for SARS-Cov-2 detection and consistently agreed with the results from conventional RT-qPCR. Furthermore, ddPCR provided quantitative data that can be used to monitor changes in the viral load of patients with COVID-19.

Non-inferior long-term outcomes of adults with Philadelphia chromosome-like acute lymphoblastic leukemia.

Philadelphia chromosome-like acute lymphoblastic leukemia (Ph-like ALL) is associated with inferior outcomes in the chemotherapy setting. We hypothesized that allogeneic hematopoietic cell transplantation (allo-HCT)-based post-remission therapy would improve outcomes of this entity. We examined the frequency and long-term outcomes of adults with Ph-like ALL, particularly focusing on allo-HCT outcomes for Ph-like ALL versus non-Ph-like ALL. Ph-like ALL was determined by anchored multiplex PCR-based targeted next-generation sequencing. Of the 344 patients, 57 (16.6%) had Ph-like ALL, 197 (57.3%) had Ph-positive ALL, and 90 (26.1%) had B-other ALL. To further evaluate the prognosis of Ph-like ALL, outcome analyses were restricted to 147 patients, excluding Ph-positive ALL. The actual allo-HCT rates in complete remission were 87.7% for Ph-like ALL, 71.4% for B-other standard-risk ALL, and 70.4% for B-other poor-risk ALL. Patients with Ph-like ALL had a higher 5-year overall survival (60.6% vs 27.1%; P = 0.008) than B-other poor-risk ALL subgroup, while no difference was observed compared with B-other standard-risk ALL subgroup. Similar results were noted in a separate analysis for patients receiving allo-HCT in complete remission. In multivariate analyses, B-other poor-risk ALL was associated with poorer outcomes. Our data showed that allo-HCT-based post-remission therapy may have contributed to non-inferior outcomes of adult Ph-like ALL.

Andersen-Tawil Syndrome With Novel Mutation in KCNJ2: Case Report.

Andersen-Tawil syndrome (ATS) is a rare autosomal dominant disorder characterized by a classic symptom triad: periodic paralysis, ventricular arrhythmias associated with prolonged QT interval, and dysmorphic skeletal and facial features. Pathogenic variants of the inwardly rectifying potassium channel subfamily J member 2 (KCNJ2) gene have been linked to the ATS. Herein, we report a novel KCNJ2 causative variant in a proband and her father showing different ATS-associated symptoms. A 15-year-old girl was referred because of episodic weakness and periodic paralysis in both legs for 2-3 months. The symptoms occurred either when she was tired or after strenuous exercise. These attacks made walking or climbing stairs difficult and lasted from one to several days. She had a short stature (142 cm, <3rd percentile) and weighed 40 kg. The proband also showed orbital hypertelorism, dental crowding, mandibular hypoplasia, fifth-digit clinodactyly, and small hands. Scoliosis in the thoracolumbar region was detected by chest X-ray. Since she was 7 years old, she had been treated for arrhythmia-associated long QT interval and underwent periodic echocardiography. Brain MRI revealed cerebrovascular abnormalities indicating absence or hypoplasia of bilateral internal carotid arteries, and compensation of other collateral vessels was observed. There were no specific findings related to intellectual development. The proband's father also had a history of periodic paralysis similar to the proband. He did not show any cardiac symptoms. Interestingly, he was diagnosed with hyperthyroidism during an evaluation for paralytic symptoms. Clinical exome sequencing revealed a novel heterozygous missense variant: Chr17(GRCh37):g.68171593A>T, NM_000891.2:c.413A>T, p.(Glu138Val) in KCNJ2 in the proband and the proband's father.

HLA-A, -B, -C, -DRB1 allele and haplotype frequencies of the Korean population and performance characteristics of HLA typing by next-generation sequencing.

INTRODUCTION: Human leukocyte antigen (HLA) identification at the allelic level is important for haematopoietic stem cell transplantation (HSCT). Next-generation sequencing (NGS) resolves ambiguous alleles by determining the phase of the polymorphisms. The aim of this study was to validate the software for HLA-SBT (sequence-based typing), assess Korean allele frequency, and characterise the performance of NGS-HLA typing. METHODS: From the 2009 to 2016 registry, 1293 unrelated healthy donors with a complete dataset of previously characterised HLA-A, -B, -C, and -DRB1 loci were selected and assessed for frequency, haplotype inference, and relative linkage disequilibrium. For performance characteristics of NGS-HLA, alleles included in 1293 cases and ambiguous or alleles assigned as new by SBT-HLA software, or unassigned alleles were included. A total of 91 and 41 quality control samples resulted in 1056 alleles (132 samples × 4 loci × 2 diploid) for analysis. The GenDx NGSgo kit was used for NGS-HLA typing using the Illumina MiSeq platform. RESULTS: A panel of 132 samples covered 231 alleles, including 53 HLA-A, 80 HLA-B, 43 HLA-C, and 55 HLA-DRB1 by HLA-SBT typing. Comparison of SBT-HLA and NGS-HLA typing showed 99.7% (1053/1056) concordance and discrepant cases were resolved by manual evaluation. Typing by NGS resulted in 67 HLA-A, 112 HLA-B, 71 HLA-C, and 72 HLA-DRB1 alleles. A total of 132 ambiguous, 4 new, and 1 unassigned alleles by HLA-SBT were resolved by NGS-HLA typing. CONCLUSIONS: NGS-HLA typing provided robust and conclusive results without ambiguities, and its implementation could support HSCT in clinical settings.

KRAS, NRAS, and BRAF mutations in plasma cell myeloma at a single Korean institute.

BACKGROUND: Plasma cell myeloma (PCM) is a genetically heterogeneous disease. The genetic spectrum of PCM has been expanded to mutations such as KRAS, NRAS, and BRAF genes in the RAS-RAF-MAPK pathway. In this study, we have evaluated the frequency of these mutations and their significance, including baseline characteristics and clinical outcomes. METHODS: We explored 50 patients who were newly diagnosed with PCM between 2009 and 2012 at a single Korean institute. Clinical and laboratory parameters were gathered through careful review of medical records. Mutation analysis was carried out using DNA from the bone marrow at the time of diagnosis. Pyrosequencing was performed to detect KRAS G12V, KRAS G13D, and NRAS G61R. BRAF V600E was analyzed by allele-specific real- time PCR. Comparison of clinical and laboratory parameters was carried out according to those mutations. RESULTS: We identified 14 patients (28%) with activating mutations in the RAS-RAF-MAPK pathway (RAS/RAF mutations): KRAS (N=3), NRAS (N=4), BRAF (N=7), and both KRAS and BRAF (N=1). RAS/RAF mutations were more frequently observed in patients with complex karyotypes and showed poorer progression free survival (PFS). Specifically, the BRAF V600E mutation had a significantly negative impact on median PFS. CONCLUSION: We first showed the frequency of RAS/RAF mutations in Korean patients with PCM. Screening of these mutations could be considered as a routine clinical test at the time of diagnosis and follow-up due to their influence on clinical outcome, as well as its potential as a therapeutic target.

Analytical and Potential Clinical Performance of Oncomine Myeloid Research Assay for Myeloid Neoplasms.

INTRODUCTION: Next-generation sequencing (NGS) panels have recently been introduced to efficiently detect genetic variations in hematologic malignancies. OBJECTIVES: Our aim was to evaluate the performance of the commercialized Oncomine™ myeloid research assay (OMA) for myeloid neoplasms. METHODS: Certified reference materials and clinical research samples were used, including 60 genomic DNA and 56 RNA samples. NGS was performed using OMA, which enables the interrogation of 40 target genes, 29 gene fusions, and five expression target genes with five expression control genes by the Ion S5 XL Sequencer. The analyzed data were compared with clinical data using karyotyping, reverse transcription polymerase chain reaction (PCR), fluorescence in situ hybridization, Sanger sequencing, customized NGS panel, and fragment analysis. RESULTS: All targets of reference materials were detected except three (two ASXL1 and one CEBPA) mutations, which we had not expected OMA to detect. In clinical search samples, OMA satisfactorily identified DNA variants, including 90 single nucleotide variants (SNVs), 48 small insertions and deletions (indels), and eight FLT3 internal tandem duplications (ITDs) (Kappa agreement 0.938). The variant allele frequencies of SNVs and indels measured by OMA correlated well with clinical data, whereas those of FLT3-ITDs were significantly lower than with fragment analysis (P = 0.008). Together, OMA showed strong ability to identify RNA gene fusions (Kappa agreement 0.961), except one RUNX1-MECOM. The MECOM gene was highly expressed in all five samples with MECOM-associated rearrangements, including inv(3), t(3;3), and t(3;21). CONCLUSION: OMA revealed excellent analytical and potential clinical performance and could be a good replacement for conventional molecular tests.

High incidence of RAS pathway mutations among sentinel genetic lesions of Korean pediatric BCR-ABL1-like acute lymphoblastic leukemia.

INTRODUCTION: Recent advances in genetic analysis have led to the discovery of novel genetic subtypes of precursor B-cell acute lymphoblastic leukemia (B-ALL) with prognostic relevance. In this study, we studied a cohort of pediatric B-ALL patients to retrospectively determine the incidence of patients harboring novel genetic subtypes, as well as their outcome. METHODS: B-ALL patients (N = 190) diagnosed in a single Korean hospital were included in the study. Patients' medical records were reviewed for data on established genetic abnormalities and outcome. CRLF2 expression was analyzed by quantitative RT-PCR. Anchored multiplex PCR-based enrichment was used to detect fusions and point mutations in 81 ALL-related genes. RESULTS: Incidence of established recurrent genetic subtypes was as follows: high hyperdiploidy (21.6%), ETV6-RUNX1 (21.6%), BCR-ABL1 (7.9%), KMT2A rearrangement (7.4%) TCF3-PBX1/TCF3-HLF (7.4%), and hypodiploidy (1.1%). Incidence of new genetic subtypes was as follows: BCR-ABL1-like (13.2%), ETV6-RUNX1-like (2.1%), EWSR1-ZNF384 (1.1%), and iAMP21 (1.1%). Median age at diagnosis of BCR-ABL1-like ALL was 6.8 years. According to type of genetic abnormality, BCR-ABL1-like ALL was divided into ABL class (12%), CRLF2 class (8%), JAK-STAT class (12%), and RAS class (68%). The 5-year event-free survival (EFS) of BCR-ABL1-like patients was significantly inferior to non-BCR-ABL1-like low- and standard-risk patients (71.5 ± 9.1% vs 92.5 ± 3.2%, P = .001) and comparable to non-BCR-ABL1-like high (75.2 ± 6.2%) and very high-risk patients (56.8 ± 7.4%). All four ETV6-RUNX1-like patients survived event-free. CONCLUSION: Analogous to previous studies, incidence of BCR-ABL1-like ALL in our cohort was 13.2% with outcome comparable to high and very high-risk patients. A significantly high number of RAS class mutations was a distinct feature of our BCR-ABL1-like ALL group.

Clinical Validity of Next-Generation Sequencing Multi-Gene Panel Testing for Detecting Pathogenic Variants in Patients With Hereditary Breast-Ovarian Cancer Syndrome.

BACKGROUND: Hereditary breast and ovarian cancer syndrome (HBOC) is caused by pathogenic variants in BRCA and other cancer-related genes. We analyzed variants in BRCA gene and other cancer-related genes in HBOC patients to evaluate the clinical validity of next-generation sequencing (NGS) multi-gene panel testing. METHODS: The BRCA1/2 NGS testing was conducted for 262 HBOC patients. Multiplex ligation-dependent probe amplification and direct Sanger sequencing were performed for confirmation. Multi-gene panel testing was conducted for 120 patients who did not possess BRCA1/2 pathogenic variants but met the National Comprehensive Cancer Network criteria. RESULTS: Pathogenic variants in BRCA1/2 were detected in 30 HBOC patients (11.5%). Additionally, four out of the 120 patients possessed pathogenic variants by multi-gene panel testing (3.3%): MSH2 (c.256G>T, p.Glu86*), PMS2 (c.1687C>T, p.Arg563*), CHEK2 (c.546C>A, p.Tyr182*), and PALB2 (c.3351-1G>C). All the four patients had a family history of cancer. CONCLUSIONS: Multi-gene panel testing could be a significant screening tool for HBOC patients, especially for those with a family history of cancer.

Comparative Evaluation of Seegene Allplex Gastrointestinal, Luminex xTAG Gastrointestinal Pathogen Panel, and BD MAX Enteric Assays for Detection of Gastrointestinal Pathogens in Clinical Stool Specimens.

CONTEXT.­: Infectious gastroenteritis is caused by various pathogens, including bacteria, viruses, and parasites. OBJECTIVE.­: To compare the performance of Seegene Allplex Gastrointestinal (24 targets: 13 bacteria, 5 viruses, and 6 parasites in 4 panels), Luminex xTAG Gastrointestinal Pathogen Panel (15 targets: 9 bacteria, 3 viruses, and 3 parasites), and BD MAX Enteric panel (5 bacteria and 3 parasites). We estimated the agreement among 3 molecular assays. DESIGN.­: A total of 858 stool samples (554 bacterial/parasite and 304 viral pathogens) were included. A consensus positive/negative was defined as concordant results from at least 2 tests. To evaluate the agreement among the assays, κ value was calculated. RESULTS.­: The overall positive percentage agreements of Seegene, Luminex, and BD MAX were 94% (258 of 275), 92% (254 of 275), and 78% (46 of 59), respectfully. For Salmonella, Luminex showed low negative percentage agreement because of frequent false positives (n = 31) showing low median fluorescent intensity. For viruses, positive/negative percentage agreements of Seegene and Luminex were 99%/96% and 93%/99%, respectively. Compared with routine microbiology testing, Seegene, Luminex, and BD MAX additionally identified 39, 40, and 12 pathogens, respectively. Sixty-one cases (16 cases with Seegene, 51 cases with Luminex, and 1 case with BD MAX) showed positive results for multiple pathogens, but only 3 were consensus positive. CONCLUSIONS.­: These multiplex molecular assays appear to be promising tools for the detection and identification of multiple gastrointestinal pathogens simultaneously. However, careful interpretation of positive results for multiple pathogens is required.

CDKN2B downregulation and other genetic characteristics in T-acute lymphoblastic leukemia.

We identified principal genetic alterations in 97.1% (99/102) of patients with T-acute lymphoblastic leukemia (T-ALL) using integrative genetic analyses, including massive parallel sequencing and multiplex ligation-dependent probe amplification (MLPA). A total of 133 mutations were identified in the following genes in descending order: NOTCH1 (66.7%), FBXW7 (19.6%), PHF6 (15.7%), RUNX1 (12.7%), NRAS (10.8%), and DNMT3A (9.8%). Copy number alterations were most frequently detected in CDKN2B, CDKN2A, and genes on 9p21.3 in T-ALL (45.1%). Gene expression data demonstrated the downregulation of CDKN2B in most cases of T-ALL, whereas CDKN2A downregulation was mainly restricted to deletions. Additional quantitative methylation analysis demonstrated that CDKN2B downregulation stemmed from deletion and hypermethylation. Analysis of 64 patients with CDKN2B hypermethylation indicated an association with an older age of onset and early T cell precursor ALL, which involved very early arrest of T cell differentiation. Genes associated with methylation and myeloid neoplasms, including DNMT3A and NRAS, were more commonly mutated in T-ALL with CDKN2B hypermethylation. In particular, a CDKN2B biallelic deletion or high methylation level (≥45%), the age of onset, and the GATA3 and SH2B3 mutations were factors associated with a poor prognosis. This study clarifies that one of the most important genetic events in T-ALL, namely, CDKN2B downregulation, occurs mechanistically via deletion and hypermethylation. Different susceptible genetic backgrounds exist based on the CDKN2B downregulation mechanism.

Performance Evaluation of the Beckman Coulter DxN VERIS Hepatitis B Virus (HBV) Assay in Comparison With the Abbott RealTime HBV Assay.

The detection and quantification of hepatitis B virus (HBV) DNA plays an important role in diagnosing and monitoring HBV infection as well as in assessing the therapeutic response. We compared the analytical performance of a random access, fully automated HBV assay-DxN VERIS Molecular Diagnostics System (Beckman Coulter, Brea, CA, USA)-with that of Abbott RealTime HBV assay (Abbott Laboratories, Des Plaines, IL, USA). The between-day precision of the VERIS assay ranged from 0.92% (mean 4.68 log IU/mL) to 4.15% (mean 2.09 log IU/mL) for pooled sera from HBV patients. HBV DNA levels measured by the VERIS HBV assay correlated with the calculated HBV DNA levels (r²=0.9994; P<0.0001). The lower limit of quantification was estimated as 8.76 IU/mL (Probit analysis, 95% confidence interval: 7.32-12.00 IU/mL). Passing-Bablok regression analysis showed good concordance between the VERIS and RealTime assays for 187 chronic HBV samples (y=-0.2397+0.9712x; r=0.981), as well as for 20 drug-resistant HBV genotype C positive samples (y=-0.5415+0.9954x; r=0.961). The VERIS assay demonstrated performance similar to the RealTime assay and is suitable for high-throughput HBV DNA monitoring in large hospital laboratories.

Passage-dependent accumulation of somatic mutations in mesenchymal stromal cells during in vitro culture revealed by whole genome sequencing.

Human mesenchymal stromal cells (MSCs) have served as a major cellular resource for cell-based immunomodulatory and regenerative therapies. However, genomic instability may accumulate during ex vivo expansion of MSCs, thereby increasing the potential of malignant transformation. Here, we performed whole genome sequencing of two peripheral blood-derived MSC lines (MSC1 and MSC2) at various passages (passage 1 [P1] to P9). The majority of single-nucleotide variations (SNVs) occurred in later passages; specifically, 90% and 70% of all SNVs in MSC1 and MSC2 were observed in P9 and P7/P9, respectively. These late-occurring SNVs were enriched with C > A transversions and were overrepresented in intronic regions compared to intergenic regions, suggesting that the mutational forces are not constant across the passages. Clonality analyses also distinguished early-occurring, subclonal SNVs from late-occurring, clonally fixed SNVs. In addition, MSCs were largely devoid of copy number alterations (CNAs) (i.e., 0-2 CNAs per passage), with one exception (MSC2-P3) harboring 29 passage-specific CNAs. Our findings suggest that the SNVs found to be abundant at later passages likely resulted from the accumulation of replication stress, which can be associated with proliferation activity. Thus, the genomic instability associated with proliferation records should be considered for clinical applications of MSCs.

Identification of large genomic rearrangement of BRCA1/2 in high risk patients in Korea.

BACKGROUND: While the majority of germline inactivating mutations in BRCA1/2 are small-scale mutations, large genomic rearrangements (LGRs) are also detected in a variable proportion of patients. However, routine genetic methods are incapable of detecting LGRs, and comprehensive genetic testing algorithm is necessary. METHODS: We performed multiplex ligation-dependent probe amplification assay for small-scale mutation negative patients at high-risk for LGR, based on previously published LGR risk criteria. The inclusion criteria for the high-risk subgroup were personal history of 1) early-onset breast cancer (diagnosed at ≤36 years); 2) two breast primaries; 3) breast cancer diagnosed at any age, with ≥1 close blood relatives (includes first-, second-, or third-degree) with breast and/or epithelial ovarian cancer; 4) both breast and epithelial ovarian cancer diagnosed at any age; and 5) epithelial ovarian cancer with ≥1 close blood relatives with breast and/or epithelial ovarian cancer. RESULTS: Two LGRs were identified. One was a heterozygous deletion of exon 19 and the other was a heterozygous duplication of exon 4-6. The prevalence of LGRs was 7% among Sanger-negative, high-risk patients, and accounted for 13% of all BRCA1 mutations and 2% of all patients. Moreover, LGRs reported in Korean patients, including our 2 newly identified cases, were found exclusively in families with at least one high-risk feature. CONCLUSIONS: Our result suggests that selective LGR screening for Sanger-negative, high-risk patients is necessary for Korean patients.

Practical informativeness of short tandem repeat loci for chimerism analysis in hematopoietic stem cell transplantation.

OBJECTIVE: Short tandem repeat (STR) loci are most frequently used for chimerism analysis after hematopoietic stem cell transplantation (HSCT). The aim of this study was to evaluate the practical informativeness of STR chimerism by integrating theoretical and analytical points. METHODS: Theoretical and practical informativess of 16 STR loci were evaluated from 1249 pairs of recipients and donors who were prepared for HSCT. RESULTS: Theoretical informativeness was influenced by genetic diversity including allele frequency and heterozygosity, and was higher in the unrelated HSCT group (90.5±5.3%) compared to the related HSCT group (66.2±4.4%). Practical informativeness was lower than theoretical (6.1±1.7%) because several STR loci were excluded due to stutter peaks and less reliable results, especially in type II-2 donor-recipient match pattern with no recipient-specific allele. We simulated an efficient STR combination for reliable chimerism analysis. Eight informative STR loci were required to analyze chimerism with at least one practically informative locus in the related HSCT group (D18S51, FGA, D2S1338, D13S317, D8S1179, D21S11, D16S539 and D7S820) while only three loci were needed in the unrelated group (D2S1338, FGA and D18S51). A minimum set of 2, 4 or 7 STR loci were required to provide at least 1, 3 or 5 practically informative loci in 95% of the unrelated HSCT group while 3, 8 or 12 loci were required in the related HSCT group. CONCLUSION: We deducted the practical informativeness of STR chimerism, identified the major influencing factors on the practical informativeness of each STR locus, and successfully simulated the efficient STR combination for reliable chimerism analysis.

Peptide nucleic acid probe-based fluorescence melting curve analysis for rapid screening of common JAK2, MPL, and CALR mutations.

BACKGROUND: We developed and evaluated the feasibility of peptide nucleic acid (PNA)-based fluorescence melting curve analysis (FMCA) to detect common mutations in myeloproliferative neoplasms (MPNs). METHODS: We have set up two separate reactions of PNA-based FMCA: JAK2 V617F &CALR p.Leu367fs*46 (set A) and MPL W515L/K &CALR p.Lys385fs*47 (set B). Clinical usefulness was validated with allele-specific real-time PCR, fragment analysis, Sanger sequencing in 57 BCR-ABL1-negative MPNs. RESULTS: The limit of detection (LOD) of PNA-based FMCA was approximately 10% for each mutation and interference reactions using mixtures of different mutations were not observed. Non-specific amplification was not observed in normal control. PNA-based FMCA was able to detect all JAK2 V617F (n=20), CALR p.Leu367fs*46 (n=10) and p.Lys385fs*47 (n=8). Three of six MPL mutations were detected except three samples with low mutant concentration in out of LOD. JAK2 exon 12 mutations (n=7) were negative without influencing V617F results. Among six variant CALR exon 9 mutations, two were detected by this method owing to invading of probe binding site. CONCLUSIONS: PNA-based FMCA for detecting common JAK2, MPL, and CALR mutations is a rapid, simple, and sensitive technique in BCR-ABL1-negative MPNs with >10% mutant allele at the time of initial diagnosis.