Annals of Laboratory Medicine最新文献

Background: Precision oncology is advancing, increasing the demand for comprehensive, non-invasive genomic profiling tools. Liquid biopsy using circulating tumor DNA (ctDNA) enables real-time molecular profiling, treatment monitoring, and early detection of resistance variants. We developed the PAN100 panel (Dxome), a hybridization capture panel targeting 101 genes, as a pan-cancer genotyping assay to detect clinically actionable variants across various cancer types. This study presents the first comprehensive validation of the PAN100 panel including both analytical and clinical performance across eight cancer types using reference materials and matched tissue samples.

Methods: For analytical validation, we assessed accuracy, limit of detection (LoD), and precision using Seraseq ctDNA v2 Reference Materials (SeraCare, Milford, MA, USA). Clinical validation was performed using plasma samples from 27 patients with eight types of cancer and 17 matched tumor samples. Positive percent agreement (PPA) between ctDNA and tissue next-generation sequencing (NGS) results was assessed using TruSight Oncology 500 and TruSight Tumor 170 assays. The limit of blank (LoB) was evaluated in 34 healthy individuals.

Results: The PAN100 panel demonstrated high precision and linearity (LoD, 0.3%; 95.0% confidence interval, 0.29-0.35) variant allele frequency. The PPA between ctDNA and tissue NGS was 73.1% for single-nucleotide variants, 80.0% for insertions/deletions, and 74.2% overall. The LoB was 0.00001%.

Conclusions: The PAN100 panel is a robust tool for detecting clinically significant variants with high concordance with tissue NGS. Its sensitivity for low-frequency variants enables real-time treatment adaptation, supporting precision oncology. Its comprehensive design is particularly valuable for challenging diagnoses and clonal evolution monitoring.

Background: Cytomegalovirus (CMV) infection is prevalent worldwide. Although Korea has historically shown high CMV IgG seropositivity (>95%), declines have been reported recently. We assessed current CMV IgG seropositivity and analyzed prevailing trends in the Korean population.

Methods: Residual samples from individuals undergoing regular health checkups were analyzed. We assessed 1,978 samples (from 937 men and 1,041 women) where the age group distribution was relatively balanced. CMV IgG levels were measured at two institutions using a commercial immunoassay (Alinity i CMV IgG Reagent Kit, Abbott) following the manufacturer's instructions. Results were interpreted as "reactive" when the CMV IgG concentration was ≥ 6.0 arbitrary units (AU)/mL or "nonreactive" when the CMV IgG concentration was <6.0 AU/mL. Seropositivity was compared by sex and across age groups (20-29, 30-39, 40-49, and 50-59 yrs).

Results: The overall CMV IgG seropositivity was 89.9% (1,778/1,978) and was significantly higher in men (91.7%, 859/937) than in women (88.3%, 919/1,041) (difference: 3.4%; 95% confidence interval: 0.8%-6.0%; P =0.012, chi-square test). No significant differences with regard to sex were found within each age group. Seropositivity increased with age, from 76.3% (347/455, 20-29 yrs) to 99.8% (448/449, 50-59 yrs) (P for trend <0.001), consistently in both sexes.

Conclusions: Our findings provide the most up-to-date estimate of CMV IgG seropositivity in the Korean adult population. Because of lower seropositivity in younger adults, continued monitoring and further education are essential for CMV control and prevention.

Cirrhosis progresses through distinct stages, with increasing severity and susceptibility to infections due to cirrhosis-associated immune dysfunction. Lymphocyte-based functional assays are considered the gold standard for assessing immune function, although none have been evaluated across different cirrhosis stages. We conducted a pilot study to assess whether performing interferon-γ (IFN-γ) release assays (IGRA), in response to a nonantigenic mitogen (phytohemagglutinin [PHA]), could help classify immune function and disease severity in 70 patients with cirrhosis awaiting liver transplantation. Disease severity ranged from chronic cirrhosis to acute-on-chronic liver failure. We measured IFN-γ release in response to PHA using a fresh heparinized whole-blood protocol. Progressive reduction in IFN-γ release correlated with increased disease severity parameters (with respect to total bilirubin level and number of organ failure instances). A moderate but highly significant correlation was observed between IFN-γ release and CD8⁺ T lymphocyte count (r=0.52, P <0.001). As a non-antigen-dependent mitogen, PHA enables the global assessment of lymphocyte IFN-γ-releasing capacity. Our whole-blood results suggest that PHA-IGRA testing may provide a useful parameter for characterizing immune dysfunction in patients with cirrhosis. These findings require further validation in larger cohorts to better define the clinical applicability of PHA-IGRA testing.

Background: Excessive repeat sequence expansion in the human genome causes neurodegenerative diseases. Conventional tandem repeat expansion detection methods often fail to amplify GC-rich repeat regions and cannot help in simultaneously detecting multiple regions. To overcome those limitations, we tested the PacBio PureTarget repeat expansion panel, a new target enrichment test without PCR amplification, and compared its performance with that of conventional repeat expansion detection methods, using clinical and reference samples.

Methods: We used the PacBio PureTarget repeat expansion panel, which targets 20 genes with clinically relevant repeat regions, to assess eight samples from the Coriell Institute for Medical Research (Camden) and six patient samples (previously tested for FMR1 repeat expansions via repeat-primed [RP] PCR). Data were analyzed using the tandem repeat genotyping tool.

Results: For all samples tested, the long-read sequencing results showed 100% concordance with the RP-PCR or Southern blotting results. Discrepancies in repeat counts were observed in a few alleles, with a maximum difference of 157 motifs in DMPK. The method successfully quantified long repeats in FMR1, demonstrating its applicability.

Conclusions: The PacBio PureTarget repeat expansion panel, which uses targeted enrichment based on clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 and long-read sequencing, is a promising approach for repeat expansion detection, overcoming the limitations of conventional methods. This approach enabled parallel analysis of multiple candidate genes implicated in neurodegenerative diseases with overlapping clinical features, supporting its potential integration into future clinical diagnostic workflows.

Background: Measurable residual disease (MRD) in B-cell ALL (B-ALL) can be detected via next-generation sequencing (NGS) of Ig gene rearrangements, particularly in Ig heavy chain (IGH) genes. Ig light chain rearrangements (Ig kappa chain [IGK] or Ig lambda chain [IGL]) are less reliable prognostic markers. We investigated why IGK/IGK-deleting element (IGKDE) rearrangements are unreliable biomarkers for NGS-based MRD monitoring.

Methods: This single-center prospective observational cohort study enrolled 506 pediatric patients newly diagnosed as having B-ALL between 2018 and 2022. NGS was performed on bone marrow samples collected at multiple time points, including the end of induction and consolidation.

Results: Unlike IGH clones, IGK/IGKDE clones persisted in patients in continuous remission; 12.4% of IGK and 20.0% of IGKDE clones remained detectable at 12 months in these patients. Of these persistent clones, 88.5% (IGK) and 94.1% (IGKDE) also appeared in healthy individuals, with probabilities >10%. Non-specific clones had N-region lengths ≤ 4 nucleotides, whereas leukemia-specific clones varied in this regard. Filtering out nonspecific clones with N-region length <4 nucleotides at diagnosis reduced the ratio of persistent IGK clones at 12 months in patients in remission from 12.4% to 2.5%.

Conclusions: Non-specific clones reduce the reliability of IGK rearrangements for NGS-based MRD monitoring in pediatric B-ALL. Filtering out these clones may enhance accu-racy by reducing false positives.

Background: HbE and α⁰-thalassemia are highly prevalent in Southeast Asia and co-inherited in asymptomatic carriers. α⁰-Thalassemia screening in HbE heterozygotes relies on HbE quantification. We evaluated the performance of a high-resolution HPLC system in quantifying HbE and HbA₂ to distinguish HbE heterozygotes with and without α⁰-thalassemia.

Methods: HbE heterozygotes (N=650) presenting EA patterns on capillary electrophoresis (CE) were analyzed using the Premier Resolution (PR) HPLC system (Trinity Biotech). HbA₂ and HbE levels were quantified, Pearson correlations with CE assessed, and diagnostic cutoffs determined. A prospective cohort of HbE heterozygotes (N=884) was used for validation. α⁰-Thalassemia genotyping was performed using gap-PCR.

Results: PR-HPLC confirmed HbE heterozygosity and successfully separated HbA₂ from HbE. The HbA₂, HbE, and combined HbA₂+HbE levels were consistently higher than but correlated with CE values (HbA₂: bias=1.213, r=0.686; HbE: bias=0.286, r=0.960; combined: bias=1.502, r=0.952). HbE and combined levels were substantially lower in α⁰-thalassemia carriers (14.65%±2.2% vs. 19.38%±2.5%); HbA₂ levels did not significantly differ. HbE cutoff <19% resulted in an area under the curve (AUC) of 0.975, with 100% sensitivity and 91.9% specificity. HbE+HbA₂ <24.0% yielded an AUC of 0.970, with 100% sensitivity and 89.7% specificity. Validation experiments supported the robust performance for HbE detection alone (100% sensitivity, 93.6% specificity, and 94.5% accuracy).

Conclusions: PR-HPLC can quantify HbE precisely, potentially aiding in robust α⁰-thalassemia diagnosis and screening without HbA₂ assessment.

Background: Elderly populations face disproportionate lung cancer burdens; however, biological variation (BV) data for key tumor markers (carcinoma embryonic antigen [CEA], neuron-specific enolase [NSE], cytokeratin 19 fragment [Cyfra21-1], progastrin-releasing peptide [ProGRP], and squamous cell carcinoma antigen [SCC-Ag]) in this demographic remain scarce, impeding personalized result interpretation. This study explored BV for these five lung cancer markers among older Chinese individuals.

Methods: We prospectively enrolled 45 healthy Chinese adults aged 60-75 yrs for six monthly blood collections. BV components (within-participant [CV_I], and between-participant [CV_G]) were estimated using nested ANOVA and Bayesian hierarchical modeling. Real-world data (N=8,003) were analyzed for CEA. Methods followed BV data critical appraisal checklist standards.

Results: All markers showed high individuality, with individuality indices (II) <1.4. CEA exhibited the lowest II of 0.18. Bayesian hierarchical modeling and nested ANOVA produced comparable BV estimates. CEA had higher CV_I (11.3%, 95% confidence interval [CI]: 10.2%-12.5%) than European data (6.4%, 95% CI: 6.0%-6.7%). Cyfra21-1 displayed sex-specific CV_I differences. The CV_I of CEA estimates remained consistent across sampling intervals in direct (1-5 months: 9.7%-12.3%) and indirect methods (1-365+ days: 17.2%-18.1%). Harris-Brown heterogeneity ratio analysis revealed substantial heterogeneity of individual within-participant BV (CV_p(i)) for CEA, supporting CV_p(i)-derived reference change values (RCVs).

Conclusions: This study establishes the first comprehensive BV database for lung cancer biomarkers in elderly Asians. Stable monthly variation enables flexible monitoring protocols. Our findings support implementing personalized RCVs for clinical interpretation and facilitate earlier therapeutic interventions for aging individuals.