Despite the clinical importance of CD4 testing for identifying advanced HIV disease, access to conventional flow cytometry remains limited in many settings. Epigenetic quantitative PCR (qPCR)-based immune cell quantifica...Despite the clinical importance of CD4 testing for identifying advanced HIV disease, access to conventional flow cytometry remains limited in many settings. Epigenetic quantitative PCR (qPCR)-based immune cell quantification represents a molecular alternative that may be compatible with simplified sample types such as dried blood spots (DBS). This study evaluated the analytical performance of an epigenetic CD4+ T-cell qPCR assay (Epimune diagnostics) using DBS samples. Residual EDTA whole blood specimens (n = 150) from HIV patients were applied to Whatman 903 DBS cards and tested following manual DNA extraction and qPCR using the iMune CD4 assay. CD4 counts derived from DBS were compared with reference flow cytometry using the AQUIOS PanLeucogating platform. Agreement was assessed using concordance correlation, Bland-Altman analysis, and percentage similarity. Assay repeatability, batch-related variability, and classification performance at clinically relevant CD4 thresholds were also evaluated. DBS-based epigenetic CD4 quantification demonstrated good agreement with flow cytometry (concordance correlation coefficient 0.91; 95% CI: 0.88-0.93) with a mean bias of -28 cells/μL (-6.9%). Repeatability was acceptable across the measurement range (coefficient of variation 4.0%-11.2%). At a threshold of 200 cells/μL, sensitivity and specificity were 93.8% and 88.9%, respectively. Increased variability was observed in larger manual extraction batches. These findings demonstrate the technical feasibility of epigenetic qPCR-based CD4 quantification from DBS and support further optimisation and validation.
Next-generation sequencing (NGS) is a first-tier test in molecular oncology, capable of detecting sequence variants (SVs) and copy number variants (CNVs). Although most amplicon-based gene panels are not designed to dete...Next-generation sequencing (NGS) is a first-tier test in molecular oncology, capable of detecting sequence variants (SVs) and copy number variants (CNVs). Although most amplicon-based gene panels are not designed to detect segmental chromosomal CNVs, they can still be inferred. This study describes a custom analysis for CNV detection using the amplicon-based Oncomine Comprehensive Assay v3 (OCAv3), for critical glioma biomarkers 1p/19q co-deletion, +7/-10, EGFR amplification and CDKN2A/B homozygous deletion. The segmental CNV calling algorithm leverages vendor normalized gene-level CNV data with inter-sample normalization to control regions. Assay performance was assessed across three cohorts: a retrospective discovery cohort (N=52) for threshold establishment, a prospective validation cohort (N=39) for threshold evaluation against parallel FISH testing, and a prospective clinical cohort (N=53) to assess workflow impact. Positive thresholds were defined to achieve 100% specificity, while permissive thresholds triggered reflex FISH to ensure 100% sensitivity. Samples with <30% tumor cellularity were excluded. Threshold evaluation in the validation cohort demonstrated 100% concordance with FISH across all biomarkers. Across glioma subtypes, OCAv3 enabled integrated classification in most cases, particularly in oligodendroglioma and glioblastoma, by simultaneously assessing SVs and CNVs. Implementation in the prospective clinical cohort reduced required FISH studies by 90%, significantly shortened turnaround times, and decreased molecular testing costs.
Hawkes D, Gurung D, Pineda E
… +10 more, Lee A, Romano J, Ting Keung MH, Silvers J, Steele A, Jayasinghe Y, Brotherton JML, Arbyn M, Wrede CD, Saville M
The first national human papillomavirus (HPV)-based cervical screening programs began in 2017. Since then, a growing list of countries have moved, or want to move, to HPV-based screening. One of the benefits of HPV-based...The first national human papillomavirus (HPV)-based cervical screening programs began in 2017. Since then, a growing list of countries have moved, or want to move, to HPV-based screening. One of the benefits of HPV-based screening is that a sample does not need to be collected from the cervix by a health care practitioner. Self-collection has been demonstrated to give equivalent accuracy as practitioner-collected specimens when a PCR-based clinically validated HPV assay is used. However, there are few clinically validated, PCR-based, HPV assays with on-label claims for self-collection. The current study, Self-Collection or Practitioner-Collection Evaluation 2 (SCoPE2), undertook a Validation of Human Papillomavirus Assays and Collection Devices for Self-Samples and Urine Samples (VALHUDES) protocol evaluation with individuals recruited in a colposcopy population in the context of an HPV-based screening program. SCoPE2 recruited 400 participants who each took a self-collected vaginal sample using a FLOQSwab. A practitioner-collected cervical sample was then collected at colposcopy. HPV testing was performed using both the cobas 4800 and the cobas HPV tests. The self-collected specimens demonstrated equivalent and noninferior relative sensitivity for histologically confirmed cervical intraepithelial neoplasia grade ≥2 (n = 58) when compared with the practitioner-collected specimen for both the cobas 4800 (0.982) and cobas (1.037), but relative specificity was inferior. Additional analyses were undertaken to resolve referral, self-collected, and practitioner-collected discordant results. SCoPE2 demonstrates that the assessed self-collection method is noninferior for the detection of cervical intraepithelial neoplasia grade ≥2 when compared with a practitioner-collected specimen.
Accurate detection of BRCA1 and BRCA2 variants is essential for breast cancer diagnosis. However, the large size of these genes poses challenges for comprehensive analysis using short-read sequencing, which is generally...Accurate detection of BRCA1 and BRCA2 variants is essential for breast cancer diagnosis. However, the large size of these genes poses challenges for comprehensive analysis using short-read sequencing, which is generally limited to coding regions and may miss deep intronic and structural variants. This study evaluated the performance of Oxford Nanopore Technology long-read sequencing (ONT-LRS) for comprehensive BRCA1 and BRCA2 analysis and compared its diagnostic yield with Ion Torrent sequencing. In this retrospective study, DNA samples from 27 individuals with breast cancer were initially analyzed using Ion Torrent sequencing, according to standard clinical workflows. Full BRCA1 and BRCA2 genes were subsequently amplified by long-range PCR and sequenced on R10.4.1 flow cells. Variants identified by ONT-LRS were compared with those detected by Ion Torrent. High concordance was observed between ONT-LRS and Ion Torrent for exonic single-nucleotide variants. Importantly, ONT-LRS identified additional variants not detected by Ion Torrent, including a deep intronic variant predicted to alter splicing and one structural variant. This study suggests that ONT-LRS extends the diagnostic capabilities of short-read sequencing by enabling accurate detection of BRCA1 and BRCA2 deep intronic and structural variants that may otherwise be overlooked, with potential implications for patient management and family counseling.
Lung cancer is one of the most prevalent and lethal malignancies worldwide. Despite recent advancements in precision medicine, early detection and therapeutic monitoring of lung cancer remain challenging. Herein, an endo...Lung cancer is one of the most prevalent and lethal malignancies worldwide. Despite recent advancements in precision medicine, early detection and therapeutic monitoring of lung cancer remain challenging. Herein, an endonuclease-assisted selective exponential amplification (ESEA) platform is presented that selectively depletes wild-type alleles through programmable endonuclease digestion (CRISPR-Cas or restriction enzymes) while simultaneously amplifying mutant alleles, enabling robust and cost-effective detection at mutant allele frequencies as low as 0.00625%. To address the protospacer adjacent motif-site limitations inherent in CRISPR-based enrichment strategies, a primer-directed restriction site programming approach that expands the theoretical coverage to more than 94% of mutations listed in COSMIC (Catalogue of Somatic Mutations in Cancer) is introduced. The ESEA system offers high sensitivity and cost-effectiveness compared with conventional methods, and enables multiplexed detection of key lung cancer hotspot mutations, including EGFR L858R, EGFR exon 19 deletions, EGFR T790M, BRAF V600E, as well as hotspots in KRAS and PIK3CA. In a small-sample-size test, the ESEA system achieved 100% sensitivity and specificity in five pleural effusion samples and a 100% circulating tumor DNA detection rate in six patients with extracranial lesions and disease progression. These results highlight its potential as a cost-effective, highly sensitive, and robust platform for dynamic, real-time companion diagnostics, as well as noninvasive monitoring of treatment response and tumor evolution.
NTRK gene fusions are rare but actionable oncogenic drivers, and their timely detection is critical for guiding tyrosine receptor kinase-targeted therapy. Conventional methods, such as immunohistochemistry, fluorescence...NTRK gene fusions are rare but actionable oncogenic drivers, and their timely detection is critical for guiding tyrosine receptor kinase-targeted therapy. Conventional methods, such as immunohistochemistry, fluorescence in situ hybridization, RT-PCR, and next-generation sequencing, can be limited by variable sensitivity, cost, and slow turnaround times. The Idylla GeneFusion Assay-a rapid, fully automated cartridge-based platform that infers NTRK1/2/3 fusions through 3' to 5' expression imbalance-was evaluated across a retrospective, fusion-enriched cohort of 193 tissue and cytology specimens from The MD Anderson Cancer Center and Mayo Clinic, all of which had prior RNA-based next-generation sequencing profiling. Of 108 NTRK gene fusions detected by next-generation sequencing profiling, 67 (62.0%) were reported as detected by the Idylla GeneFusion Assay. When analysis was restricted to definitive detected and not detected results (n = 147), the assay demonstrated high concordance with next-generation sequencing, with positive percentage agreement and negative percentage agreement of 95.7% and 100%, respectively, with the strongest performance for NTRK3. Including equivocal results as positive increased sensitivity (positive percentage agreement, 96.3%) with a modest reduction in specificity (negative percentage agreement, 93.9%; n = 190). Most invalid results were attributable to RNA degradation and resolved with freshly cut sections. These findings support the Idylla GeneFusion Assay as a rapid and practical frontline tool for NTRK fusion detection, particularly in settings with limited tissue or need for expedited decision-making, with reflex next-generation sequencing recommended for equivocal cases or when fusion partner identification is required.
MET exon 14 skipping mutations in non-small-cell lung cancer are important biomarkers for targeted therapy, making accurate detection essential. This study analyzed 379 non-small-cell lung cancers with mutations in MET e...MET exon 14 skipping mutations in non-small-cell lung cancer are important biomarkers for targeted therapy, making accurate detection essential. This study analyzed 379 non-small-cell lung cancers with mutations in MET exon 14 and adjacent splice sites using DNA- and RNA-based next-generation sequencing (NGS). The 379 samples contained 171 distinct mutations around MET exon 14, highlighting the diversity of these mutations. A total of 114 mutations were analyzed with both a DNA- and an RNA-based NGS assay. Two large deletions and one synonymous splice site causing exon 14 skipping were only detected by RNA-based NGS. Seven single-nucleotide variations in MET exon 14 did not induce skipping. A total of 57 variants could not be analyzed by RNA-based NGS because of insufficient material or RNA quality; among them, 18 were previously reported as skipping mutations, 30 were splice-site insertions/deletions, and 9 remained unclassified. Additionally, data from the first multinational external quality assessment schemes for MET exon 14 skipping mutation testing in formalin-fixed, paraffin-embedded tissue and liquid biopsies, organized by the lead panel institute and Quality in Pathology GmbH, are presented. They showed high success rates (98%) for formalin-fixed, paraffin-embedded tissue, whereas liquid biopsy tests had lower rates (37.5% in 2022, and 63% in 2024), primarily because of low allelic fractions and intronic deletions. This study highlights the importance of analyzing both DNA and RNA, urging improvements in sensitivity, coverage, and bioinformatics.
Acute leukemias of ambiguous lineage (ALALs) are rare acute leukemias with poor prognosis, encompassing acute undifferentiated leukemia and mixed-phenotype acute leukemia. The fifth edition of the World Health Organizati...Acute leukemias of ambiguous lineage (ALALs) are rare acute leukemias with poor prognosis, encompassing acute undifferentiated leukemia and mixed-phenotype acute leukemia. The fifth edition of the World Health Organization classification of hematolymphoid tumors recommends excluding ALAL cases with myelodysplasia-associated mutations and/or cytogenetic abnormalities from classification as acute undifferentiated leukemia or mixed-phenotype acute leukemia, instead categorizing them as acute myeloid leukemia with myelodysplasia (AML-MR; hereafter referred to as AML-MR/ALAL), a distinct entity with limited genetic and clinical characterization to date. In this study, the genetic and clinical characteristics of AML-MR/ALAL were investigated in comparison with those of ALAL and AML-MR. RUNX1 (7/11; 63.6%), WT1 (3/11; 27.3%), DNMT3A (2/11; 18.2%), TET2 (2/11; 18.2%), BCORL1 (2/11; 18.2%), and TP53 (2/11; 18.2%) were identified as the most frequently mutated genes in AML-MR/ALAL. Moreover, AML-MR/ALAL shared genetic features with AML-MR and was distinct from ALAL. Although no significant difference in overall survival was observed between AML-MR/ALAL and ALAL, patients with AML-MR/ALAL showed a trend toward worse prognosis. In summary, these findings suggest that AML-MR/ALAL may be more appropriately classified as AML-MR; however, validation in larger cohorts is required.
Yang Y, Hammond NA, Tong PW
… +19 more, Ho C, Watson N, Liao L, Tan T, Chen L, Dumas K, Fisk D, Grove ME, Hale C, Ng Z, Park Y, Reavey C, Smith EE, Weymouth KS, White S, Spiteri E, Qiao W, Ashley EA, Scott SA
Genome sequencing is a commonly used platform for genetic disease research; however, most clinical laboratories use enrichment-based targeted sequencing for diagnostic panels and exome testing. To facilitate the implemen...Genome sequencing is a commonly used platform for genetic disease research; however, most clinical laboratories use enrichment-based targeted sequencing for diagnostic panels and exome testing. To facilitate the implementation of clinical genome-based panel testing, short-read genome sequencing (≥40×) was subjected to analytical validation, including single-nucleotide variant (SNV), insertion/deletion (indel), and copy number variant (CNV) accuracy and reproducibility/repeatability, and specimen types (blood, saliva, and assisted saliva). Recall and precision for SNVs/indels (<50 bp) within nondifficult genome regions across Genome in a Bottle/National Institute of Standards and Technology reference materials were >99.9%, and nonreference genotype concordance for SNVs/indels within nondifficult Reference Sequence CDS regions across all reproducibility/repeatability assessments was >99%. In addition, genome sequencing of CNV (1.1 to 7270 kilobase) control specimens had clinical sensitivity, specificity, and accuracy for deletions (n = 14) and duplications/triplications (n = 11) of >99.9%. Nonreference genotype concordance values for deletions and duplications (≥50 bp) within nondifficult genome regions across all reproducibility/repeatability assessments were >97% and >81.0%, respectively. Contamination thresholds and mosaicism detection were assessed by sequencing mixed reference material DNAs, which determined that >99% SNV/indel accuracy was maintained up to contamination levels of approximately 9% and that 95% of small variants were detected at a level of approximately 30% mosaicism. Taken together, these data indicate that germline SNV/indel and CNV detection by short-read genome sequencing (≥40×) is accurate and robust, which supports its use as a platform for diagnostic genome-based panel and exome testing.
Non-small cell lung cancer (NSCLC) and colorectal cancer (CRC) are malignancies with numerous actionable mutations. Accurate mutation identification is essential for targeted therapies, highlighting the need for next-gen...Non-small cell lung cancer (NSCLC) and colorectal cancer (CRC) are malignancies with numerous actionable mutations. Accurate mutation identification is essential for targeted therapies, highlighting the need for next-generation sequencing (NGS). This study evaluated the Oncomine Precision Assay (OPA) as an alternative to single-gene panel (SGP) and send-out NGS (SO-NGS) testing in NSCLC and CRC. Turnaround time (TAT), quantity not sufficient (QNS) rates, and detection of National Comprehensive Cancer Network recommended alterations were compared. NSCLC alterations included EGFR, MET exon 14 skipping, ROS1, ALK fusions, RET fusions, ERBB2 mutations, NTRK1/2/3 fusions, BRAF, and KRAS p.G12C. CRC alterations included RET fusions, ERBB2 amplification, NTRK1/2/3 fusions, BRAF, KRAS, NRAS, and microsatellite instability. A total of 74 NSCLC and 72 CRC cases were analyzed concurrently with OPA and SGP and compared with historical SO-NGS data from 163 NSCLC and 49 CRC cases. OPA demonstrated broader mutation coverage than SGP, detecting all evaluated NSCLC alterations and most CRC alterations, while SO-NGS provided the most comprehensive coverage overall. In both NSCLC and CRC, OPA mean TAT was shorter when compared with SGP and SO-NGS. OPA also demonstrated lower QNS rates and comparable or improved detection rates, supporting its use for community-based molecular testing in NSCLC and CRC.
Srinivasan A, Martin R, Allen G
… +35 more, Price C, Miller D, Mittal S, Ledo C, Alvarado E, Arroyo Y, Foltz F, Halle P, Hambalek JA, Abebe A, Liu Y, Sun A, Janwari H, Allen K, Lu HW, Gutierrez N, Nako J, Martinez B, Walsh P, Johnsen N, Ross C, Casarez N, Fiore A, Perez L, Wright D, Babikian S, McDonald J, Kido H, Kuriyama S, Davis R, Heeren T, Miller B, Gill F, Okrongly D, Peytavi R
RT-PCR is the gold standard to determine the etiology of respiratory tract viral infections caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), influenza A, influenza B, and respiratory syncytial viru...RT-PCR is the gold standard to determine the etiology of respiratory tract viral infections caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), influenza A, influenza B, and respiratory syncytial virus (RSV). Multiplex testing panels can test for infections or coinfections with these viruses at the point of care. Treatment using antiviral drugs and/or monoclonal antibodies and public health measures using vaccines and specific infection prevention practices depend on accurate diagnoses. The AMDI Fast PCR Mini Respiratory Panel is a 10-minute RT-PCR test for use at the point of care to detect these viruses from a single anterior nasal swab. It is run on the Fast PCR Instrument with integrated cloud connectivity for data management. The clinical performance of the Fast PCR Mini Respiratory Panel test was established in a multicenter clinical study of 1906 participants using the Cepheid Xpert Xpress CoV-2/Flu/RSV plus test as the comparator. The study population included 242 influenza A-, 77 influenza B-, 71 RSV-, and 87 SARS-CoV-2-positive samples and had an overall percentage agreement of 97.2%, 99.7%, 99.3%, and 99.3%, respectively. Of the total 85 (4.4%) discrepant results, 61 (72%) were associated with a low viral load. The AMDI Fast PCR Mini Respiratory Panel has excellent clinical performance, providing clinically useful information at the point of care in <10 minutes.
Quantitative real-time PCR assays often lack harmonization, leading to variability in viral load reporting. Differences in calibration materials, assay design, extraction methods, and gene targets further complicate stan...Quantitative real-time PCR assays often lack harmonization, leading to variability in viral load reporting. Differences in calibration materials, assay design, extraction methods, and gene targets further complicate standardization. This study evaluated the Qiagen QIAcuityDx digital PCR (dPCR) platform for absolute quantification of adenoviruses and compared its performance with Bio-Rad droplet dPCR and the standard-of-care quantitative real-time PCR assays. Viral loads of commercially available quality control materials, calibrators, and clinical samples were assessed. Whether adenovirus genotype affects quantification precision across platforms was also examined. Replicates of the human adenovirus (HAdV) verification panel and control materials (Bio-Rad HAdV E4 and ZeptoMetrix HAdV B3) were tested using both dPCR systems. Both demonstrated excellent correlation and equivalent analytical sensitivity at 1000 copies/mL. Remnant clinical samples representing 16 HAdV genotypes were tested in serial dilutions with both digital PCR methods and the standard-of-care assay. Strong agreement was observed across assays for viral loads ≥1000 copies/mL, regardless of genotype. The Qiagen QIAcuityDx platform showed an average bias of -0.31 (SD, 0.44) log copies/mL relative to quantitative real-time PCR. These findings demonstrate that dPCR enables accurate and reproducible HAdV quantification and support its utility for calibration and standardization in clinical molecular testing. Despite minor platform differences, both dPCR systems were highly concordant with each other and with standard-of-care quantification.
Acute respiratory infections in children contribute to a significant volume of pediatric emergency department (PED) visits annually. Rapid molecular respiratory pathogen panels (RPPs) have increasingly been integrated in...Acute respiratory infections in children contribute to a significant volume of pediatric emergency department (PED) visits annually. Rapid molecular respiratory pathogen panels (RPPs) have increasingly been integrated into diagnostic workup. This study aims to investigate RPPs' impact on clinical management of children presenting to the PED and the effect of implementing restrictive criteria for RPP orders. Retrospective analysis of PED RPP orders from June 2023 through June 2024 was performed with a randomized subset of 400 patients in two cohorts (infants <1 year old and children 1 to 5 years old) selected for in-depth chart abstraction, including clinical presentation and management, with even distribution of RPP-positive and RPP-negative patients. Of 2052 RPPs performed from children age 5 years or younger, 1464 (71.3%) were positive for one or more targets. Regardless of RPP result or patient age, no significant differences in disease severity (mean Emergency Severity Index of 3.7 versus 3.7) or clinical management (infants, 6.0%; young children, 5.0%) were observed. Applying stewardship criteria retrospectively to both cohorts demonstrated a potential 48% reduction in unnecessary testing by RPP. RPP results, positive or negative for one or more targets, had limited impact on clinical management in the PED. The data support the potential value of stewardship intervention to reduce cost while maintaining critical pathogen identification.
As rates of human papillomavirus-positive (HPV) oropharynx cancer increase, there is increasing need for accurate biomarkers for diagnosis, treatment, and surveillance. The analytical performance of MyHPVscore, a droplet...As rates of human papillomavirus-positive (HPV) oropharynx cancer increase, there is increasing need for accurate biomarkers for diagnosis, treatment, and surveillance. The analytical performance of MyHPVscore, a droplet digital PCR laboratory-developed test, was characterized to detect circulating tumor DNA from multiple high-risk HPV (hrHPV) types (16, 18, 31, 33, 35, and 39) in plasma from patients with HPVcancer. Using Clinical and Laboratory Standards Institute guidelines, MyHPVscore was developed and validated in a Clinical Laboratory Improvement Amendments-certified laboratory. Analytical controls and plasma samples from patients with HPVoropharyngeal squamous cell carcinoma and noncancer controls were used to evaluate sensitivity, specificity, linearity, and analyte stability. MyHPVscore demonstrated high analytical performance for detecting multiple hrHPV types. Limits of blank were 2.7 (HPV16) and 2.6 (other hrHPV types) positive droplets/reaction; limits of detection were 15.2 and 20.8 positive droplets/reaction, respectively. The linear range was 15 to 62,500 targets/mL plasma (HPV16) and 12 to 100,000 targets/mL (other hrHPV types) with strong correlation (R > 0.99). Analytes were stable for 96 hours at -20°C and yielded consistent qualitative results after >400 days of storage. No cross-reactivity was observed between hrHPV types or low-risk types (HPV6 and HPV11). These results support the use of MyHPVscore as a laboratory-developed test for detecting HPVoropharynx cancer. This method establishes an approach and standard controls that can benchmark other emerging circulating tumor DNA assays.
Although structural heart abnormalities are not typically associated with short QT syndrome (SQTS)-related sudden unexpected death, few autopsy studies have examined the underlying pathology and genetic factors of SQTS....Although structural heart abnormalities are not typically associated with short QT syndrome (SQTS)-related sudden unexpected death, few autopsy studies have examined the underlying pathology and genetic factors of SQTS. Therefore, comprehensive pathologic examinations and whole-exome sequencing were conducted in four men (aged 24, 28, 31, and 45 years) with sudden unexpected death and a short QT interval (sQT). No variants were identified in genes currently known to be associated with SQTS. An enrichment analysis was performed to identify potential genetic causes and mechanisms. None of the men had a history of cardiovascular disease, familial sudden death, or arrhythmia. Rare variants in SCN10A, ANK2, KCNQ2, and CACNA1H were detected, potentially associated with cardiac electrophysiology. One case exhibited apical hypertrophic cardiomyopathy with a rare PLEC variant. The other three displayed left ventricular hypertrabeculation with poor compaction, deep recess formation, myocardial fibrosis, micronecrosis, and minimal inflammatory cell infiltration. The enrichment analysis indicated that these variants were associated with cardiac electrophysiology and morphogenesis. These results showed that individuals with sQT may be at risk of sudden death even without a clinical or family history. This risk may be increased by cardiomyopathy-related gene variants in preclinical or early disease stages. Electrocardiographic evaluation to identify sQT cases followed by morphologic and genetic evaluations improves the assessment of a sudden death risk in individuals with sQT.
Post-transplant engraftment monitoring is essential to assess the risk of complications after allogeneic hematopoietic transplantation, such as graft failure and disease relapse. It is based on the analysis of the percen...Post-transplant engraftment monitoring is essential to assess the risk of complications after allogeneic hematopoietic transplantation, such as graft failure and disease relapse. It is based on the analysis of the percentage of chimerism detected in the recipient. Chimerism analysis has been routinely performed using short tandem repeats (STRs). Next-generation sequencing (NGS) has made possible the use of other genetic markers, such as single-nucleotide polymorphisms and insertions/deletions (indels). This study evaluated the performance characteristics of the NGStrack assay from GenDx to verify its accuracy, sensitivity, and reproducibility. It was determined that the assay can detect DNA contributed by donor and recipient within the range of 0.5% to 99.5%. The results of this assay were compared with an STR-based method, and examples of clinical utilization of the assay were provided using patient cases, including chimerism analysis on different cell subsets. It was confirmed that chimerism analysis using indel markers provides an increased sensitivity with respect to the reported sensitivity of assays using STR. The sensitivity for STR-based methods is in the range of 1% to 5%, whereas the sensitivity for the indel NGS-based method assessed here is 0.5%. The increased sensitivity and precise correlation between chimerism results with clinical events validates the usefulness of this assay for early diagnosis of disease relapse and graft failure and allows for more precise clinical management.
Interpretation of somatic variants in clinical oncology requires integration of gene-specific biology, tumor context, and multiple evidence sources. Although large language models (LLMs) can assist variant interpretation...Interpretation of somatic variants in clinical oncology requires integration of gene-specific biology, tumor context, and multiple evidence sources. Although large language models (LLMs) can assist variant interpretation, concerns remain regarding reproducibility, safety, and dependence on opaque model behavior. A rule-constrained LLM-based decision support framework, Oncology Logic-Informed Variant Evaluator (OLIVE), was developed and evaluated for somatic variant interpretation in a clinical molecular pathology service. OLIVE summarizes multiple data sources and applies explicit gene-specific guidance files to generate structured prompts for final classification and interpretation. Performance was assessed on 200 consecutive clinical tumor next-generation sequencing cases comprising 1437 variant observations (1228 unique variants). Concordance with historical laboratory classifications was evaluated for reportable (pathogenic/likely pathogenic) versus variant of uncertain significance determinations. Across three replicates, mean concordance with historical interpretation was 97.5% (range, 97.4% to 97.6%). Forty-two variants (2.9%) showed discordance in at least one replicate, with most discordances being consistent across runs. Blinded post hoc expert adjudication of discordant variants demonstrated interpretive variability favoring the laboratory or OLIVE classification in equal proportions (21 versus 21 variants). Discordances predominantly reflected intrinsic interpretive ambiguity, evidence evolution, and rule stringency rather than model instability. Similar results with a different LLM indicate performance is not model dependent and is primarily driven by expert guidance. These results indicate that OLIVE can reproducibly support expert somatic variant interpretation in a real-world clinical setting.