Journal of Molecular Diagnostics最新文献

Cell-free DNA of ovarian tumor origin can be detected in samples from the gynecologic tract. This study aims to evaluate how pre-analytical handling, and storage conditions affect DNA profile and integrity in Pap tests, to optimize its potential for detection of ovarian cancers (OC). Analysis of archived Pap tests from OC patients, kept at RT for 48h and stored at -80°C was complemented by in vitro experiments. Temperature-associated effects on DNA fragmentation were evaluated in samples stored at 4°C, -20°C or -80°C. Time-dependent DNA degradation at RT was evaluated in comparison to storage at 4°C (0-96h). Results were validated in prospectively collected Pap tests. The DNA integrity was assessed by fragment analysis. Accumulation of short DNA fragments was observed in archived Pap tests from OC patients. In vitro, fragments of 100-350bp increased 11.5-fold within 48h at RT compared to 1.7-fold when stored at 4°C. Consistent with the in vitro findings, prospectively collected samples showed reduced fragmentation when stored at 4°C compared to RT (p=0.007). Long-term storage at 4°C had a significant negative effect on DNA stability (p=0.013), while freezing slowed down fragmentation. This study highlights the need for optimization of pre-analytical handling for cfDNA analysis. Immediate storage at 4°C after sampling markedly reduces DNA degradation suggesting a simple way to decrease unwanted fragmentation for cfDNA analysis in Pap tests.

Single-nucleotide variants (SNVs) and polymorphisms are characteristic biomarkers in various biological contexts, including pathogen drug resistances and human diseases. Tools that lower the implementation barrier of molecular SNV detection methods would provide greater leverage of the expanding single-nucleotide polymorphism/SNV database. The oligonucleotide ligation assay (OLA) is a highly specific means for detection of known SNVs and is especially powerful when coupled with PCR. Yet, the OLA design process remains intensive, and criteria for success are uncertain. To assist in the design process, this study describes OLAgen, an open-source tool to automate development of OLAs and their coupled PCR assays. The software facilitates alignment of sequences surrounding SNVs and generates ligation probes while screening for dimerization potential. OLAgen successfully produced ligation probes that closely matched previously validated designs for HIV-1, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and KRAS, confirming its reliability and potential for clinical applications. The tool was used to generate new assays targeting Mycobacterium tuberculosis drug resistance and variants in the human JAK2, BRAF, and factor V genes, all of which demonstrated 100% sensitivity and specificity in controlled laboratory experiments. The OLAgen predicted assay designs detected mutant frequencies as low as 1% to 5% in wild-type backgrounds in proof-of-concept laboratory studies. OLAgen represents a significant advancement in accessible assay design, promoting the broader application of OLA technology in clinical and research settings.

To help guide treatment decisions and clinical trial matching, tumor genomic profiling is an essential precision oncology tool. Liquid biopsy, a complementary approach to tissue testing, can assess tumor-specific DNA alterations circulating in the blood. Labcorp Plasma Complete is a next-generation sequencing, cell-free DNA comprehensive genomic profiling test that identifies clinically relevant somatic variants across 521 genes in advanced and metastatic solid cancers. Over 800 unique sequencing libraries across 27 cancer types were evaluated to establish analytical sensitivity, specificity, accuracy, and precision, reproducibility, and repeatability. Sensitivity was verified for each variant type, with a median variant allele frequency (VAF) of 1.25% and 1.27% for panel-wide single-nucleotide variants and insertions/deletions (sequence variants), respectively, with <1% VAF sensitivity observed for clinically actionable variants, 1.72-fold for copy number amplifications, 0.48% fusion read fraction for translocations, and 0.47% sequence mutation VAF for microsatellite instability-high. Analytical specificity was 99.9999% for single-nucleotide variants and 100% for all other variant types. Precision, reproducibility, and repeatability resulted in 94.9% average positive agreement and 99.9% average negative agreement for sequence variants and 100% average positive agreement and average negative agreement for copy number amplifications, translocations, and microsatellite instability. Orthogonal assays were used to assess accuracy, demonstrating an aggregate analytical concordance of 97.4% positive percentage agreement and >99.99997% negative percentage agreement for all variants. Overall, the test demonstrates high sensitivity, specificity, accuracy, and robustness to enable informed clinical decision-making.

Current clinical testing approaches for individuals with suspected imprinting disorders are complex, often requiring multiple tests performed in a stepwise manner to make a precise molecular diagnosis. We investigated whether whole-genome long-read sequencing could be used as a single data source to simultaneously evaluate copy number variants, single-nucleotide variants, structural variants, and differences in methylation in a cohort of individuals known to have either Prader-Willi or Angelman syndrome. We evaluated 25 individuals sequenced to an average depth of coverage of 36× on an Oxford Nanopore Technologies PromethION. A custom one-page report was generated that could be used to assess copy number, single-nucleotide variants, and methylation patterns at select CpG sites within the 15q11.2-q13.1 region and prioritize candidate pathogenic variants in UBE3A. After training with three positive controls, three analysts blinded to the known clinical diagnosis arrived at the correct molecular diagnosis for 22 of 22 cases (20 true positive, 2 negative controls). Our findings demonstrate the utility of long-read sequencing as a single, comprehensive data source for complex clinical testing, offering potential benefits, such as reduced testing costs, increased diagnostic yield, and shorter turnaround times, in the clinical laboratory.

A noninvasive test for earlier detection of pancreatic cancer in individuals at higher risk is currently unavailable. We devised PancSure, a laboratory-developed test based on the protein biomarkers lymphatic vessel endothelial hyaluronan receptor 1 (LYVE1) and regenerating family member 1 β (REG1B), measured in urine by enzyme-linked immunosorbent assay, and commonly used serum/plasma carbohydrate antigen 19.9 (CA19.9), with an updated PancRISK algorithm for data interpretation. The test was validated in 565 patients: 117 asymptomatic patients without any known pancreatic condition or malignancies (21%), 242 symptomatic patients with benign pancreatic diseases (43%), and 206 patients with confirmed cancers (36%); 161 (77.5%) had stage I to II disease, and 45 (22.5%) had stage III to IV disease. PancSure passed all specifications during analytical validation and distinguishes early-stage resectable cancer from asymptomatic individuals with area under the receiver operating characteristic curve (AUC) of 0.93 (95% CI, 0.89-0.97) and 85% to 90% sensitivity (SN) and 78% to 87% specificity (SP); from symptomatic patients with AUC of 0.86 (95% CI, 0.81-0.91) and 83% to 85% SN and 72% to 83% SP; and from all noncancer patients (pooled controls) with AUC of 0.89 (95% CI, 0.84-0.93) and 83% to 85% SN and 78% to 87% SP. PancSure is a noninvasive clinical-grade test with a 48-hour turnover, ready for implementation, providing a viable solution for the earlier detection of pancreatic cancer in at-risk groups for improved patient care.

Testing for somatic mutations in JAK2, MPL, and CALR genes is critical in the diagnosis of myeloproliferative neoplasms (MPNs). However, this testing may have inadvertently led to increased requests to rule out MPN, including clinical situations with low pretest probability. This article examines JAK2, MPL, and CALR testing by next-generation sequencing (NGS) with the goal of formulating practical guidelines to make test use more efficient and effective. NGS results from 1482 patients tested between 2015 and March 2022 were retrieved, along with corresponding bone marrow biopsies and complete blood cell count results performed within 90 days before NGS, and 245 cases (16.5%) were positive for pathogenic variants in JAK2, MPL, or CALR genes. The findings showed an increase in the proportion of positive cases with patient age, and a statistically significant difference in red blood cell counts and platelet counts among patients with positive versus negative results. Using these factors, simple algorithms were constructed to predict positive results with a maximum sensitivity of 91%, while potentially eliminating 28% of negative test results. However, these models still failed to identify approximately 9% of patients with MPNs. Among these missed patients, many had either primary myelofibrosis or myelodysplastic syndrome/MPN. Considering a simple triage model to help guide MPN testing could represent a more cost-effective approach, particularly if missed patients could be further reduced.

Next-generation sequencing (NGS) has applications in research, epidemiology, oncology, and infectious disease diagnostics. Wide variability exists in NGS wet laboratory techniques and dry laboratory analytical considerations. Thus, many questions remain unanswered when NGS methods are implemented in laboratories for infectious disease testing. Although this review is not intended to answer all questions, the most pressing questions from a public health and clinical hospital-based laboratory perspective will be addressed. The authors of this review are laboratory professionals who perform and interpret severe acute respiratory syndrome coronavirus 2 NGS results. Considerations for pre-analytical, analytical, and postanalytical NGS will be explored. This review highlights challenges for molecular laboratory professionals considering adopting or expanding NGS methods.

Medical exome sequencing pipelines consist of various preprocessing steps to prioritize credible causal variants before a pathologist or variant curation scientist manually interprets potential findings that are then reported to patients. The variant allele frequency (VAF), reported as the fraction of sequencing reads supporting a variant call, can be used to screen for technical artifacts, yet a specific filtering threshold has yet to be established. A total of 13,122 manually curated variants, sequenced from 289 patients using the Agilent SureSelect Focused Exome enrichment kit at the University of Kentucky Clinical Genomics laboratory from October 2019 to May 2023, were evaluated. Totals of 278 single-nucleotide polymorphisms (SNPs) and 3340 SNPs as technical artifacts are clinically reported. All reported variants had a VAF between 0.33 and 0.63, and 82% (2725/3340) of sequencing artifacts had a VAF of <0.33. It is proposed that removing SNPs in which the VAF is less than approximately 0.30 reduces manual curation time by approximately 20% while capturing all medically relevant variants in medical exome sequencing data sets.