Liquid biopsy offers a promising noninvasive alternative for tissue sampling in solid cancers. Saliva, an easily accessible biofluid, can harbor tumor DNA, yet its clinical utility compared with plasma circulating tumor...Liquid biopsy offers a promising noninvasive alternative for tissue sampling in solid cancers. Saliva, an easily accessible biofluid, can harbor tumor DNA, yet its clinical utility compared with plasma circulating tumor DNA (ctDNA) in head and neck squamous cell carcinoma (HNSCC) remains uncertain. Tumor samples from patients with HNSCC underwent next-generation sequencing to identify TP53 mutations. Matched plasma and saliva cell-free DNA (cfDNA) samples were analyzed for the presence of tumor-specific mutations. Pathologic features and survival data were evaluated in relation to mutation detectability in biofluids. Overall, TP53 mutations were detected in 64 of 85 tumors (75%). Liquid biopsy analysis included 36 plasma and 21 saliva samples from 40 patients. Plasma ctDNA was detected in 68% of oral cavity squamous cell carcinoma compared with 29% of laryngeal cancer cases, and it was associated with nodal metastases (P = 0.034). Detection of ctDNA showed a trend toward worse progression-free survival (37.4 versus 68.5 months; P = 0.134). Tumor mutation was identified in saliva cfDNA in 57% of cases, irrespective of disease stage or presence of regional metastases. Plasma ctDNA emerged as a potential prognostic marker in HNSCC, whereas mutated saliva cfDNA, although frequently detectable, lacked prognostic value or correlation with adverse pathologic features. Further research is warranted to elucidate the mechanisms governing tumor DNA shedding into saliva and to define its clinical applications.
Fragile X syndrome (FXS) is the most common cause of intellectual disability. It is usually caused by the expansion of the CGG trinucleotide repeat (>200 repeats) in FMR1, resulting in DNA hypermethylation and gene silen...Fragile X syndrome (FXS) is the most common cause of intellectual disability. It is usually caused by the expansion of the CGG trinucleotide repeat (>200 repeats) in FMR1, resulting in DNA hypermethylation and gene silencing. Conventional FXS diagnosis is based on a combination of PCR and Southern blot (SB) analysis, which is technically challenging and labor-intensive. Methylation-specific triplet-primed PCR (msTP-PCR) has been proposed to overcome these limitations in detecting FMR1 expansion and methylation status. However, its performance in FXS diagnosis in clinical laboratory settings has not been extensively studied. An msTP-PCR assay was validated, implemented, and compared versus the conventional diagnostic protocol in clinical samples. A total of 420 clinical samples (339 male subjects and 81 female subjects) previously genotyped for FMR1 CGG repeat expansion using reference methods (PCR-based methods and/or SB analysis) were investigated using msTP-PCR. The results showed high concordance with the reference results for allele categorization, repeat number correlation, and methylation status. However, discordant results were observed in rare cases of female patients with complex mosaic normal, premutation, and full mutation alleles, which need further confirmation via SB analysis. Nonetheless, these results confirm that the msTP-PCR method is a useful alternative technique for FXS diagnosis and prenatal testing, as it is rapid, reliable, cost-effective, and can potentially reduce the requirement for SB analysis.
Next-generation sequencing (NGS) has become integral to the clinical workup of blood cancers. However, there remain barriers to implementing clinical NGS, including the separate workflows required for DNA and RNA sequenc...Next-generation sequencing (NGS) has become integral to the clinical workup of blood cancers. However, there remain barriers to implementing clinical NGS, including the separate workflows required for DNA and RNA sequencing, complex bioinformatics analyses, and long turnaround times. Duoseq has been developed as a comprehensive, kitted solution to these barriers and implemented as a genomic profiling tool for blood cancers to simultaneously detect single-nucleotide variants (SNVs), small insertions/deletions (indels), and structural variants (SVs; translocations and fusions). Additionally, Duoseq can evaluate numerous other blood cancer diagnostic markers, including gene expression, copy number alterations, cell of origin, oncogenic viral status (eg, Epstein-Barr virus), B-/T-cell receptor clonality, Ig heavy chain mutational status, and diffuse large B-cell lymphoma subtypes. Analytical and clinical validation of Duoseq was performed in parallel at two clinical institutions. Limit of detection was confirmed as 5% variant allele frequency for SNVs, 10% variant allele frequency for indels, and ≥20% tumor purity for SVs. Repeatability studies showed >99% intrarun and interrun positive predictive value and >96% percentage positive agreement. Duoseq was run on formalin-fixed, paraffin-embedded biopsy specimens (N = 197), using NGS and/or fluorescence in situ hybridization as orthogonal comparison. SNVs, indels, and SVs achieved accuracy of >95%. These results establish Duoseq as a genomic profiling tool for blood cancers that can enable laboratories to provide critical diagnostic information in a time- and cost-effective manner.
This retrospective study was designed to demonstrate the clinical utility of two diagnostic tests, the FoundationOneCDx (F1CDx) and Exact Sciences Resolution homologous recombination deficiency (HRD; Resolution HRD assay...This retrospective study was designed to demonstrate the clinical utility of two diagnostic tests, the FoundationOneCDx (F1CDx) and Exact Sciences Resolution homologous recombination deficiency (HRD; Resolution HRD assay) as clinical trial-enrollment assays to identify patients with metastatic castration-resistant prostate cancer harboring homologous recombination repair (HRR) gene alterations. Tumor tissue and plasma collected from patients with metastatic castration-resistant prostate cancer in the phase 3 MAGNITUDE study were tested using the F1CDx tissue assay and/or the Resolution HRD plasma assay. Patients with HRR alterations were randomized (1:1) to receive niraparib (NIRA) and abiraterone acetate (AA) + prednisone (NIRA + AAP) or placebo and AAP (PBO + AAP; NCT03748641). Efficacy was based on radiographic progression-free survival (primary end point), time tosymptomatic progression, time to cytotoxic chemotherapy, and overall survival as secondary end points (interim analysis 1). Of 423 HRR patients, 291 (68.8%) were HRR positive (HRR) by F1CDx [BRCA: 162 (38.2%)], and 38 (8.9%) were HRR negative by F1CDx but HRR by Resolution HRD assay. Also, 277 of 423 (65.5%) were HRR by Resolution HRD assay [BRCA: 150 (35.5%)], and 124 (29.3%) were HRR negative by Resolution HRD assay but HRR by F1CDx assay. Clinically meaningful benefits for all end points were comparable for BRCA and HRR patients detected by either tissue or plasma assays. These results demonstrated the clinical utility of both tissue and plasma assays in identifying patients for NIRA + AAP treatment.
Jani K, McMillen T, Lee C
… +6 more, Miranda E, Rizzo S, Aslam A, Bubb T, Kamboj M, Babady NE
J Mol Diagn
· 2025 Dec · PMID 41130562
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Candida auris is a high-priority multidrug-resistant yeast. In New York, C. auris is reportable, and surveillance screening is recommended for high-risk patients. This study provides a retrospective review of the yield a...Candida auris is a high-priority multidrug-resistant yeast. In New York, C. auris is reportable, and surveillance screening is recommended for high-risk patients. This study provides a retrospective review of the yield and characteristics of C. auris cases detected through active surveillance testing at a tertiary cancer care center. Testing for C. auris was performed using a laboratory-developed real-time PCR test on patients admitted to the intensive care unit. Sample sources included axilla, groin, and nares swabs. Candida auris-positive PCR samples were further tested by culture with recovered isolates identified and further characterized by whole-genome sequencing and antifungal susceptibility testing. From 2019 to 2023, 27,299 samples were tested, with 139 positive samples (0.5%) on 16 unique patients. Positive swabs included 40 of 139 (28.7%) axilla, 44 of 139 (31.7%) nares, and 55 of 139 (39.5%) groin. A total of 134 of 139 (96.4%) samples were cultured, and 76 of 134 (56.7%) were positive in culture. An increase in positive swabs was noted. Four patients developed disseminated infections following a positive surveillance swab. Whole-genome sequencing classified all isolates as clade I, except for one isolate identified as clade III. Resistance to fluconazole was detected in 80% of isolates. Although the positivity rate remained low in this patient population, the recent increase in cases of C. auris nationwide underlies the need for active surveillance to prevent spread of this multidrug-resistant organism.
Liquid biopsies quantifying mutations in circulating tumor DNA by targeted next-generation sequencing have been gaining popularity. They are performed by various library preparation methods, each with distinct advantages...Liquid biopsies quantifying mutations in circulating tumor DNA by targeted next-generation sequencing have been gaining popularity. They are performed by various library preparation methods, each with distinct advantages and limitations. This work introduces Bridge Capture, a novel technology that goes beyond the advantages of market-leading liquid biopsy technologies, eliminating the need to compromise between scalability, cost-efficiency, sensitivity, or panel size. Twenty-four matched contrived colorectal biospecimens mimicking circulating tumor DNA were analyzed by Bridge Capture, Archer LIQUIDPlex, and AmpliSeq CHP version 2 for Illumina to compare variant allele frequency (VAF) detection. Bridge Capture was evaluated for sequencing depth requirement, interlaboratory reproducibility, automatization, and panel scalability. Of all methods, Bridge Capture detected the lowest VAF, and all VAFs strongly correlated with Archer LIQUIDPlex (R = 0.995) and AmpliSeq CHPv2 for Illumina (R = 0.988). Owing to its unique design, the Bridge Capture is compatible with the commonly used next-generation sequencing platforms and effectively uses sequencing capacity, permitting affordable and sensitive variant detection. The method demonstrated high reproducibility across independent laboratories and between automated and manual workflow. The panel size was increased by 300% and had negligible impact on performance and cross-reactivity of the probes, implying high multiplexing capabilities. Taken together, Bridge Capture is a cost-efficient, simple, rapid, and sensitive cancer diagnostics tool that has a potential to significantly improve the detection of mutations.
Preanalytical tissue assessment is an important step in cancer molecular testing; however, its impact on molecular test results has not been systematically evaluated. This study describes a quality-improvement project in...Preanalytical tissue assessment is an important step in cancer molecular testing; however, its impact on molecular test results has not been systematically evaluated. This study describes a quality-improvement project in which routine histology review was implemented at a US molecular diagnostics laboratory. The effects of implementation on laboratory compliance and the analytical performance of a targeted POLE assay were measured as changes in tumor cellularity documentation, tumor sample enrichment (in samples with <40% tumor cellularity), POLE mutation rate, tumor signal intensity, and repeat-testing rate. Endometrial carcinoma samples (N = 1752) and tested for POLE mutations using a multiplex PCR assay. POLE mutation rates were 6.3% and 5.0% before and after intervention, respectively (P = 0.25), with the mutations most commonly detected being p.Pro286Arg (47%) and p.Val411Leu (21%). Documentation of tumor cellularity increased from 29% to 100%, and the rate of tumor enrichment increased from 1.4% to 31.5% (both, P < 0.0001). Mutation signal intensity increased from 0.32 to 0.58, and the repeat-testing rate decreased from 8.8% to 2.3% (P = 0.004 and <0.0001, respectively). Systematic preanalytical histology review was associated with improved analytical performance of a targeted POLE assay, accompanied by compliance in tumor cellularity documentation, increased tumor enrichment, and decreased repeated testing, supporting preanalytical assessment in improving somatic mutation detection in pathology specimens with low tumor content.
Saint-Charles A, Masliah-Planchon J, Saberi-Ansari E
… +45 more, Bellini A, Bernkopf M, Font de Mora J, Noguera R, Van Roy N, Goodman A, Vicha A, Attignon V, Combaret V, Beiske K, Martinsson T, Schoumans J, Rossing M, Tops B, Westermann F, Cotteret S, Fischer M, Birger Y, Mazzocco K, Chesler L, Betts D, Cowley M, Capasso M, Bobin C, Iddir Y, Zaidi S, Carcaboso AM, Vandermeulen J, Loontiens S, Gaarder J, Ibrahim RR, Rosswog C, Hameiri-Grossman M, Shichrur K, Trahair T, Barahona P, Eggert A, Deubzer HE, Delattre O, Pasqualini C, George S, Tytgat G, Tweddle DA, Taschner-Mandl S, Schleiermacher G
In high-risk neuroblastoma, identification of ALK activating genetic alterations is considered for clinical decision-making at relapse or more recently in frontline treatment. The accurate diagnosis of genetic alteration...In high-risk neuroblastoma, identification of ALK activating genetic alterations is considered for clinical decision-making at relapse or more recently in frontline treatment. The accurate diagnosis of genetic alterations requires harmonization of molecular techniques and reporting, especially when these concern inclusion criteria for clinical trials. Analysis and validation of 14 DNA samples harboring distinct ALK alterations were performed across the 21 SIOPEN (International Society of Paediatric Oncology Europe Neuroblastoma) molecular diagnostic laboratories. These included ALK mutations at or outside hotspots in the tyrosine kinase domain with variant allele frequencies (VAFs) of 1% to 91% or ALK genomic amplification. Each laboratory used their own techniques: ALK amplifications were detected by pan-genomic copy number techniques or fluorescence in situ hybridization, and ALK mutations were characterized by next-generation sequencing techniques. All laboratories correctly identified high-level ALK amplification and ALK mutations within the known hotspots with VAF >5%, with the exception of two cases. Differences in interpretation and reporting were apparent for samples harboring mutations with a VAF <5% or outside known hotspots. These results highlight the importance of standard operating procedures, standardized reporting, and the robustness of ALK genetic testing in the SIOPEN laboratories, and the need for expert discussions regarding atypical ALK alterations, to validate eligibility for ALK targeted treatment in clinical trials.
Gene expression signatures are important for classifying lymphoid malignancies, although routine diagnostic workflows predominantly use immunohistochemical staining and fluorescence in situ hybridization. These tradition...Gene expression signatures are important for classifying lymphoid malignancies, although routine diagnostic workflows predominantly use immunohistochemical staining and fluorescence in situ hybridization. These traditional methods are labor intensive and may misclassify the underlying oncogenic signatures, leading to inaccurate prognostication. To address this issue, an RNA expression panel was developed, the Lymphoma Expression Analysis (LExA120) 120 gene expression panel, using the NanoString platform for rapid, modular analysis of various lymphoma subtypes. The LExA120 panel targets 95 genes and 25 housekeeping genes to evaluate aggressive B-cell lymphomas, including: diffuse large B-cell lymphoma cell-of-origin, dark zone, and primary mediastinal large B-cell lymphoma signatures; Epstein-Barr virus (EBV) status; and a classical Hodgkin lymphoma posttransplant risk. Fifty-four formalin-fixed, paraffin-embedded tissue samples were tested with known diagnoses and 51 samples with known EBV status. The panel showed high concordance with previously validated methods according to Pearson correlation coefficients of the signature scores. The assay also displayed high reproducibility in repeated tests and across different clinical laboratories. This study confirmed the panel's ability to stratify EBV-positive and EBV-negative lymphomas with high diagnostic certainty. Although EBER in situ hybridization confirmation was needed in approximately 12% of cases, synergizing with traditional techniques may facilitate more rapid and cost-effective diagnoses. The LExA120 panel offers a multiplexed approach to lymphoma classification, enhancing the efficiency and accuracy for subtyping lymphomas.
Maqsood M, Toubia J, Wadham C
… +11 more, Shanmuganathan N, Shahrin NH, Fernandes A, McConnell J, Saunders V, Kaczorowski D, Kenyon RR, Lin M, Hughes TP, Kok CH, Branford S
RNA-based targeted sequencing aids the detection of several types of variants in hematologic and other malignancies, including splice-altering variants. However, accurately identifying clinically relevant mis-splicing ev...RNA-based targeted sequencing aids the detection of several types of variants in hematologic and other malignancies, including splice-altering variants. However, accurately identifying clinically relevant mis-splicing events remains challenging because of the inherent complexity of the human transcriptome and the high prevalence of false-positive splice junctions in deep RNA-sequencing data. To address these challenges, SpliceChaser and BreakChaser were developed, which are bioinformatics tools designed to enhance the detection and characterization of relevant splice-altering events. SpliceChaser improves the identification of clinically relevant atypical splicing by analyzing read length diversity within flanking sequences of the mapped reads around the splice junctions. BreakChaser processes soft-clipped sequences and alignment anomalies to enhance the detection of targeted deletion breakpoints associated with atypical splice isoforms generated from intrachromosomal gene deletions. These tools were developed and validated using a cohort of >1400 RNA-sequencing samples from patients with chronic myeloid leukemia. Collectively, SpliceChaser and BreakChaser achieved a positive percentage agreement of 98% and a positive predictive value of 91% for the detection of clinically relevant atypical splice-altering variants or gene deletions in the targeted regions. By integrating splicing and breakpoint detection with robust filtering strategies, these tools facilitate precise identification of clinically relevant variants, paving the way for improved diagnostics and therapeutic strategies in chronic myeloid leukemia and other malignancies.
Corné J, Godey F, Legros A
… +12 more, Castéra L, Krieger S, Chérel M, Cochet A, Le Du F, Bourien H, Deleuze A, Crouzet L, Perrin C, Lefeuvre-Plesse C, Diéras V, De la Motte Rouge T
Analyzing somatic mutations in liquid biopsies poses a real challenge in treating patients with breast cancer. Because of the high sensitivity required to detect circulating tumor DNA, which may be present at low levels,...Analyzing somatic mutations in liquid biopsies poses a real challenge in treating patients with breast cancer. Because of the high sensitivity required to detect circulating tumor DNA, which may be present at low levels, digital PCR analysis seems highly appropriate. However, new targeted next-generation sequencing solutions are now available, enabling highly sensitive multigene analysis that could benefit patients. This study compared the analytical performance of multiplex digital PCR and targeted next-generation sequencing in detecting somatic erb-b2 receptor tyrosine kinase 2 (ERBB2), estrogen receptor 1 (ESR1), and phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit α (PIK3CA) mutations in a series of 32 plasma samples from patients with metastatic breast cancer. Forty-four mutations were detected, with an overall concordance of 95% (90/95) and a high degree of correlation (R = 0.9786). Interestingly, two ESR1 mutations detected in multiplex drop-off digital PCR were also detected by targeted next-generation sequencing with comparable mutant allele frequencies, enabling the identification of these specific variants (p.D538N and p.536LYD>P). Moreover, an additional PIK3CA mutation (p.P539R) was first detected using targeted next-generation sequencing and later confirmed with a newly designed digital PCR assay. Although more expensive than multiplex digital PCR, these new types of small targeted next-generation sequencing gene panels could provide a rapid answer to a specific clinical question with a ready-to-use solution, which could benefit patients.
Genomic mosaicism is underdiagnosed owing to its variable tissue distribution and the limitations of single-tissue testing. Cytogenomic techniques applied across multiple tissues can uncover clinically actionable variant...Genomic mosaicism is underdiagnosed owing to its variable tissue distribution and the limitations of single-tissue testing. Cytogenomic techniques applied across multiple tissues can uncover clinically actionable variants and clarify genotype-phenotype relationships. DNA from 21 patients (14 females and 7 males) with pigmentary mosaicism and global developmental delay was analyzed. Each patient underwent G-band karyotyping and array (Infinium CytoSNP-850K) on peripheral blood, skin fibroblasts, and buccal mucosa samples. Pathogenic or likely pathogenic variants were found in 13 of 21 patients (62%). Of these 13 patients, 10 (77%) exhibited mosaicism, with variant allele fractions as low as 15%. On the basis of tissue distribution, 3 cases were classified as germline events and 10 as somatic mosaicism. Patients with positive findings were subdivided into: i) mosaic numerical chromosomal alterations (n = 6), ii) pathogenic copy number variations (n = 5), and iii) structural rearrangements (n = 2). Notably, several mosaic variants-particularly aneuploidies-were detected exclusively in fibroblast DNA, underlining the added diagnostic yield of multitissue sampling. In this cohort, a multitissue cytogenomic approach achieved a 62% overall diagnostic rate and identified mosaicism in 77% of positive cases. These results support the routine incorporation of genomic arrays with multisample analysis into diagnostic workflows for rare developmental disorders, enhancing detection sensitivity and enabling precise genotype-phenotype correlations.
Bladder cancer (BC) is a lethal urological malignancy, with current diagnostic and follow-up methods being invasive and costly. Cell-free DNA (cfDNA) in liquid biopsies has shown promise in cancer diagnostics, but its fr...Bladder cancer (BC) is a lethal urological malignancy, with current diagnostic and follow-up methods being invasive and costly. Cell-free DNA (cfDNA) in liquid biopsies has shown promise in cancer diagnostics, but its fragmentation and integrity in urine remain underexplored in BC, becoming the aim of this study. cfDNA was isolated from the urine of 156 patients with BC of most stages and 79 matched controls without renal or bladder conditions. The amount of a large (>250-bp) and a nested small (<125-bp) fragment of ACTB, AR, MYC, BCAS1, and STOX1 was quantified by quantitative real-time PCR. Fragmentation and integrity (ratio of large/small) were analyzed with ordinal logistic regression. The increase in the ratio of large/small ACTB fragments and the small fragments of AR and MYC may represent a valuable tool to diagnose and stage BC when classified as both non-muscle-invasive and muscle-invasive BC or considering grades and stages separately. The small fragment of MYC, leading the effect observed, displayed a valuable diagnostic capacity [area under the receiver operating characteristic (ROC) curve = 0.7221; 95% CI, 0.6527-0.7915; P < 0.0001; sensitivity = 50%; specificity = 95%], particularly for muscle-invasive BC (area under the ROC curve = 0.8098; 95% CI, 0.6674-0.9523; P < 0.0001; sensitivity = 70%; specificity = 97%). Herein, the analysis of urine cfDNA fragmentation and integrity of these surrogate markers is proposed as noninvasive biomarkers to diagnose and stage BC. Once validated, the proposed biomarkers could improve patient management by reinforcing or substituting current invasive and expensive techniques.
Abel HJ, Mahgoub M, Davarapalli N
… +13 more, Kodgule R, Miller CA, Fulton RS, Fronick C, Markovic C, Heath S, Payton JE, Jacoby MA, Link DC, Walter MJ, Duncavage EJ, Ley TJ, Spencer DH
J Mol Diagn
· 2025 Dec · PMID 41016628
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Whole-genome sequencing (WGS) is a comprehensive approach for the genomic evaluation of acute myeloid leukemia (AML) and myelodysplastic syndromes (MDS). We recently described a streamlined tumor-only WGS assay (ChromoSe...Whole-genome sequencing (WGS) is a comprehensive approach for the genomic evaluation of acute myeloid leukemia (AML) and myelodysplastic syndromes (MDS). We recently described a streamlined tumor-only WGS assay (ChromoSeq) that uses Illumina short-read sequencing with targeted analysis to detect the full range of clinically relevant somatic mutations. Here we sought to determine the performance of this targeted analysis approach using long-read sequencing data from Oxford Nanopore Technologies and Pacific Biosciences. Samples from 26 patients with AML and MDS were sequenced to a mean of 52× coverage. Head-to-head comparison of reportable somatic variants to standard WGS revealed more than 96% recall and 91% precision for single nucleotide variants for both long-read platforms. Performance was lower for insertion/deletions (66% recall and 42% precision), especially in regions with few phased reads that facilitate accurate variant detection. The long-read platforms were 95% accurate for copy number calls, and they detected all recurrent structural variants with no false-positive findings. In addition, long reads properly identified intronic insertions near repetitive elements that were incorrectly identified as interchromosomal structural rearrangements by standard WGS. These results indicate that targeted, tumor-only analysis of long-read sequence data is a feasible approach for the genomic evaluation of myeloid cancers, and they show the utility of incorporating variants discovered via long-read sequencing to improve variant interpretation in short-read WGS.
Gibson J, El Achi H, Altenburger D
… +11 more, Brown N, Clark AS, Coleman J, Emmadi R, Fussell AM, Hameed M, Jordan D, Laudadio J, Provenzano A, Temple-Smolkin RL, Suciu CG
Despite the increasing availability of next-generation sequencing (NGS) gene panel analysis for cancers, published reports suggest underutilization of testing, citing the shortage of credentialed professionals available...Despite the increasing availability of next-generation sequencing (NGS) gene panel analysis for cancers, published reports suggest underutilization of testing, citing the shortage of credentialed professionals available to assist with the interpretation of test results among the key barriers. Obtaining a multidisciplinary consensus regarding a shared best practice NGS molecular biomarker reporting section template may facilitate introduction and/or increased testing for institutions that adopt similar report structures by improving report effectiveness and efficiency for both health care providers and laboratory professionals, leading to improved patient care. To address this challenge, the Association for Molecular Pathology convened a multidisciplinary collaborative expert working group to identify and utilize best practices from current reporting guidelines and approaches to develop an NGS biomarker report template to optimally present complex molecular profiling information for efficient use by oncologists and other health care providers. Seventeen non-small-cell lung cancer NGS biomarker reports from public, private, and academic laboratories were reviewed, and specific components (eg, report length, color use, formatting, presentation order of information, specific information included or omitted, tables, and figures) were assessed for their ability to be considered provider friendly. Based on this review, public and stakeholder input, available literature, and cumulative professional experience of the working group members, a guideline-concordant reporting template was developed based on expert opinion consensus and made freely available online with planned implementation assessment.
Liquid biopsy assays are transforming precision oncology by providing a less invasive alternative to tissue biopsies. These assays screen for tumor genetic alterations in circulating free DNA, which typically requires de...Liquid biopsy assays are transforming precision oncology by providing a less invasive alternative to tissue biopsies. These assays screen for tumor genetic alterations in circulating free DNA, which typically requires detecting variants at low allele frequencies and, therefore, a high sensitivity and specificity. This international, multicenter analytical performance study evaluated the Hedera Profiling 2 circulating tumor DNA test panel (HP2), a hybrid capture-based next-generation sequencing assay for the detection of somatic alterations in circulating free DNA. Covering 32 genes, HP2 enables the detection of single-nucleotide variants (SNVs), insertions and deletions (Indels), fusions, copy number variations, and microsatellite instability status from a single DNA-only workflow. The analytical performance was assessed using reference standards and a diverse cohort of 137 clinical samples precharacterized by orthogonal methods. In reference standards with variants spiked in at 0.5% allele frequency, sensitivity and specificity were 96.92% and 99.67%, respectively, for SNVs/Indels and 100% for fusions. In clinical samples, SNV/Indel detection showed high concordance (94% for European Society for Medical Oncology Scale of Clinical Actionability for Molecular Targets level I variants) with orthogonal methods. Evidence for solid sensitivity was also found for copy number variation detection and microsatellite instability status determination. Overall, the HP2 assay showed significant potential as a sensitive and efficient pan-cancer test for liquid biopsy testing in a decentralized molecular pathology laboratory setting.
The feasibility of circulating tumor (ct)-DNA assays in first-approach pan-cancer genomic profiling is not well established. Furthermore, low ctDNA levels limit assay sensitivity, which challenges adaptation to clinical...The feasibility of circulating tumor (ct)-DNA assays in first-approach pan-cancer genomic profiling is not well established. Furthermore, low ctDNA levels limit assay sensitivity, which challenges adaptation to clinical genomic profiling. In this study, a 33-gene next-generation sequencing-based ctDNA panel was validated, and these issues were investigated using real-world clinical data. The cohorts included 123 patients who underwent first-approach ctDNA testing, and 48 patients for whom matched tissue was tested at the same time-point. The overall ctDNA assay failure rate was 0%. Insufficient tumor tissue was the main reason for liquid biopsy (69%). The most common primary cancer profiled was lung (39.0%), followed by colon (13.8%), bile duct (8.9%), pancreas (8.1%), and breast and prostate (each 4.1%). Tier I variants were detected in 33.3% of patients, and Tier I or II variants were detected in 65.0% (including 54.5% cholangiocarcinomas, in which tissue biopsy may be challenging due to anatomic location). Compared with matched tissue, ctDNA showed 76% sensitivity for Tier I variants. Actionable variants were increased by 14.3% with ctDNA versus tissue testing alone. ctDNA results preceded tissue results by an average of 21 days. High feasibility, actionability, and sensitivity support ctDNA assays as a potential first-line genomic test, especially in specific tumor types for advanced tumors with insufficient or unavailable tissue.
Baden J, Sausen M, Anfora AT
… +14 more, D'Auria KM, Dickey J, Godsey JH, Guan L, Lococo JS, Mansfield E, Meier KL, Merriam D, Pawloski T, Shukla S, Stetson D, Stewart MD, Wenz P, Leiman LC
J Mol Diagn
· 2025 Oct · PMID 40998494
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Immunotherapies have changed the treatment paradigm for patients with advanced and metastatic solid tumors, with tumor mutation burden representing one approach to identify patients who may experience clinical benefit. C...Immunotherapies have changed the treatment paradigm for patients with advanced and metastatic solid tumors, with tumor mutation burden representing one approach to identify patients who may experience clinical benefit. Circulating tumor DNA-based approaches have been developed for comprehensive analyses of clinically actionable biomarkers; however, blood tumor mutation burden (bTMB) represents a novel, complex biomarker. Although the clinical utility of bTMB is an evolving area of active development and has not led to consistent conclusions across studies, robust analytical validation of the underlying test is important to ensure that technical and biological limitations do not confound clinical interpretation of these results. To this end, the BLOODPAC bTMB Analytical Validation Working Group sought to identify key technical and biological issues associated with analytical validation of bTMB tests, along with a conceptual framework to address these challenges. This publication provides guidance for device manufacturers to demonstrate analytical performance of their test with the understanding that these data would be accompanied by an appropriately designed clinical validation study to demonstrate performance within the intended use population. Therefore, the specific algorithm to determine the bTMB result, along with the associated cutoff, is out of scope of this Perspective. Device manufacturers should also ensure that appropriate pre-analytical variables are accounted for and methods are incorporated to differentiate tumor-specific alterations from those associated with germline polymorphisms or clonal hematopoiesis.
Bellad A, Rangiah K, Chavan S
… +3 more, Warade J, Das B, Pandey A
J Mol Diagn
· 2025 Oct · PMID 40998492
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Hemoglobinopathies are the most common inherited disorders worldwide. Accurate analysis of hemoglobin variants is critical for diagnosis of hemoglobinopathies. Although high-performance liquid chromatography and capillar...Hemoglobinopathies are the most common inherited disorders worldwide. Accurate analysis of hemoglobin variants is critical for diagnosis of hemoglobinopathies. Although high-performance liquid chromatography and capillary zone electrophoresis are widely used as screening tools, they possess inherent ambiguities that often preclude accurate detection of hemoglobin variants. The goal was to develop and optimize a sensitive and specific mass spectrometry-based assay for screening and diagnosis of hemoglobinopathies. A catalog of canonical globin-chain specific peptides as well as mutant peptides corresponding to common hemoglobin variants was generated, and their corresponding heavy synthetic peptide versions were used as internal standards for quantification and calculation of globin chain ratios. Targeted mass spectrometry analysis was performed by coupling liquid chromatography to a triple quadrupole mass spectrometer, which is the most common mass spectrometer used in clinical diagnostics. Dried blood spots from a cohort of 716 individuals (including 211 patients with hemoglobinopathy) were analyzed. The α:β-globin ratios showed a significant difference between normal patients and patients with β-thalassemia, particularly when the disease was homozygous or admixed with structural variants (compound heterozygous). The method presented here permits identification of variants in their homozygous, heterozygous, or compound heterozygous states. The intra-assay and interassay precision CV were both <20%. We envision that such mass spectrometry-based assays could be used as first-line screening assay for hemoglobin variants, including sickle cell disease as well as thalassemias.