Wojtaszewska M, Szelest M, Szarawarska M
… +27 more, Grzyb J, Blajer-Olszewska B, Gniot M, Jaskuła E, Dybko J, Soin M, Wasilewska K, Czekalska S, Zawada M, Wojtas M, Solarska I, Kwak A, Wójcik P, Nowak-Ozimek E, Kowalik A, Stokłosa T, Chudy A, Rzepecka IK, Tysarowski A, Szymanowska S, Rewus J, Morgut-Klimkowska M, Donarska E, Własiuk P, Dudziński M, Skoczylas T, Markiewicz M
Somatic hypermutation (SHM) status of IGHV gene, despite being a mature diagnostic biomarker in chronic lymphocytic leukemia (CLL), poses serious methodological problems for molecular laboratories. They may choose betwee...Somatic hypermutation (SHM) status of IGHV gene, despite being a mature diagnostic biomarker in chronic lymphocytic leukemia (CLL), poses serious methodological problems for molecular laboratories. They may choose between inefficient Sanger sequencing protocols and expensive, recently developed next-generation sequencing-based methods. The performance of both types of methods seemed incomparable, and concerted validation of different protocols between laboratories was inconsiderable. Here, a new tagmentation-based approach to sequencing of IGHV locus is presented, which is agnostic to the amplification protocol used and enables direct comparison of the amplicons and libraries dedicated to different platforms (Sanger, IonTorrent, and Illumina). To demonstrate its potential, the 12 associated molecular diagnostics laboratories were asked to amplify an artificially prepared oligoclonal DNA sample containing a near-equimolar mixture of IGHV clonotypes from six different classes. The PCR products collected from laboratories were then tagmented and sequenced according to the common TAG-CLL workflow. The productivity, degree of germline identity, and SHM status concordance between laboratories have been analyzed. Moreover, systematic biases toward uneven amplification of different clonotypes and the prevalence of accidental artifacts in vitro and in silico have been evaluated, providing a framework for future validation of IGHV SHM methods and next-generation sequencing immunoinformatic pipeline benchmarking.
Cervical cancer screening is evolving, with guidelines increasingly favoring high-risk human papillomavirus (hrHPV) nucleic acid amplification tests (NAATs) as a primary method. Although hrHPV NAATs offer high sensitivit...Cervical cancer screening is evolving, with guidelines increasingly favoring high-risk human papillomavirus (hrHPV) nucleic acid amplification tests (NAATs) as a primary method. Although hrHPV NAATs offer high sensitivity for HPV-driven cervical cancers, Papanicolaou (Pap) cytology can detect additional gynecologic malignancies, including HPV-independent ones. This study evaluated the Hologic Aptima hrHPV NAAT and Pap co-testing using 61,089 samples from patients aged ≥21 years screened between 2018 and 2023. The cohort was predominantly White (78.4%), with 16.5% Black, 4.1% Asian, and 38.9% Hispanic subjects. Overall percentage agreement (PA) between hrHPV NAAT and Pap was 87.9%, with high negative PA (93.6%) and lower positive PA (52.3%). Excluding atypical squamous/glandular cell abnormalities improved overall and positive PA to 93.0% and 78.3%, respectively. The Aptima hrHPV NAAT showed high sensitivity for high-grade squamous intraepithelial lesions (98.3%) and squamous cell carcinoma (90.0%) but lower sensitivity for low-grade lesions (73.3%), adenocarcinoma (13.3%), and other malignancies (28.6%). Notably, 29 high-grade lesions or malignant cases had abnormal Pap but were hrHPV negative; 24 (82.8%) were histologically confirmed, including endometrial, cervical, ovarian, and fallopian tube carcinomas (mostly non-HPV-related). These findings emphasize cytology's role in detecting malignancies potentially missed by hrHPV testing alone and support co-testing in diverse populations. The Aptima hrHPV NAAT is not approved by the US Food and Drug Administration for primary HPV screening, reinforcing this need.
Nodal T follicular helper cell lymphoma (nTFHL) can be difficult to diagnose because it often shows features of immune dysregulation and can have a low number of neoplastic T cells in involved lymph nodes. The analysis o...Nodal T follicular helper cell lymphoma (nTFHL) can be difficult to diagnose because it often shows features of immune dysregulation and can have a low number of neoplastic T cells in involved lymph nodes. The analysis of T-cell receptor (TR) gene rearrangements by next-generation sequencing (NGS) and the conventional EuroClonality/BIOMED-2 were performed to compare their performance on this challenging diagnosis. DNA was extracted from 32 formalin-fixed, paraffin-embedded nTFHL samples from two pathology archives. NGS amplicon-based analysis of TRBV-TRBD-TRBJ, TRBD-TRBJ, and TRGV-TRGJ rearrangements was performed using the two-step PCR protocols developed by EuroClonality. The nucleotide sequences were analyzed for abundance, clonotype, and functionality. Both the NGS-based and the conventional clonality assays resulted in a high detection of clonality (97% and 94%, respectively), including both monoclonal and biclonal cases. There was an overrepresentation of TRBV20-1 and TRBV19 gene use that was in line with the frequent use of these genes in T cells of reactive lymph nodes and tonsils. The NGS-based approach detected two or more clonal targets in all clonal samples, whereas the conventional assay detected a single (isolated) dominant rearrangement in three cases. Hence, NGS enables more reliable detection of even small clones, as is frequent in nTFHL. NGS-based TR rearrangement analysis provides the abundance, the sequences, clonotypes, and productivity of the clonal rearrangements and thus stresses the need for novel guidelines for interpretation.
Fluorescence in situ hybridization (FISH) using a break-apart probe (BAP) design is a rapid, clinically useful method for targeted evaluation of gene rearrangements in formalin-fixed, paraffin-embedded tumors. Although c...Fluorescence in situ hybridization (FISH) using a break-apart probe (BAP) design is a rapid, clinically useful method for targeted evaluation of gene rearrangements in formalin-fixed, paraffin-embedded tumors. Although clinically validated BAP FISH assays usually yield unequivocal positive or negative results, rare tumors yield equivocal FISH results. This study had two aims: to summarize typical and atypical BAP FISH results on 56,584 formalin-fixed, paraffin-embedded solid tumors over approximately one decade of clinical testing; and to investigate the clinical utility of RNA sequencing (RNA-seq) for tumors with equivocal FISH results. Of 8586 (15.2%) cases with abnormal FISH results reported, 748 (8.7%) were equivocal. RNA-seq was performed on 113 tumors, and oncogenic fusions involving the gene of interest were detected in 46 of 113 tumors (40.7%). Of the 106 tumors with equivocal FISH results, RNA-seq detected a fusion involving the expected gene target in 37 of 62 (59.7%) tumors with isolated probe signals corresponding to the active side of the gene region but only 4 of 44 (9.1%) tumors with other atypical signal patterns. This study provides a useful framework for categorizing atypical BAP FISH results and demonstrates the clinical utility of follow-up RNA-seq testing on tumors with equivocal FISH results.
Following recommendations from various consortia and professional societies, the double colon symbol (::) has become an integral part of gene fusion nomenclature (eg, EML4::ALK). Although widely adopted, its use presents...Following recommendations from various consortia and professional societies, the double colon symbol (::) has become an integral part of gene fusion nomenclature (eg, EML4::ALK). Although widely adopted, its use presents technical challenges, as many common operating systems restrict the use of colons (:) in file and directory names. Consequently, the double colon (::) is often replaced with an underscore (_) or other allowed characters, introducing ambiguity and inconsistency. The first objective of this work is to raise awareness of this issue. The second objective is to propose a simple technical solution, that is, replacing the command-carrying symbol with a visually similar and functionally distinct ASCII character. The technical solution includes confirmation of functional compatibility and visual compliance with the established fusion nomenclature. The proposal also includes using Unicode characters to replace the slash (/) for alternative variants, the greater than symbol (>) for substitutions, and the asterisk (∗) for nonsense variants, for example. To support consistency, keyboard shortcuts or custom scripts may be used to automate these substitutions. The straightforward solution presented in this paper counterbalances unintended technical consequences and thereby promotes harmonization towards a unified genomic variant nomenclature.
Jani K, Stephen T, Lee C
… +4 more, Pinto A, McMillen T, Chan JL, Babady NE
J Mol Diagn
· 2025 Sep · PMID 40902724
·
Full text
Human adenoviruses (HAdVs) can result in significant morbidity and mortality in immunocompromised patients. The AltoStar Adenovirus PCR Kit 1.5 (AltoStar HAdV PCR) is a real-time PCR test for the detection and quantifica...Human adenoviruses (HAdVs) can result in significant morbidity and mortality in immunocompromised patients. The AltoStar Adenovirus PCR Kit 1.5 (AltoStar HAdV PCR) is a real-time PCR test for the detection and quantification of HAdV DNA. In this study, the performance of the AltoStar HAdV PCR was evaluated compared with a laboratory-developed HAdV PCR based on MultiCode HAdV reagents (MultiCode HAdV PCR) using plasma and stool specimens. Performance of the AltoStar HAdV PCR was established by determining the linearity, lower limit of detection, precision, specificity, accuracy, and inclusivity of the assay. Accuracy was determined by testing plasma and stool clinical samples previously tested by the MultiCode HAdV PCR and inclusivity evaluated by using several HAdV genotypes. A time-and-motion study was performed to compare the workflows of both PCRs. The limit of detection was <200 copies/mL for both sample types. Overall agreement with the MultiCode HAdV PCR was 91% for plasma samples and 85.5% for stool samples. Quantitative agreement between the two tests was moderate. Specificity and precision in both sample types were high. All major HAdV species tested were detected. Hands-on-time was significantly less for the semi-automated AltoStar HAdV PCR. The AltoStar HAdV PCR showed great performance for the detection and/or quantitation of HAdV in clinical samples.
Roy S, Tremblay MW, Lockhart E
… +14 more, Aradhya S, Bayrak-Toydemir P, Bowser M, DaRe J, Gibson K, Kennemer M, Krueger C, Lebo M, Mao R, Nussbaum R, O'Fallon B, Rosato A, Kalman LV, Funke B
J Mol Diagn
· 2025 Aug · PMID 40846269
·
Full text
Clinical implementation of whole-genome and whole-exome sequencing by next-generation sequencing (NGS) allows for comprehensive detection of genomic alterations. However, with the growing number of clinically relevant ge...Clinical implementation of whole-genome and whole-exome sequencing by next-generation sequencing (NGS) allows for comprehensive detection of genomic alterations. However, with the growing number of clinically relevant genes and variants, there is an urgent need for reference materials to optimize, validate, and quality control NGS tests. This pilot study documents the paucity of physical reference materials for widely tested genes and demonstrates the utility of in silico mutagenized reference materials to supplement physical samples when developing NGS tests. We examined published, expert curated lists of clinically relevant variants for these widely tested genes and found that publicly available reference materials were available for only 29.4%. We outline the steps for generating in silico resources and used 49 curated variants to conduct a blinded proof-of-concept study with three experienced NGS laboratories. One laboratory detected all added variants, and two detected all but one. This study revealed common scenarios that could lead to false-negative results when common pathogenic variants cannot be tested during analytical validation. This work highlights the need to establish centralized knowledge bases for common, pathogenic variants, demonstrates the utility of in silico reference materials, and provides guidance for generating in silico reference materials in-house. Additional work will be needed to generate turnkey processes for novice laboratories without in-house bioinformatics expertise.
Short-read next-generation sequencing is widely used for clinical diagnosis but faces limitations in accurately detecting variants in complex genomic regions, such as segmental duplications, guanine-cytosine rich areas,...Short-read next-generation sequencing is widely used for clinical diagnosis but faces limitations in accurately detecting variants in complex genomic regions, such as segmental duplications, guanine-cytosine rich areas, and repeat sequences. These challenging regions comprise only 3% to 5% of the exome, yet their stochastic library preparation and bioinformatics analysis impacts variant detection reproducibility. Evaluating reproducibility is time-consuming, and variants in these regions require validation through sequencing replicates and using orthogonal methods like long-range PCR or Sanger sequencing, thereby increasing costs and turnaround times for clinical laboratories. To address these challenges, ClinRay was developed; it is a novel, generalizable bioinformatics method that uses the concept of digital twins to synthetically enhance the data distribution for variants in regions with suspected poor reproducibility. ClinRay predicts the reproducibility of detected variants by short-read next-generation sequencing probes in these difficult-to-sequence genomic regions. The model was developed using alignment data from the binary format of the sequence alignment/map files of eight replicates of the Genome in a Bottle HG002 Coriell cell and publicly available genomic context annotation resources. The model achieved an area under the receiver-operating characteristic curve of 0.89 (95% CI, 0.88-0.90) on the test data and 0.85 (95% CI, 0.84-0.86) on an independent validation data set.
This study aimed to establish a two-dimensional PCR (2D-PCR) methodology capable of simultaneously identifying HLA-A∗31:01, HLA-B∗15:02, HLA-B∗57:01, and HLA-B∗58:01 alleles to prevent adverse drug reactions and guide sa...This study aimed to establish a two-dimensional PCR (2D-PCR) methodology capable of simultaneously identifying HLA-A∗31:01, HLA-B∗15:02, HLA-B∗57:01, and HLA-B∗58:01 alleles to prevent adverse drug reactions and guide safe clinical medication use. To achieve this goal, key nucleotide sites were first selected through multiple sequence alignment for the purpose of identifying HLA alleles: c.243T and c.290T for HLA-A∗31:01; c.206C, c.256G, and c.285G for HLA-B∗58:01; c.362T and c.419C for HLA-B∗57:01; and c.1012 + 104T (rs144012689) for HLA-B∗15:02. Using universal tag sequences linked to the 5' end of primers and base-quenched probes, high-throughput identification of multiple target genes was achieved through PCR amplification and melting curve analysis, followed by methodologic optimization and evaluation. Results indicated that the 2D-PCR method has a detection limit of approximately 10 copies and achieves high specificity and accuracy. The entire detection process can be completed in approximately 100 minutes, with a cost of less than $1 (USD). Furthermore, 2D-PCR overcomes the limitations of traditional fluorescent channels, providing technical support for the identification of multiple target genes. In conclusion, this study shows that 2D-PCR offers a convenient and rapid approach for human leukocyte antigen allele identification to prevent adverse drug reactions and holds potential for clinical application.
Mata DA, Kou AA, Heilmann AM
… +23 more, Honecker J, Kroger BR, Wieland T, Williams EA, Yang SR, Benhamida JK, Vanderbilt C, Marcus C, Shyu I, Ho C, Hiemenz MC, Janovitz T, Sokol ES, Fleischmann Z, Jin D, Graf RP, Hickman RA, Lin DI, Ma P, Albacker LA, Tse JY, Ritterhouse LL, Decker B
This study evaluated a UV mutational signature classifier applied to circulating tumor DNA (ctDNA) in cell-free DNA liquid biopsies, using the FoundationOne Liquid CDx platform. Among 73,790 samples, 12.9% (9527) met the...This study evaluated a UV mutational signature classifier applied to circulating tumor DNA (ctDNA) in cell-free DNA liquid biopsies, using the FoundationOne Liquid CDx platform. Among 73,790 samples, 12.9% (9527) met the variant threshold for signature analysis, with UV signatures detected in 3.8% (365) of cases. Of these, 51.5% (188) were initially submitted as cutaneous, 8.5% (31) as unspecified primary, and 40.0% (146) as extracutaneous tumors. The UV classifier demonstrated high specificity (99.7%) and variable sensitivity, reaching up to 37.5% and 67.6% in samples with elevated ctDNA levels and mutational loads, respectively. Molecular pathologist review confirmed that 74.0% (270/365) of UV calls were true positives and enabled resolution of false positives and ambiguous findings through integration of genomic and clinicopathologic features. This refined sensitivity estimates to 41.9% and 70.2% in cases with elevated ctDNA levels and mutational loads, respectively. Confirmed UV-positive tumors exhibited genomic alterations characteristic of sun-exposed skin cancers. In 175 matched liquid and tissue biopsy pairs, the positive percent agreement for UV signature detection was 38.7% overall, increasing to 87.5% and 90.6% in subsets with elevated ctDNA levels and mutational load, respectively. These results underline the utility of FoundationOne Liquid CDx, complemented by molecular pathologist oversight, in identifying cancers of cutaneous origin to refine diagnoses and guide treatment for patients with advanced cancers.
Fang H, Eacker SM, Wu Y
… +5 more, Paschal C, Wood M, Nelson B, Muratov A, Liu Y
J Mol Diagn
· 2025 Aug · PMID 40846267
·
Full text
Structural variants are critical to genetic diversity and disease, yet their detection remains challenging with conventional cytogenetic techniques, including karyotyping, fluorescence in situ hybridization, and chromoso...Structural variants are critical to genetic diversity and disease, yet their detection remains challenging with conventional cytogenetic techniques, including karyotyping, fluorescence in situ hybridization, and chromosome microarray analysis. These methods often lack the resolution and sensitivity needed for comprehensive characterization of chromosomal aberrations. To address these limitations, we implemented genomic proximity mapping (GPM), a genome-wide chromosome conformation capture technology, in a clinical setting. Here, we applied GPM to a cohort of 123 individuals with constitutional disorders, achieving a 100% concordance in detecting 110 copy number variants (>25 kb) and 27 structural rearrangements, in addition to novel findings. GPM demonstrated unique advantages, such as resolving chromosomal rearrangements with precise (<5 kb) breakpoint resolution, maintaining robust performance with challenging samples, including formalin-fixed, paraffin-embedded tissues, and detecting mosaicism with high sensitivity. Furthermore, GPM reliably provided copy number and loss-of-heterozygosity profiles, streamlining workflows. GPM also uncovered 12 novel structural variants missed by standard methods, highlighting its superior detection capability. This analysis revealed that cases with more than two chromosomal rearrangements identified by traditional cytogenetics often harbor additional, cryptic rearrangements that remain undetected by standard-of-care methods. GPM represents a transformative tool for genomic diagnostics, offering a high-resolution, comprehensive approach to detecting genomic alterations. Its ability to address limitations of conventional cytogenetics methods positions GPM as a needed advance in the diagnosis, prognosis, and therapeutic management of genetic disorders.
Michel C, Simon A, Witz A
… +7 more, Hanriot I, Dardare J, Betz M, Brun M, Gilson P, Merlin JL, Harlé A
J Mol Diagn
· 2025 Oct · PMID 40783114
·
Full text
Homologous recombination deficiency (HRD) affects 50% of ovarian cancers and influences poly (ADP-ribose) polymerase inhibitor efficacy. Although one-third of HRD tumors harbor a deleterious BRCA1/2 mutation, these mutat...Homologous recombination deficiency (HRD) affects 50% of ovarian cancers and influences poly (ADP-ribose) polymerase inhibitor efficacy. Although one-third of HRD tumors harbor a deleterious BRCA1/2 mutation, these mutations are not the sole cause of HRD. Promoter methylation of BRCA1 and RAD51C contributes to 19% and 2% of cases, respectively. A cohort of 224 patients with ovarian cancer tested for HRD validated the droplet digital PCR (ddPCR) technique. DNA was extracted from formalin-fixed, paraffin-embedded tissue, and the Genomic Instability Index (GII) was assessed using the SOPHiA DDM HRD Solution. BRCA1 and RAD51C promoter methylation was analyzed by ddPCR following enzymatic conversion, with a 10% threshold for methylation classification. Homologous recombination status was assessed in 194 patients. A positive GII (>0) was identified in 77 cases (39.7%), with 24 (31.2%) harboring a deleterious BRCA1/2 mutation, and 2 (2.6%) a RAD51C mutation. Methylation analysis revealed BRCA1 promoter methylation in 32 patients (41.6%) and RAD51C promoter methylation in 1 patient (1.3%), clarifying HRD status in 28 additional cases (36.3%). The study confirms HRD extends beyond BRCA1/2 mutations, with promoter methylation playing a significant role in HRD detection. ddPCR effectively identified methylation-driven HRD, explaining deficiency in 36.4% additional patients with a positive GII score. These findings highlight the importance of incorporating methylation analysis into HRD testing to improve patient stratification for poly (ADP-ribose) polymerase inhibitor therapy.
Formalin-fixed, paraffin-embedded (FFPE) tumor tissue is the standard in pathology due to logistical and quality constraints of fresh-frozen samples. Although whole-genome sequencing (WGS) offers diagnostic promise, its...Formalin-fixed, paraffin-embedded (FFPE) tumor tissue is the standard in pathology due to logistical and quality constraints of fresh-frozen samples. Although whole-genome sequencing (WGS) offers diagnostic promise, its validity and utility in FFPE samples remain underexplored. This study bridges the gap by comparing FFPE-derived tumor WGS with next-generation sequencing results from FoundationOneCDx (F1CDx) and a melanoma-specific panel (MelArray) in 78 metastatic melanoma samples from the Swiss Tumor Profiler Study. A diagnostic pipeline was developed for quality control, variant annotation, and clinical actionability using public and commercial databases. FFPE-derived WGS displayed robust analytical validity, detecting 95% of somatic single nucleotide variants, 98% of multinucleotide variants, 90% of insertions/deletions, 76% of amplifications, and 96% of homozygous deletions identified by F1CDx. Tumor mutational burden strongly correlated with F1CDx (R = 0.98), particularly at the clinical threshold of ≥10 mutations per megabase, crucial for treatment decisions. WGS detected complex biomarkers such as UV-associated mutational signatures and genome-wide copy number alterations, aiding melanoma subtype distinction. Clinically, WGS suggested treatments or trials for all cases, identifying additional markers in 38% and 71% compared with F1CDx and MelArray, respectively. Novel therapeutic opportunities were found in 18% and 56% of cases. FFPE-derived WGS closely matches targeted panels in performance while providing comprehensive insights, enhancing therapeutic options. With decreasing costs, WGS could become a powerful routine diagnostic tool.
Sboner A, Benhamida J, Hirschhorn JW
… +4 more, Temple-Smolkin RL, Zhang W, Leon A, Association for Molecular Pathology Informatics Subdivision Leadership
Xu S, Luo X, Xiao B
… +12 more, Liu H, Xu T, Chen L, Yang T, Xu N, Fan Y, Qiu W, Wang R, Zhang H, Chen Y, Yu Y, Sun Y
J Mol Diagn
· 2025 Oct · PMID 40712995
·
Full text
Short tandem repeats (STRs) are associated with 70 genetic diseases. Because of the short read length of exome sequencing (ES), STR analysis is not routinely analyzed in clinical ES. So far, there has been limited system...Short tandem repeats (STRs) are associated with 70 genetic diseases. Because of the short read length of exome sequencing (ES), STR analysis is not routinely analyzed in clinical ES. So far, there has been limited systematic evaluation using large-scale clinical ES data to assess the diagnostic yield of pathogenic STR expansion. This study retrospectively analyzed 9580 exomes referred to our genetic laboratory between July 2019 and June 2024. The samples were divided into two groups: a genetically undiagnosed cohort (n = 4692) and a reference cohort with a low probability of carrying pathogenic STR expansions (n = 4888). An analysis pipeline was developed on the basis of the combination of multiple algorithms to analyze STRs detected in 30 known disease-related loci, achieving a precision of 54.9% and a sensitivity of 100%. STR verification by capillary electrophoresis analysis of STR confirmed 28 cases (0.6%) with pathogenic STR expansions in known disease-related loci. Fourteen of these cases (0.3%) could be explained by the STR findings, including seven neonates with DMPK expansions. The pipeline showed the potential to identity abnormal STR expansions at novel sites. In conclusion, this study demonstrates the clinical utility of ES-based STR analysis and advocates for its incorporation into the clinical ES workflow in genetic laboratories.
J Mol Diagn
· 2025 Oct · PMID 40712994
·
Full text
Diagnostic analysis of mRNA is essential because altered splicing is a frequent cause of genetic diseases. High-throughput splicing studies remain difficult to implement in routine diagnostics. This is why SEALigHTS (spl...Diagnostic analysis of mRNA is essential because altered splicing is a frequent cause of genetic diseases. High-throughput splicing studies remain difficult to implement in routine diagnostics. This is why SEALigHTS (splice and expression analyses by ligation and high throughput sequencing), a cost-effective and easy-to-implement technique designed for simultaneous analysis of RNA from multiple patients on a panel of genes, was developed using probes designed at exon extremities. After reverse transcription and probing on cDNA, neighboring probes are ligated and the number of ligations quantified by using unique molecular identifiers and sequencing. A panel covering 42 genes (ie, 2195 probes) involved in breast/ovarian and colorectal cancer predispositions was designed. After a training phase on 40 samples, SEALigHTS was validated in another laboratory on 56 samples carrying various splicing variations previously characterized by RNA sequencing, with a sensitivity of 96% and specificity of 94%. Subsequently, in a series of 37 selected patients and 114 consecutive patients from the genetics clinic with a concomitant DNA panel, five new diagnoses were made, revealing the impact on splicing of a cryptic genomic variant (deep intronic, retrotransposon insertion), and the unexpected impact on splicing of six genomic variations was unmasked. Beyond increased diagnostic yield and classification of variants of uncertain significance, this comprehensive DNA and RNA combined approach highlights unexpected splicing defects and addresses genotype-phenotype correlation issues.
Henkel PS, Aas E, Russnes HG
… +6 more, Dyvik I, Fagereng GL, Helland Å, Røgenes H, Lien TG, Pedersen K
J Mol Diagn
· 2025 Oct · PMID 40712993
·
Full text
Detailed cost analyses of genomic profiling for precision cancer medicine can inform strategic planning and cost-effectiveness analysis. A flexible costing framework was developed in this study to conduct microcosting of...Detailed cost analyses of genomic profiling for precision cancer medicine can inform strategic planning and cost-effectiveness analysis. A flexible costing framework was developed in this study to conduct microcosting of genomic profiling in precision cancer medicine using the broad gene panel TruSight Oncology 500 and accounting for its integration into the molecular tumor board within the national Infrastructure for Precision Diagnostics in Norway. The framework enables calculation of costs per sample, by workflow steps and cost categories. Site visits and discussions with staff at Oslo University Hospital informed the diagnostic workflow, validation of the framework, and resource use inputs. Sensitivity analysis addressed alternative resource use estimates, higher batch sizes, and investment costs for automation of the library preparation step. Total costs per sample were $2944 USD, ranging from $2366 to $4307 when considering uncertainties in estimates. Consumables and personnel were the most resource-intensive cost categories across analyses. Automating the resource-intensive library preparation step enabled a higher weekly batch size with slightly lower costs per sample ($2881) despite the additional equipment costs. The dynamic costing framework highlights how the choice of equipment and batch sizes affects sample costs and personnel needs for genomic profiling. Consumables and personnel offer the largest potential for costs savings, but potential personnel bottlenecks need to be considered when further upscaling capacity.
Honecker J, Paffenholz D, Jungmann J
… +16 more, Wieland T, Kou AA, Yang SR, Vanderbilt CM, Shanmugam V, Montesion M, Graf RP, Albacker LA, Lin DI, Heilmann A, Lakis S, Hickman RA, Decker B, Robertson A, Ritterhouse LL, Mata DA
J Mol Diagn
· 2025 Oct · PMID 40712992
·
Full text
Accurate detection of Epstein-Barr virus (EBV) in tumors is essential for refining diagnosis and guiding management of EBV-associated cancers. This validation study evaluated the performance of the FoundationOne CDx (F1C...Accurate detection of Epstein-Barr virus (EBV) in tumors is essential for refining diagnosis and guiding management of EBV-associated cancers. This validation study evaluated the performance of the FoundationOne CDx (F1CDx) and FoundationOne Heme (F1H) next-generation sequencing assays for detecting EBV compared with the reference standard EBV-encoded RNA (EBER) in situ hybridization (ISH). A total of 413 samples of gastric carcinoma, squamous cell carcinoma (predominantly nasopharyngeal carcinoma), and diffuse large B-cell lymphoma was assessed. The assays demonstrated a sensitivity of 94.0%, specificity of 99.1%, and accuracy of 98.3% with an area under the curve of 0.97 for EBV detection, showing high concordance with EBER ISH. Precision was 95.5%, and reproducibility across multiple runs was strong (CV = 4.7%). Dilution experiments demonstrated a linear relationship between EBV viral reads per million and the degree of cell culture dilution (Pearson's R > 0.99, P < 0.001), supporting assay linearity over a broad dynamic range. Characteristic mutational and cytogenetic profiles, including losses at 9p21 and gains at 11q13, were observed in EBV-positive samples, supporting the clinical utility of the method. These results indicate that F1CDx and F1H provide a reliable, in-parallel approach for detecting EBV along with somatic genomic alterations in a single assay. This method offers a promising alternative to conventional ISH, especially for identifying EBV in tumors without initial clinical suspicion, potentially streamlining diagnostic workflows.
Manning DK, Ananda G, Eifert C
… +28 more, Rojas-Rudilla V, Swanton W, Keefe S, Garcia EP, D'Eletto M, Holdmore E, Mishra S, Borziak K, Lukasse P, Davineni P, Manam MD, Shivdasani P, Ghazani AA, Wang AL, Bastepe M, Beltran H, Janeway KA, Church A, Pollaci A, Chittenden AB, Koeller DR, Schwartz Levine A, Garber JE, Johnson BE, Lindeman NI, Nowak JA, Sholl LM, MacConaill LE
J Mol Diagn
· 2025 Oct · PMID 40706989
·
Full text
Genomic profiling of cancers informs diagnostic and prognostic classification and aids in selection of targeted therapeutics. Targeted, next-generation sequencing of cancer-specific genes is clinically feasible and enabl...Genomic profiling of cancers informs diagnostic and prognostic classification and aids in selection of targeted therapeutics. Targeted, next-generation sequencing of cancer-specific genes is clinically feasible and enables comprehensive somatic reporting; without a matched germline specimen, germline alterations can confound analyses of the somatic profile and generate uncertainty in interpretation. This work reports the validation and implementation of optional matched tumor/germline sequencing in a precision cancer medicine program. DNA from 63 patient samples was analyzed using OncoPanel, a hybrid capture-based sequencing assay of 461 genes. Three analytical pipelines were implemented: tumor only, matched tumor/germline, and germline only. For matched tumor/germline, germline alterations in 19 genes with actionable/therapeutic implications were rescued. Retrospective analysis of the first 1600 matched cases was done to determine the potential clinical utility of this approach. Limit of detection for point mutations/insertions and deletions was 3% allele fraction; reproducibility was >98%. Matched tumor/germline concordance across 938 somatic calls was 100%. The average tumor mutational burden (TMB) was approximately 4 mutations/Mb lower than tumor-only sequencing. TMB-high patients were accurately reclassified as TMB-low in 14% of cases. Twenty-five percent of validation cases (14% after launch) had a pathogenic or likely pathogenic germline variant conferring cancer susceptibility; 14% of validation cases (7% after launch) harbored a germline variant of therapeutic significance. Matched tumor/germline sequencing is more accurate than tumor-only sequencing, while still encompassing all genomic findings that inform targeted therapy selection.