Yeung CCS, Narava SK, Chang TC
… +18 more, Saeed M, Aicher L, Beppu LW, Majano MS, Taylor EM, Camalier CE, Sandhuria P, Sala-Torra O, Li J, Yee LM, McShane LM, Karlovich C, Little RF, Harris L, Doroshow JH, Williams PM, Radich JP, Jiwani S
J Mol Diagn
· 2025 Aug · PMID 40381914
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myeloMATCH is a National Cancer Institute (NCI) precision medicine clinical trial initiative to evaluate treatments for acute myeloid leukemia and myelodysplastic syndrome based on a leukemia's diagnostic molecular-genet...myeloMATCH is a National Cancer Institute (NCI) precision medicine clinical trial initiative to evaluate treatments for acute myeloid leukemia and myelodysplastic syndrome based on a leukemia's diagnostic molecular-genetic profile. The NCI myeloid assay version 2 (NMAv2) uses the Genexus System, an automated platform with <48-hour turnaround from specimen receipt to reporting, to provide harmonized regulatory-compliant use for myeloMATCH across two independent clinical laboratories. Using clinical specimens, cell lines, and contrived reference materials, NMAv2 exhibited 99% sensitivity for 291 known mutations and 100% specificity. High reproducibility detecting all reportable variants was observed, with >98% mean positive percentage agreement and 100% negative percent agreement across six reproducibility assessments. Reproducibility experiments of companion diagnostic biomarkers (1 to 1.5× clinical limit of reporting) showed 100% positive percentage agreement and negative percent agreement. The limit of detection was 0.06% for hotspot single-nucleotide variants, 0.16% for non-hotspot single-nucleotide variants, 0.51% for hotspot insertion/deletions, approximately 1% for non-hotspot insertion/deletions, 0.23% for FLT3-internal tandem duplications, and ≤40 reads at 0.1% tumor content for fusions. Concordance of 99.39% was observed in orthogonal assays testing 76 blinded myeloid specimens in the sensitivity study, and 100% concordance was observed in testing 54 FLT3-internal tandem duplication specimens. The results show that NMAv2 has high specificity, sensitivity, accuracy, and reproducibility, and can rapidly characterize genomic alterations in acute myeloid leukemia and myelodysplastic syndrome.
Targeted gene panel sequencing that measures genomic variation at different depths has potential diagnostic application. A targeted gene panel, smart nonuniformity sequencing, was developed to detect somatic variants ass...Targeted gene panel sequencing that measures genomic variation at different depths has potential diagnostic application. A targeted gene panel, smart nonuniformity sequencing, was developed to detect somatic variants associated with clonal hematopoiesis of indeterminate potential (CHIP), which requires an optimal sequencing depth of >500×; and germline variants requiring a lower ≥50× depth (panel 1). This was achieved by adjusting probe ratios for genomic regions relevant to identifying CHIP in comparison to those relevant to germline variation analysis. An additional custom panel containing only the genomic regions relevant to the identification of CHIP (panel 2) was also manufactured to confirm that panel 1 did not miss variants because of the complex design. Both panels were used to sequence 150 blood-derived DNAs; 94 DNAs from research participants aged 64 to 75 years; 16 DNAs with known germline variants; 16 DNAs with known germline variants (titrated from 0% to 100%); 24 DNAs from individuals aged <40 years; and 3 commercial CHIP controls and 3 high-molecular-weight DNA controls. The sequencing median depth ratio between the CHIP and germline relevant genomic regions was 4.7:1. Fourteen CHIP-associated variants were called in both panel 1 (1382× median variant depth) and panel 2 (1665× median variant depth). All known germline variants were identified (251× median variant depth). Smart nonuniformity sequencing reliably detects variants with allele frequency in the range >0.01 to 1 in one workflow.
As the demand for health services among sexual and gender diverse (SGD) individuals rises, there is a growing need for comprehensive and equitable standards of care across the health care system. Despite progress in vari...As the demand for health services among sexual and gender diverse (SGD) individuals rises, there is a growing need for comprehensive and equitable standards of care across the health care system. Despite progress in various research areas, there is a relative lag in genetics and genomics. In this Perspective, the Association for Molecular Pathology Working Group presents survey data on how the sex and gender identity of patients, including SGD individuals, is collected, interpreted, and reported within current genomic laboratory practices during the preanalytical, analytical, and postanalytical phases. Recommendations and guidelines related to the care of the SGD community are explored, identifying knowledge and practice gaps in each phase. On the basis of the survey results, review of existing available literature, and collective professional experience, the Working Group provides future considerations to enhance affirmative and inclusive processes, improve test quality, advance health equity, and enhance patient outcomes.
Clinical bioinformaticians have come to play an essential role in clinical molecular diagnostic laboratories. However, the core knowledge needed for the clinical practice and training of this emerging group of profession...Clinical bioinformaticians have come to play an essential role in clinical molecular diagnostic laboratories. However, the core knowledge needed for the clinical practice and training of this emerging group of professionals has not been previously established. Clinical laboratories are subject to a complex set of legal and accreditation requirements from numerous governmental and nongovernmental bodies that cover the generation, processing, storage, and distribution of patient data in the form of test results and intermediate data files. Clinical bioinformaticians are intimately involved in the development and maintenance of systems that perform these activities. This third article in the Association for Molecular Pathology's Clinical Bioinformatician Body of Knowledge Core series presents a body of knowledge for the clinical bioinformatician describing relevant laboratory regulations and data security in the domains of hardware, software, networks, and interoperability. Although this article does not substitute for legal counsel, it provides a resource for clinical bioinformaticians to identify and familiarize themselves with regulations affecting their professional functions within the laboratory.
Li S, Geng S, Chen Y
… +19 more, Ren Q, Luan Y, Liang W, Chang Y, Zhang L, Zhu D, Wu W, Zhang Y, Zhang L, Wang Y, Zhong G, Wei B, Ma J, Chang Y, Wang X, Li Z, Duan C, Long G, Mao M
Recent studies highlight the promise of blood-based multicancer early detection (MCED) tests for identifying asymptomatic patients with cancer. However, most focus on a single cancer hallmark, thus limiting effectiveness...Recent studies highlight the promise of blood-based multicancer early detection (MCED) tests for identifying asymptomatic patients with cancer. However, most focus on a single cancer hallmark, thus limiting effectiveness because of cancer's heterogeneity. Here, a blood-based multi-omics test named SeekInCare for MCED is reported. SeekInCare incorporates multiple genomic and epigenetic hallmarks, including copy number aberration, fragment size, end motif, and oncogenic virus, via shallow whole-genome sequencing from cell-free DNA, alongside seven protein tumor markers in one tube of blood. Artificial intelligence algorithms were developed to distinguish patients with cancer from individuals without cancer and to predict the likely affected organ. The retrospective study included 617 patients with cancer and 580 individuals without cancer, covering 27 cancer types. SeekInCare achieved 60.0% sensitivity at 98.3% specificity, resulting in an area under the curve of 0.899. Sensitivities were 37.7%, 50.4%, 66.7%, and 78.1% in patients with stage I, II, III, and IV disease, respectively. Additionally, SeekInCare was evaluated in a prospective cohort consisting of 1203 individuals who received the test as a laboratory-developed test (median follow-up time, 753 days) in which it achieved 70.0% sensitivity at 95.2% specificity. The performances of SeekInCare in both retrospective and prospective studies demonstrate that SeekInCare is a blood-based MCED test, showing comparable performance to the other tests currently in development. These findings support its potential clinical utility as a cancer screening test in high-risk populations.
Boulos H, Lo C, Zhu W
… +14 more, Driessen TM, Yamada-Hanff J, Harding T, Lozac'hmeur A, Pereira T, Sonnenschein A, Och J, Jin A, Patel N, Blidner R, Tell R, Freaney J, Beaubier N, Mahon B
Liquid biopsies are an increasingly important tool for the real-time monitoring of biomarkers, cancer recurrence, and disease burden in oncology practice. Tempus xF+ is a liquid biopsy assay that detects cell-free DNA in...Liquid biopsies are an increasingly important tool for the real-time monitoring of biomarkers, cancer recurrence, and disease burden in oncology practice. Tempus xF+ is a liquid biopsy assay that detects cell-free DNA in blood samples of patients with advanced solid tumors. The xF+ panel covers 523 genes spanning approximately 1.8 Mb of the human genome and can detect single-nucleotide variants and insertions-deletions in 522 genes. It also detects copy number gains in 7 genes and translocations (gene rearrangements) in 10 genes. Furthermore, the larger panel size allows for the calculation of blood tumor mutational burden. This work highlights the analytical validation performed for the xF+ assay, comparing it with a smaller panel liquid biopsy assay, calculating blood tumor mutational burden, and exploring its potential clinical utility.
Clinical bioinformaticians play a critical role in clinical molecular diagnostics laboratories as developers of data analysis pipelines, tools, and databases. They also contribute to a variety of other tasks, such as gen...Clinical bioinformaticians play a critical role in clinical molecular diagnostics laboratories as developers of data analysis pipelines, tools, and databases. They also contribute to a variety of other tasks, such as genomic data interpretation, database administration, hardware engineering, informatics, information technology, infrastructure support, and software engineering. To effectively perform these functions, the clinical bioinformaticians must possess a strong foundational knowledge of molecular biology, genetics, genomics, computational biology, and the relevant federal, state, and/or regional regulations, laboratory accreditation requirements, and other standards and best practices. This first article in the Association for Molecular Pathology's Clinical Bioinformatician Body of Knowledge series provides a comprehensive core knowledge base on molecular biology, genetics, genomics, clinical laboratory practices, sequencing technologies, databases, and clinical applications. This resource serves not only to equip clinical bioinformaticians for their professional roles but also as a valuable reference for laboratorians.
Nie Q, Schilter KF, Hernandez KM
… +15 more, Adams JN, Jagadish R, Acevedo A, Larson A, Domagala BA, Vo SA, Khurana S, Mitchell K, Ellis D, Muhammedov B, Wang Y, Douville C, Coe B, Bettegowda C, Reddi HV
J Mol Diagn
· 2025 Jul · PMID 40280408
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In contrast to most solid tumors, cancers of the central nervous system (CNS) pose a unique challenge for effective detection and tracking via plasma because of the blood-brain barrier. Informed diagnosis of primary and...In contrast to most solid tumors, cancers of the central nervous system (CNS) pose a unique challenge for effective detection and tracking via plasma because of the blood-brain barrier. Informed diagnosis of primary and metastatic CNS tumors can be facilitated using a liquid biopsy assay that evaluates tumor-derived DNA from the cerebrospinal fluid (CSF), potentially increasing the efficacy of diagnosis and reducing the uncertainty and morbidities associated with the current standard of care that involves neurosurgical procedures. The Belay Summit assay involves tumor-derived DNA-based genomic profiling of CSF to inform diagnosis of CNS tumors. The analytical sensitivity of Summit for single-nucleotide/multinucleotide variants and insertions/deletions is 96% at a 95% limit of detection of 0.30% variant allele fraction. Analytical sensitivity for chromosomal arm-level aneuploidy is 91% at abs(log2r) of 0.09 limit of detection. Clinical sensitivity across a cohort of 124 specimens, including primary and metastatic CNS tumors, was demonstrated to be 90% with a specificity of 95%, supporting the potential for positive clinical utility. These results demonstrate that the Belay Summit assay can accurately and reproducibly be used to inform the diagnosis of primary and metastatic CNS tumors using CSF.
Herpes simplex virus 1 (HSV-1), HSV 2 (HSV-2), and varicella-zoster virus (VZV) cause nondescript cutaneous and mucocutaneous lesions requiring rapid, differential identification for appropriate diagnosis and patient cou...Herpes simplex virus 1 (HSV-1), HSV 2 (HSV-2), and varicella-zoster virus (VZV) cause nondescript cutaneous and mucocutaneous lesions requiring rapid, differential identification for appropriate diagnosis and patient counseling. Decentralized multiplex molecular assays may provide more rapid results than existing methodologies but require clinical validation. This multicenter study evaluated the clinical performance of the Savanna HSV 1+2/VZV Assay against the high-complexity Lyra Direct HSV 1+2/VZV real-time PCR nucleic acid test for the detection of HSV-1, HSV-2, and VZV from clinical specimens. The Savanna HSV 1+2/VZV Assay is an automated, moderate-complexity, real-time PCR assay recently cleared by the US Food and Drug Administration for the simultaneous detection and differentiation of HSV-1, HSV-2, and VZV DNA isolated from lesion swabs. In this study, 744 clinical specimens (531 female, 213 male) were evaluated by Savanna and compared with Lyra. Discrepant result analysis was conducted with the moderate-complexity Solana HSV 1+2/VZV isothermal nucleic acid test. For 744 clinical samples, Savanna exhibited overall, positive, and negative percent agreement of 99.5%, 100%, and 99.3% for HSV-1; 99.9%, 100%, and 99.8% for HSV-2; and 100%, 100%, and 100% for VZV. The Savanna HSV 1+2/VZV Assay exhibited excellent performance in a multicenter, clinical study. Savanna can provide laboratory-equivalent results outside of the central laboratory with the potential to deliver accurate results during the patient visit.
Fragile X syndrome (FXS) is the leading cause of monogenic autism spectrum disorder and inherited intellectual disabilities. Although the value of population-based FXS carrier screening has been acknowledged, appropriate...Fragile X syndrome (FXS) is the leading cause of monogenic autism spectrum disorder and inherited intellectual disabilities. Although the value of population-based FXS carrier screening has been acknowledged, appropriate screening methods are urgently required to establish and implement screening programs. We developed a nanopore sequencing-based assay that includes data analysis software to identify FXS carriers. Reference and clinical samples were used to evaluate the performance of the nanopore sequencing assay. Triplet-primed PCR and PacBio sequencing assays were used for comparisons. Nanopore sequencing identified reference carrier samples with a full range of premutation alleles in single-, 10-, and 100-plex assays, and identified AGG interruptions in an allele-specific manner. Moreover, nanopore sequencing revealed no size preference for amplicons containing different-length CGG repeat regions. Finally, nanopore sequencing successfully identified three carriers among 10 clinical samples for preliminary clinical validation. The observed variation in CGG repeat region size resulted from the base calling process of nanopore sequencing. In conclusion, the nanopore sequencing assay is rapid, high-capacity, inexpensive, and easy to perform, thus providing a promising tool and paving the way for population-based FXS carrier screening.
Martín Castillo I, Mora E, Hernani R
… +11 more, Cervera JV, Fernandez MJ, Ferrer-Lores B, Such E, Calabuig M, Abellán R, Díaz-Beyá M, Hernández-Boluda JC, Solano C, Villamón E, Tormo M
VEXAS (vacuoles, E1 enzyme, X-linked, autoinflammatory, somatic) is a severe adult-onset autoinflammatory disease associated with hematologic conditions, such as myelodysplastic syndrome. VEXAS is mostly due to an acquir...VEXAS (vacuoles, E1 enzyme, X-linked, autoinflammatory, somatic) is a severe adult-onset autoinflammatory disease associated with hematologic conditions, such as myelodysplastic syndrome. VEXAS is mostly due to an acquired mutation affecting methionine 41 (p.M41) of the UBA1 gene, which is present in >90% of patients and usually at a high burden. Treatment strategies are diverse, but many aim to suppress the UBA1 mutant clone with hypomethylating agents or by allogeneic hematopoietic cell transplantation. In the present study, we have developed a high-resolution melting tool for rapid detection of UBA1 p.M41 mutations, useful in diagnostic discrimination, and three sensitive real-time allele-specific oligonucleotide PCRs to determine the variant allele frequency of p.M41T/V/L mutations, applicable in the molecular monitoring of the disease.
Escherichia coli causes >400,000 annual deaths in children aged <5 years worldwide, with morbidity and mortality exacerbated by antimicrobial-resistant strains. A high-throughput multiplexing assay called digital multipl...Escherichia coli causes >400,000 annual deaths in children aged <5 years worldwide, with morbidity and mortality exacerbated by antimicrobial-resistant strains. A high-throughput multiplexing assay called digital multiplex ligation assay (dMLA) was developed to detect simultaneously 43 priority genes in E. coli related to the following: antibiotic resistance (n = 19), virulence factors (n = 16), and phylogroup markers (n = 6) with controls (uidA, gapdh). Genes are detected via PCR amplification of adjacent probe pairs that ligate in the presence of target gene-specific DNA, followed by sequencing of amplicons on short-read sequencers. The assay was tested in technical replicates on 63 synthetic DNA controls, and applied to 58 E. coli, 2 Staphylococcus aureus, 2 Klebsiella pneumoniae, 1 Klebsiella oxytoca, 1 Vibrio cholera, 1 Pseudomonas lurida, and 1 Salmonella enterica isolates in duplicate. Whole-genome sequencing was used to assess specificity and sensitivity. dMLA showed 100% sensitivity and >99.9% specificity and balanced accuracy on synthetic DNA. Balanced accuracy, calculated as the average of sensitivity and specificity, accounts for imbalanced data sets where negative outcomes are significantly more prevalent than positive ones. dMLA achieved a balanced accuracy of 90% for bacterial isolates. The results underline dMLA's effectiveness in high-throughput characterization of E. coli libraries for antimicrobial resistance genes and virulence factors, leveraging sequencing for massively parallel multiplexing of gene regions on multiple samples simultaneously, and are extendable to targets beyond E. coli.
Lara Gutierrez A, Halbwedl I, Sauer S
… +8 more, Regitnig P, Petru E, Seeböck R, Schubert S, Peternell C, Bodó K, Prein K, Kashofer K
J Mol Diagn
· 2025 Jun · PMID 40188947
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Genomic instability scars are markers for detecting homologous recombination deficiency (HRD) status in patients with ovarian cancer and predicting the response to poly (ADP-ribose) polymerase inhibitor treatment. Curren...Genomic instability scars are markers for detecting homologous recombination deficiency (HRD) status in patients with ovarian cancer and predicting the response to poly (ADP-ribose) polymerase inhibitor treatment. Currently, only a few reliable and validated assays are available, with the Myriad myChoice CDx being the most commonly used commercial assay for genomic instability scar score determination. Given the need for a more straightforward, accessible, and reliable method for detecting genomic instability scars methods, in this work, we describe the feasibility of using the microarray OncoScan copy number variant assay and open-source software packages to quantify genomic instability scores, and the development of an open-access online platform for genomic instability score calculation. The laboratory-developed test accurately classified homologous recombination-proficient and recombination-deficient samples based on genomic instability scores derived from the OncoScan copy number variant assay. Internally evaluated genomic instability scores demonstrated a 92% overall agreement and a higher sample success rate compared with externally analyzed genomic instability scar scores. The availability of HRD determination has doubled the number of patients eligible for poly (ADP-ribose) polymerase therapy. The assay can be conveniently performed on individual samples, and the open-access online platform facilitates HRD determination without the need for specialized bioinformatics support.
Wei S, Lattin MT, Morgan S
… +9 more, DiBianco L, Chen J, Galloway S, Karipcin S, Wapner R, Landau C, Forman EJ, Chung WK, Williams Z
J Mol Diagn
· 2025 Jun · PMID 40158886
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Sperm mosaicism, the presence of a pathogenic variant in a subset of sperm, is an important cause of heritable genetic disease. However, clinical testing for sperm mosaicism outside research has been limited by the lack...Sperm mosaicism, the presence of a pathogenic variant in a subset of sperm, is an important cause of heritable genetic disease. However, clinical testing for sperm mosaicism outside research has been limited by the lack of Clinical Laboratory Improvement Amendments (CLIA)-validated results deliverable to patients. We developed the Sensitive Assay for Mosaicism (SAM), a two-phase method for sperm mosaicism detection. In phase 1, sperm DNA undergoes deep sequencing using next-generation sequencing or nanopore-based sequencing with unique molecular identifiers (UMIs) to improve accuracy. In phase 2, PCR primers specific to UMI sequences generate amplicons for CLIA-validated Sanger sequencing, providing patient-ready results. SAM's performance was characterized and tested on semen samples from 14 participants, each with a prior offspring with a de novo pathogenic variant. SAM demonstrated a detection limit of approximately 0.005%. The UMI strategy improved sequencing accuracy on next-generation sequencing and nanopore platforms from 99.9% to >99.999%, and from 93% to >99.99%, respectively. Sperm mosaicism was identified in two tested cases: FAM111A (5.51%) and FGFR3 (0.0129%), with FGFR3 exhibiting selfish mutation validated in unrelated individuals showing varying mosaicism levels. SAM provides sensitive detection of low-level sperm mosaicism with CLIA-validated results for patients, enabling recurrence risk assessment and guiding risk mitigation strategies such as in vitro fertilization with preimplantation genetic testing for monogenic disease, sperm donation, and prenatal diagnosis.
Galbo PM, Klees RF, Burgher B
… +3 more, Miles KM, Morrison CM, Glenn ST
J Mol Diagn
· 2025 Jun · PMID 40158885
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The US Food and Drug Administration's recent move to regulate laboratory-developed tests as in vitro diagnostics has raised significant interest and concerns regarding its implementation. The New York State Department of...The US Food and Drug Administration's recent move to regulate laboratory-developed tests as in vitro diagnostics has raised significant interest and concerns regarding its implementation. The New York State Department of Health's Clinical Laboratory Evaluation Program (CLEP) provides a useful framework for understanding laboratory-developed test oversight, particularly through its guidelines for next-generation sequencing assays. These CLEP requirements for analytical validation are widely recognized as a national standard, yet there is limited peer-reviewed literature detailing the studies needed for CLEP approval. This study presents the validation of the Rapid Pan-Heme (RPPH) assay, a genomic profiling tool for hematopoietic neoplasms, developed in compliance with CLEP standards. The RPPH assay features a comprehensive next-generation sequencing panel targeting >400 genes with clinically relevant variants, including single-nucleotide variants, insertions and deletions, and fusions critical for classifying hematopoietic malignancies. CLEP approval mandates detailed documentation, quality control metrics, validation studies (accuracy, precision, and reproducibility), and compliant clinical reporting. It is demonstrated that RPPH achieves CLEP's stringent analytical sensitivity and reproducibility criteria. Variants were orthogonally validated, and proper controls were implemented. Additionally, it is outlined how RPPH's clinical reports align with CLEP requirements. Overall, this study establishes RPPH as a robust molecular diagnostic tool and provides actionable insights for researchers navigating regulatory compliance and assay validation in clinical settings.
Spinal muscular atrophy (SMA) is one of the most common recessive disorders, for which several life-saving treatment options are available. It is therefore essential to establish universal SMA screening and diagnostic pr...Spinal muscular atrophy (SMA) is one of the most common recessive disorders, for which several life-saving treatment options are available. It is therefore essential to establish universal SMA screening and diagnostic programs using scalable, cost-effective, and accessible platforms to accurately identify all variation types. This task is complicated by high sequence homology between the SMN1 and SMN2 genes. Toward this goal, a dual-mode PCR-based target-enrichment method was developed, optimized, and evaluated in an external laboratory as a proof-of-concept for scalable and deployable any-length nanopore sequencing. The assay generates 2.7- to 11.2-kb amplicons spanning exons 3 to 8 of the SMN1 and SMN2 genes, which are then analyzed using a variant calling model that reports sequence and copy number variants specific to each gene from paralog-specific sequences and read-depth data. Overall, the assay detected single-nucleotide variants, insertions/deletions, and copy number variants with >98% genotype agreement across >750 samples, including cell lines, residual presumed-normal whole-blood donors, and patients with known SMN1 and SMN2 genotypes. The assay also demonstrated a dynamic sample throughput, 9-hour turnaround time, and 4-hour hands-on time. Together with the modest capital investment and consumable costs per sample, this assay can help to increase access to SMA testing in low- and middle-income settings. As a result, this PCR/Nanopore sequencing assay and analysis pipeline has the potential for universal implementation in SMA carrier screening and diagnostic programs.
T-cell receptor (TCR) gene rearrangement clonality studies help resolve atypical T-cell proliferations in the context of suspected malignancy. However, the interpretation criteria for this assay using a next-generation s...T-cell receptor (TCR) gene rearrangement clonality studies help resolve atypical T-cell proliferations in the context of suspected malignancy. However, the interpretation criteria for this assay using a next-generation sequencing (NGS) platform have not been extensively explored and standardized. Thus, this project assessed the current performance of the Stanford Health Care in-house NGS-based TCR clonality diagnostic testing with the goal of optimizing the interpretation criteria and identifying recurrent analytical challenges. The current assay identifies a predominant clonotype when its sequence comprises at least 2.5% of the total reads with at least 5× fold change from the background. Using concurrent pathology reports as the clinical truth, this project analyzed 619 cases and determined that the current assay performs at 74% sensitivity and 85% specificity. Receiver operating characteristic analysis identified an optimized interpretation criterion that only improved the diagnostic yield marginally compared with the preexisting algorithm. Further clinicopathologic evaluation of discordant cases revealed that discrepancies mostly arose from technical limitations or the underlying nuanced biology of the diagnosis. Overall, this study provides an objective approach in establishing the interpretation criteria of the current NGS-based TCR clonality test and offers a roadmap for other laboratories considering implementing a similar assay.
The genomic evaluation of multiple myeloma in routine diagnostics involves isolating plasma cells expressing CD138, usually followed by fluorescence in situ hybridization analyses. However, cell sorting often yields a li...The genomic evaluation of multiple myeloma in routine diagnostics involves isolating plasma cells expressing CD138, usually followed by fluorescence in situ hybridization analyses. However, cell sorting often yields a limited number of cells, restricting the number of probes that can be used and limiting the analysis to a few markers required for minimal prognostic classification. Optical genome mapping is a high-resolution technology capable of identifying structural variants and copy number variations across the entire genome; however, it currently requires 1 million cells. To overcome this constraint, an innovative strategy was implemented in this work based on mixing CD138-positive and CD138-negative fractions from the same patient, optimizing the use of available CD138-positive cells for genome-wide analysis. First, dilution experiments demonstrated that a 50% CD138-positive mix was sufficient to achieve complete detection of clonal structural and copy number variants, while establishing a detection threshold of 24% for copy number variants. Using this optimized protocol, 13 additional samples from 13 patients were analyzed. Optical genome mapping achieved 93% (13/15) concordance with fluorescence in situ hybridization for clonal anomalies and revealed >22 additional genomic variations not detected by fluorescence in situ hybridization. This strategy consolidated multiple analyses into a single test, minimized material requirements, and addressed critical prognostic and increasingly described anomalies, providing refined stratification for patients with multiple myeloma.