Petersen LM, Sainger R, Sanchez P
… +4 more, Burke J, Wemmer JD, Patay B, Miller JE
J Mol Diagn
· 2025 Oct · PMID 40706988
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KMT2A fusions are a critical oncogenic driver in 5% to 10% of patients with acute myeloid leukemia (AML) and are associated with poor prognosis. Currently, there are no published somatic guidelines for fusions in AML, an...KMT2A fusions are a critical oncogenic driver in 5% to 10% of patients with acute myeloid leukemia (AML) and are associated with poor prognosis. Currently, there are no published somatic guidelines for fusions in AML, and developing methods to accurately classify fusions, especially those involving KMT2A, is essential for patient care. Therefore, the Laboratory for Personalized Molecular Medicine (LabPMM) KMT2A Fusions Workflow was developed utilizing the framework of the somatic guidelines by Horak et al, where classification of oncogenicity is based on points awarded for varying types of evidence. Fusions previously detected by LabPMM's CAP/CLIA-certified MyAML and MyMRD gene panels were used to test this workflow. A total of 100 KMT2A fusions were reassessed, and 97 of these had a breakpoint in the major breakpoint cluster region. There were 20 distinct partner genes for KMT2A, and the most common partners were MLLT3, ELL, AFDN, MLLT10, and AFF1. Five KMT2A fusions had a novel partner (MYB, RC3H1, SNAPC3, STPG1, and HPSE2). Breakpoints in the partner genes were assessed to better understand their potential role in driving leukemogenesis. Of the 100 fusions reassessed, 9 had a classification change. This LabPMM KMT2A Fusions Workflow provides a points-based system for curation that allows for standardization and clarity both within and among genetic diagnostic laboratories reporting on KMT2A fusions in AML.
Dickson A, Cone KR, Fortini BK
… +7 more, Goldstein J, Thompson ML, Wilke MVMB, Hurst ACE, Schroeder MC, Polonis K, Bowling KM
J Mol Diagn
· 2025 Oct · PMID 40706987
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Genomic testing has proven utility in disease diagnostics, guiding clinical management and improving outcomes. Use of high-throughput sequencing by clinical laboratories has generated opportunities and challenges in data...Genomic testing has proven utility in disease diagnostics, guiding clinical management and improving outcomes. Use of high-throughput sequencing by clinical laboratories has generated opportunities and challenges in data analysis, resulting in the emergence of a laboratory role termed variant scientist. The aim of this study was to characterize this laboratory role. A 30-item survey was developed to collect information describing the current demographic landscape, salary ranges, work environments, training options, and professional development of variant scientists. The survey was disseminated to individuals conducting variant analysis in the United States from November 6, 2023, to March 15, 2024. Survey responders (n = 87) were predominantly female (78%), aged ≤40 years (64%), hold advanced degrees (38% master's, 47% doctoral), and report ≥4 years of experience (75%). Responders report involvement in a diverse set of laboratory tasks and received relevant training on the job (78%). This workforce is satisfied with their career path (70%) and reports adequate support from employers, but perceives that resources and recognition from professional organizations are currently lacking. Characterization of this workforce will be of interest to individuals working as variant scientists, individuals interested in careers in variant science, and laboratory directors seeking assistance for effectively maintaining and efficiently growing clinical laboratory operations.
Cancer treatment costs pose a significant global economic burden. By facilitating treatment plans tailored to the genomic profile of patients' cancer, genomic testing has the potential to reduce health care costs. Using...Cancer treatment costs pose a significant global economic burden. By facilitating treatment plans tailored to the genomic profile of patients' cancer, genomic testing has the potential to reduce health care costs. Using real-world evidence, this study compared the cost-effectiveness of comprehensive genomic profiling (CGP) versus small panel (SP) testing in patients with advanced non-small-cell lung cancer in the United States and Germany. A partitioned survival model was developed to estimate the life years and drug acquisition costs associated with CGP and SP testing in patients receiving matched targeted therapy, matched immunotherapy, or no matched therapy/untreated. Key model parameters were informed by real-world data derived from the Syapse study. Scenario and sensitivity analyses were conducted. CGP improved the average overall survival by 0.10 years compared with SP. CGP was associated with higher health care costs because of a higher percentage of patients receiving targeted therapies. The estimated incremental cost-effectiveness ratio (ICER) of CGP versus SP was $174,782 and $63,158 per life-year gained in the United States and Germany, respectively. Increasing the number of patients receiving treatment decreased the ICERs ($86,826 in the United States and $29,235 in Germany), while switching from immunotherapy plus chemotherapy to chemotherapy alone increased the ICERs ($223,226 in the United States and $83,333 in Germany). Altogether, CGP has the potential to improve patient outcomes and is more cost-effective than SP.
Asian-type DEL (RHD, c.1227G>A) is relatively prevalent in East Asians and is essential to detect in serologic rhesus (Rh) D-negative individuals. Despite the recognized importance of identifying Asian-type DEL, traditio...Asian-type DEL (RHD, c.1227G>A) is relatively prevalent in East Asians and is essential to detect in serologic rhesus (Rh) D-negative individuals. Despite the recognized importance of identifying Asian-type DEL, traditional detection methods, such as adsorption-elution and Sanger sequencing, are complex and time-consuming. A reliable method is required for the rapid and precise detection of Asian-type DEL. A novel multiplex real-time PCR assay using allele-specific TaqMan hydrolysis probes was developed to target the Asian-type DEL variant. Analytical specificity and sensitivity were evaluated using RHD or RHCE synthetic DNAs with 1227G or 1227A. The assay was tested on 315 clinical samples, and the results were compared with Sanger sequencing to assess diagnostic performance. Analytical specificity evaluations confirmed that the assay selectively amplified RHD, 1227A, or 1227G without cross-reactivity to RHCE. Additionally, clinical sample tests showed the assay maintained high specificity and sensitivity even at high nucleic acid concentrations. The Asian-type DEL multiplex real-time PCR assay demonstrated 100% sensitivity and specificity, with complete concordance across all samples compared with reference methods. Compared with Sanger sequencing, the multiplex real-time PCR assay had a shorter analysis time. The assay developed in this study offers a fast, reliable, and accurate approach for detecting Asian-type DEL. This method significantly improves efficiency over conventional techniques and provides a valuable tool for managing transfusion safety and RhD alloimmunization risks in RhD-negative populations.
Molecular syndromic assays have improved respiratory diagnostics by enabling the simultaneous detection of multiple pathogens from a single sample. However, fixed-panel designs may not align with age-specific prevalence...Molecular syndromic assays have improved respiratory diagnostics by enabling the simultaneous detection of multiple pathogens from a single sample. However, fixed-panel designs may not align with age-specific prevalence patterns or evolving epidemiologic trends, limiting clinical utility and reimbursement viability. In this study, 1520 positive nasopharyngeal swabs from symptomatic individuals collected during the 2022 to 2023 respiratory season were analyzed by using the LIAISON PLEX Respiratory Flex Assay to evaluate the benefits of customizable, tiered testing strategies. Diagnostic yields from a standard-of-care panel were compared with tiered (core-plus-reflex) frameworks across pediatric (age ≤21 years), adult (age 22 to 64 years), and elderly (age ≥65 years) cohorts. Weighted analyses revealed that 99.8% of cases were viral, while bacterial pathogens accounted for <1%. The most commonly detected viruses included severe acute respiratory syndrome coronavirus 2 (28.2%), human enterovirus/rhinovirus (17.1%), influenza A (11.9%), and human coronavirus (7.4%). Age-related differences were observed, with human enterovirus/rhinovirus and adenovirus more common in pediatric patients, whereas severe acute respiratory syndrome coronavirus 2 and influenza A predominated in adults and the elderly. Standard-of-care panels captured only 58% of infections overall and 33% in pediatric patients; the tiered testing approach identified ≥99% of infections using flexible core-plus-reflex panels. Moreover, core panel targets alone accounted for >76% of all detections. These findings underscore the diagnostic, clinical, operational, and cost management value of age-informed, customizable testing frameworks to improve detection, reduce unnecessary testing, and support stewardship.
J Mol Diagn
· 2025 Sep · PMID 40578551
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Next-generation RNA sequencing (RNA-seq) enables comprehensive transcriptomic profiling for disease characterization, biomarker discovery, and precision medicine. Despite its potential, RNA-seq has not yet been widely ad...Next-generation RNA sequencing (RNA-seq) enables comprehensive transcriptomic profiling for disease characterization, biomarker discovery, and precision medicine. Despite its potential, RNA-seq has not yet been widely adopted for clinical applications, and a key barrier to its adoption is the variability introduced during processing and analysis. Quality controls (QCs) must be considered through all stages of biomarker discovery. This study describes a comprehensive QC framework for effective RNA-seq biomarker discovery. Multilayered quality metrics were established across preanalytical, analytical, and postanalytical processes. Total RNA-seq was performed by using RNA isolated from whole blood (PAXgene Blood RNA tubes). Bulk RNA controls were incorporated to monitor sequencing batches. This framework was applied to a catalog of prospectively collected or biobanked clinical specimens spanning multiple disease indications. Among all QCs, preanalytical metrics (specimen collection, RNA integrity, and genomic DNA contamination) exhibited the highest failure rates and resulted in the addition of a secondary DNase treatment, which reduced genomic DNA levels. The additional DNase treatment significantly lowered intergenic read alignment and provided sufficient RNA for downstream sequencing and analysis. This end-to-end QC framework for RNA-seq biomarker discovery was developed and implemented to enhance the confidence and reliability of results. To advance the clinical adoption of RNA-seq, developing and implementing standards will improve reliability, accelerate biomarker discovery, and facilitate its translation into clinically actionable diagnostics and therapeutics.
LaFramboise WA, Petrosko P, Gallo PH
… +19 more, Gil L, Lam TL, Barr RM, Schumacher PE, Kharoud HK, Taylor KM, Dalton E, Bapat B, Patel S, Nakayama J, Hilton CJ, Ercolano LB, Zaidi AH, Allen CJ, Rachman T, Carja O, Schwartz R, Wagner PL, Bartlett DL
J Mol Diagn
· 2025 Sep · PMID 40578550
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Blood collection, plasma processing, and cell-free DNA (cfDNA) purification were optimized to capture circulating tumor DNA without blood cell background DNA among 874 patients with cancer. cfDNA comprised predominantly...Blood collection, plasma processing, and cell-free DNA (cfDNA) purification were optimized to capture circulating tumor DNA without blood cell background DNA among 874 patients with cancer. cfDNA comprised predominantly mononucleosomal fragments [n = 874; mean (x¯) ± SD = 166 ± 5 bp] that generated comparably sized sequencing reads (x¯ ± SD = 162 ± 25 bp). Despite a vast range of cfDNA concentrations (0.50 to 1132.9 ng/mL) across 21 tumor types, matched tumor and blood specimens (n = 430 patients) revealed high concordance for coding (median = 97%) and clinical oncogenic mutations (median = 88% concordance). Therapeutically actionable mutations were identified in 233 patients by both assays, whereas 126 patients had oncogenic mutations without an established pharmacotherapeutic agent. An additional 48 patients (11%) had actionable mutations detected only in cfDNA assays, whereas 23 patients (5%) had mutations in tumor only. Concordance was high in both prevalent (lung, breast, and colon) and rare tumors (appendiceal, sarcoma). Cell-free DNA levels from diagnostic blood specimens were a strong indicator of patient survival duration independent of age, sex, tumor type, and stage, demonstrative of a potentially important role as a prognostic biomarker. Mutations in established oncogenes and tumor suppressors were readily detectable across all tumor types in circulating tumor DNA, indicating a diagnostic role for cfDNA from blood extending beyond the identification of companion therapeutics to patient screening and monitoring.
Neurogenetic disorders are a large group of genetic and phenotypically heterogeneous diseases, making diagnosis challenging. Sequencing hundreds of disease genes concurrently using massively parallel sequencing is, there...Neurogenetic disorders are a large group of genetic and phenotypically heterogeneous diseases, making diagnosis challenging. Sequencing hundreds of disease genes concurrently using massively parallel sequencing is, therefore, invaluable for diagnosis of these disorders. The PathWest neuromuscular disease gene panels include all known genes associated with neurologic and muscle disorders. Initially implemented in 2013, covering 336 genes, the gene panel has undergone various updates in chemistries and seen the addition of many newly described neurogenic disease genes. The results from versions 3 and 5 of the panel are reported, which included 644 and 830 genes, respectively. In total, 3961 patients were tested across 20 phenotypic subpanels: 2740 on version 3 and 1221 on version 5. Overall diagnostic success was 23.0%, with 8.4% of diagnoses attributed to newly added genes. Diagnostic success varied greatly between phenotypic subpanels, from 63.4% for the congenital muscular dystrophy subpanel to 2.6% for the Alzheimer disease/frontotemporal dementia subpanel. The five most frequently reported genes, DMD, RYR1, SPG7, PMP22, and NOTCH3, accounted for 22% of all diagnoses. Changing chemistries improved coverage of regions that were previously not well resolved. This enabled improved copy number variant calling, with 10.5% of diagnoses from version 5 attributed to copy number variants. The data generated have enabled identification of factors broadly affecting diagnosis of neuromuscular disorders and potential limitations hampering diagnostic success.
Genetic testing, as part of the medicolegal autopsy in cases of suspected sudden cardiac death, has been recommended for several years; however, it is rarely performed. The aim was to assess the value of postmortem genet...Genetic testing, as part of the medicolegal autopsy in cases of suspected sudden cardiac death, has been recommended for several years; however, it is rarely performed. The aim was to assess the value of postmortem genetic testing in unexplained sudden death in the young. This is a prospective study including all cases with unexplained natural sudden death cases in individuals aged ≤50 years undergoing a legal autopsy. Postmortem genetic testing was routinely performed in cases aged ≤35 years and aged >35 years only when no cause of death was identified or there was suspicion of a possible inherited cardiac phenotype after a complete autopsy. In cases aged ≤35 years, genetic testing showed a positivity rate of 7.6%. The most striking finding has been the positivity rate of thoracic aorta aneurysms and myocarditis cases at 33%. In cases between the ages of 36 and 50 years, the positivity rate was 4.9%. If this group was approached with direct genetic analysis, as was done with the younger cohort, the yield of positive genetic testing would decrease to 2.5%. This is the largest study of postmortem genetic testing in the young to date, and the first to address its value in consecutive cases, free of selection bias.
Accurate genomic characterization of respiratory syncytial virus (RSV) is crucial for studies of epidemiology and viral evolution, including monitoring potential escape from newly authorized vaccines and prophylactic mon...Accurate genomic characterization of respiratory syncytial virus (RSV) is crucial for studies of epidemiology and viral evolution, including monitoring potential escape from newly authorized vaccines and prophylactic monoclonal antibodies. A viral genome tiling amplicon panel (UW-ARTIC) was adapted to develop a custom bioinformatic pipeline for high-throughput, cost-effective sequencing of both RSV-A and RSV-B subgroups. Genome acceptability criteria were established and the performance characteristics of the panel were determined, including assay sensitivity, specificity, breadth of genome recovery, accuracy, and precision, using contrived and remnant clinical specimens. High-quality genomes (>95% genome completeness; >500× and >1000× average depth for whole genome and fusion gene, respectively) were recovered from samples with cycle threshold ≤ 30 (approximately 594 and 2004 copies per reaction for RSV-A and RSV-B, respectively). Minor variants were accurately identified at >5% allele frequency. The assay showed high accuracy when compared with Sanger, shotgun metagenomic, and hybridization capture-based sequencing, as well as high repeatability and reproducibility. The UW-ARTIC RSV panel has utility for cost-effective RSV genome recovery in public health, clinical, and research applications. It has been used to generate US Food and Drug Administration-reportable data for clinical trials of RSV antiviral products, with robust performance in global samples from as recently as the 2023/2024 season. Continued genomic surveillance and future updates to primers will be essential for continued recovery of genomes as RSV continues to evolve.
Glucose-6-phosphate dehydrogenase (G6PD) deficiency is a common inherited disorder worldwide and is particularly prevalent in historically malaria-endemic countries. This study established a matrix-assisted laser desorpt...Glucose-6-phosphate dehydrogenase (G6PD) deficiency is a common inherited disorder worldwide and is particularly prevalent in historically malaria-endemic countries. This study established a matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) assay for G6PD mutation detection. This MALDI-TOF-MS assay with single-base extension was developed to efficiently and accurately test 19 common G6PD variants in the Chinese population. The MALDI-TOF-MS assay was used to analyze a total of 2205 peripheral blood samples, including 1111 normal individuals and 1094 G6PD gene mutation carriers. All 2205 sample results were validated by Sanger sequencing in a blinded study. The MALDI-TOF-MS assay developed in this study was applied to detect G6PD mutations in 300 uncharacterized blood donor samples, of which 17 (5.67%) were found to carry G6PD gene mutations. The baseline and follow-up characteristics of the 17 blood donors were summarized. In this study, a MALDI-TOF-MS assay was applied to detect common G6PD mutations in samples from blood donors in Guangdong, China, which provided a new concept for establishing the information regarding the blood bank database of G6PD-deficient donors.
Homologous recombination deficiency (HRD) is a genomic feature present in some malignant neoplasms and is attributed to the failure of the homologous recombination repair pathway. Tumors with an HRD-positive status may h...Homologous recombination deficiency (HRD) is a genomic feature present in some malignant neoplasms and is attributed to the failure of the homologous recombination repair pathway. Tumors with an HRD-positive status may have a distinct prognosis and/or response to therapies, including poly (ADP-ribose) polymerase inhibitors. The Association for Molecular Pathology assembled an expert panel to examine current practice and perform a scoping review of the medical literature pertaining to the molecular detection of HRD in the clinical setting. The expert panel examined the following topics: components of existing and proposed HRD and genomic instability biomarkers (including mutational signatures, loss of heterozygosity, mutations in homologous recombination repair-associated genes, and epigenetic silencing of RAD51C, BRCA1, or BRCA2); technical considerations for identifying genomic scars from tumor and germline next-generation sequencing results; guidelines on interpretation and caveats when reporting assessments of genomic instability and HRD scores; and the clinical significance of HRD. The panel formulated a set of expert consensus opinion recommendations regarding HRD assay design and validation to guide laboratories in developing HRD tests to ensure high-quality and reproducible results.
Evaluating robustness of somatic mutation detections is essential when using whole-exome sequencing (WES) for treatment decision-making. A comprehensive evaluation was conducted using tumor WES from the US Food and Drug...Evaluating robustness of somatic mutation detections is essential when using whole-exome sequencing (WES) for treatment decision-making. A comprehensive evaluation was conducted using tumor WES from the US Food and Drug Administration-led Sequencing Quality Control Phase 2 project, in which multiple library kits sequenced identical DNA materials across three laboratories to benchmark analytical validity. These workflows included various read aligner (BWA, Bowtie2, DRAGEN-Aligner, DRAGMAP, and HISAT2) and mutation caller (Mutect2, TNscope, DRAGEN-Caller, and DeepVariant) combinations. The results revealed that DRAGEN exhibited superior performance, achieving mean F1 scores of 0.966 and 0.791 for single-nucleotide variant and insertion/deletion detection, respectively. Among open-source software, BWA Mutect2 and HISAT2 Mutect2 combinations showed the highest mean F1 scores for single-nucleotide variant (0.949) and insertion/deletion (0.722), respectively. The analyses indicated that high-quality data can be analyzed as having worse results, and vice versa. Evaluations of Catalog of Somatic Mutations in Cancer reported mutations unveiled discrepancies across enrichment kits. Integrated DNA Technologies enrichment kits showed a higher false-negative rate, whereas Agilent WES kits tended to miss mutations in CBL and IDH1, and Roche library kits tended to miss the mutations in PIK3CB. Sentieon TNscope tended to underestimate tumor mutation burden and overlook FLT3:c.G1879A for cytarabine resistance in leukemia and MAP2K1:c.G199A for BRAF inhibitors in melanoma. The findings highlight the importance of robust bioinformatic analysis in guiding clinical decision-making.
Decalcification of bone-containing tumor samples serves to soften tissues before histologic processing. However, it can lead to nucleic acid degradation, resulting in next-generation sequencing failures that impede diagn...Decalcification of bone-containing tumor samples serves to soften tissues before histologic processing. However, it can lead to nucleic acid degradation, resulting in next-generation sequencing failures that impede diagnostic solutions for patients. The Morphological Bone Score (MBS) described herein optimizes the assessment of decalcified tissue samples, consequently improving both diagnostic accuracy and cost efficiency in molecular genetic laboratories. The MBS, constructed using five key morphologic features, assigns scores from 0 to 11, reflecting low to high tissue damage and direct proportionality with nucleic acid yields per cell. The MBS threshold can be adjusted depending on the aims of a specific analysis while balancing between sensitivity and accuracy. In our next-generation sequencing workflow, the exclusion of poor-quality samples from downstream processing using MBS led to a savings of $1500 per sample. The MBS provides a cost-effective approach for maximizing tissue utilization and optimizing downstream profiling in precision oncology because its objectivity and consistency in evaluating pathologic samples ensure reliable and reproducible outcomes. With additional verification, this tool could be implemented in computational models for converting morphologic features into measurable units.
Human papillomavirus (HPV) is linked to various cancers, including cervical, anal, and head and neck cancers. Conventional methods for HPV genotyping and commercial platforms are limited to detecting high-risk HPV genoty...Human papillomavirus (HPV) is linked to various cancers, including cervical, anal, and head and neck cancers. Conventional methods for HPV genotyping and commercial platforms are limited to detecting high-risk HPV genotypes primarily in gynecologic samples. Because of changing trends in the epidemiology and pathogenesis, there is a growing need for HPV genotyping techniques applicable to emerging clinical contexts involving diverse sample types, such as head and neck or anal samples, particularly for formalin-fixed, paraffin-embedded (FFPE) tissues. This study aimed to validate amplicon-based sequencing with Oxford Nanopore Technologies (ONT) for the detection and genotyping of HPV in 181 samples, including FFPE head and neck samples, and ThinPrep liquid-based cytology samples from anal and gynecologic tissues. Sanger sequencing was used as a reference for genotyping accuracy. The ONT sequencing method demonstrated a limit of detection of 1 copy/μL for HPV16 and HPV18. Perfect agreement (κ coefficient = 1.0) was observed for HPV detection across all sample types. Genotyping accuracy exceeded 95%, and ONT identified additional genotypes in certain anal and gynecologic samples that were undetected by Sanger sequencing. The assay showed high reproducibility, with consistent results across intrarun and interrun analyses. This study is the first to validate ONT sequencing for HPV genotyping in FFPE head and neck samples. ONT provides a rapid, cost-effective method for comprehensive HPV genotyping in diverse sample types.
This study compared the performance of 5-fold genome sequencing (GS) with single nucleotide polymorphism (SNP) array technology in detecting chromosomal abnormalities, particularly in the context of prenatal and postnata...This study compared the performance of 5-fold genome sequencing (GS) with single nucleotide polymorphism (SNP) array technology in detecting chromosomal abnormalities, particularly in the context of prenatal and postnatal diagnostics. A total of 42 samples, previously analyzed by SNP array, were re-examined using 5-fold GS to evaluate the detection of clinically significant copy number variations (CNVs), mosaicism, and absence of heterozygosity (AOH). The results revealed a 100% concordance between the two methods for the identification of clinically relevant CNVs, with both technologies detecting similar CNV size ranges. However, 5-fold GS demonstrated better precision in defining CNV breakpoints and exhibited a lower false-positive rate, as confirmed by quantitative PCR validation. Additionally, 5-fold GS detected mosaicism with comparable sensitivity to SNP array, capturing mosaic levels as low as 17%, whereas SNP array identified levels between 15% and 84%. For AOH detection, 5-fold GS identified all candidate AOH regions with a slightly better sensitivity, achieving a detection size limit of 4.8 Mb compared with SNP array's 5.08 Mb. Overall, 5-fold GS shows potential as a reliable method for chromosomal abnormality detection, offering high accuracy and clinical utility in both prenatal and postnatal genetic testing.
Ikoma H, Carreras J, Kikuti YY
… +12 more, Miyaoka M, Nagase S, Kondo Y, Ito A, Orita M, Tomita S, Hiraiwa S, Kawada H, Garcia JF, Roncador G, Campo E, Nakamura N
It was recently reported that follicular lymphoma (FL) with BCL6 rearrangement (R) is associated with favorable progression-free survival, whereas BCL2-R and BCL2-6-R cases are associated with disease progression. Howeve...It was recently reported that follicular lymphoma (FL) with BCL6 rearrangement (R) is associated with favorable progression-free survival, whereas BCL2-R and BCL2-6-R cases are associated with disease progression. However, the pathologic mechanism remained unexplored. This study analyzed the mutational landscape and immune microenvironment of 31 FL cases, including 16 BCL2-R, 11 BCL6-R, and 4 BCL2-6-R FL cases. The method included an in-house next-generation targeted sequencing panel of 168 genes associated with aggressive B-cell lymphoma and FL, whole genome copy number change microarray (OncoScan), and immunohistochemistry for the immune microenvironment focused on M2-like tumor-associated macrophages, regulatory T lymphocytes, and programmed cell death protein 1 (PDCD1; alias PD-1)-positive follicular T helper cells. The resulting mutational profile was compatible with a previously reported conventional FL series featuring frequent mutations in CREBBP, KMT2D, TNFRSF14, STAT6, and CD36. Moreover, BCL6-R cases had mutations in ARID1B, ARID5B, and RHOA; low frequency of mutations in other genes, such as OSBPL10, PTPRD, ATM, and HLA-B; 6q loss; and absence of disease progression. In comparison with BCL6-R cases, BCL2-R and BCL2-6-R cases had mutations in EZH2, chromosome 18 copy number gain, and disease progression in some cases. The immune microenvironment profile was heterogeneous; however, BCL6-R cases demonstrated higher infiltration of colony-stimulating factor 1 receptor- and leukocyte immunoglobulin like receptor B3 (LILRB3; alias CD85a)-positive cells. In conclusion, compared with BCL2-R, FL with BCL6-R exhibited some differences in mutational profiles and immune microenvironment.
With the evolution of next-generation sequencing-based testing in molecular diagnostics laboratories, the clinical role of bioinformaticians has also evolved. The Association for Molecular Pathology's Clinical Bioinforma...With the evolution of next-generation sequencing-based testing in molecular diagnostics laboratories, the clinical role of bioinformaticians has also evolved. The Association for Molecular Pathology's Clinical Bioinformatician Body of Knowledge aims to define the various roles the clinical bioinformatician operates individually or within a clinical bioinformatics team, along with proficiencies and skill sets that may be required or desirable across these roles. One of the most common professional responsibilities of a clinical bioinformatician is to implement bioinformatics pipelines, either vendor supplied or custom built for the assays in the molecular diagnostics laboratory, along with analysis and quality control of clinical genomics data. This second article in the series describes the various stages in the life cycle of a clinical bioinformatics pipeline and the considerations, areas of expertise, and skill sets required in each stage. This information may help laboratory professionals to better work with clinical bioinformaticians and laboratory directors to hire the appropriate expertise based on the specific needs of the laboratory.
Ewalt MD, DiNapoli SE, Mullaney K
… +16 more, Urvalek A, Uh MK, Sukhadia P, Killian JK, Zaidinski M, Jung HJ, McFarlane T, Rios-Papachristos K, Drilon A, Kris MG, Nafa K, Arcila ME, Ladanyi M, Zehir A, Offin M, Benayed R
J Mol Diagn
· 2025 Aug · PMID 40381915
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Tissue availability is often a limiting factor in obtaining comprehensive genomic profiling to identify actionable oncogenic drivers in tumors from patients with cancer. The utility of performing complementary DNA and RN...Tissue availability is often a limiting factor in obtaining comprehensive genomic profiling to identify actionable oncogenic drivers in tumors from patients with cancer. The utility of performing complementary DNA and RNA sequencing to better identify targetable gene fusions was previously reported. Here, we report our experience using RNA recovered from lysate material, following DNA extraction, to perform targeted RNA sequencing and identify gene fusions and oncogenic transcript variants in a large cohort of patients with solid tumors. To validate this approach, RNA-sequencing results of lysate-extracted RNA and direct formalin-fixed, paraffin-embedded (FFPE) extracted RNA from the same tumors were compared. After finding equivalent identification of oncogenic gene fusions and transcript variants, efforts were expanded to a larger cohort across more diverse tumor types. Lysate-extracted RNA performed comparably to freshly FFPE extract RNA, with 97% and 96% success rates, respectively. Within the lysate-extracted group, it was documented that lysate was the only material available for RNA extraction (n = 1862, 42% of all tested samples) and, within this subgroup, 364 (20%) samples were positive for actionable fusions or oncogenic isoforms. Using RNA recovered from lysate can permit sequential or simultaneous comprehensive DNA/RNA sequencing from scant FFPE samples in laboratories where dual sample extraction is not logistically possible, allowing more complete profiling to enhance the identification of actionable oncogenic gene fusions to guide care.