Development and Validation of Clinical Whole-Exome and Whole-Genome Sequencing for Detection of Germline Variants in Inherited Disease

Context.-With the decrease in the cost of sequencing, the clinical testing paradigm has shifted from single gene to gene panel and now whole-exome and whole-genome sequencing. Clinical laboratories are rapidly implementing next-generation sequencing-based whole-exome and whole-genome sequencing. Because a large number of targets are covered by whole-exome and whole-genome sequencing, it is critical that a laboratory perform appropriate validation studies, develop a quality assurance and quality control program, and participate in proficiency testing. Objective.-To provide recommendations for wholeexome and whole-genome sequencing assay design, validation, and implementation for the detection of germline variants associated in inherited disorders. Data Sources.-An example of trio sequencing, filtration and annotation of variants, and phenotypic consideration to arrive at clinical diagnosis is discussed. Conclusions.-It is critical that clinical laboratories planning to implement whole-exome and whole-genome sequencing design and validate the assay to specifications and ensure adequate performance prior to implementation. Test design specifications, including variant filtering and annotation, phenotypic consideration, guidance on consenting options, and reporting of incidental findings, are provided. These are important steps a laboratory must take to validate and implement whole-exome and whole-genome sequencing in a clinical setting for germline variants in inherited disorders

Development and Validation of Targeted Next-Generation Sequencing Panels for Detection of Germline Variants in Inherited Diseases.

Context.-The number of targeted next-generation sequencing (NGS) panels for genetic diseases offered by clinical laboratories is rapidly increasing. Before an NGS-based test is implemented in a clinical laboratory, appropriate validation studies are needed to determine the performance characteristics of the test. Objective.-To provide examples of assay design and validation of targeted NGS gene panels for the detection of germline variants associated with inherited disorders. Data Sources.-The approaches used by 2 clinical laboratories for the development and validation of targeted NGS gene panels are described. Important design and validation considerations are examined. Conclusions.-Clinical laboratories must validate performance specifications of each test prior to implementation. Test design specifications and validation data are provided, outlining important steps in validation of targeted NGS panels by clinical diagnostic laboratories

Quality control material for the detection of somatic mutations in fixed clinical specimens by next-generation sequencing

Background Targeted next generation sequencing (NGS) technology to assess the mutational status of multiple genes on formalin-fixed, paraffin embedded (FFPE) tumors is rapidly being adopted in clinical settings, where quality control (QC) practices are required. Establishing reliable FFPE QC materials for NGS can be challenging and/or expensive. Here, we established a reliable and cost-effective FFPE QC material for routine utilization in the Ion AmpliSeq™ Cancer Hotspot Panel v2 (CHP2) assay. Methods The performance characteristics of the CHP2 assay were determined by sequencing various cell line mixtures and 55 different FFPE tumors on the Ion Torrent PGM platform. A FFPE QC material was prepared from a mixture of cell lines derived from different cancers, comprising single nucleotide variants and small deletions on actionable genes at different allelic frequencies. Results The CHP2 assay performed with high precision and sensitivity when custom variant calling pipeline parameters where established. In addition, all expected somatic variants in the QC material were consistently called at variant frequencies ranging from 9.1 % (CV = 11.1 %) to 37.9 % (CV = 2.8 %). Conclusions The availability of a reliable and cost-effective QC material is instrumental in assessing the performance of this or any targeted NGS assay that detects somatic variants in fixed solid tumor specimens

Quality control material for the detection of somatic mutations in fixed clinical specimens by next-generation sequencing

Background: Targeted next generation sequencing (NGS) technology to assess the mutational status of multiple genes on formalin-fixed, paraffin embedded (FFPE) tumors is rapidly being adopted in clinical settings, where quality control (QC) practices are required. Establishing reliable FFPE QC materials for NGS can be challenging and/or expensive. Here, we established a reliable and cost-effective FFPE QC material for routine utilization in the Ion AmpliSeq™ Cancer Hotspot Panel v2 (CHP2) assay. Methods: The performance characteristics of the CHP2 assay were determined by sequencing various cell line mixtures and 55 different FFPE tumors on the Ion Torrent PGM platform. A FFPE QC material was prepared from a mixture of cell lines derived from different cancers, comprising single nucleotide variants and small deletions on actionable genes at different allelic frequencies. Results: The CHP2 assay performed with high precision and sensitivity when custom variant calling pipeline parameters where established. In addition, all expected somatic variants in the QC material were consistently called at variant frequencies ranging from 9.1 % (CV = 11.1 %) to 37.9 % (CV = 2.8 %). Conclusions: The availability of a reliable and cost-effective QC material is instrumental in assessing the performance of this or any targeted NGS assay that detects somatic variants in fixed solid tumor specimens

Multilaboratory Evaluation of Real-Time PCR Tests for Hepatitis B Virus DNA Quantification

The performance characteristics of four different assays for hepatitis B virus (HBV) quantification were assessed: the Abbott RealTime HBV IUO, the Roche Cobas AmpliPrep/Cobas TaqMan HBV test, the Roche Cobas TaqMan HBV test with HighPure system, and the Qiagen artus HBV TM ASR. Limit of detection (LOD), linear range, reproducibility, and agreement were determined using a serially diluted plasma sample from a single chronically infected subject. Each assay was tested by at least three laboratories. The LOD of the RealTime and two TaqMan assays was approximately 1.0 log10 IU/ml; for artus HBV (which used the lowest volume of extracted DNA), it was approximately 1.5 log10 IU/ml. The linear range spanned 1.0 to at least 7.0 log10 IU/ml for all assays. Median values were consistently lowest for artus HBV and highest for Cobas AmpliPrep/Cobas TaqMan HBV. Assays incorporating automated nucleic acid extraction were the most reproducible; however, the overall variability was minor since the standard deviations for the means of all tested concentrations were ≤0.32 log10 IU/ml for all assays. False-positive results were observed with all assays; the highest rates occurred with tests using manual nucleic acid extraction. The performance characteristics of these assays suggest that they are useful for management and therapeutic monitoring of chronic HBV infection

Application of a correlation correction factor in a microarray cross-platform reproducibility study

Background Recent research examining cross-platform correlation of gene expression intensities has yielded mixed results. In this study, we demonstrate use of a correction factor for estimating cross-platform correlations. Results In this paper, three technical replicate microarrays were hybridized to each of three platforms. The three platforms were then analyzed to assess both intra- and cross-platform reproducibility. We present various methods for examining intra-platform reproducibility. We also examine cross-platform reproducibility using Pearson\u27s correlation. Additionally, we previously developed a correction factor for Pearson\u27s correlation which is applicable when X and Y are measured with error. Herein we demonstrate that correcting for measurement error by estimating the disattenuated correlation substantially improves cross-platform correlations. Conclusion When estimating cross-platform correlation, it is essential to thoroughly evaluate intra-platform reproducibility as a first step. In addition, since measurement error is present in microarray gene expression data, methods to correct for attenuation are useful in decreasing the bias in cross-platform correlation estimates

Oral Presentation No. 121. Aortic stiffness descriptors by cardiac magnetic resonance are correlated with mechanical testing of ex-vivo aortic aneurysms specimens

Background Aortic stiffness independently predicts mayor adverse cardiovascular events and mortality in the general population. Cardiovascular magnetic resonance (CMR) permits the assessment of a number of parameters theoretically linked to aortic stiffness, such as distensibility (AD), pulse wave velocity (PWV) and proximal aorta longitudinal strain. However, no previous study validates these parameters as descriptors of aortic wall stiffness against ex-vivo mechanical testing. Materials and methods Ascending aorta (AAo) specimens were collected from 20 patients undergoing AAo replacement for aneurysms. Patients underwent a CMR protocol in the days leading to the surgery, including 4D flow CMR. Two 15×5 mm specimens (one oriented in the circumferential and the other in the longitudinal aortic direction) were extracted during surgery, and later tested controlling for extension force. Elongation was measured by laser video extensometer and the tangent of the stress-strain curve at diastolic pressure was extracted. AAo PWV and the Eh product (E being Young modulus and h wall thickness) were measured from 4D flow CMR while AD and AAo longitudinal were quantified from cine images. Results and conclusions Marked correlations were found between circumferential elastic modulus and AAo AD (R = −0.502), PWV(R = 0.652) and Eh (R = 0.602). Similarly, strong correlation was identified between AAo longitudinal strain and longitudinal elastic modulus(R = −0.513). In conclusion, PWV and the Eh product are positively related to aortic wall stiffness while aortic distensibility and strain show negative relationships. Thus, these biomarkers are a reliable expression of aortic wall stiffness

Diagnostic value of quantitative parameters for myocardial perfusion assessment in patients with suspected coronary artery disease by single- and dual-energy computed tomography myocardial perfusion imaging

To compare performance of visual and quantitative analyses for detecting myocardial ischaemia from single- and dual-energy computed tomography (CT) in patients with suspected coronary artery disease (CAD). Eighty-four patients with suspected CAD were scheduled for dual-energy cardiac CT at rest (CTA) and pharmacological stress (CTP). Myocardial CT perfusion was analysed visually and using three parameters: mean attenuation density (MA), transmural perfusion ratio (TPR) and myocardial perfusion reserve index (MPRI), on both single-energy CT and CT-based iodine images. Significant CAD was defined in AHA-segments by concomitant myocardial hypoperfusion identified visually or quantitatively (parameter < threshold) and coronary stenosis detected by CTA. Single-photon emission CT and invasive coronary angiography were used as reference. Perfusion-parameter cut-off values were calculated in a randomly-selected subgroup of 30 patients. The best-performing thresholds for TPR, MPRI and MA were 0.96, 23 and 0.5 for single-energy CT and 0.97, 47 and 0.3 for iodine imaging. For both CT-imaging modalities, TPR yielded the highest area under receiver operating characteristic curve (AUC) (0.99 and 0.97 for single-energy CT and iodine imaging, respectively, in vessel-based analysis) compared to visual analysis, MA and MPRI. Visual interpretation on iodine imaging resulted in higher AUC compared to that on single-energy CT in per-vessel (AUC: 0.93 vs 0.86, respectively) and per-patient (0.94 vs 0.93) analyses. Transmural perfusion ratio on both CT-imaging modalities is the best-performing parameter for detecting myocardial ischaemia compared to visual method and other perfusion parameters. Visual analysis on CT-based iodine imaging outperforms that on single-energy CT

Tumor Organoids for Primary Liver Cancers: A Systematic Review of Current Applications in Diagnostics, Disease Modeling, and Drug Screening

Background/AimsLiver cancer ranks third in cancer-related deaths globally, projected to exceed one millionannually by 2030. Existing therapies have significant limitations, including severe sideeffects and inconsistent efficacy. Innovative therapeutic approaches to address primary livercancer (PLC) have led to the ongoing development of tumor-derived organoids. These aresophisticated three-dimensional structures capable of mimicking native tissue architectureand function in vitro, improving our ability to model in vivo homeostasis and disease.MethodsThis systematic review consolidates known literature on human and mouse liver organoidsacross all PLC subtypes, emphasizing diagnostic precision, disease modeling, and drugscreening capabilities.ResultsAcross all 39 included studies, organoids were frequently patient derived organoids (PDO),closely followed by cancer cell line derived organoids (CCO). The literature concentrated onHepatocellular Carcinoma (HCC) and Intrahepatic Cholangiocarcinoma (ICC), whileexploration of other subtypes was limited. These studies demonstrate a valuable role for PLCorganoid cultures in biomarker discovery, disease modeling, and therapeutic exploration.ConclusionsEncouraging advancements such as organoid-on-a-chip and co-culturing systems presentpromising prospects in advancing treatment regimens for PLC. Standardizing in vitroprotocols is crucial to integrate research breakthroughs into practical treatment strategies forPLC.Impact and ImplicationsThis review underscores the expanding utility of PLC organoids across therapeutic discovery,diagnostics, and disease modeling. PDOs replicate many tumor characteristics. Novel genesfrom HCC organoids offer promising biomarkers for personalized treatments. Innovativemethodologies, like microfluidic chips, enhance organoid culture reproducibility. Despitelimitations, co-culturing, and organ-on-a-chip show potential in better mimicking the in vivo tumor microenvironment. These advancements position PLC organoids as crucial tools forpersonalized cancer therapy, biomarker discovery, and disease modeling, with ongoingprotocol standardization efforts essential for clinical applications.<br/

Co-Administration Of The MTORC1/TORC2 Inhibitor INK128 And The Bcl-2/Bcl-XL Antagonist ABT-737 Kills Human Myeloid Leukemia Cells Through Mcl-1 Down-Regulation And AKT Inactivation

Effects of concurrent inhibition of mTORC1/2 and Bcl-2/Bcl-xL in human acute myeloid leukemia cells were examined. Tetracycline-inducible Bcl-2/Bcl-xL dual knockdown markedly sensitized acute myeloid leukemia cells to the dual TORC1/2 inhibitor INK128 in vitro as well as in vivo. Moreover, INK128 co-administered with the Bcl-2/xL antagonist ABT-737 sharply induced cell death in multiple acute myeloid leukemia cell lines, including TKI-resistant FLT3-ITD mutants and primary acute myeloid leukemia blasts carrying various genetic aberrations e.g., FLT3, IDH2, NPM1, and Kras, while exerting minimal toxicity toward normal hematopoietic CD34+ cells. Combined treatment was particularly active against CD34+/CD38−/CD123+ primitive leukemic progenitor cells. The INK128/ABT-737 regimen was also effective in the presence of a protective stromal microenvironment. Notably, INK128 was more potent than the TORC1 inhibitor rapamycin in down-regulating Mcl-1, diminishing AKT and 4EBP1 phosphorylation, and potentiating ABT-737 activity. Mcl-1 ectopic expression dramatically attenuated INK128/ABT-737 lethality, indicating an important functional role for Mcl-1 down-regulation in INK128/ABT-737 actions. Immunoprecipitation analysis revealed that combined treatment markedly diminished Bax, Bak, and Bim binding to all major anti-apoptotic Bcl-2 members (Bcl-2/Bcl-xL/Mcl-1), while Bax/Bak knockdown reduced cell death. Finally, INK128/ABT-737 co-administration sharply attenuated leukemia growth and significantly prolonged survival in a systemic acute myeloid leukemia xenograft model. Analysis of subcutaneous acute myeloid leukemia-derived tumors revealed significant decrease in 4EBP1 phosphorylation and Mcl-1 protein level, consistent with results obtained in vitro. These findings demonstrate that co-administration of dual mTORC1/mTORC2 inhibitors and BH3-mimetics exhibits potent anti-leukemic activity in vitro and in vivo, arguing that this strategy warrants attention in acute myeloid leukemia