Technical Validation Study Research Articles

High-Throughput Hybridization Assay as First-Line Diagnostic Test for Sarcomas: Clinical Assessment in a Tertiary Referral Center.

Sarcomas are rare and highly heterogeneous mesenchymal tumors with deceptive morphologic features that pose a challenge for precise diagnostics. Chromosomal rearrangements generating pathognomonic gene fusions are useful diagnostic markers, traditionally tested using single-plex standard of care assays with limited diagnostic yield. NanoString nCounter technology has emerged as a robust solution with multiplexing capabilities. To optimize NanoString effective coverage of specific entities and conduct a validation study to support its clinical implementation. We reconfigured a NanoString's codeset by including a set of probes for detecting gene fusion variants of solitary fibrous tumors, low-grade fibromyxoid sarcomas/sclerosing epithelioid fibrosarcomas, and undifferentiated small round cell sarcomas, totaling 188 probes. A technical validation study was conducted with 96 retrospective samples. Additionally, 76 prospective samples were evaluated to assess the assay's clinical performance. Both technical and clinical validation studies showed that NanoString's codeset reached >88% sensitivity and 100% specificity, compared with standard of care methods, and superior diagnostic yield as a first-line test. Our design enabled the detection of almost all fusion variants of NGFI-A binding protein 2 (NAB2) with signal transducer and activator of transcription 6 (STAT6) in solitary fibrous tumors, as well as cAMP responsive element binding protein 3 like 1/2 (CREB3L1/2) rearrangements in all low-grade fibromyxoid sarcoma/sclerosing epithelioid fibrosarcoma cases. Identification of specific gene fusions of undifferentiated small round cell sarcoma was also improved, but additional strategies are necessary to attain full coverage. The NanoString platform demonstrated good sensitivity, specificity, and superior diagnostic yield. It is a cost-effective assay with rapid turnaround time, low sample consumption, streamlined analysis, and easy customization. Therefore, it is a promising alternative first-line diagnostic tool for routine sarcoma testing.

Low-pass whole genome sequencing is a reliable and cost-effective approach for copy number variant analysis in the clinical setting.

Next generation sequencing technology has greatly reduced the cost and time required for sequencing a genome. An approach that is rapidly being adopted as an alternative method for CNV analysis is the low-pass whole genome sequencing (LP-WGS). Here, we evaluated the performance of LP-WGSto detect copy number variants (CNVs) in clinical cytogenetics. DNA samples with known CNVs detected by chromosomal microarray analyses (CMA) were selected for comparison and used as positive controls; our panel included 44 DNA samples (12 prenatal and 32 postnatal), comprising a total of 55 chromosome imbalances. The selected cases were chosen to provide a wide range of clinically relevant CNVs, the vast majority being associated with intellectual disability or recognizable syndromes. The chromosome imbalances ranged in size from 75kb to 90.3Mb, including aneuploidies and two cases of mosaicism. All CNVs were successfully detected by LP-WGS, showing a high level of consistency and robust performance of the sequencing method. Notably, the size of chromosome imbalances detected by CMA and LP-WGS were compatible between the two different platforms, which indicates that the resolution and sensitivity of the LP-WGS approach are at least similar to those provided by CMA. Our data show the potential use of LP-WGS to detect CNVs in clinical diagnosis and confirm the method as an alternative for chromosome imbalances detection. The diagnostic effectiveness and feasibility of LP-WGS, in this technical validation study, were evidenced by a clinically representative dataset of CNVs that allowed a systematic assessment of the detection power and the accuracy of the sequencing approach. Further, since the software used in this study is commercially available, the method can easily be tested and implemented in a routine diagnostic setting.

Abstract 6604: Analytical development and validation of a personalized hybrid capture based assay for high sensitivity monitoring minimal residual disease (MRD)

Abstract Introduction: 5-30% of patients with early-stage cancer relapse, even though most patients had undergone radical surgery. There is increasing evidence that circulating tumor DNA (ctDNA) minimal residual disease (MRD) predicts relapse following treatment for solid tumors. However, liquid biopsy analyses are challenged by the very low concentrations of ctDNA in blood samples. Existing ctDNA methods lack the sensitivity needed for detecting minimal residual disease (MRD) following therapy. Here, we describe a tumor-informed personalized hybrid capture based method using next-generation sequencing (NGS) to highly sensitive and specific detect MRD and predict recurrence in plasma cell-free DNA. To validate the performance of the assay, we present a technical validation study on DNA from cancer cell line material, including lung cancer cell line, reference material, Onco gDNA reference standard and wild type gDNA reference standard. Method: The performance of the assay was assessed using samples from Onco gDNA reference standard and lung cancer cell line. To estimate number of monitoring single nucleotide variants (SNVs) at input volume of 20-80ng, Onco gDNA reference standard was diluted into 8 concentrations (10, 20, 30, 40, 50, 60, 150 and 750 ppm with a variant allele frequency [VAF] range of 0.001% to 0.075%).To estimate limit of detection (LoD) of the assay at input volume of 20ng and 40ng, lung cancer cell line was diluted into 5 concentrations (10, 20, 40, 80 and 160 ppm with a variant allele frequency [VAF] range of 0.001% to 0.016%).10 samples were performed for each concentration, as well as 10 negative reference samples. Personalized panel was performed using ultradeep (100,000X) NGS with customized 55 or 60 SNVs. Result: Validation results of Onco gDNA reference standard: using 40 variants, sensitivity of input volume of 20ng and 40ng are 95% and 98% at 40 ppm, with a specificity of 100%, respectively. Using 40 variants, sensitivity of input volume of 60ng are 95% at 20 ppm, with a specificity of 100%. Using 50 variants, sensitivity of input volume of 60ng are 70% at 10 ppm, with a specificity of 100%. Validation results of lung cancer cell line: using 40 variants, sensitivity of input volume of 20ng and 40ng are 98% and 100% at 50 ppm, with a specificity of 100%, respectively. Conclusion: The assay was validated by sequentially diluting of lung cancer cell line and Onco gDNA reference standard. With input volume of 20ng, sensitivity of the assay is 95% at 40ppm when monitoring 40 variants, while with input volume of 60ng the LoD reached as low as 10 ppm with sensitivity of 70% when monitoring 50 variants. The assay demonstrates high sensitivity, while maintaining specificity above 99%. Citation Format: Xiaoqiang Wang, Lei Ye, Chao Zhang, Wanglong Deng, Yongjie Wei, Jing Chen, Xuan Tang, Xiaoxuan Wang, Chao Song, Qing Xu, Yong Ren. Analytical development and validation of a personalized hybrid capture based assay for high sensitivity monitoring minimal residual disease (MRD) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6604.

Acceptability of wearable devices for measuring mobility remotely: Observations from the Mobilise-D technical validation study.

This study aimed to explore the acceptability of a wearable device for remotely measuring mobility in the Mobilise-D technical validation study (TVS), and to explore the acceptability of using digital tools to monitor health. Participants (N = 106) in the TVS wore a waist-worn device (McRoberts Dynaport MM + ) for one week. Following this, acceptability of the device was measured using two questionnaires: The Comfort Rating Scale (CRS) and a previously validated questionnaire. A subset of participants (n = 36) also completed semi-structured interviews to further determine device acceptability and to explore their opinions of the use of digital tools to monitor their health. Questionnaire results were analysed descriptively and interviews using a content analysis. The device was considered both comfortable (median CRS (IQR; min-max) = 0.0 (0.0; 0-20) on a scale from 0-20 where lower scores signify better comfort) and acceptable (5.0 (0.5; 3.0-5.0) on a scale from 1-5 where higher scores signify better acceptability). Interviews showed it was easy to use, did not interfere with daily activities, and was comfortable. The following themes emerged from participants' as being important to digital technology: altered expectations for themselves, the use of technology, trust, and communication with healthcare professionals. Digital tools may bridge existing communication gaps between patients and clinicians and participants are open to this. This work indicates that waist-worn devices are supported, but further work with patient advisors should be undertaken to understand some of the key issues highlighted. This will form part of the ongoing work of the Mobilise-D consortium.

Study of Discriminating Markers for the Diagnosis of Chronic Lymphoid Leukemia and Evaluation of Matutes Scores

The study of the expression level by lymphoma cells (MFI: mean fluorescence intensity) completed by ROC curve for diagnostic markers is a valuable aid for the daily interpretation of lymphocyte immunophenotyping as well as the differential diagnosis with other chronic lymphoproliferative B syndromes. The aim is to provide diagnostic guidance, increase the power of the diagnostic test based on the discriminating capacity of each marker and validate the value of the Matutes score adapted to our study population. Materials and Methods: This is a technical validation study with retrospective recruitment of 91 patients diagnosed with B-CLL according to clinical, cytological and biological results. Statistical analysis is performed by the SPSS software. Results: The ROC curve shows the discriminative power of each diagnostic marker. The combination of discriminatory markers (CD23, CD5, CD43) either in double or triple marking did not increase the power of the test; The diagnosis of B-CLL according to our threshold values is changed to a score value greater than or equal to 3. Conclusion: The analysis of each marker, using the ROC curve allows us to identify the most discriminating markers useful to orient the diagnosis by elaborating a diagnostic score

SWALKIT: A generic augmented walker kit to provide haptic feedback navigation assistance to people with both visual and motor impairments.

Feedback solutions are a privileged form of assistance in order to increase mobility and independence of people with both motor and visual impairments. Indeed, it empowers the ability of the person to make decisions and take actions based on the provided information. Moreover, it maintains the use of the walker, and thus the residual locomotor skills. We here propose the SWALKIT, an open-source, cost-efficient, lightweight, easy to install and generic augmented walker kit. The SWALKIT can be equipped on any walker without requiring modifications of the structure or advanced technical knowledge. Vibrotactile feedback is provided through the handles to indicate the proximity of obstacles on the way of the user. The open source project is reproducible thanks to the online repository https://github.com/IH2A/Swalkit. In this paper, we present the design of the SWALKIT based on a user-centered approach following target users and therapists guidelines. Then, we present a technical validation study performed with 14 able-bodied blindfolded participants on a cardboard circuit. They were asked to use a standard walker with and without activation of the SWALKIT system. Results of this pilot study showed the efficiency and reliability of the proposed solution. Finally, we provide feedback after 2 months of daily life use by a target user.

Measurements of illuminance in simulated daylight photodynamic therapy.

Simulated daylight photodynamic therapy (SDL-PDT) is a new treatment alternative for actinic keratosis. The aim of this study was to show how the illuminance that reaches the target skin area during SDL-PDT depends on the spatial positioning of the patient. In this technical validation study, illuminance from the SDL-PDT system IndoorLux© was measured at different angles, directions, and distances from the light sources corresponding to potential target skin areas. Using two different photometers, data from 63 measuring points at seven specific distances from the ceiling were collected at 0°, 45°, and 90° angles, respectively. Illuminance levels ≥12,000 lux were regarded as adequate. Hotspots were defined as adequate measurements in all directions at a specific measuring point at distances of 1.3, 1.5, and 1.8m from the light sources (i.e., the most common patient treatment positions). Adequate illuminance levels were more common with photometer 1 (73%) than photometer 2 (57%). Almost all illuminance levels were adequate at a 0° angle with both photometers. Adequate illuminance levels were observed at 82-93% of the measuring points at a 45° angle and 22-47% at a 90° angle. Hotspots were registered with both photometers at all measuring points at 0°; 59-79% of the measuring points at 45°; and 0-21% at 90°. Patient positioning is important during SDL-PDT. Adequate illuminance is achieved if target skin areas are positioned at 0°-45° angles relative to the light sources, but not at 90° angles.

Accuracy and precision of apparent diffusion coefficient measurements on a 1.5 T MR-Linac in central nervous system tumour patients

Background and purposeMRI linear accelerators (MR-Linacs) may allow treatment adaptation to be guided by quantitative MRI including diffusion-weighted imaging (DWI). The aim of this study was to evaluate the accuracy and precision of apparent diffusion coefficient (ADC) measurements from DWI on a 1.5 T MR-Linac in patients with central nervous system (CNS) tumours through comparison with a diagnostic scanner. Materials and methodsCNS patients were treated using a 1.5 T Elekta Unity MR-Linac. DWI was acquired during MR-Linac treatment and on a Philips Ingenia 1.5 T. The agreement between the two scanners on median ADC over the gross tumour/clinical target volumes (GTV/CTV) and in brain regions (white/grey matter, cerebrospinal fluid (CSF)) was computed. Repeated scans were used to estimate ADC repeatability. Daily changes in ADC over the GTV of high-grade gliomas were characterized from MR-Linac scans. ResultsDWI from 59 patients was analyzed. MR-Linac ADC measurements showed a small bias relative to Ingenia measurements in white matter, grey matter, GTV, and CTV (bias: –0.05 ± 0.03, –0.08 ± 0.05, –0.1 ± 0.1, –0.08 ± 0.07 μm2/ms). ADC differed substantially in CSF (bias: –0.5 ± 0.3 μm2/ms). The repeatability of MR-Linac ADC over white/grey matter was similar to previous reports (coefficients of variation for median ADC: 1.4%/1.8%). MR-Linac ADC changes in the GTV were detectable. ConclusionsIt is possible to obtain ADC measurements in the brain on a 1.5 T MR-Linac that are comparable to those of diagnostic-quality scanners. This technical validation study adds to the foundation for future studies that will correlate brain tumour ADC with clinical outcomes.

HPV Sequencing Facilitates Ultrasensitive Detection of HPV Circulating Tumor DNA.

Human papillomavirus (HPV) DNA offers a convenient circulating tumor DNA (ctDNA) marker for HPV-associated malignancies, but current methods, such as digital PCR (dPCR), provide insufficient accuracy for clinical applications in patients with low disease burden. We asked whether a next-generation sequencing approach, HPV sequencing (HPV-seq), could provide quantitative and qualitative assessment of HPV ctDNA in low disease burden settings. We conducted preclinical technical validation studies on HPV-seq and applied it retrospectively to a prospective multicenter cohort of patients with locally advanced cervix cancer (NCT02388698) and a cohort of patients with oropharynx cancer. HPV-seq results were compared with dPCR. The primary outcome was progression-free survival (PFS) according to end-of-treatment HPV ctDNA detectability. HPV-seq achieved reproducible detection of HPV DNA at levels less than 0.6 copies in cell line data. HPV-seq and dPCR results for patients were highly correlated (R 2 = 0.95, P = 1.9 × 10-29) with HPV-seq detecting ctDNA at levels down to 0.03 copies/mL plasma in dPCR-negative posttreatment samples. Detectable HPV ctDNA at end-of-treatment was associated with inferior PFS with 100% sensitivity and 67% specificity for recurrence. Accurate HPV genotyping was successful from 100% of pretreatment samples. HPV ctDNA fragment sizes were consistently shorter than non-cancer-derived cell-free DNA (cfDNA) fragments, and stereotyped cfDNA fragmentomic patterns were observed across HPV genomes. HPV-seq is a quantitative method for ctDNA detection that outperforms dPCR and reveals qualitative information about ctDNA. Our findings in this proof-of-principle study could have implications for treatment monitoring of disease burden in HPV-related cancers. Future prospective studies are needed to confirm that patients with undetectable HPV ctDNA following chemoradiotherapy have exceptionally high cure rates.

Test\u2013retest assessment of non-contrast MRI sequences to characterise and quantify the small bowel wall in healthy participants

ObjectiveQuantitative Magnetic Resonance Imaging sequences have been investigated as objective imaging biomarkers of fibrosis and inflammation in Crohn’s disease.AimTo determine the repeatability and inter- and intra-observer agreement of these measures in the prepared small bowel wall.MethodsTen healthy participants were scanned at 3 T on 2 separate occasions using T1 and T2 relaxometry, IVIM-DWI and MT sequences. Test–retest repeatability was assessed using the coefficient of variation (CoV) and intra-class correlation coefficients (ICCs) were used to evaluate the intra- and inter-observer agreementResultsTest–retest repeatability in the bowel wall was excellent for apparent diffusion coefficient (ADC), magnetisation transfer ratio (MTR), T1, and diffusion coefficient D (CoV 5%, 7%, 8%, and 10%, respectively), good for perfusion fraction (PF) (CoV 20%) and acceptable for T2 (CoV 21%). Inter-observer agreement was good for the T2, D and ADC (ICC = 0.89, 0.86, 0.76, respectively) and moderate for T1 (ICC = 0.55). Intra-observer agreement was similar to inter-observer agreement.DiscussionThis study showed variable results between the different parameters measured. Test–retest repeatability was at least acceptable for all parameters except pseudo-diffusion coefficient D*. Good inter- and intra-observer agreement was obtained for T2, ADC and D, with these parameters performing best in this technical validation study.

Technical validation studies of a dual-wavelength LED-based photoacoustic and ultrasound imaging system

Recent advances in high power, pulsed, light emitting diodes (LEDs) have shown potential as fast, robust and relatively inexpensive excitation sources for photoacoustic imaging (PAI), yet systematic characterization of performance for biomedical imaging is still lacking. We report here technical and biological validation studies of a commercial dual-wavelength LED-based PAI and ultrasound system. Phantoms and small animals were used to assess temporal precision. In phantom studies, we found high temporal stability of the LED-based PAI system, with no significant drift in performance observed during 6 h of operation or over 30 days of repeated measurements. In vivo dual-wavelength imaging was able to map the dynamics of changes in blood oxygenation during oxygen-enhanced imaging and reveal the kinetics of indocyanine green contrast agent inflow after intravenous administration (Tmax∼6 min). Taken together, these studies indicate that LED-based excitation could be promising for future application in functional and molecular PAI.

Abstract A45: HPV sequencing facilitates ultrasensitive detection of HPV circulating tumor DNA

Abstract Background: Human papillomavirus (HPV)-associated cancers often present with locoregionally confined disease and are treated with curative intent. An emerging treatment paradigm includes radical therapy (i.e., chemoradiotherapy or surgery) followed by adjuvant treatment. Accurate detection of minimal residual disease (MRD) following radical therapy could enable personalized use of adjuvant treatment. The HPV genome offers a convenient circulating tumor (ct)DNA marker for HPV-associated cancers, but current methods such as digital (d)PCR provide insufficient accuracy for accurate MRD detection and for other clinical applications in patients with low disease burden. We asked whether a next-generation sequencing approach (HPV-seq) could provide quantitative and qualitative assessment of HPV ctDNA in low-disease-burden settings. Methods: We conducted preclinical technical validation studies on HPV-seq using cervix cancer cell lines. We then applied HPV-seq retrospectively to a prospective multicenter cohort study of locally advanced cervix cancer patients accrued from 2015 to 2016 (NCT02388698). Patients were treated with radical chemoradiotherapy with blood obtained at baseline, end of treatment, and 3 months post-treatment. Median follow-up was 27.5 months. In 38 plasma samples from 17 patients, HPV-seq results were compared with dPCR. The primary outcome was progression-free survival according to end-of-treatment HPV ctDNA detectability. Results: HPV-seq achieved reproducible detection of HPV DNA at levels <0.01%. HPV-seq and dPCR results were highly correlated (R=0.98, p=1.8E-22) with HPV-seq detecting ctDNA at levels down to 0.001% in dPCR-negative post-treatment samples. Detectable HPV ctDNA at the end-of-treatment timepoint was associated with inferior progression-free survival (log-rank p=0.05) with 100% sensitivity and 67% specificity for recurrence. Accurate HPV genotyping was successful from 100% of pretreatment plasma samples. HPV ctDNA fragment sizes were consistently shorter than non-cancer-derived cell-free DNA fragments (median fragment size difference: 22 bp) regardless of HPV genotype or clinical setting. Conclusions: HPV-seq is a quantitative method for ctDNA detection that outperforms dPCR. HPV-seq also reveals qualitative information about ctDNA fragments such as HPV genotype and ctDNA fragment length distribution. Our findings will have implications for MRD detection in HPV-related cancers. Future prospective studies are needed to confirm that patients with undetectable HPV ctDNA following chemoradiotherapy have exceptionally high cure rates. Citation Format: Eric Leung, Kathy Han, Jinfeng Zou, Zhen Zhao, Yangqiao Zheng, Ting Ting Wang, Lillian L. Siu, Trevor J. Pugh, Scott V. Bratman. HPV sequencing facilitates ultrasensitive detection of HPV circulating tumor DNA [abstract]. In: Proceedings of the AACR Special Conference on Advances in Liquid Biopsies; Jan 13-16, 2020; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(11_Suppl):Abstract nr A45.

On‐the‐Fly Calculation of Absorbed and Equivalent Atmospheric Radiation Dose in A Water Phantom with the Atmospheric Radiation Interaction Simulator (AtRIS)

AbstractComputation of the ionization and radiation dose in arbitrary (exo‐) planetary atmospheres due to energetic particles is recently becoming more important due to several reasons that are either correlated with the detection of trace gases for life on exoplanets or with computing dose rates at arbitrary altitudes in the Earth atmosphere. We previously presented Atmospheric Radiation Interaction Simulator, a new Geant4‐based code tailored specifically to enable parametric studies of radiation propagation through exoplanetary atmospheres (Banjac et al., 2019 https://doi.org/10.1029/2018JA026042). Therein, the calculation of ion‐electron pair production rates, which are a mandatory input for chemical and atmospheric modeling, has been presented and validated against Earth measurements and also other, similar, but solar‐system‐specific Geant4‐based codes (PLANETOCOSMICS). In addition to providing input for atmospheric modeling of exoplanets, with AtRIS we aim to directly characterize the habitability by calculating the absorbed dose. In this technical validation study, after showing a detailed analysis of the secondary particles contributing to the atmospheric radiation, we describe a feature of the code which makes direct parametric studies of the interrelation of incident radiation and the resulting absorbed dose throughout the atmosphere possible. In a validation case study configured using an atmospheric model obtained with NRLMSISE‐00 and a primary proton and helium GCR flux calculated using a recent improvement of the force‐field approach, we have compared simulation results with measurements obtained with the Flight Radiation Environment Detector (FRED). We show that Atmospheric Radiation Interaction Simulator (AtRIS) can reproduce the measured dose rate dependence on altitude.

Cell-free DNA analysis in healthy individuals by next-generation sequencing: a proof of concept and technical validation study

Pre-symptomatic screening of genetic alterations might help identify subpopulations of individuals that could enter into early access prevention programs. Since liquid biopsy is minimally invasive it can be used for longitudinal studies in healthy volunteers to monitor events of progression from normal tissue to pre-cancerous and cancerous condition. Yet, cell-free DNA (cfDNA) analysis in healthy individuals comes with substantial challenges such as the lack of large cohort studies addressing the impact of mutations in healthy individuals or the low abundance of cfDNA in plasma. In this study, we aimed to investigate the technical feasibility of cfDNA analysis in a collection of 114 clinically healthy individuals. We first addressed the impact of pre-analytical factors such as cfDNA yield and quality on sequencing performance and compared healthy to cancer donor samples. We then confirmed the validity of our testing strategy by evaluating the mutational status concordance in matched tissue and plasma specimens collected from cancer patients. Finally, we screened our group of healthy donors for genetic alterations, comparing individuals who did not develop any tumor to patients who developed either a benign neoplasm or cancer during 1–10 years of follow-up time. To conclude, we have established a rapid and reliable liquid biopsy workflow that allowed us to study genomic alterations with a limit of detection as low as 0.08% of variant allelic frequency in healthy individuals. We detected pathogenic cancer mutations in four healthy donors that later developed a benign neoplasm or invasive breast cancer up to 10 years after blood collection. Even though larger prospective studies are needed to address the specificity and sensitivity of liquid biopsy as a clinical tool for early cancer detection, systematic screening of healthy individuals will help understanding early events of tumor formation.

Abstract 4591: Integration of genomic analysis and assessment of pre-analytic variables in the HD-SCA workflow: A technical validation study

Abstract The liquid biopsy allows assessment of multiple biological analytes over time to provide dynamic temporal information with the potential for improving clinical management and guiding treatment decisions. While the promise of liquid biopsies for prediction and response monitoring are intensely investigated, the pre-analytic variables are of primary concern for its implementation in diagnostic clinical medicine, including such categories as collection method, shipping conditions, and sample storage. Here we utilize an integrated high-definition single cell analysis (HD-SCA) workflow for genomic analysis of rare cells and cfDNA from the liquid biopsy to characterize the effects of pre-analytical variation and reproducibility of data analysis from the same cohort of patients. The results presented here confirm consistent rare cell enumeration and morphometric characterization between 24h and 48h time to assay (TTA), with a high efficiency and capacity for both copy number variation (CNV) and single nucleotide variation (SNV) analysis at the single cell level. Additionally, the freezing process neither diminishes or increases the DNA quantity, nor does it affect the DNA quality for CNV or SNV analysis. The integration of genomic information is imperative to reveal essential disease-implicated mutational profiles, but effective molecular diagnostic tests require reproducible coverage over a broad dynamic range. The performance of the HD-SCA platform quantified here may be utilized as a guide for implementation into patient care and/or research biorepository processes. Citation Format: Stephanie Nicole Shishido, Mariam Rodriguez_Lee, Anand Kolatkar, Liya Xu, Sara Restrepo-Vassalli, Lisa Welter, Anders Carlsson, Emily Greenspan, Shelley Hwang, Kathryn Waitman, Jorge Nieva, Kelley Bethel, James Hicks, Peter Kuhn. Integration of genomic analysis and assessment of pre-analytic variables in the HD-SCA workflow: A technical validation study [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4591.

TRAnexamic acid in hemorrhagic CESarean section (TRACES) randomized placebo controlled dose-ranging pharmacobiological ancillary trial: study protocol for a randomized controlled trial

BackgroundEvidence increases that a high or a standard dose of tranexamic acid (TA) reduces postpartum bleeding. The TRACES pharmacobiological substudy aims to establish a therapeutic strategy in hemorrhagic (H) Cesarean section (CS) with respect to the intensity of fibrinolysis by using innovative assays.Method/DesignThe TRACES trial is a multicenter, randomized, double-blind, placebo-controlled, TA dose-ranging study that measures simultaneously plasmatic and uterine and urine TA concentrations and the plasmin peak inhibition tested by a simultaneous thrombin plasmin generation assay described by Van Geffen (novel hemostasis assay [NHA]). Patients undergoing H CS (>800 mL) will receive blindly TA 0.5 g or 1 g or placebo. A non-hemorrhagic (NH) group will be recruited to establish plasmin generation profile. Venous blood will be sampled before, at the end, and then at 30, 60, 120, and 360 min after injection. Uterine bleeding will be sampled after injection. Urine will be sampled 2 h and 6 h after injection. The number of patients entered into the study will be 114 H + 48 NH out of the 390 patients of the TRACES clinical trial.DiscussionTo explore the two innovative assays, a preliminary pilot study was conducted. Blood samples were performed repeatedly in patients undergoing either a H (>800 mL) or NH (<800 mL) CS and in non-pregnant women (NP). H patients received TA (0–2 g). Dose-dependent TA plasmatic concentrations were determined by LC-MS/MS quantification. Plasmin generation and its inhibition were tested in vitro and in vivo using the simultaneous thrombin–plasmin generation assay (STPGA). The pilot study included 15 patients in the H group, ten patients in the NH group, and seven patients in the NP group. TA plasmatic concentration showed a dose-dependent variation. STPGA inter-assay variation coefficients were < 20% for all plasmin parameters. Inter-individual dispersion of plasmin generation capacity was higher in H and NH groups than in NP group. Profile evolution over time was different between groups. This preliminary technical validation study allows TRACES pharmacobiological trial to be conducted.Trial registrationClinicalTrials.gov, NCT02797119. Registered on 13 June 2016.

Defining the performance parameters of a rapid screening tool for myotonic dystrophy type 1 based on triplet-primed PCR and melt curve analysis

ABSTRACTBackground: DMPK CTG-repeat expansions that cause myotonic dystrophy type 1 (DM1) can be detected more rapidly, cost-effectively, and simply by combining triplet-primed PCR (TP-PCR) with melting curve analysis (MCA). We undertook a detailed technical validation study to define the optimal operational parameters for performing bidirectional TP-PCR MCA assays.Methods: We determined the assays’ analytic specificity and sensitivity, assessed the effect of reaction volumes, DNA diluents, and common contaminants on melt peak temperature, determined the assays’ sensitivity in detecting low-level mosaicism for repeat expansion, and evaluated their performance on two real-time PCR platforms.Results: Both assays were highly specific and sensitive, and performed optimally under a broad range of parameters. Bidirectional TP-PCR MCA analysis also reduces the risk of generating false-negative results associated with the rare CCG-interruptions that may be present at either end of expanded alleles.Conclusion: The DMPK TP-PCR MCA is a highly specific, sensitive, and significantly cost-saving screening tool for DM1.

First report of the point-of-care TEG: A technical validation study of the TEG-6S system

Thrombelastography (TEG) measured by the TEG5000 Hemostasis Analyzer is an established but the labor-intensive method for assessing global hemostasis. The first true point-of-care TEG, the TEG6s system, uses resonance-frequency viscoelasticity measurements and a disposable multi-channel microfluidic cartridge to assess hemostasis and response to antiplatelet therapy. TEG assays (n = 5,100) were performed on the blood of healthy volunteers (n = 157) and patients undergoing coronary revascularization at three hospitals (n = 300). The results from the TEG6s were compared with the conventional TEG5000 in accordance with Clinical and Laboratory Standards Institute (CLSI) and FDA recommendations. Precision testing was conducted using blood from healthy donors, all assays were run for 5 consecutive days in duplicate using multiple operators, lots, and instruments. Reference ranges were comparable between the TEG systems.Deming regression analysis demonstrated a strong correlation between the two systems for the standard hemostasis tests (R r = 0.932, MA r = 0.972, LY30 r = 0.938). Method comparison analysis showed an acceptable agreement between PlateletMapping (PM) assays for measuring arachidonic acid (indicator of aspirin response)- and adenosine diphosphate (indicator of P2Y12 inhibitor response)-induced platelet aggregation (total agreement = 90%, and 72%, respectively). TEG6s precision testing yielded low variability (CV 0–13%) in all measures. The new point-of-care TEG6s is associated with greater ease of use than the TEG5000 and provides precise results. The results correlated between methods for all variables. TEG6s is a promising device for near-patient hemostasis monitoring and future trials of personalized therapy designed to reduce bleeding and thrombosis.

Enzymatic Screening and Diagnosis of Lysosomal Storage Diseases.

Lysosomal storage diseases (LSDs) are a group of more than 50 genetic disorders. Clinical symptoms are caused by the deficiency of specific enzyme (enzymes) function and resultant substrate accumulation in the lysosomes, which leads to impaired cellular function and progressive tissue and organ dysfunction. Measurement of lysosomal enzyme activity plays an important role in the clinical diagnosis of LSDs. The major enzymatic testing methods include fluorometric assays using artificial 4-methylumbelliferyl (4-MU) substrates, spectrophotometric assays and radioactive assays with radiolabeled natural substrates. As many effective treatment options have become available, presymptomatic diagnosis and early intervention are imperative. Many methods were developed in the past decade for newborn screening (NBS) of selective LSDs in dried blood spot (DBS) specimens. Modified fluorometric assays with 4-MU substrates, MS/MS or LC-MS/MS multiplex enzyme assays, digital microfluidic fluorometric assays, and immune-quantification assays for enzyme contents have been reported in NBS of LSDs, each with its own advantages and limitations. Active technical validation studies and pilot screening studies have been conducted or are ongoing. These studies have provided insight in the efficacy of various methodologies. In this review, technical aspects of the enzyme assays used in clinical diagnosis and NBS are summarized. The important findings from pilot NBS studies are also reviewed.

Fragile X–related element 2 methylation analysis may provide a suitable option for inclusion of fragile X syndrome and/or sex chromosome aneuploidy into newborn screening: a technical validation study

We show that a novel fragile X-related epigenetic element 2 FMR1 methylation test can be used along with a test for sex-determining region Y (SRY) to provide the option of combined fragile X syndrome and sex chromosome aneuploidy newborn screening. Fragile X-related epigenetic element 2, SRY, and FMR1 CGG repeat analyses were performed on blood and saliva DNA, and in adult and newborn blood spots. The cohort consisted of 159 controls (CGG <40), 187 premutation (CGG 56-170), and 242 full-mutation (CGG ~200-2,000) males and females, 106 sex chromosome aneuploidy individuals, and 151 cytogenetically normal controls. At the 0.435 threshold, fragile X-related epigenetic element 2 analysis in males was robust on both blood DNA and newborn blood spots, with specificity and sensitivity of ~100% for full-mutation genotype. In females, the specificity was 99%, whereas half of full-mutation females were above the 0.435 threshold in both blood DNA and newborn blood spots. Furthermore, at this threshold, the test could not differentiate individuals with Klinefelter syndrome from female controls without using the SRY marker. When combined with SRY analysis, the test was consistent with most results for sex chromosome aneuploidies from karyotyping. Setting specific thresholds for fragile X-related epigenetic element 2 analysis and including the SRY marker provides the option to either include or exclude detection of sex chromosome aneuploidies as part of fragile X syndrome newborn screening.